Genetically engineered mouse models of PI3K signaling in breast cancer

Abstract

Breast cancer is the most common type of cancer in women. A substantial fraction of breast cancers have acquired mutations that lead to activation of the phosphoinositide 3-kinase (PI3K) signaling pathway, which plays a central role in cellular processes that are essential in cancer, such as cell survival, growth, division and motility. Oncogenic mutations in the PI3K pathway generally involve either activating mutation of the gene encoding PI3K (PIK3CA) or AKT (AKT1), or loss or reduced expression of PTEN. Several kinases involved in PI3K signaling are being explored as a therapeutic targets for pharmacological inhibition. Despite the availability of a range of inhibitors, acquired resistance may limit the efficacy of single-agent therapy. In this review we discuss the role of PI3K pathway mutations in human breast cancer and relevant genetically engineered mouse models (GEMMs), with special attention to the role of PI3K signaling in oncogenesis, in therapeutic response, and in resistance to therapy. Several sophisticated GEMMs have revealed the cause-and-effect relationships between PI3K pathway mutations and mammary oncogenesis. These GEMMs enable us to study the biology of tumors induced by activated PI3K signaling, as well as preclinical response and resistance to PI3K pathway inhibitors.

Figure 1

Simplified representation of the PI3K signaling pathway. Proteins that activate signaling are depicted in red; proteins that inhibit in green. Upon ligand binding to the receptor tyrosine kinase (RTK), PI3K is activated, leading to formation of PI(3,4,5)3. AKT is phosphorylated at threonine 308 (TP) by PDK1, and on serine 473 (SP) by mTORC2. Note that most kinases are activated by phosphorylation, whereas 4EBP1 is inactivated by phosphorylation.

Figure 2

Types of GEMMs of PI3K‐driven breast cancer. Wildtype tissues are represented in white, while knockout tissues are depicted in gray. Tissues with expression of a transgene or knock in gene are depicted in blue. (A) Conventional knockout mice show tumor suppressor gene (TSG) inactivation in all tissues. (B) A conditional tumor suppressor gene (TSG) knockout can be engineered by flanking one or more critical exons with loxP sequences, which are substrates for the Cre site‐specific recombinase. Tissue specificity for a conditional knockout may be achieved by placing the Cre gene under control of a tissue‐specific promoter. (C) A hypomorphic mouse model can be engineered by intronic insertion of a neomycin cassette, which leads to transcriptional interference and lower expression of the targeted TSG. (D) A constitutive transgene can be inserted into the host genome in a construct containing a tissue‐specific promoter (TSP) to achieve tissue specific expression of the gene of interest (GOI). (E) In a conditional transgenic system, loxP sites (red triangles) flank a transcriptional termination sequence (represented as a STOP sign). Recombination by Cre leads to removal of the STOP sequence and tissue‐specific expression of the GOI. (F) Doxycycline‐inducible ‘tet‐on’ systems are controlled by tissue‐specific expression of a transactivator (rtTA), which can only bind the Tet operator (TetO) and drive expression of the GOI in the presence of doxycycline (dox).

Figure 3

(A) Outline of the GEMM‐ESC strategy. Embryonic stem cell (ESC) lines are derived from genetically engineered mouse models (GEMMs) and equipped with vectors containing FRT sequences that permit rapid and reproducible introduction of GOI via FLP recombinase‐mediated cassette exchange (RMCE). (B) Time line and costs of introduction of a conditional GOI allele in a multi‐allele GEMM using conventional gene targeting vs. the GEMM‐ESC strategy. The conventional approach takes around 20 months and involves introduction of the allele via homologous recombination in wildtype ESCs, generation of chimeras, germline transmission of the mutant allele and subsequent breeding of the F1 mice to the GEMM model. One or more additional rounds of breeding are required to generate mice that are homozygous for all conditional TSG alleles present in the original GEMM. The GEMM‐ESC approach takes only 6 months and involves accelerated introduction of the conditional GOI allele into GEMM‐ESCs via recombinase‐mediated cassette exchange (RMCE) and direct production of an experimental cohort of chimeras via microinjection of the modified GEMM‐ESCs into blastocysts. Compared to the conventional approach, the GEMM‐ESC approach will deliver a considerable reduction in time and costs as well as full control over genetic background, as no breeding is required.