Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

LINE-1 retrotransposons are abundant repetitive elements of viral origin, which in normal cells are kept quiescent through epigenetic mechanisms. Activation of LINE-1 occurs frequently in cancer and can enable LINE-1 mobilization but also has retrotransposition-independent consequences. We previously reported that in cancer, aberrantly active LINE-1 promoters can drive transcription of flanking unique sequences giving rise to LINE-1 chimeric transcripts (LCTs). Here, we show that one such LCT, LCT13, is a large transcript (>300 kb) running antisense to the metastasis-suppressor gene TFPI- 2. We have modelled antisense RNA expression at TFPI-2 in transgenic mouse embryonic stem (ES) cells and demonstrate that antisense RNA induces silencing and deposition of repressive histone modifications implying a causal link. Consistent with this, LCT13 expression in breast and colon cancer cell lines is associated with silencing and repressive chromatin at TFPI-2. Furthermore, we detected LCT13 transcripts in 56% of colorectal tumours exhibiting reduced TFPI-2 expression. Our findings implicate activation of LINE-1 elements in subsequent epigenetic remodelling of surrounding genes, thus hinting a novel retrotransposition-independent role for LINE-1 elements in malignancy

Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

<p>Hypomethylation of LINE-1 repeats in cancer has been proposed as the main mechanism behind their activation; this assumption, however, was based on findings from early studies that were biased toward young and transpositionally active elements. Here, we investigate the relationship between methylation of 2 intergenic, transpositionally inactive LINE-1 elements and expression of the LINE-1 chimeric transcript (LCT) 13 and LCT14 driven by their antisense promoters (L1-ASP). Our data from DNA modification, expression, and 5′RACE analyses suggest that colorectal cancer methylation in the regions analyzed is not always associated with LCT repression. Consistent with this, in HCT116 colorectal cancer cells lacking DNA methyltransferases DNMT1 or DNMT3B, LCT13 expression decreases, while cells lacking both DNMTs or treated with the DNMT inhibitor 5-azacytidine (5-aza) show no change in LCT13 expression. Interestingly, levels of the H4K20me3 histone modification are inversely associated with LCT13 and LCT14 expression. Moreover, at these LINE-1s, H4K20me3 levels rather than DNA methylation seem to be good predictor of their sensitivity to 5-aza treatment. Therefore, by studying individual LINE-1 promoters we have shown that in some cases these promoters can be active without losing methylation; in addition, we provide evidence that other factors (e.g., H4K20me3 levels) play prominent roles in their regulation.</p

DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.</p

Rationalising the role of Keratin 9 as a biomarker for Alzheimer’s disease

Keratin 9 was recently identified as an important component of a biomarker panel which demonstrated a high diagnostic accuracy (87%) for Alzheimer’s disease (AD). Understanding how a protein which is predominantly expressed in palmoplantar epidermis is implicated in AD may shed new light on the mechanisms underlying the disease. Here we use immunoassays to examine blood plasma expression patterns of Keratin 9 and its relationship to other AD-associated proteins. We correlate this with the use of an in silico analysis tool VisANT to elucidate possible pathways through which the involvement of Keratin 9 may take place. We identify possible links with Dickkopf-1, a negative regulator of the wnt pathway, and propose that the abnormal expression of Keratin 9 in AD blood and cerebrospinal fluid may be a result of blood brain barrier dysregulation and disruption of the ubiquitin proteasome system. Our findings suggest that dysregulated Keratin 9 expression is a consequence of AD pathology but, as it interacts with a broad range of proteins, it may have other, as yet uncharacterized, downstream effects which could contribute to AD onset and progression

miR-21 expression in primary colorectal tissue samples, calculated relatively to that of the control miRNA RNU44, was studied in primary tumour and matched adjacent normal mucosa (n = 45; A); in Dukes' A (n = 9), B (n = 23) and C (n = 13) stages (B); in lymph node negative (No; n = 32) versus lymph node positive (Yes, n = 13) cases (C); and in tumours displaying lower (pT1 & pT2; n = 11) versus higher (pT3 & pT4; n = 34) depth of invasion (D).

<p>Column bar graphs indicate the median and whiskers demonstrate the IQR of miR-21 expression. Note that significant increase in miR-21 expression is observed in tumours, and this increases further with worsening stage, lymphnode involvement and depth of invasion. Statistical significance was calculated using the Wilcoxon signed-rank test in A, the Kruskall-Wallis test in B, and the Mann-Whitney U test in C and D.</p

Inflammation and MiR-21 Pathways Functionally Interact to Downregulate PDCD4 in Colorectal Cancer

<div><p>Inflammation plays a direct role in colorectal cancer (CRC) progression; however the molecular mechanisms responsible for this effect are unclear. The inflammation induced cyclooxygenase 2 (COX-2) enzyme required for the production of Prostaglandin E2 (PGE₂), can promote colorectal cancer by decreasing expression of the tumour suppressor gene Programmed Cell Death 4 (PDCD4). As PDCD4 is also a direct target of the oncogene microRNA-21 (miR-21) we investigated the relationship between the COX-2 and miR-21 pathways in colorectal cancer progression. Gene expression profile in tumour and paired normal mucosa from 45 CRC patients demonstrated that up-regulation of COX-2 and miR-21 in tumour tissue correlates with worse Dukes' stage. In vitro studies in colonic adenocarcinoma cells revealed that treatment with the selective COX-2 inhibitor NS398 significantly decreased miR-21 levels (p = 0.0067) and increased PDCD4 protein levels (p<0.001), whilst treatment with PGE₂ up-regulated miR-21 expression (p = 0.019) and down-regulated PDCD4 protein (p<0.05). These findings indicate that miR-21 is a component of the COX-2 inflammation pathway and that this pathway promotes worsening of disease stage in colorectal cancer by inducing accumulation of PGE₂ and increasing expression of miR-21 with consequent downregulation of the tumour suppressor gene PDCD4.</p></div

miR-21 and <i>PDCD4</i> expression in HCA-7 cells treated with the COX-2 inhibitor NS398.

<p>Following 72 hours treatment significant decrease in miR-21 levels is seen in NS398 treated cells (<b>A</b>). <i>PDCD4</i> mRNA levels are not affected by NS398 treatment (<b>B</b>). Western blot analysis of PDCD4 (<b>C</b> top panel for a representative blot) reveals a significant increase in protein levels in NS398 treated cells as compared to untreated or vehicle alone (DMSO) treated cells, when quantified relatively to beta actin protein (BACT) as a loading control (<b>C</b> bottom panel). The column bar graph indicates the mean and the whiskers demonstrate the standard error of the mean (SEM). Statistical significance was calculated using the unpaired t-test. Experiments were repeated three times and analysed in triplicate. Untreated = cells cultured in media; DMSO = cells cultured in media supplemented with 0.1% DMSO vehicle; NS398 = cells cultured in media supplemented with NS398 prepared in DMSO to a final concentration of 100 µM NS398 and 0.1% DMSO.</p

Proposed model of how the inflammation pathway can promote colorectal cancer progression by linking inflammation to downregulation of PDCD4 via miR-21 induction mediated by COX-2 driven accumulation of PGE₂.

<p>Persistent inflammation at the tumour site drives increase in COX-2 expression leading to accumulation of PGE₂. This in turn leads to the increase in expression of miR-21 and subsequent downregulation of PDCD4 protein levels facilitating progressive increase to more invasive and metastatic forms of colorectal cancer (from Dukes' A to B, C and D). Our experiments indicate that this pathway can be blocked by COX-2 inhibitors (NS398 and Aspirin) by preventing accumulation of PGE₂ as well as by miR-21 inhibitors. ↑ Activation, Inhibition. See text for further details.</p

miR-21 and <i>PDCD4</i> expression in HCA-7 cells treated with miR-21 inhibitor.

<p>Following 72 hours from transfection significant decrease in miR-21 levels are seen only in cells transfected with miR-21 inhibitor (<b>A</b>). <i>PDCD4</i> mRNA levels were not affected by inhibition of miR-21 (<b>B</b>). Western blot analysis of PDCD4 (<b>C</b> top panel for a representative blot) reveals a significant increase in protein levels in cells transfected with the inhibitor as compared to untreated or scramble control treated cells, when quantified relatively to beta actin protein (BACT) as a loading control (<b>C</b> bottom panel). The column bar graph indicates the mean and the whiskers demonstrate the standard error of the mean (SEM). Statistical significance was calculated using the unpaired t-test. Experiments were repeated three times and analysed in duplicate. Untreated = cells culture in media; scramble = cells transfected with scramble RNA inhibitors; miR-21 inhibitor = cells treated with the miR-21 RNA inhibitor.</p

Relative expression of miR-21 with common clinical-pathological features of colorectal cancer.

<p>Relative expression of miR-21 with common clinical-pathological features of colorectal cancer.</p