Antje Hascher

The long noncoding MALAT-1 RNA indicates a poor prognosis in non-small cell lung cancer and induces migration and tumor growth.

Introduction: The functions of large noncoding RNAs (ncRNAs) have remained elusive in many cases. Metastasis-Associated-in-Lung-Adenocarcinoma-Transcript-1 (MALAT-1) is an ncRNA that is highly expressed in several tumor types. Methods: Overexpression and RNA interference (RNAi) approaches were used for the analysis of the biological functions of MALAT-1 RNA. Tumor growth was studied in nude mice. For prognostic analysis, MALAT-1 RNA was detected on paraffin-embedded non-small cell lung cancer (NSCLC) tissue probes (n = 352) using in situ hybridization. Results: MALAT-1 was highly expressed in several human NSCLC cell lines. MALAT-1 expression was regulated by an endogenous negative feedback loop. In A549 NSCLCs, RNAi-mediated suppression of MALAT-1 RNA suppressed migration and clonogenic growth. Forced expression of MALAT-1 in NIH 3T3 cells significantly increased migration. Upon injection into nude mice, NSCLC xenografts with decreased MALAT-1 expression were impaired in tumor formation and growth. In situ hybridization on paraffin-embedded lung cancer tissue probes revealed that high MALAT-1 RNA expression in squamous cell carcinoma of the lung was associated with a poor prognosis. On genetic level, MALAT-1 displays the strongest association with genes involved in cancer like cellular growth, movement, proliferation, signaling, and immune regulation. Conclusions: These data indicate that MALAT-1 expression levels are associated with patient survival and identify tumor-promoting functions of MALAT-1.

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

S100A2 Induces Metastasis in Non–Small Cell Lung Cancer

Purpose: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non–small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC. Experimental Design: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2. Results: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021). Conclusions: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

Genome wide analysis of histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene

With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34(+) progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc-induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.

Adjuvant Therapy with Small Hairpin RNA Interference Prevents Non–Small Cell Lung Cancer Metastasis Development in Mice

Development of distant metastasis is the major reason for cancer-related deaths worldwide. Adjuvant therapy approaches after local therapies are most effective when specific targets are inhibited. Recently, we identified S100P overexpression as a strong predictor for metastasis development in early-stage non-small cell lung cancer (NSCLC) patients. Here, we show that S100P overexpression increased angiogenesis in and metastasis formation from s.c. xenotransplants of NSCLC cells. Plasmid-derived short hairpin RNAs (shRNA) were developed as specific adjuvant therapy. I.v. injected shRNA against S100P significantly decreased S100P protein expression in xenograft tumors and inhibited tumor angiogenesis in vivo. Metastasis formation 8 weeks after primary tumor resection was significantly reduced. Lung metastases developed in 31% of mice treated with S100P-targeting shRNAs compared with 64% in control shRNA-treated mice (P < 0.05). These findings suggest that RNA interference-based therapy approaches can be highly effective in the adjuvant setting.

The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors, 5-azacytidine and CP-4200, in human leukemia cells

The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [14C]5-azacytidine demonstrated Na+-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5′-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.

DNA Methyltransferase Inhibition Reverses Epigenetically Embedded Phenotypes in Lung Cancer Preferentially Affecting Polycomb Target Genes

Purpose: Cancer cell phenotypes are partially determined by epigenetic specifications, such as DNA methylation. Metastasis development is a late event in cancerogenesis and might be associated with epigenetic alterations. Experimental Design: An in vivo selection approach was used to generate highly aggressive non–small cell lung cancer (NSCLC) cell lines (A549 and HTB56) followed by genome-wide DNA methylation analysis. Furthermore, the therapeutic effects of the epigenetic agent azacytidine on DNA methylation patterns and the in vivo phenotypes were explored. Results: Widespread changes of DNA methylation were observed during development of highly aggressive cell lines. Up to 2.5% of the CpG-rich region was differentially methylated as identified by reduced representation bisulfite sequencing compared with the less aggressive parental cell lines. DNA methyltransferase inhibition by azacytidine reversed the prometastatic phenotype; this was highly associated with the preferential loss of DNA methylation at sites that were hypermethylated during the in vivo selection. Of note, polycomb (PRC2) binding sites were particularly affected by DNA methylation changes after azacytidine exposure that persisted over time. Conclusions: We could show that metastatic capability of NSCLC is closely associated with DNA methylome alterations. Because inhibition of DNA methyltransferase reversed metastasis-prone phenotype, epigenetic modulation seems to be a potential therapeutic approach to prevent metastasis formation. Clin Cancer Res; 20(4); 814–26. ©2013 AACR.

Epigenetic dysregulation of KCa 3.1 channels induces poor prognosis in lung cancer.

Epigenomic changes are an important feature of malignant tumors. How tumor aggressiveness is affected by DNA methylation of specific loci is largely unexplored. In genome‐wide DNA methylation analyses, we identified the KCa3.1 channel gene (KCNN4) promoter to be hypomethylated in an aggressive non–small‐cell lung carcinoma (NSCLC) cell line and in patient samples. Accordingly, KCa3.1 expression was increased in more aggressive NSCLC cells. Both findings were strong predictors for poor prognosis in lung adenocarcinoma. Increased KCa3.1 expression was associated with aggressive features of NSCLC cells. Proliferation and migration of pro‐metastatic NSCLC cells depended on KCa3.1 activity. Mechanistically, elevated KCa3.1 expression hyperpolarized the membrane potential, thereby augmenting the driving force for Ca2+ influx. KCa3.1 blockade strongly reduced the growth of xenografted NSCLC cells in mice as measured by positron emission tomography–computed tomography. Thus, loss of DNA methylation of the KCNN4 promoter and increased KCa3.1 channel expression and function are mechanistically linked to poor survival of NSCLC patients.

Mutations of the EPHB6 receptor tyrosine kinase induce a pro-metastatic phenotype in non-small cell lung cancer

Alterations of Eph receptor tyrosine kinases are frequent events in human cancers. Genetic variations of EPHB6 have been described but the functional outcome of these alterations is unknown. The current study was conducted to screen for the occurrence and to identify functional consequences of EPHB6 mutations in non-small cell lung cancer. Here, we sequenced the entire coding region of EPHB6 in 80 non-small cell lung cancer patients and 3 tumor cell lines. Three potentially relevant mutations were identified in primary patient samples of NSCLC patients (3.8%). Two point mutations led to instable proteins. An in frame deletion mutation (del915-917) showed enhanced migration and accelerated wound healing in vitro. Furthermore, the del915-917 mutation increased the metastatic capability of NSCLC cells in an in vivo mouse model. Our results suggest that EPHB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer.