Type II Endometrial Cancer Overexpresses NILCO: A Preliminary Evaluation.

OBJECTIVE: The expression of NILCO molecules (Notch, IL-1, and leptin crosstalk outcome) and the association with obesity were investigated in types I and II endometrial cancer (EmCa). Additionally, the involvement of NILCO in leptin-induced invasiveness of EmCa cells was investigated. METHODS: The expression of NILCO mRNAs and proteins were analyzed in EmCa from African-American (n = 29) and Chinese patients (tissue array, n = 120 cases). The role of NILCO in leptin-induced invasion of Ishikawa and An3ca EmCa cells was investigated using Notch, IL-1, and leptin signaling inhibitors. RESULTS: NILCO molecules were expressed higher in type II EmCa, regardless of ethnic background or obesity status of patients. NILCO proteins were mainly localized in the cellular membrane and cytoplasm of type II EmCa. Additionally, EmCa from obese African-American patients showed higher levels of NILCO molecules than EmCa from lean patients. Notably, leptin-induced EmCa cell invasion was abrogated by NILCO inhibitors. CONCLUSION: Type II EmCa expressed higher NILCO molecules, which may suggest it is involved in the progression of the more aggressive EmCa phenotype. Obesity was associated with higher expression of NILCO molecules in EmCa. Leptin-induced cell invasion was dependent on NILCO. Hence, NILCO might be involved in tumor progression and could represent a new target/biomarker for type II EmCa.

Nanoparticle-linked antagonist for leptin signaling inhibition in breast cancer.

AIM: To develop a leptin peptide receptor antagonist linked to nanoparticles and determine its effect on viability of breast cancer cells. METHODS: The leptin antagonist, LPrA2, was coupled via EDAC [1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide] to iron oxide nanoparticles (IONP-LPrA2) to increase its efficacy. IONP-LPrA2 conjugation was confirmed by Western blot and nanoparticle tracking analysis. Human triple negative breast cancer (TNBC) MDA-MB-231, HCC1806 and estrogen receptor positive (ER+) MCF-7 cells were analyzed for the expression of the leptin receptor, Ob-R. The effects of leptin and antagonist on levels of leptin-induced STAT3 phosphorylation and cyclin D1, cell cycle progression, cell proliferation, and tumorsphere formation in breast cancer cells were determined. Doses of the chemotherapeutics [cisplatin (Cis), cyclophosphamide (CTX), doxorubicin (Dox) and paclitaxel (PTX)] to effectively reduce cell viability were calculated. The effects of combination treatments of IONP-LPrA2 and chemotherapeutics on cell viability were determined. RESULTS: Western blot analysis of coupling reaction products identified IONP-LPrA2 at approximately 100 kD. IONP-LPrA2 significantly decreased leptin-induced pSTAT3 levels in HCC1806 cells and drastically decreased cyclin D1 levels in all cell lines. IONP-LPrA2 significantly reduced leptin-induced S phase progression and cell proliferation in all breast cancer cell lines and the formation of tumorspheres in MDA-MB-231 cells. Also, IONP-LPrA2 showed an additive effect on the reduction of breast cancer cell survival with chemotherapeutics. Cis plus IONP-LPrA2 produced a significant reduction in the survival of MDA-MB-231 and HCC1806 cells. CTX plus IONP-LPrA2 caused a significant decrease in the survival of MDA-MB-231 cells. Dox plus IONP-LPrA2 caused a marked reduction in the survival of HCC1806 cells. Although, PTX plus IONP-LPrA2 did not have a major effect on the viability of the breast cancer cells when compared to PTX alone. CONCLUSION: Present data indicate that IONP-LPrA2 may be a useful adjuvant for chemotherapeutic treatment of breast cancer, particularly for TNBC which lacks targeted therapeutic options.

Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation.

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin's actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin's actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin's effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.

Obesity induced a leptin-Notch signaling axis in breast cancer.

To investigate whether obesity induces a leptin-Notch signaling axis in breast cancer (BC), leptin-induced Notch was determined in human MCF-7 and MDA-MB231 and mouse E0771 cells and in E0771-BC hosted by syngeneic lean and diet-induced obesity (DIO) C57BL/6J female mice. Lean and DIO mice were treated for 3 weeks with leptin inhibitor (PEG-LPrA2) 1 week after the inoculation of E0771 cells. Leptin induced Notch1, 3 and 4 in BC cells, but Notch2 expression showed opposite pattern in MCF-7 compared to MDA-MB231 cells. Notch loss-of-function (DAPT and dominant negative [R218H] RBP-Jk [CSL/CBF1]) showed that a functional leptin-Notch signaling axis was involved in the proliferation and migration of E0771 cells. E0771-BC onset was affected by obesity (lean mice7/10 [70%] vs. DIO mice: 11/12 [92%]; Pearson χ(2) : p = 0.06]). PEG-LPrA2 significantly reduced BC growth (untreated: 19/42; [45%] vs. treated: 8/42 [19%]; Pearson χ(2) : p = 0.008). PEG-LPrA2 did not influence the caloric intake of mice but increased carcass and/or body weights of lean and DIO mice inoculated with E0771 cells, which could be related to the improvement of health conditions (less aggressive disease). Importantly, BC from obese mice had higher levels of Notch3, JAG1 and survivin than lean mice. Inhibition of leptin signaling reduced protein levels of Notch (NICD1, NICD4, Notch3, JAG1 and survivin) and significantly decreased mRNA expression of Notch receptors, ligands and targets. PEG-LPrA's effects were more prominent in DIO mice. Present data suggest that leptin induces Notch, which could be involved in the reported higher incidence and aggressiveness and, poor prognosis of BC in obese patients.

Leptin's Pro-Angiogenic Signature in Breast Cancer.

Obesity is linked to increased incidence of breast cancer. The precise causes and mechanisms of these morbid relationships are unknown. Contradictory data on leptin angiogenic actions have been published. However, accumulating evidence would suggest that leptin's pro-angiogenic effects in cancer play an essential role in the disease. Leptin, the main adipokine secreted by adipose tissue, is also abnormally expressed together with its receptor (OB-R) by breast cancer cells. Leptin induces proliferation and angiogenic differentiation of endothelial cells upregulates VEGF/VEGFR2 and transactivates VEGFR2 independent of VEGF. Leptin induces two angiogenic factors: IL-1 and Notch that can increase VEGF expression. Additionally, leptin induces the secretion and synthesis of proteases and adhesion molecules needed for the development of angiogenesis. Leptin's paracrine actions can further affect stromal cells and tumor associated macrophages, which express OB-R and secrete VEGF and IL-1, respectively. A complex crosstalk between leptin, Notch and IL-1 (NILCO) that induces VEGF/VEGFR2 is found in breast cancer. Leptin actions in tumor angiogenesis could amplify, be redundant and/or compensatory to VEGF signaling. Current failure of breast cancer anti-angiogenic therapies emphasizes the necessity of targeting the contribution of other pro-angiogenic factors in breast cancer. Leptin's impact on tumor angiogenesis could be a novel target for breast cancer, especially in obese patients. However, more research is needed to establish the importance of leptin in tumor angiogenesis. This review is focused on updated information on how leptin could contribute to tumor angiogenesis.