HDAC6通过PTPN1调控错误蛋白降解在肢端雀斑样黑色素瘤中的作用机制

Acral lentiginous melanoma (ALM) is the most common subtype (about 50%-70%, compared with 5% in Caucasian) in China, with distinct epidemiological, clinical and mutational profiles. Yet few systemic analysis and mechanism exploration for the oncogenesis of ALM had been presented. Utilizing clinical tissue samples for proteomic research, our group have previously demonstrated that that ALM has different molecular characteristics comparing other subtypes, indicating that ALM might have unique mechanism in tumorigenesis and pathogenesis. Histone deacetylases 6 (HDAC6) have recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment. This includes deacetylation of multiple non-histone targets, such as members of oncogenic- and metabolic- related pathways. In recent work, using ALM tissue for genetic screening and clinical research, we found the prominent role of HDAC6 in the key biological processes of melanoma. Further, protein spectrum analysis demonstrated that Tyrosine-Protein Phosphatase Non-Receptor Type 1 (PTPN1) may play as potential partner of HDAC6. Proved by our subsequent work, organizationally and cellularly, HDAC6 could regulate the occurrence and development of melanoma through PTPN1, and bioinformatics analysis showed that one of its most important functions may be as a key regulator of protein degradation. Unfolded/misfolded protein degradation encompassed various pathways which melanoma cells were heavily reliant upon for its growth and survival. The rapid cell cycle and the highest mutation burden of melanoma cells made them more dependent on the degradative aspect of unfolded/misfolded protein. However, the molecular mechanism that illustrated these fundamental degradative process remains mysterious. Our preliminary experiment included that there was a positive correlation between Beclin-1, NPL4, UFD1, and PSMA4 with PTPN1. Thus, we hypothesized that HDAC6 may regulate unfolded/misfolded protein degradation in ALM through PTPN1. To test this hypothesis, this project intends to research the precise molecular mechanism of HDAC6 regulate unfolded/misfolded protein degradation pathway through PTPN1. We plan: A) To determine the impacts of HDAC6 regulate PTPN1 in ALM unfolded/misfolded protein degradation. B) To characterize the site of HDAC6-PTPN1 interaction. C) To investigate the molecular mechanism underlying regulation of HDAC6-PTPN1 in ALM unfolded/misfolded protein degradation. This study is conformity to the National Natural Science Foundation requirements as “specific metabolic patterns of tumor cells and their biological behavior”, More significantly, this study will explore our understanding of the mechanism of unfolded/misfolded protein degradation in ALM, and lay a theoretical basis for the prognosis and individualized treatment of ALM targeting HDAC6.

肢端雀斑样黑色素瘤（ALM）是我国临床最常见的病理类型，错误蛋白降解是影响ALM发生发展的重要环节，但少有针对ALM错误蛋白降解作用机制的研究。申请人前期工作发现组蛋白去乙酰化酶6（HDAC6）可能是调控黑色素瘤错误蛋白降解的关键，后续研究进一步证明HDAC6可通过酪氨酸蛋白磷酸酶非受体1型（PTPN1）影响黑色素瘤细胞的生物学功能。申请人通过预实验发现PTPN1与错误蛋白降解过程的关键分子存在正相关趋势，提示PTPN1可能是HDAC6调控ALM错误蛋白降解的靶分子。据此我们提出假说：ALM中，HDAC6可通过PTPN1调控关键蛋白表达参与错误蛋白降解的调控。本项目将系统探讨ALM错误蛋白降解的调控形式，从而揭示HDAC6通过PTPN1调控错误蛋白降解在ALM的作用及分子机制。这将有助于深入理解ALM错误蛋白降解的精细过程，为靶向HDAC6分子的ALM预后判断及个体化治疗奠定理论基础。

肢端雀斑样黑色素瘤（ALM）是我国最常见的病理类型，但少有针对ALM发生发展机制的研究。项目首先应用黑色素瘤单病种数据库和开源的大数据技术，证明组蛋白去乙酰化酶6（HDAC6）和酪氨酸蛋白磷酸酶非受体1型（PTPN1）在肢端雀斑样黑色素瘤中特征性高表达；并发现PTPN1高表达与肢端雀斑样黑色素瘤转移和不良预后相关。项目通过蛋白质谱、Co-IP、构建突变体等技术，证明HDAC6直接促进PTPN1表达，该过程不依赖HDAC6的组蛋白去乙酰化活性。项目通过CCK-8、划痕实验、垂直迁移实验、Transwell实验等体外研究证明，PTPN1能促进黑色素瘤的特征性垂直生长，并促进黑色素瘤的转移。项目进一步通过PET-CT检测裸鼠动物模型，证明PTPN1促进黑色素瘤转移。在机制研究中，项目通过Phospho-Kinase Array分析、Western blot实验、Co-IP实验和Transwell实验等技术，发现PTPN1可能通过去磷酸化修饰Src的Tyr530位点促进黑色素瘤的侵袭转移；并发现PTPN1可以修饰细胞骨架蛋白ACTN4，调控细胞的侵袭能力。利用成熟技术，项目还发现了PTPN1与PD-L1具有潜在相互作用。项目按照计划书实施，不仅取得了预期成果，且有所拓展，对我们深入理解ALM的发生发展机制具有重要科学意义，并提供了PTPN1这一肢端雀斑样黑色素瘤的潜在治疗靶点。该项目的结果紧密结合临床需求，具有转化应用潜力。