CROSSTALK BETWEEN R1175 METHYLATION AND Y1173 PHOSPHORYLATION NEGATIVELY MODULATES EGFR-MEDIATED ERK ACTIVATION

Post-translational protein modifications are critical regulators of protein functions as they expand the signaling potentials of the modified proteins, leading to diverse physiological consequences. Currently, increasing evidence suggests that protein methylation is as important as other post-translational modifications in the regulation of various biological processes. This drives us to ask whether methylation is involved in the EGFR (epidermal growth factor receptor) signaling, a biological process extensively regulated by multiple post-translational modifications including phosphorylation, glycosylation and ubiquitination. We found that EGFR R1175 is methylated by a protein arginine methyltransferase named PRMT5. During EGFR activation, PRMT5-mediated R1175 methylation specifically enhances EGF-induced EGFR autophosphorylation at Y1173 residue. This novel modification crosstalk increases SHP1 recruitment to EGFR and suppresses EGFR-mediated ERK activation, resulting in inhibition of cell proliferation, migration, and invasion of EGFR-expressing cells. Based on these findings, we provide the first link between arginine methylation and tyrosine phosphorylation and identify R1175 methylation as an inhibitory modification specifically against EGFR-mediated ERK activation.

ABERRATIONS OF A PUTATIVE TUMOR SUPPRESSOR GENE SEL1L IN PANCREATIC DUCTAL ADENOCARCINOMA

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death among males and females in the United States. Sel-1-like (SEL1L) is a putative tumor suppressor gene that is downregulated in a significant proportion of human pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that SEL1L expression could be down-modulated by somatic mutation, loss of heterozygosity (LOH), CpG island hypermethylation and/or aberrantly expressed microRNAs (miRNAs). Material and methods: In 42 PDAC tumors, the SEL1L coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR), and analyzed by agarose gel electrophoresis and sequenced to search for mutations. Using fluorescent fragment analysis, two intragenic microsatellites in the SEL1L gene region were examined to detect LOH in a total of 73 pairs of PDAC tumors and normal-appearing adjacent tissues. Bisulfite DNA sequencing was performed to determine the methylation status of the SEL1L promoter in 41 PDAC tumors and 6 PDAC cell lines. Using real-time quantitative PCR, the expression levels of SEL1L mRNA and 7 aberrantly upregulated miRNAs that potentially target SEL1L were assessed in 42 PDAC tumor and normal pairs. Statistical methods were applied to evaluate the correlation between SEL1L mRNA and the miRNAs. Further the interaction was determined by functional analysis using a molecular biological approach. Results: No mutations were detected in the SEL1L coding region. More than 50% of the samples displayed abnormally alternate or aberrant spliced transcripts of SEL1L. About 14.5% of the tumors displayed LOH at the CAR/CAL microsatellite locus and 10.7% at the RepIN20 microsatellite locus. However, the presence of LOH did not show significant association with SEL1L downregulation. No methylation was observed in the SEL1L promoter. Statistical analysis showed that SEL1L mRNA expression levels significantly and inversely correlated with the expression of hsa-mir-143, hsa-mir-155, and hsa-mir-223. Functional analysis indicated that hsa-mir-155 acted as a suppressor of SEL1L in PL18 and MDAPanc3 PDAC cell lines. Discussion: Evidence from these studies suggested that SEL1L was possibly downregulated by aberrantly upregulated miRNAs in PDAC. Future studies should be directed towards developing a better understanding of the mechanisms for generation of aberrant SEL1L transcripts, and further analysis of miRNAs that may downregulate SEL1L.

Prognostic and predictive roles of MGMT protein expression and promoter methylation in sporadic pancreatic neuroendocrine neoplasms.

BACKGROUND/AIMS O(6)-methylguanine-methyltransferase (MGMT) is an important enzyme of DNA repair. MGMT promoter methylation is detectable in a subset of pancreatic neuroendocrine neoplasms (pNEN). A subset of pNEN responds to the alkylating agent temozolomide (TMZ). We wanted to correlate MGMT promoter methylation with MGMT protein loss in pNEN, correlate the findings with clinico-pathological data and determine the role of MGMT to predict response to TMZ chemotherapy. METHODS We analysed a well-characterized collective of 141 resected pNEN with median follow-up of 83 months for MGMT protein expression and promoter methylation using methylation-specific PCR (MSP). A second collective of 10 metastasized, pretreated and progressive patients receiving TMZ was used to examine the predictive role of MGMT by determining protein expression and promoter methylation using primer extension-based quantitative PCR. RESULTS In both collectives there was no correlation between MGMT protein expression and promoter methylation. Loss of MGMT protein was associated with an adverse outcome, this prognostic value, however, was not independent from grade and stage in multivariate analysis. Promoter hypermethylation was significantly associated with response to TMZ. CONCLUSION Loss of MGMT protein expression is associated with adverse outcome in a surgical series of pNET. MGMT promoter methylation could be a predictive marker for TMZ chemotherapy in pNEN, but further, favourably prospective studies will be needed to confirm this result and before this observation can influence clinical routine.

TWIST1 and TWIST2 promoter methylation and protein expression in tumor stroma influence the epithelial-mesenchymal transition-like tumor budding phenotype in colorectal cancer.

Tumor budding in colorectal cancer is likened to an epithelial-mesenchymal transition (EMT) characterized predominantly by loss of E-cadherin and up-regulation of E-cadherin repressors like TWIST1 and TWIST2. Here we investigate a possible epigenetic link between TWIST proteins and the tumor budding phenotype. TWIST1 and TWIST2 promoter methylation and protein expression were investigated in six cell lines and further correlated with tumor budding in patient cohort 1 (n = 185). Patient cohort 2 (n = 112) was used to assess prognostic effects. Laser capture microdissection (LCM) of tumor epithelium and stroma from low- and high-grade budding cancers was performed. In colorectal cancers, TWIST1 and TWIST2 expression was essentially restricted to stromal cells. LCM results of a high-grade budding case show positive TWIST1 and TWIST2 stroma and no methylation, while the low-grade budding case was characterized by negative stroma and strong hypermethylation. TWIST1 stromal cell staining was associated with adverse features like more advanced pT (p = 0.0044), lymph node metastasis (p = 0.0301), lymphatic vessel invasion (p = 0.0373), perineural invasion (p = 0.0109) and worse overall survival time (p = 0.0226). Stromal cells may influence tumor budding in colorectal cancers through expression of TWIST1. Hypermethylation of the tumor stroma may represent an alternative mechanism for regulation of TWIST1.

Methylation of PITX2, HOXD3, RASSF1 and TDRD1 predicts biochemical recurrence in high-risk prostate cancer

PURPOSE To explore differential methylation of HAAO, HOXD3, LGALS3, PITX2, RASSF1 and TDRD1 as a molecular tool to predict biochemical recurrence (BCR) in patients with high-risk prostate cancer (PCa). METHODS A multiplexed nested methylation-specific PCR was applied to quantify promoter methylation of the selected markers in five cell lines, 42 benign prostatic hyperplasia (BPH) and 71 high-risk PCa tumor samples. Uni- and multivariate Cox regression models were used to assess the importance of the methylation level in predicting BCR. RESULTS A PCa-specific methylation marker HAAO in combination with HOXD3 and a hypomethylation marker TDRD1 distinguished PCa samples (>90 % of tumor cells each) from BPH with a sensitivity of 0.99 and a specificity of 0.95. High methylation of PITX2, HOXD3 and RASSF1, as well as low methylation of TDRD1, appeared to be significantly associated with a higher risk for BCR (HR 3.96, 3.44, 2.80 and 2.85, correspondingly) after correcting for established risk factors. When DNA methylation was treated as a continuous variable, a two-gene model PITX2 × 0.020677 + HOXD3 × 0.0043132 proved to be the best predictor of BCR (HR 4.85) compared with the individual markers. This finding was confirmed in an independent set of 52 high-risk PCa tumor samples (HR 11.89). CONCLUSIONS Differential promoter methylation of HOXD3, PITX2, RASSF1 and TDRD1 emerges as an independent predictor of BCR in high-risk PCa patients. A two-gene continuous DNA methylation model "PITX2 × 0.020677 + HOXD3 × 0.0043132" is a better predictor of BCR compared with individual markers.

Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

Alginate-coated chitosan nanogels differentially modulate class-A and class-B CpG-ODN targeting of dendritic cells and intracellular delivery.

UNLABELLED CpG-oligodeoxynucleotides (CpG-ODNs) interact with dendritic cells (DCs), but evidence is less clear for CpG-ODN admixed with or incorporated into vaccine delivery vehicles. We loaded alginate-coated chitosan-nanogels (Ng) with class-A or class-B CpG-ODN, and compared with the same CpG-ODNs free or admixed with empty Ng. Experiments were performed on both porcine and human blood DC subpopulations. Encapsulation of class-A CpG-ODN (loading into Ng) strongly reduced the CpG-ODN uptake and intracellular trafficking in the cytosol; this was associated with a marked deficiency in IFN-α induction. In contrast, encapsulation of class-B CpG-ODN increased its uptake and did not influence consistently intracellular trafficking into the nucleus. The choice of CpG-ODN class as adjuvant is thus critical in terms of how it will behave with nanoparticulate vaccine delivery vehicles. The latter can have distinctive modulatory influences on the CpG-ODN, which would require definition for different CpG-ODN and delivery vehicles prior to vaccine formulation. FROM THE CLINICAL EDITOR This basic science study investigates the role of class-A and class-B CpG-oligodeoxynucleotides loaded into alginate-coated chitosan nanogels, demonstrating differential effects between the two classes as related to the use of these nanoformulations as vaccine delivery vehicles.

Chemistry of pyrrocorphins: methylative opening of the macrocycle between rings A and D, a side reaction in the peripheral C-methylation of a 20-methyl-pyrrocorphinate

One of the minor products from the previously described peripheral -methylation of a magnesium()-20-methyl--pyrrocorphinate is a C-19-methylated 19,20-seco-corphinoid derivative which, on complexation with nickel() acetate, recyclizes to a nickel()-tetradehydro-corrinate.

DNA Methylation-Guided Prediction of Clinical Failure in High-Risk Prostate Cancer.

BACKGROUND Prostate cancer (PCa) is a very heterogeneous disease with respect to clinical outcome. This study explored differential DNA methylation in a priori selected genes to diagnose PCa and predict clinical failure (CF) in high-risk patients. METHODS A quantitative multiplex, methylation-specific PCR assay was developed to assess promoter methylation of the APC, CCND2, GSTP1, PTGS2 and RARB genes in formalin-fixed, paraffin-embedded tissue samples from 42 patients with benign prostatic hyperplasia and radical prostatectomy specimens of patients with high-risk PCa, encompassing training and validation cohorts of 147 and 71 patients, respectively. Log-rank tests, univariate and multivariate Cox models were used to investigate the prognostic value of the DNA methylation. RESULTS Hypermethylation of APC, CCND2, GSTP1, PTGS2 and RARB was highly cancer-specific. However, only GSTP1 methylation was significantly associated with CF in both independent high-risk PCa cohorts. Importantly, trichotomization into low, moderate and high GSTP1 methylation level subgroups was highly predictive for CF. Patients with either a low or high GSTP1 methylation level, as compared to the moderate methylation groups, were at a higher risk for CF in both the training (Hazard ratio [HR], 3.65; 95% CI, 1.65 to 8.07) and validation sets (HR, 4.27; 95% CI, 1.03 to 17.72) as well as in the combined cohort (HR, 2.74; 95% CI, 1.42 to 5.27) in multivariate analysis. CONCLUSIONS Classification of primary high-risk tumors into three subtypes based on DNA methylation can be combined with clinico-pathological parameters for a more informative risk-stratification of these PCa patients.

Heterogeneity analysis of Metastasis Associated in Colon Cancer 1 (MACC1) for survival prognosis of colorectal cancer patients: a retrospective cohort study.

BACKGROUND Metastasis of colorectal cancer (CRC) is directly linked to patient survival. We previously identified the novel gene Metastasis Associated in Colon Cancer 1 (MACC1) in CRC and demonstrated its importance as metastasis inducer and prognostic biomarker. Here, we investigate the geographic expression pattern of MACC1 in colorectal adenocarcinoma and tumor buds in correlation with clinicopathological and molecular features for improvement of survival prognosis. METHODS We performed geographic MACC1 expression analysis in tumor center, invasive front and tumor buds on whole tissue sections of 187 well-characterized CRCs by immunohistochemistry. MACC1 expression in each geographic zone was analyzed with Mismatch repair (MMR)-status, BRAF/KRAS-mutations and CpG-island methylation. RESULTS MACC1 was significantly overexpressed in tumor tissue as compared to normal mucosa (p < 0.001). Within colorectal adenocarcinomas, a significant increase of MACC1 from tumor center to front (p = 0.0012) was detected. MACC1 was highly overexpressed in 55% tumor budding cells. Independent of geographic location, MACC1 predicted advanced pT and pN-stages, high grade tumor budding, venous and lymphatic invasion (p < 0.05). High MACC1 expression at the invasive front was decisive for prediction of metastasis (p = 0.0223) and poor survival (p = 0.0217). The geographic pattern of MACC1 did not correlate with MMR-status, BRAF/KRAS-mutations or CpG-island methylation. CONCLUSION MACC1 is differentially expressed in CRC. At the invasive front, MACC1 expression predicts best aggressive clinicopathological features, tumor budding, metastasis formation and poor survival outcome.