Oocyte maturation (OM) in teleosts is under precise hormonal control by estrogens and progestins. We show here that estrogens activate an epidermal growth factor receptor (EGFR) signaling pathway through the G protein-coupled estrogen receptor (GPER) to maintain meiotic arrest of full-grown zebrafish (Danio rerio) oocytes in an in vitro germinal vesicle breakdown (GVBD) bioassay. A GPER- specific agonist decreased OM and a GPER-specific antagonist increased spontaneous OM, whereas specific nuclear estrogen receptor (ERα and ERβ) agonists did not affect OM, which suggests the inhibitory action of estrogens on OM are solely mediated through GPER. Furthermore, a peptide-bound estrogen, which cannot enter the oocyte, decreased GVBD, showing that these estrogen actions are mediated through a membrane receptor. Treatment of oocytes with actinomycin D, a transcription inhibitor, did not block the inhibitory effects of estrogens on OM, indicating that estrogens act via a nongenomic mechanism to maintain oocyte meiotic arrest. EGFR mRNA was detected in denuded zebrafish oocytes by reverse transcription polymerase chain reaction (RT-PCR). Therefore, the potential role of transactivation of EGFR in estrogen inhibition of OM was investigated. The matrix metalloproteinase inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor (HB-EGF), increased spontaneous OM. Moreover, specific EGFR1 (ErbB1) inhibitors and inhibitors of extracellular-related kinase 1 and 2 (ERK1/2) increased spontaneous OM. Previously, estrogens have been shown to increase 3’-5’-cyclic adenosine mono phosphate (cAMP) levels through GPER in zebrafish oocytes during meiotic arrest. Taken together these present results suggest that estrogens also act through GPER to maintain meiotic arrest through a second signaling pathway involving transactivation of EGFR and activation of ERK 1 and 2. / text

The introduction of tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) inhibitors to the clinic has resulted in an improvement in the treatment of non small cell lung cancer (NSCLC). However, many patients treated with EGFR TKIs do not respond to therapy. The burden of failed treatment is largely placed on the healthcare field, limiting the effectiveness of EGFR TKIs. Furthermore, responses are hindered by the emergence of resistance. Thus, two questions must be addressed to achieve maximum benefit of EGFR inhibitors: How can patients who will benefit from EGFR TKIs be selected a priori? How can patients who respond achieve maximal benefit? To answer these questions, two hypotheses were formed. First, the EGFR-dependent phenotype, which is displayed by the tumors cells of those patients who respond clinically to EGFR TKIs, can be captured by genomic profiling of NSCLC cell lines stratified by sensitivity to EGFR TKIs. This gene signature may be used to predict the outcome of EGFR TKI therapy in unknown samples. Secondly, the predictive signature of response to EGFR TKI could provide insights into the underlying biology of the phenotype of EGFR-dependency. This information could be exploited to identify inhibitors which could be combined with EGFR inhibitors to elicit a greater effect, thereby minimizing resistance. The work herein describes the testing of these hypotheses. Pharmacogenomics was utilized to define a signature of EGFR-dependency which effectively predicted response to EGFR TKI in vitro and in vivo. Furthermore, the signature was analyzed by bioinformatic approaches to identify the RAS/MAPK pathway as a candidate target in EGFR-dependent NSCLC. The RAS/MAPK pathway regulates expression and activation of EGF-like ligands. Furthermore, the RAS/MAPK pathway modulates EGFR stability in the EGFR-dependent phenotype. Further biochemical analyses demonstrated that the RAS/MAPK pathway mediates proliferation and survival of EGFR-dependent NSCLC cells. Finally, combinatorial treatment of EGFR-dependent NSCLC cell lines with small molecules targeting EGFR and the RAS/MAPK pathway yielded cytotoxic synergy. Thus, we have used pharmacogenomics methods to potentially improve NSCLC treatment by developing a method of predicting response and identifying an additional target to combine with EGFR TKIs to maximize responses.

Feline oral squamous cell carcinomas (FOSCCs) are locally aggressive tumours and a common cause of mortality and morbidity. Current treatment options are rarely successful and animals are frequently euthanised upon diagnosis due to their grave prognosis. Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase receptor which is frequently dysregulated in SCC of the head and neck (HNSCC) in man. Recent advances in human medicine have identified EGFR as a therapeutic target in HNSCC. In this study the role of EGFR in FOSCC was investigated. Sixty seven biopsy samples were immunohistochemically labelled for EGFR and Ki67, a proliferation marker. The tyrosine kinase region of feline EGFR was cloned and sequenced, and six small interfering RNAs (siRNAs) targeting the tyrosine kinase region were developed. The most effective siRNA as well as an EGFR specific tyrosine kinase inhibitor, gefitinib, was then used on a feline SCC cell line (SCCF1), and the effect of EGFR targeting alone, or in combination with irradiation, on the cell line was determined. The majority of the biopsy samples were labelled positively for EGFR and Ki67, and high proliferation corresponded with poor prognosis. The siRNA caused reduction in EGFR mRNA by Real-Time Polymerase Chain Reaction and protein levels as assessed by western blot analysis. Reduced cell proliferation and migration were also observed by proliferation assays and scratch assays respectively. Combining EGFR knockdown with irradiation caused an additive effect on the ability of the cell line to form colonies. These results support the role of EGFR as a potential therapeutic target in FOSCCs.

Cell polarity is an important regulator of cellular processes and is vital in helping to prevent metaplasia and tumorigenesis. There are three many polarity complexes that regulate and maintain epithelial cellular polarity. The Par and Crumbs complexes locate to the apical membrane of the cell, while the Scribble complex is located basolaterally. Of the Scribble complex components, the polarity protein Hugl1, also known as Mgl1 in mice, is especially important in helping to maintain apical basolateral and planar polarity, and is lost in multiple types of cancer. When Hugl1 expression is lost in epithelial cells, it results in a mesenchymal phenotype. We now show that the loss of Hugl1 fundamentally shifts the cellular phenotype and specifically alters EGFR trafficking and signaling. Loss of Hugl1 results in the nuclear translocation of Taz and Slug, increased migration, and the mislocalization of EGFR (Epidermal Growth Factor Receptor), driving cellular growth. Hugl1 regulates the expression of multiple cell identity markers and its loss results in stem cell characteristics, including the increased expression of CD44, and a decrease of CD49f and CD24 expression. The loss of Hugl1 also results in increased growth in soft-agar and prolonged survival when transplanted into NOD-SCID mice; its loss also results in EGF-dependent migration which aids in increasing mammosphere survival. Furthermore, isolated EGFR mislocalization via a point mutation (P667A) also drives these same phenotypes, including activation of Akt and Taz nuclear translocation, indicating the importance of Hugl1 in the regulation of EGFR localization and its signaling. In mice, the loss of total Mgl1 is lethal within days of birth due to hydrocephaly and results in the formation of rosette like structures in the brain that are reminiscent of neuroectodermal tumors. We designed a targeted Mgl1 knockout in the mammary epithelial cells using the Cre/Lox system to evaluate the effects of Mgl1 loss in murine mammary gland development and tumorigenesis. The loss of Mgl1 expression in mice inhibits ductal outgrowth, increases side branching and epithelial layers, and results in the mislocalization of EGFR. While overt mammary tumors did not develop, some individuals did develop hyperplastic nodules that could progress into cancer. The knockdown of Hugl1 in vitro and Mgl1 in vivo reveal how the loss of polarity and presence of Hugl1 results in cancer stem cell characteristics, increased migration, and abnormal signaling due to the mislocalization of EGFR. While these changes result in metaplasia and a potential pre-cancerous state, the loss of Hugl1 alone is not enough to drive the cancer progression, indicating that other mutations or factors are necessary for the development of breast cancer. Because of the key role polarity plays in the prevention of breast cancer development we investigated if the addition of Hugl1 back into breast cancer cells could revert the cancerous cells to a normal epithelial phenotype. Most of the breast cancer cells transfected with Hugl1 expression did not survive, indicating that the re-expression of polarity regulators forces cancer cells to die. The small percentage of cells that did survive re-expression of Hugl1 had retarded growth in soft agar and a decrease in EGFR expression. Together, these data indicate that Hugl1 expression and EGFR activity are closely related and that Hugl1 is required for the proper localization and signaling of EGFR. When Hugl1 is lost, EGFR is mislocalized and fails to be degraded properly, promoting pre-neoplastic changes.

Mechanistic insights on molecular and cellular mechanisms whereby KSHV induces Kaposi?s sarcoma (KS) are key for our understanding of KS tumors and for the development of new therapies. We have previously developed an animal model for KSHV induced KS using murine bone marrow cells transfected with a KSHVBac36. We found that although these cells lacked attributes of transformed cells in vitro, they were able to cause KS-like tumors in vivo. In vivo tumorigenesis correlated with upregulation of both KSHV lytic genes and host angiogenesis suggesting that that cues provided from the microenvironment played a major role in regulating viral and host gene expression related with KSHV-induced tumorigenesis. Our goal thus, was to identify these molecular cues regulating pathogenic gene expression in KSHV infected cells in vivo. An important difference between cells kept in vitro versus in vivo is the lack of environmental extracellular matrix (ECM) signals. Therefore the mECK36 cells were cultured in vitro in matrigel, a basement membrane preparation rich in ECM proteins and its individual components, to discern the effect of host signaling by the ECM on KSHV infected cells. Investigation of gene expression through Real Time RT-PCR identified several viral and host genes associated with tumorigenesis such as KSHV vGPCR and angiogenesis associated VEGF and EGF- receptors were upregulated in response to this environment. Further analysis of the molecular activity of the cell indicated the change in transcription was due to the activation of integrin signaling, as assessed by phosphorylation of the Focal Adhesion Kinase (FAK) protein. Our results show that integrin signaling occurring in vivo through interaction with ECM serves to enhance the pathogenic viral and host gene expression of KSHV infected cells and that EGFR upregulation can be correlated with these conditions. These results points to the integrin signaling pathway or the EGF-Receptor as promising targets for therapy and prevention of KS tumors.

Most of tumor consists of a heterogeneous population of tumor cells among a tumor initiating and chemo or radiation resistant subpopulation, called cancer stem cells (CSCs), which have become increasingly important new anticancer targets. CD133 has been recently identified as a prominent marker for CSCs in pancreatic and other tumors; however, the signaling cascade of this cancer stem cell marker has not been fully explored. This study shows increased cell proliferation, colony formation, adhesion, and migration following CD133 overexpression in pancreatic ductal adenocarcinoma (PDAC) cells. Signaling studies have indicated that CD133 overexpression increases the epidermal growth factor receptor (EGFR) activation and phosphorylation of PI3K/Akt and MAPK/ ERK pathways. An in vivo xenograft study confirmed that overexpression of CD133 has higher tumorgentic ability than control mice. Molecular studies have found that CD133 physically associates with EGFR and promotes EGFR protein level and its phosphorlyation, which might be critical for PDAC tumor progression and chemoresistance. The data also showed that CD133 overexpression suppresses the EGF mRNA expression, which may imply that CD133 induces EGFR activation through an EGF ligand-independent process. The findings here point to an important mechanism of action for CD133 in PDAC. The EGFR inhibitor has potent anti-CD133 activity, and the current results have important implications for developing targeting CD133 activity as a novel cancer therapy strategy and the inhibitor approach presented here identifies the inhibition of CD133 activity by the EGFR inhibitor and sheds light on developing a new cancer therapeutic that functions by targeting CD133 activity in human cancer.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on Feb. 18, 2010). Includes bibliographical references.

Cancer has become one of the leading causes of death today. The early detection of cancer may lead to desired therapeutic management of cancer and to decrease the mortality rate through effective therapeutic strategies. Advances in materials science have enabled the use of nanoparticles for added contrast in various imaging techniques. More recently there has been much interest in the use of gold nanoparticles as optical contrast agents because of their strong absorption and scattering properties at visible and near-infrared wavelengths. Highly proliferative cancer cells overexpress molecular markers such as epidermal growth factor receptor (EGFR). When specifically targeted gold nanoparticles bind to EGFR they tend to cluster thus leading to an optical red-shift of the plasmon resonances and an increase in absorption in the red region. These changes in optical properties provide the foundation for photoacoustic imaging technique to differentiate cancer cells from surrounding benign cells. In photoacoustic imaging, contrast mechanism is based on the optical absorption properties of the tissue constituents. Studies were performed on tissue phantoms, ex-vivo and in-vivo tumor models to evaluate molecular specific photoacoustic imaging technique. The results indicate that highly sensitive and selective detection of cancer cells can be achieved by utilizing the plasmon resonance coupling effect of EGFR targeted gold nanoparticles and photoacoustic imaging. In conclusion, the combined ultrasound and photoacoustic imaging technique has the ability to image molecular signature of cancer using bioconjugated gold nanoparticles. / text

Lung cancer is the leading cause of cancer-related deaths among men and women. Non-Small Cell Lung Cancer (NSCLC) constitutes the most common type of lung cancer and is frequently diagnosed at advanced stages. In the past decade, discovery of Epidermal Growth Factor Receptor (EGFR) mutations have heralded a new paradigm of personalized treatment for NSCLC. Clinical studies have shown that molecular targeted therapies increase survival and improve quality of life in patients. Despite these advances, the realization of personalized therapies for NSCLC faces a number of challenges including the integration of clinical and genetic data and a lack of clinical decision support tools to assist physicians with patient selection. This thesis demonstrates the development of a predictive computational model for personalized therapeutic interventions in advanced NSCLC. The findings suggest that the combination of clinical and genetic data significantly improves the model’s predictive performance for tumor response than clinical data alone.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 132-165.