RhoA and Rac1 GTPases play major and differential roles in stromal cell-derived factor-1-induced cell adhesion and chemotaxis in multiple myeloma

Abstract

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) milieu plays a crucial role in MM pathogenesis. Stromal cell-derived factor-1 (SDF1) regulates homing of MM cells to the BM. In this study, we examined the role of RhoA and Rac1 GTPases in SDF1-induced adhesion and chemotaxis of MM. We found that both RhoA and Rac1 play key roles in SDF1-induced adhesion of MM cells to BM stromal cells, whereas RhoA was involved in chemotaxis and motility. Furthermore, both ROCK and Rac1 inhibitors reduced SDF1-induced polymerization of actin and activation of LIMK, SRC, FAK, and cofilin. Moreover, RhoA and Rac1 reduced homing of MM cells to BM niches. In conclusion, we characterized the role of RhoA and Rac1 GTPases in SDF1-induced adhesion, chemotaxis, and homing of MM cells to the BM, providing the framework for targeting RhoA and Rac1 GTPases as novel MM therapy.

Figure 1

Gene expression of the GTPases RhoA, Rac1, and CDC42 and expression of RhoA and Rac1 in MM cell lines and patient samples. (A) Expression of RhoA and Rac1 in MM cell lines (MM1S, OPM2, and RPMI8226) and 3 MM patient samples demonstrating similar expression of both GTPases in all cell lines and patient samples. (B) Gene expression of the GTPases RhoA, Rac1, and CDC42, based on NIH Gene Expression Omnibus database under the accession number GSE6691, demonstrating significant overexpression of RhoA and Rac1, but not CDC42, GTPases in MM samples compared with normal subjects.

Figure 2

Effect of ROCK and Rac1 inhibitors on the SDF1-induced adhesion. Shown is the effect of ROCK and Rac1 inhibitors on the SDF1-induced adhesion of (A) MM cell lines and (B) plasma cells from MM patient samples to fibronectin and the adhesion of MM cell lines to BMSCs (C). Both the ROCK and Rac1 inhibitors decreased the adhesion of MM cells to fibronectin and BMSCs, and no additive effect was shown for their combination. Panels A and B: Groups compared with the corresponding SDF1-treated group. Panel C: Groups compared with the control, *P < .05; #P < .01.

Figure 3

The effect of ROCK and Rac1 inhibitors on expression and function of adhesion molecules. (A) Effect of SDF1, ROCK, and Rac1 inhibitors on the expression of CXCR4, LFA1, and VLA4 detected by flow cytometry. It was shown that none of the inhibitors or their combination affected the internalization of CXCR4 induced by SDF1, and neither SDF1 nor any of the inhibitors affected the expression of LFA1 or VLA4 on MM1S cells. (B) The expression of ICAM and VCAM on BMSCs indicates that both molecules were expressed on BMSCs. (C) The adhesion of MM1S cells to plates coated with recombinant ICAM and VCAM shows that MM1S cells adhered to VCAM more than ICAM and that the inhibitors reduced only the adhesion of MM1S to VCAM. (D) The effect of ROCK and Rac1 inhibitors on the adhesion of MM1S cell to recombinant fibronectin in the presence or absence of VLA4 blocking antibody shows that none of the inhibitors had an additive effect beyond that of VLA4 blocking antibody. Panel C: groups compared with SDF1-treated group. *P < .05; #P < .01.

Figure 4

The effect of Rock and Rac1 inhibitors on chemotaxis and actin polymerization. (A) Expression of CXCR4 on MM cell lines (MM1S, OPM2, and RPMI8226) detected by flow cytometry showing that MM1S, OPM2, and RPMI8226 had low, intermediate, and high expression of CXCR4, respectively. (B) The effect of ROCK and Rac1 inhibitors on SDF1-induced chemotaxis of MM cell lines that was shown to correlate with the expression of CXCR4; ROCK, but not Rac1, inhibitor prevents the SDF1-induced migration of MM cells. (C) The effect of ROCK and Rac1 inhibitors on the SDF1-induced chemotaxis in MM cells from patient samples showing similar result to those obtained with the cell lines. (D) The effect of ROCK and Rac1 inhibitors on SDF1-induced actin polymerization and polarization detected by confocal microscopy and (E) flow cytometry. It was shown that both inhibitors reduced actin polymerization, but the ROCK inhibitor showed a more profound effect, and only the ROCK inhibitor prevented actin polarization. Panels A-C: Groups compared with SDF1-treated group. *P < .05; #P < .01.

Figure 5

The effect of down-regulation of RhoA and Rac1 on MM1s adhesion and chemotaxis. The effect of transient transfection of MM1s cells with siRNA on the expression of Rac1 (A) and RhoA (B) detected by immunoblotting and adhesion to fibronectin (C) and chemotaxis induced by SDF1. *P < .05; #P < .01.

Figure 6

Characterization of the role of RhoA and Rac1 in cytoskeletal signaling. (A) The effect of SDF1 on the activation of key proteins in cytoskeletal signaling including RhoA, Rac1, LIMK, FAK, SRC, cofilin, MLC and AKT detected by immunoblotting. SDF1 was shown to induce fast activation of those proteins 1 minute after treatment. (B) The effect of ROCK and Rac1 inhibitors on the activation of cytoskeletal proteins, detected by immunoblotting. (C) The effect of inhibitors of coupling of G-protein, PI3K, and AKT on the SDF1-induced activation of RhoA and Rac1 GTPases, detected by immunoblotting. (D) The effect of inhibitors of coupling of G-protein, PI3K, and AKT on the SDF1-induced chemotaxis of MM1S cells. The effect of ROCK and Rac1 inhibitors on MM cell (E) chemotaxis and (F) adhesion to fibronectin induced by SDF1 in the presence or absence of PI3K inhibitor. *P < .05; #P < .01.

Figure 7

The effect of ROCK and Rac1 inhibitors on adhesion to endothelial cells in vitro and extravasation and homing in vivo. (A) The effect of ROCK and Rac1 inhibitors on SDF1-induced MM1S cell adhesion to plates coated with HUVEC cells in vitro showing that both inhibitors reduced the adhesion with no additive effect for the combination. (B) The effect of ROCK and Rac1 inhibitors on MM1S cell homing after tail vein injection in mice detected by detection of circulating MM1S cells with time showing that both inhibitors reduced the adhesion with no additive effect for the combination. (C) The effect of ROCK and Rac1 inhibitors on MM1S cell homing to the BM niches in mouse skull after tail vein injection demonstrating that both inhibitors prevented homing of MM1S cell to BM, whereas ROCK inhibitor had a more profound effect. (D) Quantification of the number of MM1S cell homed to BM in panel C. (E) The suggested mechanism for SDF1-induced MM cell adhesion and chemotaxis involving RhoA and Rac1. Panel A: Groups compared with the SDF1-treated group. Panel D: Groups compared with the control, *P < .05; #P < .01.