Fanmao Zhang

Aurora B kinase phosphorylates and instigates degradation of p53

Aurora B is a mitotic checkpoint kinase that plays a pivotal role in the cell cycle, ensuring correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in many types of human cancers, which has made it an attractive target for cancer therapies. Tumor suppressor p53 is a genome guardian and important negative regulator of the cell cycle. Whether Aurora B and p53 are coordinately regulated during the cell cycle is not known. We report that Aurora B directly interacts with p53 at different subcellular localizations and during different phases of the cell cycle (for instance, at the nucleus in interphase and the centromeres in prometaphase of mitosis). We show that Aurora B phosphorylates p53 at S183, T211, and S215 to accelerate the degradation of p53 through the polyubiquitination–proteasome pathway, thus functionally suppressing the expression of p53 target genes involved in cell cycle inhibition and apoptosis (e.g., p21 and PUMA). Pharmacologic inhibition of Aurora B in cancer cells with WT p53 increased p53 protein level and expression of p53 target genes to inhibit tumor growth. Together, these results define a mechanism of p53 inactivation during the cell cycle and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise the tumor suppressor function of p53. We have elucidated the antineoplastic mechanism for Aurora B kinase inhibitors in cancer cells with WT p53.

Subunit 6 of the COP9 signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers

The mammalian constitutive photomorphogenesis 9 (COP9) signalosome (CSN), a protein complex involved in embryonic development, is implicated in cell cycle regulation and the DNA damage response. Its role in tumor development, however, remains unclear. Here, we have shown that the COP9 subunit 6 (CSN6) gene is amplified in human breast cancer specimens, and the CSN6 protein is upregulated in human breast and thyroid tumors. CSN6 expression positively correlated with expression of murine double minute 2 (MDM2), a potent negative regulator of the p53 tumor suppressor. Expression of CSN6 appeared to prevent MDM2 autoubiquitination at lysine 364, resulting in stabilization of MDM2 and degradation of p53. Mice in which Csn6 was deleted died early in embryogenesis (E7.5). Embryos lacking both Csn6 and p53 survived to later in embryonic development (E10.5), which suggests that loss of p53 could partially rescue the effect of loss of Csn6. Mice heterozygous for Csn6 were sensitized to γ-irradiation-induced, p53-dependent apoptosis in both the thymus and the developing CNS. These mice were also less susceptible than wild-type mice to γ-irradiation-induced tumorigenesis. These results suggest that loss of CSN6 enhances p53-mediated tumor suppression in vivo and that CSN6 plays an important role in regulating DNA damage-associated apoptosis and tumorigenesis through control of the MDM2-p53 signaling pathway.

Effects of Obesity on Transcriptomic Changes and Cancer Hallmarks in Estrogen Receptor–Positive Breast Cancer

Background Obesity increases the risk of cancer death among postmenopausal women with estrogen receptor–positive (ER+) breast cancer, but the direct evidence for the mechanisms is lacking. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating this epidemiologic phenomenon. Methods We analyzed transcriptomic profiles of pretreatment biopsies from a prospective cohort of 137 ER+ breast cancer patients. We generated transgenic (MMTV-TGFα;A y /a) and orthotopic/syngeneic (A y /a) obese mouse models to investigate the effect of obesity on tumorigenesis and tumor progression and to determine biological mechanisms using whole-genome transcriptome microarrays and protein analyses. We used a coculture system to examine the impact of adipocytes/adipokines on breast cancer cell proliferation. All statistical tests were two-sided. Results Functional transcriptomic analysis of patients revealed the association of obesity with 59 biological functional changes (P < .05) linked to cancer hallmarks. Gene enrichment analysis revealed enrichment of AKT-target genes (P = .04) and epithelial–mesenchymal transition genes (P = .03) in patients. Our obese mouse models demonstrated activation of the AKT/mTOR pathway in obesity-accelerated mammary tumor growth (3.7- to 7.0-fold; P < .001; n = 6–7 mice per group). Metformin or everolimus can suppress obesity-induced secretion of adipokines and breast tumor formation and growth (0.5-fold, P = .04; 0.3-fold, P < .001, respectively; n = 6–8 mice per group). The coculture model revealed that adipocyte-secreted adipokines (eg, TIMP-1) regulate adipocyte-induced breast cancer cell proliferation and invasion. Metformin suppress adipocyte-induced cell proliferation and adipocyte-secreted adipokines in vitro. Conclusions Adipokine secretion and AKT/mTOR activation play important roles in obesity-accelerated breast cancer aggressiveness in addition to hyperinsulinemia, estrogen signaling, and inflammation. Metformin and everolimus have potential for therapeutic interventions of ER+ breast cancer patients with obesity.

14-3-3σ Exerts Tumor-Suppressor Activity Mediated by Regulation of COP1 Stability

Constitutive photomorphogenic 1 (COP1) is a p53-targeting E3 ubiquitin ligase that is downregulated by DNA damage through mechanisms that remain obscure. Here, we report that COP1 is not downregulated following DNA damage in 14-3-3σ null cells, implicating 14-3-3σ as a critical regulator in the response of COP1 to DNA damage. We also identified that 14-3-3σ, a p53 target gene product, interacted with COP1 and controlled COP1 protein stability after DNA damage. Mechanistic studies revealed that 14-3-3σ enhanced COP1 self-ubiquitination, thereby preventing COP1-mediated p53 ubiquitination, degradation, and transcriptional repression. In addition, we found that COP1 expression promoted cell proliferation, cell transformation, and tumor progression, manifesting its role in cancer promotion, whereas 14-3-3σ negatively regulated COP1 function and prevented tumor growth in a mouse xenograft model of human cancer. Immunohistochemical analysis of clinical breast and pancreatic cancer specimens demonstrated that COP1 protein levels were inversely correlated with 14-3-3σ protein levels. Together, our findings define a mechanism for posttranslational regulation of COP1 after DNA damage that can explain the correlation between COP1 overexpression and 14-3-3σ downregulation during tumorigenesis.

Abstract A93: Genetic variations in aging related genes/pathways and colorectal cancer risk

Advanced age is arguably the most important risk factor for developing cancer. About 77% of all cancers are diagnosed in individuals 55 years of age and older. Aging is especially a dominant risk factor for colorectal cancer (CRC), with 91% of cases diagnosed in individuals 50 years of age and older. A number of genes and signaling pathways have been indicated in the aging process in model organisms including mammals, e.g. the insulin/IGF-1 signaling pathway, mTOR pathway and telomere and telomerase pathway. Recently, genome-wide association studies also identified several single nucleotide polymorphisms (SNPs) associated with longevity. Therefore, we hypothesized that genetic variations of the genes and pathways related to aging might be associated with CRC risk. We systematically identified 189 putative functional SNPs in 109 aging-related genes and 20 longevity-associated SNPs from GWAS. In the discovery phase, we genotyped these SNPs in 797 Caucasian CRC cases and 797 matched healthy control subjects. In main effect analysis, 10 SNPs were significantly associated with altered CRC risk. When evaluating combined effects of these SNPs, we found a significant gene dosage effect for increased CRC risk with increasing number of unfavorable genotypes. Compared with individuals carrying fewer than three unfavorable genotypes, individuals with six and more than six of these unfavorable genotypes exhibited a 2.06-fold (95% CI: 1.24-3.43, P = 0.006) and a 2.44-fold (95% CI: 1.76-3.38, P = 7.56×10−8) increased risk of CRC, respectively (P for trend 1.25×10−9). Higher order gene-gene interaction analysis also revealed potential relationships among SNPs in TP53, ERCC2, GPR133, PRKAG3, TEP1, TSC1 and LMNA that further defined risk groups for CRC. Our results suggest that genetic polymorphisms in aging-related genes and pathways may modify CRC susceptibility both individually and jointly. Replication is currently underway to validate these findings. Citation Format: Fanmao Zhang, Cathy Eng, Moubin Lin, Michelle A.T. Hildebrandt, Yonggang He, Jie Lin, Maosheng Huang, Jian Gu, Xifeng Wu. Genetic variations in aging-related genes/pathways and colorectal cancer risk. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A93.

Abstract 296: Pre-treatment clinical laboratory tests indicative of metabolic status are associated with survival in patients with non-small cell lung cancer

The value of serum pre-treatment laboratory tests for prognostic purposes in non-small cell lung cancer (NSCLC) is mixed, with inconsistent results. This study was designed to identify pre-treatment routine laboratory tests indicative of a patient9s overall metabolic status, as predictors of NSCLC survival. We assessed seven pre-treatment serum laboratory test levels, including alanine aminotransferase, albumin, alkaline phosphatase, glucose, lactate dehydrogenase, total bilirubin, and total protein in 2,675 NSCLC patients, among which 2,201 patients were Caucasians. There were 623 early stage and 2,052 advanced stage NSCLC patients. To minimize the effect of treatment heterogeneity, we focused on subgroups of patients with similar treatment regimens. We studied 978 advanced stage NSCLC patients who were treated with platinum-based chemotherapy. Compared to those with pre-treatment laboratory test levels within the normal range, lower than normal levels of albumin [adjusted hazard ratio (HR) = 1.47, 95% confidence interval (CI): 1.15-1.87, P Citation Format: Fanmao Zhang, Qing H. Meng, Yuanqing Ye, Michelle AT Hildebrandt, Maosheng Huang, Xifeng Wu. Pre-treatment clinical laboratory tests indicative of metabolic status are associated with survival in patients with non-small cell lung cancer. [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 296. doi:10.1158/1538-7445.AM2014-296

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 LB-27: Metabolomic profiling identifies bilirubin as a novel serum marker for lung cancer.

Background. Lung cancer is the leading cause of cancer death in the United States. Cost-effective screening and early detection is essential for reducing lung cancer mortality. Serum metabolites are emerging as promising biomarkers for improving risk prediction and early detection of lung cancer. Methods. In the discovery phase, we performed global, unbiased metabolomic profiling in serum samples from 20 healthy controls, 20 early stage, and 20 late stage lung cancer patients who were matched on age and gender. We then selected three differentially expressed metabolites for validation in two additional case control populations, followed by final validation of one metabolite in a large Taiwanese prospective cohort of 435,985 individuals. Results. A total of 403 named metabolites were identified by metabolomic profiling in the discovery phase, with 306 metabolites remaining after initial quality assurance. Of these, 29 metabolites exhibited a significant trend when comparing normal controls, early stage and late stage lung cancer cases. Three of the top differentially expressed metabolites, gamma-glutamylalanine, bilirubin and allantoin, were selected and validated in two additional sets of subjects, one set comprised of 50 controls, 50 early stage and 50 late stage lung cancer cases, and the second comprised of 123 controls, 123 early stage and 123 late stage lung cancer cases. The most promising metabolite, bilirubin was further validated in a large prospective cohort. In this cohort, the incidence rate of lung cancer per 10,000 person-years in men was 3.73 (95% confidence interval [CI], 3.13-4.43) in the highest quartile of bilirubin level compared to 7.02 (95% CI, 6.16-7.99) in the lowest quartile, which translates into a 38% increase in lung cancer incidence for the low bilirubin group (P=0.003). The corresponding lung cancer specific mortality rate in men was 2.46 (95% CI, 2.02-3.00) in the highest quartile compared to 4.84 (95% CI, 4.19-5.60) in the lowest quartile, a 53% increase in lung cancer specific mortality for the low bilirubin group (P Conclusions. Smokers with low bilirubin levels had significantly increased risks for both lung cancer incidence and mortality. Serum bilirubin is a valuable biomarker for lung cancer risk prediction in ever-smokers. Citation Format: Fanmao Zhang, Chi Pang Wen, Dong Liang, Heath Skinner, Jian Gu, Wong-Ho Chow, Yuanqing Ye, Michelle A.T. Hildebrandt, Xia Pu, Maosheng Huang, Min Kuang Tsai, Chwen Keng Tsao, Xifeng Wu. Metabolomic profiling identifies bilirubin as a novel serum marker for lung cancer. [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 LB-27. doi:10.1158/1538-7445.AM2013-LB-27

Abstract 4485: Prediction of esophageal cancer risk through quantification of gamma-H2AX levels in peripheral blood lymphocytes

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Increased susceptibility to DNA damage has been shown to be associated with increased risk of many cancers. Accumulation of gamma-H2AX (γ-H2AX) in cultured cells after stimulation by ionizing radiation or other genotoxic agents is a sensitive indicator of DNA double-strand breaks (DSBs) induction. We hypothesize that higher level of DSBs, as measured by γ-H2AX levels in peripheral blood lymphocytes (PBLs), may be able to predict higher cancer risk. We first developed an objective, high-throughput, and image-based method to quantify immunofluorescence intensities of γ-H2AX in cultured PBLs. To test this hypothesis, using an ongoing esophageal cancer case control study, we measured the γ-H2AX levels at baseline and after gamma-radiation in the PBLs from 245 newly diagnosed, previously untreated esophageal cancer patients and 400 healthy control subjects, and calculated the ratio of radiation-induced to the baseline γ-H2AX levels as a parameter of DSBs. The mean γ-H2AX ratio was significantly higher in cases (1.48±0.14) than controls (1.39±0.11) (P =1.47×10-14). When dichotomized at the median level of the γ-H2AX ratio in control subjects, individuals in the high γ-H2AX ratio group exhibited a significantly increased risk of esophageal cancer (odds ratio [OR]=2.83, 95% confidence intervals [CI]: 2.00-4.00). When subjects were categorized into tertiles defined by the distribution of the relative γ-H2AX ratio in control subjects, a significant dose-response relationship was observed between γ-H2AX ratio and risk of esophageal cancer. Compared to subjects in the lowest tertile of γ-H2AX ratio, those in the middle and the highest tertiles exhibited increased risks of esophageal cancer with ORs of 1.60 (95% CI: 1.00-2.55) and 3.72 (95% CI: 2.43-5.70), respectively (P for trend =2.24×10-10). Quartile analysis also showed similar dose-response relationship. Our data suggest that higher ratio of gamma-radiation-induced to baseline γ-H2AX is significantly associated with an increased risk of esophageal cancer. This study supports that measurement of this ratio may be used as a novel phenotypic assay to identify individuals at high risk for esophageal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4485. doi:1538-7445.AM2012-4485

Mutation and functional analysis of PIK3CA and PIK3R1 in endometrial cancer

The phosphatidylinositol 3 kinase (PI3K) pathway is the most frequently activated pathway across multiple tumor lineages and this is a potential therapeutic target. As a critical step to accelerate therapeutic development in endometrial cancer, we performed a comprehensive analysis of mutation and function of PI3K pathway members in a set of 243 highly characterized endometrial tumors. Whole gene resequencing revealed the highest frequencies of mutation in the PI3K pathway of any tumor lineage including PTEN (44%), PIK3CA (40%; encoding the p110α catalytic subunit of PI3K) and PIK3R1 (20%; encoding the p85a regulatory subunit). Indeed, when complete protein loss is considered, almost 80% of endometrioid endometrial cancers demonstrate an aberration in the PI3K pathway. Remarkably, mutations in the PI3K pathway were not mutually exclusive and indeed co-existence of PIK3CA or PIK3R1 mutation with heterozygous PTEN mutation occurred at frequencies higher than predicted by the frequency of each lesion alone. High-throughput reverse-phase protein array suggested that the dominant signaling effects occurred with PTEN protein loss and that PIK3CA or PIK3R1 mutation functionally mimic PTEN protein loss as indicated by protein markers including phosphorylated Akt and stathmin. Thus it appears likely that co-mutations in the PI3K pathway are selected to compensate haploinsufficiency of PTEN due to PTEN heterozygous mutation. To determine the functional consequences of mutations in PIK3R1, we demonstrated that several somatic PIK3R1 mutations tested conferred cytokine-independent growth on interleukin-3-dependent Ba/F3 cells and induced Akt phosphorylation in endometrial cancer cell lines indicating that they were gain of function mutations likely acting as oncogenes. Strikingly two of the gain of function mutations (E160* and R348*) lacked the ability to bind to the p110 catalytic subunit and E160* failed to bind to both p110 and PTEN. We demonstrate that E160* disrupts a novel mechanism of pathway regulation wherein p85 binds and stabilizes PTEN. Together, the data indicate that the PI3K pathway is targeted in the vast majority of endometrioid endometrial cancers representing a novel opportunity for implementation of targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2011-LB-247