Explore the alterations of downstream molecular pathways caused by ARID1A mutation/knockout in human endometrial cancer cells.

Abstract

As one of the most common gynecologic malignancies, endometrial cancer (EC) is driven by multiple genetic alterations that may be targeted for treatments. AT-rich interaction domain 1A (ARID1A) gene mutations were reported as early events in endometrial carcinogenesis. To explore the alterations of downstream molecular pathways caused by ARID1A mutations and the associated therapeutic implications, we edited ARID1A gene in human endometrial cancer cell line Ishikawa using the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-Associated Proteins (CRISPR/Cas9) technology. We successfully constructed a stable Ishikawa cell line with a confirmed 10bp deletion on the ARID1A gene, which resulted in a code-shift mutation and gene knockout. Compared with unedited wild-type cells, ARID1A knockout (KO) led to reduced apoptosis, accelerated transformation from G0/G1 to S phase, and enhanced cell proliferation. ARID1A deficiency would reduce the protein levels of p21, caspase 7, and caspase 9 in Ishikawa endometrial cancer cells compared with the wild-type cells. In addition, ARID1A KO resulted in high levels of microsatellite instability (MSI-H). Moreover, transcriptomic analyses showed that ARID1A KO can lead to activated phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling. Furthermore, experimental analyses demonstrated that ARID1A KO cells had reduced expression of genetic instability-associated markers mutL homologue 1 (MLH1) and progesterone receptor B (PR) and increased p-Akt expression. These findings support further exploration of ARID1A as a therapeutic target for EC and provide insight into developing more effective treatments in EC, such as the combinatory use of immune checkpoint inhibitors.