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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