Role of Connexin43 in Triple Negative Breast Cancer and Skin Regeneration After Oncological Treatments

Laburpena

Breast cancer is the most diagnosed cancer worldwide and represents the fifth cause of cancer mortality globally. It is a highly heterogeneous disease, and one of its subtypes, triple negative breast cancer (TNBC), is characterized by the highest aggressiveness and poorest prognosis, currently lacking efficient targeted therapies. Among emerging strategies to treat TNBC, EGFR-targeted therapies and immunotherapy are being critical. Of particular interest within TNBC are BRCA1/2 mutated tumours, which are deficient in DNA damage repair by homologous recombination. The promising new strategy represented by PARP inhibitors (PARPi) can take advantage of this situation and lead to DNA damage accumulation and cell death in BRCA1/2 mutated tumours. However, PARPi have also been associated with both de novo and acquired resistance, accentuating the need to design new therapeutic strategies to address this issue. Connexins (Cxs) are hemichannel and gap junction (GJ)-forming proteins involved in cellular communication and channel-independent functions. Cx43 has been reported to be highly dysregulated in breast cancer and suggested as a conditional tumour suppressor. However, there is still controversy and its role in the pathogenesis and development of breast cancer depends on the subtype and is far from being elucidated. The main aim of this thesis was to investigate the role of Cx43 (i) in TNBC, (ii) in the interaction of TNBC with the tumour microenvironment such as fibroblasts and natural killer (NK) cells and (iii) in the response to PARPi olaparib. Cx43 was found to be deeply downregulated in human breast cancer patient-derived samples and cell lines, and even more in TNBC. Upon restoration of Cx43 by plasmid transfection of TNBC cells, Cx43 mainly localized perinuclearly and to some extent at the plasma membrane, where it formed functional GJs. Cx43-restituted TNBC HCC1937 and MDAMB231 cells showed reduced 2D and 3D proliferation, colony formation and migration, epithelialmesenchymal transition reversion, resensitization to Anoikis and improved adhesion to extracellular matrix protein collagen I. Cx43-restored TNBC BRCA1 mutated HCC1937 cells triggered a more cytotoxic antitumour response of primary and EGFR-CAR-NK cells than wild type tumour cells, presumably in a GJ-independent manner. Moreover, Cx43 restitution in these cells distinctly resensitized them to olaparib, to which the wild type cells are de novo resistant, via reduced proliferation and colony formation, DNA damage and senescence accumulation, enhanced reactive oxygen species production and eventually increased caspase 3-mediated apoptosis. These results were further recapitulated in BRCA1 mutated ovarian carcinoma cells. An additional objective of this thesis was to address the unsolved clinical burden of wound healing impairment in oncological patients after radio- or chemo-therapy. The proposed strategy, based on the combination of natural polyphenol oleuropein and p38 inhibitor BIRB796, targets Cx43 expression/function and cellular senescence in irradiated human fibroblasts, reducing Cx43 but restoring its functionality, ameliorating overstimulated cellular senescence-SASP production and improving in vitro migration. These findings support Cx43 as a double therapeutic target capable of reducing tumoral malignancy and resensitizing TNBC BRCA1 mutated cells to NK immunotherapy and PARPi, and of improving wound healing after radiotherapy.