TMEM147 aggravates the progression of HCC by modulating cholesterol homeostasis, suppressing ferroptosis, and promoting M2 polarization of TAMs

doi:10.21203/rs.3.rs-2955183/v1

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TMEM147 aggravates the progression of HCC by modulating cholesterol homeostasis, suppressing ferroptosis, and promoting M2 polarization of TAMs

https://doi.org/10.21203/rs.3.rs-2955183/v1

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Background: The endoplasmic reticulum (ER) regulates critical processes, including lipid synthesis, which can be affected by transmembrane proteins localized in the ER membrane. One of them, the transmembrane protein 147 (TMEM147) has been recently implicated for its role in hepatocellular carcinoma (HCC) tumorigenesis, though the mechanisms remain unclear. Here, we investigated the role of TMEM147 in HCC and its underlying mechanisms.

Methods: Expression of TMEM147 was examined in human HCC and adjacent non-tumorous tissues using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The effects of TMEM147 on HCC progression were investigated both in vitro and in vivo. Proteins interacting with TMEM147 were identified using RNAseq analysis, immunoprecipitation, and mass spectrometry analyses. Lipidomic analysis and enzyme-linked immunosorbent assay (ELISA) were employed to identify and analyze cholesterol and 27-hydroxycholesterol (27HC) contents. Comprehensive experimental methods were used to demonstrate ferroptosis in HCC cells. The fatty acid content of macrophages affected by TMEM147 was quantified using ELISA. Macrophage phenotypes were determined using various assays, such as immunofluorescence assay and flow cytometry analysis.

Results: TMEM147 mRNA and protein levels were upregulated in HCC, and increased TMEM147 expression was associated with poor survival. TMEM147 promoted tumor cell proliferation and metastasis in vitro and in vivo. It was found to interact with the key sterol reductase DHCR7, which affected cellular cholesterol homeostasis and increased extracellular 27HC levels in HCC. TMEM147 promoted the expression of DHCR7 by enhancing the activity of the transcription factor, STAT2. 27HC upregulated the expression of glutathione peroxidase 4 in HCC, leading to ferroptosis resistance and promotion of HCC proliferation. HCC cell-derived 27HC activated PPARγ signaling and enhanced lipid metabolism in macrophages, thereby activating M2 polarization, and then promoted the invasion and migration of HCC.

Conclusions: Our results indicated that TMEM147 confers ferroptosis resistance and M2 macrophage polarization, which are mainly dependent on the upregulation of cellular cholesterol homeostasis and 27HC secretion, leading to cancer growth and metastasis. Our findings suggest that the TMEM147/STAT2/DHCR7/27HC axis in the tumor microenvironment may serve as a promising therapeutic target for HCC.

Hepatocellular carcinoma

TMEM147

Cholesterol metabolites

Ferroptosis

Tumor-associated macrophages

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and a leading cause of cancer-related death worldwide. Though multiple treatment modalities exist, surgical resection has been found to be most effective for early-stage HCC. [1] However, the prognosis of HCC remains poor because of its propensity for metastatic progression and poor response to therapeutic treatments. Therefore, there is need for identification of new diagnostic and therapeutic targets to improve the prognosis in patients with HCC.

Transmembrane Protein 147 (TMEM147) is a small, fairly recently discovered 25 kDa protein that localizes to both the nuclear envelope (NE) and the endoplasmic reticulum (ER), which hosts critical processes, including lipid synthesis.[2, 3] TMEM147 has seven transmembrane domains, each of which fully spans the ER membrane, with the N-terminus facing the ER lumen and the C-terminus facing the cytosolic side of the membrane.[4] Its expression in many tissues suggests that it has general and wide-ranging physiological role.[5] TMEM147 has been studied in various human diseases, including prostate cancer,[6] rheumatoid arthritis,[7] and colon cancer[8]; however, its role in HCC has not been explored in HCC. Our research showed that TMEM147 plays a crucial role in the progression and prognosis of HCC. Further mechanistic exploration revealed that the downstream target is the gene encoding 7-dehydrocholesterol reductase (DHCR7). DHCR7 catalyzes the conversion of 7-dehydrocholesterol to cholesterol, which is the final step of cholesterol synthesis in the Kandutsch-Russell pathway.[9] Previous studies have shown that DHCR7 has vital functions in several malignant tumors, such as cervical [10], bladder ,[11] ovarian ,[12] gastric ,[13] and colorectal cancers[14]; however, the role of DHCR7 in HCC is still unclear and requires more investigations. The present study revealed that DHCR7 promotes the production of cholesterol esters and the downstream metabolite 27-hydroxycholesterol (27HC), which plays an essential part in TMEM147 role in progression of HCC. It has been reported that DHCR7 could be regulated by many transcription factors, such as sterol-regulatory element binding protein 1/2 (SREBP), [15, 16] osteogenic transcription factor (Runx2) [17] and E2F transcription factor 1 (E2F1)[18]. We found that transcription factor 2 (STAT2) was activated by TMEM147, thereby promoting DHCR7 transcription.

Ferroptosis is an intracellular iron-dependent form of cell death that is distinct from apoptosis, necrosis, and autophagy. It has been found to a crucial role in the progression of HCC.[19] Multiple signaling pathways dictate the susceptibility of cells to ferroptosis. For instance, the cyst(e)ine- glutathione (GSH)-recombinant glutathione peroxidase 4 (GPX4) axis is the main ferroptosis-regulating pathway in mammals together with the E-cadherin-NF2-Hippo-YAP pathway, which dictates ferroptosis sensitivity, has important implications. Studies have shown that the chronic exposure of breast cancer cells to 27HC increases their resistance to ferroptosis. [20] However, whether TMEM147, an upstream regulator of 27HC, affects ferroptosis in HCC has not yet been reported.

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME) and play a key role in tumor-associated inflammation. TAMs undergo two forms of activation in response to TME signaling. M1 macrophages are antitumorigenic, whereas M2 macrophages are pro-tumorigenic.[21] It has been shown that various cancer-derived substances can regulate the M2 polarization of TAMs, such as the lipid molecule phosphatidylserine,[22] succinate, [23], lactate[24], exosomal, [25, 26] and tumor-secreted protein S (Pros1).[27] Recent studies reveal that 27HC affects cellular functions in the TME, such as those of T cells, [28] osteoclast, [29], myeloid cells[30]; while it is unclear whether 27HC regulates the polarization of TAMs.

We demonstrated for the first time that TMEM147 increases the phosphorylation of the transcription factor STAT2 and promotes the transcription of the downstream gene DHCR7, consequently contributing to HCC proliferation and metastasis. DHCR7 alters cholesterol homeostasis in HCC by increasing the levels of the downstream metabolite 27HC. 27HC protects HCC cells from ferroptosis via the GPX4 pathway. It can also be discharged into the TME, activating fatty acid oxidation (FAO) metabolic reprogramming in TAM, and then promoting M2 polarization. M2 polarized TAM can promote HCC invasion and metastasis in a positive feedback manner. Our study demonstrates that TMEM147 can serve as a diagnostic biomarker and a crucial therapeutic target for HCC, and targeting its downstream pathways and metabolites may bring aid in developing therapeutic strategies for HCC therapy.

HCC specimens

Only patients with pathological diagnosis of HCC were included in this study. We collected paired HCC and adjacent non-cancerous liver tissue samples from patients who underwent liver resection at the First Affiliated Hospital of Harbin Medical University between January 2012 and August 2017. Senior pathologists were invited to perform pathological diagnoses on the paraffin sections. All patients provided written informed consent, and the study was approved by the Research Ethics Committee of the First Affiliated Hospital of Harbin Medical University.

Cell lines and cell culture

Human HCC cell lines SK-Hep-1, Huh7, HepG2, HCCLM3, and BEL-7402 were obtained from the Chinese Academy of Science (Shanghai, China). The normal liver cell line WRL-68 was obtained from AcceGen (Fairfield, USA). The cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin. Human monocytic leukemia cells, THP-1, were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 100 U/mL penicillin, and 100 µg/mL streptomycin. THP-1 cells were treated with 100 nM phorbol myristate acetate (PMA) (Sigma-Aldrich, USA) for 48 h to induce differentiation into macrophages. All cells were incubated at 37°C in a humidified atmosphere (5% CO₂).

Lentivirus infection

Lentiviral vectors for human TMEM147 gene overexpression (lenti-TMEM147) and downregulation (lenti-TMEM147), and DHCR7 and GPX4 gene downregulation (lenti-sh DHCR7 and lenti-sh GPX4, respectively) were obtained from Hanbio Biotechnology Co. Ltd. (Shanghai). Corresponding empty vectors (lenti-Con and lenti-shcon) were used as negative controls. (Hanbio, Shanghai, China).

The transfection was performed with a multiplicity of infection (MOI) of 10–30 in the presence of polybrene (5 µg/ml). After lentiviral infection, single-cell clones were selected with 3.5 µg/ml puromycin (Sigma-Aldrich) after incubation for 2 weeks. Stably transfected clones were isolated and used for in vitro and in vivo experiments.

Immunohistochemical staining

The tissue sections were deparaffinized, rehydrated, blocked with 10% normal goat serum, and incubated with primary antibodies overnight at 4°C. The slides were then incubated sequentially, first with a secondary antibody (Vector lab, Burlingame, CA) for 1h and then with Vectastain Elite ABC reagent (Vector Lab) for 30 min. The tissue sections were stained with diaminobenzidine (DAB kit; Vector Laboratories) and counterstained with hematoxylin (Sigma-Aldrich). The percentage score was defined as follows:0: 0–5%; 1: 5–25%; 2:, 26–50%; 3: 51–75%; 4: 76–100%; and the staining intensity was defined as 1: weak; 2: moderate; 3: strong and provided a Multiple index (MI) score (MI = intensity × percentage) as follows: MI = 0, scored as 1; MI = 1–4, scored as 2; MI = 5–8, scored as 3; MI = 9 or 12, scored as 4. Samples with a score ≥ 3 were considered to exhibit high expression, whereas those with a score ≤ 2 were classified as exhibiting low expression.

Western Blotting

Protein samples extracted from tissues or cells were separated by gel electrophoresis, and transferred onto nitrocellulose membranes (Invitrogen, Carlsbad, USA). Subsequently, the membrane was blocked with 5% skim milk and incubated with primary antibodies overnight at 4 ℃. Finally, the membrane was incubated with IRDye 800CW secondary antibody (LI-COR, USA) (1:10,000 dilution) at room temperature for 1 h, and the Odyssey® Imaging System (LI-COR, USA) was used to visualize and analyze the proteins. Details of the primary antibodies against the target proteins are listed in Additional file 1: Table S3.

Quantitative Real-time polymerase chain reaction (PCR)

Total RNA was isolated from freshly frozen tissue and logarithmically growing cells using a Total RNA Miniprep Kit (Axygen Scientific, Inc., USA) according to the manufacturer’s instructions and reverse-transcribed into cDNA using a qPCR RT Kit (TOYOBO, Shanghai, China) after RNA quantification. Real-time PCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO, Shanghai, China) on an ABI PRISM 7500HT instrument (Applied Biosystems). The expression level of the target mRNA was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level and was determined according to the 2^−ΔΔCT method. Primers used are listed in Additional file 1: Table S2.

Cell counting Kit-8 (CCK-8) and colony formation assays

1000–1500 stably transfected cells were seeded onto a 96-well plate, and cultured for 4 h for attachment. Subsequently, the culture solution was replaced with a solution containing CCK-8 reagent and the optical density (OD) value was determined. Colony formation was assayed by plating 1000 cells in petri dishes with a diameter of 6 cm and culturing them for 14 days. The medium was then removed, and the colonies were fixed with 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet.

Wound-healing assay

Stably transfected cells were seeded in a 6-well plate at 30 × 10⁴ cells/well and cultured until cell fusion occurred. A 10-µl pipette tip was used to scratch a straight cut at the bottom of the plate. The floating cells were washed away with phosphate buffered saline (PBS) and cultured in serum-free medium, and wound closure was photographed at 0 and 24 h.

Transwell migration and invasion assay

Matrigel-coated (BD Biosciences, Franklin Lakes, NJ, USA) or non-Matrigel-coated Transwell plates were used to examine the invasion and migration abilities of the cells. Cells were inoculated into the upper chamber of the transwell and serum-free medium was added. Normal media was injected into the plate wells. After a 48 h incubation period, we discarded the cells from the upper layer filter, and fixed, stained and counted the cells in the bottom layer.

Immunofluorescence (IF) assay

Cells were seeded on polylysine-coated coverslips, cultured for 24 h, and fixed with 4% paraformaldehyde, followed by 0.1% Triton-X-100 permeabilization. Next, cells were incubated with primary antibodies, secondary antibodies (Invitrogen). and DAPI (Vector Laboratories) in sequence. The images were captured with a camera attached to a microscope.

Co-immunoprecipitation (IP) assay

Cells were harvested and then lysed in 500 µL co-IP buffer containing a protease inhibitor cocktail (Sigma-Aldrich). After centrifugation, cell lysates were collected and precleared by incubating with 20 µL immobilized protein A/G beads for 1h at 4°C. The beads were then discarded using a magnetic frame, and the lysates incubated with primary antibody or control immunoglobulin (Ig)G on a rotator at 4°C overnight. On the following day, 20 µL of immobilized protein A/G beads were added to precipitate the protein complex at 4°C for 4 h. Subsequently, samples were washed five times, the beads boiled in loading buffer, and the proteins were prepared for Western blot as described above.

Mass spectrometry analysis

Similar to the IP assay, cellular protein extracts from HCC cells were incubated with anti-Flag-TMEM147 followed by and protein A/G agarose beads. Recovered proteins associated with Flag-TMEM147 or IgG were resolved by gel electrophoresis. The bands specifically bind to Flag-TMEM147 were excised, and proteomics screening was accomplished by mass spectrometry analysis on a MALDI-TOF-MS instrument (Bruker Daltonics).

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed using a ChIP Kit (Beyotime, Shanghai, China). Briefly, cells were treated with 1% formaldehyde solution for 10 min and quenched in glycine for 5 min at room temperature to generate DNA–protein cross-links. Cell lysates were sonicated to produce chromatin fragments of 200–1000 bp and immunoprecipitated with anti-DHCR7, anti-STAT2, or IgG antibodies. The immunoprecipitated DNAs was analyzed using qRT-PCR. Information on the primers and antibodies used is provided in Additional File 1: Table S2.

Dual‑luciferase reporter assay

The full-length promoter of DHCR7 carrying mutant or wild-type sequences was cloned into pGLO4.10 vectors (Promega, Madison, WI, USA) and co-transfected with a STAT2 overexpression vector or mock vector into Huh7 cells, using Lipofectamine TM 2000 (Invitrogen, CA, USA). After 48 h of culture, firefly and Renilla luciferase activities were measured using a dual-luciferase reporter gene assay system (Beyotime, Shanghai, China) in accordance with the manufacturer’s protocols.

Enzyme-linked immunosorbent assay (ELISA) assay

Cholesteryl Esters’ (CEs) concentrations were measured using a Cholesterol/Cholesterol Ester Quantification Kit (ab65359, Abcam). 27HC concentrations were measured with a Human 27-Hydroxycholesterol ELISA Kit (EH4025, FineTest). Lipid content were measured using a lipid ELISA Kit (SBJ-M0736-96T, GoldenRain/sbj). ELISA was performed according to the manufacturer’s instructions. All experiments were performed in triplicates.

Glutathione (GSH) Assay

A reduced glutathione (GSH) assay kit (A006-2-1, Nanjing Jiancheng Bioengineering Institute) was used to measure intracellular GSH levels. Cells (3 × 10⁵) were seeded into a 6-well plate and treated with erastin or RSL3 for 24 h. The cells were harvested, and 0.3 mL PBS was added to the homogenization medium. Next, a 100 µL supernatant was removed for GSH determination. Simultaneously, a standard GSH concentration curve was also generated. The plate was incubated for 5 min after mixing the sample and reagents, and absorbance was measured at 405 nm. The exact GSH concentration in the different cell lines was then calculated based on the GSH standard curve, following the manufacturer’s instructions.

Mitochondrial iron, malondialdehyde, and membrane potential assays

The mitochondria were isolated using a mitochondrial isolation kit (Thermo Fisher Scientific). Ferrous iron levels in cells or mitochondria were measured using an iron assay kit (Sigma-Aldrich). Lipid peroxidation was assessed in HCC cell lysates by measuring the concentration of malondialdehyde (MDA), an end product of lipid peroxidation, using a lipid peroxidation assay kit (Abcam, Cambridge, MA, USA). Mitochondrial membrane potential (MMP) was measured by staining the cells with 200 Nm tetramethylrhodamine ethyl ester (TMRE, Thermo Fisher Scientific) for 20 min. The mean fluorescence intensity of each group was normalized to that of the control group.

Reactive oxygen species (ROS) assay

Intracellular ROS generation was detected by reactive oxygen species assay kit (Beyotime Biotechnology, Haimen, China). Before the experiment, 1×10⁴ cells per well were seeded in a 96-well black plate and incubated for 24 h. After series of indicated experimental treatments, cells were loaded with 10 µM of fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA) in serum-free DMEM medium for 20 min at 37 ℃. The cells were then washed thrice with serum-free medium. DCF fluorescence intensity was detected using a Tecan Infinite 200 PRO microplate reader to quantify the ROS levels.

Transmission electron microscopy (TEM)

TEM was used for ultrastructural analysis of mitochondria. HCC cells were fixed with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) at 4°C for 2.5 h, washed three times with 0.1 M PBS, and post-fixed in 1% OsO₄ for 2 h at 4°C. The samples were dehydrated using an ethanol gradient and embedded in Spurr’s resin. Ultrathin sections were collected, stained with uranyl acetate or lead citrate, and examined under a JEOL 1200EX transmission electron microscope.

Flow cytometry analysis

PMA-stimulated THP-1 cells that had been co-cultured with HCC cell supernatants for 24 h were stained with PE-conjugated mouse anti-human CD86 (560957, BD Biosciences) or CD206 (555954, BD Biosciences) antibodies according to the manufacturer's instructions. The cells were then fixed and permeabilized using a fixation and permeabilization solution (554722; BD Biosciences). After washing with BD Perm/Wash buffer (554723; BD Biosciences), the cells were stained with FITC-conjugated anti-human CD68 antibody (562117; BD Biosciences). After 30 min, the cells were washed thrice with PBS and resuspended in 1 mL of FACS buffer for flow cytometry analysis.

Oxygen consumption rate (OCR) determination

Cells (2 × 10⁴ cells/well) were seeded in XFe24 seahorse cell culture microplates (Seahorse Bioscience) for 24 h, and the XFe24 sensor cartridges were hydrated overnight. The cells were treated with a cystine-free medium for 8 h, then the cell medium was replaced with basic seahorse DMEM supplemented with glucose (10 mM), sodium pyruvate (1 mM), and glutamine (2 mM). Subsequently, the cell culture microplate was kept in the CO₂-free incubator for 1 h, and OCR was measured in real-time with the sequential injection of oligomycin (1.5 µM), carbonyl cyanide chlorophenylhydrazone (CCCP) (2.0 µM), and Antimycin A/Retenone (0.5 µM) by the Seahorse XFe 24 Bioanalyzer (Seahorse Bioscience).

Animal model

All experimental protocols involving animals were approved by the Animal Ethics Committee of Harbin Medical University, China.

The subcutaneous xenograft model was established by injection of 2 × 10⁶ HCC cells in 200 µL PBS into the flanks of mice. Tumor volume was calculated at the 6th week when all mice were euthanized. Subcutaneous xenograft tumors were cut into 1 mm³ cubes and transplanted into the livers (left lobes) of mice to establish an orthotopic tumor model. Tumor size was assessed weekly with NIGHTOWL LB983 system (Berthold Technologies, Wildbad, Germany).

The pulmonary metastasis model was established as follows: 4 × 10⁶ cells dissolved in 0.15 mL PBS were injected into the tail vein of each mouse. After 6 weeks, the mice were euthanized and their lungs were collected.

All animals were euthanized to collect tumor tissues at the 6th week, and their livers were resected for IHC staining and ELISA.

Statistical analysis

Differences between groups were calculated using Student’s t-test, the chi-square test, or the Fisher exact test. The probability of differences in survival was ascertained by the Kaplan–Meier method with a log-rank test for significance. Analyses were performed with SPSS version 23.0 and Graphpad Prism 7.0 statistical analysis software. Representative data are shown as the mean ± SD. P < 0.05 was considered statistically significant.

High expression level of TMEM147 protein is associated with poor survival in HCC.

To elucidate the relationship between TMEM147 and HCC progression, we first searched the GEO database and mined TCGA transcriptome datasets to compare the levels of TMEM147 between HCC tissues and normal tissues on the GAPIA website and found that TMEM147 mRNA was significantly upregulated in HCC tissues (Fig. 1A and 1B).

We measured TMEM147 expression in 72 HCC tissues and 28 non-tumor liver tissues, and found that the TMEM147 protein exhibited a high staining rate of 83.3% (60/72) in HCC tissues, which was markedly higher than the staining rate of 10.7% (3/28) in normal liver tissues (Fig. 1C), and the protein expression level of TMEM147 in the HCC cohort was significantly correlated with patient survival (Fig. 1I). Detailed clinicopathological characteristics are shown in additional file 1: Table S1. Likewise, qRT-PCR in 24 pairs of HCC and peritumoral tissues revealed that the relative mRNA expression of TMEM147 was significantly upregulated in HCC tissues compared to that in adjacent non-tumorous tissues (Fig. 1D). Accordingly, Western blot analysis was conducted on 24 pairs of HCC tissues, and the results revealed that TMEM147 protein levels were higher in HCC tissues than in non-malignant samples (Fig. 1F and S1A). In agreement with these results, the mRNA and protein expression levels of TMEM147 in HCC cell lines were significantly higher than those in normal liver cells (Fig. 1E and 1G).

Moreover, Kaplan–Meier survival curve analysis of TCGA datasets demonstrated that HCC patients with increased TMEM147 protein expression had poorer overall five-year survival (Fig. 1H).

TMEM147 boosts tumor cell proliferation and metastasis

To investigate the effect of TMEM147 on HCC, we constructed lentivirus vectors to upregulate and silence TMEM147 in Huh7 and HepG2 cells; Western blot and qRT-PCR were applied to confirm the overexpression and knockdown efficiency of TMEM147 (Fig S2A and S2B). According to growth curve analysis and colony formation assay, deletion of TMEM147 significantly decreased HCC cell proliferation, whereas TMEM147 overexpression significantly increased HCC cell proliferation (Fig. 2A and 2B).

Next, we sought to determine the role of TMEM147 in HCC metastasis, migration, and invasion by performing Transwell and wound healing assays in vitro were performed. The results showed that overexpression of TMEM147 promoted the migration and invasion of Huh7 and HepG2 cells (Fig. 2C and S2C), whereas TMEM147 knockdown exerted the opposite effect (Fig. 2D and S2D). Collectively, these results indicate that elevated TMEM147 protein level in HCC boosts tumor cell proliferation and metastasis.

To evaluate the tumorigenic function of TMEM147 in vivo, a subcutaneous xenograft tumor model was established. Consistent with our in vitro data, the tumor volume and weight in mice implanted with Huh7- TMEM147 cells were markedly higher than those in the control group; and those in HepG2-shTMEM147 mice were drastically lower than those in the control group (Fig. 2E). To determine whether TMEM147 affects HCC metastasis in vivo, stably transfected cells (Huh7-TMEM147 and controls) were injected into the tail vein to establish a lung metastasis model. Six weeks after the intervention, the number and volume of lung metastases decreased in the TMEM147 silencing group, but increased in the TMEM147 overexpression group. (Fig. 2F). In summary, these results provided further evidence that TMEM147 is involved in the exacerbation of HCC growth and metastasis.

TMEM147 facilitates 27HC expression by activating DHCR7 to accelerate the growth and metastasis of HCC

To identify the target genes regulated by TMEM147, we knocked down TMEM147 in Huh7 cells. RNA-seq analysis revealed that the transcriptome profiles of TMEM147-silenced cells were distinct from those of the control cells (Fig. 3A). Gene ontology (GO) analysis showed that the differentially expressed genes were significantly enriched in cholesterol metabolism pathways (Fig. 3B). Several key enzymes in this pathway were further assessed, including methylsterol monooxygenase 1 (MSMO1), emopamil binding protein (EBP), Hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), type 2 isopentenyl diphosphate isomerase (IDI2), DHCR7, farnesyl-diphosphate farnesyltransferase (FDFT1), and squalene epoxidase (SQLE); DHCR7 was identified as the most differentially expressed (Fig. 3C and S3A), implying that the expression of DHCR7 may be modulated by TMEM147.

To confirm the role of TMEM147 in the regulation of DHCR7, the expression of DHCR7 in HCC cells was detected after TMEM147 was knocked down or overexpressed. TMEM147 overexpression increased DHCR7 transcription and protein levels, whereas DHCR7 levels were significantly reduced in the absence of TMEM147 (Fig. 3D and 3E).

CEs, the intracellular storage forms of excess cholesterol, are of central importance to cholesterol homeostasis; their formation is a measure of the availability of cellular free cholesterol, and CE levels are consistent with sterol reductase activity.[31] ELISA was employed to quantify the cholesteryl esters in cell lysates and subcutaneous tumors from mice. The results indicate that the total levels of CEs are greatly reduced after TMEM147 knockdown and are significantly upregulated after TMEM147 overexpression (Fig. 3F). A study showed that, in breast cancer, cholesterol mediates the metastatic effects via its oxysterol metabolite, 27-hydroxycholesterol.[28] Therefore, we next detected the content of 27HC in cells and the cell culture medium by ELISA assay. As anticipated, the content of 27HC was consistent with CEs and TMEM147 (Fig. 3G).

CYP27A1 is the enzyme responsible for the rate-limiting step in 27HC biosynthesis, and we found that the protein and mRNA levels of CYP27A1 were closely correlated with TMEM147 levels (Fig. 3H and S3B). To explore the role of DHCR7 in the tumor-promoting function of TMEM147, the effect of DHCR7 knockdown on the tumorigenic potential of TMEM147, RNA lentivirus stably transfected into Huh7 and HepG2 cells was examined (Fig S3C and S3D). Downregulation of DHCR7 expression markedly suppressed the proliferative, migratory, and invasive capabilities of HCC cells, whereas the addition of exogenous 27HC partially restored the inhibitory effect of DHCR7 silencing. The function of 27HC was impeded by the CYP27A1 inhibitor, GW297X (Fig. 3I, S3E, 3 J, and S3F).

A xenograft model showed that DHCR7 knockdown inhibited the growth of subcutaneous tumors, whereas 27HC supplementation reversed the inhibitory effect of DHCR7 downregulation on tumor growth. Additionally, DHCR7 knockdown decreases lung metastasis in a mouse model. However, 27HC supplementation neutralized the inhibition of lung metastasis by DHCR7 knockdown, whereas the injection of the CYP27A1 inhibitor GW297X in cells overexpressing TMEM147 restrained tumor progression and metastasis, and supplementation with 27HC offset the effect of GW297X (Fig S3G and S3H). Collectively, the in vitro and in vivo results suggest that DHCR7 is involved in the oncogenic effect of TMEM147 in HCC and that the function of DHCR7 depends on increased 27HC levels.

TMEM147-mediated promotion of DHCR7 expression is dependent on the transcription factor STAT2

In Huh7 and HepG2 cell lines, protein binding to TMEM147 was analyzed by mass spectrometry using CO-IP. It was found that the target gene STAT2, which encodes a transcription factor was closely bound to TMEM147 in both cell lines (Fig. 4A). The interaction between TMEM147 and STAT2 was further confirmed by CO-IP. IP assays confirmed that STAT2 existed in complexes and was precipitated with an antibody against Flag-TMEM147, unlike with the control IgG (Fig. 4B, upper). The binding of endogenous STAT2 to TMEM147 was validated by an IP assay using an antibody against STAT2 (Fig. 4B, lower). Using JASPAR and UCSC database analyses, we found that the DHCR7 promoter contained putative binding sites for the transcription factor STAT2 (Fig. 4C). We speculated that TMEM147 might regulate DHCR7 expression through the transcription factor STAT2.

As shown in Fig. 3, TMEM147 altered the mRNA and protein expression of DHCR7. Western blotting revealed that STAT2 protein expression levels did not significantly correlate with those of TMEM147 in HCC cells. However, transcription 2 (STAT2) at tyrosine residue 690 (Tyr690) and phosphorylated-STAT2(Tyr690) changed upon TMEM147 treatment (Fig. 4D). Consistent with this observation, a chromatin immunoprecipitation assay (ChIP) showed that the STAT2-bound DHCR7 promoter was enriched in TMEM147 overexpressed cells but diminished in TMEM147-knocked down cells (Fig. 4E). The luciferase reporter assay showed that STAT2 overexpression significantly increased the luciferase activity of reporters containing wild-type binding sites compared to that of NC-vector cells (Fig. 4F). However, no significant change in luciferase activity was observed upon binding of STAT2 to the mutant DHCR7 promoter (Fig. 4F). To determine whether STAT2 influenced DHCR7 expression, we transfected STAT2 into HCC cells. We found that STAT2 significantly promoted the expression levels of DHCR7 in HCC cells; in contrast, elimination of STAT2 significantly decreased DHCR7 expression (Fig. 4G). When TMEM147 was overexpressed, si-STAT2 inhibited DHCR7 expression, indicating that TMEM147’s promoting function of DHCR7 depended on STAT2 (Fig. 4H). A moderate correlation between TMEM147 and STAT2, and DHCR7 and STAT2 was detected in human HCC (Fig. 4I), suggesting that TMEM147 promotes the phosphorylation of STAT2, enhances its transcriptional activity, and then increases the expression of DHCR7. To assess the effect of STAT2 on HCC, we transfected si-STAT2 into HCC cells and found si-STAT2 significantly suppressed HCC cell proliferation, invasion, and migration (Fig. 4J and 4K).

TMEM147 induces ferroptosis by promoting 27HC/GPX4 pathway in HCC.

Previous studies have shown that chronic exposure to 27HC increases lipid accumulation in cells and enhances the resistance of breast cancer cells to ferroptosis, whereas GPX4 inhibition reverses this effect.[20] To explore the effects of TMEM147 and 27HC on ferroptosis, we measured the levels of GSH, which plays an essential role in the repair of oxidative damage. As shown in Fig. 5A, erastin and RSL3 significantly decreased GSH levels in HCC cells, and TMEM147 overexpression rescued the decreased GSH levels caused by erastin and RSL3. When GW297X was added, the GSH value decreased significantly. Correspondingly, ferrous ion (Fe²⁺) and MDA levels were reduced when TMEM147 was overexpressed but increased in cells with TMEM147 overexpression subjected to GW297X treatment (Fig S5A and S5B). MMP increased over time, with a maximum at approximately 8 h after erastin treatment (Fig. 5B); however, MMP was overwhelmingly suppressed by TMEM147, and completely rescued by GW297X. Correspondingly, as shown in Fig. 5C, TMEM147 reduced ROS production via 27HC, whereas ROS generation was significantly enhanced in GW297X treated cells.

TEM analysis further revealed that erastin and RSL3 treated HCC cells contained shrunken mitochondria with elevated membrane density, a typical morphological feature of ferroptosis. TMEM147 overexpression protected cells from ferroptosis, whereas treatment with GW297X aggravated ferroptosis. This indicates that TMEM147 confers ferroptosis resistance by promoting 27HC. (Fig. 5D)

In parallel with our observation in vitro, in comparison with erastin group, tumor size and weight were significantly increased by TMEM147 overexpression, whereas GW297X pretreatment markedly reversed this TMEM147-mediated oncogenic effect (Fig. 5E). Tissues from the control and GW297X injected tumors, exhibited increased levels of 4-hydroxy-2-noneal (4HNE, a biomarker of lipid peroxidation), whereas TMEM147 overexpression reduced the levels of 4HNE, as determined by IHC (Fig. 5F). These in vivo results further support the idea that the resistance to ferroptosis induced by TMEM147 is mainly dependent on 27HC.

To further elucidate the mechanism of ferroptosis triggered by TMEM147, the iron metabolism-related proteins GPX4, HMOX1, SLC7A11, NCOA4, NRF2, and CP were analyzed. GPX4 was markedly altered by TMEM147 and GW297X (Fig. 5G and S5C). The expression of GPX4 in HCC cells was assessed by qRT-PCR and western blot analysis, and it was found that GPX4 expression was downregulated upon the knockdown of TMEM147. Indeed, we demonstrated that the TMEM147-dependent GPX4 downregulation was dramatically rescued by the addition of exogenous 27HC. (Fig. 5H and 5I). In summary, these results suggested that TMEM147 promotes GPX4 expression via 27HC to reduce the resistance of HCC cells to ferroptosis.

HCC cell-derived 27HC enhances lipid metabolism in macrophages and drives M2 polarization

Previous studies have shown that pretreatment of macrophages with 27HC can inhibit T cell function .[30] However, it remains unclear whether tumor-associated macrophages, which are important immune cells in the tumor microenvironment, are also regulated by 27HC. To further substantiate this hypothesis, the following experiments were conducted.

THP-1 induced macrophages were incubated with CM gathered from HCC cells (Fig. 6A), and the expression of TAM markers arginase 1 (ARG1) and CD206 was further analyzed. CM from TMEM147 overexpression cells or the addition of exogenous 27HC, but not the control medium, increased the expression of the M2 markers ARG1 and CD206, whereas GW297X deterred this conversion (Fig. 6B and S6A). These results suggest that endogenous 27HC mediates M2 macrophage polarization. The results of cell immunofluorescence and flow cytometry further confirmed that tumor-derived 27HC was responsible for cancer CM-induced M2 macrophage polarization (Fig. 6C and 6D).

In animal studies, IHC staining for the M2 marker ARG1 showed that the accumulation of M2 macrophages was greater in MΦs cultured with the CM obtained from TMEM147 overexpression cells or with exogenous 27HC (Fig. 6E).

Pan Su et.al. reported that the reprogramming of fatty acid oxidative metabolism plays a crucial role in M2 polarization of TMA. Macrophages from both human and murine tumor tissues are enriched with lipids owing to increased lipid uptake by macrophages.[32] In our study, as expected, compared with control MΦs, MΦs cultured with CM collected from TMEM147 overexpression cells or added exogenous 27HC were enriched with more lipids (Fig. 6F), genes involved in fatty acid β-oxidation including FAO rate-limiting enzymes CPT1A and PPARγ, were significantly increased (Fig. 6B and S6A). In metabolic assay experiments, TAMs co-cultured with TMEM147 overexpression cells and exogenous 27HC supplementation displayed enhanced mitochondrial OCR and markedly increased spare respiratory capacity (SRC), in contrast to TMEM147 knockdown cells co-cultured with GW297X-treated macrophages (Fig. 6G and 6H). Thus, we validated that 27HC promotes the accumulation of lipids and fatty acid-oxidation in TAMs, which are crucial for M2 macrophage polarization.

In summary, we highlighted the importance of HCC cell-derived 27HC in enhancing the lipid metabolism of macrophages and promoting M2 polarization.

27HC- induced polarized macrophages potentiate migration of HCC cells

As cancer cell-derived 27HC can promote the M2 polarization of TAMs, we hypothesized that TAMs with increased M2 polarization could further promote the migration of HCC cells. CM-induced polarized macrophages were co-cultured with HCC cells (Fig. 7A), and the invasion and migration of HCC cells in each group were consistent with M2 polarization of macrophages (Fig. 7B and 7C). In vivo, we injected co-cultured cells through the tail vein of nude mice and monitored lung metastatic nodules (Fig. 7D), which showed that polarized macrophages induced by 27HC could significantly exacerbate the metastasis of HCC cells.

HCC is one of the most common life-threatening neoplasias, with a high incidence and mortality worldwide. In the present study, we identified TMEM147 as a potent oncogene responsible for HCC proliferation and metastasis. TMEM147 can enhance the transcription of DHCR7 (the key enzyme for cholesterol synthesis) through the transcription factor STAT2 and alter cholesterol homeostasis in HCC. Consequently, 27HC, a primary metabolite of cholesterol, increases both intracellularly and extracellularly. 27HC, as a key hub, confers ferroptosis resistance to tumor cells and is expelled out of the cell to promote the polarization of M2 macrophages, which forms a positive feedback loop and promotes the progression of HCC.

In this study, we selected TMEM147 as a promising research target through GEO and TCGA data analysis of patients with HCC. Accordingly, our data showed that high TMEM147 expression was linked to worse OS and DFS in patients with HCC. We confirmed that the knockdown of TMEM147 expression arrests HCC cell proliferation and reduces their migration and invasion abilities, both in vitro and in vivo. Conversely, we found that DHCR7 expression was regulated by TMEM147. A previous study has reported a correlation between TMEM147 and DHCR7 expression in HeLa cells; however, the underlying mechanism remains unclear. Our results were similar to those in HeLa cells, and we explored the potential underlying mechanisms. DHCR7 has crucial functions in several malignant tumors such as gastric cancer[13] and colorectal cancer.[14] In this study, we discovered that the positive effect of TMEM147 on the proliferation, invasion, and migration of HCC cells mainly relies on DHCR7, which may be related to cholesterol ester metabolism modulated by DHCR7.

Ota et al. found that TMEM147 plays a role in NF-κB activation in human primary chondrocytes. There are multiple phosphorylation sites on NF-κB molecules. For instance, phosphorylation of p65 S276 is important for DNA binding, and TMEM147 can phosphorylate p65 S529.[7] At the molecular level, our investigation revealed that TMEM147 increased the phosphorylation of the transcription factor STAT2 and promoted the transcriptional activity of STAT2, which binds to the transcription promoter regions of DHCR7 and further accelerates the transcription of DHCR7. DHCR7 promotes accumulation of cholesteryl esters in cells and increases 27HC levels in the tumor microenvironment.

Our research validated that 27HC is an important lipid metabolite regulated by TMEME147. 27HC plays a contradictory role in the progression of various cancers; for example, 27HC can increase ER-dependent growth and LXR-dependent metastasis in mouse models of breast cancer,[30, 33, 34] 27HC promotes lung cancer cell proliferation via ERβ and PI3K-Akt signaling, [35] whereas in colorectal cancer, increased production of 27HC inhibits cancer cell survival and infiltration. [36] Therefore, it is unclear whether 27HC promotes or suppresses malignant tumors. This study revealed that 27HC promotes HCC growth. Ferroptosis is a newly discovered mode of cell death, and multiple signaling pathways dictate the susceptibility of cells to ferroptosis. Unrestrained lipid peroxidation is the hallmark of ferroptosis. The cyst(e)ine-GSH-GPX4 axis is considered the main ferroptosis-regulating system in mammals.[37] FSP1 is another prominent ferroptosis suppressor.[38] The role of the E-cadherin-NF2-Hippo-YAP pathway in ferroptosis sensitivity has important implications.[39] Hypoxia can drive sensitivity to ferroptosis; hence,–HIF2α-HILPDA drives sensitization to ferroptosis.[40] However, upstream regulatory pathways involved in ferroptosis remain unclear. Additionally, a study reported that 27HC affects the pathogenesis of breast cancer by selecting cells that are resistant to ferroptosis.[20] A previous study has reported that ZMYND8 is a master regulator of 27HC, which promotes the tumorigenicity of breast cancer stem cells. Our study demonstrated that TMEM147 regulates 27HC production mainly by activating DHCR7. Furthermore, TMEM147 confers resistance to ferroptosis which is is primarily dependent on 27HC. To further address the specific mechanism by which 27HC inhibits ferroptosis, we detected key proteins involved in several ferroptotic pathways when TMEM147 was altered. Ultimately, GPX4 was identified as a downstream target of 27HC, which is consistent with previous studies on other tumors.

Recent studies have revealed that 27HC affects cellular functions in the tumor microenvironment. 27HC increases γδT cells and polymorphonuclear neutrophils but decreases CD8 + T cells at distal metastatic sites in breast cancer.[28] 27HC also facilitates osteoclast differentiation by activating the STAT3 signaling pathway, thus providing an appropriate bone microenvironment for the colonization of lung cancer cells.[29] Additionally, evidence suggests that 27HC is critical for promoting the pro-tumorigenic properties of myeloid cells and the abundance and plasticity of macrophages within the tumor microenvironment.[30] Further research revealed that 27HC could be released into the TME and participate in the FAO metabolic reprogramming of TAMs. To date, no studies have explored whether 27HC affects TAM polarization. Macrophages are the most abundant mesenchymal cells in HCC, and macrophage polarization plays a significant regulatory role in HCC progression .[41, 42] We speculated that the release of 27HC induced by TMEM147 may further orchestrate the polarization of TAMs; therefore, we altered the expression of TMEM147 and 27HC, and cultured macrophages with CM from the treated HCC cells to simulate the TME in vivo. Finally, consistent with our hypothesis, HCC cell-derived 27HC significantly increased the M2 polarization, which further confirmed the potential mechanism of TMEM147 in HCC cells that TMEM147 influenced TAM M2 polarization by regulating the release of 27HC. Recent research has reported that fatty acid oxidative metabolic reprogramming plays a crucial role in M2 polarization of TAMs. [32] Therefore, we detected key proteins in the FAO pathway in macrophages and found that the M2 polarization induced by 27HC was indeed caused by FAO metabolic reprogramming. We further cocultured macrophages with HCC cells and confirmed that M2 polarized macrophage aggravated HCC invasion and metastasis in a positive feedback manner.

Our results confirm that TMEM147-mediated metabolic reprogramming and the inflammatory microenvironment play vital roles in HCC progression. Future studies should further screen or develop novel compounds that interrupt the TMEM147/STAT2/DHCR7/27HC pathway, which may be a new strategy for therapy and HCC immunotherapy.

We demonstrated, for the first time, that TMEM147 is a critical regulator of HCC cell proliferation and metastasis. TMEM147 binds to the transcription factor STAT2 and increases its phosphorylation; phosphorylated STAT2 directly bind to the predicted promoter region of the DHCR7 gene, further promoting the transcription of DHCR7. This alters cholesterol homeostasis in HCC, increasing 27HC levels. 27HC leads to increased GPX4 and protects cells from ferroptosis in HCC cells, furthermore, it can also be discharged into the TME, activating PPAR/CPTA1 pathway, causing lipid accumulation in macrophages, resulting in TAM metabolic reprogramming, and then promoting M2 polarization. Collectively, these results provide a foundation for understanding the mechanisms underlying HCC progression, and may contribute to the identification of new biomarkers and therapeutic targets for HCC.

TMEM147: Transmembrane protein 147; HCC: Hepatocellular carcinoma; TCGA: The Cancer Genome Atlas; TAM: Tumor-associated macrophages qRT-PCR: Quantitative real-time polymerase chain reaction; Elisa: Enzyme-linked immunosorbent assay; 27HC:27-hydroxycholesterol; DHCR7:7-dehydrocholesterol reductase; STAT2: transcription 2; GPX4: glutathione peroxidase 4; FAO: fatty acid oxidation; PMA: phorbol myristate acetate; IHC: Immunohistochemistry; CCK-8: Cell counting Kit-8; GO: Gene ontology; IP: Immunoprecipitation; ChIP: Chromatin immunoprecipitation; CEs: Cholesteryl Esters; GSH: Glutathione; ROS: Reactive oxygen species; OCR: Oxygen consumption rate; TME: tumor microenvironment.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the First affiliated Hospital of Harbin Medical University, and was performed according to the Declaration of Helsinki. All patients recruited into the study signed written informed consent forms.

Consent for publication

All authors read and approved the final manuscript.

Availability of data and materials

All datasets on which the conclusions of the paper rely are available to readers.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by Chen Xiao-Ping Foundation For The Development Of Science And Technology Of Hubei Province (grant CXPJJH12-2020023)，the Excellent Young Medical Talents Training Fund of the First Affiliated Hospital of Harbin Medical University (grant 2021Y06), Heilongjiang Postdoctoral Scientific Research Developmental Fund (grant LBH-Q18089), The University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (grant UNPYSCT-2018070), Chen Xiao-Ping Foundation For The Development Of Science And Technology Of Hubei Province (grant CXPJJH12-2021029).

Author contributions

JH, HP, and JS performed the experiments, analyzed the data, finalized the figures, and drafted the manuscript. JW, QX, SK, SL and ZL performed the experiments. YH, ZF, BY, and BQ assisted with the data analysis and figure design. HP, HJ and WD designed and supervised the study. All the authors contributed to and approved the final version of the manuscript.

Acknowledgements

Not applicable

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No competing interests reported.

Additionalfile1.docx
FigureS1.tif
FigureS2.tif
Fig. S2 (A) qRT-PCR analysis of TMEM147 expression after lentiviral transfection. (B) Western blot analysis of TMEM147 expression after lentiviral transfection. (C) Wound healing assays show that motility is enhanced in Huh7-TMEM147 cells, whereas TMEM147 knockdown suppresses motility in Huh7 and HepG2 cells.
FigureS3.tif
Fig. S3 (A) The mRNA level of key enzymes in cholesterol metabolism pathways in TMEM147-silenced and control Huh7 cells. (B) CYP27A1 mRNA level of TMEM147 overexpression and silenced and control cells. (C) qRT-PCR analysis of DHCR7 expression after siRNA transfection. (D) DHCR7 protein level of the indicated conditions. (E) Representative images of colony formation and statistical analysis of colony numbers. (F) Wound healing assay for the indicated cells. Scale bars, 100 μm. (G) Representative images of subcutaneous xenograft derived from indicated HCC cells. (H) Representative images of lung metastasis specimens of lung metastasis derived from tail injection with indicated cells.
FigureS4.tif
FigureS5.tif
Fig. S5 (A) Ferrous ion (Fe2+) levels were assayed in in the indicated erastin or RSL3 treated cells. (B) Mitochondrial malondialdehyde (MDA) levels were assayed in in the indicated erastin or RSL3 treated cells. (C) The iron metabolism-related mRNA level in the indicated cells.
FigureS6.tif
Fig. S6 (A) The mRNA level of M2 macrophage marker arginase 1 (ARG1) and CD206, FAO rate-limiting enzyme CPT1A and PPARγ in the indicated macrophages.

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TMEM147 aggravates the progression of HCC by modulating cholesterol homeostasis, suppressing ferroptosis, and promoting M2 polarization of TAMs

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Abstract

Figures

Background

Methods

HCC specimens

Cell lines and cell culture

Lentivirus infection

Immunohistochemical staining

Western Blotting

Quantitative Real-time polymerase chain reaction (PCR)

Cell counting Kit-8 (CCK-8) and colony formation assays

Wound-healing assay

Transwell migration and invasion assay

Immunofluorescence (IF) assay

Co-immunoprecipitation (IP) assay

Mass spectrometry analysis

Chromatin immunoprecipitation (ChIP)

Dual‑luciferase reporter assay

Enzyme-linked immunosorbent assay (ELISA) assay

Glutathione (GSH) Assay

Mitochondrial iron, malondialdehyde, and membrane potential assays

Reactive oxygen species (ROS) assay

Transmission electron microscopy (TEM)

Flow cytometry analysis

Oxygen consumption rate (OCR) determination

Animal model

Statistical analysis

Results

TMEM147 boosts tumor cell proliferation and metastasis

TMEM147 facilitates 27HC expression by activating DHCR7 to accelerate the growth and metastasis of HCC

TMEM147-mediated promotion of DHCR7 expression is dependent on the transcription factor STAT2

HCC cell-derived 27HC enhances lipid metabolism in macrophages and drives M2 polarization

27HC- induced polarized macrophages potentiate migration of HCC cells

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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