hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsa-mir183, the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication.

ERK and AKT phosphorylation status in lung cancer and emphysema using nanocapillary isoelectric focusing.

BACKGROUND: Emphysema is an independent risk factor for the development of lung cancer in smokers. Activation of oncogenic signalling proteins AKT and ERK by phosphorylation has an established role in the development of lung cancer and has also been implicated in the pathogenesis of emphysema. The aim of this study was to compare the protein level and phosphorylation status of AKT and ERK in paired lung cancer and emphysema tissue using a highly sensitive phosphoprotein analysis approach. METHODS: An antibody-based, nanocapillary isoelectric focusing (cIEF) assay was used to determine the relative quantities and phosphorylation status of AKT and ERK in tumour and matched lung tissue from patients, with or without evidence of emphysema, undergoing curative resection for non-small cell lung cancer. RESULTS: 20 patients with adenocarcinoma (n=9) or squamous cell carcinoma (n=11) of the lung were included (mean age 67.3 years (SD 7.5, range 47-80 years)), 12 were men and all were current (n=10) or former smokers (n=10). Paired macroscopically normal lung tissue was either histologically normal (n=7) or showed emphysema (n=13). Total and phosphorylated AKT levels were fourfold (p=0.0001) and fivefold (p=0.001) higher in tumour compared with matched lung, respectively. There was no correlation with tumour histology, stage or differentiation; however, total AKT signal in tumour was significantly correlated with fluorodeoxyglucose avidity on positron emission tomography-CT scan (r=0.53, p=0.035). Total ERK was not differentially expressed, but doubly phosphorylated (activated) ERK was threefold higher in emphysema (23.5%, SD 9.2) than either matched tumour (8.8%, SD 8.6) or normal lung tissue (8.3%, SD 9.0) and correlated with the histological severity of emphysema (p=0.005). CONCLUSIONS: cIEF offers opportunities for quantifying subtle shifts in the phosphorylation status of oncoproteins in nanogram amounts of lung tissue. ERK activation is a feature of emphysema.

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein CrkL, a major substrate of the oncogenic tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <10(4) cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-CrkL and the protein tyrosine phosphatase PTPRC/CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents.

The antiproliferative activity of kinase inhibitors in chronic myeloid leukemia cells is mediated by FOXO transcription factors.

Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL-specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR-ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over-expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA-mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI-induced apoptosis. These data demonstrate that TKI-induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network.

Monocyte-derived dendritic cells from chronic myeloid leukaemia have abnormal maturation and cytoskeletal function that is associated with defective localisation and signalling by normal ABL1 protein.

OBJECTIVES: Mature dendritic cells (DCs) may be derived from the BCR/ABL1 expressing monocytes in chronic myeloid leukaemia. These cells have potential therapeutic applications, but are recognised to have defective function. In normal DCs, activation and maturation depend on ABL1 dependent signals. We therefore tested the hypothesis that in the DCs of chronic myeloid leukaemia, the presence of the BCR/ABL1 molecule disrupts normal ABL1 signal pathways, and contributes to the observed functional defects of the cells. METHODS: We employed in vitro culture of clinical samples, combining microscopic and biochemical techniques with a phosphoproteomic approach to compare and characterise DCs from normal individuals and chronic myeloid leukaemia patients. RESULTS AND CONCLUSIONS: We identified an altered intracellular localisation for ABL1 within DCs derived from the monocytes of chronic myeloid leukaemia. The protein was found in the perinuclear region co-distributed with the adapter-protein CRKL and the BCR/ABL1 protein. This altered distribution was associated with defective generation of ABL1-dependent maturation signals, and a dislocation of ABL1 from the F-actin cytoskeleton. We suggest that abnormal ABL1-dependent signals contribute to the recognised functional defects affecting chronic myeloid leukaemia DCs.

A specific PTPRC/CD45 phosphorylation event governed by stem cell chemokine CXCL12 regulates primitive hematopoietic cell motility.

CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells.

Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

A technically detailed and pragmatic protocol for quantitative serum proteomics using iTRAQ.

Blood is recognised as a highly important source of disease-related biomarkers, and proteomic approaches for identifying novel blood-borne biomarkers are in demand. The complexity and dynamic protein concentration range of plasma/serum however complicates the analysis process. A number of strategies for simplification of blood prior to proteomic analysis have been developed. In addition, methods for quantifying the levels of proteins in samples, such as isobaric tags for relative and absolute quantification (iTRAQ) are emerging. However, the successful application of these procedures is not always straightforward and technical hurdles must be overcome. Here we provide a technically detailed working protocol for iTRAQ-based quantification of serum proteins following immunodepletion of high abundance proteins. To improve the number of proteins identified and quantified we have introduced several modifications to the standard iTRAQ protocol. We report identifications of 217 proteins (5773 peptides) with a false discovery rate of 1% or 254 proteins with 95% confidence, respectively. Relative quantification data were obtained for 234 (95% confidence) serum proteins, including species present in the concentration range of tissue leakage factors. The samples described here relate to pancreatic cancer; however the protocol can be applied to serum from other control or disease types.

Pancreatic cancer: proteomic approaches to a challenging disease.

PURPOSE OF REVIEW: To describe progress in the application of proteomic approaches to advance our understanding of the biology of pancreatic cancer as well as contribute potential protein biomarkers for this disease. RECENT FINDINGS: Here we review proteomic studies relating to pancreatic cancer that have been published in the past 12 months. We describe novel techniques for the simplification of complex protein samples, focusing particularly on emerging methods for reducing the complexity of blood. We provide examples, where possible, of the application of these novel technologies to pancreatic cancer research. SUMMARY: Both the range of proteomic-based approaches and their sensitivities for the detection of low-abundance proteins has increased. This provides promise that further research will yield insight into pancreatic cancer, including valuable information on proteins that may ultimately serve as biomarkers for pancreatic cancer.

The pancreatic cancer proteome - recent advances and future promise.

Of the common cancers, pancreatic ductal adenocarcinoma is one of the most lethal. The cancer's aggressive biology, leading to rapid dissemination, combined with a lack of clearly recognisable symptoms means that for many patients, the disease is at an advanced stage when diagnosed. The prognosis is consequently very poor, with as few as 3-5% of patients surviving 5 years. Recently, proteomic technologies have been employed in an effort to identify protein biomarkers, therapeutic targets and disease response markers for pancreatic cancer. Research has primarily relied upon pancreatic tissue samples, and body fluids such as pancreatic juice and blood serum. In this article, we will highlight the current proteomic techniques, qualitative and quantitative, employed in the field of pancreatic cancer research. We will review both the progress made and the challenges ahead, in elaborating the biology of pancreatic cancer and identifying novel biomarkers.

In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants.

Dissipation of excess light energy in plant photosynthetic membranes plays an important role in the response of plants to the environment, providing short-term balancing between the intensity of sunlight and photosynthetic capacity. The carotenoid zeaxanthin and the photosystem II subunit PsbS play vital roles in this process, but the mechanism of their action is largely unexplained. Here we report that the isolated photosystem II subunit PsbS was able to bind exogenous zeaxanthin, the binding resulting in a strong red shift in the absorption spectrum, and the appearance of characteristic features in the resonance Raman spectrum and a distinct circular dichroism spectrum, indicating pigment-protein, as well as specific pigment-pigment, interaction. A strong shift in the absorption spectrum of PsbS phenylalanine residues after zeaxanthin binding was observed. It is concluded that zeaxanthin binding to PsbS is the origin of the well known energy dissipation-related 535-nm absorption change that we showed in vivo to arise from activation of 1-2 molecules of this pigment. The altered properties of zeaxanthin and PsbS that result from this interaction provide the first direct indication about how they regulate energy dissipation.