The role of JAK2, STAT3 and ERBB2 in ovarian cancer

Background Ovarian cancer is the most lethal gynaecological malignancy, accounting for an estimated 140,000 deaths per year worldwide. Five year survival rates have not increased significantly in the last 10 years and the acquisition of resistance to chemotherapy remains a significant barrier to improving patient survival. Isogenic cell line models of in vivo acquired resistance to chemotherapy were used to examine cellular responses to cisplatin and identify differences between sensitive and resistant pairs that might be exploited to sensitise cells to treatment. Results Microarray analysis of the isogenic paired sensitive/resistant high grade serous ovarian cell lines PEO1 and PEO4 revealed IL6 expression is induced by cisplatin exposure. This result was replicated by QRT-PCR and validated in the additional isogenic pair PEA1/PEA2. Western blotting demonstrated the lack of a correlation between IL6 expression and phosphorylation of either Y1007/1008 JAK2 or Y705 STAT3 levels, suggesting IL6 is not driving the constitutive activation of these proteins. Cells did however display dose dependant changes in STAT3, JAK2 and ERBB2 activation in response to cisplatin that differed between sensitive and resistant isogenic pairs. Both sensitive clones increased their activation of JAK2 and ERBB2 when exposed to low dose, 2µM, cisplatin but reversed these increases at higher concentrations. Resistant clones, PEO4 and PEA2, experienced no low dose increases in ERBB2 JAK2 or STAT3 activation instead reducing the activation of these proteins with greater sensitivity to cisplatin dose. Common to all cell lines was a high degree of correlation in the levels of activated JAK2 and ERBB2. Interfering with cisplatin dependent STAT3 deactivation using IL6 treatment was able to both sensitise cells and reduce cisplatin IC50, suggesting a functional role for STAT3 in both response, and acquired resistance, to cisplatin. Overexpression and knockdown of STAT3 demonstrated it promotes proliferation and the expression of cyclin D1 and BCL xL/S. STAT3 knockdown increased cisplatin resistance as quantified by IC50 whereas STAT3 overexpression was able to potentiate cisplatin induced apoptosis and decrease cisplatin IC50. Similarly the overexpression and knockdown of JAK2 demonstrated it also promotes proliferation, in part by regulating the activity of STAT3. The inhibition of JAK2 activity also increased resistance to cisplatin; small molecule inhibition of JAK2 both lowered background levels of apoptosis as well as attenuating cisplatin induced apoptosis. In common with STAT3 ablation JAK2 knockdown also increased cisplatin IC50. Surprisingly knockdown, overexpression and inhibition of JAK2 were all associated with changes in the activation of ERBB2. Knockdown and inhibition were associated with decreases in Y1248 phosphorylated ERBB2 whereas overexpression was associated with an increase, changes in activation appear to be driven by changes at the level of total protein. GP130 was investigated for due to its role in IL6 signalling and STAT3 activation, mRNA overexpressed was detected in the resistant pair of 2/3 isogenic cell lines. GP130 overexpression was associated with growth promotion and cisplatin resistance as revealed by siRNA knockdowns, which had no effect in cisplatin sensitive non overexpressing cells lines. Knockdown also revealed different pathways to constitutive STAT3 activation, in each high grade serous line assessed there was no effect on pSTAT3 levels, which were almost completely abolished in SKOV3. RNAi of GP130 was also associated with an increase in ERK1/2 activation which was recapitulated by JAK2 knockdown, inhibition as well as cisplatin and doxorubicin exposure. Treatment with a MEK1/2 inhibitor was able to reverse cisplatin and doxorubicin dependent activation and attenuate cytotoxic induced apoptosis. MEK1/2 inhibition was associated with an increase in JAK2, pSTAT3 and pERBB2 which was capable of reversing cisplatin dependent down regulation of these protein, highlighting a mutual feedback mechanism between the GP130/JAK2 and MAPK pathways. Central Conclusion Transcriptional regulation of JAK2 in response to cisplatin exposure drives differential behaviour of paired isogenic cell lines. Greater sensitivity of cisplatin resistant cells lines, in their deactivation of STAT3 and ERBB2 is regulated by their greater extent of JAK2 downregulation upon cisplatin exposure. Downregulation of JAK2 and its commensurate reduction in STAT3 activation were associated with reduced proliferation rates, rendering cells in a state refractory to cisplatin toxicity. This may be due to either or both of reducing the accumulation of DNA double stranded breaks associated with cell division and allowing more time for the repair of single stranded DNA adducts before cell division. While STAT3 has been suggested as a target for adjuvant chemotherapy, data presented here suggests that in combination with cisplatin STAT3 abrogation might actually reduce the effectiveness of treatment.Imperial Users Onl

Genetic Predisposition to Chronic Lymphocytic Leukemia Is Mediated by a BMF Super-Enhancer Polymorphism

SummaryChronic lymphocytic leukemia (CLL) is an adult B cell malignancy. Genome-wide association studies show that variation at 15q15.1 influences CLL risk. We deciphered the causal variant at 15q15.1 and the mechanism by which it influences tumorigenesis. We imputed all possible genotypes across the locus and then mapped highly associated SNPs to areas of chromatin accessibility, evolutionary conservation, and transcription factor binding. SNP rs539846 C>A, the most highly associated variant (p = 1.42 × 10−13, odds ratio = 1.35), localizes to a super-enhancer defined by extensive histone H3 lysine 27 acetylation in intron 3 of B cell lymphoma 2 (BCL2)-modifying factor (BMF). The rs539846-A risk allele alters a conserved RELA-binding motif, disrupts RELA binding, and is associated with decreased BMF expression in CLL. These findings are consistent with rs539846 influencing CLL susceptibility through differential RELA binding, with direct modulation of BMF expression impacting on anti-apoptotic BCL2, a hallmark of oncogenic dependency in CLL

HDAC4-regulated STAT1 activation mediates platinum resistance in ovarian cancer

Ovarian cancer frequently acquires resistance to platinum chemotherapy, representing a major challenge for improving patient survival. Recent work suggests resistant clones exist within a larger drug sensitive cell-population prior to chemotherapy, implying that resistance is selected for rather than generated by treatment. We sought to compare clinically-derived, intra-patient paired models of initial platinum response and subsequent resistant relapse to define molecular determinants of evolved resistance. Transcriptional analysis of a matched cell-line series from three patients with high-grade serous ovarian cancer before and after development of clinical platinum resistance (PEO1/PEO4/PEO6, PEA1/PEA2, PEO14/PEO23) identified 91 up- and 126 down-regulated genes common to acquired resistance. Significantly enhanced apoptotic response to platinum treatment in resistant cells was observed following knockdown of HDAC4, FOLR2, PIK3R1 or STAT1 (p<0.05). Interestingly, HDAC4 and STAT1 were found to physically interact. Acetyl-STAT1 was detected in platinum sensitive but not HDAC4 over-expressing platinum resistant cells from the same patient. In resistant cells, STAT1 phosphorylation/nuclear translocation was seen following platinum exposure, whereas silencing of HDAC4 increased acetyl-STAT1 levels, prevented platinum induced STAT1 activation and restored cisplatin sensitivity. Conversely, matched sensitive cells were refractory to STAT1 phosphorylation on platinum treatment. Analysis of 16 paired tumor biopsies taken before and after development of clinical platinum resistance showed significantly increased HDAC4 expression in resistant tumors (n=7/16[44%]; p=0.04). Therefore, clinical selection of HDAC4 overexpressing tumor cells upon exposure to chemotherapy promotes STAT1 deacetylation and cancer cell survival. Together, our findings identify HDAC4 as a novel, therapeutically tractable target to counter platinum resistance in ovarian cancer

Genetic predisposition to chronic lymphocytic leukemia is mediated by a BMF super-enhancer polymorphism

Chronic lymphocytic leukemia (CLL) is an adult B cell malignancy. Genome-wide association studies show that variation at 15q15.1 influences CLL risk. We deciphered the causal variant at 15q15.1 and the mechanism by which it influences tumorigenesis. We imputed all possible genotypes across the locus and then mapped highly associated SNPs to areas of chromatin accessibility, evolutionary conservation, and transcription factor binding. SNP rs539846 C>A, the most highly associated variant (p = 1.42 × 10(-13), odds ratio = 1.35), localizes to a super-enhancer defined by extensive histone H3 lysine 27 acetylation in intron 3 of B cell lymphoma 2 (BCL2)-modifying factor (BMF). The rs539846-A risk allele alters a conserved RELA-binding motif, disrupts RELA binding, and is associated with decreased BMF expression in CLL. These findings are consistent with rs539846 influencing CLL susceptibility through differential RELA binding, with direct modulation of BMF expression impacting on anti-apoptotic BCL2, a hallmark of oncogenic dependency in CLL

Deciphering colorectal cancer genetics through multi-omic analysis of 100,204 cases and 154,587 controls of European and east Asian ancestries

In the version of this article initially published, the author affiliations incorrectly listed “Candiolo Cancer Institute FPO-IRCCS, Candiolo (TO), Italy” as “Candiolo Cancer Institute, Candiolo, Italy.” The change has been made to the HTML and PDF versions of the article

Fine-mapping analysis including over 254,000 East Asian and European descendants identifies 136 putative colorectal cancer susceptibility genes

Genome-wide association studies (GWAS) have identified more than 200 common genetic variants independently associated with colorectal cancer (CRC) risk, but the causal variants and target genes are mostly unknown. We sought to fine-map all known CRC risk loci using GWAS data from 100,204 cases and 154,587 controls of East Asian and European ancestry. Our stepwise conditional analyses revealed 238 independent association signals of CRC risk, each with a set of credible causal variants (CCVs), of which 28 signals had a single CCV. Our cis-eQTL/mQTL and colocalization analyses using colorectal tissue-specific transcriptome and methylome data separately from 1299 and 321 individuals, along with functional genomic investigation, uncovered 136 putative CRC susceptibility genes, including 56 genes not previously reported. Analyses of single-cell RNA-seq data from colorectal tissues revealed 17 putative CRC susceptibility genes with distinct expression patterns in specific cell types. Analyses of whole exome sequencing data provided additional support for several target genes identified in this study as CRC susceptibility genes. Enrichment analyses of the 136 genes uncover pathways not previously linked to CRC risk. Our study substantially expanded association signals for CRC and provided additional insight into the biological mechanisms underlying CRC development

Absolute and relative generality

This thesis is concerned with the debate between absolutists and relativists about generality. Absolutists about quantification contend that we can quantify over absolutely everything; relativists deny this. The introduction motivates and elucidates the dispute. More familiar, restrictionist versions of relativism, according to which the range of quantifiers is always subject to restriction, are distinguished from the view defended in this thesis, an expansionist version of relativism, according to which the range of quantifiers is always open to expansion. The remainder of the thesis is split into three parts. Part I focuses on generality. Chapter 2 is concerned with the semantics of quantifiers. Unlike the restrictionist, the expansionist need not disagree with the absolutist about the semantics of quantifier domain restriction. It is argued that the threat of a certain form of semantic pessimism, used as an objection against restrictionism, also arises, in some cases, for absolutism, but is avoided by expansionism. Chapter 3 is primarily engaged in a defensive project, responding to a number of objections in the literature: the objection that the relativist is unable to coherently state her view, the objection that absolute generality is needed in logic and philosophy, and the objection that relativism is unable to accommodate ‘kind generalisations’. To meet these objections, suitable schematic and modal resources are introduced and relativism is given a precise formulation. Part II concerns issues in the philosophy of mathematics pertinent to the absolutism/relativism debate. Chapter 4 draws on the modal and schematic resources introduced in the previous chapter to regiment and generalise the key argument for relativism based on the set-theoretic paradoxes. Chapter 5 argues that relativism permits a natural motivation for Zermelo-Fraenkel set theory. A new, bi-modal axiomatisation of the iterative conception of set is presented. It is argued that such a theory improves on both its non-modal and modal rivals. Part III aims to meet a thus far unfulfilled explanatory burden facing expansionist relativism. The final chapter draws on principles from metasemantics to offer a positive account of how universes of discourse may be expanded, and assesses the prospects for a novel argument for relativism on this basis.</p

The Caesar problem – a piecemeal solution

The Caesar problem arises for abstractionist views, which seek to secure reference for terms such as ‘the number of Xs’ or #X by stipulating the content of ‘unmixed’ identity contexts like ‘#X = #Y’. Frege objects that this stipulation says nothing about ‘mixed’ contexts such as ‘#X = Julius Caesar’. This article defends a neglected response to the Caesar problem: the content of mixed contexts is just as open to stipulation as that of unmixed contexts

Absolute and relative generality

This thesis is concerned with the debate between absolutists and relativists about generality. Absolutists about quantification contend that we can quantify over absolutely everything; relativists deny this. The introduction motivates and elucidates the dispute. More familiar, restrictionist versions of relativism, according to which the range of quantifiers is always subject to restriction, are distinguished from the view defended in this thesis, an expansionist version of relativism, according to which the range of quantifiers is always open to expansion. The remainder of the thesis is split into three parts. Part I focuses on generality. Chapter 2 is concerned with the semantics of quantifiers. Unlike the restrictionist, the expansionist need not disagree with the absolutist about the semantics of quantifier domain restriction. It is argued that the threat of a certain form of semantic pessimism, used as an objection against restrictionism, also arises, in some cases, for absolutism, but is avoided by expansionism. Chapter 3 is primarily engaged in a defensive project, responding to a number of objections in the literature: the objection that the relativist is unable to coherently state her view, the objection that absolute generality is needed in logic and philosophy, and the objection that relativism is unable to accommodate ‘kind generalisations’. To meet these objections, suitable schematic and modal resources are introduced and relativism is given a precise formulation. Part II concerns issues in the philosophy of mathematics pertinent to the absolutism/relativism debate. Chapter 4 draws on the modal and schematic resources introduced in the previous chapter to regiment and generalise the key argument for relativism based on the set-theoretic paradoxes. Chapter 5 argues that relativism permits a natural motivation for Zermelo-Fraenkel set theory. A new, bi-modal axiomatisation of the iterative conception of set is presented. It is argued that such a theory improves on both its non-modal and modal rivals. Part III aims to meet a thus far unfulfilled explanatory burden facing expansionist relativism. The final chapter draws on principles from metasemantics to offer a positive account of how universes of discourse may be expanded, and assesses the prospects for a novel argument for relativism on this basis.This thesis is not currently available in ORA

The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line

Reference cell lines are often used for quality assessment; however, their effectiveness can be encumbered by the lack of standardized culture protocols. This results in variation to a supposed standard reference, inherently causing variation in measurement and analysis of the cells of interest. This is problematic when reference marker stability and characteristics are affected by use of non-standardized culture procedures. The overarching aim of this work was to apply defined parameter changes to an in-house protocol adapted from the National Institute for Biological Standards and Control. This was to investigate the impact of cell culture parameter changes on process output variation i.e. variability of growth rate and cell phenotype. The use of defined seeding densities and time-defined passage points sought to mitigate human-based sources of variation. Work was undertaken using the embryonic carcinoma 2102Ep reference line. highlighted the requirement for robust, well-characterised and standardized culture protocols. Initially, a systematic approach utilising "quick hit" experiments demonstrated significant variability introduced into culture systems resulting from slight changes to culture conditions (culture route A). This formed the basis for longitudinal experiments investigating long-term effects of culture parameters including seeding density and feeding regime (culture route B). Our results demonstrated that the specific growth rate (SGR) of passage 59 (P59) cells seeded at 20,000 cells/cm2 and subjected to a medium exchange after 48 hours prior to reseeding at 72 hours (route B2) on average was marginally higher than, P55 cells cultured under equivalent conditions (culture route A1); where the SGR values were (0.021 ±0.004) and (0.019 ±0.004) respectively. Cell viability was higher in route B2 over 10 passages with average cell viability reported as (86.3 % ±8.1) compared to route A1 (83.3 ±8.8). The metabolite data demonstrated that both culture route B1 (P57 cells seeded at 66,667 cells/cm2) and B2 had a consistent specific metabolite rate (SMR) for glucose metabolism over the 10 passages but glucose SMR values of route B1 was consistently lower than route B2 (0.00001 mmol. cell-1.d-1 and 0.000025 respectively). The present work noted that cell behaviour differences and characteristics are based not only on density, but also feeding regimes. Results revealed an interaction between phenotype, SMR and feeding regime that may not be accurately reflected by growth rate or observed morphology. This infers implies that current schemes of protocol control do not adequately account for variability, since key cell characteristics, including phenotype and SMR, change regardless of standardized seeding densities. This highlights the need to control culture parameters through defined protocols, for processes that involve cell culture for therapeutic use, biologics production, and reference lines. For the latter, this is imperative as operator-defined protocol changes result in cell characteristic variation, abating the effective use as reference lines for process control and product validation