Expression of RASSF1A, DIRAS3, and AKAP9 Genes in Thyroid Lesions: Implications for Differential Diagnosis and Prognosis of Thyroid Carcinomas.

Thyroid carcinoma is the primary endocrine malignancy worldwide. The preoperative examination of thyroid tissue lesion is often unclear. Approximately 25% of thyroid cancers cannot be diagnosed definitively without post-surgery histopathological examination. The assessment of diagnostic and differential markers of thyroid cancers is needed to improve preoperative diagnosis and reduce unnecessary treatments. Here, we assessed the expression of RASSF1A, DIRAS3, and AKAP9 genes, and the presence of BRAF V600E point mutation in benign and malignant thyroid lesions in a Polish cohort (120 patients). We have also performed a comparative analysis of gene expression using data obtained from the Gene Expression Omnibus (GEO) database (307 samples). The expression of RASSF1A and DIRAS3 was decreased, whereas AKAP9's was increased in pathologically changed thyroid compared with normal thyroid tissue, and significantly correlated with e.g., histopathological type of lesion papillary thyroid cancer (PTC) vs follicular thyroid cancer (FTC), patient's age, tumour stage, or its encapsulation. The receiver operating characteristic (ROC) analysis for the more aggressive FTC subtype differential marker suggests value in estimating RASSF1A and AKAP9 expression, with their area under curve (AUC), specificity, and sensitivity at 0.743 (95% CI: 0.548-0.938), 82.2%, and 66.7%; for RASSF1A, and 0.848 (95% CI: 0.698-0.998), 54.8%, and 100%, for AKAP9. Our research gives new insight into the basis of the aggressiveness and progression of thyroid cancers, and provides information on potential differential markers that may improve preoperative diagnosis.

DIRAS3 regulates autophagy in an endometriosis epithelial cell line.

RESEARCH QUESTION: What is the role of DIRAS3 in endometriosis pathogenesis? DESIGN: Prospective patient cohort study combined with experiments in the 12Z human endometriosis epithelial cell line model to determine the role of DIRAS3 in endometriosis. Endometrium and endometriosis lesion samples were collected from premenopausal women from 24 control and 40 endometriosis patients by laparoscopic surgery. The role of DIRAS3 in endometriosis was assessed by siRNA knockdown in 12Z cells followed by proliferation, apoptosis, invasion and autophagy assays. Autophagy was induced by serum starvation and the levels of autophagy determined by assessing changes in the expression levels and localization of autophagy marker proteins, such as LC3. RESULTS: DIRAS3 mRNA showed a large increase in expression in ectopic endometriosis lesions compared with endometrium from control patients, with expression largely localized to the epithelium. DIRAS3 knockdown in 12Z endometriosis epithelial cells caused a significant reduction in the number of proliferating cells (1.6-fold, adjusted P = 0.0007) and increased apoptosis (AnnexinV/7AAD double-positive cells +48%, P = 0.01), indicating an effect on cell proliferation. Induction of autophagy by serum starvation caused significant upregulation in DIRAS3 expression after 24 h (mRNA +2.4-fold [adjusted P = 0.017], protein +8.1-fold (adjusted P = 0.029), reduced LC3I/LC3II ratio (-2.2-fold, adjusted P = 0.044) and an increase in the number of double positive LC3/DIRAS3 puncta (+2.3-fold, P = 0.02). Knockdown of DIRAS3 in serum-starved cells led to a reduction in autophagy, indicated by an overall decrease in LC3 expression and significant increase in LC3I/LC3II ratio. CONCLUSIONS: DIRAS3 is highly upregulated in endometriosis lesions. Studies in an endometriosis epithelial cell line indicate that DIRAS3 facilitates cell survival in this context by inducing autophagy.

Five crucial prognostic-related autophagy genes stratified female breast cancer patients aged 40-60 years.

BACKGROUND: Autophagy is closely related to the progression of breast cancer. The aim at this study is to establish a prognostic-related model comprised of hub autophagy genes (AGs) to assess patient prognosis. Simultaneously, the model can guide clinicians to make up individualized strategies and stratify patients aged 40-60 years based on risk level. METHODS: The hub AGs were identified with univariate COX regression and LASSO regression. The functions and alterations of these selected AGs were analyzed as well. Moreover, the multivariate COX regression and correlation analysis between hub AGs and clinicopathological parameters were done. RESULTS: Totally, 33 prognostic-related AGs were obtained from the univariate COX regression (P < 0.05). SERPINA1, HSPA8, HSPB8, MAP1LC3A, and DIRAS3 were identified to constitute the prognostic model by the LASSO regression. The survival curve of patients in the high-risk and low-risk groups was statistically significant (P < 0.05). The 3-year and 5-year ROC displayed that their AUC value reached 0.762 and 0.825, respectively. Stage and risk scores were independent risk factors relevant to prognosis. RB1CC1, RPS6KB1, and BIRC6 were identified as the most predominant mutant genes. It was found that AGs were mainly involved in regulating the endopeptidases synthesis and played important roles in the ErbB signal pathway. SERPIN1, risk score was closely related to the stage (P < 0.05); HSPA8, risk score were closely related to T stag (P < 0.05); HSPB8 was closely related to N stag (P < 0.05). CONCLUSIONS: Our prognostic model had the relatively robust predictive ability on prognosis for patients aged 40-60 years. If the stage was added into the prognostic model, the predictive ability would be more powerful.

Elimination of dormant, autophagic ovarian cancer cells and xenografts through enhanced sensitivity to anaplastic lymphoma kinase inhibition.

BACKGROUND: Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. METHODS: By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. RESULTS: Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. CONCLUSIONS: Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.

Oncogenic DIRAS3 promotes malignant phenotypes of glioma by activating EGFR-AKT signaling.

Epidermal growth factor receptor (EGFR)-Akt signaling cascade activation plays a pivotal role in gliomas malignant phenotype, especially in Classical and Mesenchymal subtype gliomas. However, the molecules and mechanisms underlying regulate and maintain the activation of EGFR-AKT signaling remains unclear. Previously reports showed that DIRAS3 inhibits cell proliferation and induces autophagy in ovarian, breast, lung and prostate cancers, which is heterozygosity loss or down-regulated in aforementioned cancers and functionally as a tumor suppressor, whereas the role of DIRAS3 in glioma is still veiled. Here, in this study, we investigated the biological function and role of DIRAS3 in gliomas, and found that DIRAS3 is up-regulated in gliomas and is positively correlated with poor prognosis of glioma patients, meanwhile, over-expressed DIRAS3 promotes glioma cells proliferation and invasion. Further mechanistic study showed that the expression level of DIRAS3 in Classical and Mesenchymal subtype GBMs is higher, and over-expression of DIRAS3 promotes EGFR-AKT signaling activation at the downstream of EGFR and increases AKT phosphorylation, meanwhile suppression of AKT by MK-2206 reverses the tumor promoting function of DIRAS3. Taken together, these findings reveal a novel oncogenic role of DIRAS3 in the development and progression of glioma, which suggest that DIRAS3 could serve as a potential diagnostic marker and a promising therapeutic target of gliomas.

DNA methyltransferase inhibitors influence on the DIRAS3 and STAT3 expression and in vitro migration of ovarian and breast cancer cells.

OBJECTIVES: Downregulation of DIRAS3 (DIRAS family, GTP-binding Ras-like 3) is related to ovarian and breast cancer progression. A possible mechanism that silences this gene is the promoter region DNA methylation. The potential reversibility of this epigenetic mechanism makes it more attractive candidate for new mode of cancer treatment. DIRAS3 regulates cell cycle, tumor dormancy and inhibits cancer cell growth and motility, all of which may indirectly depend on interaction with STAT3 (Signal Transducer and Activator of Transcription 3) classified as a potential oncogene. The restoration of DIRAS3 expression could inhibit cell proliferation and invasiveness. MATERIAL AND METHODS: Human ovarian carcinoma cell line (A2780) and human breast cancer cell line (MCF7) were exposed to two DNA methyltransferase inhibitors (DNMTi): decitabine (5-aza-2'-deoxycytidine) [25 µM and 12.5 µM] and RG108 [150 µM and 100 µM]. In vitro migration changes of cancer cells were examined with wound healing assay. After 7 days of DNMTi treatment cells were harvested and DNA and RNA was isolated. The methylation status of the promoter sequences of DIRAS3 and STAT3 genes was determined using methylation specific PCR (MS-PCR). Level of target genes' expression was quantified using quantitative reverse transcription PCR (QRT-PCR). RESULTS AND CONCLUSIONS: The in vitro wound healing assay showed changes in the migration rate of both adherent cell lines after DNMTi treatment compared to the untreated cells. Relative balance between methylated and unmethylated variants of DIRAS3 after MS-PCR was shifted towards unmethylated version after DNMTi treatment in A2780 cells. Statistically significant dose dependent effect of decitabine and RG108 on DIRAS3 expression in A2780 cells was observed.

DIRAS3 induces autophagy and enhances sensitivity to anti-autophagic therapy in KRAS-driven pancreatic and ovarian carcinomas.

Pancreatic ductal adenocarcinoma (PDAC) and low-grade ovarian cancer (LGSOC) are characterized by the prevalence of KRAS oncogene mutations. DIRAS3 is the first endogenous non-RAS protein that heterodimerizes with RAS, disrupts RAS clustering, blocks RAS signaling, and inhibits cancer cell growth. Here, we found that DIRAS3-mediated KRAS inhibition induces ROS-mediated apoptosis in PDAC and LGSOC cells with KRAS mutations, but not in cells with wild-type KRAS, by downregulating NFE2L2/Nrf2 transcription, reducing antioxidants, and inducing oxidative stress. DIRAS3 also induces cytoprotective macroautophagy/autophagy that may protect mutant KRAS cancer cells from oxidative stress, by inhibiting mutant KRAS, activating the STK11/LKB1-PRKAA/AMPK pathway, increasing lysosomal CDKN1B/p27 localization, and inducing autophagic gene expression. Treatment with chloroquine or the novel dimeric chloroquine analog DC661 significantly enhances DIRAS3-mediated inhibition of mutant KRAS tumor cell growth in vitro and in vivo. Taken together, our study demonstrates that DIRAS3 plays a critical role in regulating mutant KRAS-driven oncogenesis in PDAC and LGSOC.Abbreviations: AFR: autophagic flux reporter; ATG: autophagy related; CQ: chloroquine; DCFDA: 2'-7'-dichlorodihydrofluorescein diacetate; DIRAS3: DIRAS family GTPase 3; DOX: doxycycline; KRAS: KRAS proto-oncogene, LGSOC: low-grade serous ovarian cancer; MiT/TFE: microphthalmia family of transcription factors; NAC: N-acetylcysteine; PDAC: pancreatic ductal adenocarcinoma; ROS: reactive oxygen species; TFEB: transcription factor EB.

DIRAS3, GPR171 and RAC2 were identified as the key molecular patterns associated with brain metastasis of breast cancer.

Objective: Brain metastasis is a primary cause of morbidity and mortality in breast cancer patients. Therefore, elucidation and understanding of the underlying mechanisms are essential for the development of new therapeutic strategies. Methods: Differential gene analysis was performed for those with and without distant metastasis in The Cancer Genome Atlas (TCGA) database and those with and without recurrence in the brain in the dataset GSE12276. The differentially expressed genes procured from the two databases were intersected to obtain the intersecting genes associated with brain metastasis. Thereafter, the intersecting genes were subjected to LASSO model construction to screen for prognostic genes. The expression of the obtained genes in metastatic breast cancer was observed, and survival analysis was performed. Finally, GSEA analysis of the obtained genes was performed, and the relationship between them and immune cells was explored. Results: A total of 335 differential genes for the occurrence of distant metastases were obtained based on the TCGA database. A total of 1070 differential genes for recurrence to the brain were obtained based on the dataset GSE12276. The Venn diagram showed 24 intersecting genes associated with brain metastasis. The LASSO prognostic model contained a total of five genes (GBP2, GPR171, DIRAS3, RAC2, and CACNA1D). Expression difference analysis showed that GBP2, GPR171, DIRAS3, and RAC2 were significantly down-regulated in expression in metastatic breast cancer compared with primary breast cancer tumors. Only GPR171, DIRAS3, and RAC2 were strongly correlated with the overall survival of breast cancer patients. Their correlation analysis with immune cells showed that the correlation coefficient between the expression levels of DIRAS3 and immune cells was low, and the expression levels of GPR171 and RAC2 were more closely correlated with B cells and macrophages. Conclusions: The expression of DIRAS3, GPR171 and RAC2, genes associated with brain metastasis, was reduced in metastatic breast cancer, and GPR171 was found to promote brain metastasis of breast cancer cells by inducing B cells and thereby.

Resveratrol Contrasts IL-6 Pro-Growth Effects and Promotes Autophagy-Mediated Cancer Cell Dormancy in 3D Ovarian Cancer: Role of miR-1305 and of Its Target ARH-I.

Tumor dormancy is the extended period during which patients are asymptomatic before recurrence, and it represents a difficult phenomenon to target pharmacologically. The relapse of tumors, for instance arising from the interruption of dormant metastases, is frequently observed in ovarian cancer patients and determines poor survival. Inflammatory cytokines present in the tumor microenvironment likely contribute to such events. Cancer cell dormancy and autophagy are interconnected at the molecular level through ARH-I (DIRAS3) and BECLIN-1, two tumor suppressors often dysregulated in ovarian cancers. IL-6 disrupts autophagy in ovarian cancer cells via miRNAs downregulation of ARH-I, an effect contrasted by the nutraceutical protein restriction mimetic resveratrol (RV). By using three ovarian cancer cell lines with different genetic background in 2D and 3D models, the latter mimicking the growth of peritoneal metastases, we show that RV keeps the cancer cells in a dormant-like quiescent state contrasting the IL-6 growth-promoting activity. Mechanistically, this effect is mediated by BECLIN-1-dependent autophagy and relies on the availability of ARH-I. We also show that ARH-I (DIRAS3) is a bona fide target of miR-1305, a novel oncomiRNA upregulated by IL-6 and downregulated by RV. Clinically relevant, bioinformatic analysis of a transcriptomic database showed that the high expression of DIRAS3 and MAP1LC3B mRNAs together with that of CDKN1A, directing a cellular dormant phenotype, predicts better overall survival in ovarian cancer patients, and this correlates with MIR1305 downregulation. The possibility of maintaining a permanent cell dormancy in ovarian cancer by the chronic administration of RV should be considered as a therapeutic option to prevent the "awakening" of cancer cells in response to a permissive microenvironment, thus limiting the risk of tumor relapse and metastasis.

GTP-binding protein Di-RAS3 diminishes the migration and invasion of non-small cell lung cancer by inhibiting the RAS/extracellular-regulated kinase pathway.

The GTP-binding protein Di-Ras3 (DIRAS3) has been established as a maternally imprinted tumor suppressor gene. Growing evidence has correlated the DIRAS3 gene with tumor progression, but its role in non-small cell lung cancer (NSCLC) is rarely reported. Accordingly, the current study sought to evaluate the role and mechanism of DIRAS3 in NSCLC cell progression. First, we uncovered that DIRAS3 was poorly expressed in NSCLC tissues and cells. Subsequently, we examined the effect of DIRAS3 over-expression or knockdown in different lung cancer cells on their malignant phenotypes, with the help of transwell cell migration and invasion assays, and Western blot analyses. It was found that the over-expression of DIRAS3 inhibited the migration and invasion of A549 cells or H520 cells, whereas knockdown of DIRAS3 led to opposing trends. In addition, over-expression of DIRAS3 attenuated the tumor growth and reduced the number of lung tumor nodules. Mechanistically, DIRAS3 may inhibit the migration and invasion of NSCLC cells by inhibiting the RAS/extracellular-regulated kinase (ERK) signaling pathway. Collectively, our findings indicate that DIRAS3 could serve as a potential therapeutic target biomarker for NSCLC.

Shensu IV prevents glomerular podocyte injury in nephrotic rats via promoting lncRNA H19/DIRAS3-mediated autophagy.

Shensu IV is a Chinese prescription well-known for its function in treating chronic kidney diseases. However, the potential mechanisms underlying how Shensu IV exerts its effects remain unclear. In the present study, we investigated the effects of Shensu IV on glomerular podocyte injury in nephrotic rats and puromycin-induced injury in cultured podocytes, and assessed the associated molecular mechanisms. Liquid chromatography-mass spectrometry (LC-MS) results showed that the main components of Shensu IV were l-Carnitine, P-lysoPC (LPC) 16:0, Coumaroyl tyramine, Tetramethylpyrazine, LPC 18:1, Choline, (S,S)-Butane-2,3-diol, and Scopoletin. We further found that nephrotic rats displayed pathological alterations in kidney tissues and ultrastructural changes in glomerular podocytes; however, these effects were reversed with Shensu IV treatment. Compared with the control, the numbers of autophagosomes were markedly reduced in the model group, but not in the Shensu IV treatment group. Furthermore, the expression of p62 was significantly higher in the model group than in the controls, whereas the LC3-II/I ratio was significantly lower; however, these changes were not observed when Shensu IV was administered. The protective effects of Shensu IV were further confirmed in podocytes displaying puromycin-induced injury. Compared with control group, the expression of long non-coding RNA (lncRNA) H19, mTOR, p-mTOR, and p62 was significantly increased in the puromycin group, whereas that of distinct subgroup of the RAS family member 3 (DIRAS3) was significantly decreased, as was the LC3-II/I ratio. The opposite results were obtained for both shH19- and Shensu IV-treated cells. Collectively, our data demonstrated that Shensu IV can prevent glomerular podocyte injury in nephrotic rats and puromycin-treated podocytes, likely via promoting lncRNA H19/DIRAS3-regulated autophagy.

Genomic Imprinting at the Porcine DIRAS3 Locus.

The epigenetic mechanisms underlying genomic imprinting include DNA methylation and monoallelic expression of genes in close proximity. Although genes imprinted in humans and mice have been widely characterized, there is a lack of detailed and comprehensive studies in livestock species including pigs. The purpose of this study was to investigate a detailed methylation status and parent-of-origin-specific gene expression within the genomic region containing an underexamined porcine DIRAS3 locus. Through whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) of porcine parthenogenetic embryos and analyses of public RNA-seq data from adult pigs, DNA methylation and monoallelic expression pattern were investigated. As a result, maternal hypermethylation at the DIRAS3 locus and hypothalamus-specific and monoallelic expression of the DIRAS3 gene were found in pigs. In conclusion, the findings from this study suggest that the presence of maternal hypermethylation, or imprints, might be maintained and related to monoallelic expression of DIRAS3 during pig development.

Re-expression of DIRAS3 and p53 induces apoptosis and impaired autophagy in head and neck squamous cell carcinoma.

BACKGROUND: p53 and DIRAS3 are tumor suppressors that are frequently silenced in tumors. In this study, we sought to determine whether the concurrent re-expression of p53 and DIRAS3 could effectively induce head and neck squamous cell carcinoma (HNSCC) cell death. METHODS: CAL-27 and SCC-25 cells were treated with Ad-DIRAS3 and rAd-p53 to induce re-expression of DIRAS3 and p53 respectively. The effects of DIRAS3 and p53 re-expression on the growth and apoptosis of HNSCC cells were examined by TUNEL assay, flow cytometric analysis and MTT. The effects of DIRAS3 and p53 re-expression on Akt phosphorylation, oncogene expression, and the interaction of 4E-BP1 with eIF4E were determined by real-time PCR, Western blotting and immunoprecipitation analysis. The ability of DIRAS3 and p53 re-expression to induce autophagy was evaluated by transmission electron microscopy, LC3 fluorescence microscopy and Western blotting. The effects of DIRAS3 and p53 re-expression on HNSCC growth were evaluated by using an orthotopic xenograft mouse model. RESULTS: TUNEL assay and flow cytometric analysis showed that the concurrent re-expression of DIRAS3 and p53 significantly induced apoptosis (P < 0.001). MTT and flow cytometric analysis revealed that DIRAS3 and p53 re-expression significantly inhibited proliferation and induced cell cycle arrest (P < 0.001). Mechanistically, the concurrent re-expression of DIRAS3 and p53 down-regulated signal transducer and activation of transcription 3 (STAT3) and up-regulated p21WAF1/CIP1 and Bax (P < 0.001). DIRAS3 and p53 re-expression also inhibited Akt phosphorylation, increased the interaction of eIF4E with 4E-BP1, and reduced the expression of c-Myc, cyclin D1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor receptor (EGFR) and Bcl-2 (P < 0.001). Moreover, the concurrent re-expression of DIRAS3 and p53 increased the percentage of cells with GFP-LC3 puncta compared with that in cells treated with control adenovirus (50.00% ± 4.55% vs. 4.67% ± 1.25%, P < 0.001). LC3 fluorescence microscopy and Western blotting further showed that DIRAS3 and p53 re-expression significantly promoted autophagic activity but also inhibited autophagic flux, resulting in overall impaired autophagy. Finally, the concurrent re-expression of DIRAS3 and p53 significantly decreased the tumor volume compared with the control group in a HNSCC xenograft mouse model [(3.12 ± 0.75) mm3 vs. (189.02 ± 17.54) mm3, P < 0.001]. CONCLUSIONS: The concurrent re-expression of DIRAS3 and p53 is a more effective approach to HNSCC treatment than current treatment strategies.

Amino Acid Deprivation-Induced Autophagy Requires Upregulation of DIRAS3 through Reduction of E2F1 and E2F4 Transcriptional Repression.

Failure to cure ovarian cancer relates to the persistence of dormant, drug-resistant cancer cells following surgery and chemotherapy. "Second look" surgery can detect small, poorly vascularized nodules of persistent ovarian cancer in ~50% of patients, where >80% are undergoing autophagy and express DIRAS3. Autophagy is one mechanism by which dormant cancer cells survive in nutrient poor environments. DIRAS3 is a tumor suppressor gene downregulated in >60% of primary ovarian cancers by genetic, epigenetic, transcriptional and post-transcriptional mechanisms, that upon re-expression can induce autophagy and dormancy in a xenograft model of ovarian cancer. We examined the expression of DIRAS3 and autophagy in ovarian cancer cells following nutrient deprivation and the mechanism by which they are upregulated. We have found that DIRAS3 mediates autophagy induced by amino acid starvation, where nutrient sensing by mTOR plays a central role. Withdrawal of amino acids downregulates mTOR, decreases binding of E2F1/4 to the DIRAS3 promoter, upregulates DIRAS3 and induces autophagy. By contrast, acute amino acid deprivation did not affect epigenetic regulation of DIRAS3 or expression of miRNAs that regulate DIRAS3. Under nutrient poor conditions DIRAS3 can be transcriptionally upregulated, inducing autophagy that could sustain dormant ovarian cancer cells.

DIRAS3-Derived Peptide Inhibits Autophagy in Ovarian Cancer Cells by Binding to Beclin1.

Autophagy can protect cancer cells from acute starvation and enhance resistance to chemotherapy. Previously, we reported that autophagy plays a critical role in the survival of dormant, drug resistant ovarian cancer cells using human xenograft models and correlated the up-regulation of autophagy and DIRAS3 expression in clinical samples obtained during "second look" operations. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26 kD GTPase with homology to RAS that inhibits cancer cell proliferation and motility. Re-expression of DIRAS3 in ovarian cancer xenografts also induces dormancy and autophagy. DIRAS3 can bind to Beclin1 forming the Autophagy Initiation Complex that triggers autophagosome formation. Both the N-terminus of DIRAS3 (residues 15-33) and the switch II region of DIRAS3 (residues 93-107) interact directly with BECN1. We have identified an autophagy-inhibiting peptide based on the switch II region of DIRAS3 linked to Tat peptide that is taken up by ovarian cancer cells, binds Beclin1 and inhibits starvation-induced DIRAS3-mediated autophagy.

DIRAS3 (ARHI) Blocks RAS/MAPK Signaling by Binding Directly to RAS and Disrupting RAS Clusters.

Oncogenic RAS mutations drive cancers at many sites. Recent reports suggest that RAS dimerization, multimerization, and clustering correlate strongly with activation of RAS signaling. We have found that re-expression of DIRAS3, a RAS-related small GTPase tumor suppressor that is downregulated in multiple cancers, inhibits RAS/mitogen-activated protein kinase (MAPK) signaling by interacting directly with RAS-forming heteromers, disrupting RAS clustering, inhibiting Raf kinase activation, and inhibiting transformation and growth of cancer cells and xenografts. Disruption of K-RAS cluster formation requires the N terminus of DIRAS3 and interaction of both DIRAS3 and K-RAS with the plasma membrane. Interaction of DIRAS3 with both K-RAS and H-RAS suggests a strategy for inhibiting oncogenic RAS function.

Distinct subgroup of the Ras family member 3 (DIRAS3) expression impairs metastasis and induces autophagy of gastric cancer cells in mice.

PURPOSE: Distinct subgroup of the Ras family member 3 (DIRAS3), also called Aplasia Ras homolog member I, is a tumor suppressor gene that induces autophagy in several cancer cell lines. METHODS: This study analyzed DIRAS3, and markers of autophagy (p62, and LC3B-II) in surgically resected GC samples from 420 patients. The promotion of autophagy by DIRAS3 in gastric cancer (GC) cells was explored, which might explain its inhibitory role in gastric cancer cells. RESULTS: DIRAS3 expression in GC was positively correlated with LC3B-II amount, and negatively with metastasis; DIRAS3 and p62 levels were independent prognostic factors in GC. Overexpression of DIRAS3 in BGC-823 cells induced autophagy, led to decreased proliferation, cell cycle arrest in G0/G1 phase, increased apoptosis, and impaired migration and invasion. While knockdown of DIRAS3 promoted proliferation and migration in MKN-45 cells. Overexpression of DIRAS3 in BGC-823 cells elevated autophagy levels in subcutaneous xenograft and inhibited tumor growth in mice; the hematogenous liver and lung metastasis of cancer cells were also suppressed. CONCLUSIONS: In conclusion, the results suggest DIRAS3 may play a role in affecting proliferation and metastatic potential of GC cells, which may be associated with its involvement in autophagy regulation.

RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells.

Among the 3 GTPases in the DIRAS family, DIRAS3/ARHI is the best characterized. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26-kDa GTPase that shares 60% homology to RAS and RAP. DIRAS3 is downregulated in many tumor types, including ovarian cancer, where re-expression inhibits cancer cell growth, reduces motility, promotes tumor dormancy and induces macroautophagy/autophagy. Previously, we demonstrated that DIRAS3 is required for autophagy in human cells. Diras3 has been lost from the mouse genome during evolutionary re-arrangement, but murine cells can still undergo autophagy. We have tested whether DIRAS1 and DIRAS2, which are homologs found in both human and murine cells, could serve as surrogates to DIRAS3 in the murine genome affecting autophagy and cancer cell growth. Similar to DIRAS3, these 2 GTPases share 40-50% homology to RAS and RAP, but differ from DIRAS3 primarily in the lengths of their N-terminal extensions. We found that DIRAS1 and DIRAS2 are downregulated in ovarian cancer and are associated with decreased disease-free and overall survival. Re-expression of these genes suppressed growth of human and murine ovarian cancer cells by inducing autophagy-mediated cell death. Mechanistically, DIRAS1 and DIRAS2 induce and regulate autophagy by inhibition of the AKT1-MTOR and RAS-MAPK signaling pathways and modulating nuclear localization of the autophagy-related transcription factors FOXO3/FOXO3A and TFEB. Taken together, these data suggest that DIRAS1 and DIRAS2 likely serve as surrogates in the murine genome for DIRAS3, and may function as a backup system to fine-tune autophagy in humans.

LncRNA H19 inhibits autophagy by epigenetically silencing of DIRAS3 in diabetic cardiomyopathy.

We previously generated a rat model of diabetic cardiomyopathy and found that the expression of long non-coding RNA H19 was downregulated. The present study was aimed to explore the pathogenic role of H19 in the development of diabetic cardiomyopathy. Overexpression of H19 in diabetic rats attenuated cardiomyocyte autophagy and improved left ventricular function. High glucose was found to reduce H19 expression and increase autophagy in cultured neonatal cardiomyocytes. The results of RNA-binding protein immunoprecipitation showed that H19 could directly bind with EZH2 in cardiomyocytes. The chromatin immunoprecipitation assays indicated that H19 knockdown could reduce EZH2 occupancy and H3K27me3 binding in the promoter of DIRAS3. In addition, overexpression of H19 was found to downregulate DIRAS3 expression, promote mTOR phosphorylation and inhibit autophagy activation in cardiomyocytes exposed to high glucose. Furthermore, we also found that high glucose increased DIRAS3 expression in cardiomyocytes and DIRAS3 induced autophagy by inhibiting mTOR signaling. In conclusion, our study suggested that H19 could inhibit autophagy in cardiomyocytes by epigenetically silencing of DIRAS3, which might provide novel insights into understanding the molecular mechanisms of diabetic cardiomyopathy.

Silencing of the small GTPase DIRAS3 induces cellular senescence in human white adipose stromal/progenitor cells.

Inhibition of Akt-mTOR signaling protects from obesity and extends life span in animals. In the present study, we analyse the impact of the small GTPase, GTP-binding RAS-like 3 (DIRAS3), a recently identified weight-loss target gene, on cellular senescence in adipose stromal/progenitor cells (ASCs) derived from human subcutaneous white adipose tissue (sWAT). We demonstrate that DIRAS3 knock-down (KD) in ASCs induces activation of Akt-mTOR signaling and proliferation arrest. DIRAS3 KD ASCs lose the potential to form colonies and are negative for Ki-67. Moreover, silencing of DIRAS3 results in a premature senescence phenotype. This is characterized by senescence-associated ß-galactosidase positive enlarged ASCs containing increased p16INK4A level and activated retinoblastoma protein. DIRAS3 KD ASCs form senescence-associated heterochromatic foci as shown by increased level of γ-H2A.X positive foci. Furthermore, these cells express a senescence-associated secretory phenotype characterized by increased interleukin-8 secretion. Human DIRAS3 KD ASCs develop also a senescence phenotype in sWAT of SCID mice. Finally, we show that DIRAS3 KD in ASCs stimulates both adipogenic differentiation and premature senescence. In conclusion, our data suggest that silencing of DIRAS3 in ASCs and subsequently hyper-activation of Akt-mTOR drives adipogenesis and premature senescence. Moreover, differentiating ASCs and/or mature adipocytes may acquire features of cellular senescence.