Molecular pathways undergoing dramatic transcriptomic changes during tumor development in the human colon

Colon carcinogenesis is a multistep process involving the gradual accumulation of genetic and epigenetic alterations. These changes promote the malignant transformation of precancerous lesions of the colorectal mucosa [1], a process reflected by progressively severe cellular dysplasia and increases in lesion size. At least two-thirds of all colorectal cancers develop from precancerous lesions with adenomatous features [2]. The “serrated” histotype characterized by cells arranged in a saw-toothed pattern [1] is somewhat less common, but in both cases, size is an important indicator of the distance the lesion has travelled on the road toward malignancy. For this reason, post-polypectomy surveillance guidelines vary depending in part on the size of the polyps removed. In fact, individuals with 3 or more adenomas on initial colonoscopy, including 1 or more measuring ≥10 mm, are significantly more likely to present with new lesions at the next colonoscopy [3].

Analysis of precancerous colorectal lesions of different sizes can thus furnish important information on the steps involved in their malignant transformation. During colonoscopy, benign lesions of all sizes are routinely removed to prevent their progression toward cancer, and this provides a valuable source of tissues for molecular studies. Efforts of this type have already identified several genetic and epigenetic changes that seem to occur at the transition from normal mucosa to precancerous lesions. Mutations involving the APC or CTNNB1 gene, for example, are considered early events that fuel epithelial-cell proliferation [4, 5]. Gain-of-function mutations in the oncogenes KRAS and BRAF are also frequent findings in early stages of transformation [6]. Additional alterations (genetic and epigenetic) are believed to be necessary for subsequent steps toward invasiveness, such as those identified with recent genome-wide analyses [7, 8].

The transcriptomes of colorectal cancers have been intensively investigated with high-throughput, array-based tools, which furnish quantitative, genome-wide descriptions of the individual gene expression levels associated with different cell phenotypes (e.g., adenoma cells vs. normal epithelial cells) [9‐12]. More recently, other methods of analyzing gene expression data have been developed to gain additional insight into the mechanisms driving the phenotypic differences. One such approach involves the analysis not of single genes but of predefined functional gene sets, that is, groups of genes that are known components of a defined molecular pathway representing a given biological process. The basic aim here is to identify those gene sets (i.e., pathways) that display enrichment for―or over-representation of― genes whose expression is substantially altered in the phenotype being investigated. We have explored several methods for quantitatively analyzing transcriptomic data for pathway enrichment [13‐15], including gene set enrichment analysis (GSEA) [16], random-set methods (RS) [17], and gene list analysis with prediction accuracy (a method developed by our group) [15]. Although these methods differ substantially from one another, all three are statistically accurate and identify relevant gene sets, and none consistently outperforms the others [14].

Our experience indicates that pathway-based analysis of gene expression data furnishes highly reproducible results that can be useful for dissecting a complex, polygenic disease like colorectal cancer. For instance, we recently used GSEA and RS analysis to identify pathway enrichment in four independent transcriptional data sets representing colorectal cancer and normal mucosa. The results of these analyses displayed substantial overlap: both of the analytical methods used revealed similar dysregulation of 53 pathways in each of the four data sets. These pathways are very likely to play important roles in the pathology of colorectal cancer [13].

In the present study, we used RS analysis to explore a large body of previously collected transcriptomic data on human colorectal tissues, including normal mucosa, preinvasive lesions of various sizes, and colorectal cancers (CRCs). Our aim was to identify biological processes that become dysregulated during the course of colorectal tumorigenesis. Because the preinvasive stages have been far less extensively explored than the cancerous phases of this process, there were no independent sets of transcriptomic data on precancerous lesions that we could use to validate our findings. To overcome this limitation, we used two strategies. First, we re-analyzed all the original data sets with GSEA and compared the results with those obtained with RS. Second, we performed RS analysis of two publicly available sets of data on CRCs and normal colorectal mucosa.

All data were analyzed in MatLab (MathWorks, Natick, MA) unless otherwise stated.

As shown in Tables 2 and 3, a total of 64 pathways were found to be significantly upregulated (n=23) or downregulated (n=41) in SPLs; 50 were upregulated (n=21) or downregulated (n=29) in LPLs; and 58 were upregulated (n=33) or downregulated (n=25) in the CRCs. The approach we used allows in-depth exploration of each instance of pathway dysregulation to characterize its evolution across the transformation process. Because this process is progressive, it was not surprising to find significant dysregulation of certain pathways in 2 or even 3 of the tumor stage-specific data sets, but other alterations were more circumscribed (Figure 1). For example, the BIOCARTA CELL CYCLE PATHWAY (Table 2, row 5) is one of the 23 gene sets that displayed significant upregulation only in the CRCs. This gene set comprises 22 genes (32 RefSeqs) encoding cyclins, cyclin-dependent kinases (CDK), cyclin-dependent kinase inhibitors (CDKI), and transcription factors, including E2F1, whose activation governs the G1-to-S phase transition of the cell cycle. The tumor suppressor RB1 (retinoblastoma protein) negatively regulates cell cycling by complexing with E2F1, and this effect is reversed by the phosphorylation of RB1 by cyclin D/CDK4, cyclin D/CDK6, and cyclin E/CDK2, which releases E2F1 from this complex and allows cell cycling to resume. For this reason, specific inhibitors of the cyclin/CDK complexes, such as p15 (CDKN2B), p16 (CDKN2A), p21 (CDKN1A), and p27 (CDKN1B), are also considered tumor suppressors. Dysregulation of this network stemming (for example) from the overexpression of certain cyclins, CDKs, or E2F1 itself, or from the downregulation of certain CDKIs, can lead to uncontrolled cell growth, which favors tumor formation and progression [20‐24].

Figure 2 (panels A, B, C) shows heat maps of the expression of the 22 genes included in the Biocarta cell cycle pathway at each stage of tumorigenesis (compared with normal mucosa). Each of the three tumor + N data sets was subjected to hierarchical clustering analysis using the 22 cell cycle-associated genes. As shown in Figure 2A, this analysis identified two clusters within the N vs. SPL data set, which showed no relation to the actual tissue labels (see column labels in Figure 2A). In the N vs. LPL data set (Figure 2B), the two tissue-type groups were more readily distinguished (only 6 LPL samples were misclassified), and in the N vs. CRC set, the two classes of tissues were separated with only three errors. Collectively, these findings point to progressive dysregulation of the cell cycle pathway, which becomes overt in the invasive stage of tumorigenesis, as highlighted by our RS analysis. Major involvement of this pathway at the CRC stage also emerged when the gene expression profiles were subjected to PCA (Figure 2D).

As shown in Figures 3A and 3B, certain cell cycle genes were already overexpressed in SPLs and LPLs, including those encoding CCND1, CCND2, and CCNE1, CDKs 2, 4, 6, and 7, and the oncogenes CDC25A and TFDP1. These changes were associated with downregulated transcription of the genes encoding the CDKI p15 (CDKN2B) and p21 (CDKN1A), an expected finding for preinvasive lesions with high proliferation rates. In contrast, CDKI p27 (CDKN1B) expression was upregulated in LPLs, but not CRCs (Figure 3C), a finding that is consistent with previously reported immunostaining profiles of adenomatous and cancerous colorectal tissues [25]. Interestingly, the tumor suppressor RB1 was also upregulated across all stages of tumorigenesis (Figure 3), whereas, in previous studies, this alteration has been documented only in the malignant phases [26‐28]. The most convincing explanation proposed for the upregulated expression of RB1 and p27 envisions these factors as possible mediators of a homeostatic mechanism that protects cells from the putatively toxic effects of excessive cyclin, CDK, or E2F1 activity [25, 28].

One of the most dramatic changes that characterized the transition to CRC (Figure 3C) was an increase in the expression of E2F1, the master regulator of the cell cycle pathway. This alteration is well known in colorectal carcinomas [22, 29], and it seems to be associated with higher tumor stages and poorer prognoses in these cancers [30] and those of other organs as well [31‐33]. Two other important cell cycle genes, those encoding the tumor suppressors p16 (CDKN2A) and the RB homolog p107 (RBL1), were also upregulated in CRCs. The expression of p16 can be silenced during tumorigenesis by gene promoter methylation, but this phenomenon is largely confined to colorectal cancers with the hypermethylator phenotype and DNA mismatch repair defects, which account for < 20% of all colorectal cancers [34‐36]. We have found p16 overexpression in ~80% of the colorectal cancers we have studied over the years (unpublished data). Like the p27 and RB1 upregulation mentioned above (or that of RBL1, which exerts inhibitory effects on E2F1-mediated trans-activation), p16 upregulation might represent a negative feedback mechanism aimed at preventing the G1-to-S transition (although E2F1 can readily overcome a p16-mediated G1 block) [37]. It is interesting to note that the trends shown in Figure 3, which are based on our analysis of transcript levels, are—on the whole—consistent with published data on the corresponding gene products.

Closer inspection of Tables 2 and 3 shows that the pathways exhibiting tumor-related downregulation were generally larger (in terms of the total number of RefSeqs they contained) than those that were upregulated in tumor tissues (mean numbers of RefSeqs in the gene sets: 69 vs. 27.9, respectively; p-value of one-tailed t-test = 2.4 · 10^-4). This finding might be related to the fact that tumor-associated downregulation was often seen in highly conserved pathways that govern normal mucosa homeostasis (e.g., cell differentiation programs). Pathways of this type have been extensively studied since the early days of molecular biology, and a relatively large number of their gene components have been identified. Consequently, the gene sets representing these pathways are likely to be larger than those of more specialized pathways, which have probably been less thoroughly explored. Nonetheless, it is also possible that fundamental pathways and networks are effectively larger as a result of relatively high-level component redundancy, a feature that would increase their robustness and versatility and ensure essential cellular functions in normal tissues under a variety of conditions.

Because the preinvasive stages of colorectal tumorigenesis analyzed in our study have been far less extensively explored than the cancerous phases, there were no independent transcriptomic data sets for precancerous lesions to use to validate our results. To overcome this limitation, we used two different approaches.

First, we re-analyzed our three data sets (N vs. SPL, N vs. LPL, and N vs. CRC) with GSEA [16], in a manner similar to that used in previous studies by our group [13]. Table 4 shows the numbers of pathways displaying significant tumor-associated enrichment in the RS and GSEA analyses. In all cases, a high percentage of the pathways found to be significantly up- or down-regulated in tumors (compared with normal mucosa) displayed the same trend in GSEA. (In both cases, a p-value cut-off of 0.05 was used to define significant enrichment.) For example, in the analysis of N vs. SPL data set, GSEA confirmed the presence of significant tumor-associated enrichment for 21 (91%) of the 23 pathways identified as enriched by our RS analysis (p-values = 0 computed by Fisher’s exact test). The number of enriched pathways identified by GSEA was always substantially higher than that obtained with RS analysis. This finding reflects the fact that in GSEA the nominal p-value of a pathway enrichment score is computed via an empirical phenotype-based permutation test procedure [16]. RS analysis uses a more stringent selection process in which the actual enrichment score of each pathway is compared with the scores obtained by the permutation of labels—an approach similar to that used in GSEA—and with the scores for sets composed of randomly selected genes [17].

Second, we validated the findings regarding CRCs by performing RS analysis of two publicly available, independent transcriptomic data sets. The first (V-set I) had been generated by Affymetrix HGU133A GeneChip analysis of 47 samples of human colorectal tissues (22 of normal mucosa, 25 CRCs) and is accessible through the ArrayExpress site (E-MTAB-57). The second (V-set II) was obtained with GeneChip Human Exon 1.0 ST array analysis of 20 paired CRC-normal mucosa samples [38]. The results of these validation analyses are shown in Table 5. The vast majority of pathways exhibiting CRC-related upregulation in the original N vs. CRC data set were also significantly upregulated in V-set I (73%, p-value = 1.1x10^-16, Fisher’s exact test) and V-set II (82%, p-value = 3.3x10^-16, Fisher’s exact test). Lower but still excellent degrees of overlap were also observed for the pathways found to be downregulated in CRCs compared with normal mucosa.

Figure 4 summarizes the most relevant tumor-related pathway dysregulations at different stages of transformation. Due to space constraints, only the upregulated pathways (Table 2) are discussed below; those that were downregulated (Table 3) are considered in detail in Additional file 1.

Our data suggest that the early preinvasive phase of colorectal tumorigenesis is characterized on the whole by upregulated activity of pathways involved in DNA replication and repair (i.e., KEGG BASE EXCISION REPAIR, KEGG HOMOLOGOUS RECOMBINATION, REACTOME ACTIVATION OF THE PRE-REPLICATIVE COMPLEX). These findings are consistent with recent reports [39, 40] showing that the progression of early precancerous lesions (in the colon and elsewhere) is curbed by cell cycle checkpoints that are activated by DNA replication “stress.” The precise nature of this stress is currently unclear, but it is probably initiated by increased expression of or gain-of-function mutations involving oncogenes (e.g., CCN1, KRAS, or MYC), which are known to be early events in tumorigenesis. Abnormal activation of the prereplicative complex entails upregulation of CDC6 and several minichromosome maintenance genes. (Our data and those described by Freeman et al. [41] might reflect an early step in this type of replicative stress.) This process is associated with stalling and/or collapse of replication forks and double-strand breaks, which slow or arrest the cell cycle to allow the DNA to be repaired (e.g., via homologous recombination). Activation of base excision repair suggests that DNA base oxidation or deamination may also be accelerated in early preinvasive lesions. Paradoxically, each of these repair processes can per se cause genomic instability [40, 42]. This would favor the onset and selection of loss-of-function mutations involving tumor suppressor genes, whose protein products drive the cell cycle checkpoints (e.g., TP53, which is often mutated in the later phases of colorectal tumorigenesis [1]), and the result would be unrestrained tumor progression.

In line with the above findings, two other pathways also appeared to be upregulated in our SPLs and LPLs. The BIOCARTA ARF PATHWAY emanates from the tumor suppressor proteins p16INK4a and p14ARF (both encoded by CDKN2A). It is a key sensor of oncogenic stress (e.g., the KRAS- or MYC-associated hyperproliferative signal documented in colorectal adenomas). Activation of the ARF pathway stabilizes TP53, thereby promoting effective checkpoint activity [43]. Both classes of preinvasive lesions also displayed upregulated nucleotide excision repair (KEGG NUCLEOTIDE EXCISION REPAIR), which targets UV- and carcinogen-induced DNA adducts [44]. In conditions of replicative stress, sustained activation of this pathway might be triggered by the complex (but poorly defined) mixture of putative carcinogens generated in the colorectum by host and bacterial metabolism.

DNA damage checkpoints and apoptosis appear to be efficient barriers that can restrain tumor growth for up to two decades [45]. Nonetheless, DNA replication stress and repair are naturally associated with increased cell proliferation rates in colorectal tumors. The need for DNA building blocks, before and after these barriers have been disrupted, explains why nucleotide metabolism is increased throughout tumorigenesis, as reflected by the early persistent upregulation we observed in the REACTOME PURINE RIBONUCLEOSIDE MONOPHOSPHATE BIOSYNTHESIS pathway and also by that of the KEGG PYRIMIDINE METABOLISM pathway. (The significance of the latter upregulation was borderline, so it is not listed in Table 2.)

DNA replication is followed by dramatic changes in the nucleus and its membrane during mitosis, so it was not surprising that the RAN/mitotic spindle pathway (BIOCARTA RANMS PATHWAY) was upregulated at all three stages of tumorigenesis. The small nuclear GTPase RAN (ras-related nuclear protein) directs the assembly of the mitotic spindle and later that of the nuclear envelope, whose nuclear pore complexes are necessary to re-establish nucleocytoplasmic transport [46]. Pathways involved in the G2-to-M transition of the cell cycle (e.g., REACTOME CYCLIN A1 ASSOCIATED EVENTS DURING G2 M TRANSITION) were also constantly upregulated during tumorigenesis, as was the REACTOME FORMATION OF TUBULIN FOLDING INTERMEDIATES BY CCT TRIC pathway, which is involved in protein folding mediated by the chaperonin containing the TCP1 complex. This complex plays central roles in the folding and assembly of numerous proteins [47], so the upregulated expression of several genes encoding its subunits could be easily ascribed to increased protein metabolism in tumor cells.

Of the 23 pathways selectively upregulated in CRCs, six pointed to the activation of the G1-to-S phase transition: SA REG CASCADE OF CYCLIN EXPR (Regulatory cascades of cyclin expression), BIOCARTA SKP2E2F PATHWAY, BIOCARTA CELLCYCLE PATHWAY, BIOCARTA P27 PATHWAY, REACTOME G1 PHASE, and BIOCARTA RB PATHWAY (see also first section of Results and Discussion). The simultaneous upregulation of these inter-related cell-cycle pathways in advanced colorectal tumors reflects the sustained proliferation that is a fundamental trait of cancer cells [48]. The invasive stages of tumorigenesis are thought to be characterized by mutations involving tumor suppressor genes like TP53 or PTEN, alterations that allow cancer cells to circumvent programs that limit proliferation (e.g., the cell-cycle checkpoints, which operate more efficiently in early-stage tumors, as discussed above). This high-proliferation environment is naturally associated with increased transcription and translation, as documented in our dataset by the upregulation of diverse RNA polymerase II and III functions, amino-acid transport across the plasma membrane, and tRNA aminoacylation (Table 2).

Over the past 20 years, important roles have emerged for nonepithelial cells in the progression of colorectal adenocarcinomas (and those involving other organs) [48]. Macrophages, for example, seem to play conflicting (but nonetheless crucial) roles in both tumor development and metastasis [49], and this is consistent with the marked upregulation of the BIOCARTA MONOCYTE PATHWAY observed in our CRC dataset. Monocyte differentiation gives rise to tumor-antagonizing and tumor-promoting macrophages. The latter cells promote angiogenesis, enhance tumor cell migration and invasion, and suppress antitumor immunity [49]. CRC-related upregulation of the BIOCARTA SET PATHWAY reflects the importance of another stromal contribution to colorectal carcinogenesis: granzyme release by cytotoxic T lymphocytes. These serine proteases (along with the multiprotein SET complex, whose components are encoded by genes frequently upregulated in our tumors) trigger apoptosis and are therefore regarded as mediators of antitumor immunity [50]. But they can also provoke inflammation and cleave extracellular matrix components [50]. Moreover, the SET protein is believed to act as an oncoprotein (given its apoptosis-inhibiting activity within the SET complex) and as a regulator of chromatin remodeling [51, 52]. On the basis of our transcriptomic data alone, it is difficult to discern what type of impact SET pathway activation has on colorectal cancer progression.

Finally, the REACTOME GLYCOLYSIS pathway was found to be upregulated in CRCs. Since its first description in 1924 by Otto Warburg [53], aerobic glycolysis has been considered the preferred pathway for metabolizing glucose in cancer cells (as opposed to the oxidative metabolism used by normal differentiated cells). Our data demonstrate that the switch to aerobic metabolism can be documented with transcriptional analysis of the genes encoding metabolic enzymes. Cancer cells appear to exploit aerobic glycolysis to produce the biomass needed for new cells, despite the pathway’s inefficient ATP generation [54]. Cancer cells’ need for nutrients to fuel biomass production is also reflected in the activation of other pathways mentioned above, such as those involving glucose and amino-acid transport, regulation of glucokinase, and purine biosynthesis.

Our exhaustive description of the sequence of critical molecular events characterizing the progression of colorectal tumors is based on a statistically robust analysis of transcriptomic data carried out at the level of functional molecular processes rather than individual genes or proteins. This analysis revealed specific pathways whose dysregulation might play a role in each transition of the transformation process. This is the first study in which such an approach has been used to gain further insights into colorectal tumorigenesis. Therefore, our findings provide a foundation for larger projects in which transcriptomic data will be integrated with (epi)genomic, proteomic, and metabolomic data from ongoing and future studies. They should open roads to experimental research aimed at providing more in-depth, systems-level understanding of colorectal tumorigenesis.