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The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients. The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.

The Telomeric Repeat binding Factor 2 (TRF2), a key protein involved in telomere integrity, is over-expressed in several human cancers and promotes tumor formation and progression. Recently, TRF2 has been also found outside telomeres where it can affect gene expression. Here we provide evidence that TRF2 is able to modulate the expression of microRNAs (miRNAs), small non-coding RNAs altered in human tumors. Among the miRNAs regulated by TRF2, we focused on miR-193b-3p, an oncomiRNA that positively correlates with TRF2 expression in human colorectal cancer patients from The Cancer Genome Atlas dataset. At the mechanistic level, the control of miR-193b-3p expression requires the cooperative activity between TRF2 and the chromatin organization factor CTCF. We found that CTCF physically interacts with TRF2, thus driving the proper positioning of TRF2 on a binding site located upstream the miR-193b-3p host-gene. The binding of TRF2 on the identified region is necessary for promoting the expression of miR-193b3p which, in turn, inhibits the translation of the onco-suppressive methyltransferase SUV39H1 and promotes tumor cell proliferation. The translational relevance of the oncogenic properties of miR-193b-3p was confirmed in patients, in whom the association between TRF2 and miR-193b-3p has a prognostic value.

In vertebrates, the telomere repeat containing long, non-coding RNA TERRA is prone to form RNA:DNA hybrids at telomeres. This results in the formation of R-loop structures, replication stress and telomere instability, but also contributes to alternative lengthening of telomeres (ALT). Here, we identify the TERRA binding proteins NONO and SFPQ as novel regulators of RNA:DNA hybrid related telomere instability. NONO and SFPQ locate at telomeres and have a common role in suppressing RNA:DNA hybrids and replication defects at telomeres. NONO and SFPQ act as heterodimers to suppress fragility and homologous recombination at telomeres, respectively. Combining increased telomere fragility with unleashing telomere recombination upon NONO/SFPQ loss of function causes massive recombination events, involving 35% of telomeres in ALT cells. Our data identify the RNA binding proteins SFPQ and NONO as novel regulators at telomeres that collaborate to ensure telomere integrity by suppressing telomere fragility and homologous recombination triggered by RNA:DNA hybrids., LncRNA TERRA forms RNA-DNA hybrids at telomere sites leading to telomere instability. Here the authors identify the RNA interacting factors NONO and SFPQ as proteins that interact with TERRA and telomere chromatin and reveal putative roles for these factors in telomere integry maintenance by interfering with RNA:DNA hybrid formation.

The Suv39h1 and Suv39h2 H3K9 histone methyltransferases (HMTs) have a conserved role in the formation of constitutive heterochromatin and gene silencing. Using a transgenic mouse model system we demonstrate that elevated expression of Suv39h1 increases global H3K9me3 levels in vivo. More specifically, Suv39h1 overexpression enhances the imposition of H3K9me3 levels at constitutive heterochromatin at telomeric and major satellite repeats in primary mouse embryonic fibroblasts. Chromatin compaction is paralleled by telomere shortening, indicating that telomere length is controlled by H3K9me3 density at telomeres. We further show that increased Suv39h1 levels result in an impaired clonogenic potential of transgenic epidermal stem cells and Ras/E1A transduced transgenic primary mouse embryonic fibroblasts. Importantly, Suv39h1 overexpression in mice confers resistance to a DMBA/TPA induced skin carcinogenesis protocol that is characterized by the accumulation of activating H-ras mutations. Our results provide genetic evidence that Suv39h1 controls telomere homeostasis and mediates resistance to oncogenic stress in vivo. This identifies Suv39h1 as an interesting target to improve oncogene induced senescence in premalignant lesions.

// Roberto Dinami 1, 2, * , Valentina Buemi 1, 3, * , Rosanna Sestito 2, *, ** , Antonina Zappone 1, 3 , Yari Ciani 4 , Miguel Mano 5 , Eleonora Petti 1, 2, 3 , Andrea Sacconi 6 , Giovanni Blandino 6 , Mauro Giacca 5 , Silvano Piazza 4 , Roberta Benetti 7, 8 and Stefan Schoeftner 1, 2, 3 1 Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Genomic Stability Unit, Trieste 34149, Italy 2 Italian National Cancer Institute, Regina Elena, Rome 00144, Italy 3 Department of Life Sciences, Universita degli Studi di Trieste, Trieste 34127, Italy 4 Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Bioinformatics and Functional Genomics Unit (BFGU), Trieste 34149, Italy 5 International Centre for Genetic Engineering and Biotechnology (ICGEB), Molecular Medicine Laboratory, Trieste 34149, Italy 6 Italian National Cancer Institute, Regina Elena, Translational Oncogenomics Group, Rome 00144, Italy 7 Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Cancer Epigenetics Unit, Trieste 34149, Italy 8 Department of Medical and Biological Sciences, Universita degli Studi di Udine, Udine 33100, Italy * These authors have contributed equally to this work ** Current address: Italian National Cancer Institute, Regina Elena, Preclinical Models and New Therapeutic Agents Unit, Via Elio Chianesi 53, Rome 00144, Italy Correspondence to: Roberta Benetti, email: roberta.benetti@uniud.it Stefan Schoeftner, email: stefan.schoeftner@lncib.it Keywords: miR-296-5p; miR-512-5p; telomerase; telomeres; breast cancer Received: August 17, 2016 Accepted: August 27, 2017 Published: September 23, 2017 ABSTRACT The catalytic subunit of the telomerase complex, hTERT, ensures unlimited proliferative potential of cancer cells by maintaining telomere function and protecting from apoptosis. Using a miRNA screening approach we identified miR-296-5p and miR-512-5p as miRNAs that target hTERT in breast cancer cells. Ectopic miR-296-5p and miR-512-5p reduce telomerase activity, drive telomere shortening and cause proliferation defects by enhancing senescence and apoptosis in breast cancer cells. In line with the relevance of hTERT expression for human cancer we found that miR-296-5p and miR-512-5p expression is reduced in human breast cancer. Accordingly, high expression of miR-296-5p and miR-512-5p target genes including hTERT is linked with significantly reduced distant metastasis free survival and relapse free survival of basal type breast cancer patients. This suggests relevance of the identified miRNAs in basal type breast cancer. Epigenetic silencing of miR-296 and miR-512 encoding genes is responsible for low levels of miR-296-5p and miR-512-5p expression in basal type breast cancer cells. Disrupting gene silencing results in a dramatic upregulation of miR-296-5p and miR-512-5p levels leading to reduced hTERT expression and increased sensitivity to the induction of apoptosis. Altogether, our data suggest that epigenetic regulatory circuits in basal type breast cancer may contribute to high hTERT levels by silencing miR-296-5p and miR-512-5p expression, thereby contributing to the aggressiveness of basal type breast cancer.

Telomeres consist of DNA tandem repeats that recruit the multiprotein complex shelterin to build a chromatin structure that protects chromosome ends. Although cancer formation is linked to alterations in telomere homeostasis, there is little understanding of how shelterin function is limited in cancer cells. Using a small-scale screening approach, we identified miR-155 as a key regulator in breast cancer cell expression of the shelterin component TERF1 (TRF1). miR-155 targeted a conserved sequence motif in the 3′UTR of TRF1, resulting in its translational repression. miR-155 was upregulated commonly in breast cancer specimens, as associated with reduced TRF1 protein expression, metastasis-free survival, and relapse-free survival in estrogen receptor–positive cases. Modulating miR-155 expression in cells altered TRF1 levels and TRF1 abundance at telomeres. Compromising TRF1 expression by elevating miR-155 increased telomere fragility and altered the structure of metaphase chromosomes. In contrast, reducing miR-155 levels improved telomere function and genomic stability. These results implied that miR-155 upregulation antagonizes telomere integrity in breast cancer cells, increasing genomic instability linked to poor clinical outcome in estrogen receptor–positive disease. Our work argued that miRNA-dependent regulation of shelterin function has a clinically significant impact on telomere function, suggesting the existence of “telo-miRNAs” that have an impact on cancer and aging. Cancer Res; 74(15); 4145–56. ©2014 AACR.

Telomeres are located at the end of chromosomes and consist of DNA tandem repeats that recruit the specialized protein complex “shelterin”. Shelterin has a crucial role in controlling chromosome end protection, telomere recombination and telomere length. Telomeres shorten with every cell division, finally leading to telomere-dysfunction and the induction of senescence or apoptosis. Cancer formation is paralleled by a change in telomere regulation. Re-activation of telomerase ensures the maintenance of telomere function to facilitate unlimited proliferative potential. Importantly, aberrant function of the shelterin complex contributes to tumor formation and genomic instability in human cancer. miRNAs are important regulators of central cancer pathways and are of high clinical relevance. We recently showed that the onco-microRNA miR-155 controls the expression of TRF1 in human breast cancer to promote increased telomere fragility and genomic instability. Importantly, low TRF1 expression correlates with poor prognosis in estrogen receptor positive cancer patients, underlining the clinical relevance of miR-155 dependent regulation of TRF1 in human breast cancer. Our work suggests that multiple miRNAs exist that control telomere function in telomere related pathologies. Identification and functional validation of “telo-miRNAs” is expected to open new avenues in the understanding of telomere related maladies such as cancer and aging.