Defense Date

2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Aylin Marz

Abstract

Overexpression of the DNA repair protein, X-ray repair cross-complementing protein 3 (XRCC3), has been shown to increase breast cell invasiveness. This raises the question of whether XRCC3 is a positive modulator of invasion or whether overexpressed XRCC3 is trapped in non-functional complexes, resulting in a dominant negative effect. To address this we transiently downregulated XRCC3 in a triple-negative breast cell line model of pre-invasive to invasive transition. Downregulation of XRCC3 in invasive T4-2 cells enhances cell invasiveness and increases FAK activation and signaling. This effect is abolished by EGF or TGFβ suggesting that XRCC3 is upstream of these factors. Conditioned media (CM) from XRCC3-downregulated T4-2 cells enhances the invasion of naïve pre-invasive and invasive cells. Mass spectrometric analysis reveals that secreted cyclophilin A (CYPA) is upregulated in the CM by XRCC3-siRNAs. The addition of recombinant CYPA to naïve T4-2 cells enhances their invasiveness, suggesting that CYPA is sufficient to induce neighboring cell invasiveness. Inhibiting the secreted CYPA by CYPA-inhibitor Cyclosporine-A (CsA) in CMs from low-XRCC3 T4-2 cells renders the CM incapable of increasing invasiveness, suggesting that CYPA is downstream of XRCC3. Furthermore, intracellular CYPA levels increase in progression from S1 to invasive T4-2 cells, consistent with previous observations of overexpression of CYPA in cancers. Overexpressing XRCC3 in S1 cells has no effect on invasion, whereas overexpressing XRCC3 in T4-2 cells increases invasion, but also viability. Overexpressing XRCC3 T241M mutation in S1 cells significantly increases their invasiveness, but in T4-2 cells it moderately increases invasion, suggesting an early role of this polymorphism in breast cancer invasion. Therefore, XRCC3 is an invasion suppressor, upstream of adhesion, growth factors, and paracrine effectors.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

10-15-2013

Available for download on Sunday, October 17, 2213

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