RESUMO
Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/ß-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate (HS) proteoglycan syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its HS chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible short hairpin RNA-mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS chains decorating syndecan-1 mediate aberrant Wnt pathway activation in MM. HS-deficient MM cells exhibited strongly decreased autocrine Wnt/ß-catenin pathway activity and reduced Wnt pathway-dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side chains of syndecan-1 and that this binding contributes to paracrine Wnt pathway activation through the Wnt receptor Frizzled (Fzd). Furthermore, in an HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Fzd degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Costimulation with R-spondin and its binding to HS chains decorating syndecan-1 are indispensable for optimal stimulation of Wnt signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/ß-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets in the treatment of MM.
Assuntos
Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Sindecana-1/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Linhagem Celular Tumoral , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sindecana-1/genética , Trombospondinas/genética , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The unrestrained growth of tumor cells is generally attributed to mutations in essential growth control genes, but tumor cells are also affected by, or even addicted to, signals from the microenvironment. As therapeutic targets, these extrinsic signals may be equally significant as mutated oncogenes. In multiple myeloma (MM), a plasma cell malignancy, most tumors display hallmarks of active Wnt signaling but lack activating Wnt-pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts emanating from the bone marrow (BM) niche. Here, we report a pivotal role for the R-spondin/leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) axis in driving aberrant Wnt/ß-catenin signaling in MM. We show that LGR4 is expressed by MM plasma cells, but not by normal plasma cells or B cells. This aberrant LGR4 expression is driven by IL-6/STAT3 signaling and allows MM cells to hijack R-spondins produced by (pre)osteoblasts in the BM niche, resulting in Wnt (co)receptor stabilization and a dramatically increased sensitivity to auto- and paracrine Wnts. Our study identifies aberrant R-spondin/LGR4 signaling with consequent deregulation of Wnt (co)receptor turnover as a driver of oncogenic Wnt/ß-catenin signaling in MM cells. These results advocate targeting of the LGR4/R-spondin interaction as a therapeutic strategy in MM.
Assuntos
Glicoproteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Interleucina-6/metabolismo , Ligantes , Camundongos , Ligação Proteica/fisiologia , Fator de Transcrição STAT3/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND & AIMS: Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. METHODS: We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (AhCre/Metfl/fl/LacZ) or ISC-specific disruption of MET (Lgr5Creert2/Metfl/fl/LacZ) and control mice (AhCre/Met+/+/LacZ, Lgr5Creert2/Met+/+/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5Creert2/Metfl/fl/Apcfl/fl and Lgr5Creert2/Met+/+/Apcfl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in AhCre/Metfl/fl/Apcfl/+ mice compared with AhCre/Met+/+/Apcfl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44+/+, Cd44-/-, Cd44s/s, or Cd44v4-10/v4-10 mice). RESULTS: Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in AhCre/Metfl/fl/LacZ mice. Lgr5Creert2/Metfl/fl/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5Creert2/Metfl/fl/Apcfl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (AhCre/Metfl/fl/Apcfl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44-/- mice did not expand to the same extent as crypts from Cd44+/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. CONCLUSIONS: In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors.
Assuntos
Adenoma/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Receptores de Hialuronatos/metabolismo , Neoplasias Intestinais/metabolismo , Intestinos/enzimologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Regeneração , Células-Tronco/enzimologia , Adenoma/genética , Adenoma/patologia , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Genótipo , Fator de Crescimento de Hepatócito/farmacologia , Homeostase , Receptores de Hialuronatos/genética , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Intestinos/efeitos da radiação , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Proteínas Proto-Oncogênicas c-met/genética , Regeneração/efeitos dos fármacos , Regeneração/efeitos da radiação , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Células-Tronco/efeitos da radiação , Fatores de Tempo , Técnicas de Cultura de Tecidos , Carga TumoralRESUMO
The recombination activating gene (RAG) 1 and RAG2 protein complex introduces DNA breaks at Tcr and Ig gene segments that are required for V(D)J recombination in developing lymphocytes. Proper regulation of RAG1/2 expression safeguards the ordered assembly of Ag receptors and the development of lymphocytes, while minimizing the risk for collateral damage. The ataxia telangiectasia mutated (ATM) kinase is involved in the repair of RAG1/2-mediated DNA breaks and prevents their propagation. The simultaneous occurrence of RAG1/2-dependent and -independent DNA breaks in developing lymphocytes exposed to genotoxic stress increases the risk for aberrant recombinations. In this study, we assessed the effect of genotoxic stress on RAG1/2 expression in pre-B cells and show that activation of the DNA damage response resulted in the rapid ATM-dependent downregulation of RAG1/2 mRNA and protein expression. We show that DNA damage led to the loss of FOXO1 binding to the enhancer region of the RAG1/2 locus (Erag) and provoked FOXO1 cleavage. We also show that DNA damage caused by RAG1/2 activity in pre-B cells was able to downmodulate RAG1/2 expression and activity, confirming the existence of a negative feedback regulatory mechanism. Our data suggest that pre-B cells are endowed with a protective mechanism that reduces the risk for aberrant recombinations and chromosomal translocations when exposed to DNA damage, involving the ATM-dependent regulation of FOXO1 binding to the Erag enhancer region.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteína Forkhead Box O1/metabolismo , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Células Precursoras de Linfócitos B/metabolismo , Transdução de Sinais , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Nucleares/metabolismoRESUMO
Aberrant activation of Wnt/ß-catenin signaling plays a central role in the pathogenesis of a wide variety of malignancies and is typically caused by mutations in core Wnt pathway components driving constitutive, ligand-independent signaling. In multiple myelomas (MMs), however, these pathway intrinsic mutations are rare despite the fact that most tumors display aberrant Wnt pathway activity. Recent studies indicate that this activation is caused by genetic and epigenetic lesions of Wnt regulatory components, sensitizing MM cells to autocrine Wnt ligands and paracrine Wnts emanating from the bone marrow niche. These include deletion of the tumor suppressor CYLD, promotor methylation of the Wnt antagonists WIF1, DKK1, DKK3, and sFRP1, sFRP2, sFRP4, sFRP5, as well as overexpression of the co-transcriptional activator BCL9 and the R-spondin receptor LGR4. Furthermore, Wnt activity in MM is strongly promoted by interaction of both Wnts and R-spondins with syndecan-1 (CD138) on the MM cell-surface. Functionally, aberrant canonical Wnt signaling plays a dual role in the pathogenesis of MM: (I) it mediates proliferation, migration, and drug resistance of MM cells; (II) MM cells secrete Wnt antagonists that contribute to the development of osteolytic lesions by impairing osteoblast differentiation. As discussed in this review, these insights into the causes and consequences of aberrant Wnt signaling in MM will help to guide the development of targeting strategies. Importantly, since Wnt signaling in MM cells is largely ligand dependent, it can be targeted by drugs/antibodies that act upstream in the pathway, interfering with Wnt secretion, sequestering Wnts, or blocking Wnt (co)receptors.
Assuntos
Mieloma Múltiplo/etiologia , Mieloma Múltiplo/metabolismo , Via de Sinalização Wnt , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Comunicação Autócrina , Biomarcadores , Doenças Ósseas/etiologia , Doenças Ósseas/metabolismo , Epigênese Genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Ligantes , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Comunicação Parácrina , Ligação Proteica , Processamento de Proteína Pós-Traducional , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Mitochondrial dysfunction is the most prominent source of oxidative stress in acute and chronic kidney disease. NLRX1 is a receptor of the innate immune system that is ubiquitously expressed and localized in mitochondria. We investigated whether NLRX1 may act at the interface of metabolism and innate immunity in a model of oxidative stress. Using a chimeric mouse model for renal ischemia-reperfusion injury, we found that NLRX1 protects against mortality, mitochondrial damage, and epithelial cell apoptosis in an oxidative stress-dependent fashion. We found that NLRX1 regulates oxidative phosphorylation and cell integrity, whereas loss of NLRX1 results in increased oxygen consumption, oxidative stress, and subsequently apoptosis in epithelial cells during ischemia-reperfusion injury. In line, we found that NLRX1 expression in human kidneys decreased during acute renal ischemic injury and acute cellular rejection. Although first implicated in immune regulation, we propose that NLRX1 function extends to the control of mitochondrial activity and prevention of oxidative stress and apoptosis in tissue injury.
Assuntos
Apoptose/fisiologia , Mitocôndrias/fisiologia , Proteínas Mitocondriais/fisiologia , Estresse Oxidativo/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Modelos Animais de Doenças , Humanos , Isquemia/fisiopatologia , Rim/irrigação sanguínea , Rim/metabolismo , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
The Wnt/ß-catenin pathway plays a crucial role in the pathogenesis of various human cancers. In multiple myeloma (MM), aberrant auto-and/or paracrine activation of canonical Wnt signaling promotes proliferation and dissemination, while overexpression of the Wnt inhibitor Dickkopf1 (DKK1) by MM cells contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/ß-catenin mediated transcription, these findings suggest that DKK1 is part of a negative feedback loop in MM and may act as a tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is low or undetectable in a subset of patients with advanced MM as well as in MM cell lines. This absence of DKK1 is correlated with enhanced Wnt pathway activation, evidenced by nuclear accumulation of ß-catenin, which in turn can be antagonized by restoring DKK1 expression. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of ß-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling.