Delineating WWOX Protein Interactome by Tandem Affinity Purification-Mass Spectrometry: Identification of Top Interactors and Key Metabolic Pathways Involved

It has become clear from multiple studies that WWOX (WW domain-containing oxidoreductase) operates as a “non-classical” tumor suppressor of significant relevance in cancer progression. Additionally, WWOX has been recognized for its role in a much wider array of human pathologies including metabolic conditions and central nervous system related syndromes. A myriad of putative functional roles has been attributed to WWOX mostly through the identification of various binding proteins. However, the reality is that much remains to be learned on the key relevant functions of WWOX in the normal cell. Here we employed a Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach in order to better define direct WWOX protein interactors and by extension interaction with multiprotein complexes under physiological conditions on a proteomic scale. This work led to the identification of both well-known, but more importantly novel high confidence WWOX interactors, suggesting the involvement of WWOX in specific biological and molecular processes while delineating a comprehensive portrait of WWOX protein interactome. Of particular relevance is WWOX interaction with key proteins from the endoplasmic reticulum (ER), Golgi, late endosomes, protein transport, and lysosomes networks such as SEC23IP, SCAMP3, and VOPP1. These binding partners harbor specific PPXY motifs which directly interact with the amino-terminal WW1 domain of WWOX. Pathway analysis of WWOX interactors identified a significant enrichment of metabolic pathways associated with proteins, carbohydrates, and lipids breakdown. Thus, suggesting that WWOX likely plays relevant roles in glycolysis, fatty acid degradation and other pathways that converge primarily in Acetyl-CoA generation, a fundamental molecule not only as the entry point to the tricarboxylic acid (TCA) cycle for energy production, but also as the key building block for de novo synthesis of lipids and amino acids. Our results provide a significant lead on subsets of protein partners and enzymatic complexes with which full-length WWOX protein interacts with in order to carry out its metabolic and other biological functions while also becoming a valuable resource for further mechanistic studies

The head and neck cancer cell oncogenome: a platform for the development of precision molecular therapies

The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-β in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.Centro de Investigaciones Inmunológicas Básicas y Aplicada

The head and neck cancer cell oncogenome: a platform for the development of precision molecular therapies

The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-β in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.Centro de Investigaciones Inmunológicas Básicas y Aplicada

Stromal Genes Add Prognostic Information to Proliferation and Histoclinical Markers: A Basis for the Next Generation of Breast Cancer Gene Signatures

BACKGROUND: First-generation gene signatures that identify breast cancer patients at risk of recurrence are confined to estrogen-positive cases and are driven by genes involved in the cell cycle and proliferation. Previously we induced sets of stromal genes that are prognostic for both estrogen-positive and estrogen-negative samples. Creating risk-management tools that incorporate these stromal signatures, along with existing proliferation-based signatures and established clinicopathological measures such as lymph node status and tumor size, should better identify women at greatest risk for metastasis and death. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the strength and independence of the stromal and proliferation factors in estrogen-positive and estrogen-negative patients we constructed multivariate Cox proportional hazards models along with tree-based partitions of cancer cases for four breast cancer cohorts. Two sets of stromal genes, one consisting of DCN and FBLN1, and the other containing LAMA2, add substantial prognostic value to the proliferation signal and to clinical measures. For estrogen receptor-positive patients, the stromal-decorin set adds prognostic value independent of proliferation for three of the four datasets. For estrogen receptor-negative patients, the stromal-laminin set significantly adds prognostic value in two datasets, and marginally in a third. The stromal sets are most prognostic for the unselected population studies and may depend on the age distribution of the cohorts. CONCLUSION: The addition of stromal genes would measurably improve the performance of proliferation-based first-generation gene signatures, especially for older women. Incorporating indicators of the state of stromal cell types would mark a conceptual shift from epithelial-centric risk assessment to assessment based on the multiple cell types in the cancer-altered tissue

SPARC 2017 retrospect & prospects : Salford postgraduate annual research conference book of abstracts

Welcome to the Book of Abstracts for the 2017 SPARC conference. This year we not only celebrate the work of our PGRs but also the 50th anniversary of Salford as a University, which makes this year’s conference extra special. Once again we have received a tremendous contribution from our postgraduate research community; with over 130 presenters, the conference truly showcases a vibrant PGR community at Salford. These abstracts provide a taster of the research strengths of their works, and provide delegates with a reference point for networking and initiating critical debate. With such wide-ranging topics being showcased, we encourage you to exploit this great opportunity to engage with researchers working in different subject areas to your own. To meet global challenges, high impact research inevitably requires interdisciplinary collaboration. This is recognised by all major research funders. Therefore engaging with the work of others and forging collaborations across subject areas is an essential skill for the next generation of researchers

Protein Kinase C Epsilon Cooperates with PTEN Loss for Prostate Tumorigenesis through the CXCL13-CXCR5 Pathway

Summary: PKCε, an oncogenic member of the PKC family, is aberrantly overexpressed in epithelial cancers. To date, little is known about functional interactions of PKCε with other genetic alterations, as well as the effectors contributing to its tumorigenic and metastatic phenotype. Here, we demonstrate that PKCε cooperates with the loss of the tumor suppressor Pten for the development of prostate cancer in a mouse model. Mechanistic analysis revealed that PKCε overexpression and Pten loss individually and synergistically upregulate the production of the chemokine CXCL13, which involves the transcriptional activation of the CXCL13 gene via the non-canonical nuclear factor κB (NF-κB) pathway. Notably, targeted disruption of CXCL13 or its receptor, CXCR5, in prostate cancer cells impaired their migratory and tumorigenic properties. In addition to providing evidence for an autonomous vicious cycle driven by PKCε, our studies identified a compelling rationale for targeting the CXCL13-CXCR5 axis for prostate cancer treatment. : Garg et al. find that PKCε overexpression cooperates with Pten loss to promote prostate cancer in mice. These two alterations together confer enhanced growth, tumorigenic, migratory and invasive capabilities to prostate epithelial cells, and promote the release of CXCL13, an effect that is mediated by the non-canonical NF-κB pathway. Keywords: PKCε, PTEN, CXCL13, NF-κB, prostate cancer, CXCR5, migration, proliferation, transgenic mic

Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes

Abstract Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specific modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic efforts had recently led to the identification of AJH-836, a DAG-lactone with preferential affinity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profiling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an effect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics efforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identified major differences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the differential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of differentially regulating PKC isozyme-specific function in cellular models