MYC phosphorylation, activation, and tumorigenic potential in hepatocellular carcinoma are regulated by HMG-CoA reductase

Abstract

MYC is a potential target for many cancers but is not amenable to existing pharmacologic approaches. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) by statins has shown potential efficacy against a number of cancers. Here, we show that inhibition of HMG-CoA reductase by atorvastatin (AT) blocks both MYC phosphorylation and activation, suppressing tumor initiation and growth in vivo in a transgenic model of MYC-induced hepatocellular carcinoma (HCC) as well as in human HCC-derived cell lines. To confirm specificity, we show that the antitumor effects of AT are blocked by cotreatment with the HMG-CoA reductase product mevalonate. Moreover, by using a novel molecular imaging sensor, we confirm that inhibition of HMG-CoA reductase blocks MYC phosphorylation in vivo. Importantly, the introduction of phosphorylation mutants of MYC at Ser62 or Thr58 into tumors blocks their sensitivity to inhibition of HMG-CoA reductase. Finally, we show that inhibition of HMG-CoA reductase suppresses MYC phosphorylation through Rac GTPase. Therefore, HMG-CoA reductase is a critical regulator of MYC phosphorylation, activation, and tumorigenic properties. The inhibition of HMG-CoA reductase may be a useful target for the treatment of MYC-associated HCC as well as other tumors.

Figure 1. Inhibition of HMG-CoA reductase by atorvastatin (AT) suppresses growth of MYC-induced hepatocellular carcinoma (HCC) in vitro and in vivo

a) AT inhibits the proliferation of a MYC-induced HCC tumor derived cell line, 3–4. MTT assay was performed every 24 hours for 4 days on HCC cells treated with 10μM AT, AT plus 100μM MV, or DMSO as a vehicle control. Cells were also treated with doxycycline (Dox) to inactivate transgenic MYC as a positive control. All experiments were repeated 3 times (p<0.0001). Error bars represent standard deviation. b) AT induces cell cycle arrest in murine HCC as assessed by FACS analysis of PI stained cells (p=0.01). Cells were treated with 10μM AT, AT plus MV, DMSO, or Dox for 48 hr. c) Immunofluorescence for Ki67 on HCC cells treated with 10μM AT for 48hr demonstrates that statin treatment inhibits HCC proliferation (p<0.0001). d) AT inhibits growth of MYC-induced HCC cells in vivo. Murine HCC cells were subcutaneously transplanted into FVB/N mice treated with PBS (n=5) or AT (n=5). p=0.0003. Error bars represent standard deviation. e) Representative images of mice treated with PBS (left), AT (middle), or AT plus MV (right) shows that AT suppresses growth of MYC-induced HCC in vivo.

Figure 2. Blocking HMG-CoA reductase via AT inhibits growth of human HCC cells in vitro and in vivo

a) MTT analysis of the human HCC cell line, Huh7, treated with 10μM AT demonstrates significant inhibition of growth in vitro (p<0.0001). Error bars represent standard deviation. b) FACS analysis of PI-stained Huh7 cells reveals that AT suppresses cell cycle progression (p=0.0003). Cells were treated with 10μM AT for 48hr. c) Huh7 cells treated with 10μM AT for 48hr were examined by immunofluorescence for Ki67, demonstrating statin-induced reduction in proliferative cells (p<0.0001). d) Huh7 cells were transplanted intraperitoneally into SCID mice, and host animals were treated with PBS, AT, or AT with MV. A Kaplan-Meier curve demonstrates a significant increase in survival of animals treated with AT (PBS versus AT, p=0.03; AT versus AT+MV, p=0.04; PBS versus AT+MV, p=0.8).

Figure 3. AT inhibition of HMG-CoA reductase suppresses MYC-induced hepatocellular tumorigenesis

a) Kaplan-Meier survival curves of adult LAP-tTA/TRE-MYC mice treated with PBS (n=8), 100mg/kg AT (n=5), or AT with MV (n=5) three times per week demonstrates a significant increase in survival of animals treated with AT (PBS versus AT, p=0.005; AT versus AT/MV, p=0.016; PBS versus AT/MV, p=0.25). b) Representative pictures from each treatment group are shown. Gross anatomy reveals inhibition of tumor onset due to AT treatment. H&E staining shows that normal hepatic structure is maintained by AT treatment (middle row), which was reversed by MV treatment (bottom row). Immunohistochemistry for Ki67 shows a significant inhibition of proliferation due to AT (right column, 8 ± 3 positive cells versus 422 ± 23 positive cells per high power field; p<0.02).

Figure 4. Inhibition of HMG-CoA reductase blocks MYC activity by reducing its phosphorylation

a) Murine HCC cells were treated with indicated concentrations of AT with or without MV for 24 hours. MYC phosphorylation and expression are suppressed by AT treatment in a dose-dependent manner (p=0.002). Values are normalized to the DMSO control. Representative immunoblots are shown. b) Primary liver tissue of transgenic animals shows an AT-dependent suppression of MYC phosphorylation (p=0.04). Representative immunoblots are shown. Error bars represent standard deviation. c) qPCR analysis of MYC and MYC target gene mRNA expression in murine HCC cells. Treatment of cells with 10μM AT for 24 hours results in reduced MYC transcriptional activity as shown by 72% and 76% reduction in expression of ODC and nucleolin, respectively (p=0.003, p=0.03). Expression is normalized to ubiquitin and values are relative to DMSO control. Error bars represent standard deviation. d) qPCR analysis of MYC and MYC target gene mRNA expression in human Huh7 cells. Cells were treated with 10μM AT for 24hr and demonstrate suppression of MYC transcriptional activity as assessed by reductions of 64% for ODC and 59% for nucleolin expression (p=0.016, p=0.008). Expression is normalized to ubiquitin and values are relative to DMSO control. Error bars represent standard deviation.

Figure 5. A novel bioluminescence c-Myc phospho-sensor noninvasively demonstrates AT-dependent inhibition of MYC phosphorylation

a) The N- and C-termini of split firefly luciferase (FL) were fused to the phospho-regulated domain of c- Myc and the corresponding domain of GSK3β, respectively. Phosphorylation of the c- Myc peptide results in FL enzymatic activity. b) Huh7 cells were transfected with the c-Myc phosphorylation sensor and treated with AT. Bioluminescent imaging (BLI) shows a dose-dependent inhibition of phospho-MYC (p<0.0001). Firefly luciferase (FL) activity was normalized to Renilla luciferase (RL) activity and plotted against AT concentration. Error bars represent standard deviation. c) Western blot analysis confirms AT-dependent suppression of phospho-c-Myc in transfected Huh7 cells. d) LAP-tTA/TRE-MYC transgenic mice were treated with AT or PBS (n=3), and hydrodynamic injection of the phospho-sensor followed by BLI shows AT-dependent inhibition of c-Myc phosphorylation in vivo (AT-treated mice day 0 versus day 15, p=0.038; PBS-treated mice day 0 versus day 15, p=0.638). e) FL activity was normalized to RL activity and plotted against days of AT treatment.

Figure 6. HCC transduced with S62A or T58A MYC phospho-mutants demonstrate reduced sensitivity to HMG-CoA reductase inhibition

a) Murine HCC cells were infected with Ad-MYC^WT (WT), Ad-MYC^S62A (S62A), or Ad-MYC^T58A (T58A) adenovirus, and HA-tagged MYC was immunoprecipitated using an antibody to the HA tag. Immunoblot analysis suggests that AT-dependent phospho-regulation of MYC is via S62. However, the inhibition of protein stability requires both S62 and T58 phospho- regulation. Cells were treated with 10μM AT for 24 hr. b) S62A partially and T58A completely abrogated AT inhibition of cell proliferation (S62A: PBS versus AT, p<0.0001; T58A: PBS versus AT p=0.8). Error bars represent standard deviation. c) HCC cells isolated from transgenic animals were transplanted into SCID mice, injected with Ad-MYC^WT, Ad-MYC^S62A, or Ad-MYC^T58A once every week, and orally treated with PBS (n=6), AT (n=5), or AT with MV (n=5) together with Dox. Tumor growth was measured three times per week. d) In vivo growth kinetics of HCC infected with Ad-MYC^WT show that AT inhibits tumor growth in vivo (Left, PBS versus AT, p=0.01; AT versus AT+MV, p=0.007). Error bars represent standard deviation. Infection with Ad-MYC^S62A partially rescues growth inhibition due to AT (Middle, PBS versus AT, p=0.02; AT versus AT+MV, p=0.03). Error bars represent standard deviation. Ad-MYC^T58A completely rescues AT-mediated growth inhibition of HCC (Right, PBS versus AT, p=0.56; AT versus AT+MV, p=0.03). Error bars represent standard deviation.

Figure 7. HMG-CoA reductase influences MYC phosphorylation through a Rac GTPase-dependent mechanism

a) Suppression of murine HCC growth in vitro upon 10μM AT treatment for 96 hours is rescued by GGPP treatment, as assessed by MTT (DMSO versus AT+MV, p=0.07; DMSO versus GGPP, p=0.053, DMSO versus FPP, p=0.002). Error bars represent standard deviation. b) GGPP treatment rescues the AT- dependent suppression in human HCC cell growth upon 10μM AT treatment (DMSO versus AT+MV, p=0.01; DMSO versus GGPP, p=0.01, DMSO versus FPP, p<0.001). Error bars represent standard deviation. c) GGPP treatment rescues AT-dependent inhibition of MYC phosphorylation. Representative immunoblots are shown. Error bars represent standard deviation. d) GGPP treatment prevents the decrease in the membrane accumulation of Rac induced by 24 hours of 10μM AT. e) AT treatment inhibits Rac activity. Rac activity was reduced by 77% as measured by pull-down assay. f) Our results suggest a model in which inhibition of HMG-CoA reductase by AT blocks prenylation and activation of small GTPases, specifically including Rac. AT-mediated inhibition of Rac likely results in reduction of phospho-S62 MYC. Dephosphorylation at S62 in the context of phospho-T58 thereby results in the ubiquitin-mediated degradation of MYC. As such, AT treatment ultimately results in the inhibition of MYC oncogenic activity and suppressed hepatocellular carcinogenesis.