Kuniko Horie-Inoue

Identification of novel androgen response genes in prostate cancer cells by coupling chromatin immunoprecipitation and genomic microarray analysis

The androgen receptor (AR) plays a key role as a transcriptional factor in prostate development and carcinogenesis. Identification of androgen-regulated genes is essential to elucidate the AR pathophysiology in prostate cancer. Here, we identified androgen target genes that are directly regulated by AR in LNCaP cells, by combining chromatin immunoprecipitation (ChIP) with tiling microarrays (ChIP-chip). ChIP-enriched or control DNAs from the cells treated with R1881 were hybridized with the ENCODE array, in which a set of regions representing approximately 1% of the whole genome. We chose 10 bona fide AR-binding sites (ARBSs) (P<1e-5) and validated their significant AR recruitment ligand dependently. Eight upregulated genes by R1881 were identified in the vicinity of the ARBSs. Among the upregulated genes, we focused on UGT1A and CDH2 as AR target genes, because the ARBSs close to these genes (in UGT1A distal promoter and CDH2 intron 1) were most significantly associated with acetylated histone H3/H4, RNA polymerase II and p160 family co-activators. Luciferase reporter constructs including those two ARBSs exhibited ligand-dependent transcriptional regulator/enhancer activities. The present study would be powerful to extend our knowledge of the diversity of androgen genetic network and steroid action in prostate cancer cells.

Amyloid Precursor Protein Is a Primary Androgen Target Gene That Promotes Prostate Cancer Growth

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.

Association of the HTRA1 gene variant with age-related macular degeneration in the Japanese population

AbstractThe purpose of this investigation was to determine whether the high-temperature requirement A-1 (HTRA1) gene polymorphism is associated with age-related macular degeneration (AMD) in native, unrelated Japanese patients. A total of 123 patients with AMD and 133 control subjects without AMD were recruited for this study. The single-nucleotide polymorphism (SNP) rs11200638 in the HTRA1 gene was assessed using a TaqMan assay. The risk A allele frequencies in the AMD cases and control patients were 0.577 and 0.380, respectively, and were associated with a significant risk of developing AMD (p=7.75×10−6). The results were more significant in subtype analyses with wet AMD (p=5.96×10−7). We conclude that the rs11200638 variant in the HTRA1 gene is strongly associated with AMD in the Japanese population. This result supports the hypothesis that the HTRA1 gene may increase susceptibility to AMD development and can participate in a potential new molecular pathway for AMD pathogenesis by extending this association across diverse ethnicities.

Vitamin K2 induces phosphorylation of protein kinase A and expression of novel target genes in osteoblastic cells.

Vitamin K is known as a critical nutrient required for bone homeostasis and blood coagulation, and it is clinically used as a therapeutic agent for osteoporosis in Japan. Besides its enzymatic action as a cofactor of vitamin K-dependent gamma-glutamyl carboxylase (GGCX), we have previously shown that vitamin K(2) is a transcriptional regulator of bone marker genes and extracellular matrix-related genes, by activating the steroid and xenobiotic receptor (SXR). To explore a novel action of vitamin K in osteoblastic cells, we identified genes up-regulated by a vitamin K(2) isoform menaquinone-4 (MK-4) using oligonucleotide microarray analysis. Among these up-regulated genes by MK-4, growth differentiation factor 15 (GDF15) and stanniocalcin 2 (STC2) were identified as novel MK-4 target genes independent of GGCX and SXR pathways in human and mouse osteoblastic cells. The induction of GDF15 and STC2 is likely specific to MK-4, as it was not exerted by another vitamin K(2) isoform MK-7, vitamin K(1), or the MK-4 side chain structure geranylgeraniol. Investigation of the involved signaling pathways revealed that MK-4 enhanced the phosphorylation of protein kinase A (PKA), and the MK-4-dependent induction of both GDF15 and STC2 genes was reduced by the treatment with a PKA inhibitor H89 or siRNA against PKA. These results suggest that vitamin K(2) modulates its target gene expression in osteoblastic cells through the PKA-dependent mechanism, which may be distinct from the previously known vitamin K signaling pathways.

Increased expression of estrogen-related receptor alpha (ERRalpha) is a negative prognostic predictor in human prostate cancer.

The nuclear receptor ERRα (estrogen‐related receptor α) is known to modulate the estrogen‐signaling pathway, but the biological significance of ERRα in the prostate remains unclear. We investigated the expression of ERRα in human prostate tissues and cancer cell lines to evaluate the potential roles of the receptor in prostate cancer (PC). Western blot analysis of ERRα was performed in three cell lines of human PC (LNCaP, DU145 and PC‐3). The expressions of ERRα in cancerous lesions (n = 106) and benign foci (n = 99) of 106 surgically obtained prostate specimens were evaluated by immunohistochemistry. The relationships between the ERRα expression and clinicopathological features were evaluated. Western blot analysis using the polyclonal anti‐ERRα antibody detected a 52 kD band in all three PC cell lines. Positive immunostaining of ERRα in the nuclei was found in 73 (69%) cancerous and 47 (47.5%) benign epithelium, whereas the stromal tissues were negative for ERRα. The mean immunoreactivity score (IR score) of the cancerous lesions (3.5 ± 2.6) was significantly higher than that of the benign foci (1.8 ± 2.1) (p < 0.0001). The IR score of the cancerous lesions significantly correlated with the Gleason score (p = 0.0135). Univariate and multivariate hazard analyses revealed significant correlations between elevated ERRα expression and poor cancer‐specific survival (p = 0.0141 and 0.0367, respectively). The enhanced expression of ERRα might play a role in the development of human PC and serve as a significant prognostic factor for the disease.

FOXP1 is an androgen-responsive transcription factor that negatively regulates androgen receptor signaling in prostate cancer cells.

Androgen and androgen receptor (AR) play important roles in the formation and the progression of prostate cancer. AR activates its target genes by recruiting various coregulators and transcriptional factors. Here we show that the FOXP1 forkhead transcription factor is a novel androgen-regulated gene. By sequencing DNA fragments obtained from chromatin immunoprecipitation (ChIP), a bona-fide AR binding site (ARBS) is identified in an intron region of FOXP1 gene. FOXP1 can be induced by androgen in hormone-sensitive prostate cancer LNCaP cells at both mRNA and protein levels. In particular, a smaller FOXP1 variant, FOXP1D, is upregulated in response to androgen. Notably, we demonstrate that FOXP1 directly interacts with AR and negatively regulates AR signaling ligand-dependently, as exemplified by the transcriptional repression of PSA gene regulated by androgen-dependent FOXP1 recruitment on its enhancer region. We show that several other forkhead transcription factors are also androgen-responsive in LNCaP cells. Our study provides a new insight to the function of forkhead transcription factors that modulates AR signaling as an androgen-regulated transcriptional factor, which would contribute to the tumorigenesis of prostate cancer.

Steroid and xenobiotic receptor mediates a novel vitamin K2 signaling pathway in osteoblastic cells.

The nuclear receptor steroid and xenobiotic receptor (SXR) is a transcriptional regulator activated by various biological and xenobiotic substances. We have recently shown that SXR is expressed in bone and that this receptor is critical for bone metabolism, particularly in osteoblastic cells. Vitamin K2, one of the critical nutrients in bone metabolism, has been demonstrated that it is a potent SXR agonist and modulates the expression of various bone-related genes in osteoblastic cells. Using microarray analysis, we identified novel SXR target genes that were activated by vitamin K2 in osteoblastic cells. Among them, a small leucine-rich repeat proteoglycan, tsukushi, has been shown to contribute to collagen accumulation, and the protein may interact with another vitamin K2-inducible SXR target, matrilin-2, a member of the matrilin family that functions as collagen adaptors. Besides functioning as a xenobiotic biosensor, our findings show that SXR is also a vitamin K2 target and an important transcriptional factor that regulates bone homeostasis in bone cells.

EBAG9 is a tumor-promoting and prognostic factor for bladder cancer

Upregulation of EBAG9 expression has been observed in several malignant tumors such as advanced breast and prostate cancers, indicating that EBAG9 may contribute to tumor proliferation. In the present study, we assess the role of EBAG9 in bladder cancer. We generated human bladder cancer EJ cells stably expressing FLAG‐tagged EBAG9 (EJ‐EBAG9) or empty vector (EJ‐vector), and investigated whether EBAG9 overexpression modulates cell growth and migration in vitro as well as the in vivo tumor formation of EJ transfectants in xenograft models of BALB/c nude mice. EBAG9 overexpression promoted EJ cell migration, while the effect of EBAG9 to cultured cell growth was rather minimal. Tumorigenic experiments in nude mice showed that the size of EJ‐EBAG9‐derived tumors was significantly larger than EJ‐vector‐derived tumors. Loss‐of‐function study for EBAG9 using small interfering RNA (siRNA) in xenografts with parental EJ cells showed that the intra‐tumoral injection of EBAG9 siRNA markedly reduced the EJ tumor formation compared with control siRNA. Furthermore, immunohistochemical study for EBAG9 expression was performed in 60 pathological bladder cancer specimens. Intense and diffuse cytoplasmic immunostaining was observed in 45% of the bladder cancer cases. Positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients (p = 0.0001) and it was an independent prognostic predictor for disease‐specific survival in multivariate analysis (p = 0.003). Our results indicate that EBAG9 would be a crucial regulator of tumor progression and a potential prognostic marker for bladder cancer.