Fig 2: TTFields activate GEF-H1 and inhibit cancer cell motility in a Rho-dependent manner (A,B) A-172 and LN-229 glioblastoma cells were treated with TTFields for the indicated times and RhoA activation was assessed. Mean + SEM; paired t-test. (C) Western blot analysis of GEF-H1 phosphorylated on Ser886 and the total GEF-H1 levels in control and TTFields-treated cells (top panel). Quantification of normalized relative levels of P-GEF-H1 compared to total GEF-H1 levels from two western blots for each condition (bottom panel). (D) ROCK activity in A-172, LN-229, and U-87 MG cells treated with TTFields for 24 h. (E,F) Migration responses of (E) T-cells and (F) dendritic cells using the modified Boyden chamber either with or without CCL19. ns, not significant. Mean + SEM; paired t-test. * p < 0.05.

Fig 3: Downregulating neural lineage marker ARHGEF2 gene expression decreased C4-2B MDVR and H660 cell viability.a FPKM value of ARHGEF2, LHX2 and EPHB2 gene expression in 22RV1, LNCaP95, MSKCC-EF1 and H660 cells from GSE154576 dataset (n = 2). b Relative mRNA level of ARHGEF2, LHX2, and EPHB2 in C4-2B parental, MDVR, and H660 cells (n = 4). c Knocking down ARHGEF2 by siRNA decreased the expression of the markers of neuroendocrine including CHGA, NSE, and SYP in C4-2B MDVR and H660 cells. C4-2B MDVR and H660 cells were transfected with siRNAs for ARHGEF2 for 48 h, total cell lysates were collected and subjected to western blot analysis. d Knocking down ARHGEF2 by siRNA inhibited cell viability of C4-2B MDVR and H660 cells. C4-2B MDVR and H660 cells were transiently transfected with siRNAs targeting ARHGEF2 and viable cells were quantified using CellTiter Glo assay (n = 5). e qPCR analysis of AR targets and neuroendocrine markers and ARHGEF2 in NEPC LuCaP49 patient-derived xenograft (PDX) tumors and prostate adenocarcinoma LuCaP35CR tumors (n = 3). f Downregulation of ARHGEF2 by siRNA inhibits the growth of organoids derived from LuCaP49 PDX tumors. LuCaP49 organoids were seeded in a 96-well plate in the format of 3D Matrigel and then transfected with 50 nM ARHGEF2 siRNA and cultured for 14 days. The viability of the organoids was analyzed by CellTiter Glo and visualized by LIVE/DEAD staining (n = 4). Green = Calcein staining of live cells, Red = Ethidium homodimer-1 staining of dead cells. Scale bar 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001 (mean and SD in a, b, d, e, f) using one-way ANOVA with multiple-comparisons test.

Fig 4: Upregulated neural lineage genes correlate with poor overall survival in prostate cancer patients.a Gene expressions of the seven neural lineage signature genes were presented in the indicated groups of Beltran, Aggarwal, Labrecque, and Wassim cohorts. Gene expression from CRPC patients in Beltran study, prostate adenocarcinoma samples from Aggarwal, Labrecque, and Wassim study were presented in blue, while that of CRPC-NE samples from Beltran study, small-cell neuroendocrine prostate cancer from Aggarwal, Labrecque and Wassim studies was presented in purple. CRPC castration-resistant prostate cancer; CRPC-NE castration-resistant prostate cancer with neuroendocrine features; Adeno; non-SC non-small-cell prostate adenocarcinoma; Small-Cell small-cell neuroendocrine prostate cancer; prostate adenocarcinoma; SCNPC small-cell neuroendocrine prostate cancer; SC + NE small-cell neuroendocrine prostate cancer. *p < 0.05, **p < 0.01, ***p < 0.001, using unpaired t-test (median, quartiles and distribution of all data points presented). b Kaplan–Meier analysis of neural lineage signature genes including ARHGEF2, LHX2, and EPHB2 for the overall survival in Wassim cohorts. c Kaplan–Meier analysis of neural lineage signature genes for disease-free survival in MSKCC cohorts using log-rank test.

Fig 5: Loss of reactive oxygen species at the leading edge of wounded GCN2KO keratinocytes is coincident with reduced RAC1-GTP and branching F-actin.A, leading edge of migrating keratinocytes at 6 h postwounding in both WT and GCN2KO cells. Top two panels demonstrate ruffled edge (black arrows) of WT keratinocytes that are absent in GCN2KO keratinocytes (phase contrast, black bar = 20 μm). Bottom four panels stained with phalloidin-FITC (F-actin, green) and DAPI (nuclei, blue). Filopodia (yellow arrows) and lamellipodia (white arrows) are seen on the leading edge keratinocytes of WT but not GCN2KO cells (white bar = 10 μm). B, ROS production in WT and GCN2KO NTERT keratinocytes subjected to HDW (top two panels). In the lower four panels, WT NTERT cells were treated with vehicle (DMSO), 5 μM GCN2iB, 5 μM NOX inhibitor VAS2870, or 50 μM RAC1-GTP inhibitor NSC23766, as indicated, and cells were stained for ROS with CellRox Green 6 h after wounding. Cells were imaged with an Opera Phenix confocal microscope (100 μM scale bar). C, WT NTERT cells were treated with NOX inhibitors 5 μM VAS2870 or 300 nM DPI, or RAC1-GTP inhibitor 50 μM NSC23766 as indicated, at the time of wounding. As a control, GCN2KO cells were wounded without treatment. Percent wound closure was measured at 16 h post wounding. One-way ANOVA Dunnet’s multiple comparisons, (n = 3). D, levels of RAC1-GTP exchange factors ARHGEF2, ARHGEF6, ARHGEF18, TIAM1, and TIAM2 mRNAs in WT versus GCN2KO cells. Values are presented as normalized read counts from our RNA-seq analyses. Multiple unpaired t-tests, (n = 5). E, WT and GCNKO NTERT cells were subjected to wounding for up to 6 h and ARHGEF2, GCN2, and GAPDH levels were measured by immunoblot analyses. F, measurements of RHOA-GTP and RAC1-GTP by affinity purification assays. GST-Rhotekin or GST-PAK1-PBD fusion protein was used to bind the activated form of GTP-bound RHO or RAC1, respectively, in equivalent amounts of lysates prepared from WT and GCN2KO NTERT cells subjected to HDW (+) or no wounding (−). Levels of bound activated RHOA and RAC1 were collected using glutathione beads then measured by immunoblot using an antibody specific to total RAC1 or RHOA proteins. The measurements for RAC1-GTP and RHOA-GTP in the WT and GCN2KO cells were carried out in the same in same immunoblot experiment and therefore are directly comparable between the two panels. Quantification is provided below the panels and is normalized to WT not subject to wounding. G, measurements of total RAC1, RHOA, and GAPDH proteins from the lysates prepared from WT and GCN2KO NTERT cells subjected to HDW (+) or no wounding (−). Error bars are SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗ represents p < 0.0001.