Skip to main content
Download PDF

HMGCL activates autophagy in osteosarcoma through β-HB mediated inhibition of the PI3K/AKT/mTOR signaling pathway

Journal of Translational Medicine volume 23, Article number: 219 (2025) Cite this article

Abstract

Background

3-hydroxy-3-methylglutaryl-coenzymOHBe A(HMG-CoA) lyase (HMGCL) catalyzes the cleavage of HMG-CoA into acetyl-CoA and acetoacetic acid and serves as a rate-limiting enzyme in the metabolism of ketone bodies. While HMGCL is involved in various biological processes, its specific role in osteosarcoma remains unclear.

Methods

Using data from a public database of osteosarcoma patients, we investigated the expression and prognostic value of HMGCL. The effects of HMGCL on the proliferation, migration, and invasion of osteosarcoma cells were assessed using CCK-8 assays, wound healing tests, and transwell invasion assays. We explored and validated the specific molecular mechanisms by which HMGCL influences osteosarcoma through transcriptome sequencing. Finally, we established a subcutaneous tumor formation model in nude mice to investigate the function of HMGCL in vivo.

Results

The expression of HMGCL is downregulated in osteosarcoma and correlates with the prognosis of osteosarcoma patients. Overexpression of HMGCL can inhibit the proliferation, migration, and invasion of osteosarcoma cells, as well as tumor growth in vivo. Through our investigation of the underlying mechanism, we found that HMGCL may inhibit the activation of the PI3K/AKT/mTOR signaling pathway via its product, β-HB. This inhibition promotes the phosphorylation of ULK1, thereby facilitating autophagy in osteosarcoma cells and enhancing the malignancy of the disease.

Conclusion

HMGCL inhibits the activation of the PI3K/AKT/mTOR signaling pathway mediated by β-HB, thereby reducing the proliferation, migration, and invasion of osteosarcoma cells while promoting autophagy. HMGCL may represent a new target for the treatment of osteosarcoma, offering new hope for patients with this disease.

Graphical Abstract

Introduction

osteosarcoma (OS) is the most common primary solid malignancy of bone, affecting 2–3 million people annually, with the highest incidence observed in individuals aged 15–19 years [1]. The metaphyseal region of long bone is the most active part of the skeleton and serves as the primary growth site for osteosarcoma [2]. Osteosarcoma typically arises during periods of rapid bone growth in adolescence, suggesting a link to the rapid proliferation of bone. OS is characterized by a tendency for lung metastasis, rapid progression, high morbidity, and mortality [3]. Despite advancements in surgery, chemotherapy, immunotherapy, and gene therapy that have improved clinical outcomes, the 5-year survival rate for osteosarcoma patients remains low, posing a significant threat to public health [4]. Therefore, there is an urgent need to identify new and effective diagnostic and prognostic biomarkers, as well as therapeutic targets, to develop novel treatment strategies aimed at improving survival rates in OS patients.

The ketogenic diet (KD) is a type of diet, and the components and mechanisms of KD waving may be different in different tumor environments [5]. β-hydroxybutyrate (β-HB) is a basic carrier of fatty acid synthesis in the liver and energy delivery to peripheral tissues when the supply of glucose is insufficient to meet the body’s energy needs [6]. Studies have shown that KD intake can significantly increase the concentration of β-HB in the blood [7]. Studies have also shown that β-HB can promote the occurrence of autophagy in tumors and inhibit the progression of tumors [8]. 3-hydroxy-3-methylglutaryl lyase (HMGCL) is a key gene in the ketogenic pathway [9]. The HMG-CoA lyase encoded by HMG-CoA directly catalyzes the cleavage of upstream substrates HMG-CoA into Acetoacetate (AcAc) and acetoacetate (CoA). Acetoylacetic acid is a precursor of β-HB and acetone (acetone), and under the action of β-hydroxybutyrate dehydrogenase (BDH), the resulting acetoacetic acid is reduced to β-HB [10]. We hypothesize that HMGCL may influence the progression of osteosarcoma by regulating β-HB [11]. In recent years, more and more attention has been paid to the role of HMGCL in tumors. For example, in lung cancer, HMGCL may be influenced by tumor necrosis factor α (TNF-α) to inhibit lung cancer progression [12]; In liver cancer, HMGCL regulates H3K9 acetylation through β-HB, thereby regulating DPP4-mediated iron death and thus playing a tumor suppressive role [11]. However, it has also been reported that HMGCL and its product β-HB can increase the aggressiveness of pancreatic cancer and contribute to the progression of pancreatic cancer [10]. In summary, HMGCL may play different roles in the progression of different tumors, but its role in the development of osteosarcoma remains unclear.

In this study, we found that the expression of HMGCL in osteosarcoma tissues and cells is down-regulated, and osteosarcoma patients with high expression of HMGCL have a better prognosis. Promoting the expression of HMGCL can inhibit the proliferation, migration, and invasion ability of osteosarcoma cells, on the contrary, inhibiting the expression of the HMGCL gene can get the opposite result. The results suggest that HMGCL may promote autophagy by regulating the PI3K/AKT/mTOR signaling pathway through β-HB. In summary, we elucidate the role of HMGCL in the progression of osteosarcoma and the potential mechanism of action. HMGCL may be a potential therapeutic target for osteosarcoma.

Materials and methods

Data acquisition

The differences in the HMGCL gene between tumor and normal tissues were investigated using the Tumor Immune Estimation Resource (TIMER) database (https://cistrome.shinyapps.io/timer/). We collected RNA sequencing data and clinical data of osteosarcoma patients from The Cancer Genome Atlas (TCGA) website (https://portal.gdc.cancer.gov/projects/TCGA-COAD) and the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), and excluded samples with no follow-up time. Finally, 139 patients were included for follow-up gene expression analysis and survival analysis.

Clinical samples

In this study, we collected 12 groups of human osteosarcoma tissues and corresponding adjacent normal tissues from Wuhan University People’s Hospital. The collection and use of human samples were approved by the Ethics Committee of Wuhan University People’s Hospital.

Cell culture and transfection

We obtained human osteosarcoma cell lines HOS, 143B, U2OS, and MG63 from Wuhan Saiwei Biotechnology Co., LTD., and human osteoblast cell lines hFOB1.19 from the National Collection of Authenticated Cell Cultures (Shanghai, China). The cells were cultured in DMEM medium containing 10%FBS in a constant temperature incubator at 37℃ and 5%CO2. To construct stable transfection cell lines, we designed HMGCL overexpression (LV-HMGCL), HMGCL knockdown (shHMGCL), and corresponding control lentiviral particles (LV-Control and shNC) using the lentivirus obtained from OBiO (Shanghai, China) and transfected them according to the instructions. The siRNA of ULK1 is also from OBiO. We purchased the PI3K inhibitor 3-MA and the mTOR agonist 3BDO from MedChemExpress (USA).

Quantitative real-time PCR (qRT-PCR)

We used the TRIzol reagent (Invitrogen, Carlsbad, CA, United States) to extract total RNA from a well-grown, stably transfected osteosarcoma cell line, according to the instructions, and then used the Promega M-MLV kit (Promega, Beijing, China) reverse transcribed the extracted RNA into cDNA. qRT-PCR was performed by SYBR®Premix Ex Taq™ (Takara, Tokyo, Japan). The priming sequences were: ULK1 forward,5’- CCAGAGCAACATGATGG-3’and reverse, 5’-CCTTCCCGTCGTAGTGCT-3’; GAPDH, forward, 5’-TGCAACCGGGAAGGAAATGA-3’ and reverse, 5’-GCATCACCCGGAGGAGAAAT-3’.

Western blot

The well-grown cells were taken and added to RIPA lysate (Servicebio, Wuhan, China), mixed solution of protease inhibitor and phosphatase inhibitor for pyrolysis on ice for 30 min, and quantified by BCA protein concentration determination kit (Servicebio, Wuhan, China). Electrophoresis with polyacrylamide gel. The protein was transferred to the PVDF membrane, sealed in 5% skim milk for 2 h, then primary antibody was added, and the PVDF membrane was incubated at 4 °C overnight. Wash with TBST 3 times, 10 min each time, and then incubate with secondary antibody (1∶10 000) for 1 h, and wash with TBST 3 times (10 min/time). ECL detection reagent was used to develop the image.

CCK-8 assay

According to the experimental design, the 96-well plate was inoculated with 1000 /100 ul cell density, and 100 ul/ well PBS buffer was added to the surrounding holes. According to the corresponding time gradient, 10 ul CCK8 reagent was added to each hole at the same time, and incubated in a constant temperature incubator at 37℃ for 1 h without light. The optical density (OD) value of each hole was detected at 450 nm.

Scratch healing assay and transwell invasion assay

The cells were inoculated into the six-well plate, and when the cells were overgrown, scratches were made in the center of the hole with the tip of a 200 ul pipette gun. 2 ml serum-free medium was added to each hole for 36 h, and the cells were observed and photographed. The scratched image is analyzed by ImageJ software.

We used the Transwell chamber and Matrigel (Corning, USA) for the Transwell invasion test. 100 ul serum-free cell suspension was added to each Transwell chamber, and 500 ul complete medium was added to the pore plate below the Transwell chamber. After 24 h of culture, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.

Immunohistochemistry (IHC)

Paraffin-embedded sections were immersed in xylene. The tissue sections were then immersed in anhydrous ethanol for hydration. Then, after antigen repair and endogenous peroxidase blockade, 1×PBS solution containing 0.3% TritonX-100 was permeable. Then the primary and secondary antibodies were incubated, DAB color development and hematoxylin staining were performed, and the tissue samples were photographed with an inverted microscope.

Animal models

We purchased 4-week-old male nude mice from Shulaibao Biotechnology Co., LTD., Wuhan, China. Six wild-type osteosarcoma cells and six HMGCL overexpressed osteosarcoma cells were inoculated. The length and width of the tumor were measured weekly, and the tumor volume was calculated as follows: V = L× W2/2, L was the tumor length, and W was the tumor width. The mice were killed after 5 weeks of injection and subsequent analysis was performed. All animal experiments were conducted by the protocols approved by the Animal Protection and Use Committee of Wuhan University People’s Hospital.

Statistical analysis

We used Graphpad Prism 8.0 software and R (4.0.1) software for statistical analysis of all data in this study. Measurement data were expressed as mean ± standard deviation. Student’s t-test or Wilcoxon test was used to analyze the differences between the two groups. One-way ANOVA compares the differences between groups. The experiment was repeated three times, and P < 0.05 was considered statistically significant.

Results

HMGCL expression is downregulated in osteosarcoma and is associated with poor prognosis

To explore the role of HMGCL in tumors, we used online sites to analyze the differences in HMGCL expression between individual tumors and normal tissues. Our analysis shows that the HMGCL gene in CHOL, COAD, KICH, KIRC, KIRP, LIHC, LUSC, and READ expression in tumor tissue is lower than in the surrounding normal tissues, on the contrary, the expression of the HMGCL gene was up-regulated in BLCA, LUAD and UCEC tumor tissues (Fig. 1A). To explore the expression and significance of HMGCL in osteosarcoma patients, we performed qRT-PCR and western blot detection on samples collected from osteosarcoma patients (Fig. 1B-D). The results showed that the expression of HMGCL in osteosarcoma tissues was lower than that in normal tissues. Similarly, we also verified the expression of HMGCL in osteosarcoma cell lines (HOS, MG63, U2OS, 143B) and human osteoblast cell lines (hFOB1.19), and the results showed that the mRNA and protein levels of HMGCL in osteosarcoma were lower than those in osteoblasts (Fig. 1E-G). To verify the value of HMGCL in the prognosis of osteosarcoma patients, we collected the data of osteosarcoma patients in TCGA and GEO databases. We then performed Kaplan-Meier analysis to demonstrate the difference in survival probability of osteosarcoma patients with different HMGCL gene expression groups (Fig. 1H-I). The horizontal axis is time, in years, indicating the length of follow-up time. The vertical axis is the probability of survival, representing the proportion of patients alive at a particular point in time. The p values marked in the figure indicate that in the TCGA (p = 0.019) and GSE21257 (p = 0.008) cohorts, the survival rate of osteosarcoma patients with high expression of HMGCL gene is significantly higher than that with low expression, suggesting that HMGCL gene expression is related to the clinical prognosis of osteosarcoma patients. Finally, univariate and multivariate COX analysis was performed on gender, age, and HMGCL gene expression in osteosarcoma patients (Fig. 1J-K). The results showed that HMGCL gene expression was an independent prognostic factor in osteosarcoma patients.

HMGCL can inhibit the proliferation, migration, and invasion of osteosarcoma

To investigate the role of HMGCL in the development of osteosarcoma, we constructed a recombinant lentivirus of HMGCL and transfected it in osteosarcoma cell lines. qRT-PCR and western blot assay proved that we had successfully constructed stable transfection cell lines with HMGCL overexpression (Fig. 2A-C). First, we conducted a CCK-8 assay to explore the effect of the HMGCL gene on the proliferation of osteosarcoma cells. The experimental results showed that the growth rate of osteosarcoma cells in the LV-HMGCL group was lower, suggesting that HMGCL could affect the malignant degree of osteosarcoma by affecting the proliferation of osteosarcoma cells (Fig. 2D-E). Similarly, the results of wound healing and transwell invasion assay showed that overexpression of the HMGCL gene can promote the migration and invasion ability of osteosarcoma cells (Fig. 2F-I). Finally, we evaluated the effect of HMGCL gene expression changes on epithelial-mesenchymal transition (EMT). The results showed that overexpression of HMGCL could inhibit the expression of EMT-related proteins N-cadherin and Vimentin, but could promote the expression of E-cadherin (Fig. 2J-L). In summary, we found that overexpression of HMGCL protein can inhibit proliferation, migration, and invasion of osteosarcoma in vitro.

Inhibition of HMGCL gene expression can promote the proliferation, migration, and invasion of osteosarcoma

According to the above experimental results, we further explored the effect of inhibiting the expression of the HMGCL gene on the malignant degree of osteosarcoma cells. First, we constructed HMGCL knockdown lentivirus and transfected it into osteosarcoma cell lines, and verified the knockdown efficiency of the virus by qRT-PCR and western blot assay (Fig. 3A-C). The results showed that we successfully constructed a stable cell line, and selected a cell line with high knockdown efficiency for subsequent assay. The results of the CCK-8 assay showed that inhibiting the expression of HMGCL could promote the growth of osteosarcoma cells (Fig. 3D-E). Similarly, the results of wound healing and transwell invasion assay also showed that down-regulation of HMGCL protein can promote the migration and invasion ability of osteosarcoma cells (Fig. 3F-I). Finally, we verified the effect of HMGCL knockdown on the EMT process by western blot (Fig. 3J-L). The results showed that N-cadherin and Vimentin protein levels were significantly increased, while E-cadherin protein expression levels were inhibited. In conclusion, inhibition of HMGCL gene expression can promote the malignancy of osteosarcoma cells.

HMGCL negatively regulates the PI3K/AKT/mTOR signaling pathway in osteosarcoma

To further investigate the specific molecular mechanism of HMGCL’s influence on the malignancy of osteosarcoma, we performed RNA-seq analysis on the cells of the control group and the LV-HMGCL group [13, 14]. Differential gene analysis showed that 468 genes were down-regulated and 423 genes were up-regulated in the LV-HMGCL group (Fig. 4A-B). We then performed KEGG functional enrichment analysis for differential genes (Fig. 4C). Gene Ratio represents the ratio of the number of genes involved in the pathway to the total number of genes. The higher the ratio, the more the bubble moves to the right, indicating higher gene enrichment in this pathway. The vertical axis of the bubble represents the P-value of the corresponding pathway, reflecting the significance of enrichment. The smaller the P-value, the higher the bubble, indicating that the enrichment of this pathway is more significant. The size of the bubble is proportional to the number of genes involved in a particular pathway, with the larger the number, the larger the bubble. The results showed that HMGCL acted more on tumor pathways, including PI3K/AKT signaling pathway, cAMP signaling pathway and autophagy signaling pathway. Studies have shown that the PI3K/AKT signaling pathway is related to the degree of malignancy of tumors, including osteosarcoma [15,16,17,18]. Therefore, we investigated the influence of changes in the expression of HMGCL protein in osteosarcoma cells on the expression of key molecules in the PI3K/AKT signaling pathway. Western blot results showed that overexpression of HMGCL inhibited the expression levels of p-PI3K, p-AKT, and p-mTOR in osteosarcoma cells (Fig. 4D-F). Similarly, inhibiting the expression of HMGCL can increase the expression levels of p-PI3K, p-AKT, and p-mTOR (Fig. 4G-I). These results suggest that HMGCL may influence the malignancy of osteosarcoma by negatively regulating the PI3K/AKT signaling pathway.

Inhibition of HMGCL on the progression of osteosarcoma can be partially reversed by PI3K inhibitors

To confirm that HMGCL can influence the malignancy of osteosarcoma by influencing the PI3K/AKT signaling pathway, we investigated the role of the PI3K inhibitor 3-MA in this process. We added 3-MA to shNC and shHMGCL osteosarcoma cell lines and then observed the proliferation, migration, and invasion ability of these four groups of cells. The results of the CCK-8 experiment showed that inhibiting the expression of the HMGCL gene can promote the proliferation of osteosarcoma cells, but the addition of 3-MA can reduce this effect (Fig. 5A-B). Wound healing and transwell invasion experiments also confirmed the above results and the ability to inhibit HMGCL gene expression to promote the migration and invasion of osteosarcoma cells could be partially reversed by 3-MA (Fig. 5C-G). In addition, western blot results showed that the addition of 3-MA partially offset the up-regulation effect of inhibiting the expression of the HMGCL gene on p-PI3K, p-AKT, and p-mTOR (Fig. 5H-J). In summary, the PI3K inhibitor 3-MA can partially reverse the enhancement of malignant degree of osteosarcoma caused by inhibition of HMGCL gene expression. This further suggests that HMGCL is likely to affect the progression of osteosarcoma by regulating the PI3K/AKT/mTOR signaling pathway.

HMGCL regulates autophagy in osteosarcoma

The above sequencing results suggest that HMGCL may affect the progression of osteosarcoma by influencing autophagy. To explore the effect of the HMGCL gene on autophagy in osteosarcoma, we analyzed the expression changes of autophagy-related proteins between the control group and the LV-HMGCL group. The results showed that overexpression of HMGCL could promote the expression of Beclin1 protein and inhibit the expression of P62 protein. On the contrary, silencing HMGCL could obtain the opposite result, indicating that overexpression of HMGCL could promote the occurrence of autophagy (Fig. 6A-E, Supplementary Figure S1 A-I). Many recent studies have reported that mTOR signal transduction is closely related to autophagy [19,20,21]. Subsequently, to explore whether HMGCL affects autophagy by affecting the PI3K/AKT/mTOR signaling pathway, we added mTOR agonist 3BDO to two cell lines respectively and observed the expression changes of autophagy-related proteins. The results of western blot and immunofluorescence showed that the regulation of HMGCL protein on autophagy-related proteins could be partially reversed by 3BDO (Fig. 6F-H), suggesting that HMGCL may regulate the autophagy level of osteosarcoma by regulating the PI3K/AKT/mTOR signaling pathway, thus affecting the malignity of osteosarcoma. In summary, overexpression of HMGCL promotes autophagy in osteosarcoma and can be partially reversed by mTOR agonists.

HMGCL influences autophagy of osteosarcoma cells through the PI3K/AKT/mTOR/ULK1 axis mediated by β-HB

In previous studies, we have explored that HMGCL may influence the occurrence of autophagy in osteosarcoma by regulating the PI3K/AKT/mTOR signaling pathway. HMGCL is involved in the production of β-HB. Some studies have shown that the production of β-HB can mediate the effect of HMGCL on the occurrence of tumors, and β-HB has also been reported to affect the autophagy process of tumors [10, 22,23,24]. Moreover, we found that the addition of β-HB to osteosarcoma cell lines inhibited the PI3K/AKT/mTOR signaling pathway, and the expression levels of p-PI3K, p-AKT, and p-mTOR were inhibited (Fig. 6I-K). Therefore, we may preliminarily conclude that HMGCL may regulate the PI3K/AKT/mTOR signaling pathway through the synthesis of β-HB, thus regulating the autophagy process of tumors. It has been reported that high mTOR activity can disrupt the interaction between Ulk1 and AMPK to prevent Ulk1 activation, and thus inhibit autophagy [25,26,27]. Therefore, we thoroughly investigated the role of ULK1 in the influence of HMGCL on autophagy in osteosarcoma. Consistent with our hypothesis, overexpression of HMGCL can promote the phosphorylation of ULK1 and thus the activation of ULK1, and mTOR agonists can reverse this result (Fig. 7A-D). Subsequently, we inhibited the expression of ULK1 in osteosarcoma cells and verified the effect on autophagy (Fig. 7E-I). Western blot results showed that ULK1 expression was successfully inhibited in osteosarcoma cell lines. Inhibition of ULK1 expression level can inhibit the expression of Beclin1 protein, promote the expression of P62 protein, and inhibit the occurrence of autophagy. Finally, we investigated in osteosarcoma that inhibition of ULK1 expression can partially reverse the autophagy promotion effect of overexpression of HMGCL (Fig. 7J-L). In summary, our experimental results suggest that HMGCL promotes the phosphorylation and activation of ULK1 through β-HB-mediated inhibition of the PI3K/AKT/mTOR signaling pathway, thus promoting the occurrence of autophagy in osteosarcoma cells.

HMGCL affects tumor growth by regulating the PI3K/AKT/mTOR signaling pathway and autophagy

To further investigate the role of HMGCL in vivo, we constructed subcutaneous xenotransplantation models of nude mice using wild osteosarcoma cells and HMGCL overexpressed cells respectively. The tumor size was measured and recorded every week after cell inoculation. The experimental results showed that HMGCL overexpression inhibited the growth rate of subcutaneous tumors in nude mice (Fig. 8A-C). Subsequently, to verify that HMGCL can also affect the PI3K/AKT/mTOR signaling pathway of osteosarcoma and the process of autophagy and EMT in vivo, we performed western blot analysis on subcutaneous tumor cells. Compared with the control group, the expression levels of p-PI3K, p-AKT, p-mTOR, N-cadherin, Vimentin, and P62 were significantly decreased, while the expression levels of E-cadherin, Beclin1, and p-ULK1 were significantly increased in the LV-HMGCL group (Fig. 8D-F). In summary, these results once again demonstrate that HMGCL affects the malignancy of osteosarcoma by regulating the PI3K/AKT/mTOR signaling pathway and autophagy.

Discussion

OS is the most common primary malignant bone tumor, which develops from the mesenchymal cell line, usually occurs in the epiphysis of the long diaphyseal in adolescents, and is prone to metastasis and recurrence [28, 29]. Although the treatment of osteosarcoma has made progress, the prognosis of patients is still not ideal due to its high metastatic characteristics, and its survival rate has remained unchanged in the past 30 years, with the 5-year survival rate of patients being only 20%, which has a huge impact on patient’s physical and mental health [1, 30, 31]. With the development of genetic characterization of osteosarcoma, it is critical to discover new biomarkers to facilitate the development of therapeutic strategies.

HMGCL is an energy-limiting enzyme that mediates the metabolism of ketone bodies and can catalyze the cleavage of HMG-CoA into acetyl-CoA and acetoacetic acid [32]. Acetyl-CoA is the main component of fatty acids (FA) synthesis. Long-chain fatty acids are metabolized in cells mainly through esterification to form fatty acid esters (triacylglycerol, phospholipids) or oxidation to carbon dioxide and ketone bodies. Ketone bodies, another product of FA metabolic transformation, are produced and released in normal epithelial cells and tumors and appear in hepatocytes [33]. Studies have shown that HMGCL plays a role in various tumors, in hepatocellular carcinoma (HCC), HMGCL can catalyze the production of β-HB and regulate the acetylation of H3K9 through β-HB, and regulate the expression of DPP4 in a dose-dependent manner, thereby leading to iron death of HCC cells and inhibiting the progression of HCC. However, in pancreatic ductal adenocarcinoma (PDA), HMGCL can affect ketone body metabolism, and can synergistically promote the development of PDA with β-HB. The results show that HMGCL plays a different role in different tumors, but its role and mechanism in osteosarcoma remain unclear [9,10,11, 34]. Our study suggests that HMGCL acts as a tumor suppressor gene in osteosarcoma. Through the analysis of clinical osteosarcoma tissues and cells, we found that HMGCL expression is reduced in osteosarcoma, and through the analysis of public databases, it was found that osteosarcoma patients with low HMGCL expression have a poor prognosis. Moreover, univariate and multivariate COX analysis showed that HMGCL was an independent prognostic factor in patients with osteosarcoma. In vitro cell experiments show that overexpression of HMGCL can inhibit the proliferation, migration, and invasion of osteosarcoma cells, on the contrary, inhibition of HMGCL expression will promote the malignant degree of osteosarcoma. In summary, our results suggest that HMGCL is a potential tumor suppressor gene in osteosarcoma and is expected to be a therapeutic target for osteosarcoma.

The PI3K/AKT signaling pathway is involved in regulating various processes of the cell cycle and promoting the occurrence and development of tumors. Several downstream molecules make up the PI3K/AKT signaling pathway. Among them, the mammalian target of rapamycin (mTOR) is one of the more important. Activated AKT can directly phosphorylate mTOR to activate it [18, 35]. Several studies have reported associations between tumors and this pathway. For example, NETO2 can promote the progression of esophageal cancer by activating the PI3K/AKT signaling pathway [36], and inhibiting the expression of TRIM47 can inhibit the activation of this pathway, thus inhibiting the occurrence and progression of breast cancer [37]. Similarly, the PI3K/AKT signaling pathway also plays an important role in osteosarcoma. It has been reported that circ_001422 can promote the progression and metastasis of osteosarcoma through the PI3K/AKT signaling pathway [38]. To investigate whether HMGCL plays a role in osteosarcoma through the PI3K/AKT signaling pathway, we evaluated the phosphorylation status of PI3K, AKT, and mTOR after changes in HMGCL gene expression. The results showed that HMGCL could inhibit the activation of the PI3K/AKT signaling pathway and thus play a role in cancer inhibition. Moreover, we found that the PI3K inhibitor 3-MA can partially reverse the activation of HMGCL expression on this pathway. Therefore, we can conclude that HMGCL can affect the malignancy of osteosarcoma by regulating the PI3K/AKT signaling pathway.

Autophagy is a catabolic process that involves chelating damaged organelles or debris into double-membrane vesicles for further degradation [39]. In general, autophagy plays a protective role in cells, but disruption of autophagy mechanisms or excessive autophagy flux usually leads to cell death [40]. In this study, we found that after overexpression of HMGCL, the expression of autophagy-related protein Beclin1 in cells increased, while the expression of P62 decreased. These results suggest that HMGCL can promote autophagy in osteosarcoma cells. To explore whether HMGCL affects autophagy through the PI3K/AKT signaling pathway, we investigated the effect of mTOR agonist use on the above results. Interestingly, mTOR agonists partially reversed the autophagy-promoting effect of overexpression of HMGCL. We also found that the addition of β-HB, the synthetic product of HMGCL, can also inhibit the activation of the PI3K/AKT signaling pathway. Therefore, we preliminically concluded that HMGCL may inhibit the activation of the PI3K/AKT signaling pathway by increasing its product’s expression, thus promoting autophagy in osteosarcoma cells. Next, we explored how the PI3K/AKT signaling pathway influences autophagy. It has been reported that mTOR can inhibit the occurrence of autophagy by inhibiting the phosphorylation of ULK1, and we hypothesize that a similar regulatory mechanism exists in osteosarcoma [25]. First, we found that overexpression of HMGCL can promote the phosphorylation level of ULK1 protein, and mTOR agonist 3BDO can partially reverse this result. Subsequently, we inhibited ULK1 expression levels in osteosarcoma cells and showed that autophagy was inhibited. Moreover, inhibition of ULK1 expression can partially reverse the activation effect of overexpression of HMGCL on the autophagy pathway. These results suggest that HMGCL may inhibit the activation of the PI3K/AKT/mTOR signaling pathway by increasing the expression of β-HB and promoting the phosphorylation of ULK1, thus promoting autophagy of osteosarcoma cells.

Limitations

Of course, there are inevitably some shortcomings in our research. First, the number of clinical samples used for analysis in this study was relatively small, including only tumor tissues and matched normal tissues of 12 groups of patients, and the small sample size may limit the statistical significance and generality of the findings. Therefore, larger samples are needed to verify these results. Secondly, the experimental results of this study are mainly based on in vitro cell experiments and mouse xenotransplantation models, which have not been verified on a large scale in clinical patients. Therefore, the actual clinical application value of HMGCL as a therapeutic target still needs to be further explored. Moreover, osteosarcoma is highly heterogeneous, and gene expression and molecular mechanisms may differ significantly between patients and within the tumor of the same patient. The role of HMGCL in different osteosarcoma subtypes was not adequately evaluated in this study, which may affect the generalization of the findings. Although our study identified the role of β-HB and HMGCL in the regulation of the PI3K/AKT/mTOR pathway, it did not further explore potential crosstalk with other signaling pathways (such as AMPK), and subsequent experiments to explore interactions with other signaling pathways could provide a more complete understanding. Finally, although this study suggests that HMGCL may be a potential therapeutic target, no specific drugs or therapies have been developed for HMGCL. As a result, the transition from laboratory research to clinical applications remains challenging.

Future directions

Future studies should collect more clinical samples covering different osteosarcoma subtypes and patient populations to further verify the expression level of HMGCL and its impact on patient prognosis. At the same time, the role of HMGCL in tumor heterogeneity can be further analyzed with single-cell sequencing technology. Based on the results of this study, HMGCL can regulate the activation of autophagy pathway through β-HB and PI3K/AKT/mTOR signaling pathways to further inhibit cancer in osteosarcoma. Therefore, we can try to develop a combined treatment strategy targeting PI3K/AKT/mTOR and autophagy to improve the therapeutic effect. In order to achieve the translation of laboratory studies into clinical applications, future prospective clinical studies are needed to evaluate the diagnostic and therapeutic potential of HMGCL in patients with osteosarcoma. At the same time, biomarkers related to HMGCL can be developed in combination with liquid biopsy and imaging technology for early diagnosis and treatment monitoring of patients.

Conclusions

In conclusion, our study illustrates the role of HMGCL as a tumor suppressor gene in osteosarcoma. The expression of HMGCL is down-regulated in osteosarcoma, and downregulation of HMGCL is associated with poor prognosis in osteosarcoma patients. Functionally, HMGCL may promote autophagy through the PI3K/AKT/mTOR signaling pathway mediated by its product β-HB, thereby inhibiting the proliferation, migration, and invasion of osteosarcoma. Therefore, HMGCL may be a molecular target for osteosarcoma therapy, which can inhibit the progression of osteosarcoma by restoring the expression of HMGCL.

Fig. 1
figure 1

HMGCL expression is down-regulated in osteosarcoma, and its low level is associated with poor patient prognosis. (A) Expression patterns of HMGCL in various tumors and normal tissues based on the TIMER database. (B) mRNA levels of HMGCL in osteosarcoma tissues and adjacent tissues of clinical patients. (C) Protein levels of HMGCL in osteosarcoma tissues and adjacent tissues of clinical patients. (D) Quantitative analysis of HMGCL protein expression. (E-G) Protein and mRNA expression levels of HMGCL in human osteoblasts and osteosarcoma cells. (H-I) Kaplan-Meier survival analysis of patients with high or low HMGCL expression in TCGA and GEO databases. (J-K) Univariate and multivariate COX analysis was performed to evaluate the prognostic ability of HMGCL. * P < 0.05, *** P < 0.001

Full size image
Fig. 2
figure 2

Overexpression of HMGCL can inhibit the malignancy of osteosarcoma. (A-C) Protein and mRNA expression levels of HMGCL in osteosarcoma cells after transfection with lentivirus. (D-E) The effect of HMGCL overexpression on the proliferation of osteosarcoma cells was evaluated by CCK-8 assay. (F-G) The effect of HMGCL overexpression on osteosarcoma cell migration was evaluated in wound healing experiments, scale bar: 200 μm. (H-I) The effect of HMGCL overexpression on the invasion ability of osteosarcoma cells was evaluated by transwell invasion assay, scale bar: 400 μm. (J-L) Effect of overexpression of HMGCL on expression of EMT-related proteins. * P < 0.05, ** P < 0.01, *** P < 0.001

Full size image
Fig. 3
figure 3

Silencing HMGCL can promote the malignancy of osteosarcoma. (A-C) Protein and mRNA expression levels of HMGCL in osteosarcoma cells after transfection with lentivirus. (D-E) The effect of silencing HMGCL on the proliferation of osteosarcoma cells was evaluated by CCK-8 assay. (F-G) The effect of silencing HMGCL on osteosarcoma cell migration was evaluated by wound healing assay, scale bar: 200 μm. (H-I) The transwell invasion assay was used to evaluate the effect of silencing HMGCL on the invasion ability of osteosarcoma cells, scale bar: 400 μm. (J-L) Effect of silencing HMGCL on expression of EMT-related proteins. * P < 0.05, ** P < 0.01, *** P < 0.001

Full size image
Fig. 4
figure 4

HMGCL inhibits the activation of the PI3K/AKT/mTOR signaling pathway in osteosarcoma cells. (A-B) Volcanic maps and heat maps showed significant changes in gene and expression patterns after overexpression of HMGCL. (C) KEGG analyzed the functional pathways of differential gene enrichment. (D-F) western blot analysis of the changes of PI3K/AKT/mTOR signaling pathway-related proteins after HMGCL overexpression. (G-I) western blot analysis was performed to detect the changes of PI3K/AKT/mTOR signaling pathway-related proteins changes after HMGCL silencing. ** P < 0.01, *** P < 0.001

Full size image
Fig. 5
figure 5

PI3K inhibitors can partially reverse the effect of silencing HMGCL on the progression of osteosarcoma. (A-B) The proliferative capacity of four groups of cells was assessed by CCK-8 assay. (C-E) The migration capacity of four groups of cells was evaluated in wound healing experiments. (F-G) The invasion ability of four groups of cells was evaluated by transwell invasion assay. (H-J) western blot analysis of PI3K/AKT/mTOR signaling pathway-related protein expression in the four groups of cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Full size image
Fig. 6
figure 6

HMGCL promotes autophagy in osteosarcoma cells. (A-C) The changes in autophagy-related protein expression after overexpression of HMGCL were detected by western blot. (D-E) The expression level of Beclin1 protein after overexpression of HMGCL was analyzed by immunofluorescence. (F-H) The effect of mTOR molecular agonist on autophagy protein after HMGCL overexpression was detected by western blot. (I-K) The expression of PI3K/AKT/mTOR signaling pathway-related proteins was detected by western blot after β-HB (8mM) treatment of osteosarcoma cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Full size image
Fig. 7
figure 7

HMGCL promotes autophagy in osteosarcoma cells through the PI3K/AKT/mTOR/ULK1 axis mediated by its product β-HB. (A-B) western blot analysis of ULK1 protein phosphorylation after HMGCL overexpression. (C-D) Effect of mTOR agonists on the phosphorylation of ULK1 after overexpression of HMGCL. (E-F) western blot analysis of ULK1 knockdown efficiency in osteosarcoma. (G-I) western blot analysis of the effect of silencing ULK1 expression on autophagy of osteosarcoma cells. (J-L) Silencing ULK1 partially reversed the overexpression of HMGCL and promoted autophagy in osteosarcoma cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Full size image
Fig. 8
figure 8

HMGCL affects the progression of osteosarcoma by regulating the PI3K/AKT/mTOR signaling pathway and autophagy in vivo. (A) Effect of overexpression of HMGCL on tumor volume. (B) Comparison of tumor in nude mice between the LV-control group and LV-HMGCL group. (C) Comparison of tumor weight between the two groups. (D-E) We performed western blot quantitative detection of HMGCL, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR, N-cadherin, E-cadherin, Vimentin, P62, Beclin1, p-ULK1 and ULK1 in the two groups of tumors. (F) Immunohistochemical analysis of HMGCL, p-mTOR, p-ULK1, Beclin1 in two groups of tumors, Scale bar: 200 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Full size image

Data availability

The published article includes all data sets generated/analyzed for this study. The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

References

  1. Chen C, Xie L, Ren T, Huang Y, Xu J, Guo W. Immunotherapy for osteosarcoma: fundamental mechanism, rationale, and recent breakthroughs. Cancer Lett. 2021;500:1–10.

    Article  CAS  PubMed  Google Scholar 

  2. Shoaib Z, Fan TM, Irudayaraj JMK. Osteosarcoma mechanobiology and therapeutic targets. Br J Pharmacol. 2022;179(2):201–17.

    Article  CAS  PubMed  Google Scholar 

  3. Yang C, Tian Y, Zhao F, Chen Z, Su P, Li Y, Qian A. Bone Microenvironment and Osteosarcoma Metastasis. Int J Mol Sci 2020, 21(19).

  4. Gill J, Gorlick R. Advancing therapy for osteosarcoma. Nat Rev Clin Oncol. 2021;18(10):609–24.

    Article  PubMed  Google Scholar 

  5. Lane J, Brown NI, Williams S, Plaisance EP, Fontaine KR. Ketogenic Diet for Cancer: critical Assessment and Research recommendations. Nutrients 2021, 13(10).

  6. Wang L, Chen P, Xiao W. beta-hydroxybutyrate as an anti-aging metabolite. Nutrients 2021, 13(10).

  7. Li K, Wang WH, Wu JB, Xiao WH. beta-hydroxybutyrate: a crucial therapeutic target for diverse liver diseases. Biomed Pharmacother. 2023;165:115191.

    Article  CAS  PubMed  Google Scholar 

  8. Cai Z, Zhang Z, Leng J, Xie M, Zhang K, Zhang J, Zhang H, Hu H, Deng Y, Bai X et al.: beta-hydroxybutyrate ameliorates osteoarthritis through activation of the ERBB3 signaling pathway in mice. J Bone Min Res 2024.

  9. Sun Y, Mu G, Zhang X, Wu Y, Wang S, Wang X, Xue Z, Wang C, Liu J, Li W, et al. Metabolic modulation of histone acetylation mediated by HMGCL activates the FOXM1/beta-catenin pathway in glioblastoma. Neuro Oncol. 2024;26(4):653–69.

    Article  CAS  PubMed  Google Scholar 

  10. Gouirand V, Gicquel T, Lien EC, Jaune-Pons E, Da Costa Q, Finetti P, Metay E, Duluc C, Mayers JR, Audebert S, et al. Ketogenic HMG-CoA lyase and its product beta-hydroxybutyrate promote pancreatic cancer progression. EMBO J. 2022;41(9):e110466.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cui X, Yun X, Sun M, Li R, Lyu X, Lao Y, Qin X, Yu W. HMGCL-induced beta-hydroxybutyrate production attenuates hepatocellular carcinoma via DPP4-mediated ferroptosis susceptibility. Hepatol Int. 2023;17(2):377–92.

    Article  PubMed  Google Scholar 

  12. Zhong C, Xiong G, Yang H, Du X, Du J, Yao F, Fang W, Deng Y. Phosphorylation by IKKbeta promotes the degradation of HMGCL via NEDD4 in Lung Cancer. Int J Biol Sci. 2023;19(4):1110–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liu Y, DiStasio M, Su G, Asashima H, Enninful A, Qin X, Deng Y, Nam J, Gao F, Bordignon P, et al. High-plex protein and whole transcriptome co-mapping at cellular resolution with spatial CITE-seq. Nat Biotechnol. 2023;41(10):1405–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fan R, Zhang D, Rodriguez-Kirby L, Lin Y, Song M, Wang L, Wang L, Kanatani S, Jimenez-Beristain T, Dang Y et al. Spatial dynamics of mammalian brain development and neuroinflammation by multimodal tri-omics mapping. Res Sq 2024.

  15. Glaviano A, Foo ASC, Lam HY, Yap KCH, Jacot W, Jones RH, Eng H, Nair MG, Makvandi P, Geoerger B, et al. PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer. Mol Cancer. 2023;22(1):138.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. He Y, Sun MM, Zhang GG, Yang J, Chen KS, Xu WW, Li B. Targeting PI3K/Akt signal transduction for cancer therapy. Signal Transduct Target Ther. 2021;6(1):425.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li H, Shen X, Ma M, Liu W, Yang W, Wang P, Cai Z, Mi R, Lu Y, Zhuang J, et al. ZIP10 drives osteosarcoma proliferation and chemoresistance through ITGA10-mediated activation of the PI3K/AKT pathway. J Exp Clin Cancer Res. 2021;40(1):340.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Yu L, Wei J, Liu P. Attacking the PI3K/Akt/mTOR signaling pathway for targeted therapeutic treatment in human cancer. Semin Cancer Biol. 2022;85:69–94.

    Article  CAS  PubMed  Google Scholar 

  19. Ballesteros-Alvarez J, Andersen JK. mTORC2: the other mTOR in autophagy regulation. Aging Cell. 2021;20(8):e13431.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kim YC, Guan KL. mTOR: a pharmacologic target for autophagy regulation. J Clin Invest. 2015;125(1):25–32.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Pei F, Ma L, Jing J, Feng J, Yuan Y, Guo T, Han X, Ho TV, Lei J, He J, et al. Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis. Nat Commun. 2023;14(1):344.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Chu Y, Hua Y, He L, He J, Chen Y, Yang J, Mahmoud I, Zeng F, Zeng X, Benavides GA, et al.: beta-hydroxybutyrate administered at reperfusion reduces infarct size and preserves cardiac function by improving mitochondrial function through autophagy in male mice. J Mol Cell Cardiol. 2024;186:31–44.

    Article  CAS  PubMed  Google Scholar 

  23. Huang M, Yu Y, Tang X, Dong R, Li X, Li F, Jin Y, Gong S, Wang X, Zeng Z, et al. 3-Hydroxybutyrate ameliorates sepsis-associated acute lung injury by promoting autophagy through the activation of GPR109alpha in macrophages. Biochem Pharmacol. 2023;213:115632.

    Article  CAS  PubMed  Google Scholar 

  24. McCarthy CG, Chakraborty S, Singh G, Yeoh BS, Schreckenberger ZJ, Singh A, Mell B, Bearss NR, Yang T, Cheng X et al. Ketone body beta-hydroxybutyrate is an autophagy-dependent vasodilator. JCI Insight 2021, 6(20).

  25. Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011;13(2):132–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lin CY, Wu KY, Chi LM, Tang YH, Huang HJ, Lai CH, Tsai CN, Tsai CL. Starvation-inactivated MTOR triggers cell migration via a ULK1-SH3PXD2A/TKS5-MMP14 pathway in ovarian carcinoma. Autophagy. 2023;19(12):3151–68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lin M, Hua R, Ma J, Zhou Y, Li P, Xu X, Yu Z, Quan S. Bisphenol A promotes autophagy in ovarian granulosa cells by inducing AMPK/mTOR/ULK1 signalling pathway. Environ Int. 2021;147:106298.

    Article  CAS  PubMed  Google Scholar 

  28. Cortini M, Avnet S, Baldini N. Mesenchymal stroma: role in osteosarcoma progression. Cancer Lett. 2017;405:90–9.

    Article  CAS  PubMed  Google Scholar 

  29. Zheng Y, Wang G, Chen R, Hua Y, Cai Z. Mesenchymal stem cells in the osteosarcoma microenvironment: their biological properties, influence on tumor growth, and therapeutic implications. Stem Cell Res Ther. 2018;9(1):22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kansara M, Teng MW, Smyth MJ, Thomas DM. Translational biology of osteosarcoma. Nat Rev Cancer. 2014;14(11):722–35.

    Article  CAS  PubMed  Google Scholar 

  31. Cersosimo F, Lonardi S, Bernardini G, Telfer B, Mandelli GE, Santucci A, Vermi W, Giurisato E. Tumor-Associated macrophages in Osteosarcoma: from mechanisms to Therapy. Int J Mol Sci 2020, 21(15).

  32. Arnedo M, Latorre-Pellicer A, Lucia-Campos C, Gil-Salvador M, Antonanzas-Perez R, Gomez-Puertas P, Bueno-Lozano G, Puisac B, Pie J. More Than one HMG-CoA lyase: the classical mitochondrial enzyme plus the Peroxisomal and the cytosolic ones. Int J Mol Sci 2019, 20(24).

  33. Chavan R, Feillet C, Costa SS, Delorme JE, Okabe T, Ripperger JA, Albrecht U. Liver-derived ketone bodies are necessary for food anticipation. Nat Commun. 2016;7:10580.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Luo W, Qin L, Li B, Liao Z, Liang J, Xiao X, Xiao X, Mo Y, Huang G, Zhang Z, et al. Inactivation of HMGCL promotes proliferation and metastasis of nasopharyngeal carcinoma by suppressing oxidative stress. Sci Rep. 2017;7(1):11954.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Tewari D, Patni P, Bishayee A, Sah AN, Bishayee A. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: a novel therapeutic strategy. Semin Cancer Biol. 2022;80:1–17.

    Article  PubMed  Google Scholar 

  36. Xu JC, Chen TY, Liao LT, Chen T, Li QL, Xu JX, Hu JW, Zhou PH, Zhang YQ. NETO2 promotes esophageal cancer progression by inducing proliferation and metastasis via PI3K/AKT and ERK pathway. Int J Biol Sci. 2021;17(1):259–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang Y, Liu C, Xie Z, Lu H. Knockdown of TRIM47 inhibits breast cancer tumorigenesis and progression through the inactivation of PI3K/Akt pathway. Chem Biol Interact. 2020;317:108960.

    Article  CAS  PubMed  Google Scholar 

  38. Yang B, Li L, Tong G, Zeng Z, Tan J, Su Z, Liu Z, Lin J, Gao W, Chen J, et al. Circular RNA circ_001422 promotes the progression and metastasis of osteosarcoma via the miR-195-5p/FGF2/PI3K/Akt axis. J Exp Clin Cancer Res. 2021;40(1):235.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liu S, Yao S, Yang H, Liu S, Wang Y. Autophagy: Regulator of cell death. Cell Death Dis. 2023;14(10):648.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Klionsky DJ, Petroni G, Amaravadi RK, Baehrecke EH, Ballabio A, Boya P, Bravo-San Pedro JM, Cadwell K, Cecconi F, Choi AMK, et al. Autophagy in major human diseases. EMBO J. 2021;40(19):e108863.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We sincerely thank the TCGA (https://portal.gdc.cancer.gov/) and GEO database (https://www.ncbi.nlm.nih.gov/geo/) for the use of their data.

Funding

This study was supported by funds from The Open Project of Hubei Key Laboratory (2023KFZZ022).

Author information

Author notes

  1. Wenda Liu and Kezhou Xia have contributed equally to this work and share the first authorship.

Authors and Affiliations

  1. Department of Orthopaedics, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan, 430060, Hubei Province, China

    Wenda Liu, Kezhou Xia, Xinghan Huang, Zhun Wei, Zicheng Wei, Xingyu Wang, Chen Xiong & Weichun Guo

Authors
  1. Wenda Liu
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Kezhou Xia
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Xinghan Huang
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Zhun Wei
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Zicheng Wei
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Xingyu Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Chen Xiong
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Weichun Guo
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

WG designed the study. WL and KX conducted bioinformatic analysis, wrote the manuscript, and was responsible for language revisions. XH, ZW, XW, CX and ZW helped with some of the experiments. All authors reviewed the manuscript.

Corresponding author

Correspondence to Weichun Guo.

Ethics declarations

Ethics approval and consent to participate

The Animal Center of Renmin Hospital of Wuhan University approved all experiments conducted on animals. Our animal experiments strictly follow the ARRIVE guidelines.

Consent for publication

All authors agree to publication.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, W., Xia, K., Huang, X. et al. HMGCL activates autophagy in osteosarcoma through β-HB mediated inhibition of the PI3K/AKT/mTOR signaling pathway. J Transl Med 23, 219 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06227-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06227-6

Keywords

Journal of Translational Medicine

ISSN: 1479-5876

Contact us