Jens Claus Hahne

The Ets-1 transcription factor is involved in the development and invasion of malignant melanoma

The Ets-1 transcription factor plays a role in tumor vascularization and invasion by regulating expression of matrix-degrading proteases in endothelial cells and fibroblasts in the tumor stroma. During early embryogenesis, Ets-1 is expressed in migrating neural crest cells from which melanocytes arise. In the present study, we analyzed Ets-1 expression in various melanocytic lesions and investigated its functional importance in malignant melanomas. We found that Ets-1 was upregulated both in vivo and in vitro in malignant melanoma, compared to benign melanocytic lesions and to primary melanocytes. Assessment of DNA-binding and transactivation assays documented a strong Ets activity in melanoma cells. Using an antisense strategy, the expression and activity of Ets-1 were reduced in the melanoma cell line Mel Im. This correlated with a diminished expression of several Ets-1 target genes known to be involved in invasion, such as MMP1, MMP3, uPA and integrin β3. In line with these findings, the invasive potential of the melanoma cells measured in a Boyden Chamber model was reduced up to 60% after Ets-1 blockade. This can be attributed to the role of Ets-1 in transcriptional regulation of factors involved in invasion of melanoma cells. We conclude that over-expression of Ets-1 during melanoma development contributes to the malignant phenotype.

Patient-derived organoids model treatment response of metastatic gastrointestinal cancers

Cancer organoids to model therapy response Cancer organoids are miniature, three-dimensional cell culture models that can be made from primary patient tumors and studied in the laboratory. Vlachogiannis et al. asked whether such “tumor-in-a-dish” approaches can be used to predict drug responses in the clinic. They generated a live organoid biobank from patients with metastatic gastrointestinal cancer who had previously been enrolled in phase I or II clinical trials. This allowed the authors to compare organoid drug responses with how the patient actually responded in the clinic. Encouragingly, the organoids had similar molecular profiles to those of the patient tumor, reinforcing their value as a platform for drug screening and development. Science, this issue p. 920 Organoids can recapitulate patient responses in the clinic, with potential for drug screening and personalized medicine. Patient-derived organoids (PDOs) have recently emerged as robust preclinical models; however, their potential to predict clinical outcomes in patients has remained unclear. We report on a living biobank of PDOs from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients recruited in phase 1/2 clinical trials. Phenotypic and genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. Molecular profiling of tumor organoids was matched to drug-screening results, suggesting that PDOs could complement existing approaches in defining cancer vulnerabilities and improving treatment responses. We compared responses to anticancer agents ex vivo in organoids and PDO-based orthotopic mouse tumor xenograft models with the responses of the patients in clinical trials. Our data suggest that PDOs can recapitulate patient responses in the clinic and could be implemented in personalized medicine programs.

Ets-1 expression promotes epithelial cell transformation by inducing migration, invasion and anchorage-independent growth

Ets-1 is the prototype of the family of ETS transcription factors. In human tumors, Ets-1 is expressed in endothelial cells and fibroblasts of the tumor stroma and is proposed to play a role in tumor vascularization and invasion by upregulating expression of matrix-degrading proteases. In human carcinomas, Ets-1 is also expressed by neoplastic cells, but little is known about the functional implications of this observation. We have addressed the role of Ets-1 in epithelial HeLa tumor cells by selecting stably Ets-1 over and underexpressing HeLa cells. Ets-1 expression increases the transformed phenotype of HeLa cells, by promoting cell migration, invasion and anchorage-independent growth, while Ets-1 downregulation reduces cell attachment. In correlation with these results, Ets-1 upregulation increases integrinβ2 expression but not that of other integrins. These results suggest that, in addition to its role in the tumor stroma, Ets-1 may also promote tumor development and progression by increasing neoplastic transformation.

Effect of Pathologic Tumor Response and Nodal Status on Survival in the Medical Research Council Adjuvant Gastric Infusional Chemotherapy Trial

Purpose The Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) trial established perioperative epirubicin, cisplatin, and fluorouracil chemotherapy as a standard of care for patients with resectable esophagogastric cancer. However, identification of patients at risk for relapse remains challenging. We evaluated whether pathologic response and lymph node status after neoadjuvant chemotherapy are prognostic in patients treated in the MAGIC trial. Materials and Methods Pathologic regression was assessed in resection specimens by two independent pathologists using the Mandard tumor regression grading system (TRG). Differences in overall survival (OS) according to TRG were assessed using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate analyses using the Cox proportional hazards method established the relationships among TRG, clinical-pathologic variables, and OS. Results Three hundred thirty resection specimens were analyzed. In chemotherapy-treated patients with a TRG of 1 or 2, median OS was not reached, whereas for patients with a TRG of 3, 4, or 5, median OS was 20.47 months. On univariate analysis, high TRG and lymph node metastases were negatively related to survival (Mandard TRG 3, 4, or 5: hazard ratio [HR], 1.94; 95% CI, 1.11 to 3.39; P = .0209; lymph node metastases: HR, 3.63; 95% CI, 1.88 to 7.0; P < .001). On multivariate analysis, only lymph node status was independently predictive of OS (HR, 3.36; 95% CI, 1.70 to 6.63; P < .001). Conclusion Lymph node metastases and not pathologic response to chemotherapy was the only independent predictor of survival after chemotherapy plus resection in the MAGIC trial. Prospective evaluation of whether omitting postoperative chemotherapy and/or switching to a noncross-resistant regimen in patients with lymph node-positive disease whose tumor did not respond to preoperative epirubicin, cisplatin, and fluorouracil may be appropriate.

The Transcription Factor ETS-1: Its Role in Tumour Development and Strategies for its Inhibition

Transcription factors are an important group of proteins. Changes in expression or activity of transcription factors result in diverse and manifold effects on the whole transcriptome of the cell. Therefore transcription factors are of special interest in physiological as well as pathological processes particularly tumour development and progression. In this review we focus on Ets-1, the prototype of the ETS family of transcription factors. ETS family members play important roles in development, differentiation and proliferation of cells in general and they are involved in apoptosis and tissue remodelling as well. Most of them are downstream nuclear targets of Ras-MAP kinase signalling and the deregulation of ets genes results in malignant transformation of different cells. Several ets genes are rearranged in human leukaemia, Ewing tumours and prostate cancer to produce chimeric oncoproteins. Furthermore, an aberrant expression of several ets genes is often observed in various types of human malignant tumours. With regard to the involvement of some ETS transcription factors, especially Ets-1, in malignant transformation and tumour progression (including invasion, metastasis and neoangiogenesis) through transactivation of cancer related genes, they are potential molecular targets for selective cancer therapy. In this review we focus on the roles of Ets-1 for tumour development and progression with special emphasis on tumour vascularization and invasion. We then discuss specific strategies for Ets-1 inhibition as a potential tool for cancer treatment.

Identification of ETS-1 target genes in human fibroblasts

The transcription factor Ets-1 plays several distinct critical roles in tumour development and progression by acting both in neoplastic cells and in the tumour stroma. Increased expression of Ets-1 in tumours is often associated with a worse prognosis. Stromal fibroblasts attribute an important part to the behaviour of malignant tumours. In this study we investigated the role of Ets-1 in the tumour stroma. It is well known that ets-1 expression in fibroblasts--one of the main components of the tumour stroma--can be induced by basic fibroblast growth factor (bFGF). We applied suppression subtractive hybridization (SSH) to identify genes that are differentially expressed between bFGF stimulated wild-type fibroblasts and fibroblasts with reduced Ets-1 expression. We selected clones up- or down-regulated in bFGF stimulated wild-type fibroblasts using SSH and functionally characterized them by reference to public databases using NCBI BLAST tools. Expression levels of genes corresponding to subtracted clones were analyzed using RT-PCR. Known genes were associated with diverse functions; novel Ets-1 regulated genes identified by SSH not only encoded components involved in matrix degradation (as cathepsin and PAI-2) but also constituents of the extracellular matrix (ECM) including α-2-Type I collagen, TGF-β induced protein, lumican and decorin. Our findings identify several potential novel target genes of Ets-1, and they provide potentially important insights into the role of Ets-1 in stromal fibroblasts for both remodelling and different functionalities of the ECM.

Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers

Objective Transcribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/β-catenin pathway in liver cancer. Design Hypomorphic Apc mice (Apcfl/fl) and thiocetamide (TAA)-treated rats developed Wnt/β-catenin dependent hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), respectively. T-UCR expression was assessed by microarray, real-time PCR and in situ hybridisation. Results Overexpression of the T-UCR uc.158− could differentiate Wnt/β-catenin dependent HCC from normal liver and from β-catenin negative diethylnitrosamine (DEN)-induced HCC. uc.158− was overexpressed in human HepG2 versus Huh7 cells in line with activation of the Wnt pathway. In vitro modulation of β-catenin altered uc.158− expression in human malignant hepatocytes. uc.158− expression was increased in CTNNB1-mutated human HCCs compared with non-mutated human HCCs, and in human HCC with nuclear localisation of β-catenin. uc.158− was increased in TAA rat CCA and reduced after treatment with Wnt/β-catenin inhibitors. uc.158− expression was negative in human normal liver and biliary epithelia, while it was increased in human CCA in two different cohorts. Locked nucleic acid-mediated inhibition of uc.158− reduced anchorage cell growth, 3D-spheroid formation and spheroid-based cell migration, and increased apoptosis in HepG2 and SW1 cells. miR-193b was predicted to have binding sites within the uc.158− sequence. Modulation of uc.158− changed miR-193b expression in human malignant hepatocytes. Co-transfection of uc.158− inhibitor and anti-miR-193b rescued the effect of uc.158− inhibition on cell viability. Conclusions We showed that uc.158− is activated by the Wnt pathway in liver cancers and drives their growth. Thus, it may represent a promising target for the development of novel therapeutics.

Prognostic role of the LCS6 KRAS variant in locally advanced rectal cancer: results of the EXPERT-C trial

KRAS mutation has been reported as a marker of radio-resistance in rectal cancer and unfavourable outcome in both colon and rectal cancer. This study suggests that a single-nucleotide polymorphism of the KRAS gene (LCS-6 variant) may predict response to neoadjuvant treatment and mitigate the poor prognosis associated with KRAS mutation in locally advanced rectal cancer.

Immune escape of AKT overexpressing ovarian cancer cells

Platinum-resistance is the most crucial problem for treatment of ovarian cancer. There is a clinical need for new treatment strategies which overcome platinum resistance. As survival is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising. Therefore a better understanding of the interaction between ovarian tumour cells and cells of the immune system is a necessary prerequisite. In the present study we aimed to enlighten the interactions between platinum resistant and platinum sensitive ovarian cancer cells and natural-killer (NK)-cells. Modified FATAL assay was used for determining the killing efficiency of NK-cells for the parental A2780 cells and the cis-platinum resistant A2780cis human ovarian cancer cells. Expression of pro- and anti-apoptotic genes as well as ligands involved in NK-cell receptor recognition were analysed by RT-PCR and flow cytometric analysis. The efficiency of NK mediated cell lysis differs between A2780 cells and the cis-platinum-resistant A2780cis cells. A2780cis cells are less accessible for NK-cell mediated killing. Based on this observation we characterized the molecular basis for resistance mechanisms. Besides an increase in anti-apoptotic genes (especially CIAP-1 and -2) that probably render A2780cis cells more resistant against apoptosis an increased amount of soluble MICA/B seems to be responsible for the lower killing rate of platinum-resistant A2780cis cells compared to their parental A2780 cells.

Functional imaging and circulating biomarkers of response to regorafenib in treatment-refractory metastatic colorectal cancer patients in a prospective phase II study

Objective Regorafenib demonstrated efficacy in patients with metastatic colorectal cancer (mCRC). Lack of predictive biomarkers, potential toxicities and cost-effectiveness concerns highlight the unmet need for better patient selection. Design Patients with RAS mutant mCRC with biopsiable metastases were enrolled in this phase II trial. Dynamic contrast-enhanced (DCE) MRI was acquired pretreatment and at day 15 post-treatment. Median values of volume transfer constant (Ktrans), enhancing fraction (EF) and their product KEF (summarised median values of Ktrans× EF) were generated. Circulating tumour (ct) DNA was collected monthly until progressive disease and tested for clonal RAS mutations by digital-droplet PCR. Tumour vasculature (CD-31) was scored by immunohistochemistry on 70 sequential tissue biopsies. Results Twenty-seven patients with paired DCE-MRI scans were analysed. Median KEF decrease was 58.2%. Of the 23 patients with outcome data, >70% drop in KEF (6/23) was associated with higher disease control rate (p=0.048) measured by RECIST V. 1.1 at 2 months, improved progression-free survival (PFS) (HR 0.16 (95% CI 0.04 to 0.72), p=0.02), 4-month PFS (66.7% vs 23.5%) and overall survival (OS) (HR 0.08 (95% CI 0.01 to 0.63), p=0.02). KEF drop correlated with CD-31 reduction in sequential tissue biopsies (p=0.04). RAS mutant clones decay in ctDNA after 8 weeks of treatment was associated with better PFS (HR 0.21 (95% CI 0.06 to 0.71), p=0.01) and OS (HR 0.28 (95% CI 0.07–1.04), p=0.06). Conclusions Combining DCE-MRI and ctDNA predicts duration of anti-angiogenic response to regorafenib and may improve patient management with potential health/economic implications.