Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth

Abstract

Afadin/AF6, an F-actin-binding protein, is ubiquitously expressed in epithelia and has a key role during development, through its regulatory role in cell-cell junction organization. Afadin loss of expression in 15% of breast carcinoma is associated with adverse prognosis and increased risk of metastatic relapse. To determine the role of afadin in breast cancer, we studied the functional consequences of afadin protein extinction using in vitro and in vivo models. Three different breast cancer cell lines representative of the major molecular subtypes were stably repressed for afadin expression (knockdown of afadin (afadin KD)) using RNA interference. Collective and individual migrations as well as Matrigel invasion were markedly increased in afadin KD cells. Heregulin-b1 (HRG-b1)-induced migration and invasion were increased by twofold in afadin KD cells. Conversely, ectopic expression of afadin in the afadin-negative T47D cell line inhibited spontaneous and HRG-b1-induced migrations. RAS/MAPK and SRC kinase pathways were activated in afadin KD cells. Activation levels positively correlated with migration and invasion strength. Use of MEK1/2 (U0126) and SRC kinases (SU6656) inhibitors reduced afadin-dependent migration and invasion. Afadin extinction in the SK-BR-3 cell line markedly accelerated tumor growth development in mouse mammary gland and lung metastasis formation. These results may explain why the loss of afadin expression in tumors correlates with high tumor size and poor metastasis-free survival in patients.

Loss of polarity protein AF6 promotes pancreatic cancer metastasis by inducing Snail expression

“…Stable AF6-overexpressing Capan-1 and Capan-2 cell lines were generated using lentiviral vector pRRL-AF6FL; this vector, which encodes full-length AF6, was generously provided by Dr M Lopez (University of the Mediterranee, Marseille, France). Lentivirus production and cell-line constructions were performed as described previously 45 ; in brief, the virus particles were produced by transient transfection into 293T cells seeded in 6-cm dishes with 2.7 mg of the packaging construct, 0.9 mg of the construct that express the vesicular stomatitis virus glycoprotein and 2.7 mg of the pRRL-AF6FL vector, and the media from the transfected cells was collected as the cells were re-fed each day for 3-4 days. The virus-containing media was pooled, filtered and used to generate AF6-overexpressing stable cell lines.…”

“…The phenotypic changes observed with these mice include disorganization of the ectoderm, impaired migration of the mesoderm and loss of somites or other structures derived from both the ectoderm and the mesoderm 10,11 . Recently, expression of the AF6 gene was found to be aberrant in multiple cancer types, including leukaemia, ovarian and breast cancers 3,4,[12][13][14][15][16] , but the specific roles that AF6 might play in tumorigenesis remain unclear.…”

PI3K/AKT/Afadin signaling pathway contributes to pathological vascularization in glioblastomas

“…Previous studies have indicated that Afadin regulates migration and cell-cell adhesion in other cell types, including fibroblasts and epithelial cells (28,29). A previous study indicated that Afadin is associated with angiogenesis through Rap1 signaling (30).…”

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

“…Occludin has four known splice variants (52), and knockout studies of the parent molecule have indicated its importance in tight junction stability and maintenance in epithelial cells (53). Additionally, occludin has been established as a regulator of epithelial cell migration, with phosphorylation at the Y473 residue resulting in PI3K activation at the leading edge of migratory cells (54).…”