Background and rationale Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and overexpressed in several malignancies but its expression in hepatocellular carcinoma (HCC) is unknown. Hepatic S-adenosylmethionine (SAMe) level falls in methionine adenosyltransferase 1A (Mat1a) knockout (KO) mice, which develop HCC, and in ethanol fed mice. Here we examined regulation of Ubc9 by SAMe in murine liver and human HCC, breast and colon carcinoma cell lines and specimens. Methods Real-time PCR and Western blotting measured gene and protein expression, respectively. Immunoprecipitation followed by Western blotting examined protein-protein interactions. Results Ubc9 expression is increased in HCC and when hepatic SAMe level falls. SAMe treatment in Mat1a KO mice reduced Ubc9 protein but not mRNA level and lowered sumoylation. Similarly, treatment of liver cancer cell lines HepG2 and Huh-7, colon cancer cell line RKO and breast cancer cell line MCF-7 with SAMe or its metabolite methylthioadenosine (MTA) reduced only Ubc9 protein level. Ubc9 post-translational regulation is unknown. Ubc9 sequence predicts a possible phosphorylation site by cell division cycle 2 (Cdc2), which directly phosphorylated recombinant Ubc9. Mat1a KO mice have higher phospho-Ubc9 level, which normalized after SAMe treatment. SAMe and MTA treatment lowered Cdc2 mRNA and protein levels, phospho-Ubc9 and protein sumoylation in liver, colon and breast cancer cells. Serine 71 of Ubc9 is required for phosphorylation, interaction with Cdc2 and protein stability. Cdc2, Ubc9 and phospho-Ubc9 levels are increased in human liver, breast and colon cancers. Conclusions 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 adenosyltransferase 2B (MAT2B) encodes for two variant proteins V1 and V2 that promote cell growth. Using in-solution proteomics, GIT1 (G-protein-coupled receptor kinase-interacting protein 1) was identified as a potential interacting partner of MAT2B. Here we examined the functional significance of this interplay. Coimmunoprecipitation experiments examined protein interactions. Tissue expression levels of proteins were examined using immunohistochemistry and Western blotting. The expression levels of the proteins were varied using transient knockdown or overexpression to observe the effect of alterations in each protein on the entire complex. Direct interaction among the individual proteins was further verified using in vitro translated and recombinant proteins. We found both MAT2B variants interact with GIT1. Overexpression of V1, V2 or GIT1 activated MEK1, ERK, raised cyclin D1 protein level and increased growth, while the opposite occurred when V1, V2 or GIT1 was knocked down. MAT2B and GIT1 require each other to activate MEK1/ERK and increase growth. MAT2B directly interacts with MEK1, GIT1 and ERK2. Expression level of V1, V2 or GIT1 directly influenced recruitment of GIT1 or MAT2B and ERK2 to MEK1, respectively. In pull down assays, MAT2B directly promoted binding of GIT1 and ERK2 to MEK1. MAT2B and GIT1 interact and are overexpressed in most human liver and colon cancer specimens. Increased expression of V1, V2 or GIT1 promoted growth in an orthotopic liver cancer model; while increased expression of either V1 or V2 with GIT1 further enhanced growth and lung metastasis. Conclusion MAT2B and GIT1 form a scaffold, which recruits and activates MEK and ERK to promote growth and tumorigenesis. This novel MAT2B/GIT1 complex may provide a potential therapeutic gateway in human liver and colon cancer.

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.

AimTransanal haemorrhoidal dearterialization (THD) has become well established for the treatment of haemorrhoids. In this study we describe a technical modification of this technique, targeted mucopexy (THD TM), and report the results for advanced haemorrhoids.MethodThe study included a prospective evaluation of patients with Grade IV (fourth-degree) haemorrhoids operated on with the THD TM technique. This consisted of an initial dearterialization when the haemorrhoidal arteries were transfixed and a second phase of mucopexy, using a different needle from that usually used in the original technique.ResultsFrom January 2007 to December 2011, 31 consecutive patients with Grade IV haemorrhoids were operated on using the THD TM technique. Postoperative pain was reported by 22 (70%) patients on day 1 and 19 (61%) on day 7, while nine (30%) did not experience any pain at all. Severe pain was reported by only nine (16%) patients. At a mean follow-up of 32 months, two (6.4%) patients required a further intervention for on-going symptoms.ConclusionTransanal haemorrhoidal dearterialization TM is effective for advanced haemorrhoids.What does this paper add to the literatureThis paper describes a technical modification of the original transanal haemorrhoidal dearterialization technique introduced by the senior author and reports the results achieved with it in patients with Grade IV haemorrhoids. We believe the modification has allowed a significant improvement in the outcome, especially when dealing with advanced haemorrhoids.