Review Article Insulin-Like Growth Factor System in …downloads.hindawi.com/journals/bmri/2015/538019.pdfReview Article Insulin-Like Growth Factor System in Cancer: Novel Targeted

Review ArticleInsulin-Like Growth Factor System in CancerNovel Targeted Therapies

Varsha P Brahmkhatri1 Chinmayi Prasanna1 and Hanudatta S Atreya12

1NMR Research Centre Indian Institute of Science Bangalore 560012 India2Solid State and Structural Chemistry Unit Indian Institute of Science Bangalore 560012 India

Correspondence should be addressed to Hanudatta S Atreya hsatreyagmailcom

Received 7 July 2014 Revised 13 October 2014 Accepted 20 October 2014

Academic Editor Eileen M McGowan

Copyright copy 2015 Varsha P Brahmkhatri et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Insulin-like growth factors (IGFs) are essential for growth and survival that suppress apoptosis and promote cell cycle progressionangiogenesis andmetastatic activities in various cancersThe IGFs actions aremediated through the IGF-1 receptor that is involvedin cell transformation induced by tumour These effects depend on the bioavailability of IGFs which is regulated by IGF bindingproteins (IGFBPs) We describe here the role of the IGF system in cancer proposing new strategies targeting this system We haveattempted to expand the general viewpoint on IGF-1R its inhibitors potential limitations of IGF-1R antibodies and tyrosine kinaseinhibitors and IGFBP actionsThis review discusses the emerging view that blocking IGF via IGFBP is a better option than blockingIGF receptors This can lead to the development of novel cancer therapies

1 Introduction

Insulin-like growth factor (IGF) is a natural growth hormoneand plays crucial role in normal growth and developmentThe IGF family is comprised of insulin and two factors similarto insulin termed IGF-1 and IGF-2These factors directly reg-ulate cellular functions by interacting with specific cell sur-face receptors and activating various intracellular signallingcascadesThe cellular responses to the IGFs are mediated pri-marily by the IGF-1 receptorThe IGF-1 receptor is a memberof the family of tyrosine kinase growth factor receptors

IGFs actions are regulated by six soluble IGF binding pro-teins (IGFBPs) and IGFBP proteasesThe IGFBPs comprise asuperfamily of six proteins (IGFBP-1-6) that bind to IGFswithhigh affinity and specificity and a family of IGFBP-relatedproteins (IGFBP-rPs) which are structurally similar to theIGFBPs but bind IGFs with much lower affinity

IGF-1 circulates in relatively high concentrations inplasma approximately 150ndash400 ng per mL where it mostlyexists as the protein-bound form The free ligand concentra-tion is very little that is less than 1 [1] IGFs in circulationare protected from degradation by forming a complex with

a family of high affinity IGF binding proteins (IGFBPs) [2]IGFBP-3 is the most abundant IGF binding protein in theblood stream followed by IGFBP-2 which is produced in theliver Most of the circulating IGF-1 and IGF-2 are associatedwith a high molecular weight complex sim150 kDa consistingof IGFBP-3 and the acid labile subunit (ALS) [2] Oncethe ternary complex dissociates the binary complexes ofIGFBP-IGF are removed from the circulation and cross theendothelium to reach the target tissues and to interact withcell surface receptors (Figure 1) In the tissues IGFBPs mayinhibit the interaction of the IGFs with their receptors as theIGFBPs have a higher affinity for the IGFs than the receptorsIn some cases IGFBPs can enhance IGF action in the localmicroenvironment by acting as a reservoir that can slowlyrelease the ligands In addition some IGFBPs can have IGF-independent effects on cells [2]

The IGFs are signalling proteins (sim75 kDa) whose actionsare mediated by the IGF-1R and access to the receptor isregulated by the IGFBPs which vary in size (sim22ndash31 kDa) andshare overall sequence and structural homology with eachotherThe IGFBPs bind strongly to IGFs (119870D sim 300ndash700 pM)and inhibit the action of IGFs by blocking their access to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 538019 24 pageshttpdxdoiorg1011552015538019

2 BioMed Research International

ProteasesIGF-1

IGF-1R

Extracellular matrix

Cytoplasm

IGFBP

IGFBP fragments

ALSIGF-1

IGF-1

IGF-1

ALS

Blood vesse

l

Proteolysis

Figure 1 The IGF axis circulating IGFs are protected fromdegradation by forming complex with IGFBPs IGFs apart fromtheir local functioning in an autocrine or a paracrine manner enterthe bloodstream where they exist as binary complexes with eachIGFBP In addition ternary complex also exists when the binarycomplexes with IGFBP-3 or IGFBP-5 interact with the acid labilesubunit (ALS) After dissociation of ternary complex the binarycomplexes of IGFBP-IGF are removed from the circulation and crossthe endothelium to reach the target tissues and to interact with cellsurface receptors

the receptors Proteolysis of the IGFBPs dissociates IGFs fromthe complex enabling them to bind and activate the cellsurface receptors Deregulation of IGF-1R signalling has beennoted to contribute to a variety of diseases including diabeticretinopathy [3] diabetic nephropathy [4] age-related macu-lar degeneration [5] cardiovascular disease and aging and ina variety of cancers [5]

IGF system is gaining tremendous interest over the lastdecade because it plays an important role in cancer The cur-rent treatment options for cancer have shifted more towardsthe targeted therapies [6 7] rather than the traditionalchemotherapy Many strategies have been exploited to targettumours The most commonly used strategy is engineeredantibodies or antibody fragments [8] Though monoclonalantibodies are very selective poor penetration inside thetumours and high production cost hinder their usage astherapeutic agents [9] Current therapeutics targeting theIGF-signalling pathways focus on blocking IGF-1R directlyandor its downstream effectors [10] However a potentialdrawback of such approaches is the resulting adverse sideeffects or toxicities due to its interference with the insulinpathway As a more efficacious alternative we propose thatIGFBPs can be developed as IGF-antagonist based cancertherapeutics serving to block the IGF-1R mediated tumourprogression Notably the IGFBPs do not bind insulin andthus do not interfere with insulin-insulin receptor interac-tions

In the current paper we will provide a brief overview onIGF systemanddiscuss some literature and experimental datareported to demonstrate the role of IGF system in cancer anddevelopment of new targeted anticancer therapies Because it

is not possible to provide a complete coverage of all publishedpapers dealing with IGF system we have mainly focusedon different strategies targeting IGF system in cancer andattempted to provide an overview on IGF system includingIGF-1R its inhibitors and potential limitations of IGF-1Rantibodies and tyrosine kinase inhibitors IGFBP actionsand blocking IGF via IGFBP (which is better option thanblocking IGF receptors) leading to development of novelcancer therapies

2 DiscoveryHistory of IGF System

The first member of IGF family to be identified was insulinwith subsequent investigation resulting in the elucidationof its role in glucose metabolism and its implication in theaetiology of diabetes mellitus This discovery effected anexplosion in the investigation of the structure function andmechanisms of action of insulin The enormous interest inthis molecule resulted in the concession of three Nobel Prizesfor the investigation of insulin in 1923 for the discovery ofits capacity to treat diabetes by Frederick Banting and J JMacleod [11] in 1958 for the first sequence of a protein byFrederick Sanger [12] and in 1963 for the first determinationof the three-dimensional structure of a protein by DorothyHodgkin [13] Hence the investigation of insulin has beena pioneer in many scientific fields Later the IGFs werediscovered and found to be intricately involved in embryonicdevelopment and postnatal growth

The existence of the IGFs was first proposed by Salmonand Daughaday in 1957 on the basis of studies indicat-ing that growth hormone (GH) did not directly stimulatethe incorporation of sulfate into cartilage but rather actedthrough a serum factor [14] In the original study by Salmonand Daughaday 35S-labeled amino acid was incorporatedinto cartilage explants and was used as a surrogate forgrowth The serum of normal rats induced 35S-amino acidincorporation into cartilage but not serum fromhypophysec-tomized rats However serum from hypophysectomized ratstreated with GH yielded serum that allowed for 35S-aminoacid incorporation indicating that a second messenger wasnecessary forGH signallingThis factorwas originally termedsulfation factor then somatomedin and ultimately insulin-like growth factor-1 and insulin-like growth factor-2 IGF-1 was not purified and characterized until more than twodecades later [15] The terminology ldquoinsulin-likerdquo was usedbecause these factors are able to stimulate glucose uptake intofat cells and muscle and indeed both IGF-1 and IGF-2 showapproximately 50 homology with insulin [15 16]

Subsequent investigation demonstrated that GH afterbinding to its transmembrane receptor initiates a signallingcascade leading to transcriptional regulation of IGF-1 andrelated genes It was originally thought that systemic growthwas promoted by GH acting mainly on the liver to stimulateIGF-1 production which then reached target tissues viathe circulation to activate mechanisms involved in tissueproliferation growth and metabolism It is now evident thatnot only does GH have independent actions that do notinvolve IGF-1 production [17] but IGF-1 synthesis occurs

BioMed Research International 3

HypothalamusPituitary

Muscles Bone Fat cells

GHRH GHIHsomatostatin

Hypothalamus

Pituitary

Liver

IGF-1

GH

Figure 2 Growth hormone-releasing hormone (GHRH) is ahormone produced by the hypothalamus which stimulates thepituitary gland to produce GH Somatostatin secreted by the cellsof hypothalamus and also by the cells of stomach intestine andpancreas that inhibits GH production When pituitary secretes GHinto the bloodstream it results in the production of IGF-1 in the liverIGF-1 is the factor that actually causes the growth of bones and othertissues of the body It also plays an important role in signalling thepituitary to reduce GH production

in many tissues under the control of a variety of local andcirculating factors whichmay ormaynot includeGH[18ndash21]Furthermore this local production of IGF-1 may be directlyresponsible for the growth promoting effects of GH ratherthan the circulating growth factor [22]

21 IGF-1 Synthesis and Secretion IGF functions as both acirculating hormone and as a tissue growth factor Liver isthe production house for the most circulating IGFs that aresubject to both hormonal and nutritional factors Growthhormone (GH) which is produced in the pituitary glandunder the control of the hypothalamic factors stimulatesIGF-1 production (Figure 2)

The insulin like growth factor binding proteins (IGFBPs)are also synthesized in the liver The IGF ligands in additionto the IGFBPs are delivered in an endocrine manner throughthe circulation from the liver to act in IGF-responsive tissuesIGFs and IGFBPs are also produced in other organs whereautocrine or paracrine mechanisms take place frequentlyinvolving interactions between stromal and epithelial cellpopulations [23]

22 Autocrine and Paracrine Actions of IGF The insulin-likegrowth factors play a major role in regulating cell prolifera-tion and inhibiting apoptosis The IGFs are expressed ubiq-uitously and act in an autocrineparacrine manner throughbinding to the IGF-1 receptor (IGF-1R) The bioavailabilityof IGF in tissues is determined by both local and systemicfactors The local factors include the levels of receptorsthat are expressed various IGF binding proteins (IGFBPs)and IGFBP proteases The systemic factors involved aremainly those that regulate the circulating levels of IGFs such

as growth hormone (GH) and various nutritional factorsStudies in cultured cells have demonstrated that the IGF-1Ris frequently overexpressed in cancer cell lines

The IGFs are not stored within cells of a specific tissuebut are present at very high levels throughout the body Theycirculate at total concentrations approximately 1000 timeshigher than those of most peptide hormones and althoughtissue levels are somewhat lower they are still present invast excess compared to that required for maximal cellularstimulation These high levels are maintained due to theirassociation with the IGFBPs which dramatically slow theirclearance The IGFBPs bind the IGFs with greater affinitythan their cell surface receptors enabling them to tightlycontrol tissue activity The IGFBP proteases modify theIGFBPs lowering the affinity with which they bind IGFs Inthe tissues the IGFs are regulators of cell survival growthmetabolism and differentiated function the complex systemconfers specificity on these actions

23 Evidence for ParacrineAutocrine IGF-1 Actions from Stud-ies of TransgenicMice Themost convincing evidence of localIGF-1 actions comes from lines of transgenic (Tg) mice madeto overexpress IGF-1 in specific tissues for example brainmammary gland andmuscle Each of these Tgmousemodelsexhibits specific overgrowth in the organ or tissue of IGF-1overexpression and none has an alteration in circulating IGF-1 levels Reports of such mouse models are summarized inTable 1 In every model studied biologic actions in the organof IGF-1 transgene expression have been demonstrated IGF-1 therefore can exert local in vivo actions

Other experiments that address IGF-1 local actions arethe generation of Tgmice that overexpress IGFBPs in specifictissues Here the expectation is that these IGFBPs will inhibitthe actions of locally expressed IGF-I Such studies haveyielded results consistent with those obtained from studiesof site-specific IGF-1 overexpression An example is theoverexpression of rat IGFBP-4 in smooth muscle driven bythe regulatory region of the 120572-actin gene [24] TransgeneIGFBP-4 expression results in smooth muscle hypoplasiaThe lack of any change in circulating IGFBP-4 or IGF-1and the restriction of hypoplasia to smooth muscle arguefor the inhibition of IGF-1 growth promoting effects onsmooth muscle Alternative but unlikely interpretations arethat IGFBP-4 inhibited the actions of IGF-1 derived fromthe circulation andor that IGFBP-4 inhibits growth bymechanisms independent of IGF-I Other Tg mouse modelshave yielded consistent results For example a number oflines of IGFBP-1 Tg mice exhibit organ growth retardationthat appears due to the capacity of IGFBP-1 to inhibit IGFactivity in specific tissues for example in brain [25ndash27]

3 IGF Receptors (IGF-Rs)

The IGF system comprises two main receptors (IGF-1R andIGF-2R) Both IGFIR and IGF-2R are transmembrane glyco-proteins that differ completely in their structure and function[19ndash21 28ndash31] IGF-1R is a tetramer which comprised twoequal 120572-subunits and two equal 120573-subunits [28 29 32] IGF-1R resembles the insulin receptor at structural level with 60


Table 1 IGF-1 transgenic mice with tissue-specific IGF-1 overexpression

Organs IGF-1 action Promoter ReferenceBrain Increased brain size characterized by increased neuron number M IGF-2 51015840 flanking region [35 36 181 182]Bone Increased trabecular bone Bovine osteocalcin [37]Heart Increased myocyte proliferation r 120572myosin heavy chain [38]Muscle skeletal Stimulates differentiation and myofibril hypertrophy Avian skeletal 120572 actin [39]

Muscle smooth

Smooth muscle hyperplasia in many flanking fragmentsorganstissuesIncreased vascular contractilityEnhanced neointimal formation after injury

r smooth muscle 120572 actin (mSMA) [183ndash185]

Ovary Increased testosterone and cyst m LH receptor [186]Prostate Epithelial neoplasia Bovine keratin-5 [187]

Thyroid When the IGF-1R is also overexpressed there is a decreasedTSH requirement and goiter Bovine thyroglobulin [188]

homology IGFs and insulin are proficient to cross-bind toeach otherrsquos receptor although with much weaker bindingaffinity than that for the preferred ligand [33 34] IGF-1R andIR can hybridize to form a heterodimer composed of one 120572-subunit and one 120573-subunit of each receptor [28 30] as shownin Figure 3 The amount of insulinIGF-1 hybrid receptorvaries significantly from tissue to tissue Since its bindingaffinity for IGF-1 is higher than that for insulin the receptoris thought to function principally as an IGF-1 receptor but itsbiologic significance remains mostly unidentified

The postreceptor signal transduction events includephosphorylation of insulin receptor substrate (IRS) family ofproteins and activation of phosphatidylinositol-3 (PI-3) andmitogen-activated protein kinases (MAPK) [19 35] This willresult in a myriad of events including the upregulation ofcyclin D1 leading to the phosphorylation of retinoblastomaprotein and expression of downstream target genes such ascyclin E [36 37] Moreover IGF-1R activation downregulatesthe cell-cycle suppressors like PTEN [38 39] indicating thatmultiple pathways are involved in producing its mitogeniceffect Activated IRSs trigger the activation of two intracellu-lar signaling networks RasRafMekErk and PI3K pathwaysThe first one is mainly involved in mediating the mitogeniceffect of insulin and IGFs while the PI3K pathway via Aktmediates both metabolic and cell growth responsesThe Akt-mediated metabolic effects are induced by the activationof enzymes involved in gluconeogenesis glucose uptakeprotein synthesis and lipogenesis whereas the cell growthresponses are mainly induced by the mTOR pathway

IGF-2R is monomeric [29 40ndash42] the largest transmem-brane receptor that is completely unrelated to the IGF-1Rand insulin receptor (IR) In the extracellular domain ofthe receptor three ligand-binding regions are found one forIGF-2 binding and two for proteins containing mannose-6-phosphate (M6P) and the dormant form of transforminggrowth factor- (TGF-) 120573 [30] Binding of IGF-2R to TGF-120573 activates the latter [40 43] IGF-2R is also called theIGF-IIM6P receptor as it can bind both IGF-2 and M6P-containing Molecules The expression of IGF-1R is regulatedby steroid hormones and growth factors [29 32] Since highIGF-1 levels result in a low levels of IGF-1R IGFs may act as

negative feedback signals to suppress expression of IGF-1R[44 45] In contradiction of the effect of IGFs other growthfactors including basic FGF PDGF and EGF stimulateIGF-1R expression [32 46 47] The expression of IGF-1Ris also stimulated by estrogens glucocorticoids GH FSHluteinizing hormone and thyroid hormones [28 32] Onthe other hand tumour suppressor gene products such aswild type p53 protein and WT1 (Wilmsrsquo tumour protein)inhibit expression of IGF-1R [11 48ndash50] IGF-1R levels are alsoaffected by nutrition [13 51 52] Not much is known aboutthe regulation of IGF-2R expression although some studies[29 30 53 54] have suggested that insulin IGFs EGF andM6P may increase the level of IGF-2R in the cell membraneBinding of IGFs to IGF-1R activates the receptorrsquos tyrosinekinase activity which starts a cascade of reactions amonga number of molecules involved in the signal transductionpathway (Figure 3)

IGF-2R acts as a scavenger for circulating IGF-2 uniquelyThe extracellular domain of the receptor disassociates uponproteolytic cleavage from the cell membrane as a solublefragment circulating in the blood with the ability to bind toIGF-2 and facilitate its degradation [55ndash60]These receptorsadditionally to the IGFBPs provide an extra control on thecirculating levels of IGF-II

4 Insulin-Like Growth Factor BindingProteins (IGFBPs)

The insulin-like growth factor binding proteins (IGFBPs)were originally discovered while purifying IGF-1 from serum[61 62] The insulin-like growth factors (IGFs) are presentin extracellular fluids bound to high affinity carrier pro-teins (Table 2) Six forms of IGF binding proteins (IGFBPs)have been cloned and their complete sequences have beenobtained [63]

IGFBPs have three domains Human IGFBPs 1ndash6 eachcontain 216ndash289 amino acids organized into three domainsof approximately equal size with the conserved N- and C-domains being joined by a ldquolinkerrdquo L-domain [2 64] IGFBPs1ndash5 have 18 conserved cysteines whereas IGFBP-6 has 16


Table 2 Human insulin-like growth factor binding proteins

IGFBPs Mass (kDa) Source of purification Relative bindingaffinity for IGFs

IGFBP1 250 Amniotic fluid placenta IGFI = IGFIIIGFBP-2 313 BRL-3A and MDBK cells human serum IGFII gt IGFIIGFBP-3 287 Plasma IGFI = IGFIIIGFBP-4 259 Human osteosarcomas prostatic carcinoma colon carcinoma and glioblastoma IGFI = IGFIIIGFBP-5 285 C2 myoblasts conditioned media human bone IGFI = IGFIIIGFBP-6 228 Cerebrospinal fluid human serum IGFII gt IGFI

Ins

IGF-2

IGF-1

Survival Proliferation Metastasis Metabolism

IR-B IR-A IRA IRB IGF-1R IGF-2RIGF-1RIGF-1R

Figure 3 IGF receptor signalling IGF-1R is a tetramer of twoidentical 120572-subunits and two identical 120573-subunits IGF-1R and IRcan hybridize to form a heterodimer composed of one 120572-subunitand one 120573-subunit of each receptor Formation of hybrid receptors isexplainedwith different colour code scheme IGF-IIR themannose-6-phosphate (M6P) receptor has high affinity for binding the IGF-IIligand but is a nonsignalling receptorThe biological activities of theIGF ligands are mediated by IGF-IR but the IGF-IIR is consideredto function as a ldquosinkrdquo that controls the local bioavailability of IGFligands for binding to the IGF-IR

[2 65] The N-domains of IGFBPs 1ndash5 contain six disulfidesand share a conserved GCGCC motif IGFBP-6 shares allof these except the two adjacent cysteines in this motifTherefore the first three N-terminal disulfide linkages ofIGFBP-6 differ from those of IGFBP-1 and by implicationthe other IGFBPs [65] By contrast the remaining N-domaindisulfides and all three C-domain disulfides are probablyconserved in all IGFBPs

The sequence alignment of IGFBPs 1ndash6 is depicted inFigure 4 where the N-domains of IGFBP 1ndash5 contain sixdisulfides and share a conserved GCGCC motif IGFBP-6 shares all of these except the two adjacent cysteines inthis motif The C-domains are known to share the highlyconserved CWCV motif But the central domains do notcontain any cysteines and exhibit little homology

The six IGF binding proteins are unrelated to the cellsurface receptors but are structurally very closely related toeach other although they are each products of distinct genesand they all have very distinct functional properties Table 3

summarizes the results of inhibiting IGFBPs activity and theirrole in cancer

41 IGFBP Proteases Ever since the discovery of IGFBP-3protease in seminal plasma [66] andhumanpregnancy serum[67] IGFBP proteases have been known to be present invarious body fluids [68] IGFBP proteases belong to a super-family of proteases with specificity towards IGFBPs therebyregulating the action of IGFBPs These proteases are primefactors in modulating the levels of IGFBPs and ultimately thebioactivity and downstream actions of IGFs [69]

IGFBP proteases broadly fall into three major superfamiliesmdashserine proteinases (kallikrein-like serine protease)matrix metalloproteinases (MMPs) and cathepsins [70 71]The work of Cohen et al demonstrating the significance ofIGFBP proteases and a descriptive review by Fowlkes talkmiles about their classification [70 71] Table 4 summarizesdifferent IGFBP proteases and their target substrates withtarget sequence specificity

Prostate specific antigen (PSA) the first IGFBP proteaseto be discovered in seminal plasma [66] and later on inpregnancy serum [72] is a serine proteinase produced bythe prostate gland and is known to degrade IGFBP-3 [66]120574-nerve growth factor (NGF) homologous to PSA is alsoknown to degrade IGFBP-3 and IGFBPs 4 5 and 6 therebyenhancing IGF actions Epidermal growth factor bindingprotein (EGFBP) human plasma kallikrein (hPK) and reninare relatively poor IGFBP proteases [71]

Matrix metalloproteinases are calcium-dependent zinc-containing endopeptidases with the capability of degradingseveral extracellular matrix molecules including collagenselastins gelatin matrix glycoproteins and proteoglycan[71 73ndash75] These extracellular degrading enzymes are alsoknown to be active against IGFBPs [74] They were firstdiscovered as IGFBP-3 proteinases in human dermal fibrob-lasts [74 76] These MMPs are known to contribute to thedegradation of IGFBPs 1 2 3 4 and 5 known from variousscientific studies including a study showing the proteolyticcleavage of IGFPB-1 and IGFBP-2 by MMP-1 in smoothmuscle airway cells [71 73 76ndash78] Research has shown thatMMP-3 andMMP-9 can cleave IGFBP-1MMP-1 andMMP-3degraded rhIGFBP-3 to much greater extent than MMP-2 invitro [74] ADAM-12 a disintegrin metalloproteinase is alsoknown to have proteolytic activity against IGFBP-3 [77]

Cathepsins belong to a family of lysosomal proteinaseswith optimal activity in acidic conditions discovered by their


sp|P08833|IBP1 HUMAN






290 300 310 320 330 340

||||||||||||

FYHSRQCETSMDGEAGLCWCVYPWNGKRIPGSPEIRGDPNCQIYFNVQN-----------

LYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ

FYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK-------------

NFHPKQCHPALDGQRGKCWCVDRKTGVKLPGGLEPKGELDCHQLADSFRE----------

FYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDG-DFQCHTFDSSNVE----------

FYRKRQCRSSQGQRRGPCWCVDRMGKSLPGSPDGNG-SSSCPTGSSG-------------

211

266

245

209

224

195







220 230 240 250 260 270 280

||||||||||||||

--DHSILWDAISTYDGSKALHVTNIKKWKEPCRIELYRVVESLAKAQET----SGEEISKFYLPNCNKNG

TEQHRQMGKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHG

DSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLN-------VLSPRGVHIPNCDKKG

TSGGKMKVNG--------APREDARPVPQGSCQSELHRALERLAAS----QSRTHEDLYIIPIPNCDRNG

TQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASP-------RMVPRAVYLPNCDRKG

----------GTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQLQTEV-------YRGAQTLYVPNCDHRG

146

196

182

151

161

141







||||||||||||||

GACVQESDAS---------APHAAEAGSPESPESTEITEEELLDNFHLMAPSEE----------------

GTCEKRRDAEYGAS---PEQVADNGDDHSEGGLVENHVDSTMNMLGGGGSAGRKPLKSGMKELAVFREKV

GLCVNASAVSRLRAYLLPAPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHSKIIIIKKGHAK

GVCMELAEIEAIQE---SLQPSD-----------KDEGDHPNNSFSPCSAHDRR---CLQKHFAKIRDRS

GVCLNEKSYR-------EQVKIERDSREHEEPTTSEMAEETYSPKIFRPKHTRISELKAEAVKKDRRKKL

GRCLP------------ARAPAVAEENPKESKPQAGTARPQDVNRRDQQRNP------ -- ----------

150 160 170 180 190 200 210

102

129

112

98

98

102







80 90 100 110 120 130 140

||||||||||||||

AVAAVAGGARMPCAELVRE----PGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGE

----------ASCSEVTRS----AGCGCCPMCALPLGAACGVATARCARGLSCRALPGEQQPLHALTRGQ

----------AVCAELVRE----PGCGCCLTCALSEGQPCGIYTERCGSGLRCQPSPDEARPLQALLDGR

----------VGCEELVRE----PGCGCCATCALGLGMPCGVYTPRCGSGLRCYPPRGVEKPLHTLMHGQ

----------LGC-ELVKE----PGCGCCMTCALAEGQSCGVYTERCAQGLRCLPRQDEEKPLHALLHGR

-----------GCVEEEDGGSPAEGCAEAEGCLRREGQECGVYTPNCAPGLQCHPPKDDEAPLRALLLGR

45

63

55

41

42

42







10 20 30 40 50 60 70

||||||||||||||

----------MSEVPVARVWLVLLLLTVQVGVT----------AGAPWQCAPCSAEKLALCPPVS-----

----------MQRARPTLWAAALTLLVLLRGPPVARAGASSAGLGPVVRCEPCDARALAQCAPPP-----

MLP--------------LCLVAALLLAAGPGP---------SLGDEAIHCPPCSEEKLARCRPP------

---------------MVLLTAVLLLLAAYAGP--------AQSLGSFVHCEPCDEKALSMCPPSP-----

------------MTPHRLLPPLLLLLALLLAAS---------PGGALARCPGCGQGVQAGCPG-------

MLPRVGCPALPLPPPPLLPLLLLLLGASGGGGG--------ARAEVLFRCPPCTPERLAACGPPPVAPPA

1

1

1

1

1

1

Figure 4 Amino acid sequence alignment of human IGFBP-1 to IGFBP-6 Alignment was performed using the ClustalW program Smallgaps were introduced to optimize alignment


Table 3 Consequences of inhibiting IGFBP activity and cancer

IGFBPs Expression Results of inhibiting IGFBP activity Reference

IGFBP-1 LiverIt can induce or inhibit the IGF actions in many types of cellsAs an example of the inhibiting activity of IGFBP-1 it inhibitedIGF-I-induced growth in MCF-7 breast cancer cells

[30 157]

IGFBP-2 Liver adipocytes reproductive systemand central nervous system

IGFBP-2 level changes were associated with the development ofdifferent types of cancer including breast and prostate cancer Inprostate cancer high level of serum IGFBP-2 was associatedwith low grade prostate cancer

[189 190]

IGFBP-3 Circulating carrier protein expressedin many tissues

IGFBP-3 plays important role in different types of humancancers IGFBP-3 can induce apoptosis by increasing the ratio ofproapoptotic to antiapoptotic proteins in breast cancer cells

[191]

IGFBP-4 Liver bone tissue and muscles

IGFBP-4 showed a strong inhibitory effect on IGF-1 bypreventing the activation of the IGF-1R when the IGFBP-4 isfound in the tissue Conversely intravenous administration ofIGFBP-4 in the presence of a protease will promote cellularproliferation

[79 192ndash195]

IGFBP-5 Mammary glands In breast cancer IGFBP-5 induced apoptosis and inhibitedcellular differentiation in an IGF-dependent manner [196 197]

IGFBP-6 Epithelial layer of human bronchialorgan

It can inhibit IGF-2 activity mediated through the IGF-1Rincluding proliferation differentiation migration and survivalin different cell lines

[198 199]

Table 4 Summary of IGFBP proteases and their proteolytic cleavage sites

Proteolytic cleavage sites IGFBP protease ReferenceIGFBP-2

Met166-Gly167 Lys168-Gly169 Tyr103-Gly104 Leu152-Ala153 Arg156-Glu157Gln165-Met166 Thr205-Met206 Arg287-Met288 Unknown protease in hemofiltrate [80]

Leu3-Phe4 Lys168-Gly169 Lys181-Leu182 Unknown in milk [200]Arg164-Gln165 Human kallikrein-2 [201]Leu152 Gly175-Leu176 Lys181-Leu182 Matrix metalloproteinase-7 [202]Gln165-Met166 PAPP-A [203]His165-Arg166 Calpain [204]

IGFBP-3Arg97-Ala98 Lys160-Val161 Plasmin [205]Arg95-Leu96 Lys160-Val16 Plasmin [206]Arg97-Ala98 Arg206-Gly207 Thrombin [205]Arg97-Ala98 Lys149-Lys150 Lys150-Gly151 Lys154-Asp155 Serum [205]Arg97-Ala98 Arg132-Val133 Tyr159-Lys160 Phe173-Ser174 Arg179-Glu180 Seminal plasma PSA [157]Arg97-Ala98 His131-Arg132 Tyr159-Lys160 Urinary PSA [207]Arg97-Ala98 Cysteine protease fromMCF-7 cells [208]Tyr99-Leu100 Leu96-Arg97 Leu141-His142 MMP-1 MMP-2 [176]Tyr99-Leu100 Asn109-Ala110 Glu176-Ser177 MMP-3 [176]

IGFBP-4

Lys120-His121 Calcium-dependent serine proteasefrom smooth muscle cells [193 209 210]

Met135-Lys136 PAPP-A [211 212]IGFBP-5

Arg138-Arg139 Serine protease from smoothmuscle cells [213]

Ser143-Lys144 (secondary cleavage site) Ser143-Lys144 PAPP-A2 [214]Gln142-Ser-143 PAPPA [214]Lys120-His121 Arg156-Ile157 Arg192-Ala193 Thrombin [215]


proteolytic activity on IGFBP-3 [70 71] Cathepsin D isa well-known IGFBP protease shown to have proteolyticactivity against IGFBPs 1ndash5 in acidified condition [70 79]In neutral conditions their proteolytic activity seems to beunclear

The central linker domain which is the least conservedregion has not been cited to be a part of the IGF bindingsite for any IGFBPs but is reported to have four majorprotease cleavage sites in IGFBP-2 determined to be betweenTyr103 and Gly104 Leu152 and Ala153 Arg156 and Glu157and Gln165 and Met166 [80] A study involving mutationof selected residues of the linker domain of IGFBP-4 ledto protease resistivity of IGFBP-4 [81] This leads to theconclusion that the proteolysis of IGFBPs occurs at specificsites by proteases in unstimulated homeostatic conditions(eg PAPP-A activity in normal cell lines) As the reportssuggest the linker domain to be most proteolysis susceptibleamong the N- C- and the linker domain it acts as thedeterminant in the release of IGF from IGFBPs Thus adetailed understanding of the interaction of L-IGFBP-2 withIGF at atomic level is important This may help to determinethe changes which can be brought about in the linker domainfor careful modulation of IGF release which could in turnprevent unwanted IGF-1R signalling controlling abnormalcellular growth and proliferation Alternatively in conditionswhere cellular proliferation is desired (eg wound healing)control on release of IGFmay facilitate IGFmediated cellulargrowth and proliferation Thus a study of the structure oflinker domain (L-IGFBP-2) and its interaction with IGF-1together with the change in dynamics in presence of IGF-1was studied in our laboratory

42 Significance of IGFBP Proteases in Cancer IGFBP pro-teases are known to target and degrade IGFBPs to smallerfragments and thus bring down the affinity of IGFBPs toIGFs This results in IGFs binding to their respective IGFreceptors resulting in signalling cell proliferation growthand cell migration Kallikreins have also been employedas biomarkers in cancer [82] Apart from the significanceof proteolysis in regulating the bioavailability of IGFs intissues and increasing the affinities of IGFs to IGF receptorsthis seems to play a significant role in tumour progressionand tumour cell survival considering the autocrine-paracrineactions in the IGF axisThus IGFBP proteases have potentialclinical implications in cancer research

A novel approach in this regard is development of mutantIGFBPs lacking the IGFBP protease cleavage sites renderingthem protease resistantThis serves as a potential therapeuticagent as it inhibits IGF signally through IGF receptors Suchstudies reported a decade ago where a protease resistantIGFBP-4 was designed and in vivo studies of this pro-tease resistant IGFBP-4 [81] were explored confirming thecomplete resistance to IGFBP-4 protease indicating that themutant IGFBP-4 resulted in greater growth inhibition thanequivalent levels of native IGFBP-4 demonstrating a role forIGFBP-4 proteolysis in the regulation of IGF-1 action and apotential implication in cancer [81] In yet another similarin vivo study protease resistant IGFBP-4 has been shown toblock IGF activity tumour growth and angiogenesis [83]

In another such recent study a novel approach hasbeen used to develop protease resistant (PR) and proteaseresistantnon-matrix-binding (PRNMB) variants of IGFBP-2 as potential tumour growth inhibitors [84] They hypoth-esized that lack of protease and matrix-binding sites renderthe IGFBP-2 devoid of the ability to promote IGF-dependentaction (through release of IGFs to the receptors) and IGF-independent action (through ECMbinding)The in vitro andin vivo studies indicate that the mutant IGFBP-2 (lacking alarge portion of the central linker domain) is able to inhibittumour growth possibly by inhibition of angiogenesis Theirstudies promise to open up new avenues for better targetingstrategies for the effectiveness of cancer treatment in the nearfuture

43 IGFBP-Related Proteins (IGFBP-rPs) The IGFBP super-family includes 6 members (IGFBP-1 to IGFBP-6) with highaffinity for IGF-1 and IGF-2 and 10 IGFBP-related pro-teins (IGFBP-rP1 to IGFBP-rP10) with low affinity for theseligands Remarkably IGFBP-related protein 1 (IGFBP-rP1)also known as insulin-like growth factor binding protein-7 (IGFBP-7) [85] is identified as a secretory and low-affinity IGFBPs It is distinct from other low-affinity IGFBP-rPs in that it can bind strongly to insulin [86] suggestingthat IGFBP-7 is likely to have distinct biological functionsfrom other IGFBPs IGFBP-related protein 1 (or IGFBP-7)has been found to have an important role in the femalereproductive system It was implicated in human endometrialreceptivity folliculogenesis as well as growth developmentand regression of the corpus luteum in higher mammals [87ndash89] Other studies showed that it could induce apoptosis inM12 prostate cancer cell line [90]

Rupp et al demonstrated that adding to IGFBP-7 tumoursuppressor function it can promote anchorage-independentgrowth of malignant mesenchymal cells and of epithelialcells with an EMT-phenotype when IGFBP-7 is expressedby the tumour cells themselves [91] Expression of IGFBP-7 in tumour-associated fibroblasts can also promote colonyformation when epithelial tumour cells are cocultured withIGFBP-7-expressing cancer-associated fibroblasts (CAFs) bysecondary paracrine tumour-stroma interactions Zhu et alrecently reviewed role of insulin-like growth factor bindingprotein-related protein 1 IGFBP-rP1 in cancer [92] Inmany cancers IGFBP-rP1 acts as a tumour suppressor geneby suppressing proliferation and inducing apoptosis andsenescence However there are some contradictory data anddifferent opinions for example IGFBP-rP1 has been reportedas promoting glioma cell growth and migration [93] It hasbeen recently reported that IGFBP-rP1 could bind to the IGF-1R and block its activation [94]

44 IGFBP Structure The structural features of IGFBPswhich carry IGFs in the circulation are very important forunderstanding their role in normal growth and develop-ment as well as in diseases The insulin-like growth factorbinding protein-2 the second most abundant IGFBP incirculation and known to form binary complexes with IGF is32 kDa (289 amino acid residues) in size with three distinct


CBP-1 CBP-2 CBP-6

Figure 5 Structures of C-terminal domains of IGFBP-1 IGFBP-2 and IGFBP-6 represented as CBP-1 (1ZT5) CBP-2 (2H7T) and CBP-6(1RMJ) respectively

regions the highly conserved N-terminal region (IGFBPhomolog domain) the highly conserved C-terminal regionwith thyroglobulin type 1 repeat [95] and the mid-regionknown as the linker domain of IGFBP-2 with multiple cleav-age sites The structures of C-terminal domains of IGFBP-1IGFBP-2 and IGFBP-6 are shown in Figure 5

Notably the C-terminal domain contains an arginine-glycine-aspartic acid (RGD) motif which can bind to inte-grins and take part in cell mediated signaling The N- and C-terminal domains are cysteine rich and are structured withboth of them having IGF binding properties capable of mod-ulating the IGFIGF receptor interactions [96] While somereports have emphasized the importance of the binding of N-terminal domain to IGFbymutagenesis experiments [97] andby iodination protection study [98] others have describedthe C-terminal region of IGFBP-2 as playing important rolein the binding to IGFs by mutagenesis experiments [99 100]and by nuclear magnetic resonance spectroscopy [101] Someothers emphasize the cooperative role which the N-terminaland the C-terminal domain play in the binding to IGF-1[102] The structural aspects of IGFBPs have been recentlyreviewed by Forbes et al [103] The important structuralfeatures for interaction of IGFBPs with extracellular matrixand integrins were described Further they highlighted theimportant structural features for bindingwith IGFs and otherpartners also

45 Structural Studies of Human IGFBP-2 Binding by NMRWhile the biological actions of IGF-1-IGFBP-IGF-1R axishave been extensively studied a complete understanding ofIGF-IGFBP interactions on a structural level is lacking Ourobjective was to elucidate the mechanistic aspects of IGF-IGFBP interactions at the atomic level in order to developIGFBPs as cancer therapeutics

A critical challenge in the structural characterizationof full-length IGFBPs has been the difficulty in expressinglarge amounts of these proteins for NMRX-ray crystal-lography analysis We have developed a method for high-yield expression of full-length recombinant human IGFBP-2(hIGFBP-2) in E coli [104] Using a single step purification

protocol we obtain hIGFBP-2 with gt95 purity The proteinexists as a monomer at the high concentrations (up to30mgmL) required for structural studies in a single confor-mation exhibiting a unique intramolecular disulfide-bondingpattern We have thus for the first time obtained high-yield expression of wild type recombinant human IGFBP-2 in E coli and initiated structural characterization of afull-length IGFBP We are currently studying the molecularinteractions of the different domains of hIGFBP-2 with IGF-1 in particular the central flexible domain which is knownto play a pivotal role in the protein function and regulationThese are described in the proceeding section

451 Study of Nanotubular Structures Formed by a Fragmentof IGFBP-2 We recently discovered that the C-terminalfragment of hIGFBP-2 (residues 249ndash289) self-assemblesspontaneously and reversibly into nanotubular structuresunder nonreducing conditions and remains as a monomerunder reducing conditionThese nanotubular structures werestudied extensively by transmission electron microscopy(TEM) NMR spectroscopy (Figures 6(a) and 6(b)) andcircular dichroism (CD) and amechanism for their formationhas been worked out [105]

452 Biomedical Applications of IGFBP-2 Nanotubes Thepresence of an RGDmotif in this polypeptide fragment offersavenues for novel biomedical applications The RGDmotif isknown to be recognized by integrinsThe design of such self-assembling polypeptide fragments containing an RGD motifcan be utilized to enhance the efficacy of cancer therapeuticsWe have explored the possibility of using these nanotubes forcancer cell imaging This is based on the idea that in manycancers integrins are expressed in large quantities on the cellsurface Thus IGFBP-2

249minus289nanotubes can be developed

to identify the location of cancer cells through their bindingto integrins via the RGD motif Towards this end we havecarried out cell-adhesion and cell-proliferation assays whichhave helped to characterize the binding of the nanotubes tointegrin via the RGD motif


105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)

Figure 6 (a) 2D [15N-1H] HSQC spectrum of purified full-length hIGFBP-2 (10mM nondeuterated) recorded at a 1H resonance frequencyof 800MHz at 285K (b) TEM images of (hollow) nanotubular structures formed by the C-terminal fragment of human IGFBP-2

5 Therapeutic Strategies Targeting IGFSystem in Cancer

Therapeutic strategies targeting various components of theIGF system with varying degree of success have been devel-oped for treatment of different types of cancer Descriptionand challenges of each targeting strategy will be enlightenedin this section

51 Targeting IGF-R Therapeutic Potential of IGF-Rs inCancer IGF-1R activation by tyrosine phosphorylation of 120573subunit results in activation of PI3KAKT and RASMAPKpathways [106 107] which in turn regulate cell survival andproliferation IGF axis is tightly regulated under normalphysiological conditions maintaining cell homeostasis andgrowth Genetic alterations of IGF-1R leading to varying lev-els of their expression are found to have a link in cancer [108]These receptors maybe activated in the tumour cells in anunregulatedmanner (mutation chromosomal translocationabnormal stimulation and loss of genomic imprinting)

IGF-1R does not solely drive tumour cell proliferationhowever most oncogenes are required in mediating anchor-age independent growth given its property to mediate prolif-eration and cell survival This is one of the key processes toachieve metastasis among tumour cells [107 109]

High levels of IGF-1 have been reported in several casesof breast and prostate cancers [110] and since IGF-2 is mater-nally imprinted [111 112] loss of this imprinting results inbiallelic expression resulting in increased IGF-2 productionand a suspected mechanism of cancer development andprogression in many conditions [111 113ndash115] These higherlevels of IGF-1 and IGF-2 promote IGF-1R signalling and

the consequently activated downstream pathways Increasesin IGF-1R have been shown in different types of cancermelanoma and carcinomas [116ndash118] Considering diseaseprognosis therapeutic approaches based on targeting IGFRsseem to be promising in cancer research

Another aspect of IGF-R is the formation of IGF-1RIRhybrids by random association of insulin half-receptor (IR-A) with an IGF half-receptor adding further complexityin receptor targeting strategy [119] IR isoform (IR-A) isoverexpressed in cancer and it is the fetal isoform of IR(while other half is IR-B involved in regulating glucoseuptake) and IGF-1R is also overexpressed in cancer Withthe overexpression of these receptors formation of IGF-1RIR hybrid receptors is expectedThese have broad bindingspecificity as they bind IGF-1 IGF-2 and also insulin [119]Targeting these hybrid receptors becomes one of the severalstrategies

There are several approaches of targeting IGF-R till datenamely small molecule tyrosine kinase inhibitors (TKIs)anti-IGF-1R antibodies and molecular agents such as anti-sense and small interfering RNAs (si-RNAs) [107 120](Figure 7)

While a lot is known on targeting IGF-Rs through TKIsand anti-IGF-1R antibodies and there are detailed multiplereviews on their targeting strategies [108 120ndash129] little isknown on targeting the former using antisense technologyand si-RNAs Tables 5 and 6 summarize few of the severaldifferent TKIs and anti-IGF-1Rs studied

Recent advancements in this approach show us that it ispossible to genetically target IGF-Rs Adenoviruses express-ing antisense IGF-1R and truncated IGF-1R nonviral vectorsexpressing truncated IGF-1R were used to successfully block


Table 5 Few examples of small molecule TKIs (tyrosine kinase inhibitors) directed against IGF receptors

Small molecule inhibitor Mode of action Effects ReferenceNVP-AEW541NVP-AEW54 in combinationwith gemcitabine

Kinase inhibition Antineoplastic tumour regression and inhibition ofmetastasis [216 217]

Picropodophyllin(PPP)

Against autophosphorylation atthe substrate level Inhibition and downregulation of IGF-1R [218ndash221]

BMS-554417 ATP-competitive dual kinaseinhibition Antiproliferative activity [222]

INSM-18 ReversibleATP-competitive

Inhibitor of transcription(blocking also cdc2 survivin and VEGF) [223]

OSI-906 ReversibleATP-competitive Derived from compound-1 also known as PQIP [223]

XL-228 (XL-2280) Inhibits bcr-abl scr and IGF-1R [224]BVP-51004 Biovitrum(Cyclolignan PPP) Non-ATP-competitive Causes IGF-1R downregulation probably through the

induction of ubiquitination [223]

TKIs

Anti-IGF-IRantibodies

Tyrosine kinase inhibitors (TKIs)

Gene silencing antisense technology

siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs

Figure 7 Various strategic approaches to targeting IGF-1R recep-tors Small-molecule TKIs inactivating anti-IGF-1R antibodiesreduction or elimination of IGF-1R protein expression by blockingIGF-1R transcription (with triple helix) or translation (antisensetechnology and siRNA) IGF-1R and mutants lacking beta-subunits(dominant-negative receptors)

IGF-1R thereby suppressing tumorigenicity in vitro and invivo and also effectively blocked both IGF-1- and IGF-2-induced activation of Akt-1

Studies in which small interfering RNAs (siRNAs) inducepotent IGF-1R gene silencing without affecting the insulinreceptor demonstrate that siRNAs block IGF signallingthereby enhancing radio and chemosensitivity and pavingyet another way of therapeutic potential and may in futuregenerate nucleic-acid-based therapeutics [125 130] The effi-cacy of IGF-1R targeting in the clinics depends on majorfactors such as the role of IFGR in itself in the tumoursinhibition potential of siRNAs and antisense therapies in vivoand compensation of other signalling pathways due to IGFRloss [130]

These studies also prove the potential genetic blockadestudies of IGF-1R and its efficacy and prognosis in several

malignancies lung colon and pancreatic carcinoma [131132] Such antisense and dominant negative strategies (trun-cated) also enhance tumour cell chemosensitivity (effectivechemo- and radiotherapy induced apoptosis) One moreprominent feature is the immune protection induced bytumour cells killed in vivo by IGF-1R-antisense techniqueMajor drawback is that antisense agents cause adequate IGF-1R downregulation and also affect insulin receptor

Cotargeting IGF-Rs along with other tumour promot-ing pathways is yet another way to effectively overcomethe limitations of resistance to conventional chemo- andendocrine therapy to single agent targets discussed in previ-ous sections as cross talk between IFG-R and RTKssteroidhormones is known to promote tumorigenesis IGF-1R isknown to interact with several pathways and moleculesreceptor tyrosine kinases (RTKs) including insulin receptor(IR) epidermal growth factor receptor (EGFR) vascularendothelial growth factor receptor (VEGFR) mesenchymal-epithelial transition factor (MET) platelet-derived growthfactor receptor (PDGFR) and fibroblast growth factor recep-tor (FGFR) and steroid hormones including estrogen recep-tors alpha and beta androgen receptor (AR) and proges-terone receptor (PR) This novel approach pertains to crosstalk cotargeting [133] Examples of such a targeting strategyinclude monoclonal antibodies and small molecule tyrosinekinase inhibitors in combination or cotargeting IGF-1R andEGFR receptors [123 134 135] where simultaneously bothreceptors are targeted making it a promising novel approachIn a recent study cotargeting the IGF system and HIF-1(hypoxia-inducible factor-1) has been shown to inhibit themigration and invasion by breast cancer cells [136] indicatingthat ligand-targeting compounds or coinhibition of the IGFand HIF-1 systems may prevent activation of compensatorysignalling (due to cross talks) thereby providing a valuableand novel addition to IGF-1R inhibitor-based therapies [136]

IGF-2R deserves a mention since studies implicatethat the mannose 6-phosphateinsulin-like growth factor-II receptor (M6PIGF-2R) functions in the intracellulartrafficking of lysosomal enzymes the activation of the potentgrowth inhibition transforming growth factor beta 2 and the


Table 6 Few examples of anti-IGF-RI monoclonal antibodies (MAbs) [223]

Monoclonal antibody Class Clinical information

CP-751871 Fully humanIgG2 mab

Ewingrsquos sarcoma family of tumoursbreast cancer single agent in metastatic CRC

IMC-A12 Fully humanIgG1 mab

Ewingrsquos sarcoma family of tumours CRC and HampNcancer

R1507 Fully humanIgG1 mab previously known as RO4858696 Pediatric patients and sarcomas

AMG-479 Fully human mab Ewingrsquos sarcoma family of tumourspancreatic cancer

SCH-717454 Fully human mabpreviously known as 19D12 (Medarex) Colorectal cancer (CRC)

AVE-1642 Humanized mab Previously known as EM164 (ImmunoGen)

MK-0646

FabreHumanized mabPreviously known as A2CHM F50035 7C10 or7H2HM

Colorectal cancer (CRC)

BIIB022Fully humannonglycosylatedIgG4P antibody

Devoid of Fc-effector function to eliminate potential Fcmediated toxicity to the normal vital organs

degradation of IGF2 (which are overexpressed in tumours)Studies have shown that M6PIGF-2R gene functions as atumour suppressor in human liver carcinogenesis [137]

52 Targeting IGFs Therapeutic Potential of IGFs in CancerThe insulin-like growth factors (IGFs) IGF-1 and IGF-2 areligands that bind to IGF receptor (IGF-1R) and regulate can-cer cell proliferation survival and metastasis Since IGF axisis involved in regulating cell metastasis the pathway plays asignificant role in cancer cell metastasis and proliferation andmany studies over a couple of decades have tried to establishthe relationship between serum IGF levels and cancer risk

Many experiments demonstrate the increase in neoplasticcell proliferation with increasing IGF-1 concentration [138]Various human epidemiological studies describe the correla-tion between circulating levels of IGF-1 coupled with IGFBPsand the risk of developing various cancers lung colonbreast and prostrate [139ndash143] Circulating IGF-1 levels playa significant role as a risk factor in the onset and developmentof mammary tumours in breast cancer [144] In vivo studiessuggest that carcinogenesis and cancer progression are influ-enced by germ line variation of genes encoding signallingmolecules in theGH-IGF-1 axis and thesemutations are oftenassociated with genetic manipulations [144] and low IGF-1levels thus tumour growth is influenced by IGF-1 physiology[145] Yet the connection between circulating IGF-1 levels andcancer risk remains inadequately hidden Two contradictoryhypotheses on relationship between IGF-1 and cancer risk areunderlined by Pollak [146]

Firstly if a cell at risk is considered (eg somatic cellmutations lead to accumulating DNA damage) IGF bioac-tivity in the cellular microenvironment influences the fateof the cell survival and evolves to malignant cell lineage orapoptosis in early carcinogenesis To balance apoptotic celldeath and survival of damaged cells might be slightly inclined

towards survival in an environment with high IGF levels andthis would favour the appearance of a malignant clone Thefate of such millions of DNA damaged cells is determinedevery hour and even a modest influence of higher IGF-1level on survival probability might lead to an associationof circulating level with cancer risk [146] Secondly theinfluence of IGF-1 level on cancer risk is somewhat related toearly carcinogenesis Higher IGF-1 levels facilitate the morerapid proliferation of early cancers to the stage at which theycan be clinically detected Such lesions would be common inall adults and cancer diagnosis would reflect the probabilityof these lesions progressing toward a detectable and clinicallysignificant size with this latter process being influenced byIGF-1 level [146]

Findings in the case of prostate cancer may be consistentwith this second hypothesis This is consistent with the viewthat the IGF-1 level is more related to the probability ofprogression of early lesions than to the actual process ofearly carcinogenesis According to Pollak both hypothesesare plausible and are not mutually exclusive also there isno definitive mechanistic evidence to support either of them[146]

IGF-2 is also a ligand for the IGF-1 receptor and is presentin serumat concentrations that are generally higher than IGF-I IGF-2R serves as a sink to IGF-2R and does not allow thesignal transduction of the latter and has the characteristics ofa tumour suppressor which is discussed in previous sectionon targeting IGF-Rs [137]

Several drug candidates that target IGF-1 signalling werefound to have antineoplastic activity by using in vitro studiesand in vivo models both as single agents and in combinationwith currently approved drugs Several high-affinity antibod-ies are developed which cross-react with both IGF-1 and IGF-2 and these are at their early developmental stage MEDI-573is one such human antibody (fully human) that neutralizesboth IGF-1 and IGF-2 thus inhibiting IGF signalling through


both the IGF-1R and IR-A pathways Studies also show thatMEDI-573 inhibited the in vivo growth of IGF-I- or IGF-II-driven tumours [147] Hypophysectomy is also thought to beone of the IGF-1 ligand lowering strategies which was alsosuccessfully employed in patients with hormone-responsivebreast cancer [148] Advantage of antiligand approach is thatit has the potential to block the action of IGF-2 at the insulinisoform A without interfering with insulin action Thisfinding is in view of various cancers where IGF-2 productionis autocrine [126]

53 Targeting IGFBPs Therapeutic Potential of IGFBPs inCancer There is accumulating evidence in the literaturestating that IGFBPs can also cause apoptosis in an IGF-independent manner [149] and they can show inhibitoryeffects towards tumour growth and cancer [150]

Although IGFBPs can prevent IGF from binding to IGF-1R because of their higher affinity to IGF than the IGF-1R itcan also induce tumour growth and progression in situationswhere the IGFBP proteases levels are high andor when IGF-BPs interact with ECM Thus modifying IGFBP depends onthe targeted tissue and the disease state For example IGFBP-3 has shown proapoptotic antiproliferative and antiangio-genic functions in in vitro tumour models [69 151] On theother side IGFBPs can promote tumour progression in thepresence of proteases IGFBP-2 and IGFBP-5 upregulation inCRPC are a good example of that In the presence of PSAand other factors affecting the IGF-IIGFBP-2 and IGFBP-5binding it will result in the delivery of the IGFs to the IGF-1R and activation of the downstream signalling 21 pathwaythus helping the progression to castration resistant disease[152 153] Recently Baxter et al reviewed IGFBPs and theircellular actions beyond their endocrine role in IGF transport[154] They suggest that IGFBPs can also function in theirpericellular and intracellular sections to regulate cell growthand survival Further they interact with many other proteinsincluding their canonical ligands IGF-1 and IGF-II Also theyhave shown that the intracellular functions of IGFBPs intranscriptional regulation induction of apoptosis and DNAdamage repair which also point to their friendly participationin tumour development progression and resistance to treat-ment

531 Cancer StimulatoryInhibitory Effects of IGFBPs

IGFBP-1 IGFBP-1 has higher IGF-1 binding affinity in variousphosphorylated forms than the unphosphorylated proteinand is inhibitory to IGF action [155] An interesting studyusing IGFBP-1 deficient mice demonstrated that IGFBP-1can function as a cell survival factor by repressing TGF120573activation [156] but the relevance of this effort for cancer cellsurvival is not understood On the whole there is no specificconfirmation that IGFBP-1 stimulates tumour growth or it isextensively a tumour growth inhibitor [157]

IGFBP-2 IGFBP-2 overexpression in mice is found to inhibitdevelopment of colorectal adenomas by reducing the tumorgrowth by inhibition of cell proliferation [158] Further thereis significant evidence for a growth promoting effect of

IGFBP-2 in many tumour systems by sequestering IGFs[159] IGFBP-2 contains an Arg-Gly-Asp motif but substi-tution of these amino acid residues did not affect the cellbinding of IGFBP-2 [160] Additionally this motif interactswith 1205725 integrin and is found to be involved in regulatingthe effect of IGFBP-2 on glioma cell migration and invasion[161 162]

IGFBP-3 IGFBP-3 can function as a cancer suppressor andis downregulated in some cancer tissues However growthpromotion by IGFBP-3 has been described by several mech-anisms which involve its overlap with other cell signalingsystems Potentiation of IGF-I dependent proliferation byIGFBP-3 that was first described in human skin fibroblasts in1988 [163] has also been revealed in breast cancer and someother cell types [68 164ndash166] Further in some cases IGFBP-3was shown to stimulate IGF-1 action even for IGF derivativesthat have negligible interaction with the binding protein[167] so the consequence is unlikely to involve IGFBP-3somehow presenting IGFs to their receptor

In patients with NSCLC the greatest activation of IGF-1R was observed in tumours that expressed high levels ofIGFBP-3 [168] although it is not clear whether this activationwas ligand dependent The high expression levels of bothEGFR and IGFBP-3 are seen in tumour tissue compared withnormal tissue in case of oesophageal cancer [72]

IGFBP-4 Cancer inhibitory effects of IGFBP-4 are generallyaccepted IGFBP-4 is found to inhibit tumour progression bysequestering IGFs [66] but some reports demonstrate thatin some circumstances it might suppress cell death [72] orstimulate cell migration In epithelial ovarian cancer IGFBP-4 mRNA is found to be highly expressed [170] but has notbeen shown to be significant for prognosis

IGFBP-5 In breast cancer models IGFBP-5 overexpres-sion was strongly tumour inhibitory in vitro and in vivo[171] whereas the opposite effects were observed in someother cancer models in which IGFBP-5 can stimulate IGF-dependent and IGF-independent cell survival and prolifer-ation [172ndash175] In noncancer cell lines similar effects havebeen reported [176 177] In prostate cancer cells downregulation of IGFBP-5 inhibited IGF-dependent growth invitro and in vivo and castration induced upregulation ofIGFBP-5 in mice accelerated the development of androgenindependence [178]

IGFBP-6As recently reviewed [82] IGFBP-6 is also known tohave inhibitory effects in cancer by blocking IGF signallingextraordinarily IGFII but there is evidence where in somecircumstances it may have oncogenic actions stimulatingmigration [179] and proliferation [70] which is mechanisti-cally stronger than for IGFBP-4 The IGFBP-6 was shownto be involved in cell surface interaction with prohibitin 2a protein found in the mitochondria and nucleus as well asin the plasma membrane thus it stimulates rhabdomyosar-coma cell migration IGFBP-6 ligation results in tyrosine-phosphorylation of Prohibitin 2 [180] Primarily IGFBP-6is tumour suppressive [82] but an ultimate link between itsactivity in vivo remains to be established


ProliferationSurvival

Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R

Side effects and toxicity due to its interferencewith insulin pathway

IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of

interactionreceptor ligand

tyrosine

IGFs

do not bind

mediated tumor progression

Figure 8 Targeting IGFBPs a novel strategy in cancer therapeuticsThe cancer therapeutics targeting the IGF-signalling pathway focuson blocking IGF-1R directly andor its downstream effect Draw-back of such approaches is the adverse side effects or toxicities dueto its interference with the insulin pathway The more efficaciousalternatives IGFBPs as IGF-antagonist based cancer therapeuticsalso contribute to block the IGF-1R mediated tumour progressionAs IGFBPs do not bind insulin they do not interfere with insulin-insulin receptor interactions

It is now clear that the IGFBPs have many effects oncell death via both IGF-dependent and IGF-independentactions Although the mechanisms underlying these latteractions are only beginning to be understood it is alreadyclear that they may provide very specific strategies forfine-tuning therapeutic interventions Current therapeuticstargeting the IGF-signalling pathway focus on blocking IGF-1R directly andor its downstream effect Potential drawbackof such approaches is the resulting adverse side effects ortoxicities due to its interference with the insulin pathway Asa more efficacious alternative we propose that IGFBPs canbe developed as IGF-antagonist based cancer therapeuticsserving to block the IGF-1R mediated tumour progression(Figure 8) The IGFBPs do not bind insulin and thus do notinterfere with insulin-insulin receptor interactions

6 Natural Products Targeting IGFSignalling Pathways

Natural products are known to have medicinal benefits fromancient history They have been used for the treatment ofvarious diseases and are gaining tremendous importance inthe area of drug discovery These natural product derivedphytochemicals have been extensively studied and haveexhibited anticarcinogenic activities by interfering at variousstages of cancer through various mechanisms includingcellular proliferation differentiation apoptosis angiogenesisand metastasis [230] We have a rich historical record fromancient physicians about the use of natural productmedicinesalone and in combination which might provide importanthints for inventing new drugs Nowadays many anticancer

drugs available in themarket are natural product phytochem-icals or their derivatives [231] and some are under clinicaltrials [232]

The natural products including curcumin (331015840-diindol-ylmethane (DIM)) isoflavone genistein (indole-3-carbinol(I3C)) epigallocatechin-3-gallate (EGCG) resveratrol lyco-pene and apigenin have been recognized as cancer chemo-preventive agents (Figure 9) because of their anticarcino-genic activity [233 234] The in vitro and in vivo stud-ies have demonstrated that these natural products haveinhibitory effects on various human and animal cancers[235ndash239] therefore many researchers have focused oninterpreting the molecular mechanisms and identifying thetargets of action of these natural products The variousnatural products perturbing IGF signalling pathways andtheirmechanism of actions have been summarised in Table 7The understanding of molecular mechanism of natural prod-uct derived phytochemical against a specific cancer type willlead to the development of novel anticancer drugs

7 Future Perspectives Challenges andOpportunities for Novel IGF Therapies

The success of targeted therapies for cancer is undisputedstrong preclinical evidence and on-going clinical trials ofsome of the drugs chemical molecules antibodies antisensetechnology si-RNA therapy against members of the IGF-axis-IGF ligands IGFBPs and IGF-Rs have resulted in theapproval of several new agents for cancer treatment Not onlytargeting of these by single substances but also the approachesof cotargeting strategies seem to be a very promising avenuewithmore andmore studies directed in this approach to solvethe complications which come across while targeting specificmolecules involved in cancer pathways

Targeting IGF ligands seems to be problematic since theIGF mediated signalling has important roles in regulatingcellular proliferation and apoptosis (role as circulating hor-mone and a tissue growth factor) apart from their increasedlevels in various cancers Another important factor to bearin mind is that higher levels of IGFBPs might increase IGF-1concentration by increasing its circulating half-life and thismay not possibly lead to increase in receptor activation atthe tissue level and the link between higher IGF levels andneoplasm seems to be unclear here

Another approach is to target IGFBPs in a way whichsequesters more and more IGFs thereby downregulating theIGFmediated signalling in cancer pathway Since IGFBPs arefurther regulated by IGFBP proteases developing mutantswhich lack proteolytic cleavage sites for these proteases canpave a way for strong interaction between IGF and IGFBPsA recent study in this regard showed that novel modifiedIGFBP-2 proteins (protease resistant alone or also lackedthe ability to bind extracellular matrix) sequestered boththe IGFs and thereby was able to inhibit tumour growthThese modified IGFBPs were found to do so by inhibitionof angiogenesis both in vitro and in vivo [84] Apart fromIGF-dependent (proteolysis) activities IGFBPs also haveIGF-independent activities in relation to cancer mutants


Apigenin

Chamomile tea

Genistein

Epigallocatechin-3-gallate (EGCG)

Resveratrol

Curcumin

Quercetin

Lycopene

Turmeric powderSoybeans

Tomatoesguavawatermelonand papaya

Fruits vegetables leaves and grains Green tea

Grapes mainly in the skin

HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C

Figure 9 Natural product derived phytochemicals with anticancer activity perturbing IGF signalling pathways

Table 7 Natural products perturbing IGF signalling pathways

Active phytochemicals Natural source Mode of action Molecular target

Curcumin [225 226] Curcuma longa (turmeric powder) Antiproliferation anticarcinogenesis cellcycle arrest apoptosis and antiangiogenesis IGF-1R

Genistein [226]Soybeans and soy products red clover(Trifolium pratense) and sicilian pistachio(Pistacia vera)

Antioxidant antiproliferationantiproliferation anticarcinogenesis cellcycle arrest apoptosis antiangiogenesis andanti-inflammation

IGF-1R

Lycopene [226]Tomatoes guava rosehip watermelonpapaya apricot and pink grapefruit mostabundant in red tomatoes

Antioxidant antiproliferation (growthinhibition cell cycle arrest and apoptosis)antiangiogenesis anti-inflammation andimmunomodulator

IGFBP-3

Apigenin [227]

Fruits and vegetables including orangesgrapefruits parsley celery onions wheatsprouts cereals of millet and wheat and insome seasonings such as coriandermarjoram oregano rosemary tarragon andchamomile tea

Inhibit cellular proliferation suppresstumorigenesis and angiogenesis and induceapoptosis

IGF axis and itsintracellularsignalling inprostate cancer

Quercetin [228] Fruits vegetables leaves and grains Inhibits the proliferation and inducesapoptosis of cancer cells IGFIR

Epigallocatechin-3-gallate[229]

Green tea Inhibits angiogenesisInhibitory effectson IGF-I-inducedVEGF expression

Resveratrol [225] Grapes (mainly in the skin) mulberriespeanuts vines and pines

Antioxidant antiproliferationanticarcinogenesis cell cycle arrestapoptosis antiangiogenesis andanti-inflammation

Suppression ofIGF-1RAktWntsignallingpathways


lacking both proteolysis andmatrix-binding activitiesmay beeffective for the treatment of cancers in the future

While IGF receptors seem to be themost favourite targetsin the IGF-axis in relation to cancer the drawbacks andchallenges in achieving this seem to add further complexitybecause of the cross talks between IGF-R mediated path-ways and other growth mediated pathways in cells Thoughvarious TKIs against IGF-1Rs seem to be in clinical trialspecificity and concentrations can be well documented invitro while their extent of in vivo roles seems to be a questionmark considering the variation in concentration amongdifferent tissues and toxicity could be another issue Anti-IGFR antibodies are advantageous over TKIs in this regardwhile blockage of IGFRs may pressurize the tumour cellsto compensate for blockade by increased signalling throughalternate receptors (eg EGFRs) In some instances IGF-2action via the IR-A also promotes resistance to anti-IGF-1Rinhibitors Thus specific therapeutic combinations can be ananswer to this problem

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] D R Clemmons ldquoValue of insulin-like growth factor sys-tem markers in the assessment of growth hormone statusrdquoEndocrinology andMetabolismClinics of North America vol 36no 1 pp 109ndash129 2007

[2] S M Firth and R C Baxter ldquoCellular actions of the insulin-likegrowth factor binding proteinsrdquo Endocrine Reviews vol 23 no6 pp 824ndash854 2002

[3] L E H Smith W Shen C Perruzzi et al ldquoRegulation ofvascular endothelial growth factor-dependent retinal neovas-cularization by insulin-like growth factor-1 receptorrdquo NatureMedicine vol 5 no 12 pp 1390ndash1395 1999

[4] M J Horney D W Shirley D T Kurtz and S A RosenzweigldquoElevated glucose increases mesangial cell sensitivity to insulin-like growth factor Irdquo American Journal of Physiology RenalPhysiology vol 274 no 6 part 2 pp F1045ndashF1053 1998

[5] M G Slomiany and S A Rosenzweig ldquoIGF-1-induced VEGFand IGFBP-3 secretion correlates with increasedHIF-1120572 expres-sion and activity in retinal pigment epithelial cell line D407rdquoInvestigative Ophthalmology amp Visual Science vol 45 no 8 pp2838ndash2847 2004

[6] K T Flaherty F S Hodi andD E Fisher ldquoFrom genes to drugstargeted strategies for melanomardquo Nature Reviews Cancer vol12 no 5 pp 349ndash361 2012

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[8] A M Scott J D Wolchok and L J Old ldquoAntibody therapy ofcancerrdquoNature Reviews Cancer vol 12 no 4 pp 278ndash287 2012

[9] P Chames M van Regenmortel E Weiss and D Baty ldquoTher-apeutic antibodies successes limitations and hopes for thefuturerdquo British Journal of Pharmacology vol 157 no 2 pp 220ndash233 2009

[10] S A Rosenzweig and H S Atreya ldquoDefining the pathway toinsulin-like growth factor system targeting in cancerrdquo Biochem-ical Pharmacology vol 80 no 8 pp 1115ndash1124 2010

[11] H Werner G G Re I A Drummond et al ldquoIncreasedexpression of the insulin-like growth factor I receptor geneIGF1R inWilms tumor is correlated with modulation of IGF1Rpromoter activity by the WT1 Wilms tumor gene productrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 90 no 12 pp 5828ndash5832 1993

[12] A O W Stretton ldquoThe first sequence fred Sanger and insulinrdquoGenetics vol 162 no 2 pp 527ndash532 2002

[13] Z Qu J C Chow P-R Ling T R Ziegler B R Bistrian andR J Smith ldquoTissue-specific effects of chronic dietary proteinrestriction and gastrostomy on the insulin-like growth factor-I pathway in the liver and colon of adult ratsrdquo MetabolismClinical and Experimental vol 46 no 6 pp 691ndash697 1997

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[20] V K M Han P K Lund D C Lee and A J DrsquoErcoleldquoExpression of somatomedininsulin-like growth factor mes-senger ribonucleic acids in the human fetus identificationcharacterization and tissue distributionrdquoThe Journal of ClinicalEndocrinology amp Metabolism vol 66 no 2 pp 422ndash429 1988

[21] W L Lowe Jr S R Lasky D LeRoith and C T RobertsJr ldquoDistribution and regulation of rat insulin-like growthfactor I messenger ribonucleic acids encoding alternative car-boxyterminal E-peptides evidence for differential processingand regulation in liverrdquo Molecular Endocrinology vol 2 no 6pp 528ndash535 1988

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[24] J Wang W Niu D P Witte et al ldquoOverexpression of insulin-like growth factor-binding protein-4 (IGFBP- 4) in smooth


muscle cells of transgenic mice through a smooth muscle 120572-actin-IGFBP-4 fusion gene induces smoothmuscle hypoplasiardquoEndocrinology vol 139 no 5 pp 2605ndash2614 1998

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[27] W Ni K Rajkumar J I Nagy and L J Murphy ldquoImpairedbrain development and reduced astrocyte response to injuryin transgenic mice expressing IGF binding protein-1rdquo BrainResearch vol 769 no 1 pp 97ndash107 1997

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[31] H Yu and T Rohan ldquoRole of the insulin-like growth factorfamily in cancer development and progressionrdquo Journal of theNational Cancer Institute vol 92 no 18 pp 1472ndash1489 2000

[32] L Sepp-Lorenzino ldquoStructure and function of the insulin-likegrowth factor I receptorrdquoBreast Cancer Research and Treatmentvol 47 no 3 pp 235ndash253 1998

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[35] D Chrysis A S Calikoglu P Ye and A J DrsquoErcole ldquoInsulin-like growth factor-I overexpression attenuates cerebellar apop-tosis by altering the expression of Bcl family proteins in adevelopmentally specific mannerrdquoThe Journal of Neurosciencevol 21 no 5 pp 1481ndash1489 2001

[36] K D Dentremont P Ye A J DrsquoErcole and J R OrsquoKuskyldquoIncreased insulin-like growth factor-I (IGF-I)expression dur-ing early postnatal development differentially increases neuronnumber and growth in medullary nuclei of the mouserdquo Devel-opmental Brain Research vol 114 no 1 pp 135ndash141 1999

[37] G Zhao M-C Monier-Faugere M C Langub et al ldquoTargetedoverexpression of insulin-like growth factor I to osteoblastsof transgenic mice increased trabecular bone volume withoutincreased osteoblast proliferationrdquo Endocrinology vol 141 no7 pp 2674ndash2682 2000

[38] K Reiss W Cheng A Ferber et al ldquoOverexpression of insulin-like growth factor-1 in the heart is coupled with myocyteproliferation in transgenic micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no16 pp 8630ndash8635 1996

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[44] Y Yang A Hoeflich O Butenandt and W Kiess ldquoOppositeregulation of IGF-I and IGF-I receptormRNA and concomitantchanges of GH receptor and IGF-IIM6P receptor mRNAin human IM-9 lymphoblastsrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1310 no 3 pp 317ndash3241996

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[74] J L Fowlkes J J Enghild K Suzuki and H Nagase ldquoMatrixmetalloproteinases degrade insulin-like growth factor-bindingprotein-3 in dermal fibroblast culturesrdquo Journal of BiologicalChemistry vol 269 no 41 pp 25742ndash25746 1994

[75] R P Verma and C Hansch ldquoMatrix metalloproteinases(MMPs) chemical-biological functions and (Q)SARsrdquo Bioor-ganic ampMedicinal Chemistry vol 15 no 6 pp 2223ndash2268 2007

[76] J L Fowlkes K M Thrailkill D M Serra K Suzuki andH Nagase ldquoMatrix metalloproteinases as insulin-like growthfactor binding protein-degrading proteinasesrdquo Cytokine andGrowth Factor Reviews vol 6 no 2ndash4 pp 255ndash263 1995

[77] H A Coppock A White J D Aplin and M West-wood ldquoMatrix metalloprotease-3 and -9 proteolyze insulin-likegrowth factor-binding protein-1rdquo Biology of Reproduction vol71 no 2 pp 438ndash443 2004

[78] R Rajah R V Nachajon M H Collins H Hakonarson M MGrunstein and P Cohen ldquoElevated levels of the IGF-bindingprotein protease MMP-1 in asthmatic airway smooth musclerdquoTheAmerican Journal of Respiratory Cell andMolecular Biologyvol 20 no 2 pp 199ndash208 1999

[79] C A Conover G F Faessen K E Ilg et al ldquoPregnancy-associated plasma protein-A is the insulin-like growth factorbinding protein-4 protease secreted by human ovarian granu-losa cells and is a marker of dominant follicle selection and thecorpus luteumrdquo Endocrinology vol 142 no 5 pp 2155ndash21582001

[80] S Mark B Kubler S Honing et al ldquoDiversity of humaninsulin-like growth factor (IGF) binding protein-2 fragmentsin plasma primary structure IGF-binding properties anddisulfide bonding patternrdquo Biochemistry vol 44 no 9 pp3644ndash3652 2005

[81] M Zhang E P Smith H KurodaW Banach S D Chernausekand J A Fagin ldquoTargeted expression of a protease-resistant


IGFBP-4 mutant in smooth muscle of transgenic mice resultsin IGFBP-4 stabilization and smooth muscle hypotrophyrdquo TheJournal of Biological Chemistry vol 277 no 24 pp 21285ndash212902002

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[84] C-L Soh KMcNeil CMOwczarek et al ldquoExogenous admin-istration of protease-resistant non-matrix-binding IGFBP-2inhibits tumour growth in a murine model of breast cancerrdquoBritish Journal of Cancer vol 110 no 12 pp 2855ndash2864 2014

[85] Y Oh S R Nagalla Y Yamanaka H-S Kim E Wilson andR G Rosenfeld ldquoSynthesis and characterization of insulin-likegrowth factor-binding protein (IGFBP)-7 recombinant humanmac25 protein specifically binds IGF-I and -IIrdquo The Journal ofBiological Chemistry vol 271 no 48 pp 30322ndash30325 1996

[86] M R Corkins J A Vanderhoof DH Slentz R GMacDonaldand J H Y Park ldquoGrowth stimulation by transfection ofintestinal epithelial cells with an antisense insulin-like growthfactor binding protein-2 constructrdquoBiochemical andBiophysicalResearch Communications vol 211 no 3 pp 707ndash713 1995

[87] O M Casey R Fitzpatrick J O McInerney D G Morris RPowell and J M Sreenan ldquoAnalysis of gene expression in thebovine corpus luteum through generation and characterisationof 960 ESTsrdquo Biochimica et Biophysica Acta Gene Structure andExpression vol 1679 no 1 pp 10ndash17 2004

[88] F Domınguez S Avila A Cervero et al ldquoA combined approachfor gene discovery identifies insulin-like growth factor-bindingprotein-related protein I as a new gene implicated in humanendometrial receptivityrdquo The Journal of Clinical Endocrinologyand Metabolism vol 88 no 4 pp 1849ndash1857 2003

[89] S-A Wandji J E Gadsby J A Barber and J M HammondldquoMessenger ribonucleic acids for mac25 and connective tissuegrowth factor (CTGF) are inversely regulated during folliculo-genesis and early luteogenesisrdquo Endocrinology vol 141 no 7 pp2648ndash2657 2000

[90] C C Sprenger M E Vail K Evans J Simurdak and S RPlymate ldquoOver-expression of insulin-like growth factor bind-ing protein-related protein-1(IGFBP-rP1mac25) in the M12prostate cancer cell line alters tumor growth by a delay in G1and cyclin A associated apoptosisrdquo Oncogene vol 21 no 1 pp140ndash147 2002

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[94] V Evdokimova C E Tognon T Benatar et al ldquoIGFBP7 bindsto the IGF-1 receptor and blocks its activation by insulin-likegrowth factorsrdquo Science Signaling vol 5 no 255 article ra922012

[95] T Fukushima and H Kataoka ldquoRoles of insulin-like growthfactor binding protein-2 (IGFBP-2) in glioblastomardquoAnticancerResearch vol 27 no 6 pp 3685ndash3692 2007

[96] F E Carrick B E Forbes and J C Wallace ldquoBIAcore analysisof bovine insulin-like growth factor (IGF)-binding protein-2 identifies major IGF binding site determinants in boththe amino- and carboxyl-terminal domainsrdquo The Journal ofBiological Chemistry vol 276 no 29 pp 27120ndash27128 2001

[97] G D Hobba A Lothgren E Holmberg B E Forbes GL Francis and J C Wallace ldquoAlanine screening mutagenesisestablishes tyrosine 60 of bovine insulin-like growth factorbinding protein-2 as a determinant of insulin-like growth factorbindingrdquoThe Journal of Biological Chemistry vol 273 no 31 pp19691ndash19698 1998

[98] G D Hobba B E Forbes E J Parkinson G L Francis and JCWallace ldquoThe insulin-like growth factor (IGF) binding site ofbovine insulin-like growth factor binding protein-2 (biGFBP-2)probed by iodinationrdquoThe Journal of Biological Chemistry vol271 no 48 pp 30529ndash30536 1996

[99] B E Forbes D Turner S J Hodge K A McNeil G Forsbergand J C Wallace ldquoLocalization of an insulin-like growth factor(IGF) binding site of bovine IGF binding protein-2 usingdisulfide mapping and deletion mutation analysis of the C-terminal domainrdquoThe Journal of Biological Chemistry vol 273no 8 pp 4647ndash4652 1998

[100] MMKibbeyM J Jameson EM Eaton and S A RosenzweigldquoInsulin-like growth factor binding protein-2 contributions ofthe C-terminal domain to insulin-like growth factor-1 bindingrdquoMolecular Pharmacology vol 69 no 3 pp 833ndash845 2006

[101] F E Carrick M G Hinds K A McNeil J C Wallace B EForbes and R S Norton ldquoInteraction of insulin-like growthfactor (IGF)-I and -II with IGF binding protein-2 mappingthe binding surfaces by nuclear magnetic resonancerdquo Journal ofMolecular Endocrinology vol 34 no 3 pp 685ndash698 2005

[102] Z Kuang S Yao K A McNeil et al ldquoCooperativity of theN- and C-terminal domains of insulin-like growth factor (IGF)binding protein 2 in IGF bindingrdquo Biochemistry vol 46 no 48pp 13720ndash13732 2007

[103] B E Forbes PMcCarthy andR SNorton ldquoInsulin-like growthfactor binding proteins a structural perspectiverdquo Frontiers inEndocrinology vol 3 article 38 2012

[104] M Swain M G Slomiany S A Rosenzweig and H S AtreyaldquoHigh-yield bacterial expression and structural characterizationof recombinant human insulin-like growth factor bindingprotein-2rdquo Archives of Biochemistry and Biophysics vol 501 no2 pp 195ndash200 2010

[105] M Swain R Thirupathi B Krishnarjuna et al ldquoSpontaneousand reversible self-assembly of a polypeptide fragment ofinsulin-like growth factor binding protein-2 into fluorescentnanotubular structuresrdquoChemical Communications vol 46 no2 pp 216ndash218 2010

[106] S Y Kim J A Toretsky D Scher and L J Helman ldquoThe role ofIGF-1R in pediatric malignanciesrdquoThe Oncologist vol 14 no 1pp 83ndash91 2009

[107] M M Chitnis J S P Yuen A S Protheroe M Pollak and VM Macaulay ldquoThe type 1 insulin-like growth factor receptorpathwayrdquo Clinical Cancer Research vol 14 no 20 pp 6364ndash6370 2008

[108] K Takeuchi and F Ito ldquoReceptor tyrosine kinases and targetedcancer therapeuticsrdquo Biological and Pharmaceutical Bulletinvol 34 no 12 pp 1774ndash1780 2011


[109] R Baserga F Peruzzi and K Reiss ldquoThe IGF-1 receptor incancer biologyrdquo International Journal of Cancer vol 107 no 6pp 873ndash877 2003

[110] A G Renehan M Zwahlen C Minder S T OrsquoDwyer S MShalet and M Egger ldquoInsulin-like growth factor (IGF)-I IGFbinding protein-3 and cancer risk systematic review andmeta-regression analysisrdquo The Lancet vol 363 no 9418 pp 1346ndash1353 2004

[111] H Cui M Cruz-Correa F M Giardiello et al ldquoLoss ofIGF2 imprinting a potential marker of colorectal cancer riskrdquoScience vol 299 no 5613 pp 1753ndash1755 2003

[112] G E Krassas N Pontikides T Kaltsas et al ldquoFree and totalinsulin-like growth factor (IGF)-I -II and IGF binding protein-1 -2 and -3 serum levels in patients with active thyroid eyediseaserdquoThe Journal of Clinical Endocrinology and Metabolismvol 88 no 1 pp 132ndash135 2003

[113] P Vorwerk H Wex C Bessert B Hohmann U Schmidt andU Mittler ldquoLoss of imprinting of IGF-II gene in children withacute lymphoblastic leukemiardquo Leukemia Research vol 27 no9 pp 807ndash812 2003

[114] C M Coutinho-Camillo M M Brentani O Butugan HTorloni and M A Nagai ldquoRelaxation of imprinting of IGFIIgene in juvenile nasopharyngeal angiofibromasrdquo DiagnosticMolecular Pathology vol 12 no 1 pp 57ndash62 2003

[115] O Ogawa D M Becroft I M Morison et al ldquoConstitutionalrelaxation of insulin-like growth factor II gene imprintingassociatedwithWilmsrsquo tumour and gigantismrdquoNature Geneticsvol 5 no 4 pp 408ndash412 1993

[116] J Zhang J M Trent and P S Meltzer ldquoRapid isolation andcharacterization of amplified DNA by chromosome microdis-section Identification of IGF1R amplification in malignantmelanomardquo Oncogene vol 8 no 10 pp 2827ndash2831 1993

[117] G Armengol S Knuutila F Lluıs G Capella R Miro andM R Caballın ldquoDNA copy number changes and evaluation ofMYC IGF1R and FES amplification in xenografts of pancreaticadenocarcinomardquoCancerGenetics andCytogenetics vol 116 no2 pp 133ndash141 2000

[118] A Almeida M Muleris B Dutrillaux and B Malfoy ldquoTheinsulin-like growth factor I receptor gene is the target for the15q26 amplicon in breast cancerrdquo Genes Chromosomes andCancer vol 11 no 1 pp 63ndash65 1994

[119] A Belfiore ldquoThe role of insulin receptor isoforms and hybridinsulinIGF-I receptors in human cancerrdquo Current Pharmaceu-tical Design vol 13 no 7 pp 671ndash686 2007

[120] E A Bohula M P Playford and V M Macaulay ldquoTargetingthe type 1 insulin-like growth factor receptor as anti-cancertreatmentrdquoAnti-Cancer Drugs vol 14 no 9 pp 669ndash682 2003

[121] J S P Yuen and V M Macaulay ldquoTargeting the type 1 insulin-like growth factor receptor as a treatment for cancerrdquo ExpertOpinion onTherapeutic Targets vol 12 no 5 pp 589ndash603 2008

[122] E Surmacz ldquoGrowth factor receptors as therapeutic targetsStrategies to inhibit the insulin-like growth factor I receptorrdquoOncogene vol 22 no 42 pp 6589ndash6597 2003

[123] I Singh H Amin B Rah and A Goswami ldquoTargeting EGFRand IGF 1R a promising combination therapy for metastaticcancerrdquo Frontiers in BiosciencemdashScholar vol 5 no 1 pp 231ndash246 2013

[124] L Mohanraj and Y Oh ldquoTargeting IGF-I IGFBPs and IGF-I receptor system in cancer the current and future in breastcancer therapyrdquo Recent Patents on Anti-Cancer Drug Discoveryvol 6 no 2 pp 166ndash177 2011

[125] J Riedemann and V M Macaulay ldquoIGF1R signalling and itsinhibitionrdquoEndocrine-RelatedCancer vol 13 no 1 pp S33ndashS432006

[126] M Pollak ldquoThe insulin receptorinsulin-like growth factorreceptor family as a therapeutic target in oncologyrdquo ClinicalCancer Research vol 18 no 1 pp 40ndash50 2012

[127] F I Arnaldez and L J Helman ldquoTargeting the insulin growthfactor receptor 1rdquo HematologyOncology Clinics of North Amer-ica vol 26 no 3 pp 527ndash542 2012

[128] A Arcaro ldquoTargeting the insulin-like growth factor-1 receptorin human cancerrdquo Frontiers in Pharmacology vol 4 article 302013

[129] D Yee ldquoInsulin-like growth factor receptor inhibitors baby orthe bathwaterrdquo Journal of theNational Cancer Institute vol 104no 13 pp 975ndash981 2012

[130] V M Macaulay ldquoThe IGF receptor as anticancer treatmenttargetrdquo Novartis Foundation Symposium vol 262 pp 235ndash2462004

[131] Y Adachi C T Lee and D P Carbone ldquoGenetic blockade ofthe insulin-like growth factor 1 receptor for humanmalignancyrdquoNovartis Foundation Symposium vol 262 pp 177ndash268 2004

[132] Y Adachi C Lee K Coffee et al ldquoEffects of genetic blockade ofthe insulin-like growth factor receptor in human colon cancercell linesrdquo Gastroenterology vol 123 no 4 pp 1191ndash1204 2002

[133] C Liu Z Zhang H Tang Z Jiang L You and Y LiaoldquoCrosstalk between IGF-1R and other Tumor Promoting Path-waysrdquo Current Pharmaceutical Design vol 20 no 17 pp 2912ndash2921 2014

[134] M Xue X Cao Y Zhong et al ldquoInsulin-like growth factor-1receptor (IGF-1R) kinase inhibitors in cancer therapy advancesand perspectivesrdquo Current Pharmaceutical Design vol 18 no20 pp 2901ndash2913 2012

[135] J van der Veeken S Oliveira R M Schiffelers G Storm P MP Van Bergen En Henegouwen and R C Roovers ldquoCrosstalkbetween epidermal growth factor receptor- and insulin-likegrowth factor-1 receptor signaling Implications for cancertherapyrdquoCurrent Cancer Drug Targets vol 9 no 6 pp 748ndash7602009

[136] M Mancini M B Gariboldi E Taiana et al ldquoCo-targetingthe IGF system and HIF-1 inhibits migration and invasion by(triple-negative) breast cancer cellsrdquo British Journal of Cancervol 110 no 12 pp 2865ndash2873 2014

[137] A T de Souza G R Hankins M K Washington T COrton and R L Jirtle ldquoM6PIGF2R gene is mutated in humanhepatocellular carcinomas with loss of heterozygosityrdquo NatureGenetics vol 11 no 4 pp 447ndash449 1995

[138] Y Myal R P C Shiu B Bhaumick and M Bala ldquoReceptorbinding and growth-promoting activity of insulin-like growthfactors in human breast cancer cells (T-47D) in culturerdquoCancerResearch vol 44 no 12 part 1 pp 5486ndash5490 1984

[139] S E Hankinson W C Willett G A Colditz et al ldquoCirculatingconcentrations of insulin-like growth factor-I and risk of breastcancerrdquoThe Lancet vol 351 no 9113 pp 1393ndash1396 1998

[140] H Yu M R Spitz J Mistry J Gu W K Hong and X WuldquoPlasma levels of insulin-like growth factor-I and lung cancerrisk a case-control analysisrdquo Journal of the National CancerInstitute vol 91 no 2 pp 151ndash156 1999

[141] J Ma M N Pollak E Giovannucci et al ldquoProspective study ofcolorectal cancer risk in men and plasma levels of insulin-likegrowth factor (IGF)-I and IGF-binding protein-3rdquo Journal of theNational Cancer Institute vol 91 no 7 pp 620ndash625 1999


[142] R Shi H J Berkel and H Yu ldquoInsulin-like growth factor-I andprostate cancer a meta-analysisrdquo British Journal of Cancer vol85 no 7 pp 991ndash996 2001

[143] J M Chan M J Stampfer E Giovannucci et al ldquoPlasmainsulin-like growth factor-I and prostate cancer risk a prospec-tive studyrdquo Science vol 279 no 5350 pp 563ndash566 1998

[144] Y Wu K Cui K Miyoshi et al ldquoReduced circulating insulin-like growth factor I levels delay the onset of chemically andgenetically induced mammary tumorsrdquo Cancer Research vol63 no 15 pp 4384ndash4388 2003

[145] N Majeed M-J Blouin P J Kaplan-Lefko et al ldquoA germ linemutation that delays prostate cancer progression and prolongssurvival in a murine prostate cancer modelrdquo Oncogene vol 24no 29 pp 4736ndash4740 2005

[146] M Pollak ldquoInsulin insulin-like growth factors and neopla-siardquo Best Practice and Research Clinical Endocrinology andMetabolism vol 22 no 4 pp 625ndash638 2008

[147] J Gao J W Chesebrough S A Cartlidge et al ldquoDual IGF-III-neutralizing antibody MEDI-573 potently inhibits IGFsignaling and tumor growthrdquo Cancer Research vol 71 no 3 pp1029ndash1040 2011

[148] G T Tindall S S Ambrose J H Christy and J M PattonldquoHypophysectomy in the treatment of disseminated carcinomaof the breast and prostate glandrdquo Southern Medical Journal vol69 no 5 pp 579ndash587 1976

[149] A Grimberg ldquop53 and IGFBP-3 apoptosis and cancer protec-tionrdquoMolecular Genetics and Metabolism vol 70 no 2 pp 85ndash98 2000

[150] M J Moreno M Ball M Rukhlova et al ldquoIGFBP-4 anti-angiogenic and anti-tumorigenic effects are associated withanti-cathepsin B activityrdquo Neoplasia vol 15 no 5 pp 554ndash5672013

[151] A Grimberg and P Cohen ldquoRole of insulin-like growth factorsand their binding proteins in growth control and carcinogene-sisrdquo Journal of Cellular Physiology vol 183 no 1 pp 1ndash9 2000

[152] T Nickerson H Miyake M E Gleave and M PollakldquoCastration-induced apoptosis of androgen-dependentShionogi carcinoma is associated with increased expression ofgenes encoding insulin-like growth factor-binding proteinsrdquoCancer Research vol 59 no 14 pp 3392ndash3395 1999

[153] T Nickerson and M Pollak ldquoBicalutamide (Casodex)-inducedprostate regression involves increased expression of genesencoding insulin-like growth factor binding proteinsrdquo Urologyvol 54 no 6 pp 1120ndash1125 1999

[154] R C Baxter ldquoIGF binding proteins in cancer mechanistic andclinical insightsrdquoNature Reviews Cancer vol 14 no 5 pp 329ndash341 2014

[155] J I Jones A Joseph DrsquoErcole C Camacho-Hubner and DR Clemmons ldquoPhosphorylation of insulin-like growth factor(IGF)-binding protein 1 in cell culture and in vivo effectson affinity for IGF-Irdquo Proceedings of the National Academy ofSciences of the United States of America vol 88 no 17 pp 7481ndash7485 1991

[156] J I Leu M A S Crissey and R Taub ldquoMassive hepaticapoptosis associated with TGF-1205731 activation after Fas ligandtreatment of IGF binding protein-1-deficient micerdquoThe Journalof Clinical Investigation vol 111 no 1 pp 129ndash139 2003

[157] D Yee J G Jackson TWKozelsky and J A Figueroa ldquoInsulin-like growth factor binding protein 1 expression inhibits insulin-like growth factor I action in MCF-7 breast cancer cellsrdquo CellGrowth amp Differentiation vol 5 no 1 pp 73ndash77 1994

[158] D Diehl E Hessel D Oesterle et al ldquoIGFBP-2 overexpressionreduces the appearance of dysplastic aberrant crypt foci andinhibits growth of adenomas in chemically induced colorectalcarcinogenesisrdquo International Journal of Cancer vol 124 no 9pp 2220ndash2225 2009

[159] A Hoeflich R Reisinger H Lahm et al ldquoInsulin-like growthfactor-binding protein 2 in tumorigenesis protector or pro-moterrdquo Cancer Research vol 61 no 24 pp 8601ndash8610 2001

[160] V C Russo B S Schutt E Andaloro et al ldquoInsulin-like growthfactor binding protein-2 binding to extracellular matrix plays acritical role in neuroblastoma cell proliferation migration andinvasionrdquo Endocrinology vol 146 no 10 pp 4445ndash4455 2005

[161] S W Song G N Fuller A Khan et al ldquoIIp45 an insulin-like growth factor binding protein 2 (IGFBP-2) binding proteinantagonizes IGFBP-2 stimulation of glioma cell invasionrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 100 no 2 pp 13970ndash13975 2003

[162] G K Wang L Hu G N Fuller and W Zhang ldquoAn interactionbetween insulin-like growth factor-binding protein 2 (IGFBP2)and integrin 1205725 is essential for IGFBP2-induced cell mobilityrdquoThe Journal of Biological Chemistry vol 281 no 20 pp 14085ndash14091 2006

[163] J S M de Mellow and R C Baxter ldquoGrowth hormone-dependent insulin-like growth factor (IGF) binding proteinboth inhibits and potentiates IGF-I-stimulated DNA synthe-sis in human skin fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 156 no 1 pp 199ndash204 1988

[164] W F Blum E W Jenne F Reppin K Kietzmann M BRanke and J R Bierich ldquoInsulin-like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-IrdquoEndocrinology vol 125 no 2 pp 766ndash772 1989

[165] J L Martin M Z Lin E M McGowan and R C BaxterldquoPotentiation of growth factor signaling by insulin-like growthfactor-binding protein-3 in breast epithelial cells requires sphin-gosine kinase activityrdquoThe Journal of Biological Chemistry vol284 no 38 pp 25542ndash25552 2009

[166] S Y Heazlewood R J Neaves B Williams D N Haylock TE Adams and S K Nilsson ldquoMegakaryocytes co-localise withhemopoietic stem cells and release cytokines that up-regulatestem cell proliferationrdquo StemCell Research vol 11 no 2 pp 782ndash792 2013

[167] C A Conover ldquoPotentiation of insulin-like growth factor(IGF) action by IGF-binding protein-3 studies of underlyingmechanismrdquo Endocrinology vol 130 no 6 pp 3191ndash3199 1992

[168] W-Y Kim M-J Kim H Moon et al ldquoDifferential impactsof insulin-like growth factor-binding protein-3 (IGFBP-3) inepithelial IGF-induced lung cancer developmentrdquo Endocrinol-ogy vol 152 no 6 pp 2164ndash2173 2011

[169] B Bartling A Koch A Simm R Scheubel R E Silber andA N Santos ldquoInsulin-like growth factor binding proteins-2 and -4 enhance the migration of human CD34-CD133+hematopoietic stem and progenitor cellsrdquo International Journalof Molecular Medicine vol 25 no 1 pp 89ndash96 2010

[170] R A Mosig M Lobl E Senturk et al ldquoIGFBP-4 tumor andserum levels are increased across all stages of epithelial ovariancancerrdquo Journal of Ovarian Research vol 5 no 1 article 3 2012

[171] A J Butt K A Dickson F McDougall and R C Bax-ter ldquoInsulin-like growth factor-binding protein-5 inhibits thegrowth of human breast cancer cells in vitro and in vivordquo TheJournal of Biological Chemistry vol 278 no 32 pp 29676ndash29685 2003


[172] B Tanno V Cesi R Vitali et al ldquoSilencing of endogenousIGFBP-5 bymicro RNA interference affects proliferation apop-tosis and differentiation of neuroblastoma cellsrdquo Cell Death andDifferentiation vol 12 no 3 pp 213ndash223 2005

[173] C McCaig C M Perks and J M P Holly ldquoIntrinsic actionsof IGFBP-3 and IGFBP-5 on Hs578T breast cancer epithelialcells inhibition or accentuation of attachment and survival isdependent upon the presence of fibronectinrdquo Journal of CellScience vol 115 no 22 pp 4293ndash4303 2002

[174] S K Johnson and R S Haun ldquoInsulin-like growth factorbinding protein-5 influences pancreatic cancer cell growthrdquoWorld Journal of Gastroenterology vol 15 no 27 pp 3355ndash33662009

[175] X L Xu T C Lee N Offor et al ldquoTumor-associated retinalastrocytes promote retinoblastoma cell proliferation throughproduction of IGFBP-5rdquoTheAmerican Journal of Pathology vol177 no 1 pp 424ndash435 2010

[176] A Sokolovic M Sokolovic W Boers R P O Elferink and P JBosma ldquoInsulin-like growth factor binding protein 5 enhancessurvival of LX2 human hepatic stellate cellsrdquo Fibrogenesis andTissue Repair vol 3 article 3 2010

[177] L J Cobb D A M Salih I Gonzalez et al ldquoPartitioningof IGFBP-5 actions in myogenesis IGF-independent anti-apoptotic functionrdquo Journal of Cell Science vol 117 part 9 pp1737ndash1746 2004

[178] H Miyake M Pollak and M E Gleave ldquoCastration-inducedup-regulation of insulin-like growth factor binding protein-5potentiates insulin-like growth factor-I activity and acceleratesprogression to androgen independence in prostate cancermodelsrdquo Cancer Research vol 60 no 11 pp 3058ndash3064 2000

[179] P Fu J AThompson and L A Bach ldquoPromotion of cancer cellmigration An insulin-like growth factor (IGF)-independentaction of IGF-binding protein-6rdquo Journal of Biological Chem-istry vol 282 no 31 pp 22298ndash22306 2007

[180] P Fu Z Yang and L A Bach ldquoProhibitin-2 binding mod-ulates insulin-like growth factor-binding protein-6 (IGFBP-6)-induced rhabdomyosarcoma cell migrationrdquo The Journal ofBiological Chemistry vol 288 no 41 pp 29890ndash29900 2013

[181] P Ye Y Xing Z Dai and A J DrsquoErcole ldquoIn vivo actions ofinsulin-like growth factor-I (IGF-I) on cerebellumdevelopmentin transgenic mice evidence that IGF-I increases proliferationof granule cell progenitorsrdquo Developmental Brain Research vol95 no 1 pp 44ndash54 1996

[182] P Ye and J DrsquoErcole ldquoInsulin-like growth factor I (IGF-I)regulates IGF binding protein-5 gene expression in the brainrdquoEndocrinology vol 139 no 1 pp 65ndash71 1998

[183] J Wang W Niu Y Nikiforov et al ldquoTargeted overexpression ofIGF-I evokes distinct patterns of organ remodeling in smoothmuscle cell tissue beds of transgenic micerdquo The Journal ofClinical Investigation vol 100 no 6 pp 1425ndash1439 1997

[184] G Zhao R L Sutliff C S Weber et al ldquoSmooth muscle-targeted overexpression of insulin-like growth factor I resultsin enhanced vascular contractilityrdquo Endocrinology vol 142 no2 pp 623ndash632 2001

[185] B Zhu G Zhao D P Witte D Y Hui and J A FaginldquoTargeted overexpression of IGF-I in smooth muscle cellsof transgenic mice enhances neointimal formation throughincreased proliferation and cell migration after intraarterialinjuryrdquo Endocrinology vol 142 no 8 pp 3598ndash3606 2001

[186] M K Dyck A F Parlow J-F Senechal M-A Sirard andF Pothier ldquoOvarian expression of human insulin-like growth

factor-I in transgenic mice results in cyst formationrdquoMolecularReproduction and Development vol 59 no 2 pp 178ndash185 2001

[187] J DiGiovanni K Kiguchi A Frijhoff et al ldquoDeregulatedexpression of insulin-like growth factor 1 in prostate epitheliumleads to neoplasia in transgenic micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3455ndash3460 2000

[188] S Clement S Refetoff B Robaye J E Dumont and SSchurmans ldquoLow TSH requirement and goiter in transgenicmice overexpressing IGF-I and IGF-I receptor in the thyroidglandrdquo Endocrinology vol 142 no 12 pp 5131ndash5139 2001

[189] L-T Busund E Richardsen R Busund et al ldquoSignificantexpression of IGFBP2 in breast cancer compared with benignlesionsrdquo Journal of Clinical Pathology vol 58 no 4 pp 361ndash3662005

[190] M L Neuhouser E A Platz C Till et al ldquoInsulin-like growthfactors and insulin-like growth factor-binding proteins andprostate cancer risk results from the prostate cancer preventiontrialrdquo Cancer Prevention Research vol 6 no 2 pp 91ndash99 2013

[191] A J Butt SM FirthM A King and R C Baxter ldquoInsulin-likegrowth factor-binding protein-3 modulates expression of Baxand Bcl-2 and potentiates p53-independent radiation-inducedapoptosis in human breast cancer cellsrdquoThe Journal of BiologicalChemistry vol 275 no 50 pp 39174ndash39181 2000

[192] N Miyakoshi X Qin Y Kasukawa et al ldquoSystemic admin-istration of insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) increases bone formation parameters in miceby increasing IGF bioavailability via an IGFBP-4 protease-dependentmechanismrdquoEndocrinology vol 142 no 6 pp 2641ndash2648 2001

[193] A Parker A Gockerman W H Busby and D R ClemmonsldquoProperties of an insulin-like growth factor-binding protein-4protease that is secreted by smoothmuscle cellsrdquoEndocrinologyvol 136 no 6 pp 2470ndash2476 1995

[194] M Iwashita Y Kudo and Y Takeda ldquoEffect of follicle stimu-lating hormone and insulin-like growth factors on proteolysisof insulin-like growth factor binding protein-4 in humangranulosa cellsrdquo Molecular Human Reproduction vol 4 no 4pp 401ndash405 1998

[195] S Mazerbourg M T Overgaard C Oxvig et al ldquoPregnancy-associated plasma protein-A (PAPP-A) in ovine bovineporcine and equine ovarian follicles involvement in IGF bind-ing protein-4 proteolytic degradation and mRNA expressionduring follicular developmentrdquo Endocrinology vol 142 no 12pp 5243ndash5253 2001

[196] S Swami N Raghavachari U R Muller Y P Bao and DFeldman ldquoVitamin D growth inhibition of breast cancer cellsgene expression patterns assessed by cDNA microarrayrdquo BreastCancer Research and Treatment vol 80 no 1 pp 49ndash62 2003

[197] H Huynh X F Yang and M Pollak ldquoA role for insulin-likegrowth factor binding protein 5 in the antiproliferative actionof the antiestrogen ICI 182780rdquo Cell Growth and Differentiationvol 7 no 11 pp 1501ndash1506 1996

[198] L A Bach ldquoIGFBP-6 five years on not so ldquoforgottenrdquordquo GrowthHormone and IGF Research vol 15 no 3 pp 185ndash192 2005

[199] L A Bach ldquoInsulin-like growth factor binding protein-6 theldquoforgottenrdquo binding proteinrdquoHormone andMetabolic Researchvol 31 no 2-3 pp 226ndash234 1999

[200] P Jean Ho and R C Baxter ldquoCharacterization of truncatedinsulin-like growth factor-binding protein-2 in human milkrdquoEndocrinology vol 138 no 9 pp 3811ndash3818 1997


[201] S Rehault P Monget S Mazerbourg et al ldquoInsulin-like growthfactor binding proteins (IGFBPs) as potential physiologicalsubstrates for human kallikreins hK2 and hK3rdquo EuropeanJournal of Biochemistry vol 268 no 10 pp 2960ndash2968 2001

[202] S Miyamoto M Nakamura K Yano et al ldquoMatrixmetalloproteinase-7 triggers the matricrine action of insulin-like growth factor-II via proteinase activity on insulin-likegrowth factor binding protein 2 in the extracellular matrixrdquoCancer Science vol 98 no 5 pp 685ndash691 2007

[203] P Monget S Mazerbourg T Delpuech et al ldquoPregnancy-associated plasma protein-A is involved in insulin-like growthfactor binding protein-2 (IGFBP-2) proteolytic degradationin bovine and porcine preovulatory follicles identification ofcleavage site and characterization of IGFBP-2 degradationrdquoBiology of Reproduction vol 68 no 1 pp 77ndash86 2003

[204] U Berg P Bang and C Carlsson-Skwirut ldquoCalpain proteolysisof insulin-like growth factor binding protein (IGFBP) -2 and -3 but not of IGFBP-1rdquo Biological Chemistry vol 388 no 8 pp859ndash863 2007

[205] B A Booth M Boes and R S Bar ldquoIGFBP-3 proteolysis byplasmin thrombin serum heparin binding IGF binding andstructure of fragmentsrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 271 no 3 pp E465ndashE4701996

[206] C Lalou S Sawamura B Segovia Y Ogawa and M BinouxldquoProteolytic fragments of insulin-like growth factor bindingprotein-3 N-terminal sequences and relationships betweenstructure and biological activityrdquo Comptes Rendus de lrsquoAcademiedes SciencesmdashSerie III vol 320 no 8 pp 621ndash628 1997

[207] E Okabe J-I Kajihara Y Usami and K Hirano ldquoThe cleavagesite specificity of human prostate specific antigen for insulin-like growth factor binding protein-3rdquo FEBS Letters vol 447 no1 pp 87ndash90 1999

[208] H Salahifar S M Firth R C Baxter and J L MartinldquoCharacterization of an amino-terminal fragment of insulin-like growth factor binding protein-3 and its effects in MCF-7breast cancer cellsrdquo Growth Hormone and IGF Research vol 10no 6 pp 367ndash377 2000

[209] S D Chernausek C E Smith K L Duffin W H Busby GWright and D R Clemmons ldquoProteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4) Localization ofcleavage site to non-homologous region of native IGFBP-4rdquoTheJournal of Biological Chemistry vol 270 no 19 pp 11377ndash113821995

[210] C Rees D R Clemmons G D Horvitz J B Clarke andW HBusby ldquoA protease-resistant form of insulin-like growth factor(IGF) binding protein 4 inhibits IGF-1 actionsrdquo Endocrinologyvol 139 no 10 pp 4182ndash4188 1998

[211] C A Conover S K Durham J Zapf F R Masiarz and M CKiefer ldquoCleavage analysis of insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis and expression ofprotease-resistant IGF-binding protein-4 mutantsrdquo Journal ofBiological Chemistry vol 270 no 9 pp 4395ndash4400 1995

[212] J B Lawrence C Oxvig M T Overgaard et al ldquoThe insulin-like growth factor (IGF)-dependent IGF binding protein-4protease secreted by human fibroblasts is pregnancy-associatedplasma protein-Ardquo Proceedings of the National Academy ofSciences of the United States of America vol 96 no 6 pp 3149ndash3153 1999

[213] Y Imai W H Busby Jr C E Smith et al ldquoProtease-resistantform of insulin-like growth factor-binding protein 5 is aninhibitor of insulin-like growth factor-I actions on porcine

smoothmuscle cells in culturerdquo Journal of Clinical Investigationvol 100 no 10 pp 2596ndash2605 1997

[214] M T Overgaard H B Boldt L S Laursen L Sottrup-Jensen C A Conover and C Oxvig ldquoPregnancy-associatedplasma protein-A2 (PAPP-A2) a novel insulin-like growthfactor-binding protein-5 proteinaserdquo The Journal of BiologicalChemistry vol 276 no 24 pp 21849ndash21853 2001

[215] B Zheng J B Clarke W H Busby C Duan and D R Clem-mons ldquoInsulin-like growth factor-binding protein-5 is cleavedby physiological concentrations of thrombinrdquo Endocrinologyvol 139 no 4 pp 1708ndash1714 1998

[216] C Garcıa-Echeverrıa M A Pearson A Marti et al ldquoIn vivoantitumor activity of NVP-AEW541mdashA novel potent andselective inhibitor of the IGF-IR kinaserdquo Cancer Cell vol 5 no3 pp 231ndash239 2004

[217] SWolf J Lorenz J Mossner andMWiedmann ldquoTreatment ofbiliary tract cancer with NVP-AEW541 mechanisms of actionand resistancerdquoWorld Journal of Gastroenterology vol 16 no 2pp 156ndash166 2010

[218] A Girnita L Girnita F Del Prete A Bartolazzi O Larssonand M Axelson ldquoCyclolignans as inhibitors of the insulin-likegrowth factor-1 receptor and malignant cell growthrdquo CancerResearch vol 64 no 1 pp 236ndash242 2004

[219] D Vasilcanu A Girnita L Girnita R Vasilcanu M Axelsonand O Larsson ldquoThe cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor Link to the phosphatidyl inositol-3 kinaseAkt apoptotic pathwayrdquo Oncogene vol 23 no 47 pp7854ndash7862 2004

[220] R Vasilcanu D Vasilcanu L Rosengren et al ldquoPicrop-odophyllin induces downregulation of the insulin-like growthfactor 1 receptor potential mechanistic involvement of Mdm2and 120573-arrestin1rdquo Oncogene vol 27 no 11 pp 1629ndash1638 2008

[221] E Menu H Jernberg-Wiklund H de Raeve et al ldquoTargetingthe IGF-1R using picropodophyllin in the therapeutical 5T2MMmouse model of multiple myeloma beneficial effects on tumorgrowth angiogenesis bone disease and survivalrdquo InternationalJournal of Cancer vol 121 no 8 pp 1857ndash1861 2007

[222] M A Economou S Andersson D Vasilcanu et al ldquoOralpicropodophyllin (PPP) is well tolerated in vivo and inhibitsIGF-1R expression and growth of uvealmelanomardquo InvestigativeOphthalmology and Visual Science vol 49 no 6 pp 2337ndash23422008

[223] J Rodon V DeSantos R J Ferry Jr and R Kurzrock ldquoEarlydrug development of inhibitors of the insulin-like growthfactor-I receptor pathway lessons from the first clinical trialsrdquoMolecular Cancer Therapeutics vol 7 no 9 pp 2575ndash25882008

[224] J A Toretsky S M Steinberg M Thakar et al ldquoInsulin-likegrowth factor type 1 (IGF-1) and IGF binding protein-3 inpatients with Ewing sarcoma family of tumorsrdquo Cancer vol 92no 11 pp 2941ndash2947 2001

[225] A P Majumdar S Banerjee J Nautiyal et al ldquoCurcuminsynergizes with resveratrol to inhibit colon cancerrdquo Nutritionand Cancer vol 61 no 4 pp 544ndash553 2009

[226] Y Li and T Zhang ldquoTargeting cancer stem cells by curcuminand clinical applicationsrdquoCancer Letters vol 346 no 2 pp 197ndash205 2014

[227] M A Babcook and S Gupta ldquoApigenin a promising anticanceragent for the modulation of the Insulin-like Growth Factor(IGF) axis in prostate cancerrdquo Biomedical Research vol 23supplement 1 pp 55ndash68 2012


[228] M Jung S Y Bu K-H Tak J-E Park and E Kim ldquoAnti-carcinogenic effect of quercetin by inhibition of insulin-likegrowth factor (IGF)-1 signaling inmouse skin cancerrdquoNutritionResearch and Practice vol 7 no 6 pp 439ndash445 2013

[229] X Li Y Feng J Liu X Feng K Zhou and X TangldquoEpigallocatechin-3-gallate inhibits IGF-I-stimulated lung can-cer angiogenesis through downregulation of HIF-1alpha andVEGF expressionrdquo Journal of Nutrigenetics and Nutrigenomicsvol 6 no 3 pp 169ndash178 2013

[230] D Chen and Q P Dou ldquoTea polyphenols and their roles incancer prevention and chemotherapyrdquo International Journal ofMolecular Sciences vol 9 no 7 pp 1196ndash1206 2008

[231] S Mondal S Bandyopadhyay M K Ghosh S MukhopadhyayS Roy and C Mandal ldquoNatural products promising resourcesfor cancer drug discoveryrdquo Anti-Cancer Agents in MedicinalChemistry vol 12 no 1 pp 49ndash75 2012

[232] N Sultana ldquoClinically useful anticancer antitumor and anti-wrinkle agent ursolic acid and related derivatives as medici-nally important natural productrdquo Journal of Enzyme Inhibitionand Medicinal Chemistry vol 26 no 5 pp 616ndash642 2011

[233] Y-J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[234] N Khan N Hadi F Afaq D N Syed M-H Kweon andH Mukhtar ldquoPomegranate fruit extract inhibits prosurvivalpathways in human A549 lung carcinoma cells and tumorgrowth in athymic nude micerdquo Carcinogenesis vol 28 no 1 pp163ndash173 2007

[235] C A Lamartiniere M S Cotroneo W A Fritz J Wang RMentor-Marcel and A Elgavish ldquoGenistein chemopreventiontiming and mechanisms of action in murine mammary andprostaterdquoThe Journal of Nutrition vol 132 no 3 pp 552sndash558s2002

[236] Y Li and F H Sarkar ldquoGene expression profiles of genistein-treated PC3 prostate cancer cellsrdquoThe Journal of Nutrition vol132 no 12 pp 3623ndash3631 2002

[237] S R Chinni Y Li S Upadhyay P K Koppolu and F H SarkarldquoIndole-3-carbinol (I3C) induced cell growth inhibition G1 cellcycle arrest and apoptosis in prostate cancer cellsrdquo Oncogenevol 20 no 23 pp 2927ndash2936 2001

[238] Y Li X Li and F H Sarkar ldquoGene expression profiles of 13C-and DIM-treated PC3 human prostate cancer cells determinedby cDNAmicroarray analysisrdquo Journal of Nutrition vol 133 no4 pp 1011ndash1019 2003

[239] K Hastak S Gupta N Ahmad M K Agarwal M L Agarwaland H Mukhtar ldquoRole of p53 and NF-120581B in epigallocatechin-3-gallate-induced apoptosis of LNCaP cellsrdquo Oncogene vol 22no 31 pp 4851ndash4859 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


ProteasesIGF-1

IGF-1R

Extracellular matrix

Cytoplasm

IGFBP

IGFBP fragments

ALSIGF-1

IGF-1

IGF-1

ALS

Blood vesse

l

Proteolysis

Figure 1 The IGF axis circulating IGFs are protected fromdegradation by forming complex with IGFBPs IGFs apart fromtheir local functioning in an autocrine or a paracrine manner enterthe bloodstream where they exist as binary complexes with eachIGFBP In addition ternary complex also exists when the binarycomplexes with IGFBP-3 or IGFBP-5 interact with the acid labilesubunit (ALS) After dissociation of ternary complex the binarycomplexes of IGFBP-IGF are removed from the circulation and crossthe endothelium to reach the target tissues and to interact with cellsurface receptors

the receptors Proteolysis of the IGFBPs dissociates IGFs fromthe complex enabling them to bind and activate the cellsurface receptors Deregulation of IGF-1R signalling has beennoted to contribute to a variety of diseases including diabeticretinopathy [3] diabetic nephropathy [4] age-related macu-lar degeneration [5] cardiovascular disease and aging and ina variety of cancers [5]

IGF system is gaining tremendous interest over the lastdecade because it plays an important role in cancer The cur-rent treatment options for cancer have shifted more towardsthe targeted therapies [6 7] rather than the traditionalchemotherapy Many strategies have been exploited to targettumours The most commonly used strategy is engineeredantibodies or antibody fragments [8] Though monoclonalantibodies are very selective poor penetration inside thetumours and high production cost hinder their usage astherapeutic agents [9] Current therapeutics targeting theIGF-signalling pathways focus on blocking IGF-1R directlyandor its downstream effectors [10] However a potentialdrawback of such approaches is the resulting adverse sideeffects or toxicities due to its interference with the insulinpathway As a more efficacious alternative we propose thatIGFBPs can be developed as IGF-antagonist based cancertherapeutics serving to block the IGF-1R mediated tumourprogression Notably the IGFBPs do not bind insulin andthus do not interfere with insulin-insulin receptor interac-tions

In the current paper we will provide a brief overview onIGF systemanddiscuss some literature and experimental datareported to demonstrate the role of IGF system in cancer anddevelopment of new targeted anticancer therapies Because it

is not possible to provide a complete coverage of all publishedpapers dealing with IGF system we have mainly focusedon different strategies targeting IGF system in cancer andattempted to provide an overview on IGF system includingIGF-1R its inhibitors and potential limitations of IGF-1Rantibodies and tyrosine kinase inhibitors IGFBP actionsand blocking IGF via IGFBP (which is better option thanblocking IGF receptors) leading to development of novelcancer therapies

2 DiscoveryHistory of IGF System

The first member of IGF family to be identified was insulinwith subsequent investigation resulting in the elucidationof its role in glucose metabolism and its implication in theaetiology of diabetes mellitus This discovery effected anexplosion in the investigation of the structure function andmechanisms of action of insulin The enormous interest inthis molecule resulted in the concession of three Nobel Prizesfor the investigation of insulin in 1923 for the discovery ofits capacity to treat diabetes by Frederick Banting and J JMacleod [11] in 1958 for the first sequence of a protein byFrederick Sanger [12] and in 1963 for the first determinationof the three-dimensional structure of a protein by DorothyHodgkin [13] Hence the investigation of insulin has beena pioneer in many scientific fields Later the IGFs werediscovered and found to be intricately involved in embryonicdevelopment and postnatal growth

The existence of the IGFs was first proposed by Salmonand Daughaday in 1957 on the basis of studies indicat-ing that growth hormone (GH) did not directly stimulatethe incorporation of sulfate into cartilage but rather actedthrough a serum factor [14] In the original study by Salmonand Daughaday 35S-labeled amino acid was incorporatedinto cartilage explants and was used as a surrogate forgrowth The serum of normal rats induced 35S-amino acidincorporation into cartilage but not serum fromhypophysec-tomized rats However serum from hypophysectomized ratstreated with GH yielded serum that allowed for 35S-aminoacid incorporation indicating that a second messenger wasnecessary forGH signallingThis factorwas originally termedsulfation factor then somatomedin and ultimately insulin-like growth factor-1 and insulin-like growth factor-2 IGF-1 was not purified and characterized until more than twodecades later [15] The terminology ldquoinsulin-likerdquo was usedbecause these factors are able to stimulate glucose uptake intofat cells and muscle and indeed both IGF-1 and IGF-2 showapproximately 50 homology with insulin [15 16]

Subsequent investigation demonstrated that GH afterbinding to its transmembrane receptor initiates a signallingcascade leading to transcriptional regulation of IGF-1 andrelated genes It was originally thought that systemic growthwas promoted by GH acting mainly on the liver to stimulateIGF-1 production which then reached target tissues viathe circulation to activate mechanisms involved in tissueproliferation growth and metabolism It is now evident thatnot only does GH have independent actions that do notinvolve IGF-1 production [17] but IGF-1 synthesis occurs





Hypothalamus

Pituitary

Liver

IGF-1

GH















Muscle smooth

















Ins

IGF-2

IGF-1




















290 300 310 320 330 340

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211

266

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220 230 240 250 260 270 280

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146

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129

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98

102







80 90 100 110 120 130 140

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45

63

55

41

42

42







10 20 30 40 50 60 70

||||||||||||||







1

1

1

1

1

1






[30 157]



[189 190]



[191]



[79 192ndash195]




[198 199]






IGFBP-4















CBP-1 CBP-2 CBP-6












105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Hypothalamus

Pituitary

Liver

IGF-1

GH















Muscle smooth

















Ins

IGF-2

IGF-1




















290 300 310 320 330 340

||||||||||||







211

266

245

209

224

195







220 230 240 250 260 270 280

||||||||||||||







146

196

182

151

161

141







||||||||||||||







150 160 170 180 190 200 210

102

129

112

98

98

102







80 90 100 110 120 130 140

||||||||||||||







45

63

55

41

42

42







10 20 30 40 50 60 70

||||||||||||||







1

1

1

1

1

1






[30 157]



[189 190]



[191]



[79 192ndash195]




[198 199]






IGFBP-4















CBP-1 CBP-2 CBP-6












105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Muscle smooth

















Ins

IGF-2

IGF-1




















290 300 310 320 330 340

||||||||||||







211

266

245

209

224

195







220 230 240 250 260 270 280

||||||||||||||







146

196

182

151

161

141







||||||||||||||







150 160 170 180 190 200 210

102

129

112

98

98

102







80 90 100 110 120 130 140

||||||||||||||







45

63

55

41

42

42







10 20 30 40 50 60 70

||||||||||||||







1

1

1

1

1

1






[30 157]



[189 190]



[191]



[79 192ndash195]




[198 199]






IGFBP-4















CBP-1 CBP-2 CBP-6












105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Ins

IGF-2

IGF-1




















290 300 310 320 330 340

||||||||||||







211

266

245

209

224

195







220 230 240 250 260 270 280

||||||||||||||







146

196

182

151

161

141







||||||||||||||







150 160 170 180 190 200 210

102

129

112

98

98

102







80 90 100 110 120 130 140

||||||||||||||







45

63

55

41

42

42







10 20 30 40 50 60 70

||||||||||||||







1

1

1

1

1

1






[30 157]



[189 190]



[191]



[79 192ndash195]




[198 199]






IGFBP-4















CBP-1 CBP-2 CBP-6












105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























290 300 310 320 330 340

||||||||||||







211

266

245

209

224

195







220 230 240 250 260 270 280

||||||||||||||







146

196

182

151

161

141







||||||||||||||







150 160 170 180 190 200 210

102

129

112

98

98

102







80 90 100 110 120 130 140

||||||||||||||







45

63

55

41

42

42







10 20 30 40 50 60 70

||||||||||||||







1

1

1

1

1

1






[30 157]



[189 190]



[191]



[79 192ndash195]




[198 199]






IGFBP-4















CBP-1 CBP-2 CBP-6












105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















[30 157]



[189 190]



[191]



[79 192ndash195]




[198 199]






IGFBP-4















CBP-1 CBP-2 CBP-6












105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























CBP-1 CBP-2 CBP-6












105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CBP-1 CBP-2 CBP-6












105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















105

110

115

120

125

130

135

1000 900 800 700 600

IGFBP-2(1ndash289)

1HN (ppm)15N

(ppm

)

(a) (b)
























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























TKIs




siRNAs

Truncated IFG-IR

Anti-IGF-IRMAbs



















MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























MK-0646




























Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Inhibitors of

kinase domainIR-A

IGFsIGFBPIGFs

IGFsIGFs

IGF-1R


IGFs IGFBPs

Block IGF-1R

IGFBPs

insulin

Inhibition of


tyrosine

IGFs

do not bind













Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Apigenin

Chamomile tea

Genistein


Resveratrol

Curcumin

Quercetin

Lycopene





HO

OO O

O

OH

CH3CH3

HO O

O OHOH

HO

O

O

OH

OH

HO

O

O

OHOH

OH

OH

HO O

O

OOH

OH

OH

OH

OH

OHOH

HO

OH

OH

CH3 CH3 CH3

CH3

CH3

CH3CH3CH3

H3C

H3C







IGF-1R



IGFBP-3

Apigenin [227]















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















References

































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































































































































































































































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