Gerhard Unteregger

The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

Antimicrobial peptides of the Cecropin-family show potent antitumor activity against bladder cancer cells.

BackgroundThis study evaluated the cytotoxic and antiproliferative efficacy of two well-characterized members of the Cecropin-family of antimicrobial peptides against bladder tumor cells and benign fibroblasts.MethodsThe antiproliferative and cytotoxic potential of the Cecropins A and B was quantified by colorimetric WST-1-, BrdU- and LDH-assays in four bladder cancer cell lines as well as in murine and human fibroblast cell lines. IC50 values were assessed by logarithmic extrapolation, representing the concentration at which cell viability was reduced by 50%. Scanning electron microscopy (SEM) was performed to visualize the morphological changes induced by Cecropin A and B in bladder tumor cells and fibroblasts.ResultsCecropin A and B inhibit bladder cancer cell proliferation and viability in a dose-dependent fashion. The average IC50 values of Cecropin A and B against all bladder cancer cell lines ranged between 73.29 μg/ml and 220.05 μg/ml. In contrast, benign fibroblasts were significantly less or not at all susceptible to Cecropin A and B. Both Cecropins induced an increase in LDH release from bladder tumor cells whereas benign fibroblasts were not affected. SEM demonstrated lethal membrane disruption in bladder cancer cells as opposed to fibroblasts.ConclusionCecropin A and B exert selective cytotoxic and antiproliferative efficacy in bladder cancer cells while sparing targets of benign murine or human fibroblast origin. Both peptides may offer novel therapeutic strategies for the treatment of bladder cancer with limited cytotoxic effects on benign cells.

Genomic and Expression Analysis of the 3q25-q26 Amplification Unit Reveals TLOC1/SEC62 as a Probable Target Gene in Prostate Cancer

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined. (Mol Cancer Res 2006;4(3):169–76)

Silencing of the SEC62 gene inhibits migratory and invasive potential of various tumor cells

Sec62 is part of the protein translocation apparatus in the membrane of the endoplasmic reticulum (ER). In yeast, Sec62 participates in the post‐translational translocation of proteins into the ER, but its function in mammals remains elusive. Previously we described the amplification and over‐expression of the SEC62 gene in prostate cancer cell lines and the protein has been described as a potential target gene in prostate cancer. In the current study we show that in the tumor tissue of prostate cancer patients Sec62 protein levels are elevated compared with tumor‐free tissue derived from the same patients or from prostates of control group patients and that the higher Sec62 protein content correlates with an increasing de‐differentiation of the cells. Therefore, up‐regulation of Sec62 protein content indeed is a phenomenon associated with prostate cancer progression. Analysis of a multi‐tissue tumor array showed that in addition to prostate cancer, overproduction of Sec62 is observed in various other tumors, most significantly in tumors of the lung and the thyroid. To examine the tumor‐related functions of Sec62, we silenced the SEC62 gene in the prostate cancer cell‐line PC3 as well as in a set of other tumor cell‐lines with two different siRNAs. In general, after silencing of SEC62 the cell migration and the invasive potential of the cells was blocked or at least dramatically reduced while cell viability was hardly affected. Thus, the SEC62 gene may indeed be considered as a target gene in the therapy of various tumors.

A new serial transfer explant cell culture system for human prostatic cancer tissues preventing selection toward diploid cells

An improved explant cell culture technique to avoid selection of prostatic adenocarcinoma cells toward diploid cells is described. This method is based on 1) histologically characterized tissue explants, 2) the use of polyethylenteraphthalate (PET) membranes as growth surface, which are part of special inserts in six-well-plates to allow 3) cocultivation with heterologous fibroblasts, and 4) coating of the membranes with elements of the extracellular matrix. The main characteristic of this particular approach is the serial transfer of the tissue explant from one membrane to the other. Up to ten serial transfer steps could be performed to produce cell monolayers growing out of the same tissue specimen. Using this approach, 21 prostatic carcinoma specimens that were obtained from 13 primary prostatic adenocarcinomas after radical prostatectomy were cultivated. Ploidy of the cells was monitored by fluorescence in situ DNA hybridization using the centromere specific DNA probes pUC1.77, p alpha 7t1, and pY3.4. Interestingly, a high aneuploidy rate of the cell cultures was found with maintainance of aneuploidy in 18 (86%) of the 21 paraffin-embedded cancer tissue specimens with proved aneuploidy. Although a slight decrease of the proportion of aneuploid cells during serial transfer was observed, significant aneuploid cell populations were retained up to a maximum of ten transfer steps. These findings indicate that selection toward diploid cells can be prevented by improved cell culture techniques that mimic the in vivo situation.

Sec62 protein level is crucial for the ER stress tolerance of prostate cancer

We previously reported that over‐expression of the SEC62 gene is a widespread phenomenon in prostate cancer. Since the use of endoplasmic reticulum (ER) stress‐inducing substances such as thapsigargin in prostate cancer therapy is widely discussed in the literature, we investigated the influence of Sec62 protein content on the cellular response to these drugs.

Selection toward diploid cells in prostatic carcinoma derived cell cultures

For elucidation of the growth‐regulatory mechanisms in prostatic carcinoma, in vitro investigations on prostatic cell cultures are required. However, one major problem of cell culturing is the selection of particular cell types such that the cell lines representing only some of the features as compared with the tumor of origin. We studied the chromosomal composition of 20 prostatic tissue‐derived cell cultures and 12 original (fresh) tissue specimens that were obtained from 13 patients with prostatic adenocarcinoma. Using fluorescence in situ DNA hybridization (FISH), evident clonal abnormalities were detected in 78% of the fresh cancer samples and in 47% of the cultured cancer samples. Of the seven cases revealing clonal abnormalities in the fresh cancer specimen, aneuploidy was detected in only two samples after cell culturing at the earliest passage studied. The aneuploid cell populations in the cultured samples were all lost during progressive subcultivation (after passage 4). Interestingly, by performing FISH on cytogenetic preparations aneuploidy was confined to the interphases, with the metaphases being found to be diploid. This finding indicates that the aneuploid cells have a proliferation disadvantage in cell culture resulting in an overgrowth of diploid cells.

Experimental orthotopic prostate tumor in nude mice: Techniques for local cell inoculation and three-dimensional ultrasound monitoring

OBJECTIVES Orthotopic prostate cancer models are of great importance for cancer research. Orthotopic models in mice have been described previously. However, these studies lack a detailed methodological description and fail to define standards for local cell inoculation. Herein, we studied the effect of different protocols on tumor growth and report for the first time the use of high resolution ultrasound for monitoring of tumor growth. MATERIALS AND METHODS Orthotopic inoculation of DU 145 MN1 prostate cancer cells was performed in 30 nude mice varying (1) the amount of cells (5 × 10(5) vs. 5 × 10(4)), (2) the number of puncture sites, and (3) the addition of matrigel. Surgical complications such as recoil of cells through the injection canal and rupture of the prostatic capsule were monitored. Animals were tracked by ultrasound imaging after 4, 5, and 6 weeks. Autopsy and histology confirmed local tumor growth. RESULTS A take rate of 27/30 (90%) was observed. Growth of orthotopic prostate tumors was increased after inoculation of a large amount of cells under the capsule of 1 dorsal prostate lobe, but inoculation of small amounts of cells still induced local tumors. Noninvasive ultrasound examination allowed to identify orthotopic tumor formation and to monitor tumor growth in vivo. Addition of matrigel did not accelerate tumor growth. Complications like recoil (6.8%) or rupture of the prostate capsule (1.4%) were rare. CONCLUSIONS Inoculation of DU 145 MN1 cells under the prostate capsule with a defined procedure results in very high take rates. Ultrasound screening is feasible to repetitively monitor tumor growth.

Two different forms of p53 localized differently within cells of urogenital tumours

We analyzed the subcellular localization of p53 in prostate and bladder carcinoma cells. Using laser scanning microscopy and PAb1620, a monoclonal antibody recognizing the wildtype conformation of p53, and another monoclonal antibody directed against the mutant conformation of the protein (PAb240), we found two different subsets of p53 within the same cell. The wildtype subgroup was found in the nucleolus, whereas the mutant protein was confined to the nucleus. The results obtained by immunofluorescence were verified by Western blot analysis and immunoprecipitation. Thus, our findings demonstrate an unusual subcellular localization pattern of p53 in prostate and bladder cancer cells which may indicate another mechanism of inactivation of p53.

Orthotopic tumorgrafts in nude mice: A new method to study human prostate cancer.

In vivo model systems in prostate cancer research that authentically reproduce tumor growth are still sparse. While orthotopic implantation is technically difficult, particularly in the mouse, most models favor subcutaneous tumor growth. This however provides little information about natural tumor growth behavior and tumor stroma interaction. Furthermore, established prostate cancer cell lines grown as in vivo xenografts are not able to reflect the variety of tumor specific growth patterns and growth behavior in men. Primary cell cultures are difficult to handle and an induction of orthotopic tumors has not been successful yet. Therefore, a tumorgraft model using tumor tissue from prostatectomy specimens was developed.