RESUMO
BACKGROUND: Tumor mutation burden (TMB) and VHL mutation play a crucial role in the management of patients with clear cell renal cell carcinoma (ccRCC), such as guiding adjuvant chemotherapy and improving clinical outcomes. However, the time-consuming and expensive high-throughput sequencing methods severely limit their clinical applicability. Predicting intratumoral heterogeneity poses significant challenges in biology and clinical settings. Our aimed to develop a self-supervised attention-based multiple instance learning (SSL-ABMIL) model to predict TMB and VHL mutation status from hematoxylin and eosin-stained histopathological images. METHODS: We obtained whole slide images (WSIs) and somatic mutation data of 350 ccRCC patients from The Cancer Genome Atlas for developing SSL-ABMIL model. In parallel, 163 ccRCC patients from Clinical Proteomic Tumor Analysis Consortium cohort was used as independent external validation set. We systematically compared three different models (Wang-ABMIL, Ciga-ABMIL, and ImageNet-MIL) for their ability to predict TMB and VHL alterations. RESULTS: We first identified two groups of populations with high- and low-TMB (cut-off point = 0.9). In two independent cohorts, the Wang-ABMIL model achieved the highest performance with decent generalization performance (AUROC = 0.83 ± 0.02 and 0.8 ± 0.04 in predicting TMB and VHL, respectively). Attention heatmaps revealed that the Wang-ABMIL model paid the highest attention to tumor regions in high-TMB patients, while in VHL mutation prediction, non-tumor regions were also assigned high attention, particularly the stromal regions infiltrated by lymphocytes. CONCLUSIONS: Our results indicated that SSL-ABMIL can effectively extract histological features for predicting TMB and VHL mutation, demonstrating promising results in linking tumor morphology and molecular biology.
Assuntos
Carcinoma de Células Renais , Aprendizado Profundo , Neoplasias Renais , Mutação , Proteína Supressora de Tumor Von Hippel-Lindau , Humanos , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Biomarcadores Tumorais/genética , IdosoRESUMO
BACKGROUND: Patients with von Hippel-Lindau (VHL) disease are at risk of developing tumors in the eye, brain, kidney, adrenal gland, and other organs based on their gene mutations. The VHL tumor suppressor gene contains pathogenic variants responsible for these events. This meta-analysis aims to investigate the genetic differences among the various types of VHL syndrome and their correlation with the location of mutations (exons and domains) in the VHL gene. METHOD: Papers eligible for publication until September 2023 were identified using the electronic databases of PubMed, Google Scholar, Scopus, and EMBASE. The Random Effect model was utilized to evaluate the genetic differences between type 1 and type 2 VHL syndromes. RESULTS: The prevalence of missense mutations (MSs) was found to be 58.9% in type 1, while it was 88.1% in type 2. Interestingly, the probability of observing MSs in type 1 was 0.42 times lower compared to type 2. The mutation hotspots of the VHL gene were R167Q/W, Y98H, R238W, and S65L, respectively. Although type 2 had a high presentation of Y98H and R238W, it did not have a higher S65L than type 1. The analysis demonstrated a statistically significant higher prevalence of truncated mutations (PTMs) in type 1. Among type 1, large/complete deletions (L/C DELs) were found in 16.9% of cases, whereas in type 2 only 3.7%. This difference was statistically significant with a p-value < 0.001. Overall, the probability of identifying mutations in domain 2 compared to domain 1 was found to be 2.13 times higher in type 1 (p-value < 0.001). Furthermore, the probability of detecting exon 1 in comparison with observing exon 2 in type 1 was 2.11 times higher than type 2 and revealed a statistically significant result (p-value < 0.001). The detection of exon 2 was 2.18 times higher in type 1 (p-value < 0.001). In addition, the likelihood of discovering exon 2 compared with others was significantly lower in type 1 compared with type 2 VHL (OR = 0.63, p-value = 0.015). CONCLUSIONS: We have revealed a comprehensive genetic difference between types 1 and 2 of VHL syndrome. The significant differences in MS, PTMs, L/C DELs, and the location of the mutations between type 1 and type 2 VHL patients in the Asian, European, and American populations emphasize the genetic heterogeneity of the syndrome. These findings may pave the way for the diagnosis, treatment, and further investigation of the mechanisms behind this complex genetic disorder.
Assuntos
Proteína Supressora de Tumor Von Hippel-Lindau , Doença de von Hippel-Lindau , Humanos , Doença de von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Mutação , Mutação de Sentido Incorreto , Predisposição Genética para DoençaRESUMO
The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( Siniperca chuatsi) VHL ( scVHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of scVHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα, scIKKß, scIκBα, or scp65. Co-immunoprecipitation (Co-IP) analysis identified scIκBα as a novel target protein of scVHL. Moreover, scVHL targeted scIκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.
Assuntos
Doenças dos Peixes , NF-kappa B , Ranavirus , Transdução de Sinais , Replicação Viral , Animais , NF-kappa B/metabolismo , NF-kappa B/genética , Replicação Viral/fisiologia , Doenças dos Peixes/virologia , Ranavirus/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Proteínas de Peixes/metabolismo , Proteínas de Peixes/genética , Proteínas I-kappa B/metabolismo , Proteínas I-kappa B/genética , Regulação da Expressão GênicaRESUMO
Renal cell carcinoma (RCC), the most common form of kidney cancer, is a heterogeneous disease with clear cell RCC (ccRCC) being the most prevalent and aggressive subtype. While most ccRCC tumors have elevated expression of angiopoietin-like4 (ANGPTL4), in our study we identified a significant subset of patients whose cancers show no increase in ANGPTL4 expression. These patients have a worse prognosis compared to the patients with high expression of ANGPTL4. These ANGPTL4-low cancers are characterized by the increased frequency of wild-type Von Hippel-Lindau(WT VHL), a gene that is commonly mutated in ccRCC, and an enrichment for genes associated with lipid metabolism. Using RCC tumor models with WT VHL, we demonstrate that ANGPTL4 behaves as a tumor suppressor. The loss of ANGPTL4 in ccRCC cell lines results in increased tumor growth and colony formation in a lysosomal acid lipase (LAL)-dependent manner, a phenotype rescued by the expression of N-terminus ANGPTL4. At the mechanistic level, the loss of ANGPTL4 increases LAL activity in ccRCC cells. These data suggest that ANGPTL4 enacts its tumor-suppressive effects in ccRCC by regulating LAL activity. Importantly, the identified patient cohort with low ANGPTL4 expression may exhibit increased reliance on lipid metabolism, which can be a point of target for future therapy. SIGNIFICANCE: Our data indicate angiopoietin-like 4 (ANGPTL4) acts as a tumor suppressor in clear cell renal cell carcinoma via regulating lipid metabolism and identifies a cohort of patients with lower expression of ANGPTL4 that are correlated with shorter survival.
Assuntos
Proteína 4 Semelhante a Angiopoietina , Carcinoma de Células Renais , Neoplasias Renais , Esterol Esterase , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Humanos , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Linhagem Celular Tumoral , Animais , Camundongos , Esterol Esterase/genética , Esterol Esterase/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Prognóstico , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , FemininoRESUMO
The histone acylation reader eleven-nineteen leukemia (ENL) plays a pivotal role in sustaining oncogenesis in acute leukemias, particularly in mixed-lineage leukemia-rearranged (MLL-r) leukemia. ENL relies on its reader domain to recognize histone lysine acylation promoting oncogenic gene expression and leukemia progression. Here, we report the development of MS41, a highly potent and selective von Hippel-Lindau-recruiting ENL degrader that effectively inhibits the growth of ENL-dependent leukemia cells. MS41-induced ENL degradation reduces the chromatin occupancy of ENL-associated transcription elongation machinery, resulting in the suppression of key oncogenic gene expression programs and the activation of differentiation genes. MS41 is well-tolerated in vivo and substantially suppresses leukemia progression in a xenograft mouse model of MLL-r leukemia. Notably, MS41 also induces the degradation of mutant ENL proteins identified in Wilms' tumors. Our findings emphasize the therapeutic potential of pharmacological ENL degradation for treating ENL-dependent cancers, making MS41 not only a valuable chemical probe but also potential anticancer therapeutic for further development.
Assuntos
Progressão da Doença , Leucemia , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Leucemia/genética , Leucemia/patologia , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
This article systematically reviewed the pathological features, molecular mechanisms, and tumor microenvironment of head and neck paragangliomaï¼HNPGLï¼, with a focus on pseudohypoxic HNPGL. It was demonstrated that pseudohypoxic HNPGL mainly involves multiple gene mutations, such as SDHx and VHL/EPAS1, which affect the stability and activity of HIF protein and exacerbate the development of the tumor. Meanwhile, the paper also analyzed the expression patterns of HIF-1α and HIF-2α in HNPGL, and found that differences in HIF activation may have an impact on the therapeutic response of specific subtypes. In addition, the paper explored the tumor microenvironment of HNPGL and found that immune cells such as macrophages, CD4âºT cells, and CD8âºT cells play an important role in the tumor, and the heterogeneity of the immune microenvironment also affects the choice of therapeutic approaches and responsiveness. Through comprehensive analysis, these findings not only contribute to a deeper understanding of the pathogenesis and developmental process of HNPGL, but also provide clues for future personalized treatments for specific subtypes.
Assuntos
Neoplasias de Cabeça e Pescoço , Subunidade alfa do Fator 1 Induzível por Hipóxia , Paraganglioma , Microambiente Tumoral , Humanos , Microambiente Tumoral/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Paraganglioma/metabolismo , Paraganglioma/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mutação , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
L-type amino acid transporter 1 (LAT1) is upregulated in various cancer types and contributes to disease progression. Previous studies have demonstrated or suggested that hypoxia-inducible factors (HIFs), the key transcription factors in hypoxic responses, control the expression of LAT1 gene in several types of cancer cells. However, this regulatory relationship has not been investigated yet in colorectal cancer (CRC), one of the cancer types in which the increased LAT1 expression holds prognostic significance. In this study, we found that neither LAT1 mRNA nor protein is induced under hypoxic condition (1% O2) in CRC HT-29 cells in vitro, regardless of the prominent HIF-1/2α accumulation and HIFs-dependent upregulation of glucose transporter 1. The hypoxic treatment generally did not increase either the mRNA or protein expression of LAT1 in eight CRC cell lines tested, in contrast to the pronounced upregulation by amino acid restriction. Interestingly, knockdown of von Hippel-Lindau ubiquitin ligase to inhibit the proteasomal degradation of HIFs caused an accumulation of HIF-2α and increased the LAT1 expression in certain CRC cell lines. This study contributes to delineating the molecular mechanisms responsible for the pathological expression of LAT1 in CRC cells, emphasizing the ambiguity of HIFs-dependent transcriptional upregulation of LAT1 across cancer cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia , Transportador 1 de Aminoácidos Neutros Grandes , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células HT29 , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Linhagem Celular Tumoral , Hipóxia Celular , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
To maximize the impact of precision medicine approaches, it is critical to identify genetic variants underlying disease and to accurately quantify their functional effects. A gene exemplifying the challenge of variant interpretation is the von Hippel-Lindautumor suppressor (VHL). VHL encodes an E3 ubiquitin ligase that regulates the cellular response to hypoxia. Germline pathogenic variants in VHL predispose patients to tumors including clear cell renal cell carcinoma (ccRCC) and pheochromocytoma, and somatic VHL mutations are frequently observed in sporadic renal cancer. Here we optimize and apply saturation genome editing to assay nearly all possible single-nucleotide variants (SNVs) across VHL's coding sequence. To delineate mechanisms, we quantify mRNA dosage effects and compare functional effects in isogenic cell lines. Function scores for 2,268 VHL SNVs identify a core set of pathogenic alleles driving ccRCC with perfect accuracy, inform differential risk across tumor types and reveal new mechanisms by which variants impact function. These results have immediate utility for classifying VHL variants encountered clinically and illustrate how precise functional measurements can resolve pleiotropic and dosage-dependent genotype-phenotype relationships across complete genes.
Assuntos
Alelos , Carcinoma de Células Renais , Edição de Genes , Neoplasias Renais , Polimorfismo de Nucleotídeo Único , Proteína Supressora de Tumor Von Hippel-Lindau , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Humanos , Edição de Genes/métodos , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Linhagem Celular Tumoral , Predisposição Genética para Doença , MutaçãoRESUMO
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
Age-related macular degeneration (AMD) is a common cause of vision loss. The aggressive form of AMD is associated with ocular neovascularization and subretinal fibrosis, representing a responsive outcome against neovascularization mediated by epithelial-mesenchymal transition of retinal pigment epithelium (RPE) cells. A failure of the current treatment (anti-vascular endothelial growth factor therapy) has also been attributed to the progression of subretinal fibrosis. Hypoxia-inducible factors (HIFs) increase gene expressions to promote fibrosis and neovascularization. HIFs act as a central pathway in the pathogenesis of AMD. HIF inhibitors may suppress ocular neovascularization. Nonetheless, further investigation is required to unravel the aspects of subretinal fibrosis. In this study, we used RPE-specific HIFs or von Hippel-Lindau (VHL, a regulator of HIFs) conditional knockout (cKO) mice, along with pharmacological HIF inhibitors, to demonstrate the suppression of subretinal fibrosis. Fibrosis was suppressed by treatments of HIF inhibitors, and similar suppressive effects were detected in RPE-specific Hif1a/Hif2a- and Hif1a-cKO mice. Promotive effects were observed in RPE-specific Vhl-cKO mice, where fibrosis-mediated pathologic processes were evident. Marine products' extracts and their component taurine suppressed fibrosis as HIF inhibitors. Our study shows critical roles of HIFs in the progression of fibrosis, linking them to the potential development of therapeutics for AMD.
Assuntos
Fibrose , Camundongos Knockout , Epitélio Pigmentado da Retina , Proteína Supressora de Tumor Von Hippel-Lindau , Animais , Camundongos , Fibrose/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/tratamento farmacológico , Retina/metabolismo , Retina/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer, but a comprehensive description of its genomic landscape is lacking. We report the whole genome sequencing of 778 ccRCC patients enrolled in the 100,000 Genomes Project, providing for a detailed description of the somatic mutational landscape of ccRCC. We identify candidate driver genes, which as well as emphasising the major role of epigenetic regulation in ccRCC highlight additional biological pathways extending opportunities for therapeutic interventions. Genomic characterisation identified patients with divergent clinical outcome; higher number of structural copy number alterations associated with poorer prognosis, whereas VHL mutations were independently associated with a better prognosis. The observations that higher T-cell infiltration is associated with better overall survival and that genetically predicted immune evasion is not common supports the rationale for immunotherapy. These findings should inform personalised surveillance and treatment strategies for ccRCC patients.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Mutação , Proteína Supressora de Tumor Von Hippel-Lindau , Sequenciamento Completo do Genoma , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/terapia , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Humanos , Neoplasias Renais/genética , Neoplasias Renais/terapia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Prognóstico , Masculino , Feminino , Variações do Número de Cópias de DNA , Pessoa de Meia-Idade , Epigênese Genética , Idoso , Regulação Neoplásica da Expressão Gênica , Imunoterapia/métodosRESUMO
The hypoxia-inducible factor-1α (HIF-1α) pathway coordinates skeletal bone homeostasis and endocrine functions. Activation of the HIF-1α pathway increases glucose uptake by osteoblasts, which reduces blood glucose levels. However, it is unclear whether activating the HIF-1α pathway in osteoblasts can help normalize glucose metabolism under diabetic conditions through its endocrine function. In addition to increasing bone mass and reducing blood glucose levels, activating the HIF-1α pathway by specifically knocking out Von HippelâLindau (Vhl) in osteoblasts partially alleviated the symptoms of streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM), including increased glucose clearance in the diabetic state, protection of pancreatic ß cell from STZ-induced apoptosis, promotion of pancreatic ß cell proliferation, and stimulation of insulin secretion. Further screening of bone-derived factors revealed that islet regeneration-derived protein III gamma (RegIIIγ) is an osteoblast-derived hypoxia-sensing factor critical for protection against STZ-induced T1DM. In addition, we found that iminodiacetic acid deferoxamine (SF-DFO), a compound that mimics hypoxia and targets bone tissue, can alleviate symptoms of STZ-induced T1DM by activating the HIF-1α-RegIIIγ pathway in the skeleton. These data suggest that the osteoblastic HIF-1α-RegIIIγ pathway is a potential target for treating T1DM.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Subunidade alfa do Fator 1 Induzível por Hipóxia , Osteoblastos , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/etiologia , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Masculino , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Estreptozocina , Apoptose/efeitos dos fármacos , Glicemia/metabolismoRESUMO
Proteolysis targeting chimeras (PROTACs) are heterobifunctional small-molecule degraders made of a linker connecting a target-binding moiety to a ubiquitin E3 ligase-binding moiety. The linker unit is known to influence the physicochemical and pharmacokinetic properties of PROTACs, as well as the properties of ternary complexes, in turn impacting on their degradation activity in cells and in vivo. Our LRRK2 PROTAC XL01126, bearing a trans-cyclohexyl group in the linker, is a better and more cooperative degrader than its corresponding cis- analogue despite its much weaker binary binding affinities. Here, we investigate how this subtle stereocenter alteration in the linker affects the ligand binding affinity to the E3 ligase VHL. We designed a series of molecular matched pairs, truncating from the full PROTACs down to the VHL ligand, and find that across the series the trans-cyclohexyl compounds showed consistently weaker VHL-binding affinity compared to the cis- counterparts. High-resolution co-crystal structures revealed that the trans linker exhibits a rigid stick-out conformation, while the cis linker collapses into a folded-back conformation featuring a network of intramolecular contacts and long-range interactions with VHL. These observations are noteworthy as they reveal how a single stereochemical inversion within a PROTAC linker impacts conformational rigidity and binding mode, in turn fine-tuning differentiated propensity to binary and ternary complex formation, and ultimately cellular degradation activity.
Assuntos
Proteína Supressora de Tumor Von Hippel-Lindau , Humanos , Sítios de Ligação , Ligantes , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteólise/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/química , Cicloexanos/químicaRESUMO
While large-scale artificial intelligence (AI) models for protein structure prediction and design are advancing rapidly, the translation of deep learning models for practical macromolecular drug development remains limited. This investigation aims to bridge this gap by combining cutting-edge methodologies to create a novel peptide-based PROTAC drug development paradigm. Using ProteinMPNN and RFdiffusion, we identified binding peptides for androgen receptor (AR) and Von Hippel-Lindau (VHL), followed by computational modeling with Alphafold2-multimer and ZDOCK to predict spatial interrelationships. Experimental validation confirmed the designed peptide's binding ability to AR and VHL. Transdermal microneedle patching technology was seamlessly integrated for the peptide PROTAC drug delivery in androgenic alopecia treatment. In summary, our approach provides a generic method for generating peptide PROTACs and offers a practical application for designing potential therapeutic drugs for androgenetic alopecia. This showcases the potential of interdisciplinary approaches in advancing drug development and personalized medicine.
Assuntos
Alopecia , Desenho de Fármacos , Peptídeos , Receptores Androgênicos , Alopecia/tratamento farmacológico , Receptores Androgênicos/metabolismo , Receptores Androgênicos/química , Humanos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Animais , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/química , MasculinoRESUMO
Renal cell carcinoma (RCC) is a significant oncological challenge due to its heterogeneous nature and limited treatment options. The PAX developmental gene family encodes nine highly conserved transcription factors that play crucial roles in embryonic development and organogenesis, which have been implicated in the occurrence and development of RCC. This review explores the molecular landscape of RCC, with a specific focus on the role of the PAX gene family in RCC tumorigenesis and disease progression. Of the various RCC subtypes, clear cell renal cell carcinoma (ccRCC) is the most prevalent, characterized by the loss of the von Hippel-Lindau (VHL) tumor suppressor gene. Here, we review the published literature on the expression patterns and functional implications of PAX genes, particularly PAX2 and PAX8, in the three most common RCC subtypes, including ccRCC, papillary RCC (PRCC), and chromophobe RCC (ChRCC). Further, we review the interactions and potential biological mechanisms involving PAX genes and VHL loss in driving the pathogenesis of RCC, including the key signaling pathways mediated by VHL in ccRCC and associated mechanisms implicating PAX. Lastly, concurrent with our update regarding PAX gene research in RCC, we review and comment on the targeting of PAX towards the development of novel RCC therapies.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Fatores de Transcrição Box Pareados , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Fator de Transcrição PAX8/genética , Fator de Transcrição PAX8/metabolismo , Transdução de Sinais/genéticaRESUMO
Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer driven by VHL loss and aberrant HIF-2α signaling. Identifying means to regulate HIF-2α thus has potential therapeutic benefit. Acetyl-CoA synthetase 2 (ACSS2) converts acetate to acetyl-CoA and is associated with poor patient prognosis in ccRCC. Here we tested the effects of ACSS2 on HIF-2α and cancer cell metabolism and growth in ccRCC models and clinical samples. ACSS2 inhibition reduced HIF-2α levels and suppressed ccRCC cell line growth in vitro, in vivo, and in cultures of primary ccRCC patient tumors. This treatment reduced glycolytic signaling, cholesterol metabolism, and mitochondrial integrity, all of which are consistent with loss of HIF-2α. Mechanistically, ACSS2 inhibition decreased chromatin accessibility and HIF-2α expression and stability. While HIF-2α protein levels are widely regulated through pVHL-dependent proteolytic degradation, we identify a potential pVHL-independent pathway of degradation via the E3 ligase MUL1. We show that MUL1 can directly interact with HIF-2α and that overexpression of MUL1 decreased HIF-2α levels in a manner partially dependent on ACSS2. These findings identify multiple mechanisms to regulate HIF-2α stability and ACSS2 inhibition as a strategy to complement HIF-2α-targeted therapies and deplete pathogenically stabilized HIF-2α.
Assuntos
Acetato-CoA Ligase , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Carcinoma de Células Renais , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais , Transdução de Sinais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/genética , Linhagem Celular Tumoral , Acetato-CoA Ligase/metabolismo , Acetato-CoA Ligase/genética , Animais , Camundongos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genéticaRESUMO
Protein homeostasis is predominantly governed through post-translational modification (PTM). UBE3B, identified as an oncoprotein, exhibits elevated protein levels in breast cancer. However, the impact of PTM on UBE3B remains unexplored. In this study, we show that VHL is a bona fide E3 ligase for UBE3B. Mechanistically, VHL directly binds to UBE3B, facilitating its lysine 48 (K48)-linked polyubiquitination at K286 and K427 in a prolyl hydroxylase (PHD)-independent manner. Consequently, this promotes the proteasomal degradation of UBE3B. The K286/427R mutation of UBE3B dramatically abolishes the inhibitory effect of VHL on breast tumor growth and lung metastasis. Additionally, the protein levels of UBE3B and VHL exhibit a negative correlation in breast cancer tissues. These findings delineate an important layer of UBE3B regulation by VHL.
Assuntos
Neoplasias da Mama , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células HEK293 , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos Nus , Metástase Neoplásica , Ligação Proteica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Estrogen is thought to have a role in slowing down aging and protecting cardiovascular and cognitive function. However, high doses of estrogen are still positively associated with autoimmune diseases and tumors with systemic inflammation. First, we administered exogenous estrogen to female mice for three consecutive months and found that the aorta of mice on estrogen develops inflammatory manifestations similar to Takayasu arteritis (TAK). Then, in vitro estrogen intervention was performed on mouse aortic vascular smooth muscle cells (MOVAS cells). Stimulated by high concentrations of estradiol, MOVAS cells showed decreased expression of contractile phenotypic markers and increased expression of macrophage-like phenotypic markers. This shift was blocked by tamoxifen and Krüppel-like factor 4 (KLF4) inhibitors and enhanced by Von Hippel-Lindau (VHL)/hypoxia-inducible factor-1α (HIF-1α) interaction inhibitors. It suggests that estrogen-targeted regulation of the VHL/HIF-1α/KLF4 axis induces phenotypic transformation of vascular smooth muscle cells (VSMC). In addition, estrogen-regulated phenotypic conversion of VSMC to macrophages is a key mechanism of estrogen-induced vascular inflammation, which justifies the risk of clinical use of estrogen replacement therapy.
Assuntos
Estrogênios , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Macrófagos , Músculo Liso Vascular , Proteína Supressora de Tumor Von Hippel-Lindau , Animais , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/efeitos dos fármacos , Feminino , Estrogênios/farmacologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Fenótipo , Aorta/patologia , Aorta/efeitos dos fármacos , Inflamação/metabolismoRESUMO
The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)-a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein - a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.
Assuntos
Autofagia , Neoplasias Cerebelares , Proteínas Hedgehog , Subunidade alfa do Fator 1 Induzível por Hipóxia , Meduloblastoma , Proteína Supressora de Tumor Von Hippel-Lindau , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Meduloblastoma/genética , Humanos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Autofagia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Linhagem Celular Tumoral , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/genética , Camundongos , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Ubiquitinação , Proteínas RepressorasRESUMO
INTRODUCTION: Von Hippel-Lindau disease (e.g. VHL) is an autosomal dominant multi-organ cancer syndrome caused by a mutation in the VHL tumour suppressor gene. In this study, we introduce a novel genetic variant found in 11 family members diagnosed initially with isolated Pheochromocytoma. Subsequent findings revealed its association with VHL syndrome and corresponds to the Type 2 C phenotype. METHODS: The VHL gene was amplified through the utilisation of the polymerase chain reaction (PCR). PCR fragments were sequenced using bidirectional Sanger sequencing, using BigDye™ Terminator v3.1 Cycle Sequencing Kit, running on the 3500 genetic analyser. Results were assembled and analysed Using Software SeqA and chromas pro. RESULTS: A heterozygous in-frame duplication of three nucleotides, specifically ATG, c.377_379dup; p.Asp126dup in exon 2, was identified in all the patients tested within the pedigree. CONCLUSION: In this study, we disclose the identification of a novel genetic variant in a Jordanian family, affecting eleven family members with pheochromocytoma associated with VHL disease. This finding underscores the importance of screening family members and contemplating genetic testing for individuals newly diagnosed with pheochromocytoma and could enhance our comprehension of the potential adverse consequences associated with VHL germline mutations.
Goal: To study a novel gene change in a family with Von Hippel-Lindau (e.g. VHL) syndrome, which increases cancer chances.Participants: 11 family members with Pheochromocytoma, a tumour linked to VHL.Methods:Used PCR to copy the VHL gene.Analysed the gene using Sanger sequencing.Findings:Found a novel gene change in all family members. This change, called an in-frame duplication, affects a protein.It's in a specific part of the gene.Conclusion:Stressing the importance of genetic testing for Pheochromocytoma patients to grasp VHL mutation risks.