Assinaturas de miRNAs envolvidos na malignidade, EMT e metástase revelam o papel do miR-322-5p na plasticidade fenotípica do melanoma
Data
2022-11-23
Autores
Perestrelo, Bruna de Oliveira 
Orientadores
Jasiulionis, Miriam Galvonas
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A compreensão das bases moleculares do melanoma é crucial para o desenvolvimento de novos alvos terapêuticos e identificação de biomarcadores. Entre as alterações moleculares observadas estão aquelas que ocorrem na classe de RNA não codificantes conhecidos como miRNAs. É reconhecida a importância dos miRNAs na progressão tumoral do melanoma. O melanoma apresenta subpopulações de células com diferentes fenótipos e assinaturas gênicas especificas, resultantes da plasticidade celular que tem como consequência a heterogeneidade tumoral e resposta incompleta às terapias. Em nosso laboratório, desenvolvemos um modelo murino de progressão do melanoma, baseado na transformação maligna de melanócitos induzida por condição sustentada de estresse, composto por várias linhagens celulares correspondendo a diferentes etapas da progressão tumoral. A partir de análises de dados prévios de expressão de um painel de miRNAs nas linhagens de melanócitos diferenciados melan-a, melanócitos pré-malignos indiferenciados e mesenquimal-like 4C, células de melanoma pouco proliferativas, indiferenciadas e mesenquimal-like 4C11- e células de melanoma altamente proliferativas, diferenciadas e metastáticas 4C11+, selecionamos o mmu-miR-322-5p, considerando seu envolvimento em processos como diferenciação e remodelamento tecidual e sua relevância na tumorigênese. Em relação ao seu papel no câncer, esse miRNA foi descrito como regulador da transição epitélio-mesênquima (EMT) no câncer de mama e de próstata e em carcinoma de células escamosas de língua. No entanto, seu papel no melanoma ainda não está claro. O objetivo deste estudo foi elucidar um possível papel desse miRNA na plasticidade celular observada no melanoma. No nosso modelo de estudo, o mmu-miR-322-5p tem expressão elevada em assinaturas de malignidade (em células 4C, 4C11- e 4C11+ comparado aos melanócitos melan-a) e EMT (em células indiferenciadas 4C e 4C11- comparado às células diferenciadas melan-a e 4C11+). A superexpressão desse miRNA na linhagem celular metástica e diferenciada 4C11+ resultou em inibição da proliferação celular e migração coletiva, mas não afetou o crescimento tumoral in vivo. Além disso, o aumento de expressão do mmu-miR-322-5p levou à redução significativa da expressão de marcadores epiteliais (E-caderina e β-catenina) e aumento do marcador mesenquimal N-caderina. Entre os diversos genes potencialmente regulados pelo hsa-miR-424, ortólogo humano do mmu-miR-322-5p, está o Cdh1 (E-caderina). Ensaios de repórter de luciferase serão realizados para determinar se Cdh1 e outros alvos selecionados por análises in sílico são alvos diretos de regulação pelo mmu-miR-322-5p em células de melanoma. Este estudo revelou um papel do mmu-miR-322-5p na regulação da mudança de fenotípicos no melanoma, afetando drasticamente a expressão de marcadores mesenquimais e epiteliais. Estratégias terapêuticas alvejando esse miRNA poderiam ser futuramente consideradas visando a indução de um estado fenotípico proliferativo mais responsivo a terapias convencionais.
Understanding the molecular basis of melanoma is crucial for the development of new therapeutic targets and the identification of biomarkers. Among the observed molecular changes are those that occur in the class of non-coding RNAs known as miRNAs. The importance of miRNAs in melanoma tumor progression is well-recognized. Melanoma presents cell subpopulations with different phenotypes and specific gene signatures, resulting from cellular plasticity, that contributes to tumor heterogeneity and incomplete response to therapies. In our laboratory, we developed a murine model of melanoma progression, based on the malignant transformation of melanocytes induced by a sustained stress condition, composed of several cell lines corresponding to different stages of tumor progression. From the analysis of previous data on the expression of a panel of miRNAs in differentiated melan-a melanocytes, LOW PROLIFERATIVE, undifferentiated and mesenchymal-like 4C pre-malignant melanocytes, low proliferative, undifferentiated and mesenchymal-like 4C11- melanoma cells, and highly proliferative, differentiated and metastatic 4C11+ melanoma cells, we selected mmu-miR-322-5p, considering its involvement in processes such as tissue differentiation and remodeling and its relevance in tumorigenesis. Regarding its role in cancer, this miRNA has been described as a regulator of the epithelial-mesenchymal transition (EMT) in breast and prostate cancer and in squamous cell carcinoma of the tongue. However, its role in melanoma is still unclear. The aim of this study was to elucidate a possible role of this miRNA in the cellular plasticity observed in melanoma. In our study model, mmu-miR- 322-5p is highly expressed in signatures of malignancy (in 4C, 4C11- and 4C11+ cells compared to melan-a melanocytes) and EMT (in 4C and 4C11- undifferentiated cells compared to differentiated melan-a and 4C11+). The overexpression of this miRNA in the metastatic and differentiated cell line 4C11+ resulted in inhibition of cell proliferation and collective migration but did not affect tumor growth in vivo. Furthermore, the increase in the expression of mmu-miR-322-5p led to a significant reduction in the expression of epithelial markers (E-cadherin and β-catenin) and an increase in the mesenchymal marker N-cadherin. Among the various genes potentially regulated by hsa-miR-424, the human ortholog of mmu-miR-322-5p, is Cdh1 (coding for E-cadherin protein). Luciferase reporter assays will be performed to determine whether Cdh1 and other targets selected by in silico analysis are direct targets of mmu-miR-322-5p in melanoma cells. This study revealed a role of mmu-miR-322-5p in the regulation of phenotypic changes in melanoma, dramatically affecting the expression of mesenchymal and epithelial markers. Therapeutic strategies targeting this miRNA could be considered in the future aimed at inducing a proliferative phenotypic state more responsive to conventional therapies.
Understanding the molecular basis of melanoma is crucial for the development of new therapeutic targets and the identification of biomarkers. Among the observed molecular changes are those that occur in the class of non-coding RNAs known as miRNAs. The importance of miRNAs in melanoma tumor progression is well-recognized. Melanoma presents cell subpopulations with different phenotypes and specific gene signatures, resulting from cellular plasticity, that contributes to tumor heterogeneity and incomplete response to therapies. In our laboratory, we developed a murine model of melanoma progression, based on the malignant transformation of melanocytes induced by a sustained stress condition, composed of several cell lines corresponding to different stages of tumor progression. From the analysis of previous data on the expression of a panel of miRNAs in differentiated melan-a melanocytes, LOW PROLIFERATIVE, undifferentiated and mesenchymal-like 4C pre-malignant melanocytes, low proliferative, undifferentiated and mesenchymal-like 4C11- melanoma cells, and highly proliferative, differentiated and metastatic 4C11+ melanoma cells, we selected mmu-miR-322-5p, considering its involvement in processes such as tissue differentiation and remodeling and its relevance in tumorigenesis. Regarding its role in cancer, this miRNA has been described as a regulator of the epithelial-mesenchymal transition (EMT) in breast and prostate cancer and in squamous cell carcinoma of the tongue. However, its role in melanoma is still unclear. The aim of this study was to elucidate a possible role of this miRNA in the cellular plasticity observed in melanoma. In our study model, mmu-miR- 322-5p is highly expressed in signatures of malignancy (in 4C, 4C11- and 4C11+ cells compared to melan-a melanocytes) and EMT (in 4C and 4C11- undifferentiated cells compared to differentiated melan-a and 4C11+). The overexpression of this miRNA in the metastatic and differentiated cell line 4C11+ resulted in inhibition of cell proliferation and collective migration but did not affect tumor growth in vivo. Furthermore, the increase in the expression of mmu-miR-322-5p led to a significant reduction in the expression of epithelial markers (E-cadherin and β-catenin) and an increase in the mesenchymal marker N-cadherin. Among the various genes potentially regulated by hsa-miR-424, the human ortholog of mmu-miR-322-5p, is Cdh1 (coding for E-cadherin protein). Luciferase reporter assays will be performed to determine whether Cdh1 and other targets selected by in silico analysis are direct targets of mmu-miR-322-5p in melanoma cells. This study revealed a role of mmu-miR-322-5p in the regulation of phenotypic changes in melanoma, dramatically affecting the expression of mesenchymal and epithelial markers. Therapeutic strategies targeting this miRNA could be considered in the future aimed at inducing a proliferative phenotypic state more responsive to conventional therapies.