Gene Summary

Gene:CCN5; cellular communication network factor 5
Aliases: CT58, WISP2, CTGF-L
Summary:This gene encodes a member of the WNT1 inducible signaling pathway (WISP) protein subfamily, which belongs to the connective tissue growth factor (CTGF) family. WNT1 is a member of a family of cysteine-rich, glycosylated signaling proteins that mediate diverse developmental processes. The CTGF family members are characterized by four conserved cysteine-rich domains: insulin-like growth factor-binding domain, von Willebrand factor type C module, thrombospondin domain and C-terminal cystine knot-like (CT) domain. The encoded protein lacks the CT domain which is implicated in dimerization and heparin binding. It is 72% identical to the mouse protein at the amino acid level. This gene may be downstream in the WNT1 signaling pathway that is relevant to malignant transformation. Its expression in colon tumors is reduced while the other two WISP members are overexpressed in colon tumors. It is expressed at high levels in bone tissue, and may play an important role in modulating bone turnover. [provided by RefSeq, Jul 2008]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:WNT1-inducible-signaling pathway protein 2
Source:NCBIAccessed: 29 August, 2019


What does this gene/protein do?
Show (11)

Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 29 August 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • CCN Intercellular Signaling Proteins
  • Repressor Proteins
  • Transfection
  • Kidney Cancer
  • beta Catenin
  • Cell Movement
  • In Situ Hybridization
  • Intercellular Signaling Peptides and Proteins
  • Cancer Gene Expression Regulation
  • Cysteine-Rich Protein 61
  • Chromosome 20
  • Proto-Oncogene Proteins
  • Western Blotting
  • Cell Division
  • Estrogen Receptors
  • Cancer RNA
  • Neoplasm Invasiveness
  • Base Sequence
  • Insulin-Like Growth Factor Binding Proteins
  • Breast
  • DNA Primers
  • Estrogen Receptor alpha
  • Breast Cancer
  • Adrenocortical Cancer
  • Immunohistochemistry
  • Gene Expression Profiling
  • Tumor Microenvironment
  • Connective Tissue Growth Factor
  • Young Adult
  • p53 Protein
  • Transcription Factors
  • Pituitary Tumors
  • Cell Proliferation
  • Estradiol
  • Signal Transduction
  • Neoplasm Proteins
  • Oligonucleotide Array Sequence Analysis
  • Receptors, Progesterone
  • Intracellular Signaling Peptides and Proteins
  • Cell Differentiation
  • Tumor Suppressor Proteins
  • Messenger RNA
Tag cloud generated 29 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CCN5 (cancer-related)

Yamada Y, Sugawara S, Arai T, et al.
Molecular pathogenesis of renal cell carcinoma: Impact of the anti-tumor miR-29 family on gene regulation.
Int J Urol. 2018; 25(11):953-965 [PubMed] Related Publications
OBJECTIVES: To identify key oncogenes and proteins that are controlled by the microRNA miR-29 family (miR-29a, miR-29b and miR-29c) in renal cell carcinoma pathogenesis.
METHODS: Genome-wide gene expression and in silico database analyses were carried out. The Cancer Genome Atlas database was used to investigate the clinical significance of gene expression data in renal cell carcinoma patients. Loss-of-function assays were applied to investigate the function of target genes.
RESULTS: We identified 47 possible target genes that might be regulated by the miR-29 family in renal cell carcinoma cells. Among the targets of the miR-29 family, high expression of 10 genes (ADAMTS14, TRIB13, SERPINH1, FCGR1B, COL1A1, LAIR2, WISP2, TREM1, TNKS1BP1 and GBP2) significantly predicted poor patient prognosis (P < 0.001). SERPINH1 was directly regulated by the miR-29 family, and its overexpression was detected in renal cell carcinoma surgical specimens and tyrosine kinase inhibitor failure autopsy specimens. High expression of SERPINH1 was significantly associated with tumor stage, pathological grade and poor prognosis (P < 0.0001). Knockdown assays showed that its expression enhanced cancer cell migration and invasive abilities.
CONCLUSIONS: Genes regulated by the anti-tumor miR-29 family are closely involved in the molecular pathogenesis of renal cell carcinoma. Our approach based on anti-tumor microRNAs might contribute to the development of new diagnostic markers and therapeutic strategies.

Jucá CEB, Colli LM, Martins CS, et al.
Impact of the Canonical Wnt Pathway Activation on the Pathogenesis and Prognosis of Adamantinomatous Craniopharyngiomas.
Horm Metab Res. 2018; 50(7):575-581 [PubMed] Related Publications

Li L, Cui Y, Ji JF, Jiang WG
Clinical Correlation Between WISP2 and β-Catenin in Gastric Cancer.
Anticancer Res. 2017; 37(8):4469-4473 [PubMed] Related Publications
BACKGROUND: Evidence indicates that wingless-type MMTV integration site family, member 1 (WNT1)-inducible signaling pathway protein 2 (WISP2) may play an important role in the development of gastric cancer (GC) by regulating the WNT/β-catenin signaling pathway. In the present study, we investigated whether there is correlation between WISP2 and β-catenin proteins, and their association with clinicopathological features in GC.
MATERIALS AND METHODS: Immunohistochemical staining was carried out on 119 paraffin-embedded gastric cancer tissues and 99 adjacent normal gastric tissues collected from patients with GC at the Beijing Cancer Hospital. Data were analyzed by Spearman rank correlation and Chi-square tests.
RESULTS: Both WISP2 and β-catenin were more highly expressed in GC tissues compared to adjacent normal tissues. Moreover, Spearman rank correlation analysis showed positive correlation between WISP2 and β-catenin (R=0.2254, p=0.0137). Additionally, their co-expression was seen in a higher proportion of patients with GC at early stage or without metastasis.
CONCLUSION: These findings suggest that the expression of WISP2 and β-catenin might be a favorable biomarker for prediction and prognosis in the early stage of GC.

Lottrup G, Belling K, Leffers H, et al.
Comparison of global gene expression profiles of microdissected human foetal Leydig cells with their normal and hyperplastic adult equivalents.
Mol Hum Reprod. 2017; 23(5):339-354 [PubMed] Related Publications
STUDY QUESTION: Do human adult Leydig cells (ALCs) within hyperplastic micronodules display characteristics of foetal LCs (FLCs)?
SUMMARY ANSWER: The gene expression profiles of FLCs and all ALC subgroups were clearly different, but there were no significant differences in expressed genes between the normally clustered and hyperplastic ALCs.
WHAT IS KNOWN ALREADY: LCs are the primary androgen producing cells in males throughout development and appear in chronologically distinct populations; FLCs, neonatal LCs and ALCs. ALCs are responsible for progression through puberty and for maintenance of reproductive functions in adulthood. In patients with reproductive problems, such as infertility or testicular cancer, and especially in men with high gonadotrophin levels, LC function is often impaired, and LCs may cluster abnormally into hyperplastic micronodules (defined as clusters of >15 LCs in a cross-section).
STUDY DESIGN, SIZE, DURATION: A genome-wide microarray study of LCs microdissected from human foetal and adult tissue samples (n = 12). Additional tissue specimens (n = 15) were used for validation of the mRNA expression data at the protein level.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Frozen human tissue samples were used for the microarray study, including morphologically normal foetal (gestational week 10-11) testis samples, and adult testis specimens with normal LC distribution, LC micronodules or LC micronodules adjacent to hCG-producing testicular germ cell tumours. Transcriptome profiling was performed on Agilent whole human genome microarray 4 × 44 K chips. Microarray data pre-processing and statistical analysis were performed using the limma R/Bioconductor package in the R software, and differentially expressed genes were further analysed for gene set enrichment using the DAVID Bioinformatics software. Selected genes were studied at the protein level by immunohistochemistry.
MAIN RESULTS AND THE ROLE OF CHANCE: The transcriptomes of FLCs and ALCs differed significantly from each other, whereas the profiles of the normally clustered and hyperplastic ALCs were similar despite morphological heterogeneity. The study revealed several genes not known previously to be expressed in LCs during early development, including sulfotransferase family 2A member 1 (SULT2A1), WNT1-inducible signalling pathway protein 2 (WISP2), hydroxyprostaglandin dehydrogenase (HPGD) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), whose expression changes were validated at the protein level.
LARGE SCALE DATA: The transcriptomic data are deposited in ArrayExpress (accession code E-MTAB-5453).
LIMITATIONS, REASONS FOR CAUTION: The small number of biological replicates and the necessity of RNA amplification due to the scarcity of human tissues, especially foetal specimens, are the main limitations of the study. Heterogeneous subpopulations of LCs within micronodules were not discriminated during microdissection and might have affected the expression profiling. The study was constrained by the lack of availability of truly normal controls. Testis samples used as 'controls' displayed complete spermatogenesis and were from patients with germ cell neoplasia but with undetectable hCG and normal hormone levels.
WIDER IMPLICATIONS OF THE FINDINGS: The changes in LC morphology and function observed in patients with reproductive disorders possibly reflect subtle changes in the expression of many genes rather than regulatory changes of single genes or pathways. The study provides new insights into the development and maturation of human LCs by the identification of a number of potential functional markers for FLC and ALC.
STUDY FUNDING AND COMPETING INTEREST(S): The study was supported by research grants from the Danish Cancer Society, the Capital Region's Research Fund for Health Research, Rigshospitalet's research funds, the Villum Kann Rasmussen Foundation, the Danish Innovation Fund, ReproUnion, Kirsten and Freddy Johansen's foundation and the Novo Nordisk Foundation. None of the funding agencies had any influence on the study. The authors declare no conflicts of interest.

Chen Z, Tang J, Cai X, et al.
HBx mutations promote hepatoma cell migration through the Wnt/β-catenin signaling pathway.
Cancer Sci. 2016; 107(10):1380-1389 [PubMed] Free Access to Full Article Related Publications
HBx mutations (T1753V, A1762T, G1764A, and T1768A) are frequently observed in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Aberrant activation of the Wnt/β-catenin signaling pathway is involved in the development of HCC. However, activation of the Wnt/β-catenin signaling pathway by HBx mutants has not been studied in hepatoma cells or HBV-associated HCC samples. In this study, we examined the effects of HBx mutants on the migration and proliferation of HCC cells and evaluated the activation of Wnt/β-catenin signaling in HBx-transfected HCC cells and HBV-related HCC tissues. We found that HBx mutants (T, A, TA, and Combo) promoted the migration and proliferation of hepatoma cells. The HBx Combo mutant potentiated TOP-luc activity and increased nuclear translocation of β-catenin. Moreover, the HBx Combo mutant increased and stabilized β-catenin levels through inactivation of glycogen synthase kinase-3β, resulting in upregulation of downstream target genes such as c-Myc, CTGF, and WISP2. Enhanced activation of Wnt/β-catenin was found in HCC tissues with HBx TA and Combo mutations. Knockdown of β-catenin effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants. Our results indicate that HBx mutants, especially the Combo mutant, allow constitutive activation of the Wnt signaling pathway and may play a pivotal role in HBV-associated hepatocarcinogenesis.

Neely BA, Wilkins CE, Marlow LA, et al.
Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma.
PLoS One. 2016; 11(4):e0154074 [PubMed] Free Access to Full Article Related Publications
Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from each domain, demonstrating the complementary nature of such an analysis. Together these results highlight the importance of the VHL/HIF1A/HIF2A axis and provide a foundation and therapeutic targets for future studies. (Data are available via ProteomeXchange with identifier PXD003271 and MassIVE with identifier MSV000079511.).

Minchenko DO, Kharkova AP, Tsymbal DO, et al.
IRE1 inhibition affects the expression of insulin-like growth factor binding protein genes and modifies its sensitivity to glucose deprivation in U87 glioma cells.
Endocr Regul. 2015; 49(4):185-97 [PubMed] Related Publications
OBJECTIVE: The aim of the present study was to investigate the effect of inhibition of endoplasmic reticulum stress signaling mediated by IRE1/ERN1 (inositol-requiring enzyme 1/endoplasmic reticulum to nucleus signaling 1) on the expression of genes encoding different groups of insulin-like growth binding proteins (IGFBP6 and IGFBP7) and CCN family (IGFBP8/CTGF/CCN2, IGFBP9/NOV/CCN3, IGFBP10/CYR61/CCN1, WISP1/CCN4, and WISP2/CCN5) and its sensitivity to glucose deprivation in U87 glioma cells.
METHODS: The expression of IGFBP6, IGFBP7, IGFBP8, IGFBP9, IGFBP10, WISP1, and WISP2 genes was studied by qPCR in control U87 glioma cells (wild-type) and its subline with IRE1 signaling enzyme loss of function upon glucose deprivation.
RESULTS: The expression of IGFBP8, IGFBP9, and WISP2 genes was up-regulated in control glioma cells upon glucose deprivation with most significant changes for IGFBP9 gene. At the same time, the expression of IGFBP6, IGFBP10, and WISP1 genes was resistant to glucose deprivation in these glioma cells, but the IGFBP7 gene expression was down-regulated. The inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells modified the sensitivity of most studied gene expressions to glucose deprivation condition: introduced sensitivity of IGFBP10 and WISP1 genes to glucose deprivation, enhanced the effect of this deprivation on IGFBP7 and IGFBP9 gene expressions, and reduced this effect on WISP2 gene and induced suppressive effect of glucose deprivation on the expression of IGFBP8 gene. Furthermore, the inhibition of IRE1 strongly affected the expression of all studied genes in glioma cells upon regular growing condition in gene specific manner: up-regulated the expression levels of IGFBP7, IGFBP8, IGFBP10, WISP1, and WISP2 genes and down-regulated the IGFBP6 and IGFBP9 genes.
CONCLUSIONS: The data of this investigation demonstrate that the expression of IGFBP7, IGFBP8, IGFBP9, and WISP2 genes are sensitive to glucose deprivation in U87 glioma cells and that inhibition of IRE1 signaling enzyme function may significantly affect the expression of all studied genes in the presence of glucose as well as modify the effect of glucose deprivation on the expression of most studied genes. These data also show that proteins encoded by these genes may participate in the regulation of metabolic and proliferative processes via IGF/INS receptors and possibly other signaling pathways as well, via IRE1 signaling, which is a central mediator of the unfolded protein response and an important component of the tumor growth and metabolic diseases.

Shang H, Braggio D, Lee YJ, et al.
Targeting the Notch pathway: A potential therapeutic approach for desmoid tumors.
Cancer. 2015; 121(22):4088-96 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Desmoid tumors (DTs) are rare mesenchymal lesions that can recur repeatedly. When it is feasible, DTs are surgically resected; however, this often results in high recurrence rates. Recently, treatment with PF-03084014, a potent γ-secretase inhibitor, has been shown to have antitumor activity in several tumor types by affecting the WNT/β-catenin pathway. Consequently, Notch pathway inhibition by PF-03084014 might be a promising approach for DT treatment.
METHODS: The expression of Notch pathway components was analyzed in DT tissues and cell strains with immunohistochemistry and Western blotting, respectively. A panel of DT cell strains was exposed to PF-03084014 and evaluated for cell proliferation. Antitumor effects were assessed via cell cycle, apoptosis, and migration and invasion analysis. Cells treated with PF-03084014 were characterized with a gene array analysis combined with Ingenuity Pathway Analysis.
RESULTS: The results showed that Notch pathway components were expressed at different levels in DTs. Hes1 (Hes Family BHLH Transcription Factor 1) was overexpressed in DT tumors versus dermal scar tissue, and PF-03084014 caused significant decreases in Notch intracellular domain and Hes1 expression in DT cell strains. PF-03084014 decreased DT cell migration and invasion and also caused cell growth inhibition in DT cell strains, most likely through cell cycle arrest. Gene array analysis combined with Ingenuity Pathway Analysis showed that Wnt1-inducible signaling pathway protein 2 possibly regulated Notch and WNT pathways after treatment with PF-03084014 through integrin.
CONCLUSION: Our findings suggest that the Notch pathway is an important DT therapeutic target. Furthermore, PF-03084014 has significant antitumor activity against DTs, and it may be an alternative strategy for DT treatment.

Ji J, Jia S, Jia Y, et al.
WISP-2 in human gastric cancer and its potential metastatic suppressor role in gastric cancer cells mediated by JNK and PLC-γ pathways.
Br J Cancer. 2015; 113(6):921-33 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: It has recently been shown that WISP proteins (Wnt-inducted secreted proteins), a group of intra- and extra-cellular regulatory proteins, have been implicated in the initiation and progression of a variety of tumour types including colorectal and breast cancer. However, the role of WISP proteins in gastric cancer (GC) cells and their clinical implications have not yet been elucidated.
METHODS: The expression of WISP molecules in a cohort of GC patients was analysed using real-time quantitative PCR and immunohistochemistry. The expression of a panel of recognised epithelial-mesenchymal transition (EMT) markers was quantified using Q-PCR in paired tumour and normal tissues. WISP-2 knockdown (kd) sublines using ribozyme transgenes were created in the GC cell lines AGS and HGC27. Subsequently, several biological functions, including cell growth, adhesion, migration and invasion, were studied. Potential pathways for the interaction of EMT, extracellular matrix and MMP were evaluated.
RESULTS: Overexpression of WISP-2 was detected in GC and significantly correlated with early tumour node-metastasis staging, differentiation status and positively correlated with overall survival and disease-free survival of the patients. WISP-2 expression was inversely correlated with that of Twist and Slug in paired samples. Kd of WISP-2 expression promoted the proliferation, migration and invasion of GC cells. WISP-2 suppressed GC cell metastasis through reversing EMT and suppressing the expression and activity of MMP9 and MMP2 via JNK and ERK. Cell motility analysis indicated that WISP-2 kd contributed to GC cells' motility and can be attenuated by PLC-γ and JNK small inhibitors.
CONCLUSIONS: Increased expression of WISP-2 in GC is positively correlated with favourable clinical features and the survival of patients with GC and is a negative regulator of growth, migration and invasion in GC cells. These findings suggest that WISP-2 is a potential tumour suppressor in GC.

Zhang H, Li W, Huang P, et al.
Expression of CCN family members correlates with the clinical features of hepatocellular carcinoma.
Oncol Rep. 2015; 33(3):1481-92 [PubMed] Related Publications
Studies have reported that the CCN family of proteins plays an important role in stimulating tumorigenesis. However, the relationship between the CCN protein family members and the features of hepatocellular carcinoma (HCC) remains unclear. The objective of this study was to determine the relationship between the expression levels of CCN protein family members and the features of HCC. Expression levels of the CCN family of proteins in 80-paired primary HCC samples and 11 normal liver samples were determined by a quantitative real-time PCR assay. Enhanced expression of nephroblastoma overexpressed protein (NOV) and decreased expression of Wnt-induced secreted protein 1 (WISP1), cysteine-rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were found in HCC samples when compared to levels in matched non-cancerous tissues. No significant difference in WISP2 was found between matched-pair samples; only a few samples showed WISP3 expression. Furthermore, the expression levels of NOV, WISP1 and CYR61 were closely correlated with certain clinical features, including venous invasion, cellular differentiation, pTNM stage, disease-free survival and overall survival. Our results suggest that HCC progression may be enhanced by NOV and suppressed by WISP1 and CYR61. Our statistical analysis suggests that these proteins may be valuable in determining the prognosis of this deadly disease and directs attention to modulating the levels of these proteins as a potential mode of therapy.

Patel P, Brooks C, Seneviratne A, et al.
Investigating microenvironmental regulation of human chordoma cell behaviour.
PLoS One. 2014; 9(12):e115909 [PubMed] Free Access to Full Article Related Publications
The tumour microenvironment is complex and composed of many different constituents, including matricellular proteins such as connective tissue growth factor (CCN2), and is characterized by gradients in oxygen levels. In various cancers, hypoxia and CCN2 promote stem and progenitor cell properties, and regulate the proliferation, migration and phenotype of cancer cells. Our study was aimed at investigating the effects of hypoxia and CCN2 on chordoma cells, using the human U-CH1 cell line. We demonstrate that under basal conditions, U-CH1 cells express multiple CCN family members including CCN1, CCN2, CCN3 and CCN5. Culture of U-CH1 cells in either hypoxia or in the presence of recombinant CCN2 peptide promoted progenitor cell-like characteristics specific to the notochordal tissue of origin. Specifically, hypoxia induced the most robust increase in progenitor-like characteristics in U-CH1 cells, including increased expression of the notochord-associated markers T, CD24, FOXA1, ACAN and CA12, increased cell growth and tumour-sphere formation, and a decrease in the percentage of vacuolated cells present in the heterogeneous population. Interestingly, the effects of recombinant CCN2 peptide on U-CH1 cells were more pronounced under normoxia than hypoxia, promoting increased expression of CCN1, CCN2, CCN3 and CCN5, the notochord-associated markers SOX5, SOX6, T, CD24, and FOXA1 as well as increased tumour-sphere formation. Overall, this study highlights the importance of multiple factors within the tumour microenvironment and how hypoxia and CCN2 may regulate human chordoma cell behaviour.

Haque I, Banerjee S, De A, et al.
CCN5/WISP-2 promotes growth arrest of triple-negative breast cancer cells through accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation.
Oncogene. 2015; 34(24):3152-63 [PubMed] Related Publications
The matricellular protein CCN5/WISP-2 represents a promising target in triple-negative breast cancer (TNBC) because treatment or induced activation of CCN5 in TNBC cells promotes cell growth arrest at the G0/G1 phase, reduces cell proliferation and delays tumor growth in the xenograft model. Our studies found that the p27(Kip1) tumor suppressor protein is upregulated and relocalized to the nucleus from cytoplasm by CCN5 in these cells and that these two events (upregulation and relocalization of p27(Kip1)) are critical for CCN5-induced growth inhibition of TNBC cells. In the absence of CCN5, p27(Kip1) resides mostly in the cytoplasm, which is associated with the aggressive nature of cancer cells. Mechanistically, CCN5 inhibits Skp2 expression, which seems to stabilize the p27(Kip1) protein in these cells. On the other hand, CCN5 also recruits FOXO3a to mediate the transcriptional regulation of p27(Kip1). The recruitment of FOXO3a is achieved by the induction of its expression and activity through shifting from cytoplasm to the nucleus. Our data indicate that CCN5 blocks PI3K/AKT signaling to dephosphorylate at S318, S253 and Thr32 in FOXO3a for nuclear relocalization and activation of FOXO3a. Moreover, inhibition of α6β1 receptors diminishes CCN5 action on p27(Kip1) in TNBC cells. Collectively, these data suggest that CCN5 effectively inhibits TNBC growth through the accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation, and thus, activation of CCN5 may have the therapeutic potential to kill TNBC.

Akalay I, Tan TZ, Kumar P, et al.
Targeting WNT1-inducible signaling pathway protein 2 alters human breast cancer cell susceptibility to specific lysis through regulation of KLF-4 and miR-7 expression.
Oncogene. 2015; 34(17):2261-71 [PubMed] Related Publications
The molecular basis for the resistance of tumor cells to cell-mediated cytotoxicity remains poorly understood and thus poses a major challenge for cancer immunotherapy. The present study was designed to determine whether the WNT1-inducible signaling pathway protein 2 (WISP2, also referred to as CCN5), a key regulator of tumor cell plasticity, interferes with tumor susceptibility to cytotoxic T-lymphocyte (CTL)-mediated lysis. We found that silencing WISP2 signaling in human breast adenocarcinoma MCF7 cells impairs CTL-mediated cell killing by a mechanism involving stem cell marker Kruppel-like factor-4 (KLF-4) induction and microRNA-7 (miR-7) downregulation. Inhibition of transforming growth factor beta (TGF-β) signaling using the A83-01 inhibitor in MCF7-shWISP2 cells resulted in a significant reversal of the epithelial-to-mesenchymal-transitioned (EMT) phenotype, the expression of KLF-4 and a partial recovery of target susceptibility to CTLs. More importantly, we showed that silencing KLF-4 was accompanied by a reduction in MCF7-shWISP2 resistance to CTLs. Using human breast cancer tissues, we demonstrated the coexpression of KLF-4 with EMT markers and TGF-β pathway signaling components. More importantly, we found that KLF-4 expression was accompanied by miR-7 inhibition, which is partly responsible for impairing CTL-mediated lysis. Thus, our data indicate that WISP2 has a role in regulating tumor cell susceptibility through EMT by inducing the TGF-β signaling pathway, KLF-4 expression and miR-7 inhibition. These studies indicate for the first time that WISP2 acts as an activator of CTL-induced killing and suggests that the loss of its function promotes evasion of immunosurveillance and the ensuing progression of the tumor.

Wu MY, Xie X, Xu ZK, et al.
PP2A inhibitors suppress migration and growth of PANC-1 pancreatic cancer cells through inhibition on the Wnt/β-catenin pathway by phosphorylation and degradation of β-catenin.
Oncol Rep. 2014; 32(2):513-22 [PubMed] Free Access to Full Article Related Publications
Cantharidin is an active constituent of mylabris, a traditional Chinese medicine, and presents strong anticancer activity in various cell lines. Cantharidin is a potent and selective inhibitor of serine/threonine protein phosphatase 2A (PP2A). Our previous studies revealed the prospect of application of cantharidin, as well as other PP2A inhibitors, in the treatment of pancreatic cancer. However, the mechanisms involved in the anticancer effect of PP2A inhibitors have not been fully explored. The Wnt/β‑catenin pathway is involved in cell migration and proliferation and participates in the progression of pancreatic cancer. If β‑catenin is phosphorylated and degraded, the Wnt/β‑catenin pathway is blocked. PP2A dephosphorylates β‑catenin and keeps the Wnt/β‑catenin pathway active. In the present study, we found that PP2A inhibitor treatment induced phosphorylation and degradation of β‑catenin. The suppression on the migration and growth of PANC‑1 pancreatic cancer cells could be attenuated by pretreatment with FH535, a β‑catenin pathway inhibitor. Microarray showed that PP2A inhibitor treatment induced expression changes in 13 of 138 genes downstream of the β‑catenin pathway. Real‑time PCR further confirmed that FH535 attenuated the expression changes induced by PP2A inhibitors in 6 of these 13 candidate genes. These 6 genes, VEGFB, Dkk3, KRT8, NRP1, Cacnalg and WISP2, have been confirmed to participate in the migration and/or growth regulation in previous studies. Thus, the phosphorylation- and degradation-mediated suppression on β‑catenin participates in the cytotoxicity of PP2A inhibitors. Our findings may provide insight into the treatment of pancreatic cancer using a targeting PP2A strategy.

Zhang YW, Zheng Y, Wang JZ, et al.
Integrated analysis of DNA methylation and mRNA expression profiling reveals candidate genes associated with cisplatin resistance in non-small cell lung cancer.
Epigenetics. 2014; 9(6):896-909 [PubMed] Free Access to Full Article Related Publications
DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.

Oliveras-Ferraros C, Vazquez-Martin A, Cuyàs E, et al.
Acquired resistance to metformin in breast cancer cells triggers transcriptome reprogramming toward a degradome-related metastatic stem-like profile.
Cell Cycle. 2014; 13(7):1132-44 [PubMed] Free Access to Full Article Related Publications
Therapeutic interventions based on metabolic inhibitor-based therapies are expected to be less prone to acquired resistance. However, there has not been any study assessing the possibility that the targeting of the tumor cell metabolism may result in unforeseeable resistance. We recently established a pre-clinical model of estrogen-dependent MCF-7 breast cancer cells that were chronically adapted to grow (> 10 months) in the presence of graded, millimolar concentrations of the anti-diabetic biguanide metformin, an AMPK agonist/mTOR inhibitor that has been evaluated in multiple in vitro and in vivo cancer studies and is now being tested in clinical trials. To assess what impact the phenomenon of resistance might have on the metformin-like "dirty" drugs that are able to simultaneously hit several metabolic pathways, we employed the ingenuity pathway analysis (IPA) software to functionally interpret the data from Agilent whole-human genome arrays in the context of biological processes, networks, and pathways. Our findings establish, for the first time, that a "global" targeting of metabolic reprogramming using metformin certainly imposes a great selective pressure for the emergence of new breast cancer cellular states. Intriguingly, acquired resistance to metformin appears to trigger a transcriptome reprogramming toward a metastatic stem-like profile, as many genes encoding the components of the degradome (KLK11, CTSF, FREM1, BACE-2, CASP, TMPRSS4, MMP16, HTRA1), cancer cell migration and invasion factors (TP63, WISP2, GAS3, DKK1, BCAR3, PABPC1, MUC1, SPARCL1, SEMA3B, SEMA6A), stem cell markers (DCLK1, FAK), and key pro-metastatic lipases (MAGL and Cpla2) were included in the signature. Because this convergent activation of pathways underlying tumor microenvironment interactions occurred in low-proliferative cancer cells exhibiting a notable downregulation of the G 2/M DNA damage checkpoint regulators that maintain genome stability (CCNB1, CCNB2, CDC20, CDC25C, AURKA, AURKB, BUB1, CENP-A, CENP-M) and pro-autophagic features (i.e., TRAIL upregulation and BCL-2 downregulation), it appears that the unique mechanism of acquired resistance to metformin has opposing roles in growth and metastatic dissemination. While refractoriness to metformin limits breast cancer cell growth, likely due to aberrant mitotic/cytokinetic machinery and accelerated autophagy, it notably increases the potential of metastatic dissemination by amplifying the number of pro-migratory and stemness inputs via the activation of a significant number of proteases and EMT regulators. Future studies should elucidate whether our findings using supra-physiological concentrations of metformin mechanistically mimic the ultimate processes that could paradoxically occur in a polyploid, senescent-autophagic scenario triggered by the chronic metabolic stresses that occur during cancer development and after treatment with cancer drugs.

Ferrand N, Gnanapragasam A, Dorothee G, et al.
Loss of WISP2/CCN5 in estrogen-dependent MCF7 human breast cancer cells promotes a stem-like cell phenotype.
PLoS One. 2014; 9(2):e87878 [PubMed] Free Access to Full Article Related Publications
It has been proposed that the epithelial-mesenchymal transition (EMT) in mammary epithelial cells and breast cancer cells generates stem cell features. WISP2 (Wnt-1-induced signaling protein-2) plays an important role in maintenance of the differentiated phenotype of estrogen receptor-positive breast cancer cells and loss of WISP2 is associated with EMT. We now report that loss of WISP2 in MCF7 breast cancer cells can also promote the emergence of a cancer stem-like cell phenotype characterized by high expression of CD44, increased aldehyde dehydrogenase activity and mammosphere formation. Higher levels of the stem cell markers Nanog and Oct3/4 were observed in those mammospheres. In addition we show that low-cell inoculums are capable of tumor formation in the mammary fat pad of immunodeficient mice. Gene expression analysis show an enrichment of markers linked to stem cell function such as SOX9 and IGFBP7 which is linked to TGF-β inducible, SMAD3-dependent transcription. Taken together, our data demonstrate that WISP2 loss promotes both EMT and the stem-like cell phenotype.

Ji J, Jia S, Ji K, Jiang WG
Wnt1 inducible signalling pathway protein-2 (WISP‑2/CCN5): roles and regulation in human cancers (review).
Oncol Rep. 2014; 31(2):533-9 [PubMed] Related Publications
Wnt1 inducible signalling pathway protein-2 (WISP‑2), also known as CCN5, CT58, CTGF-L, CTGF-3, HICP and Cop1, is one of the 3 WNT1 inducible proteins that belongs to the CCN family. This family of members has been shown to play multiple roles in a number of pathophysiological processes, including cell proliferation, adhesion, wound healing, extracellular matrix regulation, epithelial-mesenchymal transition, angiogenesis, fibrosis, skeletal development and embryo implantation. Recent results suggest that WISP-2 is relevant to tumorigenesis and malignant transformation, particularly in breast cancer, colorectal cancer and hepatocarcinoma. Notably, its roles in cancer appear to vary depending on cell/tumour type and the microenvironment. The striking difference in the structure of WISP-2 in comparison with the other 2 family members may contribute to its difference in functions, which leads to the hypothesis that WISP-2 may act as a dominant-negative regulator of other CCN family members. In the present review, we summarise the roles, regulation and underlying mechanism of WISP-2 in human cancers.

Frewer KA, Sanders AJ, Owen S, et al.
A role for WISP2 in colorectal cancer cell invasion and motility.
Cancer Genomics Proteomics. 2013 Jul-Aug; 10(4):187-96 [PubMed] Related Publications
BACKGROUND: WNT inducible secreted protein 2 (WISP2) has been linked with a variety of human cancer types and may contribute to cancer metastasis. The current study investigated the importance of WISP2 in colorectal cancer cells, examining the impact of targeting WISP2 on Caco-2 cell invasion and motility together with potential mechanisms of action.
MATERIALS AND METHODS: WISP2 expression was targeted in Caco-2 cells using a ribozyme transgene system and successful knockdown was verified using reverse transcription-polymerase chain reaction (RT-PCR). The impact of WISP2 knockout (Caco-2(WISP2 KO)) on cell growth, adhesion, motility and invasion was examined using a number of in vitro functional assays. In vitro invasion assays were repeated in the presence of wingless-type MMTV integration site family (WNT) inhibitors (FH535 and IWP-2) to investigate the role of the WNT-signalling pathway in the regulation of cell invasion by WISP2. Quantitative-PCR was conducted to measure matrix metalloproteinase (MMP) expression in control [wild-type (Caco-2(WT)) and cells containing the empty pEF6 plasmid (Caco-2(pEF6))] and Caco-2(WISP2 KO) cells.
RESULTS: WISP2 knockout resulted in a significant increase in Caco-2 cell invasion and motility (p<0.05 in comparison to wild-type and plasmid control Caco-2 cells). WISP2 knockout had no significant effect on Caco-2 cell growth rate in 3- and 5-day incubation and no significant impact on Caco-2 cell-matrix adhesion rates (p>0.05). Expression analysis of a number of MMPs indicated an insignificant up-regulation of MMP2, MMP9 (p>0.05) but significant up-regulation of MMP7 (p=0.025) in Caco-2(WISP2 KO) cells compared to controls. Inhibition of WNT signalling using FH535 and IWP-2 brought about a significant or borderline significant decrease in Caco-2(WISP2 KO) cell invasion (FH535 p=0.065) and (IWP-2 p=0.002) and negated the pro-invasive effect of targeting WISP2 in Caco-2 cells.
CONCLUSION: WISP2 knockout significantly increased Caco-2 cell invasion and motility. Up-regulation of MMP2, -7 and -9 may indicate that WISP2 regulates invasion and motility through MMPs. Regulation of invasion by WISP2 may involve the WNT signalling pathway.

Wierer M, Verde G, Pisano P, et al.
PLK1 signaling in breast cancer cells cooperates with estrogen receptor-dependent gene transcription.
Cell Rep. 2013; 3(6):2021-32 [PubMed] Related Publications
Polo-like kinase 1 (PLK1) is a key regulator of cell division and is overexpressed in many types of human cancers. Compared to its well-characterized role in mitosis, little is known about PLK1 functions in interphase. Here, we report that PLK1 mediates estrogen receptor (ER)-regulated gene transcription in human breast cancer cells. PLK1 interacts with ER and is recruited to ER cis-elements on chromatin. PLK1-coactivated genes included classical ER target genes such as Ps2, Wisp2, and Serpina3 and were enriched in developmental and tumor-suppressive functions. Performing large-scale phosphoproteomics of estradiol-treated MCF7 cells in the presence or absence of the specific PLK1 inhibitor BI2536, we identified several PLK1 end targets involved in transcription, including the histone H3K4 trimethylase MLL2, the function of which on ER target genes was impaired by PLK1 inhibition. Our results propose a mechanism for the tumor-suppressive role of PLK1 in mammals as an interphase transcriptional regulator.

Tomimaru Y, Koga H, Yano H, et al.
Upregulation of T-cell factor-4 isoform-responsive target genes in hepatocellular carcinoma.
Liver Int. 2013; 33(7):1100-12 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The Wnt/β-catenin signalling pathway regulates genes involved in cell proliferation, survival, migration and invasion through regulation by T-cell factor (TCF)-4 transcription factor proteins. However, the role of TCF-4 isoforms generated by alternative splicing events in hepatocellular carcinoma (HCC) is unknown.
AIM: Here, we investigated TCF-4 isoforms (TCF-4J and K)-responsive target genes that are important in hepatic oncogenesis and tumour development.
METHODS: Gene expression microarray was performed on HCC cells overexpressing TCF-4J and K isoforms. Expression level of selected target genes was evaluated and correlations were made between their expression level and that of TCF-4 isoform in 47 pairs of human HCC tumours.
RESULTS: Comparison by gene expression microarray revealed that 447 genes were upregulated and 343 downregulated more than 2.0-fold in TCF-4J compared with TCF-4K expressing cells. We validated expression of 18 selected target genes involved in Wnt/β-catenin, insulin/IGF-1/IRS1 and Notch signalling pathways in 47 pairs of human HCCs and adjacent uninvolved liver tissues. It was observed that 13 genes (CLDN2, STK17B, SPP1, AXIN2, WISP2, MMP7, IRS1, ANXA1, CAMK2N1, ASPH, GPR56, CD24 and JAG1) activated by TCF-4J isoform in HCC cells, were also upregulated in HCC tumours compared with adjacent peritumour tissue; more importantly, 10 genes exhibited a significant correlation with the TCF-4J expression level in tumour.
CONCLUSION: TCF-4 isoforms (TCF-4J and K) activated different downstream target genes in HCC. The biological consequence of TCF-4J isoform expression was upregulation of genes associated with tripartite Wnt/β-catenin, insulin/IGF-1/IRS1 and Notch signal transduction pathway activation, which contribute to the pathogenesis of HCC.

Colli LM, Saggioro F, Serafini LN, et al.
Components of the canonical and non-canonical Wnt pathways are not mis-expressed in pituitary tumors.
PLoS One. 2013; 8(4):e62424 [PubMed] Free Access to Full Article Related Publications
INTRODUCTION: Canonical and non-canonical Wnt pathways are involved in the genesis of multiple tumors; however, their role in pituitary tumorigenesis is mostly unknown.
OBJECTIVE: This study evaluated gene and protein expression of Wnt pathways in pituitary tumors and whether these expression correlate to clinical outcome.
MATERIALS AND METHODS: Genes of the WNT canonical pathway: activating ligands (WNT11, WNT4, WNT5A), binding inhibitors (DKK3, sFRP1), β-catenin (CTNNB1), β-catenin degradation complex (APC, AXIN1, GSK3β), inhibitor of β-catenin degradation complex (AKT1), sequester of β-catenin (CDH1), pathway effectors (TCF7, MAPK8, NFAT5), pathway mediators (DVL-1, DVL-2, DVL-3, PRICKLE, VANGL1), target genes (MYB, MYC, WISP2, SPRY1, TP53, CCND1); calcium dependent pathway (PLCB1, CAMK2A, PRKCA, CHP); and planar cell polarity pathway (PTK7, DAAM1, RHOA) were evaluated by QPCR, in 19 GH-, 18 ACTH-secreting, 21 non-secreting (NS) pituitary tumors, and 5 normal pituitaries. Also, the main effectors of canonical (β-catenin), planar cell polarity (JNK), and calcium dependent (NFAT5) Wnt pathways were evaluated by immunohistochemistry.
RESULTS: There are no differences in gene expression of canonical and non-canonical Wnt pathways between all studied subtypes of pituitary tumors and normal pituitaries, except for WISP2, which was over-expressed in ACTH-secreting tumors compared to normal pituitaries (4.8x; p = 0.02), NS pituitary tumors (7.7x; p = 0.004) and GH-secreting tumors (5.0x; p = 0.05). β-catenin, NFAT5 and JNK proteins showed no expression in normal pituitaries and in any of the pituitary tumor subtypes. Furthermore, no association of the studied gene or protein expression was observed with tumor size, recurrence, and progressive disease. The hierarchical clustering showed a regular pattern of genes of the canonical and non-canonical Wnt pathways randomly distributed throughout the dendrogram.
CONCLUSIONS: Our data reinforce previous reports suggesting no activation of canonical Wnt pathway in pituitary tumorigenesis. Moreover, we describe, for the first time, evidence that non-canonical Wnt pathways are also not mis-expressed in the pituitary tumors.

Ferrand N, Stragier E, Redeuilh G, Sabbah M
Glucocorticoids induce CCN5/WISP-2 expression and attenuate invasion in oestrogen receptor-negative human breast cancer cells.
Biochem J. 2012; 447(1):71-9 [PubMed] Related Publications
CCN5 (cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed 5)/WISP-2 [WNT1 (wingless-type MMTV integration site family, member 1)-inducible signalling pathway protein 2] is an oestrogen-regulated member of the CCN family. CCN5 is a transcriptional repressor of genes associated with the EMT (epithelial-mesenchymal transition) and plays an important role in maintenance of the differentiated phenotype in ER (oestrogen receptor)-positive breast cancer cells. In contrast, CCN5 is undetectable in more aggressive ER-negative breast cancer cells. We now report that CCN5 is induced in ER-negative breast cancer cells such as MDA-MB-231 following glucocorticoid exposure, due to interaction of the endogenous glucocorticoid receptor with a functional glucocorticoid-response element in the CCN5 gene promoter. Glucocorticoid treatment of MDA-MB-231 cells is accompanied by morphological alterations, decreased invasiveness and attenuated expression of mesenchymal markers, including vimentin, cadherin 11 and ZEB1 (zinc finger E-box binding homeobox 1). Interestingly, glucocorticoid exposure did not increase CCN5 expression in ER-positive breast cancer cells, but rather down-regulated ER expression, thereby attenuating oestrogen pathway signalling. Taken together, our results indicate that glucocorticoid treatment of ER-negative breast cancer cells induces high levels of CCN5 expression and is accompanied by the appearance of a more differentiated and less invasive epithelial phenotype. These findings propose a novel therapeutic strategy for high-risk breast cancer patients.

Ouelaa-Benslama R, De Wever O, Hendrix A, et al.
Identification of a GαGβγ, AKT and PKCα signalome associated with invasive growth in two genetic models of human breast cancer cell epithelial-to-mesenchymal transition.
Int J Oncol. 2012; 41(1):189-200 [PubMed] Related Publications
The epithelial-to-mesenchymal transition (EMT) confers an aggressive subtype associated with chemotherapy resistance in epithelial cancers. However, the mechanisms underlying the EMT and its associated signaling dysfunctions are still poorly understood. In two genetic models of MCF-7 breast cancer cells induced to EMT by WISP-2 silencing and Snail transformation, we investigated the status of several signaling elements downstream of G-protein receptors (GPR) and their functional roles in the invasive growth potential. We report that the E-cadherin repressors Slug, Zeb1/2 and Twist are overexpressed in these EMT cells characterized by a triple negative phenotype (loss of estrogen ERα and progesterone PRA/PRB receptors, no HER2 amplification), combined with loss of the alternative GPR30 estrogen receptor and induction of the invasive growth in collagen type I gels. Ectopic Snail expression suppressed WISP-2 transcripts and down-regulated WISP-2 gene promoter expression in transfected cells. Accordingly, WISP-2 transcripts and Wisp-2 protein were depleted in these two convergent models of BC cell EMT. The EMT caused dominance of several proinvasive pathways downstream of GPR, including GαGβγ subunits, PKCα, AKT and c-Jun induction, constitutive activation of the actin-remodeling GTPase Rac1, coupled with growth responses (more cells at S and G2/M phases of the cell cycle), in line with inhibition of the p27kip1/cyclin-dependent kinase CDK3 cascade. RNA interference or selective inhibitors targeting GαGβγ subunits (BIM-46187, gallein), PKCα (Gö6976, MT477, sh-RNAs) and PI3K-AKT (wortmannin) alleviated the invasive phenotype. In contrast, MCF-7 cells in EMT showed signaling independence to inhibitors of HER family tyrosine kinases and the mitogen- and stress-activated protein kinases. Our study suggests that the signaling protagonists GαGβγ, PKCα and PI3K-AKT are promising candidates as predictive molecular biomarkers and therapeutic targets in the management of clinical BC in EMT.

Liu J, Ding X, Tang J, et al.
Enhancement of canonical Wnt/β-catenin signaling activity by HCV core protein promotes cell growth of hepatocellular carcinoma cells.
PLoS One. 2011; 6(11):e27496 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The Hepatitis C virus (HCV) core protein has been implicated as a potential oncogene or a cofactor in HCV-related hepatocellular carcinoma (HCC), but the underlying mechanisms are unknown. Overactivation of the Wnt/β-catenin signaling is a major factor in oncogenesis of HCC. However, the pathogenesis of HCV core-associated Wnt/β-catenin activation remains to be further characterized. Therefore, we attempted to determine whether HCV core protein plays an important role in regulating Wnt/β-catenin signaling in HCC cells.
METHODOLOGY: Wnt/β-catenin signaling activity was investigated in core-expressing hepatoma cells. Protein and gene expression were examined by Western blot, immunofluorescence staining, RT-qPCR, and reporter assay.
PRINCIPAL FINDINGS: HCV core protein significantly enhances Tcf-dependent transcriptional activity induced by Wnt3A in HCC cell lines. Additionally, core protein increases and stabilizes β-catenin levels in hepatoma cell line Huh7 through inactivation of GSK-3β, which contributes to the up-regulation of downstream target genes, such as c-Myc, cyclin D1, WISP2 and CTGF. Also, core protein increases cell proliferation rate and promotes Wnt3A-induced tumor growth in the xenograft tumor model of human HCC.
CONCLUSIONS/SIGNIFICANCE: HCV core protein enhances Wnt/β-catenin signaling activity, hence playing an important role in HCV-associated carcinogenesis.

Stiehl DP, Bordoli MR, Abreu-Rodríguez I, et al.
Non-canonical HIF-2α function drives autonomous breast cancer cell growth via an AREG-EGFR/ErbB4 autocrine loop.
Oncogene. 2012; 31(18):2283-97 [PubMed] Related Publications
Tumor progression is intrinsically tied to the clonal selection of tumor cells with acquired phenotypes allowing to cope with a hostile microenvironment. Hypoxia-inducible factors (HIFs) master the transcriptional response to local tissue hypoxia, a hallmark of solid tumors. Here, we report significantly longer patient survival in breast cancer with high levels of HIF-2α. Amphiregulin (AREG) and WNT1-inducible signaling pathway protein-2 (WISP2) expression was strongly HIF-2α-dependent and their promoters were particularly responsive to HIF-2α. The endogenous AREG promoter recruited HIF-2α in the absence of a classical HIF-DNA interaction motif, revealing a novel mechanism of gene regulation. Loss of AREG expression in HIF-2α-depleted cells was accompanied by reduced activation of epidermal growth factor (EGF) receptor family members. Apparently opposing results from patient and in vitro data point to an HIF-2α-dependent auto-stimulatory tumor phenotype that, while promoting EGF signaling in cellular models, increased the survival of diagnosed and treated human patients. Our findings suggest a model where HIF-2α-mediated autocrine growth signaling in breast cancer sustains a state of cellular self-sufficiency, thereby masking unfavorable microenvironmental growth conditions, limiting adverse selection and improving therapy efficacy. Importantly, HIF-2α/AREG/WISP2-expressing tumors were associated with luminal tumor differentiation, indicative of a better response to classical treatments. Shifting the HIF-1/2α balance toward an HIF-2-dominated phenotype could thus offer a novel approach in breast cancer therapy.

Leal LF, Mermejo LM, Ramalho LZ, et al.
Wnt/beta-catenin pathway deregulation in childhood adrenocortical tumors.
J Clin Endocrinol Metab. 2011; 96(10):3106-14 [PubMed] Related Publications
CONTEXT: CTNNB1/β-catenin mutations and activation of Wnt/β-catenin pathway are frequent in adult adrenocortical tumors (ACT), but data on childhood ACT are lacking.
OBJECTIVE: The aim of the study was to investigate the presence of Wnt/β-catenin pathway abnormalities in childhood ACT.
PATIENTS AND METHODS: Clinicopathological findings and outcome of 62 childhood ACT patients were analyzed regarding CTNNB1 mutations and the expression of Wnt-related genes (CTNNB1; WNT4, a Wnt ligand; SFRP1, DKK3, and AXIN1, Wnt inhibitors; TCF7, a transcription factor; and MYC and WISP2, target genes) by quantitative PCR and immunohistochemistry.
RESULTS: CTNNB1-activating mutations were found in only four of 62 ACT (6%), all of them harboring TP53 mutation. There was association between the presence of CTNNB1 mutations and death (P = 0.02). Diffuse β-catenin accumulation was found in 71% of ACT, even in ACT without CTNNB1 mutations. Compared to normal adrenals, ACT presented increased expression of CTNNB1 (P = 0.008) and underexpression of Wnt inhibitor genes: DKK3 (P < 0.0001), SFRP1 (P = 0.05), and AXIN1 (P = 0.04). With regard to Wnt/β-catenin target genes, ACT presented increased expression of WISP2 but lower expression of MYC. Higher overall survival was associated with underexpression of SFRP1 (P = 0.01), WNT4 (P = 0.004), and TCF7 (P < 0.01).
CONCLUSIONS: CTNNB1 mutations are not common in childhood ACT but appear to associate with poor prognosis. Nevertheless, most ACT exhibit increased expression of β-catenin and WISP2 and reduced expression of Wnt inhibitor genes (DKK3, SFRP1, and AXIN1). Thus, in addition to CTNNB1 mutations, other genetic events affecting the Wnt/β-catenin pathway may be involved in childhood adrenocortical tumorigenesis.

Guillon-Munos A, Oikonomopoulou K, Michel N, et al.
Kallikrein-related peptidase 12 hydrolyzes matricellular proteins of the CCN family and modifies interactions of CCN1 and CCN5 with growth factors.
J Biol Chem. 2011; 286(29):25505-18 [PubMed] Free Access to Full Article Related Publications
Kallikrein-related peptidases (KLKs) are an emerging group of secreted serine proteases involved in several physiological and pathological processes. We used a degradomic approach to identify potential substrates of KLK12. MDA-MB-231 cells were treated either with KLK12 or vehicle control, and the proteome of the overlying medium was analyzed by mass spectrometry. CCN1 (cyr61, ctgf, nov) was among the proteins released by the KLK12-treated cells, suggesting that KLK12 might be responsible for the shedding of this protein from the cell surface. Fragmentation of CCN1 by KLK12 was further confirmed in vitro, and the main cleavage site was localized in the hinge region between the first and second half of the recombinant protein. KLK12 can target all six members of the CCN family at different proteolytic sites. Limited proteolysis of CCNs (cyr61, ctgf, nov) was also observed in the presence of other members of the KLK family, such as KLK1, KLK5, and KLK14, whereas KLK6, KLK11, and KLK13 were unable to fragment CCNs. Because KLK12 seems to have a role in angiogenesis, we investigated the relations between KLK12, CCNs, and several factors known to be involved in angiogenesis. Solid phase binding assays showed that fragmentation of CCN1 or CCN5 by KLK12 prevents VEGF(165) binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-β1 and FGF-2, either as intact or truncated forms, was found to be concentration-dependent. These findings suggest that KLK12 may indirectly regulate the bioavailability and activity of several growth factors through processing of their CCN binding partners.

Sabbah M, Prunier C, Ferrand N, et al.
CCN5, a novel transcriptional repressor of the transforming growth factor β signaling pathway.
Mol Cell Biol. 2011; 31(7):1459-69 [PubMed] Free Access to Full Article Related Publications
CCN5 is a member of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family and was identified as an estrogen-inducible gene in estrogen receptor-positive cell lines. However, the role of CCN5 in breast carcinogenesis remains unclear. We report here that the CCN5 protein is localized mostly in the cytoplasm and in part in the nucleus of human tumor breast tissue. Using a heterologous transcription assay, we demonstrate that CCN5 can act as a transcriptional repressor presumably through association with histone deacetylase 1 (HDAC1). Microarray gene expression analysis showed that CCN5 represses expression of genes associated with epithelial-mesenchymal transition (EMT) as well as expression of key components of the transforming growth factor β (TGF-β) signaling pathway, prominent among them TGF-βRII receptor. We show that CCN5 is recruited to the TGF-βRII promoter, thereby providing a mechanism by which CCN5 restricts transcription of the TGF-βRII gene. Consistent with this finding, CCN5, we found, functions to suppress TGF-β-induced transcriptional responses and invasion that is concomitant with EMT. Thus, our data uncovered CCN5 as a novel transcriptional repressor that plays an important role in regulating tumor progression functioning, at least in part, by inhibiting the expression of genes involved in the TGF-β signaling cascade that is known to promote EMT.

Davies SR, Davies ML, Sanders A, et al.
Differential expression of the CCN family member WISP-1, WISP-2 and WISP-3 in human colorectal cancer and the prognostic implications.
Int J Oncol. 2010; 36(5):1129-36 [PubMed] Related Publications
The WISPs (Wnt-inducted secreted proteins, WISP-1, WISP-2 and WISP-3) are part of the CCN family. These molecules are known to play a diverse role in cells but their role in cancer cells remains controversial. We analysed the expression of the three WISP molecules at the mRNA and protein levels in a cohort of 94 human colorectal tumours and 80 normal colorectal tissues and correlated the results with the pathological features and clinical outcome of the patients. WISP-1 transcripts were found at higher levels in the tumour samples than in the normal tissue (p=0.0015); higher in patients with Dukes stage B and C compared to Dukes A (p=0.017 and p=0.024, respectively); higher in patients with moderately and poorly differentiated cancers compared to the well differentiated cancers (p=0.020 and p=0.076, respectively and p=0.0035 when combined); higher in node positive tumours compared with the node negative (p=0.11) and in the patients with higher TNM staging (TNM 2, 3 and 4 compared to TNM 1 p=0.037). WISP-2 showed the opposite pattern with lower levels of expression in cancer cells compared to normal (p=0.082). Although no significant differences were found within the cancer group when indices of a more aggressive tumour were compared to the normal tissue a significant reduction in expression was found (Dukes C p=0.044, poorly differentiated p=0.019, TNM 3 p=0.020 and node positive disease p=0.048). WISP-3 transcript levels showed no significant differences between groups. WISPs may play important but contrasting roles in colorectal cancer with WISP-1 appearing to act as a factor stimulating aggressiveness, WISP-2 as a tumour suppressor and WISP-3 having no definable beneficial or detrimental role.

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