Human Immunology Pathophysiology & Immunotherapy (HIPI), Université Paris Cité, INSERM U976 - Hôpital Saint Louis - 1 avenue Claude Vellefaux, Paris, 75010, France.
Université Paris Cité, AP-HP Dermato-oncology and CIC, Cancer institute APHP.nord Paris Cité, INSERM U976, Saint Louis Hospital, Paris, F-75010, France.
Cell Commun Signal. 2024 Nov 21;22(1):559. doi: 10.1186/s12964-024-01941-y.
Phosphodiesterase type 4D (PDE4D) breaks down cyclic AMP (cAMP) reducing the signaling of this intracellular second messenger which plays a major role in melanocyte pathophysiology. In advanced melanoma, expression of PDE4D is increased, plays a role in tumor invasion and is negatively associated with survival. In the current work, we investigated the role of PDE4D in the resistance of BRAF-mutated melanoma to mitogen-activated protein kinase (MAPK) pathway-targeted therapy.
Established human melanoma cell line sensitive and resistant to BRAF and MEK inhibitors and tumor tissues from melanoma patients were used in this study. Immunoblotting was used to analyze protein expression and quantitative reverse transcription-PCR was used to analyze mRNA expression. DNA methylation analysis was evaluated via bisulfite treatment followed by quantitative PCR. Cell viability was measured by clonogenic assays or spheroid cultures. Cell xenograft experiments in immunodeficient mice were used to validate the results in vivo.
Analysis of baseline tumors from patients with BRAFV600E-mutated melanoma treated with MAPK inhibitors showed that higher PDE4D expression in situ predicted worse survival in patients. Furthermore, acquired resistance to BRAF and MEK inhibitors was associated with overexpression of PDE4D in situ and ex vivo. The overexpression of the PDE4D5 isoform in melanoma cells resistant to targeted therapies was explained by demethylation or deletion of a CpG island located upstream of the PDE4D5 promoter. We further showed that PDE4D overexpression allowed RAF1 activation, promoting a switch from BRAF to RAF1 isoform in BRAF-mutated melanoma, favoring resistance to BRAF and MEK inhibitors. As a result, pharmacological inhibition of PDE4 activity impeded the proliferation of resistant cells ex vivo and in vivo. The anti-tumorigenic activity of PDE4 inhibitor was achieved via inhibition of the Hippo pathway which plays an important role in resistance to targeted therapies.
In summary, our research showed that PDE4D drives rewiring of the MAPK pathway in BRAF-mutated melanoma resistant to MAPK inhibitors and suggests that PDE4 inhibition is a novel therapeutic option for treatment of BRAF-mutated melanoma patients.
磷酸二酯酶 4D（PDE4D）分解环腺苷酸（cAMP），降低这种细胞内第二信使的信号转导，该信使在黑素细胞病理生理学中起着重要作用。在晚期黑色素瘤中，PDE4D 的表达增加，在肿瘤侵袭中起作用，并与生存呈负相关。在目前的工作中，我们研究了 PDE4D 在 BRAF 突变黑色素瘤对丝裂原活化蛋白激酶（MAPK）通路靶向治疗的耐药中的作用。
本研究使用了对 BRAF 和 MEK 抑制剂敏感和耐药的人黑色素瘤细胞系以及黑色素瘤患者的肿瘤组织。免疫印迹用于分析蛋白表达，定量逆转录-PCR 用于分析 mRNA 表达。通过亚硫酸氢盐处理 followed by quantitative PCR 评估 DNA 甲基化分析。通过集落形成测定或球体培养来测量细胞活力。使用免疫缺陷小鼠的细胞异种移植实验在体内验证结果。
对接受 MAPK 抑制剂治疗的 BRAFV600E 突变黑色素瘤患者的基线肿瘤进行分析表明，原位 PDE4D 表达较高预示着患者的生存较差。此外，对 BRAF 和 MEK 抑制剂的获得性耐药与原位和体外 PDE4D 的过表达有关。靶向治疗耐药的黑色素瘤细胞中 PDE4D5 同工型的过表达是由位于 PDE4D5 启动子上游的 CpG 岛的去甲基化或缺失引起的。我们进一步表明，PDE4D 的过表达允许 RAF1 激活，从而促进 BRAF 突变黑色素瘤中 RAF1 同工型的转换，有利于对 BRAF 和 MEK 抑制剂的耐药。结果，PDE4 活性的药理学抑制阻碍了耐药细胞在体外和体内的增殖。PDE4 抑制剂的抗肿瘤活性是通过抑制 Hippo 通路实现的，该通路在对靶向治疗的耐药中起着重要作用。
总之，我们的研究表明，PDE4D 驱动 BRAF 突变黑色素瘤对 MAPK 抑制剂耐药时 MAPK 通路的重布线，并表明 PDE4 抑制是治疗 BRAF 突变黑色素瘤患者的一种新的治疗选择。
