Iveland Tobias S, Hagen Lars, Sharma Animesh, Sousa Mirta M L, Sarno Antonio, Wollen Kristian Lied, Liabakk Nina Beate, Slupphaug Geir
Department of Clinical and Molecular Medicine, Faculty of Medicine and Health, Norwegian University of Science and Technology, 7491, Trondheim, Norway.
Cancer Clinic, St. Olav's Hospital, Trondheim, Norway.
J Transl Med. 2020 Apr 7;18(1):159. doi: 10.1186/s12967-020-02318-8.
HDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used in the treatment of lymphocyte-derived malignancies, but their mechanisms of action remain poorly understood. Here we aimed to identify novel protein targets of HDACi in B- and T-lymphoma cell lines and to verify selected candidates across several mammalian cell lines.
Jurkat T- and SUDHL5 B-lymphocytes were treated with the HDACi SAHA (vorinostat) prior to SILAC-based quantitative proteome analysis. Selected differentially expressed proteins were verified by targeted mass spectrometry, RT-PCR and western analysis in multiple mammalian cell lines. Genomic uracil was quantified by LC-MS/MS, cell cycle distribution analyzed by flow cytometry and class switch recombination monitored by FACS in murine CH12F3 cells.
SAHA treatment resulted in differential expression of 125 and 89 proteins in Jurkat and SUDHL5, respectively, of which 19 were commonly affected. Among these were several oncoproteins and tumor suppressors previously not reported to be affected by HDACi. Several key enzymes determining the cellular dUTP/dTTP ratio were downregulated and in both cell lines we found robust depletion of UNG2, the major glycosylase in genomic uracil sanitation. UNG2 depletion was accompanied by hyperacetylation and mediated by increased proteasomal degradation independent of cell cycle stage. UNG2 degradation appeared to be ubiquitous and was observed across several mammalian cell lines of different origin and with several HDACis. Loss of UNG2 was accompanied by 30-40% increase in genomic uracil in freely cycling HEK cells and reduced immunoglobulin class-switch recombination in murine CH12F3 cells.
We describe several oncoproteins and tumor suppressors previously not reported to be affected by HDACi in previous transcriptome analyses, underscoring the importance of proteome analysis to identify cellular effectors of HDACi treatment. The apparently ubiquitous depletion of UNG2 and PCLAF establishes DNA base excision repair and translesion synthesis as novel pathways affected by HDACi treatment. Dysregulated genomic uracil homeostasis may aid interpretation of HDACi effects in cancer cells and further advance studies on this class of inhibitors in the treatment of APOBEC-expressing tumors, autoimmune disease and HIV-1.
组蛋白去乙酰化酶抑制剂（HDACi）属于一类新型化疗药物，越来越多地用于治疗淋巴细胞源性恶性肿瘤，但其作用机制仍知之甚少。在这里，我们旨在确定HDACi在B细胞和T淋巴瘤细胞系中的新蛋白靶点，并在多个哺乳动物细胞系中验证选定的候选靶点。
在基于稳定同位素标记氨基酸的细胞培养定量蛋白质组分析之前，用HDACi SAHA（伏立诺他）处理Jurkat T细胞和SUDHL5 B淋巴细胞。通过靶向质谱、RT-PCR和western分析在多个哺乳动物细胞系中验证选定的差异表达蛋白。通过LC-MS/MS对基因组尿嘧啶进行定量，通过流式细胞术分析细胞周期分布，并通过FACS监测小鼠CH12F3细胞中的类别转换重组。
SAHA处理分别导致Jurkat和SUDHL5中125和89种蛋白质的差异表达，其中19种受到共同影响。其中包括几种以前未报道受HDACi影响的癌蛋白和肿瘤抑制因子。几种决定细胞dUTP/dTTP比率的关键酶被下调，并且在两种细胞系中我们都发现UNG2（基因组尿嘧啶清除中的主要糖基化酶）大量减少。UNG2的减少伴随着超乙酰化，并由蛋白酶体降解增加介导，与细胞周期阶段无关。UNG2降解似乎是普遍存在的，并且在不同来源的几种哺乳动物细胞系和几种HDACi中都观察到。UNG2的缺失伴随着自由循环的HEK细胞中基因组尿嘧啶增加30 - 40%，以及小鼠CH12F3细胞中免疫球蛋白类别转换重组减少。
我们描述了几种以前在转录组分析中未报道受HDACi影响的癌蛋白和肿瘤抑制因子，强调了蛋白质组分析在鉴定HDACi治疗的细胞效应器方面的重要性。UNG2和PCLAF明显普遍的减少将DNA碱基切除修复和跨损伤合成确立为受HDACi治疗影响的新途径。基因组尿嘧啶稳态失调可能有助于解释HDACi在癌细胞中的作用，并进一步推进对这类抑制剂在治疗表达APOBEC的肿瘤、自身免疫性疾病和HIV - 1方面的研究。
