Crystal C. Lipsey

Oncogenic role of leptin and Notch interleukin-1 leptin crosstalk outcome in cancer.

Obesity is a global pandemic characterized by high levels of body fat (adiposity) and derived-cytokines (i.e., leptin). Research shows that adiposity and leptin provide insight on the link between obesity and cancer progression. Leptins main function is to regulate energy balance. However, obese individuals routinely develop leptin resistance, which is the consequence of the breakdown in the signaling mechanism controlling satiety resulting in the accumulation of leptin. Therefore, leptin levels are often chronically elevated in human obesity. Elevated leptin levels are related to higher incidence, increased progression and poor prognosis of several human cancers. In addition to adipose tissue, cancer cells can also secrete leptin and overexpress leptin receptors. Leptin is known to act as a mitogen, inflammatory and pro-angiogenic factor that induces cancer cell proliferation and tumor angiogenesis. Moreover, leptin signaling induces cancer stem cells, which are involved in cancer recurrence and drug resistance. A novel and complex signaling crosstalk between leptin, Notch and interleukin-1 (IL-1) [Notch, IL-1 and leptin crosstalk outcome (NILCO)] seems to be an important driver of leptin-induced oncogenic actions. Leptin and NILCO signaling mediate the activation of cancer stem cells that can affect drug resistance. Thus, leptin and NILCO signaling are key links between obesity and cancer progression. This review presents updated data suggesting that adiposity affects cancer incidence, progression, and response to treatment. Here we show data supporting the oncogenic role of leptin in breast, endometrial, and pancreatic cancers.

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.

Leptin-Notch signaling axis is involved in pancreatic cancer progression.

Pancreatic cancer (PC) shows a high death rate. PC incidence and prognosis are affected by obesity, a pandemic characterized by high levels of leptin. Notch is upregulated by leptin in breast cancer. Thus, leptin and Notch crosstalk could influence PC progression. Here we investigated in PC cell lines (BxPC-3, MiaPaCa-2, Panc-1, AsPC-1), derived tumorspheres and xenografts whether a functional leptin-Notch axis affects PC progression and expansion of pancreatic cancer stem cells (PCSC). PC cells and tumorspheres were treated with leptin and inhibitors of Notch (gamma-secretase inhibitor, DAPT) and leptin (iron oxide nanoparticle-leptin peptide receptor antagonist 2, IONP-LPrA2). Leptin treatment increased cell cycle progression and proliferation, and the expression of Notch receptors, ligands and targeted molecules (Notch1-4, DLL4, JAG1, Survivin and Hey2), PCSC markers (CD24/CD44/ESA, ALDH, CD133, Oct-4), ABCB1 protein, as well as tumorsphere formation. Leptin-induced effects on PC and tumorspheres were decreased by IONP-LPrA2 and DAPT. PC cells secreted leptin and expressed the leptin receptor, OB-R, which indicates a leptin autocrine/paracrine signaling loop could also affect tumor progression. IONP-LPrA2 treatment delayed the onset of MiaPaCa-2 xenografts, and decreased tumor growth and the expression of proliferation and PCSC markers. Present data suggest that leptin-Notch axis is involved in PC. PC has no targeted therapy and is mainly treated with chemotherapy, whose efficiency could be decreased by leptin and Notch activities. Thus, the leptin-Notch axis could be a novel therapeutic target, particularly for obese PC patients.

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.

Abstract C52: Targeting the obesity/leptin link to TNBC: A breast cancer disparity problem

Triple Negative Breast Cancer (TNBC) is an aggressive cancer associated with poor prognosis and is difficult to treat. Standard therapy for TNBC includes a combination of chemotherapeutic drugs: doxorubicin (DOX), paclitaxel (TAX), and cyclophosphamide (CTX). Current epidemiological data show that obesity increases TNBC incidence, which is more relevant for African American women. Recent findings suggest that leptin (an adipokine increased in obesity) is involved in TNBC acquired drug resistance via induction of breast cancer stem cells (BCSCs). Understanding the role of obesity in TNBC is vital to the development of new and effective therapies. Chemotherapeutic drugs exhibit shortcomings that may be due to increased drug resistance, survival, proliferation, and angiogenic signal redundancy induced by leptin. Our lab has developed and tested a novel, specific inhibitor of leptin signaling, LPrA2. A pegylated derivative of LPrA2 (PEG-LPrA2) has previously shown to enhance, 68 fold, LPrA29s bioavailability in mouse breast cancer models. The objective of this study was to determine the adjuvant effects of PEG-LPrA2 on TNBC in vitro. We hypothesized that PEG-LPrA2 can serve as an adjuvant for standard TNBC chemotherapeutics. This adjuvant would allow for the reduction of chemoresistance and TNBC recurrence. It could also reduce chemotherapeutic dose and undesirable side effects associated with standard therapy for the disease. Two human TNBC cell lines were cultured: MDA-MB-231 (highly metastatic) and MDA-MB-468 (minimally invasive). Cellometer imaging technologies were used to determine S-phase progression in cells treated with PEG-LPrA2 and leptin. Flow cytometry technologies were used to determine the effects of leptin on BCSC surface markers. Additionally, in vitro toxicity of PEG-LPrA2 was tested in non-malignant mammary cells (MCF-10A) via MTT assay. Our data shows that leptin (1.2 nM, equivalent to serum levels of overweight individuals) induced S-phase progression and increased BCSC markers and the formation of TNBC tumorspheres. Remarkably, PEG-LPrA2 (1.2 nM) abrogated leptin induction of S-phase, BCSC markers, and tumorspheres. It also showed no toxicity in non-malignant cells. These results suggest leptin-signaling inhibition could be used as an adjuvant strategy for chemotherapy of TNBC. These results are most relevant for obese African American women suffering from TNBC. Citation Format: Courtney D. Dill, Adriana Harbuzariu, Tia L. Harmon, Crystal C. Lipsey, Ayobami Loye, Ruben Rene Gonzalez-Perez. Targeting the obesity/leptin link to TNBC: A breast cancer disparity problem. [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 C52.

Abstract 3540: Targeting obesity-related cancer progression with novel leptin receptor antagonists

Obesity is a global health issue that has been identified as a risk factor for several types of cancer. High levels of body fat and circulating leptin are typical identifiers of obesity in humans and animals. Leptin is a 16kD protein hormone which is secreted by adipocytes, and maybe secreted from cancer cells, that functions to control satiation via leptin receptor binding. However, obese individuals often develop “leptin resistance”, which is a mechanism that leads to the accumulation of excess leptin. Increased binding of leptin to its receptor (OB-R) due to leptin resistance has been associated with disease progression and poor prognosis in human cancers. Our group has previously shown that leptin-mediated cancer cell proliferation is inhibited by the LPrA2 (leptin peptide receptor antagonist 2). We have prepared novel leptin antagonists and tested their ability to block leptin-induced survival and chemoresistance to Paclitaxel (TAX) and Gemcitabine (GEM) in cancer cells and derived tumorspheres (pancreatic: PANC-1, MiaPaca2 and triple negative breast cancer: MDA-MB231 and MDA-MB468). Western blot protein analyses showed the ability of the antagonists to specifically inhibit leptin-induced phosphorylation of STAT3, and expression of cyclin D, and Notch1 in cancer cells. Additionally, potential toxicity of antagonists was tested using MTT assay with concentrations up to 100X higher than the effective dose in non-malignant breast cells (MCF-10A). Data generated show no toxicity of the novel antagonists in vitro. Leptin-induced proliferation of breast cancer and pancreatic cancer cells (120-160%) was significantly inhibited by the novel antagonists. In addition, leptin-mediated progression of S-phase was also reduced by the antagonists. Leptin increased TAX and GEM chemoresistance in cells and tumorspheres that were efficiently inhibited by the antagonists. These data suggest that the new antagonists could be equally or more effective than LPrA2 for adjuvant treatment of cancer. Acknowledgements: This work has been supported by Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP, and facilities and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR 03034 and 1C06 RR18386). Citation Format: Crystal C. Lipsey, Adriana Harbuzariu, Ruben R. Gonzalez-Perez. Targeting obesity-related cancer progression with novel leptin receptor antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2017-3540

Abstract 3544: Leptin-Notch crosstalk axis: A novel target for pancreatic cancer

Background: Pancreatic cancer (PC) has consistently been the fourth cancer-related cause of death in United States for more than 10 years. Its dismal prognosis is due to the aggressive behavior, late detection, lack of reliable biomarkers and low response to chemotherapy. Obesity, characterized by high levels of leptin, correlates to low PC survival. Notch and leptin signaling have been associated with increased tumor growth and chemoresistance. We have previously shown that leptin induces proliferation of PC cell lines, increases Notch expression, PC stem cells (PCSC) and tumorsphere formation. Hypothesis: Leptin increases PC chemoresistance through its crosstalk to Notch signaling pathway that is essential for the expansion of PCSC. Methods: To test whether a leptin-Notch crosstalk axis is involved in PC-chemoresistance, human PC (MiaPaCa-2, Panc-1 and BxPC-3) cells were cultured in serum-free medium containing leptin, Gemcitabine and 5-Fluorouracil (5-FU) in combination with DAPT (γ-secretase inhibitor) and leptin signaling inhibitor bound to iron-oxide nanoparticles (IONP-LPrA2). PC cells were cultured with leptin and allow to form primary and secondary tumorspheres, enriched in PCSC. Tumorspheres were treated with the compounds described for 7-14 days. Number and size of tumorspheres were recorded, and expression of leptin receptor, OB-R and secretion of leptin were determined by Western blot (WB) and ELISA, respectively. Additionally, the levels of Notch, PCSC and EMT markers, pluripotency associated genes (Oct-4, Sox-2 and Nanog) and ATP-binding cassette transporters (ABCB1, ABCG2 and ABCC5) were determined using flow cytometry and WB. Results: PC cells expressed Leptin/OB-R system, suggesting an autocrine role in PC progression and chemoresistance. Leptin induced the formation of primary and secondary tumorspheres and increased their expression of Notch, EMT and pluripotency markers. Additionally, chemotherapeutics induced PC survival and the expression of ATP-binding cassette transporters in tumorspheres that was reinforced by leptin actions. Blockade of leptin signaling via IONP-LPrA2 and Notch activation via DAPT reduced leptin-induced molecular effects, PCSC and tumorsphere growth. Conclusion: Present data suggest that obesity, through leptin-induced Notch signaling, could increase chemoresistance in PC patients. Inhibition of leptin-Notch axis may be novel therapeutic strategy for PC, which may improve chemotherapeutic efficacy and increase survival in PC patients, particularly in obese contexts. Acknowledgements: this work has been supported by a Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP, and facilities and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR 03034 and 1C06 RR18386). Citation Format: Adriana Harbuzariu, Crystal C. Lipsey, Ruben R. Gonzalez-Perez. Leptin-Notch crosstalk axis: A novel target for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3544. doi:10.1158/1538-7445.AM2017-3544

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 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.