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

Abstract

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.

Keywords: 27-Hydroxycholesterol; Cholesterol metabolism; DHCR24; Melanoma.

Fig.1

DHCR24 is highly expressed in melanoma and essential for tumor growth. A, B Representative images and quantitative bar chart of immunohistochemistry staining against DHCR24 in normal skin tissue, primary and metastatic malignant melanoma samples. C, D Western blot and quantitative bar chart of DHCR24 knock down efficiency in melanoma cell lines using lentivirus harboring scramble shRNA (shNC) and DHCR24 shRNAs (sh6 and sh7). E Transwell assay of A375 and A2058 melanoma cells harboring scramble shRNA (shNC) and DHCR24 shRNAs (sh6 and sh7). F, G Cell cycle assay determined by flow cytometry of A375 and A2058 melanoma cells harboring scramble shRNA (shNC) and DHCR24 shRNAs (sh6 and sh7). H NOD-SCID mice were subcutaneously injected with 1 × 10⁷ sh6 and sh7 A375 cells (n = 5) compared with the wild type and shNC groups (n = 5 each). I The tumor volume among four groups. J–K Representative images and quantitative bar chart of immunohistochemistry staining against Ki67 in xenograft tumor tissues. Different colors represent different groups, *p < 0.05, **p < 0.01, ***p < 0.001, asterisks (*) stand for significance levels

Fig. 2

DHCR24 is associated with vemurafenib resistance and promotes melanoma spheroid propagation. A, B Western blot and quantitative bar chart of DHCR24 overexpression in melanoma cell lines. C, D Percentage of melanoma cell viability under the treatment of vemurafenib (1, 5, 10 and 20 μM) in presence of DHCR24 overexpression or not for 48 h determined by CKK-8. E, F Representative images and quantitative bar chart of spheroid propagation in melanoma cells with DHCR24 overexpression. G, H The stemness of spheroid is evaluated by stem markers (Sox2, CD133, Nanog and ABCB5) mRNA expression. I Chemical equation of cholesterol synthesis from desmosterol catalyzed by DHCR24, which reduces the double bond at C24–C25 position of desmosterol. J, K Representative images and quantitative bar chart of spheroid propagation in A375 and A2058 in presence of cholesterol with gradient concentration. L The stemness of spheroid is evaluated by Rhodamine 123. M, N Percentage of melanoma cell viability under vemurafenib treatment (1, 2, 4 and 10 μM) in presence of cholesterol (5 and 10 μM) or not for 48 h. O–P Apoptosis rate of melanoma cell viability under vemurafenib treatment (10 μM) in presence of cholesterol (5 and 10 μM) or not for 48 h determined by flow cytometry

Fig.3

DHCR24 activates Rap1-AKT signaling induces cellular 27HC accumulation. A Heatmap of spheroids induced by DHCR24 overexpressed A375 cell (134 genes, FDR-corrected P value < 0.05 and ≥ twofold change cut-off), totally 38 downregulated genes and 96 upregulated genes. B KEGG enrichment analysis (representative pathways) of 96 upregulated genes in A375 spheroids with DHCR24 overexpression. C–E Western blots and quantitative bar chart of Rap1A/Rap1B, AKT and pAKT in spheroids induced by DHCR24 overexpressed A375 and A2058 cells. F–G Representative images and quantitative bar chart of spheroid propagation of A375 and A2058 melanoma cells with wortmannin (PI3K inhibitor) treatment. H, I Western blots and quantitative bar chart of Rap1A/Rap1B, AKT and pAKT in A375 melanoma cell treated by Rap1 agonist HJC0197. J Heatmap of 9 paneled cholesterol metabolites quantitation of melanoma cells with DHCR24 overexpression. K Cellular 27HC quantitation in melanoma cells with DHCR24 overexpression determined by ELISA. ***p < 0.001, asterisks (*) stand for significance levels

Fig. 4.

27HC promotes melanoma spheroids propagation and increases vemurafenib resistance. A, B Representative images and quantitative bar chart of spheroid propagation of A375 and A2058 in presence of 27HC with gradient concentration. C Diagram of cholesterol centered metabolic pathway. D Expression levels of CYP27A1 in Skin Cutaneous Melanoma (SKCM) samples compared with normal tissue, the RNA-Seq datasets is based on the UCSC Xena project (http://xena.ucsc.edu), and visualized by GEPIA (http://gepia.cancer-pku.cn). E DHCR24 induces CYP27A1 mRNA upregulation in A2058 melanoma cells. F–G Western blot and quantitative bar chart of CYP27A1 overexpression in A375 and A2058 cells. H Cellular 27HC quantitation in melanoma cells with CYP27A1 overexpression determined by ELISA. I, J Percentage of melanoma cell viability under the treated with vemurafenib (1, 5, 10 and 20 μM) in presence of CYP27A1 overexpression or not for 48 h determined by CKK-8. K–J Representative images and quantitative bar chart of spheroid propagation in melanoma cells with CYP27A1 overexpression. *p < 0.05, ***p < 0.001, asterisks (*) stand for significance levels

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

Fig. 6.

27HC is promising therapeutic target of vemurafenib resistant melanoma. A Morphological images of A375 and A375 vemurafenib resistant melanoma cell (A375VR) treated by gradient concentration of vemurafenib (2, 4, 8,15 and 20 μM). B IC50 determination of vemurafenib on A375 and A375VR cells. C The mRNA expression level of CYP27A1 and HMGCR in A375 and A375VR cells. Combinational use vemurafenib with D Dafadine-A or E Mevastatin on A375VR cells. F–G Percentage of A375 cell viability under the treatment of dafadine-A or mevastatin for 48 h determined by CKK-8. H, I Western blot and quantitative bar chart of Rap1A/Rap1B, AKT and pAKT in A375 cells under the treatment of vemurafenib. J, K Western blot and quantitative bar chart of Rap1A/Rap1B, AKT and pAKT in A375VR cells with combinational treatment vemurafenib and dafadine-A or mevastatin. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, asterisks (*) stand for significance level