Christoph Minichsdorfer

Genome-Wide miRNA Expression Profiling Identifies miR-9-3 and miR-193a as Targets for DNA Methylation in Non–Small Cell Lung Cancers

Purpose: The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on miRNA silencing in non–small cell lung cancers (NSCLC). Experimental Design: We conducted microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. miRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding nonmalignant lung tissue samples of 101 patients with stage I–III NSCLC. Results: By comparing microarray data of untreated and drug-treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analyzed. Moreover, miR-9-3 and miR-193a were found to be tumor specifically methylated in patients with NSCLC. We observed a shorter disease-free survival of patients with miR-9-3 methylated lung squamous cell carcinoma (LSCC) than patients with miR-9-3 unmethylated LSCC by multivariate analysis [HR = 3.8; 95% confidence interval (CI), 1.3–11.2, P = 0.017] and a shorter overall survival of patients with miR-9-3 methylated LSCC than patients with miR-9-3 unmethylated LSCC by univariate analysis (P = 0.013). Conclusions: Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in patients with LSCC. Clin Cancer Res; 18(6); 1619–29. ©2012 AACR.

Autocrine amplification loop in statin-induced apoptosis of human melanoma cells

Background and purpose:  Beside their cholesterol lowering effect, statins exert pleiotropic effects, which include anti‐inflammatory, immunosuppressive and anti‐proliferative actions. In higher concentrations, statins trigger apoptosis in primary cells and tumour cells. In particular, melanoma cells have been found to be susceptible to statin‐induced apoptosis, although only after longer incubation times. The molecular mechanisms behind this delayed drug‐induced apoptosis are still unclear.

Autocrine secretion of 15d‐PGJ2 mediates simvastatin‐induced apoptotic burst in human metastatic melanoma cells

Despite new therapeutic approaches, metastatic melanomas still have a poor prognosis. Statins reduce low‐density lipoprotein cholesterol and exert anti‐inflammatory and anti‐proliferative actions. We have recently shown that simvastatin triggers an apoptotic burst in human metastatic melanoma cells by the synthesis of an autocrine factor.

Tocilizumab unmasks a stage-dependent interleukin-6 component in statin-induced apoptosis of metastatic melanoma cells.

The interleukin (IL)-6 inhibits the growth of early-stage melanoma cells, but not metastatic cells. Metastatic melanoma cells are susceptible to statin-induced apoptosis, but this is not clear for early-stage melanoma cells. This study aimed to investigate the IL-6 susceptibility of melanoma cells from different stages in the presence of simvastatin to overcome loss of growth arrest. ELISA was used to detect secreted IL-6 in human melanoma cells. The effects of IL-6 were measured by western blots for STAT3 and Bcl-2 family proteins. Apoptosis and proliferation were measured by caspase 3 activity, Annexin V staining, cell cycle analysis, and a wound-healing assay. Human metastatic melanoma cells A375 and 518A2 secrete high amounts of IL-6, in contrast to early-stage WM35 cells. Canonical IL-6 signaling is intact in these cells, documented by transient phosphorylation of STAT3. Although WM35 cells are highly resistant to simvastatin-induced apoptosis, coadministration with IL-6 enhanced the susceptibility to undergo apoptosis. This proapoptotic effect of IL-6 might be explained by a downregulation of Bcl-XL, observed only in WM35 cells. Furthermore, the IL-6 receptor blocking antibody tocilizumab was coadministered and unmasked an IL-6-sensitive proportion in the simvastatin-induced caspase 3 activity of metastatic melanoma cells. These results confirm that simvastatin facilitates apoptosis in combination with IL-6. Although endogenous IL-6 secretion is sufficient in metastatic melanoma cells, exogenously added IL-6 is needed for WM35 cells. This effect may explain the failure of simvastatin to reduce melanoma incidence in clinical trials and meta-analyses.

Statins induce apoptosis in human melanoma cells via multiple pathways

Melanoma is one of the most resistant cancer types to chemotherapy. This is mostly mediated by its resistance against apoptosis. Beside their cholesterol-lowering effects, HMG-CoA-reductase inhibitors have a wide range of pleiotropic effects. Most importantly, they induce cell cycle arrest and apoptosis in various tumour cells including rhabdomyosarcoma [1]. Therefore, we investigated the susceptibility of two human melanoma cell lines to statin treatment. We treated the melanoma cell lines A375 and 518A2 with simvastatin and other statin derivates. In both cell lines apoptosis was executed by caspase 3 by different statins in a time and concentration-dependent manner according to their lipophilicity. In addition, we investigated mechanisms leading to caspase activation in more detail. Most interestingly, an apoptotic burst was observed after 48 hours of treatment. Delayed caspase 8 activation after 24 hours was attributable to an amplification loop. The tremendous boost in caspase 3 and 9 activation was mediated by caspase 8 via cleavage of Bid in 518A2 cells. This observation favours the conjecture that an autocrine factor is responsible for caspase 8 activation. To check for this, we designed a medium change experiment. Caspase 8 activation was decreased by 80% in 518A2 cells by adding of fresh medium every 4 hours, which confirms the hypothesis that a suicide factor is secreted into the medium. In the Western blot full length Bid degradation and cleavage of procaspase 8 also followed the wash protocol. Co-treatment with cycloheximide, but not lactacystin, abrogated caspase 3 and 8 activation in both cell lines, which proves that statin-exerted stress needs transcriptional activity to induce apoptosis in melanoma cells. In this work we highlight statins as activators of multiple apoptotic pathways in melanoma cells. Moreover, it is feasible to initiate a suicide factor which leads to a caspase 8-mediated apoptotic burst.

SERPINB7 Expression Predicts Poor Pancreatic Cancer Survival Upon Gemcitabine Treatment

Stratification of patients with pancreatic ductal adenocarcinoma (PDAC) remains a key challenge in the field of clinical oncology. No predictive biomarkers have yet been found for any available treatment options. Previously, we identified SERPINB7 as a putative biomarker for PDAC and thus, herein, we aimed to validate our previous findings and assessed the predictive value of SERPINB7. Patients who underwent surgery and received gemcitabine (gem) or gemcitabine plus nab-paclitaxel (gem/nab) as adjuvant therapy, between 2011 and 2017, were included in this study (n = 57). Expression level of SERPINB7 was assessed in tumor tissue by immunohistochemistry (IHC) and RNA in situ hybridization (RNA ISH). Its association with disease-free survival (DFS) and overall survival (OS) was investigated. While IHC did not show any correlation between survival and the protein level of SERPINB7, RNA ISH revealed that expression of SERPINB7 was associated with a poor DFS (P = .01) and OS (P = .002) in the gem group but not in the gem/nab. Adjusted Cox-regression analysis confirmed the independent predictive value of SERPINB7 on OS (P = .006, HR: 3.47; 95% CI: 1.49–8.09) in the gem group. In conclusion, SERPINB7 was identified as the first predictive RNA biomarker for PDAC. This study suggests that patients who expressed SERPINB7 might receive another treatment than gem alone.

Abstract 336: IL-6 primes melanoma cells from early stages to statin induced apoptosis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Statins trigger apoptosis in tumour cells in vitro and, particularly, melanoma cells are susceptible to statin induced apoptosis. Growth of normal melanocytes and early stage melanoma can be inhibited by IL-6, whereas metastatic melanoma cells are mostly resistant to the anti-proliferative effects of IL-6. It has been shown that nearly 50% of metastatic melanoma secrete IL-6 into the supernatant. Nevertheless until now the stage dependant effects of IL-6 on melanoma cells are not fully understood. Methods: The secretion of IL-6 by A375, 518A2 human metastatic melanoma cells was compared with WM 35 cells which derive from an early lesion (radial growth phase) and investigated with ELISA. The sensitivity toward statin induced caspase 3 activation was investigated in these cells in the absence and presence of IL-6 or an anti IL-6 receptor antibody (tocilizumab). Apoptosis was also confirmed by FACS analyses for AnnexinV/PI staining and proliferation by cell cycle analysis using PI staining in such treated cells. Phosphorylation of Stat3 and the regulation of pro- (Bax, Bak) and anti-apoptotic proteins (Bcl-2, Bxl-XL) were studied by Western blot. Results: Melanoma cells derived from late stage lesions (A375, 518A2) secrete high amounts of IL-6 in contrast to WM35 cells. IL-6 signalling is intact in A375, 518A2 and WM 35 cells indicated by IL-6 triggered phosphorylation of Stat-3. Most interestingly, the A375 and 518A2 cells show a high sensitivity to simvastatin induced apoptosis with EC50 of 0,52 μM (518A2) and 1,7 μM (A375) in contrast to WM35 cells which are more resistant (EC50 16 μM). Co-treatment with IL-6 leads to an augmentation of the statin induced apoptosis in WM35 cells. Statin induced apoptosis can be mediated by the intrinsic apoptotic pathway which requires the loss of mitochondrial membrane potential. This is often alleviated by a downregulation of antiapoptotic Bcl-2 family members. Interestingly IL-6 treatment led to a marked decrease in the amount of Bcl-2 and Bcl-XL in WM35 cells which may explain the increased activation of apoptosis. Since A375 and 518A2 secrete high amounts of IL-6 we investigated the effects of the anti IL-6 receptor antibody tocilizumab. Conversely interruption of the IL-6 signalling with tocilizumab had no effect on the proliferation of 518A2 and A375 cells. Moreover the simvastatin induced caspase 3 activation was not decreased by blockage of the IL-6 signalling. Conclusion: Taken together our data prove different sensitivities of melanoma cells from various stages to statin induced apoptosis. Moreover, IL-6 facilitates apoptosis in early stage melanoma cells by the down-regulation of anti-apoptotic proteins. Nevertheless, these pro-apoptotic effects of IL-6 are not observed in metastatic melanoma cells indicating a stage specific role of IL-6 in the development and tumorigenesis of melanoma. Citation Format: Christoph Minichsdorfer, Christine Wasinger, Martin Hohenegger. IL-6 primes melanoma cells from early stages to statin induced apoptosis. [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 336. doi:10.1158/1538-7445.AM2014-336

Abstract 2940: Statin induced apoptosis in human melanoma cells is prevented by inhibition of caspase 2 and translational activity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Statins may trigger apoptosis in tumour cells in vitro and, particularly, melanoma cells are susceptible to statin induced apoptosis. Lipophilic statins (e.g. Atorvastatin, Simvastatin) induce strong caspase 3 activation after 48h of treatment, whereas hydrophilic statins (e.g. Pravastatin, Rosuvastatin) do not trigger caspase activation. Recently, we demonstrated the presence of an autocrine amplification loop which increases apoptosis by a caspase 8 dependent pathway after treatment with lipophilic statins (Minichsdorfer and Hohenegger, Br J Ph 2009). It is known that activation of caspase 2 may prime melanoma cells for death receptor induced apoptosis, however, although an association between caspase 2 activation and RhoB upregulation was shown, the significance of this interaction is still not clear. Methods: We investigated the activity of caspase 2, 3 and 8 in A375 and 518A2 human metastatic melanoma cells after statin treatment in the presence and absence of a caspase 2 inhibitor (Z-VDVAD-FMK) or cycloheximide by cleavage of specific fluorescent caspase substrates. Upregulation of RhoB after statin treatment as well as the influence of the co-treatment with the isoprenoids farnesylpyrophosphate (FPP) or geranylgeranylpyrophosphate (GGPP) was studied by Western blot. Results: Exposure of these cells to simvastatin led to increased caspase 2 activation which is abrogated by co-application of the caspase 2 inhibitor Z-VDVAD-FMK. Moreover, treatment with Z-VDVAD-FMK also prevented caspase 8 and caspase 3 activation. We could show that production of an autocrine factor needs an intact translation machinery. Inhibition of translation by co-administration of cycloheximide prevented apoptotic morphological changes as well as caspase 3 and caspase 8 activation in A375 and 518A2 cells. Since caspase 2 may interact with RhoB, we also investigated the effect of statin treatment on RhoB expression and observed an upregulation of RhoB which was inhibited by the addition of cycloheximide in both cell lines. However, isoprenoids were not able to reverse RhoB upregulation or PARP cleavage in 518A2 cells. Conclusion: Overall, our data provide evidence for an important role of caspase 2 in statin induced apoptosis of 2 human metastatic melanoma cell lines. These findings may help to understand the function of caspase 2 in the induction of apoptosis. Moreover, we observed upregulation of RhoB following statin treatment in these cell lines. However, additional experiments are necessary to fully understand the role of RhoB in statin induced apoptosis. Citation Format: Christoph Minichsdorfer, Christine Wasinger, Evelyn Sieczkowski, Atil Bihter, Gerwin Heller, Sabine Zochbauer-Muller, Martin Hohenegger. Statin induced apoptosis in human melanoma cells is prevented by inhibition of caspase 2 and translational activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2940. doi:10.1158/1538-7445.AM2013-2940

Abstract 122: MiR-9-3 and miR-193a are targets for DNA methylation in non-small cell lung cancers

DNA methylation (referred to as methylation) is part of the epigenetic gene regulation complex which is relevant for the pathogenesis of different malignant diseases including non-small cell lung cancers (NSCLC). Recently, it has been reported that besides protein encoding genes also microRNA (miRNA) encoding genes may be targets for methylation in NSCLCs, however, the number of known methylated miRNA genes is still small. To investigate the role of methylation on miRNA gene silencing in NSCLCs, we performed microarray expression analyses of 856 miRNA genes in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. Comparing microarray data of untreated and drug treated A549 cells, we identified 33 miRNA genes whose expression was upregulated after drug treatment and which are associated with a CpG island. Methylation of these miRNA genes was determined in 11 NSCLC cell lines by methylation-sensitive high resolution melting (MS-HRM) analysis and 30 of 33 (91%) miRNA genes were found to be methylated in at least 1 NSCLC cell line. In addition, methylation of the miRNA genes miR-9-3 and miR-193a was also analysed in primary tumors and corresponding non-malignant lung tissue samples of 101 stage I-III NSCLC patients and was found to be tumor-specific. Moreover, we compared miR-9-3 and miR-193a methylation results and clinico-pathological characteristics of NSCLC patients. We found a shorter disease-free survival of miR-9-3 methylated lung squamous cell carcinoma (LSCC) patients compared to miR-9-3 unmethylated LSCC patients by multivariate analysis (HR = 3.8, 95% CI = 1.3 to 11.2, p = 0.017) and a shorter overall survival of miR-9-3 methylated LSCC patients compared to miR-9-3 unmethylated LSCC patients by univariate analysis (p = 0.013). In conclusion, our results suggest that methylation is an important mechanism for silencing certain miRNA genes in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in LSCC patients. 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 122. doi:1538-7445.AM2012-122

IL-6-mediated migration of human metastatic melanoma cells is reduced by simvastatin treatment

Background In 1987 the HMG-CoA reductase inhibitors, statins, were first marketed and are now widely used as well-tolerated therapeutics for hypercholesterolemia. High interleukin 6 (IL-6) plasma levels in melanoma patients are linked to a higher tumour burden and reduced overall survival. We have recently shown that simvastatin triggers apoptosis in human metastatic melanoma cells which is associated with concentration-dependent changes in autocrine IL-6 secretion. Here, we investigated IL-6 signalling with respect to proliferation and migration in human metastatic melanoma cells under statin application.