Abstract 1112: CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1

Abstract

Background: Breast cancer is one of the leading causes of cancer death among women and the pathological status of axillary lymph nodes is an important predictor of prognosis. However, the mechanism involved in this early stage of metastasis remains largely unknown. Methods: We used microarray analysis to carry out differential genomics analyses between matched pairs of sentinel lymph node metastatic tissues and breast primary tumors. We found 182 up-regulated genes in metastatic tissues (fold change≥1.5). We used CRISPR/Cas9 gene editing system to knockout these genes, each with 6 unique sgRNAs, and screened in vivo by transplanting loss-of-function cell pool into immunocompromised mice. MAGeCK was used to analyze the screening. Immunohistochemistry was performed to examine protein expressions in 212 cases of breast cancer tissues using tissue microarray (TMA) technology. Gene and protein expressions were monitored by qPCR and western blot. The relationship between coiled-coil domain containing 102B (CCDC102B) and receptor for activated C kinase 1 (RACK1) was examined using Co-IP, mass spectrometry, nuclear protein extraction assay and dual-luciferase reporter assay. Functional studies were detected in vitro by cell proliferation, wound-healing and migration, and in vivo by lung metastasis in immunocompromised mice injected CCDC102B-overexpressed cells into tail vein. The primary biological functions and pathways related to CCDC102B were enriched by RNA sequencing. The regulation of NF-κB signaling by CCDC102B and RACK1 was detected using dual-luciferase reporter assay and western blot. Results: We identified CCDC102B through screening and found that it was significantly up-regulated in breast cancer patients with poor prognosis. The expression of CCDC102B were independent predictors in both DFS and OS ( P =0.009; P =0.014). CCDC102B expression was found to promote the proliferation and migration of breast cancer cells MDA-MB-231 and BT549, and lung metastasis in mice. Moreover, we found that overexpression of CCDC102B decreased RACK1 expression whereas knockdown of CCDC102B increased RACK1 expression at both protein and mRNA levels, and the aggressive behavior of CCDC102B in breast cancer cells could be reversed by the expression of RACK1. According to gene set enrichment analysis (GSEA), CCDC102B was correlated with significant enrichment focused on NF-κB pathway. The mechanistic study revealed that gain of CCDC102B resulted in decreased expression of RACK1, which increased levels of epithelial mesenchymal transition (EMT) and phosphorylated Rel-A (p65), leading to the activation of NF-κB pathway and progression of breast cancer metastasis. Conclusion: Taken together, our findings suggest that CCDC102B is strongly associated with negative clinical outcomes and may serve as a potential metastasis promotor by regulating the activation of NF-κB pathway via RACK1. Citation Format: Jing Si, Rong Guo. CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1112.