THE ROLE OF UV IN MELANOMA INDUCTION IN XERODERMA ... · T TC A T* T T C C TA C T A TA G * G G G G...
Transcript of THE ROLE OF UV IN MELANOMA INDUCTION IN XERODERMA ... · T TC A T* T T C C TA C T A TA G * G G G G...
THE ROLE OF UV IN THE ROLE OF UV IN MELANOMA INDUCTION IN MELANOMA INDUCTION IN
XERODERMA PIGMENTOSUM PATIENTS XERODERMA PIGMENTOSUM PATIENTS
Yun Wang, John J DiGiovanna, Yun Wang, John J DiGiovanna, Jere B Stern, Thomas J Hornyak, Jere B Stern, Thomas J Hornyak,
Mark Raffeld and Kenneth H KraemerMark Raffeld and Kenneth H Kraemer
Basic Research Laboratory, Basic Research Laboratory, Dermatology Branch, Pathology Branch, Dermatology Branch, Pathology Branch, National Cancer Institute, Bethesda, MD National Cancer Institute, Bethesda, MD
Department of Dermatology, Brown Med School, Department of Dermatology, Brown Med School, Providence, RI.Providence, RI.
Main questionMain question
What is the role of UV radiation in What is the role of UV radiation in induction of melanomas in XP induction of melanomas in XP patients?patients?
Puzzling relationship betweenPuzzling relationship betweenUV exposure and melanomaUV exposure and melanoma
The site distribution of melanomas is The site distribution of melanomas is different from that of BCC and SCC different from that of BCC and SCC and does not correspond to the most and does not correspond to the most sun exposed areas of the body (face, sun exposed areas of the body (face, head and neck)head and neck)
Similar sites of BCC and SCC Similar sites of BCC and SCC in XP and normalsin XP and normals
Kraemer et al, Arch Dermatol 130: 1018 (1994)
Similar sites of melanoma in XP Similar sites of melanoma in XP and normalsand normals
Kraemer et al, Arch Dermatol 130:1018 (1994)
Puzzling relationship between UV Puzzling relationship between UV exposure and melanomaexposure and melanoma
The site distribution of melanomas is The site distribution of melanomas is different from that of BCC and SCC different from that of BCC and SCC and does not correspond to the most and does not correspond to the most sun exposed areas of the body (face, sun exposed areas of the body (face, head and neck)head and neck)People with intermittent intense sun People with intermittent intense sun exposure have greater melanoma exposure have greater melanoma risk than people with constant risk than people with constant exposure such as farmers and sailorsexposure such as farmers and sailors
Approach IApproach I
UV radiation results in DNA damage at UV radiation results in DNA damage at sites of adjacent pyrimidines (for example sites of adjacent pyrimidines (for example TT, CC, TC) forming photoproducts. TT, CC, TC) forming photoproducts. XP patients have defective DNA repair and XP patients have defective DNA repair and a 1000a 1000--fold increase in melanomas.fold increase in melanomas.Unrepaired UV photoproducts may result Unrepaired UV photoproducts may result in characteristic mutations (for example C in characteristic mutations (for example C to T mutations).to T mutations).Mutations in tumor suppressor genes may Mutations in tumor suppressor genes may result in inactivation leading to cancer. result in inactivation leading to cancer.
Approach IIApproach IIPTEN tumor suppressor gene has PTEN tumor suppressor gene has been found to be mutated in many been found to be mutated in many cancers. cancers. Analysis of base substitution Analysis of base substitution mutations in the PTEN gene in mutations in the PTEN gene in melanomas may provide evidence for melanomas may provide evidence for UV induction of the mutations and UV induction of the mutations and thereby demonstrate a role of UV in thereby demonstrate a role of UV in causation of melanomas.causation of melanomas.
Signals from receptor tyrosine kinases can promote proliferationSignals from receptor tyrosine kinases can promote proliferation through the MAP through the MAP kinase pathway (left branch) and survival through the PI3 kinasekinase pathway (left branch) and survival through the PI3 kinase pathway (right pathway (right branch). Phosphatase and tensin homolog (PTEN), a negative regulbranch). Phosphatase and tensin homolog (PTEN), a negative regulator of the ator of the pathway, may be a somatic target in melanoma.pathway, may be a somatic target in melanoma.
The MitogenThe Mitogen--Activated Protein (MAP) Kinase and Activated Protein (MAP) Kinase and Phosphatidylinositol 3' Kinase (PI3K) PathwaysPhosphatidylinositol 3' Kinase (PI3K) Pathways
Curtin et al NEJM 353:2135 (2005)
RESULTSRESULTS
Melanoma on left upper arm.
Melanoma in situ from 52 y/o patient XP295BE
Dermoscopic image showing asymmetrically hyperpigmented lesion (Tumor 1).
PixCell II laser capture microdissectorPixCell II laser capture microdissector
Laser capture microdissection of melanoma tumor 1Laser capture microdissection of melanoma tumor 1
Atypical melanocytes are partly arranged in nests.
About 300 melanocytes captured and DNA sequencing performed
After capture of the melanoma
cells , the remaining tissue can be inspected and the transfer efficiency of the
captured cells can be evaluated.
Melanoma cells expressing
Melan-A
High frequency of melanomasHigh frequency of melanomaswith base substitution mutations in the PTEN genewith base substitution mutations in the PTEN gene
33 (6)33 (6)59 (12) 59 (12) 8 patients8 patientsTotTot
10 (2)10 (2)14 (2)14 (2)XPCXPC45/F45/FXP1BEXP1BE88
2 (0)2 (0)3 (0)3 (0)XPCXPC44/F*44/F*XP376BEXP376BE33
4 (1)4 (1)9 (4)9 (4)XPCXPC23/F23/FXP21BEXP21BE44
3 (0)3 (0)6 (0)6 (0)XPCXPC29/F29/FXP24BEXP24BE55
0 (0)0 (0)2 (1)2 (1)VARVAR60/M60/MXP31BEXP31BE77
56%(50%)56%(50%)100%100%FreqFreq
10 (3)10 (3)18 (5)18 (5)XPDXPD32/M32/MXP29BEXP29BE66
1 (0)1 (0)2 (0)2 (0)XPCXPC52/F*52/F*XP86BEXP86BE22
3 (0)**3 (0)**5 (0)**5 (0)**XPCXPC49/F*49/F*XP295BEXP295BE11
Number of Number of melanomas melanomas with PTEN with PTEN mutationsmutations
Number of Number of MelanomasMelanomas
CGCGAge/ SexAge/ SexPatientPatient
*Members of same kindred **Invasive melanoma
8 XP patients had 59 melanomas8 XP patients had 59 melanomas
High frequency of melanomasHigh frequency of melanomaswith base substitution mutations in the PTEN genewith base substitution mutations in the PTEN gene
33 (6)33 (6)59 (12) 59 (12) 8 patients8 patientsTotTot
10 (2)10 (2)14 (2)14 (2)XPCXPC45/F45/FXP1BEXP1BE88
2 (0)2 (0)3 (0)3 (0)XPCXPC44/F*44/F*XP376BEXP376BE33
4 (1)4 (1)9 (4)9 (4)XPCXPC23/F23/FXP21BEXP21BE44
3 (0)3 (0)6 (0)6 (0)XPCXPC29/F29/FXP24BEXP24BE55
0 (0)0 (0)2 (1)2 (1)VARVAR60/M60/MXP31BEXP31BE77
56%(50%)56%(50%)100%100%FreqFreq
10 (3)10 (3)18 (5)18 (5)XPDXPD32/M32/MXP29BEXP29BE66
1 (0)1 (0)2 (0)2 (0)XPCXPC52/F*52/F*XP86BEXP86BE22
3 (0)**3 (0)**5 (0)**5 (0)**XPCXPC49/F*49/F*XP295BEXP295BE11
Number of Number of melanomas melanomas with PTEN with PTEN mutationsmutations
Number of Number of MelanomasMelanomas
CGCGAge/ SexAge/ SexPatientPatient
*Members of same kindred **Invasive melanoma
56%56% of the melanomas had PTEN mutationsof the melanomas had PTEN mutations
High frequency of XP melanomas with multiple High frequency of XP melanomas with multiple base substitution mutations in the PTEN genebase substitution mutations in the PTEN gene
66
33 33 (100%)(100%)
AllAll
00
1 1 (3%)(3%)
44
5 5 (15%)(15%)8 8 (24%)(24%)19 (58%)19 (58%)Number of Number of melanomasmelanomas
2 (33%)2 (33%)1 (17%)1 (17%)3 (50%)3 (50%)MMMM
332211Number of Number of mutations mutations
per per melanomamelanoma
42% of the melanomas with mutations42% of the melanomas with mutationshad multiple had multiple mutations, amutations, a feature of UV feature of UV mutagenesismutagenesis
Significant frequency of UV type base Significant frequency of UV type base substitution mutations in the PTEN gene in substitution mutations in the PTEN gene in
XP melanomasXP melanomas
1387 bp 1387 bp (100%)(100%)
641 bp 641 bp (46%)(46%)
746 bp 746 bp (54%)(54%)
PTEN PTEN sequence sequence (expected (expected frequency*)frequency*)
54 54 (100%)(100%)
6 6 (11%)(11%)
4848****(89%)(89%)
Number of Number of mutations mutations (observed (observed frequency)frequency)
TotalTotalNot Not UV typeUV type
UV type UV type
**P=0.0001*frequency of dipyrimidines
Significant increase in UV type mutations compared to expected frequency
Significant frequency of UV type mutations in Significant frequency of UV type mutations in PTEN gene in XP melanomasPTEN gene in XP melanomas
compared to compared to internal cancers in normal patientsinternal cancers in normal patients
2020(100%)(100%)
3(15%)3(15%)17 17 (85%)(85%)Melanoma of Melanoma of non XP*non XP*
179179 (100%)(100%)9393 (52%)(52%)8686** (48%)** (48%)Cancer of Cancer of endometrium*endometrium*
272 (100%)272 (100%)101 (37%)101 (37%)171171** (63%)** (63%)Cancer of Cancer of central nervous central nervous
system*system*
1387 bp 1387 bp (100%)(100%)
641 bp (46%)641 bp (46%)746 bp (54%)746 bp (54%)PTEN sequence PTEN sequence (expected (expected frequency)frequency)
54 (100%)54 (100%)6 (11%)6 (11%)48 48 (89%)(89%)Melanoma of Melanoma of XPXP
TotalTotalNot UV typeNot UV typeUV type UV type Number of Number of mutations mutations ((obsobs freq)freq)
**P=0.0001 vs Melanoma*from Sanger database
Significant increase in UV type mutations compared to internal cancers
PTEN mutations frequently change PTEN mutations frequently change amino acids in XP melanomasamino acids in XP melanomas
33 (61%)33 (61%)2 (4%)2 (4%)31 (57%)31 (57%)MissenseMissense
TotalTotal
No AA No AA changechange
AA changeAA change
37 (69%)37 (69%)2 (4%)2 (4%)35 (65%)35 (65%)TotalTotal
17 (31%)17 (31%)4 (7%)4 (7%)13 (24%)13 (24%)
54 (100%)54 (100%)6 (11%)6 (11%)48 (89%)48 (89%)
2 (4%)2 (4%)002 (4%)2 (4%)SpliceSplice
2 (4%)2 (4%)002 (4%)2 (4%)Nonsense Nonsense (stop)(stop)
TotalTotalNot Not UV typeUV type
UV typeUV type
37 (69%) of the 54 mutations change the amino acid
The MitogenThe Mitogen--Activated Protein (MAP) Kinase and Activated Protein (MAP) Kinase and Phosphatidylinositol 3' Kinase (PI3K) PathwaysPhosphatidylinositol 3' Kinase (PI3K) Pathways
Curtin et al NEJM 353:2135 (2005)
Functional Assay of Phosphorylation of AKT by PTEN Mutants
NCI-H1155
(PTEN-null cells)
HA-AKT
mutant-PTENOR
w.t-PTEN
PTEN HA-AKT
HA-AKT
PTEN
Western blotmoc
k
w.t
.
muta
nt
Ser 473 p-AKT
AKT
PTEN
24hrs
HA-IP
Cell lysis
Western blot of PTEN, P-AKT and AKT
PTEN
AKT
Ser 473 p-AKT
1 2 3 64 5 7 8 9 10
moc
kw
.t.
w.t
.
F154L
L325F
P95S
Q110R
moc
k
D301N
S362L
PTEN mutants
Functional assay of phosphorylation of AKT by selected PTEN mutants
X X X
3/6 (50%) of the mutations reduce PTEN function
404
PTEN cDNA
1
UV-TYPE MUTATIONS IMPAIR PTEN FUNCTION
Exon 5 Exon 7 Exon 8
CCG
TA216
222283
C T
T
T
TTTTT TC
A
T*
TT
CC CT A
T
T AA
G*
G
G
G
G
292309 329
311 342285
447401
C
C
C
707688668
462
A AAA801735
728
GT
750
T
T T
T
T
G
G G
903872815C
973946
936
C C
CT
A996
G T C
UV type mutation
Exon 4 Exon 9
1212C
1084
T
1085
T TT TTTA
A
G26
164
C
7367
CC
7148 901
209
AA
G
G
T
CA
C
G GGA
A
AAA
703131 247
744
AA
G
GC
2501162
1207
Not UV type mutation
PTEN protein
S*
SS
UV type mutation
1
25 179 190 347123
134
R
E
D
Q K
104 114110 154149 223
M Q
T
F
H RL* F
G
X
D
G
F
S
Q
H N
Q
E L
G
236230 245 272267243
291 325 362S
LL
R
A
V
K
7023555
E44
3019
DG HL
NNN
DD
D40324
F
S
P95
L RC84
83
G IK
E
388
V
P R GK
Not UV type mutation
X
X X
XX X
Protein phosphatase Calcium / lipid binding region
ConclusionsConclusionsUltraviolet radiation damage plays a direct role in Ultraviolet radiation damage plays a direct role in induction of PTEN mutations in melanomas in XP induction of PTEN mutations in melanomas in XP patientspatients
Frequent PTEN mutations in Frequent PTEN mutations in in situin situ melanomas melanomas indicate that it is an early event in melanoma indicate that it is an early event in melanoma inductioninduction
The variety of mutations indicate that The variety of mutations indicate that each each melanoma arose independentlymelanoma arose independently
These mutations frequently altered the PTEN amino These mutations frequently altered the PTEN amino acid sequenceacid sequence
Some UVSome UV--type mutations impaired PTEN functiontype mutations impaired PTEN function
Thank you!Thank you!
Wang et al PNAS 106: 6269 (2009)
These data provide solid mechanistic support These data provide solid mechanistic support for UV protection for prevention of melanoma.for UV protection for prevention of melanoma.