IL-4-induced Stat6 activities affect apoptosis and gene expression in breast cancer cells
Introduction
Signal transducer and activator of transcription 6 (Stat6) is a molecule with dual function capable of signaling and transcription. Stat6 is activated in response to interleukin (IL)-4 and IL-13 stimulation. The IL-4/Stat6 signaling pathway is composed of at least six associated molecules including IL-4, IL-4Rα, common γ chain (γc), Jak1, Jak3, and Stat6. IL-4 addition results in the activation of the signaling pathway via a cascade of interactions including activation of Jak1 and Jak3, tyrosine phosphorylation of IL-4Rα and Stat6, and the dimerization of phosphorylated Stat6 followed by its subsequent translocation to the nucleus [1]. Activated Stat6 binds to the promoter of various genes and plays an important role in target cells through the modification of cell differentiation and growth, and the induction of resistance to apoptosis [2].
Functionally active IL-4/Stat6 signaling pathway is important in a variety of cell types, including immune cells and cancer cells [3]. In cancer cells, including those of breast and colon cancer, IL-4/Stat6 signaling induces the expression of 3β-hydroxysteroid dehydrogenase type 1 [4]. IL-4 may also upregulate cell surface molecules such as CD23, MHC class II, and IL-4Rα [1], and simultaneously downregulate pro-inflammatory cytokines, including IL-12 and tumor necrosis factor-α (TNF-α) [5]. The number of Stat6-regulated genes has been increasing rapidly and there are at least 35 genes known to be regulated via Stat6 pathway [6]. Interestingly, Stat6 signaling upregulates the expression of suppressor of cytokine signaling 1 (SOCS1) which in turn downregulates the Stat6 signaling, forming a negative feedback [7].
The Stat6 pathway has been extensively studied in knockout animals. Mice deficient in Stat6 exhibit defective Th2 cell development [8], [9] similar to IL-4Rα-deficient mice [10], consistent with the role of the IL-4/Stat6 pathway in T cell differentiation [1]. It is very interesting that mice lacking Stat6 also manifest enhanced tumor immunity to both primary and metastatic mammary carcinomas [11], [12] and induce spontaneous rejection of implanted tumors [13]. In humans, Stat6 protein has been found to be constitutively active in several cancer types including prostate cancer [14], cutaneous T cell lymphoma [15], Hodgkin’s lymphoma [16], and primary mediastinal large B cell lymphoma [17], strongly supporting the hypothesis that a functional Stat6 signaling may be beneficial to tumor growth possibly by several mechanisms, including gaining resistance to apoptosis and escaping the immune surveillance.
Using a semiquantitative gel shift assay and EBV-B lymphoblastoid cell lines, we have previously defined three naturally occurring IL-4-induced Stat6 activational phenotypes, termed as Stat6high, Stat6low, and Stat6null, which are hypothesized to be genetically determined [18]. Interestingly, Western blot analysis has demonstrated that cells carrying differential Stat6 phenotypes express similar levels of constitutive Stat6 protein, suggesting that the formation of Stat6 activational phenotypes may be due to mechanism(s) other than constitutive Stat6 expression [18]. Nevertheless, the Stat6null is a defective phenotype which is correlated with decreased expression of CD23 [18], increased expression of IL-12, IFN-γ, and TNF-α [19], and accelerated cell apoptosis [20]. Furthermore, using RNAi technology to partially silencing the expression of Stat6 in HT-29 colon cancer cell line, which carries an active Stat6high phenotype, we have been able to produce a defective Stat6 signaling which also exhibits accelerated spontaneous apoptosis [21] similar to that seen in Stat6null EBV-B cells [20], demonstrating that Stat6 signaling is involved in the regulation of cell apoptosis [2], a feature that may possibly cross cell types.
Variations in Stat6 activity may further contribute to the pathophysiology of cancers and autoimmune diseases due to altered cell functions, which may create an advantageous or disadvantageous state in the host depending on disease types. In the present study of breast cancer cells, we show that (1) IL-4-induced defective Stat6null phenotype correlates with increased spontaneous apoptosis; (2) IL-4 mediates the expression of several novel genes previously unknown to be IL-4 responsive; and (3) constitutively expressed genes involved in biological functions such as apoptosis and metastasis vary dramatically depending on Stat6 phenotypes.
Section snippets
Cell lines
Epstein–Barr virus (EBV)-transformed lymphoblastoid B (EBV-B) cell lines were established and phenotyped for Stat6 activities previously [18]. Breast cancer cell lines ZR-75-1 and BT-20 were obtained from the American Type Culture Collection (ATCC) and cultured at 37 °C with 5% CO2.
Preparation of nuclear extracts
Cells (2 × 106 per line) were stimulated with 10 ng/ml (or as indicated) recombinant human IL-4 (hIL4, Sigma, St. Louis, MO, USA) for 30 min at 37 °C; unstimulated cells served as controls. Cell lysates and nuclear
Phenotyping of IL-4-induced Stat6 activity in breast cancer cell lines
By reference to an EBV-B cell line with confirmed Stat6high phenotype as standard control established previously [18] we were able to assign Stat6 activational phenotypes for breast cancer cell lines using EMSA assay. Based on the criteria for Stat6 phenotyping, ZR-75-1 was assigned as Stat6high phenotype as the cell yielded a T/C OD ratio of 1.05, indicating a similar density to the standard. BT-20 was assigned as Stat6null phenotype as the cell yielded a T/C OD ratio of 0.06, falling below
Discussion
The IL-4/Stat6 signaling pathway involves at least six genes, with the activational status of Stat6 serving as a pivotal reporter capable of revealing the functional fitness of the IL-4/Stat6 pathway. In human EBV-B cell lines, we have shown that there are at least three major IL-4-induced Stat6 activational phenotypes detectable by EMSA assay, which may be genetically determined [18]. In the same study, we have observed no difference in constitutive Stat6 protein expression among the three
Acknowledgments
We wish to thank Maggie Slattery for help in cell culture, and Dr. Qing He Meng for assistance in RNA extraction. This work was supported by a grant to W.J. Zhang from the National Natural Science Foundation of China (NSFC No. 30470981).
References (30)
- et al.
The role of constitutively active Stat6 in leukemia and lymphoma
Crit Rev Oncol Hematol
(2006) - et al.
Crucial role of cytokines in sex steroid formation in normal and tumoral tissues
Mol Cell Endocrinol
(2001) - et al.
Signaling mechanisms, interaction partners, and target genes of STAT6
Cytokine Growth Factor Rev
(2006) - et al.
Selective activation of members of the signal transducers and activators of transcription family in prostate carcinoma
J Urol
(2002) - et al.
Constitutive and interleukin-7- and interleukin-15-stimulated DNA binding of STAT and novel factors in cutaneous T cell lymphoma cells
J Invest Dermatol
(2001) - et al.
Signal transducer and activator of transcription 6 is frequently activated in Hodgkin and Reed–Sternberg cells of Hodgkin lymphoma
Blood
(2002) - et al.
Constitutive STAT6 activation in primary mediastinal large B-cell lymphoma
Blood
(2004) - et al.
Genetic control of interleukin-4-induced activation of the human signal transducer and activator 6 signaling pathway
Hum Immunol
(2003) - et al.
Stat6null phenotype human lymphocytes exhibit increased apoptosis
J Surg Res
(2004) - et al.
STAT6 specific shRNA inhibits proliferation and induces apoptosis in colon cancer HT-29 cells
Cancer Lett
(2006)
Eotaxin-3/CCL26 gene expression in intestinal epithelial cells is up-regulated by interleukin-4 and interleukin-13 via the signal transducer and activator of transcription
Int J Biochem Cell Biol
Identification of critical residues required for suppressor of cytokine signaling-specific regulation of interleukin-4 signaling
J Biol Chem
IL-4/Stat6 activities correlate with apoptosis and metastasis in colon cancer cells
Biochem Biophys Res Commun
Regulation of Th2 differentiation and Il4 locus accessibility
Annu Rev Immunol
Inducible resistance to Fas-mediated apoptosis in B cells
Cell Res
Cited by (55)
STAT family of transcription factors in breast cancer: Pathogenesis and therapeutic opportunities and challenges
2022, Seminars in Cancer BiologyCombined p53- And PTEN-deficiency activates expression of mesenchyme homeobox 1 (MEOX1) required for growth of triple-negative breast cancer
2020, Journal of Biological ChemistryPrimary cilia distribution and orientation during involution of the bovine mammary gland
2016, Journal of Dairy ScienceInterplay between signal transducers and activators of transcription (STAT) proteins and cancer: involvement, therapeutic and prognostic perspective
2023, Clinical and Experimental MedicineHematopoietic Prostaglandin D<inf>2</inf>Synthase Controls Tfh/Th2 Communication and Limits Tfh Antitumor Effects
2022, Cancer Immunology ResearchRole of Lymphocytes in Cancer Immunity and Immune Evasion Mechanisms
2022, Biomarkers of the Tumor Microenvironment, Second Edition
- 1
These authors contributed equally to this work.