Claudin 1 expression in basal-like breast cancer is related to patient age

Abstract

Background: Defects in tight junctions, gate-keepers of the integrity of the epidermal barrier function, are known to contribute to cancer development. As such, enhancing our understanding of how the expression of proteins involved in these junctions is regulated in cancer, remains a priority. Although the expression of one of these proteins, claudin 1, is down regulated in most invasive human breast cancers (HBC), we have recently shown that high levels of claudin 1, characterized tumors belonging to the very aggressive basal-like breast cancer (BLBC) subtype. In these tumors, the claudin 1 protein, usually localized in the cell membrane, is often mislocalized to the cytoplasm.

Methods: To examine the clinical relevance of this observation, we have generated and analyzed an invasive HBC tissue microarray consisting of 151 breast tumor samples; 79 of which presented a basal-like phenotype (i.e. ER-ve, PR-ve HER2-ve, CK5/6 or EGFR+ve). We also interrogated the outcome of claudin 1 knockdown in a human BLBC cell line, BT-20.

Results: Immunohistochemical analysis of this patient cohort revealed a significant association between high claudin 1 expression and BLBCs in women 55 years of age and older. Interestingly, no significant association was found between claudin 1 and nodal involvement, tumor grade or tumor size. Regression analysis however, showed a significant positive association between claudin 1 and claudin 4, even though claudin 4 did not significantly correlate with patient age. Claudin 1 knockdown in BT-20 cells resulted in decreased cell migration. It also significantly altered the expression of several genes involved in epithelial-mesenchymal-transition (EMT); in particular, SERPINE 1 (PAI1) and SSP1 (osteopontin), known to inhibit EMT and cancer cell migration. Conversely, genes known to maintain EMT through their interaction, SNAIL2, TCF4 and FOXC2 were significantly down regulated.

Conclusions: The association of high claudin 1 protein levels observed in tumors derived from older women with BLBC, suggests that claudin 1 has the potential to serve as a marker which can identify a specific subgroup of patients within the BLBC subtype and thus, further contribute to the characterization of these ill-defined breast cancers. More importantly, our studies strongly suggest that claudin 1 directly participates in promoting breast cancer progression, possibly through the alteration of expression of EMT genes.

Figure 1

Kaplan-Meier graphs for survival and recurrence in basal-like tumors. Univariate survival analyses were performed using Cox regression. Symbols on the graph lines represent censored data. No significant association was found between claudin 1 expression and patient survival (p=0.93), nor recurrence of the disease (p=0.29); although a trend appeared towards significance for disease recurrence. A. Survival n = 79; low claudin 1 (H≤40) events = 13, high claudin 1(H>40) events = 12; B. Recurrence n = 79; low claudin 1 (H≤40) events = 16, high claudin 1(H>40) = 10. Cldn1 = claudin 1.

Figure 2

Localization of claudin 1 and claudin 4 proteins in human invasive breast cancers. A,B: Tumors showing both membrane and cytoplasmic staining with the claudin 1 antibody. C,D: Tumors showing cytoplasmic staining alone with the claudin 1 antibody E: Tumor showing both membrane and cytoplasmic staining with the claudin 4 antibody. F: Tumor showing cytoplasmic staining alone with the claudin 4 antibody (black arrows, membrane staining; red arrows, cytoplasmic staining). Scale bars represent 50μm.

Figure 3

Subcellular localization of claudin 1 protein in BT-20 cells. Subcellular fractions of control BT-20 cells were analyzed by Western blot using the claudin 1 antibody. A. Short exposure shows claudin 1 in the membrane fraction only, B. longer exposure reveals some protein in the cytoskeletal and to a lesser extent, the nuclear fraction. The arrow indicates the 21kD claudin 1 protein. C. Immunofluorescent staining with the claudin 1 antibody (left panels) shows positive fluorescence for claudin 1 in the cell membrane and the cytoplasm of a control clonal cell line and reduced fluorescence in the claudin 1 knockdown clone (clone 3).

Figure 4

Knockdown of claudin 1 protein in stably transfected BT-20 HBC cells. Cells were transfected with a control sequence (C1, C2) and two different shRNA constructs targeting claudin 1 (3,4). The shRNA sequence 4 shows partial knockdown whereas sequence 3 shows >90% knockdown of the claudin 1 protein. (β-actin loading control; Western blot analysis; Cldn1 = claudin 1).

Figure 5

Claudin 1 knockdown results in a decrease in cell migration rate in the BT-20 HBC cell line. Representative light microscopic images of wound healing assays for claudin 1 knocked-down and control BT-20 cells used in evaluating migration rate into a cell free area are shown. Cells were grown to confluency and a scratch made through the cell monolayer. Measurements of the wound areas at time 0 (left panels) and 18h (right panels) were compared using the Image-J program which measured the surface area covered by migrating cells. A. BT-20 cells stably transfected with the control sh-RNA sequence; B. BT-20 cells stably transfected with the sh-RNA-claudin 1 vector. C. BT-20 cells stably transfected with a control shRNA sequence (control 1, n=12) migrated faster than the claudin 1 knockdown clones (clone 3, n=8; clone 4, n=12; mean ± S.E; ANOVA p=0.0054).* p<0.05, **p,0.01 Bonferroni’s Multiple Comparison Test.

Figure 6

Silencing of claudin 1 in HBC cells altered expression of genes involved in EMT. Expression of genes historically linked to EMT was assessed in BT-20 HBC cells in which claudin 1 was knocked down using real-time PCR arrays. Five housekeeping genes were used as controls for each gene expression calculation. Only genes significantly (p<0.02) differentially expressed >2 fold are shown.