Targeting Epithelial Mesenchymal Plasticity for Therapy Resistance in Breast Cancer

Background:
The MDA-MB-468 human breast cancer (BC) model is one of several that demonstrates intrinsic epithelial mesenchymal plasticity (EMP) in vitro and in vivo (Cursons, Leuchowius et al. 2015, Hugo, Gunasinghe et al. 2017), and represents a unique model in which to search for EMP-associated factors that influence therapy resistance. Similarly, the PMC42-LS cells harbour intrinsic EMP in vitro and in vivo (Bhatia, Monkman et al. 2019), as do HCC-38 cells (Yamamoto, Sakane et al. 2017).

A polarity / EMP - enriched shRNA library screening was performed in these cells to identify and target the genes that might contribute to drug resistance against three currently employed BC drugs: doxorubicin, docetaxel and eribulin. ShRNAs that were depleted across all the three drug treatments included TGFB2, RUNX1, DLG1, CCDC80, HYOU1 and RPS6KB1. Two hundred and forty nine shRNAs were depleted with doxorubicin treatment (at p < 0.01), as compared to 65 with docetaxel and 15 with eribulin treatment. Using Connectivity Map, we identified partner inhibitors targeting these gene families that may induce cell death in combination with doxorubicin. Inhibitors of MDM, TGFBR and FGFR were identified as the top pharmaceutical perturbagens for doxorubicin and synergistic validations verified combinatorial benefits of selected inhibitors.

Hypotheses: That novel therapy opportunities can be identified using shRNA screeing in the EMP-positive MDA-MB-468 and PMC42-LA

Aim 1. To further assess both the individual gene product targets (shRNA / small molecules) and C-Map-identified candidates from the MDA-MB-468 BC screen models for therapy responses in MDA-MB-468, PMC42-LA and HCC-38 cultures
Aim 2. To extend these studies into appropriate in vivo models
Aim 3. To assess material obtained clinically through the CPAC collaboration

Research plan:
For Aim 1, 2D culture systems used to date will be extended to 3D PEG-heparin gel models as developed by Laura Bray, and the shRNA screen will be repeated in 3D. Both MDA-MB-468 and PMC42-LA, each of which carry the EMP-targeted shRNA library will be tested. Individual gene product candidates will be assessed using shRNA, while C-Map identified drugs will be tested directly with support from Pam Pollock.

In vivo testing (Aim 2) will be carried out in NSG mice with different breast cancer cell lines (as above those showing EMP) as well as PDX models available through External Supervisor Christine Chaffer

For Aim 3, we anticipate the availability of breast cancer metastasis-derived 3D tumouroid models via CPAC and Laura Bray, which will be used as clinically-representative testing.

Clinical significance and/or commercial impact:
Therapy resistance is a critical factor underpinning the metastatic progression and treatment failure resulting in death of ~2,200 women annually in Australia from breast cancer. New treatment options are urgently needed to increase survival and reduce suffering. Our shRNA screen strategy has rationally identified novel treatment combinations that could benefit these patients. The project has commercial potential due to novelty and opportunity to develop IP.