Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways

Abstract

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

Keywords: PAK1; cell migration; cell proliferation; drug resistance; genetic interaction; glioma; kinase; molecular cell biology; signal transduction.

Figure 1.

Evaluation of PAK1 genetic interactions in organotypic brain slices. A, experimental timeline. DiI-labeled spheroids were implanted onto brain slice cultures on day 1. Confocal imaging and inhibitor treatment were performed at the given time points. B–F, GL26 glioma spheroids stably expressing empty vector or PAK1 (GL26-PAK1-1 and GL26-PAK1-2) were implanted onto the slice cultures followed by treatment with vehicle or pharmacological inhibitors of potential PAK1 interaction partners: fumagillin, 1 nm (B); monastrol, 1 μm (C); MPS1-IN-1, 100 nm (D); rapamycin, 100 nm (E); vildagliptin, 250 nm (F). The Z-stack images were superimposed into a single image representing the entire spheroid. Invasive cell density was quantified as shown in the graphs. Scale bar, 200 μm. *, p < 0.05 (Student's t test comparing empty versus PAK1 groups, n = 3); #, p < 0.05 (Student's t test comparing vehicle versus inhibitor groups, n = 3); NS, not significant. Error bars, S.D.

Figure 2.

Evaluation of PAK1 genetic interactions in an orthotopic model of glioma. A, experimental timeline. C57BL/6 mice were injected with 9 × 10⁵ empty vector– or PAK1–expressing GL26-effLuc cells in the striatum. Glioma-implanted mice were injected with inhibitors (MPS1-IN-1, rapamycin, or vildagliptin) or vehicle, and IVIS images were obtained at the indicated time points to assess glioma growth. B–D, IVIS images were collected on days 2, 6, 11, and 16, and glioma growth in the presence of vehicle or inhibitors was quantified in the graphs: MPS1-IN-1, 100 nm, 5 µl (B); rapamycin, 100 nm, 5 µl (C); vildagliptin, 250 nm, 5 µl (D). Photon intensity of the images is directly related to tumor size. Scale bar, 5 mm. *, p < 0.05 (Student's t test comparing empty versus PAK1-1 group, n = 5); #, p < 0.05 (Student's t test comparing vehicle versus inhibitor groups, n = 5); NS, not significant. Error bars, S.D.

Figure 3.

Assessment of tumor volume and invasiveness in an orthotopic model of glioma. After IVIS imaging (as in Fig. 2), mice (n = 5) from each group (empty vector– or PAK1–transfected GL26 glioma cells implanted; vehicle or inhibitors injected) were euthanized to obtain brain tissues, and the tumor volume (A and B) and invasiveness (C) were evaluated. A, representative hematoxylin-stained images from serial sections confirm viable regions of tumor. B, tumor volume was calculated by quantitative digital image analysis of the serial sections containing tumor. C, overall tumor invasion was quantified by particle analysis of threshold images prepared from tumor-bearing sections of engrafted animals. Tumor invasion in each group of animals was assessed by counting the number of tumor particles invading the brain parenchyma per tumor rim length. Scale bar, 2 mm. *, p < 0.05 (Student's t test, n = 5); #, p < 0.05 (Student's t test, n = 5); NS, not significant. Error bars, S.D.

Figure 4.

Role of AKT1 in the genetic interaction between PAK1 and TTK. A, experimental timeline. NIH3T3 cells were transiently transfected with empty vector, PAK1, or TTK cDNAs and treated with inhibitors (TTK inhibitor, MPS1-IN-1; PAK1 inhibitor, IPA3), and signaling pathway or cell migration was analyzed by Western blotting or a wound-healing assay, respectively. B, phospho-AKT (p-AKT), total AKT (t-AKT), or β-actin protein levels were measured by Western blotting. Quantification of the band intensities is presented in the adjacent graphs. *, p < 0.05 (Student's t test, n = 3); #, p < 0.05 (Student's t test, n = 3). C, quantification of cell migration based on the wound-healing assays. *, p < 0.05 (Student's t test comparing empty vector–transfected versus TTK- or PAK1-transfected group, n = 5); #, p < 0.05 (Student's t test, n = 5). Error bars, S.D. D, schematic representation of the PAK1, TTK, and AKT pathways. The PAK1-TTK-AKT1 axis appears to play an important role in glioma migration/invasion, drug resistance, and proliferation. PAK1-mTOR or PAK1-DPP4 may play a similar role through either AKT1-dependent or -independent pathways.