Deregulation of the pRb-E2F4 axis alters epidermal homeostasis and favors tumor development

E2F/RB activity is altered in most human tumors. The retinoblastoma family of proteins plays a key role in regulating the progression of the cell cycle from the G1 to S phases. This is achieved through negative regulation of E2F transcription factors, important positive regulators of cell cycle entry. E2F family members are divided in two groups: activators (E2F1-E2F3a) and repressors (E2F3b-E2F8). E2F4 accounts for a large part of the E2F activity and is a main E2F repressor member in vivo. Perturbations in the balance from quiescence towards proliferation contribute to increased mitotic gene expression levels frequently observed in cancer. We have previously reported that combined Rb1-Rbl1 and Rb1-E2F1 ablation in epidermis produces important alterations in epidermal proliferation and differentiation, leading to tumor development. However, the possible roles of E2F4 in this context are still to be determined. Here we show the absence of any discernible phenotype in the skin of mice lacking of E2F4. In contrast, the inducible loss of Rb1 in the epidermis of E2F4-null mice produced multiple skin abnormalities including altered differentiation and proliferation, spontaneous wounds, carcinoma in situ development and stem cell perturbations. All these phenotypic alterations are associated with extensive gene expression changes, the induction of c-myc and the Akt activation. Moreover, the whole transcriptome analyses in comparison with previous models generated also revealed extensive changes in multiple repressive complexes and in transcription factor activity. These results point to E2F4 as a master regulator in multiple steps of epidermal homeostasis in Rb1 absence. Gene expression was compared between skin from transgenic Rbf/f;K14creERTM, Rbf/f;K14creERTM;E2F1-/-, Rbf/f;K14creERTM;E2F4-/- and Rbf/f, E2F4-/- mice. All mice were treated with tamoxifen.

大多数人类肿瘤中，E2F/视网膜母细胞瘤（Retinoblastoma, RB）活性均发生异常改变。视网膜母细胞瘤蛋白家族在调控细胞周期由G1期向S期进展的过程中发挥关键作用，该功能通过负向调控E2F转录因子（E2F transcription factors）实现，而E2F转录因子正是细胞周期进入的重要正向调控因子。E2F家族成员可分为两类：激活型亚基（E2F1-E2F3a）与抑制型亚基（E2F3b-E2F8）。E2F4在E2F活性中占比极高，是体内主要的E2F抑制型亚基。细胞由静息状态向增殖状态的平衡紊乱，会导致癌症中常见的有丝分裂相关基因表达水平升高。本团队此前曾报道，在表皮中同时敲除Rb1（Retinoblastoma 1, Rb1）-Rbl1（Retinoblastoma-like 1, Rbl1）与Rb1-E2F1（E2F1），会导致表皮增殖与分化发生显著异常，进而引发肿瘤发生。然而，E2F4在此类肿瘤发生过程中的潜在作用仍有待阐明。本研究显示，E2F4敲除小鼠的皮肤未出现任何可检测到的表型异常。与之相反，在E2F4敲除（E2F4-null）小鼠的表皮中诱导性敲除Rb1，会引发多种皮肤异常，包括分化与增殖异常、自发性伤口、原位癌发生以及干细胞稳态紊乱。所有上述表型异常均与广泛的基因表达改变、c-myc（c-Myc）基因激活以及Akt（蛋白激酶B, Akt）活化密切相关。此外，与此前构建的模型进行全转录组分析对比后发现，多种抑制性复合物以及转录因子活性均发生了广泛改变。上述结果表明，在Rb1缺失的情况下，E2F4是表皮稳态多个环节的核心调控因子。本研究对以下转基因小鼠的皮肤组织进行了基因表达对比：Rbf/f、Rbf/f;K14creERTM、Rbf/f;K14creERTM;E2F1-/-、Rbf/f;K14creERTM;E2F4-/-以及Rbf/f;E2F4-/-小鼠，所有小鼠均经他莫昔芬（Tamoxifen）处理。