Dataset Information

Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

ABSTRACT: Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel therapeutic strategy to treat advanced bladder cancer.

SUBMITTER: Kang M

PROVIDER: S-EPMC5343857 | biostudies-other | 2017 Feb

REPOSITORIES: biostudies-other

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Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

Kang Minyong M Lee Kyoung-Hwa KH Lee Hye Sun HS Jeong Chang Wook CW Kwak Cheol C Kim Hyeon Hoe HH Ku Ja Hyeon JH

International journal of molecular sciences 20170204 2

Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquin ...[more]

PMID: 28165387

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Project description:Background: The 14-3-3? protein, which acts as a putative oncoprotein, has been found to promote the proliferation, metastasis, and chemoresistance of cancer cells in several cancers including lung adenocarcinoma (LUAD); however, its significance in epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance remains unknown. Methods: The Cancer Genome Atlas (TCGA) database was used to determine 14-3-3? expression in pancancer and LUAD. 14-3-3? and ID1 expression was then examined in clinical LUAD samples by immunohistochemistry (IHC). Lentiviral transfection with 14-3-3?-specific small hairpin RNA (shRNA) was used to establish stable 14-3-3? knockdown gefitinib-resistant PC9 (PC9/GR) and H1975 cell lines. The effect of 14-3-3? knockdown on reversing EGFR-TKI resistance was determined in vitro by Cell Counting Kit-8 (CCK-8), wound healing, Transwell assays, and flow cytometry. A xenograft tumor model was established to evaluate the role of 14-3-3? in EGFR-TKI resistance. Microarray analysis results showed multiple pathways regulated by 14-3-3?-shRNA. Results: In the present study, we demonstrated that based on the TCGA, pancancer and LUAD 14-3-3? expression was elevated and predicted unfavorable prognosis. In addition, high 14-3-3? expression was associated with advanced T stage, TNM stage, presence of lymph node metastasis and, importantly, poor treatment response to EGFR-TKIs in LUAD patients with EGFR-activating mutations. 14-3-3? shRNA sensitized EGFR-TKI-resistant human LUAD cells to gefitinib and reversed epithelial-to-mesenchymal transition (EMT). After 14-3-3? depletion, bone morphogenetic protein (BMP) signaling activation was decreased in EGFR-TKI-resistant cells in microarray analysis, which was further validated by Western blot analysis. Furthermore, the expression of 14-3-3? positively correlates with ID1 expression in human EGFR-mutant LUAD patient samples. In vivo, there was a reduction in the tumor burden in mice treated with 14-3-3? shRNA and gefitinib compared to mice treated with gefitinib alone. Conclusion: Our work uncovers a hitherto unappreciated role of 14-3-3? in EGFR-TKI resistance. This study might provide a potential therapeutic approach for treating LUAD patients harboring EGFR mutations.

Dataset Information

Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

Publications

Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

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