May tumor microenvironment cooperate with a mutant ERalpha to promote breast cancer progression?

Surrounding stromal cells and adipokines, as leptin, strongly influence the phenotypic behavior of malignant breast epithelial cells and elevated serum levels of leptin in obesity is a risk factor for breast cancer incidence, mortality and drug resistance. We identified a lysine to arginine transition at residue 303 (K303R) within estrogen receptor (ERα) in premalignant and invasive breast cancers, which confers estrogen hypersensitivity and resistance to hormone therapy.Microarray analysis showed increased leptin receptor mRNA levels in K303R-expressing MCF-7 breast cancer cells. We hypothesized that K303R ERα, as an amplified effector of leptin signaling, may potentiate tumor/stroma microenvironment stimulatory effects on breast cancer progression.We used as experimental models for breast cancer ERα-positive MCF-7 and ERα-negative SKBR3 cells stably transfected with wild-type (WT) or K303R ERα and for stromal cells primary fibroblasts isolated from human breast carcinoma (CAFs), that we showed secreting leptin. We found, in mutant cells, an increase in leptin receptor isoforms, and in its signalling activation. Leptin also enhanced phosphorylation and transcriptional activity of K303R ERα.Leptin treatment 100-1000ng/ml increased anchorage-dependent and independent cell growth, and in vitro cell motility and invasiveness, evaluated by scratch, migration and matrigel invasion assays, in WT and K303R clones, but in higher extent in mutant cells. Low physiological leptin doses had effects only in K303R clones. The JAK2/STAT3 inhibitor AG490 and the ER antagonist ICI 182,760 abrogated these effects.Conditioned medium (CM) isolated from CAFs was more effective in stimulating proliferation and migration of both MCF-7 and SKBR3 K303R ERα cells compared to WT cells. AG490 and ICI reversed these effects. To specifically define leptin contribution, we immunodepleted leptin from stroma-derived CM using specific antibodies and found a reduction on proliferation and migration especially in mutant cells.Understanding the molecular dynamics of stroma-tumor networks may help to improve decision-making for some ER-positive breast cancer patients. Since the K303R mutation was identified in 30% of typical hyperplasia, we could speculate that the pressure of the microenvironment in the presence of this mutation hypersensitive to leptin signaling may promote or accelerate the development of cancers from premalignant breast lesions, further increasing risk in obese women.