We have recently identified a novel gene, hepaCAM, in liver that encodes a cell adhesion molecule of the immunoglobulin superfamily. In this study, we examined the characteristics of hepaCAM protein and the relationship between its structure and function, in particular its adhesive properties. The wild-type and the cytoplasmic domain-truncated mutants of hepaCAM were transfected into the human breast carcinoma MCF7 cells, and the physiological and biological properties were assessed. Biochemical analyses revealed that hepaCAM is an N-linked glycoprotein phosphorylated in the cytoplasmic domain and that it forms homodimers through cis-interaction on the cell surface. The subcellular localization of hepaCAM appears density-dependent; in well spread cells, hepaCAM is distributed to cell protrusions, whereas in confluent cells, hepaCAM is predominantly accumulated at the sites of cell-cell contacts on the cell membrane. In polarized cells, hepaCAM is recruited to the lateral and basal membranes, and lacking physical interaction, hepaCAM is shown to co-localize with Ecadherin at the lateral membrane. Cell adhesion and motility assays demonstrated that hepaCAM increased cell spreading on the matrices fibronectin and matrigel, delayed cell detachment, and enhanced wound healing. Furthermore, when the cytoplasmic domain was deleted, hepaCAM mutants did not affect cell surface localization and dimer formation. Cell-matrix adhesion, however, was less significantly increased, and cell motility was almost unchanged when compared with the effect of the wild-type hepaCAM. Taken together, the cytoplasmic domain of hepaCAM is essential to its function on cell-matrix interaction and cell motility.

MiRNAs are endogenous ~22 nt RNAs which play critical regulatory roles in a wide range of biological and pathological processes, which can act as oncogenes or tumor suppressor genes depending on their target genes. We have recently shown that ANXA1 inhibits the expression of miRNAs including miR196a. Here, we show that miR196a was highly expressed in ER+ MCF-7 breast cancer cells when compared to normal mammary gland cells, with expression levels negatively correlating to ANXA1. ANXA1 inhibits the biogenesis of oncogenic miR-196a by suppressing primary-miR196a indirectly through the stimulation of c-myc and NFkB expression and activity in breast cancer cells. In a negative feedback loop, miR-196a directly inhibits ANXA1 and enhances breast cancer cell proliferation in vitro. Finally, miR196a promotes breast tumor growth in vivo. This study reports a novel regulatory circuit between ANXA1, NF-kB, c-myc and miR-196a which regulates breast cancer cell proliferation and tumor growth.

Previously, we reported the identification of a novel immunoglobulin-like cell adhesion molecule hepaCAM that is frequently downregulated and inhibits cell growth in hepatocellular carcinoma. In this study, we show that the expression of hepaCAM is suppressed in diverse human cancers. Aiming to evaluate the biological role of hepaCAM in breast cancer, we stably transfected the MCF7 cell line with either wild-type hepaCAM or its mutant hCAM-tailless that lacked the cytoplasmic domain. We found that hepaCAM inhibited colony formation and cell proliferation and arrested cells in the G(2)/M phase. Intriguingly, hepaCAM was capable of inducing cellular senescence as defined by the enlarged cell morphology and increased beta-galactosidase activity. Furthermore, hepaCAM elevated the expression levels of senescence-associated proteins including p53, p21 and p27. In contrast, cell growth inhibition and senescence were less apparent in cells overexpressing hCAM-tailless mutant. To determine if the p53-mediated pathway was involved in hepaCAM-induced senescence, we used the small-interfering RNA system to knock down endogenous p53 expression. In the presence of hepaCAM, downregulation of p53 resulted in a clear reduction of p21, insignificant change in p27 and alleviated senescence. Together, the results suggest that the expression of hepaCAM in MCF7 cells not only inhibits cell growth but also induces cellular senescence through the p53/21 pathway.

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