SRC FAMILY KINASES MEDIATE INDIVIDUAL CELL RESPONSES TO RECEPTOR TYROSINE KINASE SIGNALING IN NEUROBLASTOMA CELLS

Receptor Tyrosine Kinases (RTKs) are involved in proliferation, differentiation, cellular metabolism, and homeostasis. More than half of the RTKs in the human genome are expressed in neuroblastoma cell lines. RKTs activate downstream signaling pathways including the canonical pathways of ERK, AKT, PLCγ, and SRC Family Kinases (SFKs). Phosphoproteomic data showed high expression and phosphorylation levels of SFK scaffold PAG1, and SFKs FYN and LYN in neuroblastoma cell lines and endosomes. We hypothesized that FYN and LYN distinguish between proliferation- and differentiation-inducing signals from different RTKs in neuroblastoma cells. To test this hypothesis, we measured changes in FYN and LYN’s intracellular location using cell fractionation in response to RTK activation. We found that FYN and LYN increased in endosomes and lysosomes in response to activation of ALK and KIT, RTKs that stimulate proliferation, whereas the FYN and LYN decreased in endosomes in response to activation of RET, which stimulates differentiation. PAG1kd cells were made using the CRISPR-CAS9 system in order to define the effect of PAG1 ablation on proliferation, differentiation, and activation of SFKs and downstream pathways. We used flow cytometry to simultaneously measure the level of intracellular phosphoproteins and DNA content in adherent neuroblastoma cells. We found that PAG1 ablation increased the levels of !V activated SFKs, as well and proliferation, as shown by increased number of cells in the S and G2-M stages of the cell cycle, and an increased absorbances in MTT assays. PAG1kd cells displayed increased number of cells with high levels of pAKT, which were located in the G2-M stages of the cell cycle for both PAG1kd and wild type cells. In addition, PAG1 ablation differentially affected the activation of the ERK pathway downstream from RTKs that induce proliferation. The levels of pERK increased after EGFR activation and were not affected by PAG1 ablation, whereas KIT activation slightly increased only long term activation of ERK, and this increase was suppressed by PAG1 ablation. We then evaluated differentiation in PAG1kd cells. PAG1kd cells did not respond to treatment with Retinoic Acid and NGF: PAG1kd cells did not change their proliferation rates or their cell cycle distribution. PAG1kd cells dramatically decrease the expression of the neuronal marker of differentiation, β III-Tubulin. In addition, PAG1 ablation prevented the activation of ERK in response to activation of TRKA and RET. Our data show that FYN and LYN increase in late endosomes and lysosomes in response to activation of RTKs that induce proliferation, such as ALK, and KIT. In addition, tumor suppressor PAG1 is a key regulator of proliferation, differentiation, and RTK mediated signaling in neuroblastoma cells. Future studies will define therapeutic potential of targeting PAG1 expression in the treatment of neuroblastoma