Anti-lymphangiogenic properties of mTOR inhibitors in head and neck squamous cell carcinoma experimental models

Ekshyyan et al. BMC Cancer 2013, 13:320 http://www.biomedcentral.com/1471-2407/13/320 RESEARCH ARTICLE Open Access Anti-lymphangiogenic properties of mTOR inhibitors in head and neck squamous cell carcinoma experimental models Oleksandr Ekshyyan1,2, Tara N Moore-Medlin1,2, Matthew C Raley1, Kunal Sonavane1,2, Xiaohua Rong1,2, Michael A Brodt1, Fleurette Abreo3, Jonathan Steven Alexander4 and Cherie-Ann O Nathan1,2* Abstract Background: Tumor dissemination to cervical lymph nodes via lymphatics represents the first step in the metastasis of head and neck squamous cell carcinoma (HNSCC) and is the most significant predictor of tumor recurrence decreasing survival by 50%. The lymphatic suppressing properties of mTOR inhibitors are not yet well understood. Methods: Lymphatic inhibiting effects of rapamycin were evaluated in vitro using two lymphatic endothelial cell (LEC) lines. An orthotopic mouse model of HNSCC (OSC-19 cells) was used to evaluate anti-lymphangiogenic effects of rapamycin in vivo. The incidence of cervical lymph node metastases, numbers of tumor-free lymphatic vessels and those invaded by tumor cells in mouse lingual tissue, and expression of pro-lymphangiogenic markers were assessed. Results: Rapamycin significantly decreased lymphatic vascular density (p = 0.027), reduced the fraction of lymphatic vessels invaded by tumor cells in tongue tissue (p = 0.013) and decreased metastasis-positive lymph nodes (p = 0.04). Rapamycin also significantly attenuated the extent of metastatic tumor cell spread within lymph nodes (p < 0.0001). We found that rapamycin significantly reduced LEC proliferation and was correlated with decreased VEGFR-3 expression in both LEC, and in some HNSCC cell lines. Conclusions: The results of this study demonstrate anti-lymphangiogenic properties of mTOR inhibitors in HNSCC. mTOR inhibitors suppress autocrine and paracrine growth stimulation of tumor and lymphatic endothelial cells by impairing VEGF-C/VEGFR-3 axis and release of soluble VEGFR-2. In a murine HNSCC orthotopic model rapamycin significantly suppressed lymphovascular invasion, decreased cervical lymph node metastasis and delayed the spread of metastatic tumor cells within the lymph nodes. Keywords: Head and neck squamous cell carcinoma, Rapamycin, mTOR inhibitors, mTOR, VEGFR-3, VEGFR-2, Lymph node metastasis Background Despite advances in treatment options, there have been no significant improvements in 5-year survival rates of head and neck squamous cell carcinoma (HNSCC) patients in the past four decades. While the 1-year survival rate is 81%, the 5-year survival rate remains at ~45% for * Correspondence: 1 Department of Otolaryngology/Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, USA 2 Feist-Weiller Cancer Center, LSUHSC, Shreveport, LA, USA Full list of author information is available at the end of the article all stages of oral cancer [1]. Metastasis to regional lymph nodes occurs in 30-40% of HNSCC cases [2], and is associated with poor prognosis and low survival [2,3]. Lymphatogenous spread of cancer cells is a significant problem in HNSCC reflecting the rich lymphatic supply in the head and neck. High risk features, such as lymphovascular invasion and extracapsular spread significantly increase the risk of both local recurrence, and distant metastasis. Consequently postoperative adjuvant chemoradiotherapy is recommended to decrease recurrence rates [4]. De Carvalho in a prospective analysis of © 2013 Ekshyyan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ekshyyan et al. BMC Cancer 2013, 13:320 http://www.biomedcentral.com/1471-2407/13/320 170 cases of previously untreated patients with laryngeal or hypopharyngeal squamous cell carcinoma found that macroscopic extracapsular tumor spread increased the risk of recurrence 3.5-fold compared with patients with no evidence of metastasis at their initial diagnosis, or patients in whom the tumor was confined to the lymph node [5]. In another study, patients with extracapsular nodal spread had significantly higher rates of recurrent disease and distant metastasis [6]. Tumor cell spread to regional lymph nodes through lymphatic vessels is known to be one of the worst prognostic factors, decreasing survival by 50%. Formation of new tumor-associated lymphatic vessels through lymphangiogenesis plays an active role in the initiation and progression of metastatic disease spread as demonstrated by the significant correlation between intratumoral lymphatic vascular density and lymph node metastasis. HNSCC is characterized by persistent activation of the Akt/mTOR pathway that triggers a cascade of molecular events central to carcinogenesis including cancer cell survival, cell cycle progression, proliferation, transcription and translation, angiogenesis, invasion, and metastasis [7,8]. The Akt/mTOR pathway is a fundamental coordinator of several signaling pathways related to cell growth and division, and mTOR inhibitors effectively reduce proliferation in cells with constitutively upregulated Akt/mTOR signaling. The mammalian target of rapamycin (mTOR) signaling pathway is dysregulated in nearly all (99%) cases of HNSCC [9]. mTOR inhibitors depress translation of several mRNAs specifically required for tumor cell cycle progression, proliferation, and angiogenesis suppressing oncogenesis [10-15]. Because these pathways are commonly dysregulated in cancer, mTOR represents an attractive anti-tumor target. The mTOR inhibitor rapamycin (sirolimus) was approved by the FDA in 1999 to prevent renal transplant rejection [16] and is a clinically approved immunosuppressive agent with promising anti-tumor properties. Chronic use of rapamycin shows a good safety profile in renal transplantion [17,18] and is well tolerated with only mild and usually reversible side effects which include herpes simplex lesions, acne-like and maculopapular rash, and nail disorders. Dose-limiting toxicities consist of mucositis/stomatitis, asthenia, thrombocytopenia and hyperlipidemia [19]. Although the role of mTOR inhibitors is well established in renal cell carcinoma and recent phase 1 and 2 studies in solid tumors hold promise, their antilymphatic properties are not well characterized. Previously in collaboration with Dr. Silvio Gutkind’s group (Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, NIH) using an orthotopic model of HNSCC generated by injection of UMSCC2 cells into the tongue of SCID/NOD mice we demonstrated significant inhibition of tumor growth, decreased lymphatic Page 2 of 9 microvessel density and a decrease in the number of invaded lymph nodes after rapamycin and RAD001 treatment [20]. In the current study (...truncated)