Metastatic Competence Can Emerge with Selection of Preexisting Oncogenic Alleles without a Need of New Mutations

Abstract

Several experimental models faithfully recapitulate many important facets of human metastatic disease. Here, we have performed whole-exome sequencing in five widely used experimental metastasis models that were independently derived through in vivo selection from heterogeneous human cancer cell lines. In addition to providing an important characterization of these model systems, our study examines the genetic evolution of metastatic phenotypes. We found that in vivo selected highly metastatic cell populations showed little genetic divergence from the corresponding parental population. However, selection of genetic variations that preexisted in parental populations, including the well-established oncogenic mutations KRAS(G13D) and BRAF(G464V), was associated with increased metastatic capability. Conversely, expression of the wild-type BRAF allele in metastatic cells inhibited metastatic outgrowth as well as tumor initiation in mice. Our findings establish that metastatic competence can arise from heterogeneous cancer cell populations without the need for acquisition of additional mutations and that such competence can benefit from further selection of tumor-initiating mutations that seed primary tumorigenesis.

Figure 1

Comparisons of allelic frequencies between parental and metastatic populations reveal selection. A, sequence variations for each sample are quantified. Red bars depict variants found at greater allelic frequencies in metastatic derivatives compared to parental lines. Blue bars depict variants found at lower allelic frequencies. B, categories of observed variant allelic frequency (VAF) shifts. Schematic depicting six categories of VAF shifts. Categories include 1) insignificant changes in VAF; 2) variants depleted in metastatic populations; 3) variants private to metastatic populations; 4) rare parental variants enriched in metastatic populations; 5) parental variants enriched in metastatic populations; 6) parental variants for which the VAF is enriched to 100% in metastatic populations. C-E, pairwise comparisons of VAF between parental and matched metastatic derivative lines in the MDA-MB-231 model system (C), the H2030 and PC9 model systems (D), and the 786-O and OS-RC model systems (E). Key for heatmap representing the density of data points (VAF for each sequence variation found) is depicted to the right of each set of graphs. F, phylogenetic tree depicting the genetic relationships between the MDA-MB-231 parental line and matched metastatic derivatives. The dashed line to control represents simulated data.

Figure 2

Selection of oncogenic mutations in metastatic populations. A, graphical representation of sequence variations found in known cancer genes (11, 19, 20). Red bars represent enriched variant alleles (metastatic variant allele frequency ≥ parental variant allele frequency + 0.25, p-value <0.001). Blue bars represent depleted variant alleles (metastatic variant allele frequency ≤ parental variant allele frequency − 0.25, p-value <0.001). B-C, Sanger sequencing (top panels) confirms BRAF (B), and KRAS (C), mutations in metastatic cell populations. Bottom panels show read counts for each allele from whole exome sequencing of each sample. D, copy number analyses of chromosomes 7 (top panel) and 12 (bottom panel). Results from aCGH data were compiled to show comparisons between metastatic derivatives and parental lines.

Figure 3

Wild-type allele of BRAF inhibits metastasis and tumor growth. A, wild-type BRAF was reintroducted into MDA231-BrM2-1 cells and compared with cells expressing an empty vector in vivo. One thousand cells were injected into the left ventricle of mice. Tumor burden was quantitated by bioluminescence imaging at 9 weeks. n = 10, empty vector; n = 7 wild-type BRAF B, one hundred MDA231-BrM2-1 cells expressing either empty vector or wild-type BRAF were suspended in matrigel and innoculated subcutaneously into the flanks of immunodeficient mice (n = 20 per condition). Tumor burden was quantitated by bioluminescence imaging at 7 weeks. P values shown was calculated by a two-tailed Wilcoxon rank sum test. * - p<0.05, ** - p<0.01.