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- Harmine and Piperlongumine revert TRIB2-mediated drug resistancePublication . Machado, Susana; Silva, Andreia; De Sousa-Coelho, Ana Luísa; Duarte, Isabel; Grenho, Inês; Santos, Bruno F; Mayoral-Varo, Victor; Megias, Diego; Sánchez-Cabo, Fátima; Dopazo, Ana; Ferreira, Bibiana I.; Link, WolfgangTherapy resistance is responsible for most relapses in patients with cancer and is the major challenge to improving the clinical outcome. The pseudokinase Tribbles homologue 2 (TRIB2) has been characterized as an important driver of resistance to several anti-cancer drugs, including the dual ATP-competitive PI3K and mTOR inhibitor dactolisib (BEZ235). TRIB2 promotes AKT activity, leading to the inactivation of FOXO transcription factors, which are known to mediate the cell response to antitumor drugs. To characterize the downstream events of TRIB2 activity, we analyzed the gene expression profiles of isogenic cell lines with different TRIB2 statuses by RNA sequencing. Using a connectivity map-based computational approach, we identified drug-induced gene-expression profiles that invert the TRIB2-associated expression profile. In particular, the natural alkaloids harmine and piperlongumine not only produced inverse gene expression profiles but also synergistically increased BEZ235-induced cell toxicity. Importantly, both agents promote FOXO nuclear translocation without interfering with the nuclear export machinery and induce the transcription of FOXO target genes. Our results highlight the great potential of this approach for drug repurposing and suggest that harmine and piperlongumine or similar compounds might be useful in the clinic to overcome TRIB2-mediated therapy resistance in cancer patients.
- Generation of drug-resistant cell lines as a model to study pancreatic cancerPublication . Grenho, Inês Filipa Acácio; Ferreira, Bibiana I.; Link, WolfgangPancreatic cancer (PC) is among the most aggressive cancers in the world, characterized by an extremely high mortality/incidence ratio. Besides its aggressiveness, PC is usually diagnosed in an advanced and metastatic stage, which limits the treatment options available. Most of these tumors are unresectable by surgery, thus chemotherapy remains the only option available for treatment. However, a majority of these patients relapse within months and have a recurrence of the disease, usually more aggressive and no longer sensitive to the initial treatment. The major responsible for this relapse is the development of acquired therapy resistance. Our work focused on generating cell lines resistant to the current first line chemotherapy drug, Gemcitabine, and on their characterization. This allowed us to generate tools that will be crucial to unveil the mechanism driving acquired resistance in PC. Previous studies from our group and others demonstrated an association between the expression profiles of TRIBBLES pseudokinases and drug resistant phenotypes in other cancers. We evaluated the sensitivity of the generated cell lines to Gemcitabine and characterized them in terms of migration ability, cell death rate under stress and the expression of TRIBBLES proteins and EMT markers. Our results show that we successfully generated Gemcitabine-resistant cell lines, and that these cell lines are phenotypically different from the sensitive ones, showing fibroblastic-like features. Furthermore, we observed a reversible phenotypic switch when these cells undergo Gemcitabine treatment. They show different migration ability and increased mRNA expression of EMT markers, a hallmark of the epithelial-to-mesenchymal transition process. Moreover, in the resistant cells we observed higher TRIB2 protein expression levels and a decrease in the TRIB3 protein expression, compared with the sensitive cell lines. Overall, the phenotype associated with the resistant cells is concordant with drug resistance development by chronic Gemcitabine exposure.