Chaperonin Containing Tailless complex polypeptide 1 (CCT) is an essential molecular chaperone required for the folding of the abundant proteins actin and tubulin. The CCT oligomer also folds a range of other proteins and participates in non-folding activities such as providing assembly support for complexes of the von Hippel Lindau tumor suppressor protein and elongins. Here we show that the oncogenic transcription factor STAT3 binds to the CCT oligomer, but does not display the early binding upon translation in rabbit reticulocyte lysate typical of an obligate CCT folding substrate. Consistent with this, depletion of each of the CCT subunits by siRNA targeting indicates that loss of CCT oligomer does not suppress the activation steps of STAT3 upon stimulation with IL-6: phosphorylation, dimerisation and nuclear translocation. Furthermore, the transcriptional activity of STAT3 is not negatively affected by reduction in CCT levels. Instead, loss of CCT oligomer in MCF7 cells leads to an enhancement of STAT3 phosphorylation at Tyr705, implicating a role for the CCT oligomer in the sequestration of non-phosphorylated STAT3. Thus, as CCT is dynamic oligomer, the assembly state and also abundance of CCT oligomer may provide a means to modulate STAT3 phosphorylation.

The von Hippel-Lindau (VHL) disease is an autosomal dominant cancer syndrome caused by mutations in the VHL tumor suppressor gene. VHL protein (pVHL) forms a complex (VBC) with Elongins B-C, Cullin2, and Rbx1. Although other functions have been discovered, the most described function of pVHL is to recognize and target hypoxia-inducible factor (HIF) for degradation. This work comprises the functional characterization of two novel variants of the VHL gene (P138R and L163R) that have been described in our center in patients with VHL disease by in vitro, in vivo, and in silico approaches. In vitro, we found that these variants have a significantly shorter half-life compared to wild-type VHL but still form a functional VBC complex. Altered fibronectin deposition was evidenced for both variants using immunofluorescence. In vivo studies revealed that both variants failed to suppress tumor growth. By means of molecular dynamics simulations, we inspected in silico the nature of the changes introduced by each variant in the VBC complex. We have demonstrated the pathogenicity of P138R and L163R novel variants, involving HIF-dependent and HIF-independent mechanisms. These results provide the basis for future studies regarding the impact of structural alterations on posttranslational modifications that drive pVHL's fate and functions.

Clear cell renal cell carcinoma (ccRCC) incidence has been rising in recent years, with strong association between differential microRNA (miRNA) expression and neoplastic progression. Specifically, overexpression of miR-155-5p has been associated with promoting aggressive cancer in ccRCC and other cancers. In this study, we further investigate the role of this miRNA and one of its protein targets, Jade-1, to better understand the mechanism behind aggressive forms of ccRCC. Jade-1, a tumor suppressor, is stabilized by Von-Hippel Lindau (VHL), which is frequently mutated in ccRCC. Experiments featuring downregulation of miR-155-5p in two ccRCC cell lines (786-O and Caki-1) attenuated their oncogenic potential and led to increased levels of Jade-1. Conversely, knockdown experiments with an anti-Jade-1 shRNA in 786-O and Caki-1 cells showed increased metastatic potential through elevated proliferation, migration, and invasion rates. In a mouse xenograft model, downregulation of miR-155 decreased the rate of tumor implantation and proliferation. Direct interaction between miR-155-5p and Jade-1 was confirmed through a 3'UTR luciferase reporter assay. These findings further elucidate the mechanism of action of miR-155-5p in driving an aggressive phenotype in ccRCC through its role in regulating Jade-1.

The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of VHL loss is currently best understood to occur through stabilization of the hypoxia-inducible factors, activation of hypoxia-induced signaling pathways, and transcriptional reprogramming towards a pro-angiogenic and pro-growth state. However, hypoxia also drives other pro-tumorigenic processes, including the development of genomic instability via down-regulation of DNA repair gene expression. Here, we find that DNA repair genes involved in double-strand break repair by homologous recombination (HR) and in mismatch repair, which are down-regulated by hypoxic stress, are decreased in VHL-deficient renal cancer cells relative to wild type VHL-complemented cells. Functionally, this gene repression is associated with impaired DNA double-strand break repair in VHL-deficient cells, as determined by the persistence of ionizing radiation-induced DNA double-strand breaks and reduced repair activity in a homology-dependent plasmid reactivation assay. Furthermore, VHL deficiency conferred increased sensitivity to PARP inhibitors, analogous to the synthetic lethality observed between hypoxia and these agents. Finally, we discovered a correlation between VHL inactivation and reduced HR gene expression in a large panel of human renal carcinoma samples. Together, our data elucidate a novel connection between VHL-deficient renal carcinoma and hypoxia-induced down-regulation of DNA repair, and identify potential opportunities for targeting DNA repair defects in human renal cell carcinoma.

The majority of renal cell carcinomas (RCCs) are characterized by loss of function of the tumor suppressor gene von Hippel Lindau (VHL), which acts as ubiquitin ligase for hypoxia-inducible factor-1α (HIF-1α). In the absence of VHL, HIF-1α protein becomes stabilized and contributes to tumorigenesis. Recent data demonstrate the antitumor efficacy of VHL promoter in RCC cells. This study demonstrates that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of VHL. NDRG2 is involved in proliferation and invasion of cancer cell, furthermore it is frequently down-regulated in renal cell carcinoma. Herein we evaluated the effect of NDRG2 overexpression on proliferation and invasion in human renal cancer cells. The human renal cancer cell line 786-O and A498 were infected with Ad-NDRG2 or Ad-LacZ. Overexpression of NDRG2 not only inhibited the growth of the cells, but also suppressed the invasion. Further study showed that the tumor suppressor gene VHL were up-regulated, whereas transcription factor HIF-1a and vascular endothelial growth factor (VEGF) were down-regulated in 786-O cells infected by Ad-NDRG2. Finally, in a nude mouse model, intratumoral injections of Ad-NDRG2 every 3 days for a total of seven times significantly inhibited the growth and angiogenesis of xenografted 786-O tumors. In conclusion, these data indicate that NDRG2 may be involved in proliferation and invasion by impacting the expression of VHL and HIF-1α. NDRG2 may be an attractive therapeutic target for renal cell carcinoma.

The mortality rate of ovarian cancer is increasing and the role of hypoxia inducible factor-1α (HIF-1α) in tumor progression has been confirmed. von Hippel-Lindau tumor suppressor protein (pVHL) binds HIF-1α and mediates proteasome degradation of HIF-1α. Besides, histone deacetylase inhibitor (HDACi) mitigates tumor growth via targeting HIF-1α, whereas underlying mechanism still requires investigation. In this research, we exposed ovarian cancer cell lines OV-90 and SKOV-3 to escalating concentrations of HDACi LBH589. As a result, cell viability was significantly suppressed and expression of HIF-1α was remarkably reduced along with decreased levels of signal molecules, including phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) (P = 0.000). Interestingly, pVHL was expressed in a notably declining tendency (P = 0.000). Chaperone heat shock protein-70 (HSP70) was expressed in an ascending manner, whereas expression of chaperonin TCP-1α was reduced clearly (P = 0.000). Besides, co-inhibition of pVHL plus HDAC did not contribute to a remarkable difference in HIF-1α expression as compared with single HDAC inhibition. Furthermore, both cell lines were transfected with plasmids of VHL plus VHL binding protein-1 (VBP-1). Consequently, the expression of HIF-1α as well as lactate dehydrogenase-A (LDHA) was remarkably decreased (P = 0.000). These findings indicate HDACi may repress expression of HIF-1α via inhibiting PI3K and GSK3β and promote degradation of HIF-1α via HSP70, independent of pVHL. Additionally, a sophisticated network of HDAC and chaperones may involve in pVHL quality control.

The Wnt/β-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 β-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/β-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/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-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/β-catenin signaling pathway activity.

The metabolic phenotype of cancer cells is reflected by the metabolites they consume and by the byproducts they release. Here, we use quantitative, extracellular metabolomic data of the NCI-60 panel and a novel computational method to generate 120 condition-specific cancer cell line metabolic models. These condition-specific cancer models used distinct metabolic strategies to generate energy and cofactors. The analysis of the models' capability to deal with environmental perturbations revealed three oxotypes, differing in the range of allowable oxygen uptake rates. Interestingly, models based on metabolomic profiles of melanoma cells were distinguished from other models through their low oxygen uptake rates, which were associated with a glycolytic phenotype. A subset of the melanoma cell models required reductive carboxylation. The analysis of protein and RNA expression levels from the Human Protein Atlas showed that IDH2, which was an essential gene in the melanoma models, but not IDH1 protein, was detected in normal skin cell types and melanoma. Moreover, the von Hippel-Lindau tumor suppressor (VHL) protein, whose loss is associated with non-hypoxic HIF-stabilization, reductive carboxylation, and promotion of glycolysis, was uniformly absent in melanoma. Thus, the experimental data supported the predicted role of IDH2 and the absence of VHL protein supported the glycolytic and low oxygen phenotype predicted for melanoma. Taken together, our approach of integrating extracellular metabolomic data with metabolic modeling and the combination of different network interrogation methods allowed insights into the metabolism of cells.

Our previous study suggested that microtubule network alteration affects the process of glycolysis in cardiomyocytes (CMs) via the regulation of hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known regarding the underlying mechanisms of microtubule network alteration-induced changes of HIF-1α. The von Hippel-Lindau tumor suppressor protein (pVHL) has been shown to mediate the ubiquitination of HIF-1α in the nuclear compartment prior to HIF-1α exportation to the cytoplasm, and pVHL dynamic nuclear-cytoplasmic trafficking is indicated to be involved in the process of HIF-1α degradation. In this study, by administering different microtubule-stabilizing and -depolymerizing interventions, we demonstrated that microtubule stabilization promoted pVHL nuclear export and drove the translocation of pVHL to the cytoplasm, while microtubule disruption prevented pVHL nuclear export in hypoxic CMs. Moreover, the ratio between nuclear and cytoplasmic pVHL was associated with HIF-1α regulation. Importantly, microtubule network alteration also affected the subcellular localization of Ran, which was involved in the regulation of pVHL nuclear-cytoplasmic trafficking. The above results suggest that the subcellular translocation of pVHL plays an important role in microtubular structure alteration-induced HIF-1α regulation. Interestingly, Ran is involved in the process of pVHL nuclear-cytoplasmic trafficking following microtubule network alteration in hypoxic CMs.

Clear cell renal cell carcinoma (ccRCC) is the most common and lethal form of urological cancer diagnosed globally. Mutations of the von Hippel-Lindau (VHL) tumor-suppressor gene and the resultant overexpression of hypoxia-inducible factor (HIF)-1α protein are considered hallmarks of ccRCC. Persistently activated HIF-1α is associated with increased cell proliferation, angiogenesis, and epithelial⁻mesenchymal transition (EMT), consequently leading to ccRCC progression and metastasis to other organs. However, the VHL status alone cannot predict the differential sensitivity of ccRCC to cancer treatments, which suggests that other molecular differences may contribute to the differential response of ccRCC cells to drug therapies. In this study, we investigated the response to metformin (an antidiabetic drug) of two human ccRCC cell lines Caki-1 and Caki-2, which express wild-type VHL. Our findings demonstrate a differential response between the two ccRCC cell lines studied, with Caki-2 cells being more sensitive to metformin compared to Caki-1 cells, which could be linked to the differential expression of HIF-1 despite both cell lines carrying a wild-type VHL. Our study unveils the therapeutic potential of metformin to inhibit the progression of ccRCC in vitro. Additional preclinical and clinical studies are required to ascertain the therapeutic efficacy of metformin against ccRCC.

Hepatocellular carcinoma (HCC) is regarded as a leading cause of cancer-related deaths, and its progression is associated with hypoxia and the induction of hypoxia-inducible factor (HIF). Meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor, induces cell death in various malignancies. However, the underlying mechanism remains to be elucidated in HCC, especially under hypoxic conditions. The alteration of COX-2 and HIF-1α oncogenicity was evaluated in HCC specimens by tissue microarray. Cell viability, angiogenesis assays, and xenografted nude mice were used to evaluate the effects of meloxicam, along with flow cytometry to detect the cell cycle, apoptosis, and mitochondrial membrane potential (ΔΨm) of HCC. qRT-PCR, Western blotting, immunofluorescence, immunohistochemistry, luciferase assay, and RNAi were carried out to determine the HIF-1α signaling affected by meloxicam. In this study, we showed that meloxicam exerts antiproliferative and antiangiogenesis efficacy in vitro and in vivo and causes disruption of mitochondrial membrane potential (ΔΨm), thus leading to caspase-dependent apoptosis under hypoxic environments. Exposure to meloxicam significantly reduced HIF-1α transcriptional activation and expression through sequestering it in the cytoplasm and accelerating degradation via increasing the von Hippel-Lindau tumor suppressor protein (pVHL) in HCC. These data demonstrated that inhibition of HIF-1α by meloxicam could suppress angiogenesis and enhance apoptosis of HCC cells. This discovery highlights that COX-2 specific inhibitors may be a promising therapy in the treatment of HCC.

Intrauterine hypoxia is a reason for impaired kidney development. The cellular and molecular pathways along which hypoxia exerts effects on nephrogenesis are not well understood. They are likely triggered by hypoxia-inducible transcription factors (HIFs), and their effects appear to be dependent on the cell compartment contributing to kidney formation. In this study, we investigated the effects of HIF activation in the developing renal stroma, which also essentially modulates nephron development from the metanephric mesenchyme. HIF activation was achieved by conditional deletion of the von Hippel-Lindau tumor suppressor (VHL) protein in the forkhead box FOXD1 cell lineage, from which stromal progenitors arise. The resulting kidneys showed maturation defects associated with early postnatal death. In particular, nephron formation, tubular maturation, and the differentiation of smooth muscle, renin, and mesangial cells were impaired. Erythropoietin expression was strongly enhanced. Codeletion of VHL together with HIF2A but not with HIF1A led to apparently normal kidneys, and the animals reached normal age but were anemic because of low erythropoietin levels. Stromal deletion of HIF2A or HIF1A alone did not affect kidney development. These findings emphasize the relevance of sufficient intrauterine oxygenation for normal renal stroma differentiation, suggesting that chronic activity of HIF2 in stromal progenitors impairs kidney development. Finally, these data confirm the concept that normal stroma function is essential for normal tubular differentiation.

Clear cell Renal Cell Carcinoma (ccRCC) formation is connected to functional loss of the von Hippel-Lindau (VHL) gene. Recent data identified its gene product, pVHL, as a multifunctional adaptor protein which interacts with HIFα subunits but also with the tumor suppressor p53. p53 is hardly expressed and rarely mutated in most ccRCC. We showed that low and absent p53 expression correlated with the severity of VHL mutations in 262 analyzed ccRCC tissues. In contrast to nonsense and frameshift mutations which abrogate virtually all pVHL functions, missense mutations may rather influence one or few functions. Therefore, we focused on four VHL missense mutations, which affect the overlapping pVHL binding sites of p53 and Elongin C, by investigating their impact on HIFα degradation, p53 expression and signaling, as well as on cellular behavior using ccRCC cell lines and tissues. TP53 mRNA and its effector targets p21, Bax and Noxa, were altered both in engineered cell lines and in tumor tissues which carried the same missense mutations. Two of these mutations were not able to degrade HIFα whereas the remaining two mutations led to HIFα downregulation, suggesting the latter are p53 binding site-specific. The selected VHL missense mutations further enhanced tumor cell survival, but had no effects on cell proliferation. Whereas Sunitinib was able to efficiently reduce cell proliferation, Camptothecin was additionally able to increase apoptotic activity of the tumor cells. It is concluded that systematic characterization of the VHL mutation status may help optimizing targeted therapy for patients with metastatic ccRCC.

A common feature of tumorigenesis is the upregulation of angiogenesis pathways in order to supply nutrients via the blood for the growing tumor. Understanding how cells promote angiogenesis and how to control these processes pharmaceutically are of great clinical interest. Clear cell renal cell carcinoma (ccRCC) is the most common form of sporadic and inherited kidney cancer which is associated with excess neovascularization. ccRCC is highly associated with biallelic mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. Although upregulation of the miR-212/132 family and disturbed VHL signaling have both been linked with angiogenesis, no evidence of a possible connection between the two has yet been made. We show that miRNA-212/132 levels are increased after loss of functional pVHL, the protein product of the VHL gene, in vivo and in vitro. Furthermore, we show that blocking miRNA-212/132 with anti-miRs can significantly alleviate the excessive vascular branching phenotype characteristic of vhl-/- mutant zebrafish. Moreover, using human umbilical vascular endothelial cells (HUVECs) and an endothelial cell/pericyte coculture system, we observed that VHL knockdown promotes endothelial cells neovascularization capacity in vitro, an effect which can be inhibited by anti-miR-212/132 treatment. Taken together, our results demonstrate an important role for miRNA-212/132 in angiogenesis induced by loss of VHL. Intriguingly, this also presents a possibility for the pharmaceutical manipulation of angiogenesis by modulating levels of MiR212/132.

Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.

Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.

The von Hippel-Lindau tumor suppressor gene (VHL) is mutated in up to 90% of clear cell renal cell carcinoma (ccRCC) cases, thus playing a key role in ccRCC pathogenesis. ccRCC can be classified as a metabolic disease in which alterations in fatty acid metabolism facilitate cancer cell proliferation. Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH) is an enzyme involved in peroxisomal fatty acid degradation. It is primarily expressed in renal proximal tubule cells, presumably the origin of ccRCC. Although EHHADH is still a relatively unexplored gene, it is known to be differentially expressed in several tumors. In this study, analysis of several databases revealed that EHHADH expression is downregulated in ccRCC samples compared to healthy kidney samples. Moreover, cell culture experiments were performed to investigate the relationship between EHHADH and VHL at the gene and protein level. qPCR and Western blot analyses using the human ccRCC cell line RCC4 revealed that EHHADH is expressed in a VHL-dependent manner. RCC4 cells reconstituted with VHL show significantly higher EHHADH mRNA and protein levels than VHL-deficient RCC4 control cells. These results indicate that the downregulation of EHHADH in ccRCC reported may be due to the loss of VHL function. This study is the first to molecularly characterize EHHADH, a key enzyme in peroxisomal ß-oxidation, in relation to VHL, suggesting a potential pathogenic interaction that is worthy of further investigation.

Evolution of clear cell renal cell carcinoma is guided by dysregulation of hypoxia-inducible transcription factor (HIF) pathways following loss of the von Hippel-Lindau tumor suppressor protein. Renal cell carcinoma (RCC)-associated polymorphisms influence HIF-DNA interactions at enhancers of important oncogenes thereby modulating the risk of developing renal cancer. A strong signal of genome-wide association with RCC was determined for the single nucleotide polymorphism (SNP) rs4903064, located on chr14q.24.2 within an intron of DPF3, encoding for Double PHD Fingers 3, a member of chromatin remodeling complexes; however, it is unclear how the risk allele operates in renal cells. In this study, we used tissue specimens and primary renal cells from a large cohort of RCC patients to examine the function of this polymorphism. In clear cell renal cell carcinoma tissue, isolated tumor cells as well as in primary renal tubular cells, in which HIF was stabilized, we determined genotype-specific increases of DPF3 mRNA levels and identified that the risk SNP resides in an active enhancer region, creating a novel HIF-binding motif. We then confirmed allele-specific HIF binding to this locus using chromatin immunoprecipitation of HIF subunits. Consequentially, HIF-mediated DPF3 regulation was dependent on the presence of the risk allele. Finally, we show that DPF3 deletion in proximal tubular cells retarded cell growth, indicating potential roles for DPF3 in cell proliferation. Our analyses suggest that the HIF pathway differentially operates on a SNP-induced hypoxia-response element at 14q24.2, thereby affecting DPF3 expression, which increases the risk of developing renal cancer.

Von Hippel-Lindau tumor suppressor protein (pVHL) functions to induce neuronal differentiation of neural stem/progenitor cells (NSCs) and skin-derived precursors (SKPs). Here we identified a neuronal differentiation domain (NDD) in pVHL. Neuronal differentiation of SKPs was induced by intracellular delivery of a peptide composed of the amino-acid sequences encoded by the NDD. Neuronal differentiation mediated by the NDD was caused by the binding between it and elongin C followed by Janus kinase-2 (JAK2) ubiquitination of JAK2 and inhibition of the JAK2/the signal transducer and activator of transcription-3(STAT)3 pathway. The NDD in pVHL contained the BC-box motif ((A,P,S,T)LXXX (A,C) XXX(A,I,L,V)) corresponding to the binding site of elongin C. Therefore, we proposed that other BC-box proteins might also contain an NDD; and subsequently also identified in them an NDD containing the amino-acid sequence encoded by the BC-box motif in BC-box proteins. Furthermore, we showed that different NDD peptide-delivered cells differentiated into different kinds of neuron-like cells. That is, dopaminergic neuron-like cells, cholinergic neuron-like cells, GABAnergic neuron-like cells or rhodopsin-positive neuron-like cells were induced by different NDD peptides. These novel findings might contribute to the development of a new method for promoting neuronal differentiation and shed further light on the mechanism of neuronal differentiation of somatic stem cells.

Dysregulation of C‑Type Lectin Domain Family 3 Member B (CLEC3B) in serum or tumor tissues has been reported in patients with various cancer types. However, the expression and function of CLEC3B in clear cell renal cell carcinoma (ccRCC) remain unknown. To examine the function of CLEC3B in ccRCC, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were examined to determine the expression of CLEC3B at the transcriptional level and it was demonstrated that CLEC3B mRNA was significantly downregulated in ccRCC compared with normal tissues (P<0.0001 and P=0.0392 in TCGA and GEO databases, respectively). The downregulation of CLEC3B was further validated at the protein level in 78.9% of ccRCCs by immunohistochemistry. To investigate the potential genetic mechanism for CLEC3B downregulation in ccRCC, copy number analysis was performed by profiling the copy number variation data from the TCGA project and it was revealed that the copy number loss of CLEC3B was prevalent in up to 88.1% of patients with ccRCC. CLEC3B genetic deletion was coupled with the well‑known genetic loss of the von Hippel‑Lindau tumor suppressor, which is a characteristic oncogenic event during ccRCC carcinogenesis. The downregulation of CLEC3B was associated with tumor progression and predicted unfavorable prognostic outcomes in the TCGA cohort. Real‑time cell analyzer system technology revealed that CLEC3B inhibited the proliferation of ccRCC cell lines in vitro and that the mitogen‑activated protein kinase pathway may contribute to this process. CLEC3B demonstrated substantial positive associations with proliferation inhibitors, but inverse associations with proliferation inducers and markers in two large ccRCC cohorts, suggesting that CLEC3B was able to identify ccRCCs with a lower proliferation capacity. In conclusion, the results of the present study propose that CLEC3B is a promising target for therapeutic intervention in ccRCC.