Identification of LRP8, CDCA7, and MLK4 as Novel Therapeutic Targets for Cancer Stem Cells in Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. TNBC is the most challenging breast cancer subtype with poor prognosis, high metastatic potential, and lack of effective targeted therapies. Currently, chemotherapy remains the major strategy to treat TNBC. However, TNBC patients with residual disease after chemotherapy have higher risk of relapse and significantly worse survival than non-TNBC patients with residual disease. Therefore, there is an imperative need to identify novel and effective targeted therapies for TNBC. Cancer stem cells, also termed tumor-initiating cells, have been considered important targets for cancer treatment due to their high metastatic potential and resistance to conventional chemotherapy. In agreement with the inherently aggressive clinical behavior of TNBC, emerging evidence has demonstrated that breast cancer stem cells (BCSCs) are enriched in TNBC. Therefore, BCSCs serve as ideal therapeutic targets for TNBC. This study aims to identify novel therapeutic targets for BCSCs in TNBC. Based on the analysis of our unpublished RNA sequencing (RNA-Seq) data and patient data sets such as METABRIC and TCGA, we identify several potential oncogenes in TNBC. Our study further demonstrates that three of the candidates, namely cell division cycle associated 7 (CDCA7), low-density lipoprotein receptor-related protein 8 (LRP8), and mixed-lineage kinase 4 (MLK4), are functionally important to the maintenance of BCSCs in TNBC. The candidate genes are highly expressed in TNBC compared to other breast cancer subtypes according to the analysis of TCGA or METABRIC datasets. Genetic silencing of the candidate genes in TNBC cell lines significantly decreased CD44+/CD24- BCSCs and mammosphere formation in vitro. Furthermore, silencing of the genes suppressed both tumor growth and tumorigenesis in vivo. By analyzing the RNA-Seq data of the siRNA transfected TNBC cells, we found that knockdown of the candidate genes inhibited epithelial-to-mesenchymal transition (EMT), an important developmental program that can enrich stemness of cancer cells. Immunofluorescence staining of the xenograft tumor biopsies further revealed that the candidate gene knockdown decreased the expression of CD44 and increased the expression of CD24 and CK8/18, confirming the inhibition of EMT. Mechanistically, our RNA-Seq data analysis and experiments reveal that LRP8 and CDCA7 are critical to Wnt signaling and PRC2-mediated epithelial gene suppression, respectively. In addition, silencing of CDCA7 and MLK4 significantly dysregulates cell cycle of TNBC cells. Collectively, this study has demonstrated the benefits of targeting CDCA7, LRP8, and MLK4 to remove BCSCs and suppress tumorigenesis in TNBC. Therefore, our study uncovers LRP8, CDCA7, and MLK4 as novel therapeutic targets for TNBC.