WISP-2/CCN5 is involved as a novel signaling intermediate in phorbol ester-protein kinase Calpha-mediated breast tumor cell proliferation

PMA and active phorbol esters stimulate the proliferation of various tumor cells, including ER-positive human breast tumor cell lines. However, the specific signaling pathways involved in the PMA-induced mitogenic effect on breast tumor cells have not been fully elucidated. In the present study, we explored the mechanisms associated with the mitogenic influence of PMA on breast tumor cells. Following an acute exposure (i.e., within 2 to 6 h) to PMA (50 nM), a mitogenic effect was observed on WISP-2/CCN5-positive breast tumor cell lines, including MCF-7, ZR-75-1 and SKBR-3 cells, and induction of WISP-2/CCN5 mRNA expression paralleled the observed mitogenic proliferation. This effect was undetected in WISP-2/CCN5 negative MDA-MB-231 breast tumor cells or human mammary epithelial cells with or without ER-alpha transfection. The mitogenic effect of PMA was perturbed by short hairpin RNA (shRNA)-mediated inhibition of WISP-2/CCN5 signaling in MCF-7 cells. Moreover, the upregulation of WISP-2/CCN5 by PMA is not ER dependent but is instead mediated through a complex PKCalpha-MAPK/ERK and SAPK/JNK signaling pathway, which leads to growth stimulation of MCF-7 breast tumor cells. These series of experiments provide the first evidence that WISP-2/CCN5 is a novel signaling molecule that critically participates in the mitogenic action of PMA on noninvasive, WISP-2/CCN5-positive breast tumor cells through PKCalpha-dependent, multiple molecular signal transduction pathways.     

WISP-2: a serum-inducible gene differentially expressed in human normal breast epithelial cells and in MCF-7 breast tumor cells

WISP-2 is a Wnt-1-induced signaling protein identified as a member of CCN growth factor family. A role for this molecule during tumorigenesis is suspected but remains unproven. Here we show that WISP-2 expression was undetectable, or minimally detectable, in nontransformed human mammary epithelial cells, but was overexpressed in MCF-7 cells. Expression of WISP-2 in MCF-7 cells was modulated by serum and correlated with the serum-induced MCF-7 tumor cell proliferation, suggesting that WISP-2 is serum responsive and may be a positive regulator of tumor cell proliferation.     

CCN5/WISP-2: A micromanager of breast cancer progression

The gain of plasticity by a subset of cancer cells is a unique but common sequence of cancer progression from epithelial phenotype to mesenchymal phenotype (EMT) that is followed by migration, invasion and metastasis to a distant organ, and drug resistance. Despite multiple studies, it is still unclear how cancer cells regulate plasticity. Recent studies from our laboratory and others’ proposed that CCN5/WISP-2, which is found intracellularly (in the nucleus and cytoplasm) and extracellularly, plays a negative regulator of plasticity. It prevents the EMT process in breast cancer cells as well as pancreatic cancer cells. Multiple genetic insults, including the gain of p53 mutations that accumulate over the time, may perturb CCN5 expression in non-invasive breast cancer cells, which ultimately helps cells to gain invasive phenotypes. Moreover, emerging evidence indicates that several oncogenic lesions such as miR-10b upregulation and activation of TGF-β-signaling can accumulate during CCN5 crisis in breast cancer cells. Collectively, these studies indicate that loss of CCN5 activity may promote breast cancer progression; application of CCN5 protein may represent a novel therapeutic intervention in breast cancer and possibly pancreatic cancer.     

CYR61 (CCN1) is essential for placental development and vascular integrity

CYR61 (CCN1) is a member of the CCN family of secreted matricellular proteins that includes connective tissue growth factor (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). First identified as the product of a growth factor-inducible immediate-early gene, CYR61 is an extracellular matrix-associated angiogenic inducer that functions as a ligand of integrin receptors to promote cell adhesion, migration, and proliferation. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. To understand the functions of CYR61 during development, we have disrupted the Cyr61 gene in mice. We show here that Cyr61-null mice suffer embryonic death: approximately 30% succumbed to a failure in chorioallantoic fusion, and the reminder perished due to placental vascular insufficiency and compromised vessel integrity. These findings establish CYR61 as a novel and essential regulator of vascular development. CYR61 deficiency results in a specific defect in vessel bifurcation (nonsprouting angiogenesis) at the chorioallantoic junction, leading to an undervascularization of the placenta without affecting differentiation of the labyrinthine syncytiotrophoblasts. This unique phenotype is correlated with impaired Vegf-C expression in the allantoic mesoderm, suggesting that CYR61-regulated expression of Vegf-C plays a role in vessel bifurcation. The genetic and molecular basis of vessel bifurcation is presently unknown, and these findings provide new insight into this aspect of angiogenesis.     

Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins alphavbeta3 and alpha6beta1 in human umbilical vein endothelial cells

CYR61 (CCN1) is an extracellular matrix-associated protein of the CCN family, which also includes CTGF (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). Purified CYR61 induces neovascularization in corneal implants, and Cyr61-null mice suffer embryonic death due to vascular defects, thus establishing that CYR61 is an important regulator of angiogenesis. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. In culture, CYR61 functions through integrin-mediated pathways to promote cell adhesion, migration, and proliferation. Here we show that CYR61 can also promote cell survival and tubule formation in human umbilical vein endothelial cells. Furthermore, we have dissected the integrin receptor requirements of CYR61 with respect to its pro-angiogenic activities. Thus, CYR61-induced cell adhesion and tubule formation occur through interaction with integrin alpha(6)beta(1) in early passage endothelial cells in which integrins have not been activated. By contrast, in endothelial cells in which integrins are activated by phorbol ester or vascular endothelial growth factor, CYR61-promoted cell adhesion, migration, survival, growth factor-induced mitogenesis, and endothelial tubule formation are all mediated through integrin alpha(v)beta(3). These findings indicate that CYR61 is an activation-dependent ligand of integrin alpha(v)beta(3) and an activation-independent ligand of integrin alpha(6)beta(1) and that these integrins differentially mediate the pro-angiogenic activities of CYR61. These findings help to define the mechanisms by which CYR61 acts as an angiogenic regulator, provide a molecular interpretation for the loss of vascular integrity and increased apoptosis of vascular cells in Cyr61-null mice, and underscore the importance of CYR61 in the development and homeostasis of the vascular system.     

Differential expression of estrogen receptor alpha (ERalpha) protein in MCF-7 breast cancer cells chronically exposed to TCDD

Estrogens play a key role in the development and evolution of breast cancer tumors. Estrogen receptor alpha (ERalpha) mediates many of the biological activities of estrogens, and its expression is associated with low invasiveness and good prognosis. Recent epidemiological reports suggest that long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is implicated in the increased incidence of breast cancer in exposed women. TCDD interferes with the expression of some ERalpha-dependent genes and inhibits estradiol (E2)- dependent growth of breast cancer cells in vitro. However, E2-dependent xenographs of MCF-7 human breast cancer cells resumed growth after a 2-week exposure to TCDD. The mechanisms involved in the resumption of cell growth are not completely understood. In this study, we show that short term-exposure (16 days) to 1 nM TCDD results in the suppression of ERalpha protein expression, while chronic exposure for more than 1 year (LTDX cells) results in the partial re-expression of the receptor. Immunocytochemistry studies showed that re-expression of ERalpha in LTDX cells occurred in some of the cells. Analysis by Western immunoblots indicated that four out of five LTDX clones expressed ERalpha at levels comparable to those in unexposed MCF-7 cells. Removal of TCDD treatment for 16 days restored the expression of ERalpha in the ERalpha-negative clonal cells. These results suggest that MCF-7 cells chronically exposed to TCDD contain at least two cell subpopulations that may respond differently to the ERalpha-mediated effects of TCDD.     

Epigenetic reprogramming of breast cancer cells by valproic acid occurs regardless of estrogen receptor status

Estrogen receptors (ERs) are a recognized prognostic factor and therapeutic target in breast cancer. The loss of ER expression relates to poor prognosis, poor clinical outcome and impairs the use of anti-estrogenic treatment. Histone deacetylase inhibitors are candidate drugs for cancer therapy. Among them, valproic acid (VPA) is a long used and safe anti-epileptic drug. We studied the biological consequences of the chromatin remodeling action of VPA in a normal human mammary epithelial cell line and in ERalpha-positive and ERalpha-negative breast cancer cell lines. In these cells and regardless of their ER status, VPA-induced cell differentiation, as shown by increased milk lipids production, decreased expression of the CD44 antigen and growth arrest in the G(0)-G(1) phase of the cell cycle. These effects were accompanied by decreased Rb phosphorylation, hyperacetylation of the p21(WAF1/CIP1) gene promoter and increased p21 protein expression. Only in breast cancer cells, cyclin B1 expression was decreased and the cells accumulated also in G(2). ERalpha expression decreased in ERalpha-positive, increased in ERalpha-negative and was unchanged in normal mammary epithelial cells, as did the expression of progesterone receptor, a physiological ERalpha target. VPA decreased the expression of the invasiveness marker pS2 in ERalpha-positive breast cancer cells, but did not cause its re-expression in ERalpha-negative cells. Overall, these data suggest that in both ERalpha-positive and -negative malignant mammary epithelial cells VPA reprograms the cells to a more differentiated and "physiologic" phenotype that may improve the sensitivity to endocrine therapy and/or chemotherapy in breast cancer patients.     

CCN3/NOV small interfering RNA enhances fibrogenic gene expression in primary hepatic stellate cells and cirrhotic fat storing cell line CFSC

Nephroblastoma overexpressed gene encodes a matricellular protein (CCN3/NOV) of the CCN family, comprising CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). CCN proteins are involved in the regulation of mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration in multiple cell types. Compared to CCN2/CTGF, known as a profibrotic protein, the biological role of CCN3/NOV in liver fibrosis remains obscure. In this study we showed ccn3/nov mRNA to increase dramatically following hepatic stellate cell activation, reaching peak levels in fully transdifferentiated myofibroblasts. In models of experimental hepatic fibrosis, CCN3/NOV increased significantly at the mRNA and protein levels. CCN3/NOV was found mainly in non-parenchymal cells along the areas of tissue damage and repair. In the bile-duct ligation model, CCN3/NOV was localized mainly along portal tracts, while the repeated application of carbon tetrachloride resulted in CCN3/NOV expression mainly in the centrilobular areas. In contrast to CCN2/CTGF, the profibrotic cytokines platelet-derived growth factor-B and -D as well as transforming growth factor-β suppressed CCN3/NOV expression. In vitro, CCN3/NOV siRNA attenuated migration in the cirrhotic fat storing cell line CFSC well in line with in vivo findings that various types of cells expressing CCN3/NOV migrate into the area of tissue damage and regeneration. The suppression of CCN3/NOV enhanced expression of profibrotic marker proteins, such as α-smooth muscle actin, collagen type I, fibronectin, CCN2/CTGF and TIMP-1 in primary rat hepatic stellate cells and in CFSC. We further found that adenoviral overexpression of CCN2/CTGF suppressed CCN3/NOV expression, while the overexpression of CCN3/NOV as well as the suppression of CCN3/NOV by targeting siRNAs both resulted in enhanced CCN2/CTGF expression. These results indicate the complexity of CCN actions that are far beyond the classic Yin/Yang interplay.     

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling,on adipocyte differentiation

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer–binding protein δ expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activator of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator–activator receptor γ expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-α did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.     

MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer

A search for regulators of estrogen receptor alpha (ERalpha) expression has yielded a set of microRNAs (miRNAs) for which expression is specifically elevated in ERalpha-negative breast cancer. Here we show distinct expression of a panel of miRNAs between ERalpha-positive and ERalpha-negative breast cancer cell lines and primary tumors. Of the elevated miRNAs in ERalpha-negative cells, miR-221 and miR-222 directly interact with the 3'-untranslated region of ERalpha. Ectopic expression of miR-221 and miR-222 in MCF-7 and T47D cells resulted in a decrease in expression of ERalpha protein but not mRNA, whereas knockdown of miR-221 and miR-222 partially restored ERalpha in ERalpha protein-negative/mRNA-positive cells. Notably, miR-221- and/or miR-222-transfected MCF-7 and T47D cells became resistant to tamoxifen compared with vector-treated cells. Furthermore, knockdown of miR-221 and/or miR-222 sensitized MDA-MB-468 cells to tamoxifen-induced cell growth arrest and apoptosis. These findings indicate that miR-221 and miR-222 play a significant role in the regulation of ERalpha expression at the protein level and could be potential targets for restoring ERalpha expression and responding to antiestrogen therapy in a subset of breast cancers.