Aldolase A Accelerates Cancer Progression by Modulating mRNA Translation and Protein Biosynthesis via Noncanonical Mechanisms

Abstract Aldolase A (ALDOA), a crucial glycolytic enzyme, is often aberrantly expressed in various types of cancer. Although ALDOA has been reported to play additional roles beyond its conventional enzymatic role, its nonmetabolic function and underlying mechanism in cancer progression remain elusive. Here, it is shown that ALDOA promotes liver cancer growth and metastasis by accelerating mRNA translation independent of its catalytic activity. Mechanistically, ALDOA interacted with insulin‐ like growth factor 2 mRNA‐binding protein 1 (IGF2BP1) to facilitate its binding to m6A‐modified eIF4G mRNA, thereby increasing eIF4G protein levels and subsequently enhancing overall protein biosynthesis in cells. Importantly, administration of GalNAc‐conjugated siRNA targeting ALDOA effectively slows the tumor growth of orthotopic xenografts. Collectively, these findings uncover a previously unappreciated nonmetabolic function of ALDOA in modulating mRNA translation and highlight the potential of specifically targeting ALDOA as a prospective therapeutic strategy in liver cancer.

The primers were listed in Table S5.All constructs were verified by DNA sequencing.

RNA extraction and RT-qPCR analysis
Total RNA from HCC cell lines, animal tumor tissues as well as clinical tissue specimens was isolated using TRIzol (Life Technologies, CA, USA) following the manufacturer's instructions.Subsequently, 5 μg of total RNA were reverse-transcribed using the PrimeScript RT Reagent kit (TaKaRa, Tokyo, Japan) at 37°C for 20 min followed by 85°C for 30s and stored at -20°C.Real-time qPCR was performed using SYBR ® Green Pro Taq HS (Accurate Biology, Changsha, China) in 7900 Real-Time PCR apparatus (Applied Biosystems, USA).ACTB was used as internal control for normalization.Primers for RT-qPCR are shown in Table S5.

Western blot analysis
Cells were lysed in RIPA buffer containing protease inhibitor cocktail and PMSF (Beyotime, Shanghai, China).Protein extracts were then separated by 10% SDS-PAGE, transferred to NC membranes followed by blocked with 5% non-milk powder in TBST.After probing with specific primary antibodies overnight at 4 °C, the membranes were washed with TBST, and then incubated in HRP-conjugated secondary antibodies in room temperature for 40 min.The membranes were visualized using the Omni-ECL TM Reagent kit (Epizyme, Shanghai, China).
Antibodies used in this study are provided in Table S7.

Cell proliferation, migration and invasion assay
Cell Counting Kit-8 (CCK-8) (MCE, Shanghai, China) and colony formation assay were used to assess cell proliferation ability.For CCK-8 assay, 2000 cells were seeded in 96-well plate.After attachment, cells were replaced with fresh medium containing 10% CCK-8 reagent, and cell viability was measured at 450 nm absorbance for indicated time points.For colony formation assay, 3000 cells were seeded in six-well plate and cultured for 7-10 days.The colonies were subsequently stained with 1% crystal violet solution and quantified using ImageJ analysis software.
The migration assay was performed using transwell chamber system (8 μm pores, Corning, USA).Briefly, 3 × 10 4 cells with different treatments were seeded in the upper chamber of an insert with 200 μl serum-free medium in 24-well plate, 600 μl DMEM medium containing 20% FBS were added to the lower chamber.After incubation at 37 °C for 24 h, the migrated cells were stained with 1% crystal violet dye and counted in four randomly selected fields with Inverted Microscopes (10×) (Olympus, Japan).The invasion assay was implemented similarly, with coating the filters with Matrigel.

Immunohistochemistry
Immunohistochemistry was conducted as the following procedure.In short, paraffin embedded slides were dewaxed in xylene followed by rehydrated in graded ethanol and distilled water sequentially.Slides were immersed in 0.3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, and blocked with 5% BSA for 20 min at room temperature.After probed with indicated primary antibodies overnight at 4 °C, the slides were subsequently washed with PBS triplicates.Next, sections were incubated with HRP-conjugated secondary antibodies at 37 °C for 30 min.Slides were then exhibited by DAB staining.The antibodies used for IHC staining are displayed in Table S7.

Dual-luciferase reporter assay
The 3'UTR sequence of eIF4G mRNA was inserted into the downstream of the Firefly luciferase coding region of the dual-luciferase reporter vector pmirGLO (Promega, USA) to construct the pmirGLO-eIF4G-3'UTR WT or pmirGLO-eIF4G-3'UTR Mut plasmid, respectively.IGF2BP1 knockout or corresponding control cells were seeded in 24-well plate and transfected with 500 ng pmirGLO-eIF4G-3'UTR WT, pmirGLO-eIF4G-3'UTR Mut or pmirGLO control plasmids.After 48 h, cells were harvested and the luciferase activity was measured employing Dual Luciferase Reporter Assay Kit (Promega, USA).The sequences inserted into the pmirGLO vector are displayed in Table S5.

Immunofluorescence staining
HCC cells with indicated treatments were seeded in eight-well chamber slides followed by fixation with 4% paraformaldehyde for 15 min at room temperature.
After washing with PBS twice, cells were permeabilized with 0.25% Triton X-100 in PBS for 15 min, and then blocked in Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min.After that, cells were incubated with primary antibodies overnight at 4 °C.Next day, cells were incubated with fluorescence-labeled secondary antibodies in PBS at room temperature for 1 h.Nuclei were stained by DAPI (Beyotime, Shanghai, China).Images were acquired with confocal microscopy (Leica, Mannheim, Germany).

Aldolase activity assay
Aldolase activity was assessed employing Aldolase Activity Colorimetric Assay Kit (BioVision, USA) following the supplier's instructions.Briefly, 1 × 10 6 HCC cells were lysed in 100 µl ice cold Aldolase Assay Buffer.After centrifugation at 10000 g for 5 min, the supernatant was collected, and added into 96-well plate with reaction mix (aldolase assay buffer, aldolase enzyme mix, aldolase developer and aldolase substrate).Absorbance at 450 nm was measured after reaction for 30 min at 37°C.

Measurement of ECAR, glucose consumption and lactate production
The glycolytic rate was examined by using XF Glycolysis Stress Test Kit (Agilent, USA).In brief, HCC cells (initial density of 2 × 10 4 cells/well) with indicated treatments were seeded into XF96-well plate.Next day, cells were changed with XF DMEM Base Medium followed by sequential injection of 10 × 10 -3 M glucose, 1 × 10 -6 M oligomycin and 50 × 10 -3 M 2-deoxyglucose (2-DG).After incubated in a CO 2 -free incubator for 1 h at 37 °C, cells were subjected to extracellular acidification rate analysis with Seahorse XF-96 Wave software.
Glucose uptake assay was conducted using Glucose Colorimetric Assay Kit (BioVision, USA).Briefly, cells were seeded in six-well plate, the culture medium was collected and mixed with glucose assay buffer coupled to glucose reaction buffer in a 96-well plate.After incubated at room temperature for 30 min, the absorbance was measured at 450 nm.For lactate production assay, cells were collected and resuspended in lactate assay buffer followed by centrifugation at 12000 g for 5 min at 4 °C.The concentration of lactate was then measured employing CheKine™ Micro Lactate Assay Kit (Abbkine, Wuhan, Hubei) according to the manufacture's procedure.

Bioinformatics analysis
The single gene loss-of-functions were calculated from Project Achilles.We collected the proliferation score of liver cancer cell lines for the individual metabolic enzyme genes knockout condition from CRISPR knockout screens (DepMap Portal: https://depmap.org/portal/).The gene-level scores were generated using CERES, and normalized per cell line so that nonessential genes have a median score of 0 and independently identified common essentials have a median score of -1.Negative scores imply cell growth inhibition following gene knockout. [1]ALDOA mRNA level was analyzed based on TCGA_LIHC dataset ( http://www.tcga-data.nci.nih.gov) and NCBI GEO database (GSE25097, GSE144269, GSE45436, GSE76427, and GSE64041).The survival analysis of ALDOA in TCGA_LIHC and CHCC_HBV was performed by GraphPad Prism 8.0 software.GO enrichment analysis was performed using online tools (DAVID: https://david.ncifcrf.gov/).IGF2BP1 eCLIP-seq data was download from ENCODE database (https://www.encodeproject.org/).