FUNCTION OF ZNF668 IN CANCER DEVELOPMENT

Human cancer develops as a result of accumulation of mutations in oncogenes and tumor suppressor genes. Zinc finger protein 668 (ZNF668) has recently been identified and validated as one of the highly mutated genes in breast cancer, but its function is entirely unknown. Here, we report two major functions of ZNF668 in cancer development. (1) ZNF668 functions as a tumor suppressor by regulating p53 protein stability and function. We demonstrate that ZNF668 is a nucleolar protein that physically interacts with both MDM2 and p53. By binding to MDM2, ZNF668 regulates MDM2 autoubiquitination and prevents MDM2-mediated p53 ubiquitination and degradation; ZNF668 deficiency impairs DNA damage-induced p53 stabilization. Notably, ZNF668 effectively suppresses breast cancer cell proliferation and transformation in vitro and tumorigenicity in vivo. Consistently, ZNF668 knockdown readily transforms normal mammary epithelial cells. Together, our studies identify ZNF668 as a novel breast tumor suppressor gene that acts at least in part by regulating the stability and function of p53. (2) ZNF668 functions as a DNA repair protein by regulating histone acetylation. DNA repair proteins need to access the chromatin by chromatin modification or remodeling to use DNA template within chromatin. Dynamic posttranslational modifications of histones are critical for cells to relax chromatin in DNA repair. However, the precise underlying mechanism mediating enzymes responsible for these modifications and their recruitment to DNA lesions remains poorly understood. We observed ZNF668 depletion causes impaired chromatin relaxation as a result of impaired DNA-damage induced histone H2AX hyper-acetylation. This results in the decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after DNA damage, albeit with the presence of a functional ATM/ATR dependent DNA-damage signaling cascade. Importantly, the impaired loading of repair proteins and the defect in DNA repair in ZNF668-deficient cells can be counteracted by chromatin relaxation, indicating that the DNA-repair defect that was observed in the absence of ZNF668 is due to impeded chromatin accessibility at sites of DNA breaks. Our findings therefore identify ZNF668 as a key molecule that links chromatin relaxation with response to DNA damage in the control of DNA repair.

CHROMOSOMAL ORIGIN OF DM-DNA

Double minutes (dm) are small chromatin particles of 0.3 microns diameter found only in the metaphase cells of human and murine tumors. Dm are unique cytogenetic structures since their numbers per cell show wide variation. At cell division, dm are retained despite the lack of centromeres. In squash preparations, dm show clustering often in association with chromosomes. Human carcinoma cell line SW613-S18 was found to have large numbers of dm and biological characteristics favorable for mitotic synchronization and chromosome isolation experiments.^ S18 cells were synchronized to mitosis with metabolic and mitotic blocking compounds. Mitotic cells were lysed to release chromosomes and dm from the mitotic spindle and the resulting suspensions were fractionated to enrich for dm. The DNA in enriched fractions was characterized. The reassociation kinetics of dm-DNA driven with placental human DNA was similar to the reassociation curve of labeled placental DNA under similar conditions. In situ hybridization of dm-DNA to tumor and normal metaphase cells showed grain localization over the entire karyotype. Dm-DNA was shown by pulse chase DNA replication experiments to replicate during early and mid S-phase of the cell cycle, but not in late S-phase. In addition, BrdUrd incorporation studies showed that dm-DNA replicates only once during the S-phase. Premature chromosome condensation studies suggest the basis of numerical heterogeneity of dm is nondisjunction, not anomalous or unscheduled DNA replication.^ These data and previous cytochemical banding studies of dm in SW613-S18 indicate that dm-DNA is chromosomal in origin. No evidence of gene amplification was found in the DNA reassociation data. It is likely that dm-DNA represents the pale-staining G-band regions of the human karyotype in this cell line. ^

Left ventricular growth from age 8 to 18 and its anthropometric determinants: Longitudinal results from Project Heartbeat!

Left ventricular mass (LVM) is a strong predictor of cardiovascular disease (CVD) in adults. However, normal growth of LVM in healthy children is not well understood, and previous results on independent effects of body size and body fatness on LVM have been inconsistent. The purpose of this study was (1) to establish the normal growth curve of LVM from age 8 to age 18, and evaluate the determinants of change in LVM with age, and (2) to assess the independent effects of body size and body fatness on LVM.^ In Project HeartBeat!, 678 healthy children aged 8, 11 and 14 years at baseline were enrolled and examined at 4-monthly intervals for up to 4 years. A synthetic cohort with continuous observations from age 8 to 18 years was constructed. A total of 4608 LVM measurements was made from M-mode echocardiography. The multilevel linear model was used for analysis.^ Sex-specific trajectories of normal growth of LVM from age 8 to 18 was displayed. On average, LVM was 15 g higher in males than females. Average LVM increased linearly in males from 78 g at age 8 to 145 g at age 18. For females, the trajectory was curvilinear, nearly constant after age 14. No significant racial differences were found. After adjustment for the effects of body size and body fatness, average LVM decreased slightly from age 8 to 18, and sex differences in changes of LVM remained constant.^ The impact of body size on LVM was examined by adding to a basic LVM-sex-age model one of 9 body size indicators. The impact of body fatness was tested by further introducing into each of the 9 LVM models (with one or another of the body size indicators) one of 4 body fatness indicators, yielding 36 models with different body size and body fatness combinations. The results indicated that effects of body size on LVM can be distinguished between fat-free body mass and fat body mass, both being independent, positive predictors. The former is the stronger determinant. When a non-fat-free body size indicator is used as predictor, the estimated residual effect of body fatness on LVM becomes negative. ^

Bayesian and survival model for tumor relapse status and disease-specific survival, with applications to breast cancer

Breast cancer is the most common non-skin cancer and the second leading cause of cancer-related death in women in the United States. Studies on ipsilateral breast tumor relapse (IBTR) status and disease-specific survival will help guide clinic treatment and predict patient prognosis.^ After breast conservation therapy, patients with breast cancer may experience breast tumor relapse. This relapse is classified into two distinct types: true local recurrence (TR) and new ipsilateral primary tumor (NP). However, the methods used to classify the relapse types are imperfect and are prone to misclassification. In addition, some observed survival data (e.g., time to relapse and time from relapse to death)are strongly correlated with relapse types. The first part of this dissertation presents a Bayesian approach to (1) modeling the potentially misclassified relapse status and the correlated survival information, (2) estimating the sensitivity and specificity of the diagnostic methods, and (3) quantify the covariate effects on event probabilities. A shared frailty was used to account for the within-subject correlation between survival times. The inference was conducted using a Bayesian framework via Markov Chain Monte Carlo simulation implemented in softwareWinBUGS. Simulation was used to validate the Bayesian method and assess its frequentist properties. The new model has two important innovations: (1) it utilizes the additional survival times correlated with the relapse status to improve the parameter estimation, and (2) it provides tools to address the correlation between the two diagnostic methods conditional to the true relapse types.^ Prediction of patients at highest risk for IBTR after local excision of ductal carcinoma in situ (DCIS) remains a clinical concern. The goals of the second part of this dissertation were to evaluate a published nomogram from Memorial Sloan-Kettering Cancer Center, to determine the risk of IBTR in patients with DCIS treated with local excision, and to determine whether there is a subset of patients at low risk of IBTR. Patients who had undergone local excision from 1990 through 2007 at MD Anderson Cancer Center with a final diagnosis of DCIS (n=794) were included in this part. Clinicopathologic factors and the performance of the Memorial Sloan-Kettering Cancer Center nomogram for prediction of IBTR were assessed for 734 patients with complete data. Nomogram for prediction of 5- and 10-year IBTR probabilities were found to demonstrate imperfect calibration and discrimination, with an area under the receiver operating characteristic curve of .63 and a concordance index of .63. In conclusion, predictive models for IBTR in DCIS patients treated with local excision are imperfect. Our current ability to accurately predict recurrence based on clinical parameters is limited.^ The American Joint Committee on Cancer (AJCC) staging of breast cancer is widely used to determine prognosis, yet survival within each AJCC stage shows wide variation and remains unpredictable. For the third part of this dissertation, biologic markers were hypothesized to be responsible for some of this variation, and the addition of biologic markers to current AJCC staging were examined for possibly provide improved prognostication. The initial cohort included patients treated with surgery as first intervention at MDACC from 1997 to 2006. Cox proportional hazards models were used to create prognostic scoring systems. AJCC pathologic staging parameters and biologic tumor markers were investigated to devise the scoring systems. Surveillance Epidemiology and End Results (SEER) data was used as the external cohort to validate the scoring systems. Binary indicators for pathologic stage (PS), estrogen receptor status (E), and tumor grade (G) were summed to create PS+EG scoring systems devised to predict 5-year patient outcomes. These scoring systems facilitated separation of the study population into more refined subgroups than the current AJCC staging system. The ability of the PS+EG score to stratify outcomes was confirmed in both internal and external validation cohorts. The current study proposes and validates a new staging system by incorporating tumor grade and ER status into current AJCC staging. We recommend that biologic markers be incorporating into revised versions of the AJCC staging system for patients receiving surgery as the first intervention.^ Chapter 1 focuses on developing a Bayesian method to solve misclassified relapse status and application to breast cancer data. Chapter 2 focuses on evaluation of a breast cancer nomogram for predicting risk of IBTR in patients with DCIS after local excision gives the statement of the problem in the clinical research. Chapter 3 focuses on validation of a novel staging system for disease-specific survival in patients with breast cancer treated with surgery as the first intervention. ^