Hernandezine acts as a CDK4 suppressor inhibiting tumor growth by the CDK4/PKM2/NRF2 axis in colon cancer

Background: The cyclin-dependent kinase 4 (CDK4) interacts with its canonical and non-canonical substrates modulating the cell cycle in tumor cells. However, the potential substrates and the beyond-cell-cycle-regulated functions of CDK4 in colon Cancer (CC) are still unknown. Hernandezine (HER) is previously verified to induce G0/G1 phase arrest and autophagic cell death in human Cancer cells, which implies that HER might target G0/G1 phase-related proteins, including CDK4.

Purpose: The present study tried to investigate the glycolytic metabolism and oxidative stress functions of CDK4 in colon Cancer. Furthermore, the inhibitory effects and potential binding sites of HER on CDK4, as well as its anti-tumor activity were investigated in CC cells.

Methods: The mass spectrometry assay was performed to identify potential endogenous substrates of CDK4 and the correlation between glycolytic metabolic rate and CDK4 level in COAD patient tissues. Meanwhile, after inhibiting the activity or the expression of CDK4, the binding capacity of CDK4 to PKM2 and NRF2 and the latter two protein distributions in cytoplasm and nucleus were detected in CC cells. In vitro, the regulatory effects of the CDK4-PKM2-NRF2 axis on glycolysis and oxidative stress were performed by ECAR, OCR, and ROS assay. The inhibitory effect of HER on CDK4 activity was explored in CC cells and the potential binding sites were predicted and testified in vitro. Furthermore, tumor growth inhibition of HER by suppressing the CDK4-PKM2-NRF2 axis was also investigated in vitro and in vivo.

Results: PKM2 and NRF2 were identified as endogenous substrates of CDK4 and, high-expressed CDK4 was associated with low-level glycolysis in COAD. In vitro, inactivated CDK4 facilitated CDK4-PKM2-NRF2 complex formation which resulted in 1) inhibited PKM2 activity and retarded the glycolytic rate; 2) cytoplasm-detained NRF2 failed to transcript anti-oxidative gene expressions and induced oxidant stress. Additionally, as a CDK4 Inhibitor, HER developed triple anti-tumor effects including induced G0/G1 phase arrest, suppressed glycolysis, and disrupted the anti-oxidative capacity of CC cells.

Conclusion: The results first time revealed that CDK4 modulated glycolytic and anti-oxidative capacity of CC cells via bound to its endogenous substrates, PKM2 and NRF2. Additionally, 140_Asp145_Asn amino acid sites of CDK4 were potential targets of HER. HER exerts anti-tumor activity by inhibited the activity of CDK4, promoted the CDK4-PKM2-NRF2 complex formation in the CC cells.

Colon cancer; Cyclin-dependent kinase 4; Glycolysis; Hernandezine; Pyruvate kinase 2.