Blood Vessel Epicardial Substance Research Articles

MiR-665-Mediated Regulation of AHCYL2 and BVES Genes in Recurrent Implantation Failure.

The primary goal of this investigation was to identify mRNA targets affected by dysregulated miRNAs in RIF. This was accomplished by comprehensively analyzing mRNA and miRNA expression profiles in two groups: female subjects with normal reproductive function (control, n = 5) and female subjects experiencing recurrent implantation failure (RIF, n = 5). We conducted transcriptome sequencing and small RNA sequencing on endometrial tissue samples from these cohorts. Subsequently, we validated a selection of intriguing findings using real-time PCR with samples from the same cohort. In total, our analysis revealed that 929 mRNAs exhibited differential expression patterns between the control and RIF patient groups. Notably, our investigation confirmed the significant involvement of dysregulated genes in the context of RIF. Furthermore, we uncovered promising correlation patterns within these mRNA/miRNA pairs. Functional categorization of these miRNA/mRNA pairs highlighted that the differentially expressed genes were predominantly associated with processes such as angiogenesis and cell adhesion. We identified new target genes that are regulated by miR-665, including Blood Vessel Epicardial Substance (BVES) and Adenosylhomocysteinase like 2 (AHCYL2). Our findings suggest that abnormal regulation of genes involved in angiogenesis and cell adhesion, including BVES and AHCYL2, contributes to the endometrial dysfunction observed in women with recurrent implantation failure (RIF) compared to healthy women.

MBNL1‑AS1 attenuates tumor cell proliferation by regulating the miR‑29c‑3p/BVES signal in colorectal cancer.

Dysregulation of long non‑coding RNAs (lncRNAs) is involved in the development of colorectal cancer (CRC). In the present study, the identification of muscle blind like splicing regulator 1 antisense RNA 1 (MBNL1‑AS1) lncRNA was reported. Firstly, Cell Counting Kit‑8, EdU and colony formation assays were uesed to explore the role of MBNL1‑AS1 in regulating the proliferation of CRC cells. According to TCGA database, it was found that MBNL1‑AS1 was correlated with microRNA (miR)‑29c‑3p and blood vessel epicardial substance (BVES) expression in CRC cells. Then, the regulation among MBNL1‑AS1, miR‑29C‑3P and BVES was detected by dual luciferase reporter assay and the function of MBNL1‑AS1/miR‑29C‑3P/BVES axis was explored by rescue assay. The results demonstrated that MBNL1‑AS1 expression was decreased in CRC and was associated with the size of tumors derived from patients with CRC. Functionally, the upregulation of MBNL1‑AS1 suppressed CRC cell proliferation invitro and inhibited tumor growth invivo, while knockdown of MBNL1‑AS1 expression caused the opposite effects. MBNL1‑AS1 expression correlated with BVES expression in CRC tissues and MBNL1‑AS1 enhanced the stability of BVES mRNA by functioning as a competing endogenous RNA to sponge miR‑29c‑3p; the latter directly targeted MBNL1‑AS1 and BVES mRNA 3'UTR. Collectively, the results indicated that MBNL1‑AS1 suppressed CRC cell proliferation by regulating miR‑29c‑3p/BVES signaling, suggesting that the MBNL1‑AS1/miR‑29c‑3p/BVES axis may be a potential therapeutic target for CRC.

Genome-wide identification, evolution and expression analysis of tight junction gene family and the immune roles of claudin5 gene in turbot (Scophthalmus maximus L.)

Tight junction proteins (TJs) are important component proteins that maintaining the structure and function of TJs, connecting to each other to form a TJ complex between cells, maintaining the biological homeostasis of the internal environment. In this study, a total of 103 TJ genes were identified in turbot according to our whole-transcriptome database. Transmembrane TJs were divided into seven subfamilies, including claudin (CLDN), occludin (OCLD), tricellulin (MARVELD2), MARVEL domain containing 3 (MARVELD3), junctional adhesion molecules (JAM), immunoglobulin superfamily member 5 (IGSF5/JAM4), blood vessel epicardial substance (BVEs). Moreover, the majority of homologous pairs of TJ genes showed highly conserved alongside length, exon/intron number and motifs. As for phylogenetic analysis for 103 TJ genes, eight of them have undergone a positive selection and JAMB-like has undergone the most neutral evolution. The expression patterns of several TJ genes showed the lowest expression levels in blood, while the highest expression levels were detected in intestine, gill and skin, which all belong to mucosal tissues. Meanwhile, most examined TJ genes showed down-regulated expression patterns during bacterial infection, while several TJ genes exhibited up-regulated expression patterns at a later stage (24 h). At the same time, several potential candidate genes (such as CLDN-15, CLDN-3, CLDN-12, CLDN-5 and OCLD) were significantly down-regulated, which may indicate their important functions that involved in the regulation of bacterial infection. Currently, there is little research on CLDN5 in the intestine, but it is highly expressed in the intestine and has significant changes in intestinal expression after bacterial infection. Thus, we knocked down CLDN5 by the method of lentiviral infection. The result showed CLDN5 was related to cell migration (wound healing) and apoptosis, and the method of dualluciferasereporterassay showed that the functions of CLDN5 could be regulated by miR-24. The study of TJs may lead to a better understanding of the function of TJs in teleost.

Defective BVES-mediated feedback control of cAMP in muscular dystrophy

Biological processes incorporate feedback mechanisms to enable positive and/or negative regulation. cAMP is an important second messenger involved in many aspects of muscle biology. However, the feedback mechanisms for the cAMP signaling control in skeletal muscle are largely unknown. Here we show that blood vessel epicardial substance (BVES) is a negative regulator of adenylyl cyclase 9 (ADCY9)-mediated cAMP signaling involved in maintaining muscle mass and function. BVES deletion in mice reduces muscle mass and impairs muscle performance, whereas virally delivered BVES expressed in Bves-deficient skeletal muscle reverses these defects. BVES interacts with and negatively regulates ADCY9’s activity. Disruption of BVES-mediated control of cAMP signaling leads to an increased protein kinase A (PKA) signaling cascade, thereby promoting FoxO-mediated ubiquitin proteasome degradation and autophagy initiation. Our study reveals that BVES functions as a negative feedback regulator of ADCY9-cAMP signaling in skeletal muscle, playing an important role in maintaining muscle homeostasis.

Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking

The Popeye domain containing (POPDC) genes encode sarcolemma-localized cAMP effector proteins. Mutations in blood vessel epicardial substance (BVES) also known as POPDC1 and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein–protein interaction. Patients carrying a novel homozygous variant, BVES (c.547G > T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. In contrast, variants such as BVES p.Q153X or POPDC2 p.W188X were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1–POPDC2 complex. Mutations in POPDC proteins that cause an impairment in membrane localization affect POPDC complex formation while mutations which leave protein–protein interaction intact likely affect some other essential function of POPDC proteins.

Cell adhesion molecule BVES functions as a suppressor of tumor cells extrusion in hepatocellular carcinoma metastasis

BackgroundTumor cells detachment from primary lesions is an early event for hepatocellular carcinoma (HCC) metastasis, in which cell adhesion molecules play an important role. The role of mechanical crowding has attracted increasing attention. Previous studies have found that overcrowding can induce live cells extrusion to maintain epithelial cell homeostasis, and normally, live extruded cells eventually die through a process termed anoikis, suggesting the potential of tumor cells resistant to anoikis might initiate metastasis from primary tumors by cell extrusion. We have demonstrated transmembrane adhesion molecule blood vessel epicardial substance (BVES) suppression as an early event in HCC metastasis. However, whether its suppression is involved in HCC cell extrusion, especially in HCC metastasis, remains unknown. This study aims to investigate the role of BVES in tumor cells extrusion in HCC metastasis, as well as the underlying mechanisms.MethodsCells extrusion was observed by silicone chamber, petri dish inversion, and three-dimensional cell culture model. Polymerase chain reaction, western blotting, immunohistochemistry, immunofluorescence, co-immunoprecipitation, and RhoA activity assays were used to explore the underlying mechanisms of cell extrusion regulated by BVES. An orthotopic xenograft model was established to investigate the effects of BVES and cell extrusion in HCC metastasis in vivo.ResultsTumor cell extrusion was observed in HCC cells and tissues. BVES expression was decreased both in HCC and extruded tumor cells. BVES overexpression led to the decrease in HCC cells extrusion in vitro and in vivo. Moreover, our data showed that BVES co-localized with ZO-1 and GEFT, regulating ZO-1 expression and localization, and GEFT distribution, thus modulating RhoA activity.ConclusionThe present study revealed that BVES downregulation in HCC enhanced tumor cells extrusion, thus promoting HCC metastasis, which contributed to a more comprehensive understanding of tumor metastasis, and provided clues for developing novel HCC therapy strategies.BDBbGbGkfGhr4TimZgb_57Video abstract

Bves maintains vascular smooth muscle cell contractile phenotype and protects against transplant vasculopathy via Dusp1-dependent p38MAPK and ERK1/2 signaling

Background and aimsVascular smooth muscle cell (VSMC) plasticity is tightly associated with the pathological process of vasculopathy. Blood vessel epicardial substance (Bves) has emerged as an important regulator of intracardiac vasculogenesis and organ homeostasis. However, the involvement and role of Bves in VSMC plasticity and neointimal lesion development remain unclear. MethodsWe used an in vivo rat model of graft arteriosclerosis and in vitro PDGF-treated VSMCs and identified the novel VSMC contractile phenotype-related gene Bves using a transcriptomic analysis and literature search. In vitro knockdown and overexpression approaches were used to investigate the mechanisms underlying VSMC phenotypic plasticity. In vivo, VSMC-specific Bves overexpression in rat aortic grafts was generated to assess the physiological function of Bves in neointimal lesion development. ResultsHere, we found that Bves expression was negatively regulated in aortic allografts in vivo and PDGF-treated VSMCs in vitro. The genetic knockdown of Bves dramatically inhibited, whereas Bves overexpression markedly promoted, the VSMC contractile phenotype. Furthermore, RNA sequencing unraveled a positive correlation between Bves and dual-specificity protein phosphatase 1 (Dusp1) expression in VSMCs. We found that Bves knockdown restrained Dusp1 expression, but enhanced p38MAPK and ERK1/2 activation, resulting in the loss of the VSMC contractile phenotype. In vivo, an analysis of a rat graft model confirmed that VSMC-specific Bves and Dusp1 overexpression in aortic allografts significantly attenuated neointimal lesion formation. ConclusionsBves maintains the VSMC contractile phenotype through Dusp1-dependent p38MAPK and ERK1/2 signaling, and protects against neointimal formation, underscoring the important role of Bves in preventing transplant vasculopathy.

Proteomic and morphological insights and clinical presentation of two young patients with novel mutations of BVES (POPDC1)

Popeye domain containing protein 1 (POPDC1) is a highly conserved transmembrane protein essential for striated muscle function and homeostasis. Pathogenic variants in the gene encoding POPDC1 (BVES, Blood vessel epicardial substance) are causative for limb-girdle muscular dystrophy (LGMDR25), associated with cardiac arrhythmia. We report on four affected children (age 7-19 years) from two consanguineous families with two novel pathogenic variants in BVES c.457C>T(p.Q153X) and c.578T>G (p.I193S). Detailed analyses were performed on muscle biopsies from an affected patient of each family including immunofluorescence, electron microscopy and proteomic profiling. Cardiac abnormalities were present in all patients and serum creatine kinase (CK) values were variably elevated despite lack of overt muscle weakness. Detailed histological analysis of skeletal muscle, however indicated a myopathy with reduced sarcolemmal expression of POPDC1 accompanied by altered sarcolemmal and sarcoplasmatic dysferlin and Xin/XIRP1 abundance. At the electron microscopic level, the muscle fiber membrane was focally disrupted. The proteomic signature showed statistically significant dysregulation of 191 proteins of which 173 were increased and 18 were decreased. Gene ontology-term analysis of affected biological processes revealed - among others - perturbation of muscle fibril assembly, myofilament sliding, and contraction as well as transition between fast and slow fibers. In conclusion, these findings demonstrate that the phenotype of LGMDR25 is highly variable and also includes younger children with conduction abnormalities, no apparent muscular problems, and only mildly elevated CK values. Biochemical studies suggest that BVES mutations causing loss of functional POPDC1 can impede striated muscle function by several mechanisms.

Inhibition of Musashi-1 enhances chemotherapeutic sensitivity in gastric cancer patient-derived xenografts.

Musashi-1 (MSI1), a neural RNA-binding protein, is considered a gastric and intestinal stem cell marker. Although the function of MSI1 in gastric cancer has attracted increasing interest, it is not known whether MSI1 can be used as a biomarker to monitor gastric cancer development and response to treatment. Here, the role of MSI1 in the chemotherapeutic sensitivity of gastric cancer was investigated. Patients with high MSI1 levels had poor outcomes, implicating the gene in the development and progression of the disease. We overexpressed and silenced MSI1 in the human gastric cancer cell lines MKN45 and HGC27, finding that knockdown reduced proliferation, invasion, and migration, while promoting apoptosis. A patient-derived xenograft gastric cancer model was constructed in which mice received chemical drugs, si-MSI1, or a drug-si-MSI1 combination. It was found that blocking MSI1 expression reduced gastric cancer drug tolerance. The combination treatment with si-MSI1 was superior to 5F-dUMP and cisplatin, either separately or in combination, indicating that including si-MSI1 was better than drug therapy alone. Transcriptome sequencing analysis showed that MSI1 altered cell cycle regulation and growth signal transduction, including that of blood vessel epicardial substance (BVES). These results suggest that MSI1 reduces the tolerance of gastric cancer to chemical drugs through modulation of MSI1/BVES signaling.

Mice Lacking the cAMP Effector Protein POPDC1 Show Enhanced Hippocampal Synaptic Plasticity.

Extensive research has uncovered diverse forms of synaptic plasticity and an array of molecular signaling mechanisms that act as positive or negative regulators. Specifically, cyclic 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent signaling pathways are crucially implicated in long-lasting synaptic plasticity. In this study, we examine the role of Popeye domain-containing protein 1 (POPDC1) (or blood vessel epicardial substance (BVES)), a cAMP effector protein, in modulating hippocampal synaptic plasticity. Unlike other cAMP effectors, such as protein kinase A (PKA) and exchange factor directly activated by cAMP, POPDC1 is membrane-bound and the sequence of the cAMP-binding cassette differs from canonical cAMP-binding domains, suggesting that POPDC1 may have an unique role in cAMP-mediated signaling. Our results show that Popdc1 is widely expressed in various brain regions including the hippocampus. Acute hippocampal slices from Popdc1 knockout (KO) mice exhibit PKA-dependent enhancement in CA1 long-term potentiation (LTP) in response to weaker stimulation paradigms, which in slices from wild-type mice induce only transient LTP. Loss of POPDC1, while not affecting basal transmission or input-specificity of LTP, results in altered response during high-frequency stimulation. Popdc1 KO mice also show enhanced forskolin-induced potentiation. Overall, these findings reveal POPDC1 as a novel negative regulator of hippocampal synaptic plasticity and, together with recent evidence for its interaction with phosphodiesterases (PDEs), suggest that POPDC1 is involved in modulating activity-dependent local cAMP–PKA–PDE signaling.

BVES-AS1 inhibits the malignant behaviors of colon adenocarcinoma cells via regulating BVES.

The underexpression of the long noncoding RNA blood vessel epicardial substance antisense RNA 1 (BVES-AS1) has been shown in colon adenocarcinoma (COAD) patients. However, its role in COAD remains to be explored. This study aimed to investigate the function and potential mechanism of BVES-AS1 in COAD. Colony formation, Cell Counting Kit-8, JC-1 mitochondrial membrane potential assay, wound healing, transwell, and western blot analyses were used to measure cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) in COAD cells. RNA pull-down, luciferase reporter, and RNA binding protein immunoprecipitation assays were used to detect the interaction of BVES-AS1 and downstream genes. BVES-AS1 was expressed at low levels in COAD cells. Overexpressed BVES-AS1 inhibited COAD cell proliferation, migration, invasion, and EMT while elevating cell apoptosis. Mechanistically, BVES-AS1 functioned as a competing endogenous RNA sponging miR-522-3p to regulate the expression of nearby gene blood vessel epicardial substance (BVES). Besides this, BVES-AS1 recruited TATA-box binding protein associated factor 15 (TAF15) to promote BVES messenger RNA stability. Taken together, our study confirmed that BVES-AS1 inhibited COAD progression via interacting with miR-522-3p and TAF15 to regulate BVES expression, which might offer a perspective for COAD treatment.

Identification of the Novel Target Genes for Osteosarcoma Therapy Based on Comprehensive Bioinformatic Analysis.

Osteosarcoma is one of the most common primary malignant tumors of the bone and tends to develop in teenage years. Although multitreatments for the diagnosis and therapy of osteosarcoma have been developed, there are still needs of new methods to prevent and treat the osteosarcoma. Here, we performed bioinformatic analysis to screen for the key genes, molecules, and pathways involved in osteosarcoma survival. Four microarray data sets (GSE99671, GSE87624, GSE65071, and GSE28423), which include data from human bone and osteosarcoma samples, were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed mRNAs and miRNAs were identified. Kyoto Encyclopedia of Genes and Genomes enriched pathways, miRNA-mRNA target, gene/disease relationship, and overall survival was elucidated using related websites and software according to bioinformatic analysis protocols. We found three critical genes miR-29c, blood vessel epicardial substance (BVES), and proteasome 20S subunit beta 2 (PSMB2) through the GEO database and predicting miRNA-mRNA target. Among these genes, BVES and PSMB2 presented a high expression level in osteosarcoma based on GSE99671 and GSE87624 data sets, while miR-29c showed a low expression level in osteosarcoma based on GSE65071 and GSE28423 data sets. Furthermore, we found that the high expression level of miR-29c and BVES associated with better prognosis, while highly expressed PSMB2 associated with poor prognosis. The abnormally expressed mRNAs and miRNAs, which were identified by integrated bioinformatic analysis, provided insights into the molecular mechanisms of osteosarcoma. Notably, we found three critical genes that could be used as novel therapeutic targets for preventing or diagnosing osteosarcoma. Finally, PSMB2 may be the target of miR-29c.

The Functional Polymorphism R129W in the BVES Gene Is Associated with Sporadic Tetralogy of Fallot in the Han Chinese Population.

Background: Tetralogy of Fallot (TOF) accounts for ∼10% of congenital heart disease cases. The blood vessel epicardial substance (BVES) gene has been reported to play a role in the function of adult hearts. However, whether allelic variants in BVES contribute to the risk of TOF and its possible mechanism remains unknown. Methods: The open reading frame of the BVES gene was sequenced using samples from 146 TOF patients and 100 unrelated healthy controls. qRT-PCR and western blot assays were used to confirm the expression of mutated BVES variants in the TOF samples. The online software Polyphen2 and SIFT were used to predict the deleterious effects of the observed allelic variants. The effects of these allelic variants on the transcriptional activities of genes were examined using dual-fluorescence reporter assays. Results: We genotyped four single nucleotide polymorphisms (SNPs) in the BVES gene from each of the 146 TOF patients. Among them, the minor allelic frequencies of c.385C>T (p.R129W) were 0.035% in TOF, but ∼0.025% in 100 controls and the Chinese Millionome Database. This allelic variant was predicted to be a potentially harmful alteration by the Polyphen2 and SIFT softwares. qRT-PCR and western blot analyses indicated that the expression of BVES in the six right ventricular outflow tract samples with the c.385C>T allelic variant was significantly downregulated. A dual-fluorescence reporter system showed that the c.385C>T allelic variant significantly decreased the transcriptional activity of the BVES gene and also decreased transcription from the GATA4 and NKX2.5 promoters. Conclusions: c.385C>T (p.R129W) is a functional SNP of the BVES gene that reduces the transcriptional activity of BVES in vitro and in vivo in TOF tissues. This subsequently affects the transcriptional activities of GATA4 and NKX2.5 related to TOF. These findings suggest that c.385C>T may be associated with the risk of TOF in the Han Chinese population.

Ten years of research on the role of BVES/ POPDC1 in human disease: a review.

Since the blood vessel epicardial substance or Popeye domain-containing protein 1 (BVES/POPDC1) was first identified in the developing heart by two independent laboratories in 1999, an increasing number of studies have investigated the structure, function, and related diseases of BVES/POPDC1. During the first 10 years following the discovery of BVES/POPDC1, studies focused mainly on its structure, expression patterns, and functions. Based on these studies, further investigations conducted over the previous decade examined the role of BVES/POPDC1 in human diseases, such as colitis, heart diseases, and human cancers. This review provides an overview of the structure and expression of BVES/POPDC1, mainly focusing on its potential role and mechanism through which it is involved in human cancers.

Blood vessel epicardial substance (BVES) reduces LRP6 receptor and cytoplasmic -catenin levels to modulate Wnt signaling and intestinal homeostasis.

Blood vessel epicardial substance (BVES, otherwise known as POPDC1) is an integral membrane protein known to regulate tight junction formation and epithelial-mesenchymal transition. BVES is underexpressed in a number of malignancies, including colorectal cancer. BVES loss leads to activation of the Wnt pathway, suggesting that decreased BVES expression functionally contributes to tumorigenesis. However, the mechanism by which BVES modulates Wnt signaling are unknown. Here we confirm that BVES loss increases -catenin protein levels, leads to Wnt pathway activation in a ligand-independent fashion, and coordinates with Wnt ligand to further increase Wnt signaling. We show that BVES loss increases levels and activation of the Wnt co-receptor, LRP6, in cell lines, murine adenoma tumoroids, and human-derived colonoids. We also demonstrate that BVES interacts with LRP6. Finally, murine tumor modelling using a Wnt-driven genetic model and a chemically-induced model of colorectal carcinogenesis demonstrates that BVES loss increases tumor multiplicity and dysplasia. Together, these results implicate BVES as an inhibitor of Wnt signaling, provide one of the first examples of a tight junction-associated protein regulating Wnt receptor levels, and expand the number of putative molecular targets for therapeutic intervention in colorectal cancer.

BVES is required for maintenance of colonic epithelial integrity in experimental colitis by modifying intestinal permeability.

Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein which modulates epithelial to mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical to maintaining colonic epithelial integrity. At baseline, Bves−/− mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves−/− mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves−/− mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.

829 - Blood Vessel Epicardial Substance (BVES) Coordinately Reduces LRP6 Receptor and Cytoplasmic β-Catenin Levels to Modulate WNT Signaling and Intestinal Homeostasis

829 - Blood Vessel Epicardial Substance (BVES) Coordinately Reduces LRP6 Receptor and Cytoplasmic β-Catenin Levels to Modulate WNT Signaling and Intestinal Homeostasis

Abstract 4124: POPDC1 inhibits breast cancer cell migration and proliferation, and is regulated by EGFR (ErbB1) and cAMP

Abstract Despite progress in the development of new treatments, breast cancer remains the second leading cause of global cancer morbidity and mortality (1). Identification and validation of effective therapeutic targets is thus essential for the development of better breast cancer treatments and improved prognoses. This project seeks to develop POPDC proteins as potential therapeutic targets for inhibiting breast cancer migration and proliferation. POPDC1 (also known as blood vessel epicardial substance or BVES), is a cAMP-binding transmembrane protein encoded by the POPDC1 gene. Loss or suppression of POPDC1 has been correlated with enhanced aggressive cancer cell behaviour (2-6). Suppression of POPDC1 promotes cell migration and invasion in hepatocellular carcinoma and tumourigenesis in colorectal cancer (2,4,5,7). Furthermore, we have previously shown that loss of POPDC1 promotes cell proliferation and migration in breast cancer and glioblastoma cells (3,8). Using a cAMP-agarose pulldown assay, we confirmed that POPDC1 interact with, and is upregulated by cAMP in luminal A (MCF7) and triple-negative (MDA231) breast cancer cells. Furthermore, immunocytochemistry and Western blotting has shown that POPDC1 expression is abrogated in MCF7 and MDA231 breast cancer cells compared to normal breast cells (HMepC). To confirm that POPDC1 negatively regulates migration and proliferation in breast cancer cells, we conducted knock-in experiments where overexpression of POPDC1 significantly inhibited cell migration and proliferation in MCF7 and MDA231 cells. Interestingly, epidermal growth factor receptor (EGFR/ErbB1) signalling negatively regulates POPDC1 expression in MCF7 and MDA231 cells and reduces the capacity of POPDC1 to inhibit cell migration. Epidermal growth factor reversed POPDC1-mediated inhibition of cell migration, an effect blocked by the AG 1478 (an EGFR inhibitor), suggesting EGFR-dependent modulation of POPDC1 activity. Furthermore, we have established for the first time using human breast tissue sections that Her2- status correlates with low POPDC1 expression while Her2+ status correlates with high POPDC1 expression. This suggests further interaction between EGF and POPDC1 and that any links between these molecules diverges at the level of the different EGFRs. Taken together, this strong dataset shows that POPDC1 plays a major role in breast cancer tumourigenesis that is at least in part, modulated by EGFR and cAMP signalling pathways. Indeed, these seem to demonstrate differential control of POPDC1 expression. This interplay and the fact that POPDC1 downregulation may be a downstream molecular target of the EGF cascade suggest that POPDC1 and its stabilisation may be a therapeutic strategy in the future of breast cancer. Citation Format: Johanna N. Amunjela, Steven J. Tucker. POPDC1 inhibits breast cancer cell migration and proliferation, and is regulated by EGFR (ErbB1) and cAMP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4124. doi:10.1158/1538-7445.AM2017-4124

Novel insights into cardiac remodelling revealed by proteomic analysis of the trout heart during exercise training

The changes in the cardiac proteome of rainbow trout (Oncorhynchus mykiss) were quantified during the early phases (4, 7, and 14d) of a typical exercise-training regime to provide a comprehensive overview of the cellular changes responsible for developing a trained heart phenotype. Enhanced somatic growth during the 14d experiment was paralleled by cardiac growth to maintain relative ventricular mass. This was reflected in the cardiac proteome by the increased abundance of contractile proteins and cellular integrity proteins as early as Day 4, including a pronounced and sustained increase in blood vessel epicardial substance – an intercellular adhesion protein expressed in the vertebrate heart. An unexpected finding was that proteins involved in energy pathways, including glycolysis, β-oxidation, the TCA cycle, and the electron transport chain, were generally present at lower levels relative to Day 0 levels, suggesting a reduced investment in the maintenance of energy production pathways. However, as the fish demonstrated somatic and cardiac growth during the exercise-training program, this change did not appear to influence cardiac function. The in-depth analysis of temporal changes in the cardiac proteome of trout during the early stages of exercise training reveals novel insights into cardiac remodelling in an important model species. Biological significanceRainbow trout hearts have a remarkable ability for molecular, structural, and functional plasticity, and the inherent athleticism of these fish makes them ideal models for studies in comparative exercise physiology. Indeed, several decades of research using exercise-trained trout has shown both conserved and unique aspects of cardiac plasticity induced by a sustained increase in the workload of the heart. Despite a strong appreciation for the outcome of exercise training, however, the temporal events that generate this phenotype are not known. This study interrogates the early stages of exercise training using in-depth proteomic analysis to understand the molecular pathways of cardiac remodelling. Two major and novel findings emerge: (1) structural remodelling is initiated very early in training, as evidenced by a general increase in proteins associated with muscle contraction and integrity at Day 4, and (2) the abundance of proteins directly involved in energy production are decreased during 14d of exercise training, which contrasts the general acceptance of an exercise-induced increase in aerobic capacity of muscle, and suggests that regulation of energy pathways occurs at a different biological level than protein abundance.

BVES regulates c-Myc stability via PP2A and suppresses colitis-induced tumourigenesis.

Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD. We determined BVES promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured BVES mRNA levels in a tissue microarray consisting of normal colons and CAC samples. Bves-/- and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels. BVES mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, BVES promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, Bves was underexpressed in experimental inflammatory carcinogenesis, and Bves-/- mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of Bves-/- tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction. Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. BVES promoter methylation status may serve as a CAC biomarker.