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ARID2 suppression promotes tumor progression and upregulates cytokeratin 8, 18 and β-4 integrin expression in TP53-mutated tobacco-related oral cancer and has prognostic implications

Abstract

Mutations in ARID2 and TP53 genes are found to be implicated in the tobacco related tumorigeneses. However, the effect of loss of ARID2 in the TP53 mutated background in tobacco related cancer including oral cancer has not been investigated yet. Hence, in this study we knockdown ARID2 using shRNA mediated knockdown strategy in TP53 mutated oral squamous cell carcinoma (OSCC) cell line and studied its tumorigenic role. Our study revealed that suppression of ARID2 in TP53 mutated oral cancer cells increases cell motility and invasion, induces drastic morphological changes and leads to a marked increase in the expression levels of cytokeratins, and integrins, CK8, CK18 and β4-Integrin, markers of cell migration/invasion in oral cancer. ARID2 suppression also showed early onset and increased tumorigenicity in-vivo. Interestingly, transcriptome profiling revealed differentially expressed genes associated with migration and invasion in oral cancer cells including AKR1C2, NCAM2, NOS1, ADAM23 and genes of S100A family in ARID2 knockdown TP53 mutated oral cancer cells. Pathway analysis of differentially regulated genes identified “cancer pathways” and “PI3K/AKT Pathway” to be significantly dysregulated upon suppression of ARID2 in TP53 mutated OSCC cells. Notably, decreased ARID2 expression and increased CK8, CK18 expression leads to poor prognosis in Head and Neck cancer (HNSC) patients as revealed by Pan-Cancer TCGA data analysis. To conclude, our study is the first to demonstrate tumor suppressor role of ARID2 in TP53 mutated background indicating their cooperative role in OSCC, and also highlights its prognostic implications suggesting ARID2 as an important therapeutic target in OSCC.

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Fig. 1: shRNA mediated ARID2 knockdown in ITOC-02 cell line using pLKO vector.
Fig. 2: Suppression of ARID2 in TP53 mutated background leads to enhanced tumorigenic potential in-vivo in OSCC.
Fig. 3: ARID2 knockdown leads to change in morphology of the cells as examined by phase contrast microscopy and transmission electron microscopy.
Fig. 4: ARID2 knockdown in TP53 mutated OSCC upregulates markers of neoplastic progression.
Fig. 5: RNA sequencing analysis revealed altered expression of genes involved in migration and invasion in ARID2 knockdown TP53 mutated OSCC.
Fig. 6: Pan-Cancer TCGA analysis revealed poor prognosis associated with decreased expression of ARID2 andincreased expression of CK8, CK18, β4-Integrin in HNSC patients.

Data availability

Most data have been incorporated into the article and its online supplementary material. Remaining data are available on reasonable request to corresponding author.

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Acknowledgements

We would like to thank Genomics & NGS facility, electron microscopy facility, animal House, biorepository, ACTREC, Dr. Pradnya Kowtal and members of Sarin Lab. We are also grateful to TCGA/ULACAN databases to make data available free for analysis. We also thank NIBMG, and grant support from Department of Biotechnology, Govt. of India (Grant Number (BT/MB/01/ICGC/2009). We are grateful to Dr. T. Mahmoudi, Netherland for providing ARID2 antibody and Dr. Sorab Dalal, ACTREC, Mumbai for providing pLKO-PURO EGFP vector. The study has been supported by grant from Department of Biotechnology, Govt. of India (Grant Number (BT/MB/01/ICGC/2009) under the International Cancer Genome Consortium (ICGC) project.

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PS conceptualized the idea, conducted cloning and molecular biology experiments, TCGA data analysis, designed and wrote the manuscript. PD performed cell culture and all functional assays and helped in animal experiments. PC supervised BSM in conducting all animal experiments. NG analyzed bioinformatics data. RS conceived the project, supervised all the experiments and edited the manuscript.

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Correspondence to Rajiv Sarin.

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Shukla, P., Dange, P., Mohanty, B.S. et al. ARID2 suppression promotes tumor progression and upregulates cytokeratin 8, 18 and β-4 integrin expression in TP53-mutated tobacco-related oral cancer and has prognostic implications. Cancer Gene Ther (2022). https://doi.org/10.1038/s41417-022-00505-x

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