Prognostic value of integrin αV expression and localization pattern in invasive breast carcinomas

Abstract

Invasion of surrounding stroma is an early event in breast cancer metastatic progression, and involves loss of cell polarity, loss of myoepithelial layer, epithelial-mesenchymal transition (EMT) and remodeling of the extracellular matrix (ECM). Integrins are transmembrane receptors responsible for cell-ECM binding, which triggers signals that regulate many aspects of cell behavior and fate. Changes in the expression, localization and pairing of integrins contribute for abnormal responses found in transformed epithelia. We analyzed 345 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of integrin αV and correlation with clinical parameters. Patients with lower levels of integrin αV staining showed reduced cancer specific survival. A subset of cases presented a peripheral staining of integrin αV surrounding tumor cell clusters, possibly matching the remaining myoepithelial layer. Indeed, the majority of ductal carcinoma in situ (DCIS) components found in the TMA presented integrin αV at their periphery, whereas this pattern was mostly lost in invasive components, even in the same sample. The lack of peripheral integrin αV correlated with decreased cancer specific survival. In addition, we observed that the presence of integrin αV in the stroma was an indicative of poor survival and metastatic disease. Consistently, by interrogating publicly available datasets we found that, although patients with higher mRNA levels of integrin αV had increased risk of developing metastasis, high co-expression of integrin αV and a myoepithelial cell marker (MYH11) mRNA levels correlated with better clinical outcomes. Finally, a 3D cell culture model of non-malignant and malignant cells reproduced the integrin αV pattern seen in patient samples. Taken together, our data indicate that both the expression levels of integrin αV and its tissue localization in primary tumors have prognostic value, and thus, could be used to help predict patients at higher risk of developing metastasis.

Keywords: 3D cell culture; Breast cancer; Integrin αV; Metastasis; Tissue microarray; Tumor stroma.

Fig. 1

Low expression of integrin αV in breast cancer tissues is linked to poor prognosis. Patients were stratified according to the expression of integrin αV: H-score under mean (= 10.34) was considered low (n=242, red) and H-score above mean, high (n=92, blue). A) Although the expression of integrin αV has not shown relevance for Overall Survival (OS) (p=0.081), cancer specific survival (CSS) (B) showed statistically significant differences pointing out that the decrease in integrin αV expression correlates with a worse prognosis (p=0.041). The expression of integrin αV did not show statistically significant relationships for both C) Disease-free survival (DFS) (p=0.347) and D) Distant disease-free survival (DDFS) (p=0.268).

Fig. 2

Representative immunohistochemistry staining for integrin αV in different breast samples. Breast tissue sections of different types of lesions submitted to immunohistochemistry for integrin αV. Normal acini and ductal hyperplasia (A) and in situ component (B) display integrin αV at the lesion peripheries. C and D, invasive ductal carcinoma (IDC). In D, integrin αV can be observed contouring individual cells. Scale bar= 50 µm.

Fig. 3

Comparison between in situ and invasive components found in the same patient TMA core. (A) Representative image of an IDC case with in situ component, the latter displaying peripheral integrin αV. Typical in situ component (indicated by an arrow) outlined by integrin αV staining. The invasive component shows almost complete absence of peripheral integrin αV. Scale bar = 100 µm. See supplementary Figure 1 for an additional representative image. (B) Detailed information of 8 cases that had both in situ and invasive components, regarding the presence and intensity of peripheral integrin αV and molecular subtype classification.

Fig. 4

Loss of peripheral integrin αV is associated with poor prognosis in human breast tumors. Kaplan-Meier survival analyses for patients with breast cancer stratified by negative (n=225, red) or positive (n=113, blue) staining for peripheral integrin αV: A) For overall survival (OS), no statistical differences were found between the groups (p=0.164). However, in B) the cancer specific survival (CSS) plot shows that patients with loss of peripheral expression of integrin αV had a worse prognosis when compared to those who maintained integrin αV in the periphery of the lesion clusters (p=0.042). For the disease-free survival (DFS) KM plot C), we did not observe statistical differences (p=0.060), although a trend of greater incidence of metastases can be observed in patients with no peripheral integrin αV. For D) Distant disease-free survival (DDFS), no significant differences were detected between the groups (p=0.439).

Fig. 5

Integrin αV is less abundant in a human basal-like breast cancer line in comparison to luminal breast cancer or non-tumoral cells. A) Western blot showing the relative amount of integrin αV in sub confluent monolayer cultures of MCF-10A, MCF-7 and MDA-MB231 cell lines. The basal-like cancer cell line MDA-MB231 expresses 50% less integrin αV than the non-tumoral cell line MCF1-0A. The luminal breast cancer cell line MCF-7 still presented high levels of integrin αV. B) ITGAV mRNA expression analysis in breast cell lines using the online tool Genevestigator revealed a decrease in ITGAV levels for both tumoral cell lines (MCF-7 and MDA-MB231). C) Immunofluorescence of integrin αV (green) in breast cell lines grown as monolayers. Integrin αV fluorescence staining was weaker in the membrane of MDA-MB231 cells. DAPI (blue) was used to stain nuclear DNA. Scale bar = 5 µm.

Fig. 6

Three-dimensional structures formed by the human breast cancer cell line MCF-10A (non-malignant) shows higher levels of peripheral integrin αV (ITGAV). A) 3D acini (MCF-10A) and tumor spheroids (MCF-7 and MDA-MB-231) immunostained for integrin αV. B) Fluorescence profile intensity plots of integrin αV of MCF-10A acini (n = 5), MCF-7 tumor-spheroids (n = 4) and MDA-MB-231 tumor-spheroids (n = 4). Scale bar = 25 μm.

Fig. 7

Representative immunohistochemistry staining images of stromal integrin αV. Breast tissue sections of different lesions arranged in negative stromal integrin αV (A) and positive stromal integrin αv (B). Scale bar = 50 µm.

Fig. 8

Presence of stromal integrin αV is associated with metastasis incidence and poor prognosis in human breast tumors. Kaplan-Meier survival curves stratified breast cancer patients of our cohort in negative stromal integrin αV (n=182, red) or positive (n=156, blue): A) For overall survival (OS) no statistical differences were found between the groups (p=0.052). However, in B) the cancer specific survival (CSS) plot shows that patients with positive stromal integrin αV had a worse prognosis when compared to negative stromal integrin αV (p=0.017). Following this trend, we also observe that stromal integrin αV not only worsens the incidence of recurrence C), represented by disease-free survival (DFS) (p=0.024), but also metastases incidence D), showed as distant disease-free survival (DDFS) (p=0.036).