The anti-HIV drug tipranavir induces gastric cancer stem cell apoptosis and exerts anticancer activity via the PRSS23–IL24 pathway

doi:10.21203/rs.3.rs-1660593/v1

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The anti-HIV drug tipranavir induces gastric cancer stem cell apoptosis and exerts anticancer activity via the PRSS23–IL24 pathway

https://doi.org/10.21203/rs.3.rs-1660593/v1

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Gastric cancer stem cells (GCSCs) contribute substantially to the refractory features of gastric cancer (GC) and are responsible for metastasis, relapse and drug resistance. However, strategies aimed at eliminating GCSCs to improve the clinical outcome of GC patients remain a challenge. Here, we report that tipranavir, a clinically used anti-HIV drug, effectively killed both GCSC and GC cell lines, and inhibited tumor growth in GCSC-derived xenograft models without apparent toxicity. Mechanistic analyses demonstrated that tipranavir induced GCSC cell apoptosis by targeting the novel serine protease PRSS23, releasing MKK3 from the PRSS23/MKK3 complex to activate p38 MAPK, and thereby activating the interleukin (IL) 24-mediated Bax/Bak mitochondrial apoptotic pathway. Tipranavir also killed other types of cancer cell lines and drug-resistant cell lines. Our findings indicate that by targeting both GCSC and GC cells, tipranavir is a promising anti-cancer drug, and the clinical development of tipranavir or other drugs specifically targeting the PRSS23/MKK3/p38MAPK–IL24 mitochondrial apoptotic pathway may offer an effective approach to combat gastric and other cancers.

Tipranavir

Gastric cancer stem cell

PRSS23

MKK3

p38 MAPK

IL24

Gastric cancer (GC) is the fifth most commonly diagnosed cancer and the third leading cause of cancer-related death globally [1]. The highest incidence and mortality rates of GC occur in East Asian countries, accounting for ~ 60% of the total new cases and deaths worldwide [2]. In China, GC is the third leading cause of cancer incidence and mortality, with an estimated 478,508 new cases and 373,789 deaths in 2020 [3]. Most patients with GC are diagnosed at an advanced stage [4]. Thus, chemotherapy is the current preferred treatment in patients with primarily unresectable, advanced-stage GC [5]. Fluoropyrimidine- and platinum-based chemotherapy regimens, such as 5-fluorouracil (5-FU) in combination with cisplatin, are the most commonly used first-line treatments for advanced GC patients [6–8]. However, the prognosis of advanced GC patients remains poor, and the five-year overall survival rate is < 20% (with a median overall survival time of less than 12 months) due to recurrence and metastasis [9–11].

Cancer stem cells (CSCs) are a subpopulation of stem cell-like cancer cells that are responsible for all aspects of cancer pathogenesis including initiation, development, drug resistance, metastasis and cancer recurrence [12–14]. Traditional chemo- and radiotherapies kill most tumor cells but leave rare CSCs. These residual cells then regenerate the tumor or spread to form new tumors in other organs, and thus lead to tumor relapse, metastasis and treatment failure. Targeting CSCs can lead to tumor regression, and combining CSC-targeted therapy with conventional chemotherapy could eradicate the tumor [15–16]. Therefore, the development of CSC-targeting drugs for cancer research and clinical application is critical and urgent.

Gastric cancer stem cells (GCSCs) were first identified from human gastric cancer cell lines [17]. We previously identified and purified GCSCs from human gastric adenocarcinoma (GAC) tumor tissue, and showed that these cells can be expanded in vitro and form tumors in mice [18]. GCSC-targeting therapies are currently being investigated in lab studies and clinical trials, including chemotherapeutic and biological agents that target GCSC surface markers, signaling pathways, and the CSC microenvironment [19, 20]. However, only a few agents that target these molecules have been identified. Further clinical studies are needed to prove the clinical significance of these agents [19, 21], and there are no therapies that target GCSCs therapies approved for clinical use. To date, no agents can efficiently eliminate both GCSCs and GC cells.

Multiple studies indicate that human immunodeficiency virus (HIV) protease inhibitors (PIs) such as nelfinavir have anti-cancer effects [22–25], suggesting they could be suitable candidates for drug repurposing for cancer.

In the present study, we found that the anti-HIV drug tipranavir killed GCSC cells by activating the PRSS23/MKK3/p38-IL24-Bax/Bak mitochondrial apoptotic pathway, and inhibited the growth of GCSC-derived tumor xenografts in nude mice with little toxicity on non-cancerous cells. Moreover, tipranavir killed GC cells as well as other types of tumor cells. Collectively, our results show that by targeting both GCSC and GC cells, tipranavir could be a promising and effective treatment for gastric cancer.

Reagents and antibodies. Atazanavir, darunavir, fosamprenavir, tipranavir, 5-FU and cisplatin were purchased from MedChemExpress. Poloxamer 188 (F68) was obtained from Shenyang Jiqi Pharmaceutical Company. Antibodies against Bcl-2 (#4223), Bcl-xL (#2764), Bax (#5023), Bak (#12105), cleaved caspase-9 (#7237), cleaved caspase-7 (#8438), cleaved caspase-3 (#9661), cleaved PARP (#5625), p38 MAPK (#8690), phosphorylated p38 MAPK (#4511), phosphorylated MKK3/MKK6 (#12280), GAPDH (#5174), and VDAC (#4866) were purchased from Cell Signaling Technology. The antibody against PRSS23 (#ab201182) was from Abcam. The antibody against CEBPβ (#sc-7962) was from Santa Cruz Biotechnology. The antibody against IL24 (#26772-1-AP), MKK3 (#13898-1-AP), MKK4 (#17340-1-AP), MKK6 (#12745-1-AP) was from Proteintech. The antibody against cytochrome c (#556433) was from BD Biosciences. The secondary antibodies anti-rabbit (#7074) and anti-mouse (#7076) were obtained from Cell Signaling Technology.

Cell culture. Freshly isolated, primary tumor-derived GCSC cell lines (GCSC1 and GCSC2) were obtained from Dr Xianming Mo (Sichuan University), and were grown and maintained as previously described [18]. AGS, HGC-27, MGC-803, BGC-823, GES-1, PC9, PC3, HCT116, MDA-MB-231 and Huh7 cells were obtained from Cobioer Biosciences. KYSE180 and KYSE520 cells were obtained from DSMZ, the German Resource Centre for Biological Material. PC3/Tax cells were provided by Dr Xiangwei Wang (Shenzhen University General Hospital) [53]. AGS, HGC-27, MGC-803, BGC-823, GES-1, PC9 and HCT116 cells were cultured in RPMI (Gibco) with 10% FBS (Gibco) and penicillin/streptomycin (Gibco). KYSE180, KYSE520, MDA-MB-231 and Huh7 cells were cultured in DMEM (Gibco) with 10% FBS and penicillin/streptomycin. PC3 cells were cultured in DMEM/F12 (Gibco) with 10% FBS and penicillin/streptomycin. PC3/Tax cells were cultured in DMEM/F12 with 10% FBS, paclitaxel (10 µM, Selleckchem), and penicillin/streptomycin. All cell lines were mycoplasma negative.

RNA extraction and qRT-PCR analysis. Total RNA was extracted using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix (Takara Biotechnology) and qPCR was conducted using TB Green Premix Ex Taq (Takara Biotechnology) on a CFX96 real-time PCR detection system (Bio-Rad Laboratories). GAPDH was used as an internal control and the 2-∆∆CT method was used to quantify the relative mRNA expression of each gene. The sequences of primers used in this study were as follows:

PRSS23-F: 5’-CAGTGTCATAAGGGAACTCCAC-3’

PRSS23-R: 5’-CCTGAGTCTCGGTGTTGGG-3’

CEBPβ-F: 5'-AGAAGACCGTGGACAAGCAC-3'

CEBPβ-R: 5'-GCTTGAACAAGTTCCGCAGG-3'

IL24-F: 5’-TTGCCTGGGTTTTACCCTGC-3’

IL24-R: 5’-AAGGCTTCCCACAGTTTCTGG-3’

GAPDH-F: 5’-CGAGATCCCTCCAAAATCAA-3’

GAPDH-R: 5’-ATCCACAGTCTTCTGGGTGG-3’

Western blot analysis. Cultured cells or in vivo tumor tissues were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined by the BCA assay (Thermo Fischer Scientific). Equal amounts of protein were separated by SDS–PAGE, transferred to PVDF membranes (Bio-Rad) and probed overnight at 4°C with the following primary antibodies: anti-Bax (1:2000), anti-Bak (1:2000), anti-Bcl-2 (1:2000), anti-Bcl-xL (1:2000), anti-cleaved caspase-9 (1:1000), anti-cleaved caspase-7 (1:1000), anti-cleaved caspase-3 (1:1000), anti-cleaved PARP (1:2000), anti-GAPDH (1:5000), anti-MKK3 (1:2000), anti-MKK4 (1:2000), anti-MKK6 (1:2000), anti-phospho MKK3/MKK6 (1:2000), anti-p38 MAPK (1:2000), anti-phospho p38 MAPK (1:2000), anti-VDAC (1:2000), anti-cytochrome c (1:2000), anti-PRSS23 (1:1000), anti-CEBPβ (1:500), anti-IL-24 (1:1000). The secondary antibodies used were anti-rabbit-HRP (1:5000) or anti-mouse-HRP (1:5000). Blots were developed using Immobilon western chemiluminescent HRP substrate (Millipore) or SuperSignal west chemiluminescent substrate (Thermo Fisher Scientific), and imaged using a ChemiDoc MP imaging system (BioRad). The band intensity was quantified using Image Lab software (Bio-Rad).

Cell viability assay. Cell viability was analyzed using a cell counting kit-8 (CCK-8) (Dojindo Kumamoto) following the manufacturer’s instructions. Exponentially growing cells were seeded into 96-well culture plates (5 × 10⁴ cells/ml) in 100 ul of medium for 24 h. Cells were treated for the indicated times. After incubation with CCK-8 reagent for 2 hours at 37°C, the absorbance was measured at 450 nm wavelength using a microplate reader (Biotek). Experiments were performed in triplicate and repeated at least three times. Cell viability percentages of treated cells were compared with untreated cell viability, which was normalized as 100%. The half-maximal inhibitory concentrations (IC50 value) were calculated using GraphPad Prism software (GraphPad Software Inc).

Apoptosis assay. Apoptotic cells were identified by staining with FITC-labelled annexin V and propidium iodide using a FITC annexin V apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions. The cells were stained with annexin V-FITC and propidium iodide in annexin V binding buffer for 15 min on ice, then analyzed by flow cytometry using CytoFLEX (Beckman Coulter), and data were analyzed using FlowJo software.

TUNEL assay. DNA fragmentation, which is indicative of apoptosis, was examined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method (TUNEL). The TUNEL assay was performed using an in situ cell death detection kit (Roche Molecular Diagnotics) according to the instructions of the manufacturer. Briefly, cells were fixed in 4% paraformaldehyde at room temperature for 1h, then rinsed with phosphate-buffered saline (PBS). The cells were incubated with 3% H₂O₂ (in methanol) at room temperature for 10 min, then rinsed with PBS. The cells were permeated with 0.1% Triton X-100 for 2 min on ice. TUNEL enzyme and label solution were mixed and applied to the prepared cell climbing slices, which were incubated in a humidified chamber for 1 h at 37°C. Slices were thoroughly rinsed with PBS, counterstained with DAPI for nuclear staining and analyzed in a drop of PBS using a fluorescence microscope. The nuclei of apoptotic cells fluoresced green (stained with FITC fluorescein-dUTP). The TUNEL positive cells (apoptotic cells) were counted. Three fields in each section were measured. The percentage of apoptotic cells was quantified by dividing the number of green cells by the total cell number x 100%.

Mitochondrial isolation. Mitochondria were isolated with a mitochondria isolation kit for cultured cells (Thermo Fisher Scientific) according to the manufacturer's instructions. Mitochondria were isolated under sterile conditions at 4°C.

Transfection. PRSS23 (NM_001293180.1) and IL24 (NM_006850.3) overexpression and control vectors were designed and provided by GeneCopoeia, and siRNA specific for PRSS23 (siPRSS23-1) (5’-CCCAGATTTGCTATTGGATTA-3’), (siPRSS23-2) (5'-CAAGCAATATCTGTCTTAT-3'), IL24 (siIL24-1) (5’-ACAAUAGAACAGUUGAAGUCATT-3’), (siIL24-2) (5’-CUGGAUGCAGAAAUUCUACAATT-3’), MKK3 (siMKK3-1) (5’-CAGAAACTTTGAGGTGGAGGCTGAT-3’), MKK3 (siMKK3-2) (5’-CCGAGTTTGTGGACTTCACTGCTCA-3’), MKK4 (siMKK4-1) (5’- CAACTTGTGCCTTACGAAGGA-3’), MKK4 (siMKK4-2) (5’- CCAAAGTGGAATAGTGTATTT-3’), MKK6 (siMKK6-1) (5’- GAGCTAATGCAACATCCATTT-3’), MKK6 (siMKK6-2) (5’- GGATCCGAGCCACAGTAAATA-3’), MKK6 (siMKK6-3) (5’- CAATGCTCTCGGTCAAGTGAA-3’) and the non-targeting siRNA control (5’-UUCUCCGAACGUGUCACGUTT-3’) were purchased from GenePharma (China). Transfection of plasmids or siRNA into cells using Lipofectamine 3000 (Thermo Fisher Scientific) was performed according to the manufacturer’s instructions. Cells were harvested for western blotting analysis to verify the expression of targeted genes 72 h after transfection.

RNA-seq analysis. GCSCs were treated with or without tipranavir (20 µM) for 24 h, and the total RNA was extracted and purified using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. RNA quantitation and quality control were performed using a Bioanalyzer 2100 (Agilent Technologies). Construction of stranded RNA-seq libraries for high-throughput sequencing was carried out on an Illumina Novaseq 6000 following the manufacturer’s protocol. Raw reads passing the Illumina RTA quality filter were pre-processed using FASTQC, version 0.11.2 for quality control of the base sequencing. RNA-seq reads were mapped to the reference genome of Illumina Ensembl genome GRCh37 using STAR version 2.4.2a. Mapped reads were summarized for each gene using RSEM version 1.2.29. Differential expression analysis was performed using edgeR version 3.2.4. P values were calculated by the Student’s t-test.

Immunoprecipitation. Cell lysates were pre-cleared, incubated in primary antibodies PRSS23 or p38 MAPK antibodies, followed by incubation with protein G beads (GE healthcare). The beads were washed six times by lysis buffer and immunoprecipitates were eluted with SDS sample buffer by boiling for 5 min followed by western blot analysis.

Animal studies. The following animal handling and procedures were approved by the Shenzhen University Animal Care and Use Committee and followed the ARRIVE guidelines. Single-cell GCSC cells were resuspended in PBS medium with Matrigel basement membrane matrix (BD Biosciences) at a 1∶1 ratio (total volume 100 µL). Cells (1 x 10⁶ cells) were then were subcutaneously injected into BALB/c nude mice (4–6 weeks old, 16.0 ± 2.0 g). Ten days after inoculation, when the tumor diameter reached about 5 mm, the mice were randomly divided into three groups (5 animals for each group, day 1). Tipranavir was freshly prepared (25 mg/kg/mouse) as described previously [54] with a minor modification. In brief, tipranavir was dissolved in 2% (v/v) ethanol and 10% (v/v) PEG-300 and maintained at 37°C in a water bath. This dissolved solution was then added dropwise to F68 solution (1 mg/ml in normal saline) at 37°C under stirring. The solution was cooled at room temperature before use. The tipranavir solution was intraperitoneally injected every day for 8 days. The control group was injected with the same volume of vehicle (2% ethanol, 10% PEG-300, 1 mg/ml F68). The 5-FU + Cis group was treated with 5-FU (20 mg/kg/mouse) and cisplatin (2 mg/kg/mouse) every day for 8 days. Tumor size and body weight were measured every day. Tumor size was measured using a caliper, and tumor volume was determined by using the formula: L×W²×0.52, where L is the longest diameter and W is the shortest diameter. After 8 days, all mice were euthanized. Tumors were collected and weighted. Organs including heart, lung, liver, spleen, kidney were harvested, collected and organ coefficients were calculated using the formula: W_organ/W_Body×100%, where W_organ is the weight of the organ and W_Body is the body weight. The harvested organs were subjected to histological examination using hematoxylin and eosin (H&E) staining.

Statistical analysis. Statistical analyses were performed using GraphPad Prism software (La Jolla, CA, USA). Results are expressed as mean ± SEM, unless indicated otherwise. Groups were compared with either a two-tailed Student t-test for analysis of two groups or a two-way ANOVA to compare multiple groups. Significance was accepted when P was less than 0.05.

Tipranavir inhibits the cell viability of both GCSC and GC cell lines. To discover potential therapeutic drugs targeting GCSCs, we tested four approved HIV-PIs (atazanavir, darunavir, fosamprenavir and tipranavir) in two GCSC cell lines (GCSC1 and GCSC2). Interestingly, we found that low concentrations of tipranavir (0–20 µM) significantly and concentration-dependently inhibited the viability of two GCSC cell lines, whereas other HIV-PIs (atazanavir, darunavir and fosamprenavir) produced little inhibition at low concentrations (Fig. 1a). The half-maximal inhibitory concentration (IC₅₀) values for tipranavir were 4.7 µM in GCSC1 cells and 6.4 µM in GCSC2, and the inhibitory effect was time-dependent (Fig. 1b and Supplementary Fig. 1). Next, we evaluated the effects of 5-fluorouracil (5-FU), cisplatin and tipranavir on two GCSC cell lines. It is well known that CSCs are resistant to conventional chemotherapeutic drugs such as 5-FU and cisplatin. Tipranavir alone or in combination with 5-FU, cisplatin, or 5-FU plus cisplatin significantly inhibited the viability of GCSCs, whereas GCSCs were more resistant to the effects of 5-FU, cisplatin, or 5-FU plus cisplatin treatments (Fig. 1c). These results indicate that tipranavir is more effective at killing GCSCs than chemotherapeutic drugs such as 5-FU and cisplatin, which are the standard first-line chemotherapy regimens for gastric cancer patients. Treatment of GCSCs with tipranavir alone or in combination with 5-FU, cisplatin, or 5-FU plus cisplatin inhibited the growth of GCSCs to a similar degree, suggesting that tipranavir and 5-FU or cisplatin did not act synergistically to inhibit the viability of GCSCs.

The effect of tipranavir on cell viability was also evaluated in a panel of GC cell lines (AGS, HGC-27, MGC-803 and BGC-823 cells) and a normal gastric epithelial cell line (GES-1). As shown in Fig. 1d, tipranavir inhibited growth to a greater extent than 5-FU plus cisplatin in the four GC cell lines. In contrast, GES-1 was insensitive to tipranavir, whereas 5-FU plus cisplatin treatment significantly reduced cell viability; this finding is consistent with the nonspecific off-target toxicities that are associated with 5-FU plus cisplatin (Fig. 1d). These data also indicate that tipranavir induces GC cell death without obvious toxicity in vitro. Furthermore, tipranavir inhibited the viability of various types of human cancer cell lines, including lung (PC9), prostate (PC3), esophageal (KYSE180 and KYSE520), colorectal (HCT116), breast (MDA-MB-231), and liver (Huh7) cancer cells (Supplementary Fig. 2). Interestingly, tipranavir significantly reduced the viability of a paclitaxel-resistant prostate cancer cell line (PC3/Tax) (Supplementary Fig. 2), indicating that tipranavir may kill chemo-resistant cancer cells. Taken together, these data show that tipranavir kills GCSCs, GC cells, and other types of cancer cells without toxicity to normal gastric epithelial cells.

Tipranavir induces GCSCs apoptosis by activating the mitochondrial apoptotic pathway. Tipranavir-treated GCSC1 and GCSC2 cells exhibited morphological changes typical of apoptotic cell death, including cytoplasmic shrinkage, nuclear fragmentation and cell disassembly into apoptotic bodies (Fig. 2a). In line with this observation, annexin V-FITC and PI staining analyses revealed that tipranavir-treated GCSC1 and GCSC2 cells had a much higher apoptotic rate than control cells (Fig. 2b, c). Moreover, TUNEL assays showed that the apoptotic rate of tipranavir-treated GCSCs was significantly higher than that of control GCSCs, confirming that tipranavir had pro-apoptotic activity (Fig. 2d, e).

Next, we studied the effects of tipranavir on the regulation of apoptotic signaling pathways. The intrinsic or mitochondrial apoptosis pathway involves changes in mitochondrial outer membrane permeabilization (MOMP) [26]. MOMP is controlled by pro- and anti-apoptotic members of the Bcl-2 protein family, which collectively determine cellular death and survival decisions [27, 28]. As shown in Fig. 2f, the pro-apoptotic Bcl-2 proteins Bax and Bak, which are the essential effectors of MOMP, were strongly upregulated in GCSC1 and GCSC2 cells after 72 h of treatment with tipranavir. However, the tipranavir also upregulated two anti-apoptotic proteins, Bcl-2 and Bcl-xL, in GCSC1 and GCSC2 cells. Nevertheless, pro-apoptotic markers including cleaved forms of caspase-9, caspase-7, caspase-3 and PARP were activated in tipranavir-treated GCSCs (Fig. 2f). In line with these results, tipranavir treatment caused the release of cytochrome c from the mitochondria into the cytosol (Fig. 2f). Collectively, these data indicate that tipranavir causes GCSC cell apoptosis by activating the mitochondrial apoptotic pathway. Interestingly, 5-FU plus cisplatin treatment had little effect on the expression of these Bcl-2 family members and pro-apoptotic markers in GCSC1 and GCSC2 cells (Supplementary Fig. 3). Moreover, the protein level of mitochondrial cytochrome c was significantly lower in tipranavir-treated GCSCs than in 5-FU plus cisplatin-treated GCSCs (Supplementary Fig. 3), and whole-cell cytochrome c levels were significantly higher in tipranavir-treated GCSCs (Supplementary Fig. 3). This result indicated that tipranavir is more effective than 5-FU plus cisplatin in inducing apoptosis of GCSCs.

Tipranavir-induced GCSC and GC apoptosis are dependent on PRSS23. To elucidate the molecular basis of apoptosis induced by tipranavir, we performed RNA sequencing (RNA-seq) analysis on GCSCs treated with or without tipranavir (Supplementary Fig. 4). Compared with the expression profile of control GCSCs without tipranavir treatment, 423 genes were up-regulated and 350 genes were down-regulated in tipranavir-treated GCSCs (using an arbitrary cut-off of the signal log ratio ≥ 2.0 or ≤-2.0; Supplementary Table 1). Notably, we observed a significant reduction of PRSS23 (fold-change = 0.31; Supplementary Table 1), a novel serine protease. GO cellular component analysis ranked this gene highest of all the genes that were down-regulated upon tipranavir treatment (Supplementary Table 2). Proteases play key roles in the mitochondrial apoptotic pathway. PRSS23 is regulated by estrogen receptor α in ER-positive breast cancer cells, and it might be a critical component of estrogen signaling, which affects cell proliferation, survival and apoptosis [29]. A previous study showed that PRSS23 knockdown inhibited gastric cancer cell proliferation and induced apoptosis, indicating that the depletion of PRSS23 confers a strong apoptosis-promoting effect on cancer cells [30]. Therefore, we explored whether PRSS23 is a therapeutic target of tipranavir in GCSCs. In line with our RNA-seq data, qPCR analysis confirmed a 0.28- and 0.24-fold downregulation of PRSS23 in GCSC1 and GCSC2 cells, respectively, upon tipranavir treatment (Fig. 3a). Western blotting analyses showed that tipranavir treatment reduced PRSS23 protein expression in GCSC1 and GCSC2 cells (Fig. 3b) and this effect was concentration-dependent (Fig. 3c). This result indicates that PRSS23 may be indispensable for tipranavir-induced GCSC apoptosis.

To determine whether tipranavir exerts its apoptogenic activity through PRSS23 in GCSCs, we examined the effect of tipranavir on the growth of GCSCs treated with PRSS23-targeting siRNAs. As expected, PRSS23 knockdown significantly reduced the cell viability of GCSC1 (Fig. 3d) and caused GCSC cell apoptosis by activating the pro-apoptotic marker cleaved PARP (Fig. 3f), which were similar to those effects produced by tipranavir treatment on GCSC1 (Fig. 3d, f). Conversely, PRSS23 overexpression in GCSC2 enhanced cell proliferation, rescued the reduction in GCSC cell viability induced by tipranavir (Fig. 3e), and attenuated the tipranavir-induced activation of the mitochondrial apoptotic pathway in GCSCs (Fig. 3g). Moreover, we also investigated whether tipranavir exerted its apoptogenic activity through PRSS23 in GC cell lines. Similar to the results in GCSCs, PRSS23 knockdown significantly reduced the viability of AGS and HGC-27 cells (Fig. 3h), and caused GC cell apoptosis by activating cleaved PARP (Fig. 3i). These results indicate that tipranavir-mediated inhibition of PRSS23 contributes to the proapoptotic effects of tipranavir, and suggest that PRSS23 could be a new therapeutic target for reducing the growth of GCSC and GC cells.

Tipranavir-inhibited PRSS23 induces IL24 expression to promote GCSC apoptosis. We found that tipranavir treatment significantly increased the mRNA level of IL24 (fold change = 6.4; Supplementary Fig. 5), which is a known apoptosis-promoting tumor suppressor in human cancer cells [31, 32]. Moreover, IL24 could target CSCs for death [33, 34]. PRSS23 knockdown enhanced the protein expression of IL24 in GCSCs (Supplementary Fig. 6), so we sought to further investigate its role in tipranavir-induced GCSC apoptosis. Real-time PCR analysis showed that tipranavir treatment increased mRNA expression of IL24 in GCSC1 and GCSC2 cells (Fig. 4a), and western blotting analyses showed that tipranavir increased IL24 protein expression (Fig. 4b) in a concentration-dependent manner in GCSCs (Fig. 4c, d). These results indicate that IL24 may be indispensable for tipranavir-induced GCSC apoptosis. To investigate whether the anti-GCSCs effects of tipranavir are dependent on IL24, we performed siRNA-mediated knockdown of IL24 in GCSC1 and GCSC2 cells. As expected, tipranavir-mediated GCSC apoptosis was inhibited by the expression of two functional siRNAs targeting IL24 (Fig. 4e). The tipranavir-induced expression of mitochondrial apoptotic pathway proteins such as Bax, Bak, cleaved caspase-9, cleaved caspase-7, cleaved caspase-3, and cleaved PARP were strongly inhibited in siRNA-treated cells but not in control cells. The tipranavir-induced cytochrome c efflux from mitochondria was blocked in si-RNA-treated GCSC1 cells (Fig. 4f). Notably, knockdown of IL24 did not alter the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL in GCSC1 cells treated with tipranavir. This result suggests that the IL24-dependent mitochondrial apoptosis pathway that is activated by tipranavir in GCSCs may be independent of Bcl-2 and Bcl-xL, but dependent upon Bax and Bak. This finding is in line with previous reports showing that Bax and Bak alone were sufficient to induce MOMP [35, 36]. Furthermore, we examined the effects of IL24 overexpression on GCSCs. Ectopic overexpression of IL24 inhibited cell growth and induced apoptosis (Fig. 4g), and also activated the mitochondrial apoptotic pathway in GCSCs (Fig. 4h). Taken together, these results indicate that tipranavir induces GCSC apoptosis by reducing the expression PRSS23, which induces IL24 expression and thereby strongly influences the expression of pro-apoptotic proteins Bax and Bak to activate the mitochondrial apoptotic pathway.

The IL24-dependent mitochondrial apoptotic pathway activated by tipranavir relies on MKK3/p38 MAPK.

A previous study showed that phosphorylated p38 MAPK regulates IL24 expression by altering IL24 mRNA stability [37]. To assess the molecular mechanisms that underlie how IL24 expression is regulated by tipranavir-induced increases in PRSS23 in expression, we first investigated whether tipranavir alters p38 MAPK activation in GCSCs and the effect of p38 MAPK activation on IL24 mRNA expression. Accordingly, western blotting analyses showed that tipranavir increased phosphorylated p38 MAPK in GCSC1 cells (Fig. 5a). Moreover, the p38 MAPK inhibitor SB203580 downregulated IL24 mRNA expression in the absence or presence of tipranavir. Conversely, treatment with the p38 MAPK agonist anisomycin significantly enhanced an increase in IL24 mRNA expression induced by tipranavir (Fig. 5b). Simultaneous inhibition of transcription (using actinomycin D) and inhibition of p38 MAPK (using SB203580) decreased IL24 mRNA expression to a level similar to that observed with actinomycin D treatment alone (Fig. 5c). Treatment with actinomycin D (an RNA polymerase inhibitor) and anisomycin, or tipranavir alone increased IL24 mRNA expression, while combined treatment with SB203580, actinomycin D and tipranavir significantly decreased IL24 mRNA expression (Fig. 5c). Hence, as anticipated, IL24 expression is regulated by phosphorylated p38 MAPK, which alters IL24 mRNA stability in GCSCs.

Next, we determined whether phosphorylation of p38 MAPK can be modulated by PRSS23. Indeed, PRSS23 knockdown activated p38 MAPK and increased the level of phosphorylated p38 MAPK in GCSCs (Fig. 5d). Overexpression of PRSS23 decreased phosphorylated p38 MAPK (Fig. 5d), indicating that PRSS23 regulates the phosphorylation of p38 MAPK.

As p38 MAPK can be phosphorylated by MKK3, MKK6, or MKK4 [38], we next determined which kinase is responsible for the p38 MAPK phosphorylation. We silenced MKK3, MKK4, or MKK6 by siRNA and measured changes in the level of phosphorylated p38 MAPK. Knockdown of MKK3 significantly reduced the level of phosphorylated p38 MAPK; however, knockdown of MKK6 or MKK4 had little effect on the level of phosphorylated p38 MAPK (Supplementary Fig. 7), suggesting that phosphorylation of p38 MAPK is mainly dependent on MKK3. In line with these results, MKK3 knockdown decreased the level of phosphorylated p38 MAPK in tipranavir-untreated GCSCs, without the alteration of the expression of PRSS23 (Fig. 5e), while tipranavir treatment increased the level of phosphorylated p38 MAPK by inhibition of PRSS23 expression (Fig. 5e), and MKK3 depletion impaired the tipranavir-induced up-regulation of phosphorylated p38 MAPK (Fig. 5e), indicating that PRSS23 regulates the level of phosphorylated p38 MAPK via MKK3.

To dissect how PRSS23 modulates the level of phosphorylated p38 MAPK via MKK3 in GCSCs, we investigated the interactions between PRSS23 and MKK3, or p38 MAPK and MKK3. In GCSCs, phophorylated MKK3 (p-MKK3) binds predominately to PRSS23 rather than p38 MAPK (Fig. 5f), which leads to the presence of unphosphorylated p38 MAPK. However, tipranavir treatment reduces the expression of PRSS23, which releases phosphorylated MKK3 and allows it to interact with and activate p38 MAPK (Fig. 5f). In GC cells, phophorylated MKK3 (p-MKK3) binds predominately to p38 MAPK with or without tipranavir treatment, indicating that PRSS23-induced GC apoptosis is not dependent on the MKK3/p38 MAPK pathway (Fig. 5g). Overall, these results suggest that tipranavir-induced activation of the IL24-dependent mitochondrial apoptotic pathway relies on the PRSS23/MKK3/p38 signaling cascade in GCSCs.

Tipranavir inhibits GCSC-derived tumor growth in vivo without apparent toxicity. To explore the anti-tumor potential of tipranavir in vivo, nude mice bearing GCSC tumors were treated intraperitoneally with tipranavir (25 mg/kg), 5-FU plus cisplatin (5-FU: 20 mg/kg, cisplatin: 2 mg/kg), or vehicle (i.p.) once every day for 8 days, receiving eight treatments (Fig. 6a). As shown in Fig. 6b and 6c, GCSC-derived tumors from the tipranavir-treated group were significantly smaller and had a lower tumor weight than tumors from the control and 5-FU plus cisplatin-treated treatment groups. Tipranavir significantly inhibited the growth of subcutaneous GCSC tumors (Fig. 6d; P < 0.0001). There was no obvious difference in the growth of GCSC tumors between the 5-FU plus cisplatin treatment group and the control group (Fig. 6d; P > 0.05). These results are consistent with our in vitro data which showed that GCSCs are resistant to 5-FU and cisplatin, and suggest that tipranavir could inhibit GCSC-derived tumors more effeminately than standard chemotherapy drugs.

We used histopathology to evaluate the toxicity of tipranavir in vivo. H&E staining results showed no apparent injury to major organs, including the heart, lung, liver, spleen and kidney (Fig. 6e and Supplementary Fig. 8). Measurement of body weights, which respond to the systemic toxicity of drugs, showed that the average body weight in of tipranavir-treated group was similar to that of the control group (Fig. 6f; P > 0.05), suggesting that tipranavir did not cause weight loss in mice. However, treatment with 5-FU plus cisplatin caused significant weight loss in mice (Fig. 6f; P < 0.0001). Moreover, there was no significant difference in the organ coefficients of the major organs between the tipranavir-treated and control groups, suggesting that tipranavir treatment had no detectable toxic effects on the heart, lung, liver, spleen, and kidney (Fig. 6g). In contrast, treatment with 5-FU plus cisplatin significantly reduced the weight of the spleen, suggesting that this drug combination may impair spleen function (Fig. 6g; P < 0.001).

To test whether tipranavir promotes mitochondrial apoptosis by decreasing PRSS23 and inducing IL24 expression in vivo, we evaluated the protein expression of PRSS23, IL24 and apoptotic marker cleaved PARP in tumor samples from tipranavir-treated and control mice. GCSCs-derived tumors from the tipranavir-treated group showed significantly lower expression of PRSS23, and higher expression of IL24 and cleaved PARP than those from the control group (Fig. 6h). This result indicates that tipranavir suppressed gastric tumor growth via the PRSS23/IL24-mediated mitochondrial apoptotic pathway in vivo. Collectively, these data suggest that tipranavir may be an effective anti-tumor drug with little toxicity.

Tipranavir, a non-peptidic HIV-1 protease inhibitor, is an FDA-approved antiviral drug that is used in combination with ritonavir to treat HIV-1 infection. It is advantageous particularly in the management of patients with multi-protease inhibitor-resistant HIV [39–41]. Our current study demonstrates that tipranavir kills GCSC cells and inhibits tumor growth in human GCSCs xenografts without any observable toxic effects. Moreover, we showed that tipranavir induces GC cell death without cytotoxicity to normal gastric epithelial cells. Emerging evidence supports the notion that conventional chemotherapy targets fast-proliferating tumor cells, leading to the emergence of residual chemo-resistant cells with CSCs-like properties that may cause tumor relapse and metastasis [42]. While killing CSCs may lead to tumor regression, the combined killing of both cancer cells and CSCs may offer the opportunity to cure cancers. The results of the present study suggest that tipranavir could effectively eliminate gastric tumors. Tipranavir is an FDA-approved drug, so repurposing it as an anti-cancer drug would be beneficial for the effective treatment and/or long-term cure for GC patients. Additionally, tipranavir might have broad-spectrum anti-cancer effects, as it inhibited the growth of lung, prostate, esophageal, colorectal, breast, and liver cancer cells. Intriguingly, tipranavir was able to kill paclitaxel-resistant prostate cancer cells, so further studies to investigate its potential in paclitaxel-resistant prostate cancer is warranted.

In the present study, we provide the first evidence that tipranavir induces GCSC death via the IL24-dependent mitochondrial apoptotic pathway. IL24 (also known as melanoma differentiation-associated gene-7; mda-7) is a tumor suppressor gene found in numerous types of cancers [32]. Overexpression of IL24 induces apoptosis in a wide variety of cancer cells without affecting normal human cells [31]. In addition, preclinical and clinical data demonstrate that IL24 is a potent, effective therapeutic agent for cancer treatment [46–48]. In this study, we demonstrated that tipranavir upregulates the expression of IL24, which induces GCSC apoptosis. This finding is consistent with a previous study showing that recombinant human IL24 induces gastric cancer cell apoptosis and suppress tumor growth in vitro and in vivo [49]. However, it is not yet fully understood how IL24 induces tumor-cell apoptosis. Previous studies indicate that IL24 modulates the mitochondrial apoptotic pathway by downregulating the expression of anti-apoptotic proteins such as Bcl-2, Bcl-xL Mcl-1, and upregulating the expression of pro-apoptotic proteins such as Bax, Bak, Bim and Bid [50], thereby increasing the ratio of pro-apoptotic to anti-apoptotic proteins [51]. In this study, we observed a dramatic upregulation of pro-apoptotic proteins, including Bax and Bak, in tipranavir-treated GCSC cells or IL24-overexpressing GCSC cells. Unexpectedly, anti-apoptotic proteins like Bcl-2 and Bcl-xL were also upregulated tipranavir-treated GCSC cells or IL24-overexpressing GCSC cells; this finding is inconsistent with previous reports. Moreover, knockdown of IL24 didn’t alter the level of Bcl-2 and Bcl-xL in tipranavir-treated GCSC cells, but significantly reduced the expression of Bax and Bak, suggesting that the IL24-mediated mitochondrial apoptotic pathway is mainly dependent on Bax and Bak. In agreement with this finding, genetic deletion of all Bcl-2 proteins and reintroduction of Bax alone was sufficient to induce MOMP [36]. It seems that the mechanism by which IL24-Bax/Bak induces apoptosis in GCSC is different from the previous reported mechanisms of IL24-mediated apoptosis in other cells. Further studies to elucidate the molecular mechanisms that underlie how IL24 regulates Bax/Bak in GCSCs will improve our understanding of the biological function of GCSCs, and possibly lead to new therapies targeting GCSCs. Taken together, our findings suggest that the IL24-induced GCSC apoptosis elicited by tipranavir can be attributed to the activity of Bax and Bak, which shifts the balance of cell fate survival to death.

How does the tipranavir-mediated decrease PRSS23 expression regulate IL24 expression in GCSCs? Previous studies demonstrated that AP1 and CEBPβ are involved in the transcriptional control of IL24 expression [52]. Based on our RNA-seq data (Supplementary Table 1) and real-time PCR analysis (Supplementary Fig. 9a), we found that tipranavir treatment significantly upregulated the expression of the CEBPβ gene. However, western blotting analysis revealed that tipranavir or PRSS23 knockdown reduced the protein expression of CEBPβ (Supplementary Fig. 9b, 9c). Additionally, our results also demonstrated that IL24 expression isn’t modulated by AP1. Mechanistically, we found that activation of the IL24-mediated mitochondrial apoptotic pathway depended on the phosphorylation of p38 MAPK by MKK3. Activated p38 MAPK can regulate IL24 expression through a mechanism involving mRNA stabilization [37], and p38 MAPK can be phosphorylated by MKK3/MKK6, or MKK4 [38]. In this study, we demonstrated that in GCSCs, MKK3/p38 MAPK activation is modulated by PRSS23 expression. Unlike the direct link between MKK3/p38 MAPK that is not depended on PRSS23 in GC cells, our findings reveal that in GCSCs, the IL24-mediated mitochondrial apoptotic pathway is regulated by a rare and complex mechanism. Within this CSCs-specific mechanism of apoptotic regulatory networks, CSCs could be more resistant than GCs to apoptosis and cell death induced by extracellular stimuli. In this perspective, our findings could provide a potentially safe, effective therapeutic strategy to kill GCSCs.

In this study, we demonstrated that tipranavir exerts its apoptogenic activity by decreasing the expression of PRSS23 in both GCSC and GC cells. PRSS23 is a novel serine protease, initially identified in mouse ovary [45]. However, few studies on the structure and function of PRSS23 have been conducted. Although molecular docking studies showed that PRSS23 is not a direct target of tipranavir (data not shown), it is plausible that tipranavir’s functional target is PRSS23. The expression of PRSS23 was concentration-dependently inhibited by tipranavir, and inhibition of PRSS23 — either by siRNA knockdown or tipranavir — produced similar apoptotic effects in both GCSC and GC cells. Conversely, PRSS23 overexpression in GCSCs protected cells from tipranavir-mediated apoptosis. Mechanistically, PRSS23 interacts with phosphorylated MKK3, and interferes with the binding of phosphorylated MKK3 to p38 MAPK in GCSCs. Depletion of PRSS23 by tipranavir releases MKK3, which activates p38 MAPK, resulting in activation of the IL24-dependent mitochondrial apoptotic pathway in GCSC cells. However,, PRSS23 doesn’t interact with phosphorylated MKK3 in GC cells, and the detail mechanism that tipranavir-induced GC cells apoptosis needs to be further investigated.

Clinically targeting the MKKs/p38 MAPK pathway is challenging because of the intrinsic pleiotropic nature of MKKs and p38 MAPK. Targeting PRSS23 could represent an effective and potentially safe therapeutic strategy to kill gastric cancer stem cells. The key molecule(s) involved in GCSC survival are still poorly understood. To the best of our knowledge, this is the first study to identify a potential molecular drug target that is expressed by both GCSC and GC cells. Recently, PRSS23 knockdown was shown to inhibit GC cell proliferation and induce apoptosis by inhibiting EIF2 signaling [30]. Our findings suggest that PRSS23 could represent a selective, safe, and effective way of targeting GCSCs. Moreover, PRSS23-targeted therapy in combination with drugs that act on other targets such as EIF4E may offer an attractive approach to combating gastric cancer.

In summary, our present study demonstrates that tipranavir induces GCSC apoptosis via the PRSS23/MKK3/p38MAPK–IL24–Bax/Bak mitochondrial apoptotic pathway (Fig. 7). Moreover, tipranavir induced GC cell death without cytotoxicity to normal cells. Our findings not only provide evidence that by targeting both GCSC and GC cells, tipranavir could be a promising gastric cancer drug but also provide the first evidence that the PRSS23–IL24-mediated mitochondrial apoptotic pathway is a new target for the treatment of gastric cancer.

GCSCs Gastric cancer stem cells

GC gastric cancer

IL interleukin

5-FU 5-fluorouracil

CSCs Cancer stem cells

GAC gastric adenocarcinoma

HIV human immunodeficiency virus

PIs protease inhibitors

CCK-8 cell counting kit-8

IC₅₀ half-maximal inhibitory concentrations

TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

PBS phosphate-buffered saline

H&E hematoxylin and eosin

MOMP mitochondrial outer membrane permeabilization

p-MKK3 phophorylated MKK3

mda-7 melanoma differentiation-associated gene-7

Ethics approval and consent to participate

This study was approved by the institutional review board of Shenzhen University. Informed consents were obtained for the original work that produced human tissue samples.

Consent for publication

All subjects have written informed consent.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing Interests

The authors declare that they have no competing interests.

Funding

This work was supported by grants from the National Key R&D Program of China (2017YFA0503900), the National Natural Science Foundation of China (82173003), the Science and Technology Foundation of Shenzhen (JCYJ20200109113810154), the Science and Technology Program of Guangdong Province in China (2019B030301009), the Industry and Information Technology Foundation of Shenzhen (20180309100135860), the SZU Top Ranking Project (86000000210), the Guangdong Basic and Applied Basic Research Foundation (2020A1515010989 and 2018A030310586), and the Medical Scientific Research Foundation of Guangdong Province (A2019405).

Authors’ contributions

Conceptualization: J.X., and L.F.; Methodology: J.X., Y.L., X.T., and L.F.; Investigation: J.X, Y.L., X.T., and T.C.; Writing: J.X., and L.F.; Funding acquisition: J.X., and L.F.; Resources: B.L.; Supervision: J.X., and L.F.

Acknowledgments

We acknowledge Dr Xianming Mo (Sichuan University) for providing the GCSC cells. We thank Dr Xiangwei Wang and Dr Yuqi Wu (Shenzhen University General Hospital) for providing the PC3/Tax cells. We also thank Dr Wen Su (Shenzhen University) for her help in histological examination.

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No competing interests reported.

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The anti-HIV drug tipranavir induces gastric cancer stem cell apoptosis and exerts anticancer activity via the PRSS23–IL24 pathway

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Abstract

Figures

Background:

Methods:

PRSS23-F: 5’-CAGTGTCATAAGGGAACTCCAC-3’

PRSS23-R: 5’-CCTGAGTCTCGGTGTTGGG-3’

IL24-F: 5’-TTGCCTGGGTTTTACCCTGC-3’

IL24-R: 5’-AAGGCTTCCCACAGTTTCTGG-3’

GAPDH-F: 5’-CGAGATCCCTCCAAAATCAA-3’

GAPDH-R: 5’-ATCCACAGTCTTCTGGGTGG-3’

Results:

Discussion:

Conclusions:

Abbreviations

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