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 consid- ered 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 exper- iments, 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).

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