Fibrillin-1, induced by Aurora-A but inhibited by BRCA2, promotes ovarian cancer metastasis

Abstract

While Aurora-A (Aur A) provokes, BRCA2 restrains primary tumorigenesis, the roles of Aur A and BRCA2 in cancer metastasis remains unclear. Here, we show that the metastatic promoting markers SLUG, FBN1, and MMP2, 9, 13 are either stimulated or suppressed by Aur A or BRCA2, but the metastatic suppressors E-cadherin, β-catenin, and p53 are either inhibited or promoted by Aur A or BRCA2, leading to enhanced or reduced cell migration and invasion. Further study suggests that FBN1 inhibits E-cadherin and β-catenin, but stimulates MMP2, 9, 13. Depletion of SLUG abrogates FBN1 and MMP9, but increases E-cadherin, while p53 decreases both SLUG and FBN1. Animal assays demonstrate that FBN1 promotes both ovarian tumorigenesis and metastasis. Clinically, overexpression of BRCA2 or Aur A in ovarian cancer tissues predicts good or poor overall and disease free survivals. High expression of SLUG or FBN1 indicates poor overall survivals, whereas high expression of FBN1 but not of SLUG predicts poor disease free survival. No significant associations between p53 expression and patient survivals were found. Overall, FBN1, acts at the downstream of Aur A and BRCA2, promotes ovarian cancer metastasis through the p53 and SLUG-associated signaling, which may be useful for ovarian cancer diagnosis and treatment.

Keywords: Aurora-A; BRCA2; FBN1; SLUG; metastasis.

Figure 1. The negative correlation between Aur A and BRCA2 regulates the expression levels of metastasis-related proteins

(A) Detection of Aurora-A or BRCA2 by Western blotting in OVCA429, OVCA429/Aur A shRNA, OVCA429/BRCA2 cDNA, OVCA420, OVCA420/Aur A cDNA, and OVCA420/BRCA2 shRNA cells. (B) Immunoblotting analyses of p53, FBN1, and other metastasis-associated proteins. β-actin was used as a loading control.

Figure 2. Regulation of cell invasion and migration by Aur A or BRCA2

(A) Detection of cell migration and invasion by using a high throughput screening multi-well insert 24-well two-chamber plates. (B) Quantitative analysis of invaded cells (P < 0.05). Error bars = 95% CIs. (C) Detection of migration by scratching assay. (D) Quantitative analysis of migration speed using migration index (P < 0.05). Error bars = 95% CIs.

Figure 3. Regulation of cell invasion and migration by FBN1

(A) Detection of FBN1 and SLUG in OVCA429, OVCA429/Aur A shRNA, OVCA429/BRCA2 cDNA, OVCA420, OVCA420/Aur A cDNA, and OVCA420/BRCA2 shRNA cells. (B) Detection of the silencing efficiency of FBN1 in OVCA429 cells with three siRNAs by Western blotting. (C) Interruption of FBN1 expression in OVCA429, OVCA420/Aur A and OVCA420/BRCA2 shRNA cells with shRNA altered the expressions of multiple proteins regulated by Aur A and BRCA2. β-actin was used as a loading control. (D) Detection of cell migration and invasion by using a high throughput screening multi-well insert 24-well two-chamber plates. (E) Detection of migration speed of FBN1-silencing cells by scratch assay. (F) Quantitative analysis of migrated and invaded cells (P < 0.05). Error bars = 95% CIs.

Figure 4. Upregulation of FBN1 by SLUG and effects of FBN1 on tumorigenesis

(A) Knockdown of SLUG with three siRNA detected by Western blotting in OVCA429. (B) Knockdown of SLUG with SiRNA detected by Western blotting in OVCA429, OVCA420/Aur A cDNA and OVCA420/BRCA2 shRNA cells and immunoblotting analysis of metastasis-associated proteins. (C) Detection of p53, SLUG and FBN1 in SKOV3 cells induced by p53 cDNA. (D–F) In vivo tumorigenesis examined by animal assays. (G) Dissection of xenograft tumors. (H) Quantitative analyses of the numbers of the nodules formed in animals (P < 0.025 or P < 0.05). Error bars = 95% CIs. (I) Quantitative analyses of the weights of the nodules dissected from mice (P < 0.025 or P < 0.05). Error bars = 95% CIs. β-actin was used as the loading control.

Figure 5. Immunohistochemical analyses of Aur A, BRCA2, FBN1 and SLUG expression and correlation in high-grade ovarian serous carcinoma and the associations of the molecules with patient survivals

Representative images from tissue microarray stained for BRCA2 and Aur A. (A) high expression of BRCA2 in nuclei is correlated with low expression of Aur A, FBN1 and SLUG in the same core of high-grade ovarian carcinoma (× 400). (B) high expressions of Aur A, FBN1 and SLUG expression are correlated with low nuclear accumulation of BRCA2 in the same core of high-grade ovarian carcinoma (× 400). (C) the favorable overall and disease-free survivals (P = 0.015, P = 0.021) are associated with the accumulated nuclear staining of BRCA2. (D) the poor overall and disease-free survivals (P = 0.027, P = 0.033) are associated with strong staining of Aur A. (E) the poor overall survival (P = 0.018) but not the disease-free survival (P = 0.076) is associated with the strong expression of SLUG. (F) the poor overall survival (P = 0.025) but not the disease-free survival (P = 0.779) is associated with the strong expression of FBN1. H, high expression; L, low expression.

Figure 6. Association of the expression ratios between Aurora-A, SLUG, or FBN1 and BRCA2 with patient survivals

(A–C) The increased ratios of Aurora-A, SLUG or FBN1 to BRCA2 (Ap/Bn, Sp/Bn, or Fp/Bn) are associated with poor overall survival (P_A = 0.002, P_F = 0.003 or Ps = 0.001) and disease-free survival (P_A = 0.010, P_F = 0.020 or Ps = 0.027), compared with the decreased ratios of Aurora-A, SLUG or FBN1 to BRCA2 (An/Bp, Sn/Bp, or Fn/Bp).

Figure 7. A schematic diagram of how Aur A and BRCA2 regulate ovarian cancer metastasis through p53/SLUG-mediated FBN1 signalling

The expression of p53, suppressed by Aur A, but promoted by BRCA2, inhibits the expressions of SLUG and FBN1, which accentuates the expressions of E-cadherin and β-catenin, but attenuates the levels of MMP2, MMP9 and MMP13 to eventually abrogate the ovarian cancer metastasis.