Investigator Grant dell'Associazione Italiana per la Ricerca sul cancro (AIRC) per il triennio 2016-2019. Background Melanoma, the deadliest form of skin cancer, is an aggressive disease rising in incidence. Despite the recent advances in therapy, available treatments are still rarely successful for metastatic forms of the disease. For these reasons, the identification of new therapeutic targets is eagerly sought. The P2X7 receptor for extracellular ATP (eATP) recently emerged as central player in oncology, thanks to its ability to stimulate tumour growth, angiogenesis, metastasis and to modulate anti-tumoral immune response. Hypothesis Our preliminary data, showing a striking reduction of experimental melanoma growth upon administration of P2X7 blockers, indicate the receptor as a possible therapeutic target for the disease. We hypothesize that P2X7 receptor could orchestrate melanoma growth and progression at various levels. Firstly, P2X7 will increase cancer growth acting at intracellular pathways. Secondly, P2X7 will influence ATP secretion both from cancer and host cells, affecting the composition of tumour microenvironment. Thirdly, P2X7 will mediate vesicular release of different molecules including ATP, miRNAs and cytokines/growth factors from tumour and immune cells . Fourthly, P2X7 will be favouring melanoma spreading worsening patients' prognosis. Aims Aims of this project will be: 1) Demonstration that P2X7 receptor is an up-stream regulator of the PI3K/Akt, GSK3β/MIFT, HIF1α/VEGF pathways in melanoma cells and that P2X7 antagonists can effectively impact on these paths. 2) Identification of P2X7 as mediator of ATP secretion in cancer. 3) Association of P2X7 activation with microvesicles secretion from cancer cells 4) Establishment of a connection between P2X7 expression and melanoma metastatization in murine models and in melanoma patients. Experimental Design To address these issues we will administer P2X7 antagonists AZ10606120 and A70003 to human and murine melanoma cells (A375 and B16) and to mice bearing in situ or disseminated melanomas, derived from the same cell lines. Alterations in the activity of PI3K, GSK3β, HIF1α pathways will be analysed in vitro and ex vivo. The role of P2X7 receptor in secretion of ATP will be explored thanks to a luciferase probe detecting eATP (PmeLUC). Melanoma cells expressing PmeLUC will be injected in WT and KO P2X7 mice, treated or not with P2X7 antagonists and eATP levels will be assessed by in vivo luminometry. Preliminary experiments shows P2X7 dependent microvesicles secretion from cancer and immune cells. The molecular content of these vesicles will be analysed to determine whether they contain ATP, miRNAs and proteins linked to cancer progression. Finally, P2X7 expression levels will be quantified in a patient population by immunohistochemistry and Real Time PCR, to perform a clinical explorative study aimed at associating P2X7 expression with melanoma progression and prognosis. Expected Results We expect to demonstrate that P2X7 receptor exerts a central role in melanoma growth and progression by affecting intracellular pathways, cell-cell crosstalk and the composition of tumour microenvironment. Taken together these actions will favour melanoma growth and dissemination and make P2X7 an ideal pharmacological target for advanced melanoma treatment. Impact On Cancer P2X7 receptor antagonists are in phase III clinical trials for immune diseases, therefore they will be soon available at patients bed offering a possible life extending, easily administrable, cost-effective anti-melanoma therapy.
Role of extracellular ATP and its receptor P2X7 in melanoma cell to cell communication and progression
ADINOLFI, Elena
2016
Abstract
Investigator Grant dell'Associazione Italiana per la Ricerca sul cancro (AIRC) per il triennio 2016-2019. Background Melanoma, the deadliest form of skin cancer, is an aggressive disease rising in incidence. Despite the recent advances in therapy, available treatments are still rarely successful for metastatic forms of the disease. For these reasons, the identification of new therapeutic targets is eagerly sought. The P2X7 receptor for extracellular ATP (eATP) recently emerged as central player in oncology, thanks to its ability to stimulate tumour growth, angiogenesis, metastasis and to modulate anti-tumoral immune response. Hypothesis Our preliminary data, showing a striking reduction of experimental melanoma growth upon administration of P2X7 blockers, indicate the receptor as a possible therapeutic target for the disease. We hypothesize that P2X7 receptor could orchestrate melanoma growth and progression at various levels. Firstly, P2X7 will increase cancer growth acting at intracellular pathways. Secondly, P2X7 will influence ATP secretion both from cancer and host cells, affecting the composition of tumour microenvironment. Thirdly, P2X7 will mediate vesicular release of different molecules including ATP, miRNAs and cytokines/growth factors from tumour and immune cells . Fourthly, P2X7 will be favouring melanoma spreading worsening patients' prognosis. Aims Aims of this project will be: 1) Demonstration that P2X7 receptor is an up-stream regulator of the PI3K/Akt, GSK3β/MIFT, HIF1α/VEGF pathways in melanoma cells and that P2X7 antagonists can effectively impact on these paths. 2) Identification of P2X7 as mediator of ATP secretion in cancer. 3) Association of P2X7 activation with microvesicles secretion from cancer cells 4) Establishment of a connection between P2X7 expression and melanoma metastatization in murine models and in melanoma patients. Experimental Design To address these issues we will administer P2X7 antagonists AZ10606120 and A70003 to human and murine melanoma cells (A375 and B16) and to mice bearing in situ or disseminated melanomas, derived from the same cell lines. Alterations in the activity of PI3K, GSK3β, HIF1α pathways will be analysed in vitro and ex vivo. The role of P2X7 receptor in secretion of ATP will be explored thanks to a luciferase probe detecting eATP (PmeLUC). Melanoma cells expressing PmeLUC will be injected in WT and KO P2X7 mice, treated or not with P2X7 antagonists and eATP levels will be assessed by in vivo luminometry. Preliminary experiments shows P2X7 dependent microvesicles secretion from cancer and immune cells. The molecular content of these vesicles will be analysed to determine whether they contain ATP, miRNAs and proteins linked to cancer progression. Finally, P2X7 expression levels will be quantified in a patient population by immunohistochemistry and Real Time PCR, to perform a clinical explorative study aimed at associating P2X7 expression with melanoma progression and prognosis. Expected Results We expect to demonstrate that P2X7 receptor exerts a central role in melanoma growth and progression by affecting intracellular pathways, cell-cell crosstalk and the composition of tumour microenvironment. Taken together these actions will favour melanoma growth and dissemination and make P2X7 an ideal pharmacological target for advanced melanoma treatment. Impact On Cancer P2X7 receptor antagonists are in phase III clinical trials for immune diseases, therefore they will be soon available at patients bed offering a possible life extending, easily administrable, cost-effective anti-melanoma therapy.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
