Inhibition of Breast Cancer Metastasis by Presurgical Treatment with an Oral Matrix Metalloproteinase Inhibitor: A Preclinical Proof-of-Principle Study

Abstract

Breast cancer has the second highest death toll in women worldwide, despite significant progress in early diagnosis and treatments. The main cause of death is metastatic disease. Matrix metalloproteinases (MMP) are required for the initial steps of metastasis, and have therefore been considered as ideal pharmacologic targets for antimetastatic therapy. However, clinical trials of MMP inhibitors were unsuccessful. These trials were conducted in patients with advanced disease, beyond the stage when these compounds could have been effective. We hypothesized that early treatment with a selective MMP inhibitor between the time of diagnosis and definitive surgery, the so-called "window-of-opportunity," can inhibit metastasis and thereby improve survival. To investigate our hypothesis, we used the 4T1 mouse model of aggressive mammary carcinoma. We treated the animals with SD-7300, an oral inhibitor of MMP-2, -9, and -13, starting after the initial detection of the primary tumor. Seven days later, the primary tumors were excised and analyzed for MMP activity, and the SD-7300 treatment was discontinued. After 4 weeks, the animals were sacrificed and their lungs analyzed histologically for number of metastases and metastatic burden (metastases' area/lung section area). SD-7300 treatment inhibited 70% to 80% of tumor-associated MMP activity (P = 0.0003), reduced metastasis number and metastatic burden by 50% to 60% (P = 0.002 and P = 0.0082, respectively), and increased survival (92% vs. 66.7%; P = 0.0409), relative to control vehicle. These results show that treatment of early invasive breast cancer with selective MMP inhibitors can lower the risk of recurrence and increase long-term disease-free survival. Mol Cancer Ther; 15(10); 2370-7. ©2016 AACR.

Figure 1. Study design

Schematic representation of the experimental protocol for assessing the anti-metastatic effect of presurgical MMP inhibition in the window of opportunity. A. Characterization of SD-7300 inhibition of the target MMP activity in the primary tumor. Four groups of 3 mice were injected with 4T1-Luc cells. Forty-eight hours later 3 groups received the indicated doses of SD-7300, and 1 group received control vehicle. After 7 days of treatment (day 10) the tumors (100–150 mm³ in size) were excised and assayed for MMP-9 activity as representative of the three target MMPs. B. Analysis of the effect of presurgical administration of SD-7300 on lung metastasis. Two groups of 10 mice (experiment 1) or 15 mice (experiment 2) were injected with 4T1-Luc cells. Forty-eight hours later 1 group received 30 mg/kg of SD-7300, and 1 group received control vehicle, BID by gavage. Seven days later (day 10) the treatment was discontinued, the tumors were excised and their weight was measured. The mice were imaged on the indicated days. On day 38 the mice were sacrificed, the lungs harvested and analyzed for number of metastases and metastatic burden as described in Materials and Methods.

Figure 2. Effect of SD-7300 on tumor associated MMP-9 activity

Mice were treated as described in the legend to Fig. 1A, and MMP-9 activity was measured as described in Materials and Methods. The histograms show MMP-9 activity normalized to μg of tumor lysate protein. Mean ± standard error of triplicate samples are shown. *: P = 0.0003 by one-way ANOVA (each SD-7300 sample vs. control). ^#: P = 0.1453 by one-way ANOVA (difference between SD-7300 samples).

Figure 3. Effect of presurgical administration of SD-7300 on local recurrence and distant metastasis after surgical excision of the primary tumor

IVIS imaging of mice at the indicated times after injection of luciferase-labeled 4T1 cells. Day 9: primary tumors after 7 days of treatment with SD-7300 or control vehicle. Day 16: local relapse 6 days after surgical excision of the primary tumor. Day 38: local relapse and metastasis 28 days after surgical excision of the primary tumor. One mouse in the vehicle group died within 24 h after tumor cell injection. The order of the mice in each row within the vehicle or SD-7300 groups is not the same as in the other rows.

Figure 4. Histological analysis of lung metastasis

Representative images of H&E stained sections of the lungs 28 days after surgical excision of the primary tumor. Similar images could be seen in lung sections from both control and SD-7300-treated mice. The 4T1 tumor metastases can be readily identified by their intense purple staining. Panel A shows lungs devoid of metastases. In the other panels arrows point to metastases of different sizes, from small nidi of tumor cells (panel B) to larger lesions. In panels E and F very large intraparenchymal metastases (arrows) can be seen in addition to extraparenchymal tumor (arrowheads). Size bars: A, D, E, F: 1 mm; B: 400 μm; C: 100 μm.

Figure 5. Effect of presurgical administration of SD-7300 on (A) number of metastases per lung, and (B) metastatic burden

The histograms show mean ± standard deviation of (A) 5 control and 10 SD-7300 treated mice (*: P = 0.002), and (B) 15 control and 15 SD-7300 treated mice (**: P = 0.0082).

Figure 6. Effect of presurgical SD-7300 treatment on primary tumor size, incidence of lung metastasis and survival

A. Primary tumor weight. Mean ± standard deviation of 24 control and 25 SD-7300 treated mice. P = 0.1394 by two-tailed unpaired t test. B. Incidence of lung metastasis. The histograms show the fraction of mice free of lung metastasis in the control and SD-7300 groups. P = 0.1117 by two-tailed Fisher’s exact test. C. Kaplan-Meier survival analysis. Mice surviving at the end of the experiment: control, 16/24 (66.7%); SD-7300 treated, 23/25 (92%; P = 0.0409).