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
