The Rho kinase inhibitor fasudil inhibits tumor progression in human and rat tumor models.

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 approximately 40% (P < 0.05). In the orthotopic breast cancer model with MDA-MB-231, there were 3-fold more tumor-free mice in the fasudil-treated group versus saline control group (P < 0.01). Fasudil has been approved for the treatment of cerebral vasospasm and associated cerebral ischemic symptoms. In patients, fasudil is well tolerated without any serious adverse reactions. Therefore, the concept of Rho kinase inhibition as an antimetastatic therapy for cancer can now be clinically explored.

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Antibodies neutralizing hepsin protease activity do not impact cell growth but inhibit invasion of prostate and ovarian tumor cells in culture.

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.

Identification of a novel prostate tumor target, mindin/RG-1, for antibody-based radiotherapy of prostate cancer.

Gene expression analysis showed that a human mindin homologue, mindin/RG-1, is expressed selectively in prostate tissues and that its expression level is elevated in some prostate tumors. Mindin/RG-1 protein expression is maintained in >80% of prostate cancers metastatic to bone or lymph nodes as well as in locally recurrent tumors in androgen-unresponsive patients. In contrast, mindin/RG-1 expression in other normal tissues is significantly lower than that seen in the prostate. A fully human antibody, 19G9, was generated against mindin/RG-1 protein and was shown to accumulate at high abundance in LNCaP tumor xenografts. Conjugates of this antibody with the chelator CHX-A''-DTPA were generated and radiolabeled with either 111In, 90Y, or 86Y. Small animal positron emission tomography imaging with the 86Y-radiolabeled conjugate showed very specific accumulation of the antibody in LNCaP tumor xenografts with clear tumor delineation apparent at 4 hours. The therapeutic efficacy of [90Y]-CHX-A''-DTPA-19G9 was evaluated in mice bearing LNCaP xenografts. A dose-finding study identified a nontoxic therapeutic dose to be approximately 75 microCi. Significant antitumor effects were seen with a single administration of radiolabeled antibody to animals bearing 200 to 400 mm3 tumors. Inhibition of tumor growth was observed in all treated animals over a 49-day period. At 49 days posttreatment, slow tumor growth recurred but this could be prevented for an additional 40-day period by a second administration of a 75 microCi dose at day 49. We conclude that [90Y]-CHX-A''-DTPA-19G9 is a novel antibody conjugate that has considerable promise for therapy of metastatic prostate cancer in androgen-unresponsive patients.

Targeting tomoregulin for radioimmunotherapy of prostate cancer.

Radiotherapy is an effective approach for the treatment of local prostate cancer. However, once prostate cancer metastasizes, radiotherapy cannot be used due to the distribution of multiple metastases to lymph nodes and bones. In contrast, radioimmunotherapy should still be efficacious in metastatic prostate cancer as radioisotopes are brought to tumor cells by targeting antibodies. Here we identify and validate a prostate-expressed molecule, tomoregulin, as a target for radioimmunotherapy of prostate cancer. Tomoregulin is a transmembrane protein selectively expressed in the brain, prostate, and prostate cancer, but not expressed in other normal tissues. Immunohistochemical studies of tomoregulin protein in clinical samples show its location in the luminal epithelium of normal prostate, benign prostatic hyperplasia, and prostatic intraepithelial neoplasia. More importantly, the tomoregulin protein is expressed in primary prostate tumors and in their lymph node and bone metastases. The nature of tomoregulin as a transmembrane protein and its tissue-specific expression make tomoregulin an attractive target for radioimmunotherapy, in which tomoregulin-specific antibodies will deliver a radioisotope to prostate tumor cells and metastases. Indeed, biodistribution studies using a prostate tumor xenograft model showed that the (111)In-labeled anti-tomoregulin antibody 2H8 specifically recognizes tomoregulin protein in vivo, leading to a strong tumor-specific accumulation of the antibody. In efficacy studies, a single i.p. dose of 150 microCi (163 microg) (90)Y-labeled 2H8 substantially inhibits the growth rate of established LNCaP human prostate tumor xenograft in nude mice but produces no overt toxicity despite cross-reactivity of 2H8 with mouse tomoregulin. Our data clearly validate tomoregulin as a target for radioimmunotherapy of prostate cancer.