SMAD3 and p300 complex scaffolding by long non-coding RNA LIMD1-AS1 promotes TGF-ß-induced breast cancer cell plasticity [CRISPR screen, inhibition]

Transforming growth factor (TGF)-ß signaling enhances cancer cell plasticity by inducing epithelial-to-mesenchymal transition (EMT). Here, we identified a TGF-ß-induced long non-coding RNA, LIMD1 Antisense RNA 1 (LIMD1-AS1) that strengthens the SMAD-mediated transcriptional response to TGF-ß. LIMD1-AS1 expression is upregulated in breast cancer tissues compared to normal breast tissues, and high LIMD1-AS1 expression is associated with poor prognosis in breast cancer patients. Depletion of LIMD1-AS1 hinders TGF-ß-induced EMT, migration, and extravasation of breast cancer cells. Mechanistically, LIMD1-AS1 promotes the interaction between SMAD3 and its transcriptional coactivator p300, thereby enhancing SMAD3 transcriptional activity and TGF-ß/SMAD signaling. We demonstrated that LIMD1-AS1 binds to the MAD homology 2 (MH2) domain of SMAD3 and the interferon-binding domain (IBiD) of p300. Displacing LIMD1-AS1 from p300 by its competitor interferon regulatory factor 3 (IRF3) suppressed the effects of LIMD1-AS1 on potentiating TGF-ß/SMAD signaling. Furthermore, blockage of p300 acetyltransferase activity with a pharmacological inhibitor A-485 reduced the ability of LIMD1-AS1 to enhance SMAD3 transcriptional activity, TGF-ß-induced EMT, and migration. This study identifies LIMD1-AS1 as a novel stimulator of TGF-ß signaling by establishing a positive feedback loop and highlights its potential as a therapeutic target for breast cancer. Overall design: CRISPR-inhibition screen for a custom set of lncRNAs. MDA-MB231 expressing a TGF-ß inducible GFP reporter and dCas9-KRAB(ZIM3) were transduced with a guide library. An early time point (ETP) control sample was taken at day 5 post-transduction. At day 7 post transduction, cells were treated with TGF-ß for 24 hours. An unsorted control sample was taken, and the 15% highest and lowest GFP expressors were sorted. The screen was performed in two replicates.