Search

Background: Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter-α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive. Methods: ITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement. Results: ITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards β1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility. Conclusions: Our results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer. [ABSTRACT FROM AUTHOR]

NDRG2, a member of the N-myc downstream-regulated gene family, is thought to be a putative tumor suppressor gene with promising clinical impact in breast cancer. Since breast cancer comprises heterogeneous intrinsic subtypes with distinct clinical outcomes we investigated the pivotal role of NDRG2 in basal-type breast cancers. Based on subtype classified tumor (n = 45) and adjacent normal tissues (n = 17) we examined NDRG2 mRNA expression and CpG-hypermethylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA). In addition, NDRG2 protein expression was evaluated immunohistochemically using a tissue micro array (TMA, n = 211). In vitro, we investigated phenotypic effects caused by NDRG2 silencing in the basal A-like HCC1806 as well as NDRG2 over-expression in basal A-like BT20 compared to luminal-type MCF7 breast cancer cells. Our tissue collections demonstrated an overall low NDRG2 mRNA expression in breast cancer subtypes compared to normal breast tissue in line with an increased CpG-hypermethylation in breast cancer tissue. Independent TCGA data sets verified a significant (P<0.001) expression loss of NDRG2 in breast tumors. Of interest, basal-like tumors more frequently retained abundant NDRG2 expression concordant with a lower CpG-hypermethylation. Unexpectedly, basal-like breast cancer revealed an association of NDRG2 expression with unfavorable patients’ outcome. In line with this observation, in vitro experiments demonstrated reduced proliferation and migration rates (~20%) in HCC1806 cells following NDRG2 silencing. In contrast, NDRG2 over-expressing luminal-type MCF7 cells demonstrated a 26% decreased proliferation rate. Until now, this is the first study investigating the putative role of NDRG2 in depth in basal-type breast cancer. Our data indicate that the described putative tumor suppressive function of NDRG2 may be confined to luminal- and basal B-type breast cancers. [ABSTRACT FROM AUTHOR]

Liquid biopsy holds great promise to complement traditional analysis on cancerous tissue during clinical management of cancer: screening of patients, (early) disease diagnosis, prognosis, therapy selection as well as early response to treatment and disease monitoring. Among emerging circulating biomarkers, cell-free miRNA (cfmiRNA) may have potential in detecting lung cancer and following the course of the disease. Furthermore, several studies highlighted the possibility to utilize these regulatory RNAs to obtain prognostic information as well as to verify patient's response towards treatment. However, despite these findings, cfmiRNA is not used in the clinical practice as biomarkers to date, since their clinical utility and validity has not been confirmed in prospective clinical studies yet. In addition, there is no consensus on standardized (pre)analytical procedures. In this review, we present an overview of cfmiRNA biomarker candidates for clinical management of lung cancer and we discuss the issue of assay standardization. [ABSTRACT FROM AUTHOR]

Over the last decade, the immune checkpoint blockade targeting the programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis has improved progression-free and overall survival of advanced non-small cell lung cancer (NSCLC) patients. PD-L1 tumor expression, along with tumor mutational burden, is currently being explored as a predictive biomarker for responses to immune checkpoint inhibitors (ICIs). However, lung cancer patients may have insufficient tumor tissue samples and the high bleeding risk often prevents additional biopsies and, as a consequence, immunohistological evaluation of PD-L1 expression. In addition, PD-L1 shows a dynamic expression profile and can be influenced by intratumoral heterogeneity as well as the immune cell infiltrate in the tumor and its microenvironment, influencing the response rate to PD-1/PD-L1 axis ICIs. Therefore, to identify subgroups of patients with advanced NSCLC that will most likely benefit from ICI therapies, molecular characterization of PD-L1 expression in circulating tumor cells (CTCs) might be supportive. In this review, we highlight the use of CTCs as a complementary diagnostic tool for PD-L1 expression analysis in advanced NSCLC patients. In addition, we examine technical issues of PD-L1 measurement in tissue as well as in CTCs. [ABSTRACT FROM AUTHOR]

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice. [ABSTRACT FROM AUTHOR]

Background: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. Methods: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. Results: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. Conclusions: Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology. [ABSTRACT FROM AUTHOR]