An RNA binding protein, HuR, in pancreatic cancer EMT, metastasis, and CSC
Authors: Dong Ruochen , Polireddy Kishore , Wu Xiaoqing , Chen Ping , Wang Tao , Dixon Dan , Xu Liang , Chen Qi 
Published: June 2021

Objectives

Epithelial‐mesenchymal transition (EMT) contributes importantly to cancer cell metastasis and formation of cancer stem cells (CSC). An RNA‐binding protein, HuR, regulates gene expressions involved in tumor cell proliferation, metastasis, anti‐apoptosis, and drug resistance. The role of HuR in cancer cell EMT and CSC has not been elucidated. Here we aim to investigate the role of HuR in regulating pancreatic cancer EMT and CSC, and develop novel HuR inhibitor to suppress pancreatic cancer EMT and CSC.

Methods

HuR was knocked down by siRNA silencing and CRISPR/Cas 9 gene deletion, and was overexpressed to examine its role in EMT/CSC and metastasis. Fluorescence Polarization assay and surface plasmon resonance assay were utilized to examine binding of a novel HuR inhibitor, namely KH‐3, to HuR. Scratching assay and Boyden Chamber trans‐well invasion assay were used for cell migration and invasion, whereas MTT assay for viability. Tumorsphere formation assay was used as an indication of CSC. Western blot and RT‐qPCR was used for protein and mRNA expression. Ribonucleoprotein‐Immunoprecipitation (RNP‐IP) assay and dual‐luciferase reporter assay was used for HuR downstream target verification.

Results

HuR knockdown inhibited migration and invasion of pancreatic cancer cells PANC‐1 and MIA PaCa‐2. Expressions of signature EMT genes in both cells were altered: the epithelial marker Claudin‐1 was significantly upregulated, the mesenchymal marker Vimentin was downregulated, and the EMT enhancing transcription factor Snail was significantly decreased. In contrast, HuR re‐expression in HuR knocked‐out MIA PaCa‐2 cells significantly enhanced the cell migration. The tumor spheroids were significantly inhibited in PANC‐1 and MIA PaCa2 cells with siHuR silencing, indicating the inhibition of CSC formation. RNPIP assay and reporter assay showed HuR bound to the 3′UTR of Snail by targeting the AU rich element (ARE).

A novel compound KH‐3 inhibited the proliferation of pancreatic cancer cell lines more potently in HuR‐high pancreatic cancer cells MIA PaCa‐2 (IC50 ~5 μM), than in HuR‐low pancreatic cancer cells BxPc‐3 and PANC‐1 (IC50 10–25 μM), and had minimal toxicity to the normal pancreas ductal epithelial cell hTERT‐HPNE which had the lowest HuR level (IC50≫40 uM). KH‐3 directly bound to HuR and inhibited binding of HuR with its target mRNAs. KH‐3 treatment mimicked the HuR knockdown effects in inhibiting cell migration, EMT, and spheroids formation. In addition, KH‐3 inhibited pancreatic cancer progression and metastasis in vivo.

Conclusion

HuR is an important regulator in pancreatic cancer EMT and CSC. HuR directly binds to the 3′UTR of Snail by targeting the AREs, thus the inhibition of HuR inhibits the EMT and CSC process. KH‐3 as a novel HuR inhibitor inhibited pancreatic cancer EMT and CSC. Further investigation is underway.

Support or Funding Information

5R21CA198265‐02

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.