Ovarian cancer is the most lethal gynecological malignancy in women. Metformin intake is associated with a reduced incidence of ovarian cancer and increased overall survival rate. We determined the effect of metformin on sphere formation, extracellular matrix invasion, and transcriptome profile of ovarian cancer cells (COVCAR) isolated from ascites of chickens that naturally developed ovarian cancer. We found that metformin treatment significantly decreased sphere formation and invasiveness of COVCAR cells. RNA-Seq data analysis revealed 0, 4, 365 differentially expressed genes in cells treated with 0.5, 1, 2 mM metformin, respectively compared to controls. Transcriptomic and ingenuity pathway analysis (IPA) revealed significant downregulation of MMP7, AICDA, GDPD2, APOC3, APOA1 and predicted inhibition of upstream regulators NFKB, STAT3, TP53 that are involved in epithelial–mesenchymal transition, DNA repair, and lipid metabolism. The analysis revealed significant upregulation of RASD2, IHH, CRABP-1 and predicted activation of upstream regulators VEGF and E2F1 that are associated with angiogenesis and cell cycle. Causal network analysis revealed novel pathways suggesting predicted inhibition of ovarian cancer through master regulator ASCL1 and dataset genes DCX, SEMA6B, HEY2, and KCNIP2. In summary, advanced pathway analysis in IPA revealed novel target genes, upstream regulators, and pathways affected by metformin treatment of COVCAR cells.

Neurofibromatosis type 1 (NF1) patients are predisposed to develop plexiform neurofibromas (PNFs). By cross-comparison of RNA sequencing and RUNX1-CHIP sequencing data on mouse PNFs, we found that transcripts encoding the NF1-interacting p97/valosin-containing protein (VCP) gene are overexpressed in PNFs. Co-immunoprecipitation confirmed that VCP bounded to neurofibromin. Western blot and immunostaining confirmed VCP overexpression in both mouse and human PNFs. Treatment of primary mouse PNF Schwann cells with CB-5083, a p97/VCP inhibitor, led to accumulation of poly-ubiquitinated proteins and generation of irresolvable proteotoxic stress. Pharmacological or genetic inhibition of VCP reduced mouse PNF cell-derived sphere number, and genetic inhibition of Vcp in Schwann cell precursors decreased tumor-like lesion numbers in a cell transplantation model. In vivo treatment with CB-5083 in Nf1^fl/fl;DhhCre PNF mice significantly inhibited cell proliferation, increased cell apoptosis and reduced PNF volume. The combination with a MEK inhibitor did not increase efficacy compared to the single agent, supporting the hypothesis that VCP functions in parallel to, and may be modulated by, RAS–MAPK signaling under stress or oncogenic conditions. The significant effects of VCP inhibition in this pre-clinical study suggest a potential novel therapy for patients with PNFs.