Targeting mutant p53 protein and the tumor vasculature: an effective combination therapy for advanced breast tumors

Abstract

Breast cancer progression depends upon the elaboration of a vasculature sufficient for the nourishment of the developing tumor. Breast tumor cells frequently contain a mutant form of p53 (mtp53), a protein which promotes their survival. The aim of this study was to determine whether combination therapy targeting mtp53 and anionic phospholipids (AP) on tumor blood vessels might be an effective therapeutic strategy for suppressing advanced breast cancer. We examined the therapeutic effects, singly, or in combination, of p53 reactivation and induction of massive apoptosis (PRIMA-1), which reactivates mtp53 and induces tumor cell apoptosis, and 2aG4, a monoclonal antibody that disrupts tumor vasculature by targeting AP on the surface of tumor endothelial cells and causes antibody-dependent destruction of tumor blood vessels, leading to ischemia and tumor cell death. Xenografts from two tumor cell lines containing mtp53, BT-474 and HCC-1428, were grown in nude mice to provide models of advanced breast tumors. After treatment with PRIMA-1 and/or 2aG4, regressing tumors were analyzed for vascular endothelial growth factor (VEGF) expression, blood vessel loss, and apoptotic markers. Individual drug treatment led to partial suppression of breast cancer progression. In contrast, combined treatment with PRIMA-1 and 2aG4 was extremely effective in suppressing tumor growth in both models and completely eradicated approximately 30% of tumors in the BT-474 model. Importantly, no toxic effects were observed in any treatment group. Mechanistic studies determined that PRIMA-1 reactivated mtp53 and also exposed AP on the surface of tumor cells as determined by enhanced 2aG4 binding. Combination treatment led to significant induction of tumor cell apoptosis, loss of VEGF expression, as well as destruction of tumor blood vessels. Furthermore, combination treatment severely disrupted tumor blood vessel perfusion in both tumor models. The observed in vitro PRIMA-1-induced exposure of tumor epithelial cell AP might provide a target for 2aG4 and contribute to the increased effectiveness of such combination therapy in vivo. We conclude that the combined targeting of mtp53 and the tumor vasculature is a novel effective strategy for combating advanced breast tumors.

Fig. 1

a PRIMA-1 converts the conformation of mtp53 in breast cancer cells into the wtp53 form. BT-474 and HCC-1428 cells were grown in 8-well chamber slides and treated with 25-μM (PM25) or 50-μM (PM50) PRIMA-1 for 3 and 8 h. Conformation-specific antibodies and imaging procedures were used as described in the “Materials and methods” section. Nuclei are stained with DAPI (blue), p53 is stained red, and in the merged images, nuclear p53 is pink. b PRIMA-1 exposes AP on the surface of breast cancer cells in vitro. Breast cancer cells were grown in 8-well chamber slides and treated with 5, 10, or 25 μM PRIMA-1 for 24 h at 37°C. Exposure of AP was examined by live-cell immunofluorescence double-staining as described in the “Materials and methods” section. Externalized AP were labeled with 2aG4 and are shown in red (rhodamine), the cytoskeleton is in green (FITC), and the overlap in the merged images ranges from yellow to orange based on the intensity captured. c Weak exposure of AP induced by PRIMA-1 on HUVEC cells. HUVEC were treated with PRIMA-1 as in (b)

Fig. 2

a Treatment protocol for human breast tumor xenografts in nude mice. b Additive effect of PRIMA-1 and 2aG4 on inhibition of BT-474 breast tumor growth in nude mice. Mice (n = 9 per group) were inoculated with 5 × 10⁶ BT-474 cells in both flanks. Tumors were measured every 3 days with a digital caliper to determine volume. (Left) Growth curves for tumors with points representing mean tumor volumes ± SEM. The combination therapy was significantly more effective than either single treatment alone. *P < 0.05 compared with the C44 group, **P < 0.05 compared with the C44 and 2aG4 groups. (Top right) Tumor weights at the end of the experiment confirming tumor volumes. (Bottom right) Animal weight following treatment. c Tumor images in situ representing loss of tumor growth in nude mice following treatment. Images show tumor size before and after 4 weeks of treatment. d Eradication of BT-474 tumors in nude mice following PRIMA-1 + 2aG4 treatment. Images in situ show that some tumors were eradicated from the original tumor sites. Numbers of tumor-free animals after 4 weeks of treatment is shown in the bar graph

Fig. 3

a–d Enhanced inhibition of HCC-1428 breast tumor growth by PRIMA-1 plus 2aG4 combination therapy. Nude mice (n = 8 per group) were injected with HCC-1,428 cells, and tumor growth was measured as described in the “Materials and methods” section. The treatment protocol was identical to that described in Fig. 2a except the PRIMA-1 dose was increased to 75 mg/kg/day. a Tumor growth curve. Points represent mean tumor volumes ± SEM in each group of mice; b bar graph representing tumor weight from the experiment shown in (a). c Representative in situ tumor image from each group at the end of the experiment. Circles represent tumor locations. d Graph of animal weight. Values represent mean ± SEM; *P < 0.05 compared with the C44 group, **P < 0.05 compared with the C44, PRIMA-1 and 2aG4 groups

Fig. 4

a, b Tumor blood vessel perfusion assay using FITC-dextran in vivo. Following 3 weeks of treatment with PRIMA-1 and 2aG4, tumor-bearing animals were injected (iv) with 0.2 ml of 25 mg/ml FITC-dextran (molecular weight 2,000,000) for 20 min. Tumors and whole blood were collected, processed, and centrifuged. Fluorescence was measured in supernatant from tumor tissue and whole body plasma. The ratio of tumor/plasma fluorescence reflects the extent of tumor perfusion. Samples were analyzed from four animals per group. Points indicate mean volumes in each group of mice, bars indicate SEM. *P < 0.05 compared with the C44 group, **P < 0.05 compared with the C44, PRIMA-1, and 2aG4 groups

Fig. 5

a–c Immunohistochemical analysis of tumor tissue for angiogenesis markers. a At the termination of the experiment, tumor tissue sections were stained for VEGF and CD-34 (biomarkers for angiogenesis-related genes). Results demonstrated a significant reduction of both molecules following combination therapy. b VEGF expression was quantified using the Fovea Pro 3.0 imaging program (Fovea Pro 3.0, Reindeer Graphics, Asheville, NC). Eight to twelve images from three to five tumors per treatment group were analyzed for VEGF. c CD-34-based vessel count. Blood vessels were counted in 8–15 individual images from three to four tumors per treatment group as described in the “Materials and methods” section

Fig. 6

a Induction of tumor cell apoptosis in xenografts by PRIMA-1, 2aG4, or the combination. Animals with BT-474 xenografts were treated with PRIMA-1 and 2aG4 alone or in combination for 4 weeks; the last treatment was administered 16 h prior to kill and tumor collection. Frozen tissue sections were processed for fluorescence double-staining as described in the “Materials and methods” section. Green TUNEL staining, Blue nuclear staining of tumor cells with DAPI. Merged colors show intense apoptosis in xenograft tissue in the presence of PRIMA-1 and 2aG4. b Induction of apoptosis in tumor endothelial cells. Animals with BT-474 xenografts were treated with PRIMA-1 and 2aG4 alone or in combination for 3 weeks. Frozen tissue sections were processed as described in (a). Red endothelial cell staining, Green TUNEL staining, Blue nuclear staining of tumor cells with DAPI. Merged colors do not show apoptosis in endothelial cells under any treatment. c Immunohistochemical staining of apoptotic markers (caspase-3 and p21) in tumor tissues. H&E staining of sections from both tumor models showed an increase in morphologically apoptotic cells in all the three treatment groups, but apoptotic cells were especially abundant following combination treatment (upper panels). Tumor tissues from experiment described in (a) were collected and embedded in paraffin. Staining was performed as described in the “Materials and methods” section. d Western blot analysis to assess Bcl-2 levels in tumors. Mice with BT-474 tumors were treated with PRIMA-1 and 2aG4 alone or in combination for 4 weeks, the last treatment occurring 16 h prior to kill. Tumor tissues were harvested, homogenized, and whole cell extracts were prepared. Protein (50 μg per group) was loaded onto NuPAGE 10% Bis–Tris Gel. The bar graph shows densitometric analysis of Bcl-2 levels normalized to β-actin internal controls. Results from two independent experiments are shown. Bar graph also shows densitometric analysis of Bcl-2 levels normalized to β-actin intensity (n = 3)