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BACKGROUND AND AIMS: Liver fibrosis represents a global health burden, given the paucity of approved antifibrotic therapies. Liver sinusoidal endothelial cells (LSECs) play a major gatekeeping role in hepatic homeostasis and liver disease pathophysiology. In early tumorigenesis, runt-related transcription factor 3 (RUNX3) functions as a sentinel; however, its function in liver fibrosis in LSECs remains unclear. This study aimed to investigate the role of RUNX3 as an important regulator of the gatekeeping functions of LSECs and explore novel angiocrine regulators of liver fibrosis. APPROACH AND RESULTS: Mice with endothelial Runx3 deficiency develop gradual and spontaneous liver fibrosis secondary to LSEC dysfunction, thereby more prone to liver injury. Mechanistic studies in human immortalized LSECs and mouse primary LSECs revealed that IL-6/JAK/STAT3 pathway activation was associated with LSEC dysfunction in the absence of RUNX3. Single-cell RNA sequencing and quantitative RT-PCR revealed that leucine-rich alpha-2-glycoprotein 1 ( LRG1 ) was highly expressed in RUNX3-deficient and dysfunctional LSECs. In in vitro and coculture experiments, RUNX3-depleted LSECs secreted LRG1, which activated HSCs throughTGFBR1-SMAD2/3 signaling in a paracrine manner. Furthermore, circulating LRG1 levels were elevated in mouse models of liver fibrosis and in patients with fatty liver and cirrhosis. CONCLUSIONS: RUNX3 deficiency in the endothelium induces LSEC dysfunction, LRG1 secretion, and liver fibrosis progression. Therefore, endothelial RUNX3 is a crucial gatekeeping factor in LSECs, and profibrotic angiocrine LRG1 may be a novel target for combating liver fibrosis.
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RUNX family members play pivotal roles in both normal development and neoplasia. In particular, RUNX1 and RUNX2 are essential for determination of the hematopoietic and osteogenic lineages, respectively. RUNX3 is involved in lineage determination of various types of epithelial cells. Analysis of mouse models and human cancer specimens revealed that RUNX3 acts as a tumor suppressor via multiple mechanisms. p53-related pathways play central roles in tumor suppression through the DNA damage response and oncogene surveillance, and RUNX3 is involved in both processes. In response to DNA damage, RUNX3 facilitates p53 phosphorylation by the ATM/ATR pathway and p53 acetylation by p300. When oncogenes are activated, RUNX3 induces ARF, thereby stabilizing p53. Here, we summarize the molecular mechanisms underlying the p53-mediated tumor-suppressor activity of RUNX3.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/genética , Genes ras/genética , Neoplasias/genética , Oncogenes/genética , Proteína Supressora de Tumor p53/genética , Animais , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , HumanosRESUMO
Core binding factor ß (Cbfß) is a partner protein of Runx family transcription factors with minimally characterized function in cartilage. Here we address the role of Cbfß in cartilage by generating chondrocyte-specific Cbfß-deficient mice (Cbfb(Δch/Δch) ) from Cbfb-floxed mice crossed with mice expressing Cre from the Col2a1 promoter. Cbfb(Δch/Δch) mice died soon after birth and exhibited delayed endochondral bone formation, shorter appendicular skeleton length with increased proliferative chondrocytes, and nearly absent hypertrophic chondrocyte zones. Immunohistochemical and quantitative real-time PCR analyses showed that the number and size of proliferative chondrocytes increased and the expression of chondrocyte maturation markers at the growth plates, including Runx2, osterix, and osteopontin, significantly diminished in Cbfb(Δch/Δch) mice compared to wild type mice. With regard to signaling pathways, both PTHrP-Ihh and BMP signaling were compromised in Cbfb(Δch/Δch) mice. Mechanistically, Cbfß deficiency in chondrocytes caused a decrease of protein levels of Runx transcription factors by accelerating polyubiquitination-mediated proteosomal degradation in vitro. Indeed, Runx2 and Runx3, but not Runx1, decreased in Cbfb(Δch/Δch) mice. Collectively, these findings indicate that Cbfß plays a critical role for chondrocyte differentiation through stabilizing Runx2 and Runx3 proteins in cartilage.
Assuntos
Diferenciação Celular/genética , Condrócitos/citologia , Condrogênese/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , Lâmina de Crescimento/metabolismo , Animais , Cartilagem/fisiologia , Subunidade beta de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteogênese/fisiologiaRESUMO
Fibroblast growth factor 2 (FGF2) signaling plays a pivotal role in bone growth/differentiation through the activation of osteogenic master transcription factor Runx2, which is mediated by the ERK/MAPK-dependent phosphorylation and the p300-dependent acetylation of Runx2. In this study, we found that Pin1-dependent isomerization of Runx2 is the critical step for FGF2-induced Runx2 transactivation function. We identified four serine or threonine residues in the C-terminal domain of Runx2 that are responsible for Pin1 binding and structural modification. Confocal imaging studies indicated that FGF2 treatment strongly stimulated the focal accumulation of Pin1 in the subnuclear area, which recruited Runx2. In addition, active forms of RNA polymerase-II also colocalized in the same subnuclear compartment. Dipentamethylene thiuram monosulfide, a Pin1 inhibitor, strongly attenuated their focal accumulation as well as Runx2 transactivation activity. The Pin1-mediated structural modification of Runx2 is an indispensable step connecting phosphorylation and acetylation and, consequently, transcriptional activation of Runx2 by FGF signaling. Thus, the modulation of Pin1 activity may be a target for the regulation of bone formation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/química , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Osteoblastos/citologia , Peptidilprolil Isomerase/metabolismo , Acetilação/efeitos dos fármacos , Animais , Sítios de Ligação , Núcleo Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Humanos , Isomerismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Peptidilprolil Isomerase de Interação com NIMA , Osteoblastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.
Assuntos
Neoplasias Ósseas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , MicroRNAs/metabolismo , Osteossarcoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Raios gama , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Osteossarcoma/genética , Osteossarcoma/patologia , Estabilidade Proteica/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/deficiênciaRESUMO
PURPOSE: RUNX3 is a tumor suppressor gene, which is inactivated in approximately 70% of lung adenocarcinomas. Nicotinamide, a sirtuin inhibitor, has demonstrated potential in re-activating epigenetically silenced RUNX3 in cancer cells. This study assessed the therapeutic benefits of combining nicotinamide with first-generation EGFR-tyrosine kinase inhibitors (TKI) for patients with stage IV lung cancer carrying EGFR mutations. PATIENTS AND METHODS: We assessed the impact of nicotinamide on carcinogen-induced lung adenocarcinomas in mice and observed that nicotinamide increased RUNX3 levels and inhibited lung cancer growth. Subsequently, 110 consecutive patients with stage IV lung cancer who had EGFR mutations were recruited: 70 females (63.6%) and 84 never-smokers (76.4%). The patients were randomly assigned to receive either nicotinamide (1 g/day, n = 55) or placebo (n = 55). The primary and secondary endpoints were progression-free survival (PFS) and overall survival (OS), respectively. RESULTS: After a median follow-up of 54.3 months, the nicotinamide group exhibited a median PFS of 12.7 months [95% confidence interval (CI), 10.4-18.3], while the placebo group had a PFS of 10.9 months (9.0-13.2; P = 0.2). The median OS was similar in the two groups (31.0 months with nicotinamide vs. 29.4 months with placebo; P = 0.2). Notably, subgroup analyses revealed a significant reduction in mortality risk for females (P = 0.01) and never-smokers (P = 0.03) treated with nicotinamide. CONCLUSIONS: The addition of nicotinamide with EGFR-TKIs demonstrated potential improvements in PFS and OS, with notable survival benefits for female patients and those who had never smoked (ClinicalTrials.gov Identifier: NCT02416739).
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Feminino , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Niacinamida/uso terapêutico , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Receptores ErbB/genéticaRESUMO
The regulation of hypoxia-inducible factor-1α (HIF-1α) during endochondral bone formation is not fully understood. Here, we investigated the cross-talk between HIF-1α and Runt-related transcription factor 2 (Runx2) in the growth plate. Runx2 caused the accumulation of HIF-1α protein in ATDC5 chondrocytes and HEK293 cells under normoxic conditions. Runx2 also increased the nuclear translocation of HIF-1α when coexpressed in HEK293 cells and interacted with HIF-1α at the oxygen-dependent degradation domain (ODDD). In addition, Runx2 competed with von Hippel-Lindau tumor suppressor protein by directly binding to ODDD-HIF-1α and significantly inhibited the ubiquitination of HIF-1α, even though Runx2 did not change the hydroxylation status of HIF-1α. Furthermore, overexpression of Runx2 resulted in the significant enhancement of vascular endothelial growth factor (VEGF) promoter reporter activity and protein secretion. Runx2 significantly increased angiogenic activity in human umbilical vein endothelial cells in vitro. In wild-type mice, HIF-1α and Runx2 were colocalized in hypertrophic chondrocytes in which the cluster of differentiation 31 (CD31) protein was expressed at embryonic day 15.5 (E15.5). In contrast, the expression of HIF-1α was markedly reduced in areas of CD31 expression in Runx2(-/-) mice. These results suggest that Runx2 stabilizes HIF-1α by binding to ODDD to block the interaction between von Hippel-Lindau protein and HIF-1α. In conclusion, Runx2, HIF-1α, and VEGF may regulate vascular angiogenesis spatially and temporally in the hypertrophic zone of the growth plate during endochondral bone formation.
Assuntos
Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Fisiológica , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Condrócitos/citologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Células HEK293 , Humanos , Hidroxilação/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Runx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/- mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation-dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteogênese/fisiologia , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Displasia Cleidocraniana/genética , Displasia Cleidocraniana/metabolismo , Displasia Cleidocraniana/fisiopatologia , Células HEK293 , Humanos , Camundongos , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteogênese/genética , Fenótipo , Fosforilação/genética , Proteólise , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Runt-related transcription factor 3 (Runx3) is essential for normal mouse development, and Runx3 knock-out (KO) mice (FVB strain), which die within 24 h after birth, show various organ defects, such as lung hyperplasia. For proper early liver development, angiogenesis and liver cell differentiation mechanisms are necessary in mammals. Previous studies have reported that various signaling molecules, such as vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and cluster of differentiation 31 (CD31), are closely related to angiogenesis in the developing liver. Proper expression levels of molecules that induce liver cell differentiation, such as phosphorylated Smad2 (pSmad2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), Wilms tumor-1 (WT-1) and CD90 (Thy-1), are necessary for fetal liver development. To confirm the pathogenesis of liver defects caused by the loss of function of Runx3, the localization of proliferating cells was examined in wild-type and Runx3 KO mouse livers at postnatal day 1 (PN1). Specimens were also stained for various liver differentiation markers to confirm the function of Runx3. Moreover, gene expression level was examined by real-time quantitative polymerase chain reaction (RT-qPCR). Our results indicate that VEGF, vWF, CD31, pSmad2, NF-kB, WT-1 and Thy-1 were markedly up-regulated by the loss of Runx3. Therefore, our results indicate that liver development is controlled by Runx3. Clarifying the mechanisms of angiogenesis and liver differentiation might aid in the design of efficient and safe antiangiogenic therapy and gene therapy for liver disorders.
Assuntos
Diferenciação Celular , Subunidade alfa 3 de Fator de Ligação ao Core/deficiência , Fígado/metabolismo , Fígado/patologia , Neovascularização Patológica/metabolismo , Animais , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
A cell cycle is a series of events that takes place in a cell as it grows and divides. At the G1 phase of cell cycle, cells monitor their cumulative exposure to specific signals and make the critical decision to pass through the restriction (R)-point. The R-point decision-making machinery is fundamental to normal differentiation, apoptosis, and G1-S transition. Deregulation of this machinery is markedly associated with tumorigenesis. Therefore, identification of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in tumor biology. RUNX3 is one of the genes frequently inactivated in tumors by epigenetic alterations. In particular, RUNX3 is downregulated in most K-RAS-activated human and mouse lung adenocarcinomas (ADCs). Targeted inactivation of Runx3 in the mouse lung induces adenomas (ADs), and markedly shortens the latency of ADC formation induced by oncogenic K-Ras. RUNX3 participates in the transient formation of R-point-associated activator (RPA-RX3-AC) complexes, which measure the duration of RAS signals and thereby protect cells against oncogenic RAS. This review focuses on the molecular mechanism by which the R-point participates in oncogenic surveillance.
Assuntos
Adenocarcinoma de Pulmão , Adenocarcinoma , Neoplasias Pulmonares , Animais , Humanos , Camundongos , Transformação Celular Neoplásica , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Neoplasias Pulmonares/genéticaRESUMO
Oncogenic K-RAS mutations occur in approximately 25% of human lung cancers and are most frequently found in codon 12 (G12C, G12V, and G12D). Mutated K-RAS inhibitors have shown beneficial results in many patients; however, the inhibitors specifically target K-RASG12C and acquired resistance is a common occurrence. Therefore, new treatments targeting all kinds of oncogenic K-RAS mutations with a durable response are needed. RUNX3 acts as a pioneer factor of the restriction (R)-point, which is critical for the life and death of cells. RUNX3 is inactivated in most K-RAS-activated mouse and human lung cancers. Deletion of mouse lung Runx3 induces adenomas (ADs) and facilitates the development of K-Ras-activated adenocarcinomas (ADCs). In this study, conditional restoration of Runx3 in an established K-Ras-activated mouse lung cancer model regressed both ADs and ADCs and suppressed cancer recurrence, markedly increasing mouse survival. Runx3 restoration suppressed K-Ras-activated lung cancer mainly through Arf-p53 pathway-mediated apoptosis and partly through p53-independent inhibition of proliferation. This study provides in vivo evidence supporting RUNX3 as a therapeutic tool for the treatment of K-RAS-activated lung cancers with a durable response.
Assuntos
Adenocarcinoma , Neoplasias Pulmonares , Animais , Humanos , Camundongos , Adenocarcinoma/patologia , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Genes ras , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Recidiva Local de Neoplasia/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The Hippo kinase cascade functions as a central hub that relays input from the "outside world" of the cell and translates it into specific cellular responses by regulating the activity of Yes-associated protein 1 (YAP1). How Hippo translates input from the extracellular signals into specific intracellular responses remains unclear. Here, we show that transforming growth factor ß (TGFß)-activated TAK1 activates LATS1/2, which then phosphorylates YAP1. Phosphorylated YAP1 (p-YAP1) associates with RUNX3, but not with TEAD4, to form a TGFß-stimulated restriction (R)-point-associated complex which activates target chromatin loci in the nucleus. Soon after, p-YAP1 is exported to the cytoplasm. Attenuation of TGFß signaling results in re-localization of unphosphorylated YAP1 to the nucleus, where it forms a YAP1/TEAD4/SMAD3/AP1/p300 complex. The TGFß-stimulated spatiotemporal dynamics of YAP1 are abrogated in many cancer cells. These results identify a new pathway that integrates TGFß signals and the Hippo pathway (TGFßâTAK1âLATS1/2âYAP1 cascade) with a novel dynamic nuclear role for p-YAP1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fator de Crescimento Transformador beta , Proteínas de Sinalização YAP , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/fisiologiaRESUMO
Hepatocellular carcinoma (HCC) can cause severe complications, resulting in a high incidence of recurrence after treatment of the primary tumor. Recently, we have shown that nicotinamide effectively inhibits the growth and progression of bladder tumors by up-regulating RUNX3 and p300 expression. Therefore, in this study, the efficacy and inhibitory mechanisms of nicotinamide against HCC were investigated in mice and HCC cell lines, respectively. To evaluate the inhibitory effects of nicotinamide on HCC development, mice were injected with diethylnitrosamine (DEN) and simultaneously treated with 1% nicotinamide. Also, the effect of nicotinamide on human HCC cell lines was assessed by measuring caspase activity, cell proliferation, and DNA content using immunoblot analysis and reverse-transcriptase polymerase chain reaction. It was found that nicotinamide significantly inhibited the development of pre-neoplastic lesions (foci and adenomas) during the early stages of HCC. Furthermore, nicotinamide inhibited cell proliferation and induced mitochondria-mediated apoptosis in HCC cell lines. It also increased the expression of p21, and the expression and acetylation of p53. These results strongly suggest that nicotinamide inhibits the progression of early-stage HCC and may contribute to the induction of apoptosis and the inhibition of proliferation of HCC cells. Taken together, the results of this study indicate that nicotinamide is a potential chemopreventive agent, i.e., it may prevent the progression of early HCC development and/or the recurrence of HCC after primary treatment.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/patologia , Niacinamida/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/prevenção & controle , Complexo Vitamínico B/farmacologiaRESUMO
As H. pylori infection progresses, intestinal metaplasia (IM), a key event in gastric carcinogenesis, develops in the stomach. The mechanism by which H. pylori infection causes the trans-differentiation of gastric cells to intestinal-type cells remains an important question. In the current study, we found that RUNX3 is deregulated in all human IM specimens examined by either down regulation or mislocalization; Aberrant localization of a gastric tumor suppressor RUNX3 is observed in most human cases of IM with concurrent H. pylori infection, and RUNX3 is down-regulated in most cases of IM without H. pylori-infection. The cytoplasmic mislocalization of a RUNX3 was associated with H. pylori-induced c-Src activation and RUNX tyrosine phosphorylation. Moreover, gastric epithelial cells of Runx3(-/-) mice expressed the intestinal markers Muc2 and Li-Cadherin, which suggests that the deregulation of Runx3 is a key event in the intestinalization of the gastric epithelium. Collectively, the results of the current study suggest that RUNX3 deregulation is associated with H. pylori-induced pathogenesis and the development of IM.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Citoplasma/metabolismo , Gastrite/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori , Animais , Linhagem Celular Tumoral , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Citoplasma/microbiologia , Citoplasma/patologia , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gastrite/genética , Gastrite/patologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/prevenção & controleRESUMO
Recent genetic screens of fly mutants and molecular analysis have revealed that the Hippo (Hpo) pathway controls both cell proliferation and cell death. Deregulation of its human counterpart (the MST pathway) has been implicated in human cancers. However, how this pathway is linked with the known tumor suppressor network remains to be established. RUNX3 functions as a tumor suppressor of gastric cancer, lung cancer, bladder cancer, and colon cancer. Here, we show that RUNX3 is a principal and evolutionarily conserved component of the MST pathway. SAV1/WW45 facilitates the close association between MST2 and RUNX3. MST2, in turn, stimulates the SAV1-RUNX3 interaction. In addition, we show that siRNA-mediated RUNX3 knockdown abolishes MST/Hpo-mediated cell death. By establishing that RUNX3 is an endpoint effector of the MST pathway and that RUNX3 is capable of inducing cell death in cooperation with MST and SAV1, we define an evolutionarily conserved novel regulatory mechanism loop for tumor suppression in human cancers.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Evolução Biológica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Drosophila , Proteínas de Drosophila/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/genética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Runt domain transcription factor 3 (RUNX3) is a tumor suppressor that is silenced in cancer via hypermethylation of its promoter. This study investigated the mechanisms involved in reactive oxygen species (ROS)-induced silencing of RUNX3 in terms of epigenetic alteration since the effects of oxidative stress in tumor suppressor gene transcription are largely unknown. RUNX3 mRNA and protein expressions were down-regulated in response to hydrogen peroxide (H(2)O(2)) in the human colorectal cancer cell line SNU-407. This down-regulation was abolished with pretreatment of the ROS scavenger, N-acetylcysteine (NAC). Moreover, methylation-specific PCR data revealed that H(2)O(2) treatment increased RUNX3 promoter methylation; however, NAC and the cytosine methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-dC), decreased it, suggesting that an epigenetic regulatory mechanism by ROS-induced methylation may be involved in RUNX3 silencing. H(2)O(2) treatment resulted in DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) up-regulation with increased expression and activity, increased binding of DNMT1 to HADC1, and increased DNMT1 binding to the RUNX3 promoter. In addition, 5-Aza-dC treatment prevented the decrease in RUNX3 mRNA and protein levels by H(2)O(2) treatment. Additionally, H(2)O(2) treatment inhibited the nuclear localization and expression of RUNX3, which was abolished by NAC treatment. Furthermore, the down-regulation of RUNX3 expression by H(2)O(2) also influenced cell proliferation. Taken together, the data suggested that ROS silenced the tumor suppressor, RUNX3, by epigenetic regulation and may therefore be associated with the progression of colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Metilação de DNA , Epigênese Genética , Estresse Oxidativo , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Citosina/química , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Inativação Gênica , Genes Supressores de Tumor , Histona Desacetilase 1/genética , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
BACKGROUND AND OBJECTIVES: We have previously reported that RUNX3 inactivation by promoter hypermethylation correlated with advanced disease and poor clinical outcome in bladder cancer. In this study, we examined primary tumors from non-muscle-invasive bladder cancer (NMIBC) patients in order to investigate the relationship between RUNX3 methylation and disease progression. METHODS: The association between the hypermethylation of RUNX3 and clinicopathological characteristics of 186 NMIBC samples was analyzed using methylation-specific polymerase chain reaction (MS-PCR). RESULTS: RUNX3 methylation was associated with increased tumor stage, grade, and number of tumors (each P < 0.05). Kaplan-Meier estimates revealed significant differences in time to recurrence and progression based on RUNX3 methylation status (P = 0.043 and 0.006, respectively). RUNX3 methylation was an independent predictor of NMIBC progression on univariate and multivariate analysis. Combining tumor grade and RUNX3 methylation status demonstrated that patients with G3 tumors with RUNX3 methylation had a worse progression-free survival compared with the patients with lower-grade or unmethylated tumors [hazard ratio (HR), 19.450]. CONCLUSIONS: RUNX3 methylation status predicted the risk of NMIBC progression independently of tumor stage. In conjunction with tumor grade, RUNX3 methylation status in patients with NMIBC strongly predicts disease progression.
Assuntos
Carcinoma in Situ/genética , Carcinoma de Células de Transição/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Metilação de DNA , Recidiva Local de Neoplasia/genética , Neoplasias da Bexiga Urinária/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma in Situ/mortalidade , Carcinoma in Situ/patologia , Carcinoma de Células de Transição/mortalidade , Carcinoma de Células de Transição/patologia , Criança , DNA de Neoplasias/genética , Progressão da Doença , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Prognóstico , Regiões Promotoras Genéticas/genética , Taxa de Sobrevida , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia , Adulto JovemRESUMO
The runt-domain transcription factor Runx3 plays crucial roles during development such as regulating gene expression. It has been shown that Runx3 is involved in neurogenesis, thymopoiesis and functions like a tumor suppressor. Runx3 null mouse die soon after birth as a result of multiple organ defects. Runx3 null mouse lung shows an abnormal phenotype and loss of Runx3 induced remodeling in the lung. Interestingly, lung adenocarcinoma is observed in Runx3 heterozygous mice at 18 months of age. During lung development various cellular and molecular events occur such as cell proliferation, cell death, differentiation and epithelial-mesenchymal transition (EMT). To understand the specific lethal events in Runx3 null mice, we examined cellular and molecular networks involved in EMT, and EMT inducers were quantified by RT-qPCR during lung development. Excessive EMT was observed in lungs at PN1 day in Runx3 null mice and PN18 months in Runx3 heterozygous mice. Pharmacologic inhibition of EMT was used to curb tumor progression. In this study, U0126 was injected to pregnant mouse for inhibition of pERK signaling. After U0126 treatment, life spans of newborn mice were increased and lung hyperplasia was partially rescued by down-regulated cell proliferation and EMT. Our data suggest that Runx3 is involved in crucial regulation of alveolar differentiation and tumor suppression in developing mouse lung.
Assuntos
Transformação Celular Neoplásica/genética , Subunidade alfa 3 de Fator de Ligação ao Core/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/crescimento & desenvolvimento , Alvéolos Pulmonares/crescimento & desenvolvimento , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Animais , Butadienos/farmacologia , Diferenciação Celular/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Pulmão/anormalidades , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Camundongos Knockout , Nitrilas/farmacologia , Gravidez , Alvéolos Pulmonares/metabolismo , Transdução de Sinais , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genéticaRESUMO
BACKGROUND & AIMS: RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear. METHODS: To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3R122C knock-in mice. Stomach tissues were harvested, followed by histologic and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and nonisthmus epithelial cells, and RNA extraction for transcriptomic analysis. RESULTS: The corpus tissue of RUNX3R122C/R122C homozygous mice showed a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia. We observed mucous neck cell hyperplasia; massive reduction of pit, parietal, and chief cell populations; as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell-cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either a proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate. CONCLUSIONS: RUNX3R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest, and development of a precancerous state. This work highlights the importance of RUNX3 in the prevention of metaplasia and gastric cancer.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/genética , Lesões Pré-Cancerosas , Neoplasias Gástricas , Animais , Carcinogênese/patologia , Mucosa Gástrica , Metaplasia/genética , Metaplasia/patologia , Camundongos , Mutação Puntual , Lesões Pré-Cancerosas/patologia , Células-Tronco/metabolismo , Neoplasias Gástricas/patologiaRESUMO
RUNX3 is a transcription factor that functions as a tumor suppressor. In some cancers, RUNX3 expression is down-regulated, usually due to promoter hypermethylation. Recently, it was found that RUNX3 can also be inactivated by the mislocalization of the protein in the cytoplasm. The molecular mechanisms controlling this mislocalization are poorly understood. In this study, we found that the overexpression of Src results in the tyrosine phosphorylation and cytoplasmic localization of RUNX3. We also found that the tyrosine residues of endogenous RUNX3 are phosphorylated and that the protein is localized in the cytoplasm in Src-activated cancer cell lines. We further showed that the knockdown of Src by small interfering RNA, or the inhibition of Src kinase activity by a chemical inhibitor, causes the re-localization of RUNX3 to the nucleus. Collectively, our results demonstrate that the tyrosine phosphorylation of RUNX3 by activated Src is associated with the cytoplasmic localization of RUNX3 in gastric and breast cancers.