Lourdes Estrada

The Hsp Organizer Protein Hop Enhances the Rate of but Is Not Essential for Glucocorticoid Receptor Folding by the Multiprotein Hsp90-based Chaperone System

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)·Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90·Hop·Hsp70·Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR·Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358–7366). Hop has been considered essential for the formation of receptor·Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR·Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.

A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro

BackgroundClassical in vitro wound-healing assays and other techniques designed to study cell migration and invasion have been used for many years to elucidate the various mechanisms associated with metastasis. However, many of these methods are limited in their ability to achieve reproducible, quantitative results that translate well in vivo. Such techniques are also commonly unable to elucidate single-cell motility mechanisms, an important factor to be considered when studying dissemination. Therefore, we developed and applied a novel in vitro circular invasion assay (CIA) in order to bridge the translational gap between in vitro and in vivo findings, and to distinguish between different modes of invasion.MethodOur method is a modified version of a standard circular wound-healing assay with an added matrix barrier component (Matrigel™), which better mimics those physiological conditions present in vivo. We examined 3 cancer cell lines (MCF-7, SCOV-3, and MDA-MB-231), each with a different established degree of aggressiveness, to test our assays ability to detect diverse levels of invasiveness. Percent wound closure (or invasion) was measured using time-lapse microscopy and advanced image analysis techniques. We also applied the CIA technique to DLD-1 cells in the presence of lysophosphatidic acid (LPA), a bioactive lipid that was recently shown to stimulate cancer cell colony dispersal into single migratory cells, in order to validate our methods ability to detect collective and individual motility.ResultsCIA method was found to be highly reproducible, with negligible levels of variance measured. It successfully detected the anticipated low, moderate, and high levels of invasion that correspond to in vivo findings for cell lines tested. It also captured that DLD-1 cells exhibit individual migration upon LPA stimulation, and collective behavior in its absence.ConclusionGiven its ability to both determine pseudo-realistic invasive cell behavior in vitro and capture subtle differences in cell motility, we propose that our CIA method may shed some light on the cellular mechanisms underlying cancer invasion and deserves inclusion in further studies. The broad implication of this work is the development of a reproducible, quantifiable, high-resolution method that can be applied to various models, to include an unlimited number of parameters and/or agents that may influence invasion.

Dependence of Invadopodia Function on Collagen Fiber Spacing and Cross-Linking: Computational Modeling and Experimental Evidence

Invadopodia are subcellular organelles thought to be critical for extracellular matrix (ECM) degradation and the movement of cells through tissues. Here we examine invadopodia generation, turnover, and function in relation to two structural aspects of the ECM substrates they degrade: cross-linking and fiber density. We set up a cellular automaton computational model that simulates ECM penetration and degradation by invadopodia. Experiments with denatured collagen (gelatin) were used to calibrate the model and demonstrate the inhibitory effect of ECM cross-linking on invadopodia degradation and penetration. Incorporation of dynamic invadopodia behavior into the model amplified the effect of cross-linking on ECM degradation, and was used to model feedback from the ECM. When the model was parameterized with spatial fibrillar dimensions that closely matched the organization, in real life, of native ECM collagen into triple-helical monomers, microfibrils, and macrofibrils, little or no inhibition of invadopodia penetration was observed in simulations of sparse collagen gels, no matter how high the degree of cross-linking. Experimental validation, using live-cell imaging of invadopodia in cells plated on cross-linked gelatin, was consistent with simulations in which ECM cross-linking led to higher rates of both invadopodia retraction and formation. Analyses of invadopodia function from cells plated on cross-linked gelatin and collagen gels under standard concentrations were consistent with simulation results in which sparse collagen gels provided a weak barrier to invadopodia. These results suggest that the organization of collagen, as it may occur in stroma or in vitro collagen gels, forms gaps large enough so as to have little impact on invadopodia penetration/degradation. By contrast, dense ECM, such as gelatin or possibly basement membranes, is an effective obstacle to invadopodia penetration and degradation, particularly when cross-linked. These results provide a novel framework for further studies on ECM structure and modifications that affect invadopodia and tissue invasion by cells.

Skeletal-Specific Expression of Fgd1 During Bone Formation and Skeletal Defects in Faciogenital Dysplasia (FGDY; Aarskog Syndrome)

FGD1 encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase Cdc42; FGD1 mutations result in Faciogenital Dysplasia (FGDY, Aarskog syndrome), an X‐linked developmental disorder that adversely affects the formation of multiple skeletal structures. To further define the role of FGD1 in skeletal development, we examined its expression in developing mouse embryos and correlated this pattern with FGDY skeletal defects. In this study, we show that Fgd1, the mouse FGD1 ortholog, is initially expressed during the onset of ossification during embryogenesis. Fgd1 is expressed in regions of active bone formation in the trabeculae and diaphyseal cortices of developing long bones. The onset of Fgd1 expression correlates with the expression of bone sialo‐protein, a protein specifically expressed in osteoblasts at the onset of matrix mineralization; an analysis of serial sections shows that Fgd1 is expressed in tissues containing calcified and mineralized extracellular matrix. Fgd1 protein is specifically expressed in cultured osteoblast and osteoblast‐like cells including MC3T3‐E1 cells and human osteosarcoma cells but not in other mesodermal cells; immunohistochemical studies confirm the presence of Fgd1 protein in mouse calvarial cells. Postnatally, Fgd1 is expressed more broadly in skeletal tissue with expression in the perichondrium, resting chondrocytes, and joint capsule fibroblasts. The data indicate that Fgd1 is expressed in a variety of regions of incipient and active endochondral and intramembranous ossification including the craniofacial bones, vertebrae, ribs, long bones and phalanges. The observed pattern of Fgd1 expression correlates with FGDY skeletal manifestations and provides an embryologic basis for the prevalence of observed skeletal defects. The observation that the induction of Fgd1 expression coincides with the initiation of ossification strongly suggests that FGD1 signaling plays a role in ossification and bone formation; it also suggests that FGD1 signaling does not play a role in the earlier phases of skeletogenesis. With the observation that FGD1 mutations result in the skeletal dysplasia FGDY, accumulated data indicate that FGD1 signaling plays a critical role in ossification and skeletal development.

Microenvironmental Independence Associated with Tumor Progression

Tumor-microenvironment interactions are increasingly recognized to influence tumor progression. To understand the competitive dynamics of tumor cells in diverse microenvironments, we experimentally parameterized a hybrid discrete-continuum mathematical model with phenotypic trait data from a set of related mammary cell lines with normal, transformed, or tumorigenic properties. Surprisingly, in a resource-rich microenvironment, with few limitations on proliferation or migration, transformed (but not tumorigenic) cells were most successful and outcompeted other cell types in heterogeneous tumor simulations. Conversely, constrained microenvironments with limitations on space and/or growth factors gave a selective advantage to phenotypes derived from tumorigenic cell lines. Analysis of the relative performance of each phenotype in constrained versus unconstrained microenvironments revealed that, although all cell types grew more slowly in resource-constrained microenvironments, the most aggressive cells were least affected by microenvironmental constraints. A game theory model testing the relationship between microenvironment resource availability and competitive cellular dynamics supports the concept that microenvironmental independence is an advantageous cellular trait in resource-limited microenvironments.

Linking changes in epithelial morphogenesis to cancer mutations using computational modeling.

Most tumors arise from epithelial tissues, such as mammary glands and lobules, and their initiation is associated with the disruption of a finely defined epithelial architecture. Progression from intraductal to invasive tumors is related to genetic mutations that occur at a subcellular level but manifest themselves as functional and morphological changes at the cellular and tissue scales, respectively. Elevated proliferation and loss of epithelial polarization are the two most noticeable changes in cell phenotypes during this process. As a result, many three-dimensional cultures of tumorigenic clones show highly aberrant morphologies when compared to regular epithelial monolayers enclosing the hollow lumen (acini). In order to shed light on phenotypic changes associated with tumor cells, we applied the bio-mechanical IBCell model of normal epithelial morphogenesis quantitatively matched to data acquired from the non-tumorigenic human mammary cell line, MCF10A. We then used a high-throughput simulation study to reveal how modifications in model parameters influence changes in the simulated architecture. Three parameters have been considered in our study, which define cell sensitivity to proliferative, apoptotic and cell-ECM adhesive cues. By mapping experimental morphologies of four MCF10A-derived cell lines carrying different oncogenic mutations onto the model parameter space, we identified changes in cellular processes potentially underlying structural modifications of these mutants. As a case study, we focused on MCF10A cells expressing an oncogenic mutant HER2-YVMA to quantitatively assess changes in cell doubling time, cell apoptotic rate, and cell sensitivity to ECM accumulation when compared to the parental non-tumorigenic cell line. By mapping in vitro mutant morphologies onto in silico ones we have generated a means of linking the morphological and molecular scales via computational modeling. Thus, IBCell in combination with 3D acini cultures can form a computational/experimental platform for suggesting the relationship between the histopathology of neoplastic lesions and their underlying molecular defects.

Co-expression network analysis identifies Spleen Tyrosine Kinase (SYK) as a candidate oncogenic driver in a subset of small-cell lung cancer

BackgroundOncogenic mechanisms in small-cell lung cancer remain poorly understood leaving this tumor with the worst prognosis among all lung cancers. Unlike other cancer types, sequencing genomic approaches have been of limited success in small-cell lung cancer, i.e., no mutated oncogenes with potential driver characteristics have emerged, as it is the case for activating mutations of epidermal growth factor receptor in non-small-cell lung cancer. Differential gene expression analysis has also produced SCLC signatures with limited application, since they are generally not robust across datasets. Nonetheless, additional genomic approaches are warranted, due to the increasing availability of suitable small-cell lung cancer datasets. Gene co-expression network approaches are a recent and promising avenue, since they have been successful in identifying gene modules that drive phenotypic traits in several biological systems, including other cancer types.ResultsWe derived an SCLC-specific classifier from weighted gene co-expression network analysis (WGCNA) of a lung cancer dataset. The classifier, termed SCLC-specific hub network (SSHN), robustly separates SCLC from other lung cancer types across multiple datasets and multiple platforms, including RNA-seq and shotgun proteomics. The classifier was also conserved in SCLC cell lines. SSHN is enriched for co-expressed signaling network hubs strongly associated with the SCLC phenotype. Twenty of these hubs are actionable kinases with oncogenic potential, among which spleen tyrosine kinase (SYK) exhibits one of the highest overall statistical associations to SCLC. In patient tissue microarrays and cell lines, SCLC can be separated into SYK-positive and -negative. SYK siRNA decreases proliferation rate and increases cell death of SYK-positive SCLC cell lines, suggesting a role for SYK as an oncogenic driver in a subset of SCLC.ConclusionsSCLC treatment has thus far been limited to chemotherapy and radiation. Our WGCNA analysis identifies SYK both as a candidate biomarker to stratify SCLC patients and as a potential therapeutic target. In summary, WGCNA represents an alternative strategy to large scale sequencing for the identification of potential oncogenic drivers, based on a systems view of signaling networks. This strategy is especially useful in cancer types where no actionable mutations have emerged.

Effects of intracellular organelles on the apparent diffusion coefficient of water molecules in cultured human embryonic kidney cells.

The apparent diffusion coefficient (ADC) of water in tissues is dependent on the size and spacing of structures in the cellular environment and has been used to characterize pathological changes in stroke and cancer. However, the factors that affect ADC values remain incompletely understood. Measurements of ADC are usually made using relatively long diffusion times; so they reflect the integrated effects of cellular structures over a broad range of spatial scales. We used temporal diffusion spectroscopy to study diffusion in packed cultured human embryonic kidney cells over a range of effective diffusion times following microtubule and actin/cytoskeleton depolymerization and disassembly of the Golgi complex. While Golgi disruption did not change ADC, depolymerization of the microtubule and the actin filament networks caused small decreases in ADC at short diffusion times only. Temporal diffusion spectroscopy provided a novel way to assess intracellular influences on the diffusion properties of tissue water. Magn Reson Med, 2011.

Altered TGF-α/β signaling drives cooperation between breast cancer cell populations

The role of tumor heterogeneity in regulating disease progression is poorly understood. We hypothesized that interactions between subpopulations of cancer cells can affect the progression of tumors selecting for a more aggressive phenotype. We developed an in vivo assay based on the immortalized nontumorigenic breast cell line MCF10A and its Ras‐transformed derivatives AT1 (mildly tumorigenic) and CA1d (highly tumorigenic). CA1d cells outcompeted MCF10A, forming invasive tumors. AT1 grafts were approximately 1% the size of CA1d tumors when initiated using identical cell numbers. In contrast, CA1d/ AT1 mixed tumors were larger than tumors composed of AT1 alone (100‐fold) or CA1d (3‐fold), suggesting cooperation in tumor growth. One of the mechanisms whereby CA1d and AT1 were found to cooperate was by modulation of TGF‐α and TGF‐β signaling. Both of these molecules were sufficient to induce changes in AT1 proliferative potential in vitro. Reisolation of AT1 tumor‐derived (AT1‐TD) cells from these mixed tumors revealed that AT1‐TD cells grew in vivo, forming tumors as large as tumorigenic CA1d cells. Cooperation between subpopulations of cancer epithelium is an understudied mechanism of tumor growth and invasion that may have implications on tumor resistance to current therapies.—Franco, O. E., Tyson, D. R., Konvinse, K. C., Udyavar, A. R., Estrada, L., Quaranta, V., Crawford, S. E., Hayward, S. W. Altered TGF‐α/β signaling drives cooperation between breast cancer cell populations. FASEB J. 30, 3441–3452 (2016). www.fasebj.org

Abstract 5216: Gene co-expression network analysis of small cell lung cancer (SCLC) identifies new subclasses and novel therapeutic targets including Src family kinase Fyn and Spleen Tyrosine Kinase Syk.

Oncogenic mechanisms in SCLC remain poorly understood leaving this tumor with the worst prognosis among all lung cancers. Unlike other cancer types, traditional genomic approaches have been of limited success in SCLC. For instance, no patterns of prevalent mutations associated with SCLC progression or targeted therapeutics have emerged. Gene expression profiling based on differential expression have yielded potentially useful signatures in SCLC, but they have proven to be independent of each other and with minimal overlap, reducing translational potential. Nonetheless, since integrative systems biology studies of SCLC are warranted, we hypothesized that gene co-expression analyses can unveil key signaling hubs of specific networks that regulate SCLC proliferation and survival. We applied weighted gene co-expression network analysis (WGCNA) to a lung cancer patient gene expression dataset comprising of normal lung (NL), SCLC and Non-SCLC (NSCLC) such as squamous cell carcinoma and adenocarcinoma, and identified hierarchical gene networks highly specific to SCLC. The hubs of these SCLC-specific networks formed a SCLC-specific hub network (SSHN) signature that: 1) classified SCLC from NSCLC and NL on 3 independent patient and 1 cell line datasets; 2) was validated in independent RNAseq and shotgun proteomic patient datasets; 3) identified 2 SCLC subtypes with high and low SSHN expression in patients and cell lines. Three top SSHN hubs, Fyn, Syk and BRCA1, are involved in oxidative stress (OSR) and DNA damage response (DDR), respectively. At the protein level, Fyn, Syk and BRCA1 were significantly co-expressed in tissue microarrays and classified SCLC patients into 2 subtypes: High and Low Fyn/Syk/BRCA1 (F/S/B). High F/S/B staining significantly correlated with longer recurrence-free survival of patients treated with standard chemotherapy, suggesting that efficient OSR and DDR may enhance chemo response. This possibility was tested in established SCLC cell lines, which also could be separated into high vs low F/S/B groups corresponding to the clinical subtypes. High F/S/B cell lines exhibited lower proliferative rates, suggesting that OSR and DDR may still exert influence over cell cycle. Syk knockdown in these cell lines caused significant reduction in cellular viability and proliferation, as well as increased levels of Reactive Oxygen Species (ROS). Induction of oxidative stress in High F/S/B cell lines resulted in early activation of Fyn, Syk, p38, Erk and Akt. Thus, OSR may be overactive in SCLC and play a role in its natural history and response to treatment. In summary, our WGCNA combinatorial approach yielded novel insights into SCLC pathogenesis, separated two novel SCLC molecular subtypes with distinct functional OSR and DDR properties, and pointed to unsuspected potential therapeutic targets. Citation Format: Akshata R. Udyavar, Megan D. Hoeksema, Jonathan Clark, Yong Zou, Ming Li, Chiu-Lan (Heidi) Chen, Rosana Eisenberg, Alexander Statnikov, Daniel C. Liebler, John Field, Yu Shyr, Lourdes Estrada, Vito Quaranta, Pierre P. Massion. Gene co-expression network analysis of small cell lung cancer (SCLC) identifies new subclasses and novel therapeutic targets including Src family kinase Fyn and Spleen Tyrosine Kinase Syk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5216. doi:10.1158/1538-7445.AM2013-5216