Upregulation of miR-3195, miR-3687 and miR-4417 is associated with castration-resistant prostate cancer.

PURPOSE: Prostate cancer (PCa) is a leading cause of cancer-related death. Upon androgen-deprivation therapy, the disease may progress further to castration-resistant PCa (CRPC) with a poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs, which play crucial roles in gene regulation. The aim of our study is to find CRPC-associated miRNAs and to evaluate their functional role. METHODS: In this study, 23 benign prostatic hyperplasia (BPH), 76 primary PCa, and 35 CRPC specimens were included. Total RNA extracted from tissue sections was used for miRNA profiling on the Affymetrix GSC 3000 platform. Subsequently, stem-loop RT-qPCR analysis was performed to validate the expression levels of selected miRNAs. PCa cell lines were transfected with miRNA mimics or inhibitors to evaluate the effects on cell proliferation, cell migration and cell invasion. RESULTS: In our profiling study, several miRNAs were found to be deregulated in CRPC compared to primary PCa tissue, of which miR-205 (- 4.5-fold; p = 0.0009), miR-92b (- 3.1 fold; p < 0.0001) were downregulated and miR-3195 (5.6-fold; p < 0.0001), miR-3687 (8.7-fold; p = 0.0006) and miR-4417 (5.0-fold; p = 0.0005) were most upregulated. While KLK3, miR-21 and miR-141 expression levels in androgen-treated VCaP and LNCaP cells were increased, the expression levels of miR-3687 and miR-4417 were reduced. None of the miRNAs were androgen-regulated in the AR-negative PC3 cell line. Overexpression of miR-3687 reduced cell migration and cell invasion, whilst miR-3195 enhanced cell migration. CONCLUSION: We have identified several novel deregulated miRNAs in CRPC tissue, including two microRNAs that are potentially involved in tumor invasion. Our data support the hypothesized involvement of miRNAs in PCa tumorigenesis and progression to CRPC. The applicability of these miRNAs as novel biomarkers for CRPC remains to be further investigated.

Expression of the cellular adhesion molecule E-cadherin is reduced or absent in high-grade prostate cancer.

E-cadherin is a Ca(2+)-dependent cell adhesion molecule which plays an important role in normal growth and development via mediation of homotypic, homophilic cell-cell interaction. Recent studies suggest that E-cadherin may be important in neoplastic progression as well, particularly as a suppressor of invasion. We have previously demonstrated that the invasive phenotype of rat prostate cancer cells is associated with the decreased expression of E-cadherin (M. J. G. Bussemakers, R. J. A. Van Moorselaar, L. A. Giroldi, T. Ichikawa, J. T. Isaacs, F. M. J. Debruyne, and J. A. Schalken, Cancer Res., 52:2916-2922, 1992). This is of particular interest, since the locus to which the human E-cadherin gene is mapped is frequently involved in allelic loss in prostate cancer (B. S. Carter, C. M. Ewing, W. S. Ward, B. F. Treiger, T. W. Aalders, J. A. Schalken, J. I. Epstein, and W. B. Isaacs, Proc. Natl. Acad. Sci. USA, 87:8751-8755, 1990; U. S. Bergerheim, K. Kunimi, V. P. Collins, and P. Ekman, Genes, Chromosomes Cancer, 3: 215-220, 1991). Impaired E-cadherin function is likely to be associated with aberrant expression of the protein. We therefore analyzed E-cadherin expression in situ by immunohistochemistry in nonmalignant and malignant specimens of human prostatic tissue. Of 92 tumor samples of either primary or metastatic deposits of prostate cancer, 46 had reduced or absent E-cadherin staining when compared to nomalignant prostate, which uniformly stained strongly positive. There was a statistically significant correlation between the decreased expression of E-cadherin and loss of tumor differentiation. Additionally, certain tumors within a histologically similar group could be distinguished by the presence of mixed populations of E-cadherin-negative and -positive cells. The percentage of tumors with aberrant E-cadherin staining increased when clinically localized tumors were compared to either tumors with extensive local progression or metastatic deposits of prostate cancer, suggesting a correlation between loss of E-cadherin and tumor progression. Taken together, these findings suggest that further exploration of E-cadherin as a candidate invasion suppressor molecule in human prostate cancer is warranted.

Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer.

In the epithelium of secretory acini of the prostate two different cell types can be discriminated on the basis of localization, morphology, and degree of differentiation, the luminal and basal cells. The possibility of a developmental relationship between basal and luminal cells has been a subject of interest in several studies. According to the stem cell model at least three cell types, i.e., stem, amplifying, and transit cells, can be discriminated in the epithelium of prostate secretory acini. We previously reported that in the process of degeneration and regeneration in normal rat prostate a population of cells could be identified as candidates for the amplifying cells. These cells showed a keratin expression profile intermediate between those of basal and luminal cells. We now show, by using keratin antibodies, that also in normal human prostate at least three subpopulations of cells can be identified, one of them putatively representing amplifying cells as defined in the stem cell model. Furthermore, these antibodies were used to obtain a better insight into the different cell types involved in the etiology and progression of prostatic carcinoma. Both primary and hormone-independent prostatic tumors were investigated. Our results indicated that the candidate stem cell population was absent in prostatic carcinoma. Unlike earlier reports on the unique presence of cells with luminal characteristics in prostatic carcinoma, we identified also a population of cells coexpressing basal and luminal cell-type cytokeratins in primary and hormone-independent prostatic carcinoma. Since amplifying cells are defined in the stem cell model as precursors of transit (luminal) cells in the hierarchical pathway of prostatic epithelium differentiation, we postulate that on the basis of the keratin expression profile this subpopulation is most likely the target for neoplastic transformation.

Tenascin-C expression in prostatic intraepithelial neoplasia (PIN): a marker of progression?

BACKGROUND: Tenascin (tenascin-C) has been suggested to be associated with active epithelial-stromal interactions. We evaluated tenascin expression in tissue remodelling processes presumably associated with PIN and prostate carcinoma (PCa). MATERIALS AND METHODS: Tenascin immunoreactivity was evaluated in 38 PIN lesions (low-grade = 5, high-grade = 33) from 27 paraffin-embedded PCa specimens, and compared with expression in pre-existent (normal) prostate, benign prostatic hyperplasia (BPH), and PCa. RESULTS: Periepithelial stromal tenascin expression was low in low-grade PIN, and similar to normal glands and BPH, whereas expression in high-grade PIN was high and partly overlapped that of well-/moderately differentiated PCa. High-grade PCa usually expressed little, if any tenascin. CONCLUSIONS: The variable periglandular tenascin expression in high-grade PIN may reflect the biologic behaviour of this lesion, and may be indicative of variable levels of tissue remodelling. In well/moderately differentiated PCa tenascin expression levels may be an indicator of tumour progression.

Differential expression of keratins in the basal and luminal compartments of rat prostatic epithelium during degeneration and regeneration.

The role of the different epithelial compartments during degeneration and regeneration of the rat prostate is examined on basis of intermediate filament protein (IFP) expression pattern. With the monoclonal antibodies RCK 103 and RGE 53, directed against specific keratins, it was possible to differentiate between the basal (RCK 103+) and luminal (RGE 53+) cells of the prostatic epithelium. After testosterone deprivation, by orchiectomy, an extensive and rapid cell loss was observed which appeared to affect mainly the luminal cells. In the process of prostate regeneration, induced by testosterone administration, using silastic implants, the luminal compartment rapidly regained its normal thickness. A heterogeneous population of morphologically luminal cells was observed showing keratin expression patterns intermediate between basal and luminal cells. These findings support the idea of a relationship between basal and luminal cells as being members of the same lineage of differentiation.

Karyometric analysis of intra-tumour heterogeneity in prostate adenocarcinoma.

The intra-tumour heterogeneity in different locations in the prostate was determined by karyometric image analysis and compared with local tumour progression in a retrospective analysis of 65 patients with localized adenocarcinoma of the prostate. In these 65 radical prostatectomy specimens 290 tumour locations were documented. In each location tumour volume was estimated and Gleason grade determined. Quantitative image analysis of nuclear size, shape and chromatin pattern was performed. Differences in Gleason grade and karyometric feature values were evaluated as measures for intra-tumour heterogeneity. Moreover, the location within the prostate was documented for each tumour area. Gleason grade and karyometric features varied widely in the prostate. Tumour locations in the apex (n = 92) had significantly larger and more irregularly shaped nuclei (P < 0.005) compared with basally located tumours (n = 49). Significant differences in nuclear shape were also found in different locations in the equatorial plane of the organ. Tumour heterogeneity in chromatin pattern features was found to correlate with local extension (seminal vesical invasion, extracapsular tumour growth, positive resection margins) and lymph node metastases. This correlation was even stronger when more pathological features were present. In view of the observed tumour heterogeneity, (karyometric) analysis of multiple tumour areas in the prostate is advisable for optimal evaluation of prostate carcinoma.

Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas.

Telomerase reverse transcriptase (hTERT) messenger RNA has been detected in 95% of bladder tumors using RT-PCR. In this study, we quantified the expression of hTERT in 35 bladder urothelial cell carcinomas and in 6 normal bladder epithelia using a real-time quantitative PCR assay. hTERT expression was detected in all 35 urothelial cell carcinomas of varying grade and stage, but not in normal tissue samples. An increase in both pathological grade and clinical stage as prognostic parameters correlated with increased hTERT expression. Using different cutoff values for grades and stages, normalized hTERT expression values could discriminate among low, medium, and high grade tumors and between superficial and muscle-invasive tumors. We conclude that standardized real-time measurement of hTERT expression can be used for early tumor detection and may be used for determination of prognosis in urothelial cell carcinomas of the bladder.

Changes in keratin expression during the development of benign prostatic hyperplasia.

OBJECTIVE: The relationship between different types of epithelial cells in the prostate and the regulatory mechanism underlying benign prostatic hyperplasia (BPH) are still obscure as is the association between BPH and prostate carcinoma (PCa.) On the basis of keratin immunophenotyping, a subpopulation of cells in normal rat prostate and human PCa have been identified as candidates for the 'amplifying cell' in the stem cell model. In this model the basal cell is described as being associated with the stem cell. From this precursor an intermediate cell type develops which may differentiate into the luminal-type cell. In this study these different cell types are investigated in the development of isolated BPH and BPH associated with PCa, using monoclonal antibodies to intermediate filaments of the keratin class. METHODS: We immunohistochemically stained 64 snap-frozen human prostatic tissues, using monoclonal antibodies against keratin 14 (marker for putative 'stem cell'), keratin 18 (marker for putative 'transit cell'), and MAb RCK103 (marker for putative 'amplifying cell' or intermediate cell). RESULTS: In basal cell hyperplasia, an atypical form of BPH, keratins previously associated with intermediate cells were frequently detected. Cells with this keratin phenotype were detected in the luminal compartment of BPH, and were more prevalent in BPH adjacent to PCa. This keratin expression pattern was similar to that of PCa. CONCLUSION: On the basis of keratin phenotyping we demonstrated that large numbers of cells with the keratin expression pattern of so-called intermediate cells were identified in BPH associated with PCa, while in isolated BPH these cells were infrequently found. This supports the concept that BPH with intermediate phenotype may have premalignant potential. Furthermore this is suggestive of an etiologic relationship between the two diseases.

Expression of periglandular tenascin-C and basement membrane laminin in normal prostate, benign prostatic hyperplasia and prostate carcinoma.

OBJECTIVE: To evaluate the structural relationship of the distribution between tenascin (tenascin-C, an extra-cellular matrix glycoprotein involved in stromal-epithelial interactions in both normal and pathological conditions) and laminin, an important component of the basement membrane, in normal and neoplastic human prostate, and to establish whether changes in the basement membrane are accompanied by changes in tenascin staining. MATERIALS AND METHODS: Seventy-five snap-frozen prostate samples representing normal glands, nodular benign prostatic hyperplasia and prostate carcinoma were stained for tenascin. From these, 15 samples were selected for dual-immunofluorescence staining and a confocal laser scan microscope was used to simultaneously visualize tenascin and laminin immunoreactivity. RESULTS: Tenascin was expressed in the extracellular matrix, mainly at the periphery of the glands, in tumour foci and blood vessels. In cases with intact basement membranes, e.g. normal glands and hyperplastic lesions, tenascin expression was weak. Low- and moderate-grade tumours were characterized by strong tenascin expression, while laminin expression was weak and/or showed discontinuities, indicating disturbances in basement membrane composition. High-grade tumours had sparse tenascin staining and a marked loss of laminin immunoreactivity. CONCLUSION: These results indicate that periglandular tenascin expression correlates with the integrity of the basement membrane in the human prostate. By influencing stromal-epithelial interactions, tenascin may play a role in maintaining tissue homeostasis in the prostate.

Allelic loss of chromosomes 16q and 10q in human prostate cancer.

Recent advances in understanding the molecular genetics of common adult tumors have indicated that multiple genetic alterations including the activation of oncogenes and the inactivation of tumor suppressor genes are important in the pathogenesis of these tumors. Loss of heterozygosity is a hallmark of tumor suppressor gene inactivation and has been used to identify chromosomal regions that contain these genes. We have examined allelic loss in the most common tumor in men, prostate cancer. Twenty-eight prostate cancer specimens have been examined for loss of heterozygosity at 11 different chromosomal arms including 3p, 7q, 9q, 10p, 10q, 11p, 13q, 16p, 16q, 17p, and 18q. Fifty-four percent (13/24) of clinically localized tumors and 4 of 4 metastatic tumors showed loss of heterozygosity on at least one chromosome. Chromosomes 16q and 10q exhibited the highest frequency of loss of heterozygosity with 30% of tumors showing loss at these chromosomes. These data demonstrate that allelic loss is a common event in prostate cancer and suggest that chromosomes 16q and 10q may contain the sites of tumor suppressor genes important in the pathogenesis of human prostate cancer.

Rapid microwave-stimulated fixation of entire prostatectomy specimens. Biomed-II MPC Study Group.

Conventional fixation of large solid surgical specimens is a slow process. Consequently, autolytic damage to tissues may occur if the fixative does not reach the central part of the specimen in time. However, as there is also a time relationship between formalin fixation and antigen masking, fixation for too long can also be detrimental. In seeking the optimum balance for fixation, microwave irradiation might be of assistance. This study set out to evaluate methods for fixing entire prostate glands within a brief period of time, using microwave-stimulated formalin fixation. The results show that entire prostates can be optimally fixed if formalin is present throughout the tissue as the temperature is increased by microwave irradiation. This is achieved by injecting the fixative into the prostate at multiple sites immediately following prostatectomy. The technique described ensures standardization of a critical step during tissue processing, leading to uniform microscopic results with both routine and immunohistochemical stains. It is a simple, rapid method, suitable for routine diagnostic use. Using this modified approach, DNA of much larger sizes can be extracted from paraffin-embedded material, which could expand the possibilities for molecular analysis.

Expression of basal cell keratins in human prostate cancer metastases and cell lines.

Within normal human prostate epithelium, basal and luminal cells can be discriminated by their expression of keratins (K). While basal cells express K5/14, luminal cells show expression of K8/18 and an intermediate cell population can be identified by co-expression of K5/18. Prostate cancer is predominantly composed of luminal and neuroendocrine cells, while a minority of cells have a basal phenotype. In order to distinguish between basal and intermediate cells, and to assess the effects of androgen deprivation on prostate cancer, 56 human prostate cancer metastases and three cancer cell lines were characterized using antibodies to K5, K14, K18, and the neuroendocrine marker chromogranin A (ChA). The staining was performed on paraffin tissue and visualized by the avidin-biotin-peroxidase complex method. Protein expression was quantified as the number of positive cells in 20 high power fields (HPF; 400x). Keratin expression in the prostate cancer cell lines LNCaP, DU145, and PC3 was analysed by immunofluorescence with triple staining and confocal laser scanning microscopy. Prostate cancer metastases were consistently positive for K18 and negative for K14, irrespective of hormonal therapy. K5 expression was displayed in 28.9% of the tumours without treatment, in 75% after androgen deprivation, and in 57.1% of hormone-escaped prostate carcinomas. After androgen deprivation, the number of K5-expressing cells increased significantly. While androgen-dependent prostate cancer showed a median of 0 cells/20 HPF (range 0-50), regressed tumours displayed 22.5 (range 0-65) and hormone-escaped tumours 7.5 (range 0-361) positive cells/20 HPF. Expression of ChA was observed in 47.4% of the androgen-dependent tumours. The number of neuroendocrine cells was not significantly affected in regressed or hormone-escaped disease. The androgen-dependent cell line LNCaP stained for K18, while the androgen-independent lines DU145 and PC3 both expressed K5 and 18. Expression of K5 in the absence of K14 identifies the existence of an intermediate cell population in prostate carcinoma. Accumulation of intermediate cells in regressed and hormone-escaped prostate cancer indicates that for their survival, these cells are androgen-independent.