linking the pigmentary response to dna repair in melanocytes · 2011. 6. 29. · •scf/c‐kit...
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John D’Orazio, M.D.,Ph.D.
Department of PediatricsThe Markey Cancer Center
The Graduate Center for ToxicologyDepartment of Molecular Pharmacology
University of Kentucky College of Medicine
23 June 2011NIH DNA Repair
Videoconference Series
Linking the pigmentary response to DNA repair in melanocytes
There are no relevant conflicts of interest or financial disclosures.
Skin Cancer• >50% of all new cancers (U.S.)
• Keratinocyte malignancies
– most common (> 1,000,000 per yr in US)
• basal cell carcinoma (BCC)
• squamous cell carcinoma (SCC)
• Malignant melanoma
– most deadly
• Account for ~¾ of the 10,000 annual US deaths from skin cancer
– Increasing prevalence across age groups (even pediatrics)
• Highest incidence in males > 50 yrs
• #1 cause of cancer deaths among women ages 20-25 yrs.
American Cancer Society’s Facts and Figures
Melanoma• Cancer of melanocytes.
• Incidence rising for several decades.
– ~ 60,000 new cases a year
– ~ 8,500 deaths
• Top-ten cancer, both sexes
• Essentially insensitive to chemotherapy or radiation therapy.
• New therapeutic advances
SEER data, National Cancer Institute, and data from the American Cancer Institute
Recent advances in Melanoma therapy
• ~50% of melanomas carry an activating mutation of the BRAF
– serine–threonine protein kinase in the MAP kinase cascade
• Treatment of BRAF-mutant melanoma with PLX4032 (BRAF inhibitor) resulted in tumor regression in the majority of patients
– Overall survival 10.1 months (vs. 6.4 months)
• BUT… the majority of patients on early trials of these drugs develop secondary resistance and subsequent disease progression
• Improved survival (by ~4 months) in patients with advanced melanomas receiving ipilimumab+ gp100vaccine
• Ipilimumab mAb that binds to CTLA-4 on cytotoxic T cells to sustain anti-cancer immunity.
• gp100 peptide vaccine + IL-2 led to better clinical responses and overall survival in metastatic melanoma patients
• 16% vs. 6% overall clinical response
• Progression-free survival (3.9 vs.1.6 mo)
• Better overall survival (17.8 vs. 11.1 mo)
Immune modulationBRAF inhibition
Flaherty et. al., NEJM, 2010
British Journal of Cancer, 2011
Hodi et. al., NEJM, 2010
Schwartzentruber et. al., NEJM, 2011
Melanoma Risk Factors• Fair skin, freckling and light hair
– Inability to tan effectively
• Defective Nucleotide Excision Repair
• Xeroderma pigmentosum (XP)
• Moles
– Dysplastic nevus syndrome
• UV radiation
– Blistering sunburns
• Immune suppression
• Family or personal history
• Age
• Inherited cancer predisposition
– Familial melanoma (p16)
American Cancer Society, 2006 http://www.dermcoll.asn.au/public/images/mole3b.jpg
Highly pigmented
Skin pigmentation phenotype – a major determinant of melanoma risk
Melanocyte• Neural crest derived cell
• Exclusive pigment producing cell in the skin
• Mainly in the skin– Leptomeninges (medulla oblongata)
– Eye (retina)
– Inner ear (ionic gradient)
• Precursor cell for malignant melanoma
Primary melanocytes in culture
The Skin
Keratinocytes
EP
IDE
RM
IS
Melanocyte
DE
RM
IS
Melanocyte
Stratum corneum
Stratum granulosum
Stratum spinosum
Stratum basale
Stratum lucidum
“Epidermal Melanin Unit”
Skin Phototype
Constitutive skin color
MED (mJ/cm2
UVB)
UVB Sensitivity Tanning/Burning History
I Ivory/pale white 15-30 ++++ Burns easily and strongly, never tans
II Very white 25-40 +++/++++ Burns easily, tans minimally with difficulty
III White 30-50 +++ Burns moderately, tans somewhat
IV Light brown, beige, olive 40-60 ++ Burns minimally, tans
moderately
V Moderate brown 60-90 + Rarely burns, tans well
VI Dark brown/black 90-150 +/- Never burns, tans profusely
Fitzpatrick TB: Soleil et peau. J Med Esthet 1975;2:33034
Skin pigmentation:
Fitzpatrick scale
LeucoDOPAchrome
HO
HO NH
COOH
DOPAchrome
O-O N
HCOOH+
DHI
HO
HO NH
Indole-5,6-quinone
O
O NH
DHICA
HO
HO NH
COOH NH
COOH
O
O
Indole-5,6-quinoneCarboxylic acid
HO
HO
COOHNH2
DOPAHO
COOH
NH2
Tyrosine DOPAquinone
O
O
COOHNH2
CysteinylDOPA
HO
HO
COOHNH2
S
H2N COOH
1,4-benzothiazinyl-alanine
HO COOHNH2
S
Tyrosinase Tyrosinase
Eumelanin Pheomelanin
CYSTEINE
(brown/black melanin)- very effective UV blocking pigment
(red/blond melanin)- less able to block UV
Melanin biosynthesis
• Skin cancer incidence correlates with pigmentation• 20-30% of all neoplasms in Caucasians.
• 2-4% of all neoplasms in persons of East Asian inheritance
• 1-2% of all neoplasms in persons of African or Asian-Indian descent
• The dose of UVR required to produce sunburn is up to 30 times greater in people of color than in Caucasians.
Eumelanin is a Terrific Natural Sunscreen!
Gloster and Neal, J. Amer. Acad. Dermatol., 2006.
% of UV Radiation that gets through the epidermis
UV-B
Darkly Pigmented Skin Lightly Pigmented Skin
17.5% 55%
7.4% 24%
UV-A
05
101520253035
MenWomen
US Melanoma Incidence by Race, 2004-08In
cide
nce
rate
(per
105
/yr)
Skin complexion is multi-genic • MATP
– Membrane-associated transport protein
– Sugar transporter in melanosome membrane
• MC1R– Melanocortin 1 receptor, Gs-coupled protein
– Binds MSH on surface of melanocytes
• SLC24A5– solute carrier family 24, member 5
– cation exchanger in melanosomes, cysteine transport
• ASIP– Agouti signaling protein
– MC1R antagonist
• P– Pink-eyed dilution protein
• Tyr– Tyrosinase
– Oculocutaneous albinism type 1
• DCT– Dopachrome tautomerase
– Oculocutaneous albinism type 4
Correlates with fair skin, red hair, freckling, inability to tan and melanoma risk in humans• P. Valverde et al., Hum Mol Genet 5, 1663 (1996).• J. L. Rees et al., Ann N Y Acad Sci 885, 134 (1999).• Z. Abdel-Malek et al., Ann N Y Acad Sci 885, 117 (1999).• N. F. Box et al., Am J Hum Genet 69, 765 (2001).• A. L. Kadekaro et al., Ann N Y Acad Sci 994, 359 (2003).• R. A. Sturm et al., Pigment Cell Res 16, 266 ( 2003).• N. K. Hayward, Oncogene 22, 3053 (2003) .• L. Pastorino et al., Hum Mutat 24, 103 (2004).• F. Rouzaud et al., Mutat Res 571, 133 (2005).• V. Chaudru et al., Cancer Epid Bio Prev 14, 2384 (2005).• A. J. Stratigos et al., J Invest Dermatol 126, 1842 (2006).• J. E. Hauser et al., Pigment Cell Res 19, 303 (2006).• M. T. Landi et al., Science, (2006).• S. Raimondi et al., Int J Cancer 122, 2753 (2008).• M. C. Fargnoli et al., J Invest Dermatol 128, 2485 (2008).• Z. A. Abdel-Malek et al., Photochem Photobiol 84, 501 (2008).• F. Demenais et al., J Natl Cancer Inst 102, 1568 (2010).• C. de Torre et al., Melanoma Res 20, 342 (2010).
UV
Keratinocytes
Melanocyte
p53 activation
POMC transcription
Mc1r
AdenylateCyclase
ATPcAMP CREB
PKA
Pigment Enzymes
Eumelanintransfer to
keratinocytes
Mechanism of Adaptive Pigmentation
cellular damage
α- MSH
Eumelaninsynthesis
D’Orazio, et. al., Nature, 2006.Cui, et. al., Cell, 2007
cellulardamage
responses
β-endorphin
ACTH
Mitf
MC1R defects correlate with:
MC1R mutations– Arg151Cys– Arg160Trp– Asp294His
• Fair-skin
• Inability to tan
• Melanoma risk
VI V IV III II I
Skin phototype (complexion); Fitzpatrick scale
Melanoma Risk
MC1R Function
Good cAMP signaling Loss of function
Eumelanin Pheomelaninmelanin found in skin
Never BurnsTans easily
Can’t tanUV sensitive
“Red Hair Color” phenotype(MC1R-defective)
ANIMAL MODEL
http://biology.plosjournals.org/archive/1545-7885/3/8/figure/10.1371_journal.pbio.0030303.g001-M.jpg
Defective MSH signaling causes fair skin
Fair skin
Mc1re/eMSH
Pheomelanin
Muted cAMP response
Dark skin
Mc1rE/EMSH
Eumelanin
Adenylatecyclase
cAMP
Robbins, et. al., Cell, 1993
Human Skin
Mouse Skin
Melanocytes in hair follicles
andbasal epidermis
Melanocytes only in
hair follicles
Stem Cell Factor Signaling
• SCF/c‐kit pathway important to melanocyte migration and survival.
• Human basal keratinocytes express c‐kit constitutively
Melanocyte
receptortyrosinekinase
c-kit
SCF
aka “steel factor”
Yoshida H, et, al. J Investig Dermatol Symp Proc. 2001 Nov;6(1):1-5.
AKT
PI3K
Apoptosis survival
pathways
K14 - Stem Cell Factor Transgene
+ - + - + -
C57BL/6
Kunisada, T. et. al., J. Exp. Med. 1998.187:1565. + -Epidermal melanocytes in the SCF background
C57BL/6 Tyrc2j/c2j
Mc1rE/EC57BL/6 Tyr+/+
Mc1re/eC57BL/6 Tyr+/+
Mc1rE/E
Wild typeExtensionAlbino
Eumelanin
0
500
1000
1500
2000
2500
3000
3500
Pigment Variant
ng/m
g dr
y w
eigh
t +/-
SD
C2J Albino Extension Wild Type
Pheomelanin
0
50
100
150
200
250
300
Pigment Variant
C2J Albino Extension Wild Type
ng/m
g dr
y w
eigh
t +/-
SD
K14-SCF+
D’Orazio, et. al., Nature, 2006
Murine model of epidermal melanocytes
Wild Type
(Mc1rE/E)
0 mJ/cm2 50 mJ/cm2 100 mJ/cm2 200 mJ/cm2
Extension
(Mc1re/e)
Adaptive melanization is Mc1r-dependent
Daily treatments, 5d/wk, one month total.
D’Orazio, et. al., Nature, 2006
Forskolin• Cell-permeable diterpenoid
• Activator of adenyl cyclase
– ↑ cytoplasmic cAMP
http://medicine.osu.edu/news/images/high_quality/Coleus_forskohlii_p1.jpg
Coleus forskohlii plant, (Plectranthus barbatus)
Skin
Col
or (L
*) +
/-SD
0 7 14 21
Experimental Day
35
40
45
50
55
60
65 UntreatedVehicle ControlForskolinUVB
(Dar
ker -
---L
ight
er)
Forsk Ctrl Forsk Ctrl Forsk Ctrl
Topical treatment
250
500
750
1000
1250
0 NT VC UVBForsk
Condition
(ng/
mg)
+/-
SD
Eumelanin
Forskolin but not UV rescues eumelanin production in mice with defective MSH signaling
D’Orazio, et. al., Nature, 2006
Control Forskolin
(Dar
ker -
----
---L
ight
er)
25
30
35
40
45
50
55
60
1 2 3 4 5 6 7 8 9 10 11 12Week of study
Skin
Col
or (L
*) +
/-SD
Topical treatments started
Topical treatments stopped
0 μmoles10 μmoles20 μmoles40 μmoles80 μmoles
0 μmoles 2 μmoles 10 μmoles 40 μmoles
D’Orazio, et. al., Nature, 2006
B.
Mc1re/e
vehicleMc1rE/E
vehicleAlbino
vehicleMc1re/e
forskolinAlbino
forskolin
Sunb
urn
cells
/cm
+/-
SD
0
20
40
60
80p=0.005
A. Mc1re/e
vehicle
*
Mel
anin
St
aini
ngH
E St
aini
ng
Mc1re/e
forskolin
*
C.Mc1re/e
VehicleMc1re/e
ForskolinMc1rE/E
VehicleAlbinoVehicle
50 m
J/cm
220
mJ/
cm2
0 m
J/cm
2
DAP
ITˇ
TD
API
TˇT
DAP
ITˇ
T
Forskolin-induced melanin protects against UV damage
D’Orazio, et. al., Nature, 2006
*9 mice in each group
02Multiple tumors
03Other tumors
68Squamous cell carcinoma
Mc1re/e forskolinMc1re/e vehicle
Tumorigenesis in UV-treated animals
A.
B.
611Total tumors
Time from start of irradiation (weeks)
0 10 3020 40 50 600
20
40
60
80
100
Cum
ulat
ive
tum
or-fr
ee s
urvi
val (
%)
UVB
Vehicle
Forskolin
p<0.001
Protection against carcinogenesis
XPC-/-
D’Orazio, et. al., Nature, 2006
Conclusions• The melanocortin 1 receptor cAMP pathway mediates
production of eumelanin by melanocytes.
– Mc1r defects lead to a fair-skinned, UV-sensitive phenotype.
• Eumelanin production is rescued in an animal model of the fair-skinned human by forskolin.– Pigmentary machinery remains intact in Mc1r-deficient state
• Forskolin-induced epidermal eumelanin is highly protective against acute and chronic UV-mediated injury.– Novel UV-protective strategy
Mc1r and Melanoma• Mc1r-defective individuals are at high risk of
melanoma.– tend to be fair-skinned and UV-sensitive.
– Having less eumelanin in the skin certainly promotes UV penetration into the basal layer of the epidermis
• Rate of UV-induced mutagenesis can be affected by rate of production and rate of clearance.
• Could Mc1r signaling also affect melanocyte DNA repair mechanisms?
UV
Keratinocytes
Melanocyte
p53 activation
POMC transcription
Mc1r
AdenylateCyclase
ATPcAMP CREB
PKA
Pigment Enzymes
Eumelanintransfer to
keratinocytes
Adaptive Pigmentation and DNA Repair in the Skin
cellular damage
α- MSH
Eumelaninsynthesis
D’Orazio, et. al., Nature, 2006.Cui, et. al., Cell, 2007
cellulardamage
responses
β-endorphin
ACTH
DNA repair
Mitf
• Melanoma incidence is on the rise.
• It may be possible to alter melanoma risk
– Novel UV- and cancer-protective strategy.
• safe, healthy tanning
• better recovery from UV damage!
Fairskin
Inabilityto tan
Blunted DNA repair
UV MC1Rmutations
cAMP modulators in practice
• Methylxanthines
– Phosphodiesterase inhibition
– Theophylline
• Asthma; relaxes smooth muscles in bronchioles
– Caffeine
• Stimulant; sed for apnea of prematurity
– Theobromine
• Caffeine-like agent in chocolate
• Other phosphodiesterase inhibitors
– Amrinone, Milrinone
• Heart failure; positive inotropic effect on heart, vasodilator
– Rolipram
• Psychiatric uses; anti-depressant, memory aid, increased wakefulness
Khaled, et. al., 2010, Genes & Development
jdorazio@uky.edu; 859-323-6238jdorazio@uky.edu; 859-323-6238
Contributors, Collaborators & Acknowledgements
Dana-Farber Cancer Inst.David FisherEmi NishimuraTetsu NobuhisaRutao CuiVivien IgrasIan Davis
Funding Sources
NIH (NCI)Univ. KY, Markey Cancer CtrAmerican Cancer SocietyWendy Will Case FoundationJennifer and David Dickens Melanoma Foundation
Gifu Univ. Grad. Sch. Med.
Takahiro Kunisada
Fujita Univ. Sch. Health Sci.Kazu WakamatsuShosuke ItoUniversity of Kentucky
Markey Cancer Ctr.Dep’t PediatricsGrad. Ctr. ToxicologyDep’t Mol Pharmacology
Mark EversJeff MoscowMary VoreDaret St. ClairMark LovellBrett Spear
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