TRAP1 modulates mitochondrial biogenesis via PGC-1α/TFAM signalling pathway in colorectal cancer cells

Abstract

Metabolic rewiring promotes cancer cell adaptation to a hostile microenvironment, representing a hallmark of cancer. This process involves mitochondrial function and is mechanistically linked to the balance between mitochondrial biogenesis (MB) and mitophagy. The molecular chaperone TRAP1 is overexpressed in 60-70% of human colorectal cancers (CRC) and its over-expression correlates with poor clinical outcome, being associated with many cancer cell functions (i.e. adaptation to stress, protection from apoptosis and drug resistance, protein synthesis quality control, metabolic rewiring from glycolysis to mitochondrial respiration and vice versa). Here, the potential new role of TRAP1 in regulating mitochondrial dynamics was investigated in CRC cell lines and human CRCs. Our results revealed an inverse correlation between TRAP1 and mitochondrial-encoded respiratory chain proteins both at transcriptional and translational levels. Furthermore, TRAP1 silencing is associated with increased mitochondrial mass and mitochondrial DNA copy number (mtDNA-CN) as well as enhanced MB through PGC-1α/TFAM signalling pathway, promoting the formation of new functioning mitochondria and, likely, underlying the metabolic shift towards oxidative phosphorylation. These results suggest an involvement of TRAP1 in regulating MB process in human CRC cells. KEY MESSAGES: TRAP1 inversely correlates with protein-coding mitochondrial gene expression in CRC cells and tumours. TRAP1 silencing correlates with increased mitochondrial mass and mtDNA copy number in CRC cells. TRAP1 silencing favours mitochondrial biogenesis in CRC cells.

Keywords: Colorectal cancer; Metabolism; Mitochondrial biogenesis; Peroxisome proliferation-activated receptor gamma coactivator α1-alpha; TNF receptor-associated protein 1; Transcription factor A mitochondrial.

Fig. 1

Inverse transcriptional correlation between TRAP1 and protein-coding mt-genes in human CRC HCT116 cells and colon adenocarcinoma tissues. a RT-qPCR analysis of protein-coding 13 mt-genes in HCT116 cells transiently transfected with TRAP1 (siTRAP1) or negative control (siNEG) siRNAs. The relative normalized expression is the mean of two independent experiments (± SEM) and p-values indicate statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001). Insert: western blot and densitometric analysis of TRAP1 protein expression in TRAP1-silenced and control HCT116 cells. b Boxplot graphs of TRAP1 (left graph) and protein-coding 13 mt-genes signature (right graph) mRNA expression comparing human normal colorectal mucosa (n = 349) and colorectal cancer (n = 275) using multiple dataset COAD matched with TCGA normal and GTEx data in GEPIA2 tool (|Log₂FC| cutoff = 1; p-value cutoff = 0.01). c Correlation analysis graph between TRAP1 and protein-coding 13 mt-genes transcript levels using TCGA COAD tumour dataset of GEPIA2. Pearson’s linear correlation was applied and p-value indicates statistically significant differences (p < 0.05)

Fig. 2

TRAP1 conversely correlates with mitochondria-encoded proteins in human CRC primary tumours and HCT116 cells. a Jitter plots of TRAP1 and 5 mt-genes protein expression comparing colorectal primary tumours (n = 97) and normal colorectal mucosa (n = 100) using CPTAC dataset of UALCAN web resource. p-value indicates statistically significant differences (*p < 0.05). b Western blot analysis of TRAP1 and mitochondrial proteins ATP8, COX2, ND1 and ND4L in HCT116 cells silenced or not for TRAP1 (upper panel). Densitometric analysis reports the mean of two independent experiments (± SD) and p-value indicates statistically significant differences (lower panel) (*p < 0.05, **p < 0.01, ***p < 0.001). GAPDH, β-ACTIN and ATP synthase β were used as housekeeping genes for protein expression normalization

Fig. 3

TRAP1 silencing correlates with increased mitochondrial mass and mt-DNA CN in human CRC HCT116 cells. a Confocal images of mitochondria from HCT116 cells silenced or not for TRAP1 (left panel): mitochondria stained (red) with MitoTracker, TRAP1 (green) stained with FITC and nuclei (blue) stained with DAPI. Statistical representation of FITC and MitoTracker percentage of fluorescence (mean ± SD) of three acquisition fields (right panel) and p-values indicate statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001). b qPCR analysis of the relative mtDNA/nDNA ratio in HCT116 cells silenced or not for TRAP1. The relative ratio is the mean of two independent experiments (± SEM) and p-values indicate statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

TRAP1 silencing favours MB via PGC1-α/TFAM axis in human CRC HCT116 cells. a Western blot analysis of TRAP1, PGC1-α, TFAM, ERK1/2 and phospho-ERK1/2 (p-ERK) proteins in HCT116 cells silenced or not for TRAP1. Densitometric analysis histogram (on the right) reports the mean of two independent experiments (± SD) and p-value indicates statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001). GAPDH, β-ACTIN and α-TUBULIN were used as housekeeping genes for protein expression normalization. b Western blot of TRAP1 and TFAM expression in both mitochondrial and cytosolic compartments in HCT116 cells silenced or not for TRAP1. Densitometric analysis histogram (below) reports the mean of two independent experiments (± SD) and p-value indicates statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001). ATP synthase β was used as mitochondrial housekeeping genes for protein expression normalization and β-ACTIN as a control of the cytosolic fraction. c Western blot analysis of TFAM, TRAP1 and p-ERK1/2 proteins in HCT116 cells silenced for TRAP1 and/or TFAM. Densitometric analysis reports the mean of two independent experiments (± SD) and p-value indicates statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001). α-TUBULIN was used as housekeeping genes for protein expression normalization. d qPCR analysis of the relative mtDNA/nDNA ratio in HCT116 cells silenced for TRAP1 and/or TFAM. The relative ratio is the mean of two independent experiments (± SEM) and p-values indicate statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001)