Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and inhibits apoptosis via Bcl-2 by unknown mechanism. Methionine adenosyltransferase 2A (MAT2A) encodes for MATα2, the catalytic subunit of the MATII isoenzyme that synthesizes S-adenosylmethionine (SAMe). Ubc9, Bcl-2 and MAT2A expression are up-regulated in several malignancies. Exogenous SAMe decreases Ubc9 and MAT2A expression and is pro-apoptotic in liver and colon cancer cells. Here we investigated whether there is interplay between Ubc9, MAT2A and Bcl-2. We used human colon and liver cancer cell lines RKO and HepG2, respectively, and confirmed key finding in colon cancer specimens. We found MATα2 can regulate Bcl-2 expression at multiple levels. MATα2 binds to Bcl-2 promoter to activate its transcription. This effect is independent of SAMe as MATα2 catalytic mutant was also effective. MATα2 also directly interacts with Bcl-2 to enhance its protein stability. MATα2's effect on Bcl-2 requires Ubc9 as MATα2's stability is influenced by sumoylation at K340, K372 and K394. Overexpressing wild type (but not less stable MATα2 sumoylation mutants) protected from 5-fluorouracil-induced apoptosis in both colon and liver cancer cells. Colon cancer have higher levels of sumoylated MATα2, total MATα2, Ubc9 and Bcl-2 and higher MATα2 binding to the Bcl-2 P2 promoter. Taken together, Ubc9's protective effect on apoptosis may be mediated at least in part by sumoylating and stabilizing MATα2 protein, which in turn positively maintains Bcl-2 expression. These interactions feed forward to further enhance growth and survival of the cancer cell.

Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70 to 75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100 pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell.

MicroRNA-34a (miR-34a) is down-regulated in colorectal cancers (CRC) and required for interleukin-6 (IL-6)-induced CRC metastasis. Mice lacking miR-34a developed more invasive cancer in a colitis-associated cancer model. In the same model, S-adenosylmethionine (SAMe) and methylthioadenosine (MTA) inhibited IL-6/STAT3 and lowered tumor burden. SAMe and MTA reduce the expression of methionine adenosyltransferase 2A (MAT2A) and there are consensus binding sites for miR-34a/b in the MAT2A 3’UTR. Here we examined whether SAMe/MTA influence miR-34a/b expression and cancer metastasis. We found SAMe and MTA raised miR-34a/b expression in CRC cell lines, inhibited migration and invasion in vitro and liver metastasis in vivo. Like CRC, MAT2A and MAT2B expression is induced in human pancreas and prostate cancers. Treatment with SAMe, MTA, miR-34a or miR-34b inhibited MAT2A expression mainly at the protein level. MAT2B protein level also fell because MAT2A and MAT2B enhance each other’s protein stability. Overexpressing miR-34a or miR-34b inhibited while MAT2A or MAT2B enhanced CRC migration and invasion. Co-expressing either miR-34a/b had minimal to no effect on MAT2A/MAT2B’s ability to increase migration, invasion and growth. Taken together, MAT2A and MAT2B are important targets of miR-34a/b and SAMe and MTA target this axis, suppressing MAT2A/MAT2B while raising miR-34a/b expression, inhibiting cancer metastasis.

Endotoxemia correlates with the degree of liver failure and may participate in worsening of liver diseases. Lipopolysaccharide (LPS, synonymous as endotoxin) treatment in mice lowered hepatic glutathione (GSH) level, which in turn is a variable that determines susceptibility to LPS-induced injury. We previously showed that LPS treatment in mice lowered hepatic expression of the rate-limiting enzyme in GSH synthesis, glutamate-cysteine ligase (GCL). The aims of our current work were to determine the molecular mechanism(s) responsible for these changes. Studies were done using RAW cells (murine macrophage), in vivo LPS treated mice, and mouse hepatocytes. We found that LPS treatment lowered GCL catalytic and modifier (Gclc and Gclm) subunit expression at the transcriptional level, which was unrelated to alteration in nitric oxide production or induction in NFkB/p65 subunit. The key mechanism was due to decreased sumoylation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and MafG, which is required for their heterodimerization and subsequent binding and trans-activation of the anti-oxidant response element (ARE) present in the promoter region of these genes that is essential for their expression. LPS treatment lowered markedly the expression of ubiquitin-conjugating enzyme 9 (Ubc9), which is required for sumoylation. Similar findings also occurred in liver after in vivo LPS treatment and LPS-treated mouse hepatocytes. Overexpression of Ubc9 protected against LPS-mediated inhibition of Gclc and Gclm expression in RAW cells and hepatocytes. Conclusions LPS-mediated lowering of GCL expression in hepatocyte and macrophage is due to lowering of sumoylation of Nrf2 and MafG, leading to reduced heterodimerization, binding and trans-activation of ARE.