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1.
Science ; 384(6700): eadk0775, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38843331

ABSTRACT

How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.


Subject(s)
Carcinoma, Pancreatic Ductal , Extracellular Signal-Regulated MAP Kinases , Gene Expression Regulation, Neoplastic , MAP Kinase Signaling System , Mutation , Pancreatic Neoplasms , Proto-Oncogene Proteins p21(ras) , Transcriptome , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Animals , Cell Line, Tumor , Mice , Drug Resistance, Neoplasm/genetics
2.
Science ; 384(6700): eadk0850, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38843329

ABSTRACT

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.


Subject(s)
Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Mutation , Pancreatic Neoplasms , Phosphoproteins , Proteome , Proto-Oncogene Proteins p21(ras) , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Phosphorylation , Mitogen-Activated Protein Kinase 1/metabolism , Phosphoproteins/metabolism , Phosphoproteins/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Cell Line, Tumor , MAP Kinase Signaling System , Animals , Mice , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/genetics
3.
Zool Res ; 45(3): 551-566, 2024 May 18.
Article in English | MEDLINE | ID: mdl-38757223

ABSTRACT

Hepatocellular carcinoma (HCC), a prevalent solid carcinoma of significant concern, is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes. The etiology and pathological progression of non-alcoholic steatohepatitis (NASH)-related HCC is multifactorial and multistage. However, no single animal model can accurately mimic the full NASH-related HCC pathological progression, posing considerable challenges to transition and mechanistic studies. Herein, a novel conditional inducible wild-type human HRAS overexpressed mouse model (HRAS-HCC) was established, demonstrating 100% morbidity and mortality within approximately one month under normal dietary and lifestyle conditions. Advanced symptoms of HCC such as ascites, thrombus, internal hemorrhage, jaundice, and lung metastasis were successfully replicated in mice. In-depth pathological features of NASH- related HCC were demonstrated by pathological staining, biochemical analyses, and typical marker gene detections. Combined murine anti-PD-1 and sorafenib treatment effectively prolonged mouse survival, further confirming the accuracy and reliability of the model. Based on protein-protein interaction (PPI) network and RNA sequencing analyses, we speculated that overexpression of HRAS may initiate the THBS1-COL4A3 axis to induce NASH with severe fibrosis, with subsequent progression to HCC. Collectively, our study successfully duplicated natural sequential progression in a single murine model over a very short period, providing an accurate and reliable preclinical tool for therapeutic evaluations targeting the NASH to HCC continuum.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Proto-Oncogene Proteins p21(ras) , Animals , Non-alcoholic Fatty Liver Disease/pathology , Non-alcoholic Fatty Liver Disease/genetics , Carcinoma, Hepatocellular/pathology , Mice , Liver Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Disease Models, Animal , Mice, Transgenic , Mice, Inbred C57BL , Humans
4.
Protein Sci ; 33(6): e5016, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38747381

ABSTRACT

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.


Subject(s)
14-3-3 Proteins , Nanostructures , Proto-Oncogene Proteins B-raf , Proto-Oncogene Proteins p21(ras) , 14-3-3 Proteins/metabolism , 14-3-3 Proteins/chemistry , 14-3-3 Proteins/genetics , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Humans , Nanostructures/chemistry , Protein Multimerization , Protein Binding , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism
5.
Gen Physiol Biophys ; 43(3): 243-253, 2024 May.
Article in English | MEDLINE | ID: mdl-38774924

ABSTRACT

Cataract, a painless and progressive disorder is manifested as the opacification of the lens that represents the most significant cause of blindness worldwide. The objective of this study is to unveil the function of Kirsten rat sarcoma (KRAS) and potential action mechanisms against cataract. The ferroptosis-associated differentially expressed genes (DEGs) and pivot genes were extracted through the comprehensive bioinformatics methods. Erastin was applied for inducing ferroptosis in hydrogen peroxide (H2O2)-treated SRA01/04 cells, and validated by detecting content of intracellular iron, glutathione (GSH), malondialdehyde (MDA). Additionally, the effects of KRAS deficiency on ferroptosis were determined by functional assays. The proteins expression related to ferroptosis and Hippo pathway were determined by Western blotting. A total of 73 ferroptosis-related DEGs were discovered, and 6 critical core genes were confirmed upregulation in cataract cell model. The H2O2-treated SRA01/04 cells exhibited decrease of cell viability and proliferation, iron accumulation, MDA increase, GSH consumption, rise of COX2 and decline of GPX4, with further aggravated under erastin treatment, while the phenomena were improved by KRAS knockdown. Additionally, KRAS deficiency was involved in the Hippo signalling pathway activation. Downregulation of KRAS might restrain ferroptosis and affect Hippo pathway in cataract.


Subject(s)
Cataract , Ferroptosis , Hippo Signaling Pathway , Proto-Oncogene Proteins p21(ras) , Signal Transduction , Ferroptosis/drug effects , Cataract/metabolism , Cataract/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Humans , Signal Transduction/drug effects , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Cell Line
6.
Int Rev Cell Mol Biol ; 386: 167-222, 2024.
Article in English | MEDLINE | ID: mdl-38782499

ABSTRACT

Historically, KRAS has been considered 'undruggable' inspite of being one of the most frequently altered oncogenic proteins in solid tumors, primarily due to the paucity of pharmacologically 'druggable' pockets within the mutant isoforms. However, pioneering developments in drug design capable of targeting the mutant KRAS isoforms especially KRASG12C-mutant cancers, have opened the doors for emergence of combination therapies comprising of a plethora of inhibitors targeting different signaling pathways. SHP2 signaling pathway, primarily known for activation of intracellular signaling pathways such as KRAS has come up as a potential target for such combination therapies as it emerged to be the signaling protein connecting KRAS and the immune signaling pathways and providing the link for understanding the overlapping regions of RAS/ERK/MAPK signaling cascade. Thus, SHP2 inhibitors having potent tumoricidal activity as well as role in immunomodulation have generated keen interest in researchers to explore its potential as combination therapy in KRAS mutant solid tumors. However, the excitement with these combination therapies need to overcome challenges thrown up by drug resistance and enhanced toxicity. In this review, we will discuss KRAS and SHP2 signaling pathways and their roles in immunomodulation and regulation of tumor microenvironment and also analyze the positive effects and drawbacks of the different combination therapies targeted at these signaling pathways along with their present and future potential to treat solid tumors.


Subject(s)
Immunomodulation , Neoplasms , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Proto-Oncogene Proteins p21(ras) , Signal Transduction , Humans , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/therapy , Signal Transduction/drug effects , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Immunomodulation/drug effects , Animals , Treatment Outcome , Molecular Targeted Therapy
7.
Int J Mol Sci ; 25(9)2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38731942

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) can originate from acinar-to-ductal metaplasia (ADM). Pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is frequently observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D regulates this process remain an enigma. Herein, we revealed a new downstream target of oncogenic Kras, cytokine CCL9, during ADM formation. Higher levels of CCL9 and its receptors, CCR1 and CCR3, were detected in ADM regions of the pancreas in p48cre:KrasG12D mice and human PDAC patients. Knockdown of CCL9 in KrasG12D-expressed pancreatic acini reduced KrasG12D-induced ADM in a 3D organoid culture system. Moreover, exogenously added recombinant CCL9 and overexpression of CCL9 in primary pancreatic acini induced pancreatic ADM. We also showed that, functioning as a downstream target of KrasG12D, CCL9 promoted pancreatic ADM through upregulation of the intracellular levels of reactive oxygen species (ROS) and metalloproteinases (MMPs), including MMP14, MMP3 and MMP2. Blockade of MMPs via its generic inhibitor GM6001 or knockdown of specific MMP such as MMP14 and MMP3 decreased CCL9-induced pancreatic ADM. In p48cre:KrasG12D transgenic mice, blockade of CCL9 through its specific neutralizing antibody attenuated pancreatic ADM structures and PanIN lesion formation. Furthermore, it also diminished infiltrating macrophages and expression of MMP14, MMP3 and MMP2 in the ADM areas. Altogether, our results provide novel mechanistic insight into how oncogenic Kras enhances pancreatic ADM through its new downstream target molecule, CCL9, to initiate PDAC.


Subject(s)
Acinar Cells , Carcinoma, Pancreatic Ductal , Metaplasia , Pancreatic Neoplasms , Proto-Oncogene Proteins p21(ras) , Reactive Oxygen Species , Animals , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Mice , Reactive Oxygen Species/metabolism , Humans , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Metaplasia/metabolism , Metaplasia/genetics , Acinar Cells/metabolism , Acinar Cells/pathology , Mice, Transgenic , Chemokines, CC/metabolism , Chemokines, CC/genetics , Macrophage Inflammatory Proteins/metabolism , Macrophage Inflammatory Proteins/genetics , Pancreas/metabolism , Pancreas/pathology
8.
Langmuir ; 40(19): 10157-10170, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38700902

ABSTRACT

I-Motif (iM) DNA structures represent among the most significant noncanonical nucleic acid configurations. iM-forming DNA sequences are found in an array of vital genomic locations and are particularly frequent in the promoter islands of various oncogenes. Thus, iM DNA is a crucial candidate for anticancer medicines; therefore, binding interactions between iM DNA and small molecular ligands, such as flavonoids, are critically important. Extensive sets of spectroscopic strategies and thermodynamic analysis were utilized in the present investigation to find out the favorable interaction of quercetin (Que), a dietary flavonoid that has various health-promoting characteristics, including anticancer properties, with noncanonical iM DNA structure. Spectroscopic studies and thermal analysis revealed that Que interacts preferentially with HRAS1 iM DNA compared with VEGF, BCL2 iM, and duplex DNA. Que, therefore, emerged as a suitable natural-product-oriented antagonist for targeting HRAS1 iM DNA. The innovative spectroscopic as well as mechanical features of Que and its specific affinity for HRAS1 iM may be useful for therapeutic applications and provide crucial insights for the design of compounds with remarkable medicinal properties.


Subject(s)
DNA , Promoter Regions, Genetic , Proto-Oncogene Proteins p21(ras) , Quercetin , Quercetin/chemistry , Quercetin/pharmacology , Quercetin/metabolism , DNA/chemistry , DNA/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/metabolism , Thermodynamics , Humans , Nucleotide Motifs , Binding Sites
9.
Nat Commun ; 15(1): 4642, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38821916

ABSTRACT

Post-translational modifications of proteins in malignant transformation and tumor maintenance of pancreatic ductal adenocarcinoma (PDAC) in the context of KRAS signaling remain poorly understood. Here, we use the KPC mouse model to examine the effect of palmitoylation on pancreatic cancer progression. ZDHHC20, upregulated by KRAS, is abnormally overexpressed and associated with poor prognosis in patients with pancreatic cancer. Dysregulation of ZDHHC20 promotes pancreatic cancer progression in a palmitoylation-dependent manner. ZDHHC20 inhibits the chaperone-mediated autophagic degradation of YTHDF3 through S-palmitoylation of Cys474, which can result in abnormal accumulation of the oncogenic product MYC and thereby promote the malignant phenotypes of cancer cells. Further, we design a biologically active YTHDF3-derived peptide to competitively inhibit YTHDF3 palmitoylation mediated by ZDHHC20, which in turn downregulates MYC expression and inhibits the progression of KRAS mutant pancreatic cancer. Thus, these findings highlight the therapeutic potential of targeting the ZDHHC20-YTHDF3-MYC signaling axis in pancreatic cancer.


Subject(s)
Acyltransferases , Carcinoma, Pancreatic Ductal , Disease Progression , Gene Expression Regulation, Neoplastic , Lipoylation , Pancreatic Neoplasms , Proto-Oncogene Proteins c-myc , Proto-Oncogene Proteins p21(ras) , Animals , Humans , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Acyltransferases/metabolism , Acyltransferases/genetics , Mice , Cell Line, Tumor , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Male , Signal Transduction , RNA Stability , Female
10.
Life Sci ; 348: 122680, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38697280

ABSTRACT

AIMS: Hepatocellular carcinoma (HCC) is still a leading cause of cancer-related death worldwide. But its chemotherapeutic options are far from expectation. We here compared H-ras targeted genetic therapy to a commercial docetaxel formulation (DXT) in inhibiting HCC in rats. MAIN METHODS: After the physicochemical characterization of phosphorothioate-antisense oligomer (PS-ASO) against H-ras mutated gene, the PS-ASO-mediated in vitro hemolysis, in vivo hepatic uptake, its pharmacokinetic profile, tissue distribution in some highly perfused organs, its effect in normal rats, antineoplastic efficacy in carcinogen-induced HCC in rats were evaluated and compared against DXT treatment. Mutated H-ras expression by in situ hybridization, hep-par-I, CK-7, CD-15, p53 expression patterns by immunohistochemical methods, scanning electron microscopic evaluation of hepatic architecture, various hepatic marker enzyme levels and caspase-3/9 apoptotic enzyme activities were also carried out in the experimental rats. KEY FINDINGS: PS-ASO showed low in vitro hemolysis (<3 %), and had a sustained PS-ASO blood residence time in vivo compared to DTX, with a time-dependent hepatic uptake. It showed no toxic manifestations in normal rats. PS-ASO distribution was although initially less in the lung than liver and kidney, but at 8 h it accumulated more in lung than kidney. Antineoplastic potential of PS-ASO (treated for 6 weeks) excelled in inhibiting chemically induced tumorigenesis compared to DTX in rats, by inhibiting H-ras gene expression, some immonohistochemical modulations, and inducing caspase-3/9-mediated apoptosis. It prevented HCC-mediated lung metastatic tumor in the experimental rats. SIGNIFICANCE: PS-ASO genetic therapy showed potential to inhibit HCC far more effectively than DXT in rats.


Subject(s)
Antineoplastic Agents , Docetaxel , Genetic Therapy , Animals , Docetaxel/pharmacology , Rats , Male , Genetic Therapy/methods , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/pathology , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Apoptosis/drug effects , Rats, Sprague-Dawley , Taxoids/pharmacology
11.
Nat Commun ; 15(1): 3741, 2024 May 03.
Article in English | MEDLINE | ID: mdl-38702301

ABSTRACT

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Drug Resistance, Neoplasm , Lung Neoplasms , Signal Transduction , Transcription Factors , YAP-Signaling Proteins , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Transcription Factors/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , YAP-Signaling Proteins/metabolism , Cell Line, Tumor , Animals , Drug Resistance, Neoplasm/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Neoplasm, Residual , Mice , Focal Adhesion Kinase 1/metabolism , Focal Adhesion Kinase 1/genetics , ErbB Receptors/metabolism , ErbB Receptors/genetics , Anaplastic Lymphoma Kinase/metabolism , Anaplastic Lymphoma Kinase/genetics , Anaplastic Lymphoma Kinase/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacology , Xenograft Model Antitumor Assays
12.
Molecules ; 29(10)2024 May 15.
Article in English | MEDLINE | ID: mdl-38792177

ABSTRACT

The phosphorylation of different sites produces a significant effect on the conformational dynamics of KRAS. Gaussian accelerated molecular dynamics (GaMD) simulations were combined with deep learning (DL) to explore the molecular mechanism of the phosphorylation-mediated effect on conformational dynamics of the GTP-bound KRAS. The DL finds that the switch domains are involved in obvious differences in conformation contacts and suggests that the switch domains play a key role in the function of KRAS. The analyses of free energy landscapes (FELs) reveal that the phosphorylation of pY32, pY64, and pY137 leads to more disordered states of the switch domains than the wild-type (WT) KRAS and induces conformational transformations between the closed and open states. The results from principal component analysis (PCA) indicate that principal motions PC1 and PC2 are responsible for the closed and open states of the phosphorylated KRAS. Interaction networks were analyzed and the results verify that the phosphorylation alters interactions of GTP and magnesium ion Mg2+ with the switch domains. It is concluded that the phosphorylation pY32, pY64, and pY137 tune the activity of KRAS through changing conformational dynamics and interactions of the switch domains. We anticipated that this work could provide theoretical aids for deeply understanding the function of KRAS.


Subject(s)
Deep Learning , Guanosine Triphosphate , Molecular Dynamics Simulation , Protein Conformation , Proto-Oncogene Proteins p21(ras) , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Phosphorylation , Guanosine Triphosphate/metabolism , Guanosine Triphosphate/chemistry , Humans , Protein Binding , Principal Component Analysis
13.
J Proteome Res ; 23(6): 2160-2168, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38767394

ABSTRACT

Resistance is a major problem with effective cancer treatment and the stroma forms a significant portion of the tumor mass but traditional drug screens involve cancer cells alone. Cancer-associated fibroblasts (CAFs) are a major tumor stroma component and its secreted proteins may influence the function of cancer cells. The majority of secretome studies compare different cancer or CAF cell lines exclusively. Here, we present the direct characterization of the secreted protein profiles between CAFs and KRAS mutant-cancer cell lines from colorectal, lung, and pancreatic tissues using multiplexed mass spectrometry. 2573 secreted proteins were annotated, and differential analysis highlighted understudied CAF-enriched secreted proteins, including Wnt family member 5B (WNT5B), in addition to established CAF markers, such as collagens. The functional role of CAF secreted proteins was explored by assessing its effect on the response to 97 anticancer drugs since stromal cells may cause a differing cancer drug response, which may be missed on routine drug screening using cancer cells alone. CAF secreted proteins caused specific effects on each of the cancer cell lines, which highlights the complexity and challenges in cancer treatment and so the importance to consider stromal elements.


Subject(s)
Cancer-Associated Fibroblasts , Secretome , Humans , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/pathology , Cell Line, Tumor , Secretome/metabolism , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Mass Spectrometry , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Proteomics/methods , Lung Neoplasms/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/genetics
14.
Bioorg Chem ; 148: 107467, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38772290

ABSTRACT

KRAS-G12C inhibitors has been made significant progress in the treatment of KRAS-G12C mutant cancers, but their clinical application is limited due to the adaptive resistance, motivating development of novel structural inhibitors. Herein, series of coumarin derivatives as KRAS-G12C inhibitors were found through virtual screening and rational structural optimization. Especially, K45 exhibited strong antiproliferative potency on NCI-H23 and NCI-H358 cancer cells harboring KRAS-G12C with the IC50 values of 0.77 µM and 1.50 µM, which was 15 and 11 times as potent as positive drug ARS1620, respectively. Furthermore, K45 reduced the phosphorylation of KRAS downstream effectors ERK and AKT by reducing the active form of KRAS (KRAS GTP) in NCI-H23 cells. In addition, K45 induced cell apoptosis by increasing the expression of anti-apoptotic protein BAD and BAX in NCI-H23 cells. Docking studies displayed that the 3-naphthylmethoxy moiety of K45 extended into the cryptic pocket formed by the residues Gln99 and Val9, which enhanced the interaction with the KRAS-G12C protein. These results indicated that K45 was a potent KRAS-G12C inhibitor worthy of further study.


Subject(s)
Antineoplastic Agents , Cell Proliferation , Coumarins , Drug Screening Assays, Antitumor , Proto-Oncogene Proteins p21(ras) , Humans , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Coumarins/chemistry , Coumarins/pharmacology , Coumarins/chemical synthesis , Structure-Activity Relationship , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Cell Proliferation/drug effects , Molecular Structure , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Discovery , Apoptosis/drug effects , Molecular Docking Simulation , Drug Evaluation, Preclinical
15.
Bioorg Chem ; 148: 107460, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38781668

ABSTRACT

A series of genipin derivatives were designed and synthesized as potential inhibitors targeted KRAS G12D mutation. The majority of these compounds demonstrated potential antiproliferative effects against KRAS G12D mutant tumor cells (CT26 and A427). Notably, seven compounds exhibited the anticancer effects with IC50 values ranging from 7.06 to 9.21 µM in CT26 (KRASG12D) and A427 (KRASG12D) cells and effectively suppressed the colony formation of CT26 cells. One representative compound SK12 was selected for further investigation into biological activity and action mechanisms. SK12 markedly induced apoptosis in CT26 cells in a concentration-dependent manner. Moreover, SK12 elevated the levels of reactive oxygen species (ROS) in tumor cells and exhibited a modulatory effect on the KRAS signaling pathway, thereby inhibiting the activation of downstream phosphorylated proteins. The binding affinity of SK12 to KRAS G12D protein was further confirmed by the surface plasmon resonance (SPR) assay with a binding KD of 157 µM. SK12 also exhibited notable anticancer efficacy in a nude mice tumor model. The relative tumor proliferation rate (T/C) of the experimental group (50 mg/kg) was 31.04 % (P < 0.05), while maintaining a commendable safety profile.


Subject(s)
Antineoplastic Agents , Cell Proliferation , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Iridoids , Mice, Nude , Proto-Oncogene Proteins p21(ras) , Humans , Iridoids/pharmacology , Iridoids/chemistry , Animals , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Cell Proliferation/drug effects , Structure-Activity Relationship , Mice , Molecular Structure , Apoptosis/drug effects , Drug Discovery , Cell Line, Tumor , Mutation , Mice, Inbred BALB C , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Neoplasms, Experimental/metabolism
16.
Int J Biol Macromol ; 270(Pt 2): 132477, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38772459

ABSTRACT

KRASG12D are the most prevalent oncogenic mutations and a promising target for solid tumor therapies. However, its inhibition exhibits tremendous challenge due to the necessity of high binding affinity to obviate the need for covalent binders. Here we report the evidence of a novel class of Imidazo[1,2-a]pyridine derivative as potentially significant novel inhibitors of KRASG12D, discovered through extensive ligand-based screening against 2-[(2R)-piperidin-2-yl]-1H-indole, an important scaffold for KRASG12D inhibition via switch-I/II (S-I/II) pocket. The proposed compounds exhibited similar binding affinities and overlapped pose configurations to 2-[(2R)-piperidin-2-yl]-1H-indole, serving as a reliable starting point for drug discovery. Comparative free energy profiles demonstrated that C4 [2-methyl-3-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)imidazo[1,2-a]pyridine] effectively shifted the protein to a stable low-energy conformation via a prominent transition state. The conformational changes across the transition revealed the conformational shift of switch-I and II to a previously known off-like conformation of inactive KRASG12D with rmsd of 0.91 Å. These conformations were even more prominent than the privileged scaffold 2-[(2R)-piperidin-2-yl]-1H-indole. The representative structure overlay of C4 and another X-ray crystallography solved BI-2852 bound inactive KRASG12D revealed that Switch-I and II exhibited off-like conformations. The cumulative variance across the first eigenvalue that accounted for 57 % of the collective variance validated this on-to-off transition. In addition, the relative interaction of C4 binding showed consistent patterns with BI-2852. Taken together, our results support the inhibitory activity of [2-methyl-3-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)imidazo[1,2-a]pyridine] by shifting active KRASG12D to an inactive conformation.


Subject(s)
Proto-Oncogene Proteins p21(ras) , Pyridines , Pyridines/chemistry , Pyridines/pharmacology , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Protein Conformation , Molecular Docking Simulation , Protein Binding , Mutation
17.
Sci Signal ; 17(836): eadd5073, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38743809

ABSTRACT

The Ras-mitogen-activated protein kinase (MAPK) pathway is a major target for cancer treatment. To better understand the genetic pathways that modulate cancer cell sensitivity to MAPK pathway inhibitors, we performed a CRISPR knockout screen with MAPK pathway inhibitors on a colorectal cancer (CRC) cell line carrying mutant KRAS. Genetic deletion of the catalytic subunit of protein phosphatase 6 (PP6), encoded by PPP6C, rendered KRAS- and BRAF-mutant CRC and BRAF-mutant melanoma cells more resistant to these inhibitors. In the absence of MAPK pathway inhibition, PPP6C deletion in CRC cells decreased cell proliferation in two-dimensional (2D) adherent cultures but accelerated the growth of tumor spheroids in 3D culture and tumor xenografts in vivo. PPP6C deletion enhanced the activation of nuclear factor κB (NF-κB) signaling in CRC and melanoma cells and circumvented the cell cycle arrest and decreased cyclin D1 abundance induced by MAPK pathway blockade in CRC cells. Inhibiting NF-κB activity by genetic and pharmacological means restored the sensitivity of PPP6C-deficient cells to MAPK pathway inhibition in CRC and melanoma cells in vitro and in CRC cells in vivo. Furthermore, a R264 point mutation in PPP6C conferred loss of function in CRC cells, phenocopying the enhanced NF-κB activation and resistance to MAPK pathway inhibition observed for PPP6C deletion. These findings demonstrate that PP6 constrains the growth of KRAS- and BRAF-mutant cancer cells, implicates the PP6-NF-κB axis as a modulator of MAPK pathway output, and presents a rationale for cotargeting the NF-κB pathway in PPP6C-mutant cancer cells.


Subject(s)
MAP Kinase Signaling System , NF-kappa B , Proto-Oncogene Proteins B-raf , Proto-Oncogene Proteins p21(ras) , Humans , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , NF-kappa B/metabolism , NF-kappa B/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , MAP Kinase Signaling System/drug effects , Animals , Cell Line, Tumor , Mutation , Mice , Protein Kinase Inhibitors/pharmacology , Cell Proliferation/drug effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Melanoma/genetics , Melanoma/metabolism , Melanoma/drug therapy , Melanoma/pathology , Xenograft Model Antitumor Assays , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , Mice, Nude
18.
Nanomedicine ; 55: 102714, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38738528

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor survival rates. Here, we evaluated iron-doped hydroxyapatite (FeHA) as a potential nanomedicine-based approach to combat PDAC. FeHA, in combination with a sublethal dose of the glutathione peroxidase 4 (GPX4) inhibitor RSL3, was found to trigger ferroptosis in KRAS mutant PANC-1 cells, but not in BxPC3 cells, while sparing normal human cells (fibroblasts and peripheral blood mononuclear cells). These findings were recapitulated in 3D spheroids generated using PDAC cells harboring wild-type versus mutant KRAS. Moreover, ferroptosis induction by FeHA plus RSL3 was reversed by the knockdown of STEAP3, a metalloreductase responsible for converting Fe3+ to Fe2+. Taken together, our data show that FeHA is capable of triggering cancer cell death in a KRAS-selective, STEAP3-dependent manner in PDAC cells.


Subject(s)
Carcinoma, Pancreatic Ductal , Ferroptosis , Iron , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Iron/chemistry , Iron/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Ferroptosis/drug effects , Cell Line, Tumor , Nanoparticles/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism
19.
Elife ; 132024 Apr 04.
Article in English | MEDLINE | ID: mdl-38573819

ABSTRACT

Oncogenic mutations in KRAS are among the most common in cancer. Classical models suggest that loss of epithelial characteristics and the acquisition of mesenchymal traits are associated with cancer aggressiveness and therapy resistance. However, the mechanistic link between these phenotypes and mutant KRAS biology remains to be established. Here, we identify STAT3 as a genetic modifier of TGF-beta-induced epithelial to mesenchymal transition. Gene expression profiling of pancreatic cancer cells identifies more than 200 genes commonly regulated by STAT3 and oncogenic KRAS. Functional classification of the STAT3-responsive program reveals its major role in tumor maintenance and epithelial homeostasis. The signatures of STAT3-activated cell states can be projected onto human KRAS mutant tumors, suggesting that they faithfully reflect characteristics of human disease. These observations have implications for therapeutic intervention and tumor aggressiveness.


Subject(s)
Pancreatic Neoplasms , Transforming Growth Factor beta , Humans , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Pancreas/metabolism , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Transforming Growth Factor beta/metabolism
20.
Stem Cell Res Ther ; 15(1): 106, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38627844

ABSTRACT

BACKGROUND: Although oncogenic RAS mutants are thought to exert mutagenic effects upon blood cells, it remains uncertain how a single oncogenic RAS impacts non-transformed multipotent hematopoietic stem or progenitor cells (HPCs). Such potential pre-malignant status may characterize HPCs in patients with RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). This study sought to elucidate the biological and molecular alterations in human HPCs carrying monoallelic mutant KRAS (G13C) with no other oncogene mutations. METHODS: We utilized induced pluripotent stem cells (iPSCs) derived from two unrelated RALD patients. Isogenic HPC pairs harboring either wild-type KRAS or monoallelic KRAS (G13C) alone obtained following differentiation enabled reliable comparative analyses. The compound screening was conducted with an established platform using KRAS (G13C) iPSCs and differentiated HPCs. RESULTS: Cell culture assays revealed that monoallelic KRAS (G13C) impacted both myeloid differentiation and expansion characteristics of iPSC-derived HPCs. Comprehensive RNA-sequencing analysis depicted close clustering of HPC samples within the isogenic group, warranting that comparative studies should be performed within the same genetic background. When compared with no stimulation, iPSC-derived KRAS (G13C)-HPCs showed marked similarity with the wild-type isogenic control in transcriptomic profiles. After stimulation with cytokines, however, KRAS (G13C)-HPCs exhibited obvious aberrant cell-cycle and apoptosis responses, compatible with "dysregulated expansion," demonstrated by molecular and biological assessment. Increased BCL-xL expression was identified amongst other molecular changes unique to mutant HPCs. With screening platforms established for therapeutic intervention, we observed selective activity against KRAS (G13C)-HPC expansion in several candidate compounds, most notably in a MEK- and a BCL-2/BCL-xL-inhibitor. These two compounds demonstrated selective inhibitory effects on KRAS (G13C)-HPCs even with primary patient samples when combined. CONCLUSIONS: Our findings indicate that a monoallelic oncogenic KRAS can confer dysregulated expansion characteristics to non-transformed HPCs, which may constitute a pathological condition in RALD hematopoiesis. The use of iPSC-based screening platforms will lead to discovering treatments that enable selective inhibition of RAS-mutated HPC clones.


Subject(s)
Induced Pluripotent Stem Cells , Humans , Cell Differentiation/genetics , Hematopoietic Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism
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