Dynamic Modeling of the JAK2/STAT5 Signal Transduction Pathway to Dissect the Specific Roles of Negative Feedback Regulators

Erythropoietin (Epo) acts as the key regulator of red blood cell development in mammals. During erythropoiesis, Epo initiates the JAK2/STAT5 signal transduction pathway that elicits pro-survival signals in erythroid progenitor cells. Therefore, the tight regulation of JAK2/STAT5 signaling is crucial for the fine-tuned balance of erythrocyte production. Recently, several factors regulating Epo-induced JAK2/STAT5 signaling have been identified. However, their relative contribution in controlling the dynamic behavior of JAK2/STAT5 signaling is poorly understood. To elucidate the specific roles of these negative regulators in attenuating the pathway, data-based mathematical modeling was employed. In this study, standardized protocols were established facilitating the generation of quantitative time-resolved data of Epo-induced JAK2/STAT5 pathway activation in primary erythroid progenitor cells and the hematopoietic cell line BaF3-EpoR, which is a frequently used model system to study EpoR signaling. For the fine-tuned overexpression of negative regulators in hematopoietic cells, an inducible Tet-On retroviral vector system was developed. Systematic comparison of stoichiometries and activation dynamics of Epo-induced JAK2/STAT5 signaling in CFU-E and BaF3-EpoR cells revealed fundamental differences between both cell types, emphasizing the importance of the use of primary cells in the investigation of EpoR signaling. Genome-wide expression profiling identified potential feedback regulators of Epo-induced JAK2/STAT5 signaling in CFU-E cells. To dissect the complex roles of negative regulators that employ different mechanisms to attenuate JAK2/STAT5 signaling, a data-based dynamic pathway model was established. Calibration of the mathematical model was performed using multiple experimental data sets of Epo-induced JAK2/STAT5 signaling monitored under different conditions. The estimated parameters were fully identifiable and displayed small confidence intervals, which are required for accurate simulations. Comprehensive model analysis identified the rapid recruitment of the phosphatase SHP-1 as major mechanism controlling the early-phase kinetics of pathway activation, while the two transcriptionally induced regulators SOCS3 and CIS were elucidated as modulators of the STAT5 steady-state phosphorylation level. Furthermore, global sensitivity analysis uncovered the concentrations of SHP-1 and JAK2 as well as the parameter SOCS3 expression as critical to control the integral signal strength of nuclear phosphorylated STAT5, which is proportionally linked to the survival of erythroid progenitor cells. In conclusion, by combining mathematical modeling with experimental data, the crucial regulators enabling the tight control of Epo-induced JAK2/STAT5 signaling were elucidated. The detailed understanding of the molecular processes and regulatory mechanisms of Epo-induced signaling during normal erythropoiesis can be further exploited to gain insights into alterations promoting erythroleukemia and related malignant hematopoietic diseases

JAK-STAT core cancer pathway : An integrative cancer interactome analysis

Through a comprehensive review and in silico analysis of reported data on STAT-linked diseases, we analysed the communication pathways and interactome of the seven STATs in major cancer categories and proposed rational targeting approaches for therapeutic intervention to disrupt critical pathways and addictions to hyperactive JAK/STAT in neoplastic states. Although all STATs follow a similar molecular activation pathway, STAT1, STAT2, STAT4 and STAT6 exert specific biological profiles associated with a more restricted pattern of activation by cytokines. STAT3 and STAT5A as well as STAT5B have pleiotropic roles in the body and can act as critical oncogenes that promote many processes involved in cancer development. STAT1, STAT3 and STAT5 also possess tumour suppressive action in certain mutational and cancer type context. Here, we demonstrated member-specific STAT activity in major cancer types. Through systems biology approaches, we found surprising roles for EGFR family members, sex steroid hormone receptor ESR1 interplay with oncogenic STAT function and proposed new drug targeting approaches of oncogenic STAT pathway addiction.Peer reviewe

Function of the Myc-binding protein Miz1 in the mouse mammary gland

The study of the expression and function of proteins important for human health in normal development provides valuable information for the design of therapeutical opportunities in the context of disease. Myc is one of the current most promising targets for a number of cancer types including triple-negative breast cancer and Miz1 has been shown to play an important role in Myc-mediated tumorigenesis. In the present work, the function of the Myc-binding protein Miz1 in the mammary gland is investigated for the first time using two different lines of transgenic mice expressing Cre-recombinase to conditionally knockout the POZ domain of Miz1 in the murine mammary gland. Deletion of this evolutionary-conserved region impedes multimerization and stable association of Miz1 with chromatin. MMTV-Cre mediated deletion was used to investigate Miz1 function in the virgin gland, considering branching morphogenesis and mammary stem/progenitor biology. Ablation under the Wap-Cre promoter provided information about alveologenesis and mammary differentiation. The mammary gland is a very suitable organ for stem cell and developmental studies as rounds of proliferation, differentiation and apoptosis occur after each pregnancy. POZ domain deletion using MMTV-Cre (Line A), already active in the embryo, led to a delayed ductal tree formation, less cellularity in knockout ducts and a Myc-independent accumulation of stem/progenitor cells in virgin mammary glands of Miz1DPOZ animals. No differences in the expression of luminal and myoepithelial markers were observed between control and Miz1DPOZ virgin mice. In addition, the delay in the development of the mammary ductal tree in knockout mice is rescued at around two months of age. Endogenous Miz1 expression in the mammary gland of control animals was found to be highly boosted during lactation by immunohistochemistry and Western blotting. Very low Miz1 levels were detected at the end of pregnancy, which increased after parturition and diminished upon cessation of pup suckling at around 48 hours of forced involution. Miz1 POZ domain ablation in luminal alveolar mammary cells during pregnancy using the WAP-Cre transgenic line resulted in a lactation defect in mutant dams during the first two pregnancies analysed. Mutant lactating glands display a reduced alveologenesis as a result of a diminished mammary cell proliferation and differentiation. These data were also confirmed in vitro using the HC11 murine mammary cell line after retroviral infection for stable knockdown of Miz1. HC11 cells with low levels of Miz1 show a reduced proliferation and a decreased expression of ß-casein after inducing differentiation by addition of a lactogenic hormone cocktail containing prolactin. Apoptosis is unaffected after either Miz1 POZ domain ablation in vivo or stable knockdown of Miz1 in vitro. Mutant glands display lower levels of activated Stat5 which lead to a reduced expression of its transcriptional targets, mainly genes which code for milk proteins like a-casein, b-casein or whey acidic protein (WAP). Gene expression of negative regulators of the Jak2/Stat5 pathway like Socs (Socs1, Socs2 and Socs3) or Caveolin-1 (Cav1) is not upregulated in Miz1DPOZ lactating glands. In contrast, the expression of receptors important for a proper phosphorylation of Stat5, like the prolactin receptor or ErbB4, is decreased in lactating mutant glands. ChIP-Seq experiments revealed that genes encoding the prolactin receptor and ErbB4 are not direct targets of Miz1. Rather, Miz1 binds to genes which regulate vesicular transport and thus alters processes like endocytosis and autophagy in mammary gland cells. A model in which the vesicular transport of these receptors in mutant glands could be disrupted is proposed. In conclusion, this work shows for the first time that Miz1 is important for mammary stem/progenitor cell regulation in the virgin gland and for a proper proliferation and differentiation in the lactating mammary gland

Dynamics of receptor and protein transducer homodimerisation

<p>Abstract</p> <p>Background</p> <p>Signalling pathways are complex systems in which not only simple monomeric molecules interact, but also more complex structures that include constitutive or induced protein assemblies. In particular, the hetero-and homo-dimerisation of proteins is a commonly encountered motif in signalling pathways. Several authors have suggested in recent times that dimerisation relates to a series of physical and biological outcomes used by the cell in the regulation of signal transduction.</p> <p>Results</p> <p>In this paper we investigate the role of homodimerisation in receptor-protein transducer interactions. Towards this end, mathematical modelling is used to analyse the features of such kind of interactions and to predict the behaviour of the system under different experimental conditions. A kinetic model in which the interaction between homodimers provokes a dual mechanism of activation (single and double protein transducer activation at the same time) is proposed. In addition, we analyse under which conditions the use of a power-law representation for the system is useful. Furthermore, we investigate the dynamical consequences of this dual mechanism and compare the performance of the system in different simulated experimental conditions.</p> <p>Conclusion</p> <p>The analysis of our mathematical model suggests that in receptor-protein interacting systems with dual mechanism there may be a shift between double and single activation in a way that intense double protein transducer activation could initiate and dominate the signal in the short term (getting a fast intense signal), while single protein activation could control the system in the medium and long term (when input signal is weaker and decreases slowly). Our investigation suggests that homodimerisation and oligomerisation are mechanisms used to enhance and regulate the dynamic properties of the initial steps in signalling pathways.</p

Investigation of the role of autocrine and paracrine growth factors in the survival and proliferation of chronic myeloid leukaemia stem and progenitor cells

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder arising in a haemopoietic stem cell (HSC) as a result of the reciprocal translocation between the long arms of chromosomes 9 and 22 (t9;22), leading to the formation of the fusion oncogene BCR-ABL. BCR-ABL has constitutive tyrosine kinase (TK) activity which drives, at least during the chronic phase (CP) of the disease, myeloid progenitor cells expansion through terminally differentiated cells and is necessary for the transformed phenotype. The introduction at the end of the last century of BCR-ABL TK inhibitors (TKI) has dramatically changed the management of newly diagnosed CP CML patients as the vast majority achieve deep molecular responses while enjoying good quality of life when treated with TKI. However about 20% of patients still show various degree of resistance to all currently available TKI while in those achieving deep responses, there is compelling evidence of persistent minimal residual disease demanding lifelong treatment which has obvious implications in terms of compliance, adverse events and costs. It is now known that the main reason for disease persistence in CML patients treated with TKI lies in the insensitivity of the most primitive CML leukaemia stem cell (LSC). More recent evidence has demonstrated that, in contrast to more mature leukaemic progenitor cells, CML LSC are not addicted to BCR-ABL kinase activity but rather rely on other stem cell intrinsic pathways for their survival. The main focus in the CML field is therefore to identify these pathways while also trying to exploit them therapeutically to achieve CML LSC eradication and as a result disease cure. Growth factor (GF) signals are known to provide survival cues to CML stem and progenitor cells (SPC) and potentially support their survival even in the presence of TKI. Moreover CML SPC are also known to produce higher levels of some GFs via an autocrine loop and support their survival and proliferation through this mechanism. In this thesis, the characterisation of the autocrine GF production by CML SPC was extended while also investigating the role of several GFs and downstream signals in survival, proliferation and self-renewal of CML SPC. Whenever possible, the consequences of therapeutic targeting of these signals on CML SPC survival and proliferation were also assessed in vitro. In particular the role of the intracellular janus kinase (JAK) 2, which relays several myeloid GF signals, such as those from interleukin (IL)-3 and granulocyte macrophage colony-stimulating factor (GM-CSF), in CML SPC survival and proliferation was investigated mainly because higher levels of autocrine expression of GM-CSF by CML SPC relative to normal were demonstrated, while autocrine IL-3 production by CML SPC had already been shown. Moreover the cognate receptor of both GM-CSF and IL-3 (CSF2RB) was also shown to be expressed at higher levels in CML SPC relative to their normal counterparts, further supporting investigations on the role of JAK2 in CML SPC biology. Indeed targeting JAK2 with small molecule inhibitors in CML SPC in vitro, particularly in the presence of maximal BCR-ABL TK inhibition, resulted in increased apoptosis, reduced proliferation and colony output of CML SPC. The JAK2 inhibitor plus TKI combination treatment, compared to either single agent, further reduced survival of the more primitive quiescent LSCs in vitro, while also reducing engraftment of primary CML CD34+ cells in vivo in immunocompromised hosts. Although a degree of toxicity to normal haemopoietic stem and progenitor cells (HSPC) was observed, this was not as great as seen in CML SPC, thus suggesting that a therapeutic window for using JAK2 inhibitors in CML patients might be present when a carefully selected concentration of these compounds is chosen. Tumour necrosis factor (TNF)-α was another GF shown to be produced in an autocrine fashion at higher levels by CML SPC relative to normal HSPC. Moreover its levels of production by CML SPC were not modulated by BCR-ABL TK activity. Using a small molecule TNF-α inhibitor and exogenous TNF-α, it was shown that autocrine TNF-α acts as a survival and proliferative signal in CML SPC. Moreover its role became even more important in the presence of TKI, as combining TNF-α inhibition with TKI led to high levels of apoptosis in CML CD34+ cells, including the more primitive quiescent population, while also causing increased apoptosis in a population enriched for CML LSCs based on its surface marker expression (CD34+ CD38-). Finally given the known importance of quiescence and self-renewal pathways in CML LSC persistence following TKI treatment, the role of transforming growth factor (TGF)-β1 and novel neurotransmitter mediated pathways in CML LSC quiescence and self-renewal was investigated based on the findings of a genome and epigenome-wide screen of primary CML LSCs and normal HSCs carried out in our laboratory. Using in vitro assays the putative role of the neuromediators norepinephrine and acethylcoline in CML LSC self-renewal was demonstrated. Moreover the role of TGF-β1 in inducing primary CML LSC quiescence mainly by modulating the AKT pathway was also demonstrated. Overall the work presented in this thesis has furthered our understanding of the role of both autocrine and paracrine known and novel regulators of haemopoiesis in several aspects of CML SPC biology such as their survival, proliferation and self-renewal. Furthermore the efficacy in eradicating CML SPC of therapeutic strategies targeting some of these GF signals has been explored in vitro, thus providing evidence supporting their subsequent testing in in vivo assays and in due course in clinical studies. It is hoped therefore that the work presented will contribute to devise novel therapeutic strategies to eradicate CML LSC and in turn lead to a cure for CML patients

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy

AIMS/HYPOTHESIS: Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. METHODS: RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. RESULTS: mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). CONCLUSIONS/INTERPRETATION: A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified

The effects of growth hormone receptor-associated ERK activation on adipocyte differentiation and function

Growth hormone (GH) modulates adipocyte function to promote lipolysis via a cell surface GH receptor (GHR) which activates multiple signalling cascades including STAT5 and p42/44 MAP kinase (MAPK) pathways. The growth promoting effects of GH are mediated primarily by STAT5 activation but little is known about pathways mediating the effects of GH on adipocyte function. We therefore studied the effect of GH on STAT5 and MAPK (ERK) activation in the 3T3-L1 mouse pre-adipocyte cell line during adipogenesis. Cells were plated, allowed to reach confluence and cultured in adipogenic medium containing the PPAR agonist, pioglitazone. GH induced activation (10 minutes exposure) of STAT5 and MAPK was analysed on days 0, 2, 5 and 8 during adipogenesis by phospho-specific western blotting and densitometry. During adipogenesis, GH progressively loses the ability to activate p42/44 MAPK despite elevated GHR and unaltered total ERK levels. In contrast, GH-stimulated STAT5 activation increases as 3T3-L1 differentiation proceeds. Subsequently we investigated possible explanations for the altered GHR signalling. The adapter protein p66Shc is thought to be necessary to link GHR activation to the ERK pathway. However levels of this protein, measured by western blotting and densitometry, did not decrease as 3T3-L1 cells underwent adipocyte differentiation. GHR levels increase with adipogenic differentiation of 3T3-L1 cells leading us to hypothesize that this may lead to preferential association with JAK2-STAT5. This was tested by overexpressing the GHR in 3T3-L1; similar GH-stimulated ERK pathway activation was obtained in cells transfected with the GHR vector and in those transfected with the empty vector. Finally, we have investigated whether changes in GHR signalling also occur during adipogenesis of primary pre-adipocytes from mice and various human depots. There was minimal GH-induced phosphorylation of ERK at all-time points before and during differentiation (required up to 15 days in primary cells) and no depot, either murine or human, demonstrated a reduction in p ERK, suggesting that this feature is unique to 3T3-L1. Furthermore, ERK phosphorylation may be the stimulus for mitotic clonal expansion which occurs in the cell line but not in human primaries. GH-stimulated STAT5 activation increases as human and mouse primary pre-adipocytes differentiation progresses, as in the 3T3-L1 cell-line, and may be the result of increased GHR transcript levels as differentiation proceeds. Future studies could investigate the mechanisms responsible for these similarities and differences