The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment. In this study, we used cultured cells derived from prostate tissue from patients with PCa to define a molecular mechanism underlying the most aggressive form of PCa that involves the functional activation of eNOS and HIFs in association with estrogen receptor β (ERβ). Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. Upon estrogen treatment, formation of ERβ/eNOS, ERβ/HIF-1α, or ERβ/HIF-2α combinatorial complexes led to chromatin remodeling and transcriptional induction of prognostic genes. Tissue microarray analysis, using an independent cohort of patients, established a hierarchical predictive power for these proteins, with expression of eNOS plus ERβ and nuclear eNOS plus HIF-2α being the most relevant indicators of adverse clinical outcome. Genetic or pharmacologic modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with bad or good outcome, respectively, highlighting the relevance of eNOS in PCa progression. Our work has considerable clinical relevance, since it may enable the earlier diagnosis of aggressive PCa through routine biopsy assessment of eNOS, ERβ, and HIF-2α expression. Furthermore, proposing eNOS as a therapeutic target fosters innovative therapies for PCa with NO inhibitors, which are employed in preclinical trials in non-oncological diseases. IntroductionIn the clinical management of prostate cancer (PCa), the second most common neoplasia in men worldwide (1), the ability to distinguish between aggressive and indolent forms of the disease is critical. Thus, therapeutic approaches would be substantially improved by the identification of the molecular mechanisms involved in tumor progression and the key biomarkers capable of improving patients' stratification at diagnosis, by discriminating between those at risk for relapse and those with indolent tumors not requiring further intervention beyond surgery.

Flat adenomas are flat or slightly elevated dysplastic lesions of the colorectal mucosa, mostly with a tubular architecture. Compared with polypoid adenomas of similar size, flat adenomas show a higher frequency of high-grade dysplasia and rapid submucosal invasion. The aim of this study was to survey whether flat colorectal lesions differ in their pattern of chromosomal aberrations from their polypoid counterparts. Six flat adenomas and 12 flat carcinomas were analysed by comparative genomic hybridization (CGH) and the pattern of chromosomal aberrations was compared with a previously published series of 112 polypoid adenomas and 82 polypoid carcinomas. In addition, multiplex ligation-dependent probe amplification (MLPA) for identifying DNA copy number changes of 25 individual genes on chromosome 20 was performed on 14 flat and 15 polypoid tumours. With CGH, flat adenomas showed on average 1.8 gains (range 1-4) and 3.2 losses (range 0-4), and the flat carcinomas 4.5 gains (range 0-8) and 3.5 losses (range 1-6). In both adenomas and carcinomas, high frequencies of 20q gain (83% and 92%, respectively) and 18q loss (83% and 92%, respectively) were found. This correlation between 20q gain and 18q loss had previously been observed in a subgroup of polypoid colorectal tumours. Both flat and polypoid colorectal tumours with 20q gains by CGH showed similar patterns of copy number ratios for the individual genes tested. TOP1, BCL2L1, and E2F1 had median copy number ratios of 2 or higher, while ZNF217 had a ratio around 3. In conclusion, flat adenomas and carcinomas of the large intestine show a similar pattern of chromosomal aberrations to that observed in a specific subgroup of polypoid lesions. The transcription factor ZNF217 is an important candidate for driving the 20q gain.

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Small adenocarcinomas of the colorectum showing no evidence of origin from an adenoma have been called de novo carcinomas, a name that implies an origin via a different molecular genetic mechanism than the usual colorectal carcinoma which develops from an adenoma. Using microsatellite analysis, 35 early (pT1) de novo and 36 pT1 ex-adenoma carcinomas were compared using 8 microsatellite loci at 6 different chromosomal loci (1p, 2p, 8p, 5q, 17p, and 18q) known or hypothesized to be important for colorectal carcinogenesis. The rate of loss of heterozygosity (LOH) at the 17p locus (near the p53 gene) was significantly higher in the de novo than in the ex-adenoma group (73 vs. 37%, P = 0.004). The rates of LOH at the other loci (including the APC and DCC genes) and the rate of MSI were not significantly different in the two groups. These results indicate that de novo carcinomas of the colorectum develop via a similar carcinogenetic pathway as conventional ex-adenoma carcinomas; however, their higher rate of LOH at 17p is evidence for a biologically more advanced lesion with more frequent p53 mutations, consistent with clinicopathological data indicating that de novo carcinomas are more aggressive than ex-adenoma carcinomas.

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Pseudoinvasion in colorectal adenomas is often difficult to distinguish from invasive carcinoma. Previous studies have indicated that expression of stromelysin-3 (ST-3), one of the metalloproteinase family of enzymes, may be useful for the identification of early invasive carcinoma. The goal of our study was to detect ST-3 expression in colorectal adenomatous polyps to see if it could be helpful for the differential diagnosis of pseudoinvasion vs. true invasion. We studied 25 polypectomy specimens which were divided histologically into 2 groups; the first consisted of 15 adenomas with invasive carcinoma, 8 of these carcinomas were more diffusely infiltrative (pT1), and 7 tended to be expansively invasive. The second group was composed of 10 adenomas with pseudoinvasion. A 35S labelled cDNA probe was used for in situ hybridization (ISH) and a monoclonal antibody (5ST-4A9) for immunohistochemistry (IHC). The distribution of ST-3 expression as detected by IHC and ISH was identical. All diffusely infiltrative carcinoma cases showed ST-3 expression, but only focally in 2 cases with marked lymphocytic infiltration. None of the expansive carcinoma or pseudoinvasion cases showed ST-3 expression. ST-3 expression seems to be an indicator of invasion, but a negative reaction for ST-3 does not rule out an expansive invasive neoplasm or a diffusely infiltrative invasive tumour with a dense lymphocytic reaction.

An apparent exception to the colorectal adenoma-carcinoma carcinogenetic pathway is the so-called "de novo" carcinoma which has no evidence of adenoma in its vicinity. Despite the fact that they are often quite small, these lesions appear to be more aggressive (i.e., greater likelihood of lymph-node metastases) than carcinomas that clearly arise from surrounding adenomas exadenoma carcinoma. The purpose of the present comparative immunohistochemical study was to compare rates of cell adhesion molecule (E-cadherin) and protease [stromelysin-3 (ST-3)] expression in groups of de novo (n=64) and ex adenoma (n=42) lesions in order to see whether their more aggressive behavior is associated with decreased cell adhesion and increased protease expression. The rates of extensive ST-3 expression and decreased E-cadherin expression were significantly higher in the de novo group (P=0.014 and 0.005, respectively). Histopathologically, the de novo group also had a significantly higher percentage of cases with an infiltrative invasion pattern. These differences highlight the more aggressive phenotype of the de novo colorectal carcinoma and fit with their greater invasive potential.