Sequences of the primers used are shown in Supplementary Table S1.
cDNA was amplified by real time quantitative PCR in a ViiA 7 Real-Time PCR system, using SYBR Select Master Mix from Applied Biosystems. RNA was isolated using the QIAGEN RNeasy Kit and retrotranscription was performed using the iScript cDNA synthesis Kit from Bio-Rad, following the manufacturer's instructions. For RNA sequencing (RNA-seq) and RT-qPCR experiments and to control PI3K signaling by Western blot, cells were treated for 4 hours, while for Western blot analyses of PTK2/focal adhesion kinase-related non-kinase (FRNK), cells were treated for 24 hours. Where indicated, cells were treated with DMSO as control or alpelisib (1 μmol/L), taselisib (100 nmol/L), GDC0077 (100 nmol/L), GDC0068/ipatasertib (1 μmol/L), or RAD001/everolimus (100 nmol/L). Twenty-four hours after seeding, cells were washed twice with PBS before adding the starvation media (without serum, EGF, and insulin). MCF10A parental and mutant cells were seeded in 6-multiwell plates in regular culture conditions to allow correct attachment and ensure ∼75% confluency at harvesting day. MCF-10A cells were maintained in DF-12 media supplemented with 5% filtered horse serum (Invitrogen), EGF (20 ng/μL Sigma), hydrocortisone (0.5 mg/mL Sigma), cholera toxin (100 mg/mL Sigma), insulin (10 μg/mL Sigma), and 1% penicillin/streptomycin. Isogenic parental and PIK3CA H1047R heterozygous mutants were purchased from Horizon Discovery. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing.
Analysis of paired tumor biopsies from patients with PIK3CA-mutated breast cancer undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. These analyses uncovered widespread alternatively spliced isoforms linked to proliferation, metabolism, and splicing in PIK3CA-mutant cells, which were reversed by inhibition of PI3Kα. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. A comprehensive elucidation of how this signaling pathway controls transcriptional and cotranscriptional processes could provide new insights into the key functions of PI3K signaling in cancer. The phosphoinositide 3–kinase (PI3K) pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers.