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471 Down-regulated TRPV1 contributes to the progression of melanoma
Melanoma and Other Skin Cancers | ABSTRACTS 471
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Down-regulated TRPV1 contributes to the progression of melanoma Y Yang, J Ma, W Guo, G Zhu, T Gao and C Li Department of Dermatology, Xijing Hospital, Xi’an, China To identify the function of TRPV1 in the progression of melanoma and investigate the underlying mechanism. Immunofluorescence, qRT-PCR and Western Blot were performed to detect the expression of TRPV1 in tissue samples of melanocytic nevus, primary melanoma, metastatic melanoma as well as in melanoma cell lines. In addition, using CCK8 assay, Edu and flow cytometry, we identified the effect of TRPV1-specific agonist, capsaicin, on the proliferation and apoptosis of melanoma cells. To identify the molecular mechanism by which TRPV1 regulates the progression of melanoma, we performed western blot assay to detect the expression of p53, PARP, Bcl2, Bax in TRPV-1 activated melanoma cells exposed to Calcinerin blocker FK506. TRPV1 expression was decreased in melanoma cell lines compared with normal melanocytes (P <0.01). Tissue TRPV1 level was drastically reduced in primary and metastatic melanoma compared with melanocytic nevus (P <0.01). Furthermore, TRPV1-specific agonist capsaicin suppressed the proliferation by inhibiting ERK signaling (P <0.05). Notably, TRPV1 activation induced the apoptosis of melanoma cells by activation of Ca2+/calcineurin-NFAT-ATF3 pathway, and resulting the elevated expression of p53 (P <0.01). TRPV1 was significantly down-regulated in melanoma, activated TRPV1 suppressed the progression of melanoma via ERK pathway and promote the apoptosis by activating calcineurin. Taken together, TRPV1 acts as a crucial repressor in melanoma progression and can be a promising target in melanoma treatment.
Loss of Laminin a3 drives SCC invasion via ROCK signalling M Caley1, V Martins1, K Moore2, M Lashari1, V Ka¨ha¨ri3, L Nissinen3, M Donaldson4, J Marshall2 and EA O’Toole1 1 Centre for Cell Biology and Cutaneous Research, Barts and the London SMD, QMUL, London, United Kingdom, 2 Barts Cancer Institute, Barts and The London School of Medicine and Dentistry, London, United Kingdom, 3 Dermatology, University of Turku, Turku, Finland and 4 Dermatology TA, GSK, Uxbridge, United Kingdom Squamous cell carcinoma (SCC) make up 20% of non-melanoma skin cancers and about 4 to 6% of SCC metastasise. In this study, we examined the role of the 3 chains of Laminin 332 (Lam332) in SCC invasion. Analysis of SCC tumours and cell lines using Lam332 chain specific antibodies identified a link between poorly differentiated SCC and reduced expression of Laminin a3 (but not the other chains of Lam332). This loss of expression of Laminin a3 is due to aberrant methylation at the LAMA3 promoter. To investigate further the role of each chain of Lam332 in SCC we generated stable knockdowns (KDs) of each Lam332 chain (LAMA3, LAMB3 & LAMC2) in cutaneous SCC cell lines. Using these cells we studied their role in cell attachment, motility, in vitro and in vivo invasion, cellular signalling and global gene expression. We observed distinct phenotypic and genotypic changes in each of our laminin chain KDs. RNA-seq analysis identified over-represented genes involved in motility, invasion and cytoskeletal remodelling with KD of LAMA3. Loss of LAMA3 decreased attachment and increased motility in vitro. Using a xenograft model we identified increased invasion and increased EMT in vivo with KD of LAMA3. The loss of LAMB3 decreased invasion and increased differentiation in vivo with increased Keratin 10 and involucrin expression. Cancer cell invasion is linked with the RHO Kinase signalling pathways. We investigated RHO signalling in our cell lines and identified that invading cells with KD of LAMA3 have a significant increase in ROCK activity characterised by increased phosphorylated myosin light chain kinase. Using RHO kinase inhibitors we confirmed that invasion in cells lacking LAMA3 is ROCK- dependent. These findings demonstrate that Lam332 is a major regulator of SCC differentiation and invasion.
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MiR-4286 inhibition alters proliferation and apoptosis in melanoma cells N Palkina, A Komina, M Aksenenko and T Ruksha Pathophysiology, Krasnoyarsk State Medical University, Krasnoyarsk, Russian Federation MicroRNAs are considered as essential regulators of gene expression in malignant tumors with prooncogenic or tumor suppressive capacities. More than 2500 types of microRNAs identified at the present time although the precise role in carcinogenesis of the most of them remains unclear. Our study aimed to compare melanoma and melanocytic nevi profiles to determine microRNAs which are characterized by different expression pattern in melanoma as compared to benign melanocytic tumors for unveiling their functional role in carcinogenesis. Melanoma (n¼23) and melanocytic nevi (n¼9) samples were obtained from patients of Krasnoyarsk Regional Oncology center. MicroRNA expression pattern was determined by GeneAtlasÔ Microarray System (Affymetrix, CA, USA). Bro melanoma cell line was grown at 37 C in 5% CO2-incubator. Cell apoptosis detection was performed by flow cytometry, melanoma cells survival was estimated by MTT-test. Migration and invasion experiments were performed by CytoSelectÔ 24-Well Cell Migration and Invasion Assay (Cell Biolabs, Inc., USA). DIANA-mirPath v.3.0 was used for the KEEG pathway analysis of miRNA signature. MiRNA targets were predicted based on TargetScan 7.0, miRWalk 2.0, and miRTarBase v.4.5. MiR-4286 was more than 17.8 times up-regulated in melanoma in comparison to benign melanocytic tumors in our study. The efficiency of cell transfection with anti-miR-4286 was estimated by the real-time PCR analysis of HMGA2 expression levels in the transfected cells. MTT assay was performed in 24, 48, 72, and 96 h after transfection. The inhibition of miR4286 activity resulted in a significant decrease of melanoma cells viability/proliferation. By flow cytometry, it was found that miR-4286 inhibition in melanoma cells led to the increase in 2.6-fold in apoptosis rate. Inhibition of miR-4286 does not alter melanoma cells migration and invasion. Our results indicate that miR-4286 is involved in regulation of melanoma cells’ apoptosis and proliferation by mechanisms requiring further investigation.
The bioinformatics workflow for epigenetics profiling of progresing melanoma K Poterlowicz and K Murat Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom The poor-prognosis tumours are often lack genetic well characterized alterations but are epigenetically heterogeneous, pointing to the important role of the multi-domain epigenetic regulation in cancer progression. In the recent years researchers studying epigenetic regulation of tumor progression generated a vast amount of high-throughput sequencing data for processes such as DNA methylation and modification of histones. Despte of it, there is a lack of multi-layers epigenetic workflows characterizing tumor progression regulation. Here we integrated the DNA methylation profiles at the different stages of melanoma and ChIP-Seq profiles for H3K27me3, H3K4me3, MITF, BRG1 to identify novel epigenetic switches responsible for metastatic progression of this tumor and correlate them with the survival rates of the patient. Results and experiences using this framework demonstrate the potential for bioinformatics solution for multi-omics cancer biomarker discovery tool.
Meta-analysis of genomic datasets to identify recurrent mutations and altered pathways in cutaneous T-cell lymphoma L Chang and L Geskin Dermatology, Columbia University, New York, NY Recent studies have used whole genome or whole exome sequencing to identify recurrently mutated genes in cutaneous T-cell lymphoma (CTCL). However, these studies usually include tumors of multiple types and stages. Due to the genomic heterogeneity of CTCL, discovering genes or pathways that play a central role in CTCL pathogenesis remains challenging. Here, we combined and co-analyzed recently published genomic sequencing datasets of cutaneous T cell lymphoma. We generated a list of 6653 single nucleotide mutations in CTCL samples, including 67 Sezary syndrome and 19 mycosis fungoides. We found 19 recurrently mutated loci, including those for R48W in PLCG1, Q575E in RLTPR and a nonsense mutation of Y331 in CCR4. When the list of mutations is used to study commonly mutated genes, we found previously reported genes as well as genes that were not deemed significantly mutated by individual studies. Importantly, frequently mutated genes in mycosis fungoides and Sezary syndrome overlap. Among the most frequently mutated genes are PCLO (20%), TP53 (16%) and FAT3 (16%). Sucrase-isomaltase (SI) a commonly mutated genes in CLL, is also identified as a recurrently mutated genes in Sezary syndrome. When mutations were analyzed in the context of biological pathways, we found genes in the T cell receptor pathway and the JAK/STAT pathway are frequently mutated in Sezary syndrome, including TNFR2 (8%), PLCG1 (11%) and CARD11 (11%). To conclude, this study validated previous reported recurrent mutations in CTCL and identified additional genes that are frequently mutated. These results provide new information on the molecular mechanism of CTCL pathogenesis.
Expression of retinoic acid receptor-related orphan receptors (ROR) a and g correlates with hypoxia in cutaneous melanomas AA Brozyna1, W Jozwicki1, M Pawlikowska2, E Wedrowska4 and A Slominski3 1 Department of Tumor Pathology and Pathomorphology, Collegium Medicum, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland, 2 Department of Immunology, Nicolaus Copernicus University, Torun, Poland, 3 Departments of Dermatology and Pathology, University of Alabama at Birmingham, Birmingham, AL and 4 Department of Gene Therapy, Collegium Medicum, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland Hypoxia is one of the hallmarks of the tumor microenvironment and up-regulation of hypoxia-inducible factor 1 alpha (HIF-1a) is considered as a negative prognostic marker. Recently the role of retinoic aciderelated orphan receptor (ROR) a and g in hypoxia signaling pathway have been identified. RORa and RORg are involved in many physiological functions in several organs. Previously we showed that RORs are expressed in human normal skin and melanocytic tumors including nevi and melanomas. Furthermore, we reported that HIF-1a expression and HIF-1a dependent pathways can be regulated by melanogenesis and are related to poorer prognosis of melanoma. In this study using immunohistochemistry, we examined the relationship between RORa and RORg and HIF-1a in 73 cutaneous melanomas (36, 35 and 2, nodular, superficial spreading and acral types, respectively) and 34 metastases. In both, primary melanomas and metastases, the HIF-1a immunostaining increased with increasing RORa expression. This relationship was more evident in advanced melanomas (pT3-pT4) and was also observed in metastases. On the contrary, HIF-1a expression was higher in RORg-negative advanced melanomas, while in pT1-pT2 melanomas there were no differences in its expression levels. In metastases, HIF-1a level was higher in RORg-positive cases. Furthermore, HIF-1a expression was elevated in primary melanomas showing brisk tumor-infiltrating lymphocytes (TILs) response. Thus, our results indicate that hypoxia defined by an up-regulated expression of HIF-1a is differentially related to the expression of RORa and RORg in melanomas. We suggest that mutual interaction between those regulators may affect behavior of human melanoma.
www.jidonline.org S241
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Down-regulated TRPV1 contributes to the progression of melanoma Y Yang, J Ma, W Guo, G Zhu, T Gao and C Li Department of Dermatology, Xijing Hospital, Xi’an, China To identify the function of TRPV1 in the progression of melanoma and investigate the underlying mechanism. Immunofluorescence, qRT-PCR and Western Blot were performed to detect the expression of TRPV1 in tissue samples of melanocytic nevus, primary melanoma, metastatic melanoma as well as in melanoma cell lines. In addition, using CCK8 assay, Edu and flow cytometry, we identified the effect of TRPV1-specific agonist, capsaicin, on the proliferation and apoptosis of melanoma cells. To identify the molecular mechanism by which TRPV1 regulates the progression of melanoma, we performed western blot assay to detect the expression of p53, PARP, Bcl2, Bax in TRPV-1 activated melanoma cells exposed to Calcinerin blocker FK506. TRPV1 expression was decreased in melanoma cell lines compared with normal melanocytes (P <0.01). Tissue TRPV1 level was drastically reduced in primary and metastatic melanoma compared with melanocytic nevus (P <0.01). Furthermore, TRPV1-specific agonist capsaicin suppressed the proliferation by inhibiting ERK signaling (P <0.05). Notably, TRPV1 activation induced the apoptosis of melanoma cells by activation of Ca2+/calcineurin-NFAT-ATF3 pathway, and resulting the elevated expression of p53 (P <0.01). TRPV1 was significantly down-regulated in melanoma, activated TRPV1 suppressed the progression of melanoma via ERK pathway and promote the apoptosis by activating calcineurin. Taken together, TRPV1 acts as a crucial repressor in melanoma progression and can be a promising target in melanoma treatment.
Loss of Laminin a3 drives SCC invasion via ROCK signalling M Caley1, V Martins1, K Moore2, M Lashari1, V Ka¨ha¨ri3, L Nissinen3, M Donaldson4, J Marshall2 and EA O’Toole1 1 Centre for Cell Biology and Cutaneous Research, Barts and the London SMD, QMUL, London, United Kingdom, 2 Barts Cancer Institute, Barts and The London School of Medicine and Dentistry, London, United Kingdom, 3 Dermatology, University of Turku, Turku, Finland and 4 Dermatology TA, GSK, Uxbridge, United Kingdom Squamous cell carcinoma (SCC) make up 20% of non-melanoma skin cancers and about 4 to 6% of SCC metastasise. In this study, we examined the role of the 3 chains of Laminin 332 (Lam332) in SCC invasion. Analysis of SCC tumours and cell lines using Lam332 chain specific antibodies identified a link between poorly differentiated SCC and reduced expression of Laminin a3 (but not the other chains of Lam332). This loss of expression of Laminin a3 is due to aberrant methylation at the LAMA3 promoter. To investigate further the role of each chain of Lam332 in SCC we generated stable knockdowns (KDs) of each Lam332 chain (LAMA3, LAMB3 & LAMC2) in cutaneous SCC cell lines. Using these cells we studied their role in cell attachment, motility, in vitro and in vivo invasion, cellular signalling and global gene expression. We observed distinct phenotypic and genotypic changes in each of our laminin chain KDs. RNA-seq analysis identified over-represented genes involved in motility, invasion and cytoskeletal remodelling with KD of LAMA3. Loss of LAMA3 decreased attachment and increased motility in vitro. Using a xenograft model we identified increased invasion and increased EMT in vivo with KD of LAMA3. The loss of LAMB3 decreased invasion and increased differentiation in vivo with increased Keratin 10 and involucrin expression. Cancer cell invasion is linked with the RHO Kinase signalling pathways. We investigated RHO signalling in our cell lines and identified that invading cells with KD of LAMA3 have a significant increase in ROCK activity characterised by increased phosphorylated myosin light chain kinase. Using RHO kinase inhibitors we confirmed that invasion in cells lacking LAMA3 is ROCK- dependent. These findings demonstrate that Lam332 is a major regulator of SCC differentiation and invasion.
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MiR-4286 inhibition alters proliferation and apoptosis in melanoma cells N Palkina, A Komina, M Aksenenko and T Ruksha Pathophysiology, Krasnoyarsk State Medical University, Krasnoyarsk, Russian Federation MicroRNAs are considered as essential regulators of gene expression in malignant tumors with prooncogenic or tumor suppressive capacities. More than 2500 types of microRNAs identified at the present time although the precise role in carcinogenesis of the most of them remains unclear. Our study aimed to compare melanoma and melanocytic nevi profiles to determine microRNAs which are characterized by different expression pattern in melanoma as compared to benign melanocytic tumors for unveiling their functional role in carcinogenesis. Melanoma (n¼23) and melanocytic nevi (n¼9) samples were obtained from patients of Krasnoyarsk Regional Oncology center. MicroRNA expression pattern was determined by GeneAtlasÔ Microarray System (Affymetrix, CA, USA). Bro melanoma cell line was grown at 37 C in 5% CO2-incubator. Cell apoptosis detection was performed by flow cytometry, melanoma cells survival was estimated by MTT-test. Migration and invasion experiments were performed by CytoSelectÔ 24-Well Cell Migration and Invasion Assay (Cell Biolabs, Inc., USA). DIANA-mirPath v.3.0 was used for the KEEG pathway analysis of miRNA signature. MiRNA targets were predicted based on TargetScan 7.0, miRWalk 2.0, and miRTarBase v.4.5. MiR-4286 was more than 17.8 times up-regulated in melanoma in comparison to benign melanocytic tumors in our study. The efficiency of cell transfection with anti-miR-4286 was estimated by the real-time PCR analysis of HMGA2 expression levels in the transfected cells. MTT assay was performed in 24, 48, 72, and 96 h after transfection. The inhibition of miR4286 activity resulted in a significant decrease of melanoma cells viability/proliferation. By flow cytometry, it was found that miR-4286 inhibition in melanoma cells led to the increase in 2.6-fold in apoptosis rate. Inhibition of miR-4286 does not alter melanoma cells migration and invasion. Our results indicate that miR-4286 is involved in regulation of melanoma cells’ apoptosis and proliferation by mechanisms requiring further investigation.
The bioinformatics workflow for epigenetics profiling of progresing melanoma K Poterlowicz and K Murat Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom The poor-prognosis tumours are often lack genetic well characterized alterations but are epigenetically heterogeneous, pointing to the important role of the multi-domain epigenetic regulation in cancer progression. In the recent years researchers studying epigenetic regulation of tumor progression generated a vast amount of high-throughput sequencing data for processes such as DNA methylation and modification of histones. Despte of it, there is a lack of multi-layers epigenetic workflows characterizing tumor progression regulation. Here we integrated the DNA methylation profiles at the different stages of melanoma and ChIP-Seq profiles for H3K27me3, H3K4me3, MITF, BRG1 to identify novel epigenetic switches responsible for metastatic progression of this tumor and correlate them with the survival rates of the patient. Results and experiences using this framework demonstrate the potential for bioinformatics solution for multi-omics cancer biomarker discovery tool.
Meta-analysis of genomic datasets to identify recurrent mutations and altered pathways in cutaneous T-cell lymphoma L Chang and L Geskin Dermatology, Columbia University, New York, NY Recent studies have used whole genome or whole exome sequencing to identify recurrently mutated genes in cutaneous T-cell lymphoma (CTCL). However, these studies usually include tumors of multiple types and stages. Due to the genomic heterogeneity of CTCL, discovering genes or pathways that play a central role in CTCL pathogenesis remains challenging. Here, we combined and co-analyzed recently published genomic sequencing datasets of cutaneous T cell lymphoma. We generated a list of 6653 single nucleotide mutations in CTCL samples, including 67 Sezary syndrome and 19 mycosis fungoides. We found 19 recurrently mutated loci, including those for R48W in PLCG1, Q575E in RLTPR and a nonsense mutation of Y331 in CCR4. When the list of mutations is used to study commonly mutated genes, we found previously reported genes as well as genes that were not deemed significantly mutated by individual studies. Importantly, frequently mutated genes in mycosis fungoides and Sezary syndrome overlap. Among the most frequently mutated genes are PCLO (20%), TP53 (16%) and FAT3 (16%). Sucrase-isomaltase (SI) a commonly mutated genes in CLL, is also identified as a recurrently mutated genes in Sezary syndrome. When mutations were analyzed in the context of biological pathways, we found genes in the T cell receptor pathway and the JAK/STAT pathway are frequently mutated in Sezary syndrome, including TNFR2 (8%), PLCG1 (11%) and CARD11 (11%). To conclude, this study validated previous reported recurrent mutations in CTCL and identified additional genes that are frequently mutated. These results provide new information on the molecular mechanism of CTCL pathogenesis.
Expression of retinoic acid receptor-related orphan receptors (ROR) a and g correlates with hypoxia in cutaneous melanomas AA Brozyna1, W Jozwicki1, M Pawlikowska2, E Wedrowska4 and A Slominski3 1 Department of Tumor Pathology and Pathomorphology, Collegium Medicum, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland, 2 Department of Immunology, Nicolaus Copernicus University, Torun, Poland, 3 Departments of Dermatology and Pathology, University of Alabama at Birmingham, Birmingham, AL and 4 Department of Gene Therapy, Collegium Medicum, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland Hypoxia is one of the hallmarks of the tumor microenvironment and up-regulation of hypoxia-inducible factor 1 alpha (HIF-1a) is considered as a negative prognostic marker. Recently the role of retinoic aciderelated orphan receptor (ROR) a and g in hypoxia signaling pathway have been identified. RORa and RORg are involved in many physiological functions in several organs. Previously we showed that RORs are expressed in human normal skin and melanocytic tumors including nevi and melanomas. Furthermore, we reported that HIF-1a expression and HIF-1a dependent pathways can be regulated by melanogenesis and are related to poorer prognosis of melanoma. In this study using immunohistochemistry, we examined the relationship between RORa and RORg and HIF-1a in 73 cutaneous melanomas (36, 35 and 2, nodular, superficial spreading and acral types, respectively) and 34 metastases. In both, primary melanomas and metastases, the HIF-1a immunostaining increased with increasing RORa expression. This relationship was more evident in advanced melanomas (pT3-pT4) and was also observed in metastases. On the contrary, HIF-1a expression was higher in RORg-negative advanced melanomas, while in pT1-pT2 melanomas there were no differences in its expression levels. In metastases, HIF-1a level was higher in RORg-positive cases. Furthermore, HIF-1a expression was elevated in primary melanomas showing brisk tumor-infiltrating lymphocytes (TILs) response. Thus, our results indicate that hypoxia defined by an up-regulated expression of HIF-1a is differentially related to the expression of RORa and RORg in melanomas. We suggest that mutual interaction between those regulators may affect behavior of human melanoma.
www.jidonline.org S241