Melanie Eichenmüller

Blocking the hedgehog pathway inhibits hepatoblastoma growth

Recent evidence has indicated that Hedgehog (Hh) signaling significantly contributes to liver development and regeneration and that activation of the pathway may contribute to growth of hepatocellular carcinoma (HCC) in adults. However, the role of Hh signaling in pediatric liver tumors remains to be elucidated. In this study, we show that Hh signaling is activated in hepatoblastoma (HB), the most common liver tumor in childhood, with most occurrences before the age of 3 years. The Hh target genes glioma‐associated oncogene homolog 1 (GLI1) and Patched (PTCH1) showed increased transcript levels in 65% and 30% of HB samples, respectively, compared with normal liver tissues. Most interestingly, the gene encoding the hedgehog interacting protein (HHIP) is transcriptionally silenced by cytosine‐phospho‐guanosine (CpG) island promoter hypermethylation in 26% of HB cases and treatment with the DNA‐demethylating agent 5‐aza‐2′‐deoxycytidine partially restored HHIP expression. Blocking Hh signaling with the antagonist cyclopamine had a strong inhibitory effect on cell proliferation of HB cell lines with an activated pathway. We further demonstrate that this decrease in cell viability is caused by a massive induction of apoptosis, as shown by morphological changes and phosphatidylserine membrane asymmetry. In cyclopamine‐exposed HB cells, caspase 3 and poly(adenosine diphosphate‐ribose) polymerase proteins were specifically activated by their proteolytic cleavage. Conclusion: This study demonstrates, for the first time, the frequent occurrence of GLI1 and PTCH1 overexpression and HHIP promoter methylation in early childhood HB, thus indicating a key role for Hh signaling activation in the malignant transformation of embryonal liver cells. (HEPATOLOGY 2009;49:482–490.)

The genomic landscape of hepatoblastoma and their progenies with HCC-like features

BACKGROUND & AIMS Hepatoblastoma (HB) is the most common childhood liver cancer and occasionally presents with histological and clinical features reminiscent of hepatocellular carcinoma (HCC). Identification of molecular mechanisms that drive the neoplastic continuation towards more aggressive HCC phenotypes may help to guide the new stage of targeted therapies. METHODS We performed comprehensive studies on genetic and chromosomal alterations as well as candidate gene function and their clinical relevance. RESULTS Whole-exome sequencing identified HB as a genetically very simple tumour (2.9 mutations per tumour) with recurrent mutations in ß-catenin (CTNNB1) (12/15 cases) and the transcription factor NFE2L2 (2/15 cases). Their HCC-like progenies share the common CTNNB1 mutation, but additionally exhibit a significantly increased mutation number and chromosomal instability due to deletions of the genome guardians RAD17 and TP53, accompanied by telomerase reverse-transcriptase (TERT) promoter mutations. Targeted genotyping of 33 primary tumours and cell lines revealed CTNNB1, NFE2L2, and TERT mutations in 72.5%, 9.8%, and 5.9% of cases, respectively. All NFE2L2 mutations affected residues of the NFE2L2 protein that are recognized by the KEAP1/CUL3 complex for proteasomal degradation. Consequently, cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signalling. Clinically, overexpression of the NFE2L2 target gene NQO1 in tumours was significantly associated with metastasis, vascular invasion, the adverse prognostic C2 gene signature, as well as poor outcome. CONCLUSIONS Our study demonstrates the importance of CTNNB1 mutations and NFE2L2-KEAP1 pathway activation in HB development and defines loss of genomic stability and TERT promoter mutations as prominent characteristics of aggressive HB with HCC features.

Betulinic acid induces apoptosis and inhibits hedgehog signalling in rhabdomyosarcoma

Background:Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood with the ability to resist apoptosis by the activation of survival promoting and anti-apoptotic proteins.Methods:Efficacy of the apoptosis-inducing agent betulinic acid (BA) was determined in RMS cell cultures and in vivo by measuring cell viability, survival, apoptosis, hedgehog signalling activity, and neovascularisation.Results:Betulinic acid had a strong cytotoxic effect on RMS cells in a dose-dependent manner. The BA treatment caused a massive induction of apoptosis mediated by the intrinsic mitochondrial pathway, which could be inhibited by the broad-range caspase inhibitor zVAD.fmk. Exposure of hedgehog-activated RMS-13 cells to BA resulted in a strong decrease in GLI1, GLI2, PTCH1, and IGF2 expression as well as hedgehog-responsive luciferase activity. Intraperitoneal injection of 20 mg BA per kg per day significantly retarded growth of RMS-13 xenografts in association with markedly higher counts of apoptotic cells and down-regulation of GLI1 expression compared with control tumours, while leaving microvascular density, cell proliferation, and myogenic differentiation unaffected.Conclusion:Our data show that induction of apoptosis and inhibition of hedgehog signalling are important features of the anti-tumourigenic effect of BA in RMS and advices this compound for the use in a multimodal therapy of this highly aggressive paediatric tumour.

Rapamycin blocks hepatoblastoma growth in vitro and in vivo implicating new treatment options in high-risk patients

Activation of the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway plays a central role in the formation of hepatoblastoma (HB), the most common liver cancer in childhood. Blocking this pathway with specific mTOR inhibitors such as the immunosuppressant rapamycin is being currently tested for a variety of cancers. Here, we report that rapamycin treatment induced a significant dose-dependent inhibition of cell viability and promoted apoptosis in HB cells in vitro. Moreover, rapamycin inhibited AKT/mTOR signalling by dephosphorylation of the downstream target p70S6 kinase (p70S6K). Most importantly, treating subcutaneous HUH6 xenograft tumour bearing mice orally with 5mg/kg/day rapamycin for three weeks resulted in a striking reduction of tumour growth, as evidenced by reduced volume and weight, and moderately lowered tumour-specific alpha-fetoprotein (AFP) serum levels. The anti-tumourigenic effect was primarily ascribed to a significantly reduced proliferation rate upon p70S6K dephosphorylation, as microvascular density of rapamycin-treated compared to vehicle-treated tumours stayed grossly unchanged. Of uttermost clinical importance, we found no evidence for a feedback-loop activation of AKT in vivo. In conclusion, we demonstrate that rapamycin effectively inhibits HB growth both in vitro and in vivo by blocking AKT/mTOR signalling at the level of p70S6K and that rapamycin should be considered to treat HB patients especially those to be indicated for liver transplantation to benefit from its anti-tumourigenic and immunosuppressive properties.

Abstract 3086: The genetic landscape of the childhood liver cancer hepatoblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Hepatoblastoma (HB) is the most common malignant liver tumor in children with a histology that resembles various stages of the developing liver. To explore the genetic origin of this cancer, we performed whole-exome sequencing of 8 tumors and matched germline tissue. In average, we detected only three sequence variants per tumor, which is the lowest mutation rate ever reported for any cancer. Mutations of s-catenin (CTNNB1) displayed the only recurrent event occurring in all tumors, thereby underscoring the significance of activated Wnt signaling in the genesis of HB. Gene ontology analysis revealed a predominance of candidate genes involved in the regulation of gene expression and transcription, including the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2). Targeted resequencing of a cohort of 50 HB cases and cell lines revealed NFE2L2 mutations in 10% of all tumors. Interestingly, all mutations were located in or adjacent to the DLG and ETGE motifs of the NFE2L2 protein that are known to be recognized from the KEAP1/CUL3 complex for proteasomal degradation. In line with this, cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signaling. Clinically, overexpression of the NFE2L2 target gene NQO1 in tumor tissues was significantly associated with high tumor stage, metastasis, and poor outcome. These data suggest that activation of NFE2L2-KEAP1 signaling through mutations in NFE2L2 is a prominent characteristic in pediatric liver tumors, thereby advocating inhibition of aberrant NFE2L2 activity as a therapeutic option, especially in high-risk patients with advanced disease. Citation Format: Roland Kappler, Melanie Eichenmuller, Franziska Trippel, Tim M. Strom, Dietrich von Schweinitz. The genetic landscape of the childhood liver cancer hepatoblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3086. doi:10.1158/1538-7445.AM2014-3086