Abstract PD15-01: Snord67 promotes lymph node metastasis through U6-mediated alternative splicing in breast cancer

Background: While the presence of lymph node (LN) metastases in solid tumor malignancies is clinically significant and associated with increased risk for distant metastases, there is little evidence that lymphatic metastasis is distinct from hematogenous dissemination. Recent studies showed that cancer cells in LNs can directly give rise to distant metastases, but the mechanisms behind how cancer cells exit lymph nodes to establish distant metastases is still unknown. Materials and Methods: We developed an immunocompetent mouse model of lymph node metastasis using the 4T1 murine breast cancer cell line. In this model, 4T1 cells micro-surgically injected into axillary lymph nodes (AxLN) are capable of spontaneously establishing distant metastases. We then performed microarray analyses of subclones derived from MFP tumors, AxLN-injected tumors (AxLN), and lung metastases that arose from AxLN-injected tumors (AxLN-LuM) to evaluate for non-coding RNAs (ncRNAs) that were differentially expressed in AxLN tumors. Results: Microarray analyses of ncRNAs expressed in MFP, AxLN, and AxLN-LuM subclones showed that a class of ncRNAs, small nucleolar RNAs (snoRNAs), were enriched in AxLN tumors compared to MFP and AxLN-LuM. We identified the snoRNA SNORD67 as a key regulator of LN metastasis. Knockout of SNORD67 by both CRISPR and antisense oligonucleotide (ASO) resulted in significantly decreased LN tumor growth and subsequent development of distant metastases. This was at least in-part due to loss of targeted 2’-O-methylation of the small nuclear RNA U6, a component of the spliceosome. RNA-Seq analyses revealed distinct alternative splicing in SNORD67 knockouts. Using rapid autopsy breast cancer cases, we found that matched primary tumor and LN metastases revealed similar alternatively spliced genes, including several genes with previously described cancer-promoting splice variants. Conclusions: Small nucleolar RNAs (snoRNAs) have long been regarded as “housekeeping genes”, important for ribosomal biogenesis and protein synthesis. However, there is increasing evidence that this largely ignored class of non-coding RNAs (ncRNAs) also have wide-ranging, non-canonical functions in diseases, including cancer. SnoRNAs have been shown to have both oncogenic and tumor suppressor roles, yet whether snoRNAs regulate metastasis is unknown. Here we show that expression of certain snoRNAs are enriched in lymph node (LN) metastases in a micro-surgical, immune-competent mouse model of breast cancer. Moreover, our results show that SNORD67 is critical for growth of LN metastases and subsequent spread to distant metastases, and suggest that snoRNA-guided modifications of the spliceosome represent a previously unappreciated, yet targetable pathway in cancer. Citation Format: Yvonne Chao, Yinzhou Zhu, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Alessandro Porrello, Amanda ED Van Swearingen, Lisa Carey, Jimena Giudice, Christopher L Holley, Chad V Pecot. Snord67 promotes lymph node metastasis through U6-mediated alternative splicing in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-01.