Original Research
LLGL1 Regulates Gemcitabine Resistance by Modulating the ERK-SP1-OSMR Pathway in Pancreatic Ductal Adenocarcinoma

https://doi.org/10.1016/j.jcmgh.2020.06.009Get rights and content
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Background & Aims

Gemcitabine resistance is rapidly acquired by pancreatic ductal adenocarcinoma (PDAC) patients. Novel approaches that predict the gemcitabine response of patients and enhance gemcitabine chemosensitivity are important to improve patient survival. We aimed to identify genes as novel biomarkers to predict the gemcitabine response and the therapeutic targets to attenuate chemoresistance in PDAC cells.

Methods

Genome-wide RNA interference screening was conducted to identify genes that regulated gemcitabine chemoresistance. A cell proliferation assay and a tumor formation assay were conducted to study the role of lethal giant larvae homolog 1 (LLGL1) in gemcitabine chemoresistance. Levels of LLGL1 and its regulating targets were measured by immunohistochemical staining in tumor tissues obtained from patients who received gemcitabine as a single therapeutic agent. A gene-expression microarray was conducted to identify the targets regulated by LLGL1.

Results

Silencing of LLGL1 markedly reduced the gemcitabine chemosensitivity in PDAC cells. Patients had significantly shorter survival (6 months) if they bore tumors expressing low LLGL1 level than tumors with high LLGL1 level (20 months) (hazard ratio, 0.1567; 95% CI, 0.05966–0.4117). Loss of LLGL1 promoted cytokine receptor oncostatin M receptor (OSMR) expression in PDAC cells that led to gemcitabine resistance, while knockdown of OSMR effectively rescued the chemoresistance phenotype. The LLGL1-OSMR regulatory pathway showed great clinical importance because low LLGL1 and high OSMR expressions were observed frequently in PDAC tissues. Silencing of LLGL1 induced phosphorylation of extracellular signal-regulated kinase 2 and specificity protein 1 (Sp1), promoted Sp1 (pThr453) binding at the OSMR promoter, and enhanced OSMR transcription.

Conclusions

LLGL1 possessed a tumor-suppressor role as an inhibitor of chemoresistance by regulating OSMR–extracellular signal-regulated kinase 2/Sp1 signaling. The data sets generated and analyzed during the current study are available in the Gene Expression Omnibus repository (ID: GSE64681).

Keywords

Pancreatic Cancer
Gemcitabine
Oncostatin M Receptor
SP1 Signaling

Abbreviations used in this paper

cDNA
complementary DNA
ChIP
chromatin immunoprecipitation
CSC
cancer stem cell
Dlg
discs large protein
EMT
epithelial-mesenchymal transition
ERK2
extracellular signal-regulated kinase 2
hENT1
human equilibrative nucleoside transporter 1
HPDE
human pancreatic ductal epithelial
IC50
median inhibitory concentration
IHC
immunohistochemistry
Lgl
lethal giant larvae
LLGL1
lethal giant larvae homolog 1
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
OSM
oncostatin M
OSMR
oncostatin M receptor
PDAC
pancreatic ductal adenocarcinoma
Pol II
RNA polymerase II
qRT-PCR
quantitative reverse-transcription polymerase chain reaction
RNAi
RNA interference
siLLGL1
small interfering lethal giant larvae homolog 1
siRNA
small interfering RNA
Sp1
specificity protein 1
SWH
Salvador/Warts/Hippo
TGF
transforming growth factor

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Conflicts of interest The authors disclose no conflicts.

Funding Supported by grants from the National Natural Science Foundation of China (81672323), the General Research Fund, Research Grants Council of Hong Kong (14171217 and 14120618), and a direct grant from The Chinese University of Hong Kong (Y.C.).

Authors contributed equally

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Authors contributed equally.