Background. The Wnt/β-catenin signaling is frequently deregulated in colorectal cancer (CRC). Germ-line (as for familial adenomatous polyposis – FAP) or somatic loss of function of APC gene are crucial events in CRC onset (1). The mTOR pathway is frequently impaired in CRC progression (2) and closely involved in a fine cross-talk together with Wnt/β-catenin signaling, which often leads to the acquisition of resistance to common single-inhibitors against the two pathways (3).
Aim. The aim of the study was to evaluate the effect of a combination (RDE) of Rapamycin (R), ω3-PUFA Docosahexaenoic Acid (D) and epigallocatechin-3-gallate (E) on PI3K/mTOR and Wnt/β-catenin pathways to define an effective combinatorial approach against APC-driven CRC onset and progression.
Methods. RDE was tested in 2D and 3D models representative of different settings: APC-mutated-pre-cancerous settings consisting of intestinal organoids (PDOs) derived from FAP patients normal-appearing mucosa (NM) and adenomas (A); APC-mutated-cancerous settings comprising sporadic-CRC-cancer (K)-PDOs and the cell lines SW480/HT-29; non-APC-mutated-CRC setting (HCT116 cell line). Cells/organoids viability, intestinal stemness (LGR5), differentiation (KRT20), target genes and effector proteins expression of Wnt and mTOR were evaluated. CHI99021 was used as Wnt-active positive control; CTRL (vehicles) and non-treated cells/organoids (NC) as negative controls.
Results. RDE significantly inhibited the mTOR pathway by downregulating phosphorylated-S6R (p-S6R) in a background-independent manner (Fig.1A-2A), and reduced cells and organoids viability (~20%) (Fig.1B-2B). In APC-mutated-pre-cancerous settings RDE enhanced the Wnt signaling activation but significantly suppressed LGR5, promoting a shift towards differentiation (upregulation of KRT20) (Fig.1C-D). In contrast, the hyperactivation of Wnt induced by RDE in CRC-settings (APC/non-APC mutated) promoted β-catenin protein degradation in a GSK3β/AXIN1-independent manner and suppressed the LGR5 expression (Fig.2C-D).
Conclusion. Taken together our results suggests that RDE represents a potential therapeutic approach for both preventive and therapeutic strategies against CRC.
References
(1) Zhao H, Ming T, Tang S, Ren S, Yang H, Liu M, Tao Q, Xu H. Wnt signaling in colorectal cancer: pathogenic role and therapeutic target. Mol Cancer. 2022 Jul 14;21(1):144. doi: 10.1186/s12943-022-01616-7. PMID: 35836256.
(2) Mossmann D, Park S, Hall MN. mTOR signalling and cellular metabolism are mutual determinants in cancer. Nat Rev Cancer. 2018 Dec;18(12):744-757. doi: 10.1038/s41568-018-0074-8. PMID: 30425336.
(3) Prossomariti A, Piazzi G, Alquati C, Ricciardiello L. Are Wnt/β-Catenin and PI3K/AKT/mTORC1 Distinct Pathways in Colorectal Cancer? Cell Mol Gastroenterol Hepatol. 2020;10(3):491-506. doi: 10.1016/j.jcmgh.2020.04.007. Epub 2020 Apr 22. PMID: 32334125.

<b>Fig.1 RDE in </b><b>APC-mutated-pre-cancerous settings. (A)</b> Phosphorylated-S6R (p-S6R) protein expression evaluated in FAP-PDOs by WB. <b>(B)</b> FAP-PDOs Organoids Viability analysis. <b>(C)</b> Total-β-Catenin protein expression evaluated in FAP-PDOs by WB. <b>(D)</b> <i>LGR5</i> and <i>KRT20</i> RNA expression in FAP-PDOs assessed by qPCR (2^-ΔΔCt). Statistical significance was tested using the T-test. CTRL was used as the reference sample. *P<0.05; **P<0.010; ***P<0.001; ****P<0.0001.

Fig.1 RDE in APC-mutated-pre-cancerous settings. (A) Phosphorylated-S6R (p-S6R) protein expression evaluated in FAP-PDOs by WB. (B) FAP-PDOs Organoids Viability analysis. (C) Total-β-Catenin protein expression evaluated in FAP-PDOs by WB. (D) LGR5 and KRT20 RNA expression in FAP-PDOs assessed by qPCR (2^-ΔΔCt). Statistical significance was tested using the T-test. CTRL was used as the reference sample. *P<0.05; **P<0.010; ***P<0.001; ****P<0.0001.

<b>Fig.2 RDE in </b><b>cancerous settings. (A)</b> Phosphorylated-S6R (p-S6R) protein expression evaluated in CRCK-PDOs, SW480 and HCT116 by WB. <b>(B)</b> CRCK-PDOs, SW480 and HCT116 viability analysis. <b>(C)</b> AXIN1, GSK3β, Total-β-Catenin protein expression evaluated in CRCK-PDOs, SW480 and HCT116 by WB. <b>(D)</b> <i>LGR5</i> RNA expression in CRCK-PDOs, SW480 and HCT116 assessed by qPCR (2^-ΔΔCt). Statistical significance was tested using the T-test. CTRL was used as the reference sample. *P<0.05; **P<0.010; ***P<0.001; ****P<0.0001.

Fig.2 RDE in cancerous settings. (A) Phosphorylated-S6R (p-S6R) protein expression evaluated in CRCK-PDOs, SW480 and HCT116 by WB. (B) CRCK-PDOs, SW480 and HCT116 viability analysis. (C) AXIN1, GSK3β, Total-β-Catenin protein expression evaluated in CRCK-PDOs, SW480 and HCT116 by WB. (D) LGR5 RNA expression in CRCK-PDOs, SW480 and HCT116 assessed by qPCR (2^-ΔΔCt). Statistical significance was tested using the T-test. CTRL was used as the reference sample. *P<0.05; **P<0.010; ***P<0.001; ****P<0.0001.

Luigi Ricciardiello

The University of Texas MD Anderson Cancer Center

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