AT-rich Interactive Domain 1A Research Articles

Microarray pathway analysis indicated that mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin growth factor 1 signaling pathways were inhibited by small interfering RNA against AT-rich interactive domain 1A in endometrial cancer.

Mutations in the gene encoding AT-rich interactive domain 1A (ARID1A) are frequently observed in endometrial cancer (EC) but the molecular mechanisms linking the genetic changes remain to be fully understood. The present study aimed to elucidate the influence of ARID1A mutations on signaling pathways. Missense, synonymous and nonsense heterozygous ARID1A mutations in the EC HEC-1-A cell line were verified by Sanger sequencing. Mutated ARID1A small interfering RNA was transfected into HEC-1-A cells. Biochemical microarray analysis revealed 13 upregulated pathways, 17 downregulated pathways, 14 significantly affected disease states and functions, 662 upstream and 512 downstream genes in mutated ARID1A-depleted HEC-1-A cells, among which the mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin-like growth factor-1 (IGF1) signaling pathways were the 2 most downregulated pathways. Furthermore, the forkhead box protein O1 pathway was upregulated, while the IGF1 receptor, insulin receptor substrate 1 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit b pathways were downregulated. Carcinoma tumorigenesis, tumor cell mitosis and tumor cell death were significantly upregulated disease states and functions, while cell proliferation and tumor growth were significantly downregulated. The results of the present study suggested that ARID1A may be a potential prognostic and therapeutic molecular drug target for the prevention of EC progression.

Arid1a regulates response to anti-angiogenic therapy in advanced hepatocellular carcinoma

AT-rich interaction domain 1a (Arid1a), a component of the chromatin remodeling complex, has emerged as a tumor suppressor gene. It is frequently mutated in hepatocellular carcinoma (HCC). However, it remains unknown how Arid1a suppresses HCC development and whether Arid1a deficiency could be exploited for therapy, we aimed to explore these questions. The expression of Arid1a in human and mouse HCCs was determined by immunohistochemical (IHC) staining. Gene expression was determined by quantitative PCR, ELISA or western blotting. Arid1a knockdown HCC cell lines were established by lentiviral-based shRNA. Tumor angiogenesis was quantified based on vessel density. The regulation of angiopoietin (Ang2) expression by Arid1a was identified by chromatin immunoprecipitation (ChIP) assay. The tumor promoting function of Arid1a loss was studied with a xenograft model in nude mice and diethylnitrosamine (DEN)-induced HCC in Arid1a conditional knockout mice. The therapeutic values of Ang2 antibody and sorafenib treatment were evaluated both in vitro and in vivo. We demonstrate that Arid1a deficiency, occurring in advanced human HCCs, is associated with increased vessel density. Mechanistically, loss of Arid1a causes aberrant histone H3K27ac deposition at the angiopoietin-2 (Ang2) enhancer and promoter, which eventually leads to ectopic expression of Ang2 and promotes HCC development. Ang2 blockade in Arid1a-deficient HCCs significantly reduces vessel density and tumor progression. Importantly, sorafenib treatment, which suppresses H3K27 acetylation and Ang2 expression, profoundly halts the progression of Arid1a-deficient HCCs. Arid1a-deficiency activates Ang2-dependent angiogenesis and promotes HCC progression. Loss of Arid1a in HCCs confers sensitivity to Ang2 blockade and sorafenib treatment. AT-rich interaction domain 1a (Arid1a), is a tumor suppressor gene. Arid1a-deficiency promotes Ang2-dependent angiogenesis leading to hepatocellular carcinoma progression. Arid1a-deficiency also sensitizes tumors to Ang2 blockade by sorafenib treatment.

Repair of Calvarial Bone Defect Using Jarid1a-Knockdown Bone Mesenchymal Stem Cells in Rats.

Histone methylation is regarded as an important epigenetic event during stem cell differentiation. Jumonji AT-rich interactive domain 1A (Jarid1a) is a histone demethylase that specifically catalyzes demethylation of dimethyl or trimethyl histone H3K4me3, which is normally associated with transcriptionally active genes. Runt-related transcription factor 2 (Runx2) has been identified as a key transcription factor in the early stage of osteogenesis. A better understanding of this epigenetic mechanism that governs osteogenic differentiation of bone mesenchymal stem cells (BMSCs) can provide new insights into BMSC-based bone tissue engineering. To define the function and regulatory mechanisms of Jarid1a in the osteogenic differentiation of BMSCs, we compared the expression of Jarid1a between undifferentiated and osteoinductive BMSCs. The expression of osteogenic transcriptional factors in BMSCs after Jarid1a knockdown was explored using western blot. To determine whether Jarid1a was associated with Runx2 during osteogenic differentiation, endogenous coimmunoprecipitation (co-IP) experiments were performed with osteoinductive BMSCs extracts. Then, we systematically evaluated the function of si-Jarid1a in enhancing BMSCs osteogenesis and the therapeutic potential of si-Jarid1a-modified BMSCs in a rat calvarial bone defect model with β-tricalcium phosphate scaffolds. Knockdown of Jarid1a by small interfering RNA enhanced osteogenic differentiation of BMSCs in vitro. Knockdown of Jarid1a significantly improved the mRNA and protein expression of bone-specific factors. Furthermore, co-IP in BMSCs lysate suggested that Jarid1a was physically and functionally associated with Runx2. The repair potential of bone defect was dramatically improved by Jarid1a-knockdown BMSCs, including increased bone volume, increased bone mineral density, and decreased scaffold residue in vivo. Altogether, this study explores the functional and regulatory role of Jarid1a in osteogenic differentiation and bone regeneration of BMSCs, and provides a new approach for bone defect repairing using epigenetic modification in vitro and in vivo.

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.

A key HDAC6 dependency of ARID1A-mutated ovarian cancer.

Mutations in AT rich interactive domain 1A (ARID1A) are common in ovarian cancers. A study now shows that HDAC6 activity is required for survival of ovarian cancer cells bearing ARID1A mutations. HDAC6 inhibition selectively promotes apoptosis of ARID1A-inactivated cells, supporting the use of HDAC6 inhibition in the treatment of clear cell ovarian cancer.

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.

Down-regulation of ARID1A is sufficient to initiate neoplastic transformation along with epigenetic reprogramming in non-tumorigenic endometriotic cells

The chromatin remodeler AT-Rich Interactive Domain 1A (ARID1A) is frequently mutated in ovarian clear cell carcinoma (OCCC) and endometriosis precursor lesions. Here, we show that knocking down ARID1A in an immortalized endometriosis cell line is sufficient to induce phenotypic changes indicative of neoplastic transformation as evidenced by higher efficiency of anchorage-independent growth, increased propensity to adhere to collagen, and greater capacity to invade basement membrane extract in vitro. ARID1A knockdown is associated with expression dysregulation of 99 target genes, and many of these expression changes are also observed in primary OCCC tissues. Further, pathway analysis indicates these genes fall within networks highly relevant to tumorigenesis including integrin and paxillin pathways. We demonstrate that the down-regulation of ARID1A does not markedly alter global chromatin accessibility or DNA methylation but unexpectedly, we find strong increases in the active H3K27ac mark in promoter regions and decreases of H3K27ac at potential enhancers. Taken together, these data provide evidence that ARID1A mutation can be an early stage event in the oncogenic transformation of endometriosis cells giving rise to OCCC.

Next-generation sequencing of urine specimens: A novel platform for genomic analysis in patients with non-muscle-invasive urothelial carcinoma treated with bacille Calmette-Guérin.

Biopsies from patients with high-risk (HR) non-muscle-invasive urothelial carcinoma (NMIUC), especially flat urothelial carcinoma in situ, frequently contain scant diagnostic material or denuded mucosa only, and this precludes further extensive genomic analysis. This study evaluated the use of next-generation sequencing (NGS) analysis of urine cytology material from patients with HR NMIUC in an attempt to identify genetic alterations that might correlate with clinical features and responses to bacille Calmette-Guérin (BCG) treatment. Forty-one cytology slides from patients with HR NMIUC treated with intravesical BCG were selected for this study. Histological confirmation was available for all cases. The specimens were subjected to NGS analysis with a customized targeted exome capture assay composed of 341 genes. In this cohort, genomic alterations were successfully identified in all cytology samples. Mutations were detected down to a 2% allele frequency and chromosomal rearrangements including copy number alterations and gene fusions were identified. The most frequently altered genes included telomerase reverse transcriptase (TERT), tumor protein 53 (TP53), Erb-B2 receptor tyrosine kinase 2 (ERBB2), and chromatin remodeling genes such as lysine demethylase 6A (KDM6A) and AT-rich interaction domain 1A (ARID1A). For patients with matched tumor tissue, cytology specimens revealed all mutations detected in tissue as well as additional mutations, and this suggested that urine might more effectively capture the full genetic heterogeneity of disease than an individual cystectomy. Alterations in multiple genes correlated with clinical and histopathological features, including responses to BCG treatment, flat architecture versus papillary architecture, and smoking history. Urine specimens can replace tissue as a substrate for NGS analysis of HR NMIUC. Several genomic alterations identified in urine specimens might be associated with histological features and clinical characteristics. Cancer Cytopathol 2017;125:416-26. © 2017 American Cancer Society.

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.

Genome-wide analysis of gynecologic cancer: The Cancer Genome Atlas in ovarian and endometrial cancer.

Cancer typically develops due to genetic abnormalities, but a single gene abnormality cannot completely account for the onset of cancer. The Cancer Genome Atlas (CGA) project was conducted for the cross-sectional genome-wide analysis of numerous genetic abnormalities in various types of cancer. This approach has facilitated the identification of novel AT-rich interaction domain 1A gene mutations in ovarian clear cell carcinoma, frequent tumor protein 53 (TP53) gene mutations in high-grade ovarian serous carcinoma, and Kirsten rat sarcoma and B-rapidly accelerated fibrosarcoma proto-oncogene, serine/threonine kinase gene mutations in low-grade ovarian serous carcinoma. Genome-wide analysis of endometrial cancers has led to the establishment of four subgroups: Polymerase ultramutated, microsatellite instability hypermutated, genome copy-number low and genome copy-number high. These results may facilitate the improvement of the prediction of patient prognosis and therapeutic sensitivity in various types of gynecologic cancer. The enhanced use of currently available therapeutic agents and the development of novel drugs may be facilitated by the novel classification of ovarian cancer based on TP53 mutations, the efficacy of poly (ADP-ribose) polymerase inhibitors for tumors with breast cancer 1/2 mutations and the effect of phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin inhibitors for tumors with mutations in the PI3K/protein kinase B signaling pathway. Important results have been revealed by genome-wide analyses; however, the pathogenic underlying mechanisms of gynecologic cancer will require further studies and multilateral evaluation using epigenetic, transcriptomic and proteomic analyses, in addition to genomic analysis.

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.

879P - Loss of ARID1A expression is associated with poor prognosis in patients with stage I/II clear cell carcinoma of the ovary

Clear cell carcinoma of the ovary (CCC) has a poor prognosis because of its resistance to conventional platinum- or taxane-based chemotherapy. Consequently, there is a need to discover biomarkers for predicting the outcome of patients with CCC and develop novel treatment strategies for this disease. Recent studies have shown that somatic mutations in the AT-rich interactive domain 1A (SWI-like) gene (ARID1A) are the most common genetic changes in CCC. This gene is located in chromosome 1p36 and encodes a member of the switch/sucrose-nonfermentable (SWI/SNF) family protein BAF250a (ARID1A). Here, we investigated whether ARID1A could be a prognostic biomarker for this disease. Paraffin-embedded specimens were collected from 220 Japanese patients with epithelial ovarian cancer, including 112 CCC and 108 high-grade serous adenocarcinoma of the ovary (HG-SAC). We analyzed the protein expression of ARID1A in these samples by immunohistochemical staining, and evaluated the association of these molecular parameters with clinical outcome. The loss of ARID1A expression was found in 39.3 % (44/112) of CCC, and strong expression of the protein was not observed. ARID1A protein was present mainly in the cell nuclei of the tumors; however, in tumors with HG-SAC, only 8 (7.4 %) tumors showed loss of ARID1A expression. The rate of absent expression of ARID1A in CCC tumors was significantly higher than in HG-SAC tumors (P < 0.0001). We found no significant association between ARID1A expression and patient age, FIGO stage, and status of residual tumor in CCC. The 5-year survival rate for FIGO stage I or II CCC patients with negative tumor expression of ARID1A was lower than those with positive tumor expression of ARID1A (74 % vs 91 %), but this difference was not observed in FIGO stage III or IV patients. Multivariable analysis revealed that FIGO stage and residual tumor were independent prognostic factors, but ARID1A expression was not. However, ARID1A expression was an independent prognostic factor in FIGO stage I or II CCC patients. The ARID1A protein may be a promising prognostic marker for FIGO stage I and II CCC.

Next-generation sequencing survey of biliary tract cancer reveals the association between tumor somatic variants and chemotherapy resistance.

Biliary tract cancers (BTCs) are uncommon and are associated with a dismal prognosis. Combinations of gemcitabine and platinum chemotherapy (gemcitabine and platinum-based therapy [GP]) form the standard approach for treating advanced BTC. To characterize the spectrum of mutations and to identify potential biomarkers for a GP response in BTC, this study evaluated the genomic landscape and assessed whether mutations affecting DNA repair were associated with GP resistance. Pretreatment, formalin-fixed, paraffin-embedded samples from 183 BTC patients treated with GP were analyzed. Cox regression models were used to determine the association between mutations, progression-free survival (PFS), and overall survival (OS). When genes with an incidence > 10% were considered, no individual gene was independently predictive of a GP response. In patients with unresectable BTC who received GP as their first-line therapy, the joint status of cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein 53 (TP53), and AT-rich interaction domain 1A (ARID1A) was associated with PFS (P = .0004) and OS (P ≤ .0001). Patients with mutations in CDKN2A and TP53 were identified as a poor-prognosis cohort with a median PFS of 2.63 months and a median OS of 5.22 months. Patients with mutant ARID1A, regardless of the single-mutation status of TP53 or CDKN2A, had similar outcomes. A patient who exhibited mutations in all 3 genes had a median PFS of 20.37 months, and OS was not reached. In the largest exploratory analysis of this kind for BTC, 3 prevalent, mutually exclusive mutations represent distinct patient cohorts. These mutations are prognostic and may represent a predictive biomarker for a GP response. Prospective studies to validate these findings are needed, and they should include the incorporation of therapies that exploit the genomic instability observed with these mutations in BTC. Cancer 2016;122:3657-66. © 2016 American Cancer Society.

Loss of ARID1A expression predicts poor survival prognosis in gastric cancer: a systematic meta-analysis from 14 studies.

The chromatin remodeling gene, AT-rich interactive domain 1A gene (ARID1A), frequently mutates inactively in gastric cancer (GC). However, its prognostic value remains controversial. To address this issue, a comprehensive meta-analysis was performed. Studies published until March 2016 were systematically searched. A total of 15 cohorts from 14 literatures involving 3183 patients were subjected to this meta-analysis. The pooled data showed that ARID1A expression loss predicted poor overall survival (OS) in GC (Hazard Ratio (HR) = 1.60; 95% Confidence Interval (CI) = 1.40–1.81; P < 0.001), with low heterogeneity among these studies (I2 = 21.5%; P = 0.214). Stratification analyses revealed that ARID1A expression loss was associated with poor OS in Asians (HR = 1.65, 95% CI = 1.44–1.89), proportion of proximal disease ≤30% subgroup (HR = 1.80, 95% CI = 1.36–2.38) and Epstein-Barr virus (EBV) (+) > 5% subgroup (HR = 1.59, 95% CI = 1.18–2.15). The robust results were suggested by sensitivity analyses and no evidence of significant publication bias was detected. This study demonstrated a significant relationship between deletion of ARID1A expression and poor OS in GC. Moreover, ethnicity, tumor location and EBV infection status might be potential key factors influencing this correlation.

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.

Deletion of Arid1a in Reproductive Tract Mesenchymal Cells Reduces Fertility in Female Mice.

Women with endometriosis can suffer from decreased fecundity or complete infertility via abnormal oocyte function or impaired placental-uterine interactions required for normal pregnancy establishment and maintenance. Although AT-rich interactive domain 1A (SWI-like) (ARID1A) is a putative tumor suppressor in human endometrial cancers and endometriosis-associated ovarian cancers, little is known about its role in normal uterine function. To study the potential function of ARID1A in the female reproductive tract, we generated mice with a conditional knockout of Arid1a using anti-Müllerian hormone receptor 2-Cre. Female Arid1a conditional knockout mice exhibited a progressive decrease in number of pups per litter, with a precipitous decline after the second litter. We observed no tumors in virgin mice, although one knockout mouse developed a uterine tumor after pregnancy. Unstimulated virgin female knockout mice showed normal oviductal, ovarian, and uterine histology. Uteri of Arid1a knockout mice showed a normal decidualization response and appropriate responses to estradiol and progesterone stimulation. In vitro studies using primary cultures of human endometrial stromal fibroblasts revealed that small interfering RNA knockdown of ARID1A did not affect decidualization in vitro. Timed pregnancy studies revealed the significant resorption of embryos at Embryonic Day 16.5 in knockout mice in the third pregnancy. In addition to evidence of implantation site hemorrhage, pregnant Arid1a knockout mice showed abnormal placental morphology. These results suggest that Arid1a supports successful pregnancy through its role in placental function.

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.

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.

Genomic characterization of sarcomatoid transformation in clear cell renal cell carcinoma

The presence of sarcomatoid features in clear cell renal cell carcinoma (ccRCC) confers a poor prognosis and is of unknown pathogenesis. We performed exome sequencing of matched normal-carcinomatous-sarcomatoid specimens from 21 subjects. Two tumors had hypermutation consistent with mismatch repair deficiency. In the remainder, sarcomatoid and carcinomatous elements shared 42% of somatic single-nucleotide variants (SSNVs). Sarcomatoid elements had a higher overall SSNV burden (mean 90 vs. 63 SSNVs, P = 4.0 × 10(-4)), increased frequency of nonsynonymous SSNVs in Pan-Cancer genes (mean 1.4 vs. 0.26, P = 0.002), and increased frequency of loss of heterozygosity (LOH) across the genome (median 913 vs. 460 Mb in LOH, P < 0.05), with significant recurrent LOH on chromosomes 1p, 9, 10, 14, 17p, 18, and 22. The most frequent SSNVs shared by carcinomatous and sarcomatoid elements were in known ccRCC genes including von Hippel-Lindau tumor suppressor (VHL), polybromo 1 (PBRM1), SET domain containing 2 (SETD2), phosphatase and tensin homolog (PTEN). Most interestingly, sarcomatoid elements acquired biallelic tumor protein p53 (TP53) mutations in 32% of tumors (P = 5.47 × 10(-17)); TP53 mutations were absent in carcinomatous elements in nonhypermutated tumors and rare in previously studied ccRCCs. Mutations in known cancer drivers AT-rich interaction domain 1A (ARID1A) and BRCA1 associated protein 1 (BAP1) were significantly mutated in sarcomatoid elements and were mutually exclusive with TP53 and each other. These findings provide evidence that sarcomatoid elements arise from dedifferentiation of carcinomatous ccRCCs and implicate specific genes in this process. These findings have implications for the treatment of patients with these poor-prognosis cancers.