Sensitivity Of Gastric Cancer Cell Line Research Articles

Anti-apoptotic protein BCL-XL as a therapeutic vulnerability in gastric cancer.

New therapeutic targets are needed to improve the outcomes for gastric cancer (GC) patients with advanced disease. Evasion of programmed cell death (apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro-survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. Here we explored the role of BCL2L1 and the encoded anti-apoptotic BCL-XL in GC. Using Droplet Digital PCR (ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL-XL inhibitors A1155463 and A1331852, pan-inhibitor ABT-263, and VHL-based PROTAC-BCL-XL was analyzed using (CellTiter-Glo) CTG assay in vitro. Western Blot (WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC-BCL-XL kills GC cells. Co-immunoprecipitation (Co-IP) was used to investigate the mechanism of A1331852 and ABT-263 kills GC cell lines. DDPCR, WB, and real-time PCR (RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. The functional assay showed that a subset of GC cell lines relies on BCL-XL for survival. In gastric cancer cell lines, BCL-XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT-263, indicating that ABT-263 is not an optimal inhibitor of BCL-XL. VHL-based PROTAC-BCL-XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time- and dose-dependent manner through the proteasome pathway. Statistical analysis showed that the BCL-XL protein level predicts the response of GC cells to BCL-XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL-XL expression. We identified BCL-XL as a promising therapeutic target in a subset of GC cases with high levels of BCL-XL protein expression. Functionally, we demonstrated that both selective BCL-XL inhibitors and VHL-based PROTAC BCL-XL can potently kill GC cells that are reliant on BCL-XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL-XL expression, but the BCL-XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL-XL-targeting compounds. Taken together, our study pinpointed BCL-XL as potential druggable target for specific subsets of GC.

Up-regulation of KLF17 expression increases the sensitivity of gastric cancer to 5-fluorouracil.

It has been reported that the expression of Krüppel-like factor 17 (KLF17) was associated with the occurrence, development, invasion, metastasis and chemotherapy resistance of various tumors. However, the detailed mechanisms by which KLF17 promotes chemotherapy resistance in gastric cancer (GC) have not been fully investigated. In the present study, we collected the GC tissues and non-tumor tissues (matched adjacent normal tissues with corresponding GC tissues) of 60 GC patients, used qRT-PCR, Western blot and immunohistochemistry assay to analyze the relationship between the expression of KLF17 and the clinical pathological data of the patients. The effect of KLF17 on the sensitivity of GC cell lines to 5-fluorouracil (5-FU), and the potential mechanism were detected by MTS assay, Flow cytometry assay, and Western blot. Compared with non-tumor tissues, the expression level of KLF17 in GC tissue was significantly down-regulated, and the expression level of KLF17 in GES-1 cell line and GC cell lines also had a similar trend. Down-regulated expression of KLF17 is related to tumor size, invasion, regional lymph node metastasis, and TNM staging. Furthermore, through upregulating the expression of KLF17, the sensitivity of BGC-823 and SGC-7901 cell lines to 5-FU was obviously increased. Mechanistically, upregulation the expression of KLF17 can inhibit the expressions of P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and B-Cell lymphoma-2 (BCL-2), which have been reported to be associated with drug resistance and cell proliferation. Collectively, these data implied that KLF17 has the biological effect of inhibiting chemotherapy resistance of GC, and it could be a potential strategy for the GC chemotherapy resistance.

Prolonged MEK inhibition leads to acquired resistance and increased invasiveness in KRAS mutant gastric cancer.

Gastric cancer (GC) is one of the most common causes of cancer-associated death. However, traditional therapeutic strategies have failed to significantly improve the survival of patient with advanced GC. While KRAS mutations have been found in some patients with gastric cancer, an effective therapy to treat KRAS-driven gastric cancer has not been established yet. To provide a rationale for clinical application of kinase inhibitors targeting RAS pathways, we first determined the sensitivity of GC cell lines harboring KRAS mutations or amplification to RAS pathway inhibitors. We found that MAPK pathway inhibitors (MEKi and ERKi) were more effective than AKT inhibitor, suggesting that KRAS-driven gastric cancer cells are dependent on MAPK pathway for survival. Further, we established a KRAS mutant GC cell line with acquired resistance to MEK inhibitors in order to mimic clinical situation of kinase inhibitor resistance. A comprehensive analysis of tyrosine phosphorylation in receptor tyrosine kinases in combination with small molecule chemical library screening revealed upregulated c-MET phosphorylation in this resistance cell line with elevated sensitivity to c-MET TKI (crizotinib) and PI3K/mTOR dual inhibitor (BEZ235). We also showed that migration and invasion of resistant cells were promoted, and crizotinib and BEZ235 could inhibit this malignant phenotype. Overall, our results indicate that prolonged MAPK pathway inhibition could result in acquired resistance which is associated with increased malignant phenotype in KRAS mutant GC and pharmacological targeting c-MET and PI3K/mTOR could overcome this problem.

Transcription Factor AP2ε: A Potential Predictor of Chemoresistance in Patients With Gastric Cancer.

Chemotherapy is a mainstay of therapy for advanced gastric cancer (GC); however, owing to drug resistances, the effectiveness of chemotherapy is not satisfactory for some patients with GC. Therefore, identification of a marker that predicts treatment response is beneficial to patients. Hypermethylation of transcription factor activating enhancer-binding protein 2∊ (TFAP2E) has been implicated in chemotherapy resistance to fluorouracil-based chemotherapy in patients with colorectal cancer, but its role in GC is still unknown. In this study, we investigated TFAP2E as a predictor of treatment response in GC. We used methylation-sensitive high-resolution melting analysis to study the methylation of TFAP2E in 141 GC tissue specimens and 45 adjacent nontumor tissue specimens. In vitro experiments, we analyzed the expression and methylation of TFAP2E and to examine the sensitivity of GC cell lines to 5-fluorouracil (5-FU). The TFAP2E methylation occurred at a significantly higher incidence rate in tumor tissues compared to adjacent nontumor tissues (chi-square [χ2] = 38.919, P < .001). Hypermethylation of TFAP2E occurred more frequently in tumors with lower differentiation grades (P < .001) and was significantly associated with nonresponse to fluorouracil-based chemotherapy (P = .010). Hypermethylation was also associated with decreased expression of TFAP2E (P < .01) and nonresponse to 5-FU exposure in vitro (P < .001). Hypermethylation of TFAP2E was associated with lack of response to fluorouracil-based chemotherapy, indicating that it might be a potential predictor of treatment response in patients with GC.

Abstract 2398: Activity of the PARP inhibitor olaparib in ATM-deficient gastric cancer: from preclinical models to the clinic

Abstract Olaparib is an oral inhibitor of the Poly-(ADP-ribose)-polymerases (PARP-1, -2 and -3), and has demonstrated clinical activity in trials of patients with BRCA-deficient tumors. The BRCA1 and BRCA2 proteins are both important for the repair of DNA double strand breaks (DSBs) by homologous recombination repair (HRR) and sensitivity to olaparib has further been shown preclinically to extend to other DNA DSB repair factor deficiencies, including those in the ataxia telangiectasia mutated (ATM) protein. We have demonstrated that a high proportion of gastric cancer (GC) cell lines (∼50%) are responsive (IC50 &amp;lt;500 nM) to single agent olaparib and that characterization of their DNA repair status reveals that, as in breast and ovarian cancer, HRR deficiencies (HRD) can account at least in part for this. However, unlike breast and ovarian cancer cell lines, there is no strong correlation with platinum sensitivity, suggesting alternative drivers of response in the GC panel to HRD. Here, we demonstrate that low levels of ATM protein expression correlate with sensitivity of GC cell lines to olaparib, and moreover can be observed in approximately 15-20% of clinical gastric cancer samples when evaluated using ATM immunohistochemistry (IHC). Based on these data, a Phase II GC trial has been carried out that enriches for patients with tumors having low or undetectable ATM IHC scores (H-score equivalent less than or equal to 10). Data from this clinical trial confirm activity (overall survival [OS] benefit HR=0.56, 95% CI: 0.35-0.87; P=0.010) in the overall population, which includes ATM tumor positive patients, and importantly, a substantially better OS benefit in the ATM-negative population (HR=0.35, 95% CI: 0.17-0.71; P=0.003). Together, these data provide an important example of a PARP inhibitor demonstrating both preclinical and clinical activity in a well defined but non-BRCA DNA repair deficient background. Citation Format: Darren Hodgson, Helen Mason, Lenka Oplustilova, Chris Harbron, Xiaolu Yin, Seock-Ah Im, Helen Jones, Lai Zhongwu, Brian Dougherty, Matthew McLoughlin, James Bradford, Andrew Dickinson, Anitra Fielding, Jane Robertson, Woo-Ho Kim, Chris Womack, Yi Gu, Yung-Jue Bang, Alan Lau, J. Carl Barrett, Mark J. O'Connor. Activity of the PARP inhibitor olaparib in ATM-deficient gastric cancer: from preclinical models to the clinic. [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 2398. doi:10.1158/1538-7445.AM2014-2398