Regorafenib sensitizes human breast cancer cells to radiation by inhibiting multiple kinases and inducing DNA damage

Abstract

Purpose

Triple-negative breast cancer (TNBC) is the most challenging and aggressive subtype of breast cancer with limited treatment options because of tumor heterogeneity, lack of druggable targets and therapy resistance. TNBCs are characterized by overexpression of growth factor receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR) making them promising therapeutic targets. Regorafenib is an FDA approved oral multi-kinase inhibitor that blocks the activity of multiple protein kinases including those involved in the regulation of tumor angiogenesis [VEGFR1-3, TIE2], tumor microenvironment [PDGFR-β, FGFR] and oncogenesis (KIT, RET, RAF-1, BRAF). In the current study, we examined the radiosensitizing effects of Regorafenib on TNBC cell lines and explored the mechanism by which Regorafenib enhances radiosensitivity.

Methods

MDA-MB-231 and SUM159PT (human TNBC cell lines) and MCF 10a (human mammary epithelial cell line) were treated with Regorafenib, ionizing radiation or a combination of both. Following treatment with Regorafenib and radiation we conducted clonogenic assay to determine radiosensitivity, immunoblot analysis to assess the effect on key signaling targets, tube formation to evaluate effect on angiogenesis and comet assay as well as western blot for γH2AX to assess DNA damage response (DDR).

Results

Regorafenib reduced cell proliferation and enhanced radiosensitivity of MDA-MB-231 and SUM159PT cell lines but had no effect on the MCF 10a cells. Clonogenic survival assays showed that the surviving fraction at 2 Gy for both MDA-MB-231 and SUM159PT was reduced from 66.4 ± 8.9 and 88.2 ± 1.7 in controls to 38.1 ± 4.9 and 75.1 ± 1.1 following a 24 hr pretreatment with 10 μM and 5 μM Regorafenib, respectively. A marked reduction in the expression of VEGFR, PDGFR, EGFR and the downstream target, ERK, was observed with Regorafenib treatment alone or in combination with radiation. We also observed a significant inhibition of VEGF-A production in the TNBC cell lines following treatment with Regorafenib. Further, the addition of conditioned medium from Regorafenib-treated tumor cells onto human umbilical vein endothelial cells (HUVEC) suppressed tube formation, indicating an inhibition of tumor angiogenesis. Regorafenib also decreased migration of TNBC cells and suppressed radiation-induced DNA damage repair in a time-dependent manner.

Conclusions

Our findings demonstrate that Regorafenib enhanced radiosensitivity of breast cancer cells by inhibiting the expression of multiple receptor tyrosine kinases, VEGF-mediated angiogenesis and DNA damage response in TNBC. Therefore, combining Regorafenib with radiation and antiangiogenic agents will be beneficial and effective in controlling TNBC.

Keywords:

• Regorafenib
• VEGF
• angiogenesis
• DNA damage
• radiotherapy
• breast cancer

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This study was supported in part by grants received from an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences [P20 GM103639] of the National Institutes of Health (NIH) (AM, RR), a pilot grant from the Stephenson Cancer Center funded by the NCI Cancer Center Support Grant [P30 CA225520] awarded to the University of Oklahoma Stephenson Cancer Center, and the OUHSC Department of Radiation Oncology Research Development Funds (AM). RR is an Oklahoma TSET Research Scholar and holds the Jim and Christy Everest Endowed Chair in Cancer Developmental Therapeutics. The content is solely the responsibility of the authors and does not represent the official views of any grant-awarding agency.

Notes on contributors

Meghna Mehta

Meghna Mehta, MS is a Research Associate in the Department of Radiation Oncology. She received her Master's degree in Microbiology from the University of Oklahoma. Her research interests include studies combining drug treatment with radiation to radio sensitize tumor cells and evaluating DNA damage response. Other areas of interests include investigating the role of mitochondrial metabolism in metabolic reprogramming of cancer cells.

James Griffith

James Griffith, BS is a Research Assistant at the University of Oklahoma Health Sciences Center. He received his Bachelor of Science in Microbiology from the University of Oklahoma at Norman. He is currently a member of a research laboratory in the Department of Surgery at OUHSC. The laboratory focuses on wound healing, fibrosis, and the progression of metastasis in colon cancer.

Janani Panneerselvam

Janani Panneerselvam, PhD is a Senior Research Associate for Center for Cancer Prevention and Drug Development and Division of Medical Oncology at the University of Oklahoma Health Sciences Center. Her research mainly focuses on molecular characterization of cancer cells, chemoprevention, target identification, and validation and drug development at the functional and molecular level. She is also assessing the role of immune cells in cancer development and progression and for anti-tumor immunity.

Anish Babu

Anish Babu is Post-Doctoral research fellow at the Stephenson Cancer Center, University of Oklahoma Health Science Center, Oklahoma City, USA. He holds a PhD degree in Biology in the area of anti-cancer therapy and nano-drug delivery from Madurai Kamaraj University, India. His current research is focused on identifying the mechanism(s) and devising therapeutic strategies to overcome therapy resistance in cancer.

Jonathan Mani

Jonathan Mani, MD is a resident physician for the Department of Radiation Oncology at the University of Oklahoma. His interests include the interactions between drugs and tissue effects with ionizing radiation.

Terence Herman

Terence Herman, MD, is Professor and Chairman of the Department of Radiation Oncology and an Adjunct Professor of Internal Medicine (Hematology/Oncology) at the University of Oklahoma Health Sciences Center. He specializes in drug/radiation interactions in patients with gastrointestinal, sarcoma, breast and lymphomatous cancers. Dr. Herman is a reviewer for several cancer related journals and is the Principle Investigator for the Radiation Therapy Oncology Group studies at the Stephenson Cancer Center and University of Oklahoma.

Rajagopal Ramesh

Rajagopal Ramesh, PhD, is a Professor in the Department of Pathology at the University of Oklahoma Health Sciences Center and Co-Leader of the Cancer Biology Program and Co-Director of the Nanomedicine Program at the Stephenson Cancer Center. His research is focused on developing novel gene-based therapeutics using viral and non-viral vectors, and nanomedicine with emphasis on translational cancer research. His research has been supported by the National Institutes of Health, Department of Defense and the Department of Veterans Affairs.

Anupama Munshi

Anupama Munshi, PhD, is an Associate Professor in the Department of Radiation Oncology at the University of Oklahoma Health Sciences Center. Her research focuses on understanding the specific molecular mechanisms and biological processes that govern tumor response to radiation and other anticancer agents. A major emphasis of her research is to develop innovative and effective approaches to sensitize tumor cells to radiation and DNA damage and study the molecular mechanism of resistance to radiation-induced cell death.