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"Cancer-Associated Fibroblasts: Development and Responses to Ionizing Radiation"

Jason Domogauer
MD/PhD Program
B.S. 2008, Rutgers University, New Brunswick, NJ

Thesis Advisor: Edouard Azzam, Ph.D.
Professor, Department of Radiology

Wednesday, May 11, 2016
12:00 P.M., Cancer Center, G-1196


Cancer remains a leading cause of death worldwide. Although substantial progress has been made in the prevention, diagnosis, and treatment options, cancer morbidity and mortality continues to increase due to an expanding and aging global population, and as developing countries adopt lifestyles with cancer-associated risks. Thus, a deeper understanding of the mechanisms contributing to the development of cancer are needed in order to improve existing therapeutic strategies and to develop new cellular and molecular-targeted approaches.
It is now widely accepted that cancer progression does not depend solely on the intrinsic properties of the cancer cells, but also on the activated tumor stroma, comprised predominantly of cancer-associated fibroblasts (CAFs). Together, cancer and stromal cells encompass the majority of the tumor microenvironment (TME), and CAFs are intimately involved in tumor initiation, progression, metastasis, and recurrence. In this thesis project, we tested the hypothesis that CAFs develop unique characteristics including an altered redox-environment and an elevated DNA repair capacity, which contributes to an increased resistance to ionizing radiation exposure. Further, development of this resistance is dependent upon direct intercellular communication via junctional channels, and through secreted factors. Additionally, CAFs are prone to genomic instability, and propagate signaling events that modulate neighboring cancerous and non-cancerous cells.
Pure populations of CAFs were generated from normal human fibroblasts through intimate co-culture with cancer cells, using a permeable microporous membrane insert system. The phenotype of these CAFs was validated by a decrease in Caveolin-1 (CAV1), a known CAF biomarker. Characterization of the CAFs revealed alterations in protein levels and signaling cascades, which were affected by the oxygen environment in which they were developed. Further analysis revealed that CAFs experience perturbations in their redox environment as demonstrated by increased protein carbonylation, increased levels of mitochondrial superoxide anion, and modulation of the antioxidants manganese superoxide dismutase (MnSOD) and catalase. Although intimate association with cancer cells is continuously required for maintenance of the CAF phenotype, fibroblasts isolated from a co-culture with breast cancer cells and propagated for 25 population-doublings demonstrated enhanced genomic instability, as judged by an increase in micronucleus formation. Strikingly, the increase in micronuclei, a surrogate form of chromosomal damage, was associated with a decrease in expression of the senescence markers, -galactosidase and p16INK4a.
Importantly, this project has shown that CAFs in co-culture with cancer cells were resistant to the clastogenic effects of 137Cs γ-rays (single acute exposures of 0.5 or 1 Gy). The response appeared to be largely general as it was observed with CAFs of different origin when in co-culture with diverse types of cancer cells (e.g., breast, brain, lung, and prostate). Incubation of the co-culture with repair inhibitors of single- or double-strand DNA breaks attenuated the resistance to -rays, suggesting that an increased ability to repair DNA damage may be a contributor to CAF radio-resistance. The results also support a role for increased activity of MnSOD in CAF radio-resistance, perhaps as an adaptive response to the increased oxidative stress experienced during CAF development.
Permeability aspects of connexin channels/hemi-channels likely participate in the resistance of CAFs to -rays. Radio-resistance was detected in MRC5 CAFs co-cultured with MCF7 breast cancer cells that expressed connexin (Cx)32, but not Cx26 or Cx43. Interestingly, AG1522 CAFs demonstrated significant resistance to radiation following co-culture with MCF7 devoid of Cx, suggesting that fibroblasts from different tissues may require different stimuli to transition into CAFs and develop radio-resistant properties.
This project also revealed that CAFs modulate cancerous and non-cancerous cells in their vicinity. Expression of CD24 and CD44 antigens in MCF7 cells was altered following co-culture with AG1522 or MRC5 CAFs. Further, MDA-MB-231 breast cancer cells developed a significantly greater migratory capacity following co-culture with CAFs. The CAFs also develop a complex secretome following co-culture with MDA-MB-231 cells: Relative to control, clear differences in the abundance of factors with a role in tumor growth and therapy response were detected, notably the levels of basic fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), heparin-binding EGF-like growth factor (HB-EGF), insulin-like growth factor-binding protein (IGFBP)-2 and -6, and vascular endothelial growth factor (VEGF), were altered.
Collectively, our data show that several mechanisms contribute to radio-resistance of CAFs. Increased ability to repair DNA damage and potentially antioxidant defense mediate this resistance. Cells in the TME are likely affected by factors that are secreted by CAFs. Understanding of the mechanisms that contribute to the development of CAFs and their radio-resistance is relevant towards improving the effectiveness of radiotherapy by introducing new targets for the treatment of human tumors.

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