Increased expression of High Mobility Group Box 1 (HMGB1) is associated with an elevated level of the anti- apoptotic c-IAP2 protein in human colon carcinomas

Kirsten Völp ¹, Marie-Luise Brezniceanu ², Susanne Bösser ³, Thomas Brabletz ⁴, Thomas Kirchner ⁴, Daniel Göttel ⁵, Stefan Joos ⁵ and Martin Zörnig ^3*

¹ Abbott GmbH&Co. KG, Germany
² Centre hospitalier de l`Universite de Montreal-Hotel-Dieu, Canada
³ Georg-Speyer-Haus, Germany
⁴ Dept. of Pathology, Univ. of Erlangen, Germany
⁵ Deutsches Krebsforschungszentrum DKFZ, Germany

^* To whom correspondence should be addressed. E-mail: zoernig{at}em.uni-frankfurt.de.

Accepted 2 August 2005

Abstract

Background: High-mobility group box 1 (HMGB1) is a non-histone chromosomal protein implicated in a variety of biologically important processes including transcription, DNA repair, V(D)J recombination, differentiation, and development. Overexpression of HMGB1 inhibits apoptosis, arguing that the molecule may act as an anti-apoptotic oncoprotein. Indeed, an increased expression of HMGB1 has been reported for several different tumor types. In this study, we analyze human colon carcinoma for HMGB1 as well as for c-IAP2 expression levels. c-IAP2 is an anti-apoptotic protein which may be upregulated as a consequence of NF-B activation via HMGB1.

Methods: A Comparative Genomic Hybridization (CGH) database comprising 1,645 cases from different human tumor types was screened to detect cytogenetic changes at the HMGB1 locus. Immunohistochemical staining of human colon tissue microarrays and tumor biopsies, as well as Western Blot analysis of tumor lysates were performed to detect elevated HMGB1 and c-IAP2 expression in colon carcinomas. The anti-apoptotic potential of HMGB1 was analyzed by measuring caspase activities, and luciferase reporter assays and quantitative PCR analysis were employed to confirm NF-B activation and c-IAP2 mRNA upregulation upon HMGB1 overexpression.

Results: According to CGH analysis, the genomic locus containing the HMGB1 gene is overrepresented in one third (35/96) of colon cancers. Correspondingly, HMGB1 protein levels were significantly elevated in 90% of the 60 colon carcinomas tested, as compared to the corresponding normal tissues evaluable from the same patients. HMGB1 increases NF-B activity and leads to co-overexpression of the anti-apoptotic NF-B target gene product c-IAP2 in vitro. Furthermore, increased HMGB1 levels correlated with enhanced amounts of c-IAP2 in colon tumors analyzed by us. Finally, we could demonstrate that HMGB1 overexpression suppresses Caspase-9 (Casp-9) and Casp-3 activity, suggesting that HMGB1 interferes with the apoptotic machinery at the level of apoptosomal Casp-9 activation.

Conclusions: We identified in vitro a molecular pathway triggered by HMGB1 to inhibit apoptosis via c- IAP2 induction. Our data indicate a strong correlation between upregulation of the apoptosis-repressing HMGB1 and c-IAP2 proteins in the pathogenesis of colon carcinoma.

Keywords: HMGB1, NF-kB, apoptosis inhibitors, c-IAP2, colorectal cancer

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