NPC1以胆固醇转运非依赖的方式控制TGFBR1的稳定性，并促进肝细胞癌的进展-动脉网

NPC1 controls TGFBR1 stability in a cholesterol transport-independent manner and promotes hepatocellular carcinoma progression

Nature 等信源发布 2025-01-07 10:18

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Abstract

摘要

Niemann-Pick disease type C protein 1 (NPC1), classically associated with cholesterol transport and viral entry, has an emerging role in cancer biology. Here, we demonstrate that knockout of

尼曼-匹克病C型蛋白1（NPC1）通常与胆固醇转运和病毒进入有关，在癌症生物学中具有新兴作用。在这里，我们证明了

Npc1

in hepatocytes attenuates hepatocellular carcinoma (HCC) progression in both DEN (diethylnitrosamine)-CCl

在肝细胞中，DEN（二乙基亚硝胺）-CCl均可减轻肝细胞癌（HCC）的进展

induced and MYC-driven HCC mouse models. Mechanistically, NPC1 significantly promotes HCC progression by modulating the TGF-β pathway, independent of its traditional role in cholesterol transport. We identify that the 692-854 amino acid region of NPC1’s transmembrane domain is critical for its interaction with TGF-β receptor type-1 (TGFBR1).

诱导和MYC驱动的HCC小鼠模型。从机制上讲，NPC1通过调节TGF-β途径显着促进HCC进展，与其在胆固醇转运中的传统作用无关。我们确定NPC1跨膜结构域的692-854个氨基酸区域对于其与TGF-β受体1型（TGFBR1）的相互作用至关重要。

This interaction prevents the binding of SMAD7 and SMAD ubiquitylation regulatory factors (SMURFs) to TGFBR1, reducing TGFBR1 ubiquitylation and degradation, thus enhancing its stability. Notably, the NPC1 (P691S) mutant, which is defective in cholesterol transport, still binds TGFBR1, underscoring a cholesterol-independent mechanism.

这种相互作用阻止了SMAD7和SMAD泛素化调节因子（SMURFs）与TGFBR1的结合，减少了TGFBR1的泛素化和降解，从而增强了其稳定性。值得注意的是，胆固醇转运缺陷的NPC1（P691S）突变体仍与TGFBR1结合，强调了不依赖胆固醇的机制。

These findings highlight a cholesterol transport-independent mechanism by which NPC1 contributes to the stability of TGFBR1 in HCC and suggest potential therapeutic strategies targeting NPC1 for HCC treatment..

这些发现突出了NPC1促进HCC中TGFBR1稳定性的胆固醇转运非依赖性机制，并提出了针对NPC1治疗HCC的潜在治疗策略。。

Introduction

简介

Liver cancer presents a significant global health challenge, with an increasing incidence worldwide

肝癌是一项重大的全球健康挑战，全球发病率不断上升

. It ranks as the sixth most common malignancy globally, with its mortality rate being the third highest among cancers

. Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancer, accounting for approximately 90% of all liver cancer cases

肝细胞癌（HCC）是最常见的原发性肝癌，约占所有肝癌病例的90%

. The treatment of HCC faces hurdles such as a high recurrence rate and a limited survival period, with existing clinical therapies yielding suboptimal results. Therefore, there is a pressing need for in-depth research into the molecular mechanisms driving HCC progression, which could lead to the discovery of innovative and effective diagnostic biomarkers and drug targets, offering avenues for treatment..

.HCC的治疗面临着诸如高复发率和有限生存期等障碍，现有的临床治疗效果不佳。因此，迫切需要深入研究驱动HCC进展的分子机制，这可能导致发现创新和有效的诊断生物标志物和药物靶标，为治疗提供途径。。

Previous research has indicated that cholesterol homeostasis is significantly disrupted in HCC

先前的研究表明，HCC中的胆固醇稳态被显着破坏

. NPC1, a protein commonly associated with cholesterol transport, has been identified as having a high prognostic risk score in HCC

NPC1是一种通常与胆固醇转运相关的蛋白质，已被确定在HCC中具有较高的预后风险评分

. NPC1 is a large protein, that spans the membrane 13 times and is predominantly localized in the late endosome/lysosome (LE/Ly) membrane. Diseases related to NPC1 include Niemann-Pick disease type C1, a rare autosomal recessive genetic disorder. Dysfunctional NPC1, resulting from mutations in the

NPC1是一种大蛋白，跨越膜13倍，主要位于晚期内体/溶酶体（LE/Ly）膜中。与NPC1相关的疾病包括C1型尼曼-皮克病，这是一种罕见的常染色体隐性遗传疾病。功能失调的NPC1，由突变引起

NPC1

gene, leads to excessive accumulation of intracellular lysosomal cholesterol. This pathogenesis is characterized by features such as hepatosplenomegaly, cognitive impairment, and progressive and disabling neurological symptoms

基因，导致细胞内溶酶体胆固醇的过度积累。这种发病机制的特征是肝脾肿大、认知障碍、进行性和致残性神经系统症状

. Furthermore, mutations in

此外，突变

NPC1

have been identified as a risk factor for childhood and adult morbid obesity

已被确定为儿童和成人病态肥胖的危险因素

. NPC1’s role extends beyond genetic disorders, as it has been implicated in various viral infections. For instance, the Ebola virus spike glycoprotein (GP) binds specifically to NPC1 in the LE/Ly, triggering cellular infection

NPC1的作用超越了遗传疾病，因为它与各种病毒感染有关。例如，埃博拉病毒刺突糖蛋白（GP）与LE/Ly中的NPC1特异性结合，引发细胞感染

. Additionally, NPC1 has been associated with other viral infections, including HIV-1

此外，NPC1还与其他病毒感染有关，包括HIV-1

, Chikungunya virus

and certain hepatoviruses

. Despite these associations, the exact role of NPC1 in the context of HCC remains unclear, warranting further investigation.

尽管存在这些关联，但NPC1在HCC中的确切作用仍不清楚，需要进一步研究。

In this work, we elucidate the critical roles of NPC1 in the development and progression of HCC using knockout (KO) mouse models. Interestingly, we find that NPC1 promotes the TGF-β pathway by stabilizing the protein TGFBR1, independent of its role in cholesterol transport. This function positions NPC1 as a facilitator of tumor progression and metastasis in HCC.

在这项工作中，我们使用敲除（KO）小鼠模型阐明了NPC1在HCC发展和进展中的关键作用。有趣的是，我们发现NPC1通过稳定蛋白质TGFBR1来促进TGF-β途径，而与其在胆固醇转运中的作用无关。该功能将NPC1定位为HCC中肿瘤进展和转移的促进剂。

Furthermore, we reveal that NPC1 interacts with TGFBR1 and impedes the binding between TGFBR1 and the SMAD7/SMURFs complex, thereby reducing the ubiquitylation of TGFBR1. These insights not only shed light on the molecular mechanisms underpinning HCC progression but also establish NPC1 as both a potential prognostic marker and therapeutic target for combating HCC..

此外，我们揭示NPC1与TGFBR1相互作用并阻碍TGFBR1与SMAD7/SMURFs复合物之间的结合，从而减少TGFBR1的泛素化。这些见解不仅阐明了支持HCC进展的分子机制，而且将NPC1确立为抗HCC的潜在预后标志物和治疗靶点。。

Results

结果

Up-regulation of NPC1 correlates with poor prognosis of HCC

NPC1上调与肝癌预后不良相关

We analyzed The Cancer Genome Atlas (TCGA) datasets and found that NPC1 was significantly upregulated in tumors compared with adjacent tissues in 52% (12 out of 23) of TCGA cancer types, including HCC (Fig.

我们分析了癌症基因组图谱（TCGA）数据集，发现在包括HCC在内的52%（23个中的12个）TCGA癌症类型中，NPC1在肿瘤中与邻近组织相比显着上调（图）。

1a级

and Supplementary Fig.

和补充图。

1a级

). We also found that both protein and mRNA levels of NPC1 were significantly elevated in HCC tissues compared with paired non-tumor tissues. Furthermore, NPC1 expression showed a significant increasing trend in correlation with the prognostically associated proteomic subtypes in HCC patients from the cohorts of Jiang et al.’s.

)。我们还发现，与配对的非肿瘤组织相比，HCC组织中NPC1的蛋白质和mRNA水平均显着升高。此外，来自Jiang等人队列的HCC患者中，NPC1表达与预后相关的蛋白质组学亚型相关，显示出显着增加的趋势。

and Gao et al.’s

和高等人的

(Fig.

（图。

1b, c

1b、c

and Supplementary Fig.

和补充图。

1b, c

1b、c

). Patients with high NPC1 expression had significantly worse overall survival (OS) and disease-free survival (DFS) than those with low NPC1 expression (Fig.

)。NPC1高表达患者的总生存率（OS）和无病生存率（DFS）明显低于NPC1低表达患者（图）。

1d–i

Fig. 1: Up-regulation of NPC1 correlates with poor prognosis of HCC.

图1:NPC1的上调与HCC的不良预后相关。

一

–

c级

Upregulation of NPC1 mRNA (

NPC1 mRNA的上调(

一

) or protein (

)或蛋白质(

b类

c级

) in paired non-tumor tissues (NT) and tumor tissues (T) in TCGA datasets (

)在TCGA数据集中成对的非肿瘤组织（NT）和肿瘤组织（T）中(

一

) (NT,

)（新台币，

= 32; T,

=32;T

= 375), Jiang et al.’s cohort (

= 375），Jiang等人的队列（

b类

) (NT,

)（新台币，

= 98; S-I,

==参考98；S-I，

= 36; S-II,

== 36;S-II，

= 32; S-III

=32;S-III

= 33) and Gao et al.’s cohort (

33）和Gao等人的队列(

c级

) (NT,

)（新台币，

= 159; S-Mb,

=159；S-Mb，

= 55; S-Me,

== 55;S-Me，

= 57; S-Pf,

== 57;S-Pf，

= 47).

== 47).

–

我

Kaplan–Meier overall survival (

Kaplan–Meier总生存期(

f级

小时

) and disease-free survival (

)和无病生存(

克

我

) curves of individuals with high or low NPC1 expression in TCGA datasets (

)TCGA数据集中NPC1表达高或低的个体的曲线(

), Jiang et al.’s cohort (

)，江等人的队列(

f级

克

) and Gao et al.’s cohort (

)和高等人的队列(

小时

我

Representative IHC staining of TMA with NPC1 antibodies in an independent cohort of HCC (

在独立的HCC队列中，用NPC1抗体对TMA进行代表性IHC染色(

= 295 biologically independent samples); scale bars, 100 μm.

=295个生物学上独立的样本）；比例尺，100微米。

Staining intensity of NPC1 between NT and T samples from TMA (

来自TMA的NT和T样品之间NPC1的染色强度(

= 295 biologically independent samples).

=295个生物学上独立的样本）。

Kaplan–Meier overall survival (

Kaplan–Meier总生存期(

) and disease-free survival (

)和无病生存(

) curves of individuals with high or low NPC1 expression. In the box plots, the middle bar represents the median, and the box represents the interquartile range; bars extend to 1.5× the interquartile range. Data are presented as the mean ± s.e.m. (

)NPC1表达高或低的个体的曲线。在箱形图中，中间条表示中位数，方框表示四分位间距；酒吧延伸到四分位间距的1.5倍。数据表示为平均值±标准误(

一

). Statistical significance was determined by Mann–Whitney U test (

)。统计学显著性由Mann-Whitney U检验确定(

一

–

c级

) or log-rank test (

)或对数秩检验(

–

我

). Source data are provided as a Source Data file.

)。源数据作为源数据文件提供。

Full size image

全尺寸图像

To further investigate the prognostic value of NPC1 in HCCs, we performed a tissue microarray (TMA)-based immunohistochemistry (IHC) study of NPC1 in HCC tumor and paired non-tumor liver tissues. High NPC1 expression was found to be significantly associated with poor prognosis in HCC patients (both OS and DFS), further suggesting that NPC1 plays a critical role in HCC (Fig. .

为了进一步研究NPC1在HCC中的预后价值，我们对HCC肿瘤和成对的非肿瘤肝组织中的NPC1进行了基于组织微阵列（TMA）的免疫组织化学（IHC）研究。发现高NPC1表达与HCC患者（OS和DFS）的预后不良显着相关，进一步表明NPC1在HCC中起关键作用（图）。

1j-m

1j-m型

and Supplementary Data

和补充数据

NPC1 promotes HCC progression

NPC1促进HCC进展

Evaluation of NPC1 function in HCC was performed both in vivo and in vitro. To investigate NPC1’s role in HCC progression, we engineered PLC/PRF/5 cells with stable overexpression of NPC1, and HepG2 and MHCC-97H cells with stable knockdown of NPC1 (Fig.

在体内和体外均进行了HCC中NPC1功能的评估。为了研究NPC1在HCC进展中的作用，我们设计了具有稳定过表达NPC1的PLC/PRF/5细胞，以及具有稳定敲低NPC1的HepG2和MHCC-97H细胞（图）。

2a级

and Supplementary Fig.

和补充图。

2a级

). Overexpression of NPC1 significantly increased the proliferation of PLC/PRF/5 cells (Supplementary Fig.

)。NPC1的过表达显着增加了PLC/PRF/5细胞的增殖（Supplementary Fig.）。

2b级

), while knockdown of NPC1 notably suppressed the proliferation of HepG2 and MHCC-97H cells (Supplementary Fig.

)。

2c, d

2c，d

). Additionally, enhanced NPC1 expression substantially increased the migration and invasion abilities of PLC/PRF/5 cells (Fig.

)。此外，增强的NPC1表达显着增加了PLC/PRF/5细胞的迁移和侵袭能力（图）。

2b, d

2b，d

), while silencing NPC1 significantly reduced these abilities (Fig.

)，而沉默NPC1显着降低了这些能力（图）。

2c, e

2c，e

and Supplementary Fig.

和补充图。

2e, f

2e，f

). To exclude potential off-target effects, we reintroduced NPC1 into NPC1-knockdown HepG2 and MHCC-97H cells (Fig.

)。为了排除潜在的脱靶效应，我们将NPC1重新引入NPC1敲低的HepG2和MHCC-97H细胞（图）。

2a级

and Supplementary Fig.

和补充图。

2a级

). Reintroduction of NPC1 almost completely restored the proliferation, migration, and invasion capacities in both cell lines (Fig.

)。NPC1的重新引入几乎完全恢复了两种细胞系的增殖、迁移和侵袭能力（图）。

2c, e

2c，e

and Supplementary Fig.

和补充图。

2c–f

). Notably, the slight reduction in proliferation rate (10%–15%, as shown in Supplementary Fig.

)。。

2g–i

) could not account for the nearly 70% decrease in migration ability observed in serum-free medium.

)不能解释在无血清培养基中观察到的迁移能力下降近70%。

Fig. 2: NPC1 promotes HCC progression.

图2:NPC1促进HCC进展。

一

Confirmation of NPC1 overexpression, NPC1 knockdown and re-expression in HCC cells.

HCC细胞中NPC1过表达，NPC1敲低和再表达的确认。

b类

-（笑声）

Transwell assay to examine the effect of NPC1 on HCC cell migration (

(

b类

c级

) or invasion (

)或入侵(

); scale bars, 100 μm.

)。

f级

–

我

1×10

Luciferase-expressing HCC cells (MHCC-97H) were injected into NOD SCID mice by tail vein. The mice were euthanized 8 weeks later by a cervical dislocation. Representative images of whole body luminescence monitoring of NOD SCID mice injected via tail vein with HCC cells 8 weeks after injection (

通过尾静脉将表达荧光素酶的HCC细胞（MHCC-97H）注射到NOD-SCID小鼠中。8周后，通过颈椎脱位对小鼠实施安乐死。注射后8周通过尾静脉注射HCC细胞的NOD-SCID小鼠全身发光监测的代表性图像(

f级

). Lung and liver tissues were isolated for analysis of IVIS imaging (

)。分离肺和肝组织用于IVIS成像分析(

克

). Representative H&E staining images of lung tissues are shown; scale bars, 500 μm; insets: fivefold magnification; scale bars, 100 μm (

)。；比例尺，500微米；插图：放大五倍；比例尺，100微米(

小时

). The incidence of lung metastasis in mice (

)。小鼠肺转移的发生率(

我

). (

= 8 mice per group). Data are presented as the mean ± s.e.m.

每组8只小鼠）。数据表示为平均值±标准误。

= 3 (

b类

–

) biologically independent samples (

)生物独立样本(

b类

–

). Statistical significance was determined by two-tailed unpaired Student’s t-test (

)。统计学显著性由两尾不成对学生t检验确定(

b类

–

一

–

Data were verified in three independent experiments. Source data are provided as a Source Data file.

数据在三个独立的实验中得到验证。源数据作为源数据文件提供。

Full size image

全尺寸图像

To further explore NPC1’s role in both tumor growth and metastasis, we utilized a subcutaneous tumor inoculation model alongside a mouse tail vein metastasis model. In the subcutaneous model, NPC1 knockdown led to a significant reduction in tumor size and weight, while reintroducing NPC1 effectively restored tumor growth (Supplementary Fig. .

为了进一步探索NPC1在肿瘤生长和转移中的作用，我们利用了皮下肿瘤接种模型和小鼠尾静脉转移模型。在皮下模型中，NPC1敲低导致肿瘤大小和重量显着减少，而重新引入NPC1可有效恢复肿瘤生长（补充图）。

2j-m

2j-m型

). In the tail vein metastasis model, NPC1 depletion resulted in a significant reduction in lung tumor metastases, further supporting NPC1’s critical role in tumor metastasis (Fig.

)。在尾静脉转移模型中，NPC1耗竭导致肺肿瘤转移显着减少，进一步支持NPC1在肿瘤转移中的关键作用（图）。

2f–i

). Collectively, these findings highlight NPC1 as a key promoter of HCC progression.

)。总的来说，这些发现突出了NPC1作为HCC进展的关键启动子。

NPC1 regulates the TGF-β pathway in a cholesterol transport-independent manner

NPC1以胆固醇转运非依赖性方式调节TGF-β途径

To investigate the mechanism through which NPC1 promotes HCC progression, we performed proteomic analysis on PLC/PRF/5 and HepG2 cells following NPC1 knockdown. Differentially expressed proteins were identified, and pathway enrichment analysis revealed that NPC1 knockdown significantly inhibited the TGF-β pathway in HCC cells (Supplementary Fig. .

为了研究NPC1促进HCC进展的机制，我们在NPC1敲低后对PLC/PRF/5和HepG2细胞进行了蛋白质组学分析。鉴定差异表达的蛋白质，途径富集分析显示NPC1敲低显着抑制HCC细胞中的TGF-β途径（补充图）。

3a, b

3a，b

). Notably, activation of the TGF-β pathway is a hallmark of S-III HCC, which is typically associated with a poor prognosis after first-line surgery

)。值得注意的是，TGF-β途径的激活是S-III型HCC的标志，这通常与一线手术后预后不良有关

. Additionally, the TGF-β pathway is closely linked to epithelial-mesenchymal transition and cancer cell invasion and metastasis

此外，TGF-β途径与上皮-间质转化和癌细胞侵袭和转移密切相关

We further validated the influence of NPC1 on TGF-β signaling in HCC cells. In cells overexpressing NPC1, there was a significant increase in the protein levels of TGFBR1, p-SMAD2 and p-SMAD3 (Fig.

我们进一步验证了NPC1对HCC细胞中TGF-β信号传导的影响。在过表达NPC1的细胞中，TGFBR1、p-SMAD2和p-SMAD3的蛋白水平显著增加（图）。

). Conversely, NPC1 knockdown led to a notable decrease in these protein levels (Fig.

)。相反，NPC1敲低导致这些蛋白质水平显着降低（图）。

3b级

and Supplementary Fig.

和补充图。

). Accordingly, NPC1 overexpression increased the mRNA levels of

)。因此，NPC1过表达增加了

MMP2

基质金属蛋白酶2

MMP9

MMP9公司

and

和

COL5A3

in PLC/PRF/5 cells (Fig.

在PLC/PRF/5细胞中（图）。

). In contrast, knockdown of NPC1 in HepG2 cells resulted in a downregulation of these genes (Fig.

)。相反，HepG2细胞中NPC1的敲低导致这些基因的下调（图）。

). Furthermore, high expression of these target genes was significantly associated with reduced OS in HCC patients, as evidenced by data from Jiang et al.’s cohort (Supplementary Fig.

)。此外，这些靶基因的高表达与HCC患者的OS降低显着相关，如Jiang等人的队列数据所证明的（Supplementary Fig.）。

3d–f

). Functionally, NPC1 knockdown reduced cell migration in HepG2 and MHCC-97H cells, independent of TGF-β1 treatment (Fig.

)。在功能上，NPC1敲低减少了HepG2和MHCC-97H细胞的细胞迁移，与TGF-β1处理无关（图）。

3e公司

and Supplementary Fig.

和补充图。

3小时

). In cells overexpressing NPC1, migration was significantly increased regardless of TGF-β1 stimulation, although TGF-β1 treatment in the control group induced higher migration compared to NPC1-overexpressing cells without TGF-β1 (Supplementary Fig.

)。在过表达NPC1的细胞中，无论TGF-β1刺激如何，迁移均显着增加，尽管与没有TGF-β1的NPC1过表达细胞相比，对照组中的TGF-β1处理诱导了更高的迁移（补充图）。

). These findings suggest that SMAD2/3 activation is a critical pathway regulated by NPC1 in promoting HCC metastasis.

)。这些发现表明SMAD2/3激活是NPC1在促进HCC转移中调节的关键途径。

Fig. 3: NPC1 regulates the TGF-β pathway in a cholesterol transport-independent manner.

图3:NPC1以胆固醇转运非依赖性方式调节TGF-β途径。

一

b类

Immunoblot analysis of TGFBR1, p-SMAD2, p-SMAD3, SMAD2, SMAD3 and NPC1 expression in PLC/PRF/5 cells with NPC1 stable overexpression (

NPC1稳定过表达的PLC/PRF/5细胞中TGFBR1，p-SMAD2，p-SMAD3，SMAD2，SMAD3和NPC1表达的免疫印迹分析(

一

) or HepG2 cells with NPC1 stable knockdown (

)或具有NPC1稳定敲低的HepG2细胞(

b类

c级

qPCR (

qPCR(

= 3 biological replicates) was used to examine the mRNA level of TGF-β target genes in NPC1-overexpression PLC/PRF/5 cells (

3个生物学重复）用于检测NPC1过表达PLC/PRF/5细胞中TGF-β靶基因的mRNA水平(

c级

) or in NPC1-knockdown HepG2 cells with/without further overexpression of NPC1 (

)(

Transwell assay was performed in NPC1-knockdown HepG2 cells with or without TGF-β1 (10 ng/mL) treatment; scale bars, 100 μm.

在有或没有TGF-β1（10ng/mL）处理的NPC1敲低的HepG2细胞中进行Transwell测定；比例尺，100微米。

f级

Immunoblot analysis of TGFBR1, p-SMAD2, SMAD2, and NPC1 expression in NPC1-knockdown HepG2 cells with further overexpression of NPC1 or NPC1 (P691S).

免疫印迹分析NPC1敲低的HepG2细胞中TGFBR1，p-SMAD2，SMAD2和NPC1的表达，并进一步过表达NPC1或NPC1（P691S）。

克

Cells related to (

与相关的单元格(

f级

) were fixed and stained with filipin to label free cholesterol accumulated in LE/Ly; scale bars, 10 μm.

)固定并用菲律宾血脂染色，以标记LE/Ly中积累的游离胆固醇；比例尺，10微米。

小时

Transwell assay was performed in cells related to (

Transwell分析是在与(

f级

); scale bars, 100 μm. Data are presented as the mean ± s.e.m.

)；比例尺，100微米。数据表示为平均值±标准误。

= 3 (

c级

小时

) biologically independent samples. Statistical significance was determined by two-tailed unpaired Student’s t-test (

)生物学上独立的样本。统计学显著性由两尾不成对学生t检验确定(

c级

小时

). All experimental data were verified in three independent experiments. Source data are provided as a Source Data file.

)。所有实验数据均在三个独立实验中得到验证。源数据作为源数据文件提供。

Full size image

全尺寸图像

Next, we explored whether NPC1’s regulation of the TGF-β pathway depends on its cholesterol transport function. To assess the cholesterol transport activity, we employed Filipin III staining, a fluorescent probe that binds specifically to unesterified cholesterol in fixed cells

接下来，我们探讨了NPC1对TGF-β途径的调节是否取决于其胆固醇转运功能。为了评估胆固醇的转运活性，我们采用了菲律宾III染色，这是一种荧光探针，与固定细胞中未酯化的胆固醇特异性结合

. As shown in Fig.

如图所示。

, NPC1 knockdown resulted in significant intracellular cholesterol accumulation. Re-expression of wild-type NPC1, which possesses intact cholesterol transport function, rescued this phenotype, reducing cholesterol accumulation to normal levels. However, the P691S mutant NPC1, which is known to lack cholesterol transport activity.

，NPC1敲低导致细胞内胆固醇大量积累。具有完整胆固醇转运功能的野生型NPC1的重新表达挽救了这种表型，将胆固醇积累降低到正常水平。但是，已知缺乏胆固醇转运活性的P691S突变体NPC1。

, did not reverse the cholesterol accumulation. Following reintroduction of NPC1 (wild-type or P691S mutant), we observed restored levels of TGFBR1 and p-SMAD2, as well as rescued cell migration capacity (Fig.

，并没有逆转胆固醇的积累。在重新引入NPC1（野生型或P691S突变体）后，我们观察到TGFBR1和p-SMAD2的水平恢复，以及挽救的细胞迁移能力（图）。

3f, h

3f，h

). We also explored the impact of cholesterol modulation on the TGF-β pathway using MβCD, a cyclic oligosaccharide commonly employed to deplete membrane cholesterol

)。我们还使用MβCD（一种通常用于消耗膜胆固醇的环状寡糖）探索了胆固醇调节对TGF-β途径的影响

. Our results showed that altering cholesterol levels with MβCD did not affect TGFBR1 expression or TGF-β pathway activity (Supplementary Fig.

我们的研究结果表明，用MβCD改变胆固醇水平不会影响TGFBR1的表达或TGF-β途径的活性（Supplementary Fig.）。

4a, b

4a，b

). Additionally, treatment with U18666A, a specific NPC1 cholesterol transport inhibitor

)。此外，用特定的NPC1胆固醇转运抑制剂U18666A治疗

did not affect TGFBR1 or p-SMAD2 levels at various concentrations (Supplementary Fig.

在不同浓度下不影响TGFBR1或p-SMAD2水平（Supplementary Fig.）。

4c, d

4c，d

). These results indicate that NPC1 regulates the TGF-β pathway in a manner independent of its cholesterol transport function.

)。这些结果表明，NPC1以独立于其胆固醇转运功能的方式调节TGF-β途径。

NPC1 increases protein stability of TGFBR1 and inhibits its ubiquitination

NPC1增加TGFBR1的蛋白质稳定性并抑制其泛素化

Subsequently, we observed that in PLC/PRF/5 cells with NPC1 overexpression, the protein levels of TGFBR1 were significantly elevated, while the corresponding mRNA levels remained unchanged (Figs.

随后，我们观察到在NPC1过表达的PLC/PRF/5细胞中，TGFBR1的蛋白水平显着升高，而相应的mRNA水平保持不变（图1和图2）。

a、，

4a级

). Similarly, in NPC1-knockdown HepG2 and PLC/PRF/5 cells, TGFBR1 protein levels were notably reduced, with no significant changes in mRNA expression (Figs.

)。。

b、，

4b级

and Supplementary Figs.

和补充图。

5a级

). Given that TGF-β receptors undergo constant internalization and recycling, independent of ligand presence

)。鉴于TGF-β受体经历不断的内在化和再循环，与配体的存在无关

, our experiments indicated that TGF-β stimulation did not affect the half-life of the TGFBR1 protein in PLC/PRF/5 and HepG2 cells (Supplementary Fig.

，我们的实验表明TGF-β刺激不影响PLC/PRF/5和HepG2细胞中TGFBR1蛋白的半衰期（Supplementary Fig.）。

5b, c

5b，c

). Cycloheximide (CHX) chase experiments demonstrated that NPC1 overexpression extended the half-life of TGFBR1 in PLC/PRF/5 cells (Fig.

)。环己酰亚胺（CHX）追踪实验表明，NPC1过表达延长了PLC/PRF/5细胞中TGFBR1的半衰期（图）。

4c级

), whereas NPC1 knockdown accelerated its degradation in HepG2 cells (Fig.

)，而NPC1敲低加速了其在HepG2细胞中的降解（图）。

4d级

), underscoring NPC1’s crucial role in stabilizing TGFBR1 protein levels. To elucidate the sustained dynamics of TGF-β signaling under NPC1 overexpression, we conducted time course experiments. In NPC1-overexpressing PLC/PRF/5 cells, SMAD2 phosphorylation was prolonged over a 24-hour period, whereas attenuation of the signal was observed in control cells (Supplementary Fig. .

)，强调了NPC1在稳定TGFBR1蛋白水平中的关键作用。为了阐明NPC1过表达下TGF-β信号传导的持续动力学，我们进行了时程实验。在过表达NPC1的PLC/PRF/5细胞中，SMAD2磷酸化在24小时内延长，而在对照细胞中观察到信号减弱（补充图）。

5e级

). This suggests that NPC1 overexpression prolongs TGF-β signaling, likely by impairing receptor downregulation.

)。这表明NPC1过表达可能通过损害受体下调来延长TGF-β信号传导。

Fig. 4: NPC1 increases protein stability of TGFBR1 and inhibits its ubiquitination.

图4:NPC1增加TGFBR1的蛋白质稳定性并抑制其泛素化。

一

b类

qPCR analysis of NPC1 and TGFBR1 mRNA levels in NPC1-overexpression PLC/PRF/5 (

NPC1过表达PLC/PRF/5中NPC1和TGFBR1 mRNA水平的qPCR分析(

一

) or NPC1-knockdown HepG2 (

)或NPC1敲低HepG2(

b类

) cells.

)细胞。

c级

PLC/PRF/5 cells with/without stable overexpression of NPC1 (

有/没有稳定过表达NPC1的PLC/PRF/5细胞(

c级

) or HepG2 cells with/without stable knockdown of NPC1 (

)或有/没有稳定敲除NPC1的HepG2细胞(

) were treated with CHX for indicated times and then analyzed by western blot.

)用CHX处理指定的时间，然后通过蛋白质印迹分析。

HepG2 cells with or without stable knockdown of NPC1 were treated with vehicle, MG132 (10 μM), or NH

用载体、MG132（10m）或NH处理有或没有稳定敲除NPC1的HepG2细胞

Cl (10 mM) for 12 hours. Cell lysates were subjected to immunoblot with TGFBR1 or NPC1 antibody.

Cl（10毫米）持续12小时。用TGFBR1或NPC1抗体对细胞裂解物进行免疫印迹。

f级

克

TGFBR1-mCherry-His stable overexpression PLC/PRF/5 (

TGFBR1-mCherry-His稳定过表达PLC/PRF/5(

f级

) and HepG2 (

)和HepG2(

克

) cells with or without NPC1 overexpression (

)有或没有NPC1过表达的细胞(

f级

) or knockdown (

)或击倒(

克

) were pretreated with MG132 (10 μM) for 8 hours before collection. Then TGFBR1-mCherry-His was pulled down by Ni-NTA and immunoblotted with anti-K48-Ubiquitin, anti-K63-Ubiquitin and anti-Ubiquitin antibody. Data are presented as the mean ± s.e.m.

)收集前用MG132（10μM）预处理8小时。然后用Ni-NTA下拉TGFBR1-mCherry-His，并用抗K48-泛素，抗K63-泛素和抗泛素抗体进行免疫印迹。数据表示为平均值±标准误。

= 3 (

一

–

) biologically independent samples. Statistical significance was determined by two-tailed unpaired Student’s t-test (

)生物学上独立的样本。统计学显著性由两尾不成对学生t检验确定(

一

b类

) or two-way analysis of variance (ANOVA) (

)或双向方差分析（ANOVA）(

c级

). All experimental data were verified in three independent experiments. Source data are provided as a Source Data file.

)。所有实验数据均在三个独立实验中得到验证。源数据作为源数据文件提供。

Full size image

全尺寸图像

To further investigate the mechanism by which NPC1 regulates TGFBR1 degradation, we examined whether it was mediated by the proteasome or lysosome pathways. Treatment with the proteasome inhibitor MG132 led to a significant increase in TGFBR1 protein levels, while no such effect was observed with the lysosomal inhibitor NH.

为了进一步研究NPC1调节TGFBR1降解的机制，我们检查了它是由蛋白酶体还是溶酶体途径介导的。用蛋白酶体抑制剂MG132处理导致TGFBR1蛋白水平显着增加，而溶酶体抑制剂NH未观察到这种作用。

Cl in HepG2 and PLC/PRF/5 cells with stable NPC1 knockdown (Fig.

具有稳定NPC1敲低的HepG2和PLC/PRF/5细胞中的Cl（图）。

4e级

and Supplementary Fig.

和补充图。

5天

). Quantitative analyses of these immunoblots confirmed that proteasomal degradation is the predominant pathway for TGFBR1 degradation in the context of NPC1 knockdown (Fig.

)。这些免疫印迹的定量分析证实，在NPC1敲低的情况下，蛋白酶体降解是TGFBR1降解的主要途径（图）。

4e级

and Supplementary Fig.

和补充图。

5天

). These results suggest that NPC1 promotes TGF-β signaling by preventing proteasome-mediated degradation of TGFBR1.

)。这些结果表明NPC1通过阻止蛋白酶体介导的TGFBR1降解来促进TGF-β信号传导。

Furthermore, we examined the ubiquitination of TGFBR1. In PLC/PRF/5 cells, NPC1 overexpression significantly reduced overall TGFBR1 polyubiquitination, whereas NPC1 knockdown in HepG2 cells had the opposite effect (Fig.

此外，我们检查了TGFBR1的泛素化。在PLC/PRF/5细胞中，NPC1过表达显着降低了总体TGFBR1多聚泛素化，而HepG2细胞中的NPC1敲低具有相反的作用（图）。

4f, g

4f，g

). Specifically, NPC1 decreased Lys 48-linked polyubiquitination, which is associated with proteasomal degradation, while Lys 63-linked polyubiquitination, typically involved in non-proteolytic functions, was unaffected (Fig.

)。具体而言，NPC1降低了与蛋白酶体降解相关的Lys 48连接的多聚泛素化，而通常参与非蛋白水解功能的Lys 63连接的多聚泛素化不受影响（图）。

4f, g

4f，g

). These data indicate that NPC1 stabilizes TGFBR1 by reducing its proteasomal degradation through inhibition of Lys 48-linked ubiquitination.

)。这些数据表明，NPC1通过抑制Lys 48连接的泛素化来减少其蛋白酶体降解，从而稳定TGFBR1。

NPC1 interacts with TGFBR1 and inhibits the binding of TGFBR1 with SMAD7/SMURFs

NPC1与TGFBR1相互作用并抑制TGFBR1与SMAD7/SMURFs的结合

We explored the potential interaction between NPC1 and TGFBR1 through co-immunoprecipitation (Co-IP) assays, which confirmed that ectopic NPC1 binds to TGFBR1 in PLC/PRF/5 and HEK-293T cells (Fig.

我们通过免疫共沉淀（co-IP）分析探讨了NPC1和TGFBR1之间的潜在相互作用，证实了异位NPC1与PLC/PRF/5和HEK-293T细胞中的TGFBR1结合（图）。

5a级

and Supplementary Fig.

和补充图。

). This interaction was further validated endogenously in PLC/PRF/5 and HepG2 cells (Fig.

)。这种相互作用在PLC/PRF/5和HepG2细胞中得到了进一步的内源性验证（图）。

5b条

). To gain insights into the subcellular localization of the TGFBR1-NPC1 complex, we examined their co-localization patterns. Although prior studies reported that TGFBR1 undergoes sustained internalization and localizes to various cytoplasmic vesicles, including compartments marked by LAMP1 or caveolin-1.

)。为了深入了解TGFBR1-NPC1复合物的亚细胞定位，我们检查了它们的共定位模式。尽管先前的研究报道TGFBR1经历持续的内在化并定位于各种细胞质囊泡，包括由LAMP1或caveolin-1标记的区室。

, our results revealed that the TGFBR1-NPC1 complex predominantly colocalizes with the lysosomal marker LAMP1 rather than with caveolin-1 (Fig.

，我们的结果显示TGFBR1-NPC1复合物主要与溶酶体标记LAMP1共定位，而不是与小窝蛋白-1共定位（图）。

5摄氏度

, Supplementary Fig.

，补充图。

6b-f

). This suggests that, within HCC cells, the TGFBR1-NPC1 complex predominantly resides in lysosomes. Notably, neither overexpression nor knockdown of NPC1 altered the lysosomal localization of TGFBR1 (Fig.

)。这表明，在HCC细胞内，TGFBR1-NPC1复合物主要存在于溶酶体中。。

5摄氏度

, Supplementary Fig.

，补充图。

6h, j, b, g, i

Fig. 5: NPC1 interacts with TGFBR1 and inhibits the binding of TGFBR1 with SMAD7/SMURFs.

图5:NPC1与TGFBR1相互作用并抑制TGFBR1与SMAD7/SMURFs的结合。

一

The lysates of PLC/PRF/5 transfected with indicated constructs were subjected to immunoprecipitation with anti-Flag (or GFP) antibody. The immunoprecipitates were then immunoblotted with anti-GFP (or Flag) antibody.

用指定的构建体转染的PLC/PRF/5裂解物用抗Flag（或GFP）抗体进行免疫沉淀。然后用抗GFP（或Flag）抗体对免疫沉淀物进行免疫印迹。

b类

PLC/PRF/5 and HepG2 cell lysates were subjected to immunoprecipitation with control IgG or anti-NPC1 antibodies.

用对照IgG或抗NPC1抗体对PLC/PRF/5和HepG2细胞裂解物进行免疫沉淀。

c级

PLC/PRF/5 cells stably overexpressing TGFBR1-mCherry-His and NPC1-HA were immunostained with antibodies against HA and LAMP1 to determine the colocalization among TGFBR1, NPC1 and LAMP1 in PLC/PRF/5 cells. Representative images from three independent experiments are shown; scale bars, 10 µm.

用抗HA和LAMP1的抗体对稳定过表达TGFBR1-mCherry-His和NPC1-HA的PLC/PRF/5细胞进行免疫染色，以确定PLC/PRF/5细胞中TGFBR1，NPC1和LAMP1之间的共定位。显示了来自三个独立实验的代表性图像；比例尺，10µm。

A schematic representation of NPC1 (

NPC1的示意图(

) or TGFBR1 (

)或TGFBR1(

) WT and deletion mutants.

)WT和缺失突变体。

f级

PLC/PRF/5 cells stably overexpressing TGFBR1-mCherry-His were transfected with various plasmids encoding NPC1-His-Flag or NPC1 deletion mutants as indicated. Cell lysates were subjected to immunoprecipitation with anti-Flag magnetic beads and immunoblot with mCherry or Flag antibody.

如所示，用编码NPC1-His-Flag或NPC1缺失突变体的各种质粒转染稳定过表达TGFBR1-mCherry-His的PLC/PRF/5细胞。用抗Flag磁珠对细胞裂解物进行免疫沉淀，并用mCherry或Flag抗体进行免疫印迹。

克

PLC/PRF/5 cells were transfected with various combinations of plasmids encoding NPC1-His-Flag and TGFBR1-Myc or TGFBR1 deletion mutants as indicated. Cell lysates were subjected to immunoprecipitation with anti-Myc magnetic beads and immunoblot with Flag or Myc antibody.

如图所示，用编码NPC1-His-Flag和TGFBR1-Myc或TGFBR1缺失突变体的质粒的各种组合转染PLC/PRF/5细胞。用抗Myc磁珠对细胞裂解物进行免疫沉淀，并用Flag或Myc抗体进行免疫印迹。

小时

Immunoblot analysis of TGFBR1 and NPC1 expression in NPC1-knockdown PLC/PRF/5 cells with further overexpression of NPC1, NPC1 (P691S), or NPC1 (Δ692-854).

。

我

Transwell assay was performed in cells related to (

Transwell分析是在与(

小时

); scale bars, 100 μm.

)。

TGFBR1-mCherry-His stable overexpression PLC/PRF/5 (

TGFBR1-mCherry-His稳定过表达PLC/PRF/5(

) and HepG2 (

)和HepG2(

) cells with or without NPC1 overexpression (

)有或没有NPC1过表达的细胞(

) or knockdown (

)或击倒(

) were subjected to immunoprecipitation with anti-His antibody. The lysates and immunoprecipitates were then blotted. Data are presented as the mean ± s.e.m.

)用抗His抗体进行免疫沉淀。然后印迹裂解物和免疫沉淀物。数据表示为平均值±标准误。

= 3 (

我

) biologically independent samples. Statistical significance was determined by two-tailed unpaired Student’s t-test (

)生物学上独立的样本。统计学显著性由两尾不成对学生t检验确定(

我

). All experimental data were verified in three independent experiments. Source data are provided as a Source Data file.

)。所有实验数据均在三个独立实验中得到验证。源数据作为源数据文件提供。

Full size image

全尺寸图像

Further mapping of the NPC1-TGFBR1 interaction domain revealed that the amino acid (aa) 692-854 region of NPC1 is responsible for binding to TGFBR1 (Fig.

NPC1-TGFBR1相互作用域的进一步定位表明，NPC1的氨基酸（aa）692-854区负责与TGFBR1结合（图）。

5d, f

5d，f

). The interaction surface of TGFBR1 with NPC1 was mapped to its transmembrane domain (Fig.

)。TGFBR1与NPC1的相互作用表面被定位到其跨膜结构域（图）。

5e, g

5e，g

). Additionally, the NPC1 (P691S) mutant retained its ability to bind TGFBR1 (Supplementary Fig.

)。此外，NPC1（P691S）突变体保留了其结合TGFBR1的能力（Supplementary Fig.）。

6百万

). Transwell assays were employed to examine the functional role of NPC1 truncations and mutants. In PLC/PRF/5 cells with stable NPC1 knockdown, reintroduction of either wild-type NPC1 or NPC1 (P691S) restored TGFBR1 expression and cell migration, whereas reintroduction of NPC1 (Δ692-854) failed to rescue these phenotypes (Fig. .

)。。在具有稳定NPC1敲低的PLC/PRF/5细胞中，野生型NPC1或NPC1（P691S）的重新引入恢复了TGFBR1的表达和细胞迁移，而NPC1（Δ692-854）的重新引入未能挽救这些表型（图。

5h, i

5小时，我

). Moreover, ectopic expression of wild-type NPC1 and NPC1 (P691S) significantly promoted cell migration, while NPC1 (Δ616-854) had no effect (Supplementary Fig.

)。此外，野生型NPC1和NPC1（P691S）的异位表达显着促进细胞迁移，而NPC1（Δ616-854）没有作用（补充图）。

6升

), aligning with the observed effects of these mutants on TGFBR1 stability (Supplementary Fig.

)，与观察到的这些突变体对TGFBR1稳定性的影响一致（Supplementary Fig.）。

6千

). These results suggest that the aa 692-854 region of NPC1 is essential for its role in promoting TGFBR1 stability and cell migration.

)。这些结果表明，NPC1的氨基酸692-854区对于其促进TGFBR1稳定性和细胞迁移的作用至关重要。

Given that SMAD7 recruits the E3 ubiquitin ligases SMURF1 and SMURF2 to TGFBR1, facilitating its ubiquitination and degradation

鉴于SMAD7将E3泛素连接酶SMURF1和SMURF2募集到TGFBR1，促进其泛素化和降解

, we hypothesized that NPC1 might suppress TGFBR1 ubiquitination by modulating its interaction with SMAD7 or SMURFs. Therefore, we speculated that NPC1 might suppress the ubiquitylation of TGFBR1 via modulating its interaction with SMAD7 or E3 enzymes. Co-IP assays revealed that NPC1 overexpression reduced the interaction between TGFBR1 and SMAD7, as well as with the E3 ligases SMURF1 and SMURF2, in PLC/PRF/5 cells (Fig. .

，我们假设NPC1可能通过调节其与SMAD7或SMURFs的相互作用来抑制TGFBR1的泛素化。因此，我们推测NPC1可能通过调节其与SMAD7或E3酶的相互作用来抑制TGFBR1的泛素化。Co-IP分析显示，NPC1过表达降低了PLC/PRF/5细胞中TGFBR1和SMAD7之间以及与E3连接酶SMURF1和SMURF2之间的相互作用（图）。

5j型

). Conversely, NPC1 knockdown in HepG2 cells enhanced these interactions (Fig.

)。相反，HepG2细胞中的NPC1敲低增强了这些相互作用（图）。

5公里

). Supporting these findings, colocalization studies of SMAD7-EGFP and TGFBR1-mCherry (Supplementary Fig.

)。支持这些发现，SMAD7-EGFP和TGFBR1-mCherry的共定位研究（Supplementary Fig.）。

7a, b

7a，b

) showed that in NPC1 knockdown cells, cytoplasmic localization of SMAD7-EGFP increased significantly and colocalized with TGFBR1-mCherry. Further analysis was conducted to determine whether NPC1 stabilizes TGFBR1 in a SMAD7-dependent manner. Knockdown of SMAD7 or inhibition of SMURF1/2 using siRNA rescued TGFBR1 protein levels in HCC cells with stable NPC1 knockdown (Supplementary Fig. .

)结果表明，在NPC1敲低细胞中，SMAD7-EGFP的细胞质定位显着增加，并与TGFBR1-mCherry共定位。。使用siRNA敲低SMAD7或抑制SMURF1/2可以挽救具有稳定NPC1敲低的HCC细胞中的TGFBR1蛋白水平（补充图）。

7c, d

). These results imply that TGFBR1 degradation in NPC1 knockdown cells is driven, at least in part, by the increased recruitment of SMAD7 and SMURFs to TGFBR1. Collectively, these findings suggest that NPC1 protects TGFBR1 from proteasomal degradation by interacting with it and inhibiting its binding with SMAD7 and SMURFs..

)。这些结果意味着NPC1敲低细胞中TGFBR1的降解至少部分是由SMAD7和SMURFs向TGFBR1的募集增加所驱动的。总的来说，这些发现表明NPC1通过与TGFBR1相互作用并抑制其与SMAD7和SMURFs的结合来保护TGFBR1免受蛋白酶体降解。。

TGFBR1 is Crucial for NPC1-Mediated Promotion of HCC Progression

TGFBR1对于NPC1介导的HCC进展的促进至关重要

We examined the correlation between NPC1 and TGFBR1 protein levels in 286 pairs of human HCC tissue samples using immunohistochemistry (IHC). Our analysis revealed a significant positive correlation between NPC1 and TGFBR1 expression (

我们使用免疫组织化学（IHC）检查了286对人类HCC组织样本中NPC1和TGFBR1蛋白水平之间的相关性。我们的分析显示NPC1和TGFBR1表达之间存在显着的正相关(

< 0.0001, R

<0.0001，R

= 0.5028) (Fig.

=0.5028）（图。

6a–c

). To further explore this relationship, we performed immunofluorescence staining on HCC tissues with antibodies specific to NPC1 and TGFBR1. The results confirmed that high NPC1 expression is positively correlated with TGFBR1 levels in these tissues (Fig.

)。为了进一步探索这种关系，我们用NPC1和TGFBR1特异性抗体对HCC组织进行了免疫荧光染色。结果证实，高NPC1表达与这些组织中TGFBR1水平呈正相关（图）。

6d–f

Fig. 6: TGFBR1 is crucial for NPC1-mediated promotion of HCC progression.

。

一

Representative images from IHC staining of NPC1 and TGFBR1 in HCC tissues (

HCC组织中NPC1和TGFBR1的IHC染色的代表性图像(

= 286 biologically independent samples); scale bars, 50 µm.

=286个生物学上独立的样本）；比例尺，50微米。

b类

The Pearson correlation analysis between NPC1 level and TGFBR1 level in HCC tissues.

HCC组织中NPC1水平与TGFBR1水平之间的Pearson相关分析。

c级

The analysis of TGFBR1 IHC score in HCC tissues with low (

(

= 174 biologically independent samples) or high (

174个生物独立样本）或高(

= 112 biologically independent samples) NPC1 level.

=112个生物学独立样本）NPC1水平。

Representative images from Immunofluorescence staining of NPC1 and TGFBR1 in HCC tissues (

肝癌组织中NPC1和TGFBR1免疫荧光染色的代表性图像(

= 10 biologically independent samples); scale bars, 20 µm.

=10个生物学上独立的样本）；比例尺，20µm。

The Pearson correlation analysis between NPC1 positive area and TGFBR1 positive area in HCC tissues (

肝癌组织中NPC1阳性区域与TGFBR1阳性区域的Pearson相关分析(

= 50 regions). These multiplexed IF staining were performed on ten HCC tissue sections from HCC patients, qualifying an average of five regions per sample.

=50个地区）。这些多重IF染色是在来自HCC患者的十个HCC组织切片上进行的，每个样品平均有五个区域。

f级

The analysis of the percentages of TGFBR1 positive area in HCC tissues with low (

肝癌组织中TGFBR1阳性区域百分比分析(

= 13 regions) or high (

=13个地区）或高(

= 37 regions) NPC1 level.

=37个地区）NPC1水平。

克

Immunoblot analysis of TGFBR1 and NPC1 expression in NPC1-overexpression PLC/PRF/5 cells with or without TGFBR1 inhibitor LY2157299 (10 μM) treatment.

在有或没有TGFBR1抑制剂LY2157299（10μM）处理的NPC1过表达PLC/PRF/5细胞中TGFBR1和NPC1表达的免疫印迹分析。

小时

Transwell assay was performed in cells related to (

Transwell分析是在与(

克

); scale bars, 100 μm.

)。

我

-（笑声）

1×10

我

). Lung and liver tissues were isolated for analysis of IVIS imaging (

)。分离肺和肝组织用于IVIS成像分析(

). Representative H&E staining images of lung tissues are shown; scale bars, 500 μm; insets: fivefold magnification; scale bars, 100 μm (

)。；比例尺，500微米；插图：放大五倍；比例尺，100微米(

). The incidence of lung metastasis in mice (

)。小鼠肺转移的发生率(

) (

= 6 mice per group). Data are presented as the mean ± s.e.m.

每组6只小鼠）。数据表示为平均值±标准误。

= 3 (

小时

) biologically independent samples. In the box plots, the middle bar represents the median, and the box represents the interquartile range; bars extend to 1.5× the interquartile range. Statistical significance was determined by two-sided Pearson correlation test (

)生物学上独立的样本。在箱形图中，中间条表示中位数，方框表示四分位间距；酒吧延伸到四分位间距的1.5倍。统计学显着性由双侧Pearson相关检验确定(

b类

), two-sided Mann–Whitney U test (

双侧曼-惠特尼U检验

c级

f级

) or two-tailed unpaired Student’s t-test (

)或两尾不成对学生t检验(

小时

克

小时

Data were verified in three independent experiments. Source data are provided as a Source Data file.

数据在三个独立的实验中得到验证。源数据作为源数据文件提供。

Full size image

全尺寸图像

To explore whether NPC1 promotes HCC progression via TGFBR1 activation, we treated NPC1-overexpressing PLC/PRF/5 cells with LY2157299 (Galunisertib), a clinical inhibitor of TGFBR1 activation that has been investigated in clinical trials involving HCC patients

为了探讨NPC1是否通过TGFBR1激活促进HCC进展，我们用LY2157299（Galunisertib）治疗了过表达NPC1的PLC/PRF/5细胞，LY2157299是一种TGFBR1激活的临床抑制剂，已在涉及HCC患者的临床试验中进行了研究

. Treatment with LY2157299 significantly attenuated the NPC1-induced enhancement of cell migration (Supplementary Fig.

用LY2157299处理显着减弱了NPC1诱导的细胞迁移增强（Supplementary Fig.）。

8a, b

8a，b

and Fig.

和图。

6g, h

6克，小时

). Additionally, in MHCC-97H cells with NPC1 knockdown and stable overexpression of TGFBR1 (Supplementary Fig.

)。此外，在具有NPC1敲低和TGFBR1稳定过表达的MHCC-97H细胞中（Supplementary Fig.）。

8摄氏度

), we observed that TGFBR1 overexpression rescued the metastatic potential of NPC1-knockdown cells in a mouse tail vein metastasis model (Fig.

)，我们观察到TGFBR1过表达在小鼠尾静脉转移模型中挽救了NPC1敲低细胞的转移潜能（图）。

6i–l

Next, we explored the interaction between NPC1 and TGFBR1 by knocking down TGFBR1 in NPC1-overexpressing PLC/PRF/5 cells. This knockdown significantly inhibited cell migration, though it did not affect cell proliferation or tumor growth in xenograft models (Supplementary Fig.

接下来，我们通过在过表达NPC1的PLC/PRF/5细胞中敲低TGFBR1来探索NPC1和TGFBR1之间的相互作用。这种敲低显着抑制细胞迁移，尽管它不影响异种移植模型中的细胞增殖或肿瘤生长（Supplementary Fig.）。

8d–i

), indicating that NPC1-driven migration depends on TGFBR1. We also found that knocking down either NPC1 or TGFBR1 reduced migration in MHCC-97H cells (Supplementary Fig.

)，表明NPC1驱动的迁移取决于TGFBR1。我们还发现敲除NPC1或TGFBR1可减少MHCC-97H细胞的迁移（Supplementary Fig.）。

8k个

). In NPC1-knockdown cells, reintroducing wild-type NPC1 or the P691S mutant rescued the migratory phenotype, while reintroducing the NPC1 (Δ692-854) mutant failed to restore it (Supplementary Fig.

)。在NPC1敲低细胞中，重新引入野生型NPC1或P691S突变体挽救了迁移表型，而重新引入NPC1（Δ692-854）突变体未能恢复它（补充图）。

8j, k

8j，k

). These phenotypes were also validated in a mouse metastasis model, where knockdowns of both NPC1 and TGFBR1 reduced metastatic potential (Supplementary Fig.

)。这些表型也在小鼠转移模型中得到验证，其中NPC1和TGFBR1的敲低降低了转移潜能（Supplementary Fig.）。

8l, m

8升，米

), underscoring the critical roles of these proteins in HCC progression.

)，强调了这些蛋白质在HCC进展中的关键作用。

Hepatic NPC1 deficiency suppresses TGF-β signaling and limits HCC progression

肝脏NPC1缺乏抑制TGF-β信号传导并限制HCC进展

To explore NPC1’s role in regulating TGF-β pathway and HCC progression in vivo, we utilized CRISPR-Cas9 system to generate liver-specific conditional

Npc1

knockout (

淘汰赛(

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

) and control mice (

)和对照小鼠(

Npc1

F/F

前/后

) (Fig.

)（图。

7a个

) for analysis in the DEN-CCl

)用于DEN CCl中的分析

induced HCC model (Fig.

诱导HCC模型（图）。

7b条

). This model is initiated by a single injection of the carcinogen diethylnitrosamine (DEN) followed by repeated administration of carbon tetrachloride (CCl

)。该模型是通过单次注射致癌物二乙基亚硝胺（DEN），然后重复施用四氯化碳（CCl）来启动的

). As expected,

)。正如预期的那样，

Npc1

mRNA and protein levels were drastically reduced in the livers of

肝脏中的mRNA和蛋白质水平急剧降低

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice (Supplementary Fig.

小鼠（补充图）。

9a, b

9a，b

). Remarkably, while control mice developed large liver tumors,

)。值得注意的是，虽然对照组小鼠出现了较大的肝肿瘤，

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice exhibited significantly smaller liver sizes and liver-to-body weight ratios at 24 weeks post-DEN injection (Fig.

DEN注射后24周，小鼠肝脏大小和肝体重比明显减小（图）。

7c, d

7c、d

and Supplementary Fig.

和补充图。

9摄氏度

). These mice also showed fewer and smaller tumors (Fig.

)。这些小鼠也显示出越来越小的肿瘤（图）。

7e, f

7e，f

and Supplementary Fig.

和补充图。

9天

). Histological analysis demonstrated preserved reticulin structure in

)。组织学分析表明保留了网状结构

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

tumors, in contrast to the disrupted reticulin patterns seen in control mice (Supplementary Fig.

肿瘤，与对照小鼠中观察到的网状蛋白模式破坏相反（Supplementary Fig.）。

9e级

). Liver injury markers-serum ALT, AST, and TBIL-were markedly reduced in

)。肝损伤标志物血清ALT，AST和TBIL显着降低

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice (Supplementary Fig.

小鼠（补充图）。

9h–j

), demonstrating ameliorated liver damage. Further IHC staining showed that

)，表明肝损伤有所改善。进一步的IHC染色显示

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

tumors had lower percentages of Ki67-positive cells (Supplementary Fig.

肿瘤Ki67阳性细胞百分比较低（Supplementary Fig.）。

9f, g

9f，g

), indicating reduced tumor cell proliferation. Tumors also displayed diminished staining for EpCAM and cytokeratin 19 (Fig.

)，表明肿瘤细胞增殖减少。肿瘤也显示EpCAM和细胞角蛋白19的染色减少（图）。

7克

), both markers of poor prognosis and invasiveness in human HCC

)，都是人类肝癌预后不良和侵袭性的标志

. GRP78, a marker of aggressive disease

GRP78，侵袭性疾病的标志

, was similarly reduced in

，在

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice (Fig.

小鼠（图）。

7克

). Importantly,

)。重要的是，

Npc1

knockout resulted in decreased expression of TGFBR1 protein and its downstream effector p-SMAD2 (Fig.

敲除导致TGFBR1蛋白及其下游效应子p-SMAD2的表达降低（图）。

7小时

), reinforcing NPC1’s role in regulating TGF-β signaling. Survival was significantly longer in

)，增强了NPC1在调节TGF-β信号传导中的作用。存活时间明显延长

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice compared to controls (Fig.

小鼠与对照组相比（图）。

7i个

Fig. 7: Hepatic NPC1 deficiency inhibits HCC tumorigenesis and progression and TGF-β pathway.

图7：肝脏NPC1缺陷抑制HCC肿瘤发生和发展以及TGF-β途径。

一

Construction of

建造

Npc1

-conditional knockout (

-条件淘汰赛(

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

) mice.

)老鼠。

b类

Npc1

F/F

前/后

and

和

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice were treated with DEN followed by twenty injections of CCl

用DEN治疗小鼠，然后注射20次CCl

to construct HCC mouse model.

。

c级

Representative images of

具有代表性的图像

Npc1

F/F

前/后

and

和

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mouse livers with HCC.

HCC小鼠肝脏。

liver to body weight ratio (

肝脏与体重比(

= 8 mice) (

8只小鼠

) numbers of nodules per liver (

)每个肝脏的结节数(

= 21 mice in

=21只小鼠

Npc1

F/F

前/后

and

和

= 13 mice in

=13只小鼠

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

groups) (

组）(

) of the indicated mice.

)指定的小鼠。

f, g

f、克

Representative H&E staining images (

代表性的H＆E染色图像(

f级

), Representative IHC staining images of EpCAM, GPR78 and Cytokeratin19 (

)，EpCAM，GPR78和细胞角蛋白19的代表性IHC染色图像(

克

) of the indicated mouse livers. H&E staining scale bars: left panels, 2.5 mm; right panels, 500 μm. IHC staining scale bars, 50 μm.

)指定的小鼠肝脏。H＆E染色比例尺：左图，2.5毫米；右侧面板，500微米。IHC染色比例尺，50μm。

小时

TGFBR1, p-SMAD2, SMAD2, and NPC1 protein expression in the indicated mouse non-tumor and tumor tissues.

TGFBR1，p-SMAD2，SMAD2和NPC1蛋白在指定的小鼠非肿瘤和肿瘤组织中的表达。

我

Kaplan–Meier survival curves for

Kaplan–Meier生存曲线

Npc1

F/F

前/后

(

= 35) or

=35）或

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

(

= 23).

== 23).

Scheme used to establish the model of spontaneous HCC with targeted

用于建立具有靶向性的自发性HCC模型的方案

Myc

Myc公司

knock-in and

敲入和

Npc1

knockout in the liver.

肝脏中的基因敲除。

Representative images of

具有代表性的图像

Cre

Cre公司

Alb

Alb公司

Myc

Myc公司

and

和

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

Myc

Myc公司

mouse livers with HCC.

HCC小鼠肝脏。

liver to body weight ratio (

肝脏与体重比(

= 5) in the indicated mice.

=5）在指定的小鼠中。

m, n

m、 n

Representative H&E staining images (

代表性的H＆E染色图像(

), Representative IHC staining images of EpCAM, GPR78 and Cytokeratin19 (

)，EpCAM，GPR78和细胞角蛋白19的代表性IHC染色图像(

) of the indicated mouse livers. H&E staining scale bars: left panels, 2.5 mm; right panels, 500 μm. IHC staining scale bars, 50 μm.

)指定的小鼠肝脏。H＆E染色比例尺：左图，2.5毫米；右侧面板，500微米。IHC染色比例尺，50μm。

TGFBR1, p-SMAD2, SMAD2, and NPC1 protein expression in

TGFBR1，p-SMAD2，SMAD2和NPC1蛋白在

WT, Cre

重量，Cre

Alb

Alb公司

Myc

Myc公司

and

和

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

Myc

Myc公司

mouse liver tissues.

小鼠肝组织。

Schematic diagram of tamoxifen-induced liver-specific

他莫昔芬诱导的肝脏特异性示意图

Npc1

knockout mouse generation and the treatment plan in the DEN-CCl

基因敲除小鼠的产生和DEN CCl的治疗计划

-induced HCC model.

-诱导HCC模型。

Representative liver images of the indicated group.

所示组的代表性肝脏图像。

r, s

r、 s

liver to body weight ratio (

肝脏与体重比(

), numbers of nodules per liver (

)，每个肝脏的结节数(

) of

)的

Npc1

F/F

前/后

mice treated with coil (CO) (

用线圈（CO）治疗的小鼠(

= 5),

CreERT2

Alb

Alb公司

Npc1

F/F

前/后

mice treated with coil or tamoxifen (TAM) (

用线圈或他莫昔芬（TAM）治疗的小鼠(

= 8).

Representative H&E staining images of the indicated mouse livers. Scale bars: left panels, 2.5 mm; right panels, 500 μm.

所示小鼠肝脏的代表性H＆E染色图像。比例尺：左侧面板，2.5毫米；右侧面板，500微米。

NPC1 protein expression in the indicated mouse tissues. Data are presented as means ± s.e.m. Statistical significance was determined by two-tailed unpaired Student’s t-test (

NPC1蛋白在指定的小鼠组织中的表达。数据以平均值±标准误表示。统计显着性由两尾不成对学生t检验确定(

d, e, l, r, s

d、 e、l、r、s

) or log-rank test (

)或对数秩检验(

我

). These experiments (

)。这些实验(

f级

克

小时

–

) were repeated three times independently with similar results. Source data are provided as a Source Data file.

)独立重复三次，结果相似。源数据作为源数据文件提供。

Full size image

全尺寸图像

We extended these observations to a MYC-driven HCC model by crossing

我们通过交叉将这些观察结果扩展到MYC驱动的HCC模型

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice with

小鼠

H11-CAG-LSL-Myc

H11 CAG LSL Myc

mice to generate homozygous

小鼠产生纯合子

Npc1

knockout mice (

基因敲除小鼠(

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

Myc

Myc公司

) (Fig.

)（图。

7j型

). In this model,

)。在这个模型中，

Npc1

knockout significantly reduced tumor sizes and weights (Fig.

敲除显着减少肿瘤大小和重量（图）。

7k–m

and Supplementary Fig.

和补充图。

9千

). Consistent with the DEN-CCl

)。与DEN CCl一致

model, hepatic

模型，肝

Npc1

knockout inhibited TGF-β signaling and tumor progression in the MYC-driven HCC model (Fig.

敲除抑制MYC驱动的HCC模型中的TGF-β信号传导和肿瘤进展（图）。

7牛

, o and Supplementary Fig.

，o和补充图。

9l–q

Using an inducible

使用诱导型

Npc1

knockout model with tamoxifen (TAM)-inducible Cre recombinase (ERT2-Cre) in DEN-CCl

DEN CCl中他莫昔芬（TAM）诱导的Cre重组酶（ERT2-Cre）的基因敲除模型

-induced HCC model, we confirmed that

-诱导HCC模型，我们证实

Npc1

deficiency for an eight-week period significantly reduced tumor burden, tumor number, and liver-to-body weight ratios (Fig.

八周的缺乏显着降低了肿瘤负荷，肿瘤数量和肝体重比（图）。

7p–s

and Supplementary Fig.

和补充图。

9r, s

9r，s

). Histological analysis further confirmed tumor regression in the

)。组织学分析进一步证实了肿瘤消退

Npc1

-knockout mice (Fig.

-敲除小鼠（图）。

7吨

). In addition, therapeutic targeting of NPC1 in MYC-driven HCC using AAV8-shNPC1 significantly reduced tumor size and number (Supplementary Fig.

)。此外，使用AAV8-shNPC1在MYC驱动的HCC中治疗靶向NPC1显着降低了肿瘤的大小和数量（Supplementary Fig.）。

9t–x

), with knockdown efficiency validated by Western blotting (Supplementary Fig.

)，通过蛋白质印迹验证了击倒效率（Supplementary Fig.）。

9年

). These findings establish NPC1 as a potential therapeutic target in HCC. Collectively, these data provide strong evidence supporting the vital role of NPC1 in regulation the TGF-β pathway and promoting HCC progression in vivo.

)。这些发现将NPC1确立为HCC的潜在治疗靶点。总的来说，这些数据提供了强有力的证据支持NPC1在调节TGF-β途径和促进体内HCC进展中的重要作用。

Discussion

讨论

NPC1 is a late endosomal/lysosome membrane protein involved in the transport of low-density lipoprotein-derived cholesterol into cells and virus entry, such as with Ebola

NPC1是一种晚期内体/溶酶体膜蛋白，参与低密度脂蛋白衍生的胆固醇向细胞的转运和病毒进入，如埃博拉病毒

. Mutations in NPC1 cause Niemann-Pick type C disease, a rare lipid storage disorder

NPC1突变导致尼曼-皮克C型疾病，这是一种罕见的脂质贮积病

. Despite its well-characterized function in cholesterol metabolism, research into NPC1’s role in cancer is limited. Only a few reports suggest its upregulation in some cancers, where it may contribute to proliferation and invasion

尽管NPC1在胆固醇代谢中具有良好的功能，但对其在癌症中的作用的研究却很有限。只有少数报道表明它在某些癌症中上调，可能导致增殖和侵袭

. Here, we identify a pro-tumorigenic function for NPC1 in hepatocellular carcinoma (HCC) using transgenic mice with

在这里，我们使用转基因小鼠鉴定了NPC1在肝细胞癌（HCC）中的促肿瘤发生功能

Npc1

deletion in hepatocytes.

肝细胞中的缺失。

This study also has several implications for both TGF-β and NPC1 biology. TGF-β signaling regulates a variety of cellular processes, including cell proliferation, differentiation, apoptosis, plasticity, and migration

这项研究对TGF-β和NPC1生物学也有一些启示。TGF-β信号调节多种细胞过程，包括细胞增殖，分化，凋亡，可塑性和迁移

. Dysregulated TGF-β signaling has been linked to cancer progression, stemness, and therapy resistance

TGF-β信号传导失调与癌症进展，干性和治疗抵抗有关

. TGF-β signaling is tightly regulated at different levels of the pathway. Regulation of TGF-β signaling critically depends on the modulation of TGFBR1 activity and its stability. The receptor’s degradation is mediated not only by caveolar-dependent endocytosis but also via clathrin-mediated internalization into early endosomes, followed by transport to late endosomes.

TGF-β信号在该途径的不同水平受到严格调控。TGF-β信号传导的调节关键取决于TGFBR1活性的调节及其稳定性。受体的降解不仅由细胞膜穴样内陷依赖性内吞作用介导，而且还通过网格蛋白介导的内化进入早期内体，然后转运到晚期内体。

The widely accepted model suggests that lipid rafts and caveolae play a negative role in TGF-β signaling by facilitating the turnover of TGF-β receptors, a process likely involving the recruitment of SMAD7 and SMURF, which subsequently induce receptor ubiquitination.

广泛接受的模型表明，脂筏和细胞膜穴样内陷通过促进TGF-β受体的更新而在TGF-β信号传导中发挥负面作用，这一过程可能涉及SMAD7和SMURF的募集，随后诱导受体泛素化。

. While literature has reported that TGFBR1 can localize to caveolae and that NPC1 may colocalize with caveolin-1 under certain conditions

虽然文献报道TGFBR1可以定位于细胞膜穴样内陷，而NPC1在某些条件下可能与细胞膜穴样内陷蛋白-1共定位

, our findings suggest that in HCC cells, the TGFBR1-NPC1 complex is primarily localized within lysosomes. This suggests a potential role for NPC1 in stabilizing TGFBR1 in lysosomes, rather than promoting receptor turnover through lipid rafts/caveolae. Previous studies indicate that SMAD7 can stably interact with TGFBR1 or SMURF2 even in the absence of exogenous ligand stimulation, with TGF-β potentially enhancing this interaction.

，我们的发现表明，在HCC细胞中，TGFBR1-NPC1复合物主要位于溶酶体内。。先前的研究表明，即使在没有外源配体刺激的情况下，SMAD7也可以与TGFBR1或SMURF2稳定相互作用，而TGF-β可能会增强这种相互作用。

Given that SMAD7 acts as an inhibitor in the early stages of TGF-β signaling.

鉴于SMAD7在TGF-β信号传导的早期阶段起到抑制剂的作用。

, our findings further suggest that NPC1 inhibits the interaction between TGFBR1 and SMAD7, thereby promoting TGFBR1 stability at a very early stage in the TGF-β signaling pathway, even without TGF-β1 stimulation (Fig.

，我们的研究结果进一步表明，NPC1抑制TGFBR1和SMAD7之间的相互作用，从而在TGF-β信号通路的早期阶段促进TGFBR1的稳定性，即使没有TGF-β1刺激（图）。

). The regulation we found occurs independent of ligand, consistent with previous studies that TGFBR1 was constantly internalized and recycled in the absence and presence of ligand

)。我们发现的调节独立于配体发生，这与先前的研究一致，即TGFBR1在不存在和存在配体的情况下不断内化和再循环

. We acknowledge that the overexpression of the TGFBR1-mCherry-His construct may have influenced the observed localization patterns. This potential limitation arises from the fact that overexpression could lead to artificial redistribution of TGFBR1, which may not fully reflect its physiological behavior.

我们承认TGFBR1-mCherry-His构建体的过表达可能影响了观察到的定位模式。这种潜在的局限性是由于过度表达可能导致TGFBR1的人工重新分布，这可能不能完全反映其生理行为。

This should be carefully considered when interpreting the data. To address this, further studies are needed to explore the role of NPC1 in TGFBR1 degradation under more physiologically relevant conditions, including using endogenous TGFBR1 levels and alternative experimental approaches..

在解释数据时应仔细考虑这一点。为了解决这个问题，需要进一步的研究来探索NPC1在更生理相关的条件下在TGFBR1降解中的作用，包括使用内源性TGFBR1水平和替代实验方法。。

Fig. 8: Schematic representation of the proposed mechanism by which NPC1 modulates the TGF-β pathway in HCC cells.

图8:NPC1调节HCC细胞中TGF-β途径的拟议机制的示意图。

NPC1 interacts with TGFBR1, preventing the binding of SMAD7 and SMURFs to TGFBR1. This interaction reduces TGFBR1 ubiquitylation and degradation, enhancing its stability and promoting TGF-β pathway activation in HCC cells.

NPC1与TGFBR1相互作用，阻止SMAD7和SMURFs与TGFBR1的结合。这种相互作用减少了TGFBR1的泛素化和降解，增强了其稳定性并促进了HCC细胞中TGF-β途径的激活。

Full size image

全尺寸图像

Structurally, NPC1 contains 13 transmembrane segments (TMs) and three luminal domains (NTD, MLD, CTD), with TMs 3-7 forming the sterol-sensing domain (SSD), which is conserved in proteins involved in cholesterol metabolism. Previous studies on NPC1’s structure show that cholesterol first binds NPC2 and is then transferred to NPC1’s NTD, eventually entering a tunnel connected to SSD.

在结构上，NPC1包含13个跨膜片段（TM）和三个腔结构域（NTD，MLD，CTD），其中TM 3-7形成固醇感应结构域（SSD），该结构域在参与胆固醇代谢的蛋白质中是保守的。先前对NPC1结构的研究表明，胆固醇首先与NPC2结合，然后转移到NPC1的NTD，最终进入与SSD连接的隧道。

. NPC1 is essential for Ebola virus infection, that the domain C of NPC1 binds to the virus glycoprotein (GP), independent of its known function in cholesterol transport

NPC1对于埃博拉病毒感染至关重要，NPC1的结构域C与病毒糖蛋白（GP）结合，与其在胆固醇转运中的已知功能无关

. Interestingly, in our study, we found that the P691S mutation in NPC1’s SSD—a classical mutation that disrupts cholesterol transport—did not affect its binding to TGFBR1. Reintroducing NPC1 (P691S) restored TGFBR1 levels and cell migration, highlighting a cholesterol-independent function for NPC1 in cancer progression.

有趣的是，在我们的研究中，我们发现NPC1 SSD中的P691S突变（一种破坏胆固醇转运的经典突变）并不影响其与TGFBR1的结合。重新引入NPC1（P691S）恢复了TGFBR1水平和细胞迁移，突出了NPC1在癌症进展中的胆固醇非依赖性功能。

Further, our truncation experiments demonstrated that the 692-854 region of NPC1’s TM domain is essential for its interaction with TGFBR1, similar to NPC1’s interaction with STING, which promotes STING degradation and inhibits its signaling.

此外，我们的截短实验表明，NPC1 TM结构域的692-854区域对于其与TGFBR1的相互作用至关重要，类似于NPC1与STING的相互作用，后者促进STING降解并抑制其信号传导。

. These interesting findings suggest that the interaction between NPC1 and other proteins may play an important role in protein stability. These results underscore that NPC1’s interaction with other proteins may regulate their stability, presenting an additional function beyond cholesterol transport..

这些有趣的发现表明，NPC1与其他蛋白质之间的相互作用可能在蛋白质稳定性中起重要作用。这些结果强调了NPC1与其他蛋白质的相互作用可能会调节其稳定性，从而在胆固醇转运之外还具有其他功能。。

Despite over a century of research on Niemann-Pick disease type C and extensive structural studies on NPC1, its function in cancer has been relatively unexplored. Our findings demonstrate that NPC1 promotes HCC progression by stabilizing TGFBR1 and facilitating tumor cell migration. NPC1’s influence on migration is likely related to its role in membrane trafficking and integrin recycling, as reported in previous studies.

尽管对C型尼曼-匹克病进行了一个多世纪的研究，并对NPC1进行了广泛的结构研究，但其在癌症中的功能尚未得到探索。我们的研究结果表明，NPC1通过稳定TGFBR1和促进肿瘤细胞迁移来促进HCC进展。。

. These studies have linked NPC1 to cell migration via regulation of cholesterol levels and focal adhesion dynamics. However, our study highlights an alternative, cholesterol-independent mechanism by which NPC1 interacts with TGFBR1 to promote cancer progression. Future research is needed to further elucidate the full scope of NPC1’s functions in cancer, particularly its interactions with other signaling pathways.

这些研究通过调节胆固醇水平和粘着斑动力学将NPC1与细胞迁移联系起来。然而，我们的研究强调了NPC1与TGFBR1相互作用以促进癌症进展的另一种不依赖胆固醇的机制。需要进一步的研究来进一步阐明NPC1在癌症中的全部功能，特别是其与其他信号通路的相互作用。

Regrettably, we did not use NPC1-specific inhibitors in this study to test its therapeutic effect on HCC. Although NPC1 SSD domain contains a U18666A-inhibitable site, while U18666A is a widely used inhibitor of NPC1, it may also have broader effects on cholesterol-related pathways.

遗憾的是，我们在这项研究中没有使用NPC1特异性抑制剂来测试其对HCC的治疗效果。尽管NPC1 SSD结构域包含一个U18666A可抑制位点，而U18666A是一种广泛使用的NPC1抑制剂，但它也可能对胆固醇相关途径产生更广泛的影响。

. To validate the therapeutic potential of NPC1, we employed an inducible

为了验证NPC1的治疗潜力，我们采用了诱导型

Npc1

knockout model and AAV8-mediated hepatic knockdown, both of which confirmed the tumor-suppressive effects of NPC1 inhibition. Our findings reveal a cholesterol-transport-independent role for NPC1 in HCC and suggest therapeutic opportunities targeting NPC1 protein for HCC patients.

敲除模型和AAV8介导的肝敲低，两者均证实了NPC1抑制的肿瘤抑制作用。我们的研究结果揭示了NPC1在HCC中的胆固醇转运独立作用，并为HCC患者提供了针对NPC1蛋白的治疗机会。

Methods

方法

Ethics statement

道德声明

The animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Center for Protein Sciences, Beijing, China (NCPSB).

动物护理和实验方案已获得中国北京国家蛋白质科学中心（NCPSB）机构动物护理和使用委员会（IACUC）的批准。

Cell Lines

Human HCC cell lines (MHCC-97H, HepG2, PLC/RPF/5) and HEK-293T cells were used in this study. MHCC-97H cells were obtained from Liver Cancer Institute, Zhongshan Hospital, Fudan University. HepG2 (ATCC, HB-8065), HEK-293T (SCSP-502) and PLC/RPF/5 (TCHu119) cells purchased from Stem Cell Bank/Stem Cell Core Facility, SIBCB, CAS.

本研究使用人HCC细胞系（MHCC-97H，HepG2，PLC/RPF/5）和HEK-293T细胞。MHCC-97H细胞获自复旦大学中山医院肝癌研究所。HepG2（ATCC，HB-8065），HEK-293T（SCSP-502）和PLC/RPF/5（TCHu119）细胞购自干细胞库/干细胞核心设施，SIBCB，CAS。

Cells were maintained in DMEM (GIBCO) supplemented with 10% (v/v) fetal bovine serum (GIBCO) at 37°C in 5% CO.

。

. All the cell lines were authenticated by STR profiling and confirmed to be free of mycoplasma contamination.

所有细胞系均通过STR分析进行鉴定，并确认无支原体污染。

Mice

老鼠

Mice were housed under specific pathogen-free (SPF) conditions in a temperature-controlled facility with a 12-hour light/dark cycle (08:00–20:00 light, 20:00–08:00 dark), and relative humidity maintained at 40-50%. Mice had free access to food and water throughout the study. All animals were maintained on normal chow diet (SPF-F02-003) until treatment.

将小鼠在无特定病原体（SPF）条件下饲养在温度控制的设施中，该设施具有12小时的光照/黑暗周期（08:00–20:00光照，20:00–08:00黑暗），相对湿度保持在40-50%。在整个研究过程中，小鼠可以自由获得食物和水。所有动物维持正常的食物饮食（SPF-F02-003）直至治疗。

Age- and sex-matched mice were used for experiments. Liver-specific conditional .

年龄和性别匹配的小鼠用于实验。肝脏特异性条件性。

Npc1

-knockout mice (

-基因敲除小鼠(

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

, 2-4 months old, 3 males and 4 females)

，2-4个月大，男3例，女4例）

Npc1

F/F

前/后

(2-4 months old, 8 males and 7 females) and

（2-4个月大，男8例，女7例）和

CreERT2

Alb

Alb公司

(6-8 weeks old, 2 males) in the C57BL/6 J background were made by Cyagen with CRISPR-Cas9. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice (5 weeks old, female) were purchased from Beijing Vital River Laboratory Animal Technology. NCG mice (5 weeks old, male) were purchased from GemPharmatech Co.

（6-8周龄，2只雄性）在C57BL/6J背景下由Cyagen用CRISPR-Cas9制成。非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠（5周龄，雌性）购自Beijing Vital River Laboratory Animal Technology。NCG小鼠（5周龄，雄性）购自GemPharmatech Co。

Ltd (Nanjing, China). .

有限公司（中国南京）。

H11-CAG-LSL-Myc

H11 CAG LSL Myc

mice (Cat. No. NM-KI-00039, 2-4 months old, 2 males and 2 females) were purchased from Shanghai Model Organisms.

小鼠（目录号NM-KI-00039，2-4个月大，2只雄性和2只雌性）购自上海模式生物。

TMA and immunohistochemistry

TMA和免疫组织化学

Four TMA chips were purchased from Shanghai Outdo Biotech (LivH180Su08, HLivH180Su15, T16-855TMAB, TFHCC-02). Immunohistochemistry was performed as previously described

四种TMA芯片购自Shanghai Outdo Biotech（LivH180Su08，HLivH180Su15，T16-855TMAB，TFHCC-02）。免疫组织化学如前所述进行

. Briefly, the tissues were exposed to primary antibodies overnight at 4 °C, after which they were incubated with secondary antibodies. Immunostaining was performed according to the protocol provided by the Ventana automated staining system (Ventana Medical Systems). The intensity of NPC1 or TGFBR1 proteins was scored according to a previously reported method.

简而言之，将组织在4℃下暴露于一抗过夜，然后将其与二抗孵育。根据Ventana自动染色系统（Ventana Medical Systems）提供的方案进行免疫染色。根据先前报道的方法对NPC1或TGFBR1蛋白的强度进行评分。

, blindly and independently, by two trained pathologists. Each specimen was assigned a score based on both the intensity of staining in the nucleus, cytoplasm, and/or membrane, and the percentage of positively stained cells. The intensity was scored as follows: no staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3.

由两位训练有素的病理学家盲目而独立地进行。根据细胞核，细胞质和/或膜中的染色强度以及阳性染色细胞的百分比为每个标本分配分数。强度评分如下：无染色=0，弱染色=1，中度染色=2，强染色=3。

The percentage of stained cells was rated as: 0% = 0, 1-25% = 1, 26-50% = 2, 51-75% = 3, and 76-100% = 4. The final immunoreactive score was calculated by multiplying the intensity score by the percentage score, resulting in a value ranging from 0 (minimum) to 12 (maximum). Image-Pro Plus software was used to quantify the number of Ki67-positive cells per field in tumor lesions.

染色细胞的百分比评定为：0%=0,1-25%=1,26-50%=2,51-75%=3和76-100%=4。通过将强度评分乘以百分比评分来计算最终的免疫反应评分，得到从0（最小）到12（最大）的值。Image-Pro Plus软件用于量化肿瘤病变中每个视野中Ki67阳性细胞的数量。

The antibodies used in the IHC assays are listed in Supplementary Data .

。

Plasmids and established stable cells

质粒并建立稳定的细胞

Lentiviruses carrying shRNA targeting human NPC1 or SMAD7 were purchased from OBiO Technology (Shanghai) Co., Ltd. Lentiviruses carrying shRNA targeting human TGFBR1 were purchased from GenePharma (Shanghai) Co.,Ltd. The shRNA sequences are listed in Supplementary Data

携带靶向人NPC1或SMAD7的shRNA的慢病毒购自OBiO Technology（Shanghai）Co.，Ltd。携带靶向人TGFBR1的shRNA的慢病毒购自GenePharma（Shanghai）Co.，Ltd。shRNA序列列于补充数据中

. HA-tagged NPC1, HA-tagged NPC1 (P691S) mutant, HA-tagged NPC1 deletion mutants, mCherry-His tagged TGFBR1, and EGFP-tagged SMAD7 overexpression lentiviral vectors and plasmids were custom-designed and synthesized by OBiO Technology (Shanghai) Co., Ltd. Information on the overexpression plasmids is provided in Supplementary Data .

HA标记的NPC1，HA标记的NPC1（P691S）突变体，HA标记的NPC1缺失突变体，mCherry-His标记的TGFBR1和EGFP标记的SMAD7过表达慢病毒载体和质粒由OBiO Technology（Shanghai）Co.，Ltd.定制设计和合成。关于过表达质粒的信息在补充数据中提供。

Small Interfering RNA (siRNA) and cell transduction

小干扰RNA（siRNA）与细胞转导

siRNAs targeting SMURF1 or SMURF2 were designed and synthesized by OBiO Technology (Shanghai) Co., Ltd. For cell transduction, Lipofectamine 2000 was used to perform the transfection according to the manufacturer’s protocol. The sequences of the siRNAs are shown in Supplementary Data

靶向SMURF1或SMURF2的siRNA由OBiO Technology（Shanghai）Co.，Ltd.设计和合成。对于细胞转导，使用Lipofectamine 2000根据制造商的方案进行转染。siRNA的序列显示在补充数据中

Proliferation assay

增殖测定

Cells were plated into 96-well plates at a density of approximately 7,000 cells per well. CCK8 reagent was then added to each well at a final concentration of 10%, and the cells were incubated for 1 h. Absorbance was measured at 450 nm. Data analysis was performed using GraphPad Prism 8 software.

将细胞以每孔约7000个细胞的密度接种到96孔板中。然后将CCK8试剂以10%的终浓度添加到每个孔中，并将细胞孵育1小时。在450 nm处测量吸光度。使用GraphPad Prism 8软件进行数据分析。

Cell migration and invasion assays

细胞迁移和侵袭测定

For the transwell migration or invasion assays, cells were trypsinized and seeded onto the upper chamber of a 24-well migration (3422, Corning) or invasion (354480, Corning) plate. The lower chamber was filled with DMEM supplemented with 10% serum. After 24 h of incubation for the migration assay or 48 h for the invasion assay, the filters were removed, and the cells on the membrane were fixed with methanol.

对于transwell迁移或侵袭测定，将细胞用胰蛋白酶消化并接种到24孔迁移（3422，Corning）或侵袭（354480，Corning）板的上室中。下室充满补充有10%血清的DMEM。在迁移测定孵育24小时或侵袭测定48小时后，除去过滤器，并用甲醇固定膜上的细胞。

Migrated cells on the underside of the membrane were stained with 0.5% crystal violet. The excess dye was washed off with water, and the cells were then examined under a microscope..

膜下侧的迁移细胞用0.5%结晶紫染色。用水洗去多余的染料，然后在显微镜下检查细胞。。

Western blotting

西方墨点法

Protein lysates from cells were prepared using RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS and 1% NP-40) with 1% protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78441). The lysates were heated at 100 °C for 10 min and then subjected to electrophoresis on 6-12% SDS-polyacrylamide gels.

使用RIPA缓冲液（50mM Tris-HCl，pH 8.0，150 mM NaCl，1 mM EDTA，0.1%SDS和1%NP-40）和1%蛋白酶和磷酸酶抑制剂混合物（Thermo Fisher Scientific，78441）。将裂解物在100℃加热10分钟，然后在6-12%SDS-聚丙烯酰胺凝胶上进行电泳。

Detailed information on the antibodies used can be found in Supplementary Data .

有关所用抗体的详细信息，请参见补充数据。

Proteome profiling of HCC cell lines with NPC1 knockdown

NPC1敲低的HCC细胞系的蛋白质组分析

HepG2 and PLC/PRF/5 cells were divided into two groups, each with three biological replicates: one group treated with scramble shRNA and the other with NPC1 shRNA. For protein extraction and trypsin digestion, cell samples were first lysed in buffer containing 1% sodium deoxycholate, 10 mM Tris (2-carboxyethyl) phosphine, 40 mM 2-chloroacetamide, and 100 mM Tris-HCl (pH 8.8).

将HepG2和PLC/PRF/5细胞分为两组，每组有三个生物学重复：一组用scramble shRNA处理，另一组用NPC1 shRNA处理。为了进行蛋白质提取和胰蛋白酶消化，首先将细胞样品在含有1%脱氧胆酸钠的缓冲液中裂解，10mM三（2-羧乙基）膦，40 mM 2-氯乙酰胺和100 mM Tris-HCl（pH 8.8）。

The lysates were then heated at 95 °C for 10 min and subjected to sonication for 3 min (3 s on, 3 s off, at 30% amplitude). After centrifugation at 16,000 g for 10 minutes at 4 °C, the supernatants were collected. For digestion, 100 μg of protein (concentration measured using Thermo Nanodrop One) was treated overnight with trypsin (Promega, V528A) at 37 °C, and the reaction was stopped with 1% formic acid.

然后将裂解物在95°C加热10分钟，并进行超声处理3分钟（3秒，。在4℃下以16000克离心10分钟后，收集上清液。为了消化，将100μg蛋白质（使用Thermo Nanodrop One测量的浓度）在37℃下用胰蛋白酶（Promega，V528A）处理过夜，并用1%甲酸终止反应。

Precipitated sodium deoxycholate was removed by centrifugation at 16,000 g for 10 min at 4 °C, and the supernatants were collected, desalted, vacuum-dried, and stored at −80 °C until further analysis..

通过在4°C下以16000 g离心10分钟除去沉淀的脱氧胆酸钠，收集上清液，脱盐，真空干燥，并保存在-80°C直至进一步分析。。

For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the dried peptides from shCTRL and shNPC1 HepG2 or PLC/PRF/5 cells were re-dissolved in Solvent A (0.1% formic acid in water) and loaded onto a homemade trap column (100 μm × 2 cm, particle size: 1.9 μm, pore size: 120 Å, SunChrom).

对于液相色谱-串联质谱（LC-MS/MS）分析，将来自shCTRL和shNPC1 HepG2或PLC/PRF/5细胞的干燥肽重新溶解在溶剂A（水中0.1%甲酸）中，并加载到自制的捕集柱（100μm×2 cm，粒径：1.9μm，孔径：120Å，SunChrom）。

The peptides were separated on a homemade 150 μm × 30 cm silica microcolumn (particle size: 1.9 μm, pore size: 120 Å, SunChrom) with a gradient of 5% to 35% mobile phase B (80% acetonitrile and 0.1% formic acid) at a flow rate of 600 nL/min over 140 minutes. After a 10-minute wash with 95% mobile phase B, the peptides were ionized at 2 kV.

肽在自制的150μm×30cm二氧化硅微柱上分离（粒径：1.9μm，孔径：。用95%流动相B洗涤10分钟后，肽在2 kV下电离。

MS was conducted using data-dependent acquisition (DDA) mode. Raw MS files were analyzed using MaxQuant (version 1.6.1.0).

MS使用数据相关采集（DDA）模式进行。使用MaxQuant（版本1.6.1.0）分析原始MS文件。

with default parameters against the human UniProt database (version 20180705). Further differencial analysis and functional enrichment analysis were performed using Perseus software

具有针对人类UniProt数据库（版本20180705）的默认参数。使用Perseus软件进行了进一步的差异分析和功能富集分析

and the WebGestalt web tool

和WebGestalt web工具

Quantitative RT-PCR

定量RT-PCR

Total RNA was extracted from cells or tissues according to the manufacturer’s instructions using TRIzol reagent (CWBIO, CW0580). Complementary DNA was synthesized from 1-3 µg of RNA with PrimeScript™ RT Master Mix (Takara, RR036A). qPCR was conducted using SYBR Green Master Mix (Vazyme, Q712-03), and primer sequences are provided in Supplementary Data .

根据制造商的说明，使用TRIzol试剂（CWBIO，CW0580）从细胞或组织中提取总RNA。用PrimeScript™RT Master Mix（Takara，RR036A）从1-3µg RNA合成互补DNA。使用SYBR Green Master Mix（Vazyme，Q712-03）进行qPCR，引物序列在补充数据中提供。

. All samples were normalized to

。所有样品均标准化为

Actb

for mouse samples or

对于鼠标样本或

ACTB

for human samples.

用于人体样本。

Filipin III staining

菲律宾III染色

A Cholesterol Cell-Based Detection Assay Kit (No. 10009779) from Cayman was used for the experiment. Filipin III staining was carried out following the manufacturer’s instructions. Fluorescent staining was visualized using an upright fluorescence microscope (Nikon, Model No. 3000102).

使用来自Cayman的基于胆固醇细胞的检测测定试剂盒（编号10009779）进行实验。按照制造商的说明进行菲律宾III染色。使用立式荧光显微镜（Nikon，型号3000102）观察荧光染色。

CHX chase assay

CHX追踪分析

Cells were incubated with 50 μg/mL cycloheximide (CHX; Sigma, C7698) for the indicated times before being lysed in lysis buffer. Protein levels was determined by western blot analysis.

将细胞与50μg/mL环己酰亚胺（CHX；Sigma，C7698）孵育指定的时间，然后在裂解缓冲液中裂解。通过蛋白质印迹分析确定蛋白质水平。

Ubiquitylation Assay

泛素化测定

TGFBR1-mCherry-His was transfected into HCC cells with stable NPC1 knockdown or overexpression. Cells were treated with MG132 for 8 h before collection. Cells were rinsed three times with prechilled phosphate-buffered saline (PBS) and subsequently lysed using immunoprecipitation buffer containing 6 M guanidine hydrochloride, 0.1 M NaH.

将TGFBR1-mCherry-His转染到具有稳定NPC1敲低或过表达的HCC细胞中。。用预冷的磷酸盐缓冲盐水（PBS）冲洗细胞三次，然后用含有6M盐酸胍的免疫沉淀缓冲液裂解，0.1百万纳。

采购订单

/Na

/不适用

HPO

, 0.01 M Tris-HCl (pH 8.0), 5 mM imidazole and 10 mM β-mercaptoethanol. The lysates were then centrifuged at 14,000

，0.01M Tris-HCl（pH 8.0），5mM咪唑和10mMβ-巯基乙醇。然后将裂解物在14000离心

克

for 20 min at 4 °C. The resulting supernatants were incubated with Ni-NTA agarose beads at room temperature for 4 h. Beads were then washed respectively with washing buffer A (6 M guanidine hydrochloride, 0.1 M NaH

在4°C下放置20分钟。将所得上清液与Ni-NTA琼脂糖珠在室温下孵育4小时。然后分别用洗涤缓冲液A（6M盐酸胍，0.1百万NaH

采购订单

/ Na

/不适用

HPO

, 0.01 M Tris-HCl, pH 8.0, and 10 mM β-mercaptoethanol), washing buffer B (8 M urea, 0.1 M NaH

，0.01M Tris-HCl，pH 8.0和10mMβ-巯基乙醇），洗涤缓冲液B（8M尿素，0.1百万NaH

采购订单

/Na

/不适用

HPO

, 0.01 M Tris-HCl, pH 8.0, and 10 mM β-mercaptoethanol), washing buffer C (8 M urea, 0.1 M NaH

，0.01M Tris-HCl，pH 8.0和10mMβ-巯基乙醇），洗涤缓冲液C（8M尿素，0.1百万NaH

采购订单

/Na

/不适用

HPO

, 0.01 M Tris-HCl, pH 6.3, 10 mM β-mercaptoethanol and 0.2% Triton X-100) and washing buffer D (8 M urea, 0.1 M NaH

，0.01M Tris-HCl，pH 6.3，10mMβ-巯基乙醇和0.2%Triton X-100）和洗涤缓冲液D（8M尿素，0.1百万NaH

采购订单

/Na

/不适用

HPO

, 0.01 M Tris-HCl, pH 6.3, 10 mM β-mercaptoethanol and 0.1% Triton X-100) for 5 min. Then the beads were incubated with buffer E (200 mM imidazole, 0.15 M Tris-HCl, pH 6.7, 30% glycerol, 0.72 M β-mercaptoethanol and 5% SDS) for 30 min at room temperature. The beads were subsequently boiled in loading buffer at 95 °C for 10 min.

，0.01M Tris-HCl，pH 6.3，，0.15 M Tris-HCl，pH 6.7，30%甘油，0.72Mβ-巯基乙醇和5%SDS）在室温下放置30分钟。随后将珠子在上样缓冲液中于95℃煮沸10分钟。

The resulting aliquots were collected and analyzed by western blot using antibodies against His, mCherry, K48-Ubiquitin, K63-Ubiquitin, or Ubiquitin..

收集所得等分试样，并使用针对His，mCherry，K48泛素，K63泛素或泛素的抗体通过蛋白质印迹进行分析。。

Immunoprecipitation

免疫沉淀法

Cells were lysed in IP buffer (NP-40, Beyotime) supplemented with a protease inhibitor cocktail and incubated on ice for 2 h. The lysates were then centrifuged at 16,000 g for 10 min at 4 °C. Immunoprecipitation was performed using the specified primary antibody and Pierce™ protein A/G Magnetic Beads (Thermo Fisher Scientific, 88803), anti-DDDDK-tag mAb magnetic agarose (MBL, M185-10R), or anti-GFP mAb magnetic agarose (MBL, D153-10) at 4 °C.

将细胞在补充有蛋白酶抑制剂混合物的IP缓冲液（NP-40，Beyotime）中裂解，并在冰上孵育2小时。然后将裂解物在4℃以16000 g离心10分钟。使用指定的一抗和Pierce™蛋白A/G磁珠（Thermo Fisher Scientific，88803），抗DDDDK标签mAb磁性琼脂糖（MBL，M185-10R）或抗GFP mAb磁性琼脂糖（MBL，D153-10）在4°C下进行免疫沉淀。

The immunocomplexes were thoroughly washed with the same buffer. Both lysates and immunoprecipitates were analyzed using the indicated primary antibodies, followed by detection with the corresponding secondary antibody and SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, 34580).

用相同的缓冲液彻底洗涤免疫复合物。使用指定的一抗分析裂解物和免疫沉淀物，然后用相应的二抗和SuperSignal™West Pico PLUS化学发光底物（Thermo Fisher Scientific，34580）进行检测。

The primary antibodies used are listed in Supplementary Data .

使用的一抗列于补充数据中。

Immunofluorescence

免疫荧光

Cells were fixed for 15 min with 4% paraformaldehyde at room temperature, washed twice with PBS and blocked in 10% normal goat serum for 1 h at room temperature. They were then incubated overnight with the primary antibody at 4°C. The antibodies are listed in Supplementary Data

将细胞在室温下用4%多聚甲醛固定15分钟，用PBS洗涤两次，并在室温下在10%正常山羊血清中封闭1小时。然后将它们与一抗在4°C孵育过夜。抗体列在补充数据中

. The secondary antibodies, conjugated with fluorescein, were incubated for 1 h at room temperature. Nuclei were then stained with 4’,6-diamidino-2-phenylindole (DAPI) for counterstaining. Confocal laser scanning was performed on a ZEISS LSM880 Confocal Microscope (Fig.

将与荧光素缀合的二抗在室温下孵育1小时。然后将细胞核用4'，6-二脒基-2-苯基吲哚（DAPI）染色以进行复染。共聚焦激光扫描在蔡司LSM880共聚焦显微镜上进行（图）。

5摄氏度

, Supplementary Fig.

，补充图。

6d, e, h

6d，e，h

) or a spinning disk confocal microscope (Supplementary Fig.

)或旋转盘共聚焦显微镜（Supplementary Fig.）。

6b, c, g

6b、c、g

). The images in Supplementary Fig.

)。补充图中的图像。

7a个

were collected using the Polar-SIM system (Airy Technology Co., Ltd., China). A 640 nm laser was used to excite mouse Alexa Fluor 633 with 2DSIM modality. The SIM reconstruction process was conducted using the Airy-SIM software with pre-processing (Dark) or post-processing (MRA). ImageJ software was applied to quantify colocalization using Mander’s coefficient..

使用Polar SIM系统（Airy Technology Co.，Ltd.，China）收集。使用640nm激光器以2DSIM模式激发小鼠Alexa Fluor 633。SIM重建过程是使用具有预处理（黑暗）或后处理（MRA）的Airy SIM软件进行的。ImageJ软件用于使用Mander系数量化共定位。。

Multiplexed Immunofluorescence (IF) Staining

多重免疫荧光（IF）染色

Multiplex immunofluorescence staining was carried out using the PANO IHC Kit (Panovue, China) following the manufacturer’s protocol. The stained slides were then scanned with the Digital Slide Scan system (AxioScan7), and individual scans for each slide were merged to generate a composite image. The resulting multilayer images were subsequently analyzed quantitatively using the ZEISS LSM880 Confocal Microscope..

按照制造商的规程，使用PANO IHC试剂盒（Panovue，China）进行多重免疫荧光染色。然后用数字载玻片扫描系统（AxioScan7）扫描染色的载玻片，并合并每个载玻片的单独扫描以生成合成图像。随后使用蔡司LSM880共聚焦显微镜对所得的多层图像进行定量分析。。

Animal studies

动物研究

For subcutaneous xenograft experiments, NCG nude mice were randomly assigned to the designated groups and subcutaneously injected with the indicated cells, which stably expressed the specified shRNAs or constructs (1×10

对于皮下异种移植实验，将NCG裸鼠随机分配到指定组，并皮下注射指定的细胞，该细胞稳定表达指定的shRNA或构建体（1×10

cells, mixed with 100 μl of Matrigel (ABW, 0827245) at a 1:1 ratio. Tumor progression was tracked, and tumor size was assessed using calipers. Tumor volume was determined using the following formula: (width

将细胞与100μlMatrigel（ABW，0827245）以1:1的比例混合。跟踪肿瘤进展，并使用卡尺评估肿瘤大小。使用以下公式确定肿瘤体积：（宽度

× length)/2.

×长度）/2。

For the NOD SCID or NCG mouse tail vein metastasis model, the mice were injected with the indicated luciferase-expressing cells through the lateral tail vein. All animals were sacrificed 8 weeks post-injection, and their lungs were surgically removed, fixed, and analyzed using hematoxylin and eosin (H&E) staining.

对于NOD SCID或NCG小鼠尾静脉转移模型，通过侧尾静脉向小鼠注射所示的荧光素酶表达细胞。注射后8周处死所有动物，通过手术切除，固定肺部，并使用苏木精和曙红（H＆E）染色进行分析。

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Center for Protein Sciences, Beijing, China (NCPSB). The tumor size did not exceed 20 mm in any direction, in accordance with the approval from our institutional review board. Mice were euthanized immediately if the tumor size exceeded 20 mm in any direction by the final measurement day.

所有动物研究均经中国北京国家蛋白质科学中心（NCPSB）机构动物护理和使用委员会（IACUC）批准。。如果在最终测量日肿瘤大小在任何方向上超过20毫米，则立即对小鼠实施安乐死。

At the study endpoint, animals were euthanized through cervical dislocation while under anesthesia..

在研究终点，在麻醉下通过颈椎脱位对动物实施安乐死。。

For the DEN/CCl

对于DEN/CCl

-induced HCC model, 2-week-old male mice (

-诱导肝癌模型，2周龄雄性小鼠(

Npc1

F/F

前/后

and

和

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

) were administered an intraperitoneal injection of DEN (50 mg/kg), followed by weekly CCl

)腹腔注射DEN（50mg/kg），然后每周CCl

injections (diluted 1:4 in corn oil) starting two weeks later at a dose of 2.5 ml/kg body weight, continuing for 20 weeks. Mice were euthanized 72 h after the final CCl

两周后开始注射（在玉米油中以1:4稀释），剂量为2.5ml/kg体重，持续20周。最终CCl后72小时对小鼠实施安乐死

injection, and their livers were collected for biochemical, histological, and molecular analyses.

注射，并收集其肝脏进行生化，组织学和分子分析。

CreERT2

Alb

Alb公司

Npc1

F/F

前/后

mice were generated by crossing

通过杂交产生小鼠

Npc1

F/F

前/后

mice with

小鼠

CreERT2

Alb

Alb公司

mice. Two-week-old male mice (

老鼠。两周大的雄性小鼠(

Npc1

F/F

前/后

and

和

CreERT2

Alb

Alb公司

Npc1

F/F

前/后

) were injected intraperitoneally with DEN (50 mg/kg), followed by CCl

)腹腔注射DEN（50mg/kg），然后注射CCl

injections (diluted 1:4 in olive oil) at a dose of 2.5 ml/kg body weight once a week for 16 weeks. At 19 weeks of age,

注射（在橄榄油中以1:4稀释），剂量为2.5ml/kg体重，每周一次，持续16周。在19周龄时，

CreERT2

Alb

Alb公司

Npc1

F/F

前/后

mice were randomly assigned to two groups and administered tamoxifen (TAM, Sigma-Aldrich, T5648, 75 mg/kg) or corn oil for 5 consecutive days via intraperitoneal injection and were sacrificed two months later. Tamoxifen was prepared by dissolving it in corn oil to a concentration of 20 mg/mL, and the solution was shaken overnight at 37 °C..

将小鼠随机分为两组，并通过腹膜内注射连续5天给予他莫昔芬（TAM，Sigma-Aldrich，T5648,75mg/kg）或玉米油，并在两个月后处死。通过将其溶解在玉米油中至20 mg/mL的浓度来制备他莫昔芬，并将溶液在37℃下振荡过夜。。

To establish the model of spontaneous HCC with targeted

建立自发性肝癌的靶向模型

Myc

Myc公司

knock-in and

敲入和

Npc1

knockout in the liver,

肝脏中的基因敲除，

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice were crossbred with

小鼠与

H11-CAG-LSL-Myc

H11 CAG LSL Myc

mice, resulting in homozygous

小鼠，导致纯合子

Npc1

knockout

淘汰赛

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

mice. Spontaneous liver cancer developed in 6- to 8-week-old mice following the mating of

老鼠。自发性肝癌发生在6至8周龄的小鼠交配后

H11-CAG-LSL-Myc

H11 CAG LSL Myc

mice with

小鼠

Cre

Cre公司

Alb

Alb公司

mice. Eight-week-old

老鼠。八周大

Cre

Cre公司

Alb

Alb公司

Myc

Myc公司

and

和

Cre

Cre公司

Alb

Alb公司

Npc1

F/F

前/后

Myc

Myc公司

mice were sacrificed, and their livers were used for biochemical, histological, and molecular analysis.

处死小鼠，并将其肝脏用于生化，组织学和分子分析。

Adeno-Associated Virus (AAV)-Mediated Gene Knockdown

腺相关病毒（AAV）介导的基因敲除

An adeno-associated virus (AAV) delivery system was used to specifically knock down murine genes in mouse hepatocyte. The AAV cloning vectors AAV-shRNA and pAAV-TBG-GdGreen-miR30shRNA-WPRE were obtained from OBiO Technology (Shanghai) Co., Ltd. AAV8-shCTRL and AAV8-shNPC1 were generated by cloning a control shRNA (5’- GAAGTCGTGAGAAGTAGAA-3’) or NPC1 shRNA (5’- CCCGTCTTACTCAGTTACATA-3’) into pAAV-TBG-GdGreen-miR30shRNA-WPRE.

腺相关病毒（AAV）递送系统用于特异性敲除小鼠肝细胞中的鼠基因。AAV克隆载体AAV-shRNA和pAAV-TBG-GdGreen-miR30shRNA-WPRE获自OBiO Technology（Shanghai）Co.，Ltd。通过将对照shRNA（5'-GAAGTCGTGAAGTAGAA-3'）或NPC1 shRNA（5'-CCCGTCTTACTCAGTTACATA-3'）克隆到pAAV-TBG-GdGreen-miR30shRNA-WPRE中来产生AAV8-shCTRL和AAV8-shNPC1。

Cre

Cre公司

Alb

Alb公司

Myc

Myc公司

mice (4-week-old, male) were transduced with AAV serotype 8 vectors (6 × 10

用AAV血清型8载体（6×10

viral genome per mouse, via the tail vein) and euthanized at 8 weeks of age. Liver tumor tissues were collected for biochemical, histological, and molecular analysis.

每只小鼠的病毒基因组，通过尾静脉），并在8周龄时安乐死。收集肝肿瘤组织用于生化，组织学和分子分析。

Histology

组织学

Livers and tumor tissues were fixed overnight in a neutral-buffered formalin solution, followed by dehydration and embedding in paraffin. The resulting sections were then used for Hematoxylin-Eosin and reticulin staining according to standard procedures.

。然后根据标准程序将所得切片用于苏木精-伊红和网状蛋白染色。

Biochemical assays

生化分析

Blood samples were collected from the retro-orbital plexus of each mouse. The samples were then left at room temperature for 30 min to allow clotting. Afterward, the blood was centrifuged at 1,500 g for 10 min to separate the serum. Serum ALT, AST, TBIL levels were measured using an automatic chemical analyzer (Toshiba Biochemical Analyzer, model TBA 40FR)..

从每只小鼠的眶后丛收集血样。然后将样品在室温下放置30分钟以使其凝结。之后，将血液以1500克离心10分钟以分离血清。使用自动化学分析仪（东芝生化分析仪，型号TBA 40FR）测量血清ALT，AST，TBIL水平。。

Statistics and reproducibility

统计和再现性

Details regarding statistical analyses and sample sizes are provided in the figures, figure legends, and source data. While the data were presumed to follow a normal distribution, formal testing was not conducted. Animal experiments were randomly assigned to groups. Except for the analysis of IHC scores, data collection and analysis were not blinded to experimental conditions.

数字，图例和源数据中提供了有关统计分析和样本量的详细信息。虽然假定数据遵循正态分布，但没有进行正式测试。动物实验被随机分组。除了IHC评分的分析外，数据收集和分析并不盲目于实验条件。

Unless otherwise stated in the figure legends, each experiment was conducted independently and yielded consistent results. Quantitative data are reported as mean ± s.e.m. Statistical analyses were performed in GraphPad Prism 8. Two-tailed unpaired Student’s t-test, Mann-Whitney U test and two-way analysis of variance (ANOVA) were used to calculate .

除非图例中另有说明，否则每个实验都是独立进行的，并产生了一致的结果。。使用双尾不成对学生t检验，Mann-Whitney U检验和双向方差分析（ANOVA）进行计算。

values. Kaplan–Meier curves, with the log-rank test, were used to depict survival function from lifetime data.

价值观。Kaplan–Meier曲线和对数秩检验用于描述寿命数据的生存函数。

values are displayed in all figures, with values below 0.05 regarded as statistically significant.

数值显示在所有数字中，低于0.05的数值被认为具有统计学意义。

Reporting summary

报告摘要

Further information on research design is available in the

有关研究设计的更多信息，请参阅

Nature Portfolio Reporting Summary

自然投资组合报告摘要

linked to this article.

链接到本文。

Data availability

数据可用性

The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium (

质谱蛋白质组学数据已保存在ProteomeXchange Consortium(

http://proteomecentral.proteomexchange.org

) via the iProX partner repository

)通过iProX合作伙伴存储库

with the dataset identifier PXD046018 (

使用数据集标识符PXD046018(

https://www.iprox.cn//page/project.html?id=IPX0007268000

). The remaining data are available within the Article, Supplementary Information or Source Data file.

)。其余数据可在文章，补充信息或源数据文件中找到。

Source data

源数据

are provided with this paper.

随本文提供。

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Acknowledgements

致谢

This work was partially supported by the National Key R&D Program of China (No. 2021YFA1301601 to Y.J. and No. 2020YFE0202200 to Y.J.) and the National Natural Science Foundation of China (No. 82273243 to Y.J., No. 92168207 to Y.J., and No. 82090051 to C.L.). We thank Nieng Yan (Department of Molecular Biology, Princeton University) for providing the pCAG-NPC1-His-Flag plasmid. We thank the Imaging Facility (J.

这项工作得到了中国国家重点研发计划（Y.J.编号2021YFA1301601，Y.J.编号2020YFE0202200）和国家自然科学基金（Y.J.编号82273243，Y.J.编号92168207，C.L.编号82090051）的部分支持。我们感谢Nieng Yan（普林斯顿大学分子生物学系）提供pCAG-NPC1-His-Flag质粒。我们感谢成像设备（J。

Chen), the Animal Facility (C. Qiu) and the Mass Spectrum Facility (S. Ji) of the National Center for Protein Sciences in Beijing (NCPSB) for their assistance..

陈），北京国家蛋白质科学中心（NCPSB）的动物设施（C.Qiu）和质谱设施（S.Ji）的帮助。。

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Author notes

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These authors contributed equally: Shuangyan Li, Lishan Yan.

。

Authors and Affiliations

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School of Life Sciences, Tsinghua University, Beijing, China

清华大学生命科学学院，北京

Shuangyan Li

李双燕

State Key Laboratory of Medical Proteomics, National Center for Protein Sciences (Beijing), Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing, China

国家蛋白质科学中心（北京）医学蛋白质组学国家重点实验室，北京生命组学研究所北京蛋白质组研究中心，北京

Shuangyan Li, Lishan Yan, Chaoying Li, Lijuan Lou, Fengjiao Cui, Xiao Yang, Fuchu He & Ying Jiang

李双艳、严丽山、李朝英、娄丽娟、崔凤娇、肖扬、何福初、蒋颖

School of Basic Medicine, Qingdao University, Qingdao, China

青岛大学基础医学院，青岛

Fengjiao Cui

崔凤娇

Research Unit of Proteomics Dirven Cancer Precision Medicine, Chinese Academy of Medical Sciences, Beijing, China

中国医学科学院蛋白质组学与癌症精准医学研究室，北京

Fuchu He

何福初

Anhui Medical University, Hefei, China

安徽医科大学，合肥

Fuchu He & Ying Jiang

何福初、蒋颖

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Shuangyan Li

李双燕

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Chaoying Li

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Contributions

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Y.J. and F.H. conceived the study and supervised experiments. S.L. designed experiments. S.L., L.Y., C.L., L.L., F.C. and X.Y. performed experiments. S.L. and L.Y. analyzed the data. S.L., Y.J., F.H., and L.Y. wrote the paper. All authors read and approved the manuscript.

Y、 J.和F.H.构思了这项研究并监督了实验。S、 L.设计实验。S、 L.，L.Y.，C.L.，L.L.，F.C.和X.Y.进行了实验。S、 L.和L.Y.分析了数据。S、 L.，Y.J.，F.H。和L.Y.写了这篇论文。所有作者都阅读并批准了手稿。

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何福初

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Ying Jiang

盈江

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The authors declare no competing interests.

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Nature Communications

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thanks Marene Landström and the other anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

感谢Marene Landström和其他匿名审稿人对这项工作的同行评审做出的贡献。可以获得同行评审文件。

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Description of Additional Supplementary Files

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Supplementary Data 1

补充数据1

Supplementary Data 2

补充数据2

Supplementary Data 3

补充数据3

Supplementary Data 4

补充数据4

Supplementary Data 5

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Li, S., Yan, L., Li, C.

李，S.，严，L.，李，C。

et al.