Poster Abstract Abstract
(Basic Science), #220524712725
Steroid Hormone Estrogen Induces Metastatic Process in Breast Cancer Through Regulation of Gene Splicing Event In Vitro
Siti Sarah Binti Hamzah, Institute for Medical Research (IMR); Dimitris K Grammatopolous, University of Warwick, UK
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Misregulation of alternative pre- mRNA splicing (AS) plays important roles in tumor progression and metastasis. The connection between AS and cancer cells metastasis was first established when specific CD44 splice variants were detected in metastatic pancreatic cancer cells that was not present in the primary tumor. Notably, estrogen signaling has been reported to involve in abnormal gene splicing which leads to metastatic phenotype change in breast cancer cells. Therefore, this study aimed to investigate the mechanism by which estrogen affects gene-splicing that promotes progression of estrogen receptor positive (ER+) breast cancer cells, in vitro.
For all experiments, ER+ breast cancer cell line, MCF7 was cultured and stimulated with 10nM estrogen (17- beta estradiol, E2) for 24hrs. Protein samples were run for proteomic analysis using LC-MS/MS, as well as for protein and gene expression by western blot and RT-PCR, respectively. For monitoring the abnormality in gene splicing, CD44 gene was used as a splicing reporter. Finally, the change in cellular behavior was monitored for 24hrs using Xcelligence real-time cell monitoring system.
Proteomic analysis showed that serine-arginine protein kinase 1 (SRPK1), one of the key kinases in regulating alternative splicing mechanism was among the ER-signaling targets, and found to be upregulated 7-fold in the stimulated cells. In addition, both SRPK1 protein and gene expression was also found upregulated. The level of CD44 splice isoform, CD44s was found increased by 50%, but no significant change detected in CD44v6 level, suggesting positive correlation between increased SRPK1 and CD44s expression. Finally, cell monitoring assay showed a slight increase in proliferation after 24hrs of estrogen treatment.
This study demonstrated that estrogen can induce overexpression of SRPK1 and triggered abnormal splicing of CD44 gene which eventually accelerates breast cancer progression through to its increased proliferation ability.