Overexpression of β1 integrin contributes to polarity reversal and a poor prognosis of breast invasive micropapillary carcinoma

Abstract

Invasive micropapillary carcinoma (IMPC) of the breast is a highly aggressive breast cancer. Polarity reversal exemplified by cluster growth is hypothesized to contribute to the invasiveness and metastasis of IMPC. In this study, we demonstrate that levels of β1 integrin and Rac1 expression were greater in breast IMPC than in invasive breast carcinoma of no specific type and paraneoplastic benign breast tissue. We show that silencing β1 integrin expression using the β1 integrin inhibitor AIIB2 partially restored polarity in IMPC primary cell clusters and downregulated Rac1. Thus, overexpression of β1 integrin upregulates Rac1. Univariate analysis showed that overexpression of β1 integrin and Rac1 was associated with breast cancer cell polarity reversal, lymph node metastasis, and poor disease-free survival in IMPC patients. Multivariate analysis revealed that polarity reversal was an independent predictor of poor disease-free survival. These findings indicate that overexpression of β1 integrin and the resultant upregulation of Rac1 contribute to polarity reversal and metastasis of breast IMPC, and that β1 integrin and Rac1 could be potential prognostic biomarkers and targets for treatment of breast IMPC.

Keywords: breast; invasive micropapillary carcinoma; metastasis; polarity reversal; β1 integrin.

Figure 1. β1 integrin and Rac1 expression and polarity of breast cancer cell lines with silencing of β1 integrin and Rac1

(A, B) β1 integrin and Rac1 mRNA in MCF-10A was downregulated after transfection with siRNA-β1 integrin and siRNA-Rac1. (C, D) Decreased β1 integrin and Rac1 protein expression in MCF-10A was detected by Western blot after transfection with siRNA-β1 integrin and siRNA-Rac1. β-actin was used as control. (E) Disordered polarity of MCF-10A cell clusters in 3D culture after treatment with siRNA-β1 integrin and siRNA-Rac1 is shown. Normal polarity was determined by MUC-1 (red) at the luminal surface of control cells. Nuclei are shown with DAPI (blue). Scale bars, 50 μm. si-ctrl: control cell line.

Figure 2. Expression of β1 integrin and Rac1 in breast cancer cell lines and primary tumor cells at 3D culture treated with β1 integrin inhibitor AIIB2 and detected by Western blot

(A) Rac1 expression in MCF-7 and MDA-MB-231 was lower than in controls. (B, C) Rac1 expression in IMPC and IDC-NST tumor cells was decreased. β-Actin was used as control. Relative gray value was defined as the ratio between the gray value of Rac1 and β-actin (student’s t-test, ^*P < 0.05). T1-T3: 3 cases of IMPC primary tumor; T4-T6: 3 cases of IDC-NST primary tumor.

Figure 3. Polarity alterations induced by the β1 integrin inhibitor AIIB2

IMPC and IDC-NST cells were stained with anti-MUC-1 antibody (red) and anti-E-cadherin antibody (green), and the nuclei were stained with DAPI (blue). Representative confocal images were taken. Scale bars, 50 μm.

Figure 4. (A) Immunohistochemical stain of β1 integrin and Rac1 in IMPC and IDC-NST (×200)

β1 integrin was predominantly expressed on the cell membrane with brown-yellow color, whereas Rac1 was primarily expressed in the nucleus and cytoplasm. (B) Reversed immunohistochemistry pattern of MUC-1 in IMPC, which was defined as the presence of complete linear reactivity on the outer surface of tumor cell clusters facing stroma (×200). (C) Nonreversed immunohistochemistry pattern of MUC-1 in IMPC, which was defined as whole cytoplasmic membrane staining of tumor cell clusters (×200).

Figure 5. Kaplan-Meier curves showing the prognostic value of β1 integrin, Rac1, and (reversal of) MUC-1 expression in IMPC

(A) High β1 integrin expression was correlated with shorter disease-free survival (DFS) (P = 0.017). (B) High Rac1expression was correlated with shorter DFS (P = 0.027). (C) MUC-1 reversed pattern was correlated with shorter DFS (P = 0.004). (D) The DFS of patients with high β1 integrin and Rac1 expression was shorter than that of patients with high expression of only one of these markers (both high vs β1 integrin high, P = 0.029; both high vs Rac1 high, P = 0.019; and both high vs both low, P = 0.15).