Defective Interleukin-4/Stat6 Activity Correlates With Increased Constitutive Expression of Negative Regulators SOCS-3,SOCS-7,and CISH in Colon Cancer Cells

Abstract

Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6^high phenotype, while Caco-2 carries a defective Stat6^null phenotype, respectively. Cancer cells with Stat6^high show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6^high HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6^high HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6^null Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2′-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6^null Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.