Cell death and differentiation. their stemness potential and increases the cytotoxic effect of conventional chemotherapeutic drugs. Being DCMI an inhibitor of the E3 ubiquitin ligase Itch, we also verified the effect of Itch deregulation on CSCs survival. We found that the siRNA-mediated depletion of Itch induces similar 12-O-tetradecanoyl phorbol-13-acetate anti-proliferative effects on lung CSCs, suggesting that DCMI 12-O-tetradecanoyl phorbol-13-acetate might exert its effect, at least in part, by inhibiting Itch. Notably, Itch expression is a negative prognostic factor in two primary lung tumors datasets, supporting the potential clinical relevance of Itch inhibition to circumvent drug resistance in the treatment of lung cancer. and expansion and characterization, which allow us testing and preclinical validation of new targeted therapies [6, 7]. A current strategy to enhance the efficacy of anticancer therapy involves the usage of drugs deregulating autophagic processes. Autophagy is a conserved lysosome-mediated process, which degrades cellular organelles and macromolecules, allowing the recycling of bioenergetics components in order to favour the survival of cells in response to diverse stress like starvation, hypoxia and endoplasmatic reticulum stress [8, 9]. Besides its role in the regulation of several biological processes, autophagy is also known to be closely involved in many human diseases, including cancer [9, 10]. However, the role of autophagy in tumor progression is controversial and may depend on various factors, such as the cancer type, the development stage and the genetic background [11-14]. Currently, several drugs targeting autophagy process has been tested and some of them are in clinical trials [15, 16]. Clomipramine is an FDA-approved drug generally used for treatment of obsessive-compulsive disorders [17, 18]. It has a long-standing record with good subject tolerance. Besides its function as noradrenergic and serotonergic reuptake inhibitor, clomipramine acts as a regulator of autophagy [19, 20]. Treating cells with clomipramine or its active metabolite desmethylclomipramine (DCMI) induces the blockade of the autophagic flux, as revealed by the increase of authophagosomal markers and a concomitant blockade of the degradation of autophagic cargo, such as p62. Importantly, DCMI increases the pro-apoptotic effects of conventional chemotherapic drugs in several cancer 12-O-tetradecanoyl phorbol-13-acetate cell lines [21]. Recently, clomipramine has been also identified as an inhibitor of Itch, an E3 ubiquitin ligase belonging to the HECT-type family of E3 ubiquitin ligase [22]. By controlling the proteasomal-dependent degradation of a subset of target proteins, Itch regulates several important biological processes, such as apoptosis, cell growth and inflammation [23-25]. Several reports have demonstrated that the expression levels of Itch affect the apoptotic response induced by the chemotherapeutic drugs [26-28]. In details, it has been shown that Itch depletion by siRNA increases the cytotoxic effect of anti-neoplastic drugs in different cancer cell lines and the administration of siRNA duplex targeting Itch mRNA is effective in sensitizing pancreatic cancer to gemcitabine [29]. The pro-apoptotic effects exerted by Itch depletion are more evident in cells with no functional p53, highlighting the importance that changes in levels of Itch may play in majority of cancers, where p53 is absent or mutated. In the present manuscript, we investigate the biological effect of DCMI on the growth properties of lung CSCs isolated from non-small-cell lung cancers (NSCLC) surgical specimens. We report that DCMI inhibits lung CSC growth, decreases their stemness potential and increases the cytotoxic effect of conventional chemotherapeutic agents. Being the DCMI an inhibitor of the E3 ubiquitin ligase Itch, we also analyzed the consequences of Itch downregulation on lung CSCs. Similarly to what we observed in DCMI treated lung CSCs, the siRNA-mediated depletion of Itch Rabbit Polyclonal to HSL (phospho-Ser855/554) decreases CSCs survival in response to gemcitabine treatment, suggesting that the pro-apoptotic effects of DCMI might be exerted, at least in part, by Itch inhibition. Notably, Itch expression is a negative prognostic factor in several primary lung cancer datasets, supporting the potential clinical relevance of Itch inhibition to circumvent drug resistance in the treatment of lung cancer. RESULTS Characterization of non-small 12-O-tetradecanoyl phorbol-13-acetate cell lung CSCs and their resistance to conventional chemotherapeutic drugs Two squamous cell carcinomas (LC1 and LC2) and one adenocarcinoma 12-O-tetradecanoyl phorbol-13-acetate (LC3) lung CSCs were isolated from NSCLC surgical samples and characterized for the presence of common genetic alterations exhibited by lung tumors and for their ability to histologically recapitulate the tumor of origin in mice (Table ?(Table1)1) [7, 30]. In serum-free medium containing EGF and basic-FGF these cells grow as tumor spheroids expressing stem cell markers such as CD133. Upon serum.