Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

Fyn, a member of the Src family kinases, is an oncogene in murine epidermis and is associated with cell-cell adhesion turnover and migration. Introduction of active H-Ras(G12V) into the HaCaT human keratinocyte cell line resulted in upregulation of FYN mRNA (200-fold) and protein, but did not increase the expression of other SFKs. We identified two nucleotides 74 bases upstream of the human FYN gene transcription start site as necessary for FYN upregulation in HaCaT-Ras cells. Further studies identified Lef-1 as the transcription factor most likely binding to this site and mediating FYN upregulation.

Transduction of active Ras or Fyn was sufficient to induce an epithelial-to-mesenchymal transition in HaCaT cells. Introduction of Ras or Fyn also resulted in upregulation of the EMT master regulator, TWIST1, demonstrating a potential mechanism driving the transition to a mesenchymal phenotype. Fyn inhibition using siRNA or the clinical SFK inhibitor Dasatinib increased cell-cell adhesion in HaCaT-Ras cells over 6-fold (p<0.001) through a rapid (5-60 min.) increase in the cortical F-actin cytoskeleton. Treatment of the HaCaT-Fyn cells with Dasatinib also resulted in upregulation of F-actin. Induced F-actin colocalized with the adherens junction proteins α-catenin, β-catenin and p120catenin in HaCaT-Ras cells, suggesting that stable adherens junctions were mediating the increase in cell-cell adhesion. Additionally, Dasatinib treatment significantly inhibited cell migration (p<0.001). Dasatinib-induced cell-cell adhesion could be blocked by Cytochalasin D, indicating that F-actin polymerization was a key initiator of cell-cell adhesion through the adherens junction. Conversely, inhibiting cell-cell adhesion with low Ca2+ media did not block Dasatinib-induced F-actin polymerization. Inhibition of the Rho effector kinase ROCK blocked Dasatinib-induced F-actin and cell-cell adhesion, implicating relief of Rho GTPase inhibition as a mechanism of Dasatinib-induced cell-cell adhesion.

Finally, topical Dasatinib treatment immediately following UV exposure significantly reduced total tumor burden in the SKH1 mouse model of skin carcinogenesis (p<0.05). Together these results identify the promotion of actin-based cell-cell adhesion as a newly described mechanism of action for Dasatinib and suggest that Fyn inhibition may be an effective therapeutic approach in treating SCC.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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