Stefan Haemmig

miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor κB (NF-κB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-κB is in part owing to downregulation of negative regulators of NF-κB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-κB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-κB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFα- and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-κB confirming that miR-125b is implicated in NF-κB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy.

miR-29b Mediates NF-κB Signaling in KRAS-Induced Non–Small Cell Lung Cancers

A global understanding of miRNA function in EGFR signaling pathways may provide insights into improving the management of KRAS-mutant lung cancers, which remain relatively recalcitrant to treatment. To identify miRNAs implicated in EGFR signaling, we transduced bronchial epithelial BEAS-2B cells with retroviral vectors expressing KRAS(G12V) and monitored miRNA expression patterns by microarray analysis. Through this approach, we defined miR-29b as an important target for upregulation by mutant KRAS in non-small cell lung cancers. Cell biologic analyses showed that pharmacologic inhibition of EGFR or MEK was sufficient to reduce levels of miR-29b, while PI3K inhibition had no effect. In KRAS(G12V)-transduced BEAS-2B cells, introduction of anti-miR-29b constructs increased the sensitivity to apoptosis, arguing that miR-29b mediated apoptotic resistance conferred by mutant KRAS. Mechanistic investigations traced this effect to the ability of miR-29b to target TNFAIP3/A20, a negative regulator of NF-κB signaling. Accordingly, overexpression of an miR-29b-refractory isoform of TNFAIP3 restored NF-κB and extrinsic apoptosis, confirming that TNFAIP3 is a functionally relevant target of miR-29b. We also noted that miR-29b could confer sensitivity to intrinsic apoptosis triggered by exposure to cisplatin, a drug used widely in lung cancer treatment. Thus, miR-29b expression may tilt cells from extrinsic to intrinsic mechanisms of apoptosis. Overall, our results reveal a complexity in cancer for miR-29b, which can act as either an oncogene or tumor suppressor gene depending on signaling context. Cancer Res; 76(14); 4160-9. ©2016 AACR.

MicroRNA dysregulation in the tumor microenvironment influences the phenotype of pancreatic cancer

Cellular interactions in the tumor microenvironment influence neoplastic progression in pancreatic ductal adenocarcinoma. One underlying mechanism is the induction of the prognostically unfavorable epithelial–mesenchymal-transition-like tumor budding. Our aim is to explore the expression of microRNAs implicated in the regulation of tumor budding focusing on the microenvironment of the invasive front. To this end, RNA from laser-capture-microdissected material of the main tumor, tumor buds, juxta-tumoral stroma, tumor-remote stroma, and non-neoplastic pancreatic parenchyma from pancreatic cancer cases with (n=7) and without (n=6) tumor budding was analyzed by qRT-PCR for the expression of a panel of miRNAs that are known to be implicated in the regulation of epithelial–mesenchymal transition, including miR-21, miR-183, miR-200b, miR-200c, miR-203, miR-205, miR-210, and miR-217. Here we show that at the invasive front of pancreatic ductal adenocarcinoma, specific microRNAs, are differentially expressed between tumor buds and main tumor cells and between cases with and without tumor budding, indicating their involvement in the regulation of the budding phenotype. Notably, miR-200b and miR-200c were significantly downregulated in the tumor buds. Consistent with this finding, they negatively correlated with the expression of epithelial–mesenchymal-transition-associated E-cadherin repressors ZEB1 and ZEB2 in the budding cells (P<0.001). Interestingly, many microRNAs were also dysregulated in juxta-tumoral compared to tumor-remote stroma suggesting that juxta-tumoral stroma contributes to microRNA dysregulation. Notably, miR-200b and miR-200c were strongly downregulated while miR-210 and miR-21 were upregulated in the juxta-tumoral vs tumor-remote stroma in carcinomas with tumor budding. In conclusion, microRNA targeting in both tumor and stromal cells could represent a treatment option for aggressive pancreatic cancer.

Abstract 189: KRAS-induced microRNA-29b attenuates apoptosis in non-small cell lung cancer

EGFR tyrosine kinase or downstream components of this pathway, e.g. KRAS, are frequently mutated in NSCLC patients. These mutations enable a constitutive active downstream signaling pathway and result in lung cancer development by uncontrolled proliferation and survival. MicroRNAs (miRNAs) are noncoding short nucleic acids, and are implicated in NSCLC progression. We aim to identify miRNAs that are regulated by EGFR downstream key molecules and to investigate their role in malignant processes of NSCLC. We demonstrate that viral transduction of bronchial epithelial cells (BEAS-2B) with mutated KRAS induces an activated downstream signaling pathway. Subsequently, a miRNA array profiling of KRAS-transduced cells identified upregulated miR-29b expression. We confirmed the upregulation of miR-29b expression in individual clones of KRAS-transduced cells. Moreover, the upregulated miR-29b expression was affirmed in NSCLC tissue harboring KRAS mutations by comparing it to the expression in matched normal tissue. Furthermore, to elucidate the downstream signaling pathway involved in the underlying regulation of miR-29b expression, NSCLC cell lines were treated with EGFR and MEK inhibitors which lead to downregulated miR-29b expression. Next, to study the role of miR-29b in NSCLC apoptosis, we overexpressed miR-29b in adenocarcinoma cells (A549) and performed cell death assays. Interestingly, and in contrast to other cell systems, A549 cells transfected with precursor miR-29b demonstrated attenuated TNF-α plus actinomycin D-induced apoptosis compared to control-transfected cells. Conversely, the downregulation of endogenous miR-29b levels by overexpressing antagomiR-29b, lead to increased sensitivity to apoptosis compared to scramble-transduced A549 cells. Consistently, KRASG12V-transduced BEAS-2B cells showed reduced sensitivity to TNF-α plus actinomycin D-induced apoptosis, while the effect was reversed in KRAS/antagomiR-29b co-transduced cells, indicating that KRAS attenuates apoptosis by overexpressing miR-29b. Furthermore, miR-29b is predicted to target the pro-apoptotic BH3-only protein Bim and the tumor necrosis alpha-induced protein 3 (TNFAIP3/A20), an important negative regulator of the NFκB signaling pathway and thereby may confer resistance to apoptosis. We plan to verify the predicted regulation of these genes by performing luciferase reporter assays and Western blot analysis. In addition, NFκB activation will be assessed by verifying nuclear and cytoplasmic fractions of cells transfected with precursor miR-29b. Finally, we plan to stably overexpress TNFAIP3 in NSCLC cell lines and assess apoptosis by overexpressing miR-29b. In summary, we provide a novel regulation mechanism of miR-29b by KRAS signaling, leading to attenuated apoptosis in NSCLC. Citation Format: Stephanie Langsch, Stefan Haemmig, Ulrich Baumgartner, Mario P. Tschan, Erik Vassella. KRAS-induced microRNA-29b attenuates apoptosis in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 189. doi:10.1158/1538-7445.AM2015-189

Abstract 3067: microRNAS implicated in the regulation of NF-κB activity in glioma tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Gliomas are among the most prognostically discouraging neoplasia in human and there is basically no curative option for glioma patients. Nuclear factor κB (NF-κB) is a key effector associated with temozolomide (TMZ) resistance in glioma tumours. This is at least in part due to down-regulation of TNFAIP3, which is a critical negative regulator of NF-κB activity. Downregulation of this gene is strongly associated with resistance to TMZ, but the molecular mechanism of dysregulation is largely unknown. We hypothesize that microRNAs that are implicated in the NF-κB pathway may be important regulators of resistance to alkylating agents in glioma tumours. TNFAIP3 is a predicted target of brain-enriched microRNAs miR-125a and miR-125b. Consistent with these findings, luciferase activity assays of reporter plasmids containing miR-125a/b target sites revealed that TNFAIP3 is directly regulated by physiological concentrations of miR-125a/b. In addition, overexpression of miR-125a/b resulted in reduced mRNA and protein levels of TNFAIP3 and, as a consequence, also in a significant induction of NF-κB activity. Consistent with these findings, glioblastoma cells overexpressing these miRNAs showed a strong upregulation of the anti-apoptotic proteins Bcl-2 and BIRC3. Interestingly, this was significantly associated with resistance of glioma cells to TNFalpha- and TRAIL-induced apoptosis and also resistance to TMZ. A siRNA directed against TNFAIP3 and miR-125a/b gave rise to similar phenotypes suggesting that TNFAIP3 is a major target of miR-125a/b. In glioblastoma tissues, high level expression of either miRNA was highly correlated with nuclear localisation of NF-κB confirming that miR-125a/b are implicated in NF-κB signalling. Most remarkably, miR-125a/b expression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that these miRNAs are important predictors of response to therapy. However, we found no correlation between miR-125a/b levels and the MGMT methylation status. In conclusion, miR-125a/b expression is a predictor of the patients response to chemotherapy. Since miR-125a/b confers resistance to TMZ, antisense miR-125a/b might potentially be used in adjuvant therapy for the treatment of glioma tumours. Citation Format: Stefan Haemmig, Erik Vassella. microRNAS implicated in the regulation of NF-κB activity in glioma tumors. [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 3067. doi:10.1158/1538-7445.AM2013-3067