Immunohistochemical analysis of patient material (S1); IHC score for TGF-beta pathway member from matched patient samples (S2); List of TGF-beta regulated genes (S3); List of EMT-related genes (S4); Results of paired t-tests for Microarray (S5); Patient information (S6); List of specific primers used for qRT-PCR gene expression analysis (S7).

Description of additional methods and procedures used in the study.

Activity of the TGF-beta pathway in tumor-free lung and lung cancer (S1); Validation of the microarray data for individual targets with qRT-PCR assay (S2); Expression of EMT-related proteins in human lung tumor tissues and tumor-free lung (S3); Secretion of TGF-beta and expression of the pathway components in NSCLC cell lines (S4); Activation of TGF-beta pathway members and expression of EMT markers in lung cancer cells with BAMBI reconstitution (S5); Verification of DNA methylation values determined by HumanMethylation450K BeadChip analysis by performing bisulfite pyrosequencing (S6); siRNA-mediated knockdown of BAMBI expression in A549 cell line enhances TGF-beta-induced signaling (S7); siRNA-mediated knockdown of BAMBI expression in the squamous cell carcinoma cell line SK-MES1 enhances TGF-beta-induced signaling (S8); Human alveolar epithelial cells type II (AECII) cells express low amount of SMAD3 (S9); GFP- and BAMBI-GFP-positive A549 cells are present in the mice lungs at the time of sacrifice (S10).

Use of the diabetes type II drug Metformin is associated with a moderately lowered risk of cancer incidence in numerous tumor entities. Studying the molecular changes associated with the tumor-suppressive action of Metformin we found that the oncogene SOX4, which is upregulated in solid tumors and associated with poor prognosis, was induced by Wnt/β-catenin signaling and blocked by Metformin. Wnt signaling inhibition by Metformin was surprisingly specific for cancer cells. Unraveling the underlying specificity, we identified Metformin and other Mitochondrial Complex I (MCI) inhibitors as inducers of intracellular acidification in cancer cells. We demonstrated that acidification triggers the unfolded protein response to induce the global transcriptional repressor DDIT3, known to block Wnt signaling. Moreover, our results suggest that intracellular acidification universally inhibits Wnt signaling. Based on these findings, we combined MCI inhibitors with H+ ionophores, to escalate cancer cells into intracellular hyper-acidification and ATP depletion. This treatment lowered intracellular pH both in vitro and in a mouse xenograft tumor model, depleted cellular ATP, blocked Wnt signaling, downregulated SOX4, and strongly decreased stemness and viability of cancer cells. Importantly, the inhibition of Wnt signaling occurred downstream of β-catenin, encouraging applications in treatment of cancers caused by APC and β-catenin mutations.

Collective cell migration is a common phenotype in epithelial cancers, which is associated with tumor cell metastasis and poor patient survival. However, the interplay between physiologically relevant pro-migratory stimuli and the underlying mechanical cell–cell interactions are poorly understood. We investigated the migratory behavior of different collectively migrating non-small cell lung cancer cell lines in response to motogenic growth factors (e.g. epidermal growth factor) or clinically relevant small compound inhibitors. Depending on the treatment, we observed distinct behaviors in a classical lateral migration assay involving traveling fronts, finger-shapes or the development of cellular bridges. Particle image velocimetry analysis revealed characteristic speed dynamics (evolution of the average speed of all cells in a frame) in all experiments exhibiting initial acceleration and subsequent deceleration of the cell populations. To better understand the mechanical properties of individual cells leading to the observed speed dynamics and the phenotypic differences we developed a mathematical model based on a Langevin approach. This model describes intercellular forces, random motility, and stimulation of active migration by mechanical interaction between cells. Simulations show that the model is able to reproduce the characteristic spatio-temporal speed distributions as well as most migratory phenotypes of the studied cell lines. A specific strength of the proposed model is that it identifies a small set of mechanical features necessary to explain all phenotypic and dynamical features of the migratory response of non-small cell lung cancer cells to chemical stimulation/inhibition. Furthermore, all processes included in the model can be associated with potential molecular components, and are therefore amenable to experimental validation. Thus, the presented mathematical model may help to predict which mechanical aspects involved in non-small cell lung cancer cell migration are affected by the respective therapeutic treatment.

Introduction: Despite recent developments in targeted therapies, non-small cell lung cancer still remains among the most fatal types of cancer. Most patients are diagnosed with an advanced state of the disease and exhibit poor prognosis independent of therapeutic efforts. The TGF-β signaling pathway is known to be involved in several different diseases as well as crucial cellular or immunological processes. Especially in malignant diseases such as lung cancer, the pathway is known to exert a double-edged role. In early stages of carcinogenesis, the tumor suppressive effects prevail, while at later stages invasiveness and metastasis are favored.

The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of the MAPK kinase MEK and its targets extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we elucidated network structures for the reversible threonine and tyrosine phosphorylation of ERK in both cell types. In addition to differences in the phosphorylation and dephosphorylation reactions, the HaCaT network model required two feedback mechanisms, which, as the experimental data suggested, involved the induction of the dual-specificity phosphatase DUSP6 and the scaffold paxillin. We assayed and modeled the accumulation of the double-phosphorylated and active form of ERK1/2, as well as the dynamics of the changes in the monophosphorylated forms of ERK1/2. Modeling the differences in the dynamics of the changes in the distributions of the phosphorylated forms of ERK1/2 suggested that different amounts of MEK activity triggered context-specific responses, with primary hepatocytes favoring the formation of double-phosphorylated ERK1/2 and HaCaT A5 cells that produce both the threonine-phosphorylated and the double-phosphorylated form. These differences in phosphorylation distributions explained the threshold, sensitivity, and saturation of the ERK response. We extended the findings of differential ERK phosphorylation profiles to five additional cultured cell systems and matched liver tumor and normal tissue, which revealed context-specific patterns of the various forms of phosphorylated ERK.

Abstract Non–small cell lung cancer (NSCLC) is characterized by early metastasis and has the highest mortality rate among all solid tumors, with the majority of patients diagnosed at an advanced stage where curative therapeutic options are lacking. In this study, we identify a targetable mechanism involving TGFβ elevation that orchestrates tumor progression in this disease. Substantial activation of this pathway was detected in human lung cancer tissues with concomitant downregulation of BAMBI, a negative regulator of the TGFβ signaling pathway. Alterations of epithelial-to-mesenchymal transition (EMT) marker expression were observed in lung cancer samples compared with tumor-free tissues. Distinct alterations in the DNA methylation of the gene regions encoding TGFβ pathway components were detected in NSCLC samples compared with tumor-free lung tissues. In particular, epigenetic silencing of BAMBI was identified as a hallmark of NSCLC. Reconstitution of BAMBI expression in NSCLC cells resulted in a marked reduction of TGFβ-induced EMT, migration, and invasion in vitro, along with reduced tumor burden and tumor growth in vivo. In conclusion, our results demonstrate how BAMBI downregulation drives the invasiveness of NSCLC, highlighting TGFβ signaling as a candidate therapeutic target in this setting. Cancer Res; 76(13); 3785–801. ©2016 AACR.

Rationale: Transforming growth factor-β (TGFβ)-induced effects are different, even opposite, depending on the cellular type and conditions. TGFβ signaling has been widely acknowledged to contribute to cancerogenesis via enhancing tumor cell metastasis. Numerous attempts to target TGFβ pathway to reduce cancer spread proved to be ineffective.

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types.

Lung cancer with its most prevalent form non-small-cell lung carcinoma (NSCLC) is one of the leading causes of cancer-related deaths worldwide and is commonly treated with chemotherapeutic drugs, e.g. cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (Epo). However, recent studies have indicated that Epo may not only induce erythropoiesis in hematopoietic cells but can also affect NSCLC cells.

Chemical dimerizers are powerful tools for non-invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small-molecule-based dimerization system and demonstrate a sufficiently fast switch-off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3-kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.

Cellular communication is mediated by extracellular stimuli that bind cellular receptors and activate intracellular signaling pathways. Principal biochemical reactions used for signal transduction are protein or lipid phosphorylation, proteolytic cleavage, protein degradation and complex formation mediated by protein-protein interactions. Within the nucleus, signaling pathways regulate transcription factor activity and gene expression. Cells differ in their competence to respond to extracellular stimuli. A deeper understanding of complex biological responses cannot be achieved by traditional approaches but requires the combination of experimental data with mathematical modeling. Following a systems biology approach, data-based mathematical models describing sub-modules of signaling pathways have been established. By combining computer simulations with experimental verification systems properties of signaling pathway including cycling behavior or threshold response could be identified. Yet, to analyze complex growth and maturation processes at a systems level and quantitatively predict the outcome of perturbations further advances in experimental and theoretical methodologies are required.

Lung cancer, with its predominant form non-small cell lung cancer (NSCLC), is the leading cause of cancer related-deaths world-wide. The key medical problem in lung cancer is early systemic spread of tumors independent of tumor size. Activating mutations or overexpression of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor (HGF) receptor (c-Met) are frequently observed. A major unresolved question is how these receptor systems interact and contribute to early spread in lung cancer. To unravel key regulatory mechanisms and to establish a quantitative link of information processing through signaling networks, target gene induction and lung cancer cell migration, we are employing a selected panel of NSCLC adenocarinoma cell lines with defined EGFR status. In monolayer cultures we are monitoring HGF induced signal transduction and activation kinetics of cMet, Akt and Erk1/2 by quantitative immunoblotting and quantitative mass spectrometry in the presence of various inhibitors. Studies at the molecular level are complemented by studies of cellular decisions in response of HGF such as proliferation, migration and apoptosis at the cell population level. The quantitative data obtained is used to establish detailed mechanistic models of signaling pathways that will be utilized to identify key regulatory mechanisms and predict strategies for targeted intervention.

Inflammation contributes to tumour growth, invasion and angiogenesis. We investigated the contribution of macrophages and their polarization to tumour progression in a model of VEGF‐A‐induced skin carcinogenesis. Transfection of the human non‐tumourigenic keratinocyte cell line HaCaT with murine VEGF‐A leads to malignant tumour growth in vivo . The resulting tumours are characterized by extensive vascularization, invasive growth and high numbers of M2‐polarized macrophages that crucially contribute to the establishment of the malignant phenotype. Accordingly, macrophage depletion from tumour‐bearing animals resulted in reduced tumour growth, inhibition of invasion, decreased proliferation and reduced angiogenesis. In vitro , VEGF‐A exerted a chemo‐attracting effect on macrophages, but did not induce M2 polarization. We identified IL‐4 and IL‐10 as the factors involved in M2 polarization. These factors were produced by tumour cells (IL‐10) and macrophages (IL‐4) in vivo . Addition of recombinant IL‐4 and IL‐10 in vitro induced a pro‐invasive M2 macrophage phenotype and inhibition of the IL‐4 receptor in vivo blocked M2 polarization of macrophages, resulting in a less aggressive tumour phenotype. Thus, we provide evidence that M2 macrophages are crucial for the development of VEGF‐A‐induced skin tumours and that VEGF‐A contributes to malignant tumour growth, not only by enhancing angiogenesis but also by establishing an anti‐inflammatory microenvironment. However, VEGF‐A alone is not sufficient to create a tumour‐promoting microenvironment and requires the presence of IL‐4 and IL‐10 to induce M2 polarization of macrophages. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ERK is a member of the MAPK pathway with essential functions in cell proliferation, differentiation, and survival. Complete ERK activation by the kinase MEK requires dual phosphorylation at T and Y within the activation motif TEY. We show that exposure of primary mouse hepatocytes to hepatocyte growth factor (HGF) results in phosphorylation at the activation motif, but not of other residues nearby. To determine the relative abundances of unphosphorylated ERK and the three ERK phospho-forms pT, pY, and pTpY, we employed an extended one-source peptide/phosphopeptide standard method in combination with nanoUPLC-MS. This method enabled us to determine the abundances of phospho-forms with a relative variability of ≤5% (SD). We observed a switch-like preference of ERK phospho-form abundances toward the active, doubly phosphorylated and the inactive, unphosphorylated form. Interestingly, ERK phospho-form profiles were similar upon growth factor and cytokine stimulation. A screening of several murine and human cell systems revealed that the balance between TY- and pTpY-ERK is conserved while the abundances of pT- and pY-ERK are more variable within cell types. We show that the phospho-form profiles do not change by blocking MEK activity suggesting that cellular phosphatases determine the ERK phospho-form distribution. This study provides novel quantitative insights into multisite phosphorylation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes tumor progression in different tumor models in an autocrine and paracrine manner. However, at the same time GM-CSF is used in cancer therapies to ameliorate neutropenia. We have previously shown in GM-CSF and G-CSF expressing or negative skin or head and neck squamous cell carcinoma that GM-CSF expression is associated with a highly angiogenic and invasive tumor phenotype. To determine the functional contribution of GM-CSF to tumor invasion, we stably transfected a GM-CSF negative colon adenocarcinoma cell line HT-29 with GM-CSF or treated the same cell line with exogenous GM-CSF. While GM-CSF overexpression and treatment reduced tumor cell proliferation and tumor growth in vitro and in vivo, respectively, it contributed to tumor progression. Together with an enhanced migratory capacity in vitro, we observed a striking increase in tumor cell invasion into the surrounding tissue concomitant with the induction of an activated tumor stroma in GM-CSF overexpressing or GM-CSF treated tumors. In a complex 3D in vitro model, enhanced GM-CSF expression was associated with a discontinued basement membrane deposition that might be mediated by the increased expression and activation of MMP-2, -9, and -26. Treatment with GM-CSF blocking antibodies reversed this effect. The increased presence and activity of these tumor cell derived proteases was confirmed in vivo. Here, expression of MMP-26 protein was predominantly located in pre- and early-invasive areas suggesting MMP-26 expression as an early event in promoting GM-CSF dependent tumor invasion.

Interleukin-6 (IL-6) is one of the major inflammatory interleukins that has been linked to cancer progression. In our model for human skin squamous cell carcinoma (SCC), IL-6 expression is strongly upregulated upon progression from benign tumors to highly malignant, metastasizing SCCs. We now demonstrate that IL-6 promotes malignant and invasive tumor growth in human skin SCCs by inducing cell type specific cytokine profiles in tumor keratinocytes and stromal fibroblasts, activating the latter towards a tumor associated fibroblast (TAF) phenotype. In three-dimensional organotypic cocultures in vitro invasive growth of IL-6 overexpressing tumor keratinocytes, is associated with increased expression of matrix metalloproteinase-2 (MMP-2), MMP-14 and tissue inhibitor of metalloproteinases-2, and clearly depends on IL-6 activated fibroblasts. IL-6-induced secretion of monocyte chemotactic protein-1 (MCP-1) in tumor keratinocytes and of hepatocyte growth factor in fibroblasts is crucial for regulating expression and activation of MMP-2. This functional role of IL-6 is confirmed in vivo. Here MMP-14 and MMP-2 expression occur exclusively in surface transplants of IL-6 overexpressing keratinocytes and fibroblasts are identified as important source of MMP-2. Our data indicate that tumor keratinocytes derived IL-6 activates stromal fibroblasts towards a TAF phenotype, promoting tumor invasion via enhanced expression and activation of MMP-2.