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 three weeks with leptin inhibitor (PEG-LPrA2) one 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 mice: 7/10 (70%) vs DIO-mice: 11/12 (92%); Pearson Chi2: P=0.06]. PEG-LPrA2 significantly reduced BC growth [untreated: 19/42; (45%) vs treated: 8/42 (19%); Pearson Chi2: 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, JAG-1 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.

BackgroundNotch, IL-1 and leptin are known pro-angiogenic factors linked to breast cancer development, tumor aggressiveness and poor prognosis. A complex crosstalk between these molecules (NILCO) has been reported in breast cancer cell lines. However, whether NILCO biomarkers are differentially expressed in estrogen responsive (ER+), unresponsive (ER-) and triple negative (TNBC) breast cancer tissues is unknown.MethodsExpression levels of nine NILCO and targets [Notch1, Notch4, JAG1, DLL4, VEGF, VEGFR2 (FLK-1), leptin, leptin receptor (OB-R) and interleukin-1 receptor type I (IL-1R tI)] were examined via immunohistochemistry in breast cancer tissue microarrays from Chinese patients (ER+, n=33; ER-, n=21; TNBC, n=13) and non-malignant breast tissue (n=5; Pantomics, Inc.) using a semi-quantitative analysis of intensity staining, HSCORE.ResultsCategorical expression of NILCO and targets (+ or -) was similar among all cancer tissues. However, TNBC showed differential localization pattern of NILCO. TNBC showed fewer nuclei and cytoplasms positive for Notch4 and JAG1, but more cytoplasms positive for leptin. In addition, fewer TNBC stromas were positive for Notch1 and Notch4, but 100% of TNBC stromas were positive for VEGFR2. Moreover, TNBC had lower DLL4 and IL-1R tI expression. TNBC and ER- showed higher expression of EGFR, but lower expression of AR. Leptin and OB-R were detected in more than 61% of samples. Leptin positively correlated to OB-R, JAG1, VEGF, and marginally to IL-1R tI. Notch1 positively correlated to IL-1R tI. EGFR and Ki67 were positively associated to Notch1, but no associations of NILCO and targets with p53 were found.ConclusionsPresent data suggest that NILCO components are differentially expressed in breast cancer. TNBC showed distinctive patterns for NILCO expression and localization. The complex crosstalk between leptin, IL-1 and Notch could differentially drive breast cancer growth and angiogenesis. Furthermore, the analysis of NILCO and targets using Pathway Studio9 software (Ariadine Genomics) showed multiple molecular relationships that suggest NILCO has potential prognostic biomarker value in breast cancer.

Introduction: Notch, IL-1 and leptin are known pro-angiogenic factors linked to breast cancer (BC) development, aggressiveness and poor prognosis. We have recently reported leptin as an inducer of IL-1 and Notch in BC cells. Furthermore, we have unveiled a novel signaling crosstalk between these factors: NILCO (Notch, IL-1, leptin crosstalk outcome). Our data show NILCO impacts on VEGF/VEGFR2 levels in BC cells. Objective: In this investigation, we aim to elucidate differences in NILCO relationships to the pathogenesis of triple negative breast cancer (TNBC) as compared to ER positive BC. We hypothesize that the expression of NILCO components correlates to TNBC etiology. Methods: Expression levels of Notch 1, 4, Jagged 1, DLL4, VEGF/VEGFR2 (FLK-1), Ob, Ob-R and IL-1R tI were examined by immunohistochemistry (IHC) in tissue microarrays (n=75/duplicate from Pantomics, Inc.) BC samples included normal/hyperplastic specimens (n=3), fibroadenomas (n=2), invasive ductal carcinomas (n=62; 13 were TNBC: ER-, PR- and HER2/neu-), ductal carcinomas in situ (n=2) and Paget's disease (n=1). IHC evaluations were conducted by two independent observers using semi-quantitative analysis of staining [HSCORE = ∑pi (i + 1), where “i’” is the intensity of staining with a value of 1, 2 or 3 (weak, moderate or strong, respectively) and ‘pi’ is the percentage of stained cells for each intensity]. The following cell lines: TNBC (MDA-MB231 and HCC1806) and ER+ (T47D and MCF-7) were treated with increasing doses of human leptin; leptin peptide receptor antagonist, LPrA2 and a γ-secretase inhibitor (DAPT: Notch pathway inhibitor). After treatments, NILCO and targets were determined by western blot (WB) and ELISA. Results: Data from BC tissue array showed NILCO components were present in the majority of samples. However, lower expression of Notch1, leptin and IL-1R tI was found in non-malignant tissues. In general, TNBC overexpressed Notch1/leptin as compared to ER positive BC. ELISA and WB analysis of cell lysates revealed that leptin increased NILCO component/activation and targets in a dose-dependent manner. Both DAPT and LPrA2 reduced leptin's effects on NILCO. Conclusions: These results support the hypothesis that NILCO may play an important role in BC pathogenesis. [This work was supported in part by NIH/NCI1SC1CA138658-03; NIH/ARRA/3SC1CA138658-02S1 and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP); CREDO (MSCR) 2R25RR017694-06A1 to L.S.C; the Morehouse School of Medicine (MSM) MBRS RISE Program (NIH/NIGMS 506 GM08248) to T.Z.M; Blacksone Academy, NIH/NIDDK Step-up HS Research Program: Short-Term Education Program for Underrepresented Persons to K.W. and facilities and support services at Morehouse School of Medicine (NIH RR03034 and 1C06 RR18386)]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 80. doi:1538-7445.AM2012-80

401 Background: HD carboplatin is an essential part of curative salvage high-dose chemotherapy (HDCT) for GCT. Although ototoxicity is a known side effect of HD carboplatin, data on severity and characteristics of HL in this population are limited. Methods: Eligible patients (pts) received salvage HD carboplatin for GCT at our center from 1993-2017 and had audiograms pre- and post-HDCT. All pts were planned for 3 sequential cycles of HD carboplatin; some had audiograms after each cycle. HL severity was classified by ASHA criteria (slight: 15-24dB; mild: 25-40dB; moderate: 41-55dB; moderately severe: 56-70dB; severe: 71-90dB; profound: >90dB). Change in hearing threshold at each frequency (0.25 – 8 kHz) was analyzed by clinical and treatment variables using the Wilcoxon rank-sum test. The proportion of pts recommended for hearing aids was also recorded. Results: Of 115 pts (median age 31, 77% nonseminoma), 96 received TI-CE and 19 received TI-TIC on a clinical trial (Feldman CGUC 2015). Pts varied by prior cisplatin exposure (77% with ≤4 vs 23% with 5-9 cycles) and number of HD carboplatin cycles (89% had 3, 10% had 2, and 2% had 1). Carboplatin AUC was 21 in 23%, 24 in 75%, and 28 in 3%. Moderate to profound HL was present in 23% pre-HD carboplatin vs 82% post, preferentially at high frequencies (Table). Of 89 pts with normal to mild HL at baseline, 78% developed moderate or worse HL post-HD carboplatin, including 61% with moderately severe or severe HL. Higher AUC (24 vs 21) and number of HD carboplatin cycles were associated with greater HL with HD carboplatin whereas prior cisplatin exposure was not. Due to moderate or severe HL in the vital speech frequency range (2 – 4kHz), 20 (17%) pts were recommended for hearing aids post-HD carboplatin. Conclusions: In the largest series to detail ototoxicity in pts receiving salvage HD carboplatin for GCT, we show HL, particularly at high frequencies, is a major dose-dependent toxicity. Most pts will have at least moderate high frequency HL after HD carboplatin with approximately 1 in 6 recommended for hearing aids. [Table: see text]