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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: DNA Repair (Amst). 2020 Jan 16;87:102802. doi: 10.1016/j.dnarep.2020.102802

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

Kayla L Conner a,†, Asra N Shaik a,†, Elmira Ekinci a, Seongho Kim a, Julie J Ruterbusch a, Michele L Cote a,b, Steve M Patrick a,b,*
PMCID: PMC7033022  NIHMSID: NIHMS1551613  PMID: 31981740

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 A3G 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.

Keywords: APOBEC3, HPV, Head and Neck Cancer, survival, cisplatin

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 [5–8]. 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 [10–16]. 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 [17–20]. A3 expression is also induced by type 1 interferons (IFN) [21–24]. 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 [27–29]. 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 [5–8]. 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 [33–36]. 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.

Materials and Methods

Chemicals

Cisplatin, oxaliplatin, and carboplatin were purchased from Sigma-Aldrich. For preparation, cisplatin, carboplatin and oxaliplatin were diluted in 1X PBS for a stock concentration of 1mM and vortexed until drug was completely dissolved followed by filtration through 0.2micron filters. Cisplatin and carboplatin were prepared fresh before each experiment. Oxaliplatin was stored at −80°C and used within six months.

Cell lines

The human pharynx squamous cell carcinoma FaDu cells and the human tongue squamous cell carcinoma Scc-25 cells were purchased from ATCC. We selected FaDu and Scc-25 based on their higher expression of A3s from the Broad Institutes cancer cell line encyclopedia (CCLE). FaDu were grown in DMEM containing 10% FBS and 1% penicillin/streptomycin. Scc-25 were grown in DMEM F-12 media containing 10% FBS and 1% penicillin/streptomycin, and supplemented with 400ng hydrocortisone.

shRNA transfection

Mission shRNA plasmid bacterial stocks targeting human MSH6, Polβ, A3B and A3C were purchased from Sigma-Aldrich. Maxi prep kit (Qiagen) was used to purify the plasmid DNA. Lentiviral particles were packaged using HEK293T cells with packaging plasmids PMD2G, PMDLG/RRE, PRSV/RRE. Lipofectamine 2000 reagent (Invitrogen) was used to tranfect the plasmid DNA. The media was changed 24 hours after transfection and viral particles were harvested 72 hours after transfection, followed by centrifugation and filtration through 0.2micron filters. Viral stocks aliquoted and stored at −80°C. Polybrene (Sigma Aldrich) was used with the viral stocks to transfect cells to knockdown the protein of interest. Cells were used 72 hours post-transduction for the associated experiments and to check for transcript expression.

siRNA transfection

ON-TARGET plus siRNAs for human A3D and the non-targeting control siRNA were purchased from Dhamacon. Transfection was carried out per Dharmacon’s protocol. Cells were plated in 6-well plates without penicillin/streptomycin. Transfection was completed with 60–70% cell density, with two transfections performed 24 hours apart. DharmaFECT 1 transfection reagent was used for FaDu and Scc-25. The cells were used at 48 and 72 hours after transfection for the associated experiments and to assess transcript expression.

Real Time PCR for transcript expression

Cells were harvested and pelleted as previously indicated post-transfection time points. RNA was extracted using TRIzol reagent (Invitrogen) using standard procedures. RNA concentration was determined using SpectraMax M5. 2μg total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Transcript levels were quantified using PowerUP SYBR Green Master Mix (Applied Biosystems), with GAPDH as an endogenous control. The percent transcript knockdown was determined from 2^−ΔΔCT values. A3 primer sequences were obtained from Refsland et. al [38].

Colony Survival Assay

Cells (400 to 800) were treated using increasing concentrations of cisplatin, carboplatin or oxaliplatin for 2 hours in serum-free media. Following treatment, complete media was added and the cells were allowed to grow for 7–14 days at 37°C with 5% CO2. Colonies were fixed and stained with 0.2% crystal violet in 20% ethanol. Colonies with greater than or equal to 50 cells were counted and colony survival was expressed as the ratio of the average number of colonies in drug treated cells compared to control (untreated) cells multiplied by 100. The experiment was done in biological and technical triplicates for each drug concentration. IC50s were calculated using CompuSyn version 1.

Modified Alkaline Comet Assay

Modified alkaline comet assay was used to analyze the repair of ICLs as previously described [33–36]. Cell suspensions containing ~10,000 cells were embedded on a microscope slide in agarose, lysed, and incubated in cold alkaline buffer for 20 minutes to allow the DNA to unwind. Electrophoresis was performed for 25 minutes at 300mA, 20–20V. Slides were neutralized and stained with SYBR Gold (Invitrogen). The comets were scored using a Nikon fluorescence microscope. At least fifty cells were analyzed per slide using Komet 5.5 Software (Kinetic Imaging, Liverpool, UK). Data are expressed as the percentage decrease olive tail moment, which corresponds to the percent remaining ICLs.

TCGA Head and Neck Cancer Dataset

HNSC clinical and RNA Seq data from the Cancer Genome Atlas (TCGA) were obtained from cbioportal (http://www.cbioportal.org). RNA Seq V2 RSEM values were used for gene expression analysis and were normalized by scaling to a mean of 0 and variance of 1, and were normally distributed. Data for the immune subtypes and cells were obtained from Throsson et al [39]. Briefly, CIBERSORT was utilized to estimate the relative fraction of immune cell types in the leukocyte compartment using the RNASeq TCGA data [39, 40]. This group identified six categories of immune subtypes based on the expression of immune cells: C1 (wound healing), C2 (IFN-γ dominant), C3 (inflammatory), C4 (lymphocyte depleted), C5 (immunologically quiet), C6 (TGF-β dominant) [39].

Statistical Analysis

Wilcoxon rank-sum tests were used to compare expression differences between HPV-positive and –negative groups. For each gene, expression was then dichotomized based on the median (high versus low) and overall survival (OS) was estimated using Kaplan-Meier method. Kaplan-Meier curves were compared using log-rank tests. Multivariable cox proportional hazard regression analysis was performed, adjusting for the following covariates, selected a priori: age at diagnosis (continuous), gender (male/female), metastasis (yes/no/unknown), tumor size (T1/T2/T3/T4/T4a/T4b), and lymph node infiltration (yes/no/unknown). Covariates that were not significantly associated with OS and not included in the multivariable model: smoking status (Never/current/former) and tissue site source (institution where the tissue was collected). This left 565 HNSC cases. The proportional hazard assumption was validated and no violation was found. Interaction between A3s and HPV-status were performed by adding the interaction term to the multivariable model. Wilcoxon rank-sum tests were used to compare deconvoluted immune cell subsets by low and high A3 expression, HPV status, and gender, as detailed by Newman et. al.[40]. Kruskal-Wallis test was used to compare differences between A3 expression and immune subtypes. Data analyses were carried out using R version 3.5.1 and RStudio: Integrated Development for R, version 1.1.463.

IC50 values were normally distributed and standard deviation values are listed next to the IC50 of each experiment. Unpaired T-test was used to determine differences in IC50 values. Unpaired T-test was used to determine significance between percent decrease in olive tail moment at each time point. Differences in RNA expression by RT-PCR were determined using T-Test. Graphs were generated using SigmaPlot version 10.0.

Results

We initially compared the expression of A3s by HPV-positive or –negative status in HNSC. A3B, A3C, A3D, A3F, A3G, and A3H had significantly higher mean expression in HPV-positive tumors than HPV-negative tumors (p-values <1e−11, Figure 1 and supplemental table 1), but A3A did not.

Figure 1.

Figure 1.

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APOBEC3 expression is higher in HPV-positive HNSC TCGA cases than HPV-negative cases. Expression is based on RNA-Seq values. p-values were determined using Wilcoxon rank-sum test. Boxplot lower and upper hinges correspond to 25th and 75th percentiles, and middle hinge correspond to the median. Boxplot whiskers represent 95% confidence intervals, with outliers being individually plotted.

Univariate analysis through Kaplan-Meier survival curves showed no statistically significant differences in OS in HNSC cases by A3 expression dichotomized by the median, except A3A (Supplemental Figure 1). Patients with high A3A had better overall survival than patients with low A3A (Supplemental Figure 1, p-value 0.014). Next, we analyzed the association of A3 expression with survival using Cox proportional hazard models. In the multivariable model, patients with high A3A had better OS (HR 0.74, 95% CI 0.58–0.96, p-value 0.021, Supplemental Table 2). Adjusting for HPV status, patients with high A3A had better OS (HR 0.74, 95% CI 0.58–0.96, p-value 0.023), whereas high A3F had worse OS (HR 1.3, 95% CI 1.0–1.7, p-value 0.03, Supplemental Table 2). It has previously been shown that HPV-positive HNSC patients have better OS than HPV-negative HNSC cases [3, 30, 41–44]. However, there is not differential expression of A3A between HPV positive or negative cells, unlike the other A3s, and patients with higher A3A expression have better OS in all cases. Therefore, we stratified HNSC cases by HPV status. In HPV-positive cases in the multivariable analysis, high A3G expression correlated with better OS (HR 0.33, 95% CI 0.11–0.93, p-value 0.04), while other A3s were not significant (Supplemental Table 2). In HPV-negative cases in the multivariable analysis, high expression of A3F correlated with worse OS (HR 1.4, 95% CI 1.1–1.8, p-value 0.02, Supplemental Table 2). In HPV-negative cases, high expression of A3A correlated with better OS (HR 0.74, 95% CI 0.57–0.96, p-value 0.02, Supplemental Table 2). Considering A3A expression does not vary by HPV status but HPV status correlates with better OS, it is likely that there is no connection with A3A expression and the mechanism that contributes to better OS with HPV positive status. In the univariate model in HPV-positive cases, high A3F expression correlated to better OS (HR 0.29, 95% CI 0.10–0.83, p-value 0.02, Supplemental Table 2). Because of the association of better OS in HPV-positive cases with A3G expression, we looked at the interaction between HPV and A3s. There was a statistically significant interaction between A3G and HPV (p-value 0.043, Supplemental Table 2). Univariate Kaplan-Meier survival curves showed similar results as the Cox proportional hazard models (Supplemental Figure 1). Univariate Kaplan-Meier survival curves also shows differential survival by A3D, A3F or A3G expression by HPV status, with higher expression in HPV-positive tumors having better survival (Figure 2).

Figure 2.

Figure 2.

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Better overall survival in HPV-positive cases with high expression of A3D, A3F, or A3G, with no difference in HPV-negative cases. Kaplan-Meier survival curves of overall survival in HSNC TCGA cases by APOBEC3 expression. Log-rank test used to determine p-values. Time is represented as months. Each A3 family member is indicated above the graphs.

Due to the role of A3s in the immune system, we used CIBERSORT to assess differences in immune cell subsets by low and high A3 expression, HPV status, and gender [40]. Resting memory CD4+ T cells, and Mast cells were significantly different by gender (p=0.017 and 0.008, respectively), however there were no differences by other immune subsets (Supplemental Table 3). There were significant differences in immune cells by HPV status, listed in Supplemental Table 3. Lymphocytes, mast cells, and macrophages were significantly different by HPV status (Supplemental Table 3, p<0.0001). Lymphocytes significantly differed by A3A, A3C, A3D, A3F, A3G and A3H expression (p<0.0001), mast cells significantly differed by A3C, A3D, A3F, A3G and A3H expression (p<0.0001), and macrophages significantly differed by A3A, A3B, A3C, A3D, A3F, A3G, and A3H (p=0.005, 0.012, 0.012, <0.0001, 0.0003, <0.0001, <0.0001, respectively, Supplemental Table 3). These differences in individual immune subsets led us to analyze immune subtypes that were identified in Throsson et al [39]. There were differences in the expression of A3A, A3C, A3D, A3G, and A3H by immune subtypes (Supplemental Figure 2). Multiple comparison with Bonferroni adjustment showed that there were differences between C1 (wound healing) and C2 (IFN-γ dominant) immune subtypes by A3A, A3D, A3G, and A3H (Supplemental Figure 2, p <0.0001), with expression of these A3s being highest in the C2 subtype, or IFN-γ dominant. Previous reports suggest that IFN-γ prevent or help clear HPV infection, likely due to driving A3 expression [45, 46]. These results indicate a strong correlation between the innate immune system function and A3 expression in response to HPV infection, which links to better OS in HPV positive cases.

Cisplatin is a common chemotherapeutic used to treat HNSC, and with the subtle differences in survival by A3 expression, we sought to assess whether A3 expression mediates response to cisplatin. To determine whether A3s mediate cisplatin response, we utilized commercial shRNA targeting A3B and A3C. Colony survival assays showed that shA3B and shA3C knockdown in FaDu cells resulted in resistance to cisplatin compared to shControl (Figure 3A). These commercial shRNAs are not specific shRNAs to the individual A3s. A3s have sequence homology that makes targeting A3s individually difficult. shA3B decreases the expression of A3B, A3C, A3D, A3F, A3G and A3H, and shA3C decreases the expression of A3C, A3D, and A3G (Supplemental Figure 3A). Given the non-specificity of these shRNAs, we utilized these shRNAs to target A3s in general to assess effects on platinum response. shA3B Scc-25 cells were also resistant to cisplatin (Supplemental Figure 4A). Based on the structure of cisplatin and carboplatin ICLs, we hypothesized that A3s can deaminate the extrahelical cytosines and activate BER through cytosine to uracil formation adjacent to the ICL. We have previously shown that BER (UNG, APE1, Polβ), and MMR (MSH6, MSH2, MLH1) mediate cisplatin sensitivity in breast cancer cells [33–36]. To determine if MMR and BER mediate cisplatin sensitivity in HNSC and connect the response observed with A3 knockdowns, colony survival assays were performed with knockdowns of MSH6 and Polβ using shRNA in FaDu and Scc-25 cells. shMSH6 and shPolβ treated FaDu cells were resistant to cisplatin compared to shControl consistent with shA3 knockdown (Figure 3B). shMSH6 and shPolβ Scc-25 cells were also resistant to cisplatin compared to shControl (Supplemental Figure 4B). Carboplatin, another platinum agent, has the same structure as cisplatin ICLs once bound to DNA, as the only difference is the chemical leaving group. Therefore, carboplatin ICLs also form extrahelical cytosines and should provide similar results as cisplatin. As expected, shA3B, shA3C, shMSH6, and shPolβ in FaDu cells were resistant to carboplatin (Figure 3C & D). shA3B, shMSH6, and shPolβ Scc-25 cells were resistant to carboplatin compared to shControl (Supplemental Figure 4C & D). Oxaliplatin is another platinum-based chemotherapeutic, that forms ICLs, but does not induce the same extent of extrahelical cytosines that cisplatin and carboplatin form. As expected, shMSH6, shPolβ, shA3B, and shA3C in FaDu and Scc-25 cells showed no difference compared to shControl with oxaliplatin treatment (Supplemental Figure 3B, Supplemental Figure 4E, respectively). To determine if A3s function in the same pathway as BER or MMR, we utilized combination knockdowns of A3s with either MSH6 or Polβ. FaDu shA3B+shMSH6 and shA3B+shPolβ did not increase resistance beyond the individual knockdowns, suggesting that A3s work within the same pathway as BER and MMR and are epistatic with respect to cisplatin response (Figure 3E, Supplemental Figure 3E). Similar to the single knockdowns, combination knockdowns did not alter oxaliplatin response (Supplemental Figure 3D). These data suggest that BER and MMR work in conjunction with A3 enzymes to mediate cisplatin and carboplatin sensitivity.

Figure 3.

Figure 3.

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Loss of A3s, Polβ and MSH6 confer resistance to cisplatin and increase the rate of ICL repair. A) FaDu cells with shA3B, shA3C or shControl were treated with increasing concentrations of cisplatin and assessed in colony survival assays. B) FaDu cells with shPolβ, shMSH6 or shControl were treated with increasing concentrations of cisplatin and assessed in colony survival assays. C) FaDu cells with shA3B, shA3C or shControl were treated with increasing concentrations of carboplatin and assessed in colony survival assays. D) FaDu cells with shPolβ, shMSH6 or shControl were treated with increasing concentrations of carboplatin and assessed in colony survival assays. E) FaDu cells with shA3B and shMSH6 or shPolβ and individual knockdowns treated with increasing concentrations of cisplatin and assessed in colony survival assays. F) Modified alkaline comet assay, FaDu knockdown (shControl, shA3B, shA3C, shMSH6, shPolβ, shA3B+shMSH6, shA3B+shPolβ) cells treated with 8μM cisplatin for 2 hours. ** p<0.01, *p<0.05. G) siA3D and siControl knockdown in FaDu cells with increasing concentrations of cisplatin using colony survival assays. H) Modified alkaline comet assay with FaDu siControl or siA3D cells treated with 8μM cisplatin for 2 hours. IC50 values are represented as mean ± S.D.

We previously showed that BER and MMR influence the rate of cisplatin ICL repair [33–36]. The modified alkaline comet assay utilizes hydrogen peroxide to induce a fixed amount of DNA strand breaks, that migrate to form comet tails during electrophoresis [34, 47]. ICLs in DNA will hinder the migration of DNA strands, resulting in shorter comet tails. Olive tail moments of hydrogen peroxide alone and hydrogen peroxide plus cisplatin treated cells are compared along with untreated cells to calculate the percent ICLs remaining in the DNA. At the 24 hour timepoint there is an increase in ICLs within the DNA of each cell (Figure 3F). This is consistent with the conversion of cisplatin mono-adducts to ICLs which reaches maximum conversion up to 18 hours post-treatment, which can result in the increase in ICL formation. At 48 hours post-cisplatin treatment, shMSH6, shPolβ, shA3B and shA3C cells had less ICLs remaining compared to shControl, suggesting that in the absence of MSH6, Polβ, and A3s, FaDu cells are able to repair ICLs at a faster rate (Figure 3F). This remains consistent at 72 hours post-cisplatin treatment, with A3, Polβ and MSH6 knockdown FaDu cells having less ICLs remaining compared to shControl (Figure 3F). The combination of shA3B with either shMSH6 or shPolβ did not further change ICL repair, supporting the model of BER and MMR functioning together with A3s (Figure 3F).

Due to the non-specificity of commerical shRNA, we wanted to determine if specific A3s can mediate cisplatin and carboplatin sensitivity. We utilized a specific siRNA to A3D, which is one of the A3s whose expression decreases the most with both shA3B and shA3C. Knockdown of A3D resulted in resistance to cisplatin in FaDu compared to control, and did not alter oxaliplatin response (Figure 3G, Supplemental Figure 3F, respectively). siA3D decreased A3D expression, and did not target other A3 members, but we did observe slight increases in expression of other A3 members which could explain the modest resistance with siA3D compared with shA3B colony assays (Supplemental Figure 3G). Further, knockdown of A3D in Scc-25 cells also resulted in resistance to cisplatin and no effect on oxaliplatin response compared to control (Supplemental Figure 4H and 4I, respectively). Knockdown efficiency of siA3D in Scc-25 is shown in Supplemental Figure 4J. To determine if the loss of A3D alters ICL DNA repair, we utilized a modified alkaline comet assay. FaDu siA3D cells had less percent remaining ICLs at 48 and 72 hours post-cisplatin treatment compared to siControl demonstrating faster ICL DNA repair at these time points (Figure 3H).

Discussion

A3s are cytidine deaminases involved in host-cell immunity to target viral DNA and protect the cell against viral infection, including HPV. We found HPV-positive HNSC cases more highly express A3s, except for A3A, compared to HPV-negative HNSC, which is consistent with an upregulation of A3s due to HPV infection. The impact of A3s on OS differed by HPV status, with HPV-positive tumors with high expression of A3G having better OS, while HPV-negative tumors with high A3F expression had worse OS. These data suggest differential roles for A3s (A3A, A3G, and A3F) in HPV-negative and –positive tumors, as high expression, correlated with better OS in HPV-positive, but worse OS in HPV-negative cases. This could be related to the overall expression of the APOBEC3 family members for each case as HPV infection significantly induces expression of all A3 family members except A3A. The mutational signature attributed to A3 activity has previously been correlated to HPV-positive HNSC TCGA cases [26].

We investigated A3 expression by immune subtype and HPV status. In our analysis, there were differences between C1 and C2 immune subtypes by the expression A3A, A3D, A3G, and A3H. The C2 immune subtype had higher expression of A3A, A3D, A3G, and A3H compared to the C1 subtype. The C2 subtype is also referred to as the IFN-γ dominant subtype and response to type I interferons. There is a link between lymph node metastasis, poor prognosis and genes within IFN-regulation in HNSC and other cancers [48, 49]. This connection is supported by previous reports of A3 expression induced by type I interferon signaling, suggesting that tumors within the C2 subtype have increases in signaling that leads to the expression of A3s.

Strengths of our study include the use of publicly available RNA sequencing data from TCGA. This dataset is relatively large and samples from several sites are well annotated. Limitations of our study stem partly from grouping squamous head and neck cancers together, due to sample size of each anatomical primary site. However, utilization of cell lines taken from two different sites does show similar results. Another limitation is the lack of variables that may confound the relationship between A3s and survival including race, alcohol use, tobacco use, and treatment details. The restricted follow-up time in this study may limit survival association estimates; however, there were enough events to examine survival associated with A3s here (number of events=255). While two HNSC cell lines are used, future studies can include more cell lines to further explore the alteration of A3 expression and response to chemotherapeutic agents. In cell culture experiments, mediation of A3 expression using shRNA and siRNA results in resistance to cisplatin and carboplatin. Further investigation is needed to directly correlate A3 expression to cisplatin response and patient outcomes to these treatments.

Cisplatin, carboplatin, and oxaliplatin are platinum-based drugs that are first line chemotherapeutics for the treatment of HNSC. We show that knockdown of A3s, BER (Polβ), and MMR (MSH6) in HNSC cells results in resistance to cisplatin and carboplatin treatment and increase the rate of ICL repair. Our data demonstrate that A3s function epistatically with BER and MMR with respect to mediating cisplatin and carboplatin response. As detailed in the mechanistic model shown in Figure 4, HPV increases A3 expression, leading to increased deamination of extrahelical cytosines formed by cisplatin and carboplatin ICLs. This cytosine deamination leads to the formation of uracil, and BER processing by UNG, APE1 and Polβ. Polβ has low fidelity, especially at the 3’ OH group adjacent to the ICL structure. Base misincorporation activates MMR (MSH6, MSH2, and MLH1) to repair the misincorporated bases. This futile pathway leads to persistent ICLs, leading to apoptosis and overall sensitivity to cisplatin and carboplatin. HPV-positive patients had higher expression of A3s, and we would expect them to respond better to cisplatin therapy. In support of this, it has been shown that HPV-positive patients respond better to cisplatin and radiation therapy and decreasing treatment intensity is being investigated for HPV-positive patients [5–8, 50]. Based on our proposed mechanistic model (Figure 4), we would expect to see changes in protein function in either BER or MMR to influence cisplatin or carboplatin response. However, RNA expression of MSH6 and Polβ are fairly consistent in HNSC cases and in other cancers (TCGA analysis). In many cancers, however, mutations in MSH6 and Polβ are most frequently associated with differences in outcomes and response to therapy [51–56]. Particularly identified in colorectal cancer; patients with mutations in MMR are resistant to cisplatin therapy. Unfortunately, there are not enough patients with mutations to determine impact on survival (four patients with MSH6 mutations and five patients with Polβ mutations). We believe that it is the APOBEC3 expression patterns that can drive the response to cisplatin and carboplatin as a consequence of deamination of the extrahelical cytosines at ICL structures and ultimately, activation of BER and MMR. Considering the APOBEC3 deaminases are critical as a defense mechanism to viral infection, we speculated that HPV infection in HNSC may drive the APOBEC3 expression and those cases which have higher APOBEC3 expression would respond better to cisplatin and/or carboplatin therapy. Our data is consistent with HPV induced A3 expression modulating the deamination of the extrahelical cytosines adjacent to cisplatin or carboplatin ICLs and altering ICL DNA repair. The activation of BER via UNG removal of deaminated cytosines results in non-productive ICL processing and persistent ICLs which ultimately improve response to cisplatin and carboplatin (Figure 4). Further studies are required to elucidate which A3s have enzymatic activity on the extrahelical cytosines and establish a causative link in HNSC patients on how A3 expression mediates response to cisplatin and carboplatin.

Figure 4.

Figure 4.

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Mechanistic model of A3 cytidine deaminases in mediating sensitivity of cells to cisplatin and carboplatin. HPV infection leads to an increase in APOBEC3 expression. Cisplatin and carboplatin form ICLs that induce extrahelical cytosines adjacent to the ICL. APOBEC3 enzymes deaminate the extrahelical cytosines adjacent to the ICLs, forming uracils. UNG, APE1, and Polβ (all required members of BER for mediating cis/carboplatin response) process and remove the uracil, cleave the phosphodiester backbone and synthesize DNA, respectively. Polβ is prone to misincorporate DNA bases during this processing as a consequence of the ICL structure, which ultimately activates the MMR proteins (MutSα: MSH2 and MSH6, along with MLH1 and PMS2) to correct the misincorporated DNA bases. The ICL remains on the DNA as a result of this non-productive processing and blocking of productive DNA repair processes (e.g. NER and HR) and cells are sensitive to cisplatin and carboplatin.

Supplementary Material

1

Highlights:

  • HPV induces APOBEC3 expression in HNSC

  • High APOBEC3G expression correlates with better overall survival

  • APOBEC3, Polβ and MSH6 knockdown results in cisplatin resistance

  • Cisplatin resistance following A3 knockdown correlates with ICL repair

Acknowledgments

This work was supported in part by the National Institute of Health Ruth L. Kirschstein National Research Service Award (T32-CA00953 to KLC, 1F31CA22133301 to ANS); Susan G. Komen for the Cure (GTDR14299438 to MLC, ANS, and KLC); and the National Institutes of Health (R01CA229535) awarded to SMP. The authors thank the members of the Patrick lab for carefully reading the manuscript. The results shown here are generated from TCGA (https://cancergenome.nih.gov).

Abbreviations:

HPV

Human papillomavirus

HNSC

head and neck squamous cell carcinomas

A3

APOBEC3

BER

Base excision repair

MMR

Mismatch repair

ICLs

interstrand crosslinks

UNG

uracil DNA glycosylase

TCGA

the cancer genome atlas

OS

overall survival

IFN

interferons

Polβ

polymerase beta

MSH6

mutS protein homolog 6

Footnotes

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Conflicts of Interest

The authors declare there are no conflicts of interest.

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