title: Integrity of the LXXLL motif in Stat6 is required for the inhibition of breast cancer cell growth and enhancement of differentiation in the context of progesterone.
abstract: BACKGROUND
  Progesterone is essential for the proliferation and differentiation of mammary gland epithelium. Studies of breast cancer cells have demonstrated a biphasic progesterone response consisting of an initial proliferative burst followed by sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the effects of progesterone on mammary cell growth and differentiation remain to be determined. Recently, it was demonstrated that signal transducer and activator of transcription 6 (Stat6) is a cell growth suppressor. Similar to progesterone-bound PR, Stat6 acts by inducing the expression of the G1 cyclin-dependent kinase inhibitors p21 and p27. The possible interaction between Stat6 and progesterone pathways in mammary cells was therefore investigated in the present study.
  
  
  METHODS
  ChIP and luciferase were assayed to determine whether Stat6 induces p21 and p27 expression by recruitment at the proximal Sp1-binding sites of the gene promoters. Immunoprecipitation and Western blotting were performed to investigate the interaction between Stat6 and PR-B. The cellular DNA content and cell cycle distribution in breast cancer cells were analyzed by FACS.
  
  
  RESULTS
  We found that Stat6 interacts with progesterone-activated PR in T47D cells. Stat6 synergizes with progesterone-bound PR to transactivate the p21 and p27 gene promoters at the proximal Sp1-binding sites. Moreover, Stat6 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. Stat6 knockdown also abolished the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. In addition, knockdown of Stat6 expression prevented the induction of breast cell differentiation markers, previously identified as progesterone target genes. Finally, Stat6 gene expression levels increased following progesterone treatment, indicating a positive auto-regulatory loop between PR and Stat6.
  
  
  CONCLUSIONS
  Taken together, these data identify Stat6 as a coactivator of PR mediating the growth-inhibitory and differentiation effects of progesterone on breast cancer cells.
authors.affiliation: Breast Department, International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030, People's Republic of China. wmhsp@126.com.
authors.forename: Min
authors.initials: M
authors.lastname: Wei
authors.forename: Qi
authors.initials: Q
authors.lastname: He
authors.forename: Zhongyin
authors.initials: Z
authors.lastname: Yang
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Wang
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Bingya
authors.initials: B
authors.lastname: Liu
authors.forename: Qinlong
authors.initials: Q
authors.lastname: Gu
authors.forename: Liping
authors.initials: L
authors.lastname: Su
authors.forename: Yingyan
authors.initials: Y
authors.lastname: Yu
authors.forename: Zhenggang
authors.initials: Z
authors.lastname: Zhu
authors.forename: Guofeng
authors.initials: G
authors.lastname: Zhang
mesh_terms: D020816:Amino Acid Motifs; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D011374:Progesterone / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011980:Receptors, Progesterone / Q000378:metabolism*; D050800:STAT6 Transcription Factor / Q000235:genetics* / Q000378:metabolism
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D011980:Receptors, Progesterone; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D050760:Cyclin-Dependent Kinase Inhibitor p27; D011374:Progesterone