Genesis of Liver Carcinomas with Oval Cell Traits

描述（由申请人提供）：尽管有令人信服的证据表明成人肝脏中存在双能祖细胞，但这些祖细胞在肝癌发生中的作用仍然是一个有争议的话题。基于我们过去对胆管细胞和卵形细胞（大多数肝癌原激活的双能性胆道祖细胞）的研究经验，我们继续努力鉴定和表征胆道树中存在的双能性祖细胞，并确定它们在肝和胆道癌发生中的作用。在过去的4年里，我们对胆管细胞标记阳性（CMP），双能性，胎儿肝上皮细胞（FLEC）的研究已经产生了新的基于单克隆抗体的分离双能性CMP-FLEC的方案。将二肽基肽酶IV （DPPIV）阳性大鼠的CMP-FLEC分离物移植到经逆转录酶/部分肝切除术（R/PH）治疗的DPPIV缺陷大鼠体内，结果发现CMP-FLEC在R/PH处理的成人肝脏中具有比胎儿肝母细胞和其他非实质细胞类型更高的生长能力。在目前的建议中，为双能性CMP-FLEC开发的分离方案将应用于分离新生儿和成年大鼠肝脏中表型等效的cmp -肝上皮细胞（CMP-LEC）。我们假设这些胎儿样的CMP-FLEC将具有类似于卵圆细胞的肝细胞分化能力，并在体外自发转化和体内进展为HCC后保持这种能力。在Specific Aim 1中，我们将采用一种用丝裂霉素C （mitoC/PH）代替逆转录酶的快速移植模型来验证以下假设：出生后2岁时首次出现的胆管细胞标志物OC.4的表达，可以识别出肝细胞分化能力大大降低的成熟CMP-LEC。在Specific Aim 2中，我们将验证自发转化的CMP-LEC和卵形细胞（而非BDEC）在mitoC/PH处理大鼠中发生不完全肝细胞分化并进展为CMP-HCC的假设。通过选择塑料和/或软琼脂，转染ErbB2或原位暴露于致癌物，将加速CMP-LEC的自发转化。在Specific Aim 3中，基因组学和蛋白质组学方法将用于绘制BDEC、CMP-LEC和卵形细胞自发转化过程中的信号通路，这些信号通路促进生存，并与ErbB2合作，赋予锚定独立/侵入性生长。特异性Aim 4将继续对BD.1进行表征，BD.1是一种170 kDa的蛋白，由胆管细胞表达，而不是卵形细胞，与微管相关蛋白CLIP170形成稳定的复合物。我们将验证BD.1缺失会改变微管/着丝点动力学从而促进非整倍体的假设。这些研究将为双能祖细胞在肝癌中的作用提供新的见解。

DESCRIPTION (provided by applicant): Although there is compelling evidence for the presence of bipotent progenitor cells in the adult liver, the role of these progenitors s in hepatocarcinogenesis is still a subject of debate. Building on our past experience with cholangiocytes and oval cells, bipotent biliary progenitors activated by most hepatocarcinogens, we have continued with our efforts to identify and characterize bipotent progenitors present in the biliary tree and to ascertain their role in hepato-and cholangio-carcinogenesis. Over the past 4 years, our studies of cholangiocyte marker positive (CMP), bipotent, fetal liver epithelial cells (FLEC) have yielded novel monoclonal antibody based schemes for isolating bipotent CMP-FLEC. Results from transplantation of CMP-FLEC isolates from dipeptidyl peptidase IV (DPPIV) positive rats into DPPIV deficient host rats treated with retrorsine/partial hepatectomy (R/PH) led to the unexpected finding that CMP-FLEC possesses a much higher capacity for growth in the R/PH treated adult liver than fetal hepatoblasts and other nonparenchymal cell types. In the current proposal, isolation schemes developed for bipotent CMP-FLEC will be applied to the isolation of phenotypically equivalent CMP-liver epithelial cells (CMP-LEC) from newborn and adult rat liver. We hypothesize that these fetal-like CMP-FLEC will possess a capacity for hepatocytic differentiation similar to oval cells and will retain this capacity following spontaneous transformation in vitro and progression to HCC in vivo. In Specific Aim 1, we will employ a rapid transplantation model that replaces retrorsine with mitomycin C (mitoC/PH) to test the hypothesis that the expression of the cholangiocyte marker OC.4, a marker first seen at 2 after birth, identifies mature CMP-LEC that have a greatly diminished capacity for hepatocytic differentiation. In Specific Aim 2, we will test the hypothesis that spontaneously transformed CMP-LEC and oval cells but not BDEC will undergo incomplete hepatocytic differentiation in mitoC/PH treated rats and progress to CMP-HCC. Spontaneous transformation of CMP-LEC will be accelerated by selection on plastic and/or soft agar, transfection with ErbB2 or exposure to carcinogen in situ. In Specific Aim 3, genomic and proteomic methodologies will be used to map the signaling pathways operative during the spontaneous transformation of BDEC, CMP-LEC and oval cells that promote survival and cooperate with ErbB2 to confer anchorage independent/invasive growth. Specific Aim 4 will continue with the characterization of BD.1, a 170 kDa protein expressed by cholangiocytes but not oval cells that forms stable complexes with CLIP170, a microtubule associated protein. We will test the hypothesis that loss of BD.1 alters microtubule/kinetochore dynamics in a manner that promotes aneuploidy. The proposed studies will provide new insights into the role of bipotent progenitors in liver cancer.