Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells
[['Lung carcinoma is one of the leading causes of cancer-related deaths worldwide .', 'The main types of lung cancer are small-cell lung carcinoma ( SCLC ) and non-small-cell lung carcinoma ( NSCLC ) .', 'NSCLC is the most frequent form with a prevalence of around 85% and can be classified in squamous-cell carcinoma , large-cell carcinoma , and adenocarcinoma which is the most prevalent subgroup ( 40% ) [1] .', 'Because lung cancer metastasizes already at early stages independent of the tumor size [2] , most of the patients receive chemotherapeutic agents such as cisplatin .', 'As a side effect of chemotherapy , as well as due to tumor-related effects , anemia frequently occurs [3] .', 'Anemia is treated either by blood transfusion or by erythropoiesis stimulating agents ( ESAs ) such as erythropoietin ( EPO ) alfa or beta [4] .', 'EPO is the key regulator of red blood cell production and ensures survival , proliferation and differentiation of erythroid progenitors at the colony forming unit-erythroid ( CFU-E ) stage in the fetal liver , the adult bone marrow and spleen .', 'Biosynthesis of EPO in the kidney is stimulated by reduced blood oxygen levels [5] .', 'Unfortunately , recent studies suggested that EPO treatment could reduce the overall survival of NSCLC patients [6] .', 'Furthermore , expression of the EPO receptor ( EPOR ) has been detected in some tumors and cancer cells including NSCLC cells [7–10] .', 'Co-expression of EPO and the EPOR has been shown to be associated with poor survival of NSCLC patients , even at stage I [11] .', 'Because of these ambivalent effects , the treatment of lung cancer patients with EPO in the context of cancer-related anemia is controversially discussed [12 , 13] .', 'In addition to clinically administered recombinant EPO , endogenous EPO produced in the kidney may also influence cancer cells .', 'It has been speculated that EPO might affect the survival of cancer cells [14] .', 'The key signaling pathway that is activated by EPO binding to the EPOR and that is involved in survival signaling is the Janus kinase ( JAK ) 2 / signal transducer and activator of transcription ( STAT ) 5 pathway .', 'EPOR is a member of the cytokine receptor superfamily and is present at the cell surface as a homodimer [15] .', 'Upon EPO binding , the receptor undergoes conformational changes and activates the pre-bound tyrosine kinase JAK2 [16] .', 'The activated JAK2 is transphosphorylated and phosphorylates ( p ) tyrosine residues on the EPOR cytoplasmic domain that serve as docking site for the interaction with Src-homology ( SH ) 2-domain containing proteins .', 'The phosphorylated EPOR-JAK2 complex is able to bind and phosphorylate the latent transcription factor STAT5 .', 'Subsequently , pSTAT5 forms dimers that translocate to the nucleus , where they induce the transcription of target genes .', 'JAK2/STAT5 target genes such as cytokine-inducible SH2-containing protein ( CISH mRNA , translated to CIS ) and suppressor of cytokine signaling 3 ( SOCS3 mRNA , translated to SOCS3 ) are expressed that act as negative regulators of JAK2/STAT5 signaling at the EPOR/JAK2 level [17] .', 'Specifically , CIS inhibits STAT5 activation by binding to the EPOR , whereas SOCS3 binds to the activated receptor and the kinase domain of JAK2 , thereby inhibiting its tyrosine kinase activity [18–21] .', 'In addition , protein tyrosine phosphatases ( PTPs ) negatively regulate the activated EPOR-JAK2 complex [21] .', 'It has been shown that the PTP SHP1 negatively regulates EPOR signaling in hematopoietic cells [22] .', 'EPO acts on the erythroid lineage , particularly on CFU-E cells residing in the bone marrow of human adults .', 'It is difficult to obtain primary human CFU-E cells in adequate numbers to perform time-resolved biochemical experiments required for mathematical modeling .', 'However , murine CFU-E cells can be readily purified from fetal mouse liver preparations subjected to negative depletion with lineage-specific antibodies [23] and thereby can be isolated in sufficient quantities for data-based mathematical modeling .', 'Therefore , we employed murine CFU-E cells as a proxy for human CFU-E cells and as representative for healthy erythroid cells .', 'Recently , an ordinary differential equation ( ODE ) model of the EPO-induced JAK2/STAT5 pathway in murine CFU-E cells was reported [19] that focused on the distinct roles of SOCS3 and CIS and linked the integrated nuclear pSTAT5 response to cell survival .', 'To investigate differences of EPO-induced JAK2/STAT5 signaling in hematopoietic and non-hematopoietic cell types like cancer cells , such an ODE model can be generalized .', 'The parameters of the generalized mathematical model can be estimated individually using time- and dose-resolved quantitative data for the studied cell types .', 'A major challenge is to identify significant differences in parameter values that are characteristic of a respective cell type .', 'In general , one could discriminate between cell type-specific parameters and cell type-independent parameters .', 'Mathematically , all combinations of parameters being either cell type-specific or cell type-independent have to be tested .', 'Finding the exact solution of this model selection task is challenging as the number of candidate models grows exponentially with the number of model parameters [24] .', 'Established approaches to approximate the solution of such a selection task have been developed mainly in classical statistics and comprise approaches such as lasso ( least absolute shrinkage and selection operator ) [25] , elastic net [26] , forward selection and backward elimination [27] , and combinations thereof [28] .', 'These approaches favor small models by penalizing increasing numbers of parameters .', 'Their main difference is the metric quantifying the model complexity .', 'In the lasso approach , the L1 metric , i . e . , the absolute value of differences to zero is used .', 'It has been shown that under certain conditions , the L1 metric produces similar results as the L0 metric that penalizes only the number of parameters [29] .', 'However , due to convexity and continuity of the L1 metric , it is considered favorable over L0 [30] .', 'The L2 metric , on the other hand , does not lead to a sparse ( parsimonious ) model and is therefore not suitable for our task .', 'Because L1 regularization techniques have been mainly developed for linear models , only a subset of existing algorithms can be employed for models that are nonlinear , which is a prevalent property of mathematical models based on coupled ODEs .', 'Additional issues arising from nonlinearity comprise local optima , non-identifiabilities , and decreased performance of numerical calculations in general .', 'To tackle these challenges , state-of-the-art implementations for optimization in nonlinear ODE systems have been developed [31] .', 'We utilize these established approaches and extend their functionality by identifying parameter differences with L1 regularization .', 'For a successful implementation , strategies to consider discontinuities in the derivatives and adaptation of convergence criteria have to be taken into account [32] .', 'Here , we show on the basis of simulated data that our implementation for modeling of nonlinear ODE systems in combination with an L1 approach is a suitable method to infer cell type-specific parameters of two cell types and demonstrate the interpretation of the results .', 'This approach is then applied to assess parameter differences in EPO-induced JAK2/STAT5 signaling between the NSCLC cell line H838 that expresses the EPOR [9 , 10 , 33] and CFU-E cells .', 'Furthermore , a sensitivity analysis of the biological readout is performed to discover potential targets for intervention that specifically reduce the effects of EPO on the cancer cells while leaving the EPO-induced survival of hematopoietic cells unaffected .'], ['We previously showed that the NSCLC cell line H838 expresses the EPOR at the mRNA and protein level , albeit at rather low levels [10] .', 'To more robustly detect EPO-induced phosphorylation of signaling components and expression of target genes in this cell line , we stably expressed high levels of HA-tagged human EPOR ( HA-hEPOR ) in H838 cells by retroviral transduction ( H838-HA-hEPOR ) .', 'The amount of EPOR protein present in the human lung cancer cell line H838 and its derivative H838-HA-hEPOR was determined by immunoprecipitation followed by quantitative immunoblotting and compared to the amount present in mouse CFU-E cells ( Fig 1A ) .', 'A protein band in the immunoblot that corresponds to the EPOR was detected in the analyzed cell types .', 'As shown in S1A and S1B Fig , the hEPOR levels in H838-HA-hEPOR cells were increased 170-fold compared to H838 cells .', 'The shift in molecular weight of the EPOR in H838-HA-hEPOR cells is most likely due to the HA-tag introduced into the EPOR .', 'For absolute quantification of receptor expression levels , signal intensities of purified protein standards of mouse or human GST-tagged EPOR [34] were used to establish calibration curves and to subsequently estimate the number of EPOR molecules per cell ( S1D Fig ) .', 'As shown in S1 Table , H838-HA-hEPOR cells harbor 620 000 ± 200 000 hEPOR molecules per cell , H838 cells 3 600 ± 1 200 hEPOR molecules per cell and CFU-E cells 4 300 ± 2 200 mEPOR molecules per cell .', 'To assess the activation of signal transduction we examined ligand-induced phosphorylation of the EPOR ( Fig 1A ) .', 'H838 and H838-HA-hEPOR cells were treated for 10 min with 10 U/ml EPO beta and CFU-E cells for 10 min with 5 U/ml EPO alfa or were left untreated .', 'In each case , a band in the immunoblot corresponding to the phosphorylated EPOR was detectable in the treated samples , but was absent in the unstimulated controls .', 'The amount of pEPOR in H838-HA-hEPOR upon stimulation with 10 U/ml EPO beta was only approximately 12-fold increased compared to H838 ( S1C Fig ) , indicating that in cells expressing elevated levels of the EPOR such as H838-HA-hEPOR possibly the transport of the receptor to the plasma membrane or phosphorylation of the EPOR by JAK2 might be limiting .', 'S2A and S2B Fig show that EPO alfa and EPO beta activate EPO-induced signaling at a comparable extent .', 'Further , we examined EPO-induced JAK2/STAT5 survival signaling in the NSCLC cell line H838 and its derivative H838-HA-hEPOR because it had been reported that EPO induces survival signaling via the JAK2/STAT5 pathway in CFU-E cells [19] .', 'In H838 cells , pJAK2 was detected upon stimulation with 100 U/ml and in H838-HA-hEPOR an EPO dose-dependent increase in JAK2 phosphorylation was observed ( Fig 1B ) .', 'Because the latent transcription factor STAT5 is a key mediator of the JAK2/STAT5 signaling pathway , we determined the degree of STAT5 phosphorylation by mass spectrometry in H838 and H838-HA-hEPOR cells treated with different doses of EPO beta ( Fig 1C ) .', 'The highest degree of STAT5 phosphorylation was observed at 10 U/ml EPO beta , with approximately 60% of STAT5 in H838-HA-hEPOR cells being phosphorylated and 3% in H838 cells .', 'As quantified in S2C and S2D Fig and summarized in S1 Table both H838 and H838-HA-hEPOR harbor approximately 1 200 JAK2 and 90 000 STAT5 molecules compared to 24 000 JAK2 and 20 000 STAT5 molecules in CFU-E .', 'To also consider the cytoplasmic and nuclear volume of the cell types analyzed , trypsinized cells were analyzed by fluorescence microscopy and the diameter of the cell and of the nucleus was determined ( S3A and S3B Fig ) .', 'CFU-E cells with a cell volume of 700 μm3 and a nuclear volume of 300 μm3 are much smaller compared to H838 cells that have a cell volume of around 14 000 μm3 and a nuclear volume of around 2 000 μm3 .', 'The respective volumes were utilized to convert number of molecules per cell into cell type-specific initial concentrations , which are summarized in S1 Table .', 'To test whether EPO influences survival and may reduce cisplatin-induced apoptosis in NSCLC cells , H838 and H838-HA-hEPOR cells were treated with 5 mg/l of the chemotherapeutic agent cisplatin in combination with 10 U/ml EPO beta or were left untreated for three days .', 'Cell viability was measured every 24 hours by the CellTiter-Blue assay ( Fig 1D , S4 Fig ) .', 'In untreated proliferating H838 and H838-HA-hEPOR cells , a two- to three-fold increase in cell numbers was observed .', 'No significant impact of EPO beta on cell viability was detected during this observation period .', 'The treatment of H838 and H838-HA-hEPOR with 5 mg/l cisplatin reduced cell viability or at least decreased proliferation compared to the untreated control .', 'Interestingly , co-treatment with 10 U/ml EPO beta decreased the impact of cisplatin in H838 and H838-HA-hEPOR at each time point .', 'While there was some variability in the response of these cells to cisplatin , the rescue effect induced by EPO beta was consistent .', 'To validate these findings , the Casper3-GR FRET ( fluorescence resonance energy transfer ) -based sensor was expressed in H838-HA-hEPOR ( H838-HA-hEPOR-Casper3-GR ) and caspase-3 activity , a key indicator for apoptotic responses , was measured by life-cell imaging in a time-resolved manner ( Fig 1E , S5 Fig ) .', 'As a positive control 10 μM staurosporine was applied that is known to rapidly induce caspase-3 .', 'Indeed , the effect of staurosporine was observed within 5 hours upon treatment whereas in untreated cells no peak of caspase-3 activation was detected .', 'Upon treatment with 5 mg/l cisplatin the maximum peak of caspase-3 activity was reached at around 24 hours followed by a strong signal decrease due to cells undergoing apoptosis .', 'Upon co-treatment with 10 U/ml or 500 U/ml EPO the amplitude of the cisplatin-induced maximal caspase-3 activation showed an EPO dose-dependent reduction , which is in line with the cell viability assay , and a peak shift to around 15 hours .', 'Treatment with EPO alone did not induce caspase-3 activation .', 'Concluding , we demonstrated that , similar to CFU-E , the EPOR expressed in H838 cells was capable of activating the JAK2/STAT5 signaling cascade .', 'Further , in line with the observation that EPO-induced activation of JAK2/STAT5 signaling correlates with survival signaling [19] , we showed in co-treatment experiments that EPO reduces the extent of cisplatin-induced apoptosis in H838 and H838-HA-hEPOR cells and thus exhibits a rescuing effect in the lung cancer cell line and its derivative .', 'Due to the similarity of the core signaling components , it is difficult to predict cell type-specific differences in the signaling network based only on experimental observations .', 'We developed a systematic mathematical modeling approach for the unbiased identification of cell type-specific differences with the aim to propose strategies to exclusively target the lung cancer cells but not the erythroid progenitor cells .', 'To predict cell type-specific model parameters between two different cell types , we propose an ODE-based mathematical modeling strategy in combination with a L1 regularization method .', 'To show that this approach is capable to identify these parameter differences , first an in silico model with simulated data was investigated .', 'The exemplary mathematical model mimics a two-step phosphorylation cascade in two different cell types , in which a protein ( Protein ) is irreversibly converted into a phosphorylated protein ( pProtein ) with rate k1 followed by another phosphorylation step resulting in a doubly phosphorylated protein ( ppProtein ) .', 'This second step can be reversed by a dephosphorylation reaction ( Fig 2 ) .', 'The ppProtein and its intermediate pProtein equilibrate depending on the ratio of the forward ( k2 ) and backward reaction ( k3 ) .', 'The corresponding differential equation system is given by:', 'd[Protein]dt=−k1⋅\u2009[Protein]d[pProtein]dt=k1⋅[Protein]−k2⋅[pProtein]\xa0+\xa0k3⋅[ppProtein]d[ppProtein]dt=k2⋅[pProtein]\xa0−\xa0k3⋅[ppProtein]', 'The model structure and the parameters k1 and k2 were chosen to be identical for cell type 1 and cell type 2 .', 'In contrast , the following two parameters were set as cell type-specific: The rate k3 was set to 0 . 5 min-1 in cell type 1 and 0 . 1 min-1 in cell type 2 and the parameter [Protein]t = 0 was defined as 1 nM in cell type 1 and 2 nM in cell type 2 .', 'The initial concentration of pProtein and ppProtein at t = 0 min were assumed to be zero in both cell types .', 'The ODE solution was calculated using the D2D software [31] and data ( black dots ) were simulated by adding normally distributed noise ( σ = 0 . 1 ) ( Fig 2 ) .', 'The model trajectories are depicted as black lines and the shading represents the noise distribution .', 'To approximate a realistic setting , only two proteins were assumed to be measurable and sampling time points were not equal .', 'Hence , data was available for Protein ( n = 21 ) and ppProtein ( n = 10 ) .', 'To estimate parameters and identify cell type-specific differences given only by the model structure and the simulated data , the model was parameterized with parameter pi for cell type 1 and ri ∙ pi for cell type 2 , with ri denoting fold-changes between both cell types .', 'After calculating the maximum likelihood estimates individually for cell type 1 and 2 , the regularization weight λ of the constrained likelihood', 'C\xa0=\xa0Lcell\xa0type\xa01+Lcell\xa0type\xa02+λ∑i|log10ri|', 'was scanned .', 'Here , ℒcell type 1 and ℒcell type 2 denote the likelihood ( i . e . the negative two-fold log-likelihood ) of the respective cell type and the last term regularizes the fold-changes ri of parameters between the cell types .', 'If the parameter pi is the same in both cell types , ri = 1 and the regularization term is zero .', 'To obtain the regularization path for all parameters , we gradually increased λ from 10−4 to 104 and re-estimated the constrained likelihood at each step .', 'With increasing regularization weight λ , the number of cell type-specific parameters decreased until all parameters were independent from the cell type ( Fig 3A ) .', 'To determine the optimal regularization weight λ , i . e . to statistically evaluate the minimum number of cell type-specific parameters that are necessary for the model to sufficiently describe the data ( parsimonious model ) , the likelihood ratio test was utilized .', 'The L1 regularization was used to select cell type-specific parameters .', 'To reduce bias , the parameters of the non-regularized parsimonious model were then estimated in a second step .', 'The likelihood ratio test statistic D = ℒλ − ℒfull was used , where ℒfull denotes the likelihood of the full model with only cell type-specific parameters ( M ) and ℒλ the likelihood of a model with N cell type-specific parameters that were selected for a given λ .', 'If D < χ2dof , α , the smaller model cannot be rejected based on the χ2-distribution for a given confidence level α and degrees of freedom ( dof = M − N ) .', 'In Fig 3B , the test statistic D is shown by a solid blue line and the statistical threshold based on the likelihood ratio test is shown as a dashed red line .', 'The crossing point of both lines defines the parsimonious model , in which only relevant parameters are defined as cell type-specific .', 'The so-called regularization path shown in Fig 3C indicates the fold-changes between cell type 1 and cell type 2 for all parameters at a given regularization weight .', 'The colors red or blue show whether a parameter is larger in cell type 1 or 2 , respectively .', 'The regularization path is also shown in S6A Fig as a line plot .', 'In our example , the parsimonious model that is indicated by the vertical dashed line ( Fig 3A , 3B and 3C ) has two cell type-specific parameters .', 'These two parameters were the rate k3 and the initial concentration of Protein [Protein]t = 0 , which could be reconstructed from the example model as cell type-specific ( indicated with asterisks in Fig 3C and S6A Fig ) .', 'We determined the relative difference and by a profile likelihood approach [35] the corresponding confidence interval ( S6B Fig ) : [Protein]t = 0 is larger in cell type 2 by a factor of 1 . 88 ± 0 . 17 ( true value is 2 ) and k3 is larger in cell type 1 by a factor of 4 . 71 ± 1 . 20 ( true value is 5 ) .', 'To illustrate how well individual models that differ in the number of cell type-specific parameters can describe the simulated data , model trajectories of three different scenarios were plotted with the simulated data ( Fig 3D ) .', 'The model with only cell type-specific parameters ( green dashed line ) , which is the largest model and has the highest degree of freedom , was capable to describe all data sets .', 'The smallest model with no cell type-specific parameters ( blue dashed line ) was not in statistical agreement with the simulated data , because e . g . for both cell types the species “ppProtein” was not correctly described .', 'In line with the likelihood ratio test , the parsimonious model with only relevant cell type-specific parameters ( solid red line ) described the simulated data to a comparable extent as the model with only cell type-specific parameters .', 'In sum , the L1 regularization was able to reconstruct cell type-specific parameters for simulated data using a single scan of the regularization weight λ , suggesting that our implementation for dynamic ODE-based mathematical modeling in combination with L1 regularization is appropriate to reveal differences between two different cell types .', 'To pinpoint cell type-specific differences in EPO-induced JAK2/STAT5 signaling in CFU-E cells and H838 & H838-HA-hEPOR cells by the approach introduced above , the previously published mathematical model of EPO-induced JAK2/STAT5 signaling in murine CFU-E cells [19] was used as reference model structure: The JAK2/STAT5 model for mCFU-E cells described the EPO-induced JAK2 activation in the EPOR-JAK2 complex , the subsequent phosphorylation of the EPO receptor , attenuation by the phosphatase SHP1 , the activation of the transcription factor STAT5 by the EPOR-JAK2 complex and the STAT5 transport from cytosol into and out of the nucleus , as well as the induced transcription and translation of the negative feedback regulators SOCS3 and CIS .', 'To describe EPO-induced JAK2/STAT5 signaling in human H838 and H838-HA-hEPOR cells , a generalized model structure was developed by adding the following components to the reference model:', '( i ) Basal RNA activation rates were included , because we observed that basal CISH mRNA and SOCS3 mRNA is also reduced by inhibiting transcription ( S7A and S7B Fig ) .', '( ii ) A dephosphorylation step of nuclear phosphorylated ( np ) STAT5 was included to be able to distinguish dephosphorylation and nuclear export of STAT5 [36] .', '( iii ) The regulation of the SOCS3 promotor binding activity by npSTAT5 was described by a Hill coefficient to allow non-linear transcriptional activity .', 'In addition , the receptor phosphatase SHP1 that is known to be restricted to hematopoietic cells was substituted by the general term PTP ( Fig 4 ) .', 'The generalized model structure was parameterized based on cell type-specific quantitative data .', 'First , the model was calibrated by directly implementing cell type-specific quantities for H838 and H838-HA-hEPOR cells such as cellular and nuclear volumes and the initial concentrations of the EPOR as well as JAK2 and STAT5 together with their respective experimental error ( summarized in S1 Table ) .', 'As the ratio of the EPOR and JAK2 was different for H838 and H838-HA-hEPOR cells possibly resulting in a saturation of the phosphatase in H838-HA-hEPOR cells , distinct properties were assumed for the phosphatase PTP in H838-HA-hEPOR cells .', 'Except for this difference and the number of receptors on the surface as shown in S1 Fig , the JAK2/STAT5 signaling models for H838 and H838-HA-hEPOR cells were parameterized identically .', 'Therefore , H838 and H838-HA-hEPOR cells were treated as the same cell type but with different conditions .', 'To calibrate the model for CFU-E cells , quantitative data of the reference model [19] was reutilized ( open circles in Fig 5 ) .', 'For the NSCLC cell line H838 and its derivative H838-HA-hEPOR , quantitative time-resolved data was generated ( black closed circles in Fig 5 ) .', 'For H838 cells , the dynamics of pEPOR , pJAK2 and pSTAT5 and the expression of CISH mRNA upon stimulation with 10 U/ml EPO beta were determined .', 'For H838-HA-hEPOR cells , the amount of total and phosphorylated STAT5 , pEpoR , pJAK2 and the expression of CISH and SOCS3 mRNA were measured upon stimulation with 10 U/ml EPO beta .', 'While the temporal changes of pEPOR were similar in CFU-E , H838-HA-hEPOR and H838 cells , the dynamics of CISH mRNA was more transient in CFU-E cells , while pSTAT5 was more sustained in H838-HA-hEPOR cells than in H838 cells .', 'SOCS3 mRNA , on the other hand , was more sustained in CFU-E cells than in H838-HA-hEPOR cells .', 'Thus , we observed substantial differences in the dynamics of signaling components in the three cell types analyzed .', 'We performed a multitude of additional measurements with different conditions ( S7 Fig ) .', 'The basal expression levels of CISH mRNA and SOCS3 mRNA were determined for CFU-E and H838-HA-hEPOR cells by applying the transcription inhibitor actinomycin D . Time- and dose-resolved protein quantification was performed by quantitative immunoblotting and relative mRNA quantification by qRT-PCR .', 'The degree of STAT5 phosphorylation ( pSTAT5 ) was measured for both the lung cancer cell line and its derivative by quantitative mass spectrometry .', 'The generalized model structure comprised 25 kinetic parameters and 3 initial concentrations ( the EPOR-JAK2 complex , STAT5 and PTP at time point 0 ) .', 'Assuming that all kinetic parameters and initial concentrations were cell type-specific ( i . e . different parameters for CFU-E and for H838 & H838-HA-hEPOR cells ) , this model was calibrated based on the experimental data of CFU-E ( 516 data points ) using 83 additional observation parameters and of H838 and H838-HA-hEPOR cells ( 625 data points ) using 77 additional observation parameters .', 'For global optimization of the likelihood , a multi-start deterministic strategy was applied [37] .', 'The model could describe all data sets of the cell types tested , shown representatively by the model trajectory of the model with only cell type-specific parameters ( dashed green line in Fig 5 ) .', 'Concluding , we identified a model structure that was able to describe the data sets of EPO-induced JAK2/STAT5 signaling in CFU-E , H838 and H838-HA-hEPOR cells .', 'Our experimental observations suggested that core reactions of the pathway operate comparable in the different cell systems .', 'We therefore tested whether it is possible to identify cell type-independent and cell type-dependent parameters to establish a parsimonious model .', 'The parsimonious model is defined as the model with the smallest number of cell type-specific parameters that is still compatible with the experimental data .', 'To infer cell type-specific parameters , the L1 regularization strategy was applied while parameterizing the mathematical model based on the experimental data established for the different cell types .', 'We tested the 25 kinetic parameters and the initial concentrations of PTP , while the initial concentrations of the EPOR-JAK2 complex and STAT5 were fixed to the measured values ( S1 Table ) .', 'As expected , by increasing the regularization weight the number of cell type-specific parameters was gradually decreased ( Fig 6A ) .', 'In analogy to the example ( Fig 3 ) , the parsimonious model was determined using the likelihood ratio test ( blue line in Fig 6B ) .', 'The statistical threshold corresponding to a significance level α = 0 . 05 is shown by the red dashed line .', 'At the crossing point of these two lines , the parsimonious model with relevant cell type-specific parameters is defined and depicted by the dashed black vertical line in all panels of Fig 6 .', 'The parsimonious model with the minimal number of cell type-specific parameters ( red line , Fig 5 ) resulted in trajectories describing the data and showed similar performance as the model with only cell type-specific parameters ( dashed green line , Fig 5 ) .', 'On the contrary , the model with only cell type-independent parameters ( dashed blue line , Fig 5 ) was not capable of representing the dynamics of the data as evidenced e . g . by the difference of the model trajectory and the experimental data of SOCS3 mRNA .', 'The parsimonious model simultaneously described all experimental data measured in CFU-E ( S8 Fig ) , H838-HA-hEPOR ( S9A and S9B Fig ) and H838 ( S9B and S9C Fig ) .', 'Seven relevant cell type-specific parameters are highlighted with an asterisk in the regularization path of the 26 parameters in Fig 6C and in an alternative representation in S10A Fig . We ensured significance by calculating the confidence intervals of these parameter differences ( S10B Fig ) .', 'Table 1 provides a description of the 26 parameters and the model-predicted differences between CFU-E cells and H838 & H838-HA-hEPOR cells .', 'The cell type-specific parameters predicted to have higher values in CFU-E cells ( depicted in red , Fig 6C ) were the CISH mRNA turnover rate ( CISHRNAturn ) , the activation rate of the EPOR by JAK2 ( EPORactJAK2 ) and the activation rate of JAK2 by EPO ( JAK2actEPO ) .', 'In contrast , the parameters that were predicted to have higher values in the H838 & H838-HA-hEPOR cells ( depicted in blue , Fig 6C ) were the parameter inversely defining the delay in SOCS3 mRNA production ( SOCS3RNAdelay ) , the SOCS3 promoter activity ( SOCS3prom ) the import rate of pSTAT5 into the nucleus ( STAT5imp ) and the deactivation rate of STAT5 in the nucleus ( nSTAT5deact ) .', 'The parameter that showed strongest evidence for a difference between both cell types was the parameter regulating npSTAT5-induced SOCS3 promoter activity ( SOCS3prom ) ( Fig 6C ) .', 'The model predicted a linear correlation of the SOCS3 mRNA production rate and the concentration of npSTAT5 for CFU-E cells ( S11A Fig ) .', 'In contrast , the same prediction analysis for H838 & H838-HA-hEPOR cells indicated a threshold behavior of the SOCS3 mRNA production rate with respect to npSTAT5 .', 'The analysis showed that in these cells a certain amount of activated STAT5 has to be present in the nucleus to induce SOCS3 mRNA synthesis ( S11B Fig ) .', 'To test whether this difference between cell types can be explained by differences in promoter binding elements or epigenetic modifications , the human and murine SOCS3 promoter was analyzed for STAT5 binding sites .', 'Indeed , the murine promoter contains four STAT5 binding sites , whereas the human harbors only two ( S11C Fig ) .', 'Additionally , we performed methylation analyses and demonstrated that the SOCS3 promoter is accessible in both CFU-E and H838 cells ( S11C Fig ) .', 'Taken together , the selected parsimonious model could simultaneously describe the data sets of CFU-E , H838 and H838-HA-hEPOR cells .', 'Seven relevant cell type-specific parameters were identified and three of these parameters had higher values in CFU-E cells and four had higher values in H838 & H838-HA-hEPOR cells .', 'The parsimonious model predicted several cell type-specific parameters comprising mRNA processing: the turnover rate of the CISH mRNA , the SOCS3 promoter activity and the SOCS mRNA delay parameter .', 'Therefore , we selected mRNA processing as a biological process that can be measured experimentally and is connected to parameters identified by the model as cell type-specific for experimental validation of the parsimonious model .', 'We hypothesized that by the application of the transcriptional inhibitor actinomycin D the dynamics of CISH mRNA and SOCS3 mRNA would be altered in a cell type-specific manner .', 'First , the dynamics of CISH mRNA and SOCS3 mRNA were predicted for CFU-E and H838-HA-hEPOR cells for 300 min upon treatment with EPO ( black ) or EPO in combination with actinomycin D ( blue ) ( Fig 7 ) .', 'We proposed by experimental design that the transcriptional inhibitor should be applied at the predicted peak of mRNA expression upon EPO stimulation , which was after 60 min for CFU-E cells treated with 5 U/ml EPO alfa and after 30 min for H838-HA-hEPOR stimulated with 10 U/ml EPO be
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['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 .']
['A major challenge in the development of therapeutic interventions is the selective inhibition of a signal transduction pathway in one cell type such as a cancer cell leaving the other cell type such as a healthy cell as unaffected as possible .', 'Here , we propose a new approach that combines mathematical modeling based on quantitative experimental data with statistical methods .', 'We demonstrate based on simulated data that our approach can determine which parameters are the same and which parameters differ in two exemplary cell types .', 'We compare a lung cancer cell line to the precursor cells of red blood cells .', 'We show that the same signal transduction network induced by erythropoietin ( EPO ) , a hormone that is frequently employed to treat anemia in cancer patients , regulates survival of both cell types .', 'Based on our experimental data in combination with our computational approach , we identify seven cell type-specific differences in this signaling pathway .', 'Our strategy allows predicting therapeutic targets that could be inhibited to interfere with survival of lung cancer cells while leaving production of red blood cells unaffected .']
['phosphorylation', 'medicine and health sciences', 'molecular probe techniques', 'messenger rna', 'cancers and neoplasms', 'immunoblotting', 'simulation and modeling', 'oncology', 'immunoprecipitation', 'molecular biology techniques', 'research and analysis methods', 'proteins', 'lung and intrathoracic tumors', 'stat signaling', 'molecular biology', 'precipitation techniques', 'biochemistry', 'rna', 'signal transduction', 'cell biology', 'post-translational modification', 'nucleic acids', 'biology and life sciences', 'non-small cell lung cancer', 'cell signaling']