Abstract 214: ARID1A and ARID1B dependent proteomics
Authors: Shoji Yutaka , Kato-Shoji Kumiko , Conrads Kelly A. , Challa Rusheeswar , Hood Brian L. , Bateman Nicholas W. , Conrads Thomas P. , Risinger John I. 
Published: July 2017

Abstract
Protein sub-units of the SWI/SNF nucleosome and chromatin remodeling complexes are frequently mutated in cancer. The ARID1A and ARID1B DNA interacting component are among these mutated proteins. Alterations in ARID1A have been reported in breast, colon, lung, kidney, pancreatic, bladder, cervical, ovarian and uterine cancers whereas ARID1B mutation is less common. Additional data suggests ARID1A mutant cancers are dependent on functional ARID1B and that targeting ARID1B may be a therapeutic strategy. However, ARID1A and 1B are often co-inactivated in aggressive un/dedifferentiated carcinomas of the ovary and endometrium suggesting tumor suppressive functions might exist not only in ARID1A but also in 1B. In this study, we examined the effects of restoring ARID1A or 1B in an undifferentiated endometrial adenocarcinoma cell line harboring inactivating mutations in both genes. ACI-98 cells derived from a stage IV undifferentiated endometrial cancer were found to lack expression of ARID1A and 1B protein. We subsequently identified two individual truncating mutations in ARID1A and 1B by genomic DNA and cDNA sequencing indicating no wild-type message was expressed. Restoration of ARID1A in ACI-98 cells using a Tet-on system revealed remarkable growth inhibition, however, ARID1B restoration showed only a moderate cell growth inhibition with cells taking on a flattened phenotype. Clones of ARID1A/1B restored cells were treated w/wo doxycycline (Dox) for 48h a time point prior to cell death or morphologic changes and protein changes catalogued using LC MS/MS. We identified 771 and 1168 differentially expressed proteins (z-score &lt;0.05) from ARID1A or 1B restored clones compared to dox free control and 138 proteins were common in these two groups (up-regulated: 57, down-regulated: 37, reverse regulation between ARID1A and 1B: 44). We performed pathway analysis of the ARID1A and 1B proteome by Advaita Pathway Guide software and this analysis identified 10 significantly impacted pathways in ARID1A and 17 pathways in ARID1B. However, there was no common pathway between the two. Wnt signaling pathway (KEGG: 04310) was among ARID1A distinguished pathways with 9 of 46 proteins being differentially expressed. Among these NKD1 a negative regulator of the Wnt-β-catenin-Tcf signaling pathway was increased in ARID1A restored cells more than 10 fold. We confirmed the differential expression of NKD1 using qRT-PCR following ARID1A expression. The p53 signaling pathway (KEGG: 04916) was significantly (11 of 34 proteins) affected following ARID1B complementation. We confirmed the differential expression of FAS was up-regulated 6 fold in proteomics and 3.4 fold in qPCR. These results suggest that restoration of ARID1A or 1B both effect cell proliferation to varying degree. The lack of overlap between the proteomes of ARID1A/1B restored cells indicates distinct pathways underlying these induced phenotypes and different function of SWI/SNFARID1A or SWI/SNFARID1B.
Citation Format: Yutaka Shoji, Kumiko Kato-Shoji, Kelly A. Conrads, Rusheeswar Challa, Brian L. Hood, Nicholas W. Bateman, Thomas P. Conrads, John I. Risinger. ARID1A and ARID1B dependent proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 214. doi:10.1158/1538-7445.AM2017-214