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CDO1 phosphorylation is required for IL-6-induced tumor cell proliferation through governing cysteine availability

Cell Communication and Signaling volume 23, Article number: 194 (2025) Cite this article

Abstract

Inflammatory pathways are often hijacked by cancer cells to favor their own proliferation and survival. Cysteine dioxygenase type 1 (CDO1), an iron-dependent thiol dioxygenase enzyme, catalyzes the rate-limiting step for cysteine oxidation, and so that functions as an important regulator of cellular cysteine availability. However, whether inflammatory environment affects CDO1 activity and cysteine oxidation and its potential impact on tumor growth remains substantially elusive. In the present study, we demonstrate that CDO1 activity and cysteine oxidation is inhibited upon IL-6 treatment, without noticeable alterations in CDO1 expression. Mechanistically, AKT1 phosphorylates CDO1 T89 under IL-6 treatment, which represses CDO1 enzymatic activity by disrupting iron incorporation. Further, AKT1-mediated CDO1 T89 phosphorylation is required for IL-6-elicited oral squamous cell carcinoma (OSCC) growth, and is associated with the progression of OSCC development. The present data discover a new mechanism by which AKT1-mediated CDO1 T89 phosphorylation governs cysteine oxidation to support OSCC growth, thereby highlighting its value as a potential anti-tumor target.

Introduction

A variety of intracellular and extracellular events, such as abnormal accumulation of factors in the tissue environment, imbalanced redox homeostasis, interact to promote carcinogenesis [1]. It is widely documented that the surrounding tissue environment plays a critical role in cancer progression by promoting the survival and multiplication of cancer cells and inhibiting the antitumor immunity [2]. Since the protracted activation of immune system and production of cytokines might promote cancer cell growth, many malignancies originate from the locations with chronic inflammation. Additionally, by releasing signaling molecules, including cytokines, cancer cells themselves can eventually form an inflammatory microenvironment [3]. Therefore, understanding the interactions between tumor cells and the inflammatory environment is critical for the development of novel anti-inflammatory medications for cancer prevention and treatment, as well as new inflammatory biomarkers to track cancer development.

Interleukin-6 (IL-6), a cytokine with diverse functions, often deposits in the microenvironment of solid tumors and hematopoietic malignancies, acting as a key pro-inflammatory agent [4]. STAT3, also known as signal transducer and activator of transcription 3, demands Janus kinases (JAKs)-dependent activation for IL-6 signal to be transduced. In particular, by binding with IL-6 receptor (IL-6R), I6 forms a heterohexamer with glycoprotein 130 (gp130) and IL-6R, which activates JAKs and launches the subsequent signaling pathway. In turn, JAKs phosphorylate gp130 at several tyrosine residues, four of which are found in the C-terminal area and serve for STAT3 docking. Following its association with gp130, STAT3 was phosphorylated by JAKs at tyrosine (Y)705. This allows STAT3 to reach the cell nucleus, functioning as transcription regulatory factor. It ultimately regulates cell phenotypes by stimulating targeting gene transcription [5].

Targeting the IL‑6/IL‑6R axis represents a promising therapeutic strategy for various inflammation-related diseases [6, 7]. IL‑6 blockade was shown as a potential way to disrupt STAT3-mediated tumor proliferation, metastasis, and immunosuppressive microenvironment remodeling [8]. Tocilizumab, an IL-6R blocker, efficiently impairs chromosomal instability-driven tumor growth, and combination of IL-6 blockade with immune checkpoint blockade enhances antitumor CD4+/CD8+ T cells, decoupling toxicity from efficacy in preclinical models [9]. Intriguingly, IL- 6 trans-signaling was recently evidenced to play an essential role in the dissemination of breast cancer cells to bone marrow. IL-6 trans-signaling was able to activate normal and premalignant cells, with an induction of a proliferative stem/progenitor-like phenotype in mammary epithelial cells, and this process in disseminated cancer cells is largely dictated by the niche cells in bone marrow [10]. Moreover, IL-6 trans-signaling was found to act upstream of the AIM2 inflammasome to augment AIM2 expression in emphysema, suggesting that targeting the cross-talk between the AIM2 inflammasome/IL-1β and IL-6 trans-signaling axes may provide potential therapeutic strategy for emphysema [11]. However, the intracellular effectors and downstream signaling cascades for IL-6 in OSCC cells remains substantially elusive.

Cysteine, a sulfur-containing amino acid, is essential in various cellular processes, including antioxidant effect, detoxifying properties and biosynthesis of proteins [12, 13]. Cysteine oxidation is an important metabolic route for the consumption of cellular cysteine. Cysteine can be firstly converted to cysteine sulfinic acid with the addition of molecular dioxygen to its thiol group, which is the first and the rate-limiting step for cysteine oxidation [14]. Cysteine sulfinic acid is then processed by cytosolic aspartate aminotransferase to produce β-Sulfinyl pyruvate, which breaks down spontaneously to produce pyruvate and sulfite, and the latter is further oxidated to sulfate by sulfite oxidase. Alternatively, cysteine sulfinate decarboxylase can convert cysteine sulfinic acid to hypotaurine, which is subsequently oxidized to taurine by hypotaurine dehydrogenase [15]. Cysteine dioxygenase type 1 (CDO1), an iron (Fe2+)-dependent thiol dioxygenase enzyme, catalyzes the cysteine dioxygenation, and so that functions as an important regulator for the metabolic flux of cysteine oxidation pathway [15]. CDO1 has been recently evidenced as a tumor suppressor, tumor cells lacking CDO1 can proliferate and become more invasive, and downregulation of mRNA and protein levels of CDO1 has been observed in a bundle of cancer cell lines and tumor samples [16]. Nevertheless, whether CDO1 and cysteine oxidation is involved in the crosstalk between cancer cell and inflammatory environment is largely unknown. Here, we demonstrate that CDO1 is phosphorylated at threonine (T)89 by AKT1 in OSCC cells in upon IL-6 treatment, which dampens CDO1 activity and cysteine oxidation. Disruption of AKT1-dependent CDO1 T89 phosphorylation retards IL-6-elicited growth for OSCC.

Materials and methods

Materials

Antibodies recognizing AKT2 (#3063), AKT3 (#14,982), AKT1 pS473 (#9018), AKT1 pT308 (#13,038), phosphothreonine (#9386) and STAT3 pY705 (#9145) were purchased from Cell Signaling Technology. Antibody recognizing phosphoserine (AB1603) was purchased from Merck. Human recombinant IL-6 (HZ-1019), TNF-α (HZ-1014) and IL-1β (HZ-1164) protein were purchased from Proteintech. Antibodies recognizing AKT1 (ab233755), AKT1 (ab238477), Tubulin (ab7291), CDO1 (ab232699), Flag (ab205606), and His (ab18184) were purchased from Abcam. Antibody recognizing CDO1 (CBMAB-C3260-YY) was purchased from CreativeBiolabs. [γ-32P]-ATP was purchased from PerkinElmer (BLU002Z001MC). MK-2206 (HY-10358), A-674563 (HY-13254), LY294002 (HY-10108), Tocilizumab (HY-P9917) and PD98059 (HY-12028) were purchased from MedChemExpress. SP600125 (S1460) and Pyridone 6 (S6789) were purchased from Selleck Chemicals. CDO1 pT89 antibody was customized from Boer Biotechnology (Chengdu, China). [1, 2, 1’, 2’-14C] L-Cystine (NEC854050UC), [14C(U)] L-Leucine (NEC279E050UC) and [14C(U)] L-Lysine (NEC280E250UC) were purchased from Revvity.

Cells and IL- 6 treatment

HSC-3 and Huh-7 cell lines were purchased from Japanese Collection of Research Bioresources (JCRB) Cell Bank. CAL-27, HCT116, and DU145 cell lines were purchased from American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum was used as the routing cell culture condition. 50 ng/mL recombinant IL-6 protein was added in the cell culture for IL-6 treatment for diverse time.

Immunoprecipitation and immunoblot

Immunoprecipitation and immunoblot was performed according to the previously reported protocol [17,18,19,20]. The sequences for blocking peptides used in this study were as follows: CDO1 pT89, SSIHDHpT89NSHCFLK; CDO1, SSIHDHT89NSHCFLK. The blocking peptides were used at 25 μg/ml for the working concentration.

shRNAs and vectors

shRNAs used in this study were generated according to the listed sequences: CDO1 shRNA, TTC TTC TGC AGT AGC TGA G (targeting non-coding region); AKT1 shRNA, AGC TGG TGA CAG ACA GCC C; AKT2 shRNA, AAA TTC ATC ATC GAA GTA C; AKT3 shRNA, TGA GGT TTC TCT TTA TAT C; STAT3 shRNA, TAC CTA AGG CCA TGA ACT T (targeting non-coding region); scramble shRNA, GCT TCT AAC ACC GGA GGT CTT. Transfection was performed following previous reports [21, 22].

DNAs were cloned in the mock Flag-pcDNA3.1, His-pcDNA3.1 or pcDNA3.1 vector. Mutations were conducted with QuikChange site-directed mutagenesis kit (Agilent).

Protein expression and purification

Expression of wild type (WT) or mutant CDO1 proteins was performed using pCold I vector, and BL21 (DE3) bacteria [23]. After IPTG stimulation, BL21 bacterial cells were cultured for 16 h at 16℃, and lysed. WT and mutant AKT1 - 3 were expressed by cloning in the pcDNA3.1-Flag vector and expression in 293T cells. AKT1 - 3 DNAs were transfected in 293T cells, and cells were lysed 72 h later.

To purify the His-CDO1 proteins, the lysates were centrifuged and the supernatants were loaded onto a Ni–NTA column (GE Healthcare Life Sciences), washed with 20 mM imidazole and the protein was eluted with 250 mM imidazole. To purify Flag-AKT1, Flag-AKT2, and Flag-AKT3 proteins, the lysates were centrifuged and the supernatants were loaded onto a column containing Anti-Flag M2 agarose affinity gel, washed with PBS and eluted with 100 μg/ml Flag peptide. The eluted protein solution was subjected to a HiPrep 16/60 Sephacryl S-200 HR gel filtration column (GE Healthcare Life Sciences) to remove the contaminants, and the fractions containing the wanted protein were collected [24].

CDO1 enzymatic activity assay

CDO1 enzymatic activity was examined following a reported method with minor modifications [25, 26]. Briefly, assay buffer (50 mM MES buffer pH 6.1, 0.3 mM ammonium iron sulfate, 2.5 mM cysteine, 62.5 μM bathocuproine disulfonate) was freshly prepared and used in this assay. Equal volumes of assay buffer and cell or tissue lysate sample or immunoprecipitation sample was mixed, followed by incubation at 37℃ with shaking for 30 min. The reaction was terminated by addition of 10% (w/v) trichloroacetic acid. Proteins were removed by centrifuge at 10,000 g for 20 min, and 200 µL supernatant was then transferred onto a Dowex minicolumn and eluted with water. 100 µL eluate was subsequently mixed with 50 µL ninhydrin reagent (Sigma Aldrich), and the mixture was incubated at 90℃ for 10 min, followed by addition of 150 µL 95% ethanol. Absorbance of the mixture was measured at 550 nm. As a measurement regarding the binding between CDO1 protein and iron, activity of CDO1 proteins derived from in vitro kinase assay was assayed at various EDTA concentration, following previous report [27].

AKT1-dependent in vitro kinase assay

The experimental procedures followed previous report with minor modifications [23]. AKT1 protein (20 ng) purified from 293T cells was incubated with CDO1 protein purified from bacteria (150 ng) in 25 μL kinase assay buffer (50 mM Tris–HCl [pH 7.5], 100 mM KCl, 5 mM MgCl2, 5% glycerol, 1 mM Na3VO4, 50 μM DTT, 100 μM ATP, 10 μCi [γ-32P]-ATP (if autoradiography performed)) at 30°C for 30 min. CDO1 protein was purified by Ni–NTA beads and separated by SDS-PAGE gels, followed by immunoblotting and autoradiographic analyses.

If ATPγS (ab138911, Abcam) was used for the in vitro kinase assay, the experiment was conducted following a reported protocol [28]. In brief, AKT1 protein and CDO1 protein were incubated in the reaction buffer (20 mM Hepes [pH 7.5], 100 mM NaCl, 1 mM ATPγS, 10 mM MgCl2) at 30°C for 30 min, and 2.5 mM p-Nitrobenzyl mesylate (ab138910, Abcam) was added in the mixture followed by incubation at 25°C for 1 h. CDO1 protein was then isolated and analyzed by immunoblotting using the thiophosphate ester specific antibody (ab92570, Abcam).

Measurement for cellular 14C-pyruvate

To measure the level of cellular pyruvate, pyruvate was firstly converted to acetyl-phosphate by pyruvate oxidase, following previous reports [29, 30]. Briefly, 20 µL cell lysate was mixed with 500 µL reaction buffer (500 µM EDTA; 100 mM potassium phosphate, pH 6.8; 1.0 mM MgSO4; 10 μM FAD, 0.2 mM thiamine pyrophosphate, and 0.2 U/ml pyruvate oxidase) for reaction at 30°C for 30 min. Acetyl-phosphate was then separated through a two-dimensional thin layer chromatography-based method using 10 cm × 10 cm EMD PEI cellulose-F plates (EMD Chemicals, La Jolla, CA). The first dimension was developed for 45 min with a solvent system containing 0.52 M LiCl and 1% (v/v) glacial acetic acid, dried in air, incubated in methanol for 15 min, dried in air again, and developed in the second dimension for another 90 min. The spot corresponding to acetyl-phosphate was evaluated using liquid scintillation counter. The relative level of cellular 14C-pyruvate was calculated based on the radioactive signal from acetyl-phosphate. The results were normalized to the cell numbers.

Determination of radioactivity in total protein

The radioactivity in total protein was measured according to a previous report [31]. After indicated labeling, cells were homogenized on ice. Sodium hydroxide was added in the cell homogenate to 0.5 M, followed by incubation for 15 min at 37°C. 10% TCA was then added in the mixture to precipitate the protein. The precipitates were transferred onto glass filters and washed with 5% TCA. The radioactive signal of the filter was evaluated using liquid scintillation counter. The values of radioactivity were normalized to the amount of total protein.

Colony formation assay

Cells were seeded in 6-well plates with medium containing or not containing 50 ng/ml IL- 6. The cells were allowed to adhere and culture for 14 days. The medium was refreshed every three days. The colonies were then fixed with methanol for 10 min, followed by staining with 0.05% (w/v) crystal violet for 10 min. The colonies were washed five times with PBS, and only those clones containing over 50 cells were selected and quantified under a microscope [32].

Measurement of BrdU incorporation

Measurement of BrdU incorporation was conducted using the BrdU Cell Proliferation ELISA Kit (colorimetric) from Abcam (cat# ab126556). Briefly, 3 × 104 cells were plated in each well of a 96-well plate. On the second day, cells were exposed to 50 ng/ml IL- 6 for 8 h. Subsequently, 20 µL diluted 1 × BrdU solution was mixed in each well, followed by incubation for 6 h. After BrdU incorporation, cells were fixed, treated with detection antibodies, and the signal was measured at 450 nm with a microplate reader.

Establishment of mouse tumor xenograft

For each mouse, 2 × 105 HSC-3 cells were implanted into 6–8 weeks old BALB/c nude mice by subcutaneous injection. After day 9, tumor volume was monitored every three days. Mice were euthanized and tumors were excised at Day 27. Tumor size was calculated using the formula: 1/2 × length × width2. Tocilizumab or control IgG was intraperitoneally administered at 10 mg/kg once per week. All animal procedures were approved by the institutional review board of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D-2022-528), and were conducted in accordance with the relevant institutional and national guidelines and regulations.

Clinical samples and immunohistochemical staining

Immunohistochemical staining was conducted using the Vectastain ABC kit (Vector Laboratories, CA). Staining was scored based on a previously reported method [24, 33, 34], where the proportion of positively stained cells was categorized as follows: 0, no cells with positive staining; 1, 1–10%; 2, 11–30%; 3, 31–70%; and 4, 71–100%. The intensity of staining was graded as 0, negative; 1, weak; 2, moderate; 3, strong; and 4, very strong. The final score for each sample was obtained by multiplying the proportion score by the intensity score. The use of human OSCC specimens was approved by the institutional review board of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D- 2022-372), in accordance with institutional and ethical regulations.

Quantification and statistical analysis

All experiments were repeated at least three times. All data showed as mean ± SD and data were analyzed using the two-sided Student’s t test or ANOVA test, unless otherwise indicated. Statistically significant was considered as P < 0.05.

Results

IL-6 treatment represses CDO1 activity and cysteine oxidation

To analyze the cysteine oxidation pathway, two OSCC cell lines, HSC-3 and CAL-27 that were documented to be IL-6-responsive [28, 35], were used as cell models. Cells were treated with 14C-labeled cystine, which are commonly used as a cysteine source in cell culture medium, and the metabolic flux was determined by measuring the generation of 14C-labeled pyruvate, an important product of cysteine oxidation (Fig. 1A). Notably, a markedly lowered level of 14C-labeled pyruvate was detected after IL-6 treatment, hinting that cysteine consumption through cysteine oxidation was repressed (Fig. 1B). Consistently, an increased level of cellular cysteine was found in the both IL-6-treated HSC-3 and CAL-27 cells (Fig. 1C). IL-6-induced accumulation of cellular cysteine was not likely due to augmented uptake of environmental cystine, as comparable radioactive signal was detected in the cell lysate from IL-6-treated or untreated cells after incubation with 14C-labeled cystine (Fig. 1D). These results suggest that cysteine oxidation was repressed in OSCC cells upon IL-6 treatment.

Fig. 1
figure 1

IL- 6 treatment inhibits CDO1 activity in OSCC cells. A Schematic illustration of cysteine oxidation pathway. ○, non-radioactive carbon; ●, 14C-labeled carbon. B-F HSC-3 and CAL-27 cells were priming with 50 ng/mL IL- 6 for the specified duration. B The amount of 14C-pyruvate was measured from cells incubated with [1, 2, 1’, 2’-14C]-cystine (0.1 μCi/ml) for 0.5 h. The data was shown as mean ± SD from three replicates. ** P < 0.01, *** P < 0.001. C The cellular level of cysteine was measured. * P < 0.05, ** P < 0.01. D The radioactive signal level was evaluated in the WCL from cells incubated with [1, 2, 1’, 2’-.14C]-cystine (0.1 μCi/ml) for 0.5 h. The data was shown as mean ± SD from three replicates. ns, not significant. WCL, whole cell lysate. E The CDO1 activity in cell lysates was measured. The data was shown as mean ± SD from three replicates. * P < 0.05, ** P < 0.01, *** P < 0.001. F Immunoblot analyses of CDO1 expression. The data were collected from three replicates. G Flag-CDO1 was transfected into HSC-3 and CAL-27 cells, and cells were then treated with IL-6 (50 ng/mL) for the specified duration. Anti-Flag M2 antibody was used for immunoprecipitation, and the CDO1 activity in the precipitates was determined. The data was shown as mean ± SD from three replicates. * P < 0.05, **P < 0.01, ***P < 0.001

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CDO1-mediated cysteine dioxygenation, which consumes oxygen and generates cysteine sulfinic acid, is the rate-limiting step for cysteine oxidation pathway [15]. IL-6 treatment reduced cysteine dioxygenation activity by more than 50% in the cell lysates, and this effect could be observed as early as 1 h under IL-6 treatment (Fig. 1E). Reduced cysteine dioxygenation activity in cell lysate could be also observed in IL-6-treated hepatocellular carcinoma cell line Huh-7, colorectal cancer cell line HCT116 and prostate cancer cell line DU145 (Supplementary Figure S1 A), or TNF-α or IL-1β-treated HSC-3 cells (Supplementary Figure S1B). In contrast, CDO1 protein expression was rarely altered (Fig. 1F), hitting that IL-6-repressed cysteine dioxygenation activity did not simply result from the changes in CDO1 protein expression. To test whether IL-6 treatment affect CDO1 enzymatic activity, equal amount of Flag-tagged CDO1 protein was precipitated from untreated or IL-6-treated cells, and revealed a markedly decreased activity for the CDO1 protein derived from IL-6-treated cells (Fig. 1G). These data indicated that IL-6 treatment inhibits CDO1 activity and cysteine oxidation in OSCC cells.

AKT1 phosphorylates CDO1 at T89 in response to IL-6 treatment

To determine the intracellular factors that was responsible for IL-6-mediated CDO1 suppression, a bundle of inflammation-responsive signaling cascades were abrogated by small-molecule inhibitors, respectively. Incubation with MK-2206, a pan inhibitor for AKT kinases, pronouncedly restored IL-6-repressed CDO1 activity (Fig. 2A). In contrast, treatment with PD98059 (MAPK inhibitor), SP600125 (JNK inhibitor), or Pyridone 6 (JAK inhibitor) only exhibited negligible effects (Fig. 2A). Three highly homologous AKT isoforms are currently known in mammalian cells, including AKT1, AKT2 and AKT3 [22, 36]. Silence of each of them by specific shRNA unveiled that only loss of AKT1 substantially restored CDO1 activity in IL-6-stimulated cells (Fig. 2B). Though more widely recognized as an important cellular effector of growth factors, it is documented that AKT1 could be also activated by IL-6 and in turn mediates downstream signal transduction of IL- 6 [37,38,39]. Indeed, treatment with IL-6 largely induced AKT1 activation in HSC-3 cells, evidenced by the increased level of AKT1 serine (S)473 and threonine (T)308 phosphorylation and TSC2 threonine (T)1462 phosphorylation, a bona fide AKT1 substrate (Fig. 2C and Supplementary Figure S2 A) [40]. Incubation of bacterially purified Flag-AKT1 protein and His-CDO1 protein revealed that these two proteins could directly bind to each other (Fig. 2D). Further, co-immunoprecipitation analyses showed that the interaction between CDO1 and AKT1 in HSC-3 and CAL-27 cells could be exceedingly enhanced upon IL-6 treatment (Fig. 2E). In contrast, interaction between CDO1 and AKT2 or AKT3 were barely detected (Supplementary Figure S2B-S2 C). AKT1 is a multifaceted protein implicated in the regulation of cell growth, mainly through functioning as a serine/threonine protein kinase, and we thus opted to perform an in vitro kinase assay to test if CDO1 was a novel substrate for AKT1. We mixed purified Flag-AKT1 protein and His-CDO1 proteins with [γ-32P]-ATP, which was used as a traceable phosphate group donor, and phosphorylation of His-CDO1 protein was determined by autoradiography. Notably, addition of [γ-32P]-ATP in the reaction system led to a significant autoradiographic band corresponding to His-CDO1, which was undetectable when Flag-AKT1 protein was not included in the reaction system (Fig. 2F). Further, this autoradiographic band was not likely due to non-covalently binding between [γ-32P]-ATP and His-CDO1 protein, since His-CDO1 protein was isolated by nickel beads pulldown and separated by SDS-PAGE before subjected to autoradiography. In line with this, this autoradiographic band could not be induced by a kinase-dead AKT1 K179M mutant protein (Fig. 2F) [41]. These results strongly suggest that AKT1 directly phosphorylates CDO1.

Fig. 2
figure 2

AKT1 phosphorylates CDO1 T89 under IL-6 treatment. A-H, J-P Immunoblotting analysis was conducted by the specified antibodies. A HSC-3 cells were pre-treated with PD98059 (20 μM), SP600125 (20 μM), Pyridone 6 (2 µM) or MK-2206 (5 µM) for 2 h, followed by incubation with IL-6 (50 ng/mL) for 1 h. The cell lysates were then immunoprecipitated using anti-Flag M2 antibody and CDO1 activity was measured. The data was shown as mean ± SD from three replicates. ** P < 0.01; ns, not significant. B Specified shRNA and/or Flag-CDO1 was transfected into HSC-3 cells. Cells were challenged with IL-6 (50 ng/mL) for 1 h. The cell lysates were then immunoprecipitated using anti-Flag M2 antibody. CDO1 activity was measured. Data was shown as mean ± SD from three replicates. ** P < 0.01; ns, not significant. C HSC-3 cells were treated with or without A-674563 (10 µM) for 2 h, and then incubated with IL-6 (50 ng/mL) for indicated time. D Purified Flag-AKT1 and His-CDO1 protein were mixed for an Ni–NTA pulldown assay. E HSC-3 and CAL-27 cells were treated with or without IL-6 (50 ng/mL) for 1 h, followed by co-immunoprecipitations using the specified antibodies. The data were collected from three replicates. WCL, whole cell lysate. F In vitro kinase assay was conducted by incubating purified recombinant His-CDO1 protein with WT Flag-AKT1 or Flag-AKT1 K179M mutant protein using [γ-32P]-ATP as the phosphate group donor, followed by Ni–NTA agarose bead pulldown and autoradiography. The data were collected from three replicates. G HSC-3 cells were treated with or without IL-6 (50 ng/mL) for 1 h. H In vitro kinase assay using purified WT His-CDO1 recombinant protein, designated mutants, and/or purified Flag-AKT1 protein was performed in the presence of ATPγS, followed by Ni–NTA agarose bead pulldown and detection using the thiophosphate ester (TE)-specific antibody. The data were collected from three replicates. I Alignment of adjacent sequence of CDO1 T89 site for the designated species. T89 is shown in red. J HSC-3 cells were incubated with IL-6 (50 ng/mL) for 1 h. Immunoblot of CDO1 T89 phosphorylation was performed in the presence or absence of CDO1 pT89 blocking peptide. The data were collected from three replicates. K HSC-3 or CAL-27 cells stably expressing WT Flag-CDO1 or Flag-CDO1 T89A mutant were treated with IL-6 (50 ng/mL) for 1 h, and then cell lysates were subjected to immunoprecipitation using anti-Flag M2 antibody. The data were collected from three replicates. L HSC-3 cells were pre-treated with A-674563 (10uM) for 2 h and cells were subsequently incubated with IL-6 (50ng/mL) for 1 h. The data were collected from three replicates. M AKT1-shRNA-expressed HSC-3 cells were exposed to IL-6 (50 ng/mL) for 1 h. The data were collected from three replicates. O HSC-3 cells were transfected with HA-Myr-AKT1. The data were collected from three biological replicates. N HSC-3 or CAL-27 cells were pre-treated with LY294002 (10 µM) for 2 h and cells were subsequently incubated with IL-6 (50ng/mL) for 1 h. The data were collected from three replicates. P We generated stable expression of STAT3 shRNA, WT His-STAT3 or His-STAT3 Y705 F in HSC-3 cells. Cells were treated with IL-6 (50ng/mL) for 1 h, followed by Ni–NTA pulldown. The shRNA constructs target STAT3 mRNA’s non-coding region. The data were collected from three replicates

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To identify the phosphorylation site, we analyzed Flag-CDO1 protein derived from untreated or IL-6-treated HSC-3 cells by immunoblot with a pan anti-phosphoserine antibody or a pan anti-phosphothreonine antibody, and found that only threonine phosphorylation was increased (Fig. 2G). Mutation of a couple of threonine residues, which were located on the protein surface, based on the reported human CDO1 protein structure (PBD code: 2IC1), showed that only mutation of T89 abolished AKT1-mediated phosphorylation (Fig. 2H). In addition, alignment analyses revealed that the flanking sequences of CDO1 T89 were highly conserved during evolution (Fig. 2I).

To better investigate this modification, we generated an antibody that recognizes CDO1 T89 phosphorylation (pT89). With this antibody, immunoblot using whole cell lysate revealed a markedly increased CDO1 T89 phosphorylation after IL-6 treatment, and no immunoreactivity was detected when a CDO1 pT89 blocking peptide was applied, indicating a good specificity for this antibody (Fig. 2J). CDO1 T89 phosphorylation could be also detected in IL-6-treated Huh-7, HCT116 and DU145 cells (Supplementary Figure S2D), or TNF-α or IL-1β-treated HSC-3 cells (Supplementary Figure S2E). In line with the aforementioned results from in vitro kinase assay, IL-6-induced CDO1 T89 phosphorylation could be blocked by replacement of T89 into a non-phosphorylatable alanine and largely attenuated by treatment with A-674563, AKT1 shRNA or a PI3K inhibitor LY294002 (Fig. 2K- N). Notably, with the expression of Myr-HA-AKT1, a constitutively active form of AKT1 [42], CDO1 T89 phosphorylation could be sharply enhanced in HSC-3 cells even in the absence of IL-6 treatment (Fig. 2O). Though STAT3 plays a crucial role for IL-6 signal transduction, STAT3 was not likely involved in IL-6-induced CDO1 T89 phosphorylation, since interception of IL-6-dependent STAT3 activation by the STAT3 Y705 F mutation barely changed the accumulation of CDO1 T89 phosphorylation in IL-6-treated cells (Fig. 2P). These results suggest that AKT1 phosphorylates CDO1 T89 in response to IL-6.

AKT1-mediated T89 phosphorylation reduces CDO1 enzymatic activity

We then examined whether T89 phosphorylation affects CDO1 enzymatic activity in the context of IL-6 treatment. We isolated wild type His-CDO1 or His-CDO1 T89A protein from an AKT1-dependent in vitro kinase assay, and found that AKT1-mediated CDO1 T89 phosphorylation reduced the activity of WT CDO1 protein by more than 60%, while the activity of CDO1 T89A mutant stayed intact (Fig. 3A). In addition, T89A mutation show no detectable effects on CDO1 activity when AKT1 protein was not included in the kinase assay (Fig. 3A). Consistently, T89A mutation substantially restored CDO1 activity in both IL-6-treated HSC-3 and CAL-27 cells (Fig. 3B), suggesting that this modification inhibits CDO1 activity.

Fig. 3
figure 3

T89 phosphorylation inhibits CDO1 by disrupting CDO1 binding with iron. A, B, E–H Immunoblotting analyses were conducted using the specified antibodies. A In vitro kinase assay with purified WT His-CDO1, His-CDO1 T89A mutant protein, and/or purified Flag-AKT1 protein was conducted. Followed by Ni–NTA pulldown, the enzymatic activity of CDO1 within the precipitates was evaluated. The data were shown as mean ± SD from three replicates. *** P < 0.001. B HSC-3 or CAL-27 cells stably expressing either WT Flag-CDO1 or Flag-CDO1 T89A was treated with IL-6 (50 ng/mL) for 1 h. CDO1 protein was isolated from cell lysates and the activity of CDO1 protein was measured. The data was shown as mean ± SD from three replicates. WCL, whole cell lysate; ** P < 0.01. C, D The catalytic site of human CDO1 protein (PDB code: 6BGF) was boxed. (C) and the binding mode for iron (shown in orange) and cysteine (shown in grey) are shown. (D) H88 residue was shown in yellow and T89 residue was shown in purple. The protein structure data was analyzed by PyMOL. E HSC-3 cells expressing WT Flag-CDO1 or Flag-CDO1 H88A were treated with IL-6 (50 ng/mL) for 1 h. Flag-CDO1 protein was isolated and CDO1 enzymatic activity was measured. The data were shown as mean ± SD from three replicates. WCL, whole cell lysate; ** P < 0.01. F Purified His-CDO1 protein was mixed with or without purified Flag-AKT1 for in vitro kinase assay. His-CDO1 recombinant proteins were isolated and subjected to CDO1 activity assay in the presence of increasing concentration of EDTA. For all groups, the CDO1 activity tested at various EDTA concentration was converted to the percentage of the CDO1 activity tested in the absence of EDTA. The data were mean ± SD from three replicates. G HSC-3 cells expressing WT Flag-CDO1 or Flag-CDO1 T89A for 1 h. Isolated Flag-CDO1 protein from cell lysates was subjected to CDO1 enzymatic activity assay in the presence of increasing concentration of EDTA. For all groups, the CDO1 activity tested at various EDTA concentration was converted to the percentage of the CDO1 activity tested in the absence of EDTA. The data was shown as mean ± SD from three replicates. H HSC-3 cells expressing WT Flag-CDO1 were treated with 10 µM A-674563 for 2 h and followed by exposure to IL-6 (50ng/mL) for 1 h. Isolated Flag-CDO1 protein from cell lysates was subjected to CDO1 activity assay in the presence of increasing concentration of EDTA. For all groups, the CDO1 activity tested at various EDTA concentration was converted to the percentage of the CDO1 activity tested in the absence of EDTA. The data were mean ± SD from three replicates

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According to the protein structure of human CDO1 that was previously generated and deposited in PDB database by other groups [43], one iron molecule and one cysteine molecule were visible in the active site (Fig. 3C). T89 was located between β sheet 3 and 4 with its side chain on the protein surface, therefore T89 was not likely to have direct contact with either the iron or the cysteine molecule (Fig. 3D). CDO1 is a non-heme iron enzyme, and incorporation of iron molecules is indispensable for its enzymatic activity [15]. CDO1 histidine (H)88, which is participating in coordinating the iron in the active site [44], was just adjacent to T89 (Fig. 3D). In addition, H88A mutation largely abolished CDO1 activity, but this effect could not be further enhanced by AKT1-mediated phosphorylation though in vitro kinase assay (Fig. 3E). These findings led to a hypothesis that T89 phosphorylation may perturbate the interaction between H88 and iron molecule, and so that it prompted us to further test whether T89 phosphorylation affect iron incorporation in CDO1. His-CDO1 protein isolated from AKT1-dependent in vitro kinase assay and its untreated counterpart were subjected to enzymatic activity assay in the presence of increasing concentration of the metal chelator EDTA. The activity of untreated His-CDO1 protein was progressively reduced with increasing EDTA concentration, and about 80% activity was abolished at EDTA concentrations above 0.5 mM (Fig. 3F). In contrast, His-CDO1 protein with T89 phosphorylation was inactivated at much lower EDTA concentration with roughly 70% decrease in activity at 0.1 mM EDTA, while only less than 10% decrease was found for untreated His-CDO1 protein at this EDTA concentration (Fig. 3F). These results indicated that the activity of COD1 protein with T89 phosphorylation was more sensitive to iron chelation, suggesting a reduced iron-binding affinity. In line with this, the curve for the activity of the CDO1 protein derived from IL-6-treated cells was more steeply changed than that from untreated cells, and this phenomenon could be substantially abolished by T89A mutation or the use of AKT1 inhibitor A-674563 (Fig. 3G,H). These results suggest that AKT1-dependent T89 phosphorylation reduces CDO1 activity by disrupting iron incorporation.

CDO1 T89 phosphorylation is required for IL-6-mediated inhibition of cysteine oxidation

To manipulate CDO1 T89 phosphorylation in OSCC cells, we replaced endogenous WT CDO1 by knocking down endogenous CDO1 with specific shRNA and expressing similar level of exogenous WT Flag-CDO1 or Flag-CDO1 T89A (Fig. 4A). This shRNA recognizes the non-coding region of CDO1 mRNA, and the targeted sequence did not exist in the mRNA corresponding to exogenous CDO1. Therefore, the expression of exogenous CDO1 was not interfered by it. Consistently, CDO1 phosphorylation was palpably induced for WT Flag-CDO1 rather than Flag-CDO1 T89A after IL-6 treatment in both HSC-3 and CAL-27 cells (Fig. 4B). By using 14C-cystine as a tracer, 14C-pyruvate production that was abated under IL-6 treatment in WT CDO1-expressed cells was largely restored in non-phosphorylatable CDO1 T89A mutant-expressed cells (Fig. 4C), suggesting that CDO1 T89 phosphorylation is required for IL-6-repressed cysteine oxidation. Accordingly, IL-6-induced accumulation of cellular cysteine was substantially alleviated by CDO1 T89A mutation (Fig. 4D). In addition, CDO1 T89A mutation showed minor effect on cysteine oxidation and cellular cysteine level in the absence of IL-6 treatment (Fig. 4C, D). The availability of amino acids sustains the protein synthesis in rapidly proliferating cells [45,46,47]. Therefore, the impact of CDO1 T89 phosphorylation on cellular cysteine availability prompted us to further test whether protein synthesis was changed. Indeed, after incubation with 14C-labeled cystine, leucine or lysine, IL-6-treatment induced much more radioactive signal in total protein extracts, which was largely abolished by CDO1 T89A mutation (Fig. 4E, Supplementary Figure S3 A-S3 B). These results suggest that CDO1 T89 phosphorylation is required for IL-6-mediated inhibition of cysteine oxidation, and supports protein synthesis.

Fig. 4
figure 4

CDO1 T89 phosphorylation is required for IL-6-mediated inhibition of cysteine oxidation. A-E CDO1 shRNA, WT Flag-CDO1 or Flag-CDO1 T89A was stably expressed in HSC-3 or CAL-27 tumor cells. The shRNA specifically targeted CDO1 mRNA’s non-coding region. A Immunoblots using the specified antibodies were conducted. The data were collected from three replicates. B Cells were challenged with IL-6 (50 ng/mL) for 1 h, and cell lysates are subjected to immunoprecipitation using anti-Flag M2 antibody. Subsequently, immunoblots with the indicated antibodies were performed. The data were collected from three replicates. C Cells were pretreated with IL-6 (50 ng/mL) for 4 h, and then incubated in the presence of [1, 2, 1’, 2’-14C]-cystine (0.1 μCi/ml) for 1 h. The amount of 14C-pyruvate was determined. The data was shown as mean ± SD from three replicates. ** P < 0.01, *** P < 0.001. D Cells were treated with IL-6 (50 ng/mL) for 4 h, followed by measurement for the cellular level of cysteine. ** P < 0.01, *** P < 0.001. E Cells were pretreated with IL-6 (50 ng/mL) for 4 h, and cultured with [1, 2, 1’, 2’-.14C]-cystine (0.1 μCi/ml) for 3 h. The cells were washed and the total protein was extracted, and the radioactive signal was evaluated. The data was shown as the mean ± SD from three replicates. * P < 0.05

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CDO1 T89 phosphorylation is required for IL-6-induced OSCC cell proliferation and tumor growth

We next determined whether CDO1 T89 phosphorylation affects OSCC cell proliferation. As expected, IL-6 treatment accentuated the proliferation in both HSC-3 and CAL-27 cells with the exogenous expression of WT CDO1, shown by both BrdU incorporation assay and colony formation assay (Fig. 5A and B). In contrast, those cells expressing non-phosphoryable CDO1T89 A mutant only showed limited augment in cell proliferation (Fig. 5A and B), suggesting that CDO1 T89 phosphorylation is required for IL-6-induced OSCC cell proliferation. To expand our findings, a mouse xenograft model was established by subcutaneous injection of HSC-3 cells with overexpression of IL-6 and reconstituted expression of WT CDO1 or CDO1 T89A. Similar to many other reports [48, 49], heightened IL-6 expression promoted tumor growth, evidenced by the larger tumor size and the increased Ki-67 signal by immunohistochemistry (IHC) staining (Fig. 5C,D). Strikingly, replacement of endogenous CDO1 with Flag-CDO1 T89A mutant, which mitigated IL-6-induced CDO1 T89 phosphorylation (Fig. 5E), counteracted IL-6-induced tumor growth (Fig. 5C, D). In addition, the immunohistochemical signal of CDO1 T89 phosphorylation in I- 6-overexpressed tumors could be clearly abolished when a CDO1 pT89 blocking peptide was used, suggesting that this antibody worked well for IHC staining (Fig. 5F). Furthermore, we also used Tocilizumab, an IL-6 receptor antagonist, to treat the tumor-bearing mice to inhibit IL-6 signaling. As results, treatment with Tocilizumab largely reduced IL-6-mediated tumor growth, Ki-67 expression, CDO1 T89 phosphorylation, and restored CDO1 activity in xenograft tissue (Supplementary Figure S4 A-S4D).

Fig. 5
figure 5

CDO1 T89 phosphorylation is required for IL-6 induced OSCC cell proliferation and tumor growth. A CDO1 shRNA, WT Flag-CDO1 or Flag-CDO1 T89A was stably expressed in HSC-3 or CAL-27 cells. Cells were treated with IL-6 (50 ng/mL) before BrdU incorporation assay. The shRNA specifically targeted CDO1 mRNA’s non-coding region. The data was shown as mean ± SD from three replicates. * P < 0.05, ** P < 0.01. B Colony formation assay of HSC-3 or CAL-27 tumor cells that stably expressed CDO1 shRNA, WT Flag-CDO1 or Flag-CDO1 T89A was performed in the presence or absence of IL-6. The shRNA specifically targeted CDO1 mRNA’s non-coding region. The data was shown as mean ± SD from three biological replicates. ** P < 0.01. C HSC-3 cells which stably expressed CDO1 shRNA, WT Flag-CDO1, Flag-CDO1 T89A or/and IL-6 were subcutaneously injected into Balb/c nude mice (n = 7). Images of two representative xenografts from each group are shown in the left panel. The tumor volume was displayed in the right panel. *** P < 0.001. D Ki-67 immunohistochemical staining. Scale bar, 60 µm. E Immunohistochemical staining for the indicated protein. Scale bar, 60 µm. F The immunohistochemical staining was performed using the anti-CDO1 pT89 antibody with or without the specified blocking peptides. Mouse tumor samples with the expression of IL-6 and WT Flag-CDO1 were used for this staining. The data was collected from three replicates. Scale bar, 80 µm. G, H Immunohistochemical staining of 50 human OSCC samples using the specified antibodies. G Images of four representative samples are shown. H Staining score were analyzed by linear regression (50 cases). If more than two samples received the same scores for both IL-6 and CDO1 pT89 staining, their corresponding dots overlap and are shown as a single dot. Scale bar, 100 µm. I Comparison of immunohistochemical staining scores between stage I/II group and stage III/IV group. J Comparison of immunohistochemical staining scores between well differentiated group and moderately/poorly differentiated group

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To examine the clinical relevance of CDO1 T89 phosphorylation, 50 clinical OSCC samples were collected. IHC staining showed that strong CDO1 T89 phosphorylation was apparently observed in those OSCC samples with high IL-6 expression (Fig. 5G). The linear regression analyses unveiled that the IL-6 expression was in positive correlation with the level of CDO1 T89 phosphorylation (Fig. 5H). Further, a high level of CDO1 T89 phosphorylation was more frequently detected in advanced TNM stages (stage III and IV) or moderately/poorly differentiated samples compared to the early stages (stage I and II) or well differentiated samples (Fig. 5I and J). These results suggest that AKT1-dependent CDO1 phosphorylation plays important role in IL-6-induced OSCC cell proliferation and tumor progression.

Discussion

OSCC is a frequent type of head and neck cancer, accounting for roughly 380,000 new cases globally each year [50, 51]. Inflammatory pathways are often hijacked by cancer cells to further their own proliferation and survival [52]. An inflammatory microenvironment is continually created when cancer cells interact with nearby matrix and inflammatory cells [53]. Additionally, within this sort of tumor microenvironment, the quick growth of cancer cells triggers homeostatic reactions by expanding the quantity of macrophages, as demonstrated by the presence of signaling pathways consisting of diverse cytokines that are reciprocally produced by one cell type and exploited by the other [3]. As one of the primary cytokines found in tumor microenvironment, IL-6 is found elevated in nearly all cancer types and is abundant in tumor tissues [54]. Consequently, investigating the interactions between cancer cells and the inflammatory environment would be ideally done by tracing IL-6-mediated signal pathway in cancer cells.

Cysteine dioxygenation catalyzed by CDO1, which consumes oxygen and generates cysteine sulfinic acid, is the rate-limiting step for cysteine oxidation pathway [14]. CDO1 has been found to function as a tumor suppressor to play a critical role in downregulating tumor cell growth and invasiveness, and deficiency of CDO1 expression has been reported in various type of cancers to date [16, 55]. In the present study, we determined the impact of IL-6 treatment on CDO1 in OSCC cells by using 14C-labeled cystine as a tracer. Treatment with IL-6 induced apparently lower level of 14C-pyruvate and increased level of cellular cysteine, indicating that cysteine consumption through oxidation was repressed. By precipitating Flag-tagged CDO1 protein, we found that enzymatic activity of the CDO1 protein derived from IL-6-treated cells decreased markedly. Hence, our data strongly suggest that IL-6 treatment represses CDO1 activity and cysteine oxidation. Since our data also show that AKT1-mediated CDO1 activity repression and T89 phosphorylation was also observed in IL-6-treated hepatocellular carcinoma cell line Huh-7, colorectal cancer cell line HCT116 and prostate cancer cell line DU145, as well as TNF-α or IL-1β-treated OSCC cells, it is reasonable to inter that AKT1-CDO1 axis might be common target for the regulation of multiple inflammatory cytokines in multiple types of cancer.

It is documented that AKT1 could be activated by IL-6 and in turn mediates downstream signal transduction of IL-6 [39]. We report here that CDO1 is a novel AKT1 phosphorylation substrate. We developed an in vitro kinase test employing purified recombinant proteins, and the results demonstrated that AKT1 could directly phosphorylate CDO1 at T89. This phosphorylation was induced by IL-6 treatment, and could be inhibited by non-phosphorylatable threonine-to-alanine mutation as well as treatment with A-674563 or AKT1 shRNA. Interestingly, with the expression of HA-Myr-AKT1, a constitutively active form of AKT1, CDO1 T89 phosphorylation could be sharply enhanced even without IL-6 treatment, further suggesting that AKT1 mediates IL-6-induced CDO1 T89 phosphorylation. Moreover, we assessed the enzymatic activity of CDO1 protein with or without T89 phosphorylation in the context of EDTA-mediated metal chelation, and revealed that AKT1-mediated T89 phosphorylation inhibits CDO1 activity by disrupting iron incorporation. Though many AKT1-mediated phosphorylation were found to happen in the motif sequence in substrate proteins, accumulating evidences have showed that AKT1-mediated phosphorylation might not be limited to the motif sequence, such as AEP T332 [56], LonP1 S181 [57], and HK1 S178 [58]. Together with these reported substrates without the traits of motif sequence, our data reflects the complexity of the mode for AKT1 substrate recognition, which requires further investigation.

Amino acids are fundamental to mammalian cells, serving as substrates for protein synthesis and supporting cell proliferation. Tumor cells, characterized by rapid growth and heightened metabolic activity, have an increased demand for cysteine for the elevated rate of protein synthesis [45,46,47, 51]. CDO1 consumes intracellular cysteine levels by converting it to cysteine sulfinic acid, thus governing cellular cysteine availability [43, 59]. In this study, we incubated HSC-3 and CAL-27 cells with 14C-labeled cystine, leucine or lysine, and found that IL-6-treatment induced much more radioactive signal in total protein extracts, which was largely abolished by CDO1 T89A mutation. This observation is consistent with previous reports showing that IL-6 is capable to enhance global protein synthesis [60, 61]. Further, we show that CDO1 T89A mutation, which is resistant to AKT1-dependent phosphorylation and maintains CDO1 enzymatic activity, retarded IL-6-induced OSCC tumor growth. Therefore, our data suggest that cellular cysteine availability might be a vulnerability of tumor cells that is exploitable in clinical treatment.

The present data reveals a novel protein phosphorylation-based mechanism that stimulates cancer cell proliferation and tumor growth (Fig. 6). AKT1-mediated CDO1 T89 phosphorylation reduces the iron-binding affinity and enzymatic activity of CDO1. This molecular event is required for IL-6-induced OSCC growth, highlighting the complex interaction between tumor cells and the inflammatory environment, and might be used as potential target for OSCC treatment.

Fig. 6
figure 6

An illustration of IL-6/AKT1 axis-mediated phosphorylation of CDO1 at T89 promoting OSCC proliferation. IL-6 stimulation activates AKT1, which phosphorylates CDO1 at T89, leading to the inhibition of CDO1 enzymatic activity and suppression of cysteine oxidation. It results in the aberrant accumulation of cysteine, thereby enhancing protein synthesis to support the proliferation of OSCC

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Data availability

Data is provided within the manuscript or supplementary information files.

Abbreviations

CDO1:

Cysteine Dioxygenase Type 1

IL-6:

Interleukin-6

IL-6R:

IL-6 receptor

JAK:

Janus kinase

OSCC:

Oral squamous cell carcinoma

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Acknowledgements

Not applicable

Funding

This work was supported by Sichuan Science and Technology Program 2023 NSFSC1924 (R.L.), and R&D Project of West China Hospital of Stomatology RD- 03–202404 (R.L.).

Author information

Author notes

  1. Xin Li and Zhe Zhao contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Chinese Academy of Medical Sciences Research Unit of Oral Carcinogenesis and Management, Department of Oral Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, 610041, China

    Xin Li, Hongping Ye, Dan Li & Rui Liu

  2. The Second Affiliated Hospital of Chengdu Medical College Nuclear Industry 416 Hospital, Chengdu, China

    Zhe Zhao

  3. Department of Urology, Xindu District People’s Hospital of Chengdu, Chengdu, 610500, People’s Republic of China

    Xiaoke Huang

  4. Department of Health Sciences, The Graduate School of Dong-a University, Busan, 49315, Republic of Korea

    Jong-Ho Lee

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  1. Xin Li
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  2. Zhe Zhao
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  4. Dan Li
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Contributions

Conceptualization: R.L., X.H. and J.-H. L.; methodology: X.L., H.Y., and D.L.; investigation: X.L., H.Y., Z.Z., D.L. X.H. and J.-H. L.; writing (original draft): R.L.; writing (review and editing): R.L.; funding acquisition: R.L.. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xiaoke Huang, Jong-Ho Lee or Rui Liu.

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Ethics approval and consent to participate

The use of human OSCC specimens was approved by the institutional review board of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D- 2022–372), in accordance with institutional and ethical regulations. All animal procedures were approved by the institutional review board of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D- 2022–528), and were conducted in accordance with the relevant institutional and national guidelines and regulations.

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The authors declare no competing interests.

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Supplementary Information

12964_2025_2189_MOESM1_ESM.zip

Supplementary Material 1: Figure S1 Treatment with inflammatory factors inhibit CDO1 activity. (A) Huh-7, HCT116 and DU145 cells were treated with 50 ng/mL IL-6 for 2 h. The CDO1 activity in cell lysates was measured. The data was shown as mean ± SD from three replicates. ** P < 0.01, *** P < 0.001. (B) HSC-3 cells were treated with 50 ng/ml TNF-α or 5 nM IL-1β for 2 h. The CDO1 activity in cell lysates was measured. The data was shown as mean ± SD from three replicates. * P < 0.05, ** P < 0.01. Figure S2 AKT1 phosphorylates CDO1 T89 under treatment with inflammatory factors. (A-E) Immunoblotting analyses was performed using indicated antibodies. (A) HSC-3 cells were treated with 50 ng/mL IL-6 for 2 h. (B) Purified Flag-AKT2 or Flag-AKT3 and His-CDO1 protein were mixed for an Ni–NTA pulldown assay. (C) HSC-3 cells were treated with or without IL-6 (50 ng/mL) for 1 h, followed by co-immunoprecipitations using the specified antibodies. WCL, whole cell lysate. (D) Huh-7, HCT116 and DU145 cells were treated with 50 ng/mL IL-6 for 2 h. (E) HSC-3 cells were treated with or without 50 ng/ml TNF-α or 5 nM IL-1β for 2 h. Figure S3 CDO1 T89 phosphorylation is required for IL-6-mediated protein synthesis. (A-B) Cells were pretreated with IL-6 (50 ng/mL) for 4 h, and cultured with [14C]-leucine (0.1 μCi/ml, A) or [14C]-lysine (0.1 μCi/ml, B) for 3 h. The cells were washed and the total protein was extracted, and the radioactive signal was evaluated. The data was shown as the mean ± SD from three replicates. ** P < 0.01. Figure S4 CDO1 T89 phosphorylation is required for IL-6 induced OSCC tumor growth. (A) HSC-3 cells stably expressed with or without IL-6 were subcutaneously injected into Balb/c nude mice (n = 7), and treated with or without 10 mg/kg Tocilizumab once per week. Images of two representative xenografts from each group are shown in the left panel. The tumor volume was displayed in the right panel. *** P < 0.001. (B) Ki-67 immunohistochemical staining. Scale bar, 30 µm. (C) Immunohistochemical staining for the indicated protein. Scale bar, 50 µm. (D) CDO1 activity in xenograft lysate (n = 7) was measured. Data was normalized to total protein concentration. *** P < 0.001.

Supplementary Material 2.

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Li, X., Zhao, Z., Ye, H. et al. CDO1 phosphorylation is required for IL-6-induced tumor cell proliferation through governing cysteine availability. Cell Commun Signal 23, 194 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12964-025-02189-w

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Keywords

Cell Communication and Signaling

ISSN: 1478-811X