Abstract
Understanding the mechanism by which sulforaphene (SFE) affects esophageal squamous cell carcinoma (ESCC) contributes to the application of this isothiocyanate as a chemotherapeutic agent. Thus, we attempted to investigate SFE regulation of ESCC characteristics more deeply. We performed gene set enrichment analysis (GSEA) on microarray data of SFE-treated ESCC cells and found that differentially expressed genes are enriched in TNFα_Signaling_via_the_NFκB_Pathway. Coupled with the expression profile data from the GSE20347 and GSE75241 datasets, we narrowed the set to 8 genes, 4 of which (C-X-C motif chemokine ligand 10 (CXCL10), TNF alpha induced protein 3 (TNFAIP3), inhibin subunit beta A (INHBA), and plasminogen activator, urokinase (PLAU)) were verified as the targets of SFE. RNA-sequence (RNA-seq) data of 182 ESCC samples from The Cancer Genome Atlas (TCGA) were grouped into two phenotypes for GSEA according to the expression of CXCL10, TNFAIP3, INHBA, and PLAU. The enrichment results proved that they were all involved in the NFκB pathway. ChIP-seq analyses obtained from the Cistrome database indicated that NFκB-p65 is likely to control the transcription of CXCL10, TNFAIP3, INHBA, and PLAU, and considering TNFAIP3 and PLAU are the most significantly differentially expressed genes, we used chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) to verify the regulation of p65 on their expression. The results demonstrated that SFE suppresses ESCC progression by down-regulating TNFAIP3 and PLAU expression in a p65-dependent manner.
Highlights
Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide, with many causes that vary by histologic type
We found that the expression levels of CXCL10, TNF alpha induced protein 3 (TNFAIP3), inhibin subunit beta A (INHBA), and PLAU were significantly correlated with each other in the The Cancer Genome Atlas (TCGA) Esophageal squamous cell carcinoma (ESCC) samples (Figure 3D), and considering that p65 of the NFκB family can regulate genes involved in the immune response, cell proliferation, differentiation, and metastasis as a transcription factor, we speculated that these 4 genes might be the direct downstream targets of p65 in ESCC
We searched publicly available Chromatin Immunoprecipitation (ChIP)-seq data related to CXCL10, TNFAIP3, INHBA, and PLAU in the Cistrome database to verify the association between p65 and its 4 possible targets
Summary
Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide, with many causes that vary by histologic type. SFE has reportedly regulated several signaling pathways involved in cell proliferation, invasion, metastasis, and apoptosis. It inhibited triple-negative breast cancer by activating the tumor suppressor Egr1 [6], induced hepatocellular carcinoma cell death by repressing keratin 8 and promoting anoikis [7], and targeted the PI3K-Akt pathway to cause lung cancer cell apoptosis [8]. Reinterpreting the data of microarray analyses by GSEA, we found the significantly differentially expressed genes in SFE-treated EC109 and KYSE510 cells were enriched in the NFκB pathway. Based on the ChIP-seq data of NFκB-p65 in multiple cancer cells and samples from the Cistrome database and subsequent verification experiments, we guessed and acknowledged that SFE could block the transcription-promoting activity of the NFκB pathway to suppress ESCC development. All our efforts were to prove that SFE is a promising chemotherapeutic agent that may be used to treat ESCC in the future
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