Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes‐associated protein (YAP). Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance. Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood. Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils. We also confirm the accumulation of amyloid‐like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta‐secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta‐secretase inhibitors as potential therapeutic approach for metastatic melanoma.
KMT2A (Lysine methyltransferase 2A) is a member of the epigenetic machinery, encoding a lysine methyltransferase responsible for the transcriptional activation through lysine 4 of histone 3 (H3K4) methylation. KMT2A has a crucial role in gene expression, thus it is associated to pathological conditions when found mutated. KMT2A germinal mutations are associated to Wiedemann–Steiner syndrome and also in patients with initial clinical diagnosis of several other chromatinopathies (i.e., Coffin–Siris syndromes, Kabuki syndrome, Cornelia De Lange syndrome, Rubinstein–Taybi syndrome), sharing an overlapping phenotype. On the other hand, KMT2A somatic mutations have been reported in several tumors, mainly blood malignancies. Due to its evolutionary conservation, the role of KMT2A in embryonic development, hematopoiesis and neurodevelopment has been explored in different animal models, and in recent decades, epigenetic treatments for disorders linked to KMT2A dysfunction have been extensively investigated. To note, pharmaceutical compounds acting on tumors characterized by KMT2A mutations have been formulated, and even nutritional interventions for chromatinopathies have become the object of study due to the role of microbiota in epigenetic regulation.
Matrix metalloproteinase (MMP) dysregulation is implicated in several diseases, given their involvement in extracellular matrix degradation and cell motility. In lymphangioleiomyomatosis (LAM), a pulmonary rare disease, MMP-2 and MMP-9 have been detected at high levels in serum and urine. LAM cells, characterized by a mutation in the tuberous sclerosis complex (TSC)1 or TSC2, promote cystic lung destruction. The role of MMPs in invasive and destructive LAM cell capability has not yet been fully understood. We evaluated MMP-2 and MMP-7 expression, secretion, and activity in primary LAM/TSC cells that bear a TSC2 germline mutation and an epigenetic modification and depend on epidermal growth factor (EGF) for survival. 5-azacytidine restored tuberin expression with a reduction of MMP-2 and MMP-7 levels and inhibits motility, similarly to rapamycin and anti-EGFR antibody. Both drugs reduced MMP-2 and MMP-7 secretion and activity during wound healing and decreased their expression in lung nodules of a LAM mouse model. In LAM/TSC cells, MMP-2 and MMP-7 are dependent on tuberin expression, cellular adhesion, and migration. MMPs appears sensitive to rapamycin and anti-EGFR antibody only during cellular migration. Our data indicate a complex and differential modulation of MMP-2 and MMP-7 in LAM/TSC cells, likely critical for lung parenchyma remodeling during LAM progression.
Senescence is a stress-response process characterized by the irreversible inhibition of cell proliferation, associated to the acquisition of a senescence-associated secretory phenotype (SASP), that may drive pathological conditions. Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells, featuring the hyperactivation of the mammalian Target of Rapamycin Complex 1 (mTORC1) for the absence of tuberin expression, cause the disruption of the lung parenchyma. Considering that LAM cells secrete SASP factors and that mTOR is also a driver of senescence, we deepened the contribution of senescence in LAM cell phenotype. We firstly demonstrated that human primary tuberin-deficient LAM cells (LAM/TSC cells) have senescent features depending on mTOR hyperactivation, since their high positivity to SA-β galactosidase and to phospho-histone H2A.X are reduced by inducing tuberin expression and by inhibiting mTOR with rapamycin. Then, we demonstrated the capability of LAM/TSC cells to induce senescence. Indeed, primary lung fibroblasts (PLFs) grown in LAM/TSC conditioned medium increased the positivity to SA-β galactosidase and to phospho-histone H2A.X, as well as p21WAF1/CIP1 expression, and enhanced the mRNA expression and the secretion of the SASP component IL-8. Taken together, these data make senescence a novel field of study to understand LAM development and progression.
Cutaneous lesions are one of the hallmarks of tuberous sclerosis complex (TSC), a genetic disease in which mTOR is hyperactivated due to the lack of hamartin or tuberin. To date, novel pharmacological treatments for TSC cutaneous lesions that are benign but still have an impact on a patient’s life are needed, because neither surgery nor rapamycin administration prevents their recurrence. Here, we demonstrated that primary TSC2-/meth cells that do not express tuberin for an epigenetic event caused cutaneous lesions and follicular neogenesis when they were subcutaneously injected in nude mice. Tuberin-null cells localized in the hair bulbs and alongside mature hairs, where high phosphorylation of S6 and Erk indicated mTOR hyperactivation. Interestingly, 5-azacytidine treatment reduced hair follicles, indicating that chromatin remodeling agents might be effective on TSC lesions in which cells lack tuberin for an epigenetic event. Moreover, we demonstrated that the primary TSC2-/meth cells had metastatic capability: when subcutaneously injected, they reached the bloodstream and lymphatics and invaded the lungs, causing the enlargement of the alveolar walls. The capability of TSC2-/meth cells to survive and migrate in vivo makes our mouse model ideal to follow the progression of the disease and test potential pharmacological treatments in a time-dependent manner.
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