ITIH5 mediates epigenetic reprogramming of breast cancer cells

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

Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling

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

Conversations under a Tung Tree

<p>Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of <i>SFRP3</i> in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate <i>SFRP3</i> expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, <i>SFRP3</i> mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of <i>SFRP3</i> in LUAD and LUSC patients. Moreover, DNA hypermethylation of <i>SFRP3</i> was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of <i>SFRP3</i> expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and <i>in vitro</i> demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of <i>CyclinD1</i> expression <i>in vitro</i>. Our results indicate that <i>SFRP3</i> acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.</p

Die Identifizierung und Validierung neuer potentieller Blut-basierter Früherkennungs-Biomarker sowie die funktionelle Charakterisierung von ITIH5 im humanen Mammakarzinom

Mammography has become standard of care in breast cancer screening but the limitations are well-recognized resulting in an ongoing discussion about the screening necessity. Regarding these limitations of mammography in population-based screening the validation of novel minimally-invasive screening tests that could complement to mammography is an important research topic of modern medicine. There is a growing confidence that the next generation of screening tests will be based on molecular biomarkers present in bodily fluids. Determination of promoter methylation of tumor suppressor genes in the circulating free DNA (cfDNA) of serum is a rapidly growing research field in cancer detection, indicating high specificity and sensitivity of these biomarkers. Based on the largest sample collection analyzed for serum based cfDNA methylation biomarkers so far, promoter methylation of the DKK3-ITIH5 gene combination was highly significant compared to initially six analyzed candidate genes. In an independent validation set the combined ITIH5- and DKK3-methylation achieved 40% sensitivity with a high specificity of 94% (AUC: 0,673, P<0,0001). In case of positivity the likelihood of an existent breast tumor was 85% (positive predictive value), while in case of negativity the likelihood of an existent breast tumor was 34% (negative predictive value: 66%). However, the cumulative DKK3-ITIH5-methylation failed in discriminating between breast and colon cancer with a reduction of specificity to 56%. In contrast, these biomarkers showed the capability to detect cfDNA methylation in the important clinical subgroup of premenopausal women revealing a higher sensitivity (52%) with a specificity of 100% (AUC: 0.762, P<0,0001). Due to the high breast cancer related mortality rate, the understanding of tumor biological and molecular consequences is mandatory to further develop and targeted breast cancer therapy. Besides the potential use of ITIH5 as an early detection biomarker, this putative metastasis suppressor gene encodes an extracellular matrix protein, being involved in extracellular matrix integrity. It is known that the DNA-methylation based loss of ITIH5-expression is associated with a reduction of patient’s survival, while the functional characterization of ITIH5-related signaling pathways remains unknown. On the basis of two cell culture based gain-of-function tumors models, reduction of proliferation and migration capability as well as an enhanced cell-to-matrix adhesion was shown. Furthermore, a gene expression microarray identified differential regulated genes in ITIH5-positive cells. Microarray analysis showed a clear classification of ITIH5-associated genes in Gene Ontology Annotation “extracellular matrix” (ECM). During malignant progression cancer cells overcome an ongoing degradation of the ECM, the central barrier of invasive cells. Consistent with this observation, a previous study revealed that ITIH5-expression in MDA-MB-231 breast tumor cells led to the disruption of stress fibers and their anchored focal adhesion sites, which are both necessary for cell movement. Therewith, ITIH5 could suppress specific mechanisms of the mesenchymal migration process that is defined as a fundamental hallmark of invasive cancer cells. In line with the different cell morphology a complete demethylation of cancer related genes, e.g. of the tumor suppressor gene NDRG2 and EHD3, was found. Furthermore, NDRG2 and ITIH5 RNA- and protein-expression correlated significant in breast cancer tissue. However, in the complex network of ITIH5-associated genes RNAi mediated NDRG2 knock-down revealed no solid influence in reactivation of the phenotype of ITIH5-positive MDA-MB-231 cells. In this context, complex interactions of focal adhesion complex with the ECM play a supporting role. Since endoglin, a co-receptor of TGF-beta1 which is known to modulate the composition of focal adhesion sites was found to be re-expressed only in cells positive for ITIH5, it could be hypothesized that endoglin represents the direct connection to the extracellular compartment. While there was an increased expression of the non-receptor focal adhesion kinase (FAK) in ITIH5-positive cells, phosphorylation state was identical unregarded of ITIH5, i.e. ITIH5 seems to influence FAK activation. In this signaling pathway the non-receptor Src-kinase, an important component of FAK signaling, seems to be degraded in the presence of ITIH5. Therewith, a higher caveolin-1 expression and a decrease in STAT1 phosphorylation fit to the putative abruption of the Src/FAK-signaling pathway in the presence of ITIH5

Die Identifizierung und Validierung neuer potentieller Blut-basierter Früherkennungs-Biomarker sowie die funktionelle Charakterisierung von ITIH5 im humanen Mammakarzinom

Mammography has become standard of care in breast cancer screening but the limitations are well-recognized resulting in an ongoing discussion about the screening necessity. Regarding these limitations of mammography in population-based screening the validation of novel minimally-invasive screening tests that could complement to mammography is an important research topic of modern medicine. There is a growing confidence that the next generation of screening tests will be based on molecular biomarkers present in bodily fluids. Determination of promoter methylation of tumor suppressor genes in the circulating free DNA (cfDNA) of serum is a rapidly growing research field in cancer detection, indicating high specificity and sensitivity of these biomarkers. Based on the largest sample collection analyzed for serum based cfDNA methylation biomarkers so far, promoter methylation of the DKK3-ITIH5 gene combination was highly significant compared to initially six analyzed candidate genes. In an independent validation set the combined ITIH5- and DKK3-methylation achieved 40% sensitivity with a high specificity of 94% (AUC: 0,673, P<0,0001). In case of positivity the likelihood of an existent breast tumor was 85% (positive predictive value), while in case of negativity the likelihood of an existent breast tumor was 34% (negative predictive value: 66%). However, the cumulative DKK3-ITIH5-methylation failed in discriminating between breast and colon cancer with a reduction of specificity to 56%. In contrast, these biomarkers showed the capability to detect cfDNA methylation in the important clinical subgroup of premenopausal women revealing a higher sensitivity (52%) with a specificity of 100% (AUC: 0.762, P<0,0001). Due to the high breast cancer related mortality rate, the understanding of tumor biological and molecular consequences is mandatory to further develop and targeted breast cancer therapy. Besides the potential use of ITIH5 as an early detection biomarker, this putative metastasis suppressor gene encodes an extracellular matrix protein, being involved in extracellular matrix integrity. It is known that the DNA-methylation based loss of ITIH5-expression is associated with a reduction of patient’s survival, while the functional characterization of ITIH5-related signaling pathways remains unknown. On the basis of two cell culture based gain-of-function tumors models, reduction of proliferation and migration capability as well as an enhanced cell-to-matrix adhesion was shown. Furthermore, a gene expression microarray identified differential regulated genes in ITIH5-positive cells. Microarray analysis showed a clear classification of ITIH5-associated genes in Gene Ontology Annotation “extracellular matrix” (ECM). During malignant progression cancer cells overcome an ongoing degradation of the ECM, the central barrier of invasive cells. Consistent with this observation, a previous study revealed that ITIH5-expression in MDA-MB-231 breast tumor cells led to the disruption of stress fibers and their anchored focal adhesion sites, which are both necessary for cell movement. Therewith, ITIH5 could suppress specific mechanisms of the mesenchymal migration process that is defined as a fundamental hallmark of invasive cancer cells. In line with the different cell morphology a complete demethylation of cancer related genes, e.g. of the tumor suppressor gene NDRG2 and EHD3, was found. Furthermore, NDRG2 and ITIH5 RNA- and protein-expression correlated significant in breast cancer tissue. However, in the complex network of ITIH5-associated genes RNAi mediated NDRG2 knock-down revealed no solid influence in reactivation of the phenotype of ITIH5-positive MDA-MB-231 cells. In this context, complex interactions of focal adhesion complex with the ECM play a supporting role. Since endoglin, a co-receptor of TGF-beta1 which is known to modulate the composition of focal adhesion sites was found to be re-expressed only in cells positive for ITIH5, it could be hypothesized that endoglin represents the direct connection to the extracellular compartment. While there was an increased expression of the non-receptor focal adhesion kinase (FAK) in ITIH5-positive cells, phosphorylation state was identical unregarded of ITIH5, i.e. ITIH5 seems to influence FAK activation. In this signaling pathway the non-receptor Src-kinase, an important component of FAK signaling, seems to be degraded in the presence of ITIH5. Therewith, a higher caveolin-1 expression and a decrease in STAT1 phosphorylation fit to the putative abruption of the Src/FAK-signaling pathway in the presence of ITIH5

Circulating Tumor Cell PD-L1 Expression as Biomarker for Therapeutic Efficacy of Immune Checkpoint Inhibition in NSCLC

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