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Globular adiponectin and its downstream target genes areup-regulated locally in human colorectal tumors:ex vivo and in vitro studies☆
Katja Kannisto Vetvika, b,⁎, Tonje Soneruda, Mona Lindebergd, f, Torben Lüdersa,Ragnhild H. Størksona, e, Kristin Jonsdottir a, Eirik Frengend, f,Kirsi H. Pietiläineng, h, Ida Bukholma, b, c
a Department of Clinical Molecular Biology and Laboratory Sciences (EpiGen), Institute for Clinical Medicine, University of Oslo, Norwayb Surgical Department, Akershus University Hospital, Lørenskog, Norwayc Institute of Health Promotion, Akershus University Hospital, Lørenskog, Norwayd Department of Medical Genetics, Oslo University Hospital, Oslo, Norwaye Surgical Department, Østfold Hospital Trust, Fredrikstad, Norwayf Department of Medical Genetics, University of Oslo, Norwayg Obesity Research Unit, Department of Medicine, Division of Endocrinology, Helsinki University Central Hospital andUniversity of Helsinki, Finlandh Institute of Molecular Medicine Finland, FIMM, University of Helsinki, Helsinki, Finland
A R T I C L E I N F O
Abbreviations: AGRP, agouti-related peptidAdipoR2, adiponectin receptor type 2; ACC,protein 39; CPT, carnitine palmitoyltransftransporter; FDR, false discovery rate; gAd,HMW, high-molecular-weight polymer; JAK,activated protein kinase; MSH, α-melanocysteatohepatitis; NPY, neuropeptide Y; pAMPCART, proopiomelanocortin/cocaine- and amsubunit; qPCR, quantitative real-time PCR;phosphatase nonreceptor type 1; siRNA, sitransport protein 1; STAT, signal transduceprotein; TFIIB, transcription factor IIB.☆ MICROARRAY DATA: Themicroarray datano. E-MTAB-833.⁎ Corresponding author at: Akershus Universi
Article history:Received 18 April 2013Accepted 3 February 2014
Objective. Low plasma adiponectin levels are linked to obesity, insulin resistance, and therisk of several types of malignancy. Despite the decline in circulating adiponectinconcentrations, the increase in the expression of adiponectin receptors AdipoR1 andAdipoR2 is greater in cancerous than in normal colonic tissue. The purpose of this study wasto obtain new information regarding local adiponectin signaling in the pathogenesis ofcolorectal cancer (CRC).
Methods.We characterized the expressions of adiponectin and several of its downstreamtargets in paired samples of tumor tissue and adjacent noncancerous mucosa in 60 surgicalpatients with colorectal adenocarcinomas.
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Results. Adiponectin was expressed in both colorectal tumors and the adjacent mucosa.The expressions of adiponectin mRNA and its globular protein variant (gAd), adiponectinreceptor type 1 and 5′AMP-activated protein kinase (AMPK)mRNAwere significantly higherin colorectal tumors than in the adjacent mucosa. This finding was accompanied byincreased mRNA expression of genes encoding proteins involved in fatty-acid traffickingand oxidation. The potential interference between adiponectin stimulation and AMPKactivation through AMPK1 was examined in an in vitro model with the aid of silencing-RNAexperiments. Furthermore, AMPK mRNA expression on tumors was positively correlatedwith a more advanced tumor stage in the patients.
Conclusion. We propose that the globular adiponectin-AMPK pathway functions in anautocrinemanner in colorectal tumors, explaining some of the beneficial changes in cellularoxidative capacity in tumors in favor of tumorigenesis.
Adipose tissue is involved in the metabolic and inflammatoryresponses of the body by secreting adipokines, which aresystemic mediators [1]. These cytokines play multiple roles incellular processes such as cell proliferation, differentiation,and apoptosis [2]. Adiponectin is a hormone secreted byadipocytes that has diverse biological functions, includingstimulation of glucose utilization, fatty-acid oxidation, andinhibition of gluconeogenesis [3]. Low levels of circulatingadiponectin are associated with obesity and insulin resis-tance, as well as increased risk for the development andprogression of several obesity-related malignancies, such asbreast, endometrial, prostate, and colon cancers [4].
The plasma levels of full-length adiponectin (fAd) (alsoknown as 30-kDa adipocyte complement-related protein)exceed 10 μg/ml and constitute ≈0.01% of the total amountof plasma protein [3,5]. Proteolytic cleavage of fAd at aminoacid 110 produces globular adiponectin (gAd) [6]. fAd candimerize and most of the adiponectin present in plasma isoligomerized to form a high-molecular-weight polymer(HMW) [7,8], whereas the abundance of the low-molecular-weight form, or gAd, is normally low [9,10]. It has beendemonstrated that oligomerization and posttranslationalmodifications of adiponectin are critical for binding to itsreceptors and to determine its biological activity. Thus,different oligomeric forms of adiponectin, or gAd versusfAd, exhibit distinct biological effects through the differen-tial activation of downstream signaling pathways [5] whichmay explain their eventual different roles in tumordevelopment [11].
Two adiponectin receptors have been described: AdipoR1and AdipoR2 [12]. These receptors mediate the signaling ofboth fAd and gAd. AdipoR1 has been shown to bind gAd withhigher affinity than fAd, whereas AdipoR2 seems to be anintermediate-affinity receptor for both forms [7,12]. AdipoR1 isubiquitously expressed, but is present most abundantly inskeletal muscle, whereas AdipoR2 is predominantlyexpressed in the liver [12]. Activation of the two receptorvariants reveals clearly opposing metabolic functions in vivo[13], as observed in studies on knockout animal models.AdipoR1 signaling occurs mainly through 5′ AMP-activatedprotein kinase (AMPK), involving increased energy consump-
tion in the form of increased glucose and fatty-acid oxidationand energy expenditure in vivo, whereas AdipoR2 acts throughperoxisome proliferator-activated receptor α-related path-ways and seems to be involved in increased storage of energy,glucose intolerance, and reduced energy expenditure. Theexpressions of both types of adiponectin receptor have beenidentified in several cancer types, whereas AdipoR1 wasrecently shown to be more ubiquitously expressed in obesity-associated cancers [14,15]. Previous studies have demonstratedincreased adiponectin receptor expression in colon cancer butnot in prostate cancer [16,17]. Investigation of the role of thesetwo receptors and the pathways that are involved wouldprovide additional information regarding the role of adiponec-tin signaling during tumor development.
It has been hypothesized that the generally beneficialmetabolic and antioxidative actions of adiponectin can beexplained in part through its activation of the AMPK pathway[18]. There is mounting evidence for both this observation andthe finding that activation of AMPK constitutes a generalintracellular response to adiponectin mainly by activationthrough AdipoR1: it has been shown that AMPK is activated inresponse to adiponectin treatment in adipocytes [19], myo-cytes and hepatocytes [20], pancreatic β-cells [21], cardiomyo-cytes [22], and endothelial cells [23], as well as in colon (HT-29)and prostate (PC-3) cancer cells [18]. AMPK activation sup-presses cell proliferation through multiple mechanisms,including suppression of the mammalian target of rapamycin(mTOR) pathway and inhibition of the enzymes implicated inthe regulation of protein and fatty-acid and triacylglycerolsynthesis [acetyl-CoA carboxylase (ACC2) and fatty-acidsynthase]. In addition, adiponectin, through activation ofAMPK, negatively regulates the downstream members in themTOR pathway, such as the p70 S6 kinase and S6 protein[20,24,25]. It is speculated that activation of the mTORpathway directly promotes colonic epithelial cell prolifera-tion, and thereby colorectal carcinogenesis [26].
The role of adiponectin in malignancies may be explainedboth by its effect at the systemic level, whereby it regulateswhole-body insulin sensitivity or inflammatory processes,and at the cellular level, whereby it acts directly on cancercells. In line with epidemiological data, there is accumulatingevidence from several previous in vitro studies that adiponec-tin is an important regulator of cell proliferation [27]. A
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previous study found that both specific inhibitors of AMPKand a specific silencing RNA (siRNA) to AdipoR1 blocked theantiproliferative effects of adiponectin [28]. Plasma adiponec-tin administration suppressed the growth of intestinaladenomas in the Apc(Min)(/+) mice [29], and reducedimplanted tumor growth and angiogenesis in tumors obtainedfrom mice fed a high-fat diet and from adiponectin-deficientC57BL/6 mice [30]. Consistent results were obtained whenapplying a high-fat diet to adiponectin-receptor-knockoutmice [26]. Despite an increasing amount of experimental data,the mechanisms underlying the antiproliferative and tumor-suppressing effects of adiponectin are not fully understood,and some of the results show opposite actions. For example,Ogunwobi and Beales demonstrated that adiponectin stimu-lates the proliferation of colon cancer HT-29 cells [31]. Thediscrepancies between the aforementioned and other previ-ous results could be partly explained by the findings of Habeebet al., which suggest that the bimodal action of adiponectindepends upon the glucose level in the culture medium, suchthat adiponectin supports cell survival in a glucose-deprivedmedium but not in a glucose-enriched medium [32]. However,most of the previous studies focused on the role of fAd ratherthan gAd stimulation in cell proliferation.
For the present study it was proposed that autocrineactivation of the genes in the gAd-AdipoR1-AMPK pathway bytumor cells could be one of the mechanisms underlying theimprovement in tumor-cell metabolism, providing a linkbetween colorectal tumor disease, obesity and the body’smetabolic state. This study is the first to examine adiponectinsecretion in tumors in vivo, and the first to reveal increasedgAd expression in tumors.
2. Methods
Details regarding the materials and methods are provided inthe Supplementary Materials and Methods section. Thematerials and methods are summarized below.
2.1. Study subjects
The study comprised samples of tumor and the adjacentnormal mucosa, which were removed surgically from 60patients with colorectal adenocarcinoma at the AkershusUniversity Hospital, Norway. All patients had read, under-stood, and signed an informed consent document before theircolorectal surgery and the collection of either samples or anypatient data. Patients eventually using any drugs that maypossess anticancer effects, such as nonsteroidal anti-inflam-matory drugs and metformin, were not excluded. Samplesfrom colorectal tumors and adjacent noncancerous mucosawere collected from the patients during open colorectalsurgery. The tumor tissue was collected from solid tumorsthat were macroscopically visible, and subsequently investi-gated microscopically and characterized histologically. Onlypatients with malignant tumors were included. Radical tumorresection was ensured by employing a resection margin ofseveral centimeters. Adjacent mucosal tissue was collectedwith a margin of several centimeters from the solid tumor,
from an area that was confirmed to be tumor free throughboth macroscopic and microscopic investigation. The prepa-ration of tissues is reported elsewhere [33]. The study wasperformed in accordance with the principles of the HelsinkiDeclaration and was approved by the local ethics committee(REK Sør-Øst, Blindern, Oslo) before any study-related activ-ities took place.
2.2. RNA isolation and quantitative real-time PCR (qPCR)
Total RNA was isolated using a Trizol/RNeasy hybrid protocol[34]. RNA was reverse transcribed to cDNA using the Super-Script First Strand Synthesis System (Invitrogen, Carlsbad, CA,USA). TaqMan gene-expression assays were purchased fromApplied Biosystems (Bedford, MA, USA) and the qPCR wasperformed with the aid of the ABI PRISM 7900 HT Fast qPCRsystem (Applied Biosystems), as reported previously [33]. Theprimers are listed in the Supplementary Materials andMethods. General transcription factor IIB [TATA-bindingprotein (TPB)/transcription factor IIB (TFIIB)] expression wasused as a reference gene (TPB/TFIIB) for normalization. All datawere calculated relative to TPB, and the ratios between theexpression in the tumor samples and normal colon samplesare presented.
2.3. Oligo microarray analysis
Oligo microarray analyses were available for 23 of the 60sample pairs. Agilent’s Human Whole Genome Oligo Micro-arrays (44 K), which contain ~41,000 probes of genes andtranscripts, were used. Data collection and quality assessmentwere performed using Agilent G2567AA Feature Extractionsoftware v8.5 (with the default parameters). Data analysis wasperformed using J-express Pro v2.7 (Molmine, Bergen, Nor-way). The LSimpute function calculated missing values forgenes with less than 30% missing values. A two-classSignificant Analysis of Microarray (SAM) [35] was used toidentify genes that were significantly differentially expressedin the tumor samples compared to the samples from adjacentnormal mucosa, implemented in J-express with 1,000 permu-tations and a false discovery rate (FDR) of <10%. Pathwayanalyses were carried out in the Database for Annotation,Visualization, and Integrated Discovery (http://david.abcc.ncifcrf.gov/), which applies the gene list found in the KyotoEncyclopedia of Genes and Genomes pathway. The microar-ray data have been deposited in ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) under accession no. E-MTAB-833.
2.4. Cell culture and transient transfection of siRNA forthe knockdown of AdipoR1
The colorectal adenocarcinoma cell line WiDr (ATCC NumberCCL-218) was purchased from LGC Promochem (Boras, Sweden).ON-TARGETplus siRNA AdipoR1 (Catalog nr J-007800-10) waspurchased from Thermo Fisher Scientific (Lafayette, CO, USA).Four different siRNAs for AdipoR1, as well as a mix of all four,were tested on WiDr colon cancer cells, and the best siRNA wasselected. Transient transfections were carried out with Dharma-fect4 transfection reagent (Thermo Fisher Scientific). Since
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AdipoR1 activation is dependent upon the presence of its ligand[5], gAd was added to the cell culture in order to reveal thedownstream effect of the AdipoR1 knockdown on AMPKphosphorylation. The transfected cells were incubated with orwithout 2.5 μg/ml gAd (AXXORA, San Diego, CA, USA) for 15 min,30 min, or 24 h before harvesting them.
2.5. Protein extraction and Western blot analysis
Detailed procedures for protein extraction from the colorectaltissue and Western blot analysis, as well as a list of theantibodies used, are provided in the Supplementary Materialsand Methods.
2.6. Statistical analyses
The mRNA expression of each gene was compared betweencolorectal tumor and normal tissue samples using pairedWilcoxon’s rank-sum tests for nonparametric data. Anunpaired Mann–Whitney U test was used for comparisons ofgene expressions between males and females. Relativechanges (tumor divided by normal tissue) in gene expressionswere used when evaluating the role of clinical pathologicalstages on changes in gene expression. The Mann–Whitney Utest was also used for comparisons between medians in geneexpressions in Dukes’ B and C stages, and Spearman’scorrelation coefficient was calculated between gene expres-sion and categorical or continuous tumor characteristics(localization, size, differentiation, and lymph nodes affected).Spearman’s correlation analyses between selected geneexpressions involved either the relative changes (tumordivided by normal tissue) or tumor values. Statistical analyseswere performed using Stata statistical software (release 9.0,Stata Corporation, College Station, TX, USA).
3. Results
3.1. Clinical characteristics
This study analyzed paired colorectal tissues (tumor andadjacent normal mucosa) from 60 patients for pathwaysinvolved in the regulation of tumor metabolism. The ratioof males/females was 40/20, and the age at surgery of theentire study population was 61.8 ± 1.5 years (mean ± SEM).The distribution of tumor location was as follows: 34% in therectum, 25% in the sigmoid colon, and the remainder in themore proximal portions of colon. The tumors were 5.0 ±0.3 cm in diameter. Histologically, most (80%) of the patientshad moderately differentiated tumors. The tumors werestaged as either Dukes’ B (51.7%) or C (48.3%), and the numberof lymph nodes affected ranged from 0 to 22, with half of thepatients having no affected nodes.
3.2. Increased mRNA expression of AMPK-related genes intumor tissue, as revealed by microarray analysis
Microarray expression arrays were first used to measuremRNA transcript profiles from 23 paired samples of colorectal
tumor and adjacent normal tissue from the same patients.The sampleswere randomly selected frommaterial consistingof paired samples. SAM with 1,000 permutations and an FDRof 0% yielded 3,738 significant genes. The regulators of theAMPK pathway were studied in order to identify the micro-matrix mRNA expression of the leptin and adiponectinpathways in tumors, which involve two adipocyte-specifichormones previously shown to activate AMPK (Fig. 1, Table 1)[19,36]. The genes encoding glucose transporter type 4 (GLUT4;FDR = 0.1), carnitine palmitoyltransferase I (CPT1; FDR = 0.0X),and carnitine palmitoyltransferase 2 (CPT2; FDR = 0.0X) weresignificantly elevated in tumors relative to the adjacentnormal colorectal tissue from the same patients. The expres-sion of AMPKwas also increased, but with a lower significance(FDR = 13.1). The expressions of the genes encoding tyrosine-protein phosphatase nonreceptor type 11 (SHP2; FDR = 0.0–0.6)and those encoding long-chain fatty acid transport protein 1(SLC27A1; FDR = 3.0) were significantly lower in the tumorsthan in the normal tissue in the paired samples.
3.3. Increased mRNA expression of AMPK-related genes intumors, as revealed by qPCR
To verify the results from the global gene-array analysis andto further identify the regulators of the AMPK pathway, qPCRanalysis was performed for the following genes, which areknown to be involved in the AMPK pathway: adiponectin,ADIPOR1, ADIPOR2, AMPK, ACC2, plasma membrane fatty-acid-binding protein (FABP-pm), fatty acid transporter (FATP),CPT1, and fatty-acid-binding protein 4 (FABP-4). qPCR analysisconfirmed the results from the gene-array analysis regardingleptin, whose expressionwas the same in normalmucosa andtumor tissue, and hence it was not therefore a candidate foran intrinsic activator of the AMPK pathway in the colorectalmucosa cells. In contrast, adiponectin mRNA expression wasdetected in colorectal epithelium, andwas significantly higherin tumor tissue than in adjacent normal mucosa from thesame patient (2.7-fold, p < 0.0001). Adiponectin exerts itseffects through its receptors AdipoR1 and AdipoR2. AdipoR1(1.6-fold, p = 0.0037) but not AdipoR2 (p = 0.42) mRNA expres-sion was significantly higher in tumor tissue than in thesample from adjacent normal mucosa from the same patient.The ratio of the expressions of AdipoR1 to AdipoR2 was higherin the tumors (1.50 ± 0.12) than in the normal samples (1.19 ±0.08, p = 0.012). It has been demonstrated that activation ofAdipoR1 leads to an increase in AMPK activation in several celltypes [18–23]. In our material, AMPK mRNA expression was1.5-fold (p = 0.034) higher in the tumor tissue than in theadjacent normal mucosa, and was significantly correlatedwith AdipoR1 mRNA expression (r = 0.92, p < 0.0001). Therewas also a significant positive correlation (r = 0.85, p < 0.0001)between AdipoR1 and AMPKmRNA expressions in the normalmucosal tissue, but with much lower expression levels.
A downstream target of AMPK, ACC2 mRNA, was 0.5-folddecreased (p < 0.0001) in the tumor tissue; this phenomenonis known to switch on fatty-acid oxidation [37]. The mRNAexpression of a downstream target of AMPK and ACC,carnitine palmitoyltransferase (CPT)1, was positively corre-lated with the AMPK expression in both tumors (r = 0.90,p < 0.0001) and normal mucosal tissue (r = 0.83, p < 0.0001).
Fig. 1 – mRNA expressions in gene arrays (n = 23) were used to measure the transcript profiles of the adiponectin pathway intumor and normal tissue samples obtained from the same patients. The genes in the adiponectin-AMPK pathway that showedsignificantly increased or decreased expression in tumors in the global gene array are marked with red stars.
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Several genes involved in the transport of fatty acids into thecells (i.e., FATP, FABP-pm, and FABP4) were simultaneouslyup-regulated in the tumor relative to the adjacent normaltissue (mRNA increase of 1.8 for FATP and FABP-pm, p =0.0229 and 0.056, respectively; mRNA increase of 1.6 fold forFABP, p < 0.001). Differential expressions of GLUT4, CPT2,SHP2, and SLC27A1 were also detected in the microarraydata. These results were not validated using qPCR.
3.4. Increased gAd versus fAd protein expression intumors, as revealed by Western blot analysis
Western blot analyses were conducted to confirm andfurther study the adiponectin protein secretion in thecolorectal samples (Fig. 2). The expressions of both the15-kDa gAd and 30-kDa fAd were quantified, since theseproteins exhibit distinct biological activities. Fifty pairedtumor and normal tissue samples were available for theseanalyses. The gAd protein was increased by 1.7-fold incolorectal tumor tissue compared pairwise with the adjacentnormal mucosa from the same patient (p = 0.0088, Fig. 3a).fAd expression in tumor tissue was decreased to 60 % of thatin the adjacent normal mucosa (p = 0.0007, Fig. 3b). The ratiobetween gAd and fAd was significantly higher in the tumorsamples (Fig. 3c). This increased relative amount of gAd wasassociated with increased AMPK mRNA expression (r = 0.34,p = 0.018) and tended to be correlated with AdipoR1 mRNAexpression (r = 0.25, p = 0.089) but not with AdipoR2 expres-sion (r = 0.01, p = 0.95). This suggests that activation ofAMPK is can be facilitated by increased sensitivity to gAdin colorectal tumors through increased AdipoR1 expression,but not AdipoR2 expression.
3.5. Correlation between the AMPK activation and gAdlevels, as revealed by RNA interference (RNAi)
The existence of a causal relationship between the expres-sion of gAd and AMPK activation in colon cancer cells wasinvestigated using an in vitro model by down-regulating theexpression of AdipoR1 using RNAi in cell-culture experi-ments. WiDr colon cancer cells were first incubated withgAd for 15 min, which resulted in a more than twofoldincrease in phosphorylation of the α-subunit of AMPK(15-min gAd), an effect that was suppressed by simulta-neous treatment of the cells with siRNA targeting the geneencoding AdipoR1 (siRNA ADIPOR1, 15 min) (Fig. 4). Thedifference in AMPK phosphorylation evident betweenuntreated and siRNA-treated WiDr colon cancer cells in thebaseline situation is thought to depend upon the alteredcircumstances in cells due to siRNA treatment. These resultsindicate that the correlation between increased gAd levelsand increased AMPK activation detected in tumor samplesmay be mediated at least in part via the activation ofADIPOR1 in colon cancer cells.
3.6. mRNA expression in tumors relative to pathologicalstaging, tumor size, lymph node involvement, andcolorectal location
No significant association between the Dukes’ stages andadiponectin expression was found. In contrast, there was agreater increase in AMPK expression in later-stage tumors.The relative change in AMPK mRNA expression (tumor/normal tissue) was significantly higher in Dukes’ class C(median, 1.8 ± 0.4) than in class B tumors (1.0 ± 0.2; p = 0.022).
Table 1 –mRNA expression of the adiponectin-AMPK pathway in tumor and normal tissues, as assessed using gene-arrayanalyses [50].
(Psoriasis-associated fatty-acid-binding protein homolog; PA-FABP), partial(53%)
8.366
Fig. 2 – Adiponectin protein expression was confirmed in both normal colorectal tissue and tumor samples from the samepatient byWestern blot analyses. The sampleswith different numbers represent different patients. N = normalmucosal tissue,T = tumor from a patient with the same patient number. The quantified expressions of both gAd (15 kDa; 60-min exposure ofthe membrane) and fAd (30 kDa; 3-min exposure of the membrane) are also shown. The red oblong illustrates the proteinexpression of gAd versus fAd in normal versus tumor tissue from a single patient.
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Fig. 3 – Protein expression of gAd was increased in colorectal tumors relative to normal colorectal tissue samples (a). Theexpression of fAd protein was lower in the tumor than in normal tissue samples (b). The ratio between gAd and fAd proteinexpressions (gAd/fAd ratio) was increased in colorectal tumors relative to normal colorectal tissue samples (c). The p valueswere obtained using pairwise Wilcoxon’s tests.
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In addition, neither tumor size nor differentiation stage wasassociated with adiponectin expression. However, the num-ber of samples was small and more than 80% of the tumorshad a moderate differentiation grade. Interestingly, theexpression of FATP changed significantly more betweennormal tissue and tumor tissue in class C tumors (1.7 ± 2.3)than in class B tumors (1.0 ± 0.3; p = 0.018). The geneexpression was correlated with the size of the tumor for
Fig. 4 –WiDr colon cancer cells were first incubated with gAdfor 15 min, which increased the phosphorylation of theα-subunit of AMPK by more than twofold (15-min gAd; thetwo first bars from left to right). This effectwas suppressed bysimultaneous treatment of the cells with siRNA ADIPOR1(15-min; the third and fourth bars from the left). Thedifference inAMPKphosphorylation seen betweenuntreatedand siRNA-treated WiDr colon cancer cells in the baselinesituation (baseline versus siRNA ADIPOR1 15-min) is thoughtto depend upon the altered circumstances in cells due tosiRNA treatment. This experiment confirms the connectionbetween AdipoR1 stimulation and AMPK phosphorylationunder in vitro conditions.
both FATP (r = 0.28, p = 0.035) and FABP-pm (r = 0.24, p = 0.066).There was no statistically significant association betweenFATP (r = 0.25, p = 0.053) and AMPK expressions (r = 0.24, p =0.065), and the number of lymph nodes affected. However, thenumber of samples here are also small, since only 50% of thepatients had lymph-node metastases. Furthermore, geneexpression patterns were not associated with tumor locationin the colon, sigmoid colon, or rectum.
4. Discussion
Adiponectin is generally believed to exert a protective roleagainst carcinogenesis [5,38]. Low plasma levels of adiponec-tin have been linked to increased colorectal cancer (CRC) riskand tumor grade, especially in male patients [38,39]. It hasbeen shown that the rs266729 single-nucleotide polymor-phism, which tags the 5′ flanking region of the adiponectingene, is associated with decreased CRC risk [40]. In 2007,Körner et al. showed that women with the highest adiponec-tin levels had a 65% reduced risk of breast cancer [41]. Thesefeatures are based on the measurement of plasma levels offAd and HMW, but neither the local expression of adiponectinin the tumor tissue nor expression of its globular form havebeen either discussed or demonstrated previously in CRC. Thepresent study is the first to show that adiponectin is expressedlocally by both colorectal tumors and the adjacent normalmucosa. In line with previous results, levels of the fAd proteinwere lower in the tumors than in the adjacent healthy colonmucosa. In contrast, the total expression of adiponectinmRNAwas increased in tumors. fAd can be cleaved to producea fragment containing the globular domain (gAd), whichexerts potent metabolic effects [6]. One especially interestingfinding of the present study is that the relative ratio betweengAd and fAd was significantly higher in the tumor samplesthan in the adjacent healthy mucosa from the same patients.These results suggest that colorectal tumors produce adipo-nectin locally and then cleave it to produce gAd to a greaterextent than in the adjacent healthy colorectal mucosa.
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While it is currently generally accepted that adiponectin isa circulating hormone that is secreted mainly by adiposetissue [3], there are a few other indications supporting thepresent finding that it is also synthesized in cell types otherthan adipocytes, and suggesting both endocrine and auto-/paracrine effects. The autocrine production of adiponectin inendothelial cells has been correlated with the antiatherogenicproperties of the hormone [42], and the expressions of bothadiponectin and its receptor AdipoR1 have been reported inairway epithelial cells [43]. In addition, proinflammatorycytokines in muscle elicit an autocrine production of adipo-nectin [44]. Furthermore, myogenesis has been shown toinduce an autocrine production of adiponectin, which canagain be increased by treatment with proinflammatorycytokines or exposure to oxidative stress, thereby suggestingautocrine-loop-sustaining myogenesis [42]. Therefore, inaddition to the results reported here, there are reasons tobelieve that the autocrine production of gAd in response tooxidative stress or as a response to proinflammatory cyto-kines could also occur in colorectal tumor cells and act as atumor-specific mechanism for achieving metabolic modifica-tions that are oxidatively beneficial for the tumor cells.
In addition to its eventual autocrine functions, there isevidence that the adiponectin response can vary according tothe local circumstances in the tissue. In the study of Habeeb etal. showing bimodal effects of adiponectin on cell survival, theeffect of gAd appeared to be more potent for supporting cellsurvival than fAd in glucose-deprived conditions [32]. Accord-ing to that study, the effect of adiponectin would changeduring the carcinogenic process by suppressing cell prolifer-ation in favorable conditions with abundant nutrients pre-sent, while supporting cell survival during stress conditionssuch as glucose deficiency [32]. The bimodal effects ofadiponectin have also been seen in nonalcoholic steatohepa-titis (NASH). Reduced serum adiponectin levels have beenimplicated in the pathogenesis of NASH; in contrast, adipo-nectin levels were shown to increase while hepatic fatdecreased during the development of liver cirrhosis [45].
The physiological effect of adiponectin is mediated by itsreceptors, whose various isoforms can be regulated byhyperinsulinemia and hyperglycemia, with the consequenceof increased sensitivity or resistance to specific forms ofadiponectin [7,12]. The different forms of adiponectin, as wellas differential expression or activation of its receptors, mayalso partly explain some previous conflicting results regardingthe downstream effects of adiponectin stimulation in tumors[5]. AdipoR1 and AdipoR2 expressions have been reported inboth CRC and normal mucosa, with the surface expressionsbeing greater in cancerous tissue than in either normalmucosa or gastrointestinal stromal tumors. This findingsupports the hypothesis implicating adiponectin in thepathogenesis of CRC [17]. A hypoadiponectinemia-inducedup-regulation of adiponectin receptors in CRC tissues maycompensate and maintain the adiponectin signaling path-ways. Again, the same mechanism has also been discussedwith respect to NASH. The specific roles of AdipoR1 andAdipoR2, and their relative amounts in advanced NASH maybe important for estimating the net response due to therelative increase in AdipoR1 expression leading to increasedfatty acid oxidation [46]. Habeeb et al. found that the
expressions of both AdipoR1 and AdipoR2 were significantlyincreased in cell culture after glucose deficiency, mimickingthe situation in nutrient-deprived tumor cells; however, theincrease in the expression of AdipoR1 was more marked [32].The expression of AdipoR1 (but not AdipoR2) mRNA was alsosignificantly increased in the tumors in the present material.Giamalas et al. revealed the same expression pattern, exceptthat in their study the expression of AdipoR2 was alsoincreased [39]. The expected beneficial effect of adiponectinin cancer disease has prompted ongoing work in designingand developing an adiponectin receptor agonist for cancertreatment [47]. However, the findings of the present studyconfirm that a fundamental understanding of the reportedbimodal effects of adiponectin with respect to the pathophys-iology of tumor development and progression is necessarywhen evaluating possible treatment options with the adipo-nectin analogs.
It has been demonstrated that gAd is a strong activator ofAMPK, mainly by inducing its phosphorylation [42]. Knock-out of AdipoR1 by siRNA in human WiDr colon cancer cellsin the present study prevented the phosphorylation ofAMPK, thus confirming the connection between AdipoR1stimulation and AMPK phosphorylation under in vitroconditions. AMPK is a central sensor of cellular energyrequirements and can also be activated through a high AMP/ATP ratio [37]. There is evidence that gradual alterations ingene expression promote metabolic changes during tumor-igenesis [48,49]. Early premalignant lesions benefit from ageneral inhibition of AMPK activity [48], which occurs instates such as obesity and insulin resistance. In contrast,oncogene-mediated changes at the beginning of the diseaseare subsequently followed by hypoxia-induced-factor-mediated gene expression. The AMPK activation benefitsadvanced neoplasms by increasing their intracellular sub-strate availability for energy breakdown processes [48]. Thisassumption is supported by several previous studies linkinghigh AMPK expression with advanced tumor disease [48].The increased gAd expression in the tumor tissue relative tothe adjacent healthy mucosa in the present study wasassociated with increased AMPK mRNA expression, andtended to be correlated with AdipoR1 but not AdipoR2 mRNAexpression. These results should be interpreted with cau-tion, since neither the protein levels of AMPK nor itsphosphorylation were measured in the patient material.gAd-induced activation of AMPK is involved in the inhibitionof ACC2 and increased fatty-acid oxidation, glucose uptake,and lactate production [20]. However, the expressions ofseveral genes involved in the transport of fatty acids intocells (i.e., FATP, FABP-pm, and FABP-4) were simultaneouslyup-regulated in the tumor tissues compared with theadjacent healthy tissue in the present study, supportingthe biological relevance of our findings. Furthermore,Habeeb et al. showed recently that during glucose depriva-tion, the AMPK–mTOR cascade leads to the activation ofmicrotubule-associated protein 1 light chain 3, resulting incell survival via activation of the autophagic machinery ofthe malignant DLD-1 cell line [32]. In the present study,AMPK expression was activated mostly in later-stagetumors; these findings are in line with previously reporteddata on AMPK activation in tumors [48].
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The present study shows for the first time that theglobular form of adiponectin is increasingly produced bycolorectal tumors ex vivo in combination with up-regulationof AdipoR1 on the cell surface. This might be a compensa-tory mechanism that occurs in response to low plasmaadiponectin levels, as reported previously in CRC, or to othercircumstances in the tumor microenvironment, such asreduced oxygen or glucose levels. Whatever the reason forthe response, this finding implicates a beneficial metabolicadvantage in the tumor cells as compared with those of thenormal mucosa. The biological relevance of these results issupported by the findings regarding several gAd targetedgenes, showing the expected increased or decreased expres-sion ex vivo. However, the major weakness of this is study isthat the results do not highlight the impact of local versussystemic changes in adiponectin signaling during tumori-genesis. The eventual bimodal actions of adiponectin shouldbe investigated during tumorigenesis, including both plasmalevels and local expression of the hormone during differentphases of the disease. The results of this study do notresolve the issue of how adiponectin expression is regulatedduring the tumorigenic process, and it is unclear whetherthese results simply represent an epiphenomenon orwhether local adiponectin activation in tumors is a signif-icant driving force for the tumorigenic process. The clinicalor translational potential of these results include a potentialtarget for pharmaceutical or lifestyle interventions, al-though activation of genes in the gAd-AdipoR1-AMPKpathway in advanced cancer disease needs to be confirmedand studied further.
Author contributions
K.K.V. designed the study, supervised the experiments, andwrote the paper. T.S. conceived and carried out experiments.K.K.V. and M.L. conceived and carried out some of theexperiments. I.B. revised the paper and coordinated thecollection of the clinical samples, and R.H.S. contributed tothe collection of clinical samples. K.J. carried out parts of theRNA isolation and the microarray experiments. K.H.P. andK.K.V. analyzed the data. T.L. analyzed the microarray data.E.F. conceived and supervised some of the experiments, andrevised the paper. All authors were involved in writing andrevising the paper, and provided their final approval of thesubmitted version.
Funding
This study was supported by grants from Akershus Univer-sity Hospital, Helsinki University Central Hospital, Jalmari jaRauha Ahokas, and Novo Nordisk Foundation (K.H.P.). E.F.was supported by The Research Council of Norway throughgrants from the Functional Genomics Program (FUGE, grantnumber 151882), Helse Sør-Øst (grant number 2007060),“Ullevål University Hospital Research Fund (VIRUUS),” and“University of Oslo Research Fund (UNIFOR).”
Acknowledgments
We thank English Science Editing for improving the use ofEnglish in this manuscript.
Conflict of interest
While working on this research article, Katja Kannisto Vetvikalso worked in parallel as Medical Director at Merck SeronoNorway, an affiliate of Merck Serono S.A. There is no conflictof interest or any linkage between this position and thecontent or results of this work. Katja Kannisto Vetvik has notreceived any commercial research grants.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.metabol.2014.02.001.
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