RESUMO
Phosphatidylinositol-3'-kinases (PI3Ks) are a family of lipid kinases that phosphorylate the 3' hydroxyl (OH) of the inositol ring of phosphatidylinositides (PI). Through their downstream effectors, PI3K generated lipids (PI3K-lipids hereafter) such as PI(3,4,5)P3 and PI(3,4)P2 regulate myriad biochemical and biological processes in both normal and cancer cells including responses to growth hormones and cytokines; the cell division cycle; cell death; cellular growth; angiogenesis; membrane dynamics; and autophagy and many aspects of cellular metabolism. Engagement of receptor tyrosine kinase by their cognate ligands leads to activation of members of the Class I family of PI3'-kinases (PI3Kα, ß, δ & γ) leading to accumulation of PI3K-lipids. Importantly, PI3K-lipid accumulation is antagonized by the hydrolytic action of a number of PI3K-lipid phosphatases, most notably the melanoma suppressor PTEN (lipid phosphatase and tensin homologue). Downstream of PI3K-lipid production, the protein kinases AKT1-3 are believed to be key effectors of PI3'-kinase signalling in cells. Indeed, in preclinical models, activation of the PI3KâAKT signalling axis cooperates with alterations such as expression of the BRAFV600E oncoprotein kinase to promote melanoma progression and metastasis. In this review, we describe the different classes of PI3K-lipid effectors, and how they may promote melanomagenesis, influence the tumour microenvironment, melanoma maintenance and progression to metastatic disease. We also provide an update on both FDA-approved or experimental inhibitors of the PI3KâAKT pathway that are currently being evaluated for the treatment of melanoma either in preclinical models or in clinical trials.
Assuntos
Melanoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Ensaios Clínicos como Assunto , Humanos , Microambiente TumoralRESUMO
In vitro polymerized type I collagen hydrogels have been used extensively as a model system for three-dimensional (3D) cell and tissue culture, studies of fibrillogenesis, and investigation of multiscale force transmission within connective tissues. The nanoscale organization of collagen fibrils plays an essential role in the mechanics of these gels and emergent cellular behavior in culture, yet quantifying 3D structure with nanoscale resolution to fully characterize fibril organization remains a significant technical challenge. In this study, we demonstrate that a new imaging modality, focused ion beam scanning electron microscopy (FIB-SEM), can be used to generate 3D image datasets for visualizing and quantifying complex nanoscale organization and morphometry in collagen gels. We polymerized gels at a number of concentrations and conditions commonly used for in vitro models, stained and embedded the samples, and performed FIB-SEM imaging. The resulting image data had a voxel size of 25 nm, which is the highest resolution 3D data of a collagen fibril network ever obtained for collagen gels. This resolution was essential for discerning individual fibrils, fibril paths, and their branching and grouping. The resulting volumetric images revealed that polymerization conditions have a significant impact on the complex fibril morphology of the gels. We segmented the fibril network and demonstrated that individual collagen fibrils can be tracked in 3D space, providing quantitative analysis of network descriptors such as fibril diameter distribution, length, branch points, and fibril aggregations. FIB-SEM 3D reconstructions showed considerably less lateral grouping and overlap of fibrils than standard 2D SEM images, likely due to artifacts in SEM introduced by dehydration. This study demonstrates the utility of FIB-SEM for 3D imaging of collagen gels and quantitative analysis of 3D fibril networks. We anticipate that the method will see application in future studies of structure-function relationships in collagen gels as well as native collagenous tissues.
Assuntos
Colágeno Tipo I/química , Hidrogéis/química , Microscopia Eletrônica de Varredura , Nanotecnologia , Animais , Imageamento Tridimensional , Multimerização Proteica , Estrutura Quaternária de Proteína , RatosRESUMO
Aberrant activation of the PI3K-AKT pathway is common in many cancers, including melanoma, and AKT1, 2 and 3 (AKT1-3) are bona fide oncoprotein kinases with well-validated downstream effectors. However, efforts to pharmacologically inhibit AKT have proven to be largely ineffective. In this study, we observed paradoxical effects following either pharmacologic or genetic inhibition of AKT1-3 in melanoma cells. Although pharmacological inhibition was without effect, genetic silencing of all three AKT paralogs significantly induced melanoma cell death through effects on mTOR. This phenotype was rescued by exogenous AKT1 expression in a kinase-dependent manner. Pharmacological inhibition of PI3K and mTOR with a novel dual inhibitor effectively suppressed melanoma cell proliferation in vitro and inhibited tumor growth in vivo. Furthermore, this single-agent-targeted therapy was well-tolerated in vivo and was effective against MAPK inhibitor-resistant patient-derived melanoma xenografts. These results suggest that inhibition of PI3K and mTOR with this novel dual inhibitor may represent a promising therapeutic strategy in this disease in both the first-line and MAPK inhibitor-resistant setting.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Linhagem Celular Tumoral , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células , Morte CelularRESUMO
Oncolytic viruses (OVs) are being developed as a type of immunotherapy and have demonstrated durable tumor responses and clinical efficacy. One such OV, Coxsackievirus A21 (CVA21), exhibited therapeutic efficacy in early phase clinical trials, demonstrating the ability to infect and kill cancer cells and stimulate anti-tumor immune responses. However, one of the major concerns in using this common cold virus as a therapeutic is the potential for innate and adaptive immune responses to mitigate the benefits of viral infection, particularly in individuals that have been exposed to coxsackievirus prior to treatment. In this study, we assess melanoma responses to CVA21 in the absence or presence of prior exposure to the virus. Melanomas were transplanted into naïve or CVA21-immunized C57BL6 mice and the mice were treated with intratumoral (IT) CVA21. We find that prior exposure to CVA21 does not dramatically affect tumor responses, nor does it alter overall survival. Our results suggest that prior exposure to coxsackievirus is not a critical determinant of patient selection for IT CVA21 interventions.
RESUMO
Mutational activation of RAC1 is detected in ~7% of cutaneous melanoma, with the most frequent mutation (RAC1C85T ) encoding for RAC1P29S . RAC1P29S is a fast-cycling GTPase that leads to accumulation of RAC1P29S -GTP, which has potentially pleiotropic regulatory functions in melanoma cell signaling and biology. However, the precise mechanism by which mutationally activated RAC1P29S propagates its pro-tumorigenic effects remains unclear. RAC1-GTP is reported to activate the beta isoform of PI3'-kinase (PIK3CB/PI3Kß) leading to downstream activation of PI3'-lipid signaling. Hence, we employed both genetic and isoform-selective pharmacological inhibitors to test if RAC1P29S propagates its oncogenic signaling in melanoma through PI3Kß. We observed that RAC1P29S -expressing melanoma cells were largely insensitive to inhibitors of PI3Kß. Furthermore, RAC1P29S melanoma cell lines showed variable sensitivity to pan-class 1 (α/ß/γ/δ) PI3'-kinase inhibitors, suggesting that RAC1-mutated melanoma cells may not rely on PI3'-lipid signaling for their proliferation. Lastly, we observed that RAC1P29S -expressing cell lines also showed variable sensitivity to pharmacological inhibition of the RAC1 â PAK1 signaling pathway, questioning the relevance of inhibitors of this pathway for the treatment of patients with RAC1-mutated melanoma.
Assuntos
Biomarcadores Tumorais/metabolismo , Terapia de Alvo Molecular , Mutação/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Linhagem Celular Tumoral , Humanos , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib in vivo. However, the emergence of drug-resistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance in vivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.
Assuntos
Benzamidas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indazóis/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Modelos Biológicos , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Receptor trkA/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Células HEK293 , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos NOD , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologiaRESUMO
Alterations in the PI3K/AKT pathway occur in up to 70% of melanomas and are associated with disease progression. The three AKT paralogs are highly conserved but data suggest they have distinct functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated E17K, E40K, and Q79K mutations in AKT1, AKT2, and AKT3 and show that mice harboring tumors expressing AKT1E17K had the highest incidence of brain metastasis and lowest mean survival. Tumors expressing AKT1E17K displayed elevated levels of focal adhesion factors and enhanced phosphorylation of focal adhesion kinase (FAK). AKT1E17K expression in melanoma cells increased invasion and this was reduced by pharmacologic inhibition of either AKT or FAK. These data suggest that the different AKT paralogs have distinct roles in melanoma brain metastasis and that AKT and FAK may be promising therapeutic targets. IMPLICATIONS: This study suggests that AKT1E17K promotes melanoma brain metastasis through activation of FAK and provides a rationale for the therapeutic targeting of AKT and/or FAK to reduce melanoma metastasis.