S-adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers

Abstract

Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and is overexpressed in several malignancies, but its expression in hepatocellular carcinoma (HCC) is unknown. Hepatic S-adenosyl methionine (SAMe) levels decrease in methionine adenosyltransferase 1A (Mat1a) knockout (KO) mice, which develop HCC, and in ethanol-fed mice. We examined the regulation of Ubc9 by SAMe in murine liver and human HCC, breast, and colon carcinoma cell lines and specimens. Real-time polymerase chain reaction and western blotting measured gene and protein expression, respectively. Immunoprecipitation followed by western blotting examined protein-protein interactions. Ubc9 expression increased in HCC and when hepatic SAMe levels decreased. SAMe treatment in Mat1a KO mice reduced Ubc9 protein, but not messenger RNA (mRNA) levels, and lowered sumoylation. Similarly, treatment of liver cancer cell lines HepG2 and Huh7, colon cancer cell line RKO, and breast cancer cell line MCF-7 with SAMe or its metabolite 5'-methylthioadenosine (MTA) reduced only Ubc9 protein level. Ubc9 posttranslational regulation is unknown. Ubc9 sequence predicted a possible phosphorylation site by cell division cycle 2 (Cdc2), which directly phosphorylated recombinant Ubc9. Mat1a KO mice had higher phosphorylated (phospho)-Ubc9 levels, which normalized after SAMe treatment. SAMe and MTA treatment lowered Cdc2 mRNA and protein levels, as well as phospho-Ubc9 and protein sumoylation in liver, colon, and breast cancer cells. Serine 71 of Ubc9 was required for phosphorylation, interaction with Cdc2, and protein stability. Cdc2, Ubc9, and phospho-Ubc9 levels increased in human liver, breast, and colon cancers.

Conclusion: Cdc2 expression is increased and Ubc9 is hyperphosphorylated in several cancers, and this represents a novel mechanism to maintain high Ubc9 protein expression that can be inhibited by SAMe and MTA.

Fig. 1. Ubc9 expression is up-regulated in HCC and ethanol fed mouse livers

Expression was measured using quantitative real-time PCR and Western Blotting analysis. (A, B) Results from 18 pairs of matched non-tumorous (N) and HCC (T) tissues are expressed as fold of non-tumorous tissue (mean ± SEM). *P<0.05 and **p<0.002 vs.. non-tumorous tissues. (C, D) Ethanol fed mice were treated with ethanol or isocaloric dextrose as described in Methods. Results are expressed as fold of control (mean ±SEM) from n=4 for each group. *P<0.02 vs. control mice.

Fig. 2. Effects of SAMe treatment on Ubc9 expression and sumoylation in Mat1a KO mice

(A) Real-time PCR analysis measured Ubc9 mRNA levels. Results are expressed as fold of WT mice (mean ± SEM) from four mice per group. *P<0.03 vs. WT; **p<0.04 vs. WT. (B) Western Blotting analysis measured Ubc9 protein levels. Results are expressed as % of WT mice (mean ± SEM) from four mice per group. *P<0.01 vs. WT; **p<0.04 vs. KO. (C) SAMe treatment normalized the increase in sumoylated RanGAP1 levels in Mat1a KO mice livers. Results are expressed as % of WT mice (mean ± SEM) from four each of WT, untreated KO, and SAMe-treated KO mice. *P<0.001 vs. WT; **p<0.001 vs. KO.

Fig. 3. SAMe and MTA treatment lowered Ubc9 protein levels in HepG2, Huh-7, RKO and MCF-7 cells

Cells were treated with SAMe (2mM) or MTA (1mM) for 24 hours. (A) Quantitative real-time PCR measured mRNA levels of Ubc9. Results are expressed as fold of control (mean ± SEM) from 3 independent experiments. (B) Western blot analysis using whole cell lysates (10μg) measured Ubc9 protein levels. The values are expressed as % of control (mean ± SEM) from 3 independent experiments. *P<0.001 vs. control of HepG2, *p<0.001 vs. control of Huh-7, *p<0.01 vs. control of RKO and *p< 0.01 vs. control of MCF-7 cells.

Fig. 4. SAMe and MTA treatment reduced Ubc9’s state of phosphorylation and sumoylation

(A) Huh-7, RKO and MCF-7 cells were treated with SAMe (2mM) or MTA (1mM) for 24 hours. Total protein phosphorylation was examined by Western blotting using anti-phospho antibody, while phospho-Ubc9 was determined by first immunoprecipitating total Ubc9, followed by Western blotting with anti-phospho antibody. Sumoylation was measured using anti-SUMO-1 antibody in Western blotting. The values are expressed as % of control cells (mean ± SEM) from 3 independent experiments. *P<0.02, **p<0.05 vs. control cells. (B) RKO cells were cultured and treated with phosphatase inhibitor cocktail (1:10000) for 24 hours and processed for immunoprecipitation using anti-Ubc9 antibody followed by Western blotting with anti-phospho antibody or anti-Ubc9 antibody. Results are expressed as % of control (mean ± SEM) from 3 independent experiments. *P<0.02 vs. control cells. (C) Prediction of potential kinases that can phosphorylate Ubc9 protein sequence using the KinasePhos, PostMod, MotifScan, Phosphonet analysis tools.

Fig. 5. Cdc2 phosphorylates Ubc9 in vitro, interacts with Ubc9 in vivo and its expression is inhibited by SAMe and MTA

(A) Cdc2/Cyclin B phosphorylates recombinant Ubc9 in vitro. Results are expressed as % control (mean ± SEM) from 3 independent experiments. *P<0.001 vs. Ubc9, †p<0.05 vs. Ubc9+Cdc2/CyclB. (B) RKO cells were treated with SAMe and MTA for 24 hours. Ubc9 expression was examined by real-time PCR and Western blotting. Results are expressed as fold or % control (mean ± SEM) from 3 independent experiments. *P<0.001 vs. control cells. (C) Effect of Cdc2 RNAi, SAMe and MTA treatments on Ubc9 and Cdc2 protein levels. RKO cells were treated with scrambled or Cdc2 RNAi or with 2mM SAMe or 1mM MTA as described in Methods. Results are expressed as % of scrambled control (mean ± SEM) from 3 independent experiments. *P<0.01, **p<0.001 vs. scrambled control. (D) Ubc9 interacts with Cdc2 in vivo. Immunoprecipitation with anti-Ubc9 antibody followed by Western blotting for phospho-Ubc9, total Ubc9, and Cdc2 in Mat1a KO livers was done as described in Methods. Results are expressed as % of WT mice (mean ± SEM) from 4 mice per group. *P < 0.001 vs. WT; **P < 0.04 vs. KO.

Fig. 6. Levels of phospho-Ubc9, total Ubc9, Cdc2 and Cdc2 that interacts with Ubc9 are increased in human colon, liver and breast cancers

Representative Western Blots of colorectal (A), HCC (B), breast (C) cancer specimens (T) and corresponding non–tumorous (N) tissues are shown. The immunoprecipitation was done as described in Methods for phosphorylated, total Ubc9 and Cdc2. Results represent mean ± SEM from 12 colorectal, 12 HCC, and 12 breast cancers expressed as % of corresponding non-tumorous (N) tissues. *P<0.01 vs. non-tumorous tissue.

Fig. 7. Mutation at Ubc9 S71 reduced interaction of Cdc2 with Ubc9 and Ubc9 protein stability

(A) RKO cells were transfected with the vector, hUbc9-WT, hUbc9-F70mut, and hUbc9-F71mut for 48 hours. Co-IP analysis was done as described in Methods for phosphorylated, total Ubc9 and Cdc2. Results are expressed as % of WT (mean ± SEM) from 4 independent experiments. *P<0.02 vs. WT. (B) Ubc9 protein stability was determined in RKO and Huh-7 cells by Western blot analyses of HA tag following cycloheximide (CHX) treatment as described in Methods. Representative blots are shown. (C) Protein stability was determined using linear regression and half-life calculated using equation indicated. Results represent mean±SEM from 3 independent experiments expressed as % of respective 0 hr level for both cell types (p<0.05 between WT and MUT).

Fig. 8. Simplified schematic of key findings from this work

Under physiological conditions, Cdc2 phosphorylates Ubc9 at a basal level. In cancer and conditions of increased oxidative stress (which occurs when hepatic SAMe level is reduced), expression of Ubc9 and Cdc2 is increased leading to higher phospho-Ubc9 level and increased Ubc9 stability, resulting in increased protein sumoylation. This pathway can be disrupted by SAMe or MTA treatment, which can inhibit Cdc2 at the mRNA level to lower Cdc2-mediated Ubc9 phosphorylation, resulting in reduced Ubc9 protein level and protein sumoylation.