Title

 

Authors

E. Paolicchi¹, F. Crea¹, L. Fornaro², G. Masi², P. Frumento³, F. Loupakis², L. Salvatore², C. Cremolini², M. Schirripa², F. Graziano⁴, M. Ronzoni⁵, V. Ricci⁵, W.L. Farrar⁶, A. Falcone² and R. Danesi¹.

1. Dept. of Internal Medicine, Division of Pharmacology, University of Pisa, Italy
2. Dept. of Oncology, Transplants and New Technologies in medicine, Division of Medical Oncology, University of Pisa, Italy
3. Sant’Anna School of Advanced Study, Pisa, Italy
4. Dept. of  Onco-Hematology, Medical Oncology Unit, Azienda Ospedaliera Ospedale Pesaro, Pesaro, Italy
5. Medical Oncology, Scientific Institute S. Raffaele, Milan, Italy
6. Laboratory of Cancer Prevention, Cancer Stem Cell Section, National Institute of Cancer-Frederick, Frederick, MD, USA

 

Abstract

Metastatic colorectal cancer (mCRC) is one of the leading causes of cancer death in Western countries (Jemal A et al 2010). For the majority of mCRC patients, doublet chemotherapy with a fluoropyrimidine plus either irinotecan or oxaliplatin combined with a biologic agent is nowadays considered the preferred treatment option in first-line (Fornaro et al 2010). In particular bevacizumab, a monoclonal humanized antibody directed against the vascular endothelial growth factor (VEGF), has been shown to extend overall survival in patients treated with 5-fluorouracil-based chemotherapy (Hurwitz H et al 2004), and is approved in combination with fluoropyrimidine-based chemotherapy for the first- or second-line treatment of mCRC patients.Cancer stem cells (CSCs) were recently shown to drive colorectal cancer (CRC) progression and chemo-resistance(Ricci-Vitiani et al 2007). Polycomb group genes (PcGs) are epigenetic modifiers involved in CSCs self-renewal through oncosuppressor gene silencing (Mathews LA et al 2009). The polycomb group protein enhancer of zeste homolog 2 (EZH2) has been reported as a Drosophila protein homolog that maintains homeotic gene repression by regulating chromatin (Chen H et al 1996). EZH2 is a PcG member that mediates gene silencing through histone-H3 lysine-27 methylation (Mathew LA et al 2009).  Expression of EZH2 mRNA is increased in colorectal cancer. High concentrations of EZH2 mRNA show a worse clinical outcome (Wang CG et al 2010). Recently, 4 EZH2 single nucleotide polymorphisms (SNPs) have been characterizedthree of them are associated to lung cancer risk (rs: 3757441, 41277434, 6950683) and one (rs2302427) is responsible for a coding sequence change.The aim of this study was to evaluate the correlation between EZH2 SNPs and outcome in mCRC patients and their prognostic and predictive role in mCRC patients. DNA was extracted from blood samples of 110 mCRC patients treated with first-line FOLFIRI plus bevacizumab. Genotyping was performed by Real-Time PCR.  Oncomine meta-analysis on microarray data showed that a set of EZH2 target genes are specifically silenced in FOLFIRI-non responder CRC patients. One allelic variant (rs3757441 C/C vs C/T or T/T) was significantly associated to shorter Progression free survival (PFS) (8.7 vs 11 months, respectively; HR=0.2689 [95%CI: 0.1024-0.7058]; p=0.0076) and Overall survival (OS) (23.8 vs 18.3 months; HR=0.329, 95% CI: 0.109-0.997; p=0.049). Variants of the other three tested SNPs were not significantly associated with outcome parameters. At multivariate analysis, the same variant resulted an independent predictor of both PFS and OS (p<0.05)Through analysis by dedicated softwares (GeneCard,  Promo 3.0) we investigated the functional relevance of the rs3757441 SNP, finding that it can affect binding to XBP transcription inhibitor. In particular, the T variant creates a binding site for XBP, thereby reducing EZH2 expression. Among 50 patients analysed for EZH2 expression and genotyped for EZH2 rs3757441 SNP, mRNA levels were significantly higher in patients harbouring the C/C genotype with respect to C/T and T/T (p<0.05), with no difference between C/T and T/T genotypes. To confirm these results, we studied the rs3757441 SNPin a parallel set of 104 mCRC patients, treated with FOLFIRI regimen. The C/C genotype was still associated to shorter PFS and OS (p<0.05, log-rank test). Our results indicate that the EZH2 rs3757441 SNP is associated to higher EZH2 expression and may be useful to predict PFS and OS in mCRC patients treated with FOLFIRI regimen.
 
Jemal A et al, CA Cancer J Clin. 2010 Jul 7.
Fornaro L et al, Expert Opin Pharmacother. 2010 Jan;11(1):63-77.
Hurwitz H et al, N Engl J Med. 2004;23:2335–42.
Ricci-Vitiani L, et al Nature. 2007 Jan 4;445(7123):111-5.
Mathews LA et al,Differentiation.2009 Jul;78(1):1-17.
 Chen H et al,Genomics1996;38:30–7.
Wang CG et al, World J Gastroenterol. 2010; 16(19):2421-7