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Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases., National Institutes of Health (U.S.) (R01EB010246), National Institutes of Health (U.S.) (UH2TR000496), Institute for Collaborative Biotechnologies (W911NF-09-0001), National Institutes of Health (U.S.) (T32GM008334), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), John and Karinne Begg Fund, Begg New Horizon Fund for Undergraduate Research at MIT, Massachusetts Institute of Technology. Biophysical Instrumentation Facility, Manton Foundation, Ludwig Postdoctoral Fellowship for Cancer Research, Swiss National Science Foundation (Postdoctoral Fellowship)

The ability of a cancer cell to migrate through the dense extracellular matrix within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment, but the basis for their effects is not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB-dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFβ1 dual blockade as an antimetastatic strategy in solid tumors., National Institutes of Health (U.S.) (Grant U01 CA202177-01)

Activation of synovial fibroblasts (SFs) contributes to rheumatoid arthritis (RA) by damaging synovial membranes and generating inflammatory cytokines that recruit immune cells to the joint. In this paper we profile cytokine secretion by primary human SFs from healthy tissues and from donors with RA and show that SF activation by TNF, IL-1α, and polyinosinic-polycytidylic acid (Poly(I:C)) cause secretion of multiple cytokines found at high levels in RA synovial fluids. We used interaction multiple linear regression to quantify therapeutic and countertherapeutic drug effects across activators and donors and found that the ability of drugs to block SF activation was strongly dependent on the identity of the activating cytokine. (5z)-7-oxozeaenol (5ZO), a preclinical drug that targets transforming growth factor-β-activated kinase 1 (TAK1), was more effective at blocking SF activation across all contexts than the approved drug tofacitinib, which supports the development of molecules similar to 5ZO for use as RA therapeutics., National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (5F32AR062931)

Introduction - Targeted cancer therapeutics have demonstrated more limited clinical efficacy than anticipated, due to both intrinsic and acquired drug resistance. Underlying mechanisms have been largely attributed to genetic changes, but a substantial proportion of resistance observations remain unexplained by genomic properties. Emerging evidence shows that receptor tyrosine kinase (RTK) reprogramming is a major alternative process causing targeted drug resistance, separate from genetic alterations. Hence, the contributions of mechanisms leading to this process need to be more rigorously assessed., National Institutes of Health (U.S.) (Grant R01-CA96504), National Institutes of Health (U.S.) (Grant U54-CA217377), United States. Army Research Office (grant W911NF-09-0001)

Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract that has limited treatment options. To gain insight into the pathogenesis of chronic colonic inflammation (colitis), we performed a multiomics analysis that integrated RNA microarray, total protein mass spectrometry (MS), and phosphoprotein MS measurements from a mouse model of the disease. Because we collected all three types of data from individual samples, we tracked information flow from RNA to protein to phosphoprotein and identified signaling molecules that were coordinately or discordantly regulated and pathways that had complex regulation in vivo. For example, the genes encoding acute-phase proteins were expressed in the liver, but the proteins were detected by MS in the colon during inflammation. We also ascertained the types of data that best described particular facets of chronic inflammation. Using gene set enrichment analysis and trans-omics coexpression network analysis, we found that each data set provided a distinct viewpoint on the molecular pathogenesis of colitis. Combining human transcriptomic data with the mouse multiomics data implicated increased p21-activated kinase (Pak) signaling as a driver of colitis. Chemical inhibition of Pak1 and Pak2 with FRAX597 suppressed active colitis in mice. These studies provide translational insights into the mechanisms contributing to colitis and identify Pak as a potential therapeutic target in IBD., Crohn's and Colitis Foundation of America (Research Fellowship), National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant 1122374), Institute for Collaborative Biotechnologies (W911NF-09-0001)

Metastases are a major cause of cancer mortality. AXL, a receptor tyrosine kinase aberrantly expressed in many tumors, is a potent oncogenic driver of metastatic cell motility and has been identified as broadly relevant in cancer drug resistance. Despite its frequent association with changes in cancer phenotypes, the precise mechanism leading to AXL activation is incompletely understood. In addition to its ligand growth arrest specific-6 (Gas6), activation of AXL requires the lipid moiety phosphatidylserine (PS). Phosphatidylserine is only available to mediate AXL activation when it is externalized on cell membranes, an event that occurs during certain physiologic processes such as apoptosis. Here, it is reported that exposure of cancer cells to phosphatidylserine-containing vesicles, including synthetic liposomes and apoptotic bodies, contributes to enhanced migration of tumor cells via a PS–Gas6–AXL signaling axis. These findings suggest that anticancer treatments that induce fractional cell killing enhance the motility of surviving cells in AXL-expressing tumors, which may explain the widespread role of AXL in limiting therapeutic efficacy. Implications: This study demonstrates that motility behavior of AXL-expressing tumor cells can be elicited by Gas6-bearing apoptotic bodies generated from tumor treatment with therapeutics that produce killing of a portion of the tumor cells present but not all, hence generating potentially problematic invasive and metastatic behavior of the surviving tumor cells. Mol Cancer Res; 15(12); 1656–66. ©2017 AACR.

Ectodomain shedding of cell-surface precursor proteins by metalloproteases generates important cellular signalingmolecules. Of importance for disease is the release of ligands that activate the EGFR, such as TGFα, which is mostly carried out by ADAM17 [a member of the A-disintegrin and metalloprotease (ADAM) domain family]. EGFR ligand shedding has been linked to many diseases, in particular cancer development, growth and metastasis, as well as resistance to cancer therapeutics. Excessive EGFR ligand release can outcompete therapeutic EGFR inhibition or the inhibition of other growth factor pathways by providing bypass signaling via EGFR activation. Drugging metalloproteases directly have failed clinically because it indiscriminately affected shedding of numerous substrates. It is therefore essential to identify regulators for EGFR ligand cleavage. Here, integration of a functional shRNA genomic screen, computational network analysis, and dedicated validation tests succeeded in identifying several key signaling pathways as novel regulators of TGFα shedding in cancer cells. Most notably, a cluster of genes with NFκB pathway regulatory functions was found to strongly influence TGFα release, albeit independent of their NFκB regulatory functions. Inflammatory regulators thus also govern cancer cell growth-promoting ectodomain cleavage, lending mechanistic understanding to the well-known connection between inflammation and cancer. Implications: Using genomic screens and network analysis, this study defines targets that regulate ectodomain shedding and suggests new treatment opportunities for EGFR-driven cancers., National Institutes of Health (U.S.) (Grant U01-CA155758), National Institutes of Health (U.S.) (Grant R01-CA096504), National Institutes of Health (U.S.) (Grant U01-CA184898), National Science Foundation (U.S.) (Grant DB1-0821391)

Tyro3, a member of TAM receptor tyrosine kinase family has been suggested to be autophosphorylated upon activation. In the current study we mapped the autophosphorylation sites of murine Tyro3 to tyrosine 723 and 756, with K540 being required for its kinase activity. Knockdown of Axl significantly decreases the tyrosyl-phosphorylation of Tyro3 in fibroblasts NR6WT, suggesting an interaction among the TAM family members. Interestingly, the carboxyl terminal region of Tyro3 is required for its stability in cells with a minimal length of 1–778 amino acids which is not conserved in murine Axl, a member of TAM. These data suggest that the autophosphorylation sites of TAM RTK members are unique although they share high similarity in amino acids within their carboxyl kinase domain. Keywords: Tyro3 receptor tyrosine kinase; Autophosphorylation; Protein stability; TAM family kinases, National Institute of General Medical Sciences (U.S.) (Award GM69668), National Institute of General Medical Sciences (U.S.) (Award GM63569)

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk., National Institutes of Health (U.S.) (grant UH3TR00069), United States. Defense Advanced Research Projects Agency (grant Microphysiological Systems Program (W911NF-12-2-00))

Tyro3, a member of TAM receptor tyrosine kinase family, has been implicated in the regulation of melanoma progression and survival. In this study, we sought the molecular mechanism of Tyro3 effects avoiding endogenous background by overexpression of Tyro3 in fibroblasts that have negligible levels of Tyro3. This introduction triggers the tyrosyl-phosphorylation of ACTN4, a member of actin binding protein family involved in motility, a behavior critical for invasive progression, as shown by siRNA to Tyro3 limiting melanoma cell migration and invasion. Tyro3-mediated phosphorylation of ACTN4 required FAK activation at tyrosine 397 and the EGF receptor cascade, but not EGFR ligand binding. Using PCR-based mutagenesis, the sites of Tyro3-mediated ACTN4 phosphorylation were mapped to ACTN4 tyrosine 11 and 13, and this occurs in conjunction with EGF-mediated phosphorylation on Y4 and Y31. Interestingly, Tyro3-mediated phosphorylation only slightly decreases the actin binding activity of ACTN4. However, this rendered the phosphorylated ACTN4 resistant to the m-calpain cleavage between Y13 and G14, a limited proteolysis that prevents growth factor regulation of ACTN4 interaction with F-actin. Overexpression of both WT ACTN4 and ACTN4Y11/13E, a mimic of ACTN4 phosphorylated at tyrosine 11 and 13, in melanoma WM983b cells resulted in a likely mesenchymal to amoeboidal transition. ACTN4Y11/13E-expressing cells were more amoeboidal, less migratory on collagen I gel coated surface but more invasive through collagen networks. In parallel, expression of ACTN4Y11/13E, in ACTN4 knockdown melanoma WM1158 cells resulted in an increase of invasion compared to WT ACTN4. These findings suggest that Tyro3-mediated phosphorylation of ACTN4 is involved in invasion of melanoma cells. Keywords: Actinin-4; Tyro3; Phosphorylation; Migration

Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes., United States. National Institutes of Health (R01DK108056), United States. National Institutes of Health (R24DK090963)

During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes., Virginia and D.K. Ludwig Fund for Cancer Research (Graduate Student Fellowship), National Institutes of Health (U.S.) (GM58801), Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology, David H. Koch Institute for Integrative Cancer Research at MIT (NCI Core Grant P30-CA14051)

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity., Author Summary The female genital tract (FGT) is a key mucosal surface in the context of HIV transmission. Lactobacillus species are beneficial to the FGT, while Garderella vaginalis and other anaerobic bacteria are detrimental. Bacterial vaginosis (BV) is an inflammatory condition characterized by an outgrowth of G. vaginalis and other anaerobes, which is linked to increased HIV acquisition rates. However, the mechanism behind this remains unknown. Here, we used a novel proteomic approach to simultaneously evaluate host and bacterial functions in the FGT. We found that women with G. vaginalis-dominated FGT bacterial communities always displayed markers of decreased epithelial barrier integrity, and decreased wound healing capacity. We also demonstrated that the abundance of proteins from G. vaginalis associated with these signatures of disrupted epithelial integrity. Finally, we showed that products derived from G. vaginalis prevented healing of wounded cell monolayers while products derived from L. iners maintained the ability of the cell monolayers to close wounds. This study provides novel mechanistic insight into the link between BV and increased HIV acquisition rates.

Signal transduction networks coordinate transcriptional programs activated by diverse extracellular stimuli, such as growth factors and cytokines. Cells receive multiple stimuli simultaneously, and mapping how activation of the integrated signaling network affects gene expression is a challenge. We stimulated colon adenocarcinoma cells with various combinations of the cytokine tumor necrosis factor (TNF) and the growth factors insulin and epidermal growth factor (EGF) to investigate signal integration and transcriptional crosstalk. We quantitatively linked the proteomic and transcriptomic data sets by implementing a structured computational approach called tensor partial least squares regression. This statistical model accurately predicted transcriptional signatures from signaling arising from single and combined stimuli and also predicted time-dependent contributions of signaling events. Specifically, the model predicted that an early-phase, AKT-associated signal downstream of insulin repressed a set of transcripts induced by TNF. Through bioinformatics and cell-based experiments, we identified the AKT-repressed signal as glycogen synthase kinase 3 (GSK3)-catalyzed phosphorylation of Ser(37) on the long form of the transcription factor GATA6. Phosphorylation of GATA6 on Ser(37) promoted its degradation, thereby preventing GATA6 from repressing transcripts that are induced by TNF and attenuated by insulin. Our analysis showed that predictive tensor modeling of proteomic and transcriptomic data sets can uncover pathway crosstalk that produces specific patterns of gene expression in cells receiving multiple stimuli.

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell–cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue “LERER” repeats. In fibroblasts, the Mena–α5 complex was required for “outside-in” α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins., National Institutes of Health (U.S.) (Grant GM58801), National Cancer Institute (U.S.) (Grant U54-CA112967), Howard Hughes Medical Institute

Individual signaling pathways operate in the context of the broader signaling network. Thus, the response of a cell to signals from the environment is affected by the state of the signaling network, such as the clinically relevant example of whether some components in the network are inhibited. The cytokine tumor necrosis factor–α (TNF-α) promotes opposing cellular behaviors under different conditions; the outcome is influenced by the state of the network. For example, in the mouse intestinal epithelium, inhibition of the mitogen-activated protein kinase (MAPK) kinase MEK alters the timing of TNF-α–induced apoptosis. We investigated whether MAPK signaling directly influences TNF-α–induced apoptosis or whether network-level effects secondary to inhibition of the MAPK pathway alter the cellular response. We found that inhibitors of the MAPK kinase kinase Raf, MEK, or extracellular signal–regulated kinase (ERK) exerted distinct effects on the timing and magnitude of TNF-α–induced apoptosis in the mouse intestine. Furthermore, even different MEK inhibitors exerted distinct effects; one, CH5126766, potentiated TNF-α–induced apoptosis, and the others reduced cell death. Computational modeling and experimental perturbation identified the kinase Akt as the primary signaling node that enhanced apoptosis in the context of TNF-α signaling in the presence of CH5126766. Our work emphasizes the importance of integrated network signaling in specifying cellular behavior in response to experimental or therapeutic manipulation. More broadly, this study highlighted the importance of considering the network-level effects of pathway inhibitors and showed the distinct effects of inhibitors that share the same target., National Institutes of Health (U.S.) (Grant R01-GM088827)

Biomimetic scaffolds have been proposed as a means to facilitate tissue regeneration by multi-potent stromal cells (MSCs). Effective scaffold colonization requires a control of multiple MSC responses including survival, proliferation, differentiation, and migration. As MSC migration is relatively unstudied in this context, we present here a multi-level approach to its understanding and control, integratively tuning cell speed and directional persistence to achieve maximal mean free path (MFP) of migration. This approach employs data-driven computational modeling to ascertain small molecule drug treatments that can enhance MFP on a given materials substratum. Using poly(methyl methacrylate)-graft-poly(ethylene oxide) polymer surfaces tethered with epidermal growth factor (tEGF) and systematically adsorbed with fibronectin, vitronectin, or collagen-I to present hTERT-immortalized human MSCs with growth factor and extracellular matrix cues, we measured cell motility properties along with signaling activities of EGFR, ERK, Akt, and FAK on 19 different substrate conditions. Speed was consistent on collagen/tEGF substrates, but low associated directional persistence limited MFP. Decision tree modeling successfully predicted that ERK inhibition should enhance MFP on collagen/tEGF substrates by increasing persistence. Thus, we demonstrated a two-tiered approach to control MSC migration: materials-based “coarse-graining” complemented by small molecule “fine-tuning”., National Institutes of Health (U.S.) (NIH grant R01-DE019523), National Institutes of Health (U.S.) (NIH Cell Migration Consortium U54-GM064346), National Institutes of Health (U.S.) (NIH grant R01-GM018336)

Variable sensitivity to T-cell-receptor (TCR)- and IL-7-receptor (IL-7R)-mediated homeostatic signals among naïve T cells has thus far been largely attributed to differences in TCR specificity. We show here that even when withdrawn from self-peptide-induced TCR stimulation, CD8+ T cells exhibit heterogeneous responses to interleukin-7 (IL-7) that are mechanistically associated with IL-7R expression differences that correlate with relative CD5 expression. Whereas CD5[superscript hi] and CD5[superscript lo] T cells survive equivalently in the presence of saturating IL-7 levels in vitro, CD5[superscript hi] T cells proliferate more robustly. Conversely, CD5[superscript lo] T cells exhibit prolonged survival when withdrawn from homeostatic stimuli. Through quantitative experimental analysis of signaling downstream of IL-7R, we find that the enhanced IL-7 responsiveness of CD5[superscript hi] T cells is directly related to their greater surface IL-7R expression. Further, we identify a quantitative threshold in IL-7R-mediated signaling capacity required for proliferation that lies well above an analogous threshold requirement for survival. These distinct thresholds allow subtle differences in IL-7R expression between CD5lo and CD5hi T cells to give rise to significant variations in their respective IL-7-induced proliferation, without altering survival. Heterogeneous IL-7 responsiveness is observed similarly in vivo, with CD5[superscript h]i naïve T cells proliferating preferentially in lymphopenic mice or lymphoreplete mice administered with exogenous IL-7. However, IL-7 in lymphoreplete mice appears to be maintained at an effective level for preserving homeostasis, such that neither CD5hi IL-7R[superscript hi] nor CD5lo IL-7R[superscript lo] T cells proliferate or survive preferentially. Our findings indicate that IL-7R-mediated signaling not only maintains the size but also impacts the diversity of the naïve T-cell repertoire., National Institutes of Health (U.S.) (grant AI69208), National Institutes of Health (U.S.) (grant GM068762)

Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines., Author summary Chronic inflammation of the gastrointestinal track is the common defect shared by inflammatory bowel diseases (IBDs), such as Crohn’s disease and ulcerative colitis, which affect many people around the world. However, the genetic and physiologic complexities of IBDs have made it difficult to identify therapeutically tractable drivers of disease that can alleviate the symptoms. We reasoned that this complexity is probably originated by a smaller number of dysregulated signaling pathways, and therefore, a “protein-centric” approach would be more suited to identify new therapeutic targets. To this end, in this study we profiled the expression and phosphorylation status of proteins that mediate signaling between and within cells in a mouse model of colitis. We found that hyperactivated mammalian target of rapamycin (mTOR) signaling interferes with the proper differentiation of epithelial cells, which promotes colitis by altering the epithelial inflammatory cytokine secretion in the colon.

Computational models are increasingly used to analyze the operation of complex biochemical networks, including those involved in cell signaling networks. Here we review recent advances in applying logic-based modeling to mammalian cell biology. Logic-based models represent biomolecular networks in a simple and intuitive manner without describing the detailed biochemistry of each interaction. A brief description of several logic-based modeling methods is followed by six case studies that demonstrate biological questions recently addressed using logic-based models and point to potential advances in model formalisms and training procedures that promise to enhance the utility of logic-based methods for studying the relationship between environmental inputs and phenotypic or signaling state outputs of complex signaling networks., National Institutes of Health (U.S.) (Grant P50- GM68762), National Institutes of Health (U.S.) (Grant U54-CA112967), United States. Dept. of Defense (Institute for Collaborative Biotechnologies)

More than 40% of colon cancers have a mutation in K-RAS or N-RAS, GTPases that operate as central hubs for multiple key signaling pathways within the cell. Utilizing an isogenic panel of colon carcinoma cells with K-RAS or N-RAS variations, we observed differences in tumor necrosis factor-α (TNFα)–induced apoptosis. When the dynamics of phosphorylated ERK response to TNFα were examined, K-RAS mutant cells showed lower activation whereas N-RAS mutant cells exhibited prolonged duration. These divergent trends were partially explained by differential induction of two ERK-modulatory circuits: negative feedback mediated by dual-specificity phosphatase 5 and positive feedback by autocrine transforming growth factor-α. Moreover, in the various RAS mutant colon carcinoma lines, the transforming growth factor-α autocrine loop differentially elicited a further downstream chemokine (CXCL1/CXCL8) autocrine loop, with the two loops having opposite effects on apoptosis. Although the apoptotic responses of the RAS mutant panel to TNFα treatment showed significant dependence on the respective phosphorylated ERK dynamics, successful prediction across the various cell lines required contextual information concerning additional pathways including IKK and p38. A quantitative computational model based on weighted linear combinations of these pathway activities successfully predicted not only the spectrum of cell death responses but also the corresponding chemokine production responses. Our findings indicate that diverse RAS mutations yield differential cell behavioral responses to inflammatory cytokine exposure by means of (a) differential effects on ERK activity via multiple feedback circuit mechanisms, and (b) differential effects on other key signaling pathways contextually modulating ERK-related dependence., National Institutes of Health (U.S.) (NIH grants U54-CA112967), National Institutes of Health (U.S.) (NIH grant P50-GM68762), American Cancer Society (grant PF-08-026-01-CCG)

Bone marrow-derived multipotent stromal cells (MSCs) offer great promise for regenerating tissue. Although certain transcription factors have been identified in association with tendency toward particular MSC differentiation phenotypes, the regulatory network of key receptor-mediated signaling pathways activated by extracellular ligands that induce various differentiation responses remains poorly understood. Attempts to predict differentiation fate tendencies from individual pathways in isolation are problematic due to the complex pathway interactions inherent in signaling networks. Accordingly, we have undertaken a multivariate systems approach integrating experimental measurement of multiple kinase pathway activities and osteogenic differentiation in MSCs, together with computational analysis to elucidate quantitative combinations of kinase signals predictive of cell behavior across diverse contexts. In particular, for culture on polymeric biomaterial surfaces presenting tethered epidermal growth factor, type I collagen, neither, or both, we have found that a partial least-squares regression model yields successful prediction of phenotypic behavior on the basis of two principal components comprising the weighted sums of eight intracellular phosphoproteins: phospho-epidermal growth factor receptor, phospho-Akt, phospho-extracellular signal-related kinase 1/2, phospho-heat shock protein 27, phospho-c-Jun, phospho-glycogen synthase kinase 3α/β, phospho-p38, and phospho-signal transducer and activator of transcription 3. This combination provides the strongest predictive capability for 21-day differentiated phenotype status when calculated from day-7 signal measurements; day-4 and day-14 signal measurements are also significantly predictive, indicating a broad time frame during MSC osteogenesis wherein multiple pathways and states of the kinase signaling network are quantitatively integrated to regulate gene expression, cell processes, and ultimately, cell fate. STEM CELLS 2009;27:2804–2814, National Institutes of Health (U.S.) (Grant NIH R01-GM059870-07), National Institutes of Health (U.S.) (Grant R01 DE019523- 10), United Negro College Fund ((UNCF)/Merck Postdoctoral Fellowship), Georgia Institute of Technology (Facilitating Academic Careers in Engineering and Sciences Fellowship)

A vital defect in the immune systems of HIV-infected individuals is the loss of CD4⁺ T cells, resulting in impaired immune responses. We hypothesized that there were CD4⁺ T cell–dependent and CD4⁺ T cell–independent alterations in the immune responses of HIV-1⁺ individuals. To test this, we analyzed the secretion of cytokines and chemokines from stimulated peripheral blood mononuclear cell (PBMC) populations from HIV+ donors, healthy donors, and healthy donors with CD4⁺ T cells experimentally depleted. Multivariate analyses of 16 cytokines and chemokines at 6 and 72 hours after three stimuli (antibody-coated beads to stimulate T cells and R848 or lipopolysaccharide to stimulate innate immune cells) enabled integrative analysis of secreted profiles. Two major effects in HIV⁺ PBMCs were not reproduced upon depletion of CD4⁺ T cells in healthy PBMCs: (i) HIV⁺ PBMCs maintained T cell–associated secreted profiles after T cell stimulation; (ii) HIV⁺ PBMCs showed impaired interferon-γ (IFN-γ) secretion early after innate stimulation. These changes arose from hyperactive T cells and debilitated natural killer (NK) cell, respectively. Modeling and experiments showed that early IFN-γ secretion predicted later differences in secreted profiles in vitro. This effect was recapitulated in healthy PBMCs by blocking the IFN-γ receptor. Thus, we identified a critical deficiency in NK cell responses of HIV-infected individuals, independent of CD4⁺ T cell depletion, which directs secreted profiles. Our findings illustrate a broad approach for identifying key disease-associated nodes in a multicellular, multivariate signaling network., Ragon Institute of MGH, MIT and Harvard, National Institutes of Health (U.S.) (AI6922694), National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award #F32-CA180586), National Institutes of Health (U.S.) (U19-AI6922694), National Cancer Institute (U.S.) (David H. Koch Center for Integrative Cancer Research. Support (Core) Grant P30-CA14051)

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling., National Institutes of Health (U.S.) (Grant R01-CA096504), National Institutes of Health (U.S.) (Grant U54-CA112967)

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena[superscript INV], which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5′ inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena[superscript INV] is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor–induced signaling. Disruption of this attenuation by Mena[superscript INV] sensitizes tumor cells to low–growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes., Breast Cancer Research Program (U.S.) (Grant W81XWH-10-1-0040), Breast Cancer Research Program (U.S.) (Grant W81XWH-13-1-0031), National Institutes of Health (U.S.) (Grant U54-CA112967), National Institutes of Health (U.S.) (Grant GM58801), Virginia and D.K. Ludwig Fund for Cancer Research

The AXL receptor is a TAM (Tyro3, AXL, MerTK) receptor tyrosine kinase (RTK) important in physiological inflammatory processes such as blood clotting, viral infection, and innate immune-mediated cell clearance. Overexpression of the receptor in a number of solid tumors is increasingly appreciated as a key drug resistance and tumor dissemination mechanism. Although the ligand-receptor (Gas6-AXL) complex structure is known, literature reports on ligand-mediated signaling have provided conflicting conclusions regarding the influence of other factors such as phosphatidylserine binding, and a detailed, mechanistic picture of AXL activation has not emerged. Integrating quantitative experiments with mathematical modeling, we show here that AXL operates to sense local spatial heterogeneity in ligand concentration, a feature consistent with its physiological role in inflammatory cell responses. This effect arises as a result of an intricate reaction-diffusion interaction. Our results demonstrate that AXL functions distinctly from other RTK families, a vital insight for the envisioned design of AXL-targeted therapeutic intervention., National Institutes of Health (U.S.) (Grants U54-CA112967 and DP5-OD019815), United States. Army Research Office (Institute for Collaborative Biotechnologies. Grant W911NF-09-0001), National Cancer Institute (David H. Koch Institute for Integrative Cancer Research at MIT. Core Support Grant P30-CA14051)

As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloproteinases (ADAMs) in particular, are known to impact the microenvironment of individual cells and have significant implications in various pathological situations including cancer, inflammatory and vascular diseases. There is great incentive to develop a high-throughput platform for single-cell measurement of pericellular protease activity, as it is essential for studying the heterogeneity of protease response and the corresponding cell behavioral consequences. In this work, we developed a microfluidic platform to simultaneously monitor protease activity of many single cells in a time-dependent manner. This platform isolates individual microwells rapidly on demand and thus allows single-cell activity measurement of both cell-surface and secreted proteases by confining individual cells with diffusive FRET-based substrates. With this platform, we observed dose-dependent heterogeneous protease activation of HepG2 cells treated with phorbol 12-myristate 13-acetate (PMA). To study the temporal behavior of PMA-induced protease response, we monitored the pericellular protease activity of the same single cells during three different time periods and revealed the diversity in the dynamic patterns of single-cell protease activity profile upon PMA stimulation. The unique temporal information of single-cell protease response can help unveil the complicated functional role of pericellular proteases., National Institutes of Health (U.S.) (Grant R01-CA096504), Singapore-MIT Alliance for Research and Technology (SMART)

Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human a-actinin-4 (ACTN4), Tyr[superscript 4] and Tyr[superscript 31] are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr[superscript 4] site. We found that introducing a phosphomimetic mutation at only Tyr[superscript 31] was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr[superscript 31] is mostly buried and that phosphorylation of Tyr[superscript 4] would increase the solvent exposure and thus kinase accessibility of Tyr[superscript 31]. In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr[superscript 4], whereas a truncated mutant representing the product of m-calpain cleavage exhibited EGF-stimulated tyrosine phosphorylation at a background amount similar to that observed for a double phosphomimetic mutant of Tyr[superscript 4] and Tyr[superscript 31]. We also found that inhibition of the receptor tyrosine kinases of the TAM family, such as AXL, blocked EGF-stimulated tyrosine phosphorylation of ACTN4. Mathematical modeling predicted that the kinetics of phosphorylation at Tyr[superscript 31] can be dictated by the kinase affinity for Tyr[superscript 4]. This study suggests that tandem-site phosphorylation within intrinsically disordered regions provides a mechanism for a site to function as a switch to reveal a nearby function-regulating site., National Institutes of Health (U.S.) (Grant R01 GM69668)

IL-2 and IL-15 are common γ-chain family cytokines involved in regulation of T cell differentiation and homeostasis. Despite signaling through the same receptors, IL-2 and IL-15 have non-redundant roles in T cell biology, both physiologically and at the cellular level. The mechanisms by which IL-2 and IL-15 trigger distinct phenotypes in T cells remain elusive. To elucidate these mechanisms, we performed a quantitative comparison of the phosphotyrosine signaling network and resulting phenotypes triggered by IL-2 and IL-15. This study revealed that the signaling networks activated by IL-2 or IL-15 are highly similar and that T cell proliferation and metabolism are controlled in a quantitatively distinct manner through IL-2/15R signal strength independent of the cytokine identity. Distinct phenotypes associated with IL-2 or IL-15 stimulation therefore arise through differential regulation of IL-2/15R signal strength and duration because of differences in cytokine–receptor binding affinity, receptor expression levels, physiological cytokine levels, and cytokine–receptor intracellular trafficking kinetics. These results provide important insights into the function of other shared cytokine and growth factor receptors, quantitative regulation of cell proliferation and metabolism through signal transduction, and improved design of cytokine based clinical immunomodulatory therapies for cancer and infectious diseases., National Institutes of Health (U.S.) (Grant U54CA11927), National Institutes of Health (U.S.) (Grant R01 AI065824), United States. Army Research Office (Institute for Collaborative Biotechnologies Grant W911NF-09-0001)

The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment., National Science Foundation (U.S.) (NSF EFRI grant 735997), National Institutes of Health (U.S.) (NIH Cell Migration Consortium grant GM06346), National Institutes of Health (U.S.) (NIH Cell Decision Processes Center grant GM68762), National Institutes of Health (U.S.) (NIH grant GM69668), National Institutes of Health (U.S.) (NIH grant GM81336)

The relationship between drug resistance, changes in signaling, and emergence of an invasive phenotype is well appreciated, but the underlying mechanisms are not well understood. Using machine learning analysis applied to the Cancer Cell Line Encyclopedia database, we identified expression of AXL, the gene that encodes the epithelial-to-mesenchymal transition (EMT)–associated receptor tyrosine kinase (RTK) AXL, as exceptionally predictive of lack of response to ErbB family receptor–targeted inhibitors. Activation of EGFR (epidermal growth factor receptor) transactivated AXL, and this ligand-independent AXL activity diversified EGFR-induced signaling into additional downstream pathways beyond those triggered by EGFR alone. AXL-mediated signaling diversification was required for EGF (epidermal growth factor)–elicited motility responses in AXL-positive TNBC (triple-negative breast cancer) cells. Using cross-linking coimmunoprecipitation assays, we determined that AXL associated with EGFR, other ErbB receptor family members, MET (hepatocyte growth factor receptor), and PDGFR (platelet-derived growth factor receptor) but not IGF1R (insulin-like growth factor 1 receptor) or INSR (insulin receptor). From these AXL interaction data, we predicted AXL-mediated signaling synergy for additional RTKs and validated these predictions in cells. This alternative mechanism of receptor activation limits the use of ligand-blocking therapies and indicates against therapy withdrawal after acquired resistance. Further, subadditive interaction between EGFR- and AXL-targeted inhibitors across all AXL-positive TNBC cell lines may indicate that increased abundance of EGFR is principally a means to transactivation-mediated signaling., United States. Dept. of Defense (Congressionally Directed Medical Research Programs, Breast Cancer Research Program (W81XWH-11-1-0088)), National Science Foundation (U.S.) (Graduate Research Fellowship), Repligen Corporation (Fellowship in Cancer Research), National Cancer Institute (U.S.). Integrative Cancer Biology Program (1-U54-CA112967), David H. Koch Institute for Integrative Cancer Research at MIT (Frontier Research Program Initiator Award), National Institutes of Health (U.S.) (NIH R01-CA96504)

Although many anticancer drugs that target receptor tyrosine kinases (RTKs) provide clinical benefit, their long-term use is limited by resistance that is often attributed to increased abundance or activation of another RTK that compensates for the inhibited receptor. To uncover common and unique features in the signaling networks of RTKs, we measured time-dependent signaling in six isogenic cell lines, each expressing a different RTK as downstream proteins were systematically perturbed by RNA interference. Network models inferred from the data revealed a conserved set of signaling pathways and RTK-specific features that grouped the RTKs into three distinct classes: (i) an EGFR/FGFR1/c-Met class constituting epidermal growth factor receptor, fibroblast growth factor receptor 1, and the hepatocyte growth factor receptor c-Met; (ii) an IGF-1R/NTRK2 class constituting insulin-like growth factor 1 receptor and neurotrophic tyrosine receptor kinase 2; and (iii) a PDGFRβ class constituting platelet-derived growth factor receptor β. Analysis of cancer cell line data showed that many RTKs of the same class were coexpressed and that increased abundance of an RTK or its cognate ligand frequently correlated with resistance to a drug targeting another RTK of the same class. In contrast, abundance of an RTK or ligand of one class generally did not affect sensitivity to a drug targeting an RTK of a different class. Thus, classifying RTKs by their inferred networks and then therapeutically targeting multiple receptors within a class may delay or prevent the onset of resistance., W. M. Keck Foundation, National Institutes of Health (U.S.) (R21 CA126720), National Institutes of Health (U.S.) (P50 GM068762), National Institutes of Health (U.S.) (RC1 HG005354), National Institutes of Health (U.S.) (U54-CA112967), National Institutes of Health (U.S.) (R01-CA096504), Alfred and Isabel Bader (Fellowship), Jacques-Emile Dubois (fellowship)

During wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKCδ/MLC-2). PKCδ is activated by growth factor-driven PLCγ1 hydrolysis of phosphoinositide bisphosphate (PIP[subscript 2]) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKCδ n-terminus mediates PKCδ localization to the membrane in relative proximity to PLCγ1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKCδ mediates the transcellular contractility of fibroblasts., National Institutes of Health (U.S.) (T32 HL094295), National Institutes of Health (U.S.) (R01 GM069668), National Institutes of Health (U.S.) (AR061395), National Institutes of Health (U.S.) (AR060920)

Computational models of cell signalling are perceived by many biologists to be prohibitively complicated. Why do math when you can simply do another experiment? Here, we explain how conceptual models, which have been formulated mathematically, have provided insights that directly advance experimental cell biology. In the past several years, models have influenced the way we talk about signalling networks, how we monitor them, and what we conclude when we perturb them. These insights required wet-lab experiments but would not have arisen without explicit computational modelling and quantitative analysis. Today, the best modellers are cross-trained investigators in experimental biology who work closely with collaborators but also undertake experimental work in their own laboratories. Biologists would benefit by becoming conversant in core principles of modelling in order to identify when a computational model could be a useful complement to their experiments. Although the mathematical foundations of a model are useful to appreciate its strengths and weaknesses, they are not required to test or generate a worthwhile biological hypothesis computationally., National Institutes of Health (U.S.) (Director's New Innovator Award Program grant number 1-DP2-OD006464), American Cancer Society (grant number 120668-RSG-11-047-01-DMC), Pew Charitable Trusts (Pew Scholars Program in the Biomedical Sciences), David & Lucile Packard Foundation, National Institutes of Health (U.S.) (NCI Integrative Cancer Biology Program, grant U54-CA112967), National Institutes of Health (U.S.) (NCI Integrative Cancer Biology Program, R24-DK090963), National Institutes of Health (U.S.) (NCI Integrative Cancer Biology Program, grant R01-EB010246)

Advances in diagnosis and treatment have rendered most solid tumors largely curable if they are diagnosed and treated before dissemination. However, once they spread beyond the initial primary location, these cancers are usually highly morbid, if not fatal. Thus, current efforts focus on both limiting initial dissemination and preventing secondary spread. There are two modes of tumor dissemination – invasion and metastasis – each leading to unique therapeutic challenges and likely to be driven by distinct mechanisms. However, these two forms of dissemination utilize some common strategies to accomplish movement from the primary tumor, establishment in an ectopic site, and survival therein. The adaptive behaviors of motile cancer cells provide an opening for therapeutic approaches if we understand the molecular, cellular, and tissue biology that underlie them. Herein, we review the signaling cascades and organ reactions that lead to dissemination, as these are non-genetic in nature, focusing on cell migration as the key to tumor progression. In this context, the cellular phenotype will also be discussed because the modes of migration are dictated by quantitative and physical aspects of the cell motility machinery., National Institute of General Medical Sciences (U.S.), National Cancer Institute (U.S.)

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine that can elicit distinct cellular behaviors under different molecular contexts. Mitogen activated protein kinase (MAPK) pathways, especially the extracellular signal-regulated kinase (Erk) pathway, help to integrate influences from the environmental context, and therefore modulate the phenotypic effect of TNF-α exposure. To test how variations in flux through the Erk pathway modulate TNF-α-elicited phenotypes in a complex physiological environment, we exposed mice with different Ras mutations (K-Ras activation, N-Ras activation, and N-Ras ablation) to TNF-α and observed phenotypic and signaling changes in the intestinal epithelium. Hyperactivation of Mek1, an Erk kinase, was observed in the intestine of mice with K-Ras activation and, surprisingly, in N-Ras null mice. Nevertheless, these similar Mek1 outputs did not give rise to the same phenotype, as N-Ras null intestine was hypersensitive to TNF-α-induced intestinal cell death while K-Ras mutant intestine was not. A systems biology approach applied to sample the network state revealed that the signaling contexts presented by these two Ras isoform mutations were different. Consistent with our experimental data, N-Ras ablation induced a signaling network state that was mathematically predicted to be pro-death, while K-Ras activation did not. Further modeling by constrained Fuzzy Logic (cFL) revealed that N-Ras and K-Ras activate the signaling network with different downstream distributions and dynamics, with N-Ras effects being more transient and diverted more towards PI3K-Akt signaling and K-Ras effects being more sustained and broadly activating many pathways. Our study highlights the necessity to consider both environmental and genomic contexts of signaling pathway activation in dictating phenotypic responses, and demonstrates how modeling can provide insight into complex in vivo biological mechanisms, such as the complex interplay between K-Ras and N-Ras in their downstream effects., National Institute of General Medical Sciences (U.S.) (Grant R01-GM088827), National Cancer Institute (U.S.) (U54-CA112967), United States. Army Research Office (Institute for Collaborative Biotechnologies Grant W911NF-09-D-000)

Angiogenesis requires coordinated dynamic regulation of multiple phenotypic behaviors of endothelial cells in response to environmental cues. Multi-scale computational models of angiogenesis can be useful for analyzing effects of cell behaviors on the tissue level outcome, but these models require more intensive experimental studies dedicated to determining the required quantitative “rules” for cell-level phenotypic responses across a landscape of pro- and anti-angiogenic stimuli in order to ascertain how changes in these single cell responses lead to emerging multi-cellular behavior such as sprout formation. Here we employ single-cell microscopy to ascertain phenotypic behaviors of more than 800 human microvascular endothelial cells under various combinational angiogenic (VEGF) and angiostatic (PF4) cytokine treatments, analyzing their dynamic behavioral transitions among sessile, migratory, proliferative, and apoptotic states. We find that an endothelial cell population clusters into an identifiable set of a few distinct phenotypic state transition patterns (clusters) that is consistent across all cytokine conditions. Varying the cytokine conditions, such as VEGF and PF4 combinations here, modulates the proportion of the population following a particular pattern (referred to as phenotypic cluster weights) without altering the transition dynamics within the patterns. We then map the phenotypic cluster weights to quantified population level sprout densities using a multi-variate regression approach, and identify linear combinations of the phenotypic cluster weights that associate with greater or lesser sprout density across the various treatment conditions. VEGF-dominant cytokine combinations yielding high sprout densities are characterized by high proliferative and low apoptotic cluster weights, whereas PF4-dominant conditions yielding low sprout densities are characterized by low proliferative and high apoptotic cluster weights. Migratory cluster weights show only mild association with sprout density outcomes under the VEGF/PF4 conditions and the sprout formation characteristics explored here., National Science Foundation (U.S.) (NSF grant EFRI-0735007), National Institutes of Health (U.S.) (NIH grant R01-GM081336), National Institutes of Health (U.S.) (NIH grant R01-EB010246)

Growth factor–induced migration is a critical step in the dissemination and metastasis of solid tumors. Although differences in properties characterizing cell migration on two-dimensional (2D) substrata versus within three-dimensional (3D) matrices have been noted for particular growth factor stimuli, the 2D approach remains in more common use as an efficient surrogate, especially for high-throughput experiments. We therefore were motivated to investigate which migration properties measured in various 2D assays might be reflective of 3D migratory behavioral responses. We used human triple-negative breast cancer lines stimulated by a panel of receptor tyrosine kinase ligands relevant to mammary carcinoma progression. Whereas 2D migration properties did not correlate well with 3D behavior across multiple growth factors, we found that increased membrane protrusion elicited by growth factor stimulation did relate robustly to enhanced 3D migration properties of the MDA-MB-231 and MDA-MB-157 lines. Interestingly, we observed this to be a more reliable relationship than cognate receptor expression or activation levels across these and two additional mammary tumor lines., National Institutes of Health (U.S.) (Grant no. R01- GM081336), National Science Foundation (U.S.). Graduate Research Fellowship, United States. Dept. of Defense. Congressionally Directed Medical Research Programs. Breast Cancer Research Program (Grant no. W81XWH-11-1-0088), United States. Dept. of Defense. Congressionally Directed Medical Research Programs. Breast Cancer Research Program (Grant no. W81XWH-10-1-0040)

Cell signaling networks propagate information from extracellular cues via dynamic modulation of protein–protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein–protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein–protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein–protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein–protein interactions in a dynamic context- and phosphorylation site-specific manner., National Institutes of Health (U.S.) (Grant U54-CA112967), National Institutes of Health (U.S.) (Grant R01-CA96504), National Institutes of Health (U.S.) (Grant P50-GM68762), National Institutes of Health (U.S.) (Grant R01 ES-015339)

Chemotaxis of tumor cells in response to a gradient of extracellular ligand is an important step in cancer metastasis. The heterogeneity of chemotactic responses in cancer has not been widely addressed by experimental or mathematical modeling techniques. However, recent advancements in chemoattractant presentation, fluorescent-based signaling probes, and phenotypic analysis paradigms provide rich sources for building data-driven relational models that describe tumor cell chemotaxis in response to a wide variety of stimuli. Here we present gradient sensing, and the resulting chemotactic behavior, in a ‘cue-signal-response’ framework and suggest methods for utilizing recently reported experimental methods in data-driven modeling ventures., United States. Dept. of Defense. Breast Cancer Research Program (U.S.) (Fellowship BC087781), National Institutes of Health (U.S.) (Grant U54-CA112967), National Institutes of Health (U.S.) (Grant R01-GM081336)

Primary rat liver sinusoidal endothelial cells (LSEC) are difficult to maintain in a differentiated state in culture for scientific studies or technological applications. Relatively little is known about molecular regulatory processes that affect LSEC differentiation because of this inability to maintain cellular viability and proper phenotypic characteristics for extended times in vitro, given that LSEC typically undergo death and detachment around 48–72 h even when treated with VEGF. We demonstrate that particular lipid supplements added to serum-free, VEGF-containing medium increase primary rat liver LSEC viability and maintain differentiation. Addition of a defined lipid combination, or even oleic acid (OA) alone, promotes LSEC survival beyond 72 h and proliferation to confluency. Moreover, assessment of LSEC cultures for endocytic function, CD32b surface expression, and exhibition of fenestrae showed that these differentiation characteristics were maintained when lipids were included in the medium. With respect to the underlying regulatory pathways, we found lipid supplement-enhanced phosphatidylinositol 3-kinase and MAPK signaling to be critical for ensuring LSEC function in a temporally dependent manner. Inhibition of Akt activity before 72 h prevents growth of SEC, whereas MEK inhibition past 72 h prevents survival and proliferation. Our findings indicate that OA and lipids modulate Akt/PKB signaling early in culture to mediate survival, followed by a switch to a dependence on ERK signaling pathways to maintain viability and induce proliferation after 72 h. We conclude that free fatty acids can support maintenance of liver LSEC cultures in vitro; key regulatory pathways involved include early Akt signaling followed by ERK signaling., National Science Foundation (U.S.) (Grant EFRI-0735997), National Institutes of Health (U.S.) (Grant R01 GM069668)

Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24–204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nm and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein β levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease., National Institutes of Health (U.S.) (Grant R01EB010246), National Institutes of Health (U.S.) (Grant R01GM081336), Heptagon Fund (London, England), Cancer Research UK, Whitehead Foundation, Duke University. School of Medicine (Bridge Funding Program), Germany. Bundesministerium für Bildung und Forschung, China (National Fellowship from the Chinese Scholarship Council), Florida State University

Following DNA damage, cells display complex multi-pathway signaling dynamics that connect cell-cycle arrest and DNA repair in G1, S, or G2/M phase with phenotypic fate decisions made between survival, cell-cycle re-entry and proliferation, permanent cell-cycle arrest, or cell death. How these phenotypic fate decisions are determined remains poorly understood, but must derive from integrating genotoxic stress signals together with inputs from the local microenvironment. To investigate this in a systematic manner, we undertook a quantitative time-resolved cell signaling and phenotypic response study in U2OS cells receiving doxorubicin-induced DNA damage in the presence or absence of TNFα co-treatment; we measured key nodes in a broad set of DNA damage signal transduction pathways along with apoptotic death and cell-cycle regulatory responses. Two relational modeling approaches were then used to identify network-level relationships between signals and cell phenotypic events: a partial least squares regression approach and a complementary new technique which we term ‘time-interval stepwise regression.’ Taken together, the results from these analysis methods revealed complex, cytokine-modulated inter-relationships among multiple signaling pathways following DNA damage, and identified an unexpected context-dependent role for Erk in both G1/S arrest and apoptotic cell death following treatment with this commonly used clinical chemotherapeutic drug., National Institutes of Health (U.S.) (Grant CA112967), National Institutes of Health (U.S.) (Grant ES015339), National Institutes of Health (U.S.) (Grant GM60594), United States. Dept. of Defense (Fellowship BC097884)