Research in my laboratory is directed towards identifying the cellular targets of hepatocarcinogens and understanding the molecular events leading to the malignant conversion of hepatocytes and liver progenitor cells. A long standing interest has been the role of cell adhesion molecules (CAMs) in liver regeneration and carcinogenesis. We have restricted our investigations to two CAMs in the Ig super gene family: CEACAM1 which is lost on liver carcinomas as well as carcinomas in the bladder, colon, and prostate; Nectin-like protein 5 (Necl-5), a CAM that is not expressed by normal resting hepatocytes but is dramatically upregulated following liver injury and is expressed constitutively at high levels on hepatocellular carcinomas. In the rat, there are two major splice variants of CEACAM1 designated CEACAM1-L (long) and CEACAM1-S (short) that differ in the length of their cytoplasmic domains. The cytoplasmic domain of CEACAM1-L, mediates signaling via the tyrosine phosphorylation sites in its ITIM residues, signaling that suppresses the growth of tumor cells when expression is restored. We have previously shown that the cytoplasmic domain is necessary and sufficient for tumor suppression, a finding we are trying to exploit for cancer therapy by developing a small peptide corresponding to the region of the 70 amino acid cytoplasmic domain determined to be necessary and sufficient for tumor suppression. Although the short isoform does not display tumor-suppressor activity, it has been shown to play a role in glandular morphogenesis of mammary epithelial cells. In addition, we have found serendipitously that restoration of CEACAM1-S expression induces tumorigenicity in a non-tumorigenic clone derived from a rat hepatocellular carcinoma. Current research is aimed at determining the role of two phosphorylation sites in the 10 amino acid CEACCAM1-S cytoplasmic domain in restoring tumorigenicity. We are also examining the involvement in tumor suppression of three GXXXG dimerization motifs and two tyrosine residues in the transmembrane domain, the latter having been shown to be required for signaling mediated by surface IgM and several other signaling molecules.
Another aspect of ongoing research is the identification of hepatic progenitor cells involved in liver regeneration and carcinogenesis. Although there is compelling evidence for the presence of bipotent progenitor cells in the adult liver, the role of these progenitors in hepatocarcinogenesis is still a subject of debate. Building on our past experience with cholangiocytes and oval cells, a bipotent biliary progenitor activated by most hepatocarcinogens, we have focused on identifying bipotent progenitors present in the biliary tree and ascertaining their role in hepato- and cholangio- carcinogenesis. Over the past 3 years, we have developed novel monoclonal antibody based isolation schemes for bipotent liver progenitors that persist in the newborn rat liver in relative high numbers. We have examined the ability of fetal liver cholangiocytes, hepatoblasts, and non-parenchymal cells isolated from dipeptidyl peptidase IV (DPPIV)-positive rats to regenerate the liver of DPPIV-deficient host rats treated with retrorsine and partial hepatectomy (R/PH). In this transplantation model, DPPIV serves as a marker for donor derived cells and retrorsine is used to compromise the ability of host liver cells to regenerate the liver after partial hepatectomy but has no effect on the regenerative response of transplanted liver cells. Results from transplantation of cholangiocyte marker positive fetal liver cells (CMP-FLEC) produced the unexpected finding that the capacity of CMP-FLEC for growth in the R/PH treated adult liver is much higher than that of fetal hepatoblasts and other nonparenchymal cell types. In ongoing studies, we are using the retrorsine transplantation model to address the question of whether immature cholangiocyte marker positive newborn liver cells (CMP-NBLC) retain an ability to differentiate into hepatocytes and regenerate the liver of R/PH treated rats. To assess the role of CMP-NBLC in hepatocarcinogenesis, we are employing a modified version of the resistant hepatocyte carcinogenesis protocol to test the hypothesis that neonatal treatment with diethylnitrosamine (DENA) generates initiated CMP-NBLC capable of progression to cholangiocyte marker positive hepatocellular carcinoma (CMP-HCC).
Another aspect of ongoing research is the delineation of changes in gene expression that are involved in the process of cholangiocarcinogenesis. We have found that at high passage, bile duct epithelial cultures (BDEC) undergo spontaneous transformation, as evidenced by the formation in vivo of desmoplastic ductular adenocarcinomas, anchorage independent growth in vivo, and elevated expression of activated ErbB2 and COX-2. Growth on soft agar and tumor formation were also acquired by high passage but not by low passage anchorage dependent BDEC following stable transduction with an activated ErbB2 retrovirus, suggesting that ErbB2 was necessary but not sufficient for neoplastic transformation. Functional genomic analysis with RNAi will be utilized in future studies to identify genes that cooperate with (high passage) or antagonize (low passage) ErbB2 mediated spontaneous transformation of bile duct epithelial cells.
We have recently discovered that neoplastic progression of BDEC in vitro is closely associated with aneuploidy, a genetic abnormality appearing after as few as 30 passages. Closely associated with the acquisition of aneuploidy is the loss of a 170 kDa protein, designated BD.1, a cytoplasmic protein originally identified using hybridoma technology as an Monoclonal antibody (MAb) defined epitope expressed by cholangiocytes but not by oval cells, stem-like liver cells that proliferate in response to a variety of carcinogens. We now have evidence that BD.1 is either identical to or complexed with one of two 170 kDa microtubule associated proteins, CLIP 170 and CLASP, a conclusion based on the down regulation of BD.1 during S phase and by co-precipitation and co-localization with CLIP 170. Our current investigations are centered on determining the identify of BD.1 using mass spectrometry and on testing the hypothesis that the loss of BD.1 alters microtubule/kinetochore dynamics in a manner that promotes aneuploidy.
A long term goal of our liver progenitor research is to ascertain whether any of the lineage markers defined in the rat have human counterparts that can be used to isolate progenitor cells from human liver. Of the lineage markers we have identified to date, OC.10 is the only surface marker currently available that recognizes the cholangiocytes forming the nascent ductal structures in fetal rat liver. MAb OC.10 has proven to be invaluable for isolating bipotent cholangiocytes from fetal and newborn rat liver. These valuable features provided a strong impetus to identify the protein bearing the epitope recognized by MAb OC.10. Using a variety of biochemical and immunochemical methods, we have determined that MAb OC.10 defines an epitope unique to a cell surface form of Hsc70 expressed by fetal, newborn, and adult cholangiocytes. Current efforts are focused on determining the structural basis for the selective reactivity of MAb OC.10 with Hsc70 on the surface of cholangiocytes but not with the cytoplasmic forms of Hsc70 expressed by hepatocytes. The ultimate goal of this analysis is to establish a rationale basis for generating MAbs against the human cholangiocyte equivalent of OC.10. Most recently, we have shown by indirect immunofluorescence that the reactivity of affinity purified polyclonal anti Hsc70 antibodies is confined to the surface of both hepatocytes and bile ducts, suggesting that the surface forms of Hsc70 in rat liver carries both common (polyclonal anti Hsc70) and cell-type specific (MAb OC.10) epitopes. In addition, we have preliminary data suggesting that the cell surface epitope recognized by MAb OC.10 is restricted to a small subset of CK19 positive ductules in human liver. We are currently using confocal microscopy to obtain three dimensional reconstructions of these MAb OC.10 reactive ductules to determine if they have a morphology and localization consistent with the canals of Hering, a specificity that could be exploited for the isolation of bipotent cholangiocyte progenitors from human liver.