Telomere dysfuydifferentiation, a

David GiDavid GiIndiana University

Department of MedicalDepartment of Medical

unction in breast aging and cancer

illey Ph Dilley Ph.D. School of Medicineand Molecular Geneticsand Molecular Genetics

Part I: The

Consequences of telqin cancer: an

capCapped telome

Uncapping event

Telomere fusion

Breakage-fusion-bridge (BFB) cyc

e hypothesis

lomere dysfunction yn early event?

ere

Genomic instability

cle - McClintock, Genetics,1941

Telomere dysfunTelomere dysfun

Knowledge gap: no ditelomere dysfunction y

A novel assay to detecunderstand and moni

nction in cancersnction in cancers

irect evidence that occurs in human cancers

ct telomere fusions toitor disease progressionp g

Detection and analysNormal

chromosomes

Dicentric chromosomeDicentric chromosome

FusionTAR Fusion PCR

primer

TAR Fusion PCR

telomere adjacent seq.

p

PCR pr

Detec

sis of fusion junction

TelomereTelomeredysfunction

point

primer

telomere adjacent seq.Multiplex: primer sets

p

roduct

ction

TAR-Fusion PCR p-arm(CCCTAA)n

Chr 1

Chr 2

TAR1

TAR1

Chr 4

Chr 5

Chr 7

TAR1

TAR1

Chr 9

Chr 10

Chr 11

TAR1

Chr 12

Chr 16

Chr 15

TAR1

Chr 17

Chr 18

Chr 19

TAR1

Chr 21

Chr X or Y

multiplex primersq-arm (TTAGGG)n

TAR1

TAR1

TAR1

TAR1

TAR1

TAR1

NOTE:TAR-PCR primers cover

40% of ends but onlyTAR1

TAR1

~40% of ends but only…~15% of possible

end-to-end combinations

TAR1

TAR1

TAR1

Telomere fusioni b t t

Primer Mix: A B

5 0(kb)

A B A B A B A

in breast tu

1.01.5

2.03.04.05.0

1 2 3 1

DCIS InvDCIS Inv

40%

50%

P=0.004

usio

n m

ors

10%

20%

30%

Telo

mer

e fu

posi

tive

tum

0%

10%

Normal D(N=24) (N

ns present early i i

A B A B A B A B A B

morigenesis

2 1 23 3

vasive Normalvasive NormalP=0.016

1. ~40% of tumor tissue

2. Only 15% of possible fusionscovered by TAR fusion PCR

3. Stabilization at or before

DCIS InvasiveN=25) (N=23)

DCIS stage

Summary: fusioybreast tum

TAR1 (TTAGGG)n (CCTelomere to Telomere

TAR1 (TTAGGG)nTelomere to subtelomere

Complex TAR1 (TTAGGG)n I

0.6 ~ 2 kb

*Hallmark of sshortened telomeres ans o te ed te o e es a

on junctions in jmor tissue

7.7% (1/13) 6.7% (1/15)

DCIS Invasive

CCTAA)n TAR1

92.3% (12/13)

0% (0/13)

80.0% (12/15)

13.3% (2/15)nsertion

TAR1

TAR1

solid tumors: nd activated telomerased act vated te o e ase

Short insertions of retrt i i b t tat invasive breast tumor

Chr.4p q

Chr.2q p

Chr.17p q

pq

Chr. X or Yp q

XpYp4q

Chr.2q:211 bp Chr.17p:374 bp

SINEXpYp telomere

4q telomere SINE

rotransposon elements ti f i j ti

p q

r tissue fusion junctions

Chr.10

p q

Chr.4

Chr.4

p q

q p

4p10q LTR

Chr.4q: 447 bp

q

4p telomere10q telomere

Conclusions: fusioConclusions: fusio Telomere fusions (dy Telomere fusions (dy

early in human bre

Potentially a highly pmarker for tumoribreast, prostate*, obreast, prostate , o

Tanaka, Abe, Huda, Tu, Beam, Gin early human breas

on junction resultson junction resultsysfunction) foundysfunction) foundeast tumors (DCIS).

prevalent genetic genesis:

ovarian*, CLL, others?ovarian , CLL, others?

Grimes and Gilley, Telomere fusionst carcinoma. PNAS (2012)

Par

Telomere plastTelomere plasthuman mammaryy

Note1) Critical to determine

repopulating subpcomparing to tum

2) Uncovering possibdisease resistance

rt II:

ticity in normalticity in normaly repopulating cellsy p p g

e: the biology of normal

populations before mor subpopulations

ble mechanisms of e and initiation

Human mammary

MammaryMammarystem cell (BC)

Lprog

Bipotentprogenitor (BC)

p g

MyoeProgen

cellular hierarchyCell of origin:

Luminal progenitors??

Luminal genitor (LP) Mature Luminal g ( )

Cells (LC)

epithelialnitors (BC)

Mature Myoepithelial Cells (BC)

(Lim et al., 2009 Nat. Med.)(Molyneux et al., (2010) Cell Stem Cell)

Subpopulatif d tifrom reduction

Enzymaticdissociation

trypsin/dispase/DNase

Si l C llSingle Cells

ion isolationl tn mammoplasty

FACS purification A

M P

E LPLC

CD31- CD45-

EpC

A

BCSC

CD49f APCs

Telomere length plasticity20 ld20 year old

Mammarystem cell (BC)

Bipotprogenito

T

9

10

11

12

ngth

(kb)

Tp <

p < 0.001

5

6

7

8

Telo

mer

e le

n

4

T

BC L

y in isolated breast subsets

Luminal M t L i l

tentor (BC)

progenitor (LP) Mature Luminal Cells (LC)

MyoepithelialProgenitors (BC)

Mature Myoepithelial Cells (BC)

TRF qPCR

9

10

11

12

ngth

(kb)

TRF qPCR0.001

p = 0.004

p = 0.004

5

6

7

8

Telo

mer

e le

n

4

TLP SCLC BC LP SCLC

Age-dependent telomerin the mature lu

2620463146

STLCLPBC

TCEEC

TCEEC

HT1

080

ladd

er

STLCLPBC

STLCLPBC

23.1

(kb)

8.07.0

9.0

6.0

5.0

4.34.0

3.0

2.4

2.0

1.5

1.0

re length shortening uminal cells .

8

10

r 2 = 0.50

b)

6

8

C T

RF

(kb

40 20 40 60 80

age (years)LC

age (years)

Telomerase activation

BC

26

BC

LP LC ST

24

HeL

a

HI CH

APS

HT1

080

BC

LP LC ST BC

LP LC ST

48

TRF2 hTERT merge

BC

LP

in luminal progenitors

LP LC ST

58

p < 0.001

4

8

12

16

RTA

(%)

IC

BC LP SCLC0

R

100o-fo

ci

hTERT / TRF2

0

20

40

60

80

quen

cy o

f co

0

Freq BC LP

Reduced activation of progenitor cprogenitor c

25 (%)

r 2 = 0.54

15

20

lom

eras

e

0

5

10

0 20 40 60 80

elat

ive

Tel

0 20 40 60 80R

age (year)

Telomerase activity decreases with age ofluminal progenitor cells

co

f telomerase in luminal cells with agecells with age

10

8

r 2 = 0.50

F (k

b)4

6

LC T

RF

0 20 40 60 80

age (year)

Telomere lengths decreases with age of differentiated luminal cellsrrelates with:

Telomere-associated DNnormal luminalnormal luminal

LP fraction

BP Fraction

T l iTelomere-associ

NA damage response in progenitor cellsprogenitor cells

MRN complexMRE11Rad50

i t d t i NBS1

RT-PCR confirmation

iated proteins

mRNA expression: Telin basal versus lumin basal versus lum

4.0

4.5

7

8

1 0

1.2 2.5p = 0.006 p = 0.012 p = 0.025 p =

1.5

2.0

2.5

3.0

3.5

3

4

5

6

0.4

0.6

0.8

1.0

1.0

1.5

2.0

0.0

0.5

1.0

1.5

0

1

2

0.0

0.2

0.0

0.5

BC

LP BC

LP BC

LP BCC C C C

MRE11 RAD50 ATM AT

lomere and DDR genesminal progenitorsminal progenitors

0.08

0 10

0.12 0.054 p = 0.002 p = 0.020 p = 0.039

0.04

0.06

0.04

0.06

0.08

0.10

0.00

0.02

0.00

0.02

LP BC

LP BC

LP BC

LPC C C

TR BLM RAP1 DNA-PKcs

Telomere dysfunction-indnormal human

TRF2 H2A

normal human

TRF2 γ-H2A

BCBC

LP

Si il lt / 53BP1 NSimilar results w/ 53BP1, N

duced DNA damage foci in mammary LPs

AX M

mammary LPs.

AX Merge

NBS1 MRE11 d RAD50NBS1, MRE11 and RAD50

Telomere-dysfunctyresponse in normal l

100

% c

ells

50

0 3 4 6 7 9 10 1

%

0

0 - 3 4 - 6 7 - 9 10 - 1

# of TIF

tion DNA damage gluminal progenitors

BC LPTRF2 / NBS1

TRF2 / MRE11TRF2 / γH2AXTRF2 / 53BP1TRF2 / RAD5053BP1 / γH2AX

2 13 15 16 18 19 21 22 24

53BP1 / γH2AX

2 13 - 15 16 - 18 19 - 21 22 - 24

Fs/nucleus

stemcell

Nd

bipotentprogenitor

dmature luminal

Longer telomeresdecreasing with age

mature

luminalprogenitor

proliferation

shortened

mature luminal

Telomerase negative

luminaltelomeres

activated telomerase decreasing

with age

Telomere dysfunctioninduced foci

Very

tumorinitiation

yRareCommon

shortentumor

suppression

Normal breast diff ti tidifferentiation:

a doublea double edged sword?edged sword?*Hallmark of solid tumors:

ed telomeres and activated telomerase

Disease consequences ofqCancer: Blood-borne and solid tum

C di l t l h t i

i f

Cardiovascular: telomere shortenin

Environmental factors &(-via telomere shortening, oxidative dama

Exogenous: smoking, obesity, socio-

Endogenous: stress, meditation, exe

Now include

g

f telomere dysfunctionymors (CLL, breast, prostate*, ovarian*)

i t d ith h t i CVD

f i

ng associated with hypertension, CVD

& telomere dysfunctionage, epigenetic remodeling, others)

-economic group, etc…

ercise, etc….

: normal cellular differentiation

Ack

David GilleyDavid Gilley(IUSM)Hiromi TanakaNazmul HudaSatoshi AbeLiRen TuMatthew BeamMatthew RehmelMatthew RehmelGabriela FigueroaKimberly SmithAmanda CampbellAlli B tAllison BatesKimberly Ho A LimZahir Sheikh

nowledgements

Breast subpopulations:Connie Eaves (Terry Fox Lab)Nagarajan Kannang j

Cancer stem

S llStem cell

Progenitor

Differentiated progeny

cell hypothesis

Adapted from Cheng L et al. Toxicol Pathol 2009;38:62-71