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BRAF抑制剂耐药黑素瘤促进CXCL1表达招募肿瘤相关成纤维细胞

BRAF inhibitor-resistant melanoma promotes CXCL1 expression to recruit cancer-associated fibroblasts

    摘要
  • 摘要:
    目的  探讨BRAF抑制剂耐药的黑素瘤招募肿瘤相关成纤维细胞（cancer-associated fibroblast, CAF）的关键分子及信号通路。
    方法  收集2024年9月至2025年9月复旦大学附属中山医院接受BRAF抑制剂靶向治疗的20例黑素瘤患者的肿瘤组织标本切片，其中耐药组10例、敏感组10例，通过免疫组织化学染色比较两组中CAF数量。通过共培养模型，比较黑素瘤细胞在与CAF共培养及非共培养模式下BRAF抑制剂的药物半数抑制浓度（IC50）。通过测序数据（GSE196434）分析，比较BRAF抑制剂敏感与耐药黑素瘤基因表达差异，筛选关键差异基因。采用实时荧光定量反转录聚合酶链反应及Western印迹法验证CXCL1在BRAF抑制剂敏感与耐药黑素瘤中的表达差异，并检测CXCL1在AKT抑制剂处理组及未处理组黑素瘤中的表达情况。采用Transwell侵袭实验，观察BRAF抑制剂耐药与敏感黑素瘤对于CAF的招募能力，并比较AKT抑制剂处理组、AKT抑制剂＋CXCL1处理组和空白对照组黑素瘤对CAF的招募能力差异。
    结果 BRAF抑制剂耐药组黑素瘤中的CAF数量多于BRAF抑制剂敏感组（P＜0.01）；黑素瘤细胞与CAF共培养组的细胞活性大于非共培养组（P＜0.01），IC50高于非共培养组。CXCL1为关键差异基因，其在BRAF抑制剂耐药的黑素瘤中表达高于BRAF抑制剂敏感黑素瘤（P＜0.01）；使用AKT抑制剂后，BRAF抑制剂耐药黑素瘤中的CXCL1表达受抑制（P＜0.01）。BRAF抑制剂耐药黑素瘤对于CAF的招募作用强于BRAF抑制剂敏感黑素瘤（P＜0.05）；使用AKT抑制剂后，BRAF抑制剂耐药黑素瘤对于CAF的招募能力下降（P＜0.05）；加入CXCL1后，该招募能力得到恢复（P＜0.05）。
    结论  BRAF抑制剂耐药黑素瘤激活AKT通路促进CXCL1招募CAF。

     

    Abstract:
    Objective To explore the key molecule and signaling pathway involved in the recruitment of cancer-associated fibroblasts (CAFs) by BRAF inhibitor (BRAFi)-resistant melanoma.
    Methods Tumor tissue sections were collected from 20 melanoma patients who received BRAFi-targeted therapy in Zhongshan Hospital, Fudan University, between September 2024 and September 2025, including 10 cases in the BRAFi-resistant group and 10 cases in the BRAFi-sensitive group. Immunohistochemical staining was performed to compare the number of CAFs in tumor tissues in the two groups. A co-culture model was established to compare the half-maximal inhibitory concentration (IC50) of BRAFi in melanoma cells under co-culture conditions with CAFs versus non-co-culture conditions. Differential gene expression between BRAFi-sensitive and BRAFi-resistant melanoma was analyzed using publicly available sequencing data (GSE196434), and key differentially expressed genes were identified. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were employed to validate the expression differences of CXCL1 between BRAFi-sensitive and BRAFi-resistant melanoma cells, and to detect CXCL1 expression in melanoma cells treated or untreated with the AKT inhibitor (AKTi). Transwell invasion assays were performed to evaluate the recruitment capability of BRAFi-resistant and BRAFi-sensitive melanoma cells toward CAFs, and to compare the differences in recruitment capability of melanoma cells toward CAFs among the AKTi-treated group, the AKTi＋CXCL1-treated group, and the control group.
    Results The number of CAFs was significantly higher in the BRAFi-resistant group than that in the BRAFi-sensitive group (P＜0.01). The cell viability (P＜0.01) and IC50 were higher in the melanoma co-culture group with CAFs than in the non-co-culture group. CXCL1 was identified as a key differentially expressed gene, with higher expression in BRAFi-resistant melanoma than in BRAFi-sensitive melanoma (P＜0.01). AKTi treatment suppressed CXCL1 expression in BRAFi-resistant melanoma (P＜0.01). BRAFi-resistant melanoma exhibited a stronger CAF-recruiting capacity than BRAFi-sensitive melanoma (P＜0.05), and AKTi treatment reduced its ability to recruit CAFs (P＜0.05). This effect was restored after the addition of exogenous CXCL1 (P＜0.05).
    Conclusions BRAFi-resistant melanoma activates the AKT pathway to upregulate CXCL1, thereby recruiting CAFs.

     

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