Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.

Sphingosine-1-phosphate, a novel second messenger involved in cell growth regulation and signal transduction, affects growth and invasiveness of human breast cancer cells

This review will focus on the role of sphingosine and its phosphorylated derivative sphingosine-1-phosphate (SPP) in cell growth regulation and signal transduction. We will show that many of the effects attributed to sphingosine in quiescent Swiss 3T3 fibroblasts are mediated via its conversion to SPP. We propose that SPP has appropriate properties to function as an intracellular second messenger based on the following: it elicits diverse cellular responses; it is rapidly produced from sphingosine by a specific kinase and rapidly degraded by a specific lyase; its concentration is low in quiescent cells but increases rapidly and transiently in response to the growth factors, fetal calf serum (FCS) and platelet derived growth factor (PDGF); it releases Ca2+ from internal sources in an InsP3-independent manner; and finally, it may link sphingolipid signaling pathways to cellular ras-mediated signaling pathways by elevating phosphatidic acid levels. The effects of this novel second messenger on growth, differentiation and invasion of human breast cancer cells will be discussed. © 1994 Kluwer Academic Publishers.

Taxation determinations as de facto regulation : private equity exits in Australia

The Australian Taxation Office (AT)) attempted to challenge both the private equity fund reliance on double tax agreements and the assertion that profits were capital in nature in its dispute with private equity group TPG. Failure to resolve the dispute resulted in the ATO issuing two taxation determinations: TD 2010/20 which states that the general anti-avoidance provisions can apply to arrangements designed to alter the intended effect of Australia's international tax agreements net; and TD 2010/21 which states that the profits on the sale of shares in a company group acquired in a leveraged buyout is assessable income. The purpose of this article is to determine the effectiveness of the administrative rulings regime as a regulatory strategy. This article, by using the TPG-Myer scenario and subsequent tax determinations as a case study, collects qualitative data which is then analysed (and triangulated) using tonal and thematic analysis. Contemporaneous commentary of private equity stakeholders, tax professionals, and media observations are analysed and evaluated within a framework of responsive regulation and utilising the current ATO compliance model. Contrary to the stated purpose of the ATO rulings regime to alleviate complexities in Australian taxation law and provide certainty to taxpayers, and despite the de facto law status afforded these rulings, this study found that the majority of private equity stakeholders and their advisors perceived that greater uncertainty was created by the two determinations. Thus, this study found that in the context of private equity fund investors, a responsive regulation measure in the form of taxation determinations was not effective.

Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens

Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17β-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17β-estradiol was withdrawn. Stimulation of invasiveness with 17β-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line which lacks estrogen receptors were not affected by estrogen or antiestrogen in terms of proliferation or invasion. These studies indicate that the invasiveness of MCF-7 cells is regulated by antiestrogens through the estrogen receptor and may be mediated by collagenase IV activity. Antiestrogens which reduce both the proliferation and invasiveness of these cells may be interesting new candidates for clinical application.

Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells

Matrix Metalloproteinase-2 (MMP-2) is secreted as a zymogen, the activation of which has been associated with metastatic progression in human breast cancer (HBC). Concanavalin A (Con A) has been found to induce activation of MMP-2 in invasive HBC cell lines. Con A effects on the expression of mRNA for membrane-type matrix metalloproteinase (MT-MMP), a newly described cell surface-associated MMP, showed a close temporal correlation with induction of MMP-2 activation. It is surprising that MT-MMP mRNA is constitutively present in the uninduced MDA-MB-231 cell, despite a lack of MMP-2 activation. We have used actinomycin D to demonstrate a partial requirement for de novo gene expression in the induction of MMP-2 activation by Con A in MDA-MB-231 HBC cells. Furthermore, this transcriptional response to Con A appeared to require the continued presence of Con A for its manifestation. The nontranscriptional component of the Con A induction manifests rapidly, is quite substantial, and persists strongly despite actinomycin D abrogation of both constitutive and Con A-induced MT-MMP. Cycloheximide analyses suggest that protein synthesis may be involved in this rapid transcription-independent response. These studies suggest that Con A induces MMP-2-activation in part by up-regulation of MT-MMP expression but has a more complicated mode of action, involving additional nontranscriptional effects, which apparently require protein synthesis.

Expression of 67 kDa laminin receptor in human breast cancer cells : regulation by progestins

The level of 67 kDa laminin receptor (67LR) expression on breast and colon tumor cell surfaces was previously shown to be correlated with the capacity of tumor cells to metastasize. In the present work we investigate the effects of progestins and estrogen on the expression of 67LR in two sublines of the T47D human breast cancer cells: weakly tumorigenic, poorly invasive parental T47D cells and a highly tumorigenic, more invasive T47Dco subclone. Inmmunoblotting with an affinity purified antibody directed against a synthetic peptide recognizes the 67LR in these cells. 67LR expression in the T47Dco subclone is 5,5-fold higher than in their parental T47D cells. Treatment of T47D cells with 1 nM of the synthetic progestin R5020 results in a 4-fold increase in 67LR protein expression. Estrogen also induced 67LR expression, but only by 1.5-fold. The progestin-stimulated expression of the 67LR correlates with a 4.3-fold increase in attachment of T47D cells to laminin. A monoclonal antibody, mAb 13, directed against β1 integrin, completely blocks the attachment of T47D cells to fibronectin, only partially inhibits the attachment of T47D cells to laminin, and appears not to affect the progestin-stimulated laminin attachment of T47D cells. A new antiprogestin, ZK 112.993, significantly inhibits both progestin-stimulated 67LR expression and the increased attachment to laminin. These results suggest a possible role for progestin in mediating one of the multiple events thought to be important in metastasis of steroid receptor positive human breast cancer cells.

Towards a global framework for advertising self regulation

One of the forces which has indelibly shaped marketing is the internet. It has not only changed the way we communicate, but our marketing practices and our advertising self-regulation process (Kerr, Mortimer, Dickinson and Waller 2012). This special session seeks to build a new global framework to regulate advertising activity in this uncharted online environment. It looks back to how advertising has been traditionally self-regulated and looks forward to identify the key issues for marketers, consumers, regulators and the media. This special session explores and reinforces the fundamental purpose of the conference, as well as addressing the urgent needs of marketers, consumers and regulators.

MT1-MMP-dependent and -independent regulation of gelatinase A activation in long-term, ascorbate-treated fibroblast cultures: Regulation by fibrillar collagen

Human skin fibroblasts were cultured long-term in the presence of ascorbic acid to allow formation of a three-dimensional collagen matrix, and the effects of this on activation of secreted matrix metalloproteinase-2 (MMP-2) were examined. Accumulation of collagen over time correlated with increased levels of both mature MMP-2 and cell-associated membrane type 1-MMP (MT1-MMP), and subsequently increased mRNA levels for MT1-MMP, providing temporal resolution of the "nontranscriptional" and "transcriptional" effects of collagen on MT-1MMP functionality. MMP-2 activation by these cultures was blocked by inhibitors of prolyl-4-hydroxylase, or when fibroblasts derived from the collagen α1(I) gene-deficient Mov-13 mouse were used. MMP-2 activation by the Mov-13 fibroblasts was rescued by transfection of a full-length α1(I) collagen cDNA, and to our surprise, also by transfection with an α1(I) collagen cDNA carrying a mutation at the C-proteinase cleavage, which almost abrogated fibrillogenesis. Although studies with ascorbate-cultured MT1-MMP-/- fibroblasts showed that MT1-MMP played a significant role in the collagen-induced MMP-2 activation, a residual MT1-MMP-independent activation of MMP-2 was seen which resembled the level of MMP-2 activation persisting when wild-type fibroblasts were cultured in the presence of both ascorbic acid and MMP inhibitors. We were also unable to block this residual activation with inhibitors specific for serinyl, aspartyl, or cysteinyl enzymes.

MTI-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression

Membrane type 1 metalloprotease (MT1-MMP) is a transmembrane metalloprotease that plays a major role in the extracellular matrix remodeling, directly by degrading several of its components and indirectly by activating pro-MMP2. We investigated the effects of MT1-MMP overexpression on in vitro and in vivo properties of human breast adenocarcinoma MCF7 cells, which do not express MT1-MMP or MMP-2. MT1-MMP and MMP-2 cDNAs were either transfected alone or cotransfected. All clones overexpressing MT1-MMP 1) were able to activate endogenous or exogenous pro-MMP-2, 2) displayed an enhanced in vitro invasiveness through matrigel-coated filters independent of MMP-2 transfection, 3) induced the rapid development of highly vascularized tumors when injected subcutanously in nude mice, and 4) promoted blood vessels sprouting in the rat aortic ring assay. These effects were observed in all clones overexpressing MT1-MMP regardless of MMP-2 expression levels, suggesting that the production of MMP-2 by tumor cells themselves does not play a critical role in these events. The angiogenic phenotype of MT1-MMP-producing cells was associated with an up-regulation of VEGF expression. These results emphasize the importance of MT1-MMP during tumor angiogenesis and open new opportunities for the development of antiangiogenic strategies combining inhibitors of MT1-MMP and VEGF antagonists. - Sounni, N. E., Devy, L., Hajitou, A., Frankenne, F., Munaut, C., Gilles, C., Deroanne, C., Thompson, E. W., Foidart, J. M., Noel, A. MT1-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression.

Regulation of breast cancer cells by hormones and growth factors : effects on proliferation and basement membrane invasiveness

The current understanding of the regulation of breast cancer cell proliferation and invasiveness by hormones and growth factors is reviewed. It has been shown that polypeptide growth factors are involved in hormone-independent breast cancer, and are sometimes oestrogen-regulated in hormone-responsive models. Basement-membrane invasiveness, relating to the metastatic potential of these cells, is also stimulated by oestrogen in hormone-dependent models, elevated in hormone-independent models, and is growth factor sensitive. Further understanding of the differential effects of growth factors on breast cancer cell proliferation and invasiveness should facilitate better therapeutic exploitation of regulation at this level.

Regulation of proliferation, invasion and growth factor synthesis in breast cancer by steroids

Endogenous ovarian estrogens and progestins appear to play a critical role in the development and progression of breast cancer. Local productions of growth factors probably also contribute to malignant proliferation, while production and activation of collagenolytic enzymes may be equally critical for local invasive processes. The current review focusses on characterization of growth factor-receptor systems operant in normal and malignant breast epithelium. In addition, the determinants of local invasion are reviewed: attachment, modality, and proteose secretion. Finally, data are discussed concerning the regulation of both proliferation and invasion by hormones and antihormonal agents in hormone-dependent breast cancer. The results suggest new potential pharmacologic targets to explore to suppress onset and progression of breast cancer.

Automatic inference of driving task demand from visual cues of emotion and attention

Sensing the mental, physical and emotional demand of a driving task is of primary importance in road safety research and for effectively designing in-vehicle information systems (IVIS). Particularly, the need of cars capable of sensing and reacting to the emotional state of the driver has been repeatedly advocated in the literature. Algorithms and sensors to identify patterns of human behavior, such as gestures, speech, eye gaze and facial expression, are becoming available by using low cost hardware: This paper presents a new system which uses surrogate measures such as facial expression (emotion) and head pose and movements (intention) to infer task difficulty in a driving situation. 11 drivers were recruited and observed in a simulated driving task that involved several pre-programmed events aimed at eliciting emotive reactions, such as being stuck behind slower vehicles, intersections and roundabouts, and potentially dangerous situations. The resulting system, combining face expressions and head pose classification, is capable of recognizing dangerous events (such as crashes and near misses) and stressful situations (e.g. intersections and way giving) that occur during the simulated drive.

Methods for sociological inquiry on emotion in educational settings

Sociological approaches to inquiry on emotion in educational settings are growing. Despite a long tradition of research and theory in disciplines such as psychology and sociology, the methods and approaches for naturalistic investigation of emotion are in a developmental phase in educational settings. In this article, recent empirical studies on emotion in educational contexts are canvassed. The discussion focuses on the use of multiple methods within research conducted in high school and university classrooms highlighting recent methodological progress. The methods discussed include facial expression analysis, verbal and non-verbal conduct, and self-report methods. Analyses drawn from different studies, informed by perspectives from microsociology, highlight the strengths and limitations of any one method. The power and limitations of multi-method approaches is discussed.