The Hic-5 protein is encoded by a transforming growth factor-β1- and hydrogen peroxide-inducible gene,hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.

Matrix metalloproteinases (MMPs) are tissue‐remodeling enzymes involved in the processing of various biological molecules. MMPs also play important roles in cancer metastasis, contributing to angiogenesis, intravasation of tumor cells, and cell migration and invasion. Accordingly, unraveling the signaling pathways controlling MMP activities could shed additional light on cancer biology. Here, we report a molecular axis, comprising the molecular adaptor hydrogen peroxide‐inducible clone‐5 (HIC‐5), NADPH oxidase 4 (NOX4), and mitochondria‐associated reactive oxygen species (mtROS), that regulates MMP9 expression and may be a target to suppress cancer metastasis. We found that this axis primarily downregulates mtROS levels which stabilize MMP9 mRNA. Specifically, HIC‐5 suppressed the expression of NOX4, the source of the mtROS, thereby decreasing mtROS levels and, consequently, destabilizing MMP9 mRNA. Interestingly, among six cancer cell lines, only EJ‐1 and MDA‐MB‐231 cells exhibited upregulation of NOX4 and MMP9 expression after shRNA‐mediated HIC‐5 knockdown. In these two cell lines, activating RAS mutations commonly occur, suggesting that the HIC‐5–mediated suppression of NOX4 depends on RAS signaling, a hypothesis that was supported experimentally by the introduction of activated RAS into mammary epithelial cells. Notably, HIC‐5 knockdown promoted lung metastasis of MDA‐MB‐231 cancer cells in mice. The tumor growth of HIC‐5–silenced MDA‐MB‐231 cells at the primary sites was comparable to that of control cells. Consistently, the invasive properties of the cells, but not their proliferation, were enhanced by the HIC‐5 knockdown in vitro. We conclude that NOX4‐mediated mtROS signaling increases MMP9 mRNA stability and affects cancer invasiveness but not tumor growth.

Hic-5 was originally isolated as an H₂O₂-inducible cDNA clone whose product was normally found at focal adhesions. In this study, we found that Hic-5 accumulated in the nucleus in response to oxidants such as H₂O₂. Other focal adhesion proteins including paxillin, the most homologous to Hic-5, remained in the cytoplasm. Mutation analyses revealed that the C- and N-terminal halves of Hic-5 contributed to its nuclear localization in a positive and negative manner, respectively. After the finding that leptomycin B (LMB), an inhibitor of nuclear export signal (NES), caused Hic-5 to be retained in the nucleus, Hic-5 was demonstrated to harbor NES in the N-terminal, which was sensitive to oxidants, thereby regulating the nuclear accumulation of Hic-5. NES consisted of a leucine-rich stretch and two cysteines with a limited similarity to Yap/Pap-type NES. In the nucleus, Hic-5 was suggested to participate in the gene expression of c-fos. Using dominant negative mutants, we found that Hic-5 was actually involved in endogenous c-fos gene expression upon H₂O₂treatment. Hic-5 was thus proposed as a focal adhesion protein with the novel aspect of shuttling between focal adhesions and the nucleus through an oxidant-sensitive NES, mediating the redox signaling directly to the nucleus.

Jumping translocation breakpoint (JTB) is a gene located on human chromosome 1 at q21 that suffers an unbalanced translocation in various types of cancers, and potentially encodes a transmembrane protein of unknown function. The results of cancer profiling indicated that its expression was suppressed in many cancers from different organs, implying a role in the neoplastic transformation of cells. Recently, we isolated JTB as a TGF-β1-inducible clone by differential screening. In this study, we characterized its product and biological functions. We found that it was processed at the N-terminus and located mostly in mitochondria. When expressed in cells, JTB-induced clustering of mitochondria around the nuclear periphery and swelling of each mitochondrion. In those mitochondria, membrane potential, as monitored with a JC-1 probe, was significantly reduced. Coinciding with these changes in mitochondria, JTB retarded the growth of the cells and conferred resistance to TGF-β1-induced apoptosis. These activities were dependent on the N-terminal processing and induced by wild-type JTB but not by a mutant resistant to cleavage. These findings raised the possibility that aberration of JTB in structure or expression induced neoplastic changes in cells through dysfunction of mitochondria leading to deregulated cell growth and/or death.

Mitochondria are considered to play an important role in oxidative stress response since they are a source of reactive oxygen species and are also targeted by these species. This study examined the mitochondrial conditions in cells of epithelial origin that were exposed to H₂O₂ and found a decline in the membrane potential along with a specific loss of UQCRC1, a sub-unit of complex III, suggesting that mitochondrial dysfunction occurs upon exposure to oxidative stress. This observation led to the hypothesis that certain cellular responses to oxidative stress occurred because of mitochondrial dysfunction. When mitochondria-less (pseudo ρ0) cells were examined as a model of mitochondrial dysfunction, striking similarities were found in their cellular responses compared with those found in cells exposed to oxidative stress, including changes in gene expression and gelatinolytic enzyme activities, thus suggesting that cellular responses to oxidative stress were partly mediated by mitochondrial dysfunction. This possibility was further validated by microarray analysis, which suggested that almost one-fourth of the cellular responses to oxidative stress were mediated by mitochondrial dysfunction that accompanies oxidative stress, thereby warranting a therapeutic strategy that targets mitochondria for the treatment of oxidative stress-associated diseases.