Background
Colorectal cancer is the third most common cancer worldwide, with a mortality of 9.2% [
1]. At present, fluorouracil and platinum-based chemotherapy is still the main treatment for advanced and recurrent colorectal cancer [
2]. Although target drugs and immune checkpoint inhibitors are successful in colorectal cancer treatment, most patients still cannot benefit from these novel drugs [
3]. Therefore, it is necessary to develop effective target drugs for colorectal cancer.
Survivin is the smallest member of inhibitor of apoptosis protein (IAP) family. It is overexpressed in most cancers including colorectal cancer, but rarely expressed in normal differentiated tissues [
4]. Survivin contains a single N-terminal baculovirus IAP repeat (BIR) domain, a zinc finger fold and a C-terminal α helical coiled-coil domain, and exists in both monomeric and dimeric forms. Through its interactions with multiple essential proteins such as Hsp90, SMAC/DIABLO, XIAP, and the components of chromosomal passenger complex (CPC), survivin exerts two major functions: (i) inhibiting apoptosis and (ii) regulating cell division, both of which play key roles in the development of cancer [
5‐
7]. Therefore, survivin is believed to have clinical significance and has been extensively studied. In glioma, a positive association was observed between survivin expression and pathological grade [
8]. In patients with non-small-cell lung cancer, the overexpression of survivin was significantly related to poor postoperative survival [
9]. It has also been reported that survivin contributes to the resistance of cancer cells to radiotherapy and chemotherapy [
10,
11]. Moreover, high survivin expression in circulating tumor cells (CTC) predicted shortened overall survival in metastatic colorectal cancer patients [
12]. Taken together, due to its specific overexpression in most cancer tissues, dual role in cell division regulation and apoptosis inhibition, and clinical significance, survivin has been established as an ideal target for anticancer therapy.
To date, a variety of drugs targeting survivin have been developed including YM155, LY2181308, LQZ-7F, shepherdin, survivin-specific SMAC mimetics and so on [
13‐
17]. Inhibiting the expression of survivin or blocking the interactions between survivin and other essential proteins to interfere with its functions are two major approaches by which survivin inhibitors exert their anticancer activity [
18]. Despite exhibiting potent anticancer effects both in vitro and in vivo, existing survivin-targeted drugs have not been successfully translated into the clinic [
19‐
22]. Thus, it is necessary to develop new drugs that target survivin in cancer cells. XIAP is another important member of the IAP family. It has been reported that survivin and XIAP can form an IAP-IAP complex to synergistically antagonize the activity of Caspase 9 [
23]. Therefore, we hypothesized that interfering with the interaction between survivin and XIAP may be a promising approach for colorectal cancer treatment.
In this study, we designed and synthesized a novel anticancer peptide Sur-X that targets the survivin-XIAP complex. Sur-X was proved to have specific and potent anticancer activity both in colorectal cancer cell lines and xenograft mouse models. Mechanistically, it was found that apart from inducing apoptosis by disrupting the survivin-XIAP complex, Sur-X also promoted necroptosis in colorectal cancer cells by interfering with the interaction between XIAP and TAB1. Thus, this study provides a potential strategy for colorectal cancer treatment.
Materials and methods
On-line databases analysis
The mRNA expression levels of IAPs in cancer tissues were compared with normal controls by Oncomine (
https://www.oncomine.org/), an on-line cancer microarray database. The thresholds of
p-value and fold change were set to 0.05 and 1.5, respectively.
GEPIA (Gene Expression Profiling Interactive Analysis) is an interactive web server for analyzing gene expression profiling of cancer and normal tissues from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) projects (
http://gepia.cancer-pku.cn/). The expression profile of BIRC5/survivin in multiple cancers and all the IAPs expression in colorectal cancer and matched normal tissues were analyzed by GEPIA, parameters were set to default values.
Reagents and antibodies
The following primary antibodies were used for Western blot: antibodies against survivin (#2803), XIAP (#14334), Caspase 8 (#9746S), Caspase 9 (#9508S), Caspase 3 (#9662S), cleaved-Caspase 3 (#9661S), PARP (#9542 L), ubiquitin (#3933S), RIP1 (#3493S), phospho-RIP1 (Ser166) (#44590), RIP3 (#13526S), phospho-RIP3 (Ser227) (#93654), phospho-MLKL (Ser358) (#91689S), TAB1 (#3226), TAK1 (#5206), phospho-TAK1 (Thr184/187) (#4508) and GAPDH (#5174) were from Cell Signaling Technology; anti-Caspase 7 antibody (#sc-28,295) was purchased from Santa Cruz and anti-MLKL antibody (#184718) was purchased from and Abcam. Z-VAD-fmk (Sigma-Aldrich, V116) and Nec-1 s (Biovision, 2535–1) were used to reveal apoptotic and necroptotic cell death, respectively. Cycloheximide (CHX, C7698-5G) was purchased from Sigma-Aldrich. MG132 (S2619) was obtained from Selleckchem.
Cell culture
Human colorectal cancer cell lines HCT116, RKO, HCT15, and HT29 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The human peritoneal mesothelial cell line HMrSV5 (SV5) was provided by Prof. Huimian Xu (Department of Surgical Oncology and General Surgery, The First Hospital of China Medical University). All cell lines were cultured with RPMI-1640 containing 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) and 1% penicillin/streptomycin. The short tandem repeats (STR) profile was used to authenticate all the cell lines. All cells were cultured for no longer than 2 months and negatively tested for mycoplasma contamination.
Peptide synthesis
The peptide with the sequence derived from the XIAP-binding region (K15-M38) of survivin and the cell-penetrating sequence from HIV Tat protein added to its N-terminal, was named Sur-X. The sequence of Sur-X is YGRKKRRQRRRKDHRISTFKNWPFLEGCACTPERM-COOH. A negative control peptide is YGRKKRRQRRRKDDGNYKTRAEVKFEGDTLVNRIE-COOH, and named Con. Peptides Sur-X and Con, as well as FITC labeled peptides FITC-Sur-X and FITC-Con were all synthesized by Synpeptide Co. Ltd. (Nanjing, China). DMSO was used to dissolve these peptides immediately before use. Final concentrations of DMSO in experiments were < 0.4%.
Confocal imaging
Cells were seeded into 2 cm glass-bottom cell culture dish (NEST, 801001) and treated by 10 μM FITC-Sur-X or FITC-Con after attachment. Hoechst 33342 (BD Biosciences, 561,908) was used to stain nuclei. After 30 min, the culture medium was discarded and cells were washed twice using ice-cold PBS and inspected under an Olympus FluoView FV1000 laser scanning confocal microscope.
Co-immunoprecipitation and Western blot analysis
When the confluency reached 90% in a 10-cm dish, cells were lysed in 1% Triton lysis buffer (1% Triton X-100, 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na
3VO
4, 1% protease inhibitor cocktail) on ice for 5 min and collected in a 1.5 ml EP tube. Retaining 10% of cell lysate as input, the remaining was subjected to co-immunoprecipitation assay and Western blot analysis, which were performed as described in our previous studies [
24,
25]. Image J (NIH, USA) was used to perform densitometric analysis.
Cell viability assay
3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect cell viability as previously described [
24].
Cells were seeded into 12-well plates and treated by 10 μM Sur-X or Con. Ten days later, cells were stained by Wright-Giemsa after 75% ethanol-fixation. The colonies in each well were counted in five fields under an optical microscope.
qRT-PCR assay
Trizol-chloroform was used to extract total RNA and qRT-PCR was performed as previously described [
26]. Primers were listed in Table S
1.
Real-time apoptosis and necrosis assay
HCT116 and RKO cells were seeded at the density of 4000 cells/well in 100 μl culture medium into Nunclon 96-Well flat white plate (Thermo Fisher Scientific). After cells were attached, 2× Sur-X of finally detected concentrations and 2× detection reagents in RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay (Promega, JA1011) were mixed in 100 μl pre-warming culture medium and the mixture was added to each well. Relative luminescence units (RLU) and relative fluorescence units (RFU) were recorded overtime up to 6 h by the Spark multimode microplate reader (Tecan Trading AG, Switzerland).
Caspase 3 activity assay
The activity of Caspase 3 was measured according to the instruction of Caspase 3 Activity Assay Kit (Cell Signaling Technology, #5723). RFU with excitation at 380 nm and emission at 440 nm was detected by Thermo Scientific Varioskan Flash.
Ubiquitination assay
HCT116 cells were incubated with 10 μM MG132 for 8 h and then treated by 10 μM Sur-X for 1 h. Subsequently, immunoprecipitation was implemented as previously described. After SDS-PAGE, the immune-precipitates were probed with indicated antibodies including anti-ubiquitin antibody. The experiment was performed for three times independently.
Annexin V-FITC/7-AAD assay
After indicated treatment, HCT116 and RKO cells were collected and prepared for flow cytometry analysis by using FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend, 640,922), according to the staining procedure recommended by the manufacture. BD Accuri C6 Flow Cytometer was used for the detection and analysis of samples.
Preparation of the structures of XIAP and Sur-X for molecular dynamics simulation
To predict the binding mode of XIAP and Sur-X, the structure of XIAP was constructed based on the crystal structures of BIR1, BIR2, BIR3 domains (PDB codes: 4OXC, 4J3Y, and 1G73) and the missing residues were filled using YASARA. The assembled XIAP structure was then subject to three step-wise rounds of energy minimization to relax the loops that connect different domains. For Sur-X, we extracted corresponding atomic coordinates (residues number 15 to 38) from the crystal structure of survivin (PDB code: 1F3H). Starting from this conformer, a total of 400 ns accelerated molecular dynamics (aMD) simulations were carried out and the center of the highest-populated cluster obtained from aMD trajectories was selected as the representative low-energy conformational state of the peptide [
27,
28]. Afterwards, the initial XIAP-peptide complexes were created by using ZDOCK [
29]. Finally, the binding poses were clustered into 27 complexes and selected for further molecular dynamics (MD) simulations.
MD simulation of interaction between Sur-X and XIAP
All MD simulations were performed by using Amber 16 program (University of California, San Francisco) on GPUs. The Amber99sb force field was used for all systems [
30]. For each binding pose, the complex structure was solvated in a cubic periodic box of TIP3P water with at least 10 Å distance from the complex surface, followed by the addition of 0.15 M NaCl ions to neutralize the system. To relax the whole system, the starting structure was then subject to energy minimization and two rounds of 500-ps constant volume (NVT) and 500-ps constant pressure (NPT) equilibration with position restraints on the solute heavy atoms. Subsequently, the production NPT simulations were conducted for up to 200 ns for each complex system. During the production runs, all bonds involving hydrogen were constrained using the SHAKE algorithm to enable an integration time-step of 2 fs. The non-bonded interaction was cut off at 1.0 nm, whereas the electrostatic interaction beyond that was treated using the particle mesh Ewald (PME) method. The Langevin Thermostat was applied to maintain the temperature at 300 K, and the Berendsen Barostat was used to maintain the pressure at 1.0 bar. The trajectories of the production runs were saved at 8 ps intervals.
We collected 675,000 coordinates from all of the 5.4 μs production runs and performed our analysis on the basis of these snapshots. The interaction residues between XIAP and Sur-X were determined by the following criterion: if the shortest distance between any heavy atoms from two residues belonging to two proteins was less than 4.5 Å, then these two residues from two proteins were considered to form an interaction residue pair. By counting the highest-frequent pairs appeared during the 60–100, 100–160, and 160–200 ns trajectories of each simulation, we determined the interaction pairs shared between the three time periods as the interface residues between XIAP and Sur-X.
Enzyme-linked immunosorbent assay (ELISA)
According to the manufacturer’s instruction, Human TNF-α ELISA kit (R & D Systems, DTA00D) was used to measure the TNF-α in culture medium secreted by colorectal cancer cells.
Plasmid and small interfering RNA transfection
TAB1 plasmid and the vector pcDNA3.1(+) were purchased from Obio Technology Corp., Ltd. (Shanghai, China). SiRNAs of TAK1 and negative control (NC) were obtained from Beijing ViewSolid Biotech, the sequences are as follows: 5′-GGUGCUGAACCAUUGCCAUTT-3′ for si#1 of TAK1; 5′-GCAACCCAAAGCGCUAAUUTT-3′ for si#2 of TAK1; 5′-UUCUCCGAACGUGUCACGUTT-3′ for NC. Cells were transfected as previously described [
25].
Xenograft mouse model
A total of 10 four-week old female BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and fed at a specific pathogen-free environment in Animal Laboratory Unit of China Medical University, with the approval (2019088) of Institutional Review Board of China Medical University for all in vivo experiments. HCT116 cells (5 × 106) were injected subcutaneously near the right scapula of mice. When the average tumor volume reached 50–75 mm3, mice were randomly divided into two groups (n = 5) and received intravenous injection of 50 mg/kg Sur-X or Con daily for a total of 14 times since then. Tumor size and body weight were measured every other day. The volume of each tumor V was calculated using V = 1/2 (length × width2). Mice were killed by cervical dislocation according to the protocol filed with the Guidance of Institutional Animal Care and Use Committee of China Medical University.
Immunohistochemistry (IHC)
Tumor tissues were fixed by formalin, embedded by paraffin and prepared for staining with haematoxylin and eosin (HE) and antibodies of survivin, XIAP, and MLKL as described in our previous studies [
31]. The staining was evaluated by scanning the entire tissue specimen under low magnification (× 10) and confirmed under high magnification (× 20 and × 40). Both staining intensity and staining area were used to classify the expression of proteins, with staining intensity scored as 0 (no), 1 (low), 2 (intermediate), and 3 (high) points and staining area scored as 0 (≤5%), 1 (5–25%), 2 (25–50%), 3 (50–75%) and 4 (> 75%) points, respectively. Histoscore was calculated as histoscore = staining intensity × staining area. Two pathologists were responsible for determining the final histoscore independently. The expression of protein with a histoscore of 0, 1–4 points and 6–12 points were defined as negative (−), weak positive (+) and strong positive (++), respectively and evaluated under 5 randomly selected, non-overlapping fields from the stained sections.
TUNEL assay
One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, China, C1088) was used to detect the apoptosis in xenograft tumors according to the manufacture’s instruction. Fluorescence microscope BX53 (Olympus, Japan) was used to visualize the stained tissue sections.
Statistical analysis
SPSS Version 16.0 (SPSS Inc., Chicago, IL) was used to analyze the results of experiments which were conducted in triplicate and presented as mean ± standard deviation (SD). The graphics were generated using GraphPad 6.0 (GraphPad Software, USA). Student’s t-test and one-way ANOVA were used to analyze the differences between two independent groups and multiple groups, respectively; Chi-square test was used to assess the differences of categorical data; p < 0.05 was considered statistically significant.
Discussion
Apoptosis evasion is one of the most important hallmarks of tumorigenesis and a major contributor of chemotherapy resistance in various cancers including colorectal cancer [
35]. Thus, inducing apoptosis has been considered as one of the fundamental strategies in cancer treatment [
36]. Survivin is believed a promising target in cancer therapy due to its apoptosis inhibition function and specific overexpression in most cancers [
37]. To date, plenty of survivin-targeted anticancer drugs have been developed to interfere with the interactions between survivin and other proteins [
18]. In particular, anticancer peptides have emerged with potential for use in cancer treatment, due to their ease of synthesis, less toxic side-effects and lower immunogenicity [
38]. Shepherdin is a potent anticancer peptidomimetic antagonist of the survivin-Hsp90 complex, which can occupy the ATP-pocket of Hsp90 [
16]. It was reported that shepherdin exerted significant pro-apoptotic effects mainly by promoting rapid degradation of survivin in multiple cancer cell lines and xenograft mouse models [
16,
39,
40]. However, since Hsp90 also chaperones some tumor suppressive proteins such as interferon regulatory factor 1, LATS1 and LATS2 kinases, shepherdin’s inhibition on Hsp90 might deregulate tumor suppressor pathways, which might be one of the reasons why shepherdin failed in clinical application [
41]. Therefore, it is necessary to develop more specific and effective peptides targeting survivin with clinical translation potential.
In this study, we focused on the interaction between survivin and XIAP. It was reported that survivin and XIAP can form a heterocomplex to stabilize both of them and synergistically antagonize the activity of Caspase 9 to inhibit apoptosis in cancer cells [
23]. Therefore, we speculated that the peptide targeting the survivin-XIAP complex might be a promising therapeutic strategy for colorectal cancer treatment. As the first peptide that targets the survivin-XIAP complex, Sur-X was proved to be able to interfere with survivin-XIAP interaction, promote ubiquitination-mediated degradation of survivin and XIAP, and hence induce Caspase 9-dependent intrinsic apoptosis in colorectal cancer cells. Of note, previous study has reported that XIAP can directly inhibit Caspase 9 through the BIR 3 domain [
42], however, MD simulations in our study showed that Sur-X mainly bound to the BIR1 of XIAP. Moreover, no reduction of XIAP-Caspase 9 interaction was detected in Sur-X-treated cells. Thus, it is plausible that Sur-X induces intrinsic apoptosis via disrupting the survivin-XIAP complex and subsequently accelerating the degradation of survivin and XIAP, rather than directly interfering with XIAP-Caspase 9 interaction.
As an alternative of caspase-dependent apoptosis, necroptosis is a novel form of regulated cell death with a necrotic morphology, mainly mediated by RIP1, RIP3 and MLKL [
43]. Our study found that apart from inducing apoptosis, Sur-X also promoted necroptosis both in colorectal cancer cells and HCT116 xenograft mouse models. In fact, shepherdin was also reported to be able to induce non-apoptotic cell death, whereas the exact form was not identified [
16]. Similar to our findings, LCL161, a SMAC mimetic compound which can bind and antagonize IAPs including XIAP, was observed to promote both apoptosis and necroptosis in breast cancer cells [
44]. BV6, another small-molecule SMAC mimetic, was able to trigger necroptosis in pancreatic cancer cells when caspase activation was blocked [
45]. Yabal et al. reported that loss of XIAP resulted in increased expression and aberrantly elevated ubiquitination of RIP1 to promote RIP3-dependent cell death [
46]. However, whether XIAP directly interacts with RIP1 and/or RIP3 remains unknown. Therefore, the exact mechanisms by which XIAP regulates necroptosis in cancer cells merit further investigation.
In the present study, no elevated expression of RIP1 was observed in Sur-X-treated cells, which excluded the assumption that Sur-X might regulate RIP1 by reducing XIAP expression. Of note, TAK1, a member of the serine/threonine protein kinase family, has recently been known as an endogenous necroptosis inhibitor [
47]. It was reported that TAK1 can directly mediate inhibitory phosphorylation of RIP1 on S321 to prevent RIP1-RIP3 interaction [
48]. The TAK1-dependent S25 phosphorylation of RIP1 by IKKα/β was also demonstrated to inhibit the kinase activity of RIP1 [
49]. Podder B and his colleagues reported that inhibiting TAK1 can promote necroptosis through RIP1 activation and necrosome formation in melanoma cells [
34]. Moreover, it was found that TAB1, an upstream adaptor for the activation of the kinase TAK1, can interact with the BIR1 domain of XIAP [
33]. In our study, the result of MD simulations indicated that Sur-X mainly bound to the BIR1 domain of XIAP. Thus, we hypothesized that Sur-X might interfere with the interaction between the BIR1 domain of XIAP and TAB1. As expected, Sur-X was found to be able to effectively inhibit the interaction between XIAP and TAB1 in the XIAP-TAB1-TAK1 complex. Interestingly, the disruption of XIAP-TAB1 interaction by Sur-X accelerated the degradation of TAK1 and hence the inhibition of TAK1 on necroptosis was attenuated in colorectal cancer cells. Taken together, apart from inducing intrinsic apoptosis by disrupting the survivin-XIAP complex, the novel anticancer peptide Sur-X can also promote necroptosis by accelerating the degradation of TAK1 via inhibiting XIAP-TAB1 interaction.
Conclusions
In summary, we designed a novel anticancer peptide, Sur-X, which targets the survivin-XIAP complex, and found that Sur-X can promote both apoptosis and necroptosis via interfering with the interaction between survivin and XIAP, XIAP and TAB1, respectively. Since survivin, XIAP, TAB1 and TAK1 are involved in the anticancer effects of Sur-X, it can be inferred that Sur-X may have broader efficacy in cancer patients with high expression of survivin, XIAP, TAB1 and/or TAK1. Moreover, Sur-X may also disrupt the interactions between other essential proteins with survivin or XIAP, which need further exploration. Similar to most other peptide agents, the poor stability of Sur-X in vivo may limit its translational application for cancer patients. Therefore, in the future study, we will utilize available manufacturing technologies to improve the bioavailability of Sur-X. In addition, conjugation with other anticancer agents is considered as another effective approach to facilitate the clinical translation of Sur-X.
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