Na Monocarboxylate Transport (SMCT) Protein Expression

Na؉͞monocarboxylate transport (SMCT) protein expression 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 State Polytechnic University, Pomona, CA 91768; ‡Molecular Oncology and Aging Group, Molecular Biology and Biochemistry Research Center, Valle Hebron Hospital 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 10467

Communicated by H. Ronald Kaback, University of California, Los Angeles, CA, March 22, 2006 (received for review January 7, 2006) We report an extensive characterization of the Na؉͞monocarboxy- major regulator of thyroid function. We determined the cellular and late transporter (SMCT), a plasma membrane protein that mediates subcellular localization of this transporter in the thyroid, kidney, active transport of monocarboxylates such as propionate and and colon. Strikingly, we show that higher levels of SMCT expres- nicotinate, and we show that SMCT may play a role in colorectal sion in Duke C colorectal cancer samples obtained from 113 cancer diagnosis. SMCT, the product of the SLC5A8 gene, is 70% patients correlated with significantly longer disease-free survival. similar to the Na؉͞I؊ symporter, the protein that mediates active I؊ uptake in the basolateral surface of thyrocytes and other cells. Results and Discussion SMCT was reported in the apical surface of thyrocytes and formerly SMCT Expression in Thyroid Cells Is TSH-Independent. Given the -proposed also to transport I؊ and was called the apical I؊ trans- homology between SMCT and NIS and based on the experimen porter. However, it is now clear that SMCT does not transport I؊. tally tested NIS secondary structure model (Fig. 1A), we have Here we demonstrate a high-affinity Na؉-dependent monocar- proposed one for SMCT (Fig. 1B), which predicts that SMCT boxylate transport system in thyroid cells, which is likely to be traverses the membrane 13 times, its N terminus faces the extra- ,SMCT. We show that, whereas thyroidal Na؉͞I؊ symporter expres- cellular milieu, and its C terminus faces the cytoplasm. Interestingly sion is thyroid-stimulating hormone (TSH)-dependent and baso- as many as 8 of the 12 cysteines found in NIS are conserved in lateral, SMCT expression is TSH-independent and apical not only in SMCT (at positions 130, 173, 270, 297, 308, 344, 396, and 480), and the thyroid but also in kidney and colon epithelial cells and in most of the NIS residues identified as critical from the study of NIS polarized Madin–Darby canine kidney cells. We determine the mutations that cause congenital iodide transport defect are also kinetic parameters of SMCT activity and show its inhibition by conserved in SMCT (Val-57, Arg-122, Thr-352, Gly-393, and .(␮M) in Xenopus laevis oocytes. SMCT was Gly-536) (12–14 9 ؎ 73 ؍ ibuprofen (Ki proposed to be a tumor suppressor in colon cancer [Li, H., et al. (2003) Because SMCT expression was initially demonstrated in the Proc. Natl. Acad. Sci. USA 100, 8412–8417]. Significantly, we show that thyroid (5), we used our affinity-purified anti-SMCT Abs against higher expression of SMCT in colon samples from 113 colorectal the last 17 amino acid residues of mouse SMCT to probe membrane cancer patients correlates with longer disease-free survival, suggest- fractions from FRTL-5 cells, a line of highly functional rat thyroid ing that SMCT expression may be a favorable indicator of colorectal cells (15). These Abs exhibited a very high affinity for SMCT (Ka Ϸ cancer prognosis. 10 nM; data not shown). Immunoreactivity was observed against a single, broad Ϸ75-kDa polypeptide (Fig. 2A, left lane), whose electrophoretic migration was slower than that predicted by the he SLC5A8 gene product is a plasma membrane transport molecular mass of SMCT (Ϸ62 kDa). This observation, coupled protein that belongs to solute carrier family 5 (SLC5A). Mem- T with the presence of two putative N-linked glycosylation sites bers of this family couple the energy released by the inward ϩ (Asn-480 and Asn-485, Fig. 1B), suggested that SMCT was a ‘‘downhill’’ translocation of Na in favor of its concentration glycoprotein. We confirmed this prediction by demonstrating that gradient to the inward active transport of another solute against its ϩ͞ Ϫ treatment of the membrane fractions with peptide N-glycosidase F, chemical gradient. The extensively characterized Na I sym- an enzyme that removes N-linked carbohydrates, caused SMCT to porter (NIS, SLC5A5), a key plasma membrane protein that Ϸ Ϫ migrate as an 60-kDa polypeptide (Fig. 2A, right lane). mediates active I transport in the thyroid and other tissues, is also The predicted cytosolic orientation of the SMCT C terminus a member of this family (1–4). SLC5A8 was originally identified as (Fig. 1B) was demonstrated by detecting immunofluorescence in a protein homologous (70% similarity and 46% identity) to NIS and Ϫ permeabilized (Fig. 2B Center) but not in nonpermeabilized also proposed to transport I . These two molecules are localized on FRTL-5 cells (Fig. 2B Left). There was no immunoreactivity when opposite surfaces of thyroid epithelial cells: NIS basolaterally and Ϫ only the secondary Ab was added (Fig. 2B Right). Further confir- the new molecule apically. The latter was thus named the apical I mation was obtained by flow cytometry (Fig. 2C): a fluorescence transporter (5). However, it has since been unequivocally demon- Ϫ shift was observed only in permeabilized cells, where the Ab had strated that the product of the SLC5A8 gene does not transport I access to the epitope (Fig. 2C Right). (6, 7), a finding we have also confirmed. Instead, because this Because NIS expression and targeting to the plasma membrane protein transports monocarboxylic acids such as lactate, pyruvate, propionate, butyrate, and nicotinate (6–9), it is now called the ϩ Na ͞monocarboxylate transporter (SMCT). Significantly, SMCT Conflict of interest statement: No conflicts declared. has also been proposed to be a tumor suppressor in gliomas and Freely available online through the PNAS open access option. colon cancer (10, 11). Abbreviations: CHC, cyano-4-hydroxycinnamate; hSMCT, human SMCT; MCT1, monocarboxy- ϩ We report here the existence of a high-affinity Na -dependent late transporter 1; MDCK, Madin–Darby canine kidney; NIS, Naϩ͞IϪ symporter; SCFA, short- monocarboxylate transport system in thyroid cells and that neither chain fatty acid; SMCT, Naϩ͞monocarboxylate transporter; TSH, thyroid-stimulating hormone. ϩ SMCT expression nor the Na -dependent monocarboxylate trans- ¶To whom correspondence should be addressed. E-mail: [email protected]. port system is regulated by thyroid-stimulating hormone (TSH), a © 2006 by The National Academy of Sciences of the USA

7270–7275 ͉ PNAS ͉ May 9, 2006 ͉ vol. 103 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602365103 Downloaded by guest on September 28, 2021 Fig. 1. Secondary structure models of NIS and SMCT. The experimentally tested NIS (A) and proposed SMCT (B) secondary structure models are compared. The 13 putative transmembrane segments are indicated by cylinders. Least conserved transmembrane segments (III and VIII) are depicted in white for SMCT. N termini face extracellularly and C termini intracellu- larly. N-linked glycosylation sites are depicted as branches. The segment of the C terminus against which the SMCT Ab was generated is indicated as a black rectangle.

are regulated by TSH in thyroid cells (16), we examined whether [14C]nicotinate as a substrate. We chose nicotinate because, unlike TSH had similar effects on SMCT. TSH was withdrawn from the other monocarboxylates, it is not transported by monocarboxylate FRTL-5 cell culture medium for 7 days, and SMCT expression was transporter 1 (MCT1), a different protein expressed in most cell assessed by immunoblotting the FRTL-5 membrane fractions. In types, including FRTL-5 cells (17, 18). In contrast to SMCT activity, marked contrast to NIS (Fig. 2D Top), SMCT was expressed in both MCT1-mediated monocarboxylate transport is not Naϩ-depen- the presence and absence of TSH (Fig. 2D Middle). Cell surface dent, but its activity is large enough to interfere with measurements biotinylation revealed that SMCT plasma membrane targeting was of SMCT-mediated transport. Although derivatives of ␣-cyanocin- also not regulated by TSH (Fig. 2D Bottom). namate such as ␣-cyano-4-hydroxycinnamate (CHC) are considered specific inhibitors of MCT1, these compounds also block the ,An Endogenous Na؉-Dependent Monocarboxylate Transport System mitochondrial pyruvate transporter and the anion exchanger AE1 Likely to Be SMCT, Is Present in Thyroid Cells. We examined the monocarboxylate transport properties of FRTL-5 cells by using (18), limiting their usefulness. When SMCT was expressed in oocytes, modest inhibition of propionate-evoked currents in the presence of 1 mM CHC was observed (6). Further, in our experiments, CHC moderately inhibited the Naϩ-dependent compo- nents of monocarboxylate uptake (not shown). Thus, to prevent MCT1-mediated activity from being factored in, we used 50 ␮M [14C]nicotinate as a substrate in steady-state transport assays in FRTL-5 cells maintained in the presence or absence of TSH for 7 days. [14C]Nicotinate uptake was clearly Naϩ-dependent and displayed very similar characteristics in the presence and absence of TSH (Fig. 2E). Determination of kinetic parameters for [14C]nicotinate uptake was performed at concentrations from 4.5 to 600 ␮M. The data displayed typical Michaelis–Menten behavior with Km ϭ 53 Ϯ 5.8 ␮M and Vmax ϭ 72 Ϯ 7.3 pmol͞␮g of DNA per 2 min in the presence and absence of TSH (Fig. 2F). The Eadie– Hofstee transformation showed linearity of data, compatible with the presence of a single transport system (not shown). In conclusion, these findings demonstrate the existence of an endogenous high- affinity Naϩ-dependent monocarboxylate uptake system in thyroid cells not regulated by TSH, characteristics that make it likely for this system to be SMCT. Further studies are necessary to reveal the specific physiological role of SMCT in the thyroid.

SMCT Is Not Inhibited by High Concentrations of Short-Chain Fatty Acids (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 Ϫ50 mV, the holding current was recorded when 1 mM propionate was added to the bath (Fig. 3A). In agreement with Fig. 2. SMCT protein expression and function in FRTL-5 cells. (A) Immunoblot previous results (6, 7, 9), the addition of propionate caused an Ϸ ␮ analysis of membrane fractions from FRTL-5 cells ( 60 g of protein) incubated inward positive current of Ϸ5–70 nA, indicating a net transfer of either with or without peptide N-glycosidase F overnight at 37°C, electrophoresed, and immunoblotted with anti-mouse SMCT Ab. (B) Indirect immunoﬂuo- positive charge into the oocyte, attributable to SMCT activity (Fig. 3A Right). Thus, SMCT-mediated propionate transport is electro- rescence of FRTL-5 cells with anti-mouse SMCT Ab followed by ﬂuorescein- ϩ conjugated anti-rabbit IgG. (Left) Nonpermeabilized conditions. (Center) genic, i.e., it involves the translocation of at least two Na ions per Permeabilized with 0.1% Triton X-100. (Right) Without primary Ab. (C) FACS propionate anion. When propionate was added in the absence of analysis of nonpermeabilized (Left) and permeabilized (Right) FRTL-5 cells with external Naϩ in the medium (choline was used to replace Naϩ), no anti-SMCT Ab. (D) Membrane fractions (20 ␮g) from FRTL-5 cells grown in the inward current was evoked, indicating that SMCT-mediated trans- presence or absence of TSH were electrophoresed and immunoblotted with port is completely Naϩ-dependent (Fig. 3A Center); in control either anti-rat NIS (Top) or anti-mouse SMCT Abs (Middle). (Bottom) Immunoblot water-injected oocytes, propionate at concentrations up to 7.5 mM analysis of biotinylated cell surface polypeptides with anti-mouse SMCT Ab. (E) [14C]Nicotinate steady-state uptake in FRTL-5 cells in the presence or absence of did not induce an inward current (Fig. 3A Left). Therefore, the TSH and in the presence of Naϩ (shaded bars) or choline (open bars). (F) Kinetic observed currents were generated by SMCT activity. We then

analysis of [14C]nicotinate uptake in FRTL-5 cells in the presence (continuous line) investigated the transport of several monocarboxylates (1 mM) BIOCHEMISTRY or absence (broken line) of Naϩ. besides propionate: nicotinate, L- and D-lactate, pyruvate, butyrate,

Paroder et al. PNAS ͉ May 9, 2006 ͉ vol. 103 ͉ no. 19 ͉ 7271 Downloaded by guest on September 28, 2021 Fig. 4. Expression and subcellular localization of SMCT in colon, kidney, and thyroid. (A) Immunoblot analysis of peptide N-glycosidase-F-treated human colon tissue lysates. (B–D) Immunoblot analysis (Upper) of membrane fractions of colon (B), kidney (C), and thyroid (D) rat tissues with the corresponding immunohistochemistries (ϫ40) (Lower) using anti-mouse SMCT Ab. Each immunoblot contains tissues from two representative animals.

observed at 4 mM (Fig. 3C) and up to 7.5 mM (not shown), demonstrating that propionate does not inhibit SMCT activity at these concentrations. These results differ from those of a previous report contending that concentrations of SCFAs Ͼ1 mM inhibited the substrates’ own transport (6). We also examined SMCT activity as a function of extracellular pH (ranging from 5 to 9). At each pH, the propionate-evoked current was normalized to that elicited at pH 7.4 in the same oocyte. At pH values lower than 6.5, transport activity decreased with decreasing pH; however, peak SMCT activity was virtually identical in the pH range 6.5–9.0 (Fig. 3D), unlike the reported narrow pH dependence of mouse SMCT (9). The nonsteroidal antiinflamma- tory drug ibuprofen inhibited the propionate-evoked current with a Ki value of 73 Ϯ 9 ␮M (Fig. 3 E and F). Further, kinetic analysis of human SMCT (hSMCT)-mediated nicotinate transport revealed a Km value of 390 Ϯ 36 ␮M (Fig. 3G), in agreement with Gopal et al. (8) (Km ϭ 296 Ϯ 88 ␮M) for murine SMCT, whereas in rat FRTL-5 cells the Km for nicotinate was Ϸ6-fold higher (Fig. 2F), probably because of species differences. Interestingly, in FRTL-5 ␮ ϩ Fig. 3. Functional properties of SMCT in X. laevis oocytes. The membrane cells 500 M ibuprofen completely inhibited the Na -dependent 14 potential was clamped at Ϫ50 mV. (A) Propionate (1 mM) did not evoke a component of [ C]nicotinate uptake as well (not shown). current in control oocytes (Left), whereas in SMCT-expressing oocytes, 1 mM propionate evoked an inward current that was 100% Naϩ-dependent (Center SMCT Is Localized on the Apical Surface of Thyroid, Colon, and Kidney and Right). (B) Currents evoked by various substrates (1 mM) were normalized Epithelial Cells. Because SMCT exon 1 was reported to be hyper- to the current elicited by 1 mM propionate in the same oocyte (n ϭ 3). (C) methylated in colon cancer (11), and SMCT transports SCFAs, ϭ Kinetic analysis of propionate transport (n 3). (D) The pH dependence of which are the major energy source of colonocytes (21), we inves- propionate-evoked inward currents was examined in the range of 5–9. At each tigated the cellular and subcellular localization of SMCT in human pH, the current evoked by 1 mM propionate was normalized with respect to and rat colon tissues. When extracts from human colon samples that elicited at pH 7.4. (E) Application of ibuprofen alone (up to 2 mM) did not Ϸ alter the holding current. When applied in the presence of propionate, were probed, an 75-kDa polypeptide was detected (Fig. 4A, left ibuprofen inhibited the 1 mM propionate-evoked current in a dose- lane), whose electrophoretic migration was slower than that pre- dependent manner. (F) Ibuprofen inhibited the propionate-evoked current dicted by the hSMCT molecular mass (Ϸ67 kDa). During treatment with a Ki of 73 Ϯ 9 ␮M(n ϭ 3). (G) Kinetic analysis of nicotinate transport. with peptide N-glycosidase F, the polypeptide migrated at an apparent molecular mass of Ϸ65 kDa (Fig. 4A, right lane), indicating that SMCT is glycosylated in human colon. Two bands were pentanoate, acetate, 2-oxobutyrate, 2-hydroxybutyrate, 3-hydroxy- detected by immunoblot analysis of rat colon tissue: a broad butyrate, 4-hydroxybutyrate, and acetoacetate, all of which evoked Ϸ85-kDa one and a narrow Ϸ54-kDa one, corresponding to the currents (Fig. 3B). We normalized these currents to the current maturely and immaturely glycosylated polypeptides, respectively elicited by propionate (100%) in the same oocyte. Significantly, (Fig. 4B Upper). The immaturely glycosylated precursor becomes biotin and thyroxine evoked no currents, demonstrating that these detectable when fractions are enriched for integral membrane are not SMCT substrates (not shown). In addition, consistent with proteins by alkaline extraction (22). Immunohistochemistry analysis Ϫ Coady et al. (6), I did not elicit currents either, disproving a revealed that SMCT expression was restricted to the apical surface Ϫ previous report contending that SMCT transported I (5). Biotin of the colonocytes and was absent from the surrounding goblet cells is the substrate of the multivitamin transporter SMVT, which is and underlying tissue layers (Fig. 4B Lower). This finding is 58% similar to SMCT. In oocytes, the kinetic analysis of propionate consistent with the notion that SMCT mediates the translocation of transport at a membrane potential of Ϫ50 mV revealed a Km value SCFAs from the colonic lumen to the epithelial cells. SMCT of 162 Ϯ 26 ␮M (Fig. 3C), in agreement with reported data (6). migrated as an Ϸ75–80-kDa band in immunoblot analyses of rat Given the high SCFA concentrations in the colonic lumen, we kidney and thyroid (Fig. 4 C and D). SMCT was also expressed reexamined the effect of high concentrations of SCFAs on SMCT apically in the brush border of renal proximal tubules (Fig. 4C activity. SMCT-mediated propionate transport continued to be Lower) and to a lesser extent, but still apically, in the distal parts of

7272 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602365103 Paroder et al. Downloaded by guest on September 28, 2021 and D5). The apical distribution of SMCT was confirmed by colocalization with gp135 (Fig. 5 D3 and D6).

Higher Expression of SMCT in Duke C Human Colorectal Cancer Correlates with Longer Disease-Free Survival. Because no high- affinity anti-SMCT Abs were available, other investigators had examined SMCT expression only at the transcriptional level (8, 9, 11). Using our Abs, we analyzed SMCT protein expression in Duke C (locally advanced lymph node-positive) human colon cancer samples (tumoral and paired peritumoral normal mucosa) by immunoblotting. Remarkably, SMCT was absent or markedly down-regulated in tumor tissue (Fig. 6 A and B, asterisks) compared with adjacent normal mucosa in 14 of 15 pairs examined. Equal loading for each tissue pair was determined with monoclonal anti-␤-tubulin (Fig. 6A)oranti-␤-actin Abs (Fig. 6B). These samples, obtained from patients treated in New York City, repre- sented a heterogeneous histopathological population (ranging from poorly to moderately differentiated adenocarcinomas). There was no correlation between the differentiation status of the tumor and the down-regulation of SMCT protein levels. To extend our findings, we assessed SMCT expression by immunohistochemistry in tumor samples from colorectal cancer patients. To control for possible confounding factors affecting patient survival, all 113 analyzed tumors were stage Duke C, and cases with Fig. 5. SMCT is apically expressed in polarized MDCK cells. (A) Immunoblot microsatellite instability were excluded. SMCT expression was analysis of SMCT expression in MDCK cells stably transfected with hSMCT and evaluated in a tissue microarray-based immunohistochemical assay in nontransfected (NT) MDCK cells. (B) Time course of [14C]nicotinate uptake in MDCK cells stably transfected with hSMCT (open squares) and nontrans- in triplicate and scored blindly on a scale from 0 (no staining) to 4 fected MDCK cells (ﬁlled squares). (C) Steady-state IϪ uptake assays (80 ␮MIϪ) (strong staining) (Fig. 6C). The average score of triplicates was used in MDCK cells stably transfected with human NIS or with hSMCT; shaded bars, in the following analyses. Tumor samples with staining intensities of assays done in the presence of Naϩ; black bars, assays done in the presence of 1.7 or less (75 of 113) were considered low-SMCT-expressing, Naϩ and perchlorate, a competitive inhibitor of NIS. (D) Immunoﬂuorescence whereas those with staining intensities higher than 1.7 (38 of 113) analysis of SMCT targeting in MDCK cells stably transfected with hSMCT. (1–6) were considered high-SMCT-expressing; 66.4% of the tumors Colocalization with an apical marker, gp135. (7–12) Absence of colocalization ϩ ϩ expressed low levels of SMCT. There was a significant correlation with a basolateral marker (Na ,K -ATPase). between staining levels and patient age (Spearman r ϭ 0.27, P ϭ 0.0018), i.e., patients with low-SMCT-expressing tumors tended to the nephron. In the thyroid, as reported in ref. 5, SMCT was be younger. There were no correlations between SMCT expression expressed apically as well (Fig. 4D Lower). and common genetic abnormalities linked to colorectal cancer (p53 and kras mutations or loss of heterozygosity in 18q) or other SMCT Is Properly Targeted to the Apical Surface in Polarized Madin– clinicopathological features [gender, grade, or tumor location (right Darby Canine Kidney (MDCK) Cells. MDCK cells have been exten- or left colon or rectum)]. sively used to investigate polarized membrane protein targeting To avoid selecting a threshold arbitrarily, we arranged the 113 (23). We generated MDCK cells stably expressing functional tumors in order of increasing SMCT expression and divided them hSMCT. SMCT expression was analyzed by FACS, followed by into two groups: low- and high-SMCT expression. Disease-free survival in both groups and log-rank P values were calculated for immunoblot analysis of membrane fractions from FACS-positive every possible grouping resulting from increasing the number of clones. An MDCK clone moderately expressing hSMCT was se- patients allocated to the low-SMCT-expressing group from 1 to 113, lected for further analysis; immunoreactivity against an Ϸ84-kDa starting with the patient with the lowest SMCT tumor level. Patients polypeptide from this clone is shown in Fig. 5A. with low-SMCT-expressing tumors showed shorter disease-free and We assessed the functionality of SMCT in this system by con- 14 overall survival in 108 of 113 groupings. These differences were ducting steady-state and kinetic analyses of [ C]nicotinate uptake. statistically significant (log-rank test P Ͻ 0.05) in 11 of these The MDCK hSMCT clone transported Ϸ9 times more [14C]nico- Ϯ groupings, suggesting that low expression of SMCT is a marker of tinate than nontransfected MDCK cells (Fig. 5B) with a Km of 284 poor prognosis in Duke C colorectal cancer (Fig. 6D). On a ␮ Ϫ 83 M. Because SMCT was initially reported to be an I trans- multivariate analysis, high SMCT protein levels were a strong porter, we examined whether SMCT-expressing MDCK cells trans- predictor of longer disease-free survival (P ϭ 0.027). Further, to Ϫ Ϫ ␮ locate I . Steady-state I uptake experiments at saturating (20 M, make sure that longer disease-free survival was not the result of ␮ Ϫ not shown) and supersaturating (80 M) I concentrations were postsurgical chemotherapy with 5-fluorouracil, we conducted sur- conducted in MDCK cells stably expressing NIS (24) or SMCT (Fig. vival analyses with 81 patients from this group who were only 5C). As expected, NIS-expressing MDCK cells displayed perchlor- treated surgically. Higher SMCT protein levels remained a strong Ϫ ate-sensitive I uptake. In contrast, SMCT-expressing MDCK predictor for disease-free survival in these patients (Fig. 6E). This Ϫ exhibited no I transport, further confirming previous reports and our finding is significant, given that it is currently impossible to predict Ϫ own observations that SMCT does not mediate I transport (Fig. 5C). accurately the probability of recurrence in Duke C colorectal cancer We analyzed SMCT polarized targeting by immunofluorescence after surgery with or without chemotherapy (26–29). Considering directly on filter membranes. Monoclonal antibodies against gp135 that SMCT expression would be easy to ascertain in colon cancer and the Naϩ,Kϩ-ATPase were used to monitor apical and baso- samples obtained in routine colonoscopic screenings, these findings lateral markers, respectively (25). The en face (xy) view revealed suggest that SMCT expression may be a significant marker for lower

clear apical localization of SMCT and gp135 (Fig. 5 D1 and D2). disease recurrence in Duke C colorectal cancer. BIOCHEMISTRY Cross sections in the xz direction confirmed this finding (Fig. 5 D4 In conclusion, we have extensively characterized the SMCT

Paroder et al. PNAS ͉ May 9, 2006 ͉ vol. 103 ͉ no. 19 ͉ 7273 Downloaded by guest on September 28, 2021 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 and peritumoral colon tissue extracts (70 ␮g each) with anti-hSMCT Ab. All samples displayed clear expression of SMCT in peritumoral tissue. Tumoral tissues in 14 of 15 cases (as indicated by asterisks) exhibited complete absence or marked down-regulation of SMCT. ␤-Tubulin (A)or␤-actin (B) served as an internal loading control. (C)(Upper) Representative examples of colorectal tumors showing increasing levels (from 1 to 4) of SMCT immunostaining. (Lower) High-power magniﬁcation of the areas outlined in Upper.(D) Overall survival (Upper) and disease-free survival (Lower) based on SMCT protein levels in Duke C colorectal cancer patients (Kaplan–Meier plots). (E) Overall survival (Upper) and disease-free survival (Lower) based on SMCT protein levels in Duke C colorectal cancer patients (Kaplan–Meier plots) who were not treated with 5-ﬂuorouracil postoperatively.

protein at multiple levels and shown that it may play a valuable role, Colon Tumor Extract and Membrane Fraction Preparation. Human not previously examined, in colon cancer management. colorectal tumors with paired adjacent normal tissue were collected after surgical resection from patients who consented according to Methods institutional guidelines; the samples were immediately snap-frozen Cloning. The hSMCT cDNA was cloned from the Human Thyroid in liquid nitrogen. Proteins were extracted by brief homogenization Gland Marathon-Ready cDNA (BD Biosciences Clontech). The on ice in lysis buffer (50 mM Tris⅐HCl, pH 7.5͞150 mM NaCl͞1% primers used to amplify SMCT cDNA were 5Ј-GTTCTCATCTG- Nonidet P-40͞0.5% sodium deoxycholate͞1 mM EDTA͞protease CTCAGGTGTCC and 5Ј-GCGTGTATTAGCCTTTCAGCAT. inhibitor mixture) followed by incubation for 30 min at 4°C with The PCR parameters were 35 cycles of 95°C for 30 s, 53°C for 30 s, gentle agitation. After incubation, samples were sonicated for 10 s and 68°C for 2.5 min, followed by 68°C for 10 min, and 4°C to cool. and centrifuged at 14,000 ϫ g for 10 min. Membrane fractions were The amplified cDNA was digested with BamHI and EcoRI and prepared as described in ref. 22. ligated into the pcDNA3 vector (Invitrogen). Sequencing primers Ј were T7 promoter primer 5 -TAATACGACTCACTATAGGG Deglycosylation Assays. Membrane protein fractions or tissue ex- (Invitrogen), 5Ј-CGCAGAATGACCGCAGTG, 5Ј-CCAGCATC- Ј tracts were deglycosylated with peptide N-glycosidase F TACGGTCTCAAC, and 5 -TTTGGGCATTTTGGTTCCC. (PROzyme, San Leandro, CA) and subjected to immunoblot analysis as described in ref. 31. Generation of Anti-SMCT Abs. High-affinity site-directed polyclonal Abs against the C-terminal sequences of the rodent (amino acids Immunoblot Analysis and Cell-Surface Biotinylation. These proce- 596–611, VELNFTDHSGKINGTRL) and human (amino acids dures were performed as described in ref. 22 with 13 nM affinity- 591–610, AFNHIELNSDQSGKSNGTRL) SMCT proteins were purified anti-mouse SMCT Ab, 7 nM affinity-purified anti-hSMCT generated. Purification was performed as described in ref. 30. Ab, and 4 nM affinity-purified anti-rat NIS Ab for1hand monoclonal anti-␤-actin 1:10,000 (Sigma) or anti-␤-tubulin 1:3,000 Cell Culture. FRTL-5 rat thyroid cells were cultured as described in ref. 16. MDCK II cells were maintained in DMEM (Invitrogen) Ab (Sigma). supplemented with 10% FBS (Gemini BioProducts, West Sacra- mento, CA), 1% glutamine, and 1% penicillin͞streptomycin (In- Immunofluorescence and Immunohistochemical Analyses. FRTL-5 cells were seeded onto polylysine-coated coverslips. Cells were vitrogen) at 37°C in a 95% air͞5% CO2 atmosphere. For polarized culture, stably transfected MDCK cells or nontransfected MDCK incubated with 70 nM primary Ab against mouse SMCT in PBS ͞ cells were plated on 12-mm (1-cm2 area) Transwell polyester filter containing 1 mM CaCl2 and 0.1 mM MgCl2 (PBS CM) with 0.2% units (0.4-␮m pore size; Costar) at a density of 150,000 cells per BSA and 0.1% Triton X-100 for 1 h and processed as described in ␮ filter unit and cultured for 5 days to allow development of polarity. ref. 14. For immunohistochemistry, 5- m colon tissue sections were deparaffinated and rehydrated and processed as described, Generation of Stable Clones. MDCK II cells in 12-well plates were with affinity-purified anti-mouse-SMCT Ab at 7 nM final concen- transfected with 2 ␮g of plasmid containing hSMCT cDNA with tration (32). Lipofectamine 2000 (Invitrogen). Stable clones were selected and maintained in a selection medium containing 1 g͞liter G418, as Immunofluorescence Microscopy of Polarized MDCK Cells. MDCK cell described in ref. 24. monolayers were fixed in 2% freshly prepared paraformaldehyde in PBS for 20 min at room temperature and quenched with 50 mM Flow Cytometry. FRTL-5 cells were incubated for1hwith100␮lof NH4Cl in PBS͞CM. For permeabilization, methanol at Ϫ20°C or PBS͞0.1% BSA͞0.2% saponin containing 9 nM anti-mouse SMCT 0.1% saponin was used. Cells were blocked in PBS͞CM containing Ab and processed as described in ref. 14. 10% goat serum (Invitrogen). The primary Ab used was a mAb

7274 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602365103 Paroder et al. Downloaded by guest on September 28, 2021 against the ectodomain of gp135 (1:50 dilution; kindly provided by morpholino)ethanesulfonic acid] or TAPS [N-tris(hydroxymethyl) E. Rodriguez-Boulan). Secondary Abs used were Alexa 488-tagged methyl-3-aminopropanesulfonic acid] replaced Hepes as appropri- anti-rabbit IgG and Alexa 568-tagged anti-mouse IgG. Images were ate. All experiments were carried out at 21 Ϯ 1°C. obtained on a Radiance 2000 laser scanning confocal microscope (Bio-Rad) with a ϫ60 oil objective. Serial (0.3-␮m) xz sections (top Tissue Microarray and Immunohistochemistry. An independent set of to bottom) were collected and processed with IMAGE J software formalin-fixed, paraffin-embedded samples from a total of 113 (National Institutes of Health) and PHOTOSHOP 5.0 (Adobe, San Duke C colorectal tumors was used for immunohistochemical Jose, CA). Images presented here show individual confocal xy assessment of SMCT expression by tissue microarray. Samples were section and the entire xz section. collected at collaborating medical institutions in southern Finland. Informed consent was obtained from each patient according to the Transport Assays. [14C]Nicotinate was obtained from Moravek Human Investigations and Ethical Committee-approved research Biochemicals (Brea, CA). Cells grown in 12-well plates were proposal. All 113 tumor samples were from patients with Duke C incubated with buffered Hanks’ balanced salt solution containing colorectal cancer: 81 of them had surgery as the only form of 50 ␮M[14C]nicotinate and processed as described in ref. 1. Results treatment, and 32 also received 5-fluorouracil-based adjuvant che- ͞ are the average of at least three separate experiments performed motherapy. After histological examination of hematoxylin eosin- in triplicate. For kinetic analysis, cells were incubated for 2 min stained tumor sections, areas containing a high proportion of tumor with 4.5–600 ␮M[14C]nicotinate. Initial-rate data were analyzed cells were selected. Sections were processed as described in ref. 26 by a nonlinear regression by using the following equation for and incubated with anti-hSMCT polyclonal Ab at a 1:100 dilution [14C]nicotinate-dependent [14C]nicotinate uptake: for 1 h. SMCT expression was evaluated in the 113 tumor samples blinded from clinical data. A semiquantitative scale from 0 to 4 was ϭ ⅐͓͓14 ͔͑͞ ϩ ͓͓14 ͔͒ used to measure the intensity of the staining. To investigate how v Vmax C]nicotinate Km C]nicotinate survival differences in the high- and low-SMCT-expression groups ϩ 0.07⅐͓͓14C]nicotinate͔ ϩ 0.41. [1] change as a function of the staining cutoff level selected, we systematically calculated the mean survival in both groups as well The terms 0.07⅐[[14C]nicotinate] ϩ 0.41 correspond to background as the hazard ratio and the log-rank P value for every possible adjusted by least squares of the data obtained with FRTL-5 cells in grouping resulting from increasing the number of patients allocated the presence of choline. Data were fitted by nonlinear least squares to the low-SMCT group from 1 to 113, starting with the patient with with the Marquard–Levenberg algorithm (33). Data were analyzed the lowest SMCT tumor level. The cutoff value of 1.7 for staining with GNUPLOT (www.gnuplot.info). Km and Vmax values are the intensity was chosen because it showed maximal differences in average of three experiments and are expressed as the mean Ϯ disease-free and overall survival between high- and low-SMCT SEM. IϪ transport assays in MDCK cells stably transfected with groups after iteratively testing every possible grouping. either hSMCT or hNIS were performed exactly as described in ref. 1. Statistical Analysis. Survival curves were constructed by using the Electrophysiological Analysis. pcDNA3 containing the hSMCT method of Kaplan and Meier (34), and survival differences were cDNA was linearized with XhoI and cRNA and transcribed in vitro assessed with the log-rank test (26). The Cox proportional-hazards by T7 polymerase. X. laevis oocytes were injected with 50 ng of model was used to assess the simultaneous contribution of the cRNA and maintained in Barth’s medium [88 mM NaCl͞1mM following covariates: sex, age, grade, tumor location (right͞left Ͻ KCl͞0.33 mM Ca(NO3)2͞0.41 mM CaCl2͞0.82 mM MgSO4͞2.4 colon), and SMCT protein tumor levels. A P value of 0.05 was mM NaHCO3͞10 mM Hepes, pH 7.4͞50 ␮g/ml gentamicin͞100 considered to indicate statistical significance. ␮g/ml streptomycin͞100 units/ml penicillin] at 18°C for 2–10 days before use in experiments. The two-microelectrode voltage clamp We thank Dr. E. Rodriguez-Boulan for providing gp135 Ab. We technique was used for the recording of whole-cell transporter- especially thank the members of the Carrasco laboratory for helpful discussions, suggestions, experimental advice, and critical reading of the mediated currents as described in ref. 19. SCFAs and͞or inhibitors ϩ manuscript. This work was supported by National Institutes of Health were added to the NaCl buffer as indicated. In Na -free solutions, Grants DK-41544 (to N.C.) and S06 GM53933 (to S.E.), National Cancer NaCl was isosmotically replaced with choline⅐Cl. In experiments Institute͞National Institutes of Health Grant CA-098390 (to N.C.), and examining the effect of pH on transport activity, Mes [2-(N- Medical Scientist Training Program Grant T32 JM007288.

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