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611 Alpha-1 antitrypsin suppresses melanoma growth by upregulating melanocyte differentiation antigens and enhancing T cell cytotoxicity
ABSTRACTS | Pigmentation & Melanoma 606
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The pH sensor soluble adenylyl cyclase regulates melanogenesis D Zhou1, R Halaban2, A Bacchiocchi2, C Nardin1 and JH Zippin1 1 Weill Cornell Medical College, New York, NY and 2 Yale University, New Haven, CT Melanin pigments are synthesized by melanocytes in highly specialized organelles called melanosomes. Two distinct types of melanin, eumelanin and pheomelanin, are produced by eu- and pheomelanosomes, respectively. While the enzymes responsible for melanin synthesis and the general mechanisms for activating melanosomes are well studied, how melanosomes are directed to become eu- vs pheomelanosomes, or how cells regulate melanosome creation vs destruction via autophagolysosomes is poorly understood. Cyclic AMP (cAMP) is a key signaling molecule in melanin synthesis and the role of the transmembrane adenylyl cyclases (tmACs) in melanogenesis is well established. It is clear that melanosomal pH is an important regulator of melanogenesis, implicated in tyrosinase activity, eumelanin/pheomelanin production ratio, as well as melanosome maturation, but how melanocytes regulate melanosome pH remains poorly understood. Soluble adenylyl cyclase (sAC), a non-canonical source of cAMP, is not responsive to G proteins (e.g. MC1R) but is a pH sensor. Our group recently demonstrated that sAC is essential for normal lysosomal pH maintenance, and since melanosomes are lysosome-related organelles, we asked if sAC affects melanogenesis. Even though cAMP normally induces melanin synthesis, sAC KO mouse melanocytes paradoxically had increased melanin levels, and RNA profiling revealed increased expression of pigmentation genes. Consistent with elevated melanin content, EM imaging revealed that sAC KO melanocytes have significantly more eu- and pheomelanosomes and a drastic increase in melanosome laden autophagolysosomes suggesting a defect in both the synthesis and degradation of melanosomes in sAC null cells. These results were confirmed in human melanocytes, and interestingly, sAC inhibition led to distinct effects depending on the skin type of the donor: in Caucasian melanocytes pigmentation increased but in African-American melanocytes it was reduced. These data reveal a novel cAMP signaling cascade important for melanogenesis.
Measurement of skin pigmentation using a chromameter in a 3-dimensional epidermal model containing functional melanocytes MA Bachelor, B Breyfogle, A Armento and M Klausner MatTek Corporation, Ashland, MA Various cosmetic or skin care pharmaceutical formulations augment skin pigmentation either for the intended purpose of skin lightening or as a side effect induced by medication. A convenient way to screen such effects utilizes the MelanoDerm tissue model, a highly differentiated, three-dimensional tissue culture model of human epidermis containing normal human melanocytes and keratinocytes. Use of this model can provide valuable in vitro data as an early screening tool prior to the commencement of costly clinical trials. In this study, pigmentation was evaluated over the course of 2-3 weeks using a tristimulus chromometer to measure brightness (L*), yellowness (b*) and redness (a*) in MelanoDerm tissue produced with normal human melanocytes from Black, Asian, or Caucasian donors. In parallel to measurements taken with the chromameter, total melanin content of tissues was also quantified. Over time, cultures became increasingly pigmented with retention of normal epithelial morphology with the expected pigmentation level of the donor tissue, i.e. Black>Asian>Caucasian when cultured in media containing alpha-MSH and beta-FGF. Several over-the-counter skin lightening products were also evaluated in cultures containing normal human melanocytes from Black donors. Over the 2-3 week treatment period, control cultures became increasingly pigmented while tissues treated topically with cosmetic skin lightening agents containing tyosinase inhibitors such as kojic acid and magnesium ascorbyl phosphate remained distinctly lighter when compared to control cultures. After 14 days in culture, total melanin content was found to inversely correlate with surface reflectance (L*). The results described herein suggest that this model is useful for evaluating effects on melanogenesis, skin lightening, and other pigmentation phenomena of skin in vitro. In particular, this study highlights two distinct endpoints, total melanin content and skin color measurement that can be used to evaluate skin pigmentation in vitro.
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Targeting the MC1R by selective tetra- and tripeptide analogs of a-Melanocortin for melanoma prevention ZA Abdel-Malek, V Swope, L Koikov, R Starner and A Kadekaro Dermatology, University of Cincinnati, Cincinnati, OH Our goal is to develop a topical formulation of small peptide analogs of a-Melanocortin (a-melanocyte stimulating hormone; a-MSH) that are highly selective for the melanocortin 1 receptor (MC1R) for prevention of photodamage in human melanocytes (HM) and ultimately melanoma. The MC1R is a melanoma susceptibility gene, and its loss-of-function variants result in red hair phenotype, reduced tanning and DNA repair capacity, and increased melanoma risk. Activation of the MC1R by its physiological agonist a-MSH stimulates eumelanin synthesis and proliferation, enhances the repair of UV-induced DNA damage and inhibits apoptosis of HM. We have designed short peptide analogs of the physiological tridecapeptide a-MSH exhibiting full MC1R agonism and excellent MC1R selectivity. Modifications of the His-D-Phe-Arg-Trp, or His-D-Phe-Arg sequence of a-MSH combined with terminal modifications resulted in stable small molecules with logD favorable for skin penetration. The tetrapeptides LK 184 and LK 467, and the tripeptide LK 514 were tested on primary cultures of HM and cultured skin substitutes (CSS). Dose-response experiments revealed that LK 184 and LK 467 were 100 fold more potent, while LK 514 was slightly less (10 fold) potent than a-MSH in stimulating cAMP formation and the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, in HM. The three peptides enhanced the repair of UV-induced DNA damage in HM. Treatment of CSS with LK 184 and LK 467 at 100 nM, or LK 514 at -5 mM for 10-15 days significantly increased pigmentation in the absence of UV exposure, and increased repair of UV-induced DNA photoproducts in melanocytes and the epidermis in the CSS or intact human skin. Prolonged treatment with these peptides had no effect on melanocyte number or histology of CSS. Given that increased pigmentation and DNA repair protect against photocarcinogenesis, we are continuing with preclinical testing of these peptides to determine their safety and efficacy as topical agents for sunless tanning and prevention of photodamage and melanoma.
Estrogen and progesterone reciprocally regulate melanin production through G protein-coupled receptors C Natale1, E Duperret1, J Zhang1, R Sadeghi1, A Dahal1, K O’Brien2, J Winkler2 and TW Ridky1 1 Dermatology, University of Pennsylvania, Philadelphia, PA and 2 Chemistry, University of Pennsylvania, Philadelphia, PA The association between pregnancy and altered skin pigmentation has been documented for over two millennia, suggesting that sex hormones regulate epidermal melanocyte (MC) homeostasis. We show that physiologic estrogen (17b-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that human MCs do not express classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G proteincoupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR to modulate melanin production. Topical delivery of a specific GPER agonist modulated pigmentation in murine skin in vivo. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics.
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The presence and distribution of PD-L1 staining in melanoma JM Donigan1, M Salama2, LR Rowe2, SR Tripp2, SR Florell1 and DA Wada1 1 Dermatology, University of Utah, Salt Lake City, UT and 2 Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT Programmed death-ligand 1 (PD-L1; also known as B7-H1 and CD274) is expressed by a variety of cell types, including immune and cancer cells, and influences tumor survival by suppressing T-lymphocyte mediated destruction of tumor cells. Anti-PD-L1 agents are currently in clinical trial for the treatment of melanoma. The aim of this study was to assess the presence and distribution of PD-L1 staining in melanoma. Twenty-eight formalin-fixed, paraffin-embedded melanoma specimens were obtained from the Department of Dermatology at the University of Utah in Salt Lake City. These included 24 primary tumors and four metastases. Tissue sections were stained for PD-L1 with an anti-human PD-L1 mouse monoclonal antibody (clone 22C3; Dako, Carpinteria, CA) developed for non-small cell lung cancer treatment with pembrolizumab (MK-3475). Staining of specimens was graded on strength of staining and percent of positive cells within the tumor and peri-tumoral mononuclear cells (presumed to be antigen presenting cells [APCs]). Specimens were deemed positive if there was staining in at least one percent of the cells. Thirteen (46%) of the specimens had positive staining within a mean of 14.4% (range 1 to 50%) of the tumor cells. Strength of staining within tumors was weak to moderate (1+ to 2+). Twenty-three (82%) of the specimens had positive staining of the APCs within an average of 12.7% (range 1 to 40%) of the cells. Strength of staining of APCs was mostly weak (range 1+ to 3+). These findings are among the first to demonstrate the intensity and distribution of this anti-PD-L1 antibody in a variety of melanoma specimens. Future studies assessing the utility of PD-L1 staining in melanoma as a marker of response to anti-PD-L1 therapy are warranted.
S108 Journal of Investigative Dermatology (2016), Volume 136
Alpha-1 antitrypsin suppresses melanoma growth by upregulating melanocyte differentiation antigens and enhancing T cell cytotoxicity DR Pearson1, Y Luo1, Z Zhai1, KL Couts1, T Azam2, CA Dinarello2 and M Fujita1,3 1 Dept of Dermatology, University of Colorado Denver SOM, Aurora, CO, 2 Dept of Medicine, University of Colorado Denver SOM, Aurora, CO and 3 Denver Veterans Affairs Medical Center, Denver, CO Inflammation within the tumor microenvironment plays a crucial role in tumor development and progression. Alpha-1 antitrypsin (AAT), a serine protease inhibitor, protects normal tissues from degradation by proteases and has been shown to inhibit inflammation. In the present study, we investigated the impact of AAT on the tumorigenesis of melanoma using a C57BL/6 strain of transgenic mice expressing human AAT under a surfactant protein C promoter (hAAT-Tg). In this model, hAAT is secreted from lung tissues and detected in circulation. Large tumors (>1,500 mm3) were formed after subcutaneous injection of B16F10 melanoma cells in wild-type (WT) mice, while only small tumors (<50 mm3) were observed in hAAT-Tg mice (Day 21; P<0.001). Suppressed melanoma growth was also seen by injection of a less aggressive primary cell line, B16F0, in hAAT-Tg mice (P<0.001). Histologic analysis of tumor tissues revealed reduced mitoses, increased apoptosis, and enhanced melanin pigmentation in tumor cells in hAAT-Tg mice compared to WT mice. However, when B16F10 cells were cultured in vitro, hAAT did not show an effect on cell viability in 2D culture or colony formation capacity in 3D culture. Since immunohistochemical analysis of tumors demonstrated enhanced TUNEL staining in tumor cells and increased CD3+ infiltrating cells in hAAT-Tg mice compared to WT mice, we speculated increased tumor cell destruction by T cells in hAAT-Tg mice. Treatment of B16F10 cells with hAAT in vitro revealed upregulated expression of melanocyte differentiation antigens TYR and TYRP1, and enhanced cytotoxicity by effector T cells. Lastly, intraperitoneal injection of recombinant hAAT confirmed suppression of tumor growth of B16F10 melanoma cells in WT mice. These results provide new insight into the mechanism of action of hAAT on the melanoma microenvironment and its potential role as a therapeutic agent.
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The pH sensor soluble adenylyl cyclase regulates melanogenesis D Zhou1, R Halaban2, A Bacchiocchi2, C Nardin1 and JH Zippin1 1 Weill Cornell Medical College, New York, NY and 2 Yale University, New Haven, CT Melanin pigments are synthesized by melanocytes in highly specialized organelles called melanosomes. Two distinct types of melanin, eumelanin and pheomelanin, are produced by eu- and pheomelanosomes, respectively. While the enzymes responsible for melanin synthesis and the general mechanisms for activating melanosomes are well studied, how melanosomes are directed to become eu- vs pheomelanosomes, or how cells regulate melanosome creation vs destruction via autophagolysosomes is poorly understood. Cyclic AMP (cAMP) is a key signaling molecule in melanin synthesis and the role of the transmembrane adenylyl cyclases (tmACs) in melanogenesis is well established. It is clear that melanosomal pH is an important regulator of melanogenesis, implicated in tyrosinase activity, eumelanin/pheomelanin production ratio, as well as melanosome maturation, but how melanocytes regulate melanosome pH remains poorly understood. Soluble adenylyl cyclase (sAC), a non-canonical source of cAMP, is not responsive to G proteins (e.g. MC1R) but is a pH sensor. Our group recently demonstrated that sAC is essential for normal lysosomal pH maintenance, and since melanosomes are lysosome-related organelles, we asked if sAC affects melanogenesis. Even though cAMP normally induces melanin synthesis, sAC KO mouse melanocytes paradoxically had increased melanin levels, and RNA profiling revealed increased expression of pigmentation genes. Consistent with elevated melanin content, EM imaging revealed that sAC KO melanocytes have significantly more eu- and pheomelanosomes and a drastic increase in melanosome laden autophagolysosomes suggesting a defect in both the synthesis and degradation of melanosomes in sAC null cells. These results were confirmed in human melanocytes, and interestingly, sAC inhibition led to distinct effects depending on the skin type of the donor: in Caucasian melanocytes pigmentation increased but in African-American melanocytes it was reduced. These data reveal a novel cAMP signaling cascade important for melanogenesis.
Measurement of skin pigmentation using a chromameter in a 3-dimensional epidermal model containing functional melanocytes MA Bachelor, B Breyfogle, A Armento and M Klausner MatTek Corporation, Ashland, MA Various cosmetic or skin care pharmaceutical formulations augment skin pigmentation either for the intended purpose of skin lightening or as a side effect induced by medication. A convenient way to screen such effects utilizes the MelanoDerm tissue model, a highly differentiated, three-dimensional tissue culture model of human epidermis containing normal human melanocytes and keratinocytes. Use of this model can provide valuable in vitro data as an early screening tool prior to the commencement of costly clinical trials. In this study, pigmentation was evaluated over the course of 2-3 weeks using a tristimulus chromometer to measure brightness (L*), yellowness (b*) and redness (a*) in MelanoDerm tissue produced with normal human melanocytes from Black, Asian, or Caucasian donors. In parallel to measurements taken with the chromameter, total melanin content of tissues was also quantified. Over time, cultures became increasingly pigmented with retention of normal epithelial morphology with the expected pigmentation level of the donor tissue, i.e. Black>Asian>Caucasian when cultured in media containing alpha-MSH and beta-FGF. Several over-the-counter skin lightening products were also evaluated in cultures containing normal human melanocytes from Black donors. Over the 2-3 week treatment period, control cultures became increasingly pigmented while tissues treated topically with cosmetic skin lightening agents containing tyosinase inhibitors such as kojic acid and magnesium ascorbyl phosphate remained distinctly lighter when compared to control cultures. After 14 days in culture, total melanin content was found to inversely correlate with surface reflectance (L*). The results described herein suggest that this model is useful for evaluating effects on melanogenesis, skin lightening, and other pigmentation phenomena of skin in vitro. In particular, this study highlights two distinct endpoints, total melanin content and skin color measurement that can be used to evaluate skin pigmentation in vitro.
608
609
Targeting the MC1R by selective tetra- and tripeptide analogs of a-Melanocortin for melanoma prevention ZA Abdel-Malek, V Swope, L Koikov, R Starner and A Kadekaro Dermatology, University of Cincinnati, Cincinnati, OH Our goal is to develop a topical formulation of small peptide analogs of a-Melanocortin (a-melanocyte stimulating hormone; a-MSH) that are highly selective for the melanocortin 1 receptor (MC1R) for prevention of photodamage in human melanocytes (HM) and ultimately melanoma. The MC1R is a melanoma susceptibility gene, and its loss-of-function variants result in red hair phenotype, reduced tanning and DNA repair capacity, and increased melanoma risk. Activation of the MC1R by its physiological agonist a-MSH stimulates eumelanin synthesis and proliferation, enhances the repair of UV-induced DNA damage and inhibits apoptosis of HM. We have designed short peptide analogs of the physiological tridecapeptide a-MSH exhibiting full MC1R agonism and excellent MC1R selectivity. Modifications of the His-D-Phe-Arg-Trp, or His-D-Phe-Arg sequence of a-MSH combined with terminal modifications resulted in stable small molecules with logD favorable for skin penetration. The tetrapeptides LK 184 and LK 467, and the tripeptide LK 514 were tested on primary cultures of HM and cultured skin substitutes (CSS). Dose-response experiments revealed that LK 184 and LK 467 were 100 fold more potent, while LK 514 was slightly less (10 fold) potent than a-MSH in stimulating cAMP formation and the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, in HM. The three peptides enhanced the repair of UV-induced DNA damage in HM. Treatment of CSS with LK 184 and LK 467 at 100 nM, or LK 514 at -5 mM for 10-15 days significantly increased pigmentation in the absence of UV exposure, and increased repair of UV-induced DNA photoproducts in melanocytes and the epidermis in the CSS or intact human skin. Prolonged treatment with these peptides had no effect on melanocyte number or histology of CSS. Given that increased pigmentation and DNA repair protect against photocarcinogenesis, we are continuing with preclinical testing of these peptides to determine their safety and efficacy as topical agents for sunless tanning and prevention of photodamage and melanoma.
Estrogen and progesterone reciprocally regulate melanin production through G protein-coupled receptors C Natale1, E Duperret1, J Zhang1, R Sadeghi1, A Dahal1, K O’Brien2, J Winkler2 and TW Ridky1 1 Dermatology, University of Pennsylvania, Philadelphia, PA and 2 Chemistry, University of Pennsylvania, Philadelphia, PA The association between pregnancy and altered skin pigmentation has been documented for over two millennia, suggesting that sex hormones regulate epidermal melanocyte (MC) homeostasis. We show that physiologic estrogen (17b-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that human MCs do not express classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G proteincoupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR to modulate melanin production. Topical delivery of a specific GPER agonist modulated pigmentation in murine skin in vivo. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics.
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The presence and distribution of PD-L1 staining in melanoma JM Donigan1, M Salama2, LR Rowe2, SR Tripp2, SR Florell1 and DA Wada1 1 Dermatology, University of Utah, Salt Lake City, UT and 2 Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT Programmed death-ligand 1 (PD-L1; also known as B7-H1 and CD274) is expressed by a variety of cell types, including immune and cancer cells, and influences tumor survival by suppressing T-lymphocyte mediated destruction of tumor cells. Anti-PD-L1 agents are currently in clinical trial for the treatment of melanoma. The aim of this study was to assess the presence and distribution of PD-L1 staining in melanoma. Twenty-eight formalin-fixed, paraffin-embedded melanoma specimens were obtained from the Department of Dermatology at the University of Utah in Salt Lake City. These included 24 primary tumors and four metastases. Tissue sections were stained for PD-L1 with an anti-human PD-L1 mouse monoclonal antibody (clone 22C3; Dako, Carpinteria, CA) developed for non-small cell lung cancer treatment with pembrolizumab (MK-3475). Staining of specimens was graded on strength of staining and percent of positive cells within the tumor and peri-tumoral mononuclear cells (presumed to be antigen presenting cells [APCs]). Specimens were deemed positive if there was staining in at least one percent of the cells. Thirteen (46%) of the specimens had positive staining within a mean of 14.4% (range 1 to 50%) of the tumor cells. Strength of staining within tumors was weak to moderate (1+ to 2+). Twenty-three (82%) of the specimens had positive staining of the APCs within an average of 12.7% (range 1 to 40%) of the cells. Strength of staining of APCs was mostly weak (range 1+ to 3+). These findings are among the first to demonstrate the intensity and distribution of this anti-PD-L1 antibody in a variety of melanoma specimens. Future studies assessing the utility of PD-L1 staining in melanoma as a marker of response to anti-PD-L1 therapy are warranted.
S108 Journal of Investigative Dermatology (2016), Volume 136
Alpha-1 antitrypsin suppresses melanoma growth by upregulating melanocyte differentiation antigens and enhancing T cell cytotoxicity DR Pearson1, Y Luo1, Z Zhai1, KL Couts1, T Azam2, CA Dinarello2 and M Fujita1,3 1 Dept of Dermatology, University of Colorado Denver SOM, Aurora, CO, 2 Dept of Medicine, University of Colorado Denver SOM, Aurora, CO and 3 Denver Veterans Affairs Medical Center, Denver, CO Inflammation within the tumor microenvironment plays a crucial role in tumor development and progression. Alpha-1 antitrypsin (AAT), a serine protease inhibitor, protects normal tissues from degradation by proteases and has been shown to inhibit inflammation. In the present study, we investigated the impact of AAT on the tumorigenesis of melanoma using a C57BL/6 strain of transgenic mice expressing human AAT under a surfactant protein C promoter (hAAT-Tg). In this model, hAAT is secreted from lung tissues and detected in circulation. Large tumors (>1,500 mm3) were formed after subcutaneous injection of B16F10 melanoma cells in wild-type (WT) mice, while only small tumors (<50 mm3) were observed in hAAT-Tg mice (Day 21; P<0.001). Suppressed melanoma growth was also seen by injection of a less aggressive primary cell line, B16F0, in hAAT-Tg mice (P<0.001). Histologic analysis of tumor tissues revealed reduced mitoses, increased apoptosis, and enhanced melanin pigmentation in tumor cells in hAAT-Tg mice compared to WT mice. However, when B16F10 cells were cultured in vitro, hAAT did not show an effect on cell viability in 2D culture or colony formation capacity in 3D culture. Since immunohistochemical analysis of tumors demonstrated enhanced TUNEL staining in tumor cells and increased CD3+ infiltrating cells in hAAT-Tg mice compared to WT mice, we speculated increased tumor cell destruction by T cells in hAAT-Tg mice. Treatment of B16F10 cells with hAAT in vitro revealed upregulated expression of melanocyte differentiation antigens TYR and TYRP1, and enhanced cytotoxicity by effector T cells. Lastly, intraperitoneal injection of recombinant hAAT confirmed suppression of tumor growth of B16F10 melanoma cells in WT mice. These results provide new insight into the mechanism of action of hAAT on the melanoma microenvironment and its potential role as a therapeutic agent.