Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Raymond R. Mattingly

Abstract

The purpose of this study is to determine the role of Rap1Gap in the progression of DCIS (ductal carcinoma in situ) to IDC (invasive ductal cancer). We employed an in-vitro three-dimensional (3D) overlay model that provides a physiologically relevant microenvironment to study mechanisms of malignant progression. Previous studies from this laboratory aimed to determine specific gene expression changes common to three models of DCIS- MCF10.DCIS, SUM 102 and SUM 225 in comparison to MCF10A cells, a model of non-transformed human mammary epithelium. The expression of 295 genes was found to be significantly altered, with 63 being increased in expression in all three DCIS cell lines.

The mRNA-Seq results were further mined by Genomatix analysis to gain an insight into common frameworks in promoter regions of these 63 up-regulated genes. 244 promoter loci were found to be associated with these 63 up-regulated genes. Enrichment analysis showed that the common framework RXRF-ZF02-ZF02-PLAG-HDBP is highly enriched [336-fold], being present in the promoters of RAP1GAP, SPRY4 and PDGFB genes.

Rap1Gap is a GTPase-activating protein (i.e., an inactivator) for the small GTPase, Rap1. It is known to be involved in regulation of cell adhesion and has been previously studied in pancreatic and thyroid cancers, where a decrease in its expression has been associated with malignant progression. Immunoblotting results show that in cells grown in 3D, Rap1Gap levels in MCF10.CA1d cells are reduced compared to those in MCF10.DCIS cells. Lentiviral shRNA silencing of Rap1Gap in DCIS induced an increase in Rap1 and MAPK activity, as determined by Rap1 activity assay and immunblotting for phosphorylated ERK1/2. Confocal immunofluorescence (staining of F-actin) of 3D structures and invasion assays reveal appearance of multicellular outgrowths, extensive cytoskeletal organization and increase in invasion. Concomitantly, increase in collagen IV degradation was observed. Lentiviral silencing of Rap1Gap also resulted in an increase in proliferation. Re-expression of Rap1Gap in DCIS-Rap1GapshRNA cells reduced Rap1 activity and suppressed the development of invasive outgrowths in 3D structures. Additionally, adherens junctions and E-cadherin levels were partially restored. Thus, we conclude that reduction of Rap1Gap in DCIS acts as a switch to progression to an invasive phenotype via deregulated Rap1 and MAPK activation.

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