In agreement with ObR overexpression, raising doses of leptin were able to stimulate to a greater extent growth and migration in AnaR than sensitive cells. sensitive cells. Moreover, leptin contributed to enhanced crosstalk between AnaR cells and macrophages within the tumor microenvironment. Indeed, AnaR, through leptin secretion, modulated macrophage profiles and increased macrophage motility through CXCR4 Bleomycin signaling, as evidenced by RNA-sequencing, real-time PCR, and immunoblotting. Reciprocally, activated macrophages increased AnaR cell growth and motility in coculture systems. In conclusion, acquired AI resistance is accompanied by the development of a leptin-driven phenotype, highlighting the potential clinical benefit of targeting this cytokine network in hormone-resistant breast cancers, Bleomycin especially in obese women. < 0.05; ** < 0.005; *** < 0.0005. First, we evaluated using real-time PCR specific transcript levels of leptin and the long and short leptin receptor isoforms (ObRl and ObRsh) in MCF-7 aro at the start of treatment with Ana and after different time points until cells acquired the resistant phenotype (~4th month). We observed that the prolonged treatment with Ana induced a progressive phenotypic shift characterized after the 4th month of treatment by increased mRNA expression of leptin and its receptors, persisting over time (Figure 2a). The increase in both ObRl and ObRsh was then confirmed by evaluating protein levels Mouse monoclonal to CRTC3 using immunoblotting in AnaR compared to MCF-7 aro cells (Figure 2b), whereas ELISA measurement in breast cancer cell-derived conditioned media showed that AnaR cells exhibited a 2.5 fold higher leptin secretion than MCF-7 aro cells (Figure 2c). These results imply that an enhanced leptin autocrine feedback loop may exist in AnaR cells. Indeed, resistant cells exhibited increased constitutive phosphorylation levels of the leptin downstream effectors JAK2, STAT3, AKT, and MAPK (Figure 2d). Accordingly, treatment with the peptide LDFI, a small peptide of the wild type sequence of leptin binding site I, that we have recently demonstrated to specifically inhibit both in vitro and in vivo the leptin signaling pathway , significantly reduced the increased basal anchorage-independent growth (Figure 2e) and motility (Figure 2f) in AnaR cells, indicating a selective dependency on leptin signaling for this cell line. Open in a separate window Figure 2 Increased leptin signaling activation in AnaR breast cancer cells. (a) Quantitative real-time PCR for mRNA expression of leptin < 0.05; ** < 0.005. 3.2. Anastrozole-Resistant Breast Cancer Cells Show Leptin Hypersensitivity The adipocyte-derived leptin, whose synthesis and plasma levels increase in parallel to total adipose tissue mass, has an Bleomycin important role in promoting breast cancer progression. Thus, we also evaluated the effects of exogenous leptin stimulation on growth in our anastrozole-resistant cell models (Figure 3a). In a dose-response study, we observed that low concentrations of leptin (10 and 100 ng/mL) were Bleomycin able to increase colony numbers in anchorage-independent growth assays only in AnaR cells. In addition, treatment with leptin at 1000 ng/mL enhanced cell growth in both sensitive and resistant cells, although to a greater extent in AnaR cells. We also evaluated the ability of increasing doses of leptin to influence cell migration in wound-healing Bleomycin scratch assays (Figure 3b). The resistant cells moved more rapidly at the front of cell migration to close the gap compared with MCF-7 aro cells. Leptin treatments at 1000 ng/mL promoted cell motility in both cells, but at a higher extent in resistant cells. Interestingly, leptin at 10 and 100 ng/mL stimulated migration only in AnaR cells. The increase in colony numbers and migration induced by leptin was reversed by the peptide LDFI (Figure 3c,d, respectively). Therefore, increased leptin sensitivity may likely support AIR in breast cancer cells. Open in a separate window Figure 3 Leptin hypersensitivity in AnaR cells. (a) Soft agar growth assays in MCF-7 aro and AnaR breast cancer cells stimulated for 14 days with vehicle (-) or increasing doses of leptin. (b) Wound healing assays in cells exposed to vehicle (-) or leptin (Lep) as indicated. Inset, time 0. Pictures.