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Through analysis of the detailed genome-wide gene expression profiles of 81 breast tumors, we identified a novel gene, G-patch domain containing 2 (GPATCH2), that was overexpressed in the great majority of breast cancer cases. Treatment of breast cancer cells MCF-7 and T47D with siRNA against GPATCH2 effectively suppressed its expression, and resulted in the growth suppression of cancer cells, suggesting its essential role in breast cancer cell growth. We found an interaction of GPATCH2 protein with hPrp43, an RNA-dependent ATPase. Their interaction could significantly enhance the ATPase activity of hPrp43 and induce a growth-promoting effect on mammalian cells. Because northern blot analyses of normal human organs implied GPATCH2 to be a novel cancer/testis antigen, targeting GPATCH2 or inhibition of the interaction between GPATCH2 and hPrp43 could be a promising novel therapeutic strategy of breast cancer. (Cancer Sci

We previously reported Frizzled homolog 10 (FZD10), a member of the Frizzled family, to be a promising therapeutic target for synovial sarcomas. In this report, we established a murine monoclonal antibody (MAb), namely, MAb 92-13 that had specific binding activity against native

We previously reported that Frizzled homologue 10 (FZD10), a member of the Wnt signal receptor family, was highly and specifically upregulated in synovial sarcoma and played critical roles in its cell survival and growth. We here report a possible molecular mechanism of the FZD10 signaling in synovial sarcoma cells. We found a significant enhancement of phosphorylation of the Dishevelled (Dvl)2/Dvl3 complex as well as activation of the Rac1-JNK cascade in synovial sarcoma cells in which FZD10 was overexpressed. Activation of the FZD10-Dvls-Rac1 pathway induced lamellipodia formation and enhanced anchorage-independent cell growth cells. FZD10 overexpression also caused the destruction of the actin cytoskeleton structure, probably through the downregulation of the RhoA activity. Our results have strongly implied that FZD10 transactivation causes the activation of the non-canonical Dvl-Rac1-JNK pathway and plays critical roles in the development/progression of synovial sarcomas.

To investigate the molecular mechanism of mammary carcinogenesis and identify novel molecular targets for breast cancer therapy, we analyzed genome-wide gene expression profiles of 81 clinical breast cancer samples. Here, we report the critical role of LGN/GPSM2 (Leu-Gly-Asn repeat-enriched protein/G-protein signaling modulator 2) in the growth of breast cancer cells. Semiquantitative RT-PCR and Northern blot analyses confirmed upregulation of LGN/GPSM2 in a large proportion of breast cancers. Immunocytochemical staining identified LGN/GPSM2 at the spindle in cells at metaphase, and at midzone and midbody in cytokinetic cells. Western blot analysis indicated the highest expression and the phosphorylated form of LGN/GPSM2 protein in G2/M phase. Treatment with small-interfering RNAs (siRNAs) targeting LGN/GPSM2 caused incompletion of cell division and resulted in significant growth suppression of breast cancer cells. We found that the 450th threonine (Thr450) of LGN/GPSM2 was phosphorylated by the serine/threonine kinase PBK/TOPK during mitosis. Overexpression of LGN/GPSM2-T450A in which Thr450 was substituted with alanine induced growth suppression and aberrant chromosomal segregation. These findings imply an important role of LGN/GPSM2 in cell division of breast cancer cells and suggest that the PBK/TOPK-LGN/GPSM2 pathway might be a promising molecular target for treatment of breast cancer.

T he Wnt signal pathway is an essential signal pathway for multicellular organisms with diverse biological consequences. A large number of components are involved in signal transduction, one of which is the Frizzled homolog protein (FZD) that serves as a seven-pass transmembrane receptor for Wnt ligands. So far 10 members of FZD family genes have been identified based on structural and functional homologies. Colorectal cancers (CRCs) are prototype tumors and the Wnt signal pathway is profoundly involved in CRC pathogenesis. There are two major intracellular pathways of Wnt signal transduction: canonical and non-canonical pathways. In the early stages of CRC progression, Wnt signal activation frequently appears due to genetic alterations of molecules in the Wnt canonical pathway including Adenomatous polyposis coli (APC) and β-catenin, resulting in the stabilization and nuclear translocation of β-catenin with the subsequent transcriptional activation of Wnt target genes.(1-6) In contrast to extensive studies related to downstream events of Wnt signal transduction, little attention has been paid to the specificity of FZD receptors in CRCs, although there have been a few reports demonstrating the up-regulation of FZD1 and FZD2 in poorly differentiated CRCs,and FZD7 expression in colon cancer cell lines with APC or β-catenin mutations. (8) We previously demonstrated that FZD10, the latest cloned member of the FZD family, was specifically up-regulated in synovial sarcomas based upon a genome-wide analysis of gene-expression profiles in soft-tissue sarcomas.(9) Since FZD10 is located in plasma membranes and its expression is very low or absent in vital organs, we focused on this molecule as a likely candidate for antibody-based therapy, and demonstrated the therapeutic potential of antibodies directed against FZD10 in synovial sarcomas.(10,11) Although FZD10 protein expression patterns in tumor tissues were not demonstrated, and the precise biological behavior of FZD10 with regard to tumorigenesis of CRCs remains obscure, the up-regulation of FZD10 mRNA was observed in two out of 11 cases with primary CRCs. (12) Using a specific monoclonal antibody directed against FZD10, we analyzed expression patterns of FZD10 in colonic polyps, primary CRCs, and metastatic liver lesions to elucidate the role of FZD10 in the progression of CRCs. In addition, we examined the relationship between expression patterns of FZD10 and β-catenin, which is the main signal transducer of the canonical Wnt pathway. Materials and MethodsTissue samples and cell lines. Tissue samples were obtained from 104 CRC patients that had undergone surgical curative resection at the Department of Surgery, Kyoto University Hospital during 1999-2001 (Table 1). All samples were approved for analysis by the ethics committee of the Faculty of Medicine of Kyoto University. Formalin-fixed and paraffin-embedded samples were obtained from all patients, and were subdivided into the three following groups: (1) only primary tumor samples were available in 57 cases; (2) p...

Abstract. Through genome-wide gene expression profile analysis of breast cancer, we identified a gene, chromosome 12 open reading frame 32 (C12orf32), to be involved in mammary carcinogenesis. Semiquantitative RT-PCR and Northern blot analysis confirmed C12orf32 overexpression in breast cancer cells and its almost undetectable level of expression in normal human tissues. Immunocytochemical staining analysis using breast cancer cell lines revealed a cell cycle-dependent subcellular localization of endogenous C12orf32 protein. Depletion of C12orf32 expression by small-hairpin RNA interference significantly suppressed the growth of breast cancer cell lines possibly due to the inhibition of G1/S transition and subsequent cell death. Western blot analysis indicated that a C12orf32 protein of 35 kDa predicted from the cDNA sequences was processed to a 16-kDa protein of (C12orf32-p16) which was accumulated in most of breast cancer cell lines examined. Our data suggest that C12orf32 is a promising molecular target for the development of novel anticancer drugs such as peptide vaccines and siRNA drugs.

Porcine cardiac myosin monomers in equilibrium with filaments under physiological conditions were observed to have two conformations, extended and folded forms, upon electron microscopy and gel filtration HPLC. The conformational state was independent of ATP and the phosphorylation of regulatory light chain. The folded monomers of cardiac myosin were mainly in an open conformation with only one bend in the tail, and may not trap the hydrolysis products of ATP, as assessed by single turnover experiments. These properties are similar to those of the folded monomers of rabbit skeletal myosin [Katoh, T., Konishi, K., and Yazawa, M. (1998) J. Biol. Chem. 273, 11436-11439]. The conformational states of skeletal and cardiac myosin monomers were not affected by pH between 7.0 and 8.5. Although significant disassembly of filaments and thus an increase in the monomer concentration were observed with an increase in pH. The results indicate that the pH-dependent change in filament assembly is due to a shift of equilibrium between the filaments and extended monomers toward filament disassembly. The Mg2+-ATPase activity of these myosin monomers decreased with a decrease in the salt concentration below approximately 0.1 M, suggestive of the formation of a closed conformation similar to the conformation of 10S smooth myosin. The results suggest that the conformational change from the extended to the folded form is a common property of various myosin IIs.