Hsa_circ_0001740 mediates trophoblast cell function via regulating miR-188-3p/ARRDC3.

Preeclampsia is an obstetric disorder and remains the leading contributor to maternal and fetal morbidity and mortality. This study was designed to explore the role of hsa_circ_0001740 in preeclampsia as well as its underlying mechanism. Real-time quantitative polymerase chain reaction was performed to examine hsa_circ_0001740 and miR-188-3p levels in trophoblast cell line HTR-8/SVneo. The proliferation, migration, invasion, and apoptosis of HTR-8/SVneo cells were detected using cell counting kit-8, colony formation, wound healing, transwell, and terminal-deoxynucleoitidyl transferase mediated nick end labeling assays, respectively. The expression of apoptosis- and Hippo signaling-related proteins were assessed by western blot. Moreover, the binding relationship between hsa_circ_0001740 and miR-188-3p, miR-188-3p and ARRDC3 were verified by luciferase report assay. The results showed that hsa_circ_001740 overexpression inhibited the proliferation, migration, and invasion, and promoted apoptosis of HTR-8/SVneo cells. Hsa_circ_0001740 was verified to bind to miR-188-3p, and ARRDC3 was demonstrated to be a target of miR-188-3p. miR-188-3p overexpression partially counteracted the suppressive effects of hsa_circ_001740 overexpression on the proliferation, migration, and invasion of HTR-8/SVneo cells. What's more, ARRDC3 expression was upregulated by hsa_circ_001740-overexpression but was downregulated by miR-188-3p overexpression. Hsa_circ_001740/miR-188-3p also mediated Hippo signaling. To summarize, hsa_circ_0001740 could maintain trophoblast cell function via downregulating miR-188-3p, providing a potential biomarker for the diagnosis and treatment of preeclampsia.

Arrestin domain-containing 3 (Arrdc3) modulates insulin action and glucose metabolism in liver.

Insulin action in the liver is critical for glucose homeostasis through regulation of glycogen synthesis and glucose output. Arrestin domain-containing 3 (Arrdc3) is a member of the α-arrestin family previously linked to human obesity. Here, we show that Arrdc3 is differentially regulated by insulin in vivo in mice undergoing euglycemic-hyperinsulinemic clamps, being highly up-regulated in liver and down-regulated in muscle and fat. Mice with liver-specific knockout (KO) of the insulin receptor (IR) have a 50% reduction in Arrdc3 messenger RNA, while, conversely, mice with liver-specific KO of Arrdc3 (L-Arrdc3 KO) have increased IR protein in plasma membrane. This leads to increased hepatic insulin sensitivity with increased phosphorylation of FOXO1, reduced expression of PEPCK, and increased glucokinase expression resulting in reduced hepatic glucose production and increased hepatic glycogen accumulation. These effects are due to interaction of ARRDC3 with IR resulting in phosphorylation of ARRDC3 on a conserved tyrosine (Y382) in the carboxyl-terminal domain. Thus, Arrdc3 is an insulin target gene, and ARRDC3 protein directly interacts with IR to serve as a feedback regulator of insulin action in control of liver metabolism.

ARRDC3 inhibits liver fibrosis and epithelial-to-mesenchymal transition via the ITGB4/PI3K/Akt signaling pathway.

OBJECTIVE: The effect of ARRDC3 has not been reported in liver fibrosis. Our study aimed to explore the molecular mechanisms by which ARRDC3 attenuates liver fibrosis. METHODS: The vectors pcDNA-ARRDC3 (which promotes ARRDC3 expression) and si-ITGB4 (which blocks IGTB4 expression) and their negative controls were constructed. The rat liver fibrosis model was established by intraperitoneal injection of CCl4 with or without intraperitoneal injection of pcDNA-ARRDC3. ELISA was used to detect the concentrations of γ-GGT, ALT, AST, and ALP in serum. HE, Masson's trichome, and Sirius red staining were used to observe the pathological changes in liver tissue. LX-2 cells were treated with TGF-ß, and pcDNA-ARRDC3 or si-ITGB4RNA was transfected to promote ARRDC3 expression or knock down ITGB4 expression. Western blotting was used to detect the expression levels of proteins. RESULTS: ARRDC3 effectively reduced liver injury, improved liver function, and decreased collagen production and deposition in the CCl4-induced rat fibrosis model. The studies showed that overexpressed ARRDC3 remarkably reduced the expression of E-cadherin and collagen-related protein and increased the expression of mesenchymal markers and EMT-related transcription factors, consequently inhibiting the activity of the ITGB4/PI3K/Akt signaling pathway. CONCLUSION: Our study shows that ARRDC3 could ameliorate CCl4-induced liver fibrosis and EMT progression via the ITGB4/PI3K/Akt signaling pathway, which provides a meaningful reference for the clinical targeted treatment of liver fibrosis.

ARRDC3 polymorphisms may affect the risk of glioma in Chinese Han.

This study ascertained to explore the potential contribution of ARRDC3 polymorphisms in the risk and prognosis of glioma. One thousand sixty-one patients and healthy controls were conducted to assess whether ARDC3 polymorphism was associated with glioma risk and prognosis. Four sites in ARRDC3 were selected and genotyped in MassARRAY platform. The calculated odd ratios and 95% confidence intervals from logistic regression were applied for risk assessment. The relationship between ARRDC3 variants and glioma prognosis was evaluated using log-rank test, Kaplan-Meier analysis, and so on. Also, false-positive report probability (FPRP) and statistical power were also assessed. Our findings suggested the negative role of ARRDC3 polymorphisms in the glioma risk. We also found the effect of candidate SNPs in ARRDC3 on the susceptibility to glioma was dependent on the age, gender, and histology of glioma patients. The results suggested that the genetic polymorphisms of ARRDC3 were related to an increased risk of glioma.

Endo-lysosomal sorting of G-protein-coupled receptors by ubiquitin: Diverse pathways for G-protein-coupled receptor destruction and beyond.

Ubiquitin is covalently attached to substrate proteins in the form of a single ubiquitin moiety or polyubiquitin chains and has been generally linked to protein degradation, however, distinct types of ubiquitin linkages are also used to control other critical cellular processes like cell signaling. Over forty mammalian G protein-coupled receptors (GPCRs) have been reported to be ubiquitinated, but despite the diverse and rich complexity of GPCR signaling, ubiquitin has been largely ascribed to receptor degradation. Indeed, GPCR ubiquitination targets the receptors for degradation by lysosome, which is mediated by the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, and the proteasome. This has led to the view that ubiquitin and ESCRTs primarily function as the signal to target GPCRs for destruction. Contrary to this conventional view, studies indicate that ubiquitination of certain GPCRs and canonical ubiquitin-binding ESCRTs are not required for receptor degradation and revealed that diverse and complex pathways exist to regulate endo-lysosomal sorting of GPCRs. In other studies, GPCR ubiquitination has been shown to drive signaling and not receptor degradation and further revealed novel insight into the mechanisms by which GPCRs trigger the activity of the ubiquitination machinery. Here, we discuss the diverse pathways by which ubiquitin controls GPCR endo-lysosomal sorting and beyond.

Mid-pregnancy PM2.5 exposure affects sex-specific growth trajectories via ARRDC3 methylation.

Prenatal particulate matter <2.5 µm (PM2.5) is associated with adverse birth growth. However, the longitudinal growth impacts have been little studied, and no mechanistic relationships have been described. We investigated the association between prenatal PM2.5 exposure and growth trajectories, and the possible role of epigenetics. We enrolled 1313 neonates with PM2.5 data measured by ordinary kriging from the COhort for Childhood Origin of Asthma and allergic diseases, followed up at 1, 3, and 5 years to evaluate growth. Differential DNA methylation and pyrosequencing of cord blood leukocytes was evaluated according to the prenatal PM2.5 levels and birth weight (BW). PM2.5 exposure during the second trimester (T2) caused the lowest BW in both sexes, further adjusted for indoor PM2.5 levels [female, aOR 1.39 (95% CI 1.05-1.83); male, aOR 1.36 (95% CI 1.04-1.79)]. Bayesian distributed lag models with indoor PM2.5 adjustments revealed a sensitive window for BW effects at 10-26 weeks gestation, but only in females. Latent class mixture models indicated that a persistently low weight-for-height percentile trajectory was more prevalent in the highest PM2.5 exposure quartile at T2 in females, compared to a persistently high trajectory (36.5% vs. 20.3%, P = 0.022). Also, in the females only, the high PM2.5 and low BW neonates showed significantly greater ARRDC3 methylation changes. ARRDC3 methylation was also higher only in females with low weight at 5 years of age. Higher fetal PM2.5 exposure during T2 may cause a decreased growth trajectory, especially in females, mediated by ARRDC3 hyper-methylation-associated energy metabolism.

Possible association of arrestin domain-containing protein 3 and progression of non-alcoholic fatty liver disease.

The prevalence of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is increasing worldwide. Several effective drugs for these diseases are now in development and under clinical trials. It is important to reveal the mechanism of the development of NAFLD and NASH. We investigated the role of arrestin domain-containing protein 3 (ARRDC3), which is linked to obesity in men and regulates body mass, adiposity and energy expenditure, in the progression of NAFLD and NASH. We performed knockdown of endogenous ARRDC3 in human hepatocytes and examined the inflammasome-associated gene expression by real-time PCR-based array. We also examined the effect of conditioned medium from endogenous ARRDC3-knockdown-hepatocytes on the apoptosis of hepatic stellate cells. We observed that free acids enhanced the expression of ARRDC3 in hepatocytes. Knockdown of ARRDC3 could lead to the inhibition of inflammasome-associated gene expression in hepatocytes. We also observed that conditioned medium from endogenous ARRDC3-knockdown-hepatocytes enhances the apoptosis of hepatic stellate cells. ARRDC3 has a role in the progression of NAFLD and NASH and is one of the targets for the development of the effective treatment of NAFLD and NASH.

GPCRs in Cancer: Protease-Activated Receptors, Endocytic Adaptors and Signaling.

G protein-coupled receptors (GPCRs) are a large diverse family of cell surface signaling receptors implicated in various types of cancers. Several studies indicate that GPCRs control many aspects of cancer progression including tumor growth, invasion, migration, survival and metastasis. While it is known that GPCR activity can be altered in cancer through aberrant overexpression, gain-of-function activating mutations, and increased production and secretion of agonists, the precise mechanisms of how GPCRs contribute to cancer progression remains elusive. Protease-activated receptors (PARs) are a unique class of GPCRs implicated in cancer. PARs are a subfamily of GPCRs comprised of four members that are irreversibly activated by proteolytic cleavage induced by various proteases generated in the tumor microenvironment. Given the unusual proteolytic irreversible activation of PARs, expression of receptors at the cell surface is a key feature that influences signaling responses and is exquisitely controlled by endocytic adaptor proteins. Here, we discuss new survey data from the Cancer Genome Atlas and the Genotype-Tissue Expression projects analysis of expression of all PAR family member expression in human tumor samples as well as the role and function of the endocytic sorting machinery that controls PAR expression and signaling of PARs in normal cells and in cancer.

Androgen receptor regulated microRNA miR-182-5p promotes prostate cancer progression by targeting the ARRDC3/ITGB4 pathway.

MicroRNAs (miRNAs) are important endogenous gene regulators that play key roles in prostate cancer development and metastasis. However, specific miRNA expression patterns in prostate cancer tissues from Chinese patients remain largely unknown. In this study, we compared miRNA expression patterns in 65 pairs of prostate cancer and para-cancer tissues by RNA sequencing and found that miR-182-5p was the most up-regulated miRNA in prostate cancer tissues. The result was validated using realtime PCR in 18 pairs of prostate cancer and para-cancer tissues. In in vitro analysis, it was confirmed that miR-182-5p promotes prostate cancer cell proliferation, invasion and migration and inhibit apoptosis. In addition, the androgen receptor directly regulated the transcription of miR-182-5p, which could target to the 3'UTR of ARRDC3 mRNA and affect the expression of ARRDC3 and its downstream gene ITGB4. For the in vivo experiment, miR-182-5p overexpression also promoted the growth and progression of prostate cancer tumors. In this regard, we suggest that miR-182-5p may be a key androgen receptor-regulated factor that contributes to the development and metastasis of Chinese prostate cancers and may be a potential target for the early diagnosis and therapeutic studies of prostate cancer.

ARRDC3 regulates the targeted therapy sensitivity of clear cell renal cell carcinoma by promoting AXL degradation.

AXL plays crucial roles in the tumorigenesis, progression, and drug resistance of neoplasms; however, the mechanisms associated with AXL overexpression in tumors remain largely unknown. In this study, to investigate these molecular mechanisms, wildtype and mutant proteins of arrestin domain-containing protein 3 (ARRDC3) and AXL were expressed, and co-immunoprecipitation analyses were performed. ARRDC3-deficient cells generated using the CRISPR-Cas9 system were treated with different concentrations of the tyrosine kinase inhibitor sunitinib and subjected to cell biological, molecular, and pharmacological experiments. Furthermore, immunohistochemistry was used to analyze the correlation between ARRDC3 and AXL protein expressions in renal cancer tissue specimens. The experimental results demonstrated that ARRDC3 interacts with AXL to promote AXL ubiquitination and degradation, followed by the negative regulation of downstream signaling mechanisms, including the phosphorylation of protein kinase B and extracellular signal-regulated kinase. Notably, ARRDC3 deficiency decreased the sunitinib sensitivity of clear cell renal cell carcinoma (ccRCC) cells in a manner dependent on the regulation of AXL stability. Overall, our results suggest that ARRDC3 is a negative regulator of AXL and can serve as a novel predictor of sunitinib therapeutic response in patients with ccRCC.

Fuzheng Kang-Ai inhibits NSCLC cell proliferation via regulating hsa_circ_0048091/hsa-miR-378g/ARRDC3 pathway.

BACKGROUND: Current treatments for lung cancer have their own deficiencies, such as severe adverse effect. Therefore, more safe and effective drugs are needed. PURPOSE: Fuzheng Kang-Ai (FZKA for short) has been applied as an adjuvant treatment in advanced Non-Small Cell Lung Cancer (NSCLC) patients for decades in China, showing a definitive effect with minimal toxicities. However, the underlying mechanism is yet to be identified. STUDY DESIGN: Both in vitro and in vivo experiments were performed in this study to identify the exact mechanism by which FZKA inhibits NSCLC cell proliferation. METHODS: MTT and CCK-8 assays were used to detect cell viability. Xenograft model was performed for in vivo experiments. CircRNA and miRNA sequencing were used to find the differentially expressed circRNAs and miRNAs, respectively. qRT-PCR was performed to check the expression levels of circRNA, miRNA and mRNA. BaseScope was carried out to observe the expression of circRNA in situ. Actinomycin D and RNase R experiments were done to show the stability of circRNA. Nuclear-cytoplasmic fractionation and FISH were used to identify the localization of circRNA and miRNA. Pull-down, RIP, and luciferase activity assays were performed to show the biding ability of circRNA, miRNA and target proteins. Flow cytometry was done to observe cell apoptosis. Western blot and IHC were done to detect the protein expression. TCGA database was used to analyze the survival rate. RESULTS: FZKA inhibits NSCLC cell proliferation both in vitro and in vivo. Hsa_circ_0048091 and hsa-miR-378g were the most differentially expressed circRNA and miRNA, respectively, after FZKA treatment. Silencing hsa_circ_0048091 and overexpressing hsa-miR-378g promoted cell proliferation and reversed the inhibition effect of FZKA on NSCLC, respectively. Hsa-miR-378g was sponged by hsa_circ_0048091, and the overexpression of miR-378g reversed the inhibition effect of hsa_ circ_0048091 on NSCLC. ARRDC3, as a target of hsa-miR-378g, was increased by FZKA treatment. Silencing ARRDC3 reversed both the inhibition effect of FZKA and miR-378g inhibitor on NSCLC. CONCLUSION: This study, for the first time, has established the function of hsa_circ_0048091, hsa- miR-378g, and ARRDC3 in lung cancer. It also shows that FZKA inhibits NSCLC cell proliferation through hsa_circ_0048091/hsa-miR-378g/ARRDC3 pathway, uncovering a novel mechanism by which FZKA controls human NSCLC cell growth.

circ_0004904 regulates the trophoblast cell in preeclampsia via miR-19b-3p/ARRDC3 axis.

Circular RNAs have been demonstrated to act as vital participants in various diseases, including preeclampsia (PE). This study aimed to research the effects of circ_0004904 on PE. The contents of circ_0004904, microRNA-19b-3p (miR-19b-3p) and arrestin domain containing 3 (ARRDC3) were quantified by quantitative real-time PCR and western blot. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 5-ethynyl-2'-deoxyuridine assays were enforced to assess cell proliferation. The transwell assay and flow cytometry were applied to detect the cell migration, invasion, and apoptosis. The liaison between miR-19b-3p and circ_0004904 or ARRDC3 was demonstrated by dual-luciferase reporter assay. Thereafter, circ_0004904 and ARRDC3 were augmented, and miR-19b-3p was restrained in PE. Circ_0004904 silencing contributed to cell proliferation, migration, and invasion, but restrained cell apoptosis in trophoblast cells. Further, miR-19b-3p was a target of circ_0004904, and miR-19b-3p could target ARRDC3. Additionally, circ_0004904 accelerated PE evolution via changing ARRDC3 level by binding to miR-19b-3p. In all, circ_0004904 encouraged PE progress via miR-19b-3p/ARRDC3 axis.

Association of ARRDC3 and NFIA variants with bovine congestive heart failure in feedlot cattle.

Background: Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease. Methods: A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar's test. Results: Two independent genomic regions were identified as having the most significant association with BCHF: the arrestin domain-containing protein 3 gene ( ARRDC3), and the nuclear factor IA gene ( NFIA, mid- p-values, 1x10 -8 and 2x10 -7, respectively). Animals with two copies of risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates with either one or zero risk alleles at both genes (CI 95 = 3-17). Further, animals with two copies of risk alleles at both genes were 28-fold more likely to have BCHF than all others ( p-value = 1×10 -7, CI 95 = 4-206). A missense variant in ARRDC3 (C182Y) represents a potential functional variant since the C182 codon is conserved among all other jawed vertebrate species observed. A two-SNP test with markers in both genes showed 29% of 273 BCHF cases had homozygous risk genotypes in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This and other DNA tests may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here. Conclusions: Although pathogenic roles for variants in the ARRDC3 and NFIA genes are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management.

Thioredoxin-Interacting Protein in Cancer and Diabetes.

Significance: Thioredoxin-interacting protein (Txnip) is an α-arrestin protein that acts as a cancer suppressor. Txnip is simultaneously a critical regulator of energy metabolism. Other alpha-arrestin proteins also play key roles in cell biology and cancer. Recent Advances: Txnip expression is regulated by multilayered mechanisms, including transcriptional regulation, microRNA, messenger RNA (mRNA) stabilization, and protein degradation. The Txnip-based connection between cancer and metabolism has been widely recognized. Meanwhile, new aspects are proposed for the mechanism of action of Txnip, including the regulation of RNA expression and autophagy. Arrestin domain containing 3 (ARRDC3), another α-arrestin protein, regulates endocytosis and signaling, whereas ARRDC1 and ARRDC4 regulate extracellular vesicle formation. Critical Issues: The mechanism of action of Txnip is yet to be elucidated. The regulation of intracellular protein trafficking by arrestin family proteins has opened an emerging field of biology and medical research, which needs to be examined further. Future Directions: A fundamental understanding of the mechanism of action of Txnip and other arrestin family members needs to be explored in the future to combat diseases such as cancer and diabetes. Antioxid. Redox Signal. 36, 1001-1022.

Upregulated ARRDC3 limits trophoblast cell invasion and tube formation and is associated with preeclampsia.

OBJECTIVE: Bioinformatics analysis indicated that the arrestin ARRDC3 was upregulated in placental tissue from patients with preeclampsia (PE). The study aimed to confirm the finding by examining placenta samples from women with and without early-onset PE and to investigate ARRDC3 roles in trophoblast function. METHODS: ARRDC3 expression level and localization in placental tissue were determined by Western blot, real-time quantitative PCR and immunohistochemistry. An in vitro hypoxia and an in vitro ischemia (hypoxia/reoxygenation) cell models were used to determine the hypoxic and ischemic effects on ARRDC3 expression in extravillous trophoblast-derived HTR/8SVneo cells and trophoblast cell activity. The role of ARRDC3 in HTR8/SVneo cell proliferation, invasion and tube formation in vitro was investigated by testing the effects of ARRDC3 gene overexpression or siRNA-based gene silencing. RESULTS: ARRDC3 expression was significantly elevated in placental tissue from women with early-onset PE compared to preterm birth pregnancies. ARRDC3 protein was localized in human placental trophoblasts. Hypoxia and ischemia both enhanced ARRDC3 protein expression in HTR8/SVneo cells. Hypoxia altered trophoblast cell activities. Overexpression of ARRDC3 in HTR8/SVneo cells suppressed cell invasion and tube formation. ARRDC3 gene silencing, by contrast, promoted invasion and tube formation under hypoxic conditions. CONCLUSION: ARRDC3 was highly expressed in placental tissues of PE patients and directly affected biological activities of trophoblasts under hypoxic conditions. In regulation of ARRDC3- protein expression, ischemia (hypoxia/reoxygenation) are also important. These findings suggest that ARRDC3 may play a clinically significant role in the pathogenesis of PE.

Methylation and serum response factor mediated in the regulation of gene ARRDC3 in breast cancer.

Breast cancer poses a serious threat to women's life and health and many factors contribute to breast cancer including gene mutation and epigenetics. Gene ARRDC3 was usually repressed in breast cancer and methylation in promoter was reported to be involved in gene ARRDC3 expression regulation. To this end, the methylation status for gene ARRDC3 promoter was assayed by the Massarray quantitative method. The results indicated that different methylation level CpG sites including CpG_6, CpG_13.14, CpG_17.18, and CpG_25 existed between the tumor tissue and the adjacent normal tissue. In order to further verify whether methylation participated in gene ARRDC3 expression, three cell lines were treated with methylation inhibitor Aza-2'-deoxycytidine including A-375, HepG2, and MDA-MB-231. The results revealed that methylation inhibition observably increased ARRDC3 mRNA expression. Then we confirmed the effective length of promoter through the fluorescence report assay used for further analysis. The results showed that the 1746 bp length promoter produced the maximum fluorescence signal. To obtain the direct evidence that methylation in gene ARRDC3 promoter mediated in ARRDC3 expression regulation, the promoter plasmid was methylated by M.SssI enzyme and subjected to the fluorescence report assay. The results showed that methylation in the promoter markedly suppressed relative luciferase activity. In addition, the ecRNA was also analyzed for the methylation regulation and results illustrated that the ecRNA did not regulate ARRDC3 promoter methylation. However, several methylation CpG sites were found to be around CpG_25 site such as TGCATGG, TTGCAA, TTCGTA, and ATAGTT. These sites provide a good clue for further research in methylation for gene ARRDC3 expression regulation. Furthermore, the possible transcription factors involved in the ARRDC3 regulation were investigated by western blot, luciferase activity analysis and ChiP assay. These results documented that gene ARRDC3 expression was improved by SRF and that the methylation affected the interaction between the promoter and SRF. Lastly, the inhibition role of gene ARRDC3 on breast cancer was probed in vivo and in vitro and our results demonstrated that ARRDC3 could inhibit breast cancer growth through the STAT3 signal pathway. In summary, Gene ARRDC3 was inhibited by promoter methylation and was promoted by transcription factor SRF by binding the promoter region and the inhibition on breast cancer growth was exerted by ARRDC3 through STAT3 signal pathway.

Therapeutic Potential of Chemically Modified miR-489 in Triple-Negative Breast Cancers.

Triple-negative breast cancers (TNBCs) lack ER, PR and her2 receptors that are targets of common breast cancer therapies with poor prognosis due to their high rates of metastasis and chemoresistance. Based on our previous studies that epigenetic silencing of a potential metastasis suppressor, arrestin domain-containing 3 (ARRDC3), is linked to the aggressive nature of TNBCs, we identified a sub-group of tumor suppressing miRNAs whose expressions were significantly up-regulated by ARRDC3 over-expression in TNBC cells. Among these tumor suppressing miRs, we found that miR-489 is most anti-proliferative in TNBC cells. miR-489 also blocked DNA damaging responses (DDRs) in TNBC cells. To define the mechanism by which miR-489 inhibits TNBC cell functions, we screened the potential target genes of miR-489 and identified MDC-1 and SUZ-12 as novel target genes of miR-489 in TNBC cells. To further exploit the therapeutic potentials of miR-489 in TNBC models, we chemically modified the guide strand of miR-489 (CMM489) by replacing Uracil with 5-fluorouracil (5-FU) so that tumor suppressor (miR-489) and DNA damaging (5-FU) components are combined into a single agent as a novel drug candidate for TNBCs. Our studies demonstrated that CMM489 shows superior effects over miR-489 or 5-FU in inhibition of TNBC cell proliferation and tumor progression, suggesting its therapeutic efficacy in TNBC models.

Arrestin Domain Containing 3 Reverses Epithelial to Mesenchymal Transition and Chemo-Resistance of TNBC Cells by Up-Regulating Expression of miR-200b.

Our previous studies demonstrated the importance of arrestin domain containing 3 (ARRDC3), a metastasis suppressor, in inhibiting invasive and metastatic potential of triple negative breast cancer (TNBC) in vitro and in vivo. However, little is known about ARRDC3 mediated transcriptional control and its target genes that are implicated in its metastatic suppressing activity. In this study, we used miRNA array and subsequent functional analyses to identify miRNAs whose expression are significantly regulated by ARRDC3 in TNBC cells. We identified miR-200b as a major target gene of ARRDC3. miR-200b played an essential role in mediating ARRDC3 dependent reversal of EMT phenotypes and chemo-resistance to DNA damaging agents in TNBC cells. Expression of miR-200b also increased the expression of ARRDC3 as well in TNBC cells, suggesting a positive feedback loop between these two molecules. In addition, we combined the therapeutic powers of miR-200b and 5-fluorourancil (5-FU) into a single compound (5-FU-miR-200b) to maximize the synergistic effects of these compounds. Chemically modified miR-200b (5-FU-miR-200b mimic) was more effective in inhibiting metastatic potentials of TNBC cells than unmodified miR-200b and does not require transfection reagents, implying its therapeutic potential in TNBC. Our studies showed the importance of therapeutic targeting ARRDC3/miR-200b pathway in TNBC.

ARRDC3 suppresses colorectal cancer progression through destabilizing the oncoprotein YAP.

Although colorectal cancer (CRC) is a prevalent malignancy of the digestive system, the underlying mechanisms of CRC tumorigenesis are still elusive. Arrestin-related domain-containing protein-3 (ARRDC3) has been reported to promote lysosome-mediated protein degradation. In the present study, we find that the expression of ARRDC3 is downregulated in CRC specimens. Mechanistically, we reveal that ARRDC3 binds and decreases expression of the oncoprotein YAP, the cotranscription factor of the Hippo pathway. The regulation of the Hippo pathway by ARRDC3 is conserved from Drosophila to mammals. Furthermore, we demonstrate that ARRDC3 plays an anti-oncogenic role in CRC progression by promoting YAP degradation. Finally, we show that ARRDC3 increases the sensitivity of CRC cells toward chemotherapeutic drugs. Taken together, our findings point to ARRDC3 as a potential target for CRC treatment.

ARRDC1 and ARRDC3 act as tumor suppressors in renal cell carcinoma by facilitating YAP1 degradation.

The α-arrestins domain-containing 1 and 3 (ARRDC1 and ARRDC3) are two members of the α-arrestins family. Yes-associated protein 1 (YAP1) is a key downstream transcription co-activator of the Hippo pathway essential for cancer initiation, progression, or metastasis in clear cell renal cell carcinoma (ccRCC). The aim of this work was to elucidate the role of the α-arrestins in ccRCC tumorigenesis by identifying molecular interacting factors and exploring potential mechanisms. In this study, we identified YAP1 as a novel ARRDC3 interacting protein in RCC cells through tandem affinity purification and mass spectrometry. We confirmed that ARRDC1 and ARRDC3, but not other α-arrestin family proteins, interact with YAP1. Binding of ARRDC1/3 to YAP1 is mediated through the WW domains of YAP1 and the PPXY motifs of ARRDC1/3. Functional analysis of ARRDC1/3 by lentiviral shRNA revealed a role for ARRDC1/3 in suppression of cell growth, migration, invasion and epithelial-mesenchymal transition in ccRCC cells, and these effects were mediated, at least in part, through YAP1. Mechanically, ARRDC1/3 negatively regulates YAP1 protein stability by facilitating E3 ubiquitin ligase Itch-mediated ubiquitination and degradation of YAP1. Moreover, ARRDC1/3 mRNA levels were significantly downregulated in ccRCC specimens. A negative correlation was identified between ARRDC3 and YAP1 expression in ccRCC specimens by immunohistochemistry. This study revealed a novel mechanism for ARRDC1/3 in the regulation of YAP1 stability and provided insight in understanding the relationship between ARRDC1/3 downregulation and aberrant Hippo-YAP1 pathway activation in ccRCC.