Recombinant CCN1 Research Articles

Human trophoblast invasion and migration are mediated by the YAP1-CCN1 pathway: defective signaling in trophoblasts during early-onset severe preeclampsia†.

The transcription coactivator YAP1 mediates the major effects of the Hippo signaling pathway. The CCN family is a small group of glycoproteins known to be downstream effectors of YAP1 in diverse tissues. However, whether CCN family members mediate the effects of YAP1 in human trophoblasts is unknown. In this study, placental expression of both YAP1 and CCN1 was found to be impaired in pregnancies complicated by early-onset severe preeclampsia. CCN1 was expressed not only in cytotrophoblasts, trophoblast columns, and mesenchymal cells, similar to active YAP1, but also in syncytiotrophoblasts of normal first-trimester placental villi; moreover, decidual staining of active YAP1 and CCN1 was found in both interstitial and endovascular extravillous trophoblasts. In cultured immortalized human trophoblastic HTR-8/SVneo cells, knockdown of YAP1 decreased CCN1 mRNA and protein expression and led to impaired cell invasion and migration. Also, CCN1 knockdown negatively affected HTR-8/SVneo cell invasion and migration but not viability. YAP1 knockdown was further found to impair HTR-8/SVneo cell viability via G0/G1 cell cycle arrest and apoptosis, while CCN1 knockdown had minimal effect on cell cycle arrest and no effect on apoptosis. Accordingly, treatment with recombinant CCN1 partially reversed the YAP1 knockdown-induced impairment in trophoblast invasion and migration but not in viability. Thus, CCN1 mediates the effects of YAP1 on human trophoblast invasion and migration but not apoptosis, and decreased placental expression of YAP1 and CCN1 in pregnancies complicated by early-onset severe preeclampsia might contribute to the pathogenesis of this disease.

Ginsenoside Rg1 ameliorates hypoxia-induced pulmonary arterial hypertension by inhibiting endothelial-to-mesenchymal transition and inflammation by regulating CCN1

Pulmonary arterial hypertension (PAH) is a chronic obstructive disease characterized by vascular remodeling. Studies have confirmed that ginsenoside Rg1 can improve pulmonary hypertension to a certain extent, but the potential mechanism by which it improves hypoxia-induced PAH remains unclear. The aim of this study was to investigate the therapeutic effect of ginsenoside Rg1 on hypoxia-induced PAH. The results showed that hypoxia promoted inflammation, EndMT, and vascular remodeling, which were accompanied by decreased CCN1 levels and increased p-NFκB p65, TGF-β1, and p-Smad 2/3 levels. Treatment with ginsenoside Rg1, recombinant CCN1, BAY-11-7082, and SB-431542 could prevent hypoxia-induced vascular remodeling, reduce the expression of the hypoxia-induced inflammatory cytokines TNF-α and IL-1β, inhibit the expression of the mesenchymal markers α-SMA and Vimentin and restore the expression of the endothelial markers CD31 and VE-cadherin to improve hypoxia-induced EndMT, which may be associated with the upregulation of CCN1 protein expression and downregulation of p-NFκB p65, TGF-β1, and p-Smad 2/3 in rats and cells. siRNA CCN1 transfection increased the expression of p-NFκB p65, TGF-β1, and p-Smad 2/3 and accelerated the occurrence and development of inflammation and EndMT after hypoxia. In summary, our study indicated that hypoxia-induced EndMT and inflammation play a role in hypoxic pulmonary hypertension (HPH). Ginsenoside Rg1 treatment could reverse hypoxia-induced EndMT and inflammation by regulating CCN1 and has potential value in the prevention and treatment of HPH.

Astragaloside IV improves pulmonary arterial hypertension by increasing the expression of CCN1 and activating the ERK1/2 pathway.

The aim of the present study was to investigate the underlying mechanism of AS-IV and CCN1 in PAH and to evaluate whether the protective effect of AS-IV against PAH is associated with CCN1 and its related signalling pathway. In vivo, male SD rats were intraperitoneally injected with monocrotaline (MCT, 60 mg/kg) or exposed to hypoxia (10% oxygen) and gavaged with AS-IV (20, 40 and 80 mg/kg/day) to create a PAH model. In vitro, human pulmonary artery endothelial cells (hPAECs) were exposed to hypoxia (3% oxygen) or monocrotaline pyrrole (MCTP, 60 μg/mL) and treated with AS-IV (10, 20 and 40 μM), EGF (10nM, ERK agonist), small interfering CCN1 (CCN1 siRNA) and recombinant CCN1 protein (rCCN1, 100 ng/mL). We identified the differences in the expression of genes in the lung tissues of PAH rats by proteomics. At the same time, we dynamically detected the expression of CCN1 by Western blot both in vivo and in vitro. The Western blot experimental results showed that the expression of CCN1 increased in the early stage of PAH and decreased in the advanced stage of PAH. The results showed that compared with the control group, MCT- and hypoxia-induced increased the hemodynamic parameters and apoptosis. AS-IV can improve PAH, as characterized by decreased hemodynamic parameters, vascular wall area ratio (WA%), vascular wall thickness ratio (WT%) and α-SMA expression and inhibition of cell apoptosis. Moreover, the improvement of PAH by AS-IV was accompanied by increased CCN1 expression, which activated the ERK1/2 signalling pathway. Meanwhile, CCN1 and p-ERK1/2 were inhibited by siCCN1 and promoted by rCCN1. EGF not only activated the ERK1/2 signalling pathway but also induced the expression of CCN1. In conclusion, AS-IV improves PAH by increasing the expression of CCN1 and activating the ERK1/2 signalling pathway. The results of our study provide a theoretical basis for additional study on the protective effect of AS-IV against PAH.

Binding of the angiogenic/senescence inducer CCN1/CYR61 to integrin α6β1 drives endocrine resistance in breast cancer cells.

CCN1/CYR61 promotes angiogenesis, tumor growth and chemoresistance by binding to its integrin receptor αvβ3 in endothelial and breast cancer (BC) cells. CCN1 controls also tissue regeneration by engaging its integrin receptor α6β1 to induce fibroblast senescence. Here, we explored if the ability of CCN1 to drive an endocrine resistance phenotype in estrogen receptor-positive BC cells relies on interactions with either αvβ3 or α6β1. First, we took advantage of site-specific mutagenesis abolishing the CCN1 receptor-binding sites to αvβ3 and α6β1 to determine the integrin partner responsible for CCN1-driven endocrine resistance. Second, we explored a putative nuclear role of CCN1 in regulating ERα-driven transcriptional responses. Retroviral forced expression of a CCN1 derivative with a single amino acid change (D125A) that abrogates binding to αvβ3 partially phenocopied the endocrine resistance phenotype induced upon overexpression of wild-type (WT) CCN1. Forced expression of the CCN1 mutant TM, which abrogates all the T1, H1, and H2 binding sites to α6β1, failed to bypass the estrogen requirement for anchorage-independent growth or to promote resistance to tamoxifen. Wild-type CCN1 promoted estradiol-independent transcriptional activity of ERα and enhanced ERα agonist response to tamoxifen. The α6β1-binding-defective TM-CCN1 mutant lost the ERα co-activator-like behavior of WT-CCN1. Co-immunoprecipitation assays revealed a direct interaction between endogenous CCN1 and ERα, and in vitro approaches confirmed the ability of recombinant CCN1 to bind ERα. CCN1 signaling via α6β1, but not via αvβ3, drives an endocrine resistance phenotype that involves a direct binding of CCN1 to ERα to regulate its transcriptional activity in ER+ BC cells.

Abstract TP11: G-quadruplexes Regulate The Senescence Factor Cysteine-rich Angiogenic Inducer-61 In Primary Cerebral Endothelial Cells

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. Whether CCN1 contributes to senescence of CEC and how it is regulated require further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. G4 stabilization regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and exhibited cognitive deficits. The expression of human CCN1 can be upregulated via the stabilization of G4s. A sequence in the 3’- untranslated region (UTR) of the CCN1 mRNA has the ability to fold in to G4 in vitro . Thus, we hypothesize that CCN1 is upregulated with aging and induces senescence, and that G4 stabilization upregulates CCN1 in cultured primary CEC. We used whole brain lysates, cerebral vessel fractions, and cultured primary CEC from young (4-months old, mo) and aged (18-mo) female mice to determine the CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA G4s for fluorescence spectra. We found that CCN1 levels were increased in the brains of aging mice (p=0.0314, n=5), in cerebral vessel fractions (p=0.001, n=5), and in cultured primary CEC derived from aged mice (p=0.012, n=3), compared with those derived from young mice. In cultured CEC, a recombinant CCN1 protein induced SA phenotypes, such as SA beta-galactosidase activity (p=0.003, n=3), and double-strand DNA damage (p=0.016, n=3). Furthermore, CCN1 levels were upregulated by a G4 ligand (p=0.0103, n=3), and a G-rich motif in the 3’-UTR of the Ccn1 mRNA folded into a G4 (p=0.008, n=4). In conclusion , we demonstrate that CCN1 induces senescence in cultured primary CEC, and we propose G4 stabilization as a novel mechanism that regulates the SASP component CCN1.

Identification and characterization of collagen-like glycosylation and hydroxylation of CCN1.

CCN1 is a secreted protein and belongs to the CCN family of matricellular proteins. CCN1 binds to various cell surface receptors; thus, CCN1 has important functions in cell proliferation, migration and angiogenesis through a variety of signaling pathways. We have reported that CCN1 is O-fucosylated and that this O-fucosylation regulates the secretion of CCN1 into the extracellular region. In this study, we detected collagen-like glycosylation and hydroxylation at Lys203 of recombinant CCN1 by mass spectrometry. We then examined the role of collagen-like glycosylation in the functions of CCN1. As a result, we found that a deficiency in collagen-like glycosylation decreased the secretion of CCN1 using wild-type CCN1- and collagen-like glycosylation-defective mutant CCN1-overexpressing cell lines. Further, knockout of lysyl hydroxylase3, a multifunctional protein with hydroxylase and glucosyltransferase activities, impaired the secretion and glycosylation level of recombinant CCN1. Previous studies reported that collagen glycosylation of Lys residues mediated by lysyl hydroxylase3 is glucosyl-galactosyl-hydroxylation, presuming that this collagen-like glycosylation detected at Lys203 of recombinant CCN1 in this study might be glucosyl-galactosyl-hydroxylation. Taken together, our results demonstrate the novel function of the collagen-like glycosylation of CCN1 and suggest that lysyl hydroxylase3-mediated glycosylation is important for CCN1 secretion.

CCN1 accelerates re-epithelialization by promoting keratinocyte migration and proliferation during cutaneous wound healing

Re-epithelialization is an essential part of wound healing and has a prominent influence on the prognosis. CCN family member 1 (CCN1 or Cysteine-rich 61, CYR61), a matricellular protein, has a potential role in the wound healing process. However, its role in re-epithelialization remains unclear. The aim of this study was to determine the expression of CCN1 in the epidermis and its effect on keratinocytes during re-epithelialization. CCN1 expression in the wounded skin was analyzed using microarray data from GEO database and detected by immunofluorescence. The results showed upregulated CCN1 during the early stages of wound healing. Human primary keratinocytes were treated with recombinant human CCN1. The results showed that CCN1 promoted keratinocyte migration and proliferation. Moreover, a full-thickness mouse skin wound model and a superficial second-degree burn mouse model treated intracutaneously with CCN1 were used for in vivo studies. Topical treatment with CCN1 protein accelerated wound closure and re-epithelialization. Additionally, longer newly-formed epithelium tongue and elevated expression of PCNA and Ki67 were detected in the CCN1-treated group 4 days post-burn. These results indicate that CCN1 accelerates re-epithelialization by promoting keratinocyte migration and proliferation, and may serve as a novel target to promote re-epithelialization.

Recombinant CCN1 prevents hyperoxia-induced lung injury in neonatal rats.

BackgroundCystein-rich protein 61 (Cyr61/CCN1) is a member of the CCN family of matricellular proteins that has an important role in tissue development and remodeling. However, the role of CCN1 in the pathogenesis of bronchopulmonary dysplasia (BPD) is unknown. Accordingly, we have investigated the effects of CCN1 on a hyperoxia-induced lung injury model in neonatal rats.MethodsIn experiment 1, newborn rats were randomized to room air (RA) or 85% oxygen (O2) for 7 or 14 days, and we assessed the expression of CCN1. In experiment 2, rat pups were exposed to RA or O2 and received placebo or recombinant CCN1 by daily intraperitoneal injection for 10 days. The effects of CCN1 on hyperoxia-induced lung inflammation, alveolar and vascular development, vascular remodeling, and right ventricular hypertrophy (RVH) were observed.ResultsIn experiment 1, hyperoxia downregulated CCN1 expression. In experiment 2, treatment with recombinant CCN1 significantly decreased macrophage and neutrophil infiltration, reduced inflammasome activation, increased alveolar and vascular development, and reduced vascular remodeling and RVH in the hyperoxic animals.ConclusionThese results demonstrate that hyperoxia-induced lung injury is associated with downregulated basal CCN1 expression, and treatment with CCN1 can largely reverse hyperoxic injury.

CCN1 suppresses pulmonary vascular smooth muscle contraction in response to hypoxia.

Pulmonary vasoconstriction and increased vascular resistance are common features in pulmonary hypertension (PH). One of the contributing factors in the development of pulmonary vasoconstriction is increased pulmonary artery smooth muscle cell (PASMC) contraction. Here we report that CCN1, an extracellular matrix molecule, suppressed PASMC contraction in response to hypoxia. CCN1 (Cyr61), discovered in past decade, belongs to the Cyr61-CTGF-Nov (CCN) family. It carries a variety of cellular functions, including angiogenesis and cell adhesion, death, and proliferation. Hypoxia robustly upregulated the expression of CCN1 in the pulmonary vessels and lung parenchyma. Given that CCN1 is a secreted protein and functions in a paracine manner, we examined the potential effects of CCN1 on the adjacent smooth muscle cells. Interestingly, bioactive recombinant CCN1 significantly suppressed hypoxia-induced contraction in human PASMCs in vitro. Consistently, in the in vivo functional studies, administration of bioactive CCN1 protein significantly decreased right ventricular pressure in three different PH animal models. Mechanistically, protein kinase A-pathway inhibitors abolished the effects of CCN1 in suppressing PASMC contraction. Furthermore, CCN1-inhibited smooth muscle contraction was independent of the known vasodilators, such as nitric oxide. Taken together, our studies indicated a novel cellular function of CCN1, potentially regulating the pathogenesis of PH.

Abstract 3572: Metformin inhibits the oncogenic potential and invasiveness of pancreatic cancer cells targeting CCN1-CXCR4 axis : A new perspective for an old antidiabetic drug

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is among the most aggressive of solid malignancies responsible for around 330,000 deaths globally and accounts for the fourth most common cause of deaths due to cancer in USA. There has been a little advancement in the treatment of PDAC patients and the mortality rate has remained unchanged and may even be climbing up. Metformin, an oral biguanide medication used to treat type-2 diabetes mellitus, has demonstrated potential therapeutic effect against PDAC. Recent meta-analysis and epidemiologic studies indicate that diabetic patients treated with metformin were less likely to develop pancreatic cancer exhibit longer overall survival than those using other oral antidiabetic medications. Although there are several reports of the possible mode of action of Metformin against PDAC, no direct cellular/ molecular target is till reported. The present study aims to identify the direct molecular target of metformin in pancreatic cancer cells. Previously, we have demonstrated that matricellular protein CCN1/ Cyr61 plays pivotal role in pancreatic cancer development, maintenance of stemness, and induction of tumor angiogenesis. Hence the goal of this study is to investigate whether CCN1/ Cyr61 signaling cascade acts as a potential target for metformin. The encouraging results we have obtained so far reveal that metformin, which reduces CCN1 expression, significantly inhibits SDF-1 induced invasion, formation of tumor spheres (pancospheres) and also results in the down regulation of CXCR4 receptor in PC cell lines (Panc-1 and AsPC-1). Interestingly we observed that CXCR4 expression is drastically down regulated in genetically engineered CCN1-knock out Panc-1 (Panc-1 KO CCN1) cells, compared to the scrambled shRNA transfected Panc-1 cells. Further, it is also observed that Panc-1 K. CCN1 cells are found to be more susceptible to metformin treatment, whereas extracellular supplementation of recombinant CCN1 protein significantly abrogates the effect of metformin on PC cells. Moreover, tumor progression was found to be drastically inhibited in metformin treated Panc-1 (CCN1+ve) tumor xenografts in nude mice model, while the anti-tumorigenic effect of metformin was found to be more drastic in Panc-1 KO CCN1 tumor xenografts. In this scenario, we conclude that CCN1 acts as a direct target for metformin in PC cells, and targeted knock down of CCN1 increases the effectiveness of metformin. [This project is funded by VA Merit Award grants (SB & SKB)] Citation Format: Amlan Das, Archana De, Inamul Haque, Gargi Maity, Sushanta Banerjee, Snigdha Banerjee. Metformin inhibits the oncogenic potential and invasiveness of pancreatic cancer cells targeting CCN1-CXCR4 axis : A new perspective for an old antidiabetic drug. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3572. doi:10.1158/1538-7445.AM2015-3572

CAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1

Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel cAMP-dependent mechanism controlling VSMC behaviour.

CCN1 secreted by tonsil-derived mesenchymal stem cells promotes endothelial cell angiogenesis via integrin αv β3 and AMPK.

CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow-derived mesenchymal stem cells (BM-MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently-established tonsil-derived MSC (T-MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T-MSC, a higher level of CCN1 was secreted by T-MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T-MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T-MSC (CCM), and these stimulatory effects were reversed by neutralization with anti-CCN1 antibody. Treatment with recombinant human CCN1 (rh-CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh-CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh-CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well-known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin α(v) β(3) with anti-integrin α(v) β(3) antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T-MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin α(v) β(3) and AMPK mediate CCN1-induced EC migration and tube formation independent of intracellular ATP levels alteration.

CYR61/CCN1 overexpression in the myeloma microenvironment is associated with superior survival and reduced bone disease

AbstractSecreted protein CCN1, encoded by CYR61, is involved in wound healing, angiogenesis, and osteoblast differentiation. We identified CCN1 as a microenvironmental factor produced by mesenchymal cells and overexpressed in bones of a subset of patients with monoclonal gammopathy of undetermined significance (MGUS), asymptomatic myeloma (AMM), and multiple myeloma (MM). Our analysis showed that overexpression of CYR61 was independently associated with superior overall survival of MM patients enrolled in our Total Therapy 3 protocol. Moreover, elevated CCN1 was associated with a longer time for MGUS/AMM to progress to overt MM. During remission from MM, high levels of CCN1 were associated with superior progression-free and overall survival and stratified patients with molecularly defined high-risk MM. Recombinant CCN1 directly inhibited in vitro growth of MM cells, and overexpression of CYR61 in MM cells reduced tumor growth and prevented bone destruction in vivo in severe combined immunodeficiency-hu mice. Signaling through αvβ3 was required for CCN1 prevention of bone disease. CYR61 expression may signify early perturbation of the microenvironment before conversion to overt MM and may be a compensatory mechanism to control MM progression. Therapeutics that upregulate CYR61 should be investigated for treating MM bone disease.

Aging increases CCN1 expression leading to muscle senescence

Using microarray analysis, we found that aging sarcopenia is associated with a sharp increase in the mRNA of the matricellular protein CCN1 (Cyr61/CTGF/Nov). CCN1 mRNA was upregulated 113-fold in muscle of aged vs. young rats. CCN1 protein was increased in aging muscle in both rats (2.8-fold) and mice (3.8-fold). When muscle progenitor cells (MPCs) were treated with recombinant CCN1, cell proliferation was decreased but there was no change in the myogenic marker myoD. However, the CCN1-treated MPCs did express a senescence marker (SA-βgal). Interestingly, we found CCN1 increased p53, p16(Ink4A), and pRP (hypophosphorylated retinoblastoma protein) protein levels, all of which can arrest cell growth in MPCs. When MPCs were treated with aged rodent serum CCN1 mRNA increased by sevenfold and protein increased by threefold suggesting the presence of a circulating regulator. Therefore, we looked for a circulating regulator. Wnt-3a, a stimulator of CCN1 expression, was increased in serum from elderly humans (2.6-fold) and aged rodents (2.0-fold) compared with young controls. We transduced C2C12 myoblasts with wnt-3a and found that CCN1 protein was increased in a time- and dose-dependent manner. We conclude that in aging muscle, the circulating factor wnt-3a acts to increase CCN1 expression, prompting muscle senescence by activating cell arrest proteins.

The matricellular protein CCN1 suppresses lung cancer cell growth by inducing senescence via the p53/p21 pathway

CCN1, a secreted matrix-associated molecule, is involved in multiple cellular processes. Previous studies have indicated that expression of CCN1 correlates inversely with the aggressiveness of non-small-cell lung carcinoma (NSCLC); however, the underlying mechanisms remain elusive. Using three NSCLC cell line systems, here we show that long-term treatment of cells with the recombinant CCN1 protein led to a permanent cell cycle arrest in G1 phase; cells remained viable as judged by apoptotic assays. CCN1-treated NSCLC cells acquired a phenotype characteristic of senescent cells, including an enlarged and flattened cell shape and expression of the senescence-associated β-galactosidase. Immunoblot analysis showed that addition of CCN1 increased the abundance of hypo-phosphorylated Rb, as well as accumulation of p53 and p21. Silencing the expression of p53 or p21 by lentivirus-mediated shRNA production in cells blocked the CCN1-induced senescence. Furthermore, a CCN1 mutant defective for binding integrin α6β1 and co-receptor heparan sulfate proteoglycans was incapable of senescence induction. Our finding that direct addition of CCN1 induces senescence in NSCLC cells provides a potential novel strategy for therapeutic intervention of lung cancers.

High CCN1 Levels Are Associated with Longer Progression-Free and Overall Survival in GEP-Defined High-Risk Myeloma

Abstract 3940 Background:An imbalance between osteoblasts and osteoclasts results in osteolytic bone disease, a major complication in multiple myeloma (MM). Using gene expression profiling (GEP), we identified CYR61 as the most significantly overexpressed gene in bone biopsies in subsets of MGUS and MM patients compared with healthy controls. CYR61 is induced during osteoblast differentiation by endothelin-1, (Clines et al., 2007) and by Wnt3a (Si et al., 2006). CYR61 also inhibits the formation of multinucleate osteoclasts in vitro (Crockett et al., 2007). GEP revealed that elevated levels of CYR61 in 90/264 (34%) of newly diagnosed total therapy 3 (TT3) patients is associated with superior overall survival (p=0.006, HR-0.78). The CYR61 protein, CCN1, is significantly elevated in 57/177 (32%) TT3 patients at diagnosis and is associated with superior overall survival (p=0.024, HR-0.1). Recombinant CCN1 reduces MM cell growth and survival in vitro, and ectopic CYR61 produced by MM cells implanted into the SCID-hu model show that CCN1 inhibits osteolysis and reduces MM tumor growth in vivo. Taken together these data suggest that elevated CCN1 may be indicative of a dysfunctional microenvironment (ME) and could serve as a biomarker to monitor the MM ME. Methods:To test whether CCN1 could serve as an MM ME biomarker, CCN1 protein was quantitated by ELISA (DRG International). In BM sera from patients with MGUS/AMM (n=84), newly diagnosed myeloma (n=239), and in paired samples at diagnosis and 48h after a test dose of velcade (n=168 pairs), in nCR/CR (n=156 pairs) or at relapse (n=41 pairs). Differences between groups were analyzed using the t-test and survival using the log-rank test. Results and Discussion:CCN1 levels in myeloma patients at diagnosis (464.5 ± 30.4, p=0.0007), near complete remission (nCR, 466.1 ± 51.9 pg/ml, p=0.0055), complete remission (CR, 510.4 ± 36.5 pg/ml, p< 0.0001) and at relapse (692.5 ± 166.2 pg/ml, p=0.0185) were significantly elevated compared with healthy donors (274.4 ± 40.1 pg/ml). In paired samples, CCN1 levels at 48h post test-dose of velcade were significantly higher than at diagnosis (p=0.005). This suggests that elevated levels of CCN1 post-velcade may reflect recovery of the MM ME. To determine whether CCN1 may be associated with CR duration, CCN1 levels at nCR/CR were compared in patients that remained in CR for < 5 years and ≥ 5 years. CCN1 levels were significantly elevated in patients in CR ≥ 5 years (563.2 ± 53.2 pg/ml, p=0.01) compared to those in CR < 5 years (411.4 ± 22.8). CCN1 levels at nCR/CR and molecularly-defined risk were examined to determine whether they were independently associated with survival. Four groups were compared: CCN1 quartiles 1–3 levels [Q1-Q3] with low- or high-risk by GEP 70 or CCN1 [Q4] with low- or high-risk. While CCN1 levels were not prognostic for low-risk patients, Q4 high-risk patients had significantly longer PFS (Figure 1a, p=0.0120) and OS (Figure 1b, p<0.0001) than those with Q1-Q3 levels (median OS 6.9 and 3.8 years, respectively).To determine whether baseline CCN1 levels are associated with time to CR, CCN1 levels in patients who had not achieved CR (368.0 ± 38.0 pg/ml n=25) were compared to those that achieved CR within 6 months (525.6 ± 60.0 pg/ml, p=0.0284) and within 1 year (548.2 ± 74.5 pg/ml n=96, p=0.033). Patients who achieve CR had significantly higher CCN1 levels than those who did not reach CR. At diagnosis, MM CCN1 levels were lower than those of MGUS/AMM (608.3 ± 71.5 pg/ml, p=0.0324). We thus examined CCN1 levels in untreated MGUS and AMM patients to determine whether they were associated with time to progression to overt MM. Patients who progressed in less than 3 years had CCN1 levels significantly lower (441.6 ± 28.5 n=38) than those who progressed later (726.7 ± 121.7 pg/ml n=46, p=0.0399). CCN1 levels at the upper quartile were associated with significantly longer time to progress to overt MM (p<0.0056).Taken together, these data suggest that elevated CCN1 levels indicate a recovery effort by the ME and due to its positive association with survival of myeloma patients may be useful in the monitoring of disease course and thus personalized therapeutic management. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Matricellular Protein Cysteine-rich Protein 61 (CCN1/Cyr61) Enhances Physiological Adaptation of Retinal Vessels and Reduces Pathological Neovascularization Associated with Ischemic Retinopathy

Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP), a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase, and recapitulated, although aberrantly, in the subsequent ischemia-induced neovessel formation phase of ROP. Yet, whereas the histopathological features of ROP are well characterized, many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed, matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably, injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion, migration, and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands, their receptors, inhibitors, and downstream targets. CCN1-induced Wnt signaling mediated, at least in part, adhesion and endothelial differentiation of cultured HSCs, and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy.

CCN1 induces a reversible epithelial–mesenchymal transition in gastric epithelial cells

CCN1 is a matricellular protein that activates many genes related to wound healing and tissue remodeling in fibroblasts, but its effect on epithelial cells remains unclear. This study examined the role of CCN1 in epithelial wound healing using rat gastric epithelial cells and rat stomach ulcer as in vitro and in vivo models, respectively. We found that CCN1 expression is highly upregulated in the epithelial cells adjacent to a wound and remains high until the wound is healed. Upregulation of CCN1 activates a transient epithelial–mesenchymal transition in the epithelial cells at the migrating front and drives wound closure. Once the wound is healed, these epithelial cells and their progeny can resume their original epithelial phenotype. We also found that CCN1-induced E-cadherin loss is not due to transcriptional regulation but rather protein degradation due to the collapse of adherens junctions, which is contributed by β-catenin translocation. CCN1-activated integrin-linked kinase mediates this process. Finally, our in vivo study showed that locally neutralizing CCN1 drastically impairs wound closure, whereas local injection of recombinant CCN1 protein induces expression of vimentin and smooth muscle α-actin in normal gastric mucosal epithelial cells and accelerates re-epithelialization during ulcer healing. In conclusion, our study indicates that CCN1 can induce reversible epithelial–mesenchymal transition, and this feature may have great value for clinical wound healing.

Integrin α11 mediates CCN1‐induced focal adhesion and β‐catenin translocation in esophageal epithelial cells

BackgroundCCN1 is a matricellular protein that plays a critical role in several normal and pathological processes by signaling through different integrin receptors in a cell‐type specific manner. Clinical studies have suggested CCN1 as a putative marker for malignant progression towards esophageal adenocarcinoma. However, little is known about its signaling mechanisms in this tissue. The aim of this study was to determine the integrin receptor that mediates CCN1 actions in esophageal epithelial cells.MethodsEsophageal epithelial cells were treated with recombinant CCN1 protein. Its effects on integrins were examined by microarray, real‐time PCR, immunoprecipitation, immunoblotting, and immunofluorescence microscopy.ResultsOut of all the known α‐ and β‐integrins, ITGA11 and ITGB5 were the only ones that responded to CCN1 treatment. Co‐immunoprecipitation assays demonstrated an association between integrin α11 and β5 in the presence of CCN1. Blocking either integrin subunit prevented CCN1‐induced focal adhesion and β‐catenin nuclear translocation. Taken together, these results provide evidence that integrin α11 can form a new partnership with β5 and that together they serve as a new receptor to mediate CCN1 actions in esophageal epithelial cells.This study was supported by the Department of Veterans Affairs of the United States and the American Heart Association.

The pro-angiogenic factor CCN1 enhances the re-endothelialization of biological vascularized matrices in vitro

A problem in generating artificial tissues is supplying nutrients to cells within 3D constructs. The use of a decellularized biological vascularized matrix with preserved pedicles (BioVaM), as a scaffold, appears to aid the generation of perfusable tissue constructs in vitro. To prevent vessel occlusion upon implantation, a functional endothelium must line the graft vessel bed. Here we tested whether the pro-angiogenic factor CCN1 could improve the re-endothelialization of BioVaM in vitro. BioVaM vessel beds were incubated with 100 ng/mL recombinant human CCN1. Human cord blood endothelial cells (hCBEC) were analysed with respect to adhesion behaviour upon CCN1 exposure and seeded onto vessel structures of CCN1 exposed BioVaM (cBioVaM). BioVaMs were fixed in a bioreactor and perfusion cultured for 4 and 14 days (d). BioVaM without CCN1 treatment served as controls. Initial seeding success and endothelialization progression were monitored by fluorescence-labelled hCBEC. During construct cultivation, pH and lactate production were measured. Degree of endothelialization and characterization of seeded cells, with respect to endothelial markers, were investigated histologically. BioVaM vessel structures showed a 78 +/- 17% increase of attached cells when pre-treated with CCN1. Evaluation of re-endothelialization (arbitrary units) was 4.0 +/- 0.8 and 2.6 +/- 0.8 after 4 d, and 5.0 +/- 0.0 and 3.0 +/- 0.5 after 14 d in cBioVaM vs. BioVaM, respectively. On day 14, lactate concentration, an indicator of metabolic activity, was increased 12-fold in cBioVaM relative to BioVaM. A preserved endothelial phenotype of seeded cells was verified in all cultures by acetylated low density lipoprotein uptake and positive immunohistochemistry against von Willebrand factor, endothelial nitric oxide synthase, and CD31. Coating of decellularized vessel structures with CCN1 supports adhesion of hCBEC and enhances re-endothelialization of BioVaM. Perfusable, endothelialized constructs may aid in solving the problem of nourishing cells inside 3D tissue-engineered constructs.