RESUMO
Bone destruction, a major source of morbidity, is mediated by heightened differentiation and activity of osteoclasts (OC), highly specialized multinucleated myeloid cells endowed with unique bone-resorptive capacity. The molecular mechanisms regulating OC differentiation in the bone marrow are still partly elusive. Here, we aimed to identify new regulatory circuits and actionable targets by comprehensive proteomic characterization of OCgenesis from mouse bone marrow monocytes, adopting two parallel unbiased comparative proteomic approaches. This work disclosed an unanticipated protein signature of OCgenesis, with most gene products currently unannotated in bone-related functions, revealing broad structural and functional cellular reorganization and divergence from macrophagic immune activity. Moreover, we identified the deubiquitinase UCHL1 as the most upregulated cytosolic protein in differentiating OCs. Functional studies proved it essential, as UCHL1 genetic and pharmacologic inhibition potently suppressed OCgenesis. Furthermore, proteomics and mechanistic dissection showed that UCHL1 supports OC differentiation by restricting the anti-OCgenic activity of NRF2, the transcriptional activator of the canonical antioxidant response, through redox-independent stabilization of the NRF2 inhibitor, KEAP1. Besides offering a valuable experimental framework to dissect OC differentiation, our study discloses the essential role of UCHL1, exerted through KEAP1-dependent containment of NRF2 anti-OCgenic activity, yielding a novel potential actionable pathway against bone loss.
Assuntos
Reabsorção Óssea , Osteólise , Animais , Camundongos , Reabsorção Óssea/metabolismo , Diferenciação Celular/genética , Enzimas Desubiquitinantes/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Osteoclastos/metabolismo , Osteólise/metabolismo , Proteômica , Ligante RANK/metabolismoRESUMO
Multiple myeloma grows by establishing multiple interactions with bone marrow cells. These include expansion of myeloid-derived suppressor cells, which drive immunoevasion via mechanisms that include arginase-1-driven depletion of L-arginine, thus indirectly promoting myeloma cell survival and tumor progression. The peculiar biology of malignant plasma cells postulates that arginine depletion may benefit their fitness also directly, e.g., by engaging the integrated stress response, or by stimulating autophagy through mTORC1 inhibition. We thus investigated the direct impact of arginine deprivation on myeloma cells and challenged its pathophysiological relevance in vitro and in vivo. First, we found that partial arginine depletion spared proliferation of human multiple myeloma cells at concentrations that arrest human T cells. Next, we asked if arginine shortage activates putative adaptive pathways in myeloma cells. Low arginine failed to activate the integrated stress response, as indicated by unmodified phosphorylation of the eukaryotic initiation factor 2α, but sizably inhibited mTORC1, as revealed by reduced phosphorylation of ribosomal protein S6. Notably, depressed mTORC1 activity was not sufficient to increase autophagy, as assessed by the lysosomal digestion rate of the autophagosome-associated protein, LC3-II. Rather, it stimulated mTORC2, resulting in increased phosphatidylinositol-3 kinase-dependent AKT phosphorylation and activity, leading to heightened inhibitory phosphorylation of the pro-apoptotic BAD protein. We then tested whether arginine depletion-activated AKT may protect malignant plasma cells from cell death. Indeed, culturing myeloma cells in low arginine medium significantly reduced the apoptotic effect of the first-in-class proteasome inhibitor, bortezomib, an outcome prevented by pharmacological inhibition of AKT phosphorylation. Finally, we challenged the relevance of the identified circuit in vivo. To gauge the pathophysiologic relevance of low arginine to myeloma growth independently of immunoevasion, we xenotransplanted human myeloma cells subcutaneously into T cell-deficient Rag2-/-γc-/- recipient mice and treated palpable tumor-bearing mice with the clinical-grade arginase inhibitor CB1158. Arginase inhibition significantly raised serum arginine concentration, reduced tumor growth by caliper assessment, and decreased intra-tumor AKT phosphorylation in vivo. Altogether, our results reveal a novel direct pro-survival effect of arginine deprivation on myeloma cells, with potential therapeutic implications.
RESUMO
FAM46C is a non-canonical poly(A) polymerase uniquely mutated in up to 20% of multiple myeloma (MM) patients, implying a tissue-specific tumor suppressor function. Here, we report that FAM46C selectively stabilizes mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, thereby concertedly enhancing the expression of proteins that control ER protein import, folding, N-glycosylation, and trafficking and boosting protein secretion. This role requires the interaction with the ER membrane resident proteins FNDC3A and FNDC3B. In MM cells, FAM46C expression raises secretory capacity beyond sustainability, inducing ROS accumulation, ATP shortage, and cell death. FAM46C activity is regulated through rapid proteasomal degradation or the inhibitory interaction with the ZZ domain of the autophagic receptor p62 that hinders its association with FNDC3 proteins via sequestration in p62+ aggregates. Altogether, our data disclose a p62/FAM46C/FNDC3 circuit coordinating sustainable secretory activity and survival, providing an explanation for the MM-specific oncosuppressive role of FAM46C and uncovering potential therapeutic opportunities against cancer.
Assuntos
Fibronectinas/metabolismo , Homeostase , Nucleotidiltransferases/metabolismo , Proteostase , Proteína Sequestossoma-1/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , Inativação Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Imunoglobulinas/metabolismo , Membranas Intracelulares/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/patologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Inibidores de Proteassoma/farmacologia , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteostase/efeitos dos fármacos , Proteína Sequestossoma-1/químicaRESUMO
To cope with intrinsic and environmental stress, cancer cells rely on adaptive pathways more than non-transformed counterparts. Such non-oncogene addiction offers new therapeutic targets and strategies to overcome chemoresistance. In an attempt to study the role of adaptive pathways in acquired drug resistance in carcinoma cells, we devised a model of in vitro conditioning to three standard chemotherapeutic agents, cisplatin, 5-fluorouracil, and docetaxel, from the epithelial cancer cell line, HEp-2, and investigated the mechanisms underlying reduced drug sensitivity. We found that triple-resistant cells suffered from higher levels of oxidative stress, and showed heightened anti-stress responses, including the antioxidant Nrf2 pathway and autophagy, a conserved pleiotropic homeostatic strategy, mediating the clearance of aggregates marked by the adapter p62/SQSTM1. As a result, re-administration of chemotherapeutic agents failed to induce further accumulation of reactive oxygen species and p62. Moreover, autophagy proved responsible for chemoresistance through the avoidance of p62 accumulation into toxic protein aggregates. Indeed, p62 ablation was sufficient to confer resistance in parental cells, and genetic and pharmacological autophagic inhibition restored drug sensitivity in resistant cells in a p62-dependent manner. Finally, exogenous expression of mutant p62 lacking the ubiquitin- and LC3-binding domains, required for autophagic engulfment, increased chemosensitivity in TDR HEp-2 cells. Altogether, these findings offer a cellular system to investigate the bases of acquired chemoresistance of epithelial cancers and encourage challenging the prognostic and antineoplastic therapeutic potential of p62 toxicity.
Assuntos
Antineoplásicos/farmacologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Epiteliais e Glandulares/metabolismo , Proteína Sequestossoma-1/metabolismo , Autofagia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Docetaxel/farmacologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Neoplasias Epiteliais e Glandulares/genética , Estresse Oxidativo , Domínios Proteicos , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/genéticaRESUMO
Chronic lymphocytic leukemia cells strongly depend on external stimuli for their survival. Both antigen receptor and co-stimulatory receptors, including Toll-like receptors, can modulate viability and proliferation of leukemic cells. Toll-like receptor ligands, and particularly the TLR9 ligand CpG, mediate heterogeneous responses in patients' samples reflecting the clinical course of the subjects. However, the molecular framework of the key signaling events underlying such heterogeneity is undefined. We focused our studies on a subset of chronic lymphocytic leukemia cases characterized by expression of CD38 and unmutated immunoglobulin genes, who respond to CpG with enhanced metabolic cell activity. We report that, while CpG induces NFKBIZ mRNA in all the samples analyzed, it induces the IκBζ protein in a selected group of cases, through an unanticipated post-transcriptional mechanism. Interestingly, IκBζ plays a causal role in sustaining CpG-induced cell viability and chemoresistance, and CpG stimulation can unleash immunoglobulin secretion by IκBζ-positive malignant cells. These results identify and characterize IκBζ as a marker and effector molecule of distinct key pathways in chronic lymphocytic leukemia.
Assuntos
Regulação Leucêmica da Expressão Gênica , Proteínas I-kappa B/genética , Imunoglobulina M/biossíntese , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas Nucleares/genética , Receptor Toll-Like 9/agonistas , Proteínas Adaptadoras de Transdução de Sinal , Autofagia , Biomarcadores , Células Cultivadas , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/patologia , Oligodesoxirribonucleotídeos/farmacologia , Processamento Pós-Transcricional do RNARESUMO
Systemic light chain (AL) amyloidosis is caused by the clonal production of an unstable immunoglobulin light chain (LC), which affects organ function systemically. Although pathogenic LCs have been characterized biochemically, little is known about the biology of amyloidogenic plasma cells (PCs). Intrigued by the unique response rates of AL amyloidosis patients to the first-in-class proteasome inhibitor (PI) bortezomib, we purified and investigated patient-derived AL PCs, in comparison with primary multiple myeloma (MM) PCs, the prototypical PI-responsive cells. Functional, biochemical, and morphological characterization revealed an unprecedented intrinsic sensitivity of AL PCs to PIs, even higher than that of MM PCs, associated with distinctive organellar features and expression patterns indicative of cellular stress. These consisted of expanded endoplasmic reticulum (ER), perinuclear mitochondria, and a higher abundance of stress-related transcripts, and were consistent with reduced autophagic control of organelle homeostasis. To test whether PI sensitivity stems from AL LC production, we engineered PC lines that can be induced to express amyloidogenic and nonamyloidogenic LCs, and found that AL LC expression alters cell growth and proteostasis and confers PI sensitivity. Our study discloses amyloidogenic LC production as an intrinsic PC stressor, and identifies stress-responsive pathways as novel potential therapeutic targets. Moreover, we contribute a cellular disease model to dissect the biology of AL PCs.
Assuntos
Amiloidose/tratamento farmacológico , Amiloidose/metabolismo , Bortezomib/farmacocinética , Cadeias Leves de Imunoglobulina/biossíntese , Plasmócitos/metabolismo , Inibidores de Proteassoma/farmacocinética , Amiloidose/patologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Plasmócitos/patologiaRESUMO
Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Inibidores de Proteassoma/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteína Sequestossoma-1 , Proteínas Ubiquitinadas/metabolismoRESUMO
The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo.
Assuntos
Apresentação de Antígeno/fisiologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas Nucleares/imunologia , Osteoclastos/imunologia , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Transativadores/imunologia , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5(-/-) differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.
Assuntos
Autofagia , Linfócitos B/metabolismo , Imunoglobulinas/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Plasmócitos/imunologia , Trifosfato de Adenosina , Animais , Formação de Anticorpos , Proteína 5 Relacionada à Autofagia , Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Diferenciação Celular , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/genética , Centro Germinativo/imunologia , Homeostase , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Plasmócitos/citologia , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fatores de Transcrição/biossínteseRESUMO
It has been proposed that in cancer, where the bulk of the genome becomes hypomethylated, there is an increase in transcriptional noise that might lead to the generation of antisense transcripts that could affect the function of key oncosuppressor genes, ultimately leading to malignant transformation. Here, we describe the computational identification of a melanoma-enriched antisense transcript, TRPM2-AS, mapped within the locus of TRPM2, an ion channel capable of mediating susceptibility to cell death. Analysis of the TRPM2-AS genomic region indicated the presence in the same region of another tumor-enriched TRPM2 transcript, TRPM2-TE, located across a CpG island shared with TRPM2-AS. Quantitative PCR experiments confirmed that TRPM2-AS and TRPM2-TE transcripts were up-regulated in melanoma, and their activation was consistent with the methylation status of the shared CpG island. Functional knock-out of TRPM2-TE, as well as over-expression of wild-type TRPM2, increased melanoma susceptibility to apoptosis and necrosis. Finally, expression analysis in other cancer types indicated that TRPM2-AS and TRPM2-TE over-expression might have an even wider role than anticipated, reinforcing the relevance of our computational approach in identifying new potential therapeutic targets.
Assuntos
Elementos Antissenso (Genética) , Melanoma/genética , Neoplasias Cutâneas/genética , Canais de Cátion TRPM/genética , Apoptose/genética , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Ilhas de CpG , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Transcrição GênicaRESUMO
Transformed stem cells have been isolated from some human cancers. We report that, unlike other brain cancers, the lethal glioblastoma multiforme contains neural precursors endowed with all of the critical features expected from neural stem cells. Similar, yet not identical, to their normal neural stem cell counterpart, these precursors emerge as unipotent (astroglial) in vivo and multipotent (neuronal-astroglial-oligodendroglial) in culture. More importantly, these cells can act as tumor-founding cells down to the clonal level and can establish tumors that closely resemble the main histologic, cytologic, and architectural features of the human disease, even when challenged through serial transplantation. Thus, cells possessing all of the characteristics expected from tumor neural stem cells seem to be involved in the growth and recurrence of adult human glioblastomas multiforme.