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652 XPA promotes autophagy to facilitate cisplatin resistance in melanoma cells through the activation of PARP1
Pigmentation & Melanoma | ABSTRACTS 648
649
TLR3 agonist poly(I:C) enhances melanosome uptake by normal human epidermal keratinocytes S Koike, K Yamasaki, T Yamauchi, K Tsuchiyama and S Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan Innate immune stimuli restlessly influence epidermis where human melanocytes reside. We have examined how innate immunity affects pigmentation and observed that TLR3 agonist poly(I:C) increased melanin release from melanocytes through increasing Rab27A expression and cell peripheral accumulation, which facilitate melanosome transportation to cell membrane of melanocytes. To further explorer machineries involved in melanosome transfer by innate immunity, we examined the effect of innate immune stimuli on keratinocytes functions involving melanosome transfer. Protease-activated receptor-2 (PAR-2) is known as a key factor of melanosome transfer in keratinocytes. We examined PAR-2 expression in keratinocytes stimulated by the panel of TLR 1-9 agonists. TLR3 agonist poly(I:C) induces PAR-2 mRNA expression about 3-fold against control. Other TLR agonists did not change mRNA level of PAR-2. To examine if keratinocyte enhance melanosome uptake by TLR agonists, we isolated melanosome from normal human melanocytes and added the isolated melanosome to the keratinocytes pre-treated with TLR agonists. Along with the increase of PAR-2 expression, keratinocytes stimulated by poly(I:C), but not other TLR agonists, increased uptake of isolated melanosome about five times as much as no stimuli control. Since UVB induces TLR3 signaling cascades by forming fragmentation of noncoding RNA in keratinocytes, we examined whether UVB-irradiated keratinocytes increases melanosome transfer. We obaserved UVB-irradiation (15 mJ/cm2) increased melanosome uptake about 4 times compared with control. These indicated that TLR3 signaling induces melanosome transfer by effect on not only melanocytes but also keratinocytes. Therefore the innate immunity may modulate the pigmentation process in skin.
OX40/OX40L and 4-1BB/4-1BBL signaling in cutaneous anti-tumoral immunity G Geidel1, M Maurer2, T Luger1 and K Loser1 1 Department of Dermatology, University of Mu¨nster, Muenster, Germany and 2 Department of Dermatology, Charite, Berlin, Germany OX40/OX40L and 4-1BB/4-1BBL are members of the TNF family. Signaling via these receptor ligand pairs controls inflammation and regulates anti-tumoral immunity. The role of OX40 and 4-1BB during cutaneous anti-tumoral immunity has not been investigated in detail and therefore, transgenic mice overexpressing OX40 or 4-1BB in the skin (K14-OX40 tg, K14-4-1BB tg) were inoculated with B16-melanoma cells. Interestingly, K14-OX40 tg and K14-4-1BB tg mice showed a significantly increased tumor growth compared to wildtype (wt) controls. FACS, histology and gene expression studies revealed increased numbers of CD8+ T cells in melanomas from tg versus wt mice, which however, had a similar tumor-specific cytotoxic activity compared to CD8+ T cells from wt tumors, thus suggesting a minor importance of CD8+ T cells in explaining the increased tumor progression in tg mice. Notably, FACS and in vitro suppression assays revealed significantly increased levels of myeloid-derived suppressor cells (MDSC) and an elevated inhibitory function of regulatory T cells (Treg). MDSC and Treg affect anti-tumoral immunity by interacting with mast cells and OX40/OX40L signaling controls the cross-talk of Treg with mast cells whereas 4-1BB signaling directly expands mast cells. Thus, we quantified the numbers of mast cells and detected markedly decreased levels of CD117+ mast cells in melanomas from tg compared to wt mice. Of note, low numbers of degranulated mast cells have been associated with reduced survival in advanced stage melanoma. To assess the role of mast cells during tumor progression in vivo K14-OX40 tg mice were bred to KitW-sh mutants lacking CD117+ mast cells. In line with our hypothesis K14-OX40 tg x KitW-sh double mutants showed an increased tumor progression compared to mast cell competent K14-OX40 tg controls. Thus, our data suggest that up-regulated OX40/OX40L and 4-1BB/4-1BBL signaling in the skin mediated the expansion of suppressor cells in B16-melanoma-bearing mice resulting in reduced numbers of tumor-protective mast cells.
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651
Intermittent high-dose UVB induces an interferon-related DNA damage response signature (IRDS) in melanocytes e Implications for early transformation to melanoma LA Cornelius and K Zhang Dermatology, Washington University School of Medicine, St. Louis, MO Intermittent burning doses of Ultraviolet Radiation (UVR) in childhood confers significant melanoma risk. Melanocytes are relatively resistant to UVR-induced apoptosis and the immediate response of increased DNA repair and tumor suppressor gene expression attenuates UVR insult. We wanted to determine whether repeated intermittent high doses of UVB instigated a distinct response that could contribute to melanomagenesis. We exposed light and medium-dark pigmented human neonatal melanocytes (lpNHM, m-dpNHM) to 100mj/cm2 of UVB 2x/week for 14 days and evaluated gene expression. As expected, UVB exposure upregulated expression of p53 at 24 hrs. By day 14, p21 was expressed, along with inflammatory mediators (IL-6, IL-8, CCL2), increased STAT1 expression and up to a 90-fold induction of IFN-related genes (IRGs) IFIT1, IFI6, MX1, OAS2, 3 and L and TRIM2 that was independent of IFNs I-III. Cells maintained in culture for an additional 14 days passaged normally (28 day time-point) and retained this IRG signature - most significantly in lpNHM with loss of p21 and p53 expression, sustained up-regulation of IL-6 and STAT1 expression. We next evaluated senescence. At the 14 day time-point, < 20% cells stained positively for SA-b-gal and cells in G0/G1 increased by 10% compared to control (84 vs 74%); returning to control levels by day 28 (76%). Finally, we subjected UV-irradiated cells to increasing doses of Doxorubicin to determine whether this IRG signature conferred survival advantage to further DNA damage and found a 10-20% increase in cell viability compared to control. In sum, we demonstrate that repeated high dose UVB induces a sustained cell autonomous pro-survival secretome (IL-6), constitutive STAT1 expression and an IRG response in lpNHMs. We propose that in addition to its mutagenic effects, repeated UVB exposure may selectively enrich for a population of melanocytes with this IRDS-like phenotype that serves to promote melanocyte survival, having implications for early melanoma development.
Self-delivering RNAi compounds for reduction of hyperpigmentation M Maxwell, K Holton, J Cardia, R Looby, M Byrne and K Bulock RXi Pharmaceuticals, Marlborough, MA An increased production of melanin by melanocytes can result in a number of hyperpigmentation disorders which are of considerable cosmetic concern with a potentially negative impact on a person’s psychological well-being. Melanin production is controlled primarily by tyrosinase, an enzyme which catalyzes the hydroxylation of tyrosine to L-DOPA and the oxidation of L-DOPA to dopaquinone. Inhibition of tyrosinase results in a reduction of pigmentation. Hydroquinone has been the gold standard for treatment of hyperpigmentation but has fallen under scrutiny in recent years leading people to seek alternative methods for skin lightening. We have developed a new class of stable, self-delivering RNAi compounds (sd-rxRNAÒ) that incorporate features of RNAi and antisense technology. sd-rxRNAs demonstrate potent activity, stability, and reduced immune stimulation, and are rapidly and efficiently taken up by cells. Tyrosinase-targeting sd-rxRNAs were designed, synthesized, and screened in the skin melanoma cell line SK-MEL-5 to identify potential lead compounds for skin lightening studies. These lead compounds were further screened using in vitro dopachrome formation and melanin content assays with moderately pigmented normal human embryonic melanocytes (NHEM-MP) to identify those which produced the greatest reduction in tyrosinase activity and pigmentation. A dose-dependent reduction in both tyrosinase activity and melanin content was seen in these cells. Studies using MelanoDerm, a 3-dimensional reconstituted human epidermal culture model, demonstrated reduction in tyrosinase mRNA expression with a corresponding visible reduction in pigmentation when treated with tyrosinase-targeting sd-rxRNAs every other day over two weeks. From these studies, our cosmeceutical candidate, RXI-231 was selected for further development. An update, including efforts to develop non-invasive topical delivery methods for RXI-231 will be presented.
652
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XPA promotes autophagy to facilitate cisplatin resistance in melanoma cells through the activation of PARP1 R Ge, L Liu, W Dai, W Zhang, Y Yang, H Wang, Q Shi, S Guo, X Yi, G Wang, T Gao, Q Luan and C Li Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Xeroderma pigmentosum group A (XPA), a key protein in nucleotide excision repair (NER) pathway, has been shown to promote the resistance of tumor cells to chemotherapeutic drugs by facilitating the DNA repair process. However, the role of XPA in the resistance to platinumbased drugs like cisplatin in melanoma is largely unknown. In this study, we initially found that XPA was expressed at higher levels in cisplatin-resistant melanoma cells than that in cisplatin-sensitive ones. Furthermore, the knockdown of XPA not only increased cellular apoptosis but also inhibited cisplatin-induced autophagy, which rendered the melanoma cells more sensitive to cisplatin. Moreover, we discovered that the increased XPA in resistant melanoma cells promoted poly (ADP-ribose) polymerase 1 (PARP1) activation, and the inhibition of PARP1 could attenuate the cisplatin-induced autophagy. At last, we proved that the inhibition on PARP1 as well as the autopagy process made resistant melanoma cells more susceptible to cisplatin treatment. In conclusion, our study demonstrates that XPA can promote cell-protective autophagy in a DNA repair-independent manner by enhancing the activation of PARP1 in melanoma cells resistant to cisplatin, and the XPA-PARP1-mediated autophagy process can be targeted to overcome cisplatin resistance in melanoma chemotherapy.
Plumbagin induces apoptosis in melanoma cells by ROS-mediated disruption of mitochondrial membrane potential and inhibition of PI3K/AKT/mTOR signaling RL Pearlman, HC Pal, CA Elmets and F Afaq Dermatology, University of Alabama at Birmingham, Birmingham, AL Melanoma is the deadliest form of skin cancer due to its tendency to aggressively metastasize. Despite advances in therapy options, the five-year survival rate for metastatic melanoma has not significantly improved. Exploration of novel therapeutic agents remains critical to the fight against melanoma progression. Recently, interest in phytochemicals as potential adjuvant or chemotherapeutic agents has risen. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naturally occurring naphthoquinone isolated from the plant, Plumbago zeylanica L., exhibits anti-proliferative, anti-tumorigenic and anti-metastatic properties. In this study, we aimed to determine the effects of plumbagin on cell proliferation and apoptosis in melanoma cells from different genetic backgrounds. Treatment of BRAF-mutated (SK-MEL-28, WM35, RPMI-7951), NRAS-mutated (SK-MEL-119), and BRAF/NRAS wild type (Hs294T) melanoma cells with plumbagin (2.5-10 mM; 24 hrs) resulted in dose-dependent (i) reduction of cell proliferation, (ii) induction of apoptosis, and (iii) disruption of mitochondrial membrane potential. Treatment of SK-MEL-28, SK-MEL-119 and Hs294T cells with plumbagin induced DNA damage as assessed by comet assay and also increased the phosphorylation of g-H2AX. In addition, plumbagin treatment caused (i) cleavage of PARP and caspase 3, (ii) release of cytochrome c, and (iii) generation of ROS. N-acetyl-L-cysteine treatment inhibited disruption of mitochondrial membrane potential and apoptotic effects of plumbagin. Furthermore, treatment of melanoma cells with plumbagin decreased the (i) protein expression of PI3K(p110a), (ii) phosphorylation of AKT at Ser473, (iii) phosphorylation of mTOR at Ser2448 and Ser2481 and (iv) protein expression of raptor and rictor. These findings suggest that plumbagin demonstrates strong therapeutic potential, and further mechanistic studies are needed to establish the use of plumbagin either alone or as an adjuvant in melanoma therapy.
www.jidonline.org S115
649
TLR3 agonist poly(I:C) enhances melanosome uptake by normal human epidermal keratinocytes S Koike, K Yamasaki, T Yamauchi, K Tsuchiyama and S Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan Innate immune stimuli restlessly influence epidermis where human melanocytes reside. We have examined how innate immunity affects pigmentation and observed that TLR3 agonist poly(I:C) increased melanin release from melanocytes through increasing Rab27A expression and cell peripheral accumulation, which facilitate melanosome transportation to cell membrane of melanocytes. To further explorer machineries involved in melanosome transfer by innate immunity, we examined the effect of innate immune stimuli on keratinocytes functions involving melanosome transfer. Protease-activated receptor-2 (PAR-2) is known as a key factor of melanosome transfer in keratinocytes. We examined PAR-2 expression in keratinocytes stimulated by the panel of TLR 1-9 agonists. TLR3 agonist poly(I:C) induces PAR-2 mRNA expression about 3-fold against control. Other TLR agonists did not change mRNA level of PAR-2. To examine if keratinocyte enhance melanosome uptake by TLR agonists, we isolated melanosome from normal human melanocytes and added the isolated melanosome to the keratinocytes pre-treated with TLR agonists. Along with the increase of PAR-2 expression, keratinocytes stimulated by poly(I:C), but not other TLR agonists, increased uptake of isolated melanosome about five times as much as no stimuli control. Since UVB induces TLR3 signaling cascades by forming fragmentation of noncoding RNA in keratinocytes, we examined whether UVB-irradiated keratinocytes increases melanosome transfer. We obaserved UVB-irradiation (15 mJ/cm2) increased melanosome uptake about 4 times compared with control. These indicated that TLR3 signaling induces melanosome transfer by effect on not only melanocytes but also keratinocytes. Therefore the innate immunity may modulate the pigmentation process in skin.
OX40/OX40L and 4-1BB/4-1BBL signaling in cutaneous anti-tumoral immunity G Geidel1, M Maurer2, T Luger1 and K Loser1 1 Department of Dermatology, University of Mu¨nster, Muenster, Germany and 2 Department of Dermatology, Charite, Berlin, Germany OX40/OX40L and 4-1BB/4-1BBL are members of the TNF family. Signaling via these receptor ligand pairs controls inflammation and regulates anti-tumoral immunity. The role of OX40 and 4-1BB during cutaneous anti-tumoral immunity has not been investigated in detail and therefore, transgenic mice overexpressing OX40 or 4-1BB in the skin (K14-OX40 tg, K14-4-1BB tg) were inoculated with B16-melanoma cells. Interestingly, K14-OX40 tg and K14-4-1BB tg mice showed a significantly increased tumor growth compared to wildtype (wt) controls. FACS, histology and gene expression studies revealed increased numbers of CD8+ T cells in melanomas from tg versus wt mice, which however, had a similar tumor-specific cytotoxic activity compared to CD8+ T cells from wt tumors, thus suggesting a minor importance of CD8+ T cells in explaining the increased tumor progression in tg mice. Notably, FACS and in vitro suppression assays revealed significantly increased levels of myeloid-derived suppressor cells (MDSC) and an elevated inhibitory function of regulatory T cells (Treg). MDSC and Treg affect anti-tumoral immunity by interacting with mast cells and OX40/OX40L signaling controls the cross-talk of Treg with mast cells whereas 4-1BB signaling directly expands mast cells. Thus, we quantified the numbers of mast cells and detected markedly decreased levels of CD117+ mast cells in melanomas from tg compared to wt mice. Of note, low numbers of degranulated mast cells have been associated with reduced survival in advanced stage melanoma. To assess the role of mast cells during tumor progression in vivo K14-OX40 tg mice were bred to KitW-sh mutants lacking CD117+ mast cells. In line with our hypothesis K14-OX40 tg x KitW-sh double mutants showed an increased tumor progression compared to mast cell competent K14-OX40 tg controls. Thus, our data suggest that up-regulated OX40/OX40L and 4-1BB/4-1BBL signaling in the skin mediated the expansion of suppressor cells in B16-melanoma-bearing mice resulting in reduced numbers of tumor-protective mast cells.
650
651
Intermittent high-dose UVB induces an interferon-related DNA damage response signature (IRDS) in melanocytes e Implications for early transformation to melanoma LA Cornelius and K Zhang Dermatology, Washington University School of Medicine, St. Louis, MO Intermittent burning doses of Ultraviolet Radiation (UVR) in childhood confers significant melanoma risk. Melanocytes are relatively resistant to UVR-induced apoptosis and the immediate response of increased DNA repair and tumor suppressor gene expression attenuates UVR insult. We wanted to determine whether repeated intermittent high doses of UVB instigated a distinct response that could contribute to melanomagenesis. We exposed light and medium-dark pigmented human neonatal melanocytes (lpNHM, m-dpNHM) to 100mj/cm2 of UVB 2x/week for 14 days and evaluated gene expression. As expected, UVB exposure upregulated expression of p53 at 24 hrs. By day 14, p21 was expressed, along with inflammatory mediators (IL-6, IL-8, CCL2), increased STAT1 expression and up to a 90-fold induction of IFN-related genes (IRGs) IFIT1, IFI6, MX1, OAS2, 3 and L and TRIM2 that was independent of IFNs I-III. Cells maintained in culture for an additional 14 days passaged normally (28 day time-point) and retained this IRG signature - most significantly in lpNHM with loss of p21 and p53 expression, sustained up-regulation of IL-6 and STAT1 expression. We next evaluated senescence. At the 14 day time-point, < 20% cells stained positively for SA-b-gal and cells in G0/G1 increased by 10% compared to control (84 vs 74%); returning to control levels by day 28 (76%). Finally, we subjected UV-irradiated cells to increasing doses of Doxorubicin to determine whether this IRG signature conferred survival advantage to further DNA damage and found a 10-20% increase in cell viability compared to control. In sum, we demonstrate that repeated high dose UVB induces a sustained cell autonomous pro-survival secretome (IL-6), constitutive STAT1 expression and an IRG response in lpNHMs. We propose that in addition to its mutagenic effects, repeated UVB exposure may selectively enrich for a population of melanocytes with this IRDS-like phenotype that serves to promote melanocyte survival, having implications for early melanoma development.
Self-delivering RNAi compounds for reduction of hyperpigmentation M Maxwell, K Holton, J Cardia, R Looby, M Byrne and K Bulock RXi Pharmaceuticals, Marlborough, MA An increased production of melanin by melanocytes can result in a number of hyperpigmentation disorders which are of considerable cosmetic concern with a potentially negative impact on a person’s psychological well-being. Melanin production is controlled primarily by tyrosinase, an enzyme which catalyzes the hydroxylation of tyrosine to L-DOPA and the oxidation of L-DOPA to dopaquinone. Inhibition of tyrosinase results in a reduction of pigmentation. Hydroquinone has been the gold standard for treatment of hyperpigmentation but has fallen under scrutiny in recent years leading people to seek alternative methods for skin lightening. We have developed a new class of stable, self-delivering RNAi compounds (sd-rxRNAÒ) that incorporate features of RNAi and antisense technology. sd-rxRNAs demonstrate potent activity, stability, and reduced immune stimulation, and are rapidly and efficiently taken up by cells. Tyrosinase-targeting sd-rxRNAs were designed, synthesized, and screened in the skin melanoma cell line SK-MEL-5 to identify potential lead compounds for skin lightening studies. These lead compounds were further screened using in vitro dopachrome formation and melanin content assays with moderately pigmented normal human embryonic melanocytes (NHEM-MP) to identify those which produced the greatest reduction in tyrosinase activity and pigmentation. A dose-dependent reduction in both tyrosinase activity and melanin content was seen in these cells. Studies using MelanoDerm, a 3-dimensional reconstituted human epidermal culture model, demonstrated reduction in tyrosinase mRNA expression with a corresponding visible reduction in pigmentation when treated with tyrosinase-targeting sd-rxRNAs every other day over two weeks. From these studies, our cosmeceutical candidate, RXI-231 was selected for further development. An update, including efforts to develop non-invasive topical delivery methods for RXI-231 will be presented.
652
653
XPA promotes autophagy to facilitate cisplatin resistance in melanoma cells through the activation of PARP1 R Ge, L Liu, W Dai, W Zhang, Y Yang, H Wang, Q Shi, S Guo, X Yi, G Wang, T Gao, Q Luan and C Li Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Xeroderma pigmentosum group A (XPA), a key protein in nucleotide excision repair (NER) pathway, has been shown to promote the resistance of tumor cells to chemotherapeutic drugs by facilitating the DNA repair process. However, the role of XPA in the resistance to platinumbased drugs like cisplatin in melanoma is largely unknown. In this study, we initially found that XPA was expressed at higher levels in cisplatin-resistant melanoma cells than that in cisplatin-sensitive ones. Furthermore, the knockdown of XPA not only increased cellular apoptosis but also inhibited cisplatin-induced autophagy, which rendered the melanoma cells more sensitive to cisplatin. Moreover, we discovered that the increased XPA in resistant melanoma cells promoted poly (ADP-ribose) polymerase 1 (PARP1) activation, and the inhibition of PARP1 could attenuate the cisplatin-induced autophagy. At last, we proved that the inhibition on PARP1 as well as the autopagy process made resistant melanoma cells more susceptible to cisplatin treatment. In conclusion, our study demonstrates that XPA can promote cell-protective autophagy in a DNA repair-independent manner by enhancing the activation of PARP1 in melanoma cells resistant to cisplatin, and the XPA-PARP1-mediated autophagy process can be targeted to overcome cisplatin resistance in melanoma chemotherapy.
Plumbagin induces apoptosis in melanoma cells by ROS-mediated disruption of mitochondrial membrane potential and inhibition of PI3K/AKT/mTOR signaling RL Pearlman, HC Pal, CA Elmets and F Afaq Dermatology, University of Alabama at Birmingham, Birmingham, AL Melanoma is the deadliest form of skin cancer due to its tendency to aggressively metastasize. Despite advances in therapy options, the five-year survival rate for metastatic melanoma has not significantly improved. Exploration of novel therapeutic agents remains critical to the fight against melanoma progression. Recently, interest in phytochemicals as potential adjuvant or chemotherapeutic agents has risen. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naturally occurring naphthoquinone isolated from the plant, Plumbago zeylanica L., exhibits anti-proliferative, anti-tumorigenic and anti-metastatic properties. In this study, we aimed to determine the effects of plumbagin on cell proliferation and apoptosis in melanoma cells from different genetic backgrounds. Treatment of BRAF-mutated (SK-MEL-28, WM35, RPMI-7951), NRAS-mutated (SK-MEL-119), and BRAF/NRAS wild type (Hs294T) melanoma cells with plumbagin (2.5-10 mM; 24 hrs) resulted in dose-dependent (i) reduction of cell proliferation, (ii) induction of apoptosis, and (iii) disruption of mitochondrial membrane potential. Treatment of SK-MEL-28, SK-MEL-119 and Hs294T cells with plumbagin induced DNA damage as assessed by comet assay and also increased the phosphorylation of g-H2AX. In addition, plumbagin treatment caused (i) cleavage of PARP and caspase 3, (ii) release of cytochrome c, and (iii) generation of ROS. N-acetyl-L-cysteine treatment inhibited disruption of mitochondrial membrane potential and apoptotic effects of plumbagin. Furthermore, treatment of melanoma cells with plumbagin decreased the (i) protein expression of PI3K(p110a), (ii) phosphorylation of AKT at Ser473, (iii) phosphorylation of mTOR at Ser2448 and Ser2481 and (iv) protein expression of raptor and rictor. These findings suggest that plumbagin demonstrates strong therapeutic potential, and further mechanistic studies are needed to establish the use of plumbagin either alone or as an adjuvant in melanoma therapy.
www.jidonline.org S115