Cancer Letters 345 (2014) 17–20

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Cancer Letters

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Mini-review

Metabolomics in diagnosis and biomarker discovery of colorectal cancer

0304-3835/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.11.011

⇑ Corresponding author. Tel./fax: +86 451 82110818.E-mail address: xijunwangls@126.com (X. Wang).

Aihua Zhang, Hui Sun, Guangli Yan, Ping Wang, Ying Han, Xijun Wang ⇑National TCM Key Lab of Serum Pharmacochemistry, Key Laboratory of Metabolomics and Chinmedomics, Heilongjiang University of Chinese Medicine, Heping Road 24,Harbin 150040, China

a r t i c l e i n f o

Article history:Received 6 November 2013Received in revised form 25 November 2013Accepted 29 November 2013

Keywords:MetabolomicsColorectal cancerBiomarkersMetabolitesEarly diagnosis

a b s t r a c t

Colorectal cancer (CRC), a major public health concern, is the second leading cause of cancer death indeveloped countries. There is a need for better preventive strategies to improve the patient outcome thatis substantially influenced by cancer stage at the time of diagnosis. Patients with early stage colorectalhave a significant higher 5-year survival rates compared to patients diagnosed at late stage. Although tra-ditional colonoscopy remains the effective means to diagnose CRC, this approach generally suffers frompoor patient compliance. Thus, it is important to develop more effective methods for early diagnosis ofthis disease process, also there is an urgent need for biomarkers to diagnose CRC, assess disease severity,and prognosticate course. Increasing availability of high-throughput methodologies open up new possi-bilities for screening new potential candidates for identifying biomarkers. Fortunately, metabolomics, thestudy of all metabolites produced in the body, considered most closely related to a patient’s phenotype,can provide clinically useful biomarkers applied in CRC, and may now open new avenues for diagnostics.It has a largely untapped potential in the field of oncology, through the analysis of the cancer metabolometo identify marker metabolites defined here as surrogate indicators of physiological or pathophysiologicalstates. In this review we take a closer look at the metabolomics used within the field of colorectal cancer.Further, we highlight the most interesting metabolomics publications and discuss these in detail; addi-tional studies are mentioned as a reference for the interested reader.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Colorectal cancer (CRC) is one of the most commonly diagnosedcancers and cause of cancer-related deaths worldwide [1]. CRCdiagnosis and therapy remain dependent upon descriptive classifi-cation and staging systems, based primarily on morphology andhistology [2,3]. Despite an increased understanding of the molecu-lar pathogenesis of CRC during the past two decades, reliable androbust biomarkers to enable screening, surveillance, and primaryprevention of this disease are lacking. Importantly however, thereare no markers currently available, to predict CRC in early diagnosis,therefore, the diagnosis and management of CRC continue to be anoverwhelming challenge [4]. Metabolomics, a dynamic portrait ofthe metabolic status of living systems, offers potential advantagesthrough discovery of a suite clinically relevant biomarker whichare simultaneously affected by the CRC [5,6]. Because small changesin body can lead to large changes in metabolite levels, the metabo-lome can be regarded as the amplified output of a biological system.Monitoring fluctuations of certain metabolite levels in body fluids,has become an important way to detect early stages in CRC [7,8].

Metabolomics represents one of the new omics sciences andcapitalizes on the unique presence and concentration of small

molecules in fluids to construct a ‘fingerprint’ that can be uniqueto the individual states [9]. By applying advanced analytical andstatistical tools, metabolomics involves the comprehensive profil-ing of the full complement of low MW compounds in a biologicalsystem and can be used to classify CRC on the basis of tumor biol-ogy, to identify new prognostic and predictive markers and to dis-cover new targets for future therapeutic interventions. Acomprehensive coverage of metabolism can be achieved by acombination of analytical approaches including mass spectrome-try and nuclear magnetic resonance (NMR) spectroscopy[10–12].

In the last decade, metabolomics has been applied towardidentifying metabolic alterations in CRC that may provide clini-cally useful biomarkers. Technology and bioinformatics have ledto the application of metabolomic profiling to CRC-the highthroughput evaluation of a large complement of metabolitesand how they are altered by disease perturbations [13]. Recently,high profile publications have drawn attention to the potential ofmetabolomic analysis to identify biomarkers for early detectionor disease progression from readily accessible body fluids[14,15]. This relatively new approach using metabolomics hasjust begun to enter the mainstream of cancer diagnostics andtherapeutics. Here we intend to explore the potential role of met-abolomics in CRC and, highlight the key values of markermetabolites.

18 A. Zhang et al. / Cancer Letters 345 (2014) 17–20

2. Metabolomics technologies: the metabolites hunter

Metabolome is a data-rich source of information concerningall the low-molecular-weight metabolites in body, which canindicate early biological changes to the host due to perturbationsin metabolic pathways. The emerging field of metabolomicspromises immense potential for early diagnosis, therapy moni-toring and for understanding the pathogenesis of many diseases[16]. The technological development is the driving force for ad-vances in identifying marker metabolites. Detecting the diseaseas early as possible is an important task in cancer medicine.Thus, many technologies have been developed for biomarker dis-covery in cancer to achieve this aim [17]. There are two majorhigh-throughput tools consisting of NMR and MS used in meta-bolomics study, and they both can provide complementary snap-shots of the metabolome of body fluids [18]. A combinedanalytical approach can improve the potential to detect meta-bolic profile alterations in a biological specimen. Multiplatformapproaches could provide a more comprehensive understandingof metabolic alterations, because no single analytical tool canaccommodate the biochemical diversity in entire metabolome[19,20]. An improved combination of MS and NMR approachesmay gain more accurate disease detection and insight into mech-anisms of CRC [21].

3. Potential role of small molecule metabolites

Metabolomics allows the simultaneous and relative quantifica-tion of thousands of different metabolites within a given sampleusing sensitive and specific methodologies. The repertoire ofsmall-molecular-weight substances in body fluids are known asthe metabolites that are an ultimate product of gene, mRNA,and protein activity. The metabolites are biological indicators ofnormal biological processes, pathological processes or pharmaco-logic responses to a therapeutic intervention in clinical practice[22,23]. Integrated analysis of these metabolites may provide apowerful platform for discovering novel biomarkers and detectingcancer [24]. Analyzing metabolic differences between unper-turbed and perturbed systems in a disease, can lead to insightsinto the underlying pathology [25]. With advances in methodsand technology, biomarker discovery is one of the newly emerg-ing innovations in the diagnosis and treatment of cancer, measur-ing the response to treatment, identifying perturbed pathways[26]. Recently, a variety of biomarkers have been developed andserve a key role in diagnosis and management, monitoring treat-ment response of human diseases [27–29]. Validation of biomark-ers may entail intensive use of labor and technology andgenerally requires a large number of study participants as wellas laboratory validation studies.

4. Metabolic characteristics of CRC

CRC is the common cause of death from cancer in the world.The limitations of the currently available methods for CRC man-agement highlight the necessity of finding novel markers. There-fore, there is an underlying necessity to discover tumor-specificmarkers that may serve as molecular targets for the imaging ofCRC. Metabonomics can be used to search for potential markersthat can provide molecular insight into human CRC, and providesa means for noninvasive screening of tumor-associated perturba-tions in metabolism. Understanding the metabolome will notonly provide insights into the critical sites of regulation, but willalso assist in identifying intermediate or surrogate cancer bio-markers for establishing preemptive/preventative or therapeuticapproaches [30]. Recently, there has been a growing applications

of metabolomics aimed to finding marker metabolites that al-lows recognition of the critical metabolic pathways in CRC,which could assist diagnosis, provide therapy guidance, and eval-uate response to CRC.

5. Bringing metabolomics into the forefront of CRC research:the sooner, the better

CRC is one of the most commonly diagnosed cancers and causeof cancer-related deaths worldwide. The five-year survival rate forCRC caught early is about 50% -but catching it early is incrediblydifficult, because symptoms typically appear only during advancedstages of disease. Once the cancer has spread, the survival ratedrops to just 1%. Ideally, screening tools and diagnostics wouldnot only be able to detect early signs of cancer, but also differenti-ate between harmless changes and abnormalities that precede thedisease. Traditional tests remain the effective means to diagnoseCRC, but this approach suffers from poor patient compliance. Manydiseases result in specific and characteristic changes in the chem-ical and biochemical profiles of biological fluids and tissues prior todevelopment of clinical symptoms [31–33]. Using biomarkers toselect the most at-risk population, to detect the disease while mea-surable and yet not clinically apparent has been the goal of manyinvestigations. Metabolomics promises to be a valuable tool inthe early detection of CRC that may enable earlier treatment andimproved clinical outcomes. Advantages of metabolomics overother ‘‘omics’’, include its high sensitivity and its ability to enablethe analysis of relatively few metabolites compared with the un-wieldy number of corresponding genes or mRNA molecules[34,35]. Potential roles for metabolomics in the clinical trials ofCRC include biomarker discovery and validation, molecular targetdiscovery, therapy decisions, and patient monitoring [36–39]. Inte-gration of metabolomics into the CRC would be the direction to en-able a revolution for future health cares, also perhaps it is time toembrace the arrival of ‘CRC–OMICS’ era.

6. Metabolomics studies on CRC

Recently, several molecules were found to be specifically ex-pressed in CRC, and these novel molecular markers are reportedto improve the sensitivity of cytology or biopsy. Serum samplesof patients suffering from colon cancer and controls were collectedto analyze metabolic alterations [40]. It revealed multiple signifi-cant disease-associated alterations in the amino acid profile withpromising diagnostic power. To improve the quality of life of CRCpatients, it is important to establish new screening methods forearly diagnosis of CRC. Nishiumi et al. had performed serummetabolome analysis using GC/MS established a CRC predictionmodel [41]. It was composed of 2-hydroxybutyrate, aspartic acid,kynurenine, and cystamine, and its AUC, sensitivity, specificity,and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively.This prediction model established via GC/MS-based serum meta-bolomic analysis is valuable for early detection of CRC and hasthe potential to become a novel screening test for CRC. Sera fromCRC patients were analyzed by NMR spectroscopy and GC–MS[42]. In CRC, the serum metabolomic profile changes markedlywith metastasis, and site of disease also appears to affect thepattern of circulating metabolites. It may have clinical utility inenhancing staging accuracy and selecting patients for surgical ormedical management.

In a study, a GC � GC/TOFMS was developed for the tissue-based global metabonomic profiling of CRC [43]. Results showedthat the metabotype associated with CRC is distinct from that ofnormal tissue and led to the identification of chemically diversemarker metabolites. Metabolic pathway mapping suggested dereg-ulation of various biochemical processes such as glycolysis, Krebs

A. Zhang et al. / Cancer Letters 345 (2014) 17–20 19

cycle, osmoregulation, steroid biosynthesis, eicosanoid biosynthe-sis, bile acid biosynthesis, lipid, amino acid and nucleotide metab-olism. Ma et al. study was designed metabolomics approach tosearch for potential diagnostic biomarkers in the serum of CRC[44]. Of the analyzed metabolites, only 6 metabolites were signifi-cantly increased or decreased in CRC group. Supervised predictivemodels allowed a separation of 93.5% of CRC patients from thehealthy controls using these metabolites. Cheng et al. had recentlyreported a urinary metabonomic study on a larger cohort of CRC[45]. A panel of metabolite markers composed of citrate, hippurate,p-cresol, 2-aminobutyrate, myristate, putrescine, and kynurenatewas selected, which was able to discriminate CRC subjects fromtheir healthy counterparts. A number of dysregulated metabolicpathways, such as glycolysis, TCA cycle, urea cycle, pyrimidinemetabolism, tryptophan metabolism, polyamine metabolism, aswell as gut microbial-host co-metabolism in CRC subjects wereidentified. ROC analysis of these markers resulted in an AUC of0.993 and 0.998 for the training set and the testing set, respec-tively. These marker metabolites provide a promising moleculardiagnostic approach for the early detection of CRC. Bertini et al.had used NMR to profile the serum metabolome in CRC patientsand determine whether a disease signature may exist that is strongenough to predict overall survival [46]. In the training set, NMRmetabolomic profiling could discriminate patients with CRC witha cross-validated accuracy of 100%. A number of metabolites con-curred with the NMR fingerprint of CRC, offering insights intoCRC metabolic pathways. These approach developed will promotethe translation of biomarkers with clinical value into routine clin-ical practice.

Using tandem-MS/MS technology, Ritchie et al. evaluated theutility of selected markers and metabolomics technology for dis-criminating between CRC and healthy subjects [47]. A compre-hensive metabolomics technology was used to identify asystemic metabolic dysregulation comprising previously un-known hydroxylated polyunsaturated ultra-long chain fatty acidmetabolites in CRC patients. These metabolites are easily measur-able in serum and a decrease in their concentration appears to behighly sensitive and specific for the presence of CRC. The mea-surement of these metabolites may represent an tool for the earlydetection of CRC. In a study, Ong et al. studied the metabolomicprofiles using highly specific GC/MS and LC/MS/MS to attain asystems-level view of the shift in metabolism path to CRC [48].Various amino acids and lipids in the polyps and tumors wereelevated, suggesting higher energy needs for increased cellularproliferation. In contrast, significant depletion of glucose and ino-sitol in polyps revealed that glycolysis may be critical in earlyCRC. GC–MS in combination with pattern recognition techniqueswas used to analyze serum metabolome in CRC patients [49].The endogenous metabolites including amino acid, fatty acid, car-bohydrate and other intermediate metabolites were identified. Itdemonstrated the GC–MS technique is an valuable tool for thecharacterization of the metabolic perturbation, and the metabolo-mic study will certainly benefit for monitoring the nutrition stateof CRC patients, the prognosis and therapy evaluation of CRC pa-tients. Monleón et al. obtained NMR metabolic profiles of fecalwater extracts from patients with CRC and healthy individuals,to characterize possible differences between them and to identifypotential diagnostic markers [50]. Concentrations of proline andcysteine, which are major components of most colonic epitheliummucus glycoproteins, also display significant changes in samplesfrom CRC. A low concentration of short-chain fatty acids, suchas acetate and butyrate, previously associated with the develop-ment of CRC, appears to be the most effective marker. It showedthat metabolic profiling of fecal water extracts is a cheap, repro-ducible, effective and high-throughput screening method fordetecting CRC.

7. Conclusions and future perspectives

Elimination of CRC in the 21st Century is likely to depend notonly on the effective individualized treatment, but also upon ear-lier detection and prevention [51]. The burden of CRC is growingworldwide and with it a more desperate need for better tools to de-tect, diagnose and monitor the disease is required. Metabolomics,the non-targeted interrogation of the small molecules in biologicalsamples, is an ideal technology for identifying diagnostic biomark-ers. It has the potential to generate novel noninvasive diagnostictests, based on biomarkers of disease, which are simple and costeffective yet retain high sensitivity and specificity characteristics.To enable a better diagnose CRC patients, a deeper understandingof global perturbations in body could provide valuable insightsabout mechanisms of CRC. Technological advances have madeand will continue to make possible earlier, more accurate, lessinvasive diagnoses, all enhancing our understanding of the rootcauses of CRC. The translational value of metabolomics in theCRC studies will eventually lead to diagnostic toolkits that willfacilitate a much more precise predictive and prognosticassessment.

Conflict of Interest

The authors have declared that they have no competinginterests.

Acknowledgments

This work was supported by grants from the Key Program ofNatural Science Foundation of State (Grant Nos. 90709019,81173500, 81373930, 81302905, 81102556, 81202639), NationalKey Technology Research and Development Program of theMinistry of Science and Technology of China (Grant Nos.2011BAI03B03, 2011BAI03B06, 2011BAI03B08), National KeySubject of Drug Innovation (Grant No. 2009ZX09502-005),Postdoctoral Science Foundation of Heilongjiang Province (LBH -Z10013), Key Project of Chinese Ministry of Education (GrantNo. 212044), Foundation of Heilongjiang University of ChineseMedicine (Grant No. 201209).

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