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Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells.

ABSTRACT: Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/?-catenin pathway, because ?-catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression.

SUBMITTER: Freeman TJ

PROVIDER: S-EPMC6356688 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells.

Freeman Taylor J TJ Sayedyahossein Samar S Johnston Danielle D Sanchez-Pupo Rafael E RE O'Donnell Brooke B Huang Kenneth K Lakhani Zameena Z Nouri-Nejad Daniel D Barr Kevin J KJ Harland Luke L Latosinsky Steven S Grant Aaron A Dagnino Lina L Penuela Silvia S

Cancers 20190116 1

Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carben ...[more]

PMID: 30654593

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Project description:Overexpression and activation of c-Jun N-terminal kinases (JNKs) have been observed in multiple cancer cell lines and tumor samples. Various JNK isoforms have been reported to promote lung and liver cancer, as well as keratinocyte transformation, suggesting an important role of JNK signaling in promoting tumor development. However, there are three JNK isoforms, and it is unclear how each individual isoform, especially the ubiquitously expressed JNK1 and JNK2, functions in melanoma. Our previous study found that C116S mutations in both JNK1 and JNK2 rendered them insensitive to the covalent pan-JNK inhibitor JNK-IN-8 while retaining kinase activity. To delineate the specific roles of JNK1 and JNK2 in melanoma cell proliferation and invasiveness, we expressed the wild type (WT) and C116S mutants in melanoma cell lines and used JNK-IN-8 to enable chemical-genetic dissection of JNK1 and JNK2 activity. We found that the JNK2C116S allele consistently enhanced colony proliferation and cell invasiveness in the presence of JNK-IN-8. When cells individually expressing WT or C116S JNK1/2 were subcutaneously implanted into immunodeficient mice, we again found that bypass of JNK-IN-8-mediated inhibition of JNK signaling by expression of JNK2C116S specifically resulted in enhanced tumor growth in vivo. In addition, we observed a high level of JNK pathway activation in some human BRAF inhibitor (BRAFi) resistant melanoma cell lines relative to their BRAFi sensitive isogenic counterparts. JNK-IN-8 significantly enhanced the response to dabrafenib in resistant cells overexpressing JNK1WT, JNK2WT, and JNK1C116S but had no effect on cells expressing JNK2C116S, suggesting that JNK2 signaling is also crucial for BRAFi resistance in a subset of melanomas. Collectively, our data show that JNK2 activity is specifically required for melanoma cell proliferation, invasiveness, and BRAFi resistance and that this activity is most important in the context of JNK1 suppression, thus providing a compelling rationale for the development of JNK2 selective inhibitors as a potential therapy for the treatment of melanoma.

Project description:BackgroundGlioblastoma (GBM) is the most common primary brain malignancy and confers a dismal prognosis. GBMs harbor glioblastoma-initiating cells (GICs) that drive tumorigenesis and contribute to therapeutic resistance and tumor recurrence. Consequently, there is a strong rationale to target this cell population in order to develop new molecular therapies against GBM. Accumulating evidence indicates that Nα-terminal acetyltransferases (NATs), that are dysregulated in numerous human cancers, can serve as therapeutic targets.MethodsMicroarrays were used to study the expression of several NATs including NAT12/NAA30 in clinical samples and stem cell cultures. The expression of NAT12/NAA30 was analyzed using qPCR, immunolabeling and western blot. We conducted shRNA-mediated knockdown of NAT12/NAA30 gene in GICs and studied the effects on cell viability, sphere-formation and hypoxia sensitivity. Intracranial transplantation to SCID mice enabled us to investigate the effects of NAT12/NAA30 depletion in vivo. Using microarrays we identified genes and biochemical pathways whose expression was altered upon NAT12/NAA30 down-regulation.ResultsWhile decreased expression of the distal 3'UTR of NAT12/NAA30 was generally observed in GICs and GBMs, this gene was strongly up-regulated at the protein level in GBM and GICs. The increased protein levels were not caused by increased levels of the steady state mRNA but rather by other mechanisms. Also, shorter 3'UTR of NAT12/NAA30 correlated with poor survival in glioma patients. As well, we observed previously not described nuclear localization of this typically cytoplasmic protein. When compared to non-silencing controls, cells featuring NAT12/NAA30 knockdown exhibited reduced cell viability, sphere-forming ability, and mitochondrial hypoxia tolerance. Intracranial transplantation showed that knockdown of NAT12/NAA30 resulted in prolonged animal survival. Microarray analysis of the knockdown cultures showed reduced levels of HIF1α and altered expression of several other genes involved in the hypoxia response. Furthermore, NAT12/NAA30 knockdown correlated with expressional dysregulation of genes involved in the p53 pathway, ribosomal assembly and cell proliferation. Western blot analysis revealed reduction of HIF1α, phospho-MTOR(Ser2448) and higher levels of p53 and GFAP in these cultures.ConclusionNAT12/NAA30 plays an important role in growth and survival of GICs possibly by regulating hypoxia response (HIF1α), levels of p-MTOR (Ser2448) and the p53 pathway.

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Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells.

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Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells.

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