MPC1 deficiency accelerates lung adenocarcinoma progression through the STAT3 pathway

Mitochondrial pyruvate carrier 1 (MPC1), a key factor that controls pyruvate transportation in the mitochondria, is known to be frequently dysregulated in tumor initiation and progression. However, the clinical relevance and potential molecular mechanisms of MPC1 in lung adenocarcinoma (LAC) progression remain to be illustrated. Herein, MPC1 was lowly expressed in LAC tissues and significantly associated with favorable survival of patients with LAC. Functionally, MPC1 markedly suppressed stemness, invasion, and migration in vitro and spreading growth of LAC cells in vivo. Further study revealed that MPC1 could interact with mitochondrial signal transducer and activator of transcription 3 (mito-STAT3), disrupting the distribution of STAT3 and reducing cytoplasmic signal transducer and activator of transcription 3 (cyto-STAT3) as well as its phosphorylation, while the activation of cyto-STAT3 by IL-6 reversed the attenuated malignant progression in MPC1-overexpression LAC cells. Collectively, we reveal that MPC1/STAT3 axis plays an important role in the progression of LAC, and our work may promote the development of new therapeutic strategies for LAC.


Introduction
Lung adenocarcinoma (LAC) represents about 40% of overall lung cancers and the leading cause of cancerrelated deaths worldwide 1 . Stemness, invasion, and subsequent metastasis, which increase the incidence of recurrence and treatment failure, are the major causes of LAC-related death 2,3 . A better understanding of the molecular mechanisms underlying LAC cell progression is crucial for developing effective treatments.
Aberrant mitochondrial pyruvate metabolism is the important feature in cancer cells, and key enzymes associated with mitochondrial pyruvate metabolism are crucial in the tumor progression [4][5][6] . Mitochondrial pyruvate carrier 1 (MPC1), which is located in the inner mitochondrial membrane, is one of the key enzymes responsible for pyruvate transportation and oxidation [7][8][9] , MPC1 deficiency or inactivation accelerates aerobic glycolysis and malignant progression in diverse types of cancer, such as colon cancer and esophageal squamous cell carcinomas 6,10 . Additionally, decreased expression of MPC1 is associated with poor prognosis in various types of tumors, including prostate cancer and colon cancer 11,12 . Collectively, these findings indicate that MPC1 probably serves as a tumor suppressor to impair tumor malignancy.
Despite the involvement of MPC1 in tumor progression, the clinical relevance and function of MPC1 in LAC remains to be investigated. In this study, we showed that MPC1 in LAC was positively correlated with overall survival of LAC patients. Functionally, overexpression of MPC1 attenuated the stemness, invasion, and migration capacities of LAC cancer cells in vitro and in vivo, while knockdown of MPC1 or using MPC inhibitor UK5099 promoted those malignant phenotypes. Mechanically, MPC1 suppressed tumor progression via interacting with mito-STAT3, disrupting STAT3 distribution and inhibiting cyto-STAT3 activation. Thus, our data revealed a critical role of MPC1 in controlling the progression of LAC and its potential therapeutic and prognostic value for LAC patients.

Patients and tissue specimen
Tumor and adjacent normal tissues from LAC patients who underwent surgical resection in Southwest Hospital, Third Military Medical University (Army Medical University) from May 2016 to June 2017, Chongqing, China, with approval from the Institutional Ethics Committee. The tissue microarray, including 78 LAC without radiotherapy or chemotherapy before surgery, was from OUTDO BIOTECH (Shanghai, China). All patients were provided with informed consent and clinical data were listed in Supplementary Table 1.

GEO database analysis
A total number of 1926 tumor samples were used to generate Kaplan-Meier curves for the survival analysis of MPC1 in LAC (http://www.kmplot.com/lung). The median expression was used as the final cutoff value.

Western blotting
Western blotting was performed as previously described 15 . The primary antibodies were as follows: anti-MPC1

Nuclear and cytoplasmic extraction assays
The NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Waltham, MA, USA) were purchased from Thermo Scientific. The detailed procedures were performed according to the manufacturer's instructions.

Isolation of mitochondrion and cytoplasm fractions
Mitochondrion and cytoplasm fractions from tumor cells were separated using Mitochondria Isolation Kit (Solarbio, Beijing, China) (number: SM0020) following the manufacturer's protocol.

Co-immunoprecipitation (Co-IP) and mass spectrometry (MS) analyses
For Co-IP, LAC cells were infected with MPC1 overexpressing lentivirus or control lentivirus, which contained a 3 × flag sequence. Anti-flag M2 Magnetic Beads (Sigma-Aldrich, St. Louis, MO, USA) were used for detection and capture of the fusion proteins. The detailed procedures were performed according to the manufacturer's instructions. For the liquid chromatography-MS analysis, the gel pieces with MPC1 immunoprecipitated complex or control were dehydrated in acetonitrile, dried in a speed vacuum, and digested with trypsin. The peptides were extracted from the polyacrylamide and were evaporated for MS analysis using LTQ-Orbitrap Elite Mass Spectrometer System (Thermo Scientific, Waltham, MA, USA).

Tumorsphere-formation assay
Cancer cell were seeded into 96-well plates (1 × 10 2 / well) by flow cytometry (Beckman Coulter, S. Kraemer Boulevard Brea, CA, USA). Cells were cultured in DMEM/F12 stem cell medium containing basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and B-27 for 1 week, the tumorspheres were photographed by a light microscope and counted to calculate the tumorsphere efficiency.

Tumor cell invasion and migration assay
Tumor cell invasion assay was performed as previously described 13 . For the migration assay, 1 × 10 4 cells were added to the transwell chambers with 8-μm pore size (Millipore, Billerica, MA, USA). After incubation for 24 h, the chambers were wiped using cotton swabs, fixed with 4% paraformaldehyde, and stained with crystal violet. The lower chamber was photographed using a light microscope, and the cells were counted.

Invadopodia immunofluorescence staining
Tumor cells grown on glass coverslips were fixed with 4% paraformaldehyde for 10 min, permeabilized by 0.3% Triton X-100 for 5 min, and then incubated with antirhodamine phalloidin (Solarbio, Beijing, China). Cells were then photographed under a laser confocal scanning microscope (Leica, Brunswick, Ohio, Germany). Cells with more than four microspikes on the surface were considered invadopodia-positive cells 16 . The cells were counted in 10 separate, randomly selected 200 × fields with a fluorescence microscopy.

Xenograft
Four-week-old female BALB/c athymic nude mice were approved by the Institutional Animal Care and Use Committee of the Southwest Hospital, TMMU. The number of 1 × 10 6 MPC1-overexpression cells or control cells were suspended in 50 μl of PBS and subcutaneously implanted in female nude mice. The size of the xenograft tumor was measured every 4 days for a month using a Vernier calliper, and the volume was calculated at different indicated intervals post-transplantation as follows: volume = shortest diameter 2 × longest diameter/2. The animals were killed 1 month after cell implantation, and the subcutaneous xenografts were weighed and analyzed by IHC. The animal experiments were approved by the Institutional Animal Care and Use Committee of Southwest Hospital, Third Military Medical University in accordance with the Guide for the Care and Use of Laboratory Animals.

Statistical analysis
All experiments were performed at least three times with triplicate samples. Data were analyzed using SPSS 16.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 6.0 (San Diego, CA, USA) and were expressed as the mean ± SD. Unless otherwise noted, the data were analyzed using Student's t test or one-way ANOVA. Significance of correlation between the expression of MPC1 and histopathological factors were determined using Pearson's χ 2 test. Kaplan-Meier plots were performed to investigate the prognostic relevance of MPC1 in univariate analysis. Multivariate analysis was performed by applying Cox proportional hazards test. Statistical difference was considered significant if P-values were < 0.05. All significant statistical differences were defined as *P<0.05; **P<0.01, and ***P<0.001.

MPC1 is downregulated in LAC and predicts favorable prognosis
To determine the status of MPC1 in LAC, we examined the expression of MPC1 in nine pairs of LAC tissues, and found that MPC1 was lowly expressed in LAC tissues as compared with corresponding adjacent non-tumor tissues (Fig. 1a, b). Furthermore, MPC1 mRNA also decreased in LAC tissues as compared with that in corresponding normal tissue (Fig. 1c), and the similar results were observed in LAC cells (A549, H1299, H1975) as compared with normal human bronchial epithelial (HBE) cells (Fig. 1d). Additionally MPC1 was negatively correlated with tumor size and TNM stages, but not related with tumor location, histological grade, EGFR mutation (Supplementary Table 2). Furthermore, the overall survivals (OS) of LAC patients with MPC1 low showed shorter than that with MPC1 high in our clinical cohort (Fig. 1e, f) and GEO database (Fig. 1g). Univariate and multivariate analyses further indicated that MPC1 was an independent prognostic indicator for OS of LAC patients (Table 1). Therefore, MPC1 might act as an indicator of favorable prognosis for patients with LAC.

MPC1 deficiency augments the stemness of LAC cells in vitro
To explore the potential relationship between MPC1 status and the stemness of LAC cells, MPC1overexpression and MPC1-knockdown cells were established (Supplementary Figure 1). The volume and number of tumorspheres derived from OEMPC1 cells were smaller than those of control cells (Fig. 2a, b), whereas knockdown of MPC1 or inhibiting MPC1 activity treated with UK5099 increased those capabilities (Fig. 2c, d). In addition, western blotting revealed that overexpression of MPC1 decreased the level of cancer stem cell (CSC) markers, including NANOG, OCT4, and SOX2 in LAC cells (Fig. 2e), while knockdown of MPC1 increased the level of these markers (Fig. 2f). Conclusively, these data indicate MPC1 deficiency increased cancer stem-like traits of LAC cells.

Loss of MPC1 accelerates the migration and invasion of LAC cells in vitro
Next, we determined whether MPC1 can influence the invasion and migration of LAC cells. The transwell assay showed that overexpression of MPC1 significantly reduced motile ability of LAC cells (Fig. 3a, b), while treatment with siMPC1 or UK5099 in H1299 cells increased their mobility (Fig. 3c, d). Invadopodia, a form of cellular projection that is embedded in or protruding from the lamellipodial actin network, and matrix metalloproteinases (MMPs) are critical in migration and invasion of cancer cells 17,18 . We revealed that the number of invadopodia in the OEMPC1 LAC cells was decreased (Fig. 3e, f), while in the siMPC1 H1299 cells, the number of invadopodia was increased (Fig. 3g, h). Additionally, western blotting showed that the expression levels of MMP2, MMP3, and MMP7 were significantly decreased in OEMPC1 group (Supplementary Figure 2a), while downregulated MPC1 increased the expression levels of those proteins (Supplementary Figure 2b). Collectively, these observations demonstrate that MPC1 decreases the invasion and migration capabilities of LAC cells through invadopodia formation and the MMPs pathway.

MPC1 interacts with mito-STAT3 to inhibit cyto-STAT3 activation in LAC cells
To further explore the underlying mechanism of MPC1 in progression of LAC cells, we screened potential MPC1-binding proteins through mass spectrometric (MS) analysis. STAT3 was identified in the intersection candidate of MPC1 interacting protein set with the KEGG pathway in cancer gene set from GSEA (Fig. 4a). Furthermore, Co-IP assays further demonstrated that MPC1 can interact with STAT3 rather than P-STAT3 (Fig. 4b, c and Supplementary Figure 3a). STAT3 is present in mitochondrial (mito-STAT3) and cytoplamic (cyto-STAT3) fractions 19 . To further define the location, where the interaction between MPC1 and STAT3 occured, we extracted the mitochondria from cytoplasm, and the Co-IP assays demonstrated that MPC1 could interact with mito-STAT3 (Fig. 4d), but not cyto-STAT3 (Supplementary Figure 3b). Additionally, overexpression of MPC1 downregulated P-STAT3(Y705), whereas knockdown of MPC1 upregulated P-STAT3(Y705), but without affecting P-STAT3(S727) and total STAT3 expression ( Fig. 4e and Supplementary Figure 3c). Since cyto-STAT3 can be recruited into the mitochondrial inner membrane by some chaperone, and MPC1 is located in the inner membrane of mitochondrial, we proposed that the binding of MPC1 with STAT3 in the mitochondria promotes the process that cyto-STAT3 is transported into the mitochondria. To test this possibility, we overexpressed MPC1 expression in two LAC cell lines and determined the protein abundance of STAT3 in mitochondria fractions and in cytoplasm fractions. Western blotting shows that overexpression of MPC1 increased the level of STAT3 anchored on mitochondrion, but decreased the level of STAT3 and P-STAT3 (Y705) in cytoplasm without mitochondria (Fig. 4f). Previous study shows that STAT3 promotes cancer progression in various types of cancer and is activated by phosphorylation at Tyr705(Y705) 20,21 , and P-STAT3 is translocated into the nuclei to induce the transcription of several target genes implicated in cancer cell malignancy 22 . Cell fractionation analysis demonstrated that overexpression of MPC1 reduced P-STAT3(Y705) in the cytoplasm and nuclei (Fig. 4g). Furthermore, IHC analysis of both MPC1 and P-STAT3(Y705) in the specimens from 20 LAC patients revealed that P-STAT3(Y705) was highly expressed in the MPC1 low group (Fig. 4h, i). Taken together, these data indicate that MPC1 interacts with mito-STAT3 and disrupts the location of STAT3 in the cytoplasm and mitochondria, which decreases P-STAT3 (Y705) into the nuclei.

MPC1/ STAT3 axis regulates the progression of LAC cells
Since MPC1 binds to mito-STAT3 and inhibits the phosphorylation of STAT3, we explored whether MPC1-mediated cancer cell progression depends on the downstream of the STAT3 pathway. Previous report 23 and our data demonstrated that IL-6 acts as the agonist for the phosphorylation of STAT3 (Fig. 5a). We found OEMPC1 LAC cells treated with IL-6 (100 ng/ml) increased the stemness (Fig. 5b, c) and mobility capacities (Fig. 5d and Supplementary Figure 4), which partially overcame the inhibitory effects of MPC1 on progression of LAC cells. Furthermore, OEMPC1 LAC cells treated with IL-6 (100 ng/ml) partially recovered the basal expression levels of SOX2 and MMP2 (Fig. 5e). Altogether, STAT3 was a   novel downstream regulator of MPC1 to mediate progression in LAC cells.

Overexpression of MPC1 inhibits tumorigenicity in vivo
To further examine the effect of MPC1 on LAC cells in vivo, the subcutaneous xenograft model was established. The xenograft tumors formed by OEMPC1 cells had a slower growth rate and smaller average volume as compared with those formed by control cells (Fig. 6a-c). Consistent with our previous data, IHC showed that the expression of P-STAT3(Y705), SOX2, and MMP2 in the OEMPC1 groups was decreased compared with the control group (Fig. 6d). Therefore, these results indicate that upregulated MPC1 inhibits the tumorigenicity of LAC cells in vivo.

Discussion
The "Warburg effect" is considered a vital hallmark of cancer cells, characterized by an altered pyruvate metabolism, indicating mitochondrial pyruvate carrier is involved in the progression of cancer 5 . MPC1, which is located at the genomic locus of 6q27 and known as BRP44L, has been reported to form a heterologous protein complex with MPC2 in the inner mitochondrial membrane and acts as a gatekeeper for pyruvate entry into the mitochondria 24,25 . Actually, MPC1 has been reported to be downregulated in several types of cancers, such as gastric cancer 26 and colon cancer 27 , but the clinical relevance and function of MPC1 in LAC remains elusive. Herein, we revealed that MPC1 was lowly expressed in LAC tissues and positively correlated with prognosis. Further studies showed that the malignancy of LAC cells were increased via depletion of MPC1 or MPC1 inhibitor, but decreased by overexpression of MPC1 in vitro and in vivo. Thus, these data indicate that MPC1 could act as a tumor suppressor and a candidate for therapeutic target as well as a prognostic biomarker in LAC.
Previous studies have shown that MPC1 can transport pyruvate into the mitochondria for oxidation, reducing lactate production in PGC1α-transduced cholangiocarcinoma cells, but elevating reactive oxygen species (ROS) production to facilitate metastatic dissemination of cholangiocarcinoma cells 28 . It has been found that MPC inhibitor UK5099 treatment in esophageal squamous epithelium cells can block pyruvate transportation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation (OXPHOS) and trigger aerobic glycolysis. In addition, UK5099-treated cells exhibit stronger invasive capacity compared with the parental cells and is more resistant to chemotherapy and radiotherapy. The HIF-1α expression and ROS production were also activated 10 . However, whether MPC1overexpression could regulate Warburg effect to affect the production of lactate in LAC cells has not been illustrated yet. Our results showed that upregulated MPC1 significantly decreased the production of lactate compared with the control groups (data not shown), suggesting that MPC1 may increase pyruvate into the mitochondria for oxidation and decreased lactate production in LAC cells.
Invasion is correlated with invadopodia formation. Actin cytoskeleton and actin-binding proteins control the dynamic changes in the formation of polarized actinbased migratory protrusions, such as lamellipodia, filopodia, apically restricted circular dorsal ruffles (CDRs), and invadopodia. Herein, we found MPC1 could act as a regulator in invadopodia to control LAC cell invasion.
Our study also revealed that MPC1 was a key inhibitory factor in sustaining the stemness capability of LAC cells by decreasing the expression of stemness biomarker. In our previous work, we have shown that IGF-1R mediated OCT4 expression to form a complex with β-catenin and SOX2, which is crucial for the stemness of LAC cells 13,29 . Despite IGF-1R, our study on MPC1 may bring forward a new underlying mechanism of the stemness traits in LAC.
STAT3, a member of the signal transducer and activator family, was phosphorylated on tyrosin 705 (Y705) and serine 727 (S727) residue to form a dimmer and then translocated into the nuclei upon binding of growth factors and cytokines with their receptors, such as IL-6 30 . P-STAT3 acts as a transcription factors to activate downstream target genes that are implicated in cancer (see figure on previous page) Fig. 4 MPC1 interacts with STAT3 in mitochondria and decreases its phosphorylation and transloacation into nuclei. a Identification of STAT3 from the intersection between MPC1 interacting protein set by MS and the KEGG pathway in cancer gene set from GSEA. b, c Co-IP analysis of STAT3 interaction with MPC1 in A549 and H1299 MPC1-overpxpression cells. d The mitochondria was isolated from the cytoplasm, and Co-IP analysis showing that STAT3 interacts with MPC1 in the mitochondria in A549 and H1299 MPC1-overpxpression cells. e Western blotting showing that the protein expression levels of STAT3, P-STAT3(S727), P-STAT3(Y705), and MPC1 in A549 and H1299 cell with stable infection of OEMPC1 or Ctrl vector. f The mitochondria was isolated from the cytoplasm, mitochondria (mito) and cytoplasm without mitochondria (cyto) were prepared, western blotting showing that the protein expression levels of P-STAT3(Y705), STAT3, MPC1, and mitochondrial reference COX IV in A549 and H1299 cell with stable infection of OEMPC1 or Ctrl vector. g The nuclear and cytoplasmic extracts were prepared. Western blotting showing that overexpression of MPC1 decreased the expression of P-STAT3(Y705) in the nuclei. h IHC showing the expression of P-STAT3(Y705) in the MPC1 low LAC tissues (n = 11) and MPC1 high (n = 9) LAC tissues, Scale bar = 50 μm. i IHC score of P-STAT3(Y705) in the MPC1 low LAC tissues (n = 11) and MPC1 high LAC tissues (n = 9). Data are expressed as the mean ± SD, *P<0.05   31 . However, robust and stable antitumor STAT3 activity has not been observed in clinical trials 32 . Recent reports showed STAT3 is not only present in the cytoplasm, but also resides in the inner mitochondrial membrane to increases activity of complex I and II of the electron transport chain to regulate cellular respiration in a transcriptional independent manner, and mitochondrial STAT3 sustained glycolytic and oxidative phosphorylation activities of cancer cells [33][34][35] . Additionally, the mitochondrial STAT3 suppresses autophagy induced by oxidative stress and may effectively preserve the mitochondria from being degraded by mitophagy 36 . Gene associated with retinoid interferon-induced cell mortality  can recruit STAT3 by the site of S727A into the mitochondria and enhance the integration of STAT3 involved in celluar respiration and glycolysis 37 . These data indicate that altering the balance between cyto-STAT3 and mito-STAT3, leading to the phosphorylation of STAT3 may be an effective way to combat cancer. Our work identified the interaction between mito-STAT3 and MPC1, which preventing STAT3 distribution in the cytoplasm and decreasing its phosphorylation in the cytoplasm and nuclei.
In summary, our study illustrates an important function of MPC1 in controlling the malignant behavior of LAC and acts as a novel prognostic indicator for LAC patients. Furthermore, we identify that MPC1/STAT3 axis is crucial in the progression of LAC (Fig. 6e), which provides a new train of thought regarding the molecular targeting of STAT3 pathway.