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BACKGROUND - We characterised the phenotypic consequence of genetic variation at the PCSK9 locus and compared findings with recent trials of pharmacological inhibitors of PCSK9.
METHODS - Published and individual participant level data (300,000+ participants) were combined to construct a weighted PCSK9 gene-centric score (GS). Seventeen randomized placebo controlled PCSK9 inhibitor trials were included, providing data on 79,578 participants. Results were scaled to a one mmol/L lower LDL-C concentration.
RESULTS - The PCSK9 GS (comprising 4 SNPs) associations with plasma lipid and apolipoprotein levels were consistent in direction with treatment effects. The GS odds ratio (OR) for myocardial infarction (MI) was 0.53 (95% CI 0.42; 0.68), compared to a PCSK9 inhibitor effect of 0.90 (95% CI 0.86; 0.93). For ischemic stroke ORs were 0.84 (95% CI 0.57; 1.22) for the GS, compared to 0.85 (95% CI 0.78; 0.93) in the drug trials. ORs with type 2 diabetes mellitus (T2DM) were 1.29 (95% CI 1.11; 1.50) for the GS, as compared to 1.00 (95% CI 0.96; 1.04) for incident T2DM in PCSK9 inhibitor trials. No genetic associations were observed for cancer, heart failure, atrial fibrillation, chronic obstructive pulmonary disease, or Alzheimer's disease - outcomes for which large-scale trial data were unavailable.
CONCLUSIONS - Genetic variation at the PCSK9 locus recapitulates the effects of therapeutic inhibition of PCSK9 on major blood lipid fractions and MI. While indicating an increased risk of T2DM, no other possible safety concerns were shown; although precision was moderate.

OBJECTIVE - In the randomised scleroderma: Cyclophosphamide Or Transplantation (SCOT trial) (NCT00114530), myeloablation, followed by haematopoietic stem cell transplantation (HSCT), led to improved clinical outcomes compared with monthly cyclophosphamide (CYC) treatment in systemic sclerosis (SSc). Herein, the study aimed to determine global molecular changes at the whole blood transcript and serum protein levels ensuing from HSCT in comparison to intravenous monthly CYC in 62 participants enrolled in the SCOT study.
METHODS - Global transcript studies were performed at pretreatment baseline, 8 months and 26 months postrandomisation using Illumina HT-12 arrays. Levels of 102 proteins were measured in the concomitantly collected serum samples.
RESULTS - At the baseline visit, interferon (IFN) and neutrophil transcript modules were upregulated and the cytotoxic/NK module was downregulated in SSc compared with unaffected controls. A paired comparison of the 26 months to the baseline samples revealed a significant decrease of the IFN and neutrophil modules and an increase in the cytotoxic/NK module in the HSCT arm while there was no significant change in the CYC control arm. Also, a composite score of correlating serum proteins with IFN and neutrophil transcript modules, as well as a multilevel analysis showed significant changes in SSc molecular signatures after HSCT while similar changes were not observed in the CYC arm. Lastly, a decline in the IFN and neutrophil modules was associated with an improvement in pulmonary forced vital capacity and an increase in the cytotoxic/NK module correlated with improvement in skin score.
CONCLUSION - HSCT contrary to conventional treatment leads to a significant 'correction' in disease-related molecular signatures.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.

RATIONALE - Atherosclerosis is, in part, caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid-specific epsins promote atherogenesis is an open and significant question.
OBJECTIVE - We will determine the role of myeloid-specific epsins in regulating lesion macrophage function during atherosclerosis.
METHODS AND RESULTS - We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO) on an ApoE background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections. Epsins deficiency hindered foam cell formation and suppressed proinflammatory macrophage phenotype but increased efferocytosis and anti-inflammatory macrophage phenotype in primary macrophages. Mechanistically, we show that epsin loss specifically increased total and surface levels of LRP-1 (LDLR [low-density lipoprotein receptor]-related protein 1), an efferocytosis receptor with antiatherosclerotic properties. We further show that epsin and LRP-1 interact via epsin's ubiquitin-interacting motif domain. ox-LDL (oxidized LDL) treatment increased LRP-1 ubiquitination, subsequent binding to epsin, and its internalization from the cell surface, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE/LysM-DKO mice onto an LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis.
CONCLUSIONS - Myeloid epsins promote atherogenesis by facilitating proinflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis.

Somatostatin receptor 2 (SSTR2) is overexpressed in a majority of neuroendocrine neoplasms, including small-cell lung carcinomas (SCLCs). SSTR2 was previously considered an inhibitory receptor on cell growth, but its agonists had poor clinical responses in multiple clinical trials. The role of this receptor as a potential therapeutic target in lung cancer merits further investigation. We evaluated the expression of SSTR2 in a cohort of 96 primary tumors from patients with SCLC and found 48% expressed SSTR2. Correlation analysis in both CCLE and an SCLC RNAseq cohort confirmed high-level expression and identified an association between NEUROD1 and SSTR2. There was a significant association with SSTR2 expression profile and poor clinical outcome. We tested whether SSTR2 expression might contribute to tumor progression through activation of downstream signaling pathways, using in vitro and in vivo systems and downregulated SSTR2 expression in lung cancer cells by shRNA. SSTR2 downregulation led to increased apoptosis and dramatically decreased tumor growth in vitro and in vivo in multiple cell lines with decreased AMPKα phosphorylation and increased oxidative metabolism. These results demonstrate a role for SSTR2 signaling in SCLC and suggest that SSTR2 is a poor prognostic biomarker in SCLC and potential future therapeutic signaling target.
© 2018 UICC.

Infection with Helicobacter pylori is one of the strongest risk factors for development of gastric cancer. Although these bacteria infect approximately half of the world's population, only a small fraction of infected individuals develops gastric malignancies. Interactions between host and bacterial virulence factors are complex and interrelated, making it difficult to elucidate specific processes associated with H. pylori-induced tumorigenesis. In this study, we found that H. pylori inhibits p14ARF tumor suppressor by inducing its degradation. This effect was found to be strain-specific. Downregulation of p14ARF induced by H. pylori leads to inhibition of autophagy in a p53-independent manner in infected cells. We identified TRIP12 protein as E3 ubiquitin ligase that is upregulated by H. pylori, inducing ubiquitination and subsequent degradation of p14ARF protein. Using isogenic H. pylori mutants, we found that induction of TRIP12 is mediated by bacterial virulence factor CagA. Increased expression of TRIP12 protein was found in infected gastric epithelial cells in vitro and human gastric mucosa of H. pylori-infected individuals. In conclusion, our data demonstrate a new mechanism of ARF inhibition that may affect host-bacteria interactions and facilitate tumorigenic transformation in the stomach.

OBJECTIVE - Homologous recombination (HR)-proficient ovarian tumors have poorer clinical outcomes and show resistance to poly ADP ribose polymerase inhibitors (PARPi). A subset of HR-proficient ovarian tumors show amplification in bromodomain and extra-terminal (BET) genes such as BRD4. We aimed to test the hypothesis that BRD4 inhibition sensitizes ovarian cancer cells to PARPi by reducing HR efficiency and increasing DNA damage.
METHODS - HR-proficient ovarian cancer cell lines (OVCAR-3, OVCAR-4, SKOV-3, UWB1.289+BRCA1) were treated with BRD4-targeting siRNA, novel (INB054329, INCB057643) and established (JQ1) BET inhibitors (BETi) and PARPi (olaparib, rucaparib). Cell growth and viability were assessed by sulforhodamine B assays in vitro, and in SKOV-3 and ovarian cancer patient-derived xenografts in vivo. DNA damage and repair (pH2AX, RAD51 and BRCA1 foci formation, and DRGFP HR reporter activity), apoptosis markers (cleaved PARP, cleaved caspase-3, Bax) and proliferation markers (PCNA, Ki67) were assessed by immunofluorescence and western blot.
RESULTS - In cultured cells, inhibition of BRD4 by siRNA or INCB054329 reduced expression and function of BRCA1 and RAD51, reduced HR reporter activity, and sensitized the cells to olaparib-induced growth inhibition, DNA damage induction and apoptosis. Synergy was observed between all BETi tested and PARPi. INCB054329 and olaparib also co-operatively inhibited xenograft tumor growth, accompanied by reduced BRCA1 expression and proliferation, and increased apoptosis and DNA damage.
CONCLUSIONS - These results provide strong rationale for using BETi to extend therapeutic efficacy of PARPi to HR-proficient ovarian tumors and could benefit a substantial number of women diagnosed with this devastating disease.
Copyright © 2018 Elsevier Inc. All rights reserved.

Neuroinflammation is consistently found in many neurological disorders, but whether or not the inflammatory response independently affects neuronal network properties is poorly understood. Here, we report that intracerebroventricular injection of the prototypical inflammatory molecule lipopolysaccharide (LPS) in rats triggered a strong and long-lasting inflammatory response in hippocampal microglia associated with a concomitant upregulation of Toll-like receptor (TLR4) in pyramidal and hilar neurons. This, in turn, was associated with a significant reduction of the dendritic hyperpolarization-activated cyclic AMP-gated channel type 1 (HCN1) protein level while Kv4.2 channels were unaltered as assessed by western blot. Immunohistochemistry confirmed the HCN1 decrease in CA1 pyramidal neurons and showed that these changes were associated with a reduction of TRIP8b, an auxiliary subunit for HCN channels implicated in channel subcellular localization and trafficking. At the physiological level, this effect translated into a 50% decrease in HCN1-mediated currents (I) measured in the distal dendrites of hippocampal CA1 pyramidal cells. At the functional level, the band-pass-filtering properties of dendrites in the theta frequency range (4-12 Hz) and their temporal summation properties were compromised. We conclude that neuroinflammation can independently trigger an acquired channelopathy in CA1 pyramidal cell dendrites that alters their integrative properties. By directly changing cellular function, this phenomenon may participate in the phenotypic expression of various brain diseases.

Estrogen receptor-α positive (ERα+) breast cancer accounts for approximately 70-80% of the nearly 25,0000 new cases of breast cancer diagnosed in the US each year. Endocrine-targeted therapies (those that block ERα activity) serve as the first line of treatment in most cases. Despite the proven benefit of endocrine therapies, however, ERα+ breast tumors can develop resistance to endocrine therapy, causing disease progression or relapse, particularly in the metastatic setting. Anti-apoptotic Bcl-2 family proteins enhance breast tumor cell survival, often promoting resistance to targeted therapies, including endocrine therapies. Herein, we investigated whether blockade of anti-apoptotic Bcl-2 family proteins could sensitize luminal breast cancers to anti-estrogen treatment. We used long-term estrogen deprivation (LTED) of human ERα+ breast cancer cell lines, an established model of sustained treatment with and acquired resistance to aromatase inhibitors (AIs), in combination with Bcl-2/Bcl-xL inhibition (ABT-263), finding that ABT-263 induced only limited tumor cell killing in LTED-selected cells in culture and in vivo. Interestingly, expression and activity of the Bcl-2-related factor Mcl-1 was increased in LTED cells. Genetic Mcl-1 ablation induced apoptosis in LTED-selected cells, and potently increased their sensitivity to ABT-263. Increased expression and activity of Mcl-1 was similarly seen in clinical breast tumor specimens treated with AI + the selective estrogen receptor downregulator fulvestrant. Delivery of Mcl-1 siRNA loaded into polymeric nanoparticles (MCL1 si-NPs) decreased Mcl-1 expression in LTED-selected and fulvestrant-treated cells, increasing tumor cell death and blocking tumor cell growth. These findings suggest that Mcl-1 upregulation in response to anti-estrogen treatment enhances tumor cell survival, decreasing response to therapeutic treatments. Therefore, strategies blocking Mcl-1 expression or activity used in combination with endocrine therapies would enhance tumor cell death.

OBJECTIVE - Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD.
DESIGN - We determined promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured mRNA levels in a tissue microarray consisting of normal colons and CAC samples. and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels.
RESULTS - mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, was underexpressed in experimental inflammatory carcinogenesis, and mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction.
CONCLUSION - Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. promoter methylation status may serve as a CAC biomarker.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

BACKGROUND AND AIMS - c-Myc is highly expressed in pancreatic multipotent progenitor cells (MPC) and in pancreatic cancer. The transition from MPC to unipotent acinar progenitors is associated with c-Myc downregulation; a role for c-Myc in this process, and its possible relationship to a role in cancer, has not been established.
DESIGN - Using coimmunoprecipitation assays, we demonstrate that c-Myc and Ptf1a interact. Using reverse transcriptase qPCR, western blot and immunofluorescence, we show the erosion of the acinar programme. To analyse the genomic distribution of c-Myc and Ptf1a and the global transcriptomic profile, we used ChIP-seq and RNA-seq, respectively; validation was performed with ChIP-qPCR and RT-qPCR. Lineage-tracing experiments were used to follow the effect of c-Myc overexpression in preacinar cells on acinar differentiation.
RESULTS - c-Myc binds and represses the transcriptional activity of Ptf1a c-Myc overexpression in preacinar cells leads to a massive erosion of differentiation. In adult mice: (1) c-Myc binds to Ptf1a, and Tcf3 is downregulated; (2) Ptf1a and c-Myc display partially overlapping chromatin occupancy but do not bind the same E-boxes; (3) at the proximal promoter of genes coding for digestive enzymes, we find reduced PTF1 binding and increased levels of repressive chromatin marks and PRC2 complex components. Lineage tracing of committed acinar precursors reveals that c-Myc overexpression does not restore multipotency but allows the persistence of a preacinar-like cell population. In addition, mutant KRas can lead to c-Myc overexpression and acinar dysregulation.
CONCLUSIONS - c-Myc repression during development is crucial for the maturation of preacinar cells, and c-Myc overexpression can contribute to pancreatic carcinogenesis through the induction of a dedifferentiated state.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.