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.

A distinct innate lymphoid cell population regulates tumor-associated T cells.

Antitumor T cells are subject to multiple mechanisms of negative regulation. Recent findings that innate lymphoid cells (ILCs) regulate adaptive T cell responses led us to examine the regulatory potential of ILCs in the context of cancer. We identified a unique ILC population that inhibits tumor-infiltrating lymphocytes (TILs) from high-grade serous tumors, defined their suppressive capacity in vitro, and performed a comprehensive analysis of their phenotype. Notably, the presence of this CD56+CD3- population in TIL cultures was associated with reduced T cell numbers, and further functional studies demonstrated that this population suppressed TIL expansion and altered TIL cytokine production. Transcriptome analysis and phenotypic characterization determined that regulatory CD56+CD3- cells exhibit low cytotoxic activity, produce IL-22, and have an expression profile that overlaps with those of natural killer (NK) cells and other ILCs. NKp46 was highly expressed by these cells, and addition of anti-NKp46 antibodies to TIL cultures abrogated the ability of these regulatory ILCs to suppress T cell expansion. Notably, the presence of these regulatory ILCs in TIL cultures corresponded with a striking reduction in the time to disease recurrence. These studies demonstrate that a previously uncharacterized ILC population regulates the activity and expansion of tumor-associated T cells.

Pneumococcal Competence Coordination Relies on a Cell-Contact Sensing Mechanism.

Bacteria have evolved various inducible genetic programs to face many types of stress that challenge their growth and survival. Competence is one such program. It enables genetic transformation, a major horizontal gene transfer process. Competence development in liquid cultures of Streptococcus pneumoniae is synchronized within the whole cell population. This collective behavior is known to depend on an exported signaling Competence Stimulating Peptide (CSP), whose action generates a positive feedback loop. However, it is unclear how this CSP-dependent population switch is coordinated. By monitoring spontaneous competence development in real time during growth of four distinct pneumococcal lineages, we have found that competence shift in the population relies on a self-activated cell fraction that arises via a growth time-dependent mechanism. We demonstrate that CSP remains bound to cells during this event, and conclude that the rate of competence development corresponds to the propagation of competence by contact between activated and quiescent cells. We validated this two-step cell-contact sensing mechanism by measuring competence development during co-cultivation of strains with altered capacity to produce or respond to CSP. Finally, we found that the membrane protein ComD retains the CSP, limiting its free diffusion in the medium. We propose that competence initiator cells originate stochastically in response to stress, to form a distinct subpopulation that then transmits the CSP by cell-cell contact.

B7-H4 expression by nonhematopoietic cells in the tumor microenvironment promotes antitumor immunity.

The B7 family plays a critical role in both positive and negative regulation of immune responses by engaging a variety of receptors on lymphocytes. Importantly, blocking coinhibitory molecules using antibodies specific for CTLA-4 and PD-1 enhances tumor immunity in a subset of patients. Therefore, it is critical to understand the role of different B7 family members since they may be suitable therapeutic targets. B7-H4 is another member that inhibits T-cell function, and it is also upregulated on a variety of tumors and has been proposed to promote tumor growth. Here, we investigate the role of B7-H4 in tumor development and show that B7-H4 expression inhibits tumor growth in two mouse models. Furthermore, we show that B7-H4 expression is required for antitumor immune responses in a mouse model of mammary tumorigenesis. We found that the expression levels of B7-H4 correlate with MHC class I expression in both mouse and human samples. We show that IFNγ upregulates B7-H4 expression on mouse embryo fibroblasts and that the upregulation of B7-H4 on tumors is dependent on T cells. Notably, patients with breast cancer with increased B7-H4 expression show a prolonged time to recurrence. These studies demonstrate a positive role for B7-H4 in promoting antitumor immunity.

Mislocalization of the cell polarity protein scribble promotes mammary tumorigenesis and is associated with basal breast cancer.

Scribble (SCRIB) localizes to cell-cell junctions and regulates establishment of epithelial cell polarity. Loss of expression of SCRIB functions as a tumor suppressor in Drosophila and mammals; conversely, overexpression of SCRIB promotes epithelial differentiation in mammals. Here, we report that SCRIB is frequently amplified, mRNA overexpressed, and protein is mislocalized from cell-cell junctions in human breast cancers. High levels of SCRIB mRNA are associated with poor clinical prognosis, identifying an unexpected role for SCRIB in breast cancer. We find that transgenic mice expressing a SCRIB mutant [Pro 305 to Leu (P305L)] that fails to localize to cell-cell junctions, under the control of the mouse mammary tumor virus long terminal repeat promoter, develop multifocal hyperplasia that progresses to highly pleomorphic and poorly differentiated tumors with basal characteristics. SCRIB interacts with phosphatase and tensin homolog (PTEN) and the expression of P305L, but not wild-type SCRIB, promotes an increase in PTEN levels in the cytosol. Overexpression of P305L, but not wild-type SCRIB, activates the Akt/mTOR/S6K signaling pathway. Human breast tumors overexpressing SCRIB have high levels of S6K but do not harbor mutations in PTEN or PIK3CA, identifying SCRIB amplification as a mechanism of activating PI3K signaling in tumors without mutations in PIK3CA or PTEN. Thus, we demonstrate that high levels of mislocalized SCRIB functions as a neomorph to promote mammary tumorigenesis by affecting subcellular localization of PTEN and activating an Akt/mTOR/S6kinase signaling pathway.

BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival.

Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor.

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.

A complementation method for functional analysis of mammalian genes.

Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies.

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference.

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-alpha, IL-1beta or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-kappaB, or on the protein levels of IkappaB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases.

Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia.

We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.

Adeno-associated virus (AAV)-mediated gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture.

BACKGROUND/PURPOSE: Since the discovery of the cystic fibrosis transmembrane regulator (CFTR) gene, cystic fibrosis has been an attractive target for gene therapy. Postnatal gene transfer in the respiratory epithelium has been difficult and particularly inefficient in the submucosal gland cells, the target cells for CFTR gene transfer. The authors hypothesized that during development, there is a favorable environment for fetal gene therapy with fewer physical barriers to efficient gene transfer and more accessible epithelial and submucosal gland precursor cells. The authors tested this hypothesis in a novel human fetal tracheal organ culture system using a serotype 2 recombinant AAV that contains an enhanced green fluorescent protein (eGFP) reporter gene (AAV-CMV-eGFP). METHODS: Human fetal tracheas at between 16 and 20 weeks' gestation age were used in this study. The distal end of each trachea was ligated and secured in an upright position with the open proximal end facing up. Only the ante-lumenal surface was exposed to culture media. 5 x 10(9) particles of AAV-CMV-eGFP were administered intratracheally through the open end. Fetal tracheas were maintained in tracheal organ culture media and harvested at either 2 weeks (n = 3) or 4 weeks (n = 7) after injection. The fetal tracheas were processed for routine H&E, standard electron microscopy (EM), and fluorescence microscopy for analysis of eGFP transgene expression. RESULTS: Histology confirmed the preservation of structural integrity out to 4 weeks of fetal tracheal organ culture. EM showed intact tight junctions of the apical respiratory epithelium. At 2 weeks after AAV-CMV-eGFP injection, there was minimal transgene expression. However, at 4 weeks there was extensive transgene expression in not only the respiratory surface epithelium but also the submucosal gland cells of the human fetal tracheal organ culture. Transgene expression was seen in nearly all cells in the submucosal glands. CONCLUSIONS: AAV-mediated gene transfer in human fetal tracheal organ culture was highly efficient with excellent transgene expression at 4 weeks in both respiratory surface epithelium and submucosal gland cells. This highly efficient gene transfer may result from fewer physical barriers and more accessible target precursor cells. These results are consistent with more efficient gene transfer in fetal tracheobronchial epithelium and show the potential for fetal gene therapy using AAV for the treatment of congenital airway disease such as cystic fibrosis.