Cellular processes of v-Src transformation revealed by gene profiling of primary cells--implications for human cancer

Abstract

Background: Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. In this study, we took advantage of several strains of the Rous sarcoma virus (RSV) to characterize the patterns of v-Src-dependent gene expression in two different primary cell types, namely chicken embryo fibroblasts (CEF) and chicken neuroretinal (CNR) cells. We identified a common set of v-Src regulated genes and assessed if their expression is associated with disease-free survival using several independent human tumor data sets.

Methods: CEF and CNR cells were infected with transforming, non-transforming, and temperature sensitive mutants of RSV to identify the patterns of gene expression in response to v-Src-transformation. Microarray analysis was used to measure changes in gene expression and to define a common set of v-Src regulated genes (CSR genes) in CEF and CNR cells. A clustering enrichment regime using the CSR genes and two independent breast tumor data-sets was used to identify a 42-gene aggressive tumor gene signature. The aggressive gene signature was tested for its prognostic value by conducting survival analyses on six additional tumor data sets.

Results: The analysis of CEF and CNR cells revealed that cell transformation by v-Src alters the expression of 6% of the protein coding genes of the genome. A common set of 175 v-Src regulated genes (CSR genes) was regulated in both CEF and CNR cells. Within the CSR gene set, a group of 42 v-Src inducible genes was associated with reduced disease- and metastasis-free survival in several independent patient cohorts with breast or lung cancer. Gene classes represented within this group include DNA replication, cell cycle, the DNA damage and stress responses, and blood vessel morphogenesis.

Conclusion: By studying the v-Src-dependent changes in gene expression in two types of primary cells, we identified a set of 42 inducible genes associated with poor prognosis in breast and lung cancer. The identification of these genes provides a set of biomarkers of aggressive tumor behavior and a framework for the study of cancer cells characterized by elevated Src kinase activity.

Figure 1

Transformation regulated (TR) genes belong to two clusters of differentially expressed genes in SR-A RSV transformed CEF. Unsupervised hierarchal clustering was performed on the Transformation-Regulated (TR) gene set revealing transformation responsive genes clustering into one of two approximately equal sized clusters. Class I includes genes up-regulated during transformation representing approximately 44% of the TR genes while class II comprises the down-regulated genes (56% TR genes). The color scale indicates standard deviations from mean centered gene expression values.

Figure 2

Euler representation of genes differentially expressed between control and v-Src transformed cells. Comparison of gene sets differentially expressed by two-fold between CEF or CNR cells infected with the temperature sensitive mutant NY72-4 RSV, or the set of Transformation-Regulated (TR) genes defined in Additional File 3. Numbers indicate total probe sets.

Figure 3

Validation of gene profiling results by northern and western blotting analyses. Steady state transcript levels for a selected set of v-Src regulated genes were determined by northern blotting analyses (A-D). CEF infected with RCASBP(A), NY315 or SR-A RSV were maintained at 41.5°C for the duration of the analysis while NY72-4 RSV infected cells were cultured at the permissive and non-permissive temperatures of 37.5°C and 41.5°C, respectively, for the indicated period of time. AB) CEF infected with NY 72-4 RSV were grown at the non-permissive temperature of 41.5°C and transferred to 37.5°C for the indicated period. C-D) CNR cells infected with NY 72-4 RSV were grown at the permissive temperature of 37.5°C and either maintained at this temperature or transferred to 41.5°C for a 24 hr period before RNA isolation. CNR cells transformed by SR-A RSV were kept at 41.5°C. RNA loading was assessed by probing for GAPDH. E) Western blotting analysis of heme oxygenase 1 (HMOX1) in CEF. Protein samples were prepared from CEF infected RCASBP(A), NY315 or SR-A RSV at 41.5°C. CEF infected with NY 72-4 RSV were either kept at the non-permissive temperature of 41.5°C or transferred to 37.5°C for 24 hrs before lysis. Erk1 was used as a control to assess protein loading.

Figure 4

Up-regulation of Twist1 in v-Src-transformed CEF. Twist1 protein levels are up-regulated in v-Src transformed CEF. Two Twist1 immunoreactive protein species of 26 kDa of molecular weight are detected in normal and v-Src transformed CEF.

Figure 5

A subset of CSR genes predicts poor prognosis in human tumors. A) Hierarchal clustering of two breast tumor datasets with respect to up-regulated CSR genes reveals tumor sample clusters associated with low disease-free survival (cluster 3 in panel i and cluster 4 in panel ii). The color scale beneath the heat maps indicates standard deviations from mean centered gene expression values. Colors of survival curves correspond to the colored clusters indicated in the heat maps above each survival plot. B) A common set of up-regulated genes associated with low-survival clusters in panel A, termed the aggressive tumor gene signature (Table 4), correlates with poor prognosis in several patient cohorts. Tumor clusters expressing high levels of the aggressive signature genes (aggressive high) correlate with lower lung and bone metastasis-free survival in patients with breast tumors (i and ii), as well as with lower disease-free survival in separate cohorts of patients with breast (iii) or lung tumors (iv).