RESUMO
Postzygotic mutations of the PIK3CA [phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha] gene constitutively activate the PI3K/AKT/mTOR pathway in PIK3CA-related overgrowth spectrum (PROS) patients, causing congenital mosaic tissue overgrowth that even multiple surgeries cannot solve. mTOR inhibitors are empirically tested and given for compassionate use in these patients. PROS patients could be ideal candidates for enrolment in trials with PI3K/AKT pathway inhibitors, considering the "clean" cellular setting in which a unique driver, a PIK3CA mutation, is present. We aimed to assess the effects of blocking the upstream pathway of mTOR on PROS patient-derived cells by using ARQ 092, a potent, selective, allosteric, and experimental orally bioavailable and highly selective AKT-inhibitor with activity and long-term tolerability, currently under clinical development for treatment of cancer and Proteus syndrome. Cell samples (i.e., primary fibroblasts) were derived from cultured tissues obtained from six PROS patients [3 boys, 3 girls; aged 2 to 17 years] whose spectrum of PIK3A-related overgrowth included HHML [hemihyperplasia multiple lipomatosis; n = 1], CLOVES [congenital lipomatosis, overgrowth, vascular malformations, epidermal nevi, spinal/skeletal anomalies, scoliosis; n = 1], and MCAP [megalencephaly capillary malformation syndrome; n = 4]. We performed the following: (a) a deep sequencing assay of PI3K/AKT pathway genes in the six PROS patients' derived cells to identify the causative mutations and (b) a pathway analysis to assess the phosphorylation status of AKT [Ser473 and Thr308] and its downstream targets [pAKTS1 (Thr246), pRPS6 (Ser235/236), and pRPS6Kß1 (Ser371)]. The anti-proliferative effect of ARQ 092 was tested and compared to other PI3K/AKT/mTOR inhibitors [i.e., wortmannin, LY249002, and rapamycin] in the six PROS patient-derived cells. Using ARQ 092 to target AKT, a critical node connecting PI3K and mTOR pathways, we observed the following: (1) strong anti-proliferative activity [ARQ 092 at 0.5, 1, and 2.5 µM blunted phosphorylation of AKT and its downstream targets (in the presence or absence of serum) and inhibited proliferation after 72 h; rapamycin at 100 nM did not decrease AKT phosphorylation] and (2) less cytotoxicity as compared to rapamycin and wortmannin. We demonstrated the following: (a) that PROS cells are dependent on AKT; (b) the advantage of inhibiting the pathway immediately downstream of PI3K to circumventing problems depending on multiple classes a PI3K kinases; and (c) that PROS patients benefit from inhibition of AKT rather than mTOR. Clinical development of ARQ 092 in PROS patients is on going in these patients.
Assuntos
Aminopiridinas/administração & dosagem , Classe I de Fosfatidilinositol 3-Quinases/genética , Fibroblastos/efeitos dos fármacos , Transtornos do Crescimento/tratamento farmacológico , Transtornos do Crescimento/genética , Imidazóis/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Adolescente , Regulação Alostérica , Criança , Pré-Escolar , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Mutação , Proteína Oncogênica v-akt/metabolismo , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacosRESUMO
The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-ß, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial-mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.
Assuntos
Células da Medula Óssea/fisiologia , Fibroblastos/fisiologia , Mieloma Múltiplo/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Quimiocina CXCL12/fisiologia , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , FenótipoRESUMO
Bone marrow (BM) angiogenesis has an important role in the initiation and progression of multiple myeloma (MM). We looked at novel mechanisms of vessel formation in patients with MM through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with active MM (MMECs) and ECs of patients with monoclonal gammopathy of undetermined significance (MGECs) and of subjects with benign anemia (normal ECs). Four proteins were found overexpressed in MMECs: filamin A, vimentin, α-crystallin B and 14-3-3ζ/δ protein, not yet linked to overangiogenic phenotype. These proteins gave a typical distribution in the BM of MM patients and in MMECs versus MGECs, plausibly according to a different functional state. Their expression was enhanced by vascular endothelial growth factor, fibroblast growth factor 2, hepatocyte growth factor and MM plasma cell conditioned medium in step with enhancement of MMEC angiogenesis. Their silencing RNA knockdown affected critical MMEC angiogenesis-related functions, such as spreading, migration and tubular morphogenesis. A gradual stabilization of 14-3-3ζ/δ protein was observed, with transition from normal ECs to MGECs and MMECs that may be a critical step for the angiogenic switch in MMECs and maintenance of the cell overangiogenic phenotype. These proteins were substantially impacted by anti-MM drugs, such as bortezomib, lenalidomide and panobinostat. Results suggest that these four proteins could be new targets for the antiangiogenic management of MM patients.
Assuntos
Mieloma Múltiplo/irrigação sanguínea , Mieloma Múltiplo/patologia , Neovascularização Patológica/genética , Proteínas 14-3-3/genética , Idoso , Idoso de 80 Anos ou mais , Anemia/genética , Anemia/patologia , Células da Medula Óssea/patologia , Movimento Celular , Proteínas Contráteis/genética , Células Endoteliais/patologia , Feminino , Filaminas , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mieloma Múltiplo/genética , Paraproteinemias/genética , Paraproteinemias/patologia , Proteômica , Vimentina/genética , alfa-Cristalinas/genéticaRESUMO
Ebastine is a well-known selective second-generation histamine H(1) receptor antagonist, which is used for seasonal and perennial allergic rhinitis and chronic urticaria. Angiogenesis plays a crucial role in the development of airway inflammation and remodelling in allergic rhinitis and asthmatic patients, in whom, indeed, the mucosa displays increased vascularity and overexpression of vascular endothelial growth factor (VEGF). The aim of the present study was to evaluate the anti-angiogenic properties of carebastine, the active metabolite of ebastine. The effects of carebastine on human umbilical vein endothelial cell (EC) (HUVEC) and human pulmonary artery EC (HPAEC) proliferation, migration and capillary-like tube formation were investigated in vitro, and in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Moreover, the effect of carebastine on phosphorylation of the cell VEGF receptor fetal liver kinase-1, or VEGF receptor 2 (VEGFR-2), and Akt kinase (Akt) was evaluated by Western blotting. Carebastine inhibited VEGF-induced HUVEC and HPAEC proliferation, migration and angiogenesis in a dose-dependent manner in vitro. Cell proliferation was inhibited by 42 and 64% in HUVECs and 62 and 75% in HPAECs upon exposure for 48 and 72 h, respectively, to 20 microM carebastine (p < or = 0.03), and even more with 30 microM carebastine. Cell migration was inhibited by 37 and 70% in HUVECs (p < or = 0.03) and 60 and 78% in HPAECs (p < or = 0.01) in the presence of 10 and 30 microM carebastine, respectively. Carebastine (20 microM) caused a significant reduction (70-86%; p<0.01) in topological parameters of the capillary network produced in vitro by both EC lines on a basement membrane extract. Carebastine (30 and 50 microM) inhibited the VEGF-induced angiogenesis in the CAM assay in vivo two- and three-fold, respectively (p<0.001). Finally, both EC lines, on exposure to 10 and 20 microM carebastine, showed a four- to six-fold reduction (p < or = 0.01) in both VEGF- and H1 receptor-induced VEGFR-2 and Akt phosphorylation. Overall, these data provide the first evidence regarding the anti-angiogenic activity of ebastine, and suggest its potential use as an anti-angiogenic molecule, besides its antihistaminic activity for the treatment of allergic diseases in which angiogenesis takes place.