Isolating and targeting the real-time plasticity and malignant properties of epithelial-mesenchymal transition in cancer

The EMT/EMP program promotes tumor cell plasticity and heterogeneous phenotypes driving tumor progression and metastasis. Here we demonstrate a novel approach to isolate and characterize EMT-phenotypes and target the real-time plasticity of EMP using a fluorescent dual-reporter suitable for HCS.

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Tumor heterogeneity and plasticity remain key problems to overcome in cancer given their ability to become drug resistant, metastasize, and evade the immune system. Therefore, our group sought to develop a novel tool capable of examining in real-time the heterogeneity and plasticity observed in cancer cells in the context of epithelial-mesenchymal transition (EMT). By pairing two putative markers of EMT, E-cadherin and vimentin, we designed a dual fluorescent reporter that measures these two proteins’ promoter activity.

The dual reporter enabled us to isolate the spectrum of quasi-EMT phenotypes epithelial (E), hybrid (E/M), and mesenchymal (M) by flow cytometry and we characterized these phenotypes based on their protein expression, cell morphology, cell metabolism, and malignant properties. After uncovering the different malignant properties of the isolated phenotypes, we sought to discover probes that modulate or inhibit epithelial-mesenchymal plasticity (EMP) or induce mesenchymal-epithelial transition (MET). Notably, the value of inducing MET was explored in our previous work1-4, in which we were able to decrease vimentin promoter activity and increase E-cadherin promoter activity while also decreasing malignancy by using novel TOP2A and CHD1L inhibitors being developed in our lab. We realized a single reporter model presented high-throughput screening limitations. Thus, we replicated our previous results using the dual reporter and even expanded the validation of induced MET using metabolomics. Interestingly, the isolated mesenchymal phenotype treated with the TOP2A inhibitor (compound 7) induced MET to the extent that it was statistically indistinguishable from the isolated epithelial phenotype by principal component analysis (PCA). However, we note that small molecule induced MET does not provide the exact quasi-epithelial phenotype. We view this as a positive effect in that the compound induced MET also carries significant anti-tumor and anti-metastatic activity beyond promoting the loss of the mesenchymal phenotype.   

Treatment of M-phenotype CRC tumor organoids with 7 induces MET by metabolomics.

Encouraged by our results, we proceeded to validate our tool for 3D high-content screening (HCS) by testing the ability of ~23,000 compounds against EMP in colorectal cancer mesenchymal tumor organoids. This appears to be one of the largest phenotypic 3D HCS campaigns reported in the literature to date. The screen discovered hundreds of probes that modulated EMT. Notably, a small subset of lead probes that induce MET and their respective targets coupled with our metabolomics data allowed us to generate a coherent mechanistic relationship of the pathways controlling EMP in colorectal cancer.

 

Taken together, the dual reporter is an effective tool capable of monitoring in real-time the heterogeneity and plasticity of EMT in cancer cell populations. The dual reporter technology coupled with cell and in vivo models should prove useful in advancing our mechanistic understanding of EMT/EMP and facilitate novel drugs and therapies effective against tumor progression and metastasis.

1          Esquer H, Zhou Q, Abraham AD, LaBarbera DV. Advanced High-Content-Screening Applications of Clonogenicity in Cancer. SLAS Discov 2020; 25: 734-743.

2          Abbott JM, Zhou Q, Esquer H, Pike L, Broneske TP, Rinaldetti S et al. First-in-Class Inhibitors of Oncogenic CHD1L with Preclinical Activity against Colorectal Cancer. Mol Cancer Ther 2020; 19: 1598-1612.

3          Abraham AD, Esquer H, Zhou Q, Tomlinson N, Hamill BD, Abbott JM et al. Drug Design Targeting T-Cell Factor-Driven Epithelial-Mesenchymal Transition as a Therapeutic Strategy for Colorectal Cancer. J Med Chem 2019; 62: 10182-10203.

4          Zhou Q, Abraham AD, Li L, Babalmorad A, Bagby S, Arcaroli JJ et al. Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer. Oncogene 2016; 35: 4990-4999.

Daniel V. LaBarbera

Professor, University of Colorado Anschutz Medical Campus