Hormonal regulation of HES-1 in breast cancer cells

Hairy and Enhancer of Split homolog 1 (HES-1) is a transcriptional repressor belonging to the basic helix-loop-helix family of proteins, and has been shown to have a pivotal role as a regulator of neurogenesis, myogenesis and hematopoiesis. HES-1 has several well conserved domains, from Drosophila to mammals, and is expressed in a variety of tissues, in both embryos and adults. The most described function of HES-1 is as the primary effector protein in Notch signaling, as an inhibitor of differentiation, where expression of HES-1 maintains precursor cells undifferentiated and in a proliferative state. In breast cancer cells, exogenous expression of HES-1 abolishes the estrogen-induced proliferation, whereas endogenous HES-1 expression is repressed in response to estrogen. These background data suggest that breast cancer cells need to repress HES-1 expression in order to proliferate in response to estrogen.

The general aim of this thesis was to determine the role of HES-1 in estrogen- and retinoic acid-regulated proliferation of human breast cancer cells.

In study I, we show that HES-1 is a mediator of retinoic acid-induced inhibition of proliferation in the human breast cancer cell line MCF-7. Increasing levels of retinoic acid caused increasing expression of HES-1, followed by decreasing proliferation. By stably expressing a dominant negative HES-1 in MCF-7 cells the endogenous activity of HES-1 was inhibited and the cells become unresponsive to retinoic acid.

In study II, we show that exogenous expression of HES-1 causes cell cycle arrest in the G1 phase of the cell cycle. We found the cell cycle factor E2F-1, critical for G1 to S-phase progression, to be regulated by HES-1, via a cis-element in the 5 regulatory region of the E2F-1 gene. HES-1 was shown to mediate the negative regulation on E2F-1 expression induced by retinoic acid, as indicated by the lack of the negative effect when expressing the dominant negative HES-1. We also found that HES-1 antagonizes the effect of another mitogen, heregulin-beta1. The heregulin-beta1 induced E2F-1 expression and the subsequent proliferation were inhibited by overexpression of HES-1.

In study III, we investigated the mechanism by which HES-1 is regulated in response to estrogen and retinoic acid. There is a retinoic acid response element 18 kb upstream of the HES-1 gene that was bound by retinoic acid receptor alpha and activated transcription in response to retinoic acid. Results from a whole-genome estrogen receptor alpha (ERalpha) binding site mapping experiment, show ERalpha binding 23 kb downstream of HES-1 gene. ERalpha binds and regulates transcription via a novel cis-element, containing one consensus estrogen response element and a half site. ChIP assays revealed that ERalpha, coactivators and corepressors were recruited to this cis-element in response to estrogen. Importantly, a decrease in acetylation of histone H3 and H4 and a decrease in recruitment of RNA POL II were observed, indicating a repression of HES-1 expression in response to estrogen.

In study IV, we show that retinoic acid mediates the induction of HES-1 gene expression via the transcription factor SOX9. We found SOX9 bound to a cis-element 3.7 kb upstream of HES-1 gene in response to retinoic acid. This cis-element regulated transcription induced by retinoic acid.

In conclusion, these results establish HES-1 as a mediator of the cell cycle arrest induced by retinoic acid, and suggest HES-1 to be an important regulator of proliferation of breast cancer cells.

List of scientific papers

I. Muller P, Kietz S, Gustafsson JA, Strom A (2002). The anti-estrogenic effect of all-trans-retinoic acid on the breast cancer cell line MCF-7 is dependent on HES-1 expression. J Biol Chem. 277(32): 28376-9. Epub 2002 Jun 21
https://pubmed.ncbi.nlm.nih.gov/12080040

II. Hartman J, Müller P, Foster JS, Wimalasena J, Gustafsson JA, Ström A (2004). HES-1 inhibits 17beta-estradiol and heregulin-beta1-mediated upregulation of E2F-1. Oncogene. 23(54): 8826-33.
https://pubmed.ncbi.nlm.nih.gov/15467735

III. Müller P, Merrel KW, Rönnlund C, Lin CY, Gustafsson JÅ, Ström A (2008). Estrogen-dependent downregulation of HES-1 gene expression in breast cancer cells is mediated via a 3 distal element. [Submitted]

IV. Müller P, Crofts JD, Newman BS, Bridgewater LC, Lin CY, Gustafsson JÅ, Ström A (2008). SOX9 mediates the retinoic acid-induced HES-1 gene expression in human breast cancer cells. [Submitted]