BACKGROUND: DNA mismatch repair (MMR) defects are a major factor in colorectal tumorigenesis in Lynch syndrome (LS) and 15% of sporadic cases. Some adenomas from carriers of inherited MMR gene mutations have intact MMR protein expression implying other mechanisms accelerating tumorigenesis. We determined roles of DNA methylation changes and somatic mutations in cancer-associated genes as tumorigenic events in LS-associated colorectal adenomas with intact MMR.
METHODS: We investigated 122 archival colorectal specimens of normal mucosae, adenomas and carcinomas from 57 LS patients. MMR-deficient (MMR-D, n = 49) and MMR-proficient (MMR-P, n = 18) adenomas were of particular interest and were interrogated by methylation-specific multiplex ligation-dependent probe amplification and Ion Torrent sequencing.
FINDINGS: Promoter methylation of CpG island methylator phenotype (CIMP)-associated marker genes and selected colorectal cancer (CRC)-associated tumor suppressor genes (TSGs) increased and LINE-1 methylation decreased from normal mucosa to MMR-P adenomas to MMR-D adenomas. Methylation differences were statistically significant when either adenoma group was compared with normal mucosa, but not between MMR-P and MMR-D adenomas. Significantly increased methylation was found in multiple CIMP marker genes (IGF2, NEUROG1, CRABP1, and CDKN2A) and TSGs (SFRP1 and SFRP2) in MMR-P adenomas already. Furthermore, certain CRC-associated somatic mutations, such as KRAS, were prevalent in MMR-P adenomas.
INTERPRETATION: We conclude that DNA methylation changes and somatic mutations of cancer-associated genes might serve as an alternative pathway accelerating LS-associated tumorigenesis in the presence of proficient MMR. FUND: Jane and Aatos Erkko Foundation, Academy of Finland, Cancer Foundation Finland, Sigrid Juselius Foundation, and HiLIFE.

(1) Background: Epithelial⁻mesenchymal plasticity (EMP) is a dynamic process whereby epithelial carcinoma cells reversibly acquire morphological and invasive characteristics typical of mesenchymal cells. Identifying the methylation differences between epithelial and mesenchymal states may assist in the identification of optimal DNA methylation biomarkers for the blood-based monitoring of cancer. (2) Methods: Methylation-sensitive high-resolution melting (MS-HRM) was used to examine the promoter methylation status of a panel of established and novel markers in a range of breast cancer cell lines spanning the epithelial⁻mesenchymal spectrum. Pyrosequencing was used to validate the MS-HRM results. (3) Results:

BACKGROUND: The prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis.
METHODS: We used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers.
RESULTS: We identified 17 genes significantly differentially expressed between mFTC and wFTC. C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC.
CONCLUSIONS: We conclude that the underexpression of CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.

Progression of solid cancers, colorectal carcinomas among them, from their primaries to metastatic lesions traditionally is thought to proceed by a stepwise acquisition of and selection for genomic aberrations. To test if patterns of genomic aberrations would be consistent with this model, we studied 10 colorectal carcinoma primary-metastasis pairs, 9 with 1 liver metastasis each and 1 with 2 metastases. Next-generation targeted sequencing (50-gene panel) with samples obtained from different regions of the primaries and their metastases demonstrated 1-11 gene mutations per lesion. But only in 2 tumors were there seen mutations in all samples from the metastasis and not any of the primaries (BRAF

In India, retinoblastoma is among the top five childhood cancers. Children mostly present with extraocular extension and high risk features that results in unsatisfactory treatment and low survival rate. In addition, lack of potential therapeutic and prognostic targets is another challenge in the management of retinoblastoma. We studied comparative proteome of retinoblastoma patients (HPV positive and negative (n=4 each) and controls (n=4), in order to identify potential retinoblastoma-specific protein targets. 2D-DIGE coupled MALDI-TOF/TOF mass spectrometry identified 39 unique proteins. Highly deregulated proteins were GFAP,RBP3,APOA1,CRYAA,CRABP1,SAG and TF. Gene ontology (Panther 7.0) revealed majority of proteins to be associated with metabolic processes (26%) and catalytic activity (38%). 8 proteins were significantly upregulated in HPV positive vis-a-vis HPV negative cases. Patient group exhibited 12 upregulated and 18 downregulated proteins compared to controls. Pathway and network analysis (IPA software) revealed CTNNB1 as most significantly regulated signalling pathway in HPV positive than HPV negative retinoblastoma. The trends in transcriptional change of 9 genes were consistent with those at proteomic level. The Western blot analysis confirmed the expression pattern of RBP3,GFAP and CRABP1. We suggest GFAP,RBP3,CRABP1,CRYAAA,APOA1 and SAG as prospective targets that could further be explored as potential candidates in therapy and may further assist in studying the disease mechanism.
SIGNIFICANCE: In this study we evaluated tumor tissue specimens from retinoblastoma patients and identified 39 differentially regulated proteins compared to healthy retina. From these, we propose RBP3, CRABP1, GFAP, CRYAA, APOA1 and SAG as promising proteomic signatures that could further be explored as efficient prognostic and therapeutic targets in retinoblastoma. The present study is not only a contribution to the ongoing endeavour for the discovery of proteomic signatures in retinoblastoma, but, may also act as a starting point for future studies aimed at uncovering novel targets for further therapeutic interventions and improving patient outcomes.

Cellular binding-proteins (BP), including CRBP1, CRBP2, CRABP1, CRABP2, and FABP5, shepherd the poorly aqueous soluble retinoids during uptake, metabolism and function. Holo-BP promote efficient use of retinol, a scarce but essential nutrient throughout evolution, by sheltering it and its major metabolite all-trans-retinoic acid from adventitious interactions with the cellular milieu, and by imposing specificity of delivery to enzymes, nuclear receptors and other partners. Apo-BP reflect cellular retinoid status and modify activities of retinoid metabolon enzymes, or exert non-canonical actions. High ligand binding affinities and the nature of ligand sequestration necessitate external factors to prompt retinoid release from holo-BP. One or more of cross-linking, kinetics, and colocalization have identified these factors as RDH, RALDH, CYP26, LRAT, RAR and PPARβ/δ. Michaelis-Menten and other kinetic approaches verify that BP channel retinoids to select enzymes and receptors by protein-protein interactions. Function of the BP and enzymes that constitute the retinoid metabolon depends in part on retinoid exchanges unique to specific pairings. The complexity of these exchanges configure retinol metabolism to meet the diverse functions of all-trans-retinoic acid and its ability to foster contrary outcomes in different cell types, such as inducing apoptosis, differentiation or proliferation. Altered BP expression affects retinoid function, for example, by impairing pancreas development resulting in abnormal glucose and energy metabolism, promoting predisposition to breast cancer, and fostering more severe outcomes in prostate cancer, ovarian adenocarcinoma, and glioblastoma. Yet, the extent of BP interactions with retinoid metabolon enzymes and their impact on retinoid physiology remains incompletely understood.

Retinol and its derivatives play an important role in epidermal growth and differentiation and represent chemopreventive agents in nonmelanoma skin cancer. Retinoic acid binding protein II (CRABP-II) is a cytoplasmic receptor that critically regulates all-trans-retinoic acid (ATRA) trafficking. We documented the marked reduced expression of CRABP-II and its promoter methylation in human poorly differentiated squamous cell carcinomas. To investigate the role of CRABP-II in skin carcinogenesis we used skin lesion induction by dimethylbenz[a]anthracene/12-O-tetradecanoyl-phorbol-13-acetate in CRABP-II-knockout C57BL/6 mice. We observed earlier and more diffuse epidermal dysplasia, greater incidence and severity of tumors, reduced expression of cytokeratin 1/cytokeratin 10 and involucrin, increased proliferation, and impaired ATRA inhibition of tumor promotion compared with wild-type animals. CRABP-II-transfected HaCaT, FaDu, and A431 cells showed expression of differentiation markers, retinoic acid receptor-β/-γ signaling, ATRA sensitivity, and suppression of EGFR/v-akt murine thymoma viral oncogene homolog 1 (AKT) pathways in a fatty acid binding protein 5/peroxisome proliferator-activated receptor-β/-δ-independent manner. The opposite was true in keratinocytes isolated from CRABP-II-knockout mice. Finally, CRABP-II accumulation induced ubiquitination-associated reduction of EGFR. Our results showed reduced CRABP-II expression in human poorly differentiated squamous cell carcinomas, and its gene deletion favored experimental skin carcinogenesis and impaired ATRA antitumor efficacy, likely modulating EGFR/AKT pathways and retinoic acid receptor-β/-γ signaling. Therapeutic interventions aimed at restoring CRABP-II-mediated signaling may amplify therapeutic retinoid efficacy in nonmelanoma skin cancer.

Retinoic acid (RA), a metabolite of vitamin A, is required for the regulation of growth and development. Aberrant expression of molecules involved in RA signaling has been reported in various cancer types including glioblastoma multiforme (GBM). Cellular retinoic acid-binding protein 2 (CRABP2) has previously been shown to play a key role in the transport of RA to retinoic acid receptors (RARs) to activate their transcription regulatory activity. Here, we demonstrate that CRABP2 is predominantly located in the cytoplasm of GBM tumors. Cytoplasmic, but not nuclear, CRABP2 levels in GBM tumors are associated with poor patient survival. Treatment of malignant glioma cell lines with RA results in a dose-dependent increase in accumulation of CRABP2 in the cytoplasm. CRABP2 knockdown reduces proliferation rates of malignant glioma cells, and enhances RA-induced RAR activation. Levels of CRYAB, a small heat shock protein with anti-apoptotic activity, and GFAP, an astrocyte-specific intermediate filament protein, are greatly reduced in CRABP2-depleted cells. Restoration of CRYAB expression partially but significantly reversed the effect of CRABP2 depletion on RAR activation. Our combined in vivo and in vitro data indicate that: (i) CRABP2 is an important determinant of clinical outcome in GBM patients, and (ii) the mechanism of action of CRABP2 in GBM involves sequestration of RA in the cytoplasm and activation of an anti-apoptotic pathway, thereby enhancing proliferation and preventing RA-mediated cell death and differentiation. We propose that reducing CRABP2 levels may enhance the therapeutic index of RA in GBM patients.

Neuroblastoma is a childhood neural crest tumor. Fenretinide, a retinoic acid analogue, induces accumulation of mitochondrial reactive oxygen species and consequent apoptosis in neuroblastoma cells. The p75 neurotrophin receptor (p75NTR) enhances the antineuroblastoma cell efficacy of fenretinide in vitro. We examined the role of the retinoid binding protein, CRABP1, in p75NTR-mediated potentiation of the efficacy of fenretinide. Knockdown and overexpression, respectively, of either p75NTR or CRABP1 were effected in neuroblastoma cell lines using standard techniques. Expression was determined by qRT-PCR and confirmed at the protein level by Western blot. Metabolic viability was determined by Alamar blue assay. While protein content of CRABP1 correlated roughly with that of p75NTR in the three neuroblastoid or epithelioid human neuroblastoma cell lines studied, manipulation of p75NTR expression resulted in cell line-dependent, variable change in CRABP1 expression. Furthermore, in some cell lines, induced expression of CRABP1 in the absence of p75NTR did not alter cell sensitivity to fenretinide treatment. The effects of manipulation of p75NTR expression on CRABP1 expression and the effects of CRABP1 expression on fenretinide efficacy are therefore neuroblastoma cell line-dependent. Potentiation of the antineuroblastoma cell effects of fenretinide by p75NTR is not mediated solely through CRABP1.

BACKGROUND: Clinical trials designed to test the efficacy of retinoic acid (RA) as an adjuvant for the treatment of solid cancers have been disappointing, primarily due to RA resistance. Estrogen receptor (ER)-negative breast cancer cells are more resistant to RA than ER-positive cells. The expression and subcellular distribution of two RA-binding proteins, FABP5 and CRABP2, has already been shown to play critical roles in breast cancer cell response to RA. CRABP1, a third member of the RA-binding protein family, has not previously been investigated as a possible mediator of RA action in breast cancer.
METHODS: CRABP1 and CRABP2 expression in primary breast tumor tissues was analyzed using gene expression and tissue microarrays. CRABP1 levels were manipulated using siRNAs and by transient overexpression. RA-induced subcellular translocation of CRABPs was examined by immunofluorescence microscopy and immunoblotting. RA-induced transactivation of RAR was analyzed using a RA response element (RARE)-driven luciferase reporter system. Effects of CRABP1 expression and RA treatment on downstream gene expression were investigated by semi-quantitative RT-PCR analysis.
RESULTS: Compared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers. Elevated CRABP1 levels are associated with poor patient prognosis, high Ki67 immunoreactivity and high tumor grade in breast cancer. The prognostic significance of CRABP1 is attributed to its cytoplasmic localization. We demonstrate that CRABP1 expression attenuates RA-induced cell growth arrest and inhibits RA signalling in breast cancer cells by sequestering RA in the cytoplasm. We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism.
CONCLUSIONS: CRABP1 is an adverse factor for clinical outcome in triple-negative breast cancer and a potent inhibitor of RA signalling in breast cancer cells. Our data indicate that CRABP1, in conjunction with previously identified CRABP2 and FABP5, plays a key role in breast cancer cell response to RA. We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.

Capicua (CIC) has been implicated in pathogenesis of spinocerebellar ataxia type-1 (SCA1) neurodegenerative disease and some types of cancer; however, the role of CIC in prostate cancer remains unknown. Here we show that CIC suppresses prostate cancer progression. CIC expression was markedly decreased in human prostatic carcinoma. CIC overexpression suppressed prostate cancer cell proliferation, invasion, and migration, whereas CIC RNAi exerted opposite effects. We found that knock-down of CIC derepresses expression of ETV5 and CRABP1 in LNCaP and PC-3 cells, respectively, thereby promoting cell proliferation and invasion. We also discovered that miR-93, miR-106b, and miR-375, which are known to be frequently overexpressed in prostate cancer patients, cooperatively down-regulate CIC levels to promote cancer progression. Altogether, we suggest miR-93/miR-106b/miR-375-CIC-CRABP1 as a novel key regulatory axis in prostate cancer progression.

Alterations in global DNA methylation and specific regulatory gene methylation are frequently found in cancer, but the significance of these epigenetic changes in EBV-associated gastric carcinoma (EBVaGC) remains unclear. We evaluated global DNA methylation status in 49 EBVaGC and 45 EBV-negative gastric carcinoma (EBVnGC) tissue samples and cell lines by 5-methylcytosine immunohistochemical staining and methylation quantification. We determined promoter methylation status and protein expression for the p16, FHIT, CRBP1, WWOX, and DLC-1 genes in tissues and studied the correlation between CpG island methylator phenotype (CIMP) class and clinicopathological characteristics. Changes in gene methylation and mRNA expression in EBVaGC cell line SNU-719 and in EBVnGC cell lines SGC-7901, BGC-823, and AGS were assessed after treatment with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA), or a combination of both, by methylation-specific PCR and quantitative real-time RT-PCR. Global genomic DNA hypomethylation was more pronounced in EBVnGC than in EBVaGC. Promoter methylation of all five genes was more frequent in EBVaGC than in EBVnGC (p < 0.05). p16 and FHIT methylation was reversely correlated with protein expression in EBVaGC. Most (41/49) EBVaGC exhibited CIMP-high (CIMP-H), and the prognosis of CIMP-H patients was significantly worse than that of CIMP-low (p = 0.027) and CIMP-none (p = 0.003) patients. Treatment with 5-aza-dC and/or TSA induced upregulation of RNA expression of all five genes in SNU-719; meanwhile, individual gene expression increased in EBVnGC cell lines. In summary, EBV-induced hypermethylation of p16, FHIT, CRBP1, WWOX, and DLC-1 may contribute to EBVaGC development. Demethylation therapy may represent a novel therapeutic strategy for EBVaGC.

Breast cancer has been considered to be a multifactorial disease with a wide array of well-characterized gene mutations and chromosomal abnormalities. However, it is becoming evident that the onset or development of breast cancer also depends on epigenetic factors, although the mechanisms have not been fully elucidated. We performed a genome-wide analysis of DNA methylation of breast carcinomatous tissues and paired normal tissues to examine the differences in methylation between them. Methylation-specific polymerase chain reaction (MSP) was used to validate the hypermethylated genes screened out by DNA methylation microarray. We found that hypomethylation and hypermethylation occurred in 2753 and 1795 genes, respectively, in breast carcinomatous tissues. Meanwhile, gene ontology analysis and ingenuity pathway analysis revealed the function and pathway of several genes whose methylation status was altered in breast carcinomatous tissues. In addition, we investigated the promoter methylation status of four genes in breast carcinomatous tissue and paired normal tissues (n=30) by MSP. Promoter hypermethylation of CRABP1, HOXB13, IFNGR2, and PIK3C3 was found in 37% (11/30), 23% (7/30), 17% (5/30), and 2% (2/30) of the carcinomas, respectively. Mutation of these four important genes was critical to many types of cancer. Our results suggest that DNA methylation mechanisms may be involved in regulating the occurrence and development of breast cancer.

CRABP1 (cellular retinoic acid binding protein 1) belongs to the family of fatty acid binding proteins. Retinoic acid binding is the only known functional activity of this protein. The role of CRABP1 in human carcinogenesis remains poorly understood. Here, for the first time we demonstrated pro-metastatic and pro-tumorigenic activity of CRABP1 in mesenchymal tumors. Further functional analysis revealed that the pro-tumorigenic effect of CRABP1 does not depend on retinoic acid binding activity. These results suggest that CRABP1 could have an alternative intracellular functional activity that contributes to the high malignancy of transformed mesenchymal cells. Microarray analysis detected CRABP1-mediated alterations in the expression of about 100 genes, including those encoding key regulatory proteins. CRABP1 is ubiquitously expressed in monophasic synovial sarcomas, while in biphasic synovial sarcomas it is expressed uniquely by the spindle cells of the aggressive mesenchymal component. High level of CRABP1 expression is associated with lymph node metastasis and poor differentiation/high grade of pancreatic neuroendocrine tumors (pNETs). Presented data suggest CRABP1 as a promising biomarker of pNETs' clinical behavior. Our results give the first evidence of pro-tumorigenic and pro-metastatic activity of CRABP1 in mesenchymal and neuroendocrine tumors.

BACKGROUND: In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP). However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer.
METHODS: Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G) were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared.
RESULTS: For 47 samples (71%), the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20%) consistently scored as CIMP positive. Only four of 31 probes (13%) investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using 'the most stringent' as compared to 'the least stringent' criteria (20% vs 46%, respectively; p<0.005) was demonstrated.
CONCLUSIONS: A statistical significant variation in the frequency of CIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition used.

INTRODUCTION: The prognosis of breast cancer is strongly influenced by the developmental stage of the breast when the tumor is diagnosed. Pregnancy-associated breast cancers (PABCs), cancers diagnosed during pregnancy, lactation, or in the first postpartum year, are typically found at an advanced stage, are more aggressive and have a poorer prognosis. Although the systemic and microenvironmental changes that occur during post-partum involution have been best recognized for their role in the pathogenesis of PABCs, epidemiological data indicate that PABCs diagnosed during lactation have an overall poorer prognosis than those diagnosed during involution. Thus, the physiologic and/or biological events during lactation may have a significant and unrecognized role in the pathobiology of PABCs.
METHODS: Syngeneic in vivo mouse models of PABC were used to examine the effects of system and stromal factors during pregnancy, lactation and involution on mammary tumorigenesis. Mammary adipose stromal cell (ASC) populations were isolated from mammary glands and examined by using a combination of in vitro and in vivo functional assays, gene expression analysis, and molecular and cellular assays. Specific findings were further investigated by immunohistochemistry in mammary glands of mice as well as in functional studies using ASCs from lactating mammary glands. Additional findings were further investigated using human clinical samples, human stromal cells and using in vivo xenograft assays.
RESULTS: ASCs present during lactation (ASC-Ls), but not during other mammary developmental stages, promote the growth of carcinoma cells and angiogenesis. ASCs-Ls are distinguished by their elevated expression of cellular retinoic acid binding protein-1 (crabp1), which regulates their ability to retain lipid. Human breast carcinoma-associated fibroblasts (CAFs) exhibit traits of ASC-Ls and express crabp1. Inhibition of crabp1in CAFs or in ASC-Ls abolished their tumor-promoting activity and also restored their ability to accumulate lipid.
CONCLUSIONS: These findings imply that (1) PABC is a complex disease, which likely has different etiologies when diagnosed during different stages of pregnancy; (2) both systemic and local factors are important for the pathobiology of PABCs; and (3) the stromal changes during lactation play a distinct and important role in the etiology and pathogenesis of PABCs that differ from those during post-lactational involution.

BACKGROUND: Diagnosis of ovarian cancer is often delayed because of subtle symptoms and a lack of a specific, sensitive test useful for the general population. The majority of cases are diagnosed at late stages, after the tumor has metastasized and implanted on many other abdominal organs and cavity surfaces. A paucity of prognostic markers makes it difficult to define which tumors will act aggressively and shorten survival. Hence, it is imperative to have new screening tests for diagnosis of ovarian cancer at earlier stages, prior to metastatic progression. Diagnosis at these early stages will dramatically increase the overall survival of women with ovarian cancer.
MATERIAL AND METHODS: Based on previously published literature on proposed molecular cell markers in ovarian carcinoma, we sought to validate the overexpression of two genes (cellular retinoic acid Binding Protein, CRABP-1, and spondin 1) through immunohistochemistry.
RESULTS: We verified the overexpression of spondin 1 in ovarian cancer. Expression of cellular retinoic acid Binding Protein, CRABP-1 in whole ovarian cancer tissue sections was higher than in the TMA tissue cores.
CONCLUSION: Our results thus demonstrate that spondin 1 is a useful marker for ovarian cancer; additionally, the high percentages of tumors that are positive for spondin 1 make it an ideal target for therapy. CRABP-1 was not expressed at high levels in any subtype of ovarian cancer, making it a poor marker.

UNLABELLED: We analysed the effects of all-trans retinoic acid (ATRA) on proliferation and changes in the global proteome of the nullipotent human embryonal carcinoma cell line 2102Ep and the pluripotent cell line NTERA2 cl.D1 (NT2). Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of proteins of the retinoid pathway. We established a proteome map of the germ cell tumor (GCT) cell line NT2 showing neuronal differentiation under ATRA treatment for 7days. Using bioinformatic analyses, we identified functional groups of altered proteins and potentially involved pathways, of which changes to the organization of the cytoskeleton and anti-apoptotic effects were the most prominent. Changes observed in the expression of factors involved in the retinoid pathway under ATRA, namely an upregulation of CRBP and CRABP2, were also reflected in GCT tissues of different histologies, providing further insight into factors involved in the differentiation of these pluripotent tumors.
BIOLOGICAL SIGNIFICANCE: Treatment of NT2 germ cell tumor cells with all-trans retinoic acid (ATRA) is a model to investigate differentiation. We analysed differentially expressed proteins by 2D-PAGE and mass spectrometry and provide a proteome map of NT2 cells under 7days of ATRA. By bioinformatic analyses, functional groups of proteins and involved pathways like changes to the cytoskeleton and anti-apoptotic effects were identified. Factors involved in the retinoid pathway, in particular upregulation of CRBP, CRABP1 and CRABP2, also showed differential expression in tumors with different histological subtypes, which provides insight into gene regulation under induced and spontaneous differentiation in germ cell tumors.

Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are associated with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 associates with a subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we determined structures of WDR5 bound to six different naturally occurring Win motif sequences at resolutions ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.

Medulloblastoma cells exhibit varied responses to therapy by all-trans retinoic acid (RA). The underlying mechanism for such diverse effects however remains largely unclear. In this study, we attempted to elucidate the molecular basis of RA resistance through the study of RA signaling components in both RA-sensitive (Med-3) and RA-resistant (UW228-2 and UW228-3) medulloblastoma cells. The results revealed that RARα/β/γ and RXRα/β/γ were found in the three cell lines. Expression of CRABP-I and CRABP-II was seen in Med-3 cells, up-regulated when treated with RA, but was absent in UW228-2 and UW228-3 cells regardless of RA treatment. Bisulfite sequencing revealed 8 methylated CG sites at the promoter region of CRABP-II in UW228-2 and UW228-3 but not in Med-3 cells. Demethylation by 5-aza-2'-deoxycytidine recovered CRABP-II expression. Upon restoration of CRABP-II expression, both UW228-2 and UW228-3 cells responded to RA treatment by forming neuronal-like differentiation, synaptophysin expression, β-III tubulin upregulation, and apoptosis. Furthermore, CRABP-II specific siRNA reduced RA sensitivity in Med-3 cells. Tissue microarray-based immunohistochemical staining showed variable CRABP-II expression patterns among 104 medulloblastoma cases, ranging from negative (42.3%), partly positive (14.4%) to positive (43.3%). CRABP-II expression was positively correlated with synaptophysin (rs = 0.317; p = 0.001) but not with CRABP-I expression (p > 0.05). In conclusion, aberrant methylation in CRABP-II reduces the expression of CRABP-II that in turn confers RA resistance in medulloblastoma cells. Determination of CRABP-II expression or methylation status may enable a personalized RA therapy in patients with medulloblastomas and other types of cancers.

The effect of all-trans retinoic acid (ATRA) on cutaneous squamous cell carcinomas (c-SCC) has been poorly described. Because the imbalance of CRABP-II-mediated anticancer signalling and FABP5-mediated growth-promoting signalling was supposed to be related with ATRA sensitivities of cancer cells, COLO16 human c-SCC cell line was selected to check underlying mechanism leading to ATRA resistance by multiple experimental approaches. The results revealed that COLO 16 cells were resistant to 15 μm ATRA treatment. FABP5 as well as the elements related with CRABP-II signalling (CYP26A1, CYP26B1, CRABP-I, RARα/β/γ and RXRα/β/γ) and with FABP5 signalling (PPARβ/δ) were expressed, but CRABP-II was undetectable in COLO 16 cells. 5-Aza treatment enhanced CRABP-II expression but further bisulfite sequencing PCR-DNA sequencing revealed no methylation in CRABP-II promoter region. Transfection of CRABP-II-expressing plasmids or FABP5 siRNA or both successfully manipulated the level(s) of target gene expression but failed to overcome ATRA resistance in the transfectants. In conclusion, CRABP-II and FABP5 expression were imbalanced in ATRA-resistant COLO 16 cells. 5-Aza-enhanced CRABP-II expression and unmethylation in CRABP-II promoter region suggest the methylation of certain CRABP-II regulatory gene(s) in COLO 16 cells. As neither restoration of CRABP-II expression nor the increased CRABP-II versus FABP5 ratio can overcome ATRA resistance of COLO 16 cells, additional ATRA-resistant mechanism(s) may present in human c-SCCs and COLO 16 cells would be of value in addressing this issue.

BACKGROUND: Pituitary tumors account for approximately 10-15% of intracranial neoplasms.
AIM: Using the cDNA microarray method, we have previously compared expression under two distinct conditions: a pool of 4 clinically non-functioning pituitary adenomas (NFPA) and a spinal cord metastasis of a non-functioning pituitary carcinoma, in order to gain biological insights into genomic changes of pituitary neoplasias. In the present study, we further investigated the mRNA expression of 3 selected genes previously described as being involved in other neoplasias based on a series of 60 pituitary adenomas: CRABP1 (cellular retinoic acid binding protein 1), GRP (gastrin-releasing peptide), and RERG (Ras-related, estrogen- regulated, growth inhibitor).
MATERIAL AND METHODS: The expression of CRABP1, GRP, and RERG was determined by quantitative RT-PCR.
RESULTS: A significantly higher content of CRABP1 mRNA was observed in NFPA compared to functioning adenomas, and PRL-secreting adenomas showed a lower expression of this gene compared to normal pituitary. A lower expression of GRP mRNA was detected in NFPA compared to normal pituitary and also to functioning adenomas. RERG mRNA was overexpressed in NFPA in comparison to functioning adenomas and to normal pituitary. Among the functioning adenomas, only the ACTH-secreting adenomas presented a higher expression of RERG mRNA compared to normal pituitary.
CONCLUSIONS: The findings of differential expression of CRABP1 in prolactinomas and of RERG in NFPA compared to normal pituitary suggests that retinoic acid and estrogen receptor, respectively, could be involved in the tumorigenesis of these adenomas subtypes. Additional studies are required to further confirm this hypothesis.

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.

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 (β-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.

BACKGROUND: Prostaglandin-endoperoxide synthase 2 (PTGS2, the HUGO Gene Nomenclature Committee-approved official symbol for cycloxygenase-2, COX-2) and its enzymatic product prostaglandin E2 have critical roles in inflammation and carcinogenesis through the G protein-coupled receptor PTGER2 (EP2). The PTGS2 (COX-2) pathway is a promising target for cancer therapy and chemoprevention. PTGS2 (COX-2) expression in colon cancer has been inversely associated with survival as well as tumoral microsatellite instability (MSI) and the CpG island methylator phenotype (CIMP). However, the prognostic significance of PTGER2 expression or its relationship with MSI, CIMP, LINE-1 hypomethylation, or PTGS2 (COX-2) remains uncertain.
METHODS: Using the database of 516 colorectal cancers in two prospective cohort studies with clinical outcome data, we detected PTGER2 overexpression in 169 (33%) tumors by immunohistochemistry. We analyzed MSI using 10 microsatellite markers; CIMP by MethyLight (real-time methylation-specific PCR) on an eight-marker panel [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1]; BRAF, KRAS, PIK3CA, and methylation in LINE-1 by Pyrosequencing; and CTNNB1 (beta-catenin) and TP53 (p53) by immunohistochemistry.
RESULTS: PTGER2 overexpression was positively associated with the mucinous component (P = 0.0016), signet ring cells (P = 0.0024), CIMP-high (P = 0.0023), and MSI-high (P < 0.0001). In multivariate analysis, the significant relationship between PTGER2 and MSI-high persisted (adjusted odds ratio, 2.82; 95% confidence interval, 1.69-4.72; P < 0.0001). PTGER2 was not significantly associated with PTGS2 (COX-2), TP53, or CTNNB1 expression, patient survival, or prognosis.
CONCLUSION: PTGER2 overexpression is associated with MSI-high in colorectal cancer.
IMPACT: Our data imply potential roles of inflammatory reaction by PTGER2 upregulation in carcinogenic process to MSI-high colorectal cancer.

BACKGROUND: Genetic markers for thyroid cancers identified by microarray analysis have offered limited predictive accuracy so far because of the few classes of thyroid lesions usually taken into account. To improve diagnostic relevance, we have simultaneously analyzed microarray data from six public datasets covering a total of 347 thyroid tissue samples representing 12 histological classes of follicular lesions and normal thyroid tissue. Our own dataset, containing about half the thyroid tissue samples, included all categories of thyroid lesions.
METHODOLOGY/PRINCIPAL FINDINGS: Classifier predictions were strongly affected by similarities between classes and by the number of classes in the training sets. In each dataset, sample prediction was improved by separating the samples into three groups according to class similarities. The cross-validation of differential genes revealed four clusters with functional enrichments. The analysis of six of these genes (APOD, APOE, CLGN, CRABP1, SDHA and TIMP1) in 49 new samples showed consistent gene and protein profiles with the class similarities observed. Focusing on four subclasses of follicular tumor, we explored the diagnostic potential of 12 selected markers (CASP10, CDH16, CLGN, CRABP1, HMGB2, ALPL2, ADAMTS2, CABIN1, ALDH1A3, USP13, NR2F2, KRTHB5) by real-time quantitative RT-PCR on 32 other new samples. The gene expression profiles of follicular tumors were examined with reference to the mutational status of the Pax8-PPARgamma, TSHR, GNAS and NRAS genes.
CONCLUSION/SIGNIFICANCE: We show that diagnostic tools defined on the basis of microarray data are more relevant when a large number of samples and tissue classes are used. Taking into account the relationships between the thyroid tumor pathologies, together with the main biological functions and pathways involved, improved the diagnostic accuracy of the samples. Our approach was particularly relevant for the classification of microfollicular adenomas.

OBJECTIVE: To determine whether endometriosis-associated endometrioid cancer (EAOC) is a specific entity compared with endometrioid cancer not associated with endometriosis (OC).
DESIGN: Case-control study.
SETTING: University hospital research laboratory.
PATIENT(S): Seven patients with endometriosis-associated ovarian cancer EAOC and five patients each with OC, ovarian endometriosis, and benign ovaries.
INTERVENTION(S): Ovarian tissue samples were collected from surgical procedures.
MAIN OUTCOME MEASURE(S): We hybridized cRNA samples to the Affymetrix HG-U133A microarray chip. Representative genes were validated by real time polymerase chain reaction.
RESULT(S): We identified two main groups of genes: The first group contained the genes SICA2, CCL14, and TDGF1. These genes were equally regulated in endometriosis and EAOC but not in OC and benign ovaries. The second group contained the genes StAR, SPINT1, Keratin 8, FoxM1B, FOLR1, CRABP1, and Claudin 7. They were equally regulated in EAOC and OC but not in ovarian endometriosis and benign ovaries.
CONCLUSION(S): That the first group is composed of the cytokines SICA2 and CCL14 and the growth factor TDGF1 indicates that the regulation of the autoimmune system and of inflammatory cytokines may be very important in the etiology of endometriosis and EAOC. That the second group is composed of genes that play a central role in cell-cell interaction, differentiation, and cell proliferation indicates that they may be important in the development of ovarian cancer in women with endometriosis.

Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked approximately 600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex.

PURPOSE: DNA methyltransferase-3B (DNMT3B) plays an important role in de novo CpG island methylation. Dnmt3b can induce colon tumor in mice with methylation in specific CpG islands. We hypothesized that cellular DNMT3B level might influence the occurrence of widespread CpG island methylation (i.e., the CpG island methylator phenotype, CIMP) in colon cancer.
EXPERIMENTAL DESIGN: Utilizing 765 colorectal cancers in two cohort studies, we detected DNMT3B expression in 116 (15%) tumors by immunohistochemistry. We assessed microsatellite instability, quantified DNA methylation in repetitive long interspersed nucleotide element-1 (LINE-1) by Pyrosequencing, eight CIMP-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and eight other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN) by real-time PCR (MethyLight).
RESULTS: Tumoral DNMT3B overexpression was significantly associated with CIMP-high [> or =6/8 methylated CIMP-specific promoters; odds ratio (OR), 3.34; 95% confidence interval, 2.11-5.29; P < 0.0001]. The relations between DNMT3B and methylation in 16 individual CpG islands varied substantially (OR, 0.80-2.96), suggesting variable locus-to-locus specificities of DNMT3B activity. DNMT3B expression was not significantly related with LINE-1 hypomethylation. In multivariate logistic regression, the significant relation between DNMT3B and CIMP-high persisted (OR, 2.39; 95% confidence interval, 1.11-5.14; P = 0.026) after adjusting for clinical and other molecular features, including p53, beta-catenin, LINE-1, microsatellite instability, KRAS, PIK3CA, and BRAF. DNMT3B expression was unrelated with patient outcome, survival, or prognosis.
CONCLUSIONS: Tumoral DNMT3B overexpression is associated with CIMP-high in colorectal cancer. Our data support a possible role of DNMT3B in nonrandom de novo CpG island methylation leading to colorectal cancer.

The class III histone deacetylase SIRT1 (sir2) is important in epigenetic gene silencing. Inhibition of SIRT1 reactivates silenced genes, suggesting a possible therapeutic approach of targeted reversal of aberrantly silenced genes. In addition, SIRT1 may be involved in the well-known link between obesity, cellular energy balance and cancer. However, a comprehensive study of SIRT1 using human cancer tissue with clinical outcome data is currently lacking, and its prognostic significance is uncertain. Using the database of 485 colorectal cancers in two independent prospective cohort studies, we detected SIRT1 overexpression in 180 (37%) tumors by immunohistochemistry. We examined its relationship to the CpG island methylator phenotype (CIMP), related molecular events, clinical features including body mass index, and patient survival. We quantified DNA methylation in eight CIMP-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) and eight other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN) by MethyLight. SIRT1 overexpression was associated with CIMP-high (> or =6 of 8 methylated CIMP-specific promoters, P=0.002) and microsatellite instability (MSI)-high phenotype (P<0.0001). In both univariate and multivariate analyses, SIRT1 overexpression was significantly associated with the CIMP-high MSI-high phenotype (multivariate odds ratio, 3.20; 95% confidence interval, 1.35-7.59; P=0.008). In addition, mucinous component (P=0.01), high tumor grade (P=0.02), and fatty acid synthase overexpression (P=0.04) were significantly associated with SIRT positivity in multivariate analysis. SIRT1 was not significantly related with age, sex, tumor location, stage, signet ring cells, cyclooxygenase-2 (COX-2), LINE-1 hypomethylation, KRAS, BRAF, BMI, PIK3CA, HDAC, p53, beta-catenin, COX-2, or patient prognosis. In conclusion, SIRT1 expression is associated with CIMP-high MSI-high colon cancer, suggesting involvement of SIRT1 in gene silencing in this unique tumor subtype.