LINE-1 encoded reverse transcriptase (RT) in the generation of new genetic information, embryonic development and tumorigenesis Corrado Spadafora Italian National Institute of Health Rome, Italy Background: mouse sperm cells can internalize exogenous DNA molecules DAPI FISH Acrosome Nucleus Merge Spadafora, BioEssays, 1998 Sperm-mediated gene transfer: molecular basis + Incubation 37°C Exogenous DNA Spadafora, BioEssays, 1998 + Sperm-mediated gene transfer: summary features 1. Sperm cells can internalize exogenous DNA with which they come in contact 2. The uptake of exogenous DNA is a highly regulated process mediated by specific factors 3. The binding of exogenous DNA activates nuclear functions that are otherwise repressed in spermatozoa 4. One of these activities is an endogenous Reverse Transcriptase (RT) Immunofluorescence detection of LINE-1 encoded RT in mouse sperm cells Anti-L1 RT DAPI a Merge b c Oocyte Anti-mouse L1 RT a b c a’ b' c‘ a b c Sperm 2ary antibody a b c Anti-human LINE1 RT Vitullo et al. 2012 Exogenous !-gal is detected in sperm cells and derived embryos 5’ LTR 3’ LTR RNA 9685bp 850 650 500 300 200 5f / 8r cDNA product Sciananna et al., BBRC 2003 RNA-mediated Sperm-mediated “Reverse” Gene Transfer 3’ LTR 5’ LTR pVLMB !-gal expression in organs of F0 and F1 pVLMB RNA-transformed animals Sciananna et al., BBRC 2003 EGFP copies are reverse-transcribed and spliced in sperm cells incubated with pBSKS-EGFP-int pBSKS EGFP-int SD SA SA Unspliced EGFP + int 1243bp Spliced EGFP 342bp Plasmid ng/106sperms SUP Nuclei Plasmid DNA (µg) 5 50 500 5 50 500 2 1 0.5 0.01 SD Time (min.) Nuclei 5’ 15’ 30’ SUP controls 5’ 15’ 30’ . EGFP+int. EGFP Sperm-derived reverse-transcribed EGFP is expressed in tissues of F0 mice Liver Liver 100x 250x Brain Kidney G! 250x 250x Conclusions (I) The sperm-mediated reverse gene transfer assays suggest that - a sperm RT-mediated mechanism is responsible for the genesis of newly reverse-transcribed genetic information, - that can be transmitted to offsprings, besides that carried by chromosomes Detection of EGFP from tumours to germ cells: experimental outline 1 A-375 human melanoma cells stably expressing EGFP whole cells released exosomes 2 RNA DNA proteins RNA DNA proteins A-375/EGFP cells xenografted in athymic mice 45 days blood exosomes RNA 45 days 3 A-375/EGFP xenograft growth mature sperm cells RNA Cossetti et al. Plos One 2014 Tumour marker RNAs in circulating exosomes and in germ cells of xenografted mice A-375 melanoma cell line A375 RNA whole cells exosomes EGFP Cossetti et al. PLoS ONE 2014 GAPDH Mice-derived blood exosomes A375 cells Mice-derived sperm cells sperm RNA blood-derived exosomes EGFP EGFP GAPDH Human exosomes are !"#$%"&'("))*+!(#"& taken up by murine spermatozoa Interaction with non labelled exosomes & Conclusions (II) - Human cancer cells xenografted in mice release tumorspecific RNA-containing nanovesicles (exosomes) in the circulating blood - RNA-mediated information flows from the soma to the germline, crossing the Weissman barrier Does the endogenous Reverse Transcriptase play a role in embryogenesis? Antisense oligonucleotides targeting active LINE-1/L1 arrest early embryo development RT activity assay 5’ UTR A 100 90 80 70 60 50 40 30 20 10 0 ORF1 B interORF ORF2 3’ UTR MS2 cDNA LINE-1 C ns-inj as-B non inj PBS inj ns inj as-C as-A as-B Bright field ns zyg 2 cell 4 cell 8 cell mor blast as-B Beraldi et al Mol Repr Dev 2006 FITC BrdU incorporation in early mouse embryos secondary antibody alone anti-BrdU antibody Phase contrast DAPI pronuclei ! BrdU pronuclei " ! " zygote 2-cell embryo zygote 2-cell embryo Vitullo et al. Mol Repr Dev 2012 Nevirapine (nonnucleoside RT Inhibitor) abolishes aphidicolin-resistant BrdU incorporation IVF Inhibitor 2h 2h Fix+IF 20 h DAPI BrdU " ! aphidicolin + nev aphidicolin Phase BrdU, Aphidicolin Vitullo et al. Mol Repr Dev 2012 LINE-1 copy number is amplified in preimplantation embryogenesis '"!!# ORF 1 LINE-1/5S copy number &"$!# ((# ((# ((# &"!!# ORF2 ((# ((# ((# %"$!# (# %"!!# !"$!# !"!!# sperm zygote 2-cell morula blasto Vitullo et al. Mol Repr Dev 2012 Conclusions (III) - RT inhibition causes a drastic arrest of embryo development (2-4 cell stages) - Reverse transcription takes place in both male and female zygotic pronuclei soon after fertilization - LINE-1s are amplified throughout preimplantation development RT activity is strictly necessary for preimplantation development Does the endogenous Reverse Transcriptase play a role in cell proliferation and tumor growth? Targeting human active LINE-1 retroelements by RNA interference LINE-1 ORF1 5’ UTR ORF2 Intergenic region 3’ UTR GAGAACGCCACAAAGAUAC Oricchio et al., Oncoegne 2007 LINE1-TARGETED RNAi REDUCES CELL PROLIFERATION AND STIMULATES DIFFERENTIATION PROLIFERATION 120 cell counts (%) A375 MELANOMA CELL CULTURES 100 80 parental cell line pS neo 60 pS-L1i 40 20 0 16 pS neo MORPHOLOGICAL DIFFERENTIATION Oricchio et al., Oncoegne 2007 23 33 days post-infection pS-L1i A375 cells interfered for LINE-1 exhibit reduced tumorigenicity in vivo Tumor volume (cm3) 4 CTR 3 2 1 pS L1-i 0 0 13 17 21 25 30 37 days Oricchio et al., Oncoegne 2007 Efavirenz inhibits proliferation in human transformed cell lines Viable cells (% of control) 120 U87 A375 100 PC3 80 SAOS-2 WI38 60 40 20 0 DMSO 10 20 30 40 50 µM EFV LINE1 ORF2p "-tubulin Sciamanna et al. Oncotarget 2013 RT inhibitors induce morphological differentiation of melanoma cells SEM Tubulin Efavirenz Nevirapine DMSO Phase contrast A-375 melanoma cells exposed to RT inhibitors acquire: - dendritic-like extensions - flattened shape - high adhesion These features are typical of melanoma cells induced to differentiate In vivo anti-tumor effectiveness of RT inhibitors Assaying RT inhibitory treatments in animal models Human tumor cell lines xenografted in nude mice: • • • • PC3 prostate carcinoma HT29 colon carcinoma A375 melanoma H69 small cell lung carcinoma Treatment with RT inhibitors started one day, or one week, after tumor xenograft Efavirenz inhibits the growth of H69 small cell lung carcinoma in nude mice Untreated 25 days 40 days Efavirenz-treated RT inhibitors reduce the growth of tumor xenografts in vivo Sciamanna et al., Oncogene 2005 A375 melanoma 2,0 2,5 1,6 2,0 1,2 1,5 0,8 1,0 0,4 0,5 0 0 7 12 17 20 23 25 28 30 33 0 0 6 9 14 18 21 23 28 31 37 45 ctrl! Efavirenz starting 1 day after tumor cell inoculation Efavirenz starting 1 week after tumor cell inoculation Efavirenz interrupted after 14 days HT29 colon carcinoma 1,6 Tumor volume (cm3) Tumor volume (cm3) H69 small lung carcinoma 3,0 PC3 prostate carcinoma 1,4 1,2 1,2 1,0 1,0 0,8 0,8 0,6 0,6 0,4 0,4 0,2 0,2 0 0 8 10 12 14 16 18 20 16 18 20 22 24 26 28 30 TESTING OF RT INHIBITORS: SUMMARY The results with animal models suggest that RT can be regarded as a target in a novel cancer differentiation therapy. A phase II trial with the RT inhibitor Efavirenz on patients with bone metastasis of primary prostate carcinoma is ongoing (Institut Bergoniè, Bordeaux, France) A “junk DNA”-based anticancer therapy? Merge with DNA LINE-1 RT RT expression and activity during breast cancer progression LINE-1 RT protein RT activty 1 Gualtieri et al. Oncotarget 2013 2 3 4 Stages 5 6 Enhanced transcription and copy number amplification of LINE-1 and SINE B1 during breast cancer progression Gualtieri et al. Oncotarget 2013 Efavirenz-modulated metastamiRs, miRNAs promoting tumor progression, invasiveness and metastasis Name EFV modulation Modulation in Biological function cancer Cancer type miR-21 down up Correlated with invasion and metastasis lung, colorectal miR-33a down up miR-181a down up Dysregulated in bone metastasis from primary prostate prostate cancer Related with shortened disease-free survival, highly osteosarcoma upregulated in osteosarcoma miR-199b down up Dysregulated in metastasis brain miR-34b up down miR-125b up down/up Downregulated in metastasis, reactivated upon drug treatment inhibits tumor growth and lymph node metastasis Downregulated in breast and upregulated in i cancer, association with cancer metastasis colorectal, melanoma, head and neck breast, colorectal miR-146a up down prostate, breast miR-148a up down miR-193b up down Inversely correlated expression with cancer progression and metastasis Downregulated in metastasis, acts as metastasis suppressor inhibiting tumor growth and lymph node metastasis Inversely correlated expression with cancer progression, invasion and metastasis miR-204 up down colorectal, melanoma, head and neck breast Highly reduced expression in cancer progression; head and neck overexpression suppresses invasiveness and acts as metastasis suppressor Sciamanna et al. Oncotarget 2013 RT control of the cancer cell transcriptome: a model Sciamanna et al. Oncotarget 2013 Identification or RNA:DNA hybrid structures in cancer cells through CsCl density gradient centrifugation (Sciamanna et al. 2013) 1,402 1,4 1,400 1,398 1,396 1,394 1,392 1,39 1,390 1,388 1,386 1,384 1,382 1,38 1,380 1,378 1,376 RNA Hybrid RNA:DNA gr/ml Circular DNA Linear DNA 6 7 8 (# A375 (# PC3 WI38 A375 EFV 8 A375 17 21 25 CN 8 DNAse I RNAse /DNAse I - PC3 A375 EFV + - + - 21 + - DNAse I RNAse /DNAse I + Alu Alu WI38 fraction 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 LINE-1 ORF2 5 LINE-1 ORF2 1000 850 650 500 400 300 200 100 -EFV - + - -EFV + - + - +EFV + - + - + Conclusions (IV) - LINE-1 and Alu elements are up-regulated, both in expression and in copy number, during tumor progression - LINE-1-encoded ORF2 protein, hence RT, increases during tumor progression - RT inhibition reduces cancer cell proliferation and promotes differentiation; also antagonizes cancer progression in animal models in vivo - RT inhibition globally reprogrammes the expression profile in cancer cells - An RT-dependent cancer-promoting mechanism plays a causative role in cancer onset and progression A. Inhibition of endogenous RT in early embryos RT inhibition Arrest of development (irreversible) Normal development Zygote Blastocyst B. Inhibition of endogenous RT in transformed cells RT inhibition Dedifferentiation Tumor progression Transformed cell Differentiated cell RT inhibitor removal Differentiated cell (reversible) Transformed cell Paola Sinibaldi-Vallebona Enrico Garaci Ilaria Sciamanna Patrizia Vitullo Cristina Cossetti Alberto Gualtieri Chiara De Luca National Institute of Health, Rome, Italy Anna Lucia Serafino Federica Andreola CNR Inst. Neurobiology and Molecular Medicine; Rome Patrizia Lavia CNR Inst. Molecular Biology and Pathology, Rome Kathleen H. Burns Dpt. of Oncology, Johns Hopkins University, Baltimore, USA
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