C12ORF49 inhibits ferroptosis in hepatocellular carcinoma cells via reprogramming SREBP1/SCD1-mediated lipid metabolism

Abstract

Altered lipid metabolism is an emerging hallmark of cancer, which is involved in various aspects of the cancer phenotypes. C12ORF49 has recently been identified as a pivotal regulator of sterol regulatory element binding proteins (SREBPs), a family of transcriptional factors that govern lipid biosynthesis. Nevertheless, the function of C12ORF49 in human cancers has not been studied. Here, we show that C12ORF49 levels are higher in HCC tissue than in nearby non-cancerous liver tissue. Additionally, increased C12ORF49 expression is linked to poorer survival outcomes in HCC patients. Functional experiments uncovered that knockdown of C12ORF49 inhibited HCC cell survival and tumor growth by inducing ferroptosis, whereas the opposites were observed upon C12ORF49 overexpression. Mechanistically, C12ORF49 promotes SREBP1/SCD-regulated production of monounsaturated fatty acids, which inhibits ferroptosis in HCC cells. Furthermore, silencing C12ORF49 combined with Sorafenib treatment showed a synergistic effect in inducing HCC cell death. Together, our findings suggest a critical role of C12ORF49 in the evasion of ferroptosis in HCC cells, highlighting the potential of targeting C12ORF49 as a therapeutic strategy to enhance the efficacy of Sorafenib treatment in HCC.

Fig. 1. C12ORF49 expression is significantly elevated and its higher level predicts poor patients’ survival in HCC.

A qRT-PCR assay for C12ORF49 expression in paired cancerous and adjacent non-cancerous tissues from a cohort of 30 HCC patients. B UALCAN analysis was conducted to compare C12ORF49 expression in human HCC tissues versus non-tumorous liver tissues. C12ORF49 expression was assessed in indicated cells through qRT-PCR (C) and western blot (D) assays. E Immunohistochemical (IHC) staining of C12ORF49 was performed on a larger cohort of 238-paired cancerous and adjacent non-cancerous tissues (Scale bar = 20 µm). Comparison of overall survival (F) in C12ORF49 high or low expression HCC patients (n = 238). G Comparison of recurrence-free survival in C12ORF49 high or low expression HCC patients (n = 238). H Prognostic relevance of C12ORF49 in HCC was evaluated through bioinformatics analysis using the UALCAN platform.

Fig. 2. C12ORF49 knocking-down inhibited the growth of HCC cells.

The effectiveness of C12ORF49 knockdown in HLF and HLE cells was validated by qRT-PCR (A) and WB (B) experiments. C, D The impact of C12ORF49 knockdown on proliferation capabilities of HLF and HLE cells were assessed by MTS and colony assays. E, F C12ORF49 knockdown on cell cycle distribution was assessed via flow cytometry and EdU incorporation assays (Scale bar = 20 µm). G Cell apoptosis was analyzed upon C12ORF49 knockdown. Values are expressed as mean ± SEM from three individual experiments (n = 3).

Fig. 3. The suppressive effect of C12ORF49 silencing on HCC growth was confirmed in mouse models.

A Subcutaneous implantation nude mice models were constructed in indicated groups. B Representative images and weight of tumors dissected from subcutaneous xenograft models. C, D C12ORF49 and Ki-67 expressions were assessed by IHC assay to assess the efficiency of C12ORF49 knockdown and its impact on cell proliferation (Scale bar = 50 µm). E TUNEL assay was employed to assess the impact of C12ORF49 knockdown on in vivo cell apoptosis (Scale bar = 50 µm). F The impact of C12ORF49 knockdown on in vivo lung metastatic capabilities of HLF cells was assessed in nude mice models (Scale bar = 50 µm).

Fig. 4. Overexpression of C12ORF49 promoted the growth of HCC.

Upregulation of C12ORF49 was validated by qRT-PCR (A) and WB (B). C, D The influence of C12ORF49 overexpression on cell viability and growth was assessed by MTS and colony-forming assays. E, F The influence of C12ORF49 overexpression on cell migration and invasion was assessed. Values are expressed as mean ± SEM from three individual experiments (n = 3).

Fig. 5. C12ORF49 promotes HCC survival by suppressing ferroptosis.

A An assessment of cell death was conducted in HLF and HLE cells treated with inhibitors targeting distinct forms of cell death (ferroptosis inhibitor Fer-1; apoptosis inhibitor ZVF; necroptosis inhibitor NEC-1, pyroptosis inhibitor VX765). B Quantification of lipid reactive oxygen species (ROS) was carried out using the C11-BODIPY 581/591 staining assay (Scale bar = 20 µm). C Intracellular levels of Fe²⁺ were measured (Scale bar = 20 µm). Cell death (D) and concentrations of lipid ROS (E) and Fe²⁺ (F) were measured in SNU-354 and SNU-739 cells treated with RSL3 (Scale bar = 20 µm). Values are expressed as mean ± SEM from three individual experiments (n = 3).

Fig. 6. C12ORF49 enhances lipogenesis by activating SREBP1/SCD1 signaling.

Concentrations of free fatty acid (FFA) (A), triglyceride (TG) (B) and phospholipids (PL) (C) were quantified. D Lipophilic BODIPY 493/503 staining assay was conducted to detect the levels of neutral lipids (Scale bar = 20 µm). The influence of C12ORF49 on the expression levels of SREBP1 and SREBP2 was evaluated by qRT-PCR (E) and WB (F) assays. G WB assay for the impact of C12ORF49 on the nuclear SREBP1 level. H WB assay was conducted for the impact of C12ORF49 on the expression of SCD. FFA (I), TG (J) and PL (K) levels were detected in HCC cells with indicated treatment. L Lipophilic BODIPY 493/503 staining assay was conducted to evaluate the neutral lipids content (Scale bar = 20 µm). M The contents of palmitoleic (C16:1) and oleic (C18:1) acids were detected in HCC cells with indicated treatment. N IHC staining assays for correlations between the expressions of C12ORF49 and SREBP1 and SCD1 in HCC tissues. Values are expressed as mean ± SEM from three individual experiments (n = 3).

Fig. 7. C12ORF49 suppresses ferroptosis by activating SREBP1/SCD1-mediated biogenesis of monounsaturated fatty acids.

A Cell death was evaluated in HCC cells under indicated treatment. Quantifications of lipid ROS (B) and Fe²⁺ (C) (Scale bar = 20 µm). D, E Cell viability and growth were assessed using MTS and clonogenic assays. Values are expressed as mean ± SEM from three individual experiments (n = 3).

Fig. 8. C12ORF49 silencing enhances the efficiency of sorafenib treatment on the suppression of HCC growth and induction of ferroptosis.

A Cell death in HLF and HLE cells was evaluated. Sorafenib (10 μM). Quantifications of lipid ROS (B) and Fe2⁺ (C) in HLF and HLE cells (Scale bar = 20 µm). MTS (D) and colony (E) assays in HLF and HLE cells. F, G qRT-PCR and WB assays were conducted to determine C12ORF49 expression in Sorafenib-resistant or -sensitive HLF and HLE cells. Values are expressed as mean ± SEM from three individual experiments (n = 3).