Stat6 activity-related Th2 cytokine profile and tumor growth advantage of human colorectal cancer cells in vitro and in vivo.

Signal transducer and activator of transcription 6 (Stat6) is critical in Th2 polarization of immune cells and active Stat6 activity has been suggested in anti-tumor immunity in animal models. The present study aims at investigating the impact of natural Stat6 activity on tumor microenvironment in human colorectal cancer cells in vitro and in vivo. Using colorectal cancer cell lines HT-29 and Caco-2 whose IL-4/Stat6 activities were known and nude mice as a model, we examined correlative relationships between Stat6 activities and gene expression profiles together with cellular behaviors in vitro and in vivo. HT-29 cells carrying active Stat6 signaling displayed spontaneous expression profiles favoring Th2 cytokines, cell cycle promotion, anti-apoptosis and pro-metastasis with increased mRNA levels of IL-4, IL-13, GATA-3, CDK4, CD44v6 and S100A4 using RT-PCR. In contrast, Caco-2 cells carrying defective Stat6 signaling exhibited spontaneous expression profiles favoring Th1 and Th17 cytokines, cell cycle inhibition, pro-apoptosis and anti-metastasis with elevated mRNA expression of IFNγ, TNFα, IL-12A, IL-17, IL-23, T-bet, CDKN1A, CDKNIB, CDKN2A and NM23-H1. Xenograft tumors of Stat6-active HT-29 cells showed a growth advantage over those of Stat6-defective Caco-2 cells. Furthermore, mice bearing HT-29 tumors expressed increased levels of Th2 cytokines IL-4 and IL-5 in the blood and pro-growth and/or pro-metastasis proteins CDK4 and CD44v6 in the tumor. To the contrary, mice bearing Caco-2 tumors expressed heightened levels of Th1 cytokines IFNγ and TNF in the blood and pro-apoptosis and anti-metastatic proteins p53 and p27(kip1) in the tumor. Colorectal cancer cells carrying active Stat6 signaling may create a microenvironment favoring Th2 cytokines and promoting expression of genes related to pro-growth, pro-metastasis and anti-apoptosis, which leads to a tumor growth advantage in vivo. These findings may imply why Stat6 pathway is constitutively activated in a number of human malignancies.

Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

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.

DNA methylation regulates constitutive expression of Stat6 regulatory genes SOCS-1 and SHP-1 in colon cancer cells.

PURPOSE: Stat6 signaling is active in cancer cells and IL-4-induced Stat6 activities or Stat6 activational phenotypes vary among cancer cells. This study aimed at investigating possible mechanism(s) involved in the formation of varying Stat6 activities/phenotypes. METHODS: Stat6 regulatory genes, SOCS-1 and SHP-1, were examined for mRNA expression using RT-PCR, and their promoter DNA methylation was assayed by methylation-specific PCR in Stat6-phenotyped colon cancer cell lines. DNA methylation was then verified by sequencing. RT-PCR assay and Western blotting were used to detect the expression of SOCS-1 and SHP-1 after demethylation using 5-aza-2'-deoxycytidine. RESULTS: Compared with Stat6(null) Caco-2 cells, Stat6(high) HT-29 cells showed decreased constitutive expression of SOCS-1 and SHP-1, which correlated with DNA hypermethylation in these genes' promoters. Interestingly, demethylation in HT-29 cells recovered the constitutive expression of SOCS-1 and SHP-1. CONCLUSIONS: These findings suggest that DNA methylation controls the constitutive expression of negative Stat6 regulatory genes, which may affect Stat6 activities.

Differential IL-4/Stat6 activities correlate with differential expression of regulatory genes SOCS-1, SHP-1, and PP2A in colon cancer cells.

PURPOSE: To investigate potential differences in the expression of Stat6 regulatory genes that may influence IL-4/Stat6 activities (phenotypes) in colon cancer cells. METHODS: RT-PCR method was employed to examine the constitutive mRNA expression of Stat6 negative regulators SOCS-1 and SHP-1, and positive regulator PP2A in colon cancer cell lines HT-29 and Caco-2. Stat6 protein expression and nuclear phosphorylation were detected using Western blotting. RESULTS: Caco-2 cells carrying inactive Stat6(null) phenotype showed normal constitutive expression of Stat6 but decreased phosphorylation of nuclear Stat6 compared with HT-29 cells carrying active Stat6(high) phenotype. Stat6(null) Caco-2 cells expressed increased levels of mRNA and protein of SOCS-1 and SHP-1, and decreased mRNA expression of PPP2CA and PPP2CB, encoding two critical subunits of PP2A. CONCLUSIONS: Constitutively increased expression of Stat6 negative regulators SOCS-1 and SHP-1, together with decreased expression of positive regulator PP2A, may play a role in forming the inactive Stat6(null) phenotype in colon cancer cells.

IL-4-induced Stat6 activities affect apoptosis and gene expression in breast cancer cells.

IL-4-induced Stat6 signaling is active in a variety of cell types, including immune cells and cancer cells, and plays an important role in the regulation of gene expression. Using EMSA gel shift assay and an antibody to Stat6, we phenotyped two breast cancer cell lines, ZR-75-1 being active Stat6(high) phenotype and BT-20 being defective Stat6(null) phenotype, respectively. Breast cancer cells carrying Stat6(null) phenotype exhibited increased spontaneous apoptosis compared with those carrying Stat6(high) phenotype. Expression microarray analyses demonstrated that IL-4 upregulated CCL26, SOCS1, CISH, EGLN3, and SIDT1, and downregulated DUSP1, FOS, and FOSB, respectively, in these breast cancer cells. Among those genes, CCL26 and SOCS1 were known genes regulated by IL-4/Stat6 pathway, but CISH, EGLN3, SIDT1, DUSP1, FOS, and FOSB were novel genes demonstrated to be IL-4 responsive for the first time. IL-4 also upregulated 38 genes unique to Stat6(null) BT-20 cells and 23 genes unique to Stat6(high) ZR-75-1 cells, respectively. Furthermore, Stat6(high) and Stat6(null) cells showed very different profiles of constitutively expressed genes relevant to apoptosis and metastasis among others, which serve as a valuable expression database and warrant for detailed studies of IL-4/Stat6 pathway in breast cancer.

IL-4/Stat6 activities correlate with apoptosis and metastasis in colon cancer cells.

IL-4-induced Stat6 signaling is active in a variety of cell types and plays a role in cell proliferation/growth and resistance to apoptosis. Using EMSA, we identified differential IL-4/Stat6 activities in colorectal cancer cell lines, HT-29 being active Stat6(high) phenotype and Caco-2 being defective Stat6(null) phenotype, respectively. Active Stat6(high) HT-29 cells exhibited resistance to apoptosis by flowcytometry and aggressive metastasis by Transwell assay compared with defective Stat6(null) Caco-2 cells. Comparing one another using RT-PCR, Stat6(high) HT-29 cells expressed more mRNA of anti-apoptotic and pro-metastatic genes Survivin, MDM2, and TMPRSS4, while Stat6(null) Caco-2 cells expressed more mRNA of pro-apoptotic and anti-metastatic genes BAX, CAV1, and P53, respectively. This is the first study describing correlations of IL-4/Stat6 activities with apoptosis and metastasis in colon cancer. These findings, together with the observation of constitutive Stat6 activation in many human malignancies, suggest that Stat6 activities could be a biomarker for cancer cell's invasive/metastatic capability.