Gerwin Heller

Genome-Wide miRNA Expression Profiling Identifies miR-9-3 and miR-193a as Targets for DNA Methylation in Non–Small Cell Lung Cancers

Purpose: The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on miRNA silencing in non–small cell lung cancers (NSCLC). Experimental Design: We conducted microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. miRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding nonmalignant lung tissue samples of 101 patients with stage I–III NSCLC. Results: By comparing microarray data of untreated and drug-treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analyzed. Moreover, miR-9-3 and miR-193a were found to be tumor specifically methylated in patients with NSCLC. We observed a shorter disease-free survival of patients with miR-9-3 methylated lung squamous cell carcinoma (LSCC) than patients with miR-9-3 unmethylated LSCC by multivariate analysis [HR = 3.8; 95% confidence interval (CI), 1.3–11.2, P = 0.017] and a shorter overall survival of patients with miR-9-3 methylated LSCC than patients with miR-9-3 unmethylated LSCC by univariate analysis (P = 0.013). Conclusions: Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in patients with LSCC. Clin Cancer Res; 18(6); 1619–29. ©2012 AACR.

A Kinase-Independent Function of CDK6 Links the Cell Cycle to Tumor Angiogenesis.

Summary In contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6’s kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6’s central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a.

Lung cancer: From single-gene methylation to methylome profiling

DNA methylation as part of the epigenetic gene-silencing complex is a universal occurring change in lung cancer. Numerous studies investigated methylation of specific genes in primary tumors, in serum or plasma samples, and in specimens from the aerodigestive tract epithelium of lung cancer patients. In most studies, single genes or small numbers of genes were analyzed. Moreover, it has been observed that methylation of certain genes can already be detected in samples from the upper aerodigestive tract epithelium of cancer-free heavy smokers. These findings indicated that methylation of certain genes may be a useful biomarker for prognosis, disease recurrence, early detection, and lung cancer risk assessment. So far, several genes were identified which seem to be of worse prognostic relevance when they were found to be methylated. In addition, it has been shown that a panel of markers may be relevant to predict disease recurrence after surgery. In comparison to analysis of single or small numbers of genes, methods for genome-wide detection of methylation were developed recently. These approaches are focused on either pharmacological re-activation of methylated genes followed by expression microarray analysis or on microarray analysis of sodium bisulfite-treated or affinity-enriched methylated DNA sequences. With currently available methods for the simultaneous detection of methylation, up to 28,000 CpG islands can be analyzed. Overall, we are just at the beginning of translating these findings into the clinic and there is hope that future patients will benefit from these results.

CDK6 as a key regulator of hematopoietic and leukemic stem cell activation.

The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in regulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk6(-/-) HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more susceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcriptional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABL(p210+) LSCs. Transplantation with BCR-ABL(p210+)-infected bone marrow from Cdk6(-/-) mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk6(-/-) BCR-ABL(p210+) LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs.

Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25‐dihydroxyvitamin D 24‐hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25‐dihydroxyvitamin D3 (1,25‐D3). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1‐specific transcription factors. We quantified CYP24A1 gene copy‐number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25‐hydroxyvitamin D 1α‐hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy‐number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25‐D3 levels. We conclude that high copy‐number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

Genome-wide CpG island methylation analyses in non-small cell lung cancer patients

DNA methylation is part of the epigenetic gene regulation complex, which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stages I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis. Overall, we identified 2414 genomic positions differentially methylated between tumor and non-malignant lung tissue samples. Ninety-seven percent of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes of which many are involved in regulation of gene transcription and cell adhesion. Tumor-specific methylation was confirmed by a gene-specific approach. In the majority of tumors, methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analyzed by gene expression microarrays suggesting that about one-third of these genes are transcriptionally regulated by methylation. Moreover, comparison of methylation results with certain clinicopathological characteristics of the patients suggests that methylation of HOXA2 and HOXA10 may be of prognostic relevance in squamous cell carcinoma (SCC) patients. In conclusion, we identified a large number of tumor-specifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Moreover, HOXA2 and HOXA10 methylation may serve as prognostic parameters in SCC patients. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs.

STAT5 Is a Key Regulator in NK Cells and Acts as a Molecular Switch from Tumor Surveillance to Tumor Promotion

UNLABELLED Natural killer (NK) cells are tightly regulated by the JAK-STAT signaling pathway and cannot survive in the absence of STAT5. We now report that STAT5-deficient NK cells can be rescued by overexpression of BCL2. Our experiments define STAT5 as a master regulator of NK-cell proliferation and lytic functions. Although NK cells are generally responsible for killing tumor cells, the rescued STAT5-deficient NK cells promote tumor formation by producing enhanced levels of the angiogenic factor VEGFA. The importance of VEGFA produced by NK cells was verified by experiments with a conditional knockout of VEGFA in NK cells. We show that STAT5 normally represses the transcription of VEGFA in NK cells, in both mice and humans. These findings reveal that STAT5-directed therapies may have negative effects: In addition to impairing NK-cell-mediated tumor surveillance, they may even promote tumor growth by enhancing angiogenesis. SIGNIFICANCE The importance of the immune system in effective cancer treatment is widely recognized. We show that the new signal interceptors targeting the JAK-STAT5 pathway may have dangerous side effects that must be taken into account in clinical trials: inhibiting JAK-STAT5 has the potential to promote tumor growth by enhancing NK-cell-mediated angiogenesis.

HIF1α Regulates mTOR Signaling and Viability of Prostate Cancer Stem Cells

Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSC), were recently identified and characterized in prostate cancer. A well-characterized murine model of prostate cancer was used to investigate the regulation of hypoxia-inducible factor 1α (HIF1A/HIF1α) in CSCs and a basal stem cell subpopulation (Lin−/Sca-1+/CD49f+) was identified, in primary prostate tumors of mice, with elevated HIF1α expression. To further analyze the consequences of hypoxic upregulation on stem cell proliferation and HIF1α signaling, CSC subpopulations from murine TRAMP-C1 cells (Sca-1+/CD49f+) as well as from a human prostate cancer cell line (CD44+/CD49f+) were isolated and characterized. HIF1α levels and HIF target gene expression were elevated in hypoxic CSC-like cells, and upregulation of AKT occurred through a mechanism involving an mTOR/S6K/IRS-1 feedback loop. Interestingly, resistance of prostate CSCs to selective mTOR inhibitors was observed because of HIF1α upregulation. Thus, prostate CSCs show a hypoxic deactivation of a feedback inhibition of AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway through HIF1α is critical for CSC quiescence and maintenance by attenuating CSC metabolism and growth via mTOR and promoting survival by AKT signaling. We also propose that prostate CSCs can exhibit primary drug resistance to selective mTOR inhibitors. Implications: This work contributes to a deeper understanding of hypoxic regulatory mechanisms in CSCs and will help devise novel therapies against prostate cancer. Mol Cancer Res; 13(3); 556–64. ©2014 AACR.

Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic myeloid leukemia

Little is known about the impact of DNA methylation on the evolution/progression of Ph+ chronic myeloid leukemia (CML). We investigated the methylome of CML patients in chronic phase (CP-CML), accelerated phase (AP-CML) and blast crisis (BC-CML) as well as in controls by reduced representation bisulfite sequencing. Although only ~600 differentially methylated CpG sites were identified in samples obtained from CP-CML patients compared with controls, ~6500 differentially methylated CpG sites were found in samples from BC-CML patients. In the majority of affected CpG sites, methylation was increased. In CP-CML patients who progressed to AP-CML/BC-CML, we identified up to 897 genes that were methylated at the time of progression but not at the time of diagnosis. Using RNA-sequencing, we observed downregulated expression of many of these genes in BC-CML compared with CP-CML samples. Several of them are well-known tumor-suppressor genes or regulators of cell proliferation, and gene re-expression was observed by the use of epigenetic active drugs. Together, our results demonstrate that CpG site methylation clearly increases during CML progression and that it may provide a useful basis for revealing new targets of therapy in advanced CML.

Epigenetic down-regulation of integrin α7 increases migratory potential and confers poor prognosis in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a devastating malignancy characterized by invasive growth and rapid recurrence. The identification and inhibition of molecular components leading to this migratory and invasive phenotype are thus essential. Accordingly, a genome‐wide expression array analysis was performed on MPM cell lines and a set of 139 genes was identified as differentially expressed in cells with high versus low migratory activity. Reduced expression of the novel tumour suppressor integrin α7 (ITGA7) was found in highly motile cells. A significant negative correlation was observed between ITGA7 transcript levels and average displacement of cells. Forced overexpression of ITGA7 in MPM cells with low endogenous ITGA7 expression inhibited cell motility, providing direct evidence for the regulatory role of ITGA7 in MPM cell migration. MPM cells showed decreased ITGA7 expressions at both transcription and protein levels when compared to non‐malignant mesothelial cells. The majority of MPM cell cultures displayed hypermethylation of the ITGA7 promoter when compared to mesothelial cultures. A statistically significant negative correlation between ITGA7 methylation and ITGA7 expression was also observed in MPM cells. While normal human pleura samples unambiguously expressed ITGA7, a varying level of expression was found in a panel of 200 human MPM samples. In multivariate analysis, ITGA7 expression was found to be an independent prognostic factor. Although there was no correlation between histological subtypes and ITGA7 expression, importantly, patients with high tumour cell ITGA7 expression had an increased median overall survival compared to the low‐ or no‐expression groups (463 versus 278 days). In conclusion, our data suggest that ITGA7 is an epigenetically regulated tumour suppressor gene and a prognostic factor in human MPM. Copyright