Abstract 2875: Knockout of ST8SIA1 inhibits tumorigenesis in triple negative breast cancer by inducing PTEN and ganglioside GM1 mediated tumor growth arrest

Abstract

Abstract We found that ganglioside GD2 is a biomarker for breast cancer stem cell (BCSCs) in triple negative breast cancer (TNBC) and that GD2 biosynthesis is tightly regulated by enzyme ST8SIA1 (GD3 synthase) in these cells. We have reported that ST8SIA1 is highly expressed in TNBC and its expression is highly correlated with TP53 mutations in primary tumors (based on TCGA data set). Here we hypothesized that ST8SIA1 has a functional role in BCSC mediated tumorigenesis in TNBC. To test this hypothesis, we deleted ST8SIA1 in SUM159 cells using CRISPR-Cas9 technology. As expected, deletion of ST8SIA1 in SUM159 cells reduced the absolute number of GD2+ cells from 17 ± 1.5% to 0.3 ± 0.1%. Although there was no significant difference in 2D cell growth, anchorage-independent growth by soft-agar and the mammosphere formation assays revealed a complete loss of colony formation in ST8SIA1-KO cells. Moreover, transplantation of ST8SIA1-KO- or Cas9 control- SUM159 cells in mammary fat pad of NSG mice revealed that ST8SIA1-KO cells failed to produce tumor formation even after a 15-wk after implantation. In addition, most of the cas9 control cell injected mice died within 4 wk after cell implantation whereas no deaths were observed in the ST8SIA1-KO cells as long as 100 d after tumor implantation. To investigate the mechanism involved in tumor growth inhibition associated with the loss of ST8SIA1 expression, we analyzed mRNA expression in ST8SIA1-KO- and Cas9 control- SUM159 cells by RNA sequencing. At p < 0.05 and fold change >2, we found 1502 genes down-regulated and 842 genes up-regulated in the ST8SIA1-KO-cells compared to controls. Ingenuity pathway analysis revealed that several stem cell-associated signaling pathways, including wnt, stat3, NFκB, nanog, and IL8, were down-regulated. Conversely, the potent tumor suppressor PTEN was induced in the ST8SIA1-KO-cells compared to controls. In addition, gene ontology by gene set enrichment analysis (GSEA) revealed inhibition of oncogenic signaling pathways including KRAS, p53, AKT, and RB. We have also identified a significant down-regulation of breast cancer-associated genes including BCL11A, PDGFRb, VCAM1, CXCR4, and wnt5a. Interestingly DKK1, which acts as an antagonist for wnt-β-catenin signaling, was up-regulated in the ST8SIA1-KO cells. These findings were validated by qRT-PCR, flow cytometry and western blot analysis. Ganglioside GM1 has been reported to have major in anti-tumor effect by inhibiting ERK signaling. Flow cytometry analysis revealed that ST8SIA-KO cells expressed ~10-fold higher GM1 compared to Cas9 control cells. In addition, pERK was down-regulated in ST8SIA-KO cells, suggesting a loss of tumorigenic potential of these cells. In conclusion, our data suggests that inhibition of ST8SIA1 in TNBC cells depletes BCSCs and inhibits tumorigenesis by down-regulating oncogenic pathways and up-regulation of PTEN and GM1 mediated signaling. Citation Format: Khoa Nguyen, Yuanqing Yan, Chandra Bartholomeusz, Naoto Ueno, Kim-Anh Do, Michael Andreeff, V. Lokesh Battula. Knockout of ST8SIA1 inhibits tumorigenesis in triple negative breast cancer by inducing PTEN and ganglioside GM1 mediated tumor growth arrest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2875. doi:10.1158/1538-7445.AM2017-2875