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Improved risk stratification of early colorectal cancer might help to better select patients for adjuvant treatment. Alterations in the transforming growth factor-β (TGF-β) pathway have frequently been found in colorectal cancer, but their impact on prognosis remains controversial. We therefore analyzed two transcriptional corepressors of the TGF-β signaling pathway with respect to prognosis and prediction of chemotherapy benefit in early colorectal cancer.
The gene copy status of SKI and SNON was analyzed by use of quantitative real-time polymerase chain reaction in 179 colorectal tumor biopsies, which had been collected from a randomized multicenter trial of the Swiss Group for Clinical Cancer Research (SAKK).
Partial or complete allelic loss was found in 41.5% and 55.2% for SKI and SNON, whereas amplification was found in 10.1% and 15.1%, respectively. Multivariate Cox analysis showed that gene amplification of SKI independently predicted reduced relapse-free [hazard ratio (HR) for relapse 2.08, P = .049] and overall survival (HR for death 2.62, P = .012). In contrast, deletion of SKI and the gene copy status of SNON were not significantly correlated with prognosis.
Amplification of SKI is a negative prognostic marker in early-stage colorectal cancer. This marker should help to improve risk stratification to better select patients for adjuvant therapy. Confirmatory investigations are warranted.
A significant proportion of patients with early-stage colorectal cancer who are undergoing surgical resection with curative intention eventually relapse and die of metastatic disease . Recurrence of high-risk tumors has been significantly reduced by adjuvant treatment and, thereby, overall survival (OS) rates have been improved. The decision to provide adjuvant treatment is based on a risk assessment, the mainstay of which is pathologic staging according to the Dukes system. This system does not take into account recent advances in our understanding of molecular biology, including the signal transduction systems in colorectal cancer.
Colorectal cancer has become a prototype malignancy to describe general principles of molecular carcinogenesis resulting from an accumulation of genetic alterations, which lead to the activation of oncogenes and inactivation of tumor-suppressor genes . Specifically, inactivation of the adenomatous polyposis coli (APC)/β-catenin pathway, mutation of the k-RAS gene, deletion of chromosome 17p with the p53 gene, and loss of chromosome 18q harboring DCC  and, more importantly, transforming growth factor-β (TGF-β) signal-transducing molecules SMAD2, SMAD4 , and SMAD7 [5,6] have been found. The importance of the TGF-β pathway in colorectal tumorigenesis has been further demonstrated by the finding of frequent inactivating mutations of the TGF-β receptor II , SMAD2 , SMAD4 , and TGF-β itself . It therefore has been hypothesized that most colorectal cancers have an alteration in at least one component of the TGF-β signaling pathway .
TGF-β is signaling through heterodimerization of TGF-β type I and type II receptors, which, in their activated state, phosphorylate SMAD2 and SMAD3. This phosphorylation allows the formation of a heterotrimeric complex with SMAD4, which translocates into the nucleus. This activated SMAD (MAD, mothers against decapentaplegic, Dresophila, homolog) complex will either activate or repress gene expression by recruiting either coactivators or corepressors . Such corepressors are the Sloan-Kettering Institute proto-oncogene (SKI)  and its relative Ski-related novel gene N (SNON = skil) . SKI binds to the nuclear transcriptional corepressors, N-CoR/SMRT, and the adaptor molecule, mSin3A, forming a macromolecular complex. Through the adaptor, mSin3A, histone deacetylases are recruited, whose effects generally lead to chromatin condensation at target gene promoters, thereby repressing transcriptional response [15,16] SNON, which is also a corepressor, interacts with SMAD, and plays a role in the suppression of SMAD signaling in the absence of TGF-β and in negative regulation after TGF-β stimulation [14,17].
In fact, these genes might be involved in the dual role TGF-β is playing during tumorigenesis by induction of cell cycle arrest in early stages and promotion of proliferation, angiogenesis, and metastasis in the late stages. We decided to evaluate the role of SKI and SNON in colorectal cancer. To this aim, we determined the gene dosage of SKI and SNON by quantitative real-time polymerase chain reaction (PCR) in 179 tumor samples, which had been collected from a randomized trial of the Swiss Group for Clinical Cancer Research (SAKK 40/81) by evaluating the effect of 5-FU-based shortterm perioperative chemotherapy in stage II and stage III colorectal cancer . We determined the prevalence of gene copy changes and correlated the gene copy status with prognosis and prediction of chemotherapy benefit.
Paired primary tumor and normal tissue biopsies were obtained from patients involved in a randomized trial of the SAKK for the benefit of 5-FU/MMC adjuvant chemotherapy (SAKK trial 40/81) versus surgery alone. The chemotherapy consisted of an immediate postoperative infusion with 5-FU (500 mg/m2) for 7 days, with one single dose of mitomycin (10 mg/m2) on day 1. The study population comprised 533 patients younger than 75 years, with a median age of 62 years. A total of 63.4% of the patients had colonic carcinoma and 36.6% had rectal carcinoma. A total of 62.4% was node-negative (stage II) and 31.1% was node-positive (stage III), and in 6.5%, the nodal status was not assessed. Details of this trial have been described previously . From this trial, 179 biopsies from a subpopulation representative of the whole study population (Table 1) were available for analysis. The trial was performed after acceptance by the local ethics committee.
Genomic DNA was extracted from formalin-fixed and paraffin-embedded tumor and normal tissue biopsies derived from the same patients using the Nucleospin C + T kit (Macherey-Nagel, Duürren, Germany), according to the manufacturer's instructions.
The gene copy status of SKI and SNON was established by gene dosage with real-time quantitative PCR using the TaqMan system on an ABI Prism® 7700 sequence detector (Applied Biosystems, Foster City, CA), as described previously . This technique is based on a two-step process consisting of PCR amplification and hybridization of an oligomer fluorescence probe to the PCR target sequence. During amplification, the probe is digested by the 5′ exonuclease activity of the Taq DNA polymerase, leading to separation of the fluorescence dye and its quencher, which results in an increase in fluorescence emission that is continuously detected during the amplification process.
The protocol for gene dosage is given in Boulay et al. . In brief, 1 µl of DNA was mixed with 0.5 µM of each primer, and the labeled probe (0.1 µm) with 12.5 µl of the Taqman® Universal PCR Master Mix (Applied Biosystems) in a total volume of 25 µl. All reactions were performed in triplicate. Cycling conditions were according to the manufacturer's instructions: 50°C for 2 minutes, 95°C for 10 minutes to activate AmpliTaq Gold® DNA Polymerase (Applied Biosystems), followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute for annealing and extension:
For each individual, the Ct value obtained for the autosomal reference gene 36B4 (calculated by the built-in software) on normal tissue was subtracted from that of tumor tissue, thus defining ΔCt 36B4. An analogous calculation was made for each gene. The gene copy status is indicated by the ΔCt value (ΔCt 36B4 - ΔCt gene) as follows: -0.45 < ΔCt < 0.45, normal diploidy; ΔCt< -0.55, deletion; and ΔCt > 0.55, amplification.
Each gene status was considered as a categorical variable with three categories: amplification, deletion, and no change. Two multiple Cox regression models were performed to test the prognostic and predictive values of the status of SKI and SNON, respectively, on disease-free (DFS) and overall survival (OS). In addition to treatment arm, known prognostic factors, namely patients' age at study entry, sex, lymph node status, tumor size, and tumor location, were included in the regression analysis to control for their influence. The results are presented with hazard ratio (HR) and 95% confidence interval. Statistical tests were carried out at the 5% significance level (two-sided).
For both genes, the interaction between treatment arm and gene copy status was included in the regression analysis. The test for prognostic effects was based on the statistical significance of the parameter estimates for the terms gene amplification and gene deletion, and the test of predictive effects on the treatment x amplification and treatment x deletion interaction terms. A significant treatment x amplification interaction would mean that the risk for patients with amplification relative to patients without change for a given gene differs between patients with and without adjuvant chemotherapy. Analogously, it also would mean that the relative risk of patients with versus without adjuvant chemotherapy differs between patients with amplification and those without change. The same logic applies for the treatment x deletion interaction. Survival curves were estimated by the Kaplan-Meier method.
We have analyzed 179 paired tumor and normal tissue DNA samples to establish the gene copy status of SKI and SNON. Of the 179 samples, 159 and 165 were informative for SKI and SNON, respectively. A partial or complete allelic loss was found in 41.5% and 55.2% for SKI and SNON, whereas an amplification was found in 10.1% and 15.1%, respectively (Table 2). The molecular analysis was performed blindly for the clinical outcome data.
The results of the multivariate Cox regressions imply that SKI amplification is a negative prognostic indicator of DFS [HR for relapse in patients with amplification relative to those with no change of gene copy number (= normal diploidy) on SKI locus: 2.08, P = .049] and OS (HR for death in patients with amplification relative to those with no change of gene copy number on SKI locus: 2.62, P = .012) in this population. Thus, patients carrying a tumor with a SKI amplification had a two times higher risk of relapse and a 2.6 times higher risk of death than patients with no change of gene copy number at the SKI locus (Table 3). A deletion at the SKI locus did not significantly change the patients' prognosis (details in Table 3). Given that patients with SKI deletion were not significantly different from patients with no change, we pooled these two groups to form the group of all patients lacking SKI amplification to be compared to those with amplification. A Cox regression with the same covariates as described above was run for each of both outcomes. The results were consistent with the ones reported above. Patients bearing SKI amplification had a risk of relapse twice as high and a risk of death two and a half times higher than patients bearing no amplification on SKI [for DFS: HR = 2.13, P = .028; for OS: HR = 2.65, P = .006].
Figure 1 shows the Kaplan-Meier curves for DFS and OS of patients carrying tumors with SKI amplification compared to tumors without amplification (normal diploidy and deletion) of SKI. As opposed to the amplification of SKI, changes in the gene copy number of SNON were not significantly associated with prognosis (Table 3 and Figure 2).
For both SKI and SNON, all interactions between gene status and treatment arm were not significant with P values > .2. Therefore, for both SKI and SNON, the risk of relapse and the risk of death were not significantly different between patients with and without adjuvant chemotherapy.
Pathological analysis of resected tumor specimens is still the best established method to predict the survival of colorectal cancer patients . Nevertheless, the clinical outcome of patients bearing tumors with the same stage is very heterogeneous. To define the prognosis and to plan appropriate adjuvant treatment, additional predictors are needed. This is particularly important for patients with stage II disease who are not routinely treated with adjuvant chemotherapy, and among whom selection of bad-risk patients who would significantly benefit from adjuvant treatment would be especially helpful. Furthermore, with the introduction of more efficient but also more toxic adjuvant therapy regimens, risk stratification becomes an issue of even higher relevance.
This study strongly suggests that the gene copy status of SKI is an independent predictor of relapse-free survival and OS in early colorectal cancer. Amplification of SKI was identified as a negative prognostic marker in stage II and stage III colorectal cancer, with a risk of relapse twice as high as for tumors bearing a normal diploidy at the SKI locus. In the multivariate analysis, this association was shown to be independent of age, sex, lymph node status, tumor size, and tumor location, and can therefore be used as an independent prognostic marker to identify high-risk situations, thus influencing the decision to provide adjuvant treatment. Based on our data, patients with stage II disease and SKI-amplified tumors should be considered to have a higher risk of relapse, representing a molecularly defined subgroup that might benefit from adjuvant treatment. However, due to the low rate of SKI amplification (10.1%), the 95% confidence interval for HR is quite large and, therefore, a confirmatory investigation in a larger sample would be important to definitely establish the role of SKI amplification.
The association of SKI amplification and unfavorable prognosis implies a causal relation to colorectal tumorigenesis. Considering, however, that it affects only 10% of the tumors, it should not be regarded as a prerequisite but as a possible lesion in the multistage colorectal tumorigenesis. This is in line with the findings of other TGF-β signaling molecules, which are frequently, but not in all cases, altered in colorectal cancer by point mutation  or deletion. SMAD2, SMAD4, and SMAD7, for example, were found to be deleted in 66%, 64%, and 48% of the colorectal tumors, respectively . Of these, SMAD7 has been shown to be a positive prognostic factor , whereas SMAD2 and SMAD4 seem to be unrelated to a prognostic effect. This is in line with the view that colorectal cancer may harbor defects in the TGF-β signaling pathway at different levels, influencing tumor biology and thereby the patient's prognosis.
Our result of an association between SKI amplification and worse prognosis is easily explained by several previous findings. As expected for a proto-oncogene, overexpression of SKI has been shown to induce morphological transformation and anchorage independence . Furthermore, it has been demonstrated that overexpression of Ski abrogates not only transcription of TGF-β-responsive genes, but also TGF-β-induced growth arrest (i.e., it has a tumor-promoting potential) [15,16]. In melanoma, SKI has been found not only in the nucleus but also in the cytoplasm, where it comes to a SKI/SMAD2/3 association that seems to prevent SMAD3 nuclear translocation in response to TGF-β . A recent report demonstrated that SKI can block TGF-β signaling by interfering with the phosphorylation of SMAD2 and SMAD3 by the activated TGF-β type I receptor. In addition, it facilitates the assembly of SMAD2/SMAD4 and SMAD3/SMAD4 complexes independent of TGF-β signaling, thereby disabling TGF-β signaling by engaging SMAD proteins in nonproductive complexes . TGF-β target gene transcription, which in early tumor stages results in cell cycle arrest, can be inhibited by all these mechanisms, thereby explaining the facilitated growth of SKI-amplified tumors. As an interesting alternative mechanism, in human melanoma cells, SKI has been shown to be a coactivator of the Wnt/β-catenin signaling pathway . This pathway has been well characterized in colorectal cancer and activation promotes tumorigenesis. Through this mechanism, the tumor-promoting activity of SKI amplification in colorectal cancer would be easily explained.
On the contrary, SKI deletion did not have any prognostic relevance in our analysis. In accordance with our study, which found SKI deletions in 47% of the samples, loss of chromosome 1p36, the chromosomal region where SKI is located, has been reported in several studies at frequencies between 30% and 80% [24–27]. Two studies reported a negative prognostic value of 1p36 deletion [28,29]. Our data, however, do not support a prognostic relevance of 1p36 and specifically not of SKI. The frequent deletions of SKI in 47% of colorectal cancers suggest that SKI might act as a tumor-suppressor gene. In fact, in Ski-deficient heterozygous mice, an increased susceptibility to tumorigenesis, mainly of lymphomas and leukemias, was observed. In that study, however, no colorectal cancers were observed . This might be due to interspecies variations in tumor susceptibility, but it could also mean that SKI deletion is not causally linked to colorectal carcinogenesis.
SNON, the other member of the SKI gene family, was also found to be frequently deleted (55% of the tumors) in our colorectal cancer samples. SNON is located at 3q36, a region with a high frequency of loss in human osteosarcoma. In the mouse, SnoN has been demonstrated to act as a tumor suppressor, but again, there were no colorectal cancers observed . This is in line with our data suggesting SNON gene copy alterations not to be significantly associated with patients' prognosis. We have to be careful when excluding a possible prognostic value of SNON deletion because our lack of statistical significance might be due to the relatively low power of our analyses. The statistical power for SNON deletion (deletion versus no change) was 0.33 in the DFS analysis and 0.29 in the OS analysis.
Our finding of a lack of significant interaction (P > .2) between treatment and gene copy status of SKI and SNON is consistent with the suggestion that the effect of chemotherapy is not mediated through genes that are regulated by the interaction of SKI or SNON with the SMAD3 and SMAD4 complex. However, due to the small sample size, the lack of significance is difficult to interpret because the statistical power to exclude an interaction is low.
In conclusion, we have identified SKI amplification as an independent prognostic factor in early-stage colorectal cancer. Our finding might become relevant in the current attempts to improve risk stratification in the decision for or against an adjuvant treatment regimen.