Our data highlights the important role that integrative approaches may have in understanding cancer biology and implications of such in the translational arena. Our current approach analyzing the genomic and epigenomic changes in colon cancer identified the role of the ECM pathway in colorectal carcinogenesis.
These findings also are potentially useful in finding novel prognostic biomarkers as highlighted by our finding of DNA methylation of IGFBP3
showing a modest effect in being associated with worse survival. As mentioned in the results, this may have direct clinical implications in stratifying the high-risk Stage 2 colon cancers patients who have worse outcomes and who may benefit from adjuvant chemotherapy; although our findings would need to be validated in other retrospective studies of carefully clinically annotated tissues or a well-designed prospective study. Our findings were validated in an internal validation cohort (JHU validation) where DNA methylation of both IGFBP3
again showed the association with worse survival while CD109
showed a trend towards worse survival. We then validated our findings in another large external NLCS for DNA methylation of EVL
associated with worse outcomes. However, IGFBP3
methylation was not associated with worse survival in this cohort. Although at present it is unclear why IGFBP3
methylation was not validated in the NLCS cohort, there are multiple possible explanations for this. This may simply reflect the different biology in the two cohorts. In fact, epigenetic alterations have been shown to vary in different populations and races and potentially affect outcomes (30
). Netherlands also has a lack of colon cancer screening program and this is reflected in the markedly different 5-yr survival of this cohort (54.8% NLCS vs
. 67.4%-JHU Training cohort or 65.3% JHU validation cohort) and the biases associated with screening (32
). Moreover, the NLCS cohort has a preponderance of left-sided cancers while the JHU cohorts reflect a distribution and survival similar to what is seen in the Surveillance, Epidemiology and End Results database (a large national cancer database in the United States) (33
). The location of colon cancer has been shown to affect both genetic and epigenetic alterations (34
) and survival (33
). In addition, adjuvant chemotherapy was not offered routinely for high risk patients in the NLCS cohort. Thus lack of validation of IGFBP3
may also reflect the fact that this is a predictive rather than a prognostic marker. Further validation studies in comparable modern-day cohorts similar to the Johns Hopkins cohort may clarify this. In particular, our data on IGFBP3
and survival is intriguing since previous studies have shown that IGFBP3
induces apoptosis and inhibits DNA synthesis in breast, prostate and non-small cell lung cancer (38
). Moreover, prior studies have also reported that high levels of circulating IGFBP3
decrease the risk of recurrent colorectal adenoma formation (39
). Further studies are needed to confirm these findings particularly the risk stratification in stage 2 colon cancers.
Previous studies have also alluded to the potential of epigenetic changes, especially DNA hypermethylation of promoter-associated CpG islands, in serving as prognostic biomarkers. Brock et al
. showed that hypermethylation of two genes, p16
, in the primary lung tumor and the mediastinal lymph nodes could predict recurrence for early stage lung cancers (5
). In another study, DNA methylation profiles of multiple methylated-in tumor (MINT) loci predicted distal recurrence in a small cohort of rectal cancer patients (40
) while ID4
methylation has also been associated with worse outcomes (41
). However most of these studies used candidate genes from the literature and tested them for prognostic significance in a cohort of patients. In our study, we have not only identified a unique pathway, the ECM pathway, that is altered by DNA methylation of many genes in CRC, but then show that dysregulation of this core pathway has prognostic significance as well. However, our current studies do not clearly distinguish between the DNA hypermethylation arising from the tumorigenic cells or from the non-tumorigenic stromal cells. This will need to be investigated in future studies.
What are the functional consequences of loss of function for the above ECM genes in CRC? We also considered, from several perspectives, our findings in another context – namely, that they might represent an example of properties which reflect the primitive cell status increasingly attributed to multiple tumor types(42
). First, multiple previous studies(3
) have stressed that some 50% of genes that become DNA hypermethylated in colon cancers are marked by a chromatin pattern, termed bivalent chromatin, that keeps these same genes in a low expression pattern in ESC and embryonic mesenchymal (EM) progenitor cells(43
). Indeed, a key chromatin component of bivalent chromatin, the presence of polycomb group (PcG) constituents around gene start sites, is present in ESC and EM cells in the promoter regions of several of the ECM genes (IGFBP3
, CPAMD8, FBN2
), based on our database analyses (unpublished data).
In summary, our results suggest that CRC may harbor defects in key ECM genes, induced infrequently by gene mutations, and even more frequently by gene promoter DNA hypermethylation. This loss of function for such genes appears to correlate with loss of cellular differentiation and to be associated with aspects of primitive, stem/progenitor cell, features of CRC (supplementary Fig. S3
). Loss of ECM gene function could foster metastatic behavior by altering events which require ECM remodelling such as allowing cells to invade through the basement membrane, migrate into the lymphovascular space, and establish metastatic foci in a distant organ. Our study suggests that this ECM alteration by hypermethylated genes may contribute to carcinogenesis, to some degree in virtually every CRC, through silencing of selected ECM pathway genes by both genetic and epigenetic alterations. Finally, our data indicate that detection of DNA hypermethylation of selected of the genes we have studied may provide a biomarker strategy for predicting the clinical behaviour of CRC. Our data emphasize the importance of integrating cancer gene mutations and DNA hypermethylation changes to uncover the molecular events disrupting cell signaling in CRC and other cancers.
We have used an exciting integrative approach of multiple whole genome analysis (genetic and epigenetic) to identify an important pathway, the extracellular matrix (ECM) maintenance and remodeling pathway, which is silenced in all colon cancers. Colon cancers that have silenced multiple genes in this pathway have a poor prognosis based on analyses of large cohort of patients. DNA Methylation of these genes is particularly useful in stratifying the low- vs. high- risk Stage 2 colon cancer patients. Colorectal cancers that have silenced multiple genes in the ECM pathway appear to show a significantly worse survival in adjusted multivariate analysis of a large cohort of colon cancer patients. Our study has important ramifications in the clinical management of colon cancer patients since 30-40% of early stage colon cancers will present with recurrence/metastases but current strategies are not helpful in stratifying this high-risk cohort. We show that our prognostic markers are the most efficacious in stratifying the high risk subset of Stage 2 colon cancers who may then benefit from adjuvant chemotherapy.