Targeting BMI-1 as an innovative approach to overcome cancer multi drug resistance

Aging is among the major risk factors for the development of cancer and the main cause of cancer relapse and therapeutic failure is due to the onset of chemoresistance. Since many anticancer therapies cause cell death by inducing oxidative stress, one of the mechanisms activated by cancer cells to adapt and survive to the cytotoxic effect of therapeutic agents, is to stimulate the antioxidant defense. In this regard, previous studies from the research group of my PhD tutor, showed that chronic exposure of cancer cells (HTLA-230) to the chemotherapeutic drug etoposide, at a dose comparable to that used in clinic, leads to the selection of a resistant cell line (HTLA-ER). Interestingly, resistant cells are characterized by efficient oxidative metabolism, display high amounts of glutathione (GSH) and overexpress the oncogenic protein BMI-1, which is capable of regulating GSH levels and is associated with cancer stemness, epithelial-mesenchymal transition (EMT), tumor invasion, metastasis and poor prognosis. Notably, both cell lines exhibit the same p53 mutation and no changes in the expression of MDM2, the endogenous inhibitor of p53. Based on this background, the aim of this thesis is to investigate new strategies that can circumvent cancer chemoresistance, mainly by reducing intracellular GSH content and reactivating mutated p53. Therefore, the effects of sulfasalazine, an xCT inhibitor, of PRIMA-1MET, a compound able to reactivate p53 functions, and of PTC596, a novel BMI-1 inhibitor, were investigated. Firstly, it has been confirmed that HTLA-ER cells are multidrug-resistant, being unaffected by the cytotoxic effects of different chemotherapeutic drugs, and not only by etoposide. Then, it has been found that PRIMA-1MET promotes apoptosis only in parental cells, whereas PTC596 does not trigger apoptosis in both cell populations. In addition, PRIMA-1MET and PTC596 inhibited the clonogenic potential of parental and resistant cells. Since sulfasalazine alone or combined was not effective on resistant cells it was excluded from the subsequent experiments. The evaluation of the expression of p53 and BMI-1, target proteins of the two tested drugs, showed that PRIMA-1MET does not modulate p53 protein levels, while PTC596 is able to significantly reduce the expression of BMI-1. Furthermore, given that EMT is involved in chemoresistance, EMT-related proteins were evaluated. In detail, PRIMA-1MET and, most importantly, PTC596 reduced the expression of n-cadherin, b-catenin and SNAIL, demonstrating an inhibition of EMT process. In addition, both compounds prevented the formation of cancer stem cells. Analysis of cellular redox state showed that treatments with PRIMA-1MET of parental cells and with PTC596 of both cell lines induced overproduction of reactive oxygen species and increased membrane lipid peroxidation. With regard to antioxidant response, it has been observed that i) resistant cells expressed a double GSH amount compared to that of parental cells and ii) PRIMA-1MET and, more markedly, PTC596 were able to deplete the thiol antioxidant content. These promising findings certainly demonstrate the involvement of cellular redox state in chemoresistance, and suggest that PTC596, the most effective compound in resistant cells, can induce ferroptosis, a cell death characterized by membrane lipid peroxidation and reduction of GSH levels. In fact, the co-treatment of PTC596 with compounds capable of inducing or inhibiting ferroptosis, confirmed the induction of this type of cell death. Therefore, an approach targeting BMI-1 and its related pathways could be a winning strategy to overcome cancer resistance to therapy.