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The ability of cancer cells to undergo invasion and migration is a prerequisite for tumor metastasis. Rho, a Ras-related small GTPase, and the Rho-associated coiled coil -containing protein kinases (Rho kinases, ROCK1 and ROCK2) are key regulators of focal adhesion, actomyosin contraction, and thus cell motility. Inhibitors of this pathway have been shown to inhibit tumor cell motility and metastasis. Here, we show that fasudil [1-(5-isoquinolinesulfonyl)-homopiperazine], an orally available inhibitor of Rho kinases, and its metabolite 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine) (fasudil-OH) modify tumor cell morphology and inhibit tumor cell migration and anchorage-independent growth. In addition, we show that fasudil inhibited tumor progression in three independent animal models. In the MM1 peritoneal dissemination model, tumor burden and ascites production were reduced by >50% (P < 0.05). In the HT1080 experimental lung metastasis model, fasudil decreased lung nodules by f40% (P < 0.05). In the orthotopic breast cancer model with MDA-MB-

Hepsin is a type II transmembrane serine protease that is expressed in normal liver, and at lower levels in kidney, pancreas, and testis. Several studies have shown that hepsin mRNA is significantly elevated in most prostate tumors, as well as a significant fraction of ovarian and renal cell carcinomas and hepatomas. Although the overexpression of mRNA in these tumors has been extensively documented, there has been conflicting literature on whether hepsin plays a role in tumor cell growth and progression. Early literature implied a role for hepsin in human tumor cell proliferation, whereas recent studies with a transgenic mouse model for prostate cancer support a role for hepsin in tumor progression and metastases. To evaluate this issue further, we have expressed an activatable form of hepsin, and have generated a set of monoclonal antibodies that neutralize enzyme activity. The neutralizing antibodies inhibit hepsin enzymatic activity in biochemical and cell-based assays. Selected neutralizing and nonneutralizing antibodies were used in cell-based assays with tumor cells to evaluate the effect of antibodies on tumor cell growth and invasion. Neutralizing antibodies failed to inhibit the growth of prostate, ovarian, and hepatoma cell lines in culture. However, potent inhibitory effects of the antibodies were seen on invasion of ovarian and prostate cells in transwell-based invasion assays. These results support a role for hepsin in tumor cell progression but not in primary tumor growth. Consistent with this, immunohistochemical experiments with a mouse monoclonal antibody reveal progressively increased staining of prostate tumors with advanced disease, and in particular, extensive staining of bone metastatic lesions.

The transition from persistent to lytic infection by the human immunodeficiency virus, HIV, is marked by a burst of viral replication and gene expression that occurs when infected cells are stimulated by physiological inducers or tumor promoters like 12‐O‐tetradecanoyl phorbol acetate (TPA). We report here that the HIV enhancer is activated specifically by TPA in several non‐lymphoid cell types, and that this transcriptional regulation can be reproduced in a cell‐free system. In vitro transcription experiments revealed a 6‐fold activation of the HIV promoter in nuclear extracts prepared from TPA‐induced HeLa tk‐ cells, whereas a control (human alpha‐globin) promoter was transcribed with equal efficiency in either induced or uninduced cell extracts. A corresponding increase in the activity of a cellular DNA‐binding protein that interacts with the HIV enhancer was detected in TPA‐treated cells with DNase I footprint experiments. This increase occurred in the absence of de novo protein synthesis, suggesting a post‐transcriptional activation mechanism. Analysis of HIV deletion mutants suggests that the enhancer is the target for the TPA effect both in vitro and in vivo. The cell‐free system described here should facilitate studies on the mechanism of phorbol ester induction of gene‐specific transcription factors.

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