Fn14 overcomes cisplatin resistance of high-grade serous ovarian cancer by promoting Mdm2-mediated p53-R248Q ubiquitination and degradation
High-grade serous ovarian cancer (HGSOC) is the most lethal of all gynecological malignancies. Patients often suffer from chemoresistance. Several studies have reported that Fn14 could regulate migration, invasion, and angiogenesis in cancer cells. However, its functional role in chemoresistance of HGSOC is still unknown.
The expression of Fn14 in tissue specimens was detected by IHC. CCK-8 assay was performed to determine changes in cell viability. Apoptosis was measured by flow cytometry. The potential molecular mechanism of Fn14-regulated cisplatin resistance in HGSOC was investigated using qRT-PCR, western blotting, and Co-IP assays. The role of Fn14 in HGSOC was also investigated in a xenograft mouse model.
In this study, we found that Fn14 was significantly downregulated in patients with cisplatin resistance. Patients with low Fn14 expression were associated with shorter progression-free survival and overall survival. Overexpression of Fn14 suppressed cisplatin resistance in OVCAR-3 cells, whereas knockdown of Fn14 did not affect cisplatin resistance in SKOV-3 cells. Interestingly, Fn14 could exert anti-chemoresistance effect only in OVCAR-3 cells harboring a p53-R248Q mutation, but not in SKOV-3 cells with a p53-null mutation. We isolated and identified primary cells from two patients with the p53-R248Q mutation from HGSOC patients and the anti-chemoresistance effect of Fn14 was observed in both primary cells. Mechanistic studies demonstrated that overexpression of Fn14 could reduce the formation of a Mdm2-p53-R248Q-Hsp90 complex by downregulating Hsp90 expression, indicating that degradation of p53-R248Q was accelerated via Mdm2-mediated ubiquitin-proteasomal pathway.
Our findings demonstrate for the first time that Fn14 overcomes cisplatin resistance through modulation of the degradation of p53-R248Q and restoration of Fn14 expression might be a novel strategy for the treatment of HGSOC.
KeywordsHGSOC Cisplatin resistance Fn14 p53-R248Q Hsp90
American Type Culture Collection
Cell Counting Kit-8
Fibroblast growth factor-inducible 14
High grade serous ovarian cancer
Heat shock protein
Tumor necrosis factor-like weak inducer of apoptosis
Ovarian cancer is the most lethal of all gynecological malignancies and the fifth most common cause of tumor-related death among women worldwide . High-grade serous ovarian cancer (HGSOC) accounts for nearly 80% of all ovarian cancers, mostly diagnosed at advanced stages with poor prognosis . Compared to other subtypes, HGSOC is more aggressive with shorter progression-free survival . Despite surgical debulking and administration of platinum-based chemotherapy, majority of the patients suffer from drug resistance and disseminated disease leading to their death in less than 5 years. In addition, efforts aimed at developing new therapeutic approaches have largely been unsuccessful. As a representative of platinum anticancer drugs, cisplatin plays a crucial role in the treatment of HGSOC in clinical chemotherapy. Therefore, an improved understanding of the molecular mechanisms underlying cisplatin-resistance in HGSOC has the potential to significantly affect patient outcomes.
Fibroblast growth factor-inducible 14 (Fn14, also known as TNFRSF12A), receptor for cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is a member of the tumor necrosis factor receptor super-family [4, 5]. Fn14 expression has been widely detected in different mammalian tissues and most prominently in immunocytes, liver, kidney and heart. Several studies have found that Fn14 is related to inflammation and autoimmune disease by regulating pro-inflammatory cytokine secretion [6, 7]. Moreover, Fn14 is expressed in most solid tumors and reports have identified that Fn14 could regulate migration, invasion and angiogenesis in cancer cells [8, 9]. These studies mainly focused on the function of Fn14 in tumor metastasis and little is known about its role in chemoresistance. Cisplatin acts by forming a platinum complex inside a cell which binds to DNA. When DNA is cross-linked in this manner, the cell undergoes systemic cell death via apoptosis. The dysregulation of apoptotic pathways could result in post-target resistance to cisplatin . Furthermore, Fn14 could effectively inhibit tumor growth by promoting apoptosis in hepatocellular carcinoma and endometrial carcinoma [11, 12], but little is known about the underlying molecular mechanisms. Based on these findings, we hypothesized that Fn14 might overcome cisplatin resistance in HGSOC by modulating cisplatin-induced apoptosis. In this study, we identified that Fn14 attenuates chemoresistance via enhancing cisplatin-induced apoptosis in HGSOC. Furthermore, we have uncovered a mechanism wherein Fn14 causes apoptosis by inducing the ubiquitylation and degradation of p53-R248Q. Our findings indicate that Fn14 might act as a therapeutic target to improve the efficacy of cisplatin resistance and prognosis in HGSOC patients with p53-R248Q mutation.
Materials and methods
Seventy-one paraffin-embedded tissue samples were collected from patients who underwent ovary debulking surgery and received systemic treatment with a cisplatin plus paclitaxel-based regimen at the Affiliated Renji Hospital of Shanghai Jiaotong University, Shanghai, China between January 2013 and December 2018. Histological characterization of all these 71 samples indicated that they were high-grade serous ovarian cancer samples. The criterion to classify these samples as cisplatin resistant or sensitive was based on the literature . All specimens were re-evaluated independently by two experienced pathologists. Signed informed consent was obtained from all the patients involved in this study, and the experimental protocols were approved by the ethical committee of Renji Hospital.
Cell lines and clinical samples
Human HGSOC cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Tumor tissues from HGSOC patients were dissociated to single cells by enzymatic digestion. According to a previously described procedure , patient-1 and patient-2 cell lines were established from primary cells derived from patient-1 and patient-2 tumor samples, respectively. Cells were cultured in DMEM (Hyclone, GE Healthcare, UT, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone) and penicillin/streptomycin antibiotic solution (1:100, Sigma Aldrich, St. Louis, MO, USA) and incubated at 37 °C in a humidified atmosphere under 5% CO2 conditions.
IHC staining was performed on 4-μm sections of paraffin-embedded HGSOC samples to determine Fn14 expression level by using an anti-Fn14 antibody (Abcam, Cambridge, UK). In brief, the sections were subjected to standard procedures . The sections were incubated with an anti-Fn14 antibody (1:100). PBS staining served as the negative control. Two pathologists conducted the IHC scoring procedures independently, in duplicate. Score criterion of IHC was performed as follows, the percentage of staining: 0, < 5%; 1, 5–25%; 2, 25–50%; 3, 51–65%; and 4, > 65%. The intensity of staining: 0 = negative staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The final score was determined by multiplying the scores of percentage of staining with the intensity of staining. Low expression was defined as a score between 0 to 4, whereas high expression was defined as a score between 5 to 12.
Eight thousand cells per well were seeded in a 96-well plate before cisplatin treatment. During detection, each well was replaced with 100 μL of fresh medium containing 10 μL of CCK-8 and incubated for 1 h. The absorbance was measured at 450 nm by a microplate reader from Thermo Scientific (Massachusetts, USA).
For analysis of cell apoptosis, an Annexin V-FITC/PI apoptosis detection kit was used (BD Bioscience, San Jose, CA, USA). According to the manufacturer’s instructions, cells were collected and washed with binding buffer and then were incubated for 15 min with 5 μL of annexin V-FITC and 5 μL of PI. The apoptosis rate of the cells was examined by FAC Scan flow cytometry from Beckman Coulter (Brea, CA, USA).
Quantitative real-time PCR
Total RNA was extracted from cells using Trizol (Takara, Japan). cDNA was synthesized using a PrimeScript RT Reagent Kit (Takara) according to the manufacturer’s instructions. Quantification of mRNA was performed using SYBR Premix Ex Taq II (Takara) and CFX96TM PCR detection system (Bio-Rad, Hercules, CA, USA). GAPDH served as a reference gene. Relative expression was calculated using the comparative ΔΔCT method. The following primers were used: p53F: 5′ TGAGCGCTTCGAGATGTTCC 3′, p53R: 5′ GACTGGCCCTTCTTGGTCTT 3′, MDR1F: 5′ ATATCAGCAGCCCACATCAT 3′, MDR1R: 5′ GAAGCACTGGGATGTCCGGT 3′, BAXF 5′ TCCACCAAGAAGCTGAGCGAG 3′, BAXR: 5′ GTCCAGCCCATGATGGTTCT 3′.
Western blot analysis
RIPA buffer was used to lyse the cells and protein concentration of the cell lysate was measured by BCA protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Protein extract (20–30 μg) was loaded on SDS-PAGE gels (10% or 12%) and the separated proteins were transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h. Antibodies were diluted as follows: anti-Fn14 (1:1000, no.4403; Cell Signaling Technology, Beverly, MA, USA), anti-Bcl-2 (1:1000, no.2872; Cell Signaling Technology), anti-Caspase-3 (1:1000, no.9662; Cell Signaling Technology), anti-MDM2 (1:1000, no.86934; Cell Signaling Technology), anti-Hsp70 (1:1000, no. 4872; Cell Signaling Technology), anti-Hsp90 (1:1000, no. 4874; Cell Signaling Technology), anti-ubiquitin (1:1000, no.3933; Cell Signaling Technology), anti-p53 (1:1000, no.sc-47,698; Santa Cruz, CA, USA), and GAPDH (1:1000, no. 2118; Cell Signaling Technology).
Co-immunoprecipitation (co-IP) and ubiquitination assay
For Co-IP, 800 μg of protein extract was incubated overnight at 4 °C with primary antibody on a rotator. Antibodies were diluted as follows: anti-p53 (3 μg per 1000 μg protein, no.sc-47,698; Santa Cruz), MDM2 (1:100, no.86934; Cell Signaling Technology). Agarose beads (20 μL) were added to the above mixture and incubated for at least 2 h. The mixture was then centrifuged and washed three times with PBS. Samples were resuspended in 20 μL of gel loading buffer and western blotting was performed as indicated above.
Sequencing of p53 in primary cells isolated from patient samples
Genomic DNA was extracted and purified according to the manufacturer’s instructions (Qiagen). The R248Q p53-coding region was sequenced using the primers as previously described .
Negative control siRNA and p53 siRNA were purchased from Integrated Biotech Solutions (Shanghai, China). Cells were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and were harvested after 48 h post-transfection. p53, Sense-1: 5′ GAGGGATGTTTGGGAGATGTA 3′, Sense-2: 5′ GAGGGATGTTTGGGAGAT-GTA 3′ Fn14, Sense-1: 5′ CAUCCAUUCUAGAGCCAGUCUTT 3′, Sense-2 5′ GAGGGAGA-AUUUAUUAAUAAATT 3′, Mdm2, Sense: 5′ AGGCAAAUGUGCAAUACCAUU 3′.
The control and overexpression Fn14 lentivirus were purchased from Gikai gene (Shanghai, China). The lentivirus was introduced into OVCAR-3 and primary cells by adding to cell growth medium. Then, stably overexpressing Fn14 cells or control cells were selected using medium containing 1 μg/ml puromycin (Sigma-Aldrich, Milwaukee, WI, USA).
Five weeks-old BALB/c nude mice were subcutaneously injected with 3 × 106 OVCAR-3 cells. Once the tumors reached a volume of approximately 80–100 mm3 (14 days post-injection), the mice were randomly divided into two groups (n = 5) and administered chemotherapy. Cisplatin (4 mg/kg) was administered intraperitoneally twice a week up to 4 weeks. The tumor volume was calculated with the formula: V = (length × width2)/2.
Sections from OVCAR-3 xenografts described above were processed using the In Situ TUNEL detection kit according to the manufacturer’s instructions (Beyotime, Shanghai, China). Positively stained cells were identified by bright fluorescence co-localized to a DAPI positive nucleus.
The analyses were performed using Prism 7.0 software (GraphPad, Inc., San Diego, CA, USA). Unpaired Student’s t test was performed for comparison between two groups. Fisher’s exact test was performed for clinicopathological data analysis. Values of p < 0.05 were considered as statistically significant. Kaplan–Meier analysis was performed to evaluate the survival. Data are presented as mean ± SEM for three independent experiments.
Loss of Fn14 coincided with chemoresistance and poor prognosis of HGSOC
Clinicopathological features of HGSOC tissues with regard to the relative expression of Fn14
Fn14 IHC (n)
(n = 48, 67.6%)
(n = 23, 43.3%)
≥ 2 cm
The effects of Fn14 on cisplatin-resistance in HGSOC cells
Fn14 inhibits cisplatin resistance in HGSOC primary cancer cells with p53-R248Q mutation
Fn14 attenuates cisplatin-resistance by down-regulation of p53-R248Q
Fn14 enhances ubiquitylation and degradation of p53-R248Q by down-regulation of heat-shock protein (Hsp) 90
Overexpression of Fn14 alleviates cisplatin resistance in vivo
In HGSOC, patients often fail to respond to available chemotherapeutic agents and suffer from recurrence in 2 years after initial anticancer treatment. Therefore, identifying the molecular mechanism of chemoresistance to improve the prognosis of patients is critical. In this study, we found that Fn14 was down-regulated in HGSOC patients with cisplatin resistance. Functional studies revealed that ectopic overexpression of Fn14 alleviated cisplatin resistance in HGSOC cells with p53-R248Q mutation both in vitro and in vivo. Mechanistically, we found that Fn14 could reduce p53-R248Q protein expression by enhancing ubiquitin-mediated degradation of p53-R248Q by down-regulation of Hsp90. Thus, our study identified that Fn14 played an important role in abolishing cisplatin resistance in HGSOC and also uncovered the mechanisms of regulation of mutant p53 degradation.
Remarkably, studies have shown that Fn14 plays a crucial role in various malignant tumors. Fn14 contributes to regulating human tumor cell migration, invasion, and metastasis. However, its role in chemoresistance has not yet been studied in HGSOC. Cisplatin-induced DNA damage leads to the activation of a multibranched signaling cascade with proapoptotic outcomes and anti-apoptosis is one of the important mechanisms of chemoresistance leading to therapeutic failure. Fn14 appears to exhibit tumor suppressive activity by inducing apoptosis in endometrial cancer, colon carcinoma and hepatocellular carcinoma [11, 12, 21], suggesting that Fn14 might affect chemoresistance. As expected, we found that patients with low expression of Fn14 in HGSOC are more likely to exhibit cisplatin resistance, suggesting that Fn14 might be a biomarker for cisplatin resistance. Moreover, in our study (n = 71), patients with high Fn14 expression had significantly longer progression-free survival and overall survival. To clarify the suppressive mechanism of Fn14 in chemoresistance, overexpression of Fn14 in OVCAR-3 cells inhibited chemoresistance by increasing cisplatin-induced DNA damage and apoptosis. Unexpectedly, knockdown of Fn14 in SKOV-3 cells had no effect on chemoresistance. This discrepancy suggests that the suppressive role of Fn14 varies in different cancer types and other unreported mechanisms may be involved in the anti-chemoresistant effects of Fn14 in HGSOC. Cisplatin is a DNA damaging agent dependent on p53 for potent activity and HGSOC patients harbor p53 mutations. Of note, we found that Fn14 lost its anti-chemoresistance function in SKOV-3 cells harboring p53 null mutation. Moreover, overexpression of Fn14 markedly affected cisplatin resistance in OVCAR-3 cells bearing p53-R248Q mutation. Taken together, these data strongly demonstrate that Fn14 inhibits chemoresistance in HGSOC with p53-R248Q mutation. To further validate these findings, we isolated and identified primary cells with p53- R248Q mutation from HGSOC patient samples. Furthermore, overexpression of Fn14 in primary cells alleviated chemoresistance by promoting DNA damage and apoptosis. Therefore, these data indicate that Fn14 attenuated cisplatin resistance in HGSOC patients with p53-R248Q mutation.
P53 mutations can demonstrate abnormal GOF properties to facilitate oncogenesis and chemoresistance and this phenomenon has been widely confirmed by many in vivo and in vitro experiments . Mutant p53 protein can misfold and form amyloid fibrillar aggregates in ovarian cancer and this aggregation promotes platinum resistance . In addition, p53 V172F mutation also promotes cisplatin resistance in ovarian cancer . In this study, knockdown of p53-R248Q significantly inhibited cisplatin resistance in OVCAR-3 cells and primary cells, which is consistent with previous studies elucidating the role of mutant p53. These findings indicate that Fn14 might affect cisplatin resistance by regulating p53-R248Q. Notably, in HGSOC cells, overexpression of Fn14 reduced the expression of p53-R248Q protein and its downstream target genes related to drug-resistance. Taken together, we demonstrated that Fn14 overcomes cisplatin resistance in HGSOC by down-regulating p53-R248Q.
Further, our study found that Fn14 decreased the expression of p53-R248Q protein, but had no effect on p53 mRNA, indicating that Fn14 might regulate p53 expression at post-translational level. Wild-type p53 (wtp53) is regulated mainly by Mdm2, an E3 ubiquitin ligase that promotes the ubiquitylation-dependent proteasomal degradation . Generally, wtp53 is short-lived and is rapidly degraded, but p53-R248Q is stable and accumulates increasingly in the nucleus by escaping MDM2-mediated degradation. The stabilization of p53-R248Q protein is a prerequisite for the manifestation of the gamut of its various gain-of-function (GOF) properties . In our study, we found that Fn14 overexpression could accelerate degradation of p53-R248Q by increasing its ubiquitination. Several studies have revealed that wtp53 undergoes transient interactions with the HSPs. However, mutant p53 in tumors could stably interact with Hsp70 and Hsp90 forming p53-R248Q-Mdm2-Hsp complex to escape Mdm2-mediated degradation . We identified that Fn14 overexpression could down-regulate the expression of Hsp90 in HGSOC cells. We thus speculated that Fn14 might disrupt the p53-R248Q-Mdm2-Hsp90 complex. Subsequent Co-IP experiments substantiated this hypothesis in OVCAR-3 and primary cells, suggesting that Fn14 overexpression could elevate Mdm2-mediated p53 ubiquitination and degradation by targeting Hsp90.
Collectively, our findings successfully demonstrate for the first time that Fn14 could overcome cisplatin resistance through modulating the degradation of p53- R248Q and restoration of Fn14 expression might be a novel strategy for the treatment of HGSOC. Certainly, apart from the role of Fn14 revealed in our study, it has a multitude of other biological functions. Further studies are warranted to investigate the mechanism of down-regulation of Hsp90 by Fn14.
We kindly thank the National Natural Science Foundation of China, Pudong New Area Municipal Commission of Health and Family Planning and Shanghai Municipal Education Commission for the funding.
This work was supported by National Natural Science Foundation of China (81272884 to Q. L. and 81874197 to H. L), Project of Pudong New Area Municipal Commission of Health and Family Planning (PW2016E-2), Project of Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (20161412) and Key Discipline Project of Shanghai Municipal Commission of Health and Family Planning (15GWZK0701).
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
LHQ, LH and WD designed the whole experiments; AYW and LYG conducted molecular biology experiments; AYW, LH and LHQ wrote the manuscript; CW, MXC and WJW conducted the clinical material collection and performed the statistical analyses. All authors read and approved the final manuscript.
Ethics approval and consent to participate
For animal experiments, the experiments were performed according to the Shanghai Medical Experimental Animal Care Guidelines. Animal protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University.
The characteristics of the patients and their tumours were collected through the review of medical records and pathologic reports. Signed informed consent was obtained from all the patients involved in this study, and the experimental protocols were approved by the ethical committee of Renji Hospital. All of the methods in this study were in accordance with the approved guidelines, and all of the experimental protocols were approved by the ethics committee of Renji Hospital.
Consent for publication
All the authors of this review give their consent for publication.
The authors declared no conflict of interest.
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