Wnt5a-induced cell migration is associated with the aggressiveness of estrogen receptor-positive breast cancer

Abstract

Elevated expression of Wnt5a is associated with malignancy, cell invasion, and metastasis. The role of Wnt5a expression in breast cancer remains elusive. We investigated the significance of Wnt5a expression in breast cancer. The relationship between Wnt5a expression and clinicopathologic factors was assessed in invasive breast cancer (n = 178) resected at Hiroshima University Hospital between January 2011 and February 2014. Wnt5a was expressed in 69 of 178 cases (39%) of invasive breast cancer and correlated strongly with estrogen receptor (ER) expression (P < 0.001). Wnt5a expression in ER-positive breast cancer correlated significantly with lymph node metastasis, nuclear grade, and lymphatic invasion. The recurrence-free survival was shorter in breast cancer patients with Wnt5a expression than in those without (P = 0.024). The migratory capacity of ER-positive breast cancer cells increased with constitutive expression of Wnt5a and decreased with Wnt5a knockdown. DNA microarray analysis identified activated leukocyte cell adhesion molecule (ALCAM) as the primary gene induced by Wnt5a. ALCAM was expressed in 69% of Wnt5a-positive but only 27% of Wnt5a-negative cancers (κ = 0.444; P < 0.001). The inhibition of ALCAM reversed the enhanced migratory effect of Wnt5a, confirming the importance of this protein in the migration of ER-positive breast cancer cells. Wnt5a expression is related to high malignancy and a poor prognosis in ER-positive breast cancer. We suspect that Wnt5a expression increases the malignancy of breast cancer by increasing the migratory capacity of cancer cells through the induction of ALCAM expression.

Keywords: ER-positive breast cancer; Wnt5a; activated leukocyte cell adhesion molecule; cell migration; prognosis.

Figure 1. Wnt5a expression in breast cancer using breast cancer specimens

(A) Immunohistochemical staining for Wnt5a in invasive breast cancer. (a) Wnt5a expression in invasive breast cancer. (b) Wnt5a expressed weakly in cytoplasm in non-tumor mammary duct. (c) Wnt5a expressed strongly in invasive breast cancer a: bar, 500 μm, magnification ×40; b, c: bar 50 μm, ×400 (B) Evaluation of Wnt5a expression was scored as 0, 1+, 2+, or 3+, taking into consideration both staining proportion and intensity. Scores 0–2+, negative; Score 3+, positive. bar, 50 μm, ×400.

Figure 2. Relationship between recurrence-free survival and Wnt5a in 153 ER-positive breast cancer patients

The recurrence-free survival rates were analyzed according to Wnt5a immunohistochemistry. The mean 5-year recurrence-free survival rates were 100% for Wnt5a negative breast cancer and 81.1% for Wnt5a negative breast cancer. Wnt5a expression was significantly associated with poor recurrence-free survival in ER-positive cancer patients (P = 0.024).

Figure 3. Migratory capacity of Wnt5a-expressing breast cancer cells

(A) The expression levels of β-catenin, ER, and cyclin D in MCF-7/Wnt5a and control MCF-7 cells. (B) The proliferative capacity in MCF-7/Wnt5a and control MCF-7 cells. Data are presented as the mean ± SE of three proliferation assays. (C, D) The migratory capacity of MCF-7, MCF-7/Wnt5a + sc, and MCF-7/Wnt5a + Wnt5a-siRNA cells. Data are presented as the mean ± SE of three migration assays. Data were evaluated using the Mann–Whitney U-test.

Figure 4. Knockdown of Wnt5a decreases the migratory capacity of MDA-MB-175-VII cells

(A) The Wnt5a expression of MDA-MB-175-VII and MDA-MB-361 cells transfected with Wnt5a-siRNAs (Wnt5a-siRNA #1 and #2) and sc. (B) The migratory capacity of MDA-MB-175-VII and MDA-MB-361 cells transfected with Wnt5a-siRNAs and sc. Data are presented as the mean ± SE of three migration assays. Data were evaluated using the Mann–Whitney U-test.

Figure 5. Co-expression of Wnt5a and ALCAM in ER-positive breast cancer tissue

(A) ALCAM expression was reduced by knockdown of Wnt5a. (B) Evaluation of ALCAM expression was scored as 0, 1+, 2+, or 3+. Scoring of ALCAM was performed with the same evaluation as Wnt5a. (C) Co-expression of Wnt5a and ALCAM in breast cancer tissue.

Figure 6. Knockdown of ALCAM decreases the Wnt5a-induced increase in migratory capacity

(A) Wnt5a and ALCAM expression of MDA-MB-175-VII cells transfected with ALCAM-siRNAs (ALCAM-siRNA #1 and #2) and sc. (B) The migratory capacity of MDA-MB-175-VII cells transfected with ALCAM-siRNAs and sc. Data are presented as the mean ± SE of three migration assays. Data were evaluated using the Mann–Whitney U-test.