ABSTRACT
Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-ß1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.
Subject(s)
Carcinoma, Pancreatic Ductal , Cell Cycle Proteins/metabolism , MAP Kinase Signaling System/drug effects , Mutation , Pancreatic Neoplasms , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins p21(ras) , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/enzymology , Carcinoma, Pancreatic Ductal/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Humans , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/genetics , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolismABSTRACT
Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis - differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.
Subject(s)
Chemotaxis , Fibronectins/pharmacology , Pseudopodia/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Animals , Chemotaxis/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Focal Adhesions/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Integrin beta1/metabolism , Mice , Models, Biological , Signal Transduction/drug effects , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , Wiskott-Aldrich Syndrome Protein/metabolism , Wiskott-Aldrich Syndrome Protein Family/metabolism , rac GTP-Binding Proteins/metabolism , src-Family Kinases/metabolismABSTRACT
G-protein ß subunits perform essential neuronal functions as part of G-protein ßγ and Gß5-regulators of G-protein signaling (RGS) complexes. Both Gßγ and Gß5-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a cochaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesis in vivo, we deleted the Phlp1 gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G-protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gßγ dimer formation was decreased 50-fold, resulting in a >10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gß5 and RGS9-1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstrate in vivo that PhLP1 is required for the folding and assembly of both Gßγ and Gß5-RGS9.
Subject(s)
Eye Proteins/metabolism , GTP-Binding Proteins/metabolism , Retina/cytology , Retinal Rod Photoreceptor Cells/metabolism , Signal Transduction/physiology , Animals , Biophysical Phenomena/genetics , Contrast Sensitivity/genetics , Dose-Response Relationship, Radiation , Electric Stimulation , Electroretinography , Eye Proteins/genetics , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Gene Expression Regulation/genetics , In Vitro Techniques , Light , Membrane Potentials/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Patch-Clamp Techniques , Photic Stimulation , RNA, Messenger/metabolism , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Retinal Degeneration/physiopathology , Visual Acuity/geneticsABSTRACT
To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-ß-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the ßIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.
Subject(s)
Crk-Associated Substrate Protein/metabolism , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Microtubules/metabolism , Pancreatic Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-myc/metabolism , Acetamides/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Calpain/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Drug Synergism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Half-Life , Humans , Microtubules/drug effects , Morpholines/pharmacology , Mutation/genetics , Organoids/drug effects , Organoids/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Pyridines/pharmacology , Transcription, Genetic/drug effects , Tubulin/metabolism , Xenograft Model Antitumor Assays , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolismABSTRACT
Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOAY42C, the most common RHOA mutation in DGC, is a gain-of-function oncogenic mutant, and that expression of RHOAY42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOAY42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOA Y42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP-TAZ, PI3K-AKT, and ß-catenin. RHOAY42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers. SIGNIFICANCE: The functional significance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOAY42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and define how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors.See related commentary by Benton and Chernoff, p. 182.This article is highlighted in the In This Issue feature, p. 161.
Subject(s)
Focal Adhesion Kinase 1/metabolism , Protein Kinase Inhibitors/administration & dosage , Quinolones/administration & dosage , Stomach Neoplasms/genetics , Sulfones/administration & dosage , rhoA GTP-Binding Protein/genetics , 3T3 Cells , Animals , Antigens, CD/metabolism , COS Cells , Cadherins/metabolism , Chlorocebus aethiops , Focal Adhesion Kinase 1/antagonists & inhibitors , Gain of Function Mutation , Gastric Mucosa/pathology , HEK293 Cells , Humans , Mice , Signal Transduction/drug effects , Signal Transduction/genetics , Stomach Neoplasms/drug therapy , Stomach Neoplasms/pathology , Xenograft Model Antitumor Assays , rhoA GTP-Binding Protein/metabolismABSTRACT
Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry-based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser62 Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.
Subject(s)
Cyclin-Dependent Kinase 9/antagonists & inhibitors , Drug Screening Assays, Antitumor/methods , Pancreatic Neoplasms/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cyclin-Dependent Kinase 9/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Molecular Structure , Mutation , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Stability , Proteolysis , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3ß signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.
Subject(s)
Carcinoma, Pancreatic Ductal/pathology , MAP Kinase Kinase 5/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinase 7/metabolism , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Carcinoma, Pancreatic Ductal/genetics , Cell Line, Tumor , ErbB Receptors/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Pancreatic Neoplasms/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , src-Family Kinases/metabolismABSTRACT
G protein ß subunits (Gß) play essential roles in phototransduction as part of G protein ßγ (Gßγ) and regulator of G protein signaling 9 (RGS9)-Gß5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2) and RGS9-Gß5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gßγ and RGS9-Gß5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gß complex formation in G protein signaling.