Hiroshi Okuda

Metabolic genes in cancer: their roles in tumor progression and clinical implications.

Re-programming of metabolic pathways is a hallmark of physiological changes in cancer cells. The expression of certain genes that directly control the rate of key metabolic pathways including glycolysis, lipogenesis and nucleotide synthesis are drastically altered at different stages of tumor progression. These alterations are generally considered as an adaptation of tumor cells; however, they also contribute to the progression of tumor cells to become more aggressive phenotypes. This review summarizes the recent information about the mechanistic link of these genes to oncogenesis and their potential utility as diagnostic markers as well as for therapeutic targets. We particularly focus on three groups of genes; GLUT1, G6PD, TKTL1 and PGI/AMF in glycolytic pathway, ACLY, ACC1 and FAS in lipogenesis and RRM2, p53R2 and TYMS for nucleotide synthesis. All these genes are highly up-regulated in a variety of tumor cells in cancer patients, and they play active roles in tumor progression rather than expressing merely as a consequence of phenotypic change of the cancer cells. Molecular dissection of their orchestrated networks and understanding the exact mechanism of their expression will provide a window of opportunity to target these genes for specific cancer therapy. We also reviewed existing database of gene microarray to validate the utility of these genes for cancer diagnosis.

miR-7 Suppresses Brain Metastasis of Breast Cancer Stem-Like Cells By Modulating KLF4

Despite significant improvement in survival rates of patients with breast cancer, prognosis of metastatic disease is still dismal. Cancer stem-like cells (CSC) are considered to play a role in metastatic progression of breast cancer; however, the exact pathologic role of CSCs is yet to be elucidated. In this report, we found that CSCs (CD24(-)/CD44(+)/ESA(+)) isolated from metastatic breast cell lines are significantly more metastatic than non-CSC populations in an organ-specific manner. The results of our microRNA (miRNA) profile analysis for these cells revealed that CSCs that are highly metastatic to bone and brain expressed significantly lower level of miR-7 and that this miRNA was capable of modulating one of the essential genes for induced pluripotent stem cell, KLF4. Interestingly, high expression of KLF4 was significantly and inversely correlated to brain but not bone metastasis-free survival of patients with breast cancer, and we indeed found that the expression of miR-7 significantly suppressed the ability of CSCs to metastasize to brain but not to bone in our animal model. We also examined the expression of miR-7 and KLF4 in brain-metastatic lesions and found that these genes were significantly down- or upregulated, respectively, in the tumor cells in brain. Furthermore, the results of our in vitro experiments indicate that miR-7 attenuates the abilities of invasion and self-renewal of CSCs by modulating KLF4 expression. These results suggest that miR-7 and KLF4 may serve as biomarkers or therapeutic targets for brain metastasis of breast cancer.

N-myc downstream regulated gene 1 modulates Wnt-β-catenin signalling and pleiotropically suppresses metastasis.

Wnt signalling has pivotal roles in tumour progression and metastasis; however, the exact molecular mechanism of Wnt signalling in the metastatic process is as yet poorly defined. Here we demonstrate that the tumour metastasis suppressor gene, NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signalling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumour cells. Importantly, restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumour cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG−/LRP+ signature has a strong predictable value for recurrence‐free survival of cancer patients. Collectively, we have identified NDRG1 as a novel negative master regulator of Wnt signalling during the metastatic progression, which opens an opportunity to define a potential therapeutic target for metastatic disease.

Resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase.

Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. Resveratrol has been shown to have strong hypolipidemic effect on normal adipocytes and as hyper-lipogenesis is a hallmark of cancer cell physiology, the effect of resveratrol on lipid synthesis in cancer stem-like cells (CD24−/CD44+/ESA+) that were isolated from both ER+ and ER− breast cancer cell lines was examined. The authors found that resveratrol significantly reduced the cell viability and mammosphere formation followed by inducing apoptosis in cancer stem-like cells. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the fatty acid synthase (FAS) gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by TOFA and by Fumonisin B1, suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal model of xenograft without showing apparental toxicity. Taken together, the results of this study indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol.

Hyaluronan Synthase HAS2 Promotes Tumor Progression in Bone by Stimulating the Interaction of Breast Cancer Stem- Like Cells with Macrophages and Stromal Cells

The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here, we report that upregulation of hyaluronan synthase 2 (HAS2) occurs in highly metastatic breast cancer stem-like cells (CSC) defined by CD44(+)/CD24(-)/ESA(+) phenotype, where it plays a critical role in the generation of a prometastatic microenvironment in breast cancer. HAS2 was critical for the interaction of CSCs with tumor-associated macrophages (TAM), leading to enhanced secretion of platelet-derived growth factor-BB from TAMs, which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone, an inhibitor of HAS, which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings show a critical role of HAS2 in the development of a prometastatic microenvironment and suggest that HAS2 inhibitors can act as antimetastatic agents that disrupt a paracrine growth factor loop within this microenvironment.

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.

Cacalol, a natural sesquiterpene, induces apoptosis in breast cancer cells by modulating Akt-SREBP-FAS signaling pathway

We previously isolated cacalol as a free radical-scavenging compound from Cacalia delphiniifolia which is a traditional Asian herbal plant and is believed to have medicinal effects on cancer. In this report, we demonstrated that cacalol has strong anti-proliferation effect on breast cancer cells and induces apoptosis by activating a pro-apoptotic pathway. We also found that a combination of cacalol and other chemotherapeutic drugs (Taxol and cyclophosphamide) synergistically induced apoptosis and partially overcame chemo-resistance. To further gain a mechanistic insight, we tested a potential inhibitory effect of cacalol on fatty acid synthase gene (FAS) in breast cancer cells, and found that cacalol significantly modulated the expression of the FAS gene, which resulted in apoptosis through activation of DAPK2 and caspase 3. We have also shown that cacalol significantly suppressed the Akt-sterol regulatory element-binding proteins (SREBP) signaling pathway and concomitant transcriptional activation of FAS. In a xenograft model of nude mouse, when cacalol was administered intraperitoneally, tumor growth was significantly suppressed. Importantly, oral administration of cacalol before implanting tumors showed significant preventive effect on tumor growth in the same animal model. Furthermore, the treatment of mice with a combination of low dose of Taxol and cacalol significantly suppressed the tumor growth. Taken together, our results indicate that cacalol induces apoptosis in breast cancer cells and impairs mammary tumor growth in vivo by blocking the expression of the FAS gene through modulation of Akt-SREBP pathway, suggesting that cacalol has potential utility as a chemopreventive and chemotherapeutic agent for breast cancer.

Abstract 3865: HAS2 promotes tumor progression by stimulating interaction of cancer stem-like cells with tumor associated macrophage and stromal cells through a paracrine loop of growth factors in the bone.

The molecular mechanism of metastatic progression, particularly the roles of microenvironment of the metastasized organs, is as yet poorly understood. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM with or without carrying a shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA to HAS2 significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. Moreover, the interaction between cancer cell and TAMs enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and in turn enhanced self-renewal of CSCs by secretion of FGF9 and FGF7. We also found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer. Citation Format: Sambad Sharma, Hiroshi Okuda, Fei Xing, Aya Kobayashi, Misako Watabe, Puspa R. Pandey, Sudha K. Pai, Wen Liu, Koji Fukuda, Megumi Iiizumi, Kounosuke Watabe. HAS2 promotes tumor progression by stimulating interaction of cancer stem-like cells with tumor associated macrophage and stromal cells through a paracrine loop of growth factors in the bone. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3865. doi:10.1158/1538-7445.AM2013-3865

Abstract C75: miR-7 suppresses brain metastasis of cancer stem-like cells by modulating Kruppel-like factor 4 in breast cancer

The majority of cancer death of breast cancer attributed to metastatic disease. Despite this clinical importance, the exact pathological process of tumor metastasis is as yet poorly understood, and deciphering the exact molecular mechanism of this process is of paramount importance in order to identify specific therapeutic targets for this devastating disease. Recent active research in microRNA identified a series of this type of molecules that are involved in tumor progression in various tumors as oncogenes and tumor suppressors. Cancer stem-like cells (CSCs) are considered to play a role in metastatic progression of breast cancer; however, the exact pathological role of CSCs is yet to be elucidated. In this report, we found that CSCs (CD24-/CD44+/ESA+) isolated from metastatic breast cell lines are significantly more metastatic than non-CSC populations in an organ specific manner. The results of our microRNA profile analysis for these cells revealed that CSCs that are highly metastatic to bone and brain expressed significantly lower level of miR-7 and that this microRNA was capable of modulating one of the essential genes for induced pluripotent stem cell, KLF4. Interestingly, high expression of KLF4 was significantly and inversely correlated to brain- but not bone-metastasis free survival of breast cancer patients, and we indeed found that the expression of miR-7 significantly suppressed the ability of CSCs to metastasize to brain but not to bone in our animal model. We also examined the expression of miR-7 and KLF4 in brain-metastatic lesions and found that these genes were significantly down- or up-regulated, respectively, in the tumor cells in brain. Furthermore, the results of our in vitro experiments indicate that miR-7 attenuates the abilities of invasion and self-renewal of CSCs by modulating KLF4 expression. These results suggest that miR-7 and KLF4 may serve as biomarkers or therapeutic targets for brain metastasis of breast cancer. Citation Format: Fei Xing, Hiroshi Okuda, Aya Kobayashi, Puspa R. Pandey, Puspa R. Pandey, Sambad Sharma, Yin-Yuan Mo, Yin Liu, Kerui Wu, Kounosuke Watabe. miR-7 suppresses brain metastasis of cancer stem-like cells by modulating Kruppel-like factor 4 in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C75.

Abstract 1416: BMP 7 regulates dormancy and recurrence of prostate cancer stem cell in bone via P38/NDRG1/P21 signaling axis.

Bone is the most common metastatic site of prostate cancer, which affects approximately 70% of patients with advanced disease. Although patients with localized lesions can be cured by radical prostatectomy or radiotherapy, more than 90% of cancer deaths are attributed to metastatic disease. Growth of the tumor cells in the bone is generally slow and they often become dormant until an appropriate microenvironment is established for their recurrence. However, how these metastatic tumor cells become dormant and how they recur at the target organs is virtually unknown. To address this critical question, we have studied an interaction of prostate cancer cells (PC3mm) and bone stromal cells (HS5).The conditioned medium of HS5 induced growth arrest and senescence to prostate cancer cells. Our results of Western blot, qRT-PCR and reporter assay showed that this effect was mediated by induction of p38 MAP kinase, the tumor metastasis suppressor gene, N-myc downstream regulated gene 1 (NDRG1) and cell cycle inhibitors. We also found that this secretory factor is bone morphogenetic protein 7 (BMP7) and that BMP7 indeed induced NDRG1 and p21 through activation of p38. Importantly, BMP7 was able to induce senescence to PC3mm cell; however, the tumor cells regained their growth ability upon withdrawal of BMP7, suggesting that BMP7-induced senescence is reversible. Notably, our results of the existing database analysis indicate that the expression level of BMPR2, one of the BMP7 receptors, showed inverse correlation with bone metastasis and recurrence of prostate cancer. Furthermore, BMP7 significantly suppressed sphere forming ability of the cancer stem-like cells isolated from PC3mm and induced senescence followed by activation of NDRG1 and p21, and this effect was reversed by withdrawal of BMP7. To determine whether BMP7 secreted from bone stromal cells induces growth arrest of cancer stem-like cells in vivo, we co-injected the cancer stem-like cells with bone stromal cells which had either scrambled shRNA (control) or shRNA for BMP7 into the tibia of nude mice. We found that the control HS5 suppressed the growth of cancer stem-like cells, while HS5 with knocked-down BMP7 failed to inhibit cancer growth in the bone. Furthermore, direct injection of BMP7 after inoculation of cancer stem-like cells into mouse tibiae significantly suppressed the tumor growth. More importantly, we found that withdrawal of BMP7 treatment led the tumor to regrow in the bone, suggesting again that BMP7 plays a critical role in determining the fate of cancer stem-like cells for dormancy and recurrence in bone metastasis of prostate cancer. Our results revealed a novel mechanism of the BMP7-NDRG1axis which plays a critical role in the regulation of dormancy and recurrence of prostate tumor cells in bone metastasis. Citation Format: Fei Xing, Aya Kobayashi, Hiroshi Okuda, Puspa Pandey, Misako Watabe, Sudha K. Pai, Sambad Sharma, Kounosuke Watabe. BMP 7 regulates dormancy and recurrence of prostate cancer stem cell in bone via P38/NDRG1/P21 signaling axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1416. doi:10.1158/1538-7445.AM2013-1416