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

Abstract

We report an extensive characterization of the Na(+)/monocarboxylate transporter (SMCT), a plasma membrane protein that mediates active transport of monocarboxylates such as propionate and nicotinate, and we show that SMCT may play a role in colorectal cancer diagnosis. SMCT, the product of the SLC5A8 gene, is 70% similar to the Na(+)/I(-) symporter, the protein that mediates active I(-) uptake in the basolateral surface of thyrocytes and other cells. SMCT was reported in the apical surface of thyrocytes and formerly proposed also to transport I(-) and was called the apical I(-) transporter. However, it is now clear that SMCT does not transport I(-). Here we demonstrate a high-affinity Na(+)-dependent monocarboxylate transport system in thyroid cells, which is likely to be SMCT. We show that, whereas thyroidal Na(+)/I(-) symporter expression is thyroid-stimulating hormone (TSH)-dependent and basolateral, SMCT expression is TSH-independent and apical not only in the thyroid but also in kidney and colon epithelial cells and in polarized Madin-Darby canine kidney cells. We determine the kinetic parameters of SMCT activity and show its inhibition by ibuprofen (K(i) = 73 +/- 9 microM) in Xenopus laevis oocytes. SMCT was proposed to be a tumor suppressor in colon cancer. Significantly, we show that higher expression of SMCT in colon samples from 113 colorectal cancer patients correlates with longer disease-free survival, suggesting that SMCT expression may be a favorable indicator of colorectal cancer prognosis.

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 intracellularly. 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.

Fig. 2.

SMCT protein expression and function in FRTL-5 cells. (A) Immunoblot analysis of membrane fractions from FRTL-5 cells (≈60 μg of protein) incubated either with or without peptide N-glycosidase F overnight at 37°C, electrophoresed, and immunoblotted with anti-mouse SMCT Ab. (B) Indirect immunofluorescence 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) FACS analysis of nonpermeabilized (Left) and permeabilized (Right) FRTL-5 cells with anti-SMCT Ab. (D) Membrane fractions (20 μg) from FRTL-5 cells grown in the presence or absence of TSH were electrophoresed and immunoblotted with either anti-rat NIS (Top) or anti-mouse SMCT Abs (Middle). (Bottom) Immunoblot 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 TSH and in the presence of Na⁺ (shaded bars) or choline (open bars). (F) Kinetic analysis of [¹⁴C]nicotinate uptake in FRTL-5 cells in the presence (continuous line) or absence (broken line) of Na⁺.

Fig. 3.

Functional properties of SMCT in X. laevis oocytes. The membrane potential was clamped at −50 mV. (A) Propionate (1 mM) did not evoke a current in control oocytes (Left), whereas in SMCT-expressing oocytes, 1 mM propionate evoked an inward current that was 100% Na⁺-dependent (Center and Right). (B) Currents evoked by various substrates (1 mM) were normalized to 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 of propionate-evoked inward currents was examined in the range of 5–9. At each pH, the current evoked by 1 mM propionate was normalized with respect to that elicited at pH 7.4. (E) Application of ibuprofen alone (up to 2 mM) did not alter 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 current with a K_i of 73 ± 9 μM (n = 3). (G) Kinetic analysis of nicotinate transport.

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 (×40) (Lower) using anti-mouse SMCT Ab. Each immunoblot contains tissues from two representative animals.

Fig. 5.

SMCT is apically expressed in polarized MDCK cells. (A) Immunoblot analysis of SMCT expression in MDCK cells stably transfected with hSMCT and in nontransfected (NT) MDCK cells. (B) Time course of [¹⁴C]nicotinate uptake in MDCK cells stably transfected with hSMCT (open squares) and nontransfected MDCK cells (filled squares). (C) Steady-state I⁻ uptake assays (80 μM I⁻) in MDCK cells stably transfected with human NIS or with hSMCT; shaded bars, assays done in the presence of Na⁺; black bars, assays done in the presence of Na⁺ and perchlorate, a competitive inhibitor of NIS. (D) Immunofluorescence analysis of SMCT targeting in MDCK cells stably transfected with hSMCT. (1–6) Colocalization with an apical marker, gp135. (7–12) Absence of colocalization with a basolateral marker (Na⁺,K⁺-ATPase).

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 μg 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. β-Tubulin (A) or β-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.