Beta1 integrin inhibitory antibody induces apoptosis of breast cancer cells, inhibits growth, and distinguishes malignant from normal phenotype in three dimensional cultures and in vivo

Abstract

Current therapeutic approaches to cancer are designed to target molecules that contribute to malignant behavior but leave normal tissues intact. beta(1) integrin is a candidate target well known for mediating cell-extracellular matrix (ECM) interactions that influence diverse cellular functions; its aberrant expression has been implicated in breast cancer progression and resistance to cytotoxic therapy. The addition of beta(1) integrin inhibitory agents to breast cancer cells at a single-cell stage in a laminin-rich ECM (three-dimensional lrECM) culture was shown to down-modulate beta(1) integrin signaling, resulting in malignant reversion. To investigate beta(1) integrin as a therapeutic target, we modified the three-dimensional lrECM protocol to approximate the clinical situation: before treatment, we allowed nonmalignant cells to form organized acinar structures and malignant cells to form tumor-like colonies. We then tested the ability of beta(1) integrin inhibitory antibody, AIIB2, to inhibit tumor cell growth in several breast cancer cell lines (T4-2, MDA-MB-231, BT474, SKBR3, and MCF-7) and one nonmalignant cell line (S-1). We show that beta(1) integrin inhibition resulted in a significant loss of cancer cells, associated with a decrease in proliferation and increase in apoptosis, and a global change in the composition of residual colonies. In contrast, nonmalignant cells that formed tissue-like structures remained resistant. Moreover, these cancer cell-specific antiproliferative and proapoptotic effects were confirmed in vivo with no discernible toxicity to animals. Our findings indicate that beta(1) integrin is a promising therapeutic target, and that the three-dimensional lrECM culture assay can be used to effectively distinguish malignant and normal tissue response to therapy.

Figure 1

Morphology and response of breast cell lines to β₁ integrin inhibition in three-dimensional lrECM. Five malignant breast cell lines (T4-2, MDA-MB-231, SKBR3, BT474, and MCF-7) and one nonmalignant breast cell line (S-1) were propagated in three-dimensional lrECM and treated with β₁ integrin inhibitory antibody, AIIB2, after colonies were formed. A, schema of the three-dimensional lrECM assay used in this study. B, top and middle, phase-contrast micrographs of cultured colonies before and after antibody treatment, respectively. Bar, 20 µm. Bottom, confocal midsections of β₁ integrin immunofluorescence () of colonies cultured in three-dimensional lrECM. Phalloidin was used to stain actin filaments (), and DAPI was used to stain individual nuclei (). Bar, 13 µm for all cell lines shown. C, whereas there were no significant changes in Ki-67 or TUNEL expressing cells among nonmalignant S-1 cells after β₁ integrin inhibition, malignant cell lines, except SKBR3, had a significant decrease in Ki-67 expressing cells and a significant increase in TUNEL-positive cells after antibody treatment. Columns, mean (n = 3); bars, SE. P < 0.05, t test.

Figure 2

β₁ Integrin, p-β₁ integrin, and p-³⁹⁷FAK are expressed at different levels in breast cell lines in three dimensions. Lysates from five breast cancer cell lines (T4-2, MDA-MB-231, SKBR3, BT474, and MCF-7) and one nonmalignant cell line (S-1) in three-dimensional lrECM were probed by Western immunoblotting for total and p-β₁ integrin and p-³⁹⁷FAK and for surface β₁ integrin expression by FACS analysis. A, total β₁ integrin levels are relatively high in S-1, T4-2, and MDA-MB-231 cells compared with SKBR3, BT474, and MCF7 cells. Conversely, p-β₁ integrin levels are relatively low in S-1, T4-2, and MDA-MB-231 cells compared with SKBR3, BT474, and MCF7 cells. Expression of p-³⁹⁷FAK is expressed among all cell lines except SKBR3, where it is undetectable by Western blots. B, surface expression of β₁ integrin by FACS analysis corresponds to that seen by immunofluorescence (Fig. 1B) and Western blots (Fig. 1A).

Figure 3

AIIB2 reduces both total cell number and average colony size in malignant cell lines. A, average number of T4-2 cells after 3 days of treatment with AIIB2. Columns, mean; bars, SE. P < 0.05, χ². B, histogram showing the average colony size decreased with AIIB2 compared with controls. Columns, mean; bars, SE. P < 0.05, t test. C, percentage of Ki-67 and TUNEL expressing nuclei among T4-2 cells 1, 24, and 72 hours after addition of AIIB2 to cultures. Columns, mean; bars, SE. P < 0.05, t test.

Figure 4

AIIB2 suppresses tumor growth in vivo by enhancing apoptosis and decreasing proliferation. A, mean tumor volumes among animals that received 1 mg/kg AIIB2 (■) or 5 mg/kg AIIB2 (△) were significantly smaller than control animals (♦). Points, mean (n = 9); bars, SE. A′, average serum concentration (µg/mL) of AIIB2 at the time of sacrifice was significantly higher in animals receiving AIIB2 compared with control vehicle. Columns, mean; bars, SD. A″, average number of animals in each treatment group that had histologic evidence of tumor at the time of sacrifice was significantly lower among treated animals compared to controls. Columns, mean for three separate experiments; bars, SE. *, P < 0.03, χ². B, micrographs of serial sections of tumors that were scored for apoptosis by the presence of TUNEL-positive nuclei and for proliferation by the presence of Ki-67 nuclear antigen. Black ovals and triangles, coregistered regions in serial sections; arrows, examples of positive staining. B′, with AIIB2 treatment, the average number of TUNEL nuclei increased, and the number of Ki-67–positive nuclei decreased. Columns, mean; bars, SE. *, P < 0.05, χ². Bar, 247 µm.

Figure 5

AIIB2 effectively induces cytostasis in established MCF-7 tumors in vivo with no toxicity. A, animals were randomized to receive treatment or control IgG 4 weeks after MCF-7 cell implantation (arrow, time of randomization at day 30). Mean tumor volumes for animals receiving 1 mg/kg AIIB2 (■) and 5 mg/kg AIIB2 (△) and nonspecific rat IgG1 (♦). Points, mean (n = 8); bars, SE. B, compared with controls, tumors from animals that received AIIB2 treatment had significantly fewer Ki-67–positive nuclei and fewer TUNEL-positive nuclei. Columns, mean; bars, SE. *, P < 0.001, χ². C, average weight of control animals, and animals that received AIIB2 treatment were not significantly different at any biweekly time point measured throughout the experiment (data not shown) and at the end of the experiment. Columns, mean; bars, SE.