Down-regulation of kappa opioid receptor promotes ESCC proliferation, invasion and metastasis via the PDK1-AKT signaling pathway

Background: As a class of the opioid receptors, the kappa Opioid Receptor (KOR) has been verified to be a potential biomarker and therapeutic target for human malignant tumors. However, a thorough understanding of whether KOR affects progression of esophageal squamous cell carcinoma (ESCC) is still lacking. This study focused on exploring the effect of knocking down KOR in ESCC and its underlying mechanism.

Methods: Bioinformatics analysis was used to compare the different expression level of OPRK1 (KOR gene) in tumor and adjacent normal tissues, and predict the relationship between KOR expression and overall survival. RNA-sequence analysis was performed to detect the altered functions and mechanisms after down regulating KOR. The in vitro and in vivo assays were used to detect the effects of down-regulated KOR on cell proliferation, migration and invasion. Substrate gel zymography and 3D Cell Culture assays were used to find the effect of KOR knockdown on the degradation of extracellular matrix (ECM), and immunefluorescence was performed to detect the altered Cytoskeleton. Western blotting and immunohistochemistry were used to explore the underlying mechanism pathway.

Results: Bioinformatics analysis revealed that the expression of OPRK1 was lower in tumor tissue than that in adjacent normal tissues, and lowered expression of KOR was associated with poorer overall survival. The in vitro assays demonstrated that down-regulation of KOR enhanced ESCC proliferation, metastasis and invasion. Western blotting revealed that down-regulation of KOR could activate PDK1-AKT signaling pathway, which actively regulated the Cancer progression. Down-regulation of KOR enhanced the formation of invadopodia, secretion of matrix metalloproteinase-2 (MMP2) and rearrangement of Cytoskeleton, which were positively related with the invasion of ESCC. KOR knockdown enhanced the tumor invasion and elevated the Akt phosphorylation in nude mice. The Akt kinase inhibition could reverse the effect of down-regulation of KOR.

Conclusion: KOR might act as a tumor suppressor in ESCC and down-regulation of KOR could enhance the ESCC tumor phenotype. Video Abstract.