Regulation of MET Kinase Inhibitor Resistance by Copy Number of MET in Gastric Carcinoma Cells
Yohei Funakoshi,* Toru Mukohara,*‡ Roudy Chiminch Ekyalongo,* Hideo Tomioka,* Yu Kataoka,* Yohei Shimono,*† Naoko Chayahara,*
Masanori Toyoda,* Naomi Kiyota,* Yutaka Fujiwara,* and Hironobu Minami*‡

*Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
†Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan
‡Cancer Center, Kobe University Hospital, Kobe, Japan

We previously established acquired resistant models for MET-tyrosine kinase inhibitors (TKIs) by continuously exposing the MET-amplified gastric cancer cell line MKN45 to MET-TKIs, PHA665752 (MKN45-PR), or GSK1363089 (MKN45-GR). We found resistant mechanisms caused by increased copy number of MET in both lines and Y1230H mutation in MKN45-PR. We also found that excessive MET signaling caused by these MET alterations resulted in intra-S-phase arrest in the absence of MET-TKIs, so that cells grew faster in the presence of MET-TKIs, a phenomenon referred to as “addiction.” In this study, to investigate reversibility of the acquired resistance and “addiction” to MET-TKIs and their causative MET alterations, we sequentially cultured MKN45-PR and MKN45-GR in decreasing concentrations of MET-TKIs until they were able to grow in a drug-free condition. These “revertant” cell lines (designated MKN45-PR-RE and MKN45-GR-RE) were comparatively analyzed. Growth assay showed that both MKN45-PR-RE and MKN45-GR-RE partially lost the property of “addiction” to MET-TKIs. MKN45-GR-RE lost the property of resistance to GSK1363089, but MKN45-PR-RE retained resistance to PHA665752. Copy numbers and expression and phosphorylation of MET protein reduced in both MKN45-PR-RE and MKN45-GR-RE compared with MKN45-PR and MKN45-GR, respectively, but Y1230H mutation and biochemical resistance to PHA665752 remained in MKN45-PR-RE. The “addiction” to MET-TKIs appeared attributable to increased copy number, and the property and the MET alteration were reversible. The Y1230H mutation appeared enough in itself to keep cells resistant to MET-TKIs and was irreversible.
Key words: Acquired resistance; Addiction; Reversibility; Gastric cancer; MET inhibitor

INTRODUCTION
While MET-tyrosine kinase inhibitors (TKIs) are being developed clinically, emergence of acquired resistance is highly expected. In our previous study, we established resis- tant models by continuously exposing the MET-amplified gastric cancer cell line MKN45 to MET-TKIs, PHA665752 (MKN45-PR), or GSK1363089 (MKN45-GR) to clarify
the mechanism of acquired resistance (1). We revealed that both MKN45-PR and MKN45-GR had a higher copy num- ber of MET than parental MKN45. In addition, Y1230H MET point mutation, which had been suggested to be a gain-of-function mutation and reduce affinity to MET- TKIs (2), was detected only in MKN45-PR. Both types of MET alterations were suggested as causing resistance to MET-TKIs. Unexpectedly, the growth rate was lower

in the absence than in the presence of MET-TKIs in either resistant cell line, suggesting “addiction” to the inhibitors. Our additional experiments suggested that the “addiction” is formed by the following mechanisms: the same MET gene alteration, Y1230H point mutation, and/or increased copy number, which cause resistance to MET-TKIs, may also cause excessive MET signaling, subsequent replica- tion stress, and DNA damage response, eventually leading to intra-S-phase arrest in the absence of MET-TKIs (1).
In the current study, to investigate the reversibility of the properties of resistance and “addiction” to MET-TKIs and their causative MET alterations, we sequentially cul- tured these resistant cell lines in decreasing concentra- tions of MET-TKIs until the cells were able to grow in a drug-free condition.

Address correspondence to Toru Mukohara, M.D., D.Med.Sci.,7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel: +81-78-382-5820; Fax: +81-78-382-5821; E-mail: [email protected]
IP: 178.159.100.131 On: Sa2t,8701 Sep 2018 10:49:24

288 FUNAKOSHI ET AL.

MATERIALS AND METHODS
Cell Cultures and Drugs
The MKN45 gastric cancer cell line, which exhib- its MET amplification (3), was obtained from RIKEN BioResource Center (Tsukuba, Japan). Cells were main- tained in RPMI 1640 (Cellgro, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gemini-Bio-Products, CA, USA), 100 units/ml penicillin, 100 units/ml strep- tomycin, and 2 mM glutamine at 37°C in a humidified atmosphere with 5% CO2 and were in the logarithmic growth phase upon initiation of experiments.
Acquired resistant cell lines against PHA665752 (MKN45-PR) and GSK1363089 (MKN45-GR) were gen-
erated by continuously culturing MKN-45 cells in increas- ing concentrations of each drug as previously reported (1). GSK1363089 was kindly provided by Glaxo SmithKline (UK). PHA665752 was purchased from Calbiochem (Darmstadt, Germany). MKN45-PR and MKN45-GR were maintained in the same cell culture media with 0.5 M PHA665752 and 20 nM GSK1363089, respectively. Sub- sequently, MKN45-PR and MKN45-GR were cultured in decreasing concentrations of MET-TKI until they were able to grow in a drug-free condition (Fig. 1). These “revertant” cell lines were designated as MKN45-PR-RE and MKN45-GR-RE, respectively.
Antibodies and Western Blotting
For Western blotting, cells were lysed as previously described (1). Briefly, protein extracts were collected and separated by electrophoresis on 7.6% polyacrylamide- sodium dodecyl sulfate gels, transferred to nitrocellulose membranes (Millipore, MA, USA), and detected by immu- noblotting using an enhanced chemiluminescence system (New England Nuclear Life Science Products, MA, USA). Phospho-MET (Tyr1234/1235) (D26) and MET antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). -Actin antibodies were purchased from Biosource International (Camarillo, CA, USA).
Cell Growth Assay
Cell growth was determined using an MTS assay kit (Promega, Madison, WI, USA), which determines the number of viable cells by a colorimetric method based on the bioreduction of 3-(4,5-dimethylthiazol-2yl)-5- (3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetra- zolium (MTS) to a soluble formazan product detected spectrophotometrically at a wavelength of 490 nm.
Cells diluted in 10% FBS containing maintenance media (160 l/well) were plated in 96-well flat-bottom plates (Corning, Inc., Corning, NY, USA) and incubated with or without the indicated drugs until final analysis. The number of cells required to obtain an optical density within the linear of the assay (1.3–2.2) was determined to be 1,500 cells/well with pilot studies (data not shown).

Quantitative PCR
Genomic DNA was extracted using a QIAamp™ DNA Mini kit (Qiagen, Valencia, CA, USA). Genomic DNA (50 ng) was amplified and analyzed using Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) by real-time quantitative PCR using ABI Prism 7900HT (Applied Biosystems). -Actin was used to nor- malize DNA levels in subsequent quantitative analyses. Specific primers for real-time quantitative PCR were previously described (4). Gene expression and gene copy number fold increases were determined using the follow- ing formula: fold increase = 2^ − ([Ct of target gene − Ct of control gene]resistant − [Ct of target gene − Ct of control gene]parental), where Ct means cycle threshold.
DNA Sequencing
Exon 19 of the MET gene was amplified from genomic DNA extracted as described above by PCR. PCR prod- ucts were purified and subjected to bidirectional sequenc- ing using a 3730 DNA Analyzer (Applied Biosystems). Primers used for PCR and sequencing were forward primer 5-CTTCCTTCAGAAGTTATGGATTTC-3 and reverse primer 5-GAAGAAAACTGGAATTGGTGGTGTTG-3.
RESULTS
Alternation of Growth Rate With or Without MET-TKIs
We first evaluate the relative growth rate of paren- tal MKN45, two resistant cell lines (MKN45-PR and MKN45-GR), and two “revertant” cell lines (MKN45- PR-RE and MKN45-GR-RE) in the presence or absence of 0.5 M PHA665752 or 20 nM GSK1363089 by serial MTS assays (Fig. 1A and B). As previously reported (1), cell growth rates of MKN45 were significantly reduced by treatment either with PHA665752 or GSK1363089. In contrast, while each resistant cell line showed a growth rate almost equivalent to untreated MKN45 in the pres- ence of each MET-TKI, their growth was impaired in the absence of the inhibitor. This result is consistent with our previous study and indicates that each resistant cell line had similarly become “addicted” to each MET-TKI in the process of acquiring resistance to it. In regard to “rever- tant” cell lines, MKN45-PR-RE and MKN45-GR-RE achieved growth rates close to untreated MKN45 in the absence of PHA665752 and GSK1363089, respectively, indicating that the “addiction” to MET-TKIs was revers- ible. Regarding the property of resistance to MET-TKIs, while MKN45-PR-RE was not significantly inhibited with 0.5 M PHA665752 (% of control on day 4; no drug vs. PHA665752, 642% vs. 747%, p = 0.25) (Fig. 1A),
the growth rate of MKN45-GR-RE was reduced by
20 nM GSK1363089 (% of control on day 4; no drug vs. GSK1363089, 1131% vs. 627%, p < 0.0005) (Fig. 1B).
To further test reversibility of the “addiction” and resistance to MET-TKIs in different drug concentrations,

Figure 1. Growth rates with or without MET-TKIs in MKN45- and MKN45-derived cell lines. MKN45, MKN45-PR, and MKN45- PR-RE (A) and MKN45, MKN45-GR, and MKN45-GR-RE (B) were treated with or without PHA665752 and GSK1303089, respec- tively, and subjected to serial MTS assay on days 0 through 4. The percentage of viable cells is shown relative to that of day 0 controls. Each data point represents the mean value and standard deviation of six replicate wells. MKN45, MKN45-PR, and MKN45-PR-RE
(C) and MKN45, MKN45-GR, and MKN45-GR-RE (D) were cultured for 5 days in the presence of various concentrations of PHA665752 and GSK1363089, respectively. The percentage of viable cells is shown relative to that of untreated controls. Each data point repre- sents the mean value and standard deviation of six wells.

cell lines were cultured for 5 days in the presence of various concentrations of PHA665752 and GSK1363089 and subjected to MTS assay. Consistent with the time course assay above, each resistant cell line showed the highest growth rate in the presence of certain amounts of

MET-TKIs (Fig. 1C and D). However, this tendency of “addiction” was at least partially reduced in each “revertant” cell line (Fig. 1C and D), suggesting again its reversibil- ity. On the other hand, MKN45-PR-RE remained resistant to PHA665752 (IC50; MKN45-PR vs. MKN45-PR-RE,

Figure 2. Variable MET gene copy number in MKN45-derived cell lines. MET copy number was determined by analysis of genomic DNA using quantitative PCR. Copy number as a ratio to that in parental MKN45 cells was plotted on the y-axis.

~0.1 M vs. 3 M) (Fig. 1C). In contrast, the growth curve for MKN45-GR-RE was virtually identical to that for parental MKN45 (Fig. 1D).
Collectively, these results indicated that MKN45- GR-RE lost the property of both “addiction” and resis- tance to GSK1363089 in the process of drug withdrawal. On the other hand, while MKN45-PR-RE also lost the property of “addiction” to PHA665752, it remained with that of resistance.
Reversibility of Mechanisms of “Addiction” and Resistance to MET-TKIs
We reported that Y1230H mutation and/or increased copy number of MET gene could be causative for cellu- lar resistance and “addiction” to MET-TKIs. To examine the reversibility of MET alterations, MET copy number was determined in each cell line by quantitative PCR. Results showed that MET copy number in MKN45-PR

and MKN45-GR was higher than that in parental MKN45, but was partly reduced in MKN45-PR-RE and MKN45-GR-RE (Fig. 2). This indicates that an increased MET copy number is a reversible gene alteration. On the other hand, when we determined the DNA sequence of exon 19 of MET gene, Y1230H mutation was detected in MKN45-PR and was retained in MKN45-PR-RE (Fig. 3). This indicates that Y1230H mutation is an irreversible gene alteration. This particular mutation was not detected in parental MKN45, MKN45-GR, or MKN45-GR-RE.
The Effect of MET-TKIs on MET Signaling
To evaluate the biochemical basis of resistance and “addiction” to MET-TKIs, expression and phosphorylation of MET were examined by Western blotting in parental MKN45 and each resistant and “revertant” cell line being treated with and without PHA665752 or GSK1363089 (Fig. 4A and B). First, compared with parental MKN45,

Figure 3. Y1230H mutation of MET in MKN45-PR and MKN45-PR-RE. Sequencing results of MET exon 19 in MKN45, each resis- tant and “revertant” cell line is shown. Arrow shows the site of mutation; substitution of T for C leading to an amino acid change of Tyr to His at amino acid 1,230 observed only in MKN45-PR and MKN45-PR-RE.

Figure 4. Expression and phosphorylation of MET in MKN45 and MKN45-derived cell lines with and without treatment of MET- TKIs. MKN45, MKN45-PR, and MKN45-PR-RE (A) and MKN45, MKN45-GR, and MKN45-GR-RE (B) were grown in the presence of increasing concentrations of PHA665752 and GSK1363089, respectively, for 24 h. Cell lysates were subjected to Western blotting for phosphorylated and total MET along with -actin as a loading control. (C) Baseline expression levels of MET protein in each cell line are quantified and represented as a ratio to that of MKN45.

baseline MET expression and MET phosphorylation (Try1234/1235) increased in MKN45-PR and MKN45-GR (Fig. 4A and B). In both MKN45-PR-RE and MKN45- GR-RE, the expression and phosphorylation of MET par- tially reduced compared with MKN45-PR and MKN45-GR, respectively. These changes in levels of protein expression and phosphorylation of MET appeared in parallel with those in copy number of MET (Figs. 2 and 4). Next, while in parental MKN45, 0.5 M of PHA665752 was enough to induce complete inhibition of phosphorylation of MET, detectable levels of MET phosphorylation were retained in MKN45-PR and MKN45-PR-RE (Fig. 4A). This result

indicated that biochemical resistance to PHA665752 was sustained in MKN45-PR-RE. In contrast, while the mag- nitude of decrease in MET phosphorylation when treated with 20 nM of GSK1363089 was minimal in MKN45-GR, that in MKN45-GR-RE was partially retrieved to that in parental MKN45 (Fig. 4B). This indicated that biochemi- cal resistance to GSK1363089 was cancelled, at least par- tially, in MKN45-GR-RE.
DISCUSSION
In this study, we suggest that the acquired “addiction” to MET-TKIs appeared attributable to increased MET

copy number, and the cellular property and MET alteration are reversible. In contrast, Y1230H MET point mutation by itself appeared enough to keep cells resistant to MET- TKIs, and this resistant MET mutation is irreversible.
Previous preclinical studies reported that the increase in copy number of target receptor tyrosine kinase (RTK) genes, including MET and epidermal growth factor recep- tor, were potential mechanisms of acquired resistance to inhibitors of RTKs (4,5). In these studies, the increase in copy number was shown to be reversible on drug with- drawal (4,5), consistent with our current finding. With persistent inhibition of RTKs, cancer cells may increase gene copy number to keep cellular signals from them. However, with removal of RTK inhibitors, the once- increased gene copy number could be disadvantageous for the cells because resulting excessive signals could cause cellular senescence (5) or S-phase arrest (1). To keep cel- lular signals at proper levels, cancer cells may be capable of varying the copy number.
MET Y1230H point mutation as the resistance mecha- nism persisted in MKN45-PR-RE even after withdrawal of PHA665752 (Fig. 3). Consistent with this observation, a previous study has reported that in a leukemia cell line that obtained ABL T315I imatinib-resistant mutation after being exposed to imatinib, the same mutation was still detectable after 25 passages through drug-free condition (6). Conversely, some clinical case studies have reported that an epidermal growth factor receptor T790M gefitinib- resistant mutation induced by treatment with gefitinib dis- appeared during drug-free interval (7,8).
Despite the presence of Y1230H mutation, which has been suggested to reduce affinity of PHA665752 to MET (2), PHA665752 inhibited phosphorylation of MET partially but significantly (Fig. 4A). This may be caused by the fact that the mutation occurs only in a small propor- tion of amplified MET alleles (Fig. 3), so that the residual phosphorylation of MET in the presence of PHA665752 seen in Figure 4A might be mainly derived from minor MET receptor dimers involving Y1230H mutant MET. Because it was shown that in cells with MET Y1230H phosphorylation of downstream molecules was kept high in the presence of PHA665752 with subtle residual phos- phorylation of MET itself (2), MET dimers involving Y1230H mutant MET may be capable of triggering down- stream signals efficiently.
At present, treatment strategies after the emergence of clinically acquired resistance to TKIs remain controver- sial. Some reports suggest the benefit of continuous use with or without dose escalation of TKIs beyond progres- sion (9–12). This strategy is supported by findings called “flare,” in which accelerated progression of a disease after drug withdrawal is observed (9,13,14). However, in clinical practice and most clinical trials, TKIs were gener- ally stopped with a classic view of treatment resistance to

cancer therapy. If our finding of acquired “addiction” is true in clinical practice, withdrawal of MET-TKIs itself could stabilize tumor growth, although there has been no such report from clinical studies thus far. Another clini- cally relevant issue is justification for rechallenging the same TKIs after transient withdrawal of a drug. Based on our current study, rechallenging MET-TKIs may be justi- fied when Y1230H has not emerged with tumor-acquiring resistance. Recent preclinical studies showed that cells lose their acquired resistance to TKIs in the absence of the drug (4,5), similar to MKN45-GR-RE in the current study. The reversibility of the resistant property after with- drawal of TKIs was also suggested by sporadic clinical reports (15–17). Further studies are necessary—in particu- lar, comparative analysis of initial pretherapy samples and rebiopsy samples on the emergence of acquired resistance. Treatment after progression to TKIs should be individu- alized based on molecular profiles, as already partially applied in initial therapy with TKI.
This study had several limitations. First, subcloning was not performed. The Y1230H mutation was observed only in a small fraction of alleles, and we speculated that the mutation is diluted in amplified MET alleles; how- ever, we could not eliminate the possibility that the muta- tion occurred only in a subpopulation of mixed clones of MKN45-PR. Second, our use of only one MET-amplified gastric cancer cell line (MKN45) precludes generalization of the results. Third, we did not perform in vivo studies.
In conclusion, the acquired “addiction” and resistance derived from increases in MET copy numbers appeared reversible, but the acquired resistance derived from MET Y1230H point mutation appeared irreversible. For selec- tion of a treatment strategy after disease progression, identification of the type of molecular changes in rebiop- sied specimens may be useful.
ACKNOWLEDGMENTS: This study was supported by the Global Centers of Excellence Program (H.M.), Grant-in-Aid for Scientific Research (C) (T.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Research Grant from the Takeda Science Foundation (T.M.). The authors declare no conflicts of interest.
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