CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance

Triple-negative breast cancer (TNBC) is an aggressive and heterogenous breast cancer subtype. RASAL2 is a RAS GTPase-activating protein (GAP) that has been associated with platinum resistance in TNBC, but the underlying mechanism is unknown. Here, we show that RASAL2 is enriched following neoadjuvant chemotherapy in TNBC patients. This enrichment is specific to the tumour compartment compared to adjacent normal tissues, suggesting that RASAL2 upregulation is tumour-selective. Analyses based on 2D/3D cultures and patient-derived xenograft models reveal that RASAL2 confers cross-resistance to common DNA-damaging chemotherapies other than platinum. Mechanistically, we found that apoptotic signalling is significantly downregulated upon RASAL2 expression. This feature is characterised by substantial alterations in the expression of anti-versus pro-apoptotic factors, pointing to heterogeneous mechanisms. In particular, RASAL2 upregulates BCL2 via activation of the oncogenic transcription co-factor YAP. CREB1, a YAP-interacting protein, was identified as the common transcription factor that binds to the promoter regions of RASAL2 and BCL2, driving their collective expression. A subset of RASAL2 colocalises with BCL2 subcellularly. Both proteins decorate mitochondria, where the high levels of mitochondrial RASAL2-induced BCL2 expression render the organelles refractory to apoptosis. Accordingly, mitochondrial outer membrane permeabilisation assay using live mitochondria from RASAL2-high/chemoresistant tumour cells demonstrated attenuated release of death signal, cytochrome c, when exposed to pro-apoptotic factors BAX and tBID. Similarly, these cells were more resilient towards chemotherapy-induced mitochondrial depolarisation. Together, this work reveals a previously undocumented molecular link between RAS GAP and apoptosis regulation, providing a new mechanistic framework for targeting a subset of chemorefractory tumours.