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Descoberta de moduladores de FOXO para tratamentos anticancerígenos e antienvelhecimento
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Abstract(s)
A localização subcelular aberrante de proteínas supressoras de tumor é uma característica de diversas doenças humanas, incluindo o cancro. FOXOs são fatores de transcrição que quando se encontram no núcleo, controlam a transcrição de genes envolvidos em processos biológicos fundamentais na proteção contra o cancro e envelhecimento. Estas proteínas pertencem à via de sinalização mais frequentemente ativa em cancros humanos- a via PI3K/Akt e encontram-se inativos e localizados no citoplasma em diversos cancros humanos devido a modificações pós-traducionais. Assim, a sua manipulação farmacológica pode ser benéfica no tratamento de cancro e de doenças associadas ao envelhecimento.
O nosso laboratório aplicou três abordagens distintas (bioinformática, screening e candidate approach) para descobrir e caracterizar diferentes compostos anticancerígenos e antienvelhecimento que modulem a localização de FOXO. Para isso, utilizámos um sistema celular previamente estabelecido que expressa de forma estável a proteína de fusão FOXO3a-GFP de forma a monitorizar a localização subcelular de FOXO. Por um lado, utilizámos uma ferramenta bioinformática, o cMAP, para obter uma lista de diferentes compostos com capacidade de produzir um perfil de expressão génica inverso ao de TRIB2, uma proteína repressora de FOXO. Harmina e Piperlongumina foram dois desses compostos e revelaram capacidade de induzir a localização nuclear de FOXO-GFP e de afetar a transcrição dos seus genes-alvo (p21, p27 e PUMA). No caso de Piperlongumina, a modulação da ativação de FOXO é através do Akt. Por outro lado, testámos 32 extratos de microalgas (Alg1-Alg18) e de haloarqueas (Alg19-Alg32) e 3 péptidos (Pep1, Pep2, Pep3) e 10 deles mostraram capacidade de induzir a localização nuclear de FOXO-GFP. Por fim, testámos fármacos com atividade e efeitos conhecidos, nomeadamente três inibidores de mTOR - Rapamicina, Torin 1 e CID613034- e um inibidor de MEK1 e MEK2- Trametinib, que mostram que Torin 1 induz a localização nuclear de FOXO-GFP.
The aberrant subcellular localization of tumour-suppressor proteins is a prominent feature of many human diseases as well as cancer. The FOXO family of proteins are transcription factors that need to be in the cell nucleus to regulate several biological processes essential for cancer and aging. FOXO proteins are part of the most frequently activated pathway in human cancer - the PI3K/Akt pathway and are inactive and located in the cytoplasm in several human cancers due to post-translational modifications. In addition, FOXO3 has been identified as the second most replicated genetic factor associated with extreme human longevity. Thus, their pharmacological manipulation might be beneficial for the treatment of cancer and other age-related diseases. The Link lab has applied three different approaches (bioinformatic, screening and candidate approach) to discover and characterize anti-cancer and anti-aging drugs that modulate FOXO localization. Therefore, we used a previously established cellular system which stably expresses a FOXO3a-GFP fusion protein, allowing us to monitor FOXO subcellular localization. Using the bioinformatic tool connectivity MAP (cMAP), we obtained a list of candidate drugs including the natural products Harmine and Piperlongumine capable of producing a gene expression profile inverse to the profile induced by the FOXO repressor TRIB2. Accordingly, Harmine and Piperlongumine induced FOXO-GFP nuclear localization and affected the transcription of FOXO target genes (p21, p27 and PUMA). Piperlongumine modulates FOXO activation through Akt. We also tested 32 microalgae (Alg1-Alg18) and haloarchaea (Alg19-Alg32) extracts and 3 peptides (Pep1, Pep2, Pep3) for their capacity to induce FOXO-GFP nuclear localization. Ten extracts and one peptide were identified to induce the nuclear accumulation of FOXO. Finally, we analyzed known drugs with known activity and effects: three mTOR inhibitors - Rapamycin, Torin 1 and CID613034- and a MEK1 and MEK2 inhibitor -Trametinib and found that Torin 1 induces FOXO-GFP nuclear localization.
The aberrant subcellular localization of tumour-suppressor proteins is a prominent feature of many human diseases as well as cancer. The FOXO family of proteins are transcription factors that need to be in the cell nucleus to regulate several biological processes essential for cancer and aging. FOXO proteins are part of the most frequently activated pathway in human cancer - the PI3K/Akt pathway and are inactive and located in the cytoplasm in several human cancers due to post-translational modifications. In addition, FOXO3 has been identified as the second most replicated genetic factor associated with extreme human longevity. Thus, their pharmacological manipulation might be beneficial for the treatment of cancer and other age-related diseases. The Link lab has applied three different approaches (bioinformatic, screening and candidate approach) to discover and characterize anti-cancer and anti-aging drugs that modulate FOXO localization. Therefore, we used a previously established cellular system which stably expresses a FOXO3a-GFP fusion protein, allowing us to monitor FOXO subcellular localization. Using the bioinformatic tool connectivity MAP (cMAP), we obtained a list of candidate drugs including the natural products Harmine and Piperlongumine capable of producing a gene expression profile inverse to the profile induced by the FOXO repressor TRIB2. Accordingly, Harmine and Piperlongumine induced FOXO-GFP nuclear localization and affected the transcription of FOXO target genes (p21, p27 and PUMA). Piperlongumine modulates FOXO activation through Akt. We also tested 32 microalgae (Alg1-Alg18) and haloarchaea (Alg19-Alg32) extracts and 3 peptides (Pep1, Pep2, Pep3) for their capacity to induce FOXO-GFP nuclear localization. Ten extracts and one peptide were identified to induce the nuclear accumulation of FOXO. Finally, we analyzed known drugs with known activity and effects: three mTOR inhibitors - Rapamycin, Torin 1 and CID613034- and a MEK1 and MEK2 inhibitor -Trametinib and found that Torin 1 induces FOXO-GFP nuclear localization.
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Keywords
Cancros humanos Envelhecimento humano Inativação de foxo Manipulação farmacológica Localização subcelular aberrante Translocação nucleocitoplasmática de proteínas