Skip to main content
NCBI home page
Molecular Oncology logoLink to Molecular Oncology
. 2015 Apr 14;9(7):1434–1446. doi: 10.1016/j.molonc.2015.04.001

KRAS and HRAS mutations confer resistance to MET targeting in preclinical models of MET‐expressing tumor cells

Dominic Leiser 1,†, Michaela Medová 1,†, Kei Mikami 1, Lluís Nisa 1, Deborah Stroka 2, Andree Blaukat 3, Friedhelm Bladt 3, Daniel M Aebersold 1, Yitzhak Zimmer 1,✉
PMCID: PMC5528816  PMID: 25933688

Abstract

The MET receptor tyrosine kinase is often deregulated in human cancers and several MET inhibitors are evaluated in clinical trials. Similarly to EGFR, MET signals through the RAS‐RAF‐ERK/MAPK pathway which plays key roles in cell proliferation and survival. Mutations of genes encoding for RAS proteins, particularly in KRAS, are commonly found in various tumors and are associated with constitutive activation of the MAPK pathway. It was shown for EGFR, that KRAS mutations render upstream EGFR inhibition ineffective in EGFR‐positive colorectal cancers. Currently, there are no clinical studies evaluating MET inhibition impairment due to RAS mutations. To test the impact of RAS mutations on MET targeting, we generated tumor cells responsive to the MET inhibitor EMD1214063 that express KRAS G12V, G12D, G13D and HRAS G12V variants. We demonstrate that these MAPK‐activating RAS mutations differentially interfere with MET‐mediated biological effects of MET inhibition. We report increased residual ERK1/2 phosphorylation indicating that the downstream pathway remains active in presence of MET inhibition. Consequently, RAS variants counteracted MET inhibition‐induced morphological changes as well as anti‐proliferative and anchorage‐independent growth effects. The effect of RAS mutants was reversed when MET inhibition was combined with MEK inhibitors AZD6244 and UO126. In an in vivo mouse xenograft model, MET‐driven tumors harboring mutated RAS displayed resistance to MET inhibition. Taken together, our results demonstrate for the first time in details the role of KRAS and HRAS mutations in resistance to MET inhibition and suggest targeting both MET and MEK as an effective strategy when both oncogenic drivers are expressed.

Keywords: MET, RAS, Mutations, Small molecule inhibitors, Resistance

Highlights

  • RAS oncogenic variants counteract MET receptor tyrosine kinase inhibition.

  • MET‐driven tumors harboring mutated RAS display resistance to MET inhibition.

  • MAPK‐activating RAS mutations differentially interfere with MET‐mediated biological effects of MET inhibition.

  • The effects of RAS mutants can be reversed when MET inhibition is combined with MEK inhibitors.

1. Introduction

The RAS‐RAF‐MAPK pathway is a major pathway that plays a key role in cell proliferation and survival and is driven by receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR) (Hynes and MacDonald, 2009; Kiel and Serrano, 2012). Accordingly, EGFR activation recruits RAS isoforms that localize at the inner surface of the plasma membrane. The KRAS, NRAS, and HRAS genes encode the various RAS isoforms and all are highly relevant to human cancer pathogenesis and progression (Chetty and Govender, 2013; Karnoub and Weinberg, 2008; Takashima and Faller, 2013). The RAS family consists of GDP/GTP‐binding proteins that act as intracellular signal transducers. The active GTP‐bound form interacts with a variety of downstream effector proteins (Drosten et al., 2013; Pylayeva‐Gupta et al., 2011). RAS recruitment is followed by direct RAF activation, which triggers a serine/threonine kinase phosphorylation cascade including MAPK kinase and extracellular signal‐regulated kinase (ERK). Phospho‐ERK (pERK) is subsequently translocated into the nucleus, where it activates transcription factors involved in cell proliferation and survival (Drosten et al., 2013).

Importantly, RAS is one of the most frequently activated oncogenes. Approximately 33% of all human tumors harbor an activating RAS gene mutation (Karnoub and Weinberg, 2008). The vast majority (greater than 90%) of oncogenic RAS mutations affect amino acid residues Gly12 or Gly13 located close to the phosphate‐binding loop and less frequent catalytic residue Gln61. These mutations cause RAS to accumulate in the active GTP‐bound state by impairing intrinsic GTPase activity and conferring resistance to GTPase‐activating proteins (Takashima and Faller, 2013). As a result, the constitutively active RAS‐GTP conformation induces and perpetuates stimuli‐independent activation. To date, the number of oncogenic mutations is discrete, there are 12 possible mutations at codons 12 and 13 described thus far. This observation combined with the mutations' inherent stability and detectability make RAS mutations an obvious diagnostic marker (Mattingly, 2013). Somatic KRAS mutations, detected in approximately 20% of all human tumors (Baines et al., 2011), have been shown to strongly impair the effectiveness of targeted anti‐EGFR therapy, particularly in colorectal cancers (Lievre et al., 2008). Consequently, current treatment guidelines (e.g., NCCN (http://www.nccn.org/professionals/physician_gls/pdf/colon.pdf)) require pre‐selection of wild‐type RAS patients prior to deciding on a treatment protocol.

Similar to EGFR, the hepatocyte growth factor (HGF) receptor tyrosine kinase MET is often deregulated in human cancer. This is primarily due to overexpression or MET amplification as well as germline mutations, as observed in hereditary papillary renal carcinoma (HPRC) (Graveel et al., 2013). MET‐expressing cancers are usually associated with poor treatment response and unfavorable prognosis (Ghiso and Giordano, 2013; Graveel et al., 2013). MET has become a primary molecular target in clinical oncology and various MET inhibitors are extensively evaluated in clinical trials (Scagliotti et al., 2013). Similar to EGFR, MET activation stimulates the RAS‐RAF‐MAPK pathway. Therefore, it is reasonable to expect an “EGFR‐like” resistance to MET inhibitors in cancer patients with both aberrant MET expression and mutated KRAS variants. Since currently MET inhibitors are still in clinical trials, a robust correlation between RAS status and patients' responses to MET inhibitor treatment has not been demonstrated yet. Additionally, the RAS oncogene is complex due to the existence of several isoforms with distinct mutations. The impact of the different mutation variants on MET inhibitor response represents an important open question with high clinical relevance.

To mimic potential clinical scenarios, we created in vitro cell systems that co‐express MET and KRAS/HRAS mutant variants. We used cell lines with oncogenic MET activity and introduced various RAS mutations. We compared their responses to MET small molecule inhibitors with MET‐expressing, wild‐type KRAS/HRAS cells. We found that constitutive MAPK pathway activation in RAS mutant cells may differentially circumvent MET inhibition effects on cellular proliferation, motility, and anchorage‐independent growth in vitro and leads to MET inhibitor resistance in an in vivo xenograft tumor model.

2. Materials and methods

2.1. Chemicals and antibodies

EMD1214063 (MSC2156119J) (Merck KGaA, Darmstadt, Germany), AZD6244 (Selumetinib; SELLECK CHEMICALS LLC, Houston), and U0126 (Sigma–Aldrich, St. Louis, MO) were dissolved in DMSO. Antibodies were purchased as follows: isoform non‐specific p21/RAS (pan‐RAS; Cat. no. 3965) and pMET (Cat. no. 3126) antibodies from Cell Signaling Technology (Boston, MA), total MET (Cat. no. sc‐8307) from Santa Cruz Biotechnology (Santa Cruz, CA), and pERK1/2 (Cat. no. 07–467) and β‐Actin (Cat. no. MAB1501) from Millipore (Billerica, MA).

2.2. Plasmids and cell lines

H1993 human lung carcinoma cells were obtained from Dr. Sunny Zachariah (UT Southwestern Medical Center, Dallas, TX) and were grown in RPMI medium (GIBCO, Invitrogen) supplemented with 5% FCS (Sigma) and antibiotic‐antimycotic (penicillin 100 U/ml, streptomycin sulfate 100 U/ml, amphotericin B as Fungizone 0.25 μg/ml; GIBCO). NIH3T3 cell lines that stably express the M1268T MET mutation (the construction of the vector expressing this mutation was previously described and its identity was verified by sequencing of both strands of DNA in the region of interest (Jeffers et al., 1997)) were kindly provided by Dr. Laura Schmidt (National Cancer Institute, Frederick, MD) and maintained in DMEM (GIBCO) supplemented with 10% FCS, antibiotic‐antimycotic, and 0.5 mg/ml Geneticin/G‐418 sulfate (GIBCO).

Using a site‐directed‐mutagenesis kit (Stratagene), according to the manufacturer's instructions, G12D and G13D mutations were introduced into the KRAS cDNA (RASK200000, cdna.org) and the mutations were confirmed by direct sequencing. The KRAS G12V plasmid 12544 (a gift from Channing Der; Khosravi‐Far et al., 1996) and HRAS G12V plasmid 1768 (a gift from Bob Weinberg), cloned into pBABE vector, were obtained from Addgene.

We ectopically expressed the RAS variant proteins in both the NIH3T3 fibroblasts harboring constitutively active MET receptor (M1268T mutation), and the H1993 cell line that overexpresses the MET receptor. The cells were transfected with a pBABE expression vector containing one of the aforementioned RAS variants using Lipofectamine™ 2000 reagent (Invitrogen) and selected with puromycin (Sigma; NIH3T3 – 1.5 ug/ml, H1993–0.5 ug/ml).

2.3. RAS binding domain pull‐down

A GST‐tagged Raf‐1 RAS binding domain containing residues 1‐149 (Raf‐1‐GST RBD 1‐149, plasmid number 13338) was purchased from Addgene (Brtva et al., 1995). The Raf‐1‐GST‐fusion protein was transformed into E. coli. Expression was induced at an OD600nm 0.6 with 0.2 mmol/l IPTG at 30 °C. The induced cells were incubated for 2 h, harvested by centrifugation, and resuspended in lysis buffer. The lysate was centrifuged at 10 000 ×g (4 °C) for 15 min. The supernatant was incubated with rotation for 1 h in the presence of GSH‐Sepharose Beads (GE Healthcare), and then washed. The washed beads were incubated at 4 °C with transfected NIH3T3 or H1993 cell lysates for 2 h with rotation and washed. RAS pull‐down was detected by Western blot.

2.4. Soft agarose growth

Cells were seeded into 6‐well plates. Following 24 h incubation, the cells were treated with MET inhibitors or DMSO (negative control) and incubated for an additional 24 h. Cells were harvested by trypsinization, counted, mixed in medium containing 0.55% Seaplaque agarose, and plated in 6‐well plates containing medium with 1% agarose in triplicate. Control cells were incubated with DMSO and treated cells with MET inhibitor. After a week, the wells were supplemented with additional MET inhibitor or DMSO. Cells were allowed to form colonies for an average period of 18 days. At the end of the experiment, colonies were stained with 0.5 ml of 0.005% crystal violet for approximately 4 h, and the excess solution was removed by aspiration. Colonies were counted with BioRad Quantity One software (Bio‐Rad Laboratories, Inc).

2.5. Western blotting

Cells were lysed, and the protein concentration was determined as described previously (Berthou et al., 2004). Proteins were separated by SDS‐PAGE, transferred onto PVDF membranes, and incubated with the indicated primary antibodies. Horseradish peroxidase‐conjugated secondary antibodies were detected using an ECL kit (Amersham Pharmacia Biotech, Little Chalfont, UK). ECL signals were quantified using Quantity One software (Bio‐Rad Laboratories Inc., Hercules, CA).

2.6. Wound healing assay

Cells were seeded in 6‐well plates at a density of 2.5 × 105 cells/well with appropriate medium containing 10% FBS. The cells were incubated for 24 h, and treated with inhibitor or DMSO. Four hours after adding inhibitor, a portion of the monolayer was removed with a pipette tip attached to a vacuum. Cells were incubated for 20 h with inhibitor or with DMSO, and examined for regrowth of the “wounded” monolayer. For quantification of wound‐healing assays, five images per condition were taken with a Leica DC 300F microscope and processed with the Leica IM‐50 software. The invaded area relative to untreated controls was determined using the “analyze particles” plugin of ImageJ (imajej.nih.gov/ij/).

2.7. Cell proliferation/viability assay

Cell proliferation and viability, with various inhibitor treatments, were determined using a tetrazolium‐based colorimetric assay (XTT Cell Proliferation Kit II, Roche Applied Science) according to the manufacturer's instructions. Briefly, cells were seeded into a 96‐well tissue culture plate, incubated 24 h, treated with the indicated inhibitor concentrations, and incubated at 37 °C in 5% CO2. After 72 h, XTT reagent was added to each well and incubated for 2–4 h at 37 °C. The absorbance was measured at 490 nm, with a 655 nm reference wavelength. Results were normalized to untreated cells, and represent the mean of at least three independent experiments.

2.8. In vivo studies

NIH3T3 cells expressing mutated MET or mutated MET with KRAS G13D (2 × 104 cells) were injected subcutaneously into the right flank of 16 week old female Rag2 common γ‐null (γc−/− RAG2−/−) mice. Tumor growth was regularly monitored by caliper measurements, and the xenograft volume was calculated by multiplying the perpendicular axis. When all the tumors became visible and palpable, mice were randomly divided into treatment (50 mg/kg body weight EMD1214063 per day) or control groups (vehicle only, Solutol HS 15, BASF ChemTrade GmbH). Treatments were administered orally, once a day for the entire trial. The animal experiment was approved by Cantonal Veterinary Authority and in accordance with the Swiss regulations for the care and use of laboratory animals.

2.9. Statistics

Statistical analysis and graphical presentation of the data was performed using Prism Graph (version 5.03). Data for each treatment group were represented as means ± SD or SEM as indicated and compared for significance using the Student t‐test or one‐way ANOVA for multiple variables followed by the Bonferroni post hoc test (multiple comparison tests). Differences with P values <0.05 were considered significant. (*, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001).

3. Results

3.1. Generation of cell lines with oncogenic MET activity and activating RAS mutations

With the ever‐increasing recognition in the importance of targeting aberrant signaling by the MET receptor for cancer therapy and since MET is a major driver of the MAPK pathway of which RAS proteins are key signaling constituents, our studies focused on mimicking potential clinical scenarios, where MET is targeted in the presence of mutated RAS. To establish in vitro models with both aberrant MET signaling and RAS mutations, we used two cellular systems: (1) NIH3T3 cells expressing the activating MET mutation M1268T that is responsive to MET inhibition and (2) H1993 human non‐small cell lung cancer that harbors MET amplification with subsequent overexpression and constitutive receptor activity, which can also be effectively inhibited by small molecule MET inhibitors.

Mutations were introduced into human KRAS and HRAS, and the mutated RAS cDNAs were cloned in a pBABE‐Puro expression vector that confers resistance to puromycin. The presence of KRAS and HRAS mutation in puromycin‐resistant clones was confirmed by direct sequencing. Once transfected into the cell lines, total lysates of cells ectopically expressing the HRAS variant G12V and cells expressing the KRAS variants G12D, G12V, or G13D exhibit considerably higher levels of RAS proteins compared with parental NIH3T3 MET M1268T fibroblasts, as detected by pan‐RAS antibody (Figure 1A). We also used a pull‐down assay consisting of a GST‐tagged RAF binding domain (RBD), which preferentially binds activated RAS proteins. Increased RAS pull‐down demonstrated higher RAS activity in the transfected cells. For H1993 cells, total cell lysates of ectopically expressed RAS showed only a slight increase in RAS protein. The activity‐detecting pull down assay showed that the parental H1993 cells already had high RAS activity when compared with beads alone. This activity increase was maintained by the ectopic expression of the KRAS G13D (Figure 1A).

Figure 1.

Figure 1

Open in a new tab

A. KRAS and HRAS mutant variants levels following stable transfection of NIH3T3 MET M1268T cells (left) and H1993 cells (right). Clones were chosen following puromycin selection. Parental cells are shown for comparison. A RAS binding domain (RBD) pull‐down was used to determine active RAS levels. RAS protein was detected using a non‐isoform specific p21/RAS (pan‐RAS) antibody. β Actin was used as a loading control. B. MET and ERK1/2 phosphorylation levels in NIH3T3 MET M1268T cells (left) and H1993 cells (right) stably transfected with mutated HRAS or KRAS proteins. The cells were treated with either vehicle or 50 nM EMD1214063 for 16 h. β Actin was used as a loading control.

3.2. MAPK pathway is constitutively active in cells harboring activating RAS mutations when inhibiting MET

We hypothesized that MET‐dependent RAS activity could be efficiently inhibited by the MET inhibitor EMD1214063 (Bladt et al., 2013; Medova et al., 2013), whereas MET‐independent RAS activity resulting from oncogenic RAS activation would not be affected. To examine the impact of MET inhibition on the MAPK pathway, we observed the phosphorylation of ERK1/2 (pERK1/2), which is downstream of RAS in the MAPK pathway, in EMD1214063‐treated cells. EMD1214063 strongly reduced pERK1/2 in parental NIH3T3 MET M1268T and H1993 cells (Figure 1B) but cells expressing KRAS G12D, KRAS G12V, KRAS G13D, or HRAS G12V displayed a higher residual MAPK activity following MET inhibition compared with the parental cells as evaluated by pERK1/2 levels.

Increased residual ERK1/2 activity in M1268T ectopically expressing KRAS as well as HRAS variants was also detected when the corresponding lines have been exposed to another MET inhibitor, PHA665752 (Supplementary Figure 1).

3.3. Impact of combined MET and MEK1/2 inhibition on proliferation of cells expressing mutated RAS variants

Next, we evaluated whether the biochemical findings were associated with phenotypic changes, further supporting the idea of mutated RAS‐dependent resistance to MET inhibitors. The MAPK pathway, of which RAS is a major effector, is tightly associated with cell proliferation. Interestingly, cells that ectopically express KRAS or HRAS mutants in addition to the active MET oncogene exhibited overall a reduced proliferation capacity (Supplementary Figure 2). Subsequently, we investigated the impact of MET inhibition on RAS variants newly generated cell lines. Parental NIH3T3 MET M1268T cells were highly sensitive to MET inhibition with a near total cell proliferation inhibition after 72 h incubation with 25 or 50 nM EMD1214063 (Figure 2A). On the other hand, cells that ectopically express the KRAS or HRAS mutations displayed compromised responses. To overcome the mutated RAS‐related resistance to EMD1214063, we used the MAPK pathway inhibitor AZD6244, which targets the MEK1/2 kinases located downstream of RAF. The impact of AZD6244 on pERK1/2 in parental as well as RAS‐mutated MET NIH3T3 and H1993 cells is provided in Suppl. Figure 3. AZD6244 alone, at either 0.5 or 1 μM (1 μM being the highest concentration to consider cells still sensitive (Troiani et al., 2012)) had minimal impact on parental NIH3T3 MET M1268T proliferation and, interestingly, displayed statistically significant (except for KRAS G13D), but smaller effects than EMD1214063 alone (except for HRAS G12V) on the various RAS‐mutated cell lines. However, AZD6244 combined with EMD1214063 in varying doses considerably restored cell proliferation inhibition in RAS‐mutated cells. Similar results were observed for the H1993 human lung cancer line: the KRAS G13D mutant showed complete resistance to EMD1214063 treatment alone (Figure 2B) while sensitivity to the MET inhibitor could be partially restored with AZD6244 treatment.

Figure 2.

Figure 2

Open in a new tab

A. XTT cell proliferation assays for parental NIH3T3 MET M1268T as well as cells stably expressing KRAS or HRAS mutants. Cells were treated with vehicle (untreated) or exposed to the MET inhibitor EMD1214063 (25 nM or 50 nM; E1 and E2, respectively), the MEK1/2 inhibitor AZD6244 (0.5 μM or 1 μM; A1 and A2, respectively), or to a combination of both inhibitors (E1/A1, E1/A2, E2/A1, E2/A2) for 72 h. B. XTT proliferation assay comparing the impact of EMD1214063 and the EMD1214063/AZD6244 combination on H1993 and H1993 KRAS G13D cells. Concentrations and protocols were the same as in A. C. Proliferation of parental NIH3T3 MET M1268T and NIH3T3 MET M1268T cells stably expressing KRAS G12D or HRAS G12V variants. Cells were treated with vehicle (untreated) or were exposed to 50 nM MET inhibitor EMD1214063, the MEK1/2 inhibitor UO126 (20 μM or 40 μM; U1 and U2, respectively), or to the combination of both for 72 h. The data represent mean ± standard deviation of three experiments. Statistical significance of the group means was measured by a one‐way ANOVA followed by Bonferroni's Multiple Comparison Test post‐hoc analysis. Overall P‐value is shown above the graph. The star (*) indicates statistical significance compared with untreated samples. The number of * indicates P‐values as described in the Materials and methods.

To confirm the results and evaluate the minimal effect of AZD6244 treatment alone on cellular proliferation (Figure 2A and 2B), we tested another MEK1/2 inhibitor, UO126 (Wauson et al., 2013). This inhibitor weakly decreased parental NIH3T3 MET M1268T cell proliferation, as well as the RAS mutants KRAS G12D and HRAS G12V in a dose‐dependent manner (Figure 2C). These data confirmed that the MET inhibitor, EMD1214063, in combination with a MEK1/2 inhibitor, could overcome RAS mutant‐mediated MET inhibitor resistance.

3.4. Distinct RAS mutations have different effects on changes in MET inhibition‐associated cellular morphology and motility

MET targeting has been shown to induce morphological changes in various cell lines with a particularly inhibitory effect on cellular motility (Crosswell et al., 2009; Harshman and Choueiri, 2013; Medova et al., 2013; Zimmer et al., 2010). Therefore, we compared EMD1214063's impact on the morphology of parental NIH3T3 MET M1268T cells versus NIH3T3 MET M1268T cells expressing RAS mutants. Parental NIH3T3 MET M1268T cells clearly showed dramatic flattening after 24 h of treatment by 50 nM EMD1214063 (Figure 3). Interestingly, cells expressing the various RAS mutations responded differently to the MET inhibitor. Whereas KRAS G12D cells showed changes similar to the parental line, cells expressing KRAS G12V displayed only moderate flattening, and KRAS G13D and HRAS G12V cells exhibited significant resistance to EMD1214063‐associated morphological changes. To reverse potential resistance to the MET inhibitor, we again included the MEK1/2 inhibitor AZD6244 (500 nM). Administration of the MEK1/2 inhibitor contributed to EMD1214063 sensitivity restoration in the EMD1214063‐resistant cells (Figure 3).

Figure 3.

Figure 3

Open in a new tab

Impact of MET (EMD1214063) and MEK1/2 (AZD6244) inhibitors and their combination on morphology of parental and RAS‐mutated NIH3T3 MET M1268T cells. T0 – time 0 h (the time point immediately prior to the start of treatment), T24 – time 24 h (24 h following the start of the treatment). Representative pictures are shown; on the right side, magnified images of red squares are provided.

Similar results were obtained when studying the impact of RAS mutant expression on MET inhibitor interference of MET‐dependent cellular motility using a wound healing assay. EMD1214063 (50 nM) demonstrated a nearly 90% inhibitory effect on the motility of parental NIH3T3 MET M1268T cells following 24 h incubation (Figure 4). Contrary, the presence of the RAS mutants compromised cellular motility inhibition to various extents, ranging between 23 and 67% of inhibition for HRAS G12V and KRAS G12V mutants, respectively. Consistent with previous results, the combination of EMD1214063 with AZD6244 was highly effective at restoring cellular motility inhibition (Figure 4).

Figure 4.

Figure 4

Open in a new tab

Impact of MET (EMD1214063) and MEK1/2 (AZD6244) inhibitors and their combination on MET‐driven cellular motility of parental and RAS‐mutated NIH3T3 MET M1268T cells using a wound healing assay. T0 – time 0 h (the time point immediately prior to the start of treatment), T24 – time 24 h (24 h following the start of the treatment). Representative pictures (top) and quantification of invaded area of 5 experiments (bottom) are shown.

3.5. Impact of MET and/or MEK1/2 inhibition on the colony‐formation capacity of RAS mutant cells

Anchorage‐independent growth is a known characteristic of many tumorigenically transformed cells and is usually demonstrated by their ability to form colonies in soft agar or agarose. As growth in soft agar has already been documented for cells with aberrant MET activity (Christensen et al., 2003; Zimmer et al., 2010), we tested EMD1214063's potential for inhibiting it in the presence or absence of RAS mutations. As expected, EMD1214063 very effectively restricted soft agar colony growth in parental H1993 and NIH3T3 MET M1268T cells (Figure 5). The anchorage‐independent growth in the presence of MET inhibitor was partially restored in H1993 KRAS G13D cells (60% inhibition of colony formation compared with 100% in H1993 parental cells). In the NIH3T3 MET M1268T cells, the KRAS mutations provided varying advantages for growth in soft agar. In this respect, the greatest effect was observed in NIH3T3 MET M1268T HRAS G12V cells, with 72% of untreated colony formation in the presence of EMD1214063. In the NIH3T3 MET M1268T cells with KRAS mutations, EMD1214063 induced anchorage‐independent growth inhibition similar to wild‐type RAS.

Figure 5.

Figure 5

Open in a new tab

Soft agarose colony‐formation capacity of parental and RAS‐mutated NIH3T3 MET M1268T and H1993 cell lines upon MET (EMD1214063) and/or MEK1/2 (AZD6244) inhibition. A. Representative pictures of colonies formed by parental and HRAS G12V NIH3T3 MET M1268T control cells (top) and cells treated with 50 nM MET inhibitor EMD1214063 (bottom). B. Impact of MET and MEK1/2 inhibitors on relative colony count in parental and RAS‐mutated H1993 cells. C. Impact of MET and MEK1/2 inhibitors on relative colony count in parental and RAS‐mutated NIH3T3 MET M1268T cells. The data represent mean ± standard deviation of three experiments. Statistical significance of the group means was measured by one‐way ANOVA followed by Bonferroni's Multiple Comparison Test post‐hoc analysis. Overall P‐value is shown above the graph. Star (*) indicates statistical significance compared with untreated. The number of stars (*) indicates P‐values as described in the Materials and Methods.

3.6. The presence of RAS mutations restricts the impact of MET inhibition on tumor growth in an in vivo model

Finally, we investigated the relevance of these findings on tumor growth in vivo, by comparing EMD1214063's ability to control MET‐driven tumor growth in cells with and without RAS mutant coexpression. To that end, we injected immunodeficient γc−/− RAG2−/− mice with either parental NIH3T3 MET M1268T or NIH3T3 MET M1268T KRAS G13D cells. Tumors were allowed to develop until all the mice had palpable nodules, approximately 0.1 cm3 in volume (day 10 post‐injection). The animals in each group were randomly divided into two subgroups with daily oral administration of either EMD1214063 (25 mg/kg body weight/day) or vehicle alone. Tumor growth was monitored daily with caliper measurements (Figure 6A). Consistent with the in vitro proliferation rate of these cell lines, tumors derived from cells expressing mutated RAS showed a three days growth delay to reach the same size compared with M1268T RAS wild‐type tumors. Figure 6B shows tumor sizes at day 15 (the last day with complete experimental groups (animals with tumor volumes that exceeded the approved protocol limit had to be euthanized at this time point)). EMD1214063 significantly reduced NIH3T3 MET M1268T‐derived tumors, whereas a much smaller, not statistically significant, reduction was observed with NIH3T3 MET M1268T cells expressing constitutively active KRAS G13D. Over the course of the experiment (22 days), faster growth was observed in MET inhibitor‐treated animals with tumors coexpressing mutated RAS compared with RAS wild‐type.

Figure 6.

Figure 6

Open in a new tab

Impact of EMD1214063 on tumor growth in parental versus KRAS G13D NIH3T3 MET M1268T cells in an in vivo mouse model. A. Growth of parental and RAS‐mutated NIH3T3 MET M1268T‐derived tumors monitored daily by caliper measurement. EMD1214063 treatment was initiated on day 10 and continued daily. The data represent mean tumor volume ± SEM with 6 animals per group. B. Relative tumor sizes at day 15 (5 days after MET inhibitor treatment initiation). Comparison with vehicle‐treated controls was done using Student's t‐test.

4. Discussion

While KRAS mutations are characterized as an established resistance mechanism to anti‐EGFR treatment in the management of colorectal cancer, their role is still debated in other common malignant disorders, for example lung cancer (Amado et al., 2008; Siena et al., 2009). Equally, little is known about their effect on targeting other highly relevant RTK systems including MET. With the exception of the rather non‐specific inhibitor cabozantinib, which was recently approved for the treatment of advanced medullary thyroid cancer (Yavuz et al., 2014), other potential MET inhibitors are currently still under evaluation in clinical trials. Consequently, the first hints of RAS mutations playing a role in MET‐driven tumors are beginning to develop. One conceptually interesting example regarding the role of aberrant RAS in that context was the Phase 3 MARQUEE trial (NCT01244191) (Scagliotti et al., 2012). This study used the inhibitor tivantinib/ARQ 197, whose MET‐dependent mode of action is currently still under debate (Basilico et al., 2013; Katayama et al., 2013; Michieli and Di Nicolantonio, 2013; Zhou et al., 2014). This trial assessed KRAS prior to randomization, and the patients were stratified according to tumor EGFR and KRAS mutation status. This study was discontinued early after a planned interim analysis, indicating that the trial would not meet its primary endpoint of improved overall survival (Scagliotti et al., 2013). However, it confirms the feasibility of preliminary RAS mutation testing also for the indications for anti‐MET treatment, as it is part of erbitux indications in colorectal cancer. Further clinical studies involving potent MET kinase inhibitors are ongoing but the impact of RAS mutations on the effectiveness of anti‐MET strategies needs further detailed explorations. To that end and with the EGFR‐mutated KRAS precedence as a background, in the current study we have created cellular systems that co‐express several clinically relevant KRAS and HRAS mutated variants along with constitutive MET signaling for investigating MET targeting using in vitro and in vivo models.

With respect to the relevance of aberrant RAS expression to intervention with MET signaling, Cepero et al. used MET‐addicted cell lines that were treated with increasing concentrations of the MET inhibitors PHA665752 and JNJ38877605, obtaining eventually clones, which stopped displaying MET dependence with consequent non‐responsiveness to MET inhibition (Cepero et al., 2010). The corresponding clones demonstrated concomitant declined MET expression and increased wild‐type KRAS expression associated with KRAS amplification. The essential role of KRAS as the ‘novel’ oncogene driver in this system was confirmed by a decrease in cellular transformation features following introduction of KRAS siRNA (Cepero et al., 2010). Along this line, Long et al. reported in 2003 that constitutively active MET in colon cancer cells increased tumorigenicity in SCID mice and that expression of dominant‐negative MET suppressed tumorigenicity only when mutated RAS was inactivated (Long et al., 2003).

Despite some early reports describing amplification of KRAS (Heighway and Hasleton, 1986; Taya et al., 1984) or NRAS (Graham et al., 1985) in some tumors and cell lines, the current bulk of experimental data supports that RAS amplification is not a common phenomenon in human cancer. Contrarily, mutated variants represent the predominant clinically‐relevant oncogenic mechanism. Additionally, and in contrast to the aforementioned studies that mimic a scenario in which tumors may acquire RAS activation during treatment due to secondary events, as KRAS amplification, primary RAS mutations might well be present already at the start of the therapy, posing resistance for targeting of an upstream RTK driver system. Therefore, a major goal of the current study was indeed in mimicking a situation that could be used as a preface to a clinical scenario in which primary mutations in RAS genes present along with MET as key tumor drivers. We simulated this scenario by co‐expressing oncogenic RAS as well as MET and subsequently exposing these cells to MET inhibition. We hypothesized that in the presence of MET inhibition RAS mutation‐bearing tumor cells have a clear growth advantage over wild‐type RAS cells and are able to grow in an unimpeded manner.

Overall, our data indicated that the introduction of mutated RAS variants into cells with MET oncogenic activity is not well supported (data not shown). This observation might be attributed to an intolerable oncogenic shock following forced co‐expression of two strong oncogenes (Sharma et al., 2006). NIH3T3 MET‐mutated fibroblasts possessing MET‐oncogenic activity due to an activating tyrosine kinase domain mutation, M1268T, tolerated mutated RAS variants, and therefore became our major model. The experimental difficulties in developing a cancer cell line expressing two independent, strong oncogenes (e.g., RAS and MET) may account for why, to our knowledge, this is the first study to successfully establish this dual‐oncogene model system.

Using our model, we showed that KRAS exon 2 mutations did indeed confer resistance to two different MET inhibitors that were used in this work. Additionally, a similar pattern was seen for cells ectopically expressing the HRAS G12V mutant. This finding is consistent with clinical data published by Douillard et al., who demonstrated that mutations, other than those in KRAS exon 2, conferred resistance to EGFR inhibitors, and that the data could be used to improve the benefit–risk profile of panitumab‐FOLFOX4 treatment in metastatic colorectal cancer (Douillard et al., 2013).

Interestingly, we observed that the introduction of different RAS proteins resulted in different biochemical or phenotypical effects (e.g. proliferation assay, would healing assay, clonogenic assay, or in vivo mouse models). Due to the generation process of these cell lines, exact equal levels of expression could not be reached. Therefore, the aforementioned observed differences might at least partially correlate with expression levels. On the other hand, they are also consistent with the published phenotypes of the respective RAS mutations. In that respect, Seeburg et al. suggested already nearly 30 years ago, that activity of different RAS mutations may result in different biological phenotypes, with the HRAS G12V mutation producing the strongest growth in soft agar (Seeburg et al., 1984). Our current data support this early report, as can be seen with NIH3T3 MET M1268T cells expressing HRAS G12V in the soft agarose assay (Figure 5). This observation suggests the need not only for RAS mutation screening, but also a biological classification of the effect of the various RAS variants and their potential interaction with the upstream major target (e.g., EGFR, MET). Based on our data, MET tyrosine kinase inhibitor resistance could be predicted, leading to more effective patient care. In this respect, studies involving multikinase small molecule inhibitors may serve as good examples. For sunitinib treatment, distinct resistance patterns were reported for different KRAS mutations in SW48 colorectal cancer, with the lowest chemosensitivity seen for the G13D mutation (Modest et al., 2013). A different sensitivity pattern was detected for ERK phosphorylation as well (Modest et al., 2013). We also observed a broad range of phenotypic divergence between the different mutant cell lines, indicating that it might be important to stratify the clinical response by the type of mutation rather than in a general mutated/non‐mutated manner. However, confirming this hypothesis in a setup of clinical trials would require large sample sizes. Even in our study, the lack of sufficient unique cell lines to evaluate the mutations has to be seen as a limitation, hampering clear statements about the complex effects of the different RAS mutations.

Our data suggest that the combination of MET inhibitors along with MAPK targeting (e.g., MEK inhibitors) may be beneficial for circumventing mutated RAS‐dependent resistance. Interestingly, in most of the assays there was minimal to no‐effect by using the MEK inhibitor alone. This demonstrates that inhibiting the RAS‐RAF‐MAPK pathway is insufficient in the presence of activated MET, as other MET signaling pathways can transmit stimuli leading to malignant transformation. In presence of both MET and RAS mutations, a combination of both inhibitors could potentially act synergistically in future real life settings, assuming that MEK inhibitors demonstrate clinical applicability.

Using the in vivo model, tumor cells expressing mutated RAS showed a growth delay compared to RAS wild‐type tumors. This slower growth rate might be related to a toxicity resulting from co‐expression of two potent oncogenes as afore discussed. This might also explain why these two mutations do not appear often in deep sequencing studies as for example reported for lung adenocarcinoma where MET activation and RAS mutation occurred at the same time only in 2 out of 183 patients (Onitsuka et al., 2010; Onozato et al., 2009; Xia et al., 2013). It is interesting to note that in our in vivo experiment tumors containing mutated RAS showed more signs of skin invasion (redness, local ulceration and bleeding, local pain) than tumors harboring wild‐type RAS (data not shown). Alternatively, this potentially also corresponds to the hypothesis that RAS mutants are more invasive then proliferative (see also Supplementary Figure 2. and Figure 4.).

Taken together, the results of the current study further clarify the role of RAS mutations on potential resistance to anti‐MET treatment. We confirmed this result in different cellular systems and with multiple assays. These results suggest that the presence of oncogenic RAS mutations may lead to MET targeting resistance.

In our cellular models, virtually any KRAS mutation has proven the ability to confer at least partial resistance to anti‐MET treatment. Importantly, we provide evidence that also an HRAS mutation can pose resistance to inhibition of MET. It is possible that such a RAS‐MET liaison is relevant only for a subset of specific tumors, similar to activated KRAS bypassing EGFR inhibition in colorectal cancer. Therefore, our data suggest the implementation of a RAS mutational status test in clinical studies in different indications involving novel MET inhibitors, as it was performed in the MARQUEE trial. In the case of MET inhibitors and similarly to administration of EGFR inhibitors for colorectal cancer, testing RAS status prior to treatment may be well required to avoid treating patients that might not experience any clinical benefit from MET targeting. Finally, it is important to note that apart the RAS mutated variants investigated in the current study, other RAS isoforms as well as BRAF mutations should be analyzed for possible deregulation as a potential explanation for non‐responsiveness of MET‐driven tumors to novel anti‐MET therapeutic approaches. Similarly and with the increasing prevalence of PI3K mutations in human cancer (Samuels and Waldman, 2010), the relevance of these genetic lesions for interference to MET targeting is indeed an emerging topic.

Authors' contributions

Conception and design: M. Medová, D. M. Aebersold, Y. Zimmer.

Development of methodology: D. Leiser, M. Medová, K. Mikami.

Acquisition of data: D. Leiser, M. Medová, K. Mikami, D. Stroka, L. Nisa.

Interpretation of data: D. Leiser, M. Medová, K. Mikami, D. M. Aebersold, Y. Zimmer, L. Nisa.

Writing, review, and/or revision of the manuscript: D. Leiser, M. Medová, D. M. Aebersold, Y. Zimmer, A. Blaukat, F. Bladt, D. Stroka.

Administrative, technical, or material support: A. Blaukat, F. Bladt, D. Stroka.

Study supervision: M. Medová, Y. Zimmer, D. M. Aebersold.

Financial support

Bernische Krebsliga, Switzerland (grant to Y. Zimmer).

Conflict of interest

A. Blaukat and F. Bladt are employees of Merck KGaA. There are no other conflicts of interest to disclose.

Supporting information

The following are the supplementary data related to this article:

Supplementary materials and methods: PHA665752, (3Z)‐5‐[(2,6‐dichlorobenzyl)sulfonyl]‐3‐[(3,5‐dimethyl‐4‐{[(2R)‐2‐(pyrrolidin‐1‐ylmethyl)pyrrolidin‐1‐yl]carbonyl}‐1H‐pyrrol‐2‐yl)methylene]‐1,3‐dihydro‐2H‐indol‐2‐one (Pfizer, La Jolla, CA) was dissolved in DMSO.

Supplementary Figure 1 MET and ERK1/2 phosphorylation levels in NIH3T3 MET M1268T cells stably transfected with mutated HRAS or KRAS variants as indicated. The cells were treated with either vehicle or 300 nM PHA665752 for 16 h β‐Actin was used as a loading control.

Click here for additional data file. (22.5KB, jpg)

Supplementary Figure 2 Proliferative capacity of parental NIH3T3 MET M1268T as well as NIH3T3 MET M1268T cells stably expressing indicated KRAS or HRAS mutants as measured by XTT cell proliferation assay.

Click here for additional data file. (58.5KB, jpg)

Supplementary Figure 3 MET and ERK1/2 phosphorylation levels in NIH3T3 MET M1268T and H1993 cells stably transfected with mutated HRAS or KRAS variants as indicated. The cells were treated with either vehicle or 500 nM AZD6244 for 16 h β‐Actin was used as a loading control.

Click here for additional data file. (56.3KB, jpg)

Acknowledgments

We cordially thank Dr. W. Blank‐Liss and Mr. B. Streit for their excellent technical support.

Supplementary data 1.

1.1.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2015.04.001.

Leiser Dominic, Medová Michaela, Mikami Kei, Nisa Lluís, Stroka Deborah, Blaukat Andree, Bladt Friedhelm, Aebersold Daniel M., Zimmer Yitzhak, (2015), KRAS and HRAS mutations confer resistance to MET targeting in preclinical models of MET-expressing tumor cells, Molecular Oncology, 9, doi: 10.1016/j.molonc.2015.04.001.

References

  1. Amado, R.G. , Wolf, M. , Peeters, M. , Van Cutsem, E. , Siena, S. , Freeman, D.J. , Juan, T. , Sikorski, R. , Suggs, S. , Radinsky, R. , Patterson, S.D. , Chang, D.D. , 2008. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J. Clin. Oncol. 26, 1626–1634. [DOI] [PubMed] [Google Scholar]
  2. Baines, A.T. , Xu, D. , Der, C.J. , 2011. Inhibition of Ras for cancer treatment: the search continues. Future Med. Chem. 3, 1787–1808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Basilico, C. , Pennacchietti, S. , Vigna, E. , Chiriaco, C. , Arena, S. , Bardelli, A. , Valdembri, D. , Serini, G. , Michieli, P. , 2013. Tivantinib (ARQ197) displays cytotoxic activity that is independent of its ability to bind MET. Clin. Cancer Res. 19, 2381–2392. [DOI] [PubMed] [Google Scholar]
  4. Berthou, S. , Aebersold, D.M. , Schmidt, L.S. , Stroka, D. , Heigl, C. , Streit, B. , Stalder, D. , Gruber, G. , Liang, C. , Howlett, A.R. , Candinas, D. , Greiner, R.H. , Lipson, K.E. , Zimmer, Y. , 2004. The Met kinase inhibitor SU11274 exhibits a selective inhibition pattern toward different receptor mutated variants. Oncogene 23, 5387–5393. [DOI] [PubMed] [Google Scholar]
  5. Bladt, F. , Faden, B. , Friese-Hamim, M. , Knuehl, C. , Wilm, C. , Fittschen, C. , Gradler, U. , Meyring, M. , Dorsch, D. , Jaehrling, F. , Pehl, U. , Stieber, F. , Schadt, O. , Blaukat, A. , 2013. EMD 1214063 and EMD 1204831 constitute a new class of potent and highly selective c-Met inhibitors. Clin. Cancer Res. 19, 2941–2951. [DOI] [PubMed] [Google Scholar]
  6. Brtva, T.R. , Drugan, J.K. , Ghosh, S. , Terrell, R.S. , Campbell-Burk, S. , Bell, R.M. , Der, C.J. , 1995. Two distinct Raf domains mediate interaction with Ras. J. Biol. Chem. 270, 9809–9812. [DOI] [PubMed] [Google Scholar]
  7. Cepero, V. , Sierra, J.R. , Corso, S. , Ghiso, E. , Casorzo, L. , Perera, T. , Comoglio, P.M. , Giordano, S. , 2010. MET and KRAS gene amplification mediates acquired resistance to MET tyrosine kinase inhibitors. Cancer Res. 70, 7580–7590. [DOI] [PubMed] [Google Scholar]
  8. Chetty, R. , Govender, D. , 2013. Gene of the month: KRAS. J. Clin. Pathol. 66, 548–550. [DOI] [PubMed] [Google Scholar]
  9. Christensen, J.G. , Schreck, R. , Burrows, J. , Kuruganti, P. , Chan, E. , Le, P. , Chen, J. , Wang, X. , Ruslim, L. , Blake, R. , Lipson, K.E. , Ramphal, J. , Do, S. , Cui, J.J. , Cherrington, J.M. , Mendel, D.B. , 2003. A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo. Cancer Res. 63, 7345–7355. [PubMed] [Google Scholar]
  10. Crosswell, H.E. , Dasgupta, A. , Alvarado, C.S. , Watt, T. , Christensen, J.G. , De, P. , Durden, D.L. , Findley, H.W. , 2009. PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells. BMC Cancer 9, 411 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Douillard, J.Y. , Rong, A. , Sidhu, R. , 2013. RAS mutations in colorectal cancer. N. Engl. J. Med. 369, 2159–2160. [DOI] [PubMed] [Google Scholar]
  12. Drosten, M. , Lechuga, C.G. , Barbacid, M. , 2013. Genetic analysis of Ras genes in epidermal development and tumorigenesis. Small GTPases 4, [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ghiso, E. , Giordano, S. , 2013. Targeting MET: why, where and how?. Curr. Opin. Pharmacol. 13, 511–518. [DOI] [PubMed] [Google Scholar]
  14. Graham, K.A. , Richardson, C.L. , Minden, M.D. , Trent, J.M. , Buick, R.N. , 1985. Varying degrees of amplification of the N-ras oncogene in the human breast cancer cell line MCF-7. Cancer Res. 45, 2201–2205. [PubMed] [Google Scholar]
  15. Graveel, C.R. , Tolbert, D. , Vande Woude, G.F. , 2013. MET: a critical player in tumorigenesis and therapeutic target. Cold Spring Harbor Perspect. Biol. 5, [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harshman, L.C. , Choueiri, T.K. , 2013. Targeting the hepatocyte growth factor/c-Met signaling pathway in renal cell carcinoma. Cancer J. 19, 316–323. [DOI] [PubMed] [Google Scholar]
  17. Heighway, J. , Hasleton, P.S. , 1986. c-Ki-ras amplification in human lung cancer. Br. J. Cancer 53, 285–287. http://www.nccn.org/professionals/physician_gls/pdf/colon.pdf [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hynes, N.E. , MacDonald, G. , 2009. ErbB receptors and signaling pathways in cancer. Curr. Opin. Cel Biol 21, 177–184. [DOI] [PubMed] [Google Scholar]
  19. Jeffers, M. , Schmidt, L. , Nakaigawa, N. , Webb, C.P. , Weirich, G. , Kishida, T. , Zbar, B. , Vande Woude, G.F. , 1997. Activating mutations for the met tyrosine kinase receptor in human cancer. Proc. Natl. Acad. Sci. United States America 94, 11445–11450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karnoub, A.E. , Weinberg, R.A. , 2008. Ras oncogenes: split personalities. Nature reviews. Mol. Cel. Biol. 9, 517–531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Katayama, R. , Aoyama, A. , Yamori, T. , Qi, J. , Oh-hara, T. , Song, Y. , Engelman, J.A. , Fujita, N. , 2013. Cytotoxic activity of tivantinib (ARQ 197) is not due solely to c-MET inhibition. Cancer Res. 73, 3087–3096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Khosravi-Far, R. , White, M.A. , Westwick, J.K. , Solski, P.A. , Chrzanowska-Wodnicka, M. , Van Aelst, L. , Wigler, M.H. , Der, C.J. , 1996. Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation. Mol. Cell. Biol. 16, 3923–3933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kiel, C. , Serrano, L. , 2012. Challenges ahead in signal transduction: MAPK as an example. Curr. Opin. Biotechnol. 23, 305–314. [DOI] [PubMed] [Google Scholar]
  24. Lievre, A. , Bachet, J.B. , Boige, V. , Cayre, A. , Le Corre, D. , Buc, E. , Ychou, M. , Bouche, O. , Landi, B. , Louvet, C. , Andre, T. , Bibeau, F. , Diebold, M.D. , Rougier, P. , Ducreux, M. , Tomasic, G. , Emile, J.F. , Penault-Llorca, F. , Laurent-Puig, P. , 2008. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J. Clin. Oncol. 26, 374–379. [DOI] [PubMed] [Google Scholar]
  25. Long, I.S. , Han, K. , Li, M. , Shirasawa, S. , Sasazuki, T. , Johnston, M. , Tsao, M.S. , 2003. Met receptor overexpression and oncogenic Ki-ras mutation cooperate to enhance tumorigenicity of colon cancer cells in vivo. Mol. Cancer Res. 1, 393–401. [PubMed] [Google Scholar]
  26. Mattingly, R.R. , 2013. Activated ras as a therapeutic target: constraints on directly targeting ras isoforms and wild-type versus mutated proteins. ISRN Oncol. 2013, 536529 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Medova, M. , Pochon, B. , Streit, B. , Blank-Liss, W. , Francica, P. , Stroka, D. , Keogh, A. , Aebersold, D.M. , Blaukat, A. , Bladt, F. , Zimmer, Y. , 2013. The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants. Mol. Cancer Ther. 12, 2415–2424. [DOI] [PubMed] [Google Scholar]
  28. Michieli, P. , Di Nicolantonio, F. , 2013. Targeted therapies: tivantinib–a cytotoxic drug in MET inhibitor's clothes? Nature reviews. Clin. Oncol. 10, 372–374. [DOI] [PubMed] [Google Scholar]
  29. Modest, D.P. , Camaj, P. , Heinemann, V. , Schwarz, B. , Jung, A. , Laubender, R.P. , Gamba, S. , Haertl, C. , Stintzing, S. , Primo, S. , Bruns, C.J. , 2013. KRAS allel-specific activity of sunitinib in an isogenic disease model of colorectal cancer. J. Cancer Res. Clin. Oncol. 139, 953–961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Onitsuka, T. , Uramoto, H. , Ono, K. , Takenoyama, M. , Hanagiri, T. , Oyama, T. , Izumi, H. , Kohno, K. , Yasumoto, K. , 2010. Comprehensive molecular analyses of lung adenocarcinoma with regard to the epidermal growth factor receptor, K-ras, MET, and hepatocyte growth factor status. J. Thorac. Oncol. 5, 591–596. [DOI] [PubMed] [Google Scholar]
  31. Onozato, R. , Kosaka, T. , Kuwano, H. , Sekido, Y. , Yatabe, Y. , Mitsudomi, T. , 2009. Activation of MET by gene amplification or by splice mutations deleting the juxtamembrane domain in primary resected lung cancers. J. Thorac. Oncol. 4, 5–11. [DOI] [PubMed] [Google Scholar]
  32. Pylayeva-Gupta, Y. , Grabocka, E. , Bar-Sagi, D. , 2011. RAS oncogenes: weaving a tumorigenic web. Nature reviews. Cancer 11, 761–774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Samuels, Y. , Waldman, T. , 2010. Oncogenic mutations of PIK3CA in human cancers. Curr. Top. Microbiol. Immunol. 347, 21–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scagliotti, G.V. , Novello, S. , Schiller, J.H. , Hirsh, V. , Sequist, L.V. , Soria, J.C. , von Pawel, J. , Schwartz, B. , Von Roemeling, R. , Sandler, A.B. , 2012. Rationale and design of MARQUEE: a phase III, randomized, double-blind study of tivantinib plus erlotinib versus placebo plus erlotinib in previously treated patients with locally advanced or metastatic, nonsquamous, non-small-cell lung cancer. Clin. Lung Cancer 13, 391–395. [DOI] [PubMed] [Google Scholar]
  35. Scagliotti, G.V. , Novello, S. , von Pawel, J. , 2013. The emerging role of MET/HGF inhibitors in oncology. Cancer Treat Rev. 39, 793–801. [DOI] [PubMed] [Google Scholar]
  36. Seeburg, P.H. , Colby, W.W. , Capon, D.J. , Goeddel, D.V. , Levinson, A.D. , 1984. Biological properties of human c-Ha-ras1 genes mutated at codon 12. Nature 312, 71–75. [DOI] [PubMed] [Google Scholar]
  37. Sharma, S.V. , Fischbach, M.A. , Haber, D.A. , Settleman, J. , 2006. “Oncogenic shock”: explaining oncogene addiction through differential signal attenuation. Clin. Cancer Res. 12, 4392s–4395s. [DOI] [PubMed] [Google Scholar]
  38. Siena, S. , Sartore-Bianchi, A. , Di Nicolantonio, F. , Balfour, J. , Bardelli, A. , 2009. Biomarkers predicting clinical outcome of epidermal growth factor receptor-targeted therapy in metastatic colorectal cancer. J. Natl. Cancer Inst. 101, 1308–1324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Takashima, A. , Faller, D.V. , 2013. Targeting the RAS oncogene. Expert Opin. Ther. Targets 17, 507–531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Taya, Y. , Hosogai, K. , Hirohashi, S. , Shimosato, Y. , Tsuchiya, R. , Tsuchida, N. , Fushimi, M. , Sekiya, T. , Nishimura, S. , 1984. A novel combination of K-ras and myc amplification accompanied by point mutational activation of K-ras in a human lung cancer. EMBO J. 3, 2943–2946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Troiani, T. , Vecchione, L. , Martinelli, E. , Capasso, A. , Costantino, S. , Ciuffreda, L.P. , Morgillo, F. , Vitagliano, D. , D'Aiuto, E. , De Palma, R. , Tejpar, S. , Van Cutsem, E. , De Lorenzi, M. , Caraglia, M. , Berrino, L. , Ciardiello, F. , 2012. Intrinsic resistance to selumetinib, a selective inhibitor of MEK1/2, by cAMP-dependent protein kinase A activation in human lung and colorectal cancer cells. Br. J. Cancer 106, 1648–1659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wauson, E.M. , Guerra, M.L. , Barylko, B. , Albanesi, J.P. , Cobb, M.H. , 2013. Off-target effects of MEK inhibitors. Biochemistry 52, 5164–5166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Xia, N. , An, J. , Jiang, Q.Q. , Li, M. , Tan, J. , Hu, C.P. , 2013. Analysis of EGFR, EML4-ALK, KRAS, and c-MET mutations in Chinese lung adenocarcinoma patients. Exp. Lung Res. 39, 328–335. [DOI] [PubMed] [Google Scholar]
  44. Yavuz, S. , Apolo, A. , Kummar, S. , Del Rivero, J. , Madan, R.A. , Shawker, T. , Reynolds, J.C. , Celi, F.S. , 2014. Cabozantinib-induced thyroid dysfunction: a review of two ongoing trials for metastatic bladder cancer and sarcoma. Thyroid : Official J. Am. Thyroid Assoc. 24, 1223–1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhou, Y. , Zhao, C. , Gery, S. , Braunstein, G.D. , Okamoto, R. , Alvarez, R. , Miles, S.A. , Doan, N.B. , Said, J.W. , Gu, J. , Phillip Koeffler, H. , 2014. Off-target effects of c-MET inhibitors on thyroid cancer cells. Mol. Cancer Ther. 13, 134–143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zimmer, Y. , Vaseva, A.V. , Medova, M. , Streit, B. , Blank-Liss, W. , Greiner, R.H. , Schiering, N. , Aebersold, D.M. , 2010. Differential inhibition sensitivities of MET mutants to the small molecule inhibitor SU11274. Cancer Lett. 289, 228–236. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

The following are the supplementary data related to this article:

Supplementary materials and methods: PHA665752, (3Z)‐5‐[(2,6‐dichlorobenzyl)sulfonyl]‐3‐[(3,5‐dimethyl‐4‐{[(2R)‐2‐(pyrrolidin‐1‐ylmethyl)pyrrolidin‐1‐yl]carbonyl}‐1H‐pyrrol‐2‐yl)methylene]‐1,3‐dihydro‐2H‐indol‐2‐one (Pfizer, La Jolla, CA) was dissolved in DMSO.

Supplementary Figure 1 MET and ERK1/2 phosphorylation levels in NIH3T3 MET M1268T cells stably transfected with mutated HRAS or KRAS variants as indicated. The cells were treated with either vehicle or 300 nM PHA665752 for 16 h β‐Actin was used as a loading control.

Click here for additional data file. (22.5KB, jpg)

Supplementary Figure 2 Proliferative capacity of parental NIH3T3 MET M1268T as well as NIH3T3 MET M1268T cells stably expressing indicated KRAS or HRAS mutants as measured by XTT cell proliferation assay.

Click here for additional data file. (58.5KB, jpg)

Supplementary Figure 3 MET and ERK1/2 phosphorylation levels in NIH3T3 MET M1268T and H1993 cells stably transfected with mutated HRAS or KRAS variants as indicated. The cells were treated with either vehicle or 500 nM AZD6244 for 16 h β‐Actin was used as a loading control.

Click here for additional data file. (56.3KB, jpg)

Articles from Molecular Oncology are provided here courtesy of Wiley

RESOURCES