Cytokines of the tumor necrosis factor (TNF) family regulate inflammation and immunity, and a subset of this family can also induce cell death in a context‐dependent manner. Although TNFα is cytotoxic to certain tumor cell lines, it induces apoptosis in normal cells only when NFκB signaling is blocked. Here we show that the matricellular protein CCN1/CYR61 can unmask the cytotoxic potential of TNFα without perturbation of NFκB signaling or de novo protein synthesis, leading to rapid apoptosis in the otherwise resistant primary human fibroblasts. CCN1 acts through binding to integrins αvβ5, α6β1, and syndecan‐4, triggering the generation of reactive oxygen species (ROS) through a Rac1‐dependent mechanism via 5‐lipoxygenase and the mitochondria, leading to the biphasic activation of JNK necessary for apoptosis. Mice with the genomic Ccn1 locus replaced with an apoptosis‐defective Ccn1 allele are substantially resistant to TNFα‐induced apoptosis in vivo. These results indicate that CCN1 may act as a physiologic regulator of TNFα cytotoxicity, providing the contextual cues from the extracellular matrix for TNFα‐mediated cell death.

No abstract

Bladder cancer, the fourth most common noncutaneous malignancy in the United States, is characterized by high recurrence rate, with a subset of these cancers progressing to a deadly muscle invasive form of disease. Exosomes are small secreted vesicles that contain proteins, mRNA and miRNA, thus potentially modulating signaling pathways in recipient cells. Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell–cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells. EMT has been implicated in the initiation of metastasis for cancer progression. We investigated the ability of bladder cancer-shed exosomes to induce EMT in urothelial cells. Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media or from patient urine or bladder barbotage samples. Exosomes were then added to the urothelial cells and EMT was assessed. Urothelial cells treated with bladder cancer exosomes showed an increased expression in several mesenchymal markers, including α-smooth muscle actin, S100A4 and snail, as compared with phosphate-buffered saline (PBS)-treated cells. Moreover, treatment of urothelial cells with bladder cancer exosomes resulted in decreased expression of epithelial markers E-cadherin and β-catenin, as compared with the control, PBS-treated cells. Bladder cancer exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pretreatment. We further showed that exosomes isolated from patient urine and bladder barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. In conclusion, the research presented here represents both a new insight into the role of exosomes in transition of bladder cancer into invasive disease, as well as an introduction to a new platform for exosome research in urothelial cells.

Integrin-mediated cell adhesion to extracellular matrix proteins is known to promote cell survival, whereas detachment from the matrix can cause rapid apoptotic death in some cell types. Contrary to this paradigm, we show that fibroblast adhesion to the angiogenic matrix protein CCN1 (CYR61) induces apoptosis, whereas endothelial cell adhesion to CCN1 promotes cell survival. CCN1 induces fibroblast apoptosis through its adhesion receptors, integrin α6β1 and the heparan sulfate proteoglycan (HSPG) syndecan-4, triggering the transcription-independent p53 activation of Bax to render cytochrome c release and activation of caspase-9 and -3. Neither caspase-8 activity nor de novo transcription or translation is required for this process. These results show that cellular interaction with a specific matrix protein can either induce or suppress apoptosis in a cell type–specific manner and that integrin α6β1-HSPGs can function as receptors to induce p53-dependent apoptosis.

SummaryWe previously showed that the cell-cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here keratinocytes. PG -/-cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PG -/-keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM-Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability. Key words: Armadillo protein, Desmosome, Extracellular matrix, KeratinocyteJournal of Cell Science (Caldelari et al., 2001) and adhesive strength (Acehan et al., 2008) in keratinocytes lacking PG. Moreover, PG-null mice and patients with pathogenic homozygous PG mutations have impaired tissue integrity associated with skin and heart defects (Aberle et al., 1995;Bierkamp et al., 1996;McKoy et al., 2000;Ruiz et al., 1996). PG is also found in the cytoplasm and nucleus (Green and Simpson, 2007;Schmidt and Koch, 2007), where it is able to act independently of its function in intercellular adhesion. Its adhesionindependent functions are still not well defined, but the data suggest that PG can regulate gene expression and protein stability (Aktary et al., 2010;Hakimelahi et al., 2000;Shimizu et al., 2008) in both a -catenin-dependent and -independent manner (Raurell et al., 2006;Teuliere et al., 2004;Yin and Green, 2004;Zhurinsky et al., 2000).Recently we demonstrated that PG not only inhibits motility of keratinocytes in contact, but also inhibits Src-dependent single cell motility (Yin et al., 2005b). The observed changes in motility and altered cell morphology of PG -/-keratinocytes suggested to us that PG could be regulating cell-substrate interactions by modulating components of the extracellular matrix (ECM), its integrin receptors and/or the molecules involved in ECM-triggered motility cues. Using a combination of live cell imaging and cross plating, we show here that PG expression has a potent impact on the organization of actin, its associated membrane protrusions, focal adhesions and Src-dependent motility, in large part through regulation of the expression levels of the underlying ECM components. In particular, the ability of PG to regulate fibronectin (FN; also know as Fn1) mRNA stability was identified as a novel mechanism contributing to PG-dependent suppression of keratinocyte motility. Further analysis indicated that PG-dependent alterations in activity of the small GTPases Rac1 and RhoA act in parallel with FN/Src-dependent regulation of cortical actin 3577 Plakoglobin regulates motility via ECM structures to fine tune the motile behavior of keratinocytes. Collectively, these results indicate that a desmosomal molecule, PG, is capable of regulating single cell motility through matrix deposition in concert with Rho GTPases, independently of its role as a cell-cell adhesion molecule. Results PG regulates keratinocyte cell polarity and single-cell ...

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays an important role in immune surveillance and preferentially induces apoptosis in cancer cells over normal cells, suggesting its potential in cancer therapy. However, the molecular basis for its selective killing of cancer cells is not well understood. Recent studies have identified the CCN family of integrin-binding matricellular proteins as important regulators of cell behavior, including cell adhesion, proliferation, migration, differentiation, and survival. We show here that CCN1 (CYR61) supports the adhesion of prostatic carcinoma cells as an adhesion substrate through integrins and heparan sulfate proteoglycans. Knockdown of CCN1 expression in PC-3 and DU-145 androgen-independent prostate cancer cells strongly inhibited their proliferation without causing apoptosis, indicating that CCN1 promotes their growth. However, CCN1 also significantly enhances TRAIL-induced apoptosis through interaction with integrins α v β 3 and α 6 β 4 and the cell-surface heparan sulfate proteoglycan syndecan-4, acting through a protein kinase Cα-dependent mechanism without requiring de novo protein synthesis. Knockdown of CCN1 expression in PC-3, DU-145, and LNCaP cells severely blunted their sensitivity to TRAIL, an effect that was reversed by exogenously added CCN1 protein. These findings reveal a functional dichotomy for CCN1 in prostate carcinoma cells, because it contributes to both cell proliferation and TRAIL-induced cell death and suggest that CCN1 expression status may be an important parameter in assessing the efficacy of TRAIL-dependent cancer therapy. (Mol Cancer Res 2009;7(7):1045-55)

Prostate cancer mortality is primarily attributed to metastatic rather than primary, organ-confined disease. Acquiring a motile and invasive phenotype is an important step in development of tumors and ultimately metastasis. This step involves remodeling of the extracellular matrix and of cell-matrix interactions, cell movement mediated by the actin cytoskeleton, and activation of focal adhesion kinase (FAK)/Src signaling. Epidemiologic studies suggest that the metastatic behavior of prostate cancer may be an ideal target for chemoprevention. The natural flavone apigenin is known to have chemopreventive properties against many cancers, including prostate cancer. Here, we study the effect of apigenin on motility, invasion, and its mechanism of action in metastatic prostate carcinoma cells (PC3-M). We found that apigenin inhibits PC3-M cell motility in a scratchwound assay. Live cell imaging studies show that apigenin diminishes the speed and affects directionality of cell motion. Alterations in the cytoskeleton are consistent with impaired cell movement in apigenin-treated cells. Apigenin treatment leads to formation of "exaggerated filopodia," which show accumulation of focal adhesion proteins at their tips. Furthermore, apigenin-treated cells adhere more strongly to the extracellular matrix. Additionally, apigenin decreases activation of FAK and Src, and phosphorylation of Src substrates FAK Y576/577 and Y925. Expression of constitutively active Src blunts the effect of apigenin on cell motility and cytoskeleton remodeling. These results show that apigenin inhibits motility and invasion of prostate carcinoma cells, disrupts actin cytoskeleton organization, and inhibits FAK/Src signaling. These studies provide mechanistic insight into developing novel strategies for inhibiting prostate cancer cell motility and invasiveness.Prostate cancer is the most common noncutaneous malignancy in American males (over 186,000 cases diagnosed yearly), and the second leading cause of cancer-related deaths in American men (28,660 estimated deaths in 2008; ref. 1). In prostate cancer, most deaths are attributed to metastatic disease rather than primary, organ-confined prostate cancer (2-5). Numerous epidemiologic studies (6) suggest that the metastatic behavior of prostate cancer may be an ideal target for pharmacologic intervention with chemopreventive agents. Because prostate cancer is typically diagnosed in older men, chemopreventive and/or chemotherapeutic strategies to delay its progression may have a substantial impact on clinical outcome. Epidemiologic studies have shown that Asian men experience a lower incidence of metastatic prostate cancer than Western men, which is attributed to dietary and/or life-style factors (3, 4). Interestingly, some studies suggest that the incidence of primary cancer may be similar in Asian and Western populations, and that the higher rate of prostate cancer-related deaths in the United States is due to a higher rate of prostate cancer metastasis (3,5).Apigenin (4′, 5, 7-trihydroxyflavo...

The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin ␣ 6 ␤ 1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel ␣ 6 ␤ 1 binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support ␣ 6 ␤ 1 -and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with ␣ 6 ␤ 1 and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished ␣ 6 ␤ 1 -HSPGmediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain ␣ v ␤ 3 -mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in ␣ 6 ␤ 1 -HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for ␣ 6 ␤ 1 -HSPG-dependent CCN1 activities and provide a strategy to investigate these ␣ 6 ␤ 1 -HSPG-specific activities in vivo.