Telomere dysfu. unction in breast. Indiana University. School of Medicine

Transcription

1 Telomere dysfu unction in breast differentiation, aging and cancer Indiana University Department of Medical David Gi illey Ph.D. School of Medicine and Molecular Genetics

2 Part I: The hypothesis Consequences of telomere dysfunction in cancer: an early event? cap Capped telomere Uncapping event Telomere fusion Genomic instability Breakage-fusion-bridge (BFB) cycle - McClintock, Genetics,1941

3 Telomere dysfun nction in cancers Knowledge gap: no direct evidence that telomere dysfunction occurs in human cancers A novel assay to detect telomere fusions to understand and monitor disease progression

4 Detection and analysis of fusion junction Normal chromosomes Telomere dysfunction Dicentric chromosome TAR Fusion PCR telomere adjacent seq. primer Fusion point Multiplex: primer sets telomere adjacent seq. primer PCR product Detection

5 TAR-Fusion PCR multiplex primers (CCCTAA)n p-arm q-arm (TTAGGG)n Chr 1 Chr 2 Chr 4 Chr 5 Chr 7 Chr 9 Chr 10 Chr 11 Chr 12 Chr 15 NOTE: TAR-PCR primers cover ~40% of ends but only ~15% of possible end-to-end combinations Chr 16 Chr 17 Chr 18 Chr 19 Chr 21 Chr X or Y

6 Telomere fusions present early Primer Mix: A B in breast tumorigenesis i A B A B A B A B A B A B A B A B (kb) DCIS 50% Inv vasive Normal Telomere fu usion positive tum mors 40% 30% 20% 10% P=0.004 P= ~40% of tumor tissue 2. Only 15% of possible fusions covered by TAR fusion PCR 3. Stabilization at or before DCIS stage 0% Normal DCIS (N=24) (N=25) Invasive (N=23)

7 Summary: fusion junctions in breast tumor tissue DCIS Invasive Telomere to Telomere (TTAGGG) n (CCCTAA) n 7.7% (1/13) 6.7% (1/15) Telomere to subtelomere (TTAGGG) n 92.3% (12/13) 80.0% (12/15) Complex (TTAGGG) n I nsertion 0% (0/13) 13.3% (2/15) 0.6 ~ 2 kb *Hallmark of solid tumors: shortened ed telomeres e es and activated telomerase e

8 Short insertions of retrotransposon elements at tinvasive i breast ttumor tissue fusion junctions Chr.4 p Chr.2 Chr.17 q q q p p p q Chr.10 Chr.4 p q p Chr.4 q q p Chr. X or Y Chr.2q:211 bp Chr.17p:374 bp Chr.4q: 447 bp 4q XpYp 10q LTR 4p 4q telomere SINE SINE XpYp telomere 10q telomere 4p telomere

9 Conclusions: fusio on junction results Telomere fusions (dy ysfunction) found early in human breast tumors (DCIS). Potentially a highly prevalent genetic marker for tumorigenesis: breast, prostate*,o ovarian*, CLL, others? Tanaka, Abe, Huda, Tu, Beam, Grimes and Gilley, Telomere fusions in early human breast carcinoma. PNAS (2012)

10 Part II: Telomere plast ticity in normal human mammary repopulating p cells Note: 1) Critical to determine the biology of normal repopulating subpopulations before comparing to tumor subpopulations 2) Uncovering possible mechanisms of disease resistancee and initiation

11 Human mammary cellular hierarchy? Cell of origin: Luminal progenitors? Mammary stem cell (BC) Bipotent progenitor (BC) Luminal prog genitor () Mature Luminal Cells (LC) Myoepithelial Progenitors (BC) Mature Myoepithelial Cells (BC) (Lim et al., 2009 Nat. Med.) (Molyneux et al., (2010) Cell Stem Cell)

12 Subpopulation isolation from reduction mammoplasty Enzymatic dissociation trypsin/dispase/dnase FACS purification CD31- CD45- EpCA AM PE LC SC BC Single Cells CD49f APC

13 Telomere length plasticity in isolated breast subsets 20 year old Mammary stem cell (BC) Bipotent progenitor (BC) Luminal progenitor () Mature Luminal Cells (LC) MyoepithelialMature Myoepithelial Cells (BC) Progenitors (BC) 12 TRF p < qpcr p = Telomere len ngth (kb) p < Telomere len ngth (kb) p = BC 4 LC SC BC LC SC

14 Age-dependent telomere length shortening in the mature luminal cells ladder HT1080 EEC TC EEC TC BC 46 LC ST BC 20 LC ST 26 BC LC ST 10 (kb) LC TRF (kb b) 8 6 r 2 = age (years)

15 Telomerase activation HT1080 HeLa HI CHAPS BC LC ST BC LC ST BC LC ST BC TRF2 htert merge BC in luminal progenitors 58 LC ST p < RTA (%) 0 BC LC SC IC htert / TRF2 0 BC Frequency of co-foci

16 Reduced activation of telomerase in luminal progenitor cells with age (%) 25 r 2 = elative Tel lomerase R020 LC TRF (kb) r 2 = age (year) age (year) Telomerase activity decreases with age of luminal progenitor cells correlates with: Telomere lengths decreases with age of differentiated luminal cells

17 Telomere-associated DNA damage response in normal luminal progenitor cells fraction BP Fraction MRN complex MRE11 Rad50 Tl Telomere-associ itd iated proteins ti NBS1 RT-PCR confirmation

18 mrna expression: Telomere and DDR genes in basal versus lum minal progenitors p = p = p = p = p = p = p = B B B B C C C C MRE11 RAD50 ATM ATR BLM RAP1 DNA-PKcs B B B C C C

19 Telomere dysfunction-induced DNA damage foci in normal human mammary s. TRF2 γ-h2a AX Merge BC Similar il results w/ 53BP1, NBS1, MRE11 and RAD50

20 Telomere-dysfunct tion DNA damage response in normal luminal progenitors % cells BC TRF2 / NBS1 TRF2 / MRE11 TRF2 / γh2ax TRF2 / 53BP1 TRF2 / RAD50 53BP1 / γh2ax # of TIFs/nucleus

21 stem cell bipotent progenitor Normal breast luminal progenitor mature luminal differentiation: proliferation shortened telomeres activated telomerase decreasing with age mature luminal mature luminal Longer telomeres decreasing with age Telomerase negative a double edged sword? Telomere dysfunction induced foci Common Very Rare tumor initiation *Hallmark of solid tumors: shortened telomeres and activated telomerase tumor suppression

22 Disease consequences of telomere dysfunction Cancer: Blood-borne and solid tumors (CLL, breast, prostate*, ovarian*) Cardiovascular: telomere shortenin ng associated with hypertension, CVD Environmental factors & telomere dysfunction (-via telomere shortening, oxidative damage, epigenetic remodeling, others) Exogenous: smoking, obesity, socio-economic group, etc Endogenous: stress, meditation, exercise, etc. Now include: normal cellular differentiation

23 Acknowledgements David Gilley (IUSM) Hiromi Tanaka Nazmul Huda Satoshi Abe LiRen Tu Matthew Beam Matthew Rehmel Gabriela Figueroa Kimberly Smith Amanda Campbell Allison Bates Kimberly Ho A Lim Zahir Sheikh Breast subpopulations: Connie Eaves (Terry Fox Lab) Nagarajan Kannan

24 Cancer stem cell hypothesis Stem cell Progenitor Differentiated progeny Adapted from Cheng L et al. Toxicol Pathol 2009;38:62-71

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