β-catenin-IRP2-primed iron availability to mitochondrial metabolism is druggable for active β-catenin-mediated cancer

Abstract

Background Although β-catenin signaling cascade is frequently altered in human cancers, targeting this pathway has not been approved for cancer treatment. Methods High-throughput screening of an FDA-approved drug library was conducted to identify therapeutics that selectively inhibited the cells with activated β-catenin. Efficacy of iron chelator and mitochondrial inhibitor was evaluated for suppression of cell proliferation and tumorigenesis. Cellular chelatable iron levels were measured to gain insight into the potential vulnerability of β-catenin-activated cells to iron deprivation. Extracellular flux analysis of mitochondrial function was conducted to evaluate the downstream events of iron deprivation. Chromatin immunoprecipitation, real-time quantitative PCR and immunoblotting were performed to identify β-catenin targets. Depletion of iron-regulatory protein 2 (IRP2), a key regulator of cellular iron homeostasis, was carried out to elucidate its significance in β-catenin-activated cells. Online databases were analyzed for correlation between β-catenin activity and IRP2-TfR1 axis in human cancers. Results Iron chelators were identified as selective inhibitors against β-catenin-activated cells. Deferoxamine mesylate, an iron chelator, preferentially repressed β-catenin-activated cell proliferation and tumor formation in mice. Mechanically, β-catenin stimulated the transcription of IRP2 to increase labile iron level. Depletion of IRP2-sequered iron impaired β-catenin-invigorated mitochondrial function. Moreover, mitochondrial inhibitor S-Gboxin selectively reduced β-catenin-associated cell viability and tumor formation. Conclusions β-catenin/IRP2/iron stimulation of mitochondrial energetics is targetable vulnerability of β-catenin-potentiated cancer.

Oncogenic β-catenin-driven liver cancer is susceptible to methotrexate-mediated disruption of nucleotide synthesis

“…Culture conditions were reported for all the cell lines used in this study. [20] Wild type (WT) T, β-catenin Δ(ex3)/ + mouse embryonic fibroblasts (MEFs) were previously constructed by our laboratory [20] , 293FT (Abclonal, Wuhan, China), HepaRG (Thermo fisher), CCC-HEL-1 (China Infrastructure of Cell Line Resource, Beijing, China), NCTC1469 (China Infrastructure of Cell Line Resource), HepG2 (China Infrastructure of Cell Line Resource), Hepa 1-6 (Procell, Wuhan, China), HCCLM3 (China Infrastructure of Cell Line Resource) and Huh7 (Procell) were cultured in DMEM, SNU886 (Cobioer biosciences, Nanjing, China) and SNU398 (Sagen tech, Guangzhou, China) were cultured in DMEM or RPMI-1640. Cells were maintained at 37°C incubator with 5% CO 2 .…”

“…Western blotting was performed as previously described. [20] The membranes were incubated with primary antibodies against β-catenin (#9587, Cell Signaling Technology, Danvers, MA, USA), AKT2 (#2964, Cell Signaling Technology), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD, #93925, Cell Signaling Technology), phospho CAD (p-CAD Ser 1859 , #7030, Cell Signaling Technology), p-CAD Ser 1406 (PTM BioLab, Hangzhou, Zhejiang, China), cyclin D1 (#A19038, Abclonal), or Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #AC002, Abclonal) overnight at 4°C and then incubated with secondary antibodies (#68071 or #32210, LI-COR Biosciences, Lincoln, NE, USA) for 2 h at the room temperature. Finally, protein bands were detected by SuperSignal enhanced chemiluminescence (LI-COR Biosciences).…”

“…Total RNA extraction and RT-qPCR protocol were performed as described previously. [20] RT-qPCR was performed using SYBR high-sensitivity qPCR SuperMix (#abs60086, Absin Biosciences, Shanghai, China). β-actin transcripts were used to normalize the expression of each gene.The primer sequences used in the study were listed in Supplementary Table 2, http://links.lww.com/CM9/B677.…”

“…[10,11] As a downstream effector of Wnt pathway, b-catenin plays critical roles in the regulation of cell metabolism, genetic stability, proliferation, and survival. [12][13][14][15][16][17] After escaping from its degradation complex, b-catenin translocates into the nucleus and interacts with transcription factors to drive target gene expression. [18] By stimulation of protein kinase B serine/threonine kinase 2 (AKT2)-mediated pyrimidine synthesis, b-catenin with gain-of-function mutation promotes cell proliferation and liver cancer development.…”