Abstract P5-07-02: ST8SIA1 regulates tumorigenesis in triple negative breast cancer
Authors: Battula VL , Nguyen K , Sun JC , Dasgupta A , Bartholomeusz C , Andreeff M 
Published: March 2017

Recurrence in breast cancer is mainly due to metastases and drug resistance in a fraction of primary tumors cells which are also known as cancer stem cells or tumor initiating cells. We found that Ganglioside GD2 identifies breast cancer stem cell (BCSCs) in triple negative breast cancer (TNBC) and that GD2 biosynthesis is tightly regulated by enzyme ST8SIA1 (GD3 synthase) in GD2+ cells. We have reported that ST8SIA1 is highly expressed in TNBC and its expression is highly correlated with p53 mutations primary tumors (Yan et al, SABCS abstract 2016). Here we hypothesize that ST8SIA1 has a functional role in BCSC mediated tumorigenesis in TNBC. To test the hypothesis, we deleted ST8SIA1 in SUM159 cells using CRISPR-Cas9 technology. As expected, deletion of ST8SIA1 in SUM159 cells reduced GD2+ cells from 17±1.5% to 0.3±0.1%. However, cell proliferation assay revealed no significant difference between ST8SIA1-KO and Cas9 control cells. In contrast, in-vitro tumorigenesis by soft-agar assays revealed a complete loss of colony formation in ST8SIA1-KO cells, whereas Cas9 control cells produced 30±10 colonies out of 1000 cells plated. To investigate tumor initiation potential, ST8SIA1-KO- or Cas9 control- SUM159 cells were transplanted in mammary fat pad of NSG mice. Cas9 control cells produced tumors within 1-2 weeks and reached the maximum allowed size by IACUC (1.5cm) within 3-4 weeks. In contrast, ST8SIA1-KO cells failed to produce any tumors even 15 weeks after injections. In addition, survival analysis by log-rank test revealed that most of the cas9 control cell injected mice died within 4 weeks after cell implantation whereas no deaths were observed in ST8SIA1-KO cells injected mice even 100 days after tumor implantation. These data indicate that loss of ST8SIA1 in TNBC cells depletes GD2+ BCSCs and inhibits in-vitro and in-vivo tumorigenesis.

To investigate gene expression changes due to loss of ST8SIA1 in CRISPR knockout cells, we analyzed mRNA expression in ST8SIA1-KO- and Cas9 control- SUM159 cells by RNAseq analysis (done in triplicates for each cell type). At p&lt;0.05 and fold change &gt;2, we found 1502 genes down-regulated and 842 genes up-regulated in ST8SIA1-KO- compared to cas9 control- cells. Ingenuity pathway analysis revealed that several stem cell associated signaling pathway including, wnt, stat3, NFκB, nanog and IL8 whereas tumor suppressor PTEN and p38 MAPK signaling were activated in ST8SIA1-KO- compared to cas9 control- cells. In specific, proteins associated with stem cell function including NOTCH3, PDGFRB, PDGFRA, VCAM1, CXCR4, CXCL12, SOX2, wnt5a were down regulated in ST8SIA1-KO cells whereas DKK1 which acts as an antagonist for wnt-β-catenin signaling, was up-regulated in ST8SIA1-KO cells. These findings were validated by flow cytometry and western blot analysis using specific antibodies. In conclusion, our data suggests that deletion of ST8SIA1 in TNBC cells depletes BCSCs and inhibits tumorigenesis in-vitro and in-vivo. Development of specific inhibitors of ST8SIA1 could be of potential therapeutic value for patients with TNBC.

Citation Format: Battula VL, Nguyen K, Sun JC, Dasgupta A, Bartholomeusz C, Andreeff M. ST8SIA1 regulates tumorigenesis in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-02.