MicroRNA profiling in clear cell renal cell carcinoma tissues potentially links tumorigenesis and recurrence with obesity.

BACKGROUND: Twenty to 40% localised RCC patients may experience recurrence after curative surgery. Limited miRNA predictors have been identified for ccRCC recurrence. METHODS: Through a multi-phase study design, we analysed miRNAs in tissues obtained from 203 ccRCC patients. Paired t-test was used for tumour-normal comparisons and Cox regression model was performed to compute hazard ratios (HRs) and corresponding 95% CIs. RESULTS: A 17-miRNA signature was identified that can concordantly classify >95% of tumour/adjacent normal samples. Significant enrichment was found as 6 out of 17 miRNAs were associated with obesity (binomial probability=0.001). Decreased levels of miR-204-5p and miR-139-5p were each associated with an approximately three-fold increased risk of recurrence (P<0.01). Risk score was generated based on expressions of miR-204-5p and miR-139-5p, and the trend test was significant in both discovery and validation sets (Pfor trend<0.05). Striking MST reduction was observed for patients with a high-risk score (high vs low: discovery, 9.4 vs >97.7 months; validation, 20.8 vs >70.3 months). Expressions of miR-204-5p were also associated with body mass index (ß=5.64, P<0.001). Significant inverse correlations were observed and validated between miR-204-5p and 13 obesity-related genes (r<0, P<0.01). CONCLUSIONS: We identified 17 miRNAs dysregulated in ccRCC tissues and showed that low expressions of miR-204-5p and miR-139-5p were associated with the higher risk of recurrence. The link between miR-204-5p and ccRCC recurrence may be partially mediated by regulating the expression of targeted obesity-related genes.

Androgen receptor expression is associated with adverse pathological features in ureteral but not in pelvicalyceal urothelial carcinomas of the upper urinary tract.

PURPOSE: This study aims to determine the significance of androgen receptor (AR) expression in urothelial carcinoma of the upper urinary tract (UTUC). METHODS: AR expression was assessed on tissue microarrays containing specimens of 737 patients with UTUC who underwent radical nephroureterectomy with curative intent. AR expression was correlated with clinical and pathological tumor features as well as recurrence-free survival (RFS), cancer-specific survival (CSS) and overall survival (OS). RESULTS: Overall, AR was expressed in 11 % of tumors. AR expression was significantly associated with tumor necrosis as well as sessile and multifocal tumor growth but not with RFS, CSS or OS. AR was detected nearly twice as often in tumors of the ureter than of the pelvicalyceal system (p = 0.005). Subgroup analyses showed that the significant associations of AR with unfavorable pathologic features were exclusively attributable to tumors located in the ureter. However, in both ureteral and pelvicalyceal tumors, AR status was independent of RFS, CSS and OS. CONCLUSIONS: In this cohort of patients treated with RNU, AR expression was found in approximately 10 % of UTUCs, twice as often in ureteral than in pelvicalyceal tumors. While AR expression had no impact on postoperative prognosis, it was significantly associated with unfavorable pathologic features in ureteral tumors. Steroid hormone signaling might be relevant for future investigations of differences between ureteral and pelvicalyceal tumors.

Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma.

Antiangiogenic therapy resistance occurs frequently in patients with metastatic renal cell carcinoma (RCC). The purpose of this study was to understand the mechanism of resistance to sunitinib, an antiangiogenic small molecule, and to exploit this mechanism therapeutically. We hypothesized that sunitinib-induced upregulation of the prometastatic MET and AXL receptors is associated with resistance to sunitinib and with more aggressive tumor behavior. In the present study, tissue microarrays containing sunitinib-treated and untreated RCC tissues were stained with MET and AXL antibodies. The low malignant RCC cell line 786-O was chronically treated with sunitinib and assayed for AXL, MET, epithelial-mesenchymal transition (EMT) protein expression and activation. Co-culture experiments were used to examine the effect of sunitinib pretreatment on endothelial cell growth. The effects of AXL and MET were evaluated in various cell-based models by short hairpin RNA or inhibition by cabozantinib, the multi-tyrosine kinases inhibitor that targets vascular endothelial growth factor receptor, MET and AXL. Xenograft mouse models tested the ability of cabozantinib to rescue sunitinib resistance. We demonstrated that increased AXL and MET expression was associated with inferior clinical outcome in patients. Chronic sunitinib treatment of RCC cell lines activated both AXL and MET, induced EMT-associated gene expression changes, including upregulation of Snail and ß-catenin, and increased cell migration and invasion. Pretreatment with sunitinib enhanced angiogenesis in 786-0/human umbilical vein endothelial cell co-culture models. The suppression of AXL or MET expression and the inhibition of AXL and MET activation using cabozantinib both impaired chronic sunitinib treatment-induced prometastatic behavior in cell culture and rescued acquired resistance to sunitinib in xenograft models. In summary, chronic sunitinib treatment induces the activation of AXL and MET signaling and promotes prometastatic behavior and angiogenesis. The inhibition of AXL and MET activity may overcome resistance induced by prolonged sunitinib therapy in metastatic RCC.

Role of partial nephrectomy as cytoreduction in the management of metastatic renal cell carcinoma.

In this review, we describe the role, feasibility and safety of partial nephrectomy in the setting of metastatic renal cell carcinoma. Partial nephrectomy is currently the preferred therapeutic modality in patients with localized renal tumors, while radical cytoreductive nephrectomy is the standard of care for appropriately selected patients with metastatic disease. Several studies have shown the prognostic value of percentage tumor removed when cytoreductive nephrectomy is done. This concept of percentage tumor removal and the associated benefit should also be applied when considering patients for cytoreductive partial nephrectomy; however, the potential adverse events after partial nephrectomy should be kept in mind, as these, when they occur, could delay time to starting systemic therapy. Several small retrospective studies have shown the feasibility of this approach in carefully selected patient groups. In well-selected patients with metastatic disease and primary tumors that are amenable to nephron sparing approaches, partial nephrectomy could offer an alternative to radical nephrectomy, with manageable adverse events, and good renal functional outcomes. Preserving renal function in this population could allow these patients to participate in clinical trial that they otherwise might not qualify for.

Cox-2 inhibition enhances the activity of sunitinib in human renal cell carcinoma xenografts.

BACKGROUND: Sunitinib (Su), a tyrosine kinase inhibitor of VEGFR, is effective at producing tumour response in clear cell renal cell carcinoma (cRCC), but resistance to therapy is inevitable. As COX-2 is a known mediator of tumour growth, we explored the potential benefit of COX-2 inhibition in combination with VEGFR inhibition in attempts at delaying tumour progression on Su. METHODS: COX-2 expression was compared with areas of hypoxia in tumours that progressed on Su vs untreated tumours. Mice bearing human cRCC xenografts were treated with Su and the COX-2 inhibitor, celecoxib, and the effects on tumour growth were assessed. Sequential vs concurrent regimens were compared. RESULTS: COX-2 expression was increased in cRCC xenografts in areas of tumour hypoxia. The combination of Su and celecoxib achieved longer times to tumour progression compared to treatment with either agent alone or to untreated control animals in four models. This effect was seen with concurrent but not with sequential therapy. CONCLUSION: COX-2 inhibition can extend the effectiveness of VEGFR inhibition. This effect is dependent on the timing of therapy. Clinical trials combining Su and COX-2 inhibitors should be considered as a means delaying time to progression on sunitinib in patients with metastatic cRCC.

Single-arc volumetric-modulated arc therapy can provide dose distributions equivalent to fixed-beam intensity-modulated radiation therapy for prostatic irradiation with seminal vesicle and/or lymph node involvement.

OBJECTIVES: Volumetric-modulated arc therapy (VMAT) is becoming an increasingly utilised modality for treating a variety of anatomical sites. However, the efficacy of single-arc VMAT to treat prostate cancer suspicious for extraprostatic extension was heretofore unknown. In this work, we report our institutional experience with single-arc VMAT and fixed-beam intensity-modulated radiation therapy (IMRT) for prostate cancer patients treated for seminal vesicle and/or lymph node involvement. METHODS: Single-arc VMAT and 7- or 9-field IMRT treatment plans were compared for 10 prostate cancer patients treated for seminal vesicle involvement and/or lymph node involvement. All treatment plans were constructed using the Philips Pinnacle treatment planning system (v.9.0, Fitchburg, WI) and delivered on an Elekta Infinity radiotherapy accelerator (Crawley, UK). Resulting plans were compared using metrics that characterised dosimetry and delivery efficiency. RESULTS: No statistically significant differences in target coverage, target homogeneity or normal tissue doses were noted between the plans (p>0.05). For prostate patients treated for seminal vesicle involvement, VMAT plans were delivered in 1.4±0.1 min (vs 9.5±2.4 min for fixed-beam IMRT) (p<0.01) and required approximately 20% fewer monitor units (p=0.01). For prostate patients treated for lymph node involvement, VMAT plans were delivered in 1.4±0.1 min (vs 11.7±1.3 min for fixed-beam IMRT) (p<0.01) and required approximately 45% fewer monitor units (p<0.01). CONCLUSION: Single-arc VMAT plans were dosimetrically equivalent to fixed-beam IMRT plans with significantly improved delivery efficiency.

Outcome of patients with metastatic renal cell carcinoma treated with targeted therapy without cytoreductive nephrectomy.

BACKGROUND: Cytoreductive nephrectomy (CN) became a standard procedure in metastatic renal cell carcinoma (mRCC) in the immunotherapy era. Historically, median overall survival (OS) of patients treated with interferon alpha (IFN-α) without CN was 7.8 months. Median OS in patients treated with targeted therapy (TT) without CN is unknown. PATIENTS AND METHODS: We retrospectively reviewed records of patients with mRCC who received TT without CN. Kaplan-Meier methods and Cox regression analysis were used to estimate median OS and identify poor prognostic factors. RESULTS: One hundred and eighty-eight patients were identified. Most patients had intermediate-risk (54.8%) or poor-risk (44.1%) disease. Median OS for all patients was 10.4 months [95% confidence interval (CI) 8.1-12.5]. By multivariable analysis, elevated baseline lactate dehydrogenase and corrected calcium, performance status of two or more, retroperitoneal nodal metastasis, thrombocytosis, current smoking, two or more metastatic sites, and lymphopenia were independent risk factors for inferior OS. Patients with four or more factors had increased risk of death (hazard ratio 8.83, 95% CI 5.02-15.5, P < 0.001) and 5.5-month median OS. Nineteen patients (10.0%) survived for 2+ years. CONCLUSIONS: These data highlight the improved OS of patients with mRCC treated with TT without CN, compared with historical IFN-α treatment, and may guide the design of trials investigating the role of CN in the TT era.

Hsa-miR-9 methylation status is associated with cancer development and metastatic recurrence in patients with clear cell renal cell carcinoma.

The long-term prognosis for clear cell renal cell carcinoma (ccRCC) is dramatically altered by the development of metastatic recurrence. However, there are very few indicators that can predict which patient will develop a recurrence. MicroRNAs regulate many cellular processes and have been shown to be associated with cancer development and recurrence. More recently it has been shown that microRNA genes can be epigenetically modified in cancer, resulting in aberrant silencing of microRNA genes with tumor suppressor functions. In this study, we show that two genes encoding for hsa-miR-9 are significantly hypermethylated in ccRCC tumors compared with adjacent normal tissues (P-value <0.001 for both miR-9-1 and miR-9-3) resulting in decreased expression, and that the methylation of these genes was more significant in DNA obtained from the primary tumor for patients who developed a recurrence (P-value: 0.012 and 0.009 for miR-9-1 and miR-9-3, respectively) than in tumors from nonrecurrent patients. Furthermore, methylation of miR-9-3 was significantly associated with an increased risk of recurrence (hazard ratio: 5.85, 95% confidence intervals: 1.30-26.35) and high methylation levels of either miR-9-1 or miR-9-3 resulted in a significant, nearly 30-month decrease in recurrence-free survival time (P-value: 0.034 and 0.007 for miR-9-1 and miR-9-3, respectively). Our results demonstrate that hsa-miR-9 is involved in the development of ccRCC while also having a role in the development of metastatic recurrence.

Loss of type III transforming growth factor-beta receptor expression is due to methylation silencing of the transcription factor GATA3 in renal cell carcinoma.

Loss of transforming growth factor-beta receptor III (TbetaRIII) correlates with loss of transforming growth factor-beta (TGF-beta) responsiveness and suggests a role for dysregulated TGF-beta signaling in clear cell renal cell carcinoma (ccRCC) progression and metastasis. Here we identify that for all stages of ccRCC TbetaRIII expression is downregulated in patient-matched tissue samples and cell lines. We find that this loss of expression is not due to methylation of the gene and we define GATA3 as the first transcriptional factor to positively regulate TbetaRIII expression in human cells. We localize GATA3's binding to a 10-bp region of the TbetaRIII proximal promoter. We demonstrate that GATA3 mRNA is downregulated in all stages, of ccRCC, mechanistically show that GATA3 is methylated in ccRCC patient tumor tissues as well as cell lines, and that inhibiting GATA3 expression in normal renal epithelial cells downregulates TbetaRIII mRNA and protein expression. These data support a sequential model whereby loss of GATA3 expression through epigenetic silencing decreases TbetaRIII expression during ccRCC progression.

Adenoviral-mediated gene therapy of human bladder cancer with antisense interleukin-8.

We previously demonstrated the importance of interleukin-8 (IL-8) as a mediator of angiogenesis, tumorigenicity, and metastasis of transitional cell carcinoma (TCC) of the bladder. In the present study, we evaluated the feasibility of adenoviral mediated antisense IL-8 gene transfer (Ad IL-8-AS) as therapy for established TCC. In vitro, Ad IL-8-AS inhibited endothelial cell proliferation and enhanced endothelial cell apoptosis. The highly metastatic human TCC cell line 253J B-V(R) was implanted into the subcutis of athymic nude mice, and intralesional therapy with Ad IL-8-AS commenced when the tumors reached a diameter between 5 and 7 mm. Tumor growth was significantly inhibited compared with therapy in controls (saline and beta-galactosidase adenovirus). Ad IL-8-AS therapy decreased the in vivo expression of IL-8 and matrix metalloproteinase type 9 (MMP-9), reduced microvessel density, and enhanced endothelial cell apoptosis. These results indicate that Ad IL-8-AS therapy targets both tumor cells and host endothelial cells resulting in endothelial cell apoptosis and significant inhibition of tumor growth.

Prostate cancer cells induce osteoblast differentiation through a Cbfa1-dependent pathway.

Metastases from prostatic adenocarcinoma (prostate cancer) are characterized by their predilection for bone and typical osteoblastic features. An in vitro model of bone metastases from prostate cancer was developed using a bicompartment coculture system of mouse osteoblasts and human prostate cancer cells. In this model, the bone-derived prostate cancer cell lines MDA PCa 2a and MDA PCa 2b induced a specific and reproducible increase in osteoblast proliferation. Moreover, these cells were able to induce osteoblast differentiation, as assessed by increased alkaline phosphatase activity, Osteocalcin expression, and calcified matrix formation. This osteoblastic reaction was confirmed in vivo by intrafemoral injection of MDA PCa 2b cells into severe combined immunodeficiency disease mice. In contrast, the highly undifferentiated, bone-derived human prostate cancer cell line PC3 did not produce an osteoblastic reaction in vitro and induced osteolytic lesions in vivo. The osteoblast differentiation induced by MDA PCa 2b cells was associated with up-regulation of the osteoblast-specific transcriptor factor Cbfa1. Moreover, treatment of osteoblasts with conditioned medium obtained from MDA PCa 2b cells resulted in up-regulation of Cbfa1 and Osteocalcin expression. In support of the differentiation studies, a microarray analysis showed that primary mouse osteoblasts grown in the presence of MDA PCa 2b cells showed a shift in the pattern of gene expression with an increase in mRNA-encoding Procollagen type I and Osteopontin and a decrease in mRNA-encoding proteins associated with myoblast differentiation, namely myoglobin and myosin light-chain 2. Taken together, these findings suggest that the bone-derived prostate cancer cells MDA PCa 2a and MDA PCa 2b promote differentiation of osteoblast precursors to an osteoblastic phenotype through a Cbfa1-dependent pathway. These results also established that soluble factors produced by prostate cancer cells can induce expression of osteoblast-specific genes. This in vitro model provides a valuable system to isolate molecules secreted by prostate cancer cells that favor osteoblast differentiation. Moreover, it allows to screen for therapeutic agents blocking the osteoblast response to prostate cancer.

Gene therapy of human bladder cancer with adenovirus-mediated antisense basic fibroblast growth factor.

We previously investigated the role of basic fibroblast growth factor (bFGF) as a mediator of angiogenesis, tumorigenicity, and metastasis of transitional cell carcinoma (TCC) of the bladder. In the present study, we determined whether adenoviral-mediated antisense bFGF gene transfer therapy (Ad bFGF-AS) would inhibit TCCs growing in the subcutis of nude mice. In vitro, Ad bFGF-AS inhibited endothelial cell proliferation and enhanced apoptosis. The highly metastatic human TCC cell line 253J-BV(R) was implanted ectopically in the subcutis of athymic nude mice, and therapy was begun when the tumors reached a diameter between 5 and 7 mm. Intralesional therapy with Ad bFGF-AS decreased the in vivo expression of bFGF and matrix metalloproteinase type 9 mRNA and protein, and reduced microvessel density and enhanced endothelial cell apoptosis. Tumor growth was significantly inhibited by Ad bFGF-AS (mean, 58 mg) compared with controls [saline (mean, 562 mg), beta-galactosidase adenovirus (mean, 586 mg), and sense bFGF adenoviral therapy (Ad bFGF-S; mean, 3012 mg)]. These results suggest that Ad bFGF-AS therapy affects endothelial cells directly and tumor cells indirectly through down-regulation of bFGF and matrix metalloproteinase type 9, resulting in endothelial cell apoptosis and significant tumor growth inhibition. Furthermore, these studies confirm that bFGF expression is a valid target for the therapy of bladder cancer.

Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model.

Prolonged exposure of the developing neonatal ovine uterus to a progestin from birth prevents uterine gland development and creates an adult endometrial phenotype characterized by the absence of glandular epithelium, the uterine gland knockout (UGKO) phenotype. This study used endometrium from normal and UGKO sheep to identify messenger RNAs (mRNAs) expressed differentially in the endometrial epithelium using the molecular techniques of mRNA differential display PCR (DD-PCR) and suppression subtractive complementary DNA (cDNA) hybridization (SSH). Sequence analyses of DD- and SSH-identified and cloned cDNAs indicated similarity of some to known mRNAs, including beta-lactoglobulin, alkaline phosphatase, type B and D endogenous sheep retroviruses, gp330/megalin, matrix Gla protein, and others. Other cDNAs were not similar to any known sequences and are considered novel, although some of these match human expressed sequence tags. In situ hybridization analyses of uteri from cyclic and pregnant ewes indicated that all DD-PCR- and SSH-identified mRNAs were expressed in either the endometrial lumenal and/or glandular epithelium, although some were also expressed in other uterine cell types. Northern and in situ hybridization analyses revealed that patterns of mRNA expression for most clones were affected by the day of the estrous cycle and pregnancy in a manner consistent with regulation by progesterone. Studies demonstrate the utility of the ovine UGKO model as a tool with which to identify known and novel uterine epithelial-specific genes. Cloned cDNAs identified here are expressed sequence tags useful for comparative and physical genetic mapping and may be used to reveal new factors and pathways regulating endometrial function.

Tumor-suppressive activity of CD66a in prostate cancer.

CD66a (human homolog of rat cell-cell adhesion molecule, also known as biliary glycoprotein) is a cell surface protein of the immunoglobulin family. CD66a has been shown to mediate homotypic cell adhesion. Aside from this, no other functions of this molecule have been demonstrated. We have observed previously that CD66a protein expression is lost in most prostate tumors, suggesting that the down-regulation of CD66a is associated with the abnormal growth of prostate cells. CD66a is homologous (65% identity) to rat cell-cell adhesion molecule, which has been shown to have tumor-suppressive activity. This homology suggests the possibility that CD66a might also be a tumor suppressor. In this report, we show that restoring CD66a expression in DU145 human prostate cancer cells by adenovirus (Ad)-mediated gene transfer dramatically altered the malignant phenotype of these cells, as evidenced by their reduced ability to form tumors in a xenograft animal model. This result suggests that loss of CD66a protein plays an important role in the development of prostate cancer, and that restoring CD66a expression might provide an effective treatment for prostate cancer. We further explored the possibility of using Ad vectors to deliver CD66a as a potential therapeutic agent for prostate cancer. Direct injection of Ad-CD66a, an Ad vector carrying the CD66a gene, into DU145 tumors in mice significantly suppressed the growth of these tumors. This antitumor activity of CD66a was found to be dose-dependent. These results suggest that CD66a has tumor-suppressive activity and that Ad-CD66a is a potential therapeutic agent for prostate cancer treatment.