AT-rich Interactive Domain 1A Expression Research Articles

Abstract 4155: ARID1A loss leads to chemotherapy resistance and is associated with a poor clinic outcome for patients with non-small cell lung cancer

Abstract Purpose: ARID1A (AT-rich interactive domain 1A) is a member of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, and plays important roles in the regulation of gene transcription and DNA damage responses. ARID1A mutations occur frequently in non-small cell lung cancer (NSCLC), but the functions of ARID1A loss in the initiation, progression and maintenance of NSCLC remains largely unknown. This study aimed to investigate the clinical, pathological, and functional significance of ARID1A in NSCLC. Experimental design: We first analyzed the prognostic value of ARID1A using a gene-expression microarray from the Director's Challenge Lung Study. Kaplan-Meier method and Log-rank tests were used to estimate the correlation between ARID1A expression and overall survival (OS). Univariate and multivariate Cox regression models were used to investigate the relationship between ARID1A and other clinical covariates. We further established stable ARID1A knockdown H1299 and H358 cell lines, and ARID1A exogenously expressing H460 and A549 cell lines. We performed functional assays to study the effects of ARID1A on proliferation, migration, invasion, cell cycle progression, sensitivity to chemotherapy, and in vivo tumor growth. Results: In the Director's Challenge Study, decreased expression of ARID1A was associated with lymph node dissemination, higher T-stage (T3/T4), and worse survival compared with high expression in patients in the whole group (n=440) and subgroup who received surgery alone and no adjuvant therapy (n=328). ARID1A ablation by shRNA suppressed cell proliferation, promoted cell mobility, increased G2/M phase cell cycle distribution, and protected NSCLC cells from chemotherapy; whereas overexpression of ARID1A showed the opposite biological effects. Consistent with in vitro findings, in vivo tumor xenografts demonstrated ARID1A depletion led to reductions in cell growth but resistance of NSCLC to chemotherapy. Conclusions: Our results suggest that loss of ARID1A suppresses NSCLC cell growth perhaps by inducing G2/M arrest, but promotes cell migration/invasion, and protect cells from chemotherapy. Further study of ARID1A is needed to better define its roles in the initiation and progression of NSCLC and response to chemotherapy. Citation Format: Linlin Yang, Changxian Shen, Xiaokui Mo, Terence M. Williams. ARID1A loss leads to chemotherapy resistance and is associated with a poor clinic outcome for patients with non-small cell lung cancer [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 4155.

Downregulation of ARID1A is correlated with poor prognosis in non-small cell lung cancer.

BackgroundNon-small cell lung cancer (NSCLC) is the main type of lung cancer and NSCLC patients always have a low 5-year survival rate. It is vital to identify a biomarker for the prognosis of NSCLC patients. AT-rich interaction domain 1a (ARID1A) is a tumor suppressor that is involved in the progression of a variety of tumors.MethodsThe ARID1A protein level in NSCLC tissues and paracancerous normal lung (PCNL) tissues were detected with immunohistochemistry (IHC) and western blotting (WB). The χ2 test and Spearman’s rank correlation analysis were carried out to examine the association between ARID1A expression and the clinicopathological features of NSCLC. The Kaplan-Meier method and log-rank test were used to compare overall survival (OS) in the ARID1A low expression group and the ARID1A high expression group.ResultsThe results of WB and IHC demonstrated that the ARID1A protein level was significantly reduced in NSCLC tissues compared with PCNL tissues (P<0.05). The low expression of ARID1A in NSCLC tissues was significantly associated with poor differentiation (P=0.005), smoking (P<0.001), lymphatic invasion (P=0.013), distant metastasis (P=0.010), and high TNM stage (P=0.001). The overall five-year survival rate of NSCLC patients was lower in the ARID1A low expression group than in the ARID1A high expression group. Multivariate analysis showed that the expression of ARID1A had an independent prognostic impact on OS (P=0.024).ConclusionsARID1A may be a novel biomarker for predicting the prognosis of NSCLC patients.

ARID1A-SIN3A drives retinoic acid-induced neuroblastoma differentiation by transcriptional repression of TERT.

Aggressive, high-risk neuroblastoma (NB) exhibits an immature differentiation state, profound epigenetic dysregulation and high telomerase activity. It has been suggested that aggressive NB may be treatable by inducing differentiation whereas therapeutic targeting of telomerase is under investigation for multiple cancer types. While epigenetic regulation of the telomerase reverse transcriptase (TERT) promoter has been described in high-risk NB, the exact molecular mechanisms are still not completely understood. Here we used quantitative real-time polymerase chain reaction (PCR), chromatin immunoprecipitation qPCR, quantitative telomeric repeat amplification protocol, and immunoblot techniques to investigate epigenetic regulation of TERT in wild-type and genetically modified NB cell lines. We demonstrated that TERT expression is reduced during 13-cis retinoic acid-induced NB differentiation and that this inversely correlated with increased expression of AT-rich interaction domain 1A (ARID1A), a subunit of the SWItch/sucrose nonfermentable chromatin remodeling complex. We showed that ARID1A directly caused suppression of TERT and was reliant on DNA binding and co-occupancy of the TERT promoter by the SIN3 transcription regulator family member A (SIN3A) repressor complex allowing NB differentiation to proceed. Finally, using data from NB patient cohorts, we reported a significant correlation between low ARID1A expression, elevated expression of TERT, and poorly differentiated, high-risk NB. These results provide insights into a key epigenetic pathway responsible for modulating TERT-driven NB progression, which could represent a target for therapeutic intervention.

ARID1a as a marker of prognosis and increased sensitivity to CDK4/6, mTOR 1/2 and Src homology region 2 phosphatase (SHP 1/2) inhibitors in breast cancer (BC).

1082 Background: ARID1a (AT Rich Interactive Domain 1A) is part of the SWI/SNF complex, which regulates gene transcription, and is believed to be a tumor suppressor gene. Low ARID1a expression has been associated with poor prognosis in BC. The aim of this study was to explore the clinical significance of ARID1a mutation and expression loss, and its potential as a therapeutic target in BC. Methods: We analyzed publicly available genomic databases to study the clinical implication of ARID1a mutations and gene expression in BC. Results: ARID1a was mutated in ~5-7 % of BCs within TCGA/METABRIC/MSK (5511 samples), but did not show differences in frequency between histology, grade, or estrogen receptor (ER)/HER2 receptor status. MSK metastatic tissue samples had higher incidence of ARID1a mutation compared to primary tumor samples (7.6% vs 4.4%, χ2 P = 0.0073). Analysis of ARID1a in KMPLOT showed that lower gene expression was associated with worse relapse-free survival and overall survival across all BCs, but the difference was primarily in molecularly classified luminal A tumors. Mutations in ARID1a did not show an association with outcomes in TCGA/METABRIC/MSK datasets. Pathway analysis of ARID1a showed it is involved in regulating ER ligand driven signaling and interacts with targets regulated by CDK4 and mTOR activity. CancerRxgene drug sensitivity analyses on BC cell lines revealed that ARID1a mutated BC cell lines were significantly more sensitive to palbociclib, SHP1/2, and mTOR1/2 inhibitors compared to ARID1a wildtype cell lines. Conclusions: Reduced activity of ARID1a in luminal BC cells may negatively affect prognosis by altering ER signaling leading to activation of druggable resistance mechanisms, particularly in metastatic tissue. Loss of function ARID1a mutations may sensitize cancer cells to CDK4/6, mTOR1/2, and SHP1/2 inhibitors in vitro. Further research in ARID1a mutated ER+ BCs using combinations of these inhibitors is warranted.

Prognostic role of ARID1A negative expression in gastric cancer

AT-rich interactive domain 1A (ARID1A) functions as a tumor suppressor and several therapeutic targets in ARID1A-mutated cancers are under development. Here, we investigated the prognostic value of ARID1A for gastric cancer and its association with expression of PD-L1 and p53. ARID1A expression was examined by immunohistochemistry and negative expression of ARID1A was detected in 39 (19.5%) of 200 cases in a test cohort and in 40 (18.2%) of 220 cases in a validation cohort. Negative expression of ARID1A was associated with worse overall survival in undifferentiated cases, particularly early-stage cases. Negative expression of ARID1A was detected in 11 (50%) of 22 PD-L1-positive cases and in 68 (17.1%) of 398 PD-L1-negative cases in a combined cohort. Negative expression of ARID1A was detected in 45 (22%) of 205 p53-positive cases and in 34 (15.8%) of 215 p53-negative cases in a combined cohort. In addition, expression of EZH2, a potential synthetic lethal target in ARID1A-mutated tumors, was detected in 79 ARID1A-negative cases. An ARID1A-knockdown gastric cancer cell line was subjected to microarray analysis, but no actionable targets or pathways were identified. The present results indicate that ARID1A may serve as an early-stage prognostic biomarker for undifferentiated gastric cancer.

ARID1A ablation leads to multiple drug resistance in ovarian cancer via transcriptional activation of MRP2

Multiple Drug Resistance (MDR) of ovarian cancer is a severe trouble for clinical treatment and always contributes to a bad prognosis. AT-rich interaction domain 1 A (ARID1A) has been recognized as a bona fide tumor suppressor gene in recent years, with the highest mutation rate in ovarian cancer. Previous study illustrated that ARID1A expression is negatively correlated with chemoresistance of ovarian cancer cases. However, the specific role of ARID1A in chemoresistance of ovarian cancer remains elusive. In this study, we showed that ARID1A knockdown in ovarian cancer cells significantly reduced their apoptosis rate and led to MDR, while ectopic expression of ARID1A showed opposite effects. ARID1A depletion transcriptionally activates the expression of multidrug resistance-associated protein 2 (MRP2) following chromatin remodeling. Furthermore, IHC analysis of ovarian cancer samples confirmed that ARID1A expression was strong negatively correlated with MRP2 expression. Both ARID1A and MRP2 expression levels are correlated with sensitivity to platinum. Collectively, our results illustrated that ARID1A loss in ovarian cancer leads to MDR through upregulation of MRP2, providing an opportunity to overcome the ARID1A loss induced chemoresistance of ovarian cancer by targeting MRP2.

Alpha-oxoglutarate inhibits the proliferation of immortalized normal bladder epithelial cells via an epigenetic switch involving ARID1A

Interstitial cystitis (IC) is a chronic urinary tract disease that is characterized by unpleasant sensations, such as persistent pelvic pain, in the absence of infection or other identifiable causes. We previously performed comprehensive metabolomics profiling of urine samples from IC patients using nuclear magnetic resonance and gas-chromatography/mass spectrometry and found that urinary α-oxoglutarate (α-OG), was significantly elevated. α-OG, a tricarboxylic acid (TCA) cycle intermediate, reportedly functions to suppress the proliferation of immortalized normal human bladder epithelial cells. Here, we identified AT-rich interactive domain 1 A (ARID1A), a key chromatin remodeler, as being hypomethylated and upregulated by α-OG treatment. This was done through EPIC DNA methylation profiling and subsequent biochemical approaches, including quantitative RT-PCR and western blot analyses. Furthermore, we found that α-OG almost completely suppresses ten-eleven translocation (TET) activity, but does not affect DNA methyltransferase (DNMT) activity. Altogether, our studies reveal the potential role of α-OG in epigenetic remodeling through its effects on ARID1A and TET expression in the bladder. This may provide a new possible therapeutic strategy in treating IC.

Loss of Chromatin-Remodeling Proteins and/or CDKN2A Associates With Metastasis of Pancreatic Neuroendocrine Tumors and Reduced Patient Survival Times

Despite prognostic grading and staging systems, it is a challenge to predict outcomes for patients with pancreatic neuroendocrine tumors (PanNETs). Sequencing studies of PanNETs have identified alterations in death domain-associated protein (DAXX) and alpha-thalassemia/mental retardation X-linked chromatin remodeler (ATRX). In tumors, mutations in DAXX or ATRX and corresponding loss of protein expression correlate with shorter times of disease-free survival and disease-specific survival of patients. However, DAXX or ATRX proteins were lost in only 50% of distant metastases analyzed. We performed whole-exome sequencing analyses of 20 distant metastases from 20 patients with a single nonsyndrome, nonfunctional PanNET. We found distant metastases contained alterations in multiple endocrine neoplasia type 1 (MEN1) (n= 8), ATRX (n= 5), DAXX (n= 5), TSC2 (n= 3), and DEP domain containing 5 (DEPDC5) (n= 3). We found copy number loss of cyclin dependent kinase inhibitor 2A (CDKN2A) in 15 metastases (75%) and alterations in genes that regulate chromatin remodeling, including set domain containing 2 (SETD2) (n= 4), AT-rich interaction domain 1A (ARID1A) (n= 2), chromodomain helicase DNA binding protein 8 (CHD8) (n= 2), and DNA methyl transferase 1 (DNMT1) (n= 2). In a separate analysis of 347 primary PanNETs, we found loss or deletion of DAXX and ATRX, disruption of SETD2 function (based on loss of H3 lysine 36 trimethylation), loss of ARID1A expression or deletions in CDKN2A in 81% of primary PanNETs with distant metastases. Among patients with loss or deletion of at least 1 of these proteins or genes, 39% survived disease-free for 5 years and 44% had disease-specific survival times of 10 years. Among patients without any of these alterations, 98% survived disease-free for 5 years and 95% had disease-specific survival times of 10 years. Therefore, primary PanNETs with loss of DAXX, ATRX, H3 lysine 36 trimethylation, ARID1A, and/or CDKN2A associate with shorter survival times of patients. Our findings indicate that alterations in chromatin-remodeling genes and CDKN2A contribute to metastasis of PanNETs.

High‐level expression of ARID1A predicts a favourable outcome in triple‐negative breast cancer patients receiving paclitaxel‐based chemotherapy

Paclitaxel‐based chemotherapy is a common strategy to treat patients with triple‐negative breast cancer (TNBC). As paclitaxel resistance is still a clinical issue in treating TNBCs, identifying molecular markers for predicting pathologic responses to paclitaxel treatment is thus urgently needed. Here, we report that an AT‐rich interaction domain 1A (ARID1A) transcript is up‐regulated in paclitaxel‐sensitive TNBC cells but down‐regulated in paclitaxel‐resistant cells upon paclitaxel treatment. Moreover, ARID1A expression was negatively correlated with the IC 50 concentration of paclitaxel in the tested TNBC cell lines. Kaplan‐Meier analyses revealed that ARID1A down‐regulation was related to a poorer response to paclitaxel‐based chemotherapy in patients with TNBCs as measured by the recurrence‐free survival probability. The pharmaceutical inhibition with p38MAPK‐specific inhibitor SCIO‐469 revealed that p38MAPK‐related signalling axis regulates ARID1A expression and thereby modulates paclitaxel sensitivity in TNBC cells. These findings suggest that ARID1A could be used as a prognostic factor to estimate the pathological complete response for TNBC patients who decide to receive paclitaxel‐based chemotherapy.

Loss of ARID1A Expression Correlates With Tumor Differentiation and Tumor Progression Stage in Pancreatic Ductal Adenocarcinoma.

Mutations in the AT-rich interactive domain 1A gene, which encodes a subunit of the Switch/Sucrose nonfermentable chromatin remodeling complex, can result in loss of protein expression and are associated with different cancers. Here, we used immunohistochemistry to investigate the significance of AT-rich interactive domain 1A loss in 73 pancreatic ductal adenocarcinoma cases with paired paracancerous normal pancreatic tissues. The relationship between levels of the AT-rich interactive domain 1A protein product, BAF250a, and clinicopathological parameters in the 73 pancreatic cancer specimens was also analyzed. We found that the expression of AT-rich interactive domain 1A in normal pancreatic tissue was higher than that in tumor tissue. Loss of AT-rich interactive domain 1A expression in pancreatic tumors was associated with tumor differentiation (P = .002) and tumor stage (P = .048). Meanwhile, BAF250a protein levels were not related to lymph node metastasis, distant metastasis, sex, or age and were not associated with survival. Transfection of the pancreatic cancer cell lines AsPC-1 and PANC-1 with small-interfering RNA specific for AT-rich interactive domain 1A resulted in elevated messenger RNA and protein expression levels of B-cell lymphoma-2 (Bcl-2), CyclinD1, and Kirsten rat sarcoma viral oncogene (KRAS). The AT-rich interactive domain 1A expression level in the cells was increased following microRNA-31 (miR-31) inhibitor transfection. Our data provide additional evidence that AT-rich interactive domain 1A might function as a tumor suppressor gene in pancreatic carcinogenesis.

AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells.

BackgroundThe At-rich interactive domain 1A (ARID1A) is frequently mutated in gastric cancers (GCs) with a poor prognosis. Growing evidence indicates that loss of ARID1A expression leads to activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by AKT phosphorylation. We aim to investigate the different sensitivity for the AKT inhibitor in ARID1A-deficient GC cells.MethodsAfter transfection using siRNA or shRNA, the effect of ARID1A knockdown on the PI3K/AKT signaling pathway was evaluated by Western blot analysis. ARID1A-knockdown cells were treated with AKT inhibitor (GSK690693), 5-fluorouracil, or cisplatin, alone or in combination. Viability and apoptosis were analyzed using EZ-CYTOX cell viability assay and flow cytometry, respectively.ResultsARID1A depletion accelerated the phosphorylation of AKT and S6 in a dose-dependent manner and led to an increased proliferation of MKN-1, MKN-28, and KATO-III GC cells (P<0.001). ARID1A-deficient cells were more vulnerable to GSK690693 in comparison to the controls (P<0.001), even at very low doses. Flow cytometry confirmed the increased apoptosis in ARID1A-deficient cells treated with GSK690693 (0.01 μmol/L; P<0.001). In contrast to our expectations, ARID1A depletion did not cause resistance to 5-fluorouracil or cisplatin. Addition of GSK690693 to the conventional chemotherapy induced more decreased cell viability in ARID1A-knockdown cells (P<0.01).ConclusionLoss of ARID1A expression is a surrogate marker for the activation of the AKT signaling pathway and is also a reliable biomarker to predict the response for the AKT inhibitor. We anticipate that appropriate patient selection based on ARID1A expression in the tumor tissue will increase the drug sensitivity for the AKT inhibition and improve the clinical outcome.

Prognostic and Clinicopathological Significance of ARID1A in Endometrium-Related Gynecological Cancers: A Meta-Analysis.

The tumor suppressor gene, AT Rich Interactive Domain 1A (ARID1A) mutation has been reported in a variety of cancers, especially the endometrium-related gynecological cancers, including the ovarian clear cell carcinoma, ovarian endometrioid carcinoma, and uterine endometrioid carcinoma. However, the prognostic value of ARID1A in endometrium-related gynecological cancers is still inconclusive. Therefore, we performed this meta-analysis to evaluate the clinical significance of ARID1A in endometrium-related gynecological cancers. By systematically searching all the relevant studies from Pubmed, Cochrane Library, and Web of Science up to September 2016, 11 studies with 1,432 patients were included. All the study characteristics and the prognostic data were extracted. Hazard ratios (HRs) and 95% confidence intervals (CIs) were pooled using the fixed-effect or random-effect model. Our results indicated that negative ARID1A expression predicted shorter Progression free survival (PFS, HR, 1.84; 95%CI, 1.32-2.57, P = 0.000) of patients with endometrium related gynecological cancers, especially the patiently with OCCC and the patients in Japan. Besides, a marginal trend towards the same direction was found in the Overall analysis (OS, HR, 1.34; 95%CI, 0.93-1.93, P = 0.112). Furthermore, the significant correlation was achieved between the negative ARID1A expression and the FIGO stage of endometrium-related gynecological cancers, but not the other characteristics. J. Cell. Biochem. 118: 4517-4525, 2017. © 2017 Wiley Periodicals, Inc.

Abstract NTOC-114: ARID1A DEFICIENCY IN OVARIAN CLEAR CELL CARCINOMAS UP–REGULATES OXIDATIVE STRESS AND SENSITIZES CELLS TO ROS–INDUCING AGENTS

Abstract Inactivating mutations in AT-rich interaction domain 1A (ARID1A) are most frequently found in ovarian clear cell carcinomas (OCCC) and ovarian endometrioid carcinomas (OEC). The functional roles of ARID1A are not completely understood and there are limited therapeutic strategies that specifically target ARID1A-mutant cancers. Given that ARID1A expression is lost in cancer, ARID1A mutations cannot be targeted directly and novel therapeutic strategies are required to target ARID1A-mutant cancers. In this study, drug responses between ARID1A-wildtype and ARID1A-mutant cell lines were compared using the ‘Genomics of Drug Sensitivities in Cancer’ database. From this analysis, we found that ARID1A-mutant cell lines are more sensitive to elesclomol, which is a reactive oxygen species (ROS)-inducing agent. This finding was validated using a panel of well-characterized ovarian and endometrial cancer cell lines. ARID1A-mutant OCCC cell lines exhibited lower IC50 values and higher apoptotic rates when treated with elesclomol. Knockdown and re-expression of ARID1A in ovarian cancer cells showed that ARID1A is required to protect cancer cells from oxidative stress. In the absence of ARID1A, intracellular ROS levels were increased as determined by flow cytometry. Immunostaining of 4-hydroxy-2-nonenals (4-HNE), one of the products from lipid peroxidation induced by ROS, in OCCC patient samples indicated an up-regulation of oxidative stress in the tumor. We also found that ARID1A negatively regulates the expression of NRF2 which is the major regulator of the antioxidant response in the cell. Knockdown of NRF2 in ovarian cancer cell lines revealed that NRF2 expression may be preferentially required for protection from oxidative stress and cell proliferation in ARID1A-mutant cells. Analysis using The Cancer Genome Atlas (TCGA) UCEC dataset revealed that ARID1A-mutant tumor samples have higher expression of NRF2 and NRF2-target genes. In summary, this study revealed novel roles of ARID1A in protecting ovarian cancer cells against oxidative stress. In the absence of ARID1A, NRF2 is up-regulated and may be required to compensate for ARID1A deficiency. These findings suggest that ARID1A-mutant ovarian cancer cells may be targeted for treatment with ROS-inducing agents and NRF2 inhibitors. Citation Format: Suet-Yan Kwan, Xuanjin Cheng, Suet-Ying Kwan, Daisy I. Izaguirre, Yvonne T.M. Tsang, Jong-Sun Choi, Hoi-Shan Kwan, David M. Gershenson and Kwong-Kwok Wong. ARID1A DEFICIENCY IN OVARIAN CLEAR CELL CARCINOMAS UP–REGULATES OXIDATIVE STRESS AND SENSITIZES CELLS TO ROS–INDUCING AGENTS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-114.

ARID1A Expression is Down-Regulated by Oxidative Stress in Endometriosis and Endometriosis-Associated Ovarian Cancer.

Oxidative stress is considered an important factor in the development of endometriosis, including its malignant transformation. Previous studies have found that AT-rich interactive domain 1A (ARID1A), a tumor suppressor gene, is frequently mutated and inactivated in endometriosis-associated ovarian cancer (EAOC), and such a change in this gene is considered an early event in malignant transformation. We observed oxidative stress status by measuring the activity of the antioxidant enzyme manganese superoxide dismutase (MnSOD), malondialdehyde (MDA), and ARID1A gene expression in tissue samples from patients with endometriosis, EAOC, or non–endometriosis-associated ovarian cancer (non-EAOC). We also induced oxidative stress in the cultured cells from patients with primary endometriosis by adding H2O2 and tested for any alteration of ARID1A gene expression based on different H2O2 concentrations. The results showed that MnSOD activity in endometriosis and EAOC was lower than in non-EAOC, but MDA levels were higher. This study also showed that oxidative stress reduced ARID1A gene expression.

ARID1A gene silencing reduces the sensitivity of ovarian clear cell carcinoma to cisplatin.

In ovarian clear cell carcinoma (OCCC), the mutation rate of the AT-rich interaction domain 1A (ARID1A) gene is 46-57%. However, the effects of ARID1A gene silencing by small interfering RNA (siRNA) on the sensitivity of OCCC to cisplatin have not been investigated. Thus, this study aimed to elucidate the association between ARID1A gene silencing and drug resistance in OCCC. Three pairs of ARID1A gene siRNA fragments (siRNA-1, siRNA-2 and siRNA-3) were designed and transiently transfected into ES2 OCCC cells using RNAi Max reagent. Western blotting results demonstrated that the transfection reduced ARID1A protein expression levels, with the siRNA-3 group having the lowest levels. The IC50 value, determined using a Cell Counting kit-8 assay, was significantly increased by siRNA-3 transfection compared with that in blank control and negative control groups. The cell survival rate following treatment with 50 µM cisplatin for 48 h was significantly increased in the siRNA-3 group compared with the blank control and negative control groups. Flow cytometric analysis revealed that the apoptosis rate for cisplatin-treated cells was significantly lower in cells with siRNA-3 transfection than in those without, and the apoptosis rate in siRNA-3-transfected cells was lower than that in the negative control group. Western blot analysis showed that the expression level of AKT in cisplatin-treated cells was significantly decreased compared with that in the negative control group, and the AKT expression level in cisplatin-treated cells was significantly higher with siRNA-3 transfection than without. Therefore, the results demonstrated that ARID1A siRNA efficiently decreased ARID1A expression, which reduced cisplatin chemosensitivity and cell apoptosis in ES2 OCCC cells via the regulation of AKT expression.

Loss of ARID1A Expression is Related to Gastric Cancer Progression, Epstein-Barr Virus Infection, and Mismatch Repair Deficiency.

The AT-rich interactive domain 1A (ARID1A) gene encodes a member of the switch/sucrose nonfermentable (SWI-SNF) chromatin remodeling complex, and is considered to work as a tumor suppressor in concert with p53. We investigated the clinical significance of ARID1A protein expression in gastric cancer (GC), and examined its association with Epstein-Barr virus-associated (EBV) GC, mismatch repair (MMR) deficiency, and p53 alteration. We performed immunohistochemistry for ARID1A in 417 GC specimens using tissue microarray. EBV infection was examined using EBV-encoded small RNA in situ hybridization. Evaluation of MMR protein deficiency and p53 alteration was performed using immunohistochemistry, and microsatellite instability status was also assessed. Loss of ARID1A expression was observed in 21.1% of GC (88/417), but was not observed in gastric adenoma tissues or non-neoplastic gastric mucosa tissues. Loss of ARID1A showed positive correlations with advanced pTNM stage and tumor invasion (P=0.029 and 0.001, respectively). Overall survival was significantly influenced by the loss of ARID1A expression in wild-type p53 group (P=0.016, log-rank test). Moreover, ARID1A loss was significantly associated with EBV positivity, loss of MMR protein expression, and microsatellite instability high status (P=0.028, <0.001, and 0.011, respectively). All of the results from our cohort were verified using data from the Cancer Genome Atlas. In conclusion, loss of ARID1A is more common in advanced GC and is related to EBV positivity and MMR deficiency.

Clinicopathologic Significance of HNF-1β, AIRD1A, and PIK3CA Expression in Ovarian Clear Cell Carcinoma: A Tissue Microarray Study of 130 Cases.

Ovarian clear cell carcinoma (CCC) is a distinct histologic subtype with relatively poor survival. No prognostic or predictive molecular marker is currently available. Recent studies have shown that AT-rich interactive domain 1A (ARID1A) and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations are common genetic changes in ovarian CCC. Hepatocyte nuclear factor-1β (HNF-1β) expression has been proven to be highly sensitive and specific for clear cell histology. However, the correlations between these biomarkers and clinicopathologic variables and survival outcomes are controversial. The immunohistochemical analysis for HNF-1β, ARID1A, and PIK3CA was performed on a tissue microarray (TMA) consisting of 130 cases of ovarian CCC (237 tissue blocks) linked with clinical information. The immunostaining results were interpreted in a manner consistent with previous publications. The associations between biomarker expression and clinical and prognostic features were examined. All statistical analyses were conducted using 2-sided tests, and a value of P < 0.05 was considered significant. HNF-1β was expressed in 92.8% of all primary ovarian tumors, while the loss of ARID1A and PIK3CA was noted in 56.2% and 45.0%, respectively. Early-stage tumors tended to have high levels of HNF-1β immunoreactivity and expression of ARID1A (P = 0.02 and P = 0.03). Most patients (76.9%, 20/26) with concurrent endometriosis stained negative for ARID1A (P = 0.02). No relation was found between PIK3CA expression and clinical features. Low-level HNF-1β expression and loss of ARID1A were more commonly observed in patients with tumor recurrence (P = 0.02 and P < 0.001). Antibody expression was not associated with platinum-based chemotherapy response. Patients with negative ARID1A expression had worse survival outcome in terms of both overall survival (OS) and progression-free survival (PFS) (P = 0.03 and P = 0.01, respectively). On the contrary, patients with high-level HNF-1β were associated with good prognosis (P = 0.02 for OS and P = 0.01 for PFS). PIK3CA expression had no impact on survival. For univariate and multivariate analyses, only HNF-1β expression seemed to be a prognostic factor for favorable OS (P = 0.04). The loss of ARID1A was correlated with late-stage and endometriosis-associated tumors. The measurement of ARID1A expression might be a method to predict the risk of recurrence. Among the 3 biomarkers, only high-level HNF-1β expression proved to be a positive predictor for OS.

Impact of biological function on ovarian clear cell carcinoma ES2 cell line with ARID1A gene expression down-regulating in vitro

To investigate the efficiency of biological function of AT rich interaction domain 1A (ARID1A) gene silenced by small interfering RNA (siRNA) on ovarian clear cell carcinoma ES2 cell line. (1) The three pairs ARID1A gene siRNA interference fragments siN1 (ARID1A-705), siN2 (ARID1A-1513), siN3 (ARID1A-2282) and one pair negative control were respectively designed, and transfected into ES2 cells by RNA interference max reagent transiently. Reverse transcription (RT)-PCR and western blot methods were used to detect the expression of ARID1A mRNA and protein in ES2 cells transfected with interference fragments respectively. So as to select the best silencing effect of siRNA interference fragment(that was siN3), and then was used in the following experiment. (2) The following experiment were divided into three groups, namely siN3 transfection group, negative control group and blank control group. The proliferative activity of three groups of cells after transient transfection (6, 24, 48, 72, 96 hours) was assessed by cell counting kit-8 (CCK-8) assay and expressed as absorbance (A) value; the apoptosis rate of three groups of cells transfected transiently with interference fragment was measured by flow cytometry with annexin V/propidium iodide (PI) staining; the ability of cellular invasion of three groups of cells transfected transiently with interference fragment was tested by transwell experiment; the expression of nuclear factor-kappa B (NF-κB), membrane type-1 matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP2) protein in ES2 cells transfected transiently with interference fragment was measured by western blot. (1) The RT-PCR results showed that the ARID1A mRNA relative expression levels in ES2 cells after transfected transiently with siN1, siN2 and siN3 were 0.007 8±0.005 7, 0.006 8±0.000 3 and 0.002 8±0.000 3 respectively. They were all apparently lower than that in the negative control group (0.034 6±0.001 3; all P<0.01). The western blot results showed that the expression levels of ARID1A protein were 0.439 4±0.000 7, 0.424 4±0.005 0 and 0.386 0±0.005 8 respectively. They were also lower than that in the negative control group (0.732 4 ±0.030 3; all P<0.01). The siN3 with the highest transfection efficiency was selected to use in the following experiment. (2) The CCK-8 method showed that the proliferative activity of siN3 transfection group cells after transfected transiently at 6 hours was not statistically significant difference compared with those in negative control group and blank control group (0.506±0.010, 0.491±0.006, 0.498±0.009, respectively; all P>0.05). However, the proliferative activity of siN3 transfection group cells after transfected transiently at 24, 48, 72, 96 hours were higher than those in negative control group and blank control group (all P<0.01). The flow cytometry results showed that the apoptosis rate of siN3 transfection group cells was (20.0±3.9)%, which was significantly lower than those in negative control group and blank control group [(31.5±5.0)%, (34.0±4.2)%, respectively; all P<0.05]. The transwell experiment showed that the penetrated cell counts of siN3 transfection group was 60.4±2.9, which was apparently higher than those in negative control group and blank control group (54.2±3.5, 52.1±3.8, respectively; all P<0.01). Western blot experiment showed that the relative expression levels of NF-κB, MT1-MMP and MMP2 protein in siN3 transfection group were respectively 1.85±0.16, 0.37±0.08, 1.38± 0.11, which were apparently higher than those in negative control group (0.93±0.11, 0.17±0.05, 0.86±0.06; all P<0.05) and blank control group (0.94 ± 0.04, 0.15 ± 0.08, 0.85 ± 0.10, respectively; all P<0.01). It would be to promote the cell doubling time, reduce cell apoptosis and increase the invasive capability in ES2 cells that ARID1A expression was down-regulating by ARID1A mRNA interference. The invasion mechanism may be related to the activation of NF-κB signal transduction pathway, up-regulation of MT1-MMP expression and then promoting the invasion of tumor cells via the up-regulation of MMP2 expression.

Abstract B02: The role of chromatin remodeler protein ARID1a in ovarian clear cell carcinoma

Abstract Ovarian cancer is the fifth leading cause of cancer death among women in the United States and ovarian clear cell carcinoma (OCCC) is an aggressive subtype of epithelial ovarian cancer. Recent whole genome and whole exome sequencing studies have revealed that the chromatin remodeler protein AT-Rich Interactive Domain 1a (ARID1a) is frequently mutated in OCCC. ARID1a loss is also often observed in endometriosis which is strongly associated with OCCC. Consequently, we hypothesize that loss of ARID1a is an early step in transformation of endometriosis into OCCC. Here, we show that knocking down ARID1a in an immortalized endometriosis line, promotes greater invasion of basement membrane in vitro, and higher efficiency of anchorage-independent growth. Using two independent genome wide approaches to assay chromatin accessibility and DNA methylation, we demonstrate that the loss of ARID1a does not significantly alter global chromatin accessibility and DNA methylation with the exception of the genomic regions containing late replicating domains. Our findings do however indicate that the loss of ARID1a contributes to altered distribution of H3K27me3 and H3K27ac marks. Specifically we find an increased deposit of the polycomb repressive mark in the knock-down line outside of promoters. Interestingly, we also find the knock-down line has a higher level of the active mark H3K27ac at the promoters and a lower level at the enhancers relative to the scrambled control. Moreover, many of the genes differentially expressed upon ARID1a knock-down are consistent with the changes of H3K27ac at the respective gene promoter. We therefore conclude that loss of ARID1a expression contributes to cellular transformation through deregulation of gene expression primarily through alteration of histone marks. Citation Format: Ranjani Lakshminarasimhan, Claudia Andreu-Vieyra, Kate Lawrenson, Simon A. Gayther, Peter A. Jones, Gangning Liang. The role of chromatin remodeler protein ARID1a in ovarian clear cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B02.

Various ARID1A expression patterns and their clinical significance in gastric cancers

AT-rich interactive domain 1A (ARID1A) is frequently mutated in gastric cancers, and loss of ARID1A expression is considered a poor prognostic factor in various cancers. However, in practice, ARID1A shows various expression patterns, and our understanding of its significance is limited. We performed immunohistochemistry for ARID1A, MLH1, and pS6 using whole tissue blocks of 350 gastric cancers and classified the ARID1A expression as follows: retained (63.7%), reduced (17.7%), complete loss (14.9%), and partial loss (3.7%). Complete/partial loss was more common in poorly differentiated histology (P < .001), and reduced or complete loss of ARID1A was frequent in cases with MLH1 loss (P < .001). The ARID1A-reduced group showed only slightly inferior disease-free survival (DFS; P = .254) and overall survival (OS; P = .377) compared to those of the ARID1A-retained group, whereas the group with complete loss showed significantly worse DFS (hazard ratio [HR], 1.732; P = .015) and OS (HR, 1.751; P = .013). Worse DFS (HR, 2.672; P = .005) and OS (HR, 2.531; P = .002) were also noted in the group with partial loss. High expression of pS6 was observed more frequently in groups showing altered ARID1A expression patterns (P < .001). In conclusion, reduced ARID1A expression is not a major prognostic determinant, although it may lead to AKT pathway activation. Tumor cells lacking ARID1A expression may influence the prognosis even if they constitute only a small proportion of the tumor sample. Our data provide an enhanced roadmap for understanding ARID1A with implications for future research and therapeutics.