Die funktionelle Charakterisierung des putativen Tumorsuppressors DKK3 im humanen Mammakarzinom sowie die Identifizierung potentieller SFRP1-Mimetika für die Brustkrebstherapie

The clinical need to develop new drugs for the treatment of human breast cancer is emphasized by the frequent occurrence of resistances against currently used pharmaceuticals as well as a lack of effective targeted therapies, especially for mammary carcinomas of the basal subtype. Targeting of the tumor-promoting WNT signaling pathway activity might represent a promising therapeutic strategy. A deregulation of this pathway is observed in many tumor entities, including breast cancer, and is associated with an unfavorable patient prognosis. Aberrant WNT pathway activation is frequently attributed to an expression loss of its antagonists, including DKK3 and SFRP1.Profound understanding of the molecular alterations underlying the particular subtype-specific breast carcinogenesis is essential for the development of new therapeutic strategies. To contribute to this goal, one aim of the present study was the comprehensive subtype-specific characterization of the putative tumor suppressor DKK3 in human breast cancer. A subtype-specific expression analysis using three independent tissue cohorts (n=30, n=463 and n=791) revealed a significant loss of DKK3 on mRNA and protein level in all clinically relevant breast cancer subtypes compared to healthy breast tissue controls, with the most abundant reduction in basal breast cancer cases respectively. To investigate the molecular mechanisms underlying the differential expression loss observed, a methylation analysis of both transcription-regulating CpG-islands in the DKK3 promoter and its proximal regions was performed using two independent patient cohorts (n=33 and n=497). While methylation in this region was significantly correlated with a loss of DKK3 expression in non-basal cases, basal carcinomas surprisingly presented the lowest methylation frequency indicating a different mechanism of DKK3 inactivation in these tumors. A significant correlation between a low DKK3 mRNA expression and a reduced recurrence-free survival of luminal and basal like breast cancer cases suggested a possible causal involvement of DKK3 in the tumorigenesis of these two breast cancer subtypes. This hypothesis was addressed by performing functional in vitro tests using two luminal and two basal stably transfected gain-of-function breast cancer models. Interestingly, ectopic DKK3 expression resulted in most pronounced effects on cell growth and apoptosis in basal breast cancer cells. In addition, DKK3 reexpression mediated inhibition of progression-associated characteristics of basal breast cancer cell lines resulting in a modified cell morphology, an enhanced cell-matrix adhesion and a reduced cell migration. These observations indicated a possible partial mesenchymal-to-epithelial transition in DKK3-transfected breast cancer cells, supported by an increased expression of epithelial markers like E-Cadherin and Claudin 1 as well as loss of the mesenchymal transcription factor Snail 1. In conclusion, the findings of this study suggest a possible functional involvement of a DKK3 expression loss especially in the tumorigenesis of basal breast cancers. These data provide the basis for future studies investigating the underlying molecular effects of DKK3 in detail and might therefore contribute to the development of targeted therapies for basal breast carcinomas.The second sub-project of the present dissertation was realized in cooperation with the Lead Discovery Center (LDC) in Dortmund. The aim of this study was the identification and functional characterization of specific substances mimicking the tumor suppressive effects of SRFP1. These so called SFRP1-mimetics should conduce to the future development of new drugs for the treatment of SFRP1-deficient breast cancers. The LDC initially identified twelve potential SFRP1-mimetics in a cellular primary screen. In the present study, these candidate substances were further validated in additional experiments regarding their potential SFRP1-analogous effects. Based on the results of an initial cell growth analysis, four promising molecules of these twelve putative SFRP1-mimetics were selected for a subsequent comprehensive functional in vitro analysis. All four candidates exhibited a differential, dose-dependent, growth-inhibitory capacity on SFRP1-negative and SFRP1-positive breast cancer cell culture models, with a pronounced effect on SFRP1-deficient cells. Moreover, all candidate substances influenced the expression of PCDH10, a marker gene of the SFRP1-function identified in a recent microarray analysis, in a dose-dependent manner. These data were in line with a potential SFRP1-analogous mode of action of the candidate molecules. In addition, further tumor-suppressive effects were verified for all four potential SFRP1-mimetics resulting in a decreased colony growth, an increased apoptosis and a reduced cell migration. However, the substances had varying effects on the different tumor cell characteristics analyzed so that further studies are needed to investigate their defined mechanisms of action. In conclusion, four promising, putative SFRP1-mimetics, namely 064, 366, 848 and 994 were identified in the present study that have the potential to be used as initial substances for the development of new drugs for the therapy of SFRP1-deficient mammary carcinomas

Next-Generation Sequencing Reveals Potential Predictive Biomarkers and Targets of Therapy for Urothelial Carcinoma in Situ of the Urinary Bladder

Bacillus Calmette-Guerin instillation after removal of the tumor is the first line of treatment for urothelial carcinoma in situ (CIS), the precursor lesion of most muscle-invasive bladder cancers. Bacillus Calmette-Guerin therapy fails in >50% of cases, and second-line radical cystectomy is associated with overtreatment and drastic lifestyle consequences. Given the need for alternative bladder-preserving therapies, we identified genomic alterations (GAs) in urothelial CIS having the potential to predict response to targeted therapies. Laser-capture microdissection was applied to isolate 30 samples (25 CIS and 5 muscle controls) from 26 fresh-frozen cystectomy specimens. Targeted next-generation sequencing of 31 genes was performed. The panel comprised genes frequently affected in muscle invasive bladder cancer of nonpapillary origin, focusing on potentially actionable GAs described to predict response to approved targeted therapies or drugs that are in registered clinical trials. Of CIS patients, 92% harbored at least one potentially actionable GA, which was identified in TP53/cell cycle pathway related genes (eg, TP53 and MDM2) in 72%, genes encoding chromatin-modifying proteins (eg, ARID1A and KDM6A) in 68%, DNA damage repair genes (eg, BRCA2 and ATM) in 60%, and phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway genes (eg, ERBB2 and FGFR1) in 36% of the cases. These data might help guide the selection of targeted therapies to be investigated in future clinical CIS trials, and they may provide a basis for future mechanistic studies of urothelial CIS pathogenesis

Loss of Dickkopf 3 Promotes the Tumorigenesis of Basal Breast Cancer

<div><p>Dickkopf 3 (DKK3) has been associated with tumor suppression of various tumor entities including breast cancer. However, the functional impact of DKK3 on the tumorigenesis of distinct molecular breast cancer subtypes has not been considered so far. Therefore, we initiated a study analyzing the subtype-specific DKK3 expression pattern as well as its prognostic and functional impact with respect to breast cancer subtypes. Based on three independent tissue cohorts including one <i>in silico</i> dataset (n = 30, n = 463 and n = 791) we observed a clear down-regulation of DKK3 expression in breast cancer samples compared to healthy breast tissue controls on mRNA and protein level. Interestingly, most abundant reduction of DKK3 expression was detected in the highly aggressive basal breast cancer subtype. Analyzing a large <i>in silico</i> dataset comprising 3,554 cases showed that low <i>DKK3</i> mRNA expression was significantly associated with reduced recurrence free survival (RFS) of luminal and basal-like breast cancer cases. Functionally, DKK3 re-expression in human breast cancer cell lines led to suppression of cell growth possibly mediated by up-regulation of apoptosis in basal-like but not in luminal-like breast cancer cell lines. Moreover, ectopic DKK3 expression in mesenchymal basal breast cancer cells resulted in partial restoration of epithelial cell morphology which was molecularly supported by higher expression of epithelial markers like E-Cadherin and down-regulation of mesenchymal markers such as Snail 1. Hence, we provide evidence that down-regulation of DKK3 especially promotes tumorigenesis of the aggressive basal breast cancer subtype. Further studies decoding the underlying molecular mechanisms of DKK3-mediated effects may help to identify novel targeted therapies for this clinically highly relevant breast cancer subtype.</p></div

Ectopic expression of DKK3 leads to a modified cell morphology, enhanced cell-matrix-adhesion and increased expression of epithelial markers in basal-like breast cancer cells.

<p>(A) Altered cell morphology after re-expression of DKK3 in MDA-MB-436 cells displayed by phase-contrast microscopy (top) and scanning electron microscopy (bottom). Cells of the <i>DKK3</i> clones were enlarged and grew in clusters. (B) Change in morphology resulted in enhanced cell-matrix-adhesion of MDA-MB-436 <i>DKK3</i> clones (n = 3) on <i>Matrigel</i> compared to mock clones (n = 3). Experiments were performed in triplicate. Boxes: 25-75% quartiles. Vertical lines: range, minimum and maximum. Repressed mRNA expression of mesenchymal (C-D) and increased mRNA expression of epithelial markers (E-H) was revealed in <i>DKK3</i> clones (dark grey) compared to the corresponding mock clones (light grey) of the basal-like cell lines MDA-MB-436 and MDA-MB-231. Values represent the SD for 5 independent <i>DKK3</i> and mock clones each. FC: fold change, ns: not significant, * <i>P</i> < 0.05, ** <i>P</i> < 0.01.</p

Down-regulation of <i>DKK3</i> mRNA expression in human breast cancer.

<p>(A) Real-time PCR-based <i>DKK3</i> mRNA expression analysis of 30 breast tumor compared to 11 healthy breast tissue samples. (B) Box plot of the samples shown in A demonstrating a down-regulation of <i>DKK3</i> mRNA in breast tumor compared to healthy breast tissue samples, especially in carcinomas of the IHC-defined triple negative breast cancer subtype (TNBC). (C) <i>In silico DKK3</i> mRNA expression analysis of 791 PAM50-defined breast tumor and 113 normal breast tissue samples depicted as heat map (left) and box plot (right). Red color: high, black: intermediate, green: low <i>DKK3</i> mRNA expression. Horizontal lines: grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, minimum and maximum. ns: not significant, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001.</p

Loss of DKK3 protein expression in human breast cancer.

<p>(A) Normal mammary epithelial cells showing moderate, predominantly cytoplasmic, DKK3 immunoreactivity whereas (B) primary antibody negative control is free of signal. (C-E) Weak DKK3 protein expression is observed in breast tumor samples, with lowest intensity in IHC-defined (C) TNBC cases compared to (D) HER2-positive and (E) luminal carcinomas (representative images). (F) Box plot analysis demonstrating a significant down-regulation of DKK3 in tumors of the TNBC (n = 54), the HER2-positive (n = 47) and luminal subtype (n = 362) compared to normal breast tissues (n = 11). Horizontal lines: grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, minimum and maximum. * <i>P</i> < 0.05, *** <i>P</i> < 0.001, IRS: immunoreactive score.</p

Loss of <i>DKK3</i> mRNA expression is associated with reduced recurrence-free survival (RFS) in breast cancer patients.

<p>Univariate Kaplan-Meier survival analysis of data obtained from the Kaplan-Meier Plotter portal illustrating RFS in patients with high <i>DKK3</i> (red curve) compared to patients with low <i>DKK3</i> mRNA expression (black curve) in (A) tumors of all subtypes, (B) basal subtype, (C) HER2-positive subtype, (D) luminal A subtype and (E) luminal B subtype. Vertical lines: censored cases. HR = Hazard ratio, 95% CI = 95% confidence interval.</p

Multidimensional phenotyping of breast cancer cell lines to guide preclinical research

Cell lines are extremely useful tools in breast cancer research. Their key benefits include a high degree of control over experimental variables and reproducibility. However, the advantages must be balanced against the limitations of modelling such a complex disease in vitro. Informed selection of cell line(s) for a given experiment now requires essential knowledge about molecular and phenotypic context in the culture dish.We performed multidimensional profiling of 36 widely used breast cancer cell lines that were cultured under standardised conditions. Flow cytometry and digital immunohistochemistry were used to compare the expression of 14 classical breast cancer biomarkers related to intrinsic molecular profiles and differentiation states: EpCAM, CD24, CD49f, CD44, ER, AR, HER2, EGFR, E-cadherin, p53, vimentin, and cytokeratins 5, 8/18 and 19.This cell-by-cell analysis revealed striking heterogeneity within cultures of individual lines that would be otherwise obscured by analysing cell homogenates, particularly amongst the triple-negative lines. High levels of p53 protein, but not RNA, were associated with somatic mutations (p\ua0=\ua00.008). We also identified new subgroups using the nanoString PanCancer Pathways panel (730 transcripts representing 13 canonical cancer pathways). Unsupervised clustering identified five groups: luminal/HER2, immortalised ('normal'), claudin-low and two basal clusters, distinguished mostly by baseline expression of TGF-beta and PI3-kinase pathway genes.These features are compared with other published genotype and phenotype information in a user-friendly reference table to help guide selection of the most appropriate models for in vitro and in vivo studies, and as a framework for classifying new patient-derived cancer cell lines and xenografts