Long Interspersed Nuclear Element 1 Retrotransposons Become Deregulated during the Development of Ovarian Cancer Precursor Lesions.

There is growing evidence that most high-grade serous ovarian carcinomas likely arise from local dissemination of precursor lesions of the fallopian tube. Evolution of these lesions from early p53 signatures to latter-stage, serous tubal intraepithelial carcinomas (STICs) is characterized by cytologic atypia, accumulation of somatic mutations, and genomic instability, the etiologies of which remain unclear. Long interspersed element 1 (LINE-1) retrotransposon is expressed in many carcinomas, including high-grade serous ovarian carcinoma, where it contributes to genomic instability; however, the timing of LINE-1 activation during this evolution has yet to be elucidated. In this study, we assessed LINE-1 open reading frame 1 protein expression in 12 p53 signature lesions, 32 STICs, and 112 various types of ovarian cancers via immunohistochemical staining and examined LINE-1 promoter methylation in representative cases. We found that 78% and 57% of STICs, with and without concurrent ovarian carcinomas, respectively, exhibited intense LINE-1 immunoreactivity compared with adjacent, normal-appearing fallopian tube epithelium. Hypomethylation of the LINE-1 promoter was found in all STICs exhibiting overexpression. None of the 12 p53 signatures demonstrated significant LINE-1 expression. In ovarian cancer, 84 (75%) of 112 ovarian carcinomas overexpressed LINE-1. Our results indicate that LINE-1 retrotransposons often become deregulated during progression of ovarian cancer precursor lesions from the p53 signature to STIC stages and remain highly expressed in carcinoma.

Identification of hub genes within the CCL18 signaling pathway in hepatocellular carcinoma through bioinformatics analysis.

Introduction: Hepatocellular carcinoma (HCC) is an aggressive malignancy, and CCL18, a marker of M2 macrophage activation, is often associated with tumor immune suppression. However, the role of CCL18 and its signaling pathway in HCC is still limited. Our study focuses on investigating the prognostic impact of CCL18 and its signaling pathway in HCC patients and biological functions in vitro. Methods: HCC-related RNA-seq data were obtained from TCGA, ICGC, and GEO. The 6 hub genes with the highest correlation to prognosis were identified using univariate Cox and LASSO regression analysis. Multivariate Cox regression analysis was performed to assess their independent prognostic potential and a nomogram was constructed. In vitro experiments, including CCK8, EdU, RT-qPCR, western blot, and transwell assays, were conducted to investigate the biological effects of exogenous CCL18 and 6 hub genes. A core network of highly expressed proteins in the high-risk group of tumors was constructed. Immune cell infiltration was evaluated using the ESTIMATE and CIBERSORT packages. Finally, potential treatments were explored using the OncoPredict package and CAMP database. Results: We identified 6 survival-related genes (BMI1, CCR3, CDC25C, CFL1, LDHA, RAC1) within the CCL18 signaling pathway in HCC patients. A nomogram was constructed using the TCGA_LIHC cohort to predict patient survival probability. Exogenous CCL18, as well as overexpression of BMI1, CCR3, CDC25C, CFL1, LDHA, and RAC1, can promote proliferation, migration, invasion, stemness, and increased expression of PD-L1 protein in LM3 and MHCC-97H cell lines. In the high-risk group of patients from the TCGA_LIHC cohort, immune suppression was observed, with a strong correlation to 21 immune-related genes and suppressive immune cells. Conclusion: Exogenous CCL18 promotes LM3 and MHCC-97H cells proliferation, migration, invasion, stemness, and immune evasion. The high expression of BMI1, CCR3, CDC25C, CFL1, LDHA, and RAC1 can serve as a biomarkers for immune evasion in HCC.

Methylomic Landscapes of Ovarian Cancer Precursor Lesions.

PURPOSE: The current paradigm in the development of high-grade serous ovarian carcinoma (HGSC) proposes that the majority of HGSCs arise from precursor serous tubal intraepithelial carcinoma (STIC) lesions of the fallopian tube. Here we survey genome-wide methylation in HGSC precursor lesions to identify genomic regions that exhibit high-specificity differential hypermethylation for potential use as biomarkers for detecting STIC and HGSC at stages when curative intervention likely remains feasible. EXPERIMENTAL DESIGN: We first identified quality control criteria for performing reliable methylomic analysis of DNA-limited tubal precursor lesions with the Illumina Infinium MethylationEPIC array. We then used this platform to compare genome-wide methylation among 12 STICs with paired adjacent-normal epithelia, one p53 signature lesion and two samples of concurrent HGSC. The resulting methylomic data were analyzed by unsupervised hierarchical clustering and multidimensional analysis. Regions of high-confidence STIC-specific differential hypermethylation were identified using selective bioinformatic criteria and compared with published MethylationEPIC data from 23 HGSC tumors and 11 healthy fallopian tube mucosae. RESULTS: Unsupervised analysis showed that STICs largely clustered with HGSCs, but were clearly distinct from adjacent-normal fallopian tube epithelia. Forty-two genomic regions exhibited high-confidence STIC-specific differential hypermethylation, of which 17 (40.5%) directly overlapped with HGSC-specific differentially methylated regions. Methylation at these shared loci was able to completely distinguish STIC and HGSC samples from normal and adjacent-normal specimens. CONCLUSIONS: Our results suggest that most STICs are epigenetically similar to HGSCs and share regions of differential hypermethylation that warrant further evaluation for potential use as biomarkers for early detection of ovarian HGSC.See related commentary by Ishak and De Carvalho, p. 6083.

Antibody incubation at 37°C improves fluorescent immunolabeling in free-floating thick tissue sections.

Fluorescent immunolabeling and imaging in free-floating thick (50-60 µm) tissue sections is relatively simple in practice and enables design-based non-biased stereology, or 3-D reconstruction and analysis. This method is widely used for 3-D in situ quantitative biology in many areas of biological research. However, the labeling quality and efficiency of standard protocols for fluorescent immunolabeling of these tissue sections are not always satisfactory. Here, we systematically evaluate the effects of raising the conventional antibody incubation temperatures (4°C or 21°C) to mammalian body temperature (37°C) in these protocols. Our modification significantly enhances the quality (labeling sensitivity, specificity, and homogeneity) and efficiency (antibody concentration and antibody incubation duration) of fluorescent immunolabeling of free-floating thick tissue sections.