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. 2019 May 24;24(4):807–816. doi: 10.1007/s12192-019-01008-9

Hippo/Mst1 overexpression induces mitochondrial death in head and neck squamous cell carcinoma via activating β-catenin/Drp1 pathway

Chao Ma 1,✉, Longkun Fan 1, Jingxian Wang 1, Lixia Hao 1, Jinqiu He 1
PMCID: PMC6629754  PMID: 31127452

Abstract

Mammalian Ste20-like kinase 1 (Mst1) is associated with cell apoptosis. In the current study, we explored the regulatory effects of Mst1 on squamous cell carcinoma of the head and neck (SCCHN) in vitro. SCCHN Cal27 cells and Tu686 cells were transfected with adenovirus-loaded Mst1 to detect the role of Mst1 in cell viability. Then, siRNA against Drp1 was transfected into cells to evaluate the influence of mitochondrial fission in cancer survival. Our data illustrated that Mst1 overexpression promoted SCCHN Cal27 cell and Tu686 cell death via activating mitochondria-related apoptosis. Cells transfected with adenovirus-loaded Mst1 have increased expression of DRP1 and higher DRP1 promoted mitochondrial fission. Active mitochondrial fission mediated mitochondrial damage, as evidenced by increased mitochondrial oxidative stress, decreased mitochondrial energy production, and reduced mitochondrial respiratory complex function. Moreover, Mst1 overexpression triggered mitochondria-dependent cell apoptosis via DRP1-related mitochondrial fission. Further, we found that Mst1 overexpression controlled mitochondrial fission via the β-catenin/DRP1 pathways; inhibition of β-catenin and/or knockdown of DRP1 abolished the pro-apoptotic effects of Mst1 overexpression on SCCHN Cal27 cells and Tu686 cells, leading to the survival of cancer cells in vitro. In sum, our results illustrate that Mst1/β-catenin/DRP1 axis affects SCCHN Cal27 cell and Tu686 cell viability via controlling mitochondrial dynamics balance. This finding identifies Mst1 activation might be an effective therapeutic target for the treatment of SCCHN.

Keywords: SCCHN, Mst1, Wnt/β-catenin pathway, DRP1, Mitochondria

Introduction

Squamous cell carcinoma of the head and neck (SCCHN), the most common cancer in the head and neck regions, accounts for ~ 500,000 cases of cancer each year throughout the world, which is a threat and burden to the public health(Desai et al. 2019). Unfortunately, SCCHN is always diagnosed at late stage and there is no effective drug to control the advanced SCCHN (Sun et al. 2019). Besides, the efficacy of present treatment regimens for SCCHN is usually attenuated due to the occurrence of multidrug resistance. Despite progress in identifying risk factors, the pathogeneses underlying SCCHN development and progression have not been fully explained (Bocci et al. 2019).

Mitochondrial dysfunction has been implicated in many diseases, including cancer (Li et al. 2018). The dynamin-related protein 1 (DRP1) (Abukar et al. 2018), located in the outer mitochondrial membrane, functions as a mitochondrial dynamics mediator (Briere et al. 2018). Increased DRP1 is required for mitochondrial fission, and this process is necessary for cell growth and proliferation (Abeysuriya et al. 2018). Normal mitochondrial fission provides sufficient mitochondria to ensure the energy requirements during cancer progression, especially tumor colonies (Angelova et al. 2018). DRP1 is also crucial for various cellular processes ranging from Ca2+ homeostasis, redox modification, and autophagy to apoptosis execution (Chrifi et al. 2019). However, uncontrolled DRP1 activation has been shown to play a cancer-killing role in many types of tumors such as gastric cancer, lung cancer, and colorectal cancer (Boga et al. 2018; Botker et al. 2018; Broche et al. 2018). Thus, DRP1-related mitochondrial division serves at the crossroads of a variety of cell survival and cell death signals, which could be conserved as a potential target to manage the biological function of SCCHN (Armartmuntree et al. 2018; Darden et al. 2019). Although the detailed actions of DRP1-related mitochondrial division have been widely explored, the upstream mediators of DRP1 and mitochondrial division have not been found in SCCHN.

Mammalian Ste20-like kinase 1 (Mst1) is considered a tumor-suppressive protein. Lower Mst1 content in patients with osteosarcoma predicts poor prognosis (Cheng et al. 2018; Davidson et al. 2018). In gastric cancer, overexpression of Mst1 has been associated with GES-1 gastric cancer cell death in a manner dependent on the JNK-Mff signaling pathway (Ba and Boldogh 2018; Brazao et al. 2018). In colorectal cancer, activation of Mst1 using tanshinone IIA impairs cancer progression and invasion via activating the INF2-mediated mitochondrial stress. Similarly, cancer epithelial-mesenchymal transition of SCCHN can be negatively affected by Mst1 via miR-650 (Coverstone et al. 2018; Farber et al. 2019). Recent studies have reported on the influence of Mst1/Hippo pathway in mitochondrial fission in lung cancer, but not in SCCHN. At the molecular level, the Wnt/β-catenin pathway has been associated with mitochondrial fission activation in a model of cerebral ischemia reperfusion injury (Dong et al. 2019; Zhao et al. 2018). Recent studies have also found that the Wnt pathway is also involved in colon cancer tumorigenesis via modulating mitochondrial homeostasis (DeLeon-Pennell et al. 2018; Wen et al. 2019). This result was also noted in SCCHN. This information indicates that deregulation of the Wnt/β-catenin pathway is connected to mitochondrial division. Therefore, the aim of our study is to determine the influence of Mst1 on DRP1-related mitochondrial division in SCCHN, with a focus on the Wnt/β-catenin pathway.

Materials and methods

Cell culture and treatment

In the present study, two kinds of SCCHN cell lines (Cal27 cells and Tu686 cells), purchased from Shanghai Cancer Institute (China), were used to explore the influences of Mst1 on cancer cell phenotype (Erland et al. 2018a). These two cell lines were incubated under Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; GIBCO BRL) in a humidified incubator at 37 °C and 5% CO2. To inhibit the activation of Wnt/β-catenin pathway, DKK1 was used 2 h before treatment according to a previous study (Eriksson et al. 2018).

Cell viability evaluation

MTT assay was used to observe the cellular viability. Cells were seeded onto a 96-well plate, and the MTT was then added to the medium (2 mg/ml; Sigma-Aldrich). Subsequently, the cells were cultured in the dark for 4 h and DMSO was added to the medium. The OD of each well was observed at A490 nm via a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA) (Faughnan et al. 2019). In TUNEL assay, cells were fixed in 4% paraformaldehyde at room temperature for 30 min. After that, a TUNEL kit (Roche Apoptosis Detection Kit, Roche, Mannheim, Germany) was used on the slices according to the instructions (Deussen 2018). Finally, the sections were amplified to × 400; the apoptotic cells in at least 10 fields were randomly chosen. The apoptotic index was the proportion of apoptotic cells to total cells according to a previous study (Cameron et al. 2018).

Immunofluorescence analysis and confocal microscopy

Cells were plated on glass slides in a 6-well plate at a density of 1 × 106 cells per well. Subsequently, cells were fixed in ice-cold 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 2% gelatine in PBS at room temperature(Fine et al. 2018). The cells were then incubated with the primary antibodies: Tom20 (1:1000, Abcam, no. ab186735), cyt-c (1:1000; Abcam, no. ab90529), p-β-catenin (1:1000, Abcam, no. ab53050) overnight at 4 °C. After being washed with PBS, the cells were incubated with secondary antibody and DAPI (1:1000 dilution in PBS) for 1 h at room temperature. Images were obtained using a fluorescence microscope (Abeysuriya et al. 2018).

Mitochondrial reactive oxygen species and mitochondrial transmembrane potential detection

Cell suspensions were collected. The liquor (50 g, digested two times) was collected, centrifuged for 2 min with the supernatant removed, supplemented with the ROS probe DCFDA, incubated at room temperature for 10 min, centrifuged, and washed with PBS. The cells were resuspended by adding binding buffer (1×) in the dark; then, the cells were incubated at room temperature for 30 min and filtered with a nylon mesh (40-μm well). The ROS production was measured by fluorescence-activated cell sorting (FACS) (Erland et al. 2018b; Fukumoto et al. 2018). Mitochondrial membrane potential was measured with JC-1 assays (Thermo Fisher Scientific Inc., Waltham, MA, USA; catalogue no. M34152). Cells were treated with 5 mM JC-1 and then cultured in the dark for 30 min at 37 °C. Subsequently, cold PBS was used to remove the free JC-1 and DAPI was used to stain the nucleus in the dark for 3 min at 37 °C. The mitochondrial membrane potential was observed under a digital microscope (IX81, Olympus) (Chandra et al. 2018).

Western blot

Total protein was extracted by RIPA (R0010, Solarbio Science and Technology, Beijing, China), and the protein concentration of each sample was detected with a bicinchoninic acid (BCA) kit (20201ES76, Yeasen Biotech Co., Ltd., Shanghai, China) (Edwards et al. 2018; Iban-Arias et al. 2018). Deionized water was added to generate 30-μg protein samples for each lane. A 10% sodium dodecyl sulfate (SDS) separation gel and concentration gel were prepared. The following diluted primary antibodies were added to the membrane and incubated overnight: p-β-catenin (1:1000, Abcam, no. ab53050), β-catenin (1:1000, Abcam, no. ab32572), complex III subunit core (CIII-core2, 1:1000, Invitrogen, no. 459220), complex II (CII-30, 1:1000, Abcam, no. ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, no. ab110268), Drp1 (1:1000, Abcam, no. ab56788), Fis1 (1:1000, Abcam, no. ab71498), Opa1 (1:1000, Abcam, no. ab42364), Mfn2 (1:1000, Abcam, no. ab56889), caspase9 (1:1000, Cell Signaling Technology, no. 9504), Bcl2 (1:1000, Cell Signaling Technology, no. 3498), survivin (1:1000, Cell Signaling Technology, no. 2808), Bax (1:1000, Cell Signaling Technology, no. 2772), Mst1 (1:1000, Cell Signaling Technology, no. 3682). The membranes were washed three times with phosphate-buffered saline (PBS) (5 min each time), supplemented with horseradish peroxidase (HRP)–marked second antibody (1: 200, Bioss, Beijing, China), oscillated, and incubated at 37 °C for 1 h. After incubation (Gianni-Barrera et al. 2018), each membrane was washed three times with PBS (5 min for each time) and reacted with enhanced chemiluminescence (ECL) solution (ECL808-25, Biomiga, CA, USA) at room temperature for 1 min; then, the extra liquor was removed and the membranes were covered with preservative film. Each membrane was observed with an X-ray machine (36209ES01, Qian Chen Biological Technology Co. Ltd., Shanghai, China) to visualize the protein expression. GAPDH was used as the internal references. The relative protein expression was the ratio of the gray value of the target band to the inner reference band (Cheignon et al. 2018; Fan et al. 2018).

Enzyme-linked immunosorbent assay

Cellular glutathione (GSH), glutathione peroxidase (GPx), and SOD were measured via ELISA according to the manufacturer’s instructions. Cellular lactate production in the medium was measured via a lactate assay kit (no. K607-100; BioVision, Milpitas, CA, USA) according to a previous study (Hatori et al. 2018). The cancer glucose uptake rate was detected via a glucose absorption assay kit (no. K606-100; BioVision) (Jung et al. 2018).

Transfection

To augment the expression of Mst1, SCCHN cell lines were cultured in 60-mm dishes overnight. Then, pDC315-Mst1 vector, purchased from Shanghai Gene-Pharma Co. (Shanghai, China), was infected into HEK293 cells (3.0 μg per 1 × 104 cells/well) for 48 h at 37 °C. Subsequently, the cell medium supernatant was collected to obtain adenovirus-Mst1 (Ad-Mst1) (Fernandez Vazquez et al. 2018), which was used to transfect into two kinds of SCCHN cell lines for 48 h at 37 °C using Opti-MEM medium and Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol (Jo et al. 2018).

Statistical analysis

All experimental data were analyzed using SPSS Statistics software 19.0 (SPSS Inc., Chicago, IL, USA, 2006). Repeated measures analysis of variance (ANOVA) was used to compare the escape latency among groups. Other data were compared using one-way ANOVA. Data are presented as the mean ± SEM. A value of p < 0.05 was considered significant.

Results

Overexpression of Mst1 promotes SCCHN cell death in vitro

To understand the influence of Mst1 in SCCHN Cal27 cell and Tu686 cell death, we infected cells with adenovirus-loaded Mst1 (ad-Mst1) to overexpress Mst1 in SCCHN cell lines. Then, cell viability was determined via CCK8 assays (Chen et al. 2018; Giatsidis et al. 2018). In Fig. 1a and b, compared with the control group, Mst1 overexpression via ad-Mst1 transfection reduced cell viability. This result was also similar to the data in LDH release assay. In Fig. 1c and d, compared with the control group, Mst1 overexpression promoted the LDH release into the medium in SCCHN Cal27 cells and Tu686 cells. Thus, these results indicated that Mst1 overexpression triggered cell death in SCCHN Cal27 cells and Tu686 cells in vitro. Next, experiments were performed to analyze whether Mst1 induced cell death via apoptosis. In Fig. 1e–h, compared with the control group, the number of TUNEL-positive cells was significantly increased in response to ad-Mst1 transfection (Kazakov et al. 2018), suggesting that Mst1 overexpression activated apoptosis in SCCHN Cal27 cells and Tu686 cells. Besides, the activity of caspase-3 activity was also elevated in response to Mst1 overexpression, when compared with the control group (Fig. 1i, j). Thus, these data illustrate that Mst1 activation mediates cell apoptosis in SCCHN Cal27 cells and Tu686 cells in vitro.

Fig. 1.

Fig. 1

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Mst1 overexpression induces apoptosis in SCCHN Cal27 cells and Tu686 cells. a, b In vitro, CCK8 assay was used to observe the alterations of cell viability in SCCHN Cal27 cells and Tu686 cells. c, d ELISA was used to measure the content of LDH in the medium which was increased due to the cell membrane breakage. e–h Apoptotic cell was stained using TUNEL assay, and the cells with green nucleus were the apoptotic cells. The number of TUNEL-positive cell was recorded to reflect the influence of Mst1 on cell death. i–j Caspase-3 activity was determined via ELISA. *p < 0.05 vs. ad-ctrl group

DRP-1-related mitochondrial fission is augmented by Mst1 overexpression

To understand the molecular mechanism underlying Mst1-mediated apoptosis in SCCHN Cal27 cells and Tu686 cells (Kaarsholm et al. 2018), we observed the changes in mitochondrial fission which has been reported recently to be a key mediator of cell apoptosis in several kinds of cancers. Western blotting demonstrated that the proteins related to mitochondrial fission were significantly elevated in response to Mst1 overexpression, as evidenced by increased DRP1 and FIS1 (Fig. 2a–j). Interestingly, the levels of anti-fission factors such as MFN2 and OPA1 were rapidly downregulated by Mst1 overexpression in SCCHN Cal27 cells and Tu686 cells (Fig. 2a–j). These results indicate that mitochondrial fission seems to be activated by Mst1 overexpression. Subsequently, the average length of mitochondria was determined to quantify mitochondrial fission. In normal cells, mitochondrial length was ~ 9.2 μm whereas Mst1 overexpression reduced the average mitochondrial length to 3.6 μm in SCCHN Cal27 cells and Tu686 cells (Fig. 2k, l). Thus, these results illustrate that mitochondrial fission is induced by Mst1 overexpression in SCCHN Cal27 cells and Tu686 cells in vitro.

Fig. 2.

Fig. 2

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Mst1 overexpression elevated DRP1-related mitochondrial division. a–j Western blotting analysis was used to verify the alterations of mitochondrial division-related proteins. FIS1 and DPR1 were pro-division proteins whereas MFN2 and OPA1 were the anti-division factors. k, l Immunofluorescence assay for mitochondrial morphology using mitochondria-specific antibody Tom-20. Mitochondrial morphology was recorded in response to Mst1 overexpression. Mitochondrial length was determined to reflect the levels of mitochondrial division. Mst1 overexpression promoted mitochondrial division, as evidenced by decreased mitochondrial length. *p < 0.05 vs. ad-ctrl group

Mst1-mediated cell death is dependent on DRP1-related mitochondrial fission

Next, experiments were conducted to verify the detailed role played by DRP1-related mitochondrial fission in Mst1-mediated cell damage (Galano and Reiter 2018). siRNA against DRP1 was transfected into Mst1-overexpressed to perform the knockdown assays of DRP1. Then, mitochondrial apoptosis was evaluated via western blotting. In Fig. 3a–j, compared with the control group, Mst1 overexpression elevated the levels of mitochondrial pro-apoptotic proteins such as Bax and caspase-9. In contrast, the levels of anti-apoptotic protein such as Bcl2 were inhibited by Mst1 overexpression. Notably, DRP1 siRNA transfection significantly inhibited the activation of mitochondrial apoptosis induced by Mst1 overexpression, as evidenced by decreased Bax/caspase-9 and increased Bcl-2. Therefore, these results indicate that mitochondrial apoptosis triggered by Mst1 overexpression is dependent on the activation of DRP1-related mitochondrial fission.

Fig. 3.

Fig. 3

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Loss of DRP1-related mitochondrial division abolished Mst1-mediated mitochondrial apoptosis. a–j Western blotting was used to observe the changes in mitochondrial apoptosis. Caspase-9 and Bax were pro-apoptotic proteins whereas Bcl-2 and survivin were anti-apoptotic factors. DRP1 siRNA and ad-Mst1 were transfected into cells in order to inhibit DRP1 expression and overexpress Mst1 content, respectively. k, l cyt-c translocation into nucleus was evaluated via immunofluorescence. The nuclear expression of cyt-c was measured. *p < 0.05 vs. ad-ctrl group; #p < 0.05 vs. ad-Mst1 + si-ctrl group

cyt-c translocation into the nucleus has been acknowledged as a primary event for the activation of mitochondrial apoptosis (Cortese-Krott et al. 2018). Through immunofluorescence, we found that the expression of nuclear cyt-c was rapidly increased in response to Mst1 overexpression in SCCHN Cal27 cells and Tu686 cells (Fig. 3k, l). Interestingly, DRP1 siRNA transfection significantly inhibited the cyt-c migration into the nucleus (Fig. 3k, l). Altogether, these results illustrate that mitochondrial apoptosis is activated by Mst1 via DRP1-related mitochondrial fission.

DRP1 knockdown sustains mitochondrial function

Mitochondrial function was further analyzed in response to Mst1 overexpression and/or DRP1 knockdown. First, cellular ATP production was inhibited by Mst1 overexpression whereas DRP1 knockdown sustained cellular ATP production in SCCHN Cal27 cells and Tu686 cells (Fig. 4a, b) (Gonzalez et al. 2018). In addition to ATP production, we also found that mitochondrial membrane potential, as evaluated via JC-1 probe, was rapidly downregulated in response to Mst1 overexpression and was reversed to near-normal levels with DRP1 knockdown (Fig. 4c, d). Moreover, ROS production, as assessed via ROS probe, was significantly increased in response to Mst1 overexpression and was attenuated by DRP1 knockdown in SCCHN Cal27 cells and Tu686 cells (Fig. 4e–h). In addition to ROS overproduction, the levels of antioxidant factors such as SOD, GSH, and GPX were significantly inhibited by Mst1 overexpression and were reversed to near-normal levels with DRP1 knockdown (Fig. 4i–n). Altogether, our data indicate that Mst1-mediated mitochondrial dysfunction is dependent on DRP1-related mitochondrial fission.

Fig. 4.

Fig. 4

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Inhibition of DRP1 sustains mitochondrial function in the presence of Mst1 overexpression. a, b ATP production was measured to reflect mitochondrial apoptosis. Mst1 overexpression reduced ATP content, and this effect could be reversed by DRP1 knockdown. c, d JC-1 probe was used to observe mitochondrial membrane potential. Red fluorescence indicates the normal mitochondria membrane potential whereas green fluorescence means the damaged mitochondrial membrane potential. e–h Immunofluorescence was used to observe mitochondrial ROS overproduction. i–n ELISA for cellular antioxidants. SOD, GSH, and GPX were antioxidants which were downregulated by Mst1 overexpression and were reversed to near-normal levels with DRP1 knockdown. *p < 0.05 vs. ad-ctrl group; #p < 0.05 vs. ad-Mst1 + si-ctrl group

Mst1 controls DRP1 via the Wnt/β-catenin pathway

Next, experiments were carried to explore the molecular mechanism by which Mst1 affected DRP1 expression. Previous studies have illustrated that mitochondrial division is modulated by the Wnt/β-catenin pathway. The relationship between Wnt/β-catenin pathway and Mst1 has been reported by several careful reports. In the present study, we investigated whether the Wnt/β-catenin pathway was implicated in Mst1-mediated DRP1 in Cal27 cells and Tu686 cells (Kiel et al. 2018). Western blotting demonstrated that the expression of phosphorylated β-catenin (inactive status) was downregulated in response to Mst1 overexpression (Fig. 5a–d), indicative of the activation of Wnt/β-catenin pathway after exposure to ad-Mst1 transfection. To confirm whether the activated Wnt/β-catenin pathway accounted for DRP1 upregulation in the presence of Mst1 overexpression, DKK1, a specific inhibitor of Wnt/β-catenin pathway, was added to Mst1-overexpressed cells. Treatment with DKK1 reversed the levels of phosphorylated β-catenin (inactive state), an effect that was accompanied by a drop in DRP1 expression (Fig. 5a–d). This information indicated that inhibition of the Wnt/β-catenin pathway could abolish Mst1-mediated DRP1 upregulation. This finding was further supported via immunofluorescence. As shown in Fig. 5e–f, compared with the control group, the fluorescence intensity of phosphorylated β-catenin was reduced in Mst1-overexpressed cells, which was followed by an increase in DRP1 fluorescence intensity. Interestingly, treatment with DKK1 reversed the content of phosphorylated β-catenin and thus prevented DRP1 upregulation in the presence of Mst1 overexpression (Fig. 5e–f) (Kim et al. 2018). Altogether, the above data indicated that the Wnt/β-catenin pathway was linked to Mst1-mediated DPR1 modulation.

Fig. 5.

Fig. 5

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Mst1 modulates DRP1 in a manner dependent on Wnt/β-catenin pathway. a–d Western blotting was used to observe the alterations of β-catenin and DRP1 in response to Mst1 overexpression. DKK1, a specific inhibitor of Wnt/β-catenin pathway, was added into the medium of Mst1-overexpressed cell to inhibit β-catenin activation (dephosphorylation). e, f Immunofluorescence assay was used to observe the alteration of β-catenin and DRP1 in response to Mst1 overexpression and DKK1 treatment. *p < 0.05 vs. ad-ctrl group; #p < 0.05 vs. ad-Mst1 group

Discussion

SCCHN, as one of the most prevalent cancers in the world, has been listed third with respect to cancer-related mortality and is also the second leading cause of death in patients with tumors (Hardeland 2018). However, there has been a big challenge to cure SCCHN which requires new targets and novel therapeutic candidate to design anti-tumor drugs. Here, using two SCCHN cell lines and gain-of-function assays for Mst1 in vitro, we demonstrated the potential of Mst1 overexpression and DRP1-related mitochondrial division activation as innovative and effective ways for the treatment of SCCHN via inducing cell viability downregulation, mitochondrial dysfunction, energy depletion, and apoptosis activation (Cremonini et al. 2018). As far as we know, this is the first investigation to explore the detailed role played by Mst1 in modulating the viability of SCCHN Tu686 cells via affecting DRP1-related mitochondrial division in a manner dependent on the Wnt/β-catenin pathway. This finding provides a new piece of evidence to support the anticancer effect of Mst1 on SCCHN Tu686 cell progression and identifies the Mst1-Wnt/β-catenin-DRP1 axis as a novel signaling pathway involving SCCHN fate (Higgs et al. 2019).

Mitochondrial division, one important intracellular signaling event, exerts a critical impact in cell physiological processes, including but not limited to mitochondrial energy metabolism, mitochondrial respiratory, mitochondrial apoptosis, calcium balance, and redox homeostasis (Meng et al. 2018; Meyer and Leuschner 2018). Excessive mitochondrial division promotes the depolarization of mitochondrial membrane potential, along with the opening of mitochondrial permeability transition pore (mPTP). Subsequently, cyt-c freely releases from mitochondria into the nucleus with the help of mPTP opening, finally activating the caspase-9-related apoptosis pathway. The above molecular cascade has been noted in several disease models, such as cardiac ischemia reperfusion injury, endothelial oxidative stress (Zhu et al. 2018), fatty liver disease, and acute kidney injury (Montoya-Zegarra et al. 2019). In the present study, we observed that mitochondrial division was activated by Mst1 overexpression, an effect that was accompanied by mitochondrial membrane potential reduction and cyt-c release. In addition, we also provide ample evidence to support the regulatory effects of mitochondrial division in mitochondrial energy metabolism (Park et al. 2018), including ATP production and mitochondrial respiratory complex expression. Therefore, crosstalk among mitochondrial division and mitochondrial dysfunction/apoptosis reveals an intrinsic cancer-killing pathway initiated by Mst1, which is necessary for clarifying the anticancer mechanisms exerted by Mst1 in SCCHN.

In the present study, Mst1 overexpression reduced cell survival rate, inhibited ATP production, hindered mitochondrial respiratory function, and activated caspase-9-related apoptotic pathway, which added more information for Mst1 as a classical anticancer factor. Therefore, our work identifies a novel role for Mst1 as a positive regulator of mitochondrial homeostasis and SCCHN fate, as well as establishes a mitochondria-dependent function of Mst1 that could be part of the intricate antitumor defensive system. Cancer utilizes a variety of strategies to escape apoptosis, and mitochondrial division has been acknowledged as an effective approach to augment apoptosis response via quenching of the antiapoptotic factors upregulation (Zhang et al. 2018). In addition, mitochondrial division also promotes the release of cyt-c into the nucleus (Han et al. 2018; Jin et al. 2018). DRP1 functions in mitochondrial division via forming the contractile ring around mitochondria with the help of F-actin. Interestingly, DRP1-related mitochondrial division has been found to be the downstream target of several anticancer drugs such as IL-2, erlotinib, matrine, and tanshinone IIA. Thus, targeting mitochondrial division by upregulating DRP1 is a rational anticancer strategy. However, more animal experiments and clinical data are required to support our findings. There are several limitations in our experiments. First, more studies are required to validate our findings in the primary SCCHN cells in addition to the cell lines. Besides, more clinical data are necessary to verify the cancer-killing effects of Mst1 and DRP1.

To conclude, we demonstrated for the first time the tumor-suppressive effect of Mst1 on SCCHN Cal27 cells and Tu686 cells via activating the Wnt/β-catenin pathway and elevating DRP1-related mitochondrial division. Our finding might provide a mechanistic basis for Mst1-based anticancer strategies for the treatment of SCCHN.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Footnotes

Publisher’s note

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