ARHI Expression Research Articles

Abstract 3717: LncRNA RNF157-AS1 inhibits autophagy in ovarian cancer cells by downregulating ARHI: The different role in proliferation and drug resistance

Abstract Ovarian cancer(OC) is the commonest cause of female cancer death. Long noncoding RNAs (lncRNAs) has been shown to have regulatory roles in the procession of many cancers, including OC. Here, in our previous study, we reported that lncRNA RNF157-AS1 was overexpression in OC tissues, which is confirmed by GSE144077 and TCGA matched GTEx normal data. Further experiments revealed that RNF157-AS1 knockdown significantly repressed the proliferation both in vitro and in vivo and overexpressed RNF157-AS1 promoted the proliferation of OC cells. However, the higher expression of RNF157-AS1 predicted a better prognosis for OC patients in TCGA database. RNA-seq analysis revealed that ARHI was significant overexpressed after RNF157-AS1 silencing and it is negative correlated with RNF157-AS1 in TCGA and matched GTEx normal data. ARHI, a autophagy-related gene, could induced autophagy in OC cells. Because of 70% of OC patients were diagnosed with advanced tumors and need to chemotherapy. Overexpression of RNF157-AS1 could inhibit autophagy-associated cell death and promote rapid proliferation of OC cells, while overexpressed RNF157-AS1 decrease autophagy and induced cells apoptosis under adverse environment. To test our hypothesis, CCK8 assay suggested overexpression of RNF157-AS1 significantly decreased the chemoresistance to cisplatin (DDP) and silencing of RNF157-AS1 increased the DDP-resistance of OC cells, which are confirmed by clone formation assay and apoptosis experiments. Western blot assay also suggested that RNF157-AS1 silencing promote ARHI, LC3II/I and beclin1 expression. To our knowledge, this is the first report revealed that the different roles of RNF157-AS1 in OC cells' proliferation and chemoresistance and our study also can provide guidance for clinical treatment of OC patients. Citation Format: Sujuan Xu, Pengfei Xu. LncRNA RNF157-AS1 inhibits autophagy in ovarian cancer cells by downregulating ARHI: The different role in proliferation and drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3717.

The Role of Methylation in the CpG Island of the ARHI Promoter Region in Cancers.

Hypermethylation can downregulate many tumor suppressor gene expressions. Aplasia Ras homologue member I (ARHI, DIRAS3) is one of the maternally imprinted tumor suppressors in the RAS superfamily. This chapter overviewed the importance of ARHI methylation and expression phenomes in various types of cancers, although the exact mechanisms remain unclear. As an imprinted gene, aberrant DNA methylation of the paternal allele of ARHI was identified as a primary inhibitor of ARHI expression. The role of methylation in the CpG islands of the ARHI promoter region vary among ovarian cancers, breast cancers, hepatocellular carcinoma, colon cancers, pancreatic cancer osteosarcoma, glial tumors, follicular thyroid carcinoma, or lung cancers. The methylation of ARHI provides a new insight to understand molecular mechanisms of tumorigenesis and progression of cancers.

Downregulation, Blood Type, Paternal Lineage, and Genetic Variants in Ovarian Cancer: A Call for Tumor Suppressor Genes Effectiveness

This article calls for a deeper look at the role of downregulation, blood type, paternal lineage, and genetic variants in ovarian cancer. While considering the importance of oncogenes, associated tumors, expression, or overexpression, signal transducers, and downregulation, it suggests that a viable platform for tumor suppressor genes effectiveness is needed for early detection. The role of DNA methylation to gene silencing, proteins, RNA alteration in the development of metastatic tissues in late stage of ovarian cancer, which require further elucidation and research, continue to suggest that methylation, and tumor suppressor genes from BRCA1 and BRCA2 mutations are exceptionally critical, and warrant further monitoring of genetic predispositions. The role of controlled induction of ARHI associated with the X chromosome, or loss of ARHI expression and faulty breast genes is another quest for added oncological inquiries. Additionally, arguments relating to ABO blood group and its association to epithelial ovarian cancer, throughout the literature postulate that ABO genes on chromosomes 9q34 are of high consideration. Reviewing blood type correlation with ovarian cancer, through a paternal lineage connection suggesting strong inheritance patterns in first degree relatives, suggests there is an X-linked variation, and X-chromosome variation in the process. A better understanding of cancer screening by means of genetic testing, as an early detection tool, and downregulation are worthy research lenses in the fight of ovarian cancer.

Expression levels of ARHI and Beclin1 in thyroid cancer and their relationship with clinical pathology and prognosis.

Expression levels of autophagy-related genes ARHI and Beclin1 in thyroid cancer and their relationship with clinical pathology and prognosis were investigated. The expression levels of ARHI and Beclin1 proteins in 80 cases of thyroid cancer and adjacent tissues were detected by western blot analysis. According to the expression levels of ARHI and Beclin1, low- and high-expression groups were determined and the relationship of the expression levels with the pathological parameters and prognosis in thyroid cancer was compared between the two groups. The correlation between the ARHI and Beclin1 protein expression level was analyzed by Pearsons correlation analysis. The levels of ARHI and Beclin1 proteins in thyroid cancer tissues were significantly lower than those in adjacent tissues (P<0.05). There was a significant difference in the expression levels of ARHI and Beclin1 in terms of pathological stage and differentiation degree of cancer tissues (P<0.001); however, there was no significant difference in the expression levels of ARHI and Beclin1 for different types of cancer tissues (P>0 05). There was a positive correlation between the expression levels of Beclin1 and ARHI (r=0.5187, P<0.001). The 3-year survival rates of patients with low-expression level of ARHI and Beclin1 proteins were significantly lower than those of patients with high expression (P<0.05). In conclusion, the expression levels of Beclin1 and ARHI were low in thyroid cancer, and were significantly associated with the pathological stage, differentiation degree and prognosis in thyroid cancer. Beclin1 and ARHI can be used as predictors for the development and prognosis of thyroid cancer.

Correlation of ARHI upregulation with growth suppression and STAT3 inactivation in resveratrol-treated ovarian cancer cells.

Aplysia ras homology member I/ARHI is known as ovarian cancer suppressor gene and potential inhibitor of signal transducer and activator of transcription 3/STAT3 signaling. Resveratrol suppresses growth and STAT3 activation of ovarian cancer cells, while its influence in ARHI expression remains unknown. The current study aims to elucidate the status of ARHI expression and its relevance with growth suppression and STAT3 inactivation of resveratrol-treated cells. ARHI expression patterns of three ovarian cancer cell lines (human CAOV-3, OVCAR-3 and rat NUTU-19) without and with 100 μM resveratrol treatment were checked by immunocytochemical staining, Western blotting and RT-PCR. The involvement of ARHI in the growth inhibition and STAT3 inactivation of resveratrol-treated OVCAR-3 cells was investigated by transfection of ARHI-specific siRNA. ARHI is expressed in low levels in three ovarian cancer cell lines, which is upregulated upon resveratrol treatment accompanied with growth arrest, extensive apoptosis, increased autophagic activity and inactivated STAT3 signaling. Specific siRNA transfection efficiently knocked down ARHI expression in resveratrol-treated CAOV-3 and OVCAR-3 cells and increased the total cell number in limited extents (P> 0.05) in comparison with that of resveratrol-treated ovarian cancer cells without any transfection or transfected with mock oligonucleotides. ARHI knockdown failed to prevent resveratrol-caused STAT3 inactivation and cell crisis. ARHI upregulation is another molecular event caused by resveratrol and one of the elements related with resveratrol's anti-ovarian cancer efficacy. Resveratrol may inactivate STAT3 signaling of ovarian cancer cells in ARHI unrelated pattern(s).

Zebularine enhances apoptosis of human osteosarcoma cells by suppressing methylation of ARHI.

ARHI is an imprinted tumor suppressor gene and its methylation suppresses ARHI transcription levels to cause the development and progression of malignant tumors. Zebularine exerts a demethylation function for tumor suppressor genes. Our study aims to investigate the effect and mechanism of action of zebularine on the epigenetic modification of the ARHI gene, and whether this effect may modulate the viability and apoptosis of human osteosarcoma cells. We found that zebularine inhibited the viability and promoted apoptosis in osteosarcoma cells. Zebularine potentiated the expression of ARHI at both the protein and mRNA level. This was related to the downregulation of methylation of ARHI caused by zebularine. Zebularine suppressed the interaction of DNA methyltransferase 1 (DNMT1) with histone methyltransferase G9a, but had no effect on G9a alone. Knockdown of DNMT1 or G9a can induce a reduction of ARHI methylation. Therefore, we inferred that zebularine was likely to directly repress DNMT1 alone, but G9a was necessary to regulate the function of DNMT1 on ARHI methylation. Moreover, knockdown of ARHI rescued cell viability and apoptosis under the zebularine‐treated condition. We showed that zebularine inhibited viability and promoted apoptosis by disturbing the interaction between DNMT1 and G9a, thereby resulting in lower ARHI methylation and elevated ARHI expression in osteosarcoma cells.

Induction of autophagy by ARHI (DIRAS3) alters fundamental metabolic pathways in ovarian cancer models.

BackgroundAutophagy is a bulk catabolic process that modulates tumorigenesis, therapeutic resistance, and dormancy. The tumor suppressor ARHI (DIRAS3) is a potent inducer of autophagy and its expression results in necroptotic cell death in vitro and tumor dormancy in vivo. ARHI is down-regulated or lost in over 60 % of primary ovarian tumors yet is dramatically up-regulated in metastatic disease. The metabolic changes that occur during ARHI induction and their role in modulating death and dormancy are unknown.MethodsWe employed Nuclear Magnetic Resonance (NMR)-based metabolomic strategies to characterize changes in key metabolic pathways in both cell culture and xenograft models of ARHI expression and autophagy. These pathways were further interrogated by cell-based immunofluorescence imaging, tracer uptake studies, targeted metabolic inhibition, and in vivo PET/CT imaging.ResultsInduction of ARHI in cell culture models resulted in an autophagy-dependent increase in lactate production along with increased glucose uptake and enhanced sensitivity to glycolytic inhibitors. Increased uptake of glutamine was also dependent on autophagy and dramatically sensitized cultured ARHI-expressing ovarian cancer cell lines to glutaminase inhibition. Induction of ARHI resulted in a reduction in mitochondrial respiration, decreased mitochondrial membrane potential, and decreased Tom20 staining suggesting an ARHI-dependent loss of mitochondrial function. ARHI induction in mouse xenograft models resulted in an increase in free amino acids, a transient increase in [18F]-FDG uptake, and significantly altered choline metabolism.ConclusionsARHI expression has previously been shown to trigger autophagy-associated necroptosis in cell culture. In this study, we have demonstrated that ARHI expression results in decreased cellular ATP/ADP, increased oxidative stress, and decreased mitochondrial function. While this bioenergetic shock is consistent with programmed necrosis, our data indicates that the accompanying up-regulation of glycolysis and glutaminolysis is autophagy-dependent and serves to support cell viability rather than facilitate necroptotic cell death. While the mechanistic basis for metabolic up-regulation following ARHI induction is unknown, our preliminary data suggest that decreased mitochondrial function and increased metabolic demand may play a role. These alterations in fundamental metabolic pathways during autophagy-associated necroptosis may provide the basis for new therapeutic strategies for the treatment of dormant ovarian tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2850-8) contains supplementary material, which is available to authorized users.

Abstract PR18: Survival of ARHI-induced dormant autophagic cell death in vivo requires permissive levels of VEGF, IL-8, and IGF-1 in the tumor microenvironment.

Abstract Among gynecological cancers, ovarian cancer has the worst prognosis. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at second look operations. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers and in cancers from several other sites. Re-expression of ARHI induces autophagy by inhibiting PI3K and mTOR signaling, displacing Bcl-2 from Beclin1to form the autophagy initiating complex, inducing ATG4, decorating the autophagosome membrane and co-localizing with LC3II. Re-expression of ARHI in ovarian cancer cells in culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced once again in vivo, xenografts grow promptly to kill the host. Outgrowth of dormant xenografts is markedly delayed, but not prevented by treatment with chloroquine, a functional inhibitor of autophagy. The clinical relevance of this dormancy model is supported by previous studies that found ARHI expression in ovarian cancer cells was significantly increased in 90% of cases where small, dormant nodules of drug resistant ovarian cancer removed from the peritoneal surface at second-look operations, but in only 40% of the primary specimens from the same patients from initial cytoreductive surgery. In this study, we have measured Ki67 and microvascular density (MVD) in the same set of primary human ovarian cancers and in matched positive second look specimens. Second look specimens exhibited significantly lower Ki-67 (p&amp;lt;0.0001) and MVD (p&amp;lt;0.0001) than did primary cancers. We have hypothesized that that decreased access to nutrients with decreased MVD could be responsible the elevation of ARHI levels and the induction of autophagy. While autophagy could sustain nutrient deprived cancer cells up to a point, excessive autophagy can also induce type II autophagic cancer cell death. Our previous studies indicated that growth factors (VEGF, IGF1) and cytokines (IL-8) present in the tumor microenvironment could rescue ovarian cancer cells in culture from autophagic death. In this study we have found that treatment with anti-VEGF, anti-IL8 and anti-IGFR1 antibodies, individually and in combination, slowed the outgrowth of dormant autophagic ovarian cancer cells. Treatment of dormant xenografts with the same monoclonal antibodies against VEGF, IL-8 and IGFR1 with or without CQ not only delayed outgrowth when DOX is withdrawn, but cured a fraction of mice. Re-expression of ARHI reduced expression of IGF-1 receptor, but not VEGF and IL8 receptors. Treatment ovarian cancer cells with VEGF, IL-8 and IGF-1 reduced ARHI's ability inducing autophagy and inhibiting AKT and mTOR activity. Our studies provide a novel model for tumor dormancy that incorporates autophagy as a mechanism for tumor cell survival in the presence of permissive levels of growth factors and cytokines. Blocking these survival factors with antibodies could provide a novel approach to eliminate dormant cells, providing more effective treatment for women with ovarian cancer. This abstract is also presented as Poster A36. Citation Format: Zhen Lu, Aaron F. Orozco, Hailing Yang, Margie N. Sutton, Yan Wang, Weiqun Mao, Douglas A. Levine, Robert C. Bast, Jr., Robert C. Bast, Jr.. Survival of ARHI-induced dormant autophagic cell death in vivo requires permissive levels of VEGF, IL-8, and IGF-1 in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR18.

EZH2-induced H3K27me3 is associated with epigenetic repression of the ARHI tumor-suppressor gene in ovarian cancer.

Epithelial ovarian cancer (EOC) is the second leading cause of death from gynecological malignancies worldwide. Enhancer of zeste homology 2 (EZH2), participating in gene expression silencing by trimethylating histone 3 lysine 27 (H3K27me3), is often up-regulated in EOC. ARHI, an imprinted tumor-suppressor gene, is markedly down-regulated or even undetectable in the majority of EOC. To explore the correlation between EZH2 and ARHI expression in EOC as well as the possible mechanism of EZH2-ARH1 interaction. We used immunohistochemical staining to evaluate the expression of EZH2 and ARHI in EOC and normal ovarian tissue specimens; western blotting, shRNA, and chromatin immunoprecipitation were used to study the expression correlation of EZH2 and ARHI in EOC and normal ovarian epithelial cells and to further explore the mechanism of EZH2 regulation of ARHI expression. Cell viability assay was used to evaluate the influence of these two genes on cell survival. (1) The expression of EZH2 inversely correlated with ARHI expression levels and predicted shorter overall survival in EOC patients; (2) EZH2 promoted repression of ARHI by catalyzing trimethylation on H3K27; (3) ARHI was synergistically silenced by DNA methylation and histone modification; and (4) DZNep, an inhibitor of EZH2, significantly reduced survival rate of EOC cells by restoring ARHI expression. EZH2-″induced H3K27me3 is associated with epigenetic repression of the ARHI tumor-suppressor gene in EOC. Suppression of EZH2 by DZNep, as a way of restoring the expression of ARHI, could be a potential treatment modality to EOC.

ARHI (DIRAS3) induces autophagy in ovarian cancer cells by downregulating the epidermal growth factor receptor, inhibiting PI3K and Ras/MAP signaling and activating the FOXo3a-mediated induction of Rab7.

The process of autophagy has been described in detail at the molecular level in normal cells, but less is known of its regulation in cancer cells. Aplasia Ras homolog member I (ARHI; DIRAS3) is an imprinted tumor suppressor gene that is downregulated in multiple malignancies including ovarian cancer. Re-expression of ARHI slows proliferation, inhibits motility, induces autophagy and produces tumor dormancy. Our previous studies have implicated autophagy in the survival of dormant ovarian cancer cells and have shown that ARHI is required for autophagy induced by starvation or rapamycin treatment. Re-expression of ARHI in ovarian cancer cells blocks signaling through the PI3K and Ras/MAP pathways, which, in turn, downregulates mTOR and initiates autophagy. Here we show that ARHI is required for autophagy-meditated cancer cell arrest and ARHI inhibits signaling through PI3K/AKT and Ras/MAP by enhancing internalization and degradation of the epidermal growth factor receptor. ARHI-mediated downregulation of PI3K/AKT and Ras/ERK signaling also decreases phosphorylation of FOXo3a, which sequesters this transcription factor in the nucleus. Nuclear retention of FOXo3a induces ATG4 and MAP-LC3-I, required for maturation of autophagosomes, and also increases the expression of Rab7, required for fusion of autophagosomes with lysosomes. Following the knockdown of FOXo3a or Rab7, autophagolysosome formation was observed but was markedly inhibited, resulting in numerous enlarged autophagosomes. ARHI expression correlates with LC3 expression and FOXo3a nuclear localization in surgical specimens of ovarian cancer. Thus, ARHI contributes to the induction of autophagy through multiple mechanisms in ovarian cancer cells.

STAT3 acetylation-induced promoter methylation is associated with downregulation of the ARHI tumor-suppressor gene in ovarian cancer

ARHI is a Ras-related imprinted tumor-suppressor gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of ovarian cancer cells, and promoter methylation is considered to be associated with its loss of expression. however, the underlying mechanisms are not well understood. Thus, the present study aimed to investigate the specific functions of ARHI and its methylation in ovarian cancer cell proliferation. Furthermore, we examined the possible role of acetylated STAT3 in modulating the expression of ARHI and its methylation. In accordance with the majority of previous studies, reduced ARHI expression was found in epithelial ovarian cancer tissues and cancer cell lines as indicated by immunohistochemistry and RT-PCR. In addition, CpG islands I and II within ARHI promoter regions were partially methylated or hypermethylated in cancer cell lines (SKOV-3 and HO-8910) as analyzed by pyrosequencing assays, resulting in enhanced proliferation of the cancer cells. This proliferation was reversed by the administration of 5-aza-2'-deoxycytidine. Subsequently, we demonstrated that STAT3 acetylation was increased in HO-8910 cells, and the methylation status of CpG I was altered in response to the acetylation of STAT3 using western blotting. Finally, chromatin immunoprecipitation (ChIP) and IP analysis indicated that acetylated STAT3 bound to the ARHI promoter and recruited DNA methyltransferase 1 for genetic modification. In conclusion, acetylated STAT3-induced promoter gene methylation accounts for the loss of ARHI expression and cancer cell proliferation.

P-0031 Pathobiological Significance of Arhi Expression in Human Gastric Carcinoma and The Precancerous Lesions as Well as Gastric Cancer Cell Lines

IntroductionAplesia Ras homologue member I (ARHI, DIRAS3) is a maternally imprinted tumor suppressor gene which is frequently lost in breast and ovarian cancers, and results in autophagic cell death of human ovarian cancer cells in vitro. There has been no report in PMC about ARHI in human gastric carcinomas and gastric cancer cell lines so far. MethodsThe ARHI expression in 309 cases of gastric carcinomas (GCs), with corresponding normal gastric mucosa and the precancerous lesions was investigated immunohistochemically, and the prognostic value of ARHI expression was analyzed. ARHI mRNA and protein expressions in gastric cancer cell lines BGC823, MGC803, SGC7901, MKN45, NCI-N87 and normal gastric epithelial cell line GES-1 were also evaluated using quantitative real time PCR(qrtPCR) and immunofluorescence (IF) methods. ResultsThe positive expression of ARHI protein in intestinal metaplasia (76.1%, 86/113), dysplasia (50.0%, 8/16) and gastric carcinoma (29.8%, 92/309) were down-regulated compared with corresponding normal gastric mucosa (87.4%, 270/309), P<0.05. The rates of ARHI up-regulated and down-regulated expression were 9.7% (30) and 67% (207), the median patients survival time of up-regulated group (62.3 months) was longer than that of down-regulated group (36.7 months). The results of qrtPCR showed that mRNA level of ARHI in the gastric carcinoma cell lines BGC-823, MGC-803 SGC-7901, MKN-45, and NCI-N87 are 0.41, 0.41, 1.39, 1.53, 0.88 fold of gastric cell line GES, respectively. ARHI protein expression detected by IF and Western blot in the cell lines mentioned above accord with the ARHI mRNA expression by qrtPCR in the study. ConclusionWe report, for the first time, that tumor suppressor ARHI expression is mainly down regulated in human gastric carcinoma and gastric dysplasia, suggesting ARHI gene plays important roles in gastric carcinogenesis, in which ARHI related autophagy may be involved. Further study is needed in this field.

Abstract 1247: ARHI downregulates PI3K and Ras-MAP signaling and induces autophagy by promoting integrin and EGFR membrane protein degradation

Abstract Autophagy selectively degrades cellular proteins, protein aggregates and organelles, providing energy in a nutrient-deprived environment. We have reported that autophagy can be induced by re-expression of ARHI (DIRAS3), a maternally imprinted Ras-related tumor suppressor gene that is down regulated in more than 60% of ovarian cancers. Whereas prolonged expression of ARHI in cultured cells induces autophagic cell death, ARHI expression in xenografts sustains dormant cancer cell survival for weeks. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. Inhibition of ARHI-induced autophagy with chloroquine dramatically reduces regrowth of xenografts, suggesting that autophagy contributes to the survival of dormant cells. Re-expression of ARHI inhibits both PI3K and Ras-MAPK signaling. Growth factors (VEGF, IL-8, IGF) and ECM components (fibronectin) found in the cancer microenvironment can rescue ovarian cancer cells from ARHI-induced autophagic cell death in culture, associated with partial reversal of ARHI-mediated inhibition of PI3K signaling. Consequently, we have hypothesized that autophagy together with growth factors and ECM in the microenvironment are all required to maintain dormancy, where autophagy provides energy for xenografts and survival factors prevent autophagic death by partially reversing inhibition of PI3K-AKT signaling. The mechanism by which ARHI regulates PI3K and Ras-MAPK signaling has not been fully elucidated. Here, we show that re-expression of ARHI in SKOv3 ovarian cancer cells inhibits expression of integrin β1 and EGFR. ARHI expression enhanced EGF-mediated receptor endocytosis and colocalization of EGFR with Rab11 and Rab7, markers for multivesicular endosomes and lysosomes, respectively. Our results suggest that ARHI enhances membrane receptor turnover, in part, by facilitating the internalization and association of membrane receptors with vesicular trafficking proteins targeted for degradation in the lysosome. ARHI-mediated downregulation of membrane receptors reduces both PI3K-AKT and Ras-MAPK signaling, ultimately contributing to the inhibition of tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1247. doi:1538-7445.AM2012-1247

The expression of ARHI in pT2a and pT2b stage gastric cancer and its clinical significance

ARHI is a novel tumor suppressor gene located on chromosome 1p31. Downregulation of ARHI expression has been detected in many types of cancer. However, the effects of ARHI in gastric cancer remain unclear. The aim of this study was to identify the relationship between ARHI expression and gastric cancer clinicopathological features. In this study, 81 pT2 stage gastric cancer specimens were subclassified by pT2a and pT2b stage. ARHI mRNA and protein levels were evaluated by real-time PCR and western blot analysis, respectively. Methylation plays an important role in suppressor gene silencing. We utilized methylation-specific PCR to identify the status of CpG islands in the ARHI gene. We used immunohistochemistry to determine the expression of the protein and analyzed clinicopathological features. The levels of ARHI mRNA in gastric cancer were lower compared to normal tissues (P<0.01). Similarly, the levels of ARHI protein in the cancer specimens were lower (P<0.05). DNA hypermethylation was identified in 79.1% of gastric cancer specimens without ARHI expression. Immunohistochemistry results were significantly correlated with the pT2 category (P<0.05). The cumulative survival rate of patients with ARHI expression was significantly higher compared to those without ARHI expression (P<0.05). ARHI as a suppressor is not only an important factor in the pathogenesis of gastric cancer, but also a potential factor for tumor aggravation. ARHI expression in gastric cancer can be employed to indicate favorable prognosis for the disease.

The Expression and Clinical Significance of GTP-Binding RAS-Like 3(ARHI)and MicroRNA 221 and 222 in Prostate Cancer

Globally, prostate cancer is the most common malignancy among men and there is no biomarker for defining tumour invasion and progression. Guanosine-5'-triphosphate (GTP)-binding RAS-like 3 (ARHI) is a tumour suppressor gene that has been found to be downregulated in the prostate cancer cell line PC-3. MicroRNA 221 and 222 have been shown to regulate ARHI expression negatively. This study evaluated tissue samples from patients with prostate cancer (n = 35) that were designated as aggressive or non-aggressive according to their Gleason grade. Expression of ARHI and microRNA 221 and 222 was measured by real-time reverse transcription-polymerase chain reaction. The level of ARHI mRNA was significantly lower in aggressive compared with non-aggressive prostate cancer tissue samples. In contrast, microRNA 221 and 222 levels were significantly higher in aggressive compared with non-aggressive prostate cancer tissue samples. Whether ARHI and microRNA 221 and 222 could be considered as biomarkers for disease progression in prostate cancer requires further investigation.

The tumor-suppressor gene ARHI (DIRAS3) suppresses ovarian cancer cell migration through inhibition of the Stat3 and FAK/Rho signaling pathways

Ovarian cancers migrate and metastasize over the surface of the peritoneal cavity. Consequently, dysregulation of mechanisms that limit cell migration may be particularly important in the pathogenesis of the disease. ARHI is an imprinted tumor-suppressor gene that is downregulated in >60% of ovarian cancers, and its loss is associated with decreased progression-free survival. ARHI encodes a 26-kDa GTPase with homology to Ras. In contrast to Ras, ARHI inhibits cell growth, but whether it also regulates cell motility has not been studied previously. Here we report that re-expression of ARHI decreases the motility of IL-6- and epidermal growth factor (EGF)-stimulated SKOv3 and Hey ovarian cancer cells, inhibiting both chemotaxis and haptotaxis. ARHI binds to and sequesters Stat3 in the cytoplasm, preventing its translocation to the nucleus and localization in focal adhesion complexes. Stat3 siRNA or the JAK2 inhibitor AG490 produced similar inhibition of motility. However, the combination of ARHI expression with Stat3 knockdown or inhibition produced greatest inhibition in ovarian cancer cell migration, consistent with Stat3-dependent and Stat3-independent mechanisms. Consistent with two distinct signaling pathways, knockdown of Stat3 selectively inhibited IL-6-stimulated migration, whereas knockdown of focal adhesion kinase (FAK) preferentially inhibited EGF-stimulated migration. In EGF-stimulated ovarian cancer cells, re-expression of ARHI inhibited FAK(Y397) and Src(Y416) phosphorylation, disrupted focal adhesions, and blocked FAK-mediated RhoA signaling, resulting in decreased levels of GTP-RhoA. Re-expression of ARHI also disrupted the formation of actin stress fibers in a FAK- and RhoA-dependent manner. Thus, ARHI has a critical and previously uncharacterized role in the regulation of ovarian cancer cell migration, exerting inhibitory effects on two distinct signaling pathways.

MicroRNAs 221/222 and Genistein-Mediated Regulation of ARHI Tumor Suppressor Gene in Prostate Cancer

ARHI is an imprinted tumor suppressor gene and is downregulated in various malignancies. However, ARHI expression, function, and mechanisms of action in prostate cancer have not been reported. Here, we report that ARHI mRNA and protein levels were downregulated in prostate cancer tissues compared with adjacent normal tissues. Overexpression of ARHI inhibited cell proliferation, colony formation, invasion, and induced apoptosis. Further studies on a new mechanism of ARHI downregulation showed a significant inverse relationship between ARHI and miR-221 and 222, which were upregulated in prostate cancer cell lines. Transfection of miR-221 and 222 inhibitors into PC-3 cells caused a significant induction of ARHI expression. A direct interaction of miR-221 or 222 with a target site on the 3'UTR of ARHI was confirmed by a dual luciferase pMIR-REPORT assay. Finally, we also found that genistein upregulates ARHI by downregulating miR-221 and 222 in PC-3 cells. In conclusion, ARHI is a tumor suppressor gene downregulated in prostate cancer, and overexpression of ARHI can inhibit cell proliferation, colony formation, and invasion. This study demonstrates for the first time that prostate cancer cells have decreased level of ARHI which could be caused by direct targeting of 3'UTR of ARHI by miR221/222. Genistein, a potential nontoxic chemopreventive agent, restores expression of ARHI and may be an important dietary therapeutic agent for treating prostate cancer.

Imprinted tumor suppressor gene ARHI induces apoptosis correlated with changes in DNA methylation in pancreatic cancer cells

Aplesia Ras homologue member I (ARHI, DIRAS3) is a Ras-related imprinted growth inhibitory gene whose expression is down-regulated in the majority of breast and ovarian cancers. This study investigated the inhibitory function of ARHI in pancreatic cancer. Six pancreatic cancer cell lines, tumor xenografts in nude mice and 20 pancreatic cancer tissue sections were analyzed. ARHI is widely expressed in ductal and acinar cells of normal pancreatic tissue, but is down-regulated or lost in approximately 50% of pancreatic cancers. Aberrant methylation of the ARHI locus was found in five pancreatic cancer cell lines, which exhibited down-regulation or loss of ARHI expression. Hypermethylation was detected in five cell lines (5/5, 100%) at CpG island I, in two cell lines (2/5, 40%) at CpG island II and in four cell lines (4/5, 80%) at CpG island III. Re-expression of ARHI significantly inhibited the growth of pancreatic cancer cells. This inhibition was associated with the induction of apoptosis. Treatment with the demethylating agent 5-aza-2'deoxycytidine (5-aza-dC) restored ARHI mRNA expression, inhibited cell growth and induced apoptosis in PANC-1 and P3 human pancreatic cancer cells in culture. In nu/nu mice, 5-aza-dC also inhibited the growth of PANC-1 xenografts and induced apoptosis, as observed by TUNEL staining. These effects were associated with the re-expression of ARHI protein. Therefore, ARHI may serve as a growth inhibitory gene in a significant fraction of pancreatic cancers. Re-expression of ARHI significantly induced the apoptosis of pancreatic cancer cells. A demethylation agent reduced human pancreatic cancer cell line growth in conjunction with ARHI re-expression.

Abstract 2090: MicroRNAs 221/222 and genistein mediated regulation of ARHI tumor suppressor gene in prostate cancer

Abstract ARHI, an imprinted tumor suppressor gene, is expressed in normal immortalized prostate epithelial cells, but is dramatically down-regulated in prostate cancer cell lines. Here we investigated the mechanisms of ARHI silencing in prostate cancer through microRNA and genistein (a dietary isoflavone from soy) mediated pathways. First, we evaluated ARHI mRNA levels by real time PCR using Q-PCR slice (48 prostate tumor and normal adjacent tissues) and protein levels by immunostaining of prostate tissue array (36 cancer, 8 matched normal adjacent tissue and 4 bone metastases). Both experiments showed down-regulation of ARHI in prostate cancer tissues compared to adjacent normal tissues. To understand the functional significance of ARHI down-regulation with regard to tumorigenesis, ARHI was over-expressed in prostate cancer PC-3 cells by introduction of ARHI plasmid followed by functional studies. Over-expression of ARHI can inhibit cell proliferation, colony formation, invasion and induced caspase-independent apoptosis. Further studies on new mechanism of ARHI down regulation showed a significant inverse relationship between ARHI and miR-221 &amp; 222 which were up-regulated in cancer cell lines. Transfection of miR-221 &amp; 222 inhibitors into PC-3 cells caused a significant induction of ARHI expression. A direct interaction of miR-221 or 222 with a target site on the 3′UTR of ARHI was confirmed by a dual luciferase pMIR-REPORT assay. Finally, we also found that genistein up-regulated ARHI by down regulating miR-221 &amp; 222 in PC-3 cells. In conclusion, ARHI is a tumor suppressor gene down regulated in prostate cancer, and over-expression of ARHI can inhibit cell proliferation, colony formation and invasion. This study demonstrates for the first time that prostate cancer cells have elevated levels of miR-221 &amp; 222 which bind to the 3′UTR of ARHI contributing to its down regulation. Genistein, a potential non-toxic chemopreventive agent restores expression of ARHI and may be an important dietary therapeutic agent for treating prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2090.

Abstract 4993: ARHI is required for induction of beclin-1/PI(3)K class III-mediated autophagy

Abstract The maternally imprinted Ras-related tumor suppressor gene Aplasia Ras homolog member I (ARHI; also known as DiRAS3) is downregulated in more than 60% of ovarian cancers. We reported that re-expression of ARHI in vitro at physiologic levels induces autophagy by blocking PI(3)K-Akt signaling and inhibiting mTOR activity, and participated directly in the formation of autophagic vesicles, co-localizing with MAP-LC3-II in the vesicle membrane. Nucleation of autophagic vesicles requires the activity of multi-protein complexes that include Beclin-1 and PI(3) kinase Class III lipid kinase [PI(3)KC3]. A number of proteins have been reported to interact with Beclin-1 and positively or negatively regulate its function in autophagy. One prominent Beclin-1-interacting protein is Bcl-2, which inhibits Beclin-1-dependent autophagy. In this study, we show by indirect immunofluorescence microscopy that ARHI is colocalized with Beclin-1 at the trans-Golgi network. Further, ARHI can be co-immunoprecipitated with Beclin-1 and elimination of ARHI expression by RNA interference compromised Beclin-1-induced autophagy. Concomitant with increased ARHI-Beclin-1 interaction, there was a decreased association between Beclin-1 and its negative regulator Bcl-2. Consistent with its role in Beclin-1-mediated autophagy, knockdown of ARHI also decreased PI(3)KC3 activity, whereas overexpression of ARHI promoted GFP-FYVE co-localization with RFP-LC3. Taken together, our results suggest that ARHI is a positive and necessary regulator of Beclin-1/PI(3)KC3 complex and that ARHI regulates Beclin-1-mediated autophagy by forming a complex at the trans-Golgi network. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4993.