Organoid modeling identifies USP3-AS1 as a novel promoter in colorectal cancer liver metastasis through increasing glucose-driven histone lactylation

Abstract

Dysregulation of long non-coding RNAs (lncRNAs) is common in colorectal cancer liver metastasis (CRLM). Emerging evidence links lncRNAs to multiple stages of metastasis from initial migration to colonization of distant organs. In this study we investigated the role of lncRNAs in metabolic reprogramming during CRLM using patient-derived organoid (PDO) models. We established five pairs of PDOs from primary tumors and matched liver metastatic lesions, followed by microarray analysis. We found that USP3-AS1 was significantly upregulated in CRLM-derived PDOs compared to primary tumors. High level of USP3-AS1 was positively associated with postoperative liver metastasis and negatively correlated with the prognosis of colorectal cancer (CRC) patients. Overexpression of USP3-AS1 significantly enhanced both sphere formation efficiency and liver metastasis in PDOs. Gene set enrichment analysis revealed that USP3-AS1 upregulation significantly enriched glycolysis and MYC signaling pathways. Metabolomics analysis confirmed that USP3-AS1 promoted glycolysis in PDOs, whereas glycolysis inhibition partially attenuated the effects of USP3-AS1 overexpression on PDO growth and liver metastasis. We revealed that USP3-AS1 stabilized MYC via post-translational deubiquitination, thereby promoting glycolysis. We demonstrated that USP3-AS1 increased the stability of USP3 mRNA, resulting in higher USP3 protein expression. The elevated USP3 protein then interacted with MYC and promoted its stability by deubiquitination. The USP3-AS1–MYC–glycolysis regulatory axis modulated liver metastasis by promoting H3K18 lactylation and CDC27 expression in CRC. In conclusion, USP3-AS1 is a novel promoter of CRLM by inducing histone lactylation.

Introduction

Colorectal cancer (CRC) is one of the most prevalent malignancies globally, with liver metastasis as its primary mode of distant spread. Despite advances in treatment, the prognosis for CRC patients with liver metastasis remains poor [1, 2]. Thus, identifying precise biomarkers and understanding the mechanisms underlying CRC liver metastasis is essential to improve prediction and treatment.

Recent studies highlight aberrant energy metabolism as a hallmark of cancer metastasis [3], with dysregulated glucose metabolism as a critical feature [4, 5]. The Warburg effect, first described by Otto Warburg in the 1920s, refers to a metabolic shift in which cancer cells prioritize glycolysis over oxidative phosphorylation for ATP production. This preference for glycolysis occurs even in the presence of oxygen [6]. This “aerobic glycolysis” enables rapid glucose consumption, lactate production, and synthesis of growth-promoting intermediates. However, the exact mechanisms driving continuous metabolic reprogramming in cancer remain unclear. Histone lactylation was newly discovered by Zhang et al. in 2019 [7]. This brand-new epigenetic modification relies on lactate produced by intracellular metabolism and regulates cell biological functions by activating downstream gene transcription and expression [8–10]. Tumor cells produce and accumulate more lactate than normal cells, making histone lactylation a promising area for further exploration in cancer. Nevertheless, the role of histone lactylation in CRC liver metastasis remaims largely unknown.

Long non-coding RNAs (lncRNAs), a class of transcripts over 200 bp with minimal protein-coding capacity, are implicated in a range of biological processes, including epigenetic and transcriptional regulation, and tumor progression [11–13]. Emerging evidence links lncRNAs to multiple stages of metastasis, from initial migration to colonization of distant organs. For instance, colon cancer-associated transcript 2 (CCAT2) exhibits upregulation in CRC, facilitating cancer cell migration and metastasis [14]. Similarly, lncRNA ITGB8-AS1 acts as a competing endogenous RNA (ceRNA), augmenting colorectal cancer growth and migration through integrin-mediated focal adhesion signaling [15]. However, the role of lncRNAs in metabolic reprogramming during metastasis remains largely unexplored.

Traditional cancer models, such as cell lines and animal models, often fail to replicate human cancer progression accurately. Patient-derived organoids (PDOs) represent a more clinically relevant model, bridging the gap between laboratory and human physiology [16, 17]. PDOs are three-dimensional cultures that closely mimic in vivo tumor characteristics, including cellular heterogeneity. As personalized models, PDOs from CRC patients better represent the disease’s development and metastatic progression [18, 19]. In this study, we performed microarray analysis on five paired PDOs derived from primary tumors and corresponding liver metastatic lesions, revealing significant upregulation of USP3-AS1 in PDOs from liver metastases. Further bioinformatics analysis of public datasets indicated that high USP3-AS1 expression is associated with glycolysis activation. These findings warrant further investigation into USP3-AS1’s role in metabolic reprogramming and its impact on CRC progression.

Materials and methods

Collection of public dataset and pathway analysis

The largest public microarray dataset GSE39582 was downloaded from the Gene Expression Omnibus (GEO) database. The data processing procedure has been described in our previous work [20]. Gene set enrichment analysis (GSEA) was performed to identify the USP3-AS1 associated biological signaling pathways, as previously described.

Human colorectal cancer samples

CRC tissue samples were collected from patients at the Fudan University Shanghai Cancer Center (FUSCC). These tissues were used for gene and protein expression analyses and tissue microarray preparation. All participants provided informed written consent in accordance with the regulations of the Institutional Review Boards of FUSCC and this study has been examined and approved by Ethics Committee of FUSCC (050432-4-2307E).

Tissue dissociation and organoid culture

The patient-derived organoids were prepared from large intestine tumors, as described previously [21]. In short, tumors were cut into pieces and two parts were processed for immunohistochemistry and RNA isolation. The remainder was cut into smaller pieces and incubated in digestion buffer Advanced DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA) medium with 2.5% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), 75 U/mL collagenase type IX (Sigma-Aldrich, St. Louis, MO, USA), 1.25 mg/mL dispase type II (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C while shaking. After incubation, Basal culture medium (Advanced DMEM/F12 supplemented with penicillin/streptomycin), 10 mM HEPES (Invitrogen, Carlsbad, CA, USA) and 2 mM GlutaMAX (Invitrogen, Carlsbad, CA, USA) was added and the mixture was put over a 100 μm cell strainer (Invitrogen, Carlsbad, CA, USA) to remove large fragments. Cells were subsequently spun at 1000 rpm for 3 min. The pellet was resuspended in basal culture medium and spun again at 1000 rpm. The procedure was repeated twice to remove debris and collagenase. The tumor material was resuspended in Basement Membrane Extract (BME; Cultrex RGF Basement Membrane Extract, Type 2; R&D Systems, Minneapolis, MN, USA), and dispensed into 24-well culture plates (40 μL BME/well). The BME was then solidified by a 20 min incubation in a 37 °C and 5% CO₂ cell culture incubator, and overlaid with 500 µL of complete human organoid media. The composition of CRC culture medium is: Basal culture medium supplemented with 20% R-spondin-1 conditioned medium, 100 ng/mL mouse recombinant Noggin (PeproTech, Rocky Hill, NJ, USA), 1× B27 (Invitrogen, Carlsbad, CA, USA), 1.25 mM n-Acetyl Cysteine (Sigma-Aldrich, St. Louis, MO, USA), 10 mM Nicotinamide (Sigma-Aldrich, St. Louis, MO, USA), 50 ng/mL EGF (PeproTech, Rocky Hill, NJ, USA), 10 nM Gastrin (Sigma-Aldrich, St. Louis, MO, USA), 500 nM A83-01 (MedChemExpress, Monmouth Junction, NJ, USA), 5 μM SB202190 (MedChemExpress, Monmouth Junction, NJ, USA), 10 nM Prostaglandine E2 (MedChemExpress, Monmouth Junction, NJ, USA) and 100 µg/mL Primocin (Invitrogen, Carlsbad, CA, USA). Complete media was subsequently refreshed every two days.

For passaging, BME was broken up by pipetting and organoids were collected in a tube. The organoids were centrifuged at 1000 rpm for 3 min and the medium was removed. 1× TrypLe Express (Thermo Fisher Scientific, Waltham, MA, USA) was added and the organoids were incubated at 37 °C for approximately 5 min. Organoids were then dissociated to small cell clusters by applying mechanical force (pipetting), washed with HBSS (Thermo Fisher Scientific, Waltham, MA, USA), pelleted (1200 rpm, 5 min, 4 °C), resuspended in BME, and re-seeded at an appropriate ratio. Mycoplasma testing was done by nested PCR. Organoids were biobanked in FBS (Thermo Fisher Scientific, Waltham, MA, USA), containing 10% DMSO (Sigma-Aldrich, St. Louis, MO, USA).

Microarray analysis

RNA quantity and quality were measured by NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was assessed by standard denaturing agarose gel electrophoresis. The Whole Human Genome Oligo Microarray was a broad view that represents all known genes and transcripts in the human genome. Sequences were compiled from a broad source survey, and then verified and optimized by alignment to the assembled human genome. Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, Santa Clara, CA, USA). Briefly, total RNA from each sample was linearly amplified and labeled with Cy3-UTP. The Labeled cRNAs were purified by RNeasy Mini Kit (Qiagen, Hilden, Germany). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 11 μL 10× Blocking Agent and 2.2 μL of 25× Fragmentation Buffer, then heated at 60 °C for 30 min, and finally 55 μL 2× GE Hybridization buffer was added to dilute the labeled cRNA. 100 μL of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 h at 65 °C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned using the Agilent DNA Microarray Scanner (part number G2505C).

Metabolomic profiling of tissue samples

A combination of gas chromatography-time-of-flight mass spectrometry (GC-TOFMS, LECO Corp., St Joseph, MI, USA) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS, Waters Corp., Milford, MA, USA) was used to quantify small molecule metabolites in the tissue samples. Metabolomics assays were conducted by Metabo-Profile Inc. (Shanghai, China) using previously published methods [22]. The metabolites were identified by comparison with an internal library built using standard reference chemicals.

CUT&Tag

The CUT&Tag assay was performed using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina according to the manufacturer’s instructions. Briefly, prepared concanavalin A-coated magnetic beads (ConA beads; Thermo Fisher Scientific, Waltham, MA, USA) were added to resuspended cells and incubated at room temperature to bind cells. The nonionic detergent digitonin was used to permeate the cell membrane. Then, H3K18la antibody (PTM-1427RM, PTM BIO, Hangzhou, China), secondary antibody and Hyperactive pA-Tn5 Transposase were incubated with the cells that were bound by ConA beads. The purified PCR products were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Finally, these libraries were sequenced on the Illumina NovaSeq6000 platform, and 150 bp paired-end reads were generated for the following analysis.

Organoid xenograft assays

All animal procedures were performed under guidelines approved by the Institutional Animal Care and Use Committee of FUSCC (FUSCC-IACUC-S20210055). All mice were obtained from the Shanghai Experimental Animal Center (Shanghai, China). The organoids were harvested using the passaging procedure described above and resuspended in 50% Matrigel (Corning, Tewksbury, MA, USA)/50% organoid culture media at a concentration of 10⁷ cells/mL. A total 200 µL (2 × 10⁶ cells) of organoid suspension was injected subcutaneously into BALB/c nude mice (6–8 weeks old, male). The tumor volumes were measured every 4 days and calculated by using the standard formula: length×width²/2. After 60 days, mice were euthanized. Subsequently, these tumor xenografts were flash-frozen and stored in liquid nitrogen until the metabolomic assays were performed.

Tracer studies in mouse xenografts of PDOs

We performed ¹³C-labeled tracer studies using a previously reported protocol with a minor modification [23]. For tracer studies in mouse xenografts, on the final day of the experiment, the tumor-bearing mice were injected via the tail vein with 100 μL 1 M [U-¹³C6]-D-fructose (Cambridge Isotope Laboratories, Tewksbury, MA, USA) for 3 times at 15-min intervals. One hour later, tumor xenografts were excised, weighed and flash-frozen in liquid nitrogen. In our metabolic experiments, the chosen time points were selected with reference to the study by Sellers et al [23], which investigated metabolic dynamics in cancer cells through stable isotope-resolved metabolomics. Similar to their design, our time points were chosen to capture key metabolic changes while minimizing systemic artifacts that might arise from extended incubation periods.

Liver metastasis induction

To develop the mouse model of hepatic metastasis, PDOs with or without USP3-AS1 silencing/overexpression (1 × 10⁶ cells) were implanted into the spleens of 6-week-old Balb/c female mice. The mice were sacrificed 8–10 weeks after injection of the tumor cells, and liver metastases were enumerated immediately.

Lactate production and ATP level analysis

The cellular lactate production and ATP levels were measured using a Fluorometric Lactate Assay Kit (Abcam, Cambridge, UK) and a Luminescent ATP Detection Assay Kit (Abcam, Cambridge, UK), respectively.

Glucose uptake assay

This experiment was conducted using PDOs. 1 × 10⁴ PDOs were cultured in 96-well plates containing glucose-free DMEM (Thermo Fisher Scientific, Waltham, MA, USA) with 10% dialyzed fetal bovine serum (dFBS; Thermo Fisher Scientific, Waltham, MA, USA) and 6 mM glucose and then transferred to a CO₂ incubator set at 37 °C and 5% CO₂ for 48 h. Spent media were collected to measure remaining fructose using a glucose colorimetric/fluorometric assay kit (Abcam, Cambridge, UK) following the manufacturer’s instruction.

Western blotting

Briefly, equal quantities of cellular proteins were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and subjected to immunoblotting using a primary antibody for β-actin (1:1000 dilution; Abcam, Cambridge, UK) and incubating the membranes overnight at 4 °C. After incubation with the secondary antibody, the blots were visualized using ECL (Pierce, Thermo Fisher Scientific, Waltham, MA, USA), and the ECL intensity was detected using a BioImaging System (Bio-Rad, Hercules, CA, USA).

mRNA decay analyses

Stable cells were directly harvested (mRNA steady-state level) or treated with 5 mM actinomycin D (Sigma-Aldrich, St. Louis, MO, USA) and harvested at the indicated time points. The total RNA was isolated at the indicated time points after actinomycin D application, and each analyzed factor was validated by quantitative RT-PCR for each experiment. All data were analyzed from at least three independent experiments, and statistical significance was validated by Student’s t test.

Statistical analysis

All described results are representative of at least three independent experiments. Statistical analysis was conducted using R software (version 3.2.5; https://www.r-project.org/). Data are expressed as mean ± standard deviation. For comparisons between two groups, the Wilcoxon rank-sum test was applied to data with a skewed distribution, while Student’s t-test was used for normally distributed data. Survival rates were estimated using the Kaplan-Meier method, and survival differences were assessed with the log-rank test. Associations between USP3-AS1 expression and other gene expressions were evaluated using the Spearman rank correlation test. Statistical significance was defined as P < 0.05.

Results

USP3-AS1 is upregulated in PDOs from liver metastatic sites and high USP3-AS1 expression is associated with poor prognosis

To identify novel lncRNAs involved in the progression of CRC, we cultured five pairs of PDOs from primary tumors and corresponding liver metastatic lesions, followed by microarray analysis. This analysis revealed that the antisense lncRNA USP3-AS1 was significantly upregulated in liver metastatic PDOs compared to primary CRC tissues (Fig. 1a, b). Real-time PCR analysis in 15 matched sets of normal, primary CRC, and liver metastatic PDOs from CRLM patients further confirmed that USP3-AS1 expression was significantly higher in liver metastatic PDOs (Fig. 1c). Additionally, in situ hybridization (ISH) assays validated the elevated expression of USP3-AS1 in liver metastatic PDOs (Fig. 1d, e).

Fig. 1. USP3-AS1 is upregulated in liver metastatic PDOs and correlates with poor prognosis.

a Volcano plot of differentially expressed mRNAs and lncRNAs in CRC-PDOs and LM-PDOs (n = 5 pairs), highlighting significantly upregulated (red) and downregulated (blue) genes. b Normalized expression of USP3-AS1 in CRC-PDOs and LM-PDOs (n = 5 pairs; *P < 0.05, two-tailed paired t-test). c Real-time PCR validation of USP3-AS1 expression in N-PDOs, CRC-PDOs, and LM-PDOs (n = 15 per group; *P < 0.05, one-way ANOVA with Tukey’s post hoc test). d Representative ISH staining of USP3-AS1 in CRC-PDOs and LM-PDOs. Scale bars, 100 μm. e Quantitative analysis of ISH staining for USP3-AS1 in CRC-PDOs and LM-PDOs (n = 15 per group; *P < 0.05, chi-squared test). f–h Kaplan-Meier survival curves showing the association of USP3-AS1 expression with overall survival (f, P < 0.0001, log-rank test), disease-free survival (g, P = 0.00028, log-rank test), and liver metastasis-free survival (h, P = 0.011, log-rank test) in colorectal cancer patients. All data are shown as mean ± SEM. or proportions, as appropriate. *P < 0.05 was considered statistically significant.

Given the observed differences between metastatic and primary sites, we hypothesized that USP3-AS1 could serve as a potential prognostic biomarker in CRC. To test this, we performed quantitative RT-PCR (qRT-PCR) on samples from 262 patients. Correlation analysis revealed that high USP3-AS1 expression was significantly associated with advanced N and M stages (Supplementary Fig. S1a). Survival analysis demonstrated that elevated USP3-AS1 levels were significantly linked to poorer overall survival, disease-free survival, and liver metastasis-free survival (Fig. 1f–h). Multivariate analysis confirmed that high USP3-AS1 expression is an independent predictor of poor overall survival, disease-free survival, and liver metastasis-free survival (Supplementary Fig. S1b, S1c).

USP3-AS1 promotes PDOs growth and liver metastasis

We assessed the RNA expression levels of USP3-AS1 in PDOs derived from normal intestinal mucosa (normal-PDO), non-metastatic CRC (nCRC-PDO), metastatic CRC from primary sites (mCRC-PDO), and liver metastasis (LM-PDO). USP3-AS1 expression was low in normal PDOs but significantly higher in LM-PDOs (Fig. 2a, b). To evaluate the role of USP3-AS1 in CRC growth and metastasis, we silenced USP3-AS1 in LM-PDO-1 and LM-PDO-2 (Supplementary Fig. S2a) and overexpressed it in nCRC-PDO-1 and nCRC-PDO-2 (Supplementary Fig. S2b). Silencing USP3-AS1 significantly reduced sphere formation efficiency (Fig. 2c) and cell viability (Fig. 2d). In contrast, overexpression of USP3-AS1 markedly enhanced colony formation (Fig. 2e) and cell viability (Fig. 2f). Further in vivo studies revealed that LM-PDO-1 with USP3-AS1 silenced exhibited slackening subcutaneous tumor growth (Fig. 2g). Instead, PDOs with enforced USP3-AS1 showed notably accelerated subcutaneous tumor growth (Supplementary Fig. S2c). In addition, the liver metastasis model showed that PDOs with USP3-AS1 silenced formed significantly less metastatic lesions than the control PDOs (Fig. 2h) while USP3-AS1 overexpression produced strikingly more metastatic lesions (Supplementary Fig. S2d).

Fig. 2. USP3-AS1 promotes PDO growth and liver metastasis in vitro and in vivo.

a, b Baseline RNA expression levels of USP3-AS1 in normal-PDOs, nCRC-PDOs, mCRC-PDOs, and LM-PDOs were analyzed by qRT-PCR a and heatmap b. c Sphere formation efficiency of LM-PDO-1 and LM-PDO-2 organoids with or without USP3-AS1 knockdown (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). Scale bars, 200 μm. d Proliferation of LM-PDO-1 and LM-PDO-2 organoids with or without USP3-AS1 knockdown was measured by cell viability assay (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). e Sphere formation efficiency of nCRC-PDO-1 and nCRC-PDO-2 organoids with or without USP3-AS1 overexpression (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). Scale bars, 200 μm. f Proliferation of nCRC-PDO-1 and nCRC-PDO-2 organoids with or without USP3-AS1 overexpression was measured by cell viability assay (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). g Representative images and relative tumor weights of subcutaneous xenografts formed by LM-PDO-1 organoids with or without USP3-AS1 knockdown in nude mice (n = 3; *P < 0.05, two-tailed t-test). Scale bars, 1 cm. h Gross images of livers and the number of liver metastatic nodules in nude mice injected with LM-PDO-1 organoids with or without USP3-AS1 knockdown (n = 3; *P < 0.05, two-tailed t-test). Scale bars, 1 cm. All data are shown as mean ± SEM. *P < 0.05 was considered statistically significant.

USP3-AS1 acts as an activator of glycolysis and lactate production in CRC

To investigate the signaling pathways influenced by USP3-AS1, we performed GSEA analysis in GSE39582, one of the largest CRC microarray datasets in the GEO database. The analysis showed that the glycolysis signaling pathway was significantly enriched in patients with high USP3-AS1 expression (Fig. 3a). Metabolite analysis of xenografts using GC-TOF-MS further revealed that USP3-AS1 overexpression significantly increased levels of glucose, lactic acid, pyruvic acid, and anabolic products of free fatty acids (FFAs) (Fig. 3b). Conversely, silencing USP3-AS1 led to decreased levels of these metabolites, suggesting that USP3-AS1 plays a crucial role in reprogramming glucose metabolism in CRC PDOs.

Fig. 3. USP3-AS1 promotes glycolysis and lactate production in CRC.

a GSEA analysis of the GSE39582 dataset showing significant enrichment of glycolysis-related pathways in patients with high USP3-AS1 expression (FDR < 0.05). b Heatmap illustrating metabolite levels, including glucose, pyruvic acid, lactate, and FFAs, in xenografts with USP3-AS1 silencing or overexpression, analyzed by GC-TOF-MS. c–e Relative levels of ¹³C-labeled metabolites (glucose, lactate, and pyruvate) derived from ¹³C-glucose in LM-PDO-1 xenografts with or without USP3-AS1 silencing (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). f–h Relative levels of ¹³C-labeled metabolites (glucose, lactate, and pyruvate) derived from ¹³C-glucose in nCRC-PDO-1 xenografts with or without USP3-AS1 overexpression (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). i–n Relative glucose uptake, lactate production, and ATP production in nCRC-PDO-1 i–k and nCRC-PDO-2 l–n organoids with or without USP3-AS1 overexpression (n = 3 biologically independent experiments). All data are shown as mean ± SEM. *P < 0.05 was considered statistically significant.

To further validate these findings, we conducted the following metabolic flux assays. We intravenously infused ¹³C-labeled glucose into xenograft tumor-bearing mice and traced the ¹³C-glucose-derived metabolites in xenografts. We observed that ¹³C-glucose, ¹³C-lactic acid and ¹³C-pyruvic acid were dramatically reduced in LM-PDO-1 tumors with USP3-AS1 silencing (Fig. 3c–e), whereas the ¹³C-labeled metabolites were significantly upregulated in nCRC-PDO-1 tumors with enforced USP3-AS1 expression (Fig. 3f–h).

We also measured glucose uptake, lactate production, and ATP production in PDOs. Overexpression of USP3-AS1 led to a significant increase in glucose uptake, lactate production, and ATP production (Fig. 3i–n; Supplementary Fig. S3a). In contrast, USP3-AS1 attenuation significantly reduced glucose uptake (Supplementary Fig. S3a), lactate production (Supplementary Fig. S3b), and ATP production (Supplementary Fig. S3c). Further analysis of critical glycolytic enzymes revealed that several key enzymes were downregulated in PDOs with USP3-AS1 knockdown (Supplementary Fig. S3d) and upregulated in PDOs with USP3-AS1 overexpression (Supplementary Fig. S3e). These findings suggest that USP3-AS1 contributes to glucose metabolism reprogramming in CRC PDOs.

USP3-AS1 activates glycolysis by stabilizing MYC

The previous GSEA conducted in GEO database have revealed that high expression of USP3-AS1 was significantly associated with MYC signalling pathway (Fig. 3a). Activation of MYC pathway has been established as a signal being able to enhance glycolysis in cancer cells. Therefore, we hypothesized that USP3-AS1 may activate glycolysis by regulating MYC pathway. Firstly, we test the effect of USP3-AS1 on the mRNA and protein level of MYC and found that the mRNA level of MYC was not influenced by USP3-AS1 (Fig. 4a; Supplementary Fig. S4a). However, the protein level of MYC increased in PDOs with USP3-AS1 overexpression while decreased in PDOs with USP3-AS1 knockdown (Fig. 4b; Supplementary Fig. S4b). We next conducted a half-life assay to determine the stability of MYC protein in PDOs by inhibiting protein synthesis with cycloheximide. In LM-PDO-1, silencing USP3-AS1 decreased MYC protein stability (Fig. 4c; Supplementary Fig. S4c), while overexpression of USP3-AS1 in nCRC-PDO-1 significantly increased MYC stability (Fig. 4d; Supplementary Fig. S4d). This prompted us to explore whether USP3-AS1 regulates MYC stability post-translationally. To investigate whether USP3-AS1 stabilizes MYC protein via the proteasome pathway, we treated PDOs with MG132, a proteasome inhibitor. The data showed that MG132 abrogated the USP3-AS1-mediated stabilization of MYC protein, suggesting that USP3-AS1 regulates MYC protein stability via the ubiquitin-proteasome pathway (Fig. 4e; Supplementary Fig. S4e). We then examined whether USP3-AS1 could directly influence the ubiquitination level of MYC. Compared to control PDOs, silencing USP3-AS1 increased the ubiquitination level of MYC (Fig. 4f; Supplementary Fig. S4f), while overexpression of USP3-AS1 decreased MYC ubiquitination (Fig. 4g; Supplementary Fig. S4g).

Fig. 4. USP3-AS1 activates glycolysis by stabilizing MYC.

a Real-time PCR showing that USP3-AS1 silencing or overexpression does not alter MYC mRNA levels in LM-PDO-1 and nCRC-PDO-1 organoids (n = 3 biologically independent experiments). b Western blot analysis showing changes in MYC protein levels with USP3-AS1 silencing or overexpression in LM-PDO-1 and nCRC-PDO-1 organoids. Western blot analysis of MYC protein stability in LM-PDO-1 c and nCRC-PDO-1 d organoids at indicated time points after cycloheximide (CHX) treatment, with or without USP3-AS1 silencing or overexpression. e Western blot showing MYC protein levels in LM-PDO-1 and nCRC-PDO-1 organoids with USP3-AS1 silencing or overexpression following treatment with MG132. Ubiquitination levels of MYC in LM-PDO-1 organoids with USP3-AS1 silencing f and in nCRC-PDO-1 organoids with USP3-AS1 overexpression g, determined by immunoprecipitation (IP) and Western blot after MG132 treatment. Sphere formation efficiency h and relative viability i of nCRC-PDO-1 organoids overexpressing USP3-AS1 with or without MYC silencing (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). Scale bars, 200 μm. Liver metastasis model showing representative liver images j and the number of metastatic nodules k in nude mice injected with nCRC-PDO-1 organoids overexpressing USP3-AS1 with or without MYC silencing (n = 3; *P < 0.05, two-tailed t-test). Scale bars, 1 cm. All data are shown as mean ± SEM. *P < 0.05 was considered statistically significant.

To confirm that MYC expression is essential for the promoting effect of USP3-AS1 on metabolic reprogramming and liver metastasis, we silenced MYC in PDOs with USP3-AS1 overexpression. Silencing MYC reversed the metabolic reprogramming (Supplementary Fig. S4h–S4j), reduced sphere formation (Fig. 4h), cell viability (Fig. 4i), and the formation of liver metastasis (Fig. 4j, k) induced by USP3-AS1 overexpression in PDOs. Collectively, these results demonstrate that USP3-AS1 stabilizes MYC protein by regulating its ubiquitination via the proteasome pathway, thereby promoting MYC-mediated glycolysis.

USP3-AS1 stabilizes USP3 mRNA and enhances MYC protein stability via USP3-mediated deubiquitination of MYC

As an lncRNA, USP3-AS1 is unlikely to directly affect the ubiquitination status of MYC. We hypothesized that ubiquitinating/deubiquitinating enzyme expression may be regulated by USP3-AS1 and then interact with and deubiquitinating MYC. Previous studies have revealed that antisense lncRNAs are involved in the tumorigenesis and development of cancer by regulating the expression of their endogenous sense genes. As the endogenous sense gene of USP3-AS1, USP3 is a key deubiquitinating enzyme. Thus, we aimed to investigate whether USP3-AS1 modulates the ubiquitination status of MYC through the regulation of USP3 expression. We first investigated the effect of lncRNA USP3-AS1 on the expression of USP3. Initially, we examined the effect of USP3-AS1 on USP3 expression. Overexpression of USP3-AS1 significantly increased both USP3 mRNA and protein levels (Fig. 5a and b), while silencing USP3-AS1 led to a reduction in both mRNA and protein levels of USP3 in PDOs (Fig. 5a and b).

Fig. 5. USP3-AS1 stabilizes USP3 mRNA, enhancing MYC protein stability via USP3-mediated deubiquitination of MYC.

a Real-time PCR showing the effects of USP3-AS1 silencing or overexpression on USP3 mRNA levels in LM-PDO-1 and nCRC-PDO-1 organoids (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). b Western blot analysis showing the effects of USP3-AS1 silencing or overexpression on USP3 protein levels in LM-PDO-1 and nCRC-PDO-1 organoids. mRNA decay assay showing changes in USP3 mRNA stability after USP3-AS1 silencing c or overexpression d in LM-PDO-1 and nCRC-PDO-1 organoids (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). Luciferase assay measuring the effects of USP3-AS1 silencing e or overexpression f on the luciferase activity of USP3 mRNA in LM-PDO-1 and nCRC-PDO-1 organoids (*P < 0.05, two-tailed t-test). g, h Co-immunoprecipitation (IP) and Western blot analyses showing interactions between exogenous USP3 and MYC proteins in LM-PDO-1 and nCRC-PDO-1 organoids. i, j Co-IP and Western blot analyses of endogenous interactions between USP3 and MYC proteins in LM-PDO-1 and nCRC-PDO-1 organoids. k, l Western blot analysis of MYC protein stability in LM-PDO-1 k and LM-PDO-2 l organoids with USP3-AS1 silencing, at indicated time points after cycloheximide (CHX) treatment, with or without USP3 overexpression. m Western blot analysis of MYC and USP3 protein levels in USP3-AS1 silenced LM-PDO-1 and LM-PDO-2 organoids with USP3 overexpression after MG132 treatment. IP and Western blot analyses showing the ubiquitination levels of MYC in USP3-AS1 silenced LM-PDO-1 n and LM-PDO-2 o organoids with USP3 overexpression after MG132 treatment for 8 h. Liver metastasis model showing representative liver images p and the number of metastatic nodules q in nude mice injected with LM-PDO-1 organoids, with USP3-AS1 silencing and/or USP3 overexpression (n = 3; *P < 0.05, two-tailed t-test). Scale bars, 1 cm. All data are shown as mean ± SEM. *P < 0.05 was considered statistically significant.

Since USP3-AS1 showed the ability to upregulate USP3 mRNA, we hypothesized that it could regulate the stability by interacting with USP3 mRNA. To begin with, we applied an RNA stability assay to detect the effect of USP3-AS1 on the stability of USP3 mRNA. After blocking RNA transcription by adding actinomycin D, USP3 mRNA exhibited an obvious decrease in its stability mediated by USP3-AS1 knockdown in PDOs (Fig. 5c). Conversely, over-expression of USP3-AS1 significantly extend the half-life of USP3 mRNA in PDOs (Fig. 5d). Next, we conducted luciferase reporter assays, which revealed that attenuation of USP3-AS1 diminished the luciferase activity of the USP3 construct, while enhancement of USP3-AS1 expression increased luciferase activity (Fig. 5e, f). These results suggest that USP3-AS1 may influence USP3 mRNA stability through direct interaction with its mRNA.

To further confirm the interactions between USP3 and MYC, we performed Co-IP assays. The results showed that exogenously overexpressed Flag-tagged USP3 interacted with exogenously overexpressed HA-tagged MYC (Fig. 5g, h). Furthermore, endogenous interactions between USP3 and MYC were confirmed in the organoids (Fig. 5i, j). Subsequently, we enforced USP3 expression in PDOs with USP3-AS1 silencing and performed a half-life assay of MYC. The result showed that over-expressing USP3 significantly increased MYC stability (Fig. 5k, l). Futher MG132 treatment abrogated the USP3 mediated stability of MYC protein, revealing that USP3 regulated MYC protein stability via the ubiquitin proteasome pathway (Fig. 5m). We next examined the effect of USP3 on the ubiquitination level of MYC. By enforcing the USP3 expression in PDOs with USP3-AS1 silencing, the ubiquitination level of MYC in PDOs were notably decreased (Fig. 5n, o). Lastly, liver metastasis formation assay showed that enhanced USP3 expression reversed inhibition of liver metastasis formation caused by USP3-AS1 silence in PDOs (Fig. 5p, q). Taken together, these results demonstrated that USP3-AS1 enhances the stability of USP3 mRNA, leading to increased MYC protein stability through USP3-mediated deubiquitination of MYC.

Glucose driven histone lactylation modulated by USP3-AS1 promotes CRC organoids growth and liver metastasis

Histone lactylation, a newly discovered modification identified by Zhang et al. in 2019, is driven by intracellular lactate produced during glycolysis or other metabolic processes. H3K18 lactylation (H3K18la) has been shown to play a pivotal role in cancer development. We assessed H3K18la levels in CRC organoids with dysregulated USP3-AS1 expression, finding that H3K18la was upregulated in organoids with enforced USP3-AS1 expression (Fig. 6a) and downregulated in organoids with silenced USP3-AS1 (Fig. 6b). To further test whether USP3-AS1 mediated histone lactylation is essential for CRC growth and liver metastasis, we used 2-Deoxy-D-glucose (2-DG, hexokinase inhibitor) and Oxamate (lactate dehydrogenase-A inhibitor) to decrease the glucose utilization and lactate production (Fig. 6c, d) in PDOs with USP3-AS1 overexpression. The results showed that 2-DG or Oxamate treatment completely abrogated the effect of USP3-AS1 overexpression on PDOs sphere formation (Fig. 6e–h) and the formation of liver metastasis (Fig. 6i, j), confirming the fundamental role of histone lactylation in promoting CRC growth and liver metastasis.

Fig. 6. Glucose-driven histone lactylation modulated by USP3-AS1 promotes CRC organoid growth and liver metastasis.

a Western blot analysis showing increased expression of H3K18la in nCRC-PDO-1 and nCRC-PDO-2 organoids with USP3-AS1 overexpression. b Western blot analysis showing decreased expression of H3K18la in LM-PDO-1 and LM-PDO-2 organoids with USP3-AS1 silencing. c Relative lactate production in CRC organoids with USP3-AS1 overexpression after treatment with 2-DG or Oxamate (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). d Western blot analysis showing decreased H3K18la expression in organoids with USP3-AS1 overexpression after treatment with 2-DG or Oxamate. e, f Sphere formation efficiency of nCRC-PDO-1 organoids with USP3-AS1 overexpression after treatment with 2-DG or Oxamate (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). Scale bars, 200 μm. g, h Sphere formation efficiency of nCRC-PDO-2 organoids with USP3-AS1 overexpression after treatment with 2-DG or Oxamate (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). Scale bars, 200 μm. Liver metastasis model showing representative liver images i and the number of metastatic nodules j in nude mice injected with nCRC-PDO-1 organoids overexpressing USP3-AS1, with or without treatment with 2-DG or Oxamate (n = 3; *P < 0.05, two-tailed t-test). Scale bars, 1 cm. All data are shown as mean ± SEM. *P < 0.05 was considered statistically significant.

CDC27 is a target of H3K18 lactylation and acts as a promoter in liver metastasis of CRC

To unmask the regulatory mechanism of H3K18la in CRLM, CUT&Tag was performed with ChIP-grade H3K18la antibody in CRC cells. As shown in Fig. 7a, H3K18la could be enriched in the promoter region of numerous genes. We then performed differential gene expression analysis between low and high USP3-AS1 patients and conduct correlation analysis of USP3-AS1 in GSE39582 dataset, and then combined these results with CUT&Tag data. Based on further qPCR validation, cell division cycle 27 (CDC27) was selected as candidate genes with the highest upregulation in USP3-AS1 overexpressed organoids (Fig. 7b). We next confirmed that the expressions of the CDC27 can be up-regulated by USP3-AS1 (Fig. 7c) and down-regulated upon 2-DG or Oxamate treatment (Fig. 7d). The signals of enriched H3K18la in the promoter regions of CDC27 was shown in Fig. 7e and further CHIP-qPCR confirmed that H3K18la was enriched in the CDC27 promoter (Fig. 7f). Finally, we validated the essential role of CDC27 in CRC and it was found that attenuated CDC27 expression reversed sphere formation (Fig. 7g, h), proliferation acceleration (Fig. 7i) and the formation of liver metastasis (Fig. 7j, k) caused by USP3-AS1 overexpression in PDOs.

Fig. 7. CDC27 is a target of H3K18 lactylation and promotes liver metastasis in CRC.

a CUT&Tag analysis with H3K18la antibodies showing enrichment of H3K18la in the promoter regions of multiple genes in CRC cells. b Flowchart illustrating the identification of downstream targets of H3K18la through CUT&Tag, correlation analysis, and qPCR validation, highlighting CDC27 as a key target. c Western blot analysis showing increased CDC27 protein expression in nCRC-PDO-1 and nCRC-PDO-2 organoids with USP3-AS1 overexpression. d Western blot analysis showing decreased CDC27 protein expression in CRC organoids treated with 2-DG or Oxamate. e Genome browser tracks showing H3K18la peaks in the promoter region of CDC27. f ChIP-qPCR analysis confirming significant binding of H3K18la to two sites in the CDC27 promoter region (*P < 0.05, two-tailed t-test). Sphere formation efficiency g, h and relative viability i of nCRC-PDO-1 organoids with USP3-AS1 overexpression and CDC27 knockdown (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). Scale bars, 200 μm. Liver metastasis model showing representative liver images j and the number of metastatic nodules k in nude mice injected with nCRC-PDO-1 organoids overexpressing USP3-AS1, with or without CDC27 knockdown (n = 3; *P < 0.05, two-tailed t-test). Scale bars, 1 cm. All data are shown as mean ± SEM. *P < 0.05 was considered statistically significant.

Interrelated expression of USP3-AS1, USP3, and MYC in CRC

To further assess the expression relevance among USP3-AS1, USP3, and MYC in CRC, we used ISH to analyze the expression of USP3-AS1 in CRC cases and divided them into low and high USP3-AS1 groups (Fig. 8a). Meanwhile, IHC was used to test the corresponding protein level of USP3 and MYC in CRC tissues (Fig. 8a). In addition, the SUVmax of each CRC tissues was used to reflect the glycolytic levels. By comparing the low and high USP3-AS1 groups, USP3, MYC and glycolytic level were significantly higher in high USP3-AS1 group than that in low USP3-AS1 group (Fig. 8b). Further correlation analysis showed that the expression level of USP3-AS1, USP3, MYC and glycolytic level were positively correlated with each other in CRC (Fig. 8c). Overall, the regulation of glycolysis by USP3-AS1 is summarized in Fig. 8d.

Fig. 8. Interrelated expression of USP3-AS1, USP3, and MYC in CRC and their roles in liver metastasis.

a Representative images showing low and high ISH staining of USP3-AS1, IHC staining of USP3 and MYC, and PET/CT SUVmax values in CRC tissues. Scale bars: ISH, 100 μm; IHC, 200 μm. b Quantitative comparison of USP3 IHC scores, MYC IHC scores, and SUVmax values between CRC patients with low and high USP3-AS1 expression levels (n = 3 biologically independent experiments; *P < 0.05, two-tailed t-test). c Correlation matrix showing significant positive relationships among USP3-AS1 levels, USP3 expression, MYC expression, and SUVmax values (**P < 0.01, ***P < 0.001). d Schematic diagram illustrating the proposed mechanism by which USP3-AS1 stabilizes USP3 mRNA, enhancing MYC protein stability through USP3-mediated deubiquitination, which promotes glycolysis, lactate accumulation, CDC27 activation, and liver metastasis in CRC. All data are shown as mean ± SEM. *P < 0.05 was considered statistically significant.

Discussion

Various oncogenic pathways contribute to the progression of colorectal cancer (CRC), yet the role of lncRNAs in CRC and metabolic reprogramming remains poorly understood. Through comprehensive genomic, biochemical, and cell biology analyses, we identified USP3-AS1 as a novel oncogenic lncRNA in CRC. While previous studies have not elucidated how USP3-AS1 influences glucose metabolism, we provide the first evidence that USP3-AS1 promotes liver metastasis in CRC by enhancing glycolysis. Our findings demonstrate that USP3-AS1 activates glycolytic pathways and histone lactylation by stabilizing MYC, a process mediated by USP3-driven deubiquitination.

Metastatic dissemination occurs in approximately 60% of CRC patients, leading to unfavorable outcomes characterized by a median overall survival of approximately 24–27 months and a 10%–15% 5-year survival rate [24, 25]. In recent years, a growing body of research has been devoted to exploring prognostic molecular markers for CRC. Emerging evidence underscores the pivotal role of long non-coding RNAs (lncRNAs) in prognosis, positioning them as potential therapeutic targets in CRC. For instance, the overexpression of lncRNA FEZF1-AS1 has been correlated with advanced T stage, lymph node metastasis, distant metastasis, and diminished overall survival in CRC patients [26]. Conversely, reduced expression of lncRNA HOXA11-AS is strongly associated with larger tumor size, advanced TNM stage, and lymph node metastasis in CRC patients [27]. In this study, we observed heightened expression of lncRNA USP3-AS1, which correlates with advanced stage, lymph node metastasis, and an unfavorable prognosis in CRC. Moreover, we have identified USP3-AS1 as an independent prognostic factor in CRC patients, suggesting its potential utility as a biomarker and therapeutic target in the management of CRC.

Cancers, characterized by uncontrolled cell proliferation, exhibit a distinctive metabolic profile marked by enhanced nutrient acquisition and increased flux through anabolic pathways. Solid tumor growth poses challenges for cancer cells to acquire sufficient oxygen and glucose, leading to hypoxia and metabolic stress. Aerobic glycolysis supports cancer cell growth by providing energy and biosynthesis building blocks. This metabolic reprogramming, coupled with a heightened demand for fatty acids, crucial for membrane construction and cellular energy, prompts tumor cells to synthesize fatty acids de novo. This involves converting pyruvate to acetyl-CoA and subsequent fatty acid synthesis through specific enzymes and pathways. Several previous studies have attempted to identify the lncRNAs being able to rewire glucose or fatty acid metabolism in CRC. For instance, lncRNA GLCC1 was found to be able to protect the c-Myc transcription factor from ubiquitination by directly interacting with the HSP90 chaperone [28]. This interaction results in a unique transcriptional modification pattern on c-Myc target genes, including LDHA, leading to the reprogramming of glycolytic metabolism and promoting CRC proliferation. Moreover, Wang et al. uncovered that lncRNA ZFAS1 orchestrates a molecular interplay with polyadenylate-binding protein 2, stabilizing SREBP1 mRNA [29]. In this current investigation, we present novel insights by revealing, for the first time, that USP3-AS1 possesses the capability to concurrently activate glycolysis and subsequent histone lactylation. This activation mechanism, in turn, propels the growth and metastasis of colorectal cancer. In this present study, we for the first time revealed that CDC27 is a target of histone lactylation and mediates the oncogenic role of USP3-AS1 in CRC. CDC27 is one of the core components of Anaphase Promoting complex/cyclosome. The main role of this protein is defined at cellular division to control cell cycle transitions. To date, lots of studies have shown that elevated CDC27 expression level can increase cell proliferation, invasiveness and metastasis in malignancies. Additionally, it has been proposed that CDC27 upregulation may increase stemness in cancer stem cells.

While our study presents intriguing findings, certain limitations warrant consideration: (1) The absence of USP3-AS1 expression analysis in metastatic sites beyond the liver, such as the lungs and peritoneum, prevents us from conclusively determining whether low USP3-AS1 expression predicts metastasis to sites other than the liver; (2) The reasons behind the upregulation of USP3-AS1 in CRC and liver metastasis remain unexplored, and uncovering these mechanisms could offer additional avenues for developing therapeutic drugs for CRC; (3) Further investigation is needed to elucidate the mechanisms of how USP3-AS1 stabilizing USP3 mRNA. (4) The mechanisms of USP3 mediated deubiquitination of MYC needs further study.

Conclusions

In summary, our study unveils, for the first time, the regulatory impact of USP3-AS1 on CRC glucose metabolism and histone lactylation using PDOs. These insights highlight an alternative mechanism governing CRC progression through the overexpression of USP3-AS1. Our novel findings contribute to a deeper understanding of lncRNA involvement in CRC advancement, potentially paving the way for innovative strategies in future drug development for CRC treatment.

Author contributions

Conceptualization: ARM, JMZ, RJW, and WXD conceptualized the study and defined the research goals and framework. Human cancer samples: RJW and WXD collected human cancer tissue samples and performed histological analyses, including ISH and IHC validation. Metabolomics assays: RJW, WXD, and YXW conducted metabolomics experiments and analyzed glycolysis-related data. Mouse study: RJW, WXD, and ZX carried out the mouse xenograft and liver metastasis experiments, including tumor imaging and quantitative analysis. Epigenetic profiling: LW and TZ performed CUT&Tag and ChIP assays to analyze H3K18 lactylation and its downstream targets. Molecular biology experiments: JMZ and WQX conducted qPCR, Western blotting, and related mechanistic experiments to validate the molecular interactions. Data analysis: YXW, YMZ, and TZ contributed to data processing, statistical analysis, and interpretation of PET/CT results. Writing – Original Draft: ARM, JMZ, RJW, and WXD wrote the original draft of the manuscript. Writing – Review & Editing: All authors reviewed and edited the manuscript, providing critical feedback to improve its quality.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

The research was approved by the Ethical Committee and Institutional Review Board of Fudan University Shanghai Cancer Center.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-024-01465-8.