Corey Gillespie

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 χ2: p = 0.06]). PEG‐LPrA2 significantly reduced BC growth (untreated: 19/42; [45%] vs. treated: 8/42 [19%]; Pearson χ2: 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‐LPrAs 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.

Potential Role of Leptin Signaling in DMBA-induced Mammary Tumors by Non-Responsive C57BL/6J Mice Fed a High-Fat Diet

Environmental carcinogens, High-Fat Diet (HFD) and elevated levels of leptin correlate to increase breast cancer incidence. To test whether these factors could affect the development of Mammary Tumors (MT) via DMBA (7,12-dimethylbenz[a]anthracene) challenge, we used C57BL/6J mice that are non-responsive to develop MT in absence of hormonal stimulation. C57BL/6J female mice without hormonal stimulation were fed HFD (55% Kcalfat) and low-fat diets (10% Kcal-fat) received DMBA (oral gavage: 1 mg/weekly) for 6 weeks. To test whether leptin signaling is involved in DMBA-MT development, a potent inhibitor, pegylated leptin peptide receptor antagonist (PEG-LPrA2; half-life 66 hours), was used for 30 weeks. As expected, irrespective of PEG-LPrA2 treatment, lean mice fed with low-fat diet did not develop MT. However, HFD induced obesity and significantly stimulated earlier onset (within 18 weeks) and marginally increased the incidence of MT (21%; 3/14) in DIO-mice (diet-induced-obesity). It appears that leptin signaling may be involved in DMBA-induced mammary carcinogenesis in obese mice because no evidence of MT was found in DIO-mice treated with PEG-LPrA2 (0% incidence; 0/14; Wilcoxon-Breslow test Chi2, p=0.03). Interestingly, PEG-LPrA2 treatment did not apparently affect body weight or food intake, but reduced protein levels of several molecules related to breast cancer [Aryl hydrocarbon Receptor (AhR), leptin receptor (OBR), interleukin 1 receptor type I (IL-1R tI), hypoxia-induced factor 1 alpha (HIF-1α), Jagged1 (JAG1) and Notch1 activated (NICD1)] within the mammary glands. Our findings reinforce the idea that obesity induced by HFD is an additional risk factor for chemical-induced breast carcinogenesis. The present study reveals some potential mechanisms involving leptin in the effects of HFD and adiposity on mammary chemical-induced carcinogenesis. Overall, present data suggest that the inhibition of leptin signaling might be a new way to prevent breast cancer induced by chemical carcinogens, especially in obese individuals.

NILCO biomarkers in breast cancer from Chinese 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.

Abstract 5297: Autocrine stimulation of VEGFR-2 by leptin is associated with Notch signaling pathway and cancer stem cell marker expression

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Obesity-related cancer is an increasing health problem. Leptin, the major adipokine, has been suggested a major factor in these relationships. Leptin is a pro-inflammatory, pro-angiogenic and mitogenic factor for breast cancer cells. Recently, we described a comprehensive mechanism for leptin induction of VEGF, and unveiled a novel crosstalk between Notch, IL-1 and leptin (NILCO) in breast cancer. NILCO regulates VEGF/VEGR-2 in breast cancer cells and could represent the integration of developmental, pro-inflammatory and pro-angiogenic signals critical for leptin oncogenic actions. However, the specific mechanisms for leptin-induced VEGFR2 are unknown. To answer this question, we re-cloned mouse VEGFR-2 promoter and prepared VEGFR-2 luc, then analyzed VEGFR-2 regulation and expression by using several kinase inhibitors, leptin peptide antagonist 2 (LPrA2), a VEGFR-2 kinase inhibitor (SU5416), as well as siRNA against VEGFR-2, Notch1, Notch3 in 4T1 cells. Our results demonstrated that leptin-induced JAK2/STAT3, MAPK/ERK 1/2, PI-3K/AKT1, PKC, p38 and JNK signaling pathways may correlate with upregulated VEGFR-2. Moreover, leptin-induced activation of Notch/VEGFR-2 was linked to the upregulation of cancer stem cell markers CD44 and ALDH1. These leptin effects were inhibited by siRNA-Notch1&3, leptin signaling antagonist (PEG-LPrA2), VEGFR2-kinase inhibitor (SU5416) and siRNA-VEGFR-2. Our results show for the first time how leptins molecular signals regulate VEGFR-2 (an autocrine survival process) that was associated to Notch and cancer stem cell marker expression. These novel findings may help to design new strategies for prevention/treatment of breast cancer through the abrogation of tumor angiogenesis and breast cancer stem cell renewal in an obesity context. [This work was supported in part by NIH/NCI1SC1CA138658-02; NIH/ARRA/3SC1CA138658-02S1 and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP)]. 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 5297. doi:1538-7445.AM2012-5297

Abstract 80: Differential expression of NILCO reveals pathogenesis of human breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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 Pagets 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 leptins 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

Abstract 377: The role of VEGFR-2 in leptin induction of Notch and angiogenic features of endothelial cells.

We have previously shown that leptin is a pro-survival factor for breast cancer (BC) cells that reduces the effects of chemotherapeutics. We have also found that leptin is a pro-angiogenic, pro-inflammatory and mitogenic factor that induces the expression of Notch and VEGF/VEGFR-2 in BC and transactivates/phosphorylates VEGFR-2 in endothelial cells (EC). We hypothesize that leptin-induced transactivation of VEGFR-2 and upregulation of Notch are essential for the development of angiogenic features in EC. To assess the role of VEGFR-2 in these leptin-induced effects we used porcine aortic endothelial cells (PAEC) and human umbilical vein endothelial cells (HUVEC). PAEC wild type (do not express VEGFR-2), PAEC-expressing VEGFR-2, PAEC-expressing VEGFR-1 (negative control) and HUVEC (VEGFR-2+) were challenged with leptin. Phosphorylation/transactivation of VEGFR-2 and VEGF expression were determined by ELISA. Leptin-induced formation of EC tubules was determined using a Matrigel Assay. In a series of experiments, inhibitors of leptin (leptin peptide receptor antagonist 2, LPrA2), VEGFR-2 (kinase inhibitor, SU5416) and Notch signaling (g-secretase inhibitor, DAPT) were added to PAEC and HUVEC cultures. Leptin-dose and time-course effects on expression of Notch (receptors: Notch1, Notch2, Notch3 and Notch4); ligands: JAG1, DLL-4 and target: Survivin were determined by Western blot and Real-Time RT-PCR. In addition, siRNA VEGFR-2 and siRNA Notch 1 and 3 were used to further determine the role of VEGFR-2 transactivation and leptin-induced Notch in the development of EC angiogenic features. Our studies reinforce the idea that leptin is a potent inducer of EC angiogenic transformation via VEGFR-2 transactivation and Notch expression. Moreover, we show that an intact Leptin-VEGFR-2/Notch crosstalk is essential for leptin pro-angiogenic actions. Data generated from the present investigations suggest that leptin secreted either by adipose tissue or BC cells could be an important factor contributing to tumor angiogenesis by acting directly on cancer cells inducing VEGF secretion or through EC inducing VEGFR-2/Notch crosstalk. Combinatory therapies targeting both Notch and leptin signaling could be a new strategy for Breast Cancer treatment. Funding: This work was supported by NIH/NCI 5SC1CA138658-04 and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP), NIH/2G12RR003034-26 and U54 MSM/TU/UAB Cancer Center Partnership. This research was also supported by facilities and support services at Morehouse School of Medicine (NIH 1C06 RR18386) and by the University of Muenster, Germany. Citation Format: Viola Lanier, Corey Gillespie, Merle Leffers, Muna Elhassey, Johannes Waltenberger, Ruben R. Gonzalez-Perez. The role of VEGFR-2 in leptin induction of Notch and angiogenic features of endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 377. doi:10.1158/1538-7445.AM2013-377

Abstract 5643: Role of leptin-induced Notch-Wnt crosstalk in proliferation/survival of triple negative breast cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Obese African American and Latina women show the high mortality rate linked to triple negative breast cancer, TNBC. The disease is commonly detected at more advance degree and associated with resistance to common therapeutic treatments. Obesity, characterized by high levels of leptin, has consistently been associated with many different cancers including breast cancer. We hypothesize that leptin signaling is instrumental in the increased proliferation and survival of breast cancer cells, which is mediated through a crosstalk between the Notch and Wnt signaling pathways. We further hypothesized that these leptin actions are more evident in TNBC cells. Objective/Methods: The effects of leptin-Notch-Wnt crosstalk on the proliferation and survival (MTT-Vybrant assay and Annexin V-flow cytometry) of breast cancer cells responsive to estrogen (MCF-7 and T47D) and TNBC (MDA-MB231 and HC1806) were investigated. The cells were treated with agonists and inhibitors of leptin-Notch-Wnt axis [i.e., leptin, Wnt1, JAG1/14-mer peptide and leptin peptide receptor antagonist 2 (LPrA2), g-secretase inhibitor and Wif]. The expression of Notch and Wnt signaling components (ligands/receptors) and levels of cyclin D1 and myc were also determined via western blot and real-time RT-PCR. In a series of experiments the cells were treated with chemotherapeutic drugs, doxorubicin and cisplatin plus leptin and the activation of caspase 3, levels of Bcl-2 and, the drug resistance marker (multidrug resistance-associated protein 1, ABCC1/MRP1) were determined using as control the etoposide resistant MCF-7/VP cell line. The relative expression of leptin, Notch and Wnt systems in breast cancer tissue arrays (n=150; Pantomics) was also investigated. Results: Leptin induced significant increase of TNBC cell proliferation, cyclin D1, bcl-2, ABCC1 and Notch/Wnt axis. Leptin also decreased the anti-cancer effects of doxorubicin and cisplatin, which were related to leptin upregulation on Notch-Wnt crosstalk. HScore analyses of breast cancer tissue arrays showed significant correlation of the expression of leptin-targeted proteins (Notch and Wnt) in breast cancer tissues. These results suggest that leptin can induce the crosstalk between Notch and Wnt and breast cancer progression. Conclusions: Targeting leptin-Notch-Wnt signaling axis could be a new strategy to treat and/or prevent breast cancer. The inhibition of leptin signaling could be a promising new way to target the most deadly TBNC, especially in obese patients. Acknowledgments: This work was supported by NIH/NCI 5SC1CA138658-04, Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP), NIH/2G12RR003034-26 and U54 MSM/TU/UAB Cancer Center Partnership, and NIH/NIGMS R25 GM058268. This research was also supported, in part, facilities and support services at Morehouse School of Medicine (NIH 1C06 RR18386). Citation Format: Tanisha Z. McGlothen, Corey Gillespie, Krysta Greenleaf, Movado Long, Laronna S. Colbert, Gary L. Sanford, Ruben R. Gonzalez-Perez. Role of leptin-induced Notch-Wnt crosstalk in proliferation/survival of triple negative breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5643. doi:10.1158/1538-7445.AM2013-5643

Abstract 1400: Obesity induced leptin-notch signaling axis in breast cancer.

Obesity leads to increased leptin levels and signaling which has been linked to the growth of breast cancer (BC). We early identified leptin as an inducer of Notch in BC cells. However, whether leptin-induction of Notch is instrumental to the development of BC is unknown. Objective: Therefore, we examined the impact of obesity and inhibition of leptin signaling (via LPrA2 antagonist) on the growth of syngeneic E0771-derived BC and tumor expression of Notch in lean and obese mice and in E0771 cell cultures. Methods: C57BL/6J female mice were fed either high-fat diet (HFD, 60% Kcal from fat) or normal diet (10% Kcal from fat). Obesity was defined as BW ≥ 20% BW Lean-control at week 5. Lean and diet-induced-obesity (DIO) mice were orthotopically inoculated with mouse E0771 cells in the lower left region of the mammary pad. One week later the mice were treated with either (1) pegylated leptin peptide receptor antagonist 2, PEG-LPrA2 or (2) PEG-Scrambled peptide (as control; 50μl i.v. tail vein/ 0.1 mM) following the two-arm design: (a) one and (b) two doses/weekly. Treatment lasted for 4 weeks. Tumor onset and growth was determined overtime and expression of Notch, its ligands, and crosstalk factors were assessed using western blot, immunohistochemistry, and ELISA. Leptin9s effects on Notch and crosstalk factors were also investigated in E0771 cell cultures. Results: Obesity induced E0771-BC onset and growth and, increased the expression of Notch in vivo. Inhibition of leptin signaling had a higher impact on DIO mice than in lean mice by delaying onset and reducing tumor growth and the expression of Notch. Therefore, high levels of leptin in DIO-mice played a positive role in tumorigenesis. Similarly, leptin induced and PEG-LPrA2 decreased Notch expression in E0771 cells in vitro. Present data further validate the idea that the consumption of a high-fat diet leads to early BC onset and growth, which was linked to the leptin-Notch axis. Our results reinforce the potential use of inhibition of leptin signaling as a novel approach to fight breast cancer, especially in obese patients showing higher levels of leptin and incidence of BC. Funding: This work was supported by NIH/NCI 5SC1CA138658-03 and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP), NIH/2G12RR003034-26; U54 MSM/TU/UAB Cancer Center Partnership; and 1R21CA153172-01A1 (to MTK). This research was also supported, in part, facilities and support services at Morehouse School of Medicine (NIH 1C06 RR18386). Citation Format: Monica Battle, Corey Gillespie, Tanisha McGlothen, Marta Torroella-Kouri, Ruben R. Gonzalez-Perez. Obesity induced leptin-notch signaling axis in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1400. doi:10.1158/1538-7445.AM2013-1400

Abstract 785: Leptin-Notch-Wnt axis affects drug resistance in breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Triple negative breast cancer (TNBC) is a particularly aggressive form of the disease whose incidence is disproportionate in obese African American and Latina women. TNBC is commonly associated with acute early onset, poor survival and acquired drug-resistance. Obesity, characterized by high levels of leptin, is frequently found among these ethnic groups. We hypothesize that intact leptin-Notch-Wnt signaling crosstalk affects the development of drug-resistance in TNBC. Objective: To determine whether leptin signaling induces Notch/Wnt pathway crosstalk, decreasing the effectiveness of cisplatin in TNBC cells. Materials & Methods: TNBC cells (MDA MB 231 and HCC1806) and ER+ MCF-7 (as control) were treated with pharmacological doses of leptin and agonists of the Notch and Wnt pathways JAG1-17 mer peptide and Wnt-1, respectively. In addition, the cells were challenged with the leptin receptor, Notch pathway, and Wnt pathway antagonists LPrA2, DAPT, and Wnt-1 respectively. The impact of these molecules on cisplatin anti-cancer effects was also determined. Activation of Wnt (total/pβ-catenin), expression of Notch (Notch 1-4 and JAG1/Dll-4 and targets: survivin/Hey2), cell proliferation and apoptosis were determined by Western blot and flow cytometry. β-catenin levels were also investigated by immunofluorescence. We also determined the impact of the inhibition of leptin signaling (via LPrA2) on the expression of Wnt and Notch in DMBA-induced breast cancer from DIO (diet-induced obese) mice. Results: We found that leptin increased the levels of β-catenin mainly in TNBC cells. Similarly, inhibition of leptin signaling decreased Wnt/Notch expression in DMBA/DIO-breast cancer. Interestingly, leptin increased expression of Notch and attenuated the detrimental effects of cisplatin on breast cancer cells. Wnt-1 affected Notch levels but its effects were less pronounced. Conclusions: Leptin is able to induce the Notch and Wnt signaling pathways in breast cancer. Cisplatin effects were attenuated by leptin in TNBC probably through its crosstalk with Wnt and Notch signaling pathways. These results are particularly relevant for TNBC and obese patients, and suggest that leptin inhibition could be useful in increasing the chemotherapeutic effectiveness in TBNC by disrupting leptin-Wnt-Notch crosstalk. [This work was supported in part by NIH/NCI1SC1CA138658-02; NIH/ARRA/3SC1CA138658-02S1 and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP); CREDO (MSCR) 2R25RR017694-06A1 (to L.S.C); NIH/NIGMS R25 GM058268 and NCRR 5P20RR11104 (to T.Z.M); and facilities and support services at MSM (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 785. doi:1538-7445.AM2012-785

Abstract 2378: Involvement of leptin-activated canonic and non-canonic kinases in VEGF regulation in breast cancer

We have previously reported that in vitro and in vivo leptin signaling up-regulates VEGF in mouse and human mammary tumor cells (MT) but the specific molecular mechanisms involved are largely unknown. Here we describe how leptin activated canonic and non-canonic signaling pathways to induce specific transcription factors (TF) essential for the transcriptional activation of VEGF gene in mouse MT (4T1, EMT6 and MMT). Basal expression of VEGF/VEGFR2, leptin/OB-R and ER-α was initially investigated in MT. Leptin dose-response and time-course effects on VEGF levels were determined by ELISA. Several pharmacological inhibitors for key leptin-target kinases and TF were used to dissect the signaling pathways involved in leptin-induced levels of VEGF protein and mRNA in MT. A series of VEGF-promoter Luc-reporters [full-length & transcription factor (TF)-binding deletions: HRE, AP1, AP2, NFκB and SP1] were transfected into MT to analyze leptin regulation of VEGF transcription. Our data show that MAPK, PI-3K (canonic) and PKC, JNK and p38 MAP kinase (non-canonic) signaling pathways were generally involved in leptin regulation of VEGF. Analysis of cis-acting elements of VEGF promoter shows that leptin activation of HIF-1α is essential for transcription in all MT. This was mainly linked to MAPK, PI-3K, PKC, JNK and p38 signaling. Leptin-induced MAPK, JNK and p38 to activate NFκB that was essential for VEGF expression in high producer MT (EMT6 and MMT). Leptin through all canonic signaling pathways activates SP1 required for VEGF regulation in 4T1 cells. In contrast, AP1 was not involved but AP2 repressed leptin-mediated transcription of VEGF. Overall, these data suggest that leptin signaling regulates VEGF gene in MT cells mainly through HIF-1α and NFκB. These results delineate for first time the mechanisms for leptin regulation of VEGF and, reinforce the idea that the disruption of leptin signaling could impact breast cancer growth by inhibiting MT proliferation and angiogenesis. This work was supported in part by NIH/NCI 1SC1CA138658-02; NIH/UAB Breast SPORE Career Development Award; BC 504370 Susan G. Komen Foundation for the Cure and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2378.