A. Kumar1, H. L. Goel1, T. J. FitzGerald2,3, and A. Mercurio1; 1University of Massachusetts Chan Medical School, Worcester, MA, 2University of Massachusetts Medical Center, Department of Radiation Oncology, Worcester, MA, 3UMass Memorial Health Care, Worcester, MA
Purpose/Objective(s): The high rate of locoregional/distant recurrence in triple-negative breast cancer (TNBC) is a major driver of the decreased survival observed in breast cancer patients. Radiation therapy is a reliable treatment modality to limit recurrence for many cancers, but recent evidence suggests that TNBC is more resistant to radiation compared to other breast cancer subtypes. In this study, we sought to identify the differential pathways in radioresistant TNBC tumors that impact recurrence. Materials/
Methods: We developed two radioresistant cell line models (468-RR and 4T1-RR) by administering a radiation dose of 50Gy over the course of 8 weeks to the human TNBC cell line, MDA-MB-468, and the mouse TNBC cell line, 4T1, respectively. Migration was assessed using the scratch-wound assay and invasion was measured using a transwell assay with Matrigel. All cell lines expressed luciferase, allowing for the in vivo assessment of experimental and spontaneous metastasis by injecting the cells in the tail vein and mammary fat pad, respectively. RNA-sequencing of the cell lines was done to identify global transcriptomic changes and regulatory pathways induced by radiation therapy. The mRNA stability of integrin ß3was assessed by inhibiting transcription using actinomycin D. Immunoblotting and RT-qPCR were used to assess protein and mRNA levels in samples. Results: The radioresistant cell lines had higher rates of migration and invasion compared to the parental cell lines. Moreover, the radioresistant cell lines had enhanced chest wall metastasis, based on luminescence, in both the experimental and spontaneous metastasis models. We utilized differential expression analysis from our RNA-seq datasets which revealed that the radioresistant cell lines were enriched for the expression of HNRNP-L, a ribonucleoprotein involved in mRNA processing. After downregulating HNRNP-L in the radioresistant cells, there was a significant decrease in their migration and invasion. Given the role of HNRNPL in integrin and extracellular matrix genes, which are key components associated with metastasis in cancer cells, we screened for these genes in our RNA-seq dataset and identified integrin ß3 as a possible target. The downstream consequence of reduced HNRNP-L was decreased integrin ß3 expression. More specifically, knocking down HNRNP-L decreased the mRNA stability of integrin ß3 as assessed by actinomycin d treatment. Conclusion: The radioresistant cells derived from TNBC cell lines have an enhanced migration and invasion capacity, which resulted in higher metastatic potential than the treatment-naive cell lines. The upregulation of HNRNP-L upon radiation increases integrin ß3 expression by stabilizing its transcripts and preventing its degradation. This novel mechanism of radiation-induced migration capacity provides an alternative therapeutic approach to minimize TNBC recurrence after radiotherapy.
