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Background: Synaptojanin 2 (SYNJ2) was reported to be a druggable mediator of metastasis. It is overexpressed and amplified in breast cancer, particularly estrogen receptor α (ERα)-positive breast cancer. SYNJ2 was also shown to promote cell migration and invasion in breast cancer xenograft cultures and lung metastasis in mice. Here, we investigated SYNJ2 mRNA expression in breast cancer patients during long-term follow-up. Materials and methods: A total of 434 invasive breast cancer tissues were analyzed for SYNJ2mRNA expression using TaqMan PCR, and the correlation of this expression with patient clinicopathological factors was determined. We also examined the expression of markers associated with tumor-initiating capacity, such as SNAI1, SNAI2, and VIM. Survival curves were analyzed using the Kaplan–Meier method. Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values. Results: The median follow-up period was 10.7 years. We found positive correlations between high expression of SYNJ2 mRNA and shorter disease-free survival in breast cancer patients (P=0.049), which was limited to ERα-positive patients (P=0.020) and not seen in ERα-negative patients (P=0.863). High SYNJ2 mRNA levels were positively correlated with high tumor grade, ERα negativity, and node positivity. Multivariate analysis indicated that high SYNJ2 mRNA expression was an independent factor for poor disease-free survival in breast cancer patients. Multivariate analysis of poor disease-free survival DFS Mutivariate All patientsp valueHR (95%CI)Tumor size2cm and fewer1540.43151 (Reference) more than 2cm280 1.19 (0.78-1.85)Lymph node metastasisNegative2190.0001 and fewer1 (Reference) Positive184 5.61 (3.59-9.05) Unknown31 2.40 (0.89-5.48)Grade1,22330.38471 (Reference) 3195 1.20 (0.79-1.81) Unknown6 0.17 (1.78-3.49)ER statusPositive3370.02621 (Reference) Negative97 2.06 (1.87-4.07)PgR statusPositive2930.39511 (Reference) Negative141 1.30 (0.70-2.28)SYNJ2 mRNA expressionlow2170.02251 (Reference) high217 1.57 (1.07-2.34) Conclusion:High SYNJ2 expression was shown to be an independent predictive factor of poor prognosis in ERα-positive breast cancer patients. SYNJ2 could therefore be used as a candidate biomarker and therapeutic target in breast cancer. Citation Format: Nishikawa S, Kondo N, Wanifuchi-Endo Y, Hisada T, Uemoto Y, Katagiri Y, Dong Y, Kato H, Takahashi S, Toyama T. The prognostic impact of synaptojanin 2 expression in estrogen receptor α-positive breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-37.

Overcoming drug resistance is one of the biggest challenges in cancer chemotherapy. In this study, we examine whether targeting the long noncoding RNA taurine upregulated gene 1 (TUG1) could be an effective therapeutic approach to overcome drug resistance in pancreatic ductal adenocarcinoma (PDAC). TUG1 was expressed at significantly higher levels across 197 PDAC tissues compared with normal pancreatic tissues. Overall survival of patients with PDAC who had undergone 5-FU–based chemotherapy was shorter in high TUG1 group than in low TUG1 group. Mechanistically, TUG1 antagonized miR-376b-3p and upregulated dihydropyrimidine dehydrogenase (DPD). TUG1 depletion induced susceptibility to 5-FU in BxPC-3 and PK-9 pancreatic cell lines. Consistently, the cellular concentration of 5-FU was significantly higher under TUG1-depleted conditions. In PDAC xenograft models, intravenous treatment with a cancer-specific drug delivery system (TUG1-DDS) and 5-FU significantly suppressed PDAC tumor growth compared with 5-FU treatment alone. This novel approach using TUG1-DDS in combination with 5-FU may serve as an effective therapeutic option to attenuate DPD activity and meet appropriate 5-FU dosage requirements in targeted PDAC cells, which can reduce the systemic adverse effects of chemotherapy. Significance: Targeting TUG1 coupled with a cancer-specific drug delivery system effectively modulates 5-FU catabolism in TUG1-overexpressing PDAC cells, thus contributing to a new combinatorial strategy for cancer treatment.

Background: The epithelial-to-mesenchymal transition (EMT) is fundamental and is required for cancer cells to metastasize. A E3 ubiquitin ligase, HECTD1,wasidentified as a potent regulator of EMT in breast cancer patients. Depletion of HECTD1 was reported to enhance EMT and migration. Here, we investigated the correlation of HECTD1 mRNA expression with prognosis in early breast cancer patients during long-term follow-up. Materials and methods: A total of 627 invasive breast cancer tissues were analyzed for HECTD1 mRNA expression using a TaqMan PCR system. We also examined correlations between HECTD1 mRNA levels and clinicopathological factors in these samples. The median follow-up period was 10.2 years. Survival curves were analyzed using the Kaplan-Meier method. Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values. Results: We identified positive correlations between low HECTD1 mRNA levels and shorter disease-free survival (DFS) and overall survival (OS) in breast cancer patients (P=0.02 and P=0.03, respectively). Low HECTD1 mRNA levels were positively correlated with high tumor grade, ERα and PgR negativity, HER2 positivity, and type of adjuvant therapy. Univariate analysis indicated that low HECTD1 mRNA levels was a factor for poor DFS and OS in breast cancer patients (hazard ratio (HR), 1.43; 95% confidence interval (CI), 1.06-1.95 and HR, 1.49; 95% CI, 1.03-2.17, respectively) (Table 1). Conclusion: We showed that low HECTD1 mRNA expression may be a predictive factor of poor prognosis in breast cancer patients. HECTD1 could therefore be a candidate biomarker and therapeutic target in breast cancer patients. Table 1. Univariate Cox regression analysis of HECTD1 mRNA expression associated with prognosis.Patients (n=617)UnivariateDisease-free survivalOverall survivalp valueHR (95%CI)p valueHR (95%CI)HectD1 mRNA expressionhigh3130.021(Reference)0.031(Reference)low3141.43(1.06-1.95)1.49(1.03-2.17) Citation Format: Yasuaki Uemoto, Naoto Kondo, Yumi Wanifuchi-Endo, Tomoko Asano, Tomoka Hisada, Sayaka Nishikawa, Yusuke Katagiri, Mitsuo Terada, Akiko Kato, Yu Dong, Hiroyuki Kato, Satoru Takahashi, Tatsuya Toyama. The prognostic impact of HECTD1 E3 ubiquitin ligase expression in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-07-03.

Targeting androgen receptor (AR) by pharmacologic intervention is one of the effective approaches for treatment of malignant prostate cancers. Histone deacetylase (HDAC) alters the epigenetic status of tumor-associated genes, including those for miRNAs (miRNA), and affects the behavior of cancers. Here, we examined the molecular effects of a HDAC inhibitor, OBP-801, on AR expression and tumor cell growth in prostate cancers. Treatment with OBP-801 efficiently suppressed cell growth of three prostate cancer lines (22Rv1, VCaP, and LNCaP), together with AR downregulation, regardless of their hormone sensitivity. Intriguingly, this effect by OBP-801 was not due to decreased transcriptional activity of the AR gene, but due to posttranscriptional regulation, namely by miRNA-mediated suppression. Among the upregulated miRNAs after OBP-801 treatment in the three prostate cancer cell lines, miR-320a, whose expression was significantly correlated with prognosis of prostate cancers (P = 0.0185), was the most closely associated with AR expression. An miR-320a mimic suppressed AR protein expression together with growth suppression, while anti-miR-320a oligonucleotide significantly abrogated the growth suppression by OBP-801 treatment. FISH analysis revealed that miR-320a was highly expressed in human normal prostate luminal cells, but was rarely expressed in prostate cancer cells. In an AR-dependent prostate tumorigenic rat model, OBP-801 treatment profoundly increased miR-320a expression and repressed prostate tumorigenesis. Our data demonstrated that OBP-801 effectively suppressed AR activity via epigenetic upregulation of miR-320a, which resulted in tumor cell growth suppression of prostate cancers. OBP-801 may be a potent AR-targeting therapeutic reagent in AR-positive prostate cancer regardless of androgen dependency. Cancer Res; 76(14); 4192–204. ©2016 AACR.

Background: Resistance to endocrine therapy and metastatic progression is a leading cause of death in estrogen receptor-alpha(ERα)-positive breast cancer patients. Recently, the scaffolding protein LLGL2 was reported to promote cellular proliferation in ERα-positive breast cancer cells. LLGL2 forms a trimetric complex with the leucine transport protein SLC7A5. LLGL2 and SLC7A5 were reported to be involved in resistance to tamoxifen treatment. Here, we investigated LLGL2 and SLC7A5 mRNA expression in breast cancer patients during long-term follow-up. Materials and methods: A total of 627 invasive breast cancer tissues was available for analysis of LLGL2 mRNA using a TaqMan PCR system. We sought correlations between clinicopathological factors and levels of LLGL2 expression in these samples. We also examined the mRNA expression of SLC7A5. The median follow-up period was 10.8 years. Survival curves were analyzed using the Kaplan-Meier method. Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values. Receiver operating characteristic (ROC) analysis was performed to determine the cut-off values of LLGL2 and SLC7A5 mRNA expression in breast cancers. Results: We found a positive correlation between high expression of LLGL2 mRNA and shorter disease-free survival (DFS) in all patients analyzed (P=0.01). We also identified positive correlations between high expression of LLGL2 mRNA and shorter DFS and overall survival (OS) in ERα-positive breast cancer patients (P=0.0006 and P=0.004, respectively), and but not in ERα-negative patients (P=0.67 and P=0.63, respectively). Positive correlations of LLGL2 mRNA expression with DFS and OS were observed in ERα-positive patients who received tamoxifen as an adjuvant therapy (P=0.02 and P=0.03, respectively). We also found positive correlations of SLC7A5 mRNA expression with DFS and OS in ERα-positive patients who received tamoxifen as an adjuvant therapy (P=0.006 and P=0.049, respectively); interestingly, this was not observed in ERα-positive patients who did not receive tamoxifen. Multivariate analysis indicated that high LLGL2 mRNA expression was an independent factor for prognosis both in breast cancer patients overall (Table 1) and ERα-positive breast cancer patients. Conclusion: LLGL2 and SLC7A5 expression levels are associated with the prognosis of ERα-positive breast cancer patients, especially those who receive tamoxifen as an adjuvant therapy. Furthermore, higher expression of LLGL2 is an independent predictive factor of poor prognosis in ERα-positive breast cancer patients. LLGL2 and SLC7A5 could be promising candidate biomarkers and therapeutic targets in ERα-positive breast cancer patients who receive tamoxifen as an adjuvant therapy. Table 1. Multivariate Cox regression analysis of factors associated with prognosis in all breast cancer patients.Variablesn (%)Multivariate (DFS)P valueHR (95% CI)Tumor size≤ 2cm241 (38)1> 2cm383 (61)0.4771.14 (0.8-1.64)Node statusNegative340 (54)1Positive253 (40) Citation Format: Tomoka Hisada, Naoto Kondo, Yumi Endo, Tomoko Asano, Yasuaki Uemoto, Sayaka Nishikawa, Yusuke Katagiri, Mitsuo Terada, Akiko Kato, Yu Dong, Hiroyuki Kato, Satoru Takahashi, Tatsuya Toyama. LLGL2 is involved in resistance to tamoxifen in estrogen receptor-alpha positive breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-11.

Over 70% of breast cancers are estrogen receptor α (ERα)-positive, and endocrine therapy targeting estrogen action decreases mortality from breast cancer. However, their efficacy of endocrine therapy is limited by intrinsic and acquired resistance. Recently, a novel protein kinase that regulates ERα activity, lemur tyrosine kinase-3 (LMTK3) has been identified. In this study, we investigated whether LMTK3 mRNA expression and its polymorphisms are associated with prognosis in Japanese breast cancer patients during long-term follow-up. First, we investigated the relationship between mRNA expression of LMTK3 and patient outcome in 242 breast cancers (median follow-up, 6.8 years). The effects of several variables on survival were tested by Cox proportional hazards regression analysis. Next, we performed to analyze LMTK3 rs9989661 and rs8108419 genotyping in 641 breast cancer tissues (median follow-up, 9.2 years) to clarify the prognostic role of these polymorphisms. We showed that high expression levels of LMTK3 mRNA were significantly associated with a shorter overall survival (OS) in all patients. We then, analyzed the impact of LMTK3 mRNA expression on the prognosis of breast cancer according to ERα status. Both disease-free survival and OS were significantly shorter in ERα-positive patients with high LMTK3 mRNA expression receiving adjuvant endocrine therapy than in those patients with low LMTK3 mRNA expression. Notably, the level of LMTK3 mRNA expression was not associated with prognosis in ERα-negative breast cancer patients. Univariate and multivariate Cox regression analysis of factors associated with OS revealed that high LMTK3 mRNA expression was an independent poor prognostic factor in ERα-positive breast cancer patients. Univariate and multivariate Cox regression analysis of factors associated with overall survival in ERα-positive breast cancer patientsUnivariateMultivariateVariablesn (%)P valueP valueHR (95% CI)Tumor size≤ 2cm51 (31)1 (reference)> 2cm112 (67)0.2640.2282.75 (0.57-23.5)Node statusNegative79 (47)1 (reference)Positive72 (43)0.0030.0025.25 (1.35-34.7)Grade1,2128 (77)1 (reference)327 (16)0.4850.5471.57 (0.32-6.04)LMTK3 mRNA expressionLow129 (77)1 (reference)High38 (22)0.0020.0124.94 (1.44-19.4)HR, hazard ratio; CI, confidence interval. We did not find any correlation between LMTK3 genotypes and prognosis of breast cancer patients in our series. Our results suggest that higher LMTK3 expression might be one possible mechanism for endocrine resistance in ERα-breast cancer patients, to be confirmed by further research. Citation Format: Tatsuya Toyama, Tomoko Asano, Shinya Sato, Nobuyasu Yoshimoto, Yumi Endo, Yukari Hato, Dong Yu, Satoru Takahashi, Yoshitaka Fujii. High expression of LMTK3 is an independent factor indicating a poor prognosis in Japanese estrogen receptor α-positive breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-22.

The incidence of breast cancer in Japanese women has doubled in all age groups over the past two decades, making it important to evaluate breast cancer risk factors in Japanese women. It is well known that mammographic density is positively associated with breast cancer risk in Western countries, and mammographic density is known to be affected by some environmental factors, serum hormones, and growth factors. We performed stepwise variable selection in a multiple regression model with fifteen independent variables as described below, based on the Akaike information criteria (AIC) to build a mammographic density prediction model using a dataset of 1191 women (913 women with breast cancer and 278 disease-free controls). The variables included were: environmental risk factors (body-mass index (BMI), age at menarche, pregnancy, age at first birth, breastfeeding, family history of breast cancer, age at menopause, use of hormone replacement therapy, alcohol intake and smoking), serum hormones and growth factors (estradiol, testosterone, prolactin, insulin-like growth factor 1 (IGF1) and IGF binding protein 3 (IGFBP3)) and mammographic density. The resulting prediction model is: Mammographic density = + 0.000476 (IGF1) −0.0605 (testosterone) − 0.0508 (IGFBP3) − 0.00683 (age) − 0.0175 (BMI) + 0.00883 (age at menarche) − 0.0153 (breast feeding), (R2 = 0.336). In this model, IGF1, testosterone, IGFBP3, age, BMI, age at menarche, and breastfeeding were considered to be important factors. IGF1 and age at menarche were positively associated with mammographic density, while on the other hand testosterone, IGFBP3, age, BMI, and breast feeding were negatively associated with mammographic density. Further studies are required to build a modified model incorporating serial measurements of serum hormones and growth factors to take into account time-dependent changes of serum hormones and growth factors, and to assess its accuracy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-12-06.

Background: Although taxanes are a mainstay of breast cancer treatment, some cases are resistant to these drugs. This is a crucial issue in breast cancer therapy. In the emerging era of next-generation sequencing, it is possible to obtain extensive genomic information on individual tumors in a very short time. Using this technology, it was reported that specific mutations might affect therapeutic efficacy and induce resistance to specific treatment. Objective: The aim of this study was to investigate the mechanisms of taxane resistance using whole exon sequencing and expression analyses in human breast cancer tissues. Materials and Methods: We selected six breast cancer patients whose tumors responded well to anthracycline treatment but suffered disease progression on taxane treatment. We then performed whole exon sequencing on these samples using HiSeq (Illumia). In this way, we identified somatic mutations of candidate genes considered to be instrumental for mediating resistance to taxanes. Next, we performed mRNA expression analyses of these candidate genes in a further 122 breast cancers treated with taxanes at our institute. Finally, we correlated mRNA expression levels of these genes with clinicopathological factors and prognosis. Results: We identified 9 mutations common to all 6 patients analyzed in this study, and a further 16 mutations shared by 5 of them. Kaplan-Meier analyses showed that high level mRNA expression of 3 of these 25 genes was significantly associated with poorer disease-free survival. Moreover, high level mRNA expression of one of these three genes was significantly associated with worse overall survival. However, there were no significant correlations between expression levels of these three genes and any clinicopathologeical features. Conclusion: Using next-generation sequencing, we have identified three candidate genes involved in resistance to taxane treatment in breast cancer. We are now analyzing the functional attributes of these three genes. Citation Format: Endo Y, Dong Y, Kondo N, Hato Y, Hisada T, Nishimoto M, Nishikawa S, Takahashi S, Toyama T. Exome sequencing of human breast cancer tissues resistant to taxanes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-07.

The incidence of breast cancer in Japanese women has doubled in all age groups over the past two decades. We have recently demonstrated that this marked increase is mostly due to an increase in the estrogen receptor (ER)-positive subtype. It is necessary to establish risk factors capable of predicting the risk of ER-positive breast cancer which will enable the efficient selection of candidates for preventive chemotherapy. We analyzed genetic factors, including 14 single nucleotide polymorphisms (SNPs), environmental risk factors (body-mass index (BMI), age at menarche, pregnancy, age at first birth, breastfeeding, family history of breast cancer, age at menopause, use of hormone replacement therapy, alcohol intake and smoking), serum hormones and growth factors (estradiol, testosterone, prolactin, insulin-like growth factor 1 (IGF1) and IGF binding protein 3 (IGFBP3)) and mammographic density in 913 women with breast cancer and 278 disease-free controls. To identify important risk factors, risk prediction models for ER-positive breast cancer in both pre- and postmenopausal women were created by logistic regression analysis. In premenopausal women, 1 SNP (CYP19A1-rs10046), age, pregnancy, breastfeeding, alcohol intake, serum levels of prolactin, testosterone and IGFBP3 were considered to be risk predictors. In postmenopausal women, 1 SNP (TP53-rs1042522), age, BMI, age at menopause, serum levels of testosterone and IGF1 were identified as risk predictors. Risk factors may differ between women of different menopausal status, and inclusion of common genetic variants and serum hormone measurements as well as environmental factors might improve risk assessment models. Further validation studies will clarify appropriate risk groups for preventive chemotherapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-10-11.

Multiple myeloma (MM) is a currently incurable neoplasm of terminally differentiated B cells. The translocation and/or overexpression of c-MAF have been observed in human MM. Although c-MAF might function as an oncogene in human MM, there has been no report thus far describing the direct induction of MM by c-MAF overexpression in vivo. In this study, we have generated transgenic (TG) mice that express c-Maf specifically in the B-cell compartment. Aged c-Maf TG mice developed B-cell lymphomas with some clinical features that resembled those of MM, namely, plasma cell expansion and hyperglobulinemia. Quantitative RT-PCR analysis demonstrated that Ccnd2 and Itgb7, which are known target genes of c-Maf, were highly expressed in the lymphoma cells. This novel TG mouse model of the human MM t(14;16)(q32;q23) chromosomal translocation should serve to provide new insight into the role of c-MAF in tumorigenesis. Cancer Res; 71(2); 339–48. ©2011 AACR.

[Background] We previously reported that angiotensin II receptor blockers (ARB), anti-hypertensive drug, have chemopreventive and chemotherapeutic potential against prostate cancer via the reduction of androgen receptor (AR) expression. It is generally known that angiotensin II binds to two kinds of receptors, AT1R and AT2R, and the biological function of AT2R signaling has the opposite effect to that of AT1R signaling in many aspects. We therefore hypothesized that AT2R agonism might attenuate the proliferative activity in prostate cancer cells. In this study, we investigated the effects of the AT2R agonist Compound 21 (C21) designed by Vicore Pharma (Sweden), which is expected to play similar roles to an ARB, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model previously established in our laboratory. [Materials and Methods] In vitro analyses of the cell growth, Western blotting and PSA-reporter gene assays were performed using LNCaP and 22RV1 cells. TRAP rats at 6 weeks of age were randomly divided into 3 groups of 12 animals each and treated with C21 at 1 or 2 mg/kg/day in drinking water for 12 weeks. They were pathologically examined the classification as prostatic intraepithelial neoplasia and non-invasive adenocarcinoma. The proteins extracted from TRAP prostate glands were examined for Western blotting. [Results] C21 reduced the proliferation activity of prostate cancer cells and down-regulated the PSA promoter activity, medium PSA levels and the AR protein expression. We discovered that C21 inhibited the progression of prostate carcinogenesis in TRAP rats and decreased the incidence of adenocarcinoma in the lateral prostate. A significant increase in the apoptotic index with activation of caspase 3 and 7 were observed by immunohistochemistry and Western blotting analyses. C21 also down-regulated the expression of AR significantly in TRAP rat prostate. C21 decreased the expression of AR and AR-relevant genes reduced the proliferation activity effectively in prostate cancer cells and TRAP rat prostate. [Conclusion] The present study demonstrated that the epoch-making AT2R agonist C21 down-regulated the proliferation activity and the expression of AR both in vitro and in vivo. C21 is a promising drug not only for hypertension but also for human prostate cancer chemoprevention. Citation Format: Hiroji Uemura, Yusuke Ito, Aya Naiki-Ito, Hiroyuki Kato, Shugo Suzuki, Toshiya Kuno, Satoru Takahashi. Chemopreventive effects of angiotensin II receptor type 2 agonist on prostate carcinogenesis by the downregulation of the androgen receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 254.

Although clinical therapies are being developed for combating pancreatic cancer, its prognosis remains dismal; consequently, chemopreventive strategies may be needed to manage this disease. Luteolin (Lut), a flavone contained in perilla, is one of the most effective antioxidant flavonoids and has demonstrated potential for the treatment of inflammatory and neoplastic disease. Hesperetin (Hes) is a flavone contained in citrus, and the Ohsaki cohort study showed that citrus consumption was correlated with reduced pancreatic cancer (RR=0.62, 95%CI=0.38-1.00) (Int.J.Cancer, 2010: Li.W). However, there are no studies dealing with their antineoplastic effects against pancreatic cancer. In the present study, to assess the chemopreventive effects of Lut and Hes for pancreatic carcinogenesis, 6 week-old syrian golden hamsters were subcutaneously injected with N-nitrsobis(2-oxopropyl)amine four times and then fed Quick Fat Diet (control, n=14) or Quick Fat Diets with Lut 100 ppm (n=13) or Hes 100 ppm, 1000 ppm (n=13,13) for 13 weeks. There were no differences in body weights or liver or kidney weights among the groups. Serum cholesterol was significantly decreased in the Lut group. The incidence and multiplicity of pancreatic cancer was significantly suppressed in the Lut group (Incidence/ Multiplicity: 23%/ 0.23±0.44; P Citation Format: Hiroyuki Kato, Aya Naiki, Shugo Suzuki, Yoriko Yamashita, Satoru Takahashi. Chemopreventive effects of flavonoids on pancreatic carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 261.

To identify methylation-silenced genes in prostate cancers, a microarray analysis for genes up-regulated by treatment with a demethylating agent, 5-aza-2′-deoxycytidine, was performed using three rat prostate cancer cell lines. Eight genes (Aebp1, Dysf, Gas6, LOC361288, Nnat, Ocm, RGD1308119, and Tgfbr2) were re-expressed at 16-fold or more, and their promoter CpG islands were shown to be densely methylated in the cancer cell lines. From the eight genes, Tgfbr2, a key mediator of transforming growth factor-β (TGF-β) signaling that has been strongly implicated in human and rat prostate carcinogenesis, was selected, and its silencing in primary samples was analyzed further. Tgfbr2 was methylated and markedly down-regulated in three of seven 3,2′-dimethyl-4-aminobiphenyl–induced invasive adenocarcinomas in the dorsolateral lobe of the rat prostate. In humans, marked down-regulation of TGFBR2 protein was observed in 12 of 20 high-grade prostatic intraepithelial neoplasia and 36 of 60 prostate cancers. DNA methylation of the human TGFBR2 promoter CpG islands repressed transcription, if present, but neither methylation nor mutation were detected in 27 human prostate cancers analyzed. Methylation silencing of rat Tgfbr2 was associated with histone H3 lysine 9 trimethylation, whereas decreased expression of human TGFBR2 was mainly due to decreased transcription activity, sometimes in concert with histone deacetylation and H3 lysine 27 trimethylation. The identification of methylation silencing of Tgfbr2 in rat prostate cancers, in accordance with TGFBR2 down-regulation in human prostate cancers, will enable us to analyze how aberrant methylation is induced in vivo and identify factors that promote and suppress the induction of aberrant methylation. [Cancer Res 2008;68(7):2112–21]

c-Maf translocation or overexpression has been observed in human multiple myeloma. Although c-maf might function as an oncogene in multiple myeloma, a role for this gene in other cancers has not been shown. In this study, we have found that mice transgenic for c-Maf whose expression was direct to the T-cell compartment developed T-cell lymphoma. Moreover, we showed that cyclin D2, integrin β7, and ARK5 were up-regulated in c-Maf transgenic lymphoma cells. Furthermore, 60% of human T-cell lymphomas (11 of 18 cases), classified as angioimmunoblastic T-cell lymphoma, were found to express c-Maf. These results suggest that c-Maf might cause a type of T-cell lymphoma in both mice and humans and that ARK5, in addition to cyclin D2 and integrin β7, might be downstream target genes of c-Maf leading to malignant transformation. (Cancer Res 2006; 66(2): 812-9)

The androgen receptor (AR) is the key driver of both early and advanced prostate cancer, making a complete understanding of its regulation important. Here, we report the identification of multiple AR-binding sites in the gene encoding the transcription factor CtBP2 (carboxyl terminal–binding protein), genetic variations of which have been associated with prostate cancer susceptibility. Notably, we found that SNPs in the human CTBP2 gene that were associated with prostate cancer development were correlated with AR-enhancer activity. High CtBP2 expression levels correlated with poor prognosis in patients, whereas CtBP2 silencing reduced tumor growth in a mouse xenograft model of human prostate cancer. Consistent with its function as a transcriptional corepressor, CtBP2 repressed tumor-suppressor genes and AR corepressors in prostate cancer cells, such as NCOR and RIP140, by binding with AR to the promoter enhancers of these genes. Global gene-expression analyses revealed a positive effect on androgen-mediated gene expression, and CtBP2 silencing was found to increase AR interactions with corepressors that limit histone modification. Overall, our results show how CtBP2 contributes to prostate cancer progression by modulating AR and oncogenic signaling. Cancer Res; 74(22); 6542–53. ©2014 AACR.

Tumor cell plasticity contributes to functional and morphologic heterogeneity. To uncover the underlying mechanisms of this plasticity, we examined glioma stem-like cells (GSC) where we found that the biologic interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of polycomb repressive complex 2 (PRC2), a complex that modifies chromatin structure. PRC2 mediates lysine 27 trimethylation on histone H3 and in GSC it affected pluripotency or development-associated genes (e.g., Nanog, Wnt1, and BMP5) together with alterations in the subcellular localization of EZH2, a catalytic component of PRC2. Intriguingly, exogenous expression of EZH2-dNLS, which lacks nuclear localization sequence, impaired the repression of Nanog expression under differentiation conditions. RNA interference (RNAi)–mediated attenuation or pharmacologic inhibition of EZH2 had little to no effect on apoptosis or bromodeoxyuridine incorporation in GSCs, but it disrupted morphologic interconversion and impaired GSC integration into the brain tissue, thereby improving survival of GSC-bearing mice. Pathologic analysis of human glioma specimens revealed that the number of tumor cells with nuclear EZH2 is larger around tumor vessels and the invasive front, suggesting that nuclear EZH2 may help reprogram tumor cells in close proximity to this microenvironment. Our results indicate that epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Thus, PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, offering a new paradigm for glioma treatment. Cancer Res; 73(14); 4559–70. ©2013 AACR.

Targeting androgen receptor (AR) by pharmacological intervention is one of the effective approaches for treatment of malignant prostate cancers. Histone deacetylase (HDAC) alters epigenetic status of tumor-associated genes, including microRNAs (miRNAs), and affects the behavior of cancers. Here we examined molecular effects of a HDAC inhibitor, OBP-801, on AR expression and tumor cell growth in prostate cancers. Treatment of OBP-801 efficiently suppressed cell growth of three prostate cancer lines (22Rv1, VCaP, and LNCaP), together with AR downregulation, regardless of their hormone sensitivity. Intriguingly, this effect by OBP-801 is not due to downregulation of transcriptional activity of the AR gene, but due to post-transcriptional regulation, namely miRNA-mediated suppression. Among the upregulated miRNAs after OBP-801 treatment in three prostate cancer cell lines, miR-320a, of which expression was significantly correlated with prognosis of prostate cancers (P = 0.0185), was the most closely associated with AR expression. miR-320a mimic suppressed AR protein expression together with growth suppression, while anti-miR320a significantly abrogated the growth suppression by OBP-801 treatment. Fluorescent in situ hybridization analysis revealed that miR-320a was highly expressed in human normal prostate luminal cells but rarely expressed in prostate cancer cells. In AR-dependent prostate tumorigenic rat model, OBP-801 treatment profoundly increased the miR-320 expression and repressed prostate tumorigenesis. Our data demonstrated that OBP-801 effectively suppressed AR activity via epigenetic upregulation of miR-320a, which resulted in tumor cell growth suppression of prostate cancers. OBP-801 may be a potent drug as an AR targeting therapy in AR positive prostate cancer regardless of androgen dependency. Citation Format: Shinya Sato, Keisuke Katsushima, Keiko Shinjo, Satoru Takahashi, Yutaka Kondo. Inhibition of histone deacetylase induces miR-320-mediated androgen receptor suppression in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1110.

Androgen receptor (AR) is a critical transcription factor that regulates various target genes and contributes to the pathophysiology of prostate cancer hormone dependently. Here, we identify amyloid precursor protein (APP) as a primary androgen target through chromatin immunoprecipitation (ChIP) combined with genome tiling array analysis (ChIP-chip). ChIP-treated DNA were obtained from prostate cancer LNCaP cells with R1881 or vehicle treatment using AR or acetylated histone H3 antibodies. Ligand-dependent AR binding was further enriched by PCR subtraction. Using chromosome 21/22 arrays, we identified APP as one of the androgen-regulated genes with adjacent functional AR binding sites. APP expression is androgen-inducible in LNCaP cells and APP immunoreactivity was correlated with poor prognosis in patients with prostate cancer. Gain-of-function and loss-of-function studies revealed that APP promotes the tumor growth of prostate cancer. The present study reveals a novel APP-mediated pathway responsible for the androgen-dependent growth of prostate cancer. Our findings will indicate that APP could be a potential molecular target for the diagnosis and treatment of prostate cancer. [Cancer Res 2009;69(1):137–42]

Gene expression alterations are essential for the process of carcinogenesis. A carcinogen may have specific mechanisms for inducing tumors, which may involve inducing characteristic gene expression alterations. In this study, we attempted to identify genes crucial for mammary carcinogenesis. For this purpose, we used human c-Ha-ras proto-oncogene transgenic rats (Hras128), which are highly sensitive to mammary carcinogens including N-methyl-N-nitrosourea, 7,12-dimethyl benz[a]anthracene, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. DNA microarray analysis revealed that glutathione peroxidase 2 (Gpx2) was commonly up-regulated in the mammary carcinomas induced by the three different carcinogens, and its up-regulation was confirmed by quantitative reverse transcriptase-PCR and Western blotting analysis. In addition, expression of GPX2 was recognized in all 41 immunohistochemically examined cases of human breast cancer. Forced suppression of GPX2 expression by siRNA resulted in significant growth inhibition in both rat and human mammary carcinoma cell lines with wild-type p53 cells. Thus, these data suggested that GPX2 may be involved in mammary carcinogenesis and cell proliferation in both rats and humans, indicating that GPX2 may be a novel target for the prevention and therapy of breast cancer. [Cancer Res 2007;67(23):11353–8]

Background: Selective estrogen receptor modulators (SERMs) can reduce the occurrence of breast cancer in high-risk women by 50%. Recently, a genome-wide association study identified SNPs in or near the ZNF423 (rs8060157) and CTSO (rs10030044) genes that were associated with breast cancer risk during SERM therapy and these SNPs were reported to be involved in estrogen-dependent induction of BRCA1 expression (Ingle JN. et al. Cancer Discovery 2013). Materials and methods: A total of 588 breast carcinomas collected between 1983 and 2003 were available for polymorphism assay. TaqMan pre-designed SNP genotyping assays for ZNF423 rs8060157 and CTSO rs10030044 were used. We investigated whether these SNPs are associated with prognosis in breast cancer patients. The effects of several variables on survival were tested by Cox proportional hazards regression analysis. Results: Estrogen receptor (ER)-positive breast cancer patients receiving adjuvant endocrine therapy with the genotype GG at CTSO rs10030044 showed significantly shorter disease-free survival (DFS) and overall survival (OS) (P = 0.0024 and P = 0.0003, respectively). On the other hand, this genotype were not associated with prognosis in ER-negative breast cancer patients. Multivariate Cox regression analysis revealed that the GG genotype at CTSO rs10030044 was an independent poor prognostic factor in ER-positive breast cancer patients receiving adjuvant endocrine therapy (OS: RR = 1.86; 95%CI, 1.18 to 2.85). Univariate and multivariate Cox regression analysis of factors associated with overall survival Variablesn (%)Univariate (P value)Multivariate (P value)Multivariate (RR :95% CI)tumor size≤2cm210 (36) 1 (reference) >2cm376 (64) The SNP, ZNF423 rs8060157, was not associated with prognosis in this study. Conclusion: We show that the genotype GG at CTSO rs10030044 is an independent factor indicating poor prognosis in ER-positive breast cancer patients receiving adjuvant endocrine therapy. Citation Format: Yukari Hato, Yumi Endo, Nobuyasu Yoshimoto, Tomoko Asano, Mina Yamaguchi, Satoru Takahashi, Tatsuya Toyama. Prognostic impact of single-nucleotide polymorphisms (SNPs) in or near the ZNF423 and CTSO genes in estrogen receptor (ER)-positive breast cancer patients receiving adjuvant endocrine therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-02.

Because the most characteristic property of ovarian cancer is i.p. spread, the majority of patients are diagnosed at an advanced stage, leading to limited availability of options for curative therapies. With an intent to identify targeted therapeutic approaches, we have observed that ∼60% of 21 ovarian cancer tissue samples express a high density of interleukin-4 receptor (IL-4R), whereas normal ovarian tissues tested (n = 7) expressed no or low levels of IL-4R. To target IL-4R, we have developed IL-4 cytotoxin, in which circular-permuted IL-4 is fused to a mutated form of Pseudomonas exotoxin. This cytotoxin is specifically and highly cytotoxic to PA-1, IGROV-1, and SK-OV3 ovarian carcinoma cell lines in vitro. In addition, it shows remarkable antitumor activities against established s.c. ovarian tumors in immunodeficient animals. i.p. administration of IL-4 cytotoxin in mice with orthotopically implanted ovarian tumors caused regression of established tumors and prevented these animals from tumor metastasis. Continuous i.p. infusion of IL-4 cytotoxin prolonged survival of tumor-bearing mice even with bulky disease. These results indicate that IL-4R–targeted cytotoxin may be a useful agent for the management of patients with ovarian cancer, and further studies need to be done to evaluate its safety, tolerability, and efficacy.

The possible mutagenic potential of exposure to 1.5 GHz electromagnetic near field (EMF) was investigated using brain tissues of Big Blue mice (BBM). Male BBM were locally exposed to EMF in the head region at 2.0, 0.67, and 0 W/kg specific absorption rate for 90 min/day, 5 days/week, for 4 weeks. No gliosis or degenerative lesions were histopathologically noted in brain tissues, and no obvious differences in Ki-67 labeling and apoptotic indices of glial cells were evident among the groups. There was no significant variation in the frequency of independent mutations of the lacI transgene in the brains. G:C to A:T transitions at CpG sites constituted the most prevalent mutations in all groups and at all time points. Deletion mutations were slightly increased in both the high and low EMF exposure groups as compared with the sham-exposed group, but the differences were not statistically significant. These findings suggest that exposure to 1.5 GHz EMF is not mutagenic to mouse brain cells and does not create any increased hazard with regard to brain tumor development.

Histone acetylation plays an important role in chromatin remodeling and gene expression. Histone deacetylase (HDAC) inhibitors regulate gene expressions through the induction of histone acetylation and have been reported to suppress growth of several malignancies including lymphoma. In this study, we examined the effects of one example on androgen dependent and independent prostate cancer growth in vivo and in vitro. In in vitro study, androgen dependent and independent prostate cancer cell lines (LNCaP and VCaP) were used. The HDAC inhibitor, OBP-801 (Generously donated by Dr. Urata, Oncolys Biopharma), significantly suppressed cell proliferation with induction of G2/M arrest, and decreased expression of cdc2, androgen receptor (AR) and PSA protein in both cell lines. OBP-801 also significantly induced apoptosis with activation of caspase 3 in LNCaP. Next, we performed two in vivo experiments. First, we transplanted an androgen independent rat prostate cancer (PLS-P) into the femurs and backs of male F344 rats, and OBP-801 was administered intravenously at doses of 0, 2.5 and 5mg/kg every other day for 2 weeks. OBP-801 significantly reduced the volume of the transplanted tumor, while inducing cleaved caspase 3 protein expression. OBP-801 also suppressed bone destruction and induction of osteoclasts in xenografts transplanted to femurs. Second, we prepared male Transgenic Rat for Adenocarcinoma of Prostate (TRAP) rats which spontaneously develop androgen dependent rat prostate cancers, and OBP-801 was given intravenously at doses of 0, 0.5 and 2mg/kg once a week for 8 weeks. As a result, body weight loss was observed in treated groups, and numbers of carcinoma acini in the lateral and ventral prostates were significantly decreased compared with the control group values. In conclusion, the obtained data suggests that OBP-801 may be a candidate therapeutic or preventing agent for androgen dependent and independent prostate cancer, although side effects such as body weight loss may occur. Elucidation of mechanisms of HDAC underlying these effects is needed. Citation Format: Shinya Sato, Shugo Suzuki, Aya Naiki-Ito, Yoriko Yamashita, Makoto Asamoto, Tomoyuki Shirai, Satoru Takahashi. Histone deacetylase inhibition has potential to suppresse growth of androgen dependent and independent prostate cancer in vitro and in vivo. [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 672. doi:10.1158/1538-7445.AM2013-672

Aberrant DNA methylation is deeply involved in prostate carcinogenesis. However, inducers of aberrant DNA methylation in the prostate are almost unknown. Here, we investigated whether androgen excess can induce aberrant DNA methylation or not, using a rat prostate cancer model. First, by analysis of reported data of expression microarray after treatment of a prostate cancer cell line with a demethylating agent, we isolated two marker genes (Mmp23, Nkx3.1) whose promoter CpG islands were aberrantly methylated in testosterone and 3,2′-dimethyl-4-aminobiphenyl-induced invasive adenocarcinomas. These two genes and three genes previously isolated (Aebp1, Amn1, Tgfbr2) [Yamashita et al. Cancer Res. 2008] were used as markers. Then, we analyzed temporal profiles of DNA methylation levels of the five genes in the dorsolateral lobe of rats treated with excessive androgen. Testosterone was administrated every five weeks for 15, 30, 40 and 50 weeks from six weeks old by subcutaneous implantation of a Silastic tube containing 40 mg testosterone propionate. Aberrant DNA methylation of two genes (Amn1, Mmp23) was gradually induced during the testosterone treatment while that of the other three genes was not. The former two genes had lower mRNA expression levels in the normal prostate than the latter three genes, which was in accordance with the known fact that genes with low transcription are susceptible to DNA methylation. Third, we investigated molecular mechanisms for aberrant DNA methylation induction by androgen excess. Androgen excess did not induce mRNA expression of DNA methyltransferases; Dnmt1, Dnmt3a or Dnmt3b in the rat prostrate. As for histone modifications, it did not induce significant changes in either H3K4me3 or H3K27me3 levels of the five genes methylated in rat prostate cancer. In contrast, it induced infiltration of lymphocytes and neutrophils in the prostate. Temporal profiles of expression levels of specific inflammation-related genes (Cxcl2, Il1b, Nos2 and Tnf) paralleled the methylation levels of Amn1 and Mmp23. Androgen excess did not induce bacterial infection as determined by copy number of bacterial DNA, but induced increase in Ki67-positive cells in regions with and without infiltration of inflammatory cells. The inflammatory response, known as a major mechanism for aberrant DNA methylation induction, along with cell proliferation was likely to be the cause of aberrant DNA methylation induction in the rat prostate with androgen excess. These results demonstrated that androgen excess can induce aberrant DNA methylation in the prostate, and suggested that inflammatory response underlies aberrant DNA methylation induction by androgen excess. Citation Format: Satoshi Yamashita, Satoru Takahashi, Yasunori Matsuda, Ken Gyobu, Toshikazu Ushijima. Androgen excess induces aberrant DNA methylation in the prostate. [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 5375. doi:10.1158/1538-7445.AM2013-5375

Introduction: Prostate cancer is major leading cause of death in western world. And elucidating the mechanisms of metastasis in prostate cancer, particularly to the bone, is a major issue for treatment of this malignancy. Reactive oxygen species (ROS) have been identified as important chemical mediators in cell growth and differentiation, and glutathione oxidation-reduction (redox) system is the main mechanism against ROS in human body. We established a new metastatic model of prostate cancer, using new cell line, and we investigated the role of glutathione redox system in the androgen-independent prostate cancer. Methods: A new cell line, PCai1, was established from the androgen-independent cancer derived from transgenic rat model for adenocarcinoma of the prostate (TRAP), and investigated its metastatic potential in nude mice. By cDNA microarray analysis in our model, glutathione S-transferase Pi (Gst-pi), which was the major component protein of glutathione redox system, had higher expression in androgen independent prostate cancer. Therefore, effects of Gst-pi knocked down in vitro with iRNA strategy were analyzed in PCai1 cells, and the quantification of ROS was performed by DCFH assay. In addition, to evaluate roles of Gst-pi in castration-resistant cell proliferation, Gst-pi siRNA transfected PCai1 cells were transplanted subcutaneously in castrated or normal nude mice. Results: Gst-pi-siRNA in vitro significantly suppressed cell proliferation rate of PCai1. In addition, high levels of intracellular ROS were recognized in the Gst-pi knockout. Strong expression of Gst-pi was realized castration-resistant prostate cancer specimen, while no tumor was stained in hormone naïve prostate cancer. PCai1 frequently formed metastatic lesions in the lung and lymph nodes after orthotopic implantation in the prostate. Intravenous injections of PCai1, metastasis to lung and bone were obvious. Tumors metastasized in the bone marrow after intravenous injection expressed high levels of Gst-pi, while metastatic lesions in the lung and lymph nodes failed to express Gst-pi as seen in cases of the orthotopic implantation. Knock down of Gst-pi expression of PCai1 significantly inhibited tumor growth of PCai1 compared to negative controls in castrated mice. On the other hand, Gst-pi expression did not affect tumor growth in non-castrated mice Conclusion: Establishment of a new metastatic prostate cancer model allows for the systematic analysis of genetic and molecular characteristics of human tumors. And these results suggest that glutathione redox system can regulate cancer cell growth in androgen free environment. Gst-pi expression of the prostate cancers are dependent on metastatic site, and that Gst-pi has an important role in adapting prostate cancer for growth and metastasis involving an alteration of ROS signals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3284. doi:1538-7445.AM2012-3284

Molecular studies reveal that over-expression of cyclooxygenase-2 (COX-2) is a prominent feature of premalignant and malignant neoplasms. The increased COX-2-mediated prostaglandin-E2 (PGE2) production has a strong association with various cancers, by promoting cell survival, cell growth, migration, invasion, angiogenesis, and immunotolerance. The use of COX-2 selective inhibitors has shown promise in the prevention and treatment of various cancers, however, the chronic use of COX-2 inhibitors is associated with an increased risk of cardiovascular adverse effects. Therefore, there is an urgent need to further understanding of the downstream mechanisms by which PGE2 promotes tumorigenesis and more effective strategies for the treatment of cancer. There are four PGE2 receptors, i.e., EP1, EP2, EP3, and EP4, involved in the pharmacology of PGE2. Accumulating evidences suggest that EP4 is the responsible receptor for PGE2 / COX-2-mediated tumorigenic signals in cancer cells and cancer microenvironment. On the other hands, signals from other prostaglandin receptors, e.g. IP and EP1, are reported to inhibit cancer proliferation or metastasis. These evidences suggest that selective blockade of EP4 would offer anti-cancer efficacy and safety advantages over COX-2 inhibition as cancer therapy. We have developed novel plural EP4 antagonists, with pA2 values of nanomolar range which are selective against other EP receptors. EP4 antagonists inhibited the PGE2-dependent productions of key molecules for cancer promotion i.e. VEGF, IL-23, and IL-6 in macrophages, dendritic cells, and PBMC, respectively. At the same time EP4 antagonist reversed PGE2 -induced TNFα and IL-12 suppression in human whole blood. As a consequence, EP4 antagonists exhibited anti-cancer activity in lung, GI, and prostate cancer models. These results indicate that selective EP4 antagonists show anti-cancer effect mediated by multifaceted mechanisms, and represent an attractive medicine for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 893. doi:1538-7445.AM2012-893

Cancer incidence rises with age, thus, it seems that advancing age is one of the most important risk factors for malignancy. Several factors have been implicated, like DNA alterations. However, there is little experimental evidence to establish the link between aging and carcinogenesis. Gap junctions have important roles in the maintenance of tissue homeostasis and the control of cell growth and differentiation. Connexins are subunits of gap junction channels, which allow intercellular exchange of small molecules, such as ions, second messengers, and cellular metabolites between contacting cells. In the liver, connexin 32 (Cx32) is a major gap junction protein. We previously demonstrated that transgenic rats carrying a dominant negative mutant of Cx32 (Cx32ΔTg) have much decreased capacity for gap junctional intercellular communication (GJIC), and increased susceptibility to diethylnitrosamine (DEN)-induced hepatocarcinogenesis as compared to littermate wild-type (wt) rats. In order to evaluate the age-dependent susceptibility to DEN-induced hepatocarcinogenesis, male Cx32ΔTg and wt rats at 10, 30 or 85 week-old (wk-old) were given a single intraperitoneal administration of DEN (40 mg/rat) and sacrificed 12 weeks later. The number and area of glutathione S-transferase placental form (GST-P)-positive preneoplastic foci were significantly increased in the liver of 10 and 30 wk-old Cx32ΔTg rats compared with age-matched wt. However, in the 85 wk-old rats, both Cx32ΔTg and wt rats had similarly large number and area of GST-P-positive foci, and the difference was not significant. In accordance with these results, the lack of a difference at 85 weeks may be supposed to the initial lower levels of Cx32 and GJIC function in older wt rats. To confirm this hypothesis, we examined alteration of GJIC function with age in the livers of wt type rats at 10, 40, 70 and 100 wk-old. Interestingly, function of hepatic GJIC was gradually reduced, and protein and mRNA expression of Cx32 were also decreased with aging in wt rats. In summary, the present study shows that GJIC function is decreased with aging in rats, and it may cause promotion of hepatocarcinogenesis in older rats. In addition, Cx32ΔTg may be a good model to study roles of gap junctions in aged liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4980. doi:1538-7445.AM2012-4980

The 14-3-3 proteins are a family of regulatory molecules that modulate the functions of their binding partners. They are involved in many processes including protein synthesis/degradation, cell cycle regulation, apoptosis, and tumor development. We have identified one of 14-3-3 protein isoforms 14-3-3zeta as a novel androgen target gene by chromatin immunoprecipitation (ChIP) coupled with microarray analysis. The expression of 14-3-3zeta was upregulated in LNCaP prostate cancer cells at both mRNA and protein levels by androgen stimulation. We generated LNCaP cells stably expressing 14-3-3zeta or control empty vector, and found that less etoposide-induced cell death was observed in 14-3-3zeta-expressing cells compared with control cells. Cell proliferation and motility were increased in 14-3-3zeta-expressing cells. Imunoprecipitation study showed that 14-3-3zeta binds to androgen receptor (AR) in these cells. Luciferase assay showed that androgen-dependent PSA promoter activity was significantly increased in 14-3-3ζ-expressing cells. Androgen-dependent PSA expression was also increased in 14-3-3ζ-expressing cells. siRNA-dependent 14-3-3zeta knockdown revealed that androgen-dependent AR transactivation was reduced in LNCaP cells. Taken together, the present study indicates that 14-3-3ζ is a bona-fide androgen-regulated gene that plays an important role in activating AR-dependent signaling, thereby promoting cell growth, motility and acquirement of apoptosis resistance in prostate cancer. 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 1705.