Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cancer, exosome secretion has been implicated in tumor growth and metastatic spread. In this study, we explored the possibility that exosomal pathways might discard tumor-suppressor miRNA that restricts metastatic progression. Secreted miRNA characterized from isogenic bladder carcinoma cell lines with differing metastatic potential were uncoupled from binding to target transcripts or the AGO2-miRISC complex. In metastatic cells, we observed a relative increase in secretion of miRNA with tumor-suppressor functions, including miR23b, miR224, and miR921. Ectopic expression of miR23b inhibited invasion, anoikis, angiogenesis, and pulmonary metastasis. Silencing of the exocytotic RAB family members RAB27A or RAB27B halted miR23b and miR921 secretion and reduced cellular invasion. Clinically, elevated levels of RAB27B expression were linked to poor prognosis in two independent cohorts of patients with bladder cancer. Moreover, highly exocytosed miRNA from metastatic cells, such as miR23b, were reduced in lymph node metastases compared with patient-matched primary tumors and were correlated with increments in miRNA-targeted RNA. Taken together, our results suggested that exosomemediated secretion of tumor-suppressor miRNA is selected during tumor progression as a mechanism to coordinate activation of a metastatic cascade. Cancer Res; 74(20); 5758-71. Ó2014 AACR.

What's known on the subject? and What does the study add?Several studies have shown that defects in DNA‐damage response are associated with good survival after chemotherapy and radiotherapy. Furthermore, loss of cell cycle regulators may be prognostic indicators of poor survival after cystectomy. However, the potential clinical impact of previous findings is hampered by insufficient validation of significant results in suitable cystectomy and radiotherapy cohorts.Here we use a large cohort of patients receiving radiotherapy to successfully validate the importance of MRE11 as a predictive marker of disease‐specific survival (DSS). Furthermore, using two independent patient cohorts we show for the first time that TIP60 is a predictive marker of DSS after cystectomy. We show that combined use of TIP60 and MRE11 may hold the potential to guide treatment decisions.OBJECTIVE To determine the association between the proteins: tat‐interactive protein 60 kDa (TIP60), p16, meiotic recombination 11 homolog (MRE11), phosphorylated ataxia telangiectasia mutated (ATM), retinoblastoma protein (Rb), Ki67, and p53 and clinical outcome in invasive lymph node‐negative bladder cancer. PATIENTS AND METHODS Protein expression was measured by immunohistochemistry in cancer specimens from two independent cohorts of patients with bladder cancer treated with cystectomy (162 patients and 273) and one cohort of patients receiving radiotherapy (148). Disease‐specific survival (DSS) was used as the outcome measure, and patients with no disease‐specific death were followed for a minimum of 36 months. RESULTS TIP60 was significantly correlated with DSS in both cystectomy cohorts (hazard ratio [HR] 0.42, 95% confidence interval [CI] 0.26–0.68, P < 0.001 and HR 0.45, 95% CI 0.28–0.72, P= 0.001). MRE11 was significantly correlated with DSS in the cohort receiving radiotherapy (HR 0.64, 95% CI 0.47–0.86, P= 0.005). P16 was significantly correlated with DSS in all three cohorts (HR 0.46, 95% CI 0.30–0.75, P= 0.032; HR 0.60, 95% CI 0.37–0.97, P= 0.032; HR 0.52, 95% CI 0.28–0.96, P= 0.001). Rb was significantly correlated with DSS in one cystectomy cohort (HR 1.71, 95% CI 1.13–2.75, P= 0.017). Ki67, p53, and pATM were not significantly correlated with DSS in any of the cohorts. CONCLUSIONS TIP60 protein expression was a predictive marker for DSS after cystectomy in two independent cohorts. This novel marker was the strongest predictive factor in multivariate analysis in patients receiving cystectomy. MRE11 was shown to be a predictive marker for DSS after radiotherapy. We have shown that TIP60 and MRE11 hold the potential to guide patients with invasive bladder cancer to either cystectomy or radiotherapy. This study was based on retrospective material and consequently we suggest that these markers should be validated in a prospective study.

One major challenge in cancer research is to understand the complex interplay between the DNA damage response (DDR), genomic integrity, and tumor development. To address these issues, we analyzed 43 bladder tumor genomes from 22 patients using single nucleotide polymorphism (SNP) arrays, and tissue expression of multiple DDR proteins, including Timeless and its interaction partner Tipin. The SNP profiles confirmed and extended known copy number alterations (CNAs) at high resolution, showed clustering of CNAs at nine common fragile sites, and revealed that most metachronous tumors were clonally related. The occurrence of many novel uniparental disomy regions (UPDs) was of potential functional importance in some tumors because UPDs spanned mutated FGFR3 and PIK3CA alleles, and also homozygous deletion of the CDKN2A tumor suppressor locus. The DDR signaling as evaluated by phospho-epitope-specific antibodies against Ser139-phosphorylated H2A histone family member X (γH2AX), ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR) was commonly activated in tumors with both moderate and high extent of accumulated genomic aberrations, the latter tumors showing a more frequent loss of ATM expression. Strikingly, the tumor genomes exhibiting the most complex alterations were associated with a high Ki67-proliferation index, abundant Timeless but not Tipin expression, aberrant p53 expression, and homozygous CDKN2A deletions. Of clinical relevance, evaluation of a tissue microarray (TMA; n=319) showed that abundant Timeless expression was associated with risk of progression to muscle-invasive disease (P<0.0005; hazard ratio, 2.4; 95% confidence interval, 1.6-3.8) and higher T stage (P<0.05). Univariate analysis confirmed this association (P=0.006) in an independent cohort (n=241) but statistical significance was not reached in a multivariate model. Overall, our results are consistent with DDR activation preceding the accumulation of genomic aberrations. Tumors with extensive genomic rearrangements were associated with inactivation of CDKN2A, excessive proliferation, and robust Timeless expression, the latter also correlating with the risk of disease progression. Moreover, we provide evidence to suggest that UPDs likely contribute to bladder tumorigenesis.

BackgroundThe standard treatment for non-metastatic muscle-invasive bladder cancer (stages T2–T4a) is radical cystectomy with lymphadenectomy. However, patients undergoing cystectomy show metastatic spread in 25% of cases and these patients will have limited benefit from surgery. Identification of patients with high risk of lymph node metastasis will help select patients that may benefit from neoadjuvant and/or adjuvant chemotherapy.MethodsRNA was procured by laser micro dissection of primary bladder tumors and corresponding lymph node metastases for Affymetrix U133 Plus 2.0 Gene Chip expression profiling. A publically available dataset was used for identification of the best candidate markers, and these were validated using immunohistochemistry in an independent patient cohort of 368 patients.ResultsGene Set Enrichment Analysis showed significant enrichment for e.g. metastatic signatures in the metastasizing tumors, and a set of 12 genes significantly associated with lymph node metastasis was identified. Tumors did not cluster according to their metastatic ability when analyzing gene expression profiles using hierarchical cluster analysis. However, half (6/12) of the primary tumor clustered together with matching lymph node metastases, indicating a large degree of intra-patient similarity in these patients. Immunohistochemical analysis of 368 tumors from cystectomized patients showed high expression of GEM (P = 0.033; HR = 1.46) and EDNRA (P = 0.046; HR = 1.60) was significantly associated with decreased cancer-specific survival.ConclusionsGEM and EDNRA were identified as promising prognostic markers for patients with advanced bladder cancer. The clinical relevance of GEM and EDNRA should be evaluated in independent prospective studies.

Aim The aim of this work was to determine the knowledge gap in the field of erectile function (EF) after colorectal cancer surgery and investigate and compare long‐term male EF in colon and rectal cancer survivors in a national population. Method Danish male patients alive without evidence of recurrence who were treated for colon or rectal cancer between May 2001 and December 2014 were invited to participate. Using the International Index of Erectile Function (IIEF) score the derived dichotomized erectile dysfunction (ED) was defined as moderate/severe or no/mild. Patients were grouped based on type of surgery [colon resection, rectal resection (RR) or local resection] and stratified for stoma, preoperative radiotherapy (RT), age and American Society of Anesthesiologists (ASA) score. Results Of 10 037 eligible patients, 4334 responded (43.18%). The EF score was significantly lower for RR (mean 12.14) compared with both colon resection (mean 15.82) and local resection (mean 14.81) (p < 0.0001). No significant difference between colon resection and local resection was found (p = 0.29). Both a stoma and the use of RT were independent risk factors for ED. After excluding patients with stoma and RT and adjusting for age and ASA score, RR still had a higher risk of ED (OR 1.42, CI 1.20–1.67) compared with colon resection. Conclusion RR has a negative affect on EF. No difference between patients who underwent colon resection and local resection was found. RT and stoma were independent risk factors for ED.