2512 - The Role of HNRNP-L on the Mitochondrial Architecture of Radioresistant Triple Negative Breast Cancer

Purpose/Objective(s): Triple-negative breast cancer continues to be one of the most aggressive cancers due to its resistance to many therapy modalities and high metastatic potential. Recently, there has been supporting evidence for the role of mitochondria structure and function in mediating cancer cell proliferation and metastasis. However, there are limited studies that have explored this in the context of resistance to radiation therapy. In this study we investigated the differences in the architecture of mitochondria in radioresistant vs parental TNBC cell lines and elucidate the mechanism responsible for these differences.

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. Fluorescence confocal microscopy with staining of mitochondria (Mitotracker Green and TMRE) and transmission electron microscopy were used to elucidate architectural differences between cell lines and conditions. RNA-sequencing of the cell lines was done to identify global transcriptomic changes induced by radiation therapy. We used Induro–reverse transcriptase (RT)–mediated circRNA-seq (IMCR-seq) to identify differences in mitochondrial circular RNAs. Immunoblotting and RT-qPCR were used to assess protein and mRNA levels in samples.

Results: Interestingly, the radioresistant cell lines had mitochondria that were elongated and clustered in branches compared to the parental cell line. In a previous study, we had identified HNRNPL, a RNA binding protein that regulates circular RNAs, as a key regulator of the metastatic capacity of radioresistant TNBC. When exploring the role of HNRNPL in the dynamics of mitochondrial structure, we observed that downregulating HNRNPL in 468-RR and 4T1-RR cells reduced the size of mitochondria and resulted in fewer branches compared to the control. By using IMCR-seq, we also identified that circular RNAs that were encoded by the mitochondrial genome were increased in the radioresistant cells compared to the HNRNPL knockdown cells.

Conclusion: The radioresistant cells derived from TNBC cell lines have branched and longer mitochondria compared to treatment-naive cell lines. The upregulation of HNRNP-L upon radiation increases mitochondrial circular RNAs that may be contributing to the structural change of the organelle. This structural difference in the architecture of mitochondria may provide details into metabolic dependencies of the radioresistant cells that can be leveraged for therapeutic targeting.