Abstract
Background
In China, colorectal cancer (CLC) is the third most common malignancy, following lung cancer and thyroid cancer. This study attempts to reveal the protective mechanism of ubiquitin-specific peptidase 11 (USP11) on Warburg effect of CLC.
Methods
The study used single-cell analysis and the Chinese Glioma Genome Atlas (CGGA) dataset. HCT-116 cells were transfected with the USP11 or small interfering (si)-USP11 plasmid, respectively.
Results
The USP11 messengerRNA(mRNA) expression in patients with CLC was elevated compared to the normal group. Patients with high USP11 expression exhibited poorer survival than those with low USP11 expression in CLC. USP11 was expressed in the epithelial cells of CLC patients. USP11 promoted Warburg effect to induce cell growth progression of CLC. USP11 upregulates the expression of HIF-1α and YAP in CLC. Mechanistically, HIF-1α mediates the effect of USP11 in CLC by driving the progression of the Warburg effect. USP11 physically interacts with the HIF-1α complex and stabilizes HIF-1α protein in CLC cells via suppressing HIF-1α ubiquitination. Additionally, the N6-methyladenosine (m6A) methyltransferase METTL3 enhances USP11 stability in a YTHDF1-dependent manner.
Conclusions
This study demonstrates that the METTL3-mediated m6A modification facilitates USP11 stabilization by YTHDF1 in CLC models, providing novel mechanistic insights and potential therapeutic targets for the clinical management of CLC and other malignant tumors.
Keywords: N6-methyladenosine (m6A), ubiquitin-specific peptidase 11 (USP11), colon cancer, Warburg effect, HIF-1α
Highlight box.
Key findings
• This study demonstrates that METTL3-mediated N6-methyladenosine (m6A) modification facilitates the stabilization of ubiquitin-specific peptidase 11 (USP11) in colorectal cancer (CLC) models in an YTHDF1-dependent manner, which provides novel mechanistic insights and identifies promising therapeutic targets for the clinical management of CLC and other malignant tumors.
What is known and what is new?
• CLC ranks as the third most prevalent malignancy in China, only after lung cancer and thyroid cancer, posing a substantial public health burden nationwide.
• The present study unravels a previously unrecognized regulatory mechanism by which USP11 exerts its protective effects on the Warburg effect in CLC, filling a critical knowledge gap in the field of tumor metabolic reprogramming.
What is the implication, and what should change now?
• Our findings establish a functional axis of METTL3-YTHDF1-USP11 that modulates the Warburg effect in CLC, offering a clarified molecular basis for understanding m6A-driven metabolic dysregulation in tumor progression.
• This work opens new avenues for the development of targeted therapeutic strategies, for improving the outcomes of CLC and other related malignancies.
Introduction
Colorectal cancer (CLC) is a prevalent malignant tumor of the digestive tract that severely endangers human health (1). Latest global epidemiological data demonstrate that CLC ranks third in incidence and second in mortality across all human malignancies worldwide. Its high morbidity and mortality rates bring severe physical suffering to patients and place a heavy economic and social burden on individual families and public healthcare systems (2). Surgical resection combined with adjuvant radiotherapy and chemotherapy constitutes the mainstream clinical treatment for CLC (3). Despite the capacity of these conventional therapeutic regimens to partially inhibit tumor progression, they frequently result in unsatisfactory long-term prognosis and inevitable systemic adverse reactions (4,5). Therefore, there is an urgent demand to explore innovative therapeutic strategies and alternative intervention approaches to improve CLC clinical outcomes.
Distinct from normal cells that rely on oxidative phosphorylation for energy production under aerobic conditions, tumor cells exhibit a unique metabolic phenotype characterized by preferential aerobic glycolysis, which is defined as the Warburg effect (1,2). As a core regulatory transcription factor, HIF-1α governs the glycolytic reprogramming of tumor cells, thereby modulating multiple malignant biological behaviors including tumor cell survival, angiogenesis, invasion and disease progression. Targeted interventions against the HIF-1α-mediated glycolytic pathway and its upstream and downstream regulatory molecules hold great promise as a novel therapeutic strategy for a broad spectrum of malignant tumors (3).
Accumulating evidence has validated the prevalence of the Warburg effect in nearly all human tumor types, wherein cancer cells preferentially utilize glycolysis for glucose catabolism even under normoxic conditions (4,5). Functional studies in glioblastoma have demonstrated that HIF-1α accelerates glycolytic flux by directly upregulating key rate-limiting enzymes involved in glucose metabolism, which sustains energy supply for tumor growth and potentiates invasive and metastatic capacities of cancer cells (6). Breast cancer research has further revealed that HIF-1α promotes the membrane localization of lactate, H+, and HCO3− transporters. The resultant intracellular lactate accumulation reduces intracellular pH, thereby remodeling the tumor immune microenvironment and suppressing anti-tumor immune responses (7). Additional mechanistic studies have verified that the oncogenic biological functions of HIF-1α are tightly dependent on glucose metabolic reprogramming, whereby HIF-1α is indispensable for maintaining the hypermetabolic state of tumor cells (8). Therefore, inhibiting glucose metabolism by inhibiting HIF-1α may be an effective measure for tumor treatment.
HIF-1α serves as a pivotal transcription factor governing tumor progression and therapeutic resistance, with its functional activity strictly modulated by ambient oxygen tension. Under normoxic conditions, HIF-1α is rapidly degraded via the ubiquitin-proteasome pathway, leading to complete functional inactivation. In contrast, hypoxic tumor microenvironments enable HIF-1α to evade ubiquitination-mediated degradation and translocate into the nucleus, triggering the activation of downstream HIF-1α signaling cascades and modulating a spectrum of tumor-associated molecular events. Aberrant overexpression of HIF-1α and its target genes drives malignant progression through multiple oncogenic mechanisms, including tumor angiogenesis, unrestricted cell proliferation, genomic instability, and chemoradiotherapy resistance. As a core regulator of tumor glycolytic phenotypic transformation, HIF-1α dominates the activation of multiple key glycolytic signaling pathways. Accordingly, the HIF-1α-centered glycolytic regulatory network has emerged as a novel and actionable target for tumor targeted therapy.
Emerging studies have identified the Hippo/YAP signaling axis as a crucial regulatory cascade responsible for balancing cell proliferation and tissue homeostasis. Ubiquitin-specific peptidase 11 (USP11), a member of the deubiquitinase family, is ubiquitously expressed in multiple human organs and tissues. Functionally, USP11 binds to diverse substrate proteins and eliminates their ubiquitin modifications, thereby preserving the stability of target protein (9). The ubiquitin-specific protease (USP) family, consisting of more than 50 identified members, constitutes the largest subfamily of deubiquitinases. Widely distributed in human tissues, USP family proteins exert fundamental regulatory effects on protein stability, subcellular localization, and intracellular signal transduction, and are closely implicated in the pathogenesis of various malignancies (10-12). Accumulating clinical and experimental evidence has confirmed that USP11 is frequently overexpressed in multiple human cancers and plays a critical oncogenic role in tumor initiation and progression. Against this background, the present study aims to elucidate the molecular mechanism by which USP11 modulates the Warburg Effect in CLC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1567/rc).
Methods
The CLCGA dataset, survival analysis and single-cell analysis
Survival analysis and single-cell analysis were analyzed using the Gene Expression Profiling Interactive Analysis or Cell-omics Data Coordinate Platform (SCDS0000202 or SCDS0000040). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.
Animals
All mice (nude moues, male, 3 weeks, 10–12 g, n=20) were purchased from Qinglongshan Experimental Animal Center and were inoculated with HCT-116 cells (1×107 cells) by sh-USP11 plasmid (sc-63193-V, Santa Cruz Biotechnology, Inc., Shanghai, China). After 2 weeks, mice were sacrificed using cervical dislocation under anesthesia, and tumor was measured. All animal experiments were performed under a project license (No. 2023-121) granted by the Animal Care and Use Committee of Wannan Medical University, in compliance with institutional guidelines for the care and use of animals.
Histological analysis, immunofluorescence and immunohistochemistry
Cancer tissue samples were fixed in 4% paraformaldehyde, and executed histological analysis, immunofluorescence and immunohistochemistry according to references (13,14). Tissue samples were incubated and stained with anti-USP11 (1:100, abcam, ab247005) at 4 ℃ overnight. Cancer tissue samples were incubated and stained with HIF-1α (1:100, abcam, ab1) or anti-USP11 (1:100, abcam, ab247005) at 4 ℃ overnight. Cancer tissue samples or clinical samples were observed using fluorescence microscope (Zeiss Axio Observer A1, Jena, Germany).
Real-time polymerase chain reaction (PCR) and coimmunoprecipitation assay
Quantitative real-time polymerase chain reaction (qPCR) were performed with the ABI Prism 7500 sequence detection system. Relative levels of the sample mRNA expression were calculated and expressed as 2−△△Ct.
The ChIP-qPCR experiment was performed as previously described (15). Cells were treated with RIP lysis buffer, supernatant was enriched with antibody- or rabbit IgG-conjugated protein A/G magnetic beads in IP buffer supplemented with RNase inhibitors and incubated overnight at 4 ℃. The immunoprecipitated RNA was digested, purified, and further analyzed by qPCR.
The N6-methyladenosine (m6A) quantification, luciferase reporter assay, m6A-RNA immunoprecipitation (MeRIP) assay and m6A sequencing
Total RNA was extracted with DNase (Sigma) to remove genomic DNA. D m6A mRNA levels are colourimetrically measured by ELISA assay with an EpiQuik m6A RNA Methylation Quantification kit and Renilla luciferase activities were determined using a Dual-Luciferase Assay kit (Promega). m6A quantification, luciferase reporter assay, m6A-RNA immunoprecipitation (MeRIP) assay and m6A sequencing were executed as literature (16).
In vitro model
NCM460 (ZY-H203, American Type Culture Collection, ATCC), SW480 (SCSP-5033, Chinese Academy of Sciences Cell Bank), SW620 (SCSP-5234, Chinese Academy of Sciences Cell Bank), HT-29 (SCSP-5032, Chinese Academy of Sciences Cell Bank) and HCT-116 (SCSP-5076, Chinese Academy of Sciences Cell Bank) cells were maintained in DMEM (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (Gibco) under a humidified 5% (v/v) CO2 atmosphere at 37 ℃. SW480 cells were transfected with USP11 plasmid (sc-403368-ACT, Santa Cruz Biotechnology, Inc.), and HCT-116 cells were transfected with si-USP11 plasmid (sc-63193-SH, Santa Cruz Biotechnology, Inc.) using Lipofectamine 3000. After 48 h of transfection, Cocl2 (Hypoxia simulator, 20 μM) to cells used other experiment.
Cell viability and EdU kit assay
Cell viability was determined by the Cell Counting Kit-8 (CCK-8) assay (C0037, Beyotime, Nanjing, China) or EdU kit (C0075S, Beyotime) as literature (17). Absorbance was measured at 450 nm using a fluorescent reader (Synergy H1 Microplate Reader, Bio Tek, Winooski). Images were visualized using a fluorescence microscope (Olympus).
Warburg effect related experiments
Warburg effect related experiments were executed according to reference (1). Glucose (F006-1-1), lactate (A020-2-2) and ATP (A095-1-1) kits from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China) were determined. ECAR and OCR were determined using a Seahorse XFe96 analyzer (Seahorse Bioscience, Agilent Technologies), and normalized to total protein content [milli-pH (mpH)/min].
Western blotting analysis and immunofluorescence
Western blotting analysis and immunofluorescence were executed as literature (18). The membranes were USP11 (ab109232, 1:1,000, Abcam, Cambridge, UK), HIF-1α (ab51608, 1:1,000, Abcam), YAP (ab205270, 1:1,000, Abcam), and β-actin (GB15003-100, 1:1,000, Servicebio, Wuhan, China) at 4 ℃ overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (GB23303 or GB23301, 1:5,000, Servicebio) for 1 h at 37 ℃. Protein was measured using an enhanced chemiluminescence system (ECL, Beyotime) and analyzed using an Image Lab 3.0 (BioRad Laboratories, Inc.).
The cells were incubated with Alexa Fluor® 488 /555 secondary antibody for 2 h at room temperature and then stained with DAPI for 15 min in the dark.
Statistical analysis
P values lower than 0.05 were considered significant as the mean ± standard error of the mean (SEM) using GraphPad Prism 8. Data were expressed and evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s posttest.
Results
USP11 expression profile in CLC patients
In this study, public network datasets were first utilized to screen and identify key target genes associated with CLC progression. Survival analysis revealed that CLC patients with high USP11 expression exhibited significantly poorer overall survival (OS), post-progression survival (PPS), and recurrence-free survival (RFS) compared with those with low USP11 expression (Figure 1A-1C). In addition, both mRNA and protein levels of USP11 were markedly elevated in CLC cell lines relative to normal control groups (Figure 1D,1E). The protein expression of USP11 in CLC cell lines was induced compared to the normal group (Figure 1E). Collectively, these clinical expression and survival data indicate that USP11 may serve as a critical biomarker for CLC tumorigenesis and prognosis.
Figure 1.
USP11 expression in colon cancer patients. OS (A), PPS (B), RFS (C) in colon cancer patients. USP11 mRNA expression in colon cancer cell lines (D). USP11 protein expression in colon cancer cell lines (Western blotting, E). **, P<0.01; ***, P<0.001. CI, confidence interval; HR, hazard ratio; OS, overall survival; PPS, post-progression survival; RFS, recurrence-free survival.
Single-cell sequencing analysis of USP11 expression in CLC
Single-cell sequencing analysis was further performed to precisely characterize the expression pattern of USP11 in CLC patient tissues (Figure 2A,2B). Single-cell profiling results verified that USP11 was predominantly enriched in CLC cancer cells characterized by canonical tumor cell markers (e.g., FOXE1 and TTF1) (Figure 2C), whereas no detectable USP11 expression was observed in tumor-infiltrating immune cells, including B cells, T cells, and macrophages (Figure 2D-2F).
Figure 2.
USP11 expression in colon cancer using single-cell sequencing data. (A) Cell information of single-cell sequencing data; (B) gene expression of single-cell sequencing data; (C) cancer cell; (D) B cells; (E) T cells; (F) macrophage cells; (G) USP11/Ki67 in cancer tissue of colon cancer. *, P<0.05. USP11, ubiquitin-specific peptidase 11.
Furthermore, flow cytometry analysis of CLC tumor tissues confirmed that the expression levels of USP11 and the proliferation marker Ki67 were significantly upregulated in CLC patient samples (Figure 2G). Taken together, these single-cell and cytological results solidify the specific expression of USP11 in CLC tumor cells rather than stromal or immune cell populations.
Sh-USP11 reduced cell growth progression of mice model of CLC
Then, CLC mice model using sh-USP11 virus was used to explore the anti-cancer effects of sh-USP11. The results demonstrated that sh-USP11 transfection significantly reduced tumor volume and tumor weight, and efficiently downregulated the expression of Ki67 in CLC tumor tissues (Figure 3A-3D). Moreover, USP11 knockdown markedly inhibited the mRNA levels of pro-inflammatory and pro-tumorigenic factors COX-2 and TNF-α, while enhancing the activity levels of pro-apoptotic caspase-3 and caspase-9 in tumor tissues (Figure 3E-3H). In summary, targeted suppression of USP11 effectively retards tumor growth progression of CLC.
Figure 3.
USP11 promotes cell growth progression of colon cancer. Tumor volume and weight (A,B), tumor tissue (C), Ki67 levels (D; immunohistochemistry, magnification: ×20), COX-2 and TNF-α mRNA levels (E,F), caspase-3 and caspase-9 activity levels (G,H). **, P<0.01; ***, P<0.001. mRNA, messenger RNA; TNF-α, tumor necrosis factor-alpha; USP11, ubiquitin-specific peptidase 11.
USP11 promotes Warburg effect to stimulate cell growth of CLC
Subsequently, this study investigated the impacts of USP11 on cell growth using an in vitro model. The USP11 plasmid enhanced the expression of USP11 mRNA, stimulated cell growth and migration, and facilitated Edu positivity and wound healing in CLC cells (Figure 4A-4E). Moreover, USP11 up-regulation augmented glucose uptake and lactate production, facilitated ATP level, HK and PFK-1 activity levels, and reduced PK activity levels in CLC cells (Figure 4F-4K). USP11 up-regulation facilitated ECAR levels, and decreased OCR levels in CLC cells (Figure 4L,4M).
Figure 4.
USP11 promotes Warburg effect progression of colon cancer. USP11 mRNA expression (A), cell proliferation (B), migration (C; staining method: Transwell chambers), EdU positive (D; EDU staining; ×20) and wound healing (E, ×20), glucose uptake (F), lactate production (G), ATP level (H), PK activity (I), HK activity (J), PFK-1 activity (K), ECAR (L), OCR levels (M) in BCPAP cells were transfected with USP11 plasmid. **, P<0.01; ***, P<0.001. ATP, adenosine triphosphate; DAPI, 4',6-diamidino-2-phenylindole; ECAR, extracellular acidification rate; EdU, 5-Ethynyl-2'-deoxyuridine; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HK, hexokinase; mRNA, messenger RNA; OCR, oxygen consumption rate; PFK-1, phosphofructokinase-1; PK, pyruvate kinase; USP11, ubiquitin-specific peptidase 11.
Conversely, the si-USP11 plasmid decreased the expression of USP11 mRNA, suppressed cell growth and migration, and promoted Edu positivity and wound healing in CLC cells (Figure 5A-5E). Additionally, USP11 down-regulation reduced glucose uptake and lactate production, inhibited ATP level, HK and PFK-1 activity levels, and facilitated PK activity levels in CLC cells (Figure 5F-5K). USP11 down-regulation decreased ECAR levels, and facilitated OCR levels in CLC cells (Figure 5L,5M). So, these data showed that USP11 promotes Warburg Effect to stimulate CLC cell growth.
Figure 5.
Si-USP11 reduces Warburg effect progression of colon cancer. USP11 mRNA expression (A), cell proliferation (B), migration (C; staining method: Transwell chambers), EdU positive (D; EDU staining ; ×20) and wound healing (E, ×20), glucose uptake (F), lactate production (G), ATP level (H), PK activity (I), HK activity (J), PFK-1 activity (K), ECAR (L), OCR levels (M) in HCT-116 cells were transfected with si-USP11 plasmid. *, P<0.05; **, P<0.01, ***, P<0.001. ATP, adenosine triphosphate; DAPI, 4',6-diamidino-2-phenylindole; ECAR, extracellular acidification rate; EdU, 5-Ethynyl-2'-deoxyuridine; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HK, hexokinase; mRNA, messenger RNA; NC, negative control; OCR, oxygen consumption rate; PFK-1, phosphofructokinase-1; PK, pyruvate kinase; USP11, ubiquitin-specific peptidase 11.
USP11 induced HIF-1α expression in CLC
This study further explored the molecular mechanism by which USP11 regulates Warburg effect progression in CLC. Accumulating evidence has confirmed that HIF-1α functions as a critical driver of the Warburg effect in multiple human malignancies breast cancer, cervical cancer, and CLC. HIF-1α/YAP expression in CLC mice model was reduced (Figure 6A). Immunohistochemical staining further validated that USP11 knockdown significantly downregulated HIF-1α protein expression in mouse CLC tissues (Figure 6B). Consistent with these findings, Western blotting results confirmed that sh-USP11 transfection suppressed the protein abundance of USP11, HIF-1α, and YAP in xenograft tumor tissues (Figure 6C). Moreover, immunofluorescence assays further verified the inhibitory effects of USP11 depletion on the protein expression of USP11 and HIF-1α in vivo (Figure 6D).
Figure 6.
USP11 induced HIF-1α expression in colon cancer. Heat map (A), HIF-1α (B; immunofluorescence; ×20) in mice model; HIF-1α expression (immunohistochemical), USP11/HIF-1α/YAP protein expression (C), USP11/HIF-1α expression (D; immunofluorescence; ×20) in mice model of colon cancer. USP11/HIF-1α/YAP protein expression (E,F), USP11/HIF-1α expression (G,H; immunofluorescence; ×50) in vitro model of colon cancer. **, P<0.01, ***, P<0.001. HIF-1α, hypoxia-inducible factor 1-alpha; USP11, ubiquitin-specific peptidase 11.
Subsequent in vitro cellular experiments yielded consistent phenotypic changes. The overexpression of USP11 markedly elevated the protein levels of USP11, HIF-1α, and YAP in CLC cells (Figure 6E), whereas siRNA-mediated USP11 knockdown significantly inhibited the expression of these three proteins (Figure 6F). Additionally, immunofluorescence staining further confirmed that USP11 overexpression upregulated, while USP11 silencing downregulated, the protein expression of USP11 and HIF-1α in CLC cells (Figure 6G,6H). Collectively, these in vivo and in vitro findings demonstrate that USP11 facilitates Warburg effect progression in CLC by upregulating HIF-1α/YAP signaling.
HIF-1α mediates the stimulatory effect of USP11 on proliferation in CLC
To elucidate the functional role of HIF-1α in USP11-driven oncogenesis, we assessed the effects of pharmacological HIF-1α modulation on USP11-mediated CLC phenotypes in vitro. Specifically, treatment with the HIF-1α inhibitor LW6 (2 μM) markedly downregulated HIF-1α and YAP protein levels in USP11-overexpressing CLC cells (Figure 7A). HIF-1α inhibitor reduced proliferation and migration in vitro model of CLC by USP11 (Figure 7B-7E).
Figure 7.
HIF-1α mediates the stimulatory effect of USP11 on proliferation and migration in colon cancer. HIF-1α/YAP protein expression (A), cell proliferation (B), migration (C, ×20), EdU positive (D, ×20) and wound healing (E, ×20) in BCPAP cells were transfected with USP11 plasmid+ HIF-1α inhibitor; HIF-1α/YAP protein expression (F), cell proliferation (G), migration (H, ×20), Edu positive (I, ×20) and wound healing (J, ×20) in HCT-116 cells were transfected with si-USP11 plasmid+ HIF-1α agonist. *, P<0.05, **, P<0.01, ***, P<0.001. EdU, 5-Ethynyl-2'-deoxyuridine; HIF-1α, hypoxia-inducible factor 1-alpha; USP11, ubiquitin-specific peptidase 11.
In reciprocal experiments, HIF-1α agonists (100 μM Deferoxamine mesylate) induced HIF-1α/YAP protein expression in vitro model of CLC by si-USP11 (Figure 7F). Likewise, HIF-1α stimulation effectively rescued the proliferation and migration defects induced by USP11 knockdown in vitro (Figure 7G-7J).
HIF-1α mediates the stimulatory effect of USP11 on warburg effect in CLC
We next investigated whether HIF-1α mediates the USP11-induced enhancement of the Warburg effect in CLC. Pharmacological inhibition of HIF-1α using LW6 significantly attenuated the Warburg effect in USP11-overexpressing CLC cells (Figure 8A-8E). Conversely, treatment with the HIF-1α agonist deferoxamine mesylate effectively restored the Warburg effect in USP11-silenced (si-USP11) CLC cells (Figure 8F-8J).
Figure 8.
HIF-1α mediates the stimulatory effect of USP11 on Warburg effect progression in colon cancer. Glucose uptake (A), lactate production (B), ATP level (C), ECAR (D), OCR levels (E) in BCPAP cells were transfected with USP11 plasmid + HIF-1α inhibitor; glucose uptake (F), lactate production (G), ATP level (H), ECAR (I), OCR levels (J) in HCT-116 cells were transfected with si-USP11 plasmid+ HIF-1α agonist. **, P<0.01, ***, P<0.001. ATP, adenosine triphosphate; ECAR, extracellular acidification rate; HIF-1α, hypoxia-inducible factor 1-alpha; OCR, oxygen consumption rate; USP11, ubiquitin-specific peptidase 11.
HIF-1α mediates the stimulatory effect of USP11 on cell growth progression in CLC
Furthermore, we evaluated the in vivo consequences of HIF-1α activation in the context of USP11 depletion. Administration of the HIF-1α agonist deferoxamine mesylate (200 mg/kg) to CLC xenograft-bearing mice transduced with sh-USP11 significantly upregulated HIF-1α and YAP protein expression, markedly increased tumor volume and weight, and elevated the proliferative marker Ki67 in tumor tissues (Figure 9A-9E). Concurrently, HIF-1α stimulation upregulated the mRNA levels of proinflammatory mediators COX-2 and TNF-α, while markedly attenuating the enzymatic activities of caspase-3 and caspase-9 within the same tumor specimens (Figure 9F-9I). Collectively, these findings indicate that USP11 facilitates CLC progression by activating the HIF-1α/YAP signaling axis, which subsequently promotes the Warburg effect and drives malignant cell growth.
Figure 9.
HIF-1α mediates the stimulatory effect of USP11 on cell growth progression in colon cancer. HIF-1α/YAP protein expression (A), tumor tissue (B), tumor volume and weight (C,D), Ki67 levels (E; immunohistochemistry; ×20), COX-2 and TNF-α mRNA levels (F,G), caspase-3 and caspase-9 activity levels (H,I). **, P<0.01; ***, P<0.001. HIF-1α, hypoxia-inducible factor 1-alpha; TNF-α, tumor necrosis factor-alpha; USP11, ubiquitin-specific peptidase 11.
USP11 interacts with HIF-1α complex and stabilizes HIF-1α in CLC
We next investigated the direct interaction between USP11 and HIF-1α, as well as the underlying regulatory mechanism. Three-dimensional structural modeling revealed a physical interconnection between the USP11 and HIF-1α proteins (Figure 10A). The corresponding mutant constructs of USP11 and HIF-1α are illustrated in Figure 10B. Co-immunoprecipitation (Co-IP) assays further demonstrated that wild-type (WT) USP11 interacted with WT HIF-1α, whereas neither the USP11 mutant nor the HIF-1α mutant was able to engage in this interaction (Figure 10C). Furthermore, we examined the effect of USP11 on HIF-1α protein stability. Notably, USP11 overexpression markedly attenuated the ubiquitination level of HIF-1α in CLC cells, whereas USP11 knockdown significantly promoted HIF-1α ubiquitination (Figure 10D). USP11 Mut protein reduced the ubiquitination of the HIF-1α protein in the in vitro model of CLC (Figure 10E). Therefore, USP11 interacts with the HIF-1α complex and stabilizes HIF-1α in CLC. Then, HIF-1α induced YAP protein expression in the in vitro model of CLC at time-dependent pattern (Figure 10F). IP also indicates that HIF-1α protein is interconnected with YAP protein in the in vitro model of CLC (Figure 10G). HIF-1α reduced the ubiquitination of the HIF-1α protein in the in vitro model of CLC (Figure 10H). HIF-1α interacts with YAP to stabilizes YAP in CLC.
Figure 10.
USP11 interacts with HIF-1α complex and stabilizes HIF-1α in colon cancer. USP11 interacts with HIF-1α complex (3D, A), USP11 WT/Mut and HIF-1α WT/Mut (B), USP11 WT/Mut interacts with HIF-1α WT/Mut complex (IP, C), HIF-1α ubiquitin (D), USP11 WT/Mut interacts with HIF-1α ubiquitin (E); YAP protein expression (F), USP11 interacts with YAP complex (G), YAP ubiquitin (H). HIF-1α, hypoxia-inducible factor 1-alpha; IgG, immunoglobulin G; IP, Immunoprecipitation; Mut, mutant; WT, wild type; USP11, ubiquitin-specific peptidase 11.
m6A/METTL3 modification increases USP11 stability by an YTHDF1
Subsequently, we sought to elucidate the molecular mechanism by which METTL3 orchestrates the Warburg effect via USP11 in CLC. Bioinformatics analysis revealed that USP11 harbors multiple putative m6A methylation motifs within its sequence (Figure 11A). Interestingly, the m6A-specific antibody significantly inhibited USP11 mRNA enrichment in CLC cells by METTL3 (Figure 11B). Simultaneously, METTL3 markedly decreased the stability of USP11 mRNA in CLC cells (Figure 11C).
Figure 11.
METTL3-mediated m6A modification increases USP11 stability in an YTHDF1-dependent manner. Methylation modification sites near (A), USP11 mRNA enrichment in colon cancer cells by METTL3 (B), the stability of USP11 mRNA in colon cancer cells (C), m6A of USP11 (D), 3'-UTR of USP11 (E), luciferase activity level (F), m6A modification (G,H). **, P<0.01, ***, P<0.001. IgG, immunoglobulin G; m6A, N6-methyladenosine; USP11, ubiquitin-specific peptidase 11; UTR, untranslated region.
MeRIP-qPCR showed that METTL3 up-regulation increased USP11 N6-methyladensine level, and METTL3 down-regulation decreased USP11 N6-methyladensine level (Figure 11D). m6A sites in the 3'-untranslated region (UTR) of USP11 and m6A were significantly enriched at sites 1, 2, and 3 (Figure 11E). METTL3 reduced luciferase activity level by wild-type (WT) of USP11, while the mutant (Mut) USP11 did not (Figure 11F). The knockdown of YTHDF1 significantly alleviated the METTL3-induced USP11 m6A modification, indicating that YTHDF1 knockdown could regulate USP112 m6A modification (Figure 11G,11H). These findings disclosed that m6A/METTL3 decreases USP11 stability by YTHDF1.
Discussion
CLC is a highly metastatic malignancy originating in the digestive tract and ranks among the most prevalent cancers worldwide (6). In 2020 alone, over 2 million new cases of CLC were diagnosed globally. Although chemotherapy and radiotherapy remain the primary treatment modalities, patient prognosis continues to be suboptimal, largely owing to the high rates of tumor metastasis and radiotherapy; however, prognosis remains poor due to its high rates of metastasis and recurrence (7). Consequently, identifying more effective therapeutic strategies has remained a critical priority in clinical research (8). In this study, the expression of USP11 was upregulated in CLC patients. Sun et al. demonstrated that USP11 promotes the growth and metastasis of CLC (12). Collectively, these results suggest that USP11 functions as a pivotal regulatory gene in colorectal carcinogenesis, offering a potential target for further mechanistic and therapeutic exploration.
CLC ranks among the most prevalent gastrointestinal malignancies, posing a significant threat to human health owing to its persistently high incidence and mortality rates worldwide (9). Surgical resection remains the cornerstone of curative treatment for early-stage CLC; however, its therapeutic efficacy diminishes considerably in advanced disease, frequently culminating in a dismal prognosis (10). The pathogenesis of CLC is notoriously complex, driven by the coordinated dysregulation of multiple genes, signaling pathways, and aberrant protein expression patterns that collectively govern tumor initiation and progression (11). Hence, the identification of robust biomarkers intimately associated with CLC evolution is of paramount importance for refining early diagnosis, prognostic stratification, and the development of targeted therapeutic interventions (12).We discovered that sh-USP11 suppressed the cell growth in the mouse model of CLC. Huang et al. demonstrated that USP11 facilitates both the proliferation and metastasis of CLC cells (19), further underscoring USP11 as a pivotal driver in colorectal carcinogenesis.
Tumor cells predominantly generate energy via glycolysis, a distinctive metabolic phenotype termed the Warburg effect (20). Unlike normal cells, which only shift to anaerobic glycolysis for energy supplementation under hypoxic conditions, tumor cells sustain robust glycolytic metabolism even in normoxic environments, characterized by excessive glucose consumption and massive lactate accumulation (21-23). A large-scale retrospective cohort study from The Cancer Genome Atlas (TCGA) database of CLC patients has confirmed significant upregulation of glycolysis-related genes and concurrent downregulation of genes associated with the tricarboxylic acid (TCA) cycle, further validating the metabolic reprogramming characteristics of CLC cells (24). In this study, USP11 promotes Warburg Effect of CLC. Qiao et al. demonstrated that USP11 promotes glycolysis in hepatocellular carcinoma by HIF-1α (25). Collectively, these findings solidify that USP11 accelerates CLC cell proliferation by potentiating HIF-1α-mediated Warburg effect reprogramming.
Normal somatic cells primarily utilize oxidative phosphorylation for energy production under aerobic conditions, whereas tumor cells exhibit a unique metabolic preference for aerobic glycolysis to sustain rapid energy supply, which constitutes a hallmark metabolic phenotype of malignant tumors (26,27). As an essential upstream regulatory factor of tumor glycolysis, HIF-1α orchestrates the glycolytic reprogramming of cancer cells and modulates multiple malignant behaviors, including cell survival, angiogenesis, tumor invasion, and disease progression (27). Therefore, targeted intervention targeting the HIF-1α-mediated glycolytic pathway and its downstream regulatory molecules represents a promising therapeutic strategy for diverse malignancies (28). In line with previous mechanistic reports, our experimental data verified that HIF-1α acts as a crucial mediator for the oncogenic function of USP11 in CLC Warburg effect progression. Mechanistically, USP11 physically interacts with the HIF-1α protein complex and enhances HIF-1α protein stability, thereby upregulating HIF-1α expression in CLC cells. A previous study by Bai et al. has also demonstrated the pivotal role of the HIF-1α signaling axis in driving the Warburg effect in gastric cancer (29). Collectively, these findings confirm that USP11 upregulates HIF-1α expression to facilitate Warburg effect reprogramming, thereby accelerating the proliferative progression of CLC cells.
Under physiological conditions, coordinated interactions among signaling molecules within the Hippo pathway maintain the dynamic balance between cell proliferation and apoptosis. Nevertheless, aberrant expression of key pathway components disrupts cellular homeostasis, perturbs the cell cycle, and ultimately facilitates malignant transformation and tumor progression. As a canonical proto-oncogene and core downstream effector of the Hippo signaling pathway, YAP1 activity is tightly regulated by pathway activation. Hippo signaling activation induces excessive YAP1 phosphorylation, leading to cytoplasmic retention of YAP1 and its failure to initiate nuclear transcriptional programs, which disrupts the equilibrium between cell proliferation and apoptosis. Accumulating evidence has validated that YAP1 suppression markedly restrains malignant phenotypes, including proliferation, invasion, and differentiation, while promoting apoptotic progression in lung cancer cells (24,30,31). Consistently, targeted YAP downregulation has been reported to alleviate the Warburg effect in laryngeal carcinoma cells, highlighting the essential role of YAP in regulating tumor metabolic reprogramming (32). Growing domestic and international studies have demonstrated that m6A RNA methylation, the most prevalent epigenetic modification of eukaryotic mRNA, is closely implicated in the pathogenesis of metabolic disorders, neurological diseases, and multiple human malignancies. Dysregulated m6A modification and abnormal expression of m6A-related regulatory enzymes are critical drivers of the initiation and progression of CLC. The activated HIF-1α/YAP signaling axis subsequently rewrites glucose metabolic patterns and exacerbates aerobic glycolysis in CLC cells (33). In the present study, we further validated that METTL3-mediated m6A modification elevates USP11 protein stability via the m6A reader YTHDF1 in CLC. The activated HIF-1α/YAP signaling axis subsequently rewrites glucose metabolic patterns and exacerbates aerobic glycolysis in CLC cells. Consistent with our findings, previous research has confirmed that USP11 modulates YAP activity to govern tumor cell growth and migration across multiple human cancers (34). So, m6A/METTL3 modification increases USP11 stability by YTHDF1 to induce HIF-1α expression in CLC cells.
Conclusions
The interaction between the active components of USP11 and HIF-1α was significant, leading to the induction of HIF-1α/YAP signaling, thereby influencing the Warburg effect-mediated cell growth and metastasis of CLC (Figure 12). Mechanistically, METTL3-mediated m6A modification enhances USP11 protein stability in a YTHDF1-dependent manner within CLC model (Figure 12). Further in-depth exploration of the biological function of USP11 and its regulatory interplay with the HIF-1α/YAP signaling axis will deepen our mechanistic understanding of CLC pathogenesis, laying a solid foundation for the development of innovative targeted therapies and optimizing clinical management strategies for CLC or other malignant tumors.
Figure 12.
The m6A/METTL3 modifies USP11 promotes Warburg effect progression of colon cancer undifferentiated by HIF-1α/YAP signaling pathway. HIF-1α, hypoxia-inducible factor 1-alpha; USP11, ubiquitin-specific peptidase 11.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were performed under a project license (No. 2023-121) granted by the Animal Care and Use Committee of Wannan Medical University, in compliance with institutional guidelines for the care and use of animals.
Footnotes
Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1567/rc
Funding: This work was supported by Yijishan Hospital Talent Introduction Research Fund Project (No. WK2023ZZD20); and Key Scientific Research Project Training Fund of Wannan Medical College (No. WK2021ZF01).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1567/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1567/dss
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