Danielle Daley-Brown

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.

Molecular cues on obesity signals, tumor markers and endometrial cancer.

Abstract Tumor markers are important tools for early diagnosis, prognosis, therapy response and endometrial cancer monitoring. A large number of molecular and pathologic markers have been described in types I and II endometrial cancers, which has served to define the main oncogenic, epidemiological, genetic, clinical and histopathological features. Ongoing attempts to stratify biological markers of endometrial cancer are presented. However, data on changes in tumor marker profiles in obesity-related endometrial cancer are scarce. Obesity is a pandemic in Western countries that has an important impact on endometrial cancers, albeit through not very well-defined mechanisms. Although endometrial cancer is more common in Caucasian women, higher mortality is found in African Americans who also show higher incidence of obesity. Here, we describe how obesity signals (estrogen, leptin, leptin induced-molecules, Notch; cytokines and growth factors) could affect endometrial cancer. Leptin signaling and its crosstalk may be associated to the more aggressive and poor prognosis type II endometrial cancer, which affects more postmenopausal and African-American women. In this regard, studies on expression of novel molecular markers (Notch, interleukin-1 and leptin crosstalk outcome) may provide essential clues for detection, prevention, treatment and prognosis.

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 B26: Leptin affects proliferation, stem cells and chemotherapeutic treatment outcome of pancreatic cancer: A link to health disparity

Background: Pancreatic cancer (PC) is an aggressive disease that is in most cases advanced at the time of diagnosis. The overall 5-year survival rate for PC patients is less than 5 %. African-Americans show the highest incidence of obesity and PC as well as mortality rate. How obesity is associated to PC incidence and health disparity is still an unanswered question. High levels of leptin shown by obese African American patients could impact negatively on PC. We hypothesize that leptin signaling induces PC proliferation, stem cells (PCSC) and drug resistance. Methods: Human PC cell lines derived from primary pancreatic adenocarcinoma (Panc-1 and MiaPaCa-2-highly aggressive, BxPC-3-less aggressive) and from metastatic tumor (AsPC-1) were challenged with leptin and chemotherapeutic agents. Cell proliferation, apoptosis rate, and the expression levels of molecular markers were analyzed. PCSC markers were determined in cell lines and tumor biopsies. Effects of leptin and chemotherapeutics on PC-tumorsphere formation were also determined. Results: Leptin stimulated proliferation of PC cells, induced the expression of PCSC markers and increased tumorsphere formation. Moreover, leptin impaired the efficacy of chemotherapeutics on PC cells. Conclusions: Present data suggest leptin is a proliferative and survival factor for PC that reduces chemotherapeutic effectiveness. These observations are of paramount importance for obese African American PC patients, who show the highest levels of leptin, poor prognosis and outcome of chemotherapeutic treatment. Acknowledgements: this work was supported by the DOD W81XWH-13-1-0382; NIH/SBIR 1R41CA183399-01A1; 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 RR03034 and 1C06 RR18386). Citation Format: Adriana Harbuzariu, Danielle S. Daley-Brown, Tia L. Harmon, Robin C. Garrison, Derrick J. Beech, Frederick D. Cason, Christopher Klug, Ruben R. Gonzalez-Perez. Leptin affects proliferation, stem cells and chemotherapeutic treatment outcome of pancreatic cancer: A link to health disparity. [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 B26.

Abstract 5044: Role of obesity in Leptin-Notch crosstalk (NILCO) in endometrial cancer from African American and Chinese women

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Endometrial cancer is the most common gynecological malignancy of the female reproductive tract. Endometrial cancer is classified into two groups: Type I, which is estrogen dependent, and Type II, which is usually associated with endometrial atrophy and is estrogen independent. Type II is the more aggressive form with a poor prognosis and is often observed more in African American women. Obesity, characterized by high levels of leptin, is a major risk factor for endometrial cancer, which suggests that leptin may play a role in endometrial cancer pathogenesis. Over-expression of Notch, IL-1 and leptin and the signaling crosstalk (NILCO) among these pro-angiogenic factors is associated with metastasis and decreased survival rates in breast cancer patients. However, much is unknown whether this association is found in endometrial cancer. Objective: In this investigation, we aim to determine whether NILCO components are differentially expressed in Type I and Type II endometrial cancer. We hypothesize that there is a positive correlation between endometrial cancer etiology and higher BMI and NILCO expression. We further hypothesize that the correlation is more evident in Type II endometrial cancer. Methods: Expression levels of Notch1, 2, 3 & 4, Jagged-1, DLL4, Survivin, Hey2, IL-1R tI, Leptin, and Ob-R were determined via immunohistochemistry (IHC) in endometrial cancer from overweight/obese African American patients and compared to endometrial cancer tissue array from Chinese women (70 cases of carcinoma and 5 cases of non-malignant duplicated cores per case, US Biomax, Inc). Staining intensity was assigned using semi-quantitative HSCORE [∑pi (i+1), where “i” is the intensity with a value of 1, 2, or 3 (weak, moderate or strong, respectively and “pi” is the percentage of stained cells for each intensity] calculated by two independent observers in 3 different fields (100 cells/each). Western Blot and qPCR analyses of tissue lysates from cancer samples and benign surrounding tissues were also used to examine the expression levels of NILCO components.Student t-test was used to determine statistical differences between samples. Results: IHC results showed that Notch1 and 4, DLL4, Survivin, and Jagged-1 were expressed higher in Type II endometrial cancer patients. IL-1R tI was expressed higher in non-malignant compared to malignant tissue samples. Western blot and qPCR analyses further corroborated these results. Conclusion: The results support our hypothesis that obesity affects the expression of NILCO components, which could be involved in endometrial cancer progression and aggressiveness. [Partially supported by NIH/NCI 1SC1CA138658-05; NIH RR03034,1C06 RR18386, NIH/NCRR 1G12RR026250-03 and MSM/Tuskegee Univ/UAB Cancer Center Partnership grant 5U54CA118638. The National Center for Advancing Translational Sciences of the NIH Award UL1TR000454]. Citation Format: Danielle Daley-Brown, Regina Lee, Gabriela Oprea-Ilies, Emerald Screws, Roland Matthews, Roland Pattillo, Ruben Rene Gonzalez-Perez. Role of obesity in Leptin-Notch crosstalk (NILCO) in endometrial cancer from African American and Chinese women. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5044. doi:10.1158/1538-7445.AM2014-5044

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.

Emerging Biomarkers and Clinical Implications in Endometrial Carcinoma

Endometrial cancer (EmCa) is the most common type of gynecological cancer. EmCa is the fourth most common cancer in the United States, which has been linked to increased incidence of obesity. EmCa can be classified into two main types: Type I and Type II, which include the major histological subtypes. Type I EmCa is hormonally driven, less aggressive, and has a more favorable prognosis. In contrast, Type II EmCa grows independently of hormonal signals, is more aggressive, and generally has an unfavor‐ able prognosis. Various tumor biomarkers [i.e., tumor suppressor p53, hypoxiainducible factor 1-alpha (HIF1-α), human epidermal growth factor receptor 2 (HER2/ neu), and vascular endothelial growth factor (VEGF)] have been identified in EmCa. Biomarkers of treatment effectiveness involve immunosuppressive factors targeted by microRNA (miRNA)-based therapy. However, there are no reliable biomarker tests for early detection of EmCa and treatment effectiveness. A potential new biomarker is Notch, Interleukin-1, leptin crosstalk outcome (NILCO) that could affect the progres‐ sion of Type II EmCa. NILCO expression in EmCa might be dependent on patient’s obesity status. This chapter presents updated information on these, and other potential emerging biomarkers for EmCa, and discusses current challenges and clinical implications on this area of research.

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.