HSP90 promotes radioresistance of cervical cancer cells via reducing FBXO6‐mediated CD147 polyubiquitination

Abstract HSP90 inhibition might be a promising strategy to overcome the radioresistance of some cancers. In the current study, we further explored the mechanisms of HSP90 in regulating the radiosensitivity of cervical cancer cells. Bioinformatic analysis was performed based on data from TCGA‐CESC. Cellular and molecular studies were conducted using CaSki and SiHa and the derived radioresistant (RR) subclones. Through a proteomics screen, we identified HSP90 chaperones (both HSP90α and HSP90β) as CD147‐binding partners supporting its stabilization. Targeting HSP90 sensitized CaSki‐RR and SiHa‐RR cancer cells to irradiation partially through CD147 destabilization. Mechanistically, HSP90 interacts with FBXO6 and reduces FBXO6‐mediated proteasomal degradation of CD147. Enforced FBXO6 overexpression also sensitized CaSki‐RR and SiHa‐RR cancer cells to irradiation. These effects were enhanced using 17‐AAG treatment but were weakened by CD147 overexpression. Survival analysis further confirmed the association between high FBXO6 expression and favorable progression‐free survival among patients with cervical cancer. In conclusion, this study showed that HSP90 promotes radioresistance of cervical cancer cells partially via reducing FBXO6 mediated CD147 polyubiquitination. These findings help to explain why HSP90 inhibitor exerts radio‐sensitizing effects in cervical cancer.

recommended for all cases receiving definitive radiotherapy. 4 However, clinical outcome was heavily hampered by radioresistance, 5,6 the underlying mechanisms of which remain to be disclosed.
Heatshock protein 90 (HSP90) is a group of inducible ATPdependent molecular chaperones in response to cellular stress. Four isoforms of HSP90 have been identified in mammalian cells, including Hsp90α (encoded by HSP90AA1) and Hsp90β (HSP90AB1) in the cytosol, Grp94 in the endoplasmic reticulum, and Trap1 in mitochondria. 7 Unlike the small HSPs (such as HS40s and HSP70s), which primarily support the degradation of irreversibly damaged proteins and reduce aggregation of misfolded proteins, HSP90 mainly enhances the stability and function of conformationally labile proteins after primary folding. 8 Hsp90α and Hsp90β are highly homologous, with 85% sequence identity. 9 Therefore, these 2 proteins share similar activities in regulating client proteins. 10  Previous studies have observed that HSP90 inhibition might be a promising strategy to overcome radioresistance of some cancer cells, such as lung cancer cells, 11 soft tissue sarcoma cells, 12 pancreatic cancer, 13 and cervical cancer. 14,15 In the current study, we further explored the mechanisms of HSP90 in regulating the radiosensitivity of cervical cancer.

| Bioinformatic analysis
The expression profile of HSP90AA1 and HSP90AB1 in patients with primary cervical cancer and their association with survival outcomes, including progression-free survival (PFS) and overall survival (OS) were analyzed based on data extracted from The Cancer Genome Atlas Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (TCGA-CESC), using the UCSC Xena Browser. 16 Kaplan-Meier survival curves were generated between patients with the top 25% and bottom 25% of gene expression.

| Gene set enrichment analysis (GSEA)
GSEA was conducted using software 4.1.0 from the Broad Institute.
Patients with primary CESC in TCGA-CESC were separated into 2 groups by median HSP90AA1 or HSP90AB1 expression. Then, single-gene GSEA was conducted within the Hallmark gene sets.
Gene set permutations were set to 1000 to obtain a normalized enrichment score (NES). Only the gene sets with NES >1, Nominal (NOM) P < .05, and a false discovery rate (FDR) q-value <0.25 were compared.

| Cell culture and treatment
Cervical cancer cell lines CaSki and SiHa were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured following the method introduced previously. 17 X-ray resistant sublines (CaSki-RR and SiHa-RR) were generated following the strategy described previously. 18 In brief, cells were cultured in 25-cm 2 culture flasks and were irradiated with a 2 Gy dose at a rate of 1.

| Western blotting and immunoprecipitation (IP)
Total protein from cellular samples was extracted using a cold ra- After blocking with 5% BSA, the membranes were incubated with primary antibodies and corresponding secondary antibodies. The protein bands were developed using BeyoECL Star chemiluminescence reagents (Beyotime).
An IP assay was conducted using the Pierce Co-Immunoprecipitation Kit (ThermoFisher Scientific), and following the manufacturer's instruction. For each co-IP assay, 10 μg antibody was used. The eluted samples were subjected to western blotting.
The primary antibodies using included rabbit anti-HSP90α

| Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
LC-MS/MS assay was conducted to analyze the protein samples immunoprecipitated by anti-HSP90α or anti-HSP90β, following the strategy introduced in 1 previous study. 19

| Quantitative real-time reverse transcription-PCR (qRT-PCR) analysis
Total RNA was extracted from cellular samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was then reversely transcribed into cDNA using the PrimeScript RT Kit (Takara, Dalian, Liaoning, China).
qRT-PCR was performed using a SYBR Green kit (Takara). β-Actin mRNA was used as a reference gene for normalization. Relative gene expression was calculated using the 2 −ΔΔCt method. Primer sequences are listed in Table S1.

| Clonogenic assay
Cells were seeded into 24-well plates and were irradiated at de- X + B × X 2 )), in which Y is the fraction survival and X is the dose. A equals −1 times the initial slope, and the initial value of B equals −0.1 times the initial slope.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.1.2.
Quantitative data were reported as mean ±standard deviation (SD).
Statistical analysis of the statistical difference between 2 groups was estimated based on the unpaired t test with Welch correction.

| HSP90AA1 and HSP90AB1 upregulation was associated with poor survival of patients with cervical cancer
With RNA-seq data acquired from GTEx-fallopian tube (FT) (n = 5), GTEx-cervix (n = 13), and primary cervical cancer tissues from TCGA (n = 304), we examined the expression profile of HSP90AA1 and HSP90AB1. Results showed that HSP90AA1 expression was significantly upregulated in the tumor group compared with normal tissue groups ( Figure 1A, left). In comparison, no significant alteration of HSP90AB1 mRNA expression was observed among these groups ( Figure 1A, right  Figure 1B). However, no significant difference was observed in OS between the 2 groups ( Figure 1C). High HSP90AB1 expression was associated with significantly shorter PFS and OS, compared with the counterparts with the bottom quartile of gene expression ( Figure 1D,E). IHC staining showed that among 20 cases of cervical tumor tissues examined, 13/20 and 12/20 cases had medium to high level of HSP90α and HSP90β expression, respectively ( Figure 1F-H).
To understand the functional role of HSP90 in cervical cancer, we conducted a GSEA assay between patients with high and low HSP90AA1 or HSP90AB1 expression in TCGA-CESC (Table S2 and S3). GSEA identified that the gene set of DNA repair was associated with higher HSP90AA1 or HSP90AB1 expression (bold front, Figure S1). As tumor cells with a strong DNA repair capacity are usually radioresistant 22 and HSP90 inhibitor has been considered a promising radio-sensitizer, 23 we decided to explore the underlying mechanisms. Radioresistant CaSki (CaSki-RR) and SiHa (SiHa-RR) cells were generated ( Figure S2A-C). These 2 subclones had significantly elevated HSP90 expression at both mRNA and protein levels ( Figure S2D,E).

| HSP90 binds to and stabilizes CD147 in radioresistant cervical cancer cells
To explore the regulatory mechanisms of HSP90 in cervical can-  Figure S3B). Therefore, these findings implied that HSP90 interacts with and stabilizes CD147 in cervical cancer cells.

| Inhibition of HSP90 sensitizes radioresistant cervical cancer cells to radiotherapy partially via CD147
Previous studies have shown that HSP90 inhibition might be a promising strategy to overcome radioresistance in multiple cancers, 11,14 but the mechanisms were still not fully understood. As HSP90 can stabilize CD147 in cervical cancer cells, we explored whether CD147 expression contributed to radioresistance.
Cells with CD147 knockdown had reduced colony formation when exposed to X-ray IR ( Figure S4F,G). In contrast, cells with CD147 overexpression had enhanced colony formation after IR ( Figure S4H,I).

Similar experiments were performed using CaSki-RR and
SiHa-RR cells. These RR cells with stable CD147 knockdown ( Figure 3A,C) exhibited reduced colony formation when exposed to X-ray IR ( Figure 3E

| Inhibition of HSP90 enhances the polyubiquitination of CD147 that leads to proteasomal degradation
To understand the molecular mechanisms through which HSP90 stabilizes CD147 in radioresistant cervical cancer cells, we In addition, this mutant also counteracted 17-AAG-induced CD147 degradation ( Figure 5H-J).

| FBXO6 degrades CD147, which consequently sensitizes radioresistant cells to IR
As we found that FBXO6 could promote CD147 degradation, we then studied whether it modulated radiosensitivity via CD147.

FBXO6 overexpression significantly increased the sensitivity of
CaSki-RR and SiHa-RR cells to IR (2 Gy), the effect of which was enhanced by 17-AAG treatment but was weakened by CD147 overexpression ( Figure 6A-C). Moreover, western blot and immunofluorescence assays FBXO6 overexpression enhanced IR-induced expression of γ-H2AX and γ-H2AX foci ( Figure 6D,E). Similarly, the effect was enhanced by 17-AAG treatment, but was weakened by CD147 overexpression (Figure 6D,E). Overall, these data indicated that FBXO6 sensitized radioresistant cervical cancer cells to IR predominantly by inducing destabilization of CD147 via proteasomal degradation. Using survival data from TCGA-CESC, we confirmed that patients with high FBXO6 mRNA expression had significantly better PFS compared with their counterparts with low FBXO6 expression ( Figure 6F). However, this trend was not confirmed in OS ( Figure 6G).

| DISCUSS ION
In this study, we confirmed that HSP90 upregulation at the protein level was associated with enhanced radioresistance of cervical  to induce ubiquitination and degradation without the involvement of SCF. 43 Considering the promising radio-sensitizing effects of FBXO6 overexpression and CD147 inhibition in cervical cancer, it is meaningful to explore the detailed mechanisms of FBXO6-mediated CD147 degradation in the future.
In conclusion, this study found that HSP90 (both HSP90α and HSP90β) bound to FBXO6 and CD147 and reduced FBXO6mediated CD147 polyubiquitination through the proteasomal pathway, thereby promoting radioresistance of cervical cancer cells.
These mechanisms further explain why the HSP90 inhibitor exerts radio-sensitizing effects.

ACK N OWLED G M ENTS
None.

CO N FLI C T S O F I NTE R E S T
None of the authors have conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.