Downregulation of TFAM inhibits the tumorigenesis of non-small cell lung cancer by activating ROS-mediated JNK/p38MAPK signaling and reducing cellular bioenergetics

Abstract

Mitochondrial transcription factor A (TFAM) is essential for the replication, transcription and maintenance of mitochondrial DNA (mtDNA). The role of TFAM in non-small cell lung cancer (NSCLC) remains largely unknown. Herein, we report that downregulation of TFAM in NSCLC cells resulted in cell cycle arrest at G1 phase and significantly blocked NSCLC cell growth and migration through the activation of reactive oxygen species (ROS)-induced c-Jun amino-terminal kinase(JNK)/p38 MAPK signaling and decreased cellular bioenergetics. We further found that TFAM downregulation in NSCLC cells led to increased apoptotic cell death and enhanced the sensitivity of NSCLC cells to cisplatin. Tissue microarray (TMA) data showed that elevated expression of TFAM was related to the histological grade and TNM stage of NSCLC patients. We also demonstrated that TFAM is an independent prognostic factor for overall survival of NSCLC patients. Taken together, our findings suggest that TFAM could serve as a potential diagnostic biomarker and molecular target for the treatment of NSCLC, as well as for prediction of the effectiveness of chemotherapy.

Keywords: TFAM; cellular bioenergetics; chemosensitivity; mitochondria; non-small cell lung cancer.

Figure 1. TFAM knockdown inhibits NSCLC cell proliferation and tumor growth

(A) Upper panel: Western blot analysis demonstrating TFAM stable knockdown in the indicated NSCLC cells transfected with shRNA specific to TFAM mRNA. Lower panel: Real-time qPCR analysis of TFAM mRNA in the indicated TFAM stable knockdown NSCLC cells transfected with shRNA specific to TFAM mRNA. Representative data were shown from three independent experiments. Data are shown as mean ± SD (n = 3, **p < 0.01). (B) At 48 hr after incubation at 37°C in a CO₂ incubator, TFAM stable knockdown and vector control NSCLC cells were harvested and stained with PI (50 μg/ml) solution. Cell cycle arrest was analyzed with a BD Accuri^™ C6 flow cytometer. Results are representative of three independent experiments. (C) Cell proliferation of stable TFAM knockdown NSCLC A549 (left panel) and H460 (right panel) cells, measured by cell number counting. The data are presented as mean ± SD (n = 3, ***p < 0.001). (D) Representative images of colony formation assay of control and TFAM knockdown stable NSCLC cell lines A549 and H460 (left panel). The graphs represent the mean ± SD of at least three independent colony formation assays each performed in triplicate (middle and right panels). (E) Representative images of transwell migration assay of NSCLC A549 (upper panel) and H460 (lower panel) cells. Migrated cells were stained with crystal violet, photographed and counted. Data are presented as mean ± SD of at least three independent experiments. (F) Representative dissected tumors and TFAM expression in tumor tissue lysates are shown.

Figure 2. TFAM knockdown promotes ROS production and apoptosis of NSCLC cells

(A) TFAM stable knockdown in NSCLC A549 cells (left) and H460 cells (right) increases p53, p-p53(Ser15), p21, p-JNK, Bax and p-p38 expression, as well as the cleavage of PARP, caspase 3 and caspase 9. (B) TFAM stable knockdown in NSCLC A549 and H460 cells increases caspase-3 activity. The data are presented as mean ± SD. n = 3, *p < 0.05; **p < 0.01. (C) TFAM stable knockdown reduces mitochondrial membrane potential (MMP) of NSCLC A549 and H460 cells. Cells were stained with JC-1 and analyzed by flow cytometry. The ratio of fluorescence intensities Ex/Em = 490/590 and 490/530 nm (FL590/FL530) were recorded to show the MMP level of each sample. Data are presented as mean ± SD. n = 3, *p < 0.05; **p < 0.01, ***p < 0.001. (D) TFAM stable knockdown increases intracellular ROS (H₂O₂) production in NSCLC A549 and H460 cells measured by Reactive Oxygen Species Assay Kit (DCFH-DA), and pre-treatment of cells with NAC (4 mM) for 48 hr resulted in reduction of intracellular ROS (H₂O₂) levels. Data are plotted as percentage of increase in median fluorescence intensity (MFI) and shown as mean ± SD (n = 3, *p < 0.05, **p < 0.01). (E) Mitochondrial superoxide levels of control and TFAM knockdown stable NSCLC A549 and H460 cells were detected by MitoSox staining and analyzed by flow cytometry. Pre-treatment of cells with NAC (4 mM) for 48 hr resulted in reduction of mitochondrial ROS production. Data are plotted as percentage of alteration in mean fluorescence intensity (MFI) and shown as mean ± SD (n = 3, *p < 0.05, **p < 0.01). (F) TFAM stable knockdown increases apoptosis rate of NSCLC A549 and H460 cells, and the pre-treatment of cells with NAC (4 mM) for 48 hr attenuates the apoptosis rate. The data shown represent results from three independent experiments (*p < 0.05; **p < 0.01). (G) Immunoblot detecting expression of PARP, cleaved PARP, p38, p-p38, JNK, p-JNK in lysates from control and TFAM stable knockdown NSCLC cells, as well as from cells treated with NAC (4 mM) for 48 hr. β-actin was used as loading control.

Figure 3. TFAM knockdown enhances chemosensitivity of NSCLC cells by facilitating ROS induced caspase-dependent apoptosis

(A) TFAM stable knockdown NSCLC A549 (upper) and H460 (lower) cells were treated with the indicated concentration of cisplatin. At 24 hr post-cisplatin or -saline treatment, cell viability was assayed by CCK-8. The data are presented as mean ± SD (n = 3, *p < 0.05; **p < 0.01; ***p < 0.001). (B) Western blot analysis of TFAM, PARP, cleaved PARP, caspase 9, cleaved caspase 9, caspase 3 and cleaved caspase 3 in lysates from control and TFAM stable knockdown NSCLC A549 and H460 cells treated with indicated concentration of cisplatin for 24 hr. β-actin was used as loading control. (C) The effects of TFAM knockdown on intracellular ROS (H₂O₂) production induced by cisplatin (10 μg/ml) in NSCLC A549 (left) and H460 (right) cells with or without NAC (4 mM) pre-treatment for 48 hr. The intracellular ROS (H₂O₂) was measured by ROS assay kit (DCFH-DA). Data are presented as mean ± SD (n = 3, *p < 0.05; **p < 0.01; ***p < 0.001). (D) The effects of TFAM knockdown on mitochondrial ROS (superoxide) production induced by cisplatin (10 μg/ml) in NSCLC A549 (left) and H460 (right) cells with or without NAC (4 mM) pre-treatment for 48 hr. The mitochondrial ROS (superoxide) was detected by MitoSox staining and analyzed by flow cytometry. Data are presented as mean ± SD (n = 3, **p < 0.01; ***p < 0.001). (E) TFAM stable knockdown increases cisplatin (10 μg/ml)-induced apoptosis rate of NSCLC A549 and H460 cells with or without NAC (4 mM) pre-treatment for 48 hr. Data represent results of three independent experiments (**p < 0.01; ***p < 0.001).

Figure 4. TFAM knockdown inhibits mitochondrial respiration and glycolysis in NSCLC cells

(A) Mitochondrial respiration profile of TFAM stable knockdown NSCLC A549 and H460 cells. The intact cellular oxygen consumption rate (OCR) was measured in real time using the Seahorse XF96 Extracellular Flux Analyzer. Basal OCR were measured at three time points, followed by sequential injection of the ATP synthase inhibitor oligomycin (1 μM), the uncoupler FCCP (1 μM), the complex I inhibitor rotenone (1 μM) and complex III inhibitor antimycin A (1 μM). The representative graph represents the mean OCR ± SD of six replicates. (B) Extracellular acidification rate (ECAR) was detected by the Seahorse XF96 Extracellular Flux Analyzer. Injection order: Glucose (10 mM), Oligomycin (1 μM), 2-DG (100 mM). (C and D) Basal and maximal respiration (OCR) of A549 and H460 cells transfected with control and TFAM shRNA. (E and F) Basal glycolytic rate and spare glycolytic capacity were analyzed by overall ECAR in control and TFAM knockdown cells. (G) ATP production of control (shCont) and shTFAM groups were calculated by the OCR of baseline minus oligomycin treatment. Data are presented as mean ± SD (n = 6, ***p < 0.001).

Figure 5. TFAM overexpression in NSCLC is closely associated with poor outcomes

(A) Representative western blots (left panel) are shown of TFAM protein expression in NSCLC tumor adjacent normal (N) and matched tumor tissues (C) (n = 30). Relative TFAM protein expression of western blotting analyses (middle panel) were quantified using Image J and normalized to the β-actin internal control (n = 30, ***p < 0.001). TFAM mRNA in NSCLC tumor adjacent normal (N) and matched tumor tissues (C) were examined by qRT-PCR (right panel, n = 30, ***p < 0.001). Expression levels of TFAM protein and mRNA are shown as mean ± SD. (B) Immunohistochemical (IHC) staining of TFAM protein in NSCLC tumor tissues. Representative TFAM IHC staining photomicrographs (400 ×) of non-cancerous, Grade 1 (G1), Grade 2 (G2) and Grade 3 (G3) NSCLC tumor tissues (upper panel), and non-cancerous, TNM stage I, II and III NSCLC tumor tissues (lower panel), are shown. (D) Kaplan-Meier overall survival curve of NSCLC cancer patients (n = 150) according to TFAM protein expression (p = 0.0008).

Figure 6. Proposed model of TFAM in modulating JNK/p38 MAPK signaling and cellular bioenergetics

TFAM protein expression is critical for NSCLC cancer cell proliferation and tumor growth. Downregulation of TFAM depolarizes the mitochondrial membrane potential, leading to reduced cellular bioenergetics, and increases ROS generation that activates ROS-mediated JNK/p38 MAPK, p53/p-p53 (ser15)/p21 signaling and attenuates cancer cell proliferation and tumor growth.