Breast cancer is recognized as the most common type of cancer in women and its development is associated with many risk factors such as diet, alcohol consumption, child bearing, breast feeding, oral contraception, as well as underlying genetic predisposition. Epidemiological studies show a rapid increase in breast cancer incidence during reproductive years that tapers around age 50, corresponding to the onset of menopause, and studies of postmenopausal breast cancer patients have found a higher level of estrogen in breast tissue compared to healthy patient tissue[1–6]. Taken together with the fact that 60-70% of human breast cancers are estrogen receptor-alpha positive, the evidence suggests an etiological significance of estrogen in breast cancer initiation and progression.
Estrogen is a sex steroid hormone produced mostly by the ovaries in women; however other tissues, including adipose, are also able to synthesize estrogen. There are a total of nine estrogens in humans of which 17β-Estradiol (E2) is the most abundant in circulation and the most biologically active. Estrogen mediates its effects by binding to its cognate estrogen receptor(s), either estrogen receptor alpha (ERα) or estrogen receptor beta (ERβ), leading to ER dimerization and association with various co-factors. Once formed, the complex translocates to the nucleus where it acts as a transcription factor by binding to the estrogen response elements (EREs) at the promoters of estrogen responsive genes[9, 10]. Besides this classical pathway, estrogen can also regulate gene transcription in ERE independent as well as nongenomic pathways by binding to membrane associated estrogen receptor leading to signaling via the PI3K/AKT pathway[11, 12]. In addition to its normal physiological roles, estrogen is also implicated in breast cancer initiation and progression. Estrogen-ER interactions have been observed to increase cell survival by signaling through the AKT pathway which leads to suppression of TNF-α induced apoptosis. Estrogen-ER signaling also induces cell proliferation by activation of the PI3K pathway, observed in breast cancer cell lines including ER+ MCF-7 cells, but not the ER- MDA-MB-231 cell line. Interestingly, estrogen is also capable of contributing to breast cancer progression by a novel role, via modulation of proteins involved in hypoxia signaling, namely hypoxia inducible factor 1 (HIF-1).
HIF-1 is also a heterodimeric transcription factor, consisting of the oxygen dependent alpha subunit and the constitutively expressed beta subunit. During normoxia, HIF-1α is rapidly degraded via the proteasomal pathway, however during hypoxia, HIF-1α is stabilized and binds HIF-1β (aryl hydrocarbon receptor nuclear translocator, ARNT), forming a transcriptional complex which translocates to the nucleus where, with other protein co-factors, it binds hypoxia responsive elements (HRE). Binding of HIF-1 to target genes leads to transcription of proangiogenic proteins including erythropoietin and VEGF, which are essential for formation of new blood vessels, or neovasculogenesis. Further, the chemotactic protein stromal derived factor 1 (SDF-1) is also hypoxia responsive, leading to development of a chemotactic gradient for bone marrow derived cells that express the cognate receptor CXCR4[17–19]. In a rat uterine model, estrogen was observed to increase HIF-1α levels in vivo and this induction lead to an increase in VEGF expression that was abrogated by PI3K inhibitors but not MAPK inhibitors[20, 21]. Chromatin immunoprecipitation assays found that this estrogen treatment lead to binding of both ER and HIF-1 to VEGF promoters. E2 also lead to up regulation of HIF-1 in ovarian cancer cells in a PI3K dependent manner[23, 24]. ER positive breast cancers have also been linked to an increased expression of HIF-1 and correlated with a more metastatic phenotype[25, 26]. The ability of estrogen to stimulate proteins involved in hypoxia signaling as well as to induce proangiogenic proteins may elucidate a novel role of estrogen in breast cancer neovasculogenesis. This novel physiological effect of estrogen in carcinogenesis progression is an understudied area and can shed light on the systemic activity of hormone induced cancers.
Neovasculogenesis, or the formation of new blood vessels, is modulated by estrogen and is necessary for tumor growth and sustainment. Studies using ER knockout mice observed reduced vascular repair and angiogenesis thus demonstrating the role of estrogen in vessel formation. In ex vivo breast tissue cultures, as well as in vivo mouse models, E2 led to an increase in secretion of the proangiogenic cytokine IL-8, which is strongly correlated with the metastatic potential of breast cancer cells. Further, E2 increased angiogenin secretion, which led to an increase in endothelial cell proliferation and was abrogated by the antiestrogen Tamoxifen. In breast tumor mouse studies, E2 was observed to increase blood vessel formation and significantly increased endothelial progenitor cell migration to tumor sites. Further, E2 also enhanced mRNA transcripts of proangiogenic angiopoietins 1 and 2, as well as metastatic modulating matrix metalloproteinase 2 and 9. In vitro models from our laboratory demonstrated E2 induced TG1-1 cell proliferation and migration, which was abrogated by anti-estrogens. In vitro tubulogenesis models have also demonstrated the role in E2 induced neovasculogenesis in breast cancer. Considering that both hypoxia and estrogen are significant determinants of breast cancer progression and can modulate vasculogenesis processes and hence the tumor microenvironment, it is important to understand their cellular modulation so that novel intervention strategies can be examined.
This study was designed to investigate the role of estrogen on HIF-1 dependent breast cancer induced neovasculogenesis. Two types of cell lines were used: the TG1-1 murine breast cancer cell line that expresses both ERα and ERβ and the human endothelial cell line human umbilical vein endothelial cell (HUVEC). Our results define the molecular interdependence of estrogen mediated intracellular activity with hypoxia and reconnect the modulatory interdependence of cellular phenotypic changes. These studies open up new avenues of estrogen based therapeutic and preventive interventions for breast cancer that is based on the tumor microenvironment.