Distinct pathways of genomic progression to benign and malignant tumors of the liver

Abstract

We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of beta-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1alpha. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of beta-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated beta-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated beta-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.

Fig. 1.

Gross pathology of the liver in a MET transgenic mouse. Shown is a liver removed from an 8-month-old line 4 LAP-tTA/TRE-MET transgenic mouse. Doxycycline was withheld for the life of the animal. The liver contained nodules of both HCA (a) and HCC (c), which were verified by histological analysis.

Fig. 2.

Morphological and molecular progression during tumorigenesis in the livers of MET transgenic mice. Doxycyline was withheld for the life of all animals. Sections of livers from LAP-tTA transgenic animals (A, F, K, and P) or line 3 LAP-tTA/TRE-MET animals at 1 month of age (B, G, L, and Q), 2 months of age (C, H, M, and R), or >3 months of age (D, E, I, J, N, O, S, and T) are shown. Livers were sectioned and analyzed by microscopy after H&E staining (A–E), immunohistochemistry with an antibody against phosphorylated Met as a surrogate for kinase activity (F–J), immunohistochemistry with an antibody against human Met (K–O), or immunohistochemistry with an antibody against β-catenin (P–T). Images represent control livers (A, F, K, and P), hyperplastic foci (B, G, L, and Q), dysplastic foci (C, H, M, and R), HCCs (D, I, N, and S), or HCAs (E, J, O, and T). hf, hyperplastic focus; df, dysplastic focus; nt, nontumor tissue; ad, HCA.

Fig. 3.

Survival of mice after hydrodynamic transfection. Six- to 8-week-old FVB/N mice were hydrodynamically transfected with the indicated constructs and observed. The number of animals in each group is indicated to the right of each listed group. The black arrowhead indicates the point of hydrodynamic transfection.

Fig. 4.

Recurrence of HCC after regression. Eight-month-old LAP-tTA/TRE-MET line 3 mice with tumors were either placed on doxycycline (E, dashed line; n = 18) or continued on a regular diet (E, solid line; n = 15) and then followed for the remainder of the experiment or killed after 6 months (A–D and F). (A–D) Livers from killed mice were sectioned and analyzed by microscopy after H&E staining (A and C) or immunohistochemistry with an antibody against β-catenin (B and D). (A and B) Putative residual tumor cells (arrowheads) embedded in scar tissue adjacent to normal parenchyma. (C and D) Cells from a recurrent tumor nodule. (E) Survival of tumor-bearing mice was analyzed after administration of doxycycline (black arrow). (F) Western Blot analysis was performed on lysates from liver with antibodies against the indicated antigens. Lane 1, nontumor tissue from a LAP-tTA/TRE-MET line 3 mouse with recurrent tumor; lanes 2–4, recurrent tumors from LAP-tTA/TRE-MET line 3 mice maintained in the presence of doxycycline for 1 year; lane 5, wild-type FVB/N control; lane 6, nontissue from a LAP-tTA/TRE-MET line 3 mouse maintained in the absence of doxycycline; lane 7, HCC from a LAP-tTA/TRE-MET line 3 mouse maintained in the absence of doxycycline.