Expression of long chain fatty acid receptor GPR40 is associated with cancer progression in colorectal cancer: A retrospective study

Transcription

1 ONCOLOGY LETTERS 15: , 2018 Expression of long chin ftty cid receptor GPR40 is ssocited with cncer progression in colorectl cncer: A retrospective study CHIE NAKASHIMA 1, KISHI SHINGO 1, RINA FUJIWARA TANI 1, YI LUO 1,2, ISAO KAWAHARA 1, KEI GOTO 1, TAKAMITSU SASAKI 1, KIYOMU FUJII 1, HITOSHI OHMORI 1 nd HIROKI KUNIYASU 1 1 Deprtment of Moleculr Pthology, Nr Medicl University, Kshihr, Nr , Jpn; 2 Key Lbortory of Neuroregenertion of Jingsu nd Ministry of Eduction, Co innovtion Center of Neuroregenertion, Nntong University, Nntong, Jingsu , P.R. Chin Received November 6, 2017; Accepted Mrch 16, 2018 DOI: /ol Abstrct. An incresed risk of colorectl cncer (CRC) is ssocited with western style diet, prticulrly hyperlipidemi. The expression of G protein coupled receptor 40 (GPR40), membrne bound receptor for long chin ftty cids (LCFAs), ws exmined in 36 cses of subserosl invding CRC nd compred with clinicopthologicl prmeters s well s triglyceride (TG) nd low density lipoprotein (LDL) levels in the blood. All ptients with CRC expressed GPR40, which ws positively ssocited with blood TG levels (P<0.0001) but not with blood LDL levels. GPR40 expression ws positively ssocited with nodl metstsis, distnt metstsis (prticulrly to the liver), stge nd poor prognosis. Ptients with high GPR40 expression nd high TG levels hd comprtively worse survivl outcomes compred with ptients with low GPR40 expression nd low TG levels. The results of the present study suggest tht ctivtion of GPR40 my be ssocited with the progression nd prognosis of CRCs. High levels of GPR40 nd/or concurrent high levels of GPR40 nd TG my be risk for CRC progression. Introduction Colorectl cncer (CRC) is the third leding cuse of cncer relted deth in Jpn (1). The number of ptients with Correspondence to: Professor Hiroki Kuniysu, Deprtment of Moleculr Pthology, Nr Medicl University, 840 Shijo cho, Kshihr, Nr , Jpn E mil: cooninh@zb4.so net.ne.jp Abbrevitions: CRC, colorectl cncer; LCFA, long chin ftty cid; GPCR or GPR, G protein coupled receptor; TG, triglyceride; LDL, low density lipoprotein; EA, elidic cid; RAGE, receptor for dvnced glyction end products; EMT, epithelil mesenchyml trnsition; EGFR, epiderml growth fctor receptor; AGE, dvnced glyction end products Key words: GPR40, long chin ftty cid, hyperlipidemi, metstsis CRC, nd the number CRC relted mortlities hve incresed in Jpn, probbly s result of the increse in populrity of western lifestyle (2,3). Some dietry ingredients such s ftty cids hve been proposed to increse the risk of CRC (4). Some long chin ftty cids (LCFAs) possess pro tumorl ctivity. Linoleic cid enhnces colon crcinogenesis nd metstsis by ctivting the receptor for dvnced glyction end products (RAGE) nd high mobility group box 1 in the zoxymethn induced rt colon cncer model (5,6). Linoleic cid lso inhibits prolifertion of cncer cells nd induces quiescence (7,8). Elidic cid (EA), trns ftty cid, enhnces metsttic potentil in colon cncer cells by induction of stemness nd promoting epithelil mesenchyml trnsition (EMT) (9,10). The LCFAs bind to, nd medite functionl effects through, specific membrne bound receptors: the G protein coupled receptor 40 (GPR40) nd GPR120 receptors (11). The GPR40 is expressed in the bet cells of the pncretic islets to ffect insulin secretion (12), nd binds to vrious LCFAs. This receptor exhibits lignd bised signling, nd binding to oleic cid cuses it to couple to Gq type of G protein, stimulting clcium ion influx nd inositol phosphte synthesis (13). In contrst, EA induced GPR40 signling trnsctivtes epiderml growth fctor receptor (EGFR) nd c SRC signling (10). Activtion of GPR40 is essentil for multifunctionl effects of LCFAs. In the body, LCFAs re supplied s prt of triglycerides (TGs) nd rech vrious tissues s well s tumor cells from circulting blood (14). Thus, evlution of GPR40 expression sttus nd plsm TG levels in CRC ptients would be useful in elucidting the role of LCFAs in CRC. In the present study, we exmined GPR40 expression nd plsm TG levels in 36 CRC ptients nd sought to understnd if ftty cids ffect CRC progression nd survivl outcomes. Ptients nd methods Ptients. We rndomly selected 36 ptients with pt3 CRC dignosed pthologiclly in the Deprtment of Moleculr Pthology, Nr Medicl University (Kshihr, Jpn) from 2012 to All cses were treted with curtive resection, without history of dibetes mellitus. Written informed

2 8642 NAKASHIMA et l: GPR40 EXPRESSION IN COLORECTAL CANCER consent ws not required s ny identifying informtion ws removed from the smples prior to nlysis, to ensure strict privcy protection (unlinkble nonymiztion). All procedures were performed in ccordnce with the Ethicl Guidelines for Humn Genome/Gene Reserch encted by the Jpnese Government, which ws pproved by the Ethics Committee of the Nr Medicl University (pprovl no. 937). Immunohistochemistry. Consecutive 4 µm sections of resected tissue were immunohistochemiclly stined using the immunoperoxidse technique described previously (15). Anti GPR40 ntibody (Abnov, Wlnut, CA, USA) ws used t concentrtion of 0.2 µg/ml. Secondry ntibodies (Medicl nd Biologicl Lbortories, Ngoy, Jpn) were used t concentrtion of 0.2 µg/ml. Tissue sections were color developed with dimine benzidine hydrochloride (DAKO, Glstrup, Denmrk), nd counterstined with Meyer's hemtoxylin (Sigm Chemicl Co., St. Louis, MO, USA). A GPR40 expression score ws clculted by multiplying the stining strength score (0 2) with the stining re (0 5), which yielded scores rnging from 0 to 10. Sttisticl nlysis. Sttisticl nlyses of experimentl dt were crried out using the Spermn r test, nlysis of vrince (ANOVA), nd the two tiled chi squred test (InStt; Grphpd Softwre Inc., L Joll, CA, USA). A Bonferoni test ws performed fter ANOVA s post hoc test. Survivl nlysis ws performed using the Kpln Meier method long with the log rnk test. Univrite nd multivrite nlyses were performed using the log rnk trend test nd the Cox's hzrd model, respectively (SPSS Sttistics, IBM Jpn, Tokyo, Jpn). Sttisticl significnce ws defined s two sided P vlue <0.05. Results Expression of GPR40. In 36 CRCs with sub serosl or sub dventitil lyer invsion (pt3), (ptients without history of dibetes mellitus), GPR40 expression ws exmined immunohistochemiclly (Fig. 1). Eighteen cses displyed reltively high GPR40 expression, nd were ttributed GPR40 high sttus, while 18 other cses with reltively low GPR40 expression were given GPR40 low sttus (Tble I). In GPR40 high smples, GPR40 immunorectivity ws identified in the plsm membrne (Fig. 1A). In contrst, in GPR40 low smples, signl of wek intensity ws observed in reltively few cells. Interestingly, GPR40 high sttus ws ssocited significntly with lymph node metstsis, liver metstsis, nd stge, but not with histologicl differentition. GPR40 expression nd blood TG levels. Next, we compred GPR40 expression with TG or low density lipoprotein (LDL) levels (Fig. 2). A GPR40 high sttus ws ssocited with higher blood TG levels, but not with blood low density lipoprotein (LDL) levels. Moreover, TG levels were ssocited with stge, wheres LDL levels were not ssocited with stge (Tble II). The smples were divided into the two groups by GPR40 expression sttus nd blood TG levels. The first group consisted of smples with both high GPR40 sttus s well s high TG Figure 1. Expression of GPR40 in colorectl cncer. GPR40 expression ws exmined by immunohistochemistry. (A) A cse of tub2, stge IV, pt3, pn2, pm1 (liver). GPR40 expression ws 10. (B) A cse of tub1, stge II, pt3, pn0, pm0. GPR40 expression ws 0.5. Scle br, 100 µm. Pthologicl prmeters were described ccording to the tumor, node, metstsis pthologicl clssifiction system (31). GPR40, G protein coupled receptor 40. levels (GPR40:high+TG:high) nd the rest of the smples constituted the second group. The two groups were compred with regrd to stge, TG levels, nd survivl outcomes (Tble III). The GPR40:high+TG:high group showed more dvnced stge nd shorter survivl periods. Survivl nlysis showed tht the GPR40:high+TG:high group showed worse prognosis compred to tht of the other group (Fig. 3). Tble IV shows the results of univrite nlysis of clinicopthologicl prmeters. The GPR40 expression sttus nd GPR40+TG levels were the two prmeters with the highest sttisticl significnce, followed by liver metstsis (pm). Tble V shows results of multivrite nlysis; the GPR40 expression sttus ws highly sttisticlly significnt, followed by the GPR40+TG prmeter, mong the clinicopthologicl prmeters nlyzed. Discussion The incidence of CRC is on the rise in Jpn (1). Life style, especilly western style diet, is implicted s n importnt custive fctor in CRC (2,3). Recent studies reveled strong reltionship between CRC nd the metbolic syndrome (16,17) nd showed tht dibetes mellitus is risk fctor for CRC (18,19). Severl fctors linking CRC risk with dibetes hve been proposed, such s incresed expression of insulin like growth fctors; oxidtive stress (18); dvnced

3 ONCOLOGY LETTERS 15: , Tble I. Expression of GPR40 nd clinicopthologicl prmeters in 36 pt3 colorectl cncer cses. GPR40 expression Chrcteristic Low High P vlue n GPR40 levels < Age (men ± SD) 68.6± ±10.8 NS Sex (M:F) 8:10 10:8 NS Histologicl grde b G1 6 6 G NS pt pn pn pm pm Stge b II 15 3 III 3 9 IV TG (men ± SD) 121.9± ± LDL (men ± SD) 124.2± ±25.6 NS Tble II. Assocition between stge nd TG or LDL in ptients with colorectl cncer. Prmeter n TG LDL Stge II ± ±15.4 III ± ±27.0 IV ± ±24.0 P vlue NS Pthologicl prmeters were described ccording to the tumor, node, metstsis pthologicl clssifiction system (32). TG, blood triglyceride (mg/ml); LDL, low density lipoprotein; NS, not significnt. GPR40 expression ws clculted s the stining strength score (0 2) multiplied by the stining re (0 5), which yielded scores (0 10). b Pthologicl prmeters were described ccording to the tumor, node, metstsis pthologicl clssifiction system (32). G1, well differentited; G2, modertely differentited; pt3, tumor invdes sub-serosl or sub-dventitil lyer; pn 1 2, metstsis in regionl lymph nodes; pm1, metstsis confined to the liver. GPR40, G protein coupled receptor 40; TG, blood triglyceride (mg/ml); LDL, low density lipoprotein (mg/ml); NS, not significnt; M, mle; F, femle; SD, stndrd devition. glyction end products (AGE; which my enhnce mlignnt potentil of colorectl cells) (5,20); nd ctivtion of the renin ngiotensin system (21). Hypertriglyceridemi or hyper low density lipoproteinemi is ssocited with colonic denoms, though the risk contribution of these fctors is controversil (22,23). The membrne bound receptors of LCFA (which re components of TGs) re G protein coupled receptors (GPCRs), of which GPR40 nd GPR120 re well known (11,24). However, the differentil biologicl ctivities of ftty cids cnnot be fully explined by the ctivity of these receptors lone. To fully explore the spectrum of ftty cid functionlity, the ctivities of cytoplsmic receptors, bioctive metbolites of ftty cids, nd those of ftty cids integrted into the plsm membrne need to be studied (7,8,25). In previous study on trns ftty cids, we showed tht EA trnsctivtes EGFR from GPR vi c SRC to increse stemness nd induce the epiderml to mesenchyml trnsition in cncer cells (10). Oleic cid lso trnsctivtes EGFR vi c SRC signling (26). The diverse ctivities of ftty cids might thus be result of ctivtion of multiple GPCR dependent nd GPCR independent pthwys. Figure 2. Assocition between GPR40 expression nd TG or low density LDL levels. (A) Assocition between GPR40 expression nd TG. (B) Assocition between GPR40 expression nd LDL. The r vlue nd P vlue were clculted by Spermn's regression model. GPR40, G protein coupled receptor 40; TG, triglyceride; LDL, low density lipoprotein. In the present study, expression of GPR40 ws ssocited with blood TG levels but not with blood LDL levels. The GPR40 expression ws lso ssocited with nodl metstsis, distnt metstsis (especilly to the liver), stge, nd poor prognosis. High GPR40 expression nd high TG levels prognosticted worse survivl outcomes in our study. These results suggest tht GPR40 expression might be linked to CRC progression. Univrite nlysis reveled tht GPR40 expression sttus lone showed stronger ssocition with ptient survivl thn did GPR40:high+TG:high sttus. This result

4 8644 NAKASHIMA et l: GPR40 EXPRESSION IN COLORECTAL CANCER Tble III. Comprison of disese stge nd survivl between ptient groups clssified by TG nd GPR40 expression sttus. GPR40:high Other Group +TG:high cses P vlue n Stge II III 8 4 IV 4 2 TG 216.9± ±21.2 < Survivl period 15.6± ± (months) Pthologicl prmeters were described ccording to the tumor, node, metstsis pthologicl clssifiction system (32). GPR40, G protein coupled receptor 40; TG, blood triglyceride (mg/ml). Tble IV. Univrite nlyses of clinicopthologic prmeters. Prmeter Chi squred P vlue Stge II, III, IV pn (0,1 2) pm (0,1) TG (high, low) GPR GPR40+TG (GPR40:high+TG:high, others) Pthologicl prmeters were described ccording to the tumor, node, metstsis pthologicl clssifiction system (32); pn1 2, metstsis in regionl lymph nodes; pm1, metstsis confined to the liver. GPR40, G protein coupled receptor 40; TG, blood triglyceride (mg/ml). Tble V. Multivrite nlyses of clinicopthologic prmeters. Hzrd Lower Upper Prmeter rtio 95% 95% P vlue GPR GPR40+TG (GPR40:high+ TG: high, others) Stge (II, III, IV) Figure 3. Overll survivl of ptients with colorectl cncer. Survivl ws compred between the two groups; GPR40:high+TG:high cses nd the rest of the cses (the other cses). Survivl nlysis ws performed by using the Kpln Meier method long with the log rnk test. TG, triglyceride; GPR40, G protein coupled receptor 40. suggests tht heterogeneity in LCFA content of TGs might be confounding fctor nd my be msking the significnt influence of GPR40 expression in the cse of ptients with GPR40:high+TG:high sttus. Our recent reports show different effect of some long chin ftty cids. Linoleic cid, EA nd oleic cid re exmined on their effects to cncer cells. Linoleic cid provides incresed stemness with dormncy (7). EA enhnced metstbility by incresed prolifertive stemness (10). In. contrst, oleic cid does not increse metstbility (10). Univrite nd multivrite nlyses showed GPR40 is more significnt thn GPR40+TG. As described bove, different ftty cid provides different effect. TGs is mixture of mny ftty cids, which might cncel the effects ech other. Then it should be needed clrifying content of ech ftty cid. It does not seem tht GPR40 nd TG levels in stge IV cses. In stge IV cses, other pro metsttic fctors, such s growth fctors might cuse more relevnt contributions to metstsis. Pthologicl prmeters were described ccording to the tumor, node, metstsis pthologicl clssifiction system (32); pn1 2, metstsis in regionl lymph nodes; pm1, metstsis confined to the liver. GPR40, G protein coupled receptor 40; TG, blood triglyceride (mg/ml). Our previous studies show tht linoleic cid (n n 6 LCFA) nd EA ( trns ftty cid), enhnce colon crcinogenesis or metstbility of CRC (5,6,9,10), wheres docoshexenoic cid (DHA) nd eicospentenoic cid (EPA) (n 3 LCFAs) suppress colon crcinogenesis nd enhnce efficcy of chemotherpy regimens (27). The n 3 LCFAs lso ctivte the hippo system vi GPR40 nd GPR120 to inhibit prolifertion of CRC (28). Although blood TG level is widely utilized s predictive clinicl prmeter, the identity of the ftty cids tht constitute the TGs might be n importnt fctor tht determines the reltionship between TGs nd cncer risk. Metbolic syndrome is collection of risk fctors, such s hypertension, higherglycemi, hyperlipidemi, nd viscerl ft bsed on insulin resistnce (29). Diets of ptients with the metbolic syndrome re reported to contin ft with high n 6/n 3 ftty cid rtio (30) s well s high content of trns ftty cids (31). From these findings, metbolic syndrome ssocited hypertriglyceridemi might be suggested to be pro tumorl for CRC. In the cses exmined here, the blood TG level ws ssocited with survivl outcomes of the ptients, which suggests tht

5 ONCOLOGY LETTERS 15: , hypertriglyceridemi needs to be closely exmined with reference to CRC progression. The ssocition between GPR40 with blood TG levels my imply tht TGs induce GPR40 expression in CRC. In contrst, LCFAs suppress GPR40 expression in pncretic islet bet cells to dversely ffect dibetes (12). The mechnism of regultion of GPR40 expression hs not been fully elucidted, though LCFAs hve been implicted. The signling pthwys leding to GPR40 overexpression nd the effect of dyslipidemi on such signling need to be exmined in future studies. Acknowledgements The uthors would like to thnk Ms. Tomomi Msutni for ssistnce with the preprtion of this mnuscript. Funding The present study ws supported by MEXT KAKENHI (grnt nos. 16H05164, 17K15648, 17K19923 nd 16K19087). Avilbility of dt nd mterils All dt generted or nlyzed during the present study re included in this published rticle. Authors' contributions HK designed the study. CN, KS, RFT nd YL collected nd nlyzed the dt. IK, KG, TS, HO nd KF nlyzed the dt. CN nd HO prepred nd hd finl pprovl of the mnuscript. Ethics pprovl nd consent to prticipte The present study ws pproved by the Ethics Committee of the Nr Medicl University (pprovl no. 937). Consent for publiction Not pplicble. Competing interests The uthors confirm tht they hve no competing interests. References 1. Wko F, Nishimoto H, Ktonod K, Tsukum H nd Mikmi H (eds): Cncer sttistics in Jpn, Ntionl Cncer Reserch Institute, Tokyo, Kimur Y, Kono S, Toyomur K, Ngno J, Mizoue T, Moore MA, Mibu R, Tnk M, Kkeji Y, Mehr Y, et l: Met, fish nd ft intke in reltion to subsite specific risk of colorectl cncer: The Fukuok colorectl cncer study. Cncer Sci 98: , Mizoue T, Tnk K, Tsuji I, Wki K, Ngt C, Otni T, Inoue M nd Tsugne S; Reserch Group for the Development nd Evlution of Cncer Prevention Strtegies in Jpn: Alcohol drinking nd colorectl cncer risk: An evlution bsed on systemtic review of epidemiologic evidence mong the Jpnese popultion. Jpn J Clin Oncol 36: , Bultmn SJ: Interply between diet, gut microbiot, epigenetic events, nd colorectl cncer. Mol Nutr Food Res 61, Shimomoto T, Luo Y, Ohmori H, Chihr Y, Fujii K, Sshir T, Dend A nd Kuniysu H: Advnced glyction end products (AGE) induce the receptor for AGE in the colonic mucos of zoxymethne injected Fischer 344 rts fed with high linoleic cid nd high glucose diet. J Gstroenterol 47: , Ohmori H, Luo Y, Fujii K, Sshir T, Shimomoto T, Dend A nd Kuniysu H: Dietry linoleic cid nd glucose enhnces zoxymethne induced colon cncer nd metstses vi the expression of high mobility group box 1. Pthobiology 77: , Ohmori H, Sshir T, Fujii K, Luo Y, Shimomoto T nd Kuniysu H: Linoleic cid induced growth suppression induces quiescent cncer cell nests in nude mice. Pthobiology 75: , Sski T, Fujii K, Yoshid K, Shimur H, Sshir T, Ohmori H nd Kuniysu H: Peritonel metstsis inhibition by linoleic cid with ctivtion of PPARgmm in humn gstrointestinl cncer cells. Virchows Arch 448: , Ohmori H, Fujii K, Kdochi Y, Mori S, Nishiguchi Y, Fujiwr R, Kishi S, Sski T nd Kuniysu H: Elidic cid, trns ftty cid, enhnces the metstsis of colorectl cncer cells. Pthobiology 84: , Fujii K, Luo Y, Fujiwr Tni R, Kishi S, He S, Yng S, Sski T, Ohmori H nd Kuniysu H: Pro metsttic intrcellulr signling of the elidic trns ftty cid. Int J Oncol 50: 85 92, Hr T, Kshihr D, Ichimur A, Kimur I, Tsujimoto G nd Hirsw A: Role of free ftty cid receptors in the regultion of energy metbolism. Biochim Biophys Act 1841: , Tomit T, Msuzki H, Iwkur H, Fujikur J, Noguchi M, Tnk T, Ebihr K, Kwmur J, Komoto I, Kwguchi Y, et l: Expression of the gene for membrne bound ftty cid receptor in the pncres nd islet cell tumours in humns: Evidence for GPR40 expression in pncretic bet cells nd implictions for insulin secretion. Dibetologi 49: , Mizut K, Zhng Y, Mizut F, Hoshijim H, Shig T, Mski E nd Eml CW Sr: Novel identifiction of the free ftty cid receptor FFAR1 tht promotes contrction in irwy smooth muscle. Am J Physiol Lung Cell Mol Physiol 309: L970 L982, Newsholme EA: The glucose/ftty cid cycle nd physicl exhustion. Cib Found Symp 82: , Kuniysu H, Ysui W, Shinohr H, Yno S, Ellis LM, Wilson MR, Bucn CD, Rikit T, Thr E nd Fidler IJ: Induction of ngiogenesis by hyperplstic colonic mucos djcent to colon cncer. Am J Pthol 157: , O'Flngn CH, Bowers LW nd Hursting SD: A weighty problem: Metbolic perturbtions nd the obesity cncer link. Horm Mol Biol Clin Investig 23: 47 57, Schoenberg MH: Physicl ctivity nd nutrition in primry nd tertiry prevention of colorectl cncer. Visc Med 32: , Ksug M, Ueki K, Tjim N, Nod M, Ohshi K, Noto H, Goto A, Ogw W, Ski R, Tsugne S, et l: Report of the Jpn dibetes society/jpnese cncer ssocition joint committee on dibetes nd cncer. Cncer Sci 104: , Shi J, Xiong L, Li J, Co H, Jing W, Liu B, Chen X, Liu C, Liu K, Wng G nd Ci K: A liner dose response reltionship between fsting plsm glucose nd colorectl cncer risk: Systemtic review nd met nlysis. Sci Rep 5: 17591, Kuniysu H, Chihr Y nd Kondo H: Differentil effects between mphoterin nd dvnced glyction end products on colon cncer cells. Int J Cncer 104: , Shimomoto T, Ohmori H, Luo Y, Chihr Y, Dend A, Sshir T, Ttsumoto N, Fujii K nd Kuniysu H: Dibetes ssocited ngiotensin ctivtion enhnces liver metstsis of colon cncer. Clin Exp Metstsis 29: , Yng MH, Rmpl S, Sung J, Choi YH, Son HJ, Lee JH, Kim YH, Chng DK, Rhee PL, Kim JJ, et l: The ssocition of serum lipids with colorectl denoms. Am J Gstroenterol 108: , Tin Y, Wng K, Li J, Wng J, Wng Z, Fn Y, Ye Y, Ji G nd Li Y: The ssocition between serum lipids nd colorectl neoplsm: A systemic review nd met nlysis. Public Helth Nutr 18: , 2015.

6 8646 NAKASHIMA et l: GPR40 EXPRESSION IN COLORECTAL CANCER 24. Yonezw T, Kurt R, Yoshid K, Murym MA, Cui X nd Hsegw A: Free ftty cids sensing G protein coupled receptors in drug trgeting nd therpeutics. Curr Med Chem 20: , Kuniysu H (ed): Linoleic cid. In: Encyclopedi of Cncer. 2nd edition, Springer Verlg, Berlin, pp , Soto Guzmn A, Robledo T, Lopez Perez M nd Slzr EP: Oleic cid induces ERK1/2 ctivtion nd AP 1 DNA binding ctivity through mechnism involving Src kinse nd EGFR trnsctivtion in brest cncer cells. Mol Cell Endocrinol 294: 81 91, Lee JY, Sim TB, Lee JE nd N HK: Chemopreventive nd chemotherpeutic effects of fish oil derived Omeg 3 polyunsturted ftty cids on colon crcinogenesis. Clin Nutr Res 6: , Zhng K, Hu Z, Qi H, Shi Z, Chng Y, Yo Q, Cui H, Zheng L, Hn Y, Hn X, et l: G protein coupled receptors medite ω 3 PUFAs inhibited colorectl cncer by ctivting the Hippo pthwy. Oncotrget 7: , World Helth Orgniztion: Definition, dignosis nd clssifiction of dibetes mellitus nd its complictions: Report of WHO Consulttion. World Helth Orgniztion, Genev, Simopoulos AP: Essentil ftty cids in helth nd chronic disese. Am J Clin Nutr 70 (3 Suppl): 560S 569S, Mozffrin D, Aro A nd Willett WC: Helth effects of trns ftty cids: Experimentl nd observtionl evidence. Eur J Clin Nutr 63 (Suppl 2): S5 S21, Brierley JD, Gospodrowicz MK nd Wittekind C: TNM Clssifiction of Mlignnt Tumours. Wiley Blckwell, Oxford, UK, This work is licensed under Cretive Commons Attribution-NonCommercil-NoDerivtives 4.0 Interntionl (CC BY-NC-ND 4.0) License.