"A woman is diagnosed with breast cancer every 18 seconds worldwide, leading to more than 600,000 breast cancer deaths in 2018 alone. Improving patient treatment outcomes demands a better understanding of the mechanisms driving mammary tumorigenesis and resistance/relapse. In particular, deregulation of the phosphoinositide 3-kinase (PI3K) pathway undoubtedly is crucial in breast cancer because the majority of the cases harbor at least one PI3K pathway-enhancing aberration. Among these changes, over-activation of the PI3K p110α subunit through PIK3CAH1047R mutation is the most frequent. This study aims to uncover genes that strongly cooperate with PIK3CAH1047R to elucidate driver events underlying carcinogenesis and treatment resistance/relapse in PIK3CA-mutated breast cancer.
To identify cooperating genetic mutations acquired during PIK3CAH1047R-driven mammary carcinogenesis in mice, mammary-specific, inducible expression of a PIK3CAH1047R transgene (iPIK) was combined with inducible mobilization of mutagenic Sleeping Beauty transposons (inducible Sleeping Beauty mutagenesis; iSBM). To test whether iSBM enhances carcinogenesis, cohorts of iPIK, iSBM, and iPIK/iSBM female mice were generated and monitored for tumor onset during chronic administration of doxycycline inducer. The ability of iSBM to augment baseline mammary cancer predisposition was examined using the following two endpoints: 1) acceleration of tumor onset and 2) increase of primary tumor multiplicity. Invasiveness was also compared using a functional tumor explantation assay in syngeneic hosts. Tumors were finally harvested and used for next-generation sequencing (NGS) analysis to mine candidate genes, which contain common insertion sites (CIS).
iSBM was insufficient to drive mammary carcinogenesis on its own since iSBM mice remained tumor-free over more than a year of monitoring, mirroring the negligible mammary tumor predisposition in control mice lacking essential transgene components. By contrast, iSBM strongly cooperated with PIK3CAH1047R transgene-initiated carcinogenesis. Whereas iPIK mice developed mammary cancers after a prolonged latency (T50=37 weeks, n=45 mice), tumors arose significantly faster in iPIK/iSBM mice (T50=24 weeks, n=32 mice; plt;0.001, Log-rank test). In addition, iSBM showed a trend toward increasing mammary tumor multiplicity (mean of 2.2 vs. 2.8 tumors/mouse for iPIK vs. iPIK/iSBM mice, p=0.474). Additionally, a significant difference in success in explantation was observed (χ2=13.01; plt;0.001). More specifically, 79% percent of iPIK-iSBM tumors (55/68) engrafted more efficiently compared to 49% in iPIK tumors (19/40).
The Sleeping Beauty Mutagenesis system cooperates with PIK3CAH1047R to enhance tumor onset and invasiveness in PIK3CAH1047R-driven tumors. NGS is ongoingly conducted to identify candidate cooperating genes. Finally, these genes will be queried for mutations using The Cancer Genome Atlas (TCGA) and Catalog of Somatic Mutations in Cancer (COSMIC) and copy number variation using TCGA and RNA-seq Analysis of Molecular Abundance (RoMA) datasets.
National Center for Advancing Translational Sciences, Grant TL1 TR002016 and Grant UL1 TR002014 and National Cancer Institute, Grant R01CA212584
Figure 1. Inducible, mammary-directed transposon mobilization cooperates with PIK3CAH1047R-driven mammary tumorigenesis. a) iSBM transgene schematic. b) iPIK transgene schematic. c) Transposition accelerates the onset of PIK3CAH1047R mammary tumors (p < 0.001, Log-rank test). Kaplan-Meier curves for the different mouse cohorts are depicted. d) Transposition enhances primary tumor multiplicity, albeit non-significantly (mean of 2.2 vs. 2.8 tumors/mouse for iPIK vs. iPIK/iSBM mice, p=0.474)."
