Multi-omics links IL-6 trans-signalling with neutrophil extracellular trap formation and Haemophilus infection in COPD.

BACKGROUND: Interleukin (IL)-6 trans-signalling (IL-6TS) is emerging as a pathogenic mechanism in chronic respiratory diseases; however, the drivers of IL-6TS in the airways and the phenotypic characteristic of patients with increased IL-6TS pathway activation remain poorly understood. OBJECTIVE: Our aim was to identify and characterise COPD patients with increased airway IL-6TS and to elucidate the biological drivers of IL-6TS pathway activation. METHODS: We used an IL-6TS-specific sputum biomarker profile (soluble IL-6 receptor (sIL-6R), IL-6, IL-1ß, IL-8, macrophage inflammatory protein-1ß) to stratify sputum data from patients with COPD (n=74; Biomarkers to Target Antibiotic and Systemic Corticosteroid Therapy in COPD Exacerbation (BEAT-COPD)) by hierarchical clustering. The IL-6TS signature was related to clinical characteristics and sputum microbiome profiles. The induction of neutrophil extracellular trap formation (NETosis) and IL-6TS by Haemophilus influenzae were studied in human neutrophils. RESULTS: Hierarchical clustering revealed an IL-6TS-high subset (n=24) of COPD patients, who shared phenotypic traits with an IL-6TS-high subset previously identified in asthma. The subset was characterised by increased sputum cell counts (p=0.0001), persistent sputum neutrophilia (p=0.0004), reduced quality of life (Chronic Respiratory Questionnaire total score; p=0.008), and increased levels of pro-inflammatory mediators and matrix metalloproteinases in sputum. IL-6TS-high COPD patients showed an increase in Proteobacteria, with Haemophilus as the dominating genus. NETosis induced by H. influenzae was identified as a potential mechanism for increased sIL-6R levels. This was supported by a significant positive correlation between sIL-6R and NETosis markers in bronchoalveolar lavage fluid from COPD patients. CONCLUSION: IL-6TS pathway activation due to chronic colonisation with Haemophilus may be an important disease driver in a subset of COPD patients.

The iron load of lipocalin-2 (LCN-2) defines its pro-tumour function in clear-cell renal cell carcinoma.

BACKGROUND: We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. METHODS: LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. RESULTS: LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. CONCLUSIONS: We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.

The RNA-binding protein HuR inhibits expression of CCL5 and limits recruitment of macrophages into tumors.

The RNA-binding protein HuR promotes tumor growth by affecting proliferation, metastasis, apoptosis, and angiogenesis. Although immune cells, especially tumor-associated macrophages, are critical components of the tumor stroma, the influence of HuR in tumors on the recruitment of immune cells remains poorly understood. In the present study, we, therefore, aimed to elucidate the impact of tumor cell HuR on the interaction between tumor cells and macrophages. To this end, we stably depleted HuR in human MCF-7 breast cancer cells. We found that HuR-deficient cells not only showed reduced proliferation, they further expressed elevated levels of the chemokine CCL5. HuR-dependent repression of CCL5 was neither caused by altered CCL5 mRNA stability, nor by changes in CCL5 translation. Instead, loss of HuR augmented transcription of CCL5, which was mediated via an interferon-stimulated response element in the CCL5 promoter. Furthermore, HuR depletion enhanced macrophage recruitment into MCF-7 tumor spheroids, an effect which was completely lost upon neutralization of CCL5. HuR expression further negatively correlated with CCL5 expression and macrophage appearance in a cohort of breast tumors. Thus, while HuR is well-characterized to support various pro-tumorigenic features in tumor cells, we provide evidence that it limits the recruitment of macrophages into tumors by repressing CCL5. As macrophage infiltration is associated with poor prognosis, our findings underline the highly cell-type and context specific role of HuR in tumorigenesis.

The expression pattern of matrix-producing tumor stroma is of prognostic importance in breast cancer.

BACKGROUND: There are several indications that the composition of the tumor stroma can contribute to the malignancy of a tumor. Here we utilized expression data sets to identify metagenes that may serve as surrogate marker for the extent of matrix production and vascularization of a tumor and to characterize prognostic molecular components of the stroma. METHODS: TCGA data sets from six cancer forms, two breast cancer microarray sets and one mRNA data set of xenografted tumors were downloaded. Using the mean correlation as distance measure compact clusters with genes representing extracellular matrix production (ECM metagene) and vascularization (endothelial metagene) were defined. Explorative Cox modeling was used to identify prognostic stromal gene sets. RESULTS: Clustering of stromal genes in six cancer data sets resulted in metagenes, each containing three genes, representing matrix production and vascularization. The ECM metagene was associated with poor prognosis in renal clear cell carcinoma and in lung adenocarcinoma but not in other cancers investigated. Explorative Cox modeling using gene pairs identified gene sets that in multivariate models were prognostic in breast cancer. This was validated in two microarray sets. Two notable genes are TCF4 and P4HA3 which were included in the sets associated with positive and negative prognosis, respectively. Data from laser-microdissected tumors, a xenografted tumor data set and from correlation analyses demonstrate the stroma specificity of the genes. CONCLUSIONS: It is possible to construct ECM and endothelial metagenes common for several cancer forms. The molecular composition of matrix-producing cells, rather than the extent of matrix production seem to be important for breast cancer prognosis.

Prognostic stromal gene signatures in breast cancer.

INTRODUCTION: Global gene expression analysis of tumor samples has been a valuable tool to subgroup tumors and has the potential to be of prognostic and predictive value. However, tumors are heterogeneous, and homogenates will consist of several different cell types. This study was designed to obtain more refined expression data representing different compartments of the tumor. METHODS: Formalin-fixed paraffin-embedded stroma-rich triple-negative breast cancer tumors were laser-microdissected, and RNA was extracted and processed to enable microarray hybridization. Genes enriched in stroma were identified and used to generate signatures by identifying correlating genes in publicly available data sets. The prognostic implications of the signature were analyzed. RESULTS: Comparison of the expression pattern from stromal and cancer cell compartments from three tumors revealed a number of genes that were essentially specifically expressed in the respective compartments. The stroma-specific genes indicated contribution from fibroblasts, endothelial cells, and immune/inflammatory cells. The gene set was expanded by identifying correlating mRNAs using breast cancer mRNA expression data from The Cancer Genome Atlas. By iterative analyses, 16 gene signatures of highly correlating genes were characterized. Based on the gene composition, they seem to represent different cell types. In multivariate Cox proportional hazard models, two immune/inflammatory signatures had opposing hazard ratios for breast cancer recurrence also after adjusting for clinicopathological variables and molecular subgroup. The signature associated with poor prognosis consisted mainly of C1Q genes and the one associated with good prognosis contained HLA genes. This association with prognosis was seen for other cancers as well as in other breast cancer data sets. CONCLUSIONS: Our data indicate that the molecular composition of the immune response in a tumor may be a powerful predictor of cancer prognosis.

Expression of miR-34c induces G2/M cell cycle arrest in breast cancer cells.

BACKGROUND: MicroRNA-34 is a family of three miRNAs that have been reported to function as tumor suppressor miRNAs and show decreased expression in various cancers. Here, we examine functions of miR-34c in basal-like breast cancer cells. METHODS: Data from The Cancer Genome Atlas (TCGA) were used for evaluation of expression in primary breast cancers. Cellular processes affected by miR-34c were investigated by thymidine incorporation, Annexin V-assays and cell cycle analysis using breast cancer cell lines. Effects on potential targets were analyzed with qPCR and Western blot. RESULTS: TCGA data revealed that miR-34c was expressed at lower levels in basal-like breast cancer tumors and low expression was associated with poor prognosis. Ectopic expression of miR-34c in basal-like breast cancer cell lines resulted in suppressed proliferation and increased cell death. Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase. We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines. In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest. However, protein levels of PRKCA, a predicted miR-34c target and a known regulator of breast cancer cell proliferation were not influenced by miR-34c. CONCLUSIONS: Together, our results support the role of miR-34c as a tumor suppressor miRNA also in breast cancer.

Regulation of PMP22 mRNA by G3BP1 affects cell proliferation in breast cancer cells.

BACKGROUND: Regulation of mRNAs is one way to control protein levels and thereby important cellular processes such as growth, invasion and apoptosis. G3BPs constitute a family of mRNA-binding proteins, shown to be overexpressed in several cancer types, including breast, colon and pancreas cancer. G3BP has been reported to both stabilize and induce degradation of specific mRNAs. RESULTS: Here, we show that G3BP1, but not G3BP2, supports proliferation of several breast cancer cell lines. Global gene expression analyses of G3BP1- and G3BP2-depleted cells indicate that primarily G3BP1, and much less G3BP2, influences mRNA expression levels. Peripheral myelin protein 22 (PMP22) was one gene that was significantly influenced by G3BP1 depletion which led to a 2-3 fold increased expression. Depletion of PMP22 resulted in increased proliferation and the G3BP1-mediated effect on proliferation was not seen upon PMP22-depletion. CONCLUSIONS: This indicates a novel role for G3BP1 in the regulation of cell proliferation in breast cancer cells, perhaps via a regulatory effect on PMP22 expression.

Prognostic implications of the expression levels of different immunoglobulin heavy chain-encoding RNAs in early breast cancer.

The extent and composition of the immune response in a breast cancer is one important prognostic factor for the disease. The aim of the current work was to refine the analysis of the humoral component of an immune response in breast tumors by quantifying mRNA expression of different immunoglobulin classes and study their association with prognosis. We used RNA-Seq data from two local population-based breast cancer cohorts to determine the expression of IGJ and immunoglobulin heavy (IGH) chain-encoding RNAs. The association with prognosis was investigated and public data sets were used to corroborate the findings. Except for IGHE and IGHD, mRNAs encoding heavy chains were generally detected at substantial levels and correlated with other immune-related genes. High IGHG1 mRNA was associated with factors related to poor prognosis such as estrogen receptor negativity, HER2 amplification, and high grade, whereas high IGHA2 mRNA levels were primarily associated with lower age at diagnosis. High IGHA2 and IGJ mRNA levels were associated with a more favorable prognosis both in univariable and multivariable Cox models. When adjusting for other prognostic factors, high IGHG1 mRNA levels were positively associated with improved prognosis. To our knowledge, these results are the first to demonstrate that expression of individual Ig class types has prognostic implications in breast cancer.

Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation.

While aberrant cells are routinely recognized and removed by immune cells, tumors eventually escape innate immune responses. Infiltrating immune cells are even corrupted by the tumor to acquire a tumor-supporting phenotype. In line, tumor-associated macrophages are well-characterized to promote tumor progression and high levels of tumor-infiltrating macrophages are a poor prognostic marker in breast cancer. Here, we aimed to further decipher the influence of macrophages on breast tumor cells and determined global gene expression changes in three-dimensional tumor spheroids upon infiltration of macrophages. While various tumor-associated mRNAs were upregulated, expression of the cytochrome P450 family member CYP1A1 was markedly attenuated. Repression of CYP1A1 in tumor cells was elicited by a macrophage-shaped tumor microenvironment rather than by direct tumor cell-macrophage contacts. In line with changes in RNA expression profiles, macrophages enhanced proliferation of the tumor cells. Enhanced proliferation and macrophage presence further correlated with reduced CYP1A1 expression in patient tumors when compared with normal tissue. These findings are of interest in the context of combinatory therapeutic approaches involving cytotoxic and immune-modulatory compounds.

Inhibition of IRES-dependent translation of caspase-2 by HuR confers chemotherapeutic drug resistance in colon carcinoma cells.

HuR plays an important role in tumor cell survival mainly through posttranscriptional upregulation of prominent anti-apoptotic genes. In addition, HuR can inhibit the translation of pro-apoptotic factors as we could previously report for caspase-2. Here, we investigated the mechanisms of caspase-2 suppression by HuR and its contribution to chemotherapeutic drug resistance of colon carcinoma cells. In accordance with the significant drug-induced increase in cytoplasmic HuR abundance, doxorubicin and paclitaxel increased the interaction of cytoplasmic HuR with the 5'untranslated region (5'UTR) of caspase-2 as shown by RNA pull down assay. Experiments with bicistronic reporter genes furthermore indicate the presence of an internal ribosome entry site (IRES) within the caspase-2-5'UTR. Luciferase activity was suppressed either by chemotherapeutic drugs or ectopic expression of HuR. IRES-driven luciferase activity was significantly increased upon siRNA-mediated knockdown of HuR implicating an inhibitory effect of HuR on caspase-2 translation which is further reinforced by chemotherapeutic drugs. Comparison of RNA-binding affinities of recombinant HuR to two fragments of the caspase-2-5'UTR by EMSA revealed a critical HuR-binding site residing between nucleotides 111 and 241 of caspase-2-5'UTR. Mapping of critical RNA binding domains within HuR revealed that a fusion of RNA recognition motif 2 (RRM2) plus the hinge region confers a full caspase-2-5'UTR-binding. Functionally, knockdown of HuR significantly increased the sensitivity of colon cancer cells to drug-induced apoptosis. Importantly, the apoptosis sensitizing effects by HuR knockdown were rescued after silencing of caspase-2. The negative caspase-2 regulation by HuR offers a novel therapeutic target for sensitizing colon carcinoma cells to drug-induced apoptosis.

Macrophage-derived lipocalin-2 transports iron in the tumor microenvironment.

While the importance of iron for tumor development is widely appreciated, the exact sources of tumor-supporting iron largely remain elusive. The possibility that iron might be provided by stromal cells in the tumor microenvironment was not taken into account so far. In the present study, we show that tumor-associated macrophages (TAM) acquire an iron-release phenotype upon their interaction with tumor cells, thereby increasing the availability of iron in the tumor microenvironment. Mechanistically, TAM expressed elevated levels of the high-affinity iron-binding protein lipocalin-2 (LCN-2), which appeared to be critical for the export of iron from TAM, and in turn enhanced tumor cell proliferation. Moreover, in PyMT-mouse tumors as well as in primary human breast tumors LCN-2 was predominantly expressed in the tumor stroma as compared to tumor cells. LCN-2 expression in the stroma further correlated with enhanced tumor proliferation in vivo. Our data suggest a dominant role of TAM in the tumor iron-management and identify LCN-2 as a critical iron transporter in this context. Targeting the LCN-2 iron export mechanism selectively in stromal cells might open for future iron-targeted tumor therapeutic approaches.

Down Regulation of CLDND1 Induces Apoptosis in Breast Cancer Cells.

Identification of targets for apoptosis induction is important to provide novel therapeutic approaches in breast cancer. Our earlier studies showed that down regulation of protein kinase C δ (PKCδ) induces death in breast cancer cells. In this study we set out to identify previously unrecognized apoptosis regulators in breast cancer cells. To identify candidates, global expression analysis with microarray was performed after down regulation of PKCδ in the basal-like breast cancer cell lines MDA-MB-231, MDA-MB-468 and BT-549. Genes that were down regulated in all cell lines were further studied for survival-supporting effects. The claudin-like CLDND1 was singled out since several independent siRNAs targeting CLDND1 induced cell death in several cell lines. The cell death induced by CLDND1 knockdown was caspase-dependent, suggesting induction of apoptosis. Nuclear fragmentation, cleavage of caspase-3 and PARP and release of cytochrome C from the mitochondria upon CLDND1 depletion demonstrated involvement of the intrinsic apoptotic pathway. Inhibition of MEK1/2 and JNK further potentiated the cell death induction by CLDND1 knockdown. However, CLDND1 down regulation augmented ERK1/2 phosphorylation, which thereby may protect against the apoptosis inducing effects of CLDND1 down regulation. A concomitant inhibition of MEK1/2 suppresses the ERK1/2 phosphorylation and markedly potentiates the cell death following CLDND1 siRNA treatment. There is today little information on the function of CLDND1. These data provide novel information on CLDND1 and highlight it as a novel survival factor in basal-like breast cancer cell lines.

PKCα binds G3BP2 and regulates stress granule formation following cellular stress.

Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCα and a novel role for PKCα in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)(+) binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCα. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCα-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCα and G3BP2 indicated that the interaction is direct and PKCα can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCα and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2α are suppressed following downregulation of PKCα by siRNA. In conclusion this study identifies novel interaction partners of PKCα and a novel role for PKCα in regulation of stress granules.