Cdk4 and Cdk6 cooperate in counteracting the INK4 family of inhibitors during murine leukemogenesis

Hepatocellular carcinoma (HCC) is a prototype of inflammation-associated cancer. Oncoprotein Gankyrin, mostly increases in HCC, plays a critical role in HCC development and metastasis. However, the exact mechanism of Gankyrin upregulation in HCC remains unclear. A Gankyrin luciferase reporter was developed to screen potential regulator for Gankyrin from a list of pro-inflammatory cytokines, and IL-1? was found as one of its activators.

In clinical pre-malignant and malignant liver diseases samples, enhanced IL-1?/IRAK-1 signaling accompanied by increased Gankyrin was observed. Lower expression of Gankyrin and phospho-IRAK-1 are favorable prognostic markers for Hcc. A similar correlation was observed in the diethylnitrosamine (DEN) model of rat hepatocarcinogenesis. The results from Gankyrin reporter activity, real-time PCR, or immunoblot further confirmed the upregulation of Gankyrin by IL-1?/IRAK-1 inflammatory signaling. Moreover, a series of Gankyrin's truncated reporters were constructed, and electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were performed to analyze the properties of Gankyrin promoter. Mechanistically, the core promoter of Gankyrin contains the binding site of NF-Y family members, which can recruit histone acetyltransferase (HAT) co-activator p300 or CBP to promote Gankyrin transcription. Conversely, knockdown of NF-Y, p300, or CBP inhibits Gankyrin expression. IL-1? stimulation causes sequential phosphorylation of IRAK-1, c-Jun N-terminal kinase (JNK) and p300, and enhances the recruitment of the p300/CBP/NF-Y complex to Gankyrin promoter. Inhibition of phospho-JNK impairs IL-1?/IRAK-1 signaling-mediated upregulation of Gankyrin. Thus, the finding of IL-1?/IRAK-1 signaling promoting Gankyrin expression via JNK and NF-Y/p300/CBP complex provides a fresh view on inflammation-enhanced hepatocarcinogenesis.

Cdk4 and Cdk6 are related protein kinases that bind d-type cyclins and regulate cell-cycle progression. Cdk4/6 inhibitors are currently being used in advanced clinical trials and show great promise against many types of tumors. Cdk4 and Cdk6 are inhibited by INK4 proteins, which exert tumor-suppressing functions.

To test the significance of this inhibitory mechanism, the researchers generated knock-in mice that express a Cdk6 mutant (Cdk6 R31C) insensitive to INK4-mediated inhibition. Cdk6(R/R) mice display altered development of the hematopoietic system without enhanced tumor susceptibility, either in the presence or absence of p53. Unexpectedly, Cdk6 R31C impairs the potential of hematopoietic progenitors to repopulate upon adoptive transfer or after 5-fluorouracil-induced damage. The defects are overcome by eliminating sensitivity of cells to INK4 inhibitors by introducing the INK4-insensitive Cdk4 R24C allele, and INK4-resistant mice are more susceptible to hematopoietic and endocrine tumors. In BCR-ABL-transformed hematopoietic cells, Cdk6 R31C causes increased binding of p16(INK4a) to wild-type Cdk4, whereas cells harboring Cdk4 R24C and Cdk6 R31C are fully insensitive to INK4 inhibitors, resulting in accelerated disease onset. Their observations reveal that Cdk4 and Cdk6 cooperate in hematopoietic tumor development and suggest a role for Cdk6 in sequestering INK4 proteins away from Cdk4.