-Catenin Regulation in Sporadic Colorectal Carcinogenesis...

Research Article120573-Catenin Regulation in Sporadic Colorectal CarcinogenesisNot as Simple as APC

Ernst Fredericks Gill Dealtry and Saartjie Roux

Department of Biochemistry and Microbiology Nelson Mandela Metropolitan University Port Elizabeth South Africa

Correspondence should be addressed to Ernst Fredericks esfredejectcoza

Received 7 April 2018 Revised 10 July 2018 Accepted 17 July 2018 Published 16 August 2018

Academic Editor Ashok Kumar

Copyright copy 2018 Ernst Fredericks et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background The wntAPC120573-catenin pathway is a critical initiator in colorectal carcinogenesis in both hereditary and sporadiccolorectal cancer (CRC) The progression of this process remains incompletely understood although inflammation is pivotalDrivers of inflammation are elevated in malignant tissue and have been shown to regulate 120573-catenin expression Interleukin-17A(IL-17A) is protumorigenic at elevated levels via COX-2 stimulation Elevated peroxisome proliferator-activated receptor 120574 (PPAR120574)expression has reduced risk of carcinogenesis and good overall prognosis in established CRC Activation of PPAR120574 has inhibitoryeffect on 120573-cateninMethods Using qPCR and IHC we compared 120573-catenin PPAR120574 COX-2 and IL-17A in the colonic mucosaof patients with sporadic CRC inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) against a normal controlpopulation Results 120573-catenin mRNA and protein expression progressively increased from the Normal group through IBS andIBD reaching statistical significance in CRC COX-2mRNA levels increased similarly with statistical significance in IBD and CRCHowever COX-2 protein expression was inverted with significant expression in the Normal and IBS groups and reduced levels inIBD and CRC PPAR120574mRNA expression was unchanged in IBD and CRC but was significantly elevated in the IBS IL-17A mRNAwas significantly reduced in IBS and CRC but unchanged in IBDThere were no differences in all parameters tested in the Normaland IBS groups Conclusion 120573-catenin is confirmed as a major driver of colorectal carcinogenesis but is controlled by many moreplayers other than APC Elevated levels of PPAR120574may have an anticarcinogenic effectThe role of COX-2 expression especially itsposttranscriptional regulation in colorectal cancer needs further elucidation

1 Introduction

Sporadic colorectal cancer (CRC) is a heterogeneous diseasewith multiple factors involved in carcinogenesis Inheritedmutations and spontaneous molecular derangements areimportant initiatorspromoters of the carcinogenic processbut cancer propagation remains unclear Although mutationshave been linked to carcinogenesis no single gene mutationhas been exclusively linked to or identified to be consistentlypresent in all cases of CRC [1] Therefore other molecularfactors must propagate colorectal carcinogenesis

The wnt120573-catenin signalling pathway important in thehereditary CRCmodel has an equally important role in spo-radic colorectal carcinogenesis Mutations in APC a tumoursuppressor gene are central in colorectal carcinogenesisthrough the wnt120573-catenin signalling pathway APC binds120573-catenin in a complex with glycogen synthase kinase-3120573

(GSK-3120573) and regulates its activity through phosphorylationand degradation APC mutation leads to nuclear accumula-tion of 120573-catenin and transcription of oncogenes resultingin carcinogenesis Somatic mutations of APC account for70 of cases of sporadic CRC while germ line mutations ofthe same gene are found in 100 of patients with FamilialAdenomatous Polyposis [2 3]120573-cateninmutations resultingin nuclear accumulation of 120573-catenin have also been noted ina minority of patients with sporadic CRC [4]

In colitis-associated cancer (CAC) an example of inflam-matory bowel disease- (IBD-) related CRC chronic inflam-mation predisposes to carcinogenesis through signallingpathways involving wnt120573-catenin K-ras nuclear factor-120581B (NF-120581B) and downstream cytokines [5] COX-2 aninducible form of cyclooxygenase serves as an interfacebetween inflammation and cancer COX-2 expression andits downstream by-product prostaglandin E

2(PGE2) are

HindawiCanadian Journal of Gastroenterology and HepatologyVolume 2018 Article ID 4379673 10 pageshttpsdoiorg10115520184379673

2 Canadian Journal of Gastroenterology and Hepatology

upregulated in sporadic CRC and IBD [6] Moreover COX-2inhibitors have been shown to reduce carcinogenesis in bothanimal and human studies [7]

Peroxisome proliferator-activated receptor 120574 (PPAR120574)a ligand-activated nuclear transcription factor has beenimplicated in colorectal carcinogenesis although its exactrole remains unclear PPAR120574 activation is associated withinhibition of cell growth in human colon cancer cell linesas well as cancer xenografts in nude mice Mice witha heterozygous deletion of PPAR120574 (PPAR120574+minus) have anincreased tendency to develop carcinogen-induced CRC [8]Activated PPAR120574 inhibits colorectal carcinogenesis throughdownstream inhibition of the 120573-catenin mediated transcrip-tion pathway [9] Recent data showed that PPAR120574 stimu-lation inhibits Th17 activation and IL-17 production [10]Elevated PPAR120574 expression confers a good prognosis in CRC[11]

Inflammatory Th17 helper T cells derive their name bytheir ability to produce IL-17 cytokines (IL-17A-F) amongothers [12] Th17 derived cytokines play a role in gutinflammation and carcinogenesis linking IBD and CRC [13]Tosolini et al recently found that increased IL-17A mRNAexpression in the tumour tissue is associated with increasedcancer recurrence and poor survival suggesting adverseprognosis [14] Mechanistically IL-17 promotes colorectalcarcinogenesis through direct stimulation of COX-2PGE

2

[15]These molecules have an intricate interrelationship dur-

ing carcinogenesis and each one influences the expressionand function of the others COX-2 has been shown tomodulate both the wnt120573-catenin and PPAR120574 pathwaysPGE2and 15-Deoxy-Prostaglandin J

2(15-D-PGJ

2) down-

stream products of the COX-2 biosynthetic pathway increaseaccumulation and nuclear translocation of 120573-catenin andPPAR120574 respectively [16 17] PPAR120574 activation has beenshown to inhibit 120573-catenin expression and IL-17 activation[18 19] IL-17 has been shown to upregulate COX-2PGE

2

[20]In this study we examined the interrelationship of 120573-

catenin COX-2 PPAR120574 and IL-17 in patients with sporadiccolon cancer compared to subjects without cancer Furthercomparisons were made in patient populations with irritablebowel syndrome (IBS) with lowCRC risk and IBD with highrisk for CRC development [21]

The study results confirm a complex relationship betweenthese role players in colorectal carcinogenesis In both theNormal and IBS groups homeostatic mechanisms remainintact with low cell turnover as evidenced by low levels of 120573-catenin and high levels of PPAR120574 Inflammation is also keptin check by sufficient COX-2 protein expression and reducedIL-17A expression These findings were inverted for IBD andCRC

2 Materials and Methods

21 Study Population The Human Ethics Committee of theNelson Mandela Metropolitan University (H07SciBCN-001)approved the study Written informed consent was obtainedfrom each participant on enrolment Patients were grouped

into (1) Normal patients attending for screening and surveil-lance colonoscopy (2) IBS patients with constipation- anddiarrhoea-predominant IBS using Rome II criteria (3) IBDCrohnrsquos disease (CD) and Ulcerative colitis (UC) patients (4)CRC

22 Tissue Collection Colon mucosal biopsies were takenfrom the normal caecum and ascending colon in the Normaland IBS groups from the inflamed mucosa in IBD and fromthe malignant tissue in CRC Samples were (i) placed inRNA-later (Sigma) and stored at minus20∘ until analysis and (ii)preserved in 4 paraformaldehyde and embedded in paraffinfollowing standard procedures

23 Quantitative Real-Time Polymerase Chain Reaction(qPCR) Total RNA was extracted (Bio-Rad Aurum RNAextraction kit) following the manufacturerrsquos instructionscDNA was synthesised from 1120583g RNA using the reversetranscription BioRad iScript cDNA synthesis kit (Bio-RadLaboratories Inc USA) The cDNA samples were frozen atminus20∘C until required

TheGeNormNormalization (PrimerDesign UK) algo-rithm was used to identify three stably expressed referencegenes These were SF3A1 and two Alu repeats ALUsq andALUsx [22] Real-time qPCR reactionswere run on an iCyclerIQ system (Bio-Rad Laboratories Inc USA)

To each well of a 96 well PCR plate 15120583l of mastermix consisting of 10120583l SYBRGreen supermix (Bio-Rad)and 500 nM forward and reverse primers was added 5 120583l ofthe appropriate cDNA (15 diluted) was added to each wellexcept for two wells with no template controls (NTC) wherethe cDNA sample was replaced with 5 120583l of nuclease-freePCR grade water (Ambion USA) Experiments were run induplicate

The protocol for all qPCR runs is comprised of 3-minuteTaqpolymerase activation at 95∘Cand 40 cycles of denaturingat 95∘C for 30 seconds 30 seconds at appropriate annealingtemperature (Table 1) and extension at 72∘C for 30 seconds

The amplification reaction was followed by a melt curveto verify the specificity of the reactionThe plate was heated to95∘C and then cooled to 1∘C below the annealing temperatureto ensure that all the DNAwas double stranded The temper-ature was increased in increments of 05∘C for 30 seconds upto 95∘C to melt the double stranded DNA

Genes of interest were analysed following the above qPCRprotocol and Cq values compared using qBase version 2software (BioGazelle) Table 1 details all genes used in thisstudy

24 Immunohistochemistry Four micrometer sections wereused for IHC staining using the Ventana ESautomatedsystem (Ventana Medical Systems) following the manufac-turerrsquos instructions The tissue sections were deparaffinisedand rehydrated following standard procedures Antigenretrieval was performed by incubating tissue sections in10mM citrate buffer in a microwave for 15 minutes Slideswere transferred to an automated slide stainer Endogenousperoxidases were quenched by incubating tissue sections

Canadian Journal of Gastroenterology and Hepatology 3

Table 1 Forward and reverse sequences of primers for all genes and their annealing temperatures

Gene of interest Forward sequence Reverse sequence Annealing temp (∘C)120573-catenin 51015840-CTGACTTTGCTTGCTTGA-3 51015840-CACTATAACTTAACACTACGAG-31015840 60COX-2 51015840-GAAGCCAATTCAGTAGGT-31015840 51015840-ACGAAGTGATGAGAAGAC-31015840 57PPAR120574 51015840-AGGTTTGCTGAATGTGAAG-31015840 51015840-AATCTGTCTGAGGTCTGTC-31015840 60IL-17A 51015840-CCACACTCCCCAAAGCAGTT-31015840 51015840-TGACATGCCATTCCTCAGGG-31015840 53Reference genes Forward sequence Reverse sequence Annealing temp (∘C)ALUSq 51015840-CATGGTGAAACCCCGTCTCTA-3 51015840-GCCTCAGCCTCCCGAGTAG-31015840 60ALUSx 51015840-TGGTGAAACCCCGTCTCTACTAA-31015840 51015840-CCTCAGCCTCCCGAGTAGCT-31015840 60SF3A1 Commercial primer obtained from GeNormNormalization kit (Primer Design UK) 60

Table 2 Demographic details of study population

Normal C-IBS D-IBS CD UC CRCTotal 26 (195) 33 (248) 15 (113) 8 (6) 14 (105) 37 (278)Age 530 480 490 435 435 660Male 10 (385) 8 (242) 5 (333) 4 (50) 6 (429) 19 (514)Female 16 (615) 25 (758) 10 (667) 4 (50) 8 (571) 18 (486)BMI 2554 2614 2816 2117 2596 2870Smoke 6 (231) 7 (212) 5 (333) 5 (625) 1 (71) 5 (135)NSAIDS 7 (269) 3 (91) 1 (67) 0 0 0FH CRC 1 (38) 3 (91) 1 (67) 0 0 6 (162)( ) = Percentage expressed for group in all subcategories except totalNSAIDS nonsteroidal anti-inflammatory drugsFH CRC family history of colorectal cancer

with 3 H2O2for 30 minutes Sections were blocked

with 10 normal goat serum in phosphate buffer for 10minutes then incubated with primary antibody for 32minutes washed and incubated with diluted biotinylatedsecondary antibody for 6 minutes The primary antibodyfor COX-2 was a rabbit monoclonal antihuman antibodyand diluted 1100 in Tris buffer (Ventana) The secondaryantibody was a combined biotinylated goat-anti-mouse IgGand IgM (lt200120583gml) and biotinylated goat-anti-rabbit IgG(lt200120583gml) (Ventana) The primary 120573-catenin antibodywas amousemonoclonal anti-human antibody (038120583g5mlVentana) The secondary antibody was a combined biotiny-lated goat-anti-mouse IgG and IgM (lt200120583gml) andbiotinylated goat-anti-rabbit IgG (lt200120583gml) (Ventana)Streptavidin horseradish peroxidase (lt300120583gml) (Ven-tana) was applied for 20 minutes Sections were visualizedusing 33-diaminobenzidine tetrahydrochloride (DAB) (2 gLVentana) as the peroxidase substrate for 5 minutes andHarrisrsquos Haematoxylin (standard solution) counterstain Forpositive controls known colorectal cancer slides positive forCOX-2 and 120573-catenin were used Negative control slides wereincubated without primary antibody

All IHC slides were reviewed and scored by an indepen-dent pathologist A scoring system based on the intensityof the reaction and the extent of the staining was used asdescribed by Xiong et al [23] Staining intensity was gradedas 0 = absent 1 = weak 2 = moderate or 3 = strong Theextent of the positive area was classified as 0 = lt 10 1 = 10ndash 40 2 = 40 ndash 70 and 3 = gt 70 Slides without stain

were considered negative This method was validated for theimmunoexpression of COX-1 and -2 in both UC and CD[24 25]

25 Statistical Analysis All statistical parameters were cal-culated as mean plusmn standard error Statistical comparisonswere examined with one-way analysis of variance (ANOVA)followed by the Mann-Whitney U test The results wereconsidered significantly different at plt005

3 Results

A total of 133 patients were recruited Their demographicdetails are listed in Table 2

31120573-Catenin 120573-cateninmRNAexpressionwas significantlyincreased only in CRC (Figure 1(a)) 120573-catenin protein how-ever as shown with IHC showed increased staining at mem-branecytoplasmic level compared with nuclear expressionacross all disease groups (Figures 1(b) and 1(c)) Althoughnuclear 120573-catenin protein expression was low in all groupsCRC had the most expression being the only group with3+ scoring In the IBD group UC showed no 120573-cateninnuclear expression while CD had only a small percentageof 2+ scoring 120573-catenin membranecytoplasmic proteinexpression showed 2+ scoring in all groups but significant3+ expression in IBD (UC) and CRC (Figure 1(c)) 120573-catenin membranecytoplasmic protein expression generallyfollowed 120573-catenin mRNA expression 120573-catenin mRNA in


Normal C-IBS D-IBS CD UC CRC0

1

2

3

4-catenin mRNA

Diagnosis

Mea

n C

NRQ

lowast

lowast lowast

(a) The expression of beta-catenin mRNA steadily increased from Normalthrough the various disease entities to reach statistical significance in CRC

050

100

150

Nuclear -catenin IHC

S

tain

ing

inte

nsity

01+

2+3+

Normal C-IBS D-IBS CD UC CRCDiagnosis

(b) Nuclear expression of beta-catenin protein was significant for CRCcompared to IBS and IBD

0

50

100

150MembraneCytoplasmic -catenin IHC

S

tain

ing

inte

nsity

01+

2+3+


(c) Membranecytoplasmic beta-catenin protein expressionwas highest in IBDand CRC

Figure 1 120573-catenin expression (a) 120573-catenin mRNA expression CNRQ = Calibrated Normalized Relative Quantities normalized to meanfor all samples Total patient number studied = seventy-five (75) with Normal group seventeen (17) patients C-IBS group twenty-two (22)patients D-IBS group eight (8) patients CD group six (6) patients UC group eight (8) patients and CRC group sixteen (16) patientslowast denotes significant different versus normal group (plt005) (b) 120573-catenin nuclear expression and (c) 120573-catenin membranecytoplasmicexpression by IHCOne-hundred and twenty (120) patient slides analysedwith breakdown as followsNormal group twenty-five (25) patientsC-IBS thirty (30) patients D-IBS fifteen (15) patients CD seven (7) patients UC twelve (12) patients and CRC thirty-three (33) patients

the IBS group was significantly lower than the Normal group120573-catenin protein expression in C-IBS was similar to theNormal group while in D-IBS the expression was slightlylower than Normal This suggests very low cell turnoverin these patient groups Figure 2 depicts 120573-catenin proteinexpression

32 COX-2 There was a progressive increase in COX-2mRNA expression from the noninflammatory groups to theinflammatory groups with statistically significant increasesin IBDandCRCcompared toNormal (Figure 3(a))Howeverthere was an inverse COX-2 protein expression pattern withmore protein expressed in the control and IBS groups and

less in the IBD and CRC groups (Figure 3(b)) All groupshad COX-2 protein expression with 2+ scoring of between60 and 80 of the total COX-2 protein expression Significant3+ scoring for COX-2 protein was noted in the Normal andIBS groups compared to no 3+ scoring for IBD and very little3+ scoring in CRC COX-2 IHC is depicted in Figure 4

33 IL-17A The expression of IL-17A mRNA was signifi-cantly reduced in the non-inflammatory IBS and surprisinglyalso in CRC (Figure 5(a)) As cytokine expression is gener-ally short-lived with rapid cellular changes IL-17A proteinexpression by IHC was not performed


(a) (b) (c)

(d) (e) (f)

Figure 2 Nuclear andmembranecytoplasmic 120573-catenin immunoexpression for 122 patients (a) Normal group (n=25) (b) C-IBS (n=30) (c)D-IBS (n=15) (d) CD (n=7) (e) UC (n=12) (f) CRC (n=33) Significant increase 120573-catenin protein expression from Normal group to CRCespecially nuclear localization Magnification x40 Scale bar = 200120583m

0

2

4

6

8COX-2 mRNA

Mea

n C

NRQ


lowast

lowast

lowast

(a) COX-2 mRNA expression steadily increased over disease enti-ties with significance in IBD and CRC

0

20

40

60

80

100COX-2 IHC

S

atin

ing

inte

nsity

01+

2+3+


(b) COX-2 protein expression was significant in the IBS groups withreduced expression in CRC and IBD

Figure 3 COX-2 expression in the various disease entities (a) COX-2 mRNA expression CNRQ = Calibrated Normalized RelativeQuantities normalized to mean for all samples Total patient number studied = seventy-four (74) with Normal group fifteen (15) patientsC-IBS group twenty-two (22) patients D-IBS group seven (7) patients CD group six (6) patients UC group eight (8) patients and CRCgroup sixteen (16) patients lowast denotes significantly different versus normal group (plt005) (b) COX-2 IHC One-hundred and fourteen (114)patient slides analysed with breakdown as follows Normal group twenty-six (26) patients C-IBS thirty-one (31) patients D-IBS thirteen(13) patients CD seven (7) patients UC twelve (12) patients and CRC twenty-five (25) patients


(a) (b) (c)

(d) (e) (f)

Figure 4 COX-2 immunoexpression in 114 patients (a) Normal group (n=26) (b) C-IBS (n=31) (c) D-IBS (n=13) (d) CD (n=7) (e) UC(n=12) (f) CRC (n=25) Significant COX-2 protein in Normal and IBD groups with lesser expression in IBD and CRC Magnification x40Scale bar = 200120583m

Table 3 The relationship between 120573-catenin COX-2 PPAR120574 andIL-17A

120573-catenin COX-2 PPAR120574 IL-17AmRNA Protein mRNA Protein mRNA mRNA

IBS darrdarr darr uarr uarruarr uarruarr darrdarr

IBD uarr uarr uarruarr uarr larrrarr larrrarr

CRC uarruarr uarruarr uarruarr uarr larrrarr darr

34 PPAR120574 PPAR120574 mRNA expression was significantlyelevated only in the IBS groups (Figure 5(b)) As PPAR120574protein expression was expected tomirror its gene expressionand because nuclear localization is transient IHC was notperformed

4 Discussion

Understanding the molecular pathways in carcinogenesis isimportant to pave the way for novel drug development tar-geting the signalling pathways involved Table 3 summarizesthe main findings of the study120573-catenin mRNA and protein expression followed

expected trends especially for IBD and CRC Serafino etal similarly found that expression of 120573-catenin proteinpredominantly had membranous localization in IBD andsporadic CRC [26] Claessen et al also noted increasedmembranous 120573-catenin localization in inflamed IBDmucosaand reduced membranous 120573-catenin staining and increasednuclear 120573-catenin staining in CAC [27] 120573-catenin mRNA

was significantly reduced in IBS in keeping with low cellturnover while the protein expression was localized to themembranecytoplasm The conclusion from this and otherdata suggest that 120573-catenin overexpression is a marker of cellproliferation and thus a driver of the carcinogenic process

COX-2 mRNA is frequently overexpressed in IBD [28]andCRC [29] Results from this study confirmed significantlyincreased COX-2mRNAexpression in both these conditionsThe protumorigenic effect of COX-2 is thought to be medi-ated by its downstream product PGE

2[30] COX-2 protein

expression by IHC in our study was reduced in both IBD andCRC compared toNormal Similar findings of reducedCOX-2 protein expression using IHC andWestern blot were shownby Lin et al in patients with CRC [31] In contrast previousinvestigators showed increased COX-2 protein expression byIHC for both UC and CD compared to the control group[24 25] A limitation of these studies was that no comparisonwas made with COX-2 mRNA expression The expression ofCOX-2 protein in normal tissue is not unusual and has alsobeen shown by both Paiotti et al and Romero et al [24 25]This is the first report evaluating COX-2 expression in IBSand it showed low COX-2 mRNA expression with relativeincreased COX-2 protein expression for both C-IBS and D-IBS A simplistic explanation for this phenomenon may bethat a certain level of COX-2 protein expression is requiredto maintain homeostasis during normalcy and disruptionof this homeostasis with reduced COX-2 protein as in IBDcauses the upregulation of COX-2 mRNA expression noted

Several studies have proven the regulation of COX-2expression by the wnt120573-catenin signalling pathway [32 33]


0

1

2

IL-17A mRNAM

ean

CN

RQ


lowast

lowastlowast

(a) IL-17A mRNA expression was significantly reduced in IBS andunchanged in CRC and IBD

00

05

10

15

20

25PPAR mRNA

Mea

n C

NRQ


lowast

lowast

(b) PPARgamma was significantly increased in IBS and unchanged inIBD and CRC

Figure 5 (a) Interleukin-17AmRNA CNRQ = CalibratedNormalized Relative Quantities normalized tomean of all samples Total patientsstudied = 65 with Normal group thirteen (13) patients C-IBS group eighteen (18) patients D-IBS group six (6) patients CD group six (6)patients UC group six (6) patients and CRC group sixteen (16) patients (b) PPAR120574 mRNA expression Total patient number studied =seventy-four (74) with Normal group fifteen (15) patients C-IBS group twenty-two (22) patients D-IBS group eight (8) patients CD groupsix (6) patients UC group seven (7) patients and CRC group sixteen (16) patients lowast denotes significantly different versus normal group(plt005)

Increased 120573-catenin in the cell translocates to the nucleusand stimulates the production of downstream genes cyclinD c-myc and c-jun among others that drive cell proliferationand carcinogenesis In addition 120573-catenin also binds to HuRa member of the RNA binding proteins with subsequentbinding to the COX-2 31015840UTR to stabilize and regulate thetranscription of COX-2 mRNA [34] However it is theposttranscriptional regulation of the COX-2 gene whichis far more complex which controls the expression of theprotein

The role of PPAR120574 in carcinogenesis remains contro-versial PPAR120574 is highly expressed in normal human colonmucosa as well as human and animal CRC [35] Ligandactivation of PPAR120574 in colon cancer cell lines is associatedwith inhibition of cell growth increased differentiation andreversal of the malignant phenotype [36] Despite this manyhuman colon cancer cell lines are ldquoresistantrdquo to growthinhibition by thiazolidinediones ligands of PPAR120574 and alsocorrelate poorly with levels of PPAR120574 expression [37 38]In our study expression of PPAR120574 mRNA in CRC was nodifferent than the Normal group This suggests that PPAR120574seemingly plays no significant role in colorectal carcino-genesis Similarly no difference in PPAR120574 expression wasnoted for IBD although there was slight increased expressionin CD and slightly reduced expression for UC comparedto Normal This data is congruent with previous reportsshowing downregulation of PPAR120574 expression in UC andunchanged expression from normal in CD [39 40] PPAR120574was significantly increased for both C-IBS and D-IBS

PPAR120574 has important interactions with both 120573-cateninand COX-2 COX-2 activation leads to the production ofprostaglandins such as 15-D-PGJ

2 a natural ligand of PPAR120574

with activation of the receptor and downstream signalling

Although not explicitly proven by this study it is reasonableto extrapolate that high levels of COX-2 protein in IBS couldresult in increased expression of 15-D-PGJ

2and ultimately

elevated PPAR120574 expression By the same token low COX-2 protein expression leading to low levels of 15-D-PGJ

2

may lead to less PPAR120574 expression or activation fuellinginflammation and carcinogenesis as noted in IBD and CRCAs previously discussed PPAR120574 directly inhibits 120573-cateninand its nuclear translocation resulting in a nonproliferativestate High levels of PPAR120574 as seen in the IBS groups maytherefore be beneficial In addition prostaglandins increasethe release of GSK-3120573 from the axin complex therebyreleasing 120573-catenin resulting in nuclear translocation andactivation of the wnt120573-catenin signalling pathway drivingproliferation [41]Thus controlling COX-2 expression and itsdownstream by-productsmay be an important mechanism tokeep PPAR120574 and 120573-catenin levels in check

PPAR120574 also has a direct inhibitory effect on IL-17 IL-17Ais a proinflammatory cytokine important in inflammationautoimmunity and carcinogenesis Li et al showed that IL-17A upregulated COX-2 mRNA and protein in cancer cellslines via NF-120581B and ERK12 signalling pathways [15] InCRCincreased IL-17 confers adverse prognosis and poor survivalXie et al noted increased IL-17A expression in humancolon carcinogenesis [42] Moreover IL-17A deficiency wasassociated with decreased colitis-associated carcinogenesis[43] IL-17A mRNA was significantly reduced in the CRCgroup in this study No clear explanation for this discrepancycan be advanced and the authors suggest a prospectiveinvestigation of this in a larger cohort IL-17A mRNA wassignificantly reduced in both IBS groups in keeping with lowinflammatory burden IL-17A mRNA expression in the IBDgroups was not different than the Normal group


COX-2

PGD2

15D-PGJ2 PGE2

IL-17A

PPAR -CATENIN

APC

TZD Dietary PUFA

PPARRXR

Target genes

Differentiation

-CateninTCF

Target genesc-myc

cyclin D

Proliferation

COX-Inh

StimulateInhibit Normal pathway

Cell cycle progression

Inflammation

Figure 6 Interaction and regulation between COX-2 120573-catenin PPAR120574 and IL-17 in colorectal carcinogenesis TZD thiazolidinedionesPUFA poly-unsaturated fatty acids TCF T cell factor RXR retinoic acid receptor and Inh inhibitor

To maintain homeostasis basal expression of thesemolecules is important for housekeeping especially to healinflamed mucosa or to prevent carcinogenesis Alteringthe expression of these molecules will disrupt homeostasissetting the tone for propagation of inflammation (IBD) andCRC As discussed COX-2 activation leads to productionof PGE

2which act via the EP receptors PGE

2upregula-

tion promotes colorectal carcinogenesis by activating thewnt120573-catenin pathway through phosphorylation of GSK-3120573 [44] COX-2 also induces the synthesis of prostaglandinD2 (PGD

2) and its end product 15-D-PGJ

2 both of which

suppresses inflammation by inhibiting NF-120581B and activatingof PPAR-120574 [45 46] IL-17A amplifies the COX-2-mediatedeffects of TNF-120572 and greatly enhances PGE

2[47] PPAR120574

reduces carcinogenesis by inhibiting both 120573-catenin and IL-17A reducing their downstream signalling and promoting celldifferentiation [9 19]

Results from this study support the complex relationshipof the various molecules as documented by other investi-gators In conditions with low inflammatory burden likeIBS and Normal groups COX-2 protein expression is highcompared to its mRNA expression Similarly PPAR120574 expres-sion is high with low expression of IL-17A and 120573-cateninThese reported changes in the expression of the investi-gatedmolecules appear protective against carcinogenesisThe

inverse is true for conditions with high inflammatory burdenlike IBD and CRC

The study is not without limitations The biggest lim-itation is its snapshot approach with only one time-pointmeasured in a disease course that has linear progressionFuture studies should test this hypothesis on animal mod-els of CRC Posttranscriptional regulation of COX-2 hasimportant implications for CRC and needs elucidation Thepotential diagnostic prognostic and therapeutic roles ofthese molecules need investigation prospectively

5 Conclusion

Although many studies have investigated the role of thesemolecules in colorectal carcinogenesis this is the first studycomparing them in various disease entities including IBSIBD and CRC Figure 6 gives schematic interaction of themolecules controlling proliferation and differentiation 120573-catenin is confirmed as a major driver of colorectal car-cinogenesis but its expression is controlled by many moreplayers than APC as shown Although APC mutation maybe an important initiator of colorectal carcinogenesis thepropagation of the process is influenced by other molecularfactors including COX-2 PPAR120574 and IL-17A Results fromthis study suggest that mitigating colorectal carcinogenesis


would require elevated PPAR120574 and reduced IL-17A lev-els In addition increased COX-2 protein expression maydownregulate 120573-catenin expression However longitudinalprospective studies are required to test this hypothesis andto evaluate gene and protein expression in affected versusunaffected tissue in the same patient

Data Availability

The data supporting the findings in the study are availablefrom the corresponding author on request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This study was supported by National Research FoundationSouth Africa Grant no 2075266

References

[1] R Agarwal B Kumar M Jayadev D Raghav and A SinghldquoCoReCG a comprehensive Database of genes associated withcolon-rectal cancerrdquo Database vol 2016 Article ID ebaw0592016

[2] M Christie R N Jorissen D Mouradov et al ldquoDifferentAPC genotypes in proximal and distal sporadic colorectalcancers suggest distinct WNT120573-catenin signalling thresholdsfor tumourigenesisrdquo Oncogene vol 32 no 39 pp 4675ndash46822013

[3] M L Leoz S Carballal L Moreira T Ocana and F BalaguerldquoThe genetic basis of familial adenomatous polyposis and itsimplications for clinical practice and risk managementrdquo TheApplication of Clinical Genetics vol 8 pp 95ndash107 2015

[4] E Akisik D Bugra S Yamaner and N Dalay ldquoAnalysis of 120573-catenin alterations in colon tumors a novel exon 3 mutationrdquoTumor Biology vol 32 no 1 pp 71ndash76 2011

[5] J E Axelrad S Lichtiger and V Yajnik ldquoInflammatory boweldisease and cancer the role of inflammation immunosuppres-sion and cancer treatmentrdquoWorld Journal of Gastroenterologyvol 22 no 20 pp 4794ndash4801 2016

[6] E Fredericks G Dealtry and S Roux ldquoMolecular aspects ofcolorectal carcinogenesis a reviewrdquo Journal of Cancer Biologyand Research vol 3 no 1 p 1057 2015

[7] FNolfo S Rametta SMarventano et al ldquoPharmacological anddietary prevention for colorectal cancerrdquo BMC Surgery vol 13no 2 article no S16 2013

[8] Joo-In Park and Jong-Young Kwak ldquoThe role of peroxisomeproliferator-activated receptors in colorectal cancerrdquo PPARResearch vol 2012 Article ID 876418 12 pages 2012

[9] T Fujisawa A Nakajima N Fujisawa et al ldquoPeroxisomeproliferator-activated receptor 120574 (PPAR120574) suppresses colonicepithelial cell turnover and colon carcinogenesis through inhi-bition of the 120573-cateninT cell factor (TCF) pathwayrdquo Journal ofPharmacological Sciences vol 106 no 4 pp 627ndash638 2008

[10] D H Kim H-J Ihn C Moon et al ldquoCiglitazone a perox-isome proliferator-activated receptor gamma ligand inhibits

proliferation and differentiation of Th17 cellsrdquo Biomolecules ampTherapeutics vol 23 no 1 pp 71ndash76 2015

[11] S Ogino K Shima Y Baba et al ldquoColorectal cancer expressionof peroxisome proliferator-activated receptor gamma (PPARGPPARgamma) is associated with good prognosisrdquo Gastroen-terology vol 136 no 4 pp 1242ndash1250 2009

[12] X O Yang B P Pappu R Nurieva et al ldquoT helper 17 lineagedifferentiation is programmed by orphan nuclear receptorsROR alpha and ROR gammardquo Immunity vol 28 no 1 pp 29ndash39 2008

[13] I Kryczek K Wu E Zhao et al ldquoIL-17+ regulatory T Cells inthe microenvironments of chronic inflammation and cancerrdquoThe Journal of Immunology vol 186 no 7 pp 4388ndash4395 2011

[14] M Tosolini A Kirilovsky B Mlecnik et al ldquoClinical impactof different classes of infiltrating T cytotoxic and helper cells(Th1Th2 TregTh17) in patientswith colorectal cancerrdquoCancerResearch vol 71 no 4 pp 1263ndash1271 2011

[15] Q Li L Liu Q Zhang S Liu D Ge and Z You ldquoInterleukin-17 indirectly promotes M2 macrophage differentiation throughstimulation of COX-2PGE2 pathway in the cancer cellsrdquoCancer Research andTreatment vol 46 no 3 pp 297ndash306 2014

[16] J Shao C Jung C Liu and H Sheng ldquoProstaglandin E2stimulates the 120573-cateninT cell factor-dependent transcriptionin colon cancerrdquo The Journal of Biological Chemistry vol 280no 28 pp 26565ndash26572 2005

[17] P Raman B L F Kaplan J T Thompson J P VandenHeuvel and N E Kaminski ldquo15-deoxy-Δ1214-prostaglandinJ2-glycerol ester a putativemetabolite of 2-arachidonyl glycerolactivates peroxisome proliferator activated receptor 120574rdquoMolecu-lar Pharmacology vol 80 no 1 pp 201ndash209 2011

[18] Z Wan W Shi B Shao et al ldquoPeroxisome proliferator-activated receptor 120574 agonist pioglitazone inhibits 120573-catenin-mediated glioma cell growth and invasionrdquo Molecular andCellular Biochemistry vol 349 no 1-2 pp 1ndash10 2011

[19] L Klotz S Burgdorf I Dani et al ldquoThe nuclear receptorPPARgamma selectively inhibits Th17 differentiation in a Tcell-intrinsic fashion and suppresses CNS autoimmunityrdquo TheJournal of Experimental Medicine vol 206 no 10 pp 2079ndash2089 2009

[20] Z Zhang A Andoh O Inatomi et al ldquoInterleukin-17 andlipopolysaccharides synergistically induce cyclooxygenase-2expression in human intestinal myofibroblastsrdquo Journal ofGastroenterology and Hepatology vol 20 no 4 pp 619ndash6272005

[21] D Wang and R N Dubois ldquoThe role of COX-2 in intestinalinflammation and colorectal cancerrdquo Oncogene vol 29 no 6pp 781ndash788 2010

[22] L Vossaert T OrsquoLeary C Van Neste et al ldquoReference locifor RT-qPCR analysis of differentiating human embryonic stemcellsrdquo BMC Molecular Biology vol 14 no 1 pp 1ndash7 2013

[23] B Xiong T-J Sun W-D Hu F-L Cheng M Mao and Y-FZhou ldquoExpression of cyclooxygenase-2 in colorectal cancer andits clinical significancerdquoWorld Journal of Gastroenterology vol11 no 8 pp 1105ndash1108 2005

[24] A P R Paiotti R Artigiani Neto N M Forones C T FOshima S JMiszputen andM Franco ldquoImmunoexpression ofcyclooxygenase-1 and -2 in ulcerative colitisrdquo Brazilian Journalof Medical and Biological Research vol 40 no 7 pp 911ndash9182007

[25] M Romero R Artigiani H Costa C T F Oshima S Misz-puten and M Franco ldquoEvaluation of the immunoexpression


of COX-1 COX-2 and p53 in Crohnrsquos diseaserdquo Arquivos deGastroenterologia vol 45 no 4 pp 295ndash300 2008

[26] A Serafino N Moroni M Zonfrillo et al ldquoWNT-pathwaycomponents as predictive markers useful for diagnosis preven-tion and therapy in inflammatory bowel disease and sporadiccolorectal cancerrdquoOncotarget vol 5 no 4 pp 978ndash992 2014

[27] M M H Claessen M E I Schipper B Oldenburg P DSiersema G J A Offerhaus and F P Vleggaar ldquoWNT-pathwayactivation in IBD-associated colorectal carcinogenesis poten-tial biomarkers for colonic surveillancerdquoCellular Oncology vol32 no 4 pp 303ndash310 2010

[28] J Hendel and O H Nielsen ldquoExpression of cyclooxygenase-2mRNA in active inflammatory bowel diseaserdquoAmerican Journalof Gastroenterology vol 92 no 7 pp 1170ndash1173 1997

[29] H M Roelofs R H Te Morsche B W van Heumen FM Nagengast and W H Peters ldquoOver-expression of COX-2mRNA in colorectal cancerrdquo BMC Gastroenterology vol 14 no1 2014

[30] J Chang J Vacher B Yao et al ldquoProstaglandin E receptor 4(EP4) promotes colonic tumorigenesisrdquo Oncotarget vol 6 no32 pp 33500ndash33511 2015

[31] P-C Lin Y-J Lin C-T Lee H-S Liu and J-C LeeldquoCyclooxygenase-2 expression in the tumor environment isassociated with poor prognosis in colorectal cancer patientsrdquoOncology Letters vol 6 no 3 pp 733ndash739 2013

[32] Y Araki S Okamura S P Hussain et al ldquoRegulation ofcyclooxygenase-2 expression by the WNT and ras pathwaysrdquoCancer Research vol 63 no 3 pp 728ndash734 2003

[33] A L Eisinger L D Nadauld D N Shelton et al ldquoTheadenomatous polyposis coli tumor suppressor gene regulatesexpression of cyclooxygenase-2 by a mechanism that involvesretinoic acidrdquo The Journal of Biological Chemistry vol 281 no29 pp 20474ndash20482 2006

[34] I Kim H Kwak H K Lee S Hyun and S Jeong ldquoΒ-Cateninrecognizes a specificRNAmotif in the cyclooxygenase-2mRNA31015840-UTR and interacts with HuR in colon cancer cellsrdquo NucleicAcids Research vol 40 no 14 pp 6863ndash6872 2012

[35] B Zou L Qiao and B C Wong ldquoCurrent understandingof the role of PPARgamma in gastrointestinal cancersrdquo PPARResearch vol 2009 Article ID 816957 8 pages 2009

[36] V PazienzaMVinciguerra andGMazzoccoli ldquoPPARs signal-ing and cancer in the gastrointestinal systemrdquo PPAR Researchvol 2012 Article ID 560846 10 pages 2012

[37] P Sarraf E Mueller D Jones et al ldquoDifferentiation and reversalof malignant changes in colon cancer through PPAR120574rdquo NatureMedicine vol 4 no 9 pp 1046ndash1052 1998

[38] D Shen C Deng and M Zhang ldquoPeroxisome proliferator-activated receptor 120574 agonists inhibit the proliferation andinvasion of human colon cancer cellsrdquo Postgraduate MedicalJournal vol 83 no 980 pp 414ndash419 2007

[39] X Dou J Xiao Z Jin and P Zheng ldquoPeroxisome proliferator-activated receptor-120574 is downregulated in ulcerative colitis and isinvolved in experimental colitis-associated neoplasiardquoOncologyLetters vol 10 no 3 pp 1259ndash1266 2015

[40] L Dubuquoy S Dharancy S Nutten S Pettersson J Auwerxand P Desreumaux ldquoRole of peroxisome proliferator-activatedreceptor 120574 and retinoid X receptor heterodimer in hepatogas-troenterological diseasesrdquo The Lancet vol 360 no 9343 pp1410ndash1418 2002

[41] Sheval Kansal I Kim Vaiphei and Navneet Agnihotri ldquoAlter-ations in lipid mediated signaling andWnt120573-catenin signaling

in dmh induced colon cancer on supplementation of fish oilrdquoBioMed Research International vol 2014 Article ID 832025 11pages 2014

[42] Z Xie Y Qu Y Leng et al ldquoHuman colon carcinogenesis isassociated with increased interleukin-17-driven inflammatoryresponsesrdquo Drug Design Development and Therapy vol 9 pp1679ndash1689 2015

[43] Y S Hyun D S Han A R Lee C S Eun J Youn and H-YKim ldquoRole of IL-17A in the development of colitis-associatedcancerrdquo Carcinogenesis vol 33 no 4 pp 931ndash936 2012

[44] E J Greenspan J P Madigan L A Boardman and D WRosenberg ldquoIbuprofen inhibits activation of nuclear 120573-cateninin human colon adenomas and induces the phosphorylation ofGSK-3120573rdquo Cancer Prevention Research vol 4 no 1 pp 161ndash1712011

[45] J U Scher and M H Pillinger ldquoThe anti-inflammatory effectsof prostaglandinsrdquo Journal of Investigative Medicine vol 57 no6 pp 703ndash708 2009

[46] M R Fernando M A Giembycz and D M McKayldquoBidirectional crosstalk via IL-6 PGE2 and PGD2 betweenmurinemyofibroblasts and alternatively activatedmacrophagesenhances anti-inflammatory phenotype in both cellsrdquo BritishJournal of Pharmacology vol 173 no 5 pp 899ndash912 2016

[47] N N Rumzhum B S Patel P Prabhala I C GelissenB G Oliver and A J Ammit ldquoIL-17A increases TNF-120572-induced COX-2 protein stability and augments PGE2 secretionfrom airway smooth muscle cells Impact on 1205732-adrenergicreceptor desensitizationrdquo Allergy European Journal of Allergyand Clinical Immunology vol 71 no 3 pp 387ndash396 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


upregulated in sporadic CRC and IBD [6] Moreover COX-2inhibitors have been shown to reduce carcinogenesis in bothanimal and human studies [7]

Peroxisome proliferator-activated receptor 120574 (PPAR120574)a ligand-activated nuclear transcription factor has beenimplicated in colorectal carcinogenesis although its exactrole remains unclear PPAR120574 activation is associated withinhibition of cell growth in human colon cancer cell linesas well as cancer xenografts in nude mice Mice witha heterozygous deletion of PPAR120574 (PPAR120574+minus) have anincreased tendency to develop carcinogen-induced CRC [8]Activated PPAR120574 inhibits colorectal carcinogenesis throughdownstream inhibition of the 120573-catenin mediated transcrip-tion pathway [9] Recent data showed that PPAR120574 stimu-lation inhibits Th17 activation and IL-17 production [10]Elevated PPAR120574 expression confers a good prognosis in CRC[11]

Inflammatory Th17 helper T cells derive their name bytheir ability to produce IL-17 cytokines (IL-17A-F) amongothers [12] Th17 derived cytokines play a role in gutinflammation and carcinogenesis linking IBD and CRC [13]Tosolini et al recently found that increased IL-17A mRNAexpression in the tumour tissue is associated with increasedcancer recurrence and poor survival suggesting adverseprognosis [14] Mechanistically IL-17 promotes colorectalcarcinogenesis through direct stimulation of COX-2PGE

2

[15]These molecules have an intricate interrelationship dur-

ing carcinogenesis and each one influences the expressionand function of the others COX-2 has been shown tomodulate both the wnt120573-catenin and PPAR120574 pathwaysPGE2and 15-Deoxy-Prostaglandin J

2(15-D-PGJ

2) down-

stream products of the COX-2 biosynthetic pathway increaseaccumulation and nuclear translocation of 120573-catenin andPPAR120574 respectively [16 17] PPAR120574 activation has beenshown to inhibit 120573-catenin expression and IL-17 activation[18 19] IL-17 has been shown to upregulate COX-2PGE

2

[20]In this study we examined the interrelationship of 120573-

catenin COX-2 PPAR120574 and IL-17 in patients with sporadiccolon cancer compared to subjects without cancer Furthercomparisons were made in patient populations with irritablebowel syndrome (IBS) with lowCRC risk and IBD with highrisk for CRC development [21]

The study results confirm a complex relationship betweenthese role players in colorectal carcinogenesis In both theNormal and IBS groups homeostatic mechanisms remainintact with low cell turnover as evidenced by low levels of 120573-catenin and high levels of PPAR120574 Inflammation is also keptin check by sufficient COX-2 protein expression and reducedIL-17A expression These findings were inverted for IBD andCRC

2 Materials and Methods

21 Study Population The Human Ethics Committee of theNelson Mandela Metropolitan University (H07SciBCN-001)approved the study Written informed consent was obtainedfrom each participant on enrolment Patients were grouped

into (1) Normal patients attending for screening and surveil-lance colonoscopy (2) IBS patients with constipation- anddiarrhoea-predominant IBS using Rome II criteria (3) IBDCrohnrsquos disease (CD) and Ulcerative colitis (UC) patients (4)CRC

22 Tissue Collection Colon mucosal biopsies were takenfrom the normal caecum and ascending colon in the Normaland IBS groups from the inflamed mucosa in IBD and fromthe malignant tissue in CRC Samples were (i) placed inRNA-later (Sigma) and stored at minus20∘ until analysis and (ii)preserved in 4 paraformaldehyde and embedded in paraffinfollowing standard procedures

23 Quantitative Real-Time Polymerase Chain Reaction(qPCR) Total RNA was extracted (Bio-Rad Aurum RNAextraction kit) following the manufacturerrsquos instructionscDNA was synthesised from 1120583g RNA using the reversetranscription BioRad iScript cDNA synthesis kit (Bio-RadLaboratories Inc USA) The cDNA samples were frozen atminus20∘C until required

TheGeNormNormalization (PrimerDesign UK) algo-rithm was used to identify three stably expressed referencegenes These were SF3A1 and two Alu repeats ALUsq andALUsx [22] Real-time qPCR reactionswere run on an iCyclerIQ system (Bio-Rad Laboratories Inc USA)

To each well of a 96 well PCR plate 15120583l of mastermix consisting of 10120583l SYBRGreen supermix (Bio-Rad)and 500 nM forward and reverse primers was added 5 120583l ofthe appropriate cDNA (15 diluted) was added to each wellexcept for two wells with no template controls (NTC) wherethe cDNA sample was replaced with 5 120583l of nuclease-freePCR grade water (Ambion USA) Experiments were run induplicate

The protocol for all qPCR runs is comprised of 3-minuteTaqpolymerase activation at 95∘Cand 40 cycles of denaturingat 95∘C for 30 seconds 30 seconds at appropriate annealingtemperature (Table 1) and extension at 72∘C for 30 seconds

The amplification reaction was followed by a melt curveto verify the specificity of the reactionThe plate was heated to95∘C and then cooled to 1∘C below the annealing temperatureto ensure that all the DNAwas double stranded The temper-ature was increased in increments of 05∘C for 30 seconds upto 95∘C to melt the double stranded DNA

Genes of interest were analysed following the above qPCRprotocol and Cq values compared using qBase version 2software (BioGazelle) Table 1 details all genes used in thisstudy

24 Immunohistochemistry Four micrometer sections wereused for IHC staining using the Ventana ESautomatedsystem (Ventana Medical Systems) following the manufac-turerrsquos instructions The tissue sections were deparaffinisedand rehydrated following standard procedures Antigenretrieval was performed by incubating tissue sections in10mM citrate buffer in a microwave for 15 minutes Slideswere transferred to an automated slide stainer Endogenousperoxidases were quenched by incubating tissue sections











3 Results





1

2

3

4-catenin mRNA

Diagnosis

Mea

n C

NRQ

lowast

lowast lowast


050

100

150


S

tain

ing

inte

nsity

01+

2+3+



0

50

100


S

tain

ing

inte

nsity

01+

2+3+









(a) (b) (c)

(d) (e) (f)


0

2

4

6

8COX-2 mRNA

Mea

n C

NRQ


lowast

lowast

lowast


0

20

40

60

80

100COX-2 IHC

S

atin

ing

inte

nsity

01+

2+3+





(a) (b) (c)

(d) (e) (f)








4 Discussion





2[30] COX-2 protein




0

1

2

IL-17A mRNAM

ean

CN

RQ


lowast

lowastlowast


00

05

10

15

20

25PPAR mRNA

Mea

n C

NRQ


lowast

lowast









2and ultimately


2




COX-2

PGD2

15D-PGJ2 PGE2

IL-17A

PPAR -CATENIN

APC

TZD Dietary PUFA

PPARRXR

Target genes

Differentiation

-CateninTCF

Target genesc-myc

cyclin D

Proliferation

COX-Inh



Inflammation




2upregula-



2 both of which


2[47] PPAR120574





5 Conclusion




Data Availability




Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























3 Results





1

2

3

4-catenin mRNA

Diagnosis

Mea

n C

NRQ

lowast

lowast lowast


050

100

150


S

tain

ing

inte

nsity

01+

2+3+



0

50

100


S

tain

ing

inte

nsity

01+

2+3+









(a) (b) (c)

(d) (e) (f)


0

2

4

6

8COX-2 mRNA

Mea

n C

NRQ


lowast

lowast

lowast


0

20

40

60

80

100COX-2 IHC

S

atin

ing

inte

nsity

01+

2+3+





(a) (b) (c)

(d) (e) (f)








4 Discussion





2[30] COX-2 protein




0

1

2

IL-17A mRNAM

ean

CN

RQ


lowast

lowastlowast


00

05

10

15

20

25PPAR mRNA

Mea

n C

NRQ


lowast

lowast









2and ultimately


2




COX-2

PGD2

15D-PGJ2 PGE2

IL-17A

PPAR -CATENIN

APC

TZD Dietary PUFA

PPARRXR

Target genes

Differentiation

-CateninTCF

Target genesc-myc

cyclin D

Proliferation

COX-Inh



Inflammation




2upregula-



2 both of which


2[47] PPAR120574





5 Conclusion




Data Availability




Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















1

2

3

4-catenin mRNA

Diagnosis

Mea

n C

NRQ

lowast

lowast lowast


050

100

150


S

tain

ing

inte

nsity

01+

2+3+



0

50

100


S

tain

ing

inte

nsity

01+

2+3+









(a) (b) (c)

(d) (e) (f)


0

2

4

6

8COX-2 mRNA

Mea

n C

NRQ


lowast

lowast

lowast


0

20

40

60

80

100COX-2 IHC

S

atin

ing

inte

nsity

01+

2+3+





(a) (b) (c)

(d) (e) (f)








4 Discussion





2[30] COX-2 protein




0

1

2

IL-17A mRNAM

ean

CN

RQ


lowast

lowastlowast


00

05

10

15

20

25PPAR mRNA

Mea

n C

NRQ


lowast

lowast









2and ultimately


2




COX-2

PGD2

15D-PGJ2 PGE2

IL-17A

PPAR -CATENIN

APC

TZD Dietary PUFA

PPARRXR

Target genes

Differentiation

-CateninTCF

Target genesc-myc

cyclin D

Proliferation

COX-Inh



Inflammation




2upregula-



2 both of which


2[47] PPAR120574





5 Conclusion




Data Availability




Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b) (c)

(d) (e) (f)


0

2

4

6

8COX-2 mRNA

Mea

n C

NRQ


lowast

lowast

lowast


0

20

40

60

80

100COX-2 IHC

S

atin

ing

inte

nsity

01+

2+3+





(a) (b) (c)

(d) (e) (f)








4 Discussion





2[30] COX-2 protein




0

1

2

IL-17A mRNAM

ean

CN

RQ


lowast

lowastlowast


00

05

10

15

20

25PPAR mRNA

Mea

n C

NRQ


lowast

lowast









2and ultimately


2




COX-2

PGD2

15D-PGJ2 PGE2

IL-17A

PPAR -CATENIN

APC

TZD Dietary PUFA

PPARRXR

Target genes

Differentiation

-CateninTCF

Target genesc-myc

cyclin D

Proliferation

COX-Inh



Inflammation




2upregula-



2 both of which


2[47] PPAR120574





5 Conclusion




Data Availability




Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b) (c)

(d) (e) (f)








4 Discussion





2[30] COX-2 protein




0

1

2

IL-17A mRNAM

ean

CN

RQ


lowast

lowastlowast


00

05

10

15

20

25PPAR mRNA

Mea

n C

NRQ


lowast

lowast









2and ultimately


2




COX-2

PGD2

15D-PGJ2 PGE2

IL-17A

PPAR -CATENIN

APC

TZD Dietary PUFA

PPARRXR

Target genes

Differentiation

-CateninTCF

Target genesc-myc

cyclin D

Proliferation

COX-Inh



Inflammation




2upregula-



2 both of which


2[47] PPAR120574





5 Conclusion




Data Availability




Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

1

2

IL-17A mRNAM

ean

CN

RQ


lowast

lowastlowast


00

05

10

15

20

25PPAR mRNA

Mea

n C

NRQ


lowast

lowast









2and ultimately


2




COX-2

PGD2

15D-PGJ2 PGE2

IL-17A

PPAR -CATENIN

APC

TZD Dietary PUFA

PPARRXR

Target genes

Differentiation

-CateninTCF

Target genesc-myc

cyclin D

Proliferation

COX-Inh



Inflammation




2upregula-



2 both of which


2[47] PPAR120574





5 Conclusion




Data Availability




Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















COX-2

PGD2

15D-PGJ2 PGE2

IL-17A

PPAR -CATENIN

APC

TZD Dietary PUFA

PPARRXR

Target genes

Differentiation

-CateninTCF

Target genesc-myc

cyclin D

Proliferation

COX-Inh



Inflammation




2upregula-



2 both of which


2[47] PPAR120574





5 Conclusion




Data Availability




Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Data Availability




Acknowledgments


References
























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















-Catenin Regulation in Sporadic Colorectal Carcinogenesis...

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Transcript of -Catenin Regulation in Sporadic Colorectal Carcinogenesis...