Jinpei Kumagai

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.

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.

Differential expression of estrogen-related receptors β and γ (ERRβ and ERRγ) and their clinical significance in human prostate cancer

(Cancer Sci 2010; 101: 646–651)

Oct1 regulates cell growth of LNCaP cells and is a prognostic factor for prostate cancer.

The androgen receptor (AR) plays a critical role in the development and the progression of prostate cancer. Alterations in the expression of AR coregulators lead to AR hypersensitivity, which is one of the mechanisms underlying the progression of prostate cancer into a castrate‐resistant state. Octamer transcription factor 1 (Oct1) is a ubiquitous member of the POU‐homeodomain family that functions as a coregulator of AR. In our study, the contribution of Oct1 to prostate cancer development was examined. Immunocytochemistry analysis showed that Oct1 is expressed in the nuclei of LNCaP cells. siRNA‐mediated silencing of Oct1 expression inhibited LNCaP cell proliferation. Immunohistochemical analysis of Oct1 expression in tumor specimens obtained from 102 patients with prostate cancer showed a positive correlation of Oct1 immunoreactivity with a high Gleason score and AR immunoreactivity (p = 0.0042 and p < 0.0001, respectively). Moreover, patients with high immunoreactivity of Oct1 showed a low cancer‐specific survival rate, and those patients with high immunoreactivities of both Oct1 and AR exhibited poorer cancer‐specific prognosis. Multivariate hazard analysis revealed a significant correlation between high Oct1 immunoreactivity and poor cancer‐specific survival (p = 0.012). These results demonstrate that Oct1 can be a prognostic factor in prostate cancer as a coregulator of AR and may lead to the development of a new therapeutic intervention for prostate cancer.

Estrogen Receptor-Binding Fragment-Associated Antigen 9 Is a Tumor-Promoting and Prognostic Factor for Renal Cell Carcinoma

The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) has been identified as a primary estrogen-responsive gene in human breast cancer MCF7 cells. A high expression of EBAG9 has been observed in invasive breast cancer and advanced prostate cancer, suggesting a tumor-promoting role of the protein in malignancies. Here we show that intratumoral (i.t.) administration of small interfering RNA against EBAG9 exerted overt regression of tumors following s.c. implantation of murine renal cell carcinoma (RCC) Renca cells. Overexpression of EBAG9 did not promote the proliferation of culture Renca cells; however, the inoculated Renca cells harboring EBAG9 (Renca-EBAG9) in BALB/c mice grew faster and developed larger tumors compared with Renca cells expressing vector alone (Renca-vector). After renal subcapsular implantation, Renca-EBAG9 tumors significantly enlarged compared with Renca-vector tumors in BALB/c mice, whereas both Renca-EBAG9 and Renca-vector tumors were developed with similar volumes in BALB/c nude mice. No apparent difference was observed in specific cytotoxic T-cell responses against Renca-EBAG9 and Renca-vector cells; nonetheless, the number of infiltrating CD8+ T lymphocytes was decreased in Renca-EBAG9 subcapsular tumors. Furthermore, immunohistochemical study of EBAG9 in 78 human RCC specimens showed that intense and diffuse cytoplasmic immunostaining was observed in 87% of the cases and positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients. Multivariate analysis revealed that high EBAG9 expression was an independent prognostic predictor for disease-specific survival (P = 0.0485). Our results suggest that EBAG9 is a crucial regulator of tumor progression and a potential prognostic marker for RCC.

Expression of Cytochrome P450 3A4 and Its Clinical Significance in Human Prostate Cancer

OBJECTIVES To evaluate CYP3A4 expression in human prostrate cancer (PCa) tissues. Enzymes of the cytochrome P450 (CYP) family are key inactivators of testosterone in the liver and prostate. We previously reported that CYP2B6 is a growth-inhibitory and prognostic factor in human PCa; however, the status of CYP3A4 in PCa remains unclear. METHODS We used immunohistochemistry to analyze CYP3A4 expression in 107 human PCa specimens obtained by radical prostatectomy. Stained slides were evaluated for the proportion and staining intensity of positively stained cells. Total immunoreactivity scores (0-8) were obtained as the sum of the proportion and intensity scores. In addition, we estimated the relationship between CYP3A4 status and clinicopathologic features. RESULTS CYP3A4 immunoreactivity was identified in the cytoplasm of prostate cells. The CYP3A4 immunoreactive PCa score (3.6+/-2.6) was significantly lower than that of benign epithelium (4.5+/-2.1; P < .0001). In addition, CYP3A4 immunoreactivity correlated inversely with the Gleason score (P < .0001). Decreased CYP3A4 immunoreactivity was significantly related to a poor prognosis in human PCa (P = .0175). CONCLUSIONS We demonstrated differential CYP3A4 expression in prostatic tissues, indicating that decreased CYP3A4 expression may contribute to the development of PCa.

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.

Abhydrolase domain containing 2, an androgen target gene, promotes prostate cancer cell proliferation and migration

BACKGROUND The androgen receptor (AR) plays a key role in the development of prostate cancer. AR signalling mediates the expression of androgen-responsive genes, which are involved in prostate cancer development and progression. Our previous chromatin immunoprecipitation study showed that the region of abhydrolase domain containing 2 (ABHD2) includes a functional androgen receptor binding site. In this study, we demonstrated that ABHD2 is a novel androgen-responsive gene that is overexpressed in human prostate cancer tissues. METHODS The expression levels of ABHD2 in androgen-sensitive cells were evaluated by quantitative reverse transcription polymerase chain reaction and western-blot analyses. LNCaP and VCaP cells with ABHD2 overexpression or short interfering RNA (siRNA) knockdown were used for functional analyses. ABHD2 expression was examined in clinical samples of prostate cancer by immunohistochemistry. RESULTS We showed that ABHD2 expression is increased by androgen in LNCaP and VCaP cells. This androgen-induced ABHD2 expression was diminished by bicalutamide. While stable expression of ABHD2 affected the enhancement of LNCaP cell proliferation and migration, siRNA-mediated ABHD2 knockdown suppressed cell proliferation and migration. In addition, the siRNA treatment significantly repressed the tumour growth derived from LNCaP cells in athymic mice. Immunohistochemical analysis of ABHD2 expression in tumour specimens showed a positive correlation of ABHD2 immunoreactivity with high Gleason score and pathological N stage. Moreover, patients with high immunoreactivity of ABHD2 showed low cancer-specific survival rates and a resistance to docetaxel-based chemotherapy. CONCLUSION ABHD2 is a novel androgen-regulated gene that can promote prostate cancer growth and resistance to chemotherapy, and is a novel target for diagnosis and treatment of prostate cancer.

Fatal non-occlusive mesenteric ischemia after laparoscopic radical nephrectomy in a hemodialysis patient

Non-occulusive mesenteric ischemia (NOMI) refers to mesenteric ischemia without evident occlusion of the mesenteric arteries. It accounts for 20–30% of acute mesenteric ischemia. Among the various types of mesenteric ischemia, NOMI has the poorest survival with a high mortality rate of 50–80%, which has been hardly improved over time. A 60-year-old man was referred for assessment of right renal tumor. He had been on hemodialysis for 15 years because of chronic renal failure as a result of glomerulonephritis. Computed tomography (CT) of the abdomen showed a 2-cm enhancing lesion in the right kidney. Transperitoneal laparoscopic radical nephrectomy of the right kidney was subsequently carried out with an uneventful perioperative course; the ascending colon and mesocolon had no abnormal findings, with an uneventful perioperative course. On postoperative day 4, the patient developed a fever higher than 38°C. CT of the abdomen showed paralytic ileus and inhomogeneous bowel wall thickening of the ascending colon (Fig. 1). No occlusion of the superior mesenteric artery or vein was observed. Consulted gastroenterological surgeons recommended conservative therapy by an ileus tube based on the absence of obvious abdominal symptoms. On postoperative day 17, he developed a sudden abdominal distension. CT of the abdomen showed a sign of intestinal perforation. Emergency laparotomy revealed total necrosis of the ascending colon and perforation of the small intestine. These operative findings led to a clinical diagnosis of NOMI. Right hemicolectomy, repair of intestinal perforation and ileostomy were carried out. The patient was managed with vasodilators and antibiotics in an intensive care unit. Seven days after the emergency operation, however, abdominal pain with peritoneal signs developed. A second emergency laparotomy showed multiple perforations of necrotic small intestine and sigmoidal colon. The patient died of multiple organ failure 1 week after the second emergency operation. NOMI, first described by Ende in 1958, generally affects patients aged over 50 years with heart failure, septic shock and cardiac arrhythmias. Risk factors for NOMI include smoking history, diabetes mellitus, hypertension, arterial occlusive disease and administration of vasoconstrictive medications or digitalis. NOMI is also likely to occur in hemodialysis patients or in the postoperative course of major surgery. Laparoscopic surgery is not considered to be an inciting factor for NOMI. The diagnosis of NOMI is difficult. There are no clinical signs specific to NOMI. Abdominal pain might be the only presenting symptom; however, pain is absent in up to 25% of patients. The diagnosis of NOMI chiefly depends on a clinical suspicion, especially in patients with risk factors. Despite the technical evolution on non-invasive methods, such as CT and magnetic resonance imaging, angiography is still the gold standard in diagnosing peripheral splanchnic vessel disease including NOMI. Immediate angiography in suspected patients might allow an early diagnosis and improved survival. Patients with signs of ischemic complications require abdominal exploration and bowel resection. Non-surgical management of NOMI includes hemodynamic support, correction of metabolic acidosis and initiation of broad-spectrum antibiotics. The difficulty in diagnosis of NOMI might delay the initiation of treatment, resulting in poor outcomes.

288 OCT1 AS A PROGNOSTIC FACTOR FOR PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Androgen receptor (AR) plays a role in prostate cancer development and progression. Alteration of AR coregulators expression leads to hypersensitivity of AR, which is supposed to be one of the mechanisms for prostate cancer progression to castrate -resistant state. Octamer transcription factor 1 (Oct1) is a ubiquitous member of the POU-homeodomain family that was found to be one of AR coregulators. Here, we examined the contribution of Oct1 in prostate cancer development. METHODS: Immunocytochemical study for Oct1 and AR was performed in human prostate cancer cell line (LNCaP). Endogenous Oct1 was knocked down by siRNA targeting Oct1, and cell growth rate was measured using a MTS proliferation assay. Immunohistochemical study for Oct1 and AR expression was performed in 102 pathological prostate cancer specimens. Immunostained slides were evaluated for the proportion of positive cells to total cells (under 200x magnification) (score 0, none; score 1, 1/100; score 2, 1/100 to 1/10; score 3, 1/ 10 to 1/3; score 4, 1/3 to 2/3; score 5, 2/3) and the intensity (score 0, none; score 1, weak; score 2, moderate; score 3, strong) of positively stained cells. RESULTS: By immunocytochemistry analysis, Oct1 was expressed in the nucleus in LNCaP cells. In addition, silencing of Oct1 expression with siRNA decreased LNCaP cell proliferation. Immunohistochemically analysis for Oct1 and AR in 102 prostate cancer specimens, the expression of Oct1 was positively correlated with high Gleason score (GS) and AR immunoreactivity (p 0.0042, and p 0.0001, respectively). Multivariate hazard analysis revealed significant correlations between strong Oct1 immunoreactivity and poor cancerspecific survival (p 0.012, respectively). In addition, cancer-specific survival in strong expression of Oct1 cases was lower than in strong expression of AR expression cases. CONCLUSIONS: Oct1 can be a prognostic factor in prostate cancer conjunctively with AR. These findings suggest that Oct1 may be a new therapeutic intervention in prostate cancer.