河田 浩敏

Therapy-resistant cancer cells are a major problem in cancer research. Recent studies suggest that the epithelial-mesenchymal transition (EMT) is a key mechanism in therapy resistance. Yet, the expressions of EMT markers, EMT core regulators, and a stem cell marker of BMI1 during chemotherapy have been poorly analyzed in clinical breast cancer specimens. In the present study, we investigated the roles of RhoC under chemotherapy to follow up on earlier findings demonstrating the involvement of RhoC in prostate cancer resistance to endocrine therapy. Immunohistochemically, E-cadherin expression was significantly lower in human breast cancer specimens analyzed after chemotherapy than specimens biopsied before chemotherapy. Significant upregulation of fibronectin, a mesenchymal EMT marker, was found in post-chemotherapy analysis. A study of the EMT core regulators of SNAIL1, SNAIL2, TWIST1, and a well-known stem cell marker of BMI1 revealed no post-chemotherapy upregulation of these molecules. In contrast, RhoC expression was significantly upregulated in post-chemotherapy breast cancer specimens. MCF-7 cells stably transfected with the constitutive active (CA) RhoC plasmid manifested a reduced level of E-cadherin at the peripheries and disorganization of actin fibers, with no accompanying upregulation of SNAIL1, SNAIL2, TWIST1, or BMI1 in Western blots. Exposure of etoposide on MCF-7 cells showed RhoC upregulation together with reduced membranous expression of E-cadherin and disorganization of actin fibers. In MTT assay, however, the CA-RhoC-expressing MCF-7 cells failed to show chemotherapy resistance under etoposide treatment. Taken in sum, RhoC may contribute to an EMT-like process in human breast cancer during chemotherapy.

BACKGROUND Endocrine resistance is a critical issue in managing patients with prostate cancer. This study is undertaken to search for a potential molecular target connected with this process using a model system of androgen-dependent and androgen-unresponsive SC-3 and SC-4 cells. METHODS Expression profiles, actin stress fiber organization, and the levels of activated Rho GTPases were compared between SC-4 and SC-3 cells using an oligonucleotide microarray, phalloidin staining, and a Rho activation assay. The cell viability was analyzed with a Rho inhibitor or by stable transfection with either a dominant-negative (DN) form of RhoC or a mutant form of NET1 (mutNET1). The expressions of RhoC, NET1, and epithelialmesenchymal transition (EMT) markers were immunohistochemically analyzed in human prostate cancer specimens after short-term endocrine therapy and in an untreated condition. RESULTS SC-4 cells exhibited mesenchymal phenotypes with activation of Rho signals. Treatment with a Rho inhibitor suppressed the cell viability in SC-4 cells, but not in SC-3 cells. The cell viability of SC-4 cells stably expressing DN-RhoC and mutNET1 was also attenuated. In the immunohistochemical analysis, NET1 and the EMT marker of N-cadherin were expressed at higher levels in prostate cancers after short-term endocrine therapy than in untreated tumors, and RhoC expression was maintained after short-term endocrine therapy. CONCLUSIONS Rho signaling is involved in the cell survival of SC-4 cells. The higher expressions of RhoC and NET1 in human prostate cancers after short-term endocrine therapy suggest that RhoC and NET1 may become therapeutic targets during endocrine therapy. Prostate 72:10711079, 2012. (c) 2011 Wiley Periodicals, Inc.

BACKGROUND The genetic cause of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), which is characterized by ischemic, non-hypertensive, cerebral small-vessel disease with associated alopecia and spondylosis, is unclear. METHODS In five families with CARASIL, we carried out linkage analysis, fine mapping of the region implicated in the disease, and sequence analysis of a candidate gene. We also conducted functional analysis of wild-type and mutant gene products and measured the signaling by members of the transforming growth factor beta (TGF-beta) family and gene and protein expression in the small arteries in the cerebrum of two patients with CARASIL. RESULTS We found linkage of the disease to the 2.4-Mb region on chromosome 10q, which contains the HtrA serine protease 1 (HTRA1) gene. HTRA1 is a serine protease that represses signaling by TGF-beta family members. Sequence analysis revealed two nonsense mutations and two missense mutations in HTRA1. The missense mutations and one of the nonsense mutations resulted in protein products that had comparatively low levels of protease activity and did not repress signaling by the TGF-beta family. The other nonsense mutation resulted in the loss of HTRA1 protein by nonsense-mediated decay of messenger RNA. Immunohistochemical analysis of the cerebral small arteries in affected persons showed increased expression of the extra domain-A region of fibronectin and versican in the thickened tunica intima and of TGF-beta 1 in the tunica media. CONCLUSIONS CARASIL is associated with mutations in the HTRA1 gene. Our findings indicate a link between repressed inhibition of signaling by the TGF-beta family and ischemic cerebral small-vessel disease, alopecia, and spondylosis.

Active metabolites of vitamin A and D are well known to act as growth inhibitors in hormone-related prostate and breast cancers. When various concentrations of 1 alpha,25-dihydroxyvitamin D3 (vitamin D3), all-trans-retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) were examined, the androgen-stimulated growth of mouse mammary carcinoma SC-3 cells was inhibited by vitamin D3 alone in a dose-dependent manner. A flow cytometer analysis showed that vitamin D3 leads SC-3 cells to relative G1-growth arrest after 72 h. Characterization of vitamin D3-responsive genes using an oligonucleotide microarray demonstrated that 220 genes were upregulated at more than three-fold, and 84 genes were downregulated to less than one-third, compared with the testosterone-stimulated SC-3 cells. Neither cyclin-dependent kinase inhibitors (CDKIs) nor the antiapoptotic bcl-2gene were induced in vitamin D3-responsive genes, with the exception of a slight induction of p15(INK4B). Importantly, fgf8 was markedly repressed in response to vitamin D3. The exogenous addition of FGF8 canceled the growth Suppression by vitamin D3 in SC-3 cells, Suggesting that the repression of fgf8 is all indispensable step in vitamin D3-mediated growth inhibition. In reporter assays using the ARE-containing artificial construct and the natural androgen-regulated PSA promoter, co-transfection of the vitamin D receptor(VDR) and androgen receptor (AR) suppressed AR-stimulated promoter activity. In addition, vitamin D3 also suppressed androgen-stimulated prornoter activity in the stably transfected SC-3 cells. Moreover, VDR repressed the core promoter activity of fgf8 in COS1 cells and in the SC-3 cells. All these findings strongly Suggest that vitamin D3 serves as a negative regulator for both androgen-related and fgf8 transcriptions.

We here characterized the transcriptional profiles of TGF-beta-responsive genes using androgen-dependent mouse mammary carcinoma SC-3 cells. Compared with the testosterone-stimulated SC-3 cells, 165 genes were up-regulated at more than 5-fold, and 78 genes were downregulated to less than one-third in response to TGF-beta. Of note,fgf8 an androgen-inducible growth factor essential to the androgen-dependent growth of SC-3 cells, was severely repressed in response to TGF-beta. Real-time PCR confirmed that the androgenic induction of the fgf8 transcripts is severely attenuated by TGF-beta. Although a considerable number of growth- suppressive genes were up-regulated in response to TGF-beta, the treatment with TGF-beta was insufficient to lead SC-3 cells to apoptosis within 24 It by both the TUNEL method and the caspase 3 activity assay. Flow cytometric analysis rather indicated the cell-static effect of TGF-beta on the androgen-stimulated SC-3 cells. In addition, TGF-beta failed to suppress the FGF8-stimulated growth of SC-3 cells, suggesting that the repression of fgf8 is required for the TGF-beta-mediated growth inhibition in SC-3 cells. In a reporter assay, androgen-responsive promoter activity was suppressed by TGF-beta in SC-3 cells. Based on this finding, it is likely that some of the androgen-inducible genes are physiological targets of the TGF-beta-mediated transcriptional control, and therefore, it is strongly suggested that the repression of fgf8 might be directly or indirectly involved in this transcriptional control by TGF-beta. (c) 2005 Elsevier Ltd. All rights reserved.

The expression of claudin-4 was investigated in human pancreas, pancreatic ductal adenocarcinomas, and intraductal papillary-mucinous tumors of the pancreas (IPMT), and compared with that of claudin-1. In human adult pancreatic specimens, both claudin-1 and claudin-4 were immunohistochemically found in main and branching pancreatic ducts, terminal ductules and acinic cells, with the exception of endocrine cells. Of 12 cases of pancreatic ductal adenocarcinoma, 11 (92%) had positive immunostaining for claudin-4, and seven (58%) for claudin-1. In 44 lesions of 22 cases of IPMT, including six hyperplastic foci distant from the main lesions, clauidin-1 was positive in three out of six (50%) hyperplastic foci, 14 out of 17 (82%) adenomas, three out of 10 (30%) borderline tumors, two out of six (33%) non-invasive carcinomas, and one out of five (20%) invasive carcinomas, producing a statistically negative correlation with histological tumor grades. In contrast, claudin-4 was negative in the six hyperplastic foci, and positive in four out of the 17 (24%) adenomas, five out of the 10 (50%) borderline tumors, five out of the six (83%) non-invasive carcinomas, and four out of the five (80%) invasive carcinomas, producing a statistically positive correlation with histological tumor grades. On study of IPMT subtypes, claudin-1 was positive in nine out of 10 (90%) clear-cell types, seven out of 20 (35%) dark-cell types, and four out of eight (50%) compact-cell types. In contrast, claudin-4 was positive in two out of the 10 (20%) clear-cell types, 13 out of the 20 (65%) dark-cell types, and three out of the eight (38%) compact-cell types. These distinct expression patterns of claudin-1 and claudin-4 suggest that both claudins serve as useful molecular markers for the tumor classification of IPMT.

Extremely well-differentiated adenocarcinoma (EWDA) is an unusual gastric cancer that is histologically too bland to be diagnosed as malignant neoplasm, particularly using biopsy. EWDA may be a gastric counterpart of 'adenoma malignum' or minimal deviation adenocarcinoma (MDA) in the uterine cervix; however, the clinicopathological features of EWDA remain less apparent than those of MDA. A 60-year-old male was complaining of dysphagia. He had been made aware of a small submucosal tumor in the cardia 2 years before the onset of this symptom. Endoscopic ultrasonographic examination revealed a large cardiac tumor consisting of thickened layers, as observed in Borrmann type IV. Three mucosal biopsies suggested only benign changes including adenoma and hyperplastic polyps. At the fourth biopsy, cytologically bland columnar cells were located in the submucosa along with stromal fibrosis and laminated stones. The possibility that non-neoplastic aberrant pancreas with lithiasis formed the tumor was denied at laparotomy by a frozen section that revealed benign-looking glands invading the diaphragm. Immunohistochemically the cancer glands were positive for CA19-9 and human gastric mucin, but not for p53 or MUC2. To our knowledge, this is a previously unknown combination of EWDA and psammomatous calcification in the stomach.