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MEDI3039, a Novel Highly Potent Tumor Necrosis Factor (TNF)-related Apoptosis-Inducing Ligand (TRAIL) Receptor 2 Agonist, Causes Regression of Orthotopic Tumors and Inhibits Outgrowth of Metastatic Triple-Negative Breast Cancer

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MEDI3039, a Novel Highly Potent Tumor Necrosis Factor (TNF)-related Apoptosis-Inducing Ligand (TRAIL) Receptor 2 Agonist, Causes Regression of Orthotopic Tumors and Inhibits Outgrowth of Metastatic Triple-Negative Breast Cancer

Yoshimi Endo Greer et al. Breast Cancer Res.

Abstract

Background: TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists are attractive anti-tumor agents because of their capability to induce apoptosis in cancer cells by activating death receptors (DR) 4 and 5 with little toxicity against normal cells. Despite an attractive mechanism of action, previous clinical efforts to use TRAIL receptor agonists have been unsuccessful. In this study, we examined MEDI3039, a highly potent multivalent DR5 agonist, in breast cancer cell lines and in vivo models.

Methods: As in vitro model systems, we used 19 breast cancer cell lines that are categorized into four subtypes: ER+, HER2 amplified, basal A (triple-negative breast cancer) TNBC, and basal B TNBC. Cell viability was analyzed by MTS and RealTime live/dead assays. As in vivo model systems, MDA-MB231T orthotopic primary tumor growth in the mammary fat pad (MFP) and two experimental lung metastasis models were used. The effect of MEDI3039 on MFP tumors was assessed with immunohistochemical analysis. Lung metastases were analyzed with Bouin's and H&E staining.

Results: MEDI3039 killed multiple breast cancer cell lines, but the sensitivity varied among different subtypes. Sensitivity was basal B TNBC >> basal A TNBC > HER2 amplified > ER+ (average IC50 = 1.4, 203, 314, 403 pM, respectively). While the pattern of relative sensitivity was similar to GST-TRAIL in most cell lines, MEDI3039 was at least two orders of magnitude more potent compared with GST-TRAIL. In the MFP model, weekly treatment with 0.1 or 0.3 mg/kg MEDI3039 for 5 weeks inhibited tumor growth by 99.05% or 100% (median), respectively, compared with the control group, and extended animal survival (p = 0.08 or p = 0.0032 at 0.1 or 0.3 mg/kg, respectively). MEDI3039-induced caspase activation was confirmed in tumors grown in MFP (p < 0.05). In an experimental pulmonary metastasis model, MEDI3039 significantly suppressed outgrowth of surface (p < 0.0001) and microscopic metastases (p < 0.05). In an established lung metastasis model, MEDI3039 significantly inhibited growth of metastases (p < 0.01 in surface [> 4 mm], p < 0.01 in tumor percentage) and extended animal survival (p < 0.0001).

Conclusion: MEDI3039 is a potent DR5 agonist in breast cancer cells in vitro and in vivo and has potential as a cancer drug in breast cancer patients, especially those with basal B TNBC.

Keywords: Agonist; Apoptosis; Breast cancer; Death receptor; TRAIL; Triple-negative breast cancer.

Conflict of interest statement

Ethics approval and consent to participate

All animal experiments were performed under approved NCI Animal Use agreements.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
MEDI3039 induces apoptotic cell death in basal B TNBC MB231 cells. a MEDI3039-induced caspase activation (left) and cell death (right) in MB231 cells. MEDI3039 was at least two orders of magnitude more potent compared with GST-TRAIL. MB231 cells were treated with drugs for 1 h for caspase assays and 72 h for MTS assays. Data is shown as average ± SEM of multiple independent experiments. EC50 and IC50 were obtained by GraphPad Prism Nonlinear regression analysis. b Z-VAD-FMK, a pan caspase inhibitor, completely blocked caspase-3/7 activation (left) and cell killing effect (right) of MEDI3039. Cells were pretreated with Z-VAD-FMK for 60 min before treated with MEDI3039. Data is shown as average ± SEM of multiple independent experiments. ****p < 0.0001, two-way ANOVA. c Z-VAD-FMK completely blocked caspase-3/7 activation (left) and cell killing effect (right) of GST-TRAIL. Data is shown as average ± SEM of multiple independent experiments. ***p < 0.001, ****p < 0.0001, two-way ANOVA
Fig. 2
Fig. 2
MEDI3039-dependent cell death is mediated by DR5. a MB231 cells were transfected with Neg Ctl. siRNA (siNeg), two separate DR4 siRNAs (siDR4 #1 and #3), or two separate DR5 siRNAs (siDR5 #1 and #6). Two days later, MEDI3039 was added and incubated for an additional 3 days. Cell viability was examined by MTS assays. Data is shown as average ± SEM of three independent experiments. ****p < 0.0001, two-way ANOVA, siNeg vs siDR5_#1, siNeg vs siDR5_#6. b The effect of MEDI3039 at 1 pM was compared among siNeg, siDR4, and siDR5. Statistical analysis was performed with Student’s t test. c Representative western blot showing effective knockdown of endogenous DR4 and DR5 proteins by corresponding siRNAs. HSC70 was used as a loading control. d Quantitative analysis of band intensities in western blot analyses. Data is shown as mean ± SEM of three independent experiments, and band intensities are shown as % relative to siNeg ctl
Fig. 3
Fig. 3
MEDI3039 sensitivity is different depending on breast cancer subtypes. IC50 of MEDI3039 (a) and IC50 of GST-TRAIL (b) were obtained by GraphPad Prism Nonlinear regression analysis of multiple MTS assays with 3 days of cell culture (at least three times per cell line). c Correlation of IC50 between MEDI3039 and GST-TRAIL in 15 breast cancer cell lines. Note that BT474 and T47D were out of scale because IC50 could not be determined due to resistance to MEDI3039 and GST-TRAIL
Fig. 4
Fig. 4
MEDI3039 inhibited tumor growth and extended animal survival in the MB231T mammary fad pad model. a Design of the experiment. MEDI3039 was administered weekly, for 5 weeks at indicated doses. Five mice in the control and 0.3 mg/kg group were sacrificed for histology analysis and 10 mice per group were followed for tumor growth and survival. b Immunohistochemistry analysis of tumor samples from MEDI3039 (0.3 mg/kg) or vehicle-injected mice. Samples were stained with either Ki67 and DAPI or CC3 (cleaved caspase 3) and DAPI. Bar = 100 μm. c Quantitative analysis of signal intensity of Ki67 and CC3, both normalized with DAPI. Data is shown as median with IQR. Numbers of mice examined was 5 (vehicle control.) and 4 (MEDI3039 0.3 mg/kg). p value was obtained by Mann–Whitney test. d Tumor growth curve in each treatment group. The bottom panel shows averages for all mice at each dose. One-way ANOVA was used to compare statistical significance between different groups. e Tumor growth inhibition curve. Data is shown as median % tumor inhibition of 0.03, 0.1, and 0.3 mg/kg MEDI3039 groups, compared with the vehicle control group. f Survival curve of mice treated with MEDI3039 at indicated doses. Median survival was 38 days (vehicle control group), 44 days (MEDI3039, 0.03 mg/kg group), 74 days (MEDI3039, 0.1 mg/kg group), 79.5 days (MEDI3039, 0.3 mg/kg group). p value, HR (hazard ratio), and 95% of CI (confidence interval) were obtained by log-rank (Mantel–Cox) test, compared with the vehicle control group
Fig. 5
Fig. 5
MEDI3039 prevented tumor metastasis outgrowth and extended animal survival in the MB231T experimental pulmonary metastasis model. a Design of the experiment. MEDI3039 (0.3 mg/kg) or vehicle was administered twice weekly, for 2 weeks. b Mice lung tissue from both vehicle- and MEDI3039-treated groups, fixed with Bouins’ solution. c Quantitative analysis of metastasized tumors in the lung. Total numbers of surface metastases (left) and large (> 3 mm) metastases (right) are shown. Data is presented as median with IQR. d Survival curve of mice treated with MEDI3039 (0.3 mg/kg) or vehicle. p value, HR, and 95% of CI were obtained with log-rank (Mantel–Cox) test, compared with the vehicle control group. e Representative images of H&E-stained lung tissue from vehicle- or MEDI3039-treated mouse. Scatter dot graph on the right shows quantitative analysis of microscopic tumors in the lung. Note that the vehicle group has only four samples, while the MEDI3039 group has five samples. One mouse in the vehicle group developed large confluent tumors covering the entire lung, making it difficult to count tumor numbers; therefore, the result is not included in the graph. Data is presented as median with IQR
Fig. 6
Fig. 6
MEDI3039 inhibited established lung metastases and extended animal survival in the MB231T lung metastasis model. a Survival curve of mice treated with MEDI3039 (0.3 mg/kg) or vehicle. p value, HR, and 95% of CI were obtained with log-rank (Mantel–Cox) test, compared with the vehicle control group. The lung from one of two sentinel mice sacrificed is shown in the inset above the graph demonstrating grossly visible established lung metastases at day 21. b Mice lung tissue from both vehicle- and MEDI3039-treated groups, fixed with Bouins’ solution. c Quantitative analysis of metastasized tumors in the lung. The number of tumors was classified according to their size. Data is presented as mean ± SEM, and Student’s t test was used for the statistical analysis. The red bars represent MEDI3039-treated mice that were sacrificed prior to the end of the experiment. The green bars represent data from 4 mice still alive at the end of the experiment. d Two representative H&E-stained lung samples per vehicle- or MEDI3039-treated group. Top: H&E-stained lung tissue. Bottom: red-colored area indicates tumor region, green-colored area indicates non-cancerous region. Areas circled with light dotted green line indicate the diaphragm and lymph nodes excluded from analysis. e Quantitative analysis of microscopic tumors in the lung. Data is presented as median area of lung involved with tumor with IQR. Note that all mice in vehicle groups were dead or euthanized on day 72 or 73, while all 5 mice in MEDI3039 were alive and euthanized at day 90

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References

    1. Prat A, Perou CM. Deconstructing the molecular portraits of breast cancer. Mol Oncol. 2011;5(1):5–23. doi: 10.1016/j.molonc.2010.11.003. - DOI - PMC - PubMed
    1. Brenton JD, Carey LA, Ahmed AA, Caldas C. Molecular classification and molecular forecasting of breast cancer: ready for clinical application? J Clin Oncol. 2005;23(29):7350–7360. doi: 10.1200/JCO.2005.03.3845. - DOI - PubMed
    1. Hurvitz S, Mead M. Triple-negative breast cancer: advancements in characterization and treatment approach. Curr Opin Obstet Gynecol. 2016;28(1):59–69. - PubMed
    1. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol. 2016;13(11):674–690. doi: 10.1038/nrclinonc.2016.66. - DOI - PMC - PubMed
    1. Cancer Genome Atlas N Comprehensive molecular portraits of human breast tumours. Nature. 2012;490(7418):61–70. doi: 10.1038/nature11412. - DOI - PMC - PubMed

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