Viola Lanier

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.

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.

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 leptins actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptins 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 leptins 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.

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.

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.

Abstract B66: Novel adjuvant therapy for obesity-related cancers

Obesity incidence has reached alarming levels, particularly in minority populations: African American (AA) and Latinos. The risk of develop several cancers (i.e., breast, pancreatic and endometrial cancers) is strongly correlated to obesity. One of the essential factors involved in this relationship is leptin, the major adipokine secreted by adipose and tumor cells. Leptin is a mitogenic, proangiogenic, antiapoptotic and inflammatory factor that induces tumor growth and metastasis. Leptin has also been shown to increase cancer stem cell population and drug resistance in tumors. We have produced a potent and specific antagonist (LPrA2) that blocks leptin signaling and impairs its effects on cancer cells. LPrA2 conjugated to iron-oxide nanoparticles (IONP-LPrA2) shows enhanced bioavailability (half-life 8x higher) and inhibition effectiveness on leptin signaling in cancer cells. It is hypothesized that IONP-LPrA2 could serve as an adjuvant that increases effectiveness and allows the reduction of dosage of chemotherapeutic drugs, particularly in obesity contexts. Breast (E0771-TAM tamoxifen resistant, Py-8119, MDA-MB231, HCC1806 and MCF-7) and pancreatic cancer cells (Panc-1, BxPC-3, MiaPaca2), and their derived tumorspheres were treated with leptin, chemotherapeutics (Cisplatin, Doxorubicin, Paclitaxel and Gemcitabine) and IONP-LPrA2. Additionally, E0771-TAM and Py-8119 cells, and MDA-MB231 and Panc-1 tumorspheres were inoculated in C57BL/6J female mice (obese and lean) and female and male CD1 nu/nu mice. The mice were treated with IONP-LPrA2 during 4 weeks. Leptin induced proliferation and cell cycle progression in all cancer cells tested. Leptin also increased the number and size of breast and pancreatic cancer tumorspheres, and the levels of cancer stem cell (ALDH1, CD44, CD24, ESA), and chemoresistant (ABCB1) and pluripotent (NANOG) markers. IONP-LPrA2 treatment increased the effects of chemotherapeutics on cancer cells, and delayed tumor onset and growth. These data suggest that leptin is a mitogenic factor that increases cancer aggressiveness and survival. IONP-LPrA2 is a promising chemotherapeutic adjuvant, especially for patients suffering from obesity-related cancers. Citation Format: Pierre Candelaria, Tia Harmon, Adriana Harbuzariu, Antonio Rampoldi, Crystal Lipsey, Mckay Mullen, Ann Kurian, Courtney Dill, Cynthia Tchio, Danielle Daley-Brown, Shakeyla Nunez, Viola Lanier, Ruben Rene Gonzalez-Perez. Novel adjuvant therapy for obesity-related cancers. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B66.

Abstract 1026: PEG-LPrA2 is a non-toxic adjuvant for triple negative breast cancer

Background: Triple Negative Breast Cancer (TNBC) is an aggressive cancer with poor prognosis and is difficult to treat. Current standard therapy for the disease includes a combination of chemotherapeutic drugs: doxorubicin (DOX), paclitaxel (TAX), and cyclophosphamide (CTX). These drugs are ineffective as they exhibit shortcomings and several side effects. TNBC patients develop chemoresistance that may be enhanced by leptin, which affects survival, proliferation, and angiogenesis. Our lab developed and tested a novel and specific inhibitor of leptin signaling, LPrA2. A pegylated derivative of LPrA2 (PEG-LPrA2), with enhanced bioavailability, was successfully used in mouse breast cancer models. Preliminary data showed that PEG-LPrA2 was non-toxic in vitro. Therefore, it is hypothesized that PEG-LPrA2 is not toxic in vivo. Methods: To determine potential toxicity of PEG-LPrA2, in vitro and in vivo assays were performed. In vitro toxicity of PEG-LPrA2 was tested in a human non-malignant mammary epithelial cell line (MCF-10A). MCF-10A cells were cultured in 96 well plates (5,000 cells/ well) and grown to 70-80% confluence. Cells were treated with PEG-LPrA2 for 24 hrs and viability was determined via MTT assay. In vivo toxicity studies were performed in obese mice. Fifty-seven, eight week old C57BL/6J mice (Charles River Laboratories) were divided into 6 groups. Control mice were fed a low fat diet (10% Kcal from fat) and the rest of the mice were fed a high fat diet (60% Kcal from fat) for 11 weeks. Obesity was characterized as body weight (BW) ≥ 25% BW of control mice. Obese mice were divided into six groups (n = 7/each). Mice were injected with 50 μL of either Sc-PEG-LPrA2 (scramble control) or active inhibitor, PEG-LPrA2, (0.1 mM, 1 mM, or 5 mM) two times a week, for eight weeks. Blood chemistries were analyzed. Additionally, heart, liver, and kidney tissue were harvested and examined to determine toxicity. The tissues were probed for OB-R via immuno histochemistry. Results: The results showed no changes in BW or food intake. Additionally, no evident changes in blood parameters and organ histology were found. Conclusions: PEG-LPrA2 is non-toxic and could serve as an adjuvant therapy for standard TNBC chemotherapeutics. Citation Format: Courtney D. Dill, Adriana Harbuzariu, Antonio Rampoldi, Crystal C. Lipsey, Viola Lanier, Tia Harmon, Danielle Daley-Brown, Cynthia Tchio, Pierre Candelaria, Ruben Rene Gonzalez-Perez. PEG-LPrA2 is a non-toxic adjuvant for triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1026.

Abstract 227: Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma

Background: The tissue microenvironment plays a major role in tumor development and recurrence. It is known that leptin secreted by cancer and adipose cells can modify the tumor stroma, which is composed of immune, fibroblast, and endothelial cells (EC). Leptin affects both normal and cancer associated fibroblasts. We have previously shown that leptin can modify EC directly via an increase of angiogenic features; which was linked to VEGFR signaling and Notch expression independent of VEGF. It is hypothesized that leptin induces a communication between fibroblast and endothelial cells via induction of Notch signaling, which may be related to exosome production. Methods: Non-expressing Ob-R fibroblasts, and fibroblasts transfected with Ob-R plasmid, were used to investigate leptin9s actions on Notch activation. Fibroblasts were challenged with leptin (0.6, 1.2, 6.2 nM) and an inhibitor of Notch signaling activation (DAPT). Fibroblast culture supernatants were added to and human umbilical vein endothelial cells (HUVEC) cultures. Notch proteins and RNA expression and molecular markers of activated fibroblasts and derived exosomes were determined via Western blot, RT-PCR, and multiplex analyses, respectively. EC angiogenic features were also determined. Results: Our findings suggest that leptin induces fibroblast activation, which triggers the expression of Notch and the development of angiogenic features by EC. Conclusions: A novel mechanism is proposed by which leptin promotes tumor stroma development via Notch activation and a crosstalk between fibroblasts and EC. Cancer therapies targeting leptin signaling could be a new strategy for interfering with cellular processes that promotes changes of tumor stroma towards a more aggressive tumor environment. Citation Format: Viola Lanier. Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 227.

Abstract 2079: IONP-LPrA2 and PEG-LPrA2 therapies for triple negative breast cancer

Background: Breast cancer (BC) is an epidemic in the US. It is estimated that there will be over 230,000 new BC diagnoses in 2016. Triple Negative Breast Cancer (TNBC) comprises ∼15% of BC cases and lacks targeted therapeutic options. Obesity and high leptin levels are associated with higher TNBC incidence and poorer patient outcomes. Overexpression of leptin and its receptor, Ob-R, induce BC cell growth and angiogenesis; therefore leptin/Ob-R may serve as a TNBC therapeutic target. We have developed a Leptin Peptide Receptor Antagonist, LPrA2, which effectively inhibits leptin signaling. To increase its efficacy LPrA2 was coupled to iron oxide nanoparticles (IONPs) and polyethylene glycol (PEG), and subsequently tested in vitro and in vivo in obese mice hosting syngeneic TNBC-like mammary tumors. Methods: The conjugation of LPrA2 to IONPs and PEG was confirmed by immunoblotting analysis. E0771, mouse BC cells, which are progesterone receptor and HER2 negative were made insensitive to estrogen stimuli by long-term treatment with Tamoxifen (TAM). Cell cycle and MTT assays were performed to determine the effectiveness of leptin signaling inhibition by conjugated LPrA2 in vitro. C57BL/6 female mice were fed a 60% fat diet to induce obesity. The obese mice were injected with E0771-TAM cells in the mammary fat pad and treated with IONP-LPrA2 and PEG-LPrA2 after tumor development. Obese mice treated with IONP-LPrA2 Scramble (Sc) and PEG-LPrA2 Sc served as negative controls. Results and Conclusion: IONP-LPrA2 and PEG-LPrA2 attenuated leptin signaling in vitro and decreased tumor growth and progression in vivo in comparison to the controls. These findings indicate that conjugated LPrA2 may serve as a targeted therapy for TNBC. IONP-LPrA2 may be especially useful in treating this more aggressive form of BC due to its ability to capture multiple LPrA2 peptides as well as its small and uniform particle size. Citation Format: Tia L. Harmon, Adriana Harbuzariu, Antonio Rampoldi, Courtney Dill, Viola Lanier, Danielle Daley-Brown, Crystal Lipsey, Cynthia Tchio, Lily Yang, Ruben Rene Gonzalez-Perez. IONP-LPrA2 and PEG-LPrA2 therapies for triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2079.

Abstract C78: NILCO: A marker for obesity-related endometrial cancer in African American women

Obesity is a pandemic in Western countries and has a significant impact on endometrial cancer (EmCa) incidence and prognosis. Although EmCa is more common in Caucasian women, higher mortality is found in African Americans who also show higher incidence of obesity. The reasons for this cancer health disparity are not completely understood. Obesity is characterized by high levels of leptin. Leptin signaling may play a key role in the progression of the more aggressive form of EmCa, Type II, which is independent from hormonal cues. We have shown that leptin induces a signaling crosstalk in breast cancer with oncogenic and angiogenic factors (NILCO: Notch, IL-1 and Leptin Crosstalk Outcome). Hence, we hypothesize that NILCO could play an important role in Type II Emca developed by obese African American women. However, no data on NILCO signaling in EmCa is currently available. Real-Time PCR, Immunohistochemistry and Western Blot analyses were used to determine whether NILCO components are differentially expressed in EmCa biopsies (Type I vs Type II EnCa) obtained from obese African American women. All tissue samples had a paired control sample from adjacent non-tumor endometrial tissue determined by pathologists. The tissue samples (n=29) were obtained from Grady Memorial Hospital, Atlanta, GA. Patient9s written informed consent was obtained for all samples collected as well as IRB approval from Morehouse School of Medicine, Atlanta, GA. In addition, commercially available EmCa tissue arrays from Chinese patients were co-examined for the expression of NILCO. Biopsy features included age, grading, and TNM staging. However, no body weight or body mass index information was available. Each array contained 150 cores, including 75 cases in duplicate. Overall, NILCO molecules were expressed higher in Type II EmCa regardless of obesity status. Notably, Type II EmCa from obese African American women showed the highest expression of NILCO. Present data suggest for the first time that NILCO could be instrumental for the development of EmCa; more specifically for obesity-related Type II EmCa, and may play a role as potential biomarkers for the disease. Citation Format: Danielle Daley-Brown, Gabriela Oprea-Ilies, Regina Lee, Kiara T. Vann, Viola Lanier, Alexander Quarshie, James Lillard, Roland Pattillo, Ruben Rene Gonzalez-Perez. NILCO: A marker for obesity-related endometrial cancer in African American women. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C78.