Notch Shapes the Innate Immunophenotype in Breast Cancer.

Notch activation, which is associated with basal-like breast cancer (BLBC), normally directs tissue patterning, suggesting that it may shape the tumor microenvironment. Here, we show that Notch in tumor cells regulates the expression of two powerful proinflammatory cytokines, IL1ß and CCL2, and the recruitment of tumor-associated macrophages (TAM). Notch also regulates TGFß-mediated activation of tumor cells by TAMs, closing a Notch-dependent paracrine signaling loop between these two cell types. We use a mouse model in which Notch can be regulated in spontaneous mammary carcinoma to confirm that IL1ß and CCL2 production, and macrophage recruitment are Notch-dependent. In human disease, expression array analyses demonstrate a striking association between Notch activation, IL1ß and CCL2 production, macrophage infiltration, and BLBC. These findings place Notch at the nexus of a vicious cycle of macrophage infiltration and amplified cytokine secretion and provide immunotherapeutic opportunities in BLBC.Significance: BLBC is aggressive and has an unmet need for effective targeted treatment. Our data highlight immunotherapeutic opportunities in Notch-activated BLBC. Effective IL1ß and CCL2 antagonists are currently in clinical review to treat benign inflammatory disease, and their transition to the cancer clinic could have a rapid impact. Cancer Discov; 7(11); 1320-35. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

An interaction between Scribble and the NADPH oxidase complex controls M1 macrophage polarization and function.

The polarity protein Scribble (SCRIB) regulates apical-basal polarity, directional migration and tumour suppression in Drosophila and mammals. Here we report that SCRIB is an important regulator of myeloid cell functions including bacterial infection and inflammation. SCRIB interacts directly with the NADPH oxidase (NOX) complex in a PSD95/Dlg/ZO-1 (PDZ)-domain-dependent manner and is required for NOX-induced reactive oxygen species (ROS) generation in culture and in vivo. On bacterial infection, SCRIB localized to phagosomes in a leucine-rich repeat-dependent manner and promoted ROS production within phagosomes to kill bacteria. Unexpectedly, SCRIB loss promoted M1 macrophage polarization and inflammation. Thus, SCRIB uncouples ROS-dependent bacterial killing activity from M1 polarization and inflammatory functions of macrophages. Modulating the SCRIB-NOX pathway can therefore identify ways to manage infection and inflammation with implications for chronic inflammatory diseases, sepsis and cancer.

SNX31: a novel sorting nexin associated with the uroplakin-degrading multivesicular bodies in terminally differentiated urothelial cells.

Uroplakins (UP), a group of integral membrane proteins, are major urothelial differentiation products that form 2D crystals of 16-nm particles (urothelial plaques) covering the apical surface of mammalian bladder urothelium. They contribute to the urothelial barrier function and, one of them, UPIa, serves as the receptor for uropathogenic Escherichia coli. It is therefore important to understand the mechanism by which these surface-associated uroplakins are degraded. While it is known that endocytosed uroplakin plaques are targeted to and line the multivesicular bodies (MVBs), it is unclear how these rigid-looking plaques can go to the highly curved membranes of intraluminal vesicles (ILVs). From a cDNA subtraction library, we identified a highly urothelium-specific sorting nexin, SNX31. SNX31 is expressed, like uroplakins, in terminally differentiated urothelial umbrella cells where it is predominantly associated with MVBs. Apical membrane proteins including uroplakins that are surface biotin-tagged are endocytosed and targeted to the SNX31-positive MVBs. EM localization demonstrated that SNX31 and uroplakins are both associated not only with the limiting membranes of MVBs containing uroplakin plaques, but also with ILVs. SNX31 can bind, on one hand, the PtdIns3P-enriched lipids via its N-terminal PX-domain, and, on the other hand, it binds uroplakins as demonstrated by co-immunoprecipitation and proximity ligation assay, and by its reduced membrane association in uroplakin II-deficient urothelium. The fact that in urothelial umbrella cells MVBs are the only major intracellular organelles enriched in both PtdIns3P and uroplakins may explain SNX31's MVB-specificity in these cells. However, in MDCK and other cultured cells transfected SNX31 can bind to early endosomes possibly via lipids. These data support a model in which SNX31 mediates the endocytic degradation of uroplakins by disassembling/collapsing the MVB-associated uroplakin plaques, thus enabling the uroplakin-containing (but 'softened') membranes to bud and form the ILVs for lysosomal degradation and/or exosome formation.

The breast cancer susceptibility gene product fibroblast growth factor receptor 2 serves as a scaffold for regulation of NF-κB signaling.

Fibroblast growth factor (FGF) receptor 2 (FGFR2) has been identified in genome-wide association studies to be associated with increased breast cancer risk; however, its mechanism of action remains unclear. Here we show that the two major FGFR2 alternatively spliced isoforms, FGFR2-IIIb and FGFR2-IIIc, interact with IκB kinase ß and its downstream target, NF-κB. FGFR2 inhibits nuclear RelA/p65 NF-κB translocation and activity and reduces expression of dependent transcripts, including interleukin-6. These interactions result in diminished STAT3 phosphorylation and reduced breast cancer cell growth, motility, and invasiveness. FGFR2 also arrests the epithelial cell-to-mesenchymal cell transition (EMT), resulting in attenuated neoplastic growth in orthotopic xenografts of breast cancer cells. Our studies provide strong evidence for the protective effects of FGFR2 on tumor progression. We propose that FGFR2 serves as a scaffold for multiple components of the NF-κB signaling complex. Through these interactions, FGFR2 isoforms can respond to tissue-specific FGF signals to modulate epithelial cell-stromal cell communications in cancer progression.

Associations of apolipoprotein B with pulse pressure and glucose in Chinese families with familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCHL), with a marked elevation of apolipoprotein B (apoB), is estimated to cause 10-20% of premature coronary artery disease. However, little data are available to demonstrate the associations of apoB with pulse pressure and glucose levels in FCHL families in China. This study was to investigate the potential influence factors for blood pressure and glucose phenotypes in FCHL families by multiple linear regression analysis. We recruited 147 FCHL relatives and 90 spouses, aged 30 to 60 years, from 42 Chinese families with FCHL. Our results showed that triglyceride and low density lipoprotein cholesterol were associated with fasting glucose levels (all P<0.05). Body mass index and glucose significantly correlated to systolic blood pressure, diastolic blood pressure, and mean arterial pressure, respectively (all P<0.05). Furthermore, apoB was significantly related to pulse pressure and glucose in FCHL families (all P<0.05). Thus, this study demonstrates that apoB is significantly associated with pulse pressure and glucose levels in FCHL families. Accordingly, our data suggest that apoB may be a candidate risk marker for pulse pressure and glucose in FCHL populations.

Mutation Glu82Lys in lamin A/C gene is associated with cardiomyopathy and conduction defect.

Dilated cardiomyopathy is a form of heart muscle disease characterized by impaired systolic function and ventricular dilation. The mutations in lamin A/C gene have been linked to dilated cardiomyopathy. We screened genetic mutations in a large Chinese family of 50 members including members with dilated cardiomyopathy and found a Glu82Lys substitution mutation in the rod domain of the lamin A/C protein in eight family members, three of them have been diagnosed as dilated cardiomyopathy, one presented with heart dilation. The pathogenic mechanism of lamin A/C gene defect is poorly understood. Glu82Lys mutated lamin A/C and wild type protein was transfected into HEK293 cells. The mutated protein was not properly localized at the inner nuclear membrane and the emerin protein, which interacts with lamin A/C, was also aberrantly distributed. The nuclear membrane structure was disrupted and heterochromatin was aggregated aberrantly in the nucleus of the HEK293 cells stably transfected with mutated lamin A/C gene as determined by transmission electron microscopy.

[A novel LMNA gene mutation E82K associated with familial dilated cardiomyopathy].

OBJECTIVE: To examine the function of the novel mutation E82K in LMNA gene identified in a Chinese family infected by dilated cardiomyopathy. METHODS: (1) One Chinese family infected by dilated cardiomyopathy was chosen for the study. Exons 1-12 of the LMNA gene were screened with both PCR method and the cycle sequencing of the PCR products. (2) cDNA of the E82K mutation or wild type of LMNA gene was transfected into HEK293 cells and the apoptosis of the cells was detected after treatment with 0.8 mmol/L H2O2. RESULTS: (1) A new mutation E82K in LMNA gene was identified in this dilated cardiomyopathy family. (2) Apoptosis was more in the HEK293 cells transfected with E82K mutation than those with empty vector or wild type LMNA gene. CONCLUSIONS: The missense mutation E82K in LMNA gene changed the polar of the amino acid. It showed a malignant phenotype of severe clinical symptoms, early onset, poor survival prognosis and might be associated with atrioventricular conduction block (II degrees-III degrees), suggesting that the E82K mutation in LMNA gene may be a candidate for nosogenesis of dilated cardiomyopathy.

InSNP: a tool for automated detection and visualization of SNPs and InDels.

Availability of high quality SNP data is a rate-limiting factor in understanding the impact of genetic variability on gene function and phenotype. Although global projects like HAPMAP generate large numbers of SNPs in an even spacing throughout the human genome, many variation studies have a more focused approach: in the follow-up of positional association findings, candidate gene studies, and functional genomics experiments, knowledge of all variations in a limited amount of sequence (e.g., a gene) is needed. This leads to a large number of resequencing experiments, for which there is a surprising lack of analysis software. We have thus developed specialized software (InSNP) for targeted mutation detection and compared its performance to Polyphred and Mutation Surveyor using 28 amplicons. Out of a total of 579 (InSNP), 644 (Polyphred), and 526 (Mutation Surveyor) SNP predictions, 39 SNPs were confirmed by human expert inspection, with five SNPs missed by Polyphred and one missed by InSNP using the default settings. For InDel detection, out of 70 (InSNP), 28 (Polyphred), and 693 (Mutation Surveyor) InDel predictions, two InDels were confirmed by human expert inspection, with one InDel missed by Polyphred. InSNP provides a user-friendly interface with better functionality for mutation detection than general-purpose sequence handling software. It provides similar SNP detection sensitivity and specificity as the public domain and commercial alternatives in the investigated dataset. We hope that InSNP lowers the barriers to the use of automated mutation detection software and aids in the improvement of the efficiency of such experiments. The Windows installer (setup) program and sample datasets are available at www.mucosa.de/insnp/.