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    • A role for YAP in mediating resistance

    2023-01-28

    A role for YAP in mediating resistance to EGFR inhibition has also been described [28], [29]. In line with these evidences, we observed increased YAP activation in all the generated EGFR TKI–resistant cells, testified by decreased phosphorylation on the inhibitory serine 127, enhanced nuclear localization, and augmented expression of its major target CTGF. Interestingly, we detected YAP activation not only in ack1 inhibitors resistant to various EGFR TKIs but also in cells resistant to inhibitors directed against other tyrosine kinases such as MET and ROS1. This suggests that YAP activation may represent a more general mechanism to sustain resistance to drugs targeting different tyrosine kinase receptors. Hsu et al. recently reported a role for YAP in mediating resistance to erlotinib in lung cancer cells [29]: they observed increased YAP expression and decreased YAP phosphorylation in HCC827 resistant versus parental cells and YAP-mediated protection to erlotinib treatment in parental cells. It has however to be noted that the reported IC50 (2.48 μM) and the used doses of TKIs were dramatically higher than those reported by others and ourselves ([8], [30], [31] and Table 1, in the range of low nM). These authors also reported that H1975 erlotinib–resistant cells bearing the second site mutation T790M became more sensitive to erlotinib upon YAP overexpression; the gain in erlotinib efficacy, however, was very poor, and the mechanism through which YAP rendered H1975 cells more sensitive to erlotinib was not addressed. To prove that YAP activation was not the consequence of resistance onset but rather a critical element in sustaining resistance, we performed experiments of genetic interference or exogenous protein expression in resistant cells, which indeed demonstrated that lowering YAP activity restored sensitivity to the different TKIs, while increasing it impaired response to the drugs. As YAP is a transcriptional coactivator, we reasoned that the observed effect could be due to the transcription of critical effector(s). Among YAP targets, we focused on AXL, a tyrosine kinase that has been identified as a mediator of YAP-dependent oncogenic functions [25] and that has been shown to contribute to resistance to targeted therapies [11]. In fact, Zhang et al. [11] showed that AXL upregulation is sufficient to sustain erlotinib acquired resistance in EGFR mutant NSCLC cellular models. In resistant cells, we observed a YAP-dependent AXL increase concomitant with an augmented expression of its ligand GAS6, resulting in autocrine activation of this kinase. Pharmacologic inhibition and genetic interference with AXL expression showed that AXL is critical in mediating YAP-induced resistance to EGFR TKIs, thus representing an actionable target to restore sensibility to targeted therapies. It is worth noting that the described resistance is apparently not due to genetic alterations but is rather sustained by an adaptive mechanism. Interestingly, AXL silencing or pharmacological inhibition is more effective than YAP silencing in reverting cancer cell resistance, suggesting that other mechanisms — in addition to YAP activation — might concur to AXL activation. It has been reported that AXL transcription can be induced by MAPK-AP1 activation [32] and by MZF1 transcriptional activity [33]. In this frame, the possible activation of MAPK or of MZF1 by other cellular stimuli might justify the primary role of AXL in mediating resistance to EGFR TKIs we reported here. Finally, we demonstrated that the activation of the YAP/AXL axis is present in selected lung cancer patients who become resistant to EGFR inhibitors. It is known that in about 50% of the cases, resistance to EGFR TKIs is due to the appearance of resistance mutations, such as the T790M. Accordingly, in seven lung cancer patients examined, we found that this mutation was present in three biopsies obtained upon resistance onset but not in the corresponding biopsies at diagnosis. In one of the patients that did not show already described genetic alterations supporting resistance, such as new EGFR mutations or ALK/ROS1 translocations or MET amplification, we observed YAP activation and AXL overexpression, recapitulating what was found in the in vitro generated resistant cells. It is worthwhile to note that the activation of the YAP-AXL pathway in one out of five patients negative for the presence of the T790M mutation might represent a relatively high percentage, in line with other mechanisms of resistance already described, such as HER2 amplification [6]. Due to the relatively low number of samples analyzed, however, other studies are needed to verify the prevalence of YAP-AXL activation to understand the translation potential of anti-AXL treatments into the clinic.