Linking the pigmentary response to DNA repair in melanocytes
John D’Orazio, M.D.,Ph.D.
Department of Pediatrics The Markey Cancer Center The Graduate Center for Toxicology Department of Molecular Pharmacology University of Kentucky College of Medicine
23 June 2011 NIH DNA Repair Videoconference Series 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
BRAF inhibition Immune modulation
• ~50% of melanomas carry an activating • Improved survival (by ~4 months) in patients mutation of the BRAF with advanced melanomas receiving ipilimumab + gp100vaccine – serine–threonine protein kinase in the MAP kinase cascade • Ipilimumab mAb that binds to CTLA-4 on cytotoxic T cells to sustain anti-cancer • Treatment of BRAF-mutant melanoma with immunity. PLX4032 (BRAF inhibitor) resulted in tumor regression in the majority of patients • gp100 peptide vaccine + IL-2 led to better – Overall survival 10.1 months (vs. 6.4 months) 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)
Flaherty et. al., NEJM, 2010 • BUT… the majority of patients on early trials of British Journal of Cancer, 2011 these drugs develop secondary resistance and Hodi et. al., NEJM, 2010 subsequent disease progression 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 Skin pigmentation phenotype – a major determinant of melanoma risk
Highly pigmented 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 “Epidermal Melanin Unit” Keratinocytes
Stratum corneum
Stratum lucidum Stratum granulosum
Stratum spinosum EPIDERMIS
Stratum basale
DERMIS Melanocyte
Melanocyte Skin pigmentation:
Fitzpatrick scale
MED Skin Constitutive skin UVB (mJ/cm2 Tanning/Burning History Phototype color Sensitivity UVB)
Burns easily and strongly, I Ivory/pale white 15-30 ++++ never tans
Burns easily, tans minimally II Very white 25-40 +++/++++ with difficulty
Burns moderately, tans III White 30-50 +++ somewhat Light brown, beige, Burns minimally, tans IV 40-60 ++ olive 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 Melanin biosynthesis
COOH HO COOH O COOH HO COOH NH NH NH 2 2 NH2 HO 2 HO O HO C S TyrosineTyrosinase DOPA DOPAquinone Y Tyrosinase H N S 2 COOH T E CysteinylDOPA HO O HO I - HO N O N+ COOH HO N COOH N E H H H HO COOH DHI DOPAchrome LeucoDOPAchrome NH2 S 1,4-benzothiazinyl- O HO O alanine
O N HO N COOH O N COOH H H H Indole-5,6-quinone DHICA Indole-5,6-quinone Carboxylic acid Eumelanin Pheomelanin (brown/black melanin) (red/blond melanin) - very effective UV blocking pigment - less able to block UV Eumelanin is a Terrific Natural Sunscreen! • 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.
% of UV Radiation that gets through the epidermis
Darkly Pigmented Skin Lightly Pigmented Skin
UV-A 17.5% 55%
UV-B 7.4% 24%
Gloster and Neal, J. Amer. Acad. Dermatol., 2006. US Melanoma Incidence by Race, 2004-08 /yr)
5 35 30 Men 25 Women 20 15 10 5
Incidence rate (per 10 Incidence rate 0 Skin complexion is multi-genic
•MATP Correlates with fair skin, red hair, – Membrane-associated transport protein freckling, inability to tan and – Sugar transporter in melanosome membrane melanoma risk in humans •MC1R •P. Valverdeet al., Hum Mol Genet 5, 1663 (1996). – Melanocortin 1 receptor, Gs-coupled protein • J. L. Rees et al., Ann N Y Acad Sci 885, 134 (1999). – Binds MSH on surface of melanocytes • Z. Abdel-Malek et al., Ann N Y Acad Sci 885, 117 (1999). • SLC24A5 •N. F. Boxet al., Am J Hum Genet 69, 765 (2001). • A. L. Kadekaro et al., Ann N Y Acad Sci 994, 359 (2003). – solute carrier family 24, member 5 • R. A. Sturm et al., Pigment Cell Res 16, 266 ( 2003). – cation exchanger in melanosomes, cysteine transport •N. K. Hayward, Oncogene 22, 3053 (2003) . •L. Pastorinoet al., Hum Mutat 24, 103 (2004). • ASIP • F. Rouzaud et al., Mutat Res 571, 133 (2005). • V. Chaudru et al., Cancer Epid Bio Prev 14, 2384 (2005). – Agouti signaling protein • A. J. Stratigos et al., J Invest Dermatol 126, 1842 (2006). – MC1R antagonist • J. E. Hauser et al., Pigment Cell Res 19, 303 (2006). •P • M. T. Landi et al., Science, (2006). • S. Raimondi et al., Int J Cancer 122, 2753 (2008). – Pink-eyed dilution protein • M. C. Fargnoli et al., J Invest Dermatol 128, 2485 (2008). • Z. A. Abdel-Malek et al., Photochem Photobiol 84, 501 (2008). •Tyr • F. Demenais et al., J Natl Cancer Inst 102, 1568 (2010). – Tyrosinase • C. de Torre et al., Melanoma Res 20, 342 (2010). – Oculocutaneous albinism type 1 • DCT – Dopachrome tautomerase – Oculocutaneous albinism type 4 Mechanism of Adaptive Pigmentation UV
cellular p53 POMC cellular damage activation transcription damage responses Keratinocytes
β-endorphin
α-MSH ACTH
Eumelanin transfer to keratinocytes Mc1r
PKA Adenylate Cyclase
cAMP CREB ATP
Mitf
Eumelanin synthesis Pigment Enzymes
Melanocyte D’Orazio, et. al., Nature, 2006. Cui, et. al., Cell, 2007 • Fair-skin MC1R defects correlate with: • Inability to tan • Melanoma risk
MC1R Function
Good cAMP signaling Loss of function
MC1R mutations – Arg151Cys Eumelaninmelanin found in skin Pheomelanin – Arg160Trp – Asp294His
VI V IV III II I Skin phototype (complexion); Fitzpatrick scale
Never Burns Can’t tan Tans easily UV sensitive
Melanoma Risk “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
Dark skin
MSH Mc1rE/E Adenylate cyclase cAMP Fair skin Eumelanin MSH Mc1re/e
Muted cAMP response
Pheomelanin
Robbins, et. al., Cell, 1993 Melanocytes in hair follicles and basal epidermis
Human Skin
Melanocytes only in hair follicles Mouse Skin Stem Cell Factor Signaling
aka “steel factor” SCF •SCF/c‐kit pathway important to receptor c-kit tyrosine melanocyte migration kinase PI3K and survival.
AKT •Human basal Apoptosis survival keratinocytes express c‐ pathways kit constitutively Melanocyte
Yoshida H, et, al. J Investig Dermatol Symp Proc. 2001 Nov;6(1):1-5. K14 - Stem Cell Factor Transgene C57BL/6
+ - + - + -
Epidermal melanocytes in the SCF background
+ - Kunisada, T. et. al., J. Exp. Med. 1998.187:1565. Murine model of epidermal melanocytes
C57BL/6 Tyrc2j/c2j C57BL/6 Tyr+/+ C57BL/6 Tyr+/+ Mc1rE/E Mc1re/e Mc1rE/E
K14-SCF+
Albino Extension Wild type
Eumelanin Pheomelanin 3500 300
3000 250
2500 200 2000 150 1500 100 1000 50 500 ng/mg dry weight +/- weight ng/mg dry SD ng/mg dry weight +/- weight ng/mg dry SD 0 0 C2J Albino Extension Wild Type C2J Albino Extension Wild Type Pigment Variant Pigment Variant
D’Orazio, et. al., Nature, 2006 Adaptive melanization is Mc1r-dependent
0 mJ/cm2 50 mJ/cm2 100 mJ/cm2 200 mJ/cm2
Wild Type (Mc1rE/E)
Extension (Mc1re/e)
Daily treatments, 5d/wk, one month total.
D’Orazio, et. al., Nature, 2006 Forskolin • Cell-permeable diterpenoid • Activator of adenyl cyclase – ↑ cytoplasmic cAMP
Coleus forskohlii plant, (Plectranthus barbatus)
http://medicine.osu.edu/news/images/high_quality/Coleus_forskohlii_p1.jpg Skin Color (L*) +/- SD Forskolin but not UV rescues eumelanin production in mice production Forskolin butnotUVrescueseumelanin (Darker ----Lighter) 35 40 45 50 55 60 65 UVB Forskolin Vehicle Control Untreated 071421 Experimental Day with defectiveMSHsignaling
(ng/mg) +/- SD 1000 1250 250 500 750 0 TV UVB VC NT os tlFrkCr os Ctrl Forsk Ctrl Forsk Ctrl Forsk Eumelanin Condition Forsk Topical treatment D’Orazio, et.al., Nature, 2006 60 Topical treatments Topical treatments started stopped 55
50 0 μmoles 2 μmoles 10 μmoles 40 μmoles
45
40 0 μmoles 35 10 μmoles
Skin Color (L*) +/- SD 20 μmoles
(Darker ------Lighter) (Darker 30 40 μmoles 80 μmoles 25 123456789101112 Week of study
Control Forskolin D’Orazio, et. al., Nature, 2006 Forskolin-induced melanin protects against UV damage
A. Mc1re/e Mc1re/e vehicle forskolin C. Mc1re/e Mc1re/e Mc1rE/E Albino * Vehicle Forskolin Vehicle Vehicle
* T ˇ 2 Melanin Staining T 50 mJ/cm DAPI HE Staining T ˇ 2 T B.
80 20 mJ/cm DAPI p=0.005
60 T ˇ 2 T
40 0 mJ/cm
20 DAPI Sunburn cells/cm +/-Sunburn cells/cm SD 0 Mc1re/e Mc1re/e Mc1rE/E Albino Albino vehicle forskolin vehicle vehicle forskolin D’Orazio, et. al., Nature, 2006 Protection against carcinogenesis
A. 100
Forskolin XPC-/- 80
60 Vehicle
40
20 UVB p<0.001 0 Cumulative tumor-free survival (%) 0 10 20 30 40 50 60 Time from start of irradiation (weeks)
B. Tumorigenesis in UV-treated animals Mc1re/e vehicle Mc1re/e forskolin Squamous cell carcinoma 8 6 Other tumors 3 0 Multiple tumors 2 0
Total tumors 11 6 *9 mice in each group 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? Adaptive Pigmentation and DNA Repair in the Skin UV
cellular p53 POMC cellular damage activation transcription damage responses Keratinocytes
β-endorphin
α-MSH ACTH
Eumelanin transfer to keratinocytes Mc1r
PKA Adenylate Cyclase
cAMP CREB ATP
Mitf
DNA repair
Eumelanin synthesis Pigment Enzymes
Melanocyte
D’Orazio, et. al., Nature, 2006. Cui, et. al., Cell, 2007 • Melanoma incidence is on the rise.
MC1R UV mutations
Fair Inability Blunted skin to tan DNA repair
• It may be possible to alter melanoma risk
– Novel UV- and cancer-protective strategy.
• safe, healthy tanning
• better recovery from UV damage! cAMP modulators in practice
• Methylxanthines • Other phosphodiesterase inhibitors – Phosphodiesterase inhibition – Amrinone, Milrinone – Theophylline • Heart failure; positive • Asthma; relaxes smooth inotropic effect on heart, muscles in bronchioles vasodilator – Caffeine – Rolipram • Stimulant; sed for apnea of • Psychiatric uses; anti- prematurity depressant, memory aid, – Theobromine increased wakefulness • Caffeine-like agent in chocolate
Khaled, et. al., 2010, Genes & Development [email protected]; 859-323-6238 Contributors, Collaborators & Acknowledgements
Funding Sources NIH (NCI) Univ. KY, Markey Cancer Ctr American Cancer Society Wendy Will Case Foundation Jennifer and David Dickens Melanoma Foundation
Dana-Farber Cancer Inst. David Fisher Emi Nishimura Tetsu Nobuhisa Rutao Cui Vivien Igras Ian Davis
Fujita Univ. Sch. Health Sci. Kazu Wakamatsu University of Kentucky Mark Evers Shosuke Ito Jeff Moscow Markey Cancer Ctr. Mary Vore Dep’t Pediatrics Gifu Univ. Grad. Sch. Med. Daret St. Clair Grad. Ctr. Toxicology Mark Lovell Takahiro Kunisada Dep’t Mol Pharmacology Brett Spear