Megumi Iiizumi

Fatty acid synthase gene is up-regulated by hypoxia via activation of Akt and sterol regulatory element binding protein-1.

The fatty acid synthase (FAS) gene is significantly up-regulated in various types of cancers, and blocking the FAS expression results in apoptosis of tumor cells. Therefore, FAS is considered to be an attractive target for anticancer therapy. However, the molecular mechanism by which the FAS gene is up-regulated in tumor cells is poorly understood. We found that FAS was significantly up-regulated by hypoxia, which was also accompanied by reactive oxygen species (ROS) generation in human breast cancer cell lines. The FAS expression was also activated by H(2)O(2), whereas N-acetyl-L-cystein, a ROS inhibitor, suppressed the expression. We also found that the hypoxia significantly up-regulated sterol regulatory-element binding protein (SREBP)-1, the major transcriptional regulator of the FAS gene, via phosphorylation of Akt followed by activation of hypoxia-inducible factor 1 (HIF1). Moreover, our results of reporter assay and chromatin immunoprecipitation analysis indicate that SREBP-1 strongly bound to the SREBP binding site/E-box sequence on the FAS promoter under hypoxia. In our xenograft mouse model, FAS was strongly expressed in the hypoxic regions of the tumor. In addition, our results of immunohistochemical analysis for human breast tumor specimens indicate that the expressions of both FAS and SREBP-1 were colocalized with hypoxic regions in the tumors. Furthermore, we found that hypoxia-induced chemoresistance to cyclophosphamide was partially blocked by a combination of FAS inhibitor and cyclophosphamide. Taken together, our results indicate that FAS gene is up-regulated by hypoxia via activation of the Akt and HIF1 followed by the induction of the SREBP-1 gene, and that hypoxia-induced chemoresistance is partly due to the up-regulation of FAS.

Molecular Features of the Transition from Prostatic Intraepithelial Neoplasia (PIN) to Prostate Cancer Genome-wide Gene-expression Profiles of Prostate Cancers and PINs

To characterize the molecular feature in prostate carcinogenesis and the putative transition from prostatic intraepithelial neoplasia (PIN) to invasive prostate cancer (PC), we analyzed gene-expression profiles of 20 PCs and 10 high-grade PINs with a cDNA microarray representing 23,040 genes. Considering the histological heterogeneity of PCs and the minimal nature of PIN lesions, we applied laser microbeam microdissection to purify populations of PC and PIN cells, and then compared their expression profiles with those of corresponding normal prostatic epithelium also purified by laser microbeam microdissection. A hierarchical clustering analysis separated the PC group from the PIN group, except for three tumors that were morphologically defined as one very-high-grade PIN and two low-grade PCs, suggesting that PINs and PCs share some molecular features and supporting the hypothesis of PIN-to-PC transition. On the basis of this hypothesis, we identified 21 up-regulated genes and 63 down-regulated genes commonly in PINs and PCs compared with normal epithelium, which were considered to be involved in the presumably early stage of prostatic carcinogenesis. They included AMACR, OR51E2, RODH, and SMS. Furthermore, we identified 41 up-regulated genes and 98 down-regulated genes in the transition from PINs to PCs; those altered genes, such as POV1, CDKN2C, EPHA4, APOD, FASN, ITGB2, LAMB2, PLAU, and TIMP1, included elements that are likely to be involved in cell adhesion or the motility of invasive PC cells. The down-regulation of EPHA4 by small interfering RNA in PC cells lead to attenuation of PC cell viability. These data provide clues to the molecular mechanisms underlying prostatic carcinogenesis, and suggest candidate genes the products of which might serve as molecular targets for the prevention and treatment of PC.

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression.

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.

Identification of the interferon regulatory factor 5 gene (IRF-5) as a direct target for p53.

Interferon regulatory factors (IRFs) regulate transcription of interferon genes through DNA sequence-specific binding to these targets. Using a differential display method for examining gene expression in p53-defective cells infected with adenovirus containing wild-type p53, we found that expression of interferon regulatory factor 5 (IRF-5) mRNA was increased in the presence of exogenous p53. An electrophoretic mobility-shift assay showed that a potential p53 binding site (p53BS) detected in exon 2 of the IRF-5 gene could in fact bind to p53 protein. Moreover, a heterologous reporter assay revealed that the p53BS possessed p53-dependent transcriptional activity. Expression of IRF-5 was induced in p53+/+ cells (MCF7 and NHDF), but not inp53−/− cells (H1299) when DNA was damaged by γ-irradiation, UV-radiation, or adriamycin treatment in a wild-type p53-dependent manner. These results suggest that IRF-5 is a novel p53-target, and that it might mediate the p53-dependent immune response.

Mechanism of Apoptosis Induced by the Inhibition of Fatty Acid Synthase in Breast Cancer Cells

Fatty acid synthase (FAS) has been found to be overexpressed in a wide range of epithelial tumors, including breast cancer. Pharmacologic inhibitors of FAS cause apoptosis of breast cancer cells and result in decreased tumor size in vivo. However, how the inhibition of FAS induces apoptosis in tumor cells remains largely unknown. To understand the apoptotic pathway resulting from direct inhibition of FAS, we treated breast tumor cells with or without FAS small interfering RNA (siRNA) followed by a microarray analysis. Our results indicated that the proapoptotic genes BNIP3, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and death-associated protein kinase 2 (DAPK2) were significantly up-regulated on direct inhibition of the FAS gene. We also found that the knockdown of FAS expression significantly increased ceramide level in the tumor cells, and this increase was abrogated by acetyl-CoA carboxylase inhibitor. In addition, carnitine palmitoyltransferase-1 (CPT-1) inhibitor up-regulated the ceramide and BNIP3 levels in these cells, whereas treatment of tumor cells with FAS siRNA in the presence of a ceramide synthase inhibitor abrogated the up-regulation of BNIP3 and inhibited apoptosis. Furthermore, we found that treatment of cells with BNIP3 siRNA significantly counteracted the effect of FAS siRNA-mediated apoptosis. Consistent with these results, a significant inverse correlation was observed in the expression of FAS and BNIP3 in clinical samples of human breast cancer. Collectively, our results indicate that inhibition of FAS in breast cancer cells causes accumulation of malonyl-CoA, which leads to inhibition of CPT-1 and up-regulation of ceramide and induction of the proapoptotic genes BNIP3, TRAIL, and DAPK2, resulting in apoptosis.

FAS expression inversely correlates with PTEN level in prostate cancer and a PI 3-kinase inhibitor synergizes with FAS siRNA to induce apoptosis.

Fatty acid synthase (FAS), a key enzyme of the fatty acid biosynthetic pathway, has been shown to be overexpressed in various types of human cancer and is, therefore, considered to be an attractive target for anticancer therapy. However, the exact mechanism of overexpression of the FAS gene in tumor cells is not well understood. In this report, we demonstrate that the expression of the tumor suppressor gene PTEN has a significant inverse correlation with FAS expression in the case of prostate cancer in the clinical setting, and inhibition of the PTEN gene leads to the overexpression of FAS in vitro. We also found that the combination of the expression status of these two genes is a better prognostic marker than either gene alone. Furthermore, our results indicate that the specific inhibition of FAS gene by siRNA leads to apoptosis of prostate tumor cells, and inhibition of PI 3-kinase pathway synergizes with FAS siRNA to enhance tumor cell death. These results provide a strong rationale for exploring the therapeutic use of an inhibitor of the PTEN signaling pathway in conjunction with the FAS siRNA to inhibit prostate tumor growth.

Drug development against metastasis-related genes and their pathways: A rationale for cancer therapy

It is well recognized that the majority of cancer related deaths is caused by metastatic diseases. Therefore, there is an urgent need for the development of therapeutic intervention specifically targeted to the metastatic process. In the last decade, significant progress has been made in this research field, and many new concepts have emerged that shed light on the molecular mechanism of metastasis cascade which is often portrayed as a succession of six distinct steps; localized invasion, intravasation, translocation, extravasation, micrometastasis and colonization. Successful metastasis is dependent on the balance and complex interplay of both the metastasis promoters and suppressors in each step. Therefore, the basic strategy of our interventions is aimed at either blocking the promoters or potentiating the suppressors in this disease process. Toward this goal, various kinds of antibodies and small molecules have been designed. These include agents that block the ligand-recepter interaction of metastasis promoters (HGF/c-Met), antagonize the metastasis-promoting enzymes (AMF, uPA and MMP) and inhibit the transcriptional activity of metastasis promoter (beta-Catenin). On the other hand, the intriguing roles of metastasis suppressors and their signal pathways have been extensively studied and various attempts have been made to potentiate these factors. Small molecules have been developed to restore the expression or mimic the function of metastasis-suppressor genes such as NM23, E-cadherin, Kiss-1, MKK4 and NDRG1, and some of them are under clinical trials. This review summarizes our current understanding of the molecular pathway of tumor metastasis and discusses strategies and recent development of anti-metastatic drugs.

The tumor metastasis suppressor gene Drg-1 down-regulates the expression of activating transcription factor 3 in prostate cancer.

The tumor metastasis suppressor gene Drg-1 has been shown to suppress metastasis without affecting tumorigenicity in immunodeficient mouse models of prostate and colon cancer. Expression of Drg-1 has also been found to have a significant inverse correlation with metastasis or invasiveness in various types of human cancer. However, how Drg-1 exerts its metastasis suppressor function remains unknown. In the present study, to elucidate the mechanism of action of the Drg-1 gene, we did a microarray analysis and found that induction of Drg-1 significantly inhibited the expression of activating transcription factor (ATF) 3, a member of the ATF/cyclic AMP-responsive element binding protein family of transcription factors. We also showed that Drg-1 attenuated the endogenous level of ATF3 mRNA and protein in prostate cancer cells, whereas Drg-1 small interfering RNA up-regulated the ATF3 expression. Furthermore, Drg-1 suppressed the promoter activity of the ATF3 gene, indicating that Drg-1 regulates ATF3 expression at the transcriptional level. Our immunohistochemical analysis on prostate cancer specimens revealed that nuclear expression of ATF3 was inversely correlated to Drg-1 expression and positively correlated to metastases. Consistently, we have found that ATF3 overexpression promoted invasiveness of prostate tumor cells in vitro, whereas Drg-1 suppressed the invasive ability of these cells. More importantly, overexpression of ATF3 in prostate cancer cells significantly enhanced spontaneous lung metastasis of these cells without affecting primary tumorigenicity in a severe combined immunodeficient mouse model. Taken together, our results strongly suggest that Drg-1 suppresses metastasis of prostate tumor cells, at least in part, by inhibiting the invasive ability of the cells via down-regulation of the expression of the ATF3 gene.

RhoC Promotes Metastasis via Activation of the Pyk2 Pathway in Prostate Cancer

RhoC is a member of the Ras-homologous family of genes which have been implicated in tumorigenesis and tumor progression. However, the exact role of RhoC is controversial and is yet to be clarified. We have examined the effect of RhoC on prostate tumor cells and found that RhoC had no effect on cell proliferation in vitro or on tumor growth in mice. However, RhoC significantly enhanced the metastatic ability of the tumor cells in these animals, suggesting that RhoC affects only the metastasis but not the growth of prostate tumor cells. The results of our immunohistochemical analyses on tumor specimens from 63 patients with prostate cancer indicate that RhoC expression had no significant correlation with Gleason grade. However, the expression of RhoC showed significant positive correlation with both lymph node and distant metastasis, and it was inversely correlated with patient survival. We also found that RhoC significantly augmented the invasion and motility of prostate tumor cells by activating matrix metalloproteinases 2 and 9 (MMP2 and MMP9) in vitro. The results of our antibody array analysis for signal molecules revealed that RhoC significantly activated kinases including mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), Akt, and Pyk2. Inhibition of Pyk2 kinase blocked the RhoC-dependent activation of FAK, MAPK, and Akt, followed by the suppression of MMP2 and MMP9. Inhibitors of both MAPK and Akt also significantly blocked the activities of these MMPs. Therefore, our results indicate that RhoC promotes tumor metastasis in prostate cancer by sequential activation of Pyk2, FAK, MAPK, and Akt followed by the up-regulation of MMP2 and MMP9, which results in the stimulation of invasiveness of tumor cells.

Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain

Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than 20%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumour cells in the brain highly expressed IL‐1β which then ‘activated’ surrounding astrocytes. This activation significantly augmented the expression of JAG1 in the astrocytes, and the direct interaction of the reactivated astrocytes and cancer stem‐like cells (CSCs) significantly stimulated Notch signalling in CSCs. We also found that the activated Notch signalling in CSCs up‐regulated HES5 followed by promoting self‐renewal of CSCs. Furthermore, we have shown that the blood‐brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis in vivo. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re‐establish their niche for their self‐renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease.