Overexpression Of RSPO2 Research Articles

Abstract 323: Rspo2 Overexpression Attenuates Hepatic Lipid Accumulation And Atherosclerosis

Aims: We have recently reported elevated RSPO2 levels in human and murine atherosclerotic arteries and discovered its anti-lymphangiogenic potential. However, the precise role of RSPO2 in atherosclerosis is not known. In this study, we investigated the effects of RSPO2 overexpression on hepatosteatosis and atherosclerosis development. Methods and Results: Atheroprone ♂ and ♀ Apoe -/- mice were injected with AAV8- GFP or AAV8- Rspo2 , fed a Western diet for 12 weeks, and analyzed for hepatic lipid accumulation and atherosclerotic lesion formation. Our quantitative RT-PCR data revealed increased Rspo2 mRNA expression in the liver, kidney and spleen of AAV8- Rspo2 -injected mice compared with control mice. Oil Red O (ORO) staining showed reduced hepatic lipid deposition in mice injected with AAV8- Rspo2 compared with AAV8- GFP mice. Additionally, there were reduced levels of hepatic triglyceride, non-esterified fatty acid, and lipid peroxidation in Rspo2 -overexpressing mice. Rspo2 overexpression reduced hepatic fibrosis and macrophage accumulation as indicated by reduced expression of SMA and CD68 in liver tissue. Additionally, decreased mRNA expression of proinflammatory and cholesterol metabolism-related genes was found in liver of AAV8- Rspo2 -injected mice. En face ORO staining of aortae and aortic root sections demonstrated suppressed atherosclerosis in mice with Rspo2 overexpression. Interestingly, AAV8- Rspo2 -injected mice had decreased weight gain over the course of Western diet feeding in comparison to control mice, however, no differences in fasting blood glucose and plasma total cholesterol levels were observed between the two groups. Conclusions: Taken together, these findings suggest that global Rspo2 overexpression in hypercholesterolemic mice suppresses hepatic lipid accumulation and reduces atherosclerosis.

Bone marrow mesenchymal stem cell-derived extracellular vesicles containing miR-497-5p inhibit RSPO2 and accelerate OPLL

AimsMuscle and adipose tissue-derived mesenchymal stem cells presented high osteogenic potentials, which modulate osteoblast function through releasing extracellular vesicles (EVs) containing miRNAs. Herein, this study evaluated the function of bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) delivering miR-497-5p in ossification of the posterior longitudinal ligament (OPLL). Main methodsThe expression level of miR-497-5p was validated in ossified posterior longitudinal ligament (PLL) tissues and BMSC-EVs. The uptake of BMSC-EVs by ligament fibroblasts was observed by immunofluorescence. miR-497-5p was overexpressed or downregulated to assess its role in osteogenic differentiation of ligament fibroblasts. Further, an OPLL rat model was established to substantiate the effect of BMSC-EVs enriched with miR-497-5p on OPLL. Key findingsOssified PLL tissues presented with high miR-497-5p expression. PLL fibroblasts were identified to endocytose BMSC-EVs. BMSC-EVs could upregulate miR-497-5p and shuttle it to ligament fibroblasts to accelerate the osteogenic differentiation. miR-497-5p targeted and inversely regulated RSPO2. Then, RSPO2 overexpression activated Wnt/β-catenin pathway and repressed the osteogenic differentiation of ligament fibroblasts. In vivo experiments further showed that miR-497-5p-containing BMSC-EVs enhanced OPLL through diminishing RSPO2 and inactivating Wnt/β-catenin pathway. SignificanceBMSC-EVs could deliver miR-497-5p to ligament fibroblasts and modulate RSPO2-mediated Wnt/β-catenin pathway, thereby accelerating OPLL.

MiR-196b-5p Promotes Proliferation, Migration and Invasion of Lung Adenocarcinoma Cells via Targeting RSPO2.

ObjectiveTo explore the biological role of miR-196b-5p/RSPO2 in the occurrence and development of lung adenocarcinoma (LUAD) and to provide a basis for finding new therapeutic targets for LUAD.MethodsDifferentially expressed genes were analyzed based on LUAD microarray, and the target gene of the target miRNA was predicted. qRT-PCR was used to detect the expression levels of miR-196b-5p and RSPO2 mRNA in normal human bronchial epithelial cell line BEAS-2B and LUAD cell lines A549, NCI-H1792 and NCI-H226. Western blot was used to evaluate protein expression. Cell proliferative, migratory and invasive abilities were detected by CCK-8 and transwell assays. Dual-luciferase assay was conducted to verify the targeting relationship between miR-196b-5p and RSPO2.ResultsThe results of qRT-PCR showed that miR-196b-5p was significantly highly expressed in LUAD cells, and the expression level of its downstream target gene RSPO2 was significantly decreased. The results of CCK-8 and transwell assays exhibited that miR-196b-5p promoted proliferation, migration and invasion of LUAD cells, while RSPO2 inhibited the malignant progression of LUAD cells. Dual-luciferase assay confirmed the targeted binding relationship between miR-196b-5p and RSPO2. Overexpression of RSPO2 partially reversed the promotion of miR-196b-5p on proliferation, migration and invasion of LUAD cells.ConclusionmiR-196b-5p promoted proliferation, migration and invasion of LUAD cells by targeting and down-regulating RSPO2, which provided ideas for searching new targets for the diagnosis and treatment of LUAD.

Expression and Prognostic Values of the Roof Plate-Specific Spondin Family in Bladder Cancer.

The roof plate-specific spondin (RSPO) family of proteins has critical roles in the tumorigenesis and progression of several carcinomas; however, little is known about their functions in bladder cancer (BLCA). This study aimed to investigate RSPO in terms of their expression levels, prognostic value, and potential mechanisms of action in BLCA. mRNA expression profiles and clinical information of BLCA patients were collected from The Cancer Genome Atlas database. Genetic alteration and DNA methylation data were obtained from cBioPortal and MethHC databases, respectively, and SurvExpress was used to determine the prognostic risk score of each RSPO. R software was used to analyze the expression levels and prognostic roles of RSPOs in BLCA. The effects of RSPO2 overexpression in BLCA cells were detected using MTT, colony formation, and Transwell invasion assays. Gene set enrichment analysis (GSEA) was used to analyze the functions of RSPOs and associated signaling pathways in BLCA. All members of the RSPO family were differentially expressed in BLCA cells compared with normal control cells. Aberrant RSPO expression levels were associated with higher histological stages and worse prognosis. The frequency of genetic alterations in RSPO genes was very high, which was related to a less favorable prognosis. Moreover, the effects of mutations in the RSPO2 gene were reversed using a Wnt/β-catenin inhibitor, IWP-2. In addition, GSEA demonstrated that RSPOs were associated with focal adhesion and immune cell infiltration, which was then confirmed by tumor immune cell infiltration analysis. RSPOs are potential biomarkers for predicting the prognosis of patients with BLCA and may serve as novel therapeutic targets. Moreover, overexpressed RSPO2 promoted BLCA cell growth and invasion through the Wnt/β-catenin pathway. In addition, RSPOs may regulate the progression of BLCA through modulating cell adhesion, focal adhesion, and CD4+ T cell and macrophage infiltration.

R-spondin 2 Drives Liver Tumor Development in a Yes-Associated Protein-Dependent Manner.

Each year, more than 25,000 people succumb to liver cancer in the United States, and this neoplasm represents the second cause of cancer‐related death globally. R‐spondins (RSPOs) are secreted regulators of Wnt signaling that function in development and promote tissue stem cell renewal. In cancer, RSPOs 2 and 3 are oncogenes first identified by insertional mutagenesis screens in tumors induced by mouse mammary tumor virus and by transposon mutagenesis in the colonic epithelium of rodents. RSPO2 has been reported to be activated by chromosomal rearrangements in colorectal cancer and overexpressed in a subset of hepatocellular carcinoma. Using human liver tumor gene expression data, we first discovered that a subset of liver cancers were characterized by high levels of RSPO2 in contrast to low levels in adjacent nontumor tissue. To determine if RSPOs are capable of inducing liver tumors, we used an in vivo model from which we found that overexpression of RSPO2 in the liver promoted Wnt signaling, hepatomegaly, and enhanced liver tumor formation when combined with loss of transformation‐related protein 53 (Trp53). Moreover, the Hippo/yes‐associated protein (Yap) pathway has been implicated in many human cancers, influencing cell survival. Histologic and gene expression studies showed activation of Wnt/β‐catenin and Hippo/Yap pathways following RSPO2 overexpression. We demonstrate that knockdown of Yap1 leads to reduced tumor penetrance following RSPO2 overexpression in the context of loss of Trp53. Conclusion: RSPO2 overexpression leads to tumor formation in the mouse liver in a Hippo/Yap‐dependent manner. Overall, our results suggest a role for Yap in the initiation and progression of liver tumors and uncover a novel pathway activated in RSPO2‐induced malignancies. We show that RSPO2 promotes liver tumor formation in vivo and in vitro and that RSPO2's oncogenic activity requires Hippo/Yap activation in hepatocytes. Both RSPO2 and YAP1 are suggested to represent novel druggable targets in Wnt‐driven tumors of the liver.

EIF3E-RSPO2 and PIEZO1-RSPO2 fusions in colorectal traditional serrated adenoma.

Traditional serrated adenoma (TSA) is an uncommon type of colorectal serrated polyp. RSPO fusions, which potentiate WNT signalling, are common and characteristic genetic alterations in TSA. The aim of this study was to further characterise the prevalence and variation of RSPO fusions in TSA. Quantitative polymerase chain reaction (PCR) analysis of 99 TSAs revealed overexpression of RSPO2 and RSPO3 in six and 29 lesions, respectively. Reverse transcription PCR identified previously reported PTPRK-RSPO3 fusion transcripts in all 29 TSAs with RSPO3 overexpression, confirming that PTPRK-RSPO3 is the predominant RSPO fusion in TSAs. Among the six lesions with RSPO2 overexpression, two overexpressed full-length RSPO2. An EIF3E-RSPO2 fusion, which is a known recurrent RSPO fusion in colorectal cancer, was detected in three lesions. In addition, rapid amplification of cDNA ends identified a novel PIEZO1-RSPO2 fusion in one TSA. All of the four TSAs with RSPO2 fusions concurrently had KRAS mutations and showed the classic histological features. The present study identified EIF3E-RSPO2 and PIEZO1-RSPO2 in TSAs. Our observations expand the spectrum of RSPO fusions in TSAs, and suggest that TSAs are precursors of colorectal cancers with these RSPO2 fusions.

R-spondin 2-LGR4 system regulates growth, migration and invasion, epithelial-mesenchymal transition and stem-like properties of tongue squamous cell carcinoma via Wnt/β-catenin signaling

BackgroundR-spondins (Rspo) and leucine-rich repeat-containing G-protein-coupled receptors (LGR) play important roles in development, stem cells survival, and tumorigenicity by activating Wnt signaling pathway. Whether R-spondins-LGR signaling affects the progression of squamous cell carcinoma (SCC) remain unknown. This study aims to uncover the role of R-spodin2/LGR4 in tongue SCC (TSCC). MethodsThe expression of Rspo2 in TSCC specimens and its correlation with TSCC clinical outcome were evaluated. Levels of Rspo2 or LGR4 were altered by pharmacological and genetic approaches, and the effects on TSCC progression were assessed. FindingsAberrantly high levels of Rspo2 were detected in TSCC specimens. Its levels were closely related with lymph node metastasis, clinical stage and survival rate in patients with tongue SCC. Exogenous Rspo2 or overexpression of Rspo2 promoted growth, migration and invasion, epithelial-mesenchymal transition (EMT) and stem-like properties in SCC both in vivo and in vitro. Silence of Rspo2 abolished these phenotypes. LGR4 was functionally upregulated by Rspo2 in TSCC. Overexpression of Rspo2 increased, whereas Rspo2 silencing decreased the expression of LGR4, leading to subsequent phosphorylation of LRP6 and nuclear translocation of β-catenin in TSCC cell lines. This nuclear translocation of β-catenin was associated with a significant alteration in TCF-1, a downstream nuclear transcription factor of β-catenin, as well as its target genes: CD44, CyclinD1 and c-Myc. InterpretationRspo2-LGR4 system regulates growth, migration and invasion, EMT and stem-like properties of TSCC via Wnt/β-catenin signaling pathway.

Clinical value of R-spondins in triple-negative and metaplastic breast cancers

Background:RSPO ligands, activators of the Wnt/β-catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC).Methods:Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT–PCR. The effect of RSPO on the Wnt/β-catenin pathway activity was determined by luciferase assay, western blotting, and qRT–PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/β-catenin pathway, was examined on the growth of an RSPO2-positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC.Results:We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO, and amplification or hypomethylation of RSPO genes. Patients with RSPO2-overexpressing tumours have a poorer metastasis-free survival (P=3.6 × 10−4). RSPO2 and RSPO4 stimulate Wnt/β-catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2-overexpressing PDX.Conclusions:RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

Abstract A242: R-spondin 2 drives Wnt signaling and tumor formation in breast and liver cancer.

Abstract Background: R-spondins are secreted agonists of Wnt signaling that function in development and promote tissue stem cell proliferation. Rspo2 was identified as a candidate oncogene in intestinal tumors via a Sleeping Beauty transposon insertional mutagenesis screen in mice (1). Further, oncogenic activation of RSPO2 and RSPO3 mediated by recurrent genomic rearrangements has been identified in human colorectal cancer (2). The purpose of the current study was to determine if R-spondins function as oncogenes in other cancer types characterized by active Wnt signaling. Methods: R-spondin mRNA expression was determined in primary human breast tumors and adjacent normal tissue by quantitative RT-PCR. Affymetrix microarrays were used to assay gene expression and classify by molecular subtype human hepatocellular carcinomas compared to pre-malignant lesions and normal livers. RSPO2 was depleted or overexpressed in breast cell lines by stable transduction with anti-RSPO2 shRNA or RSPO2 cDNA. Resulting changes in gene expression and proliferation were measured by qRT-PCR and MTS assay respectively. RSPO2 was somatically overexpressed in murine liver by hydrodynamic injection and Fah selection in an Fah-null model. Subsequent gene expression studies were conducted by qRT-PCR and immunohistochemistry. Results: RSPO2 was highly expressed in 12% of primary human breast tumors compared to adjacent normal tissue. Similarly, RSPO2 expression was elevated in the subset of primary human liver cancers with activated Wnt/beta-catenin signaling. In human breast cancer cells with elevated RSPO2 expression, RSPO2 knockdown decreased Wnt signaling and proliferation, while RSPO2 overexpression in a non-tumorigenic breast epithelial cell line potentiated Wnt signaling. Overexpression of RSPO2 in the mouse liver increased Wnt signaling and promoted an enlarged liver phenotype and tumor formation. Conclusions: These data strongly suggest that RSPO2 is a driver of human breast and liver cancer. Future work will further characterize signaling pathways implicated in RSPO-driven phenotypes, and develop targeted therapy to inhibit RSPO signaling. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A242. Citation Format: Caitlin B. Conboy, Bobbi R. Tschida, Hsiangyu Hu, Michael B. Burns, Nuri A. Temiz, Timothy Kuka, Vincent W. Keng, Sara Toffanin, Reuben S. Harris, Josep Llovet, Timothy K. Starr, David A. Largaespada. R-spondin 2 drives Wnt signaling and tumor formation in breast and liver cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A242.

The Role of R-Spondin2 in Keratinocyte Proliferation and Epidermal Thickening in Keloid Scarring

Keloids are found only in humans and the underlying biochemical mechanisms of their pathogenesis remain unknown. R-spondins (Rspos) are a relatively new group of secreted proteins known to be Wnt/β-catenin signaling agonists, but their role in keloids has yet to be elucidated. We investigated the expression levels of R-spondin2 (Rspo2) in cell lysates and conditioned media of monocultures and co-cultures of fibroblasts and keratinocytes derived from keloids and normal skin. In this study we found increased protein expression and secretion of Rspo2 in respective monocultures of keloid fibroblasts and keratinocytes when compared with their normal counterparts. Double-chamber co-culture experiments implicated the role of keloid keratinocytes (KKs) in the induction of Rspo2 secretion from fibroblasts because of epithelial-mesenchymal interactions. Addition of recombinant human Rspo2 in culture increased the proliferation of keratinocytes and it acted synergistically with Wnt3a through the canonical Wnt/β-catenin pathway. Overexpression of Rspo2 in normal fibroblasts brought about thicker epidermis when compared with control fibroblasts in a skin organotypic culture model. This observation coincides with the hyperproliferative phenotype of thickened epidermis seen in keloids. Taken together, the results suggest the possible double paracrine action of KKs in inducing higher expression of Rspo2 in fibroblasts that promotes keratinocyte proliferation and epidermal thickening.

Wnt11 Promotes Osteoblast Maturation and Mineralization through R-spondin 2

Wnt11 signals through both canonical (beta-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases beta-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/beta-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through beta-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.

R-spondin2 is a secreted activator of Wnt/β-catenin signaling and is required for Xenopus myogenesis: Kazanskaya O, Glinka A, del BarcoBarrantes I, Stannek P, Niehrs C, Wu W, Division of Molecular Embryology, Deutsches Krebsforschungszentrum, Heidelberg, Germany

We have carried out a small pool expression screen for modulators of the Wnt/β-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate β-catenin. Analyses of functional interaction with components of the Wnt/β-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/ β-catenin pathway, Rspo2 overexpression blocks activin, nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-β pathway. Antisense morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/β-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/β-catenin cascade.

R-Spondin2 Is a Secreted Activator of Wnt/β-Catenin Signaling and Is Required for Xenopus Myogenesis

We have carried out a small pool expression screen for modulators of the Wnt/β-catenin pathway and identified Xenopus R-spondin2 ( Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate β-catenin. Analyses of functional interaction with components of the Wnt/β-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/β-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-β pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/β-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/β-catenin cascade.