PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

Simple Summary There are still controversies about the roles of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and leucyl-tRNA synthetase 1 (LARS1) in cancer. In this study, we examined whether the effects of PGC-1α on cell proliferation and invasion were mediated by modulation of LARS1. Our results showed that PGC-1α regulated cell proliferation and invasion by regulating the LARS1/AKT/GSK3β/β-catenin axis in human colorectal cancer cells. These data suggest that LARS1 might be a potential therapeutic target for PGC-1α-overexpressing human colorectal cancer. Abstract Although mounting evidence has demonstrated that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) can promote tumorigenesis, its role in cancer remains controversial. To find potential target molecules of PGC-1α, GeneFishingTM DEG (differentially expressed genes) screening was performed using stable HEK293 cell lines expressing PGC-1α (PGC-1α-HEK293). As results, leucyl-tRNA synthetase 1 (LARS1) was upregulated. Western blot analysis showed that LARS1 was increased in PGC-1α overexpressed SW480 cells but decreased in PGC-1α shRNA knockdown SW620 cells. Several studies have suggested that LARS1 can be a potential target of anticancer agents. However, the molecular network of PGC-1α and LARS1 in human colorectal cancer cells remains unclear. LARS1 overexpression enhanced cell proliferation, migration, and invasion, whereas LARS1 knockdown reduced them. We also observed that expression levels of cyclin D1, c-Myc, and vimentin were regulated by LARS1 expression. We aimed to investigate whether effects of PGC-1α on cell proliferation and invasion were mediated by LARS1. Our results showed that PGC-1α might modulate cell proliferation and invasion by regulating LARS1 expression. These results suggest that LARS1 inhibitors might be used as anticancer agents in PGC-1α-overexpressing colorectal cancer. Further studies are needed in the future to clarify the detailed molecular mechanism by which PGC-1α regulates LARS1 expression.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the binding of amino acids to their respective tRNAs and play an important role in the maintenance of cell survival [11]. Among them, leucyl-tRNA synthetase 1 (LARS1) catalyzes the covalent binding of leucine to tRNA Leu during polypeptide synthesis. It also acts as a leucine sensor in activating the mechanistic target of rapamycin complex 1 (mTORC1) [12,13]. mTORC1 is a serine/threonine kinase, integrating signals from amino acids, growth factors, and the energy level of the cells, and plays an important role in cell growth and protein synthesis [14,15]. It can phosphorylate its downstream targets, ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1), regulating protein synthesis [16]. A previous study demonstrated that knockdown of LARS1 inhibits cell migration and colony-forming ability in lung cancer [17]. In addition, it was reported that inhibition of LARS1 may reduce cancer cell proliferation via the p21 signaling pathway and cause apoptosis [18]. In recent, many investigators have focused on LARS1 as a potential target of anticancer agents [18][19][20][21][22][23][24]. However, its role and the molecular mechanism in colorectal cancer are not clearly defined yet.
Until now, the molecular network of PGC-1α and LARS1 in human colorectal cancer cells are not explored. Thus, we aimed to investigate whether PGC-1α actions on cell proliferation and invasion are mediated by LARS1.

RNA Isolation and First-Strand cDNA Synthesis
Total RNA isolation and first-strand cDNA synthesis were performed as described previously [8]. In brief, total RNAs were isolated from pcDNA-HEK293 cells and PGC-1α-HEK293 cells using an RNAeasy mini kit (Qiagen, Valencia, CA, USA). DNase I treatment of total RNAs and reverse transcription were then performed to obtain first-strand cDNAs using GeneFishing TM DEG kits (Seegene, Seoul, Republic of Korea) following the manufacturer's protocol. First-strand cDNAs were diluted by adding 80 µL of ultra-purified water for GeneFishing TM PCR (Seegene, Seoul, Republic of Korea). They were stored at −20 • C until use.

Annealing Control Primer (ACP)-Based GeneFishing PCR
Differentially expressed genes between pcDNA-HEK293 cells and PGC-1α-HEK293 cells were screened using the ACP-based PCR method [25] with GeneFishing DEG kits (Seegene, Seoul, Republic of Korea). In brief, second-strand cDNA synthesis was performed at 50 • C during one cycle of first-stage PCR in a final reaction volume of 20 µL containing 3-5 µL (~50 ng) of diluted first-strand cDNA, 1 µL of dT-ACP2 (10 µM), 1 µL of 10 µM arbitrary ACP, and 10 µL of 2X Master Mix (Seegene, Seoul, Republic of Korea). In total, 120 different types of arbitrary ACPs were used. The PCR condition for second-strand synthesis was one cycle at 94 • C for 1 min, followed by 50 • C for 3 min and 72 • C for 1 min. After second-strand DNA synthesis was completed, the second-stage PCR amplification was performed using 40 cycles of 94 • C for 40 s, followed by 65 • C for 40 s and 72 • C for 40 s and a 5-min final extension at 72 • C. Amplified PCR products were separated on a 2% agarose gel stained with ethidium bromide. Amplified cDNA fragments with >2-fold differential band intensities were re-amplified and extracted from the gel using a GeneClean II kit (Qbiogene, Solon, OH, USA) and directly sequenced with an ABI PRISM 310 Gene Analyzer (Applied Biosystems, Waltham, MA, USA).

Cell Counting
Cells were cultured at a density of 1.5 × 10 5 /well in 6-well plates. pCMV6-, LARS1-2, LARS1-4-SW480 cells, NC shRNA-, LARS1 shRNA-4, and LARS1 shRNA-5 SW620 cells were cultured for 24, 48, and 72 h, respectively. In addition, PGC-1α shRNA-SW620 cells after transfection with LARS1 shRNA or with NC shRNA were seeded at a density of 1.5 × 10 5 /well in 6-well plates and were incubated for 24, 48, or 72 h. Then, the cells were harvested by trypsinization using trypsin/EDTA and stained with trypan blue. A hemocytometer was used to count and calculate the average number of cells in each group. All experiments were repeated three times.

Transwell Migration and Invasion Assays
Transwell migration and invasion assays were performed as previously described [10]. For transwell migration assays, cells (1 × 10 5 ) in serum-free medium were plated into the upper chamber of the inserts (24-well cell culture plate transwell inserts; 353097, Corning Inc., Corning, NY, USA) with 8 µm pore filters and complete medium was added to the lower chamber. After incubation for 48 h, the cells on the upper surface were removed with cotton tips and the cells migrated to the underside of the membrane were fixed with 4% formaldehyde in PBS for 30 min at RT, stained with 0.1% crystal violet (Sigma Aldrich, St. Louis, MO, USA) for 20 min and washed with PBS three times. For transwell invasion assays, cells (1 × 10 5 ) were suspended in 200 µL of serum free medium and plated into the upper chambers of the inserts (24-well cell culture plate transwell inserts; 353097, Corning Inc., Corning, NY, USA) that were pre-coated with 50 µL of Matrigel (1 µg/µL; BD Biosciences, San Jose, CA, USA). The lower chamber had 600 µL of DMEM with 10% FBS added, and the plate was incubated for 48 h at 37 • C. After incubation, the cells on the upper surface were removed with cotton tips and the cells migrated to the underside of the membrane were fixed with 4% formaldehyde in PBS for 30 min at RT, stained with 0.1% crystal violet (Sigma Aldrich, St. Louis, MO, USA) for 20 min and washed with PBS three times. The number of cells was calculated from five random fields at a magnification of ×200, using an inverted microscope (Nikon Eclipse TS100; Nikon, Tokyo, Japan) and was indicated as the average number of cells/field of view. The mean value was calculated form three independent experiments.

Western Blot Analysis
Cell lysis and Western blot analyses were performed as described previously [10]. In brief, cells were lysed and electrophoresed on 8, 10, 12, or 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then blotted to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). Blots were blocked for 1 h in 5% skim milk in PBS at room temperature (RT). They were then probed with the appropriate primary antibody overnight at 4 • C. After washing, blots were probed with horseradish peroxidase-conjugated secondary antibodies for 2 h. After another wash, signals were detected with ECL (Amersham, Buckinghamshire, UK) according to the manufacturer's instructions. Blots were also probed with a monoclonal anti-β-actin antibody, which served as an internal loading control. Bands were quantified using Image Studio Lite Ver 3.1. (LI-COR, Inc., Lincoln, NE, USA).

Immunofluorescence Staining and Fluorescence Quantification
Immunofluorescence staining was performed as previously described [10]. In brief, cells were cultured on Lab-Tek ® Chamber Slides TM (Nalge Nunc, Inc., Rochester, NY, USA), and then fixed with formaldehyde (3%), permeabilized with Triton X-100 (0.3%), and blocked for 30 min with bovine serum albumin (BSA, 5%) at RT. Next, cells were washed with PBS and incubated with a series of primary antibodies as indicated, followed by staining with Alexa Fluor 488 conjugated donkey anti-rabbit IgG (#A-21206, Invitrogen, Carlsbad, CA, USA). Samples were then mounted using glycerol and analyzed using a laser confocal microscope (LSM800; Carl Zeiss, Jena, Germany) at the Neuroscience Translational Research Solution Center (Busan, Republic of Korea). Negative control staining was performed using only secondary antibodies. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA). Mean fluorescence intensity (MFI) for PGC-1α and LARS1 of immunofluorescence images was quantified using Image J software (NIH; v.1.53t). To obtain region of interest (ROI) values of each marker in three different fields, the 'Free Hand Selection' mode was used. In addition, background ROI values were obtained. Then, the background ROI value was subtracted from three measured ROI values. The resulting values were averaged, and statistical analysis was performed.

Statistical Analysis
All statistical analyses were performed with PASWStatistics 18 software (SPSS, Chicago, IL, USA). Data are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and unpaired Student's t-test were used to determine statistical significance. Statistical significance was defined as p < 0.05.

Overexpression of PGC-1α Leads to Upregulation of LARS1 in Human Embryonic Kidney 293 (HEK293) Cells
Previously, we have reported that PGC-1α overexpression promotes cell proliferation and tumorigenesis of HEK293 cells [8]. In order to identify genes that increased proliferation and invasion by PGC-1α overexpression in HEK293 cells, ACP-based GeneFishing PCR was performed. Band densities between PGC-1α-overexpressed HEK293 cells (PGC-1α-HEK293 cells) and empty vector-expressing HEK293 cells (control HEK293 cells) were then compared. One fragment showing >2-fold different densities between two cell lines was observed by densitometric analysis of amplified cDNA fragments ( Figure 1A). The amplified band was eluted from agarose gel, re-amplified, and sequenced. Sequences were used in a BLAST search to identify its gene annotations. BLAST analysis identified LARS1. In order to confirm increased expression of LARS1 in PGC-1α-HEK293 cells, qRT-PCR, Western blot analysis, and immunofluorescence staining were performed. The expression levels of LARS1 mRNA and protein were increased in PGC-1α-HEK293 cells ( Figure 1B,C). These data suggest that PGC-1α may regulate the expression of LARS1.

LARS1 Overexpression Enhances Cell Proliferation, Migration, and Invasion of SW480 Cells
Previously, we observed that the expression of PGC-1α in human colorectal cancer SW620 cells was higher than in SW480 cells [10]. We established stable PGC-1αoverexpressing SW480 cells (PGC-1α-1-and PGC-1α-2-SW480 cells) and examined the mRNA levels of PGC-1α and LARS1 in these cells by qRT-PCR. As shown in Figure 2A, the expression levels of PGC-1α and LARS1 were significantly increased in PGC-1α-1-and -2 -SW480 cells. As we observed increased expression of LARS1 in PGC-1α-HEK293 cells, we examined expression levels of LARS1 in SW480 and SW620 cells. As expected, the expression level of LARS1 in SW620 cells was higher than in SW480 cells (Figure 2A). To investigate whether LARS1 expression affected cell proliferation, migration, and invasion of SW480 cells, we established stable LARS1-overexpressing SW480 cells (LARS1 low expression) and confirmed that LARS1 expression was increased in stable LARS1-overexpressing SW480 cells (LARS1-2-, -4-SW480 cells) by Western blot analysis (Figure 2A). We then examined cell proliferation by performing cell counting and MTT assay. As shown in Figure 2A,C, LARS1 overexpression enhanced cell proliferation in a time-dependent manner. In addition, results of transwell migration and invasion assays showed that numbers of migrating and invading LARS1-SW480 cells were significantly higher than those of control SW480 cells ( Figure 2D,E). β-actin is used as an endogenous control. Middle and Right: The protein levels of PGC-1α and LARS1 in control pcDNA-HEK293 and PGC-1α-HEK293 cells. Protein lysates were prepared and subjected to Western blot analysis as described in Materials and Methods. Equal protein loading was ensured by showing uniform β-actin expression. Densitometry results are indicated above the bands. Representative data of three independent experiments are shown. Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, pcDNA-HEK293 cells. (C,D) Left panel: Immunofluorescence staining was performed as described in Materials and Methods using anti-PGC-1α ((C), green) and anti-LARS1 ((D), green) antibodies. Right panel: Mean Fluorescence Intensity (MFI) of PGC-1α (C) and LARS1 (D) of immunofluorescence images were quantified using Image J software (v.1.53t). GP, arbitrary general primer; 1, control HEK293 cells; 2, PGC-1α-HEK293 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images ( Figure S3).

LARS1 Knockdown Reduces Cell Proliferation, Migration, and Invasion of SW620 Cells
To confirm whether the LARS1 expression is regulated by PGC-1α expression in SW620 cells, we examined the mRNA levels of PGC-1α and LARS1 in PGC-1α shRNA-1-, -2-SW620 cells by qRT-PCR. As shown in Figure 3A, the expression levels of PGC-1α and LARS1 mRNAs were decreased in PGC-1α shRNA-1-and -2-SW620 cells. Since we observed that SW620 cells highly expressed LARS1 (Figure 2A), we established stable LARS1 shRNA-knocked down SW620 cells (LARS1 shRNA-4, -5-SW620 cells) and then confirmed that LARS1 expression was reduced in these stable LARS1 shRNA-knocked down SW620 cells by Western blot analysis ( Figure 3A). However, PGC-1α expression levels in LARS1 shRNA-4-SW620 and LARS1 shRNA-5-SW620 cells were not changed compared to those in NC shRNA-SW620 cells, suggesting that PGC-1α acted upstream of LARS1. We then examined cell proliferation by performing cell counting and MTT assay. As shown in Figure 3B,C, LARS1 knockdown decreased cell proliferation in a time-dependent manner. In addition, results of transwell migration and invasion assays showed that numbers of migrating and invading cells of LARS1 shRNA-SW620 cells were significantly lower than those of NC shRNA-SW620 cells ( Figure 3D,E).

PGC-1α
Regulates Cell Proliferation, Migration, and Invasion of PGC-1α-HEK293, SW480, and SW620 Cells by Regulating LARS1 Expression Based on the above observations, we hypothesized that PGC-1α could regulate cell proliferation, migration, and invasion through the modulation of LARS1 expression. To test this hypothesis, we examined the cell proliferation, migration, and invasion of PGC-1α-HEK293 and PGC-1α-1-SW480 cells by transfecting NC shRNA or LARS1 shRNA, respectively. As shown in Supplementary Figure S1A and Figure 4A, the cell proliferation of PGC-1α-HEK293 and PGC-1α-1-SW480 cells by NC shRNA transfection was higher than that of pcDNA-HEK293 and control SW480 (pcDNA-SW480) cells, respectively. The increase in cell proliferation of PGC-1α-HEK293 and PGC-1α-1-SW480 cells was decreased by LARS1 shRNA transfection, respectively. In addition, cell numbers of migrating and invading cells of PGC-1α-HEK293 and PGC-1α-1-SW480 cells after NC shRNA transfection were higher than those of pcDNA-HEK293 and control SW480 (pcDNA-SW480) cells, respectively. However, such increases of migrating and invading cells of PGC-1α-HEK293 and PGC-1α-1-SW480 cells were decreased by LARS1 shRNA transfection, respectively (Supplementary Figure S1B and Figure 4B,C). As shown in Figure 4D, cell proliferation of PGC-1α shRNA-1-SW620 cells after pCMV6 transfection was decreased compared to that of control SW620 (NC shRNA-SW620) cells. However, such a decrease in cell proliferation of PGC-1α-shRNA-1-SW620 cells was reversed by LARS1 transfection. In addition, numbers of migrating and invading cells of PGC-1α-shRNA-1-SW620 cells after pCMV6 transfection were decreased compared to those of control SW620 (NC shRNA-SW620) cells. However, such decreases in migrating and invading cells of PGC-1α-shRNA-1-SW620 cells were increased by LARS1 transfection (Figure 4E,F). As shown in Figure 4G, cell proliferation of PGC-1α-shRNA-1-SW620 cells after NC shRNA transfection was decreased compared to that of control SW620 (NC shRNA-SW620) cells. However, such a decrease in cell proliferation of PGC-1α shRNA-1-SW620 cells was not changed by LARS1 shRNA transfection.

Discussion
There are still controversies about the role of PGC-1α in cancer, although intensive studies have been performed. Its actions and the underlying molecular mechanisms are very complicated depending on the cell type, its posttranslational modifications (such as phosphorylation, acetylation, ubiquitination, and so on), and the presence of its interacting proteins [4]. Our previous study has revealed that PGC-1α functions as a tumor promoter through the activation of AKT/GSK-3β/β-catenin signaling [10]. However, we did not clarify the underlying mechanism by which PGC-1α regulated the AKT/GSK-3β/β-catenin pathway, although we observed a direct interaction of PGC-1α and AKT by co-immunoprecipitation. In the present study, we identified LARS1 as one of candidate downstream targets of PGC-1α by using GeneFishing TM DEG (Seegene, Seoul, Republic of Korea). LARS1 mRNA and protein expressions were increased in PGC-1α-HEK293, PGC-1α-1-, and -2-SW480 cells and in HT-29 cells by PGC-1α transfection, whereas the expressions of LARS1 mRNA and protein were decreased in PGC-1α shRNA-1-and -2-SW620 cells and in SNU-C4 cells by PGC-1α shRNA transfection. These results prompted us to hypothesize that the effects of PGC-1α on cell proliferation, migration, and invasion might be mediated by LARS1 expression. In order to investigate this possibility, first we established stable LARS1-overexpressing SW480 cells (LARS1-SW480; LARS1-2-and -4-SW480) and stable LARS1 shRNA-knocked down SW620 (LARS1 shRNA-SW620; LARS1 shRNA-4and -5-SW620) cells and found that LARS1-SW480 cells showed higher cell proliferation, migration, and invasion than pCMV6-SW480 cells. In addition, LARS1 shRNA-SW620 cells showed less cell proliferation, migration, and invasion than NC shRNA-SW620 cells. These findings are consistent with previous reports showing that LARS1 expression is related to the growth and migration of lung cancer cells, is essential for survival in osteosarcoma, and is required for cell proliferation in tuberous sclerosis complex (TSC)-null cells [17,29,30]. However, we did not examine the expression of TSC in SW480 or SW620 cells. Further studies are needed to investigate the expression of TSC1/2 in SW480 and SW620 cells. Very surprisingly, in contrast to our results, Passarelli et al. have demonstrated that LARS1 has a tumor suppressive effect by repressing the codon-dependent translation of epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5) in breast cancer [31]. These results implicate that the effects of LARS1 on tumor progression are variable depending on the cellular context.
As previously mentioned, the effects of LARS1 on cell proliferation and invasion are very similar to those of PGC-1α. Our mechanistic studies showed that LARS1 was required for effects of PGC-1α on cell proliferation and invasion using LARS1 shRNA knockdown or LARS1 overexpression in PGC-1α-SW480 cells and PGC-1α shRNA-SW620 cells, respectively. In addition, decreased cell proliferation and invasion in PGC-1α shRNA-SW620 cells were not changed by LARS1 shRNA transfection. These results support the hypothesis that PGC-1α regulates cell proliferation and invasion via LARS1. Interestingly, we have found that the LARS1 promoter/enhancer has a Sp1 binding site using GeneCards (http://genecards.org; accessed on 22 December 2022), and we have previously shown that PGC-1α enhances cell proliferation and tumorigenesis of HEK293 cells through the upregulation of Sp1 and ACBP [8]. This suggests that LARS1 expression is likely to be regulated by PGC-1α via Sp1. In the near future, we will investigate whether PGC-1α increases the expression of LARS1 via the binding of Sp1 to the promoter of LARS1. Activation of the AKT/GSK-3β/β-catenin axis, mTOR, S6K1, and 4EBP1 was observed in both PGC-1α-SW480 and LARS1-SW480 cells, whereas inhibition of those molecules was observed in both PGC-1α shRNA-and LARS1 shRNA-SW620 cells.
GSK-3β is a negative regulator of β-catenin [32]. Increasing evidence has shown that GSK-3β is a downstream molecule of AKT. It can be inactivated by AKT-mediated phosphorylation at its Ser9 residue [33][34][35]. Previous studies and the present results demonstrated that PGC-1α/LARS1 overexpression upregulated whereas PGC-1α/LARS1 knockdown downregulated phosphorylated (activated) AKT and phosphorylated (inactivated) GSK-3β levels. Furthermore, inhibition of AKT by AKT inhibitor IV in LARS1-SW480 cells reversed the ability of LARS1 to induce GSK-3β inactivation, β-catenin pathway activation, cell proliferation, migration, and invasion. These data reveal that LARS1-mediated AKT activation can lead to inactivation of GSK-3β and consequent β-catenin pathway activation. The present study suggests that AKT activation is required for increased cell proliferation and invasion by LARS1 overexpression. However, our results showed that LARS1 activated ATK, different from other studies demonstrating that LARS1 expression is inversely related to p-AKT in non-small cell lung cancer cell lines and tissues [19] and that LARS1 inhibits myogenic differentiation through the Rag-mTORC1 pathway and subsequent inhibition of the IRS1-PI3K-AKT pathway [36]. These differential effects of LARS1 on the activation of AKT are dependent on the cellular context.
Our previous study has demonstrated that decreased cell proliferation and invasion by PGC-1α knockdown are reversed by constitutive AKT expression [10]. Additionally, our present findings suggest that PGC-1α acts upstream of LARS1. Altogether, these results implicate that PGC-1α regulates cell proliferation and invasion through modulation of the LARS1/AKT/GSK3β/β-catenin axis and that LARS1 might be a potential therapeutic target for PGC-1α-overexpressing human colorectal cancer as depicted in a schematic diagram ( Figure 7). However, our study has some limitations. First, we did not evaluate the in vivo effect of the PGC-1α/LARS1 axis on tumor progression of human colorectal cancer. Thus, further research is warranted in the context of using in vivo models to evaluate whether modulation of LARS1 expression by LARS1 overexpression or LARS1 knockdown can reverse PGC-1α actions on tumor progression. Second, the underlying molecular mechanisms by which PGC-1α regulates LARS1 and how LARS1 regulates AKT were not clarified in this study. Thus, further exploration is needed to dissect its detailed mechanisms. Third, we did not evaluate the crosstalk between mTORC1 and GSK-3β in this study. Further studies about the molecular network between mTORC1 and GSK-3β are required in the future. Figure 7. Potential molecular mechanism by which PGC-1α regulates cell proliferation, migrati and invasion of human colorectal cancer cells. In summary, PGC-1α regulates cell proliferation, gration, and invasion via regulation of LARS1/AKT/GSK-3β/β-catenin axis.

Conclusions
To the best of our knowledge, this is the first report showing that PGC-1α regula cell proliferation and invasion through modulation of LARS1 expression. Further stud evaluating the clinical significance of the PGC-1α/LARS1/AKT/GSK-3β/β-catenin axis human colorectal cancer patients and therapeutic potentials of several LARS1 inhibit in PGC-1α-overexpressing colorectal cancer are needed.

Conclusions
To the best of our knowledge, this is the first report showing that PGC-1α regulates cell proliferation and invasion through modulation of LARS1 expression. Further studies evaluating the clinical significance of the PGC-1α/LARS1/AKT/GSK-3β/β-catenin axis in human colorectal cancer patients and therapeutic potentials of several LARS1 inhibitors in PGC-1α-overexpressing colorectal cancer are needed.