Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

10.1038/cddis.2011.37Cell Death and Disease25

Triggering necroptosis in cisplatin and IAP antagonist-resistant ovarian carcinoma.

Ovarian cancer patients are typically treated with carboplatin and paclitaxel, but suffer a high rate of relapse with recalcitrant disease. This challenge has fostered the development of novel approaches to treatment, including antagonists of the 'inhibitor of apoptosis proteins' (IAPs), also called SMAC mimetics, as apoptosis-inducing agents whose action is opposed by caspase inhibitors. Surprisingly, IAP antagonist plus caspase inhibitor (IZ) treatment selectively induced a tumor necrosis factor-α (TNFα)-dependent death among several apoptosis-resistant cell lines and patient xenografts. The induction of necroptosis was common in ovarian cancer, with expression of catalytically active receptor-interacting protein kinase-3 (RIPK3) necessary for death, and in fact sufficient to compromise survival of RIPK3-negative, necroptosis-resistant ovarian cancer cells. The formation of a necrosome-like complex with a second critical effector, receptor-interacting serine-threonine kinase-1 (RIPK1), was observed. RIPK1, RIPK3 and TNFα were required for the induction of death, as agents that inhibit the function of any of these targets prevented cell death. Abundant RIPK3 transcript is common in serous ovarian cancers, suggesting that further evaluation and targeting of this RIPK3-dependent pathway may be of clinical benefit

TIFA, an inflammatory signaling adaptor, is tumor suppressive for liver cancer.

TIFA (TNF receptor associated factor (TRAF)-interacting protein with a Forkhead-associated (FHA) domain), also called T2BP, was first identified using a yeast two-hybrid screening. TIFA contains a FHA domain, which directly binds phosphothreonine and phosphoserine, and a consensus TRAF6-binding motif. TIFA-mediated oligomerization and poly-ubiquitinylation of TRAF6 mediates signaling downstream of the Tumor necrosis factor alpha receptor 1 (TNFaR-I) and interleukin-1/Toll-like receptor 4 (TLR4) pathways. Examining TIFA expression in hepatocellular carcinoma (HCC) tissues microarrays, we noted marked decreases TIFA reactivity in tumor versus control samples. In agreement, we found that HCC cell lines show reduced TIFA expression levels versus normal liver controls. Reconstituting TIFA expression in HCC cell lines promoted two independent apoptosis signaling pathways: the induction of p53 and cell cycle arrest, and the activation of caspase-8 and caspase-3. In contrast, the expression of a non-oligomerizing mutant of TIFA impacted cells minimally, and suppression of TIFA expression protected cells from apoptosis. Mice bearing TIFA overexpression hepatocellular xenografts develop smaller tumors versus TIFA mutant tumors; terminal deoxynucleotidyl transferase dUTP nick end labeling staining demonstrates increased cell apoptosis, and decreased proliferation, reflecting cell cycle arrest. Interestingly, p53 has a greater role in decreased proliferation than cell death, as it appeared dispensable for TIFA-induced cell killing. The findings demonstrate a novel suppressive role of TIFA in HCC progression via promotion of cell death independent of p53

Caspase-8 association with the focal adhesion complex promotes tumor cell migration and metastasis

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors

The Relative Importance of Topography and RGD Ligand Density for Endothelial Cell Adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×102–6×1011 RGD/mm2. We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×105 RGD/mm2 on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×108 RGD/mm2 irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry

The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

The differentiation and senescence programs of metazoans play key roles in regulating normal development and preventing aberrant cell proliferation, such as cancer. These programs are intimately associated with both the mitotic and apoptotic pathways. Caspase-8 is an apical apoptotic initiator that has recently been appreciated to coordinate non-apoptotic roles in the cell. Most of these functions are attributed to the catalytic domain, however, the amino-terminal death effector domains (DED)s, which belong to the death domain superfamily of proteins, can also play key roles during development. Here we describe a novel role for caspase-8 DEDs in regulating cell differentiation and senescence. Caspase-8 DEDs accumulate during terminal differentiation and senescence of epithelial, endothelial and myeloid cells; genetic deletion or shRNA suppression of caspase-8 disrupts cell differentiation, while re-expression of DEDs rescues this phenotype. Among caspase-8 deficient neuroblastoma cells, DED expression attenuated tumor growth in vivo and proliferation in vitro via disruption of mitosis and cytokinesis, resulting in upregulation of p53 and induction of differentiation markers. These events occur independent of caspase-8 catalytic activity, but require a critical lysine (K156) in a microtubule-binding motif in the second DED domain. The results demonstrate a new function for the DEDs of caspase-8, and describe an unexpected mechanism that contributes to cell differentiation and senescence

Integrins α2β1 and α11β1 regulate the survival of mesenchymal stem cells on collagen I

Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors – α1β1, α2β1 and α11β1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2β1- or α11β1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I

Candidate pathways and genes for prostate cancer: a meta-analysis of gene expression data

<p>Abstract</p> <p>Backgound</p> <p>The genetic mechanisms of prostate tumorigenesis remain poorly understood, but with the advent of gene expression array capabilities, we can now produce a large amount of data that can be used to explore the molecular and genetic mechanisms of prostate tumorigenesis.</p> <p>Methods</p> <p>We conducted a meta-analysis of gene expression data from 18 gene array datasets targeting transition from normal to localized prostate cancer and from localized to metastatic prostate cancer. We functionally annotated the top 500 differentially expressed genes and identified several candidate pathways associated with prostate tumorigeneses.</p> <p>Results</p> <p>We found the top differentially expressed genes to be clustered in pathways involving integrin-based cell adhesion: integrin signaling, the actin cytoskeleton, cell death, and cell motility pathways. We also found integrins themselves to be downregulated in the transition from normal prostate tissue to primary localized prostate cancer. Based on the results of this study, we developed a collagen hypothesis of prostate tumorigenesis. According to this hypothesis, the initiating event in prostate tumorigenesis is the age-related decrease in the expression of collagen genes and other genes encoding integrin ligands. This concomitant depletion of integrin ligands leads to the accumulation of ligandless integrin and activation of integrin-associated cell death. To escape integrin-associated death, cells suppress the expression of integrins, which in turn alters the actin cytoskeleton, elevates cell motility and proliferation, and disorganizes prostate histology, contributing to the histologic progression of prostate cancer and its increased metastasizing potential.</p> <p>Conclusion</p> <p>The results of this study suggest that prostate tumor progression is associated with the suppression of integrin-based cell adhesion. Suppression of integrin expression driven by integrin-mediated cell death leads to increased cell proliferation and motility and increased tumor malignancy.</p

Vascular tube formation on matrix metalloproteinase-1-damaged collagen

Connective tissue damage and angiogenesis are both important features of tumour growth and invasion. Here, we show that endothelial cells maintained on a three-dimensional lattice of intact polymerised collagen formed a monolayer of cells with a cobblestone morphology. When the collagen was exposed to organ culture fluid from human basal cell tumours of the skin (containing a high level of active matrix metalloproteinase-1 (MMP-1)), degradation of the collagen matrix occurred. The major degradation products were the $3over 4$- and $1over 4$-sized fragments known to result from the action of MMP-1 on type I collagen. When endothelial cells were maintained on the partially degraded collagen, the cells organised into a network of vascular tubes. Pretreatment of the organ culture fluid with either tissue inhibitor of metalloproteinase-1 (TIMP-1) or neutralising antibody to MMP-1 prevented degradation of the collagen lattice and concomitantly inhibited endothelial cell organisation into the vascular network. Purified (activated) MMP-1 duplicated the effects of skin organ culture fluid, but other enzymes including MMP-9 (gelatinase B), elastase or trypsin failed to produce measurable fragments from intact collagen and also failed to promote vascular tube formation. Together, these studies suggest that damage to the collagenous matrix is itself an important inducer of new vessel formation

Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.

A considerable body of research indicates that mammary gland branching morphogenesis is dependent, in part, on the extracellular matrix (ECM), ECM-receptors, such as integrins and other ECM receptors, and ECM-degrading enzymes, including matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). There is some evidence that these ECM cues affect one or more of the following processes: cell survival, polarity, proliferation, differentiation, adhesion, and migration. Both three-dimensional culture models and genetic manipulations of the mouse mammary gland have been used to study the signaling pathways that affect these processes. However, the precise mechanisms of ECM-directed mammary morphogenesis are not well understood. Mammary morphogenesis involves epithelial 'invasion' of adipose tissue, a process akin to invasion by breast cancer cells, although the former is a highly regulated developmental process. How these morphogenic pathways are integrated in the normal gland and how they become dysregulated and subverted in the progression of breast cancer also remain largely unanswered questions