Assessing Tumor Progression Factors by Somatic Gene Transfer into a Mouse Model: Bcl-xL Promotes Islet Tumor Cell Invasion

Abstract

Tumors develop through multiple stages, implicating multiple effectors, but the tools to assess how candidate genes contribute to stepwise tumor progression have been limited. We have developed a novel system in which progression of phenotypes in a mouse model of pancreatic islet cell tumorigenesis can be used to measure the effects of genes introduced by cell-type-specific infection with retroviral vectors. In this system, bitransgenic mice, in which the rat insulin promoter (RIP) drives expression of both the SV40 T antigen (RIP-Tag) and the receptor for subgroup A avian leukosis virus (RIP-tva), are infected with avian viral vectors carrying cDNAs encoding candidate progression factors. Like RIP-Tag mice, RIP-Tag; RIP-tva bitransgenic mice develop isolated carcinomas by ∼14 wk of age, after progression through well-defined stages that are similar to aspects of human tumor progression, including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. When avian retroviral vectors carrying a green fluorescent protein marker were introduced into RIP-Tag; RIP-tva mice by intra-cardiac injection at the hyperplastic or early dysplastic stage of tumorigenesis, approximately 20% of the TVA-positive cells were infected and expressed green fluorescent proteins as measured by flow cytometry. Similar infection with vectors carrying cDNA encoding either of two progression factors, a dominant-negative version of cadherin 1 (dnE-cad) or Bcl-xL, accelerated the formation of islet tumors with invasive properties and pancreatic lymph node metastasis. To begin studying the mechanism by which Bcl-xL, an anti-apoptotic protein, promotes invasion and metastasis, RIP-Tag; RIP-tva pancreatic islet tumor cells were infected in vitro with RCASBP-Bcl-xL. Although no changes were observed in rates of proliferation or apoptosis, Bcl-xL altered cell morphology, remodeled the actin cytoskeleton, and down-regulated cadherin 1; it also induced cell migration and invasion, as evaluated using two-chamber transwell assays. In addition, myosin Va was identified as a novel Bcl-xL-interacting protein that might mediate the effects of Bcl-xL on tumor cell migration and invasion. Cancer cells accumulate multiple genetic alterations. Some of these contribute to tumor development while others are a mere by-product of genomic instability. To determine whether a candidate gene can promote tumor development, we have developed a novel experimental system using engineered viruses to deliver genes into premalignant lesions. We used genetically engineered mice in which both an oncogene (SV40 T antigen) and a specific docking molecule for the virus are produced in β cells in the pancreatic islets of Langerhans. Tumors form in only a subset of the islets expressing this oncogene, indicating that tumor development requires other events. Since these precancerous cells also express the virus docking molecule, we could deliver candidate progression genes via the virus to see whether they contributed to tumor progression. We show that genes encoding two proteins (a survival factor, Bcl-xL, and an inhibitory form of the cell adhesion molecule cadherin 1) can be delivered into premalignant β cells and thereby enhance tumorigenesis. Both of these proteins were previously implicated in tumor progression, confirming that our technique can identify such progression genes. Moreover, we find that Bcl-xL promotes tumor cell migration and invasion by a mechanism distinct from its known role in cell survival.