Interventional targeting of Cyclin E1 during hepatocarcinogenesis limits stem cell traits and hepatic myeloid cell homing and attenuates cancer progression

Background & Aims:

Hepatocellular carcinoma (HCC) is one of the most severe tumor diseases with increasing incidence and limited treatment options. HCC initiation and progression are associated with persistent proliferation of hepatocytes and non-parenchymal cells (e.g. hepatic stellate cells, immune cells). Proliferation is related to cell cycle activity, which is basically regulated by cyclins and Cyclin-dependent kinases (Cdks). E-Type cyclins (Cyclin E1, E2) and their canonical binding partner Cdk2 are key mediators of early cellular DNA replication (S-phase). Own work demonstrated an essential role of Cyclin E1 and Cdk2 specifically for initiation of HCCs in a murine prevention model (Sonntag et al., PNAS 2018 Sep 11;115(37):9282 – 9287). In the present study, we investigated the therapeutic benefit of Cyclin E1 or Cdk2 gene targeting after onset of hepatocarcinogenesis (intervention model).

Methods:

In this study, conditional Cyclin E1 (CcnE1f/f) or Cdk2 (Cdk2f/f) mice in a C57B6/J background with inducible expression of Cre-recombinase under control of the Mx-gene promoter were used. For HCC induction, 14 days old male mice were intraperitoneally (i.p.) injected with a single dose of diethylnitrosamine (DEN). Cre-negative littermates were used as controls. After 22 weeks (reflecting a stage of early HCC development), interventional inactivation of Cyclin E1 or Cdk2 was performed by three i.p. injections of poly-I:poly-C, inducing gene deletion in hepatocytes and in the hematopoietic cell compartment. Two weeks and 16 weeks after intervention, mice were analyzed for tumor number and size in order to determine immediate and long term effects of the treatment. Additionally, dissected liver and tumor tissues were mechanistically investigated for proliferation, stemness and DNA repair. The hepatic microenvironment was analyzed via flow cytometry.

Results:

At the age of 40 weeks, all DEN-treated mice developed HCC. However, interventional inactivation of Cyclin E1 resulted in a significant reduction of tumor numbers and size compared to DEN-treated control mice. This finding was associated with a decreased overall hepatic proliferation and intratumoral down-regulation of cell cycle activators, tumor markers, stem cell traits and vascularization markers. Already two weeks after intervention, Cyclin E1 deletion significantly reduced the expression of pro-proliferative genes. Importantly, Cyclin E1-independent growth in remnant tumors was associated with sustained expression of DNA repair genes. Moreover, Cyclin E1 inactivation also changed the composition of the myeloid HCC microenvironment (e.g. myeloid-derived suppressor cells) pointing to a new role of Cyclin E1 for immune cell progenitor cell homing during liver cancer development. In sharp contrast, interventional inactivation of Cdk2 after onset of hepatocarcinogenesis did not reveal any beneficial effect on tumor burden.

Conclusions:

Cdk2 is essential for HCC initiation, but surprisingly fully dispensable for HCC progression. However, interventional inactivation of Cyclin E1 during early HCC progression attenuates disease development. Hence, Cyclin E1 presents a promising therapeutic target for treatment of HCC patients.