IER2-induced senescence drives melanoma invasion through osteopontin.

Expression of the immediate-early response gene IER2 has been associated with the progression of several types of cancer, but its functional role is poorly understood. We found that increased IER2 expression in human melanoma is associated with shorter overall survival, and subsequently investigated the mechanisms through which IER2 exerts this effect. In experimental melanoma models, sustained expression of IER2 induced senescence in a subset of melanoma cells in a p53/MAPK/AKT-dependent manner. The senescent cells produced a characteristic secretome that included high levels of the extracellular phosphoglycoprotein osteopontin. Nuclear localization of the IER2 protein was critical for both the induction of senescence and osteopontin secretion. Osteopontin secreted by IER2-expressing senescent cells strongly stimulated the migration and invasion of non-senescent melanoma cells. Consistently, we observed coordinate expression of IER2, p53/p21, and osteopontin in primary human melanomas and metastases, highlighting the pathophysiological relevance of IER2-mediated senescence in melanoma progression. Together, our study reveals that sustained IER2 expression drives melanoma invasion and progression through stimulating osteopontin secretion via the stochastic induction of senescence.

Ex vivo modelling of drug efficacy in a rare metastatic urachal carcinoma.

BACKGROUND: Ex vivo drug screening refers to the out-of-body assessment of drug efficacy in patient derived vital tumor cells. The purpose of these methods is to enable functional testing of patient specific efficacy of anti-cancer therapeutics and personalized treatment strategies. Such approaches could prove powerful especially in context of rare cancers for which demonstration of novel therapies is difficult due to the low numbers of patients. Here, we report comparison of different ex vivo drug screening methods in a metastatic urachal adenocarcinoma, a rare and aggressive non-urothelial bladder malignancy that arises from the remnant embryologic urachus in adults. METHODS: To compare the feasibility and results obtained with alternative ex vivo drug screening techniques, we used three different approaches; enzymatic cell viability assay of 2D cell cultures and image-based cytometry of 2D and 3D cell cultures in parallel. Vital tumor cells isolated from a biopsy obtained in context of a surgical debulking procedure were used for screening of 1160 drugs with the aim to evaluate patterns of efficacy in the urachal cancer cells. RESULTS: Dose response data from the enzymatic cell viability assay and the image-based assay of 2D cell cultures showed the best consistency. With 3D cell culture conditions, the proliferation rate of the tumor cells was slower and potency of several drugs was reduced even following growth rate normalization of the responses. MEK, mTOR, and MET inhibitors were identified as the most cytotoxic targeted drugs. Secondary validation analyses confirmed the efficacy of these drugs also with the new human urachal adenocarcinoma cell line (MISB18) established from the patient's tumor. CONCLUSIONS: All the tested ex vivo drug screening methods captured the patient's tumor cells' sensitivity to drugs that could be associated with the oncogenic KRASG12V mutation found in the patient's tumor cells. Specific drug classes however resulted in differential dose response profiles dependent on the used cell culture method indicating that the choice of assay could bias results from ex vivo drug screening assays for selected drug classes.

SNAIL1 employs ß-Catenin-LEF1 complexes to control colorectal cancer cell invasion and proliferation.

The transcription factor SNAIL1 is a master regulator of epithelial-to-mesenchymal transition (EMT), a process entailing massive gene expression changes. To better understand SNAIL1-induced transcriptional reprogramming we performed time-resolved transcriptome analysis upon conditional SNAIL1 expression in colorectal cancer cells. Gene set variation analyses indicated that SNAIL1 strongly affected features related to cell cycle and Wnt/ß-Catenin signalling. This correlated with upregulation of LEF1, a nuclear binding partner of ß-Catenin. Likewise, transcriptomes of cell lines and colorectal cancers, including poor-prognosis mesenchymal tumours, exhibit positively correlated SNAI1 and LEF1 expression, and elevated LEF1 levels parallel increased patient mortality. To delineate the functional contribution of LEF1 to SNAIL1-induced EMT, we used the CRISPR/Cas9 system to knock-out LEF1 in colorectal cancer cells, and to engineer cells that express LEF1 mutants unable to interact with ß-Catenin. Both complete LEF1-deficiency and prevention of the ß-Catenin-LEF1 interaction impaired the ability of SNAIL1 to elicit expression of an alternative set of Wnt/ß-catenin targets, and to promote cancer cell invasion. Conversely, overexpression of wildtype, but not of mutant LEF1, stimulated alternative Wnt/ß-Catenin target gene expression, and caused cell-cycle arrest. Moreover, like SNAIL1, LEF1 retarded tumour growth in xenotransplantations. Thus, LEF1 phenocopies SNAIL1 with respect to several critical aspects of EMT. Indeed, comparative transcriptomics suggested that 35% of SNAIL1-induced transcriptional changes are attributable to LEF1. However, LEF1 did not autonomously induce EMT. Rather, LEF1 appears to be a strictly ß-Catenin-dependent downstream effector of SNAIL1. Apparently, SNAIL1 employs ß-Catenin-LEF1 complexes to redirect Wnt/ß-Catenin pathway activity towards pro-invasive and anti-proliferative gene expression.

Circulating DNA as prognostic biomarker in patients with advanced hepatocellular carcinoma: a translational exploratory study from the SORAMIC trial.

BACKGROUND: Liquid biopsy based on cell-free DNA circulating in plasma has shown solid results as a non-invasive biomarker. In the present study we evaluated the utility of circulating free DNA (cfDNA) and the sub-type tumor DNA (ctDNA) in hepatocellular cancer (HCC) patients to assess therapy response and clinical outcome. METHODS: A cohort of 13 patients recruited in the context of the SORAMIC trial with unresectable, advanced HCC and different etiological and clinicopathological characteristics was included in this exploratory study. Plasma samples were collected between liver micro-intervention and beginning of sorafenib-based systemic therapy and then in correspondence of three additional follow-ups. DNA was isolated from plasma and next generation sequencing (NGS) was performed on a panel of 597 selected cancer-relevant genes. RESULTS: cfDNA levels showed a significant correlation with the presence of metastases and survival. In addition cfDNA kinetic over time revealed a trend with the clinical history of the patients, supporting its use as a biomarker to monitor therapy. NGS-based analysis on ctDNA identified 28 variants, detectable in different combinations at the different time points. Among the variants, HNF1A, BAX and CYP2B6 genes showed the highest mutation frequency and a significant association with the patients' clinicopathological characteristics, suggesting a possible role as driver genes in this specific clinical setting. CONCLUSIONS: Taken together, the results support the prognostic value of cfDNA/ctDNA in advanced HCC patients with the potential to predict therapy response. These findings support the clinical utility of liquid biopsy in advanced HCC improving individualized therapy and possible earlier identification of treatment responders.

SNAIL1 combines competitive displacement of ASCL2 and epigenetic mechanisms to rapidly silence the EPHB3 tumor suppressor in colorectal cancer.

EPHB3 is a critical cellular guidance factor in the intestinal epithelium and an important tumor suppressor in colorectal cancer (CRC) whose expression is frequently lost at the adenoma-carcinoma transition when tumor cells become invasive. The molecular mechanisms underlying EPHB3 silencing are incompletely understood. Here we show that EPHB3 expression is anti-correlated with inducers of epithelial-mesenchymal transition (EMT) in primary tumors and CRC cells. In vitro, SNAIL1 and SNAIL2, but not ZEB1, repress EPHB3 reporter constructs and compete with the stem cell factor ASCL2 for binding to an E-box motif. At the endogenous EPHB3 locus, SNAIL1 triggers the displacement of ASCL2, p300 and the Wnt pathway effector TCF7L2 and engages corepressor complexes containing HDACs and the histone demethylase LSD1 to collapse active chromatin structure, resulting in rapid downregulation of EPHB3. Beyond its impact on EPHB3, SNAIL1 deregulates markers of intestinal identity and stemness and in vitro forces CRC cells to undergo EMT with altered morphology, increased motility and invasiveness. In xenotransplants, SNAIL1 expression abrogated tumor cell palisading and led to focal loss of tumor encapsulation and the appearance of areas with tumor cells displaying a migratory phenotype. These changes were accompanied by loss of EPHB3 and CDH1 expression. Intriguingly, SNAIL1-induced phenotypic changes of CRC cells are significantly impaired by sustained EPHB3 expression both in vitro and in vivo. Altogether, our results identify EPHB3 as a novel target of SNAIL1 and suggest that disabling EPHB3 signaling is an important aspect to eliminate a roadblock at the onset of EMT processes.

Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma-carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/ß-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

Class I and III HDACs and loss of active chromatin features contribute to epigenetic silencing of CDX1 and EPHB tumor suppressor genes in colorectal cancer.

Aberrant Wnt/ß-catenin signaling is a driving force during initiation and progression of colorectal cancer. Yet, the Wnt/ß-catenin targets CDX1, EPHB2, EPHB3 and EPHB4 (EPHB2-4) act as tumor suppressors in intestinal epithelial cells and frequently appear to be transcriptionally silenced in carcinomas. The molecular mechanisms which underlie the apparent loss of expression of a subset of Wnt/ß-catenin targets in a background of persistent pathway activity are largely unknown. To gain insight into this, we quantified expression of CDX1 and EPHB2-4 in human tissue specimens of case-matched colorectal normal mucosa, adenoma and invasive carcinoma. In particular EPHB2-4 display biphasic, albeit not strictly coincident, expression profiles with elevated levels in adenomas and decreased transcription in approximately 30% of the corresponding carcinomas. Consistent with their divergent and variable expression we observed considerable heterogeneity among the epigenetic landscapes at CDX1 and EPHB2-4 in a model of colorectal carcinoma cell lines. Unlike the inactive CDX1 locus, EPHB2-4 maintain DNA hypomethylation of their promoter regions in the silent state. A strong reduction of active histone modifications consistently parallels reduced expression of CDX1 and EPHB3 and to some extent of EPHB2. Accordingly, treatment with inhibitors for DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) restored CDX1 and EPHB2-4 expression depending upon epigenetic features at their promoters but also upon cellular background. Overall our findings show that downregulation of CDX1 and EphB receptor genes occurs independently and that different branches of epigenetic control systems including class I and III HDACs contribute to epigenetic silencing of Wnt/ß-catenin targets during colorectal tumorigenesis.