MET amplification as a potential therapeutic target in gastric cancer.

Our aim was to investigate both the prevalence of MET amplification in gastric cancer as well as the potential of this genetic alteration to serve as a therapeutic target in gastric cancer. MET amplification was assessed by initial screening with a PCR-based copy number assay followed by confirmatory FISH analysis in formalin-fixed, paraffin-embedded specimens of gastric cancer obtained at surgery. The effects of MET tyrosine kinase inhibitors (MET-TKIs) in gastric cancer cells with or without MET amplification were also examined. The median MET copy number in 266 cases of gastric cancer was 1.7, with a range of 0.41 to 21.3. We performed FISH analysis for the 15 cases with the highest MET copy numbers. MET amplification was confirmed in the four assessable cases with a MET copy number of at least 4, whereas MET amplification was not detected in those with a gene copy number of less than 4. The prevalence of MET amplification was thus 1.5% (4 out of 266 cases). Inhibition of MET by MET-TKIs resulted in the induction of apoptosis accompanied by attenuation of downstream MET signaling in gastric cancer cell lines with MET amplification but not in those without this genetic change. MET amplification identifies a small but clinically important subgroup of gastric cancer patients who are likely to respond to MET-TKIs. Furthermore, screening with a PCR-based copy number assay is an efficient way to reduce the number of patients requiring confirmation of MET amplification by FISH analysis.

Human papillomavirus DNA and p16 expression in Japanese patients with oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV) is a major etiologic factor for oropharyngeal squamous cell carcinoma (OPSCC). However, little is known about HPV-related OPSCC in Japan. During the study, formalin-fixed, paraffin-embedded OPSCC specimens from Japanese patients were analyzed for HPV DNA by the polymerase chain reaction (PCR) and for the surrogate marker p16 by immuno-histochemistry. For HPV DNA-positive, p16-negative specimens, the methylation status of the p16 gene promoter was examined by methylation-specific PCR. Overall survival was calculated in relation to HPV DNA and p16 status and was subjected to multivariate analysis. OPSCC cell lines were examined for sensitivity to radiation or cisplatin in vitro. The study results showed that tumor specimens from 40 (38%) of the 104 study patients contained HPV DNA, with such positivity being associated with tumors of the tonsils, lymph node metastasis, and nonsmoking. Overall survival was better for OPSCC patients with HPV DNA than for those without it (hazard ratio, 0.214; 95% confidence interval, 0.074-0.614; P = 0.002). Multivariate analysis revealed HPV DNA to be an independent prognostic factor for overall survival (P = 0.015). Expression of p16 was associated with HPV DNA positivity. However, 20% of HPV DNA-positive tumors were negative for p16, with most of these tumors manifesting DNA methylation at the p16 gene promoter. Radiation or cisplatin sensitivity did not differ between OPSCC cell lines positive or negative for HPV DNA. Thus, positivity for HPV DNA identifies a distinct clinical subset of OPSCC with a more favorable outcome in Japanese.

Activation of HER family signaling as a mechanism of acquired resistance to ALK inhibitors in EML4-ALK-positive non-small cell lung cancer.

PURPOSE: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib show marked efficacy in patients with non-small cell lung cancer positive for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown. EXPERIMENTAL DESIGN: We established lines of EML4-ALK-positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK-positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristics. RESULTS: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of epidermal growth factor receptor (EGFR), HER2, and HER3 was upregulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal-regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion. CONCLUSIONS: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK.

Antitumor action of the MET tyrosine kinase inhibitor crizotinib (PF-02341066) in gastric cancer positive for MET amplification.

Therapeutic strategies that target the tyrosine kinase MET hold promise for gastric cancer, but the mechanism underlying the antitumor activity of such strategies remains unclear. We examined the antitumor action of the MET tyrosine kinase inhibitor crizotinib (PF-02341066) in gastric cancer cells positive or negative for MET amplification. Inhibition of MET signaling by crizotinib or RNA interference-mediated MET depletion resulted in induction of apoptosis accompanied by inhibition of AKT and extracellular signal-regulated kinase phosphorylation in gastric cancer cells with MET amplification but not in those without it, suggesting that MET signaling is essential for the survival of MET amplification-positive cells. Crizotinib upregulated the expression of BIM, a proapoptotic member of the Bcl-2 family, as well as downregulated that of survivin, X-linked inhibitor of apoptosis protein (XIAP), and c-IAP1, members of the inhibitor of apoptosis protein family, in cells with MET amplification. Forced depletion of BIM inhibited crizotinib-induced apoptosis, suggesting that upregulation of BIM contributes to the proapoptotic effect of crizotinib. Crizotinib also exhibited a marked antitumor effect in gastric cancer xenografts positive for MET amplification, whereas it had little effect on those negative for this genetic change. Crizotinib thus shows a marked antitumor action both in vitro and in vivo specifically in gastric cancer cells positive for MET amplification.

Overcoming erlotinib resistance in EGFR mutation-positive non-small cell lung cancer cells by targeting survivin.

Loss of PTEN was recently shown to contribute to resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in EGFR mutation-positive non-small cell lung cancer (NSCLC) through activation of the protein kinase AKT. We previously showed that downregulation of the expression of the antiapoptotic protein survivin by EGFR-TKIs contributes to EGFR-TKI-induced apoptosis in EGFR mutation-positive NSCLC cells. We have now investigated the role of survivin expression in EGFR-TKI resistance induced by PTEN loss. The EGFR-TKI erlotinib did not affect survivin expression or induce apoptosis in EGFR mutation-positive NSCLC cells with PTEN loss. Downregulation of survivin either by transfection with a specific short interfering RNA or by exposure to the small-molecule survivin suppressor YM155 reversed erlotinib resistance in such cells in vitro. Furthermore, combination therapy with YM155 and erlotinib inhibited the growth of tumors formed by EGFR mutation-positive, PTEN-deficient NSCLC cells in nude mice to a greater extent than did treatment with either drug alone. These results thus indicate that persistent activation of signaling by the AKT-survivin pathway induced by PTEN loss underlies a mechanism of resistance to erlotinib-induced apoptosis in EGFR mutation-positive NSCLC. They further suggest that the targeting of survivin has the potential to overcome EGFR-TKI resistance in EGFR mutation-positive NSCLC.

MET tyrosine kinase inhibitor crizotinib (PF-02341066) shows differential antitumor effects in non-small cell lung cancer according to MET alterations.

INTRODUCTION: Tyrosine kinase inhibitors (TKIs) targeted to MET are undergoing clinical trials in patients with solid tumors, but the precise mechanism of the antitumor activity of these drugs remains unclear. We examined the antitumor action of the MET-TKI crizotinib (PF-02341066) in lung cancer cells that are positive or negative for MET amplification or mutation. METHODS: The antitumor action of crizotinib was evaluated on the basis of signal transduction, cell proliferation, apoptosis, and progression of tumor xenografts. RESULTS: Inhibition of MET signaling by crizotinib or by RNA interference-mediated MET depletion resulted in the induction of apoptosis accompanied by inhibition of AKT and extracellular signal-regulated kinase phosphorylation in lung cancer cells with MET amplification but not in cells with a MET mutation or in those without amplification or mutation of MET. These results suggest that MET signaling is essential for the survival of cells with MET amplification but not for that of cells without this genetic change, including those with a MET mutation. Crizotinib up-regulated the expression of BIM, a proapoptotic member of the Bcl-2 family, and down-regulated that of survivin, a member of the inhibitor of apoptosis protein family, in cells with MET amplification. Forced depletion of BIM and expression of survivin each inhibited crizotinib-induced apoptosis, suggesting that both up-regulation of BIM and down-regulation of survivin contribute to the proapoptotic effect of crizotinib. CONCLUSIONS: Crizotinib shows a marked antitumor action in MET amplification-positive lung cancer cells but not in cells without MET amplification, including those with a MET mutation.

Role of survivin in EGFR inhibitor-induced apoptosis in non-small cell lung cancers positive for EGFR mutations.

The molecular mechanism by which epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) induce apoptosis in non-small cell-lung cancer (NSCLC) cells that are positive for activating mutations of the EGFR remains unclear. In this study, we report the effects of the EGFR-TKI gefitinib on expression of the antiapoptotic protein survivin that have functional consequences in EGFR mutation-positive NSCLC cells. Immunoblot analysis revealed that gefitinib downregulated survivin expression, likely through inhibition of the PI3K-AKT signaling pathway, in NSCLC cells positive for EGFR mutation. Stable overexpression of survivin attenuated gefitinib-induced apoptosis and also inhibited the antitumor effect of gefitinib in human tumor xenografts. Furthermore, the combination of survivin overexpression with inhibition of the gefitinib-induced upregulation of the proapoptotic protein BIM attenuated gefitinib-induced apoptosis to a greater extent than either approach alone. Our results indicate that downregulation of survivin plays a pivotal role in gefitinib-induced apoptosis in EGFR mutation-positive NSCLC cells. Furthermore, they suggest that simultaneous interruption of the PI3K-AKT-survivin and MEK-ERK-BIM signaling pathways is responsible for EGFR-TKI-induced apoptotic death in these cells.

TAK-701, a humanized monoclonal antibody to hepatocyte growth factor, reverses gefitinib resistance induced by tumor-derived HGF in non-small cell lung cancer with an EGFR mutation.

Most non-small cell lung cancer (NSCLC) tumors with an activating mutation of the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (TKI) such as gefitinib but ultimately develop resistance to these drugs. Hepatocyte growth factor (HGF) induces EGFR-TKI resistance in NSCLC cells with such a mutation. We investigated strategies to overcome gefitinib resistance induced by HGF. Human NSCLC cells with an activating EGFR mutation (HCC827 cells) were engineered to stably express HGF (HCC827-HGF cells). HCC827-HGF cells secreted large amounts of HGF and exhibited resistance to gefitinib in vitro to an extent similar to that of HCC827 GR cells, in which the gene for the HGF receptor MET is amplified. A MET-TKI reversed gefitinib resistance in HCC827-HGF cells as well as in HCC827 GR cells, suggesting that MET activation induces gefitinib resistance in both cell lines. TAK-701, a humanized monoclonal antibody to HGF, in combination with gefitinib inhibited the phosphorylation of MET, EGFR, extracellular signal-regulated kinase, and AKT in HCC827-HGF cells, resulting in suppression of cell growth and indicating that autocrine HGF-MET signaling contributes to gefitinib resistance in these cells. Combination therapy with TAK-701 and gefitinib also markedly inhibited the growth of HCC827-HGF tumors in vivo. The addition of TAK-701 to gefitinib is a promising strategy to overcome EGFR-TKI resistance induced by HGF in NSCLC with an activating EGFR mutation.

Enhanced anticancer effect of the combination of BIBW2992 and thymidylate synthase-targeted agents in non-small cell lung cancer with the T790M mutation of epidermal growth factor receptor.

Most non-small cell lung cancer (NSCLC) tumors with activating mutations of the epidermal growth factor receptor (EGFR) are initially responsive to first-generation, reversible EGFR tyrosine kinase inhibitors (TKI) such as gefitinib, but they subsequently develop resistance to these drugs through either acquisition of an additional T790M mutation of EGFR or amplification of the proto-oncogene MET. We have now investigated the effects of combination treatment with thymidylate synthase (TS)-targeting drugs and the second-generation, irreversible EGFR-TKI BIBW2992 on the growth of NSCLC cells with the T790M mutation. The effects of BIBW2992 on EGFR signaling and TS expression in gefitinib-resistant NSCLC cells were examined by immunoblot analysis. The effects of BIBW2992 and the TS-targeting agents S-1 (or 5-fluorouracil) or pemetrexed on the growth of gefitinib-resistant NSCLC cells were examined both in vitro and in vivo. The combination of BIBW2992 with 5-fluorouracil or pemetrexed synergistically inhibited the proliferation of NSCLC cells with the T790M mutation in vitro, whereas an antagonistic interaction was apparent in this regard between gefitinib and either of these TS-targeting agents. BIBW2992 induced downregulation of TS in the gefitinib-resistant NSCLC cells, implicating depletion of TS in the enhanced antitumor effect of the combination therapy. The combination of BIBW2992 and either the oral fluoropyrimidine S-1 or pemetrexed also inhibited the growth of NSCLC xenografts with the T790M mutation to an extent greater than that apparent with either agent alone. The addition of TS-targeting drugs to BIBW2992 is a promising strategy to overcome EGFR-TKI resistance in NSCLC with the T790M mutation of EGFR.

Identification of c-Src as a potential therapeutic target for gastric cancer and of MET activation as a cause of resistance to c-Src inhibition.

Therapeutic strategies that target c-Src hold promise for a wide variety of cancers. We have now investigated both the effects of dasatinib, which inhibits the activity of c-Src and several other kinases, on cell growth as well as the mechanism of dasatinib resistance in human gastric cancer cell lines. Immunoblot analysis revealed the activation of c-Src at various levels in most gastric cancer cell lines examined. Dasatinib inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and induced G(1) arrest, as revealed by flow cytometry, in a subset of responsive cell lines. In other responsive cell lines, dasatinib inhibited both ERK and AKT phosphorylation and induced apoptosis, as revealed by an increase in caspase-3 activity and cleavage of poly(ADP-ribose) polymerase. Depletion of c-Src by RNA interference also induced G(1) arrest or apoptosis in dasatinib-responsive cell lines, indicating that the antiproliferative effect of dasatinib is attributable to c-Src inhibition. Gastric cancer cell lines positive for the activation of MET were resistant to dasatinib. Dasatinib had no effect on ERK or AKT signaling, whereas the MET inhibitor PHA-665752 induced apoptosis in these cells. The subsets of gastric cancer cells defined by a response to c-Src or MET inhibitors were distinct and nonoverlapping. Our results suggest that c-Src is a promising target for the treatment of gastric cancer and that analysis of MET amplification might optimize patient selection for treatment with c-Src inhibitors.

Effects of Src inhibitors on cell growth and epidermal growth factor receptor and MET signaling in gefitinib-resistant non-small cell lung cancer cells with acquired MET amplification.

The efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors such as gefitinib and erlotinib in non-small cell lung cancer (NSCLC) is often limited by the emergence of drug resistance conferred either by a secondary T790M mutation of EGFR or by acquired amplification of the MET gene. We now show that the extent of activation of the tyrosine kinase Src is markedly increased in gefitinib-resistant NSCLC (HCC827 GR) cells with MET amplification compared with that in the gefitinib-sensitive parental (HCC827) cells. In contrast, the extent of Src activation did not differ between gefitinib-resistant NSCLC (PC9/ZD) cells harboring the T790M mutation of EGFR and the corresponding gefitinib-sensitive parental (PC9) cells. This activation of Src in HCC827 GR cells was largely abolished by the MET-TKI PHA-665752 but was only partially inhibited by gefitinib, suggesting that Src activation is more dependent on MET signaling than on EGFR signaling in gefitinib-resistant NSCLC cells with MET amplification. Src inhibitors blocked Akt and Erk signaling pathways, resulting in both suppression of cell growth and induction of apoptosis, in HCC827 GR cells as effectively as did the combination of gefitinib and PHA-665752. Furthermore, Src inhibitor dasatinib inhibited tumor growth in HCC827 GR xenografts to a significantly greater extent than did treatment with gefitinib alone. These results provide a rationale for clinical targeting of Src in gefitinib-resistant NSCLC with MET amplification.