Date of Graduation
5-2015
Document Type
Dissertation (PhD)
Program Affiliation
Cancer Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Don Gibbons
Committee Member
Andrew Gladden
Committee Member
Faye Johnson
Committee Member
Yang Xia
Committee Member
Jonathan Kurie
Abstract
The microRNA-200 family is known to be a master regulator of the epithelial-to-mesenchymal transition, partially through its double-negative feedback loop with the transcriptional repressor Zeb1, yet the mechanisms on how miR-200 controls the invasive phenotype are not fully understood. Recent studies have shown that the miR-200/Zeb1 axis regulates cell-cell and cell-matrix interactions, but it has also been demonstrated that cell-intrinsic changes are insufficient to drive cancer cell invasion, leading us to focus on specific cell-matrix interactions required to activate tumor cell invasion and metastases. We have shown through 3D studies that the Integrin β1-collagen I contact is critical in mediating the invasive phenotype in cells with miR-200 loss or Zeb1 overexpression. Furthermore, those genetic changes enhanced the cells responsiveness to the ECM through the FAK/Src pathway. The importance of this pathway in our Kras and p53 mouse model and in human lung cancer cell lines was further studied using pharmacological inhibitors and an shRNA-based knockdown approach, which exhibited a significant suppression of migration and invasion in Boyden chambers and 3D invasion assays. Furthermore, pharmacological inhibition of Src prevented distant metastases in vivo. We found that miR-200 regulates the activation of the FAK/Src pathway through direct targeting of CRKL, an integrin adaptor molecule. Our studies suggest that CRKL is critical in enhancing the outside-in signaling through Itgβ1 but also involved in the inside-out signaling by maintaining the cell-matrix contact required for continuous cell invasion. Those findings highlight the importance of the ECM composition, in addition to cell-intrinsic changes, that regulate the activation of intracellular signaling pathways required for tumor cell invasion and metastases that allow for targeting at multiple levels.
Keywords
miR-200, lung cancer, metastases, EMT, Integrin b1, FAK, CRKL