{'fraction_non_alphanumeric': 0.037392867016864804, 'fraction_numerical': 0.021150124412496543, 'mean_word_length': 5.329396325459317, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Mammalian STE20‑like kinase 1 regulates pancreatic cancer cell survival and migration through Mfn2‑mediated mitophagy.

Mammalian STE20-like kinase 1 (MST1) plays an important role in pancreatic cancer progression, but its downstream targets are still unknown. In the present study, our results indicated that MST1 expression was significantly downregulated in pancreatic cancer cell lines (PANC-1, BxPC-3 and HPAC) compared with that in the normal ductal epithelial cell line (hTERT-HPNE). Moreover, MST1 overexpression in PANC-1 cells led to increased apoptosis as determined by MTT and TUNEL assays and inhibited cellular migration. Mechanistically, upregulation of MST1 expression caused mitochondrial dysfunction, decreased ATP production, and activation of the mitochondrial-dependent apoptotic pathway via inhibition of mitofusin 2 (Mfn2)-mediated mitophagy, which ultimately resulted in increased cellular apoptosis and decreased cellular migration. Collectively, the present study demonstrated that MST1 may regulate pancreatic cancer PANC-1 cell survival, invasion and migration through Mfn2-mediated mitophagy, laying the foundation for the exploration of novel therapeutic targets for pancreatic cancer.


Introduction
Pancreatic cancer is a highly malignant gastrointestinal tumor and is the fourth leading cause of cancer-related death in humans (1,2). although the development of therapeutic techniques in the past 30 years has provided more strategies for the cure of pancreatic cancer, the overall 5-year survival rate of patients with pancreatic cancer is still <5% (3,4). The only effective treatment for pancreatic cancer is segmental resection (5,6). However, due to the high viability and invasiveness of pancreatic cancer cells, the probability of recurrence and metastasis after surgery remains quite high (7). Therefore, exploring the molecular mechanisms that regulate pancreatic cancer cell survival, invasion and migration is essential for seeking effective intervention targets for pancreatic cancer treatment and improving the prognosis of patients.
Mammalian sterile 20-like kinase 1 (MST1) is one of the core members of the Hippo pathway in the FaS signaling pathway (8,9). Highly conserved in drosophila, yeast, mouse and human, MST1 regulates embryo growth and development, and inhibits tumor growth (10,11). MST1 also plays a crucial role in many physiological processes such as cell migration, differentiation and angiogenesis (12)(13)(14). recent studies have confirmed that MST1 exerts important effects on the development of pancreatic cancer (15)(16)(17). although MST1 has become a research 'hotspot' for tumor-targeted therapy, its downstream targets remain unclear.
autophagy is a process that maintains the homeostasis of the microenvironment inside cells via non-selective degradation and phagocytosis of abnormal organelles, proteins and lipids in the cytoplasm (18,19). Mitofusin 2 (Mfn2)-mediated mitophagy is a process by which cells selectively remove damaged or dysfunctional mitochondria via autophagy to maintain the balance between mitochondrial quantity and quality (20)(21)(22). numerous studies have confirmed that Mfn2-mediated mitophagy plays a crucial role in tumor origin, homeostasis, invasiveness and drug resistance (23,24). Nonetheless, the specific role of Mfn2-mediated mitophagy in pancreatic cancer progression has not been reported. Mfn2-mediated mitophagy is generally considered to have a protective effect on tumor cell survival. Moreover, several studies have identified the close relationship between MST1 and mitophagy (11,25). in myocardial ischemia-reperfusion injury, gene knockout of MST1 was found to reduce cardiomyocyte apoptosis via the inhibition of mitophagy (26). Hence, we hypothesized in this study that MST1 may regulate pancreatic cancer cell survival, invasion and migration via Mfn2-mediated mitophagy.

Materials and methods
Cell culture and treatments. The human pancreatic cancer cell lines (Panc-1, BxPc-3 and HPac) and normal pancreatic ductal epithelial cell line (hTerT-HPne) were purchased from the american Type culture collection (aTcc).
MTT assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and determination of caspase-3 activity. The cells were seeded in 96-well plates at a density of 8x10 3 cells/well and incubated overnight. after treatment, MTT (5 mg/ml) was added to each well and incubated for 4 h, and then supernatants were removed. The cells were solubilized in 200 µl dimethyl sulfoxide (dMSo) and the absorbance was recorded with a microplate reader at the wavelength of 490 nm. The Tunel assay was used to detect apoptosis. a one-step Tunel kit (Beyotime institute of Biotechnology) was used for Tunel staining, according to the manufacturer's protocol. The cells were incubated with fluorescein-dUTP (Invitrogen; Thermo Fisher Scientific, Inc.) to stain apoptotic cell nuclei and with daPi (5 mg/ml) to stain all cell nuclei at room temperature for 3 min. The number of Tunel-positive cells was calculated by counting at least 5 random separate fields as the ratio of the experimental samples to the control samples. a caspase-3 activity kit (Beyotime institute of Biotechnology) was used to measure caspase-3 activity according to the manufacturer's instructions.
Cell migration and wound healing assay. after treatment, the cells were seeded in 6-well plates at a density of 0.5x10 6 cells/well. The cells (1x10 5 ) from each group were seeded into the upper chamber of a Transwell chamber containing 200 µl serum-free dMeM medium. in addition, 500 µl dMeM medium supplemented with 10% FBS was added into the lower chamber. after 24 h, the chamber was removed and the cells in the upper chamber were wiped off. After being fixed with 4% formaldehyde for 10 min at room temperature, the membrane was stained using the Giemsa method for 15 min at room temperature and 5 random fields were observed using a light microscope (magnification, x200; olympus dX51; olympus corporation). cell migration was also analyzed using the wound healing assay (24 wells with 8-µm pores and polycarbonate membranes) as previously described (30).
ATP production, mitochondrial potential and mPTP opening.
The cellular aTP levels were measured using a firefly luciferase-based ATP assay kit (Beyotime) based on a fluorescence technique (Genmed Scientifics Inc.) as previously described (31). The Jc-1 kit (Beyotime) was applied to assess changes in the mitochondrial membrane potential (ΔΨm). The mPTP opening was observed as a rapid dissipation of tetramethylrhodamine ethyl ester (TMRE) fluorescence according to a previous study (28).
Statistical analysis. experiments were repeated three times and data are expressed as the mean ± standard error of the mean (SeM). Statistical analyses were performed using one-way analysis of variance with the Bonferroni test for post hoc comparisons. additionally, statistical analyses were also performed using GraphPad Prism 5.0 (GraphPad Software, inc.). P<0.05 was considered to indicate statistical significance.

MST1 is downregulated in pancreatic cancer cell lines and promotes apoptosis in PANC-1 cells.
First, MST1 expression was determined by western blotting in the pancreatic cancer cell lines (Panc-1, BxPc-3 and HPac) and normal pancreatic ductal epithelial cell line (hTerT-HPne). as shown in Fig. 1A and B, MST1 expression was significantly reduced in pancreatic cancer cell lines (Panc-1, BxPc-3 and HPac) compared with that noted in the normal pancreatic ductal epithelial cell line (hTerT-HPne). To investigate the role of MST1 in pancreatic cancer progression, we stably enhanced MST1 expression in the Panc-1 cell line via adenovirus vector transfection (ad-MST1). The transfection efficiency was measured by western blot analysis (Fig. 1c and d). The effect of MST1 on Panc-1 cell viability was detected. MTT assay showed that upregulation of MST1 in PANC-1 cell significantly decreased cell viability (Fig. 1e). consistent with this result, overexpression of MST1 promoted Panc-1 cell apoptosis as indicated by significantly increased cleaved caspase-3  expression ( Fig. 1F and G), significantly upregulated caspase-3 activity (Fig. 1H) and significantly increased percentage of Tunel-positive cells ( Fig. 1i and J). These results demonstrated that reintroduction of MST1 promoted apoptosis in the pancreatic cancer cells.

MST1 overexpression promotes PANC-1 cell death by inducing mitochondrial injury.
To explore the mechanism by which MST1 regulates apoptosis in Panc-1 cells, we examined mitochondrial damage in vitro. as shown in Fig. 2a, MST1 overexpression significantly reduced ATP generation compared with that noted in the control group. Furthermore, mitochondrial electrochemical gradient (ΔΨm) revealed that MST1 impaired ΔΨm (Fig. 2B and C). MST1 overexpression also significantly increased the mPTP opening rate compared with that of the control group (Fig. 2d). additionally, proteins associated with mitochondrial damage were evaluated by western blotting. overexpression of MST1 significantly increased the expression of caspase-9 and Bax and reduced the expression of Bcl2 in the Panc-1 cells, indicating activation of mitochondrial-related apoptosis pathways (Fig. 2e-H). These data identified that MST1 promotes apoptosis in PANC-1 cells by inducing mitochondrial damage.

MST1 overexpression impairs PANC-1 cell migration in vitro.
We further investigated the effects of MST1 overexpression on Panc-1 cell migration. as shown in Fig. 3a and B, Transwell assays showed that Panc-1 cell migration was greatly reduced when MST1 was overexpressed in Panc-1 cells, which was consistent with the wound-healing assay results (Fig. 3c  and d). in pancreatic cancer, epithelial-to-mesenchymal transition (eMT) contributes to metastasis (32)(33)(34). Thus, western blotting was performed to examine the expression of e-cadherin, n-cadherin and Snail. overexpression of MST1 significantly increased E-cadherin expression but decreased n-cadherin and Snail expression in the Panc-1 cells (Fig. 3e-H). These results suggest that MST1 overexpression impairs Panc-1 cell migration.

MST1 inhibits PANC-1 cell survival and migration via
Mfn2-mediated mitophagy. There is increasing evidence that Mfn2-mediated mitophagy plays a central and multifunctional role in malignant tumor progression. To further explore the underlying mechanism by which MST1 regulates mitochondrial-associated apoptosis and migration inhibition, western  blotting was used to test proteins related with mitophagy. as shown in Fig. 4A-F, MST1 overexpression significantly reduced the expression of Mfn2 and markers related to mitophagy including mito-lc3ii, atg5, Beclin1 and p62, suggesting the inactivation of Mfn2-mediated mitophagy. To explore the role of mitophagy in MST1 expression, FccP, an activator of mitophagy, was used to treat the MST1-overexpressing cells to activate mitophagy (35). FCCP significantly increased the expression of Mfn2, mito-lc3ii, atg5, Beclin1 and p62 in the MST1-overexpressing cells, suggesting the activation of Mfn2-mediated mitophagy. Furthermore, FccP promoted cell survival as indicated by MTT assay (Fig. 4G) and caspase-3 activity (Fig. 4H) and increased the migratory ability ( Fig. 4i-J) when MST1 was upregulated in Panc-1 cells. collectively, these results suggested that MST1 inhibits Panc-1 cell survival and migration by regulating Mfn2-mediated mitophagy.

Discussion
according to a previous study, mammalian STe20-like kinase 1 (MST1) is expressed at low levels in human pancreatic cancer tissues (36). MST1 overexpression in tumor cells restrains tumor progression by inhibiting cell differentiation and survival. However, the specific regulatory mechanism is still unclear. in the present study, we demonstrated that MST1 inhibits Panc-1 cell survival and migration and restrains pancreatic cancer progression via inhibiting mitofusin 2 (Mfn2)-mediated mitophagy (Fig. 5). These data identify the MST1/Mfn2/mitophagy pathway as a potential target for the treatment of pancreatic cancer and highlight a new strategy for treating pancreatic cancer involving MST1 protein and Mfn2-mediated mitophagy. However, more clinical data are needed to support our theory. as a cytoprotective mechanism, mitophagy plays an important role in tumor development and progression (37). Previous studies have reported that inhibition of mitophagy significantly suppressed tumor progression before it caused damage to normal tissues in a mouse model of lung cancer (38)(39)(40). This suggests that mitophagy is a very potential anticancer therapeutic target. Our findings are consistent with those of previous studies. In the present study, we found that inhibition of mitophagy significantly reduced pancreatic cancer Panc-1 cell viability and migration. in future studies, the role of mitophagy in pancreatic cancer progression needs to be further identified by performing animal experiments.
Mitochondria are the main organelles that mediate energy production, oxidative stress and apoptosis, and their morphology, quantity, synthesis and degradation are precisely regulated (37). The regulatory mechanism of mitophagy is very complicated. The currently reported receptors involved in the regulation of mitophagy include PinK (PTen-inducible kinase) 1/Parkin pathway, BniP (Bcl2 interacting protein)3/niX (nip3-like protein X), Fundc (Fun14 domain containing) 1, Mfn2 and drp1 (41,42). Mitophagy has been shown to be involved in the progression of endometriosis (43), colon cancer (44), and gastric cancer (23). However, the upstream regulatory signals of mitophagy in pancreatic cancer remain unknown. our research has filled this theoretical gap. In the present study, MST1 overexpression in Panc-1 cells inhibited Mfn2-mediated mitophagy, thereby activating mitochondrial-dependent apoptotic pathways and inhibiting cell migration.
in conclusion, our study elucidated the mechanism of MST1 in pancreatic cancer progression; that is, MST1 regulates pancreatic cancer cell survival and migration via inhibition of mitophagy. However, our research also has some limitations. How MST1 regulates Mfn2-mediated mitophagy in pancreatic cancer progression is still unclear. This issue needs to be further explored in future experiments. Furthermore, to clarify the roles of MST1, we will perform overexpression and inhibition assay using two cell lines.