Kazuko Sakai

Antibody-dependent cellular cytotoxicity of cetuximab against tumor cells with wild-type or mutant epidermal growth factor receptor

Cetuximab (Erbitux, IMC‐C225) is a monoclonal antibody targeted to the epidermal growth factor receptor (EGFR). To clarify the mode of antitumor action of cetuximab, we examined antibody‐dependent cellular cytotoxicity (ADCC) activity against several tumor cell lines expressing wild‐type or mutant EGFR. ADCC activity and complement‐dependent cytolysis activity were analyzed using the CytoTox 96 assay. ADCC activities correlated with the EGFR expression value (R = 0.924). ADCC activities were detected against all tumor cell lines, except K562 cells in a manner dependent on the cellular EGFR expression level, whereas complement‐dependent cytolysis activity was not detected in any of the cell lines. The ADCC activity mediated by cetuximab was examined in HEK293 cells transfected with wild‐type EGFR (293W) and a deletional mutant of EGFR (293D) in comparison with the mock transfectant (293M). ADCC activity was detected in 293W and 293D cells, in a cetuximab dose‐dependent manner, but not in 293M cells (<10%). These results indicate that ADCC‐dependent antitumor activity results from the degree of affinity of cetuximab for the extracellular domain of EGFR, independent of EGFR mutation status. These results suggest ADCC activity to be one of the modes of therapeutic action of cetuximab and to depend on EGFR expression on the tumor cell surface. (Cancer Sci 2007; 98: 1275–1280)

FOXQ1 is overexpressed in colorectal cancer and enhances tumorigenicity and tumor growth.

Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21(CIP1/WAF1) expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 +/- 301 versus 1735 +/- 769 mm3, P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family.

Thymidylate synthase as a determinant of pemetrexed sensitivity in non-small cell lung cancer.

Background:Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in non-small cell lung cancer (NSCLC). We have now investigated the effect of TS overexpression on pemetrexed sensitivity in NSCLC cells.Methods:We established NSCLC cell lines that stably overexpress TS and examined the effects of such overexpression on the cytotoxicity of pemetrexed both in vitro and in xenograft models. We further examined the relation between TS expression in tumour specimens from NSCLC patients and the tumour response to pemetrexed by immunohistochemical analysis.Results:The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells. The inhibition of DNA synthesis and induction of apoptosis by pemetrexed were also greatly attenuated by forced expression of TS. Furthermore, tumours formed by TS-overexpressing NSCLC cells in nude mice were resistant to the growth-inhibitory effect of pemetrexed observed with control tumours. Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed.Conclusion:A high level of TS expression confers a reduced sensitivity to pemetrexed. TS expression is thus a potential predictive marker for response to pemetrexed-based chemotherapy in NSCLC patients.

Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

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.

Sorafenib Inhibits the Hepatocyte Growth Factor–Mediated Epithelial Mesenchymal Transition in Hepatocellular Carcinoma

The epithelial mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Sorafenib, a VEGFR inhibitor with activity against RAF kinase, is active against hepatocellular carcinoma (HCC); however, the possible involvement of sorafenib in the EMT remains unclear. Here, we examined the effect of sorafenib on the EMT. Hepatocyte growth factor (HGF) induced EMT-like morphologic changes and the upregulation of SNAI1 and N-cadherin expression. The downregulation of E-cadherin expression in HepG2 and Huh7 HCC cell lines shows that HGF mediates the EMT in HCC. The knockdown of SNAI1 using siRNA canceled the HGF-mediated morphologic changes and cadherin switching, indicating that SNAI1 is required for the HGF-mediated EMT in HCC. Interestingly, sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced morphologic changes, SNAI1 upregulation, and cadherin switching, whereas the PI3 kinase inhibitor wortmannin did not. Collectively, these findings indicate that sorafenib downregulates SNAI1 expression by inhibiting mitogen-activated protein kinase (MAPK) signaling, thereby inhibiting the EMT in HCC cells. In fact, a wound healing and migration assay revealed that sorafenib completely canceled the HGF-mediated cellular migration in HCC cells. In conclusion, we found that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in HCC. Our findings may provide a novel insight into the anti-EMT effect of tyrosine kinase inhibitors in cancer cells. Mol Cancer Ther; 10(1); 169–77. ©2011 AACR.

Pertuzumab, a novel HER dimerization inhibitor, inhibits the growth of human lung cancer cells mediated by the HER3 signaling pathway

A humanized anti‐HER2 monoclonal antibody pertuzumab (Omnitarg, 2C4), binding to a different HER2 epitope than trastuzumab, is known as an inhibitor of heterodimerization of the HER receptors. Potent antitumor activity against HER2‐expressing breast and prostate cancer cell lines has been clarified, but this potential is not clear against lung cancers. The authors investigated the in vitro anti‐tumor activity of pertuzumab against eight non‐small cell lung cancer cells expressing various members of the HER receptors. A lung cancer 11_18 cell line expressed a large amount of HER2 and HER3, and its cell growth was stimulated by an HER3 ligand, heregulin (HRG)‐α. Pertuzumab significantly inhibited the HRG‐α‐stimulated cellular growth of the 11_18 cells. Pertuzumab blocked HRG‐α‐stimulated phosphorylation of HER3, mitogen‐activated protein kinase (MAPK), and Akt. In contrast, pertuzumab failed to block epidermal growth factor (EGF)‐stimulated phosphorylation of EGF receptor (EGFR) and MAPK. Immunoprecipitation showed that pertuzumab inhibited HRG‐α‐stimulated HER2/HER3 heterodimer formation. HRG‐α‐stimulated HER3 phosphorylation was also observed in the PC‐9 cells co‐overexpressing EGFR, HER2, and HER3, but the cell growth was neither stimulated by HRG‐α nor inhibited by pertuzumab. The present results suggest that pertuzumab is effective against HRG‐α‐dependent cell growth in lung cancer cells through inhibition of HRG‐α‐stimulated HER2/HER3 signaling. (Cancer Sci 2007; 98: 1498–1503)

Impact of serum hepatocyte growth factor on treatment response to epidermal growth factor receptor tyrosine kinase inhibitors in patients with non-small cell lung adenocarcinoma.

Purpose: The epidermal growth factor receptor (EGFR) mutation status has emerged as a validated biomarker for predicting the response to treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI) in patients with non–small cell lung cancer. However, the responses to EGFR-TKIs vary even among patients with EGFR mutations. We studied several other independently active biomarkers for EGFR-TKI treatment. Experimental Design: We retrospectively analyzed the serum concentrations of 13 molecules in a cohort of 95 patients with non–small cell lung adenocarcinoma who received EGFR-TKI treatment at three centers. The pretreatment serum concentrations of amphiregulin, β-cellulin, EGF, EGFR, epiregulin, fibroblast growth factor-basic, heparin-binding EGF-like growth factor, hepatocyte growth factor (HGF), platelet-derived growth factor β polypeptide, placental growth factor, tenascin C, transforming growth factor-α, and vascular endothelial growth factor (VEGF) were measured using enzyme-linked immunosorbent assay and a multiplex immunoassay system. The associations between clinical outcomes and these molecules were evaluated. Results: The concentrations of HGF and VEGF were significantly higher among patients with progressive disease than among those without progressive disease (P < 0.0001). HGF and VEGF were strongly associated with progression-free survival (PFS) and overall survival (OS) in a univariate Cox analysis (all tests for hazard ratio showed P < 0.0001). A stratified multivariate Cox model according to EGFR mutation status (mutant, n = 20; wild-type, n = 23; unknown, n = 52) showed that higher HGF levels were significantly associated with a shorter PFS and OS (P < 0.0001 for both PFS and OS). These observations were also consistent in the subset analyses. Conclusions: Serum HGF was strongly related to the outcome of EGFR-TKI treatment. Our results suggest that the serum HGF level could be used to refine the selection of patients expected to respond to EGFR-TKI treatment, warranting further prospective study. Clin Cancer Res; 16(18); 4616–24. ©2010 AACR.

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.

Detection of epidermal growth factor receptor T790M mutation in plasma DNA from patients refractory to epidermal growth factor receptor tyrosine kinase inhibitor

A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR‐tyrosine kinase inhibitors (EGFR‐TKIs). A non‐invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non‐small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR‐TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression‐free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M‐positive patients (n = 11) was significantly shorter than that of the T790M‐negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR‐TKI therapy.