Four PTEN-targeting co-expressed miRNAs and ACTN4- targeting miR-548b are independent prognostic biomarkers in human squamous cell carcinoma of the oral tongue.

The purpose of this study was to determine the prognostic value and oncogenic pathways associated to miRNA expression in squamous cell carcinoma of the oral tongue and to link these miRNA candidates with potential gene targets. We performed a miRNA screening within our institutional cohort (n = 58 patients) and reported five prognostic targets including a cluster of four co-expressed miRNAs (miR-18a, miR-92a, miR-103, and miR-205). Multivariate analysis showed that expression of miR-548b (p = 0.007) and miR-18a (p = 0.004, representative of co-expressed miRNAs) are independent prognostic markers for squamous cell carcinoma of the oral tongue. These findings were validated in The Cancer Genome Atlas (TCGA) cohort (n = 131) for both miRNAs (miR-548b: p = 0.027; miR-18a: p = 0.001). Bioinformatics analysis identified PTEN and ACTN4 as direct targets of the four co-expressed miRNAs and miR-548b, respectively. Correlations between the five identified miRNAs and their respective targeted genes were validated in the two merged cohorts and were concordantly significant (miR-18a/PTEN: p < 0.0001; miR-92a/PTEN: p = 0.0008; miR-103/PTEN: p = 0.008; miR-203/PTEN: p = 0.019; miR-548b/ACTN4: p = 0.009).

Mutations of POLR3A encoding a catalytic subunit of RNA polymerase Pol III cause a recessive hypomyelinating leukodystrophy.

Leukodystrophies are a heterogeneous group of inherited neurodegenerative disorders characterized by abnormal white matter visible by brain imaging. It is estimated that at least 30% to 40% of individuals remain without a precise diagnosis despite extensive investigations. We mapped tremor-ataxia with central hypomyelination (TACH) to 10q22.3-23.1 in French-Canadian families and sequenced candidate genes within this interval. Two missense and one insertion mutations in five individuals with TACH were uncovered in POLR3A, which codes for the largest subunit of RNA polymerase III (Pol III). Because these families were mapped to the same locus as leukodystrophy with oligodontia (LO) and presented clinical and radiological overlap with individuals with hypomyelination, hypodontia and hypogonadotropic hypogonadism (4H) syndrome, we sequenced this gene in nine individuals with 4H and eight with LO. In total, 14 recessive mutations were found in 19 individuals with TACH, 4H, or LO, establishing that these leukodystrophies are allelic. No individual was found to carry two nonsense mutations. Immunoblots on 4H fibroblasts and on the autopsied brain of an individual diagnosed with 4H documented a significant decrease in POLR3A levels, and there was a more significant decrease in the cerebral white matter compared to that in the cortex. Pol III has a wide set of target RNA transcripts, including all nuclear-coded tRNA. We hypothesize that the decrease in POLR3A leads to dysregulation of the expression of certain Pol III targets and thereby perturbs cytoplasmic protein synthesis. This type of broad alteration in protein synthesis is predicted to occur in other leukoencephalopathies such as hypomyelinating leukodystrophy-3, caused by mutations in aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1).

STAT1-associated intratumoural TH1 immunity predicts chemotherapy resistance in high-grade serous ovarian cancer.

High-grade serous ovarian carcinoma (HGSC) accounts for 70% of all epithelial ovarian cancers but clinical management is challenged by a lack of accurate prognostic and predictive biomarkers of chemotherapy response. This study evaluated the role of Signal Transducer and Activator of Transcription 1 (STAT1) as an independent prognostic and predictive biomarker and its correlation with intratumoural CD8+ T cells in a second independent biomarker validation study. Tumour STAT1 expression and intratumoural CD8+ T cell infiltration were assessed by immunohistochemistry as a multicentre validation study conducted on 734 chemotherapy-naïve HGSCs. NanoString-based profiling was performed to correlate expression of STAT1 target genes CXCL9, CXCL10 and CXCL11 with CD8A transcript expression in 143 primary tumours. Multiplexed cytokine analysis of pre-treatment plasma from resistant and sensitive patients was performed to assess systemic levels of STAT1-induced cytokines. STAT1 was validated as a prognostic and predictive biomarker in both univariate and multivariate models and its expression correlated significantly with intra-epithelial CD8+ T cell infiltration in HGSC. STAT1 levels increased the prognostic and predictive value of intratumoural CD8+ T cells, confirming their synergistic role as biomarkers in HGSC. In addition, expression of STAT1 target genes (CXCL9, CXCL10 and CXCL11) correlated significantly with levels of, and CD8A transcripts from intratumoural CD8+ T cells within the resistant and sensitive tumours. Our findings provide compelling evidence that high levels of STAT1, STAT1-induced chemokines and CD8+ T cells correlate with improved chemotherapy response in HGSC. These results identify STAT1 and its target genes as novel biomarkers of chemosensitivity in HGSC. These findings provide new translational opportunities for patient stratification for immunotherapies based on emerging biomarkers of inflammation in HGSC. An improved understanding of the role of interferon-inducible genes will be foundational for developing immunomodulatory therapies in ovarian cancer.

Cell cycle-dependent localization of CHK2 at centrosomes during mitosis.

BACKGROUND: Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle. RESULTS: Here, we show that CHK2 only localizes to centrosomes during mitosis. Using wild-type and CHK2-/- HCT116 human colon cancer cells and human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs we show that several CHK2 antibodies are non-specific and cross-react with an unknown centrosomal protein(s) by immunofluorescence. To characterize the localization of CHK2, we generated cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with the centrosomes in a Polo-like kinase 1-dependent manner during mitosis, from early mitotic stages until cytokinesis. CONCLUSION: Our findings demonstrate that a subpopulation of CHK2 localizes at the centrosomes in mitotic cells but not in interphase. These results are consistent with previous reports supporting a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis.

Nuclear autoantigen CENP-B transactivation of the epidermal growth factor receptor via chemokine receptor 3 in vascular smooth muscle cells.

OBJECTIVE: We have previously found that the CENP-B nuclear autoantigen, which is specifically targeted by autoantibodies in the limited cutaneous form of systemic sclerosis, behaved as a potent migratory factor for human pulmonary artery smooth muscle cells (PASMCs). Other recent studies have shown that several disease-associated autoantigens induced cell migration by interacting with various chemokine receptors. Prompted by this hypothesis, we undertook this study to determine whether CENP-B interacts with chemokine receptors on the surface of human PASMCs, to explore the relevant signaling pathways, and to characterize the effects of anti-CENP-B binding on SMC stimulation. METHODS: To demonstrate the expression of specific chemokine receptors by human PASMCs at both the messenger RNA and protein levels, reverse transcription-polymerase chain reaction, immunoblotting, and flow cytometry analyses were performed. Desensitization studies and specific inhibitors were used to further identify the CENP-B target on the surface of human PASMCs. RESULTS: Our data strongly suggested that CENP-B used chemokine receptor 3 (CCR3) to mediate human PASMCs signaling. Moreover, several lines of evidence indicated that CENP-B binding subsequently stimulated the cross-talk between CCR3 and epidermal growth factor receptor (EGFR) via a matrix metalloprotease-dependent mechanism that involved the processing of heparin-binding EGF-like growth factor. Transactivation of the EGFR through CCR3 was found to be a critical pathway that elicits MAP kinase activation and secretion of cytokines such as interleukin-8. Finally, anti-CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular smooth muscle cells. CONCLUSION: The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti-CENP-B autoantibodies.

Direct binding of anti-DNA topoisomerase I autoantibodies to the cell surface of fibroblasts in patients with systemic sclerosis.

OBJECTIVE: Fibroblasts play a crucial role in the development of systemic sclerosis (SSc), and antifibroblast antibodies (AFAs) capable of inducing a proinflammatory phenotype in fibroblasts have been detected in the sera of SSc patients. This study examined the prevalence of AFAs in SSc and other diseases and the possible correlation between AFAs and known antinuclear antibody specificities in SSc patients. METHODS: Sera from 99 patients with SSc, 123 patients with other autoimmune and nonautoimmune diseases, and 30 age- and sex-matched healthy controls were examined. AFA prevalence was assessed by flow cytometry and further characterized by indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Anti-topoisomerase I (anti-topo I) from SSc sera were purified by affinity chromatography on topo I. RESULTS: AFAs were more common in SSc patients (26.3%) than in any other disease groups studied. The presence of AFA was significantly associated with pulmonary involvement and death. AFA-positive sera from SSc patients bound to all human and rodent fibroblasts tested, but not to human primary endothelial cells or smooth muscle cells. All SSc AFAs strongly reacted with topo I by ELISA and immunoblotting. The binding intensity of SSc AFAs correlated strongly with reactivity against topo I on immunoblots of fibroblast extracts and with the immunofluorescence pattern typical of anti-topo I on permeabilized cells. Total IgG and affinity-purified anti-topo I from AFA-positive SSc sera were found to react with the surface of unpermeabilized fibroblasts by flow cytometry as well as by immunofluorescence and confocal microscopy. CONCLUSION: This is the first report establishing that AFAs in SSc are strongly correlated with anti-topo I and, furthermore, that anti-topo I antibodies themselves display AFA activity by reacting with determinants at the fibroblast surface.