Cisplatin (CDDP) resistance is the foremost obstacle in the treatment of ovarian cancer patients. According to the cancer stem cell hypothesis, the recurrence and chemoresistance are presumed to be linked to cancer stem/progenitor cells. Here, we investigated the cancer stem cell like phenotypes and mechanism of chemoresistance in CDDP resistant ovarian cancer cells.
A well-established CDDP sensitive ovarian cancer cell line A2780 and its resistant clone A2780 Cp were used and we also developed a supra resistant clone (SKOV3 Cp) from a naturally CDDP resistant cell line SKOV3 by six shocks treatment. Sensitiveness to CDDP was evaluated by MTT assay and self renewal efficiency by sphere formation, noble agar and clonogenic assays. A few cancer hallmarks and stem-cell markers were characterized using flow cytometry. Molecular analysis was performed using quantitative PCR and western blotting. For the silencing of selected genes small interfering RNA (siRNA) based approaches was utilized.
Both CDDP resistant A2780 Cp and supra resistant SKOV3 Cp cell lines showed significantly (P < 0.05) higher self-renewal ability in sphere formation, noble agar and clonogenic assays than their parental counterparts. They also showed significant resistance to apoptosis and sub-G1 arrest upon CDDP treatment. Stem cell marker ALDH1 positivity rates were higher both in A2780 Cp and SKOV3 Cp cell lines, quantified by ALDEFLUORTM assay kit using flow cytometry. Hoechst 33342 dye effluxing side populations were increased about five folds in A2780 Cp cells and two folds in SKOV3 Cp cells compared to A2780 and SKOV3 cells respectively. Among major stemness related genes (OCT4, SOX2, NANOG, NESTIN, BMI1, KLF4 and ALDH1A1) quantitative PCR analysis showed significantly higher expression of ALDH1A1 and KLF4 genes in both resistant and supra resistant cells than their parental clones. Of two genes, silencing ALDH1A1 in A2780 and A2780 Cp cells using siRNA greatly reduced the stem cell population as detected by ALDEFLUORTM assay and sensitized cells to CDDP. Moreover, silencing of ALDH1A1 reduce the level of its downstream target NEK-2. We also observed the downregulation of ABC transporters (ABCB1, ABCG2 and ABCC1) by silencing either ALDH1A1 or NEK-2 gene.
Taken together, present study showed for the first time that stemness gene ALDH1A1 can be involved in CDDP resistance via the upregulation of NEK-2 in ovarian or solid cancer.
Citation Format: Md. Hafiz Uddin, Yong Sang Song. Association of ALDH1A1 with cisplatin resistance via modulation of NEK-2 in ovarian cancer stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2511.