A novel FOXM1 isoform, FOXM1D, promotes epithelial-mesenchymal transition and metastasis through ROCKs activation in colorectal cancer

Abstract

Epithelial-mesenchymal transition (EMT) is a critical event in metastasis of colorectal cancer (CRC). Rho/ROCKs signaling has a pivotal role in orchestrating actin cytoskeleton, leading to EMT and cancer invasion. However, the underlying mechanisms for ROCKs activation are not fully understood. Here, we identified FOXM1D, a novel isoform of Forkhead box M1 (FOXM1) that has a pivotal role in ROCKs activation by directly interacting with coiled-coil region of ROCK2. FOXM1D overexpression significantly polymerizes actin assembly and impairs E-cadherin expression, resulting in EMT and metastasis in xenograft mouse model and knockdown of FOXM1D has the opposite effect. Moreover, a high FOXM1D level correlates closely with clinical CRC metastasis. FOXM1D-induced ROCKs activation could be abrogated by the ROCKs inhibitors Y-27632 and fasudil. These observations indicate that the FOXM1D-ROCK2 interaction is crucial for Rho/ROCKs signaling and provide novel insight into actin cytoskeleton regulation and therapeutic potential for CRC metastasis.

Figure 1

Identification of FOXM1D. (a) Diagram of FOXM1 exons, the primers FP1, RP1 and RP2 for identifying FOXM1D, and the primers FP2 and RP3 for amplifying full-length FOXM1D. (b) Amplified fragments using the primers FP1 and RP1 in the first-round PCR. The arrows indicate the different amplified products (a–d) as templates in the second-round PCR. (c) The arrows indicate the amplified 533 and 488 bp PCR products corresponding to FOXM1D (e) and FOXM1A (f), which were amplified by the primers FP1 and RP2 in the second-round PCR. (d and e) Sequencing result (d) of band e in (c) indicates FOXM1D, which retains the VIIa exon that is absent in FOXM1B (e). (f–i) mRNA levels of FOXM1A (f), FOXM1B (g), FOXM1C (h) and FOXM1D (i) in human colorectal cancer cell lines as detected by qRT–PCR. (j) Diagram and sequences of the epitopes for polyclonal antibody preparation. CP11, joint region of exon V and VI; CP12, part of exon VIIa; DBD, DNA-binding domain; TAD, transactivation domain. (k) Protein level of FOXM1D in human colorectal cancer cell lines as detected by the customized polyclonal antibodies CP11 and CP12. The ratios of detected target proteins against actin control were analyzed and shown under each immunoblot image. The data are represented as mean±s.d., n=3. See also Supplementary Figure S1.

Figure 2

FOXM1D Promotes EMT and Metastasis in vitro and in vivo. (a and b) IF staining for E-cadherin (green) and nuclei (DAPI, blue) was performed in SW-480 cells transfected with control or FOXM1D-expressing (a) or in LoVo cells transfected with control, FOXM1D shRNA or FOXM1D shRNA with simultaneous FOXM1D-restored vector (b) (scale bar, 25 μm). FOXM1D sufficiency decreased and FOXM1D insufficiency increased E-cadherin level. The cell morphological changes between the FOXM1D-sufficient and -insufficient cells, as determined by microscopy, are shown in the right panel (scale bars, 200 μm and 40 μm, respectively). (c) A transwell assay determined that FOXM1D sufficiency promotes and FOXM1D insufficiency inhibits the invasion capability of the indicated cells. The quantified results are shown in Supplementary Figure S4f. (d–e) FOXM1D sufficiency promotes and FOXM1D insufficiency impairs cancer progression and metastasis. SW-480 cells transfected with the control vector or with FOXM1D-expressing vector (d, upper panel) or LoVo cells transfected with shRNA-control or shRNA-FOXM1D (e, upper panel) were implanted in the foot pad or orthotopic cecum of BALB/c nude mice, respectively. The metastasis incidence and signal intensities in each group are shown at the bottom. The red arrows indicate orthotopic tumor formation and metastatic liver nodules (d and e, bottom right). The efficacy of overexpressing or knocking down FOXM1D was determined by IB assay (d and e, bottom left). The images are representative of at least seven implanted mice. **P<0.01 vs control groups. See also Supplementary Figures S2.

Figure 3

FOXM1D Directly Interacts with ROCKs. (a) ROCK2 was identified as one of FOXM1D-binding proteins. 293 T cells were transfected with Flag-tagged FOXM1D-expressing or control plasmid. IP was performed with the cell extracts using anti-Flag antibody. Subsequently, the IP complex was subjected to LC-MS/MS analysis. (b and c) GST-FOXM1D binds to ROCK2. GST pull-down was performed using GST-FOXM1D and total cell extracts of LoVo (b) or SW-480 (c) cells, followed by IB with anti-ROCK2 antibody. (d) Flag-tagged FOXM1D binds to ROCK2. 293 T cells were transfected with Flag-FOXM1D-expressing or control plasmid, followed by IP using anti-Flag antibody and IB with anti-ROCK2 antibody. (e) Reciprocal IP was performed in the indicated cells using anti-ROCK2 antibody, followed by IB with CP11 and CP12 antibodies. (f) Schematic diagram of Re-IP, that is, two-step IP using CP11 and CP12 antibodies. (g) Two round co-IP was performed using the indicated cell extracts. A greater amount of ROCK2 was captured in SW-480-FOXM1D cells. (h) FOXM1D colocalizes with ROCK2. Triple IF staining for Flag-FOXM1D (green), HA-ROCK2 (red), and nuclei (DAPI, blue) was performed in 293 cells that were transiently co-transfected with pFLAG-CMV-4-FOXM1D and pCMV-C-HA-ROCK2. Scale bar, 25 μm. (i) GST-FOXM1D also binds to ROCK1. GST pull-down was performed using GST-FOXM1D and LoVo total cell extracts, followed by IB with anti-ROCK1 antibody. (j) Flag-tagged FOXM1D also binds to ROCK1. 293 T cells were transfected with Flag-tagged FOXM1D-expressing or control plasmid, followed by IP using anti-Flag and IB with anti-ROCK1 antibody. See also Supplementary Figure S7.

Figure 4

FOXM1D is crucial for the ROCKs' ability to regulate actin arrangement and EMT in a Rho-dependent manner. (a–b) Triple IF staining for F-actin (phalloidin, green), phospho-MLC (red) and nuclei (DAPI, blue) in SW-480 cells transfected with control and FOXM1D-expressing vector (a) or in LoVo cells transfected with shRNA-control, shRNA-FOXM1D or shRNA-FOXM1D and restored FOXM1D-expressing vector (b). The cell morphological changes and F-actin assembly among these cells are shown. Scale bar, 25 μm. (c) IB assay. The expression and/or phosphorylation of ROCKs downstream targets in the indicated cells. (d–e) Triple IF staining for F-actin (phalloidin, green), phospho-MLC (red) and nuclei (DAPI, blue) was performed to detect cytoskeletal changes. SW-480-FOXM1D cells were treated with Rho inhibitor (1 μg/ml) for 4 h (d). LoVo-shRNA-FOXM1D cells were treated with Rho activator (1 U/ml) for 30 min (e). Scale bar, 25 μm. (f) IB assay. The expression and/or phosphorylation of E-cadherin, ROCKs downstream targets in the indicated cells treated with Rho inhibitor or Rho activator. (g) ROCKs activity was measured by ELISAs. In SW-480 cells, FOXM1D overexpression enhanced ROCKs activity, which was inhibited by Rho inhibitor. In LoVo cells, FOXM1D knockdown decreased ROCKs activity, and Rho activator treatment induced a slightly increase in ROCKs activity in FOXM1D knockdown cells. Restored FOXM1D expression recovered ROCKs activity. The data are represented as mean±s.d. for triplicate samples. *P<0.05 and **P<0.01. See also Supplementary Figure S8.

Figure 5

ROCKs Inhibitors Abrogate FOXM1D-Induced ROCKs Activation. (a–b) Triple IF staining for F-actin (phalloidin, green), phospho-MLC (a) or E-cadherin (b) (red) and nuclei (DAPI, blue) showed decreased F-actin and phosphorylated MLC, increased E-cadherin and altered cell shape and size in SW-480-FOXM1D cells treated with the ROCKs inhibitor Y-27632 or fasudil (both 10 μM, for 24 h). Scale bar, 25 μm. (c) Y-27632 significantly reduced ROCKs activity in SW-480-FOXM1D cells as measured by ELISAs. The data are represented as mean±s.d. for triplicate samples. * P<0.05. (d) IB assay. The expression and/or phosphorylation of E-cadherin and ROCKs downstream targets in SW-480-FOXM1D cells treated with Y-27632. (e) Y-27632 does not affect the binding of FOXM1D to ROCK2, indicating that the binding site of FOXM1D differs from that of Y-27632 on ROCK2. SW-480-FOXM1D cells were treated with Y-27632 (10 μM, for 24 h), and then, IP was performed using anti-ROCK2 antibody, followed by IB with CP12 antibody. See also Supplementary Figure S9.

Figure 6

Identification of interacting sites of FOXM1D and ROCK2. (A) Schematic illustration of truncated mutants of ROCK2 (Aai) and its coiled-coil region (686–1145 aa) (Aaii). (B) A series of HA-tagged ROCK2 and its truncated mutants (mutants A–F in figure ai, and mutants G-I in figure aii, all of which were marked by asterisks in figure bi and bii) were expressed in 293 T cells and then subjected to IP using anti-HA antibody, followed by IB with CP12 antibody. In (Bbi), only intact ROCK2 (A) and three mutants (C–E) captured FOXM1D, and further, only mutant g captured FOXM1D using IP and GST pull-down assay, respectively (Bbii and Bbiii). All of these constructs are marked as ‘+' in (A). (C) Schematic illustration of minimal FOXM1D-binding domain and its site-mutated mutants in (Cci). The mutants were expressed in 293 T cells and subjected to IP using anti-HA antibody as previously mentioned. Mutants 3 and 4 showed a subtle binding with FOXM1D in (Ccii). (D) Triple IF staining for HA-ROCK2 deletion mutants (red), Flag-FOXM1D (green) and nuclei (DAPI, blue) showed the cellular colocalization between FOXM1D and the ROCK2 truncated mutants indicated in (bi). Scale bar, 25 μm. (E) Schematic illustration of FOXM1D and its truncated mutants. (F) A series of Flag-tagged FOXM1D and its truncated mutants (A–E marked by asterisks) were expressed in 293 T cells and then subjected to IP using anti-Flag antibody, followed by IB with ROCK2 antibody. Only intact FOXM1D (A) and its mutants (B, C and possibly D) captured ROCK2; these constructs are marked as ‘+' in (E). (G) ROCKs activity in 293 T cells that expressed intact FOXM1D or its mutants was measured by ELISAs. The data are represented as mean±s.d. for triplicate samples. *P<0.05 vs blank. See also Supplementary Figure S10.

Figure 7

Schematic diagram of the role of FOXM1D in ROCKs activation. The simultaneous binding of FOXM1D to FBD and active Rho-GTP to RBD in the ROCKs coiled-coil region activates ROCKs. ROCKs activation impairs E-cadherin level and phosphorylates LIMK1/2, cofilin and MLC that regulate F-actin rearrangement and cell shape collaboratively, eventually leading to EMT and metastasis. PH, pleckstrin homology domain; CRD, cysteine-rich domain; RBD, Rho-binding domain (ROCK2 residues 934–1015); FBD, FOXM1D-binding domain (ROCK2 residues 686–824), which interacts with the FOXM1D region coded by exons V, VI, VII, VIIa.