Molecular targets of leptin signaling in estradiol responsive and non-responsive breast cancer

A2 Despite the fact that leptin is now recognized as a growth promoting factor in breast cancer (BC), the molecular targets of its signaling pathways remain unknown. Hypothesis:Leptin signaling increases the levels of pro-angiogenic/inflammatory factors that positively affect the growth of estradiol responsive and non-responsive mammary tumors (MT). Objectives: 1) To investigate whether leptin signaling could regulate molecules involved in pro-angiogenic and inflammatory events in E2-responsive and non-responsive human BC xenografts in SCID mice. 2) Determine if the inhibition of leptin signaling would negatively impact the levels of target molecules and tumor growth. Methods: MCF-7(ER+) and MDA-MB231(ER-) were orthotopically inoculated into ovariectomized (for ER+/sub-dermally implanted with 2 mg, 17s-E2 and 1.6 mg, cholesterol) or non-ovariectomized (for ER-) SCID mice (6 weeks old). Mice were either untreated (control) or treated one week before cell inoculation with one of the following compounds: a pegylated leptin peptide receptor antagonist (PEG)-LPrA2/0.2 mM/200μl, scrambled -PEG-wLPrA2 (non-specific treatment control) and PBS (sham-control). Treatment injections were delivered every 48h into the tail vein and continued for 50 (MCF-7) or 28 days (MDA-MB231). MT growth was determined by calipers and weight after euthanasia. MT lysates were produced and their level of proteins was determined. Concentrations of VEGF, leptin and IL-1 (alpha and beta) in serum and in MT lysates were determined by ELISA. Levels of VEGF, VEGFR2 and IL-1R tI were determined in MT lysates by Western blot. Immunohistochemical analysis of angiogenic-related antigens PECAM, CD31 and CD68 in MT lysates was also carried out. To assess whether the LPrA’s treatment would impact mouse health, carcass and body weight (BW), food intake (FI) and serum levels of glucose and insulin were determined. Results:Leptin signaling had a higher impact on the levels of angiogenic- and inflammatory-related compounds in MCF-7 than in MDA-MB231 MT. Treatment with the leptin receptor inhibitor (PEG-wLPrA2) reduced the levels of VEGF (11.1 %-lysates; 8.3 %-serum) and IL-1α (58.3 %) in mice hosting MCF-7 tumors versus VEGF (62.8 %-lysates; 34.7 %-serum) and IL-1α (75.3 %) in mice hosting MDA-MB231 tumors. Densitometry and Western blot analysis of MT lysates showed that leptin signaling is related to the increased levels of VEGFR2 and IL-1R tI, within ER+ or ER- tumors. However, the levels of these antigens were reduced to a lesser extent in MCF-7 than MDA-MB231 tumors. Similar results were found for the expression of PECAM and CD68. Remarkably, PEG-wLPrA treatment simultaneously reduces the growth of MCF-7 tumors and levels of the aforementioned target molecules. Moreover, the onset of MCF-7 tumors was significantly delayed by leptin signaling inhibition. In contrast, no significant effects on onset/growth of ER- MT were found after PEG-wLPrA treatment. In addition, no significant difference in the levels of glucose/insulin, body/carcass weight or food intake between treatments was found. Conclusions:These results suggest that leptin signaling in E2-responsive or non-responsive MT impacts angiogenic/inflammatory-related molecules, which in turn contribute to the growth of MT. However, ER+ MT were more sensitive to the effects of leptin signaling inhibition.