C/EBP alpha mediates the growth inhibitory effect of progestins on breast cancer cells

Cathrin Brisken, Valentina Scabia
WILEY, 2019
Article

Résumé

Steroid hormones are key gene regulators in breast cancer cells. While estrogens stimulate cell proliferation, progestins activate a single cell cycle followed by proliferation arrest. Here, we use biochemical and genome-wide approaches to show that progestins achieve this effect via a functional crosstalk with C/EBP alpha. Using ChIP-seq, we identify around 1,000 sites where C/EBP alpha binding precedes and helps binding of progesterone receptor (PR) in response to hormone. These regions exhibit epigenetic marks of active enhancers, and C/EBP alpha maintains an open chromatin conformation that facilitates loading of ligand-activated PR. Prior to hormone exposure, C/EBP alpha favors promoter-enhancer contacts that assure hormonal regulation of key genes involved in cell proliferation by facilitating binding of RAD21, YY1, and the Mediator complex. Knockdown of C/EBP alpha disrupts enhancer-promoter contacts and decreases the presence of these architectural proteins, highlighting its key role in 3D chromatin looping. Thus, C/EBP alpha fulfills a previously unknown function as a potential growth modulator in hormone-dependent breast cancer.

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https://infoscience.epfl.ch/record/271047?ln=fr

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Progestins used for hormonal contraception in Switzerland: study of their effects on the breast epithelium

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Breast cancer is the most commonly diagnosed cancer and the most common cause of cancer death among women. Hormones have been shown to have a key role in breast development and carcinogenesis. Repeated activation of progesterone receptor (PR) signaling as it occurs during menstrual cycles is important in cancer risk. Through hormonal contraception many women are exposed to synthetic PR agonists, progestins, either on their own or in combination with the synthetic estrogen receptor (ER) agonist, ethinyl estradiol (EE). Progestins vary in their chemical structures and biological activities. Epidemiological studies show that hormonal contraceptions increase the risk of breast cancer but to date there have been few studies that analyze specific progestins regarding the breast cancer risk they confer. We hypothesized that different progestins have distinct abilities to induce cell proliferation and to modulate gene expression in the breast epithelium and this may ultimately have different effects on breast cancer risk. To test the hypothesis that different progestins currently used in hormonal contraceptions in Switzerland have distinct abilities to induce the expression of important mediators of PR action, receptor activator of NF-ÎºB ligand (RANKL) and Wnt4, we employed mouse mammary gland organoids and human breast tissue microstructures. We find that androgenic progestins strongly induce RANKL and Wnt4 whereas anti-androgenic progestins fail to do so. To test the ability of different progestins currently used in hormonal contraceptions in Switzerland to induce cell proliferation in human breast epithelium, we humanized mouse mammary gland using intraductal injection of human breast epithelial cells obtained from reduction mammoplasty surgery. We show that breast epithelial cells maintain hormone receptor expression and remain hormone responsive in this approach. We find that the androgenic progestins, Gestodene and Levonorgestrel, promote cell proliferation over two months more than progesterone. Furthermore, we show that Androgen receptor (AR) inhibition abrogates promegestone-induced RANKL induction and reduces cell proliferation induced by androgenic progestin, LNG. We also show that activation of AR signaling through DHT exposure is sufficient to induce proliferation of human breast epithelial cells. Thus, different progestins have different biological effects on the breast epithelium that appear to be related to their androgenic activities.

EPFL2018

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The female reproductive hormones estrogens, progesterone, and prolactin control postnatal breast development and are important to breast carcinogenesis. In particular, exposure to progesterone is tightly linked to breast cancer risk. Using the mouse as a model system to study the mechanisms through which hormones elicit morphogenetic changes in the mammary gland in vivo, we demonstrated that progesterone acts by a paracrine mechanism and identified Wnt4 as a target of progesterone important in mediating side branch formation. The mechanisms underlying Wnt4 function remains poorly understood. To understand the pattern of canonical Wnt signaling activation during mammary gland development and to address which cellular compartment of the mammary gland is activated by canonical Wnt signaling, a reporter mouse model was utilized in which the lacZ gene is under the control of endogenous Axin2 promoter, classical target promoter of canonical Wnt signaling. Analysis of reporter mice at different developmental stages of mammary gland development showed the highest reporter activity during early pregnancy which correlated with the time of side branch formation. At this stage, β-galactosidase activity was found exclusively in myoepithelial cells. Overexpression of Wnt1 from MMTV promoter increases reporter activity in the myoepithelium. Gene expression analysis on FACS sorted luminal and myoepithelial cells shows that known canonical Wnt signaling pathway components such as receptors and co receptors as well as targets are specifically expressed in the myoepithelial compartment further supporting the notion that myoepithelial cells are the target cells for Wnt1 and Wnt4 secreted by luminal cells. Consistent with a model whereby progesterone induced Wnt4 secretion results in canonical Wnt signaling activation, reporter activity was induced by progesterone treatment and lacZ expression increased during diestrus when serum progesterone levels are high under physiological conditions. Analysis of contralateral mammary glands engrafted with PR-/-.Axin2 lacZ and PR+/-.Axin2 lacZ epithelium during pregnancy revealed that intact PR signaling is required to activate canonical Wnt signaling. Similar experiments with Wnt4-/-.Axin2 lacZ and Wnt4+/+.Axin2 lacZ mammary epithelium showed that reporter activation relies on Wnt4. Histological analysis on mammary glands in which β-catenin was ablated using MMTV-Cre revealed that β-catenin deleted cells were present only in the luminal epithelial compartment and showed that β-catenin deleted myoepithelial cells cannot participate in side branch formation. Similarly, ectopic expression of dominant negative β-catenin in wt MECs reduced side branching indicating that canonical Wnt signaling mediated by β-catenin is required for side branching. Furthermore, constitutive activation of β-catenin in the mammary epithelium was sufficient to induce side branch formation in virgin mice. In Wnt4 mutant epithelium, proliferation of progesterone receptor positive and negative cells were significantly reduced in response to progesterone treatment. Wnt signaling has been implicated in stem/progenitor activation in several tissues including mammary gland. Consistent with Wnt4 activating stem/progenitors, serial transplantation assays revealed that Wnt4-/- epithelium failed to reconstitute already at 2nd to 3rd generation when the wt has the ability to reconstitute up to seven generations indicating that Wnt4 is required for stem cell function while Wnt4 is dispensable for embryonic and rudimentary mammary gland formation. Histology and gene expression analysis in late pregnancy showed that Wnt4 mutant epithelia were able to differentiate and produce milk proteins, further supporting the thought that Wnt4 function is important for stem/progenitor cell activity and not required for differentiation. Taken together, I report that Wnt4 induces canonical Wnt signaling in myoepithelial cells and activation of canonical Wnt signaling is required and sufficient for side branching. Moreover, Wnt4 function is essential for mammary stem/progenitor cells.

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