RESUMO
Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.
Assuntos
Proteínas de Bactérias/genética , HIV/genética , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/genética , Proteínas Proto-Oncogênicas c-cbl/genética , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/imunologia , Linhagem Celular Tumoral , Chlamydia trachomatis/genética , Chlamydia trachomatis/imunologia , Regulação da Expressão Gênica , HIV/imunologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Humanos , Linfócitos/microbiologia , Linfócitos/virologia , Macrófagos/microbiologia , Macrófagos/virologia , Camundongos , Mycobacterium tuberculosis/imunologia , Cultura Primária de Células , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-cbl/deficiência , Proteínas Proto-Oncogênicas c-cbl/imunologia , Células RAW 264.7 , Transdução de Sinais , Fatores de Virulência/imunologiaRESUMO
Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b Importantly, primary human CBL mutated (CBLmut ) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies.
Assuntos
Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/fisiopatologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Citocinas/metabolismo , Estabilidade Enzimática , Células-Tronco Hematopoéticas/enzimologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Janus Quinase 2/genética , Leucemia Mieloide Aguda/genética , Proteínas de Membrana , Camundongos , Mutação , Proteólise , Proteínas Proto-Oncogênicas c-cbl/genética , Transdução de Sinais/genética , UbiquitinaçãoRESUMO
Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express second-site point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr â Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr â Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBL-Y371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-of-function in which mutant CBL proteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyper-activate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.
Assuntos
Proteínas Mutantes/química , Mutação , Oncogenes , Proteínas Proto-Oncogênicas c-cbl/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Separação Celular , Citocinas/metabolismo , Citometria de Fluxo , Regulação Leucêmica da Expressão Gênica , Glutationa Transferase/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Knockout , Mutagênese , Proteínas Mutantes/genética , Fenótipo , Fenilalanina/química , Fosforilação , Prolina/química , Domínios Proteicos , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Recombinantes de Fusão/química , Transdução de Sinais , Tirosina/químicaRESUMO
The C-terminal Eps15 homology domain-containing (EHD) proteins play a key role in endocytic recycling, a fundamental cellular process that ensures the return of endocytosed membrane components and receptors back to the cell surface. To define the in vivo biological functions of EHD1, we have generated Ehd1 knockout mice and previously reported a requirement of EHD1 for spermatogenesis. Here, we show that approximately 56% of the Ehd1-null mice displayed gross ocular abnormalities, including anophthalmia, aphakia, microphthalmia and congenital cataracts. Histological characterization of ocular abnormalities showed pleiotropic defects that include a smaller or absent lens, persistence of lens stalk and hyaloid vasculature, and deformed optic cups. To test whether these profound ocular defects resulted from the loss of EHD1 in the lens or in non-lenticular tissues, we deleted the Ehd1 gene selectively in the presumptive lens ectoderm using Le-Cre. Conditional Ehd1 deletion in the lens resulted in developmental defects that included thin epithelial layers, small lenses and absence of corneal endothelium. Ehd1 deletion in the lens also resulted in reduced lens epithelial proliferation, survival and expression of junctional proteins E-cadherin and ZO-1. Finally, Le-Cre-mediated deletion of Ehd1 in the lens led to defects in corneal endothelial differentiation. Taken together, these data reveal a unique role for EHD1 in early lens development and suggest a previously unknown link between the endocytic recycling pathway and regulation of key developmental processes including proliferation, differentiation and morphogenesis.
Assuntos
Endocitose , Cristalino/embriologia , Cristalino/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Catarata/complicações , Catarata/embriologia , Catarata/genética , Catarata/patologia , Diferenciação Celular , Polaridade Celular , Sobrevivência Celular , Embrião de Mamíferos/patologia , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Células Epiteliais/patologia , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/patologia , Camundongos Knockout , Microftalmia/complicações , Microftalmia/embriologia , Microftalmia/genética , Fenótipo , Proteínas de Transporte Vesicular/deficiênciaRESUMO
Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B-lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell regulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl-knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5 Hi ISCs with transient expansion of the Lgr5 Lo transit-amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single-cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.
RESUMO
Introduction: Atherosclerosis is a lipid-driven inflammatory disease of the arterial wall, and the underlying cause of the majority of cardiovascular diseases. Recent advances in high-parametric immunophenotyping of immune cells indicate that T cells constitute the major leukocyte population in the atherosclerotic plaque. The E3 ubiquitin ligase Casitas B-lymphoma proto-oncogene-B (CBL-B) is a critical intracellular regulator that sets the threshold for T cell activation, making CBL-B a potential therapeutic target to modulate inflammation in atherosclerosis. We previously demonstrated that complete knock-out of CBL-B aggravated atherosclerosis in Apoe-/- mice, which was attributed to increased macrophage recruitment and increased CD8+ T cell activation in the plaque. Methods: To further study the T cell specific role of CBL-B in atherosclerosis, Apoe-/- CD4cre Cblb fl/fl (Cbl-bcKO) mice and Apoe-/-CD4WTCblbfl/fl littermates (Cbl-bfl/fl) were fed a high cholesterol diet for ten weeks. Results: Cbl-bcKO mice had smaller atherosclerotic lesions in the aortic arch and root compared to Cbl-bfl/fl, and a substantial increase in CD3+ T cells in the plaque. Collagen content in the plaque was decreased, while other plaque characteristics including plaque necrotic core, macrophage content, and smooth muscle cell content, remained unchanged. Mice lacking T cell CBL-B had a 1.4-fold increase in CD8+ T cells and a 1.8-fold increase in regulatory T cells in the spleen. Splenic CD4+ and CD8+ T cells had increased expression of C-X-C Motif Chemokine Receptor 3 (CXCR3) and interferon-γ (IFN-γ), indicating a T helper 1 (Th1)-like/effector CD8+ T cell-like phenotype. Conclusion: In conclusion, Cbl-bcKO mice have reduced atherosclerosis but show increased T cell accumulation in the plaque accompanied by systemic T cell activation.
Assuntos
Aterosclerose , Linfoma , Placa Aterosclerótica , Animais , Camundongos , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Linfócitos T CD8-Positivos , Camundongos Knockout , Placa Aterosclerótica/patologia , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel targetable pathways that contribute to tumor progression in PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC is unexplored. Here, we show that GD2 is expressed in a small subpopulation of PC cells in a subset of patients and a higher proportion of metastatic tumors. Variable levels of cell surface GD2 expression were seen on many PC cell lines, and the expression was highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction was enriched upon growth of PC cells as tumorspheres and GD2high fraction was enriched in tumorsphere-forming ability. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2high CRPC cell models markedly impaired the in vitro oncogenic traits and growth as bone-implanted xenograft tumors and reduced the cancer stem cell and epithelial-mesenchymal transition marker expression. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.
Assuntos
Carcinogênese , Gangliosídeos , Células-Tronco Neoplásicas , Sialiltransferases , Masculino , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Sialiltransferases/metabolismo , Sialiltransferases/genética , Animais , Linhagem Celular Tumoral , Gangliosídeos/metabolismo , Camundongos , Carcinogênese/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Feniltioidantoína/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Benzamidas/farmacologia , Nitrilas/farmacologiaRESUMO
Among the signaling pathways that control the stem cell self-renewal and maintenance vs. acquisition of differentiated cell fates, those mediated by receptor tyrosine kinase (RTK) activation are well established as key players. CBL family ubiquitin ligases are negative regulators of RTKs but their physiological roles in regulating stem cell behaviors are unclear. While hematopoietic Cbl/Cblb knockout (KO) leads to a myeloproliferative disease due to expansion and reduced quiescence of hematopoietic stem cells, mammary epithelial KO led to stunted mammary gland development due to mammary stem cell depletion. Here, we examined the impact of inducible Cbl/Cblb double-KO (iDKO) selectively in the Lgr5-defined intestinal stem cell (ISC) compartment. Cbl/Cblb iDKO led to rapid loss of the Lgr5 Hi ISC pool with a concomitant transient expansion of the Lgr5 Lo transit amplifying population. LacZ reporter-based lineage tracing showed increased ISC commitment to differentiation, with propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro , Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single cell RNAseq analysis of organoids revealed Akt-mTOR pathway hyperactivation in iDKO ISCs and progeny cells, and pharmacological inhibition of the Akt-mTOR axis rescued the organoid maintenance and propagation defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.
RESUMO
With nearly all cancer deaths a result of metastasis, elucidating novel pro-metastatic cellular adaptations could provide new therapeutic targets. Here, we show that overexpression of the EPS15-Homology Domain-containing 2 (EHD2) protein in a large subset of breast cancers (BCs), especially the triple-negative (TNBC) and HER2+ subtypes, correlates with shorter patient survival. The mRNAs for EHD2 and Caveolin-1/2, structural components of caveolae, show co-overexpression across breast tumors, predicting shorter survival in basal-like BC. EHD2 shRNA knockdown and CRISPR-Cas9 knockout with mouse Ehd2 rescue, in TNBC cell line models demonstrate a major positive role of EHD2 in promoting tumorigenesis and metastasis. Mechanistically, we link these roles of EHD2 to store-operated calcium entry (SOCE), with EHD2-dependent stabilization of plasma membrane caveolae ensuring high cell surface expression of the SOCE-linked calcium channel Orai1. The novel EHD2-SOCE oncogenic axis represents a potential therapeutic target in EHD2- and CAV1/2-overexpressing BC.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Camundongos , Animais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Transformação Celular Neoplásica/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
Overexpression of EPS15 Homology Domain containing 1 (EHD1) has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD mRNA expression specifying shorter patient survival. ShRNA and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified the IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.
RESUMO
Overexpression of the EPS15 Homology Domain containing 1 (EHD1) protein has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD1 mRNA expression specifying shorter patient survival. ShRNA-knockdown and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.
Assuntos
Sarcoma de Ewing , Camundongos , Animais , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Membrana Celular/metabolismo , Transdução de Sinais/fisiologia , Carcinogênese/genética , Carcinogênese/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismoRESUMO
While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel, targetable, pathways that contribute to tumor progression of PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC has been only little explored. Here, we show that GD2 is expressed on a small subpopulation of PC cells in a subset of patients, especially in metastatic PC. Variable levels of cell surface GD2 expression are seen in most PC cell lines, and the expression is highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction is enriched upon growth of PC cells as tumorspheres and GD2high fraction is enriched in tumorsphere growth. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2-high CRPC cell models led to marked impairment of their in vitro oncogenic traits, reduced cancer stem cell (CSC) and epithelial-mesenchymal transition (EMT) marker expression and growth as bone-implanted xenograft tumors. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.
RESUMO
Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC-mediated glucose metabolism. IMPLICATIONS: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism.
Assuntos
Neoplasias da Mama , Carcinogênese , Carcinógenos , Proteínas de Transporte , Glucose , Proteínas Proto-Oncogênicas c-myc , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/genética , Carcinogênese/patologia , Proteínas de Transporte/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Glucose/metabolismo , Humanos , Hiperplasia/genética , Hiperplasia/patologia , Neoplasias Pulmonares/secundário , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro , Transdução de Sinais , Regulação para CimaRESUMO
Overexpression of the epidermal growth factor receptor (EGFR) family member ErbB2 (HER2) drives oncogenesis in up to 25% of invasive breast cancers. ErbB2 expression at the cell surface is required for oncogenesis but mechanisms that ensure the optimal cell surface display of overexpressed ErbB2 following its biosynthesis in the endoplasmic reticulum are poorly understood. ErbB2 is dependent on continuous association with HSP90 molecular chaperone for its stability and function as an oncogenic driver. Here, we use knockdown and overexpression studies to show that the HSP90/HSC70-interacting negative co-chaperone CHIP (C-terminus of HSC70-Interacting protein)/STUB1 (STIP1-homologous U-Box containing protein 1) targets the newly synthesized, HSP90/HSC70-associated, ErbB2 for ubiquitin/proteasome-dependent degradation in the endoplasmic reticulum and Golgi, thus identifying a novel mechanism that negatively regulates cell surface ErbB2 levels in breast cancer cells, consistent with frequent loss of CHIP expression previously reported in ErbB2-overexpressing breast cancers. ErbB2-overexpressing breast cancer cells with low CHIP expression exhibited higher endoplasmic reticulum stress inducibility. Accordingly, the endoplasmic reticulum stress-inducing anticancer drug Bortezomib combined with ErbB2-targeted humanized antibody Trastuzumab showed synergistic inhibition of ErbB2-overexpressing breast cancer cell proliferation. Our findings reveal new insights into mechanisms that control the surface expression of overexpressed ErbB2 and suggest that reduced CHIP expression may specify ErbB2-overexpressing breast cancers suitable for combined treatment with Trastuzumab and ER stress inducing agents.
RESUMO
Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.
Assuntos
Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Comunicação Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Proliferação de Células/fisiologia , Receptores ErbB/metabolismo , Humanos , Camundongos , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
OBJECTIVES: Mutations in Cbl or Cbl-b gene occur in 10% of myeloproliferative disorder (MPD) patients and are associated with poor prognosis. Hematopoietic Cbl/Cbl-b double knockout (DKO) leads to a disease in mice phenotypically similar to human MPDs. The aim of this study was to evaluate the anti-MPD activity of a clinically safe drug, Fasudil, identified in an in vitro kinase inhibitor as an inhibitor of proliferation of DKO mouse hematopoietic stem/progenitor cells (HSPCs). METHODS: Fasudil exhibited relatively selective anti-proliferative activity against Cbl/Cbl-b DKO vs. control murine bone marrow HSPCs. We established a mouse model with uniform time of MPD onset by transplanting Cbl/Cbl-b DKO HSPCs into busulfan-conditioned NOD/SCID/gamma chain-deficient mice. Four weeks post-transplant, mice were treated with 100 mg/kg fasudil (13 mice) or water (control, 8 mice) daily by oral gavage, followed by blood cell count every 2 weeks. RESULTS: By 2 weeks of treatment, total white cell and monocyte counts were significantly lower in mice treated with fasudil. We observed a trend towards improved survival in fasudil-treated mice that did not reach statistical significance. Notably, prolonged survival beyond 27 weeks was observed in two fasudil-treated mice, nearly twice the 16-week average life-span in the Cbl/Cbl-b DKO MPD model. CONCLUSIONS: Our results suggest a therapeutic potential for fasudil, a clinically safe drug with promising results in vascular diseases, in the treatment of MPDs or other mutant Cbl-driven myeloid disorders.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Transtornos Mieloproliferativos , Proteínas Proto-Oncogênicas c-cbl/deficiência , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
CBL and CBL-B ubiquitin ligases play key roles in hematopoietic stem cell homeostasis and their aberrations are linked to leukemogenesis. Mutations of CBL, often genetically-inherited, are particularly common in Juvenile Myelomonocytic Leukemia (JMML), a disease that manifests early in children. JMML is fatal unless corrected by bone marrow transplant, which is effective in only half of the recipients, stressing the need for animal models that recapitulate the key clinical features of this disease. However, mouse models established so far only develop hematological malignancy in adult animals. Here, using VAV1-Cre-induced conditional CBL/CBL-B double knockout (DKO) in mice, we established an animal model that exhibits a neonatal myeloproliferative disease (MPD). VAV1-Cre induced DKO mice developed a strong hematological phenotype at postnatal day 10, including severe leukocytosis and hepatomegaly, bone marrow cell hypersensitivity to cytokines including GM-CSF, and rapidly-progressive disease and invariable lethality. Interestingly, leukemic stem cells were most highly enriched in neonatal liver rather than bone marrow, which, along with the spleen and thymus, were hypo-cellular. Nonetheless, transplantation assays showed that both DKO bone marrow and liver cells can initiate leukemic disease in the recipient mice with seeding of both spleen and bone marrow. Together, our results support the usefulness of the new hematopoietic-specific CBL/CBL-B double KO animal model to study JMML-related pathogenesis and to further understand the function of CBL family proteins in regulating fetal and neonatal hematopoiesis. To our knowledge, this is the first mouse model that exhibits neonatal MPD in infancy, by day 10 of postnatal life.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Leucemia Mielomonocítica Juvenil/genética , Mutação/genética , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-vav/genética , Animais , Animais Recém-Nascidos , Transplante de Medula Óssea , Células Cultivadas , Hematopoese , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling.
Assuntos
Cílios/genética , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Morfogênese , Tubo Neural/embriologia , Tubo Neural/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Animais , Cílios/patologia , Desenvolvimento Embrionário/genética , Feminino , Fibroblastos/metabolismo , Deleção de Genes , Expressão Gênica , Genes Letais , Patrimônio Genético , Genótipo , Masculino , Camundongos , Camundongos Knockout , Morfogênese/genética , Família Multigênica , Ligação Proteica , Transporte Proteico , Receptor Smoothened/metabolismoRESUMO
Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling.
Assuntos
Células-Tronco Hematopoéticas/enzimologia , Leucemia/tratamento farmacológico , Leucemia/enzimologia , Proteínas Proto-Oncogênicas c-cbl/deficiência , Animais , Fluoruracila/farmacologia , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Transdução de SinaisRESUMO
Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of (125)I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl-CIN85-endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.