linking the pigmentary response to dna repair in melanocytes · 2011. 6. 29. · •scf/c‐kit...

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.


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


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


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


*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-/-


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

[email protected]; [email protected]; 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

linking the pigmentary response to dna repair in melanocytes · 2011. 6. 29. · •scf/c‐kit...

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