Cuomo, Gaia (2014) Neuroendocrine aspects of cutaneous melanoma: focus on somatostatin receptors (SSTRs) expression and role of the pan-SSTR-agonist Pasireotide on cell proliferation. [Tesi di dottorato]

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Tipologia del documento: Tesi di dottorato
Lingua: English
Titolo: Neuroendocrine aspects of cutaneous melanoma: focus on somatostatin receptors (SSTRs) expression and role of the pan-SSTR-agonist Pasireotide on cell proliferation
Autori:
AutoreEmail
Cuomo, Gaiagaia.cuomo@libero.it
Data: 31 Marzo 2014
Numero di pagine: 87
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Medicina Molecolare e Biotecnologie Mediche
Scuola di dottorato: Medicina molecolare
Dottorato: Oncologia ed endocrinologia molecolare
Ciclo di dottorato: 26
Coordinatore del Corso di dottorato:
nomeemail
Santoro, Massimomasantor@unina.it
Tutor:
nomeemail
Pivonello, Rosario[non definito]
Colao, Annamaria[non definito]
Data: 31 Marzo 2014
Numero di pagine: 87
Parole chiave: melanoma, neuroendocrine, somatostatin
Settori scientifico-disciplinari del MIUR: Area 06 - Scienze mediche > MED/13 - Endocrinologia
Depositato il: 11 Apr 2014 10:26
Ultima modifica: 15 Lug 2015 01:02
URI: http://www.fedoa.unina.it/id/eprint/9967

Abstract

Cutaneous malignant melanoma (CMM) is an aggressive malignancy whose incidence and mortality has increased worldwide. CMM is the most common cause of death from skin cancer. Wide surgical excision of early stage melanoma remains the main curative treatment. Unresectable advanced melanoma presents an aggressive behavior, tendency to rapidly metastasize and an intrinsic resistance to chemotherapy. The only targeted therapy approved for melanoma is vemurafenib, a small molecule targeting BRAF particularly when affected by common mutations in the nucleotides encoding for the aminoacid V600. These evidences suggest that novel therapeutic options for advanced CMM are still required. Melanocytes derive from neural crest cells and melanoma cells can express somatostatin receptors (SSTRs) suggesting that at least a subgroup of melanomas could have a neuroendocrine differentiation. The role of somatostatin (SST) pathway in CMM has been scantly investigated. The aim of this project was to evaluate SSTRs expression and to define the effects of SST analogs in relation to SSTR protein expression in in vitro models of CMM, exploring the role of SST pathway as a potential therapeutic target in human CMM. With this propose four cutaneous melanoma cell lines: A375, HMCB, COLO38 and M14 were used as in vitro models of CMM. The expression of SSTRs was evaluated by retro transcriptase quantitative polymerase chain reaction (RT-qPCR) and immunocitochemisty (ICC) in all four cell lines. The in vitro effects of daily administration of SST analogs pasireotide and octreotide and the BRAF inhibitor vemurafenib on cell viability, proliferation and cell cycle were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, DNA assay and fluorescence-activated cell sorting (FACS), respectively. Additionally the in vitro effects of daily administration of pasireotide in combination with vemurafenib, on cell viability, proliferation and cell cycle were investigated in two of the four cell lines (A375 and M14) that resulted sensitive to the effects of pasireotide. All tested melanoma cell lines express SSTR mRNA and proteins. At mRNA level, SSTR2 was the most expressed receptor followed by SSTR1, SSTR3 and SSTR5. The protein expression of SSTR1 was strong in A375, COLO38, M14 and moderate for HMCB; protein expression of SSTR2 was mild for A375 and COLO38, moderate for COLO38 and strong for M14; protein expression of SSTR5 was strong for A375 and M14, moderate for HMCB and mild for COLO38. The expression of SSTRs by ICC showed a predominant cytoplasmic localization in all melanoma cell lines used. Moreover, a perinucear staining for SSTR2 in COLO38 cells and for SSTR5 in HMCB and COLO38 cells was observed. Pasireotide significantly inhibited in a dose dependent-manner viability in A375 and M14 melanoma cell lines (maximal effects observed at dose of 10-7M: 41% p<0.01 and 44% p<0.001 vs control, respectively). Octreotide significantly inhibited cell viability only in A375 cells (maximal effects observed at dose of 10-6M; 54.15% p<0.001 vs control). Vemurafenib significantly inhibited A375 cell viability in a dose and time-dependent manner (maximal effects observed at dose of 10-6M: 82.89%, p< 0.001 vs control). Pasireotide, and vemurafenib but not octreotide significantly inhibited cell proliferation in A375 (maximal effects observed at dose of 10-6M: 20.57% and 21% p<0.05 vs control, with pasireotide and vemurafenib respectively; IC50 3*10-10M and 2.6*10-8M, respectively) and M14 cells (maximal effects observed at dose of 10-6M: 20.57% and 21% p<0.05 vs control, with pasireotide and vemurafenib respectively; IC50 3.8*10-8M and 1.228*10-7M, respectively). Combined treatment with vemurafenib and pasireotide had additive inhibitory effects in A375 only on cell viability (maximal effects observed at dose of vemurafenib 10-8M + pasireotide 10-9M: 41.79% p>0.001 vs control). The antiproliferative effects of pasireotide were observed only in cell lines presenting a strong SSTR5 protein expression (A375 and M14), suggesting that this pattern of SSTR protein expression could be predictive of response to this drug in CMM. Preliminary results of the FACS analysis suggest that the antiploferative effects of pasireotide in A375 and M14 could at least in part depend by an inhibition of cell cycle. Preliminary results of western blotting experiments, exploring the subcellular localization of SSTR2 and 5 in basal condition and after pasireotide or octreotide, suggest that the different trafficking of SSTR2 and 5 might explain the stronger antiproliferative effects observed with pasireotide compared to octreotide in these two melanoma cell lines. In conclusion this study firstly described the protein expression of SSTRs and suggested that the antiproliferative effects of pasireotide in human cutaneous melanoma cell lines could be related to a particular pattern of SSTR protein expression. This study has a potential translational value since the expression of SSTRs might indicate the potential use of SST analogs, radio-labeled SST analogs, SST analogs conjugate with chemotherapic agents and SSTR scintigraphy in the management of a subset of patients with CMM. This study encourages further studies to better define the role of SST pathway in diagnosis, prognosis and as potential target for treatment in human CMM.

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