Mong-Hong Lee

Cytoplasmic localization of p21 Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu -overexpressing cells

Amplification or overexpression of HER-2/neu in cancer cells confers resistance to apoptosis and promotes cell growth. The cellular localization of p21Cip1/WAF1 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Here we show that HER-2/neu-mediated cell growth requires the activation of Akt, which associates with p21Cip1/WAF1 and phosphorylates it at threonine 145, resulting in cytoplasmic localization of p21Cip1/WAF1. Furthermore, blocking the Akt pathway with a dominant-negative Akt mutant restores the nuclear localization and cell-growth-inhibiting activity of p21Cip1/WAF1. Our results indicate that HER-2/neu induces cytoplasmic localization of p21Cip1/WAF1 through activation of Akt to promote cell growth, which may have implications for the oncogenic activity of HER-2/neu and Akt.

Oncogenic Signals of HER-2/neu in Regulating the Stability of the Cyclin-dependent Kinase Inhibitor p27

Overexpression and activation of HER-2/neu, a proto-oncogene, play a pivotal role in cancer formation. Strong expression of HER-2/neu in cancers has been associated with poor prognosis. Reduced expression of p27Kip1, a cyclin-dependent kinase inhibitor, correlates with poor clinical outcome in many types of carcinomas. Because many cancers with the overexpression of HER-2/neuoverlap with those affected by reduced p27 expression, we studied the link between HER-2/neu oncogenic signals and p27 regulation. We found that down-regulation of p27 correlates with HER-2/neu overexpression. To address the molecular mechanism of this inverse correlation, we found that reduction of p27 is caused by enhanced ubiquitin-mediated degradation, and the HER-2/Grb2/MAPK pathway is involved in the decrease of p27 stability. Also, HER-2/neu activity causes mislocation of p27 andJun activation domain-binding protein 1 (JAB1), an exporter of p27, into the cytoplasm, thereby facilitating p27 degradation. These results reveal that HER-2/neu signals reduce p27 stability and thus present potential points for therapeutic intervention in HER-2/neu-associated cancers.

The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming

Summary Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumourigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programs by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anti-cancer metabolism therapy development in future.

Deficiency of metabolic regulator FGFR4 delays breast cancer progression through systemic and microenvironmental metabolic alterations

BackgroundEndocrine FGF21 and FGF19 target adipocytes and hepatocytes through betaKlotho (KLB) and FGFR tyrosine kinases effecting glucose, lipid and energy metabolism. Both factors alleviate obesity and metabolic abnormalities which are contributing factors to breast tumor progression. Genomic manipulation of hepatic FGFR4 has uncovered roles of endocrine FGF signaling in both metabolic and cellular homeostasis. Here we determined whether systemic and microenvironmental metabolic alterations caused by the FGFR4 deficiency affect tumorigenesis in breast where FGFR4 is negligible. Breast tumors were induced in the bigenic mice with ablation of FGFR4 and overexpression of TGFα that activates Her2 in the ductal and lobular epithelium surrounded by adipocytes. Mammary tumorigenesis and alterations in systemic and breast microenvironmental metabolic parameters and regulatory pathways were analyzed.ResultsAblation of FGFR4 had no effect on cellular homeostasis and Her2 activity of normal breast tissue. However, the absence of FGFR4 reduced TGFα–driven breast tumor incidence and progression and improved host survival. Notable increases in hepatic and serum FGF21, ileal FGF15/19, adiponectin and adipsin, and decreases in systemic Fetuin A, IGF-1, IGFBP-1, RBP4 and TIMP1 were observed. The ablation affected adipogenesis and secretory function of adipocytes as well as lipogenesis, glycolysis and energy homeostasis associated with the functions of mitochondria, ER and peroxisomes in the breast and tumor foci. Treatment with a chemical inhibitor of NAMPT involved in the pathways inhibited the growth and survival of breast tumor cells and tumor-initiating cell-containing spheres. The FGFR4 ablation also caused elevation of inflammatory factors in the breast.ConclusionsAlthough the primary role of FGFR4 in metabolism occurs in hepatocytes, its ablation results in a net inhibitory effect on mammary tumor progression. We suggest that the tumor-delaying effect of FGFR4 deficiency may be in large part due to elevated anti-obesogenic FGF21 that triggers tumor-suppressing signals from both peripheral and breast adipocytes. The predominant changes in metabolic pathways suggested roles of metabolic effects from both peripheral and breast adipocytes on metabolic reprogramming in breast epithelial cells that contribute to the suppression of tumor progression. These results provide new insights into the contribution of systemic and microenvironmental metabolic effects controlled by endocrine FGF signaling to breast carcinogenesis.

Abstract B20: Obesity-induced Nlrc4 inflammasome promotes angiogenesis in breast cancer

Obesity, which impacts 36% of adults in the U.S., is associated with an increased risk of ER-positive breast cancer in postmenopausal women, increased incidence of the more aggressive triple negative breast cancer, increased risk of developing invasive ductal carcinoma (IDC) versus ductal carcinoma in situ (DCIS; non-invasive) and a worse clinical outcome. There have been many hypotheses to explain this link between obesity and breast cancer, but a causal mechanism is still largely missing. Here, using two syngeneic orthotopic transplant models in C57Bl/6 mice, we examined the role of obesity-associated inflammasome activation/IL-1 signaling in obesity-driven breast cancer progression. Our results show that the tumor microenvironment under the conditions of obesity promotes Nlrc4 inflammasome activation in tumor-infiltrating myeloid cells, which in turn leads to IL-1β activation which promotes tumor progression. Further studies show that obesity-induced Nlrc4 inflammasome activation/IL-1β promotes increased angiogenesis and upregulation of Vegfa, primarily in adipocytes. Finally, treatment of mice with Metformin inhibited obesity-associated angiogenesis and breast cancer progression. This data provides evidence for obesity-induced Nlrc4 inflammasome mediated activation of IL-1β as a causal mechanism for obesity-driven breast cancer progression and a rationale for blockade of IL-1 signaling, the use of anti-angiogenesis therapies, or treatment with metformin in obese breast cancer patients. Citation Format: Ryan Kolb, Liem Phan, Nicholas Borcherding, Yinghong Liu, Fang Yuan, Ann M. Janowski, Qing Xie, Kathleen R. Markan, Wei Li, Matthew J. Potthoff, Enrique Fuentes-Mattei, Leslie G. Ellies, Michael Knudson, Mong-Hong Lee, Sai-Ching Jim Yeung, Suzanne L. Cassel, Fayyaz S. Sutterwala, Weizhou Zhang. Obesity-induced Nlrc4 inflammasome promotes angiogenesis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B20.

Abstract 1922: Target mutation comparasion of WGA circulating tumor cell and primary tumor in epithelial ovarian cancer cell line using NGS

Introduction Epithelial ovarian cancer is the third most common gynecologic cancer in the United States but a leading cause of gynecologic cancer deaths. Previous finding that numbers of circulating tumor cells (CTCs) in the blood of ovarian cancer patients are prognostically significant. Moreover, comprehensive analyses of ovarian cancer genomes may provide more information for precise cancer treatments. Genomic analysis of circulating tumor cells (CTC) can provide possible approach for EOC patients who lack primary tumor tissue or present with un-resectable metastatic diseases. The discordance between whole genome amplification of circulating tumors and primary tumor should be validated firstly and so we conducted a pilot analysis of CTC capture and targeted NGS in EOC cell lines. Methods and materials Under IRB approval, EOC cell lines, different numbers of SKOV-3 (serous adenocarcinoma) and ES-2 (clear cell carcinoma) were spiked in fresh blood samples (20 ml) of healthy donor and captured by CytoQuestTM CR system or negative enrichment method (Abnova, Taipei, Taiwan). CTCs were identified by pan-CK, CD45 and DAPI expression and stained with CEP8 FISH for aneuploidy. Spiked CTCs of two cell lines were captured and underwent whole genome amplification by PicoPLEX™ WGA kit (Rubicon Genomic, MI, USA). The WGA DNA product and DNA of two primary cell lines were prepared libraries for TruSight(TM) Tumor sequencing panel (Illumina Inc.MI,USA) according to manufacturer9s instruction. Sequencing procedure were performed on MiSEQ system(Illumina Inc.) by a local service provider. Results SKOV-3 and ES-2 can be stained and captured with these antibodies. Mean recovery of these cells were more than 80% by both systems. However, ES-2 is less Pan-CK stained but still could be captured by this system. WGA products of each one, three and five ES-2 cells and one SKOV3 cell were prepared well for library. We choose one cell WGA product and DNA extracted from primary SKOV-3 and ES2 were subjected to targeted 26 genes mutational analysis composed in TruSight Tumor panel. Average coverage depth for each amplicon is minimum 1000x with 7000x means. We would like to show if any discordance appear due to the acquisition of DNA WGA in the single cells, of which the analysis is currently ongoing. DISCUSSION Our study would like to demonstrate the condition of variants calls of single tumor cells isolated from whole blood samples and primary tumor in a targeted mutation analysis using NGS after a highly automated isolation workflow. This approach provides a pilot workflow for studying the heterogeneity within the CTC population in blood samples of patients with tumor and their primary tumor and the results can also document the possibility of WGA-induced bias of a recently commercialized WGA kit. Citation Format: Pi-Lin Sung, Chi-Ying F. Huang, Michael Hsiao, Kuo-Chang Wen, Ming-Shyen Yen, Mong-Hong Lee. Target mutation comparasion of WGA circulating tumor cell and primary tumor in epithelial ovarian cancer cell line using NGS. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2015-1922

Abstract 5171: EpCAM expression governs tumor growth or distant metastasis of human endometrial cancer: grow or go theory

Endometrial cancer is the most common gynecologic malignancy in United States. The prognosis is generally better because most patients are diagnosed at early stage. However, the overall survival rate declines to 10∼20% at advanced stage. There are no effective treatment in the management of patient with late stage of endometrial cancer or distant metastasis. EpCAM (also called TACSTD1 or CD326), a transmembrane glycoprotein, can mediate cell-cell adhesion. Its overexpression negatively correlates with clinical survival in gallbladder cancer, ampullary carcinoma, and squamous cell carcinoma of head &. neck. However, a controversial role of EpCAM is reported in Lynch syndrome and rectal cancer as a tumor suppressor gene. To study the role of EpCAM in endometrial cancer, we manipulate the EpCAM expression in loss-of-function experiment in vitro; the results demonstrate that decrease of EpCAM expression is significantly related to increase invasion ability and diminish cell proliferation. These finding can be reversed after gain-of-function in EpCAM knock-down endometrial cancer cells. EpCAM knock-down is indeed linked to the cell signaling pathway of proliferation (regulation of cell proliferation GO:0042127, regulation of cell death:GO:0010941, regulation of apoptosis: GO:0042981) and migration (biological adhesion:GO:0022610, cell adhesion:GO:0007155, cell migration:GO:0016477) from microarray analysis. Significant more number of lymph node metastasis can be found in vivo at mice with IUI (intra-uterine injection) of EpCAM knock-down cells; moreover, smaller tumor size is observed in subcutaneously injected mice. Among the patients with endometrial cancer, the clinicopathologic characteristics, such as stage, lymph node metastasis, CA-125 value, and overall survival, are significantly related to the low EpCAM expression. Conclusively, diminishing EpCAM can promote endometrial cancer invasion; furthermore, the EpCAM expression is an significant prognostic factor in human endometrial cancer. These data has important implication that development targeting EpCAM in the treatment of endometrial cancer is the new application in future. Citation Format: Kuo-Chang Wen, Peng-Hui Wang, Pi-Lin Sung, Mong-Hong Lee, Cheng-Wen Wu. EpCAM expression governs tumor growth or distant metastasis of human endometrial cancer: grow or go theory. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5171. doi:10.1158/1538-7445.AM2015-5171

Abstract 3927: Obesity induces changes in adipokines profile and activates Akt/mTOR signaling accelerating breast carcinogenesis and tumor growth

Obesity increases the risk of cancer death among postmenopausal women with estrogen receptor-positive (ER+) breast cancer (BC), but the direct evidence for the mechanisms is lacking. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating this epidemiological phenomenon. Transcriptomic profiles of pretreatment biopsies from a prospective cohort of 137 ER+ BC patients were analyzed. Transgenic and an orthotopic/syngeneic obese mouse models were created to phenocopy obese patients and evaluate the effect of obesity on breast carcinogenesis and tumor progression, and to explore further direct mechanisms. We used co-culture system to examine the impact of adipocytes and adipokines on BC cell proliferation. Functional transcriptomic analysis of patients revealed the association of obesity with many of the functional changes linked to cancer hallmarks. Our transgenic and orthotopic/syngeneic obese-mouse models recapitulated the functional transcriptomic landscape of obesity-associated changes seen in human patients and demonstrated the role of the Akt/mTOR pathway in obesity-induced breast carcinogenesis and tumor progression. Metformin and everolimus can suppress obesity-induced adipokines secretion and breast tumor formation and growth. An in vitro co-culture model revealed that adipocyte-secreted adipokines (e.g., TIMP-1) regulate adipocyte-induced BC cell proliferation and invasion. Metformin suppress adipocytes-induced cell proliferation and adipocytes-secreted adipokines in vitro. In conclusion, the patients9 data provided evidence for the mechanistic involvement of adipokines in addition to estrogen, insulin and IGF-1 signaling in the link between obesity and ER+ BC. Our animal experiments provide strong evidence for the role of obesity on the accelerated breast carcinogenesis and obesity-induced tumor growth by activation of the Akt/mTOR signaling. Metformin and everolimus may be use as alternatives therapeutic interventions for BC patients with obesity. Citation Format: Enrique Fuentes-Mattei, Guermarie Velazquez-Torres, Liem Phan, Fanmao Zhang, Ping-Chieh Chou, Ji-Hyun Shin, Hyun-Ho Choi, Jiun-Sheng Chen, Ruiying Zhao, Jian Chen, Chris Gully, Colin Carlock, Yuan Qi, Ya Zhang, Yun Wu, Francisco Esteva, Yongde Luo, Wallace L. McKeehan, Joe E. Ensor, Gabriel N. Hortobagyi, Lajos Pusztai Pusztai, W. Fraser Symmans, Mong-Hong Lee, Sai-Ching J. Yeung. Obesity induces changes in adipokines profile and activates Akt/mTOR signaling accelerating breast carcinogenesis and tumor growth. [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 3927. doi:10.1158/1538-7445.AM2014-3927

Abstract B29: Case-control study of metabolic syndrome and breast cancer in Puerto Rico

Background: Breast cancer is the leading cancer type among women in the United States (US) and Puerto Rico (PR); incidence rates of breast cancer have increased in both of these populations in the last decades. In fact, Puerto Rico has highest prevalence of diabetes among all US states and territories. Diabetes, obesity and the metabolic syndrome (MS) are rapidly increasing in Hispanic populations, and research on the impact of these conditions on breast cancer among Hispanics is scarce. Objectives: This study assessed the association of MS with breast cancer among Hispanic women in PR. The secondary aim was to assess the association of the individual components of the MS (hyperglycemia, dyslipidemia, abdominal obesity and elevated blood pressure) with breast cancer. Methods: A matched clinic-based case-control study of 226 Hispanic women was performed between 2011 and 2013. One hundred thirteen cases and 113 controls were recruited in four clinics/hospitals in Puerto Rico and were individually matched by age (±5 years), menopausal status and recruitment site (response rate of 83% for cases and 84% of controls). Data collection procedures included a structured questionnaire, anthropometric and blood pressure measurements. According to the AHA/NHLBI definition, a woman was defined to have the MS if she had at least 3 of the following risk factors: waist circumference ≥35 inches, 2) triglycerides ≥150 mg/dL, 3) HDL Results: Mean age of participants was 54.5 ±12.6 years. Prevalence of the MS was 53.2% among cases and 42.0% among controls. No significant differences were observed between cases and controls with regard to income, health care coverage, parity or smoking habits. Nonetheless, cases had significantly (p Conclusions: This pilot study found marginal evidence of an association of the MS and elevated fasting glucose with breast cancer in a population of Hispanic origin. Future studies should assess this association using a larger sample size and considering the impact of tumor subtypes on breast cancer risk. Grant Support: The project was supported by U54CA96297 and U54CA96300 from the UPR/MDACC Partnership for Excellence in Cancer Research, NCI & by the RCMI-Clinical Research Center grant G12RR03051 from the UPR Medical Sciences Campus. Citation Format: Ana Patricia Ortiz, Cynthia Perez, Edna Mora, Damarys Santiago, Carola T. Sanchez, Cristina Munoz, Marievelisse Soto, Mong-Hong Lee. Case-control study of metabolic syndrome and breast cancer in Puerto Rico. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr B29. doi:10.1158/1538-7755.DISP13-B29

Abstract 2046: TGF-β drives tumor-progression via JAK/STAT3-signaling in advanced HCC.

Background: More than 80% of Hepatocellular carcinoma (HCC) patients present at advanced stages not amenable to curative therapies. Currently existing palliative therapies achieve a minimal survival benefit of 3 months. Hence, there is an urgent need for novel therapeutic strategies. Tumor Growth Factor-β (TGF-β) is a potent tumor suppressor via its Smad2/3 signaling axis. Inactivation of this axis has been implicated in hepatocarcinogenesis. In patients with advanced HCC, TGF- β serum concentrations are elevated and correlated to disease stage, metastases and worse prognosis. Our aims were to evaluate the status and function of the TGF-β/Smad2 signaling axis in advanced HCC. Material and Methods: Immunohistochemical analysis for pSer465/467-Smad2 in human HCC samples was performed using Tissue Micro Arrays. In vitro studies were performed using established human HCC cell lines (HepG2, Hep3B, PLC/PRF/5, HUH-7, SNU-398, SNU-449). Pathway activation following TGF-β stimulation was evaluated by immunoblot analysis and immunofluorescence microscopy. Cell proliferation was assessed by cell quantification and MTS-assay. Apoptosis was quantified morphologically by DAPI stain and fluorescence microscopy, and biochemically by caspase-3/7 assay. Migration was assessed by gap closure assay, and invasion by Matrigel invasion assay. Results: Intranuclear pSer465/467-Smad2 was identified in up to 51% of advanced HCC (n=73) and in 89% of HCC metastases (n=9). In 50% of human HCC cell lines, TGF-β treatment (10 ng/mL) resulted in Ser465/467-phosphorylation and nuclear translocation of the transcription factor Smad2. Exclusively in HCC cells with an intact TGF-β/Smad2-signaling cascade, TGF-β stimulation induced Tyr705-phosporylation of the transcription factor STAT3. TGF-β induced STAT3 Tyr705-phosporylation is inhibited by Smad2-directed gene silencing. Further, pyridine 6 mediated Janus Kinase-1/2 (JAK1/2) inhibition prevented TGF-β induced Tyr705-phosporylation of STAT3. Cell proliferation of HCC cells was inhibited by TGF-β. However, TGF-β treatment significantly induced invasion and migration in HCC cell lines with intact TGF-β/Smad2 signaling. Further, TGF-β induced resistance to TRAIL-induced apoptosis in these cells. However, co-treatment with the STAT3-inhibitor Stattic (10 nM) completely abrogates TGF-β mediated HCC cell migration and invasion, and sensitizes these HCC cell lines to TRAIL-mediated apoptosis. Conclusion: In advanced HCC, the TGF-β/Smad signaling axis is constitutively activated in the majority of primary HCC tumors and HCC metastases. The function of TGF-β/Smad2 in human HCC cell lines has switched from tumor inhibitor to a driver of tumor progression via JAK/STAT3 signaling. STAT3-inhibition reverses the TGF-β tumor-promoting effects. Hence, JAK/STAT3 targeting might be a promising new strategy to restore TGF-β tumor suppressor function in advanced HCC. Citation Format: Andres Rojas, Jessica Cromheecke, Mong-Hong Lee, Mien-Chie Hung, Boris Blechacz. TGF-β drives tumor-progression via JAK/STAT3-signaling in advanced HCC. [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 2046. doi:10.1158/1538-7445.AM2013-2046