The Bi-Loop, a new general four-stranded DNA motif

The crystal structure of the cyclic octanucleotide d contains two independent molecules that form a novel quadruplex by means of intermolecular Watson-Crick A.T pairs and base stacking. A virtually identical quadruplex composed of G.C pairs was found by earlier x-ray analysis of the linear heptamer d(GCATGCT), when the DNA was looped in the crystal. The close correspondence between these two structures of markedly dissimilar oligonucleotides suggests that they are both examples of a previously unrecognized motif. Their nucleotide sequences have little in common except for two separated 5'-purine-pyrimidine dinucleotides forming the quadruplex, and by implication these so-called 'bi-loops' could occur widely in natural DNA. Such structures provide a mechanism for noncovalent linking of polynucleotides in vivo. Their capacity to associate by base stacking, demonstrated in the crystal structure of d(GCATGCT), creates a compact molecular framework made up of four DNA chains within which strand exchange could take place

EPHB2 germline variants in patients with colorectal cancer or hyperplastic polyposis

BACKGROUND: Ephrin receptor B2 (EPHB2) has recently been proposed as a novel tumor suppressor gene in colorectal cancer (CRC). Inactivation of the gene has been shown to correlate with progression of colorectal tumorigenesis, and somatic mutations have been reported in both colorectal and prostate tumors. METHODS: Here we have analyzed the EPHB2 gene for germline alterations in 101 individuals either with 1) CRC and a personal or family history of prostate cancer (PC), or 2) intestinal hyperplastic polyposis (HPP), a condition associated with malignant degeneration such as serrated adenoma and CRC. RESULTS: Four previously unknown missense alterations were observed, which may be associated with the disease phenotype. Two of the changes, I361V and R568W, were identified in Finnish CRC patients, but not in over 300 Finnish familial CRC or PC patients or more than 200 population-matched healthy controls. The third change, D861N, was observed in a UK HPP patient, but not in additional 40 UK HPP patients or in 200 UK healthy controls. The fourth change R80H, originally identified in a Finnish CRC patient, was also found in 1/106 familial CRC patients and in 9/281 healthy controls and is likely to be a neutral polymorphism. CONCLUSION: We detected novel germline EPHB2 alterations in patients with colorectal tumors. The results suggest a limited role for these EPHB2 variants in colon tumor predisposition. Further studies including functional analyses are needed to confirm this

Germline EPHB2 Receptor Variants in Familial Colorectal Cancer

Familial clustering of colorectal cancer occurs in 15–20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer

Epigenetic silencing of EphA1 expression in colorectal cancer is correlated with poor survival

Aberrant expression of Eph and ephrin proteins has well-established functions in oncogenesis and tumour progression. We describe EphA1 expression in 6 colorectal cancer (CRC) cell lines, 18 controls and 125 CRC specimens. In addition, a well-characterised cohort of 53 paired normal colon and CRCs was also assessed. Expression of EphA1 mRNA was assessed by quantitative real-time PCR and correlated with protein expression by flow cytometry, immunoprecipitation, western blotting and immunohistochemistry. Significant upregulation (2- to 10-fold) of EphA1 was seen in over 50% of cases (P=0.005) whereas many of the remainder showed downregulation of EphA1. Intriguingly, EphA1 over-expression was more prevalent in stage II compared to stage III CRCs (P=0.02). Low EphA1 expression significantly correlated with poor survival (P=0.02). Epigenetic silencing appeared to explain the loss of EphA1 expression as methylation of the EphA1 CpG island strongly correlated with low EphA1 expression (P<0.01). Furthermore, EphA1 re-expression could be induced by treatment with demethylating agents. Our findings identify EphA1 as a potential prognostic marker in CRC. Although therapies targeting high EphA1 expression seem plausible in CRC, the loss of expression in advanced disease suggests a potential risk that targeted therapy, by selecting for loss of expression, might contribute to disease progression

18q loss of heterozygosity in microsatellite stable colorectal cancer is correlated with CpG island methylator phenotype-negative (CIMP-0) and inversely with CIMP-low and CIMP-high

<p>Abstract</p> <p>Background:</p> <p>The CpG island methylator phenotype (CIMP) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high) and <it>BRAF </it>mutations. 18q loss of heterozygosity (LOH) commonly present in colorectal cancer with chromosomal instability (CIN) is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and <it>MLH1 </it>methylation) in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation) and CIMP-0 (CIMP-negative), determined by quantitative DNA methylation analysis.</p> <p>Methods:</p> <p>Utilizing MethyLight technology (real-time PCR), we quantified DNA methylation in 8 CIMP-specific promoters {<it>CACNA1G</it>, <it>CDKN2A </it>(p16), <it>CRABP1, IGF2</it>, <it>MLH1, NEUROG1, RUNX3 </it>and <it>SOCS1</it>} in 758 non-MSI-high colorectal cancers obtained from two large prospective cohorts. Using four 18q microsatellite markers (D18S55, D18S56, D18S67 and D18S487) and stringent criteria for 18q LOH, we selected 374 tumors (236 LOH-positive tumors with ≥ 2 markers showing LOH; and 138 LOH-negative tumors with ≥ 3 informative markers and no LOH).</p> <p>Results:</p> <p>CIMP-0 (0/8 methylated promoters) was significantly more common in 18q LOH-positive tumors (59% = 139/236, p = 0.002) than 18q LOH-negative tumors (44% = 61/138), while CIMP-low/high (1/8–8/8 methylated promoters) was significantly more common (56%) in 18q LOH-negative tumors than 18q LOH-positive tumors (41%). These relations persisted after stratification by sex, location, or the status of MSI, p53 expression (by immunohistochemistry), or <it>KRAS/BRAF </it>mutation.</p> <p>Conclusion:</p> <p>18q LOH is correlated positively with CIMP-0 and inversely with CIMP-low and CIMP-high. Our findings provide supporting evidence for relationship between CIMP-0 and 18q LOH as well as a molecular difference between CIMP-0 and CIMP-low in colorectal cancer.</p

TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study

<p>Abstract</p> <p>Background</p> <p>Histological phenotype and clinical behaviour of malignant tumours are not only dependent on alterations in the epithelial cell compartment, but are affected by their interaction with inflammatory cells and tumour-associated stroma. Studies in animal models have shown influence of tumour-associated macrophages (TAM) on histological grade of differentiation in colon carcinoma. Disruption of transforming growth factor beta (TGF-beta) signalling in tumour cells is related to more aggressive clinical behaviour. Expression data of components of this pathway in tumour-associated stroma is limited.</p> <p>Methods</p> <p>Tissue micro arrays of 310 colon carcinomas from curatively resected patients in UICC stage II and III were established. In a first step we quantified amount of CD68 positive TAMs and expression of components of TGF-beta signalling (TGF-beta1, TGF-beta receptors type 1 and 2, Smad 3 and 4) in tumour and associated stroma. Further we analyzed correlation to histological and clinical parameters (histological grade of differentiation (low-grade (i.e. grade 1 and 2) vs. high-grade (i.e. grade 3 and 4)), lymph node metastasis, distant metastasis, 5 year cancer related survival) using Chi-square or Fisher's exact test, when appropriate, to compare frequencies, Kaplan-Meier method to calculate 5-year rates of distant metastases and cancer-related survival and log rank test to compare the rates of distant metastases and survival. To identify independent prognostic factors Cox regression analysis including lymph node status and grading was performed.</p> <p>Results</p> <p>High-grade tumours and those with lymph node metastases showed higher rates of TAMs and lower expression of TGF-beta1. Loss of nuclear Smad4 expression in tumor was associated with presence of lymph node metastasis, but no influence on prognosis could be demonstrated. Decrease of both TGF-beta receptors in tumour-associated stroma was associated with increased lymph node metastasis and shorter survival. Stromal TGF-beta receptor 2 expression was an independent prognostic factor for cancer related survival.</p> <p>Conclusion</p> <p>Histological phenotype and clinical behaviour of colon cancer is not only influenced by mutational incidents in tumour cells but also affected by interaction of tumour tissue with inflammatory cells like macrophages and associated stroma and TGF-beta signalling is one important part of this crosstalk. Further studies are needed to elucidate the exact mechanisms.</p

Ultradeep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability

Ultradeep sequencing of genomes permits the detection of very low-level genomic instability in non-neoplastic tissues of patients with the most common form of inherited colorectal cancer