p16Ink4a enhances the migration and metastasis phenotype of hepatocellular carcinoma cells

[[abstract]]Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. The occurrence of tumor metastasis to the extra-hepatic region of the portal vein, lymph nodes, lungs or bones contributes to the high mortality seen in HCC; yet, the molecular mechanisms for HCC metastasis remain unclear. We have previously shown that concomitant loss of Ink4a/Arf locus accelerated tumor formation and metastasis in mice with liver specific Trp53 deletion, and demonstrated that loss of p19Arf enhances the migration and invasion activities of HCC cells. In this study, we characterized the role of p16Ink4a in tumorigenesis- and metastasis-related phenotypes in HCC cells. Re-introduction of p16 into Trp53 and Ink4a/Arf double null HCC cells unexpectedly led to an increase in cell migration. Similar observations were made after ectopic expression of p16 in three additional HCC cell lines, including HepG2, indicating that this is not a cell line-specific phenomenon. Importantly, knockdown of p16 in a p16 wild-type HCC cell line reduced cell migration, providing support for a significant role of p16 in this process. Consistent with its role as a tumor suppressor, ectopic p16 expression reduced soft agar colony formation in MM189 cells, although surprisingly it did not significantly reduced cell proliferation and subcutaneous tumor growth in immune-comprised mice. Analysis of p16 mutants indicated that interaction with Cdk4, and to a lesser degree Cdk6, was required for its stimulation of HCC cell migration. Furthermore, using p16 tagged with either a nuclear export signal (NES) or nuclear localization signal (NLS), we found that nuclear-cytoplasmic shuttling of p16 was required for p16-stimulated migration. Our results, consistent with published finding regarding the functions of another Cdk inhibitor, p27, suggest a novel role for p16 in regulating HCC cell migration