Ceri M Wiggins

Babraham Institute, Cambridge, England, United Kingdom

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Publications (8)47.81 Total impact

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    ABSTRACT: Studies to identify predictive biomarkers can be carried out in isogenic cancer cell lines, which enable interrogation of the effect of a specific mutation. We assessed the effects of four drugs, the PI3K–mammalian target of rapamycin inhibitor dactolisib, the PI3K inhibitor pictrelisib, and the MEK (MAPK/ERK Kinase) inhibitors PD 0325901 and selumetinib, in isogenic DLD1 parental, KRAS+/−, KRASG13D/−, PIK3CA+/− and PIK3CAE545K/− colorectal carcinoma cell lines. Importantly, we found substantial differences in the growth of these cells and in their drug sensitivity depending on whether they were studied under 2D (standard tissue culture on plastic) or 3D (in vitro soft agar and in vivo xenograft) conditions. DLD1 KRAS+/− and DLD1 PIK3CA+/− cells were more sensitive to MEK inhibitors than parental, DLD1 KRASG13D/− and DLD1 PIK3CAE545K/− cells under 2D conditions, whereas DLD1 KRASG13D/− and DLD1 PIK3CAE545K/− xenografts were sensitive to 10 mg/kg daily ×14 PD 0325901 in vivo (p ≤ 0.02) but tumours derived from parental DLD1 cells were not. These findings indicate that KRAS and PIK3CA mutations can influence the response of DLD1 colorectal cancer cell lines to MEK and PI3K inhibitors, but that the effect is dependent on the experimental model used to assess drug sensitivity.
    No preview · Article · Mar 2016 · European journal of cancer (Oxford, England: 1990)
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    ABSTRACT: No molecularly targeted therapy has yet been identified for KRAS mutant cancers. As oncogenic mutations reduce RAS enzymatic activity, classic small molecule approaches are ineffective, hence most work has focussed on drugging RAS-effector pathways. Multiple inhibitors of MEK, RAF and PI3K have been identified but toxicity issues and pathway adaptation have stymied their success against KRAS-driven cancers. An alternative approach is to exploit "non-oncogene addiction" by identifying targets with synthetic lethal or co-dependence interactions with KRAS. A number of siRNA and shRNA screens have identified targets that exhibit differential dependencies between KRAS mutant and KRAS wild-type tumours, but there is poor overlap between the different published studies. This discordance may arise from (1) the noise inherent in using cell line panels differing in much more than their KRAS mutant/wild-type status and (2) the use of RNA interference methodologies driving incomplete knockdown and associated with substantial off-target effects. Next generation screens that exploit both isogenic cell lines and cell line panels, and use a combination of knockdown and knock-out (i.e. CRISPR/Cas9-sgRNA) methodologies, may be better suited for identifying novel targets that withstand validation. However, if we are to detect co-dependence as well as synthetic lethal interactions, screens must be performed under conditions where mutant KRAS alleles are essential for growth. A library of siRNAs targeting proposed KRAS synthetic lethal targets was assembled and screened under conditions where proliferation is dependent on KRAS status. DLD1 cells harbour an activating KRASG13D mutation dispensable for proliferation in 2D, but essential for proliferation under 3D (soft agar) conditions. Knockdown of several targets including KRAS itself, PLK1, TBK1, BCL-XL & RAF1 proved more anti-proliferative under 3D conditions. This screen was extended to a panel of KRAS-mutant colon lines, with varying levels of KRAS sensitivity, where we found the requirement for RAF1 highly correlated with the requirement for KRAS. With the advent of CRISPR we are now able to design sgRNA libraries capable of probing the effect of ‘knocking out’ rather than ‘knocking down’ targets, providing a potentially superior alternative to RNA interference. Data from mouse models indicates RAF1 is required for the initiation of lung cancer by oncogenic KRAS. Although we found good correlation between sensitivity to KRAS and RAF1 depletion, we were unable to unambiguously validate RAF1 as a target in human lung cancer cells using RNA interference methodologies. However, using CRISPR-Cas9, we found complete loss of RAF1 expression was anti-proliferative in A549 cells, supporting the concept of targeting RAF1 in a KRAS mutant lung cancers. These results demonstrate that a more penetrant sgRNA based screening approach may identify novel KRAS synthetic lethal or co-dependent interactions.
    No preview · Conference Paper · Apr 2015
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    ABSTRACT: BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.
    Full-text · Article · Mar 2011 · Journal of Cell Science
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    Ceri M Wiggins · Mark Johnson · Simon J Cook
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    ABSTRACT: The pro-apoptotic protein BIM(EL) is phosphorylated by ERK1/2 and this targets the protein for poly-ubiquitination and degradation by the proteasome as a survival mechanism. To define in greater detail the sequence determinants required for BIM(EL) turnover we have compared various BIM splice variants and truncation mutants. Of the naturally occurring splice variants BIMbeta1, which lacks the C-terminal hydrophobic domain, the BH3 domain and is cytosolic, exhibited the fastest turnover rate. Indeed, neither the C-terminus, the BH3 domain nor the DLC1 binding region was required for poly-ubiquitination and turnover of BIM. However, we demonstrate that a region consisting of the ERK1/2 docking domain, ERK1/2 phosphorylation sites and either of the two potential ubiquitin-acceptor lysine residues is sufficient to allow poly-ubiquitination and turnover of BIM. In the process we demonstrate that the C-terminal hydrophobic domain, previously suggested to be important in membrane localisation, is as important as the BH3 domain for BIM to induce cell death; similarly, the pro-death BH3-domain can also confer correct mitochondrial localisation in the absence of the C-terminus. These results refine the minimal sequence for ERK1/2-driven degradation and further define the functional importance of key regions within BIM(EL), highlighting the complexity of this pro-apoptotic protein.
    Full-text · Article · May 2010 · Cellular Signalling
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    ABSTRACT: The BCL-2 homology domain 3 (BH3)-only protein, B-cell lymphoma 2 interacting mediator of cell death (BIM) is a potent pro-apoptotic protein belonging to the B-cell lymphoma 2 protein family. In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal-regulated kinase 1/2 and protein kinase B pathways. In tumour cells these oncogene-regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival. In parallel, a new generation of targeted therapeutics has been developed, which show remarkable specificity and efficacy in tumour cells that are addicted to particular oncogenes. It is now apparent that the expression and activation of BIM is a common response to these new therapeutics. Indeed, BIM has emerged from this marriage of basic and applied biology as an important mediator of tumour cell death in response to such drugs. The induction of BIM alone may not be sufficient for significant tumour cell death, as BIM is more likely to act in concert with other BH3-only proteins, or other death pathways, when new targeted therapeutics are used in combination with traditional chemotherapy agents. Here we discuss recent advances in understanding BIM regulation and review the role of BIM as a mediator of tumour cell death in response to novel oncogene-targeted therapeutics.
    Full-text · Article · Sep 2009 · FEBS Journal
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    Ceri M Wiggins · Hamid Band · Simon J Cook
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    ABSTRACT: Bim(EL) the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim(EL). It has been proposed that the RING finger protein Cbl binds to Bim(EL) and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim(EL) is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim(EL) turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim(EL) is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim(EL) does not involve its tyrosine phosphorylation; indeed, Bim(EL) is not a tyrosine phosphoprotein. Furthermore, Bim(EL) fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim(EL) turnover proceeds normally in Cbl-containing or Cbl-/- fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim(EL) turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.
    Full-text · Article · Jan 2008 · Cellular Signalling
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    Katherine E Ewings · Ceri M Wiggins · Simon J Cook
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    ABSTRACT: Bim (Bcl-2-interacting mediator of cell death) is a BH3-only protein (BOP), a pro-apoptotic member of the Bcl-2 protein family. The Bim mRNA undergoes alternate splicing to give rise to the short, long and extra long protein variants (Bim(S), Bim(L) and Bim(EL)). These proteins have distinct potency in promoting death and distinct modes of regulation conferred by their interaction with other proteins. Quite how Bim and other BOPs promote apoptosis has been the subject of some debate. Bim was isolated by it's interaction with pro-survival proteins such as Bcl-2 and it has been suggested that this is key to the ability of Bim to induce apoptosis. However, an alternative model argues that some forms of Bim can bind directly to the pro-apoptotic Bax and Bak proteins to initiate apoptosis. A new study may finally put this debate to rest as it provides strong evidence to suggest that Bim and other BOPs act primarily by binding to pro-survival Bcl-2 proteins, thereby releasing Bax or Bak proteins to promote apoptosis. The importance of the interaction between Bim and the pro-survival Bcl-2 proteins is underlined by our demonstration that it is regulated by ERK1/2-dependent phosphorylation of Bim(EL). ERK1/2-dependent dissociation of Bim(EL) from pro-survival proteins is the first step in a process by which the pro-survival ERK1/2 pathway promotes the destruction of this most abundant Bim splice variant. In this review we outline the significance of these new studies to our understanding of how BOPs such as Bim initiate apoptosis and how this process is regulated by growth factor-dependent signalling pathways.
    Full-text · Article · Aug 2007 · Cell cycle (Georgetown, Tex.)
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    ABSTRACT: The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.
    Full-text · Article · Jul 2007 · The EMBO Journal