WNT-CTNN1B signaling promotes cancer cell proliferation and stemness. Furthermore, recent evidence indicates that macroautophagy/autophagy regulates WNT signaling. Here we investigated the impact of inhibiting WNT signaling on autophagy in glioblastoma (GBM), a devastating brain tumor. Inhibiting TCF, or silencing TCF4 or CTNNB1/β-catenin upregulated SQSTM1/p62 in GBM at transcriptional and protein levels and, in turn, autophagy. DKK1/Dickkopf1, a canonical WNT receptor antagonist, also induced autophagic flux. Importantly, TCF inhibition regulated autophagy through MTOR inhibition and dephosphorylation, and nuclear translocation of TFEB, a master regulator of lysosomal biogenesis and autophagy. TCF inhibition or silencing additionally affected GBM cell proliferation and migration. Autophagy induction followed by its blockade can promote cancer cell death. In agreement with this notion, halting both TCF-CTNNB1 and autophagy pathways decreased cell viability and induced apoptosis of GBM cells through a SQSTM1-dependent mechanism involving CASP8 (caspase 8). In vivo experiments further underline the therapeutic potential of such dual targeting in GBM.

Glioblastoma multiforme (GBM) is a commonly occurring brain tumor with a poor prognosis. GBM can develop both “de novo” or evolve from a previous astrocytoma and is characterized by high proliferation and infiltration into the surrounding tissue. Following treatment (surgery, radiotherapy, and chemotherapy), tumors often reappear. Glioma-initiating cells (GICs) have been identified in GBM and are thought to be responsible for tumors initiation, their continued growth, and recurrence.β-catenin, a component of the cell-cell adhesion complex and of the canonical Wnt pathway, regulates proliferation, adhesion, and migration in different cell types.β-catenin and components of the Wnt canonical pathway are commonly overexpressed in GBM. Here, we review previous work on the role of Wnt/β-catenin signalling in glioma initiation, proliferation, and invasion. Understanding the molecular mechanisms regulating GIC biology and glioma progression may help in identifying novel therapeutic targets for GBM treatment.

Tyrosine phosphorylation of β-catenin, a component of adhesion complexes and of the Wnt pathway, affects cell adhesion, migration and gene transcription. By reducing β-catenin availability using shRNA-mediated gene silencing or expression of intracellular N-cadherin, we show that β-catenin is required for axon growth downstream of brain-derived neurotrophic factor (BDNF) signalling and hepatocyte growth factor (HGF) signalling. We demonstrate that the receptor tyrosine kinases (RTKs) Trk and Met interact with and phosphorylate β-catenin. Stimulation of Trk receptors by neurotrophins (NTs) results in phosphorylation of β-catenin at residue Y654, and increased axon growth and branching. Conversely, pharmacological inhibition of Trk or expression of a Y654F mutant blocks these effects. β-catenin phosphorylated at Y654 colocalizes with the cytoskeleton at growth cones. However, HGF, which also increases axon growth and branching, induces β-catenin phosphorylation at Y142 and a nuclear localization. Interestingly, dominant-negative ΔN-TCF4 abolishes the effects of HGF in axon growth and branching, but not that of NTs. We conclude that NT- and HGF-signalling differentially phosphorylate β-catenin, targeting this protein to distinct compartments to regulate axon morphogenesis by TCF4-transcription-dependent and -independent mechanisms. These results place β-catenin downstream of growth-factor–RTK signalling in axon differentiation.

Wnt factors regulate neural stem cell development and neuronal connectivity. Here we investigated whether Wnt‐3a and Wnt‐3, expressed in the developing spinal cord, regulate proliferation and the neuronal differentiation of spinal cord neural precursors (SCNP). Wnt‐3a promoted a sustained increase of SCNP proliferation and decreased the expression of cyclin‐dependent kinase inhibitors. In contrast, Wnt‐3 transiently enhanced SCNP proliferation and increased neurogenesis through β‐catenin signaling. Furthermore, both Wnt‐3a and Wnt‐3 stimulated neurite outgrowth in SCNP‐derived neurons through β‐catenin‐ and TCF4‐dependent transcription. Glycogen synthase kinase‐3β inhibitors mimicked Wnt signaling and promoted neurite outgrowth in established cultures. We conclude that Wnt‐3a and Wnt‐3 factors signal through the canonical Wnt/β‐catenin pathway to regulate different aspects of SCNP development. These findings may be of therapeutic interest for the treatment of neurodegenerative diseases and nerve injury.

Focal adhesion kinase (FAK) is a central component of focal adhesions that regulate cancer cell proliferation and migration. Here, we studied the effects of FAK inhibition in glioblastoma (GBM), a fast growing brain tumor that has a poor prognosis. Treating GBM cells with the FAK inhibitor PF-573228 induced a proliferative arrest and increased cell size. PF-573228 also reduced the growth of GBM neurospheres. These effects were associated with increased p27/CDKN1B levels and β-galactosidase activity, compatible with acquisition of senescence. Interestingly, FAK inhibition repressed the expression of the autophagy cargo receptor p62/SQSTM-1. Moreover, depleting p62 in GBM cells also induced a senescent-like phenotype through transcriptional upregulation of p27. Our results indicate that FAK inhibition arrests GBM cell proliferation, resulting in cell senescence, and pinpoint p62 as being key to this process. These findings highlight the possible therapeutic value of targeting FAK in GBM.

T-type Ca2+ channels (TTCC) have been identified as key regulators of cancer cell cycle and survival. In vivo studies in glioblastoma (GBM) murine xenografts have shown that drugs able to block TTCC in vitro (such as tetralol derivatives mibefradil/NNC-55-096, or different 3,4-dihydroquinazolines) slow tumor progression. However, currently available TTCC pharmacologic blockers have limited selectivity for TTCC and are unable to distinguish between TTCC isoforms. Here we analyzed the expression of TTCC transcripts in human GBM cells and show a prevalence of Cacna1g/Cav3.1 mRNAs. Infection of GBM cells with lentiviral particles carrying short hairpin RNA against Cav3.1 resulted in GBM cell death by apoptosis. We generated a murine GBM xenograft via subcutaneous injection of U87-MG GBM cells and found that tumor size was reduced when Cav3.1 expression was silenced. Furthermore, we developed an in vitro model of temozolomide-resistant GBM that showed increased expression of Cav3.1 accompanied by the activation of macroautophagy. We confirmed a positive correlation between Cav3.1 and autophagic markers in both GBM cultures and biopsies. Of note, Cav3.1 knockdown resulted in transcriptional downregulation of p62/SQSTM1 and deficient autophagy. Together, these data identify Cav3.1 channels as potential targets for slowing GBM progression and recurrence based on their role in regulating autophagy. Significance: These findings identify Cav3.1 calcium channels as a molecular target to regulate autophagy and prevent progression and chemotherapeutic resistance in glioblastoma.