Abstract
Objective: Metastasis and therapeutic resistance are the major determinants of lung cancer progression and high mortality. Epithelial–mesenchymal transition (EMT) plays a key role in the metastasis and therapeutic resistance. Highly expressed glucose-regulated protein 78 (GRP78) is a poor prognostic factor in lung cancer and possibly correlated with EMT. This study aims to examine whether the up-regulation of GRP78 is involved in EMT in lung adenocarcinoma and explore the underlying downstream molecular pathways.Study Design: EMT was assessed by analysis of cell morphology and expression of EMT protein markers in A549 cells under normoxia, hypoxia and silencing GRP78 conditions. The expression levels of Smad2/3, Src, and MAPK (p38, ERK, and JNK) proteins were examined by Western blot analysis under hypoxia and treatments with phosphorylation inhibitors.Results: Under hypoxic conditions, the EMT morphology significantly changed and the GRP78 expression was significantly up-regulated in A549 cells compared with those in normoxia control. The expression and phosphorylation levels of smad2/3, Src, p38, ERK, and JNK were also upregulated. When GRP78 was silenced, EMT was inhibited, and the levels of phospho-smad2/3, phospho-Src, phospho-p38, phospho-ERK, and phospho-JNK were suppressed. When the activation of Smad2/3, Src, p38, ERK, and JNK was inhibited, EMT was also inhibited. The inhibition effect on EMT by these phosphorylation inhibitors was found to be weaker than that of GRP78 knockdown.Conclusions: Hypoxia-induced EMT in A549 cells is regulated by GRP78 signaling pathways. GRP78 promotes EMT by activating Smad2/3 and Src/MAPK pathways. Hence, GRP78 might be a potential target for treatment of lung adenocarcinoma.
Highlights
Lung cancer is the leading cause of cancer death worldwide; according to the estimated data from GLOBOCAN in 2012, one of five cancer deaths is due to lung cancer (1.59 million deaths, 19.4% of the total cancer deaths) [1]
A549 cells cultured under hypoxia condition for 72 h showed morphological changes, from oblate fusiform-shaped epithelial cells to elongated spindle-shaped mesenchymal cells (Figure 1A)
The expression and location of the GRP78 protein in A549 cells under hypoxia and normoxia conditions were determined by immunofluorescence staining
Summary
Lung cancer is the leading cause of cancer death worldwide; according to the estimated data from GLOBOCAN in 2012, one of five cancer deaths is due to lung cancer (1.59 million deaths, 19.4% of the total cancer deaths) [1]. Despite significant progress in the development of new therapies for lung cancer, metastasis and therapeutic resistance remain the major determinants of lung cancer progression and high mortality [2]. Mounting evidence demonstrated that epithelial– mesenchymal transition (EMT) is involved in the metastasis and therapeutic resistance of lung cancer. EMT refers to the biological process by which epithelial cells are transformed into mesenchymal phenotypes through specific procedures. EMT can be induced by various factors, such as TGF-beta, which increases the expression of key nuclear transcription factors including Twist, Snail and ZEB [3] and causes phenotypic changes by activating intrinsic cellular signal molecules including Src, MAPK, Smad2/3, and other signals [4,5,6]
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