Timp1 Promotes Cell Survival by Activating the PDK1 Signaling Pathway in Melanoma

High TIMP1 expression is associated with poor prognosis in melanoma, where it can bind to CD63 and beta 1 integrin, inducing PI3-kinase pathway and cell survival. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), generated under phosphatidylinositol-3-kinase (PI3K) activation, enables the recruitment and activation of protein kinase B (PKB/AKT) and phosphoinositide-dependent kinase 1 (PDK1) at the membrane, resulting in the phosphorylation of a host of other proteins. Using a melanoma progression model, we evaluated the impact of Timp1 and AKT silencing, as well as PI3K, PDK1, and protein kinase C (PKC) inhibitors on aggressiveness characteristics. Timp1 downregulation resulted in decreased anoikis resistance, clonogenicity, dacarbazine resistance, and in vivo tumor growth and lung colonization. In metastatic cells, pAKT(Thr308) is highly expressed, contributing to anoikis resistance. We showed that PDK1(Ser241) and PKC beta IISer660 are activated by Timp1 in different stages of melanoma progression, contributing to colony formation and anoikis resistance. Moreover, simultaneous inhibition of Timp1 and AKT in metastatic cells resulted in more effective anoikis inhibition. Our findings demonstrate that Timp1 promotes cell survival with the participation of PDK1 and PKC in melanoma. In addition, Timp1 and AKT act synergistically to confer anoikis resistance in advanced tumor stages. This study brings new insights about the mechanisms by which Timp1 promotes cell survival in melanoma, and points to novel perspectives for therapeutic approaches.Fundacao de Amparo a Pesquisa do Estado de Sao PauloConselho Nacional de Desenvolvimento Cientifico e TecnologicoUniv Fed Sao Paulo, Dept Pharmacol, BR-04039032 Sao Paulo, BrazilUniv Sao Paulo, Sch Med, Canc Inst Sao Paulo, Ctr Translat Invest Oncol LIM 24, BR-01246000 Sao Paulo, BrazilFac Med Santa Casa Sao Paulo, BR-01221020 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Pharmacol, BR-04039032 Sao Paulo, Brazil|FAPESP: 2010/18715-8FAPESP: 2011/12306-1FAPESP: 2014/13663-0CNPq: 470681/2012-8Web of Scienc

Endothelial nitric oxide synthase uncoupling as a key mediator of melanocyte malignant transformation associated with sustained stress conditions

Melanoma cell lines and cells corresponding to premalignant melanocytes were established by our group after subjecting a nontumorigenic murine melanocyte lineage, melan-a, to sequential cycles of anchorage blockade. Previous results showed that in melan-a cells the superoxide level increases after such procedure. Superoxide production during melanocyte de-adhesion was inhibited by L-sepiapterin, the precursor of eNOS cofactor BH(4), and increased by the inhibitor of BH(4) synthesis, DAHP, hence indicating a partial uncoupling state of eNOS. the eNOS uncoupling seems to be maintained in cells derived from melan-a, because they present decreased nitric oxide and increased superoxide levels. the inhibition of superoxide production in Tm5 melanoma cells with L-sepiapterin reinforces their eNOS-uncoupled state. the maintenance of oxidative stress seems to be important in melanoma apoptosis resistance because Mn(III)TBAP, a superoxide scavenger, or L-sepiapterin renders Tm5 cells more sensitive to anoikis and chemotherapy. More importantly, eNOS uncoupling seems to play a pivotal role in melanocyte malignant transformation induced by sustained anchorage impediment, because no malignant transformation was observed when L-NAME-treated melanocytes were subjected to sequential cycles of de-adhesion. Our results show that uncoupled eNOS contributes to superoxide production during melanocyte anchorage impediment, contributing to anoikis resistance and malignant transformation. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Disciplina Imunol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Farmacol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Nefrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Imunol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Farmacol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Nefrol, BR-04023900 São Paulo, BrazilFAPESP: 06/61293-1FAPESP: 05/60334-3FAPESP: 08/50366FAPESP: 09/03335-8Web of Scienc

Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

Background: Anoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. in our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet.Methods: the beta 1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and beta 1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR.Results: Differential association among Timp1, CD63 and beta 1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. in human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity.Conclusions: Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and beta 1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. in addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Dept, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilLudwig Inst Canc Res, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Dept, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilFAPESP: 2011/12306-1FAPESP: 2010/18715-8CAPES: 2867/10Web of Scienc

Determinação de vias de sinalização ativadas por timp1 ao longo da genese do melanoma

Embora o melanoma maligno seja o câncer de pele menos incidente, ele é o que apresenta pior prognóstico, sendo que esta característica está relacionada ao seu alto poder de formar metástases e por ser altamente quimioresistente. Em nosso laboratório, foi desenvolvido um modelo linear de gênese do melanoma, que nos permite estudar diferentes etapas da progressão do melanoma. Melanócitos murinos melana foram submetidos a 1, 2, 3 e 4 ciclos de impedimento de adesão ao substrato e estas células apresentaram morfologia diferente e crescimento independente de PMA e foram chamadas de 1C, 2C, 3C e 4C, respectivamente. Diferentes linhagens de melanoma foram estabelecidas após submeter esferóides dos pelo bloqueio de adesão da linhagem 4C à diluição limitante (entre elas, células de melanoma não metastático 4C11- e células de melanoma metastático 4C11+). Uma das características de células transformadas é a resistência ao anoikis e esta característica está relacionada à formação de metástases. Dados prévios do nosso grupo mostraram aumento da expressão de Timp1 nas células derivadas dos bloqueios de ancoragem dos melanócitos melan-a. O aumento de expressão de Timp1 nestas células está correlacionado com a resistência ao anoikis. Embora tenha sido demonstrada a interação de CD63, Timp1 e ?1-integrinas somente em células de melanoma em nosso modelo, o mecanismo de sinalização envolvido nesse processo ainda não está claro. Portanto, neste trabalho analisamos o papel de Timp1 na progressão do melanoma e as vias de sinalização envolvidas no processo de resistência ao anoikis. Através da análise dos resultados, podemos concluir que Timp1 modula a via de sinalização PI3K. Essa modulação parece ser através de PDK1 nas linhagens de melanoma 4C11- e 4C11+. Essa modulação confere, ao longo da progressão maligna, maior resistência ao anoikis e fenótipo mais agressivo in vivo na linhagem de melanoma metastático 4C11+. Assim, o presente trabalho contribui com o entendimento de como Timp1 regula resistência ao anoikis ao longo da transformação maligna de melanócitos.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Effects of treatment with low dose lithium to spatial memory of SAMR-1 and SAMP-8.

Spatial memory was evaluated in the Barnes maze and the time to enter the escape box (A-C) and the number of wrong holes explored (D-F) were recorded. Graphs A and D show the observation during learning time of groups SAMR-1 (n = 11) and SAMP-8 (n = 24), as indicated by the legend in D. Along the mice aging process, the behaviour of non-treated SAMP-8 was compared to SAMR-1 (B and E, as indicated by the legend in E) and SAMP-8 Li+ (C and F, as indicated by the legend in F). Along the 8 months’ period of observations, some animals have died (for different reasons) and the final number of animals in each group was 9 animals in the SAMR-1, 9 animals in the SAMP-8 and 7 animals in the SAMP-8-Li group. Symbols and vertical bars are means ± SEM. *: p #: p < 0.0001.</p

Images of original membranes with antibody and Ponceau reagent markings.

Images of original membranes with antibody and Ponceau reagent markings.</p

Senile plaques in the different animals’ hippocampus.

Labeling was done with Thioflavin-S 1% (green) in the hippocampus of SAMR-1 (A–C), SAMP-8 (D–F) and SAMP-8 Li (G–I). Cellular nuclei were labeled with DAPI (blue). Images were obtained using an inverted DMi8 microscope (Leica, Wetzlar, Germany) with a 20× objective. Scale bar: 100μm. J: Quantification of the total number of plaques in the hippocampus. Histograms and vertical bars are means ± SEM of samples from 5 animals per group. *: p < 0.05; **: p < 0.01.</p