RESUMO
Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.
Assuntos
Bortezomib/farmacologia , Processos Neoplásicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , TYK2 Quinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Linhagem Celular Tumoral , Feminino , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mieloma Múltiplo , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Neoplasias de Mama Triplo Negativas/patologia , Quinases DyrkRESUMO
Bone fractures are a major cause of morbidity and mortality, particularly in patients with diabetes, who have a high incidence of fractures and exhibit poor fracture healing. Coordinated expression of osteoblast-derived vascular endothelial growth factor (VEGF) and bone morphogenic proteins (BMPs) is essential for fracture repair. The NO/cGMP/protein kinase G (PKG) signaling pathway mediates osteoblast responses to estrogens and mechanical stimulation, but the pathway's role in bone regeneration is unknown. Here, we used a mouse cortical-defect model to simulate bone fractures and studied osteoblast-specific PKG1-knockout and diabetic mice. The knockout mice had normal bone microarchitecture but after injury exhibited poor bone regeneration, with decreased osteoblasts, collagen deposition, and microvessels in the bone defect area. Primary osteoblasts and tibiae from the knockout mice expressed low amounts of Vegfa and Bmp2/4 mRNAs, and PKG1 was required for cGMP-stimulated expression of these genes. Diabetic mice also demonstrated low Vegfa and Bmp2/4 expression in bone and impaired bone regeneration after injury; notably, the cGMP-elevating agent cinaciguat restored Vegfa and BMP2/4 expression and full bone healing. We conclude that PKG1 is a key orchestrator of VEGF and BMP signaling during bone regeneration and propose pharmacological PKG activation as a novel therapeutic approach to enhance fracture healing.
Assuntos
Regeneração Óssea , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Diabetes Mellitus Experimental , Consolidação da Fratura , Osteoblastos , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Fraturas Ósseas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
NO/cGMP signaling is important for bone remodeling in response to mechanical and hormonal stimuli, but the downstream mediator(s) regulating skeletal homeostasis are incompletely defined. We generated transgenic mice expressing a partly-activated, mutant cGMP-dependent protein kinase type 2 (PKG2R242Q) under control of the osteoblast-specific Col1a1 promoter to characterize the role of PKG2 in post-natal bone formation. Primary osteoblasts from these mice showed a two- to three-fold increase in basal and total PKG2 activity; they proliferated faster and were resistant to apoptosis compared to cells from WT mice. Male Col1a1-Prkg2R242Q transgenic mice had increased osteoblast numbers, bone formation rates and Wnt/ß-catenin-related gene expression in bone and a higher trabecular bone mass compared to their WT littermates. Streptozotocin-induced type 1 diabetes suppressed bone formation and caused rapid bone loss in WT mice, but male transgenic mice were protected from these effects. Surprisingly, we found no significant difference in bone micro-architecture or Wnt/ß-catenin-related gene expression between female WT and transgenic mice; female mice of both genotypes showed higher systemic and osteoblastic NO/cGMP generation compared to their male counterparts, and a higher level of endogenous PKG2 activity may be responsible for masking effects of the PKG2R242Q transgene in females. Our data support sexual dimorphism in Wnt/ß-catenin signaling and PKG2 regulation of this crucial pathway in bone homeostasis. This work establishes PKG2 as a key regulator of osteoblast proliferation and post-natal bone formation.