Dysregulation of the Met Pathway in Non-Small Cell Lung Cancer: Implications for Drug Targeting and Resistance

Review Article

Dysregulation of the Met pathway in non-small cell lung cancer: implications for drug targeting and resistance

Joseph J. Sacco1, Michael J. Clague2

1Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, UK; 2Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK. Correspondence to: Joseph J. Sacco. Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, L69 3GA, UK. Email: [email protected].

Abstract: The receptor tyrosine kinase, Met, orchestrates a complex signalling network that physiologically drives a programme of ‘invasive growth’. In cancer however, this process may be co-opted to promote proliferation, survival and metastasis of cancer cells. Met is thus a key therapeutic target, not least in non- small cell lung cancer (NSCLC) where it is one of the most commonly dysregulated driver oncogenes. Identifying robust biomarkers that allow the selection of patients most likely to respond to Met targeted therapies will however be essential to realising their potential. This has been underlined recently by the early termination of three pivotal phase III trials investigating Met targeted agents in NSCLC, all of which failed to show clinical benefit. In contrast to these trials, which were relatively unselective, a couple of early phase trials have recently been instigated that select patients on the basis of Met amplification. While still at an early stage, interim results are relatively encouraging and strengthen the rationale for using Met amplifaction as a biomarker. Here we will discuss this and other aberrations in Met signalling in relation to their significance in the therapeutic targeting of Met.

Keywords: Hepatocyte growth factor (HGF); lung cancer; Met; non-small cell lung cancer (NSCLC); epidermal growth factor receptor (EGFR)

Submitted Feb 03, 2015. Accepted for publication Feb 05, 2015. doi: 10.3978/j.issn.2218-6751.2015.03.05 View this article at: http://dx.doi.org/10.3978/j.issn.2218-6751.2015.03.05

Introduction generally remains chemotherapy, for which survival is relatively unchanged. While there has been recent progress The last decade or so has seen the management of in targeting RET and ROS1 (2,3), mutations in these two metastatic non-small cell lung cancer (NSCLC) emerge as genes occur very infrequently, and there is thus a clear a paradigm for personalized medicine (1). This principally need to develop drugs against alternative molecular targets, follows the development and clinical validation of among which Met is a leading example. inhibitors against two key oncogenes; epidermal growth The receptor tyrosine kinase, Met [also known as factor receptor (EGFR) and anaplastic lymphoma kinase hepatocyte growth factor receptor (HGFR)], was first (ALK). Significantly, the clinical efficacy of inhibiting discovered 30 years ago as part of a fusion gene, TPR-MET, these two kinases is almost entirely restricted to patients that was isolated from a osteosarcoma cell line derived with constitutive activation of the relevant kinase, through through chemical carcinogenesis (4). It has since been mutation (EGFR) or translocation (ALK). This in turn shown to have key physiological roles, driving a programme has led to a stratified approach to therapy, with molecular of “invasive growth” that is vital in development and analysis at diagnosis and the use of EGFR or ALK tissue repair (5). Aberrant activation of Met is common inhibitors if indicated. However, for the majority of patients in malignancy, most notably hereditary papillary renal who do not have mutations in either gene, treatment carcinoma in which activating mutations in Met are

Table 1 Abnormalities in Met signalling in NSCLC amplification or exon 14 skipping. The latter increases Met Number in study Abnormality abundance by decreasing turnover rate (Figure 1). Both Aberration Ref. (histology) (%) these aberrations were mutually exclusive with other known Met oncogenes, supporting oncogene driver status. Notably only K-RAS, EGFR and BRAF mutations were more frequent. Over-expression 42 (NSCLC) 25.0 (7) Interest in the clinical potential of targeting Met has also 32 (NSCLC) 61.0 (8) been heightened by studies in which Met amplification was 40 (NSCLC) 40.0 (9) shown to be one of the principle mechanisms by which † 47 (adenocarcinoma) 72.3 (10) NSCLC escapes EGFR inhibition (14,18). † 52 (squamous cell) 38.5 (10) In contrast to K-RAS, which is the most commonly 130 (adenocarcinoma) 36.1 (11) mutated driver gene in NSCLC, Met is readily druggable, 682 (adenocarcinoma) 27.3 (12) with a wide range of Met targeted drugs already in clinical 149 (squamous cell) 0.7 (12) development. These fall into several different classes; Amplification 230 (adenocarcinoma) 2.2 (13) including small molecule inhibitors, decoy molecules which 62 (adenocarcinoma)‡ 3.0 (14) prevent binding of HGF to Met, and monoclonal antibodies 97 (squamous) 8.2§ (15) that inhibit either Met or HGF (19,20). Preclinical evidence § for these inhibitors is promising, with Met amplified NSCLC 72 (adenocarcinoma) 4.2 (15) cell lines in particular showing exquisite sensitivity (21). 655 (adenocarcinoma) 11.5 (12) However, to date this promise has not been borne out in 142 (squamous) 0.7 (12) clinical trials, which have been relatively disappointing. In the 148 (adenocarcinoma) 1.4 (16) last two years, three landmark phase III trials investigating 28 (squamous) 0.0 (16) Met targeted agents in combination with erlotinib (an Exon 14 skipping 230 (adenocarcinoma) 4.3 (13) EGFR inhibitor) in pre-treated lung cancer were suspended 87 (adenocarcinoma) 3.4 (17) following interim analyses that indicated no improvement 211 (adenocarcinoma)¶ 3.3 (16) in survival and/or safety concerns (22-24). Further trials HGF are however ongoing with these drugs and it is feasible that Over-expression 42 (NSCLC) 55.0 (7) subgroup analysis will identify patients who benefit from one 130 (NSCLC) 31.5 (11) or other combination. In addition, several other agents are in † development including crizotinib (small molecule inhibitor , Any Met protein expression detected on immunoblotting of Met and ALK kinase) which has shown early evidence of was reported as positive; immunoblotting of adjacent normal ‡ activity in Met amplified NSCLC (25). tissue was negative for Met; , EGFR mutant tumours that had § Nevertheless, the results of the recent negative trials are not been treated with EGFR inhibitors; , difference between ¶ sobering and highlight significant gaps in our knowledge, histologies not statistically different; , no exon 14 skipping not least in patient selection. It is clear that patient identified in 51 non adenocarcinomas. Abbreviations: NSCLC, stratification to identify patients most likely to benefit from non-small cell lung cancer; Ref., reference; HGF, hepatocyte Met inhibitors will be vital (26), and to this end the ability growth factor. to detect when Met is acting as a driver oncogene is crucial. In this review we will discuss the various aberrations in Met signalling in NSCLC, and how these may impact on common (6), but also in other malignancies including responses to Met inhibitors. NSCLC, where, in contrast, signalling is generally driven by increased Met abundance (Table 1). Overview of Met signalling The relative importance of Met in NSCLC has recently been underlined by the findings of a large scale Met signaling has been extensively reviewed elsewhere (5,20), comprehensive molecular profiling study conducted by The and we will cover only a few salient points here. Both Met Cancer Genome Atlas (TCGA) in lung adenocarcinoma (13). and its ligand HGF are synthesised as single polypeptide This identified Met as a key targetable driver gene, with chains that are proteolytically cleaved to form the mature approximately 7% of tumours either exhibiting Met protein, each consisting of two polypeptide chains linked by

A B Extracellular Wild-type Exon 14 Compartment Met skipping

Internalisation

Signalling Signalling

Sorting endosome

MVB Cessation of signaling/degradation Cytoplasm

Figure 1 Met is stabilised by loss of Tyr-1003. (A) Activation of wild-type Met is coupled with its internalisation and ubiquitylation by CBL allowing efficient sorting to the multivesicular body (MVB), and subsequent degradation by the lysosome; (B) following exon 14 skipping however, the juxtamembrane domain, including Tyr-1003 is deleted. This prevents recruitment of CBL and thus Met ubiquitylation; as a consequence Met is not sorted to the MVB, instead being recycled back to the cell surface. Abbreviations: HGF, hepatocyte growth factor; p, phosphate; Ub, ubiquitin. a disulphide bond. Notably, while pro-HGF is capable of the endosome, and is dependent on phosphorylation of Tyr- high affinity binding to Met, only mature HGF can activate 1003 in the Met juxtamembrane domain, which leads to Met signalling (27,28). HGF binding to the extracellular binding of the CBL tyrosine kinase binding (TKB) domain domains of Met (29) leads to its homodimerisation, and and CBL activation (42). Internalised Met continues to transphosphorylation of tyrosine kinase residues Tyr-1234 signal from the endosomal platform, although the signalling and Tyr-1235 in the catalytic domain. This is followed by output is qualitatively different due to the changing palette autophosphorylation of residues Tyr-1349 and Tyr-1356 of substrates present in different subcellular compartments in the c-terminus, which act as a platform for the binding (38,43). Notably, Met receptor in which Tyr-1003 is missing of adaptor proteins. Met has fewer pTyr binding sites than (through exon 14 skipping for example) or mutated, is not other receptor tyrosine kinases such as EGFR. However, the directed to the MVB, but is instead trafficked back to the adapter protein GAB1 expands the palette of sites and is a cell surface (Figure 1B) (41,44). key co-ordinator of Met signalling, acting as a scaffold for the docking of signalling molecules that include GRB2, PLC, Met signalling in NSCLC SRC, SHP2 and PI3K (30-33). This leads to activation of a number of downstream pathways that have been shown to In NSCLC, aberrant activation of the Met pathway in be involved in oncogenesis, most notably PI3K, MAPK and NSCLC may occur through a variety of mechanisms, STAT3 (34-37). the most important of which are summarised in Table 1. Physiological Met signalling is tightly regulated, with Over-expression of Met protein is the most commonly activation of Met being directly and acutely coupled reported, with rates of between 25% and 75% in different to its degradation (Figure 1A). Activated Met is rapidly case series (7-11). Several factors are likely to contribute internalised and delivered to the sorting endosome, from to this large variability in the literature. Expression of which a proportion is recycled back to the membrane, Met is generally assessed through immunohistochemistry while the rest is directed to the multivesicular body (MVB) and/or immunoblotting, both of which are subject to a and then undergoes degradation in the lysosome (38-42). large degree of experimental variability, and which are in Ubiquitylation of Met is required for efficient sorting by addition often quantitatively non-linear. The level of Met

© Translational lung cancer research. All rights reserved. www.tlcr.org Transl Lung Cancer Res 2015;4(3):242-252 Translational lung cancer research, Vol 4, No 3 June 2015 245 expression is a continuous variable, and thus reported rates important oncogenic event, several studies have shown of over-expression also depend partly on the choice of that Met amplification is associated with phosphorylation cut-off. The variability in reported rates may additionally and thus activation of Met in cell lines and tumour samples reflect true biological differences, for example between (12,48,49). Met amplification has also been shown to lead to histological subtypes, with some studies suggesting over- transphosphorylation of other receptor tyrosine kinases such expression is more frequent in adenocarcinomas than as EGFR and ERBB3 (49). Significantly, studies have shown in squamous cell carcinoma (8,10,12). However, even that Met amplified cell lines are sensitive to Met inhibition assuming the lower end of range, Met over-expression is a (21,50). These included a study which profiled 500 cell lines common event in NSCLC. Interestingly, although studies for sensitivity against a panel of kinase inhibitors in which have shown a correlation between Met over-expression the 7 which exhibited greatest sensitivity to Met inhibitors and phosphorylation [assessed by Immunohistochemistry were all Met amplified (5 gastric and 2 NSCLC) (21). (IHC)], not all cases with Met over-expression were positive Interestingly, a few cell lines with Met amplification for phosphorylation or vice versa (11,12). This suggests were not sensitive to the Met inhibitor; these either did that Met over-expression may not always be a marker for not express Met protein or failed to show activation of activated Met signalling. downstream survival signals (21). Met gene amplification is a well-established mechanism However, Met activation is not observed solely in the by which Met overexpression occurs. Most studies suggest presence of amplification, suggesting other mechanisms of this occurs in about 2-4% of lung adenocarcinomas, and activation of signalling. As previously discussed, impaired potentially at lower rates in squamous carcinomas (Table 1). Met degradation due to exon 14 skipping is a further likely In the large-scale, comprehensive TCGA study in oncogenic driver (Figure 1, Table 1). Splice mutants of Met that adenocarcinoma, 2.2% of cases exhibited amplification (13) lead to skipping of exon 14 and thus lose the juxtamembrane compared to only 1% of cases in the comparable study on region and Tyr1003 show enhanced stability, prolonged lung squamous cell carcinomas [curated data from (45) signalling and oncogenic capacity (44). Mutations leading to viewed in cBioPortal (46)]. Other smaller studies are exon 14 skipping have now been reported in around 3-4% generally comparable although there are exceptions (Table 1). of NSCLC cases (13,16), indicating this to be an important Met amplification has also been shown to be a major mechanism driving Met overexpression in lung cancer. mechanism by which cancers develop resistance to EGFR Two Met mutations that increase the rate of endocytosis inhibitors. In one study, an EGFR exon 19 mutant and recycling to the membrane, and reduce rates of NSCLC cell line was exposed to gefitinib at increasing degradation leading to Met accumulation have previously concentrations over 6 months leading to the generation of been described (51). Both mutations, D1246N and M1268T, gefitinib resistance (18). Unlike the parental cell line, the involve the catalytic domain, lead to constitutive activation resistant cell line (and six single cell clones) maintained and were identified in papillary renal cell carcinoma (6). A phosphorylation of ERBB3 and Akt in the presence of search of the COSMIC and cBioportal databases revealed gefitinib. Copy number analysis revealed a 5-10 fold neither of these mutations in lung cancer, and it is unlikely amplification of Met, and combined inhibition of Met and that either play a significant role in NSCLC. Intriguingly EGFR restored drug sensitivity. This finding was confirmed mutations that lead to constitutive activation of Met are in cancer tissue samples, with 4 out of 18 cases of NSCLC almost unheard of in NSCLC. While non-synonymous that had developed resistance to gefitinib demonstrating mutations in other Met domains have been described, these Met amplification (18). Further work has shown that either represent germline polymorphisms or are rare and treatment with EGFR inhibitors may positively select likely of low oncogenicity (52-54). existing clones with Met amplification, and that EGFR The discrepancy between the combined rates of kinase resistance due to either Met amplification or HGF Met amplification and exon 14 skipping (7%), and Met autocrine secretion, can be overcome with the use of Met overexpression (at least 25%) is likely due to several inhibitors (47). This is supported by a study in which Met other mechanisms, many of which remain to be defined. amplification was observed in only 3% (2 of 62) of untreated These include repression of microRNAs leading in turn controls, which increased to 21% (9 of 43) in patients with to increased expression of Met. mir27a is perhaps the best acquired resistance to EGFR inhibitors (14). studied in this setting, and has been shown to regulate In further evidence supporting Met amplification as an Met expression in NSCLC both directly, and indirectly

© Translational lung cancer research. All rights reserved. www.tlcr.org Transl Lung Cancer Res 2015;4(3):242-252 246 Sacco and Clague. Dysregulation of Met signaling in NSCLC through sprouty2 (55). Interestingly miR27a also regulates signalling. Examples of this approach include ficlatuzumab EGFR and thus may be involved in cross talk between these (AV299, Aveo) and rilotumumab (AMG-102, Amgen) two receptors (55). In addition, reduced serum expression which are both monoclonal antibodies, and CGEN241 of mir27a has also been reported in patients with early (Compugen), which is a soluble truncated Met receptor. NSCLC (56). Interestingly, a host of other miRNAs have The first of these, ficlatuzumab, showed synergism with been implicated in the regulation of Met in NSCLC the EGFR inhibitors erlotinib and cetuximab in a NSCLC (miR-449a and miR-7515) or other malignancies (57-74); xenograft model, prompting a phase I study in which however their relative importance and interplay remains to ficlatuzumab was assessed either alone or in combination be deciphered. Importantly, up regulation of Met through with erlotinib in solid tumours (78). The combination was this mechanism is unlikely to be targetable through the well tolerated, however there were no responses, and only 2 use of Met inhibitors, as most miRNAs affect multiple out of 8 patients in the combination cohort had stable disease genes. Instead expression of Met could be repressed at first assessment. A phase II trial followed in which 188 through the use of miRNA mimetics. The first in class, Asian patients with treatment-naïve NSCLC were recruited MRX34, which regulates over 20 genes including Met, is and randomised to gefitinib alone or in combination with currently undergoing investigation in a phase I trial that ficlatuzumab. The results of this trial have been presented is investigating its efficacy in patients with HCC or liver and show no benefit for the combination (80). metastases from other malignancies (75). This class of drugs The second HGF antibody, rilotumumab is currently clearly has immense promise, however many challenges being investigated in a phase II trial (again in combination remain including drug delivery to target organs as well as with an EGFR inhibitor), while CGEN241 remains in potential toxicity issues. preclinical development. A proportion of Met overexpression and/or activation undoubtedly occurs as a consequence of activation of other Met monoclonal antibodies oncogenic pathways, which may alter Met transcription, translation or degradation, or indeed directly transactivate Binding of HGF to Met involves interactions between Met receptor. EGFR is the best-studied example of the multiple surfaces on both proteins, with a recent study latter, however a host of other molecules can also affect identifying four different hotspots that can be inhibited the activation of Met (76). Met activation may also occur by antibodies (81). There are several antibodies in through HGF over-expression and autocrine secretion development, however only onartuzumab (Genentech) is in (Table 1), which has also been shown to contribute to gefitinib late clinical development. Met antibodies have been shown resistance (77). However, while these processes may facilitate to lead to reduced Met signalling, apoptosis and shrinkage oncogenesis, at present there is limited evidence to suggest of xenografts in a range of tumour types. In addition Met that over-expression of Met (apart from that driven by antibodies may drive Met degradation, thus also leading to amplification or exon 14 skipping) or HGF acts as a driver. a reduction in abundance at the membrane (82). Onartuzumab is a humanised monovalent antibody raised against Met that has recently been assessed in Targeting Met combination with the EGFR inhibitor erlotinib in NSCLC Pharmaceutical companies have invested heavily in in two trials. The first was a randomised placebo controlled developing drugs against Met, with most large companies phase II clinical trial, which compared the combination now including one or more such drugs in their pipeline. of onartuzumab and erlotinib against erlotinib alone in Several comprehensive reviews of Met inhibitors have been patients with recurrent NSCLC who had been treated published recently (19,20). There are three main classes of with one or two systemic regimens, and who had not had drug, examples of which are summarised in Table 2. significant prior exposure to EGFR directed therapy (83). Overall the trial showed no benefit in progression free survival (PFS) or overall survival (OS). However, in patients HGF monoclonal antibodies and decoys who were positive for Met by IHC (n=66), both PFS and In the first approach, antibodies against HGF or soluble Met OS were significantly improved, in contrast to Met negative fragments act as decoys, binding HGF and thus reducing patients whose outcomes were worse with onartuzumab. the concentration of free HGF available to activate Met These results prompted the institution of a phase III clinical

Table 2 Representative examples of Met targeted agents in clinical development Trial Class and Drug n Design Outcome Ref. phase HGF monoclonal antibody Ficlatuzumab (Aveo) II 188 Gefitinib with or without fliclatuzumab in first line No improvement in PFS in (78) unselected Asian patients lung adenocarcinoma patients receiving combination Met monoclonal antibody Onartuzumab III 499 Onartuzumab in combination with erlotinib versus Stopped early due to futility. (22) (Genentech/Roche) erlotinib alone in previously treated MET-positive Median OS 6.8 (combination) vs. advanced NSCLC. Met positivity defined by IHC (2+ 9.1 months (P=0.068) or 3+) Small molecule Met inhibitors Tivantinib (Arqule) III 307 Erlotinib combined with tivantinib or placebo in Stopped early due to safety (23) previously treated Asian patients with wild-type concerns. Median OS 12.9 vs. EGFR non-squamous NSCLC 11.2 months (P=0.427) III 1,048 Erlotinib combined with tivantinib or placebo in Stopped early due to futility. (24) previously treated non-squamous NSCLC Median OS 8.5 vs. 7.8 months (P=0.81) INC280 IB/II 41+ INC280 (capmatinib) in combination with gefitinib in Ongoing dose escalation. (79) (Novartis/Incyte) EGFR mutant Met positive NSCLC, previously 6 responses (15%) treated with EGFR inhibitors. Met positive defined as Met amplified or Met overexpression by IHC Crizotinib (Pfizer) I 16+ Crizotinib in Met amplified NSCLC. Three categories Ongoing. Response rate 0% (low), (25) of amplification defined; low, intermediate and high 20% (intermediate) and 50% (high) Abbreviations: NSCLC, non-small cell lung cancer; Ref., reference; HGF, hepatocyte growth factor; OS, overall survival; PFS, progression-free survival; IHC, immunohistochemistry; EGFR, epidermal growth factor receptor.

trial with a similar design to the phase II study, with the compounds that inhibit Met have been licenced for clinical exception that Met positivity was mandated. Unfortunately use, however both of these (crizotinib and cabozantinib) the study was suspended following an interim futility have activity against multiple other kinases. Crizotinib is analysis, which showed no improvement in either OS or an inhibitor of ALK kinase and has been licenced for use PFS (22). in NSCLC in which there are ALK translocations, while A related approach that may prove fruitful is the use of cabozantinib is a multi targeted kinase inhibitor that has been conjugated antibodies that are capable of delivering either licenced in prostate cancer. chemotherapy or radioisotopes to Met expressing cells. An In a case report, treatment with crizotinib resulted example is anti-Met antibody fragment (FAB) conjugated to in a rapid and sustained response in a patient with Met doxorubicin which has demonstrated efficacy in preclinical amplified NSCLC, with reduction in summated diameters HCC model (84). Proof of principle for this approach of over 50% (86). The very fact that this case is widely is provided from experience with the conjugated Her2 cited is illustrative of the paucity of evidence to date. There antibody, trastuzumab emtansine, which has demonstrated is however an ongoing phase I clinical trial of crizotinib significant activity in Her2 positive breast cancer (85). in Met amplified NSCLC that was presented at ASCO this year; this has recruited 16 patients, 4 of whom have responded to treatment with a 35 weeks median response. Small molecule inhibitors While this trial is still at a very early stage with only a very Over a dozen different small molecule inhibitors with varying small number of patients recruited, the degree of Met selectivity for Met are in clinical development (19). Two amplification correlated closely with response rate, with no

© Translational lung cancer research. All rights reserved. www.tlcr.org Transl Lung Cancer Res 2015;4(3):242-252 248 Sacco and Clague. Dysregulation of Met signaling in NSCLC responses in those classed as low level amplification, and investigating crizotinib, where there also appears to be a response rates of 20% and 50% in those classed medium or strong correlation between the degree of Met amplification high respectively. and response, although the results are as yet preliminary (25). Tivantinib is a specific small molecule inhibitor of Met Notably, only patients with high-level amplification (defined that is currently in late phase investigation. A phase III trial as a Met to centromere 7 ratio of 5 or more) showed exploring the efficacy of combining tivantinib and erlotinib significant response to crizotinib (87). This may explain why in pre-treated metastatic non-squamous lung cancer was Met amplified patients did not show improved outcomes in a halted last year after a preliminary analysis showed it would subset analysis of the pivotal onartuzumab trial (22). not achieve its primary objective of increasing overall Rare subgroups that occur at very low frequencies survival (24). Notably, this trial very closely mirrors the can however be prohibitive for drug development. In the study of onartuzumab, which similarly failed to support crizotinib study for example, only 0.8% of the population activity of the combination. A full subgroup analysis is screened had high level amplification (25). While this is awaited, and will be extremely valuable in determining clearly a challenge it is not insurmountable with the use whether the combination of a Met targeted drug in of innovative trial designs including adaptive studies such combination with erlotinib is a valid strategy. as the BATTLE study (90). In addition exon 14 skipping would intuitively be expected to have a similar effect on sensitivity to Met inhibitors, and combined with Met Future perspectives amplification would allow selection of a significantly higher Met remains an exciting target for future drug development proportion of patients. While this has not been tested with significant potential. However, the failure of the three prospectively as yet, evidence to support (or refute) exon largest trials to date raises significant questions. The results 14 skipping as a predictive biomarker may well be obtained have not been fully published and as such any hypotheses from retrospective analyses of the completed phase III trials. are only tentative. It is feasible that the combination of Met Overall, we remain optimistic that Met inhibition will inhibitor and EGFR inhibitor were antagonistic in vivo, prove a valuable addition to the therapeutic armamentarium or led to increased toxicity (23) and thus reduced dose in NSCLC, albeit for a small proportion of patients. density. Alternatively, the setting may have contributed; in Important lessons have been learnt from the recent negative two out of three trials the patients had been pretreated with trials; these should strongly influence the design of the chemotherapy and thus are more likely to have developed next generation of trials, which will need to be rigorously tumour heterogeneity and/or drug generic resistance evidence based, and highly selective if they are to unlock the mechanisms. However, perhaps the most likely contributory potential of this therapeutic strategy. factor is patient selection. Most trials investigating Met have been non-selective or have included all patients with Acknowledgements Met (over)expression by IHC. Utilising overexpression as a biomarker is however fraught with difficulties. As Disclosure: The authors declare no conflict of interest. described earlier, IHC is subject to considerable variability between users. While it is possible to overcome this with References standardisation, expression is a continuous variable, and thus any cutoff level is to some degree arbitrary and not 1. Black A, Morris D. Personalized medicine in metastatic biologically driven. In the onartuzumab study for example, non-small-cell lung cancer: promising targets and current where selection was performed on the basis of Met clinical trials. Curr Oncol 2012;19:S73-85. expression, half of all patients screened were enrolled on the 2. Bos M, Gardizi M, Schildhaus HU, et al. Activated study, which is far higher a proportion than are likely to be RET and ROS: two new driver mutations in lung Met dependent (87). adenocarcinoma. Transl Lung Cancer Res 2013;2:112-21. Thus far the strongest evidence for a biomarker of 3. Shaw AT, Ou SH, Bang YJ, et al. Crizotinib in ROS1- response is Met amplification, which predicts for increased rearranged non-small-cell lung cancer. N Engl J Med sensitivity to Met inhibition in preclinical work (21,88,89). 2014;371:1963-71. These studies suggest that tumours with Met amplification 4. Cooper CS, Park M, Blair DG, et al. Molecular cloning of display oncogene addiction, and this is supported by the trial a new transforming gene from a chemically transformed

human cell line. Nature 1984;311:29-33. activating ERBB3 signaling. Science 2007;316:1039-43. 5. Birchmeier C, Birchmeier W, Gherardi E, et al. Met, 19. Cui JJ. Targeting receptor tyrosine kinase MET in cancer: metastasis, motility and more. Nat Rev Mol Cell Biol small molecule inhibitors and clinical progress. J Med 2003;4:915-25. Chem 2014;57:4427-53. 6. Schmidt L, Duh FM, Chen F, et al. Germline and somatic 20. Gherardi E, Birchmeier W, Birchmeier C, et al. Targeting mutations in the tyrosine kinase domain of the MET MET in cancer: rationale and progress. Nat Rev Cancer proto-oncogene in papillary renal carcinomas. Nat Genet 2012;12:89-103. 1997;16:68-73. 21. McDermott U, Sharma SV, Dowell L, et al. Identification 7. Olivero M, Rizzo M, Madeddu R, et al. Overexpression of genotype-correlated sensitivity to selective kinase and activation of hepatocyte growth factor/scatter factor inhibitors by using high-throughput tumor cell line in human non-small-cell lung carcinomas. Br J Cancer profiling. Proc Natl Acad Sci U S A 2007;104:19936-41. 1996;74:1862-8. 22. Spigel DR, Edelman MJ, O'Byrne K, et al. Onartuzumab 8. Ma PC, Jagadeeswaran R, Jagadeesh S, et al. Functional plus erlotinib versus erlotinib in previously treated stage expression and mutations of c-Met and its therapeutic IIIb or IV NSCLC: Results from the pivotal phase III inhibition with SU11274 and small interfering RNA in randomized, multicenter, placebo-controlled METLung non-small cell lung cancer. Cancer Res 2005;65:1479-88. (OAM4971g) global trial. J Clin Oncol 2014;abstr 8000. 9. Ma PC, Tretiakova MS, MacKinnon AC, et al. Expression 23. Azuma K, Yoshioka H, Yamamoto N, et al. Tivantinib plus and mutational analysis of MET in human solid cancers. erlotinib versus placebo plus erlotinib in Asian patients Genes Chromosomes Cancer 2008;47:1025-37. with previously treated nonsquamous NSCLC with wild- 10. Ichimura E, Maeshima A, Nakajima T, et al. Expression type EGFR: First report of a phase III ATTENTION of c-met/HGF receptor in human non-small cell lung trial. J Clin Oncol 2014;abstr 8044. carcinomas in vitro and in vivo and its prognostic 24. ESMO News. Available online: http://www.esmo.org/ significance. Jpn J Cancer Res 1996;87:1063-9. Conferences/Past-Conferences/European-Cancer- 11. Nakamura Y, Niki T, Goto A, et al. c-Met activation in Congress-2013/News/A-Phase-III-Study-of-Tivantinib- lung adenocarcinoma tissues: an immunohistochemical Plus-Erlotinib-Did-Not-Meet-a-Primary-Endpoint- analysis. Cancer Sci 2007;98:1006-13. in-Patients-with-Locally-advanced-or-Metastatic-Non- 12. Tsuta K, Kozu Y, Mimae T, et al. c-MET/phospho-MET squamous-NSCLC, accessed on Jan 22, 2015. protein expression and MET gene copy number in non- 25. Camidge DR, Ou SI, Shapiro G, et al. Efficacy and safety small cell lung carcinomas. J Thorac Oncol 2012;7:331-9. of crizotinib in patients with advanced c-MET-amplified 13. Cancer Genome Atlas Research Network. Comprehensive non-small cell lung cancer (NSCLC). J Clin Oncol molecular profiling of lung adenocarcinoma. Nature 2014;abstr 8001. 2014;511:543-50. 26. Koeppen H, Rost S, Yauch RL. Developing biomarkers 14. Bean J, Brennan C, Shih JY, et al. MET amplification to predict benefit from HGF/MET pathway inhibitors. J occurs with or without T790M mutations in EGFR Pathol 2014;232:210-8. mutant lung tumors with acquired resistance to gefitinib 27. Naldini L, Tamagnone L, Vigna E, et al. Extracellular or erlotinib. Proc Natl Acad Sci U S A 2007;104:20932-7. proteolytic cleavage by urokinase is required for activation 15. Go H, Jeon YK, Park HJ, et al. High MET gene copy of hepatocyte growth factor/scatter factor. EMBO J number leads to shorter survival in patients with non-small 1992;11:4825-33. cell lung cancer. J Thorac Oncol 2010;5:305-13. 28. Lokker NA, Mark MR, Luis EA, et al. Structure-function 16. Onozato R, Kosaka T, Kuwano H, et al. Activation analysis of hepatocyte growth factor: identification of of MET by gene amplification or by splice mutations variants that lack mitogenic activity yet retain high affinity deleting the juxtamembrane domain in primary resected receptor binding. EMBO J 1992;11:2503-10. lung cancers. J Thorac Oncol 2009;4:5-11. 29. Niemann HH. Structural insights into Met receptor 17. Seo JS, Ju YS, Lee WC, et al. The transcriptional activation. Eur J Cell Biol 2011;90:972-81. landscape and mutational profile of lung adenocarcinoma. 30. Weidner KM, Di Cesare S, Sachs M, et al. Interaction Genome Res 2012;22:2109-19. between Gab1 and the c-Met receptor tyrosine kinase 18. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET is responsible for epithelial morphogenesis. Nature amplification leads to gefitinib resistance in lung cancer by 1996;384:173-6.

31. Schaeper U, Gehring NH, Fuchs KP, et al. Coupling 45. Cancer Genome Atlas Research Network. Comprehensive of Gab1 to c-Met, Grb2, and Shp2 mediates biological genomic characterization of squamous cell lung cancers. responses. J Cell Biol 2000;149:1419-32. Nature 2012;489:519-25. 32. Rodrigues GA, Falasca M, Zhang Z, et al. A novel positive 46. Gao J, Aksoy BA, Dogrusoz U, et al. Integrative analysis feedback loop mediated by the docking protein Gab1 and of complex cancer genomics and clinical profiles using the phosphatidylinositol 3-kinase in epidermal growth factor cBioPortal. Sci Signal 2013;6:pl1. receptor signaling. Mol Cell Biol 2000;20:1448-59. 47. Turke AB, Zejnullahu K, Wu YL, et al. Preexistence and 33. Gual P, Giordano S, Williams TA, et al. Sustained clonal selection of MET amplification in EGFR mutant recruitment of phospholipase C-gamma to Gab1 is NSCLC. Cancer Cell 2010;17:77-88. required for HGF-induced branching tubulogenesis. 48. Kubo T, Yamamoto H, Lockwood WW, et al. MET gene Oncogene 2000;19:1509-18. amplification or EGFR mutation activate MET in lung 34. Maroun CR, Naujokas MA, Holgado-Madruga M, et al. cancers untreated with EGFR tyrosine kinase inhibitors. The tyrosine phosphatase SHP-2 is required for sustained Int J Cancer 2009;124:1778-84. activation of extracellular signal-regulated kinase and 49. Tanizaki J, Okamoto I, Sakai K, et al. Differential roles of epithelial morphogenesis downstream from the met trans-phosphorylated EGFR, HER2, HER3, and RET as receptor tyrosine kinase. Mol Cell Biol 2000;20:8513-25. heterodimerisation partners of MET in lung cancer with 35. Rosário M, Birchmeier W. How to make tubes: signaling MET amplification. Br J Cancer 2011;105:807-13. by the Met receptor tyrosine kinase. Trends Cell Biol 50. Tanizaki J, Okamoto I, Okamoto K, et al. MET tyrosine 2003;13:328-35. kinase inhibitor crizotinib (PF-02341066) shows 36. Kermorgant S, Parker PJ. Receptor trafficking controls differential antitumor effects in non-small cell lung weak signal delivery: a strategy used by c-Met for STAT3 cancer according to MET alterations. J Thorac Oncol nuclear accumulation. J Cell Biol 2008;182:855-63. 2011;6:1624-31. 37. Lee YY, Kim HP, Kang MJ, et al. Phosphoproteomic 51. Joffre C, Barrow R, Ménard L, et al. A direct role analysis identifies activated MET-axis PI3K/AKT and for Met endocytosis in tumorigenesis. Nat Cell Biol MAPK/ERK in lapatinib-resistant cancer cell line. Exp 2011;13:827-37. Mol Med 2013;45:e64. 52. Tengs T, Lee JC, Paez JG, et al. A transforming MET 38. Clague MJ. Met receptor: a moving target. Sci Signal mutation discovered in non-small cell lung cancer 2011;4:pe40. using microarray-based resequencing. Cancer Lett 39. Hammond DE, Urbé S, Vande Woude GF, et al. Down- 2006;239:227-33. regulation of MET, the receptor for hepatocyte growth 53. Krishnaswamy S, Kanteti R, Duke-Cohan JS, et al. Ethnic factor. Oncogene 2001;20:2761-70. differences and functional analysis of MET mutations in 40. Hammond DE, Carter S, McCullough J, et al. Endosomal lung cancer. Clin Cancer Res 2009;15:5714-23. dynamics of Met determine signaling output. Mol Biol 54. Tyner JW, Fletcher LB, Wang EQ, et al. MET receptor Cell 2003;14:1346-54. sequence variants R970C and T992I lack transforming 41. Abella JV, Peschard P, Naujokas MA, et al. Met/ capacity. Cancer Res 2010;70:6233-7. Hepatocyte growth factor receptor ubiquitination 55. Acunzo M, Romano G, Palmieri D, et al. Cross-talk suppresses transformation and is required for Hrs between MET and EGFR in non-small cell lung cancer phosphorylation. Mol Cell Biol 2005;25:9632-45. involves miR-27a and Sprouty2. Proc Natl Acad Sci U S A 42. Peschard P, Fournier TM, Lamorte L, et al. Mutation of 2013;110:8573-8. the c-Cbl TKB domain binding site on the Met receptor 56. Heegaard NH, Schetter AJ, Welsh JA, et al. Circulating tyrosine kinase converts it into a transforming protein. micro-RNA expression profiles in early stage nonsmall cell Mol Cell 2001;8:995-1004. lung cancer. Int J Cancer 2012;130:1378-86. 43. Barrow R, Joffre C, Ménard L, et al. Measuring the role 57. Luo W, Huang B, Li Z, et al. MicroRNA-449a is for Met endosomal signaling in tumorigenesis. Methods downregulated in non-small cell lung cancer and inhibits Enzymol 2014;535:121-40. migration and invasion by targeting c-Met. PLoS One 44. Kong-Beltran M, Seshagiri S, Zha J, et al. Somatic 2013;8:e64759. mutations lead to an oncogenic deletion of met in lung 58. Lee JM, Yoo JK, Yoo H, et al. The novel miR-7515 cancer. Cancer Res 2006;66:283-9. decreases the proliferation and migration of human

lung cancer cells by targeting c-Met. Mol Cancer Res multiple targets. Mol Vis 2012;18:537-46. 2013;11:43-53. 73. Reid JF, Sokolova V, Zoni E, et al. miRNA profiling in 59. Jiang C, Chen H, Shao L, et al. MicroRNA-1 functions as colorectal cancer highlights miR-1 involvement in MET- a potential tumor suppressor in osteosarcoma by targeting dependent proliferation. Mol Cancer Res 2012;10:504-15. Med1 and Med31. Oncol Rep 2014;32:1249-56. 74. Tsuruta T, Kozaki K, Uesugi A, et al. miR-152 is a 60. Huang J, Dong B, Zhang J, et al. miR-199a-3p inhibits tumor suppressor microRNA that is silenced by DNA hepatocyte growth factor/c-Met signaling in renal cancer hypermethylation in endometrial cancer. Cancer Res carcinoma. Tumour Biol 2014;35:5833-43. 2011;71:6450-62. 61. Takeyama H, Yamamoto H, Yamashita S, et al. Decreased 75. Bouchie A. First microRNA mimic enters clinic. Nat miR-340 expression in bone marrow is associated with Biotechnol 2013;31:577. liver metastasis of colorectal cancer. Mol Cancer Ther 76. Avan A, Quint K, Nicolini F, et al. Enhancement of the 2014;13:976-85. antiproliferative activity of gemcitabine by modulation 62. Menges CW, Kadariya Y, Altomare D, et al. Tumor of c-Met pathway in pancreatic cancer. Curr Pharm Des suppressor alterations cooperate to drive aggressive 2013;19:940-50. mesotheliomas with enriched cancer stem cells via a p53- 77. Yano S, Wang W, Li Q, et al. Hepatocyte growth factor miR-34a-c-Met axis. Cancer Res 2014;74:1261-71. induces gefitinib resistance of lung adenocarcinoma with 63. Yang X, Zhang XF, Lu X, et al. MicroRNA-26a suppresses epidermal growth factor receptor-activating mutations. angiogenesis in human hepatocellular carcinoma by Cancer Res 2008;68:9479-87. targeting hepatocyte growth factor-cMet pathway. 78. Patnaik A, Weiss GJ, Papadopoulos KP, et al. Phase I Hepatology 2014;59:1874-85. ficlatuzumab monotherapy or with erlotinib for refractory 64. Hagman Z, Haflidadottir BS, Ansari M, et al. The tumour advanced solid tumours and multiple myeloma. Br J suppressor miR-34c targets MET in prostate cancer cells. Cancer 2014;111:272-80. Br J Cancer 2013;109:1271-8. 79. Wu Y, Yang JC, Kim D, et al. Safety and efficacy of INC280 65. Xu X, Chen H, Lin Y, et al. MicroRNA-409-3p inhibits in combination with gefitinib (gef) in patients with EGFR- migration and invasion of bladder cancer cells via targeting mutated (mut), MET-positive NSCLC: A single-arm phase c-Met. Mol Cells 2013;36:62-8. lb/ll study. J Clin Oncol 2014; abstr 8017. 66. Hu Z, Lin Y, Chen H, et al. MicroRNA-101 suppresses 80. Mok TSK, Park K, Geater SL, et al. A randomized phase motility of bladder cancer cells by targeting c-Met. (PH) 2 study with exploratory biomarker analysis of Biochem Biophys Res Commun 2013;435:82-7 ficlatuzumab (F) a humanized hepatocyte growth factor 67. Wang LG, Ni Y, Su BH, et al. MicroRNA-34b functions (HGF) inhibitory mAb in combination with gefitinib (G) as a tumor suppressor and acts as a nodal point in the versus G in Asian patients (pts) with lung adenocarcinoma feedback loop with Met. Int J Oncol 2013;42:957-62. (LA). ESMO 2012;Abstract 2342. 68. Luo C, Tetteh PW, Merz PR, et al. miR-137 inhibits 81. Basilico C, Hultberg A, Blanchetot C, et al. Four the invasion of melanoma cells through downregulation individually druggable MET hotspots mediate HGF- of multiple oncogenic target genes. J Invest Dermatol driven tumor progression. J Clin Invest 2014;124:3172-86. 2013;133:768-75. 82. Lee JM, Kim B, Lee SB, et al. Cbl-independent 69. Chen L, Zhang J, Feng Y, et al. MiR-410 regulates MET degradation of Met: ways to avoid agonism of bivalent to influence the proliferation and invasion of glioma. Int J Met-targeting antibody. Oncogene 2014;33:34-43. Biochem Cell Biol 2012;44:1711-7. 83. Spigel DR, Ervin TJ, Ramlau RA, et al. Randomized phase 70. Buurman R, Gürlevik E, Schäffer V, et al. Histone II trial of Onartuzumab in combination with erlotinib in deacetylases activate hepatocyte growth factor signaling patients with advanced non-small-cell lung cancer. J Clin by repressing microRNA-449 in hepatocellular carcinoma Oncol 2013;31:4105-14. cells. Gastroenterology 2012;143:811-20. 84. Chen X, Ding G, Gao Q, et al. A human anti-c-Met 71. Yan K, Gao J, Yang T, et al. MicroRNA-34a inhibits the Fab fragment conjugated with doxorubicin as targeted proliferation and metastasis of osteosarcoma cells both in chemotherapy for hepatocellular carcinoma. PLoS One vitro and in vivo. PLoS One 2012;7:e33778. 2013;8:e63093. 72. Dong F, Lou D. MicroRNA-34b/c suppresses uveal 85. Verma S, Miles D, Gianni L, et al. Trastuzumab emtansine melanoma cell proliferation and migration through for HER2-positive advanced breast cancer. N Engl J Med

2012;367:1783-91. lines harboring MET gene amplification are dependent on 86. Ou SH, Kwak EL, Siwak-Tapp C, et al. Activity of Met for growth and survival. Cancer Res 2007;67:2081-8. crizotinib (PF02341066), a dual mesenchymal-epithelial 89. Matsubara D, Ishikawa S, Oguni S, et al. Molecular transition (MET) and anaplastic lymphoma kinase (ALK) predictors of sensitivity to the MET inhibitor PHA665752 inhibitor, in a non-small cell lung cancer patient with de in lung carcinoma cells. J Thorac Oncol 2010;5:1317-24. novo MET amplification. J Thorac Oncol 2011;6:942-6. 90. Kim ES, Herbst RS, Wistuba II, et al. The BATTLE 87. Garber K. MET inhibitors start on road to recovery. Nat trial: personalizing therapy for lung cancer. Cancer Discov Rev Drug Discov 2014;13:563-5. 2011;1:44-53. 88. Lutterbach B, Zeng Q, Davis LJ, et al. Lung cancer cell

Cite this article as: Sacco JJ, Clague MJ. Dysregulation of the Met pathway in non-small cell lung cancer: implications for drug targeting and resistance. Transl Lung Cancer Res 2015;4(3):242-252. doi: 10.3978/j.issn.2218-6751.2015.03.05