Comparative proteogenomic analysis of right-sided colon cancer, left-sided colon cancer and rectal cancer reveals distinct mutational profiles
Right-sided colon cancer (RCC) has worse prognosis compared to left-sided colon cancer (LCC) and rectal cancer. The reason for this difference in outcomes is not well understood. We performed comparative somatic and proteomic analyses of RCC, LCC and rectal cancers to understand the unique molecular features of each tumor sub-types. Utilizing a novel in silico clonal evolution algorithm, we identified common tumor-initiating events involving APC, KRAS and TP53 genes in RCC, LCC and rectal cancers. However, the individual role-played by each event, their order in tumor development and selection of downstream somatic alterations were distinct in all three anatomical locations. Some similarities were noted between LCC and rectal cancer. Hotspot mutation analysis identified a nonsense mutation, APC R1450* specific to RCC. In addition, we discovered new significantly mutated genes at each tumor location, Further in silico proteomic analysis, developed by our group, found distinct central or hub proteins with unique interactomes among each location. Our study revealed significant differences between RCC, LCC and rectal cancers not only at somatic but also at proteomic level that may have therapeutic relevance in these highly complex and heterogeneous tumors.
KeywordsRight-sided colon cancer Left-sided colon cancer Rectal cancers Clonal evolution Proteomics Hotspot mutations
Akaike information criterion
Bayesian information criterion
Cancer progression inference algorithm
Kernel density estimator
Left colon cancer
Memorial sloan kettering cancer center
Pipeline for cancer inference
Right colon cancer
The cancer genome atlas
The cancer proteome atlas
Often grouped as one disease, right-sided colon cancer (RCC, originating from cecum, ascending colon, hepatic flexure) and left-sided colon cancer (LCC, originating from splenic flexure, descending colon, sigmoid colon) represent clinically distinct entities with significant differences in their prognosis and treatment outcomes [1, 2]. Therefore, given their anatomic continuity, the reason for these clinical differences presumably lie at the molecular level delineated by embryological origin. Previous studies have sought to identify these differences by analyzing significantly mutated genes and RNA expression [3, 4]. However, molecular differences including significant specific amino acid alterations (hot spots), proteomic differences and order of mutations in clonal evolution of these tumors have not been studied. We used somatic and proteomic data of colorectal cancers from The Cancer Genome Atlas (TCGA) [4, 5], Memorial Sloan Kettering Cancer Center (MSKCC)  and The Cancer Proteome Atlas (TCPA)  to study proteogenomic differences in these tumors (See Additional files 1 and 2).
Results and discussion
Clonal evolution trajectories
In RCC (n = 135; Fig. 1a; Additional file 4: Table S1), APC somatic mutations and TP53 somatic mutations were independent events. APC somatic mutations ‘selected’ for KRAS mutations or amplifications. APC somatic mutations also ‘selected’ for SMAD4 somatic mutations or deletions, BRAF mutations and amplification. KRAS and BRAF showed mutual exclusivity. Interestingly, alterations in FBWX7, TCF7L2, and SMAD2 clustered in RCC tumors harboring APC and PIK3CA mutations. With respect to TP53, alterations in this location were associated with CTNNB1, MYC or/and BRCA2 mutations.
In LCC (n = 143; Fig. 1b; Additional file 4: Table S2), KRAS somatic mutations ‘selected’ for BRCA2 amplification, PTEN deletions or somatic mutations, PIK3CA somatic mutations, IGF2 amplification or somatic mutations and ERBB2 amplification or somatic mutations. Unlike RCC, alterations in PIK3CA were a late event in LCC and IGF2 amplification via CTNNB1. APC seemed to ‘select’ for TP53, but this did not reach statistical significance (p = 0.06). Similarly, APC somatic mutations ‘selected’ for BRCA2 mutations and TCF7L2 somatic mutations or deletions, but this association also did not reach statistical significance (p = 0.3 and p = 0.2, respectively).
In rectal cancers (n = 76; Fig. 1c; Additional file 4: Table S3), key initial mutations are split between TP53 and KRAS. TP53 ‘selects’ for MYC amplification, SMAD4 deletion and BRCA2 somatic mutation or amplification. KRAS ‘selects’ for PTEN deletion or somatic mutations, PIK3CA somatic mutations, IGF2 amplification and ERBB2 amplification or somatic mutations. Among rectal cancer patients with AURKA mutations there is clustering of NRAS amplifications.
Our model shows significant differences in the mutational profiles of genes between RCC and LCC; the early common somatic gene mutations are associated with the ‘selection’ of different subsequent genomic events in RCC compared to LCC. Our results suggest that although LCC and rectal cancers have some similarities in the tumor progression model wherein KRAS ‘selected’ for several genes in common (such as PIK3CA, IGF2, and ERBB2 alterations), significant differences were also noted between these two sites. Taken altogether, our results show non-adherence to the established Vogelstein linear progression model of colorectal cancer progression from normal mucosa to adenoma to carcinoma . Further, our data suggest that RCC, LCC and rectal cancers have distinct mutational behavior in the context of their evolutionary trajectories, mutational timing during cancer development and progression. However, initial events such as mutation in the gatekeeper gene, APC, appear to be similar in colorectal cancers irrespective of location.
Mutation hotspot analysis
Several hub proteins that might have a major role in RCC were identified: BAP1 (tumor suppressor gene) CASP8 (apoptosis) PCNA (DNA repair) NRAS (RTK-RAS pathway) PEA15 (apoptosis and RET signaling) DVL3 (cell proliferation and ATM-dependent DNA damage response) and PDPK1 (growth regulation) (Fig. 3a). The potentially significant hub proteins in LCC were: BAP1, BAK1 (apoptosis and prognostic in breast cancer) COG3 (protein glycosylation/golgi function) CCNB1 (mitosis and prognosis in breast cancer) SRSF1 (RNA splicing and prognosis in small cell lung cancer) DIRAS3 (tumor suppressor gene) and LCK (resistance to apoptosis) (Fig. 3b). Hub proteins unique to rectal cancers were: IGF1R (proliferation, invasion, migration), TSC1 (cell growth) BRCA2 (DNA repair) and COPS5 (multiple pathways) (Fig. 3c).
BAP1 was found to have a prominent role in both RCC and LCC. Although there are several conserved interactions, the BAP1 interactome of LCC diverges from that of RCC. Among the conserved interacting proteins are: BRD4, ADAR, GAB2, SLC1A5, EIF4G1, ERCC5 and TP53BP1, BRD4, ADAR, MSH6, FOXM1 and XRCC5. Specific to LCC, BAP1 showed interactions with ERCC1, PRKCA, GATA6, JAK2, RAD51, TSC1, RSC1, NOTCH1, BCL2, KIT, PRKCD, CDH2, ARID1A, ASNS, SQSTM1 and DVL3. Specific to RCC, BAP1 was noted to interact with CDH1, MAPK14, MRE11A, MET, YAP1, STK11, ERBB3, PIK3CA, PXN, CHEK1, CTNNB1, STAT5A, EEF2K, G6PD, COG3, RBM15, BCL2A1, SYK, RELA and ANXA1.
Our results suggest BAP1 may have an essential role in carcinogenesis of colon cancer with conserved as well as divergent evolutionary interactions with other proteins in RCC and LCC that are largely absent in rectal cancers.
A somewhat surprising observation from this analysis is that the protein hubs and their interactomes are distinct for each of the anatomically defined tumor sites examined. Further, these protein signatures are not necessarily concordant with the somatic tumor profiles. Identifying alterations in tumor DNA and RNA have been of paramount importance. Clarifying post-transcriptional events and protein-protein interactions will also be highly relevant to understanding the variations in tumor biology and clinical behavior of these tumors. Prospective studies are needed to validate our findings and their implications in the clinical outcomes.
Part of A.H.’s time was supported by a Merit Review Award (I01 BX000545), Medical Research Service, Department of Veterans Affairs.
Availability of data and materials
TCGA somatic mutation data for colorectal cancers can be obtained from the GDC legacy archive and Broad GDAC Firehose. Proteomic data can be downloaded from The Cancer Proteome Atlas (TCPA). The datasets used and analyzed in the current study are also available from the corresponding author in response to reasonable requests.
RI and AM contributed to the design of study, involved in acquisition, analysis and interpretation of data; involved in drafting the manuscript and revising it critically for important intellectual content. ZA, OMT, CE, AK, PC, JC, KK, NM, BD involved in data acquisition, analysis and interpretation of data and involved in drafting the manuscript. LSC, SR, RM, BE, TP, AH, JS were involved in revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
Ashiq Masood Advisory board and speaker Bureau Bristol-Myers Squibb and Boehringer Ingelheim, Honorarium Biocept. Janakiraman Subramanian Advisory board - Astra Zeneca, Pfizer, Boehringer Ingelheim, Alexion, Paradigm, Bristol-Myers Squibb Speakers Bureau - Astra Zeneca, Boehringer Ingelheim, Lilly Research Support - Biocept and Paradigm. Arif Hussain Advisory board – Novartis, Bayer, Astra Zeneca, Consultant – Bristol-Myers-Squibb. All other authors declare that they have no competing interest.
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