Sonja Sievers

Frequent promoter hypermethylation of Wnt pathway inhibitor genes in malignant astrocytic gliomas

Aberrant activation of wingless (Wnt) signaling is involved in the pathogenesis of various cancers. Recent studies suggested a role of Wnt signaling in gliomas, the most common primary brain tumors. We investigated 70 gliomas of different malignancy grades for promoter hypermethylation in 8 genes encoding members of the secreted frizzled‐related protein (SFRP1, SFRP2, SFRP4, SFRP5), dickkopf (DKK1, DKK3) and naked (NKD1, NKD2) families of Wnt pathway inhibitors. All tumors were additionally analyzed for mutations in exon 3 of the β‐catenin gene (CTNNB1). While none of the tumors carried CTNNB1 mutations, we found frequent promoter hypermethylation of Wnt pathway inhibitor genes, with at least one of these genes being hypermethylated in 6 of 16 diffuse astrocytomas (38%), 4 of 14 anaplastic astrocytomas (29%), 7 of 10 secondary glioblastomas (70%) and 23 of 30 primary glioblastomas (77%). Glioblastomas often demonstrated hypermethylation of 2 or more analyzed genes. Hypermethylation of SFRP1, SFRP2 and NKD2 each occurred in more than 40% of the primary glioblastomas, while DKK1 hypermethylation was found in 50% of secondary glioblastomas. Treatment of SFRP1‐, SFRP5‐, DKK1‐, DKK3‐, NKD1‐ and NKD2‐hypermethylated U87‐MG glioblastoma cells with 5‐aza‐2′‐deoxycytidine and trichostatin A resulted in increased expression of each gene. Furthermore, SFRP1‐hypermethylated gliomas showed significantly lower expression of the respective transcripts when compared with unmethylated tumors. Taken together, our results suggest an important role of epigenetic silencing of Wnt pathway inhibitor genes in astrocytic gliomas, in particular, in glioblastomas, with distinct patterns of hypermethylated genes distinguishing primary from secondary glioblastomas.

IGF2/H19 imprinting analysis of human germ cell tumors (GCTs) using the methylation-sensitive single-nucleotide primer extension method reflects the origin of GCTs in different stages of primordial germ cell development

Previous studies have demonstrated biallelic expression of the imprinted genes H19 and IGF2 and loss of DNA methylation of the SNRPN gene, indicating a common precursor cell of human germ cell tumors (GCTs), namely, the primordial germ cell (PGC). In this study, we applied the methylation‐sensitive single‐nucleotide primer extension (MS‐SNuPE) technique to the analysis of the IGF2/H19 imprinting control region (ICR) in 55 GCTs from representative clinical and histologic subgroups. Most GCTs showed low methylation at the IGF2/H19 ICR. All 8 ovarian GCTs, 9 of 10 testicular seminomas, 7 of 10 testicular nonseminomas (all in adolescents/adults), 6 of 9 testicular yolk sac tumors (YSTs), and 12 of 14 nongonadal GCTs (all in infants/children) were hypomethylated. The highest methylation was observed in three childhood YSTs (boys) and 2 of 4 spermatocytic seminomas. The latter are derived from more advanced stages of germ‐cell development. The predominantly low methylation of most of the other GCTs correlates with studies that demonstrated erasure of the methylation imprint of the IGF2/H19 ICR during embryonal PGC migration and development. These findings suggest that the IGF2/H19 methylation status in GCTs might reflect preservation of the physiologic imprinting erasure in PGCs rather than a loss of imprinting in a sense that is accepted for somatic tumors. Furthermore, this study indicates that imprinting control mechanisms other than the proposed CTCF (CCCTC binding factor) boundary model regulate IGF2 expression during this stage of PGC development as well as in GCTs derived from PGC.

Molecular genetic analysis of central nervous system germ cell tumors with comparative genomic hybridization

The limited information available to date regarding the genetic alterations in germ cell tumors of the central nervous system has raised concerns about their biologic relationship to other germ cell tumor entities. We investigated fresh-frozen or archival tumor samples from 19 patients with central nervous system germ cell tumors (CNS-GCTs), including seven germinomas, eight malignant nongerminomatous germ cell tumors and four teratomas, using chromosomal comparative genomic hybridization to determine recurrent chromosomal imbalances. All 15 malignant CNS-GCTs and two of four teratomas showed multiple chromosomal imbalances. Chromosomal gains (median: 4 gains/tumor, range: 0–9 gains/tumor) were observed more frequently than losses (median: 1.6 losses/tumor, range: 0–6 losses/tumor). Gain of 12p, which is considered characteristic for germ cell tumors of the adult testis, was detected in 11 of 19 tumors and 10 of 15 malignant CNS-GCTs. In one tumor, gain of 12p was confined to an amplicon at 12p12, corresponding to the commonly amplified region on 12p. Other common gains were found on chromosome arms 1q and 8q (n=9, each). Among the chromosomal losses, parts of chromosome 11 (n=5), 18 (n=4), and 13 (n=3) were deleted most frequently. Notably, we observed no difference in the genetic profiles of germinomatous and nongerminomatous CNS-GCTs; however, the average number of imbalances was higher in the latter group. A meta-analysis comparing 116 malignant gonadal and extragonadal germ cell tumors revealed that the genomic alterations in CNS-GCTs are virtually indistinguishable from those found in their gonadal or other extragonadal counterparts of the corresponding age group. These data strongly argue in favor of common pathogenetic mechanisms in gonadal and extragonadal germ cell tumors.

Highly Enantioselective Catalytic Synthesis of Neurite Growth-Promoting Secoyohimbanes

Natural products endowed with neuromodulatory activity and their underlying structural scaffolds may inspire the synthesis of novel neurotrophic compound classes. The spirocyclic secoyohimbane alkaloid rhynchophylline is the major component of the extracts of Uncaria species used in Chinese traditional medicine for treatment of disorders of the central nervous system. Based on the structure of rhynchophylline, a highly enantioselective and efficient organocatalyzed synthesis method was developed that gives access to the tetracyclic secoyohimbane scaffold, embodying a quaternary and three tertiary stereogenic centers in a one-pot multistep reaction sequence. Investigation of a collection of the secoyohimbanes in primary rat hippocampal neurons and embryonal stem cell-derived motor neurons led to discovery of compounds that promote neurite outgrowth and influence the complexity of neuronal network formation.

Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization

Caseinolytic protease P (ClpP) represents a central bacterial degradation machinery that is involved in cell homeostasis and pathogenicity. The functional role of ClpP has been studied by genetic knockouts and through the use of beta-lactones, which remain the only specific inhibitors of ClpP discovered to date. Beta-lactones have served as chemical tools to manipulate ClpP in several organisms; however, their potency, selectivity and stability is limited. Despite detailed structural insights into the composition and conformational flexibility of the ClpP active site, no rational efforts to design specific non-beta-lactone inhibitors have been reported to date. In this work, an unbiased screen of more than 137 000 compounds was used to identify five phenyl ester compounds as highly potent ClpP inhibitors that were selective for bacterial, but not human ClpP. The potency of phenyl esters largely exceeded that of beta-lactones in ClpP peptidase and protease inhibition assays and displayed unique target selectivity in living S. aureus cells. Analytical studies revealed that while phenyl esters are cleaved like native peptide substrates, they remain covalently trapped as acyl-enzyme intermediates in the active site. The synthesis of 36 derivatives and subsequent structure-activity relationship (SAR) studies provided insights into conserved structural elements that are important for inhibition potency and acylation reactivity. Moreover, the stereochemistry of a methyl-substituent at the alpha position to the ester, resembling amino acid side chains in peptide substrates, impacted ClpP complex stability, causing either dissociation into heptamers or retention of the tetradecameric state. Mechanistic insights into this intriguing stereo switch and the phenyl ester binding mode were obtained by molecular docking experiments.

Imbalances of chromosome arm 1p in pediatric and adult germ cell tumors are caused by true allelic loss: A combined comparative genomic hybridization and microsatellite analysis

Previous studies on childhood germ cell tumors (GCTs) report highly variable frequencies of losses at chromosome arm 1p. Since deletions at 1p portend a poor prognosis in other embryonal tumors, this study aims to clarify the question of the frequency of true allelic loss at 1p and whether it constitutes a prognostic parameter. We analyzed 13 GCTs from different gonadal and extragonadal sites of children (4 teratomas, 9 malignant GCTs) and 18 GCTs of adolescents and adults (3 teratomas; 15 malignant GCTs) using automated microsatellite analysis with 23 polymorphic markers and chromosomal “high resolution” comparative genomic hybridization (HR‐CGH). With this combined approach, we detected loss of heterozygosity (LOH) at 1p in 8/9 childhood malignant GCTs with concordant data from HR‐CGH and microsatellite analyses. In contrast, LOH at 1p was not detected in childhood teratomas (0/4) and constituted a rare event in GCTs of adolescence and adulthood (3/18). The commonly deleted region was located at distal 1p36‐pter, with a proximal boundary between the markers D1S450 and D1S2870. These data unequivocally demonstrate that deletion at 1p is common in childhood GCTs and results in allelic loss. This observation argues for the presence of a classical tumor suppressor at distal 1p. Considering the high frequency of LOH at 1p and the overall favorable prognosis of childhood GCTs, a prognostic impact of LOH at 1p in childhood GCTs appears unlikely. However, since two postpubertal tumors with LOH at 1p progressed, a prognostic relevance in this age group seems possible, warranting a prospective evaluation.

Oxidative regioselective amination of chromones exposes potent inhibitors of the hedgehog signaling pathway

A transition metal-free, oxidative, regioselective cross-coupling between non-functionalized azoles and chromones at C2-position was developed. A broad reaction scope and further transformation of products were demonstrated. The biological evaluation of products revealed a novel class of hedgehog signaling pathway inhibitors.

Analysis of the adenomatous polyposis coli (APC) gene in childhood and adolescent germ cell tumors

Aberrant Wnt signaling due to deregulation of Wnt regulators is implicated in the development and progression of numerous embryonal tumors. This study addresses the questions if activation of Wnt signaling in germ cell tumors (GCTs) arising during childhood and adolescence is associated with aberrations of the tumor suppressor adenomatous polyposis coli (APC), and whether APC aberrations might be responsible for progression from benign teratoma to malignant yolk sac tumor (YST).

Hypermethylation of the APC promoter but lack of APC mutations in myxoid/round-cell liposarcoma.

The adenomatous polyposis coli (APC) protein is a key component of the WNT signalling pathway wherein it acts as a scaffolding protein in controlling the level of the proto‐oncoprotein β‐catenin. Although APC has been shown to be genetically or epigenetically inactivated in a variety of carcinomas, little is known about its role in sarcoma. Liposarcomas (LPSs) are the second most common soft tissue sarcoma in adults. Despite different histology and malignancy, the myxoid and round‐cell LPSs belong to one tumour entity characterized by a specific chromosomal translocation. We assessed the extent of genetic and epigenetic inactivation of the APC gene in myxoid/round‐cell LPS. Sequencing of the mutation cluster region, the protein truncation test and a loss of heterozygosity (LOH) analysis did not reveal any genetic alterations of the APC gene in all of the liposarcoma samples. Methylation of the APC promoter was detected by methylation‐specific PCR in 9 of 20 (45%) tumours. Analysis of APC expression by semiquantitative RT‐PCR in a subset of the samples demonstrated that tumours with a methylated APC promoter showed a downregulation of the APC transcript. However, APC downregulation was not correlated with a stabilisation of the β‐catenin protein. Thus, the epigenetic regulation of the APC gene might play an important role in the pathogenesis of myxoid/round‐cell LPS. However, the impact of APC methylation on liposarcoma development is quite likely not mediated through WNT signalling.

Absolute beta-catenin concentrations in Wnt pathway-stimulated and non-stimulated cells

Abstract The intracellular level of the proto-oncoprotein β-catenin is a parameter for the activity of the Wnt pathway, which has been linked to carcinogenesis. The paper introduces a novel sandwich-based ELISA for the determination of the β-catenin concentration in lysates from cells or tissues. The advantages of the method were proven by determining β-catenin levels in cell lines and in cells after activation of the Wnt pathway. Analysis revealed high β-catenin concentrations in the cell lines HeLa, KB, HT1080, MCF-7, U-87 and U-373, which had not been described before. β-Catenin concentrations were compared in HEK293 and C57MG cells after activation of the Wnt pathway. The β-catenin concentrations increased by different factors depending on whether the Wnt pathway was activated by incubation with LiCl or with Wnt-3a-conditioned medium. This finding indicated that the β-catenin level depends on the way and level of Wnt pathway activation. The quantitative analysis of β-catenin in colorectal tumours revealed high β-catenin levels in tumours with truncating mutations in the APC gene.