Differential Redox Regulation of Ca²âº Signaling and Viability in Normal and Malignant Prostate Cells.

In prostate cancer, reactive oxygen species (ROS) are elevated and Ca(2+) signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca(2+) signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca(2+) and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H2O2 induces an initial Ca(2+) increase, which in prostate cancer cells is blocked at high concentrations of H2O2. Upon depletion of intracellular Ca(2+) stores, store-operated Ca(2+) entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H2O2 compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector TH cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca(2+) channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca(2+) signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.

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

BACKGROUND: 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. METHODS: Balb/c nude mice were used to graft fresh prostate tumor tissue by renal subcapsular and orthotopic implantation. Testosterone propionate was supplemented. Animals were tracked by means of 30 MHz ultrasound to monitor tumor engraftment and growth. Autopsy, histology, PSA measurements as well as immunostaining and PCR for human tissue were performed to confirm orthotopic tumor growth. RESULTS: Renal subcapsular engraftment was seen in 2 of 3 mice. Orthotopic engraftment was observed in 7 of 11 animals (63.6%) with an overall engraftment of 5 out of 9 patient specimens (55.6%). Ultrasound confirmed the tumor growth over time. Of interest, the tumorgrafts not only retained essential features of the parental tumors, but also stained positive for tumor specific markers such as AR, PSA, and AMACR. Tumor positive animals showed highly elevated serum PSA levels with confirmation of a human specific PCR sequence and a human endothelial cell lining in the tumor vessels. CONCLUSIONS: Standardized implantation of fresh tumor tissue in nude mice prostates generates tumorgrafts with histological properties of organ-confined prostate cancer. These tumorgrafts display a new approach for an optimized in vivo model of prostate cancer and will allow further investigations on specific pathways of tumor initiation and progression as well as therapeutic response.

High-resolution ultrasound allows percutaneous initiation and surveillance of prostate cancer in an orthotopic murine model.

INTRODUCTION: Prostate cancer xenografts should prefer orthotopic growth to subcutaneous tumors as the former more closely mimics the natural tumor environment. However, these models are technically demanding and require an invasive laparotomy. To overcome these problems, we evaluated a minimally invasive approach by performing percutaneous prostate puncture under the control of high-resolution ultrasound imaging. MATERIALS AND METHODS: Orthotopic tumor cell inoculation was performed in two groups of mice, i.e. in 10 nude mice via ultrasound-guided inoculation and in another 10 nude mice via an open surgical approach. Tumor growth was monitored after 4, 5 and 6 weeks by means of a high-resolution ultrasound system. RESULTS: High-resolution ultrasound allowed exact tumor growth monitoring. After ultrasound-guided inoculation, 8 of 10 animals showed tumor engraftment. The surgical procedure was successful in 9 of 10 animals. Tumor volume was slightly but not significantly greater after surgical tumor induction. Our work demonstrates that tumor cell inoculation via percutaneous puncture of the prostate is feasible, less time-consuming and minimally invasive compared to an open surgical approach. This reduces the animal burden. CONCLUSION: Although the tumor size and the precision of inoculation is lower compared to the open surgical technique, this novel procedure enables real-time prostate punctures, suggesting the feasibility of other procedures including biopsy and local drug applications.

Sec62 bridges the gap from 3q amplification to molecular cell biology in non-small cell lung cancer.

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

Molecular Characterization of Cancer Associated Fibroblasts in Prostate Cancer.

BACKGROUND: Stromal components surrounding epithelial cancer cells seem to play a pivotal role during epithelial-to-mesenchymal transition (EMT), tumor invasion, and metastases. To identify the molecular mechanisms underlying tumor-stroma interactions may yield novel therapeutic targets for prostate cancer. METHODS: Gene expression profile of prostate-cancer associated fibroblast (PCAF) and prostate non-cancer associated fibroblast (PNAF) cells isolated from radical prostatectomy was performed by Illumina, analyzed, and further processed by Ingenuity®: IPA® software. qRT-PCR was performed on an independent set of 17 PCAF, 12 PNAF, and 12 fibroblast cell lines derived from patients with benign prostatic hyperplasia (BPHF). RESULTS: Using microarray analysis, we found six upregulated genes and two downregulated genes in PCAFs compared to PNAFs. To validate microarray results, we performed qRT-PCR for the most significantly regulated genes involved in the modulation of proliferation and androgen resistance on an independent set of PNAF, PCAF, and BHPF samples. We confirmed the increased expression of SCARB1, MAPK3K1, and TGF-ß as well as the decreased expression of S100A10 in PCAFs compared to PNAFs and BPHFs. CONCLUSIONS: These results provide strong evidence that the observed changes in the gene expression profile of PCAFs can contribute to functional alteration of adjacent prostate cancer cells.

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.

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

BACKGROUND: 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. METHODS: Growth effects were analyzed by real-time cell analysis and viability tests in DU145-cells representing an increased SEC62 expression or PC3- and LNCaP-cells representing a similar SEC62 expression compared to non-tumor cells. Ca(2+) -imaging in an established HeLa-system with fluorescent dye was used to study molecular effects of Sec62 depletion. RESULTS: We found a lower propensity toward apoptotic cell death after thapsigargin treatment for DU145 cells compared to PC3 or LNCaP and siRNA-mediated silencing of SEC62 resulted in a reduced viability of thapsigargin-treated PC3 cells, indicating that Sec62 functions in cellular stress response. Measurement of cytosolic [Ca(2+) ] demonstrated the influence of Sec62 on the cellular response to thapsigargin on a molecular level. Using real-time cell analysis, we observed the loss of androgen stimulation of LNCaP cells in the presence of thapsigargin, and an additional negative effect on cell growth of Sec62 depletion. Also, for PC3- and DU145-cells Sec62 depletion inhibited growth after thapsigargin treatment. CONCLUSIONS: Our data indicate a crucial function of Sec62 in the response to thapsigargin-induced ER stress. This will be of great significance on the background of elevated Sec62 protein levels in prostate cancer cells when treatment with thapsigargin analogs is considered.

The LARK/RBM4a protein is highly expressed in cerebellum as compared to cerebrum.

The RNA binding motif protein 4 genes RBM4a and RBM4b are located on human chromosome 11q13.2 and encode highly similar proteins of 363 and 359 amino acids, respectively. They contain two RNA recognition motifs (RRMs) and a retroviral-type Zn-finger. RBM4a binds RNA, is involved in alternative splicing and is also a part of the microRNA-processing RISC complex. In particular, RBM4a is involved in exon 10 inclusion of the tau protein. The function of RBM4b is unknown. With new monoclonal antibodies we show that RBM4a is detectable in virtually all tissues and cell lines tested while RBM4b was only found in kidney and liver. Both RBM4a and RBM4b are nuclear phosphoproteins with half-lives of 2.5h and 4.5h, respectively. To our knowledge, this is the first description of RBM4b protein in human tissue. In human brain, expression of RBM4a was strongly up-regulated in cerebellum as compared to forebrain.

Protein phosphatase and TRAIL receptor genes as new candidate tumor genes on chromosome 8p in prostate cancer.

BACKGROUND: Allelic losses on chromosome 8p are common in prostate carcinoma, but it is not known exactly how they contribute to cancer development and progression. MATERIALS AND METHODS: Expression of 12 genes located across chromosome 8p, including established tumor suppressor candidates (CSMD1, DLC1, NKX3.1), and others from a new microarray-based comparison was studied by quantitative RT-PCR in 45 M0 prostate carcinomas and 13 benign prostate tissues. RESULTS: Significantly reduced expression was observed for two protein phosphatase subunit genes (PPP2CB, PPP3CC) and two TRAIL decoy receptors (TNFRSF10C/DcR1, TNFRSF10D/DcR2), but not for the three established candidates nor for TRAIL death receptor genes. Low expression of PPP3CC and TNFRSF10C located at 8p21.3 was highly significantly associated with tumor recurrence. In addition to allele loss, down-regulation of TNFRSF10C and TNFRSF10D was found to be associated with hypermethylation, although bisulfite sequencing usually revealed it to be partial. CONCLUSION: Our data strongly support a recent proposal that a segment at 8p21.3 contains crucial prostate cancer tumor suppressors. In addition, they raise the paradoxical issue of why TRAIL decoy receptors rather than death receptors are down-regulated in aggressive prostate cancer.

Factor interaction analysis for chromosome 8 and DNA methylation alterations highlights innate immune response suppression and cytoskeletal changes in prostate cancer.

BACKGROUND: Alterations of chromosome 8 and hypomethylation of LINE-1 retrotransposons are common alterations in advanced prostate carcinoma. In a former study including many metastatic cases, they strongly correlated with each other. To elucidate a possible interaction between the two alterations, we investigated their relationship in less advanced prostate cancers. RESULTS: In 50 primary tumor tissues, no correlation was observed between chromosome 8 alterations determined by comparative genomic hybridization and LINE-1 hypomethylation measured by Southern blot hybridization. The discrepancy towards the former study, which had been dominated by advanced stage cases, suggests that both alterations converge and interact during prostate cancer progression. Therefore, interaction analysis was performed on microarray-based expression profiles of cancers harboring both alterations, only one, or none. Application of a novel bioinformatic method identified Gene Ontology (GO) groups related to innate immunity, cytoskeletal organization and cell adhesion as common targets of both alterations. Many genes targeted by their interaction were involved in type I and II interferon signaling and several were functionally related to hereditary prostate cancer genes. In addition, the interaction appeared to influence a switch in the expression pattern of EPB41L genes encoding 4.1 cytoskeleton proteins. Real-time RT-PCR revealed GADD45A, MX1, EPB41L3/DAL1, and FBLN1 as generally downregulated in prostate cancer, whereas HOXB13 and EPB41L4B were upregulated. TLR3 was downregulated in a subset of the cases and associated with recurrence. Downregulation of EPB41L3, but not of GADD45A, was associated with promoter hypermethylation, which was detected in 79% of carcinoma samples. CONCLUSION: Alterations of chromosome 8 and DNA hypomethylation in prostate cancer probably do not cause each other, but converge during progression. The present analysis implicates their interaction in innate immune response suppression and cytoskeletal changes during prostate cancer progression. The study thus highlights novel mechanisms in prostate cancer progression and identifies novel candidate genes for diagnostic and therapeutic purposes. In particular, TLR3 expression might be useful for prostate cancer prognosis and EPB41L3 hypermethylation for its detection.

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.

Preclinical antitumor activity of the oral platinum analog satraplatin.

PURPOSE: Satraplatin is an orally available platinum analog. The purpose of this study was to better characterize satraplatin's preclinical antitumor efficacy in a variety of sensitive and resistant human tumor cell lines and in a prostate cancer xenograft model and to evaluate the effect of satraplatin on PSA expression and/or secretion in a prostate cancer cell line. METHODS: Satraplatin and its primary metabolite JM-118 were preclinically tested for their cytotoxic activity in a range of cancer cells including: human prostate, those forming the NCI drug screening panel, and those resistant to anti-cancer drugs. Also, the antiproliferative efficacy of satraplatin was tested in vivo in a human prostate cancer model. The effect of satraplatin and JM-118 on PSA transcription was measured by quantitative real time PCR. RESULTS: Satraplatin and JM-118 inhibited in vitro and in vivo the growth of prostate cancer cells in a dose-dependent fashion. The IC50 cytotoxicity values for satraplatin ranged from 1 to 3 microM for androgen-insensitive cells and was 11 microM for the androgen-sensitive cell line. Interestingly, JM-118 was up to 16-fold more potent than satraplatin. Oral administration of satraplatin to nude mouse PC-3 xenograft models inhibited the growth of these human tumors. Satraplatin had no direct effect on PSA transcription and the observed decrease in secreted PSA correlated with a decrease in cell number. When evaluated in the NCI drug-screening panel, satraplatin was most active in leukemia and small cell lung cancer cell lines. Both satraplatin and JM-118 were tested on cells resistant to chemotherapeutic agents. Satraplatin and JM-118 were equally active in the cisplatin-resistant A129cp80 ovarian carcinoma cell line, with activity comparable to that observed in the parent line. Neither expression of MDR1, BCRP, MRP1, nor altered tubulin or topoisomerase I were found to mediate resistance to satraplatin or JM-118. Although these resistance mechanisms contribute to drug resistance for a number of chemotherapeutics, they do not appear to play a role in satraplatin resistance. CONCLUSIONS: These results demonstrate that satraplatin and JM-118 have preclinical antitumor activity in human prostate cancer and other tumor types as well, including several cell lines displaying drug resistance to cisplatin, docetaxel and mitoxantrone. In addition, the results suggest that PSA should be further evaluated as a relevant marker of clinical response in patients with prostate cancer treated with satraplatin.

Analysis of Argonaute Complex Bound mRNAs in DU145 Prostate Carcinoma Cells Reveals New miRNA Target Genes.

Posttranscriptional gene regulation by microRNAs (miRNAs) contributes to the induction and maintenance of prostate carcinoma (PCa). To identify mRNAs enriched or removed from Ago2-containing RISC complexes, these complexes were immunoprecipitated from normal prostate fibroblasts (PNFs) and the PCa line DU145 and the bound mRNAs were quantified by microarray. The analysis of Ago complexes derived from PNFs or DU145 confirmed the enrichment or depletion of a variety of mRNAs already known from the literature to be deregulated. Novel potential targets were analyzed by luciferase assays with miRNAs known to be deregulated in PCa. We demonstrate that the mRNAs of the death effector domain-containing protein (DEDD), the tumor necrosis factor receptor superfamily, member 10b protein (TNFRSF10B), the tumor protein p53 inducible nuclear protein 1 (TP53INP1), and the secreted protein, acidic, cysteine-rich (SPARC; osteonectin) are regulated by miRNAs miR-148a, miR-20a, miR-24, and miR-29a/b, respectively. Therefore, these miRNAs represent potential targets for therapy. Surprisingly, overexpression of miR-24 induced focus formation and proliferation of DU145 cells, while miR-29b reduced proliferation. The study confirms genes deregulated in PCa by virtue of their presence/absence in the Ago2-complex. In conjunction with the already published miRNA profiles of PCa, the data can be used to identify miRNA-regulated mRNAs.

Genetic heterogeneity of the MYC oncogene in advanced juvenile angiofibromas.

Despite their benign histological appearance, juvenile angiofibromas sometimes exhibit an aggressive growth behavior. Molecular and genetic analyses have detected beta-catenin mutations and androgen receptor gene gains in this tumor. Because intensive cross-talk among beta-catenin, androgen receptor, and C-MYC has been detected recently, we analyzed expression of the C-MYC protooncogene (MYC) on the genetic, transcriptional and translational level in seven sporadic juvenile angiofibromas. Two-color in situ hybridization analyses for chromosome 8 and MYC found in all seven juvenile angiofibromas significant MYC losses. In the three advanced juvenile angiofibromas of this series (Fisch stages III and IV) additional significant MYC gains were observed demonstrating a genetic heterogeneity for the MYC protooncogene. In cases of genetic MYC heterogeneity, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, Western blot investigations, and immunohistology showed increased C-MYC mRNA and protein levels. Semiquantitative RT-PCR analyses from laser microdissected endothelial cells and fibroblasts found no differences of C-MYC mRNA levels, leaving open the question of the neoplastic cell in juvenile angiofibromas. The finding of genetic MYC heterogeneity associated with C-MYC overexpression on the mRNA and protein level in advanced juvenile angiofibromas indicates involvement of the MYC oncogene in aggressive growth behavior.

p53 and Her-2/neu in juvenile angiofibromas.

The pathogenesis of juvenile angiofibroma (JA) remains unsolved. Further, it is unknown whether this fibrovascular tumour arises from the endothelial or stromal cells. Comparative genomic hybridisation analysis of these tumours revealed deletions of chromosome 17, including regions for the tumour suppressor gene p53 as well as the Her-2/neu oncogene, which are altered in many human tumours. In order to analyse if they are also important for progression of JA, the p53 gene and Her-2/neu gene were evaluated in 7 tumours by two-colour in situ hybridisation analysis using probes for the centromer of chromosome 17 either with a specific probe against p53 or Her-2/neu. In 5 out of 7 JAs, gene losses were detected for both genes ranging from 10.5 to 31.5%, respectively. Gene amplifications were not observed. Semi-quantitative RT-PCR analysis from laser microdissected single endothelial cells and fibroblasts showed up-regulated p53 mRNA levels in 4 out of the 7 JAs analysed in both investigated cell types and in one case in only endothelial cells. Her-2/neu mRNA was noted to be up-regulated in 2 JAs and down-regulated in 1 JA for both cell types. Western blot analysis as well as immunohistochemistry detected no p53 protein in the 5 investigated JAs, indicating absence of mutated p53. Our findings indicate that chromosomal losses on chromosome 17 imply p53 gene and Her-2/neu gene losses in JAs. However, comparison of p53 and Her-2/neu mRNA levels in laser microdissected endothelial and stromal cells were not conclusive to answer the question of the tumour cell of origin in JA.

Detailed chromosomal characterization of the breast cancer cell line MCF7 with special focus on the expression of the serine-threonine kinase 15.

Although the cell line MCF7 is widely used in breast cancer research, its cytogenetic properties have not been thoroughly investigated so far. As conventional G-banding analysis cannot resolve the complex chromosome aberrations, we investigated MCF7 cells using molecular-cytogenetic methods, with particular attention to the DNA amplification site on chromosome 20q. With spectral karyotyping we found numerous unbalanced chromosome translocations, and with comparative genomic hybridization we detected many quantitative genomic imbalances. Furthermore, we analyzed the amplified region at 20q with the candidate tumour susceptibility gene STK15 in detail by fluorescence in situ hybridization, whole chromosome painting, immunohistochemistry, Western blot and expression analysis. In MCF7 interphase cells we found increased copy number of the STK15 gene associated with overexpression of STK15 mRNA. Accordingly, STK15 protein is overexpressed as compared to normal human fibroblasts in Western blot analysis. Overexpression of STK15 mRNA and protein is disproportionally stronger than that expected from the single additional copy of the STK15 gene. These data indicate that the highly increased level of STK15 protein in MCF7 cannot be explained by gene amplification alone. Apparently, secondary mechanisms of gene up-regulation are involved. This observation may be of general interest with regard to the activation of oncogenes in tumour cells.

Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells.

Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer.We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.

Overexpression and amplification of STK15 in human gliomas.

The serine/threonine kinase 15 (STK15) at chromosome 20q13.2 is frequently shown to be amplified and overexpressed in several human cancers. STK15 has been reported to act as a cell cycle regulator and its overexpression induces centrosome amplification and aneuploidy. Recently we showed that STK15 even plays a role in human malignant brain tumours and we described an amplification of the gene in 31% of the investigated gliomas. In this study we scrutinized the correlation of increased STK15 on DNA and mRNA levels in gliomas of different histological grades. Southern blotting confirmed the amplification frequency of the STK15 gene, which had been previously detected by comparative PCR. In total, DNA gains were found in 26% of the investigated gliomas. Interestingly, we detected overexpression of STK15 mRNA in 60% of the analyzed brain tumours. The elevated expression does not strongly correlate with gains on DNA level, but all cases with an amplification of the STK15 gene display overexpression. Gains of the STK15 gene seem to occur irrespectively of the histological grades of the tumours, so that STK15 probably is not a progression associated factor. Amplification and overexpression of the kinase rather represent a primary alteration in human gliomas, which could play an important role as an early event in all glioma subtypes.

Evidence for gains at 15q and 20q in brain metastases of prostate cancer.

Many detailed genetic studies have been reported on prostate carcinogenesis. A major shortcoming of these studies, however, is the fact that most data have been gained from investigations that were performed at a single point of time during tumor development. Only little is known on the dynamic process of genetic changes during the course of the disease. We performed comparative genomic hybridization in two cases of prostate cancer brain metastases. Tissue samples from the primary tumors, the locally recurrent tumor in one case, and the brain metastases from both cases were available for analysis. The number of chromosome abnormalities was found to be increased in the metastases. This contrasts to a remarkably stable chromosome composition of the primary tumor over several years, even in an androgen-depleted environment. When focusing on these changes, which either emerged as new common aberrations in both brain metastases, or which were commonly present in the primary and metastatic tumors, we were able to delineate five chromosomal sites that are assumed to be related to prostate cancer metastasis: 8q21 approximately q22, 8q24, 15q24 approximately q26, 20q12 approximately q13.1, and Xq12 approximately q21. These findings provide new evidence for a putative role of genes at 15q and 20q in the metastatic process of prostate cancer.

Numerical sex chromosome aberrations in juvenile angiofibromas: genetic evidence for an androgen-dependent tumor?

Juvenile angiofibromas (JAs) are rare benign tumors arising almost exclusively in the posterior nasal cavity of adolescent males. While male sex predominance and tumor manifestation during adolescence are well known clinical features, their genetic causes are still unknown. Observation of an increased androgen binding rate to the cytosol of JAs and immunohistological finding of strong AR expression have suggested involvement of the androgen receptor (AR) in JA biology. In the present study, we investigated sex chromosome distribution and the expression of the AR gene in JAs of 7 males using two-color in situ hybridizations. Probes specific for the centromeres of chromosomes 1, X, and Y as well as a probe specific for the AR gene were used for investigation of paraffin-embedded JA tissue. Significant aberrations of the chromosome 1 were not observed. In 6 out of 7 analysed JAs derived from male patients we observed a significant loss of the chromosome Y in 11.5 to 63.8% (mean value: 31.3%). A gain of chromosome X was seen in 5 out of 7 JAs with the finding of two chromosomes X in 12 to 34% (mean value: 25%) of the analyzed nuclei. As each chromosome X revealed nearly almost one AR gene signal without evidence for amplifications, in 11.5 to 30% of the JA nuclei (mean value: 23.8%) two copies of the AR gene were observed. Our data indicate a significant loss of chromosome Y in combination with a gain of chromosome X in JAs. A gain of chromosome X leads to AR gene gain indicating that JAs are androgen-dependent tumors. This is supported by the finding that beta-catenin known to be overexpressed in JAs acts as a co-activator of the AR.