The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells

INTRODUCTION: The membrane cytoskeletal crosslinker ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by ezrin in breast cancer metastasis has not been clearly delineated. METHODS: We examined the role of ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type ezrin or a dominant-negative amino-terminal domain of ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model. RESULTS: Parental AC2M2 cells and cells overexpressing wild-type ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal ezrin caused strong membrane localization of cadherin, with increased cell–cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type ezrin. We further found that overexpression of amino-terminal ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system. CONCLUSION: Our findings indicate that ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis

C(naphthyl)-H Bond Activation by Rhodium: Isolation, Characterization and TD-DFT Study of the Cyclometallates

The C1(naphthyl)-H, C2(naphthyl)-H, C3(naphthyl)-H and C8(naphthyl)-H bonds of the naphthyl group present in a group of naphthylazo-2\u27-hydroxyarenes (H 2L) have been activated by [Rh(PPh 3) 3Cl] in a toluene medium. Here the cyclometallation is accompanied by metal centered oxidation [Rh(i)?Rh(iii)]. All the resulting cyclometallates [Rh(PPh 3) 2(L)Cl] (2-5) have been isolated in a pure form. The characterization of the cyclometallates [Rh(PPh 3) 2(L)Cl] have been done on the basis of spectral (IR, UV-vis, and FAB mass) data. The structures of the representative cyclometallates 2a, 3a, 4a, 4b and 5b have been determined by X-ray diffraction. In all the cyclometallates, rhodium(iii) is coordinated to naphthylazo-2\u27-hydroxyarenes via terdentate C(naphthyl), N(diazene), O(phenolato/ naphtholato) donor centers & one chloride ion in a plane along with two axial trans PPh 3 molecules. Intermolecular association in the solid state is observed due to C-H...p and p...p interactions. Compounds show an oxidative response within 0.93 to 1.11 V (vs. SCE) and a reductive response at ~ -1.0 V (vs. SCE). Both the responses are based on the coordinated diazene function and are irreversible in nature, indicating limited stability of the oxidized and reduced species. The electronic structures of selected cyclometallates have been calculated using a TD-DFT model and the simulated spectra are consistent with the observed spectra of those cyclometallates

Ezrin phosphorylation on tyrosine 477 regulates invasion and metastasis of breast cancer cells

Background The membrane cytoskeletal crosslinker, ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells. Our group previously showed that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in deregulation of cell-cell contacts and scattering of epithelial cells. In particular, ezrin phosphorylation on Y477 by Src is specific to ezrin within the ERM family, and is required for HGF-induced scattering of epithelial cells. We therefore sought to examine the role of Y477 phosphorylation in ezrin on tumor progression. Methods Using a highly metastatic mouse mammary carcinoma cell line (AC2M2), we tested the effect of over-expressing a non-phosphorylatable form of ezrin (Y477F) on invasive colony growth in 3-dimensional Matrigel cultures, and on local invasion and metastasis in an orthotopic engraftment model. Results AC2M2 cells over-expressing Y477F ezrin exhibited delayed migration in vitro, and cohesive round colonies in 3-dimensional Matrigel cultures, compared to control cells that formed invasive colonies with branching chains of cells and numerous actin-rich protrusions. Moreover, over-expression of Y477F ezrin inhibits local tumor invasion in vivo. Whereas orthotopically injected wild type AC2M2 tumor cells were found to infiltrate into the abdominal wall and visceral organs within three weeks, tumors expressing Y477F ezrin remained circumscribed, with little invasion into the surrounding stroma and abdominal wall. Additionally, Y477F ezrin reduces the number of lung metastatic lesions. Conclusions Our study implicates a role of Y477 ezrin, which is phosphorylated by Src, in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic/predictive marker or treatment target for invasive human breast cancer.Canadian Breast Cancer Research Alliance (BEE, 017374)Canadian Institutes of Health Research (BEE, 102644)Physicians Society Inc.Association pour le développement de la recherche sur le cancer (France

Estrogen and progesterone receptor levels in nonneoplastic breast epithelium of breast cancer cases versus benign breast biopsy controls

<p>Abstract</p> <p>Background</p> <p>Previous studies and biological mechanisms of carcinogenesis suggest that the steroid receptor content of benign breast epithelium may be related to breast cancer risk. The objective in this study was to compare the levels of estrogen receptor-α (ER) and progesterone receptor (PR) in nonneoplastic breast epithelium between breast cancer cases and biopsy controls.</p> <p>Methods</p> <p>Between 1995 and 1997 at two sites (Women's College Hospital in Toronto and Kingston General Hospital), 667 women who were scheduled for diagnostic excisional breast biopsies completed a questionnaire providing personal information and agreed to allow analysis of routinely resected tissue. Histological slides with nonneoplastic epithelium were available for 101 cancer cases and 200 biopsy controls in Toronto and for 105 cancer cases and 119 controls in Kingston. Nonneoplastic epithelium was examined with immunohistochemical assays to determine the percent of epithelial cells staining for ER and PR. Unconditional logistic regression was used to calculate odds ratios (OR) stratified by study site.</p> <p>Results</p> <p>The ER content of nonneoplastic tissue was higher in cases than biopsy controls in unadjusted analyses; after adjustment for age, however, a weak association remained in only one of the study sites. After adjustment for age, the PR content of nonneoplastic tissue was slightly lower in breast cancer cases than controls in one study site. Furthermore, this inverse association was confined to women with PR negative breast cancer in comparison to the controls. No interaction between ER and PR content of nonneoplastic tissue was observed in relation to the odds of having breast cancer.</p> <p>Conclusion</p> <p>The results of this study are consistent with only a slight indication of increased ER levels in nonneoplastic tissue in breast cancer cases relative to controls. This study contributes to the understanding of breast cancer by examining both ER and PR in nonneoplastic tissue. Limitations remain, however, such as the necessity of using as controls women with benign breast changes, difficulties in selecting the appropriate tissue for analysis, and tissue sampling concurrent to diagnosis.</p

Natural and Synthetic Lactones Possessing Antitumor Activities

Cancer is one of the leading causes of death globally, accounting for an estimated 8 million deaths each year. As a result, there have been urgent unmet medical needs to discover novel oncology drugs. Natural and synthetic lactones have a broad spectrum of biological uses including anti-tumor, anti-helminthic, anti-microbial, and anti-inflammatory activities. Particularly, several natural and synthetic lactones have emerged as anti-cancer agents over the past decades. In this review, we address natural and synthetic lactones focusing on their anti-tumor activities and synthetic routes. Moreover, we aim to highlight our journey towards chemical modification and biological evaluation of a resorcylic acid lactone, L-783277 (4). We anticipate that utilization of the natural and synthetic lactones as novel scaffolds would benefit the process of oncology drug discovery campaigns based on natural products

Alteration in MicroRNA Expression Governs the Nature and Timing of Cellular Fate Commitment

In the central nervous system, the expression level of transcriptional repressor Hes1 (hairy and enhancer of split-1) tightly controls the alternative cell fate commitment during differentiation as well as the time required for such cellular transitions. A microRNA, miR-9, that interacts with Hes1 in a mutually antagonistic manner, influences both the process of lineage specification and timing of differentiation significantly, but the impact of the miR-9 in guiding these events still remains poorly understood. Here, we proposed a stochastic mathematical model of the miR-9/Hes1 double-negative feedback interaction network that at the outset shows how alternative cell fate such as quiescence, progenitor, and neuronal states can be accomplished through fine-tuning the Hes1 dynamics by altering the expression level of miR-9. The model simulations further foretell a correlated variation of the period of oscillation of Hes1, and the time delay observed between <i>Hes1</i> mRNA and protein as the transcription rate of miR-9 increases during the neural progenitor state attainment. Importantly, the model simulations aided by the systematic sensitivity analysis predict that the timing of differentiation to the neuronal state crucially depends on the negative regulators (miR-9 and Hes6) of the Hes1. Our results indicate that miR-9/Hes1 interaction network can be effectively exploited for an efficient and well-timed neuronal transformation

Various gene regulatory networks under consideration.

<p><b>(A)</b> Positive feedback with additional negative feedback motif, where X (total protein) activates the synthesis of another protein Y_P which negatively regulates X population level. <b>(B)</b> Double positive feedback motifs, where X (total protein) activates the synthesis of another protein Y_P which positively regulates the synthesis of X. In all the cases X autoregulates its own synthesis positively. Here M_P denotes mRNA of P, Y_P denotes another protein and Y_M is the mRNA of protein Y_P. Detailed mechanistic models are given in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136668#pone.0136668.s006" target="_blank">S6 Fig</a>).</p

Are Quasi-Steady-State Approximated Models Suitable for Quantifying Intrinsic Noise Accurately?

<div><p>Large gene regulatory networks (GRN) are often modeled with quasi-steady-state approximation (QSSA) to reduce the huge computational time required for intrinsic noise quantification using Gillespie stochastic simulation algorithm (SSA). However, the question still remains whether the stochastic QSSA model measures the intrinsic noise as accurately as the SSA performed for a detailed mechanistic model or not? To address this issue, we have constructed mechanistic and QSSA models for few frequently observed GRNs exhibiting switching behavior and performed stochastic simulations with them. Our results strongly suggest that the performance of a stochastic QSSA model in comparison to SSA performed for a mechanistic model critically relies on the absolute values of the mRNA and protein half-lives involved in the corresponding GRN. The extent of accuracy level achieved by the stochastic QSSA model calculations will depend on the level of bursting frequency generated due to the absolute value of the half-life of either mRNA or protein or for both the species. For the GRNs considered, the stochastic QSSA quantifies the intrinsic noise at the protein level with greater accuracy and for larger combinations of half-life values of mRNA and protein, whereas in case of mRNA the satisfactory accuracy level can only be reached for limited combinations of absolute values of half-lives. Further, we have clearly demonstrated that the abundance levels of mRNA and protein hardly matter for such comparison between QSSA and mechanistic models. Based on our findings, we conclude that QSSA model can be a good choice for evaluating intrinsic noise for other GRNs as well, provided we make a rational choice based on experimental half-life values available in literature.</p></div