Oseltamivir phosphate monotherapy ablates tumor neovascularization, growth, and metastasis in mouse model of human triple-negative breast adenocarcinoma

Abstract

Background

Triple-negative breast cancers (TNBCs) lack the estrogen, progesterone, and epidermal growth factor (EGF) receptor-2 (HER2/neu) receptors. Patients with TNBC have typical high grading, more frequent relapses, and exhibit poorer outcomes or prognosis compared with the other subtypes of breast cancers. Currently, there are no targeted therapies that are effective for TNBC. Preclinical antitumor activity of oseltamivir phosphate (OP) therapy was investigated to identify its role in tumor neovascularization, growth, invasiveness, and long-term survival in a mouse model of human TNBC.

Methods

Live cell sialidase, water soluble tetrazolium, WST-1 cell viability, and immunohistochemistry assays were used to evaluate sialidase activity, cell survival, and the expression levels of tumor E-cadherin, N-cadherin, and host endothelial CD31+/PECAM-1 cells in archived paraffin-embedded TNBC MDA-MB-231 tumors grown in RAGxCγ double mutant mice.

Results

OP, anti-Neu1 antibodies, and matrix metalloproteinase-9-specific inhibitor blocked Neu1 activity associated with EGF-stimulated TNBC MDA-MB-231 cells. OP treatment of MDA-MB-231 and MCF-7 cells and their long-term tamoxifen-resistant clones reproducibly and dose-dependently reduced the sialidase activity associated with EGF-stimulated live cells and the cell viability after 72 hours of incubation. Combination of 1 μM cisplatin, 5-FU, paclitaxel, gemcitabine, or tamoxifen with OP dosages ≥300 μg/mL significantly reduced cell viability at 24, 48, and 72 hours when compared to the chemodrug alone. Heterotopic xenografts of MDA-MB-231 tumors developed robust and bloody tumor vascularization in RAG2xCγ double mutant mice. OP treatment at 30 mg/kg daily intraperitoneally reduced tumor vascularization and growth rate as well as significantly reduced tumor weight and spread to the lungs compared with the untreated cohorts. OP treatment at 50 mg/kg completely ablated tumor vascularization, tumor growth and spread to the lungs, with significant long-term survival at day 180 postimplantation, tumor shrinking, and no relapses after 56 days off-drug. OP 30 mg/kg cohort tumors expressed significantly reduced levels of human N-cadherins and host CD31+ endothelial cells with concomitant significant expression of E-cadherins compared to the untreated cohorts.

Conclusion

OP monotherapy may be the effective treatment therapy for TNBC.

Keywords:

• triple-negative breast cancer
• chemoresistance

Acknowledgments

This work was supported in part by grants to MR Szewczuk and RJ Neufeld from the Natural Sciences and Engineering Research Council of Canada and private sector cancer funding from the Josefowitz Family to MR Szewczuk. The cancer contents of this report are covered by a Patent Cooperation Treaty international patent application number, PCT/CA2011/050690 (filing November 4, 2011).

We thank Lacey Brodhagen and Alexandra Morris, veterinarian technologists, for their expertise and assistance with the animal work.

Author contributions

F Alghamdi and S Allison performed the sialidase assay; L Brodhagen and S Allison performed the WST-1 assay; F Haxho performed the immunohistochemistry and the immunostaining for host CD31+ cells in tumor tissues and the liver and lung metastatic tumor analyses; S Abdulkhalek and V Kuta generated and maintained chemoresistant breast cancer cell lines; All authors read and critically revised the manuscript. F Haxho and MR Szewczuk wrote the paper. MR Szewczuk supervised the research design and MR Szewczuk and RJ Neufeld co-supervised F Haxho who prepared the first draft of the manuscript.

Disclosure

S Abdulkhalek is a recipient of the RS McLaughlin scholarship, the Ontario Graduate scholarship, and the Canadian Institutes of Health Research Doctoral award (Frederick Banting and Charles Best Canada Graduate scholarship). F Alghamdi was the recipient of the King Abdullah Scholarship from the Ministry of Higher Education, Saudi Arabia. The authors report no conflicts of interest in this work.