SCUBE3 promotes osteogenic differentiation and mitophagy in human bone marrow mesenchymal stem cells through the BMP2/TGF-ß signaling pathway.

In clinical settings, addressing large bone defects remains a significant challenge for orthopedic surgeons. The use of genetically modified bone marrow mesenchymal stem cells (BMSCs) has emerged as a highly promising approach for these treatments. Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a multifunctional secreted glycoprotein, the role of which remains unclear in human hBMSCs. This study used various experimental methods to elucidate the potential mechanism by which SCUBE3 influences osteogenic differentiation of hBMSCs in vitro. Additionally, the therapeutic efficacy of SCUBE3, in conjunction with porous GeLMA microspheres, was evaluated in vivo using a mouse bone defect model. Our findings indicate that SCUBE3 levels increase significantly during early osteogenic differentiation of hBMSCs, and that reducing SCUBE3 levels can hinder this differentiation. Overexpressing SCUBE3 elevated osteogenesis gene and protein levels and enhanced calcium deposition. Furthermore, treatment with recombinant human SCUBE3 (rhSCUBE3) protein boosted BMP2 and TGF-ß expression, activated mitophagy in hBMSCs, ameliorated oxidative stress, and restored osteogenic function through SMAD phosphorylation. In vivo, GELMA/OE treatment effectively accelerated bone healing in mice. In conclusion, SCUBE3 fosters osteogenic differentiation and mitophagy in hBMSCs by activating the BMP2/TGF-ß signaling pathway. When combined with engineered hydrogel cell therapy, it could offer valuable guidance for the clinical management of extensive bone defects.

MMP-2-mediated Scube2 degradation promotes blood-brain barrier disruption by blocking the interaction between astrocytes and endothelial cells via inhibiting Sonic hedgehog pathway during early cerebral ischemia.

We have previously demonstrated a rapid secretion of matrix metalloproteinase-2 (MMP-2) in the ischemic brain. Since Scube2 can interact with Sonic hedgehog (Shh) to maintain blood-brain barrier (BBB) integrity via regulating the interaction between brain capillary endothelial cells (ECs) and perivascular astrocytes, and it is also a substrate of MMP-2, we hypothesized that the secreted MMP-2 could degrade Scube2 and contribute to ischemic BBB disruption. Using an in vitro ischemic model of 90-min oxygen-glucose deprivation/3-h reoxygenation (OGD/R) and an in vivo mouse stroke model of 90-min middle cerebral artery occlusion (MCAO) with 3-h reperfusion, we established an important role of MMP-2-mediated Scube2 degradation in early ischemic BBB disruption. Exposure of C8-D1A cells and bEnd.3 cells to OGD/R increased MMP secretion in both cells, and C8-D1A cells appeared to secrete more MMPs than bEnd.3 cells. Co-IP and double-immunostaining revealed that Scube2 co-localized well with MMP-2 in C8-D1A cells and could be pulled down by MMP-2 antibodies. In MCAO mice, Scube2 protein showed a drastic reduction in ischemic brain tissue, which was accompanied by suppressed expression of Shh and its downstream molecules. Of note, specific knockdown of astrocytic Scube2 with AAV-shScube2 augmented MCAO-induced Shh suppression and exacerbated BBB leakage and inflammatory reactions in the ischemic brain. Last, incubation of bEnd.3 cells with conditioned medium derived from OGD-treated C8-D1A cells led to a significant inhibition of the Shh pathway in bEnd.3 cells and degradation of VE-cadherin and ZO-1. Inhibition of MMP-2 with SB-3CT or over-expression of Scube2 with plasmids in C8-D1A cells alleviated the above effect of C8-D1A cells-derived conditioned medium. Taken together, our data indicate that ischemia-induced secretion of MMP-2 may contribute to early BBB disruption in ischemic stroke via interrupting the shared Scube2-Shh pathway between brain capillary ECs and perivascular astrocytes.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

SCUBE1 Promotes Gestational Diabetes Mellitus: A Bioinformatics and Experimental Investigation.

Gestational diabetes mellitus (GDM) is one of the most common metabolic diseases in pregnant women, posing significant risks to the life and health of both mothers and fetuses. With improving living standards, the incidence of GDM is increasing rapidly. Therefore, understanding the underlying mechanism of GDM is of paramount importance. We downloaded two datasets from the Gene Expression Omnibus (GEO) database, containing sequencing data specifically related to "gestational diabetes" and "placenta". By merging these two datasets, a mRNA expression dataset was obtained and subjected to bioinformatics analyses. To screen out corresponding genes, differential analysis and weighted correlation network analysis (WGCNA) were carried out. Lasso, support vector machine and random forest analyses were subsequently performed for identifying key genes from the differentially expressed genes (DEGs) jointly screened out through differential analysis and WGCNA. Afterwards, immunoinfiltration and correlation analysis were performed to screen immune cells that play a role in disease progression and explore the correlation between the screened key genes and immune cells, after which Western Blot, quantitative real-time polymerase chain reaction, Immunohistochemistry, methyl thiazolyl tetrazolium, flow cytometry, scratch and Transwell assays were, respectively, performed for verification. For further verification, we found that the expression levels of MAP6D1 and SCUBE1 in embryonic tissues of GDM patients was higher compared to those of healthy pregnant women, which was consistent with the results of bioinformatics analysis. Consequently, SCUBE1 was selected for follow-up experiment. In order to explore the role of SCUBE1 in the development of GDM, we treated the trophoblastic cells HTR-8/SVneo with high glucose, and on this basis downregulated the expression of SCUBE1. Through further analysis, we observed that SCUBE1 had a role in reducing cell activity, migration and invasion, and promoting cell apoptosis. In summary, SCUBE1 promotes the development of GDM by increasing cell apoptosis and reducing cell activity, migration, and invasion.

Biochemical study of ZNF76 rs10947540 and SCUBE3 rs1888822 single nucleotide polymorphisms in the Egyptian patients with systemic lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a common autoimmune disease marked by the formation of apoptotic debris and the presence of autoantibodies that target nuclear components. At this moment, the actual cause of SLE is uncertain. Genetic variables have been well proven to have a significant role in the propensity of SLE. This study aimed to investigate the effect of (ZNF76) rs (10947540) and (SCUBE) rs (1888822) gene polymorphism in patients with systemic lupus erythematosus. A case control study has been carried out at Medical Biochemistry & Molecular biology and Rheumatology unit of Internal Medicine Departments, Faculty of Medicine, Menoufia University, Egypt, for 1-year duration between 1 June 2022 and 1 June 2023. Sixty patients were females (75%) and twenty patients were males (25%). Their ages ranged from 19 to 53 years. Their disease durations ranged from 7 months to 20 years. The findings indicated that the TC genotype of the ZNF76 rs10947540 gene increases the risk of SLE by 2.274-fold, while the dominant TC + CC increases the risk by 2.472-fold, and the C allele increases the risk by 2.115-fold. Additionally, the results showed that the TT genotype of the SCUBE3 rs1888822 gene increases the risk of SLE by 3.702-fold, the dominant GT + TT increases the risk by 2.304-fold, and the T allele increases the risk by 2.089-fold, while the GT genotype increases the risk by 1.918-fold. The study revealed significant associations between the genotypes of these polymorphisms and certain clinical parameters in SLE patients. These findings highlight the potential genetic contributions to SLE susceptibility and its clinical manifestations, providing valuable insights for future research and potential personalized approaches to the management of this complex autoimmune disease.

The biology of SCUBE.

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.

Hedgehog lipids: Promotors of alternative morphogen release and signaling?: Conflicting findings on lipidated Hedgehog transport and signaling can be explained by alternative regulated mechanisms to release the morphogen.

Two posttranslational lipid modifications present on all Hedgehog (Hh) morphogens-an N-terminal palmitate and a C-terminal cholesterol-are established and essential regulators of Hh biofunction. Yet, for several decades, the question of exactly how both lipids contribute to Hh signaling remained obscure. Recently, cryogenic electron microscopy revealed different modes by which one or both lipids may contribute directly to Hh binding and signaling to its receptor Patched1 (Ptc). Some of these modes demand that the established release factor Dispatched1 (Disp) extracts dual-lipidated Hh from the cell surface, and that another known upstream signaling modulator called Scube2 chaperones the dual-lipidated morphogen to Ptc. By mechanistically and biochemically aligning this concept with established in vivo and recent in vitro findings, this reflection identifies remaining questions in lipidated Hh transport and evaluates additional mechanisms of Disp- and Scube2-regulated release of a second bioactive Hh fraction that has one or both lipids removed.

TGF-ß superfamily co-receptors in cancer.

Transforming growth factor-ß (TGF-ß) superfamily signaling via their cognate receptors is frequently modified by TGF-ß superfamily co-receptors. Signaling through SMAD-mediated pathways may be enhanced or depressed depending on the specific co-receptor and cell context. This dynamic effect on signaling is further modified by the release of many of the co-receptors from the membrane to generate soluble forms that are often antagonistic to the membrane-bound receptors. The co-receptors discussed here include TßRIII (betaglycan), endoglin, BAMBI, CD109, SCUBE proteins, neuropilins, Cripto-1, MuSK, and RGMs. Dysregulation of these co-receptors can lead to altered TGF-ß superfamily signaling that contributes to the pathophysiology of many cancers through regulation of growth, metastatic potential, and the tumor microenvironment. Here we describe the role of several TGF-ß superfamily co-receptors on TGF-ß superfamily signaling and the impact on cellular and physiological functions with a particular focus on cancer, including a discussion on recent pharmacological advances and potential clinical applications targeting these co-receptors.

CAFs-derived SCUBE1 promotes malignancy and stemness through the Shh/Gli1 pathway in hepatocellular carcinoma.

BACKGROUND: The tumour microenvironment and cirrhotic liver are excellent sources of cancer-associated fibroblasts (CAFs), which participate in carcinogenesis. Thus, it is important to clarify the crosstalk between CAFs and HCC cells and the related mechanism in regulating carcinogenesis. METHODS: Human hepatocellular carcinoma (HCC) tissues and matched adjacent normal tissues were obtained from HCC patients. Immunohistochemistry, Western blotting (WB) and RT-qPCR were performed to detect the expression of SCUBE1. The roles of SCUBE1 in inducing stemness features in HCC cells were explored and investigated in vitro and in vivo. Student's t tests or Mann-Whitney U tests were used to compare continuous variables, while chi-square tests or Fisher's exact tests were used to compare categorical variables between two groups. RESULTS: SCUBE1 was confirmed to be highly expressed in CAFs in HCC and had a strong connection with stemness and a poor prognosis. In addition, CAFs were found to secrete SCUBE1 to enhance the malignancy of HCC cells and increase the proportion of CD133-positive cells. Silencing SCUBE1 expression had the opposite effect. The Shh pathway was activated by SCUBE1 stimulation. Inhibition of cyclopamine partially reversed the stimulating effect of SCUBE1 both in vivo and in vitro. Moreover, based on the RT-qPCR, ELISA and WB results, a high SCUBE1 expression level was found in HCC tissue and serum. CONCLUSION: This study revealed that CAFs-derived SCUBE1 can enhance the malignancy and stemness of HCC cells through the Shh pathway. This study aims to provide new perspectives for future HCC studies and provide new strategies for HCC treatment.

SCUBE3 downregulation modulates hepatocellular carcinoma by inhibiting CCNE1 via TGFß/PI3K/AKT/GSK3ß pathway.

OBJECTIVES: We aimed to verify the role of signal peptide-CUB-EGF-like domain-containing protein3 (SCUBE3) in the hepatocellular carcinoma (HCC) progression. METHODS: The role of SCUBE3 in HCC cell proliferation, apoptosis, and cell cycle in vitro were detected using MTT assay, colony formation assay, 5-ethynyl-2´-deoxyuridine assay (EDU), Celigo cell counting assay, Caspase3/7 activity assay, and flow cytometry. The effect of SCUBE3 on HCC cell proliferation in vivo was inspected by a xenograft tumour model in nude mice. The related mechanisms were further studied. RESULTS: The level of SCUBE3 was upregulated in HCC tissues and cell lines. Knockdown of SCUBE3 inhibited proliferation, promoted apoptosis, and induced cell cycle arrest in HCC cell lines in vitro and in vivo. Screening of cell cycle-related proteins revealed that CCNL2, CDK6, CCNE1, and CCND1 exhibited a significantly different expression profile. We found that SCUBE3 may promote the proliferation of HCC cells by regulating CCNE1 expression. The pathway enrichment analysis showed that the TGFß signalling pathway and the PI3K/AKT signalling pathway were significantly altered. Co-immunoprecipitation results showed that SCUBE3 binds to the TGFßRII receptor. SCUBE3 knockdown inhibited the PI3K/AKT signalling pathway and the phosphorylation of GSK3ß to inhibit its kinase activity. CONCLUSIONS: SCUBE3 promotes HCC development by regulating CCNE1 via TGFß/PI3K/AKT/GSK3ß pathway. In addition, SCUBE3 may be a new molecular target for the clinical diagnosis and treatment of HCC.

The Multifaceted Role of Signal Peptide-CUB-EGF Domain-Containing Protein (SCUBE) in Cancer.

Signal peptide, CUB, and EGF-like domain-containing proteins (SCUBE) are secretory cell surface glycoproteins that play key roles in the developmental process. SCUBE proteins participate in the progression of several diseases, including cancer, and are recognized for their oncogenic and tumor suppressor functions depending on the cellular context. SCUBE proteins promote cancer cell proliferation, angiogenesis, invasion, or metastasis, stemness or self-renewal, and drug resistance. The association of SCUBE with other proteins alters the expression of signaling pathways, including Hedgehog, Notch, TGF-ß/Smad2/3, and ß-catenin. Further, SCUBE proteins function as potential prognostic and diagnostic biomarkers for breast cancer, renal cell carcinoma, endometrial carcinoma, and nasopharyngeal carcinoma. This review presents key features of SCUBE family members, and their structure and functions, and highlights their contribution in the development and progression of cancer. A comprehensive understanding of the role of SCUBE family members offers novel strategies for cancer therapy.

SCUBE3 serves as an independent poor prognostic factor in breast cancer.

BACKGROUND: Accumulating evidences indicate that the signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) plays a key role in the development and progression of many human cancers. However, the underlying mechanism and prognosis value of SCUBE3 in breast cancer are still unclear. METHODS: The clinical data of 137 patients with breast cancer who underwent surgical resection in Taizhou Hospital of Zhejiang Province were retrospectively analyzed. We first conducted a comprehensive study on the expression pattern of SCUBE3 using the Tumor Immune Estimation Resource (TIMER) and UALCAN databases. In addition, the expression of SCUBE3 in breast tumor tissues was confirmed by immunohistochemistry. The protein-protein interaction analysis and functional enrichment analysis of SCUBE3 were analyzed using the STRING and Enrichr databases. Moreover, tissue microarray (TMA) was used to analyze the relationship between SCUBE3 expression levels and clinical-pathological parameters, such as histological type, grade, the status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2). We further supplemented and identified the above results using the UALCAN and bc-GenExMiner v4.4 databases from TCGA data. The correlation between the expression of SCUBE3 and survival was calculated by multivariate Cox regression analysis to investigate whether SCUBE3 expression may be an independent prognostic factor of breast cancer. RESULTS: We found that the expression level of SCUBE3 was significantly upregulated in breast cancer tissue compared with adjacent normal tissues. The results showed that the distribution of breast cancer patients in the high expression group and the low expression group was significantly different in ER, PR, HER2, E-cadherin, and survival state (p < 0.05), but there was no significant difference in histologic grade, histologic type, tumor size, lymph node metastasis, TMN stage, subtypes, or recurrence (p > 0.05). In addition, the high expression of SCUBE3 was associated with relatively poor prognosis of ER- (p = 0.012), PR- (p = 0.029), HER2 + (p = 0.007). The multivariate Cox regression analysis showed that the hazard ratio (HR) was 2.80 (95% CI 1.20-6.51, p = 0.0168) in individuals with high SCUBE3 expression, and HR was increased by 1.86 (95% CI 1.06-3.25, p = 0.0300) for per 1-point increase of SCUBE3 expression. CONCLUSIONS: These findings demonstrate that the high expression of SCUBE3 indicates poor prognosis in breast cancer. SCUBE3 expression may serve as a potential diagnostic indicator of breast cancer.

Evaluation of SCUBE-1 levels as a placental dysfunction marker at gestational diabetes mellitus.

Objective: To evaluate the alteration of plasma levels of signal peptide-CUB-EGF domain-containing protein (SCUBE)-1 as a marker of endothelial dysfunction and vascular injury in gestational diabetes mellitus (GDM) in comparison to healthy pregnant controls.Methods: A prospective study conducted at an antenatal outpatient clinic of a University hospital. Fifty pregnancies with GDM and thirty healthy pregnancies as controls were enrolled in the study.Results: There was no statistically significant difference between the groups in terms of age, gravidity, weight and BMI from pre-pregnancy until delivery, total weight gain, fetal weight and other hematological and biochemical parameters. SCUBE-1 levels were significantly higher in GDM patients (p = .007).Conclusions: Hyperglycemia predisposes to endothelial injury and vascular remodeling at GDM, and therefore, SCUBE-1 could be a predictor of vascular injury during pregnancy. Our study is the first to illustrate increased SCUBE-1 levels in GDM as a marker of placental endothelial dysfunction.

Serum SCUBE-1 levels in patients with diabetic retinopathy.

PURPOSE: Signal peptide-CUB-Epidermal growth factor-like domain-containing protein 1 (SCUBE-1) is a protein expressed on the cell surface of endothelial cells and platelets. We aimed to determine the alterations in SCUBE-1 levels in patients at different stages of diabetic retinopathy (DR). MATERIALS AND METHODS: Participants were grouped into 4 regarding the presence and stage of diabetic retinopathy as follows: control group (n 32), patients without DR (n 39), patients with background DR (n 42) and patients with proliferative DR (n 55). Age, gender, duration of diabetes, fasting blood glucose levels, serum hemoglobin A1c (HbA1c), SCUBE-1 and malondialdehyde (MDA) levels of the patients were recorded. Central macular thickness and central macular volume were recorded and compared between groups. RESULTS: Totally 136 patients with diabetes mellitus and 32 control cases were included in the study. There is a significant increase in SCUBE-1 and MDA levels with an advance in DR. In correlation analysis, there was only a significant correlation present between serum SCUBE-1 and MDA levels (p 0.001) but not with other parameters. CONCLUSION: For the first time in literature, we determined a significant increase in SCUBE-1 levels in patients with DR. Moreover, with an advance in DR, SCUBE-1 levels were also increasing.

Inhibition of Endothelial SCUBE2 (Signal Peptide-CUB-EGF Domain-Containing Protein 2), a Novel VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) Coreceptor, Suppresses Tumor Angiogenesis.

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.

Upregulated SCUBE2 expression in breast cancer stem cells enhances triple negative breast cancer aggression through modulation of notch signaling and epithelial-to-mesenchymal transition.

BACKGROUND: Metastatic and/or recurrent breast carcinomas are leading causes of cancer-related death worldwide. Breast cancer stem cells (BCSCs) have been implicated in cancer metastases and progression, thus, the need for the discovery and development of effective BCSCs-specific therapies against metastatic and triple negative breast cancer (TNBC). The expression of SCUBE2, originally identified in vascular endothelia, then in several non-endothelial cell types, is downregulated in invasive breast carcinomas. However, the role of SCUBE2 in BCSCs remains unknown. This present study investigated the probable involvements of SCUBE2 in BCSCs and TNBC metastasis. METHODS: The mRNA expression of SCUBE2, stemness and EMT markers in MDA-MB-231 and Hs578T tumorspheres or adherent cells were evaluated by qRT-PCR and microarray analyses. Using gene overexpression, in vitro migration and invasion assays, as well as in vivo bioluminescence imaging, we evaluated the role of SCUBE2 in MDA-MB-231 or Hs578T BCSCs. Western blot and cytotoxicity assays helped identify and validate SCUBE2 molecular target(s) and inhibitor(s). RESULTS: Concurrently increased SCUBE2 expression and cell motility were observed in TNBC tumorspheres compared to the parental adherent cells. SCUBE2 overexpression augmented BCSCs motility in vitro, and enhanced TNBC metastasis in vivo. While SCUBE2 overexpression activated Notch signaling its downregulation suppressed Notch signal effectors NICD, Jagged 1, HEY1, and HES1. CONCLUSIONS: We demonstrate that SCUBE2 expression is upregulated in BCSCs, promote EMT and enhance TNBC metastasis by activating Notch signaling. This reveals a potential druggable molecular target and an effective therapeutic strategy against metastatic and aggressive TNBC.

The Inhibitory Effect of (-)-Epigallocatechin-3-Gallate on Breast Cancer Progression via Reducing SCUBE2 Methylation and DNMT Activity.

Epigenetic modifications are important mechanisms responsible for cancer progression. Accumulating data suggest that (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, may hamper carcinogenesis by targeting epigenetic alterations. We found that signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor) domain-containing protein 2 (SCUBE2), a tumor suppressor gene, was hypermethylated in breast tumors. However, it is unknown whether EGCG regulates SCUBE2 methylation, and the mechanisms remain undefined. This study was designed to investigate the effect of EGCG on SCUBE2 methylation in breast cancer cells. We reveal that EGCG possesses a significantly inhibitory effect on cell viability in a dose- and time-dependent manner and presents more effects than other catechins. EGCG treatment resulted in enhancement of the SCUBE2 gene, along with elevated E-cadherin and decreased vimentin expression, leading to significant suppression of cell migration and invasion. The inhibitory effect of EGCG on SCUBE2 knock-down cells was remarkably alleviated. Further study demonstrated that EGCG significantly decreased the SCUBE2 methylation status by reducing DNA methyltransferase (DNMT) expression and activity. In summary, this study reported for the first time that SCUBE2 methylation can be reversed by EGCG treatment, finally resulting in the inhibition of breast cancer progression. These results suggest the epigenetic role of EGCG and its potential implication in breast cancer therapy.

The value of plasma SCUBE1 and oxidative stress parameters in the early diagnosis of acute ovarian torsion.

AIM: The purpose of our study was to determine the diagnostic value of plasma SCUBE1, malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant status (TAS) investigated on the 4th hour of ovarian torsion, and at the same time to determine the relation between these markers and histopathological damage occurring in the ovary. MATERIALS AND METHODS: Sixteen rats were included, eight in the torsion group and eight in the control group. The right ovaries of the rats in the torsion group were rotated clockwise 720 degrees, after which they were fixed and attached to the abdominal wall. The abdominal walls of the control group rats were opened for 1 min and then closed. Relaparotomy was performed 4 h after surgery in both groups, blood specimens were collected for biochemical analyses, and the right ovaries were surgically extracted. RESULTS: No statistically significant difference was determined in the control and torsion groups' plasma SCUBE1, SOD and TAS values (p = 0.987, p = 0.33, and p = 0.244, respectively). Torsion group plasma MDA values were significantly higher than those of the control group (p = 0.039). At histopathological evaluation, histological score, apoptotic index and Caspase-3 index values of the torsion group were significantly higher compared to the control group. CONCLUSION: Plasma SCUBE1 is not useful in the early diagnosis of ovarian torsion. Similarly, use of the oxidative stress markers SOD and TAS does not appear useful in early diagnosis, although MDA may be considered for that purpose (Tab. 2, Fig. 3, Ref. 23).

Endothelial SCUBE2 Interacts With VEGFR2 and Regulates VEGF-Induced Angiogenesis.

OBJECTIVE: Vascular endothelial growth factor (VEGF), a major mediator of angiogenesis, exerts its proangiogenic action by binding to VEGFR2 (VEGF receptor 2), the activity of which is further modulated by VEGFR2 coreceptors such as neuropilins. However, whether VEGFR2 is regulated by additional coreceptors is not clear. To investigate whether SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2), a peripheral membrane protein expressed in vascular endothelial cells (ECs) known to bind other signaling receptors, functions as a VEGFR2 coreceptor and to verify the role of SCUBE2 in the VEGF-induced angiogenesis. APPROACH AND RESULTS: SCUBE2 lentiviral overexpression in human ECs increased and short hairpin RNA knockdown inhibited VEGF-induced EC growth and capillary-like network formation on Matrigel. Like VEGF, endothelial SCUBE2 was upregulated by hypoxia-inducible factor-1α at both mRNA and protein levels. EC-specific Scube2 knockout mice were not defective in vascular development but showed impaired VEGF-induced neovascularization in implanted Matrigel plugs and recovery of blood flow after hind-limb ischemia. Coimmunoprecipitation and ligand-binding assays showed that SCUBE2 forms a complex with VEGF and VEGFR2, thus acting as a coreceptor to facilitate VEGF binding and augment VEGFR2 signal activity. SCUBE2 knockdown or genetic knockout suppressed and its overexpression promoted the VEGF-induced activation of downstream proangiogenic and proliferating signals, including VEGFR2 phosphorylation and mitogen-activated protein kinase or AKT activation. CONCLUSIONS: Endothelial SCUBE2 may be a novel coreceptor for VEGFR2 and potentiate VEGF-induced signaling in adult angiogenesis.

Hedgehog receptor function during craniofacial development.

The Hedgehog signalling pathway plays a fundamental role in orchestrating normal craniofacial development in vertebrates. In particular, Sonic hedgehog (Shh) is produced in three key domains during the early formation of the head; neuroectoderm of the ventral forebrain, facial ectoderm and the pharyngeal endoderm; with signal transduction evident in both ectodermal and mesenchymal tissue compartments. Shh signalling from the prechordal plate and ventral midline of the diencephalon is required for appropriate division of the eyefield and forebrain, with mutation in a number of pathway components associated with Holoprosencephaly, a clinically heterogeneous developmental defect characterized by a failure of the early forebrain vesicle to divide into distinct halves. In addition, signalling from the pharyngeal endoderm and facial ectoderm plays an essential role during development of the face, influencing cranial neural crest cells that migrate into the early facial processes. In recent years, the complexity of Shh signalling has been highlighted by the identification of multiple novel proteins that are involved in regulating both the release and reception of this protein. Here, we review the contributions of Shh signalling during early craniofacial development, focusing on Hedgehog receptor function and describing the consequences of disruption for inherited anomalies of this region in both mouse models and human populations.