RESUMO
In solid tumors, drug concentrations decrease with distance from blood vessels. However, cellular adaptations accompanying the gradated exposure of cancer cells to drugs are largely unknown. Here, we modeled the spatiotemporal changes promoting chemotherapy resistance in breast cancer. Using pairwise cell competition assays at each step during the acquisition of chemoresistance, we reveal an important priming phase that renders cancer cells previously exposed to sublethal drug concentrations refractory to dose escalation. Therapy-resistant cells throughout the concentration gradient display higher expression of the solute carriers SLC38A7 and SLC46A1 and elevated intracellular concentrations of their associated metabolites. Reduced levels of SLC38A7 and SLC46A1 diminish the proliferative potential of cancer cells, and elevated expression of these SLCs in breast tumors from patients correlates with reduced survival. Our work provides mechanistic evidence to support dose-intensive treatment modalities for patients with solid tumors and reveals two members of the SLC family as potential actionable targets.
Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Animais , Humanos , Feminino , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Mama/metabolismo , Transportador de Folato Acoplado a PrótonRESUMO
Multiple myeloma (MM) is a hematological malignancy that emerges from antibody-producing plasma B cells. Proteasome inhibitors, including the US Food and Drug Administration-approved bortezomib (BTZ) and carfilzomib (CFZ), are frequently used for the treatment of patients with MM. Nevertheless, a significant proportion of patients with MM are refractory or develop resistance to this class of inhibitors, which represents a significant challenge in the clinic. Thus, identifying factors that determine the potency of proteasome inhibitors in MM is of paramount importance to bolster their efficacy in the clinic. Using genome-wide CRISPR-based screening, we identified a subunit of the mitochondrial pyruvate carrier (MPC) complex, MPC1, as a common modulator of BTZ response in 2 distinct human MM cell lines in vitro. We noticed that CRISPR-mediated deletion or pharmacological inhibition of the MPC complex enhanced BTZ/CFZ-induced MM cell death with minimal impact on cell cycle progression. In fact, targeting the MPC complex compromised the bioenergetic capacity of MM cells, which is accompanied by reduced proteasomal activity, thereby exacerbating BTZ-induced cytotoxicity in vitro. Importantly, we observed that the RNA expression levels of several regulators of pyruvate metabolism were altered in advanced stages of MM for which they correlated with poor patient prognosis. Collectively, this study highlights the importance of the MPC complex for the survival of MM cells and their responses to proteasome inhibitors. These findings establish mitochondrial pyruvate metabolism as a potential target for the treatment of MM and an unappreciated strategy to increase the efficacy of proteasome inhibitors in the clinic.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Estados Unidos , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Antineoplásicos/uso terapêutico , Transportadores de Ácidos Monocarboxílicos/uso terapêutico , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Piruvatos/uso terapêuticoRESUMO
Chemotherapy resistance is a critical barrier in cancer treatment. Metabolic adaptations have been shown to fuel therapy resistance; however, little is known regarding the generality of these changes and whether specific therapies elicit unique metabolic alterations. Using a combination of metabolomics, transcriptomics, and functional genomics, we show that two anthracyclines, doxorubicin and epirubicin, elicit distinct primary metabolic vulnerabilities in human breast cancer cells. Doxorubicin-resistant cells rely on glutamine to drive oxidative phosphorylation and de novo glutathione synthesis, while epirubicin-resistant cells display markedly increased bioenergetic capacity and mitochondrial ATP production. The dependence on these distinct metabolic adaptations is revealed by the increased sensitivity of doxorubicin-resistant cells and tumor xenografts to buthionine sulfoximine (BSO), a drug that interferes with glutathione synthesis, compared with epirubicin-resistant counterparts that are more sensitive to the biguanide phenformin. Overall, our work reveals that metabolic adaptations can vary with therapeutics and that these metabolic dependencies can be exploited as a targeted approach to treat chemotherapy-resistant breast cancer.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Epirubicina/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Cancer cachexia is a paraneoplastic syndrome that causes profound weight loss and muscle mass atrophy and is estimated to be the cause of up to 30% of cancer deaths. Though the exact cause is unknown, patients with cancer cachexia have increased muscle protein catabolism. In healthy muscle, injury activates skeletal muscle stem cells, called satellite cells, to differentiate and promote regeneration. Here, we provide evidence that this mechanism is inhibited in cancer cachexia due to persistent expression of CCAAT/Enhancer Binding Protein beta (C/EBPß) in muscle myoblasts. C/EBPß is a bzip transcription factor that is expressed in muscle satellite cells and is normally downregulated upon differentiation. However, in myoblasts exposed to a cachectic milieu, C/EBPß expression remains elevated, despite activation to differentiate, resulting in the inhibition of myogenin expression and myogenesis. In vivo, cancer cachexia results in increased number of Pax7+ cells that also express C/EBPß and the inhibition of normal repair mechanisms. Loss of C/EBPß expression in primary myoblasts rescues differentiation under cachectic conditions without restoring myotube size, indicating that C/EBPß is an important inhibitor of myogenesis in cancer cachexia.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Caquexia/metabolismo , Caquexia/patologia , Desenvolvimento Muscular/fisiologia , Neoplasias/metabolismo , Neoplasias/patologia , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Animais , Caquexia/etiologia , Carcinoma Pulmonar de Lewis/complicações , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/patologia , Neoplasias/complicações , RegeneraçãoRESUMO
The process of adipocyte differentiation is driven by a highly coordinated cascade of transcriptional events that results in the development of the mature adipocyte and in lipid accumulation. One of the early events of differentiation is the up-regulation of CCAAT/enhancer-binding protein beta (C/EBPbeta) expression. C/EBPbeta then acts to up-regulate the expression of adipogenic factors such as C/EBPalpha, which control the late stage of adipogenesis. Retinoic acid (RA) is a potent inhibitor of adipogenesis, and its action appears to block C/EBPbeta transcriptional potential early during differentiation. Using preadipocytes and mesenchymal stem cell models, we show that RA specifically blocks the occupancy of C/EBPbeta of the Cebpa promoter, thereby abrogating the differentiation process. RA does not act directly on C/EBPbeta but rather stimulates the expression of the transforming growth factor beta-effector protein Smad3, which can interact with C/EBPbeta via its Mad homology 1 domain and can interfere with C/EBPbeta DNA binding. The RA-induced increase in Smad3 expression results in increased cytoplasmic and nuclear Smad3, an important event as ectopic expression of Smad3 in preadipocytes in the absence of RA treatment only modestly inhibits adipogenesis and C/EBPbeta DNA binding, suggesting that Smad3 alone is not sufficient to completely recapitulate the effects of retinoic acid treatment during differentiation. However, in the absence of Smad3, RA is not able to inhibit adipocyte differentiation or to elicit a decrease in C/EBPbeta DNA occupancy suggesting that Smad3 is necessary to convey the inhibitory effects of retinoic acid during adipogenesis.
Assuntos
Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Antineoplásicos/farmacologia , Células-Tronco Mesenquimais/metabolismo , Proteína Smad3/metabolismo , Tretinoína/farmacologia , Células 3T3-L1 , Adipogenia/fisiologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Regiões Promotoras Genéticas/fisiologia , Proteína Smad3/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Runx2/CBFA1/AML3 is a master regulator of the osteoblast lineage and has been shown to directly control the transcription of numerous osteoblast-specific genes including alkaline phosphatase, osteopontin, and type I collagen. In its absence, ossification does not occur during development resulting in animals with cartilaginous skeletons and no osteoblasts. In humans, loss of one copy of Runx2 causes cleidocranial dysplasia characterized by malformations of the facial and cranial bones and the clavicle. Despite its important role in osteoblast biology, relatively little is known about the transcriptional regulation of the Runx2 gene. In the present study, we show that CCAAT/enhancer binding protein beta (C/EBPbeta) is a negative regulator of Runx2 expression and acts by directly binding a C/EBP element located at -591/-576 within the osteoblast-specific Runx2 P1 promoter. Ectopic expression of C/EBPbeta in C3H10T1/2 cells causes a reduction in Runx2 expression concomitant with a decrease in osteogenic potential during all-trans retinoic acid (ATRA)-induced differentiation. In nondifferentiating cells, C/EBPbeta can be found occupying the C/EBP negative response element within the Runx2 P1 promoter. ATRA, the effects of which are mediated by retinoic acid receptor alpha and gamma in C3H10T1/2 cells, stimulates the dissociation of C/EBPbeta from this element and promotes Runx2 expression. Thus, ATRA initiates osteoblastic differentiation of C3H10T1/2 cells, at least in part, by triggering the dissociation of C/EBPbeta from the Runx2 promoter.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Regulação para Baixo/fisiologia , Osteoblastos/citologia , Tretinoína/antagonistas & inibidores , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Camundongos Endogâmicos C3H , Células NIH 3T3RESUMO
Protein design is currently used for the creation of new proteins with desirable traits, which include a superior nutritional value. One of the challenges of protein design in this area is to achieve the production of stable native-like proteins that resist the proteolytic pressure of the organism used for its production (the bioreactor). We report here the identification of a specific peptide bond sensitive to E. coli proteolysis in the designer protein MB-1Trp. In an attempt to reduce proteolysis, we have created a MB-1TrpHis gene library in which the two amino acids surrounding the peptide bond, N44 and L45, were randomized using degenerated oligonucleotides. The initial characterization of MB-1TrpHis N44E/L45V and MB-1TrpHis N44E/L45M, 2 variants of the library that were more resistant than the parent protein, was performed in order to investigate the nature of the mutants' resistance. Our results suggest that the mutants behaved like MB-1Trp regarding folding and thermal stability, and that proteolytic resistance is due to the elimination of the protease recognition site.