Figure S1. Expression of ZNF326 a in human breast cancer tissues. The images show a representative scoring intensities following immunostaining with anti-ZNF326 antibody (Range of low, moderate and high). Figure S2. RT-PCR analysis of the candidate cancer genes downregulated by shRNA-pools in the (A) HCC70 and (B) MDA-MB-231 breast cancer cell lines. Figure S3. Stable ZNF326 knockdown in (A) HCC70, (B) HCC1569 and (C) MDA-MB-231 TNBC cell lines using two independent shRNAs was confirmed by qRT-PCR. (D) Ectopic expression of ZNF326 in MDA-MB-231 cells transduced with lentivirus particles expressing ZNF326 cDNA and empty vector (control) respectively. ZNF326 mRNA levels were quantified by qRT-PCR. Figure S4. Western blot analysis of mammosphere cell lysates of HCC70 (A) and MDA-MB-231 (B) with stable shRNA (left panel) and ectopic overexpressing ZNF326 (right panel). Figure S5. Gene expression-based outcome for breast cancer online (GOBO) was used to obtain the expression analysis of (A) DACH1 and (B) GATA3 in different breast cancer subtypes.
ARTICLE 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.
