Beta1 integrin inhibition dramatically enhances radiotherapy efficacy in human breast cancer xenografts

Abstract

Beta(1) integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of beta(1) integrin signaling. We previously showed that beta(1) integrin inhibitory antibodies (e.g., AIIB2) enhance apoptosis and decrease growth in human breast cancer cells in three-dimensional laminin-rich extracellular matrix (lrECM) cultures and in vivo. Here, we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used three-dimensional lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in three-dimensional lrECM. Colonies were assayed for apoptosis and beta(1) integrin/Akt signaling pathways were evaluated using Western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in three-dimensional lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down-regulating Akt in breast cancer colonies in three-dimensional lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared with either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We previously showed that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.

Figure 1

β₁ Integrin inhibition enhances apoptosis post-IR in an Akt-dependent manner in T4-2 breast cancer cells but not S-1 structures in three-dimensional lrECM. A, S-1 nonmalignant structures were treated with IR (0, 2, or 8 Gy) with or without AIIB2 in three-dimensional lrECM. The percentage of apoptotic cells was not significantly changed despite IR alone, AIIB2 alone, or a combination of IR plus AIIB2. Columns, mean (n = 3); bars, SE. B, Western blot from total cell lysates shows no up-regulation in p-AKT after IR or down-regulation after addition of AIIB2. Quantitation is relative to β-actin. C, T4-2 control colonies were treated with IR (0, 2, or 8 Gy) with or without AIIB2 in three-dimensional lrECM. The percentage of apoptotic cells significantly increased with the addition of AIIB2 post-IR by >80%. Columns, mean (n = 3); bars, SE; *, P < 0.05, χ². D, Western blot for phosphorylated S473 Akt 6 h after treatment showed an IR-dose dependent increase in Akt activity (two times post 8 Gy IR) that is down-regulated post-IR with the addition of AIIB2. Quantitation is relative to β-actin.

Figure 2

β₁ Integrin inhibition optimally enhances apoptosis post-IR in MCF-7 breast cancer cells in three-dimensional lrECM. A, experimental schema: MCF-7 cells were allowed to form malignant colonies in three-dimensional lrECM for 3 d and then were treated with IR ± AIIB2 as shown. B, apoptosis was measured by counting TUNEL-positive nuclei. Columns, mean (n = 3); bars, SE *, P < 0.05, χ². C, total levels of AKT and pS473 Akt were detected by Western blotting of total cell lysates. Quantitation is relative to β-actin.

Figure 3

Akt-mediated resistance to apoptosis post-IR is abrogated by AIIB2 in a dose-dependent manner. A, Western blot shows the presence of constitutively active Akt in transfected T4-2 cells versus vector controls. B, T4 myr-AKT cells are resistant to apoptosis after IR and 0.08 mg/mL AIIB2. Columns, mean; bars, SE. C, apoptosis is significantly increased with doses of 0.24 mg/mL AIIB2 and greater (three times the doses necessary to induce apoptosis in vector control cells). Columns, mean; bars, SE. *, P < 0.05, χ². D, Western blot (and quantification) of phospho-Akt and phospho-Thr1462 TSC2 was increased with IR or AIIB2 alone, but decreased when treated with a combination of 0.24 mg/mL of AIIB2 and IR.

Figure 4

IR (2 Gy) followed by AIIB2 is more effective than AIIB2 or 2 Gy alone. A, experimental schema: MCF-7 xenograft-bearing mice (n = 14) were randomized to receive either IR plus control antibody, IR plus AIIB2, or AIIB2 plus IR. B, comparison of tumor growth curves shows that 2 Gy IR→AIIB2 resulted in greater time to tumor progression (400 mm³) than 2 Gy alone (*, log-rank P < 0.014) and was similar in efficacy to 8 Gy alone. C, Western blot shows that IR plus AIIB2 is associated with down-regulation of p-S473Akt activity in vivo.