Figure 1.

NF1 deficiency increases cell proliferation and increases RAS and PI3K/AKT pathway signaling. A) Schematic of the NF1-deficient human breast cancer MCF7 model. Schematic of wild-type and mutant neurofibromin in the MCF7 NF1-EV and NF1-45 respectively. The G1 and G2 arrows within the GRD denote the CRISPR guide positioning. Non-GRD domains shown include the cysteine/serine-rich domain (CSDR), Sec14 homologous domain and pleckstrin homologous domain (SEC14/PH), and c-terminal domain (CTD). B) Schematic of Nf1-deficient rat and rat-derived fibroblast and tumor cell line models. Schematic of neurofibromin in the Nf1-WT, Nf1-IF, Nf1-PS, Nf1-IFPS rat models. The G1 and G2 arrows within the GRD denote thesCRISPR guide positioning. C) NF1 deficiency increases cell proliferation in MCF7 cells and rat mammary fibroblasts, n = 2, 24 tech reps per n. The bar represents the geometric mean, and the black, horizontal lines represent the 95 % confidence interval boundaries. D) NF1-deficient MUT-45 spheroids grow larger than NF1-EV spheroids when grown in Matrigel. (n = 3+, 1 representative technical replicate shown above, day 10 of 3D culture). E) Schematic description of the role of neurofibromin in the ER, RAS, and PI3K/AKT signaling pathways and relevant targeted inhibitors: Cobimetinib (COB), Tamoxifen (4OHT), and Everolimus (EVE). F) NF1 indels alter neurofibromin expression. Western blot analysis of total neurofibromin protein. Immunoblotting was carried out using antibodies against neurofibromin (H12, Bethyl, iNF-07E) and β-tubulin (n = 3+, 1 representative biological replicate shown). G) NF1 deficiency increases RAS and AKT/PI3K pathway signaling. Western blot analysis of ER, RAS, and PI3K/AKT phospho-signaling. Immunoblotting was carried out using antibodies against neurofibromin (Bethyl), pERα S118, ERα, pERK T202/Y204, ERK, pAKT S473, AKT, and β-tubulin (n = 3, 1 representative biological replicate shown).