Mutsuo Furihata

Novel lipogenic enzyme ELOVL7 is involved in prostate cancer growth through saturated long-chain fatty acid metabolism.

A number of epidemiologic studies have indicated a strong association between dietary fat intake and prostate cancer development, suggesting that lipid metabolism plays some important roles in prostate carcinogenesis and its progression. In this study, through our genome-wide gene expression analysis of clinical prostate cancer cells, we identified a novel lipogenic gene, ELOVL7, coding a possible long-chain fatty acid elongase, as overexpressed in prostate cancer cells. ELOVL7 expression is regulated by the androgen pathway through SREBP1, as well as other lipogenic enzymes. Knockdown of ELOVL7 resulted in drastic attenuation of prostate cancer cell growth, and it is notable that high-fat diet promoted the growth of in vivo tumors of ELOVL7-expressed prostate cancer. In vitro fatty acid elongation assay and fatty acid composition analysis indicated that ELOVL7 was preferentially involved in fatty acid elongation of saturated very-long-chain fatty acids (SVLFA, C20:0 approximately ). Lipid profiles showed that knockdown of ELOVL7 in prostate cancer cells affected SVLFAs in the phospholipids and the neutral lipids, such as cholesterol ester. Focusing on cholesterol ester as a source of de novo steroid synthesis, we show that ELOVL7 affected de novo androgen synthesis in prostate cancer cells. These findings suggest that EVOLV7 could be involved in prostate cancer growth and survival through the metabolism of SVLFAs and their derivatives, could be a key molecule to elucidate the association between fat dietary intake and prostate carcinogenesis, and could also be a promising molecular target for development of new therapeutic or preventive strategies for prostate cancers.

Common variants at 11q12, 10q26 and 3p11.2 are associated with prostate cancer susceptibility in Japanese

We have previously reported multiple loci associated with prostate cancer susceptibility in a Japanese population using a genome-wide association study (GWAS). To identify additional prostate cancer susceptibility loci, we genotyped nine SNPs that were nominally associated with prostate cancer (P < 1 × 10−4) in our previous GWAS in three independent studies of prostate cancer in Japanese men (2,557 individuals with prostate cancer (cases) and 3,003 controls). In a meta-analysis of our previous GWAS and the replication studies, which included a total of 7,141 prostate cancer cases and 11,804 controls from a single ancestry group, three new loci reached genome-wide significance on chromosomes 11q12 (rs1938781; P = 1.10 × 10−10; FAM111A-FAM111B), 10q26 (rs2252004; P = 1.98 × 10−8) and 3p11.2 (rs2055109; P = 3.94 × 10−8). We also found suggestive evidence of association at a previously reported prostate cancer susceptibility locus at 2p11 (rs2028898; P = 1.08 × 10−7). The identification of three new susceptibility loci should provide additional insight into the pathogenesis of prostate cancer and emphasizes the importance of conducting GWAS in diverse populations.

Comparison between intravesical and oral administration of 5-aminolevulinic acid in the clinical benefit of photodynamic diagnosis for nonmuscle invasive bladder cancer

This study was undertaken to evaluate the clinical value of photodynamic diagnosis (PDD) with intravesical and oral instillation of 5‐aminolevulinic acid (ALA) (ALA‐PDD), and transurethral resection of bladder tumor (TURBT) guided by ALA‐PDD (PDD‐TURBT) for nonmuscle invasive bladder cancer.

Zeb1-mediated T-cadherin repression increases the invasive potential of gallbladder cancer

Here, we report that the transcriptional regulator Zeb1 repressed the transcription of T‐cadherin, to increase the invasive activity of gallbladder cancer cells. Zeb1 physically bound to the promoter of T‐cadherin, repressed promoter activity in E‐box‐like sequence‐dependent fashion, and suppressed T‐cadherin expression. In gallbladder cancer tissues, Zeb1 was expressed at the cancer invasion front, whereas T‐cadherin was exclusively expressed in non‐invasive foci. Collagen gel invasion assay showed that T‐cadherin was a negative regulator for gallbladder cancer invasion. These findings suggest that Zeb1 represses T‐cadherin expression and thus increases the invasive activity of gallbladder cancer.

Thrombomodulin Protects Endothelial Cells From a Calcineurin Inhibitor–Induced Cytotoxicity by Upregulation of Extracellular Signal–Regulated Kinase/Myeloid Leukemia Cell-1 Signaling

Objective—We have recently reported that recombinant human soluble thrombomodulin (rTM) counteracted capillary leakage associated with engraftment, as well as sinusoidal obstructive syndrome after hematopoietic stem cell transplantation. These observations prompted us to explore whether rTM possessed cytoprotective effects on endothelial cells. Methods and Results—Exposure of human umbilical vein endothelial cells to rTM induced expression of antiapoptotic protein myeloid leukemia cell-1 through the activation of extracellular signal–regulated kinase in these cells. Additional studies found that exposure of human umbilical vein endothelial cells to cyclosporine A and FK506, an immunosuppressant used for the individuals receiving hematopoietic stem cell transplantation, induced apoptosis, which was attenuated when human umbilical vein endothelial cells were exposed to these agents in the presence of rTM. Further studies using deletion mutants of thrombomodulin (TM) identified that the epidermal growth factor domain of TM possessed cytoprotective effects. A single nucleotide substitution at codon 376 or 424 of TM, which impairs the ability of TM to produce activated protein C or bind to thrombin, respectively, did not hamper the cytoprotective effects of TM, which suggested that cytoprotective effects of rTM were distinctive from those of activated protein C. Conclusion—TM may be useful for prevention, as well as treatment of endothelial cell damage after hematopoietic stem cell transplantation.

Chemokine CCL2/MCP-1 negatively regulates metastasis in a highly bone marrow-metastatic mouse breast cancer model

Bone is the most frequent site of breast cancer metastasis, and once such metastasis occurs, complete remission is extremely difficult to achieve. In an effort to define the mechanisms underlying metastatic spread of breast cancer to bone, we previously developed and characterized the highly bone metastatic 4T1E/M3 mouse breast cancer cells. We found that following injection into mice, 4T1E/M3 cells exhibited greater bone metastasis and greater in vitro anchorage-independent growth and cell migration than their parental cells (4T1E). We also found that expression of intracellular adhesion molecule-1 (ICAM-1) is crucially involved in these metastatic activities of 4T1E/M3 cells. In the present study, our analysis of gene and protein expression revealed that production of chemokine CCL2 (MCP-1) is dramatically reduced in 4T1E/M3 cells, and that restoration of CCL2 expression in 4T1E/M3 cells diminishes their metastasis to bone and lung. Overexpression of CCL2 in 4T1E/M3 cells significantly reduced not only in vitro anchorage-independent cell growth and cell migration, but also mRNA and cell surface expression of ICAM-1. Conversely, knocking down CCL2 in 4T1E parental cells augmented their metastatic spread to spine and lung. The expression of ICAM-1 was also upregulated in 4T1E-derived CCL2 knockdown cells. Taken together, these results suggest that CCL2 expression may negatively regulate breast cancer metastasis to bone marrow and lung in our model and that expression of ICAM-1 plays a crucial role in that process.

A ubiquitin ligase, skeletrophin, is a negative regulator of melanoma invasion

Skeletrophin (mindbomb homolog 2 (MIB2)) is a RING (Really Interesting New Gene) finger-dependent ubiquitin ligase, which targets the intracellular region of Notch ligands. A previous immunohistochemical study demonstrated that skeletrophin was downregulated in many melanomas. In the present study, we have identified a promoter region of skeletrophin on a CpG island and detected aberrant methylation of this region in six of 31 invasive melanomas, but in none of 25 benign nevi or five non-invasive superficial spreading melanomas. Subsequently, we found that a zinc-finger transcriptional factor Snail, which is overexpressed in many melanoma cells, repressed the skeletrophin promoter activity via an E-box-related element and was involved in downregulation of skeletrophin. An activator protein-2, which has a tumor suppressor-like role in melanoma, increased skeletrophin expression. Interestingly, exogenously expressed skeletrophin reduced melanoma cell invasion in vitro and in vivo. Colony formation in soft agar was also reduced in a RING motif-dependent manner, without affecting cell growth. We also found that skeletrophin downregulated transcription of the Met oncogene, which encodes the hepatocyte growth factor receptor and plays a role in the determination of the invasive phenotype of many malignant tumors. Finally, exogenously expressed skeletrophin, but not its RING mutant, increased transcription of Hes1 gene, a downstream effector of Notch pathway in melanoma cells. The present findings indicate that skeletrophin might be a novel suppressor factor for melanoma invasion.

Analysis of Aurora B kinase in non-Hodgkin lymphoma

This study explored the levels of Aurora B, a key regulator of mitosis, in 71 lymph nodes and tumor specimens excised operatively from individuals with various types of non-Hodgkin lymphoma (NHLs). Immunohistochemical examination found that diffuse large B-cell lymphoma (10/21, 48%) and Burkitt lymphoma (BL) (6/7, 86%) cells highly (percentage of positive cells, >20%) expressed Aurora B in their nuclei. On the other hand, none of the low-grade B-cell lymphoma (n=20), except for one case of follicular lymphoma, highly expressed this protein kinase, suggesting that levels of Aurora B correlated with histological grade in B-cell NHLs (P<0.01). Exposure of BL/leukemia cells to AZD1152-HQPA in vitro, a selective inhibitor of Aurora B kinase, potently induced growth arrest and apoptosis in a caspase-dependent, as well as -independent manner. Moreover, AZD1152 synergistically enhanced the effects of vincristine (VCR) to induce growth arrest of these cells. Further experiments found that VCR increased levels of the p-Aurora B through the activation of c-Jun N-terminal kinase, which was blocked in the presence of AZD1152-HQPA.

Augmented autocrine bone morphogenic protein (BMP) 7 signaling increases the metastatic potential of mouse breast cancer cells

As malignant breast cancers progress, they acquire the ability to spread to other regions of the body, including bone and lung, but the molecular mechanism underlying the increase in metastatic potential is not fully understood. Here we studied murine 4T1E/M3 highly bone marrow metastatic breast cancer cells, which we established previously. These cells show upregulated expression of bone morphogenetic protein (BMP) 7 and BMP receptors, as well as augmented phosphorylation of Smad1/5/8. Both anchorage-independent cell growth measured in colony forming assays and cell migration measured in wound healing assays were suppressed in 4T1E/M3 cells following treatment with a neutralizing anti-BMP7 antibody or knockdown of BMP7 gene expression. In addition, metastasis of 4T1E/M3 cells to the spine and lung and intracellular levels of phosphorylated Smad1/5/8 were suppressed by knocking down BMP7. Conversely, overexpression of BMP7 in the weakly metastatic parental 4T1E cells augmented their anchorage-independent growth, migration and metastasis to spine and lung. Taken together, our results strongly suggest that augmented autocrine BMP7 signaling leads to increases in the anchorage-independent cell growth, migration and metastatic potential in our bone marrow metastatic breast cancer model.

Photodynamic diagnosis of positive margin during radical prostatectomy: preliminary experience with 5-aminolevulinic acid.

Objectives:  To investigate the feasibility of intraoperative photodynamic diagnosis (PDD) by 5‐aminolevulinic acid (ALA) for the identification of positive surgical margins (PSM) during retropubic radical prostatectomy (RRP) in patients with prostate cancer (PCa).