ABSTRACT
Novel components in the noncanonical Hippo pathway that mediate the growth, metastasis, and drug resistance of breast cancer (BC) cells need to be identified. Here, we showed that expression of SAM and SH3 domain-containing protein 1 (SASH1) is negatively correlated with expression of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) in a subpopulation of patients with luminal-subtype BC. Downregulated SASH1 and upregulated MAP4K4 synergistically regulated the proliferation, migration, and invasion of luminal-subtype BC cells. The expression of LATS2, SASH1, and YAP1 and the phosphorylation of YAP1 were negatively regulated by MAP4K4, and LATS2 then phosphorylated SASH1 to form a novel MAP4K4-LATS2-SASH1-YAP1 cascade. Dephosphorylation of Yes1 associated transcriptional regulator (YAP1), YAP1/TAZ nuclear translocation, and downstream transcriptional regulation of YAP1 were promoted by the combined effects of ectopic MAP4K4 expression and SASH1 silencing. Targeted inhibition of MAP4K4 blocked proliferation, cell migration, and ER signaling both in vitro and in vivo. Our findings reveal a novel MAP4K4-LATS2-SASH1-YAP1 phosphorylation cascade, a noncanonical Hippo pathway that mediates ER signaling, tumorigenesis, and metastasis in breast cancer. Targeted intervention with this noncanonical Hippo pathway may constitute a novel alternative therapeutic approach for endocrine-resistant BC.
Subject(s)
Adaptor Proteins, Signal Transducing , Breast Neoplasms , Intracellular Signaling Peptides and Proteins , Protein Serine-Threonine Kinases , Transcription Factors , Tumor Suppressor Proteins , YAP-Signaling Proteins , Humans , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/genetics , Female , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , YAP-Signaling Proteins/metabolism , YAP-Signaling Proteins/genetics , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Animals , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Phosphoproteins/metabolism , Phosphoproteins/genetics , Mice , Signal Transduction , Neoplasm Metastasis , Cell Movement , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/metabolism , Neoplasm Proteins/genetics , Phosphorylation , Mice, Nude , Carcinogenesis/genetics , Carcinogenesis/metabolismABSTRACT
MAP4K4 is a serine/threonine kinase of the STE20 family involved in the regulation of actin cytoskeleton dynamics and cell motility. It has been proposed as a target of angiogenesis and inhibitors show potential in cardioprotection. MAP4K4 also mediates cell invasion in vitro, is overexpressed in various types of cancer, and is associated with poor patient prognosis. Recently, MAP4K4 has been shown to be overexpressed in pancreatic cancer, but its role in tumour initiation, progression, and metastasis is unknown. Here, using the KrasG12D Trp53R172H Pdx1-Cre (KPC) mouse model of pancreatic ductal adenocarcinoma (PDAC), we show that deletion of Map4k4 drives tumour initiation and progression. Moreover, we report that the acceleration of tumour onset is also associated with an overactivation of ERK and AKT, two major downstream effectors of KRAS, in vitro and in vivo. In contrast to the accelerated tumour onset caused by loss of MAP4K4, we observed a reduction in metastatic burden with both the KPC model and in an intraperitoneal transplant assay indicating a major role of MAP4K4 in metastatic seeding. In summary, our study sheds light on the dichotomous role of MAP4K4 in the initiation of PDAC onset, progression, and metastatic dissemination. It also identifies MAP4K4 as a possible druggable target against pancreatic cancer spread, but with the caveat that targeting MAP4K4 might accelerate early tumorigenesis. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Animals , Mice , Humans , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , MAP Kinase Signaling System , Cell Line, Tumor , Pancreatic Neoplasms/pathology , Carcinoma, Pancreatic Ductal/pathology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Serine , Intracellular Signaling Peptides and Proteins/metabolismABSTRACT
RASopathies represent a distinct class of neurodevelopmental syndromes caused by germline variants in the Ras/MAPK pathways. Recently, a novel disease-gene association was implicated in MAPK kinase kinase kinase 4 (MAP4K4), which regulates the upstream signals of the MAPK pathways. However, to our knowledge, only two studies have reported the genotype-phenotype relationships in the MAP4K4-related disorder. This study reports on a Korean boy harboring a novel de novo missense variant in MAP4K4 (NM_001242559:c.569G>T, p.Gly190Val), revealed by trio exome sequencing, and located in the hotspot of the protein kinase domain. The patient exhibited various clinical features, including craniofacial dysmorphism, language delay, congenital heart defects, genitourinary anomalies, and sagittal craniosynostosis. Our study expands the phenotypic association of the MAP4K4-related disorder to include syndromic craniosynostosis, thereby providing further insights into the role of the RAS/MAPK pathways in the development of premature fusion of calvarial sutures.
Subject(s)
Craniosynostoses , Genetic Association Studies , Mutation, Missense , Humans , Male , Craniosynostoses/genetics , Craniosynostoses/pathology , Exome Sequencing , Genetic Predisposition to Disease , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Mutation, Missense/genetics , Phenotype , Protein Serine-Threonine Kinases/genetics , SyndromeABSTRACT
Dynamin-related protein 1 (Drp1) is a crucial regulator of mitochondrial dynamics, the overactivation of which can lead to cardiovascular disease. Multiple distinct posttranscriptional modifications of Drp1 have been reported, among which S-nitrosylation was recently introduced. However, the detailed regulatory mechanism of S-nitrosylation of Drp1 (SNO-Drp1) in cardiac microvascular dysfunction in diabetes remains elusive. The present study revealed that mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) was consistently upregulated in diabetic cardiomyopathy (DCM) and promoted SNO-Drp1 in cardiac microvascular endothelial cells (CMECs), which in turn led to mitochondrial dysfunction and cardiac microvascular disorder. Further studies confirmed that MAP4K4 promoted SNO-Drp1 at human C644 (mouse C650) by inhibiting glutathione peroxidase 4 (GPX4) expression, through which MAP4K4 stimulated endothelial ferroptosis in diabetes. In contrast, inhibition of MAP4K4 via DMX-5804 significantly reduced endothelial ferroptosis, alleviated cardiac microvascular dysfunction and improved cardiac dysfunction in db/db mice by reducing SNO-Drp1. In parallel, the C650A mutation in mice abolished SNO-Drp1 and the role of Drp1 in promoting cardiac microvascular disorder and cardiac dysfunction. In conclusion, our findings demonstrate that MAP4K4 plays an important role in endothelial dysfunction in DCM and reveal that SNO-Drp1 and ferroptosis activation may act as downstream targets, representing potential therapeutic targets for DCM.
Subject(s)
Diabetic Cardiomyopathies , Dynamins , Endothelial Cells , Signal Transduction , Animals , Humans , Male , Mice , Cells, Cultured , Coronary Circulation , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/genetics , Diabetic Cardiomyopathies/physiopathology , Diabetic Cardiomyopathies/pathology , Diabetic Cardiomyopathies/enzymology , Diabetic Cardiomyopathies/etiology , Disease Models, Animal , Dynamins/metabolism , Dynamins/genetics , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelial Cells/enzymology , Endothelial Cells/drug effects , Ferroptosis/drug effects , Intracellular Signaling Peptides and Proteins , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Mitochondria, Heart/enzymology , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/geneticsABSTRACT
The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.
Subject(s)
Bacillus thuringiensis , Insecticide Resistance , Moths , Pest Control, Biological , Animals , Bacillus thuringiensis/genetics , Insecticide Resistance/genetics , Moths/microbiology , Moths/genetics , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Brazil , Insect Proteins/genetics , Insect Proteins/metabolism , Bacterial Proteins/geneticsABSTRACT
Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) has recently emerged as a promising therapeutic target in cancer. In this study, we explored the biological function of MAP4K4 in radioresistant breast cancer cells using two MAP4K4 inhibitors, namely PF06260933 and GNE-495. Radioresistant SR and MR cells were established by exposing SK-BR-3 and MCF-7 breast cancer cells to 48-70 Gy of radiation delivered at 4-5 Gy twice a week over 10 months. Surprisingly, although radioresistant cells were derived from two different subtypes of breast cancer cell lines, MAP4K4 was significantly elevated regardless of subtype. Inhibition of MAP4K4 with PF06260933 or GNE-495 selectively targeted radioresistant cells and improved the response to irradiation. Furthermore, MAP4K4 inhibitors induced apoptosis through the accumulation of DNA damage by inhibiting DNA repair systems in radioresistant cells. Notably, Inhibition of MAP4K4 suppressed the expressions of ACSL4, suggesting that MAP4K4 functioned as an upstream effector of ACSL4. This study is the first to report that MAP4K4 plays a crucial role in mediating the radioresistance of breast cancer by acting upstream of ACSL4 to enhance DNA damage response and inhibit apoptosis. We hope that our findings provide a basis for the development of new drugs targeting MAP4K4 to overcome radioresistance.
Subject(s)
Breast Neoplasms , Humans , Female , Breast Neoplasms/genetics , Cell Line, Tumor , Radiation Tolerance/genetics , DNA Repair , MCF-7 Cells , Apoptosis/radiation effects , Protein Serine-Threonine Kinases/metabolism , Intracellular Signaling Peptides and Proteins/metabolismABSTRACT
Gastric cancer (GC) is a serious malignant tumour with a high mortality rate and a poor prognosis. Recently, emerging evidence has suggested that N6-methyladenosine (m6A) modification plays a crucial regulatory role in cancer progression. However, the exact role of m6A regulatory factors FTO in GC is unclear. First, the expression of m6A methylation-related regulatory factors in clinical samples and the clinical data of the corresponding patients were obtained from The Cancer Genome Atlas (TCGA-STAD) dataset, and correlation analysis between FTO expression and patient clinicopathological parameters was subsequently performed. qRT-PCR, immunohistochemistry (IHC) and western blotting (WB) were used to verify FTO expression in GC. CCK-8, EdU, flow cytometry and transwell assays were used to evaluate the effect of FTO on the behaviour of GC cells. Transcriptome sequencing and RNA immunoprecipitation analysis were used to explore the potential regulatory mechanisms mediated by FTO. FTO was highly expressed in GC tissues and cells, and high expression of FTO predicted a worse prognosis than low expression. Functionally, overexpression of FTO promoted the proliferation, migration and invasion of GC cells but inhibited cell apoptosis. Mechanistically, we found that FTO is upregulated in GC and promotes GC progression by modulating the expression of MAP4K4. Taken together, our findings provide new insights into the effects of FTO-mediated m6A demethylation and could lead to the development of new strategies for GC monitoring and aggressive treatment.
Subject(s)
Adenine , Stomach Neoplasms , Humans , Stomach Neoplasms/pathology , Prognosis , Gene Expression Regulation, Neoplastic , Demethylation , Protein Serine-Threonine Kinases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolismABSTRACT
Cancer metastasis is the leading cause of mortality in patients with hepatocellular carcinoma (HCC). To meet the rapid malignant growth and transformation, tumor cells dramatically increase the consumption of nutrients, such as amino acids. Peptide transporter 1 (PEPT1), a key transporter for small peptides, has been found to be an effective and energy-saving intracellular source of amino acids that are required for the growth of tumor cells. Here, the role of PEPT1 in HCC metastasis and its underlying mechanisms is explored. PEPT1 is upregulated in HCC cells and tissues, and high PEPT1 expression is associated with poor prognosis in patients with HCC. PEPT1 overexpression dramatically promoted HCC cell migration, invasion, and lung metastasis, whereas its knockdown abolished these effects both in vitro and in vivo. Mechanistic analysis revealed that high PEPT1 expression increased cellular dipeptides in HCC cells that are responsible for activating the MAP4K4/G3BP2 signaling pathway, ultimately facilitating the phosphorylation of G3BP2 at Thr227 and enhancing HCC metastasis. Taken together, these findings suggest that PEPT1 acts as an oncogene in promoting HCC metastasis through dipeptide-induced MAP4K4/G3BP2 signaling and that the PEPT1/MAP4K4/G3BP2 axis can serve as a promising therapeutic target for metastatic HCC.