Cardiac glycosides as potential therapeutics for colorectal cancer

Cardiac glycosides (CGs), molecules used for the treatment of congestive heart failure and arrhythmias, have recently emerged as anti-cancer agents. These compounds target the Na+/K+ ATPase (NKA), a key regulator of ion homeostasis in cells. Beyond their role in regulating ion transport, CGs modulate the levels of MYC, an oncoprotein linked to proliferation and tumorigenesis. MYC has been identified as an inhibitor of the loading of double-stranded RNA onto the endosomal receptor TLR3. The activation of TLR3 triggers a cascade of intracellular innate immune defence mechanisms, including TBK1, essential for the immune response to viral infections. Additionally, the binding of CGs to the NKA activates several signalling pathways in a pump-independent manner. The aim of this thesis was to investigate the effects of the treatment with the CG cymarin on colorectal cancer (CRC) cells, including its potential to activate innate immune mechanisms and whether this is mediated by MYC. The effects of cymarin on a treatment resistant murine cancer cells and on a treatment-sensitive mutated version of this cell line were analysed. To identify the regions of the UTRs responsible for the downregulation of MYC following cymarin treatment, and so the effect of the treatment on MYC translation, six cell lines were utilised, each containing constructs with different regions of MYC’s UTRs. Additionally, an fCLIP (formaldehyde Cross-Linking and ImmunoPrecipitation) assay was performed to evaluate the relationship between the MYC downregulation and dsRNA loading onto TLR3. The results allowed to identify the 3’UTR as the region responsible for the cymarin dependent MYC downregulation in cancer cells. The mutation in the α1 subunit of the ATPase, which makes murine cells sensitive to cymarin, results in the activation of TBK1 and loading of dsRNA onto TLR3 when the cells are treated with CGs, as it happens in human CRC cells. The treatment with cymarin, as it happens for all CGs, induces the activation of the MAPK signalling pathway. Which is an important regulator of MYC stability. In conclusion, cymarin treatment not only inhibits the proliferation of CRC cells, but also elicits several cellular mechanisms that require further study. Full understanding of the molecular actions of CGs is essential for developing novel therapeutic strategies for CRC.