Ryuichi Fukuyama

Breast Cancer Metastasis Suppressor 1 Inhibits Gene Expression by Targeting Nuclear Factor-κB Activity

Breast cancer metastasis suppressor 1 ( BRMS1 ) functions as a metastasis suppressor gene in breast cancer and melanoma cell lines, but the mechanism of BRMS1 suppression remains unclear. We determined that BRMS1 expression was inversely correlated with that of urokinase-type plasminogen activator ( uPA ), a prometastatic gene that is regulated at least in part by nuclear factor-κB (NF-κB). To further investigate the role of NF-κB in BRMS1-regulated gene expression, we examined NF-κB binding activity and found an inverse correlation between BRMS1 expression and NF-κB binding activity in MDA-MB-231 breast cancer and C8161.9 melanoma cells stably expressing BRMS1. In contrast, BRMS1 expression had no effect on activation of the activator protein-1 transcription factor. Further, we showed that suppression of both constitutive and tumor necrosis factor-α–induced NF-κB activation by BRMS1 may be due to inhibition of IκBα phosphorylation and degradation. To examine the relationship between BRMS1 and uPA expression in primary breast tumors, we screened a breast cancer dot blot array of normalized cDNA from 50 breast tumors and corresponding normal breast tissues. There was a significant reduction in BRMS1 mRNA expression in breast tumors compared with matched normal breast tissues (paired t test, P uPA gene expression ( P

Mutated in Colorectal Cancer, a Putative Tumor Suppressor for Serrated Colorectal Cancer, Selectively Represses β-catenin-Dependent Transcription

Mutated in colorectal cancer (MCC) was originally identified as a candidate gene for familial adenomatous polyposis (FAP) but further study identified adenomatous polyposis coli (APC) as responsible for FAP and the physiologic/pathologic roles of MCC remained poorly understood. Recently, MCC promoter methylation was discovered as a frequent early event in a distinct subset of precursor lesions and colorectal cancer (CRC) associated with the serrated CRC pathway. Here we provide the first evidence of the biological significance of MCC loss in CRC and the molecular pathways involved. We show MCC expression is dramatically decreased in many CRC cell lines and the distinct subset of sporadic CRC characterized by the CpG island methylator phenotype and BRAFV600E mutation due to promoter methylation as reported previously. Importantly, we find MCC interacts with β-catenin and that reexpression of MCC in CRC cells specifically inhibits Wnt signaling, β-catenin/T-cell factor/lymphoid-enhancer factor-dependent transcription and cellular proliferation even in the presence of oncogenic mutant APC. We also show that MCC is localized in the nucleus and identify two functional nuclear localization signals. Taken together, MCC is a nuclear, β-catenin-interacting protein that can act as a potential tumor suppressor in the serrated CRC pathway by inhibiting Wnt/β-catenin signal transduction.

Role of IKK and oscillatory NFκB kinetics in MMP‐9 gene expression and chemoresistance to 5‐fluorouracil in RKO colorectal cancer cells

Nuclear factor kappa B (NFκB) is a central participant in the metastasis and chemoresistance of colorectal cancer (CRC). However, it is not fully understood to what extent NFκB contributes to induction of the metastasis‐associated matrix metalloprotease‐9 (MMP‐9) gene and sensitivity to the commonly used chemotherapeutic 5‐fluorouracil (5‐Fu) in CRC. Using the RKO human CRC cell line and two NFκB signaling deficient RKO mutants, we investigated NFκBs role in the induction of MMP‐9 and 5‐Fu sensitivity in RKO CRC cells. NFκB plays a predominant role in MMP‐9 gene induction in RKO cells, as evidenced by the failure of tumor necrosis factor alpha (TNFα) to induce MMP‐9 in either of the NFκB signaling mutants. RKO cells exhibit a robust, oscillatory NFκB activity in response to TNFα not seen in either of the NFκB mutant cell lines, which instead demonstrate diminished, nonoscillatory NFκB activation. Analysis of TNFα‐induced phosphorylation and MMP‐9 promoter recruitment of the p65 NFκB subunit revealed a significant reduction in p65 phosphorylation as well as reduced and altered recruitment of p65 to the MMP‐9 gene promoter in the mutants compared to the parental RKO cell line. 5‐Fu only activated NFκB in the parental RKO cells through induction of IκB‐kinase (IKK) activity and increased sensitivity to 5‐Fu is observed in both NFκB mutant lines. Our results suggest that TNFα‐dependent induction of MMP‐9 gene expression is tightly regulated by oscillatory/cumulative activation of NFκB and that 5‐Fu stimulates NFκB and RKO CRC cell survival through induction of IKK activity.

BRMS1 contributes to the negative regulation of uPA gene expression through recruitment of HDAC1 to the NF-κB binding site of the uPA promoter

The BRMS1 metastasis suppressor was recently shown to negatively regulate NF-κB signaling and down regulate NF-κB-dependent uPA expression. Here we confirm that BRMS1 expression correlates with reduced NF-κB DNA binding activity in independently derived human melanoma C8161.9 cells stably expressing BRMS1. We show that knockdown of BRMS1 expression in these cells using small interfering RNA (siRNA) leads to the reactivation of NF-κB DNA binding activity and re-expression of uPA. Further, we confirm that BRMS1 expression does not alter IKKβ kinase activity suggesting that BRMS1-dependent uPA regulation does not occur through inhibition of the classical upstream activators of NF-κB. BRMS1 has been implicated as a corepressor of HDAC1 and consistent with this, we show that BRMS1 promotes HDAC1 recruitment to the NF-κB binding site of the uPA promoter and is associated with reduced H3 acetylation. We also confirm that BRMS1 expression stimulates disassociation of p65 from the NF-κB binding site of the uPA promoter consistent with its reduced DNA binding activity. These data suggest that BRMS1 recruits HDAC1 to the NF-κB binding site of the uPA promoter, modulates histone acetylation of p65 on the uPA promoter, leading to reduced NF-κB binding activity on its consensus sequence, and reduced transactivation of uPA expression.

Enhanced Antitumor Effect of Coincident Intravesical Gemcitabine Plus BCG Therapy in an Orthotopic Bladder Cancer Model

OBJECTIVES To evaluate the antitumor effect of the coincident administration of intravesical gemcitabine (Gem) plus bacillus Calmette-Guérin (BCG) in an orthotopic bladder cancer model. METHODS We evaluated the cytotoxic effect of gemcitabine against MBT-2 cells in vitro. Orthotopic tumors were established by implanting MBT-2 cells into the bladder of syngeneic female C3H mice. Intravesical Gem administration was evaluated at various doses: 0 mg (control); 1, 2, 4, and 8 mg (n = 8 for each group). Next, a comparative evaluation of tumor growth among the control, Gem-alone, BCG-alone, and combined Gem + BCG groups was performed (n = 16 for each group). Therapy was administered at 3-day intervals starting on day 5 and repeated 6 times. To evaluate the proliferative activity among the groups, Ki-67 immunostaining of the tumor was performed. RESULTS Gemcitabine exhibited a dose-dependent antitumor effect. Of the 8 mice in each group treated with a dose of 0, 1, 2, 4, or 8 mg of Gem, 1, 4, 4, 4, 5, and 4 mice failed to develop tumors and survived, respectively. The combination of Gem + BCG (54.1 ± 9.4 days) provided a significant survival advantage compared with BCG-alone (39.0 ± 16.4 days) (P = .02). Ki-67 expression, representing tumor proliferation, was significantly lower in the combined Gem + BCG group than in the BCG-alone group (P < .01). CONCLUSIONS Our results suggest that intravesical Gem + BCG treatment induces an enhanced antitumor effect against bladder tumors.

Abstract 2697: Enhanced antitumor effect of combination intravesical BCG plus Docetaxel therapy in an orthotopic bladder cancer model

Introduction and Objective: Little progress in the management of non-muscle invasive bladder cancer (NMIBC) has been made. We explored the combination of intravesical BCG plus Doc in an orthotopic model. Methods: An orthotopic murine bladder cancer model was used. To assess the antitumoral effect of Doc, intravesical Doc therapy was administered at various dose-escalating concentrations: 0 μg (control: PBS), 25 μg, 50 μg and 100 μg (n = 12 for each group; total of 4 groups). Next, a comparative evaluation of tumor growth among the control group, the BCG-alone group, the Doc-alone group, and three combined BCG + Doc groups (a coincident group [CO] and two sequential groups) was performed (n = 12 for each group; total of 6 groups). The sequential combined groups consisted of SA group (SA; Doc administration followed by BCG administration on the next day) and an SB group (SB; BCG administration followed by Doc administration on the next day). Intravesical therapy was administered at 3-day intervals starting on day 5 and was repeated 5 times. To evaluate proliferation, T cell infiltration and apoptosis among the different groups, the tumor cells in the animal model were stained for Ki-67, CD8 and Cleaved caspase 3 after 4 instillations performed every 3 days. Results: In the Doc groups (12 mice each) treated with a dose of 0 μg (survival period: 32.3 ± 9.4 days), 25 μg (36.8 ± 11.4), 50 μg (38.1 ± 11.0) or 100 μg (40.0 ± 12.7), one, two, two and three mice failed to develop tumors and survived, respectively. The survival advantage for intravesical Doc in the 100 μg administration group was statistically significant when compared with that in the control group (p = 0.04). Out of the 12 mice in each group treated with control, Doc-alone, BCG-alone or combined Doc + BCG (CO, SA, SB), 1, 2, 1, 4, 4 and 1 mice failed to develop tumors and survived, respectively. Significant survival advantages were observed in the combined BCG + Doc group (CO: 50.7 ± 9.0, SA: 51.5 ± 6.9) compared with that in the BCG-alone group (33.1 ± 10.4) and the Doc-alone group (38.8 ± 11.5). The Ki-67 index of the cancer cells was significantly lower in the combined group, compared with that in the BCG-alone group (p Conclusions: Our results suggested that the combination of BCG and Doc treatment decreased the tumor appearance rate and improved the survival period. The enhancement in tumor growth inhibition enabled by combination regimens was presumably associated with a reduction in the tumor proliferation rate and might not depend on either CD8 + T cells or apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2697. doi:10.1158/1538-7445.AM2011-2697

Abstract 1903: Enhanced antitumor effect of coincident combination intravesical MMC plus BCG therapy in an orthotopic bladder cancer model

Introduction and Objective: Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is currently the most successful adjuvant agent for the treatment and/or prophylaxis of superficial bladder tumors. However, superficial bladder tumors recur in 60 to 70% of all cases, and 30 % of these recurrent tumors present with a higher grade with invasive properties. We evaluated the combination of intravesical MMC plus BCG treatment against bladder tumors in a model of orthotopic murine bladder cancer. Methods: An orthotopic murine bladder cancer model was established using the simple instillation of 1 × 10 6 MBT-2 cells into the lumen of the bladder of a female C3H/HeN mouse through a urethral catheter. To assess the antitumoral effect of MMC, intravesical MMC therapy was administered at various dose-escalating concentrations: 0μg (control: PBS), 25μg, 50μg, 100μg, and 200μg, (n = 8 for each group; a total of 5 groups). Next, a comparative evaluation of tumor growth in a control group(PBS), a MCC-alone group, a BCG-alone group(100μg), and a combined MMC plus BCG (100μg) group were performed (n= 8 for each group; a total of 4 groups). Intravesical therapy was administered at 3-day intervals starting on day 5 and repeated 5 times. On day 60 after the initial implantation of the MBT-2 cells, all the surviving mice were sacrificed and necropsied. To evaluate macrophage infiltration in the tumor and the proliferative activity among the different groups, immunohistochemical staining analysis of the tumor cells utilizing CD68 and Ki-67 was performed in animal model. Results: In the MMC groups of 8 mice each treated with a dose of 0μg (survival; 26.4 ± 3.2 days), 25μg (34.8 ± 7.0 days) and 50μg (40.0 ± 13.2 days), 0, 0 and 2 of the mice failed to develop tumor and survived, respectively. In both the 100μg (17.3 ± 2.8 days) and 200μg (11.9 ± 3.0 days) groups, all eight mice died from adverse events. A significant survival advantage was observed in the combined MMC (50μg) plus BCG (100μg) group (51.5 ± 8.1 days), compared with that in the BCG-alone group (33.4 ± 16.8 days; p = 0.03) and the MMC-alone group (39.5 ± 13.5 days; p = 0.04). BCG significantly suppressed while MMC increased macrophage infiltration in the tumors, compared with in the control groups. The Ki-67 expression level, which is regarded as a proliferation index was significantly lower in the combined MMC (50μg) plus BCG (100μg) group (31.1 ± 5.2%) than in the BCG-alone group (50.9 ± 4.1%; p Conclusion: Combining BCG plus MMC treatment decreased the tumor appearance rate, improved the survival period and reduced the proliferation rate in tumors, as compared with BCG-alone and MMC-alone treatment, possibly providing a greater therapeutic benefit over either agent alone. Immunochemotherapy with a combination of intravesical MMC plus BCG may be a promising alternative treatment for bladder tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1903.

ENHANCED ANTITUMOR EFFECT OF INTRAVESICAL GEMICITABINE THERAPY PLUS BCG IN AN ORTHOTOPIC MURINE BLADDER CANCER MODEL

INTRODUCTION AND OBJECTIVES: Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is the most successful adjuvant agent for superficial bladder tumors. However, tumors recur in 60 to 70% of the cases, and 30 % of these recurrent tumors present with a higher grade with invasive properties.Gemicitabine is a strong and specific deoxycytidine analog with activity in bladder tumors. To treat superficial bladder tumors that are resistant, we evaluated the combination treatment strategies with intravesical BCG plus Gemicitabine for bladder tumors in a model of orthotopic murine bladder cancer(MBT-2). METHODS: Superficial murine bladder cancer was established by simple instillation of 1 x 106 MBT-2 cells into the lumen of the bladder of female C3H mouse. To assess the antitumoral effect of gemicitabine, intravesical gemicitabine therapy was administered at various doseescalating concentrations; 0(control), 1mg, 2mg, 4 mg and 8mg (n = 8 for each group). Intravesical therapy was administered at 3-days intervals starting on day 5 and repeated 6 times. Next, comparative evaluation of tumor growth among the control group, Gemicitabine-alone group, Gemicabine plus BCG group and BCG-alone group were performed (n= 16 for each group). On day 60 after the initial implantation of the MBT-2 cells, all the surviving mice were sacrificed and necropsied. To evaluate the proliferative activity among the group, Ki67 staining of the tumor cells was performed following corresponding single intavesical administration in an orthotopic bladder tumor model. RESULTS: In the Gemicitabine-only groups of 8 mice each at the dose of 0(control), 1mg, 2mg, 4 mg and 8mg, one, four, four, four, five and four of the mice, respectively, survived. In the 8mg group, three of the eight mice were died due to adverse events. There was no significant difference in the tumor growth rate between any doses of Gemicitabine groups. There was a significant survival advantage in the Gemecitabine plus BCG group (54.1 ± 9.4 days) as compared with that in the BCG-alone group (39.0 ± 16.4 days) (p = 0.02). The Ki67 expression level as the proliferation index was significantly decreased in the Gemecitabine plus BCG group as compared with that in the BCG-alone group (p <0.05). CONCLUSIONS: Our results suggest that intravesical BCG plus Gemicitabine exhibited augmented antitumor effect against bladder tumors. Combined BCG plus Gemicitabine therapy was associated with a reduced proliferation rate as compared to BCG-alone therapy in the orthotopic bladder tumor model.