Cdk4 Regulates Glioblastoma Cell Invasion and Stemness and Is Target of a Notch Inhibitor Plus Resveratrol Combined Treatment

Abstract

Glioblastoma multiforme (GBM) is one of the most aggressive types of cancer characterized by poor patient outcomes. To date, it is believed that the major cause of its recurrence and chemoresistance is represented by the enrichment of GBM stem cells (GSCs) sustained by the abnormal activation of a number of signaling pathways. In this study, we found that in GBM cells, treatment with low toxicity doses of the γ-secretase inhibitor RO4929097 (GSI), blocking the Notch pathway activity, in combination with resveratrol (RSV) was able to reverse the basal mesenchymal phenotype to an epithelial-like phenotype, affecting invasion and stemness interplay. The mechanism was dependent on cyclin D1 and cyclin-dependent kinase (CDK4), leading to a reduction of paxillin (Pxn) phosphorylation. Consequently, we discovered the reduced interaction of Pxn with vinculin (Vcl), which, during cell migration, transmits the intracellular forces to the extracellular matrix. The exogenous expression of a constitutively active Cdk4 mutant prevented the RSV + GSI inhibitory effects in GBM cell motility/invasion and augmented the expression of stemness-specific markers, as well as the neurosphere sizes/forming abilities in untreated cells. In conclusion, we propose that Cdk4 is an important regulator of GBM stem-like phenotypes and invasive capacity, highlighting how the combined treatment of Notch inhibitors and RSV could be prospectively implemented in the novel therapeutic strategies to target Cdk4 for these aggressive brain tumors.

Glioblastoma Therapy: Past, Present and Future

“…The G1-S phase cell cycle checkpoint is mainly controlled by the kinases pathway, and thus improper formation of the cyclin D-1 complexes with CDK 4/6 leads to the promotion of cell-proliferation-involved carcinogenesis. Homozygous deletion of the CDKN2A-p16 INK4α gene in chromosome 9p is one of the three genetic traits of primary GB; in fact, activation of CDK4 is commonly observed in GB cells leading to cell invasion and stemness [ 394 , 395 , 396 , 397 ]. Three CDK4/6 inhibitors, including palbociclib, ribociclib and abemaciclib, have been approved by the FDA as monotherapy for treating breast cancers and are under clinical investigation in GB patients ( Table 3 ) [ 398 ].…”

Mechanism of Notch Signaling Pathway in Malignant Progression of Glioblastoma and Targeted Therapy

“…Notch2 transactivates TNC genes and promotes GBM invasion in an RBPJK-dependent manner [81]. Combining Notch inhibitors with resveratrol to target CDK4 can modulate the invasiveness of glioblastoma [82,83]. The downregulation of CBF1, a primary tran-scriptional regulator [84], can reduce Notch signaling activity and thereby attenuate GBM invasion [85].…”

“…The exogenous expression of CDK4 mutants negates the inhibitory effect of RSV and RO4929097 on the motility/invasion of glioblastoma cells and enhances the expression of stemness-specific markers GFAP, CD13.3, and SOX2, and the EMT markers TWIST and SNAIL, as well as the size/formation capability of the neurosphere [83]. The deletion of the α-kinase structural domain in TRPM7 (M7-DK) and the K1648R point mutation (M7-KR) affect Notch activity and the expression of CD133 and ALDH1 in GSCs [113].…”

“…L-685,458 is able to significantly reduce CXCR4 mRNA in endothelial cells stimulated with rhDLL4 [164]. Under the combined treatment of Resveratrol (RSV) and RO4929097, CDK4 inhibits the population of glioblastoma stem cells with a more invasive phenotype, also triggers apoptosis by pathways blocking the autophagic flux [83,165]. The treatment was well tolerated, and the combination of RO4929097, temozolomide, and RT had a specific decrease in the CD133+ CIS population [154].…”

Targeting Notch signaling pathways with natural bioactive compounds: a promising approach against cancer

“…It has the potential to inhibit cell cycle and growth ( Pinchot et al, 2011 ; Yu X. M. et al, 2013 ; Zhang et al, 2014 ), stimulate apoptosis and re-differentiation ( Yu XM. et al, 2013 ), prevent tumor recurrence ( Giordano et al, 2023 ), and impede autophagy ( Giordano et al, 2021 ) in cancer through downregulation of Notch receptors and ligands, including Notch1, Notch2, Notch4, DLL1, DLL4, and Jagged1. These studies indicated that resveratrol could hinder the activation of the Notch pathway through repression of Notch receptors and ligands in several tumors.…”