MST1 Suppresses Pancreatic Cancer Progression Via ROS-Induced

Published OnlineFirst February 22, 2019; DOI: 10.1158/1541-7786.MCR-18-0910

Cell Fate Decisions Molecular Cancer Research MST1 Suppresses Pancreatic Cancer Progression via ROS-Induced Pyroptosis Jiujie Cui1,2, Zhuqing Zhou3, Haiyan Yang1,2, Feng Jiao1,2, Ning Li4, Yong Gao5, Liwei Wang1,2, Jingde Chen5, and Ming Quan5

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a deadly of the Hippo pathway, but mediated by reactive oxygen disease, and its incidence is increasing annually. It is critical species (ROS). And ROS scavenger N-acetyl-cysteine atten- to reveal and delineate the molecular mechanism promot- uated the activation of caspase-1 induced by MST1 and the ing PDAC development and progression. Mammalian effect of MST1 in PDAC cell death, proliferation, migration, STE20-like kinase 1 (MST1) is a proapoptotic cytoplasmic and invasion. Collectively, our study demonstrated that kinase and also one of the core components of the Hippo MST1 suppressed the progression of PDAC cells at least pathway. Here, we showed that MST1 expression was partly through ROS-induced pyroptosis. decreased in PDAC, and restored expression of MST1 promoted PDAC cell death and suppressed the proliferation, Implications: In this study, we identiﬁed a new mechanism of migration, invasion, and cell spheroid formation of PDAC MST1 in inhibiting PDAC development and progression and via caspase-1–induced pyroptosis. Further studies demon- revealed that MST1 would be a potential prognostic and strated that pyroptosis induced by MST1 was independent therapeutic target for PDAC.

Introduction Mammalian STE20-like kinase 1 (MST1), also called Serine/ Threonine Kinase 4 (STK4), and MST2 share 76% identity in Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% amino acid sequence, and they are class II germinal center of all pancreatic malignancy and is often called pancreatic cancer. kinases (4). MST1 and MST2 are the closest mammalian homo- Despite decades of efforts, its 5-year survival rate remains about logs of the Drosophila Hippo pathway kinase and also core 8% and has been called the king of cancer (1). Recently, PDAC is components of the pathway. MST1 was first reported as a proa- projected to be the second leading cause of death related to cancer poptotic cytoplasmic kinase and critical for amplifying the apo- by the year 2030 (2). It has been demonstrated that many genes ptotic signals (5). Recent studies have indicated that MST1 was a and signaling pathways played essential roles in PDAC develop- putative tumor suppressor in many kinds of cancers, including ment and progression, including KRAS, TP53, CDKN2A, and head and neck cancer, colon cancer, liver cancer, glioma, prostate SMAD4 genes, and the Wnt, Notch, and Hippo pathways (3). cancer, and breast cancer (6–11). In pancreas, conditional dele- However, the mechanism of PDAC initiation and progression is tion of MST1 in mice pancreas resulted in acinar-to-ductal meta- far to be fully demonstrated, and it is still highly important to plasia, which was a precursor of pancreatic intraepithelial neo- reveal and delineate the molecular mechanism and pathways and plasia (PanIN) lesions that could progress to PDAC (12, 13). identify new targets for the treatment of PDAC. However, the roles and mechanisms of MST1 in suppressing 1Department of Medical Oncology, Renji Hospital, School of Medicine, Shanghai pancreatic cancer development and progression need further Jiao Tong University, Shanghai, China. 2State Key Laboratory of Oncogene and demonstration. Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Pyroptosis, a type of inflammatory form of programmed cell Shanghai Jiao Tong University, Shanghai, China. 3Department of Gastroenter- death, is marked by pore formation in membrane, cellular swell- ological Surgery, Shanghai East Hospital, Tongji University School of Medicine, fl 4 ing and lysis, and release of proin ammatory (14, 15). Although it Shanghai, China. Department of Oncology, First People's Hospital, School of shares many morphologic characteristic of apoptosis and necro- Medicine, Shanghai Jiao Tong University, Shanghai, China. 5Department of Oncology and Tumor Institute, Shanghai East Hospital, Tongji University School sis, pyroptosis remains a unique process that is mediated by of Medicine, Shanghai, China. inflammatory caspases, especially caspase-1 (16). Caspase-1 further cleaves proinflammatory cytokines interleukin-1b (IL1b) and Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). interleukin-18 (IL18; ref. 17). Recent studies have revealed the roles of pyroptosis in several types of cancers, including colon J. Cui and Z. Zhou contributed equally to this article. cancer, gastric cancer, lung cancer, breast cancer, liver cancer, and Corresponding Authors: Jiujie Cui, Department of Oncology and Tumor glioma. However, no studies have focused on the pyroptosis in Institute, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, PDAC (18–23). China. Phone: 86-21-61569716; E-mail: [email protected]; and Ming Quan, [email protected] In the present study, we found that the expression of MST1 was decreased in PDAC, and overexpression of MST1 sup- Mol Cancer Res 2019;17:1316–25 pressed PDAC proliferation, migration, and invasion via apo- doi: 10.1158/1541-7786.MCR-18-0910 ptosis and pyroptosis. Apoptosis has been widely reported in 2019 American Association for Cancer Research. PDAC. Consequently, we further investigated the mechanism

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of MST1-promoted pyroptosis in PDAC and found that MST1- Transfection and retroviral infection induced pyroptosis was independent of the Hippo signaling Lipofectamine 2000 CD and Lipofectamine LTX (Invitrogen) pathway, but via increasing the levels of reactive oxygen species were, respectively, used for the transfection of plasmids or shRNA. (ROS). These findings facilitate the understanding of PDAC Functional assays were performed after 48 hours of transfection. development and progression and identify a potential thera- Mock controls were PDAC cells that were treated with the trans- peutic strategy of PDAC. fection reagents. To generate MST1-expressing stable PDAC cells, BxPC-3 were infected with hexadimethrine bromide (Polybrene; 5 mg/mL) and a mixture of retroviruses. Thirty-six hours after Materials and Methods infection, cells were selected with 2 mg/mL puromycin in culture Human PDAC tissues and immunohistochemistry medium. The expression of MST1 was tested using tissue microarray (TMA), which was described previously (24). The TMA contains Antibodies and Western blot 154 PDAC tissues, 34 cancer adjacent tissues, and 22 normal Anti-MST-1 (HPA015270) was performed from Sigma. Anti- pancreas tissues. Clinical and demographic information includes MST2(#3952), anti-caspase-1 (#3866), anti-cleaved caspase-1 age, gender, T stages, N stages, M stages, TNM stages, and differ- (#4199), anti-IL1b (#12703), anti-mature.IL1b (#83186), anti- entiation grades. Immunohistochemical (IHC) analysis was con- cleaved caspase-3 (#9664), anti-cleaved caspase-7 (#9491), ducted with anti-MST1 (HPA015270, Sigma-Aldrich; diluted anti-YAP (#12395), and anti-pYAP (#13008) antibodies 1:150) antibodies. The evaluation of IHC was performed as were performed from Cell Signaling Technology. Anti-IL18 reported previously (25). (ab207323) was performed from Abcam. The internal control anti–b-actin antibody and secondary anti-mouse IgG or anti- Cell lines and reagents rabbit IgG antibodies were obtained from Santa Cruz Biotech- The human PDAC cell lines PANC-1, AsPC-1, MiaPaCa-2, nology. Standard Western blot was performed using total cell CoPan-1, and BxPC-3 were obtained from the ATCC. Colo357, lysates. Quantity One analysis software program was used to FG, L3.7, and MDA28 cells were previously described (26). All the quantify the bands (version 4.6; Bio-Rad). cancer cell lines were cultured in DMEM (Invitrogen) containing 10% FBS (Invitrogen), 50 mg/mL penicillin/streptomycin (P/S), and supplemented with a vitamin solution, sodium pyruvate, Measurement of PDAC cell migration, invasion, and colony L-glutamine, and nonessential amino acids (Flow Laboratories). formation and spheroid colony formation assays The cancer cell lines, which were obtained directly from ATCC, PDAC cells were treated as indicated groups. Transwell cham- were passaged no longer than 6 months in the laboratory. bers were used to measure PDAC cell migration and invasion The ROS scavenger N-acetyl-cysteine (NAC; Sigma-Aldrich) following the manufacturer's instructions, and the colony forma- was used with a concentration of 5 mmol/L. The caspase-1 tion assay and spheroid colony formation assay were performed inhibitor VX-765 was purchased from BioChemPartner and was as previously described (31, 32). used with a concentration of 5 mmol/L. Lactate dehydrogenase (LDH) release assay Plasmids and short hairpin RNAs (shRNA) PDAC cells, treated as indicated groups, were incubated with Full-length of MST1 was cloned from the plasmid pJ3H-MST1 50 mL Triton X-100 at a concentration of 9% (weight/volume) (plasmid #12203; Addgene) into the pcDNA3.0 as pMST1 (27). A for 30 minutes under room temperature. The CytoTox 96 BamHI–EcoRI fragment of pMST1 was cloned into the retroviral NonRadioactive Cytotoxicity Assay kit was purchased from expression vector pBABEpuro as pBABE-MST1. HEK293 cells Promega, and total LDH lysis was tested following the man- were transfected with a three-plasmid system that contained ufacturer's instructions. The absorbance value was measured at pCMV-VSV-G (plasmid #8454; Addgene), pUMVC (plasmid 450 nm. #8449; Addgene), and the pBABE-MST1 or pBABEpuro vector. Retroviruses were stored at 20C or frozen at 80C for long- term storage. The plasmid Flag-tagged yes-associated protein Viability assay (YAP) was also obtained from Addgene (plasmid #66853; ref. 28). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo. SiRNA to silence MST1 (siMST1) targeted the human MST1 The cell viability assay was performed following the manufac- fl mRNA sequences (50-CCT CCA GGA GAT AAT CAA A-30)as turer's instructions. Brie y, PDAC cells, which were treated as reported previously (29). Sequencing was used to verify all the indicated groups, were cultured in 96-well plates for 12 hours. m m amplified fragments of DNA. CCK-8 reagent (10 L) with 90 L medium were added into each well, and the cells were further cultured for 1.5 hours at 37 C. The RNA isolation and real-time PCR optical density values were tested at 450 nm. Total RNA was isolated with TRIzol reagent (Invitrogen). cDNA was synthesized and was subjected to real-time PCR with MST1 TUNEL staining and b-actin–specific primers in the presence of SYBR Green The TUNEL fluorescence FITC kit was purchased from Roche. reagent with an ABI Prism 7000HT sequence detection system PDAC cells in indicated groups were seeded into inside culture (Applied Biosystems; ref. 30). The sequences of the PCR primers plates for 24 hours, and the DNA fragmentation of PDAC cells was were as follows: MST1, 50-ACA AAT CCT CCT CCC ACA TTC CG-30 tested following the manufacturer's instructions. DAPI was used (forward) and 50-CAC TCC TGA CAA ATG GGT GCT G-30 to stain the nuclear, and fluorescence microscopy was used to (reverse); b-actin, 50-AGC CGG GCT CTT GCC AAT-30 (forward) measure the TUNEL staining. PDAC cells that were both stained and 50-AGT TAG CGC CCA AAG GAC CA-30 (reverse). with TUNEL and DAPI were positive.

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Dual-luciferase reporter assay the subcutaneous of mice. The size of the tumor was measured PDAC cells were cotransfected with pMST1 or control vector, each week. On day 35, mice were sacrificed and subcutaneous the 8GTIIC-luciferase, which is the YAP/TAZ luciferase reporter tumors were weighed. and obtained from Addgene (Plasmid #34615), and the b-actin/ Renilla luciferase reporter as indicated groups (33). After trans- Statistical analysis fecting for 24 hours, the dual-luciferase assay system (Promega) The Pearson correlation coefficient, two-tailed c2 test or Fisher was used to test the luciferase activity in each group cells. exact test were used to analyze the data of TMA. In vitro and in vivo experiments were performed twice or three times, and one rep- Measurement of ROS levels resentative result of two or three were presented. The Student t test Intracellular ROS levels were tested with flow cytometry by (two-tailed) or one-way ANOVA was used to test the significance DCFH-DA. Briefly, PDAC cells in indicated groups were seeded at of the in vitro and in vivo data. P < 0.05 was defined as statistically the density of 5 105 into 6-well plates for 12 hours. At 37C, significant. 10 mmol/L DCFH-DA was used to stain the cells in each group for 30 minutes in the dark. Then, we collected the cells and analyzed the dihydrodichlorofluorescein (DCF) fluorescence with FACS- Results fl Calibur ow cytometer (BD Biosciences). Reduced expression of MST1 and its direct association with PDAC pathologic features PDAC growth in vivo To reveal the critical roles of MST1 underlying PDAC, the Pathogen-free female athymic nude mice were obtained from expression of MST1 in PDAC cell lines and tissues was analyzed the Shanghai Laboratory Animal Center, Chinese Academy of at first. We found that the protein levels of MST1 were markedly Sciences. The mice were housed according to the protocols of the decreased in most PDAC cell lines as compared with normal Shanghai Cancer Institute Commission. PDAC cells (1 106)in HEK293 cells (Fig. 1A). We then tested the mRNA levels of MST1 indicated groups were diluted in 0.1 mL HBSS and were injected in in 14 paired PDAC and adjacent normal pancreatic tissues. The

Figure 1. Expression of MST1 in and its association with clinicopathologic features of PDAC. A, Expression of MST1 protein in PDAC cell lines. B, mRNA was extracted from 14 paired PDAC and adjacent normal pancreas tissues for quantitative PCR analysis (, P < 0.05). C, The mRNA levels of 14 PDAC tissues were negatively associated with clinical TNM stages. TMA PDAC specimens were immunostained with a specific anti-MST1 antibody. D, Representative images of MST1 expression were shown (left represent, normal tissue; middle, cancer tissue with MST1-negative expression; right, cancer tissue with MST1 strong expression). E, The expression of MST1 was significantly lower in tumors (TT) than in adjacent normal tissues (TN) and normal tissues (NN), whereas there was no difference between TN and NN. F, Verification of the efficiency of MST1 overexpression vectors and siRNA in PDAC cell lines via Western blot. BxPC-3 and FG cells were transfected with pMST1 or control vectors. AsPC-1 and PANC-1 cells were transfected with siMST1 or control siRNAs. BxPC-3 cells were infected with retroviruses containing MST1 (pBABE-MST1) and empty retroviral expression vector (pBABE-puro).

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results showed that the mRNA levels of MST1 were lower in PDAC the mRNA and protein levels of MST1 via real-time PCR and tissues in 10 of 14 cases (Fig. 1B; , P < 0.05). Furthermore, the Western blot. The results showed that the expression of MST1 was mRNA levels were negatively associated with clinical TNM stages much lower in PDAC cancer tissues as compared with its adjacent in these 14 paired tissues (Fig. 1C). With another four paired normal pancreatic tissues. Furthermore, the mRNA levels of MST1 PDAC tissues and its adjacent normal pancreatic tissues, we tested in PDAC tissues and its adjacent normal pancreatic tissues were

Figure 2. MST1 suppressed PDAC cells proliferation, migration, and invasion in vitro and in vivo. A and B, Colony formation assay was performed with BxPC-3 and PANC-1 cells in 24-well plates and the numbers of colonies were counted 14 days after. C, Spheroid colony formation assay was performed with BxPC-3 cells, and overexpression of MST1 decreased the number and size of the ﬁrst and second generations of spheroids. BxPC-3 cells were transfected with pMST1 or control vectors, and PANC-1 cells were transfected with siMST1 or control siRNAs. D–G, The migration and invasion of BxPC-3 and PANC-1 cells were determined as described in Materials and Methods. Representative tumor cell migrated or invaded were photographed; data, mean SD of triplicates. BxPC-3 cells with overexpression of MST1 were injected subcutaneously (1 106 per mouse, n ¼ 5) into the right and left scapular region of the nude mice. Tumor-bearing mice were sacriﬁced on day 35. H, Representative photo of mouse and gross tumors in the mice. I, The weight of the tumors was measured.

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correlated with protein levels (Supplementary Fig. S1A and S1B). of PANC-1 cells. Furthermore, the role of MST1 in PDAC cells We then measured the protein levels of MST1 in PDAC TMA by spheroid formation was tested. Overexpression of MST1 signifi- IHC. The results showed that MST1 mainly positively stained in cantly reduced the number and size of the first and second the cytoplasm of normal and adjacent normal pancreas tissues generations of spheroids in BxPC-3 cells (Fig. 2C). These data and some PDAC tissues (Fig. 1D). And the levels of MST1 revealed the suppressor roles of MST1 in PDAC cell proliferation expression in the normal and adjacent normal pancreas tissues and tumorigenesis. were statistically significantly higher than those in the PDAC To further assess the impact of altered MST1 levels on migration tissues (Fig. 1E). The correlation between MST1 expression levels and invasion of PDAC cells, BxPC-3 and PANC-1 cells were and PDAC clinicopathologic parameters was analyzed. The results transfected with pMST1 and siMST1, respectively. Similarly, over- revealed that MST1 expression was negatively correlated with expression of MST1 inhibited migration and invasion of BxPC-3 disease T stages (P < 0.001; Supplementary Table S1) and TNM cells (Fig. 2D and F), whereas siMST1 promoted migration and stages (P < 0.001, Supplementary Table S1). Furthermore, decreas- invasion of PANC-1 cells (Fig. 2E and G). Then, a subcutaneous ing MST1 expression associated with decreased levels of cancer xenotransplanted tumor model was used to determine the roles of differentiation (P < 0.001; Supplementary Table S1). MST1 in PDAC growth. The results showed that overexpression of These data demonstrated the tumor suppressor roles of MST1 in MST1 significantly inhibited the growth of subcutaneous tumors PDAC and lost expression of MST1 might promote the develop- (Fig. 2H and I). ment and progression of PDAC. Collectively, these results clearly demonstrated that MST1 suppressed the proliferation, tumorigenesis, migration, and inva- MST1 suppressed PDAC cell growth, migration, and invasion sion of PDAC cells, and supporting the notion that MST1 func- To investigate the impact of MST1 on PDAC biology, we tioned as a tumor suppressor of PDAC. transfected or infected BxPC-3 and FG cells that express low levels of endogenous MST1 with expression vectors (BxPC-3/FG-pMST1 MST1 promotes cell death via caspase-1–induced pyroptosis in and BxPC-3-pBABE-MST1) and the empty expression vector was PDAC used as control (BxPC-3/FG-Control and BxPC-3-pBABEpuro). MST1 was first reported as a proapoptotic cytoplasmic kinase; AsPC-1 and PANC-1, which express relatively high levels of MST1, we then investigated the effect of MST1 in PDAC cell death. were transfected with MST1-specific siRNA. MST1 protein levels in TUNEL staining was used to measure DNA damage; thus, we these cells were confirmed with Western blot (Fig. 1F). performed TUNEL staining experiments in PDAC cells. Figure 3A To investigate the roles of altered expression of MST1 in PDAC showed fluorescent signals of DAPI and TUNEL staining of BxPC- cell proliferation, we used BxPC-3 and PANC-1 cells to perform 3 cells. In the control group, there were a small number of the colony formation assay. Figure 2A and B shows that over- dead cells. But the cell death rate was significantly increased in expression of MST1 markedly suppressed the colony formation of MST1 overexpression groups. However, the increase was signif- BxPC-3 cells, but knockdown of MST1 increased colony number icantly reduced with cotransfected with pMST1 and siMST1. To

Figure 3. MST1 promoted cell death in PDAC. BxPC-3 cells were transfected with control vector, pMST1, and pMST1 and siMST1 for 48 hours. A, TUNEL (red) positive cells were increased in the pMST1 group and attenuated in the pMST1 and siMST1 group. The nuclei were stained blue with DAPI. Magniﬁcation: 200. B, The cell viability was determined as described in Materials and Methods. BxPC-3 and FG cells were transfected with control or pMST1 vectors for 48 hours. C, LDH release was determined as described in Materials and Methods. D, The protein levels of cleaved caspase-1, caspase-3, and caspase-7 were upregulated in pMST1 groups, as indicated by Western blot results (b-actin was used as an internal control).

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further confirm these results, CCK-8 assay and LDH release assay the levels of cleaved caspase-1, cleaved caspase-3, and cleaved were performed. Consistently, overexpression of MST1 induced caspase-7. Caspase-3 and caspase-7 are mostly involved in apo- BxPC-3 cell death and siMST1 could attenuate the effect (Fig. 3B). ptosis (34, 35). Caspase-1 is the inflammatory caspase essential to Also, overexpression of MST1 led to increased LDH release in canonical inflammasome-mediated pyroptosis (36). The data BxPC-3 and FG cells (Fig. 3C). These data showed that MST1 indicated that MST1 might regulate pyroptosis. MST1 was a induced PDAC cell death. proapoptotic cytoplasmic kinase, and the role of MST1 in apo- We then tested caspases involved in the MST1-induced PDAC ptosis has been widely studied. However, the role of MST1 in cell death. As shown in Fig. 3D, overexpression of MST1 increased pyroptosis is not reported. Caspase-1 is activated during

Figure 4. MST1 suppressed PDAC cell proliferation, migration, and invasion via caspase-1–mediated pyroptosis. A, BxPC-3 and FG cells were transfected with control or pMST1 vectors for 48 hours. Different forms of caspase-1, IL1b and IL18 expression levels in the two groups were determined with Western blot (b-actin was used as an internal control). BxPC-3 cells were treated as indicated groups (transfected with control vectors, transfected with pMST1 vectors, transfected with pMST1 vectors, and treated with caspase-1 inhibitor VX-765). B–D, Colony formation assay, migration, and invasion of BxPC-3 cells were determined as described in Materials and Methods. Representative tumor cell colony, migrated or invaded, was photographed; data, mean SD of triplicates.

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pyroptosis and cleaves the IL1b and IL18, which are the proin- Hippo pathway (37). YAP and transcriptional coactivator with flammatory cytokines (17). Also, expression levels of mature.IL1b PDZ-binding motif (TAZ) functioned as transcriptional coactiva- and mature.IL18 are markers of the activation of caspase-1. We tors and promoted PDAC progression (38). We then investigated then further tested the expression of IL1b and IL-18 in the over- the roles of the Hippo pathway in mediating MST1-induced expression groups of MST1 in PDAC cells. As shown in Fig. 4A, pyroptosis in PDAC. In PDAC cells, overexpression of MST1 led overexpression of MST1 in BxPC-3 and FG cells had little effect on to phosphorylation of YAP and decreased protein levels of YAP the protein levels of pro-caspase-1, pro-IL1b, and pro-IL18, but (Fig. 5A). Furthermore, overexpression of MST1 suppressed the led to the increase of mature.IL1b and mature.IL18. These findings transcriptional activity of the Hippo pathway (Fig. 5B). These data revealed that loss of expression of MST1 in PDAC induced revealed that the Hippo pathway was at least one of the mechan- pyroptosis dependent on caspase-1. We tested expression levels isms involved in the MST1's suppressor roles in PDAC. We then of caspase-1 in PDAC cell lines. The results showed that the further tested the function of the Hippo pathway in mediating endogenous expression of caspase-1 was associated with MST1 MST1-induced pyroptosis in PDAC. As shown in Fig. 5C, cotrans- in most PDAC cell lines (Supplementary Fig. S2A–S2C). We fection of YAP overexpression vector with MST1 did not attenuate then analyzed the protein level of caspase-1 in the serial PDAC the cleaved protein levels of caspase-1, mature.IL1b, and mature. TMA using IHC. We found that caspase-1 was also positively IL18, which were induced by MST1. These results indicated that stained in the cytoplasm (Supplementary Fig. S3A). And the MST1-induced pyroptosis of PDAC was independent of the expression of caspase-1 was much higher in the normal and Hippo pathway. adjacent normal pancreas tissues than that in the PDAC tissues (Supplementary Fig. S3B). Furthermore, the expression of caspase-1 was negatively associated with T stage (P < 0.001; MST1 suppresses proliferation, migration, and invasion via Supplementary Table S2) and TNM stages (P < 0.001; Supple- ROS-induced pyroptosis in PDAC mentary Table S2). Furthermore, decreasing caspase-1 expres- It has been reported that MST1 interacts with ROS and plays sion was associated with decreased levels of cancer differenti- critical roles in apoptosis, autophagy, and tumor suppres- – ation (P ¼ 0.013; Supplementary Table S2). We then further sion (39 42). Recently, studies have also demonstrated that analyzed the correlation of MST1 and caspase-1 with the same ROS induces pyroptosis and suppresses cancer progres- fi cohort. As shown in Supplementary Fig. S3C and S3D, positive sion (43, 44). Thus, we rst assess the levels of ROS on correlation between MST1 and caspase-1 expression was found pyroptosis in PDAC cells. BxPC-3 and FG cells were treated b in PDAC tissues (r ¼ 0.639, P < 0.001). with H2O2 and the levels of cleaved caspase-1, mature.LI1 , To further investigate whether MST1-induced PDAC prolifer- and mature.IL-18 via Western blot. The results revealed that b ation, migration, and invasion were pyroptosis dependent, we H2O2 increased the levels of cleaved caspase-1, mature.LI-1 , performed a caspase-1 inhibitory experiment. The results revealed and mature. IL-18 in a dose-dependent manner (Supplemen- that VX-765 (caspase-1 selective inhibitor) attenuated the sup- tary Fig. S4). We then tested the endogenous levels of ROS in pressive effect of MST1 in PDAC cell proliferation, migration, and PDAC cell lines. As shown in Supplementary Fig. S2D, endog- invasion (Fig. 4B–D). These findings demonstrated that loss of enous levels of ROS were related with MST1 and caspase-1 in expression of MST1 promoted PDAC development and progres- most PDAC cell lines. We further investigated the effect of sion via caspase-1–medicated pyroptosis. overexpression of MST1 on ROS levels in PDAC cell lines. As shown in Fig. 6A, overexpression of MST1 increased the levels of ROS. With TUNEL staining, we found that overexpression of MST1 induces PDAC cell pyroptosis independent of the Hippo MST1 increased PDAC cell death, but ROS scavenger NAC pathway attenuated the effect (Fig. 6B). Furthermore, NAC also atten- MST1 is one of core cytoplasmic kinases of the Hippo pathway uated the suppressive effect of MST1 in PDAC cell proliferation, and inhibits the oncogenic transcriptional module containing the migration, and invasion (Fig. 6C–E). These data suggested that

Figure 5. MST1-induced PDAC cell pyroptosis was independent of the Hippo pathway. A, BxPC-3 and FG cells were transfected with control or pMST1 vectors for 48 hours. The protein levels of MST1, YAP, and pYAP were determined by Western blot (b-actin was used as an internal control). B, The 8 GTIIC-Luciferase reporter which is the YAP/TAZ-responsive synthetic promoter driving luciferase reporter was transfected into BxPC-3 and FG cells in triplicate with MST1 restored expression or control vectors for 24 hours. The promoter activity was examined by a dual-luciferase assay kit. C, BxPC-3 and FG cells were transfected with control vector, pMST1, and pMST1 and pYAP for 48 hours. Western blot was used to determine the protein levels of MST1, YAP, cleaved caspase-1, cleaved caspase-3, cleaved caspase-7, mature IL1b,and mature IL18 (b-actin was used as an internal control).

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the suppressor roles of MST1 were at least partly through sis. First, we revealed that MST1 expression was decreased regulation of ROS. We then further tested whether ROS medi- in PDAC cell lines and tissues, and negatively associated ated MST1 induced pyroptosis. As shown in Fig. 6F, NAC with disease T stages, TNM stages, and poorer tumor differenti- decreased the levels of cleaved caspase-1, mature.LI1b,and ation. Second, overexpression of MST1 suppressed the growth, mature.IL18 induced by overexpression of MST1 in PDAC cells. cell spheroid formation, migration, and invasion of PDAC cells All these data demonstrated that the suppressor roles of MST1 in vitro and in vivo. These results conﬁrmed the suppressor roles of in PDAC proliferation, invasion, and migration were at least MST1 in PDAC. Third, overexpression of MST1 induced cell death partly mediated by ROS-induced pyroptosis. via the activation of caspase-1, caspase-3, and caspase-7, and the expression of MST1 was positively associated with caspase-1 in PDAC cell lines and tissues. These data indicated that MST1 might Discussion induce pyroptosis as caspase-1 was essential to canonical inﬂam- In the present study, we provided six lines of evidence masome-mediated pyroptosis. Fourth, overexpression of MST1 to demonstrate the critical roles of the MST1 in suppression of increased the levels of mature.IL1b and mature.IL18, which were proliferation and migration in PDAC via ROS-induced pyropto- markers of caspase-1 activation. Furthermore, caspase-1–selective

Figure 6. MST1 suppressed PDAC cell proliferation, migration, and invasion via ROS-induced pyroptosis. A, BxPC-3 and FG cells were transfected with control or pMST1 vectors for 48 hours. The ROS levels of indicated groups were determined as described in Materials and Methods. BxPC-3 cells were treated as indicated groups (transfected with control vectors, transfected with pMST1 vectors, transfected with pMST1 vectors and treated with ROS scavenger NAC). B, TUNEL (red) positive cells were increased in the pMST1 group and attenuated in the pMST1 plus NAC group. The nuclei were stained blue with DAPI. Magniﬁcation: 200. C–E, Colony formation assay, migration, and invasion of BxPC-3 cells were determined as described in Materials and Methods. Representative tumor cell colony, migrated or invaded, was photographed; data, mean SD of triplicates. F, Different forms of caspase-1, IL1b, and IL18 expression levels in the indicated groups were determined with Western blot (b-actin was used as an internal control).

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inhibitor VX-765 significantly attenuated the suppressive effect of been reported that ROS interacted with MST1 and also ROS MST1 in PDAC cell proliferation, migration, and invasion. These played critical roles in pyroptosis (44). Thus, we first tested the data demonstrated that MST1 inhibited PDAC development and effects of MST1 in the generation of ROS in PDAC. The results progression via suppressing caspase-1–medicated pyroptosis. showed that overexpression of MST1 increased the levels of MST1 Fifth, pyroptosis induced by MST1 was independent of the Hippo in PDAC cells. And ROS scavenger NAC attenuated the effect of pathway, as overexpression of YAP did not attenuate the activa- MST1 on PDAC cell death and the activation of caspase-1. tion of caspase-1 by MST1. Sixth, overexpression of MST1 Furthermore, NAC also attenuated the inhibitory effect of MST1 increased the levels of ROS. Furthermore, ROS scavenger NAC on PDAC cell proliferation, migration, and invasion. Our results attenuated the activation of caspase-1 induced by MST1 and the demonstrated an essential role of ROS in PDAC cell pyroptosis. effect of MST1 in PDAC cell death, proliferation, invasion, and ROS is a sign of oxidative stress that is mostly involved in the migration. Collectively, these findings demonstrated that MST1 metabolism of cells. Uncontrolled production of ROS causes cell suppressed the proliferation, migration, and invasion of PDAC death and is related to the pathogenesis in a series of diseases, cells at least partly through ROS-induced pyroptosis. including many types of cancers (48, 49). Thus, our results Previous studies focused on MST1 mostly demonstrated its indicated that metabolism might also participate in pyroptosis role in apoptosis and as a core member of the Hippo pathway. in PDAC, which needs further study. Collectively, all these results In PDAC, MST1 was reported as a tumor suppressor in dis- demonstrated that the inhibitory effects of MST1 on PDAC rupting the Hippo–YAP pathway's oncogenic effects, and MST1 proliferation, invasion, and migration were at least partly through mediated gemcitabine induced PDAC cell death (45, 46). These ROS-induced pyroptosis. studies demonstrated that MST1 was a tumor suppressor and In summary, our study provided both clinical and mecha- participated in cell death in PDAC. However, the role and nistic evidence to determine the roles and demonstrate mechanism of MST1 in pyroptosis have never been reported. the mechanism of MST1 in pyroptosis. We identified a new Pyroptosis is a kind of programmed cell death and was mechanism of MST1 in inhibiting PDAC development and first reported in macrophages that were infected with Shigella progression and demonstrated that MST1 would be a valuable flexneri (47). Recent studies have also demonstrated the impor- biomarker and promising molecular target for PDAC, which is tance of pyroptosis in a series of cancers. However, the role of worth further study. pyroptosis in PDAC has never been reported. In our study, we provided evidence that the expression of MST1 was decreased Disclosure of Potential Conflicts of Interest in PDAC and functioned as a tumor suppressor, and over- No potential conflicts of interest were disclosed. expression of MST1 inhibited PDAC cell proliferation, migration, invasion, and cell death. Further studies showed that Authors' Contributions overexpression of MST1 increased the levels of cleaved Conception and design: J. Cui, Y. Gao, L. Wang, M. Quan caspace-1, caspase-3, and caspase-7. Activation of caspase-3 Development of methodology: J. Cui, Z. Zhou, H. Yang, F. Jiao, J. Chen, and caspased-7 was mostly involved in apoptosis, which has M. Quan been widely studied. We then focused on the roles of MST1 on Acquisition of data (provided animals, acquired and managed patients, activation of caspase-1 by testing the levels of mature.IL1b and provided facilities, etc.): J. Cui, N. Li, J. Chen, M. Quan mature.IL18, which were the markers of caspase-1 activation. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The result showed that overexpression of MST1 increased the computational analysis): J. Cui, Z. Zhou, H. Yang, F. Jiao, L. Wang, M. Quan b b Writing, review, and/or revision of the manuscript: J. Cui, F. Jiao, N. Li, J. Chen, mature levels of IL1 and IL18 in PDAC cells. IL1 and IL18 are M. Quan inflammatory cytokines that could be released into the cancer Administrative, technical, or material support (i.e., reporting or organizing microenvironment and mediated the interaction between can- data, constructing databases): J. Cui, Z. Zhou, M. Quan cer cells and inflammatory cells. The regulatory effect of MST1 Study supervision: J. Cui, Y. Gao, L. Wang, J. Chen, M. Quan on the cleavage of IL1b and IL18 indicated that MST1 might also play roles in the PDAC inflammatory microenvironment. Acknowledgments We might conduct further studies in the future. Collectively, This work was supported by grants 81502018 (to J. Cui), 81502043 – (to M. Quan), 81502017 (to F. Jiao), and 81572315 (to L. Wang) from the these data revealed the roles of MST1 in caspase-1 mediated National Natural Science Foundation of China. M. Quan was supported by pyroptosis. We then further studied the mechanism of MST1 in The Natural Science Foundation of China and by Fundamental Research regulating pyroptosis. Funds for the Central Universities 22120180029. This study was funded by MST1 is one of the core components of the Hippo pathway, the Outstanding Clinical Discipline Project of Shanghai Pudong (Grant No. which plays essential roles in PDAC formation and development. PWYgy2018-02). So we first investigated whether the Hippo pathway mediated the MST1-induced pyroptosis in PDAC. The results showed that The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked reexpression of MST1 indeed activated the Hippo pathway as advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate YAP was phosphorylated and its transcriptional activity was this fact. decreased. But cotransfected YAP with MST1 could not attenuate the activation of caspase-1. These results suggested that pyroptosis Received August 26, 2018; revised December 16, 2018; accepted February 18, induced by MST1 was not mediated by the Hippo pathway. It has 2019; published first February 22, 2019.

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www.aacrjournals.org Mol Cancer Res; 17(6) June 2019 1325

MST1 Suppresses Pancreatic Cancer Progression via ROS-Induced Pyroptosis

Jiujie Cui, Zhuqing Zhou, Haiyan Yang, et al.

Mol Cancer Res 2019;17:1316-1325. Published OnlineFirst February 22, 2019.

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