Studies Of The Inhibitory Effect On Stem-like Cancer Cell Proliferation By ITE, An Endogenous Ligand Of AhR

Tumor is a multistep development disease involving dynamic changes in the genome. Many targets for tumor precaution and treatment has been found by scientists. One of the target is aryl hydrocarbon receptor(AhR), which has been highly controversial. AhR is a cytosolic ligand-activated transcription factor, which is best known for its role in mediating toxic ligands in first4years. As physiological roles of AhR and endogenous ligands being found, roles of AhR in tumor have new breakthrough. Besides, a few researches has revealed that AhR may be associated with Oct4signal pathway.2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), an endogenous ligand for AhR, has been identified in2002. ITE has been found deleting transplanted tumor of human cancer cell lines in mice. However, the mechanism is unclear. Therefore, we attempt to illustrate how ITE deletes tumor and whether it is associated with Oct4.By test AhR RNA of eleven human tissues, we found that AhR is a ubiquitously expressed protein in human tissues. Then we analyzed ten human cell lines, and we found that AhR has much more higher expression levels in differentiated cells while Oct4was on the contrary. When pluripotent stem cells(embryonic stem cells HI and embryonal carcinoma cells NCCIT) were induced to differentiation, AhR expression levels were increased. Meanwhile, Oct4expression levels were decreased. These indicated that there may exist some negative control between AhR and Oct4.On account of that AhR is an transcription factor, and there is a potential target site on promoter of OCT4gene reported by Bunaciu, R. P. In2011, we asked whether AhR can target Oct4promoter. Using Genomatic software, we found that AhR can target on POU5F1promoter region potentially. By using electrophoretic mobility shift assay(EMSA)and chromosome immunoprecipitation (ChIP), we confirmed that ITE can promote AhR binding to promoter of Oct4A gene in vitro and in vivo. By using qRT-PCR, we tested the function of AhR binding to Oct4promoter. We found that in U87cells, in which AhR is highly expressed and Oct4expression is low, ITE can inhibit Oct4transcription and protein levels of Oct4and pOct4-T235. In NCCIT cells, in which AhR expression is low and Oct4expression is high, Oct4transcription was inhibited when AhR was overexpressed. These data confirmed that activated by ITE, AhR is recruited to the POU5F1promoter and inhibits Oct4transcription. Next, we focused on whether activating AhR with ITE can inhibit Oct4transcription in stem-like cancer cells and lead to their differentiation. By using sphere formation assay, we found that pathway changes in U87sphere cells are stem-like. Then we found that ITE reduces Oct4in U87sphere cells as well as many genes that maintain undifferentiation state by genome-wide DNA microarray. The effect of differentiation inhibits the sphere formation and the proliferation of U87parental and sphere cells.At last, we asked whether ITE can suppress tumor growth in mice. We found that ITE effectively suppresses growth of tumor which derived from U87cells implanted to mouse brain. Then we found that ITE suppresses liver cancer cell and prostate cancer cell-derived xenografts. Furthermore, ITE eliminates inoculated cancer cells in a syngeneic mouse tumor model.In conclusion, we report for the first time the mechanism of how ITE inhibits tumor, which provides clues for theoretical foundation of ITE being an antitumor drug. This work revealed a novel connection between AhR and Oct4. Thus we promote the role of AhR in stem cell and link environment toxicity, stem cell and tumor. This work also provided new clues for AhR being a target for cancer therapy.