Cholesterol neutralized vemurafenib treatment by promoting melanoma stem-like cells via its metabolite 27-hydroxycholesterol

Vemurafenib has been used as first-line therapy for unresectable or metastatic melanoma with BRAFV600E mutation. However, overall survival is still limited due to treatment resistance after about one year. Therefore, identifying new therapeutic targets for melanoma is crucial for improving clinical outcomes. In the present study, we found that lowering intracellular cholesterol by knocking down DHCR24, the limiting synthetase, impaired tumor cell proliferation and migration and abrogated the ability to xenotransplant tumors. More importantly, administration of DHCR24 or cholesterol mediated resistance to vemurafenib and promoted the growth of melanoma spheroids. Mechanistically, we identified that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol synthesized by the enzyme cytochrome P450 27A1 (CYP27A1), reproduces the phenotypes induced by DHCR24 or cholesterol administration and activates Rap1-PI3K/AKT signaling. Accordingly, CYP27A1 is highly expressed in melanoma patients and upregulated by DHCR24 induction. Dafadine-A, a CYP27A1 inhibitor, attenuates cholesterol-induced growth of melanoma spheroids and abrogates the resistance property of vemurafenib-resistant melanoma cells. Finally, we confirmed that the effects of cholesterol on melanoma resistance require its metabolite 27HC through CYP27A1 catalysis, and that 27HC further upregulates Rap1A/Rap1B expression and increases AKT phosphorylation. Thus, our results suggest that targeting 27HC may be a useful strategy to overcome treatment resistance in metastatic melanoma. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05267-3.


Introduction
Malignant melanoma, which develops from the pigment cells of the skin mucosa, is the most serious skin cancer with high mortality.The V600E mutation of the B-Raf protooncogene, a serine/threonine kinase (BRAF), is the most common driver mutation in melanoma patients.It leads to constitutive activation of mitogen-activated protein kinase (MAPK), which promotes cell proliferation [1].Although a number of important scientific milestones have been achieved in the fight against melanoma, the FDA approval of BRAF V600E and MEK inhibitors for example [2][3][4][5], the 5-year survival rate of metastatic individuals remains poor [6].The surviving cancer stem cells (CSCs) contributed drug resistance is known to be the main issue for patients' relapse [7,8].There is increasing evidence that cancer cells alter their cholesterol metabolism during tumorigenesis and treatment and that cholesterol and its metabolites contribute to CSC population by providing energy and proliferation Xiaohong Wang, Feiliang Zhong and Tingting Chen have contributed equally.signals [9][10][11].Therefore, the identification of key mediators involved in metabolic dysregulation in specific contexts may provide novel therapeutic targets and highlight the importance of metabolic reprogramming in tumor biology.
In addition to chronic exposure to ultraviolet (UV) radiation from sunlight, obesity and type 2 diabetes have been shown to increase the risk of melanoma [12].Gross and colleagues recently highlighted the link between UV exposure and metabolic disorders in melanoma biology by reporting a novel pathway in which UV radiation acts on cholesterol biosynthesis to control Ca2 + influx through Orai1, resulting in protein O-GlcNAcylation that promotes conversion to invasive melanoma [13].DHCR24, 3β-Hydroxysterol Δ24-reductase, catalyzes the final step of cholesterol biosynthesis.Dysregulation of DHCR24 is associated with numerous diseases.Dhcr24 knockout mice died within hours of birth from a series of skin developmental defects related to desmosterol accumulation in the epidermis [14], and mutations in the DHCR24 gene lead to desmosterolosis, an autosomal recessive disease characterized by developmental and growth retardation [15].As a key regulator of cellular cholesterol synthesis, DHCR24 was found to be downregulated in Alzheimer's disease (AD) vulnerable regions and involved in AD-related pathological activities [16], and DHCR24 reverses AD-related pathology and cognitive impairment by increasing cholesterol levels in the hippocampus of 5xFAD mice [17].In contrast, reduced expression of DHCR24 is associated with increased apoptosis in adrenocortical cells due to inhibition of caspase-3 [18].More importantly, DHCR24 promotes the growth of CSC-like populations by activating the Hedgehog signaling pathway in breast cancer and protects melanoma cells from apoptosis [19][20][21], suggesting a possible role of DHCR24 or cholesterol in cancer metastasis and recurrence.However, DHCR24 is lower expressed in advanced tumors than in early tumors and normal tissues, and force expressed DHCR24 inhibits cell growth in prostate cancer [22,23], suggesting that that cholesterol or its metabolites play a promoting or inhibitory role in tumorigenesis, depending on the type and stage of cancer [24,25].
T h e r e a r e e p i d e m i o l o g i c l i n k s b e t we e n 27-hydroxycholesterol (27HC) and the progression of melanoma in obese or hypercholesterolemic individuals [26,27].27HC is a primary metabolite of cholesterol synthesized by the enzyme cytochrome P450 27A1 (CYP27A1), and its role in breast and prostate cancer (PCa) has been extensively characterized [28,29].Nelson et al. found that the cholesterol metabolite 27HC promotes tumor growth and metastasis in mouse models of breast cancer by serving as a partial agonist for the estrogen receptor and liver X receptor [27].In its role as a selective estrogen receptor modulator (SERM) [30,31], 27HC has been shown to increase cell proliferation and modulate resistance to docetaxel-induced cytotoxicity in non-tumorigenic RWPE-1 prostate epithelial cells [32] and in LNCaP and PC3 cancer cells, 27HC increases cell proliferation by upregulating ERβ expression [32].Although increasing CYP27A1, the rate-limiting enzyme in the formation of 27HC, led to a corresponding increase in the 27HC content of LNCaP and 22RV1 PCa cells and conditioned medium, the increase in 27HC content directly attenuated the proliferation of LNCaP and 22RV1-derived xenografts, by decreasing intracellular cholesterol as a consequence of attenuating SREBP2 activity [33], while the expression of CYP7B1, the rate-limiting enzyme in the degradation of 27HC, increases with the development and progression of PCa [34].The differential effects of ERβ isoforms regulated by 27HC on EMT and metastasis in PCa may explain the equivocal role of 27HC.Similarly, it has been reported that hepatocyte-derived 27HC could stimulate ER in melanoma cells and control proliferation and differentiation via AKT and mitogen-activated protein kinase (MAPK) signaling pathways [21].Indeed, there is an autoregulatory feedback loop to tightly regulate cholesterol homeostats.Physiologically, 27HC binds to insulin-induced proteins (INSIGs) to inhibit de novo cholesterol synthesis via SREBPs, and LXRs promote cholesterol efflux via ABCA1 and ABCG1, while decreasing uptake by downregulating LDL in the presence of excess cholesterol [35].However, whether the feedback loop works in cancers that are drug resistant is not known.
In the present study, based on IHC results from clinical tissue microarray, DHCR24 was found to be highly expressed in melanoma patients, and knocking down DHCR24 blocked the cells in S phase and inhibited the proliferation and migration of melanoma cells.Next, we found that DHCR24 induced vemurafenib resistance in BRAF-mutated cells by promoting melanoma spheroid propagation.Targeted metabolomics analyses on DHCR24-inducing spheroids identified that 27HC was significantly upregulated upon DHCR24 expression, consistent with the fact that only CYP27A1 was highly expressed among cholesterol-degrading enzymes in the TCGA SKCM cohort.More importantly, in a model of drug resistance, we demonstrated that withdrawal of cholesterol or inhibition of CYP27A1 by dafadine-A abrogated the property of vemurafenib resistance (VR) cells and decreased Rap1A/Rap1B expression and AKT thr308/309 phosphorylation.Therefore, we concluded that our results have revealed a novel mechanism underlying cholesterolmediated vemurafenib resistance in melanoma and proved that the cholesterol-27HC-Rap1 axis is a potential target for the treatment of vemurafenib-resistant melanoma.

Cell lines and cell culture
A375 and 293T cells were obtained from Cell Resource Center of Peking Union Medical College (IBMS, CAMS/ PUMC) and C1861 and VMM39 cells were purchased from Shanghai Fuyu Biotechnology.The A2058 melanoma cell was a kindly gift of Dr. Fang from the Beijing Institute of Genomics.Cells were grown in high-glucose DMEM medium (Gibco, 11995500BT) supplemented by 100 U/ ml penicillin and 100 μg/ml streptomycin (TransGen Biotech), and 10% fetal bovine serum (FBS, Gibco, 10091-148) at 37 °C with 5% CO 2 .

Immunohistochemistry on tissue microarrays (TMAs)
Skin cancer tissue Microarray (K063Me01) was purchased from Xi'an bioaitech Co., Ltd.(Xi'an, China).Protein expression was detected using IHC and analyzed according to standard method and microarray instruction.The TMAs were examined and scored independently by two pathologists.Tumor stages of the specimens on the tissue microarray were categorized according to the tumor-nodemetastasis (TNM) system by American Joint Committee on cancer (AJCC) [36].

Proliferation assay
Cell proliferation was quantified by Cell-counting kit-8 (CCK-8; TransGen, Beijing, China) following the manufacturer's instructions.6 μl CCK-8 kit solution was added to the medium after a total of 3 × 10 3 cells seeded in each well of 96-well plates.The absorbance value (OD) was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).Each individual measurement was repeated three times.

Cell cycle analysis
Flow cytometric analysis was performed to determine the effect of DHCR24 on cell cycle distribution.Briefly, cells grown in 6-well plates were treated with shRNA for 48 h.Then, cells were harvested and fixed in 75% ethanol solution.After centrifugation, cells were washed twice with cold PBS, then incubated with RNase A and stained with propidium iodide for 30 min in the dark.Cell cycle distribution was analyzed by flow cytometry (NovoCyte 2040R; ACEA Bioscience, Inc.; Agilent Technologies).

Lentivirus packaging and infection
The lentivirus was packaged with the helper plasmids (PSPAX2, PMD2.G) by co-transfection into 293 T cells.The supernatant of the 293 T cells that packages lentivirus was harvested.The A375 and A2058 cells were infected with the lentivirus in the presence of 8 μg/ml polybrene.The cells were incubated at 37 °C in a CO 2 incubator for 48 h, then selected with 1 μg/ml puromycin for 2 weeks.

Transwell assay
The matrigel (ABW® Matrigengel, 082704) was diluted with medium (1:20), and spread evenly on the chamber for 1 h at 37 ℃.Then the cells suspended in serum-free medium were added to the upper chamber, and medium containing 10% FBS was filled in the lower chamber.After incubation for 24 h, the upper chamber was fixed in 70% methanol for 30 min.The images of chamber were captured and quantified after staining with 2.5% crystal violet.After removal of the cells on the upper membrane, the cells remained on the bottom membrane were identified as invasive cells.

Subcutaneous tumor formation
Six-week-old NOD-SCID female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China) and fed under SPF conditions in Institute of Radiation Medicine, Chinese Academy of Medical Science & Peking Union Medical College (Tianjin, China).NOD SCID female mice were injected subcutaneously with 1 × 10 7 cells (0.1 ml PBS), then mice weighed twice a week.The volume of tumor was measured after three weeks, and tumor tissues were fixed in 4% paraformaldehyde for hematoxylin-eosin (HE) staining and Immunohistochemistry (IHC).Tumor volume size was calculated by the equation (length × width2/2).

Real-time quantitative PCR analyses
Total RNA was prepared from cells using EasyPure® RNA Kit (TransGen Biotech) and reversely transcribed using TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech) according to the manufacturer's protocol.Real-Time quantitative PCR was performed using SYBR SuperMix (TransGen Biotech) and specific primers are shown in Table 1.All the samples were normalized to the housekeeping gene, GAPDH.Data were calculated using the 2 − ΔΔCt method.

Rhodamine transport assay
The cell lines were incubated with 15 μM Rhodamine 123 (MedChemExpress, HY-D0816) at 37 °C for 5 min and washed three times with the culture medium.Afterwards, cells were incubated in the culture medium with varying lapatinib concentrations at 37 °C for 30 min.The cell lines were then analyzed by flow cytometry.(BD Biosciences, San Jose, CA, USA).

Flow cytometry analysis
For cell cycle assay, the cells were fixed with 70% ethanol at 4 °C overnight.The cells were added RNase A (100 μg/ ml) for 5 min at room temperature, then stained with PI (50 μg/ml, US EVERBRIGHT, Y6002) for 30 min at room temperature.All samples were detected using a flow cytometer (NovoCyte 2040R; Agilent Technologies) and analyzed using the NovoExpress 1.4.1 software (Agilent Technologies).

27HC and cholesterol determination
Human 27HC ELISA Kit was obtained from Wuhan Fine Biotech Co., Ltd (Wuhan, China).Cells (2 × 10 6 ) were collected and detected for 27HC content by Human 27HC ELISA Kit following manufacturer's protocol.The OD value was measured by a Microplate Reader (Thermo) at 450 nm.The concentration of cellular 27HC was obtained according to standards.
Cholesterol was measured with the Amplex Red cholesterol assay kit (Thermo Fisher Scientific, A12216) according to the manufacturer's instruction.Measured the fluorescence in a fluorescence microplate reader using excitation at 530 nm and emission detection at 590 nm.The concentration of cellular cholesterol was obtained according to standards.

Membrane fluidity assay
The membrane fluidity of cells was measured using the lipophilic pyrene probe, a lipid analogue probe that underwent excimer formation upon spatial interaction with the cellular membrane (Membrane Fluidity Kit, Abcam, #ab189819).Cells were treated for 48 h, followed by 1 h incubation with 2 µM PDA and 0.08% pluronic F-127.PDA fluorescence was monitored by exciting at 350 nM and taking emission values at 400 nM (monomer) and 470 nM (excimer).Relative membrane fluidity is a ratio of excimer to monomer fluorescence (Ie/Im).

Determination of cholesterol metabolites
1 × 10 7 cells were collected and added 400 μl methanol: water: chloroform (5:2:2, v/v/v) mixed solution, then precool at −20 °C for 20 min.The samples were homogenized in a micro-homogenizer for 3 min, and extracted ultrasonically for 2 times (10 min each time) in an ice-water bath.The supernatant was collected at 13,000 rpm (4 ℃) for 10 min and concentrated with a centrifugal concentration dryer.Dried lipids were dissolved with 200 μl methanol: water Liquid chromatography was performed using an UPLC system (Nexera UHPLC LC-30A).The chromatographic column was the Waters UPLC HSS C18 Column (2.1 mm × 100 mm, 1.7 µm).The mobile phase A was water containing 0.1% formic acid, and the mobile phase B was acetonitrile.The temperature of column was 45 °C, and the flowing rate of mobile phase was 0.4 ml/min.Gradient conditions were as follows: 0-0.5 min, 40 B; 0.5-1 min, 40%-55%B; 1-8 min, 50%-90% B; 8-9.2 min, 90%-40% B; 9.2-10 min, 40% B. Mass spectrometry analyzed using an AB Sciex Qtrap 5500 system containing an electrospray ionization (ESI) source.The capillary voltage was set to 5500 V for positive mode and 4500 V for negative mode.The mass spectra were acquired in a mass range of m/z 100-1000, and collision-activated dissociation (CAD) was medium.Other parameters were as follow: ion source gas 1, 60 psi; ion source gas 2, 50 psi; curtain gas (CUR), 35 psi; and turbo ion spray source temperature, 500 °C.
All standards were purchased from Sigma and prepared at the concentrations of 0.05 ng/ml, 0.13 ng/ml, 0.33 ng/ ml, 0.82 ng/ml, 2.05 ng/ml, 5.12 ng/ml, 12.80 ng/ml, 32.00 ng/ml, 80.00 ng/ml, 200.00 ng/ml, 500.00ng/ml and 1000.00 ng/ml.The content of cholesterol and its metabolites in different cells were obtained according to standards.

Cell morphological analysis
Cell morphological was on showed with Wright-Giemsa Stain Kit (Solarbio, G1021) according to the manufacturer's instruction.Observation was made under inverted fluorescence microscope.

Cancer spheroid assay
For spheroid assay, single cell suspensions harvested and seeded in 6-well ultralow adherent cell culture plates, at a density of 1000 cells/ml in serum-free DMEM/F12 medium supplemented with 1% L-glutamine, 1% penicillin/ streptomycin, 2% B27 (Invitrogen, Carlsbad, CA), 20 ng/ ml epidermal growth factor (EGF, Sigma, St. Louis, MO) and 20 ng/ml basic fibroblast growth factor (bFGF, Invitrogen, Carlsbad, CA).7 days after seeding, spheroids with diameter > 30 μm were counted using an inverted microscope (× 100) and the number of colony formation was calculated.Each experiment was repeated twice with consistency.

Histology and morphometric analysis
Tumors were collected and fixed in 10% neutral-buffered formalin.Tissues were sectioned and stained with hemoxylin and eosin (HE).Pictures were observed under an optical microscope (BX51, Olympus, Tokyo, Japan) to evaluate pathological morphology.

Whole-transcriptome sequencing
Total RNA was extracted and lysed in 500 μl TRIzol reagent (MRC, Carrollton, OH) and sent to China's Shenzhen based BGI (Shenzhen, China) for further analysis.The RNA-seq library was created using the Illumina TruSeq RNA Sample preparation Kit v2 with standard protocol.Genes with a P adjusted value (false-discover rate) < 0.05 were selected for the Gene Ontology (GO) analysis and for the heatmap.Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Statistics
Statistical analyses were performed using Prism 8 software (GraphPad, La Jolla, CA, USA).For comparison between two groups, a two-tailed Student t-test was used.Kaplan-Meier survival analysis was performed for the comparison of survival curves.The statistical significance of protein associations in the TMA data set was evaluated using the Pearson chi-square test.Statistically significant levels were defined as ns (not significant, P > 0.05), * P < 0.05, ** P < 0.01, *** P < 0.001.All data were presented as mean ± SD.

DHCR24 is upregulated in melanoma and is essential for cancer cell survival
To investigate the role of DHCR24 in melanoma tumorigenesis, immunohistochemical (IHC) staining was performed using tissue chip containing normal skin tissue (n = 15), primary melanoma (n = 38) and metastatic melanoma patient samples (n = 10) (see Supplementary Table 1 for details).
The results showed that the expression of DHCR24 was significantly higher in malignant melanoma, especially in metastatic melanoma, compared with control (Fig. 1A, B).
Subsequently, by using two different shRNA constructs targeting DHCR24 (Fig. 1C, D), we found that knockdown of DHCR24 significantly slowed cell proliferation at 48-96 h (Fig. S1A) and significantly decreased the invasion and migration ability of the knockdown group compared with the control group (Fig. 1E).Flow cytometry analysis showed that knockdown of DHCR24 decreased the number of cells in the G1 phase, while the number of cells in the S phase increased (Fig. 1F-G), indicating cell cycle arrest.
To illustrate the effect of DHCR24 on tumorigenic capacity in vivo, a xenograft assay was performed in mice with DHCR24 knockdown and overexpression of melanoma cells.
In the knockdown group, tumor volume decreased significantly (Fig. 1H-I), which was consistent with IHC staining Ki67 (Fig. 1J-K) after 21 days of injection.Conversely, overexpression of DHCR24 resulted in a dramatic increase in tumor volume but no significant change in body weight (Fig. S1C, D).These data indicate that DHCR24 plays an oncogenic role in melanoma cell survival and migration.

DHCR24 is associated with vemurafenib resistance and promotes melanoma spheroid propagation
To further investigate the function of DHCR24 in melanoma therapy, the proliferation assay was performed under vemurafenib treatment in DHCR24-expressing melanoma cells (Fig. 2A, B).The result showed that DHCR24 partially rescued the proliferation blockade caused by vemurafenib (Fig. 2C, D).Using an in vitro tumor sphere formation assay, we found that DHCR24 contributed to the proliferation of stem-like melanoma cell populations (Fig. 2E, F), and the stem cell property of the spheroids was confirmed by the expression of Sox2, CD133, Nanog and ABCB5 (Fig. 2G-H).Considering that DHCR24 is the final enzyme of the cholesterol biosynthetic pathway that converts desmosterol to cholesterol by catalyzing the reduction of C24 = C25 unsaturation in the side chain (Fig. 2I).Therefore, we next treated the A375 and A2058 melanoma cells with a gradient of concentrated cholesterol (0, 5, 10, 50 μM) on the poly-HEMA-coated 12-well plates under tumor sphere culture conditions for 7-10 days.As shown in Fig. 2F, the number and size of spheroids in melanoma cells increased significantly after cholesterol administration compared to the solvent control.In addition, rhodamine123 staining showed that more "negative" cell populations appeared in two BRAF-V600E-mutated melanoma cells with increasing cholesterol levels (Fig. 2J-L).Next, we validated that cholesterol rescued vemurafenib-induced proliferation blockade and apoptosis (Fig. 2M-P) as DHCR24 forced expression, highlighting the importance of cholesterol in melanoma therapy.

DHCR24 induces Rap1-AKT activation and cellular 27HC accumulation
To decipher the molecular mechanism underlying DHCR24, spheroids induced by DHCR24 and a control group were subjected to whole transcriptome sequencing.The heatmap showed that 134 genes were differentially expressed, including 38 down-regulated genes and 96 up-regulated genes (fold change ≥ 2 and a P value (t-test) < 0.05) (Fig. 3A).KEGG enrichment analysis of 96 upregulated genes demonstrated that the Rap1 signaling pathway, which is thought to be upstream of the PI3K/AKT signaling pathway [37], was significantly enriched in DHCR24-induced melanoma spheroids (Fig. 3B).Western blot results showed that Rap1A/ Rap1B expression and AKT phosphorylation (thr 308/309) were significantly increased (Fig. 3C-E), and the PI3K inhibitor wortmannin markedly attenuated spheroids under tumor sphere culture conditions (Fig. 3F-G), confirming that PI3K/AKT activation contributes to melanoma stem cell formation.To investigate the relationship between Rap1 and AKT signaling, HIC0197 was used to activate Rap1 signaling and then AKT phosphorylation was determined by Western blot.The results demonstrated that both AKT and AKT phosphorylation were upregulated with the increase in Rap1A/ Rap1B expression (Fig. 3H, I).Therefore, we hypothesized that Rap1-AKT signaling is downstream of DHCR24 regulation and positively related to cancer stem cells.Although DHCR24 is the final limiting enzyme of cholesterol synthesis, the catabolic fate of cholesterol in melanoma cells was not yet understood.Therefore, the profile of 17 metabolites downstream of cholesterol was characterized by LC-MS after forced DHCR24 expression.In melanoma cell A2058, 8 of 17 metabolites, including cholesterol, were detected and quantified.Among these metabolites, 27-hydroxycholesterol (27HC) was increased by 41.39%, which was statistically significant (n = 6, p = 0.0000751, see Table 2 and Fig. 3J).The increase in 27HC was further verified using a human 27HC ELISA kit (Fig. 3K).These results indicated that 27HC might be final effector of DHCR24 induced vemurafenib resistance and tumor spheroid propagation.

27HC promotes spheroid propagation in melanoma and increases vemurafenib resistance
To validate the role of 27HC in melanoma, a tumor spheroid assay was also performed on wild-type melanoma cells treated with gradient concentrations (0, 5, 10 and 20 μM) of 27HC.As shown in Fig. 4A, B the number and size of tumor spheroids were significantly increased in A375 and A2058 melanoma cells after 7-10 days of treatment with 27HC compared to solvent (ethanol as control).Next, we examined the genes encoding enzymes involved in the cholesterol-centered metabolic pathway (Fig. 4C).Only CYP27A1, encoding 27-hydroxylase, which catalyzes the conversion of cholesterol to 27HC, was highly expressed in melanoma patients compared to normal patients and melanocytes according to the TCGA-SKCM cohorts (Fig. 4D).We also found that DHCR24 strongly upregulated the expression of CYP27A1 in melanoma cells (Fig. 4E), indicating a positive feedback loop leading to cellular accumulation of 27HC.Accordingly, forced expression of CYP27A1 only in melanoma cells (Fig. 4F-G) increased cellular 27HC content (Fig. 4H), caused vemurafenib resistance (Fig. 4I, J), and promoted melanoma spheroid propagation (Fig. 4K, L) in both A375 and A2058 melanoma cells, independent of DHCR24 induction.These data confirm that 27HC as the main downstream effector of DHCR24 promotes melanoma spheroid propagation and increases vemurafenib resistance.

Depletion of 27HC reduces melanoma spheroids and improves treatment with vemurafenib
Next, we sought to determine the effect of dafadine-A, a novel inhibitor of DAF-9 cytochrome P450 in the nematode Caenorhabditis elegans and the mammalian ortholog of DAF-9 (CYP27A1), on melanoma cells (Fig. 5A).We found that dafadine-A did not affect the proliferation of adherent melanoma cells (Fig. 5B), but dramatically inhibited cholesterolinduced melanoma spheroid propagation (Fig. 5C, D), with downregulation of Rap1A/Rap1B expression and phosphorylated AKT1-thr308/309 (Fig. 5E-G).Accordingly, HIC0197 was able to abrogate the inhibition of melanoma spheroid propagation induced by dafadine-A (Fig. 5H, I), confirming that Rap1 is functional upstream of AKT signaling.When used in combination, we observed that dafadine-A significantly enhanced vemurafenib-induced apoptosis (Fig. 5J, K).These data emphasized the indispensable role of CYP27A1 in melanoma stemness and the potential targeting property.

Cholesterol metabolism is a promising therapeutic target for vemurafenib-resistant melanoma
To further investigate the potential role of targeted therapy by inhibiting the cholesterol-27HC-Rap1 axis in vemurafenib-resistant melanoma, a vemurafenib-resistant A375 cell line (A375-VR) was established by continuous treatment with vemurafenib for weeks.The adaptive resistance of A375-VR was confirmed by Giemsa staining under serial vemurafenib treatment compared to the A375 wildtype counterpart (Fig. 6A), and the IC50 was calculated by CKK8 determinations (Fig. 6B).The value of A375-VR was almost 75-fold higher than that of parental A375 cells (8.593 μM vs. 0.12 μM), demonstrating the vemurafenibresistant property of A375-VR cells.Interestingly, we found that A375-VR cells exhibited higher expression of CYP27A1 and the cholesterol synthesis-limiting enzyme HMGCR than the parental cells (Fig. 6C).Dafadine-A and mevastatin (HMGCR inhibitor) were therefore used in combination with vemurafenib.Compared to the moderate decrease induced by vemurafenib, dafadine-A and mevastatin dramatically abrogated the inhibitory effects mediated by vemurafenib (Fig. 6D, E), while A375 VR cells proliferation was unaffected (Fig. 6F, G).Mechanistically, after the addition of vemurafenib, the expression of Rap1A/Rap1B and phosphorylated AKT1-thr308/309 was decreased in wildtype cells (Fig. 6H, I), whereas the expression of Rap1A/ Rap1B and phosphorylated AKT1-thr308/309 was not altered in A375-VR cells, but was decreased in cells receiving vemurafenib and dafadine-A or mevastatin in combination (Fig. 6J, K).

Discussion
The persistent activation of oncogenic signals enables cancer cells to synthesize their own cholesterol to provide energy for rapid growth.Accordingly, numerous studies show that statins, inhibitors of HMG-CoA reductase (HMGCR), directly delay cancer development and progression in cell and animal models [38,39].In addition, survival analyzes using the Cancer Genome Atlas (TCGA) show that increased activity of the cholesterol synthesis pathway correlates with lower patient survival in sarcoma, acute myeloid leukemia and melanoma [40].By directly regulating signaling molecules in the membrane [41,42], or as signaling molecules via certain metabolites [43], cholesterol has a significant impact on the biological behavior of cancer and promotes or suppresses cancer development, depending on the cell context and the steps of metabolic perturbation [44,45].Therefore, the question arises as to how metabolic reprogramming can be integrated into a therapeutic procedure to cancer.Melanoma is a malignant invasive tumor, and the global incidence rate is increasing.The BRAFV600E mutation is found in approximately 50% of melanoma patients, facilitating the development of targeted drugs.Vemurafenib (Zelboraf; Plexxikon/Roche) is the first drug approved for the treatment of BRAF mutation metastatic melanoma in the United States in August 2011 and in the European Union in February 2012.Together with significant advances in immunotherapy, it has significantly improved the fiveyear survival rate of patients with metastatic melanoma.However, the long-term prognosis of patients with metastatic melanoma is still unsatisfactory, which is probably related to drug resistance, the major obstacle of cancer therapy [46].Drug resistance is far from being sufficiently researched, as it is a multifactorial process involving a number of mechanisms in which cancer stem cells (CSCs) may play a crucial role [47][48][49].
CSCs are defined as cancer cells that exhibit certain characteristics of embryonic stem cells [50,51], which give rise to the entire set of cell types from a single cell and high expression of OCT4, SOX2 and so forth stemness markers [52].Besides, CSCs stably express high levels of the ATP-binding cassette (ABC) transporter family, such as ABCG2, which is an important transporter responsible for pumping out chemotherapeutic drugs [53,54].It facilitating that CSCs escaped from most conventional chemotherapeutic agents.Given the importance of cancer stem cells (CSCs) in tumor metastasis [55,56], we ought to ask which enzymes are contribute to melanoma cell stemness and response to targeted therapy.Generally, we assessed the mRNA expression-based stem cell index (mRNAsi) of melanomas using the OCLR algorithm  [57].The genes responsible for cholesterol uptake (LDLR), biosynthesis (SREBF2, HMGCR, and DHCR24), and efflux (ABCA1 and ABCG1) were selected and divided into groups of 25% and 25%, respectively, according to their own expression level in the TCGA-SKCM cohort.As shown in Supplementary material Fig. S2A, patients with activated cholesterol biosynthesis genes showed higher stemness indices, and those with higher expression of efflux genes (ABCA1, ABCG1) correlated with lower stemness score, whereas the groups with high and low LDLR expression were comparable.When looking at the Spearman correlation between the mRNAsi scores and the genes related to cholesterol metabolism, only DHCR24, ABCA1, and ABCG1 were shown to be statistical significance (Supplementary material Fig. S2A).The mRNAsi results showed that elevated intracellular cholesterol was positively associated with melanoma stemness, through activation of DHCR24 in cholesterol biosynthesis or inhibition of efflux by ABCA1, and ABCG1.In line with this, our tissue microarray showed that DHCR24 was upregulated in malignant melanomas compared with normal skin tissue and which was consistent with previous conclusions that DHCR24 expression was increased in metastatic melanomas and that high DHCR24 levels were associated with greater tumor cell growth ability [20].We observed that forced expression of DHCR24 promoted tumor growth in mouse xenografts (Fig. S1C), whereas knockdown of DHCR24 blocked cells in S phase and resulted in significant inhibition of invasion and migration in melanoma cells.More importantly, DHCR24 reproduced the cholesterol phenotype by promoting the melanoma spheroid propagation, indicating the oncogenic role of DHCR24 in melanoma cells.
Targeting cholesterol metabolism is currently being used as a potential therapeutic approach for cancer therapy.Inhibition of mevalonate metabolism, silencing of the low-density lipoprotein receptor, and conjugation of cholesterol components with anticancer drugs have shown attractive effects on slowing cell growth and inducing apoptosis in various cancers [58][59][60].However, cholesterol may not be the ultimate effector of cancer cell survival.Intermediate metabolites of cholesterol have been shown to have various functions in tumorigenesis.We and others have previously shown that 7-dehydrocholesterol and vitamin K3 inhibit melanoma cell proliferation and induce apoptosis via AKT /MAPK signaling pathways [61][62][63], while Nelson et al. found that the cholesterol metabolite 27HC promoted tumor growth and metastasis in mouse models of breast cancer by serving as a partial agonist for the estrogen receptor and liver X receptor [27].Moreover, 27HC has been reported to promote cisplatin resistance in glioblastoma cells, and high 27HC concentration is associated with poor patient outcome [64].Indeed, our data showed that forced expression of DHCR24 increases cellular 27HC content without statistically affecting cholesterol content in melanoma cells and, moreover, is associated with melanoma spheroid expansion and vemurafenib resistance.At the same time, our data show that DHCR24 induces CYP27A1 expression and increases HMGCR and CYP27A1 expression in vemurafenibresistant melanoma cell lines, suggesting an enhanced regulatory loop mechanism.Therefore, we finally demonstrated that targeting CPY27A1 has a dramatic inhibitory effect on vemurafenib-resistant melanoma cells when used in combination with vemurafenib.We observed that mevastatin had a similar effect on slowing the proliferation of vemurafenib-resistant melanoma as dafadine-A.We believed that this was likely due to the fact that mevastatin targets HMGCR, resulting in the absence of a 27HC synthesis source.
Rap1 (Ras-like GTPases), the closest relative of the small GTPase Ras, was identified as a suppressor of Ras transformation in rodent fibroblasts in a tissue culturebased genetic screening assay [65].Ishida et al. suggested that Rap1 is particularly important for the maintenance of normal myelopoiesis and may provide an oncogenic signal in cooperation with RAS/ERK signaling [66].The posttranslational addition of a geranylgeranyl group to the processed Rap1 carboxyl terminus binds Rap1 to the membrane, including intracellular membranes in the perinuclear region, endocytic and exocytic vesicles, and the plasma membrane [67], suggesting a role for Rap1 in recruiting components for transport and activation of membrane-coupled receptor kinase.In this work, we found that DHCR24 and DHCR24-induced 27HC significantly upregulated Rap1A/Rap1B expression and further activated the PI3K/AKT signaling pathway in melanoma spheroids, while Rap1 agonists or AKT inhibitors reproduced the stem cell phenotypes.Interestingly, we observed that in vitro administration of 27HC to melanoma cells significantly increased membrane fluidity (Fig. S3A), suggesting activation of signaling transduction.Compared to wildtype melanoma cells, Rap1A/Rap1B expression remains unchanged upon treatment with vemurafenib, but is downregulated by combined treatment with a CYP27A1 inhibitor in vemurafenib-resistant melanoma cell lines, highlighting the potential of cholesterol metabolism in vemurafenib-resistant melanoma.

Fig. 5 .
Fig. 5. 27HC depletion decreases melanoma spheroids and enhances vemurafenib treatment.A Chemical formula of dafadine-A (CYP27A1 inhibitor).B Morphological images of A375 melanoma cells were treated with vemurafenib alone or combinational use with dafadine-A for 48 h.C, D Dafadine-A alone or combinational use with cholesterol (20 μM) on A375 and A2058 melanoma cells for evaluating the contribution of dafadine-A on melanoma spheroid f propagation.E-G Western blot and quantitative bar chart of Rap1A/ Rap1B, AKT and pAKT in A375 and A2058 melanoma cells with dafadine-A treatment.I Dafadine-A alone or combinational use with HJC0197 (25 μM) on A375 and A2058 melanoma cells for evaluating the contribution of Rap1 downstream 27HC on melanoma spheroid propagation.J, K Dafadine-A combinational use with vemurafenib on A375 and A2058 for 48 h determined by CKK-8 ◂

Table 1
Clinical-pathological information and TNM staging of human melanoma specimens (n = 63) used in this study (4:1, v/v) mixture solution and vortex for 30 s or ultrasonicate in ice water bath until mixture was homogenous.Filtering with 0.22 μm organic phase pinhole filter for LC-MS analysis.