Protein kinases mediate ligand-independent derepression of sumoylated progesterone receptors in breast cancer cells

Abstract

In advanced breast tumors, protein kinases are upregulated and steroid hormone receptors often function independently of ligand. Herein, we explored mechanisms of ligand-independent progesterone receptor (PR) activity. We showed previously that growth factorinduced phosphorylation of PR Ser-294 blocks PR Lys-388 sumoylation. SUMO-deficient mutant PR-B (K388R) thus provides a model receptor for the study of PR function in the context of high kinase activities. T47D cells stably expressing K388R PR-B exhibited increased ligand-independent proliferation and growth in soft agar relative to cells expressing wt PR-B or phospho-mutant (sumoylated) S294A PR-B. Expression of selected PR target genes (HB-EGF, IRS-1, and STC1) was significantly elevated in cells containing desumoylated (K388R) PR-B. Basal PR transcriptional activity occurred independently of progestins, was increased by activated CDK2, and attenuated by RU486. Notably, ChIP assays demonstrated that K388R PR-B and SRC1 were constitutively recruited to the STC1 promoter in the absence of progestin; PR Lys-388 sumoylation was required for HDAC3 recruitment. Knock-down of STC1 inhibited proliferation of cells expressing K388R PR-B. These data suggest a mechanism whereby phosphorylated, and thus desumoylated, PRs mediate increased expression of growth promoting genes. Our data explain why breast cancer models often remain insensitive to progestins, but are growth-inhibited by antiprogestins, and underscore the need to target PR-B and associated kinase activities as part of breast cancer therapy.HDAC ͉ phosphorylation ͉ sumoylation P rogesterone receptors (PR) mediate lobulo-alveolar proliferation during breast development and contribute to breast cancer progression, in part by synergizing with peptide growth factors (1-3). Regulation of PR activity occurs via the integration of ligand activated rapid signaling events and transcriptional activation (4). Peptide growth factors diminish the requirement for steroid hormone ligands by activation of protein kinases that directly target PR and its co-activators (i.e., a function of rapid signaling events). For example, direct phosphorylation of PR by MAPK or CDK2 alters transcriptional activity in part by modulating other posttranslational modifications including ubiquitination and sumoylation (3,5). Crosstalk between growth factor pathways and steroid receptors provides an exquisite mechanism for mammary epithelial cells to sense a dynamic range of hormone concentrations and selectively regulate specific promoters.Sumoylation of steroid hormone receptors represses transcriptional activity (6). We showed that EGF and protein kinasedependent (MAPK, CDK2) phosphorylation of PR-B on Ser-294 blocks sumoylation of PR Lys-388 (5). Undersumoylated receptors respond to low concentrations of ligand and are transcriptionally hyperactive at a subset of PRE (progesterone response element) containing target genes (HB-EGF). Sumoylation of PR is thus a key modulator of both the progesterone/PR dose-response curve and PR target gene ...

Histone Modification and Breast Cancer

“…Although the knowledge of their functions and mechanisms is still little, some studies have showed that they are also associated with breast cancer and other human cancers. The regulation of gene expression by phosphorylated and undersumoylated PRs is a novel form of hormone independent PR action that is predicted to contribute to breast cancer cell growth and survival (Daniel et al, 2009). Recent studies revealed that E3 ubiquitin ligases play important roles in breast carcinogenesis.…”

Evolution and functions of stanniocalcins in cancer

“…23 However, few studies show that the expression of STC1 is not an independent and unfavorable prognostic factor for esophageal squamous cell carcinoma (ESCC). 27 …”

“…26 Breast cancer growth is insensitive to progestins, and knockdown of STC1 inhibits proliferation of tumor cells expressing progesterone receptor (PR), suggesting that STC1 may play a potential role in therapy of breast cancer. 27 …”

Progesterone receptor modulators in breast cancer

“…Subtle changes at the cellular level can have major effects. The phosphorylation and sumolyation of PR by intracellular mechanisms can impact PR action (Daniel and Lange, 2009). Fibroids are understood to be clonal in nature and thus may vary from one cell line to another.…”