Introduction
Pancreatic cancer (PC) is emerging as one the most lethal types of cancer with only a 10% five-year survival rate [1]. Pancreatic ductal adenocarcinoma (PDAC) being the most common among PC. Low survival rate is attributed to the fact that most incidences of PDACs are diagnosed in advanced or metastatic stages [2]. In such cases, gemcitabine remains one the first-line drugs used for treatment [2] but the efficacy of the treatment can fall short due to PDAC tumour heterogeneity and its acquired resistance to said chemotherapy [3]. The aryl hydrocarbon receptor (AHR) is a transcription factor that is commonly upregulated in pancreatic ductal adenocarcinoma [4]. AHR can be linked to Human antigen R (ELAVL1) whose expression in cell cytoplasm is positively associated with PDAC response to gemcitabine [5]. AHR can block ELAVL1 from shuttling from nucleus to cytoplasm [6] where it stabilizes its target messenger RNAs (mRNAs) and increases protein expression of said mRNAs. Among those target mRNAs are those induced by gemcitabine treatment. Frequently with upregulated AHR expression, ELAVL1 would be sequestered in the nucleus which would lead to increased chemoresistance, however the relationship between AHR and ELAVL1 in PDAC is still poorly understood.
Aims & Methods
The aim of the study was to investigate how AHR and ELAVL1 genes and proteins interacts with each other and change their cellular localization in PDAC cells.
Two PDAC cell lines were used (BxPC-3, Su.86.86). AHR and ELAVL1 genes were silenced for 24-hours by lipofectamine mediated siRNA transfection. Cellular localization change of AHR and ELAVL1 proteins was illucidated by fluorescent microscopy. After silencing of AHR or ELAVL1 genes, RNA and proteins were extracted and real-time polymerase chain reaction (RT-PCR) and Western Blot (WB) analysis were performed. Direct binding between ELAVL1 protein and AHR mRNA was investigated by immunoprecipitation assay followed by RT-PCR and WB assays.
Results
The results of gene silencing showed that silencing of AHR can increase ELAVL1 mRNA and protein and silencing of ELAVL1 can decrease AHR mRNA and protein expression. The results of immunoprecipitation assay showed that ELAVL1 protein binds AHR mRNA resulting in mRNA stabilization. This shows a direct link between these two molecules. Moreover, silencing of AHR resulted in increased ELAVL1 protein concentration in cytoplasm which indicates that AHR blocks ELAVL1 shuttling from nucleus to cytoplasm. Silencing of ELAVL1 did not alter the cellular localization of AHR protein.
Conclusion
A direct relationship between AHR and ELAVL1 can be seen in PDAC. Moreover, by decreasing AHR expression it is possible to increase ELAVL1 shuttling from nucleus to cytoplasm which might contribute to PDAC cells responding better to chemotherapy.
References
[1] Hu JX, Zhao CF, Chen WB, Liu QC, Li QW, Lin YY, Gao F. Pancreatic cancer: A review of epidemiology, trend, and risk factors. World journal of gastroenterology. 2021 Jul 7;27(27):4298.
[2] Kamisawa T, Wood LD, Itoi T, Takaori K. Pancreatic cancer. The Lancet. 2016 Jul 2;388(10039):73-85.
[3] Juiz NA, Iovanna J, Dusetti N. Pancreatic cancer heterogeneity can be explained beyond the genome. Frontiers in oncology. 2019 Apr 5;9:246.
[4] Koliopanos A, Kleeff J, Xiao Y, Safe S, Zimmermann A, Büchler MW, Friess H. Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer. Oncogene. 2002 Sep;21(39):6059-70
[5] Richards, N. G., Rittenhouse, D. W., Freydin, B., Cozzitorto, J. A., Grenda, D., Rui, H., ... & Witkiewicz, A. K. (2010). HuR status is a powerful marker for prognosis and response to gemcitabine-based chemotherapy for resected pancreatic ductal adenocarcinoma patients. Annals of surgery, 252(3), 499-506.
[6] Zago, M., Sheridan, J. A., Nair, P., Rico de Souza, A., Gallouzi, I. E., Rousseau, S., ... & Baglole, C. J. (2013). Aryl hydrocarbon receptor-dependent retention of nuclear HuR suppresses cigarette smoke-induced cyclooxygenase-2 expression independent of DNA-binding. PLoS One, 8(9), e74953.
