RacGAP 1 Is a Novel Downstream Effector of E 2 F 7-Dependent Resistance to Doxorubicin and Is Prognostic for Overall Survival in Squamous Cell Carcinoma

We have previously shown that E2F7 contributes to drug resistance in head and neck squamous cell carcinoma (HNSCC) cells. Considering that dysregulation of responses to chemotherapy-induced cytotoxicity is one of themajor reasons for treatment failure in HNSCC, identifying the downstream effectors that regulate E2F7-dependent sensitivity to chemotherapeutic agents may have direct clinical impact. We used transcriptomic profiling to identify candidate pathways that contribute toE2F7-dependent resistance to doxorubicin. We thenmanipulated the expression of the candidate pathway using overexpression and knockdown in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and RacGAP1 in a custom tissuemicroarray (TMA) generated fromHNSCCpatient samples. Transcriptomic profiling identified RacGAP1 as a potential mediator of E2F7-dependent drug resistance. We validated E2F7-dependent upregulation of RacGAP1 in doxorubicininsensitive SCC25 cells. Extending this, we found that selective upregulation of RacGAP1 induced doxorubicin resistance in previously sensitive KJDSV40. Similarly, stable knockdown of RacGAP1 in insensitive SCC25 cells induced sensitivity to doxorubicin in vitro and in vivo. RacGAP1 expression was validated in a TMA, and we showed that HNSCCs that overexpress RacGAP1 are associated with a poorer patient overall survival. Furthermore, E2F7-induced doxorubicin resistance was mediated via RacGAP1-dependent activation of AKT. Finally, we show that SCC cells deficient in RacGAP1 grow slower and are sensitized to the cytotoxic actions of doxorubicin in vivo. These findings identify RacGAP1 overexpression as a novel prognostic marker of survival and a potential target to sensitize SCC to doxorubicin. Mol Cancer Ther; 14(8); 1939–50. 2015 AACR. Introduction Cutaneous squamous cell carcinomas (CSCC) and head and neck SCC (HNSCC) are among the most common malignancies afflictingman (1, 2). Current treatment options for advanced SCC include adjuvant chemotherapy with platinum-based drugs, such as taxanes, 5-Fluorouracil, or therapeutic antibodies against EGFR (3, 4). However, the response is generally transient and characterizedby thedevelopment of drug resistance. Thus, there is aneed to identify new therapeutic strategies that can bypass the emergence of a drug-resistant phenotype. The E2F transcription factor complex comprises a family of activating (E2F1, 2, 3a) or repressive/inhibitory (E2F3b, 4, 5, 6, 7, or 8) E2Fs that regulate key cellular functions, such as transcription, differentiation, and apoptosis. In the context of keratinocytes (KC), the E2F transcription factor family has been shown to control (i) proliferation, (ii) differentiation, (iii) stress responses, and (iv) apoptosis (5–10). Consistent with their roles in KCs, dysregulationofE2F is a commonoccurrence in SCC(11, 12), and overexpression of E2Fs, such as E2F1 and E2F7, occurs in the majority of CSCCs and HNSCCs (12–15). E2F1 and E2F7 are known to have opposing actions in the regulation of proliferation (5), differentiation (14), and apoptosis (9, 14). For example, recent reports have shown that treatment of wild-type cells with DNA-damaging agents, such as doxorubicin or etoposide, induces E2F7 protein levels and subsequent inhibition of the E2F1-mediated DNA damage response (9, 10). In the context of KCs, E2F7 was shown to causally modify responses to conventional chemotherapeutics (15) and UV responses in vitro (14). Thus, sensitivity to common cytotoxic agents and stimuli appear to be regulated by the relative ratio of E2F1 to E2F7 in the tissue. Given that both E2F1 and E2F7 are known to be overexpressed in SCC (14, 15), it is reasonable to speculate that this may also 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 Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Current address for P. Mukhopadhyay: The QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia. Corresponding Author: Nicholas A. Saunders, The University of Queensland Diamantina Institute, Translational Research Institute, 37 Kent St, Woolloongabba,Queensland4102, Australia. Phone: 61-7-34437098; Fax: 61-7-34436966; E-mail: nsaunders@uq.edu. au doi: 10.1158/1535-7163.MCT-15-0076 2015 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals. org 1939 on July 10, 2017. © 2015 American Association for Cancer Research. mct.aacrjournals. org Downloaded from Published OnlineFirst May 27, 2015; DOI: 10.1158/1535-7163.MCT-15-0076