Mechanisms of Resistance to Targeted Therapies.2012 Genel

99910.2217/FON.12.86 2012 Future Medicine Ltd ISSN 1479-6694Future Oncol. (2012) 8(8), 9991014

Futu

re O

nc

olo

gy

part of

The relatively new paradigm of rationally tar-geted cancer drug therapies has dramatically impacted the practice of medical oncology, with the discovery and development of personalized cancer medicines that produce remarkable clini-cal responses in a subset of patients with advanced systemic disease. This has been particularly evident with several of the recently developed kinase inhibitors that target oncogenic forms of EGFR, HER2, BCRABL, ALK, JAK2 and BRAF, where clinical activity has been tightly linked to the presence of mutationally activated alleles of the genes encoding the target kinase in a subset of genotype-defined patients. In addition to the clinical successes associated with selec-tive kinase inhibitors, other examples of geno-type-associated treatment response have been observed; for example, in the case of treatment of BRCA-mutant breast and ovarian cancers with PARP inhibitors, and in basal cell carcinoma patients defined by mutational activation of the Hh pathway upon treatment with inhibitors of the pathway component SMO. The deep inter-rogation of a deluge of recently generated cancer genome data, together with ever-accelerating efforts to discover and develop drugs that target a variety of signaling pathways associated with states of oncogene addiction, has paved the way for scientifically guided therapeutic strategies to exploit specific tumor cell vulnerabilities.

While there is rapidly growing enthusiasm for this new paradigm of personalized cancer ther-apy and the opportunity for prospective selection

of biomarker-defined patient populations most likely to benefit, there is also the sobering realiza-tion that these smart therapies suffer from the same major limitation associated with traditional chemotherapy drugs in the metastatic disease setting; that is, the duration of any observed clinical benefit is invariably short lived, owing to the relatively rapid acquisition of drug resistance.

In the case of the commonly used chemo-therapy drugs, establishing specific molecular mechanisms of resistance has been very chal-lenging, and while numerous candidate mecha-nisms have been described, thus far none of these have led to the discovery of second-generation agents that can effectively manage such acquired drug resistance. In part, this challenge reflects the relatively nonspecific nature of the antitumor mechanisms associated with these drugs, which exert their actions on complex and fundamen-tal processes including nucleotide metabolism, DNA synthesis and repair, and mitosis. On the other hand, mechanisms of acquired resis-tance to pathway-targeted drugs, such as the various clinically active kinase inhibitors, have been somewhat more straightforward to eluci-date and, in a few cases, the discovery of such mechanisms has prompted the development of drugs specifically designed to overcome them.

Mechanisms underlying acquired resistance to pathway-targeted drugs are being pursued largely through two basic strategies. Preclinically, can-cer cell line models are being used to recapitu-late the clinical experience, providing paired

Mechanisms of acquired resistance to targeted cancer therapies

Mark R Lackner1, Timothy R Wilson1 & Jeff Settleman*21Department of Oncology Biomarker Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA, USA 2Department of Discovery Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, USA*Author for correspondence: [email protected]

Drugs that target genomically defined vulnerabilities in human tumors have now been clinically validated as effective cancer therapies. However, the relatively rapid acquisition of resistance to such treatments that is observed in virtually all cases significantly limits their utility and remains a substantial challenge to the clinical management of advanced cancers. As molecular mechanisms of resistance have begun to be elucidated, new strategies to overcome or prevent the development of resistance have begun to emerge. In some cases, specific mutational mechanisms contribute directly to acquired drug resistance, and in other cases it appears that nonmutational and possibly epigenetic mechanisms play a significant role. This article discusses the various genetic and nongenetic mechanisms of acquired drug resistance that have been reported in the context of rationally targeted drug therapies.

Keywords

n cancer n drug resistance n epigenetics n kinase n kinase inhibitor n mutation

Revie

wFor reprint orders, please contact: [email protected]

Future Oncol. (2012) 8(8)1000 future science group

Review Lackner, Wilson & Settleman

samples of pretreatment drug-sensitive cells and post-treatment cells that manage to escape the consequences of drug exposure, and these largely isogenic cell line pairs can be compared in order to identify molecular mechanisms of acquired drug resistance. In addition, tumor biopsies collected prior to treatment and following post-treatment response and subsequent progression can be similarly compared. In the setting of non-small-cell lung cancer (NSCLC) and melanoma, these two strategies have revealed a seemingly overlapping set of mechanisms derived from each approach, thereby validating the utility of this strategy. For example, secondary mutations in the EGF receptor (EGFR) kinase domain (T790M) in EGFR-mutant NSCLC patients or gain-of-function mutations in NRAS in BRAF-mutant melanoma patients promote drug resis-tance by reactivating key prosurvival pathways [1,2]. Significantly, such analysis has revealed that acquired drug resistance mechanisms are likely to fall into two fundamentally distinct catego-ries: genetic and nongenetic (or epigenetic). The following sections give an overview of both of these classes of mechanisms of acquired resis-tance to pathway-targeted anticancer drugs. This is not intended to be a comprehensive review of this important topic, for which there has been a virtual explosion in the number of recently published reports. Rather, the intent is to pro-vide several examples of genetic and nongenetic mechanisms of acquired resistance to the various molecularly targeted oncology drugs that have demonstrated clinical activity derived from both preclinical cell line modeling studies and from the analysis of clinical specimens and to discuss how these findings are impacting strate-gies in which combination drug treatments can be used to overcome, or possibly to prevent, the acquisition of drug resistance.

Genetic mechanisms of acquired drug resistance

The emergence of -omics technologies capable of yielding deep molecular profiles of cancer genomes has enabled the elucidation of somatic genetic alterations associated with resistance to targeted therapies. This section covers notable examples of genetic alterations that have been associated with resistance to a variety of targeted therapies (Table 1).

EGFR-driven cancersPatients with advanced NSCLC, whose tumors harbor activating mutations in the gene encod-ing the EGFR, are exquisitely sensitive to EGFR

inhibition with the EGFR tyrosine kinase inhibi-tors (TKIs) erlotinib and gefitinib [3,4]. Most patients with cancers harboring activating EGFR mutations show marked and sometimes durable responses to EGFR-targeted therapy; despite these initial responses, acquired resistance is a pervasive problem and the median time to pro-gression for patients on EGFR-targeted therapies is approximately 12 months [5]. Resistance can be mediated through events acting at the level of the target EGFR itself, through compensatory acti-vation of other receptor tyrosine kinases (RTKs), or through activation of downstream signaling pathways. Both genetic and nongenetic resis-tance mechanisms have been described. A promi-nent genetic mechanism is the acquisition of a secondary missense mutation, EGFR T790M, affecting the so-called gatekeeper r esidue of the catalytic domain, leading to increased affinity of the mutant kinase for ATP [6]. This finding spurred the development of irreversible EGFR inhibitors, with the rationale that such binding would result in greater occupancy of the ATP binding site, thereby inhibiting T790M-mutated EGFR despite its enhanced ATP binding. More recently, it has been shown that amplification of the T790M allele can cause acquired resistance even to an irreversible EGFR inhibitor [7], sug-gesting that additional strategies will be required to treat T790M-harboring cancers.

Compensatory mutational activation of other RTKs also appears to be an important mecha-nism of resistance to EGFR TKIs. In one exam-ple, HER2 kinase domain mutations have been shown to confer resistance to EGFR TKIs by activating the AKTPI3K signaling axis and uncoupling signaling from EGFR [8]. In addi-tion, amplification of the gene encoding the MET RTK has been identified as an acquired resistance mechanism to EGFR TKIs in a small subset of EGFR-mutant NSCLCs [9], with more recent studies suggesting that low-level MET amplification pre-exists in a subset of tumor cells and can be clonally selected for in the presence of EGFR inhibitors [10]. Activation of down-stream signaling pathways that can uncouple EGFR from downstream signaling and confer resistance has also been described; for example, loss of the tumor suppressor PTEN, leading to PI3K pathway activation [11]. This study also revealed PTEN loss in primary NSCLC speci-mens, albeit at a low frequency, suggesting that it may c onstitute an innate resistance mechanism.

Given that multiple mechanisms of resis-tance to EGFR TKIs have been described, and that different mechanisms suggest different

www.futuremedicine.com 1001future science group

Mechanisms of acquired resistance to targeted cancer therapies Review

follow-on therapies (e.g., HER2-targeted agents for HER2 mutations and MET inhibitors for MET mutations), it is crucial to understand the mechanism at play in a given patients tumor. An important recent study by Sequist and colleagues analyzed serial biopsies from NSCLC patients as they progressed on EGFR-targeted therapies [12]. The authors observed mechanisms that had been previously identified, including T790M (49%) and MET amplification (5%), as well as additional candidate mechanisms such as trans-formation to a small-cell lung cancer phenotype and acquisition of PI3K mutations. Diagnostic evaluation of tumor biopsies collected at the time of progression on EGFR TKI therapy will therefore be essential in identifying the most appropriate candidate follow-on therapy. Importantly, a resistance-associated alteration could not be identified in approximately 30% of the patients in this study, suggesting that addi-tional efforts will be required to elucidate all of the various mechanisms of acquired resistance to EGFR TKIs.

EML4ALK inhibitor resistanceThe EML4ALK fusion protein, which results from a specific chromosomal translocation, has been identified as an important driver mutation in approximately 5% of NSCLCs, and clinical testing has demonstrated impressive clinical

benefit upon treatment with the dual ALK and MET inhibitor crizotinib in translocation-harboring patients [13]. Mechanisms of acquired resistance to crizotinib have been elucidated both from drug-sensitive ALK-translocated cell lines selected for resistance, as well as from patients who have progressed on crizotinib therapy. Similar to EGFR inhibitors, a promi-nent resistance mechanism involves mutations in the kinase domain of ALK, which cluster into five distinct regions around the active site and impair inhibitor binding [1418]. Selection of resistant cell lines has suggested that resis-tance at intermediate drug concentrations may be mediated by ALK gene amplification, with subsequent acquisition of mutations in the ALK kinase domain as the cells become highly resis-tant [18]. Importantly, several of these studies suggested that inhibitors with a greater potency than crizotinib, such as TAE684, can potentially overcome the resistance mediated by some of these m utations [14,18].

One promising general strategy to overcome genetic resistance is to target the trafficking of mutant oncoproteins to the cell membrane by inhibiting the protein chaperone HSP90. EML4ALK is a HSP90 client protein and stud-ies in H3122 crizotinib-resistant NSCLC cells have shown that the HSP90 inhibitor 17-AAG can overcome crizotinib resistance and suppress

Table 1. Genetic mechanisms of drug resistance.

Target Drug(s) Indication(s) Resistance mechanisms Ref.

EGFR Erlotinib NSCLC MET amplification, T790M gatekeeper mutation, PTEN loss, HER2 kinase domain mutations, IGF1R

[612]

HER2 Trastuzumab Breast PTEN loss, SRC, AXL, EGFR, IGF1R, cyclin E amplification, erythropoietin, HSP90, p95HER2, MUC4

[27,3138,41,42]

MET Crizotinib Gastric MET mutations, BRAF fusion protein, EGFR [2024]

ALK Crizotinib NSCLC Secondary ALK mutations, ALK amplification, EGFR, HSP90 [1319]

BRAF Vemurafenib, MEKi

Melanoma, CRC NRAS, CRAF, COT, PDGFR, IGF, aberrantly spliced BRAF, MEK mutation, BRAF amplification

[2,4450]

MEK MEKi Melanoma, CRC MEK1 mutations, BRAF amplification, KRAS amplification, PI3K [5154]

PI3K PI3Ki Breast Notch, Myc, MET [5759]

BCRABL Imatinib, nilotinib CML Secondary BCRABL mutations, BCRABL amplification, P-glycoprotein, MDR, LYN

[6569]

KIT Imatinib, sunitinib GIST Secondary KIT mutations, KIT amplification, MDR, AXL, FAK [7075]

ER Endocrine therapy Breast PI3K, HER2, PTEN loss [8991,93,94]

AR Antiandrogens Prostate AR mutations, AR amplification, AR splice variants, TMPRSS2ERG, WNT, PTEN loss

[98106]

Hh Vismodegib, LDE225

Medulloblastoma SMO mutations, PI3K, cyclin D1 [6264]

AR: Androgen receptor; CML: Chronic myeloid leukemia; CRC: Colorectal cancer; EGFR: EGF receptor; ER: Estrogen receptor; GIST: Gastrointestinal stromal tumor; IGF1R: IGF1 receptor; MEKi: MEK inhibitor; NSCLC: Non-small-cell lung cancer; PDGFR: PDGF receptor; PI3Ki: PI3K inhibitor.



cell growth [18]. Moreover, a Phase I clinical study of the HSP90 inhibitor IPI-504 suggested that this agent may have antitumor activity in patients with EML4ALK r earrangements [19].

MET inhibitorsThe RTK MET plays a key role in mediating resistance to EGFR inhibitors, but also seems to play an important role in cancer cell sur-vival and proliferation in its own right, and hence has attracted interest as a therapeutic target [20]. Described mechanisms of resistance to MET inhibition implicate both upstream and downstream pathway components. A mass spectrometry-based proteomic approach identified BRAF gene amplification and gene fusions as being associated with MET inhibitor resistance in MET-amplified gastric cell lines, suggesting that downstream BRAF activation can render cell lines MET independent [21]. Another study with small-molecule inhibitors of MET in gastric cancer cell lines identified mutations in MET as an important acquired resistance mechanism; specifically, substitution of Y1230, a key residue in the activation loop [22]. Analogous mutations were observed using a random mutagenesis approach [23]. Intriguingly, the former study also identified overexpression of the EGFR ligand TGF-a concurrently in the same resistant lines [22], suggesting that activa-tion of EGFR signaling can bypass the need for MET signaling. Similarly, studies of MET small-molecule inhibitor resistance in NSCLC cell lines revealed upregulation of EGFR signal-ing as a prominent resistance mechanism [24]. Moreover, initial combination treatment with MET and EGFR inhibitors effectively sup-pressed the emergence of resistant clones. Taken together, these findings suggest that EGFR and MET signaling are intimately linked in lung cancer, and that cancer cells engage in recipro-cal upregulation of one receptor to overcome inhibition of the other, a potentially nongenetic resistance mechanism described in more detail below. The clinical implication is that upfront combination therapy with MET- and EGFR-targeted therapies may be useful in combating resistance to these agents.

HER2-targeted therapiesHER2 is a RTK that belongs to the EGFR family of receptors and is a clinically validated drug target. Trastuzumab, a monoclonal anti-body that binds to the extracellular domain of HER2, is thought to function by inhibit-ing downstream PI3KAKT signaling, HER2

shedding, and possibly by activating an immune cell-mediated antibody-dependent cellular cyto-toxicity response [25]. Trastuzumab is approved for the treatment of HER2-positive breast can-cer, and is used either as a single agent or in combination with chemotherapies including taxanes and anthracylines. The small-molecule inhibitor lapatinib, which is designed to inhibit EGFR and HER2 kinase activity, is approved as a therapeutic in HER2-positive patients with advanced breast cancer who have previ-ously received trastuzumab in combination with anthracycline- or taxane-based therapies [26]. HER2 overexpression and chromosomal amplification are clinically validated predic-tive biomarkers and they are routinely used to select patients for HER2-targeted therapies. Trastuzumab and lapatinib therapy combined with diagnostic testing for HER2-positive disease have substantially increased survival rates and defined a clinically relevant subtype of breast cancer; however, it is estimated that approximately 70% of HER2-positive breast cancer patients are either intrinsically resistant to HER2-targeted therapies or develop acquired resistance at some point during treatment [25]. Similar to EGFR-targeted therapies, described resistance mechanisms include events acting at the level of the target HER2 itself, through com-pensatory activation of other RTKs or through activation of downstream signaling pathways.

Expression of a truncated version of HER2, p95HER2, which lacks the extracellular domain of HER2 and hence cannot be inhibited by trastuzumab, is one mechanism that acts at the level of HER2 itself [27]. p95HER2 fragments arise either by metalloprotease-mediated cleav-age of the extracellular domain or translation initiation from internal AUG codons; impor-tantly, p95 protein fragments retain kinase activity and are sensitive to inhibition by lapa-tinib (reviewed in [27]). Retrospective analyses of cohorts of patients treated with HER2-targeted agents have suggested that p95HER2 expres-sion is associated with resistance to trastuzumab [28,29] but does not predict resistance to lapatinib [30]. p95HER2 expression could also conceiv-ably contribute to resistance to HER2-targeted agents by inducing expression of EGFR path-way components such as EGFR and TGF-a, thereby reactivating the PI3K pathway [31]. A major downstream pathway implicated in resis-tance to trastuzumab is the PI3KAKT path-way. Specifically, loss of PTEN protein expres-sion has been shown to be predictive of poor clinical response to trastuzumab [32]. PTEN



has also been implicated in resistance through genome-wide shRNA screens, and retrospective analyses have suggested that combined analy-sis of PIK3CA and PTEN status is superior to analysis of PTEN alone in identifying patients at risk of early progression on trastuzumab [33]. Additional reported resistance mechanisms that can reactivate the PI3K pathway include increased activity of RTKs, such as AXL and IGF receptor, as well as the nonreceptor kinase SRC [3436]. Cyclin E amplification and overex-pression has also been implicated as a resistance mechanism in this setting [37], as has expres-sion of the mucin MUC4, which interferes with trastuzumab binding [38].

A promising means of overcoming lapa-tinib or trastuzumab resistance arising from various mechanisms is the development of the antibodydrug conjugate trastuzumabDM1, which was designed to combine the therapeu-tic effects of trastuzumab with a potent anti-mitotic drug in order to deliver tumor-targeted chemotherapy [39]. TrastuzumabDM1 is cur-rently in clinical development and has been shown, in preclinical models, to be active in lapatinib- and trastuzumab-resistant tumor models. As discussed above, reactivation of the prosurvival pathways either by PTEN loss or RTK upregulation (e.g., AXL) is a common resistance mechanism to anti-HER2 targeted agents [34]; consequently, administration of a cytotoxic chemotherapeutic may be more effica-cious in this setting. Phase I and Phase II clini-cal trials of trastuzumabDM1 as a single agent and in combination with paclitaxel, docetaxel and pertuzumab have shown clinical activity and a favorable safety profile in patients with HER2-positive metastatic breast cancer who had received prior HER2-targeted therapy [40]. Another promising mechanism of overcoming trastuzumab resistance is co-administration of HSP90 inhibitors, which block trafficking of HER2 to the cell membrane. The HSP90 inhibitor IPI-504 has shown excellent antitu-mor activity in trastuzumab-resistant xenograft models [41], and early clinical studies with the HSP90 inhibitor 17-AAG in combination with trastuzumab have shown signs of clinical activ-ity in patients who previously progressed on trastuzumab [42].

RASRAFMAPK pathwayRecently, a number of reports have eluci-dated resistance mechanisms to BRAF and/or MEK inhibition through in vitro studies, some of which have been followed up with

confirmation in a small number of clinical sam-ples (Figure 1). BRAF is an essential kinase in the RASRAFMAPK cascade that acts directly downstream of the RAS oncogenes, while MEK is a key downstream effector of BRAF. BRAFV600E activating mutations are found in up to 50% of malignant melanomas and selec-tive BRAF inhibitors have shown great prom-ise in the management of this disease [43]. As with other targeted therapies, initial responses almost invariably give way to progressive disease owing to acquired resistance. Broadly speak-ing, the resistance mechanisms associated with BRAF inhibitors can be either MAPK pathway-dependent or MAPK-independent (reviewed in [44]). MAPK-dependent mechanisms result in reactivation of phospho-ERK in the presence of BRAF or MEK inhibitor concentrations that are normally sufficient to suppress cell growth. Similar to other targeted therapies, mutations that alter the inhibitors ability to interact with the mutated kinase can cause resistance. These include mutations in the gatekeeper residue of BRAF that confer resistance to a selective BRAF inhibitor [45], as well as mutations in BRAF that abolish dimerization or cause aberrant BRAF mRNA splicing [46]. Other MAPK-dependent mechanisms of BRAF inhibitor resistance include molecular changes as diverse as ampli-fication of the BRAF gene [47], acquired muta-tions in NRAS [2], overexpression of CRAF [48] or the MAPK COT1 [49], or mutations in MEK that increase catalytic activity [50]. In the case of BRAF amplification, it was observed that com-bined treatment with MEK and BRAF inhibi-tors could overcome resistance to BRAF or MEK inhibitors, suggesting that inhibition of linearly linked nodes in a pathway may be a promis-ing therapeutic strategy [47]. ERK-independent mechanisms of resistance to BRAF inhibitors generally involve compensatory signaling by alternate pathways and are described below.

MEK inhibitors have also received wide-spread attention as anticancer agents; however, thus far, they have not been as impressive as BRAF inhibitors in the treatment of BRAFV600E-mutant melanoma. As with BRAF inhibitors, acquired resistance to MEK inhibitors can occur via MAPK-dependent or -independent mechanisms. In the case of mutations, both preclinical modeling and follow-up studies in patient samples have identified mutations in MEK1 that alter the allosteric binding pocket for arylamine MEK inhibitors [51], and in some cases also increase the intrinsic activity of the mutated kinase [52]. In addition, amplification



of the upstream oncogene KRAS has been shown to confer resistance to selective MEK inhibitors by increasing signaling through the ERK1/2 axis [53]. It has recently been shown that ERK inhibition can overcome acquired resistance to MEK inhibitors caused by diverse genetic mechanisms, suggesting that perhaps the com-bination of MEK and ERK inhibitors may play a role in combating clinical MEK resistance [54].

PI3KAKT pathway inhibitorsThe PI3KAKT pathway is a major effector pathway that functions downstream of RTKs, and serves to transduce survival and prolif-eration signals. This pathway is the subject of

intensive drug development efforts, with numer-ous inhibitors targeting different nodes of the pathway in various stages of clinical develop-ment [55]. Mutant KRAS has been shown to contribute to intrinsic resistance to dual PI3K/mTOR inhibitors, and the combination of MEK and PI3K inhibitors is required to effectively inhibit KRAS-mutant tumors [56]. Less is known about acquired resistance to PI3K inhibitors, although several recent reports have identified candidate mechanisms from preclinical studies. Studies of a mouse model engineered to con-ditionally express PIK3CA(H1047R) revealed that tumors that recurred after PIK3CA inac-tivation harbored focal amplification of either MET or c-MYC [57]. While the MET-amplified tumors could be inhibited with a selective PI3K inhibitor, the c-MYC-amplified tumors became independent of the PI3K pathway and refractory to treatment with a PI3K inhibitor. c-MYC was also independently identified as a candidate resistance mechanism in genetically defined mammary epithelial cells selected for resistance to the dual inhibitor BEZ-235 [58]; this latter study also identified amplification of eIF4E as a candidate resistance mechanism. Finally, a chemical genetic screen identified c-MYC as well as Notch1 as being involved in resistance mechanisms in engineered isogenic cancer cells [59].

Hh pathway inhibitorsThe Hh pathway is aberrantly activated via mutations in malignant medulloblastoma brain tumors and drugs that inhibit Hh signaling are being developed for the treatment of this disease [60]. The agent GDC-0449 inhibits the activ-ity of the serpentine receptor SMO and has shown promise in early-phase clinical trials [61]. Characterization of tumor tissue collected from a medulloblastoma patient who progressed on GDC-0449 treatment revealed a D473H muta-tion of the SMO protein, and follow-up func-tional studies showed that the alteration had no effect on Hh signaling but disrupted the ability of GDC-0449 to bind SMO and suppress sig-naling [62]. Subsequent alanine scanning muta-genesis studies by the same group identified the E518 site as another candidate resistance site, and identified additional agents that inhibit SMO despite the presence of resistance muta-tions [63]. However, this study also identified focal amplification of the downstream transcrip-tion factor Gli2 and the Hh target gene Ccnd1 in two additional resistant models, suggesting that downstream pathway activation may play a role

Mechanisms of resistance to vemurafenib

Mechanisms of resistance to MEKi

Vemurafenib

MEKi

Q61K

PDGFRIGF1REGFR

AKT

HRAS NRASKRAS

BRAFV600E ARAF CRAF

Cell proliferation/survival

MEKF129L C121S

COT

ERK

P124L

Figure 1. Genetic mechanisms of acquired drug resistance showing RAF and MEK inhibitors as an example. Drug-treated BRAF-mutant cancer cells can reactivate the RASRAFMEKERK signaling pathway by acquiring gain-of-function mutations in NRAS (Q61K) and MEK (C121S and F/P124L) or by increased expression of CRAF or COT (MAP3K8). Amplification of the target (i.e., BRAFV600E or KRASG12D) has been shown to promote resistance to MEKi. Increased signaling through receptor tyrosine kinases, such as IGF1R, PDGFRb and EGFR, may also promote resistance through increased PI3KAKT signaling. (A) indicates reported mechanisms of resistance to vemurafenib and (B) indicates reported mechanisms of resistance to MEKis. EGFR: EGF receptor; IGF1R: IGF1 receptor; MEKi: MEK inhibitor; PDGFRb: PDGF receptor-b.



in resistance to Hh pathway antagonists. Studies by another group using the Hh antagonist NVP-LDE225 also implicated SMO mutations and amplification of Gli2 as resistance mecha-nisms, and further identified upregulation of the PI3K pathway as a possible escape pathway [64].

BCRABL pathway inhibitorsChronic myeloid leukemia is a myeloproliferative disease characterized by the translocation of the Philadelphia chromosome resulting in expres-sion of the BCRABL fusion protein, in which the ABL tyrosine kinase is dysregulated. Despite impressive clinical responses observed on treat-ment with the first-generation inhibitor imatinib, chronic myeloid leukemia patients, especially those with later-stage disease, eventually relapse. The most frequent resistant mechanisms asso-ciated with imatinib are the gatekeeper muta-tions (e.g., T315I) found within the ABL kinase domain, which prevent drug binding [65,66], and amplification of the BCRABL fusion gene [65,67]. In addition to mutational activation of ABL, increased expression of drug transporters, such as P-glycoprotein and MDR1 proteins, have been reported to promote resistance to imatinib [67,68]. Second-generation BCRABL inhibitors, such as nilotinib and dasatinib, which are more potent inhibitors of BCRABL, have shown clinical activity in imatinib-refractory chronic myeloid leukemia patients, except in the context of resistance mediated by the T315I gatekeeper mutation. Similarly, in vitro-derived resistance mechanisms to nilotinib have been reported, including increased expression of P-glycoprotein, increased activity of the SRC family kinase LYN and overexpression of BCRABL [69].

Gastrointestinal stromal tumorGastrointestinal stromal tumors are driven by the mutationally activated tyrosine kinase recep-tors KIT or PDGF receptor. Despite initial clini-cal activity following treatment with imatinib (which inhibits both kinases), gastrointestinal stromal tumor patients invariably experience relapse. As with other inhibitors, mutations in both KIT and PDGF receptor that prevent drug binding have been associated with both innate and acquired resistance to imatinib [70]. Secondary mutations within the KIT gene have also been reported as an acquired resistance mechanism [71], as well as amplification of KIT [72], overexpression of AXL [73] and FAK [74], and increased expression of MDR [75]. The multiki-nase inhibitor sunitinib is currently used for the treatment of patients who have progressed on

imatinib. Similarly to imatinib resistance, KIT mutations that result in a constitutively activated kinase conformation have been reported as a resistance mechanism [76].

Nongenetic mechanisms of acquired drug resistance

Substantial recent advances in DNA sequenc-ing and additional genomic technologies have played a major role in the elucidation of many of the genetic mechanisms of acquired drug resistance described above. On the other hand, technologies to explore potential nongenetic mechanisms of acquired drug resistance are con-siderably less developed thus far, significantly hampering efforts to examine, for example, the potential role of epigenetics, alternative RNA splicing, metabolic changes or specific protein modifications that are not driven by mutations. Nevertheless, such mechanisms appear to play a significant role in the acquisition of resistance to cancer drugs, and while they remain poorly characterized thus far, evidence highlighting their importance is beginning to emerge (Table 2).

Addiction switching as a resistance mechanismMany of the molecularly targeted cancer drug therapies that have demonstrated clinical activ-ity inhibit the function of various kinases whose oncogenic function is associated with their abil-ity to transduce signals via a common set of key nodes, such as PI3K and MAPK [77]. For example, the cell proliferation and cell survival effects of activated forms of the EGFR, HER2, MET and ALK kinases are mediated through these pathways [78]. Consequently, one potential mechanism by which acquired resistance to such inhibitors could develop is through the activa-tion of a kinase that can generate redundant oncogenic signals via activation of the same pathways whose function has been disrupted by drug treatment (Figure 2) [79]. Indeed, as described above, genomic amplification of the MET RTK has been established as a mechanism of acquired resistance to EGFR kinase inhibition in approx-imately 5% of treated cases of EGFR-mutant NSCLC [12]. In addition to such genetic mecha-nisms, a variety of cell culture resistance model-ing studies have revealed similar mechanisms of acquired drug resistance, including examples involving nonmutational mechanisms; and, in some cases, these appear to be clinically relevant.

In a gastric cancer cell line model exhibiting addiction to amplified MET, resistance model-ing revealed a potential resistance mechanism



in which EGFR activation, via increased pro-duction of the EGFR ligand TGF-a, was able to overcome MET dependency [24]. In a simi-lar study, in which a MET-amplified NSCLC cell line was used to model resistance to MET kinase inhibition, the resulting resistance mech-anism appeared to involve EGFR activation via overexpression of the EGFR ligand amphiregu-lin [22]. Modeling of acquired resistance to an ALK inhibitor in ALK-translocated NSCLC cells similarly revealed a resistance mechanism associated with EGFR activation via increased amphiregulin production [17]. Modeling of acquired resistance to EGFR in several mod-els has revealed nonmutational mechanisms of resistance associated with the activation of other RTKs. Thus, IGF1R activation appears to be a widely relevant mechanism of acquired resis-tance to inhibitors of EGFR [80], HER2 [35,81] and BRAF [82]. In the case of the EML4ALK inhibitor crizotinib, compensatory EGFR acti-vation was identified as a resistance mechanism that could not be overcome with more potent ALK inhibitors, suggesting that dual inhibition of EGFR and EML4ALK may be an effec-tive therapeutic combination [17]. Resistance to HER2-targeted therapies can also be medi-ated by compensatory activation of EGFR or its ligands [31]. An analogous mechanism has recently been reported implicating EGFR signal-ing in innate resistance to RAF/MEK inhibitors

in BRAF-mutant colorectal cancer cells [83,84]. In all of these cases, it appears that cancer cells have undergone an addiction-switching mechanism in which they have changed their dependency on one activated kinase to another with shared effectors, or become co-dependent on more than one convergent pathway. Such resistance might result from the selection of rare cancer cells within the population with a distinct profile of kinase engagement, or pos-sibly from an epigenetic mechanism that results in the autocrine production of a RTK ligand. Clinical data now support the significance of many of these in vitro findings [10].

Feedback loops & pathway crosstalk as resistance mechanismsSignaling pathways in cancer cells often dis-play robust homeostatic mechanisms and can respond to inhibition by feedback by upregu-lating key signaling components or activating parallel signaling pathways. In contrast to addiction switching, as described above, which may only occur in a small percentage of the tumor cells, resistance promoted by feedback loops and crosstalk can occur acutely in the majority of tumor cells in response to anticancer agents. An example of the latter phenomenon has been described in the context of the PI3KAKT pathway; several studies have suggested that inhibition of PI3K or AKT with selective

Table 2. Nongenetic mechanisms of drug resistance.

Target Drug(s) Indication(s) Resistance mechanisms Ref.

EGFR Erlotinib NSCLC Addiction switching: IGF1R, METEMTCancer stem cells: reversible drug tolerance

[12,80][108111]

[117]

BRAF Vemurafenib Melanoma Addiction switching: IGF1R, EGFR (CRC) [8284]

HER2 Trastuzumab Breast Addiction switching: IGF1R, EGFR, EGF-like ligands [31,35,81]

MET Crizotinib Gastric, NSCLC Addiction switching: EGF-like ligands [22,24]

ALK Crizotinib NSCLC Addiction switching: EGF-like ligands [17]

PI3KAKT MK-2206, XL-147 Breast Pathway crosstalk: HER3 [85,86]

ER Antiestrogen Breast Pathway crosstalk: PI3K [9092]

AR Antiandrogen Prostate Pathway crosstalk: PI3K [103106]

AR Antiandrogen Prostate EMT [112]

N/A Chemotherapy Breast Cancer stem cells [119]

N/A 16,000-compound library Breast Cancer stem cells: exquisite sensitivity to salinomycin [113]

N/A Chemotherapy Glioblastoma Cancer stem cells: IAPs [122]

N/A Chemotherapy N/A Cancer stem cells: ABCs [120]

N/A Chemotherapy Breast Cancer stem cells: ALDH1 [121]

ABC: ATP-binding cassette; AR: Androgen receptor; CRC: Colorectal cancer; EGFR: EGF receptor; EMT: Epithelialmesenchymal transition; ER: Estrogen receptor; IGF1R: IGF1 receptor; N/A: Not applicable due to multiple or unknown targets; NSCLC: Non-small-cell lung cancer.



inhibitors abrogates negative feedback loops and causes dramatic upregulation of upstream signaling components, notably RTKs such as MET, EGFR and HER3 [59,85,86]. Such hyperac-tivation of upstream signaling may conceivably limit the single-agent efficacy of PI3K inhibi-tors and suggests that rational combinations with agents targeting upstream signaling (e.g., HER2/HER3 antagonists) may help overcome intrinsic resistance due to upstream activation.

Antiestrogen therapies in breast cancerApproximately 75% of breast cancers are posi-tive for estrogen receptor (ER) and/or proges-terone receptor expression, and expression of these proteins is routinely used as a diagnostic to select patients for antiestrogen therapies. These include agents, such as tamoxifen that antagonize ligand binding, ER downregulators, such as fulvestrant, and aromatase inhibitors, which work by blocking estrogen biosynthe-sis [87]. These agents, collectively described as endocrine therapies, are a model example of successful targeted therapy; nevertheless, many patients experience either innate or acquired resistance to these agents [88]. HER2 over expression is now widely accepted as a resistance mechanism and has been reviewed extensively in other publications [89]. An inter-esting finding that has emerged from these clinical studies is that combined targeting of HER2 and ER is inferior to treating patients with HER2-targeting agents and chemother-apy, suggesting that combining with the tar-get proposed to mediate resistance does not always produce superior results. Several other pathways have been implicated in resistance to hormonal therapies in ER-positive breast can-cer, mostly based on nongenetic upregulation of compensatory signaling pathways, and these are described below.

Pathway crosstalk as a mechanism of resis-tance to targeted therapies is exemplified by interactions between PI3K signaling and hor-mone receptor signaling in breast and prostate cancer. Studies in ER-positive breast cancer revealed a prominent role for the PI3K pathway in mediating resistance to endocrine therapy [90,91]. These findings have been summarized in detail in a recent review [92], so this article concentrates on several of the most recent find-ings and their clinical implications. One study established four long-term estrogen-deprived ER-positive cell lines that could grow in the absence of estrogens, and used proteomic pro-filing to identify PI3K and mTOR pathway

components as being significantly upregulated in these cells. Consistent with this observation, treatment with a dual PI3K/mTOR inhibitor prevented the emergence of resistance and, moreover, resulted in apoptosis of resistant cells [93]. Similar effects on apoptotic response in long-term estrogen-deprived cells were reported by a separate group, who also found that the level of ER expression played a role in predict-ing enhanced combination effects, since cells with high ER expression were more sensitive to combined fulvestrant and PI3K inhibition [90]. Similarly, loss of PTEN and thus activa-tion of the PI3K pathway has also been shown

Pretreatment On treatment Progression

Pretreatment On treatment Post-treatment

Pretreatment On treatment

Epithelial cancer cell

Mesenchymal cell

Cancer stem cell

Stromal cell

RTK ligand

Progression

Figure 2. Nongenetic mechanisms of drug resistance. (A) A solid tumor often consists of a heterogeneous population of both epithelial and mesenchymal cancer cells. Although slow growing, mesenchymal cancer cells are intrinsically resistant to anticancer agents and may be selected following the drug-promoted eradication of the epithelial cancer cell population. (B) Similarly, tumors seem to harbor a small number of cancer stem cells that are resistant to anticancer therapies. Following cessation of treatment, the surviving cancer stem cells can divide asymmetrically to generate both a cancer stem cell and a daughter epithelial cancer cell to repopulate the relapsed tumor. (C) The tumor microenvironment contains a plethora of RTK ligands that can provide survival signals by both autocrine tumor cell production and paracrine signaling from the tumor stroma. In the presence of anticancer therapies, the microenvironment may provide a prosurvival niche that promotes the eventual outgrowth of a ligand-responsive subset of tumor cells. RTK: Receptor tyrosine kinase.



to promote resistance to antiestrogen thera-pies [94]. In line with these preclinical results, a recent pivotal Phase III clinical trial of the aromatase inhibitor exemestane, in combina-tion with the mTORC1 inhibitor everolimus, showed a dramatic increase in progression-free survival compared with patients who received exemestane alone. All patients in this study had received prior aromatase inhibitor therapy [95]. Interestingly, a recent report has impli-cated impaired ER binding to chromatin as an a dditional mechanism of resistance [96].

Antiandrogen therapies in prostate cancerThe growth and proliferation of early-stage prostate cancers is fueled in large part by stim-ulation of androgen receptor (AR) via endog-enous androgens. Initial therapies for advanced prostate cancer are thus focused on reducing or blocking the effects of androgens, or antagoniz-ing AR or downstream target genes. Although effective initially, almost all patients with met-astatic prostate cancer will develop resistance and progress to a state referred to as castration-resistant prostate cancer [97]. Alterations in AR itself have been shown to underlie resistance to antiandrogen therapy. These events include amplification [98] or mutation [99] of the target; more recently, several studies have suggested that alternate splicing of AR can result in trun-cated isoforms that are constitutively active and function as ligand-independent transcription factors (reviewed in [100]). Ligand-independent AR signaling has been implicated in the activa-tion of WNTb-catenin signaling, implicating this pathway as an adaptation mechanism in castration-resistant prostate cancer [101]. The TMPRSS2ERG fusion protein has also been shown to be reactivated in castration-resistant prostate cancer, suggesting a possible role in tumor progression [102]. As in ER-positive breast cancer, activation of the PI3KAKT pathway appears to be an important mecha-nism of resistance, particularly via loss of the tumor suppressor PTEN [103,104]. Perhaps related to the role of PI3K, the insulinIGF1R pathway has been implicated as a mechanism of escape from hormone dependence [105], and an IGF1R gene expression signature was associated with recurrence-free survival in a cohort of tamoxifen-treated patients in the same study. Numerous examples have been described wherein the RASMEK pathway and PI3K pathways show reciprocal upregulation of one another, effectively blunting single-agent

efficacy and suggesting a need for combination therapies [106].

Epithelialmesenchymal transition as a resistance mechanismEpithelial tumors are typically comprised of cells that have the capacity to undergo an epi-thelialmesenchymal transition (EMT), which is associated with an increased migratory/inva-sive capacity, as well as a largely unexplained resistance to a variety of cancer drugs [107]. Consequently, tumors can exhibit varying pro-portions of epithelial and mesenchymal cells, as observed histologically, and recently accu-mulating evidence seems to link this feature of tumors both to their initial response to treat-ment and to their ability to acquire resistance to treatment (Figure 2). Thus, the epithelial ver-sus mesenchymal character of NSCLC tumors can predict their response to EGFR inhibitors [108]. Notably, EMT is not associated with a genetic alteration, but can be reversible, and appears to be driven by epigenetic mechanisms. Several studies of cancer cell lines have revealed that residual surviving cells following a drug treatment that is largely toxic to the population tend to yield a population that exhibits EMT features [12,109]. Moreover, EMT has also been implicated as an innate resistance mechanism to EGFR TKIs in NSCLC cell lines [108], and several recent studies have also suggested that cell lines selected for resistance to gefitinib or erlotinib undergo EMT, upregulating vimentin and downregulating E-cadherin and showing concomitant increases in invasion, migration and anchorage-independent growth [109111]. One of these reports found that TKI-resistant cells with a mesenchymal phenotype showed strong sensitivity to MEK inhibitors [111], sug-gesting a possible therapeutic option in tumors that have undergone EMT. Beyond the kinase inhibitors, a recent report demonstrated that androgen-deprived prostate tumor explants undergo EMT [112], associated with drug resistance, implicating EMT in the response to androgen-blocking agents as prostate cancer therapy. Such findings imply that the ability of tumor cells to potentially oscillate between these phenotypically distinct states could lead to the selection of mesenchymal cells through a nongenetic mechanism. It is worth noting, however, that in some treatment contexts it appears that the mesenchymal population may, in fact, exhibit greater sensitivity to certain drugs [113]. Such findings have prompted great interest in combination drug treatments aimed



at targeting these distinct cell populations in solid tumors.

Cancer stem cells & drug resistanceCancer stem cells have been defined by many as the self-renewing subpopulation of stem-like (expressing normal stem cell markers) cells from which tumors originate, and which are required to maintain the long-lived properties of tumors [114116]. Despite much controversy about the role of these cells in tumor initiation, there have been numerous reports implicating such cells in the acquisition of drug resistance (Figure 2). This can be seen, for example, by examining stem cell markers in subpopulations of cultured tumor cells that survive drug treatment [117,118]. In several such cases, involving a variety of can-cer types and drug treatments, the residual cell population that survives treatment can be found to express cancer stem cell markers that are only seen at low frequency within the untreated cell population. Similarly, studies in which tumor cell populations are sorted on the basis of their expression profile of surface stem cell markers, and subsequently assayed for drug sensitivity, have demonstrated that stem cell-like subpopu-lations are generally more refractory to drug treatment [113,119]. Furthermore, a recent report demonstrated that such subpopulations of can-cer cells may transiently exhibit a reversible abil-ity to tolerate drug exposure; these cells exhibit a distinct chromatin state and vulnerability to inhibition of certain chromatin-modifying enzymes, implicating an epigenetic mechanism in their reversible resistance to cancer drugs [117]. Moreover, this mechanism required the activity of the histone demethylase KDM5A, and culturing the resistant cells in the absence of drug slowly restored their sensitivity to the primary agent, further supporting the concept of reversible drug tolerance. One can imagine that such a subpopulation may be selected dur-ing the treatment of tumors with agents that spare these cells, thereby possibly contributing to the acquisition of drug resistance. Other reported mechanisms of drug resistance by can-cer stem cells include upregulation of the ATP-binding cassette drug efflux transporters [120], increased activity of the detoxifying enzyme alcohol dehydrogenase 1 [121] and inhibition of apoptosis through increased expression of the IAP family of proteins [122].

Notably, this study and several related studies have called into question the widely described hierarchical model of cancer stem cells, in which stem cells can only arise from other stem

cells, with the demonstration that nonstem cells can spontaneously acquire stem cell properties [123,124]. The fact that cancer stem cells and cells that have undergone EMT share proper-ties associated with drug resistance, as well as expression of certain protein markers, has led to studies that have revealed a likely relation-ship between these phenomena [125,126]. Indeed, Weinberg and colleagues have reported that epithelial cells undergoing an EMT can acquire stem cell properties [127,128]. Collectively, such findings highlight the likely importance of a nongenetically determined dynamic heteroge-neity within tumor cell populations, and the likely role of such heterogeneity in the response to drug treatments.

ConclusionMolecularly targeted therapies have shown promise in the management of patients with advanced cancers, often resulting in dramatic tumor responses and extending lives. However, such therapies are rarely curative and, in most cases, resistance emerges relatively rapidly. Molecular characterization of resistant cell line models and tumor samples collected at dis-ease progression have revealed a diverse array of mechanisms, both genetic and nongenetic. Prominent among the genetic mechanisms are mutational alterations of the drug target that abrogate therapeutic binding or hyperactivate signaling. Additional genetic mechanisms include downstream or bypass activation of other components of signaling pathways, which acts epistatically to maintain critical cell sur-vival and proliferative signals despite upstream blockade. Nongenetic mechanisms of resistance include oncogene switching, wherein a different kinase substitutes for the drug target, or com-pensatory activation of other signaling pathways. Recent studies have also shed light on nonge-netic mechanisms that may have a reversible, epigenetic component, such as EMT or drug-tolerant cancer stem cells. Taken together, these observations highlight a pressing need to further elucidate the various mechanisms that drive dis-ease progression during drug treatment as a key step towards developing therapeutic strategies to prevent or overcome such drug resistance in individual patients, according to the specific molecular characteristics of their tumor.

Future perspectiveTechnologies for genome-wide assessment of genetic and epigenetic alterations in cancer genomes are developing at a rapid pace and in



the near future it should be possible to obtain complete profiles of cancer genomes before and after drug treatment, particularly at the time of disease progression. Serial pharmacodynamic assessment of tumor samples with panels of molecular assays will become more common clinically, and will enable the pinpointing of acquired resistance mechanisms and selection of the most appropriate subsequent therapy.

Financial & competing interests disclosureThe authors are employees of Genentech and sharehold-ers of Roche Pharmaceuticals. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

ReferencesPapers of special note have been highlighted as:n of interestnn of considerable interest

1. Bell DW, Gore I, Okimoto RA et al. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat. Genet. 37(12), 13151316 (2005).

2. Nazarian R, Shi H, Wang Q et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468(7326), 973977 (2010).

3. Lynch TJ, Bell DW, Sordella R et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 350(21), 21292139 (2004).

4. Paez JG, Janne PA, Lee JC et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304(5676), 14971500 (2004).

5. Rosell R, Moran T, Queralt C et al. Screening for epidermal growth factor receptor mutations in lung cancer. N. Engl. J. Med. 361(10), 958967 (2009).

6. Yun CH, Mengwasser KE, Toms AV et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl Acad. Sci. USA 105(6), 20702075 (2008).

7. Ercan D, Zejnullahu K, Yonesaka K et al. Amplification of EGFR T790M causes

resistance to an irreversible EGFR inhibitor. Oncogene 29(16), 23462356 (2010).

8. Wang SE, Narasanna A, Perez-Torres M et al. HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell 10(1), 2538 (2006).

9. Engelman JA, Zejnullahu K, Mitsudomi T et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316(5827), 10391043 (2007).

nn First report to identify MET gene amplification as a mechanism of acquired resistance to EGF receptor tyrosine kinase inhibitors.

10. Turke AB, Zejnullahu K, Wu YL et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell 17(1), 7788 (2010).

11. Sos ML, Koker M, Weir BA et al. PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. Cancer Res. 69(8), 32563261 (2009).

12. Sequist LV, Waltman BA, Dias-Santagata D et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci. Transl Med. 3(75), 75ra26 (2011).

n Describes the clinical experience associated with acquired resistance to EGF receptor tyrosine kinase inhibitors in the

setting of non-small-cell lung cancer, revealing both genetic and nongenetic resistance mechanisms.

13. Shaw AT, Yeap BY, Mino-Kenudson M et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J. Clin. Oncol. 27(26), 42474253 (2009).

14. Zhang S, Wang F, Keats J et al. Crizotinib-resistant mutants of EML4-ALK identified through an accelerated mutagenesis screen. Chem. Biol. Drug Des. 78(6), 9991005 (2011).

15. Choi YL, Soda M, Yamashita Y et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N. Engl. J. Med. 363(18), 17341739 (2010).

16. Sasaki T, Okuda K, Zheng W et al. The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers. Cancer Res. 70(24), 1003810043 (2010).

17. Sasaki T, Koivunen J, Ogino A et al. A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors. Cancer Res. 71(18), 60516060 (2011).

18. Katayama R, Khan TM, Benes C et al. Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK. Proc. Natl Acad. Sci. USA 108(18), 75357540 (2011).

Executive summary

n Resistance to molecularly targeted therapies is a pervasive problem in the management of advanced cancers.n Resistance can be mediated either by genetic or nongenetic mechanisms.n Genetic mechanisms of resistance to kinase inhibitors include mutations in the target receptor and mutations or gene amplification of

compensatory receptors or other signaling effectors.n Receptors such as EGFR, HER2, MET and ALK interface with common downstream signaling pathways and addiction switching can

occur, either by the inhibition of one receptor triggering a switch to an alternate receptor engaging the same downstream pathway or through the selection of subpopulations of tumor cells with distinct pathway dependencies.

n Abrogation of negative feedback loops that normally dampen upstream signaling, or upregulation of interacting pathways, can also occur when kinases such as AKT or PI3K are inhibited, potentially causing resistance.

n Epithelialmesenchymal transition triggered by epigenetic mechanisms can result in resistance to targeted therapies.n Cancer stem cells form a self-renewing subpopulation of tumors and have been shown to be refractory to certain drug treatments; they

may in some instances transiently exhibit the ability to tolerate drug exposure, which can underlie drug resistance.

www.futuremedicine.comfuture science group 1011


19. Sequist LV, Gettinger S, Senzer NN et al. Activity of IPI-504, a novel heat-shock protein 90 inhibitor, in patients with molecularly defined non-small-cell lung cancer. J. Clin. Oncol. 28(33), 49534960 (2010).

20. Sierra JR, Tsao MS. c-MET as a potential therapeutic target and biomarker in cancer. Ther. Adv. Med. Oncol. 3(Suppl. 1), S21S35 (2011).

21. Dillon R, Nilsson CL, Shi SD, Lee NV, Krastins B, Greig MJ. Discovery of a novel B-Raf fusion protein related to c-Met drug resistance. J. Proteome Res. 10(11), 50845094 (2011).

22. Qi J, McTigue MA, Rogers A et al. Multiple mutations and bypass mechanisms can contribute to development of acquired resistance to MET inhibitors. Cancer Res. 71(3), 10811091 (2011).

23. Tiedt R, Degenkolbe E, Furet P et al. A drug resistance screen using a selective MET inhibitor reveals a spectrum of mutations that partially overlap with activating mutations found in cancer patients. Cancer Res. 71(15), 52555264 (2011).

24. McDermott U, Pusapati RV, Christensen JG, Gray NS, Settleman J. Acquired resistance of non-small cell lung cancer cells to MET kinase inhibition is mediated by a switch to epidermal growth factor receptor dependency. Cancer Res. 70(4), 16251634 (2010).

n Describes cell culture modeling of resistance to a MET kinase inhibitor to demonstrate the ability of cancer cells to switch their dependency from one receptor tyrosine kinase to another.

25. Spector NL, Blackwell KL. Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2-positive breast cancer. J. Clin. Oncol. 27(34), 58385847 (2009).

26. Ryan Q, Ibrahim A, Cohen MH et al. FDA drug approval summary: lapatinib in combination with capecitabine for previously treated metastatic breast cancer that overexpresses HER-2. Oncologist 13(10), 11141119 (2008).

27. Arribas J, Baselga J, Pedersen K, Parra-Palau JL. p95HER2 and breast cancer. Cancer Res. 71(5), 15151519 (2011).

28. Sperinde J, Jin X, Banerjee J et al. Quantitation of p95HER2 in paraffin sections by using a p95-specific antibody and correlation with outcome in a cohort of trastuzumab-treated breast cancer patients. Clin. Cancer Res. 16(16), 42264235 (2010).

29. Scaltriti M, Rojo F, Ocana A et al. Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-

HER2 therapies in breast cancer. J. Natl Cancer Inst. 99(8), 628638 (2007).

30. Scaltriti M, Chandarlapaty S, Prudkin L et al. Clinical benefit of lapatinib-based therapy in patients with human epidermal growth factor receptor 2-positive breast tumors coexpressing the truncated p95HER2 receptor. Clin. Cancer Res. 16(9), 26882695 (2010).

31. Ritter CA, Perez-Torres M, Rinehart C et al. Human breast cancer cells selected for resistance to trastuzumab in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network. Clin. Cancer Res. 13(16), 49094919 (2007).

32. Nagata Y, Lan KH, Zhou X et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6(2), 117127 (2004).

nn Implicates the tumor suppressor PTEN in the clinical response and acquired resistance to the HER2-targeted antibody trastuzumab.

33. Berns K, Horlings HM, Hennessy BT et al. A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 12(4), 395402 (2007).

34. Liu L, Greger J, Shi H et al. Novel mechanism of lapatinib resistance in HER2-positive breast tumor cells: activation of AXL. Cancer Res. 69(17), 68716878 (2009).

35. Lu Y, Zi X, Zhao Y, Mascarenhas D, Pollak M. Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). J. Natl Cancer Inst. 93(24), 18521857 (2001).

36. Zhang S, Huang WC, Li P et al. Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways. Nat. Med. 17(4), 461469 (2011).

37. Scaltriti M, Eichhorn PJ, Cortes J et al. Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+ breast cancer patients. Proc. Natl Acad. Sci. USA 108(9), 37613766 (2011).

38. Nagy P, Friedlander E, Tanner M et al. Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a Herceptin-resistant, MUC4-expressing breast cancer cell line. Cancer Res. 65(2), 473482 (2005).

39. Junttila TT, Li G, Parsons K, Phillips GL, Sliwkowski MX. Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer. Breast Cancer Res. Treat. 128(2), 347356 (2011).

40. Lorusso PM, Weiss D, Guardino E, Girish S, Sliwkowski MX. Trastuzumab emtansine: a unique antibody-drug conjugate in development for human epidermal growth factor receptor 2-positive cancer. Clin. Cancer Res. 17(20), 64376447 (2011).

41. Scaltriti M, Serra V, Normant E et al. Antitumor activity of the Hsp90 inhibitor IPI-504 in HER2-positive trastuzumab-resistant breast cancer. Mol. Cancer Ther. 10(5), 817824 (2011).

42. Modi S, Stopeck AT, Gordon MS et al. Combination of trastuzumab and tanespimycin (17-AAG, KOS-953) is safe and active in trastuzumab-refractory HER-2 overexpressing breast cancer: a Phase I dose-escalation study. J. Clin. Oncol. 25(34), 54105417 (2007).

43. Flaherty KT, Hodi FS, Bastian BC. Mutation-driven drug development in melanoma. Curr. Opin Oncol. 22(3), 178183 (2010).

44. Corcoran RB, Settleman J, Engelman JA. Potential therapeutic strategies to overcome acquired resistance to BRAF or MEK inhibitors in BRAF mutant cancers. Oncotarget 2(4), 336346 (2011).

45. Whittaker S, Kirk R, Hayward R et al. Gatekeeper mutations mediate resistance to BRAF-targeted therapies. Sci. Transl Med. 2(35), 35ra41 (2010).

46. Poulikakos PI, Persaud Y, Janakiraman M et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480(7377), 387390 (2011).

47. Corcoran RB, Dias-Santagata D, Bergethon K, Iafrate AJ, Settleman J, Engelman JA. BRAF gene amplification can promote acquired resistance to MEK inhibitors in cancer cells harboring the BRAF V600E mutation. Sci. Signal. 3(149), ra84 (2011).

48. Montagut C, Sharma SV, Shioda T et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res. 68(12), 48534861 (2008).

49. Johannessen CM, Boehm JS, Kim SY et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468(7326), 968972 (2010).

50. Wagle N, Emery C, Berger MF et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J. Clin. Oncol. 29(22), 30853096 (2011).

51. Emery CM, Vijayendran KG, Zipser MC et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc. Natl Acad. Sci. USA 106(48), 2041120416 (2009).



52. Wang H, Daouti S, Li WH et al. Identification of the MEK1(F129L) activating mutation as a potential mechanism of acquired resistance to MEK inhibition in human cancers carrying the B-RafV600E mutation. Cancer Res. 71(16), 55355545 (2011).

53. Little AS, Balmanno K, Sale MJ et al. Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells. Sci. Signal. 4(166), ra17 (2011).

54. Hatzivassiliou G, Liu B, OBrien C et al. ERK inhibition overcomes acquired resistance to MEK inhibitors. Mol. Cancer Ther. 11(5), 11431154 (2012).

55. Ihle NT, Powis G. Take your PIK: phosphatidylinositol 3-kinase inhibitors race through the clinic and toward cancer therapy. Mol. Cancer Ther. 8(1), 19 (2009).

56. Engelman JA, Chen L, Tan X et al. Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat. Med. 14(12), 13511356 (2008).

57. Liu P, Cheng H, Santiago S et al. Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms. Nat. Med. 17(9), 11161120 (2011).

58. Ilic N, Utermark T, Widlund HR, Roberts TM. PI3K-targeted therapy can be evaded by gene amplification along the MYC-eukaryotic translation initiation factor 4E (eIF4E) axis. Proc. Natl Acad. Sci. USA 108(37), E699E708 (2011).

59. Muellner MK, Uras IZ, Gapp BV et al. A chemical-genetic screen reveals a mechanism of resistance to PI3K inhibitors in cancer. Nat. Chem. Biol. 7(11), 787793 (2011).

60. Lorusso PM, Rudin CM, Reddy JC et al. Phase I trial of hedgehog pathway inhibitor vismodegib (GDC-0449) in patients with refractory, locally advanced or metastatic solid tumors. Clin. Cancer Res. 17(8), 25022511 (2011).

61. Rudin CM, Hann CL, Laterra J et al. Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N. Engl. J. Med. 361(12), 11731178 (2009).

62. Yauch RL, Dijkgraaf GJ, Alicke B et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 326(5952), 572574 (2009).

n A molecular mechanism of acquired resistance to a Hh pathway inhibitor in the treatment of medulloblastoma was identified in which the gene encoding the

pathway component SMO was mutationally altered.

63. Dijkgraaf GJ, Alicke B, Weinmann L et al. Small molecule inhibition of GDC-0449 refractory Smoothened mutants and downstream mechanisms of drug resistance. Cancer Res. 71(2), 435444 (2011).

64. Buonamici S, Williams J, Morrissey M et al. Interfering with resistance to Smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci. Transl Med. 2(51), 51ra70 (2010).

65. Gorre ME, Mohammed M, Ellwood K et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293(5531), 876880 (2001).

nn Demonstrates that acquired resistance to the ABL kinase inhibitor imatinib in chronic myeloid leukemia patients is associated with secondary mutational alterations of the BCRABL oncogene.

66. Roumiantsev S, Shah NP, Gorre ME et al. Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. Proc. Natl Acad. Sci. USA 99(16), 1070010705 (2002).

67. Mahon FX, Deininger MW, Schultheis B et al. Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 96(3), 10701079 (2000).

68. Peng XX, Tiwari AK, Wu HC, Chen ZS. Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells. Chin. J. Cancer 31(2), 110118 (2012).

69. Mahon FX, Hayette S, Lagarde V et al. Evidence that resistance to nilotinib may be due to BCR-ABL, Pgp, or Src kinase overexpression. Cancer Res. 68(23), 98099816 (2008).

70. Heinrich MC, Owzar K, Corless CL et al. Correlation of kinase genotype and clinical outcome in the North American Intergroup Phase III trial of imatinib mesylate for treatment of advanced gastrointestinal stromal tumor: CALGB 150105 study by Cancer and Leukemia Group B and Southwest Oncology Group. J. Clin. Oncol. 26(33), 53605367 (2008).

71. Antonescu CR, Besmer P, Guo T et al. Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation. Clin. Cancer Res. 11(11), 41824190 (2005).

72. Debiec-Rychter M, Cools J, Dumez H et al. Mechanisms of resistance to imatinib

mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants. Gastroenterology 128(2), 270279 (2005).

73. Mahadevan D, Cooke L, Riley C et al. A novel tyrosine kinase switch is a mechanism of imatinib resistance in gastrointestinal stromal tumors. Oncogene 26(27), 39093919 (2007).

74. Sakurama K, Noma K, Takaoka M et al. Inhibition of focal adhesion kinase as a potential therapeutic strategy for imatinib-resistant gastrointestinal stromal tumor. Mol. Cancer Ther. 8(1), 127134 (2009).

75. Theou N, Gil S, Devocelle A et al. Multidrug resistance proteins in gastrointestinal stromal tumors: site-dependent expression and initial response to imatinib. Clin. Cancer Res. 11(21), 75937598 (2005).

76. Dinitto JP, Deshmukh GD, Zhang Y et al. Function of activation loop tyrosine phosphorylation in the mechanism of c-Kit auto-activation and its implication in sunitinib resistance. J. Biochem. 147(4), 601609 (2010).

77. Moritz A, Li Y, Guo A et al. Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases. Sci. Signal. 3(136), ra64 (2010).

78. Sharma SV, Settleman J. Oncogene addiction: setting the stage for molecularly targeted cancer therapy. Genes Dev. 21(24), 32143231 (2007).

79. Engelman JA, Settleman J. Acquired resistance to tyrosine kinase inhibitors during cancer therapy. Curr. Opin Genet. Dev. 18(1), 7379 (2008).

80. Guix M, Faber AC, Wang SE et al. Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins. J. Clin. Invest. 118(7), 26092619 (2008).

81. Nahta R, Yuan LX, Zhang B, Kobayashi R, Esteva FJ. Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells. Cancer Res. 65(23), 1111811128 (2005).

82. Villanueva J, Vultur A, Lee JT et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 18(6), 683695 (2010).

83. Prahallad A, Sun C, Huang S et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature 483(7387), 100103 (2012).

84. Corcoran RB, Ebi H, Turke AB et al. EGFR-mediated re-activation of MAPK

www.futuremedicine.comfuture science group 1013


signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov. 2(3), 227235 (2012).

85. Chandarlapaty S, Sawai A, Scaltriti M et al. AKT inhibition relieves feedback suppression of receptor tyrosine kinase expression and activity. Cancer Cell 19(1), 5871 (2011).

86. Chakrabarty A, Sanchez V, Kuba MG, Rinehart C, Arteaga CL. Feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors. Proc. Natl Acad. Sci. USA 109(8), 27182723 (2011).

87. Osborne CK, Schiff R. Estrogen-receptor biology: continuing progress and therapeutic implications. J. Clin. Oncol. 23(8), 16161622 (2005).

88. Gonzalez-Angulo AM, Morales-Vasquez F, Hortobagyi GN. Overview of resistance to systemic therapy in patients with breast cancer. Adv. Exp. Med. Biol. 608, 122 (2007).

89. Prat A, Baselga J. The role of hormonal therapy in the management of hormonal-receptor-positive breast cancer with co-expression of HER2. Nat. Clin. Pract. Oncol. 5(9), 531542 (2008).

90. Sanchez CG, Ma CX, Crowder RJ et al. Preclinical modeling of combined phosphatidylinositol-3-kinase inhibition with endocrine therapy for estrogen receptor-positive breast cancer. Breast Cancer Res. 13(2), R21 (2011).

91. Crowder RJ, Phommaly C, Tao Y et al. PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivation in estrogen receptor-positive breast cancer. Cancer Res. 69(9), 39553962 (2009).

92. Miller TW, Balko JM, Arteaga CL. Phosphatidylinositol 3-kinase and antiestrogen resistance in breast cancer. J. Clin. Oncol. 29(33), 44524461 (2011).

93. Miller TW, Hennessy BT, Gonzalez-Angulo AM et al. Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor-positive human breast cancer. J. Clin. Invest. 120(7), 24062413 (2010).

94. Miller TW, Perez-Torres M, Narasanna A et al. Loss of phosphatase and tensin homologue deleted on chromosome 10 engages ErbB3 and insulin-like growth factor-I receptor signaling to promote antiestrogen resistance in breast cancer. Cancer Res. 69(10), 41924201 (2009).

95. Baselga J, Campone M, Piccart M et al. Everolimus in postmenopausal hormone-

receptor-positive advanced breast cancer. N. Engl. J. Med. 366(6), 520529 (2011).

96. Ross-Innes CS, Stark R, Teschendorff AE et al. Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature 481(7381), 389393 (2012).

97. Chen Y, Sawyers CL, Scher HI. Targeting the androgen receptor pathway in prostate cancer. Curr. Opin Pharmacol. 8(4), 440448 (2008).

98. Visakorpi T, Hyytinen E, Koivisto P et al. In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat. Genet. 9(4), 401406 (1995).

99. Taplin ME, Bubley GJ, Shuster TD et al. Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer. N. Engl. J. Med. 332(21), 13931398 (1995).

100. Dehm SM, Tindall DJ. Alternatively spliced androgen receptor variants. Endocr. Relat. Cancer 18(5), R183R196 (2011).

101. Schweizer L, Rizzo CA, Spires TE et al. The androgen receptor can signal through Wnt/beta-catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens. BMC Cell Biol. 9, 4 (2008).

102. Cai C, Wang H, Xu Y, Chen S, Balk SP. Reactivation of androgen receptor-regulated TMPRSS2:ERG gene expression in castration-resistant prostate cancer. Cancer Res. 69(15), 60276032 (2009).

103. Mulholland DJ, Tran LM, Li Y et al. Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth. Cancer Cell 19(6), 792804 (2011).

104. Carver BS, Chapinski C, Wongvipat J et al. Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer. Cancer Cell 19(5), 575586 (2011).

105. Fox EM, Miller TW, Balko JM et al. A kinome-wide screen identifies the insulin/IGF-I receptor pathway as a mechanism of escape from hormone dependence in breast cancer. Cancer Res. 71(21), 67736784 (2011).

106. Rexer BN, Ghosh R, Arteaga CL. Inhibition of PI3K and MEK: it is all about combinations and biomarkers. Clin. Cancer Res. 15(14), 45184520 (2009).

107. Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29(34), 47414751 (2010).

108. Yauch RL, Januario T, Eberhard DA et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer

patients. Clin. Cancer Res. 11(24 Pt 1), 86868698 (2005).

109. Suda K, Tomizawa K, Fujii M et al. Epithelial to mesenchymal transition in an epidermal growth factor receptor-mutant lung cancer cell line with acquired resistance to erlotinib. J. Thorac. Oncol. 6(7), 11521161 (2011).

110. Chung JH, Rho JK, Xu X et al. Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs. Lung Cancer 73(2), 176182 (2011).

111. Morgillo F, Cascone T, DAiuto E et al. Antitumour efficacy of MEK inhibitors in human lung cancer cells and their derivatives with acquired resistance to different tyrosine kinase inhibitors. Br. J. Cancer 105(3), 382392 (2011).

112. Sun Y, Wang BE, Leong KG et al. Androgen deprivation causes epithelial-mesenchymal transition in the prostate: implications for androgen-deprivation therapy. Cancer Res. 72(2), 527536 (2011).

113. Gupta PB, Onder TT, Jiang G et al. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138(4), 645659 (2009).

114. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. USA 100(7), 39833988 (2003).

115. Singh SK, Clarke ID, Terasaki M et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 63(18), 58215828 (2003).

116. Roesch A, Fukunaga-Kalabis M, Schmidt EC et al. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth. Cell 141(4), 583594 (2010).

117. Sharma SV, Lee DY, Li B et al. A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 141(1), 6980 (2010).

n Describes the existence of a subpopulation of cancer cells that transiently exhibit a phenotype associated with the ability to tolerate potentially lethal drug exposures.

118. Bao S, Wu Q, McLendon RE et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444(7120), 756760 (2006).

119. Li X, Lewis MT, Huang J et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J. Natl Cancer Inst. 100(9), 672679 (2008).



120. Elliott A, Adams J, Al-Hajj M. The ABCs of cancer stem cell drug resistance. IDrugs 13(9), 632635 (2010).

121. Ginestier C, Hur MH, Charafe-Jauffret E et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1(5), 555567 (2007).

122. Liu G, Yuan X, Zeng Z et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol. Cancer 5, 67 (2006).

123. Chaffer CL, Brueckmann I, Scheel C et al. Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state.

Proc. Natl Acad. Sci. USA 108(19), 79507955 (2011).

124. Quintana E, Shackleton M, Foster HR et al. Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized. Cancer Cell 18(5), 510523 (2010).

125. Guo W, Keckesova Z, Donaher JL et al. Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 148(5), 10151028 (2012).

126. Scheel C, Weinberg RA. Phenotypic plasticity and epithelial-mesenchymal transitions in cancer and normal stem cells? Int. J. Cancer 129(10), 23102314 (2011).

127. Mani SA, Guo W, Liao MJ et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133(4), 704715 (2008).

n Describes the ability of epithelial cells to give rise to cells with stem cell-like properties upon epithelialmesenchymal transition, thereby challenging the dogma of the hierarchical stem cell model.

128. Blick T, Hugo H, Widodo E et al. Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44hi/CD24lo/- stem cell phenotype in human breast cancer. J. Mammary Gland Biol. Neoplasia 15(2), 235252 (2010).

Documents

Mechanisms of Resistance to Targeted Therapies.2012 Genel