Oncogene (2010) 29, 876–887 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE A ‘DNA replication’ signature of progression and negative outcome in colorectal cancer M-J Pillaire1,7, J Selves2,7, K Gordien2, P-A Gouraud3, C Gentil3, M Danjoux2,CDo3, V Negre4, A Bieth1, R Guimbaud2, D Trouche5, P Pasero6,MMe´chali6, J-S Hoffmann1 and C Cazaux1 1Genetic Instability and Cancer Group, Department Biology of Cancer, Institute of Pharmacology and Structural Biology, UMR5089 CNRS, University of Toulouse, University Paul Sabatier, Toulouse, France; 2INSERM U563, Federation of Digestive Cancerology and Department of Anatomo-pathology, University of Toulouse, University Paul Sabatier, Toulouse, France; 3Service of Epidemiology, INSERM U558, Faculty of Medicine, University of Toulouse, University Paul Sabatier, Alle´es Jules Guesde, Toulouse, France; 4aCGH GSO Canceropole Platform, INSERM U868, Val d’Aurelle, Montpellier, France; 5Laboratory of Cellular and Molecular Biology of Cell Proliferation Control, UMR 5099 CNRS, University of Toulouse, University Paul Sabatier, Toulouse, France and 6Institute of Human Genetics UPR1142 CNRS, Montpellier, France Colorectal cancer is one of the most frequent cancers Introduction worldwide. As the tumor-node-metastasis (TNM) staging classification does not allow to predict the survival of DNA replication in normal cells is regulated by an patients in many cases, additional prognostic factors are ‘origin licensing’ mechanism that ensures that it occurs needed to better forecast their outcome. Genes involved in just once per cycle. Once cells enter the S-phase, the DNA replication may represent an underexplored source stability of DNA replication forks must be preserved to of such prognostic markers. Indeed, accidents during avoid susceptibility to DNA lesions or non-B DNA DNA replication can trigger ‘replicative stress’, one of the conformation. The requirement of faithful genome main features of cancer from earlier stages onward. In this duplication in dividing cells makes DNA replication study, we assessed the expression of 47 ‘DNA replication’ an important factor in cancer by limiting cancer risk genes in primary tumors and adjacent normal tissues from through preservation of genome integrity (Kunkel, 2003; a homogeneous series of 74 patients. We found that genes Hanawalt, 2007). Owing to this cardinal importance of coding for translesional (TLS) DNA polymerases, initia- DNA replication in cancer, many anticancer drugs tion of DNA replication, S-phase signaling and protection target various aspects of DNA replication. of replication forks were significantly deregulated in The key role of DNA replication in tumor prolifera- tumors. We also observed that the overexpression of tion is illustrated by many observations. Indeed, here- either the MCM7 helicase or the TLS DNA polymerase ditary forms of colon, breast, ovary and skin cancers can POLQ (if also associated with a concomitant over- be caused by mutations in DNA replication genes, such expression of firing genes) was significantly related to as translesion synthesis (for example, POLH), intra- poor patient survival. Our data suggest the existence of a S-phase signaling (for example, BRCA1/2) or mismatch ‘DNA replication signature’ that might represent a source repair genes (for example, hMLH1) (Sancar, 1994; of new prognostic markers. Such a signature could help in Marra and Boland, 1995; Bertwistle and Ashworth, understanding the molecular mechanisms underlying 1998; Masutani et al., 1999). In somatic cancers, such tumor progression in colorectal cancer patients. early mutations probably become ‘diluted’ during the Oncogene (2010) 29, 876–887; doi:10.1038/onc.2009.378; progression of the disease, making the relationship less published online 9 November 2009 obvious. However, many studies show that an alteration of genes involved in genome replication or supervision Keywords: DNA replication; S-phase checkpoint; color- promotes or favors ‘acquired’ cancers (for reviews, see ectal cancer; genetic instability; prognosis markers Kunkel, 2003; Mitchell et al., 2003). In mice, perturba- tion of the catalytic activity of the replicative DNA polymerase d increases genomic instability and accel- erates tumorigenesis (Venkatesan et al., 2007). In addi- tion, mouse fibroblasts expressing variable levels of MCM proteins, which are involved in DNA replication Correspondence: Professor C Cazaux, Institute of Pharmacology and firing, or of the MCM loader Cdt1 show numerous Structural Biology, IPBS UMR5089, CNRS University of Toulouse, University Paul Sabatier, 205 route de Narbonne, Toulouse, cedex 4, chromosomal abnormalities and form tumors in nude Midi Pyrenees 31300, France. mice (Arentson et al., 2002; Honeycutt et al., 2006). E-mail: [email protected] Colorectal cancer is one of the most frequent cancers C Cazaux and JS Hoffmann share the leadership of the Genetic worldwide, with genetic instability having a driving role Instability and Cancer Group. in this neoplasia. To clarify the importance of DNA 7These authors have equally contributed. Received 20 August 2008; revised 15 July 2009; accepted 5 October 2009; replication in colorectal adenocarcinomas, we analysed published online 9 November 2009 the variation of expression of 47 DNA replication genes DNA replication signature in colorectal cancer M-J Pillaire et al 877 in tumor versus adjacent normal colorectal tissues and MCM2, MCM7, MCM8 and SLD5) were overexpressed compared these data with disease progression and in tumors. In contrast, GEMININ, the S-phase-dependent clinical features. Our cohort included 74 patients with inhibitor of CDT1, was significantly underexpressed. We microsatellite instability-negative tumors who were not next assessed whether these modifications in gene expres- treated with neo-adjuvant therapies. sion were linked to a specific stage of disease progression. We observed that the genes involved in either the We determined that DBF4, CDC7 and CYCE1 were firing of the replication process or the maintenance of significantly overexpressed mostly in early-stage tumors, the replication fork structure were mostly overexpressed whereas GEMININ inhibition was significant in later in tumors compared with adjacent control tissues. In stages (Supplementary Table S2). contrast, genes involved in translesional (TLS) replica- Regarding the DNA polymerase family, although the tion, S-phase checkpoint were mostly repressed. Statis- expression of the replicative POLD and POLE was not tical analysis showed that some genes (POLQ, SLD5, significantly affected in tumors, we found a defective BRCA1, CYCE1, CDC7 and RUVBL1) were mainly expression of genes encoding the mitochondrial POLg deregulated in early-stage tumors, whereas others and the ‘non conventional’ TLS DNA polymerases (GEMININ, RAD9) were predominantly deregulated POLZ,i,k,l and Rev3/z (Table 1 and Figure 2), with the in late-stage carcinomas. exception of POLQ, which was significantly overex- We also observed that MCM7 overexpression was pressed mainly at early stages (Supplementary Table S2). correlated with negative outcome in colorectal cancer. We also found a lower expression of genes that sense This was also the case of a cluster of genes characterized DNA damage occurring in the S-phase, such as ATM, by concomitant overexpression of at least three ‘firing’ 53BP1, RAD17 and RAD9 (Table 1 and Figure 3a) and, genes and the translesional DNA polymerase POLQ. specifically in stage 4 tumors, RAD9 (Supplementary Interestingly, the levels of POLQ and MCM7 expression Table S2). The new MCM2-8 family member, MCM9, were not associated with the level of PCNA immuno- which is likely to have a role in an S-phase checkpoint staining, indicating that such an upregulation did not pathway, was also downregulated. In contrast, the depend on the proliferation status. S-phase checkpoint gene products that are involved in Taken together, these data suggest that, in colorectal the downstream protection of stalled DNA replication cancer, deregulation of DNA replication genes may forks RAD51, RUVBL1, BLM, BRCA1 and BRCA2, have a significant prognostic value and may be more with BRCA1 being significantly upregulated at early useful for clinicians than standard biomarkers or stages (Supplementary Table S2), as well as the the anatomo-pathological classifications. S-phase signaling mediators, CHK1 and CHK2, were overexpressed (Table 1). Finally, genes involved in the repair of replication- Results induced DNA breaks (TIP 60), cohesion of chromatids (ECO1) and resolution of replication intermediates Subsets of DNA replication genes are deregulated in (ERCC1, MUS81) were all repressed (Figure 3c). colorectal cancers TIP60 and MUS81 were downregulated in almost all Microarray studies have shown that DNA replication tumors at all stages (Supplementary Table S2). genes are often poorly expressed in human tissues To investigate whether the misregulation of replica- (NCBI GEO data sets). Therefore, we used a real-time tion genes was related to the proliferating status of PCR-based approach to generate gene expression pro- cancer tissues, we used a nonparametric Spearman’s files of 74 coupled primary colorectal carcinomas at dif- correlation test to compare the expression of 34 ferent stages of progression (Supplementary Table S1). representative replication genes with the part of We selected 47 genes known to have a role in DNA proliferating cells in tissues. The proliferation status synthesis, intra-S-phase checkpoint