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. 2010 May;138(5):1920-30.
doi: 10.1053/j.gastro.2010.01.007. Epub 2010 Jan 25.

Proliferative Suppression by CDK4/6 Inhibition: Complex Function of the Retinoblastoma Pathway in Liver Tissue and Hepatoma Cells

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Free PMC article

Proliferative Suppression by CDK4/6 Inhibition: Complex Function of the Retinoblastoma Pathway in Liver Tissue and Hepatoma Cells

Dayana B Rivadeneira et al. Gastroenterology. .
Free PMC article

Abstract

Background & aims: Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide; current chemotherapeutic interventions for this disease are largely ineffective. The retinoblastoma tumor suppressor (RB) is functionally inactivated at relatively high frequency in hepatocellular carcinoma and hepatoma cell lines. Here, we analyzed the ability of CDK4/6 inhibition to inhibit hepatocyte proliferation and the effect of RB status on this process.

Methods: Hepatoma cell lines and xenograft models harboring RB knockdown and mice harboring liver-specific Rb deletion were used to define the role of RB function in response to CDK4/6 inhibition.

Results: Our study shows that CDK4/6-dependent cell cycle progression in hepatoma cells was readily arrested by inhibition of CDK4/6 by PD-0332991 or p16ink4a irrespective of RB status. Interestingly, upon CDK4/6 inhibition, p107 protein stability was dramatically increased as a function of RB loss. This engagement of compensatory mechanisms was critical for cell cycle inhibition in the absence of RB, because both the E1A oncoprotein and overexpression of E2F proteins were capable of overcoming the effect of CDK4/6 inhibition. These findings were recapitulated in xenograft models. Furthermore, to determine how these findings relate to hepatocyte proliferation in vivo, mice were exposed to carbon tetrachloride to induce liver regeneration followed by treatment with PD-0332991. This treatment significantly inhibited hepatocyte proliferation. Strikingly, this facet of PD-0332991 function was retained even in RB-deficient livers.

Conclusions: These data show that CDK4/6 inhibition is a potent mediator of cytostasis and that RB loss can be readily compensated for in the context of both hepatoma cell lines and liver tissue.

Conflict of interest statement

Potential conflict of interest: Nothing to report.

Figures

Figure 1
Figure 1. CDK4/6 inhibitor causes cell cycle response in HCC cells
(A) Indicated cells were treated with 1µM PD-0332991 for 24 hours. Cells were pulsed with BrdU for one hour before harvest. BrdU incorporation was analyzed by bivariate flow cytometry. (B and C) Cell lysates were analyzed by immunoblot for indicated proteins. (D) Cells were infected with miRB or miNS retroviruses. Cells were harvested and lysates were analyzed by immunoblot for the indicated proteins. (E) Relative BrdU incorporation from at least three independent experiments; bars, SD.
Figure 2
Figure 2. RB-independent cell cycle arrest of hepatoma cells by CDK4/6 inhibition
(A) HepG2 and Huh7 cells models were treated with 1µM PD-0332991 for 24 hours. BrdU incorporation was analyzed by bivariate flow cytometry, data are from at least three independent experiments; bars, SD. (B) Huh7 cells were infected with Ad-p16ink4a or Ad-GFP for 24 hours. BrdU incorporation was analyzed as in (A). (C) Hep3B cells were treated and analyzed as in (A). (D) Following treatment, cell lysates were analyzed by immunoblot for the indicated proteins. (E) Cells were treated as in (A). Cell lysates were analyzed by immunoblot for the indicated proteins. (F) Hep3B cells were treated with 40µM Roscovitine (RVT) for 24 hours. BrdU incorporation was analyzed as in (A). Cell lysates were immunoblotted for indicated proteins.
Figure 3
Figure 3. G1/S phase arrest by transcriptional repression of RB/E2F target genes is independent of RB status after PD-0332991 treatment
(A) Cells were treated with increasing concentrations of PD-0332991 for 24 hours. Reverse transcription-PCR was done targeting the indicated genes. (B,C) Cells were treated as in (A) and cell lysates were analyzed by immunoblot for the indicated proteins. (D) Cells were transfected with luciferase reporter constructs pCycA-Luc, pCycA-CCREmut-Luc and CMV-ß-galactosidase expression plasmid. After transfection, cells were treated as in (A) and luciferase activity was measured. Average relative luciferase activities from at least three independent experiments; bars, SD.
Figure 4
Figure 4. p107 protein levels and promoter occupancy is differentially regulated in RB-deficient cells exposed to PD-0332991
(A) Cells treated with PD-0332991 were harvested and lysates were analyzed by immunoblot for indicated proteins. (B) Chromatin was isolated from cells treated as in (A). CHIP assays were performed using antibodies for p107 and Dbf4 (negative control). Input and immunoprecipitated DNA were amplified by PCR with primers for the promoters of the indicated genes. (C) HepG2miNS/miRB cells were treated with 0.1mM cycloheximide for twelve hours. Lysates were analyzed by immunoblot for indicated proteins. Rate of p107 protein levels were plotted relative to p107 protein levels in untreated cells; bars, SD. Cells were treated with 1µM PD-0332991 for 24h. Twelve hours later, CHX (0.1mM) was added. Cells were harvested and analyzed as in (A). (D) Cells where treated with 1µM PD-0332991 and 1µM MG132 for 24h. Cells were harvested and lysates were analyzed by immunoblot for the indicated proteins. (E) HepG2 cells were transfected with HA-tagged ubiquitin and treated as in (D). Protein extracts were immunoprecipitated with anti-p107 and analyzed by immnublot for the indicated proteins.
Figure 5
Figure 5. E1A or E2F overexpression reverses cell cycle arrest in RB cells exposed to PD-0332991
(A) Cells were co-transfected with H2B-GFP expression plasmid and indicated E1A expression plasmid, and treated with 1µM PD-0332991 for 24h. The percentage of BrdU-positive cells was determined from three independent experiments; bars, SD. (B) Cells were infected with adenoviruses harboring either GFP (vector) or E2F2. Cells were treated with 1µM PD-0332991 for 24 hours. Cells were pulsed with BrdU one hour before harvest. Cell cycle was analyzed by bivariate flow cytometry. (C) Infected cells treated 1µM PD-0332991 were harvested and analyzed by immunoblot for indicated proteins.
Figure 6
Figure 6. PD-0332991 treatment is highly effective in xenograft models irrespective of RB status
(A) Huh7-miNS/miRB xenografts where treated with 150mg/kg PD-0332991. Nude mice where injected with BrdU 1 hour prior to sacrifice. Sectioned tumors were immunohistochemically stained and scored for BrdU. Relative BrdU incorporation from at least three independent experiments; bars, SD. Representative images of BrdU staining at 10x magnification. (B) Tumor samples were harvested and lysates were analyzed by immunoblot for the indicated proteins.
Figure 7
Figure 7. Cell cycle arrest independent of RB status in vivo after PD-0332991 treatment
(A) Cre-mediated recombination of the floxed Rb locus (RbexonΔ19) was detected by PCR analysis of genomic DNA. (B, E) Mice were injected with CCl4 by i.p. 24 hours later treated with five doses of 150mg/kg PD-0332991. Mice where injected with BrdU 1 hour prior to sacrifice. BrdU incorporation was detected and scored. Percentage of BrdU incorporation from at least three independent experiments; bars, SD. Representative images of BrdU staining are shown at 10X magnification. (C) Liver tissue samples were harvested and lysates were analyzed by immunoblot for indicated proteins. (D) Liver tissue sections were stained with H&E. Representative images were taken of necrotic areas of the livers at 10X magnification.

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