Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many ...physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-β signaling during breast cancer progression remains largely unknown.
In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-β-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-β1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model.
Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-β-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-β receptor complexes might be core fucosylated by FUT8 to facilitate TGF-β binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis.
Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-β signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.
The biology of SCUBE Lin, Yuh-Charn; Sahoo, Binay K; Gau, Shiang-Shin ...
Journal of biomedical science,
05/2023, Letnik:
30, Številka:
1
Journal Article
Recenzirano
Odprti dostop
The SCUBE Signal peptide-Complement C1r/C1s, Uegf, Bmp1 (CUB)-Epithelial growth factor domain-containing protein family consists of three proteins in vertebrates, SCUBE1, 2 and 3, which are highly ...conserved in zebrafish, mice and humans. Each SCUBE gene encodes a polypeptide of approximately 1000 amino acids that is organized into five modular domains: (1) an N-terminal signal peptide sequence, (2) nine tandem epidermal growth factor (EGF)-like repeats, (3) a large spacer region, (4) three cysteine-rich (CR) motifs, and (5) a CUB domain at the C-terminus. Murine Scube genes are expressed individually or in combination during the development of various tissues, including those in the central nervous system and the axial skeleton. The cDNAs of human SCUBE orthologs were originally cloned from vascular endothelial cells, but SCUBE expression has also been found in platelets, mammary ductal epithelium and osteoblasts. Both soluble and membrane-associated SCUBEs have been shown to play important roles in physiology and pathology. For instance, upregulation of SCUBEs has been reported in acute myeloid leukemia, breast cancer and lung cancer. In addition, soluble SCUBE1 is released from activated platelets and can be used as a clinical biomarker for acute coronary syndrome and ischemic stroke. Soluble SCUBE2 enhances distal signaling by facilitating the secretion of dual-lipidated hedgehog from nearby ligand-producing cells in a paracrine manner. Interestingly, the spacer regions and CR motifs can increase or enable SCUBE binding to cell surfaces via electrostatic or glycan-lectin interactions. As such, membrane-associated SCUBEs can function as coreceptors that enhance the signaling activity of various serine/threonine kinase or tyrosine kinase receptors. For example, membrane-associated SCUBE3 functions as a coreceptor that promotes signaling in bone morphogenesis. In humans, SCUBE3 mutations are linked to abnormalities in growth and differentiation of both bones and teeth. In addition to studies on human SCUBE function, experimental results from genetically modified mouse models have yielded important insights in the field of systems biology. In this review, we highlight novel molecular discoveries and critical directions for future research on SCUBE proteins in the context of cancer, skeletal disease and cardiovascular disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
OBJECTIVE—SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2), expressed on the endothelial cell surface, functions as a novel coreceptor for VEGFR2 (vascular endothelial growth factor ...receptor 2) and enhances VEGF-induced signaling in adult angiogenesis. However, whether SCUBE2 plays a role in pathological angiogenesis and whether anti-SCUBE2 antibody is an effective strategy for blocking tumor angiogenesis remain unknown. The aim of this study was to investigate the pathological role and targeting therapy of SCUBE2 in tumor vasculature.
APPROACH AND RESULTS—Immunohistochemistry revealed that SCUBE2 is highly expressed in endothelial cells of numerous carcinomas. Genetic endothelial cell knockout of SCUBE2 and pharmacological inhibition with the anti-SCUBE2 monoclonal antibody SP.B1 significantly reduced xenograft tumor growth, decreased tumor vascular density, increased apoptosis, and decreased the proliferation of tumor cells. Mechanistic studies revealed that SP.B1 binds to SCUBE2 and induces its internalization for lysosomal degradation, thereby reducing its cell surface level and inhibiting the binding of and downstream signaling of VEGF, including VEGFR2 phosphorylation and AKT/MAPK (mitogen-activated protein kinase) activation. Importantly, dual combination therapy with anti-SCUBE2 monoclonal antibody and anti-VEGF antibody or chemotherapy was more effective than single-agent therapy.
CONCLUSIONS—Endothelial cell surface SCUBE2 is a VEGFR2 coreceptor essential for pathological tumor angiogenesis, and anti-SCUBE2 monoclonal antibody acting as an internalization inducer may provide a potent combination therapy for tumor angiogenesis.
Hair follicle stem cells are regulated by dermal papilla fibroblasts, their principal signaling niche. Overactivation of Hedgehog signaling in the niche dramatically accelerates hair growth and ...induces follicle multiplication in mice. On single-cell RNA sequencing, dermal papilla fibroblasts increase heterogeneity to include new Wnt5a
states. Transcriptionally, mutant fibroblasts activate regulatory networks for Gli1, Alx3, Ebf1, Hoxc8, Sox18, and Zfp239. These networks jointly upregulate secreted factors for multiple hair morphogenesis and hair-growth-related pathways. Among these is non-conventional TGF-β ligand Scube3. We show that in normal mouse skin, Scube3 is expressed only in dermal papillae of growing, but not in resting follicles. SCUBE3 protein microinjection is sufficient to induce new hair growth, and pharmacological TGF-β inhibition rescues mutant hair hyper-activation phenotype. Moreover, dermal-papilla-enriched expression of SCUBE3 and its growth-activating effect are partially conserved in human scalp hair follicles. Thus, Hedgehog regulates mesenchymal niche function in the hair follicle via SCUBE3/TGF-β mechanism.
Transmembrane guanylyl cyclases (GCs), with activity regulated by peptide ligands and/or calcium‐binding proteins, are essential for various physiological and sensory processes. The mode of ...activation of the GC subtype GC‐G, which is expressed in neurons of the Grueneberg ganglion that respond to cool temperatures, has been elusive. In searching for appropriate stimuli to activate GC‐G, we found that its enzymatic activity is directly stimulated by cool temperatures. In this context, it was observed that dimerization/oligomerization of GC‐G, a process generally considered as critical for enzymatic activity of GCs, is strongly enhanced by coolness. Moreover, heterologous expression of GC‐G in cultured cells rendered these cells responsive to coolness; thus, the protein might be a sensor for cool temperatures. This concept is supported by the observation of substantially reduced coolness‐induced response of Grueneberg ganglion neurons and coolness‐evoked ultrasonic vocalization in GC‐G‐deficient mouse pups. GC‐G may be a novel thermosensory protein with functional implications for the Grueneberg ganglion, a sensory organ responding to cool temperatures.
Synopsis
Many membrane proteins respond to extracellular ligands, but sensory nerves use specialized sensors also to report environmental temperatures. The transmembrane receptor guanylyl cyclase subtype GC‐G, expressed in Grueneberg ganglion neurons of the murine nose, is a novel type of molecular sensor for cool ambient temperatures in mammals.
GC‐G expression in mouse is confined to Grueneberg ganglion neurons of the murine nose.
Cool temperature stimulates the enzymatic activity of the GC‐G guanylyl cyclase by inducing its dimerization/oligomerization.
Coolness‐induced stimulation of the thermosensory GC‐G guanylyl cyclase activates the Grueneberg ganglion neurons by increasing the cGMP concentration and opening cGMP‐dependent Ca2+‐permeable channels for elevating intracellular Ca2+ levels.
Independent of more classical sensors like the coolness‐activated TRPM8 ion channel, GC‐G is critical for coolness‐evoked ultrasound‐vocalization in abandoned mouse pups to elicit maternal‐care behaviors.
The membrane‐spanning receptor guanylyl cyclase subtype GC‐G, specifically expressed in the Grueneberg ganglion neurons in the nose, is a novel thermosensor for cool ambient temperatures in mammals in vivo.
Activation of metastatic reprogramming is critical for tumour metastasis. However, more detailed knowledge of the underlying mechanism is needed to enable targeted intervention. Here, we show that ...paraspeckle component 1 (PSPC1), identified in an aberrant 13q12.11 locus, is upregulated and associated with poor survival in patients with cancer. PSPC1 promotes tumorigenesis, epithelial-to-mesenchymal transition (EMT), stemness and metastasis in multiple cell types and in spontaneous mouse cancer models. PSPC1 is the master activator for transcription factors of EMT and stemness and accompanies c-Myc activation to facilitate tumour growth. PSPC1 increases transforming growth factor-β1 (TGF-β1) secretion through an interaction with phosphorylated and nuclear Smad2/3 to potentiate TGF-β1 autocrine signalling. Moreover, PSPC1 acts as a contextual determinant of the TGF-β1 pro-metastatic switch to alter Smad2/3 binding preference from tumour-suppressor to pro-metastatic genes. Having validated the PSPC1-Smads-TGF-β1 axis in various cancers, we conclude that PSPC1 is a master activator of pro-metastatic switches and a potential target for anti-metastasis drugs.
A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal ...is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.
Signal peptide-CUB-EGF domain-containing protein 2 (SCUBE2) belongs to a secreted and membrane-associated multi-domain SCUBE protein family. We previously demonstrated that SCUBE2 is a novel ...breast-tumor suppressor and could be a useful prognostic marker. However, the role of SCUBE2 in breast-cancer cell migration and invasion and how it is regulated during the epithelial-mesenchymal transition (EMT) remain undefined. In this study, we showed that ectopic SCUBE2 overexpression could enhance the formation of E-cadherin-containing adherens junctions by β-catenin-SOX-mediated induction of forkhead box A1 (a positive regulator of E-cadherin) and upregulation of E-cadherin, which in turn led to epithelial transition and inhibited migration and invasion of aggressive MDA-MB-231 breast-carcinoma cells. SCUBE2 expression was repressed together with that of E-cadherin in TGF-β-induced EMT; direct expression of SCUBE2 alone was sufficient to inhibit the TGF-β-induced EMT. Furthermore, quantitative DNA methylation, methylation-specific PCR, and chromatin immunoprecipitation analyses revealed that SCUBE2 expression was inactivated by DNA hypermethylation at the CpG islands by recruiting and binding DNA methyltransferase 1 during TGF-β-induced EMT. Together, our results suggest that SCUBE2 plays a key role in suppressing breast-carcinoma-cell mobility and invasiveness by increasing the formation of the epithelial E-cadherin-containing adherens junctions to promote epithelial differentiation and drive the reversal of EMT.
Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: ...an NH2-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH2-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH2-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH2-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the β-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the β-catenin pathway in breast cancer cells.
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