Regulation of telomeres by mirnas in human cancer

Regulation of telomeres by mirnas in human cancer Stefan Schoeftner, PhD LNCIB, Trieste stefan.schoeftner@lncib.it SIES Discutiamo Insieme Florence, 2.11.214

Shelterin components and telomerase are key players in human cancer Shelterin Telomerase Replicative immortality Telomere maintenance Shelterin TRF1 TRF2 POT1 TPP1 TINF2 Telomerase TERT DKC1 TERC TERRA Mouse models Progeria, Cancer Progeria, Cancer Progeria, Cancer Progeria, Cancer Progeria; Cancer Progeria; Cancer Progeria; Cancer Human Cancer Cancer Progeria Progeria; Cancer Progeria; Cancer Progeria; Cancer ICF Syndrome How is telomerase/shelterin function regulated in human cancer? Can we identify mirnas that control telomere function in human cancer?? - telo-mirnas -

Regulation of telomeres by mirnas in human cancer 1. mir-155 drives telomere fragility in human breast cancer Dinami et al. Cancer Research 214 2. Silencing of mir-296 and mir-512 releases TERT expression in basal type breast cancer Dinami et al. Manuscript in preparation

luciferase reporter activity A high-throughput approach to identify mirnas with targeting specificity for telomere regulators 1. 2. 3. mirna target prediction for 3 UTRs of TRF1, TRF2, POT1, TERT, DKC1 mir-vector library firefly renilla 3 UTR TRF1 transient transfection mirna/reporter HT luciferase reporter assay 2,5 2 TRF1 onco-mir-155 1,5 1,5

A role for TRF1 and mir-155 in human cancer TRF1, a telomere master regulator * Telomere repeat binding protein * Negative regulator of telomere length * Promoted telomere replication * Mitotic fidelity * Chromosome end protection *Telomere recombination TRF1 -/- TRF1 -/- TRF1-/- Cancer +* TRF1 -/- - Down-regulation in breast cancer - Deletion of TRF1 promotes cancer in the absence of tumorsuppression mir-155, a multipotent mirna BIC locus mir-155 mir155hg Oncomir in neoplastic disease leukemia, B-cell lymphoma breast, thyroid, colon,cervical, lung cancer Cardiovascular diseases Haematopoiesis Inflammation

control mir-155 control mir-155 Relative luciferase reporter activity (control set 1 ) control mir-155 antagomir-155 mir-155 targets a partially conserved sequence motif in the 3 UTR of TRF1 125 1 75 5 25 p=.1 p=.532 TRF1 actin TRF2 actin POT1 actin wt-trf1 3 UTR D mir-155 TRF1-3 UTR

expression (log base 2 scale) High mir-155 expression correlates with low TRF1 levels in luminal breast cancer mir-155 up-regulation in breast cancer TRF1 downregulation in breast cancer TRF1 high low TRF1 in samples with high mir-155 luminal cancer with reduced TRF1 levels 39% 28% 1% mir-155-33% 75% mir-155 +/++ tumor peritumoral tumor peri- tumor peritumoral tumoral luminal triple HER2 + 2 negative n=6 p=8.14e-5 p=.2 p=.2 mir-155 high TRF1 low High (3) Medium-low (2) Low (1) negative () 5% 25% % p=.3

Low TRF1 expression correlates with poor prognosis is luminal breast cancer Low TRF1 results in poor prognosis mir-155 target genes TRF1 low TRF1 high TRF1 low TRF1 high mir-155 dependent expression of TRF1 is part of a mir-155 signature that predicts poor distant metastasis free and relapse free survival in luminal breast cancer

control mir-155 sitrf1 mir-155 modulates telomere telomere structure and homeostasis mir-155 controls TRF1 abundance at telomeres mir-155 promotes telomere elongation p<,1 p<,1 control mir-155 telomere fluorecenece units (a.f.u.) 3 2 1 595 ± 64 N=3 n=2143 67 ± 88 N=3 n=243 663 ± 22 N=3 n=2213 C B A antagomir-155 +* mir-155 sitrf1 +*

Telomere fragility Telomere sister chromatid fusions mir-155 induces chromosomal aberrations T- loop and G-stacks: obstacles to DNA replication MCF-7 + mir-155 DNA pol 1 2 spontaneous loss of DNA sister chromatid fusions abnormally shaped telomeres multiple telomere signals telomere sister chromatid fusions

mir-155 drives telomere fragility in human breast cancer by targeting TRF1 mir-155 ONCO-miRNA +* mir-155 high TRF1 - low Breast Cancer proliferation/metastasis SOCS1 TP53INP1 Telomere maintenance Telomere instability (fragility, fusions) genomic instability poor prognosis Telomere maintenance Telomere +* fragility Telomere sister chromatid fusions Dinami et al. Cancer Research 214

normal basal luma lumb her2 normal basal luma lumb her2 expression (log base 2 scale) expression (log base 2 scale) hsa-mir-541 hsa-mir-637 hsa-mir-127-5p hsa-mir-16-1* hsa-mir-661 hsa-mir-68 hsa- hsa-mir-512-5p hsa-mir-1234 hsa-mir-491-5p hsa-mir-296-3p hsa-mir-423-5p hsa-mir-299-3p hsa-mir-532-3p hsa-mir-1323 hsa-mir-1275 hsa-mir-138 hsa-mir-76 hsa-mir-663 hsa-mir-548a-3p hsa-mir-548f hsa-mir-1249 hsa-mir-548e hsa-mir-1182 hsa-mir-1266 hsa-mir-548o hsa-mir-137 hsa-mir-632 hsa-mir-1268 luciferase reporter activity and mir-512-5p target htert and are down-regulated in breast cancer mir-vector library firefly renilla 3 UTR 4 3,5 3 2,5 2 1,5 1,5 htert - mir-512-5p - transient transfection mirna/reporter mir-512-5p

TERT and mir-296/mir-512 are relevant for basal type breast cancer htert signature mir-512-5p signature ALL TUMORS (DMSF) Expression low high Expression low high Expression low high htert logrank P=.49 signature logrank P=.45 mir-512-5p signature logrank P=.15 BASAL- TYPE (DMSF) Expression low high Expression low high basal Expression low high basal

si-con si-tert mir-con mir-512-5p Control mir-296 mir-512 mir-512 TRAP activity; controls set 1 mir-296 1 si-con si-tert 1 h.i. 1 5 25 h.i. 1 5 25 h.i. 1 5 25 TSR8 mir-con mir-512-5p Control and mir-512-5p are negative regulators of telomere length and mir-512-5p target htert and reduce telomerase activity Over-expression of mir-296 or mir-512 cause telomere shortening DAPI/ Telomere Telomere p<,1 p<,1 12 1 8 6 4 2 p=.2 p=.3 n=9 n=9 n=9 74nt 68nt 62nt 56nt 5nt 8312 ± 161.8 6432 N=1491 ± 14.7 n=65 N=1344 n=67 7163 ± 142.9 N=1378 n=7 S-IC

MCF-7 SK-BR-3 T-47D MDA-MB-231 MDA-MB-468 MDA-MB-157 expression normalized to RNU49 cell number (x1 4 ) MCF-7 SK-BR-3 T-47D MDA-MB-231 MDA-MB-468 MDA-MB-157 mir-512-5p expression normalized to RNU49 cell number (x1 4 ) Control mir-296 mir-512 mir-512 and mir-296 reduce proliferation by driving apoptosis in MDA-MB-231 cells 2 15 1 5 mir-512-5p 6 4 2 Reduced proliferation mir-control mir-512 antago-mir-512 MDA-MB-231 *** ** mir-296/512 induce apoptosis 1 3 6 PARP cleaved 15 125 1 75 5 25 6 4 2 mir-control mir-296 antago-mir-296 MDA-MB-231 ** ** MDA-MB-231 Caspase cleaved actin 1 3 6 luminal basal

DMSO TSA SAHA TSA+SAHA expression (untreated set 1 ) mir-512-5p expression normalized to RNU49 expression (untreated set 1 ) mir-512-5p expression (untreated set 1 ) Epigenetic silencing of mir-512 and mir-296 in basal type MDA-MB-231 cells DNMT mir-296 DNMT mir-512 8 6 4 2 6 5 4 3 2 1 p=.1 p=.6 p=.1 1 3 1 p=,7 p=.3 p=.4 - + - + - - + + mm 5-Aza TSA SAHA 12 1 8 6 4 2 3 25 2 15 1 5 p=.3 p=.3 p=.6 1 3 1 p=,1 p=,8 p=,3 - - + - - + + mm 5-Aza HDAC mir-296 HDAC mir-512 + TSA SAHA MDA-MB-231 (basal) DNMT MDA-MB-231 (basal) HDAC HDAC HDAC HDAC Silencing of mir-296/512 loci DNMT silent silent Silencing of mir-296/512 loci Expressed Expressed

htert expression normalized to actin (DMSO set 1 ) htert expression Epigenetic silencing of mir-512 and mir-296 in basal type MDA-MB-231 cells ensures htert expression HDAC X htert 1 8 6 4 2 p=,4 p=,4 p=,4 - - + - - + + + TSA SAHA HDAC 14 12 1 8 6 4 2 mirna TSA/ SAHA mir-512-5p p=.3 p=.9 con con antago- 1 2 296/512 3 - + + htert HDAC DNMTs mir-512-5p htert poor prognosis

A mirna network that underlies telomere regulation in human cancer telo-mirnas control telomeres HDAC DNMT Integrating telo-mirnas into hallmark features of cancer mir-512-5p mir-155 tumor-promotion +* a telo-mirna network to be discovered

Acknowledgements The Team: Roberto Dinami Rosanna Sestito Eleonora Petti Valentina Buemi Federica Cioffi Collaborators Giovanni Blandino IRE, Rome Reuven Agami, NKI Amsterdam Marcella Mottolese IRE Rome Mauro Giacca, ICGEB Trieste Maria A. Blasco CNIO, Madrid Roberta Benetti LNCIB, Trieste Claudio Schneider LNCIB, Trieste LNCIB, Area Science Park, Trieste University of Trieste

mir-512-5p htert luciferase reporter activity 12 1 8 6 p=.2 p=.6 p=.1 p=.5 12 1 8 6 p=.3 p=.1 4 4 2 2 + - + - + - + - - + - + - + - + Con-miR + - + - Con-miR - + - + mir-512-5p wt- 3 UTR D83-85 3 UTR D479-481 3 UTR D83-85, 479-481 3 UTR wt- 3 UTR D17-3 UTR