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Na+/monocarboxylate transport (SMCT) protein expression correlates with survival in colon cancer: Molecular characterization of SMCT

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Na+/monocarboxylate transport (SMCT) protein expression correlates with survival in colon cancer: Molecular characterization of SMCT
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  Na   monocarboxylate transport (SMCT) proteinexpression correlates with survival in colon cancer:Molecular characterization of SMCT Viktoriya Paroder*, Shelly R. Spencer † , Monika Paroder*, Diego Arango ‡ , Simo Schwartz, Jr. ‡ , John M. Mariadason § ,Leonard H. Augenlicht § , Sepehr Eskandari † , and Nancy Carrasco* ¶ *Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461;  † Biological Sciences Department, California StatePolytechnic University, Pomona, CA 91768;  ‡ Molecular Oncology and Aging Group, Molecular Biology and Biochemistry Research Center, Valle HebronHospital Research Institute, Passeig Valle d’Hebron 119-129, 08035 Barcelona, Spain; and  § Department of Oncology, Montefiore Medical Center,Albert Einstein Cancer Center, 111 East 210th Street, Bronx, NY 10467Communicated by H. Ronald Kaback, University of California, Los Angeles, CA, March 22, 2006 (received for review January 7, 2006) WereportanextensivecharacterizationoftheNa   monocarboxy-latetransporter(SMCT),aplasmamembraneproteinthatmediatesactive transport of monocarboxylates such as propionate andnicotinate, and we show that SMCT may play a role in colorectalcancer diagnosis. SMCT, the product of the  SLC5A8   gene, is 70%similar to the Na   I  symporter, the protein that mediates activeI  uptake in the basolateral surface of thyrocytes and other cells.SMCTwasreportedintheapicalsurfaceofthyrocytesandformerlyproposed also to transport I  and was called the apical I  trans-porter. However, it is now clear that SMCT does not transport I  .Here we demonstrate a high-affinity Na  -dependent monocar-boxylate transport system in thyroid cells, which is likely to beSMCT. We show that, whereas thyroidal Na   I  symporter expres-sion is thyroid-stimulating hormone (TSH)-dependent and baso-lateral,SMCTexpressionisTSH-independentandapicalnotonlyinthe thyroid but also in kidney and colon epithelial cells and inpolarized Madin–Darby canine kidney cells. We determine thekinetic parameters of SMCT activity and show its inhibition byibuprofen ( K  i    73    9   M) in  Xenopus laevis  oocytes. SMCT wasproposedtobeatumorsuppressorincoloncancer[Li,H., etal. (2003) Proc.Natl.Acad.Sci.USA 100,8412–8417].Significantly,weshowthathigher expression of SMCT in colon samples from 113 colorectalcancer patients correlates with longer disease-free survival, suggest-ing that SMCT expression may be a favorable indicator of colorectalcancer prognosis. T he  SLC5A8  gene product is a plasma membrane transportprotein that belongs to solute carrier family 5 (SLC5A). Mem-bers of this family couple the energy released by the inward‘‘downhill’’ translocation of Na  in favor of its concentrationgradient to the inward active transport of another solute against itschemical gradient. The extensively characterized Na   I  sym-porter (NIS, SLC5A5), a key plasma membrane protein thatmediates active I  transport in the thyroid and other tissues, is alsoa member of this family (1–4). SLC5A8 was srcinally identified asaproteinhomologous(70%similarityand46%identity)toNISandalsoproposedtotransportI  .Thesetwomoleculesarelocalizedonopposite surfaces of thyroid epithelial cells: NIS basolaterally andthe new molecule apically. The latter was thus named the apical I  transporter (5). However, it has since been unequivocally demon-strated that the product of the  SLC5A8  gene does not transport I  (6, 7), a finding we have also confirmed. Instead, because thisprotein transports monocarboxylic acids such as lactate, pyruvate,propionate, butyrate, and nicotinate (6–9), it is now called theNa   monocarboxylate transporter (SMCT). Significantly, SMCThas also been proposed to be a tumor suppressor in gliomas andcolon cancer (10, 11).We report here the existence of a high-affinity Na  -dependentmonocarboxylate transport system in thyroid cells and that neitherSMCT expression nor the Na  -dependent monocarboxylate trans-port system is regulated by thyroid-stimulating hormone (TSH), amajorregulatorofthyroidfunction.Wedeterminedthecellularandsubcellular localization of this transporter in the thyroid, kidney,and colon. Strikingly, we show that higher levels of SMCT expres-sion in Duke C colorectal cancer samples obtained from 113patients correlated with significantly longer disease-free survival. Results and Discussion SMCT Expression in Thyroid Cells Is TSH-Independent.  Given thehomology between SMCT and NIS and based on the experimen-tally tested NIS secondary structure model (Fig. 1  A ), we haveproposed one for SMCT (Fig. 1  B ), which predicts that SMCTtraverses the membrane 13 times, its N terminus faces the extra-cellularmilieu,anditsCterminusfacesthecytoplasm.Interestingly,as many as 8 of the 12 cysteines found in NIS are conserved inSMCT (at positions 130, 173, 270, 297, 308, 344, 396, and 480), andmost of the NIS residues identified as critical from the study of NISmutations that cause congenital iodide transport defect are alsoconserved in SMCT (Val-57, Arg-122, Thr-352, Gly-393, andGly-536) (12–14).Because SMCT expression was initially demonstrated in thethyroid (5), we used our affinity-purified anti-SMCT Abs againstthelast17aminoacidresiduesofmouseSMCTtoprobemembranefractions from FRTL-5 cells, a line of highly functional rat thyroidcells (15). These Abs exhibited a very high affinity for SMCT (  K  a  10 nM; data not shown). Immunoreactivity was observed againsta single, broad   75-kDa polypeptide (Fig. 2  A , left lane), whoseelectrophoretic migration was slower than that predicted by themolecular mass of SMCT (  62 kDa). This observation, coupled with the presence of two putative N - linked glycosylation sites(Asn-480 and Asn-485, Fig. 1  B ), suggested that SMCT was aglycoprotein. We confirmed this prediction by demonstrating thattreatment of the membrane fractions with peptide  N  -glycosidase F,an enzyme that removes N - linked carbohydrates, caused SMCT tomigrate as an  60-kDa polypeptide (Fig. 2  A , right lane).The predicted cytosolic orientation of the SMCT C terminus(Fig. 1  B ) was demonstrated by detecting immunofluorescence inpermeabilized (Fig. 2  B Center  ) but not in nonpermeabilizedFRTL-5 cells (Fig. 2  B Left ). There was no immunoreactivity whenonly the secondary Ab was added (Fig. 2  B Right ). Further confir-mation was obtained by flow cytometry (Fig. 2 C ): a fluorescenceshift was observed only in permeabilized cells, where the Ab hadaccess to the epitope (Fig. 2 C Right ).Because NIS expression and targeting to the plasma membrane Conflict of interest statement: No conflicts declared.Freely available online through the PNAS open access option.Abbreviations:CHC,cyano-4-hydroxycinnamate;hSMCT,humanSMCT;MCT1,monocarboxy-late transporter 1; MDCK, Madin–Darby canine kidney; NIS, Na   I  symporter; SCFA, short-chainfattyacid;SMCT,Na   monocarboxylatetransporter;TSH,thyroid-stimulatinghormone. ¶ To whom correspondence should be addressed. E-mail: carrasco@aecom.yu.edu.© 2006 by The National Academy of Sciences of the USA 7270–7275    PNAS    May 9, 2006    vol. 103    no. 19 www.pnas.org  cgi  doi  10.1073  pnas.0602365103  are regulated by TSH in thyroid cells (16), we examined whetherTSH had similar effects on SMCT. TSH was withdrawn from theFRTL-5 cell culture medium for 7 days, and SMCT expression wasassessed by immunoblotting the FRTL-5 membrane fractions. InmarkedcontrasttoNIS(Fig.2  DTop ),SMCTwasexpressedinboththe presence and absence of TSH (Fig. 2  D Middle ). Cell surfacebiotinylation revealed that SMCT plasma membrane targeting wasalso not regulated by TSH (Fig. 2  D Bottom ). An Endogenous Na  -Dependent Monocarboxylate Transport System,Likely to Be SMCT, Is Present in Thyroid Cells.  We examined themonocarboxylate transport properties of FRTL-5 cells by using[ 14 C]nicotinate as a substrate. We chose nicotinate because, unlikeother monocarboxylates, it is not transported by monocarboxylatetransporter 1 (MCT1), a different protein expressed in most celltypes,includingFRTL-5cells(17,18).IncontrasttoSMCTactivity,MCT1-mediated monocarboxylate transport is not Na  -depen-dent,butitsactivityislargeenoughtointerferewithmeasurementsof SMCT-mediated transport. Although derivatives of    -cyanocin-namate such as   -cyano-4-hydroxycinnamate (CHC) are consid-ered specific inhibitors of MCT1, these compounds also block themitochondrial pyruvate transporter and the anion exchanger AE1(18), limiting their usefulness. When SMCT was expressed inoocytes, modest inhibition of propionate-evoked currents in thepresence of 1 mM CHC was observed (6). Further, in our exper-iments, CHC moderately inhibited the Na  -dependent compo-nents of monocarboxylate uptake (not shown). Thus, to preventMCT1-mediated activity from being factored in, we used 50   M[ 14 C]nicotinate as a substrate in steady-state transport assays inFRTL-5 cells maintained in the presence or absence of TSH for 7days. [ 14 C]Nicotinate uptake was clearly Na  -dependent and dis-played very similar characteristics in the presence and absence of TSH (Fig. 2  E ). Determination of kinetic parameters for [ 14 C]ni-cotinate uptake was performed at concentrations from 4.5 to 600  M. The data displayed typical Michaelis–Menten behavior with  K  m  53  5.8   M and  V  max   72  7.3 pmol   g of DNA per 2min in the presence and absence of TSH (Fig. 2  F  ). The Eadie–Hofstee transformation showed linearity of data, compatible withthepresenceofasingletransportsystem(notshown).Inconclusion,these findings demonstrate the existence of an endogenous high-affinity Na  -dependent monocarboxylate uptake system in thyroidcellsnotregulatedbyTSH,characteristicsthatmakeitlikelyforthissystem to be SMCT. Further studies are necessary to reveal thespecific physiological role of SMCT in the thyroid. SMCT Is Not Inhibited by High Concentrations of Short-Chain FattyAcids (SCFAs).  We analyzed SMCT expressed in  Xenopus laevis oocytes during injection of SMCT cRNA by using the two-microelectrode voltage clamp technique (19, 20). When SMCT-expressing oocytes were placed in a NaCl buffer and voltage-clamped at  50 mV, the holding current was recorded when 1 mMpropionate was added to the bath (Fig. 3  A ). In agreement withprevious results (6, 7, 9), the addition of propionate caused aninward positive current of   5–70 nA, indicating a net transfer of positive charge into the oocyte, attributable to SMCT activity (Fig.3  A Right ). Thus, SMCT-mediated propionate transport is electro-genic, i.e., it involves the translocation of at least two Na  ions perpropionate anion. When propionate was added in the absence of external Na  in the medium (choline was used to replace Na  ), noinward current was evoked, indicating that SMCT-mediated trans-port is completely Na  -dependent (Fig. 3  A Center  ); in control water-injected oocytes, propionate at concentrations up to 7.5 mMdid not induce an inward current (Fig. 3  A Left ). Therefore, theobserved currents were generated by SMCT activity. We theninvestigated the transport of several monocarboxylates (1 mM)besidespropionate:nicotinate, L  -and D -lactate,pyruvate,butyrate, Fig. 1.  Secondary structure models of NIS and SMCT. TheexperimentallytestedNIS(  A )andproposedSMCT( B )secondarystructure models are compared. The 13 putative transmem-brane segments are indicated by cylinders. Least conservedtransmembranesegments(IIIandVIII)aredepictedinwhiteforSMCT. N termini face extracellularly and C termini intracellu-larly. N - linked glycosylation sites are depicted as branches. Thesegment of the C terminus against which the SMCT Ab wasgenerated is indicated as a black rectangle. Fig. 2.  SMCT protein expression and function in FRTL-5 cells. (  A ) Immunoblotanalysis of membrane fractions from FRTL-5 cells (  60  g of protein) incubatedeither with or without peptide  N  -glycosidase F overnight at 37°C, electropho-resed, and immunoblotted with anti-mouse SMCT Ab. ( B ) Indirect immunofluo-rescence of FRTL-5 cells with anti-mouse SMCT Ab followed by fluorescein-conjugated anti-rabbit IgG. ( Left  ) Nonpermeabilized conditions. ( Center  )Permeabilized with 0.1% Triton X-100. ( Right  ) Without primary Ab. ( C  ) FACSanalysis of nonpermeabilized ( Left  ) and permeabilized ( Right  ) FRTL-5 cells withanti-SMCT Ab. ( D ) Membrane fractions (20   g) from FRTL-5 cells grown in thepresence or absence of TSH were electrophoresed and immunoblotted witheitheranti-ratNIS( Top )oranti-mouseSMCTAbs( Middle ).( Bottom )Immunoblotanalysis of biotinylated cell surface polypeptides with anti-mouse SMCT Ab. ( E  )[ 14 C]Nicotinate steady-state uptake in FRTL-5 cells in the presence or absence ofTSH and in the presence of Na  (shaded bars) or choline (open bars). ( F  ) Kineticanalysisof[ 14 C]nicotinateuptakeinFRTL-5cellsinthepresence(continuousline)or absence (broken line) of Na  . Paroder  et al  . PNAS    May 9, 2006    vol. 103    no. 19    7271      B     I     O     C     H     E     M     I     S     T     R     Y  pentanoate, acetate, 2-oxobutyrate, 2-hydroxybutyrate, 3-hydroxy-butyrate, 4-hydroxybutyrate, and acetoacetate, all of which evokedcurrents (Fig. 3  B ). We normalized these currents to the currentelicited by propionate (100%) in the same oocyte. Significantly,biotin and thyroxine evoked no currents, demonstrating that theseare not SMCT substrates (not shown). In addition, consistent withCoady  et al.  (6), I  did not elicit currents either, disproving aprevious report contending that SMCT transported I  (5). Biotinis the substrate of the multivitamin transporter SMVT, which is58%similartoSMCT.Inoocytes,thekineticanalysisofpropionatetransport at a membrane potential of   50 mV revealed a  K  m  valueof 162    26   M (Fig. 3 C ), in agreement with reported data (6).Given the high SCFA concentrations in the colonic lumen, wereexamined the effect of high concentrations of SCFAs on SMCTactivity. SMCT-mediated propionate transport continued to beobserved at 4 mM (Fig. 3 C ) and up to 7.5 mM (not shown),demonstrating that propionate does not inhibit SMCT activity atthese concentrations. These results differ from those of a previousreport contending that concentrations of SCFAs  1 mM inhibitedthe substrates’ own transport (6).We also examined SMCT activity as a function of extracellularpH (ranging from 5 to 9). At each pH, the propionate-evokedcurrentwasnormalizedtothatelicitedatpH7.4inthesameoocyte. At pH values lower than 6.5, transport activity decreased withdecreasingpH;however,peakSMCTactivitywasvirtuallyidenticalin the pH range 6.5–9.0 (Fig. 3  D ), unlike the reported narrow pHdependence of mouse SMCT (9). The nonsteroidal antiinflamma-tory drug ibuprofen inhibited the propionate-evoked current witha  K  i  value of 73  9   M (Fig. 3  E  and  F  ). Further, kinetic analysisofhumanSMCT(hSMCT)-mediatednicotinatetransportrevealeda  K  m  value of 390  36   M (Fig. 3 G ), in agreement with Gopal  et al.  (8) (  K  m    296    88   M) for murine SMCT, whereas in ratFRTL-5 cells the  K  m  for nicotinate was  6-fold higher (Fig. 2  F  ),probably because of species differences. Interestingly, in FRTL-5cells 500   M ibuprofen completely inhibited the Na  -dependentcomponent of [ 14 C]nicotinate uptake as well (not shown). SMCT Is Localized on the Apical Surface of Thyroid, Colon, and KidneyEpithelial Cells.  Because SMCT exon 1 was reported to be hyper-methylated in colon cancer (11), and SMCT transports SCFAs, which are the major energy source of colonocytes (21), we inves-tigated the cellular and subcellular localization of SMCT in humanand rat colon tissues. When extracts from human colon samples were probed, an  75-kDa polypeptide was detected (Fig. 4  A , leftlane), whose electrophoretic migration was slower than that pre-dictedbythehSMCTmolecularmass(  67kDa).Duringtreatment with peptide  N  -glycosidase F, the polypeptide migrated at anapparent molecular mass of    65 kDa (Fig. 4  A , right lane), indi-cating that SMCT is glycosylated in human colon. Two bands weredetected by immunoblot analysis of rat colon tissue: a broad  85-kDa one and a narrow   54-kDa one, corresponding to thematurely and immaturely glycosylated polypeptides, respectively(Fig. 4  B Upper  ). The immaturely glycosylated precursor becomesdetectable when fractions are enriched for integral membraneproteinsbyalkalineextraction(22).Immunohistochemistryanalysisrevealed that SMCT expression was restricted to the apical surfaceofthecolonocytesandwasabsentfromthesurroundinggobletcellsand underlying tissue layers (Fig. 4  B Lower  ). This finding isconsistentwiththenotionthatSMCTmediatesthetranslocationof SCFAs from the colonic lumen to the epithelial cells. SMCTmigrated as an   75–80-kDa band in immunoblot analyses of ratkidney and thyroid (Fig. 4  C  and  D ). SMCT was also expressedapically in the brush border of renal proximal tubules (Fig. 4 C Lower  ) and to a lesser extent, but still apically, in the distal parts of  Fig. 3.  Functional properties of SMCT in  X. laevis  oocytes. The membranepotential was clamped at   50 mV. (  A ) Propionate (1 mM) did not evoke acurrent in control oocytes ( Left  ), whereas in SMCT-expressing oocytes, 1 mMpropionateevokedaninwardcurrentthatwas100%Na  -dependent( Center  and Right  ).( B )Currentsevokedbyvarioussubstrates(1mM)werenormalizedto the current elicited by 1 mM propionate in the same oocyte ( n    3). ( C  )Kinetic analysis of propionate transport ( n  3). ( D ) The pH dependence ofpropionate-evokedinwardcurrentswasexaminedintherangeof5–9.AteachpH, the current evoked by 1 mM propionate was normalized with respect tothatelicitedatpH7.4.( E  )Applicationofibuprofenalone(upto2mM)didnotalter the holding current. When applied in the presence of propionate,ibuprofen inhibited the 1 mM propionate-evoked current in a dose-dependent manner. ( F  ) Ibuprofen inhibited the propionate-evoked currentwith a  K  i  of 73  9   M ( n  3). ( G ) Kinetic analysis of nicotinate transport. Fig.4.  ExpressionandsubcellularlocalizationofSMCTincolon,kidney,andthyroid. (  A ) Immunoblot analysis of peptide  N  -glycosidase-F-treated humancolontissuelysates.( B – D )Immunoblotanalysis( Upper  )ofmembranefractionsof colon ( B ), kidney ( C  ), and thyroid ( D ) rat tissues with the correspondingimmunohistochemistries (  40) ( Lower  ) using anti-mouse SMCT Ab. Each im-munoblot contains tissues from two representative animals. 7272    www.pnas.org  cgi  doi  10.1073  pnas.0602365103 Paroder  et al  .  the nephron. In the thyroid, as reported in ref. 5, SMCT wasexpressed apically as well (Fig. 4  D Lower  ). SMCT Is Properly Targeted to the Apical Surface in Polarized Madin–Darby Canine Kidney (MDCK) Cells.  MDCK cells have been exten-sively used to investigate polarized membrane protein targeting(23). We generated MDCK cells stably expressing functionalhSMCT. SMCT expression was analyzed by FACS, followed byimmunoblot analysis of membrane fractions from FACS-positiveclones. An MDCK clone moderately expressing hSMCT was se-lected for further analysis; immunoreactivity against an  84-kDapolypeptide from this clone is shown in Fig. 5  A .We assessed the functionality of SMCT in this system by con-ducting steady-state and kinetic analyses of [ 14 C]nicotinate uptake.The MDCK hSMCT clone transported  9 times more [ 14 C]nico-tinatethannontransfectedMDCKcells(Fig.5  B )witha  K  m of284  83   M. Because SMCT was initially reported to be an I  trans-porter,weexaminedwhetherSMCT-expressingMDCKcellstrans-locateI  .Steady-stateI  uptakeexperimentsatsaturating(20  M,not shown) and supersaturating (80   M) I  concentrations wereconductedinMDCKcellsstablyexpressingNIS(24)orSMCT(Fig.5 C ). As expected, NIS-expressing MDCK cells displayed perchlor-ate-sensitive I  uptake. In contrast, SMCT-expressing MDCK exhibited no I  transport, further confirming previous reports and ourown observations that SMCT does not mediate I  transport (Fig. 5 C ).We analyzed SMCT polarized targeting by immunofluorescencedirectly on filter membranes. Monoclonal antibodies against gp135and the Na  ,K   -ATPase were used to monitor apical and baso-lateral markers, respectively (25). The  en face  (  xy ) view revealedclear apical localization of SMCT and gp135 (Fig. 5  D1  and  D2 ).Cross sections in the  xz  direction confirmed this finding (Fig. 5  D4 and  D5 ). The apical distribution of SMCT was confirmed bycolocalization with gp135 (Fig. 5  D3  and  D6 ). Higher Expression of SMCT in Duke C Human Colorectal CancerCorrelates with Longer Disease-Free Survival.  Because no high-affinity anti-SMCT Abs were available, other investigators hadexamined SMCT expression only at the transcriptional level (8, 9,11). Using our Abs, we analyzed SMCT protein expression in DukeC (locally advanced lymph node-positive) human colon cancersamples (tumoral and paired peritumoral normal mucosa) byimmunoblotting. Remarkably, SMCT was absent or markedlydown-regulated in tumor tissue (Fig. 6  A  and  B , asterisks) com-pared with adjacent normal mucosa in 14 of 15 pairs examined.Equalloadingforeachtissuepairwasdeterminedwithmonoclonalanti-  -tubulin (Fig. 6  A ) or anti-  -actin Abs (Fig. 6  B ). Thesesamples, obtained from patients treated in New York City, repre-sentedaheterogeneoushistopathologicalpopulation(rangingfrompoorly to moderately differentiated adenocarcinomas). There wasno correlation between the differentiation status of the tumor andthe down-regulation of SMCT protein levels.To extend our findings, we assessed SMCT expression by immu-nohistochemistryintumorsamplesfromcolorectalcancerpatients.To control for possible confounding factors affecting patient sur- vival, all 113 analyzed tumors were stage Duke C, and cases withmicrosatellite instability were excluded. SMCT expression wasevaluated in a tissue microarray-based immunohistochemical assayin triplicate and scored blindly on a scale from 0 (no staining) to 4(strongstaining)(Fig.6 C ).Theaveragescoreoftriplicateswasusedinthefollowinganalyses.Tumorsampleswithstainingintensitiesof 1.7 or less (75 of 113) were considered low-SMCT-expressing, whereas those with staining intensities higher than 1.7 (38 of 113) were considered high-SMCT-expressing; 66.4% of the tumorsexpressed low levels of SMCT. There was a significant correlationbetween staining levels and patient age (Spearman  r   0.27,  P   0.0018), i.e., patients with low-SMCT-expressing tumors tended tobe younger. There were no correlations between SMCT expressionandcommongeneticabnormalitieslinkedtocolorectalcancer(  p53 and  k ras mutations or loss of heterozygosity in 18q) or otherclinicopathologicalfeatures[gender,grade,ortumorlocation(rightor left colon or rectum)].To avoid selecting a threshold arbitrarily, we arranged the 113tumors in order of increasing SMCT expression and divided theminto two groups: low- and high-SMCT expression. Disease-freesurvival in both groups and log-rank  P   values were calculated forevery possible grouping resulting from increasing the number of patientsallocatedtothelow-SMCT-expressinggroupfrom1to113,startingwiththepatientwiththelowestSMCTtumorlevel.Patients withlow-SMCT-expressingtumorsshowedshorterdisease-freeandoverall survival in 108 of 113 groupings. These differences werestatistically significant (log-rank test  P     0.05) in 11 of thesegroupings, suggesting that low expression of SMCT is a marker of poor prognosis in Duke C colorectal cancer (Fig. 6  D ). On amultivariate analysis, high SMCT protein levels were a strongpredictor of longer disease-free survival (  P     0.027). Further, tomake sure that longer disease-free survival was not the result of postsurgical chemotherapy with 5-fluorouracil, we conducted sur- vival analyses with 81 patients from this group who were onlytreated surgically. Higher SMCT protein levels remained a strongpredictor for disease-free survival in these patients (Fig. 6  E ). Thisfinding is significant, given that it is currently impossible to predictaccuratelytheprobabilityofrecurrenceinDukeCcolorectalcancerafter surgery with or without chemotherapy (26–29). Consideringthat SMCT expression would be easy to ascertain in colon cancersamplesobtainedinroutinecolonoscopicscreenings,thesefindingssuggestthatSMCTexpressionmaybeasignificantmarkerforlowerdisease recurrence in Duke C colorectal cancer.In conclusion, we have extensively characterized the SMCT Fig. 5.  SMCT is apically expressed in polarized MDCK cells. (  A ) ImmunoblotanalysisofSMCTexpressioninMDCKcellsstablytransfectedwithhSMCTandin nontransfected (NT) MDCK cells. ( B ) Time course of [ 14 C]nicotinate uptakein MDCK cells stably transfected with hSMCT (open squares) and nontrans-fectedMDCKcells(filledsquares).( C  )Steady-stateI  uptakeassays(80  MI  )inMDCKcellsstablytransfectedwithhumanNISorwithhSMCT;shadedbars,assaysdoneinthepresenceofNa  ;blackbars,assaysdoneinthepresenceofNa  and perchlorate, a competitive inhibitor of NIS. ( D ) ImmunofluorescenceanalysisofSMCTtargetinginMDCKcellsstablytransfectedwithhSMCT.( 1–6  )Colocalizationwithanapicalmarker,gp135.( 7–12 )Absenceofcolocalizationwith a basolateral marker (Na  ,K  -ATPase). Paroder  et al  . PNAS    May 9, 2006    vol. 103    no. 19    7273      B     I     O     C     H     E     M     I     S     T     R     Y  proteinatmultiplelevelsandshownthatitmayplayavaluablerole,not previously examined, in colon cancer management. Methods Cloning.  The hSMCT cDNA was cloned from the Human ThyroidGland Marathon-Ready cDNA (BD Biosciences Clontech). Theprimers used to amplify SMCT cDNA were 5  -GTTCTCATCTG-CTCAGGTGTCC and 5  -GCGTGTATTAGCCTTTCAGCAT.The PCR parameters were 35 cycles of 95°C for 30 s, 53°C for 30 s,and 68°C for 2.5 min, followed by 68°C for 10 min, and 4°C to cool.The amplified cDNA was digested with BamHI and EcoRI andligated into the pcDNA3 vector (Invitrogen). Sequencing primers were T7 promoter primer 5  -TAATACGACTCACTATAGGG(Invitrogen), 5  -CGCAGAATGACCGCAGTG, 5  -CCAGCATC-TACGGTCTCAAC, and 5  -TTTGGGCATTTTGGTTCCC. Generation of Anti-SMCT Abs.  High-affinity site-directed polyclonal Abs against the C-terminal sequences of the rodent (amino acids596–611, VELNFTDHSGKINGTRL) and human (amino acids591–610, AFNHIELNSDQSGKSNGTRL) SMCT proteins weregenerated. Purification was performed as described in ref. 30. Cell Culture.  FRTL-5 rat thyroid cells were cultured as described inref. 16. MDCK II cells were maintained in DMEM (Invitrogen)supplemented with 10% FBS (Gemini BioProducts, West Sacra-mento, CA), 1% glutamine, and 1% penicillin  streptomycin (In- vitrogen) at 37°C in a 95% air  5% CO 2  atmosphere. For polarizedculture, stably transfected MDCK cells or nontransfected MDCK cells were plated on 12-mm (1-cm 2 area) Transwell polyester filterunits (0.4-  m pore size; Costar) at a density of 150,000 cells perfilter unit and cultured for 5 days to allow development of polarity. Generation of Stable Clones.  MDCK II cells in 12-well plates weretransfected with 2   g of plasmid containing hSMCT cDNA withLipofectamine 2000 (Invitrogen). Stable clones were selected andmaintained in a selection medium containing 1 g  liter G418, asdescribed in ref. 24. Flow Cytometry.  FRTL-5 cells were incubated for 1 h with 100  l of PBS  0.1%BSA   0.2%saponincontaining9nManti-mouseSMCT Ab and processed as described in ref. 14. Colon Tumor Extract and Membrane Fraction Preparation.  Humancolorectaltumorswithpairedadjacentnormaltissuewerecollectedafter surgical resection from patients who consented according toinstitutional guidelines; the samples were immediately snap-frozenin liquid nitrogen. Proteins were extracted by brief homogenizationon ice in lysis buffer (50 mM Tris  HCl, pH 7.5  150 mM NaCl  1%Nonidet P-40  0.5% sodium deoxycholate  1 mM EDTA   proteaseinhibitor mixture) followed by incubation for 30 min at 4°C withgentle agitation. After incubation, samples were sonicated for 10 sandcentrifugedat14,000   g  for10min.Membranefractionswereprepared as described in ref. 22. Deglycosylation Assays.  Membrane protein fractions or tissue ex-tracts were deglycosylated with peptide  N  -glycosidase F(PROzyme, San Leandro, CA) and subjected to immunoblotanalysis as described in ref. 31. Immunoblot Analysis and Cell-Surface Biotinylation.  These proce-dures were performed as described in ref. 22 with 13 nM affinity-purified anti-mouse SMCT Ab, 7 nM affinity-purified anti-hSMCT Ab, and 4 nM affinity-purified anti-rat NIS Ab for 1 h andmonoclonal anti-  -actin 1:10,000 (Sigma) or anti-  -tubulin 1:3,000 Ab (Sigma). Immunofluorescence and Immunohistochemical Analyses.  FRTL-5cells were seeded onto polylysine-coated coverslips. Cells wereincubated with 70 nM primary Ab against mouse SMCT in PBScontaining 1 mM CaCl 2  and 0.1 mM MgCl 2  (PBS  CM) with 0.2%BSA and 0.1% Triton X-100 for 1 h and processed as described inref. 14. For immunohistochemistry, 5-  m colon tissue sections were deparaffinated and rehydrated and processed as described, with affinity-purified anti-mouse-SMCT Ab at 7 nM final concen-tration (32). ImmunofluorescenceMicroscopyofPolarizedMDCKCells. MDCKcellmonolayerswerefixedin2%freshlypreparedparaformaldehydeinPBS for 20 min at room temperature and quenched with 50 mMNH 4 Cl in PBS  CM. For permeabilization, methanol at  20°C or0.1% saponin was used. Cells were blocked in PBS  CM containing10% goat serum (Invitrogen). The primary Ab used was a mAb Fig. 6.  Higher levels of SMCT correlate with longer disease-free survival in Duke C colorectal cancer patients. (  A  and  B ) Immunoblot analysis of tumoral andperitumoral colon tissue extracts (70   g each) with anti-hSMCT Ab. All samples displayed clear expression of SMCT in peritumoral tissue. Tumoral tissues in 14of15cases(asindicatedbyasterisks)exhibitedcompleteabsenceormarkeddown-regulationofSMCT.  -Tubulin(  A )or  -actin( B )servedasaninternalloadingcontrol. ( C  ) ( Upper  ) Representative examples of colorectal tumors showing increasing levels (from 1 to 4) of SMCT immunostaining. ( Lower  ) High-powermagnification of the areas outlined in  Upper  . ( D ) Overall survival ( Upper  ) and disease-free survival ( Lower  ) based on SMCT protein levels in Duke C colorectalcancer patients (Kaplan–Meier plots). ( E  ) Overall survival ( Upper  ) and disease-free survival ( Lower  ) based on SMCT protein levels in Duke C colorectal cancerpatients (Kaplan–Meier plots) who were not treated with 5-fluorouracil postoperatively. 7274    www.pnas.org  cgi  doi  10.1073  pnas.0602365103 Paroder  et al  .
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