A combination of rapamycin, ω3-PUFA docosahexaenoic acid and epigallocatechin-3-gallate for the simultaneous suppression of PI3K/mTOR pathway and Wnt/β-catenin signalling in colorectal carcinogenesis

Loss of function mutations in the APC gene represent a critical hit for colorectal cancer (CRC) onset in both sporadic and inherited settings (e.g. FAP). This gene encodes a tumor suppressor protein that regulates the Wnt/β-catenin pathway. Together with the PI3K/mTOR signalling, Wnt/β-catenin is frequently deregulated in the early stages of CRC. Relevant cross-talks between the two pathways have been described, and numerous inhibitors have been developed and tested against CRC. However, long-term treatment often fails due to the acquisition of resistance mechanisms. The aim of the study was to evaluate the effect of a combination (RDE) of Rapamycin, ω3-PUFA Docosahexaenoic Acid (DHA) and epigallocatechin-3-gallate (EGCG) on PI3K/mTOR and Wnt/β-catenin pathways in a combinatorial approach against APC-driven CRCs. RDE was tested in different intestinal genetic backgrounds: (i) APC-mutated including FAP-morphologically-normal-appearing mucosa (NM) and adenomas (A)-organoids; sporadic-CRC-cancer (K) organoids, and SW480 and HT-29 cell lines; (ii) non-APC-mutated CRC comprising HCT116 (CTNNB1 mutated) cells; HEK293STF cells, healthy FIT+ NM/ CRC-NM-derived organoids as non-mutated (iii). Cells/organoids viability, stemness (LGR5)/differentiation (KRT20), were evaluated together with target genes and effector proteins of the two pathways. We found that RDE inhibited effectively PI3K/mTOR by suppressing the downstream effectors protein P70S6K and S6R in a background-independent manner. However, targeting Wnt/β-catenin pathway appeared more challenging and deranged-Wnt/β-catenin-dependent. Indeed, in pre-cancerous settings (FAP NM/A organoids) RDE enhanced the Wnt signalling activation; in APC-mutated and non-APC-mutated CRC settings RDE induced β-catenin protein degradation through non-canonical mechanisms. Despite the upregulation of Wnt/β-catenin pathway, RDE affected cell/organoids viability and significantly suppressed LGR5, promoting a shift towards differentiation. Our results suggests that RDE is a potential therapeutic approach for both preventive and therapeutic strategies against APC-driven CRC.