The cholesterol biosynthesis enzyme FAXDC2 couples Wnt/β-catenin to RTK/MAPK signaling
Babita Madan, Shawn R. Wadia, Siddhi Patnaik, Nathan Harmston, Emile Tan, Iain Bee Huat Tan, W. David Nes, Enrico Petretto, David M. Virshup
Babita Madan, Shawn R. Wadia, Siddhi Patnaik, Nathan Harmston, Emile Tan, Iain Bee Huat Tan, W. David Nes, Enrico Petretto, David M. Virshup
View: Text | PDF
Research Article Metabolism Oncology

The cholesterol biosynthesis enzyme FAXDC2 couples Wnt/β-catenin to RTK/MAPK signaling

Abstract

Wnts, cholesterol, and MAPK signaling are essential for development and adult homeostasis. Here, we report that fatty acid hydroxylase domain containing 2 (FAXDC2), a previously uncharacterized enzyme, functions as a methyl sterol oxidase catalyzing C4 demethylation in the Kandutsch-Russell branch of the cholesterol biosynthesis pathway. FAXDC2, a paralog of MSMO1, regulated the abundance of the specific C4-methyl sterols lophenol and dihydro-T-MAS. Highlighting its clinical relevance, FAXDC2 was repressed in Wnt/β-catenin–high cancer xenografts, in a mouse genetic model of Wnt activation, and in human colorectal cancers. Moreover, in primary human colorectal cancers, the sterol lophenol, regulated by FAXDC2, accumulated in the cancerous tissues and not in adjacent normal tissues. FAXDC2 linked Wnts to RTK/MAPK signaling. Wnt inhibition drove increased recycling of RTKs and activation of the MAPK pathway, and this required FAXDC2. Blocking Wnt signaling in Wnt-high cancers caused both differentiation and senescence; and this was prevented by knockout of FAXDC2. Our data show the integration of 3 ancient pathways, Wnts, cholesterol synthesis, and RTK/MAPK signaling, in cellular proliferation and differentiation.

Authors

Babita Madan, Shawn R. Wadia, Siddhi Patnaik, Nathan Harmston, Emile Tan, Iain Bee Huat Tan, W. David Nes, Enrico Petretto, David M. Virshup

×

Figure 2

FAXDC2 is a C4-demethylase in the KR branch of the cholesterol biosynthesis pathway.

Options: View larger image (or click on image) Download as PowerPoint
FAXDC2 is a C4-demethylase in the KR branch of the cholesterol biosynthe...
(A) Postlanosterol cholesterol biosynthesis via Bloch and KR pathways, highlighting key steps and associated enzymes. *Proposed location of FAXDC2 in the pathway. (B) Conversion of T-MAS to zymostenol, highlighting reduction at C24 and demethylation at C4 involving sterol methyl oxidase (SMO). *SMO indicates either MSMO1 or FAXDC2 sterol methyl oxidase. (C) MSMO1 and FAXDC2 structures predicted by AlphaFold show considerable homology (root-mean-square deviation of atomic positions 1.36 Å). (D) Like MSMO1, FAXDC2 colocalizes with NSDHL in the SER. Epitope-tagged MSMO1, NSDHL, and FAXDC2 constructs were expressed in HeLa cells, and their localization was visualized by staining with fluorescence-tagged anti-epitope antibodies. (E–G) Combined knockdown of FAXDC2 and MSMO1 reduces total cholesterol levels and prevents cellular proliferation. HPAF-II cells, HPAF-II cells with doxycycline-inducible (DOX-inducible) single-guide (isg) RNAs targeting FAXDC2 or MSMO1 alone, and FAXDC2-KO cells with DOX-isg targeting MSMO1 were cultured for 10 days. (E) Representative images of crystal violet staining from 3 independent experiments. (F) Crystal violet dye was solubilized, and absorbance was measured at 570 nm. (G) Total cholesterol levels were significantly reduced in the FAXDC2 and MSMO1 double-KO cells compared with the single knockouts. (H and I) ETC-159 treatment reduces C4-methyl sterols in HPAF-II orthotopic tumors with high FAXDC2 expression. Sterol levels in mice treated with vehicle or ETC-159 were measured by GC-MS. Data from individual tumor samples (n = 4–5 per group) from 2 independent experiments were combined to calculate P values, controlling for batch effects. (J–L) Stabilized β-catenin, which represses FAXDC2, increased C4-methyl sterol levels independent of upstream Wnt signaling inhibition. C4-methyl sterol levels in HPAF-II tumors with and without stabilized β-catenin from mice treated with ETC-159 or vehicle were analyzed by GC-MS. Each point denotes an individual tumor, n = 4–5 per group. (M) C4-methyl sterols are reduced in TCF7L2-KO HCT116 xenografts compared with control tumors. Sterol levels were measured by GC-MS. Each point depicts an individual tumor sample. P values were calculated by Mann-Whitney U test (G and M) and unpaired t test (J–L). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.