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.

Methionine adenosyltransferases (MATs) are essential for cell survival because they catalyze the biosynthesis of the biological methyl donor S-adenosylmethionine (SAMe) from methionine and adenosine triphosphate (ATP). Mammalian cells express two genes, MAT1A and MAT2A, which encode two MAT catalytic subunits, α1 and α2, respectively. The α1 subunit organizes into dimers (MATIII) or tetramers (MATI). The α2 subunit is found in the MATII isoform. A third gene MAT2B, encodes a regulatory subunit β, that regulates the activity of MATII by lowering the inhibition constant (Ki) for SAMe and the Michaelis constant (Km) for methionine. MAT1A expressed mainly in hepatocytes maintains the differentiated state of these cells whereas MAT2A and MAT2B are expressed in non-parenchymal cells of the liver (hepatic stellate cells [HSCs] and Kupffer cells) and extrahepatic tissues. A switch from the liver-specific MAT1A to MAT2A has been observed during conditions of active liver growth and de-differentiation. Liver injury, fibrosis, and cancer are associated with MAT1A silencing and MAT2A/MAT2B induction. Even though both MAT1A and MAT2A are involved in SAMe biosynthesis, they exhibit distinct molecular interactions in liver cells. This review provides an update on MAT genes and their roles in liver pathologies.

MATα1 catalyzes the synthesis of S-adenosylmethionine, the principal biological methyl donor. Lower MATα1 activity and mitochondrial dysfunction occur in alcohol-associated liver disease. Besides cytosol and nucleus, MATα1 also targets the mitochondria of hepatocytes to regulate their function. Here, we show that mitochondrial MATα1 is selectively depleted in alcohol-associated liver disease through a mechanism that involves the isomerase PIN1 and the kinase CK2. Alcohol activates CK2, which phosphorylates MATα1 at Ser114 facilitating interaction with PIN1, thereby inhibiting its mitochondrial localization. Blocking PIN1-MATα1 interaction increased mitochondrial MATα1 levels and protected against alcohol-induced mitochondrial dysfunction and fat accumulation. Normally, MATα1 interacts with mitochondrial proteins involved in TCA cycle, oxidative phosphorylation, and fatty acid β-oxidation. Preserving mitochondrial MATα1 content correlates with higher methylation and expression of mitochondrial proteins. Our study demonstrates a role of CK2 and PIN1 in reducing mitochondrial MATα1 content leading to mitochondrial dysfunction in alcohol-associated liver disease.

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and occurs predominantly in patients with underlying chronic liver disease and cirrhosis. The large spectrum of protein post-translational modification (PTM) includes numerous critical signaling events that occur during neoplastic transformation. PTMs occur to nearly all proteins and increase the functional diversity of proteins. We have reviewed the role of two major PTMs, SUMOylation and phosphorylation, in the altered signaling of key players in HCC. SUMOylation is a PTM that involves addition of a small ubiquitin-like modifiers (SUMO) group to proteins. It is known to regulate protein stability, protein-protein interactions, trafficking and transcriptional activity. The major pathways that are regulated by SUMOylation and may influence HCC are regulation of transcription, cell growth pathways associated with B-cell lymphoma 2 (Bcl-2) and methionine adenosyltransferases (MAT), oxidative stress pathways [nuclear erythroid 2-related factor 2 (Nrf2)], tumor suppressor pathways (p53), hypoxia-inducible signaling [hypoxia-inducible factor-1 (HIF-1)], glucose and lipid metabolism, nuclear factor kappa B (NF-κB) and β-Catenin signaling. Phosphorylation is an extensively studied PTM in HCC. The mitogen-activated protein kinase (MAPK), phosphatidyl inositol/AK-strain transforming (PI3K/AKT), and C-SRC pathways have been extensively studied for deregulation of kinases and alteration in signaling of targets through phosphorylation of their substrates. Cross-talk between phosphorylation and SUMOylation is known to influence transcriptional activity of proteins and protein-protein interactions. In HCC, several SUMOylation-dependent phosphorylation events have been studied such as MAPK activation and c-SRC activity that have been reviewed in this work. The drastic effects of site-specific phosphorylation or SUMOylation on enzyme activity of signaling players and its effect on growth and tumorigenesis suggests that these PTMs are novel targets for therapeutic intervention in HCC.

Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Here, we describe the novel regulation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, whereby Vitis vinifera L. cv Vermentino leaf hydroalcoholic extract lowers its protein stability that is regulated by small ubiquitin-like modifier (SUMO)ylation. The phenolic compounds characterization was performed by liquid chromatography–mass spectrometry (LC–MS), whereas mass spectrometry (LC–MS/MS), Western blotting/co-immunoprecipitation (Co-IP) and RT-PCR, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenicity assays, and FACS analysis were used to measure the expression of targets and tumorigenicity. Vermentino extract exhibits antitumorigenic effects, and we went on to determine that FASN and ubiquitin-conjugating enzyme 9 (UBC9), the sole E2 enzyme required for SUMOylation, were significantly reduced. Moreover, FASN was found SUMOylated in human breast cancer tissues and cell lines, and lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability. These results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism, whereas Vermentino extract inhibits these effects which supports the additional validation of the therapeutic value of this compound in breast cancer.