HIN-1, an inhibitor of cell growth, invasion, and AKT1 activation
© BioMed Central 2005
Published: 17 June 2005
High in normal 1 (HIN-1) is a small, secreted protein that was initially identified as a protein the expression of which is lost in the vast majority of breast cancers. The silencing of HIN-1 expression is due to methylation of its promoter, which in addition to breast cancer also occurs in a significant fraction of many other types of solid tumors including prostate cancer, lung cancer, pancreas cancer, and retinoblastoma, suggesting a potential tumor suppressor function. Consistent with this hypothesis, in non-small-cell lung cancer, downregulation of HIN-1 expression was found to be the most significant independent predictor of poor clinical outcome in stage I disease, suggesting loss of HIN-1 expression is a functionally important event. The receptor of HIN-1 is unknown, but ligand-binding studies indicate the presence of high-affinity cell surface HIN-1 binding sites on the same epithelial cells that express HIN-1, suggesting that HIN-1 functions as an autocrine factor.
To further explore the function and mechanism of action of HIN-1, we examined the effect of HIN-1 on immortalized mammary epithelial cells and a panel of breast cancer cell lines in vitro. HIN-1 was delivered via multiple expression systems, including a tetracycline-regulated expression system, recombinant adenovirus, and recombinant HIN-1 fusion protein.
We find that HIN-1 is a potent inhibitor of anchorage-dependent and anchorage-independent cell growth. Expression of HIN-1 in MDA-MB-435 cells causes reversion of their invasive morphology when these cells are grown in a three-dimensional culture system. As further evidence that HIN-1 can influence invasive behavior, expression of HIN-1 markedly inhibited the invasion and migration of breast cancer cell lines in Boyden chamber assays. Expression of HIN-1 in synchronized cells inhibits cell cycle re-entry and the phosphorylation of the retinoblastoma protein (Rb), while in exponentially growing cells HIN-1 induces apoptosis without apparent cell cycle arrest or an effect on Rb phosphorylation. To begin to dissect the mechanism by which HIN-1 suppresses growth, we analyzed the activation status of various signal transduction pathways involved in cell proliferation and survival using activation state specific antibodies. This investigation revealed that mitogen-induced phosphorylation of Akt (Ser 473) is inhibited in HIN-1-expressing cells. Expression of HIN-1 also inhibits Akt-mediated retention of p27 in the cytoplasm. Further supporting the role of Akt in HIN-1-mediated growth inhibition, expression of constitutively activated Akt abrogates HIN-1-mediated growth arrest.
Taken together, these studies provide further evidence that HIN-1 possesses tumor suppressor functions and suggest that these activities may be mediated through the Akt signaling pathway.