Variable Expression of PIK3R3 and PTEN in Ewing Sarcoma Impacts Oncogenic Phenotypes

Ewing Sarcoma is an aggressive malignancy of bone and soft tissue affecting children and young adults. Ewing Sarcoma is driven by EWS/Ets fusion oncoproteins, which cause widespread alterations in gene expression in the cell. Dysregulation of receptor tyrosine kinase signaling, particularly involving IGF-1R, also plays an important role in Ewing Sarcoma pathogenesis. However, the basis of this dysregulation, including the relative contribution of EWS/Ets-dependent and independent mechanisms, is not well understood. In the present study, we identify variable expression of two modifiers of PI3K signaling activity, PIK3R3 and PTEN, in Ewing Sarcoma, and examine the consequences of this on PI3K pathway regulation and oncogenic phenotypes. Our findings indicate that PIK3R3 plays a growth-promotional role in Ewing Sarcoma, but suggest that this role is not strictly dependent on regulation of PI3K pathway activity. We further show that expression of PTEN, a well-established, potent tumor suppressor, is lost in a subset of Ewing Sarcomas, and that this loss strongly correlates with high baseline PI3K pathway activity in cell lines. In support of functional importance of PTEN loss in Ewing Sarcoma, we show that re-introduction of PTEN into two different PTEN-negative Ewing Sarcoma cell lines results in downregulation of PI3K pathway activity, and sensitization to the IGF-1R small molecule inhibitor OSI-906. Our findings also suggest that PTEN levels may contribute to sensitivity of Ewing Sarcoma cells to the microtubule inhibitor vincristine, a relevant chemotherapeutic agent in this cancer. Our studies thus identify PIK3R3 and PTEN as modifiers of oncogenic phenotypes in Ewing Sarcoma, with potential clinical implications.     

Mechanism of Oncogenic Transformation by Rous Sarcoma Virus III. Role of Proviral DNA in Morphologic Conversion of Chicken Embryo Fibroblasts

Multiplication of Rous sarcoma virus and morphological conversion of chicken embryo fibroblasts are mediated by a DNA provirus. The role of the provirus in induction of morphological conversion has been shown by experiments of light inactivation of bromodeoxyuridine (BUdR)-sensitized proviral DNA. In the experiments reported here, inactivation of focus formation by BUdR and light could be obtained in cells in which the ability to produce virus has become resistant to X irradiation. This property is considered here to reflect the integrated state of the provirus. These experiments indicate that the role of proviral DNA extends beyond induction of morphological conversion and that an intact provirus is required for the maintenance of the transformed state. These experiments also indicate that no irreversible process leading to morphological conversion is initiated by a nonintegrated or by an integrated provirus.     

Quantification of the Heterogeneity of Prognostic Cellular Biomarkers in Ewing Sarcoma Using Automated Image and Random Survival Forest Analysis

Driven by genomic somatic variation, tumour tissues are typically heterogeneous, yet unbiased quantitative methods are rarely used to analyse heterogeneity at the protein level. Motivated by this problem, we developed automated image segmentation of images of multiple biomarkers in Ewing sarcoma to generate distributions of biomarkers between and within tumour cells. We further integrate high dimensional data with patient clinical outcomes utilising random survival forest (RSF) machine learning. Using material from cohorts of genetically diagnosed Ewing sarcoma with EWSR1 chromosomal translocations, confocal images of tissue microarrays were segmented with level sets and watershed algorithms. Each cell nucleus and cytoplasm were identified in relation to DAPI and CD99, respectively, and protein biomarkers (e.g. Ki67, pS6, Foxo3a, EGR1, MAPK) localised relative to nuclear and cytoplasmic regions of each cell in order to generate image feature distributions. The image distribution features were analysed with RSF in relation to known overall patient survival from three separate cohorts (185 informative cases). Variation in pre-analytical processing resulted in elimination of a high number of non-informative images that had poor DAPI localisation or biomarker preservation (67 cases, 36%). The distribution of image features for biomarkers in the remaining high quality material (118 cases, 10⁴ features per case) were analysed by RSF with feature selection, and performance assessed using internal cross-validation, rather than a separate validation cohort. A prognostic classifier for Ewing sarcoma with low cross-validation error rates (0.36) was comprised of multiple features, including the Ki67 proliferative marker and a sub-population of cells with low cytoplasmic/nuclear ratio of CD99. Through elimination of bias, the evaluation of high-dimensionality biomarker distribution within cell populations of a tumour using random forest analysis in quality controlled tumour material could be achieved. Such an automated and integrated methodology has potential application in the identification of prognostic classifiers based on tumour cell heterogeneity.     

SS18-SSX,the Oncogenic Fusion Protein in Synovial Sarcoma,Is a Cellular Context-Dependent Epigenetic Modifier

The prevalence and specificity of unique fusion oncogenes are high in a number of soft tissue sarcomas (STSs). The close relationship between fusion genes and clinicopathological features suggests that a correlation may exist between the function of fusion proteins and cellular context of the cell-of-origin of each tumor. However, most STSs are origin-unknown tumors and this issue has not yet been investigated in detail. In the present study, we examined the effects of the cellular context on the function of the synovial sarcoma (SS)-specific fusion protein, SS18-SSX, using human pluripotent stem cells (hPSCs) containing the drug-inducible SS18-SSX gene. We selected the neural crest cell (NCC) lineage for the first trial of this system, induced SS18-SSX at various differentiation stages from PSCs to NCC-derived mesenchymal stromal cells (MSCs), and compared its biological effects on each cell type. We found that the expression of FZD10, identified as an SS-specific gene, was induced by SS18-SSX at the PSC and NCC stages, but not at the MSC stage. This stage-specific induction of FZD10 correlated with stage-specific changes in histone marks associated with the FZD10 locus and also with the loss of the BAF47 protein, a member of the SWI/SNF chromatin-remodeling complex. Furthermore, the global gene expression profile of hPSC-derived NCCs was the closest to that of SS cell lines after the induction of SS18-SSX. These results clearly demonstrated that the cellular context is an important factor in the function of SS18-SSX as an epigenetic modifier.     

The Role of Cellular and Extracellular Matrix Adhesion Proteins in Organ Transplantation

The specific adhesion of cells to other cells or to particular tissue microenvirorvments is a basic function of cell migration and recognition, and underlines many biologic processes including embryogenesis, repair and immunity. Leukocytes express an array of surface receptors broadly known as “accessory adhesion molecules.” which mediate most cell -cell interactions, direct lymphocyte traffic between anatomical compartments, and facilitate cellular adhesion to the inflammation or alloantigenic sites (Springer 1990). In addition, adhesion molecules are involved in the process of antigen recognition, and may costimulate cell activation and transformation. These proteins are thought to affect the very early antigen independent events between host leukocytes and vascular endothelium. Because of these activities, the subject of adhesion molecules is gaining interest in the field of organ transplantation, in both conceptualization and development of novel therapeutic strategies (de Sousa et al. 1991, Kupiec-Weglinski et al. 1993a, Heemann et al. 1993).     

The Role of Cellular and Extracellular Matrix Adhesion Proteins in Organ Transplantation

The specific adhesion of cells to other cells or to particular tissue microenvirorvments is a basic function of cell migration and recognition, and underlines many biologic processes including embryogenesis, repair and immunity. Leukocytes express an array of surface receptors broadly known as “accessory adhesion molecules.” which mediate most cell -cell interactions, direct lymphocyte traffic between anatomical compartments, and facilitate cellular adhesion to the inflammation or alloantigenic sites (Springer 1990). In addition, adhesion molecules are involved in the process of antigen recognition, and may costimulate cell activation and transformation. These proteins are thought to affect the very early antigen independent events between host leukocytes and vascular endothelium. Because of these activities, the subject of adhesion molecules is gaining interest in the field of organ transplantation, in both conceptualization and development of novel therapeutic strategies (de Sousa et al. 1991, Kupiec-Weglinski et al. 1993a, Heemann et al. 1993).     

A Novel Group of Moraxella catarrhalis UspA Proteins Mediates Cellular Adhesion via CEACAMs and Vitronectin

Moraxella catarrhalis (Mx) is a common cause of otitis media and exacerbation of chronic obstructive pulmonary disease, an increasing worldwide problem. Surface proteins UspA1 and UspA2 of Mx bind to a number of human receptors and may function in pathogenesis. Genetic recombination events in the pathogen can generate hybrid proteins termed UspA2H. However, whether certain key functions (e.g. UspA1-specific CEACAM binding) can be exchanged between these adhesin families remains unknown. In this study, we have shown that Mx can incorporate the UspA1 CEACAM1-binding region not only into rare UspA1 proteins devoid of CEACAM-binding ability, but also into UspA2 which normally lack this capacity. Further, a screen of Mx isolates revealed the presence of novel UspA2 Variant proteins (UspA2V) in ∼14% of the CEACAM-binding population. We demonstrate that the expression of UspA2/2V with the CEACAM-binding domain enable Mx to bind both to cell surface CEACAMs and to integrins, the latter via vitronectin. Such properties of UspA2/2V have not been reported to date. The studies demonstrate that the UspA family is much more heterogeneous than previously believed and illustrate the in vivo potential for exchange of functional regions between UspA proteins which could convey novel adhesive functions whilst enhancing immune evasion.     

Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma

Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.Subject terms: Nuclear organization, Cancer     

Mechanism of Oncogenic Transformation by Rous Sarcoma Virus: I. Intracellular Inactivation of Cell-Transforming Ability of Rous Sarcoma Virus by 5-Bromodeoxyuridine and Light

Chick embryo fibroblasts brought into stationary phase of growth by maintenance in serum-free Eagle's MEM medium were infected with the Bryan strain of Rous sarcoma virus (B-RSV) and incubated for 18 hr in the presence of 5-bromo-deoxyuridine (BUdR). The cells were then allowed to resume growth and deoxyribonucleic acid (DNA) synthesis by addition of an enriched F12 medium containing serum and RSV antibody to prevent spread of viral infection. After 48 hr, the cultures were exposed for various periods to visible light, overlaid with solid culture medium, and observed for the appearance of foci of transformed cells. In cultures treated with BUdR at the time of infection, exposure to light resulted in a suppression of focus formation of from 50 to 90% in various experiments. Treatment with BUdR for 18 hr before infection or on the day after infection, followed by exposure to light, had no effect on focus formation. In cultures in which almost all cells were infected, treatment with BUdR followed by exposure to light did not result in cell death. This suggests that suppression of transformation is not due to selective killing of infected cells by this treatment but rather to the intracellular inactivation of the transforming ability of Rous sarcoma proviral DNA.     

Activation of Phosphatidylinositol 3-Kinase Is Sufficient for Cell Cycle Entry and Promotes Cellular Changes Characteristic of Oncogenic Transformation

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.     

Dissection of the C-Terminal Region of E1A Redefines the Roles of CtBP and Other Cellular Targets in Oncogenic Transformation

Human adenovirus E1A makes extensive connections with the cellular protein interaction network. By doing so, E1A can manipulate many cellular programs, including cell cycle progression. Through these reprogramming events, E1A functions as a growth-promoting oncogene and has been used extensively to investigate mechanisms contributing to oncogenesis. Nevertheless, it remains unclear how the C-terminal region of E1A contributes to oncogenic transformation. Although this region is required for transformation in cooperation with E1B, it paradoxically suppresses transformation in cooperation with activated Ras. Previous analysis has suggested that the interaction of E1A with CtBP plays a pivotal role in both activities. However, some C-terminal mutants of E1A retain CtBP binding and yet exhibit defects in transformation, suggesting that other targets of this region are also necessary. To explore the roles of these additional factors, we performed an extensive mutational analysis of the C terminus of E1A. We identified key residues that are specifically required for binding all known targets of the C terminus of E1A. We further tested each mutant for the ability to both localize to the nucleus and transform primary rat cells in cooperation with E1B-55K or Ras. Interaction of E1A with importin α3/Qip1, dual-specificity tyrosine-regulated kinase 1A (DYRK1A), HAN11, and CtBP influenced transformation with E1B-55K. Interestingly, the interaction of E1A with DYRK1A and HAN11 appeared to play a role in suppression of transformation by activated Ras whereas interaction with CtBP was not necessary. This unexpected result suggests a need for revision of current models and provides new insight into transformation by the C terminus of E1A.     

Novel Metabolic Transformation Pathway for Cyclic Imides in Blastobacter sp. Strain A17p-4

The metabolic transformation pathway for cyclic imides in microorganisms was studied in Blastobacter sp. strain A17p-4. This novel pathway involves, in turn, hydrolytic ring opening of a cyclic imide to yield a monoamidated dicarboxylate, hydrolytic deamidation of the monoamidated dicarboxylate to yield a dicarboxylate, and dicarboxylate transformation similar to that in the tricarboxylic acid cycle. The initial step is catalyzed by a novel enzyme, imidase. Imidase and subsequent enzymes involved in this metabolic pathway are induced by some cyclic imides, such as succinimide and glutarimide. Induced cells metabolize various cyclic imides.     

Elucidating the Crucial Role of Poly N-Acetylglucosamine from Staphylococcus aureus in Cellular Adhesion and Pathogenesis

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.     

CRF2 Signaling Is a Novel Regulator of Cellular Adhesion and Migration in Colorectal Cancer Cells

Stress has been proposed to be a tumor promoting factor through the secretion of specific neuromediators, such as Urocortin2 and 3 (Ucn2/3), however its role in colorectal cancer (CRC) remains elusive. We observed that Ucn2/3 and their receptor the Corticotropin Releasing Factor receptor 2 (CRF2) were up-regulated in high grade and poorly differentiated CRC. This suggests a role for CRF2 in the loss of cellular organization and tumor progression. Using HT-29 and SW620 cells, two CRC cell lines differing in their abilities to perform cell-cell contacts, we found that CRF2 signals through Src/ERK pathway to induce the alteration of cell-cell junctions and the shuttle of p120ctn and Kaiso in the nucleus. In HT-29 cells, this signaling pathway also leads to the remodeling of cell adhesion by i) the phosphorylation of Focal Adhesion Kinase and ii) a modification of actin cytoskeleton and focal adhesion complexes. These events stimulate cell migration and invasion. In conclusion, our findings indicate that CRF2 signaling controls cellular organization and may promote metastatic potential of human CRC cells through an epithelial-mesenchymal transition like process. This contributes to the comprehension of the tumor-promoting effects of stress molecules and designates Ucn2/3-CRF2 tandem as a target to prevent CRC progression and aggressiveness.     

A Novel Role for Autophagy in Neurodevelopment

We recently showed that Ambra1, a WD40-containing ~130 KDa protein, is a novel activating molecule in Beclin 1-regulated autophagy and plays a role in the development of the nervous system. Ambra1 binds to Beclin 1 and favors Beclin 1/Vps34 interaction. At variance with these factors, Ambra1 is highly conserved among vertebrates only, and its expression is mostly confined to the neuroepithelium during early neurogenesis. Ambra1 functional inactivation in mouse led to lethality in utero (starting from embryonic day 14.5), characterized by severe neural tube defects associated with autophagy impairment, unbalanced cell proliferation, accumulation of ubiquitinated proteins, and excessive apoptosis. We also demonstrated that hyperproliferation was the earliest detectable abnormality in the developing neuroepithelium, followed by a wave of caspase-dependent cell death. These findings provided in vivo evidence supporting the existence of a complex interplay between autophagy, cell proliferation and cell death during neural development in mammals. In this Addendum, we review our findings in the contexts of autophagy and neurodevelopment and consider some of the issues raised.

Addendum to:

Ambra1 Regulates Autophagy and the Development of the Nervous System

G.M. Fimia, A. Stoykova, A. Romagnoli, L. Giunta, S. Di Bartolomeo, R. Nardacci, M. Corazzari, C. Fuoco, A. Ucar, P. Schwartz, P. Gruss, M. Piacentini, K. Chowdhury and F. Cecconi

Nature 2007; In press     

Differential Roles of Akt, Rac, and Ral in R-Ras-Mediated Cellular Transformation, Adhesion, and Survival

Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.     

细丝蛋白A在细胞黏附和迁移中的作用

细胞迁移在发育、伤口愈合、炎症反应和肿瘤转移等多种病理生理过程中发挥重要作用。细丝蛋白A（filamin A,FlnA）是一种在各组织细胞中广泛表达的微丝结合蛋白,其表达异常导致细胞迁移功能障碍。该文回顾了相关的文献,首先介绍生理情况下细丝蛋白A的功能,接着介绍细丝蛋白A基因突变和表达异常导致的多种遗传性疾病及其与肿瘤转移的关系,突出细丝蛋白A对迁移的影响在这些疾病发病中的作用,最后深入探讨了细丝蛋白A影响细胞迁移和黏附的可能机制。     

Patterns in Space: Coordinating Adhesion and Actomyosin Contractility at E-cadherin Junctions

Abstract

Cadherin adhesion receptors are fundamental determinants of tissue organization in health and disease. Increasingly, we have come to appreciate that classical cadherins exert their biological actions through active cooperation with the contractile actin cytoskeleton. Rather than being passive resistors of detachment forces, cadherins can regulate the assembly and mechanics of the contractile apparatus itself. Moreover, coordinate spatial patterning of adhesion and contractility is emerging as a determinant of morphogenesis. Here we review recent developments in cadherins and actin cytoskeleton cooperativity, by focusing on E-cadherin adhesive patterning in the epithelia. Next, we discuss the underlying principles of cellular rearrangement during Drosophila germband extension and epithelial cell extrusion, as models of how planar and apical–lateral patterns of contractility organize tissue architecture.