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Molekulare Mechanismen sekundärer Lebertumore

Die Rolle der Expression leberspezifischer Transkriptionsfaktoren für die Tumorbiologie, metabolische Kompetenz und Differenzierung hepatischer Metastasen leberfremder Primärtumoren

 

In der Behandlung von Lebermetastasen werden gegenwärtig multimodale Therapiekonzepte evaluiert, da von einer Tumorresektion nur wenige Patienten dauerhaft profitieren. Gerade deshalb wäre es wünschenswert mit Hilfe von modernen genetischen Testmethoden Informationen zur Diagnose, Prognose und zum erwarteten Krankheitsverlauf frühzeitig zu erhalten, um eine optimale Behandlungsstrategie entwickeln zu können. Für die Zelldifferenzierung sind die leberspezifischen Transkriptionsfaktoren von großer Bedeutung. Bis heute wurden ca. 2500 unterschiedliche Transkriptionsfaktoren im menschlichen Genom identifiziert. An den wesentlichen Stoffwechselprozessen der Leber sind bislang fünf verschiedene Familien von Transkriptionsfaktoren – C/EBP, HNF-1, HNF-3, HNF4 und HNF6 - beteiligt. Wünschenswert wäre das frühzeitige Erkennen von Mustern charakteristischer Transkriptionsfaktoren, die bei der Entstehung von Lebermetastasen von Bedeutung sind und unweigerlich mit Stoffwechselveränderungen in der Leber einschließlich Zelldedifferenzierung einhergehen. Die quantitative Bestimmung der Genexpression ermöglicht eine Einschätzung des hepatozellulären Differenzierungsgrades und nachgeschaltet der metabolischen Kompetenz.

Ziel unserer Untersuchungen ist deshalb das Erkennen spezifischer Muster der Expression der HNFs sowie C/EBPs in der Lebermetastase im Vergleich zu gesunden Lebergewebe und Primärtumor. Die hieraus gewonnenen Ergebnisse ermöglichen ein besseres Verständnis der Tumorbiologie metastasierender epithelialer Tumore woraus sich langfristige neue Konzepte in der Therapiestrategie ableiten lassen.

 

Projektleiter: Dr. F. Lehner in Kooperation mit Professor Dr. rer. nat. Jürgen Borlak, Lehrstuhl für Pharmako- und Toxikogenomik der Medizinschen Hochschule Hannover und Institut für Toxikologie und Experimentelle Medizin – Fraunhofer Institut Hannover

 

 

 

Aktuelle Publikationen aus dem Projekt:

 

 
Lehner F, Kulik U, Klempnauer J, Borlak J
Inhibition of the Liver Enriched Protein FOXA2 Recovers HNF6 Activity in Human Colon Carcinoma and Liver Hepatoma Cells.
PLoS ONE 2010  5(10): e13344. doi:10.1371/journal.pone.0013344

Recently, we demonstrated that the transcription factors HNF6 and FOXA2 function as key regulators in human colorectal liver metastases. To better understand their proposed inhibitory crosstalk, the consequences of functional knockdown of FOXA2 on HNF6 and C/EBPα activity were investigated in the human colon Caco-2 and HepG2 carcinoma cell lines. Specifically, siRNA-mediated gene silencing of FOXA2 repressed transcript expression by >80%. This resulted in a statistically significant 6-, 3-, 4-, and 8-fold increase in mRNA expression of HNF6 and of genes targeted by this transcription factor, e.g., HSP105B, CYP51, and C/EBPα, as determined by qRT-PCR. Thus, functional knockdown of FOXA2 recovered HNF6 activity. Furthermore, with nuclear extracts of Caco-2 cells no HNF6 DNA binding was observed, but expression of HNF1α, FOXA2, FOXA3, and HNF4α protein was abundant. We therefore transfected a plasmid encoding HNF6 into Caco-2 cells but also employed a retroviral vector to transfect HNF6 into HepG2 cells. This resulted in HNF6 protein expression with DNA binding activity being recovered as determined by EMSA band shift assays. Furthermore, by flow cytometry the consequences of HNF6 expression on cell cycle regulation in transfected cells was studied. Essentially, HNF6 inhibited cell cycle progression in the G2/M and G1 phase in Caco-2 and HepG2 cell lines, respectively. Here, proliferation was reduced by 80% and 50% in Caco-2 and HepG2 cells, respectively, as determined by the BrdU labeling assay. Therefore functional knockdown of FOXA2 recovered HNF6 activity and inhibited growth of tumor-cells and may possibly represent a novel therapeutic target in primary and secondary liver malignancies.

 

Lehner F, Kulik U, Klempnauer J, Borlak J
Mapping of liver-enriched transcription factors in the human intestine
World J Gastroenterol. 2010 August 21; 16(31): 3919–3927.

AIM: To investigate the gene expression pattern of hepatocyte nuclear factor 6 (HNF6) and other liver-enriched transcription factors in various segments of the human intestine to better understand the differentiation of the gut epithelium.

METHODS: Samples of healthy duodenum and jejunum were obtained from patients with pancreatic cancer whereas ileum and colon was obtained from patients undergoing right or left hemicolectomy or (recto)sigmoid or rectal resection. All surgical specimens were subjected to histopathology. Excised tissue was shock-frozen and analyzed for gene expression of liver-enriched transcription factors by semiquantitative reverse transcription polymerase chain and compared to the human colon carcinoma cell line Caco-2. Protein expression of major liver-enriched transcription factors was determined by Western blotting while the DNA binding of HNF6 was investigated by electromobility shift assays.

RESULTS: The gene expression patterning of liver-enriched transcription factors differed in the various segments of the human intestine with HNF6 gene expression being most abundant in the duodenum (P < 0.05) whereas expression of the zinc finger protein GATA4 and of the HNF6 target gene ALDH3A1 was most abundant in the jejunum (P < 0.05). Likewise, expression of FOXA2 and the splice variants 2 and 4 of HNF4α were most abundantly expressed in the jejunum (P < 0.05). Essentially, expression of transcription factors declined from the duodenum towards the colon with the most abundant expression in the jejunum and less in the ileum. The expression of HNF6 and of genes targeted by this factor, i.e. neurogenin 3 (NGN3) was most abundant in the jejunum followed by the ileum and the colon while DNA binding activity of HNF4α and of NGN3 was confirmed by electromobility shift assays to an optimized probe. Furthermore, Western blotting provided evidence of the expression of several liver-enriched transcription factors in cultures of colon epithelial cells, albeit at different levels.

CONCLUSION: We describe significant local and segmental differences in the expression of liver-enriched transcription factors in the human intestine which impact epithelial cell biology of the gut.

 

 

Lehner F, Kulik U, Klempnauer J, Borlak J.

The hepatocyte nuclear factor 6 (HNF6) and FOXA2 are key regulators in colorectal liver metastases.

FASEB J. 2007 Feb 5; [Epub ahead of print]

PMID: 17283222 [PubMed - as supplied by publisher]

 

The molecular causes leading to secondary liver malignancies are unknown. Here we report regulation of major hepatic nuclear factors in human colorectal liver metastases and primary colonic cancer. Notably, the genes coding for HNF6, HNF1beta, and C/EBPgamma were selectively regulated in liver metastases. We therefore studied protein expression of regulated transcription factors and found unacetylated HNF6 to be a hallmark of colorectal liver metastases. For its known interaction with HNF6, we investigated expression of FOXA2, which we found to be specifically induced in colorectal liver metastases. By electromobility shift assay, we examined DNA binding of disease regulated transcription factors. Essentially, no HNF6 DNA binding was observed. We also searched for sequence variations in the DNA binding domains of HNF6, but did not identify any mutation. Furthermore, we probed for expression of 28 genes targeted by HNF6. Mostly transcript expression was repressed except for tumor growth. In conclusion, we show HNF6 protein expression to be driven by the hepatic environment. Its expression is not observed in healthy colon or primary colonic cancer. HNF6 DNA binding is selectively abrogated through lack of post-translational modification and interaction with FOXA2. Targeting of FOXA2 and HNF6 may therefore enable mechanism-based therapy for colorectal liver metastases.-- Lehner, F., Kulik, U., Klempnauer, J., Borlak, J. The hepatocyte nuclear factor 6 (HNF6) and FOXA2 are key regulators in colorectal liver metastases.