Twist promotes tumor metastasis in basal-like breast cancer by transcriptionally upregulating ROR1

Abstract

Rationale: Twist is a key transcription factor for induction of epithelial-mesenchymal transition (EMT), which promotes cell migration, invasion, and cancer metastasis, confers cancer cells with stem cell-like characteristics, and provides therapeutic resistance. However, the functional roles and targeted genes of Twist in EMT and cancer progression remain elusive. Methods: The potential targeted genes of Twist were identified from the global transcriptomes of T47D/Twist cells by microarray analysis. EMT phenotype was detected by western blotting and immunofluorescence of marker proteins. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional induction of ROR1 by Twist. A lung metastasis model was used to study the pro-metastatic role of Twist and ROR1 by injecting MDA-MB-231 cells into tail vein of nude mice. Bio-informatics analysis was utilized to measure the metastasis-free survival of breast cancer patients. Results: Twist protein was proved to directly activate the transcription of ROR1 gene, a receptor of Wnt5a in non-canonical WNT signaling pathway. Silencing of ROR1 inhibited EMT process, cell migration, invasion, and cancer metastasis of basal-like breast cancer (BLBC) cells. Knockdown of ROR1 also ameliorated the pro-metastatic effect of Twist. Furthermore, analyses of clinical specimens indicated that high expression of both ROR1 and Twist tightly correlates with poor metastasis-free survival of breast cancer patients. Conclusion: ROR1 is a targeted gene of Twist. Twist/ROR1 signaling is critical for invasion and metastasis of BLBC cells.

Keywords: EMT; ROR1; Twist; basal-like breast cancer (BLBC); tumor metastasis.

Figure 1

Twist promotes the expression of ROR1. (A-B) Representative heatmaps from global comparative transcriptome analysis indicating genes that are up-regulated upon Twist overexpression. (C) The relative mRNA levels of the indicated genes were normalized to the GAPDH level in the T47D cells stably transfected with pLenti6.3/V5-vector or pLenti6.3/V5-Twist as determined by qRT-PCR. The results are expressed as the mean ± SD of three independent experiments. * P < 0.05 using Student's t-test. (D) The indicated proteins were analyzed by Western blotting in T47D cells stably expressing pLenti6.3/V5-vector or pLenti6.3/V5-Twist, as indicated. (E) T47D cells stably expressing pLenti6.3/V5-vector or pLenti6.3/V5-Twist were analyzed by immunofluorescence as indicated. Scale bars, 50μm.

Figure 2

Twist directly upregulates ROR1 by occupying its promoter. (A) The indicated cells overexpressing empty vector or Twist were transfected with ROR1 promoter linked to luciferase, and, after 48 h, luciferase activity was assayed. (B) Illustration of the ROR1 promoter region with the indicated Twist-binding capability. "+" indicates binding and "-" indicates no binding based on the results shown in (C). (C) MDA-MB-231 cells were analyzed by ChIP using anti-Twist antibody. (D) Schematic illustration of wild-type ROR1 promoter and its mutants. (E) ROR1 promoters (wild-type or mutant) linked to luciferase were transfected into 293T cells, and, after 48 h, luciferase activity was assayed. (F) The indicated cells overexpressing empty vector or Twist were transfected with ROR1 promoter (wild-type or mutant) linked to luciferase, and, after 48 h, luciferase activity was assayed. (G, I) The indicated cells stably transfected with control or Twist shRNA were used to analyze the mRNA level of the indicated molecules by qRT-PCR. (H, J) The indicated cells stably transfected with control or Twist shRNA were used to analyze the protein levels by Western blot.

Figure 2

Twist directly upregulates ROR1 by occupying its promoter. (A) The indicated cells overexpressing empty vector or Twist were transfected with ROR1 promoter linked to luciferase, and, after 48 h, luciferase activity was assayed. (B) Illustration of the ROR1 promoter region with the indicated Twist-binding capability. "+" indicates binding and "-" indicates no binding based on the results shown in (C). (C) MDA-MB-231 cells were analyzed by ChIP using anti-Twist antibody. (D) Schematic illustration of wild-type ROR1 promoter and its mutants. (E) ROR1 promoters (wild-type or mutant) linked to luciferase were transfected into 293T cells, and, after 48 h, luciferase activity was assayed. (F) The indicated cells overexpressing empty vector or Twist were transfected with ROR1 promoter (wild-type or mutant) linked to luciferase, and, after 48 h, luciferase activity was assayed. (G, I) The indicated cells stably transfected with control or Twist shRNA were used to analyze the mRNA level of the indicated molecules by qRT-PCR. (H, J) The indicated cells stably transfected with control or Twist shRNA were used to analyze the protein levels by Western blot.

Figure 2

Twist directly upregulates ROR1 by occupying its promoter. (A) The indicated cells overexpressing empty vector or Twist were transfected with ROR1 promoter linked to luciferase, and, after 48 h, luciferase activity was assayed. (B) Illustration of the ROR1 promoter region with the indicated Twist-binding capability. "+" indicates binding and "-" indicates no binding based on the results shown in (C). (C) MDA-MB-231 cells were analyzed by ChIP using anti-Twist antibody. (D) Schematic illustration of wild-type ROR1 promoter and its mutants. (E) ROR1 promoters (wild-type or mutant) linked to luciferase were transfected into 293T cells, and, after 48 h, luciferase activity was assayed. (F) The indicated cells overexpressing empty vector or Twist were transfected with ROR1 promoter (wild-type or mutant) linked to luciferase, and, after 48 h, luciferase activity was assayed. (G, I) The indicated cells stably transfected with control or Twist shRNA were used to analyze the mRNA level of the indicated molecules by qRT-PCR. (H, J) The indicated cells stably transfected with control or Twist shRNA were used to analyze the protein levels by Western blot.

Figure 3

ROR1 positively regulates migration, invasion and cancer metastasis in BLBC. (A-B) The indicated molecules were analyzed by Western blot in the Hs578T and MDA-MB-231 cells with stable ROR1 knockdown. (C-F) Cell migration and invasion were determined in the indicated stable cell lines. These experiments were repeated at least three times. The results are expressed as the mean ± SD. * p < 0.05 and ** p < 0.01, *** p < 0.001 using Student's t-test. (G) MDA-MB-231 cells stably transfected with control or shROR1 were injected into the lateral tail vein of nude mice. The left panel presents macroscopic appearances of metastatic lung tumors; central and right panels present the H&E staining. Scale bars, 40×,500μm; 200×,100μm. (H) Illustration of the statistical results (n=6). The results are expressed as the mean ± SD. ** P < 0.01 using Student's t test.

Figure 3

ROR1 positively regulates migration, invasion and cancer metastasis in BLBC. (A-B) The indicated molecules were analyzed by Western blot in the Hs578T and MDA-MB-231 cells with stable ROR1 knockdown. (C-F) Cell migration and invasion were determined in the indicated stable cell lines. These experiments were repeated at least three times. The results are expressed as the mean ± SD. * p < 0.05 and ** p < 0.01, *** p < 0.001 using Student's t-test. (G) MDA-MB-231 cells stably transfected with control or shROR1 were injected into the lateral tail vein of nude mice. The left panel presents macroscopic appearances of metastatic lung tumors; central and right panels present the H&E staining. Scale bars, 40×,500μm; 200×,100μm. (H) Illustration of the statistical results (n=6). The results are expressed as the mean ± SD. ** P < 0.01 using Student's t test.

Figure 3

ROR1 positively regulates migration, invasion and cancer metastasis in BLBC. (A-B) The indicated molecules were analyzed by Western blot in the Hs578T and MDA-MB-231 cells with stable ROR1 knockdown. (C-F) Cell migration and invasion were determined in the indicated stable cell lines. These experiments were repeated at least three times. The results are expressed as the mean ± SD. * p < 0.05 and ** p < 0.01, *** p < 0.001 using Student's t-test. (G) MDA-MB-231 cells stably transfected with control or shROR1 were injected into the lateral tail vein of nude mice. The left panel presents macroscopic appearances of metastatic lung tumors; central and right panels present the H&E staining. Scale bars, 40×,500μm; 200×,100μm. (H) Illustration of the statistical results (n=6). The results are expressed as the mean ± SD. ** P < 0.01 using Student's t test.

Figure 4

The promotion of cell migration, invasion and cancer metastasis after Twist overexpression primarily depends on ROR1. (A-B) The indicated molecules were analyzed by Western blot in the Hs578T and MDA-MB-231 cells stably expressing control, ROR1 shRNA, Twist, or both, as indicated. (C-F) Cell migration and invasion were determined in the indicated stable cell lines. The results are expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 using Student's t-test. (G-H) An in vivo lung metastasis model was established in nude mice using MDA-MB-231 cells stably expressing control, ROR1 shRNA, Twist, or both, as indicated. (G) Representative results of gross and H&E staining (middle scale: 40×; right scale: 200×) of metastatic lung nodules. Scale bars, 40×,500μm; 200×,100μm. (H) Illustration of the statistical results (n = 6). The results are expressed as the mean ± SD. * P < 0.05, *** P < 0.001 using Student's t-test.

Figure 4

The promotion of cell migration, invasion and cancer metastasis after Twist overexpression primarily depends on ROR1. (A-B) The indicated molecules were analyzed by Western blot in the Hs578T and MDA-MB-231 cells stably expressing control, ROR1 shRNA, Twist, or both, as indicated. (C-F) Cell migration and invasion were determined in the indicated stable cell lines. The results are expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 using Student's t-test. (G-H) An in vivo lung metastasis model was established in nude mice using MDA-MB-231 cells stably expressing control, ROR1 shRNA, Twist, or both, as indicated. (G) Representative results of gross and H&E staining (middle scale: 40×; right scale: 200×) of metastatic lung nodules. Scale bars, 40×,500μm; 200×,100μm. (H) Illustration of the statistical results (n = 6). The results are expressed as the mean ± SD. * P < 0.05, *** P < 0.001 using Student's t-test.

Figure 4

The promotion of cell migration, invasion and cancer metastasis after Twist overexpression primarily depends on ROR1. (A-B) The indicated molecules were analyzed by Western blot in the Hs578T and MDA-MB-231 cells stably expressing control, ROR1 shRNA, Twist, or both, as indicated. (C-F) Cell migration and invasion were determined in the indicated stable cell lines. The results are expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 using Student's t-test. (G-H) An in vivo lung metastasis model was established in nude mice using MDA-MB-231 cells stably expressing control, ROR1 shRNA, Twist, or both, as indicated. (G) Representative results of gross and H&E staining (middle scale: 40×; right scale: 200×) of metastatic lung nodules. Scale bars, 40×,500μm; 200×,100μm. (H) Illustration of the statistical results (n = 6). The results are expressed as the mean ± SD. * P < 0.05, *** P < 0.001 using Student's t-test.

Figure 5

The expression of Twist and ROR1 correlates with clinical prognosis in breast cancer. (A) Representative immunohistochemical staining of Twist and ROR1 from 134 paraffin-embedded breast cancer tissues. Scale bars, 100μm. (B) Correlation of the expression of ROR1 and Twist in TCGA breast cancer datasets. (C-D) Graphs were derived from published data available through the PubMed GEO database (GSE16446 and GSE19615). Kaplan-Meier curves depict the prognostic impact of Twist (C) and ROR1 (D) expression on distant metastasis-free survival (DMFS), respectively. Statistical differences were determined by log-rank test.