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Profiling the tyrosine phosphoproteome of different mouse mammary tumour models reveals distinct, model-specific signalling networks and conserved oncogenic pathways.

ABSTRACT: INTRODUCTION: Although aberrant tyrosine kinase signalling characterises particular breast cancer subtypes, a global analysis of tyrosine phosphorylation in mouse models of breast cancer has not been undertaken to date. This may identify conserved oncogenic pathways and potential therapeutic targets. METHODS: We applied an immunoaffinity/mass spectrometry workflow to three mouse models: murine stem cell virus-Neu, expressing truncated Neu, the rat orthologue of human epidermal growth factor receptor 2, Her2 (HER2); mouse mammary tumour virus-polyoma virus middle T antigen (PyMT); and the p53-/- transplant model (p53). Pathways and protein-protein interaction networks were identified by bioinformatics analysis. Molecular mechanisms underpinning differences in tyrosine phosphorylation were characterised by Western blot analysis and array comparative genomic hybridisation. The functional role of mesenchymal-epithelial transition factor (Met) in a subset of p53-null tumours was interrogated using a selective tyrosine kinase inhibitor (TKI), small interfering RNA (siRNA)-mediated knockdown and cell proliferation assays. RESULTS: The three models could be distinguished on the basis of tyrosine phosphorylation signatures and signalling networks. HER2 tumours exhibited a protein-protein interaction network centred on avian erythroblastic leukaemia viral oncogene homologue 2 (Erbb2), epidermal growth factor receptor and platelet-derived growth factor receptor ?, and they displayed enhanced tyrosine phosphorylation of ERBB receptor feedback inhibitor 1. In contrast, the PyMT network displayed significant enrichment for components of the phosphatidylinositol 3-kinase signalling pathway, whereas p53 tumours exhibited increased tyrosine phosphorylation of Met and components or regulators of the cytoskeleton and shared signalling network characteristics with basal and claudin-low breast cancer cells. A subset of p53 tumours displayed markedly elevated cellular tyrosine phosphorylation and Met expression, as well as Met gene amplification. Treatment of cultured p53-null cells exhibiting Met amplification with a selective Met TKI abrogated aberrant tyrosine phosphorylation and blocked cell proliferation. The effects on proliferation were recapitulated when Met was knocked down using siRNA. Additional subtypes of p53 tumours exhibited increased tyrosine phosphorylation of other oncogenes, including Peak1/SgK269 and Prex2. CONCLUSION: This study provides network-level insights into signalling in the breast cancer models utilised and demonstrates that comparative phosphoproteomics can identify conserved oncogenic signalling pathways. The Met-amplified, p53-null tumours provide a new preclinical model for a subset of triple-negative breast cancers.

SUBMITTER: Ali NA

PROVIDER: S-EPMC4303118 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Profiling the tyrosine phosphoproteome of different mouse mammary tumour models reveals distinct, model-specific signalling networks and conserved oncogenic pathways.

Ali Naveid A NA Wu Jianmin J Hochgräfe Falko F Chan Howard H Nair Radhika R Ye Sunny S Zhang Luxi L Lyons Ruth J RJ Pinese Mark M Lee Hong Ching HC Armstrong Nicola N Ormandy Christopher J CJ Clark Susan J SJ Swarbrick Alexander A Daly Roger J RJ

Breast cancer research : BCR 20140909 5

<h4>Introduction</h4>Although aberrant tyrosine kinase signalling characterises particular breast cancer subtypes, a global analysis of tyrosine phosphorylation in mouse models of breast cancer has not been undertaken to date. This may identify conserved oncogenic pathways and potential therapeutic targets.<h4>Methods</h4>We applied an immunoaffinity/mass spectrometry workflow to three mouse models: murine stem cell virus-Neu, expressing truncated Neu, the rat orthologue of human epidermal growt ...[more]

PMID: 25200860

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Project description:BackgroundThe circadian clock governs a large variety of fundamentally important physiological processes in all three domains of life. Consequently, asynchrony in timekeeping mechanisms could give rise to cellular dysfunction underpinning many disease pathologies including human neoplasms. Yet, detailed pan-cancer evidence supporting this notion has been limited.MethodsIn an integrated approach uniting genomic, transcriptomic and clinical data of 21 cancer types (n?=?18,484), we interrogated copy number and transcript profiles of 32 circadian clock genes to identify putative loss-of-function (ClockLoss) and gain-of-function (ClockGain) players. Kaplan-Meier, Cox regression and receiver operating characteristic analyses were employed to evaluate the prognostic significance of both gene sets.ResultsClockLoss and ClockGain were associated with tumour-suppressing and tumour-promoting roles respectively. Downregulation of ClockLoss genes resulted in significantly higher mortality rates in five cancer cohorts (n?=?2914): bladder (P?=?0.027), glioma (P?<?0.0001), pan-kidney (P?=?0.011), clear cell renal cell (P?<?0.0001) and stomach (P?=?0.0007). In contrast, patients with high expression of oncogenic ClockGain genes had poorer survival outcomes (n?=?2784): glioma (P?<?0.0001), pan-kidney (P?=?0.0034), clear cell renal cell (P?=?0.014), lung (P?=?0.046) and pancreas (P?=?0.0059). Both gene sets were independent of other clinicopathological features to permit further delineation of tumours within the same stage. Circadian reprogramming of tumour genomes resulted in activation of numerous oncogenic pathways including those associated with cancer stem cells, suggesting that the circadian clock may influence self-renewal mechanisms. Within the hypoxic tumour microenvironment, circadian dysregulation is exacerbated by tumour hypoxia in glioma, renal, lung and pancreatic cancers, resulting in additional death risks. Tumour suppressive ClockLoss genes were negatively correlated with hypoxia inducible factor-1A targets in glioma patients, providing a novel framework for investigating the hypoxia-clock signalling axis.ConclusionsLoss of timekeeping fidelity promotes tumour progression and influences clinical outcomes. ClockLoss and ClockGain may offer novel druggable targets for improving patient prognosis. Both gene sets can be used for patient stratification in adjuvant chronotherapy treatment. Emerging interactions between the circadian clock and hypoxia may be harnessed to achieve therapeutic advantage using hypoxia-modifying compounds in combination with first-line treatments.

Project description:Inflammatory mechanisms influence tumorigenesis and metastatic progression even in cancers whose aetiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. For instance, prostate cancer metastasis is associated with the infiltration of lymphocytes into advanced tumours and the upregulation of two tumour-necrosis-factor family members: receptor activator of nuclear factor-?B (RANK) ligand (RANKL) and lymphotoxin. But the source of RANKL and its role in metastasis have not been established. RANKL and its receptor RANK control the proliferation of mammary lobuloalveolar cells during pregnancy through inhibitor of nuclear factor-?B (I?B) kinase-? (IKK-?), a protein kinase that is needed for the self-renewal of mammary cancer progenitors and for prostate cancer metastasis. We therefore examined whether RANKL, RANK and IKK-? are also involved in mammary/breast cancer metastasis. Indeed, RANK signalling in mammary carcinoma cells that overexpress the proto-oncogene Erbb2 (also known as Neu), which is frequently amplified in metastatic human breast cancers, was important for pulmonary metastasis. Metastatic spread of Erbb2-transformed carcinoma cells also required CD4(+)CD25(+) T cells, whose major pro-metastatic function was RANKL production. Most RANKL-producing T cells expressed forkhead box P3 (FOXP3), a transcription factor produced by regulatory T cells, and were located next to smooth muscle actin (SMA)(+) stromal cells in mouse and human breast cancers. The dependence of pulmonary metastasis on T cells was replaceable by exogenous RANKL, which also stimulated pulmonary metastasis of RANK(+) human breast cancer cells. These results are consistent with the adverse impact of tumour-infiltrating CD4(+) or FOXP3(+) T cells on human breast cancer prognosis and suggest that the targeting of RANKL-RANK can be used in conjunction with the therapeutic elimination of primary breast tumours to prevent recurrent metastatic disease.

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Profiling the tyrosine phosphoproteome of different mouse mammary tumour models reveals distinct, model-specific signalling networks and conserved oncogenic pathways.

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Profiling the tyrosine phosphoproteome of different mouse mammary tumour models reveals distinct, model-specific signalling networks and conserved oncogenic pathways.

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