Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.

Mutations in FGFR2 are common in a subset of endometrial carcinomas. Given the emergence of small molecule inhibitors specific to this receptor tyrosine kinase, FGFR2 is an attractive therapeutic target. However, compensatory and adaptation mechanisms limit the clinical utility of compounds that target nodes in the receptor tyrosine kinase network. Here, we analysed the impact of FGFR inhibition in endometrial cancer cells and observed the emergence of a resistant population in an FGFR2-mutant cell line. To understand the mechanisms underlying this adaptation response, we used a phosphoproteomics approach to measure the kinase network in an unbiased manner. These experiments led to the identification of an AKT-related compensatory mechanism underpinning this resistance. Further dissection of this resistance mechanism utilising gene expression analysis showed PHLDA1, a negative regulator of AKT, was significantly down-regulated in resistant cells. This was further confirmed at the protein level. siRNA knockdown of PHLDA1 conferred immediate drug resistance in the FGFR2-mutant endometrial cancer cell line. Therefore, we identified PHLDA1 down-regulation as a mediator of drug resistance in FGFR2 mutant cancer cells, the first demonstration of the role of PHLDA1 in the acquisition and maintenance of drug resistance. Using a 3D physiomimetic model, we demonstrated that AKT inhibition alone also led to generation of a drug-resistant population. Most importantly, dual-drug therapy inhibited proliferation and induced cell death. Our data highlight how mass spectrometry and microarray gene expression analysis can complement each other in the identification of novel resistance mechanisms in cancer cells. These data suggest that combination treatment of FGFR2-mutant endometrial cancers, targeting both FGFR2 and AKT, represents a promising therapeutic approach.

Thisbe (Ths) and Pyramus (Pyr) are the ligands for the Fibroblast-Growth-Factor (FGF)receptor Heartless (Htl), which is expressed in all mesodermal cells during gastrulation. To understand how these two FGFs orchestrate mesoderm spreading in gastrulation and mesoderm differentiation during organogenesis, loss and gain of function studies were performed. In an approach of functional analysis, a single mutant allele of ths was generated, ths759, for comparison of the single mutant conditions of ths and the null mesodermal cells to migrate and differentiate in a precise pattern.

Recent genome-wide association studies identified FGFR2 as one of breast cancer susceptibility genes. FGFR2 expression was down-regulated in breast carcinomas when compared with paired normal epithelium. Stable retroviral transduction of FGFR2-IIIb and its alternatively spliced FGFR2-IIIc variants was achieved in breast cancer MDA-MB-231, T47D and near normal MCF-10A cells. Our findings revealed a direct reduction of breast cancer cell growth and motility, a significant arrest of transformed morphogenetic changes including the Epithelial to Mesenchymal transition (EMT), anchorage independent growth, and the formation of growth-arrested 3D acinar architectures, and suppressive actions on orthotopically xenografted epithelial neoplasms and surrounding tumor stroma. These tumor protective effects were concordant with physical interactions between the two FGFR2 isoforms and IKKβ. Consistent with these interactions we noted FGFR2 to inhibit NF-κB signaling, including decreased nuclear RelA/p65 NF-κB localization, down-regulation of a transfected NF-κB luciferase reporter, reduced production of NF-κB-dependent transcripts, Interleukin-6 and p-STAT3.

Recent genome-wide association studies identified FGFR2 as one of breast cancer susceptibility genes. FGFR2 expression was down-regulated in breast carcinomas when compared with paired normal epithelium. Stable retroviral transduction of FGFR2-IIIb and its alternatively spliced FGFR2-IIIc variants was achieved in breast cancer MDA-MB-231, T47D and near normal MCF-10A cells. Our findings revealed a direct reduction of breast cancer cell growth and motility, a significant arrest of transformed morphogenetic changes including the Epithelial to Mesenchymal transition (EMT), anchorage independent growth, and the formation of growth-arrested 3D acinar architectures, and suppressive actions on orthotopically xenografted epithelial neoplasms and surrounding tumor stroma. These tumor protective effects were concordant with physical interactions between the two FGFR2 isoforms and IKKβ. Consistent with these interactions we noted FGFR2 to inhibit NF-κB signaling, including decreased nuclear RelA/p65 NF-κB localization, down-regulation of a transfected NF-κB luciferase reporter, reduced production of NF-κB-dependent transcripts, Interleukin-6 and p-STAT3.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-109).

Thesis (M.S.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on January 29, 2009). Research advisor: Dr. Michael A. Dyer, Ph.D. Document formatted into pages (vi, 47 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 41-44).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 82-96).

Made available in DSpace on 2014-06-11T19:28:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-03-01Bitstream added on 2014-06-13T18:07:24Z : No. of bitstreams: 1 silverio_kg_me_arafo.pdf: 1644294 bytes, checksum: 5d943591d5c51a3846c75163db9a2ebb (MD5) / O objetivo do presente estudo foi avaliar in vitro o efeito do condicionamento radicular com fator de crescimento de fibroblastos básico (b-FGF) sobre a morfologia e densidade de fibroblastos. Para tal, blocos de dentina com 4 mm2 de área de superfície foram obtidos de raízes de dentes humanos extraídos devido severo envolvimento periodontal, sendo instrumentados manualmente e autoclavados. Noventa amostras foram selecionadas aleatoriamente e distribuídas em 3 grupos segundo o tratamento de superfície prévio ao condicionamento com o b-FGF: sem tratamento - controle; 50 mg/mL de cloridrato de tetraciclina e EDTA a 24%. As 30 amostras de cada um destes 3 grupos foram distribuídas em 3 subgrupos quanto à dose de b-FGF: 0 æg/mL - controle; 50 æg/mL e 125 æg/mL. Após os tratamentos, as amostras foram incubadas a 37º C e 98% de umidade com 2mL de meio Eagle, sendo 1mL com fibroblastos de linhagem contínua (células McCoy) na concentração de 1 x 105 células/mL e 1mL meio sem células, por 24 h. Após as 24 h, as amostras foram submetidas a preparo de rotina para MEV e então, fotomicrografadas nos aumentos de 500X (densidade celular) e 1000X (morfologia celular). Em seguida, as fotomicrografias foram avaliadas por 3 examinadores treinados, calibrados, independentes e cegos, os quais verificaram morfologia e densidade celular segundo os escores propostos por Gamal et al. (1998) e Jenkins et al. (1988), respectivamente. A aplicação da Análise de Regressão pela Técnica da Árvore demonstrou haver diferenças estatisticamente significantes para a densidade celular (p<0,0001) entre os grupos EDTA, tetraciclina e controle, sendo que também houve diferenças entre as doses de 0/50 æg e 125 æg de b-FGF nas amostras condicionadas com EDTA (p<0,0001) e entre as doses de 0 e 50/125 æg de b-FGF nas amostras condicionadas com tetraciclina... . / The aim of this study was to evaluate in vitro the effect of the root surface conditioning with basic fibroblast growth factor (b-FGF) about morphology and density of fibroblasts. Dentin slices of with 4 mm2 of surface area were obtained from roots of teeth extracted due to severe periodontal involvment. These were scaled and sterilized. Ninety samples were randomly distributed into 3 groups according to treatment before application of b-FGF: non-treated - control; 50mg/mL of tetracycline HCl and EDTA 24%. The thirty samples of each group were distributed into 3 subgroups according to the concentration of b-FGF: 0 æg/mL - control; 50 æg/mL and 125 æg/mL. After treatments, the samples were incubated at 37ºC and 98% humidity with 1mL of Eagle Medium with 1 x 105 cells/mL of fibroblast from continuos lineage (McCoy Cells) plus 1mL this solution without cells during 24 hours. The samples were submitted to routine preparation for SEM and photographed at 500x (density celular) and 1000x (morphology celular). Three independent and blind examiners evaluated the fibroblast`s morphology and density, according to Gamam et al. (1998) and Jenkins et al. (1998), respectively. Classification and Regression Trees test results indicated significant differences on the density (p<0,0001) among EDTA, tetracycline and control groups with also differences between concentrations of 0/50æg and 125æg of b-FGF at the samples conditioning with EDTA (p<0,0001) and between concentrations of 0 and 50/125 æg of b-FGF at the samples conditioning with tetracycline(p<0,0001). The results of this test to morphology indicated significant differences between treatment or non-treatment with b-FGF, and that concentration of 125 æg demonstrated to be more favorable than the concentration of 50 æg. In conclusion, the treatment of root surfaces with b-FGF influenced the density... (Complete abstract, click electronic address below).

Comprehensive genomic profiling has an emerging role in cancer therapeutics. As treatment options remain needed for advanced cancers, patients are relying increasingly more on tumor genomic alterations as possible targets for cancer treatment. Frequent tumor fibroblast growth factor receptor (FGFR) alterations are seen in many cancers, and include genetic amplifications, mutations, rearrangements and fusions. FGFR inhibitors target these receptor alterations and show promise as a drug class. Currently 2 medications are currently FDA approved: erdafitinib and pemigatinib. Through the FDA accelerated approval process, erdafitinib is indicated to treat metastatic urothelial carcinoma with FGFR2 and FGFR3 alterations, whereas pemigatinib is indicated to treat unresectable cholangiocarcinoma with FGFR2 alterations. Despite growing knowledge about such advanced cancers, treatment is usually palliative. With multiple FGFR inhibitors in the pipeline, further FDA approvals are possible, and it is likely their role in therapy will extend to other cancer types. This review outlines erdafitinib, pemigatinib, their role in cancer, as well as outlining the possible future use of other FGFR inhibitors in urothelial carcinoma, cholangiocarcinoma, and other malignancies.