3,3'-Diindolylmethane modulates aryl hydrocarbon receptor of esophageal squamous cell carcinoma to reverse epithelial-mesenchymal transition through repressing RhoA/ROCK1-mediated COX2/PGE2 pathway

J Exp Clin Cancer Res. 2020 Jun 16;39(1):113. doi: 10.1186/s13046-020-01618-7.

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive tumors in the world. Aryl hydrocarbon receptor (AHR) has been reported to promote tumor metastasis and epithelial-mesenchymal transition (EMT) is a vital process of conferring cancer cells capabilities of migration and invasion. However, the mechanism by which modulation of AHR can inhibit tumor metastasis remains unknown. Thus, we aim to investigate the underlying mechanism regarding reversing EMT process of ESCC through modulation of AHR.

Methods: We used AHR selective modulator 3,3'-diindolylmethane (DIM) to treat ESCC cell lines TE1 and KYSE150 so as to examine alterations of migration and invasion by wound healing and Transwell assay. Western blotting (WB) and qPCR were performed to detect relative genes and proteins changes regarding EMT process. Cell transfection was utilized for confirming pathways involved in DIM-induced reversal of EMT and in vivo assay was conducted for verification of the underlying mechanism. Co-IP assay was conducted for detecting protein-protein interactions.

Results: AHR was overexpressed in ESCC and modulation of AHR by DIM could inhibit migration and invasion as well as downregulate mesenchymal cell markers β-Catenin, Vimentin and Slug and upregulate epithelial cell marker Claudin-1. Meanwhile, synergically overexpression of AHR, RhoA and ROCK1 correlated with poor clinical outcomes. DIM could inhibit COX2/PGE2 pathway by targeting AHR, and COX2 selective inhibitor Celecoxib could suppress EMT and metastasis. Results of PGE2 treatment were opposite to that of Celecoxib. Meanwhile, blockade of RhoA/ROCK1 pathway also exerted prohibitive effects on EMT and metastasis. WB results showed COX2/PGE2 pathway could be regulated by RhoA/ROCK1 pathway and DIM could inhibit RhoA/ROCK1 pathway through modulation of AHR. In vivo assay verified the results in vitro. Co-IP results showed DIM could modulate AHR to reverse EMT directly through inhibition of interaction between AHR and EGFR (epidermal growth factor receptor) so as to block RhoA/ROCK1-mediated COX2/PGE2 pathway which was connected by NF-κB.

Conclusions: In brief, modulation of AHR by DIM can reverse EMT process and inhibit metastasis of ESCC through repressing RhoA/ROCK1-mediated COX2/PGE2 pathway.

Keywords: AHR; COX2; DIM; EMT; ESCC; PGE2; ROCK1; RhoA.

MeSH terms

  • Animals
  • Anticarcinogenic Agents / pharmacology
  • Apoptosis
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Biomarkers, Tumor / genetics
  • Biomarkers, Tumor / metabolism
  • Cell Movement
  • Cell Proliferation
  • Cyclooxygenase 2 / chemistry*
  • Cyclooxygenase 2 / genetics
  • Cyclooxygenase 2 / metabolism
  • Dinoprostone / metabolism*
  • Epithelial-Mesenchymal Transition*
  • Esophageal Neoplasms / drug therapy*
  • Esophageal Neoplasms / metabolism
  • Esophageal Neoplasms / pathology
  • Esophageal Squamous Cell Carcinoma / drug therapy
  • Esophageal Squamous Cell Carcinoma / metabolism
  • Esophageal Squamous Cell Carcinoma / pathology
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Indoles / pharmacology*
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mice, Nude
  • Neoplasm Invasiveness
  • Receptors, Aryl Hydrocarbon / genetics
  • Receptors, Aryl Hydrocarbon / metabolism*
  • Tumor Cells, Cultured
  • Xenograft Model Antitumor Assays
  • rho-Associated Kinases / antagonists & inhibitors*
  • rho-Associated Kinases / genetics
  • rho-Associated Kinases / metabolism
  • rhoA GTP-Binding Protein / antagonists & inhibitors*
  • rhoA GTP-Binding Protein / genetics
  • rhoA GTP-Binding Protein / metabolism

Substances

  • AHR protein, human
  • Anticarcinogenic Agents
  • Basic Helix-Loop-Helix Transcription Factors
  • Biomarkers, Tumor
  • Indoles
  • Receptors, Aryl Hydrocarbon
  • RHOA protein, human
  • Cyclooxygenase 2
  • PTGS2 protein, human
  • ROCK1 protein, human
  • rho-Associated Kinases
  • rhoA GTP-Binding Protein
  • Dinoprostone
  • 3,3'-diindolylmethane