Marion Gerschpacher

TUSC3 loss alters the ER stress response and accelerates prostate cancer growth in vivo.

Prostate cancer is the most prevalent cancer in males in developed countries. Tumor suppressor candidate 3 (TUSC3) has been identified as a putative tumor suppressor gene in prostate cancer, though its function has not been characterized. TUSC3 shares homologies with the yeast oligosaccharyltransferase (OST) complex subunit Ost3p, suggesting a role in protein glycosylation. We provide evidence that TUSC3 is part of the OST complex and affects N-linked glycosylation in mammalian cells. Loss of TUSC3 expression in DU145 and PC3 prostate cancer cell lines leads to increased proliferation, migration and invasion as well as accelerated xenograft growth in a PTEN negative background. TUSC3 downregulation also affects endoplasmic reticulum (ER) structure and stress response, which results in increased Akt signaling. Together, our findings provide first mechanistic insight in TUSC3 function in prostate carcinogenesis in general and N-glycosylation in particular.

The 4-hydroxyestrone: Electron emission, formation of secondary metabolites and mechanisms of carcinogenesis

4-Hydroxyestrone (4-OHE(1)), a typical cancer-inducing metabolite, originating from 17beta-estradiol (17beta-E2), was chosen as a model for the studies. The aim was to get a deeper insight in the mechanisms of its ability to initiate cancer. It was found, that 4-OHE(1) can eject electrons (e(aq)(-)), when excited in the singlet state by monochromatic UV-light (lambda=254 nm) in polar media (water:ethanol=40:60 vol.%). The quantum yield Q(e(aq)(-)), determined for various 4-OHE(1) concentrations, is found to be as high as that previously observed for 17beta-E2. It decreases with increasing substrate concentration, but it is enhanced at higher temperature. The ability of 4-OHE(1) to eject as well as to consume and to transfer electrons to other biological systems, classifies it as an electron mediator, similar to 17beta-E2. The 4-OHE(1) transients resulting of the electron emission process are leading to the formation of secondary metabolites. Surprisingly, it was established that the secondary metabolites possess likewise the ability to eject as well as to consume electrons. Hence, they behave similar like 17beta-E2. However, the structure of the secondary formed metabolites, which determinates their biological properties and carcinogenity, depends on the nature of the available reaction partners involved in their formation. A probable reaction mechanism explaining the subject matter is discussed.

Circulating endothelial progenitor cells in castration resistant prostate cancer: a randomized, controlled, biomarker study.

Background Endothelial progenitor cells (CEPs) and circulating endothelial cells (CECs) are potential biomarkers of response to anti-angiogenic treatment regimens. In the current study, we investigated the effect of docetaxel and sunitinib on CEP/CEC kinetics and clinical response in castration resistant prostate cancer (CRPC) patients. Patients and methods Chemonaive patients with CRPC were enrolled in this study to receive either sunitinib (37.5 mg/d), in combination with docetaxel (75 mg/m2) or docetaxel alone. CEP and CEC kinetics were analyzed for every cycle. The primary objective was to compare CEP/CEC pharmacodynamics between both treatment arms. We also investigated if CEC/CEP spikes, induced by MTD docetaxel, are suppressed by sunitinib in patients treated with docetaxel/sunitinib relative to docetaxel monotherapy. Results A total of 27 patients were enrolled. We observed a significant increase of CEP/CEC (total/viable) counts over time within each cycle (coefficients 0.29233, 0.22092 and 0.26089, respectively; p<0.001). However, no differences between the treatment groups, in terms of CEP and CEC kinetics, were detected. In the docetaxel monotherapy arm 4 (30%) patients responded to therapy with a 50% PSA decline, while 9 (64%) patients showed a PSA decline in the combination group (n.s.). The median PFS in the docetaxel monotherapy group was 3.1 months (2.6–3.6 months, 95% CI) and 6.2 months (4.9–7.4 months, 95% CI; p = 0.062) in the combination arm. Sunitinib/docetaxel was reasonably well tolerated and toxicity manageable. Conclusion In summary, no significant differences in CEC and CEP kinetics between the treatment arms were observed, although a highly significant increase of CEPs/CECs within each cycle over time was detected. These results mirror the challenge we have to face when employing anti-angiogenic strategies in CRPC. Additional preclinical research is needed to elucidate the underlying molecular mechanisms. However, docetaxel/sunitinib therapy resulted in a better response in terms of PSA decline and a trend towards improved PFS. Trial Registery clinicaltrialsregister.eu EudraCT 2007-003705-27

Electron emission and product analysis of estrone: progesterone interactions studied by experiments in vitro.

Recent studies showed that hormones like progesterone, testosterone, etc. can eject (solvated electrons). By means of electron transfer processes via the brain, the hormones communicate with other biological systems in the organism. The present study proves that also estrone is able to emit electrons. Their yield strongly depends on the concentration of the hormone, temperature and on the absorbed energy. The metabolites resulting from this process are likewise able to generate electrons, however with much smaller yields. The formation of the estrone metabolites is studied by HPLC-analyses. In vitro experiments with MCF-7 cells demonstrate the distinct effect of progesterone on the carcinogenity of estrone metabolites. Probable reaction mechanisms for explanation of the observed effects are postulated.

The effect of progesterone on the electron emission and degradation of testosterone.

Based on recent findings that hormones can emit electrons () from their excited singlet state in polar media, it was of importance to study a possible mutual interaction of progesterone (PRG) and testosterone (TES) in this respect. Hormones of highest purity were dissolved in an air-free mixture of 40% triply distilled water and 60% ethanol, because the hormones are unsoluble in water. As energy source for substrate excitation in singlet state served a monochromatic UV-light (254 nm), the emitted electrons were scavenged by chloroethanol, whereby the quantum yield of produced Cl− ions, Q (Cl−), is equal to Q(). Hormone degradation initiated by the electron emission was studied by HPLC method, using a Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm). The quantum yield of emitted , Q(), from TES was 3.6 times higher than that from PRG, which is explained by the different molecular structures of the hormones. Observed 2nd and 3rd maxima of electron emission indicate the ability of TES and PRG products to also eject , but with lower yield. It can be stated that a part of the emitted electrons from TES are consumed by PRG·+ leading to a partial regeneration of hormone. The present results offer a deeper insight in the biological behavior of hormones.

Abstract 3997: TUSC3 (N33) affects protein N-glycosylation and proliferation of prostate cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Prostate cancer is the most prevalent cancer in males in developed countries. Molecular aberrations in the pathogenesis of this disease are numerous, though losses on the short arm of chromosome 8 are highly prevalent. In our previous studies, hypermethylation and diminished TUSC3 expression turned out to be significantly associated with poor progression free and overall survival in ovarian cancer. Additionally, expression profiling of TUSC3 negative cancer cell lines revealed deregulation of genes involved in ‘cell structure and motility’ and ‘development processes’, further supporting our hypothesis of TUSC3 as a tumor suppressor. In silico prediction of TUSC3 shows a high homology of TUSC3 to the Ost3p subunit of the oligosaccharyltransferase (OST) complex in yeast, hinting to its role in protein glycosylation. In this project, we characterized TUSC3 (N33), a putative tumor suppressor gene on 8p, in prostate cancer. To study the prognostic role of TUSC3 in prostate cancer we used a comprehensive prostate cancer tissue microarray, comprised of samples from 143 prostate cancer patients. Serum DNA methylation was studied in an additional cohort of 67 prostate cancer patients. Statistical analysis demonstrated low or missing TUSC3 protein expression in 56.6% of prostate cancer patients based on immunohistochemical staining. Methylation of the TUSC3 promoter was observed in 38.8% of prostate cancer patients’ serum DNA, confirming our previous findings of TUSC3 promoter hypermethylation as a possible epigenetic regulatory mechanism. In our prostate cancer cohorts, however, neither expression nor hypermethylation of TUSC3 had a significant effect on overall survival, possibly due to the relatively short follow-up period. Next, we studied the function of TUSC3 in N-glycosylation and in particular its influence on carcinogenesis using a cell culture model. We downregulated TUSC3 expression in prostate cancer cell lines DU145 and PC3 using shRNA mediated knockdown and analyzed them for their proliferative and carcinogenic properties in vitro. We could show that loss of TUSC3 expression confers growth advantage of prostate cancer cell lines, in particular under conditions of serum starvation. We also observed increased migratory properties of prostate cancer cell lines upon TUSC3 knockdown. Further, influence of TUSC3 downregulation on several putative targets of N-linked glycosylation has been analyzed in vitro. In our study, we characterized TUSC3 expression and/or methylation in prostate cancer patients and obtained a first mechanistic insight into its function in N-glycosylation and carcinogenesis. 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 3997. doi:1538-7445.AM2012-3997