Erina Hatashita

Radiosensitizing Effect of YM155, a Novel Small-Molecule Survivin Suppressant, in Non–Small Cell Lung Cancer Cell Lines

Purpose: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effect of YM155, a small-molecule inhibitor of survivin expression, on the sensitivity of human non–small cell lung cancer (NSCLC) cell lines to γ-radiation. Experimental Design: The radiosensitizing effect of YM155 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced DNA damage was evaluated on the basis of histone H2AX phosphorylation and foci formation. Results: YM155 induced down-regulation of survivin expression in NSCLC cells in a concentration- and time-dependent manner. A clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to γ-radiation in vitro. The combination of YM155 and γ-radiation induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of histone γ-H2AX also showed that YM155 delayed the repair of radiation-induced double-strand breaks in nuclear DNA. Finally, combination therapy with YM155 and γ-radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone. Conclusions: These results suggest that YM155 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of YM155 is likely attributable, at least in part, to the inhibition of DNA repair and enhancement of apoptosis that result from the down-regulation of survivin expression. Combined treatment with YM155 and radiation warrants investigation in clinical trials as a potential anticancer strategy.

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. Clin Cancer Res; 18(22); 6219–26. ©2012 AACR.

Enhancement of the antitumor activity of ionising radiation by nimotuzumab, a humanised monoclonal antibody to the epidermal growth factor receptor, in non-small cell lung cancer cell lines of differing epidermal growth factor receptor status

The expression and activity of the epidermal growth factor receptor (EGFR) are determinants of radiosensitivity in several tumour types, including non-small cell lung cancer (NSCLC). However, little is known of whether genetic alterations of EGFR in NSCLC cells affect the therapeutic response to monoclonal antibodies (mAbs) to EGFR in combination with radiation. We examined the effects of nimotuzumab, a humanised mAb to EGFR, in combination with ionising radiation on human NSCLC cell lines of differing EGFR status. Flow cytometry revealed that H292 and Ma-1 cells expressed high and moderate levels of EGFR on the cell surface, respectively, whereas H460, H1299, and H1975 cells showed a low level of surface EGFR expression. Immunoblot analysis revealed that EGFR phosphorylation was inhibited by nimotuzumab in H292 and Ma-1 cells but not in H460, H1299, or H1975 cells. Nimotuzumab augmented the cytotoxic effect of radiation in H292 and Ma-1 cells in a clonogenic assay in vitro, with a dose enhancement factor of 1.5 and 1.3, respectively. It also enhanced the antitumor effect of radiation on H292 and Ma-1 cell xenografts in nude mice, with an enhancement factor of 1.3 and 4.0, respectively. Nimotuzumab did not affect the radioresponse of H460 cells in vitro or in vivo. Nimotuzumab enhanced the antitumor efficacy of radiation in certain human NSCLC cell lines in vitro and in vivo. This effect may be related to the level of EGFR expression on the cell surface rather than to EGFR mutation.

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. Mol Cancer Ther; 9(10); 2785–92. ©2010 AACR.

Sorafenib Inhibits Non–Small Cell Lung Cancer Cell Growth by Targeting B-RAF in KRAS Wild-Type Cells and C-RAF in KRAS Mutant Cells

Sorafenib is a multikinase inhibitor whose targets include B-RAF and C-RAF, both of which function in the extracellular signal-regulated kinase (ERK) signaling pathway but which also have distinct downstream targets. The relative effects of sorafenib on B-RAF and C-RAF signaling in tumor cells remain unclear, however. We have now examined the effects of sorafenib as well as of B-RAF or C-RAF depletion by RNA interference on cell growth and ERK signaling in non-small cell lung cancer (NSCLC) cell lines with or without KRAS mutations. Sorafenib inhibited ERK phosphorylation in cells with wild-type KRAS but not in those with mutant KRAS. Despite this difference, sorafenib inhibited cell growth and induced G(1) arrest in both cell types. Depletion of B-RAF, but not that of C-RAF, inhibited ERK phosphorylation as well as suppressed cell growth and induced G(1) arrest in cells with wild-type KRAS. In contrast, depletion of C-RAF inhibited cell growth and induced G(1) arrest, without affecting ERK phosphorylation, in cells with mutant KRAS; depletion of B-RAF did not induce G(1) arrest in these cells. These data suggest that B-RAF-ERK signaling and C-RAF signaling play the dominant roles in regulation of cell growth in NSCLC cells with wild-type or mutant KRAS, respectively. The G(1) arrest induced by either C-RAF depletion or sorafenib in cells with mutant KRAS was associated with down-regulation of cyclin E. Our results thus suggest that sorafenib inhibits NSCLC cell growth by targeting B-RAF in cells with wild-type KRAS and C-RAF in those with mutant KRAS.

Synergistic antitumor effect of S-1 and the epidermal growth factor receptor inhibitor gefitinib in non-small cell lung cancer cell lines: role of gefitinib-induced down-regulation of thymidylate synthase

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the therapeutic response to EGFR tyrosine kinase inhibitors (TKI) in patients with advanced non-small cell lung cancer (NSCLC). The response rate to these drugs remains low, however, in NSCLC patients with wild-type EGFR alleles. Combination therapies with EGFR-TKIs and cytotoxic agents are considered a therapeutic option for patients with NSCLC expressing wild-type EGFR. We investigated the antiproliferative effect of the combination of the oral fluorouracil S-1 and the EGFR-TKI gefitinib in NSCLC cells of differing EGFR status. The combination of 5-fluorouracil and gefitinib showed a synergistic antiproliferative effect in vitro in all NSCLC cell lines tested. Combination chemotherapy with S-1 and gefitinib in vivo also had a synergistic antitumor effect on NSCLC xenografts regardless of the absence or presence of EGFR mutations. Gefitinib inhibited the expression of the transcription factor E2F-1, resulting in the down-regulation of thymidylate synthase at the mRNA and protein levels. These observations suggest that gefitinib-induced down-regulation of thymidylate synthase is responsible, at least in part, for the synergistic antitumor effect of combined treatment with S-1 and gefitinib and provide a basis for clinical evaluation of combination chemotherapy with S-1 and EGFR-TKIs in patients with solid tumors. [Mol Cancer Ther 2008;7(3):599–606]

Marked anti-tumour activity of the combination of YM155, a novel survivin suppressant, and platinum-based drugs

Background:Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effects of the combination of YM155, a novel small-molecule inhibitor of survivin expression, and platinum compounds (cisplatin and carboplatin) on human non-small cell lung cancer (NSCLC) cell lines.Methods:The anti-cancer efficacy of YM155 in combination with platinum compounds was evaluated on the basis of cell death and progression of tumour xenografts. Platinum compound-induced DNA damage was evaluated by immunofluorescence analysis of histone γ-H2AX.Results:Immunofluorescence analysis of histone γ-H2AX showed that YM155 delayed the repair of double-strand breaks induced in nuclear DNA by platinum compounds. The combination of YM155 and platinum compounds also induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Finally, combination therapy with YM155 and platinum compounds delayed the growth of NSCLC tumour xenografts in nude mice to an extent greater than that apparent with either treatment modality alone.Conclusion:These results suggest that YM155 sensitises tumour cells to platinum compounds both in vitro and in vivo, and that this effect is likely attributable to the inhibition of DNA repair and consequent enhancement of apoptosis.

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. Mol Cancer Ther; 11(7); 1557–64. ©2012 AACR.

Inhibition of Insulin-Like Growth Factor 1 Receptor by CP-751,871 Radiosensitizes Non–Small Cell Lung Cancer Cells

Purpose: Therapeutic strategies that target the insulin-like growth factor I receptor (IGF-1R) hold promise for a wide variety of cancers. We have now investigated the effect of CP-751,871, a fully human monoclonal antibody specific for IGF-IR, on the sensitivity of human non–small cell lung cancer (NSCLC) cell lines to radiation. Experimental Design: The radiosensitizing effect of CP-751,871 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced damage was evaluated by immunofluorescence analysis of the histone γ-H2AX and Rad51. Results: A clonogenic survival assay revealed that CP-751,871 increased the sensitivity of NSCLC cells to radiation in vitro. CP-751,871 inhibited radiation-induced IGF-IR signaling, and potentiated the radiation-induced increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of the histone γ-H2AX and Rad51 also showed that CP-751,871 inhibited the repair of radiation-induced DNA double-strand breaks. Finally, combination therapy with CP-751,871 and radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone. Conclusions: These results show that CP-751,871 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of CP-751,871 is likely attributable to the inhibition of DNA repair and enhancement of apoptosis that result from attenuation of IGF-IR signaling. Combined treatment with CP-751,871 and radiation thus warrants further investigation in clinical trials as a potential anticancer strategy. (Clin Cancer Res 2009;15(16):5117–25)

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. (Cancer Sci 2009)