Maximilian Marhold

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.

HIF1α Regulates mTOR Signaling and Viability of Prostate Cancer Stem Cells

Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSC), were recently identified and characterized in prostate cancer. A well-characterized murine model of prostate cancer was used to investigate the regulation of hypoxia-inducible factor 1α (HIF1A/HIF1α) in CSCs and a basal stem cell subpopulation (Lin−/Sca-1+/CD49f+) was identified, in primary prostate tumors of mice, with elevated HIF1α expression. To further analyze the consequences of hypoxic upregulation on stem cell proliferation and HIF1α signaling, CSC subpopulations from murine TRAMP-C1 cells (Sca-1+/CD49f+) as well as from a human prostate cancer cell line (CD44+/CD49f+) were isolated and characterized. HIF1α levels and HIF target gene expression were elevated in hypoxic CSC-like cells, and upregulation of AKT occurred through a mechanism involving an mTOR/S6K/IRS-1 feedback loop. Interestingly, resistance of prostate CSCs to selective mTOR inhibitors was observed because of HIF1α upregulation. Thus, prostate CSCs show a hypoxic deactivation of a feedback inhibition of AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway through HIF1α is critical for CSC quiescence and maintenance by attenuating CSC metabolism and growth via mTOR and promoting survival by AKT signaling. We also propose that prostate CSCs can exhibit primary drug resistance to selective mTOR inhibitors. Implications: This work contributes to a deeper understanding of hypoxic regulatory mechanisms in CSCs and will help devise novel therapies against prostate cancer. Mol Cancer Res; 13(3); 556–64. ©2014 AACR.

Recent developments and translational aspects in targeted therapy for metastatic breast cancer.

Biologically distinct subtypes of metastatic breast cancer (MBC) have been defined by multiple efforts in recent years, showing broad heterogeneity at the molecular level of disease. Throughout this endeavour, oncogenic drivers within MBC were identified as potential therapeutic targets. With recent results from clinical trials targeting these well-known cancer-promoting pathways, this review is trying to elucidate as well as summarise current new therapeutic aspects in MBC and shed light on translational aspects within this entity.

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

Abstract 2697: Drug combinations for breast cancer based on synthetic lethality screens in yeast

Synthetic lethality describes an interdependent relationship between two genes, where the loss of either one alone can be compensated, while the simultaneous loss of both genes causes a non-viable phenotype. In recent years, first therapeutics based on this concept entered the clinic, most notably the PARP inhibitors for BRCA1/2-mutated cancers. In the present study, we analyzed synthetic lethal interactions in yeast to identify new and potentially synergistic drug combinations for breast cancer therapy. We were able to confirm significantly enhanced cytotoxicity for predicted drug pairs in breast cancer cell lines in vitro. First, a predictor built from publicly available yeast genetic interactions in the Data Repository of Yeast Genetic INteraction (DRYGIN) was used to predict potential synthetic lethal genetic interactions in human. Independently, a data set containing all pharmacological approaches, targeted or cytostatic, in breast cancer therapy was created. These drug combinations and their respective targets were then analyzed regarding their coverage of predicted synthetic lethal interactions. New drug combinations, previously unused in breast cancer therapy, were identified in silico by combining drugs already in use for breast cancer therapy (individually or in other combinations) in such a manner that their combination covers one or more potential synthetic lethal gene pairs. From this set of new drug combination and their synthetic lethal gene pairs we further pursued the predicted interdependencies between farnesyl diphosphate synthase (FDPS) and tubulin, beta 1 (TUBB1) and between FDPS and phosphoinositide-dependent kinase-1 (PDPK1) as well as prostaglandin-endoperoxide synthase 2 (PTGS2). Drugs targeting these genes are celecoxib (PDPK1, PTGS2), an anti-inflammatory drug, and zoledronic acid (FDPS), a bone degradation inhibitor, as well as the cytotoxic agent paclitaxel (TUBB1). We performed cell viability and cytotoxicity assays to determine therapeutic effects of celecoxib, zoledronic acid and paclitaxel alone and in combination on selected breast cancer cell lines. Our results showed statistically significant decreases in cell viability for the combinational treatment with zoledronic acid and paclitaxel as well as with the per se non-cytotoxic combination of zoledronic acid and celecoxib when compared to single agent treatment. In conclusion, we present a bioinformatics approach to predict potentially synergistic gene interactions based on synthetic lethality found in yeast and a strategy for utilizing these interactions for identifying new potentially synergistic drug combinations. Citation Format: Michael Schwarz, Erwin Tomasich, Maximilian Marhold, Andreas Heinzel, Paul Perco, Peter Horak, Bernd Mayer, Michael Krainer. Drug combinations for breast cancer based on synthetic lethality screens in yeast. [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 2697.

Abstract 2407: Neuroendocrine and luminal progenitors drive cancer progression in prostate cancer

Neuroendocrine differentiation of prostate cancer (PCa) occurs frequently during the development of castration resistance and rarely in primary tumors. Using the transgenic mouse model of prostate cancer (TRAMP) in a B6/C57 background, we were able to detect, isolate and further characterize basal, luminal and neuroendocrine subsets of cancer cells. We performed allograft experiments in NSG mice to ensure cellular stem and progenitor properties as well as metastatic potential, and thus created a murine model of neuroendocrine prostate cancer. Whilst all three previously described cell populations were present in approximately three quarters of primary TRAMP tumors, some tumors lacked basal stem cells and showed a more aggressive phenotype. These tumors, mainly consisting of small cancer cells, expressed markers of neuroendocrine differentiation such as synaptophysin and chromogranin A as shown by immunohistochemistry and are further referred to as neuroendocrine carcinomas (NECs), as ruled by an experienced uropathologist of our institution. Adenocarcinoma-like tumors (ACs), in contrast, showed high expression of cytokeratins and retained glandular histology. Using fluorescence-activated cell sorting (FACS) against newly discovered NEC markers within the TRAMP model, we found a relative increase of neuroendocrine progenitors in prostate NECs compared to ACs (approximately 75 vs. 33%, respectively). In ACs, on the other hand, luminal progenitors were found to be the predominant drivers of cancer progression. To further evaluate this, we transplanted single-cell-suspensions into NSG mice without androgen supplementation and observed successful engraftment of both non-basal cell populations. Additionally, we were able to passage the resulted tumors for at least two generations and observed maintenance of histology and biological features for tumors of both luminal and neuroendocrine origin. Based on a RNA sequencing, we were able to define gene signatures for neuroendocrine and luminal progenitors, uncovering a number of novel potential therapeutic targets. Ultimately, we were able to evaluate the prognostic value of the signatures obtained from mice in the human disease by in-silico analyses of publically available gene expression profile databases. In conclusion, we created and characterized a murine model of neuroendocrine prostate cancer using flow cytometry and murine allografts. Further, we established gene expression signatures of luminal and neuroendocrine progenitors and translated them to the human disease. Our findings foster the understanding of neuroendocrine differentiation in prostate cancer and may help in developing new targeted approaches in this entity. Citation Format: Maximilian Marhold, Erwin Tomasich, Simon Udovica, Gerwin Heller, Corinna Altenberger, Andreas Spittler, Reinhard Horvat, Peter Horak, Michael Krainer. Neuroendocrine and luminal progenitors drive cancer progression in prostate cancer. [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 2407.

Abstract 2772: SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

DNA methylation is one of the major epigenetic mechanisms regulating transcriptional activity and is involved in the pathogenesis of non-small cell lung cancers (NSCLC). In a recent study we identified a large number of tumor-specifically methylated genes in NSCLCs (Heller et al., Carcinogenesis 2013). For further detailed analyses we selected the genes SPAG6 (Sperm Associated Antigen 6) and L1TD1 (LINE-1 Type Transposase Domain Containing 1). By analysing publically available IlluminaHiSeq RNA-seq data we observed frequent downregulation of SPAG6 and L1TD1 mRNA expression in primary tumor (TU) samples compared to corresponding non-malignant lung tissue (NL) samples of NSCLC patients. In addition, we investigated SPAG6 and L1TD1 mRNA expression in 5 NSCLC cell lines and found SPAG6 as well as L1TD1 mRNA expression frequently downregulated in all of these cell lines compared to normal human bronchial epithelial cells (NHBECs). Subsequently, we treated cells of NSCLC cell lines which did not express SPAG6 or L1TD1 with the epigenetically active drugs 5-aza-2′-deoxycytidine and Trichostatin A and observed re-expression of both genes suggesting that transcriptional regulation of SPAG6 and L1TD1 is mediated by DNA methylation in NSCLCs. Bisulfite genomic sequencing of parts of the 5′ region of SPAG6 and L1TD1 revealed that the vast majority of CpG sites indeed are methylated in NSCLC cells in contrast to NHBECs. Moreover, we analysed SPAG6 and L1TD1 methylation in TU and NL samples of 147 stage I-III NSCLC patients using the gene-specific approach methylation-sensitive high resolution melt analysis (MS-HRM). Differences in SPAG6 as well as in L1TD1 methylation between TU and NL samples were statistically significant for both genes and confirmed that SPAG6 and L1TD1 are tumor-specifically methylated in NSCLCs. Additionally, we investigated SPAG6 and L1TD1 protein expression in TU and NL samples of 35 NSCLC patients by immunohistochemistry. In the vast majority of SPAG6 or L1TD1 methylated TU samples, protein expression of these genes was downregulated in tumor cells. Moreover, we performed cell proliferation, cell viability and colony formation assays in vitro and observed that ectopic expression of L1TD1 but not of SPAG6 reduced tumor cell proliferation, viability and the ability of NSCLC cells to form colonies. To further investigate the role of L1TD1 in the pathogenesis of NSCLCs, in vivo studies are being carried out. Overall, our results demonstrate that DNA methylation is the major mechanism for frequent downregulation of SPAG6 and L1TD1 expression in NSCLCs. Citation Format: Corinna Altenberger, Gerwin Heller, Barbara Ziegler, Erwin Tomasich, Maximilian Marhold, Leonhard Mullauer, Gyorgy Lang, Adelheid End-Pfutzenreuter, Balazs Dome, Britt-Madeleine Arns, Walter Klepetko, Christoph C. Zielinski, Sabine Zochbauer-Muller. SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers. [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 2772.

Abstract 3047: Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tumor initiating subpopulations of cancer cells, also known as cancer progenitor or cancer stem cells (CSCs) were recently identified and characterized in prostate cancer. Emerging evidence suggests that hypoxia-inducible factor 1 alpha (HIF1A) contributes to CSC maintenance and influences the PI3K/AKT/mTOR axis, a pathway which is frequently altered in prostate cancer. In our study, we assessed the effects of hypoxia on PI3K/AKT/mTOR signaling in a subset of basal prostate cancer stem cells in a murine model of prostate cancer. First, we successfully isolated Lin-/Sca-1+/CD49f+ basal stem cell subpopulations from primary prostate tumors of TRAMP (transgenic adenocarcinoma of mouse prostate) mice. We observed a consistent decrease in S6 phosphorylation within the CSC subpopulation, in agreement with an mTOR pathway inhibition. However, AKT phosphorylation in the CSC subpopulation was simultaneously elevated, thus prompting further investigations. To analyze the molecular pathways leading to this deregulation, we used a cell line model and isolated Sca-1+/CD49f+ CSC-like subpopulations from the murine prostate cancer cell line TRAMP-C1 and CD44+/CD49f+ CSC-like subpopulations from the androgen independent human prostate cancer cell line DU145. We used sphere formation assays and expression of Notch 1 and Oct 3/4 to confirm the stem and progenitor cell properties of the sorted subpopulations. We could confirm the decrease in mTOR/S6 signaling and identify the upregulation of AKT through IRS-1 mediated feedback loop in prostate CSC-like cells under hypoxic conditions. This AKT/mTOR deregulation was promptly reversed in normoxia, suggestive of the involvement of hypoxia inducible factors. We found elevated HIF1A protein levels as well as increased target gene expression in CSC subpopulations in vitro and in vivo. Further, lentiviral knockdown of HIF1A restored AKT/mTOR activity in CSC-like cells. We could show that CSC-like prostate cancer cells with elevated expression of basal stem cell markers, such as CD44, Sca-1 or CD49f, show a hypoxic deactivation of feedback inhibition on AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway via HIF1A may be critical for cancer stem cell quiescence and maintenance, slowing down cancer stem cell metabolism and growth via mTOR and promoting their survival via AKT signaling. Citation Format: Maximilian Marhold, Erwin Tomasich, Zuzana Pernicova, Radek Fedr, Karel Soucek, Andreas Spittler, Michael Krainer, Peter Horak. Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2014-3047

Abstract LB-201: Prostate cancer stem cells display low mTOR activity and resistance to mTOR inhibitors in hypoxia

Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSCs), were recently identified and characterized in prostate cancer (PCa). We hypothesized that one of the frequently altered molecular signaling pathways in PCa, the PI3K/AKT pathway, and mammalian target of rapamycin (mTOR) in particular, play a role in PCa stem cell maintenance. In addition, hypoxia is known to be involved in cancer stem cell maintenance and to have a regulating effect on mTOR signaling. In our study, we evaluated the effects of mTOR inhibitors on proliferation of human as well as murine CSCs in hypoxic conditions in vitro. We isolated CSC like subpopulations from the androgen independent human prostate cancer cell line DU145 using fluorescence activated cell sorting according to their expression of the stem cell marker CD44. Further, we sorted the murine prostate cancer cell line TRAMP-C1 based on the expression of the stem cell markers Sca-1 and CD49f. We used sphere formation assays to confirm the stem and progenitor cell properties of the sorted subpopulations. Next, we treated the CSC and non-CSC cell populations with mTOR inhibitors rapamycin and everolimus under normoxic (20% O2) and hypoxic (3% O2) conditions in order to determine their viability at time points up to 72 hours. Our results suggest that CSC like prostate cancer cells with elevated expression of stem cell markers, such as CD44, Sca-1 or CD49f, are more resistant to mTOR inhibitors when compared to their non-CSC counterparts. Most interestingly, while the non-CSC subpopulation of prostate cancer cells displays increased sensitivity to mTOR inhibitors under hypoxic conditions, the viability of CSCs remains essentially unaffected. Immunoblotting reveals lower mTOR activity in prostate cancer stem cells compared to non-CSCs. Hypoxia in this context leads, as expected, to a decrease in mTOR activity in both subpopulations, though this effect is significantly pronounced in CSCs like subpopulations of both cancer cell lines in our study. At the same time, we do not observe alterations in signaling through other PI3K downstream effectors, such as AKT. We propose that prostate cancer stem cells might be resistant against small molecule inhibitors of mTOR and that inhibitors targeting multiple kinases along the PI3K/AKT/mTOR axis would be more effective in this setting. We also hypothesize that intratumoral hypoxia might contribute to the mTOR resistance of prostate cancer stem cells by inhibiting mTOR signaling and promoting cell growth through other, PI3K dependent pathways. These findings could be helpful to assess the impact of mTOR targeted therapy in prostate cancer, notably in light of ongoing clinical trials of mTOR inhibitors in this entity. 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 LB-201. doi:1538-7445.AM2012-LB-201