- Email: [email protected]
499 MicroRNA-203 inhibits SLUG and suppresses melanoma cell motility, tumor growth and lung-metastasis
Melanoma and Other Skin Cancers | ABSTRACTS 495
496
Hyperactive NRAS downstream signaling induces specific transcriptome changes eesiRNA based identification of new therapeutic targets in NRAS mutant melanoma identifies the noncoding RNA 7SL as a major proliferation enhancer I Vujic1, M Vujic1, M Sanlorenzo2, C Posch1, A Preschitz2, R Esteve-Puig2, K Lai2, W Ho2, K Rappersberger1 and S Ortiz-Urda2 1 Dermatology, KA Rudolfstiftung, Vienna, Austria and 2 Dermatology, University of California, San Francisco, San Francisco, CA Benign naevi and malignant melanomas both can have oncogenic mutations in the NRAS gene but naevi only rarely progress to cancer. Such NRAS mutations should lead to specific transcriptome changes. The knowledge of these changes can A) identify new therapeutic targets and B) explain why some naevi have NRAS mutations but stay benign. Here, we introduce NRAS(Q61) mutant plasmids in a pool of human melanocytes to overactivate NRAS downstream pathways. We perform deep RNASeq and compare transcriptome changes in melanocytes with and without NRAS mutation. We list differentially expressed coding and noncoding transcripts by filtering our results with transcriptomes from 2 NRAS mutant melanoma cell lines and 89 NRAS mutant patient tumors. Next we use esiRNA (Endoribonuclease-prepared siRNA) libraries to knock down these transcripts and perform proliferation assays to identify potential targets. For most promising candidates we perform further siRNA knockdowns, cell based assays and take a closer look at their mechanistics. Our approach identified 237 transcripts, of which 2 coding and 6 noncoding transcripts played an important role in the proliferation of NRAS mutant melanoma. The knockdown of each of these transcripts led to cell proliferation decreases of 30-60%. We focused on the noncoding RNA RN7SL1 and identified its up-stream regulation (MAPK pathway) and its downstream effectors (p53). In conclusion, we identify new therapeutic targets which might be used in the battle against NRAS mutant melanoma.
Radiation therapy in melanoma patients with poor clinical characteristics e a collaborative experience with 107 patients C Posch1, C Steffal2, A Haider1, B Flechl2, T Kann2, F Weihsengruber1, A Schratter-Sehn2 and K Rappersberger1 1 Dermatology, The Rudolfstiftung Hospital, Vienna, Austria and 2 Radiooncology, Kaiser-Franz-JOsef Hospital, Vienna, Austria Radiation therapy (RTX) is an important part of melanoma management. Even though it is effective for local disease control, the lack of improved overall survival often dampens enthusiasm for this therapeutic modality. We describe our collaborative experience treating 107 stage III and stage IV melanoma patients managed at a dermatology department and a radio-oncology center using therapeutic or adjuvant radiation. Our cohort comprised a subset of melanoma patients with unfavorable clinical characteristics (median Breslow thickness: 2.8mm (range 0.5-18mm); clinical stage at diagnosis: III+IV n¼95). The median age at diagnosis was 60.3 (13.6-90.0) years the median age at start of irradiation was 66.2 (20.691.5) years. The axillary area was the most frequent anatomical site for irradiation (n¼30) followed by bone irradiation (n¼20). The majority of patients received additional systemic therapy including immunotherapy (n¼60), chemotherapy (n¼50) and targeted therapy (n¼7). Average overall survival (OS) was calculated with 57.3 (SD52.1) months. Average overall survival after RTX (OSaRTX) was 17.1 (SD18.7) months. Loco-regional recurrence (LRR) of melanoma was observed in 17 irradiated areas. No difference in OS or OSaRTX and locoregional recurrence was found: OS without LRR 57.6 (SEM 5.7) years; OS with LRR 55.6 (SEM 9.2) years; OSaRTX without LRR 16.2 (SEM 1.8) years, OSaRTX with LRR 20.6 (SEM 3.8) years (all p>0.05). LDH serum levels did not correlate with local recurrence (r¼0.07). Subgroup analyses revealed that OSaRTX for axillary irradiation was 26.3 (SD24.6) months. RTX was well tolerated with the majority of acute adverse events being grade 1 (CTCAE v4.02). 11 patients experienced late irradiation adverse effects (all grade 1; RTOG/EORTC score). From our data we conclude that RTX is a well-tolerated therapeutic modality with the potential for high local disease control, particularly in the axillary region.
497
498
Eleven years of melanoma patient management e observations and trends from a single-center study in Austria C Posch, V Feichtenschlager, F Weihsengruber and K Rappersberger Dermatology, The Rudolfstiftung Hospital, Vienna, Austria Recent studies indicate a constant increase of melanoma incidence rates over the last decades. Mortality remained largely unchanged, which is thought to be due to improved early detection and better treatment. The aim of this study was to characterize melanoma patients managed at a single-center institution in Vienna between 2000 and 2010. 1329 patients with a mean age of 59.116.7 years at diagnosis were analyzed. Women were significantly younger than men at the time of diagnosis (\57.217.8 years vs. _61.0 15.2 years; p<0.001). Only a small number of patients were younger than 31 years (5.6%). Superficial spreading melanoma (39.5%) was the most frequent histological subtype, followed by nodular melanoma (14.9%), lentigo maligna melanoma (5.2%) and acral melanoma (2.6%). In 25.8% of patients the histological subtype could not be determined. The mean Breslow thickness (BT) was calculated with 1.81mm and consistently increased with the age of patients (age group 31-40:1.211.42mm; age group 71-80:2.322.63mm). The vast majority of tumors were detected at early tumor stages (TMIS:7.4%; T1a/b:41.5%). None of the TMIS and 3.1% of patients with tumor stage IA progressed. Out of all SLN biopsies, 17.3% of patients had a positive SLN; of those, 38.3% progressed. 12.9% of sentinel negative patients also progressed. In total, 11.3% of patients had disease progression of which 70.7% succumbed to melanoma. The number of patients with progressive disease increased from 3,1% when diagnosed at clinical stage IA to 50,0% for clinical stage IIIC. Women had a better 5-year overall survival compared to men (75.8% vs. 63.6%; p¼0.025). Findings of this study highlight that early detection is effective for preventing metastatic spread. We did not observe a decrease of median BT at diagnosis during the study period of 11 years. This might be explained by the high number of stage IB patients who require hospitalization for SLN biopsy. Alternatively, this could also indicate that melanoma awareness campaigns of the recent past need to be refocused.
C3 and complement factor B regulate growth of cutaneous squamous cell carcinoma P Riihila¨1, L Nissinen1, M Farshchian1, M Kallajoki2, A Kivisaari1, S Meri3, T Pihlajaniemi4, R Heljasvaara4, S Peltonen1 and V Ka¨ha¨ri1 1 Department of Dermatology, University of Turku and Turku University Hospital, Turku, Finland, 2 Department of Pathology, Turku University Hospital, Turku, Finland, 3 Haartman Institute, University of Helsinki, Helsinki, Finland and 4 Biocenter Oulu and the Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland The incidence of cutaneous squamous cell carcinoma (cSCC) and its precancerous forms is rising globally. Here, we studied the role of complement components C3 and complement factor B (CFB) in the progression of cSCC. Analysis of cSCC cell lines (n¼8) and normal human epidermal keratinocytes (n¼11) with quantitative RT-PCR and western blotting showed significant overexpression of C3 and CFB in cSCC cells. In cSCC tumors (n¼6) the mRNA levels for C3 and CFB were markedly higher than in normal skin (n¼10). Immunohistochemical analysis for C3 and CFB showed stronger intensity of staining in invasive sporadic cSCCs (n¼71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (RDEBSCC, n¼11) than in premalignant epidermal lesions (actinic keratoses, n¼65), in cSCC in situ (n¼69) and normal skin (n¼5) in vivo. Significant up-regulation of C3 and CFB mRNA expression was seen in chemically induced mouse skin cSCCs (n¼27) compared to benign papillomas (n¼17). The expression of C3 and CFB was higher in aggressive Ha-ras -transformed cell line (RT-3) than in normal epidermal keratinocytes or in less tumorigenic HaCaT cell lines (HaCaT, A5, II-4) at mRNA and protein level. The basal expression level was markedly up-regulated by IFN-g and TNF-a in cSCC cells. Knockdown of CFB with specific siRNA inhibited migration and proliferation of cSCC cells and this was associated with potent inhibition of ERK1/2 activation. Moreover, knockdown of C3 and CFB significantly inhibited the growth of human cSCC xenograft tumors in vivo. The results provide evidence for the role of C3 and CFB in progression of cSCC and identify C3 and CFB as putative therapeutic targets in cSCC.
499
500
MicroRNA-203 inhibits SLUG and suppresses melanoma cell motility, tumor growth and lung-metastasis W Lohcharoenkal, K Das Mahapatra, L Zhang, N Lande´n, L Girnita, M Mona Sta˚hle, E Sonkoly and A Pivarcsi Karolinska Institutet, Stockholm, Sweden MicroRNAs (miRNAs) are a class of small non-coding RNAs, -22nt in length, which can regulate gene expression at the post-transcriptional level by binding to the 3’UTR of the target mRNAs and subsequently leading to their degradation or translation arrest. We and others have shown that miR-203 functions as a tumor suppressor in several cancer types. In this study, we investigated the function of miR-203 in malignant melanoma using in vitro and in vivo models of tumor growth and metastasis. We observed that miR-203 was expressed at low level in melanoma cell lines and that its overexpression suppressed cell migration and invasion. Moreover, miR-203 decreased the colony-forming ability and angiogenesis-inducing capacity of melanoma cells. Transcriptome profiling revealed that miR-203 suppressed gene sets related to KRAS-signaling and Epithelial-to-Mesenchymal Transition (EMT) in melanoma cells. MiR-203 inhibited the expression of SLUG, a member of the Snail family C2H2-type zinc finger transcription factors, both at mRNA and protein level and we identified it as a direct target of miR-203 for post-transcriptional gene silencing. Specific knock-down of SLUG led to similar results in terms of melanoma cell motility as overexpression of miR-203. In vivo, miR-203 suppressed primary tumor growth and lymphatic metastasis to inguinal lymph nodes in xenograft model. Moreover, miR-203 inhibited metastasis to the lung in mouse model of melanoma metastasis. These results indicate that miR-203 suppress motility and metastasis of melanoma by down-regulating SLUG, and that miR-203 may become a novel therapeutic target in melanoma.
Exosomal microRNAs as putative predictive biomarkers for targeted therapy in stage IV cutaneous malignant melanoma W Lohcharoenkal1, FC Svedman2, E Sonkoly3, SE Brage2, J Hansson2, A Pivarcsi1 and H Eriksson2 1 Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, 2 Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden and 3 Unit of Dermatology, Karolinska University Hospital, Stockholm, Sweden MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the posttranscriptional level. Exosomes are nanometer-sized extracellular vesicles secreted by all types of cells and found in body fluids. MiRNAs are carried within exosomes and has been suggested to be involved in intercellular communication. MAP-kinase inhibitors (MAPKi) are widely used to treat patients with incurable cutaneous malignant melanoma (CMMs) harboring BRAFV600 mutations, inducing rapid tumor responses but limited durable benefit due to the emergence of resistance. Our aim was to identify possible biomarkers of durable treatment response to MAPKi in stage IV CMM. Plasma samples were obtained before and during therapy from 28 CMM patients undergoing MAPKi-therapy. Expression profiling of exosomal miRNAs identified five miRNAs (miR-125b-5p, miR-99a-5p, miR-497-5p, miR145-5p and miR-26a-5p) that were significantly decreased while one (miR-20a-5p) that was significantly elevated in non-responder patients in comparison with responders. The level of exosomal miR-125b-5p in plasma samples collected during treatment was able to predict response to MAPKi therapy with 76.5% specificity and 100% sensitivity. A low miR-125b-5plevel was associated with shorter progression-free survival with a median survival of 288 vs. 96.5 days in the patient groups with high and low miR-125b-5p-level, respectively. Changes in the level of exosomal miRNAs in the plasma of melanoma patients during MAPKi therapy may be predictive biomarkers for treatment outcome.
www.jidonline.org S245
496
Hyperactive NRAS downstream signaling induces specific transcriptome changes eesiRNA based identification of new therapeutic targets in NRAS mutant melanoma identifies the noncoding RNA 7SL as a major proliferation enhancer I Vujic1, M Vujic1, M Sanlorenzo2, C Posch1, A Preschitz2, R Esteve-Puig2, K Lai2, W Ho2, K Rappersberger1 and S Ortiz-Urda2 1 Dermatology, KA Rudolfstiftung, Vienna, Austria and 2 Dermatology, University of California, San Francisco, San Francisco, CA Benign naevi and malignant melanomas both can have oncogenic mutations in the NRAS gene but naevi only rarely progress to cancer. Such NRAS mutations should lead to specific transcriptome changes. The knowledge of these changes can A) identify new therapeutic targets and B) explain why some naevi have NRAS mutations but stay benign. Here, we introduce NRAS(Q61) mutant plasmids in a pool of human melanocytes to overactivate NRAS downstream pathways. We perform deep RNASeq and compare transcriptome changes in melanocytes with and without NRAS mutation. We list differentially expressed coding and noncoding transcripts by filtering our results with transcriptomes from 2 NRAS mutant melanoma cell lines and 89 NRAS mutant patient tumors. Next we use esiRNA (Endoribonuclease-prepared siRNA) libraries to knock down these transcripts and perform proliferation assays to identify potential targets. For most promising candidates we perform further siRNA knockdowns, cell based assays and take a closer look at their mechanistics. Our approach identified 237 transcripts, of which 2 coding and 6 noncoding transcripts played an important role in the proliferation of NRAS mutant melanoma. The knockdown of each of these transcripts led to cell proliferation decreases of 30-60%. We focused on the noncoding RNA RN7SL1 and identified its up-stream regulation (MAPK pathway) and its downstream effectors (p53). In conclusion, we identify new therapeutic targets which might be used in the battle against NRAS mutant melanoma.
Radiation therapy in melanoma patients with poor clinical characteristics e a collaborative experience with 107 patients C Posch1, C Steffal2, A Haider1, B Flechl2, T Kann2, F Weihsengruber1, A Schratter-Sehn2 and K Rappersberger1 1 Dermatology, The Rudolfstiftung Hospital, Vienna, Austria and 2 Radiooncology, Kaiser-Franz-JOsef Hospital, Vienna, Austria Radiation therapy (RTX) is an important part of melanoma management. Even though it is effective for local disease control, the lack of improved overall survival often dampens enthusiasm for this therapeutic modality. We describe our collaborative experience treating 107 stage III and stage IV melanoma patients managed at a dermatology department and a radio-oncology center using therapeutic or adjuvant radiation. Our cohort comprised a subset of melanoma patients with unfavorable clinical characteristics (median Breslow thickness: 2.8mm (range 0.5-18mm); clinical stage at diagnosis: III+IV n¼95). The median age at diagnosis was 60.3 (13.6-90.0) years the median age at start of irradiation was 66.2 (20.691.5) years. The axillary area was the most frequent anatomical site for irradiation (n¼30) followed by bone irradiation (n¼20). The majority of patients received additional systemic therapy including immunotherapy (n¼60), chemotherapy (n¼50) and targeted therapy (n¼7). Average overall survival (OS) was calculated with 57.3 (SD52.1) months. Average overall survival after RTX (OSaRTX) was 17.1 (SD18.7) months. Loco-regional recurrence (LRR) of melanoma was observed in 17 irradiated areas. No difference in OS or OSaRTX and locoregional recurrence was found: OS without LRR 57.6 (SEM 5.7) years; OS with LRR 55.6 (SEM 9.2) years; OSaRTX without LRR 16.2 (SEM 1.8) years, OSaRTX with LRR 20.6 (SEM 3.8) years (all p>0.05). LDH serum levels did not correlate with local recurrence (r¼0.07). Subgroup analyses revealed that OSaRTX for axillary irradiation was 26.3 (SD24.6) months. RTX was well tolerated with the majority of acute adverse events being grade 1 (CTCAE v4.02). 11 patients experienced late irradiation adverse effects (all grade 1; RTOG/EORTC score). From our data we conclude that RTX is a well-tolerated therapeutic modality with the potential for high local disease control, particularly in the axillary region.
497
498
Eleven years of melanoma patient management e observations and trends from a single-center study in Austria C Posch, V Feichtenschlager, F Weihsengruber and K Rappersberger Dermatology, The Rudolfstiftung Hospital, Vienna, Austria Recent studies indicate a constant increase of melanoma incidence rates over the last decades. Mortality remained largely unchanged, which is thought to be due to improved early detection and better treatment. The aim of this study was to characterize melanoma patients managed at a single-center institution in Vienna between 2000 and 2010. 1329 patients with a mean age of 59.116.7 years at diagnosis were analyzed. Women were significantly younger than men at the time of diagnosis (\57.217.8 years vs. _61.0 15.2 years; p<0.001). Only a small number of patients were younger than 31 years (5.6%). Superficial spreading melanoma (39.5%) was the most frequent histological subtype, followed by nodular melanoma (14.9%), lentigo maligna melanoma (5.2%) and acral melanoma (2.6%). In 25.8% of patients the histological subtype could not be determined. The mean Breslow thickness (BT) was calculated with 1.81mm and consistently increased with the age of patients (age group 31-40:1.211.42mm; age group 71-80:2.322.63mm). The vast majority of tumors were detected at early tumor stages (TMIS:7.4%; T1a/b:41.5%). None of the TMIS and 3.1% of patients with tumor stage IA progressed. Out of all SLN biopsies, 17.3% of patients had a positive SLN; of those, 38.3% progressed. 12.9% of sentinel negative patients also progressed. In total, 11.3% of patients had disease progression of which 70.7% succumbed to melanoma. The number of patients with progressive disease increased from 3,1% when diagnosed at clinical stage IA to 50,0% for clinical stage IIIC. Women had a better 5-year overall survival compared to men (75.8% vs. 63.6%; p¼0.025). Findings of this study highlight that early detection is effective for preventing metastatic spread. We did not observe a decrease of median BT at diagnosis during the study period of 11 years. This might be explained by the high number of stage IB patients who require hospitalization for SLN biopsy. Alternatively, this could also indicate that melanoma awareness campaigns of the recent past need to be refocused.
C3 and complement factor B regulate growth of cutaneous squamous cell carcinoma P Riihila¨1, L Nissinen1, M Farshchian1, M Kallajoki2, A Kivisaari1, S Meri3, T Pihlajaniemi4, R Heljasvaara4, S Peltonen1 and V Ka¨ha¨ri1 1 Department of Dermatology, University of Turku and Turku University Hospital, Turku, Finland, 2 Department of Pathology, Turku University Hospital, Turku, Finland, 3 Haartman Institute, University of Helsinki, Helsinki, Finland and 4 Biocenter Oulu and the Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland The incidence of cutaneous squamous cell carcinoma (cSCC) and its precancerous forms is rising globally. Here, we studied the role of complement components C3 and complement factor B (CFB) in the progression of cSCC. Analysis of cSCC cell lines (n¼8) and normal human epidermal keratinocytes (n¼11) with quantitative RT-PCR and western blotting showed significant overexpression of C3 and CFB in cSCC cells. In cSCC tumors (n¼6) the mRNA levels for C3 and CFB were markedly higher than in normal skin (n¼10). Immunohistochemical analysis for C3 and CFB showed stronger intensity of staining in invasive sporadic cSCCs (n¼71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (RDEBSCC, n¼11) than in premalignant epidermal lesions (actinic keratoses, n¼65), in cSCC in situ (n¼69) and normal skin (n¼5) in vivo. Significant up-regulation of C3 and CFB mRNA expression was seen in chemically induced mouse skin cSCCs (n¼27) compared to benign papillomas (n¼17). The expression of C3 and CFB was higher in aggressive Ha-ras -transformed cell line (RT-3) than in normal epidermal keratinocytes or in less tumorigenic HaCaT cell lines (HaCaT, A5, II-4) at mRNA and protein level. The basal expression level was markedly up-regulated by IFN-g and TNF-a in cSCC cells. Knockdown of CFB with specific siRNA inhibited migration and proliferation of cSCC cells and this was associated with potent inhibition of ERK1/2 activation. Moreover, knockdown of C3 and CFB significantly inhibited the growth of human cSCC xenograft tumors in vivo. The results provide evidence for the role of C3 and CFB in progression of cSCC and identify C3 and CFB as putative therapeutic targets in cSCC.
499
500
MicroRNA-203 inhibits SLUG and suppresses melanoma cell motility, tumor growth and lung-metastasis W Lohcharoenkal, K Das Mahapatra, L Zhang, N Lande´n, L Girnita, M Mona Sta˚hle, E Sonkoly and A Pivarcsi Karolinska Institutet, Stockholm, Sweden MicroRNAs (miRNAs) are a class of small non-coding RNAs, -22nt in length, which can regulate gene expression at the post-transcriptional level by binding to the 3’UTR of the target mRNAs and subsequently leading to their degradation or translation arrest. We and others have shown that miR-203 functions as a tumor suppressor in several cancer types. In this study, we investigated the function of miR-203 in malignant melanoma using in vitro and in vivo models of tumor growth and metastasis. We observed that miR-203 was expressed at low level in melanoma cell lines and that its overexpression suppressed cell migration and invasion. Moreover, miR-203 decreased the colony-forming ability and angiogenesis-inducing capacity of melanoma cells. Transcriptome profiling revealed that miR-203 suppressed gene sets related to KRAS-signaling and Epithelial-to-Mesenchymal Transition (EMT) in melanoma cells. MiR-203 inhibited the expression of SLUG, a member of the Snail family C2H2-type zinc finger transcription factors, both at mRNA and protein level and we identified it as a direct target of miR-203 for post-transcriptional gene silencing. Specific knock-down of SLUG led to similar results in terms of melanoma cell motility as overexpression of miR-203. In vivo, miR-203 suppressed primary tumor growth and lymphatic metastasis to inguinal lymph nodes in xenograft model. Moreover, miR-203 inhibited metastasis to the lung in mouse model of melanoma metastasis. These results indicate that miR-203 suppress motility and metastasis of melanoma by down-regulating SLUG, and that miR-203 may become a novel therapeutic target in melanoma.
Exosomal microRNAs as putative predictive biomarkers for targeted therapy in stage IV cutaneous malignant melanoma W Lohcharoenkal1, FC Svedman2, E Sonkoly3, SE Brage2, J Hansson2, A Pivarcsi1 and H Eriksson2 1 Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, 2 Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden and 3 Unit of Dermatology, Karolinska University Hospital, Stockholm, Sweden MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the posttranscriptional level. Exosomes are nanometer-sized extracellular vesicles secreted by all types of cells and found in body fluids. MiRNAs are carried within exosomes and has been suggested to be involved in intercellular communication. MAP-kinase inhibitors (MAPKi) are widely used to treat patients with incurable cutaneous malignant melanoma (CMMs) harboring BRAFV600 mutations, inducing rapid tumor responses but limited durable benefit due to the emergence of resistance. Our aim was to identify possible biomarkers of durable treatment response to MAPKi in stage IV CMM. Plasma samples were obtained before and during therapy from 28 CMM patients undergoing MAPKi-therapy. Expression profiling of exosomal miRNAs identified five miRNAs (miR-125b-5p, miR-99a-5p, miR-497-5p, miR145-5p and miR-26a-5p) that were significantly decreased while one (miR-20a-5p) that was significantly elevated in non-responder patients in comparison with responders. The level of exosomal miR-125b-5p in plasma samples collected during treatment was able to predict response to MAPKi therapy with 76.5% specificity and 100% sensitivity. A low miR-125b-5plevel was associated with shorter progression-free survival with a median survival of 288 vs. 96.5 days in the patient groups with high and low miR-125b-5p-level, respectively. Changes in the level of exosomal miRNAs in the plasma of melanoma patients during MAPKi therapy may be predictive biomarkers for treatment outcome.
www.jidonline.org S245