采用新型转基因小鼠模型探讨E2F3扩增和Rb缺失在膀胱肿瘤发生发展中的作用机制

Bladder cancer is the most urinary tract malignancy in China.The challenge of bladder cancer research now is how to decipher and study the wealth of genetic and molecular data that is evolving from the characterization of human bladder tumors to identify genetic events that cooperate and are critically important to the cancer phenotype. However, the number of existing mouse models and how well they represent human bladder cancer lag far behind models in many other solid malignancies, even malignancies with far lower incidence. Classic mouse tumor models, which used transgenic mice or conventional knockouts are now being superseded by models that are based on conditional knockouts and mice that carry regulatable oncogenes. In these mice, somatic mutations can be induced in a tissue-specific and time-controlled fashion, which more faithfully mimics sporadic tumour formation. These newer generation models provide exciting new opportunities to gain insight into the contribution of known and unknown genes in cancer and mimic human cancer better than ever before..Transcription factor E2F3 has a critical role in controlling cell cycle in G1/S phase transition. In G0, E2F3 is bound by the tumor suppressor pRb. In G1, pRb is phosphorylated and E2F3 is released to control DNA synthesis. Inactivation of the RB pathway is common in cancer and this includes bladder cancer. Roughly, 1 in 3 invasive bladder tumors have E2F3 overexpression beginning at the earliest stages of invasion. We performed fluorescence in situ hybridization to analyze the E2F3 gene locus in bladder tumors , and identified E2F3 overexpression is invariably due to gain or amplification. Further analysis of the same tumors identified pRb inactivation in all tumors by either absence or hyperphosphorylation of RB protein. Why do RB inactivation and E2F3 amplification coexist in the same tumor? We hypothesize the deregulation of E2F3 activity through the loss of Rb function and the further increase in E2F3 expression through gene amplification represent separate molecular events with different physiological and phenotypical outcomes. In this context RB mutation or hyperphosphorylation would provide a proliferative advantage to tumor cells whereas E2F3 hyperactivity would provide functions that extend beyond the traditional control of the cell cycle and be instrumental in the cancer phenotype. This may include cell differentiation or invasion as well as other functions. .To test this hypothesis two specific aims will be developed i) To construct a bladder-specific, time-specific E2F3 inducible mouse. ii) To determine whether increased expression of E2F3 can transform the phenotype of an H-Ras mouse model of superficial bladder cancer alone or in combination with loss of RB function. If successful this proposal will develop a desperately needed mouse model of bladder cancer that closely.

转录因子E2F3在细胞周期G1/S过渡中起着关键作用，我们前期对膀胱癌研究表明Rb失活使E2F3活化发挥其传统生物学作用，但E2F3基因扩增则是具有不同生理学和表型的独立的分子事件，其突破传统的细胞周期控制，具有肿瘤表型改变的细胞分化或侵袭等功能。因此本课题通过建立具有膀胱上皮组织特异性、时间可控性的E2F3转基因小鼠模型，研究明确E2F3扩增或联合RB失活与改变浅表性膀胱肿瘤（H-ras基因小鼠模型）表型的关联；利用该模型与Rb基因敲除小鼠系、uroplakin II-H-Ras小鼠系杂交，获得不同基因型小鼠，采用免疫组化、实时定量PCR、Western blot等手段观察E2F3、Rb及有关基因在不同时间点不同基因型小鼠中的表达变化，旨在揭示E2F3在膀胱肿瘤中的新型非细胞周期功能及其与RB和Ras-MAPK信号通路的协同和相互作用机制，为膀胱癌新型治疗药物的筛选提供实验依据。

E2F3是一种调节细胞增殖的转录激动因子，一般认为其活性受肿瘤抑制因子RB的调节和控制。我们在对人体膀胱癌研究的基础上提出了在膀胱肿瘤的发生发展过程中，E2F3扩增和Rb缺失实际上是各自独立的基因异常事件，但是它们可能又共同参与膀胱肿瘤发生发展机制的假设。本课题拟通过建立的具有时间可控性、膀胱上皮组织特异性的E2F3a转基因小鼠模型与Rb基因敲除小鼠系、uroplakin II-H-Ras小鼠系杂交，获得不同基因型小鼠，通过免疫组化、实时定量PCR、Western blot等手段检测E2F3a、Rb及其细胞周期相关基因在不同时间点不同基因型小鼠中的表达变化，以验证假设。若该假设得以验证，将是对传统的Rb /E2F通路的补充，同时为E2F3在肿瘤中的机制研究另辟蹊径，具有重要的理论意义。该研究不但有望初步阐明E2F3和RB在膀胱肿瘤发生发展中的机制，而且还可为膀胱癌的靶向治疗提供药物靶点。.通过本课题的研究，我们成功建立了具有时间可控性、膀胱上皮组织特异性的E2F3a转基因小鼠模型。采用RNAi技术使E2F3在浸润性膀胱癌细胞系中低表达，通过蛋白质印迹法、PCR进行检测及后续实验，通过CHIP实验等观察E2F3在HT1376和TCCSUP细胞系中与信号转导及转录激活因子3（STAT3）和Ets1之间及其相关侵袭细胞因子的调节关系。发现E2F3过表达在人膀胱癌细胞系中通过Ets1和STAT3调节免疫相关基因的表达，E2F3的过表达促进Ets1和STAT3的表达。通过免疫组织化学法验证E2F3和Rb在112例膀胱癌组织中的表达，分析其表达水平与患者的临床分期、病理分级之间的关系。发现E2F3在浸润性膀胱癌中高表达，并且与Rb失活存在着密切的关系，同时E2F3高表达的膀胱肿瘤患者预后更差。另外，应用IHC对132例膀胱肿瘤组织标本研究发现，部分肿瘤表达PD-L1但浸润肿瘤的免疫细胞不表达PD-L1，部分肿瘤本身不表达PD-L1但浸润肿瘤的免疫细胞表达PD-L1，部分肿瘤和浸润肿瘤的免疫细胞均表达PD-L1，部分肿瘤和浸润肿瘤的免疫细胞均不表达PD-L1。E2F3的表达水平与PD-L1的表达存在正相关。通过ChIP-seq在人体膀胱肿瘤细胞系的研究中证实，E2F3的ChIP-seq的信号在PDL1的启动子附近存在富集现象。