Alter Cell Behavior Research Articles

Selective drug-free cancer apoptosis by three-dimensional self-targeting magnetic nickel oxide nanomatrix.

To develop a drug-free strategy addressing limitations of current cancer therapy. A 3D self-assembled magnetic nickel oxide (NiO) nanomatrix is synthesized using femtosecond pulsed laser to mimic extracellular matrix. The tunable laser pulse-interaction time and repetition rate aided in generating programmable NiO nanomatrix chemistry. The nanomatrix mimicked extracellular matrix in physical configuration and properties presenting favorable cues to cancerous HeLa cell and fibroblast cell adhesion and proliferation without cytotoxicity. The 3D nanomatrix structure altered HeLa cell behavior and induced apoptosis cancer apoptosis with an evidence of increased endocytosis when compared with fibroblast cells. The results demonstrate the availability of new potential avenues for magnetic drug-free cancer therapeutics.

Neonatal Micro-RNA Profile Determines Endothelial Function in Offspring of Hypertensive Pregnancies.

Offspring of hypertensive pregnancies are at increased risk of developing hypertension in adulthood. In the neonatal period they display endothelial cell dysfunction and altered microvascular development. MicroRNAs, as important endothelial cellular regulators, may play a role in this early endothelial dysfunction. Therefore we identified differential microRNA patterns in endothelial cells from offspring of hypertensive pregnancies and determined their role in postnatal vascular cell function. Studies were performed on human umbilical vein endothelial cell (HUVECs) samples from 57 pregnancies. Unbiased RNA-sequencing identified 30 endothelial-related microRNAs differentially expressed in HUVECs from hypertensive compared to normotensive pregnancies. Quantitative reverse transcription PCR (RT-qPCR) confirmed a significant higher expression level of the top candidate, miR-146a. Combined miR-146a targeted gene expression and pathway analysis revealed significant alterations in genes involved in inflammation, angiogenesis and immune response in the same HUVECs. Elevated miR-146a expression level at birth identified cells with reduced ability for in vitro vascular tube formation, which was rescued by miR-146a inhibition. In contrast, miR-146a overexpression significantly reduced vascular tube formation in HUVECs from normotensive pregnancies. Finally, we confirmed that mir146a levels at birth predicted in vivo microvascular development during the first three postnatal months. Offspring of hypertensive pregnancy have a distinct endothelial regulatory microRNA profile at birth, which is related to altered endothelial cell behaviour, and predicts patterns of microvascular development during the first three months of life. Modification of this microRNA profile in vitro can restore impaired vascular cell function.

Abstract 4037: Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells

Abstract Evidence suggests that growth of cancer stem cells (CSCs) drives intratumor heterogeneity, a major clinical problem that results in drug resistance, recurrence of tumors, and metastatic progression of breast cancer. Therapeutic strategies to eliminate CSCs require identification of signaling pathways that facilitate the emergence and/or expansion of CSCs. These signaling pathways are implicated in altering cell behavior, as well as promoting the expression of genes involved in epithelial-mesenchymal transition (EMT), cell migration and invasion. Previous work demonstrated that a cooperative STAT3/SMAD3 gene transcription program increased the expression of Hyaluronan Synthase 2 (HAS2), an enzyme that produces Hyaluronic Acid (HA) which serves as a CD44 ligand and facilitates EMT. We hypothesize that STAT3 is a critical regulator of HA synthesis and signaling which enhances EMT and stemness of human mammary epithelial cells, driving tumor progression. We tested this hypothesis in a TGFβ triggered model of EMT. Immortalized human mammary epithelial cells (HMLE) expressing either a control vector (pWZL-GFP) or a vector containing TGFβ1 (pWZL-TGFβ1-GFP) were characterized with respect to STAT3 signaling, HA synthesis, and mammosphere formation. Our data revealed that HMLE-pWZL-TGFβ1-GFP cells have significantly higher expression of active STAT3 (Y705) compared to control cells, but total STAT3 gene and protein expression were similar in the two cell lines. HMLE-pWZL-TGFβ1-GFP cells exhibited a significant increase in expression of HAS2 and HAS2-AS1, a long non-coding RNA that enhances the transcription of HAS2. Preliminary results show that overexpression of TGFβ1 increased HA secretion in media compared to control cells. HMLE-pWZL-TGFβ1-GFP cells demonstrated significantly higher mammosphere forming ability compared to control cells, suggesting that TGFβ increases the percentage of stem cells in the population. Initial results demonstrated changes in mammosphere size and morphology of HMLE-pWZL-TGFβ1-GFP cells treated with the HA synthesis inhibitor 4-Methylumbelliferone (4MU). Together, these results suggest STAT3 and TGFβ1 co-regulate HAS2 expression and HA production in a model of EMT supporting the use of the HMLE-pWZL cell series as a promising model to study STAT3 regulation of the HA-CD44 signaling pathway. Citation Format: Brenda G. Trevizo-Barresi, JoEllen Welsh. Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4037.

Nucleolar Stress: hallmarks, sensing mechanism and diseases.

The nucleolus is a prominent subnuclear compartment, where ribosome biosynthesis takes place. Recently, the nucleolus has gained attention for its novel role in the regulation of cellular stress. Nucleolar stress is emerging as a new concept, which is characterized by diverse cellular insult-induced abnormalities in nucleolar structure and function, ultimately leading to activation of p53 or other stress signaling pathways and alterations in cell behavior. Despite a number of comprehensive reviews on this concept, straightforward and clear-cut way criteria for a nucleolar stress state, regarding the factors that elicit this state, the morphological and functional alterations as well as the rationale for p53 activation are still missing. Based on literature of the past two decades, we herein summarize the evolution of the concept and provide hallmarks of nucleolar stress. Along with updated information and thorough discussion of existing confusions in the field, we pay particular attention to the current understanding of the sensing mechanisms, i.e., how stress is integrated by p53. In addition, we propose our own emphasis regarding the role of nucleolar protein NPM1 in the hallmarks of nucleolar stress and sensing mechanisms. Finally, the links of nucleolar stress to human diseases are briefly and selectively introduced.

Cell Mimicking Microparticles Influence the Organization, Growth, and Mechanophenotype of Stem Cell Spheroids.

Substrate stiffness is known to alter cell behavior and drive stem cell differentiation, though most research in this area has been restricted to traditional, two-dimensional culture systems rather than more physiologically relevant, three-dimensional (3D) platforms. In this study, we utilized polymer-based, cell mimicking microparticles (CMMPs) to deliver distinct, stable mechanical cues to human adipose derived stem cells in 3D spheroid culture to examine changes in adipogenic differentiation response and mechanophenotype. After 21days of adipogenic induction, spheroids containing CMMPs (composite spheroids) stiffened in accordance with CMMP elasticity such that spheroids containing the stiffest, ~ 10kPa, CMMPs were over 27% stiffer than those incorporating the most compliant, ~ 0.25kPa CMMPs. Adipogenically induced, cell-only spheroids were over 180% larger and 50% more compliant than matched controls. Interestingly, composite spheroids cultured without chemical induction factors dissociated when presented with CMMPs stiffer than ~ 1kPa, while adipogenic induction factors mitigated this behavior. Gene expression for PPARG and FABP4 were upregulated more than 45-fold in adipogenically induced samples compared to controls but were unaffected by CMMP elasticity, attributed to insufficient cell-CMMP contacts throughout the composite spheroid. In summary, mechanically tuned CMMPs influenced whole-spheroid mechanophenotype and stability but minimally affected differentiation response.

CRISPR‐Cas9 knock out of gravin variant 1 impairs in vitro angiogenic sprouting

Angiogenesis is a highly regulated physiological process that is controlled by several signaling pathways including Src dependent and cAMP dependent pathways. Currently, the mechanisms underlying the coordination and integration of these signaling pathways are unknown, but based on several knockdown studies, we postulate that gravin (AKAP12), which is expressed as two variants in endothelial cells, may serve as a scaffold to coordinate Src and cAMP dependent signaling during angiogenesis. To investigate this hypothesis, we are using gene editing and protein expression approaches to identify the role of each gravin variant and the role of the Src and PKA binding domains in gravin in angiogenesis. Here, we report on the effect of knocking out gravin variant 1 using CRISPR‐Cas9 on angiogenic sprouting in a 3D‐collagen gel model. Immortalized human umbilical vein endothelial cells (HUVEC‐TERT2) were transfected with a CRISPR‐Cas9 construct in which Cas9 was targeted to exon 3. An RFLP assay designed to identify changes in an HpyCH4III site at the targeted double stranded break point identified a positive clone which lacked the restriction site. Subsequent sequencing revealed a single base deletion at the targeted site in exon 3 of the AKAP12 gene. Reverse transcriptase PCR showed a partial loss of variant 1 mRNA and sequencing of the RT‐PCR product confirmed the single base deletion. Western blotting demonstrated loss of variant 1 in the CRISPR‐Cas9 modified cells while immunofluorescence microscopy using an anti‐gravin antibody that recognized the C‐terminal region of both variants revealed approximately a 50% decrease in fluorescence intensity in CRISPR‐Cas9 treated cells. This CRISPR modified cell line, expressing only variant 2 of gravin, showed altered cell morphology and behavior compared to wild type cells. When cultured on collagen gels, variant 1 knockout cultures showed significantly fewer sprouts than wild type cultures. Confocal microscopy revealed that fewer variant 1 knockout cells migrated into collagen gels, resulting in shorter sprouts with fewer branches, and that these cells displayed fewer filopodia than wild type cells. These results provide evidence that variant 1 of gravin plays a role in invasive cell migration and angiogenic sprouting, which are essential cellular events that regulate angiogenesis. Future studies will investigate the mechanism by which variant 1 contributes to angiogenic sprouting and the role that each variant of gravin plays in Src and cAMP dependent signaling in endothelial cell migration, adhesion and angiogenic sprouting.Support or Funding InformationSupported by NIH grants P30GM103329, P20GM103442 and P20GM113123This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA‑142‑5p modulates breast cancer cell proliferation and apoptosis by targeting phosphatase and tensin homolog.

A total of 60 breast cancer (BC) tissues and adjacent healthy tissues from patients who underwent surgery in Renmin Hospital of Wuhan University were collected for analysis in the present study. Results from reverse transcription-quantitative polymerase chain reaction (RT-qPCR) demonstrated that, compared with the adjacent healthy tissues, the expression levels of microRNA (miR)-142-5p were significantly elevated in BC tissues. Bioinformatics analysis was performed using TargetScan for the prediction of potential target sites that matched the seed region of miR-142-5p; phosphatase and tensin homolog (PTEN) exhibited the highest score and was selected for further analysis. Results of RT-qPCR analysis demonstrated that, compared with the adjacent healthy tissues, the mRNA expression levels of PTEN were significantly decreased in breast cancer tissues. miR-142-5p and PTEN expression levels were positively and negatively associated, respectively, with patient tumor size and metastasis. MDA-MB-231 cells were divided into three groups including the Control group, the miR-NC inhibitor group and the miR-142-5p inhibitor group. As for alterations in cell behavior, including cell viability and cell apoptosis, and protein expression levels, there were no significant differences between Control and miR-NC inhibitor groups. MTT assay results revealed that, compared with Control and miR-NC inhibitor groups, miR-142-5p inhibitor reduced MDA-MB-231 cell proliferation. Flow cytometric analysis demonstrated that, compared with Control and miR-NC inhibitor groups, miR-142-5p inhibitor treatment induced MDA-MB-231 cell apoptosis. Western blotting results demonstrated that, compared with Control and miR-NC inhibitor groups, miR-142-5p inhibitor treatment significantly increased the expression of PTEN, reduced the activation of phosphatidylinositol-4,5-bisphosphate 3-kinase/RACα serine/threonine-protein kinase signaling. Finally, PTEN was demonstrated to interact with miR-142-5p from the results of dual-luciferase reporter assay in the present study. The findings of the present study suggested that miR-142-5p may be a potential therapeutic target for the future investigations and insights for breast cancer.

Expansion of Airway Basal Cells and Generation of Polarized Epithelium.

Airway basal stem cells are the progenitor cells within the airway that exhibit the capacity to self-renew and give rise to multiple types of differentiated airway epithelial cells. This stem cell-derived epithelium displays organized architecture with functional attributes of the airway mucosa. A protocol has been developed to culture and expand human airway basal stem cells while preserving their stem cell properties and capacity for subsequent mucociliary differentiation. This achievement presents a previously unrealized opportunity to maintain a durable supply of progenitor cells derived from healthy donors to differentiate into human primary airway epithelium for cellular and molecular-based studies. Further, basal stem cells can be harvested from patients with a specific airway disease, such as cystic fibrosis, enabling investigation of potentially altered behavior of disease-specific cells in the appropriate context of the airway mucosa. Here we describe, in detail, a protocol for the serial expansion of airway basal stem cells to enable the generation of nearly unlimited airway basal cells that can be stored and readily available for subsequent culturing and differentiation. In addition, we describe culturing and differentiation of airway basal stem cells on permeable transwell filters at air-liquid interface to create functional mucociliary pseudostratified polarized airway epithelial mucosa.

Effect of Bovine Serum Albumin on Red Blood Cell Optical Anisotropy Probed Through the Optomechanical Response in an Optical Trap

The dynamics of trapped entities in an Optical Trap (OT) can yield information with regards to their viscoelastic response as well as optical anisotropy, if any. Detailed analysis of such dynamics correlated with parameters which affect the response can yield additional clues to the exact effect of these on the trapped entities. In this work, we illustrate this point by showing how the altered behavior of Red Blood Cells (RBC) treated with Bovine Serum Albumin (BSA) yields information about the nature of action of BSA, on which there is no current consensus in literature. We conclude from our studies that BSA treatment leads to a change in the birefringence of the RBCs, a conclusion arrived at from the altered optomechanical response of such cells in a linearly polarized Gaussian beam OT. Furthermore, we argue that the observed changes in cellular optical anisotropy may be thought of as due to changes in the curvature of the RBC membrane. We also note that BSA action could help mimic pathological conditions that result in an altered cell shape.

Substrate stiffness regulated migration and angiogenesis potential of A549 cells and HUVECs.

Tumor tissue tends to stiffen during solid tumor progression. Substrate stiffness is known to alter cell behaviors, such as proliferation and migration, during which angiogenesis is requisite. Mono- and co-culture systems of lung cancer cell line A549 and human umbilical vein endothelial cells (HUVECs), on polydimethylsiloxane substrates (PDMS) with varying stiffness, were used for investigating the effects of substrate stiffness on the migration and angiogenesis of lung cancer. The expressions of matrix metalloproteinases (MMPs) and angiogenesis-related growth factors were up-regulated with the increase of substrate stiffness, whereas that of tissue inhibitor of matrix metalloproteinase (TIMPs) were down-regulated with increasing substrate stiffness. Our data not only suggested that stiff substrate may promote the migration and angiogenesis capacities of lung cancer, but also suggested that therapeutically targeting lung tumor stiffness or response of ECs to lung tumor stiffness may help reduce migration and angiogenesis of lung tumor.

Differentially expressed genes: OCT-4, SOX2, STAT3, CDH1 and CDH2, in cultured mesenchymal stem cells challenged with serum of women with endometriosis.

Endometriosis is a common chronic gynecological disorder defined as the presence of ectopic functional endometrial tissues, outside uterine cavity, primarily on the pelvic peritoneum and the ovaries. Several studies revealed a correlation between aberrant stem-cell activity in the endometrium and endometriosis. Yet the molecular and cellular behaviors of mesnchymal stem cells in development of endometriosis are hampered by lack of invitro experiments. Our aim was to explore morphological and molecular changes associated with mesenchymal stem cells (MSCs) exposition to serum derived from women with severe endometriosis. Two cell cultures of MSCs isolated from endometrial tissues of two endometriosis-free women. Each cell culture was treated individually with the serum of women with endometriosis (experimental group/n = 7), and serum of women without endometriosis (control group/ n = 4) for 14 days. Quantitative Real-Time PCR was performed later to reveal expression of OCT-4, CDH1 and CDH2, STAT3 and SOX2 genes. Morphologically, cells showed no significant changes. However from molecular point of view, we found increased expression in OCT-4, CDH1 and CDH2. For STAT3 and SOX2 we did not find a significant difference. This study shows that endometriosis serum induced molecular changes in human endometrial MSCs (EnMSCs) that might be related to altered cell behavior which may be a step in differentiation that may be completed invivo by other factors to complete the process of transition. Further researches are needed for optimization to reach differentiation.

The impact of transient heat transfer on tissue culture cell distribution

It is common practice to subculture adherent eukaryotic cells in multi-well plates at room temperature before 37 °C incubation for growth. Under these conditions, cell distribution was non-uniform with a higher density of cells near the edges of the wells. Non-uniform cell density can alter cell behaviour in numerous biological assays and can result in variability when using automated plate readers on intact cells. This study investigated the possibility that the non-uniform cell distribution was caused by temperature gradients in the growth medium and well walls. Cell density analyses revealed significantly greater cell densities near well walls. Temperature distribution was documented using infrared imaging and temperature-sensitive LCD films, and a transient heat transfer mathematical model was developed to characterise the system and compared to the cell density results. The model predicts that an initial less-than-1 °C temperature gradient is present in the well shortly after initiating 37 °C incubation, leading to preferential cell adhesion to the warmer edges of wells in the first ∼30 min of incubation. Techniques to remediate non-uniform cell distribution were evaluated, and a simple method proved effective to promote uniform cell densities across wells of 24-well plates.

Activating the unfolded protein response in osteocytes causes hyperostosis consistent with craniodiaphyseal dysplasia.

Bone remodeling is a balanced process between bone synthesis and degradation, maintaining homeostasis and a constant bone mass in adult life. Imbalance will lead to conditions such as osteoporosis or hyperostosis. Osteoblasts build bone, becoming embedded in bone matrix as mature osteocytes. Osteocytes have a role in sensing and translating mechanical loads into biochemical signals, regulating the differentiation and activity of osteoblasts residing at the bone surface through the secretion of Sclerostin (SOST), an inhibitor of WNT signaling. Excessive mechanical load can lead to activation of cellular stress responses altering cell behavior and differentiation. The unfolded protein response (UPR) is a shared pathway utilized by cells to cope with stress stimuli. We showed that in a transgenic mouse model, activation of the UPR in early differentiating osteocytes delays maturation, maintaining active bone synthesis. In addition, expression of SOST is delayed or suppressed; resulting in active WNT signaling and enhanced periosteal bone formation, and the combined outcome is generalized hyperostosis. A clear relationship between the activation of the unfolded protein response was established and the onset of hyperostosis that can be suppressed with a chemical chaperone, sodium 4-phenobutyrate (4-PBA). As the phenotype is highly consistent with craniodiaphyseal dysplasia (CDD; OMIM 122860), we propose activation of the UPR could be part of the disease mechanism for CDD patients as these patients are heterozygous for SOST mutations that impair protein folding and secretion. Thus, therapeutic agents ameliorating protein folding or the UPR can be considered as a potential therapeutic treatment.

Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells.

The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure. Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function, whereas its homologue GDE2 fails to attack uPAR. GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells, resulting in a less transformed phenotype, it slows tumor growth in a xenograft model and correlates with prolonged survival in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, furthermore, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior.

Cryopreservation of canine cardiosphere-derived cells: Implications for clinical application.

The clinical application of cardiosphere-derived cells (CDCs) to treat cardiac disease has gained increasing interest over the past decade. Recent clinical trials confirm their regenerative capabilities, although much remains to be elucidated about their basic biology. To develop this new treatment modality, in a cost effective and standardized workflow, necessitates the creation of cryopreserved cell lines to facilitate access for cardiac patients requiring urgent therapy. Cryopreservation may however lead to alterations in cell behavior and potency. The aim of this study was to investigate the effect of cryopreservation on canine CDCs. CDCs and mesenchymal stem cells (MSCs) isolated from five dogs were characterized. CDCs demonstrated a population doubling time that was unchanged by cryopreservation (fresh vs. cryopreserved; 57.13 ± 5.27 h vs. 48.94 ± 9.55 h, P = 0.71). This was slower than for MSCs (30.46 h, P < 0.05). The ability to form clones, self-renew, and commit to multiple lineages was unaffected by cryopreservation. Cryopreserved CDCs formed larger cardiospheres compared to fresh cells (P < 0.0001). Fresh CDCs showed a high proportion of CD105+ (89.0% ± 4.98) and CD44+ (99.68% ± 0.13) cells with varying proportions of CD90+ (23.36% ± 9.78), CD34+ (7.18% ± 4.03) and c-Kit+ (13.17% ± 8.67) cells. CD45+ (0.015% ± 0.005) and CD29+ (2.92% ± 2.46) populations were negligible. Increasing passage number of fresh CDCs correlated with an increase in the proportion of CD34+ and a decrease in CD90+ cells (P = 0.003 and 0.03, respectively). Cryopreserved CDCs displayed increased CD34+ (P < 0.001) and decreased CD90+ cells (P = 0.042) when compared to fresh cells. Overall, our study shows that cryopreservation of canine CDCs is feasible without altering their stem characteristics, thereby facilitating their utilization for clinical trials. © 2017 International Society for Advancement of Cytometry.

Estrogen receptor beta as epigenetic mediator of miR-10b and miR-145 in mammary cancer

Even though the role of estrogen receptor alpha (ERα) in the modulation of breast cancer cells' behavior is thoroughly studied, the biological functions of its isoform, ERβ, are less elucidated. The suppression of ERβ in the aggressive ERα-negative MDA-MB-231 breast cancer cells resulted in the inhibition of epithelial to mesenchymal transition (EMT) and major changes in the basic functional properties and expression levels of certain matrix components of breast cancer cells. This arrest in metastatic potential of breast cancer cells suggests the contribution of ERβ in the induction of a more aggressive phenotype in MDA-MB-231 breast cancer cells. The epigenetic alterations are responsible for the ability of the tumor cells to metastasize. Here, we report for the first time that the suppression of ERβ in MDA-MB-231 breast cancer cells leads to significant changes in the expression profiles of specific microRNAs, including miR-10b, miR-200b and miR-145. Growth of MCF-7 and MDA-MB-231 cells in estrogen-free medium has a diverse impact on miRNA expression and the behavior of these cells, suggesting the specific effect of estradiol on the miRNA expression profile depending on the ER status of breast cancer cells. Enhanced miR-10b expression or silencing of miR-145 clearly revealed that these microRNAs can regulate the functional properties, EMT program and the expression of major matrix components known to be implicated in breast cancer aggressiveness. Our data revealed that miR-10b is strongly implicated in the regulation of functional properties, EMT program and Erk1/2 signaling in shERβ MDA-MB-231 cells, thus affecting the extracellular matrix (ECM) composition, including syndecan-1, proteolytic behavior, especially MMP2, MMP7 and MMP9 expression and subsequently the aggressiveness of these cells. Accordingly, the inhibition of miR-145 expression significantly increased the aggressiveness of shERβ MDA-MB-231 cells and induced EMT. Moreover, miR-145 inhibition resulted in important changes in the gene and protein levels of ECM mediators, such as HER2 and several MMPs, whereas it significantly increased the phosphorylated levels of Erk1/2 kinases in these cells, suggesting the crucial role of miR-145 in this signaling pathway. These novel results suggest that the alterations in cell behavior and in ECM composition caused by the suppression of ERβ in MDA-MB-231 cells are closely related to certain epigenetic miRNA-induced alterations. Targeting the ERβ-regulated miR-10b and miR-145 is a promising tool for diagnosis and pharmaceutical targeting in breast cancer.

Cancer Hallmarks Analytics Tool (CHAT): a text mining approach to organize and evaluate scientific literature on cancer.

MotivationTo understand the molecular mechanisms involved in cancer development, significant efforts are being invested in cancer research. This has resulted in millions of scientific articles. An efficient and thorough review of the existing literature is crucially important to drive new research. This time-demanding task can be supported by emerging computational approaches based on text mining which offer a great opportunity to organize and retrieve the desired information efficiently from sizable databases. One way to organize existing knowledge on cancer is to utilize the widely accepted framework of the Hallmarks of Cancer. These hallmarks refer to the alterations in cell behaviour that characterize the cancer cell.ResultsWe created an extensive Hallmarks of Cancer taxonomy and developed automatic text mining methodology and a tool (CHAT) capable of retrieving and organizing millions of cancer-related references from PubMed into the taxonomy. The efficiency and accuracy of the tool was evaluated intrinsically as well as extrinsically by case studies. The correlations identified by the tool show that it offers a great potential to organize and correctly classify cancer-related literature. Furthermore, the tool can be useful, for example, in identifying hallmarks associated with extrinsic factors, biomarkers and therapeutics targets.Availability and implementationCHAT can be accessed at: http://chat.lionproject.net. The corpus of hallmark-annotated PubMed abstracts and the software are available at: http://chat.lionproject.net/aboutSupplementary information Supplementary data are available at Bioinformatics online.

Abstract 2969: Paracrine effect of the endothelium on prostate cancer cells

Abstract Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Androgen deprivation therapy (ADT) is the standard treatment for advanced or metastatic CaP. However, during ADT, CaP progresses from an androgen-sensitive (AS-CaP) to a more aggressive, and eventually lethal, castration-resistant (CRPC) phenotype. There is evidence to suggest that the prostate tumor mass is under tight control of endothelial microvasculature due to an increase in angiogenesis by tumor cells. Nevertheless, now there is evidence to support that this influence is not one-directional and that the endothelial cells secrete a large number of active substances (angiocrine factors), which may directly or indirectly influence tumor growth and progression. However, the direct impacts of the endothelium on prostate tumor progression or the molecular mechanisms that are involved in this communication remain unclear. Here we investigated the potential influence of endothelium-derived paracrine factors on prostate cancer biology and the role of connexins in these interactions, since connexins play a major role in cell-cell communication and form a bidirectional signaling pathway to assemble gap junctions and alter cell behaviors. We measured the effect of conditioned media (CM) obtained from a primary culture of human endothelial cells isolated from umbilical vein (HUVEC) on viability, proliferation, migration and invasion of CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) and in the metastatic potential by in vivo assays using co-injection of CaP cell with HUVEC or injection of CaP cells pre-incubated with CM from HUVEC in a zebrafish embryo model. Finally, we studied the expression and the role of connexins on this stimulation using pharmacological (GJIC inhibitors) approaches. All together, our results showed that CM from endothelial cell induces an increases in the viability and proliferation in all CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) but only increases migration of the CRPC cell lines (LNCaP-C4-2 and PC3). We also observed in our in vivo model that endothelial cells either through cell-cell interaction or by paracrine communication increases the metastatic ability of the CaP cells. Moreover, the increase in viability and migration of CaP cells observed with CM from endothelial cells was blocked using inhibitors of gap junctions. Real-time PCR analyses detected an up-regulation of Cx43 mRNA after exposition to CM from endothelial cells. Our data suggest that angiocrine communication between endothelial cells and CaP cells increases proliferation and migration of more aggressive CaP cells which could be important for the acquisition of the aggressive phenotype of the disease, and this interaction could be mediated by Cx43. Delineation of such critical players may culminate in identifying therapeutic targets or biomarkers to counteract CaP, especially advanced CaP. Citation Format: Verónica Torres-Estay, Patricia Fuenzalida, Catalina Ascencio, Carla Cembrano, Daniela Carreño, Néstor Corro, Viviana Montecinos, Gareth Owen, Xavier Figueroa, Julio Amigo, Juan Carlos Saéz, Alejandro Godoy. Paracrine effect of the endothelium on prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2017-2969

Effects of ErbB2 Overexpression on the Proteome and ErbB Ligand-specific Phosphosignaling in Mammary Luminal Epithelial Cells

Most breast cancers arise from luminal epithelial cells, and 25–30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1β treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis.

Neural Wiskott-Aldrich syndrome protein (nWASP) is implicated in human lung cancer invasion

Lung cancer is one of the most commonly diagnosed cancers with survival much lower in patients diagnosed with distal metastases. It is therefore imperative to identify pathways involved in lung cancer invasion and metastasis and to consider the therapeutic potential of agents that can interfere with these molecular pathways. This study examines nWASP expression in human lung cancer tissues and explores the effect of nWASP inhibition and knockdown on lung cancer cell behaviour.MethodsQPCR has been used to measure nWASP transcript expression in human lung cancer tissues. The effect of wiskostatin, an nWASP inhibitor, on A-549 and SK-MES-1 lung carcinoma cell growth, adhesion, migration and invasion was also examined using several in vitro functional assays, including ECIS, and immunofluorescence staining. The effect of nWASP knockdown using siRNA on particular behaviours of lung cancer cells was also examined.ResultsPatients with high levels of nWASP expression in tumour tissues have significantly lower survival rates. nWASP transcript levels were found to correlate with lymph node involvement (p = 0.042). nWASP inhibition and knockdown was shown to significantly impair lung cancer cell growth. nWASP inhibition also affected other cell behaviours, in SK-MES-1 invasion and A-549 cell motility, adhesion and migration. Paxillin and FAK activity are reduced in lung cancer cell lines following wiskostatin and nWASP knockdown as shown by immunofluorescence and western blot.ConclusionsThese findings highlight nWASP as an important potential therapeutic target in lung cancer invasion and demonstrate that inhibiting nWASP activity using the inhibitor wiskostatin can significantly alter cell behaviour in vitro.