Clonal Selection in Malignant Transformation of Human Fibroblasts Transduced with Defined Cellular Oncogenes

Alka M Mahale,Zahid A.T Khan,Gouri J Nanjangud,Makoto Igarashi,Stuart A Aaronson,Rui Fang Qiao,Shen Yao,Sam W Lee

doi:10.1158/0008-5472.can-07-3021

Abstract

Recent evidence has implied that disruption of a limited number of defined cellular pathways is necessary and sufficient for neoplastic conversion of a variety of normal human cell types in tissue culture. We show instead that malignancy in such models results from an iterative process of clonal selection in vitro and/or in vivo. Normal human fibroblasts underwent malignant transformation after transduction with telomerase, cyclin-dependent kinase 4, dominant-negative p53, and activated Ras or MEK. Furthermore, culture conditions favoring overgrowth resulted in clonal selection, which with added Ras or MEK oncogenes led to the emergence of tumorigenic clones. Such tumors showed variable degrees of malignancy with some even exhibiting metastasis. SV40 small t antigen (ST) has been reported to be necessary and sufficient to convert human fibroblasts with these pathway aberrations to a polyclonal tumor. However, we observed that clonal tumors emerged even with ST addition. Genomic instability was markedly increased by p53 and Rb pathway abrogation. Under the same conditions, fibroblasts with these alterations failed to induce tumors, implying that genomic instability may be necessary but not sufficient for malignant transformation. These findings indicate that the minimum number of events required for malignant transformation of human fibroblasts is greater than has been enumerated by such oncogene addition strategies and support a stochastic cancer progression model initiated by four defined cellular alterations.

Highlights

Human malignancies are thought to arise by a stepwise series of genetic and/or epigenetic alterations, which select for increasingly malignant clones [1, 2]
human diploid fibroblasts (HDF) were sequentially infected with retroviruses containing hTERT to immortalize the cells, cyclin-dependent kinase 4 (CDK4) to inactivate the Rb pathway, and dominant-negative p53 to inactivate the p53 pathway
HDFs transduced with retroviruses containing hTERT, CDK4, dominantnegative p53, and oncogenic Ras induced late-appearing tumors, regardless of the order of gene introduction (Table 1, experiments 1–2)

Summary

Introduction

Human malignancies are thought to arise by a stepwise series of genetic and/or epigenetic alterations, which select for increasingly malignant clones [1, 2]. Advances in identification of signaling pathways commonly altered in cancer cells have led to efforts aimed at defining the minimum number of genetic alterations required for malignant transformation of human diploid cells in vitro [3, 4]. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Retroviral transduction of normal human fibroblasts or epithelial cells with three elements, SV40 early region (SV40ER), catalytic subunit of telomerase (hTERT), and oncogenic Ras (Ras) was reported to be sufficient for acquisition of the malignant phenotype [7]

Methods

Results

Conclusion