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To identify novel coding association signals and facilitate characterization of mechanisms influencing glycemic traits and type 2 diabetes risk, we analyzed 109,215 variants derived from exome array genotyping together with an additional 390,225 variants from exome sequence in up to 39,339 normoglycemic individuals from five ancestry groups. We identified a novel association between the coding variant (p.Pro50Thr) in and fasting plasma insulin (FI), a gene in which rare fully penetrant mutations are causal for monogenic glycemic disorders. The low-frequency allele is associated with a 12% increase in FI levels. This variant is present at 1.1% frequency in Finns but virtually absent in individuals from other ancestries. Carriers of the FI-increasing allele had increased 2-h insulin values, decreased insulin sensitivity, and increased risk of type 2 diabetes (odds ratio 1.05). In cellular studies, the AKT2-Thr50 protein exhibited a partial loss of function. We extend the allelic spectrum for coding variants in associated with disorders of glucose homeostasis and demonstrate bidirectional effects of variants within the pleckstrin homology domain of .
© 2017 by the American Diabetes Association.
We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor , and the most up-regulated gene in SC was () in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.
Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1 cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the PC marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Expansion of the PC compartment in ErbB3-null intestines was accompanied with elevated ER stress and inflammation markers, raising the possibility that negative regulation of PCs by ErbB3 is necessary to maintain homeostasis. Taken together, our data suggest that ErbB3 restricts PC numbers through PI3K-mediated suppression of Atoh1 levels leading to inhibition of PC differentiation, with important implications for regulation of the ISC niche.
Wilms tumor (WT) is the most common renal neoplasm of childhood and affects 1 in 10 000 children aged less than 15 years. These embryonal tumors are thought to arise from primitive nephrogenic rests that derive from the metanephric mesenchyme during kidney development and are characterized partly by increased Wnt/β-catenin signaling. We previously showed that coordinate activation of Ras and β-catenin accelerates the growth and metastatic progression of a murine WT model. Here, we show that activating KRAS mutations can be found in human WT. In addition, high levels of phosphorylated AKT are present in the majority of WT. We further show in a mouse model and in renal epithelial cells that Ras cooperates with β-catenin to drive metastatic disease progression and promotes in vitro tumor cell growth, migration, and colony formation in soft agar. Cellular transformation and metastatic disease progression of WT cells are in part dependent on PI3K/AKT activation and are inhibited via pharmacological inhibition of this pathway. Our studies suggest both KRAS mutations and AKT activation are present in WT and may represent novel therapeutic targets for this disease.
© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA518-526-specific CD8+T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4+T-cells showed increased IL-2 production, CD11c+ dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8+T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8+T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8+T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8+T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8+T-cell effector function in the tumor microenvironment.
Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large B-cell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cγ), but had low α-BCR-induced signaling. This contrasted MCL tumors, where α-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, α-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors.
© 2017 by The American Society of Hematology.
Macrophage apoptosis and the ability of macrophages to clean up dead cells, a process called efferocytosis, are crucial determinants of atherosclerosis lesion progression and plaque stability. Environmental stressors initiate endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). Unresolved ER stress with activation of the UPR initiates apoptosis. Macrophages are resistant to apoptotic stimuli, because of activity of the PI3K/Akt pathway. Macrophages express 3 Akt isoforms, Akt1, Akt2 and Akt3, which are products of distinct but homologous genes. Akt displays isoform-specific effects on atherogenesis, which vary with different vascular cell types. Loss of macrophage Akt2 promotes the anti-inflammatory M2 phenotype and reduces atherosclerosis. However, Akt isoforms are redundant with regard to apoptosis. c-Jun NH-terminal kinase (JNK) is a pro-apoptotic effector of the UPR, and the JNK1 isoform opposes anti-apoptotic Akt signaling. Loss of JNK1 in hematopoietic cells protects macrophages from apoptosis and accelerates early atherosclerosis. IκB kinase α (IKKα, a member of the serine/threonine protein kinase family) plays an important role in mTORC2-mediated Akt signaling in macrophages, and IKKα deficiency reduces macrophage survival and suppresses early atherosclerosis. Efferocytosis involves the interaction of receptors, bridging molecules, and apoptotic cell ligands. Scavenger receptor class B type I is a critical mediator of macrophage efferocytosis via the Src/PI3K/Rac1 pathway in atherosclerosis. Agonists that resolve inflammation offer promising therapeutic potential to promote efferocytosis and prevent atherosclerotic clinical events. (Circ J 2016; 80: 2259-2268).
OBJECTIVE - is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between infection, proinflammatory NF-κB activation and regulation of DARPP-32.
DESIGN - The study used and experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, infection, tissue microarrays and immunohistochemical assays were used.
RESULTS - Our results indicated that infection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level and . To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the promoter into the luciferase reporter (pGL3-Luc). Both infection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of promoter and ChIP assay, we demonstrated that the sequence -996 to -1008 bp containing putative NF-κB-binding sites is the most active region. The induction of DARPP-32 expression by infection counteracted -induced cell death through activation of serine/threonine-specific protein kinase (AKT), as determined by ATP-Glo and clonogenic survival assays. Immunohistochemistry analysis demonstrated a significant positive correlation between NF-κB and DARPP-32 expression levels in gastric cancer tissues (r=0.43, p<0.01).
CONCLUSIONS - Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following infection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis.
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/.
Metastatic breast cancer is an incurable disease and identification of novel therapeutic opportunities is vital. Triple-negative breast cancer (TNBC) frequently metastasizes and high levels of activated p90RSK (RSK), a downstream MEK-ERK1/2 effector, are found in TNBC. We demonstrate, using direct pharmacologic and genetic inhibition of RSK1/2, that these kinases contribute to the TNBC metastatic process in vivo Kinase profiling showed that RSK1 and RSK2 are the predominant kinases targeted by the new inhibitor, which is based on the natural product SL0101. Further evidence for selectivity was provided by the observations that silencing RSK1 and RSK2 eliminated the ability of the analogue to further inhibit survival or proliferation of a TNBC cell line. In vivo, the new derivative was as effective as the FDA-approved MEK inhibitor trametinib in reducing the establishment of metastatic foci. Importantly, inhibition of RSK1/2 did not result in activation of AKT, which is known to limit the efficacy of MEK inhibitors in the clinic. Our results demonstrate that RSK is a major contributor to the TNBC metastatic program and provide preclinical proof-of-concept for the efficacy of the novel SL0101 analogue in vivo Mol Cancer Ther; 15(11); 2598-608. ©2016 AACR.
©2016 American Association for Cancer Research.
Ewing sarcoma is driven by characteristic chromosomal translocations between the EWSR1 gene with genes encoding ETS family transcription factors (EWS-ETS), most commonly FLI1. However, direct pharmacological inhibition of transcription factors like EWS-FLI1 remains largely unsuccessful. Active gene transcription requires orchestrated actions of many epigenetic regulators, such as the bromodomain and extra-terminal domain (BET) family proteins. Emerging BET bromodomain inhibitors have exhibited promising antineoplastic activities via suppression of oncogenic transcription factors in various cancers. We reasoned that EWS-FLI1-mediated transcription activation might be susceptible to BET inhibition. In this study, we demonstrated that small molecule BET bromodomain inhibitors repressed EWS-FLI1-driven gene signatures and downregulated important target genes. However, expression of EWS-FLI1 was not significantly affected. Repression of autocrine IGF1 by BET inhibitors led to significant inhibition of the IGF1R/AKT pathway critical to Ewing sarcoma cell proliferation and survival. Consistently, BET inhibitors impaired viability and clonogenic survival of Ewing sarcoma cell lines and blocked EWS-FLI1-induced transformation of mouse NIH3T3 fibroblast cells. Selective depletion of individual BET genes partially phenocopied the actions of BET inhibitors. Finally, the prototypical BET inhibitor, JQ1, significantly repressed Ewing sarcoma xenograft tumor growth. These findings suggest therapeutic potential of BET inhibitors in Ewing sarcoma and highlight an emerging paradigm of using epigenetic agents to treat cancers driven by fusion transcription factors.