Dual-Luciferase Reporter Assay for Prescreening CRISPR (d)Cas9-Mediated Epigenetic Editing on a Plant Promoter Using Human Cells.

Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.

Effective Targeting of Melanoma Cells by Combination of Mcl-1 and Bcl-2/Bcl-xL/Bcl-w Inhibitors.

Recent advances in melanoma therapy have significantly improved the prognosis of metastasized melanoma. However, large therapeutic gaps remain that need to be closed by new strategies. Antiapoptotic Bcl-2 proteins critically contribute to apoptosis deficiency and therapy resistance. They can be targeted by BH3 mimetics, small molecule antagonists that mimic the Bcl-2 homology domain 3 (BH3) of proapoptotic BH3-only proteins. By applying in vitro experiments, we aimed to obtain an overview of the possible suitability of BH3 mimetics for future melanoma therapy. Thus, we investigated the effects of ABT-737 and ABT-263, which target Bcl-2, Bcl-xL and Bcl-w as well as the Bcl-2-selective ABT-199 and the Mcl-1-selective S63845, in a panel of four BRAF-mutated and BRAF-WT melanoma cell lines. None of the inhibitors showed significant effectiveness when used alone; however, combination of S63845 with each one of the three ABTs almost completely abolished melanoma cell survival and induced apoptosis in up to 50-90% of the cells. Special emphasis was placed here on the understanding of the downstream pathways involved, which may allow improved applications of these strategies. Thus, cell death induction was correlated with caspase activation, loss of mitochondrial membrane potential, phosphorylation of histone H2AX, and ROS production. Caspase dependency was demonstrated by a caspase inhibitor, which blocked all effects. Upregulation of Mcl-1, induced by S63845 itself, as reported previously, was blocked by the combinations. Indeed, Mcl-1, as well as XIAP (X-linked inhibitor of apoptosis), were strongly downregulated by combination treatments. These findings demonstrate that melanoma cells can be efficiently targeted by BH3 mimetics, but the right combinations have to be selected. The observed pronounced activation of apoptosis pathways demonstrates the decisive role of apoptosis in the loss of cell viability by BH3 mimetics.

Biomarker RIPK3 Is Silenced by Hypermethylation in Melanoma and Epigenetic Editing Reestablishes Its Tumor Suppressor Function.

For several decades, cancers have demonstrably been one of the most frequent causes of death worldwide. In addition to genetic causes, cancer can also be caused by epigenetic gene modifications. Frequently, tumor suppressor genes are epigenetically inactivated due to hypermethylation of their CpG islands, actively contributing to tumorigenesis. Since CpG islands are usually localized near promoters, hypermethylation of the promoter can have a major impact on gene expression. In this study, the potential tumor suppressor gene Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) was examined for an epigenetic regulation and its gene inactivation in melanomas. A hypermethylation of the RIPK3 CpG island was detected by bisulfite pyrosequencing and was accompanied by a correlated loss of its expression. In addition, an increasing RIPK3 methylation rate was observed with increasing tumor stage of melanomas. For further epigenetic characterization of RIPK3, epigenetic modulation was performed using a modified CRISPR/dCas9 (CRISPRa activation) system targeting its DNA hypermethylation. We observed a reduced fitness of melanoma cells by (re-)expression and demethylation of the RIPK3 gene using the epigenetic editing-based method. The tumor suppressive function of RIPK3 was evident by phenotypic determination using fluorescence microscopy, flow cytometry and wound healing assay. Our data highlight the function of RIPK3 as an epigenetically regulated tumor suppressor in melanoma, allowing it to be classified as a biomarker.

BRCA1 Promoter Hypermethylation in Malignant Breast Tumors and in the Histologically Normal Adjacent Tissues to the Tumors: Exploring Its Potential as a Biomarker and Its Clinical Significance in a Translational Approach.

The hypermethylation status of the promoter region of the breast cancer 1 (BRCA1), a well-known tumor suppressor gene, has been extensively investigated in the last two decades as a potential biomarker for breast cancer. In this retrospective study, we investigated the prevalence of BRCA1 promoter methylation in 84 human breast tissues, and we correlated this epigenetic silencing with the clinical and histopathological parameters of breast cancer. We used methylation-specific PCR (MSP) to analyze BRCA1 promoter hypermethylation in 48 malignant breast tumors (MBTs), 15 normal adjacent tissues (NATs), and 21 benign breast lesions (BBLs). The results showed that BRCA1 promoter hypermethylation was higher in MBTs (20/48; 41.67%) and NATs (7/15; 46.67%) compared to BBLs (4/21; 19.05%). The high percentage of BRCA1 hypermethylation in the histologically normal adjacent tissues to the tumors (NATs) suggests the involvement of this epigenetic silencing as a potential biomarker of the early genomic instability in NATs surrounding the tumors. The detection of BRCA1 promoter hypermethylation in BBLs reinforces this suggestion, knowing that a non-negligible rate of benign breast lesions was reported to evolve into cancer. Moreover, our results indicated that the BRCA1 promoter hypermethylated group of MBTs exhibited higher rates of aggressive features, as indicated by the SBR III grade (14/19; 73.68%), elevated Ki67 levels (13/16; 81.25%), and Her2 receptor overexpression (5/20; 25%). Finally, we observed a concordance (60%) in BRCA1 promoter hypermethylation status between malignant breast tumors and their paired histologically normal adjacent tissues. This study highlights the role of BRCA1 promoter hypermethylation as a potential useful biomarker of aggressiveness in MBTs and as an early marker of genomic instability in both histological NATs and BBLs.

Enhanced Apoptosis and Loss of Cell Viability in Melanoma Cells by Combined Inhibition of ERK and Mcl-1 Is Related to Loss of Mitochondrial Membrane Potential, Caspase Activation and Upregulation of Proapoptotic Bcl-2 Proteins.

Targeting of MAP kinase pathways by BRAF inhibitors has evolved as a key therapy for BRAF-mutated melanoma. However, it cannot be applied for BRAF-WT melanoma, and also, in BRAF-mutated melanoma, tumor relapse often follows after an initial phase of tumor regression. Inhibition of MAP kinase pathways downstream at ERK1/2, or inhibitors of antiapoptotic Bcl-2 proteins, such as Mcl-1, may serve as alternative strategies. As shown here, the BRAF inhibitor vemurafenib and the ERK inhibitor SCH772984 showed only limited efficacy in melanoma cell lines, when applied alone. However, in combination with the Mcl-1 inhibitor S63845, the effects of vemurafenib were strongly enhanced in BRAF-mutated cell lines, and the effects of SCH772984 were enhanced in both BRAF-mutated and BRAF-WT cells. This resulted in up to 90% loss of cell viability and cell proliferation, as well as in induction of apoptosis in up to 60% of cells. The combination of SCH772984/S63845 resulted in caspase activation, processing of poly (ADP-ribose) polymerase (PARP), phosphorylation of histone H2AX, loss of mitochondrial membrane potential, and cytochrome c release. Proving the critical role of caspases, a pan-caspase inhibitor suppressed apoptosis induction, as well as loss of cell viability. As concerning Bcl-2 family proteins, SCH772984 enhanced expression of the proapoptotic Bim and Puma, as well as decreased phosphorylation of Bad. The combination finally resulted in downregulation of antiapoptotic Bcl-2 and enhanced expression of the proapoptotic Noxa. In conclusion, combined inhibition of ERK and Mcl-1 revealed an impressive efficacy both in BRAF-mutated and WT melanoma cells, and may thus represent a new strategy for overcoming drug resistance.

Epigenetically silenced apoptosis-associated tyrosine kinase (AATK) facilitates a decreased expression of Cyclin D1 and WEE1, phosphorylates TP53 and reduces cell proliferation in a kinase-dependent manner.

Silencing of the Apoptosis associated Tyrosine Kinase gene (AATK) has been described in cancer. In our study, we specifically investigated the epigenetic inactivation of AATK in pancreatic adenocarcinoma, lower grade glioma, lung, breast, head, and neck cancer. The resulting loss of AATK correlates with impaired patient survival. Inhibition of DNA methyltransferases (DNMTs) reactivated AATK in glioblastoma and pancreatic cancer. In contrast, epigenetic targeting via the CRISPR/dCas9 system with either EZH2 or DNMT3A inhibited the expression of AATK. Via large-scale kinomic profiling and kinase assays, we demonstrate that AATK acts a Ser/Thr kinase that phosphorylates TP53 at Ser366. Furthermore, whole transcriptome analyses and mass spectrometry associate AATK expression with the GO term 'regulation of cell proliferation'. The kinase activity of AATK in comparison to the kinase-dead mutant mediates a decreased expression of the key cell cycle regulators Cyclin D1 and WEE1. Moreover, growth suppression through AATK relies on its kinase activity. In conclusion, the Ser/Thr kinase AATK represses growth and phosphorylates TP53. Furthermore, expression of AATK was correlated with a better patient survival for different cancer entities. This data suggests that AATK acts as an epigenetically inactivated tumor suppressor gene.

Alectinib treatment improves photodynamic therapy in cancer cell lines of different origin.

BACKGROUND: Photodynamic therapy with a photosensitizer such as protoporphyrin-IX, a light sensitive metabolite of heme synthesis, is a highly selective treatment for various carcinomas. In previous studies, we found a significant down regulation of the relevant enzyme ferrochelatase in gastrointestinal carcinomas leading to an accumulation of protoporphyrin-IX within the tumor cells. Recent studies showed that a novel anti-cancer drug, Alectinib, an orally available, highly selective, potent second-generation inhibitor of anaplastic lymphoma tyrosinkinase binds to ferrochelatase. Therefore, we were interested to see whether Alectinib treatment might lead to an accumulation of protoporphyrin IX. METHODS: Tumor cells of different origin were cultured, treated with LED-light and Alectinib. Results were gained by flow cytometry, immunohistochemistry and western blotting. Apoptosis was determined by flow cytometric analysis of Annexin V-FITC stained cells. In addition, cells were counterstained with propidium iodide to distinguish early apoptotic cells and late apoptotic/necrotic cells. RESULTS: Here, we report that photodynamic treatment of tumor cell lines of different origin in combination with Alectinib increased protoporphyrin-IX specific fluorescence and concomitantly cell death. CONCLUSIONS: The usage of Alectinib could be another step for enhancing the effectiveness of photodynamic therapy. Further experiments will show whether photodynamic therapy in combination with Alectinib could be a new strategy for the treatment of e.g. peritoneal disseminated carcinomas.

Deregulated expression of circular RNAs in acute myeloid leukemia.

Circular RNAs (circRNAs) are dynamically regulated during differentiation and show cell type-specific expression, which is altered in cancer and can have a direct impact on its various hallmarks. We hypothesized that circRNA expression is deregulated in acute myeloid leukemia (AML) and that circRNA candidates might contribute to the pathogenesis of the disease. To identify leukemia-associated and differentiation-independent changes in circRNA expression, we determined the circular RNAome of 61 AML patients and 16 healthy hematopoietic stem and progenitor cell (HSPC) samples using ribosomal RNA-depleted RNA sequencing. We found hundreds of circRNAs that were differentially expressed between AML and healthy HSPCs. Gene set analysis found that many of these circRNAs were transcribed from genes implicated in leukemia biology. We discovered a circRNA derived from the T-cell transcription factor gene B cell CLL/lymphoma 11B, circBCL11B, which was exclusively expressed in AML patients, but not detected in healthy HSPCs, and associated with a T-cell-like gene expression signature. We were able to validate this finding in an independent cohort of 332 AML patients. Knockdown of circBCL11B had a negative effect on leukemic cell proliferation and resulted in increased cell death of leukemic cells, thereby suggesting circBCL11B as a novel functionally relevant candidate in AML pathogenesis. In summary, our study enables comprehensive insights into circRNA expression changes upon leukemic transformation and provides valuable information on the biology of leukemic cells and potential novel pathway dependencies that are relevant for AML therapy.

Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis.

Iroquois homeobox (IRX) encodes members of homeodomain containing genes which are involved in development and differentiation. Since it has been reported that the IRX1 gene is localized in a lung cancer susceptibility locus, the epigenetic regulation and function of IRX1 was investigated in lung carcinogenesis. We observed frequent hypermethylation of the IRX1 promoter in non-small cell lung cancer (NSCLC) compared to small cell lung cancer (SCLC). Aberrant IRX1 methylation was significantly correlated with reduced IRX1 expression. In normal lung samples, the IRX1 promoter showed lower median DNA methylation levels (<10%) compared to primary adenocarcinoma (ADC, 22%) and squamous cell carcinoma (SQCC, 14%). A significant hypermethylation and downregulation of IRX1 was detected in ADC and SQCC compared to matching normal lung samples (p < 0.0001). Low IRX1 expression was significantly correlated with impaired prognosis of ADC patients (p = 0.001). Reduced survival probability was also associated with higher IRX1 promoter methylation (p = 0.02). Inhibition of DNA methyltransferase (DNMT) activity reactivated IRX1 expression in human lung cancer cell lines. Induced DNMT3A and EZH2 expression was correlated with downregulation of IRX1. On the cellular level, IRX1 exhibits nuclear localization and expression of IRX1 induced fragmented nuclei in cancer cells. Localization of IRX1 and induction of aberrant nuclei were dependent on the presence of the homeobox of IRX1. By data mining, we showed that IRX1 is negatively correlated with oncogenic pathways and IRX1 expression induces the proapoptotic regulator BAX. In conclusion, we report that IRX1 expression is significantly associated with improved survival probability of ADC patients. IRX1 hypermethylation may serve as molecular biomarker for ADC diagnosis and prognosis. Our data suggest that IRX1 acts as an epigenetically regulated tumor suppressor in the pathogenesis of lung cancer.

The ZAR1 protein in cancer; from epigenetic silencing to functional characterisation and epigenetic therapy of tumour suppressors.

ZAR1, zygote arrest 1, is a zinc finger protein (C-terminus), which was initially identified in mouse oocytes. Later it was found that its expression is present in various human tissues e.g. lung and kidney. Interestingly, it was observed that in various tumour types the ZAR1 transcript is missing due to hypermethylation of its CpG island promoter, but not ZAR2. Since methylation of the ZAR1 promoter is described as a frequent event in tumourigenesis, ZAR1 could serve as a useful diagnostic marker in cancer screens. ZAR1 was described as a useful prognostic/diagnostic cancer marker for lung cancer, kidney cancer, melanoma and possibly liver carcinoma. Furthermore, ZAR1 was reactivated as a tumour suppressor by epigenetic therapy using CRISPR-dCas9 method. This method holds the potential to precisely target not only ZAR1 and reactivate tumour suppressors in a tailored cancer therapy. ZAR1 is highly conserved amongst vertebrates, especially its zinc finger, which is the relevant domain for its protein and RNA binding ability. ZAR1 is implicated in various cellular mechanisms including regulation of oocyte/embryo development, cell cycle control and mRNA binding, though little was known about the underlying mechanisms. ZAR1 was reported to regulate and activate translation through the binding to TCS translation control sequences in the 3'UTRs of its target mRNA the kinase WEE1. ZAR1 has a tumour suppressing function by inhibiting cell cycle progression. Here we review the current literature on ZAR1 focusing on structural, functional and epigenetic aspects. Characterising the cellular mechanisms that regulate the signalling pathways ZAR1 is involved in, could lead to a deeper understanding of tumour development and, furthermore, to new strategies in cancer treatment.

RASSF10 is frequently epigenetically inactivated in kidney cancer and its knockout promotes neoplasia in cancer prone mice.

Kidney cancer incidences are rising globally, thereby fueling the demand for targeted therapies and precision medicine. In our previous work, we have identified and characterized the Ras-Association Domain Family encoding ten members that are often aberrantly expressed in human cancers. In this study, we created and analyzed the Rassf10 knockout mice. Here we show that Rassf10 haploinsufficiency promotes neoplasia formation in two established mouse cancer models (Rassf1A-/- and p53-/-). Haploinsufficient Rassf10 knockout mice were significantly prone to various diseases including lymphoma (Rassf1A-/- background) and thymoma (p53-/- background). Especially Rassf10-/- and p53-deficient mice exhibited threefold increased rates of kidney cysts compared with p53-/- controls. Moreover, we observed that in human kidney cancer, RASSF10 is frequently epigenetically inactivated by its CpG island promoter hypermethylation. Primary tumors of renal clear cell and papillary cell carcinoma confirmed that RASSF10 methylation is associated with decreased expression in comparison to normal kidney tissue. In independent data sets, we could validate that RASSF10 inactivation clinically correlated with decreased survival and with progressed disease state of kidney cancer patients and polycystic kidney size. Functionally, we revealed that the loss of Rassf10 was significantly associated with upregulation of KRAS signaling and MYC expression. In summary, we could show that Rassf10 functions as a haploinsufficient tumor suppressor. In combination with other markers, RASSF10 silencing can serve as diagnostic and prognostic cancer biomarker in kidney diseases.

RASSF10 Is a TGFß-Target That Regulates ASPP2 and E-Cadherin Expression and Acts as Tumor Suppressor That Is Epigenetically Downregulated in Advanced Cancer.

The Ras Association Domain Family (RASSF) encodes members of tumor suppressor genes which are frequently inactivated in human cancers. Here, the function and the regulation of RASSF10, that contains a RA (Ras-association) and two coiled domains, was investigated. We utilized mass spectrometry and immuno-precipitation to identify interaction partners of RASSF10. Additionally, we analyzed the up- and downstream pathways of RASSF10 that are involved in its tumor suppressive function. We report that RASSF10 binds ASPP1 (Apoptosis-stimulating protein of p53) and ASPP2 through its coiled-coils. Induction of RASSF10 leads to increased protein levels of ASPP2 and acts negatively on cell cycle progression. Interestingly, we found that RASSF10 is a target of the EMT (epithelial mesenchymal transition) driver TGFß (Transforming growth factor beta) and that negatively associated genes of RASSF10 are significantly over-represented in an EMT gene set collection. We observed a positive correlation of RASSF10 expression and E-cadherin that prevents EMT. Depletion of RASSF10 by CRISPR/Cas9 technology induces the ability of lung cancer cells to proliferate and to invade an extracellular matrix after TGFß treatment. Additionally, knockdown of RASSF10 or ASPP2 induced constitutive phosphorylation of SMAD2 (Smad family member 2). Moreover, we found that epigenetic reduction of RASSF10 levels correlates with tumor progression and poor survival in human cancers. Our study indicates that RASSF10 acts a TGFß target gene and negatively regulates cell growth and invasion through ASPP2. This data suggests that epigenetic loss of RASSF10 contributes to tumorigenesis by promoting EMT induced by TGFß.

Epigenetic therapy of novel tumour suppressor ZAR1 and its cancer biomarker function.

BACKGROUND: Cancer still is one of the leading causes of death and its death toll is predicted to rise further. We identified earlier the potential tumour suppressor zygote arrest 1 (ZAR1) to play a role in lung carcinogenesis through its epigenetic inactivation. RESULTS: We are the first to report that ZAR1 is epigenetically inactivated not only in lung cancer but also across cancer types, and ZAR1 methylation occurs across its complete CpG island. ZAR1 hypermethylation significantly correlates with its expression reduction in cancers. We are also the first to report that ZAR1 methylation and expression reduction are of clinical importance as a prognostic marker for lung cancer and kidney cancer. We further established that the carboxy (C)-terminally present zinc-finger of ZAR1 is relevant for its tumour suppression function and its protein partner binding associated with the mRNA/ribosomal network. Global gene expression profiling supported ZAR1's role in cell cycle arrest and p53 signalling pathway, and we could show that ZAR1 growth suppression was in part p53 dependent. Using the CRISPR-dCas9 tools, we were able to prove that epigenetic editing and reactivation of ZAR1 is possible in cancer cell lines. CONCLUSION: ZAR1 is a novel cancer biomarker for lung and kidney, which is epigenetically silenced in various cancers by DNA hypermethylation. ZAR1 exerts its tumour suppressive function in part through p53 and through its zinc-finger domain. Epigenetic therapy can reactivate the ZAR1 tumour suppressor in cancer.

Pan-class I PI3-kinase inhibitor BKM120 induces MEK1/2-dependent mitotic catastrophe in non-Hodgkin lymphoma leading to apoptosis or polyploidy determined by Bax/Bak and p53.

Constitutive signaling of PI3K/Akt/mTOR plays a prominent role in malignant transformation and progression of B-cell non-Hodgkin lymphomas (B-NHL) underscoring the need for PI3K targeted therapies. The pan-class I PI3-kinase inhibitor BKM120 has shown preclinical activity in distinct malignancies and is currently tested in clinical trials. Intratumor heterogeneity is an intrinsic property of cancers that contributes to drug resistance and tumor recurrence. Here, we demonstrate that inhibition of PI3-kinases by BKM120 attenuates growth and survival of B-NHL cell lines by inducing mitotic arrest with subsequent induction of intrinsic apoptosis. BKM120-mediated downregulation of Cyclin A and activation of the CDK1/Cyclin B1 complex facilitates mitotic entry. In addition, concomitant BKM120-mediated upregulation of Cyclin B1 expression attenuates completion of mitosis, which results in mitotic catastrophe and apoptotic cell death. In Bax and Bak deficient B-NHL, which are resistant to BKM120-induced apoptosis, BKM120-induced mitotic catastrophe results in polyploidy. Upon re-expression of wt p53 in these p53 mutated cells, BKM120-induced polyploidy is strongly reduced demonstrating that the genetic status of the cells determines the outcome of a BKM120-mediated pathway inhibition. Mitotic catastrophe and unfavorable induction of polyploidy can be prevented in this setting by additional inhibition of MEK1/2 signaling. Combining MEK1/2 inhibitors with BKM120 enhances the anti-tumor effects of BKM120, prevents prognostic unfavorable polyploidy and might be a potential strategy for the treatment of B-NHL.

On-surface synthesis on a bulk insulator surface.

On-surface synthesis has rapidly emerged as a most promising approach to prepare functional molecular structures directly on a support surface. Compared to solution synthesis, performing chemical reactions on a surface offers several exciting new options: due to the absence of a solvent, reactions can be envisioned that are otherwise not feasible due to the insolubility of the reaction product. Perhaps even more important, the confinement to a two-dimensional surface might enable reaction pathways that are not accessible otherwise. Consequently, on-surface synthesis has attracted great attention in the last decade, with an impressive number of classical reactions transferred to a surface as well as new reactions demonstrated that have no classical analogue. So far, the majority of the work has been carried out on conducting surfaces. However, when aiming for electronic decoupling of the resulting structures, e.g. for the use in future molecular electronic devices, non-conducting surfaces are highly desired. Here, we review the current status of on-surface reactions demonstrated on the (10.4) surface of the bulk insulator calcite. Besides thermally induced C-C coupling of halogen-substituted aryls, photochemically induced [2 + 2] cycloaddition has been proven possible on this surface. Moreover, experimental evidence exists for coupling of terminal alkynes as well as diacetylene polymerization. While imaging of the resulting structures with dynamic atomic force microscopy provides a direct means of reaction verification, the detailed reaction pathway often remains unclear. Especially in cases where the presence of metal atoms is known to catalyze the corresponding solution chemistry reaction (e.g. in the case of the Ullmann reaction), disclosing the precise reaction pathway is of importance to understand and generalize on-surface reactivity on a bulk insulator surface. To this end, density-functional theory calculations have proven to provide atomic-scale insights that have greatly contributed to unravelling the details of on-surface synthesis on a bulk insulator surface.

Reversible and Efficient Light-Induced Molecular Switching on an Insulator Surface.

Prototypical molecular switches such as azobenzenes exhibit two states, i.e., trans and cis, with different characteristic physical properties. In recent years various derivatives were investigated on metallic surfaces. However, bulk insulators as supporting substrate reveal important advantages since they allow electronic decoupling from the environment, which is key to control the switching properties. Here, we report on the light-induced isomerization of an azobenzene derivative on a bulk insulator surface, in this case calcite (101̅4), studied by atomic force microscopy with submolecular resolution. Surprisingly, cis isomers appear on the surface already directly after preparation, indicating kinetic trapping. The photoisomerization process is reversible, as the use of different light sources results in specific molecular assemblies of each isomer. The process turns out to be very efficient and even comparable to molecules in solution, which we assign to the rather weak molecular interaction with the insulator surface, in contrast to metals.

The tumor suppressor RASSF1A induces the YAP1 target gene ANKRD1 that is epigenetically inactivated in human cancers and inhibits tumor growth.

The Hippo pathway regulates organ size, growth and comprises several tumor related factors, including the oncoprotein YAP1 and the tumor suppressor RASSF1A. RASSF1A is frequently epigenetically inactivated in cancer. In our study, we analyzed the effect of RASSF1A on the function of YAP1. Expression of YAP1 resulted in the downregulation of several tumor suppressor genes and induction of S-phase. Co-expression with RASSF1A normalized the expression levels of these tumor suppressors and induced a G0-G1 arrest and apoptosis. This effect was associated with the reduction of MDM2 and the increase of p53. These data suggest that the tumor suppressor RASSF1A inhibits the oncogenic potential of YAP1. Additionally, we could show that ANKRD1 is a YAP1 target gene that is induced by RASSF1A. Further analysis revealed that ANKRD1 is epigenetically inactivated in human cancer. ANKRD1 expression induced the expression of TP53 as well as BAX and CDKN1A and reduced colony formation of cancer cells. We found that ANKRD1 interacts with p53 and is involved in the destabilization of MDM2. Additionally, our data indicate that the tumor-suppressive effect of ANKRD1 depends on the presence of p53. These results suggest that ANKRD1 is a tumor-suppressive downstream target of the Hippo pathway that is epigenetically silenced in human cancer.

Impact of Natural Compounds on DNA Methylation Levels of the Tumor Suppressor Gene RASSF1A in Cancer.

Epigenetic inactivation of tumor suppressor genes (TSG) is a fundamental event in the pathogenesis of human cancer. This silencing is accomplished by aberrant chromatin modifications including DNA hypermethylation of the gene promoter. One of the most frequently hypermethylated TSG in human cancer is the Ras Association Domain Family 1A (RASSF1A) gene. Aberrant methylation of RASSF1A has been reported in melanoma, sarcoma and carcinoma of different tissues. RASSF1A hypermethylation has been correlated with tumor progression and poor prognosis. Reactivation of epigenetically silenced TSG has been suggested as a therapy in cancer treatment. In particular, natural compounds isolated from herbal extracts have been tested for their capacity to induce RASSF1A in cancer cells, through demethylation. Here, we review the treatment of cancer cells with natural supplements (e.g., methyl donors, vitamins and polyphenols) that have been utilized to revert or prevent the epigenetic silencing of RASSF1A. Moreover, we specify pathways that were involved in RASSF1A reactivation. Several of these compounds (e.g., reseveratol and curcumin) act by inhibiting the activity or expression of DNA methyltransferases and reactive RASSF1A in cancer. Thus natural compounds could serve as important agents in tumor prevention or cancer therapy. However, the exact epigenetic reactivation mechanism is still under investigation.

ZAR1 is a novel epigenetically inactivated tumour suppressor in lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths with 1.8 million new cases each year and poor 5-year prognosis. Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby can promote cancer development and progression. RESULTS: In this study, we analysed ZAR1 (zygote arrest 1), which has been said to be a maternal-effect gene and its expression mostly limited to certain reproductive tissues. Our study shows that ZAR1 is expressed in normal lung but inactivated by promoter methylation in lung cancer. ZAR1 is hypermethylated in primary lung cancer samples (22% small cell lung carcinoma (SCLC) and 76% non-small cell lung carcinoma (NSCLC), p < 0.001) vs. normal control lung tissue (11%). In lung cancer cell lines, ZAR1 was significantly methylated in 75% of SCLC and 83% of NSCLC vs. normal tissue (p < 0.005/0.05). In matching tumours and control tissues, we observed that NSCLC primary tumour samples exhibited a tumour-specific promoter methylation of ZAR1 in comparison to the normal control lung tissue. Demethylation treatment of various lung cancer cell lines reversed ZAR1 promoter hypermethylation and subsequently re-established ZAR1 expression. In addition, we could show the growth inhibitory potential of ZAR1 in lung cancer cell lines and cancer cell lines. Exogenous expression of ZAR1 not only inhibited colony formation but also blocked cell cycle progression of cancer cell lines. CONCLUSIONS: Our study shows for the first time the lung tumour-specific epigenetic inactivation of ZAR1 due to DNA methylation of its CpG island promoter. Furthermore, ZAR1 was characterised by the ability to block tumour growth through the inhibition of cell cycle progression in cancer cell lines. We propose that ZAR1 could serve as an epigenetically inactivated biomarker in lung cancer.

Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma.

Renal cell carcinoma (RCC) is polyresistant to chemo- and radiotherapy and biologicals, including TNF-related apoptosis-inducing ligand (TRAIL). Sorafenib, a multikinase inhibitor approved for the treatment of RCC, has been shown to sensitize cancer cells to TRAIL-induced apoptosis, in particular by down-regulation of the Bak-inhibitory Bcl-2 family protein Mcl-1. Here we demonstrate that sorafenib overcomes TRAIL resistance in RCC by a mechanism that does not rely on Mcl-1 down-regulation. Instead, sorafenib induces rapid dissipation of the mitochondrial membrane potential (ΔΨm) that is accompanied by the accumulation of reactive oxygen species (ROS). Loss of ΔΨm and ROS production induced by sorafenib are independent of caspase activities and do not depend on the presence of the proapoptotic Bcl-2 family proteins Bax or Bak, indicating that both events are functionally upstream of the mitochondrial apoptosis signaling cascade. More intriguingly, we find that it is sorafenib-induced ROS accumulation that enables TRAIL to activate caspase-8 in RCC. This leads to apoptosis that involves activation of an amplification loop via the mitochondrial apoptosis pathway. Thus, our mechanistic data indicate that sorafenib bypasses central resistance mechanisms through a direct induction of ΔΨm breakdown and ROS production. Activation of this pathway might represent a useful strategy to overcome the cell-inherent resistance to cancer therapeutics, including TRAIL, in multiresistant cancers such as RCC.