HPV induction of APOBEC3 enzymes mediate overall survival and response to cisplatin in head and neck cancer

Highlights

• HPV induces APOBEC3 expression in HNSC.

• High APOBEC3 G expression correlates with better overall survival.

• APOBEC3, Polβ and MSH6 knockdown results in cisplatin resistance.

• Cisplatin resistance following A3 knockdown correlates with ICL repair.

Abstract

Human papillomavirus (HPV) is associated with the development of head and neck squamous cell carcinomas (HNSC). Cisplatin is used to treat HNSC and induces DNA adducts including interstrand crosslinks (ICLs). Previous reports have shown that HPV positive HNSC patients respond better to cisplatin therapy. Our previous reports highlight that loss of base excision repair (BER) and mismatch repair (MMR) results in cisplatin resistance. Of importance, uracil DNA glycosylase (UNG) is required to initiate the BER response to cisplatin treatment and maintain drug sensitivity. These previous results highlight that specific cytidine deaminases could play an important role in the cisplatin response by activating the BER pathway to mediate drug sensitivity. The APOBEC3 (A3) family of cytidine deaminases are enzymes that restrict HPV as part of the immune defense to viral infection. In this study, the Cancer Genome Atlas (TCGA) HNSC data were used to assess the association between the expression of the seven proteins in the A3 cytidine deaminase family, HPV-status and survival outcomes. Higher A3 G expression in HPV-positive tumors corresponds with better overall survival (OS) (HR 0.33, 95 % CI 0.11-0.93, p = 0.04). FaDu and Scc-25 HNSC cell lines were used to assess alterations in A3, BER and MMR expression in response to cisplatin. We demonstrate that A3, Polβ, and MSH6 knockdown in HNSC cells results in resistance to cisplatin and carboplatin as well as an increase in the rate of ICL removal in FaDu and Scc-25 HNSC cells. Our results suggest that A3s activate BER in HNSC, mediate repair of cisplatin ICLs and thereby, sensitize cells to cisplatin which likely contributes to the improved patient responses observed in HPV infected patients.

Introduction

Head and neck squamous cell carcinomas (HNSC) encompass cancers of the oral cavity, pharynx, larynx, nasal cavity, paranasal sinuses and salivary glands. The overall 5-year survival rate for oral and pharynx cancers is 65 %, but improves to 84 % for cancers diagnosed at a local stage [1]. Concurrent cisplatin and radiation, along with surgery, is the preferred regimen for the treatment of locally advanced HNSC [2].

Tobacco and alcohol use are common, modifiable risk factors associated with HNSC risk. Human papillomavirus (HPV) infection is associated with the development of several cancers including HNSCs [1,3,4]. Patients with HPV-positive tumors have better prognosis than HPV-negative tumors, due partly to an increase in sensitivity to chemotherapy and radiation therapy [[5], [6], [7], [8]]. Molecular differences between HPV-positive and -negative tumors include differences in immune cell subtypes and tumor metabolism [9]. The APOBEC3 (A3) family of proteins are antiviral cytidine deaminases that are part of the immune system, restricting HPV through the deamination of cytosines that leads to viral DNA mutations [[10], [11], [12], [13], [14], [15], [16]]. There are seven proteins in this family, denoted: A3A, A3B, A3C, A3D, A3F, A3G and A3H. Previous reports suggest that A3A and A3B are upregulated with HPV infection, either directly by E6 and E7, or through dysregulation of transcription [[17], [18], [19], [20]]. A3 expression is also induced by type 1 interferons (IFN) [[21], [22], [23], [24]]. Off-target A3 deamination leads to a specific mutational signature enriched in HPV-positive cases [25,26]. This mutational signature is also found in other cancer types and thought to be part of tumor development [[27], [28], [29]]. The A3 mutational signature has been shown to be associated with HPV-positive HNSC cases in TCGA data [26].

Cisplatin is part of standard treatment of HNSC, either in combination with radiotherapy or adjuvant therapy following surgery [2]. A clinical trial detailed by Ang et al. showed that HPV status was a strong prognostic indicator of survival of oropharyngeal cancer patients treated with cisplatin in combination with standard or accelerated-fractionation radiotherapy [30]. Current research is focusing on decreasing treatment doses in HPV-positive patients to decrease side effects, as HPV-positive cases tend to have better survival and response to therapy [[5], [6], [7], [8]]. While both HPV-positive and –negative patients respond to cisplatin, there are significant disparities in the responses between HPV-status groups [31]. A recent study found that HPV-negative patients obtain a survival benefit with higher doses of cisplatin, whereas HPV-positive patients have survival benefit at lower doses [32]. Platinum agents, including cisplatin, form DNA adducts that lead to apoptosis if not repaired. Cisplatin and carboplatin form monoadducts, intrastrand adducts and interstrand crosslinks (ICLs). ICLs form at dGpC sites and are covalently linked between the guanines on opposing strands of DNA. We have previously shown in several cancer cell types that base excision repair (BER) and mismatch repair (MMR) proteins sensitize cells to cisplatin and carboplatin by preventing the removal of ICLs [[33], [34], [35], [36]]. BER and MMR physically prevent nucleotide excision repair and homologous recombination from removing ICLs by non-productively processing DNA base damage adjacent to these ICLs. Cisplatin and carboplatin ICLs distort the DNA helix that forces the cytosines that were bonded to the guanines to be extrahelical [37]. This specific structure may be opportune for enzymatic deamination of the extrahelical cytosines, resulting in uracils and thus forming substrates for BER activation. Of importance, we have previously shown that uracil DNA glycosylase (UNG) is required for subsequent BER processing to mediate cisplatin and carboplatin sensitivity as well as inhibiting ICL DNA repair [33].

Due to the induction and subsequent deamination activity of A3 enzymes in HPV infection and better survival in HPV-positive patients, we investigated whether expression of A3s alters survival in HNSC and alter cisplatin and carboplatin sensitivity. Since A3 expression is increased with HPV infection, we propose that this increase in A3 expression may be a factor in the better survival of HPV-positive patients following standard treatment with cisplatin. We also tested whether A3s sensitize HNSC cells to cisplatin and carboplatin, as we hypothesize that their deamination activity activates BER and MMR, ultimately altering ICL DNA repair.