Wnt5a-Ror2 signaling in mesenchymal stem cells promotes proliferation of gastric cancer cells by activating CXCL16-CXCR6 axis

Abstract

Wnt5a-Ror2 signaling has been shown to play important roles in promoting aggressiveness of various cancer cells in a cell-autonomous manner. However, little is known about its function in cancer-associated stromal cells, including mesenchymal stem cells (MSCs). Thus, we examined the role of Wnt5a-Ror2 signaling in bone marrow-derived MSCs in regulating proliferation of undifferentiated gastric cancer cells. Coculture of a gastric cancer cell line, MKN45, with MSCs either directly or indirectly promotes proliferation of MKN45 cells, and suppressed expression of Ror2 in MSCs prior to coculture inhibits enhanced proliferation of MKN45 cells. In addition, conditioned media from MSCs, treated with control siRNA, but not siRNAs against Ror2, can enhance proliferation of MKN45 cells. Interestingly, it was found that expression of CXCL16 in MSCs is augmented by Wnt5a-Ror2 signaling, and that recombinant chemokine (C-X-C motif) ligand (CXCL)16 protein can enhance proliferation of MKN45 cells in the absence of MSCs. In fact, suppressed expression of CXCL16 in MSCs or an addition of a neutralizing antibody against CXCL16 fails to promote proliferation of MKN45 cells in either direct or indirect coculture with MSCs. Importantly, we show that MKN45 cells express chemokine (C-X-C motif) receptor (CXCR)6, a receptor for CXCL16, and that suppressed expression of CXCR6 in MKN45 cells results in a failure of its enhanced proliferation in either direct or indirect coculture with MSCs. These findings indicate that Wnt5a-Ror2 signaling enhances expression of CXCL16 in MSCs and, as a result, enhanced secretion of CXCL16 from MSCs might act on CXCR6 expressed on MKN45, leading to the promotion of its proliferation.

Keywords: CXCL16; CXCR6; Wnt5a-Ror2 signaling; gastric cancer cells; mesenchymal stem cells (MSCs).

Figure 1

Coculture with mesenchymal stem cells (MSCs) promotes proliferation of MKN45‐Luc cells. (a) MKN45‐Luc cells were either cultured singly (MSCs−) or directly with MSCs (MSCs+). (b,c) MKN45‐Luc cells were either cultured singly (MSCs−) or indirectly with MSCs (MSCs+) in Transwell chambers in which MKN45‐Luc and MSCs were seeded in the lower and upper chambers, respectively (b) or in the upper and lower chambers, respectively (c). Luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD (n = 3). *P < 0.005; **P < 0.001, t‐test.

Figure 2

Expression of Ror2 in mesenchymal stem cells (MSCs) is required for the ability of MSCs to promote proliferation of MKN45‐Luc cells in coculture. (a) Suppressed expression of Ror2 in MSCs treated with siRNAs for Ror2. Cells were transfected with either control (ctrl) siRNA or three different siRNAs against Ror2 (#1, #2, #3) and cultured for 5 or 9 days. Expression levels of Ror2 mRNA were measured by quantitative RT‐PCR analyses. (b,c) MKN45‐Luc cells were cultured singly (monoculture) or cocultured with siRNA‐transfected MSCs either directly (b) or indirectly (c). After 9 days in culture, luciferase activities were measured. Data are expressed as mean ± SD (n = 3). *P < 0.005; **P < 0.001, t‐test.

Figure 3

Conditioned media from mesenchymal stem cells (MSCs) promote proliferation of MKN45‐Luc cells in a manner dependent on Ror2 expression in MSCs. (a) Conditioned media from MSCs promotes proliferation of MKN45‐Luc cells. MKN45‐Luc cells were cultured in the presence or absence of MSC‐conditioned media. Luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD (n = 3). *P < 0.05; **P < 0.001, t‐test. (b) MKN45‐Luc cells were cultured in the presence of conditioned media from MSCs pretreated with either ctrl siRNA or three different siRNAs against Ror2 (#1, #2, #3). After 9 days in culture, luciferase activities were measured. Data are expressed as mean ± SD (n = 3). *P < 0.05; **P < 0.001, t‐test. (c) MSCs were transfected with either ctrl or Ror2 (#2) siRNA. After 6 days in culture, conditioned media were collected and subjected to chemokine array analysis. Boxed spots (B4) indicate protein levels of CXCL16, which decreased significantly and reproducibly following suppressed expression of Ror2. Other spots with different intensities between si‐Ctrl and si‐Ror2 groups include C4 (interleukin [IL]‐8), C9 (CCL2), and E6 (CXCL12). A1, A10, and G1 represent reference spots. (d) Expression of CXCL16 is downregulated by suppressed expression of Ror2 in MSCs. Mesenchymal stem cells were transfected with either ctrl or Ror2 siRNAs. After 6 days in culture, mRNA levels of CXCL16 (left panel) and Ror2 (right panel) were measured by quantitative RT‐PCR analyses. (e) MSCs were transfected with either ctrl or Ror2 (#1) siRNA. After 6 days in culture, conditioned media were collected to measure relative amounts of CXCL16 protein by ELISA. Data are expressed as mean ± SD (n = 3). *P < 0.05, t‐test.

Figure 4

Expression of CXCL16 in mesenchymal stem cells (MSCs) is required for the ability of MSCs to promote proliferation of MKN45‐Luc cells in coculture. (a) Recombinant CXCL16 (rCXCL16) promotes proliferation of MKN45‐Luc cells. MKN45‐Luc cells were cultured in the absence (vehicle) or presence of 1 ng/mL rCXCL16, and luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD (n = 3). **P < 0.001, t‐test. (b,c) Suppressed expression of CXCL16 in MSCs. MSCs were transfected with either ctrl or CXCL16 siRNA. After 6 days in culture, mRNA levels of CXCL16 were measured by quantitative RT‐PCR analyses (b). Conditioned media from the siRNA‐treated MSCs were collected to measure relative amounts of CXCL16 protein by ELISA (c). (d,e) MKN45‐Luc cells were cultured singly or cocultured with siRNA‐treated MSCs either directly (d) or indirectly (e). Luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD (n = 3). **P < 0.001, t‐test. (f,g) Effect of neutralizing antibody against CXCL16 on proliferation of MKN45‐Luc cells cocultured with MSCs. MKN45‐Luc cells were cocultured with MSCs either directly (f) or indirectly (g) in the presence of anti‐CXCL16 neutralizing antibody or control IgG. Luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD (n = 3). **P < 0.001, t‐test.

Figure 5

Expression of CXCR6 in MKN45‐Luc cells is required for the ability of mesenchymal stem cells (MSCs) to promote proliferation of MKN45‐Luc cells in coculture. (a,b) Suppressed expression of CXCR6 in MKN45‐Luc cells. MKN45‐Luc cells were transfected with either ctrl or CXCR6 siRNA. After 6 days in culture, mRNA levels of CXCR6 were measured by quantitative RT‐PCR analyses (a). Cell surface expression of CXCR6 protein was measured by flow cytometry (b). (c,d) MKN45‐Luc cells transfected with either ctrl or CXCR6 siRNA were cocultured with MSCs either directly (c) or indirectly (d). Untreated MKN45‐Luc cells were also cultured singly (monoculture). Luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD (n = 3). *P < 0.005; **P < 0.001 between si‐Ctrl and si‐CXCR6 groups, t‐test.