RNA-seq of NIK Kockdown and control cells in lung metastatic human breast cancer cells established from MDA-MB-231 by orthotopic xenograft model

Orthotopic xenograft (OX) model can mimics early process of spontaneous metastasis comparing than intra-cardiac injection (ICI) or tail vein injection (TVI) model. However, many gene signatures in orthotopic breast cancer metastasis have not yet been studied in detail. Here we have established new highly lung metastatic breast cancer cells from MDA-MB231 by OX model.Comparative analysis between Parent cells (MDA-MB231) and the highly lung metastatic breast cancer cells (LM05) revealed that Non-canonical NF-?B pathway was constitutively activated by NIK up-regulation in protein level. In addition, the NIK up-regulation was ascribed to a decrease in translation of cIAP1 which degrade NIK by ubiquitination. Next, we investigated the NIK function in LM05 and found the NIK up-regulation contributed to tumorigenicity. To further analyze NIK funcion of tumorigenicity, we conducted RNA-seq and immunohistochemistry staining of NIK Knockdown cells and control cells. As these results,the suppression tumorigenicity caused by NIK knockdown is attributed to the down-regulation of attraction of CAFs by decreased in some cytokines and chemokines expressions. Finally, we investigated the relationship between NIK protein levels and breast cancer malignancy. From this analysis, NIK expression in breast cancer revealed that normal breast tissue was hardly detectable NIK protein. On the other hand, the expression of NIK protein was increased in malignancy tumors. In conclusion, our results demonstrated that a new role and up-regulation mechanism for NIK in breast cancer progression. Overall design: NIK knockdown cell lines were established by retroviral infection of pSUPER- target shRNA knockdown vectors and selected with 3 µg/ml puromycin for 3 days. Total RNA was extracted from control cell line (shGFP) and NIK knockdown cell lines (shNIK no.1, no.2). RNA-seq was performed to characterize these cells in detail.