Radiosensitivity in HNSCCs correlates with TRIP12 and USP7 expression and is HPV status dependent
To examine the mechanism of HPV-mediated radiosensitization, we utilized a panel of HNSCC HPV(+) and HPV(-) cell lines. To confirm previously published results, we examined the surviving fraction after 2 Gy of irradiation (SF2) for our panel and found that the HPV(+) cell lines (UMSCC-47, UPCI:SCC152, UPCI:SCC154) were significantly more sensitive to radiation than the HPV(-) (Detroit562, UMSCC-1, HN5, FaDu, HN30, HN31) lines (Fig. 1A). As expected, the HPV(-) cell lines expressed negligible levels of p16, the surrogate marker of HPV infection (Fig. 1B). In agreement with our recently published report, p16 protein levels were inversely correlated with TRIP12 protein levels (Fig. 1B). Because we previously demonstrated that p16 regulates TRIP12 in a posttranslational fashion8, we examined the deubiquitinase ubiquitin-specific protease 7 (USP7), which has been shown to bind to TRIP1219. The immunoblot of USP7 from our cell line panel demonstrated a USP7 protein expression pattern proportional to that of TRIP12 (Fig. 1B). Additionally, densitometric analysis showed that both USP7 and TRIP12 protein expression was highly correlated with radioresistance (Fig. 1C).
USP7 is downregulated by p16 through ubiquitination
To understand whether the protein level correlation was a direct consequence of p16 induction, we examined the impact of direct p16 modulation on USP7 levels. The protein levels of USP7 decreased following forced expression of p16 in HPV(-) HN5 and HN31 cells (Fig. 2A). Moreover, forced expression of p16 in a panel including both HNSCC and non-small cell lung carcinoma (NSCLC) cell lines led to an approximately 50% reduction in USP7 protein expression (Fig. 2B). This panel included p53 wild-type H460 and HN30 cells, which is suggestive of a p53-independent mechanism across multiple cell types. The converse was observed when p16 expression was inhibited via CRISPR in HPV(+) UM-SCC-47 cells, with USP7 levels increasing following p16 KO (Fig. 2C).
Next, we sought to determine the mechanism by which p16 regulates USP7. We found that forced p16 expression in HPV(-) HN5, HN30 and HN31 cells had no effect on USP7 mRNA (Supplemental Fig. 1A) despite the reduction seen at the protein level (Fig. 2A & B); this led us to suspect that the USP7 decrease may occur through posttranslational modification. To test this hypothesis, we performed cycloheximide chase assays to determine the effect of p16 expression on the stability of USP7. The presence of p16 significantly destabilized USP7 protein in p53 mutant HN5 (Fig. 2D) and p53 wild-type HN30 (Supplemental Fig. 1B). To determine whether this destabilization occurred through ubiquitination of USP7, we tested whether the addition of the proteasome inhibitor MG132 could rescue USP7 expression following forced expression of p16. We found that MG132 was able to at least partially rescue the p16-induced reduction in USP7 protein expression in all three lines tested (Fig. 2E & Supplemental Fig. 1C), which indicated that the mechanism depended on ubiquitination of USP7. To confirm the role of ubiquitin in the destabilization of USP7 by p16, HN5 cells were cotransfected with control or p16 and lenti-HA-ubiquitin expression vectors. Cells were then either immunoprecipitated (IP) with HA-tagged ubiquitin and immunoblotted for USP7 or the reverse (Fig. 2F), both of which showed an increase in ubiquitination of USP7 in the presence of p16 expression. Furthermore, we showed by IP that the ubiquitination of USP7 was K48-linked and not K63-linked (Fig. 2G). Given that K-48-linked ubiquitination is generally associated with degradation, this suggests that the p16-dependent ubiquitination indeed marks the USP7 protein for degradation20.
USP7 stabilizes TRIP12 through deubiquitination
Prior work suggested that USP7 binds to TRIP12; however, data are conflicting as to whether this interaction serves to regulate TRIP12 or USP721. Therefore, we directly asked whether USP7 was responsible for the observed impact of p16 on TRIP12. We reasoned that if USP7 was the link between p16 and TRIP12, modulating USP7 could abrogate the effect of p16 on TRIP12 expression. First, we confirmed that USP7 indeed bound to TRIP12 in HN5 cells by immunoprecipitating TRIP12 and immunoblotting for USP7 as well as the reverse (Fig. 3A). In addition, direct targeting of USP7 via shRNA resulted in significant depression of TRIP12 protein levels in HPV(-) HN5, HN30 and HN31 cells (Fig. 3B). Inhibition of TRIP12 had no effect on USP7 expression (Supplemental Fig. 2A), indicating that USP7 likely regulates TRIP12 in this model and not the converse. The regulation of TRIP12 by USP7 was confirmed in HPV(+) UM-SCC-47 and SCC-154 cells, where forced expression of USP7 led to significant upregulation of TRIP12 (Fig. 3C), despite the presence of p16, suggesting that p16 regulates TRIP12 indirectly through USP7. While inhibition of USP7 reduced TRIP12 protein expression, it did not reduce TRIP12 gene expression (Supplemental Fig. 2B), providing further evidence that TRIP12 regulation occurs through posttranslational modification. Cycloheximide chase assays in p53 mutant HN5 and p53 wild-type HN30 cells both showed a reduced half-life of TRIP12 after USP7 knockdown (Fig. 3D-E), which supports a p53-independent mechanism of TRIP12 stabilization by USP7. Treatment with MG132 at least partially reversed the reduction in TRIP12 protein expression induced by USP7 knockout in HN5, HN30 and HN31 cells (Fig. 3F and Supplemental Fig. 2C), which indicated that USP7 stabilized TRIP12 by deubiquitination. This mechanism of TRIP12 stabilization by USP7 was confirmed by co-IP with HA-tagged ubiquitin. This experiment showed that forced expression of p16 caused increased ubiquitination of TRIP12, which was nearly abolished upon coexpression with USP7 (Fig. 3G), confirming that the mechanism was indeed through deubiquitination by USP7.
USP7 plays an integral role in p16-induced radiosensitivity
As the p16-USP7-TRIP12 axis has not been reported thus far, especially in the context of the radiation response, we tested the effects of modulation of USP7 in combination with radiation both in vitro and in vivo. Knockdown of USP7 with shRNA sensitized HN5 cells to radiation (Fig. 4A) and reduced BRCA1 expression (Fig. 4B). Immunocytochemical (ICC) analysis of HN5 cells showed that targeting USP7 decreased the formation of BRCA1 foci following radiation exposure, suggesting that in the absence of USP7, the repair of radiation-induced DNA damage was compromised (Fig. 4C). This reduction in BRCA1 foci seen after inhibition of USP7 causes the cells to progress into mitosis with unrepaired DNA damage leading to aberrant mitosis, including increased centrosomes and micronuclei per cell, markers of mitotic death (Fig 4D-F).
To further characterize USP7 in the context of radiation, we overexpressed p16 in HN5 cells, which resulted in significantly enhanced radiosensitivity (Fig. 4G) comparable to our previous results8, as well as marked downregulation of USP7, TRIP12, and BRCA1 (Fig. 4H). Forced expression of both p16 and USP7, on the other hand, partially reversed p16-induced radiosensitization (Fig. 4G) and p16-induced downregulation of both TRIP12 and BRCA1 (Fig. 4H), demonstrating that loss of USP7 is a factor in conferring radiosensitivity in HPV(+) tumors.
To examine the role USP7 plays in the HPV(-) tumor response to radiation, we performed a tumor growth delay assay with HN5 xenografts in nude mice. Prior to inoculation, the cells were either infected with control shRNA or USP7 shRNA to mimic USP7 deficiency in HPV(+) tumors. The animals were treated with 4 Gy for 5 consecutive days, and tumor diameters were measured every two days. At the end of the study, tumors were excised and analyzed by western blot, which confirmed shRNA knockdown of USP7 and showed reduced BRCA1 expression consistent with prior in vitro results (Supplemental Fig. 3). Moreover, we found that inhibition of USP7 led to radiosensitization of HN5 tumors (Fig. 4I), further supporting that targeting USP7 could be a viable radiosensitization strategy for HPV(-) head and neck tumors.
USP7 is a druggable target for increasing the radiosensitivity of HPV(-) HNSCC
It is necessary to find druggable targets that can be utilized for sensitization of HPV(-) radioresistant tumors. Several USP7 inhibitors have been developed and are available for research purposes, with one, P5091, currently in clinical trials. Here, we tested three USP7 inhibitors: P22077, P5091 and GNE-6640. P22077 and P5091 inhibit USP7 but also USP10 and USP4722,23. GNE-6640 is a more selective inhibitor of USP7 that inhibits the deubiquitinase activity of USP7 with selectivity over a highly structurally similar deubiquitinase (USP47) and a highly active deubiquitinase (USP5)24.
Modulating USP7 activity via the chemical inhibitor GNE-6640 resulted in decreased TRIP12 expression (Fig. 5A-B) and increased radiosensitivity in HPV(-) HN5 cells (Fig. 5C). Furthermore, radiosensitization by GNE-6640 was only achieved when dose and duration were sufficient to reduce TRIP12 expression and BRCA1 foci formation. TRIP12 expression was only reduced with greater than 6 hours of treatment (Fig. 5A) and at doses greater than 1 µM (Fig. 5B), which explains the lack of radiosensitization seen at 1 or 10 µM with 6 hours of treatment prior to irradiation (Supplemental Fig. 4A) or at 1 µM with 48 hours of pretreatment (Fig. 5C). Similarly, BRCA1 foci were only reduced after treatment with 10 µM GNE-6640 for 48 hours prior to radiation (Fig. 5D) and not at any of the other doses or schedules tested (Supplemental Fig. 4B). Additionally, similar radiosensitization was achieved using P5091 in HN5 and FaDu cells (Supplemental Fig. 4C-D) as well as with P22077 in UMSCC25 and FaDu cells at various doses and schedules (data not shown). These data suggest that targeting USP7 could partially recapitulate favorable HPV-induced radiosensitivity in head and neck cancer.
HUWE1 is identified as an E3 ligase for USP7 and is transcriptionally regulated by p16, potentially via SP1
While we determined that USP7 linked p16 and TRIP12 and that USP7 appeared to be regulated via ubiquitination, it remained unclear how p16 modulated this cellular cascade. We performed IP mass spectrometry (IP/MS) in 3 HPV(+)/p16(+) and 3 HPV(-)/p16(-) cell lines to discover binding partners of USP7. From this analysis (schema in Fig. 6A), we identified three E3 ubiquitin ligases (HUWE1, TRIM21 and RNF168) binding USP7 in all HPV(-) cell lines tested (Fig. 6B). For two of the HPV(+) cell lines, SCC-152 and SCC-154, USP7 had to be overexpressed prior to IP/MS due to insufficient levels of endogenous USP7. The HPV(+) cell lines also had a similar set of binding partners with HUWE1 and TRIM21 binding USP7 in all 3 cell lines and RNF168 in one of the three (Fig. 6B). IP/MS evaluating proteins bound to HUWE1 confirmed its binding with USP7 in all cell types examined (Fig. 6C).
To further explore the relationship of these USP7 binding partners, we performed western blots for HUWE1, TRIM21, USP7, TRIP12 and p16 in all six cell lines. Basal expression of HUWE1 showed an inverse relationship to USP7 and TRIP12 and correlated with p16/HPV positivity, suggesting that it could be an E3 ubiquitin ligase limiting USP7 expression, while TRIM21 levels trended towards a proportional correlation to USP7, though this effect was not consistent across all cell lines (Fig. 7A).
When examining the relative gene expression of HUWE1 and TRIM21 in the 6 cell lines, we found that HUWE1 gene expression also correlated with p16/HPV positivity, indicating that HUWE1 is likely transcriptionally regulated by p16 (Fig. 7B). On the other hand, the gene expression of TRIM21 was comparable to its basal protein expression levels and did not show the predicted inverse correlation (Fig. 7C). To investigate the implied transcriptional regulation of HUWE1 by p16, we expressed p16 in HN5 cells, which led to an increase in both HUWE1 protein and mRNA levels (Fig. 7D-E), confirming that p16 regulates HUWE1 at the transcriptional level. Conversely, inhibition of p16 in HPV(+)/p16(+) SCC154 cells led to a decrease in HUWE1 gene expression (Fig. 7F), with a similar pattern observed in UMSCC47 cells (data not shown).
To further characterize this newly discovered p16-USP7-TRIP12 pathway, HN5 cells with forced expression of p16 were cotransfected with HUWE1, TRIM21, TRIP12 or USP7 siRNA. Western blots showed that HUWE1 expression increased with forced p16 expression (Fig. 7G), which suggested that p16 may be responsible for its transcriptional upregulation. TRIM21 did not appear to be affected by p16 expression (Fig. 7G). Most importantly, p16-induced downregulation of USP7 was rescued by cotransfection of HUWE1 siRNA, confirming HUWE1 as an E3 ligase for USP7 (Fig. 7G). In addition, TRIP12 levels correlated with those of USP7, further confirming USP7 as a deubiquitinase for TRIP12 (Fig. 7G). Stable expression of shRNA to HUWE1 reversed the repression of USP7 and TRIP12 in response to p16 (Fig. 7H). Moreover, in SCC-154 HPV(+)/p16(+) cells, inhibition of HUWE1 led to an increase in USP7 protein levels (Fig. 7I). Additionally, forced expression of p16 in HN5 cells led to ubiquitination of USP7, which was rescued by cotransfection with HUWE1 shRNA, thus confirming that p16 regulates USP7 through its ubiquitination by HUWE1 (Fig. 7J).
To understand how p16 controls HUWE1 transcription, we examined the HUWE1 promoter region, which contains binding sites for multiple enhancers and promoters, including specificity protein 1 (SP1). SP1 is a transcription factor and has been found to bind to p16, leading to increased transcriptional activity of the target gene but not total expression level25. To determine whether SP1 is the mediator of p16-driven upregulation of HUWE1, we inhibited SP1 expression in either HPV(-) cells forced to express p16 (Fig. 7K) or HPV(+)/p16(+) cells (Fig. 7L). In HN5 p16+ cells, HUWE1 levels increased as expected upon p16 expression, but this increase was completely reversed by SP1 inhibition (Fig. 7K). We recorded a similar reduction in HUWE1 expression in HPV(+) cells in which SP1 was inhibited (Fig. 7L). Thus, our results suggest that SP1 transcriptional activity is responsible for p16-driven HUWE1 upregulation.
HUWE1 expression is associated with disease-free survival in HPV(-) HNSCC
HUWE1 is mutated in approximately 10% of HNSCC. Based on this observation as well as the pathway we identified in our investigation, we examined the association between HUWE1 and outcome in the TCGA HNSCC patient cohort. We initially examined HUWE1 expression and found it to be elevated in HPV(+) tumors and markedly reduced in the few tumors with truncating HUWE1 mutations (Supplemental Figure 5). In HPV(-) patients, higher HUWE1 expression as a continuous variable was associated with improved DFS in univariate analysis (p=0.018) and remained significant in multivariate analysis, including tumor site and clinical stage (p=0.016). When divided into groups by HUWE1 expression (upper tertile vs. others), higher HUWE1 expression was associated with improved disease-free survival (DFS) (p=0.048) (Fig. 8A). Additionally, truncating mutations in HUWE1 led to a median survival of 9.4 months compared to 67.7 months in the remaining patients (p=0.008) (Fig. 8C). Interestingly, in HPV(+) patients, neither HUWE1 mutation nor its gene expression was associated with survival (Fig. 8B & D).