Snord67 promotes breast cancer metastasis by guiding U6 modification and modulating the splicing landscape

Previously considered “housekeeping” genes, small nucleolar RNAs (snoRNAs) are increasingly understood to have wide-ranging functions in cancer, yet their role in metastasis remains unknown. Here, we identify the snoRNA Snord67 as a regulator of lymph node (LN) metastasis in female breast cancer. Snord67 expression is enriched in LN metastases in an immune-competent mouse model of breast cancer. In an orthotopic breast cancer model, loss of Snord67 decreases LN metastasis. In a model of lymphatic metastasis, Snord67 loss decreases LN tumor growth and distant metastases. In breast cancer cell lines, Snord67 knockout results in loss of targeted 2'-O-methylation on U6 small nuclear RNA, as well as widespread changes in splicing. Together, these results demonstrate that Snord67 regulates splicing and promotes the growth of LN metastases and subsequent spread to distant metastases. SnoRNA-guided modifications of the spliceosome and regulation of splicing may represent a previously unappreciated, potentially targetable pathway in cancer. Overall design: To investigate changes in gene expression and alternative splicing upon Snord67 knockout in the murine triple-negative breast cancer cell lines 4T1 and MDA-MB-231-D3H2LN-luc (D3H2) cells, we performed RNA sequencing of polyA-selected RNA from triplicates of 4T1 wild-type cells, two 4T1 Snord67 knockout clones, D3H2 wild-type cells, and one D3H2 Snord67 knockout clone.