Adrenomedullin expression in epithelial ovarian cancers and promotes HO8910 cell migration associated with upregulating integrin α5β1 and phosphorylating FAK and paxillin

Abstract

Background: Epithelial ovarian cancer (EOC) is one of the leading causes of cancer deaths in women worldwide. Adrenomedullin (AM) is a multifunctional peptide which presents in various kinds of tumors.

Methods: In this study, we characterized the expression and function of AM in epithelial ovarian cancer using immunohistochemistry staining. Exogenous AM and small interfering RNA (siRNA) specific for AM receptor CRLR were treated to EOC cell line HO8910. Wound healing assay and flow cytometry were used to measure the migration ability and expression of integrin α5 of HO8910 cells after above treatments. Western blot was used to examine the phosphorylation of FAK and paxillin.

Results: We found that patients with high AM expression showed a higher incidence of metastasis, larger residual size of tumors after cytoreduction and shorter disease-free and overall survival time. Exogenous AM induced ovarian cancer cell migration in time- and dose- dependent manners. AM upregulated the expression of integrin α5 and phosphorylation of FAK, paxillin as well.

Conclusions: Our results suggested that AM contributed to the progression of EOC and had additional roles in EOC cell migration by activating the integrin α5β1 signaling pathway. Therefore, we presumed that AM could be a potential molecular therapeutic target for ovarian carcinoma.

Figure 1

AM expression in EOC samples. Immunohistochemical analysis of AM expression in EOCs. EOCs: FIGO III stage serous (i), FIGO I stage serous (ii), mucinous (iii), clear-cell (iv), endometrioid (v) ovarian cancer, malignant Brenner tumor of ovary (vi).

Figure 2

Correlation between AM status and EOC patient prognosis. Kaplan-Meier curves for disease-free survival rate of EOC patients according to the AM expression status (A); Kaplan-Meier total survival curves of EOC patients according to the AM expression status (B). Both survival time and disease-free time of patients were linked to AM expression status (Log-rank test, P = 0.020; P = 0.030).

Figure 3

Enhanced migration by AM in time-dependent and dose-dependent manners.

Figure 4

Down-regulation of CRLR expression in HO8910 cells inhibited influence of exogenous AM on cell migration. Reduced CRLR mRNA expression (A) and protein expression (B) were determined by real-time PCR analysis or western blot in CRLR siRNA transfected cells, compared with scrambled siRNA transfected cells. After cells were transfected by CRLR siRNA, the effect of AM on cells migration was decreased consequently (C).

Figure 5

Exogenous AM promoted cell migration with increased integrin α5β1 activation. FACS flow analysis showed increased expression of integrin α5 in AM treated HO8910 cells than in non-treated cells (A). Blocking antibody of integrin α5β1 inhibited the effect of AM on cell migration (B). Exogenous AM promoted FAK and paxillin phosphorylation at different time point (C). Blocking antibody of integrin α5β1 abolished the AM promotion on FAK, paxillin phosphorylation (D).