Genesis of Liver Carcinomas with Oval Cell Traits

描述(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S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圆细胞的经验，我们继续努力识别和鉴定胆管树中存在的双能前体细胞，并确定它们在肝和胆管癌变中的作用。在过去的4年里，我们对胆管细胞标志物阳性(CMP)、双能胎肝上皮细胞(FLEC)的研究已经产生了新的基于单抗的分离双能CMP-FLEC的方案。将来自二肽基肽酶IV(DPPIV)阳性大鼠的CMP-FLEC分离株移植到DPPIV缺陷大鼠的逆转录/部分肝切除(R/PH)治疗的宿主大鼠体内，结果意外地发现，在R/PH治疗的成人肝脏中，CMP-FLEC具有比胎肝母细胞和其他非实质细胞类型更高的生长能力。在目前的提案中，为双能CMP-FLEC开发的分离方案将被应用于从新生和成年大鼠肝脏分离表型等同的CMP-肝上皮细胞(CMP-LEC)。我们推测，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肝细胞癌后保持这种能力。在具体目标1中，我们将使用一种快速移植模型，用丝裂霉素C(mitoC/PH)取代retrorine，以检验胆管细胞标记物OC.4的表达，该标记物在出生后2岁时首次出现，表明成熟的CMP-LEC具有极大的肝细胞分化能力。在特定的目标2中，我们将检验这样的假设，即自发转化的CMP-LEC和卵圆细胞，而不是BDEC，将在MitoC/PH处理的大鼠中经历不完全的肝细胞分化，并进展为CMP-HCC。在塑料和/或软琼脂上进行选择、ErbB2基因转染或原位致癌物暴露可加速CMP-LEC的自发转化。在具体目标3中，将使用基因组和蛋白质组方法来定位在BDEC、CMP-LEC和卵圆细胞自发转化过程中起作用的信号通路，这些信号通路促进存活并与ErbB2合作实现锚定非依赖性/侵袭性生长。具体目标4将继续BD.1的特征，BD.1是一种170 kDa的蛋白，由胆管细胞表达，而不是由卵圆细胞表达，它与微管相关蛋白CLIP170形成稳定的复合体。我们将检验这一假设，即BD.1的丢失以促进非整倍体的方式改变微管/动粒动力学。拟议的研究将为双能前体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.