Identifying Therapies to Combat Epithelial Mesenchymal Plasticity-Associated Chemoresistance to Conventional Breast Cancer Therapies Using An shRNA Library Screen

Abstract

Background: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. Methods: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute’s Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. Results: In total, 259 shRNAs were depleted with doxorubicin treatment (at p < 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. Conclusions: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.

COMMD3 loss drives invasive breast cancer growth by modulating copper homeostasis

“…T-47D cells were transduced at < 0.5 MOI to reduce the chance of multiple integration of shRNA vectors per cell and puromycin selected for 3 days, followed by culturing on GFR Matrigel for 14 days. To identify changes in shRNA abundance, genomic DNA isolated from day zero (starting material) and day 14 acini was used to amplify shRNA sequences as per standard methods [ 31 , 35 ]. Relative hairpin read counts were used to quantify enrichment of shRNAs from day zero to day 14 of 3D culture, and when normalised across the screen (Table S 1 , see method section), allowed us to calculate the relative enrichment of specific shRNAs on day 14 compared to day zero (Fig.…”

COMMD3 loss drives invasive breast cancer growth by modulating copper homeostasis

“…T-47D cells were transduced at < 0.5 MOI, maintained at high representation, puromycin selected for 3 days, followed by, culturing on growth factor reduced Matrigel for 14 days. To identify changes in shRNA abundance, genomic DNA isolated from day 0 (starting material) and day 14 acini were used to amplify shRNA sequences that had integrated into the genome using primers targeting the barcoded shRNA sequences as previously reported 29,32 . Relative plasmid read counts were used to quantify enrichment of shRNAs from day zero to day 14 of 3D culture, and when normalised across the screen, allowed us to calculate the relative enrichment of speci c shRNAs on day 14 compared to day zero (Fig.…”

Local Release of TGF‐β Inhibitor Modulates Tumor‐Associated Neutrophils and Enhances Pancreatic Cancer Response to Combined Irreversible Electroporation and Immunotherapy

“…In the meantime, SB525334 was reported to have mild cytotoxicity to cancer cells, and showed synergistic effects with gemcitabine and doxorubicin. [ 43 ] To investigate the possibility that the cytotoxicity of SB525334 enhanced the efficacy of IRE, we examined the in vitro cytotoxicity of reversible electroporation and SB525334. This setup was to mimic the tumor regions where SB525334 was present but the pulse intensity was below the threshold of IRE.…”