Ahmed Group Lecture 15 CANCER LECTURE 15 Ahmed Group Lecture 15 Cancer as a Genetic Disease Oncogenes Tumor suppressor genes Telomeric changes in cancer

Ahmed GroupAhmed GroupLecture 15Lecture 15

CANCER

LECTURE 15


• Cancer as a Genetic Disease• Oncogenes• Tumor suppressor genes• Telomeric changes in cancer• Epigenetic changes in cancer: e.g. hypermethylation• Multi-step nature of carcinogenesis• Repair genes in carcinogenesis• The processes of metastasis• Molecular profiling and staging of cancer: Gene

expression profiling and proteomics• Signaling abnormalities in cancer: Effects of signaling abnormalities on radiation responses• Prognostic significance of tumor characteristics• Therapeutic targets and strategies for intervention• Monoclonals, small molecule inhibitors, gene therapy

Cancer


Gain if function mutation in an

oncogene

Loss of function mutation in a tumor

suppressor gene

Malignant Transformation

The process of malignant transformation may result from either a gain-of-function mutation that activates an oncogene, or a loss-of-function mutuation in a tumor-suppressor gene.

Cancer as a Genetic Disease

Neoplastic Development


The way in which the concept of oncogenes provides a ready answer for how agents as diverse as viruses, radiations, and chemicals all can induce tumors that are essentially indistinguishable from another. The retrovirus inserts a gene; a chemical may activate an endogenous oncogene by a point mutation; radiation may do the same by, for example a translocation.

Augmented Expression

Normal Expression

Inappropriate Expression

Neoplastic Growth

Normal Growth & Development

Radiation (Translocation)

Chemicals (Point Mutation)Retrovirus

inserts gene

Activated oncogene




Cancer

A gene that causes cancer is called an oncogene

Humans contain genes that can be converted to oncogenes

A normal gene with the potential to become an oncogene is called a proto-oncogene

Many proto-oncogenes code for growth factors.

Oncogenes were first discovered from study of retroviruses.

There are two types of Tumor Viruses

DNA Tumor viruses.

RNA Tumor viruses.

ONCOGENE

Conversion of a protooncogene into an oncogene

Deletion or point mutation in coding sequence

Gene amplificationChromosome rearrangement

Hyperactive protein made in normal amounts

Normal protein greatly overproduced

Near strong enhancer overexpression of normal protein

Fusion protein is hyperactive

Protooncogene to Oncogene

1. A point mutation, eg., K-Ras2. Chromosomal rearrangement3. Gene AmplificationChronic Myelogenous Lymphoma

Burkitt’s Lymphoma

Between 8 and 14

Acute myelocytic leukemia7:15, 9:18, 11:15:17

Myc gene has IgG Promoter

Protooncogene to Oncogene

1. A point mutation2. Chromosomal rearrangement3. Gene Amplification

N-myc is a characteristic ofmany neuroblastomas

+

+

+

-

+

+

Myc


Cooperating genes


Chromosomal Changes Leading to Oncogene Activation and the Human Malignancies Associated With Them




Cancer


How hybrid cells can be used to demonstrate the way in which tumorigenicity may result from the loss of a suppressor gene. HeLa cells are tumorigenic; normal human fibroblasts are not. HeLa cells are transformed, as indicated by their ability to grow in soft agar, and tumorigenic, as evidenced by their ability to form tumors in immune-suppressed animals. Normal human fibroblasts do not show either property of malignancy. A hybrid formed by the fusion of a HeLa cell and a normal human fibroblast grows in soft agar but does nor form tumors: The malignancy. A hybrid formed by the fusion of a HeLa cell and a normal human fibroblast grows in soft agar but does not form tumors: The malignant phenotype of the HeLa cell is suppressed by the normal fibroblast. If, however, chromosome 11 from the normal fibroblast is lost, tumorigencity is restored, ingene is located on that chromosome.

Tumor-suppressor genes

It was discovered in the 1970s that the tumorigenicity of some tumor cells could be suppressed by hybridization with normal cells


Effects of Suppressor

Illustrating the effect of the suppressor gene on human chromosome 11. HeLa cells are tumorigenic in nude mice. If a microcell is introduced containing chromosome 11 from a mormal human fibroblast, the malignant phenotype of the HeLa cell is suppressed. If durig culture, chromosome 11 is lost, tumorigenicity is restored. The chromosome in the microcell is number 11 translocated with part of the X chromosome containing the HGPRT gene as a marker of the presence or absence of the chromosome in the HeLa cell.


Rb Mutations

Rb mutations in familial and sporadic retinoblastoma. In familial retinoblastoma, one normal and one mutated Rb are inherited (1) Subsequent mutation in any retinal cell inactivates the remaining Rb (2), leading to loss of growth control in a clone of tumor cells (3). In sporadic retinoblastoma, two normal Rb are inherited (4). First, a mutation inactivates one copy of Rb (5). A subsequent mutation within the same retinal cell inactivates the remaining copy of Rb (6), leading to loss of growth control in a clone of tumor cells.


Currently Identified Tumor Suppressor Genes


Gain of function mutation in an

oncogene

Loss of function mutation in a tumor

suppressor gene

AutosomalDominant

Recessiveat the

cellular level

Dominant in a

pedigree

Gain-of-function mutations that result in the activation of an oncogene require only one copy to be activated; that is, oncogenes act in a dominant fashion. Loss-of-function mutations that inactivate a tumor-suppressor gene require both copies to be inactivated for the malignant phenotype to be expressed; that is, tumor-suppressor genes act in a recessive fashion. This may be true at a cellular level; however, in viewing the pedigree of a cancer-prone family, the loss of a tumor-suppressor gene may appear to be inherited as a dominant mutation.

Mutations required for the oncogenes and tumor-suppressor genes to be expressed


Somatic Homozygosity

The process of somatic homozygosity. In a normal cell, ther are two copies of each chromosome. One inherited from each parent. For a given suppressor gene to be ina ctivated, the copy must be lost from both chromosomes. This could, of course, occur by independent deletions from the two chromosomes, but in practice it is more common fro a single deletion to occur in one chromosome while the second chromosome is lost completely. The remaining chromosome, with the deletion, then replicates. The cell is thus homozygous, rather than heterozygous, for that chromosome.




Cancer


Telomeres and Cancer

Telomeres cap and protect the ends of chromosomes.Mammalian telomeres consist of long arrays of the repeat sequenceTTAGGG that range in length anywhere from 1.5 to 150 kilobasesEach time a normal somatic cell divides, the terminal end of thetelomere is lost. Telomere length has been described asthe “molecular clock”, because it shortens with age.Cancer cells avoid this process of aging by activating the enzymetelomerase.However, the tumors have been identified with stable telomerelength but undetectable telomerase activity.The questions thus remains: Is immortalization or carcinogenesisintimately associated with telomerase expression or not?

Telomeres and Cell Senescence




Cancer


At the outset, it is important that the term epigenetics be defined. Although this term had been coined before, Holliday provided definition for this concept in reviews 15 years or so ago. As used today, this term refers to a change in gene expression that is heritable (ie, that is can be passed on through cell division) but that does not involve a change in DNA sequence.

This contrasts with a true genetic alteration. In this context, it is helpful to look at the entire repertoire of changes that accomplish the gains or losses of gene function that result in the transformed phenotype. These changes are outlined in the next slide and span from those that are completely permanent to those that are very dynamic. Epigenetic changes are semi-permanent.

Epigenetic changes in cancer


Epigenetic changes in cancer


• Genetic alterations are a hallmark of human cancer. • Changes in DNA methylation, an epigenetic modification that is present in

mammalian cells, are also characteristic of human cancer.

• The CpG dinucleotide, which is usually underrepresented in the genome, is clustered in the promoter regions of some genes.

• These promoter regions have been termed CpG islands. CpG islands are protected from methylation in normal cells, with the exception of genes on the inactive X chromosome and imprinted genes.

• This protection is critical, since the methylation of promoter region CpG islands is associated with a loss of expression of these genes.

• The following three different alterations in DNA methylation are common in human cancer: (1) global hypomethylation, often seen within the body of genes; (2) dysregulation of DNA methyltransferase I, the enzyme involved in maintaining methylation patterns, andpotentially other methyltransferases; and (3) regional hypermethylation in normally ummethylated CpG islands.

Epigenetic changes in cancer: e.g. methylation


Epigenetic changes in cancer: e.g. methylation




Cancer


Illustrating the multistep nature of carcinogenesis and the concept of the phenotype. The first step in carcinogenesis by radiation or any other agent may be a mutation in one of the gene families responsible for the stability of the genome. This may be a DNA repair gene, a mismatch repair gene, or a gene in a family as yet unidentified. This leads to the mutator phenotype, with multiple mutations possible in both oncogenesand tumor-suppressor genes. This leads to a series of steps that result in an invasive metastatic cancer. Not all the same mutations need to be present in every case.

Multistep Process Of Cancer

Mechanisms of Multistep

Tumorigenesis


Multistep Process Of Cancer

Cancer has long been thought to be a multistep process and has been described with operational terms such as initiation, promotion, and progression. In at least one human malignancy, namely, colon cancer, the molecular events during the progress of the disease have been identified. (Based on the work of Vogelstein)

Hallmarks of Cancer Cells

Self-maintained replicationLonger survivalGenetic instabilityCapable of inducing

neo-angiogenesisCapable of invasion and metastasis


Radiation Carcinogenesis

• A stochastic late effect.• No threshold, an all or none effect.• Severity is not dose related.• Probability of carcinogenesis is dose dependent.• Leukemia has the shortest latency period of ~5 years.

Solid tumors have a latency period of ~20 to 30 years.• Total cancer risk for whole body irradiation is one

death per 104 individuals exposed to 1 rem.• For every leukemia induced there are 3 to 4 sarcoma

induced in the same irradiated population.




Cancer

DNA Repair

Oncogenes

Activation

Tumor Suppressor Genes

Inactivation

Differentiation Apoptosis/Proliferation

CANCER

Alterations of Specific Cellular Functions in Alterations of Specific Cellular Functions in CancerCancer




Human Mismatch Repair Genes

Gene Chromosomal location

Germline Mutations in HNPCC Cases (Reported family

Studies), %

hMSH2 2p21-22 (2p16?) 60

hMLH1 3p21 30

hPMS1 2q31-33 4

hPMS2 7p21 4

GTBP 2p16 (2p21-22)?) 0

Repair genes in carcinogenesis




Cancer


The processes of metastasis

Cancer cells breaking away from their original site and spread to other parts of the body. The cells spread through blood stream and lymphatic system.

Tumors of the same histologic type arising in different patients differ widely in growth rate. By contrast, metastases arising in the same patient tend to have similar rates of growth. The latter observation is the basis for using patients with multiple skin orpulmonary metastases to test and compare new treatment modalities, such as high linear energy transfer radiations or hyperthermia.


The processes of metastasisData have been accumulated comparing the growth rate of primary breast and bronchial cancers to that of metastases in the same person. The primary carcinomas grew significantly more slowly than the metastases. No entirely satisfactory explanation has been suggested for the more rapid proliferation of metastases. It may be a question of selection or a function of the favorable milieu into which secondary tumors tend to be seeded.




Cancer


• Sequencing of the human genome, • Advances in robotics• Advances in computing • Advances in imaging technologies Microarray and

Protenomics Technology

(1) The classification and identification of tumor classes (2) Gene discovery, (3) Determining mechanisms of drug action, and (4) Predicting drug response





Cancer


Cell Cycle Regulation

The current concept of the cycle and its regulation by protein kinases, activated by cyclins.


Progression through the cell cycle from one phase to the next is governed by protein kinases, activated by cyclins. In mammals, cyclins A through H have been described: each cyclin protein is synthesized at a discrete phase of the cell cycle. Cyclin levels oscillate with phase of cycle, as shown schematically in this figure


Signal Transduction Pathways

Ionizing radiation can activate both nuclear and membrane-cytoplasmic signal transduction pathways. Their interactions are illustrated in the figure, with the dotted lines representing more hypothetical or controversial effects. The major outcomes of the activation of these pathways are listed in the box on the righthand side of the figure.


Ras mediates its effect on cellular proliferation, at least in partr, by the activation of a cascade of kinases. Ras is a GDP/GTP-regulated binary switch that resides at the inner surface of the plasma membrane and acts to relay extracellular ligand-stimulated signals to cytoplasmic signaling cascades. A linear pathway in which ras functions downstream of receptor tyrosine kinases and upstream of cascade of serine-threonine kinases provides a complete link between the cell surface and the nucleus. Activated ERKs can translocate into the nucleus to phosphorylate and activate transcription factors, such as Elk-1, Activated ERKs also phosphorylate substrates in the cytoplasm, including the Mnk kinase, and thus contribute to translation initiation of mRNAs with structured 5’ – untranslated regions. This is an oversimplified illustration, because there are at least three signaling pathways that lie downstream of ras.


Illustrating the protein kinase C pathway, which processes extracellular signals and transmits tham to the nucleus. Extracellular growth factors (such as PDF) activate a receptor, which in turn activates a G protein that in turn activates the phopholipase C. In less than a second, the enzyme cleaves PIP2 to genrate two products, inositol triphosphate and diacylglycerol. IP3 is a as the endoplasmic reticulum. The enzyme activated by diacylglycerol is protein kinase C, called such because it is Ca2+ dependent. Protein kinase C phosphorylates specific serine and threonine residues on cellular proteins in addition to activating the plasma membrane Na+H+ exchanger that controls intracellular pH. Also, the calcium enters the nucleus and activates the transcription of growth-regulating genes, such as myc and fos.


The role of p53 in transduction of the signal from radiation damage. The transcriptional activity of p53 is modulated in response to DNA damage by the activity of a number of kinases including ATM and DNApk), as well as by protein : protein interactions, resulting in either cell-cycle arrest or apoptosis.



ATM ATM

ATMP

ATM

CHK2

PATM Nbs1 P

ATMPBrca1

P

P

ATMP

p53P

Bax Cell Death

p21 waf1/cip1G1 Arrest

DNA Repair

Ionizing radiation Chromatin

changes

ReactiveOxygenSpecies

EGR-1

Ras AKT/PI3-K

NFB

Bcl-2

TNF-

MDR1

Chemo-Resistance

SurvivalProliferation

Caspaseactivation

Autophosphorylation

Substratephosphorylation

PFocus Formation

Radiation induced signaling response: wild type p53 background


ATM ATM

ATMP

ATMP

Bax

p21 waf1/cip1

DNA Repair

Ionizing radiation Chromatin

changes

ReactiveOxygenSpecies

Mutant p53

Autophosphorylation

Substratephosphorylation

Ras AKT/PI3-K

NFB

Bcl-2

TNF-

MDR1

Chemo-Resistance

SurvivalProliferation

Induced RadiationResistance

Focus Formation

Radiation induced signaling response: mutant p53 background

ATM Nbs1 P

ATMPBrca1

P

P


Grb2

SOSRas

Raf

MEK1/2

ERK1/2

ERK1/2

ERK1/2

ERK1/2

p90rsk

p90rsk

mTOR

AKT/pKB

CREB

c-FosSRF

c-Jun

STAT1/3ELK-1 Ets

PI3K

4E-BP1 p70S6K

PKC

TPAPlasma Membrane

Cytoplasm

Nucleus

Growth factor

Receptor (ex.: EGFR)Growth Factor

(ex.: EGF)

Growthdifferentiation

Survival

MAPK

pp

NFB

NFB

Other cell survival pathways that may affect radiation sensitivity

Ionizing

radiation


Effect of radiation on DU-145 transfectants overexpressing EGR-1 or dominant-negative

mutant of EGR-1

BB

BB

BB

JJ

JJ

JJ

H

HH

HH

H

0.01

0.1

1

0 1 2 3 4 5 6Dose (Gy)

B DU-145/Vector J DU-145/CMV-WT1-EGR1H DU-145/CMV-EGR1DU-145/VectorSF2 : 0.609D0 : 400 cGy

DU-145/CMV-WT1-EGR1SF2 : 0.66D0 : 509 cGy

DU-145/CMV-EGR1SF2 : 0.34D0 : 164 cGy


Ionizing Radiation Induces EGR-1 Protein Which Transactivates via the GC –rich Binding

site in Egr-1+/- and Egr-1 +/+ MEF Cells.

UT 15 m 30 m 1h 3h 6h

UT 15 m 30 m 1h 3h 6h

EGR-1

β-actin

-1EGR

β-actin

5 Gy

5 Gy

-1Egr + / -

-1Egr - / -

Western blot analysisEgr-1 - / - Egr-1 + / -

EBS-CATRADIATIONCMV-EGR-1

+ ++- -+

- + -

+-

-

+-

+

++

-

EBS-CATIRCMV-EGR-1

++-+

+-- +-

Egr-1 + / +

PERCENTCONVERSION 5.3 28.6 10

PERCENTCONVERSION 0 0 58.7 1.8 14 56.6

EBS-CAT reporter assay


Ionizing Radiation Caused Enhanced Cell Death in Egr-1+/- Cells

Untreated 5 Gy (24 hours)

0

10

20

30

Egr-1 (+/-)

Egr-1 (-/-)

TUNEL staining

Gate %GatedR1 18.74R2 9.29R3 18.31R4 14.49R5 2.38

MC540

Ho342

MC540

Ho342

Gate %GatedR1 20.08R2 11.57R3 13.99R4 11.77R5 1.74

Untreated

5 Gy (48 hours)

Egr-1- / - MEFsEgr-1+ / - MEFs

R5

R4R3

R2R1

R5R4R3

R2R1

MC540

Ho342

Untreated

5 Gy (48 hours)

MC540

Ho342

Gate %GatedR1 19.14R2 11.08R3 24.11R4 24.67R5 11.16

Gate %GatedR1 3.42R2 1.27R3 41.88R4 31.75R5 13.95

R5

R4

R3

R2R1

R5

R4

R3

R2R1

MC540 and Hoechst (flow cytometry)


Ionizing Radiation Causes down-regulation of p53 Protein in Egr-1-/- MEF Cells.

Western blot analysis


Transfection of CMV-EGR-1 in Egr-1-/- Cells Lead to Restoration of Sensitivity to

Radiation-Induced Apoptosis.

UT 1h 3h 6h 12h UT 1h 3h 6h 12h

5Gy 5Gy

Egr-1-/-/CMV.EGR1 Egr-1-/-/Vector

p53

β-actin

Western blot analysis

UT 5 Gy (24h) 5 Gy (48h)0

10

20

30

40Egr1-/- (Vector)Egr1-/-(CMV.EGR1))

TUNEL staining

Egr-1 +/- Egr-1 -/-0

10

20

30

40

50

60

Untreated5 Gy

p53-CAT reporter assay


B

J

J

JJ

J

J

J

H

H

H H

H

HH

0.001

0.01

0.1

1

0 1 2 3 4 5 6

Dose (Gy)

SCC-61 (Radiation alone)

J SCC-61(Radiation + Paclitaxel-0.5nM)

H SCC-61(Radiation + Paclitaxel-1nM)

Effects of Radiation alone versus combination of Paclitaxel + Radiation in SCC-61 and SQ-20B cells

C

J

J J

JJ

JJ

H

H

HH

HH

H

0.001

0.01

0.1

1

0 1 2 3 4 5 6

Dose (Gy)

SQ-20B (Radiation alone)

J SQ-20B (Radiation + Paclitaxel-0.5nM)

H SQ-20B (Radiation + Paclitaxel-1nM)


A BHCT-116 HT-29

p53p53 p21waf1/cip1p21waf1/cip1

COLORECTAL CANCER CELL LINES

UT 1h 3h 6h

5 Gy

UT 1h 3h 6h

5 Gy

p53

p21waf1/cip1

β-actin

HCT-116 HT-29





Cancer


Breast Cancer

• Markers of tumor proliferation• Steroid hormone receptors• Factors of epidermal cell growth• Plasminogen activators and inhibitors• Factors influencing angiogenesis and apoptosis• Genome characteristics are considered • Ki-67, ER, PR and Her-2/neu recommended for

clinical use.




Cancer


Therapeutic targets and strategies for intervention

Problems with traditional cancer treatment regimens:

• nonselective• cytotoxicity in normal as well as in malignant cells• often not well tolerated

In developing novel anticancer agents, the goal is to target specific molecular lesions within tumor cells, leading to improved cure rates and reducing cytotoxicity in normal cells. Advances in the understanding of tumor pathobiology and molecular biology have allowed the development of targeted therapies.

Ahmed GroupAhmed GroupLecture 15Lecture 15Copyright ©2004 AlphaMed Press

The HER family of receptors is the target of several targeted therapies for breast cancer

Esteva, F. J. Oncologist 2004;9:4-9

The human epidermal growth factor receptor (HER-2, ErbB-2) is overexpressed in 20-25% of invasivebreast cancers. It is considered an important therapeutic target, and several types of moleculartherapies have been developed to target the HER family of receptors.


Therapeutic agent Molecular weight (Daltons)

Tyrosine kinase inhibitor (e.g., gefitinib, erlotinib) ~400Peptide vaccine ~1,000Monoclonal antibody (e.g., trastuzumab, cetuximab) ~150,000

Size comparison of molecularly targeted therapies

The molecular wiegth of these types of therapies ranges from 400 daltons to approximately 150,000, which has implications in terms oftumor penetration


Example: Humanized anti-ErbB monoclonal antibodies in development for the treatment of various types of cancers

Antibody Specificity Selected tumor types Development phase

Trastuzumab (Herceptin®; Genentech, Inc.;South San Francisco, CA) HER-2 (ErbB-2) HER-2+ metastatic breast Approved

Cetuximab (Erbitux®;ImClone Systems, Inc.; EGFR (ErbB-1) Colorectal, NSCLC, IIINew York, NY) pancreatic, breast, HNSCC

ABX-EGF (Amgen;Thousand Oaks, CA) NSCLC, EGFR (ErbB-1) NSCLC, olorectal, prostate, II

renal, HNSCC EMD 72000 (EMD Pharma;Durham, NC) EGFR (ErbB-1) NSCLC, colorectal, ovarian, II

HNSCCPertuzumab (OmnitargTM; HER-2 (ErbB-2), Prostate, ovarian, breast, NSCLC I/IIGenentech, Inc. (ligand-dependent signaling)

Abbreviations: HNSCC = head and neck squamous cell carcinoma; NSCLC = non-small cell lung cancer

Monoclonal antibodies


Example: HER tyrosine kinase (TK) inhibitors

Inhibitor Specificity Selected tumor types Development phase

Gefitinib (Iressa®; Astra ErbB-1 TK Locally advanced or metastatic NSCLC, Approved Zeneca; Wilmington, DE) head and neck, breast, prostate,

ovarian ,glioma, pancreatic, colorectal

Erlotinib (TarcevaTM; ErbB-1 TK NSCLC, head & neck, breast, ovarian, IIIGenentech, Inc.; CA) prostate, pancreatic, colorectal, glioma

EKB-569 (Wyeth-Ayerst ErbB-1/2 TK NSCLC, breast, other ErbB-dysregulated I/IILaboratories, Inc.; PA) solid tumors

CI-1033 (Pfizer Inc.; CT) ErbB-1 to –4 NSCLC, breast, other ErbB-dysregulated I/II TK solid tumors

Lapatinib(GlaxoSmithKline; NC) ErbB-1/2 TK ErbB-dysregulated solid tumors II

Abbreviation: NSCLC = non-small cell lung cancer

Small molecules


Radiolabeled Immunoglobulins

• Human antibodies attached to a radioactive isotope

• Purpose is to deliver the radioisotope to the tumor using an antibody which is specific to the tumor



Small molecule inhibitors

Historically, certain classes of molecules have been particularly amenable to the modulation of their activity by small molecule drugs. These classes include receptors and enzyme.

Receptor Tyrosine KinasesA family of proteins collectively referred to as receptor tyrosine kinases (RTKs) provides an attractive group of new molecular targets for cancer therapy. The role of some of these targets in the pathology of cancer cells is well established, and much progress has recently been made in the identification of small-molecule drugs directed against representatives of this class of receptors. Some of these molecules were identified at OSI, like TarcevaTM, which targets the Epidermal Growth Factor Receptor (HER1/EGFR), and CP-724,714 and CP-547,632, both small molecule inhibitors that target the receptor for the Vascular Endothelium Growth Factor (VEGF) (included in angiogenesis) and HER-2/neu, a related protein to HER1/EGFR that is particularly important in breast cancer. These are currently undergoing clinical evaluation (Tarceva™ in partnership with Genentech and Roche, and VEGF inhibitor and HER2/neu inhibitor trials conducted by Pfizer). Several other RTKs are also implicated in various aspects of cancer biology. These RTKs might provide more targets for small-molecule drug discovery and are the subject of active research.


Regulatory Enzymes

Another class of proteins particularly amenable to small-molecule drug discovery are regulatory enzymes. There are many examples of small molecule drugs that disrupt transfer of small chemical groups in reactions involving enzymes. Different enzyme classes catalyze these group transfer reactions: kinases, methyltransferases, acetyltransferases, deacetylases and phosphatases, to name a few. These enzymes can act to modify proteins in post-translational modification events that regulate key cellular processes necessary for continued cell division and growth. Broad-spectrum inhibition of cGMP phosphodiesterases has been shown to lead to elevated in cGMP levels and sustained activation of Protein-Kinase G (PKG). This in turn directly phosphorylates MEKK1, leading to activation of the N-terminal cJun Kinase (JNK-1) pathway, a known pro-apoptotic signaling pathway. Kinases catalyze the transfer of a phosphate group to proteins.

Small molecule inhibitors


Deregulated Signaling Pathways in Cancer

Within the processes of proliferation or apoptosis examples are three key signaling axes (see next slide), namely the Ras-Raf-Mek-Erk axis (pathway 1); the PI-3K-PDK-PKB axis (pathway 2) and the PKG-MEKK1-JNK1 axis (pathway 3). These three pathways are thought to be critical in driving either cancer cell proliferation (EGFR or c-kit via pathway 1) or in protecting cancer cells from undergoing apoptosis (IGF-1R or PKB via pathway 2, and PDEs and PKG via pathway 3). Importantly these signaling pathways also form a framework that allows combination approaches to be designed and evaluated such that OSI can leverage the maximal therapeutic impact of novel targeted therapies (e.g. Tarceva, OSI-461).

Potential therapeutic targets


Proteins such as PDK-1 and PKB as potential targets, as they are critical downstream elements of the IGF-1R signal transduction path essential in suppression of apoptosis.One example of our approach to inhibit cellular proliferation is via inhibition of the Epidermal Growth Factor Receptor (EGFR). This receptor is over-expressed or mutated in many human cancers, contributing critically to their aberrant signaling and the development of tumors. Our lead small-molecule inhibitor of the HER1/EGFR, TarcevaTM, received approval in November 2004.attempts to modulate apoptosis, is through activation of Protein Kinase G (PKG) signaling. Broad-spectrum inhibition of cGMP phosphodiesterases has been shown to lead to elevated of cGMP levels and sustained activation of PKG. This in turn directly phosphorylates MEKK1, leading to activation of the N-terminal cJun kinase (JNK-1) pathway and apoptosis of cancer cells.


Cancer gene-therapy is the transfer of nucleic acids into tumor/normal cells to eliminate or reduce tumor burden by:

•Direct cell-killing

•Immunomodulation

•Correcting genetic errors to reverse malignant state

Cancer gene therapy


In 1990: First experiment in Gene Therapy to treat Adenosine Deaminase Deficiency

By the end of 1993: 45 trials by US

Recombinant DNA Advisory Committee

30 to treat cancer


TechnologiesGene therapy for the treatment of

cancer has a wide variety of potential uses. There are several

potential strategies for gene therapy in the treatment of

cancer.


Germinal Gene Therapy

Somatic Gene Therapy

Ex-vivo

In-vivo

TWO TYPES OF GENE THERAPY(ON THE TISSUE -SPECIFIC)


Strategies of Gene Therapy for Cancer

• Enhancing the immunogenicity of the tumor, for example: introducing genes that encode foreign antigens.

• Enhancing immune cells to increase anti-tumor activity, for example: introducing genes that encode cytokines.

• Inserting a "sensitivity" or suicide' gene into the tumor, for example: introducing the gene that encodes HSV-tk.



• Blocking the expression of oncogenes, for example: introducing the gene that encodes antisense K-RAS message.

• Inserting a wild-type tumor suppressor gene, for example: p53 or the gene involved in Wilms' tumor.

• Protecting stem cells from the toxic effects of chemotherapy, for example: introducing the gene that confers MDR-1.



• Blocking the mechanisms by which tumors evade immunological destruction, for example: introducing the gene that encodes antisense IGF-l message.

• Killing tumor cells by inserting toxin genes under the control of a tumor-specific promoter, for example: gene that encodes diphtheria A chain.


Ex – Vivo Gene Transfer

Tumor

Induction of significant systemic immune response

Kill tumor cells

No recurrence

Tumor cells

In tissue culture

Re-inject

Transfer with gene

ImmunostimulationIL-1b IL-2IL-4 IL-6TNF- GM-CSFV-interferon


Tumor Tumor cells Irradiate

FibroblastIrradiated tumors & Fibroblast containing the gene

Transfectwith viral vectors containing genes such as IL-2, TNF, KM-CSF or

Block IRF-1 Expressions

RE-INJECT

Fibroblast

Ex – Vivo Gene Transfer


Anti-sense approach


HSV-skSuicide genein viral vector

Toxic

Ganciclovir

P

P

P

Not toxic

Not toxicGanciclovir

given systematically

INSERTION OF SUCIDE GENE


INSERTION OF A SENSITIVITY GENE

Anti-Herpes DrugGanciclovir (GCV)

PhosphorylatesGCV-MP

ThymidineKinase (TK)

GCV-TP

Inhibition of DNA Polymerases

Cell Death


TK gene & Herpes simplex virus (on vector) [HSVtk]

Infect The Tumor

Patient Is Given GCV

GCV Kills Selectively

The tumor cells carrying HSVtk

gene and also cause bystander effect (kills adjacent cells too)

INSERTION OF A SENSITIVITY GENE


Another Example5 –Fluorocytosine

5 – Fluorouracil (Cytotoxic)

• Cytosine Deaminase

Cell Death


Cholangiocarcinoma

AdCD gene therapy + radiation +FC



Table 1: Schedule of delivery of viral vector (Ad. CMV.CD) at a dose of 108

plaque forming units (PFU/injection, 5-FC at a dose of 800mg/kg body weight, and x-irradiation (XRT) at a dose of 5 Gy/day.


• Gene coding for Cytosine Deaminase has been used in animal Gene Therapy Trials

• Some studies have used in combination with Radiation to produce enhanced killing since 5-FU is a Radiosensitizer


Restoration of G1 checkpointAdp53 therapy

Nasal Vestibule

Radio sensitive human fibroblast


Induction of apoptosis. The percentage of apoptotic cells was determined by cytometry in a JSQ-3 population, 72 hours after exposure to 2 Gy of radiation.


Adp53 therapyAdp53 therapy


Breast Tumor Isolate tumor cells

Isolate MDR-1 gene

Clone in viral vector

Infect MDR-1 HSC2

Re-Inject

PheresisStem cells

Treat with high-doses of Taxol.

PROTECTION OF HEMATOPOIETICSTEM CELLS (HSC2)


Radiation Inducible Gene Therapy

Physics of Radiation Targeting

Molecular Aspectsof Gene Therapy

Radiation InduciblePromoter

CytotoxicAgent

Egr-1Promoter-enhancer

TNF-alphaalpha

+

+

+

Adenovirus


Tumor CellInduces

TNF-alpha GeneExpression

Cells killed orradiosensitizedby TNF-alpha

Adenoviruscontaining

EGR-TNF-alpha

IntratumorInjection

X-rays


RADIO-GENE THERAPY

Fig. 1 Reduction of SQ-20B tumour volumes following combined treatment. SQ-20B xenografts were injected with 1x108 PFU of Ad5 (null virus) or 2 x 108 PFU of Ad.Egr-TNF (twice a week for two weeks). Control tumours were not injected. Tumours were irradiated (5 Gy per day, 4 days per week) to a total dose of 40 or 50 Gy. A, Volumes of xenografts are shown following treatment with: Control (untreated); radiation alone at 50 Gy; Ad.Egr-TNF alone; Ad.Egr-TNF and 50 Gy. Data are calculated as the percent of original (day 0) tumour volume and graphed as fractional tumour volume+ s.e.m. b, Volumes of xenografts are shown following treatment with: 40Gy; Ad5 (null virus); Ad5 (null virus) + 40Gy.

Replace tumor deficient

A5 Egr-TNF


Fig. 2: Treatment effects on large versus small tumors. A, Mean volumes of large (160 mm3) and small xenografts after treatment with radiation alone. The mean of large tumours at day 0 was 234 + 25 mm3 and the mean of small tumours at day 0 was 114 + 6 mm3 . B, Mean volumes of large (.160 mm3 ) and small xenografts after treatment with Ad.Egr-TNF and radiation. The mean of large tumours at day 0 was 218 + 14 mm3 and the mean of small tumours was 96 + 12mm3.

Documents

Ahmed Group Lecture 15 CANCER LECTURE 15 Ahmed Group Lecture 15 Cancer as a Genetic Disease Oncogenes Tumor suppressor genes Telomeric changes in cancer