Πέμπτη 28 Μαρτίου 2019

Anatomy Pathology

 IL‐8 and CXCR1 expression is associated with cancer stem cell‐like properties of clear cell renal cancer

ABSTRACT

Recent studies suggests that clear cell renal cell carcinoma ccRCC possesses a rare population of cancer stem cells (CSCs) that might contribute to tumor heterogeneity, metastasis and therapeutic resistance. Nevertheless, their relevance for renal cancer is still unclear. In this study, we successfully isolated CSCs from established human ccRCC cell lines. CSCs displayed high expression of the chemokine IL‐8 and its receptor CXCR1. While recombinant IL‐8 significantly increased CSC number and properties in vitro, CXCR1 inhibition using an anti‐CXCR1 antibody or repertaxin significantly reduced these features. After injection into immune‐deficient mice, CSCs formed primary tumors that metastasized to the lung and liver. All xenografted tumors in mice expressed high levels of IL‐8 and CXCR1. Further, IL‐8/CXCR1 expression significantly correlated with decreased overall survival in ccRCC patients. These results suggest that the IL‐8/CXCR1 phenotype is associated with CSC‐like properties in renal cancer.

 TET1 reprograms the epithelial ovarian cancer epigenome and reveals casein kinase 2α as a therapeutic target

Abstract

Ten‐eleven translocation methylcytosine dioxygenase‐1, TET1, takes part in active DNA demethylation. However, our understanding of DNA demethylation in cancer biology and its clinical significance remain limited. This study showed that TET1 expression correlated with poor survival in advanced‐stage epithelial ovarian carcinoma (EOC), and with cell migration, anchorage‐independent growth, cancer stemness, and tumorigenicity. In particular, TET1 was highly expressed in serous tubal intraepithelial carcinoma (STIC), a currently accepted type II EOC precursor, and inversely correlated with TP53 mutations. Moreover, TET1 could demethylate the epigenome and activate multiple oncogenic pathways, including an immunomodulation network having casein kinase II subunit alpha (CK2α) as a hub. Patients with TET1highCK2αhigh EOCs had the worst outcomes, and TET1‐expressing EOCs were more sensitive to a CK2 inhibitor, both in vitro and in vivo. Our findings uncover the oncogenic and poor prognostic roles of TET1 in EOC and suggest an unexplored role of epigenetic reprogramming in early ovarian carcinogenesis. Moreover, the immunomodulator CK2α represents a promising new therapeutic target, warranting clinical trials of the tolerable CK2 inhibitor, CX4945, for precision medicine against EOC.

 Morpholino‐induced exon skipping stimulates cell‐mediated and humoral responses to dystrophin in mdx mice

Abstract

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic morpholino (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin‐specific autoimmune response. Here, two independent cohorts of dystrophic mdxmice were treated chronically with either 800 mg/kg/month PMO for 6 months (n=8) or 100 mg/kg/week PMO for 12 weeks (n=11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum‐circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin‐specific antibodies were observed in the control saline‐treated mdx cohorts (n=8) or in aged (12‐month‐old) mdx mice with expanded "revertant" dystrophin‐expressing fibers. Reactive antibodies recognized both full‐length and truncated, exon‐skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen‐specific T‐cell cytokine responses (e.g., IFN‐g, IL‐2) in dystrophin antibody‐positive mice than in dystrophin antibody‐negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin‐expressing fibers along with CD8+ and perforin‐positive T cells in the vicinity, suggesting an activation of cell‐mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody‐negative mice than on those of dystrophin antibody‐positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell‐mediated and humoral immune responses in mdx mice. Our data highlight the need to further investigate the autoimmune response and its long‐term consequences after exon‐skipping therapy.

 Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

Abstract

Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis‐splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2exons 2 to 9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD‐based minigene with exons 2–9 was constructed and validated in MCF‐7 cells, producing the expected transcript (1016‐nt/V1‐BRCA2_exons_2‐9‐V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE‐variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF‐7 cells. Thus, we identified nine spliceogenic ESE‐rich intervals where ESE‐variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty‐three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double‐effect. Notably, nine spliceogenic‐ESE variants were located within ESE‐containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a non‐canonical GC‐donor 4‐nt upstream. Thirty‐six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants.

In conclusion, a large fraction of BRCA2 variants (~64%) provoked splicing anomalies lending further support to the high prevalence of this disease‐mechanism. The low accuracy of ESE‐prediction algorithms may be circumvented by functional ESE‐mapping that represents an optimal strategy to identify spliceogenic ESE‐variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants.


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