Migration of breast cancer cell lines in response to pulmonary laminin 332

Abstract

Because tumor cell motility is a requirement for metastasis, we hypothesized that lung tissue harbors substances that induce tumor cell migration. MCF-7 breast carcinoma cells exposed to small airway epithelial cells and conditioned medium exhibited dose-dependent tumor cell migration. Among the extracellular matrix proteins in the conditioned medium identified by mass spectrometry, laminin 332 (LM332) had the greatest contribution to the migration of MCF-7 cells. Immunoblotting and immunohistochemistry for LM332-specific chains identified LM332 in the lung and in pulmonary epithelial cells. Antibodies to either LM332 or its integrin receptor inhibited MCF-7 motility, and knockdown of LM332 chains also reduced its migration-inducing activity. Taken together, these findings implicate LM332 as a component of lung tissue that can induce motility in breast carcinoma cells that have been transported to lung during metastasis. Earlier studies on LM332 in tumor progression have examined LM332 expression in tumor cells. This investigation, in comparison, provides evidence that the tumor promoting potential of LM332 may originate in the lung microenvironment rather than in tumor cells alone. Furthermore, this study provides evidence that the motility-inducing properties of the microenvironment can reside in epithelial cells. The findings raise the possibility that LM332 plays a role in the pulmonary metastases of breast carcinoma and may provide a target for antimetastasis therapy.

Keywords: Laminin 332; pulmonary epithelium; tumor cell migration; tumor microenvironment.

Figure 1

MCF‐7 cells transfected with GFP grown in standard culture conditions (A), and with SAEC labeled red with SNARF ^®‐1 carboxylic acid, acetate succinimidyl ester (B). MCF‐7 cells separate from the clusters and display pseudopodia and lamellipodia (arrow). Original magnification 400×, scale bar = 50 μm. MCF‐7 scattering at different densities of SAEC (C), revealing dose‐dependent scattering with increasing numbers of SAEC cells. Error bars show standard errors of the mean. SAEC, small airway epithelial cells; GFP, green fluorescent protein.

Figure 2

Dose‐dependent scattering of MCF‐7 in the presence of increasing of conditioned medium (CM) of small airway epithelial cells (SAEC) (A). Nonmotile MCF‐7 cells in the absence of SAEC CM (B) and displaying a scattering response in the presence of 20 μL of SAEC CM (C). Hematoxylin and eosin stain, scale bar = 50 μm.

Figure 3

Boyden chamber assays showing significant induction of motility by small airway epithelial cells (SAEC) CM in three breast carcinoma cell lines. Error bars show standard errors of the mean.

Figure 4

Motility dose–response studies of MCF‐7 cells to extracellular matrix proteins in small airway epithelial cells (SAEC)‐conditioned medium: laminin 332, tenascin C, thrombospondin 1 (A), laminin 511, fibronectin, and TGF β‐ig‐h3 (B). Error bars show standard error of the mean.

Figure 5

Immunoblot of small airway epithelial cells (SAEC)‐conditioned media probed with LM332 chain antibodies (A). Immunoblot of SAEC and MCF‐7 lysates (B). For each experiment, all lanes were run simultaneously on the same gel.

Figure 6

Immunohistochemistry of cultured SAEC on polyester membranes (A,C,E) and human lung (B,D,F) for laminin β3 (A, B), and γ2 (C, D) chains, and mouse antibody negative controls (E.F). Both chains are expressed in the cytoplasm and basement membranes (arrows) of cultured cells and in tissue. Original magnification 1000×, scale bar = 50 μm. SAEC, small airway epithelial cells; SAGM‐conditioned medium (CM)—conditioned small airway growth medium of SAEC, 184A1 CM conditioned medium of 184A1 cells, LM332‐ laminin 332

Figure 7

Antibody inhibition of small airway epithelial cells (SAEC)‐induced motility. Antibodies to LM332 and both integrin components significantly inhibited conditioned medium (CM)‐induced motility to the same extent to which they inhibited LM332‐induced motility (A). P‐values were determined using one‐way ANOVA with Holm–Sidak correction. Knockdown of LM332. (B). Column 1, (LM332 siRNA mix) refers to lysates of cells transfected with an equal amount of each of the siRNA directed against the three chains of laminin 332 in one reaction. Column 2 (siRNA Control) has a nonspecific sequence, in a concentration equal to the total siRNA mix, Column 3 (184A1 CM) provides a positive LM332 control. Motility of MCF‐7 cells treated with conditioned media from SAEC transfected with laminin chain siRNA or control siRNA (C), showing significantly reduced motility from CM of siRNA‐treated SAEC cells. siRNA against the α3 chain only on a immunoblot reacted with both anti‐α3 and actin antibodies (D). The α3 siRNA resulted in CM with a reduced ability to induce MCF‐7 migration (E), while the control sequence in the same or double the siRNA concentration did not knockdown α3 chain expression (D) and their CM did not affect motility (E). Error bars show standard errors of the mean. For each experiment, all lanes were run simultaneously on the same gel.