Ring Finger Protein 43 Research Articles

Hsa_circ_0097009 regulates proliferation, apoptosis, migration and invasion of hepatocellular carcinoma cells via miR-568/RNF38 axis.

Circular RNAs (circRNAs), a class of non-coding RNAs, has been proved as an essential driver of hepatocellular carcinoma (HCC) progression. However, the aim of this study is to explore the role of hsa_circ_0097009 (circ_0097009) in HCC progression. Ki67 expression in HCC tissues was labeled and examined by IHC assay. Quantitative real-time PCR (qRT-PCR) was applied to assess the expression of circ_0097009, miR-568 and RING finger protein 38 (RNF38). Western blot assay was conducted to analyze protein expression. HCC cell colony formation, proliferation, migration, invasion, angiogenesis ability and apoptosis were determined by colony formation assay, 5-ethynyl-29-deoxyuridine (EdU) assay, wound healing assay, trans well assay, angiogenesis assay and flow cytometry. The interaction between circ_0097009 and miR-568 as well as miR-568 and RNF38 was probed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. We found that circ_0097009 and RNF38 expressions were markedly higher, but miR-568 expression was significantly lower in HCC. Circ_0097009 knockdown blocked HCC cell cloning, proliferation, migration, invasion, angiogenesis and facilitated apoptosis. MiR-568 was a target of circ_0097009 in HCC cells. MiR-568 interference could attenuate the circ_0097009 knockdown-induced inhibition on HCC cells progress. MiR-568 depressed cell clonal formation, proliferation, migration, invasion, angiogenesis and promotion of apoptosis, by targeting RNF38, and RNF38 overexpression reversed the suppression of miR-568 in HCC cells. Also, circ_0097009 knockdown blocked tumor growth in vivoCONCLUSION: Together, this study indicated that the oncogenic function of circ_0097009 in HCC, and circ_0097009/miR-568/RNF38 axis was expected to be a novel therapeutic option for HCC patients (Tab. 1, Fig. 7, Ref. 33). Text in PDF www.elis.sk Keywords: circ_0097009, miR-568, RNF38, hepatocellular carcinoma.

RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy.

Triple-negative breast cancer (TNBC) has higher molecular heterogeneity and metastatic potential and the poorest prognosis. Because of limited therapeutics against TNBC, irradiation (IR) therapy is still a common treatment option for patients with lymph nodes or brain metastasis. Thus, it is urgent to develop strategies to enhance the sensitivity of TNBC tumors to low-dose IR. Here, the authors report that E3 ubiquitin ligase Ring finger protein 126 (RNF126) is important for IR-induced ATR-CHK1 pathway activation to enhance DNA damage repair (DDR). Mechanistically, RNF126 physically associates with the MRE11-RAD50-NBS1 (MRN) complex and ubiquitinates MRE11 at K339 and K480 to increase its DNA exonuclease activity, subsequent RPA binding, and ATR phosphorylation, promoting sustained DDR in a homologous recombination repair-prone manner. Accordingly, depletion of RNF126 leads to increased genomic instability and radiation sensitivity in both TNBC cells and mice. Furthermore, it is found that RNF126 expression is induced by IR activating the HER2-AKT-NF-κB pathway and targeting RNF126 expression with dihydroartemisinin significantly improves the sensitivity of TNBC tumors in the brain to IR treatment in vivo. Together, these results reveal that RNF126-mediated MRE11 ubiquitination is a critical regulator of the DDR, which provides a promising target for improving the sensitivity of TNBC to radiotherapy.

Ring finger protein 126 promotes breast cancer metastasis and serves as a potential target to improve the therapeutic sensitivity of ATR inhibitors

Background/aimsThis study explores the relationship between the E3 ubiquitin ligase Ring finger protein 126 (RNF126) and early breast cancer metastasis and tests the hypothesis that RNF126 determines the efficacy of inhibitors targeting Ataxia telangiectasia mutated and Rad3-related kinase (ATR).MethodsVarious metastasis-related genes were identified by univariable Cox proportional hazards regression analysis based on the GSE11121 dataset. The RNF126-related network modules were identified by WGCNA, whereas cell viability, invasion, and migration assays were performed to evaluate the biological characteristics of breast cancer cells with or without RNF126 knockdown. MTT, immunoblotting, immunofluorescence, and DNA fiber assays were conducted to determine the efficiency of ATR inhibitor in cells with or without RNF126 knockdown.ResultsRNF126 was associated with early breast cancer metastasis. RNF126 promoted breast cancer cell proliferation, growth, migration, and invasion. ATR inhibitors were more effective at killing breast cancer cells with intact RNF126 due to replication stress compared with the corresponding cells with RNF126 knockdown. Cyclin-dependent kinase 2 (CDK2) was involved in regulating replication stress in breast cancer cells with intact RNF126.ConclusionA high level of expression of RNF126 in early breast cancer patients without lymph node metastases may indicate a high-risk type of metastatic disease, possibly due to RNF126, which may increase breast cancer cell proliferation and invasion. RNF126-expressing breast cancer cells exhibit CDK2-mediated replication stress that makes them potential targets for ATR inhibitors.

Cancer-associated fibroblasts promote the stemness and progression of renal cell carcinoma via exosomal miR-181d-5p

The mechanisms underlying the effects of cancer-associated fibroblasts (CAFs) on cancer stemness and tumor progression in renal cell carcinoma (RCC) have not been elucidated yet. In the present study, we found that the enrichment of CAFs was positively associated with tumor progression and cancer stemness in RCC. Further investigation revealed that CAFs could enhance cancer stemness through delivering exosomes to RCC cells, and miR-181d-5p was identified as the critical exosomal miRNA in CAF-secreted exosomes by small RNA sequencing and subsequent screening assays. Mechanistically, exosomal miR-181d-5p transferred from CAFs to RCC cells directly suppressed the expression of ring finger protein 43 (RNF43) and activated Wnt/β-catenin signaling pathway, thus promoted cancer stemness and tumor progression. Overexpression of RNF43 strongly suppressed stemness properties and the effects could be reverted by miR-181d-5p. Overall, our findings revealed a crucial mechanism by which CAF-secreted exosomal miRNAs to enhance cancer stemness and thus promote RCC progression, suggesting a new avenue based on CAF-secreted miRNAs for more effective targeted therapies.

Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer.

BACKGROUNDGastric cancer (GC) is one of the most prevalent malignant tumors that endangers human health. Early diagnosis is essential for improving the prognosis and survival rate of GC patients. Ring finger protein 180 (RNF180) is involved in the regulation of cell differentiation, proliferation, apoptosis, and tumorigenesis, and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC. Thus, methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIMTo use droplet digital polymerase chain reaction (ddPCR) to quantify the methylation level of the RN180 gene. A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODSThe primer and probe were designed and selected, the conversion time of bisulfite was optimized, the ddPCR system was adjusted by primer concentration, amplification temperature and amplification cycles, and the detection limit of ddPCR was determined.RESULTSThe best conversion time for blood DNA was 2 h 10 min, and that for plasma DNA was 2 h 10 min and 2 h 30 min. The results of ddPCR were better when the amplification temperature was 56 °C and the number of amplification cycles was 50. Primer concentrations showed little effect on the assay outcome. Therefore, the primer concentration could be adjusted according to the reaction system and DNA input. The assay required at least 0.1 ng of input DNA.CONCLUSIONIn summary, a ddPCR assay was established to detect methylated RNF180, which is expected to be a new diagnostic biomarker for GC.

LncRNA KCNQ1OT1 enhances the radioresistance of lung squamous cell carcinoma by targeting the miR-491-5p/TPX2-RNF2 axis.

Lung cancer, especially lung squamous cell carcinoma (LUSC), is one of the most common malignant tumors worldwide. Currently, radiosensitization research is a vital direction for the improvement of LUSC therapy. Long non-coding RNAs (lncRNAs) can be novel biomarkers due to their multiple functions in cancers. However, the function and mechanism of lncRNA KCNQ1OT1 in the radioresistance of LUSC remain to be elucidated. The clonogenic assay was employed to determine the radioresistance of SK-MES-1R and NCI-H226R cells. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were conducted for the detection of gene expression. Cell proliferation was determined by the methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine (EdU) staining, and cell apoptosis was assessed by flow cytometry. The relationships between genes were also evaluated by applying the luciferase reporter and radioimmunoprecipitation (RIP) assays. Radioresistant LUSC cells (SK-MES-1R and NCI-H226R) had strong resistance to X-ray irradiation, and lncRNA KCNQ1OT1 was highly expressed in SK-MES-1R and NCI-H226R cells. Moreover, knockdown of lncRNA KCNQ1OT1 prominently suppressed proliferation, attenuated radioresistance, and accelerated the apoptosis of SK-MES-1R and NCI-H226R cells. More importantly, we verified that miR-491-5p was a regulatory target of lncRNA KCNQ1OT1, and Xenopus kinesin-like protein 2 (TPX2) and RING finger protein 2 (RNF2) were the target genes of miR-491-5p. The rescue experiment results also demonstrated that miR-491-5p was involved in the inhibition of cell proliferation and the downregulation of TPX2 and RNF2 expression mediated by lncRNA KCNQ1OT1 knockdown in SK-MES-1R and NCI-H226R cells. LncRNA KCNQ1OT1 was associated with the radioresistance of radioresistant LUSC cells, and the lncRNA KCNQ1OT1/miR-491-5p/TPX2-RNF2 axis might be used as a therapeutic target to enhance the radiosensitivity of radioresistant LUSC cells.

Combining expression of RNF43 and infiltration level of CD163+ tumor associated macrophage predicts prognosis of clear cell renal cell carcinoma.

Searching for reliable indicators for evaluating prognosis diagnosed with clear cell renal cell carcinoma (ccRCC) is crucial for improving clinical therapies. However, current researches have looked mainly at the prognostic value of a single intratumoral indicator, neglecting tumor-infiltrating immune cells (TIICs) in the microenvironment. This study examined whether the integration of Ring finger protein 43 (RNF43) expression and CD163+ tumor-associated macrophage (TAM) infiltration in combination with clinical indexes forecast ccRCC patient outcome with relatively high accuracy. Firstly, the expression of RNF43 and CD163 were detected with immunohistochemistry. Totally, 346 ccRCC patients were random separated evenly into training and validation datasets to make further analyses. We found that RNF43 expression was negatively correlated with infiltration level of CD163+ TAM in ccRCC, which was closely associated with the TNM stage and outcome of these patients. The multiple regression analysis demonstrated that RNF43, CD163, and TNM stage could function as independent risk factors in overall survival (OS) and progression-free survival (PFS) prediction of ccRCC. Furthermore, a better postoperative prognosis index for ccRCC patients was obtained by combining RNF43 and CD163+ TAMs, which assessed with time-dependent C-index analyses and a nomogram. Consequently, combining RNF43 and CD163+ TAMs along with TNM stage acquired robust accuracy in forecasting outcome of patients with ccRCC. In conclusion, combining intratumoral RNF43 expression, CD163+ TAM infiltration, and TNM stage could significantly enhance the veracity in forecasting postoperative outcomes.

RNF2 mediates pulmonary fibroblasts activation and proliferation by regulating mTOR and p16-CDK4-Rb1 signaling pathway.

Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease with unknown etiology, associated with increasing morbidity and pessimistic prognosis. Pulmonary fibroblasts (PFbs) are the key effector cells of PF, in which abnormal activation and proliferation is an important pathogenesis of PF. Ring finger protein 2 (RNF2), is identified as the catalytic subunit of poly-comb repressive complex 1, which is closely related to occurrence and development of lung cancer, but its function in PF has not been revealed. In this paper, we sought to identify the regulatory role of RNF2 in lung fibrogenesis and its underlying mechanisms. The expression of RNF2 in lung fibrosis tissue (human and Bleomycin-induced mouse) and cell model (TGF-β1-induced HFL1 cells) was examined by immunoblotting analysis and immunofluorescence. Western blot, qRT-PCR were performed to evaluate the expression of pro-fibrogenic cytokines (including α-SMA, ECM and MMPs/ TIMPs) induced by TGF-β1 in HFL1 cells. Cell proliferation, cycle progression and apoptosis were examined by fow cytometric. Molecular interactions were tested by Co-IP assays. RNF2 expression was elevated in PF tissues compared to normal adjacent tissues and in PFbs (HFL1) induced by TGF-β1. Furthermore, knockdown of RNF2 could evidently inhibit the abnormal expression of pro-fibrogenic cytokines (including α-SMA, ECM and MMPs/TIMPs) induced by TGF-β1 in HFL1 cells. Functionally, RNF2 silencing could significantly suppress TGF-β1-induced anomalous proliferation, cell cycle progression, apoptosis and autophagy in HFL1 cells. Mechanistically, RNF2 deficiency could effectively inhibit the abnormal activation of mTOR signaling pathway in TGF-β1-induced HFL1 cells, and mTOR pathway had feedback regulation on the expression of RNF2. Further studies RNF2 could regulate the phosphorylation level of RB1 through interacting with p16 to destroy the binding of p16 and CDK4 competitively. Simultaneously, overexpression of RNF2 could show the opposite results. These results indicated that RNF2 is a potent pro-fibrogenic molecule for PFbs activation and proliferation through mTOR and p16-CDK4-Rb signaling pathways, and RNF2 inhibition will be a potential therapeutic avenue for treating PF.

Glaucocalyxin A Inhibits the Malignancies of Gastric Cancer Cells by Downregulating MDM2 and RNF6 via MiR-3658 and the SMG1-UPF mRNA Decay Pathway.

Gastric cancer (GC) ranks as the most common gastrointestinal cancer and is among the leading causes of cancer death worldwide. Glaucocalyxin A (GLA), an entkauranoid diterpene isolated from Rab-dosia japonica var., possesses various bioactivities. To date, the data on the effect of GLA on GC are still minimal, and the molecular mechanisms remain largely unknown. Herein, we found that GLA could significantly inhibit the proliferation, cell adhesion, and invasion of HGT-1, SNU-1, SNU-6, and NCI-N87 GC cells in a dose-dependent manner. GLA enhanced the apoptosis of the GC cells as evidenced by the increased caspase-3 activity and the elevated levels of cleaved caspase-3 and cleaved PARP in GC cells in the presence of GLA. We then showed that the downregulation of Murine Double Minute Clone 2 (MDM2) and Ring Finger Protein 6 (RNF6) by GLA was implicated in the GLA-induced inhibition of the GC cells. Furthermore, MDM2 and RNF6 were identified as the targets of miR-3658 that was downregulated in the GC cells and upregulated by GLA. Moreover, it was shown that miR-3658 was hypermethylated in the GC cells, and GLA could rescue the expression of miR-3658 via demethylation by abrogating EZH2-mediated epigenetic silencing. In addition to the miR-3658-MDM2/RNF6 regulatory axis, activation of the SMG1-UPF mRNA decay pathway contributed to the downregulation of MDM2 and RNF6 by GLA in the GC cells. The inhibitory effect of GLA on gastric cancer and the expression of MDM2 and RNF6 was also validated in in vivo study. Our findings suggest that has the therapeutic potential for GC by downregulating oncogenes via posttranscriptional regulation.

Abstract 2101: RNF2 ablation reprograms the tumor-immune microenvironment and stimulates durable NK and CD4+ T cell-dependent anti-tumor immunity

Abstract Expanding the utility of immune-based cancer treatments is a clinical challenge due to tumor-intrinsic factors that suppress the immune response. Here we report the identification of tumoral Ring Finger Protein 2 (RNF2), the core subunit of the Polycomb Repressor Complex 1 (PRC1), as a negative regulator of antitumor immunity in various human cancers, including breast cancer. In syngeneic murine models of triple negative breast cancer, we found that deleting genes encoding PRC1 subunits Rnf2 or BMI1 proto-oncogene, polycomb ring finger (Bmi1), or the downstream effector of Rnf2, Remodeling and Spacing Factor 1 (Rsf1), was sufficient by itself to induce durable tumor rejection and establish immune memory by enhancing infiltration and activation of NK and CD4+ T-cells, but not CD8+ T-cells, into the tumor and enabled their cooperativity. These findings uncover an epigenetic reprogramming of the tumor-immune microenvironment which fosters durable antitumor immunity and memory. Citation Format: Zhuo Zhang, Lin Luo, Chuan Xing, Yu Chen, Peng Xu, Mao Li, Ling Zeng, Chao Li, Sadashib Ghosh, Deborah Della Manna, Tim Townes, William J. Britt, Narendra Wajapeyee, Barry P. Sleckman, Zechen Chong, Jianmei Wu Leavenworth, Eddy Yang. RNF2 ablation reprograms the tumor-immune microenvironment and stimulates durable NK and CD4+ T cell-dependent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2101.

A multi-arm, phase 2, open-label study to assess the efficacy of RXC004 as monotherapy and in combination with nivolumab in patients with ring finger protein 43 (RNF43) or R-spondin (RSPO) aberrated, metastatic, microsatellite stable colorectal cancer following standard treatments.

TPS3637 Background: The Wnt pathway is a critical driver of cancer. RXC004 is a potent, selective, orally active inhibitor of the key Wnt pathway regulator, Porcupine. Inhibition of Porcupine blocks the release of all Wnt ligands from cells, preventing both tumour growth and tumour immune evasion. Wnt pathway alterations, including loss-of-function (LoF) RNF43 mutations and RSPO gene fusions, increase expression of the Wnt receptor Frizzled (Fzd) on the tumour cell surface, driving Wnt-ligand signalling. These alterations are present in ̃8% (Gao, 2013; Cerami, 2012; Shesagiri, 2012; Shinmura, 2014; Kleeman, 2019) of colorectal cancers (CRC). LoF RNF43 mutations are associated with poor prognosis in MSS CRC (Yaeger, 2018 ). Preclinical genetically selected CRC models showed disease stabilisation, differentiation towards a normal colonic phenotype with increased mucin secretion, and reduced metabolic activity on FDG-PET. In a Phase 1 study in patients with advanced solid tumours (NCT03447470), RXC004 was safe and tolerated at doses up to 2mg QD, the recommended phase 2 dose (RP2D), and showed a differentiated efficacy signal in Wnt-ligand dependent tumours (Cook, 2021 ). Methods: The PORCUPINE (NCT04907539) trial is a 2-arm Phase 2 trial of RXC004 monotherapy (Arm A) and RXC004 in combination with nivolumab (Arm B). 20 evaluable patients will be enrolled into each Arm. The study initially opened with Arm A; Arm B will be opened once the RP2D for RXC004 in combination with nivolumab is established in a separate phase 1 study. Once Arm B is opened, patients in Arm A may be treated with RXC004 + nivolumab if they have progressive disease on the first RECIST assessment scan. To be eligible for this study, patients must have metastatic microsatellite stable (MSS) CRC that has progressed following standard therapies. Tumours must have a LoF RNF43 mutation, or an RSPO2/3 fusion. As Wnt inhibition can affect bone metabolism, patients undergo a screening DEXA scan and receive prophylactic denosumab throughout the treatment period. The primary endpoint for Arm A is the disease control rate (DCR= CR+PR+SD at 16 wks), and for Arm B is objective response rate (ORR). Secondary endpoints are Safety and PK. Exploratory endpoints include FDG-PET changes and on-treatment changes in protein and gene expression in tumour biopsies. For Arm A, a target value (TV) of 60% DCR is considered a clinically significant improvement over standard of care against a lower reference value (LRV) of 40% DCR (Grothey, 2013; Mayer, 2015 ). For Arm B, a TV of 30% ORR is considered clinically significant against a LRV of 10% ORR (Eng, 201 9). RXC004 is also being investigated in a second Phase 2 trial, PORCUPINE 2 (NCT04907851), in Biliary Tract Cancers and RNF-43 mutated Pancreatic Cancers. Clinical trial information: NCT04907539.

Neuroprotective Effect of E3 Ubiquitin Ligase RNF8 Against Ischemic Stroke via HDAC2 Stability Reduction and Reelin-Dependent GSK3β Inhibition.

Loss of E3 ubiquitin ligase RING finger protein 8 (RNF8) may lead to neuronal DNA damage and apoptosis. In order to expand on our knowledge on the mechanistic basis underlying neuronal death in ischemic stroke, the present study sought to investigate the potential role of E3 ubiquitin ligase RNF8 on ischemic stroke and explore the underlying downstream mechanism. Middle cerebral artery occlusion (MCAO) in mice and oxygen–glucose deprivation/reoxygenation (OGD/R) in neurons were induced to simulate an ischemic stroke environment. It was found that downregulation of RNF8 and Reelin occurred in MCAO mice and OGD/R-exposed neurons. Silencing of RNF8 enhanced the MCAO-induced neuronal apoptosis and oxidative stress. Mechanistically, RNF8 enhanced the ubiquitination and degradation of HDAC2, thus attenuating OGD/R-induced neuronal apoptosis and oxidative stress. Moreover, HDAC2 inhibited Reelin expression through deacetylation of H3K27me3 in its promoter, causing reduced glycogen synthase kinase-3beta (GSK3β)-Ser9 phosphorylation and the resultant elevated GSK3β activity. By this mechanism, RNF8 alleviated ischemic stroke. Coherently, this study suggests that RNF8 plays a neuroprotective effect against ischemic stroke by downregulating HDAC2 expression and inducing Reelin-induced GSK3β inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-022-02880-w.

Ring finger protein 180 suppresses cell proliferation and energy metabolism of non-small cell lung cancer through downregulating C-myc

BackgroundNon-small cell lung cancer (NSCLC) causes numerous deaths worldwide. however, biomarkers for NSCLC prognosis are scarce for its heterogeneity. Proteins containing the RING finger domain RING finger protein 180 (RNF180) is a key mediator for ubiquitination, which controls cell cycle and regulates progression in certain human tumors. However, the detailed function of RNF180 in NSCLC remains unclear. In the present study, we aimed to investigate the role of RNF180 and its molecule network in NSCLC.MethodsQuantitative real-time polymerase chain reaction and immunohistochemical staining were used to analyze RNF180 levels. RNA interference and lentiviral-mediated vector transfections were performed to silence and overexpress RNF180 in NSCLC cells. Furthermore, Cell Counting Kit-8 was used for assessing biological function of RNF180 in cell proliferation and a xenograft model for examining its function in vivo. The activity of glycolysis was determined by examining the level of the extracellular acidification rate (ECAR).ResultsRNF180 expression decreased in NSCLC tissues, and its expression was positively correlated with the survival rate of patients with NSCLC. Moreover, RNF180 overexpression suppressed the proliferation and glycolytic activities in NSCLC cells and restricted its tumorigenicity in vivo. Furthermore, RNF180 silencing promoted the proliferation and glycolysis metabolism of NSCLC cells, whereas C-myc inhibitor disrupted these effects. The underlying anti-oncogene of RNF180 involved in C-myc downregulation via ubiquitin-dependent degradation.ConclusionsTogether, these results firstly indicated the anti-tumor properties of RNF180 and its correlation with NSCLC progression, thereby endorsing the potential role of RNF180 as an efficient prognostic biomarker for tumor recurrence.

RNF43 R117fs mutant positively regulates Wnt/\u03b2-catenin signaling by failing to internalize FZD expressed on the cell surface

RING finger protein 43 (RNF43) encodes the transmembrane E3 ubiquitin ligase, which targets the Wnt receptor Frizzled (FZD). RNF43 mutations have been discovered in various human cancers including colon, pancreatic, stomach, ovarian, and liver cancers. Functional studies on RNF43 missense mutations have shown that they negatively regulate Wnt signaling; however, there are few functional studies on RNF43 frameshift mutations. In this study, we showed that R117fs and P441fs mutants enhanced Wnt/β-catenin signaling, whereas Q409fs and G659fs mutants retained the ability to suppress Wnt/β-catenin signaling. Specifically, R117fs was unable to ubiquitinate FZD5 due to lack of the RING domain, although it was able to interact with FZD5. Immunofluorescence showed that R117fs failed to internalize FZD5 expressed on the cell surface. We also showed that LGK974, a potent Wnt inhibitor, decreased the Wnt/β-catenin activity by R117fs and P441fs mutations. Together, these results demonstrate that RNF43 frameshift mutations retain normal functionality; thus, targeted anti-cancer therapy can be developed according to the mutation type of RNF43.

BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

SummaryNeurodegeneration is associated with the aggregation of proteins bearing solvent-exposed hydrophobicity as a result of their misfolding and/or proteolytic cleavage. An understanding of the cellular protein quality control mechanisms which prevent protein aggregation is fundamental to understanding the etiology of neurodegeneration. By examining the metabolism of disease-linked C-terminal fragments of the TAR DNA-binding protein 43 (TDP43), we found that the Bcl-2 associated athanogene 6 (BAG6) functions as a sensor of proteolytic fragments bearing exposed hydrophobicity and prevents their intracellular aggregation. In addition, BAG6 facilitates the ubiquitylation of TDP43 fragments by recruiting the Ub-ligase, Ring finger protein 126 (RNF126). Authenticating its role in preventing aggregation, we found that TDP43 fragments form intracellular aggregates in the absence of BAG6. Finally, we found that BAG6 could interact with and solubilize additional neurodegeneration-associated proteolytic fragments. Therefore, BAG6 plays a general role in preventing intracellular aggregation associated with neurodegeneration.

Regulation of p27 (Kip1) by Ubiquitin E3 Ligase RNF6

The cyclin-dependent kinase inhibitor p27 (Kip1) is an important regulator of the G1/S checkpoint. It is degraded by the SCF-SKP2 complex in late G1 thereby allowing cells to progress to the S phase. Here we investigated the role of the E3 ubiquitin ligase RNF6 (Ring Finger Protein 6) in cell cycle progression in prostate cancer cells. Our data demonstrate that RNF6 can promote cell cycle progression by reducing the levels of p27. Knockdown of RNF6 led to an increase in the stability of p27 and to the arrest of cells in the G1 phase. RNF6 interacted with p27 via its KIL domain and this interaction was found to be phosphorylation independent. RNF6 enhanced ubiquitination and subsequent degradation of p27 in the early G0/G1 phase of the cell cycle. Knockdown of RNF6 expression by short hairpin RNA led to inhibition of the CDK2/Cyclin E complex thereby reducing phosphorylation of Retinoblastoma protein (Rb) and to a subsequent decrease in cell cycle progression and proliferation. Our data suggest that RNF6 acts as a negative regulator for p27kip1 leading to its proteasome-dependent degradation in the early G0/G1 phase of the cell cycle.

Porcupine Inhibition Disrupts Mitochondrial Function and Homeostasis in WNT Ligand-Addicted Pancreatic Cancer.

WNT signaling promotes pancreatic ductal adenocarcinoma (PDAC) through diverse effects on proliferation, differentiation, survival, and stemness. A subset of PDAC with inactivating mutations in ring finger protein 43 (RNF43) show growth dependency on autocrine WNT ligand signaling and are susceptible to agents that block WNT ligand acylation by Porcupine O-acyltransferase, which is required for proper WNT ligand processing and secretion. For this study, global transcriptomic, proteomic, and metabolomic analyses were performed to explore the therapeutic response of RNF43-mutant PDAC to the Porcupine inhibitor (PORCNi) LGK974. LGK974 disrupted cellular bioenergetics and mitochondrial function through actions that included rapid mitochondrial depolarization, reduced mitochondrial content, and inhibition of oxidative phosphorylation and tricarboxylic acid cycle. LGK974 also broadly altered transcriptional activity, downregulating genes involved in cell cycle, nucleotide metabolism, and ribosomal biogenesis and upregulating genes involved in epithelial-mesenchymal transition, hypoxia, endocytosis, and lysosomes. Autophagy and lysosomal activity were augmented in response to LGK974, which synergistically inhibited tumor cell viability in combination with chloroquine. Autocrine WNT ligand signaling dictates metabolic dependencies in RNF43-mutant PDAC through a combination of transcription dependent and independent effects linked to mitochondrial health and function. Metabolic adaptations to mitochondrial damage and bioenergetic stress represent potential targetable liabilities in combination with PORCNi for the treatment of WNT ligand-addicted PDAC.

RING-Finger Protein 6 promotes Drug Resistance in Retinoblastoma via JAK2/STAT3 Signaling Pathway.

Chemotherapy is the first-line treatment for human retinoblastoma (RB), but the occurrence of drug resistance greatly limited its efficacy in practice. RING-finger protein 6 (RNF6) is an E3 ubiquitin ligase that is aberrantly upregulated in a range of cancers and plays important roles in cancer progression. However, the role of RNF6 in RB is largely unknown. In this study, we investigated the role of RNF6 in RB drug resistance. Two carboplatin-resistant RB cells, Y-79/CR and SO-Rb50/CR, were generated based on Y-79 and SO-Rb50 cells. RT-PCR and western blot analyses showed that RNF6 expression on both mRNA and protein levels was significantly increased in Y-79/CR and SO-Rb50/CR cells comparing to their parental cells. Knockdown of RNF6 using siRNA in Y-79/CR and SO-Rb50/CR cells resulted in cells sensitive to carboplatin on a RNF6 siRNA dose dependent manner. Similarly, RNF6 overexpression in parental Y-79 and SO-Rb50 cells could help cells gain resistance to carboplatin on a RNF6 expression dependent manner. Signaling pathway analyses revealed that JAK2/STAT3 pathway was involved in the RNF6-induced carboplatin resistance in RB cells. We further revealed that RNF6 expression in both Y-79 and SO-Rb50 cells could render cells resistant to multiple anti-cancer drugs including carboplatin, vincristine and etoposide, an implication of RNF6 as a biomarker for RB drug resistance. Taken together, our study has revealed that RNF6 is upregulated in drug-resistant RB cells and RNF6 promotes drug resistance through JAK2/STAT3 signaling pathway. The importance of RNF6 in RB cells drug resistance may represent this protein as a potential biomarker and treatment target for drug resistance in RB.

Ring finger protein 6 enhances chemo-resistance by transcriptionally activating proliferating cell nuclear antigen expression and attenuating DNA damage in lung adenocarcinoma

The E3 ubiquitin ligase RING finger protein 6 (RNF6) is elevated in several cancers, including prostate and colorectal cancers. Here, we extended the finding of elevated RNF6 expression levels and its association with poor prognosis in patients with lung adenocarcinoma (LUAD). Genome-wide RNA sequencing in H3255 cells with RNF6 knockdown, followed by analysis of differentially expressed genes using Clusters of Orthologous Groups and gene set enrichment analysis revealed aberrations in genes related to DNA repair, especially double-strand break (DSB) repair. RNF6 knockdown increased γH2AX foci, a biomarker for DSBs in H3255 and A549 LUAD cells, and enhanced DNA damage induced by chemotherapy in cisplatin-resistant A549/CDDP cells. In a series of experiments in cultured cells, as well as in nude mice carrying xenografts, RNF6 knockdown restored the sensitivity of A549/CDDP cells to cisplatin treatment. Mechanistically, RNA sequencing in RNF6-knockdown cells revealed the significant downregulation of proliferating cell nuclear antigen (PCNA), an oncogene that promotes DNA repair. Re-chromatin immunoprecipitation assay results suggested the formation of a RNF6-TCF4 complex that binds to the PCNA promoter to activate its transcription. Downregulation of RNF6 reduced TCF4 recruitment to PCNA promoters in H3255 and A549 cells, indicating that RNF6 regulates PCNA transcription to a certain extent by regulating TCF4 binding to PCNA promoters. The collective results implicate RNF6 overexpression as a molecular target in the management of cisplatin-resistant LUAD.

SBDS interacts with RNF2 and is degraded through RNF2-dependent ubiquitination

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder caused by mutation in the Shwachman-Bodian-Diamond syndrome (SBDS) gene that has a variety of clinical features, including exocrine pancreatic insufficiency and hematological dysfunction. The SBDS protein is considered to be involved in ribosome biogenesis, ribosomal RNA metabolism, stabilization of mitotic spindles and cellular stress responses, yet the function of SBDS in detail is still incompletely understood. The multiple functions imply that certain proteins might associate with SBDS and affect its function. In this study, we identified Ring finger protein 2 (RNF2) as a candidate for the SBDS interactor by yeast two-hybrid screening. Moreover, we confirmed the interaction by GST-pull down assay using recombinant proteins and co-immunoprecipitation in HEK293T cells overexpressing RNF2. In addition, it is shown that RNF2 ubiquitinates SBDS and promotes its proteasomal degradation in HEK293T cells. These findings provide new insights into the regulation of SBDS.