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American Journal of Cancer Research logoLink to American Journal of Cancer Research
. 2021 Mar 1;11(3):640–667.

IAP-1 promoted cisplatin resistance in nasopharyngeal carcinoma via inhibition of caspase-3-mediated apoptosis

Xiangwan Miao 1,*, Zeyi Deng 1,*, Siqi Wang 1, Huanhuan Weng 1, Xinting Zhang 1, Hailiang Li 2, Huifen Xie 1, Juan Zhang 1, Ying Zhong 2, Bohui Zhang 3, Quanming Li 2, Minqiang Xie 1,2
PMCID: PMC7994165  PMID: 33791146

Abstract

Recurrent/metastatic nasopharyngeal carcinoma (NPC) is known for having a poor prognosis due to its unfavorable response to chemoradiotherapy. However, the specific processes involved remain poorly understood. This study focused on the cisplatin-resistance mechanism in NPC to help understand the occurrence of advanced NPC and aims to explore the potential therapeutic target for cisplatin-resistant NPC. Two cisplatin-resistant NPC cell lines, HNE-1/DDP and CNE-2/DDP, were established and the differentially expressed genes (DEGs) between parental and cisplatin-resistance cell lines, filtering from high-throughput sequencing results, were analyzed. Next, the effects of IAP-1 on cisplatin-resistant nasopharyngeal cancer cell proliferation, apoptosis, drug resistance and associated cell signaling were evaluated in vitro and in vitro. From our bioinformatic results, more than 15,000 differentially expressed genes (DEGs) were found between parental and resistant cell lines. Nine related DEGs were found in the classic platinum resistance pathway, three of which (ATM, IAP-1, and IAP-2) also appeared in the top five differentially expressed pathways, with elevated IAP-1 showing the highest fold change. Further studies revealed that high IAP-1 expression can lead to an increased cisplatin inhibitory concentration and apoptosis inhibition. IAP-1 silencing can induce upregulation of the caspase-3 and enhance the antiproliferation and proapoptotic effects of cisplatin. Clinical data also showed that IAP-1 overexpression was associated with a worse survival status. In summary, in vitro and in vivo experiments demonstrated that IAP-1 plays a vital role in cisplatin resistance by regulating caspase induced apoptosis and serve as a potential novel therapeutic target and a prognostic indicator for advanced NPC.

Keywords: Nasopharyngeal carcinoma (NPC), cisplatin, drug resistance, IAP-1, apoptosis

Introduction

Nasopharyngeal carcinoma (NPC) is a common malignancy of the head and neck, especially in South China, Southeast Asia, and North Africa [1]. According to the National Comprehensive Cancer Network (NCCN) guidelines 2020, radiation therapy combined with scheduled cisplatin-based chemotherapy is still the main treatment for NPC [2]. Outcomes in patients with primary NPC have improved because of advances in radiotherapy combined with chemotherapy, producing a 3-year survival rate of approximately 85% [3-5].

However, recurrence rates after primary treatment range from 15% to 58% [6-8], and the overall survival (OS) of patients with recurrent or metastatic NPC is very poor, with a median OS of approximately 20 months [9]. Traditionally, cisplatin has been regarded as the standard first-line treatment for recurrent NPC because of statistically significant improvement in progression-free survival (PFS) [10]. Resistance to chemotherapy may occur initially or later after the first line of chemotherapy [11]. Few clinical trials have assessed the intrinsic and acquired resistance of chemotherapy drugs, especially cisplatin [12-14], and treatment options are scarce after first-line chemotherapy failure [2]. Therefore, clarifying the mechanism of chemoresistance in NPC will contribute to early diagnosis, developing appropriate therapy, and improving survival and quality of life for NPC patients.

Many mechanisms may help explain cisplatin resistance, including reduced drug absorption, increased DNA repair, activated anti-apoptotic pathways, restricted formation of cisplatin-DNA adducts and others [15]. However, regardless of the pathway, the final step is to inhibit the programmed cell death pathway, which is known as apoptosis [16]. Inhibition of apoptosis enhances the survival of cancer cells and facilitates their escape from immune surveillance and cytotoxic therapies [17].

The principal molecules contributing to caspase-induced apoptosis inhibition belong to the anti-apoptosis protein family, which consists of eight members, five of which have been widely studied in cancers, including IAP-1 (also known as (aka) MIHC/c-IAP2), IAP-2 (aka MIHB/c-IAP1), X-IAP (aka MIHA/HILP), livin (aka ML-IAP/KIAP) and survivin [18]. According to recent reports, IAP (inhibitor of apoptosis protein) family proteins are overexpressed in various types of tumors, such as medulloblastoma [19], testicular germ cell tumors [20], pancreatic cancer [21] and multiple other cancers [22]. Previous studies have shown that elevated IAP-1/IAP-2 indicated a poor prognosis, while ectopic X-IAP suggested improved survival [19,23-27]. Several mechanisms are involved in IAP-regulated cell apoptosis, including Fas, TNF-α [26], FAK [24], NF-kB [18,28] and other pathways [19,27,29]. In addition to regulating caspase-induced apoptosis, IAPs can also regulate apoptosis by controlling necrosis [30], cell proliferation, the cell cycle, and other processes [18,31-34].

To better understand the molecular mechanisms of drug resistance in NPC, we established cisplatin-resistant NPC cell lines (HNE-1/DDP and CNE-2/DDP) and a xenograft mouse model in this study. Then, we performed mRNA sequencing to screen differentially expressed mRNAs and investigate therapeutically actionable targets or pathways in drug-resistant NPC cell lines, and we found that IAP-1 overexpression contributed to cisplatin resistance in NPC. Next, we explored the biological function of IAP-1 in the chemotherapy response both in vitro and in vivo and revealed the regulated mechanism associated with apoptosis inhibition by regulating caspase-3 activation. Taken together, our results identified a novel biomarker in promoting chemoresistance through caspase-3-induced apoptosis inhibition and suggest that IAP-1 may serve as a predictor of drug resistance and a potential therapeutic target in NPC.

Material and methods

Cell lines and tissue samples

The human NPC cell lines HNE-1, CNE-1, CNE-2, HONE-1, and SUNE-1 were kept in our laboratory and maintained in RPM1640 medium (Gibco, Grand Island, NY, USA) containing L-glutamine with 10% fetal bovine serum (Bioind, Kibbutz Beit Haemek, Israel), penicillin (100 U/mL) and streptomycin (100 U/mL) in an incubator at 37°C with 5% CO2. Cisplatin-resistant HNE-1/DDP or CNE-2/DDP cells were obtained by culturing cells in gradually increasing doses of cisplatin up to 1.0 μg/ml for a total of 7 months in our laboratory. Primary tissue samples (nasopharyngeal carcinoma, n = 30; normal, n = 12) were anonymized and obtained in accordance with the Zhuhai People’s Hospital (Zhuhai, Guangdong, China) Institutional Review Board.

In vitro assays in NPC cell lines

Cell viability, cell proliferation and drug resistance were evaluated using an MTT assay. A wound-healing assay was performed for cell motility analysis. A colony formation assay was conducted to test the colony formation ability. Flow cytometry was used to determine apoptosis and the cell cycle distribution. Caspase-3 activity was measured using a caspase-3 assay kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. A proliferation assay was performed using a commercial EdU kit according to the manufacturer’s instructions [kFluor488 Click-iT EdU Imaging Assay kit, KeyGen, Nanjing, Jiangsu, China]. The RNA and protein levels of the target genes were examined by real-time PCR (qRT-PCR) and Western blot (The primers (Sangon, Shanghai, Shanghai, China) are listed in supplementary material Table S1). After establishing the resistant cell lines, RNA-seq analysis was used to screen differentially expressed genes (DEGs) with the following default criteria: fold change ≥2 and divergence probability ≥0.8. (They will be provided during review.).

Nude mouse xenograft and in vivo assays

All murine studies were performed under the ethical regulation of the institutional guidelines of Guangdong Province and China animal welfare regulations. In vivo experiments were performed in strict agreement with the institutionally approved protocol. All experiments were approved by the Southern Medical University Experimental Animal Ethics Committee. Male BALB/c nude mice aged 4-6 weeks (n = 20) were purchased from the Laboratory Animal Center of Southern Medical University. Each mouse was inoculated subcutaneously in the right flank with NPC cells with different treatments (4 groups, 5 in each group). Body weight, tumor growth and the general behavior of the mice were monitored. For in vivo chemosensitivity assays, the animals were treated with cisplatin via intraperitoneal injection (4 mg/kg body weight etoposide [once every 2 d]). At the end of the experiment, the mice were sacrificed, and the tumors and organs were surgically dissected. Tumors and organs were analyzed by immunohistochemistry and TUNEL assay.

Gene ontology and pathway enrichment analysis of DEGs

Gene Ontology (GO) annotation analysis was performed for screened DEGs. After obtaining GO annotations for DEGs, WEGO software was used for GO functional classifications. The KEGG database was used for pathway enrichment analysis of DEGs. The calculated P-values were Bonferroni-corrected, and a corrected P-value ≤0.05 was set as the threshold. Terms fulfilling this condition are defined as significantly enriched terms in DEGs.

Statistical analyses

SPSS statistics version 13.0 for Windows (SPSS Inc., Chicago, Ill., USA) was used for all analyses. To compare possible differences between groups, an independent-sample t-test or one-way ANOVA was used. The data are presented as the mean ± SD of at least three independent experiments. Differences with P-values See the Supplementary Data for additional methods.

Results

Establishment of the cisplatin-resistant NPC cell lines HNE-1/DDP and CNE-2/DDP

To better understand the mechanism behind drug resistance in NPC, stable in vitro models were first acquired by traditional continuous culture methods. By continuously culturing the NPC cell lines HNE-1 and CNE-2 for 7 months in gradually increasing doses of cisplatin up to 1 µg/ml, we established cisplatin-resistant NPC cell lines (HNE1/DDP and DNE-2/DDP). The relative cisplatin resistance was verified by the IC50 through MTT assay. The resistance index showed that the established HNE-1/DDP and CNE-2/DDP cells were 10- to 40-fold more resistant to cisplatin than their parental cell lines (Figure 1A; Table 1). By three months of culture without cisplatin, the IC50 of HNE-1/DDP and CNE-2/DDP cells had decreased to approximately 84% and 78%, respectively, of that of HNE-1/DDP and CNE-2/DDP cells cultured continuously in the presence of 1 µg/ml cisplatin (Table 2). In addition, the resistance of cells recovered from liquid nitrogen remained at approximately 90% of that of cells grown continuously in cisplatin. These results showed that HNE-1/DDP and CNE-2/DDP cells present stable resistance to cisplatin.

Figure 1.

Figure 1

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Establishment of the cisplatin-resistant NPC cell lines HNE-1/DDP and CNE-2/DDP. A. MTT assays showed that resistant cell lines had increased IC50 values (*P<0.05). B, C. Cell cycle analysis showed longer G0/G1 and S periods and a shorter G2/M period (HNE-1 vs HNE-1/DDP *P<0.05; CNE-2 vs CNE-2/DDP *P<0.05). D. The growth curve indicated that the cisplatin-resistant cell lines had an increased cell growth time compared with their parental cell lines (HNE-1 vs HNE-1/DDP *P<0.05; CNE-2 vs CNE-2/DDP *P<0.05). E, F. Flow cytometry showed that resistant cell lines presented lower percentages of apoptotic cells (HNE-1 vs HNE-1/DDP *P<0.05; CNE-2 vs CNE-2/DDP *P<0.05). G. Morphology between the groups showed no significant differences. Original magnification ×100, bar = 100 µm; insets ×400, bar = 20 µm. H, I. The wound-healing assay showed no notable change (HNE-1 vs HNE-1/DDP P P-value = 0.1; CNE-2 vs CNE-2/DDP P = 0.70). J, K. Colony formation assays showed that resistant cell lines presented fewer cell colonies (HNE-1 vs HNE-1/DDP *P<0.05; CNE-2 vs CNE-2/DDP *P<0.05). Data are presented as the mean ± SD of at least three independent experiments. Statistical significance is indicated by *P<0.05.

Table 1.

IC50 values of the common chemotherapy drugs in the established cell lines

Drugs IC50/(µg/ml) Resistance Index, RI IC50/(µg/ml) Resistance Index, RI
HNE-1 HNE-1/DDP CNE-2 CNE-2/DDP
Cisplatin 0.594 7.08 11.92 0.453 21.341 47.11
Paclitaxel 0.16 0.161 1.01 0.079 0.522 6.61
5-Fluorouracil 0.539 0.55 1.02 0.728 0.933 1.28
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Table 2.

Stability of the established cell lines

Periods IC50/(µg/ml) Resistance Index, RI IC50/(µg/ml) Resistance Index, RI
HNE-1 HNE-1/DDP CNE-2 CNE-2/DDP
Established 0.594 7.08 11.92 0.453 21.341 47.11
3 months without CDDP - 5.979 10.07 - 16.717 36.90
6 months’ storage in liquid nitrogen 0.679 8.26 12.16 0.459 20.252 44.12
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Furthermore, to determine the characteristics of the established cisplatin-resistant cell lines, we detected the cell cycle, proliferation, apoptosis, motility, and other biological characteristics of our resistant cell lines. As shown in Figure 1, HNE-1/DDP and CNE-2/DDP cells had a longer DNA synthesis phase (Figure 1B, 1C and Table S2). HNE-1/DDP and CNE-2/DDP cells had a slower growth time with doubling times of approximately 67 hours and 71 hours, respectively, compared to HNE-1 (50 hours) and CNE-2 (43 hours) cells (Figure 1D and Table S3). We also observed 5% and 3% reductions in the apoptosis ratios of HNE-1/DDP and CNE-2/DDP cells, respectively, compared to the ratios in their parent cell lines (Figure 1E, 1F), but no significant changes were noted in their morphology and proliferation ability (Figures 1G and S1). Then, we evaluated the motility of cells by wound-healing assay, and the results indicated no notable change (Figure 1H, 1I). In addition, colony formation results also indicated that the growth time of the cisplatin-resistant cell lines was relatively longer than that of the parental cell lines (Figure 1J, 1K and Table S4), which is similar to the multiple-resistance promyelocytic leukemia cell line HL60R [35] but different from the trends for other resistant cancer cell lines such as H69AR [36]. In summary, the established cisplatin-resistant cell lines (HNE-1/DDP and CNE-2/DDP) showed lower apoptosis ratios, slower growth rates and a higher IC50, which is consistent with the characteristics of clinical drug-resistant samples.

Cisplatin-resistant NPC cells presented differential gene expression

To explore the molecular targets and potential mechanisms associated with cisplatin resistance in NPC, total mRNA sequencing was performed to analyze differences in mRNA expression between parental and cisplatin-resistant cells (group 1: HNE-1 vs HNE-1/DDP and group 2: CNE-2 vs CNE-2/DDP). Among the 16,351 differentially expressed mRNAs in the HNE-1 group, 948 of them showed significant differences after NoiseSeq filtering, and 458 of them were upregulated (Figures 2A, S2, S3). Similarly, 16,222 DEGs were found in the CNE-2 vs CNE-2/DDP groups (Figure 2B), including 402 upregulated and 250 downregulated DEGs.

Figure 2.

Figure 2

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Cisplatin-resistant NPC cells presented differential gene expression. A, B. DEGs in the HNE-1 vs HNE-1/DDP groups and CNE-2 vs CNE-2/DDP groups with the following default criteria: fold change ≥2 and divergence probability ≥0.8. C-E. Molecular function, biological process, and cellular component results of the GO analysis in two comparison groups (HNE-1 vs HNE-1/DDP groups and CNE-2 vs CNE-2/DDP groups). F. KEGG analysis results of both groups. The calculated P-value was Bonferroni-corrected, and a corrected P-value ≤0.05 was set as the threshold. G, H. PCA of the downregulated and upregulated DEGs between the two comparison groups.

Then, GO enrichment analysis was used to identify key molecular functions, biological processes, and cellular components between parental and resistant cell lines (HNE-1 vs HNE-1/DDP or CNE-2 vs CNE-2/DDP) (Figure 2C-E), while KEGG analysis was used to identify key pathways (Figure 2F). As shown in Figure 2C, the main enriched biological processes in DEGs were cellular process, metabolic process, and biological regulation. For molecular functions, most of the DEGs were enriched in the terms binding activity, catalytic activity, and transporter activity (Figure 2D). Cell part, organelle and membrane were the most differentially expressed cellular components between resistant and parental cell lines (Figure 2E). The top 5 differentially expressed pathways were complement and coagulation, ECM-receptor interaction, lysosome, transcription misregulation in cancer and focal adhesion (Figure 2F). As shown, there were 105 and 150 identical DEGs in the downregulated and upregulated DEGs between the two comparison groups (Figure 2G and 2H).

Next, by using Cytoscape, we filtered 49 clustered key nodes among all the DEGs (see Table S5). The network structure diagrams of downregulated and upregulated DEGs are shown in Figure 3A and 3B, respectively. Moreover, a network with 3 DEGs (ATM, Birc-3: a gene coding the protein IAP-1, Birc-2: a gene coding the protein IAP-2) was also expressed in the platinum drug resistance pathway. Then, we conducted a specific study on the DEGs involved in the classic platinum drug resistance pathway in the KEGG database and found that 9 DEGs in our data belonged to the platinum drug resistance pathway. Unexpectedly, consistent with the clustering results, three DEGs (ATM, Birc-3, and Birc-2) from the 9 DEGs also appeared in the top five differentially expressed pathways (Figure 2F and Table S6) in our bioinformatics results (Figure 3C), while elevated IAP-1 had the highest fold change (log2 ratio = 4.22). Here, since the other three pathways had no DEGs associated with the platinum resistance pathway, we present only the results of the platinum resistance pathway with focal adhesion and transcriptional dysregulation pathways. Based on the high-throughput screening results, our findings revealed that IAP-1 might be a potential molecule contributing to the regulation of cisplatin resistance in NPC.

Figure 3.

Figure 3

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Birc3 gene (Protein: IAP-1) showed the highest fold change among the DEGs. A. Downregulated DEGs of 49 clustered key nodes. B. Upregulated DEGs of 49 clustered key nodes. C. Three DEGs in the classic platinum drug resistance pathway (ATM, Birc-3 for IAP-1, and Birc-2 for IAP-2) also appeared in the top 5 differentially expressed pathways in the HNE-1 vs HNE-1/DDP group analysis results (IAP-1 log2 ratio = 4.22, probability = 0.95; IAP-2 log2 ratio = 1.10, probability = 0.88; ATM log2 ratio = 1.03, probability = 0.82).

IAP-1 overexpression was involved in the NPC oncogenesis mechanism

After establishing cisplatin-resistant cell lines and screening differentially expressed genes through total mRNA sequencing, we then compared the expression of IAP-1 in open-source clinical databases to further verify our previous findings. Oncomine and GEO2R were used to analyze the GEO and Oncomine databases. As shown in Figure 4A, IAP-1 expression in all 520 head and neck carcinoma cases was much higher than that in the normal group, which is similar to the results in the NPC group (Figure 4B). Meanwhile, multiple tumor results demonstrated relevance between tumor types and IAP-1’s biological function. As shown in Figure 4C, oropharyngeal carcinoma showed the highest IAP-1 expression. Furthermore, IAP-1 overexpression was also correlated with an advanced clinical stage and a higher tumor grade (Figure 4D, 4E).

Figure 4.

Figure 4

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IAP-1 overexpression was involved in the NPC oncogenesis mechanism. A, B. IAP-1 was relatively highly expressed in head and neck carcinoma and NPC in the Oncomine database (A. All head and neck carcinoma *P<0.05; B. Sengupta nasopharyngeal carcinoma *P<0.05). C. IAP-1 was relatively high in NPC and pharyngeal carcinoma in multiple tumor types (1. Oropharyngeal carcinoma vs Normal *P<0.05; 2. Tonsillar carcinoma vs Normal *P<0.05; 3. Floor of the Mouth Carcinoma vs Normal *P<0.05; 4. Tongue carcinoma vs Normal *P<0.05). D. The results showed higher IAP-1 expression at advanced clinical stages (Normal vs Stage 3 *P<0.05; Normal vs Stage 4 *P<0.05). E. The results showed higher IAP-1 expression with higher tumor grades (Normal vs Grade 2 *P<0.05; Normal vs Grade 3 *P<0.05; Normal vs Grade 4 *P<0.05). F. Kaplan-Meier analysis of head and neck carcinoma patients in the Oncomine database showed that high IAP-1 expression was associated with a worse outcome (-, negative; +, positive; *P<0.05). G. IAP-1 expression was higher in nasopharyngeal carcinoma patients in GDS-3341 from the GEO database (Control vs Cancer: *P<0.05). H-J. IAP-1 was relatively highly expressed in other head and neck carcinomas in the Oncomine database (H. Ginos *P<0.05; I. Toruner *P<0.05; J. Peng *P<0.05). K. IAP-1 expression levels in various tumor types in the Oncomine database. Statistical significance is indicated by *P<0.05.

In addition, high IAP-1 expression was also related to poor survival status in NPC and HNC (Figure 4F), which was consistent with the GEO results (Figure 4G) and other head and neck carcinoma results from the TCGA database (Figure 4H-J). Notably, IAP-1 was not always overexpressed in every tumor type but was upregulated in HNSC (Figure 4K). Taken together, these results suggest a strong correlation between elevated IAP-1 expression and poor survival in head and neck carcinomas, especially in NPC.

Overexpression of IAP-1 promotes cisplatin resistance in NPC

To comprehensively determine the specific regulatory mechanism of IAP-1 in the cisplatin resistance of NPC, we upregulated IAP-1 in the parental cell lines HNE-1 and CNE-2 by lentivirus-mediated stable overexpression and downregulated IAP-1 expression in HNE-1/DDP and CNE-2/DDP cells by shRNA-mediated stable knockdown. As shown in Figure 5A and 5B, the lentivirus showed high transfection efficiency (>80%), and there were no significant differences in cell morphology among all the groups after transfection (Figure 5C). We then needed to explore the efficiency of transfection using qRT-PCR and Western blot. The results showed that IAP-1 was upregulated in HNE-1/DDP and CNE-2/DDP cells, with 5-fold and 2.3-fold increases in RNA expression levels (Table 3; Figure 5D, 5E) and 12.34-fold and 5.12-fold increases in protein expression levels (Figure 5G, 5H), respectively.

Figure 5.

Figure 5

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The expression levels of IAP-1 were successfully upregulated and downregulated by lentivirus-mediated stable overexpression and knockdown. A. Transfection efficiency of the lentivirus. Original magnification ×200, bar = 20 µm. B. Transfection percentage of the lentivirus. C. Morphology between the groups showed no significant differences. Original magnification ×100, bar = 100 µm; insets ×400, bar = 20 µm. D. RT-PCR results of the IAP-1 mRNA expression level in the HNE-1 groups (HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs IAP-1-HNE-1 *P<0.05; IAP-1-HNE-1 vs HNE-1 *P<0.05). E. RT-PCR results of the IAP-1 mRNA expression level in the CNE-2 groups (CNE-2 vs CNE-2/DDP *P<0.05; shIAP-1-CNE-2 vs CNE-2/DDP *P<0.05; shIAP-1-CNE-2 vs IAP-1-CNE-2 *P<0.05; IAP-1-CNE-2 vs CNE-2 *P<0.05). F. MTT assays showed that IAP-1-HNE-1 had an increased IC50 value (*P<0.05) and shIAP-1-HNE-1/DDP showed a decreased IC50 value (*P<0.05). G, H. Western blot assay results of the protein expression level of IAP-1 in two different groups (The membranes have been cropped). (HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs IAP-1-HNE-1 *P<0.05; IAP-1-HNE-1 vs HNE-1 *P<0.05. CNE-2 vs CNE-2/DDP *P<0.05; shIAP-1-CNE-2 vs CNE-2/DDP *P<0.05; shIAP-1-CNE-2 vs IAP-1-CNE-2 *P<0.05; IAP-1-CNE-2 vs CNE-2 *P<0.05). Data are presented as the mean ± SD of at least three independent experiments. Statistical significance is indicated by *P<0.05.

Table 3.

IC50 values of the established cell lines after lentivirus transfection (Unit: µg/ml)

Parental Resistance shIAP-1-HNE-1 IAP-1
IC50 IC50 RI IC50 RI IC50 RI
HNE-1 0.68 8.26 12.16 1.59 2.34 5.5 8.10
CNE-2 0.46 20.25 44.12 2.75 5.99 11.29 24.60
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These results indicated that the expression levels of IAP-1 were successfully upregulated and downregulated, respectively. We then examined the alteration of IAP-1 expression on biological characteristics (such as drug resistance and apoptosis) in NPC cell lines. IC50 results revealed that resistance to cisplatin was increased from 0.679 µg/ml and 0.459 µg/ml to 5.5 µg/ml and 11.29 µg/ml in IAP-1-upregulated HNE-1 and CNE-2 cells (Figures 5F and 6A, 6B). Similarly, cisplatin resistance in HNE-1/DDP and CNE-2/DDP cells with shRNA-IAP-1 silencing was decreased from 8.26 µg/ml and 20.25 µg/ml to 1.59 µg/ml and 2.75 µg/ml, respectively. These results suggested that altering IAP-1 expression contributed to cisplatin resistance in NPC.

Figure 6.

Figure 6

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Overexpression of IAP-1 promotes cisplatin resistance in NPC. A, B. IC50 results by MTT assays after transfection (HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs IAP-1-HNE-1 *P<0.05; IAP-1-HNE-1 vs HNE-1 *P<0.05. CNE-2 vs CNE-2/DDP *P<0.05; shIAP-1-CNE-2 vs CNE-2/DDP *P<0.05; shIAP-1-CNE-2 vs IAP-1-CNE-2 *P<0.05; IAP-1-CNE-2 vs CNE-2 *P<0.05). C, D. Flow cytometry showed that elevated IAP-1 resulted in lower apoptosis percentages, while silencing IAP-1 had the opposite effect (HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs HNE-1/DDP *P<0.05; IAP-1-HNE-1 vs HNE-1 *P<0.05; shIAP-1-HNE-1 vs IAP-1-HNE-1 *P<0.05; CNE-2 vs CNE-2/DDP *P<0.05; shIAP-1-CNE-2 vs CNE-2/DDP *P<0.05; shIAP-1-CNE-2 vs IAP-1-CNE-2 *P<0.05; IAP-1-CNE-2 vs CNE-2 P<0.05). E, F. The colony forming assay showed no significant difference between treated and untreated cells in the IAP-1-HNE-1 group (HNE-1 *P<0.05; HNE-1/DDP *P<0.05; IAP-1-HNE-1 P = 0.2887; shIAP-1-HNE-1/DDP *P<0.05). G, H. The wound healing assay showed no significant differences between treated and untreated cells in the IAP-1-HNE-1 and HNE-1/DDP groups (HNE-1 *P<0.05; HNE-1/DDP P = 0.2309; IAP-1-HNE-1 P = 0.0691; shIAP-1-HNE-1/DDP *P<0.05). Data are presented as the mean ± SD of at least three independent experiments. Statistical significance is indicated by *P<0.05.

Furthermore, we analyzed the apoptosis percentages for IAP-1-overexpressing chemosensitive cell lines (IAP-1-HNE-1 and IAP-1-CNE-2) and IAP-1-silenced resistant cell lines. The results showed that the percentages of apoptotic cells decreased from 10.36% and 12.81% to 6.92% and 7.19% in the IAP-1-HNE-1 and IAP-1-CNE-2 groups, respectively (Figures 6C, 6D and S4). Meanwhile, the percentage of apoptotic cells in IAP-1-silenced resistant cell lines increased from 5.64% and 10.51% to 8.39% and 10.7% (Figure 6C, 6D). All these results indicated that IAP-1 negatively regulates apoptosis in NPC cells.

According to our previous studies, more than 90% of NPC samples expressed folate receptor, which is a typical marker in NPC. Among all of the NPC cell lines, HNE-1 is the only cell line that showed similar expression of folate receptor to that in NPC samples [37]. Thus, HNE-1-related models were adopted to determine the following colony formation and motility ability changes with cisplatin treatment. Our previous results showed that the HNE-1/DDP cell line had a longer growth time and a similar motility to that in the parental cell line (Figure 1H-K). Interestingly, when 0.5 µg/ml cisplatin was added to the culture medium, the IAP-1-overexpressing cell line showed better tolerance to the chemical treatment, as no significant difference was found between the cisplatin-treated groups and untreated groups in the IAP-1-upregulated HNE-1 groups (Figure 6E-H). In summary, all the above results illustrated that the upregulated expression of IAP-1 is involved in cisplatin resistance of NPCs in vitro.

Inhibitors of IAP as a therapeutic method for cisplatin resistance in NPCs

Considering the elevated IAP-1 level in NPC cells followed by cisplatin resistance, we hypothesized that IAP-1 as a potential therapeutic target might help to increase cisplatin sensitivity in resistant NPC cells. Therefore, we next explored whether an IAP inhibitor could enhance sensitivity to cisplatin. We treated the 4 different HNE-1 cell lines with a combination of cisplatin and small-molecule IAP-1 inhibitors (CUDC-427, LCL161, AZD5582 and polygalacin D (PGD)) at varying drug concentrations and measured cellular viability after 72 hours of incubation with 0.5 µg/ml cisplatin. The results showed that combined with CUDC427 and LCL161, cisplatin showed greater cytotoxicity in IAP-1-HNE-1 cell lines (Figure 7A, 7B), as IAP-1-HNE-1 groups showed worse survival rates in all HNE-1 groups. Similar effects were observed with two other IAP family inhibitors, AZD5582 and polygalacin D (PGD) (Figure 7C, 7D). Taken together, these results showed that IAP inhibition can enhance the efficiency of cisplatin cytotoxicity in NPC and that an IAP-1 inhibitor may become a potential therapeutic option in NPC treatments.

Figure 7.

Figure 7

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Inhibitors of IAP as a therapeutic method for cisplatin resistance in NPCs. A-D. Cell viability changes at 72 hours when using IAP inhibitors combined with cisplatin among the four HNE-1 groups. (A. CUDC427; B. LCL161; C. AZD5582; D. PGD.) Data are presented as the mean ± SD of at least three independent experiments. Statistical significance is indicated by *P<0.05.

The resistant cell lines showed better tolerance to cisplatin in vivo

To better confirm the effectiveness of cisplatin resistance models in vivo, HNE-1/DDP cells were subcutaneously injected into nude mice to establish an animal xenograft model, while HNE-1 cells were used as controls. After tumor formation, the same doses of cisplatin were given to all the animal models. Before cisplatin treatment, the HNE-1 group showed a similar tumor volume growth rate. However, after cisplatin treatment, the tumor volume decreased significantly in the HNE-1 group compared with the HNE-1/DDP group. These results illustrated that resistant cell lines have better tolerance to cisplatin in vivo (Figure 8A, 8B), which is consistent with our in vitro results (Figure 1A-F). As shown in Figure 7, although HNE-1/DDP cells grew slightly slower, they exhibited stronger resistance after cisplatin administration with no significant difference in mouse weights between the groups (Figure 8C, 8D). Therefore, compared to the parental cell line, the HNE-1/DDP cell line showed better tolerance to cisplatin both in vitro and in vivo.

Figure 8.

Figure 8

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The resistant cell lines showed better tolerance to cisplatin in vivo. A. Growth curves of tumor volume in parental and resistant groups (*P<0.05). B. Growth curves of tumor volume in all 4 groups (IAP-1-HNE-1 and shIAP-1-HNE-1/DDP *P<0.05; IAP-1-HNE-1 vs HNE-1 *P<0.05; shIAP-1-HNE-1/DDP vs HNE-1/DDP *P<0.05; HNE-1 vs HNE-1/DDP *P<0.05). C, D. Weight changes of the mice (HNE-1 and HNE-1/DDP group) showed no significant difference (P = 0.874). E, F. The growth curves of lentivirus transfection groups and their parental groups (IAP-1-HNE-1 vs HNE-1 *P<0.05; shIAP-1-HNE-1/DDP vs HNE-1/DDP *P<0.05). G, H. Weights in the lentivirus-transfected mouse groups did not show significant differences (P = 0.3432). I. The effect of cisplatin chemotherapy on the tumor growth of NPC cell lines in vivo. J. Tumor volume changes in all four groups after cisplatin chemotherapy. The volume changes showed significant differences among the different groups (IAP-1-HNE-1 and shIAP-1-HNE-1/DDP *P<0.05; IAP-1-HNE-1 vs HNE-1 *P<0.05; shIAP-1-HNE-1/DDP vs HNE-1/DDP *P<0.05; HNE-1 vs HNE-1/DDP *P<0.05). Data are presented as the mean ± SD of at least three independent experiments. Statistical significance is indicated by *P<0.05.

Upregulated IAP-1 expression led to a worse chemotherapeutic response in vivo

To further determine whether IAP-1 confers cisplatin resistance in vivo, two transfected cell lines (IAP-1-HNE-1 and shIAP-1-HNE-1/DDP) were subcutaneously transplanted into nude mice, which were then treated with cisplatin. The chemosensitivity between the different IAP-1 expression groups (IAP-1-HNE-1 and shIAP-1-HNE-1/DDP) was tested by tumor volume changes. As shown in our in vivo results, each group showed a different therapeutic response to chemotherapy (Figure 8E, 8F), and the weights of all mice gradually decreased, but no significant differences were found between the groups (Figure 8G, 8H). After cisplatin treatment, the IAP-1-HNE-1 group showed the fastest growth rate and the worst reaction to cisplatin, while the shIAP-1 group exhibited the best response to cisplatin (Figure 8I, 8J). TUNEL assay results showed fewer apoptotic cells in the IAP-1-upregulated xenograft groups (HNE-1/DDP and IAP-1-HNE-1) than in the chemosensitive (shIAP-1-HNE-1/DDP or HNE-1) xenograft groups (Figure 9A, 9B). Moreover, no significant difference in tumor metastasis was identified between the groups (Figure S5). Taken together, we revealed that elevated IAP-1 was responsible for cisplatin resistance in vivo.

Figure 9.

Figure 9

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Xenograft results suggested that upregulated IAP-1 correlated with a reduced apoptosis rate and cleaved caspase-3 expression. A, B. TUNEL assay results showed that apoptosis was lower in the elevated IAP-1 groups and higher in the shIAP-1 groups (IAP-1-HNE-1 vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs IAP-1-HNE-1 *P<0.05; HNE-1/DDP vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs HNE-1/DDP *P<0.05). Original magnification ×400, bar = 50 µm. (White arrows indicate the apoptosis cells). C, D. Histopathologic features, and representative immunohistochemistry staining of IAP-1, Ki67, cleaved caspase-3 and caspase-9 in NPC cell line xenografts. The IAP-1/HNE-1 and HNE-1/DDP groups showed higher IAP-1 expression and lower cleaved caspase-3 expression (IAP-1: IAP-1-HNE-1 vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs IAP-1-HNE-1 *P<0.05; HNE-1/DDP vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs HNE-1/DDP *P<0.05. Caspase-3: IAP-1-HNE-1 vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs IAP-1-HNE-1 *P<0.05; HNE-1/DDP vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs HNE-1/DDP *P<0.05. Caspase-9: IAP-1-HNE-1 vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs IAP-1-HNE-1 *P<0.05; HNE-1/DDP vs IAP-1-HNE-1 *P<0.05. Ki 67: All groups NS). Original magnification ×400, bar = 100 µm. (Black arrows indicate the positive staining cells) Data are presented as the mean ± SD of at least three independent experiments. Statistical significance is indicated by *P<0.05.

IAP-1 affects the sensitivity of NPC cells to cisplatin by regulating the activity of caspase-3

Immunohistochemistry was used to determine the expression of IAP-1, Ki 67, caspase-3 and caspase-9 in xenografts, and the results suggested that IAP-1-HNE-1 and HNE-1/DDP cells showed upregulated expression of IAP-1 and reduced expression of cleaved caspase-3 (Figure 9C, 9D). At the same time, there were no significant differences between groups in Ki-67 and caspase-9.

Then, we further investigated the clinicopathologic significance of IAP-1 in NPC samples (Table 4). The results of the clinical tissues showed that there were 50% IAP-1-positive samples in all 30 NPC tissue samples (Table 5), and IAP-1 overexpression showed a negative correlation with cleaved caspase-3 expression. (R = -0.658; P<0.5) (Figure 10A, 10B). There was no significant relationship between IAP-1 and the remaining caspase family members (Figure 10A; Table S7). Similar to previous results, we found that cleaved caspase-3 activities decreased when IAP-1 expression was elevated (Figures 5D and 10C).

Table 4.

Clinicopathological features of patients

Clinicopathological features NPC (n = 30) Normal (n = 12)
Age (years)
    Mean ± SD 50.50 ± 11.89 42.17 ± 11.74
    Range 24-76 29-70
Gender
    Female, n (%) 12 (40%) 7 (58%)
    Male, n (%) 18 (60%) 5 (42%)
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Table 5.

Protein expression in NPC (Percentage)

Protein Positive rates 0 1+ 2+ 3+
IAP-1 50% 15% 35% 0%
Cleaved caspase-3 0% 10% 70% 20%
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Figure 10.

Figure 10

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IAP-1 affects the sensitivity of NPC cells to cisplatin by regulating the activity of caspase-3. A. Present immunohistology results of IAP-1, cleaved caspase-3, caspase-7, caspase-8 and caspase-9 expression in NPC and normal tissues. Original magnification ×200, bar = 100 µm. (Black arrows indicate the positive staining cells). B. Relationship between IAP-1 and cleaved caspase-3 in NPC tissues (N = 30; R = -0.658; P<0.5). C. Caspase-3 cleavage activities of the cell lines in the HNE-1 group (IAP-1-HNE-1 vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs IAP-1-HNE-1 *P<0.05; HNE-1/DDP vs shIAP-1-HNE-1/DDP *P<0.05; HNE-1 vs HNE-1/DDP *P<0.05). D. Relative mRNA expression of IAP family members in the HNE-1 groups (IAP-1: HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs HNE-1/DDP *P<0.05; shIAP-1-HNE-1 vs IAP-1-HNE-1 *P<0.05; IAP-1-HNE-1 vs HNE-1 *P<0.05). Data are presented as the mean ± SD of at least three independent experiments. Statistical significance is indicated by *P<0.05.

We next evaluated the effect of IAP-1 on apoptotic cell death together with other IAP family proteins. However, for IAP-2, X-IAP, livin and survivin, the cells did not show significant upregulation in cisplatin-resistant cell lines (HNE-1/DDP and IAP-1-HNE-1) (Figure 10D). These results revealed that IAP-1, but not X-IAP, IAP-2, survivin or livin, contributed to cleaved caspase-3-related apoptosis, which had a functional effect on cisplatin resistance in NPC.

Discussion

Although the prognosis of NPC has significantly improved due to advances in radiation therapy and concurrent chemotherapy [5,38,39], the overall survival of patients in advanced disease stage is still poor [40]. Currently, the standard treatment for recurrent NPC still consists of platinum-containing multiagent chemotherapy [2]. Despite numerous clinical trials, the development of new systemic therapies for recurrent NPC in the past 20 years has been limited [41]. Thus, few alternatives are available when platinum drugs are tolerated. Identifying the mechanism underlying cisplatin resistance and searching for potential effective therapies in cisplatin-resistant NPC are urgent tasks. In addition, a lack of preclinical tools, such as stable NPC-resistant cell line models, for predicting the clinical effect of therapeutic strategies in cancer restricts progress in oncology [42]. Therefore, we established cisplatin-resistant NPC models both in vitro and in vivo, which provided a simple, repeatable, stable experimental model and may be an efficient tool in drug screening-related fields.

In this study, we screened and analyzed DEGs between the drug-resistance model and parental cell lines. We found that the focal adhesion (FAK) pathway, which is frequently reported to function in cancer cell behavior [43,44], had 2 DEGs (IAP-1 and IAP-2) that were also highly expressed in the classic platinum resistance pathway, while IAP-1 had the highest mRNA expression level among the IAP family members. Previous studies showed that FAK overexpression protects cells against various apoptotic stimuli by upregulating IAP gene expression [24], which is consistent with our bioinformatics results (Figure 3C).

Therefore, we hypothesized that IAP-1 modulated cisplatin resistance in NPC. However, the IAP family includes 8 members, which are frequently upregulated in cancers and associated with poor prognoses [45-47]. Hence, we must determine whether other IAP members are involved in cisplatin resistance. In this paper, we mainly focused on the expression levels of IAP-1, IAP-2, X-IAP, survivin and livin, which play predominant roles in regulating apoptosis, particularly in malignant cells [48]. In our results, X-IAP, livin and survivin showed low expression in the resistant cell lines, while no significant difference was found for IAP-2 (Figure 10D). IAP-1 showed high expression with the most pronounced fold change among the IAP family members. Additionally, studies in various tumors also showed that IAP-1 overexpression can increase the resistance of tumor cells to chemotherapeutic drugs [23-27,49], which is similar to our bioinformatics results in Figure 3. All these results demonstrated that IAP-1, but not X-IAP or survivin, contributed to cisplatin resistance in NPC, which is consistent with our hypothesis. However, more clinical data are needed in recurrent NPC because only one result was found in the Oncomine database (Figure S6).

Additionally, we studied IAP-1 expression in open-source clinical databases (Figure 4) and found results consistent with our experimental data indicating that elevated IAP-1 modulated chemoresistance both in vivo and in vitro and correlated with poor survival in NPC (Figures 5, 6, 7, 8, 9 and 10). In addition, some related studies on medulloblastoma, leukemia and other tumors are consistent with our results showing that IAP-1 overexpression promotes cancer cell survival and chemoresistance [19,23-27]. Notably, elevated IAP-1 strongly indicates a poor prognosis in various virus-associated tumors, such as HPV-related cervical cancers [49] and HPV-related lung cancer [23]. Some viral proteins, such as HPV protein-E6/E7 and HLTV-1 protein-Tax, have been reported to help prevent apoptosis by upregulating IAP proteins [50-52]. EBV, as a risk factor in NPC [53], has been associated with several lymphoid and solid tumors [54]. The viral protein LMP1 in EBV has been reported to activate a signaling cascade that results in constitutive activation of the JNK, NFKB and MAPK pathways [55]. Combining the above findings and the results in this article, we hypothesize that activation of these key signaling pathways, which increase cellular growth and promote survival, may be mediated by upregulation of IAP-1 in NPC. Thus, IAP-1 serves as a prognostic indicator in NPC.

Therefore, we next observed the effect of IAP inhibitors on NPC cells and whether IAP-1 inhibition can reverse this resistance. Currently, most IAP inhibitors are the second mitochondria-derived activator of caspase (SMAC), an endogenous IAP antagonist that mimics the IAP-binding motif [54]. Among them, we selected 3 representative SMACs (CUDC-427, LCL 161 and AZD5582) for our study. The results in Figure 7 show that all these IAP antagonists had a significant killing effect when combined with cisplatin, with CUDC-427 showing the most significant effect. This drug has entered phase I clinical research on refractory solid tumors or lymphomas (NCT01226277) [54]. Apart from SMAC, we also found that small-molecule PGD, an inhibitor of IAP-1, IAP-2 and survivin [56], could also significantly increase the killing effect on NPC cells (Figure 7D). These results are consistent with a variety of studies suggesting the potential of IAP inhibitors in tumor therapy [54,56-58]. Likewise, our in vivo results showed that with downregulated IAP-1 expression, cisplatin can significantly reduce the tumor volume, reflecting the potential use of IAP inhibitors in NPC. These findings should motivate further research on combining IAP-1 antagonists with other treatments. To further investigate how IAP inhibitors enhance the effect of antineoplastic drugs on killing NPC cells, additional tests are required.

As proteins known to participate in apoptotic inhibition, the IAP family could bind to the active sites of caspases, by keeping the caspases away from their substrates or by promoting degradation of active caspases [59]. All members of IAP family contains at least one copy of a so-called BIR (baculovirus iap repeat) domain, a zinc binding fold, which could directly affect caspases, especially caspase-3/7/9 [17]. Our results have shown that IAP-1 upregulation could lead to apoptosis decrease in tumor cells (Figures 1C, 6C and 9A) and we next wanted to reveal the most functional caspases member here. The in vitro assay and xenograft assay showed consistently that active caspase-3 (cleaved caspase-3) decreased when IAP-1 expression was elevated (Figures 5D, 5G, 9C, 9D, 10C and S7, S8). What’s more, the results from clinical samples showed that IAP-1 negatively regulated the expression of cleaved caspase-3, but there was no significant relationship between IAP-1 and caspase-7, 8, and 9 (Figure 10A, 10B). Thus, we confirmed that IAP-1 overexpression contributed to cisplatin resistance in NPC by inhibiting the caspase-3-related apoptosis pathway. However, IAPs can regulate apoptosis through many other pathways [15,26-31], more related research of IAP regulation is required in the future.

In summary, we successfully established drug-resistant cell line models of NPC in vitro and an animal model of NPC in vivo. Then, we performed a series of studies, including DEG screening and bioinformatics analysis, to search for the possible therapeutic target of cisplatin resistance in NPC and identified the differential expression of FAK, ECM and other pathways. The results revealed that the altered IAP family (especially IAP-1) in the FAK pathway had a significant effect on cisplatin resistance in NPC. Our paper provides the first direct evidence that IAP-1 is responsible for cisplatin resistance by inhibiting caspase-3 induced apoptosis activity in NPC (Figure 11). Therefore, we conclude that IAP-1 is a promising clinical biomarker for NPC prognosis and chemotherapy response and that a specific inhibitor of IAP-1 may be a potential strategy for treatment in NPC patients.

Figure 11.

Figure 11

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Illustrations of the function of IAP-1 in cisplatin resistance by regulating the cleavage of caspase-3 in NPC cells.

Acknowledgements

We thank American Journal Experts (AJE) for English language editing. This study was financially supported by grants from the National Natural Science Foundation of China [Grant Number 81372477, 81673013], the Science and Technology Project of Guangdong Province, China [Grant number 2017A010103010] and the Natural Science Foundation of Guangdong Province, China [Grant number 2017A030313511].

Disclosure of conflict of interest

None.

Supporting Information

References

  • 1.Wackym PA. Ballenger’s Otorhinolaryngology Head and Neck Surgery. 2008 [Google Scholar]
  • 2.Pfister DG, Spencer S, Adelstein D, Adkins D, Darlow SD. National comprehensive cancer network. Head and Neck Cancers Version v2.2020. 2020 [Google Scholar]
  • 3.Blanchard P, Lee A, Marguet S, Leclercq J, Ng WT, Ma J, Chan AT, Huang PY, Benhamou E, Zhu G, Chua DT, Chen Y, Mai HQ, Kwong DL, Cheah SL, Moon J, Tung Y, Chi KH, Fountzilas G, Zhang L, Hui EP, Lu TX, Bourhis J, Pignon JP. Chemotherapy and radiotherapy in nasopharyngeal carcinoma: an update of the MAC-NPC meta-analysis. Lancet Oncol. 2015;16:645–655. doi: 10.1016/S1470-2045(15)70126-9. [DOI] [PubMed] [Google Scholar]
  • 4.Perri F, Della VSG, Buonerba C, Di Lorenzo G, Longo F, Muto P, Schiavone C, Sandomenico F, Caponigro F. Combined chemo-radiotherapy in locally advanced nasopharyngeal carcinomas. World J Clin Oncol. 2013;4:47–51. doi: 10.5306/wjco.v4.i2.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Yang L, Hong S, Wang Y, Chen H, Liang S, Peng P, Chen Y. Development and external validation of nomograms for predicting survival in nasopharyngeal carcinoma patients after definitive radiotherapy. Sci Rep. 2015;5:15638. doi: 10.1038/srep15638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Li JX, Lu TX, Huang Y, Han F, Chen CY, Xiao WW. Clinical features of 337 patients with recurrent nasopharyngeal carcinoma. Chin J Cancer. 2010;29:82–86. doi: 10.5732/cjc.009.10412. [DOI] [PubMed] [Google Scholar]
  • 7.Chang JT, See LC, Liao CT, Ng SH, Wang CH, Chen IH, Tsang NM, Tseng CK, Tang SG, Hong JH. Locally recurrent nasopharyngeal carcinoma. Radiother Oncol. 2000;54:135–142. doi: 10.1016/s0167-8140(99)00177-2. [DOI] [PubMed] [Google Scholar]
  • 8.Mould RF, Tai TH. Nasopharyngeal carcinoma: treatments and outcomes in the 20th century. Br J Radiol. 2002;75:307–339. doi: 10.1259/bjr.75.892.750307. [DOI] [PubMed] [Google Scholar]
  • 9.Wei WI, Sham JS. Nasopharyngeal carcinoma. Lancet. 2005;365:2041–2054. doi: 10.1016/S0140-6736(05)66698-6. [DOI] [PubMed] [Google Scholar]
  • 10.Perri F, Della VSG, Caponigro F, Ionna F, Longo F, Buonopane S, Muto P, Di Marzo M, Pisconti S, Solla R. Management of recurrent nasopharyngeal carcinoma: current perspectives. Onco Targets Ther. 2019;12:1583–1591. doi: 10.2147/OTT.S188148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Picon H, Guddati AK. Mechanisms of resistance in head and neck cancer. Am J Cancer Res. 2020;10:2742–2751. [PMC free article] [PubMed] [Google Scholar]
  • 12.Peng F, Zhang H, Du Y, Tan P. miR-23a promotes cisplatin chemoresistance and protects against cisplatin-induced apoptosis in tongue squamous cell carcinoma cells through Twist. Oncol Rep. 2015;33:942–950. doi: 10.3892/or.2014.3664. [DOI] [PubMed] [Google Scholar]
  • 13.Xu H, Zeng L, Guan Y, Feng X, Zhu Y, Lu Y, Shi C, Chen S, Xia J, Guo J, Kuang C, Li W, Jin F, Zhou W. High NEK2 confers to poor prognosis and contributes to cisplatin-based chemotherapy resistance in nasopharyngeal carcinoma. J Cell Biochem. 2019;120:3547–3558. doi: 10.1002/jcb.27632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Rodrigues-Junior DM, Tan SS, Lim SK, Leong HS, Melendez ME, Ramos CRN, Viana LS, Tan DSW, Carvalho AL, Iyer NG, Vettore AL. Circulating extracellular vesicle-associated TGFbeta3 modulates response to cytotoxic therapy in head and neck squamous cell carcinoma. Carcinogenesis. 2019;40:1452–1461. doi: 10.1093/carcin/bgz148. [DOI] [PubMed] [Google Scholar]
  • 15.Dempke W, Voigt W, Grothey A, Hill BT, Schmoll HJ. Cisplatin resistance and oncogenes--a review. Anticancer Drugs. 2000;11:225–236. doi: 10.1097/00001813-200004000-00001. [DOI] [PubMed] [Google Scholar]
  • 16.Barry MA, Behnke CA, Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem Pharmacol. 1990;40:2353–2362. doi: 10.1016/0006-2952(90)90733-2. [DOI] [PubMed] [Google Scholar]
  • 17.Hassan M, Watari H, AbuAlmaaty A, Ohba Y, Sakuragi N. Apoptosis and molecular targeting therapy in cancer. Biomed Res Int. 2014;2014:150845. doi: 10.1155/2014/150845. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 18.Gordon GJ, Mani M, Mukhopadhyay L, Dong L, Yeap BY, Sugarbaker DJ, Bueno R. Inhibitor of apoptosis proteins are regulated by tumour necrosis factor-alpha in malignant pleural mesothelioma. J Pathol. 2007;211:439–446. doi: 10.1002/path.2120. [DOI] [PubMed] [Google Scholar]
  • 19.Keating J, Tsoli M, Hallahan AR, Ingram WJ, Haber M, Ziegler DS. Targeting the inhibitor of apoptosis proteins as a novel therapeutic strategy in medulloblastoma. Mol Cancer Ther. 2012;11:2654–2663. doi: 10.1158/1535-7163.MCT-12-0352. [DOI] [PubMed] [Google Scholar]
  • 20.Weikert S, Schrader M, Krause H, Schulze W, Müller M, Miller K. The inhibitor of apoptosis (IAP) survivin is expressed in human testicular germ cell tumors and normal testes. Cancer Lett. 2005;223:331–337. doi: 10.1016/j.canlet.2004.10.038. [DOI] [PubMed] [Google Scholar]
  • 21.Chen HD, Guo N, Song SS, Chen CH, Miao ZH, He JX. Novel mutations in BRCA2 intron 11 and overexpression of COX-2 and BIRC3 mediate cellular resistance to PARP inhibitors. Am J Cancer Res. 2020;10:2813–2831. [PMC free article] [PubMed] [Google Scholar]
  • 22.Santarelli A, Mascitti M, Lo Russo L, Sartini D, Troiano G, Emanuelli M, Lo Muzio L. Survivin-based treatment strategies for squamous cell carcinoma. Int J Mol Sci. 2018;19:971. doi: 10.3390/ijms19040971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Wu HH, Wu JY, Cheng YW, Chen CY, Lee MC, Goan YG, Lee H. cIAP2 upregulated by E6 oncoprotein via epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT pathway confers resistance to cisplatin in human papillomavirus 16/18-infected lung cancer. Clin Cancer Res. 2010;16:5200–5210. doi: 10.1158/1078-0432.CCR-10-0020. [DOI] [PubMed] [Google Scholar]
  • 24.Andjilani M, Droz JP, Benahmed M, Tabone E. Down-regulation of FAK and IAPs by laminin during cisplatin-induced apoptosis in testicular germ cell tumors. Int J Oncol. 2006;28:535–542. [PubMed] [Google Scholar]
  • 25.Gordon GJ, Mani M, Mukhopadhyay L, Dong L, Edenfield HR, Glickman JN, Yeap BY, Sugarbaker DJ, Bueno R. Expression patterns of inhibitor of apoptosis proteins in malignant pleural mesothelioma. J Pathol. 2007;211:447–454. doi: 10.1002/path.2121. [DOI] [PubMed] [Google Scholar]
  • 26.Notarbartolo M, Cervello M, Dusonchet L, Cusimano A, D’Alessandro N. Resistance to diverse apoptotic triggers in multidrug resistant HL60 cells and its possible relationship to the expression of P-glycoprotein, Fas and of the novel anti-apoptosis factors IAP (inhibitory of apoptosis proteins) Cancer Lett. 2002;180:91–101. doi: 10.1016/s0304-3835(01)00834-5. [DOI] [PubMed] [Google Scholar]
  • 27.Piro G, Giacopuzzi S, Bencivenga M, Carbone C, Verlato G, Frizziero M, Zanotto M, Mina MM, Merz V, Santoro R, Zanoni A, De Manzoni G, Tortora G, Melisi D. TAK1-regulated expression of BIRC3 predicts resistance to preoperative chemoradiotherapy in oesophageal adenocarcinoma patients. Br J Cancer. 2015;113:878–885. doi: 10.1038/bjc.2015.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Notarbartolo M, Poma P, Perri D, Dusonchet L, Cervello M, D’Alessandro N. Antitumor effects of curcumin, alone or in combination with cisplatin or doxorubicin, on human hepatic cancer cells. Analysis of their possible relationship to changes in NF-kB activation levels and in IAP gene expression. Cancer Lett. 2005;224:53–65. doi: 10.1016/j.canlet.2004.10.051. [DOI] [PubMed] [Google Scholar]
  • 29.Wang Q, Chen W, Bai L, Chen W, Padilla MT, Lin AS, Shi S, Wang X, Lin Y. Receptor-interacting protein 1 increases chemoresistance by maintaining inhibitor of apoptosis protein levels and reducing reactive oxygen species through a microRNA-146a-mediated catalase pathway. J Biol Chem. 2014;289:5654–5663. doi: 10.1074/jbc.M113.526152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.McCabe KE, Bacos K, Lu D, Delaney JR, Axelrod J, Potter MD, Vamos M, Wong V, Cosford ND, Xiang R, Stupack DG. Triggering necroptosis in cisplatin and IAP antagonist-resistant ovarian carcinoma. Cell Death Dis. 2014;5:e1496–e1496. doi: 10.1038/cddis.2014.448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Samuel T, Okada K, Hyer M, Welsh K, Zapata JM, Reed JC. cIAP1 Localizes to the nuclear compartment and modulates the cell cycle. Cancer Res. 2005;65:210–218. [PubMed] [Google Scholar]
  • 32.de Graaf AO, de Witte T, Jansen JH. Inhibitor of apoptosis proteins: new therapeutic targets in hematological cancer? Leukemia. 2004;18:1751–1759. doi: 10.1038/sj.leu.2403493. [DOI] [PubMed] [Google Scholar]
  • 33.Liston P, Fong WG, Korneluk RG. The inhibitors of apoptosis: there is more to life than Bcl2. Oncogene. 2003;22:8568–8580. doi: 10.1038/sj.onc.1207101. [DOI] [PubMed] [Google Scholar]
  • 34.Plenchette S, Cathelin S, Rebe C, Launay S, Ladoire S, Sordet O, Ponnelle T, Debili N, Phan TH, Padua RA, Dubrez-Daloz L, Solary E. Translocation of the inhibitor of apoptosis protein c-IAP1 from the nucleus to the Golgi in hematopoietic cells undergoing differentiation: a nuclear export signal-mediated event. Blood. 2004;104:2035–2043. doi: 10.1182/blood-2004-01-0065. [DOI] [PubMed] [Google Scholar]
  • 35.Su GMI, Davey MW, Davey RA, Kidman AD. Development of extended multidrug resistance in HL60 promyelocytic leukaemia cells. Br J Haematol. 1994;88:566–574. doi: 10.1111/j.1365-2141.1994.tb05075.x. [DOI] [PubMed] [Google Scholar]
  • 36.Mirski SE, Gerlach JH, Cole SP. Multidrug resistance in a human small cell lung cancer cell line selected in adriamycin. Cancer Res. 1987;47:2594–2598. [PubMed] [Google Scholar]
  • 37.Xie M, Zhang H, Xu Y, Liu T, Chen S, Wang J, Zhang T. Expression of folate receptors in nasopharyngeal and laryngeal carcinoma and folate receptor-mediated endocytosis by molecular targeted nanomedicine. Int J Nanomedicine. 2013;8:2443–2451. doi: 10.2147/IJN.S46327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Zhang MX, Li J, Shen GP, Zou X, Xu JJ, Jiang R, You R, Hua YJ, Sun Y, Ma J, Hong MH, Chen MY. Intensity-modulated radiotherapy prolongs the survival of patients with nasopharyngeal carcinoma compared with conventional two-dimensional radiotherapy: a 10-year experience with a large cohort and long follow-up. Eur J Cancer. 2015;51:2587–2595. doi: 10.1016/j.ejca.2015.08.006. [DOI] [PubMed] [Google Scholar]
  • 39.Sun X, Su S, Chen C, Han F, Zhao C, Xiao W, Deng X, Huang S, Lin C, Lu T. Long-term outcomes of intensity-modulated radiotherapy for 868 patients with nasopharyngeal carcinoma: an analysis of survival and treatment toxicities. Radiother Oncol. 2014;110:398–403. doi: 10.1016/j.radonc.2013.10.020. [DOI] [PubMed] [Google Scholar]
  • 40.Yi JL, Gao L, Huang XD, Li SY, Luo JW, Cai WM, Xiao JP, Xu GZ. Nasopharyngeal carcinoma treated by radical radiotherapy alone: ten-year experience of a single institution. Int J Radiat Oncol Biol Phys. 2006;65:161–168. doi: 10.1016/j.ijrobp.2005.12.003. [DOI] [PubMed] [Google Scholar]
  • 41.Peng H, Chen L, Chen YP, Li WF, Tang LL, Lin AH, Sun Y, Ma J. The current status of clinical trials focusing on nasopharyngeal carcinoma: a comprehensive analysis of ClinicalTrials.gov database. PLoS One. 2018;13:e0196730. doi: 10.1371/journal.pone.0196730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Wang Q, Zeng F, Sun Y, Qiu Q, Zhang J, Huang W, Huang J, Huang X, Guo L. Etk interaction with PFKFB4 modulates chemoresistance of small-cell lung cancer by regulating autophagy. Clin Cancer Res. 2018;24:950–962. doi: 10.1158/1078-0432.CCR-17-1475. [DOI] [PubMed] [Google Scholar]
  • 43.Vogel V, Sheetz M. Local force and geometry sensing regulate cell functions. Nat Rev Mol Cell Biol. 2006;7:265–275. doi: 10.1038/nrm1890. [DOI] [PubMed] [Google Scholar]
  • 44.McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC. The role of focal-adhesion kinase in cancer a new therapeutic opportunity. Nat Rev Cancer. 2005;5:505–515. doi: 10.1038/nrc1647. [DOI] [PubMed] [Google Scholar]
  • 45.Yang L, Cao Z, Yan H, Wood WC. Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy. Cancer Res. 2003;63:6815–6824. [PubMed] [Google Scholar]
  • 46.Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S, Scudiero DA, Tudor G, Qui YH, Monks A, Andreeff M, Reed JC. Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. Clin Cancer Res. 2000;6:1796–1803. [PubMed] [Google Scholar]
  • 47.Ndubaku C, Cohen F, Varfolomeev E, Vucic D. Targeting inhibitor of apoptosis proteins for therapeutic intervention. Future Med Chem. 2009;1:1509. doi: 10.4155/fmc.09.116. [DOI] [PubMed] [Google Scholar]
  • 48.Bai L, Smith DC, Wang S. Small-molecule SMAC mimetics as new cancer therapeutics. Pharmacol Ther. 2014;144:82–95. doi: 10.1016/j.pharmthera.2014.05.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Rada M, Nallanthighal S, Cha J, Ryan K, Sage J, Eldred C, Ullo M, Orsulic S, Cheon DJ. Inhibitor of apoptosis proteins (IAPs) mediate collagen type XI alpha 1-driven cisplatin resistance in ovarian cancer. Oncogene. 2018;37:4809–4820. doi: 10.1038/s41388-018-0297-x. [DOI] [PubMed] [Google Scholar]
  • 50.James MA, Lee JH, Klingelhutz AJ. Human papillomavirus type 16 E6 activates NF-kappaB, induces cIAP-2 expression, and protects against apoptosis in a PDZ binding motif-dependent manner. J Virol. 2006;80:5301–5307. doi: 10.1128/JVI.01942-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Yuan H, Fu F, Zhuo J, Wang W, Nishitani J, An DS, Chen IS, Liu X. Human papillomavirus type 16 E6 and E7 oncoproteins upregulate c-IAP2 gene expression and confer resistance to apoptosis. Oncogene. 2005;24:5069–5078. doi: 10.1038/sj.onc.1208691. [DOI] [PubMed] [Google Scholar]
  • 52.Xiao G, Cvijic ME, Fong A, Harhaj EW, Uhlik MT, Waterfield M, Sun SC. Retroviral oncoprotein Tax induces processing of NF-kappaB2/p100 in T cells: evidence for the involvement of IKKalpha. EMBO J. 2001;20:6805–6815. doi: 10.1093/emboj/20.23.6805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Tsao SW, Tsang CM, Lo KW. Epstein-Barr virus infection and nasopharyngeal carcinoma. Philosophical transactions of the Royal Society of London. Philos Trans R Soc Lond B Biol Sci. 2017;372:20160270. doi: 10.1098/rstb.2016.0270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Cong H, Xu L, Wu Y, Qu Z, Bian T, Zhang W, Xing C, Zhuang C. Inhibitor of apoptosis protein (IAP) antagonists in anticancer agent discovery: current status and perspectives. J Med Chem. 2019;62:5750–5772. doi: 10.1021/acs.jmedchem.8b01668. [DOI] [PubMed] [Google Scholar]
  • 55.Eliopoulos AG, Caamano JH, Flavell J, Reynolds GM, Murray PG, Poyet JL, Young LS. Epstein-Barr virus-encoded latent infection membrane protein 1 regulates the processing of p100 NF-kappaB2 to p52 via an IKKgamma/NEMO-independent signalling pathway. Oncogene. 2003;22:7557. doi: 10.1038/sj.onc.1207120. [DOI] [PubMed] [Google Scholar]
  • 56.Seo YS, Kang OH, Kong R, Zhou T, Kim SA, Ryu S, Kim HR, Kwon DY. Polygalacin D induces apoptosis and cell cycle arrest via the PI3K/Akt pathway in non-small cell lung cancer. Oncol Rep. 2018;39:1702–1710. doi: 10.3892/or.2018.6230. [DOI] [PubMed] [Google Scholar]
  • 57.Lee EK, Jinesh G G, Laing NM, Choi W, McConkey DJ, Kamat AM. A Smac mimetic augments the response of urothelial cancer cells to gemcitabine and cisplatin. Cancer Biol Ther. 2014;14:812–822. doi: 10.4161/cbt.25326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Scott DE, Bayly AR, Abell C, Skidmore J. Small molecules, big targets: drug discovery faces the protein-protein interaction challenge. Nat Rev Drug Discov. 2016;15:533–550. doi: 10.1038/nrd.2016.29. [DOI] [PubMed] [Google Scholar]
  • 59.Saralamma VVG, Lee HJ, Raha S, Lee WS, Kim EH, Lee SJ, Heo JD, Won C, Kang CK, Kim GS. Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein. Oncotarget. 2018;9:5993–6006. doi: 10.18632/oncotarget.23202. [DOI] [PMC free article] [PubMed] [Google Scholar]

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