Abstract 5133: The RNA-binding protein HuR facilitates proliferation and metastasis in human pancreatic ductal adenocarcinoma

Abstract

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer-related death in the Unites States, with an overall 5-year survival rate of <6%. The ubiquitously expressed RNA-binding protein HuR (ELAVL1) has been implicated in numerous cancer types as a post-transcriptional facilitator of tumorigenesis, through its stabilization of various proliferation-, survival-, and metastasis-associated mRNA transcripts. Our lab has previously demonstrated that elevated HuR expression in human resected PDA samples is associated with poor prognostic factors (e.g. increased T stage). Here, we explored the role of HuR expression in human PDA growth and metastasis by generating isogenic PDA cell lines with induced silencing or overexpression of HuR. Methods: Stable MiaPaCa2 cell lines were generated that produce two different shRNAs against the HuR coding sequence (labeled Mia.sh290 and Mia.sh700) in response to doxycycline (DOX) induction. HuR silencing was validated by RT-qPCR and Western blotting. Similarly, a DOX-inducible MiaPaCa2 cell line that overexpresses HuR cDNA (Mia.HuR) was generated and validated. Sustained ∼50% knockdown and 1.5-2-fold overexpression were achieved, respectively. Following characterization of HuR expression in these cell lines under ±DOX conditions, we performed a 7-day cell proliferation (PicoGreen) assay and assessed anchorage-independence over a 4-week period by soft agar colony formation assay. Metastatic potential was measured by Matrigel invasion and in vitro scratch test assays. Results: Upon DOX induction, Mia.sh290 and Mia.sh700 cell lines showed significantly reduced cell proliferation over 7 days (22% and 15% reduction compared to non-induced in sh290 and sh700 respectively, p<0.001). Accordingly, HuR overexpression in Mia.HuR showed slightly increased cell proliferation over 7 days (15% increase compared to non-induced, p<0.001). HuR silencing resulted in a dramatic reduction of colony formation in soft agar over 4 weeks (57% and 71% reduction compared to non-induced control cells, p<0.05). HuR silencing also reduced cells’ ability to invade through Matrigel (41% and 56% reduction compared to non-induced control cells, p<0.05), whereas HuR overexpression (Mia.HuR) resulted in a dramatic increase in invasion (1,250% increase compared to control, p<0.05). In a complementary in vitro scratch test, cell migration was reduced by HuR silencing and increased by HuR overexpression (p<0.01 for both). Conclusion: Taken together with our previous findings, our work demonstrates that manipulation of HuR expression affects key characteristics of tumorigenesis (i.e. proliferation, invasion, and migration). These findings, along with ongoing in vivo work, continue to support the notion that targeting HuR in PDA cells may be a promising therapeutic strategy. Citation Format: Masaya Jimbo, Fernando F. Blanco, Brad Screnci, Gabriela Cosma, Vitali Alexeev, Yu-Hung Huang, Jordan M. Winter, Charles J. Yeo, Janet A. Sawicki, Jonathan R. Brody. The RNA-binding protein HuR facilitates proliferation and metastasis in human pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5133. doi:10.1158/1538-7445.AM2015-5133