Cancer Therapy: Preclinical A Novel E2F/Sphingosine Kinase 1 Axis Regulates Anthracycline Response in Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have amortality rate of 45%.We have previously shown that E2F7 may contribute to drug resistance in SCC cells. However, the mechanism and pathways involved remain unknown. Experimental Design: We used transcriptomic profiling to identify candidate pathways that may contribute to E2F7dependent resistance to anthracyclines. We then manipulated the activity/expression of the candidate pathway using overexpression, knockdown, and pharmacological inhibitors in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and a downstream effector in a tissue microarray (TMA) generated from HNSCC patient samples. Results: E2F7-deficient keratinocytes were selectively sensitive to doxorubicin and this was reversed by overexpressing E2F7. Transcriptomic profiling identified Sphingosine kinase 1 (Sphk1) as a potential mediator of E2F7-dependent drug resistance. Knockdown and overexpression studies revealed that Sphk1 was a downstream target of E2F7. TMA studies showed that E2F7 overexpression correlated with Sphk1 overexpression in human HNSCC. Moreover, inhibition of Sphk1 by shRNA or the Sphk1specific inhibitor, SK1-I (BML-EI411), enhanced the sensitivity of SCC cells to doxorubicin in vitro and in vivo. Furthermore, E2F7induced doxorubicin resistance was mediated via Sphk1-dependent activation of AKT in vitro and in vivo. Conclusion: We identify a novel drugable pathway in which E2F7 directly increases the transcription and activity of the Sphk1/ S1P axis resulting in activation of AKT and subsequent drug resistance. Collectively, this novel combinatorial therapy can potentially be trialed in humans using existing agents.Clin Cancer Res; 21(2); 1–11. 2014 AACR. Introduction Head and neck squamous cell carcinomas (HNSCC) arise from stratified squamous epithelia of the mucosae of the upper aerodigestive tract. At present, the mainstay of treatment for advanced HNSCC is surgery and/or radiotherapy plus adjuvant chemotherapy (1). The use of adjuvant chemotherapy provides modest improvements to overall survival but are not considered curative in their own right (1). Thus, if we are to improve outcomes in patients with advanced HNSCC, we need to develop systemic therapies that target novel pathways activated in HNSCC cells. HNSCC is a complex cancer associated with a large mutational burden (2, 3) and accompanied by dysregulation of proliferation, differentiation, and apoptosis. HNSCC is also accompanied by dysregulationof themain functions of the E2F transcription factor family (4, 5). E2F refers to a family of 10 gene products from eight genes (E2Fs 1, 2, 3a, 3b, 4, 5, 6, 7a, 7b, 8) that have been broadly divided into activators (E2F1–E2F3a) and inhibitors (E2F3b and E2F4–E2F8; ref. 6). The E2F family regulates a diverse array of functions such as proliferation, differentiation, apoptosis, and stress responses (7, 8). The way in which the E2F family coordinate such diversity of action is through isoform-specific functions of the individual E2Fs (e.g., activators vs. inhibitors) coupledwith context-specific interacting partner proteins such as pocket proteins andHDACs (7, 8). In the context of keratinocytes, it has been shown that normal human and murine keratinocytes express all members of the E2F family with the exception of E2F6 (9, 10). It has been shown that proliferation and differentiation of keratinocytes are regulated by the opposing actions of E2F1 and E2F7 (4, 9, 11, 12). Significantly, E2F1 and E2F7 are overexpressed in patient SCCs (10) and contribute to the development of cutaneous SCC (13, 14). In addition to the role of E2Fs in proliferation and differentiation, E2Fs are key regulators of apoptosis and stress responses (7, 8). For example, E2F1 has been shown to have potent Epithelial Pathobiology Group, University of Queensland Diamantina Institute, PrincessAlexandraHospital,Translational Research Institute, Woolloongabba, Queensland, Australia. Department of Pathology, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia. DepartmentofMedicalOncology,PrincessAlexandraHospital,Woolloongabba, Queensland, Australia. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Current address for P. Mukhopadhyay: The QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia. Corresponding Author: Nicholas Saunders, Epithelial Pathobiology Group, University of Queensland Diamantina Institute, Princess Alexandra Hospital, Translational Research Institute, 37 Kent Street, Woollongabba, Queensland 4102, Australia. Phone: 61-7-3443-7098; Fax: 61-7-3443-6966; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-14-1962 2014 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org OF1 Research. on April 14, 2017. © 2014 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Published OnlineFirst November 19, 2014; DOI: 10.1158/1078-0432.CCR-14-1962