Brandi B. Knight

Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

Obesity is an independent risk factor for breast cancer, and obese breast cancer patients exhibit a higher risk for larger tumor burden and increased metastasis. Obesity, as associated with metabolic syndrome, results in an increase in circulating insulin-like growth factor (IGF), which acts as a mitogen. In addition, higher plasma level of adipocytokine leptin is associated with obesity. In the present study, we show that cotreatment with leptin and IGF-I significantly increases proliferation as well as invasion and migration of breast cancer cells. We found a novel bidirectional crosstalk between leptin and IGF-I signaling; IGF-I induced phosphorylation of leptin receptor (Ob-Rb) and leptin induced phosphorylation of IGF-I receptor (IGF-IR), whereas cotreatment induced synergistic phosphorylation and association of Ob-Rb and IGF-IR along with activation of downstream effectors, Akt and extracellular signal-regulated kinase. Leptin increased phosphorylation of IGF signaling molecules insulin-receptor substrate (IRS)-1 and IRS-2. Interestingly, we found that leptin and IGF-I cotreatment synergistically transactivated epidermal growth factor receptor (EGFR), depending on the proteolytic release of EGFR ligands, as the broad-spectrum matrix metalloproteinase inhibitor GM6001 could inhibit this effect. Using clinically relevant EGFR inhibitors, erlotinib and lapatinib, we found that inhibition of EGFR activation effectively inhibited leptin- and IGF-I-induced invasion and migration of breast cancer cells. Taken together, these data suggest a novel bidirectional crosstalk between leptin and IGF-I signaling that transactivates EGFR and promotes the metastatic properties as well as invasion and migration of breast cancer cells. Our findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the procancerous effects of leptin and IGF-I in breast cancers.

Survivin upregulation, dependent on leptin-EGFR-Notch1 axis, is essential for leptin-induced migration of breast carcinoma cells

Obese breast cancer patients exhibit a higher risk for larger tumor burden and an increased likelyhood of metastasis. The molecular effects of obesity on carcinogenesis are mediated by the autocrine and paracrine effects of the adipocytokine leptin. Leptin participates in the tumor progression and metastasis of human breast. We show that leptin induces clonogenicity and increases the migration potential of breast cancer cells. We found that survivin expression is induced in response to leptin. In this study, we examine the role and leptin-mediated regulation of survivin. Leptin treatment leads to survivin upregulation, due in part to the activation of Notch1 and the release of a transcriptionally active Notch1 intracellular domain (NICD). Chromatin immunoprecipitation analysis shows that NICD gets recruited to the survivin promoter at the CSL (CBF1/RBP-Jk, Su(H), Lag-1) binding site in response to leptin treatment. Inhibition of Notch1 activity inhibits leptin-induced survivin upregulation. Leptin-induced transactivation of epidermal growth factor receptor (EGFR) is involved in leptin-mediated Notch1 and survivin upregulation, demonstrating a novel upstream role of leptin-EGFR-Notch1 axis. We further show that leptin-induced migration of breast cancer cells requires survivin, as overexpression of survivin further increases, whereas silencing survivin abrogates leptin-induced migration. Using a pharmacological approach to inhibit survivin, we show that 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase inhibitors, such as lovastatin, can effectively inhibit leptin-induced survivin expression and migration. Importantly, leptin increased breast tumor growth in nude mice. These data show a novel role for survivin in leptin-induced migration and put forth pharmacological survivin inhibition as a potential novel therapeutic strategy. This conclusion is supported by in vivo data showing the overexpression of leptin and survivin in epithelial cells of high-grade ductal carcinomas in situ and in high-grade invasive carcinomas.

Abstract 974: Fellowships in Research and Science Teaching (FIRST): Innovative postdoctoral training for aspiring academic scientists

Fellowships in Research and Science Teaching (FIRST) is a successful, three-year postdoctoral program at Emory University that combines traditional postdoctoral research training with mentored teaching instruction. Supported by the Institutional Research and Academic Career Development Award (IRACDA, K12) in the Division of Minority Opportunities in Research (MORE), FIRST encourages teaching experiences at minority serving institutions (MSIs) in the Atlanta University Center (Clark-Atlanta University, Morehouse College, and Spelman College). Fellows receive formalized instruction in pedagogy through a course entitled A Class within a Class, which provides training in innovative teaching techniques, use of technology in the classroom, course development and assessment, as well as peer-evaluation of lecture styles. Participation in the FIRST program creates a unique training experience that prepares fellows to become successful biomedical researchers as well as effective instructors. Despite having dual responsibilities during postdoctoral training, a study comparing 114 FIRST fellows to 500 traditional postdocs showed that FIRST fellows continue to publish in peer-reviewed journals at the same rate or above traditional postdoctoral fellows. In addition, FIRST fellows continue to pursue academic research careers and receive NIH grant funding. Along with the goals of the IRACDA programs, FIRST fellows have established research affiliations with Emory University and MSIs thus creating a collaborative network between research-intensive intuitions and under-represented minority liberal arts colleges. NIH/NIGMS IRACDA Grant 5K12GM000680 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 974.

Abstract LB-224: Leptin-induced breast cancer cell migration requires upregulation of antiapoptotic protein, survivin

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Leptin, an adipocytokine secreted by adipose tissue, is responsible for many biological functions including appetite regulation, energy homeostasis, bone formation, and more recently, carcinogenesis. Levels of circulating leptin are typically elevated in obese states and have been associated with increased tumor proliferation, angiogenesis, and increased metastatic behavior. Therefore, obesity has been clearly associated with increased risk of cancer and metastasis. Breast cancer remains as the second leading cause of cancer-related deaths to American women. Many epidemiological studies have shown that obese women are more at risk for developing breast cancer compared to non-obese women. Therefore, it is imperative to understand the contribution of leptin to breast cancer development and progression. Methods and Results: In this study, we used MCF-7 and MDA-MB-231 breast cancer cell lines to examine the relationship between leptin and survivin, a dual-function protein. Survivin has become an interesting target in cancer research because it is highly expressed in cancerous tissues while being nearly undetectable in normal tissues. We observed that leptin increases mRNA and protein expression of survivin in a time-dependent manner. Earlier studies from our lab demonstrated that leptin increases breast cancer cell migration and invasion and underlying mechanisms involved a bidirectional crosstalk between leptin and IGF1 leading to transactivation of EGFR. Examining the underlying mechanisms, we found that blocking EGFR activation decreased leptin-induced survivin expression indicating the involvement of EGFR. To examine the role of survivin in leptin-induced migration of breast cancer cells, we transfected both cell lines with wild-type or p-silencer-survivin and subjected the cells to scratch and electric cell-substrate impedance sensing (ECIS) migration assays. We found that survivin overexpression increased migration potential of breast cancer cells compared to untransfected cells. In addition, leptin treatment further enhanced migration of breast cancer cells with survivin overexpression. Inhibition of survivin inhibited migration of breast cancer cells alone and even in the presence of leptin. These studies showed the importance of survivin in leptin-induced migration of breast cancer cells. For therapeutic purposes, we treated the breast cancer cells with lovastatin, commonly used as an HMG-CoA reducatase inhibitor. Lovastatin has been previously shown to inhibit survivin expression. We treated cells with lovastatin in a dose-dependent manner and observed a complete inhibition of survivin expression. Combination treatment with leptin did not restore survivin protein expression. Importantly, lovastatin treatment efficiently inhibited migration of breast cancer cells even in the presence of leptin. Conclusion: In conclusion, we have established a novel role for survivin in leptin-induced migration of breast cancer cells. Targeting survivin using lovastatin may provide new therapeutic targets for the treatment of metastatic breast carcinogenesis in obese patients. 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 LB-224.

Abstract LB-269: Activation of energy sensor AMP-activated protein kinase by rosiglitazone: A novel means to rescue triple-negative breast cancer cells from metastasis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: A recent epidemiological study reported that triple-negative (TN) breast cancers occurred in association with obesity, which itself has been associated with increased risk for breast cancer. Obesity was present in 49.6% of patients with TN cancer but only in 35.8% of those with non-TN cancer. Considering the increasing epidemic of obesity this 13.8% increase is highly significant. Molecular effects of obesity on breast cancer growth and metastasis are mediated by dysregulation of adipocytokines, leptin and adiponectin. Our lab has previously shown that increased leptin signaling increases breast cancer metastasis whereas another adipocytokine, adiponectin inhibits metastatic potential of breast cancer cells. In the present study, we investigated the potential of thiazolidinediones (TZD) as adiponectin-mimetic in breast carcinogenesis and metastasis. Methods: TN breast carcinoma cell lines, MDA-MB-231 and MDA-MB-468, were used to study the effects on TZDs on TN breast cancer cell viability, anchorage-dependent tumor growth and anchorage-independent growth using the XTT colorimetric assay, clonigenecity assays, and soft-agar assays, respectively. Cell migration (wound-healing) assays were performed to assess the effects of TZDs on cell motility. Reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting were used to detect mRNA and protein expression levels in TZD treated cells, respectively. Results: Various Thiazolidinediones (TZD) have been developed including Rosiglitazone (RZG), Troglitazone (TGZ), and Pioglitazone (PZG). We found that rosiglitazone (RZG), a novel class of TZD increases adiponectin levels in TN breast cancer cells. We show that RGZ inhibits cell proliferation, anchorage-dependent and -independent growth of TN breast cancer cells in a dose-dependent manner. The ability of a cancer cell to migrate is an essential prerequisite for metastasis. Importantly, RZG treatment inhibits migration potential of TN breast cancer cells. Further examination of the underlying molecular mechanisms revealed that RGZ treatment increases phosphorylation of energy sensor AMP-activated protein kinase in TN breast cancer cells. Increased phosphorylation of AMPK increases its activity as evident by activation of its downstream target, ACC. Intriguingly, we discovered that RGZ treatment also decreases the activation of p-S6K indicating mTOR inhibition. Using AMPK-WT and AMPK-null fibroblasts, we further show that AMPK is required for RZG-mediated inhibition of metastatic potential of TN breast cancer cells. Conclusions: Our studies show that inhibition of metastatic properties of TN breast cancer cells by RGZ involves increased adiponectin levels, elevated AMPK (pAMPK) expression and activity. Our study suggests that RGZ can be used as a novel therapeutic agent for the treatment of aggressive, metastatic TN breast tumors that are unresponsive to current therapeutic regiments. 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 LB-269.

Abstract 1078: Rosiglitazone inhibits breast cancer growth and proliferation via cyclin D1 inhibition.

Breast cancer ranks as the most commonly diagnosed and second leading cause of cancer death in North American women. Thiazolidinediones (TZDs) are a class of anti-diabetic drugs primarily used to treat type 2 diabetes mellitus by increasing insulin sensitivity; these compounds are also known to possess anti-tumor effects in several cancer cells including lung and breast cancer. The present study was designed to evaluate the antiproliferative effects of Rosiglitazone on triple negative breast cancer cells. MDA-MB-231 breast cancer cells were treated with Rosiglitazone and compared with negative control (without treatment) for modulation of molecules involved in cell-cycle regulation and apoptosis, including cyclin D1, caspase-3 and −9, Bad, and Bax. Our results show that Rosigliatzone suppresses the proliferation of highly-metastatic human breast cancer MDA-MB-231 cell line by increasing the number of cells in G1 and reducing the number of cells progressing through the S-phase of the cell cycle. Further investigation revealed that the mechanism by which Rosiglitazone arrested cells in the G1-phase was via inhibition of cyclin D1 (a cell cycle regulatory protein that is essential for progression through the G1-phase of the cell cycle and has an central pathogenetic role in breast cancer). Our lab has previously shown an essential role of cyclin D1 upregulation in breast cacner growth and proliferation. Collectively, our results suggest that Rosiglitazone inhibits cancer cell proliferation by modulating expression of cyclin D1 and may provide a novel role for the use of Rosiglitazone as a therapeutic antitumorigenic agent. 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 1078.

Adiponectin activates tumor suppressor LKB1 and AMP-activated protein kinase signaling to inhibit malignant properties of breast cancer cells.

Abstract #3067 Introduction: The prevalence of obesity in the developed world has reached epidemic proportions in recent years. Obese breast cancer patients exhibit a higher risk for metastasis, larger tumor burden and poorer response to endocrine therapy. Therefore, it is very important to understand the adverse effects of obesity on breast cancer in order to devise appropriate new approaches to their treatment. Obesity is considered an endocrine disorder and serum levels of adiponectin, an adipocytokine proposed to have therapeutic potential, get significantly lowered with obesity. More recently, low serum adiponectin levels were associated with increased risk and more aggressive phenotype (larger tumor size and high histological grade) of breast cancer in women. Recently, anti-proliferative and pro-apoptotic response of adiponectin was shown in breast cancer cells but the molecular mechanisms governing the anti-cancer effects of adiponectin largely remain unknown. In the present study, we found a novel mechanism underlying adiponectin action involving upregulation of tumor suppressor LKB1 and activation of AMPK (AMP-activated protein kinase) signaling to inhibit malignant properties of breast cancer cells.
 Methods and Results: Adiponectin inhibited malignant properties such as invasion and migration of breast cancer cells in matrigel invasion, scratch migration and quantitative Electric Cell-Substrate Impedance Sensing (ECIS) invasion and migration assays. Interestingly, adiponectin induced phosphorylation of AMPK at Thr 172 in breast cancer cells. Adiponectin treatment also affected downstream targets of mTOR signaling pathway, including decreased phosphorylation of p70S6K showing the involvement of mTOR-S6K axis. Intriguingly, we found that adiponectin increased the expression of tumor suppressor LKB1 in breast cancer cells. Co-immunoprecipitation assays revealed that LKB1 co-immunoprecipitated with tumor suppressor TSC2. Increase in expression of upstream kinase LKB1 might directly regulate adiponectin induced activation of AMPK. We also found that breast cancer cells transfected with wild-type LKB1 plasmids increased phosphorylation of AMPK and decreased phosphorylation of p70S6K showing decrease in mTOR activity. In contrast, breast cancer cells transfected with kinase dead LKB1 decreased phosphorylation of AMPK and increased activation of p70S6K.
 Discussion: Taken together these data indicate a novel crosstalk between adiponectin and tumor suppressor LKB1 that modulates AMPK-S6K axis and inhibits metastatic properties, invasion and migration of breast cancer cells. The influence of adipocytokine, adiponectin on breast carcinogenesis is just beginning to be elucidated and our study is the first to show the direct involvement of tumor suppressor genes LKB1 and TSC2 in anti-cancer effects of adiponectin. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3067.

Co-targeting leptin and insulin-like growth factor I signaling: dramatic effects of epidermal growth factor receptor inhibitors on triple negative breast cancer cells.

Abstract #3044 Introduction: Obesity is an independent risk factor for breast cancer and obese breast cancer patients exhibit a higher risk for larger tumor burden, increased metastasis and poor response to endocrine therapy. Obesity affects breast carcinogenesis by autocrine and paracrine effects of adipocytokine leptin and IGF-1. We have previously shown that leptin induces growth stimulation of breast cancer cells by recruiting histone acetyltransferases and mediator complex to cyclin D1 promoter via activation of Stat3. In the present study, we found a novel bidirectional crosstalk between IGF-1 and leptin signaling that promotes invasion and migration of triple-negative (ER, PR, HER2 negative) breast cancer cells; MDA-MB-231, MDA-MB-468, MDA-MB-435 and HCC-1806.
 Methods and Results: IGF-1 induced significant tyrosine phosphorylation of leptin receptor (Ob-Rb) and leptin induced tyrosine phosphorylation of IGF-1 Receptor (IGF-1R). Combined treatment of leptin and IGF-1 induced synergistic activation of both Ob-Rb and IGF-1R along with activation of Akt and ERK. Furthermore, IGF-1 and leptin synergistically transactivated epidermal growth factor receptor (EGFR) and induced proliferation of triple negative breast cancer cells. We also found that tyrosine phosphorylation of EGFR induced by combined treatment of leptin and IGF-1 was significantly inhibited not only by EGFR tyrosine kinase inhibitor, AG1478 but also with a broad-spectrum matrix metalloproteinase inhibitor, GM6001 indicating that leptin and IGF-1 induced EGFR transactivation is dependent on proteolytic release of EGFR ligands. Intriguingly, we also found that combined treatment of leptin and IGF-1 potently induced invasion of triple negative breast cancer cells in Matrigel invasion and quantitative Electric Cell-Substrate Impedance Sensing invasion assays. Inhibition of leptin and IGF-1 mediated EGFR activation using a highly potent, reversible inhibitor of HER1/EGFR tyrosine kinase, erlotinib or dual epidermal growth factor receptor (EGFR)/human EGFR-2 (HER2) kinase inhibitor, lapatinib effectively inhibited leptin and IGF-1 induced invasion. Also, inhibition of EGFR activation using erlotinib and lapatinib reduced leptin- and IGF-1-induced migration of triple negative breast cancer cells.
 Discussion: Taken together these data indicate a novel bidirectional crosstalk between leptin and IGF-1 signaling that transactivates EGFR and promotes metastatic properties, invasion and migration of triple negative breast carcinoma cells. Our novel findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the pro-cancerous effects of leptin and IGF-1 signaling in triple negative breast cancers. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3044.