Stepwise evolution of chemoresistance: therapy-induced epigenetic reprogramming drives repopulating cells towards acquisition of cell resistance

Cytotoxic anticancer therapies, although effective in reducing tumor mass, may promote Cancer tissue Repopulation and Acquired Cell-resistance (CRAC), a major cause of patient death. During CRAC, tumor cells increase malignancy, metastasize and acquire drug resistance, suggesting that the therapy itself promotes tumor progression. It is often assumed that CRAC depends on the selection of genetically mutated clones with greater fitness, constituting an intrinsic limitation of therapies. However, a more complex scenario is emerging: in fact, tumor tissues injured by therapy react by organizing a series of aberrant responses through epigenetic regulatory mechanisms, aimed at repair and repopulation of tumor cells, promoting malignancy. The wounded tumor cells therefore play an active signaling role, promoting the epigenetic reprogramming of surviving cells, activating preset signaling "packages", such as the epithelium-mesenchymal transition (EMT) and the regenerative pathway "Phoenix rising", which through the caspase-3 / COX-2 / PGE-2 / STAT3 signaling axis, promotes compensatory proliferation of surviving cells. Overall, “pre-set” signaling promotes the coordinated expression of genes involved in cell resistance, proliferation, and motility. Since both pathways are adjustable and repressible, this scenario opens an unexpected window for an innovative therapeutic intervention, aiming to exploit the benefits of chemotherapy by limiting its undesirable effects. In literature, there is no acronym that puts these events together, therefore we felt the need to group them as cancer repopulation and acquired cell-resistance (CRAC), combining the two phenomena as a single, complex response to cytotoxic therapy; furthermore, there is no effective in vitro model for studying the development of CRAC that allows an integrated view of the various signaling processes involved. Therefore, the main aim of my project was to develop a "onepot" model of CRAC, in which tumor cells, insulted by a chemotherapy treatment, reacting with apoptosis or epigenetic reprogramming, act as both emitter and recipient of the paracrine signaling responsible for post-therapy repopulation, allowing a real-time observation of repopulation, thus allowing the study of events that occur during and immediately after chemotherapeutic treatment. Prostate cancer cells with high or low malignancy (PC3 and LNCaP) and normal (RWPE-1) are treated for 24h with clinically relevant drugs (etoposide, treosulfan or docetaxel). After a robust apoptotic phase, PC3 set a tissue-like response: profound phenotypic transformation through trans-differentiation and creation of a paracrine signaling network. A very small fraction of surviving cells repopulates through or the “escape-from-senescence” phenomenon, acquiring EMT; repopulation occurs via Phoenix Rising, and requires epigenetic reprogramming. The repopulation induced by the 3 drugs occurs in the same way, showing that it depends on a cellular response mechanism in which the various stress responses induced by the compounds converge. However, there are different implications, e.g., induced EMT is much stronger for etoposide, which is also the only drug to promote resistance to subsequent treatment, suggesting that the ability to repair the different types of damage may be the discriminating factor between CRAC and progression-free-repopulation. As for the different prostate cell lines, RWPE-1 repopulate without reprogramming or acquiring malignancy or resistance, indicating that only cancer cells require reprogramming to repopulate. CRAC requires activation of intra and extracellular signaling pathways, which include activation of the DNA damage response (DDR) and inflammatory response via lipoxygenase (LOX) and cyclooxygenase (COX) and signaling pathways that are inhibited by nuclear receptors such as pioglitazone and melatonin. I then focused on the mechanisms for acquiring resistance induced by etoposide in PC3. I have shown that resistance evolves during subsequent treatment cycles and occurs through epigenetic mechanisms rather than from the selection of pre-existing or damage-induced mutations; it initially consists of the inability to block the cell cycle after DNA damage, and cells are then induced to proliferate in the presence of DNA damage. This effect is not cell-intrinsic, as it is lost upon re-plating the culture, indicating that it needs signals from the microenvironment. Following a subsequent cycle of etoposide, which is followed by a repopulation still dependent on reprogramming, resistance is strengthened by associating the inability to block the cycle, evasion of apoptosis: now, the two characteristics are cell-intrinsic, because they remain even after re-plating. This suggests that at least two reprogramming cycles, perhaps with different epigenetic mechanisms, are required to arrive at intrinsic resistance, even if not genetically determined. The role of epigenetics and the microenvironment is therefore fundamental from the very first steps; their nature means that these can be inhibited, e.g., acting by modulation of nuclear transcription factor activity or epigenetically to their targets, indicating that CRAC can theoretically be countered pharmacologically, thus allowing a "CRAC-free" or at least "progression-free" cytotoxic treatment.