Abstract
Although genomic sequencing has provided a better understating of the genetic landmarks in triple-negative breast cancer (TNBC), functional validation of candidate cancer genes (CCG) remains unsolved. In this study, we used a transposon mutagenesis strategy based on a two-step sleeping beauty (SB) forward genetic screen to identify and validate new tumor suppressors (TS) in this disease. We generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Among CCG, whose SB-mediated genetic mutation resulted in increased cellular proliferation in all cell lines tested, the genes ADNP, AP2B1, TOMM70A, and ZNF326 showed TS activity in tumor xenograft studies. Subsequent studies showed that ZNF326 regulated expression of multiple epithelial-mesenchymal transition and cancer stem cell (CSC) pathway genes. It also modulated expression of TS genes involved in the regulation of migration and cellular invasion and was a direct transcriptional activator of genes that regulate CSC self-renewal. ZNF326 expression associated with TNBC patient survival, with ZNF326 protein levels showing a marked reduction in TNBC. Our validation of several new TS genes in TNBC demonstrate the utility of two-step forward genetic screens in mice and offer an invaluable tool to identify novel candidate therapeutic pathways and targets. Cancer Res; 77(15); 4089-101. ©2017 AACR.
Highlights
Breast cancer is one of the leading causes of cancer-related deaths in women worldwide
Pten was used as a sensitizing mutation in the screen because loss of PTEN has been implicated in breast cancer progression, is clonally selected in Triple-negative breast cancer (TNBC), and favors the activation of the Epithelial–mesenchymal transition (EMT) pathway to promote metastasis [7, 25]
We asked whether downregulation of these genes increased cell proliferation, as this is one of the most commonly altered processes promoting tumorigenesis observed in tumors with mutations in tumor suppressors (TS) genes [26]
Summary
Breast cancer is one of the leading causes of cancer-related deaths in women worldwide. Triple-negative breast cancer (TNBC), which accounts for 10%–20% of all breast cancers, does not express estrogen or progesterone receptors and lacks HER2 amplification. Patients diagnosed with TNBC have a higher risk of disease relapse within 5 years than patients treated for other breast cancer subtypes [1, 2]. TNBC is the most aggressive, with a high relapse incidence and the most undifferentiated phenotype. Cancer Research Program, Houston Methodist Research Institute, Houston, Texas. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/)
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