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Hyaluronan, CD44, and emmprin regulate lactate efflux and membrane localization of monocarboxylate transporters in human breast carcinoma cells - PubMed

  • ️Thu Jan 01 2009

Hyaluronan, CD44, and emmprin regulate lactate efflux and membrane localization of monocarboxylate transporters in human breast carcinoma cells

Mark G Slomiany et al. Cancer Res. 2009.

Abstract

Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin) is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

B.P. Toole is an inventor on a patent related to this article. The other authors disclosed no potential conflicts of interest.

Figures

Figure 1
Figure 1

Inhibition of lactate efflux by emmprin siRNA and hyaluronan oligomers. A, emmprin is necessary for lactate efflux. MCF7 human breast carcinoma cells were grown to 70%confluence , transfected for 24 h with control or emmprin siRNA, and subject to ECAR analysis as described in Materials and Methods. Equal cell density was adjusted according to µg protein per well. The inset shows the decreased level of emmprin associated with cells treated with emmprin siRNA (right) compared with control siRNA (left); emmprin was visualized by immunolabeling. B–D, hyaluronan oligomers decrease lactate efflux. MCF-7 (B), MDA-MB231 (C), and MDA-MB436 (D) human breast carcinoma cells were grown to 70%confluence. After baseline acquisition, cells were treated with 100 µg/mL hyaluronan oligomers (o-HA) and subject to ECAR analysis, as described in Materials and Methods. Error bars, SDs. Significant differences were observed (*, P < 0.05, compared with control). A–D, representative of three or more independent experiments.

Figure 2
Figure 2

CD44 forms complexes with MCTs and emmprin. A, MCF-7 and MDA-MB231 cells express CD44 and MCTs. Cells were grown to 70%conf luence (48 h). After treatment with and without 100 µg/mL hyaluronan oligomers for 1 h, whole-cell extracts were prepared, as described in Materials and Methods, and 50 µg lysates were resolved on a 4%to 20% SDS-gel and processed for Western blot analysis, as described in Materials and Methods. Left, MCF-7 and MB231 cells express standard CD44 (s, molecular weight of ~85 kDa) and CD44 splice variants (v, molecular weight of ~130 kDa). Right, MCF-7 cells express predominantly MCT1; MB231 cells express predominantly MCT4. Multiple experiments showed that treatment of the cells with hyaluronan oligomers (o-HA) had no significant effect on MCT1 or MCT4 expression. B, CD44 forms complexes with MCT1, MCT4, and β-actin. Whole-cell extracts were prepared identically to the blots in A from MB231 cells that had been pretreated with and without 100 µg/mL hyaluronan oligomers for 1 h. The extracts were immunoprecipitated (IP) with antibodies to MCT1 (left), MCT4 (middle), or CD44 (“pan-CD44”; right), then processed for Western blotting as indicated. Multiple experiments showed that mainly variant CD44 coimmunoprecipitated with the MCTs and that treatment with hyaluronan oligomers reproducibly reduced the amount of this CD44 in the MCT immunoprecipitates (left and middle). Note that standard CD44 was immunoprecipitated preferentially with the “pan-CD44” antibody (right), although more variant CD44 than standard is present in these extracts (A, left); also note that cross-reaction occurred between MCT secondary antibodies and this “pan-CD44” antibody (lane Ab). The hyaluronan oligomers decreased interaction of actin with the MCTs (left and middle) but not with CD44 (right). C, CD44 interacts with emmprin. MB231 cells were treated, and whole-cell extracts immunoprecipitated with antibody against CD44 as in B, then processed for immunoblotting as indicated. Although less variant CD44 than standard CD44 was immunoprecipitated, the ~130-kDa variant form included the v3 exon product. Emmprin, mainly the high molecular form, was coimmunoprecipitated with CD44 (ns, nonspecific band). The immunoblot in the inset shows high and low molecular forms of emmprin in a whole cell extract (WCE) from nontreated cells. D, emmprin forms complexes with MCTs and CD44. Whole-cell extracts from MB231 cells, prepared as in A, were immunoprecipitated with an antibody to emmprin. Mainly variant CD44 was coimmunoprecipitated with emmprin. Hyaluronan oligomer treatment of the cells decreased emmprin-MCT interaction but did not affect emmprin-CD44 interaction significantly. A–D, representative of three or more independent experiments.

Figure 3
Figure 3

CD44 and emmprin colocalize with MCT1 and MCT4 at the plasma membrane in a hyaluronan-dependent manner. A, MCT1; B, MCT4. MB231 cells were treated with and without 100 µg/mL hyaluronan oligomers for 1 h. After fixation, permeabilization, and blocking of nonspecific binding, the cells were immunolabeled for CD44 (green), MCT1 or MCT4 (red), and emmprin (blue) and visualized by confocal microscopy at a Z plane corresponding to the approximate center of the cell. Arrows, areas shown at higher magnification in the insets. a, b, and b’, triple labeling for all three components; colocalization appears as a white signal. Triple colocalization is clearly seen in the plasma membrane of the untreated cells but not in the membrane of oligomer-treated cells. Increased intracellular staining for CD44 is seen in the oligomer-treated cells in b. b’ in A is the same as in b, but is shown at higher gain than in a and b, to illustrate internalization of MCT1, as well as CD44; increased gain is necessary for clear visualization because the MCT becomes highly dispersed in the cytoplasm after oligomer treatment. Similar oligomer-induced internalization was observed for MCT4 (not shown). Emmprin seems to remain at the cell surface after hyaluronan oligomer treatment. c and d, double staining for CD44 and MCT1 (A) or MCT4 (B), further illustrating their colocalization (yellow signal in c) and hyaluronan oligomer-induced internalization of CD44 (d). e and f, CD44 only; g and h, the MCTs only. Increased gain revealed intracellular dispersion of MCT1 (A, h’) and MCT4 (not shown). The figure is representative of three or more independent experiments.

Figure 4
Figure 4

Hyaluronan oligomers, but not hyaluronan polymer, induce internalization of CD44, CD44v3, and MCT4. A, MB231 cells were treated for 1 h with and without 100 µg/mL hyaluronan oligomer (oHA) or polymer (HA). After fixation, permeabilization, and blocking of nonspecific binding, the cells were immunolabeled for CD44 (green) and MCT4 (red) and visualized by confocal microscopy at a Z plane corresponding to the center of the cell. Treatment with hyaluronan oligomer, but not polymer, results in internalization of CD44 and MCT4 from the plasma membrane. B, MB231 cells were treated and processed as in A but were immunolabeled for CD44 (green; a, b, c) and CD44v3 (red; d, e, f). g, h, i, double labeling. Arrows, areas shown at higher magnification in j, k, and l . CD44, including CD44v3, is present at the plasma membrane in control and hyaluronan polymer-treated cells. CD44v3 seems to be internalized preferentially in response to hyaluronan oligomer treatment (h, k, and b versus e). Representative of three or more independent experiments.

Figure 5
Figure 5

Emmprin siRNA inhibits membrane localization of MCT1 and MCT4 but not CD44. A, MCT1; B, MCT4. MB231 cells were transfected with emmprin siRNA and then processed in the same way as in Fig. 3. A and B, a and b (and b’) are triple labeled for CD44 (green), MCT1 or MCT4 (red), and emmprin (blue); c and d are double labeled for CD44 and MCT. Arrows, areas shown at higher magnification in the insets. Note that the levels of emmprin are highly reduced after treatment with emmprin siRNA (b) compared with control siRNA (a). a and c show colocalization in a similar fashion to that shown in Fig. 3. e and f show CD44 only; g and h show the MCTs only. b and d show that CD44 is not significantly internalized after knockdown of emmprin, in contrast to the effect of hyaluronan oligomers shown in Fig. 3. As explained in Fig. 3, increased gain (A, b’ and h’) is necessary to show dispersion of MCT1 in the cytoplasm; a similar result was obtained for MCT4 (not shown). The increased gain used in b’ also shows that emmprin was not completely eliminated by siRNA treatment. The figure is representative of three or more independent experiments.

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