Functional characterization of BCL2A1 in ovarian cancer development and progression

Abstract

Ovarian cancer is the most lethal gynecological malignancy worldwide. Different from other human cancers, ovarian cancer metastasis occurs primarily through the peritoneal dissemination, which is usually associated with poor prognosis. Ovarian cancer cells have to surmount various stress factors from the tumor microenvironment during peritoneal metastasis. Hypoxia is one of the most common stress factors due to its crucial role in regulating cancer progression in tumor microenvironment. The hypoxic niche of ascites has been reported promoting ovarian cancer metastasis. Thus, hypoxic-cell targeted therapy has currently become a promising therapeutic approach to impede peritoneal metastases in ovarian cancer. To this end, a cDNA microarray profiling was performed and identified BCL2A1, a BCL2 family member, as a hypoxia inducible gene. Besides, BCL2A1 could also be induced by other physiological stresses. BCL2A1 was induced immediately by stresses, followed by a gradual reduction in expression, indicating there is an auto-regulation system in ovarian cancer cells for BCL2A1 expression. Functional studies revealed that BCL2A1 enhanced foci formation ability in serum starvation medium, anoikis resistance, tumor colony formation capacity in both soft agar and ex vivo cultured mice omenta, indicating that BCL2A1 is involved in cell survival of ovarian cancer cells especially under stressed conditions. Cytochrome c detection by Western blot analysis and in situ PLA assay indicated that this process is mediated by the intervention on the intrinsic apoptotic pathway, where BCL2A1 interacts with and inhibits the BH3 only proteins Bid/Bad/HRK. Consistently, the in vivo peritoneal tumor dissemination assay showed that the depletion of BCL2A1 expression significantly impaired the tumor peritoneal colonization ability of ovarian cancer cells, as well as increased the survival probability of the host SCID mice. Moreover, using primary cancer cells isolated from the ovarian cancer patient samples, BCL2A1 was found to have higher expression in the cancer cells derived from ascites than that from the primary solid tumor. This verifies that the metastatic ovarian cancer cells require a higher BCL2A1 expression for survival in stressed ascites microenvironment. Interestingly, the prolonged overexpression of BCL2A1 retarded the cell cycle progression by G1 arrest in ovarian cancer cells in normal culture, whereas depletion of endogenous BCL2A1 promoted the G1/S transition. These results suggested that high level of BCL2A1 inhibited cell proliferation cultured in normal condition. Western blot analysis showed that this process is mediated by the high expression of proteins p27 and p18 in BCL2A1 expressing cells. Consistently, Western blot analysis of cultured cells and immunohistochemical staining of ovarian tumor xenograft showed that the adaptation or removal of stresses restored the BCL2A1 expression to a normal basal level, probably serving to relieve this cell cycle progression inhibition. Additionally, an array of biochemical studies revealed that BCL2A1 is a transcription target gene of NF-κB, and the highly regulation of BCL2A1 by hypoxia is mediated by NF-κB signaling. Taken together, these findings suggest that BCL2A1 plays dual roles in ovarian cancer metastasis, and could serve as a promising therapeutic target for impeding peritoneal metastases of ovarian cancers.