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A Novel Rnaseh2c-Immune Response Axis in Breast Cancer Alters Tumor Progression and Metastasis Open Access

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Breast cancer is the second leading cause of cancer-related deaths in women in the United States. Despite 98% survival for patients with localized disease, current therapies are largely ineffective against metastatic disease, reducing survival to 25%. Thus, a better understanding of the mechanisms influencing metastasis is needed. Using genetically heterogeneous mouse models, our lab has identified and validated numerous genes that affect metastasis through changes in expression. Using this approach, we identified the candidate gene Rnaseh2c. We first confirmed Rnaseh2c as a metastasis modifier: shRNA knockdown resulted in changes in primary tumor mass and fewer pulmonary metastases, and increased expression produced more metastases. RNASEH2C is a scaffolding subunit of the RNase H2 enzyme which removes ribonucleotides from DNA substrates. Surprisingly, despite a 70% reduction in enzyme activity upon knockdown, no evidence was observed for changes in cell proliferation or apoptosis in the tumor, cell cycle progression, or a DNA damage response in cultured cells. Interestingly, a comparable knockdown of the catalytic subunit, Rnaseh2a, did not affect metastasis, despite a 40% reduction in enzyme activity. This may suggest the effect of Rnaseh2c expression on metastasis could be through a novel enzyme-independent function. Given that mutations in RNASEH2C cause Aicardi-Goutières Syndrome (AGS), a neurological autoinflammatory disorder that triggers an anti-viral-like interferon response, we hypothesized that altered expression of Rnaseh2c in breast cancer cells affects metastasis by engaging the immune system. Abolishment of the knockdown effect on metastasis in T cell-deficient mice and an increase in CD8+ T cell infiltration at the tumor and metastatic lung in immunocompetent mice receiving knockdown cells confirmed a critical role for the adaptive immune response in this mechanism. Pathway analysis of RNA sequencing data from tumors revealed an induction of T cell-mediated immune response pathways, further supporting a novel Rnaseh2c-immune response axis in breast cancer metastasis. A lack of IRF3 activation upon knockdown showed that nucleic acid sensing via STING, the proposed mechanism of AGS, was not the inducer of immunity; exome sequencing ruled out production of neoantigens and further analysis also eliminated sensing of dsRNA and transcription of L1 elements. Mass spectrometry analysis of RNASEH2C suggested effects on ribosome biogenesis. Ribosomal RNA transcription was found to be increased upon Rnaseh2c knockdown, and immunofluorescence indicated an increase in rRNA processing proteins in the nucleolus. These results highlight a potential new mode of immune activation in metastatic breast cancer and suggest a role for RNASEH2C in facilitating ribosome biogenesis. These data may also indicate the potential importance of ribosome biogenesis in AGS or related autoimmune pathologies.

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