Explore the alterations of downstream molecular pathways caused by ARID1A mutation/knockout in human endometrial
Baoling Xing1, Xiaoying Zhang2, Xia Gu2
1Department of Pathology, Affiliated Zhoupu Hospital of Shanghai University of Medicine and Health Sciences, Shanghai, 201318, China. 793506861@qq.com.
Purpose:
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.
Methods:
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 10 bp deletion on the ARID1A gene, which resulted in a code-shift mutation and gene knockout.
Results:
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.
Conclusion:
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.
Insights
ARID1A gene mutations promote endometrial cancer (EC) development. Knocking out ARID1A in EC cells enhanced proliferation and microsatellite instability, suggesting ARID1A as a potential therapeutic target.
Area of Science:
- Gynecologic Oncology
- Cancer Genetics
- Molecular Biology
Background:
- Endometrial cancer (EC) is a common gynecologic malignancy driven by genetic alterations.
- AT-rich interaction domain 1A (ARID1A) gene mutations are early events in endometrial carcinogenesis.
- Targeting these genetic alterations offers potential therapeutic strategies for EC.
Purpose of the Study:
- To investigate the downstream molecular pathway alterations caused by ARID1A mutations.
- To explore the therapeutic implications of ARID1A alterations in endometrial cancer.
- To establish an ARID1A-deficient endometrial cancer cell line for further study.
Main Methods:
- Utilized CRISPR/Cas9 technology to edit the ARID1A gene in the human endometrial cancer cell line Ishikawa.
- Successfully created a stable Ishikawa cell line with a confirmed 10 bp deletion in ARID1A, resulting in gene knockout (KO).
- Analyzed cell proliferation, apoptosis, cell cycle, protein expression, microsatellite instability, and signaling pathway activation.
Main Results:
- ARID1A KO cells exhibited reduced apoptosis, accelerated G0/G1 to S phase transition, and enhanced proliferation compared to wild-type cells.
- ARID1A deficiency decreased p21, caspase 7, and caspase 9 protein levels and led to high microsatellite instability (MSI-H).
- Transcriptomic and experimental analyses revealed activated phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling, reduced MLH1 and PR expression, and increased p-Akt in ARID1A KO cells.
Conclusions:
- ARID1A deficiency impacts key cellular processes including proliferation, apoptosis, and DNA repair in endometrial cancer.
- The findings highlight ARID1A as a promising therapeutic target for endometrial cancer treatment.
- Suggests potential for combination therapies, such as with immune checkpoint inhibitors, for enhanced EC treatment efficacy.
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