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Cancers 14(12), 2943 (2022). doi:10.3390/cancers14122943 special issue: "Special Issue "Bio-Pathological Markers for the Diagnosis and Therapy of Cancers, Volume II" / Special Issue Editors: Dr. Lucia Salvatorelli, Guest Editor; Dr. Giuseppe Broggi, Guest Editor", Published by MDPI, Basel

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.

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. Prostate 75:1526–1537, 2015. © 2015 Wiley Periodicals, Inc.

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.

Labelled 5α-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.

Introduction Emerging evidence suggest that microRNAs could serve as non-invasive biomarker for cancer patients. Our study was designed to analyze circulating serum microRNAs in patients with renal cell carcinoma (RCC). Materials and methods Serum RNA was isolated from patients with clear cell RCC (ccRCC) and non-malignant disease; an artificial microRNA (cel-miR-39) was spiked-in prior the isolation procedure to control isolation efficiency. The levels of miR-26a-2*, miR-191, miR-337-3p and miR-378 in serum were determined using quantitative real-time PCR; the microRNA levels were normalized to cel-miR-39. Results First, miR-26a-2*, miR-191, miR-337-3p and miR-378 were quantified in serum of each 25 patients with ccRCC and non-malignant disease. The level of miR-378 was significantly increased in ccRCC patients, and thus chosen for validation. The analysis of miR-378 in the validation cohort with 117 RCC patients and 123 control subjects did not confirm a different level of miR-378. Also, miR-378 was not correlated to pT-stage, lymph node/distant metastasis, vascular invasion and Fuhrman grade. Conclusions The analysis of circulating serum levels of miR-26a-2*, miR-191, miR-337-3p and miR-378 is unlikely to provide helpful diagnostic/prognostic information in RCC patients.

The clinical course of prostate cancer, the most common cancer in men, is very variable. Despite intense research activities over the years and besides histopathological criteria, prognostic markers that reliably predict tumor behavior and the necessity for treatment are still missing. A likely explanation for this fact is the lack of good tumor models, mimicking the in vivo situation. These models are not only essential for a better understanding of the pathogenesis of prostate cancer but also play an important role in the development of new therapeutic strategies. Since results of permanent cell culture experiments reflect only in part real tumor behavior and primary cultures from patient material cannot be grown indefinitely, novel approaches need to be developed to achieve reliable and clinically relevant prostate cancer research.In this work the development of several approaches for culturing primary prostate cancer tissue is illustrated and a forecast of future research plans utilizing xenograft models in mice is made.

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. Ca2+-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 [Ca2+] 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. Prostate 71:1074–1083, 2011. © 2011 Wiley-Liss, Inc.

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.

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.

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.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.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.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.

BACKGROUND Fibulins, encoded by FBLN genes, are extracellular matrix proteins influencing cell adhesion and migration. Altered expression of fibulins is associated with progression of several cancer types, but has not been studied in prostate cancer. METHODS Expression of FBLN1 (major splice forms C and D), FBLN4, FBLN5, SPOCK1, and TENC was compared between 47 prostate cancer samples and 13 benign prostatic tissues by quantitative RT-PCR. Fibulin-1 and fibulin-5 expression was studied by immunohistochemistry. Effects of androgens and the DNA methylation inhibitor 5-aza-2′-deoxycytidine on fibulin expression were investigated in different prostate cancer cell lines. RESULTS Our recent microarray analysis suggested downregulation of three fibulins, FBLN1, FBLN4, and FBLN5, in prostate cancer, while two further ECM genes, SPOCK1 (testican) and TENC (tenascin C), appeared upregulated or unchanged. These observations were corroborated by quantitative RT-PCR. Accordingly, FBLN1 and FBLN4 were weakly expressed in carcinoma lines compared to normal prostate epithelial cells (PrECs). Only FBLN4 was induced by 5-aza-2′-deoxycytidine, but its promoter was unmethylated. Androgen did not affect expression of FBLN genes. The FBLN1C and FBLN1D splice forms were coordinately expressed. Fibulin-1 protein was weakly detectable in benign PrECs, but tended to accumulate in cancer cells. Fibulin-5 was predominantly located in the stroma with a strong gradient from the periurethral to the peripheral zone, and lost in cancers. CONCLUSIONS Three FBLN genes are significantly downregulated in prostate cancer, whereas SPOCK1 is often upregulated. FBLN5 downregulation fits its postulated anticancerous function, whereas FBLN1 and FBLN4 behave different than in certain other cancers. Prostate 67: 1770–1780, 2007. © 2007 Wiley-Liss, Inc.

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)

BACKGROUND Recent immunohistochemical data have shown that invasive prostate cancer cells are separated from the host tissue by basement membranes (BM), and express associated adhesive molecules that bind to these de novo synthesized extracellular matrices. METHODS In the present study, we used in situ hybridization techniques to determine steady-state levels of genes coding BM components (alpha1 chain of collagen IV, laminin beta1 chain, and S-laminin) in prostate tissue obtained from 15 radical prostatectomy specimens and 5 lymph node metastases of common prostatic adenocarcinomas. RESULTS In benign prostate tissue, transcripts of these genes were detected predominantly in the basal cell layer, indicating that components of epithelial BMs are synthesized by basal cells and not by stromal cells. The cancerous lesions investigated revealed increasing collagen IV, laminin beta1 chain, and S-laminin mRNA levels when compared with benign prostate tissue. The highest steady-state levels were found in high grade (primary Gleason grade 4 and 5) carcinoma and lymph node metastases, and were predominantly localized in epithelial compartments of the cancerous tissue. CONCLUSIONS These findings indicate that neoplastic BM in prostatic adenocarcinoma derive from tumor cells and not from the host tissue. Increasing transcriptional activities of genes coding BM components detected in poorly differentiated and metastatic lesions may accelerate the BM-forming process, which probably contributes to the ability of tumor cells to penetrate the extracellular matrix during the process of stromal invasion and metastasis.

Introductory Sentence Orthotopic xenografts are increasingly used as preclinical in-vivo models for the study of renal cell carcinoma (RCC). However, monitoring of these tumors requires sophisticated imaging techniques. In this study, we comparatively evaluated modern small animal imaging tools like high-resolution 3D-ultrasonography (3D-US), contrast-enhanced in-vivo micro-CT (CE-CT) and 9,4T MRI (MRI) to non-invasively monitor tumor growth and progression in an orthotopic RCC xenograft model. Material and Methods 106 CAKI-1 cells were injected under the renal capsule of 18 Balb/c-nude mice. Every 14 days from week 4 imaging was performed by CE-CT and 3D-US. 10 weeks after tumor cell inoculation, all animals additionally underwent multiparametric MRI (T1, T2, T2*, DWI). At autopsy, tumor volumes were determined using a caliper and compared to in-vivo imaging results. CE-CT, MRI and 3D-US were evaluated regarding tumor detection, analysis of tumor volume, radiographic imaging properties and examination time. Finally, similar experiments were performed with the RCC cell lines KTCTL30 (n = 2) and 786-0 (n = 1) to test their applicability for the orthotopic xenograft model. Results Tumors were detected in 16/18 animals (89%) 4 weeks after orthotopic inoculation. All methods enabled to adequately visualize orthotopic tumors and to display their growth over time. While the tumors had a homogenously radiolucent signal in CT scans, sonography and MRI were better able to visualized intratumoral structures (e.g. solid areas or intratumoral bleedings) and surrounding soft tissue. CT had the best spatial resolution, followed by 3D-US and MRI. The median examination time was 4.8min per 3D-US, 12.5min per CT and 37.9min per MRI, respectively. Of interest, tumor volumes determined by KM-CT and ultrasonography showed strong correlation with each other (n = 85, R = 0.985) as well as with caliper measurements at autopsy (CT: n = 16. R = 0.922; sonography: n = 16, R = 0.934). Similarly, tumor volumes measured with T2-weighted MRI correlated well with those determined by CT, sonography and caliper. No side effects due to radiation exposure were observed. Mice inoculated with KTCTL30 cells developed partly cystic, partly solid tumors. 786-O cells yielded purely solid, exponentially growing tumors. Conclusion All three imaging modalities are feasible tools for the non-invasive monitoring of orthotopic tumor growth and show excellent correlation with each other. While sonography allows for a fast analysis of tumor volume with excellent resolution and no radiation exposure, MRI provides the best visualization of soft tissue and can give information about tumor biology when acquiring additional sequences. CT has the best spatial resolution of all methods and enables simultaneous screening for bone and lung metastases but relies on the use of contrast agent and ionizing radiation. Citation Format: Johannes Linxweiler, Christina Körbel, Andreas Müller, Volker Jung, Eva Jüngel, Stefan Siemer, Michael Stöckle, Kerstin Junker, Michael D. Menger, Matthias Saar. Experimental imaging in orthotopic xenograft models of renal cell carcinoma: comparative evaluation of high-resolution ultrasonography, in vivo micro-CT, and 9.4T MRI. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4216.

INTRODUCTION AND OBJECTIVES: Animal models that authentically reproduce tumor growth are still sparse in prostate cancer research for several reasons: Whilst orthotopic implantation is technically difficult, most models favor subcutaneous tumor growth that provides little information about natural tumor growth behaviour and tumor stroma interaction. Established prostate cancer cell lines as in vivo xenografts are not able to reflect the variety of tumor specific growth patterns and growth behaviour. 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 recently developed. METHODS: Balb/c mice were used to graft fresh prostate tumor tissue by surgical orthotopic implantation. Tissue implantation was performed at four inoculation sides in each animal and testosterone propionate was supplemented. Animals were tracked after 12, 16 and 20 weeks by means of 30MHz ultrasound to monitor tumor engraftment. Autopsy, histology and PSA measurements were used to confirm orthotopic tumors after 13, 21 and 28 weeks. RESULTS: To date, tumor implantation was performed in 17 mice. 3 animals were excluded due to early dead or repetitive loss of the testosterone pellet. After successful testosterone supplementation PSA measurement was possible in 12 of 14 mice (86%, range: 0,009 80ng/ml). So far, orthotopic tumor growth was histologically verified in 6 of 14 mice. Ultrasound seems feasible to detect tumors. Of interest, tumorgrafts maintain essential features of the original tumors as tumor growth pattern, PSA expression, androgen receptor expression and high proliferation using human Ki67 immunostaining. CONCLUSIONS: The tumorgrafts resembled histological properties of organ confined prostate cancer. Measurements of serum PSA and three-dimensional ultrasound were useful for monitoring of tumor engraftment and growth. Further studies like comparative genomic hybridization to genetically characterize tumorgrafts are necessary. Besides their role as a bank of tumor samples for repetitive in vitro studies, tumorgrafts display a new approach for an optimized in vivo model of prostate cancer and will allow further investigations on specific components of tumor initiation and progression, including the interactions of prostate cancer cells with the surrounding stroma. Whether this model is also applicable for metastatic progression needs to be further investigated.

INTRODUCTION AND OBJECTIVES: Over the last years the influence of surrounding cellular microenviroment on cancer cell development and behavior has gained more interest. In this context, cancer associated fibroblasts (CAFs) adjacent to a tumor seem to be important modulators of tumor cell proliferation, migration and invasion. So far, only little is known about the differences between CAFs and normal, non-cancer-associated fibroblasts (NAFs) in prostate cancer on a molecular level. Therefore the aim of the present study is to compare CAFs and NAFs from primary prostate cancer samples applying cytoand molecular genetic methods. METHODS: Small tissue pieces from cancerous and cancer free areas of the prostate were obtained from 5 prostatectomies by an uropathologist. CAFs and NAFs were selectively cultivated from these tissue samples in vitro. Cultivated fibroblasts were genetically characterized by CGH analysis. Additionally, expression of androgen receptor (AR), PDGRFA, SMA, TGF, -catenin and E-cadherin was quantitated in early cell cultures passages (passage 4-6) by qRT-PCR. Fibroblasts were further characterized by immunofluorescence staining with antibodies against SMA, vimentin and CK18. RESULTS: CGH analysis revealed cytogenetic normal karyotypes without any chromosomal alterations in NAFs as well as in CAFs. Immunostaining demonstrated a vimentin expression in all fibroblasts, whereas CK18 was not detected. Positivity for SMA staining varied between 5 and 50% in NAFs and only 5-10% in CAFs. In qRT-PCR analysis, decreased expression levels of SMA were also seen in the CAFs, whereas TGFand PDGFRA expression was markedly increased compared to NAFs. A high -catenin and low AR expression was at comparable levels in CAFs and NAFs, respectively. E-Cadherin was neither in CAFs nor in NAFs detectable. CONCLUSIONS: CAFs and NAFs seem not to have gross cytogenetic abnormalities in cancer bearing prostates. Nevertheless differences in gene expression of SMA, TGF, -catenin and PDGFRA can be detected, when comparing CAFs and NAFs. Whether this differential expression pattern is caused by the adjacent tumor cells or originated by the CAFs itself to modulate tumor cell behavior needs to be clarified in further studies. Our results underline the need for a better understanding of tumor stroma interactions and a possible role of stromal cells as potential therapeutic targets in prostate cancer.

Fabian Janke*, Susanne Fuessel, Joerg Hofmann, Matthias Meinhardt, Dresden, Germany; Arndt Hartmann, Erlangen, Germany; Bastian Gunawan, Goettingen, Germany; Christine G. Stoehr, Erlangen, Germany; Sabine Brookman-May, Regensburg, Germany; Frank Becker, Homburg, Germany; Jens Bedke, Tuebingen, Germany; Karen Bluemke, Paolo Fornara, Halle, Germany; Mieczyslaw Gajda, Jena, Germany; Volker Jung, Homburg, Germany; Axel S. Merseburger, Hannover, Germany; Arne Strauss, Goettingen, Germany; Bernhard Walter, Erlangen, Germany; Heiko Wunderlich, Jena, Germany; Manfred P. Wirth, Dresden, Germany; Kerstin Junker, Jena, Germany

BACKGROUND: MicroRNA expression is altered in cancer cells, and microRNAs could serve as diagnostic/prognostic biomarker for cancer patients. Our study was designed to analyze circulating serum microRNAs in patients with renal cell carcinoma (RCC). METHODOLOGY/PRINCIPAL FINDINGS: We first explored microrna expression profiles in tissue and serum using taqman low density arrays in each six malignant and benign samples: Although 109 microRNAs were circulating at higher levels in cancer patients' serum, we identified only 36 microRNAs with up-regulation in RCC tissue and serum of RCC patients. Seven candidate microRNAs were selected for verification based on the finding of up-regulation in serum and tissue of RCC patients: miR-7-1*, miR-93, miR-106b*, miR-210, miR-320b, miR-1233 and miR-1290 levels in serum of healthy controls (n = 30) and RCC (n = 33) patients were determined using quantitative real-time PCR (TaqMan MicroRNA Assays). miR-1233 was increased in RCC patients, and thus validated in a multicentre cohort of 84 RCC patients and 93 healthy controls using quantitative real-time PCR (sensitivity 77.4%, specificity 37.6%, AUC 0.588). We also studied 13 samples of patients with angiomyolipoma or oncocytoma, whose serum miR-1233 levels were similar to RCC patients. Circulating microRNAs were not correlated with clinical-pathological parameters. CONCLUSIONS/SIGNIFICANCE: MicroRNA levels are distinctly increased in cancer patients, although only a small subset of circulating microRNAs has a tumor-specific origin. We identify circulating miR-1233 as a potential biomarker for RCC patients. Larger-scaled studies are warranted to fully explore the role of circulating microRNAs in RCC.

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 × 105 vs. 5 × 104), (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.

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.

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.

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.

Searching for amplifications in low grade and high grade gliomas we observed an interesting correlation between the recurrence and progression of astrocytic low grade gliomas and the amplification of the STK15 gene located in the chromosomal region 20q13. Chromosome copy gains in this region have been reported previously in astrocytic gliomas and glioma cell lines and in many cancer types including breast, colorectal and ovarian cancers. The putative serine/threonine kinase STK15 has been reported to be amplified and overexpressed in breast cancer cell lines and colorectal cancer. Another candidate gene located in this region is PTPN1, a protein tyrosine phosphatase non-receptor type 1 that might play a role in cell cycle control. We used comparative PCR for quantitative DNA analysis to search for STK15 and PTPN1 amplification in gliomas previously characterized by CGH. Five out of 16 tumors (31%) of different WHO grade (1x grade II, 1x grade III, 3x grade IV) showed DNA amplification of STK15 whereas we did not detect amplification of PTPN1. We hypothesize that amplification of the STK15 gene may be a non-random genetic alteration in human gliomas playing a role in the genetic pathways of tumorigenesis.

Squamous cell carcinoma of the head and neck exhibit a highly variable picture of chromosomal aberrations. In the present study the clearly defined anatomical region of the tongue was analyzed for potentially specific patterns of chromosomal alterations. Fresh tumor samples from 18 patients afflicted by squamous cell carcinoma of the tongue constituted the clinical basis of the present investigation. The tumor samples were analyzed on the basis of comparative genomic hybridization (CGH), a molecular cytogenetic FISH-approach. Gains in DNA copy numbers were detected as the predominant imbalance on chromosomes 7q (9/18), 3q (48/18), 16p (7/18) and 20q (7/18). The regions of minimal overlap on these chromosomes were mapped to 7q11.2q11.3 and 3q26. A conspicuous finding was the frequent detection of amplifications in the 7q11 region. Gains in the 7q region have been rarely reported in CGH studies of tumors derived from different regions of the head and neck. Amplifications on 7q could thus be specifically linked with the tongue region and could correlate with specific clinical factors of this tumor entity.

Introduction: Aside from its normal function in the embryonic development fibulin-5 seems to be involved in tumor formation by controlling proliferation and cell motility. In several malignancies including those from prostate, lung, breast and kidney predominantly down regulation of fibulin-5 was reported. But even enhanced expression in some minor cell populations was published. Since fibulin-5 mediates Epithelial-Mesenchymal Transition (EMT) in mammary epithelial cells its function during invasion seems to be likely. To evaluate the role of this matrix-protein in tumor progression we analyzed fibulin-5 expression in primary prostate cancer samples cultivated in our previously published 3D-invasion model. Experimental procedures: Fresh biopsies from 11 radical prostatectomies were subjected to our 3D-invasion model which allows for an expansion of the invading growing cell population. Expression of fibulin-5 was assessed by qRT- PCR in non-invading (top) and invasive growing (bottom) tumor cells. To investigate the role of Fibulin-5 on tumor cell migration, Fluoroblock® membranes were coated with 3 different Fibulin-5 peptides: two NH2-terminal peptides harbouring the RGD motif and one C-terminal peptide without this sequence. PC-3, LNCaP, DU-145 and A549 cells were applied onto these inserts and migration was quantified after 24 hrs by fluorescence reading using calcein staining. Results: No fibulin-5 signals were revealed by qRT-PCR and immunostaining in LNCaP and PC-3 cell lines. In contrast, the expression in DU-145 was comparable to A 549, a lung carcinoma cell line which serves as a positive control. Invasive growing primary prostate tumor cells exhibit in all cases lower fibulin-5 expression as compared to the non-invasive cell population. After coating with fibulin-5 peptides migration of the cell lines was inhibited differentially with the most pronounced effect in LNCaP and Du145; migration of PC-3 and A 549 were not affected. Migration was less inhibited in all cell lines by the C-terminal peptide. Conclusion: Our results confirmed the down regulation of fibulin-5 in prostate cancer samples in our 3D- invasion model. Interestingly, we observed differences in the expression of this matrix protein between the non-invading cell population and the more aggressive invading tumor cells, both originating from the same biopsy sample. Since all invasive cells exhibit a reduced fibulin-5 expression as compared to the non-invading cells we conclude that down-regulation of fibulin-5 expression favors tumor invasion and metastasis. This assumption is supported by the results of the migration experiments. Thus, the expression profile of fibulin-5 may reflect the invasive potential of individual tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2351. doi:10.1158/1538-7445.AM2011-2351

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify the underlying mechanisms we have analyzed microRNA (miRNA) expression profiles of primary prostate cancers and non-cancer prostate tissue. Using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs and to analyze the physiological consequences of miRNA deregulation using cell culture models. Material and Methods: We performed comparative miRNA expression profiling using deep sequencing of cDNA libraries and microarray analyses. Potential target genes of deregulated miRNAs were identified by database analysis and subsequently validated in vitro using reporter gene constructs. The impact of identified target genes on cell growth was examined by a standardized wound healing assay. Results: Two of the miRNAs consistently deregulated in primary prostate cancer samples were miR-200c and miR-375 which were upregulated more than 1.5-fold in deep sequencing and 2.9-fold and 3.9-fold, respectively, in microarray experiments (p Conclusion: Our findings demonstrate that the 3’-UTR of Sec23a is one regulative target of miR-200c and miR-375, which are commonly deregulated in prostate cancer. Sec23a is reduced in the majority of tumor samples analyzed and has a vital impact on cell growth as determined by wound healing experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3961. doi:10.1158/1538-7445.AM2011-3961

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify and characterize the underlying molecular mechanisms, we performed microRNA (miRNA) profiling of primary prostate cancers and non-cancer prostate tissue. Furthermore, using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs. Material and Methods: We performed comparative miRNA expression profiling in ten prostate cancer specimens and ten non-cancer prostate tissue samples using deep sequencing of cDNA libraries. The deregulation of selected miRNAs was validated in established prostate cancer cell lines by Northern blotting as well as in 26 paired samples of prostate cancer tissue and adjacent normal tissue by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The regulative capabilities of selected miRNAs on potential target genes were analyzed in vitro using reporter gene constructs and transfection of miRNA expression vectors Results: Using deep sequencing we found that 16 miRNAs were up-regulated more than 1.5-fold and 17 miRNAs were down regulated more than 1.5-fold. The most pronounced deregulation was noted for miRNAs miR-375 and miR-200c which showed an up-regulation of more than 4.5-fold and miR-143 and miR-145 which were down regulated 4-fold each. The differential expression of these miRNAs could be confirmed by Northern blot analysis of established prostate cancer cell lines. By analyzing 26 primary prostate cancer samples by qRT-PCR we could detect a significant deregulation of the miRNAs miR-375, miR-143 and miR-145 (p Conclusion: Our findings indicate that specific miRNAs are reproducibly found to be deregulated in different cohorts of prostate cancer specimens using independent experimental methods. This is the first report to show that miRNAs miR-143 and miR-145 are capable of negatively regulating the protein expression of Myosin VI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4036.

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. 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. 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.