Ario Takeuchi

Clusterin Mediates TGF-β–Induced Epithelial–Mesenchymal Transition and Metastasis via Twist1 in Prostate Cancer Cells

TGF-β promotes epithelial-mesenchymal transition (EMT) and induces clusterin (CLU) expression, linking these genes to cancer metastasis. CLU is a pleiotropic molecular chaperone that confers survival and proliferative advantage to cancer cells. However, the molecular mechanisms by which TGF-β regulates CLU expression and CLU affects metastasis remain unknown. In this study, we report that the transcription factor Twist1 mediates TGF-β-induced CLU expression. By binding to E-boxes in the distal promoter region of CLU gene, Twist1 regulated basal and TGF-β-induced CLU transcription. In addition, CLU reduction reduced TGF-β induction of the mesenchymal markers, N-cadherin and fibronectin, thereby inhibiting the migratory and invasive properties induced by TGF-β. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, our findings indicate that CLU is an important mediator of TGF-β-induced EMT, and suggest that CLU suppression may represent a promising therapeutic option for suppressing prostate cancer metastatic progression.

IL-17 production by γδ T cells is important for the antitumor effect of Mycobacterium bovis bacillus Calmette-Guérin treatment against bladder cancer.

Intravesical inoculation of Mycobacterium bovis bacillus Calmette‐Guérin (BCG) has been used for the treatment of bladder cancer. Recent studies implied the requirement of neutrophil infiltration for the antitumor effect. In this study, we found that IL‐17 was produced in the bladder after BCG treatment, preceding the infiltration of neutrophils. Neutrophils in the bladder after BCG treatment were reduced in IL‐17‐deficient mice, in which BCG‐induced antitumor effect against intravesically inoculated bladder cancer was abolished. Notably, the level of IL‐17 production and the number of neutrophils in BCG‐treated bladder was reduced in γδ T‐cell‐deficient mice but not in CD4‐depleted mice. Survival of bladder cancer‐inoculated γδ T‐cell‐deficient mice was not improved by BCG treatment. These results suggest that IL‐17‐producing γδ T cells play a key role in the BCG‐induced recruitment of neutrophils to the bladder, which is essential for the antitumor activity against bladder cancer.

Y-box binding protein-1 promotes castration-resistant prostate cancer growth via androgen receptor expression

The androgen receptor (AR) is well known to play a central role in the pathogenesis of prostate cancer (PCa). In several studies, AR was overexpressed in castration-resistant PCa (CRPC). However, the mechanism of AR overexpression in CRPC is not fully elucidated. Y-box binding protein-1 (YB-1) is a pleiotropic transcription factor that is upregulated in CPRC. We aimed to elucidate the role of YB-1 in castration resistance of PCa and identify therapeutic potential of targeting YB-1. Using immunohistochemistry, we found that nuclear YB-1 expression significantly correlated with the Gleason score and AR expression in PCa tissues. In PCa cells, YB-1 regulated AR expression at the transcriptional level. Furthermore, YB-1 expression and nuclear localization were upregulated in CRPC cells. Overexpression of AR, as well as YB-1, conferred castration-resistant growth in LNCaP and 22Rv1 cells. Conversely, knocking down YB-1 resulted in suppressed cell growth and induced apoptosis, which was more efficient than knocking down AR in LNCaP cells. In other types of PCa cells, such as CRPC cells, knocking down YB-1 resulted in a significant reduction of cell growth. In conclusion, these findings suggested that YB-1 induces castration resistance in androgen-dependent PCa cells via AR expression. Thus, YB-1 may be a promising therapeutic target for PCa, as well as CRPC.

Antioxidant Therapy Alleviates Oxidative Stress by Androgen Deprivation and Prevents Conversion From Androgen Dependent to Castration Resistant Prostate Cancer

PURPOSE Prostate cancer progression from androgen dependence to castration resistance results at least in part from oxidative stress induced by androgen deprivation therapy. We elucidated the state and the role of oxidative stress induced by androgen deprivation therapy and the possibility of antioxidant therapy in human prostate cancer. MATERIALS AND METHODS We investigated 4-HNE (4-hydroxy-2-nonenal histidine adduct) staining, and Twist1, YB-1 and androgen receptor expression by immunohistochemistry in prostate cancer samples treated with or without neoadjuvant androgen deprivation therapy. Intracellular reactive oxygen species and protein expression were examined by CM-H(2)DCFDA and Western blot analysis, respectively. A cell proliferation assay and a mouse xenograft model were used to assess tumor growth. RESULTS Androgen deprivation therapy increased oxidative stress, as shown by 4-HNE staining in human prostate cancer tissue. Twist1 and YB-1 expression was up-regulated by androgen deprivation, resulting in androgen receptor over expression. In LNCaP and 22Rv1 cells androgen deprivation increased intracellular reactive oxygen species and evoked Twist1, YB-1 and androgen receptor over expression, resulting in cell growth in a castration resistant manner. Growth was alleviated by N-acetyl-cysteine, an electrophile that supports glutathione production. N-acetyl-cysteine also decreased LNCaP and 22Rv1 tumor growth in castrated and noncastrated mice. CONCLUSIONS Androgen deprivation therapy induced oxidative stress in in vitro and human prostate cancer. Antioxidant therapy using N-acetyl-cysteine appears to be a promising therapeutic modality for prostate cancer.

Oral administration of xanthan gum enhances antitumor activity through Toll-like receptor 4

PURPOSE Xanthan gum (XG) is a complex exopolysaccharide produced by the plant-pathogenic bacterium Xanthomonas campestris pv. and is widely used as a thickener or viscosifier. We examined in this study the antitumor effects of XG. EXPERIMENTAL DESIGN Cytokine production by XG-stimulated murine macrophage cell lines, J772 and RAW264.7, and peritoneal adherent cells from wild type C57BL/6 mice, TLR2 or MyD88-deficient mice, C3H/HeN, and TLR4-mutant C3H/HeJ mice were examined. In order to examine in vivo antitumor effects of XG, mice were inoculated subcutaneously with tumor cells and administered orally with XG once every 5days from 1day before the tumor inoculation. Tumor growth, mouse survival, NK activity, and tumor-specific cytotoxicity were examined. RESULTS In vitro culture with XG induced interleukin-12 and tumor necrosis factor-alpha production from macrophages. XG stimulated macrophages in a MyD88-dependent manner and was mainly recognized by Toll-like receptor 4 (TLR4). Oral administration of XG significantly retarded tumor growth and prolonged survival of the mice inoculated subcutaneously with B16K(b) melanoma cells. NK activity as well as tumor-specific cytotoxicity of CD8 T cells was augmented in the XG-treated mice. The in vivo antitumor effects of XG were also dependent on TLR-4, as C3H/HeJ mice, which lack TLR4 signaling, exhibited no effect of XG on the growth of syngeneic bladder tumor, MBT-2. CONCLUSION These results suggest beneficial effects of oral administration of XG on immune-surveillance against neoplasms.

Interaction between docetaxel resistance and castration resistance in prostate cancer: implications of Twist1, YB-1, and androgen receptor.

Taxanes, including docetaxel, are currently the only cytotoxic chemotherapeutic agents proven to confer survival benefit in patients with castration‐resistant prostate cancer (CRPC). However, the merits of taxanes remain modest, and efforts are needed to improve their therapeutic efficacy.

Statins reduce the androgen sensitivity and cell proliferation by decreasing the androgen receptor protein in prostate cancer cells

Statins (3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase inhibitors) are cholesterol‐lowering drugs that are widely used to prevent and treat atherosclerotic cardiovascular disease. Recent epidemiological studies suggest that statins reduce serum prostate‐specific antigen (PSA) levels and decrease the risk of prostate cancer. In the present study, we determined the molecular mechanisms related to the regulation of PSA, androgen receptor (AR) and cell proliferation in prostate cancer cell lines by statins.

Peroxiredoxin 2 in the nucleus and cytoplasm distinctly regulates androgen receptor activity in prostate cancer cells.

Currently, few therapies are effective against castration-resistant prostate cancer. Increased activation of the androgen/androgen receptor (AR) signaling pathway is thought to promote castration-resistant prostate cancer. Herein, we report that peroxiredoxin (Prx) gene expression in castration-resistant prostate cancer and hydrogen peroxide-resistant cells was upregulated. Prx2 was overexpressed in castration-resistant prostate cancer at the mRNA and protein levels and was localized to the nucleus and cytoplasm. Overexpression of Prx2 increased AR transactivation, whereas Prx2 overexpression in the nucleus suppressed AR transactivation. These effects of Prx2 on AR activity were abolished by the introduction of function-disrupting mutations into Cys⁵¹ and Cys¹⁷². Silencing Prx2 reduced the expression of androgen-regulated genes and suppressed the growth of AR-expressing prostate cancer cells by inducing cell-cycle arrest at the G1 phase. Furthermore, Prx2 knockdown also suppressed cell growth in castration-resistant prostate cancer cells. These findings indicate that Prx2 is involved in the proliferation of AR-expressing prostate cancer cells by modulating AR activity. Designing therapeutics targeting Prx2 may offer a novel strategy for developing treatments for prostate cancer, including castration-resistant prostate cancer, which is dependent on AR signaling.

Inhibition of Protein Kinase C/Twist1 Signaling Augments Anticancer Effects of Androgen Deprivation and Enzalutamide in Prostate Cancer

Purpose: The progression of prostate cancer to metastatic and castration-resistant disease represents a critical step. We previously showed that the transcription factor Twist1, which promotes epithelial–mesenchymal transition, was involved in castration-resistant progression. Similarly, protein kinase C (PKC) has been implicated in both metastatic progression and castration resistance in prostate cancer. Experimental Design: In this study, we aimed to elucidate the role of PKC/Twist1 signaling in castration resistance, and to apply this information to the development of a novel therapeutic concept using PKC inhibitor Ro31-8220 against prostate cancer using various prostate cancer cell lines. Results: Androgen deprivation and the next-generation antiandrogen enzalutamide induced PKC activation and Twist1 expression, which were reversed by the PKC inhibitor Ro31-8220. Ro31-8220 suppressed cell proliferation in androgen-dependent prostate cancer LNCaP cells, which was augmented by its combination with androgen deprivation or enzalutamide. The favorable anticancer effects of the combination of Ro31-8220 and enzalutamide were also observed in castration-resistant C4-2 and 22Rv1 cells. Furthermore, PKC phosphorylation was elevated in castration-resistant and enzalutamide-resistant cells compared with their parental cells, leading to persistent sensitivity to Ro-31-8220 in castration- and enzalutamide-resistant cells. Conclusions: Taken together, these findings indicate that PKC/Twist1 signaling contributes to castration resistance as well as enzalutamide resistance in prostate cancer, and suggest that therapeutics targeting PKC/Twist1 signaling, such as PKC inhibitors, represent a promising novel therapeutic strategy for prostate cancer, especially castration-resistant prostate cancer, when combined with enzalutamide. Clin Cancer Res; 20(4); 951–61. ©2013 AACR.

Androgen receptor signaling regulates cell growth and vulnerability to doxorubicin in bladder cancer

PURPOSE There are several reports of androgen receptor in bladder cancer cases but androgen receptor expression and the function of androgen/androgen receptor signaling in bladder cancer remain unclear. We investigated androgen receptor expression and the role of androgen/androgen receptor signaling in bladder cancer. MATERIALS AND METHODS We evaluated AR mRNA expression in bladder cancer tissue by quantitative real-time polymerase chain reaction. The role of androgen receptor in cell growth and drug sensitivity was also evaluated in vitro and in vivo in several bladder cancer cell lines. RESULTS AR mRNA expression inversely correlated with bladder cancer grade, stage and spread. Of several bladder cancer cell lines UMUC3 and MBT-2 markedly expressed androgen receptor transcript and protein. In each cell line androgen/androgen receptor signaling blockade using androgen deprivation, blockade knockdown and antiandrogen agents decreased cell growth, colony formation and cell viability. Androgen receptor expression was implicated in doxorubicin resistance. Inversely androgen receptor deprivation and knockdown made UMUC3 cells sensitive to doxorubicin. Finally, castration slightly suppressed UMUC3 tumor growth in vivo, although this did not attain statistical significance. CONCLUSIONS AR transcript expression inversely correlates with bladder cancer clinicopathological characteristics. Androgen/androgen receptor signaling has an important role in the growth and vulnerability to doxorubicin of bladder cancer cells that express androgen receptor. Androgen/androgen receptor signaling might be a possible prophylactic and therapeutic target for bladder cancer that shows androgen receptor expression.