[Show abstract][Hide abstract] ABSTRACT: The loss of function of Von Hippel-Lindau (VHL) tumor suppressor leads to the development of hyper-vascular tumours, exemplified by clear cell type renal cell carcinoma (RCC). VHL governs the adaptive responses to fluctuation of oxygen levels largely through the regulated suppression of hypoxia inducible factors (HIFs). Here, we combine proteome and phospho-proteomic analysis of isogenic 786-O RCC (± VHL) cells, to compare signatures that reflect hypoxia and/or loss of VHL. VHL-independent hypoxic responses, notably include up-regulation of phosphorylation at Ser232 on pyruvate dehydrogenase α sub-unit that is known to promote glycolysis. Hypoxic responses governed by VHL include up-regulation of known biomarkers of RCC (e.g. GLUT1, NDRG1) and the signaling adaptor molecule IRS-2. Notably we also observe down-regulation of linked-components associated with the Jacobs-Stewart cycle, including the intracellular carbonic anhydrase II (CA2) which governs cellular response to CO2 fluctuations that often accompany hypoxia in tumours. Further studies indicate an unusual mechanism of control for CA2 expression, that at least in part, reflects enhanced activity of the NFκB pathway, that is associated with loss of VHL.
Journal of Proteome Research 10/2015; DOI:10.1021/acs.jproteome.5b00692 · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The receptor tyrosine kinase, Met, orchestrates a complex signalling network that physiologically drives a programme of 'invasive growth'. In cancer however, this process may be co-opted to promote proliferation, survival and metastasis of cancer cells. Met is thus a key therapeutic target, not least in non-small cell lung cancer (NSCLC) where it is one of the most commonly dysregulated driver oncogenes. Identifying robust biomarkers that allow the selection of patients most likely to respond to Met targeted therapies will however be essential to realising their potential. This has been underlined recently by the early termination of three pivotal phase III trials investigating Met targeted agents in NSCLC, all of which failed to show clinical benefit. In contrast to these trials, which were relatively unselective, a couple of early phase trials have recently been instigated that select patients on the basis of Met amplification. While still at an early stage, interim results are relatively encouraging and strengthen the rationale for using Met amplifaction as a biomarker. Here we will discuss this and other aberrations in Met signalling in relation to their significance in the therapeutic targeting of Met.
[Show abstract][Hide abstract] ABSTRACT: Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Oncogenic mutations of Ras at codons 12, 13 or 61, that render the protein constitutively active, are found in ~16% of all cancer cases. Amongst the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumour type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phospho-proteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbour specific KRAS mutations (G12V, G12D or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker Double Cortin-Like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS.
Journal of Proteome Research 01/2015; 14(3). DOI:10.1021/pr501191a · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Defined signals that dictate the architecture of cellular boundaries in confluent cultures are poorly characterized. Here, we report dramatic remodeling, invoked by long-term epidermal growth factor (EGF) withdrawal from mammary-derived MCF10A cells. Such intervention generates an interdigitated, desmosome-rich monolayer, wherein cells project actin-containing protrusions deep into neighboring cells. These changes protect cellular sheets from mechanical disruption and dramatically restrict the freedom of cells to roam within the monolayer. Ectopic expression of activated Rac counteracts interdigitation and induces membrane ruffling, but cells remain confined by their interdigitated neighbors. Interdigitations are rapidly dissolved by acute EGF application in a process that is sensitive to actin depolymerization and myosin II inhibition. These assays for formation and dissolution of interdigitations provide a platform for the dissection of novel signaling pathways that are highly specific to EGF receptor (EGFR) activation.
[Show abstract][Hide abstract] ABSTRACT: DNA double-strand breaks (DSBs) are perhaps the most toxic of all DNA lesions, with defects in the DNA-damage response to DSBs being associated with various human diseases. Although it is known that DSB repair pathways are tightly regulated by ubiquitylation, we do not yet have a comprehensive understanding of how deubiquitylating enzymes (DUBs) function in DSB responses. Here, by carrying out a multidimensional screening strategy for human DUBs, we identify several with hitherto unknown links to DSB repair, the G2/M DNA-damage checkpoint and genome-integrity maintenance. Phylogenetic analyses reveal functional clustering within certain DUB subgroups, suggesting evolutionally conserved functions and/or related modes of action. Furthermore, we establish that the DUB UCHL5 regulates DSB resection and repair by homologous recombination through protecting its interactor, NFRKB, from degradation. Collectively, our findings extend the list of DUBs promoting the maintenance of genome integrity, and highlight their potential as therapeutic targets for cancer.
[Show abstract][Hide abstract] ABSTRACT: Platinum-based chemotherapy is widely used to treat various cancers, but many patients ultimately relapse due to drug resistance. We employed phosphoproteomic analysis and functional assays of the response of SK-OV-3 ovarian cancer cells to cisplatin as a strategy to identify kinases as candidate "druggable" targets to sensitize cells to platinum. A SILAC-based approach combined with TiO2-based phosphopeptide enrichment allowed the direct identification of ERK1/2, p90RSK and ERBB2 as kinases whose phosphorylation is regulated by cisplatin. Bioinformatic analysis revealed enrichment in linear phosphorylation motifs predicted to be targets of p38MAPK, CDK2 and PIM2. All three PIM kinases were found expressed in a panel of 10 ovarian cancer cell lines, the oncogenic PIM2 being the most commonly induced by cisplatin. Targeting PIM2 kinase by either biochemical inhibitors or RNA interference impaired cell growth, decreased cisplatin-triggered BAD phosphorylation and sensitized ovarian cancer cells to drug-induced apoptosis. Over-expression of PIM2 triggered anchorage independent growth and resulted in increased BAD phosphorylation and cell resistance to DNA damaging agents. Data show that the PIM2 kinase plays a role in the response of ovarian cancer cells to platinum drugs and suggest that PIM inhibitors may find clinical application as an adjunct to platinum-based therapies.
Journal of Proteome Research 08/2014; 13(11). DOI:10.1021/pr500651n · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction
Receptor tyrosine kinases (RTKs) are found on the surface of normal and cancerous cells. Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor 2 (HER2) are RTKs that are often overexpressed in breast cancer. RTK upregulation leads to enhanced activation of downstream signalling pathways that inhibit apoptosis and promote proliferation, migration and angiogenesis. Breast Cancer Associated gene 2 (BCA2) is a poorly studied E3 ligase that is overexpressed in over 50% of breast cancers. Depletion of BCA2 has been shown to reduce cell growth and invasion while overexpression increases proliferation. BCA2 has been implicated in EGFR endocytosis, however there is conflicting evidence regarding its influence on the biology of this receptor. This project aims to elucidate the role of BCA2 in RTK endocytosis, downregulation and breast cancer.
Materials and Methods
Immunodetection was used to analyse BCA2 and EGFR levels in the breast cancer cell lines MCF-7, MDA-MB-231, T47D and BT474 and in the non-cancerous MCF10A line. HeLa cervical carcinoma cells were also studied as EGFR trafficking in this cell line is very well characterised. Transient overexpression experiments were performed in HeLa cells using a vector for HA-tagged BCA2. EGFR levels and trafficking in BCA2 overexpressing cells were analysed by Western blotting and fluorescence microscopy. The effect of BCA2 overexpression on EGFR degradation was examined by treating HA-BCA2 or mock transfected HeLa cells with EGF for between 0 and 60 min. To explore the relationship between BCA2 expression and breast cancer outcome, Kaplan-Meier curves were generated with the KMPLOT biomarker analysis tool.
Results and Discussion
By immunodetection BCA2 manifests as two bands (34 and 37kDa) and we show high expression variability across a panel of breast-derived cell lines. We find that transient BCA2 overexpression significantly decreases EGFR levels in HeLa cells. In contrast to this we find that BCA2 overexpressing cells stimulated with EGF show reduced degradation of both receptor and ligand in lysosomal compartments. This is likely to reflect a role for BCA2 in the endolysosomal system and in agreement with this we show colocalisation between BCA2 and the late endosomal marker Rab7a. Levels of the non-RTK transferrin receptor and uptake of transferrin are unaffected by BCA2 overexpression suggesting the effects on trafficking may be confined to EGFR or a distinct class of receptor. Kaplan-Meier analyses show that high levels of BCA2 are associated with reduced relapse free survival (P<0.05) in HER2+ breast cancer subtypes.
Our findings present an interesting insight into BCA2-mediated EGFR regulation via altered endocytosis and suggest that BCA2 may affect inter-tumoural growth factor signalling and patient survival. The regulatory effect of BCA2 on different EGFR family members and on other oncogenic plasma membrane receptors remains to be determined.
23rd Biennial Congress of the European Association for Cancer Research, Munich; 07/2014
[Show abstract][Hide abstract] ABSTRACT: The endosomal deubiquitylase USP8 has profound effects on endosomal morphology and organisation. Previous reports have proposed both positive (EGFR, MET) and negative roles in the down-regulation of receptors (Frizzled, Smoothened). Here we report an additional influence of USP8 on the retromer-dependent shuttling of ci-M6PR between the sorting endosome and biosynthetic pathway. Depletion of USP8 leads to a steady state redistribution of ci-M6PR from the Trans-Golgi Network (TGN) to endosomal compartments. Consequently we observe a defect in sorting of lysosomal enzymes, evidenced by increased levels of unprocessed Cathepsin D, which is secreted into the medium. The normal distribution of receptor can be restored by expression of siRNA-resistant USP8 but not by a catalytically inactive mutant or a truncated form, lacking a MIT domain required for endosomal localisation. We suggest that effects of USP8 depletion may reflect the loss of ESCRT-0 components which associate with retromer components Vps35 and SNX1, whilst failure to efficiently deliver lysosomal enzymes may also contribute to the observed block in receptor tyrosine kinase degradation.
[Show abstract][Hide abstract] ABSTRACT: Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.
Current biology: CB 03/2014; 24(6):R215-20. DOI:10.1016/j.cub.2014.02.002 · 9.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
[Show abstract][Hide abstract] ABSTRACT: The phosphatidylinositol-3-kinase (PI3K) pathway is commonly hyperactivated in cancer. One mechanism by which this occurs is by silencing of the phosphatase and tensin homolog (PTEN), a tumor suppressor and major antagonist of the pathway, through genetic, epigenetic or posttranscriptional mechanisms. Here, we used an unbiased siRNA screen in non-small-cell lung cancer cells to identify deubiquitylases (DUBs) that have an impact on PI3K signaling by regulating the abundance of PTEN. We found that PTEN expression was induced by depleting any of three members of the Josephin family DUBs: ataxin 3 (ATXN3), ataxin 3-like (ATXN3L) and Josephin domain containing 1 (JOSD1). However, this effect is not mediated through altered PTEN protein stability. Instead, depletion of each DUB increases expression of both the PTEN transcript and its competing endogenous RNA, PTENP1. In ATXN3-depleted cells, under conditions of transcriptional inhibition, PTEN and PTENP1 mRNAs rapidly decay, suggesting that ATXN3 acts primarily by repressing their transcription. Importantly, the PTEN induction observed in response to ATXN3 siRNA is sufficient to downregulate Akt phosphorylation and hence PI3K signaling. Histone deacetylase inhibitors (HDACi) have been suggested as potential mediators of PTEN transcriptional reactivation in non-small-cell lung cancer. Although PTEN exhibits a very limited response to the broad-spectrum HDACi Vorinostat (SAHA) in A549 cells, we find that combination with ATXN3 depletion enhances PTEN induction in an additive manner. Similarly, these interventions additively decrease cell viability. Thus, ATXN3 provides an autonomous, complementary therapeutic target in cancers with epigenetic downregulation of PTEN.Oncogene advance online publication, 2 December 2013; doi:10.1038/onc.2013.512.
[Show abstract][Hide abstract] ABSTRACT: Ubiquitylation is a major posttranslational modification that controls most complex aspects of cell physiology. It is reversed through the action of a large family of deubiquitylating enzymes (DUBs) that are emerging as attractive therapeutic targets for a number of disease conditions. Here, we provide a comprehensive analysis of the complement of human DUBs, indicating structural motifs, typical cellular copy numbers, and tissue expression profiles. We discuss the means by which specificity is achieved and how DUB activity may be regulated. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from ubiquitin-mediated degradation, and 3) control the dynamics of ubiquitin-mediated signaling events. Functional roles of individual DUBs from each of five subfamilies in specific cellular processes are highlighted with an emphasis on those linked to pathological conditions where the association is supported by whole organism models. We then specifically consider the role of DUBs associated with protein degradative machineries and the influence of specific DUBs upon expression of receptors and channels at the plasma membrane.
[Show abstract][Hide abstract] ABSTRACT: Reversible ubiquitylation of proteins contributes to their integrity, abundance and activity. The RE1-silencing transcription factor (REST) plays key physiological roles and is dysregulated in a spectrum of disease. It is rapidly turned over and is phosphorylated, polyubiquitylated and degraded en masse during neuronal differentiation and cell cycle progression. Through siRNA screening we identified the deubiquitylase USP15 as a key regulator of cellular REST. Both antagonism of REST polyubiquitylation and rescue of endogenous REST levels are dependent on the deubiquitylase activity of USP15. However, USP15 depletion does not destabilize pre-existing REST, but rather specifically impairs de novo REST synthesis. Indeed, we find that a small fraction of endogenous USP15 is associated with polysomes. In accordance with these findings, USP15 does not antagonize the degradation of phosphorylated REST at mitosis. Instead it is required for the rapid accumulation of newly synthesized REST on mitotic exit, thus playing a key role in its cell cycle oscillations. Importantly, this study reveals a novel role for a DUB in specifically promoting new protein synthesis.
[Show abstract][Hide abstract] ABSTRACT: Reversible oxidation of amino-acid residues can directly regulate the
activity of cellular enzymes. This principle has now been extended to
deubiquitinating enzymes, with implications for cell signalling and
[Show abstract][Hide abstract] ABSTRACT: The opposing regulators of ubiquitylation status, E3-ligases and deubiquitylases (DUBs), are often found to be associated in complexes. Here we report on a novel interaction between the E3-ligase BRAP (also referred to as IMP), a negative regulator of the MAPK scaffold protein KSR, and two closely related DUBs USP15 and USP4. We map the interaction to the amino-terminal DUSP-UBL domain of USP15 and the coiled coil region of BRAP. USP15 as well as USP4 oppose the auto-ubiquitylation of BRAP, whilst BRAP promotes the ubiquitylation of USP15. Importantly, USP15 but not USP4 depletion destabilizes BRAP by promoting its proteasomal degradation, and BRAP-protein levels can be rescued by reintroducing catalytically active but not inactive mutant USP15. Unexpectedly, USP15 depletion results in a decrease in amplitude of MAPK signalling in response to EGF and PDGF. We provide evidence for a model in which the dominant effect of prolonged USP15 depletion upon signal amplitude is due to a decrease in CRAF levels, whilst allowing for the possibility that USP15 may also function to dampen MAPK signaling through direct stabilization of a negative regulator, the E3-ligase BRAP.