Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. Development of new diagnostic tools incorporating tumor biomarkers could enhance early detection by providing molecular-level insight into the biochemical and cellular changes associated with oral carcinogenesis. The work presented in this doctoral dissertation aims to address this clinical need through the development of new automated cellular analysis methods, incorporating lab-on-a-chip sensor techniques, for examination of molecular and morphological biomarkers associated with oral carcinogenesis. Using the epidermal growth factor receptor (EGFR) as a proof-of-principle biomarker, the sensor system demonstrated capacity to support rapid biomarker analysis in less than one-tenth the time of traditional methods and effectively characterized EGFR biomarker over-expression in oral tumor-derived cell lines. Successful extension from in vitro tumor cell lines to clinically relevant exfoliative brush cytology was demonstrated, providing a non-invasive method for sampling abnormal oral epithelium. Incorporation of exfoliative cytology further helped to define the important assay and imaging parameters necessary for dual molecular and morphological analysis in adherent epithelium. Next, this new sensor assay and method was applied in a small pilot study in order to secure an initial understanding of the diagnostic utility of such biosensor systems in clinical settings. Four cellular features were identified as useful indicators of cancerous or pre-cancerous conditions including, the nuclear area and diameter, nuclear-to-cytoplasm ratio, and EGFR biomarker expression. Further examination using linear regression and ROC curve analysis identified the morphological features as the best predictors of disease while a combination of all features may be ideal for classification of OSCC and pre-malignancy with high sensitivity and specificity. Further testing in a larger sample size is necessary to validate this regression model and the LOC sensor technique, but shows strong promise as a new diagnostic tool for early detection of oral cancer. / text

Darbo tikslas nustatyti skirstymo baltymo GAB1 ryšį su anti-apoptoziniu PI3K/Akt bei mitogeniniu Ras/MAPK signalo perdavimo keliais ir įvertinti GAB1 įtaką šių kelių sąveikai EGFR signalo perdavimo tinkle. Darbo uždaviniai: įvertinti epitelinių ląstelių endogeninio GAB1 veiksmingumą EGF signalo perdavimo metu; nustatyti sąveikos pobūdį tarp PI3K/Akt ir Ras/MAPK kelių EGF signalo metu; kiekybiškai įvertinti GAB1 svarbą EGF signalo perdavimui per PI3K/Akt ir Ras/MAPK kelių in vivo, rezultatus lyginant su matematinio modelio prognozėmis in silico; nustatyti GAB1 veiksmingumo ir jo reguliacijos grįžtamaisiais ryšiais įtaką PI3K-MAPK sąveikos stiprumui priklausomai nuo EGF dozės ir laiko; ištirti GAB1 svarbą EGFR ir insulino receptoriaus signalo perdavimo tinklų sąveikai; modifikuoti Westerno pernašos metodą palyginamajai kiekybinei ir kokybinei baltymų analizei. Darbo išvados: stimuliavus EGFR, skirstymo baltymas GAB1 tampa veiksmingu; EGF lemia reciprokinę PI3K/Akt ir Ras/MAPK signalo perdavimo kelių sąveiką per GAB1; GAB1 yra pagrindinis teigiamo atgalinio ryšio elementas PI3K kelyje, padedąs pagreitinti, stiprinti ir išlaikyti MEK/ERK kinazių atsaką; PI3K-MAPK sąveikos stiprumas kinta laike ir yra atvirkščiai proporcingas EGF signalo stiprumui; GAB1 reikalingas sinergistiškai stiprinti insulinu ir mažomis EGF dozėmis stimuliuojamų ląstelių Ras/MAPK atsaką; sukurtas „Multi-juostelių“ imunopernašos metodas yra tinkamas palyginamajai kiekybinei ir kokybinei baltymų analizei... [toliau žr. visą tekstą] / The aim of the thesis was to determine a connection of endogenous docking protein GAB1 with anti-apoptotic PI3/Akt and Ras/MAPK signaling pathways and to estimate GAB1 contribution to their crosstalk in EGFR signaling network. The tasks: to evaluate GAB1 efficacy upon EGFR stimulation; to examine the nature of crosstalk between PI3K/Akt and Ras/MAPK pathways; to evaluate the contribution of GAB1 to EGF signaling via PI3K/Akt and Ras/MAPK pathways in vivo comparing the results with prognosis in silico; to estimate the EGF dose- and time-dependent impact of GAB1 efficacy and its feedback regulation on the strength of PI3K-MAPK interaction; to investigate the role of GAB1 for crosstalk of EGFR and insulin receptor signaling networks; to modify Western blotting procedure for comparative quantitative and qualitative protein analysis. The conclusions: the docking protein GAB1 is functional upon EGFR stimulation; PI3K/Akt and Ras/MAPK signaling pathways crosstalk reciprocally via GAB1 in response to EGF; GAB1 is major positive feedback element in PI3K pathway amplifying and sustaining MEK/ERK response to EGF; the strength of PI3K-MAPK interaction depends on time and is inversely proportional to EGF signal strength; GAB1 is required to synergistically potentate the Ras/MAPK response to tandem cell treatment with insulin and low EGF doses; the developed Multistrip immunoblotting method is suitable for comparative quantitative and qualitative protein analysis. In comparison with a... [to full text]

The altered protein expression and activity of receptor tyrosine kinases (TK) are implicated in the progression of various types of cancers. One such dysfunction is the overexpression of the epidermal growth factor receptor (EGFR) that correlates with aggressive tumor progression and poor prognosis. On the other hand, c-Src non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been strongly linked to progression to distant metastases. c-Src-induced phosphorylation of EGFR is required for EGF-mediated mitogenesis, tumorigenesis and tumour invasiveness. Thus we surmised that molecules termed "combi-molecules" designed to block both EGFR and c-Src should not only possess significant growth inhibitory potency but also strong anti-invasive properties. In this thesis, we utilized molecular modeling to design molecules containing two moieties: one that straddles the structure of the known Src inhibitor PP2 and the other that mimics the backbone of Iressa, a potent EGFR inhibitor. Of all the molecules synthesized, only SB163 containing the longest spacer between the two moieties was capable of inducing a dose dependent inhibition of both Src and EGFR. More importantly, SB163 blocked cell motility in the wound healing assay and showed significantly greater anti-invasive activity than a PP2+Iressa combination. The observation that SB163 was a less potent EGFR or Src inhibitor than Iressa and PP2 suggests that its superior potency when compared with the PP2+ Iressa combination may be at least partially attributed to mechanisms other than EGFR or Src blockade. This was also corroborated by the fact that SB163, despite its significant bulkiness (>700) could induce dose dependent inhibition of other kinase such PDFGR and Abl. The results in toto suggest that conferring multiple kinase targeting properties to single molecules can lead to highly anti-proliferative and anti-invasive agents. Traditionally, multi-kinase targeted molecules were discovered serendipitously through multi-kinase testing. Here we initiated a more rational approach to the design of single multi-targeted molecules. Cancer being a complex disease driven by tumours characterized by multiple disordered signaling pathways, this approach may well represent a novel avenue in the therapy of refractory malignancies.

Overall survival of the metastatic forms of epithelial derived cancers, especially head and neck squamous cell carcinomas (HNSCC), has not significantly improved even with the application of aggressive combined modality approaches incorporating radiation and chemotherapy. Cumulative evidence implicates the epidermal growth factor receptor (EGFR) as an important therapeutic target in HNSCC. We have previously demonstrated that the combination of lovastatin, a potent inhibitor of the mevalonate pathway, with EGFR tyrosine kinase inhibitors induced robust synergistic cytotoxicity. However, the use of high dose statins in our clinical trial was associated with significant toxicities including higher than anticipated rate of muscle pathologies. Our goal was to uncover novel downstream targets of the mevalonate pathway that may enhance the efficacy or limit toxicities of this novel combination therapeutic approach. In this study we have demonstrated that GGTI-298, an inhibitor of protein geranylgeranylation, through its ability to disrupt the actin cytoskeleton, inhibits EGFR dimerization and cellular trafficking. This novel mechanism targeting the EGFR has clinical implications as GGTI-298 in combination with tarceva, a clinically relevant EGFR inhibitor, showed enhanced cytotoxicity and inhibitory effects on EGFR activation and its downstream signaling.

[Truncated abstract] Bone remodeling is an important process to maintain mechanical integrity. It is accomplished by two important steps, bone resorption followed by new bone formation. Osteoclasts and osteoblasts are the principal cells in bone resorption and bone formation, respectively. A multitude of local and systemic factors regulates this process by controlling the cellular activities in bone remodeling compartments (BRC). An imbalance of osteoblastic bone formation and osteoclastic bone destruction will result in the development of skeletal diseases. Recent studies suggested that angiogenesis is closely associated with bone remodeling. The vasculature in bone is important for skeletal development, growth and repair. During endochondral ossification, cartilage is invaded by blood vessels which bring in osteoblast and osteoclast precursor cells, nutrients, growth factors and differentiation factors. During fracture repair, it has been demonstrated that mature osteoclasts produce heparanase which can degrade heparin sulfate proteoglycans, a major component in extracellular matrix (ECM). The process leads to the release of heparin-binding growth factors including vascular endothelial growth factor (VEGF), a potent angiogenic factor which contributes largely to local angiogenesis. In recent studies, endothelial cells have been found to produce bone morphogenetic protein (BMP)-2 and BMP-4 when they are subjected to mechanical stimuli, or a hypoxia environment. Conversely, inhibition of angiogenesis has been shown to prevent fracture healing. In a distraction osteogenesis model, either inhibition of angiogenesis or disruption of the mechanical environment prevents normal osteogenesis and results in fibrous nonunion. .... A total of 42 mice from F1 and F2 generations were genotyped as transgene positive. Preliminary analysis using radiography did not reveal any difference between the gross structures of transgenic and wild type mice. Interestingly, the preliminary histology revealed a decrease in trabecular bone and an increase of lipid space in metaphysis of transgenic mice overexpressing EGFL6. However, further studies will need to be carried out to investigate the role of EGFL6 in angiogenesis and adipogenesis using a transgenic mice model. This will be a prime focus of future work. Collectively, the results presented in this thesis have identified EGFL6, a member of the EGF-like family, as a potential angiogenic factor which may play an important role in bone remodeling. EGFL6 has been found to be expressed highly in calvarial osteoblasts and upregulated during primary murine osteoblast differentiation. EGFL6 has been 8 characterized to be a secreted homomeric complex. More importantly, EGFL6 has been shown to induce angiogenic activity in endothelial cell migration, tube formation and in vivo chick embryo chorioallantoic membrane assay. Furthermore, conditioned medium containing the EGFL6 recombinant protein was shown to induce phosphorylation of ERK in endothelial cells. Inhibition of ERK impaired EGFL6-induced ERK activation and endothelial cell migration. Taken together these studies raise the possibility that EGFL6 has a potential role in angiogenesis, and mediates a paracrine mechanism of cross-talk between vascular endothelial cells and osteoblasts during osteogenesis. An understanding of this process offers the potential to facilitate the development of therapeutic treatments for bone disease.

Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references. Also available online in PDF format.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / [a] in title is the Greek letter alpha. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 161-184).

Introduction : Le traitement des cancers bronchiques non à petites cellules (CBNPC) EGFR mutés repose sur les inhibiteurs de tyrosine kinase (ITK) du récepteur de l’Epidermal Growth Factor (EGFR). Cependant tous les patients traités par ITK EGFR finissent par présenter une progression tumorale, du fait de mécanismes de résistance comme l’amplification du gène codant pour le récepteur tyrosine kinase MET. Il n’existe actuellement aucune donnée sur les modifications phénotypiques induites par l’activation de MET dans ce contexte. L’objectif de cette thèse est de déterminer si l’amplification de MET, lors de la résistance aux ITK EGFR dans les CBNPC EGFR mutés, confère aux cellules tumorales un phénotype plus agressif et modifie l’histoire naturelle de la maladie.Méthodes : Les capacités de prolifération, de croissance sans ancrage, de formation de sphéroïdes, de résistance à l’anoïkis et de migration ont été étudiées in vitro dans la lignée HCC827, dérivée d’un CBNPC EGFR muté, et dans sa lignée fille HCC827-GR6 (GR6) devenue résistante aux ITK EGFR via une amplification du gène MET. L’expression de la vimentine, de ZEB1, et de la E-cadherine a également été étudiée dans les deux lignées cellulaires afin d’évaluer l’impact de l’amplification de MET sur la transition épithélio-mésenchymateuse (TEM). In vivo la croissance tumorale et le potentiel métastatique ont respectivement été analysés dans des modèles murins de xénogreffe ectopique et d’injection intracardiaque. Enfin les données cliniques de patients issus de 15 centres avec un CBNPC EGFR muté métastatique, présentant une forte surexpression de MET en immunohistochimie (score 3+) ou une amplification de MET en FISH sur une re-biopsie réalisée après la progression sous ITK EGFR ont été analysées rétrospectivement. Résultats : In vitro, l’amplification de MET induisait une augmentation significative de la prolifération, de la croissance sans ancrage, de la formation de sphéroïdes, de la résistance à l’anoïkis et de la migration. En présence d’un inhibiteur de MET, le PHA-665752, ces différentes propriétés biologiques étaient réduites de façon significative dans les cellules GR6 porteuses de l’amplification de MET. Il était également mis en évidence dans les cellules GR6 une augmentation de l’expression de la vimentine et de ZEB1. In vivo, l’amplification de MET augmentait significativement la croissance tumorale et le potentiel métastatique. Un traitement par crizotinib, ITK ciblant MET, diminuait de façon significative le potentiel métastatique des cellules porteuses de l’amplification de MET. Enfin les patients atteints d’un CBNPC EGFR muté, porteur d’une amplification de MET à la résistance à l’ITK EGFR, présentaient une durée jusqu’à apparition de nouvelles métastases plus courte après progression sous ITK EGFR que les patients avec une forte surexpression de MET sans amplification génique. Conclusion L’amplification de MET dans un contexte de résistance aux ITK EGFR est associée à un phénotype tumoral plus agressif. Ces résultats plaident en faveur d’une utilisation précoce d’inhibiteurs de MET en association avec les ITK EGFR afin d’éviter l’émergence d’un clone tumoral résistant plus agressif. / Introduction: Treatment of Epidermal Growth Factor Receptor (EGFR) mutated non-small cell lung cancers (NSCLC) relies on EGFR tyrosine kinase inhibitors (TKI). However, all patients treated with EGFR TKI eventually present tumor progression, due to mechanisms of resistance such as the MET amplification. There is currently no data on phenotypic changes induced by MET activation in this context. The objective of this thesis is to determine whether the MET amplification during EGFR TKI resistance in the EGFR mutated NSCLC induces a more aggressive phenotype in tumor cells and alters the natural history of the disease.Methods: Proliferation, anchorage independent growth, spheroid formation, anoïkis resistance and migration were studied in vitro in the HCC827 cell line, derived from an EGFR mutated NSCLC, and in its daughter cell line HCC827-GR6 (GR6) which became resistant to EGFR TKI through MET amplification. The expression of vimentin, ZEB1, and E-cadherin was evaluated in these cellular models in order to investigate an epithelial to mesenchymal transition (EMT) process induced by the MET amplification. In vivo, the tumor growth and the metastatic potential were analyzed by subcutaneous xenograft and intracardiac injection in mouse models. Finally, the clinical data of patients from 15 centers with a metastatic EGFR mutated NSCLC, exhibiting high MET overexpression in immunohistochemistry (score 3+) or MET amplification assessed by FISH on a re-biopsy performed after TKI EGFR progression were analyzed retrospectively.Results: In vitro, the MET amplification induced a significant increase in proliferation, anchorage independent growth, spheroid formation, anoïkis resistance and migration. Treatment with PHA-665752, a MET TKI, significantly reduced these biological properties in the GR6 cells harboring the MET amplification. An increase in the expression of vimentin and ZEB1 was also observed in the GR6 cells. In vivo, the MET amplification significantly increased the tumor growth and the metastatic potential. Treatment with crizotinib, another MET TKI, significantly decreased the metastatic potential of cells carrying MET amplification. Finally, patients with an EGFR mutated NSCLC, displayed a time to new metastases after TKI EGFR progression shorter than patients with high MET overexpression without MET amplification.Conclusion: The MET amplification during EGFR TKI resistance is associated in EGFR muted NSCLC with a more aggressive tumor phenotype. These results argue for the early use of MET inhibitors in combination with EGFR TKIs to avoid the emergence of a more aggressive resistant tumor clone.