Abstract P5-07-03: GD2-mediated FAK signaling regulates breast cancer stem cell function in triple negative breast cancer
Authors: Nguyen K , Sun JC , Hortobagyi GN , Andreeff M , Battula VL 
Published: March 2017

Ganglioside GD2 identifies breast cancer stem cells (BCSCs, Battula et al., JCI, 2012) and expression of GD2 is tightly regulated by GD3 synthase (GD3S). GD3S is highly expressed in GD2+ cells and inhibition of GD3S inhibits tumor formation and metastasis of breast cancer cells. However, the mechanism of GD2-mediated regulation of BCSC function is not known. Here we hypothesize that GD2 regulates signaling pathways involved in cell adhesion, migration and invasion of breast cancer cells. To identify these signaling pathways, antibody micro-arrays were used with 850 validated antibodies specific to total or phosphorylated proteins. Interestingly, focal adhesion kinase (FAK) was the most significantly phosphorylated protein in GD2+ compared to GD2- cells (S910 and S722). In addition, expression of FAK downstream mediators including Csk, PKCq, PKCl/I, Pyk2, and p38MAPK, was up-regulated in GD2+ compared to GD2- cells. Western blot analysis of FACS sorted SUM159 cells also revealed increased phosphorylation of FAK &gt;80% at Y397 and &gt;25% at Y861 in GD2+ compared to GD2- cells. FAK downstream targets including paxillin, p130 Cas, pERK were also up-regulated in GD2+ cells compared to GD2- cells indicating definitive activation of FAK signaling in GD2+ BCSCs. To investigate the functional role of GD2 in FAK mediated functions, we genetically deleted GD3S using the CRISPR knock-out system in SUM159 cells. only &lt;1% GD2expression compared to &gt;20% in parental cells was observed. GD3S-KO cells grew 5-10% slower in cell culture mostly because of the reduction (15±5%) in adherence. Trans-well assays revealed 3-5 fold reduction in migration and invasion of GD3S-KO compared to parental cells. These data indicate that GD2 and GD3S are not only the markers of BCSCs but also regulators of their function. Finally, we tested the effect of FAK inhibitor (PF-573228) against GD2+ BCSCs and GD3S-KO SUM159 cells. PF-573228 treatment decreased the number of mammospheres generated by GD2+ cells 3-4 fold in a dose dependent manner (100nM-1µm). In addition, treatment of PF-573228, inhibited migration and invasion of GD2+ cells 2 and 3 fold, respectively. However, treatment with PF-573228 on GD3S-KO cells further reduced their ability to migrate and invade by over 70% compared to untreated cells. In addition, GD3S-KO cells failed to form any mammospheres when cultured under low adherence conditions (p&lt;0.00001), whereas the parental cells formed 15-20 mammospheres per 1,000 cells plated. In conclusion, our data demonstrate that FAK signaling is activated in GD2+ cells but that FAK inhibition alone may not be sufficient to inhibit BCSC function. Combined FAK and GD3S inhibition may exert highly synergistic effects against BCSCs.

Citation Format: Nguyen K, Sun JC, Hortobagyi GN, Andreeff M, Battula VL. GD2-mediated FAK signaling regulates breast cancer stem cell function 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 P5-07-03.