Joern Kamradt

Detection of novel amplicons in prostate cancer by comprehensive genomic profiling of prostate cancer cell lines using oligonucleotide-based arrayCGH.

Background The purpose of this study was to prove the feasibility of a longmer oligonucleotide microarray platform to profile gene copy number alterations in prostate cancer cell lines and to quickly indicate novel candidate genes, which may play a role in carcinogenesis. Methods/Results and Findings Genome-wide screening for regions of genetic gains and losses on nine prostate cancer cell lines (PC3, DU145, LNCaP, CWR22, and derived sublines) was carried out using comparative genomic hybridization on a 35,000 feature oligonucleotide microarray (arrayCGH). Compared to conventional chromosomal CGH, more deletions and small regions of gains, particularly in pericentromeric regions and regions next to the telomeres, were detected. As validation of the high-resolution of arrayCGH we further analyzed a small amplicon of 1.7 MB at 9p13.3, which was found in CWR22 and CWR22-Rv1. Increased copy number was confirmed by fluorescence in situ hybridization using the BAC clone RP11-165H19 from the amplified region comprising the two genes interleukin 11 receptor alpha (IL11-RA) and dynactin 3 (DCTN3). Using quantitative real time PCR (qPCR) we could demonstrate that IL11-RA is the gene with the highest copy number gain in the cell lines compared to DCTN3 suggesting IL11-RA to be the amplification target. Screening of 20 primary prostate carcinomas by qPCR revealed an IL11-RA copy number gain in 75% of the tumors analyzed. Gain of DCTN3 was only found in two cases together with a gain of IL11-RA. Conclusions/Significance ArrayCGH using longmer oligonucleotide microarrays is feasible for high-resolution analysis of chomosomal imbalances. Characterization of a small gained region at 9p13.3 in prostate cancer cell lines and primary prostate cancer samples by fluorescence in situ hybridization and quantitative PCR has revealed interleukin 11 receptor alpha gene as a candidate target of amplification with an amplification frequency of 75% in prostate carcinomas. Frequent amplification of IL11-RA in prostate cancer is a potential mechanism of IL11-RA overexpression in this tumor type.

Telomerase Activity and Telomerase Subunit Gene Expression Levels Are Not Related in Prostate Cancer: A Real-Time Quantification and In Situ Hybridization Study

Because the mechanisms of telomerase activation in prostate cancer are mainly unknown, we investigated the relationships between telomerase activity and expression levels of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in benign and malignant alterations of the human prostate gland. Using the LightCycler technology, hTERT mRNA expression was quantified in 46 radical prostatectomy and 10 benign prostatic hyperplasia (BPH) cases; hTR expression was quantified in a subset of these tissue samples. Telomerase activity was measured using a quantitative telomeric repeat amplification protocol ELISA assay. Similar to hTR, which was expressed in all tissue samples tested, hTERT mRNA was detected in 98% of the prostate cancer samples and in 30% of the BPH samples. Regarding clinicopathologic variables, telomerase activity was significantly correlated with Gleason score (<7 vs ≥7, p = 0.02). No relationships emerged between normalized hTR or hTERT expression levels and tumor stage, Gleason score, lymph node status, or preoperative serum prostate-specific antigen. Remarkably, one third of all cancer and BPH tissue samples with hTR and hTERT expression lack telomerase activity. Quantitative analyses contradict the assumption that a certain threshold level of hTR or hTERT mRNA is required for telomerase activation, thus indicating that telomerase regulation in prostate cancer occurs more likely on a posttranscriptional level. Nevertheless, the observation that hTR and hTERT mRNA levels are significantly (p < 0.002) correlated suggests some common mechanisms in the up-regulation of hTR and hTERT expression. Because in situ hybridization revealed strong hTERT expression in all cells of the tumor glands but also in high-grade prostatic intraepithelial neoplasia foci, this up-regulation seems to occur early in prostate carcinogenesis.

Abstract 5231: Zoledronic acid encapsulated into liposomes and nanoparticles: Enhanced antitumor activity in 3D-prostate carcinoma spheroids

Introduction: Zoledronic acid is currently used for the treatment of bone metastases in hormone-resistant prostate cancer patients. The rapid plasma half-life and the high affinity for Osteoclasts, the primary targets of bisphosphonates, limit the use for extra-skeletal tissues. Moreover, it remains still unclear, whether cancer-associated fibroblasts (CAF) influence the toxicity of ZOL. The use of nanomedicine is one promising strategy to improve the availability of drugs within the tumor tissue. To investigate the benefit of such approach, we used two different nanovectors encapsulating ZOL on 3D-spheroids from prostate carcinoma cells and CAF in combination with standard toxicity assays. Material and Methods: Stealth liposomes (PGNP_ZOL) and a new generation of nanovector, named self-assembly nanoparticles (NP_ZOL), were prepared using standard protocols already described. The prostate carcinoma cell lines PC-3, DU-145 and LNCaP were cultivated in 96-well MTP9s and as Spheroids on agarose coated 96-well MTP9s. Heterologous spheroids were prepared by mixing tumor cells with different ratios of normal (NAF) or tumor-associated (CAF) fibroblasts. Cell cultures/spheroids were incubated with ZOL, NP_ZOL/PGNP_ZOL for up to 120 h. Changes in growth behavior and toxicity (apoptosis/necrosis) were quantified from spheroid-cultures by modifications of standard assays (CDD+, LDH+). The adaptability of these techniques for 3D- cultures was assessed using thapsygargin/tunicamycin as apoptosis-inducing agents. Results: Standard-viability assays revealed a significant enhancement of antitumor activity of NP_ZOL/PGNP_ZOL as compared to the free drug in cell cultures of DU-145 and LNCaP, whereas these effects were less pronounced in PC-3 cells. The antitumor efficacy of NP_ZOL/PGNP_ZOL was caused by a strong inhibition of tumor cell proliferation as revealed by the BrDU-assay; on the other hand, both CDD+ and LDH+ failed to detect both apoptosis and necrosis after drug treatment for up to 120 h. The average IC 50 values in the tumor cells ranges from 10-20µM/L, but the fibroblasts exhibit complete growth inhibition at concentrations between 2-5µm/L ZOL. Conclusion: These results confirm the hypothesis that the use of nanomedicine enables the extension in therapeutic applications of drugs such as bisphosphonates. In this context, ZOL seems to exert benefits even onto the primary tumor cells. Homo-/heterologeous 3D-spheroids offer advanced models mimicking the microenvironment within a tumor tissue grows thus bridging the gap between cell culture and live tissue. Moreover, the rapid generation of single-tumor spheroids allows for a high-throughput cell function and toxicity analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5231. doi:1538-7445.AM2012-5231

Abstract 2351: Down-regulation of Fibulin-5 in invasive growing primary prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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