Simultaneous Combination of the CDK4/6 Inhibitor Palbociclib With Regorafenib Induces Enhanced Anti-tumor Effects in Hepatocarcinoma Cell Lines

Abstract

Advanced hepatocarcinoma (HCC) is an aggressive malignancy with poor prognosis and limited treatment options. Alterations of the cyclin D-CDK4/6-Rb pathway occur frequently in HCC, providing the rationale for its targeting at least in a molecular subset of HCC. In a panel of HCC cell lines, we investigated whether the CDK4/6 inhibitor palbociclib might improve the efficacy of regorafenib, a powerful multi-kinase inhibitor approved as second-line treatment for advanced HCC after sorafenib failure and currently under clinical investigation as first-line therapy in combination with immunotherapy. In Rb-proficient cells, the simultaneous drug combination, but not the sequential schedules, inhibited cell proliferation, either in short or in long-term experiments, and induced cell death more strongly than individual treatments. Moreover, the combination significantly reduced spheroid cell growth and inhibited cell migration/invasion. The superior efficacy of palbociclib plus regorafenib emerged also under hypoxia and was associated with a significant down-regulation of CDK4/6-Rb-myc and mTORC1/p70S6K signaling. Moreover, regorafenib suppressed palbociclib-induced expression of cyclin D1 contributing to the cytotoxic effects of the combination. Besides these inhibitory effects on cell viability/proliferation, palbociclib and regorafenib reduced glucose uptake, although this effect was dependent on the cell model and on the oxygen availability (normoxia or hypoxia). Palbociclib and regorafenib combination impaired glucose uptake and utilization, down-regulating basal and hypoxia-induced expression of HIF-1α, HIF-2α, GLUT-1, and MCT4 proteins as well as the activity/expression of glycolytic enzymes (HK2, PFKP, aldolase A, PKM2). In addition, regorafenib alone reduced mitochondrial respiration. The combined treatment impaired glucose metabolism and respiration without enhancing the effects of the single agents. Our findings provide pre-clinical evidence for the effectiveness of palbociclib and regorafenib combination in HCC cell models.

Current status of ctDNA in precision oncology for hepatocellular carcinoma

“…Inactivation of the cyclin-dependent kinase inhibitor, CDKN2A, emerges in 7% of advanced HCC patients based on digital ctDNA sequencing and leads to overexpression of CDK4/6 [89]. With mutated CDKN2A, the upregulated CDK4/6 accelerates the G1/S phase transition in the cell cycle through thee CDK4/6-Rb-myc pathway and eventually promotes cell proliferation [70]. Additionally, patients with CDKN2A silencing correlate with an advanced stage and aggressive biological behavior [90].…”

“…Additionally, patients with CDKN2A silencing correlate with an advanced stage and aggressive biological behavior [90]. Thus, CDK4/6 inhibitors leading to cell cycle arrest and cell death induction, such as palbociclib, ribociclib, and abemaciclib, can provide an effective target treatment for HCC patients with CDKN2A loss of function [70].…”

The Combination of the CDK4/6 Inhibitor, Palbociclib, With the Vitamin D3 Analog, Inecalcitol, Has Potent In Vitro and In Vivo Anticancer Effects in Hormone-Sensitive Breast Cancer, But Has a More Limited Effect in Triple-Negative Breast Cancer

“…Interestingly, treatment with 1,25(OH) 2 D 3 or its analogs is able to suppress this pathway in multiple cell types, including BC cells, resulting in decreased phosphorylation of the PI3K/Akt/ mTOR-downstream targets P70S6K and 4E-BP1, thereby inhibiting ribosomal translation and protein synthesis (44)(45)(46)(47). Also, palbociclib therapy is able to decrease mTOR activation and inhibits P70S6K phosphorylation (48)(49)(50). In contrast, other studies reported that palbociclib activates the mTOR pathway (27,36).…”

Biological Hallmarks and New Therapeutic Approaches for the Treatment of PDAC

“…The p16INK4a protein, which belongs to the INK4 family of cyclin-dependent kinase inhibitors, prevents the formation of the complex cyclin D1/CDK4/6 that regulates Rb function. CDK4/cyclin D1, by phosphorylating Rb1 protein, induces the release of the transcription factor E2F, which activates genes involved in the progression from G1 to S phase [ 37 , 38 , 39 ]. In contrast, Rb in its hypophosphorylated active form leads to G1 cell cycle arrest by sequestering the E2F protein.…”