Breast cancer cells in three-dimensional culture display an enhanced radioresponse after coordinate targeting of integrin alpha5beta1 and fibronectin

Abstract

Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that beta1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific alpha heterodimer of beta1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare alpha-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of alpha5beta1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of alpha5beta1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of alpha5beta1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of alpha5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and alpha5beta1-integrin as targets for breast cancer therapy.

Figure 1

α5β1-Integrin heterodimers are strikingly upregulated in malignant T4-2 cells compared with nonmalignant S1 cells in 3D lrECM. A, Western blot for α2-, α3-, α5-integrin subunits from total cell lysates shows upregulation of α5-integrin in malignant T4-2 cells compared with nonmalignant S1 cells cultured in 3D lrECM. Equal amounts of protein were loaded and subjected to immunoblotting. Columns, mean intensity of Western blot analysis (n = 4); bars, SEM. ***, P < 0.001. B, α5β1-integrin complex are upregulated in T4-2 cells compared with S1 cells cultured in 3D lrECM. Lysates were subjected to immunoprecipitation with anti–β1-integrin and subsequent immunoblotting using antibodies, as indicated. IP, immunoprecipitation. C, elevated α5-integrin gene expression is associated with significantly decreased long-term survival in patients with breast cancer. A Kaplan-Meier survival analysis of 295 human breast cancers stratified by ITGA5 expression is shown. The highest quartile of ITGA5 expression is significantly associated with decreased survival. P values (log-rank) between upper and lower or interquartile are <0.007.

Figure 2

Upregulation of fibronectin and its splice variant EDA+ are associated with coordinate upregulation of α5β1-integrin in malignant breast cancer cells in 3D lrECM. A, upregulation of α5-integrin is not dependent on activation by ECM ligands on two-dimensional culture but required three-dimensional culture conditions. B, fibronectin (FN) secretion is upregulated in T4-2 cells compared with S1 cells in 3D lrECM. Twenty microliters of conditioned-medium (CM) from S1 or T4-2 cells were subjected to immunoblotting using fibronectin antibodies. The signals of immunoblotting were normalized with total protein of S1 or T4-2 cell lysate. Columns, mean (n = 3); bars, SEM. **, P < 0.01; ***, P < 0.001. C, quantification of mRNA levels of total and EDA+ fibronectin in S1 and T4-2 cells cultured in 3D lrECM assessed by real-time PCR. The levels of total fibronectin are relative to 18S rRNA. Columns, mean (n = 3); bars, SEM. *, P < 0.05; **, P < 0.01. D, coordinate upregulation of α5β1-integrin and total and EDA+ fibronectin occurs in several highly aggressive metastatic breast cell lines, MDA-MB-436, MDA-MB-231, and Hs578T. Lower levels were observed in less aggressive cell lines, SK-BR-3, MDA-MB-361, and MCF7. Immunofluorescence microscopy shows morphologies of breast cell lines cultured in 3D lrECM. Green, filamentous actin; red, nuclei. Scale bar, 50 μm.

Figure 3

Specific inhibition of the α5β1-integrin-fibronectin interaction induces apoptosis in malignant breast cancer cells in 3D lrECM. A, experimental schema. B, phase-contrast micrographs of S1 and T4-2 cell colonies cultured in 3D lrECM with ATN-161 are shown. Nuclei were stained with 4′,6-diamidino-2-phenylindole (insets). Scale bar, 50 μm. C, T4-2 colonies, but not S1 structures, showed a significant increase in apoptosis, measured by TUNEL assay. Columns, mean (n = 3); bars, SEM. **, P < 0.01; ***, P < 0.001; n.s., not significant. D, specific inhibition of α5β1-integrin-fibronectin interaction using anti–α5-integrin inhibitory antibodies, IIA1 and P1D6, induces apoptosis in T4-2 cells, but not S1 cells, cultured in 3D lrECM. Apoptosis was measured by TUNEL assay. Columns, mean (n = 3); bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4

Akt activity mediates survival downstream of α5β1-integrin pathway in malignant T4-2 cells in 3D lrECM. A, Akt kinase activity was downregulated by inhibition of α5β1-integrin and fibronectin interactions. Cells were treated with or without 0.5 mg/mL ATN-161. Akt kinase activity was measured by phosphorylation of the GSK-3α/β fusion protein. Columns, mean (n = 3); bars, SEM. **, P < 0.01. B, ATN-161 treatment (0.5 mg/mL) of T4-2 cells in three-dimensional culture resulted in decreased expression of α5- and β1-integrins and pAkt. The level of phosphorylated Akt relative to total Akt is shown. Columns, mean (n = 3); bars, SEM. ***, P < 0.001. C, ATN-161–induced apoptosis is totally suppressed in T4-2 cells stably transfected with a constitutively active form of Akt (T4-2 myr-Akt). Columns, mean (n = 3); bars, SEM. ***, P < 0.001.

Figure 5

α5β1-Integrin heterodimer is upregulated after IR exposure in malignant T4-2 cells in 3D lrECM. A, cell surface expression of α5-integrins is upregulated after 2-4 Gy IR exposures in T4-2 cells cultured in 3D lrECM. Surface proteins of T4-2 cells were labeled with biotin and cell lysates were subjected to immunoprecipitation of α5-integrin and subsequent immunoblotting using HRP-conjugated streptavidin. Cell surface expression of α5-integrins was measured using streptavidin blots. Columns, mean (n = 3); bars, SEM. *, P < 0.05. B, immunofluorescence confocal localization of α5-integrins after IR exposure in T4-2 cells in 3D lrECM is shown. Increased cell surface α5-integrin expression is detected after 2 to 4 Gy IR. Green, α5-integrin; red, nuclei. Scale bar, 20 μm.

Figure 6

IR exposure in combination with α5β1-integrin inhibition enhances apoptosis in T4-2 and MDA-MB-231 breast cancer cell lines in 3D lrECM. A, experimental schema. Apoptosis of breast cancer cells was measured by TUNEL assay. B, IR exposure in combination with α5β1-integrin inhibition enhances apoptosis in T4-2 cells in 3D lrECM compared with either ATN-161 or IR alone. Columns, mean (n = 3); bars, SEM. *, P < 0.05. C, IR exposure followed by α5β1-integrin inhibition enhances apoptosis in MDA-MB-231 cells in 3D lrECM. Columns, mean (n = 3); bars, SEM. *, P < 0.05; **, P < 0.01.