Tribo-Excited Chemical Reaction Using an EuIII Complex with a Stacked Anthracene Framework.

Invited for the cover of this issue are Yuichi Kitagawa, Yasuchika Hasegawa, and co-workers at Hokkaido University. The image depicts tribo-excited chemical reaction using trivalent lanthanide complexes with stacked anthracene ligands. Read the full text of the article at 10.1002/chem.202104401.

Tribo-Excited Chemical Reaction Using an EuIII Complex with a Stacked Anthracene Framework.

A spin-selective tribo-chemical reaction using a dinuclear lanthanide complex is demonstrated for the first time. The dinuclear complex is composed of two EuIII ions, hexafluoroacetylacetonato ligands, and anthracene-based phosphine oxide bridges. Single-crystal analysis revealed a face-to-face-type anthracene dimer structure in the dinuclear EuIII complex. Mechanical stimulus on the dinuclear EuIII complex induced selective formation of oxidized anthracene. The tribo-chemical reaction is based on a characteristic energy-transfer pathway for the selective formation of an excited triplet state.

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

CXCL13 is a differentiation- and hypoxia-induced adipocytokine that exacerbates the inflammatory phenotype of adipocytes through PHLPP1 induction.

Hypoxia in adipose tissue is regarded as a trigger that induces dysregulation of the secretory profile in adipocytes. Similarly, local dysregulation of adipocytokine secretion is an initial event in the deleterious effects of obesity on metabolism. We previously reported that CXCL13 is highly produced during adipogenesis, however little is known about the roles of CXCL13 in adipocytes. Here, we found that hypoxia, as modeled by 1% O2 or exposure to the hypoxia-mimetic reagent desferrioxamine (DFO) has strong inductive effects on the expression of CXCL13 and CXCR5, a CXCL13 receptor, in both undifferentiated and differentiated adipocytes and in organ-cultured white adipose tissue (WAT). CXCL13 was also highly expressed in WAT from high fat diet-fed mice. Hypoxic profile, typified by increased expression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) and decreased expression of adiponectin, was significantly induced by CXCL13 treatment during adipogenic differentiation. Conversely, the treatment of adipocytes with a neutralizing-antibody against CXCL13 as well as CXCR5 knockdown by specific siRNA effectively inhibited DFO-induced inflammation. The phosphorylation of Akt2, a protective factor of adipose inflammation, was significantly inhibited by CXCL13 treatment during adipogenic differentiation. Mechanistically, CXCL13 induces the expression of PHLPP1, an Akt2 phosphatase, through focal adhesion kinase (FAK) signaling; and correspondingly we show that CXCL13 and DFO-induced IL-6 and PAI-1 expression was blocked by Phlpp1 knockdown. Furthermore, we revealed the functional binding sites of PPARγ2 and HIF1-α within the Cxcl13 promoter. Taken together, these results indicate that CXCL13 is an adipocytokine that facilitates hypoxia-induced inflammation in adipocytes through FAK-mediated induction of PHLPP1 in autocrine and/or paracrine manner.

Constitutive activation of p46JNK2 is indispensable for C/EBPδ induction in the initial stage of adipogenic differentiation.

Adipogenic differentiation plays a vital role in energy homeostasis and endocrine system. Several transcription factors, including peroxisome proliferator-activated receptor gamma 2 and CCAAT-enhancer-binding protein (C/EBP) α, ß, and δ, are important for the process, whereas the stage-specific intracellular signal transduction regulating the onset of adipogenesis remains enigmatic. Here, we explored the functional role of c-jun N-terminal kinases (JNKs) in adipogenic differentiation using in vitro differentiation models of 3T3-L1 cells and primary adipo-progenitor cells. JNK inactivation with either a pharmacological inhibitor or JNK2-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the down-regulation of Adiponectin, fatty acid protein 4 (Fabp4), Pparg2, and C/ebpa expressions. Conversely, increased adipogenesis was observed by the inducible overexpression of p46JNK2 (JNK2-1), whereas it was not observed by that of p54JNK2 (JNK2-2), indicating a distinct role of p46JNK2. The essential role of JNK appears restricted to the early stage of adipogenic differentiation, as JNK inhibition in the later stages did not influence adipogenesis. Indeed, JNK phosphorylation was significantly induced at the onset of adipogenic differentiation. As for the transcription factors involved in early adipogenesis, JNK inactivation significantly inhibited the induction of C/ebpd, but not C/ebpb, during the initial stage of adipogenic differentiation. JNK activation increased C/ebpd mRNA and protein expression through the induction and phosphorylation of activating transcription factor 2 (ATF2) that binds to a responsive element within the C/ebpd gene promoter region. Taken together, these data indicate that constitutive JNK activity is specifically required for the initial stage differentiation events of adipocytes.

Aspirin use, tumor PIK3CA mutation, and colorectal-cancer survival.

BACKGROUND: Regular use of aspirin after a diagnosis of colon cancer has been associated with a superior clinical outcome. Experimental evidence suggests that inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2) (also known as cyclooxygenase-2) by aspirin down-regulates phosphatidylinositol 3-kinase (PI3K) signaling activity. We hypothesized that the effect of aspirin on survival and prognosis in patients with cancers characterized by mutated PIK3CA (the phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene) might differ from the effect among those with wild-type PIK3CA cancers. METHODS: We obtained data on 964 patients with rectal or colon cancer from the Nurses' Health Study and the Health Professionals Follow-up Study, including data on aspirin use after diagnosis and the presence or absence of PIK3CA mutation. We used a Cox proportional-hazards model to compute the multivariate hazard ratio for death. We examined tumor markers, including PTGS2, phosphorylated AKT, KRAS, BRAF, microsatellite instability, CpG island methylator phenotype, and methylation of long interspersed nucleotide element 1. RESULTS: Among patients with mutated-PIK3CA colorectal cancers, regular use of aspirin after diagnosis was associated with superior colorectal cancer-specific survival (multivariate hazard ratio for cancer-related death, 0.18; 95% confidence interval [CI], 0.06 to 0.61; P<0.001 by the log-rank test) and overall survival (multivariate hazard ratio for death from any cause, 0.54; 95% CI, 0.31 to 0.94; P=0.01 by the log-rank test). In contrast, among patients with wild-type PIK3CA, regular use of aspirin after diagnosis was not associated with colorectal cancer-specific survival (multivariate hazard ratio, 0.96; 95% CI, 0.69 to 1.32; P=0.76 by the log-rank test; P=0.009 for interaction between aspirin and PIK3CA variables) or overall survival (multivariate hazard ratio, 0.94; 95% CI, 0.75 to 1.17; P=0.96 by the log-rank test; P=0.07 for interaction). CONCLUSIONS: Regular use of aspirin after diagnosis was associated with longer survival among patients with mutated-PIK3CA colorectal cancer, but not among patients with wild-type PIK3CA cancer. The findings from this molecular pathological epidemiology study suggest that the PIK3CA mutation in colorectal cancer may serve as a predictive molecular biomarker for adjuvant aspirin therapy. (Funded by The National Institutes of Health and others.).

SMO expression in colorectal cancer: associations with clinical, pathological, and molecular features.

BACKGROUND: Smoothened, frizzled family receptor (SMO) is an important component of the hedgehog signaling pathway, which has been implicated in various human carcinomas. However, clinical, molecular, and prognostic associations of SMO expression in colorectal cancer remain unclear. METHODS: Using a database of 735 colon and rectal cancers in the Nurse's Health Study and the Health Professionals Follow-up Study, we examined the relationship of tumor SMO expression (assessed by immunohistochemistry) to prognosis, and to clinical, pathological, and tumor molecular features, including mutations of KRAS, BRAF, and PIK3CA, microsatellite instability, CpG island methylator phenotype (CIMP), LINE-1 methylation, and expression of phosphorylated AKT and CTNNB1. RESULTS: SMO expression was detected in 370 tumors (50 %). In multivariate logistic regression analysis, SMO expression was independently inversely associated with phosphorylated AKT expression [odds ratio (OR) 0.48; 95 % confidence interval (CI) 0.34-0.67] and CTNNB1 nuclear localization (OR 0.48; 95 % CI 0.35-0.67). SMO expression was not significantly associated with colorectal cancer-specific or overall survival. However, in CIMP-high tumors, but not CIMP-low/0 tumors, SMO expression was significantly associated with better colorectal cancer-specific survival (log-rank P = 0.012; multivariate hazard ratio, 0.36; 95 % CI 0.13-0.95; P interaction = 0.035, for SMO and CIMP status). CONCLUSIONS: Our data reveal novel potential associations between the hedgehog, the WNT/CTNNB1, and the PI3K (phosphatidylinositol-4,5-bisphosphonate 3-kinase)/AKT pathways, supporting pivotal roles of SMO and hedgehog signaling in pathway networking. SMO expression in colorectal cancer may interact with tumor CIMP status to affect patient prognosis, although confirmation by future studies is needed.

Gene alterations in the nuclear transport receptor superfamily: A study of head and neck cancer.

In cancer cells, the nuclear transport system is often disrupted, leading to abnormal localization of nuclear proteins and altered gene expression. This disruption can arise from various mechanisms such as mutations in genes that regulate nuclear transport, altered expression of transport proteins, and changes in nuclear envelope structure. Oncogenic protein build-up in the nucleus due to the disturbance in nuclear transport can also boost tumor growth and cell proliferation. In this study, we performed bioinformatic analyses of 23 key nuclear transport receptors using genomic and transcriptomic data from pancancer and head and neck squamous cell carcinoma (HNSCC) datasets from The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia and found that the total alteration frequency of 23 nuclear transport receptors in 2691 samples of the PCAWG Consortium was 42.1% and a high levels of genetic alterations was significantly associated with poor overall survival. Amplification was the most common type of genetic alterations, and results in the overexpression of nuclear transport receptors in HNSCC compared to normal tissues. Furthermore, our study revealed that seven out of eight cell cycle genes (CDK1, CDK2, CDK4, CDK6, CCNA1, CCNB1, and CCNE2) were significantly and positively correlated with nuclear transport receptor genes in TCGA pancancer and CCLE datasets. Additionally, functional enrichment analysis showed that nuclear transport receptor genes were mainly enriched in the adhesion junction, cell cycle, ERBB, MAPK, MTOR and WNT signaling pathways.

A prospective study of duration of smoking cessation and colorectal cancer risk by epigenetics-related tumor classification.

The effect of duration of cigarette smoking cessation on colorectal cancer risk by molecular subtypes remains unclear. Using duplication-method Cox proportional-hazards regression analyses, we examined associations between duration of smoking cessation and colorectal cancer risk according to status of CpG island methylator phenotype (CIMP), microsatellite instability, v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation, or DNA methyltransferase-3B (DNMT3B) expression. Follow-up of 134,204 individuals in 2 US nationwide prospective cohorts (Nurses' Health Study (1980-2008) and Health Professionals Follow-up Study (1986-2008)) resulted in 1,260 incident rectal and colon cancers with available molecular data. Compared with current smoking, 10-19, 20-39, and ≥40 years of smoking cessation were associated with a lower risk of CIMP-high colorectal cancer, with multivariate hazard ratios (95% confidence intervals) of 0.53 (0.29, 0.95), 0.52 (0.32, 0.85), and 0.50 (0.27, 0.94), respectively (Ptrend = 0.001), but not with the risk of CIMP-low/CIMP-negative cancer (Ptrend = 0.25) (Pheterogeneity = 0.02, between CIMP-high and CIMP-low/CIMP-negative cancer risks). Differential associations between smoking cessation and cancer risks by microsatellite instability (Pheterogeneity = 0.02), DNMT3B expression (Pheterogeneity = 0.03), and BRAF (Pheterogeneity = 0.10) status appeared to be driven by the associations of CIMP-high cancer with microsatellite instability-high, DNMT3B-positive, and BRAF-mutated cancers. These molecular pathological epidemiology data suggest a protective effect of smoking cessation on a DNA methylation-related carcinogenesis pathway leading to CIMP-high colorectal cancer.

Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress.

Altered RNA processing is an underlying mechanism of amyotrophic lateral sclerosis (ALS). Missense mutations in a number of genes involved in RNA function and metabolisms are associated with ALS. Among these genes is angiogenin (ANG), the fifth member of the vertebrate-specific, secreted ribonuclease superfamily. ANG is an angiogenic ribonuclease, and both its angiogenic and ribonucleolytic activities are important for motor neuron health. Ribonuclease 4 (RNASE4), the fourth member of this superfamily, shares the same promoters with ANG and is co-expressed with ANG. However, the biological role of RNASE4 is unknown. To determine whether RNASE4 is involved in ALS pathogenesis, we sequenced the coding region of RNASE4 in ALS and control subjects and characterized the angiogenic, neurogenic, and neuroprotective activities of RNASE4 protein. We identified an allelic association of SNP rs3748338 with ALS and demonstrated that RNASE4 protein is able to induce angiogenesis in in vitro, ex vivo, and in vivo assays. RNASE4 also induces neural differentiation of P19 mouse embryonal carcinoma cells and mouse embryonic stem cells. Moreover, RNASE4 not only stimulates the formation of neurofilaments from mouse embryonic cortical neurons, but also protects hypothermia-induced degeneration. Importantly, systemic treatment with RNASE4 protein slowed weight loss and enhanced neuromuscular function of SOD1 (G93A) mice.

Characteristic stacked structures and luminescent properties of dinuclear lanthanide complexes with pyrene units.

A novel design strategy of stacked organic fluorophores using dinuclear lanthanide (Ln(III)) complexes is demonstrated for the formation of excimer. The dinuclear Ln(III) complexes are composed of two Ln(III) (Eu(III) or Gd(III)) ions, six hexafluoroacetylacetonate (hfa), and two pyrene-based phosphine oxide ligands. Single-crystal analysis revealed a rigid pyrene-stacked structure via CH-F (pyrene/hfa) intramolecular interactions. The rigid aggregation structures of the two-typed organic ligands around Ln(III) resulted in high thermal stability (decomposition temperature: 340°C). The aggregated ligands exhibited excimer-type green emission from the stacked pyrene-center. The change in the Ln(III) ion promotes effective shifts of excimer emissions (Gd(III):500 nm, Eu(III):490 nm). The organic aggregation system using red-luminescent Eu(III) also provides temperature-sensitive ratiometric emission composed of π-π* and 4f-4f transitions by energy migration between aggregated ligands and Eu(III).

Thermally-assisted photosensitized emission in a trivalent terbium complex.

Luminescent lanthanide complexes containing effective photosensitizers are promising materials for use in displays and sensors. The photosensitizer design strategy has been studied for developing the lanthanide-based luminophores. Herein, we demonstrate a photosensitizer design using dinuclear luminescent lanthanide complex, which exhibits thermally-assisted photosensitized emission. The lanthanide complex comprised Tb(III) ions, six tetramethylheptanedionates, and phosphine oxide bridge containing a phenanthrene frameworks. The phenanthrene ligand and Tb(III) ions are the energy donor (photosensitizer) and acceptor (emission center) parts, respectively. The energy-donating level of the ligand (lowest excited triplet (T1) level = 19,850 cm-1) is lower than the emitting level of the Tb(III) ion (5D4 level = 20,500 cm-1). The long-lived T1 state of the energy-donating ligands promoted an efficient thermally-assisted photosensitized emission of the Tb(III) acceptor (5D4 level), resulting in a pure-green colored emission with a high photosensitized emission quantum yield (73%).

Reliability of online dental final exams in the pre and post COVID-19 era: A comparative study.

Amidst the fourth COVID-19 wave in Viet Nam, national lockdowns necessitated the closure of numerous dental schools. To assess DDS (Doctor of Dental Surgery) graduation exams, this study analyzed their 2021 implementation in comparison to onsite exams conducted in 2020 and 2022 at the Faculty of Odonto-Stomatology, University of Medicine and Pharmacy at Ho Chi Minh City, Viet Nam (FOS-UMPH). The final online examination comprises two main sessions: a synchronous online examination using FOS-UMPH e-Learning for theories (consisting of 200 MCQs and 3 written tests with 3 clinical situations needed be solved) and a synchronous online examination using Microsoft Teams for practicum (comprising of 12 online OSCE stations). The final grades were evaluated using the same metrics in face-to-face final examinations in 2022 and 2020. A total of 114, 112 and 95 students were recruited for the first-time exams in 2020, 2021 and 2022, respectively. In order to analyze the reliability, histogram and k-mean clustering were employed. The histograms from 2020, 2021 and 2022 showed a striking similarity. However, fewer students failed in 2021 and 2022 (13% and 12.6%, respectively) compared to 2020 (28%), with clinical problem-solving part grades (belonging to theory session) being notably higher in 2021 and 2022. Intriguingly, the MCQ Score results showed the identical patterns. The courses of orthodontics, dental public health, and pediatrics subjects (in the group of prevention and development dentistry) stood out for their exceptional accuracy across both sessions. After examining data gathered over three years, we identified three distinct clusters: the first comprised of scattered average and low scores, the second characterized by high scores but unstable and scattered and the third cluster boasting consistently high and centered scores. According to our study, online and onsite traditional graduation exam results are relatively equivalent, but additional measures are necessary to standardize the final examination and adapt to the new normal trend in dental education.

Searching for New Molecular Targets for Oral Squamous Cell Carcinoma with a View to Clinical Implementation of Precision Medicine.

Head and neck cancer, including oral squamous cell carcinoma (OSCC), is the eighth most common malignancy globally and is characterized by local invasiveness and high nodal metastatic potential. The OSCC incidence is also increasing, and the number of deaths is also rising steadily in Japan. The development of molecular markers to eradicate OSCC is an urgent issue for humankind. The increase in OSCC despite the declining smoking rate may be due to several viral infections through various sexual activities and the involvement of previously unfocused carcinogens, and genetic alterations in individual patients are considered to be more complicated. Given this situation, it is difficult to combat OSCC with conventional radiotherapy and chemotherapy using cell-killing anticancer drugs alone, and the development of precision medicine, which aims to provide tailor-made medicine based on the genetic background of each patient, is gaining attention. In this review article, the current status of the comprehensive search for driver genes and biomarkers in OSCC will be briefly described, and some of the candidates for novel markers of OSCC that were found will be outlined.

Periodontitis promotes the expression of gingival transmembrane serine protease 2 (TMPRSS2), a priming protease for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

OBJECTIVES: The oral cavity is one of the main entry sites for SARS-CoV-2. Gingival keratinocytes express transmembrane serine protease 2 (TMPRSS2), responsible for priming the SARS-CoV-2 spike protein. We investigated whether periodontitis increased the expression of TMPRSS2. METHODS: To investigate gene expression in periodontitis, we analyzed the expression of specific genes from (1) the Gene Expression Omnibus (GEO) dataset of 247 human gingival tissues and (2) an experimentally-induced periodontitis mouse model. Human gingival tissues with or without periodontitis were immunohistochemically stained using an anti-TMPRSS2 antibody. Analysis of the TMPRSS2 promoter was performed using a ChIP-Atlas dataset. TMPRSS2 expression was detected in cultured human keratinocytes using quantitative reverse transcription (qRT)-PCR and Western blot analysis. RESULTS: GEO dataset analysis and an experimentally-induced periodontitis model revealed increased expression of TMPRSS2 in periodontitis gingiva. The keratinocyte cell membrane in periodontitis gingiva was strongly immunohistochemically stained for TMPRSS2. Using ChIP-Atlas and GEO datasets, we screened for transcription factors that bind to the TMPRSS2 promoter region. We found one candidate, estrogen receptor 1 (ESR1), highly expressed in periodontitis gingiva. Analysis of the GEO dataset revealed a correlation between ESR1 and TMPRSS2 expression in gingival tissues. An ESR1 ligand induced TMPRSS2 expression in cultured keratinocytes. CONCLUSIONS: Periodontitis increases TMPRSS2 expression in the cell membrane of gingival keratinocytes.

IFN-γ‒Induced APOBEC3B Contributes to Merkel Cell Polyomavirus Genome Mutagenesis in Merkel Cell Carcinoma.

Merkel cell polyomavirus (MCPyV) is the causative agent of an aggressive skin tumor, Merkel cell carcinoma. The viral genome is integrated into the tumor genome and harbors nonsense mutations in the helicase domain of large T antigen. However, the molecular mechanisms by which the viral genome gains the tumor-specific mutations remain to be elucidated. Focusing on host cytosine deaminases APOBEC3s, we find that A3A, A3B, or A3G introduces A3-specific mutations into episomal MCPyV genomes in MCPyV-replicating 293-derivative cells. Sequence analysis of MCPyV genomes retrieved from the NCBI database revealed a decrease of TpC dinucleotide, a preferred target for A3A and A3B, in the 3'-region of the large T antigen‒coding sequence. The viral DNA isolated from tumors contained mutated cytosines, with a remarkable bias toward TpC dinucleotide. Analysis of publicly available microarray data showed that expression of IFN-γ and cytotoxic T lymphocyte markers was positively correlated with the A3A, A3B, and A3G levels in MCPyV-positive but not in MCPyV-negative tumors. Finally, IFN-γ treatment induced A3B and A3G expression in the MCPyV-positive Merkel cell carcinoma cell line MS-1. These results suggest that the IFN-γ-A3B axis plays pivotal roles in evolutionally shaping MCPyV genomic sequences and in generating tumor-specific large T antigen mutations during development of Merkel cell carcinoma.

Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: Cohort study and literature review.

A cyclin-dependent kinase inhibitor CDKN2A (p16/Ink4a) is a tumor suppressor and upregulated in cellular senescence. CDKN2A promoter methylation and gene silencing are associated with the CpG island methylator phenotype (CIMP) in colon cancer. However, prognostic significance of CDKN2A methylation or loss of CDKN2A (p16) expression independent of CIMP status remains uncertain. Using a database of 902 colorectal cancers in 2 independent cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study), we quantified CDKN2A promoter methylation and detected hypermethylation in 269 tumors (30%). By immunohistochemistry, we detected loss of CDKN2A (p16) expression in 25% (200/804) of tumors. We analyzed for LINE-1 hypomethylation and hypermethylation at 7 CIMP-specific CpG islands (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); microsatellite instability (MSI); KRAS, BRAF and PIK3CA mutations; and expression of TP53 (p53), CTNNB1 (ß-catenin), CDKN1A (p21), CDKN1B (p27), CCND1 (cyclin D1), FASN (fatty acid synthase) and PTGS2 (cyclooxygenase-2). CDKN2A promoter methylation and loss of CDKN2A (p16) were associated with shorter overall survival in univariate Cox regression analysis [hazard ratio (HR): 1.36, 95% CI: 1.10-1.66, p = 0.0036 for CDKN2A methylation; HR: 1.30, 95% CI: 1.03-1.63, p = 0.026 for CDKN2A (p16) loss] but not in multivariate analysis that adjusted for clinical and tumor variables, including CIMP, MSI and LINE-1 methylation. Neither CDKN2A promoter methylation nor loss of CDKN2A (p16) was associated with colorectal cancer-specific mortality in uni- or multivariate analysis. Despite its well-established role in carcinogenesis, CDKN2A (p16) promoter methylation or loss of expression in colorectal cancer is not independently associated with patient prognosis.

Hypomethylation of the IGF2 DMR in colorectal tumors, detected by bisulfite pyrosequencing, is associated with poor prognosis.

BACKGROUND & AIMS: The insulin-like growth factor 2 (IGF2) gene is normally imprinted. Constitutive loss of imprinting (LOI) of IGF2 has been associated with increased risks of colon cancer and adenoma, indicating its role in carcinogenesis. The conventional LOI assay relies on a germline polymorphism to distinguish between 2 allelic expression patterns but results in many uninformative cases. IGF2 LOI correlates with hypomethylation at the differentially methylated region (DMR)-0. An assay for methylation of the DMR0 could overcome the limitations of the conventional IGF2 LOI assay. METHODS: We measured methylation at the IGF2 DMR0 using a bisulfite-pyrosequencing assay with 1178 paraffin-embedded colorectal cancer tissue samples from 2 prospective cohort studies. A Cox proportional hazard model was used to calculate mortality hazard ratio (HR); calculations were adjusted for microsatellite instability; the CpG island methylator phenotype; LINE-1 methylation; and KRAS, BRAF, and PIK3CA mutations. RESULTS: Methylation at the IGF2 DMR0 was successfully measured in 1105 (94%) of 1178 samples. Colorectal tumors had significantly less methylation at the DMR0 compared with matched, normal colonic mucosa (P < .0001; N = 51). Among 1033 patients eligible for survival analysis, hypomethylation of the IGF2 DMR0 was significantly associated with higher overall mortality (log-rank P = .0006; univariate HR, 1.41; 95% confidence interval, 1.16-1.71; P = .0006; multivariate HR, 1.33; 95% confidence interval, 1.08-1.63; P = .0066). CONCLUSIONS: A bisulfite-pyrosequencing assay to measure methylation of the IGF2 DMR0 is robust and applicable to paraffin-embedded tissue. IGF2 DMR0 hypomethylation in colorectal tumor samples is associated with shorter survival time, so it might be developed as a prognostic biomarker.

MGMT promoter methylation, loss of expression and prognosis in 855 colorectal cancers.

OBJECTIVE: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme. MGMT promoter hypermethylation and epigenetic silencing often occur as early events in carcinogenesis. However, prognostic significance of MGMT alterations in colorectal cancer remains uncertain. METHODS: Utilizing a database of 855 colon and rectal cancers in two prospective cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study), we detected MGMT promoter hypermethylation in 325 tumors (38%) by MethyLight and loss of MGMT expression in 37% (247/672) of tumors by immunohistochemistry. We assessed the CpG island methylator phenotype (CIMP) using eight methylation markers [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and LINE-1 (L1) hypomethylation, TP53 (p53), and microsatellite instability (MSI). RESULTS: MGMT hypermethylation was not associated with colorectal cancer-specific mortality in univariate or multivariate Cox regression analysis [adjusted hazard ratio (HR) = 1.03; 95% confidence interval (CI), 0.79-1.36] that adjusted for clinical and tumor features, including CIMP, MSI, and BRAF mutation. Similarly, MGMT loss was not associated with patient survival. MGMT loss was associated with G>A mutations in KRAS (p = 0.019) and PIK3CA (p = 0.0031). CONCLUSIONS: Despite a well-established role of MGMT aberrations in carcinogenesis, neither MGMT promoter methylation nor MGMT loss serves as a prognostic biomarker in colorectal cancer.

Novel application of structural equation modeling to correlation structure analysis of CpG island methylation in colorectal cancer.

The CpG island methylator phenotype (CIMP-high, CIMP1) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent evidence suggests the presence of KRAS mutation-associated CIMP subtype (CIMP-low, CIMP2). We used cluster analysis, principal component analysis (PCA), and structural equation modeling (SEM), a novel strategy, to decipher the correlation structure of CpG island hypermethylation. Using a database of 861 colon and rectal cancers, DNA methylation at 16 CpG islands [CACNA1G, CDKN2A (p16/ink4a), CHFR, CRABP1, HIC1, IGF2, IGFBP3, MGMT, MINT-1, MINT-31, MLH1, NEUROG1, p14 (CDKN2A/arf), RUNX3, SOCS1, and WRN] was quantified by real-time PCR. Tumors were categorized into three groups: Group 1 with wild-type KRAS/BRAF (N = 440); Group 2 with mutant KRAS and wild-type BRAF (N = 308); and Group 3 with wild-type KRAS and mutant BRAF (N = 107). Tumors with mutant KRAS/BRAF (N = 6) were excluded. In unsupervised hierarchical clustering analysis, all but six markers (CACNA1G, IGF2, RUNX3, MGMT, MINT-1, and SOCS1) were differentially clustered with CIMP-high and CIMP-low according to KRAS and BRAF status. In SEM, the correlation structures between CIMP, locus-specific CpG island methylation, and MSI differed according to KRAS and BRAF status, which was consistent with PCA results. In conclusion, KRAS and BRAF mutations appear to differentially influence correlation structure of CpG island methylation. Our novel data suggest two distinct perturbations, resulting in differential locus-specific propensity of CpG methylation.