Comparative analysis of SV40 17kT and LT function in vivo demonstrates that LT’s C-terminus re-programs hepatic gene expression and is necessary for tumorigenesis in the liver

Abstract

Transformation by Simian Virus 40 (SV40) large T antigen (LT) is mediated in large part by its interaction with a variety of cellular proteins at distinct binding domains within LT. While the interaction of LT's N-terminus with the tumor suppressor Rb is absolutely required for LT-dependent transformation, the requirement for the interaction of LT's C-terminus with p53 is less clear and cell- and context-dependent. Here, we report a line of transgenic mice expressing a doxycycline-inducible liver-specific viral transcript that produces abundant 17kT, a naturally occurring SV40 early product that is co-linear with LT for the first 131 amino acids and that binds to Rb, but not p53. Comparative analysis of livers of transgenic mice expressing either 17kT or full length LT demonstrates that 17kT stimulates cell proliferation and induces hepatic hyperplasia but is incapable of inducing hepatic dysplasia or promoting hepatocarcinogenesis. Gene expression profiling demonstrates that 17kT and LT invoke a set of shared molecular signatures consistent with the action of LT's N-terminus on Rb-E2F-mediated control of hepatocyte transcription. However, 17kT also induces a unique set of genes, many of which are known transcriptional targets of p53, while LT actively suppresses them. LT also uniquely deregulates the expression of a subset of genes within the imprinted network and rapidly re-programs hepatocyte gene expression to a more fetal-like state. Finally, we provide evidence that the LT/p53 complex provides a gain-of-function for LT-dependent transformation in the liver, and confirm the absolute requirement for LT's C-terminus for liver tumor development by demonstrating that phosphatase and tensin homolog (PTEN)-deficiency readily cooperates with LT, but not 17kT, for tumorigenesis. These results confirm independent and inter-dependent functions for LT's N- and C-terminus and emphasize differences in the requirements for LT's C-terminus in cell-type dependent transformation.

Tumor-Specific T Cell Dysfunction Is a Dynamic Antigen-Driven Differentiation Program Initiated Early during Tumorigenesis

“…However, we also identified many genes and pathways that were uniquely dysregulated in tumor-specific T cells, including the transcription factors Foxo1 , Tcf7 , Foxp1 , Blimp1 , and Batf and inhibitory receptors Pdcd1 , Cd244 , 2B4 , Tim3 (Figures 3C). Thus, context-specific signals that operate in pre-malignant liver lesions comprised of transformed hepatocytes appear to have distinct consequences on the differentiation program and fate of T cells encountering antigen, compared to normal hepatocytes (Comerford et al, 2012). …”

Acetate Dependence of Tumors

“…Cohorts of male and female TAg mice with and without ACSS2 were generated as described in Experimental Procedures and provided with drinking water supplemented with 10 µg/mL doxycycline for 42–45 days. This regimen promotes reproducible and robust multifocal tumor development in a background of hepatic hyperplasia (Comerford et al, 2012). Post-sacrifice, livers were scored for tumor development using a non-linear tumor-burden scale based on the number and size of visible tumors on the surface of the liver, % liver/body weight, and the relative amount of tumor-free liver as described in detail (Table S2).…”

Hepatoblastoma modeling in mice places Nrf2 within a cancer field established by mutant β-catenin

“…Preparation of liver proteins and total liver RNA and for Northern and Western blotting was performed as previously described (23). Antibodies used for Western blotting are listed in Supplemental Table 10.…”