Knock-down of the 37kDa/67kDa laminin receptor LRP/LR impedes telomerase activity.

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85-90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA-MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.This work was supported by the National Research Foundation, the Republic of South Africa.NCS201

Mre11 modulates the fidelity of fusion between short telomeres in human cells

The loss of telomere function can result in the fusion of telomeres with other telomeric loci, or non-telomeric double-stranded DNA breaks. Sequence analysis of fusion events between short dysfunctional telomeres in human cells has revealed that fusion is characterized by a distinct molecular signature consisting of extensive deletions and micro-homology at the fusion points. This signature is consistent with alternative error-prone end-joining processes. We have examined the role that Mre11 may play in the fusion of short telomeres in human cells; to do this, we have analysed telomere fusion events in cells derived from ataxia-telangiectasia-like disorder (ATLD) patients that exhibit hypomorphic mutations in MRE11. The telomere dynamics of ATLD fibroblasts were indistinguishable from wild-type fibroblasts and they were proficient in the fusion of short telomeres. However, we observed a high frequency of insertion of DNA sequences at the fusion points that created localized sequence duplications. These data indicate that Mre11 plays a role in the fusion of short dysfunctional telomeres in human cells and are consistent with the hypothesis that as part of the MRN complex it serves to stabilize the joining complex, thereby controlling the fidelity of the fusion reaction

Extensive telomere erosion is consistent with localised clonal expansions in Barrett’s metaplasia

Barrett’s oesophagus is a premalignant metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma. However, only a minor fraction of Barrett’s oesophagus patients progress to adenocarcinoma and it is thus essential to determine bio-molecular markers that can predict the progression of this condition. Telomere dysfunction is considered to drive clonal evolution in several tumour types and telomere length analysis provides clinically relevant prognostic and predictive information. The aim of this work was to use high-resolution telomere analysis to examine telomere dynamics in Barrett’s oesophagus. Telomere length analysis of XpYp, 17p, 11q and 9p, chromosome arms that contain key cancer related genes that are known to be subjected to copy number changes in Barrett’s metaplasia, revealed similar profiles at each chromosome end, indicating that no one specific telomere is likely to suffer preferential telomere erosion. Analysis of patient matched tissues (233 samples from 32 patients) sampled from normal squamous oesophagus, Z-line, and 2 cm intervals within Barrett’s metaplasia, plus oesophago-gastric junction, gastric body and antrum, revealed extensive telomere erosion in Barrett’s metaplasia to within the length ranges at which telomere fusion is detected in other tumour types. Telomere erosion was not uniform, with distinct zones displaying more extensive erosion and more homogenous telomere length profiles. These data are consistent with an extensive proliferative history of cells within Barrett’s metaplasia and are indicative of localised clonal growth. The extent of telomere erosion highlights the potential of telomere dysfunction to drive genome instability and clonal evolution in Barrett’s metaplasia

Fusion of short telomeres in human cells is characterized by extensive deletion and microhomology, and can result in complex rearrangements

Telomere fusion is an important mutational event that has the potential to lead to large-scale genomic rearrangements of the types frequently observed in cancer. We have developed single-molecule approaches to detect, isolate and characterize the DNA sequence of telomere fusion events in human cells. Using these assays, we have detected complex fusion events that include fusion with interstitial loci adjacent to fragile sites, intra-molecular rearrangements, and fusion events involving the telomeres of both arms of the same chromosome consistent with ring chromosome formation. All fusion events were characterized by the deletion of at least one of the telomeres extending into the sub-telomeric DNA up to 5.6 kb; close to the limit of our assays. The deletion profile indicates that deletion may extend further into the chromosome. Short patches of DNA sequence homology with a G:C bias were observed at the fusion point in 60% of events. The distinct profile that accompanies telomere fusion may be a characteristic of the end-joining processes involved in the fusion event

Shorter telomere length profiles are observed in Barrett’s metaplasia but no differences are detected at four different chromosome ends.

<p>A, an example of STELA at the XpYp telomere in two patients with matched normal squamous epithelium (S), Barrett’s metaplasia (B) and normal gastric mucosa (G). The mean and standard deviation of telomere length profiles are detailed below. B, example of the same samples analysed with STELA at the 17p telomere. C-F, telomere length profiles obtained from patients #1-#8, as detailed above, depicted as scatter plots obtained by STELA at the telomeres of XpYp (C), 17p (D), 11q (E) and 9p (F). Statistically significant differences between the Barrett’s metaplasia samples with either patient matched squamous or gastric samples are illustrated with asterisks above the plots (two-tailed, Mann-Whitney; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001), error bars represent 95% confidence intervals. Patients in which the Barrett’s metaplasia sample displayed the shortest, or equal shortest, telomere-length profiles are highlighted in green. G, scatter plot displaying the mean telomere lengths determined for each chromosome end, error bars represent SD.</p

Telomere erosion in Barrett’s metaplasia occurs in zonal patches.

<p>A-B, examples of STELA of multiple tissues as indicated above from separate two patients. Mean and standard deviation are detailed below. C, scatterplot depicting STELA data from the XpYp (black) and 17p (green) telomeres from multiple tissues derived from the same patient.</p

Effect of LRP/LR on telomerase activity in HEK293 and MDA_MB231 cells.

<p>The expression level in HEK293 and MDA_MB231 cells was investigated using TRAPEZE Telomerase kit (Merck Millipore) and qPCR. Analysis of the concentrations revealed a significant (*** p < 0.001) reduction in telomerase activity once LRP was knockdown in A) HEK293, B) and C) MDA_MB231 cells, respectively, compared to control non-transfected cells and negative control siRNA transfected cells. Non-significant (ns) at p>0.05.</p

siRNA-mediated knock-down of LRP/LR in HEK293 and MDA_MB231 cells.

<p>The expression level in HEK293 and MDA_MB231 cells was investigated 72h post-transfection with siRNA-LAMR1. Densitometric analysis of western blot signals revealed a significant (*** p < 0.001) 90.48% and 92.59% reduction in LRP protein expression in A) HEK293 and B) MDA_MB231 cells, respectively, compared to control non-transfected cells (set at 100%).</p

Flag^® Immunoprecipitation assays confirming an interaction between LRP/LR and hTERT.

<p>Pull down assays were used to detect LRP::FLAG as well as any associated proteins bound to the anti-M2 flag beads. A loading control of crude HEK293 lysate was incorporated to ensure the validity of the blots. Panel C indicates the positive and negative controls, where the Bound protein shows the detection of the BAP fusion protein (50 kDa) to the anti-FLAG beads. Panel B indicates that the LRP::FLAG protein was only present in the HEK293 transfected samples, where FLAG was detected on the anti-FLAG beads (Bound protein). Panel A illustrates the detection of a ±140 kDa band (Bound protein) showing a pull down of hTERT for the HEK293 transfected cell line, whereas no signal was detected for the non-transfected HEK293 cell line.</p

Confocal microscopy analysis of the interaction between LRP/LR and hTERT on MDA_MB231 and HEK293 cells.

<p>A) Intracellular LRP/LR and hTERT on immunolabelled HEK293 cells. (B) Endogenous cell surface LRP/LR and hTERT on immunolabelled HEK293. (C) Intracellular LRP/LR and hTERT on immunolabelled MDA_MB231 cells. (D) Endogenous cell surface LRP/LR and hTERT on immunolabelled MDA_MB231. hTERT was detected using anti-telomerase reverse transcriptase and anti-goat to mouse-APC antibodies. LRP/LR was detected employing anti-IgG-iS18 and anti-human-FITC antibodies. Merged images verified the co-localization. The yellow staining indicates areas of co-localization. Secondary antibody controls are shown beneath each panel. Fluorescence was detected and images were acquired using the Olympus IX71 Immunofluorescence Microscope and Analysis Get It Research Software. Scale bars are 20μm. White arrows point to areas of co-localization.</p