(233a) CRISPR-Mediated Rewiring of Epithelial-to-Mesenchymal Transition Regulatory Networks

Cancer metastasis, defined as the proliferation of malignant cells from primary tumors to local and distant locations, remains a challenge in cancer biology and contributes to unmet clinical needs. Epithelial-to-Mesenchymal Transition (EMT) has emerged as a critical process implicated in the acquisition of invasive metastatic properties by oncogenic cells. EMT is characterized by the loss of epithelial features and the acquisition of mesenchymal traits, including enhanced motility, invasiveness, and resistance to apoptosis. Examples of key signaling pathways involved in manifestation of these various EMT phenotypes include, but are not limited to, TGFβ, Wnt/β-catenin, and Notch, and transcription factors (TFs) such as Snail, Zeb, and Twist. MicroRNAs (miRNAs), small, non-coding molecules that modulate gene expression by targeting mRNA transcripts, coordinate cellular interactions that underlie EMT phenotypes by modulating the activity of these proteins. CRISPR (Clustered Regularly Interspaced Palindromic Repeats) technology has revolutionized the field of genetic engineering. Various CRISPR-mediated DNA editing technologies, such as the type II CRISPR/spCas9 and type V CRISPR/Cas12 systems, have been extensively studied. We utilized the spCas9 endonuclease to generate stable cell models harboring distinct mutations that perturb the interactions between various miRNA to EMT-related transcription factors. These genetically stable EMT “network edge” modifications followed by thorough cell behavior characterization will contribute to our understanding of the complex network underlying the EMT and identify potential targets for therapeutic strategies that prevent the ability of cancer cells to acquire metastatic phenotypes, ultimately improving patient outcomes.

A549, human adenocarcinoma alveolar basal epithelial cells, was utilized as the EMT model. To generate mutant derivatives of the EMT model, guide RNAs (gRNAs) were designed to target and knock-out the miRNA target sites via non-homologous end joining (NHEJ) located at the 3’ untranslated regions (UTRs) of EMT related transcription factors. To assess the impact of EMT network edge modification on the EMT, we perform morphological and genetic evaluation. The established EMT models were subjected to cell characterization assays to assess the impact of miRNA to EMT-TFs edge modifications. Microscopy captured morphological changes as the cell models undergo EMT. Morphological and expression profiles were observed via flow cytometry following antibody staining for key epithelial and mesenchymal surface markers. Reverse transcription-quantitative PCR (RT-qPCR) measures relative mRNA transcript level of the EMT-TFs. Immunocytochemistry (ICC) provides qualitative expression comparison of EMT related genes between the models. Proliferation and wound healing assays assess the degree of metastatic potential, their motility and growth rates, of the EMT models.

It is widely accepted that EMT related metastasis is a driver of cancer progression. The acquisition of mesenchymal cell markers is responsible for cancer metastasis, which enhance their invasive and migratory capabilities. Our results show that, through CRISPR-mediated targeted ablation of regulatory network edges between miR and key EMT-related genes, the mutant A549 cells acquire differential mesenchymal features and potentially modulating their metastatic potential. We demonstrate that targeting of miRNA to EMT-TF edges can provide useful insight on the hierarchy of specific interactions that dictate EMT and, subsequently cancer metastasis, thus advancing the field of cancer biology and a step closer to the development of cancer therapeutics.