Martina Tinzl

Multiple Cellular Mechanisms Related to Cyclin A1 in Prostate Cancer Invasion and Metastasis

BACKGROUND Cyclin A1 is a cell cycle regulator that has been implicated in the progression of prostate cancer. Its role in invasion and metastasis of this disease has not been characterized. METHODS Immunohistochemistry and cDNA microarray analyses were used to assess protein and mRNA expression of cyclin A1 and proteins with roles in metastasis, including vascular endothelial growth factor (VEGF), metalloproteinase 2 (MMP2), and MMP9, in human prostate cancer. Transient transfection and infection with viral vectors expressing cyclin A1 and short hairpin RNA (shRNA) targeting cyclin A1 were used to study the effects of altered cyclin A1 expression in PC3 prostate cancer cells. The BrdU assay, annexin V staining, and invasion chambers were used to examine cyclin A1 effects on proliferation, apoptosis, and invasion, respectively. The role of cyclin A1 and androgen receptor (AR) in transcription of VEGF and MMP2 was assessed by promoter mutation and chromatin immunoprecipitation. The effect of cyclin A1 expression on tumor growth and metastasis was analyzed in a mouse model of metastasis. All statistical tests were two-sided. RESULTS Cyclin A1 protein and mRNA expression were statistically significantly higher in prostate cancers than in adjacent benign tissues. A statistically significant correlation between expression of cyclin A1 and of MMP2, MMP9, and VEGF was observed in prostate tumors from 482 patients (P values from Spearman rank correlation tests < .001). PC3 cells that overexpressed cyclin A1 showed increased invasiveness, and inhibition of cyclin A1 expression via shRNA expression reduced invasiveness of these cells. Eight of 10 mice (80%) bearing PC3 cells overexpressing cyclin A1 had infiltration of tumor cells in lymph node, liver, and lung, but all 10 mice bearing tumors expressing control vector were free of liver and lung metastases and only one mouse from this group had lymph node metastasis (P values from Fisher exact tests < .001). Cyclin A1, in concert with AR, bound to and increased expression from the VEGF and MMP2 promoters. CONCLUSIONS Cyclin A1 contributes to prostate cancer invasion by modulating the expression of MMPs and VEGF and by interacting with AR.

Serotonin activates MAP kinase and PI3K/Akt signaling pathways in prostate cancer cell lines

PURPOSE This study was conducted to examine the effects of 5-HT on extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt pathways in prostate cancer (PC) cells. METHODS PC cell lines PC-3, Du145, and LNCaP stimulated with 5-HT in the presence of MEK or PI3K inhibitors and 5-HT receptor subtype 1A antagonist were analyzed by Western blotting and immunofluorescence. The proliferation assay BrdU and Boyden chamber were used to determine proliferation and migration, respectively. RESULTS 5-HT dose-dependently induced rapid activation of Erk1/2 in PC-3 and Du145 cells, whereas in LNCaP cells, Erk1/2 phosphorylation was slow and sustained for up to 18 h. Similarly, 5-HT induced phosphorylation of Akt within 1 hour of stimulation, however, Akt phosphorylation was more pronounced in Du145 cells compared with PC-3 or LNCaP cells. The action of 5-HT was inhibited to varying degrees by inhibitors of MAPK and PI3K as well as by a 5-HT receptor subtype 1A antagonist. In addition to proliferation, 5-HT induced migration of PC-3 and Du145 cells, which were alleviated by the aforementioned inhibitors. The effects of 5-HT on LNCaP cells appeared to be related to neuroendocrine-phenotype acquisition and chromogranin A and neuron specific enolase expression. CONCLUSIONS This study addresses the role of 5-HT in Erk1/2 and Akt activation in PC cells. The data presented here identify 5-HT receptors as a novel target in castration-resistant PC. Furthermore, our observations are in line with previous studies, which point towards neuroendocrine factors facilitating progression and migration of prostatic cancer cells in an androgen-deficient environment. Nonetheless, additional studies are warranted to corroborate the role of 5-HTR antagonists as a potential target for anticancer therapy.

Properties and effects of a novel liquid crystal nanoparticle formulation of docetaxel in a prostate cancer mouse model

Treatment with docetaxel is the standard of care as first line chemotherapy in castration resistant prostate cancer. Due to serious side effects from the commercially available Taxotere formulation, we aimed to develop a safe and effective nanoparticle formulation of docetaxel. Liquid crystal nanoparticles (LCNPs), based on phosphatidyl choline, glycerol dioleate and polysorbate 80 dispersed in excess aqueous solution, were produced by simple procedures as carriers of docetaxel. Their effect on tumor growth in male SCID mice inoculated with PC-3 cells was compared to the effect of Taxotere and empty LCNP vehicle. Immunohistochemistry was performed to evaluate cell proliferation, angiogenesis and apoptosis in tumor tissue. Docetaxel and lipid excipients were dispersed into well-defined LCNP, stable during long-term storage. Mice subjected to LCNP/docetaxel formulation showed a better tumor regression than mice treated with Taxotere, with an indication of better tolerability. Immunohistochemical staining showed a decreased expression of Ki-67 in tumors from LCNP/docetaxel treated animals, especially in the cores of the tumors, suggesting better penetration/absorption compared to Taxotere. A new lipid-based nanoparticle formulation has been developed as carrier for docetaxel. Treatment effects in SCID mice indicate that this may be an interesting alternative to the current marketed formulation product.

Abstract B15: Androgen receptor recruitment to c-Jun promoter region regulates chemosensitivity of prostate cancer cells to taxane therapy

Introduction & Objectives. We have previously reported that response of prostate cancer (PC) to taxane therapy is based on the interplay between androgen receptor (AR) and c-jun However, the cellular mechanisms of taxane treatment in prostate cancer cells are not fully elucidated. The aim of this study was to investigate the effect of sustained down-regulation of c-jun on the outcome of taxane therapy using LNCaP cells transfected with c-jun shRNA, and to characterize the nature of the c-jun and AR interaction in regard to their corresponding promoter regions. Material & Methods. Chromatin immunoprecipitation (CHIP) assay was performed to examine gene recruitment. LNCaP cells were treated with Docetaxel (Doc) or Paclitaxel (Pac) and resulting chromatin preparations were analyzed by PCR. To analyse the impact of c-jun downregulation on AR and prostate cancer response to therapy we used LNCaP cells harboring stable knocked down c-jun (LNsiJun). Cells were exposed to Doc, Pac DHT for different time-points and the medium were subjected for PSA analysis using DELFIA 1234 fluorometer and cell response to drugs assessed by MTT assay. Results. Taxane treatment of LNsiJun cells led to a statistically significant decrease of cell viability compared to parental LNCaP cells (p Conclusions. In conclusion, down-regulation of endogenous c-jun enhances sensitivity of LNCaP cells to taxane treatment. Binding of AR to the c-jun promoter is taxane specific and results in different expression levels of AR and PSA. Citation Format: Nishtman Dizeyi, Martina V. Tinzl, Marieke van der Molen, Julius Semenas, Per-Anders Abrahamsson. Androgen receptor recruitment to c-Jun promoter region regulates chemosensitivity of prostate cancer cells to taxane therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B15.

Interaction between c-jun and androgen receptor determines the outcome of taxane therapy in castration resistant prostate cancer.

Taxane based chemotherapy is the standard of care treatment in castration resistant prostate cancer (CRPC). There is convincing evidence that taxane therapy affects androgen receptor (AR) but the exact mechanisms have to be further elucidated. Our studies identified c-jun as a crucial key player which interacts with AR and thus determines the outcome of the taxane therapy given. Docetaxel (Doc) and paclitaxel (Pac) agents showed different effects on LNCaP and LNb4 evidenced by alteration in the protein and mRNA levels of c-jun, AR and PSA. Docetaxel-induced phophorylation of c-jun occurred before JNK phosphorylation which suggests that c-jun phosphorylation is independent of JNK pathways in prostate cancer cells. A xenograft study showed that mice treated with Pac and bicalutamide showed worse outcome supporting our hypothesis that upregulation of c-jun might act as a potent antiapoptotic factor. We observed in our in vitro studies an inverse regulation of PSA- and AR-mRNA levels in Doc treated LNb4 cells. This was also seen for kallikrein 2 (KLK 2) which followed the same pattern. Given the fact that response to taxane therapy is measured by PSA decrease we have to consider that this might not reflect the true activity of AR in CRPC patients.

Abstract C226: Phosphorylation of ser63/73 in c-jun is required to respond to docetaxel treatment in prostate cancer cells.

Background: Docetaxel (doc) is currently used as first-line therapy in castration-resistant prostate cancer (CRPC). However, a significant number of patients are irresponsive to the drug. To better understand this inter-individual variability, we studied the impact of c-jun phosphorylation, a major pathway of doc-induced apoptosis, in prostate cancer (PCa) cells. Furthermore, we validated the effect of doc treatment on the androgen receptor (AR) and c-jun gene alteration. Methods: All in vitro experiments were performed on human PCa cell lines- PC-3, LNCaP and LNCaP cells cultured in bicalutamide for 4 weeks (LNb4). To examine the role of c-jun in doc therapy, AR-negative PC-3 cells were transfected with c-jun or c-jun, mutated at serine 63/73 (c-junA) or co-transfected with AR and c-jun or c-junA. Quantitative real time PCR (qPCR) of c-jun, AR, PSA, KLK2, NKX3.1 and c-Myc mRNA was used to determine the impact of doc treatment on gene expression. Proliferation and Western blotting assays were performed to analyze the treatment efficacy and protein expression, respectively. Results: A pronounced increase in cell proliferation, in c-junA-transfected cells, in presence or absence of doc was observed, suggesting that phosphorylation of c-jun at Ser63/73 might be necessary to respond to doc treatment. Moreover, cells that were co-transfected with AR seemed to be more responsive to doc treatment compared to cells transfected with c-junA or c-jun alone. Western blot analysis revealed that cells which were co-transfected with AR and c-jun exhibit a strong band for AR compared to other transfection groups. Time-dependent gene expression alterations after exposure to doc were observed in LNCaP and LNb4 cells. In LNCaP cells, doc treatment lead to a decreased in AR, but an increased in PSA mRNA. In contrast, a significant increase in AR mRNA was observed in LNb4 cells, whereas PSA mRNA levels gradually decreased after an initial flare. Expression pattern of KLK2 mRNA was similar to that of PSA in both cell types. NKX3.1 mRNA expression in LNCaP cells showed a significant increase after 48h, whereas in LNb4 cells, NKX3.1 expression significantly increased already after 6h but then gradually decreased throughout 48h of exposure to doc. No significant changes in the mRNA expression of c-jun or c-Myc were observed. Conclusions: Our results indicate functional significance of phosphorylated c-jun and AR in doc treated PCa cells. Moreover, we also provide further evidence of AR role in treatment resistance mechanisms. Conclusively, our study shows promising results that merits further in-depth investigation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C226. Citation Format: Nishtman Dizeyi, Martina Viktoria Tinzl, Shao-yong Chen, Julius Semenas, Per-Anders Abrahamsson. Phosphorylation of ser63/73 in c-jun is required to respond to docetaxel treatment in prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C226.

Intermittent DHT administration enhances effect of docetaxel in a xenograft model by modulation of ER beta, AR and NEK2

prostate cancer adding another established drug with known and manageable 830 AN EXPERIMENTAL STUDY IN NUDE MICE ON THE ELECTROMAGNETIC DIAGNOSIS OF TUMOURS Tubaro A., De Nunzio C., Stoppacciaro A., Leonetti C. , Zupi G. , Miano L. Ospedale Sant’Andrea, “La Sapienza University”, Dept. of Urology, Rome, Italy, Ospedale Sant’Andrea, “La Sapienza University”, Dept. of Pathology, Rome, Italy, National Cancer Institute “Regina Elena”, Laboratory for Preclinical Experimental Chemotherapy, Rome, Italy