Reactivation of the silenced BASP1 gene suppresses oncogenic WNT signaling in human colorectal cancer cells

Abstract

Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC and silenced BASP1, we generated stable cell lines overexpressing BASP1, either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR based system. BASP1 encodes a neuronal signaling protein and transcriptional co-repressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.

Proteome Analysis

For proteome analysis of the SW480-B and SW480-V cell lines, proteins were obtained from simultaneous proteo-metabolome liquid-liquid extraction (SPM-LLE), with the protein interphase being dissolved in the urea-containing buffer. For proteomics, sample purification by reversed phase solid phase extraction (RP-SPE) and consecutive proteome analysis by LC-MS/MS was performed using a nanoUHPLC System (Dionex UltiMate 3000 RSLCnano pro flow, Thermo Fisher Scientific) coupled to an orbitrap tribrid mass spectrometer (Orbitrap Fusion Lumos, Thermo Fisher Scientific) as described. LC-MS/MS raw data were processed using Thermo Scientific Proteome Discoverer 2.4 SP1.