Overexpression of beta1-chain-containing laminins in capillary basement membranes of human breast cancer and its metastases

Abstract

Introduction: Laminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the alpha4 chain from predominantly laminin-9 (alpha4beta2gamma1) to predominantly laminin-8 (alpha4beta1gamma1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast.

Method: In the present study the expressions of laminin isoforms alpha1-alpha5, beta1-beta3, gamma1, and gamma2 were examined during progression of breast cancer. Forty-five clinical samples of breast tissues including normal breast, ductal carcinomas in situ, invasive ductal carcinomas, and their metastases to the brain were compared using Western blot analysis and immunohistochemistry for various chains of laminin, in particular laminin-8 and laminin-9.

Results: Laminin alpha4 chain was observed in vascular basement membranes of most studied tissues, with the highest expression in metastases. At the same time, the expression of laminin beta2 chain (a constituent of laminin-9) was mostly seen in normal breast and carcinomas in situ but not in invasive carcinomas or metastases. In contrast, laminin beta1 chain (a constituent of laminin-8) was typically found in vessel walls of carcinomas and their metastases but not in those of normal breast. The expression of laminin-8 increased in a progression-dependent manner. A similar change was observed from laminin-11 (alpha5beta2gamma1) to laminin-10 (alpha5beta1gamma1) during breast tumor progression. Additionally, laminin-2 (alpha2beta1gamma1) appeared in vascular basement membranes of invasive carcinomas and metastases. Chains of laminin-5 (alpha3beta3gamma2) were expressed in the ductal epithelium basement membranes of the breast and diminished with tumor progression.

Conclusion: These results suggest that laminin-2, laminin-8, and laminin-10 are important components of tumor microvessels and may associate with breast tumor progression. Angiogenic switch from laminin-9 and laminin-11 to laminin-8 and laminin-10 first occurs in carcinomas in situ and becomes more pronounced with progression of carcinomas to the invasive stage. Similar to high-grade brain gliomas, the expression of laminin-8 (and laminin-10) in breast cancer tissue may be a predictive factor for tumor neovascularization and invasion.

Figure 1

Immunohistochemistry of human breast tissues including normal, DCIS, primary IDC and metastases. (a) Panels A–D: hematoxylin and eosin staining of normal breast, DCIS, IDC and metastatic tissues, respectively. Panels E–H: double immunostaining with laminin α4 (red) and an endothelial marker, von Willebrand factor/factor-8 (F8; green). Panels I–L: double immunostaining with laminin β1 (red) and an endothelial marker von Willebrand factor (F8, green). Panels M–P: double immunostaining for laminin β2 (red) and F8 (green). For each representative case, serial sections are shown. (b) Panels A–D: hematoxylin and eosin staining (same as in Fig. 1a, panels A–D). Panels Q–T: double immunostaining for laminin α4 chain (red) and lining epithelium markers cytokeratins (CK)-8/18 (green). Panels U–X: double immunostaining for laminin β1 (red) and CK-8/18 (green). For each case, serial sections to Fig. 1a are shown. Because of lack of appropriate antibodies, no double staining could be performed for laminin β2 chain and CK-8/18. In normal breast tissues, laminin-9 chains α4 and β2 are expressed in BMs of mammary gland ducts (arrows in Fig. 1a, panels E and M, and Fig. 1b, panel Q) and blood vessels. In DCIS laminin α4 chain starts disappearing from ductal BMs (Fig. 1a, panel F, and Fig. 1b, panel R) but β2 chain is present (Fig. 1a, panel N [arrows]). Laminin-8 chains α4 and β1 and laminin-9 chains α4 and β2 colocalize in some microvessels. In all invasive ductal carcinomas, laminin-8 α4 and β1 chains are both found in BMs of F8-positive microvessels (Fig. 1a, panels G and K). Laminin-9 is absent (no β2 chain; Fig. 1a, panel O). In metastases of breast carcinoma, laminin-8 chains are seen in microvascular BMs (Fig. 1a, panels H and L; Fig. 1b, panels T and X) but laminin-9 is absent again (no β2 chain; Fig. 1a, panel P). BM, basement membrane; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma.

Figure 2

Western blot analysis. Shown are eight out of 28 samples (two normal breast samples, two DCIS, two IDC and two breast cancer metastases to brain) subjected to Western blot analysis for laminin α4, β1 and β2 chains. Gel loading was normalized by β-actin (lower row). The expression of laminin α4 chain, a constituent of laminin-8 and laminin-9, varies in normal and tumor tissues, with the highest expression detected in metastases. Laminin β2 chain, a constituent of laminin-9, is highly expressed in normal tissues, but its expression is very low in breast cancer tissues. In contrast, expression of laminin β1 chain, a constituent of laminin-8, is high in brain metastases and IDC but low in DCIS and absent in normal tissues. Laminin α4 chain migrates at 200 kDa, β1 chain at 230 kDa, β2 chain at 190 kDa, and β-actin at 47 kDa. The T98G glioblastoma cell line, which is known to express α4 and β1 chains of laminin-8 but no β2 chain, is used as a positive control.