Obesity/Type 2 Diabetes-Associated Liver Tumors Are Sensitive to Cyclin D1 Deficiency

Abstract

Type 2 diabetes, which is mainly linked to obesity, is associated with increased incidence of liver cancer. We have previously found that in various models of obesity/diabetes, hyperinsulinemia maintains heightened hepatic expression of cyclin D1, suggesting a plausible mechanism linking diabetes and liver cancer progression. Here we show that cyclin D1 is greatly elevated in human livers with diabetes and is among the most significantly upregulated genes in obese/diabetic liver tumors. Liver-specific cyclin D1 deficiency protected obese/diabetic mice against hepatic tumorigenesis, whereas lean/nondiabetic mice developed tumors irrespective of cyclin D1 status. Cyclin D1 dependency positively correlated with liver cancer sensitivity to palbociclib, an FDA-approved CDK4 inhibitor, which was effective in treating orthotopic liver tumors under obese/diabetic conditions. The antidiabetic drug metformin suppressed insulin-induced hepatic cyclin D1 expression and protected against obese/diabetic hepatocarcinogenesis. These results indicate that the cyclin D1-CDK4 complex represents a potential selective therapeutic vulnerability for liver tumors in obese/diabetic patients. SIGNIFICANCE: Obesity/diabetes-associated liver tumors are specifically vulnerable to cyclin D1 deficiency and CDK4 inhibition, suggesting that the obese/diabetic environment confers cancer-selective dependencies that can be therapeutically exploited.

Figure 1.. Cyclin D1 is aberrantly upregulated in type 2 diabetic liver and HCC.

A. Cyclin D1 protein expression is significantly increased in liver of dietary and genetic obese/type 2 diabetic mice. Mice were kept with either chow/low-fat diets (Chow) or high-fat diets (HFD) for nine months. Lepr^db mice, referred as db/db for short, were used as a genetic model of obesity/diabetes. B. Cyclin D1 expression is elevated in clinical liver specimens from individuals with diabetes, as determined by immunohistochemistry (IHC). Physiologically normal liver samples were used as control. Representative images were shown for the staining pattern and intensity. Scale bar represents 100 μm. C-D. Expression levels of Cyclin D1 are significantly enhanced in obese/diabetic HCC, as determined by qPCR (C) and immunoblotting (D). Liver cancers were derived in mice administered with chemical carcinogen diethylnitrosomine (DEN) at postnatal day 14, followed by either chow or HFD feeding for 30 weeks.

Figure 2.. Cyclin D1 deficiency protects mice from carcinogen-induced HCC formation in the context of obesity/type 2 diabetes.

A. Deletion of Cyclin D1 in hepatocytes protects mice from liver cancer under diet-induced obese/type 2 diabetic conditions. In the diet-induced obesity/diabetes model, DEN-injected mice were subjected to HFD for the whole span of the experiments. Representative liver phenotype at the endpoint, tumor burden and liver histology between Cyclin D1 WT and liver-specific KO (LKO) mice were shown (WT, n=12; LKO, n=19). B. Deletion of Cyclin D1 in hepatocytes protects mice from liver cancer under genetic obese/type 2 diabetic conditions. The Lepr^db alleles were introduced to genetically induce obesity/diabetes in DEN-injected mice. Representative liver phenotype at the endpoint, tumor burden and liver histology between Cyclin D1 WT and LKO mice were shown (WT, n=5; LKO, n=7). C. Hepatic Cyclin D1 is dispensable for liver cancer formation in lean condition. Representative liver phenotype at the endpoint, tumor burden and liver histology between Cyclin D1 WT and LKO mice kept on chow/LFD were shown (WT, n=17; LKO, n=11). Tumor burden was presented as average surface tumor number per mouse. Dash lines outlined tumor nodules in each H&E image. Scale bar represents 500 μm.

Figure 3.. Cyclin D1 correlates with sensitivity to CDK4 inhibition in obese/diabetic liver cancer.

A. Dependency on CCND1 gene correlates positively with sensitivity to CDK4 inhibitor palbociclib across a panel of human liver cancer cell lines. Dependency on CCND1 gene was determined by CRISPR gene editing from the Broad’s Project Achilles DepMap Public 19Q3 dataset. Palbociclib sensitivity was presented as IC50 from Sanger’s Genomics of Drug Sensitivity in Cancer dataset. B. CDK4 inhibitor palbociclib is effective in suppressing the progression of obese/diabetic liver tumors (n=5). Diagram depicts the protocol for orthotopic liver cancer and treatment model. Murine hepatoma was established in the liver of db/db mice via intrasplenic injection. Upon tumor established at day 10 post injection, daily treatment was given via oral gavage for six days, followed by tumor assessment. Tumor loads were measured by subtracting the weight of normal liver from the weights of tumor-bearing livers, or by quantifying the microscopic cancerous areas of the H&E slides. C. Liver cancers developed in obese/diabetic mice are more vulnerable to CDK4 inhibitor (n=5). Upon hepatoma development in lean or obese/diabetic mice, treatments with either vehicle (Veh) or palbociclib were given at a relatively lower dose, followed by tumor assessment.

Figure 4.. Metformin reduces Cyclin D1 expression and prevented HCC initiation in obese/diabetic mice.

A. Metformin prevents insulin-induced Cyclin D1 protein levels in hepatocytes. Overnight serum starved AML12 murine hepatocytes were treated with control, metformin (10 mM), insulin (200 nM) or both for 4 h, followed by immunoblotting to detect Cyclin D1 expression. B. Design and metabolic phenotypes of the metformin treatment experiment. Metformin was given at 50 mg/kg for 27 weeks. Endpoint blood insulin level and liver histology of the db/db mice were shown. Scale bar represents 500 μm. C. Metformin treatment prevents the induction of Cyclin D1 in type 2 diabetic liver as detected by immunoblotting. D. Metformin treatment protects mice from DEN-induced liver cancer in diabetic mice (water, n=5; metformin, n=4).