Transforming growth factor {beta} (TGF-{beta})-Smad target gene protein tyrosine phosphatase receptor type kappa is required for TGF-{beta} function

Transforming growth factor beta (TGF-beta) inhibits proliferation and promotes cell migration. In TGF-beta-treated MCF10A mammary epithelial cells overexpressing HER2 and by chromatin immunoprecipitation, we identified novel Smad targets including protein tyrosine phosphatase receptor type kappa (PTPRK). TGF-beta up-regulated PTPRK mRNA and RPTPkappa (receptor type protein tyrosine phosphatase kappa, the protein product encoded by the PTPRK gene) protein in tumor and nontumor mammary cells; HER2 overexpression down-regulated its expression. RNA interference (RNAi) of PTPRK accelerated cell cycle progression, enhanced response to epidermal growth factor (EGF), and abrogated TGF-beta-mediated antimitogenesis. Endogenous RPTPkappa associated with EGF receptor and HER2, resulting in suppression of basal and ErbB ligand-induced proliferation and receptor phosphorylation. In MCF10A/HER2 cells, TGF-beta enhanced cell motility, FAK phosphorylation, F-actin assembly, and focal adhesion formation and inhibited RhoA activity. These responses were abolished when RPTPkappa was eliminated by RNA interference (RNAi). In cells expressing RPTPkappa RNAi, phosphorylation of Src at Tyr527 was increased and (activating) phosphorylation of Src at Tyr416 was reduced. These data suggest that (i) RPTPkappa positively regulates Src; (ii) HER2 signaling and TGF-beta-induced RPTPkappa converge at Src, providing an adequate input for activation of FAK and increased cell motility and adhesion; and (iii) RPTPkappa is required for both the antiproliferative and the promigratory effects of TGF-beta.     

Relationships between Signaling Pathway Usage and Sensitivity to a Pathway Inhibitor: Examination of Trametinib Responses in Cultured Breast Cancer Lines

Cellular signaling pathways involving mTOR, PI3K and ERK have dominated recent studies of breast cancer biology, and inhibitors of these pathways have formed a focus of numerous clinical trials. We have chosen trametinib, a drug targeting MEK in the ERK pathway, to address two questions. Firstly, does inhibition of a signaling pathway, as measured by protein phosphorylation, predict the antiproliferative activity of trametinib? Secondly, do inhibitors of the mTOR and PI3K pathways synergize with trametinib in their effects on cell proliferation? A panel of 30 human breast cancer cell lines was chosen to include lines that could be classified according to whether they were ER and PR positive, HER2 over-expressing, and “triple negative”. Everolimus (targeting mTOR), NVP-BEZ235 and GSK2126458 (both targeting PI3K/mTOR) were chosen for combination experiments. Inhibition of cell proliferation was measured by IC₅₀ values and pathway utilization was measured by phosphorylation of signaling kinases. Overall, no correlation was found between trametinib IC₅₀ values and inhibition of ERK signaling. Inhibition of ERK phosphorylation was observed at trametinib concentrations not affecting proliferation, and sensitivity of cell proliferation to trametinib was found in cell lines with low ERK phosphorylation. Evidence was found for synergy between trametinib and either everolimus, NVP-BEZ235 or GSK2126458, but this was cell line specific. The results have implications for the clinical application of PI3K/mTOR and MEK inhibitors.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.     

The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes

The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY¹¹¹², the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12.     

A multiscale modeling approach to investigate molecular mechanisms of pseudokinase activation and drug resistance in the HER3/ErbB3 receptor tyrosine kinase signaling network

Multiscale modeling provides a powerful and quantitative platform for investigating the complexity inherent in intracellular signaling pathways and rationalizing the effects of molecular perturbations on downstream signaling events and ultimately, on the cell phenotype. Here we describe the application of a multiscale modeling scheme to the HER3/ErbB3 receptor tyrosine kinase (RTK) signaling network, which regulates critical cellular processes including proliferation, migration and differentiation. The HER3 kinase is a topic of current interest and investigation, as it has been implicated in mechanisms of resistance to tyrosine kinase inhibition (TKI) of EGFR and HER2 in the treatment of many human malignancies. Moreover, the commonly regarded status of HER3 as a catalytically inactive 'pseudokinase' has recently been challenged by our previous study, which demonstrated robust residual kinase activity for HER3. Through our multiscale model, we investigate the most significant molecular interactions that contribute to potential mechanisms of HER3 activity and the physiological relevance of this activity to mechanisms of drug resistance in an ErbB-driven tumor cell in silico. The results of our molecular-scale simulations support the characterization of HER3 as a weakly active kinase that, in contrast to its fully-active ErbB family members, depends upon a unique hydrophobic interface to coordinate the alignment of specific catalytic residues required for its activity. Translating our molecular simulation results of the uniquely active behavior of the HER3 kinase into a physiologically relevant environment, our HER3 signaling model demonstrates that even a weak level of HER3 activity may be sufficient to induce AKT signaling and TKI resistance in the context of an ErbB signaling-dependent tumor cell, and therefore therapeutic targeting of HER3 may represent a superior treatment strategy for specific ErbB-driven cancers.     

Differential gene expression profiling of human epidermal growth factor receptor 2-overexpressing mammary tumor

Human epidermal growth factor receptor 2 (HER2) is highly expressed in approximately 30% of breast cancer patients, and substantial evidence supports the relationship between HER2 overexpression and poor overall survival. However, the biological function of HER2 signal transduction pathways is not entirely clear. To investigate gene activation within the pathways, we screened differentially expressed genes in HER2-positive mouse mammary tumor using two-directional suppression subtractive hybridization combined with reverse dot-blotting analysis. Forty genes and expressed sequence tags related to transduction, cell proliferation/growth/apoptosis and secreted/extracellular matrix proteins were differentially expressed in HER2-positive mammary tumor tissue. Among these, 19 were already reported to be differentially expressed in mammary tumor, 11 were first identified to be differentially expressed in mammary tumor in this study but were already reported in other tumors, and 10 correlated with other cancers. These genes can facilitate the understanding of the role of HER2 signaling in breast cancer.     

The Matricellular Protein Cyr61 Is a Key Mediator of Platelet-derived Growth Factor-induced Cell Migration

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an “outside-in” signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.     

Loss of PTEN permits CXCR4-mediated tumorigenesis through ERK1/2 in prostate cancer cells

Loss of PTEN is frequently observed in androgen-independent prostate cancer, resulting in the deregulation of metastatic events. SDF1α activation of CXCR4 induces signaling pathways that have been implicated in prostate metastasis and progression to an advanced disease. The pathways of CXCR4 and PTEN converge, leading to the promotion and regulation of tumorigenesis, respectively. However, loss of PTEN may permit CXCR4 to progress prostate cancer to an advanced disease. In the present study, we investigated the involvement of PTEN in CXCR4-mediated tumorigenesis. When screening advanced metastatic prostate cancer cell lines for PTEN, we observed a loss of expression in PC3 and LNCaP cells whereas Du145 expressed wild-type PTEN. All three cell lines were positive for surface expression of CXCR4. Reconsitution of PTEN induced a mesenchymal to epithelial like morphologic change and inhibited CXCR4-mediated migration and proliferation in PC3 cells. Downregulation of PTEN by siRNA enhanced the CXCR4-mediated migratory behavior of Du145 cells. By Western blot analysis, we observed that PTEN inhibited basal AKT phosphorylation but not ERK1/2 phosphorylation in PTEN-expressing cells. Upon CXCR4 stimulation, PTEN inhibited ERK1/2 phosphorylation but not phosphorylation of AKT. The CXCR4-mediated migration of PC3 cells was through the ERK1/2 pathway, as confirmed by chemical inhibitors. On the basis of these studies, we suggest that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells through the ERK1/2 pathway. Antagonizing CXCR4 and downstream signaling cascades may provide an efficient approach for treating patients with advanced prostate cancer when hormone therapy fails to the stop the growth and containment of tumors.     

Targeting the Sheddase Activity of ADAM17 by an Anti-ADAM17 Antibody D1(A12) Inhibits Head and Neck Squamous Cell Carcinoma Cell Proliferation and Motility via Blockage of Bradykinin Induced HERs Transactivation

A disintegrin and metalloproteinase 17 (ADAM17) regulates key cellular processes including proliferation and migration through the shedding of a diverse array of substrates such as epidermal growth factor receptor (EGFR) ligands. ADAM17 is implicated in the pathogenesis of many diseases including rheumatoid arthritis and cancers such as head and neck squamous cell carcinoma (HNSCC). As a central mediator of cellular events, overexpressed EGFR is a validated molecular target in HNSCC. However, EGFR inhibition constantly leads to tumour resistance. One possible mechanism of resistance is the activation of alternative EGFR family receptors and downstream pathways via the release of their ligands. Here, we report that treating human HNSCC cells in vitro with a human anti-ADAM17 inhibitory antibody, D1(A12), suppresses proliferation and motility in the absence or presence of the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Treatment with D1(A12) decreases both the endogenous and the bradykinin (BK)-stimulated shedding of HER ligands, accompanied by a reduction in the phosphorylation of HER receptors and downstream signalling pathways including STAT3, AKT and ERK. Knockdown of ADAM17, but not ADAM10, also suppresses HNSCC cell proliferation and migration. Furthermore, we show that heregulin (HRG) and heparin-binding epidermal growth factor like growth factor (HB-EGF) predominantly participate in proliferation and migration, respectively. Taken together, these results demonstrate that D1(A12)-mediated inhibition of cell proliferation, motility, phosphorylation of HER receptors and downstream signalling is achieved via reduced shedding of ADAM17 ligands. These findings underscore the importance of ADAM17 and suggest that D1(A12) might be an effective targeted agent for treating EGFR TKI-resistant HNSCC.     

FoxO转录因子的活性调节及对哺乳动物细胞进程的调控

FoxO转录因子在哺乳动物的细胞分化、增殖和细胞存活中发挥着重要调控作用,其转录活性受PI3K通路、非PI3K依赖通路、乙酰化和泛素化作用等多种途径调控.FoxO受到上游信号分子PI3K/Akt、SGK等的激活,调节靶基因的转录,从而调节哺乳动物细胞周期的进程和凋亡事件.FoxO已成为肿瘤、癌症科学研究的热点之一.     

Slug Controls Stem/Progenitor Cell Growth Dynamics during Mammary Gland Morphogenesis

Background

Morphogenesis results from the coordination of distinct cell signaling pathways controlling migration, differentiation, apoptosis, and proliferation, along stem/progenitor cell dynamics. To decipher this puzzle, we focused on epithelial-mesenchymal transition (EMT) “master genes”. EMT has emerged as a unifying concept, involving cell-cell adhesion, migration and apoptotic pathways. EMT also appears to mingle with stemness. However, very little is known on the physiological role and relevance of EMT master-genes. We addressed this question during mammary morphogenesis. Recently, a link between Slug/Snai2 and stemness has been described in mammary epithelial cells, but EMT master genes actual localization, role and targets during mammary gland morphogenesis are not known and we focused on this basic question.

Methodology/Principal Findings

Using a Slug–lacZ transgenic model and immunolocalization, we located Slug in a distinct subpopulation covering about 10–20% basal cap and duct cells, mostly cycling cells, coexpressed with basal markers P-cadherin, CK5 and CD49f. During puberty, Slug-deficient mammary epithelium exhibited a delayed development after transplantation, contained less cycling cells, and overexpressed CK8/18, ER, GATA3 and BMI1 genes, linked to luminal lineage. Other EMT master genes were overexpressed, suggesting compensation mechanisms. Gain/loss-of-function in vitro experiments confirmed Slug control of mammary epithelial cell luminal differentiation and proliferation. In addition, they showed that Slug enhances specifically clonal mammosphere emergence and growth, cell motility, and represses apoptosis. Strikingly, Slug-deprived mammary epithelial cells lost their potential to generate secondary clonal mammospheres.

Conclusions/Significance

We conclude that Slug pathway controls the growth dynamics of a subpopulation of cycling progenitor basal cells during mammary morphogenesis. Overall, our data better define a key mechanism coordinating cell lineage dynamics and morphogenesis, and provide physiological relevance to broadening EMT pathways.     

Loss of lkb1 Expression Reduces the Latency of ErbB2-Mediated Mammary Gland Tumorigenesis,Promoting Changes in Metabolic Pathways

The tumor suppressor kinase LKB1 is mutated in a broad range of cancers however, the role of LKB1 mammary gland tumorigenesis is not fully understood. Evaluation of human breast cancer tissue microarrays, indicate that 31% of HER2 positive samples lacked LKB1 expression. To expand on these observations, we crossed STK11^fl/fl mice with mice genetically engineered to express activated Neu/HER2-MMTV-Cre (NIC) under the endogenous Erbb2 promoter, to generate STK11^−/−/NIC mice. In these mice, the loss of lkb1 expression reduced the latency of ErbB2-mediated tumorigenesis compared to the latency of tumorigenesis in NIC mice alone. Analysis of STK11^−/−/NIC mammary tumors revealed hyperactivation of mammalian target of rapamycin (mTOR) through both mTORC1 and mTORC2 pathways as determined by the phosphorylation status of ribosomal protein S6 and AKT. Furthermore, STK11^−/−/NIC mammary tumors had elevated ATP levels along with changes in metabolic enzymes and metabolites. The treatment of primary mammary tumor cells with specific mTOR inhibitors AZD8055 and Torin1, that target both mTOR complexes, attenuated mTOR activity and decreased expression of glycolytic enzymes. Our findings underscore the existence of a molecular interplay between LKB1-AMPK-mTORC1 and ErbB2-AKT-mTORC2 pathways with mTOR at its epicenter, suggestive that loss of LKB1 expression may serve as a marker for hyperactivated mTOR in HER2 positive breast cancer and warranting further investigation into therapeutics that target LKB1-AMPK-mTOR and glycolytic pathways.     

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

A conformationally constrained peptidomimetic binds to the extracellular region of HER2 protein

Human epidermal growth factor receptor 2 (HER2) is a member of the human epidermal growth factor receptor kinases (other members include EGFR or HER1, HER3, and HER4) that are involved in signaling cascades for cell growth and differentiation. It is well established that HER2-mediated heterodimerization has important implications in cancer. Deregulation of signaling pathways and overexpression of HER2 is known to occur in cancer cells, indicating a role of HER2 in tumorigenesis. Therefore, blocking HER2-mediated signaling has potential therapeutic value. We have designed several peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of the compounds (HERP5, Arg-beta Naph-Phe) exhibited antiproliferative activity with IC(50) values in the micromolar-to-nanomolar range in breast cancer cell lines. Binding of fluorescently labeled HERP5 to HER2 protein was evaluated by fluorescence assay, microscopy, and circular dichroism spectroscopy. Results indicated that HERP5 binds to the extracellular region of the HER2 protein. Structure of the peptidomimetic HERP5 was studied by NMR and molecular dynamics simulations. Based on these results a model was proposed for HER2-EGFR dimerization and possible blocking by HERP5 peptidomimetic using a protein-protein docking method.     

Bayesian analysis of signaling networks governing embryonic stem cell fate decisions

MOTIVATION: Signaling events that direct mouse embryonic stem (ES) cell self-renewal and differentiation are complex and accordingly difficult to understand in an integrated manner. We address this problem by adapting a Bayesian network learning algorithm to model proteomic signaling data for ES cell fate responses to external cues. Using this model we were able to characterize the signaling pathway influences as quantitative, logic-circuit type interactions. Our experimental dataset includes measurements for 28 signaling protein phosphorylation states across 16 different factorial combinations of cytokine and matrix stimuli as reported previously. RESULTS: The Bayesian network modeling approach allows us to uncover previously reported signaling activities related to mouse ES cell self-renewal, such as the roles of LIF and STAT3 in maintaining undifferentiated ES cell populations. Furthermore, the network predicts novel influences such as between ERK phosphorylation and differentiation, or RAF phosphorylation and differentiated cell proliferation. Visualization of the influences detected by the Bayesian network provides intuition about the underlying physiology of the signaling pathways. We demonstrate that the Bayesian networks can capture the linear, nonlinear and multistate logic interactions that connect extracellular cues, intracellular signals and consequent cell functional responses.     

A Mathematical Model for the Effects of HER2 Overexpression on Cell Proliferation in Breast Cancer

We present a mathematical model to study the effects of HER2 over-expression on cell proliferation in breast cancer. The model illustrates the proliferative behavior of cells as a function of HER2 and EGFR receptors numbers, and the growth factor EGF. This mathematical model comprises kinetic equations describing the cell surface binding of EGF growth factor to EGFR and HER2 receptors, coupled to a model for the dependence of cell proliferation rate on growth factor receptors binding. The simulation results from this model predict: (1) a growth advantage associated with excess HER2 receptors; (2) that HER2-over-expression is an insufficient parameter to predict the proliferation response of cancer cells to epidermal growth factors; and (3) the EGFR receptor expression level in HER2-over-expressing cells plays a key role in mediating the proliferation response to receptor-ligand signaling. This mathematical model also elucidates the interaction and roles of other model parameters in determining cell proliferation rate of HER2-over-expressing cells.     

Model-Based Analysis of HER Activation in Cells Co-Expressing EGFR,HER2 and HER3

The HER/ErbB family of receptor tyrosine kinases drives critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation, and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panel of human mammary epithelial cells expressing varying levels of EGFR/HER1, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of human mammary epithelial cells lines with known HER expression levels in response to stimulations with ligands EGF and HRG. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2 dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2-HER3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.     

CXCL12 and [N33A]CXCL12 in 5637 and HeLa cells: regulating HER1 phosphorylation via calmodulin/calcineurin

In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals via both G-protein-dependent and β-arrestin-dependent pathways responsible for inducing a rapid and a late wave, respectively, of ERK1/2 phosphorylation. In contrast, the structural variant [N33A]CXCL12 triggered no β-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1) Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2) Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3) In cells knockdown of β-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4) HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate β-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1.