Loss of AF-6/afadin induces cell invasion, suppresses the formation of glandular structures and might be a predictive marker of resistance to chemotherapy in endometrial cancer

Abstract

BackgroundAF-6/afadin plays an important role in the formation of adherence junctions. In breast and colon cancer, loss of AF-6/afadin induces cell migration and cell invasion. We aimed to elucidate the role of AF-6/afadin in human endometrial cancer.MethodsMorphology and AF-6/afadin expression in endometrial cancer cell lines was investigated by 3-dimensional culture. We used Matrigel invasion assay to demonstrate AF-6/afadin knockdown induced invasive capability. Cell proliferation assay was performed to estimate chemoresistance to doxorubicin, paclitaxel and cisplatin induced by AF-6/afadin knockdown. The associations between AF-6/afadin expression and clinicopathological status were determined by immunohistochemical analysis in endometrial cancer tissues. Informed consent was obtained from all patients before the study.ResultsThe majority of cell clumps in 3-dimensional cultures of Ishikawa cells that strongly expressed AF-6/afadin showed round gland-like structures. In contrast, the cell clumps in 3-dimensional cultures of HEC1A and AN3CA cells—both weakly expressing AF-6/afadin—showed irregular gland-like structures and disorganized colonies with no gland-like structures, respectively. AF-6/afadin knockdown resulted in reduced number of gland-like structures in 3-dimensional cultures and enhancement of cell invasion and phosphorylation of ERK1/2 and Src in the highly AF-6/afadin-expressing endometrial cancer cell line. Inhibitors of MAPK/ERK kinase (MEK) (U0126) and Src (SU6656) suppressed the AF-6/afadin knockdown-induced invasive capability. AF-6/afadin knockdown induced chemoresistance to doxorubicin, paclitaxel and cisplatin in Ishikawa cells, not in HEC1A. Immunohistochemical analysis showed that AF-6/afadin expression was significantly associated with myometrial invasion and high histological grade.ConclusionsAF-6/afadin regulates cell morphology and invasiveness. Invasive capability is partly regulated through the ERK and Src pathway. The inhibitors to these pathways might be molecular-targeted drugs which suppress myometrial invasion in endometrial cancer. AF-6/afadin could be a useful selection marker for fertility-sparing therapy for patients with atypical hyperplasia or grade 1 endometrioid adenocarcinoma with no myometrial invasion. AF-6/afadin knockdown induced chemoresistance especially to cisplatin. Therefore, loss of AF-6/afadin might be a predictive marker of chemoresistance to cisplatin.

Anti-tumor effect of estrogen-related receptor alpha knockdown on uterine endometrial cancer

“…The research protocol was approved by the Institutional Review Board and an informed consent was obtained from all patients prior to the beginning of the study. Immunohistochemical staining was performed as previously described [43]. Furthermore, the expression level of ERRα was assessed by H-score, a commonly used method to measure the strength of ER- and ERR-staining, which semi-quantitatively evaluates both the intensity and the percentage of cells stained at each intensity [24].…”

“…Migration and invasion assays were performed using uncoated and Matrigel-coated double-chamber systems (BD BioCoat TM Matrigel TM Invasion Chamber, BD Biosciences, Bedford, MA, USA) as previously described [43]. Briefly, cancer cells were seeded into 24-well lower chamber filled with 10% FBS contained medium and, after 24 h, cells were transfected with siRNAs.…”

Listeria monocytogenesInlP interacts with afadin and facilitates basement membrane crossing

“…Conditional knockouts in the mouse intestine lead to increases in intestinal permeability (65), consistent with the phenotype we have described here of afadin loss causing permeabilization of cell-cell junctions in MDCK cells. Whether afadin also plays a role in cell-ECM connections is unclear, but in certain cell types, removal of afadin can enhance cell migration (66) and invasive capacity (67), or suppress neuronal axon branching (68). While these cellular behaviors might involve specific changes in cell-ECM interactions, it is also likely in these cases that the effects of afadin disruption are mediated indirectly through its interactions with critical signaling molecules, Rap1, Src and R-Ras respectively (66-68).…”

The Contribution of Ig-Superfamily and MARVEL D Tight Junction Proteins to Cancer Pathobiology

“…Occludin is widely expressed in both endothelial and epithelial cells (55), and has been identified in multiple tissues including kidney, skin, blood vessels, liver and bladder (56).…”

“…In order to locally invade as an early pre-requisite for metastasis to distant sites, one paradigm suggests that primary tumor cells must develop the ability to loosen TJs, and accordingly decreased expression of occludin has been associated with some cancers (56). Loss of occludin expression has been associated with altered TJ function and increased invasiveness in breast cancer cell lines, and expressional downregulation of occludin protein (but not mRNA) has been noted in conjunction with increasing stage in breast cancer tissue (55).…”