Final Report Summary - NUMIRDT (NUCLEOLAR-DEPENDENT SECRETION OF MICRORNA IN A MODEL OF DOXORUBICIN AND TRASTUZUMAB CARDIOMYOPATHY.) IntroductionBreast cancer (BC) is worldwide the most common cancer in women. Combined Doxorubicin (Dox) and trastuzumab (Trz) anticancer therapy is highly effective but associated with significant cardiotoxicity. The pathophysiological mechanisms of Dox/Trz are still unclear (Adão R et al., 2013) but recent experimental evidence suggests that anticancer drugs can target resident cardiac progenitor cells (CPCs) further decreasing heart tissue homeostasis and repair process (De Angelis A et al., 2010; Barth AS et al., 2012; Duran JM et al.,2014). We have previously shown that the nucleolar protein nucleophosmin (NPM) acts as a sensor of stress in cardiac cells treated with Dox (Avitabile D et al., 2011). NPM is essential during DNA damage response and its function could be affected by Trz (Le X et al., 2006; Koike A et al., 2010; Neganova I et al., 2011; Sekhar KR et al., 2011). Moreover, recent reports, have shown that NPM could be found in the extracellular space under particular stress conditions (Nawa Y et al., 2009; Wang k et al., 2010) protecting micro RNAs (miRNAs) from degradation (Wang k et al., 2010). The initial hypothesis of NUMIRDT was that Dox and Trz treatments induce the expression and eventually the active secretion of specific miRNAs by Cardiac Progenitor Cells (CPCs), as an early response to stress. This phenomenon was predicted to be regulated, at least in part, by the nucleuolus and associated with the active release of NPM in the extracellular space. Main ResultsDuring the granted period the in vivo studies resulted in a mouse model that simulates the current standard clinical protocol of Dox/Trx associated therapy and recapitulates the pathological effects of Dox/Trz on cardiac function. In vitro, the studies on human CPCs demonstrated for the first time that Dox/Trz treatments induced early molecular events associated with the active secretion of NPM. The rapid secretion of NPM was also observed in response to UV- induced DNA damage in hCPCs. Moreover, our results indicate that secreted NPM could act as an alarm signal from the damaged cells to the surrounding tissue and that NPM facilitate the intracellular uptake of external miRNAs. We are currently approaching the identification of a miRNAs signature associated to the secreted NPM in response to Dox/Trz. NPM secretion was also studied in different cell types and in response to more physiological stress. In endothelial cells, NPM was actively secreted in response to serum starvation and displayed pro-inflammatory and pro-angiogenic function both in vitro and in vivo. Moreover, NPM levels were found to be elevated in the plasma of diabetic patients where it co-immuprecipitated with the alarmin HMGB1. These results suggest that NPM could behave similarly to an alarmin being elevated in the plasma under chronic inflammation. Finally our study identified a previously uncharacterized mechanism of response to Dox in hCPCs. In particular Dox, induced the miR-200c /Zeb1 pathway, this ultimately resulting in the upregulation of the Sdf-1 receptor CXCR4 and temporary protection from Dox-induced apoptosis in hCPCs.ConclusionSo far, cardiomyocytes have been considered as the main target population of anthracyclines and trastuzumab – mediated cardiotoxicity, which has been ascribed to increased myofibrillar disarray, apoptosis and ROS production (Sawyer DB et al., 2010). According to the most recent literature (De Angelis et al., 2011; Barth AS et al. 2012), our in vivo and in vitro results indicate that CPCs could represent an important target population of Dox/Trz mediated cardiotoxicity. The absence of HER2 expression on human cardiomyocytes has been already reported by others (Fuchs IB et al. 2003). In vitro, our data demonstrate that hCPCs are responsive to both Dox and Trz and that these drugs induce an early stress response that is associated with the rapid secretion of the nucleolar protein NPM. This phenomenon seems to be a generalized response to DNA damage. Our results also show for the first time that NPM have an important role in the process of delivery and uptake of miRNAs. Moreover, our results in the vascular setting, suggest that NPM has important autocrine/paracrine role being implicated in the regaulation of inflammatory and angiogenenic processes. Taken together, the results of our study strongly suggest NPM as a new signaling molecule similar to an alarmin, able to mediate miRNA delivery in response to different stressor including genotoxic injury. ImpactThe increased life expectancy is having a huge impact on the social cost related to the onset of cardiovascular pathology in western countries. Moreover, due to the improved efficacy of the anticancer therapies the number of long-term survivors, experiencing anthracycline-induced cardiotoxicity is expected to grow. Therefore it is a priority for the EU to sustain the research in cardiovascular and cardioncology fields. The results of our study reveal new molecular mechanisms of Dox/Trz-induced cardiotoxicity that will potentially permit the improvement of the current therapeutic approaches or even the development of new ones. So far, the Dox/Trz interaction has been mainly focused on its toxic effects on breast cancer cells. However our results suggest hCPCs as an important target population of the drugs. In this context, the role of NPM as mediator of the delivery and the intracellular miRNAs uptake is highly innovative. The definition of a specific miRNAs signature associated with NPM in the plasma of patients under chemotherapy regimen could result in the identification of subclinical cardiac dysfunction and permit the application of cardioprotective strategies and a better management of the anticancer therapy. Despite circulating miRNAs have been widely investigated as sensitive biomarkers in cancer and cardiovascular contexts, they have never been tested for the detection of early subclinical cardiotoxicity induced by Atr/Trz. Moreover, the results obtained in the vascular setting may yield new insights into the pathogenesis of vascular complications opening the way for the discovery of novel therapeutic targets and therapeutic approaches.
