Abstract
Epithelial ovarian cancer is the most lethal gynecological cancer mainly due to late diagnosis, easy spreading and rapid development of chemoresistance. Cancer stem cells are considered to be one of the main mechanisms for chemoresistance, as well as metastasis and recurrent disease. To explore the stemness characteristics of ovarian cancer stem cells, we successfully enriched ovarian cancer stem-like cells from an established ovarian cancer cell line (SKOV-I6) and a fresh ovarian tumor-derived cell line (OVS1). These ovarian cancer stem-like cells possess important cancer stemness characteristics including sphere-forming and self-renewing abilities, expressing important ovarian cancer stem cell and epithelial–mesenchymal transition markers, as well as increased drug resistance and potent tumorigenicity. Microarray analysis of OVS1-derived sphere cells revealed increased expression of amphiregulin (AREG) and decreased expression of its conserved regulatory microRNA, miR-34c-5p, when compared with the OVS1 parental cells. Overexpression of AREG and decreased miR-34c-5p expression in SKOV-I6 and OVS1 sphere cells were confirmed by quantitative real-time PCR analysis. Luciferase reporter assay and mutant analysis confirmed that AREG is a direct target of miR-34c-5p. Furthermore, AREG-mediated increase of sphere formation, drug resistance toward docetaxel and carboplatin, as well as tumorigenicity of SKOV-I6 and OVS1 cells could be abrogated by miR-34c-5p. We further demonstrated that miR-34c-5p inhibited ovarian cancer stemness through downregulation of the AREG-EGFR-ERK pathway. Overexpression of AREG was found to be correlated with advanced ovarian cancer stages and poor prognosis. Taken together, our data suggest that AREG promotes ovarian cancer stemness and drug resistance via the AREG-EGFR-ERK pathway and this is inhibited by miR-34c-5p. Targeting AREG, miR-34c-5p could be a potential strategy for anti-cancer-stem cell therapy in ovarian cancer.
Highlights
Considering AREG is the ligand of epidermal growth factor receptor (EGFR) and the promotion effects of AREG on ovarian cancer stemness and drug resistance, we further investigated if miR-34c-5p inhibited ovarian cancer stemness and drug resistance through the AREG-EGFR-ERK axis
We found increase of several proteins in the AREG-EGFR-ERK pathway including AREG and phosphorylated forms of EGFR, Raf, MEK and ERK in SKOV-I6 and OVS1 sphere cells compared with their parental cells (Figure 6a)
We examined if miR-34c-5p inhibited ovarian cancer stemness and drug resistance through downregulation of the AREG-EGFR-ERK pathway
Summary
Epithelial ovarian cancer is the most lethal gynecological cancer.[1,2] Its high mortality rate is mainly due to late diagnosis, easy spreading, and rapid development of chemoresistance.[1,2,3] Cancer stem cells are considered to be in part account for chemoresistance, as well as metastasis and recurrent disease.[4,5,6] Cancer stem cells are defined as a very small subpopulation of tumor cells possessing the ability to self-renew and differentiate leading to the formation of heterogeneous progeny forming the tumor.[4,7] number of unique genes and microRNAs (miRNAs) have been found to regulate ovarian cancer stem cells, effective and clinically applicable inhibitors against ovarian cancer stem cells are yet to be developed.[8,9,10,11,12,13,14]. Altered expression levels of AREG and miR-34c-5p were found in those ovarian cancer stem-like cells. The effects of AREG and miR-34c-5p on ovarian cancer stemness and drug resistance were investigated for the first time. Our study has demonstrated that AREG has an important role in promoting ovarian cancer stemness and drug resistance. We are the first to identify that miR-34c-5p inhibits ovarian cancer stemness and drug resistance through downregulation of the AREG-EGFR-ERK pathway. These results provided important evidence to support miR-34c-5p and AREG as promising candidates for anti-cancer-stem cell therapy in ovarian cancer. None of the mice implanted with OVS1 parental cells formed xenograft tumors (Table 1). All xenograft tumors derived from OVS1 sphere cells were resected for pathological examination and were categorized as adenocarcinoma resembling the tumor phenotype of the fresh ovarian tumor which OVS1 cell line was derived from (Figure 1g; Supplementary Figure 4)
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