In the present work, we report the novel finding of a positive association of the MACC1 tagging SNP rs1990172 with reduced overall survival in patients with CRC. To our best knowledge this is the first study investigating the influence of MACC1 polymorphisms on the prognosis of CRC. Several studies have investigated the influence of MACC1 mRNA levels on the clinical outcome in patients with colorectal [4
], gastric [5
] and lung cancer [6
], respectively, providing evidence that MACC1 overexpression is a crucial prognostic factor for tumor recurrence, metastasis, and survival. Our finding that a tagging SNP in the MACC1 gene is significantly associated with patient's survival indicates that beside MACC1 mRNA levels also the genetic diversity of the MACC1 locus influences the prognosis of CRC. Therefore, our results emphasize the relevance of MACC1 as a prognostic marker also at the DNA level for clinical outcome prediction.
Overexpression of MACC1 may be caused by aneuploidy of chromosome 7, where MACC1 is located (position 7p21.1). Indeed, as shown by Galimi and colleagues [13
], expression of MACC1 correlates with polysomy of chromosome 7 or with ploidy of the p-arm in metastatic CRC. However, in this study polysomy of chromosome 7 or ploidy of the p-arm mainly appeared at a low-level, with an average of 3.3 and 3.4 copies, respectively. Other, yet unknown molecular mechanisms may also contribute to MACC1 overexpression or function. SNPs located in coding or regulatory sequences have the potential to modify the biological activity of MACC1. Several putative deleterious SNPs are located at the MACC1 locus: One SNP leads to a premature stop codon at residue 390 (rs2108292) and two SNPs (rs36106647, rs35043094) shift the reading frame after amino acid 670, leading to a translational stop codon after 11 additional residues [20
]. However, according to NCBI SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP
), these genetic variants are rare and will cause low statistical power, especially if penetrance is low, or even might be absent in a patient cohort of limited sample size. On the other hand, hundreds of common variants at the MACC1 locus of still unknown function are listed in the NCBI SNP database. Selection of a set of tagging SNPs based on stringent criteria like a high r2
value allows covering most of this given genetic variance [21
Here, we report the significant association of the MACC1 tagging SNP rs1990172 with reduced overall survival in CRC patients. Variant rs1990172 is located within an intronic region of the MACC1 gene (Figure ) and does not affect any splice site of a coding exon. Therefore, it is unclear, if rs1990172 is the causative SNP responsible for the observed effects. Notably, the variant is in strong linkage disequilibrium with a non-synonymous SNP, rs975263 (r2
= 0.858; http://www.hapmap.org
), which is leading to an exchange of leucine to serine at codon 515, fifth exon. However, in silico analysis using software tools PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2
] and SIFT (http://sift.jcvi.org
], respectively, does not suggest an adverse impact of this amino acid substitution on the structure or function of the MACC1 protein (data not shown). Both SNPs are located in a large haplotype spanning at least 50 kb of the MACC1 sequence, comprising three coding exons of the MACC1 gene. This indicates that also other SNPs in this haplotype may contribute to the prognosis of CRC.
Of note, according to data of HapMap recombination rates [24
], two strong recombination hotspots outside the MACC1 locus around positions 20,091,000 and 20,607,000 (NCBI build 36) are defining a huge haplotype block including beside MACC1 another metastasis-associated gene, namely ITGB8 [25
]. Therefore, an association between a SNP within the MACC1 locus and the clinical outcome might be linked to its correlation to causal genetic variants of the ITGB8 locus (position 20,337,000-20,420,000, NCBI build 36). However, linkage disequilibria between SNPs in the MACC1 and ITGB8 loci are at best of weak extend (e.g. r2
values between rs1990172 and SNPs within the ITGB8 locus ranging from 0.00 up to 0.089; http://www.hapmap.org
). Consequently, observed effect of the tagging SNP rs1990172 on overall survival appears predominantly caused by the genetic diversity of the MACC1 gene.
Genotyped SNPs rs3114446, rs10275612, rs3095007, rs3095009, and rs7780032 were not associated with overall survival. These variants are all located in non-coding or non-regulatory regions and are not in high linkage disequilibrium with any functional SNP given in HapMap SNP database [16
]. Therefore, among investigated SNPs, variant rs1990172 remains the only one which is linked to a potentially functional region, which might explain the fact that among investigated SNPs solely rs1990172 is significantly associated with the prognosis of CRC.
In vitro studies have shown that MACC1 acts a key regulator of its transcriptional target gene MET [4
], which encodes for the Met tyrosine kinase receptor for hepatocyte growth factor (HGF) [9
]. Aberrant activation of MET deregulates the HGF/MET signaling pathway, leading to increased cell proliferation, invasion, and metastasis [9
]. Consequently, high expression of MET in colorectal cancers is linked to the development of distant metastases and represents a strong prognostic indicator for poor survival [11
]. These findings have led to the development of agents that can effectively disrupt HGF/MET signaling through direct inhibition of the receptor (anti-MET antibodies), through inactivation of its ligand HGF, by interfering with HGF binding to MET, or by inhibiting MET kinase activity [26
]. Several phase I and II clinical trials addressing the therapeutic efficacy of these agents are currently under way. Amplification of the MET gene responds to Met inactivation with growth impairment gene in vitro and may therefore predict treatment outcome in vivo [13
]. In a similar way, functional SNPs at the MACC1 locus associated with MACC1 activity or function may act as easily detectable predictive markers in anti-HGF/MET treatment in the future.
It is important to consider the potential limitations of our study. Overall survival was defined as the sole clinical end point evaluated. MACC1 is associated with colon cancer metastasis. Therefore, metastasis-free survival would represent a more accurate endpoint. However, the metastatic process is directly linked to patient survival [3
] and, therefore, our observation that a MACC1 polymorphism is significantly associated with reduced overall survival also indicates its role in metastatic dissemination. Selected tagging SNPs captured 75% of variants with a MAF ≥ 0.15 and, therefore, 25% of common SNPs remained unexplored. Further, less frequent polymorphisms (MAF <0.15) were not considered by selection criteria. However, it is questionable if a probably moderate association between a polymorphism of low frequency and overall survival would have reached statistical significance due to given sample size. Further, it remains unclear, if the tagging variant rs1990172 or another variant, which is highly correlated with it, is the causative SNP responsible for the observed effects. Additional studies, such as fine mapping studies and directed functional studies to determine the molecular consequences of genetic variation at this locus are needed. Finally, we have not accounted for multiple testing in our study. Of note, the Bonferroni correction would probably have been too conservative owing to the given correlation among the tests performed. Therefore, associations between rs1990172 and clinical outcome appear significant only at a nominal significance level. Notably, the observed significant association between variant rs1990172 and overall survival would have survived Bonferroni correction in the adjusted additive model of inheritance (pcorrected
= 0.042). Replication of our observations in independent studies is necessary to clarify the prognostic relevance of MACC1 polymorphisms to the clinical course of CRC.