HKDC1 promotes ovarian cancer progression through boosting lipid metabolism and immune escape by stabilizing G6PC/G6PC2

Wang, Ying; Chen, Juan; Wang, Zhan; Luo, Xia; Wu, Nayiyuan; Wang, Jing

doi:10.1038/s42003-025-08031-w

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Published: 16 April 2025

HKDC1 promotes ovarian cancer progression through boosting lipid metabolism and immune escape by stabilizing G6PC/G6PC2

Ying Wang ORCID: orcid.org/0000-0001-8168-3314¹,
Juan Chen²,
Zhan Wang³,
Xia Luo¹,
Nayiyuan Wu¹ &
…
Jing Wang¹

Communications Biology volume 8, Article number: 615 (2025) Cite this article

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Abstract

Ovarian cancer (OC) is a significant health challenge, yet the mechanisms driving its progression remain unclear. This study explored the role of hexokinase domain-containing protein 1 (HKDC1) in OC, focusing on tumor growth, lipid metabolism, and immune evasion. Human OC cell lines (SKOV3 and HEY) and the murine OC cell line (ID8) were used to knock down and overexpress HKDC1. An ID8-based epithelial OC mouse model was established to validate the in vitro findings. Our results demonstrated that HKDC1 was upregulated in OC and promoted cell proliferation, migration, and invasion. HKDC1 enhanced lipid accumulation by elevating levels of free fatty acids (FFA), triglycerides, phospholipids, cholesterol, and neutral lipid, while upregulating key enzymes (ACC1, FASN, SCD1, HMGCS1, and HMGCR). It promoted immune escape through PD-L1 upregulation, inhibiting T cell proliferation and reducing IFN-γ, granzyme B, and perforin levels while increasing PD-1 levels. HKDC1 knockdown reversed these effects, which were restored by adding FFA. Mechanistically, HKDC1 interacted with and stabilized glucose-6-phosphatase catalytic subunits (G6PC/G6PC2), supporting its tumor-promoting functions. These findings were confirmed in an OC mouse model, highlighting HKDC1 as a key driver of OC progression through lipid biosynthesis and immune suppression, offering potential therapeutic targets.

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Introduction

Ovarian cancer (OC) is recognized as the most lethal malignancy among tumors of the female reproductive system, with epithelial OC (EOC) accounting for more than 90% of cases^1,2. EOC is highly aggressive, prone to metastasis, and often results in complications such as ascites and intestinal obstruction, significantly threatening patient survival³. Due to the subtle nature of early symptoms and the absence of effective screening methods, most EOC cases are diagnosed at an advanced stage, complicating treatment and contributing to a higher mortality rate⁴. The standard treatment for advanced EOC, which involves cytoreductive surgery followed by cisplatin and paclitaxel-based chemotherapy, provides limited improvement in long-term survival despite initial benefits^5,6. Therefore, identifying therapeutic strategies and targets remains a pressing need for improving outcomes in EOC treatment.

One of the major metabolic alterations in cancer is the dysregulation of lipid metabolism, particularly within the tumor microenvironment. Tumor cells have been shown to modulate nutrient availability and utilize lipid metabolism to support their rapid proliferation, survival, migration, invasion, and metastasis⁷. Studies have shown that lipid metabolic pathways not only drive the growth and progression of OC but are also associated with poor prognosis^8,9. For instance, Ladanyi et al. demonstrated that inhibiting cluster of differentiation 36 (CD36) reduces fatty acid (FA) uptake from the tumor microenvironment in OC cells, thereby suppressing adipocyte-mediated invasion, migration, and tumor growth¹⁰. Similarly, Zhao et al. identified mitochondrial elongation factor 2 as a key player in FA synthesis and cholesterol (CHO) biosynthesis, both of which are critical to OC progression¹¹. Another potential therapeutic target, stanniocalcin 1, has been shown to promote lipid metabolism via the forkhead box C2/integrin αvβ6 signaling axis, particularly offering therapeutic benefits in platinum-resistant OC patients¹².

Lipid metabolism plays an essential role in cancer cell immune escape mechanisms. Dysregulated lipid metabolism in cancer cells can impair immune cell activation, infiltration, and effector functions, promoting immune evasion and weakening anti-tumor immune responses^13,14. Experimental studies have demonstrated that Arf1-mediated lipid metabolism is essential for cancer cell survival, and its inhibition can trigger anti-cancer immune responses in murine models¹⁵. In addition, lipid metabolism dysfunction has been associated with tumorigenesis and immune escape in breast cancer, further supporting the role of metabolic reprogramming in cancer progression¹⁶. In OC, the C-C chemokine ligand 5 (CCL5) has been implicated in the dysregulation of the arachidonic acid-leukotriene A4 hydrolase axis, contributing to reduced infiltration of tumor-infiltrating CD8⁺ T cells and facilitating immune escape¹⁷. Despite these findings, the precise mechanisms linking metastasis, lipid metabolism, and immune escape in OC remain unclear, indicating the need for further investigation. Thus, a comprehensive understanding of these interactions could inform the development of more effective therapeutic strategies and improve patient outcomes.

Hexokinases represent a class of enzymes responsible for extracellular glucose phosphorylation, essential for intracellular glucose utilization¹⁸. Hexokinase domain-containing protein 1 (HKDC1), considered the fifth hexokinase, plays an important role in systemic glucose homeostasis^19,20. However, aberrant HKDC1 expression has been implicated in the progression of various diseases and malignancies²¹. For instance, elevated HKDC1 levels are associated with glucose metabolism disorders and poor prognosis in patients with alcoholic hepatitis²². Moreover, HKDC1 has emerged as a potential oncogene in several cancers, including lymphoma²³, hepatocellular carcinoma²⁴, breast cancer²⁵, colorectal cancer²⁶, and lung adenocarcinoma²⁷. Notably, in gastric cancer, HKDC1 promotes metastasis and chemoresistance through the reprogramming of lipid metabolism via ribonucleoprotein complex formation²⁸. Despite the growing evidence suggesting an oncogenic role of HKDC1 in several malignancies, its specific role and underlying mechanisms in OC remain largely unexplored. In this study, we used OC cell lines and EOC mouse models to study the functional role of HKDC1 in OC and whether it affects OC progression by regulating lipid metabolism and immune escape, with a view to providing valuable insights into its potential as a therapeutic target.

Results

HKDC1 is overexpressed in OC cells

Figure 1 showed increased HKDC1 mRNA and HKDC1 protein expression in OC cell lines (HEY, SKOV3, ES2, and OVCAR3) compared to the normal ovarian epithelial cell line IOSE80. Among these, HKDC1 expression was highest in SKOV3 cells and lowest in HEY cells. Additionally, we examined the expression of G6PC, G6PC2, G6PC3, and several key regulators involved in lipid metabolism across different cell lines. As shown in Supplementary Fig. 1A–C, the expression of G6PC, G6PC2, and G6PC3, along with markers of FA synthesis (ACC1, FASN, SCD1), CHO biosynthesis (HMGCS1, HMGCR), FA uptake (CD36), FA oxidation (CPT1A), and immune evasion (PD-L1), was consistently elevated in OC cells compared to IOSE80 cells. Notably, G6PC, G6PC2, and G6PC3 exhibited the highest expression levels in SKOV3 cells and the lowest in HEY cells. However, no significant differences were observed in the expression of ACC1, FASN, SCD1, HMGCS1, HMGCR, CD36, CPT1A, or PD-L1 among the various OC cell lines. Based on these findings, SKOV3 cells, which showed relatively high expression levels of HKDC1 and related genes, and HEY cells, with comparatively lower expression, were selected for further experimentation.

HKDC1 promotes proliferation, migration, invasion, and lipid metabolism of OC cells in vitro

To investigate the role of HKDC1 in OC cell function, we performed transient knockdown of HKDC1 in SKOV3 cells and overexpressed HKDC1 in HEY cells, and the efficacy was confirmed by qRT-PCR and WB analyses, which demonstrated significant changes in HKDC1 mRNA and HKDC1 protein expression (Fig. 2A). Among the knockdown constructs, si-HKDC1#1 exhibited the most pronounced reduction in HKDC1 levels and was therefore selected for further experiments. In HEY cells, HKDC1 overexpression resulted in a marked increase in expression (Fig. 2A). Cell proliferation was evaluated using CCK-8 and colony formation assays. The results showed that HKDC1 knockdown significantly reduced OC cell growth, while its overexpression enhanced proliferation (Fig. 2B, C and Supplementary Fig. 2A). Migration and invasion were assessed using wound healing and Transwell assays, respectively. HKDC1 knockdown impaired both migration and invasion in SKOV3 cells, whereas HKDC1 overexpression in HEY cells led to substantial improvements in these traits (Fig. 2D, E and Supplementary Figs. 2B, C). These findings suggest that HKDC1 positively regulates OC cell growth and motility. Given the role of PD-L1 in immune evasion²⁹, we further explored the relationship between HKDC1 and PD-L1 expression. WB analysis revealed that PD-L1 levels decreased upon HKDC1 knockdown in SKOV3 cells, while overexpression of HKDC1 in HEY cells resulted in increased PD-L1 expression (Fig. 2F).

Next, we investigated the involvement of HKDC1 in lipid metabolism. In SKOV3 cells, HKDC1 knockdown led to a significant reduction in intracellular levels of FFA, TG, PL, and CHO, while the overexpression of HKDC1 in HEY cells increased these lipid levels (Fig. 2G). Neutral lipid staining using BODIPY 493/503 dye confirmed that HKDC1 elevated the intracellular content of neutral lipids (Fig. 2H and Supplementary Fig. 2D). The observed increase in lipid content suggests enhanced lipid biosynthesis, increased FA uptake, and decreased lipid degradation³⁰. To further investigate, we examined key molecules involved in FA synthesis (ACC1, FASN, and SCD1), CHO biosynthesis (HMGCS1, HMGCR), FA uptake (CD36), and FA oxidation (CPT1A). HKDC1 knockdown reduced the mRNA and protein expression of enzymes responsible for FA and CHO biosynthesis, whereas HKDC1 overexpression led to their significant upregulation (Fig. 2I). However, the expression of key regulators of FA uptake and oxidation remained unchanged (Fig. 2I). These results demonstrate that HKDC1 plays a pivotal role in regulating lipid metabolism in OC cells. The enhancement of lipid biosynthesis and the suppression of lipid degradation likely contributed to the increased lipid content observed in HKDC1-overexpressing cells. Overall, these findings indicate the potential role of HKDC1 in promoting OC cell growth, migration, invasion, and lipid metabolism, making it a potential target for therapeutic intervention.

HKDC1 promotes the immune escape of OC cells in vitro

To investigate the involvement of HKDC1 in immune cell modulation during OC progression, we co-cultured PBMCs with SKOV3 or HEY cells, modifying HKDC1 expression through knockdown or overexpression. Flow cytometry was used to analyze the proportions of CD4⁺/CD8⁺ T cells, NK cells, and Treg cells within PBMCs. As shown in Fig. 3A and Supplementary Figs. 3, both the co-SKOV3 and co-HEY groups exhibited significant reductions in the proportions of T cells (CD45⁺CD4⁺/CD8⁺) and NK cells (CD3⁻CD16⁺CD56⁺) compared to the Control group. Conversely, Treg cells (CD4⁺CD25⁺FOXP3⁺) were significantly increased. HKDC1 knockdown led to an increase in the proportions of CD4⁺/CD8⁺ T cells and NK cells, along with a decrease in Treg cells within PBMCs. In comparison, HKDC1 overexpression in HEY cells resulted in the opposite effect, reducing T cell and NK cell populations while increasing Treg cell levels. To further clarify how HKDC1 influences immune cell behavior, we measured PD-1 expression in T cells and NK cells, as well as IFN-γ levels in CD4⁺/CD8⁺ T cells and GZMB and perforin levels in NK cells. Following co-culture with SKOV3 and HEY cells, PD-1 levels increased, while IFN-γ, GZMB, and perforin levels decreased (Fig. 3B–D and Supplementary Fig. 4). HKDC1 knockdown significantly reduced PD-1 expression while enhancing IFN-γ, GZMB, and perforin levels (Fig. 3B–D and Supplementary Fig. 4). Conversely, HKDC1 overexpression in HEY cells produced the opposite effect, with increased PD-1 expression and decreased IFN-γ, GZMB, and perforin levels (Fig. 3B–D and Supplementary Fig. 4). These results indicate that HKDC1 plays a critical role in promoting immune escape by suppressing T cell and NK cell activity while enhancing Treg populations, suggesting that HKDC1 creates an immunosuppressive environment that facilitates OC cell survival and progression in vitro.

**Fig. 3: HKDC1 promotes immune escape of OC cells in vitro.**

HKDC1 promotes proliferation, migration, invasion, and immune escape of OC cells through lipid metabolism in vitro

To further explore the relationship between lipid metabolism, cell growth, and immune escape in OC, as well as the role of HKDC1 in this process, we knocked down HKDC1 in SKOV3 and ID8 cells and treated them with FFA. qRT-PCR and WB analyses confirmed successful HKDC1 knockdown, and FFA treatment did not affect HKDC1 mRNA and HKDC1 protein expression in either cell line (Fig. 4A). However, FFA treatment counteracted the decrease in FFA, TG, PL, and CHO levels that occurred following HKDC1 knockdown (Fig. 4B). BODIPY 493/503 staining further confirmed the restoration of neutral lipid content in HKDC1-knockdown cells upon FFA treatment (Fig. 4C and Supplementary Fig. 2E). FFA also reversed the HKDC1 knockdown-induced reduction in the mRNA and protein expression of lipid biosynthetic enzymes, including ACC1, FASN, SCD1, HMGCS1, and HMGCR, although no changes were observed in the levels of CD36 and CPT1A (Fig. 4D). Functional assays demonstrated that FFA treatment significantly restored the cell proliferation, migration, and invasion that had been inhibited by HKDC1 knockdown (Fig. 4E–H and Supplementary Fig. 2F–H). These findings indicate that HKDC1 promotes cell migration and invasion through its impact on lipid metabolism. In addition, FFA reversed the downregulation of PD-L1 expression observed in HKDC1-knockdown cells (Fig. 4I), highlighting the potential link between lipid metabolism and immune escape mechanisms in OC cells.

Next, we investigated the interaction among HKDC1, lipid metabolism, and immune escape by co-culturing PBMCs with SKOV3 or ID8 cells transfected with si-HKDC1 and treated with FFA. Flow cytometry results revealed an increase in the proportions of CD4⁺/CD8⁺ T cells and NK cells, along with a reduction in Treg cell (CD4⁺CD25⁺FOXP3⁺) frequency, in the co-SKOV3/ID8^si-HKDC1 group compared to the co-SKOV3/ID8^si-NC group (Fig. 4J and Supplementary Fig. 5). However, in the co-SKOV3/ID8^si-HKDC1+FFA group, CD4⁺/CD8⁺ T cell and NK cell populations were decreased, while Treg cell numbers were increased (Fig. 4J and Supplementary Fig. 5). Moreover, co-culture of PBMCs with HKDC1-knockdown SKOV3 or ID8 cells led to a reduction in PD-1 levels in T cells and NK cells, accompanied by increased IFN-γ expression in CD4⁺/CD8⁺ T cells and elevated GZMB and perforin levels in NK cells (Fig. 4K–M and Supplementary Fig. 6). FFA treatment, however, significantly increased PD-1 expression and reduced IFN-γ, GZMB, and perforin levels (Fig. 4K–M and Supplementary Fig. 6). These findings suggest that HKDC1 plays a crucial role in facilitating immune escape in OC cells by modulating lipid metabolism. The ability of FFA to restore immune suppression further highlights the influence of lipid metabolism on the immune microenvironment in OC.

HKDC1 promotes proliferation, migration, invasion, and lipid metabolism of OC cells through interacting with and stabilizing G6PC/G6PC2

To identify proteins potentially linked to HKDC1, we utilized the STRING database (https://string-db.org/), which revealed possible interactions between HKDC1 and G6PC, G6PC2, and G6PC3. G6PC, the catalytic subunit of glucose-6-phosphatase, hydrolyzes glucose-6-phosphate (G6P) to release glucose. A deficiency in G6PC leads to G6P accumulation, disrupting metabolic homeostasis in the liver and kidneys³¹. Initial WB analysis demonstrated that HKDC1 knockdown in SKOV3 and ID8 cells resulted in reduced G6PC and G6PC2 expression, while overexpression of HKDC1 in HEY cells increased their levels (Fig. 5A). However, G6PC3 expression remained unaffected by these modifications (Fig. 5A).

**Fig. 5: HKDC1 promotes proliferation, migration, and invasion of OC cells by interacting with and stabilizing G6PC/G6PC2.**

Co-IP assays confirmed that HKDC1 directly binds to G6PC and G6PC2, while no interaction with G6PC3 was detected (Fig. 5B). WB analysis further revealed that HKDC1 knockdown led to the downregulation of HKDC1, G6PC and G6PC2, and that this effect could be reversed by the proteasome inhibitor MG132 (Fig. 5C). In HEY cells overexpressing HKDC1, MG132 treatment enhanced the expression of these proteins (Fig. 5C). Additionally, incubation of HKDC1-modified SKOV3/ID8 cells or HEY cells with cycloheximide (CHX) over 0 h, 1 h, 2 h, and 3 h³² showed that HKDC1 knockdown reduced the stability of G6PC and G6PC2 proteins, while HKDC1 overexpression improved their stability (Fig. 5D). These findings suggest that HKDC1 promotes the stability and expression of G6PC and G6PC2 by binding to these proteins.

IF staining further confirmed the co-localization of HKDC1 with G6PC and G6PC2 in SKOV3, ID8, and HEY cells (Fig. 5E). To investigate the interplay between HKDC1 and G6PC/G6PC2, we transfected SKOV3/ID8 or HEY cells with oe-G6PC/oe-G6PC2 or si-G6PC/si-G6PC2 plasmids. As shown in Fig. 5F, altering G6PC/G6PC2 mRNA expression did not significantly impact HKDC1 protein levels. Functional assays revealed that HKDC1 knockdown markedly inhibited the proliferation, migration, and invasion of SKOV3 and ID8 cells, while G6PC/G6PC2 overexpression reversed these effects (Fig. 5G–J and Supplementary Fig. 7A–C). Conversely, G6PC/G6PC2 knockdown reduced the increase of these cellular functions induced by HKDC1 overexpression in HEY cells (Fig. 5G–J and Supplementary Fig. 7A–C). Furthermore, HKDC1 knockdown in SKOV3 and ID8 cells reduced intracellular PD-L1 expression, which was restored by G6PC/G6PC2 overexpression (Fig. 6A). Similarly, HKDC1 overexpression increased PD-L1 expression in HEY cells, but this effect was mitigated by G6PC/G6PC2 knockdown (Fig. 6A).

**Fig. 6: HKDC1 promotes lipid metabolism of OC cells by interacting with and stabilizing G6PC/G6PC2.**

To assess the role of G6PC/G6PC2 in HKDC1-regulated lipid metabolism, we measured lipid levels and BODIPY staining intensity. HKDC1 knockdown in SKOV3 and ID8 cells reduced FFA, TG, PL, and CHO levels, as well as BODIPY staining, but these effects were reversed by G6PC/G6PC2 overexpression (Fig. 6B, C and Supplementary Fig. 7D). In HEY cells, HKDC1 overexpression increased these lipid indicators, but G6PC/G6PC2 knockdown attenuated these increases (Fig. 6B, C and Supplementary Fig. 7D). We also assessed the mRNA and protein expression of enzymes involved in lipid metabolism, including ACC1, FASN, SCD1, HMGCS1, HMGCR, CD36, and CPT1A. CD36 and CPT1A levels remained unchanged regardless of HKDC1 or G6PC/G6PC2 modulation (Fig. 6D). However, the reduction in lipid biosynthesis enzymes (ACC1, FASN, SCD1, HMGCS1, HMGCR) caused by HKDC1 knockdown in SKOV3 and ID8 cells was reversed by G6PC/G6PC2 overexpression (Fig. 6D). Similarly, the elevation of these enzymes induced by HKDC1 overexpression in HEY cells was mitigated by G6PC/G6PC2 knockdown (Fig. 6D). Overall, our findings indicate that HKDC1 promotes proliferation, migration, invasion, and lipid metabolism of OC cells through interacting with and stabilizing G6PC and G6PC2.

HKDC1 promotes immune escape by interacting with G6PC/G6PC2

We investigated the role of G6PC and G6PC2 in immune evasion facilitated by HKDC1. The results showed that in SKOV3 and ID8 cells, HKDC1 knockdown increased the proportions of T cells (CD45⁺CD4⁺/CD8⁺) and NK cells (CD3⁻CD16⁺CD56⁺) in PBMCs while reducing the proportion of Treg cells (CD4⁺CD25⁺FOXP3⁺) (Fig. 7A and Supplementary Fig. 8). Overexpression of G6PC or G6PC2 reversed these changes, restoring Treg cell proportions and decreasing T cell and NK cell populations (Fig. 7A and Supplementary Fig. 8). Conversely, HKDC1 overexpression in HEY cells reduced T cell and NK cell numbers and increased Treg cell proportions, effects that were weakened by the knockdown of G6PC/G6PC2 (Fig. 7A and Supplementary Fig. 8). Further analysis revealed that HKDC1 knockdown decreased PD-1 expression in T cells and NK cells, while increasing IFN-γ and GZMB levels in CD4⁺/CD8⁺ T cells and enhancing perforin levels in NK cells (Fig. 7B–D and Supplementary Fig. 9). These changes were reversed by G6PC/G6PC2 overexpression, restoring PD-1 levels and reducing IFN-γ, GZMB, and perforin expression (Fig. 7B–D and Supplementary Fig. 9). Conversely, HKDC1 overexpression in HEY cells increased PD-1 expression and reduced IFN-γ, GZMB and perforin levels, and these effects were mitigated by G6PC/G6PC2 knockdown (Fig. 7B–D and Supplementary Fig. 9). These findings suggest that the interaction between HKDC1 and G6PC/G6PC2 supports immune evasion by altering immune cell proportions and activity.

HKDC1 promotes tumor growth in vivo

To confirm the role of HKDC1 in vivo, we established an EOC mouse model. As shown in Fig. 8A–C, the body weights of mice in the four groups (sh-NC, sh-HKDC1, sh-HKDC1 + oe-G6PC, and sh-HKDC1 + oe-G6PC2) increased consistently, with no significant differences between the groups. However, tumor growth was significantly reduced in the sh-HKDC1 group compared to the sh-NC group (Fig. 8A–C). Additionally, tumors in the sh-HKDC1 + oe-G6PC and sh-HKDC1 + oe-G6PC2 groups were found to be larger and heavier than those in the sh-HKDC1 group (Fig. 8A–C). Histological analysis revealed extensive necrosis in tumors from the sh-HKDC1 group, a feature absent in the sh-HKDC1 + oe-G6PC and sh-HKDC1 + oe-G6PC2 groups (Fig. 8D). qRT-PCR and WB analysis confirmed that HKDC1 mRNA and HKDC1 protein expression was significantly reduced in the sh-HKDC1 group compared to the sh-NC group, with no changes following G6PC/G6PC2 overexpression (Fig. 8E). Serum analysis showed that levels of FFA, TG, PL, and CHO were reduced in the sh-HKDC1 group compared to the sh-NC group, but these reductions were reversed following G6PC/G6PC2 overexpression (Fig. 8F). Furthermore, the mRNA and protein expression of lipid metabolism enzymes (ACC1, FASN, SCD1, HMGCS1, and HMGCR) was reduced in tumors from the sh-HKDC1 group, while the expression of CD36 and CPT1A remained unchanged. In addition, overexpression of G6PC/G6PC2 restored the expression of these lipid-related enzymes (Fig. 8G). Taken together, these results indicate that HKDC1 promotes OC progression in vivo by enhancing lipid metabolism and tumor growth, mediated through its interaction with G6PC and G6PC2.

HKDC1 promotes immune escape of OC in vivo

To further investigate the role of HKDC1 in modulating immune cells in vivo, we analyzed CD3 T cell infiltration in tumor tissues 28 days after intervention. Suppression of HKDC1 led to a notable increase in CD3 T cell infiltration (Fig. 9A). However, this increase was reversed by the overexpression of G6PC or G6PC2 (Fig. 9A). Compared to the sh-NC group, tumors from the sh-HKDC1 group showed elevated levels of CD8 T cells, GZMB, and perforin, along with a reduction in PD-L1 expression (Fig. 9B). These effects were diminished when G6PC or G6PC2 were overexpressed in HKDC1-suppressed tumors (Fig. 9B), indicating that the immune effects induced by HKDC1 knockdown were modulated by G6PC/G6PC2. Flow cytometry analysis revealed significant increases in T cells (CD45⁺CD4⁺/CD8⁺) and NK cells (CD3⁻CD16⁺CD56⁺), as well as elevated levels of IFN-γ in the sh-HKDC1 group (Fig. 9C, D Supplementary Figs. 10A, B). Comparatively, Treg cells (CD4⁺CD25⁺FOXP3⁺) were significantly reduced in these tumors (Fig. 9C, D and Supplementary Fig. 10A, B). However, overexpression of G6PC or G6PC2 substantially reversed these changes, restoring the immune cell populations and IFN-γ levels to those observed in the sh-NC group (Fig. 9C, D and Supplementary Fig. 10A, B). These findings demonstrate that HKDC1 plays a role in promoting immune escape in OC in vivo by modulating immune cell infiltration and activity. Collectively, the reversal of immune responses by G6PC/G6PC2 overexpression suggests their involvement in HKDC1-mediated immune evasion.

Discussion

OC is one of the most lethal gynecologic malignancies, with late diagnosis and the emergence of chemotherapy resistance limiting the effectiveness of treatment^1,4,33. Although HKDC1 has been implicated in tumor progression, its specific roles in tumor growth, lipid metabolism, and immune escape in OC have remained unclear. In this study, we demonstrated that HKDC1 is upregulated in OC cells and promotes tumor growth, lipid metabolism, and immune escape by interacting with and stabilizing G6PC and G6PC2, contributing to OC progression. These findings provide valuable insights into the molecular mechanisms of HKDC1, highlighting its potential as a therapeutic target in OC.

Cell-based experiments revealed significant upregulation of HKDC1 in OC cell lines, suggesting that HKDC1 has oncogenic properties in OC. This upregulation identified HKDC1 as a potential target for therapy. In vitro and in vivo experiments further established the causal relationship between HKDC1 and OC cell proliferation, migration, and invasion. Knockdown of HKDC1 inhibited these cellular processes, while HKDC1 overexpression promoted them. Additionally, in vivo experiments confirmed that HKDC1 knockdown significantly suppressed tumor growth, emphasizing its role in tumor progression.

The reprogramming of lipid metabolism, primarily characterized by the upregulation of FA synthesis, is increasingly recognized as a hallmark of cancer³⁴. Enhanced lipogenesis, encompassing both FA synthesis and CHO biosynthesis, not only provides essential energy storage but also generates signaling molecules and structural membrane components that promote tumor growth and metastasis^30,35. Previous studies on OC have reported the upregulation of enzymes involved in FA and CHO biosynthesis, including ACC1, FASN, SCD1, HMGCR, and HMGCS1, highlighting their roles in driving tumor growth and metastasis¹¹. The induction of FA uptake has been identified as a common mechanism employed by cancer cells to support proliferation and confer resistance to therapies, with CD36 recognized as a key receptor in this process. Cells exhibiting elevated lipid uptake generally show enhanced FA oxidation, which further contributes to proliferation, migration, survival, and drug resistance³⁶. Our findings align with these observations, demonstrating that HKDC1 promotes lipid biosynthesis in OC cells by upregulating essential enzymes involved in FA synthesis (ACC1, FASN, SCD1) and CHO biosynthesis (HMGCS1, HMGCR). However, our study did not identify a significant effect of HKDC1 on FA uptake or FA oxidation, suggesting that while HKDC1 contributes to lipid biosynthesis, it does not promote lipolysis.

Defects in the immune system can lead to the onset and progression of cancer, including OC³⁷, which is considered an immunogenic tumor that relies heavily on immunotherapy targeting immune checkpoints^38,39. Among these, PD-L1 is a key immune checkpoint that inhibits T cell activation, facilitating tumor progression and correlating with poor prognosis in OC patients^40,41. Previous studies have demonstrated that the HOXA transcript at the distal tip upregulates PD-L1 expression, thereby suppressing T cell activity and promoting the immune escape of OC cells⁴². In contrast, cullin 3 facilitates the degradation of PD-L1 through the formation of a complex with its binding partner speckle-type POZ protein, leading to enhanced T cell proliferation, activity, and cytotoxicity, which inhibits immune escape and increases OC cell sensitivity to cisplatin⁴³. Our study revealed that HKDC1 upregulates PD-L1 expression, resulting in heightened immune suppression characterized by reduced T cell activation and decreased expression of key immune markers, including IFN-γ, GZMB, and perforin, as well as increased PD-1 levels. These findings were further validated through in vivo experiments, confirming that HKDC1 promotes immune escape in OC cells by enhancing PD-L1 expression. More significantly, our research further validates that HKDC1 primarily promotes proliferation, migration, invasion, and immune escape of OC cells by regulating lipid synthesis, thereby excluding the possibility of achieving these functions through other non-lipid metabolic pathways, such as by directly regulating glycolysis or mitochondrial function.

Mechanistically, our findings demonstrate that HKDC1 interacts with G6PC and G6PC2, which contribute to tumor growth, lipid metabolism, and immune escape in OC. The G6PC gene family consists of three members: G6PC1 (also referred to as G6PC), G6PC2, and G6PC3. G6PC is a key enzyme involved in gluconeogenesis and glycogenolysis⁴⁴. Previous studies have reported that G6PC expression is upregulated in OC, promoting cell proliferation and invasive phenotypes, suggesting an oncogenic role for G6PC in this context⁴⁵. G6PC2, which encodes the catalytic subunit of the glucose-6-phosphatase enzyme localized in the endoplasmic reticulum, has an unclear role in OC⁴⁶. Our study represents the first to demonstrate that the protein expression of G6PC and G6PC2 is regulated by HKDC1. Co-IP assays confirmed the interaction between HKDC1 and G6PC/G6PC2, and IF analysis showed that these proteins co-localize in OC cells. Crucially, we discovered that the binding of HKDC1 remarkably enhances the stability of the G6PC/G6PC2 protein, thereby fostering their expression. These results indicate that HKDC1 interacts with and stabilizes G6PC/G6PC2 in OC cells. To further explore the functional consequences of this interaction, we conducted a series of experiments. Overexpression of G6PC/G6PC2 reversed the inhibitory effects of HKDC1 downregulation on cell proliferation, migration, invasion, lipid metabolism, and immune escape. Conversely, the knockdown of G6PC/G6PC2 mitigated the promotive effects of HKDC1 overexpression on these cellular processes, which were also validated through in vivo experiments, confirming the impact of the HKDC1-G6PC/G6PC2 interaction on OC progression. Overall, HKDC1 was found to interact with and stabilize G6PC and G6PC2 to enhance their expression and activity, thereby playing an important role in promoting tumor growth, lipid metabolism, and immune evasion in OC, highlighting the importance of HKDC1 in these pathological processes.

Despite the promising results reported in this study, several limitations should be clarified. First, although our findings highlight the role of G6PC and G6PC2 in regulating lipid synthesis-related factors and PD-L1 expression, the precise molecular mechanisms involved require further investigation. Second, due to the minimal formation of ascites in the intraperitoneal tumor model, we were unable to assess the impact of HKDC1 on OC metastasis directly. Future experiments could focus on optimizing experimental conditions to evaluate this aspect better. Lastly, as the findings of this study were primarily derived from cellular and animal models, their applicability to human tumors requires further validation. In future research, these limitations could be addressed by conducting clinical experiments or utilizing public tumor databases, such as TCGA, to further explore the relationship between elevated HKDC1 expression, lipid synthesis enzymes, and PD-L1 levels.

Conclusion

HKDC1 is significantly overexpressed in OC cells. Through its interaction with and stabilization of G6PC and G6PC2, HKDC1 facilitates tumor growth, enhances lipid metabolism, and promotes immune escape, thereby contributing to the progression of OC. These findings provide valuable insights into the molecular mechanisms underlying OC pathogenesis and support the potential of HKDC1 as a promising therapeutic target for OC treatment.

Methods and materials

Cell culture

The human normal ovarian epithelial cell line IOSE80 (AW-CNH242), along with the human OC cell lines HEY (AW-CCH220), SKOV3 (AW-CCH110), and the mouse OC cell line ID8 (AW-CCM395), were obtained from Abiowell Company (Changsha, China). The other human OC cell lines, ES-2 (CL-0079) and OVCAR3 (CL-0178), were purchased from Procell (Wuhan, China). IOSE80 cells were cultured in RPMI-1640 medium (AW-MC002, Abiowell), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). HEY cells were grown in a DMEM medium (AW-M003, Abiowell) containing 10% FBS and 1% P/S. SKOV3 and ES-2 cells were maintained in McCoy’s 5A medium (AW-M007, Abiowell), also supplemented with 10% FBS and 1% P/S. OVCAR3 cells were cultured in RPMI-1640 medium (CM-0079, Procell), enriched with 20% FBS, 10 μg/mL insulin, and 1% P/S. The ID8 cells were maintained in a complete DMEM medium, supplemented with 10% FBS and 1% P/S, before preparing cell suspensions for mouse injections. All cell lines were incubated under standard culture conditions of 37 °C, 5% CO₂, and full humidity. Cells in the logarithmic growth phase were used for subsequent experiments.

For SKOV3 and ID8 cells, the following experimental groups were established: (1) si-NC group (transfected with si-NC), (2) si-NC + MG132 (transfected with si-NC and treated with 10 μM MG132 for 12 h⁴⁷), (3) si-HKDC1 group (transfected with si-HKDC1), (4) si-HKDC1 + MG132 (transfected with si-HKDC1 and treated with 10 μM MG132 for 12 h), (5) si-HKDC1 + FFA (transfected with si-HKDC1 and treated with 150 μM free fatty acids (FFA) for 24 h⁴⁸), (6) si-HKDC1 + oe-G6PC group (co-transfected with si-HKDC1 and oe-G6PC), and (7) si-HKDC1 + oe-G6PC2 group (co-transfected with si-HKDC1 and oe-G6PC2).

For HEY cells, the following experimental groups were established: (1) oe-NC group (transfected with oe-NC), (2) oe-NC + MG132 (transfected with oe-NC and treated with 10 μM MG132 for 12 h), (3) oe-HKDC1 group (transfected with oe-HKDC1), (4) oe-HKDC1 + MG132 (transfected with oe-HKDC1 and treated with 10 μM MG132 for 12 h), (5) oe-HKDC1 + si-G6PC group (co-transfected with oe-HKDC1 and si-G6PC), and (6) oe-HKDC1 + si-G6PC2 group (co-transfected with oe-HKDC1 and si-G6PC2).

FFA preparation

Palmitic acid (PA, 39756-30-4, Sigma-Aldrich, St. Louis, Missouri, USA) was initially dissolved in a 50% ethanol solution to prepare a 150 mM stock solution. This solution was subsequently mixed with 5% bovine serum albumin (BSA) to make a 7.5 mM PA working solution. Similarly, an oleic acid (OA) solution was diluted with 5% BSA to prepare a 15 mM OA working solution. Lastly, these two working solutions were mixed in a 1:1 volume ratio to obtain the FFA solution⁴⁸.

Cell transfection

Small interfering RNAs (siRNAs) targeting HKDC1 (HG-shHO025130), G6PC (HG-shHO000151), and G6PC2 (HG-shHO021176), along with overexpression constructs for HKDC1 (HG-HO025130), G6PC (HG-HO000151), and G6PC2 (HG-HO021176), and their respective negative controls, were obtained from HonorGene (Changsha, China). Transfections into SKOV3 and HEY cells were performed following the manufacturer’s protocol using Lipofectamine 2000 (11668019, Invitrogen, Waltham, MA, USA). After incubation at 37 °C for 12 h, the culture medium was replaced with a fresh medium. The cells were harvested for quantitative real-time PCR (qRT-PCR) analysis 48 h post-transfection.

Purification of T cells from peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation and incubated with anti-CD3 antibody (1 µg/mL, 12-0038-42, eBioscience, Waltham, MA, USA) for 60 min, followed by treatment with FlowComp immunomagnetic beads (11365D, Invitrogen) for 30 min. CD3 T cells were then purified using magnetic bead-based selection columns. The isolated cells were cultured in RPMI 1640 medium containing 10% FBS and stimulated with anti-CD3 (2 µg/mL) and anti-CD28 antibodies (2 µg/mL, 46-0037-42, eBioscience) to promote T lymphocyte proliferation. After 48 h, the activated T lymphocytes formed spheroids and were pelleted via centrifugation. Next, the cells were cultured in RPMI 1640 medium containing anti-CD3 and anti-CD28 for use in downstream experiments⁴³.

Flow cytometry analysis

Flow cytometry analysis was conducted using antibodies purchased from eBioscience, which included anti-CD45 (25-0451-82), anti-CD4 (MA5-17451), anti-CD8 (17-0088-42), anti-CD3, anti-CD16 (17-0168-42), anti-CD56 (11-0566-42), anti-CD25 (17-0257-42), anti-FOXP3 (53-4776-42), anti-PD-L1 (17-5983-42), and anti-IFN-γ (17-7311-82). After incubating PBMCs with antibodies in the dark for 30 min, cells were washed twice with wash buffer, fixed with 4% paraformaldehyde (N1012, NCM Biotech, Suzhou, China), and then analyzed using flow cytometry. CD4⁺/CD8⁺ T cell, regulatory T (Treg) cell, and natural killer (NK) cell sorting strategies were as follows: CD4⁺/CD8⁺ T cells were CD45⁺CD4⁺/CD8⁺, Treg cells were CD4⁺/CD25⁺/FOXP3⁺, and NK cells were CD3⁻/CD16⁺/CD56⁺. PD-L1 and IFN-γ expression in these cells was measured post-antibody incubation.

For the flow cytometric analysis of mouse tumor tissues, tissue sections were treated with trypsin digestion solution (AWC0232, Abiowell) and filtered to obtain single-cell suspensions. Red blood cell lysis buffer (C3702, Beyotime, Shanghai, China) was used to remove erythrocytes, and the cells were then centrifuged and resuspended in a medium containing 10% FBS. These cells were then stimulated with Cell Stimulation Cocktail, including protein transport inhibitors, at 37 °C for 4 h. After washing with phosphate-buffered saline (PBS) and centrifugation, the cells were stained with anti-IFN-γ antibody (25-7311-41, eBioscience) for 30 min in the dark. Lastly, the cells were washed with 0.5% BSA-PBS and analyzed by flow cytometry to quantify IFN-γ expression.

Cell co-culture

The co-culture experiments were conducted using Transwell plates with 0.4 µm pore-sized membranes. Briefly, the cells were resuspended in RPMI-1640 medium supplemented with 10% FBS. OC cells (2 × 10⁵ cells/well) were seeded in the lower chambers of 24-well plates, while isolated PBMCs or purified T cells (6 × 10⁵ cells/well) were placed in the upper Transwell chambers. The co-cultures were incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 16 h to allow cellular interactions.

Cell counting kit-8 (CCK-8)

Cell proliferation was assessed using the CCK-8 assay kit (NU679, Dojindo, Kumamoto, Japan). The treated cells were seeded into 48-well plates at a density of 1 × 10⁴ cells per well, with each group plated in triplicate. After 48 h of incubation, 100 µL of CCK-8 solution was added to each well and incubated for an additional 4 h at 37 °C. The optical density (OD) was measured at 450 nm using a microplate reader (MB-530, Heleas, Shenzhen, China) to determine cell viability.

Colony formation assay

The cells were seeded at an initial density of 200 cells per well in 6-well plates and incubated at 37 °C with 5% CO₂ for 2–3 weeks. Then, the supernatant was removed, and the cells were fixed with 4% paraformaldehyde for 15 min. The fixed cells were stained with 0.5% crystal violet solution for 30 min, and the excess stain was removed by rinsing the wells with PBS. The plates were allowed to air dry before capturing images. Colonies were quantified by counting the number of colonies per well.

Wound healing assay

Cell migration was assessed using a wound-healing assay. Briefly, the cells (5 × 10⁵) were seeded into 6-well plates and grown to confluence. A straight scratch was created in the cell monolayer using a sterile 200 µL pipette tip, following the cells were incubated in a serum-free F12K medium. Wound closure was documented at 0 h, 24 h, and 48 h post-wounding by capturing images. The relative migration distances were quantified using ImageJ software.

Cell invasion assay

For the cell invasion assay, Matrigel (356234, Corning Inc, Corning, NY, USA) was diluted 1:8 in serum-free F12K medium and applied to the upper chamber of the Transwell insert. The Matrigel was allowed to solidify at 37 ℃ for 30 min, after which the excess medium was removed. Cells (1 × 10⁵) suspended in 300 µL of serum-free DMEM were added to the upper chamber, while 500 µL of DMEM supplemented with 10% FBS was placed in the lower chamber. After 24 h, the cells on the lower surface of the Matrigel-coated membrane were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The number of invaded cells was counted using an inverted microscope (DSZ2000X, Cnmicro, Beijing, China).

Biochemical analysis

Cell-free supernatants were collected to measure granzyme B (GZMB, CSB-E08718h, CUSABIO, Wuhan, China) and perforin (CSB-E09313h, CUSABIO) concentrations according to the respective kit protocols. The levels of FFA (A042-2-1), triglycerides (TG; A110-1-1), and CHO (A111-1-1) were quantified in cells and serum following protocols from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Phospholipid (PL) concentrations were measured using a biochemical assay kit (JL-T2427, Jianglai Bio, Shanghai, China).

Neutral lipid fluorescent staining

The intracellular accumulation of neutral lipids was visualized using BODIPY 493/503 dye (D3922, Invitrogen) following the manufacturer’s instructions. OC cells were cultured in 10 cm dishes, fixed with 4% paraformaldehyde for 15 min, and incubated with BODIPY 493/503 dye (1 μg/mL) at 37 °C for 1 h. Fluorescence images were captured using an Olympus FV-1000 confocal microscope. The intensity of the neutral lipid signal was quantified using ImageJ software.

qRT-PCR

Total RNA was extracted using the TRIzol Total RNA Extraction Kit (15596026, Invitrogen) following the manufacturer’s protocol and quantified using a NanoDrop ND-1000 UV Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using the mRNA Reverse Transcription Kit (CW2569, CWBIO, Taizhou, China). Gene expression was analyzed using the Ultra SYBR Mixture (CW2601, CWBIO) on a QuantStudio 1 fluorescence PCR instrument (Thermo Fisher Scientific), with β-actin as the internal control. Relative gene expression levels were calculated using the 2^−ΔΔCt method. Primer sequences were obtained from NCBI, designed using Primer5 software, and synthesized by the Beijing Genomics Institute. Detailed primer sequences are listed in Table 1.

Table 1 Primer sequences

Full size table

Western blot (WB)

Proteins were extracted from tissues and cells using RIPA lysis buffer (AWB0136, Abiowell), and their concentrations were determined following the instructions of the bicinchoninic acid (BCA) Protein Assay Kit (BL521A, Biosharp, Hefei, China). Proteins were separated by electrophoresis at 75 V for 130 min until the bromophenol blue dye reached the bottom of the gel. The proteins were transferred onto a nitrocellulose membrane using a 300-mA current. The membrane was blocked with 5% skimmed milk dissolved in PBS containing 0.05% Tween-20 (PBST) for 90 min. The membrane was then incubated with the primary antibody overnight at 4 °C, followed by incubation with the appropriate secondary horseradish peroxidase (HRP)-conjugated antibody for 90 min at room temperature. β-actin was used as the internal loading control. After washing the membrane three times with PBST, the protein bands were visualized by enhanced chemiluminescence (ECL) after 60 s of exposure. The intensity of the protein bands was quantified using ImageJ software. Antibody details are listed in Table 2.

Table 2 Antibody information

Full size table

Co-immunoprecipitation (Co-IP) assay

Cells were lysed using RIPA buffer for 10 min, and the resulting lysates were incubated overnight at 4 °C with either anti-HKDC1 or control rabbit IgG antibody (B900610, Proteintech). Then, 10 µL of protein A/G agarose beads were added, and the samples were incubated for an additional 2 h at 4 °C, following which the beads were washed three times with lysis buffer to remove non-specific interactions and centrifuged at 3000 rpm for 3 min. The immunoprecipitated complexes were then eluted by heating the beads with sample buffer for 5 min, and the resulting supernatant was used for WB analysis.

Animal experiments

Female C57BL/6J mice (6-week-old, 20 ± 2 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. (China). The mice were acclimated for one week under controlled conditions (temperature of 25 ± 2 °C, relative humidity of 60 ± 10%, and a light/dark cycle of 12 h). Mice were randomly assigned to four experimental groups (n = 5): sh-NC, sh-HKDC1, sh-HKDC1 + oe-G6PC, and sh-HKDC1 + oe-G6PC2. ID8 cells were stably transduced with lentiviral vectors encoding sh-HKDC1, oe-G6PC, oe-G6PC2, or a non-targeting control. Transfected cells were resuspended in a 1:1 mixture of PBS and growth factor-reduced Matrigel® Matrix (Corning Inc.), and 7 × 10⁶ cells in 150 µL of the suspension were injected subcutaneously into the right flank of each mouse to establish EOC models^49,50. Tumor size was measured and volume was estimated using the formula V = 0.5 (Length × Width²). Mice were euthanized through intravenous injection of 150 mg/kg of a 3% pentobarbital sodium solution when the tumor did not exceed the maximum permitted volume of 1 cm³ within 28 days post-transplantation. Tumor tissues and serum were collected for further analysis. All the procedures were approved by the Animal Ethics Committee of Hunan Cancer Hospital (no. 2022187). We have complied with all relevant ethical regulations for animal use. Moreover, all experimental procedures were conducted in accordance with the ARRlVE guidelines, ensuring adherence to the principle of humane endpoints and animal welfare throughout the study. The maximal tumor measurements and volumes that were permitted by the Animal Ethics Committee of Hunan Cancer Hospital were not exceeded in the animal experiments.

Immunofluorescence (IF) assay

Dual-label IF was performed to assess the co-localization of HKDC1 with G6PC and G6PC2 in OC cells. The cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton X-100 at 37 °C for 30 min, and blocked with 3% hydrogen peroxide for 25 min at room temperature. Then, 5% BSA was applied for 1 h to prevent non-specific binding. To assess the expression of CD8⁺PD-L1, GZMB, and perforin in mouse tumor tissues, a triple-label IF kit (AWI0693a, Abiowell) was employed. Tissue sections were deparaffinized, subjected to antigen retrieval in EDTA solution, and blocked with peroxidase inhibitor. Primary antibodies, including HKDC1 (25874-1-AP, 1:50, Proteintech), G6PC (A20193, 1:50, ABclonal, Wuhan, China), G6PC2 (720253, 1:50, Invitrogen), CD8 (ab217344, 1:200, Abcam), PD-L1 (17952-1-AP, 1:200, Proteintech), GZMB (ab255598, 1:200, Abcam) and perforin (bs-7128R, 1:200, Bioss, USA), were used to incubate the sections overnight at 4 °C. After washing, the sections were treated with goat anti-rabbit IgG polymer and fluorescent dyes (TSA-520, TSA-570, TSA-690). Imaging was performed using a fluorescence microscope (BA410T, Motic, Xiamen, China).

Immunohistochemical (IHC) assay

IHC staining was performed to evaluate CD3 expression in tumor tissues. The sections were deparaffinized with xylene and rehydrated using an ethanol gradient. Antigen retrieval was performed, then incubated for 10 min with 1% periodic acid at room temperature. Next, the sections were incubated overnight at 4 °C with a primary antibody against CD3 (17617-1-AP, 1:300, Proteintech). After washing, sections were treated with an anti-rabbit IgG-HRP polymer for 30 min at 37 °C. Color development was achieved using diaminobenzidine (DAB) for 1–5 min at room temperature, followed by hematoxylin counterstaining. The sections were dehydrated, cleared in xylene for 10 min, and mounted with neutral gum. Visualization was performed under a light microscope.

Hematoxylin-eosin (H&E) staining

Paraffin-embedded tumor tissue sections (5 µm) were prepared for histological analysis. The sections were first deparaffinized in xylene and then rehydrated through a graded ethanol series (80%, 95%, and 100%). Hematoxylin staining was performed for 1–10 min, followed by a rinse with distilled water. After treatment with PBS, the sections underwent eosin staining for 1–5 min, followed by another rinse with distilled water. The sections were rapidly dehydrated through an ascending ethanol series and immersed in xylene for 10 min. Finally, the tissue sections were mounted with neutral resin and examined under a light microscope.

Statistical and reproducibility

All experiments were independently performed at least three times to ensure reproducibility. Data are expressed as mean ± standard deviation. Statistical analyses were conducted using GraphPad Prism 8 software. The Shapiro–Wilk test was applied to assess data normality, and Levene’s test was used to evaluate the homogeneity of variances. For comparisons between two groups, unpaired two-tailed t-tests were employed. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to identify significant differences among groups. P-value < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data discovered and analyzed during this study are included in this paper and its supplementary files. The source data for the graphs presented in the main figure can be found in Supplementary Data 1 (collection of raw data). Representative images of colony formation, wound healing, transwell invasion, and BODIPY staining assays are shown in Supplementary Figs. 2 and 7. Representative plots of flow cytometry can be found in Supplementary Figs. 3–6 and 8–10. The original immunoblot images can be found in Supplementary Fig. 11.

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Acknowledgements

This work was supported by the Scientific Research Project of Hunan Provincial Health Commission (no. 202105011313), the Hunan Key Laboratory of Cancer Metabolism (no. 2020TP1018), and the Natural Science Foundation of Hunan Province (no. 2023JJ60333).

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Authors and Affiliations

Department of the Central Laboratory, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital, Changsha, P. R. China
Ying Wang, Xia Luo, Nayiyuan Wu & Jing Wang
Department of Pharmacy, Xiangya Hospital, Central South University, Changsha, P. R. China
Juan Chen
Lung Cancer and Gastrointestinal Unit, Department of Medical Oncology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital, Changsha, P. R. China
Zhan Wang

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Ying Wang
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Contributions

Conceived and designed the experiments: Y.W. and J.W. Performed the experiments: Y.W., N.W., and X.L. Analyzed the data: Y.W. Contributed reagents/materials/analysis tools: Y.W. and X.L. Wrote the paper: Y.W. Reviewed/edited the manuscript: Y.W. and J.W. All authors have read and approved the final manuscript.

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Correspondence to Ying Wang or Jing Wang.

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Communications Biology thanks Biana Godin and Nobuhiro Okada for their contribution to the peer review of this work. Primary Handling Editors: Keith Syson Chan and Johannes Stortz. A peer review file is available.

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Wang, Y., Chen, J., Wang, Z. et al. HKDC1 promotes ovarian cancer progression through boosting lipid metabolism and immune escape by stabilizing G6PC/G6PC2. Commun Biol 8, 615 (2025). https://doi.org/10.1038/s42003-025-08031-w

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Received: 23 April 2024
Accepted: 02 April 2025
Published: 16 April 2025
Version of record: 16 April 2025
DOI: https://doi.org/10.1038/s42003-025-08031-w

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