ABERRATIONS OF A PUTATIVE TUMOR SUPPRESSOR GENE SEL1L IN PANCREATIC DUCTAL ADENOCARCINOMA

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death among males and females in the United States. Sel-1-like (SEL1L) is a putative tumor suppressor gene that is downregulated in a significant proportion of human pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that SEL1L expression could be down-modulated by somatic mutation, loss of heterozygosity (LOH), CpG island hypermethylation and/or aberrantly expressed microRNAs (miRNAs). Material and methods: In 42 PDAC tumors, the SEL1L coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR), and analyzed by agarose gel electrophoresis and sequenced to search for mutations. Using fluorescent fragment analysis, two intragenic microsatellites in the SEL1L gene region were examined to detect LOH in a total of 73 pairs of PDAC tumors and normal-appearing adjacent tissues. Bisulfite DNA sequencing was performed to determine the methylation status of the SEL1L promoter in 41 PDAC tumors and 6 PDAC cell lines. Using real-time quantitative PCR, the expression levels of SEL1L mRNA and 7 aberrantly upregulated miRNAs that potentially target SEL1L were assessed in 42 PDAC tumor and normal pairs. Statistical methods were applied to evaluate the correlation between SEL1L mRNA and the miRNAs. Further the interaction was determined by functional analysis using a molecular biological approach. Results: No mutations were detected in the SEL1L coding region. More than 50% of the samples displayed abnormally alternate or aberrant spliced transcripts of SEL1L. About 14.5% of the tumors displayed LOH at the CAR/CAL microsatellite locus and 10.7% at the RepIN20 microsatellite locus. However, the presence of LOH did not show significant association with SEL1L downregulation. No methylation was observed in the SEL1L promoter. Statistical analysis showed that SEL1L mRNA expression levels significantly and inversely correlated with the expression of hsa-mir-143, hsa-mir-155, and hsa-mir-223. Functional analysis indicated that hsa-mir-155 acted as a suppressor of SEL1L in PL18 and MDAPanc3 PDAC cell lines. Discussion: Evidence from these studies suggested that SEL1L was possibly downregulated by aberrantly upregulated miRNAs in PDAC. Future studies should be directed towards developing a better understanding of the mechanisms for generation of aberrant SEL1L transcripts, and further analysis of miRNAs that may downregulate SEL1L.

THE ROLE OF RECEPTOR TYROSINE KINASE AXL IN PANCREATIC DUCTAL ADENOCARCINOMA AND ITS REGULATION BY HEMATOPOIETIC PROGENITOR KINASE 1

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies with less than 5% of five year survival rate. New molecular markers and new therapeutic targets are urgently needed for patients with PDA. Oncogenic receptor tyrosine kinase Axl has been reported to be overexpressed in many types of human malignancies, including diffuse glioma, melanoma, osteosarcoma, and carcinomas of lung, colon, prostate, breast, ovary, esophagus, stomach, and kidney. However, the expression and functions of Axl in PDA are unclear. We hypothesized that Axl contributes to the development and progression of PDA. We examined Axl expression in 54 human PDA samples and their paired benign pancreatic tissue by immunohistochemistry, we found that Axl was overexpressed in 70% of stage II PDAs, but only 22% of benign ducts (P=0.0001). Axl overexpression was associated with higher frequencies of distant metastasis and was an independent prognostic factor for both poor overall and recurrence-free survivals in patients with stage II PDA (p = 0.03 and 0.04). Axl silencing by shRNA in pancreatic cancer cell lines, panc-28 and Panc-1, decreased tumor cell migration and invasion and sensitized PDA cells to apoptosis stimuli such as γ-irradiation and serum starvation. In addition, we found that Axl-mediated Akt and NF-κB activation and up regulation of MMP2 were involved in the invasion, migration and survival of PDA cells. Thus, we demonstrate that Axl plays an important role in the development and progression of PDA. Targeting Axl signaling pathway may represent a new approach for the treatment of PDA. To understand the molecular mechanisms of Axl overexpression in PDA, we found that Axl expression was down-regulated by hematopoietic progenitor kinase 1 (HPK1), a newly identified tumor suppressor in PDA. HPK1 is lost in over 95% of PDAs. Restoration of HPK1 in PDA cells down-regulated Axl expression. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1, and monensin, suggesting that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway. HPK1 interacted with and phosphorylated dynamin, a critical component of endocytosis pathway. Overexpression of dominant negative form of dynamin blocked the HPK1-mediated Axl degradation. Therefore we concluded that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway and loss of HPK1 expression may contribute to Axl overexpression in PDAs.

Proliferation index in ductal carcinoma in situ of the breast: Relation to estrogen, progesterone and HER2/neu receptors

Background. Ductal carcinoma in situ (DCIS) is the most prevalent precursor to invasive breast cancer (IBC), the second leading cause of death in women in the United States. The three most important prognostic markers for IBC are Estrogen receptor (ER), Progesterone receptor (PR) and HER2/neu. The four groups (IBC) defined as (1) ER and/or PR positive and HER2/neu negative, (2) ER and/or PR positive and HER2/neu positive (3) ER and/or PR negative and HER2/neu positive and (4) negative for all three of these receptors (Triple negative). However, they have not been well studied in DCIS. This is an exploratory study with a primary objective to examine the prevalence of ER, PR, and HER2/neu in DCIS, to explore if the defined groups of IBC occur in DCIS and to consider the biological relationship between these four groups and the proliferative activity of the tumor. A secondary goal of this study is to examine the relationship between grade and proliferative activity. Methods. Using immunohistochemistry, I have measured Ki-67, ER, PR and HER2/neu positivity for a series of cases of DCIS. Results. 20 ER and/or PR positive and HER2/neu negative (50%) with average PI of 0.05, 7 ER and/or PR positive and HER2/neu positive (17.5%) with average PI of 0.14, 10 ER and/or PR negative and HER2/neu positive (25%) with average PI of 0.18, and three triple negative (7.5%) with average PI of 0.18. ER and/or PR positive and HER2/neu positive group has the highest PI (p<0.001). Further, the ER and/or PR positive and HER2/neu positive group show a linear relationship between PI and average ER/PR positivity (R=0.6). PI increases with higher grades. Conclusion. PI appears to depend upon the average fraction of positive ER/PR tumor cells, possibly with a synergistic dependence when HER2/neu is positive. If ER/PR is negative, then both HER2/neu positive and the triple negative cases appear to cluster around an average PI that is higher than the average PI in HER2/neu negative ER/PR positive negative cases. In the triple negative tumors there must be another driver of proliferation.^

Role and Regulation of EphA2 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cancer cause of death in the US. Gemcitabine is the first-line therapy for this disease, but unfortunately it shows only very modest benefit. The focus of the current study was to investigate the role and regulation of EphA2, a receptor tyrosine kinase expressed in PDAC, to further understand this disease and identify new therapeutic targets. The role of EphA2 was determined in PDAC by siRNA mediated silencing. In combination with gemcitabine, silencing of EphA2 caused a dramatic increase in apoptosis even in highly resistant cells in vitro. Furthermore, EphA2 silencing was found to be useful in 2 orthotopic models in vivo: 1) shRNA-pretreated Miapaca-2 cells, and 2) in vivo delivery of siRNA to established MPanc96 tumors. Silencing of EphA2 alone reduced tumor growth in Miapaca-2 cells. In MPanc96, only the combination treatment of gemcitabine plus siEphA2 significantly reduced tumor growth, as well as the number of lung and liver metastases. Taken together, these observations support EphA2 as a target for combination therapies for PDAC. The regulation of EphA2 was further explored with a focus on the role of Ras. K-Ras activating mutations are the most important initiating event in PDAC. We demonstrated that Ras regulates EphA2 expression through activation of MEK2 and phosphorylation of ERK. Downstream of ERK, silencing of the transcription factor AP-1 subunit c-Jun or inhibition of the ERK effector RSK caused a decrease in EphA2 expression, supporting their roles in this process. Further examination of Ras/MEK/ERK pathway modulators revealed that PEA-15, a protein that sequesters ERK to the cytoplasm, inhibited expression of EphA2. A significant inverse correlation between EphA2 and PEA-15 levels was observed in mouse models of PDAC. In cells where an EGFR inhibitor reduced phospho-Erk, expression of EphA2 was also reduced, indicating that changes in EphA2 levels may allow monitoring the effectiveness of anti-Ras/MEK/ERK therapies. In conclusion, EphA2 levels may be a good prognostic factor for anti-EGFR/anti-Ras therapies, and EphA2 itself is a relevant target for the development of new therapies.

Altered Responses to Endoplasmic Reticulum Stress in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth most common cause of cancer-associated death in the United States. Little progress has been made in understanding how proteotoxic stress affects rapidly proliferating pancreatic tumor cells. Endoplasmic reticulum (ER) stress occurs when protein homeostasis in the ER lumen is perturbed. ER stress activates the unfolded protein response (UPR) to reduce the protein load in the ER. Under conditions of moderate ER stress, the UPR promotes cell cycle arrest which allows time for successful protein load reduction and enables cell survival. However, under conditions of high levels of ER stress the UPR induces cellular apoptosis. In this dissertation, I investigated the role of endoplasmic reticulum (ER) stress and its effects on the cell cycle in pancreatic cancer cells. Activation of the unfolded protein response after ER stress induction was determined by comparing expression of key UPR mediators in non-tumorigenic pancreatic ductal cells to pancreatic cancer cells. Two arms of the UPR were assessed: eIF2α/ATF4/CHOP and IRE1α/XBP1s. Pancreatic cancer cells exhibited altered UPR activation characterized by a delay in both phosphorylation of eIF2α and induction of spliced XBP1. Further evaluation of the UPR-mediated effects on cell cycle progression revealed that pancreatic cancer cells showed a compromised ability to inhibit G1 to S phase progression after ER stress. This reduced ability to arrest proliferation was found to be due to an impaired ability to downregulate cyclin D1, a key gatekeeper of the G1/S checkpoint. Abrogation of cyclin D1 repression was mediated through a slow induction of phosphorylation of eIF2α, a critical mediator of translational attenuation in response to ER stress. In conclusion, pancreatic cancer cells demonstrate a globally compromised ability to regulate the unfolded protein response. This deficiency results in reduced cyclin D1 repression through an eIF2α-mediated mechanism. These findings indicate that pancreatic cancer cells have increased tolerance for elevated ER stress compared to normal cells, and this tolerance results in continued tumor cell proliferation under proteotoxic conditions.

THE MECHANISM OF TUMORIGENESIS IN THE IMMORTALIZED HUMAN PANCREATIC CELL LINES: CELL CULTURE MODELS OF HUMAN PANCREATIC CANCER

The mechanism of tumorigenesis in the immortalized human pancreatic cell lines: cell culture models of human pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in the world. The most common genetic lesions identified in PDAC include activation of K-ras (90%) and Her2 (70%), loss of p16 (95%) and p14 (40%), inactivation p53 (50-75%) and Smad4 (55%). However, the role of these signature gene alterations in PDAC is still not well understood, especially, how these genetic lesions individually or in combination contribute mechanistically to human pancreatic oncogenesis is still elusive. Moreover, a cell culture transformation model with sequential accumulation of signature genetic alterations in human pancreatic ductal cells that resembles the multiple-step human pancreatic carcinogenesis is still not established. In the present study, through the stepwise introduction of the signature genetic alterations in PDAC into the HPV16-E6E7 immortalized human pancreatic duct epithelial (HPDE) cell line and the hTERT immortalized human pancreatic ductal HPNE cell line, we developed the novel experimental cell culture transformation models with the most frequent gene alterations in PDAC and further dissected the molecular mechanism of transformation. We demonstrated that the combination of activation of K-ras and Her2, inactivation of p16/p14 and Smad4, or K-ras mutation plus p16 inactivation, was sufficient for the tumorigenic transformation of HPDE or HPNE cells respectively. We found that these transformed cells exhibited enhanced cell proliferation, anchorage-independent growth in soft agar, and grew tumors with PDAC histopathological features in orthotopic mouse model. Molecular analysis showed that the activation of K-ras and Her2 downstream effector pathways –MAPK, RalA, FAK, together with upregulation of cyclins and c-myc were involved in the malignant transformation. We discovered that MDM2, BMP7 and Bmi-1 were overexpressed in the tumorigenic HPDE cells, and that Smad4 played important roles in regulation of BMP7 and Bmi-1 gene expression and the tumorigenic transformation of HPDE cells. IPA signaling pathway analysis of microarray data revealed that abnormal signaling pathways are involved in transformation. This study is the first complete transformation model of human pancreatic ductal cells with the most common gene alterations in PDAC. Altogether, these novel transformation models more closely recapitulate the human pancreatic carcinogenesis from the cell origin, gene lesion, and activation of specific signaling pathway and histopathological features.

Estrogen action during early mouse mammary gland development

Estrogens have been implicated in the normal and neoplastic development of the mammary gland. Although estradiol is essential for early mammary differentiation, its role in postnatal ductal morphogenesis is poorly defined. We have found that neonatal estradiol exposure promotes precocious ductal outgrowth and terminal end bud formation in 21 day-old female mice. In contrast to this precocious phenotype, day 21 estradiol-treated epithelium, transplanted into control host fatpads, grows more slowly than control epithelium. Western and immunohistochemical (IHC) analyses indicate that neonatally-estrogenized glands have significantly less total ER than controls at days 7 and 21, and significantly more stromal ER at day 35. Estrogen receptor α (ER) is present in the gland when treatment is initiated at day 1. We propose that the premature activation of ER by neonatal estradiol exposure, during this critical perinatal period, is a key factor in the alteration of mammary growth and ER expression. ^ To address the role of ER function in mammary morphogenesis, we have developed an in vitro system to study the effect of estradiol exposure in vivo. Keratin and ER-positive mammary epithelial cell lines from 7, 21 and 35 day-old oil or estradiol treated mice have been established. Cell lines derived from estradiol-treated mice grow significantly slower than cells from control glands. Although the level of ER expressed by each cell line is correlated to its rate of growth, epithelial growth in vitro is estradiol-independent and antiestrogen-insensitive. Estradiol-induced transcription from an ERE-reporter in transiently-transfected cell lines confirms the functionality of the ER detected by western and IHC. However, there are no differences in estradiol-stimulated transcription between cell lines. ^ In conclusion, neonatal estradiol treatment alters the pattern of ER expression in mammary epithelial and stromal cells in vivo, and the growth of mammary epithelial cells in vivo and in vitro. When grown outside of the estrogenized host, exposed epithelium grows more slowly than the control. Therefore, an extra-epithelial factor is necessary for enhanced epithelial growth. Our model, which couples an in vivo-in vitro approach, can be used in the future to identify factors involved in the period of early mammary outgrowth and carcinogen susceptibility. ^

Molecular mechanisms of NF-kappaB activation in metastatic pancreatic cancer cells

Pancreatic adenocarcinoma is the fourth leading cause of adult cancer death in the United States. At the time of diagnosis, most patients with pancreatic cancer have advanced and metastatic disease, which makes most of the traditional therapeutic strategies are ineffective for pancreatic cancer. A better understanding of the molecular basis of pancreatic cancer will provide the approach to identify the new strategies for early diagnosis and treatment. NF-κB is a family of transcription factor that play important roles in immune response, cell growth, apoptosis, and tumor development. We have shown that NF-κB is constitutively activated in most human pancreatic tumor tissues and cell lines, but not in the normal tissues and HPV E6E7 gene-immortalized human pancreatic ductal epithelial cells (HPDE/E6E7). By infecting the pancreatic cancer cell line Aspc-1 with a replication defective retrovirus expressing phosphorylation-defective IκBα (IκBαM), the constitutive NF-κB activation is blocked. Subsequent injection of this Aspc-1/IκBαM cells into the pancreas of athymic nude mice showed that liver metastasis is suppressed by the blockade of NF-κB activation. Current studies showed that an autocrine mechanism accounts for the constitutive activation of NF-κB in metastatic human pancreatic cancer cell lines, but not in nonmetastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1α (IL-1α) was the primary cytokine secreted by these cells that activates NF-κB. Inhibition of IL-1α activity suppressed the constitutive activation of NF-κB and the expression of its downstream target gene, uPA, in metastatic pancreatic cancer cell lines. Even though IL-1α is one of the previously identified NF-κB downstream target genes, our results demonstrate that regulation of IL-1α expression is independent of NF-κB and primarily dependent on AP-1 activity, which is in part induced by overexpression of EGF receptors and activation of MAP kinases. In conclusion, our findings suggest a possible mechanism by which NF-κB is constitutively activated in metastatic human pancreatic cancer cells and a possible missing mechanistic links between inflammation and cancer. ^

14-3-3 zeta overexpression in early stage breast disease and tumor progression

Recent progress in diagnostic tools allows many breast cancers to be detected at an early pre-invasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. 14-3-3 is a family of highly conserved and ubiquitously expressed proteins that are expressed in all eukaryotic organisms. In mammals there are seven isoforms, which bind to phosphor-serine/threonine residues regulating essential cellular processes such as signal transduction, cell cycle progression, and apoptosis. Our laboratory has discovered that a particular 14-3-3 family member, Zeta, is overexpressed in over 40% of breast tumor tissues. Furthermore, I examined the stage of breast disease in which 14-3-3ζ overexpression occurs and found that increased expression of 14-3-3ζ begins at the stage of atypical ductal hyperplasia, a very early stage of breast disease that confers increased risk for progress toward breast cancer. To determine whether 14-3-3ζ overexpression is a decisive early event in breast cancer, I overexpressed 14-3-3ζ in MCF10A cells, a non-transformed mammary epithelial cell (MEC) line and examined its impact on acini formation in a three dimensional (3D) culture model which simulates a basic unit of structure in the mammary gland. I discovered that 14-3-3ζ overexpression severely disrupted the acini architecture resulting in the disruption of polarity and luminal filling. Both are critical morphological events in the pre-neoplastic breast disease. This thesis focuses on the molecular mechanism of luminal filling. Proper lumen formation is a result of anoikis, a specific type apoptosis of cells not attached to the basement membrane. I found that 14-3-3ζ overexpression conferred a resistance to anoikis. Additionally, 14-3-3ζ overexpression in MCF10A cells and in MECs from 14-3-3ζ transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3ζ induced hyperactivation of the PI3K/Akt pathway which led to phosphorylation and translocation of the MDM2 to the nucleus resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3ζ overexpressing MCF10A acini in 3D cultures. These data suggest that 14-3-3ζ overexpression is a critical event in early breast disease and down-regulation of p53 is one of the mechanisms by which 14-3-3ζ alters MEC acini structure and may increase the risk of progression to breast cancer. ^

Establishment of a complex inflammatory and protumorigenic microenvironment in mouse pancreas through cyclooxygenase-2 overexpression

Chronic inflammation is an established risk factor in the pathogenesis of many cancers. Pancreatic ductal adenocarcinoma, a malignancy with a particularly dismal prognosis, is no exception. Cyclooxygenase-2, a key enzyme induced by tissue injury, has a critical role in the generation of bioactive lipids known as prostaglandins. COX-2 overexpression is a frequent finding in pancreatic cancer, chronic pancreatitis and pancreatic intraepithelial neoplasias. To explore mechanisms through which chronic inflammation establishes and maintains a protumorigenic environment, we designed a mouse model overexpressing COX-2 in pancreatic parenchyma (BK5.COX-2 mice). We discovered that constitutive expression of COX-2 has a number of important sequelae, including upregulation of additional eicosanoid-generating enzymes and proinflammatory cytokines. Many of these molecular alterations precede the onset of significant histopathological changes. Increased levels of prostaglandins E2, D2, and F2α, 5-, 12-, and 15-hydroxyeiosatetraenoic acid (HETEs) were documented in tumors and pancreata of younger transgenic mice. Using a TaqMan™ Mouse Immune Panel, we detected elevated mRNAs for a number of proinflammatory cytokines (e.g., TNFα, IL-1β, IL-6). ^ Histological examination revealed early changes in the pancreas with similarities to human chronic pancreatitis, including loss of acinar cells, appearance of metaplastic ducts, and increased deposition of stroma. As the lesions progress, features typical of dysplastic and neoplastic cells emerged within the metaplastic ductal complexes, including cellular and nuclear atypia, crowding of cells, and loss of normal tissue architecture. The amount of fibroinflammatory stroma increased considerably; numerous small vessels were evident. A number of immunocytes from both the myeloid and lymphoid lineages were identified in transgenic pancreata. Neutrophils were the earliest to infiltrate, followed shortly by macrophages and mast cells. B and T cells generally began to appear by 8–12 weeks, and organized aggregates of lymphoid cells were often found in advanced lesions. ^ We tested the efficacy of several chemopreventive agents in this model, including celecoxib, a COX-2 selective inhibitor, pentoxifylline, a cytokine inhibitor, curcumin, a polyphenol with antioxidant and anti-inflammatory properties, and GW2974, a dual EGFR/ErbB2 inhibitor. Effects on lesion development were modest in the GW2974 and pentoxifylline treated groups, but significant prevention effects were observed with curcumin and celecoxib. ^

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. ^

Breast cancer risk with aromatase inhibition and phytoestrogen-free diet

In this thesis a mouse model was used to examine the effect of pubertal estrogen inhibition and a phytoestrogen-free diet on the development of mammary glands. The study question was does treatment with aromatase inhibitor during puberty increase susceptibility to breast cancer among cohorts that consumed a diet free of phytoestrogens. The study design consisted of a cohort of mice treated with aromatase inhibitor, letrozole, during puberty and a vehicular group that was used as a control. Both groups were fed a diet free of phytoestrogens from the time of weaning until sacrifice during adulthood. The study aimed to assess mammary gland development in terms of breast cancer risk. The methods employed in this research included morphological and histological analysis of mammary glands, as well as estradiol, RNA and protein analysis. The main finding of the study was that mice exposed to aromatase inhibitor during puberty developed mammary glands with specific characteristics suggestive of vulnerability to oncogenesis such as increased lateral branching, increased number of glands, increase ductal hyperplasia, and diminished expression of TGFβ and p27 protein levels. The conclusions suggest that puberty is a critical period in which the mammary gland is susceptible to environmental threats that may result in deleterious epigenetic effects leading to an increased breast cancer risk in adulthood. This study has several public health implications; the most significant is that environmental threats during puberty may result in adverse mammary gland development and that phytoestrogen sources in the diet are necessary for normal maturation of the mammary glands.^

EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE Publication No.________Kiera Rycaj, B.S.Supervisory Professor: Feng Wang-Johanning, Ph.D., M.D. Ovarian cancer (OC) is the fourth most common cancer in women, and the most lethal gynecologic malignancy in the United States. Adequate screening methodologies are currently lacking and most women first present with either stage III or IV disease. To date, there has been no substantial decrease in death rates and the majorities of patients relapse and die from their disease despite response to first-line therapy. Several proteins, such as CA-125, are elevated in OC, but none has proven specific and sensitive enough to serve as a screening tool or for tumor cell recognition and lysis. It has been proposed that human endogenous retrovirus sequences (HERVs) may play a role in the etiology of certain cancers. In a previous study, we showed that HERV-K envelope (env) proteins are widely expressed in human invasive breast cancer (BC) and ductal carcinoma in situ (DCIS), and elicit both serologic and cell-mediated immune responses in BC patients. We also reported the expression of multiple HERV genes and proteins in OC cell lines and tissues. In this study, we strengthened our previous data by determining that HERV-K env mRNAs are expressed in 69% of primary OC tissues (n=29), but in only 24% of benign tissues (N=17). Immmunohistochemistry (IHC) staining revealed HERV-Kpositivecancer cells detected in endometrioid adenocarcinoma and serous adenocarcinoma but not in benign cyst or normal epithelium biopsies. Immunofluorescence staining (IFS) showed greater cell surface expression of HERV-K in OC samples compared to adjacent uninvolved samples. Enzyme-linked immunosorbent assay (ELISA) data confirmed that a humoral immune response is elicited against HERV-K in OC patients. T-cell responses against HERV-K in lymphocytes from OC patients stimulated with autologous HERV-K pulsed dendritic cells included induction of T-cell proliferation and IFN-γ production. HERV-K–specific cytolytic T cells induced greater specific lysis of OC target cells compared to benign and adjacent uninvolved target cells. Finally, upon T regulatory cell (T-reg) depletion, 64% of OC patients displayed an increase in the specific lysis of target cells expressing HERV-K env protein. These findings suggest that HERV-K env protein is a tumor-associated antigen capable of activating both T-cell and B-cell responses in OC patients, and has great potential in the development of immunotherapy regimens against OC.