Keiko Ohgino

Activation of the FGF2-FGFR1 Autocrine Pathway: A Novel Mechanism of Acquired Resistance to Gefitinib in NSCLC

Patients with non-small cell lung cancer (NSCLC) that harbors epidermal growth factor receptor (EGFR) mutations initially respond to EGFR-tyrosine kinase inhibitors (TKI) but eventually experience relapse. Acquired resistance to EGFR-TKIs is strongly associated with patient mortality. Thus, elucidation of the mechanism of acquired resistance to EGFR-TKIs is of great importance. In this study, gefitinib-resistant cell line models were established by long-term exposure to gefitinib using the gefitinib-sensitive lung cancer cell lines, PC9 and HCC827. Expression analyses indicated that both FGFR1 and FGF2 were increased in PC9 gefitinib-resistant (PC9 GR) cells as compared with PC9 naïve (PC9 na) cells. Importantly, proliferation of gefitinib-resistant cells was dependent on the FGF2 -FGFR1 pathway. Mechanistically, inhibition of either FGF2 or FGFR1 by siRNA or FGFR inhibitor (PD173074) restored gefitinib sensitivity in PC9 GR cells. These data suggest that FGF2 -FGFR1 activation through an autocrine loop is a novel mechanism of acquired resistance to EGFR-TKIs. Mol Cancer Res; 11(7); 759–67. ©2013 AACR.

Activation of EGFR Bypass Signaling by TGFα Overexpression Induces Acquired Resistance to Alectinib in ALK-Translocated Lung Cancer Cells

Alectinib is a highly selective ALK inhibitor and shows promising efficacy in non–small cell lung cancers (NSCLC) harboring the EML4-ALK gene rearrangement. The precise mechanism of acquired resistance to alectinib is not well defined. The purpose of this study was to clarify the mechanism of acquired resistance to alectinib in ALK-translocated lung cancer cells. We established alectinib-resistant cells (H3122-AR) from the H3122 NSCLC cell line, harboring the EML4-ALK gene rearrangement, by long-term exposure to alectinib. The mechanism of acquired resistance to alectinib in H3122-AR cells was evaluated by phospho-receptor tyrosine kinase (phospho-RTK) array screening and Western blotting. No mutation of the ALK-TK domain was found. Phospho-RTK array analysis revealed that the phosphorylation level of EGFR was increased in H3122-AR cells compared with H3122. Expression of TGFα, one of the EGFR ligands, was significantly increased and knockdown of TGFα restored the sensitivity to alectinib in H3122-AR cells. We found combination therapy targeting ALK and EGFR with alectinib and afatinib showed efficacy both in vitro and in a mouse xenograft model. We propose a preclinical rationale to use the combination therapy with alectinib and afatinib in NSCLC that acquired resistance to alectinib by the activation of EGFR bypass signaling. Mol Cancer Ther; 15(1); 162–71. ©2015 AACR.

Overcoming EGFR bypass signal-induced acquired resistance to ALK tyrosine kinase inhibitors in ALK-translocated lung cancer

Activation of the EGFR pathway is one of the mechanisms inducing acquired resistance to anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib and alectinib. Ceritinib is a highly selective ALK inhibitor and shows promising efficacy in non–small cell lung cancers (NSCLC) harboring the ALK gene rearrangement. However, the precise mechanism underlying acquired resistance to ceritinib is not well-defined. This study set out to clarify the mechanism in ALK-translocated lung cancer and to find the preclinical rationale overcoming EGFR pathway–induced acquired resistance to ALK-TKIs. To this end, ceritinib-resistant cells (H3122-CER) were established from the H3122 NSCLC cell line harboring the ALK gene rearrangement via long-term exposure to ceritinib. H3122-CER cells acquired resistance to ceritinib through EGFR bypass pathway activation. Furthermore, H3122 cells that became resistant to ceritinib or alectinib through EGFR pathway activation showed cross-resistance to other ALK-TKIs. Ceritinib and afatinib combination treatment partially restored the sensitivity to ceritinib. Implications: This study proposes a preclinical rationale to use ALK-TKIs and afatinib combination therapy for ALK-translocated lung cancers that have acquired resistance to ALK-TKIs through EGFR pathway activation. Mol Cancer Res; 15(1); 106–14. ©2016 AACR.

Methylation‐induced downregulation of TFPI‐2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non‐small‐cell lung carcinoma

We identified transmembrane protease, serine 4 (TMPRSS4) as a putative, druggable target by screening surgically resected samples from 90 Japanese non‐small‐cell lung cancer (NSCLC) patients using cDNA microarray. TMPRSS4 has two druggable domains and was upregulated in 94.5% of the lung cancer specimens. Interestingly, we found that TMPRSS4 expression was associated with tissue factor pathway inhibitor 2 (TFPI‐2) expression in these clinical samples. In contrast to TMPRSS4, TFPI‐2 expression was downregulated in NSCLC samples. The in vitro induction of TFPI‐2 in lung cancer cell lines decreased the expression of TMPRSS4 mRNA levels. Reporter assay showed that TFPI‐2 inhibited transcription of TMPRSS4, although partially. Knockdown of TMPRSS4 reduced the proliferation rate in several lung cancer cell lines. When lung cancer cell lines were treated with 5‐aza‐2′‐deoxycytidine or trichostatin A, their proliferation rate and TMPRSS4 mRNA expression levels were also reduced through the upregulation of TFPI‐2 by decreasing its methylation in vitro. The TFPI‐2 methylation level in the low TMPRSS4 group appeared to be significantly low in NSCLC samples (P = 0.02). We found a novel molecular mechanism that TFPI‐2 negatively regulates cell growth by inhibiting transcription of TMPRSS4. We suggest that TMPRSS4 is upregulated by silencing of TFPI‐2 through aberrant DNA methylation and contributes to oncogenesis in NSCLC.

Prognostic implication of PTPRH hypomethylation in non-small cell lung cancer

PTPRH is a receptor-type protein tyrosine phosphatase thought to be a potential regulator of tumorigenesis. The aim of the present study was to clarify the significance of PTPRH expression and its regulation by DNA methylation in non-small cell lung cancer (NSCLC), especially in lung adenocarcinoma (LADC). PTPRH mRNA expression was examined in 89 NSCLC and corresponding non-cancerous tissues. The correlation between DNA methylation and PTPRH gene expression was investigated in another cohort that consisted of 145 patients with LADC, a major NSCLC subtype. Gene regulation by DNA methylation was assessed using a DNA methylation inhibitor. PTPRH mRNA expression was significantly upregulated in NSCLC. PTPRH DNA methylation was reduced in LADC samples and inversely correlated with mRNA expression. 5-Aza-2′-deoxycytidine treatment of lung cancer cell lines with low PTPRH expression, restored mRNA PTPRH expression levels. Furthermore, low PTPRH methylation was associated with shorter recurrence-free survival (P=1.64×10−4) and overall survival (P=5.54×10−5). Multivariate analysis revealed that PTPRH DNA methylation was an independent prognostic factor (P=6.88×10−3). It was confirmed that PTPRH is overexpressed in NSCLC. Furthermore, we determined that PTPRH is epigenetically regulated by DNA hypomethylation, with prognostic implications for LADC.

A Phase II study of S-1 and irinotecan combination therapy in previously treated patients with advanced non-small cell lung cancer

OBJECTIVE This Phase II study was conducted to evaluate the efficacy and safety of S-1 and irinotecan combination therapy as a second-line treatment in patients with advanced non-small cell lung cancer. METHODS Irinotecan was administered at 60 mg/m(2) on Days 1 and 8. Oral S-1 was administered on Days 1-14 every 3 weeks at 80 mg/day for patients with a body surface area of <1.25 m(2), 100 mg/day for patients with a body surface area of 1.25-1.5 m(2) and 120 mg/day for patients with a body surface area of >1.5 m(2). The primary endpoint was response rate, while the secondary endpoints were progression-free survival, overall survival and safety. RESULTS Thirty-one patients were enrolled in this study. The response and disease control rates were 6.5 and 58.1%, respectively. Progression-free survival and median survival time were 2.8 and 12.6 months, respectively. Grade 3-4 adverse events were reported for 29.0% of the patients. Hematological toxicities of Grade 3 or 4 included leukopenia (9.7%), neutropenia (9.7%), febrile neutropenia (3.2%), thrombopenia (3.2%) and anemia (6.5%). Non-hematological toxicities of Grade 3 or 4 included pneumonitis (6.5%), diarrhea, colitis, dyspnea, rash, oral mucositis, anorexia and pulmonary thromboembolism/deep vein thrombosis (3.2% each). CONCLUSIONS S-1 and irinotecan combination therapy at the present dose and schedule exhibited only modest efficacy with mild toxicities in previously treated patients with non-small cell lung cancer. No further clinical investigation with current dose and schedules is warranted for patients with non-small cell lung cancer who failed first-line platinum-based doublet chemotherapy.

Erlotinib as second‑ or third‑line treatment in elderly patients with advanced non‑small cell lung cancer: Keio Lung Oncology Group Study 001 (KLOG001)

The aim of this study was to assess the efficacy and safety of erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), as second- or third-line treatment for elderly Japanese patients with non-small-cell lung cancer (NSCLC). The patients eligible for this phase II trial were aged ≥70 years, had stage III/IV or recurrent NSCLC, and had previously received 1 or 2 chemotherapy regimens that did not include EGFR-TKIs. The patients received erlotinib at a dose of 150 mg/day. The primary endpoint was overall response rate (ORR), and the secondary endpoints were progression-free survival (PFS), overall survival (OS) and toxicity. A total of 38 patients with a median age of 76 years were enrolled. The majority of the patients were men (66%), had an Eastern Cooperative Oncology Group performance status of 1 (58%), stage IV disease (66%) and adenocarcinoma (74%). Of the 35 patients, 13 (34%) had tumors with EGFR mutations. The ORR was 26.3% (95% confidence interval: 12.1-40.5%) and the disease control rate was 47.4%. The median PFS was 3.7 months and the median OS was 17.3 months. The grade 3 adverse events observed included rash (13%), diarrhea (5%), interstitial pneumonitis (5%), anorexia (3%) and gastrointestinal bleeding (3%). Grade 4 or 5 adverse events were not observed. The median OS did not differ significantly between patients aged <75 years (14.9 months) and those aged ≥75 years (19.0 months; P=0.226). Therefore, erlotinib was found to be effective and well-tolerated in elderly patients with previously treated NSCLC.

Abstract 4: ABT-263 is effective in a subset of non-small cell lung cancer cell lines

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rationale: ABT-263 (Navitoclax) is one of the BH3 mimetics targeting anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins such as Bcl-2, Bcl-XL, and Bcl-w, thereby inducing apoptosis. It has been reported that the response to ABT-263 is associated with expressions of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein. Given its effectiveness as a single agent in preclinical studies, ABT-263 is currently being evaluated in clinical trials for small cell lung cancer (SCLC) and leukemia. However, the efficacy of ABT-263 in non-small cell lung cancer (NSCLC) has not been fully evaluated. We examined the effect of ABT-263 on cell proliferation of NSCLC cell lines and investigated the underlying mechanisms. Methods: The following 9 NSCLC cell lines were examined: SK-LU-1, A549, H358, Calu3, H3122, H1975, H460, H441, and BID007. The effects of ABT-263 in NSCLC cell lines were evaluated by MTS assay. Apoptosis was examined by flowcytometry using staining for annexin V and propidium iodide (PI), and also western blotting for cleaved PARP. Quantitative RT-PCR was carried out to assess the mRNA expression levels of anti-apoptotic genes and pro-apoptotic genes. Immunoprecipitation and western blotting were performed to compare the levels of anti-apoptotic and pro-apoptotic proteins between the sensitive and resistant cell lines. In addition, knockdown of Mcl-1 was performed by siRNA. Results: By screening 9 NSCLC cell lines using MTS assay, we found Calu3 and BID007were sensitive to ABT-263. We also confirmed that apoptosis was induced only in the ABT-263 sensitive lines but not in the ABT-263 resistant cell lines after ABT-263 treatment. However, the expression levels of Bcl-2 family proteins, including Mcl-1, did not differ significantly among the ABT-263 sensitive and resistant cell lines. Unlike the results in previous reports regarding SCLC, Mcl-1 was not decreased in the sensitive cell lines. The ABT-263 resistant cell lines became sensitive to ABT-263 after knockdown of Mcl-1 by siRNA, while the ABT-263 sensitive cell lines maintained the same sensitivity. Conclusion: We found that Calu3 and BID007 were sensitive to ABT-263. In the sensitive NSCLC cell lines, ABT-263 induces apoptosis irrespective of Mcl-1 expression levels. Citation Format: Aoi Kuroda, Keiko Ohgino, Hiroyuki Yasuda, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Tetsuo Tani, Shigenari Nukaga, Ichiro Kawada, Katsuhiko Naoki, Kenzo Soejima, Tomoko Betsuyaku. ABT-263 is effective in a subset of non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4. doi:10.1158/1538-7445.AM2015-4

Abstract 5195: Claudin-1, a novel target of miR-375 in non-small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MicroRNAs, the small and noncoding RNAs, regulate the translation of specific protein-coding genes. Accumulated evidence strongly suggests that microRNAs play important and complex roles in human cancers, including lung cancer. We previously reported that miR-375 expression was low in squamous cell carcinoma (SCC) and high in adenocarcinoma (AC) of lung cancer. The target gene of miR-375 in non-small cell lung cancer (NSCLC) has not been elucidated. The purpose of this study was to identify a target of miR-375 and clarify the function of miR-375 in NSCLC. Candidate genes of miR-375 targets were determined using the prediction databases and also previous findings about the different gene expression between SCC and AC. We focused on claudin-1 (CLDN1), which has four putative target sites of miR-375 in its 3′-untranslated region (UTR). CLDN1 was reported to express high in SCC and low in AC opposite to miR-375. We evaluated miR-375 and CLDN1 expression levels by quantitative polymerase chain reaction (qPCR) and Western blotting in 12 NSCLC cell lines. The effect of miR-375 overexpression upon the CLDN1 expression was evaluated in 5 NSCLC cell lines by transfecting miR-375 precursor. It showed that the expression of CLDN1 messenger RNA and protein were attenuated by miR-375 overexpression. Luciferase reporter assay was performed to confirm direct interaction between miR-375 and CLDN1. We cloned 3′-UTR of CLDN1 cDNA into the downstream of a luciferase reporter gene and co-transfected this vector into A549 cells with miR-375 precursor. MiR-375 overexpression resulted in a 3-fold repression of luciferase activity (p < 0.001). To ascertain the clinical validity, we analyzed the relationship between miR-375 and CLDN1 expression in 63 clinical samples of NSCLC. There was a negative correlation between miR-375 and CLDN1 expression (r = -0.35, p = 0.005). In addition, we analyzed the correlation between miR-375 expression and overall survival in the same samples. High miR-375 expression correlated with poor survival in NSCLC (p = 0.043). To investigate the reason why high miR-375 expression lead to poor survival, wound healing assay was performed to evaluate the effect of miR-375 overexpression on the cell migration in SK-MES-1 cells. The cell migration was promoted by miR-375 overexpression, suggesting the high potential of invasion and metastasis in NSCLC expressing high level of miR375. In conclusion, we found that CLDN1 is a novel target of miR-375, and high miR-375 expression leads to poor survival in NSCLC. Citation Format: Satoshi Yoda, Kenzo Soejima, Hiroyuki Yasuda, Takashi Sato, Daisuke Arai, Keiko Ohgino, Kota Ishioka, Tetsuo Tani, Ayano Oashi, Aoi Kuroda, Makoto Nishino, Masayoshi Miyawaki, Junko Hamamoto, Katsuhiko Naoki, Tomoko Betsuyaku. Claudin-1, a novel target of miR-375 in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5195. doi:10.1158/1538-7445.AM2014-5195

Abstract 414: Aberrant DNA methylation and expression of mRNA in EGFR-mutant lung cancer cell line with long-term exposure to gefitinib

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Many of the patients with non-small cell lung cancer (NSCLC) who initially responded well to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) will eventually relapse. The mechanisms of resistance to EGFR-TKIs is not yet fully clarified. The suppression of genes by DNA methylation is reported to be involved in the mechanism of tolerance to cytotoxic drugs. The purpose of this study is to identify epigenetically regulated genes and to clarify the contribution of epigenetic alteration to the acquired resistance to EGFR-TKI. We established gefitinib-resistant PC-9, which was originally gefitinib-sensitive lung cancer cell line, by serial long term exposure to gefitinib. We collected RNA and DNA from both gefitinib-sensitive and -resistant PC-9 cells and performed comprehensive analysis for DNA methylation and mRNA expression using infinium array and cDNA microarray, respectively. We identified 640 genes those DNA methylations were increased in gefitinib-resistant cell line compared to parental cell line. Then, we selected 29 candidate genes those mRNA expression were decreased in resistant PC-9. We are currently underway to elucidate the specific function of each gene and updated data will be presented at the meeting. Citation Format: Hideki Terai, Kenzo Soejima, Katsuhiko Naoki, Hiroyuki Yasuda, Takashi Sato, Daisuke Arai, Keiko Ohgino, Kota Ishioka, Aoi Kuroda, Tetsuo Tani, Ayano Ohashi, Makoto Nishino, Masayoshi Miyawaki, Junko Hamamoto, Tomoko Betsuyaku. Aberrant DNA methylation and expression of mRNA in EGFR-mutant lung cancer cell line with long-term exposure to gefitinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 414. doi:10.1158/1538-7445.AM2014-414