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BMC Medicine volume 20, Número do artigo: 292 (2022) Cite este artigo

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Embora o metabolismo do colesterol seja uma via comum para o desenvolvimento de fármacos antitumorais, não existem alvos e fármacos específicos para uso clínico. Aqui, com base em nosso estudo anterior de esterol O-aciltransferase 1 (SOAT1) no carcinoma hepatocelular, procuramos rastrear uma droga direcionada eficaz para o tratamento preciso do carcinoma hepatocelular e, do ponto de vista do metabolismo do colesterol, esclarecer a relação entre a regulação do colesterol e tumorigênese e desenvolvimento.

Neste estudo, desenvolvemos uma tecnologia de triagem de afinidade integrada de triagem virtual para triagem de drogas de proteína-alvo. Uma série de experimentos in vitro e in vivo foram utilizados para verificação da atividade da droga. A análise multi-ômica e a análise por citometria de fluxo foram usadas para explorar os mecanismos antitumorais. A análise comparativa de proteoma e transcriptoma combinada com informações de acompanhamento de sobrevivência dos pacientes revela o potencial terapêutico clínico dos medicamentos rastreados.

Selecionamos três compostos, nilotinib, ABT-737 e evacetrapib, que exibiram ligação ideal com SOAT1. Em particular, o nilotinibe apresentou uma alta afinidade pela proteína SOAT1 e inibiu significativamente a atividade do tumor in vitro e in vivo. A análise multi-ômica e a análise de citometria de fluxo indicaram que os compostos direcionados a SOAT1 reprogramaram o metabolismo do colesterol em tumores e aumentaram as células T CD8+ e os neutrófilos para suprimir o crescimento do tumor.

Em conjunto, relatamos vários ligantes SOAT1 de alta afinidade e demonstramos seu potencial clínico na terapia de precisão do câncer de fígado, além de revelar o potencial mecanismo antitumoral de compostos direcionados a SOAT1.

Relatórios de revisão por pares

Estudos anteriores descobriram que a reprogramação do metabolismo do colesterol tumoral ocorre durante a tumorigênese e desenvolvimento [1, 2]. As características do metabolismo do colesterol dos tumores incluem tanto a regulação positiva da síntese e absorção de colesterol quanto o acúmulo de vários derivados do colesterol, como ésteres de colesterol e colesterol oxidado [3]. No entanto, a reprogramação do metabolismo do colesterol em tumores envolve muitos processos, incluindo síntese, captação, esterificação, efluxo e conversão. Assim, elucidar o papel desses processos na tumorigênese e no desenvolvimento de potenciais drogas terapêuticas tem sido um desafio.

Muitos estudos clínicos e pré-clínicos recentes mostraram que direcionar o metabolismo do colesterol em células tumorais e células imunes é uma maneira óbvia de tratar tumores [4]. Os alvos existentes incluem enzimas-chave na síntese do colesterol e vias de transporte, bem como fatores-chave de transcrição envolvidos no metabolismo do colesterol. Por exemplo, as estatinas exercem efeitos anticancerígenos inibindo a atividade da enzima limitante chave 3-hidroxi-3-metilglutaril-coenzima A redutase (HMGCR) na via de sinalização da síntese de colesterol [5], e o itraconazol liga-se diretamente ao esterol- domínio sensível do transportador de colesterol intracelular NPC 1 (NPC1), reduzindo assim o crescimento tumoral e a angiogênese [6]. Além disso, LXR-623, GW3965, outros inibidores de moléculas pequenas contra o receptor X do fígado (LXR) e outras drogas de moléculas pequenas também estão sendo atualmente investigados como drogas antitumorais [7].

Espera-se que a descoberta de novos alvos de drogas relacionadas ao colesterol acelere o desenvolvimento de drogas que visam a homeostase do colesterol intracelular para terapia tumoral. A proteína SOAT1 pode converter o excesso de colesterol intracelular em éster de colesterol e armazená-lo em gotículas lipídicas. O nocaute ou inibição de SOAT1 pode inibir uma variedade de tumores, regulando a homeostase do colesterol em células tumorais [8, 9] e o estado imunológico do microambiente tumoral [10, 11]. Anteriormente, descobrimos que o SOAT1 pode ser usado para estratificar pacientes com CHC com mau prognóstico e como alvo de drogas para o tratamento de pacientes com esse subtipo. Inibidores direcionados a SOAT1 demonstraram exercer efeitos antitumorais ideais tanto in vitro quanto in vivo. No entanto, existem apenas alguns inibidores SOAT1 conhecidos - apenas avasimibe, nevanimibe e CI-976 foram relatados - o que limita muito suas perspectivas de desenvolvimento de medicamentos.

95%) can be divided and stored at − 80 ℃ for future use./p> 10 (orange histogram), including three positive controls (red star), 16 compounds that did not bind to the protein (black histogram), and 7 other compounds had signal errors (gray histogram). f The KD value of the screened ligands was calculated by SPR/p> 95%, and the correlation coefficient of all samples was > 85% (Figure S3b). In total, 3960 proteins were quantified from 7307 identified proteins (Figure S3c, full of proteome data shown in separate additional file 3). After correcting for multiple testing by setting the P value at 0.01, false discovery rate (FDR) at 0.05, and fold change > 2, 226 differential proteins (180 downregulated and 48 upregulated) were simultaneously dysregulated in the proteome of two compounds targeting SOAT1 (Fig. 3a and Figure S3d). Then, we submitted all differentially expressed proteins to Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. GO and KEGG analyses showed that the dysregulated proteins were related to steroid metabolic processes or cholesterol metabolic processes, and the corresponding molecular functions were also closely related to sterol transporter activity, lipid transfer activity, cholesterol binding, and cholesterol transfer activity (Fig. 3b). These results indicated that the most dysregulated proteins were generally related to cholesterol metabolism. Further research on cholesterol metabolism signaling pathway proteins found 29 cholesterol metabolism–related proteins significantly altered after treatment with SOAT1-targeting compounds (Table S2). Classification and analysis of these differential proteins showed that most of the cholesterol synthesis–related proteins in the cholesterol metabolism signaling pathway, such as phosphomevalonate kinase(PMVK), lanosterol 14-alpha demethylase(CYP51A1), 7-dehydrocholesterol reductase(DHCR7), lanosterol synthase(LSS), and squalene monooxygenase(SQLE), were significantly downregulated. Additionally, cholesterol transport and esterification–related proteins, including NPC1, SOAT1, phospholipid transfer protein (PLTP), and neutral cholesterol ester hydrolase 1(NCEH1), were also significantly downregulated. In contrast, cholesterol conversion–related proteins, such as sterol 26-hydroxylase (CYP27A1), steroid 17-alpha-hydroxylase/17,20 lyase (CYP17A), aldo–keto reductase family 1 member (D1AKR1D1), and translocator protein (TSPO), were significantly upregulated (Fig. 3c). Cholesterol synthesis, esterification, transportation (including uptake and efflux), and conversion are the main biological processes that maintain the stability of cholesterol metabolism [23]. Numerous previous clinical and preclinical studies have shown that successful treatment of tumors can be achieved by interfering with the cholesterol metabolism of tumor cells and immune cells [24]. Our proteomics analysis showed that compounds targeting SOAT1 not only block the production of cholesterol esters, but also inhibit the synthesis and uptake of cholesterol and advance the conversion of cholesterol to bile acids. Thus, SOAT1-targeting compounds systematically restored the cholesterol metabolism in tumor cells (Fig. 3d)./p> 2410 differential metabolites (239 downregulated and 171 upregulated) were detected in the metabolome of nevanimibe and nilotinib (Fig. 4a and Figure S4d). To more accurately identify differential changes in metabolites associated with cholesterol metabolism pathways, we further performed targeted metabolome analysis using metabolite standards. As a result, we found that 26 cholesterol metabolism pathway metabolites changed after administration (Table S3). Heat map cluster analysis showed that hydroxysterol, bile acid, and sterol hormone compounds were significantly upregulated, while pre-sterol and cholesterol ester were significantly downregulated (Fig. 4b). Specifically, hydroxysterol, bile acids, and steroid hormone metabolites, such as 20-α, 22-β-dihydroxycholesterol, 24-hydroxycholesterol, lithocholic acid, and pregnenolone, were significantly increased, while pre-sterol and cholesterol ester metabolites, such as lanosterol, 4,4-dimethyl-5-α-cholesta-8, 14-demethyllanosterol, 18:0 cholesterol ester, and 20:0 cholesterol ester were significantly reduced (Fig. 4c). These results further confirmed that compounds targeting SOAT1 can inhibit cholesterol synthesis, esterification, and transport, and promoted cholesterol conversion./p>

0.9; range, 0.85–1). (c) Coverage of identified and quantified proteins. In total, 3,960 proteins were quantified from 7,307 identified proteins. (d) UpSet Venn diagram of each pairwise comparison. 226 differential proteins (180 downregulated and 48 upregulated) were simultaneously dysregulated in the proteome of nevanimibe and nilotinib (n=3, p<0.01). Figure S4. Metabolome sample preparation and data analysis. (a) Workflow of metabolome sample preparation and data collection. (b) Scatter plots and Pearson correlation coefficients for replicate metabolome profiling of two SOAT1-targeted compounds (nevanimibe and nilotinib). The x- and y-axes represent the metabolome intensities in each pairwise comparison. Notably, repeat experiments with the same samples have good reproducibility, with a high level of correlation (average > 0.9; range, 0.85–1). (c) Coverage of detected mass spectral peaks and quantitative and qualitative mass spectral. Overall, 1890 compounds were quantified from 19,671 identified MS/MS spectra, and 662 metabolites were classified. (d) UpSet Venn diagram of each pairwise comparison. 410 differential metabolites (239 downregulated and 171 upregulated) were detected in the metabolome of nevanimibe and nilotinib (n=6, p<0.01). Figure S5. The relationship between drug-regulated cholesterol dysregulation proteins and the survival of liver cancer patients. Figure S6. Schematic of multicolor flow cytometry analysis. (a) Schematic diagram of a xenograft model derived from Hepa1-6 cells transplanted into C57BL/6 mice. (b) Multicolor flow cytometry analysis of cell-derived xenograft models. (c) Histogram overlays show changes in expression profiles between samples. The table shows the groups and cell counts. (d) Fluorescence correlation analysis heat map of each color. The cells labeled with each fluorescent antibody are clearly distinguished, indicating that the analysis system is robust. Table S1. Four software molecar docking screenings yielded 62 potential compounds. Table S2. 29 cholesterol metabolism signal pathway proteins changed after administration. Table S3. Targeting identified 26 cholesterol metabolism signaling pathway metabolites changed after administration./p>