Dysregulated HAI-2 Plays an Important Role in Renal Cell Carcinoma Bone Metastasis through Ligand-Dependent MET Phosphorylation.

MET, a c-met proto-oncogene product and hepatocyte growth factor (HGF) receptor, is known to play an important role in cancer progression, including bone metastasis. In a previous study, we reported increased expression of MET and matriptase, a novel activator of HGF, in bone metastasis. In this study, we employed a mouse model of renal cell carcinoma (RCC) bone metastasis to clarify the significance of the HGF/MET signaling axis and the regulator of HGF activator inhibitor type-2 (HAI-2). Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of mice to prepare the mouse model of bone metastasis. The formation of bone metastasis was confirmed by whole-body bioluminescent imaging, and specimens were extracted. Expression of HGF/MET-related molecules was analyzed. Based on the results, we produced HAI-2 stable knockdown 786-O cells, and analyzed invasiveness and motility. Expression of HGF and matriptase was increased in bone metastasis compared with the control, while that of HAI-2 was decreased. Furthermore, we confirmed increased phosphorylation of MET in bone metastasis. The expression of matriptase was upregulated, and both invasiveness and motility were increased significantly by knockdown of HAI-2. The significance of ligand-dependent MET activation in RCC bone metastasis is considered, and HAI-2 may be an important regulator in this system.

Plasma macrophage-stimulating protein and hepatocyte growth factor levels are associated with prostate cancer progression.

Hepatocyte growth factor (HGF) is a well-known multifunctional growth factor, and evidence has accumulated indicating that the HGF/MET (HGF receptor) signaling axis is involved in the progression of cancer. Macrophage-stimulating protein (MSP) is also known as a growth factor which activates not only macrophages but also cancer cells and osteoclasts through the activation of the specific Receptor d'origine nantais (RON). Pro-HGF and pro-MSP lack biological activity and, therefore, require proteolytic activation for conversion to an active two-chain form by HGF activator (HGFA). Although, there are several studies on HGF/MET signaling with castration-resistant prostate cancer (CRPC) and bone metastasis, reports on plasma protein are rare. In addition, the MSP/RON signaling axis in PC is not well understood. Here, we analyzed associations between PC progression and plasma HGF and MSP levels. We tested plasma samples from 58 patients with PC: 36 with castration-resistant (CR) PC and 22 with pretreatment for PC as control. We used enzyme-linked immunosorbent assay (ELISA) kit to determine plasma levels of HGF, MSP and HGFA, and examined correlations with clinicopathological characteristics such as Gleason grade and bone metastasis. PCR was used to evaluate HGF and MSP-related molecules in PC cell lines. Plasma levels of HGF, MSP and HGFA in the CRPC group were higher than in the control group (HGF: P < 0.001; MSP: P = 0.008; HGFA: P < 0.001). HGF and MSP levels were significantly correlated (P = 0.003). In the CRPC group, plasma HGF and MSP levels and Gleason score were not correlated; however, high plasma MSP level correlated with bone metastasis. (P = 0.016). In cell lines, PC3 expressed significantly more HGF, MET and RON than did LNCaP (P < 0.001), and both cell lines expressed MSP. Plasma concentrations of HGF, MSP and HGFA are significantly elevated in patients with CRPC. Also, as plasma MSP levels are significantly associated with bone metastasis in CRPC patients, MSP may be a candidate for serum marker of bone metastasis. Our results show the importance of the HGF/MET and MSP/RON signaling systems in CRPC.

Significant Association of Caveolin-1 and Caveolin-2 with Prostate Cancer Progression.

BACKGROUND/AIM: Up-regulation of caveolin (CAV)-1 is associated with aggressive prostate cancer. Recently, it has been inferred that CAV2, a co-factor sub-type of CAV1, cross-talks with CAV1 and promotes tumor growth. We previously reported that plasma CAV1 levels are elevated in patients with castration-resistant prostate cancer (CRPC), but not in hormone-sensitive prostate cancer (non-CRPC), implying that CAV1 may be a therapeutic target for CRPC. However, a correlation of CAV1 and CAV2 expression in PC has not yet been reported. Herein, we analyzed associations between PC progression and plasma CAV1 and -2 in Japanese men, and expression of CAV1 and -2 in PC3 (CRPC) and LNCaP (non-CRPC) cell lines. MATERIALS AND METHODS: We investigated plasma samples from 36 patients with CRPC and 22 with non-CRPC. We used enzyme-linked immunosorbent assay (ELISA) to determine plasma levels of CAV1 and -2, and examined correlations with clinicopathological characteristics such as Gleason grade and clinical T stage. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to evaluate CAV1 and CAV2 mRNA in PC cell lines. We also introduced CAV1- and CAV2-specific small interfering (siRNA) into PC3 cells to knock-down (KD) both molecules, and examined its influence on the expression of these genes between PC3 CAV1 and -2 KD cells and control cells. RESULTS: Plasma CAV1 and -2 levels in patients with CRPC were significantly higher than in those with non-CRPC (CAV1, p=0.003; CAV2, p<0.001). Plasma levels of CAV1 and -2 were significantly correlated (p<0.001). However, we did not find any significant relationship between CAV1 or CAV2 expression and clinicopathological factors. ELISA and real-time qRT-PCR showed that both proteins and mRNAs in PC3 cells were significantly over-expressed compared to LNCaP cells (p<0.001). In PC3 CAV1 KD cells, expression of CAV2 was suppressed and confirmed the linkage of CAV2 KD and suppression of CAV1 expression. CONCLUSION: There was a significant correlation between plasma CAV-1 and -2 levels and progression of PC. CAV1 and -2 were highly expressed in the PC3 compared to the LNCaP cell line. Our findings support the potential of these molecules as therapeutic targets for CRPC.

Expression of human kallikrein 1-related peptidase 4 (KLK4) and MET phosphorylation in prostate cancer tissue: immunohistochemical analysis.

Resistance to or relapse after androgen deprivation therapy (ADT) remains a significant problem in patients with prostate cancer. Several studies have hypothesized that the overexpression of MET and the activating signaling axis in prostate cancer cells are associated with castration-resistant prostate cancer (CRPC). On the other hand, the expression of human kallikrein 1-related peptidase (KLK) 4, which activates plasma HGF activator zymogen, in prostate cancer patients with bone metastasis or advanced stage has also been reported. In this study, we analyzed the expression and phosphorylation of MET along with KLK4 by immunohistochemistry in 62 formalin-fixed paraffin-embedded sections of prostate cancer collected by needle biopsy at our hospital between 2006 and 2011. As a result, the phosphorylation of MET was observed in 56% (35 of 62 cases) and positively correlated with worsening PSA relapse rate in a group of ADT-treated patients (P = 0.0445), suggesting significant correlation with CRPC. Overexpression of KLK4 was observed in patients with high T stage (P = 0.0001) and high Gleason score (P = 0.0146), and the expression was correlated with the phosphorylation of MET (P = 0.0002). Pathologically, strong MET phosphorylation observed in specific architectures in prostate cancer, such as cribriform structures, ill-defined glands or solid patterns, suggested the significance of MET activation in promoting the architectural formation of prostate cancer. In addition, high positivity rate (80%) of phospho-MET staining at high-grade prostatic intraepithelial neoplasia (HGPIN) may indicate the importance of the activating signaling axis in the carcinogenesis of prostate cancer.

Matriptase and MET are prominently expressed at the site of bone metastasis in renal cell carcinoma: immunohistochemical analysis.

High MET expression in renal cell carcinoma (RCC) and MET activation in bone metastases are reportedly important in progression of several cancers. To find new treatment targets in bone metastasis, we immunohistochemically analyzed expression levels of MET and matriptase (specific cellular activator of hepatocyte growth factor). We obtained nephrectomy specimens from 17 RCC patients with metastasis, and bone metastases specimens from 7 RCC patients who underwent metastasectomies, and who were treated at our hospital between 2008 and 2012. We tested the samples with anti-human MET polyclonal antibody and anti-human matriptase polyclonal antibody, and compared postoperative overall survival (OS) rates between positive and negative groups. High MET expression was seen at primary sites in 8/17 (47%) nephrectomy specimens, and 6/7 (86%) bone specimens. Matriptase was expressed in 6/17 (35%) nephrectomy specimens, and all 7 (100%) bone specimens. Interestingly, matriptase was strongly expressed in osteoclasts of 5/7 bone specimens. Postoperative OS rate was significantly higher in the MET(-) group than the MET(+) group. The high MET and matriptase expression seen in RCC cells in bone metastasis accompanied by matriptase expression in osteoclasts indicates their importance in bone metastasis.

Effect of dutasteride on microvessel density in benign prostatic hyperplasia.

AIM: Dutasteride, a dual 5α-reductase inhibitor, is used to treat benign prostatic hyperplasia (BPH). However, its histopathological effects on the morphometrics of blood vessels and glands are still controversial. This study aimed to assess the histopathological effects of dutasteride in cases of BPH in a retrospective manner. PATIENTS AND METHODS: Patients with BPH were administered 0.5 mg of dutasteride daily or left untreated prior to undergoing holmium laser enucleation of the prostate (HoLEP). After HoLEP, remaining prostatic peripheral tissue at the bladder neck and the apex was resected. Each specimen was subjected to hematoxylin/eosin and immunohistochemical staining for CD31, and microvessel density (MVD) was analyzed. RESULTS: In the dutasteride-treated group (n=14), the mean duration of administration was 7.07±2.46 weeks. MVD was significantly lower at the bladder neck side in the dutasteride-treated group than in the control group (p=0.018). CONCLUSION: The present study, to our knowledge for the first time, assessed MVD by evaluating the bladder neck and apex sides of the remaining prostatic peripheral tissue after HoLEP, allowing evaluation of MVD in more detail without intraoperative damage of the peripheral tissue, such as through heat denaturation. Dutasteride reduces MVD in the bladder neck side of the prostate among patients with BPH and may lead to decreased risk of perioperative prostatic urethral bleeding.

Cyclooxygenase 2 genotypes influence prostate cancer susceptibility in Japanese Men.

This study aims to evaluate the relationship between the cyclooxygenase 2 (COX2) G1195A (rs689465) polymorphism and the risk of prostate cancer in a Japanese population and the associations between COX2 polymorphisms and clinicopathological characteristics, including Gleason grade and prostate-specific antigen (PSA) grade. We recruited 134 patients with prostate cancer and 86 healthy controls matched for age and smoking status. The COX2 G1195A polymorphism status was determined by polymerase chain reaction and restriction fragment length polymorphism analysis. Genotype distributions (p = 0.028) and allelic frequencies (p = 0.014) differed significantly between prostate cancer and control groups in terms of the COX2 G1195A polymorphism (Pearson's χ (2) test). Logistic regression analysis of case and control outcomes showed an odds ratio between the GG and AA genotypes of 3.15 (95% confidence interval = 1.27-8.08, p = 0.014), indicating an increased risk of prostate cancer associated with the AA genotype. Subset analysis revealed no significant associations between this polymorphism and clinicopathological characteristics of prostate cancer. This study demonstrated a relationship between the COX2 G1195A variant and prostate cancer risk. This polymorphism may merit further investigation as a potential genomic marker for the early detection of prostate cancer. Our results support the hypothesis that rs689465 influences susceptibility to prostate cancer; however, prostate cancer progression was not associated with rs689465 in a Japanese population.

[Analysis of multimodal treatment outcome in renal cell carcinoma patients with bone metastasis].

We retrospectively reviewed the treatment outcome of 12 renal cell carcinoma (RCC) patients with bone metastasis who had undergone targeted therapy along with a regimen of multimodal treatment. We performed metastatectomy following nephrectomy on patients with resectable foci. Patients with unresectable bone metastasis underwent radiation therapy. With the exception of two patients, zoledronic acid was also administered. As a result, the overall survival rates of 83.3% at 1 year and 72.9% at 2 years in our study compared favorably with reported cases. Patients with complete resection of solitary bone metastasis with or without extrabone lesions had favorable outcomes. As differences in the response of the targeted therapy can be observed between bone and extrabone metastases, control of bone metastasis may be a key factor for the prognosis of metastatic RCC patients.

Increased plasma caveolin-1 levels are associated with progression of prostate cancer among Japanese men.

AIM: Up-regulation of caveolin-1 (CAV1) is associated with aggressive prostate cancer. Among Caucasian and African-American patients, plasma CAV1 levels are elevated in patients with castration-resistant prostate cancer (CRPC), but not in those with hormone-sensitive prostate cancer (non-CRPC), which implies that CAV1 could be a therapeutic target for CRPC. Here, we evaluated associations between plasma CAV1 levels and these types of cancer in Japanese men, and CAV1 expression in PC3 (CRPC) and LNCaP (non-CRPC) cell lines. MATERIALS AND METHODS: Plasma samples were obtained from 58 patients with prostate cancer: 36 with CRPC and 22 with non-CRPC. Enzyme-linked immuno sorbent assay (ELISA) kits were used to determine CAV1 plasma levels; qRT-PCR and western blots were used to evaluate the expression of CAV1 mRNA and protein in cell lines. RESULTS: Plasma CAV1 levels in patients with CRPC were greatly higher than in those with non-CRPC (1.46±1.37 ng/ml in CRPC; 0.56±0.32 ng/ml in non-CRPC, p<0.004). Western blot and real-time qRT-PCR showed CAV1 protein and mRNA in PC3 cells to be significantly overexpressed compared to its expression in LNCaP cells (p<0.0001). CONCLUSION: Our results showed a relationship between CAV1 expression and prostate cancer progression, and support the possibility of CAV1 as a therapeutic target for CRPC.

Functional polymorphism in the CAV1 T29107A gene and its association with prostate cancer risk among Japanese men.

AIM: To evaluate the relationship between the Caveolin-1 (CAV1) T29107A (rs7804372) polymorphism and the risk of prostate cancer among Japanese populations, and the associations between CAV1 polymorphisms and clinicopathological characteristics, including Gleason grade and prostate-specific antigen (PSA) grade. MATERIALS AND METHODS: We recruited 134 patients with prostate cancer and 86 healthy controls matched for age and smoking status. The CAV1 T29107A polymorphism status was determined by polymerase chain reaction and restriction fragment-length polymorphism analysis. RESULTS: Genotype distributions (p=0.0045) and allelic frequencies (p=0.0018) differed between prostate cancer and control groups in terms of the CAV1 T29107A polymorphism (Pearson's χ(2) test). Logistic regression analysis of case and control outcomes showed an odds ratio of 0.35 (95% Condifence interval=0.13-0.91, p=0.033) between the TT and AA polymorphisms, indicating a reduced risk of prostate cancer to be associated with the AA polymorphism. Subset analysis revealed no significant associations between this polymorphism and clinicopathological characteristics of prostate cancer. CONCLUSION: The results of this study demonstrated a relationship between the CAV1 T29107A variant and risk of prostate cancer. This polymorphism thus, merits further investigation as a potential genomic marker for the early detection of prostate cancer. Our results support the hypothesis that the CAV1 T29107A (rs7804372) polymorphism may influence susceptibility to prostate cancer; however, prostate cancer progression was not associated with this polymorphism in a Japanese population.