RESUMO
von Hippel-Lindau (VHL) is a critical tumor suppressor in clear cell renal cell carcinomas (ccRCCs). It is important to identify additional therapeutic targets in ccRCC downstream of VHL loss besides hypoxia-inducible factor 2α (HIF2α). By performing a genome-wide screen, we identified Scm-like with four malignant brain tumor domains 1 (SFMBT1) as a candidate pVHL target. SFMBT1 was considered to be a transcriptional repressor but its role in cancer remains unclear. ccRCC patients with VHL loss-of-function mutations displayed elevated SFMBT1 protein levels. SFMBT1 hydroxylation on Proline residue 651 by EglN1 mediated its ubiquitination and degradation governed by pVHL. Depletion of SFMBT1 abolished ccRCC cell proliferation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq, RNA-seq, and patient prognosis identified sphingosine kinase 1 (SPHK1) as a key SFMBT1 target gene contributing to its oncogenic phenotype. Therefore, the pVHL-SFMBT1-SPHK1 axis serves as a potential therapeutic avenue for ccRCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Prognóstico , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Proteínas Repressoras/genética , Células Tumorais Cultivadas , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The tumor suppressor von Hippel-Lindau, pVHL, is a multifaceted protein. One function is to dock to the hypoxia-inducible transcription factor (HIF) and recruit a larger protein complex that destabilizes HIF via ubiquitination, preventing angiogenesis and tumor development. pVHL also binds to the tumor suppressor p53 to activate specific p53 target genes. The oncogene Mdm2 impairs the formation of the p53-pVHL complex and activation of downstream genes by conjugating nedd8 to pVHL. While Mdm2 can impact p53 and pVHL, how pVHL may impact Mdm2 is unclear. Like p53 somatic mutations, point mutations are evident in pVHL that are common in renal clear cell carcinomas (RCC). In patients with RCC, Mdm2 levels are elevated, and we examined whether there was a relationship between Mdm2 and pVHL. TCGA and DepMap analysis revealed that mdm2 gene expression was elevated in RCC with vhl point mutations or copy number loss. In pVHL reconstituted or deleted isogenetically match RCC or MEF cell lines, Mdm2 was decreased in the presence of pVHL. Furthermore, through analysis using genetic and pharmacological approaches, we show that pVHL represses Mdm2 gene expression by blocking the MAPK-Ets signaling pathway and blocks Akt-mediated phosphorylation and stabilization of Mdm2. Mdm2 inhibition results in an increase in the p53-p21 pathway to impede cell growth. This finding shows how pVHL can indirectly impact the function of Mdm2 by regulating signaling pathways to restrict cell growth.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Proteínas Proto-Oncogênicas c-mdm2 , Transdução de Sinais , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor Von Hippel-Lindau , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Humanos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Hypoxia-inducible factor-1 (HIF-1) is a critical transcription factor that regulates the expression of genes involved in cellular adaptation to low oxygen levels. Aberrant regulation of the HIF-1 signaling pathway is linked to various human diseases. Previous studies have established that HIF-1α is rapidly degraded in a von Hippel-Lindau protein (pVHL)-dependent manner under normoxic conditions. In this study, we find that pVHL binding protein 1 (VBP1) is a negative regulator of HIF-1α but not HIF-2α using zebrafish as an in vivo model and in vitro cell culture models. Deletion of vbp1 in zebrafish caused Hif-1α accumulation and upregulation of Hif target genes. Moreover, vbp1 was involved in the induction of hematopoietic stem cells (HSCs) under hypoxic conditions. However, VBP1 interacted with and promoted the degradation of HIF-1α in a pVHL-independent manner. Mechanistically, we identify the ubiquitin ligase CHIP and HSP70 as new VBP1 binding partners and demonstrate that VBP1 negatively regulated CHIP and facilitated CHIP-mediated degradation of HIF-1α. In patients with clear cell renal cell carcinoma (ccRCC), lower VBP1 expression was associated with worse survival outcomes. In conclusion, our results link VBP1 with CHIP stability and provide insights into underlying molecular mechanisms of HIF-1α-driven pathological processes.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Animais , Humanos , Peixe-Zebra/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Fatores de Transcrição/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas do Citoesqueleto , Chaperonas MolecularesRESUMO
The von Hippel-Lindau tumor suppressor pVHL is an E3 ligase that targets hypoxia-inducible factors (HIFs). Mutation of VHL results in HIF up-regulation and contributes to processes related to tumor progression such as invasion, metastasis, and angiogenesis. However, very little is known with regard to post-transcriptional regulation of pVHL. Here we show that WD repeat and SOCS box-containing protein 1 (WSB1) is a negative regulator of pVHL through WSB1's E3 ligase activity. Mechanistically, WSB1 promotes pVHL ubiquitination and proteasomal degradation, thereby stabilizing HIF under both normoxic and hypoxic conditions. As a consequence, WSB1 up-regulates the expression of HIF-1α's target genes and promotes cancer invasion and metastasis through its effect on pVHL. Consistent with this, WSB1 protein level negatively correlates with pVHL level and metastasis-free survival in clinical samples. This work reveals a new mechanism of pVHL's regulation by which cancer acquires invasiveness and metastatic tendency.
Assuntos
Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica , Proteínas/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Células HEK293 , Células HT29 , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Invasividade Neoplásica/genética , Neoplasias/genética , Neoplasias/fisiopatologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas/genética , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
The oncoprotein transcription factor paired box 2 (PAX2) is aberrantly expressed in cancers, but the underlying mechanism remains elusive. Prolyl hydroxylase 3 (PHD3) hydroxylates the proline residue of HIFα, mediating HIFα degradation. The von Hippel-Lindau protein (pVHL) is an E3 ligase which mediates ubiquitination and degradation of hydroxylated HIFα. PHD3 and pVHL are found to inhibit the expression of PAX2, however, the molecular mechanism is unclear. Here we demonstrate that PHD3 hydroxylates PAX2 at proline 9, which is required for pVHL to mediate PAX2 ubiquitination and degradation. Overexpression of PHD3 enhances prolyl hydroxylation, ubiquitination and degradation of PAX2 with little effect on those of PAX2(P9A). PHD3 does not influence PAX2 expression in VHL-null cells. pVHL binds to PAX2 and enhances PAX2 ubiquitination and degradation. However, pVHL does not bind with PAX2(P9A) and cannot enhance its ubiquitination and degradation. Our results suggest that proline 9 hydroxylation is a prerequisite for PAX2 degradation by pVHL. Functional studies indicate that introduction of PAX2 into PAX2-null COS-7 cells promotes cell proliferation, which is suppressed by co-expression of PHD3 but not by hydroxylase-deficient PHD3(H196A). PHD3 inhibits PAX2-induced, but not PAX2(P9A)-induced proliferation of COS-7 cells. These results suggest that PHD3 hydroxylates PAX2, followed by pVHL-mediated PAX2 ubiquitination and degradation. This study also suggests that PHD3 inhibits cell proliferation through downregulating PAX2.
Assuntos
Prolina , Prolil Hidroxilases , Proliferação de Células , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Psoriasis is a chronic inflammatory disease distinguished by an excessive proliferation and abnormal differentiation of keratinocytes. Immune cells, such as T lymphocytes and neutrophils, and inflammatory cytokines, such as Tumor Necrosis Factor-α (TNF-α) and interleukin 17 (IL-17), are essential for maintaining psoriatic lesions. Additionally, a hypoxic milieu present in the skin promotes the expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α). This protein regulates the expression of angiogenic and glycolytic factors, such as vascular endothelial grown factor and lactate dehydrogenase (LDH), both relevant in chronic inflammation. The von Hippel-Lindau protein (pVHL) is a negative regulator of HIF-1α. Previously, we found that pVHL was almost absent in the lesions of psoriasis patients; therefore, we investigated the impact of rescue pVHL expression in lesional skin. We used the imiquimod-induced psoriasis-like mouse model as an adenoviral vector that allowed us to express pVHL in the skin. Our data show that, in lesional skin, pVHL expression was reduced, whereas HIF-1α was increased. Remarkably, the retrieval of pVHL prevented psoriatic lesions, diminishing erythema, scale, and epidermal and vascular thickness. Furthermore, pVHL expression was capable of reducing HIF-1α, LDH, TNF-α and immune cell infiltration (mainly IL-17+ neutrophils). In conclusion, our results demonstrate that pVHL has a protective role to play in the pathophysiology of psoriasis.
Assuntos
Dermatite , Psoríase , Animais , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imiquimode/efeitos adversos , Inflamação , Interleucina-17/genética , Camundongos , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Fator de Necrose Tumoral alfa/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Hypoxia-inducible factors are heterodimeric transcription factors that play a crucial role in a cell's ability to adapt to low oxygen. The von Hippel-Lindau tumor suppressor (pVHL) acts as a master regulator of HIF activity, and its targeting of prolyl hydroxylated HIF-α for proteasomal degradation under normoxia is thought to be a major mechanism for pVHL tumor suppression and cellular response to oxygen. Whether pVHL regulates other targets through a similar mechanism is largely unknown. Here, we identify TET2/3 as novel targets of pVHL. pVHL induces proteasomal degradation of TET2/3, resulting in reduced global 5-hydroxymethylcytosine levels. Conserved proline residues within the LAP/LAP-like motifs of these two proteins are hydroxylated by the prolyl hydroxylase enzymes (PHD2/EGLN1 and PHD3/EGLN3), which is prerequisite for pVHL-mediated degradation. Using zebrafish as a model, we determined that global 5-hydroxymethylcytosine levels are enhanced in vhl-null, egln1a/b-double-null, and egln3-null embryos. Therefore, we reveal a novel function for the PHD-pVHL pathway in regulating TET protein stability and activity. These data extend our understanding of how TET proteins are regulated and provide new insight into the mechanisms of pVHL in tumor suppression.
Assuntos
Metilação de DNA , DNA/metabolismo , Dioxigenases/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Motivos de Aminoácidos , Animais , DNA/genética , Dioxigenases/genética , Células HEK293 , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Proteínas Supressoras de Tumor/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The Wnt/ß-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the ß-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/ß-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/ß-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/ß-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/ß-catenin signaling pathway activity.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Chaperonas Moleculares/metabolismo , Fatores de Transcrição TCF/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , Proliferação de Células , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Embrião não Mamífero/metabolismo , Humanos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição TCF/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Ativação Transcricional , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
SHANK-associated RH domain interacting protein (SHARPIN) plays an important role in carcinogenesis, as well as inflammation and immunity. Our study explored the effects and underlying mechanisms of SHARPIN in clear cell renal cell carcinoma (ccRCC). By analyzing The Cancer Genome Atlas database, we found that upregulated SHARPIN in patients with ccRCC led to a poor prognosis. Semiquantitative immunohistochemical analysis of clinical samples was carried out and the results suggested the positive association between SHARPIN and hypoxia-induced factor-2α (HIF-2α). Von Hippel-Lindau protein (pVHL) is a tumor suppressor that contributes to degrading HIF-2α. Mechanically, SHARPIN promoted the ubiquitination and proteasomal degradation of pVHL, resulting in the sustained activation of HIF-2α. The α and ß domains of pVHL and ubiquitin-like domain of SHARPIN are required for the interaction. The knockdown of SHARPIN effectively inhibited acquired sorafenib resistance in ccRCC cell lines and tumor growth in xenograft models. In conclusion, our work reveals a novel posttranslational regulation of SHARPIN on pVHL, indicating that SHARPIN could be a potential target for ccRCC treatment.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/etiologia , Neoplasias Renais/etiologia , Ubiquitinas/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Inativação Gênica , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteólise , RNA Interferente Pequeno , Distribuição Aleatória , Sorafenibe/farmacologia , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo , Regulação para CimaRESUMO
BACKGROUND: von Hippel-Lindau (VHL) disease is a familial neoplasia syndrome that results from the germline mutation of VHL. Pathogenic VHL mutations include deletion, frameshift, nonsense and missense mutations. Synonymous mutations are expected to be phenotypically silent and their role in VHL disease remains poorly understood. CASE PRESENTATION: We report a Caucasian male with a family history of pheochromocytoma and the synonymous VHL mutation c.414A > G (p.Pro138Pro). At 47-years, MRI revealed pheochromocytoma in the left adrenal gland and hemangioblastomas in the spine and brain. Pheochromocytoma was treated by adrenalectomy. Radiotherapy, followed by craniotomy and resection were needed to reduce hemangioblastomas to residual lesions. Two of three of the proband's children inherited the mutation and both presented with retinal hemangioblastomas without pheochromocytoma at age 7: one twin needed four laser treatments. Primary skin fibroblasts carrying the heterozygous mutation or wild type VHL were established from the family. Mutant fibroblasts downregulated full-length VHL mRNA and protein, and upregulated the short VHL mRNA isoform (a result of exon 2 skipping in splicing) at the mRNA level but not at the protein level. CONCLUSIONS: Our study shows that the synonymous VHL mutation c.414A > G can within 7 years induce pediatric retinal hemangioblastoma in absence of pheochromocytoma. This highlights the need to include splicing-altering synonymous mutations into the screening for VHL disease. This is also the first report on detecting and validating a synonymous VHL mutation using patient-derived fibroblasts. The mutation c.414A > G translates to p.Pro138Pro, yet it is not functionally silent, because it causes aberrant splicing by skipping exon 2. The reduced but not completely abolished pVHL protein in a loss-of-heterozygosity genetic backdrop may underlie the etiology of VHL disease.
Assuntos
Neoplasias Cerebelares/genética , Hemangioblastoma/genética , Splicing de RNA/genética , Mutação Silenciosa , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/diagnóstico , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias Cerebelares/complicações , Neoplasias Cerebelares/diagnóstico , Criança , Pré-Escolar , Família , Feminino , Mutação da Fase de Leitura/genética , Mutação em Linhagem Germinativa , Hemangioblastoma/complicações , Hemangioblastoma/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/diagnóstico , Neoplasias Primárias Múltiplas/genética , Linhagem , Feocromocitoma/complicações , Feocromocitoma/diagnóstico , Feocromocitoma/genética , Prolina/genética , Neoplasias da Retina/complicações , Neoplasias da Retina/diagnóstico , Neoplasias da Retina/genética , Neoplasias da Coluna Vertebral/complicações , Neoplasias da Coluna Vertebral/diagnóstico , Neoplasias da Coluna Vertebral/genética , Doença de von Hippel-Lindau/complicações , Doença de von Hippel-Lindau/genéticaRESUMO
Hepatitis B virus (HBV) X protein (HBx) is associated with hepatocarcinogenesis. E2-EPF ubiquitin carrier protein (UCP) catalyzes ubiquitination of itself and von Hippel-Lindau protein (pVHL) for degradation and associates with tumor growth and metastasis. However, it remains unknown whether HBx modulates the enzyme activity of UCP and thereby influences hepatocarcinogenesis. Here, we show that UCP is highly expressed in liver tissues of HBx-transgenic mice, but not non-transgenic mice. UCP was more frequently expressed in HBV-positive liver cancers than in HBV-negative liver cancers. HBx binds to UCP specifically and serotype independently, and forms a ternary complex with UCP and pVHL. HBx inhibits self-ubiquitination of UCP, but enhances UCP-mediated pVHL ubiquitination, resulting in stabilization of hypoxia-inducible factor-1α and -2α. HBx and UCP stabilize each other by mutually inhibiting their ubiquitination. HBx promotes cellular proliferation and metastasis via UCP. Our findings suggest that UCP plays a key role in HBV-related hepatocarcinogenesis.
Assuntos
Carcinoma Hepatocelular/secundário , Hepatite B/complicações , Neoplasias Hepáticas/patologia , Transativadores/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Animais , Apoptose , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Proliferação de Células , Progressão da Doença , Feminino , Hepatite B/virologia , Vírus da Hepatite B/isolamento & purificação , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Estabilidade Proteica , Transdução de Sinais , Transativadores/genética , Células Tumorais Cultivadas , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Posttranslational protein modifications play an important role in regulating protein stability and cellular function. There are at least eight Cullin family members. Among them, Cullin-2 forms a functional E3 ligase complex with elongin B, elongin C, RING-box protein 1 (RBX1, also called ROC1), as well as the substrate recognition subunit (SRS) to promote the substrate ubiquitination and degradation. In this book chapter, we will review Cullin-2 E3 ligase complexes that include various SRS proteins, including von Hippel Lindau (pVHL), leucine-rich repeat protein-1 (LRR-1), preferentially expressed antigen of melanoma (PRAME), sex-determining protein FEM-1 and early embryogenesis protein ZYG-11. We will focus on the VHL signaling pathway in clear cell renal cell carcinoma (ccRCC), which may reveal various therapeutic avenues in treating this lethal cancer.
Assuntos
Carcinoma de Células Renais/enzimologia , Proteínas Culina/metabolismo , Neoplasias Renais/enzimologia , Animais , Carcinoma de Células Renais/tratamento farmacológico , Humanos , Neoplasias Renais/tratamento farmacológico , Ligação Proteica , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
The mechanisms controlling degradation of cytosolic ß-catenin are important for regulating ß-catenin co-transcriptional activity. Loss of von Hippel-Lindau protein (pVHL) has been shown to stabilize ß-catenin, increasing ß-catenin transactivation and ß-catenin-mediated cell proliferation. However, the role of phosphoinositide 3-kinase (PI3K)/Akt in the regulation of ß-catenin signaling downstream from pVHL has never been addressed. Here, we report that hyperactivation of PI3K/Akt in cells lacking pVHL contributes to the stabilization and nuclear accumulation of active ß-catenin. PI3K/Akt hyperactivation is facilitated by the up-regulation of 14-3-3ζ and the down-regulation of 14-3-3ε, 14-3-3η and 14-3-3θ. Up-regulation of 14-3-3ζ in response to pVHL is important for the recruitment of PI3K to the cell membrane and for stabilization of soluble ß-catenin. In contrast, 14-3-3ε and 14-3-3η enhanced PI3K/Akt signaling by inhibiting PI3K and PDK1, respectively. Thus, our results demonstrated that 14-3-3 family members enhance PI3K/Akt/ß-catenin signaling in order to increase proliferation. Inhibition of Akt activation and/or 14-3-3 function strongly reduces ß-catenin signaling and decreases cell proliferation. Thus, inhibition of Akt and 14-3-3 function efficiently reduces cell proliferation in 786-0 cells characterized by hyperactivation of ß-catenin signaling due to pVHL loss.
Assuntos
Proteínas 14-3-3/biossíntese , Proliferação de Células/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , beta Catenina/metabolismo , Proteínas 14-3-3/genética , Animais , Cães , Humanos , Células Madin Darby de Rim Canino , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , beta Catenina/genéticaRESUMO
FOXO3a, a member of the forkhead homeobox type O (FOXO) family of transcriptional factors, regulates cell survival in response to DNA damage, caloric restriction, and oxidative stress. The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of the E3 ubiquitin ligase complex that mediates hypoxia-inducible factor α degradation under aerobic conditions, thus acting as one of the key regulators of hypoxia signaling. However, whether FOXO3a impacts cellular hypoxia stress remains unknown. Here we show that FOXO3a directly binds to the VHL promoter and up-regulates VHL expression. Using a zebrafish model, we confirmed the up-regulation of vhl by foxo3b, an ortholog of mammalian FOXO3a Furthermore, by employing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9) technology, we deleted foxo3b in zebrafish and determined that expression of hypoxia-inducible genes was affected under hypoxia. Moreover, foxo3b-null zebrafish exhibited impaired acute hypoxic tolerance, resulting in death. In conclusion, our findings suggest that, by modulating hypoxia-inducible factor activity via up-regulation of VHL, FOXO3a (foxo3b) plays an important role in survival in response to hypoxic stress.
Assuntos
Proteína Forkhead Box O3/metabolismo , Elementos de Resposta , Transdução de Sinais , Proteínas Supressoras de Tumor/biossíntese , Regulação para Cima , Proteína Supressora de Tumor Von Hippel-Lindau/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/metabolismo , Animais , Hipóxia Celular , Proteína Forkhead Box O3/genética , Células HEK293 , Humanos , Proteínas Supressoras de Tumor/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Patients with tongue cancer frequently show loss of heterozygosity (LOH) of the von Hippel-Lindau (VHL) tumor suppressor gene. However, expression of VHL protein (pVHL) in tongue cancer has rarely been investigated and remains largely unknown. We performed immunohistochemical staining of pVHL in tongue tissues and dysplasia, and examined the association with LOH and its clinical significance. METHODS: Immunohistochemical staining of pVHL in formalin-fixed, paraffin-embedded sections of cancerous and other tissues from 19 tongue cancer patients showed positivity for LOH of VHL in four samples, negativity in four samples, and was non-informative in 11 samples. The staining pattern of pVHL was also compared with those of cytokeratin (CK) 13 and CK17. RESULTS: In normal tongue tissues, pVHL staining was localized to the cytoplasm of cells in the basal layer and the area of the spinous layer adjacent to the basal layer of stratified squamous epithelium. Positive staining for pVHL was observed in the cytoplasm of cancer cells from all 19 tongue cancer patients. No differences as a result of the presence or absence of LOH were found. Notably, cytoplasm of poorly differentiated invasive cancer cells was less intensely stained than that of well and moderately differentiated invasive cancer cells. pVHL staining was also evident in epithelial dysplasia lesions with pVHL-positive cells expanding from the basal layer to the middle of the spinous layer. However, no CK13 staining was noted in regions of the epithelium, which were positive for pVHL. In contrast, regions with positive staining for CK17 closely coincided with those positive for pVHL. CONCLUSIONS: Positive staining for pVHL was observed in cancerous areas but not in normal tissues. pVHL expression was also detected in lesions of epithelial dysplasia. These findings suggest that pVHL may be a useful marker for proliferative lesions.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias da Língua/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/biossíntese , Adulto , Idoso , Carcinoma de Células Escamosas/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma de Células Escamosas de Cabeça e Pescoço , Língua/metabolismo , Língua/patologia , Neoplasias da Língua/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/análise , Adulto JovemRESUMO
Autophagy is a regulated process that sequesters and transports cytoplasmic materials such as protein aggregates via autophagosomes to lysosomes for degradation. Dapper1 (Dpr1), an interacting protein of Dishevelled (Dvl), antagonizes Wnt signaling by promoting Dishevelled degradation via lysosomes. However, the mechanism is unclear. Here, we show that Dpr1 promotes the von Hippel-Lindau tumor suppressor (VHL)-mediated ubiquitination of Dvl2 and its autophagic degradation. Knockdown of Dpr1 decreases the interaction between Dvl2 and pVHL, resulting in reduced ubiquitination of Dvl2. Dpr1-mediated autophagic degradation of Dvl2 depends on Dvl2 aggregation. Moreover, the aggregate-prone proteins Dvl2, p62, and the huntingtin mutant Htt103Q promote autophagy in a Dpr1-dependent manner. These protein aggregates enhance the Beclin1-Vps34 interaction and Atg14L puncta formation, indicating that aggregated proteins stimulate autophagy initiation. Ubiquitination is not essential for the aggregate-induced autophagy initiation as inhibition of the ubiquitin-activation E1 enzyme activity did not block the aggregate-induced Atg14L puncta formation. Our findings suggest that Dpr1 promotes the ubiquitination of Dvl2 by pVHL and mediates the protein aggregate-elicited autophagy initiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Animais , Autofagia , Linhagem Celular , Proteínas Desgrenhadas , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Microscopia de Fluorescência , Mutação , Plasmídeos/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Ratos , Ubiquitina/metabolismo , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Via de Sinalização WntRESUMO
NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.
Assuntos
Regulação Enzimológica da Expressão Gênica , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Cílios/metabolismo , Perfilação da Expressão Gênica , Humanos , Rim/enzimologia , Neoplasias Renais/metabolismo , Masculino , Quinases Relacionadas a NIMA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão , TransfecçãoRESUMO
In our study, we demonstrate that ccRCC cell lines with impaired function of pVHL to degrade HIFα express elevated levels of PD-L1. In vitro analysis provided evidence that both reconstitution of pVHL and silencing of HIF2α, but not of HIF1α, lead to reduced PD-L1 expression. The strong correlation of expression between the HIF2α-specific HIF target Glut1 and PD-L1 confirmed this finding in ccRCC cell lines and tissue. Soluble PD-L1 levels remained constant in the sera of ccRCC patients regardless of the PD-L1 expression status in their tumors. In conclusion, our data suggest PD-L1 as HIF2α target, which is upregulated in pVHL deficient ccRCC. The combination of PD-L1 targeting drugs with HIF inhibiting agents may be an additional option for the treatment of ccRCC.
Assuntos
Antígeno B7-H1/metabolismo , Carcinoma de Células Renais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/metabolismo , Antígeno B7-H1/sangue , Antígeno B7-H1/genética , Carcinoma de Células Renais/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imuno-Histoquímica , Neoplasias Renais/genética , Mutação , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
BACKGROUND: The VHL protein (pVHL) is a multiadaptor protein that interacts with more than 30 different binding partners involved in many oncogenic processes. About 70 % of clear cell renal cell carcinoma (ccRCC) have VHL mutations with varying impact on pVHL function. Loss of pVHL function leads to the accumulation of Hypoxia Inducible Factor (HIF), which is targeted by current targeted treatments. In contrast to nonsense and frameshift mutations that highly likely nullify pVHL multipurpose functions, missense mutations may rather specifically influence the binding capability of pVHL to its partners. The affected pathways may offer predictive clues to therapy and response to treatment. In this study we focused on the VHL missense mutation pattern in ccRCC, and studied their potential effects on pVHL protein stability and binding partners and discussed treatment options. METHODS: We sequenced VHL in 360 sporadic ccRCC FFPE samples and compared observed and expected frequency of missense mutations in 32 different binding domains. The prediction of the impact of those mutations on protein stability and function was assessed in silico. The response to HIF-related, anti-angiogenic treatment of 30 patients with known VHL mutation status was also investigated. RESULTS: We identified 254 VHL mutations (68.3 % of the cases) including 89 missense mutations (35 %). Codons Ser65, Asn78, Ser80, Trp117 and Leu184 represented hotspots and missense mutations in Trp117 and Leu 184 were predicted to highly destabilize pVHL. About 40 % of VHL missense mutations were predicted to cause severe protein malfunction. The pVHL binding domains for HIF1AN, BCL2L11, HIF1/2α, RPB1, PRKCZ, aPKC-λ/ι, EEF1A1, CCT-ζ-2, and Cullin2 were preferentially affected. These binding partners are mainly acting in transcriptional regulation, apoptosis and ubiquitin ligation. There was no correlation between VHL mutation status and response to treatment. CONCLUSIONS: VHL missense mutations may exert mild, moderate or strong impact on pVHL stability. Besides the HIF binding domain, other pVHL binding sites seem to be non-randomly altered by missense mutations. In contrast to LOF mutations that affect all the different pathways normally controlled by pVHL, missense mutations may be rather appropriate for designing tailor-made treatment strategies for ccRCC.
Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Mutação de Sentido Incorreto , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Inibidores da Angiogênese/uso terapêutico , Sítios de Ligação , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Ligação Proteica , Estabilidade Proteica , Análise de Sequência de DNA , Resultado do Tratamento , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.