Background
The incidence of thyroid carcinoma, the most common endocrine malignancy, has significantly increased over the past decades. More than 50,000 new cases of thyroid carcinoma are currently diagnosed annually in the United States. Several risk factors have been introduced to explain the development of thyroid cancer, including sex, age, genetics, radiation exposure, a low-iodine diet, and race. Although many advances have been made in the early diagnosis and treatment of thyroid carcinoma, the pathogenesis of thyroid carcinoma has not been fully addressed.
Recently, studies have found a close interaction between the Hippo pathway and cancer progression. The Hippo pathway was originally identified as a novel antitumor signaling pathway that modulates tissue growth. The core Hippo pathway components include mammalian STE20-like protein kinase 1, yes-associated protein (YAP), and large tumor suppressor 1 (LATS1). Interestingly, these three Hippo kinases have various functions on cancer fate. For example, Mst1 has been found to promote cell death in gastric cancer, colorectal cancer, lung cancer, pancreatic cancer, and breast cancer [
1‐
5]. In contrast, Yap has emerged as a growth promoter in cancer by modulating tumor aggressive behaviors, chemotherapy resistance, cancer stem cell differentiation, and tumor epithelial–mesenchymal transition [
6‐
8]. There is little evidence to explain the exact role of LATS1 in cancer progression. Notably, several reports have indicated the impacts of Yap [
9] and Mst1 [
10] in controlling the viability of thyroid cancer cells. Loss of Yap sensitizes thyroid cancer to chemotherapy [
11], whereas Mst1 overexpression augments papillary thyroid carcinoma apoptosis [
10]. Considering the different roles played by Mst1 and Yap in the cancer biological phenotype, we asked whether Mst1 overexpression in combination with Yap knockdown could further promote the death of thyroid cancer cells.
Mitochondria extensively control various critical pathophysiological processes involving cancer metabolism, growth, proliferation, movement, differentiation, survival and metastasis [
12‐
15]. As the major consumers of oxygen and glucose, mitochondria produce sufficient ATP, which is required for cancer behaviors [
16,
17]. However, damaged mitochondria impair cancer metabolism and even initiate mitochondria-related apoptotic pathway activity [
18,
19]. For example, damaged mitochondria produce excessive ROS, which induces oxidative stress to mediate cellular senescence [
20]. Moreover, injured mitochondria cannot generate enough energy, which is associated with the inability of cancer cells to adhere and invade [
21]. More seriously, poorly structured mitochondria release proapoptotic factors such as cyt-c and HtrA2/Omi to initiate caspase-mediated apoptotic signals [
22,
23]. Accordingly, mitochondria play a main role in both the survival and death of cancer cells. Notably, mitochondrial elongation factor 1 (MIEF1) has been found to be a novel mitochondrial homeostasis mediator [
24]. Increased MIEF1 expression impairs mitochondrial dynamics, leading to mitochondrial fragmentation, which has been acknowledged as an early event in mitochondrial apoptosis initiation. For example, in lung cancer, MIEF1-dependent activation of mitochondria promotes mitochondrial stress and augments mitochondrial apoptosis in A549 lung cancer cells [
25]. In addition, reperfusion-mediated cardiomyocyte death and endothelial damage are also tightly controlled by MIEF1 in a manner dependent on mitochondrial fission [
26]. However, there is no evidence to indicate the influence of MIEF1-related mitochondrial fission on thyroid cancer cell viability. Considering that the Yap/Hippo pathway has been reported to be an upstream mediator of MIEF1-related mitochondrial fission, we asked whether differential regulation of Mst1 and Yap could further activate MIEF1-related mitochondrial fission and thus promote the death of thyroid cancer cells.
At the molecular levels, mitochondrial fission is primarily modulated by JNK pathway. For example, in tongue cancer [
27], mitochondrial fission is highly modulated by the JNK-Fis1 pathway. In non-small cell lung cancer, mitochondrial fission is signaled by Hippo pathway in a manner dependent on the activity of JNK [
28]. Similar results were also noted in liver cancer in response to cytokine-based therapy [
29,
30]. This finding is also validated in thyroid cancer that activation of JNK pathway enhances mitochondrial fission and promotes cancer cell death [
31,
32]. Based on the above information, we wanted to know whether JNK pathway was involved in mitochondrial fission in Mst1/Yap-modified cell viability in MDA-T32 cell in vitro. Altogether, the goal of our study is to figure out the synergistic effects of Mst1 overexpression and Yap knockdown on thyroid cancer death via modulating MIEF1-related mitochondrial fission and the JNK pathway.
Materials and methods
Cell culture and transfection
Human thyroid carcinoma MDA-T32 (ATCC
® CRL-3351™) and MDA-T68 (ATCC
® CRL-3353™) cell lines was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). To overexpress the Mst1, adenovirus-Mst1 (ad-Mst1) was transfected into MDA-T32 cells. In brief, pDC315-Mst1 vector was obtained from Shanghai Gene-Pharma Co. (Shanghai, China) and then HEK293 cells (ATCC
® CRL-3216™, American Type Culture Collection, Manassas, VA, USA) were infected with pDC315-Mst1 vector to obtain the Ad-Mst1 according to a previous study [
33]. In brief, a total of 1 × 10
5 cells/well were infected with 50 multiplicity of infection (MOI) adenovirus (Ad)-Mst1 in serum-free RPMI-1640 for 12 h at 37 °C. To silence the expression of Yap, shRNA against Yap (sh-Yap) was used to transfect into MDA-T32 cells. The sh-Yap was purchased from Shanghai Gene-Pharma Co. (Shanghai, China) and transfection was performed with the help of Opti-MEM medium and Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol [
34]. GFP-labelled adenovirus was used as a preliminary study to observe the infection efficiency (Additional file
1: Figure S1A). To prevent the activation of JNK pathway, SP600125 (25 μM, Selleck Chemicals, Houston, TX, USA) was added into the medium of MDA-T32 cells [
35].
Cell proliferation assay and MTT assay
Cellular proliferation was evaluated via EdU assay. Cells were seeded onto a 6-well plate, and the Cell-Light™ EdU Apollo
®567 In Vitro Imaging Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalogue No. A10044) was used to observe the EdU-positive cells according to the manufacturer’s instructions [
36]. 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) [
37]. 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) [
38].
Immunofluorescence analysis and confocal microscopy
Cells were plated on glass slides in a 6-well plate at a density of 1 × 10
6 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. The cells were then incubated with the primary antibodies: [cyt-c (1:1000; Abcam; #ab90529), Tom20 (mitochondrial marker, 1:1000, Abcam, #ab186735), LAMP1 (lysosome marker, 1:1000, Abcam, #ab24170), MIEF1 (1:1000, Abcam, #ab89944)] [
39].
Mitochondrial membrane potential measurement and ATP detection
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 [
40]. 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) [
41]. Cellular ATP content was measured according to a previous report via ELISA assay. Cells were washed with PBS and then collected at room temperature. Subsequently, a luciferase-based ATP assay kit (Celltiter-Glo Luminescent Cell Viability assay; Promega, Madison, WI, USA; Catalogue No. A22066) was used according to the instructions.
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) [
42]. Deionized water was added to generate 30-µg protein samples for each lane. A 10% sodium dodecyl sulphate (SDS) separation gel and concentration gel were prepared [
43]. The following diluted primary antibodies were added to the membrane and incubated overnight: JNK (1:1000; Cell Signaling Technology, #4672), p-JNK (1:1000; Cell Signaling Technology, #9251), Mst1 (1:1000, Cell Signaling Technology, #3682), Yap (1:1000; Cell Signaling Technology, #14,074), Mfn2 (1:1000, Abcam, #ab56889), Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), Cyclin D (1:1000, Abcam, #ab134175), CDK4 (1:1000, Abcam, #ab137675), Drp1 (1:1000, Abcam, #ab56788), Fis1 (1:1000, Abcam, #ab71498), Opa1 (1:1000, Abcam, #ab42364), Mff (1:1000, Cell Signaling Technology, #86668).
RNA isolation and qPCR
TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA from cells. Subsequently, the Reverse Transcription kit (Kaneka Eurogentec S.A., Seraing, Belgium) was applied to transcribe RNA (1 μg in each group) into cDNA at room temperature (~ 25 °C) for 30 min. The qPCR was performed with primers using SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Inc. Cat. No. 4309155). The following were primers used in the present study: ROCK-1 (Forward: 5′-ACCTGTAACCCAAGGAGATGTG-3′, Reverse 5′-CACAATTGGCAGGAAAGTGG-3′), and Rac1 (Forward 5′-ATGCAGGCCATCAAGTGTGTGG-3′, Reverse: 5′-TTACAACAGCAGGCATTTTCTC-3′), and GAPDH, (Forward: 5′-AAGTTGTGFATTAGTCA-3′, Reverse 5′-AGAATAGTCCTATAATCA-3′).
ELISA
The Caspase 9 Activity Assay Kit (Beyotime, China, Cat. No: C1158) was used to measure the activity of caspase-9 according to the manufacturer’s instructions. The concentrations of GSH, SOD and GPX were evaluated using commercial kits (Cellular Glutathione Peroxidase Assay Kit, Beyotime, China, Cat. No: S0056; Glutathione Reductase Assay Kit, Beyotime, China, Cat. No: S0055; Total Superoxide Dismutase Assay Kit, Beyotime, China, Cat. No: S0101, respectively). ATP production was measured using a luciferase-based ATP assay kit (Beyotime Institute of Biotechnology) with a microplate reader [
44].
Cellular proliferation evaluation and LDH release assay
Cellular proliferation was evaluated via EdU assay. Cells were seeded onto a 6-well plate, and the Cell-Light™ EdU Apollo
®567 In Vitro Imaging Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalogue No. A10044) was used to observe the EdU-positive cells according to the manufacturer’s instructions. Cellular lactate production in the medium was measured via a lactate assay kit (#K607-100; BioVision, Milpitas, CA, USA) according to a previous study [
45].
Transwell assay
For Transwell migration assays, the upper chambers of 24-well Transwell assay plates were seeded with 2 × 103 cells in 200 μL serum-free medium per well. The lower chambers were filled with 600 μL medium containing 0.5% FBS. After a 24-h incubation in a humidified incubator at 37 °C, 5% CO2, cells that had migrated to the underside of the membranes were fixed and stained with 0.1% crystal violet. After washing with distilled water, pictures of each chamber were randomly taken using a 200× microscope field, and these images were used to quantify the total number of migrated cells.
Statistical analysis
SPSS 21.0 software (IBM Corp., Armonk, New York, USA) was applied for data analysis. All experiments were repeated 3 times in each group. The mean value of the measurement data was expressed as the mean and SEM. Comparisons among groups were by one-way analysis of variance (ANOVA), and multiple comparisons between the average number of samples were performed by LSD analysis. p < 0.05 indicated that the difference was statistically significant.
Discussion
During the last two decades, most studies have focused on the pathogenesis of thyroid cancer development and progression. In the present study, we found that the viability of thyroid cancer cells was associated with mitochondrial function, Mst1 expression, Yap levels and JNK-MIEF1 pathway activity. Molecular studies indicated that Mst1 overexpression or Yap knockdown reduced the viability of thyroid cancer MDA-T32 cells in vitro and that this process was closely associated with mitochondrial stress, as evidenced by the observed mitochondrial malfunction and mitochondrial structural disorder. Further, we found that the combination of Mst1 upregulation and Yap inhibition further increased the apoptotic rate of thyroid cancer MDA-T32 cells in vitro by augmenting mitochondrial damage and activating the JNK-MIEF1 pathway. However, blockade of the JNK pathway abolished the regulatory effects of Mst1/Yap modification in MDA-T32 cells. To our knowledge, this study is the first to investigate the synergistic effects of Mst1 overexpression and Yap knockdown on the viability of MDA-T32 cells. In this report, we show how to differentially modify the components of the Hippo pathway in order to further enhance cancer cell death, and the findings described in this manuscript are particularly applicable for designing new drugs to treat thyroid carcinoma by targeting the Hippo pathway.
Many experiments have been conducted to understand the biological significance of the Hippo pathway in tumorigenesis. Dysregulation of the Hippo pathway has been found to be an effective way to limit tumor progression. For example, dephosphorylation of Yap interrupts glucose uptake through the Bcl-XL/GLUT1 pathway in human gastric cancer [
49]. In addition, the levels of Yas could be used as an early marker to evaluate breast cancer progression [
50]. Moreover, modulation of Yap via ailanthone inhibits bladder cancer in a manner dependent on Nfr2 downregulation and c-Myc inhibition [
51]. In liver cancer, loss of Yap attenuates cancer metastasis and mobilization through impairing lamellipodium formation and inactivating the JNK–Bnip3–SERCA–CaMII pathway [
52]. With respect to Mst1, overexpression of Mst1 via tanshinone IIA increases the therapeutic sensitivity of colorectal cancer to IL-2-mediated cytokine therapy. In lung cancer, Mst1 upregulation impairs mitochondrial energy metabolism and ultimately impedes cancer migration and movement via the ROCK1/F-actin pathway [
53]. In addition, the antitumor effect of marine-origin compounds could be abolished by Mst1 inhibition in liver cancer [
54]. Therefore, the above data indicate that Yap and Mst1 seem to play different roles in regulating the cancer phenotype. However, no study has explored the synergistic or antagonistic molecular effects mediated by Yap and Mst1 in thyroid cancer. In the present study, we found that Mst1 overexpression induced cancer cell death, an effect that was similar to that of Yap knockdown. Interestingly, Mst1 overexpression in combination with Yap knockdown further promoted cancer cell death by exacerbating mitochondrial stress. This result indicates that differential regulation of the core components in the Hippo pathway is potentially a novel therapeutic tool for the treatment of thyroid cancer.
At the molecular level, we found that mitochondrial dysfunction, activated by Mst1/Yap modification, was implicated in cancer cell death. After Yap loss and Mst1 overexpression, mitochondrial membrane potential was reduced, an effect that was followed by mitochondrial ROS overproduction. In addition, mitochondrial dynamics were disturbed by Mst1/Yap modification, as evidenced by mitochondrial fragmentation. This result indicates that mitochondria could be considered a potential target of the Hippo pathway. Our data were in accordance with those of previous studies. In glioblastoma [
55], gastric cancer, and rectal cancer, Yap dysfunction is associated with mitochondrial damage, including mitochondrial apoptosis activation, mitochondrial fission initiation and mitochondrial oxidative stress [
56]. Similarly, in lung cancer and liver cancer, mitochondrial injury is triggered by Mst1 activation. This study showed that mitochondria are a potential target for thyroid cancer therapy, and further research should be undertaken to facilitate this therapeutic application.
Finally, we reported that the JNK-MIEF1 pathway was activated by Mst1/Yap modification. At the molecular level, MIEF1 is a novel mitochondrial damage mediator [
57]. In cardiac ischemia reperfusion stress, MIEF1 is upregulated, and the levels of MIEF1 are tightly correlated with the degree of myocardial injury [
58]. In addition, after exposure to UV radiation, MIEF1 expression is deregulated, and this alteration has been demonstrated to play a decisive role in initiating epidermal cell death [
59]. In the present study, we provide evidence to support the influence of MIEF1 on mitochondrial damage in thyroid cancer [
60]. MIEF1 expression was increased in response to Mst1 overexpression and/or Yap knockdown via the JNK pathway. However, the detailed role of MIEF1 in cancer cell death and mitochondrial damage has not been fully explained. More studies are required to determine the detailed role played by MIEF1 in biological functions in cancer [
61].
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