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Omics Integration Analysis Unravel the Landscape of Driving Mechanisms of

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DOI: 10.31557/APJCP.2020.21.12.3539

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 DOI:10.31557/APJCP.2020.21.12.3539
Omics Integration Analysis Unravel the Landscape of Driving Mechanisms of Colorectal Cancer

RESEARCH ARTICLE Editorial Process: Submission:06/22/2020 Acceptance:11/30/2020

Omics Integration Analysis Unravel the Landscape of Driving

Mechanisms of Colorectal Cancer
Fatemeh Nikmanesh1,2,3, Shamim Sarhadi1,4, Mehdi Dadashpour1,Yazdan Asgari3,
Nosratollah Zarghami1,5*
Abstract
Colorectal cancer (CRC) is one of the most malignant cancers and results in a substantial rate of morbidity and
mortality. Diagnosis of this malignancy in early stages increases the chance of effective treatment. High-throughput
data analyses reveal omics signatures and also provide the possibility of developing computational models for early
detection of this disease. Such models would be able to use as complementary tools for early detection of different types
of cancers including CRC. In this study, using gene expression data, the Flux balance analysis (FBA) applied to decode
metabolic fluxes in cancer and normal cells. Moreover, transcriptome and genome analyses revealed driver agents of
CRC in a biological network scheme. By applying comprehensive publicly available data from TCGA, different aspect
of CRC regulome including the regulatory effect of gene expression, methylation, microRNA, copy number aberration
and point mutation profile over protein levels investigated and the results provide a regulatory picture underlying CRC.
Compiling omics profiles indicated snapshots of changes in different omics levels and flux rate of CRC. In conclusion,
considering obtained CRC signatures and their role in biological operating systems of cells, the results suggest reliable
driver regulatory modules that could potentially serve as biomarkers and therapeutic targets and furthermore expand
our understanding of driving mechanisms of this disease.

Keywords: Colorectal cancer- flux balance analysis- omics integration- regulome

Asian Pac J Cancer Prev, 21 (12), 3539-3549

Introduction Antigen (CEA) test, but their sensitivity is not desirable

(Zou et al., 2015; Sheervalilou et al., 2016; Maasomi
Cancer is the leading cause of death worldwide (Rasouli et al., 2017; Sadeghzadeh et al., 2017). There are also
et al., 2020). There are several type of cancer, such as lung, some common methods to screen susceptible individuals
stomach, colorectal, liver, and breast (Sheervalilou et al., such as endoscopic, histopathological examination of
2016; Maasomi et al., 2017; Sadeghzadeh et al., 2017). biopsies, and surgically removed specimens that are
Among the various cancers, gastrointestinal cancers occur undesirable in terms of sensitivity and also are painful
mainly in men and women living in developing countries which bring complications for patients (Zheng et al.,
(Lotfi-Attari et al., 2017, Mohammadian et al., 2016a; 2013). Generating biological high-throughput data and
Mohammadian et al., 2016b). optimization of computational methods obviate obstacles
Colorectal cancer (CRC) is the third most common to study biological systems using systems biology
cancer among men and women in the US and affects as a multidisciplinary approach (Ideker et al., 2001).
all racial groups (Boyle and Ferlay, 2005; Siegel et al., While omics puzzle is being completed in parallel with
2016). The reports indicated 50,260 deaths during 2017 evolving data integration and computational technics
(Siegel et al., 2016; Siegel et al., 2017). Similar to other (Machado and Herrgård, 2014), numerous studies have
types of cancers, CRC could be prevented and treated tried to find biomarkers and enhanced the chance of early
with higher probability if detection occurs at the early detection to improve diagnosis and prognosis of patients
stage of tumor initiation (Levin et al., 2008). There (Levin et al., 2008; Huang et al., 2010; Vatandoost et
are several common tests to detect this malignancy al., 2016). Since in an up to bottom approach in omics
including colonoscopy and serum Carcinoembryonic levels, metabolome perturbation is the final change,

1
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2Department of Medical Biotechnology, Faculty of
Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. 3Iranian Blood Transfusion Organization-Research
Center, Iranian Blood Transfusion Organization, IBTO blg., Hemmat Exp. Way, Teheran, Iran. 4Department of Medical
Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. 5Department
of Clinical Biochemistry and Laboratory Medicine, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
*For Correspondence: [email protected]. Fatemeh Nikmanesh and Shamim Sarhadi have equal contribution in this study.
Asian Pacific Journal of Cancer Prevention, Vol 21 3539
Fatemeh Nikmanesh et al

it could provide a clearer vision of cell abnormalities E-Flux method (Colijn et al., 2009). E-Flux approach
status during initiation and progression of disease due uses pre-processed gene expression data, finds metabolic
to alteration in genomic, post-genomic, transcriptomic, genes through them, and considers the corresponding
and proteomic profiles (O’Connell, 2012; Zhang et expression values. Next, all values were replaced into the
al., 2014). Besides, it has been shown that metabolic gene-reaction association matrix available in the human
perturbation is one of the common characteristics of metabolic model. The new gene-reaction association
cancerous cells. Numerous studies have tried to develop matrix now includes some changed elements due to
metabolic models by integrating gene expression data mathematical operators and some unchanged elements
in the metabolic framework of target cells and design because they did not have any metabolic genes. Then, the
predictive cancerous metabolic models which could help gene-reaction association matrix values were rescaled to
to understand differences between tumor and normal [0,1], multiplied by the current upper bound values of the
samples (Hagland and Søreide, 2015; Mika et al., 2017). model (values for the human metabolic model), and set
Exploring such alterations in genome, transcriptome, and as the new upper bound values of the model. The lower
metabolome could pave the way of finding biomarkers and bound values were negative values of the new upper bound
understanding metabolic alterations that may be useful for values (for reversible reactions). Therefore, this method
diagnosis at the early stage of cancer. Study of underlying constrains the upper and lower bounds of each reaction
molecular mechanisms of CRC has conducted in many according to its corresponding gene expression level. We
research by systems biology approach (Madhavan et al., wrote a Mathematica script to apply E-Flux algorithm to
2013). Accumulating changes in CNA, point mutations, the human metabolic model. So, we have reconstructed
gene expression and flux balance alteration of metabolic normal and cancer colorectal metabolic models. The
drivers indicated to promote the cancer initiation and biomass and the medium compositions (RPMI-1640)
progression (Corti et al., 2019). In this study, we tried added to the models are shown in the supplementary file 1.
to unravel the landscape of metabolic, genomic and All normal and cancer metabolic models are also available
transcriptomic alterations and their integrated role in in the supplementary file 2 as MATLAB structure files.
fine-tuning of regulome for initiation and progression of
CRC by systems biology approach and using the huge Flux Balance Analysis
numbers of publicly available data. Since there have Flux Balance Analysis (FBA) considers appropriate
been very few studies that simultaneously considered constraints whereas the system is in its steady-state.
changes in different omics levels for CRC, in this study In FBA, a metabolic network is considered as a
we applied data from different sources to obtain signatures stoichiometric set of equations (in a matrix format
of each omics levels that could be applied to understand including the stoichiometric (S) and the flux (V) matrices
the molecular mechanisms of CRC. (Masoudi-Nejad and Asgari, 2015). Since a higher number
of reactions in comparison to metabolites is a common
Materials and Methods feature of most metabolic networks, this property prevents
the system of linear equations to be solved analytically.
Data collection One approach is using linear programming in which it
Gene expression microarray data related to colorectal tries to solve a system of equations in association with
cancer were downloaded from the Gene Expression minimization/maximization of an objective function as
Omnibus (GEO) database. Microarray data platform follows:
was Affymetrix Human Genome U133 Plus 2.0 Array
(GPL570) for both datasets. To analyzing copy number min/max: cT.v
aberration and point mutations related to CRC, next subject to: S.v = 0 a < v < b
generation sequencing data retrieved through Cbioportal
(Cerami et al., 2012; Gao et al., 2013). Moreover, TCGA where cT is a transposed vector of stoichiometric
data including gene expression, copy number alteration, coefficients of metabolites incorporating with the
point mutation, microRNA, methylation, and protein objective function, and v is a vector of fluxes which
profiles used to portrait regulome signatures of CRC. will be determined. Vectors a and b are also lower and
Microarray raw data were preprocessed using MATLAB upper bounds of all reaction, respectively. The solution
software (version R2015b). First, a preprocessing of gene of the metabolic fluxes is underdetermined when a
expression raw data has been performed by transferring system is unconstrained. Applying additional constraints
all gene expression values to log2 scale, and by doing (eg. a < v < b) would decrease the solution space. In such
normalization step (using quantilenorm command in a condition, one could obtain the optimal set of the flux
MATLAB based on Median). This procedure was repeated distribution while an objective function is optimized.
for both data types (normal and cancer). Therefore, FBA would turn into a linear programming
(LP) problem. For this study, we used the COBRA
Metabolic Model Reconstruction toolbox (Constraints Based Reconstruction and Analysis)
We have used the corrected human metabolic model for evaluating the metabolic models by maximization
(Shlomi et al., 2011) which includes 2766 metabolites, of the biomass equation (Feist and Palsson, 2010). The
3748 reactions, and 1905 genes to work with. Gene glpk solver was used for linear programming problems.
expression values corresponding to metabolic genes For FBA, running the optimizeCbModel function builds
have been mapped into the human metabolic model using four main output structures: f, x, w, and y, where f is the
3540 Asian Pacific Journal of Cancer Prevention, Vol 21
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Omics Integration Analysis Unravel the Landscape of Driving Mechanisms of Colorectal Cancer
objective value, x includes reaction fluxes, w is a vector transcriptomic levels of CRC, signatures corresponding
of reduced costs, and y is a vector of shadow prices. to DEGs, point mutations and CNAs were constructed by
Every reaction belongs to a known metabolic subsystem. data retrieved from the DifferentialNet database (Basha et
For example, there are 99 subsystems presented in the al., 2017). The DEGs network created by DEGs with signal
human metabolic model according to metabolic pathway to noise ratio (SNR) more than 0.5. Also, in case of point
classification (Lehninger et al., 2005). Subsystems mutation and CNA signatures frequency of 5% defined
information are available through the vector called as a cut-off to select the genes for network construction.
subSystems in the MATLAB model structure. All FBA Construction and enrichment analyses of networks were
results related to normal and cancer metabolic models are done using networkanalyst (Xia et al., 2015).
available in the supplementary file3.
Regulome analyses of CRC
Differentially expression gene and gene enrichment To this end, we retrieved 621 samples related to CRC
analysis by GSEA from TCGA through Regulome Explorer (Uddin et al.,
To find differentially expressed genes (DEGs) in CRC 2011; Kannan et al., 2015). To unfold the regulatory
cells in comparison with the normal cells, gene expression effects of different omics levels on protein level as a
microarray data sets preprocessed by FRMA package final manifest of central dogma process, we investigated
(McCall et al., 2010) in R and then limma (Smyth, 2005) the correlation between methylation, microRNA, point
was used to develop a linear model for finding DEGs. mutation, copy number alteration and gene expression
Obtained DEGs for each dataset combined by fisher profiles with protein levels in CRC.
method. To find pathways that over and under-expressed
in CRC samples mentioned datasets merged by COMBAT Results
method to remove study bias. In the next step, a dataset
consisting of DEGs and their expression values and The total number of gene expression microarray
Reactom geneset fed into the GSEA (Subramanian et samples for normal and cancer cells were 56 and 67,
al., 2007). To run the software, permutation type set to respectively (Sabates-Bellver et al., 2007; Uddin et al.,
gene-set and the number of permutations set to 1000. 2011). Moreover, 1596 samples with point mutation
After finding enriched pathways, 20 top-ranked pathways data, 1354 samples with CNA data and 621 samples that
selected for leading edge analysis that clusters genes applied in regulome analysis retrieved from cBioportal
according to the number of genes involved in common and TCGA (Table 1).
gene-sets.
Flux balance analysis reveals metabolic subsystems in
Network analysis of genomic and transcriptomic CRC
signatures of CRC Impacted subsystems in colorectal cancer showed in
To illustrate tissue-specific networks in genomic and Table 2. As it has been demonstrated in the supplementary

Figure 1. Differentially Expressed Genes in CRC Cells. Heatmap of 100 top ranked gene with altered expression
Asian Pacific Journal of Cancer Prevention, Vol 21 3541
Fatemeh Nikmanesh et al

Figure 2. Point Mutation and Copy Number Aberration Hallmarks of CRC. (a) Heatmap of point mutations and, (b)
CNAs show the genomic signatures related to colorectal cancer

file 3 in details, in the cancer metabolic model, the uridylyl transferase reactions with the values of -95 and
total number of 503 reactions had an increment in their -80, respectively.
flux values whereas 560 reactions had lower flux rates However, FBA results demonstrated some subsystems
compared to normal metabolic models. Note that we in cancerous model with an increment in their reactions.
ignored values smaller than 1×10-10 in the results. For example, in the “Nucleotides” and “Pyruvate
According to FBA results the highest flux rate drop Metabolism” subsystems, there were increment with
was in retinol dehydrogenase reaction in the “Vitamin the value of 280 and 142, respectively which were
A Metabolism” subsystem with the value of -1875. The due to nucleoside-diphosphate kinase and L-lactate
notable flux value of -1398 was calculated for bicarbonate dehydrogenase pathways. Also, Adenosine deaminase as
transport which is part of the” Transport Extracellular”
subsystem. The results showed that in the “Pyrimidine
Table 1. Summary of CRC Data Used in This Study
Catabolism” subsystem, Cytosine deaminase reaction
flux decreased in colorectal cancerous cells with the Gene expression microarray data
value of -529. Also, Glutathione peroxidase reaction in Source Number of samples
the “Glutathione Metabolism” subsystem represented a GSE8671 64 (32 normal, 32 cancer)
reduction in the colorectal cancer model with the value GSE23878 59 (24 normal, 35 cancer)
of -439. There was also a decrement in the “Transport Copy number aberration data
Mitochondrial” subsystem in which the value of ADP/
Source Number of samples
ATP transporter reaction was -382.
There was a reduction in the “Fatty acid elongation” Cbioportal 1354
subsystem with the value of -147, which was calculated Point mutation data
for palmitoyl-CoA desaturase reaction. In the “Glutamate Source Number of samples
Metabolism” subsystem, there was a reduction in glutamine Cbioportal 1596
synthetase reaction with the value of -129. Moreover, two
Data applied for regulome analyses
other subsystems (“Oxidative Phosphorylation” and
“Galactose Metabolism”) decreased in colorectal cancer Source Number of samples
model for ATP synthase and UTP-glucose-1-phosphate TCGA 621

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Omics Integration Analysis Unravel the Landscape of Driving Mechanisms of Colorectal Cancer
a reaction in the “Purine Catabolism” subsystem showed pathways were related to cell cycle and involved pathways
increment in the colorectal cancer model with the value with cell growth that represent as up-regulated pathways
of 56. In” Glycolysis/Gluconeogenesis” subsystem, the and conversely some pathways related to the immune
highest flux value was for glucose-6-phosphate isomerase system, rhodopsin-like receptors (class A/1) that serve
pathway with the value of about 53. Moreover, our results as components of hormones, light, and neurotransmitter
presented that hyaluronan synthase reaction with the value receptors are examples of down-regulated pathways in
of 21 was increased in the “Hyaluronan Metabolism” CRC cells. Figure 3 and Table 3 represent top enriched
subsystem. pathways. All enriched pathways are also available in the
supplementary file 4 and 5.
Differentially expression genes, point mutations and CNAs
Differentially expression gene analysis shows the CRC Tissue-specific network analysis
gene signature including 396 genes with signal/noise ratio Analysis of three different networks constructed for
more than 0.5 and FDR<0.05. Figure 1 shows the heatmap DEG, point mutation and CNA signatures show different
of 100 top-ranked genes that altered in CRC cells. driver nodes related to CRC. The results show that finding
Genomic data of 1,596 samples with point mutation driver nodes should be investigated in different level
data and 1,354 samples with CNA data retrieved from of omics network to obtain high reliable centrality for
eight studies through cBioportal to find point mutation hub nodes of CRC. The result of PPIN of DEGs shows
and CNA signatures corresponding to colorectal cancer. although this network illustrates a part of affected driver
Analyzing the retrieved data indicated 7 genes with CNA nodes of CRC, some CRC hallmarks cannot be found in
(gain or lose area) and 37 genes with point mutations. this network. For example, in PPIN constructed by DEG,
Figure 2 shows the heatmaps of CNAs and point mutations the P53 is not indicated as a driver node, while in point
related to CRC. mutation network it was the most important node in the
network. As a result, to find the CRC reliable hub nodes,
Pathway enrichment analysis results applying comprehensive information from all of the omics
Enrichment analysis results revealed pathways that levels is important. Figure 4 shows affected PPI networks
contributed to CRC phenotype. Some of the most impacted for DEG, point mutation and CNA signatures. Also, the

Figure 3. Gene Set Enrichment Analysis (GSEA) Results Show Impacted Pathway in CRC Cells. (a), Leading edge
analysis results of 10 top overexpressed and 10 tops under expressed pathways that represented by a clustergram. (b),
shows each enriched gene and the number of subsets in which it appears. (c), graphical view of the enrichment score
of top three over and under expressed pathways that represented by GSEA plot. Peak of GSEA plot shows enrichment
score for the gene set (FDR<0.05).
Asian Pacific Journal of Cancer Prevention, Vol 21 3543
Fatemeh Nikmanesh et al

Figure 4. Constructed Networks of Genomic and Transcriptomic Signatures of CRC. (a) Network of DEGs that
illustrate transcriptomic driver nodes in CRC. (b and c) PPIN of CRC that indicated nodes that affected by point
mutations and CNA profile of CRC.

GO biological process enrichment analysis of networks a positive correlation of hsa-miR-155-5p and CASP7 as
created by genomic and transcriptomic signatures that the most important microRNAs regulatory modules in
mapped on tissue-specific protein-protein interaction CRC regulome, (Figure 6a and supplementary file 6). Also,
network provides a perspective from all biological process considering correlation results between gene expression
underlying CRC (Figure 5). The most important hub nodes and protein levels show a positive correlation between
in each network presented in Table 4. IGFBP2, BCL2L1, INPP4B and CCNE1 gene expression
with their corresponding protein levels. Furthermore,
Regulome analyses results COL10A1 gene expression level has a positive correlation
Role of microRNA profile on protein level in with FN1 protein level, (Figure 6b and supplementary file
CRC screened and indicated negative correlation of 7). The regulatory effect analysis of methylation profile
hsa-miR-148a-3p and hsa-miR-192-5p with FN1, a in CRC shows a positive correlation between methylation
positive correlation of hsa-miR-223-3p and CHEK1, and in 5pUTR of RBM47, DGKA, ALKBH7 and TRAK1,

Table 2. Altered Reactions in the Subsystems for Colorectal Cancerous Model Compared to the Normal Model
Altered Subsystem Altered Reaction Alteration type (Tumor Model)
Vitamin A Metabolism Retinol Dehydrogenase Decreased
Transport Extracellular Bicarbonate Transport Decreased
Pyrimidine Catabolism Cytosine Deaminase Decreased
Glutathione Metabolism Glutathione Peroxidase Decreased
Transport Mitochondrial ADP/ATP Transporter Decreased
Fatty Acid Elongation Fatty Acyl-CoA Desaturase Decreased
Glutamate Metabolism Glutamine Synthetase Decreased
Oxidative Phosphorylation ATP Synthase Decreased
Galactose Metabolism UTP-Glucose-1-Phosphate Uridylyltransferase Decreased
Nucleotides Metabolism Nucleoside-Diphosphate Kinase Increased
Pyruvate Metabolism L-Lactate Dehydrogenase Increased
Purine Metabolism Adenosine Deaminase Increased
Glycolysis/Gluconeogenesis Glucose-6-Phosphate Isomerase Increased
Hyaluronan Metabolism Hyaluronan Synthase Increased

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Omics Integration Analysis Unravel the Landscape of Driving Mechanisms of Colorectal Cancer
and YAP1 protein level, negative correlation between
LOC100130987 methylation and CTNNB1 protein level
and positive correlation of TMEM156 methylation with
FN1 protein level (Figure 6c and supplementary file 8).
The results of correlation between CNAs and protein
level in the regulome framework suggest that CNAs
have a low level of impact on the regulatory operating
system of tumor cells in CRC to regulate protein level.
But the most important of these regulatory interactions
indicated for a positive correlation between chr20q12,
20q11, chr20q11 with BCL2L1 protein level as the most
important correlations (figure 6d and supplementary file 9).
Finally, considering the correlation results between point
mutations and protein levels in CRC, TP53-missense has a
positive correlation with TP53 level, PTEN-All mutations
have a positive correlation with WWTR1, ACVR2A-all
mutations have a positive correlation with CASP7, and
ACVR2A-all mutations have a positive correlation with
RAD51, (Figure 6e and supplementary file 10).

Discussion
Identification of altered agents in omics levels that are
causally implicated in malignancy has been an overriding
goal in understanding the cancer phenomenon. The
growing body of high-throughput data coupled with the
development of analyzing tools provided an opportunity
for deciphering malignancy drivers and signaling
pathways involved in tumorigenesis (Gao et al., 2013).
Application of flux balance analysis in cancer is rapidly
developing into a considerable scientific field and has been
noticed as a helpful method for cancer diagnosis (Schulze
and Harris, 2012). Indeed, the feasibility of data mining
and exploration of subsystem fluxes provided a reliable
explanation of metabolism alteration during cancer
development (Schulze and Harris, 2012; Uhlen et al.,
2017). In this study, we proposed a new metabolic model
which is useful for cancer diagnosis and treatment. The
results showed miscellaneous metabolic pathways in
which flux changes were associated with the mechanism
of cancer. According to FBA results, the highest reduction
Figure 5. Clustergram of Network Enrichment Analysis. in flux levels for the CRC samples was in the “Extracellular
GO (BP) enriched terms by DEGs, point mutations and
CNAs networks. This figure provides a snapshot from all Transport” subsystem. Such decrement has been discussed
biological process that affected in CRC. by Netti et al., (2000) which is caused due to cellular

Table 3. The most Impacted Pathways that Enriched by Reactom Geneset

Under-expressed pathways Over expressed pathways
NAME FDR (q-val) NAME FDR q-val
Reactome_immunoregulatory_interactions_between_a_ 0 Reactome_dna_replication 0
lymphoid_and_a_non_lymphoid_cell
Reactome_g_alpha_s_signalling_events 0.000434 Reactome_mitotic_m_m_g1_phases 0
Reactome_cgmp_effects 0.002634 Reactome_cell_cycle_mitotic 0
Reactome_class_a1_rhodopsin_like_receptors 0.002183 Reactome_cell_cycle 0
Reactome_gpcr_downstream_signaling 0.001746 Reactome_g2_m_checkpoints 0
Reactome_nitric_oxide_stimulates_guanylate_cyclase 0.001455 Reactome_mitotic_prometaphase 0
Reactome_generation_of_second_messenger_molecules 0.00283 Reactome_activation_of_atr_in_response_to_replication_stress 0
Reactome_tcr_signaling 0.003662 Reactome_dna_strand_elongation 0
Reactome_gpcr_ligand_binding 0.003533 Reactome_s_phase 0
Reactome_glucagon_type_ligand_receptors 0.003693 Reactome_activation_of_the_pre_replicative_complex 0

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Fatemeh Nikmanesh et al

Table 4. The Most Important Hub Nodes in Network Created by Transcriptomic and Genomic Signatures
DEGs network Point mutation network CNA network
Label Degree Betweenness Label Degree Betweenness Label Degree Betweenness
ITGA4 402 819295.4 TP53 556 596332.47 MYC 849 492887.81
TRIP13 75 146383.85 EP300 293 252816.94 BCL2L1 66 54377.5
PTP4A3 73 138377.42 CREBBP 196 127599.6 DNMT3B 35 28506.19
CCNB1 57 127533.74 CTNNB1 194 182161.38 RBFOX1 29 25624.5
NR3C1 54 159978.78 SMARCA4 100 93783.27 GNAS 28 21732.5
PTN 49 79172.5 SMAD4 100 78005.5 ASXL1 11 5978
THRB 46 89620.76 APC 89 96969.15 FLT3 8 5973.5
EPB41L3 45 94762.82 FBXW7 82 85781.6 POLR2A 3 6998.88
PCK1 45 60748.27 MTOR 68 59152.88 EZH2 3 1577.2

transfer reductions in the extracellular matrix (ECM). effects in cells particularly are related to ECM. Some of
There is a controlling mechanism for molecular traffics these functional components in cells promote tumor
in ECM. Barriers in molecular transport in ECM play an progression (Mott and Werb, 2004). Also, flux value for
important role in tumor cells viability, for example, to the “Vitamin A Metabolism” reduced in colorectal cancer
prevent penetration of some therapeutic agents (Netti et cells. This result was in concordance with a previous study
al., 2000). Moreover, initiation of pro- or anti-apoptotic indicated the decrease of retinoic acid production

Figure 6. Regulome Scheme of CRC. This figure represents correlation of protein levels with the (a) microRNAs
expression, (b) gene expression, (c) methylations, (d) copy number aberrations and (e) somatic point mutations in
CRC. Yellow lines show top regulome interactions.
3546 Asian Pacific Journal of Cancer Prevention, Vol 21
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Omics Integration Analysis Unravel the Landscape of Driving Mechanisms of Colorectal Cancer
consequently results in tumor immune evasion in roles in PPINs were highlighted in the enrichment results
colorectal cancer (Huynh et al., 2013). In addition to the of networks (Figure 5). The obtained PPINs from mapping
FBA result that shows the reduction in the production of transcriptomic and genomic signatures also showed
retinoic acid (which leads to tumor immune evasion), different functional network modules (Figures 4 and 5).
enrichment analysis of CRC’s DEG signature shows the These different cancerous network modules promote
“immune response” and “Class_A1_Rhedopsin_like_ cancer in the framework of interconnected omics
receptors” as examples of under-expressed pathways that interactions. The goal of pan-omics in cancer studies is to
were in concordance with the FBA results. Moreover, FBA identify driver agents that may be useful for finding
results showed the Nucleotides Metabolism as an activated diagnostic, prognostic and other markers related to
subsystem that is in concordance with the GSEA pathway different outcomes of disease. Screening in the result of
analysis results (including activation of cell growth and regulome section of this study showed correlation between
involved pathways), (Table 2, 3 and Figure 3). Major PTEN mutation and WWTR1 protein level (figure 6e and
alterations in energy metabolism occurred in cancerous supplementary file 10). This result also observed in a
cells. Mitochondria is the center for these changes in which previous study that indicated the WWTR1 protein level
the Warburg e-ect provides pyruvate for fermentation and was high in cancer cells with up-regulated PI3K signaling
oxidative phosphorylation process (Vander Heiden et al., in PTEN mutant tumor cells (Huang et al., 2012). A
2009). Also, amino acid and lipid biosynthesis are the literature search was done to find a correlation between
other biochemical pathways run through mitochondria WWTR1 mutation and CASP7 protein level that indicated
(Rizzuto et al., 2012; Andalib et al., 2013). It is expected in our results (Figure 6e and supp file 10), but we could
when cells become cancerous, changes occur in not find a correlation between WWTR1mutation and
biochemical transportation of cancer cells (Lytovchenko CASP7 protein level in previous studies. As reported
and Kunji, 2017). It has been previously confirmed that previously, ACVR2A harboring genomic instability in
because of the increment in glycolysis and lactate CRC cells and was correlated with RAD51 that involves
pathways in tumors, the suitable condition would be in DNA damage repair process (Brough et al., 2012; Kim
achieved for tumor growth (Solaini et al., 2011). In this et al., 2013). Subsequently, the correlation between
study, the FBA results explicitly represented a high level ACVR2A mutations and RAD51 protein level indicated
of flux response in the “Glycolysis” subsystem as it was in regulome results of this study. Analyzing the methylation
expected. Previous studies also demonstrated lipid effect on regulome network of CRC revealed the
metabolism alterations in colon tumors (Keshk et al., significant regulatory effect of RBM47 methylation over
2014), and fatty acid elongation may serve as a therapeutic YAP1 protein level. Since YAP1 is a key component of
and detection marker in colorectal cancer (Yan et al., Epithelial–Mesenchymal Transition (EMT) regulation
2016). FBA results showed a reduction in the “Fatty acid (Fisher et al., 1994), the results suggest that RBM47
elongation” subsystem. Moreover, former studies contributes to EMT through regulating YAP1 level in CRC
underscored the role of hyaluronidases in colorectal that may trigger metastasis process (De Craene and Berx,
cancer. Indeed, tissue distribution of hyaluronidase 2013). The results highlighted YAP1 as a key node in
reaction and the concept of certain isoforms in each tumor methylation regulatory network that affected by
stages indicated a new evidence of involving in the methylation of DGKA, TRAK1, ALKBH7, TM4SF4,
mechanism of colorectal cancer progression (Bouga et al., MCOLN3, MDM1 and a long list of genes that represented
2010). Analyzing the genomic data including point in supplementary file 8. Evaluating the regulatory effect
mutation and CNA data also revealed signatures related of micro-RNA profile of CRC on protein levels indicated
to CRC that could not be found by analyzing single omics correlation of hsa-miR-148a-3p with FN1, correlation of
level. As an example, P53 that plays a role as a cancer hsa-miR-155-5p with CASP7 and correlation of hsa-miR-
hallmark was not detected in transcriptome analysis. 143-3p with PEA15 (Figure 6 and supplementary File 6).
Hence, the findings of this study show that it is necessary This result was in concordance with the previous study
to study cancer as a systemic and complicated disease that profiled microRNA expression and regulatory effect
through all omics levels simultaneously. Figures 1, 2 and of top-ranked microRNA in CRC (Kara et al., 2015).
4 and Table 4 show different signatures in gene expression, While data mining of one specific omics data is a
point mutation and copy number alteration profile of CRC. promising approach to find predictive signatures of
Among these signatures, Adenomatous Polyposis Coli different biological states, the promise will be limited if
(APC) burdens high frequency of inactivating mutations findings are not considered the interaction of omics levels
(76%) in 1,596 analyzed samples (these mutations mostly in an interconnected biological network. Findings of this
included truncated mutations and deep deletions) and it study likely would help the future studies to understand
serves as an early driver for CRC (Genomic map in Figure molecular phenomena of colorectal cancer.
2). One of the most important driver pathways in CRC is In conclusion, considering paradigm shift of cancer
an abnormality of the WNT signaling pathway which studies that focus on the personalized study of cancers
extensively studied in relation with APC mutations which ignore the heterogeneity of tumors and also paucity
(Müller et al., 2016). Moreover, the driver role of the APC of multi-omics studies of colorectal cancer, this study
represented in the network which indicated as a hub node directed to provide a reliable population-based perspective
in different functional modules. Moreover, KRAS, TP53, from molecular mechanisms underlying CRC. These
and SMAD4 are the other hallmarks of CRC that serve results obtained from analysis of huge and different types
important roles in progression of CRC. Their functional of data to uncover alterations that are difficult to explain
Asian Pacific Journal of Cancer Prevention, Vol 21 3547
Fatemeh Nikmanesh et al

by analysis of one omics level in CRC. Vitamin A Metabolism in Colorectal Cancer Stromal Cells:
Role in Tumor Immune Evasion. Gastroenterology, 144,
S-71.
Acknowledgments Ideker T, Galitski T, Hood L (2001). A new approach to decoding
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The authors thank the Faculty of Advanced Medical 343-72.
Sciences, Tabriz University of Medical Sciences, Tabriz, Kannan L, Ramos M, Re A, et al (2015). Public data and open
Iran. source tools for multi-assay genomic investigation of
disease. Brief Bioinformatics, 17, 603-15.
Declaration of interest Kara M, Yumrutas O, Ozcan O, et al (2015). Differential
None. expressions of cancer-associated genes and their regulatory
miRNAs in colorectal carcinoma. Gene, 567, 81-6.
Keshk WA, Zineldeen DH, El-Khadrawy OH (2014). Fatty acid
Funding source
synthase/oxidized low-density lipoprotein as metabolic
Stem Cell Research Center, Tabriz University of oncogenes linking obesity to colon cancer via NF-kappa B
Medical Sciences, Tabriz, Iran. in Egyptians. Med Oncol, 31, 192.
Kim T-M, Laird PW, Park PJ (2013). The landscape of
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