High NOV/CCN3 expression during high-fat diet pregnancy in mice affects GLUT3 expression and the mTOR pathway

NOV/CCN3 regulates glucose homeostasis in mice during pregnancy. NOV/CCN3 upregulates GLUT3 expression and affects the mTOR pathway in the GDM environment in vivo and in vitro.

CCN3 is dynamically regulated by treatment and disease state in multiple sclerosis

Background Multiple sclerosis (MS) is an immune-mediated disease that damages myelin in the central nervous system (CNS). We investigated the profile of CCN3, a known regulator of immune function and a potential mediator of myelin regeneration, in multiple sclerosis in the context of disease state and disease-modifying treatment. Methods CCN3 expression was analysed in plasma, immune cells, CSF and brain tissue of MS patient groups and control subjects by ELISA, western blot, qPCR, histology and in situ hybridization. Results Plasma CCN3 levels were comparable between collective MS cohorts and controls but were significantly higher in progressive versus relapsing-remitting MS and between patients on interferon-β versus natalizumab. Higher body mass index was associated with higher CCN3 levels in controls as reported previously, but this correlation was absent in MS patients. A significant positive correlation was found between CCN3 levels in matched plasma and CSF of MS patients which was absent in a comparator group of idiopathic intracranial hypertension patients. PBMCs and CD4+ T cells significantly upregulated CCN3 mRNA in MS patients versus controls. In the CNS, CCN3 was detected in neurons, astrocytes and blood vessels. Although overall levels of area immunoreactivity were comparable between non-affected, demyelinated and remyelinated tissue, the profile of expression varied dramatically. Conclusions This investigation provides the first comprehensive profile of CCN3 expression in MS and provides rationale to determine if CCN3 contributes to neuroimmunological functions in the CNS.

Dynamic CCN3 expression in the murine CNS does not confer essential roles in myelination or remyelination

CCN3 is a matricellular protein that promotes oligodendrocyte progenitor cell differentiation and myelination in vitro and ex vivo. CCN3 is therefore a candidate of interest in central nervous system (CNS) myelination and remyelination, and we sought to investigate the expression and role of CCN3 during these processes. We found CCN3 to be expressed predominantly by neurons in distinct areas of the CNS, primarily the cerebral cortex, hippocampus, amygdala, suprachiasmatic nuclei, anterior olfactory nuclei, and spinal cord gray matter. CCN3 was transiently up-regulated following demyelination in the brain of cuprizone-fed mice and spinal cord lesions of mice injected with lysolecithin. However, CCN3−/−mice did not exhibit significantly different numbers of oligodendroglia or differentiated oligodendrocytes in the healthy or remyelinating CNS, compared to WT controls. These results suggest that despite robust and dynamic expression in the CNS, CCN3 is not required for efficient myelination or remyelination in the murine CNS in vivo.

CCN3/NOV as a functional driver and prognostic biomarker of prostate cancer bone metastasis.

182 Background: Prostate cancer commonly metastasizes to the bone, resulting in pathological fractures and poor prognosis. CCN3/NOV (Nephroblastoma overexpressed) has been implicated in promoting the formation of osteolytic prostate cancer (PC) bone metastases. The C-terminal domain of CCN3 binds growth factors, heparin sulfate proteoglycans, activates Notch signaling and promotes dimerization of CCN family members. We hypothesize that the CCN3 CT domain is required to promote osteolytic PC bone metastasis and that CCN3 represents a prognostic biomarker in primary PC tumors to predict recurrence to bone. Methods: CCN3WT and CCN3∆CT were overexpressed in LNCaP C4-2 cells. The role of CCN3 was assessed with in vitro proliferation, migration and invasion assays, and in vivo through intracardiac injection in male Nude mice (Nu/Nu). Ex vivo µCT scans were performed on bone specimens. CCN3 expression was assessed in two unique tissue microarrays (TMA) comprising over 1,500 primary prostate tumor using different anti-CCN3 antibodies with immunohistochemistry and immunohistofluorescnece, respectively. Results: While CCN3WT and CCN3∆CT had little effect in vitro on cell proliferation, migration or invasion, intracardiac injection of CCN3WT resulted in increased incidence of bone metastasis compared to empty vector control and CCN3∆CT. Ex vivo µCT revealed decreased bone mineral density in bones from mice injected with CCN3WT cells compared to control and CCN3∆CT expressing cells. In both TMAs studied, high CCN3 expression in tumor epithelium correlated with increased risk of biochemical relapse and bone metastasis at 5 years and 15 years post-resection, respectively. Conclusions: CCN3 requires its C-terminal domain for its bone metastatic function, and CCN3 is correlated with aggressive disease biology in prostate cancer. These findings point to CCN3 as a biomarker that can be useful to predict prostate cancer aggressiveness, while providing clarity on its role as a functional mediator of prostate cancer bone metastasis.

Stat5 Is Essential for BCR-ABL-Transformed Chronic Myeloid Leukemia (CML) Associated with Increased CCN3 Gene Expression.

Abstract 3271 Poster Board III-1 Introduction: Signal transducers and activators of transcription5 (Stat5) proteins are involved in many cellular processes through mediated cytokine, hormone, and growth factor signaling. But its role in disease pathogenesis has not been fully elucidated. Recently, activation of BCR-ABL has been reported to regulate a novel gene, CCN3 in cell lines and primary cells derived from chronic myeloid leukemia (CML) patients. To investigate the function of Stat5 in CML initiation and maintenance and determine the downstream target genes on Jak-Stat5 pathway, we developed a BCR-ABL - induced CML - like disease model by using retro-viral infection in Cre-mediated Stat5 knockout transgenic mice and analyzed the progress of CML. We also used Stat5 knockout (Stat5 KO) mice to perform gene profiling. Results: Our study showed that loss of Stat5 resulted in increased survival rate and remission of CML. Microarray analysis showed that CCN3 expression was down-regulated in KL cells derived from Stat5 KO mice. BCR-ABL-activated Stat5 increased expression level of CCN3 in CML cells. We further determined that Stat5 binds to CCN3 promoter region in IL-3 stimulated 32D cells and BCR-ABL-induced CML cells. Conclusions: Our study suggested that Stat5 is essential for BCR-ABL transformed CML and that CCN3 is involved in normal hematopoiesis and CML development. Further study will be necessary to uncover the function of CCN3 and more targets of Stat5 pathway in CML development and discover the therapeutic significance. Disclosures: No relevant conflicts of interest to declare.

CCN3, a Novel Growth Inhibitory Factor for Chronic Myeloid Leukemia

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we identified down-regulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity. Reduced CCN3 expression is a prominent feature in both primary human CML cells and cell lines. We now show that CCN3 is growth inhibitory and enhances imatinib induced growth inhibition. To evaluate the biological consequence of CCN3 expression in CML, K562 cells were stably transfected with a construct containing CCN3 (pCMV82-23) and growth characteristics and activation of signaling pathways were compared to cells transfected with empty vector (control). CCN3 expression was undetected by Real-time PCR in control cells whilst pCMV82-23 cells expressed 2.25 × 106 copies per 50ng of cDNA. pCMV82-23 cells showed reduced colony formation capacity (p=0.003) and reduced cell growth over a period of five days (p=0.005). Investigation of cellular signaling showed CCN3 expression resulted in significant down-regulation of three major signaling pathways and demonstrated reduced phosphorylation of ERK (p=0.002), pAKT (p=0.017) and pSTAT5 (p=0.005) compared to control cells. Protein levels for total ERK, AKT and STAT5 were unaffected by CCN3 expression. Flow cytometry showed that sustained CCN3 expression resulted in an accumulation of cells within the subG0 stage of the cell cycle (11.4% ± 3 (p=0.040)). To determine if CCN3 expression could influence sensitivity to the BCR-ABL kinase inhibitor, imatinib, pCMV82-23 cells and control cells were treated with imatinib (5uM) for 48h. Control cells treated with imatinib showed moderate growth inihibition (19.6% ± 2.5) compared to untreated control. pCMV82-23 cells showed a significant increase in the magnitude of imatinib induced growth inhibition (63.3% ± 10.5 (p=0.043)). This was associated with an increased accumulation of cells in the subG0 area of the cell cycle, 34.6% ± 5 for pCMV82-23 cells compared to control cells (21.7% ± 8) in response to imatinib treatment (p=0.006). To then determine if these effects could be reproduced using recombinant CCN3 (rCCN3), K562 cells were treated with imatinib (5uM) alone or in combination with rCCN3 (10nM) for 48h. K562 cells treated with the combination of rCCN3 and imatinib showed enhanced growth inhibition (71.8% ± 7.9) compared to cells treated with imatinib alone (81.1% ± 9.2 (p=0.008)). Loss of CCN3 is consistent with properties associated with the CML phenotype. Sustained expression of CCN3 in K562 cells restores growth control and re-establishes induction of apoptosis. Both increased expression of CCN3 or addition of the recombinant protein provide additional benefit for imatinib induced growth inhibition thus providing a novel avenue for therapeutic intervention.