Annemarie Koch

Changes in histone deacetylase (HDAC) expression patterns and activity of HDAC inhibitors in urothelial cancers

OBJECTIVE To determine histone deacetylase (HDAC) isoenzyme expression patterns in urothelial cancer tissues and cell lines and investigate their potential to predict the efficacy of the HDAC inhibitor vorinostat. MATERIALS AND METHODS Expression of HDAC mRNAs was determined by quantitative RT-PCR in 18 urothelial cancer cell lines (UCC), normal uroepithelial controls (NUC), 24 urothelial cancer tissues, and 12 benign controls. Results were compared with published microarray data. Effects of pan-HDAC inhibitor vorinostat and on UCCs were determined by viability and apoptosis assays, cell cycle analysis, and measurements of p21(CIP1), thymidylate synthase (TS), and EZH2. In addition, protein expression levels of HDACs were investigated in UCCs. RESULTS Prominent changes in UCCs were HDAC2 and/or HDAC8 up-regulation in 11 of 18 cell lines and decreased expression of HDAC4, HDAC5, and/or HDAC7 mRNA in 15 of 18 cell lines. In cancer tissues, HDAC8 was likewise significantly up-regulated (P = 0.002), whereas HDAC2 up-regulation was detected only in a subset of tumors (9/24, P = 0.085). Overexpression of HDAC2 and HDAC8 mRNA did not correspond with the protein level. Vorinostat induced G2/M arrest, an increase in the sub-G1 fraction, up-regulation of p21, and down-regulation of TS in all UCC. Effects on EZH2 and PARP cleavage as well as activation of caspase 3/7 differed between cell lines. Associations between the overall sensitivity to the pan-HDACi vorinostat and overexpression of HDAC2 and HDAC8 mRNA were not observed. CONCLUSIONS In urothelial cancer, up-regulation of HDAC2 and HDAC8 and down-regulation of HDAC4, HDAC5, and HDAC7 mRNA are common findings. The treatment effect of the pan-HDAC inhibitor vorinostat was variable in UCCs and up-regulation of HDAC2 and HDAC8 was not predictive for treatment response. Whether selective targeting of HDAC2, HDAC8, or other HDACs deregulated in urothelial cancer (e.g., HDAC4, HDAC5, and HDAC7) result in a more consistent treatment response needs further investigation.

HERV-K and LINE-1 DNA Methylation and Reexpression in Urothelial Carcinoma

Changes in DNA methylation frequently accompany cancer development. One prominent change is an apparently genome-wide decrease in methylcytosine that is often ascribed to DNA hypomethylation at retroelements comprising nearly half the genome. DNA hypomethylation may allow reactivation of retroelements, enabling retrotransposition, and causing gene expression disturbances favoring tumor development. However, neither the extent of hypomethylation nor of retroelement reactivation are precisely known. We therefore assessed DNA methylation and expression of three major classes of retroelements (LINE-1, HERV-K, and AluY) in human urinary bladder cancer tissues and cell lines by pyrosequencing and quantitative reverse transcription–polymerase chain reaction, respectively. We found substantial global LINE-1 DNA hypomethylation in bladder cancer going along with a shift toward full-length LINE-1 expression. Thus, pronounced differences in LINE-1 expression were observed, which may be promoted, among others, by LINE-1 hypomethylation. Significant DNA hypomethylation was found at the HERV-K_22q11.23 proviral long terminal repeat (LTR) in bladder cancer tissues but without reactivation of its expression. DNA methylation of HERVK17, essentially absent from normal urothelial cells, was elevated in cell lines from invasive bladder cancers. Accordingly, the faint expression of HERVK17 in normal urothelial cells disappeared in such cancer cell lines. Of 16 additional HERV-Ks, expression of 7 could be detected in the bladder, albeit generally at low levels. Unlike in prostate cancers, none of these showed significant expression changes in bladder cancer. In contrast, expression of the AluYb8 but not of the AluYa5 family was significantly increased in bladder cancer tissues. Collectively, our findings demonstrate a remarkable specificity of changes in expression and DNA methylation of retroelements in bladder cancer with a significantly different pattern from that in prostate cancer.

Histone deacetylase 8 is deregulated in urothelial cancer but not a target for efficient treatment

BackgroundPrevious studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.MethodsWe conducted siRNA-mediated knockdown and specific pharmacological inhibition of HDAC8 with the three different inhibitors compound 2, compound 5, and compound 6 in several urothelial carcinoma cell lines with distinct HDAC8 expression profiles. Levels of HDAC and marker proteins were determined by western blot analysis and mRNA levels were measured by quantitative real-time PCR. Cellular effects of HDAC8 suppression were analyzed by ATP assay, flow cytometry, colony forming assay and migration assay.ResultsEfficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%. The HDAC8 specific inhibitors compound 5 and compound 6 significantly reduced viability of all urothelial cancer cell lines (IC50 9 – 21 μM). Flow cytometry revealed only a slight increase in the sub-G1 fraction indicating a limited induction of apoptosis. Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected. The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.ConclusionsDeregulation of HDAC8 is frequent in urothelial cancer, but neither specific pharmacological inhibition nor siRNA-mediated knockdown of HDAC8 impaired viability of urothelial cancer cell lines in a therapeutic useful manner. Accordingly, HDAC8 on its own is not a promising drug target in bladder cancer.

Specific changes in the expression of imprinted genes in prostate cancer--implications for cancer progression and epigenetic regulation.

Epigenetic dysregulation comprising DNA hypermethylation and hypomethylation, enhancer of zeste homologue 2 (EZH2) overexpression and altered patterns of histone modifications is associated with the progression of prostate cancer. DNA methylation, EZH2 and histone modifications also ensure the parental-specific monoallelic expression of at least 62 imprinted genes. Although it is therefore tempting to speculate that epigenetic dysregulation may extend to imprinted genes, expression changes in cancerous prostates are only well documented for insulin-like growth factor 2 (IGF2). A literature and database survey on imprinted genes in prostate cancer suggests that the expression of most imprinted genes remains unchanged despite global disturbances in epigenetic mechanisms. Instead, selective genetic and epigenetic changes appear to lead to the inactivation of a sub-network of imprinted genes, which might function in the prostate to limit cell growth induced via the PI3K/Akt pathway, modulate androgen responses and regulate differentiation. Whereas dysregulation of IGF2 may constitute an early change in prostate carcinogenesis, inactivation of this imprinted gene network is rather associated with cancer progression.

Discovery of TP53 splice variants in two novel papillary urothelial cancer cell lines

BackgroundUsing a novel cell culture technique, we established two new cell lines, BC44 and BC61, from papillary urothelial carcinoma and analyzed them for genetic changes typical of this tumor type.Methods and resultsKaryotyping revealed aneuploid karyotypes with loss of chromosome 9 and rearranged chromosome 5p. Molecular analysis showed CDKN2A deletions but wild-type PIK3CA. BC61 contained a G372C FGFR3 mutation. TP53 was not mutated in either cell line and BC61 expressed normal full-length protein. In contrast, BC44 exclusively expressed cytoplasmic and nuclear p53Δ40 and 133 isoforms from the alternative promoter P2 as revealed by Western blotting, immunocytochemistry and PCR. The only discernible difference in TP53 in BC44 was homozygosity for the deletion allele of the rs17878362 polymorphism in the P2 promoter. Expression of p53 isoforms was also detected in a few other urothelial carcinoma cell lines and tumor cultures and in 4 out of 28 carcinoma tissues.ConclusionIn urothelial cancers, TP53 is typically inactivated by mutations in one allele and loss of the wildtype allele and more frequently in invasive compared to papillary carcinomas. We show that some urothelial carcinomas may predominantly or exclusively express isoforms which are not detected by commonly used antibodies to epitopes located in the p53 TA amino-terminal region. Expression of these isoforms may constitute a further mode of p53 inactivation in urothelial carcinoma. Our findings raise the question to which extent this mechanism may compromise wildtype p53 function in papillary tumors in particular, where point mutations in the gene are rare.

[Signal transduction in urothelial cancer: how exactly do we know the targets for targeted therapy?].

ZusammenfassungMit zielgerichteter Tumortherapie konnte in den letzten Jahren die Lebensqualität vieler Krebspatienten verbessert und sogar ihr Überleben verlängert werden. Möglich wurde dies durch das wachsende Verständnis tumorspezifischer Signalwege. Auch beim Urothelkarzinom wurden spezifische Veränderungen von Tumorsignalwegen identifiziert. Dazu gehören Mutationen von FGFR3, HRAS und PIK3CA, die insbesondere in papillären Tumoren zu einer Überaktivierung des MAP-Kinase- und des Akt-Signalweges führen. Im Vergleich dazu sind Veränderungen des RB1- und des p53-Regulationssystems, welche einen unmittelbaren Einfluss auf die Zellzykluskontrolle haben, häufiger in invasiven Karzinomen anzutreffen. Dass eine zielgerichtete Tumortherapie beim Urothelkarzinom sich dennoch bisher als wenig erfolgreich erwiesen hat, mag wesentlich durch die noch unvollständige Erforschung von Signaltransduktionswegen bei dieser Tumorart bedingt sein. Zielgene tumorspezifischer Signalwege werden durch epigenetische Mechanismen kontrolliert und ihre Induzierbarkeit determiniert. Daher ist die Entschlüsselung dieser Kontrollmechanismen wichtig für die Entwicklung zielgerichteter Therapien beim Urothelkarzinom.AbstractTargeted therapies have helped to improve the quality of life and prolong the survival of many cancer patients. This progress is based on the growing understanding of cellular signal transduction pathways and regulatory systems in human cancers. In urothelial carcinoma, a number of specific alterations have been identified. These include mutations in FGFR3, HRAS, and PIK3CA leading to overactivity of MAPK and Akt signaling pathways especially in papillary tumors. In comparison, the RB1 and p53 regulatory systems that act more directly on cell cycle control are more commonly compromised in invasive carcinomas. Nevertheless, targeted therapies have shown little efficacy in the treatment of urothelial carcinoma so far, owing presumably to our incomplete knowledge of molecular changes affecting signal transduction pathways in this cancer type. Target genes of cancer pathways are regulated by epigenetic mechanisms, which determine their inducibility. Elucidating these control mechanisms could therefore prove important for developing targeted therapies for urothelial carcinoma.Targeted therapies have helped to improve the quality of life and prolong the survival of many cancer patients. This progress is based on the growing understanding of cellular signal transduction pathways and regulatory systems in human cancers. In urothelial carcinoma, a number of specific alterations have been identified. These include mutations in FGFR3, HRAS, and PIK3CA leading to overactivity of MAPK and Akt signaling pathways especially in papillary tumors. In comparison, the RB1 and p53 regulatory systems that act more directly on cell cycle control are more commonly compromised in invasive carcinomas. Nevertheless, targeted therapies have shown little efficacy in the treatment of urothelial carcinoma so far, owing presumably to our incomplete knowledge of molecular changes affecting signal transduction pathways in this cancer type. Target genes of cancer pathways are regulated by epigenetic mechanisms, which determine their inducibility. Elucidating these control mechanisms could therefore prove important for developing targeted therapies for urothelial carcinoma.

Transforming growth factor β1-mediated functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia

Mesenchymal stromal cells are involved in the pathogenesis of myelodysplastic syndromes and acute myeloid leukemia, but the underlying mechanisms are incompletely understood. To further characterize the pathological phenotype we performed RNA sequencing of mesenchymal stromal cells from patients with myelodysplastic syndromes and acute myeloid leukemia and found a specific molecular signature of genes commonly deregulated in these disorders. Pathway analysis showed a strong enrichment of genes related to osteogenesis, senescence, inflammation and inhibitory cytokines, thereby reflecting the structural and functional deficits of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia on a molecular level. Further analysis identified transforming growth factor β1 as the most probable extrinsic trigger factor for this altered gene expression. Following exposure to transforming growth factor β1, healthy mesenchymal stromal cells developed functional deficits and adopted a phenotype reminiscent of that observed in patient-derived stromal cells. These suppressive effects of transforming growth factor β1 on stromal cell functionality were abrogated by SD-208, an established inhibitor of transforming growth factor β receptor signaling. Blockade of transforming growth factor β signaling by SD-208 also restored the osteogenic differentiation capacity of patient-derived stromal cells, thus confirming the role of transforming growth factor β1 in the bone marrow microenvironment of patients with myelodysplastic syndromes and acute myeloid leukemia. Our findings establish transforming growth factor β1 as a relevant trigger causing functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia and identify SD-208 as a candidate to revert these effects.

High-precision FRET analysis of the G-protein coupled receptor TGR5 in live cells

Background TGR5 is a widely expressed and highly conserved G protein coupled receptor. Its activity and functionality is commonly modulated by bile acids, especially by lithocholic acid. As true for all ligand activated G protein coupled receptors a G protein subunit is released from TGR5 after ligand binding and initiates a signaling cascade resulting in a cell type specific response. Current investigations suggest an involvement of TGR5 in bile homeostasis, inflammatory responses and hepatobiliary diseases. Therefore a targeted therapy involving site specific inhibition of TGR5 is of immense interest. However, up to date no solved structure of TGR5 exists and oligomerization properties are largely unknown. To determine structural changes and oligomerization properties of TGR5 we designed a three way strategy including TGR5 plasmids coupled with (I) fluorescent proteins (FPs) at the C-terminus or (II) a N-terminal peptide tag (ACP) for subsequent labeling with a fluorescent dye and (III) unnatural amino acids (UAAs) for site specific extracellular labeling.

Signaltransduktion im Urothelkarzinom@@@Signal transduction in urothelial cancer: Wie genau kennen wir die Ziele für eine zielgerichtete Therapie?@@@How exactly do we know the targets for targeted therapy?

ZusammenfassungMit zielgerichteter Tumortherapie konnte in den letzten Jahren die Lebensqualität vieler Krebspatienten verbessert und sogar ihr Überleben verlängert werden. Möglich wurde dies durch das wachsende Verständnis tumorspezifischer Signalwege. Auch beim Urothelkarzinom wurden spezifische Veränderungen von Tumorsignalwegen identifiziert. Dazu gehören Mutationen von FGFR3, HRAS und PIK3CA, die insbesondere in papillären Tumoren zu einer Überaktivierung des MAP-Kinase- und des Akt-Signalweges führen. Im Vergleich dazu sind Veränderungen des RB1- und des p53-Regulationssystems, welche einen unmittelbaren Einfluss auf die Zellzykluskontrolle haben, häufiger in invasiven Karzinomen anzutreffen. Dass eine zielgerichtete Tumortherapie beim Urothelkarzinom sich dennoch bisher als wenig erfolgreich erwiesen hat, mag wesentlich durch die noch unvollständige Erforschung von Signaltransduktionswegen bei dieser Tumorart bedingt sein. Zielgene tumorspezifischer Signalwege werden durch epigenetische Mechanismen kontrolliert und ihre Induzierbarkeit determiniert. Daher ist die Entschlüsselung dieser Kontrollmechanismen wichtig für die Entwicklung zielgerichteter Therapien beim Urothelkarzinom.AbstractTargeted therapies have helped to improve the quality of life and prolong the survival of many cancer patients. This progress is based on the growing understanding of cellular signal transduction pathways and regulatory systems in human cancers. In urothelial carcinoma, a number of specific alterations have been identified. These include mutations in FGFR3, HRAS, and PIK3CA leading to overactivity of MAPK and Akt signaling pathways especially in papillary tumors. In comparison, the RB1 and p53 regulatory systems that act more directly on cell cycle control are more commonly compromised in invasive carcinomas. Nevertheless, targeted therapies have shown little efficacy in the treatment of urothelial carcinoma so far, owing presumably to our incomplete knowledge of molecular changes affecting signal transduction pathways in this cancer type. Target genes of cancer pathways are regulated by epigenetic mechanisms, which determine their inducibility. Elucidating these control mechanisms could therefore prove important for developing targeted therapies for urothelial carcinoma.Targeted therapies have helped to improve the quality of life and prolong the survival of many cancer patients. This progress is based on the growing understanding of cellular signal transduction pathways and regulatory systems in human cancers. In urothelial carcinoma, a number of specific alterations have been identified. These include mutations in FGFR3, HRAS, and PIK3CA leading to overactivity of MAPK and Akt signaling pathways especially in papillary tumors. In comparison, the RB1 and p53 regulatory systems that act more directly on cell cycle control are more commonly compromised in invasive carcinomas. Nevertheless, targeted therapies have shown little efficacy in the treatment of urothelial carcinoma so far, owing presumably to our incomplete knowledge of molecular changes affecting signal transduction pathways in this cancer type. Target genes of cancer pathways are regulated by epigenetic mechanisms, which determine their inducibility. Elucidating these control mechanisms could therefore prove important for developing targeted therapies for urothelial carcinoma.

Signaltransduktion im Urothelkarzinom

ZusammenfassungMit zielgerichteter Tumortherapie konnte in den letzten Jahren die Lebensqualität vieler Krebspatienten verbessert und sogar ihr Überleben verlängert werden. Möglich wurde dies durch das wachsende Verständnis tumorspezifischer Signalwege. Auch beim Urothelkarzinom wurden spezifische Veränderungen von Tumorsignalwegen identifiziert. Dazu gehören Mutationen von FGFR3, HRAS und PIK3CA, die insbesondere in papillären Tumoren zu einer Überaktivierung des MAP-Kinase- und des Akt-Signalweges führen. Im Vergleich dazu sind Veränderungen des RB1- und des p53-Regulationssystems, welche einen unmittelbaren Einfluss auf die Zellzykluskontrolle haben, häufiger in invasiven Karzinomen anzutreffen. Dass eine zielgerichtete Tumortherapie beim Urothelkarzinom sich dennoch bisher als wenig erfolgreich erwiesen hat, mag wesentlich durch die noch unvollständige Erforschung von Signaltransduktionswegen bei dieser Tumorart bedingt sein. Zielgene tumorspezifischer Signalwege werden durch epigenetische Mechanismen kontrolliert und ihre Induzierbarkeit determiniert. Daher ist die Entschlüsselung dieser Kontrollmechanismen wichtig für die Entwicklung zielgerichteter Therapien beim Urothelkarzinom.AbstractTargeted therapies have helped to improve the quality of life and prolong the survival of many cancer patients. This progress is based on the growing understanding of cellular signal transduction pathways and regulatory systems in human cancers. In urothelial carcinoma, a number of specific alterations have been identified. These include mutations in FGFR3, HRAS, and PIK3CA leading to overactivity of MAPK and Akt signaling pathways especially in papillary tumors. In comparison, the RB1 and p53 regulatory systems that act more directly on cell cycle control are more commonly compromised in invasive carcinomas. Nevertheless, targeted therapies have shown little efficacy in the treatment of urothelial carcinoma so far, owing presumably to our incomplete knowledge of molecular changes affecting signal transduction pathways in this cancer type. Target genes of cancer pathways are regulated by epigenetic mechanisms, which determine their inducibility. Elucidating these control mechanisms could therefore prove important for developing targeted therapies for urothelial carcinoma.Targeted therapies have helped to improve the quality of life and prolong the survival of many cancer patients. This progress is based on the growing understanding of cellular signal transduction pathways and regulatory systems in human cancers. In urothelial carcinoma, a number of specific alterations have been identified. These include mutations in FGFR3, HRAS, and PIK3CA leading to overactivity of MAPK and Akt signaling pathways especially in papillary tumors. In comparison, the RB1 and p53 regulatory systems that act more directly on cell cycle control are more commonly compromised in invasive carcinomas. Nevertheless, targeted therapies have shown little efficacy in the treatment of urothelial carcinoma so far, owing presumably to our incomplete knowledge of molecular changes affecting signal transduction pathways in this cancer type. Target genes of cancer pathways are regulated by epigenetic mechanisms, which determine their inducibility. Elucidating these control mechanisms could therefore prove important for developing targeted therapies for urothelial carcinoma.