BackgroundThe enhancer of zeste homolog 2 (EZH2) gene exerts oncogene-like activities and its (over)expression has been linked to several human malignancies. Here, we studied a possible association between EZH2 expression and prognosis in patients with renal cell carcinoma (RCC).MethodsEZH2 protein expression in RCC specimens was analyzed by immunohistochemistry using a tissue microarray (TMA) containing RCC tumor tissue and corresponding normal tissue samples of 520 patients. For immunohistochemical assessment of EZH2 expression, nuclear staining quantity was evaluated using a semiquantitative score. The effect of EZH2 expression on cancer specific survival (CSS) was assessed by univariate and multivariate Cox regression analyses.ResultsDuring follow-up, 147 patients (28%) had died of their disease, median follow-up of patients still alive was 6.0 years (range 0-16.1 years). EZH2 nuclear staining was present in tumor cores of 411 (79%) patients. A multivariate Cox regression analysis revealed that high nuclear EZH2 expression was an independent predictor of poor CSS (> 25-50% vs. 0%: HR 2.72, p = 0.025) in patients suffering from non-metastatic RCC. Apart from high nuclear EZH2 expression, tumor stage and Fuhrman's grading emerged as significant prognostic markers. In metastatic disease, nuclear EZH2 expression and histopathological subtype were independent predictive parameters of poor CSS (EZH2: 1-5%: HR 2.63, p = 0.043, >5-25%: HR 3.35, p = 0.013, >25%-50%: HR 4.92, p = 0.003, all compared to 0%: HR 0.36, p = 0.025, respectively).ConclusionsThis study defines EZH2 as a powerful independent unfavourable prognostic marker of CSS in patients with metastatic and non-metastatic RCC.

The Enhancer of Zeste 2 (EZH2) protein has been reported to stimulate cell growth in some cancers and is therefore considered to represent an interesting new target for therapeutic intervention. Here, we investigated a possible role of EZH2 for the growth control of colon cancer cells. RNA interference (RNAi)-mediated intracellular EZH2 depletion led to cell cycle arrest of colon carcinoma cells at the G1/S transition. This was associated with a reduction of cell numbers upon transient transfection of synthetic EZH2-targeting siRNAs and with inhibition of their colony formation capacity upon stable expression of vector-borne siRNAs. We furthermore tested whether EZH2 may repress the growth-inhibitory p27 gene, as reported for pancreatic cancer. However, expression analyses of colon cancer cell lines and colon cancer biopsies did not reveal a consistent correlation between EZH2 and p27 levels. Moreover, EZH2 depletion did not re-induce p27 expression in colon cancer cells, indicating that p27 repression by EZH2 may be cell- or tissue-specific. Whole genome transcriptome analyses identified cellular genes affected by EZH2 depletion in colon cancer cell lines. They included several cancer-associated genes linked to cellular proliferation or invasion, such as Dag1, MageD1, SDC1, Timp2, and Tob1. In conclusion, our results demonstrate that EZH2 depletion blocks the growth of colon cancer cells. These findings might provide benefits for the treatment of colon cancer.

Renal cell carcinomas associated with Xp11.2 translocations have recently been identified as a distinct biological entity. The translocation results in the fusion of the transcription factor TFE3 to one of several different fusion partners including PRCC, PSF, NONO, ASPL or CTLC with consecutive overexpression of the chimeric protein. As the true frequency of these neoplasms as well as the biological properties of TFE3 activation in renal cell carcinomas are largely unknown, we have examined TFE3 expression as well as the underlying genetic alterations in a large, hospital-based series of renal cell carcinomas with long-term follow-up information. Out of a total of 876 tumours, TFE3 translocations were detected in five cases (0.6%). Three additional cases were identified in a second series of cases comprising of renal cell carcinomas developing in patients before the age of 50. However, using immunohistochemistry, 9% of all renal cell carcinomas showed some degree of TFE3 reactivity. Interestingly, these cases were associated with high nuclear grade, greater tumour extent and metastatic disease as well as an unfavourable patient outcome on uni-and multivariate analysis. Fluorescence in situ hybridisation (FISH) revealed TFE3 amplifications as an additional, novel mechanism leading to increased TFE3 expression levels. In conclusion, our data show that Xp11 translocation renal cell carcinomas are uncommon tumours accounting for o1% of adult renal cell carcinomas and that the diagnosis of Xp11 translocation renal cell carcinomas needs to be verified using molecular techniques. In turn, TFE3 overexpressing tumours show an aggressive behaviour and Xp11 translocation is only one of several possible underlying genomic alterations.

OBJECTIVE To assess whether under‐ or overweight at the time of surgery has any effect on the survival of the patients with renal cell carcinoma (RCC), as obesity increases the risk of developing RCC. PATIENTS AND METHODS We prospectively evaluated 780 patients who had nephrectomy for RCC between 1990 and 2005. We used uni‐ and multivariate Cox proportional hazards models to assess the effect of body mass index (BMI), tumour stage, Fuhrman grade, age, sex, histological type and performance status on cancer‐specific survival (CSS). Patients were grouped according to BMI (in kg/m2), as underweight (<18.5), normal (18.5–<25), overweight (25–<30) and obese (≥30). RESULTS The median (range) follow‐up was 5.3 (0.5–15.4) years, the patients being followed until June 2006; 254 patients died during the follow‐up. Multivariate analyses of all patients showed that tumour stage, Fuhrman grade, Karnofsky performance status, age, sex and BMI were independent prognostic factors for CSS. While underweight patients had a significantly worse prognosis than those of normal weight, overweight or obese patients had a similar outcome to that of patients of normal weight. In a subgroup analyses including patients with localized RCC only, there was a strong tendency to less aggressive disease in the overweight group (P = 0.081). CONCLUSIONS Being underweight is an unfavourable and new risk factor for CSS in patients with RCC treated by nephrectomy. Although not significant, there seems to be a limited favourable prognostic effect of overweight on CSS in patients with localized RCC.

Purpose: The death ligand tumor necrosis factor^related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R) are involved in immune surveillance and tumor development. Here, we studied a possible association between the expression of TRAIL/TRAIL-Rs and the prognosis in patients with renal cell carcinomas (RCC). Experimental Design: A tissue microarray containing RCC tumor tissue samples and corresponding normal tissue samples from 838 patients was generated. Expression of TRAIL and TRAIL-Rs was examined by immunohistochemistry and the effect of TRAIL and TRAIL-R expression on disease-specific survival was assessed. Results: High TRAIL-R2 expression levels were associated with high-grade RCCs (P < 0.001) and correlated negatively with disease-specific survival (P = 0.01). Similarly, highTRAIL expression was associated with a shorter disease-specific survival (P = 0.01). In contrast, lowTRAIL-R4 expression was associated with high-stage RCCs (P < 0.001) as well as with the incidence of distant metastasis (P = 0.03) and correlated negatively with disease-specific survival (P = 0.02). In patients without distant metastasis, multivariate Cox regression analyses revealed that TRAIL-R2 and TRAIL are independent prognostic factors for cancer-specific survival (in addition to tumor extent, regional lymph node metastasis, grade of malignancy, and type of surgery). Conclusion: High TRAIL-R2, high TRAIL, and low TRAIL-R4 expression levels are associated with a worse disease-specific survival in patients with RCCs. Therefore, the assessment of TRAIL/TRAIL-R expression offers valuable prognostic information that could be used to select patients for adjuvant therapy studies. Moreover, our findings are of relevance for a potential experimental therapeutic administration of TRAIL-R agonists in patients with RCCs.

DcR3 expression is an independent prognostic factor of RCC progression and mortality. Therefore, the assessment of DcR3 expression levels offers valuable prognostic information that could be used to select patients for adjuvant therapy studies.

The enhancer of zeste homolog 2 (EZH2) gene has been recently linked to human malignancies where it may serve as a new target for cancer therapy. Here, we analyzed EZH2 expression in primary renal cell carcinoma (RCC) specimens and in nontumorous tissue samples from adult kidney. EZH2 transcripts were detectable in all RCC specimens examined. Expression levels were significantly higher in tumor tissue (p 0.0001) than in samples from normal adult kidney. Moreover, inhibition of endogenous EZH2 expression in RCC cell lines by RNA interference (RNAi) led to reduced proliferation and increased apoptosis in RCC cells. These data show that EZH2 is overexpressed in RCC. Furthermore, they indicate that the EZH2 gene plays a role for both the proliferation and the apoptosis resistance of RCC cells. Targeted inhibition of EZH2 could therefore represent a novel strategy to improve the therapeutic response of RCC. ' 2008 Wiley-Liss, Inc.Key words: renal cell carcinoma; enhancer of zeste homolog 2 (EZH2); RNA interference; tumor therapy Renal cell carcinoma (RCC) is estimated to account for more than 51,000 new cases and almost 13,000 cancer-related deaths in the United States in 2007, making it the second most lethal of the urological cancers. 1 RCCs typically are highly resistant toward chemotherapy with a concomitant poor prognosis in advanced stages. 2 Therefore, the identification of novel therapeutic targets and the development of new strategies for RCC treatment are urgently required.The enhancer of zeste homolog 2 (EZH2) gene encodes a polycomb group (PcG) protein, which acts as a histone methyltransferase 3-5 and also can directly control DNA methylation. 6 EZH2 is involved in several key regulatory mechanisms within eukaryotic cells, such as control of embryonal development or cell proliferation. 7,8 Moreover, there is accumulating evidence indicating that EZH2 may also play a pivotal role in the etiology of several tumor forms, which include prostate cancer 9,10 and breast cancer. 10,11 For both of these cancers, EZH2 expression is often observed in proliferative and more aggressive tumor subgroups and has diagnostic and/or prognostic value. [9][10][11] Notably, however, EZH2 appears to be not only a potential tumor marker but may itself contribute to the deregulation of cell growth as a bona fide oncogene. Overexpression of EZH2 conferred cellular growth advantage in vitro, 11-13 promoted invasion 11 and exhibited oncogenic properties in nude mice. 14 Vice versa, inhibition of EZH2 expression by antisense constructs or RNA interference (RNAi) did result in growth inhibition of some cancer cells. 9,15 Furthermore, RNAi-mediated inhibition of EZH2 expression induced anoikis in circulating prostate carcinoma precursor cells 16 and apoptotic cell death in breast cancer cells. 17 A possible role of EZH2 for RCC has not been studied so far. Here, we analyzed EZH2 expression in primary RCC specimens and in nontumorous tissue. In addition, we investigated the functional role of EZH2 for the proliferation control and apop...

Background:The B-cell translocation gene 2 (BTG2) is considered to act as a tumour-suppressor gene because of its antiproliferative and antimigratory activities. Higher levels of BTG2 expression in tumour cells have been linked to a better clinical outcome for several cancer entities. Here, we investigated the expression and function of BTG2 in bladder cancer.Methods:The expression of BTG2 in bladder cancer cells was silenced by RNA interference. Cell motility was investigated by wound healing and Boyden chamber assays. The protein expression of BTG2 in bladder cancer was studied by immunohistochemistry.Results:We observed that targeted suppression of BTG2 by RNA interference did not result in growth stimulation but led to a substantial inhibition of bladder cancer cell motility. Tissue microarray analyses of bladder cancer cystectomy specimens revealed that higher BTG2 expression levels within the tumours correlated strongly with a decreased cancer-specific survival for bladder cancer patients.Conclusion:These results indicate that endogenous BTG2 expression contributes to the migratory potential of bladder cancer cells. Moreover, high levels of BTG2 in bladder cancers are linked to decreased cancer-specific survival. These findings question the conception that BTG2 generally acts as a tumour suppressor and typically represents a favourable clinical marker for cancer patients.