15 research outputs found

    PKCĪµ Is an Essential Mediator of Prostate Cancer Bone Metastasis.

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    UNLABELLED: The bone is a preferred site for metastatic homing of prostate cancer cells. Once prostate cancer patients develop skeletal metastases, they eventually succumb to the disease; therefore, it is imperative to identify key molecular drivers of this process. This study examines the involvement of protein kinase C epsilon (PKCĪµ), an oncogenic protein that is abnormally overexpressed in human tumor specimens and cell lines, on prostate cancer cell bone metastasis. PC3-ML cells, a highly invasive prostate cancer PC3 derivative with bone metastatic colonization properties, failed to induce skeletal metastatic foci upon inoculation into nude mice when PKCĪµ expression was silenced using shRNA. Interestingly, while PKCĪµ depletion had only marginal effects on the proliferative, adhesive, and migratory capacities of PC3-ML cells in vitro or in the growth of xenografts upon s.c. inoculation, it caused a significant reduction in cell invasiveness. Notably, PKCĪµ was required for transendothelial cell migration (TEM) as well as for the growth of PC3-ML cells in a bone biomimetic environment. At a mechanistic level, PKCĪµ depletion abrogates the expression of IL1Ī², a cytokine implicated in skeletal metastasis. Taken together, PKCĪµ is a key factor for driving the formation of bone metastasis by prostate cancer cells and is a potential therapeutic target for advanced stages of the disease. IMPLICATIONS: This study uncovers an important new function of PKCĪµ in the dissemination of cancer cells to the bone; thus, highlighting the promising potential of this oncogenic kinase as a therapeutic target for skeletal metastasis

    Transcriptional regulation of oncogenic protein kinase CĪµ (PKCĪµ) by STAT1 and Sp1 proteins

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    Overexpression of PKCĪµ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCĪµ expression and its up-regulation in cancer, we cloned an āˆ¼1.6-kb promoter segment of the human PKCĪµ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and-105 bp (region A) and-921 and-796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and-269/-247 as well as STAT1 sites in positions -880/-869 and- 793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCĪµ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCĪµ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCĪµ and its effectors in cancer therapeutics.Centro de Investigaciones InmunolĆ³gicas BĆ”sicas y AplicadasFacultad de Ciencias MĆ©dica

    Transcriptional regulation of oncogenic protein kinase CĪµ (PKCĪµ) by STAT1 and Sp1 proteins

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    Overexpression of PKCĪµ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCĪµ expression and its up-regulation in cancer, we cloned an āˆ¼1.6-kb promoter segment of the human PKCĪµ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and-105 bp (region A) and-921 and-796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and-269/-247 as well as STAT1 sites in positions -880/-869 and- 793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCĪµ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCĪµ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCĪµ and its effectors in cancer therapeutics.Centro de Investigaciones InmunolĆ³gicas BĆ”sicas y AplicadasFacultad de Ciencias MĆ©dica

    NAC blocks Cystatin C amyloid complex aggregation in a cell system and in skin of HCCAA patients.

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    To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadHereditary cystatin C amyloid angiopathy is a dominantly inherited disease caused by a leucine to glutamine variant of human cystatin C (hCC). L68Q-hCC forms amyloid deposits in brain arteries associated with micro-infarcts, leading ultimately to paralysis, dementia and death in young adults. To evaluate the ability of molecules to interfere with aggregation of hCC while informing about cellular toxicity, we generated cells that produce and secrete WT and L68Q-hCC and have detected high-molecular weight complexes formed from the mutant protein. Incubations of either lysate or supernatant containing L68Q-hCC with reducing agents glutathione or N-acetyl-cysteine (NAC) breaks oligomers into monomers. Six L68Q-hCC carriers taking NAC had skin biopsies obtained to determine if hCC deposits were reduced following NAC treatment. Remarkably, ~50-90% reduction of L68Q-hCC staining was observed in five of the treated carriers suggesting that L68Q-hCC is a clinical target for reducing agents.Artic Therapeutics LLC Autonomous Community of Madrid (CAM). Spai

    Contribution of C3G and other GEFs to liver cancer development and progression

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    Primary liver cancers constitute the fourth leading cause of cancer mortality worldwide, due to their high morbidity, late diagnosis and lack of effective treatments. Hepatocellular carcinoma (HCC) represents 80% and cholangiocarcinoma (CCA) 15% of liver cancers. Several genetic and epigenetic gene alterations (e.g., TERT, TP53 or CTNNB1) are HCC drivers, although many additional gene alterations contribute to HCC initiation and/or progression. Rho and Ras GTPases have been widely implicated in tumorigenesis and their activators (GEFs) have recently emerged as putative key players in liver cancer. The Ras GEF, C3G (RAPGEF1), a GEF mainly for Rap proteins, has recently been uncovered as a relevant gene in HCC. Its upregulation promotes tumor growth, although a decrease in C3G levels favors migration/invasion and lung metastasis. Rap1A/1B/2A/2B are overexpressed in HCC tumors, but their effects are controversial and not equivalent to those of C3G. The C3G partner, CRKL, is also overexpressed in HCC, promoting proliferation, migration and invasion. Various Rho GEFs are also deregulated in liver cancer. Tiam1 and Tiam2 expression is upregulated in HCC, promoting proliferation, migration and metastasis. In addition, ARHGEF-10L/9/19/39 are overexpressed in HCC tumors, facilitating migration, invasion, metastasis and proliferation. Another Rho GEF, Vav2, is also involved in metastasis. Little is known about the participation of these GEFs and GTPases in CCA. However, analysis of cancer databases uncovered deregulations or genetic alterations in several of these genes, in both CCA and HCC. Hence, GEFs function appear essential for liver homeostasis, although future studies are needed to define their precise function in liver cancer

    C3G Protein, a New Player in Glioblastoma

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    C3G (RAPGEF1) is a guanine nucleotide exchange factor (GEF) for GTPases from the Ras superfamily, mainly Rap1, although it also acts through GEF-independent mechanisms. C3G regulates several cellular functions. It is expressed at relatively high levels in specific brain areas, playing important roles during embryonic development. Recent studies have uncovered different roles for C3G in cancer that are likely to depend on cell context, tumour type, and stage. However, its role in brain tumours remained unknown until very recently. We found that C3G expression is downregulated in GBM, which promotes the acquisition of a more mesenchymal phenotype, enhancing migration and invasion, but not proliferation. ERKs hyperactivation, likely induced by FGFR1, is responsible for this pro-invasive effect detected in C3G silenced cells. Other RTKs (Receptor Tyrosine Kinases) are also dysregulated and could also contribute to C3G effects. However, it remains undetermined whether Rap1 is a mediator of C3G actions in GBM. Various Rap1 isoforms can promote proliferation and invasion in GBM cells, while C3G inhibits migration/invasion. Therefore, other RapGEFs could play a major role regulating Rap1 activity in these tumours. Based on the information available, C3G could represent a new biomarker for GBM diagnosis, prognosis, and personalised treatment of patients in combination with other GBM molecular markers. The quantification of C3G levels in circulating tumour cells (CTCs) in the cerebrospinal liquid and/or circulating fluids might be a useful tool to improve GBM patient treatment and survival

    Ī²3-Chimaerin, a novel member of the chimaerin Rac-GAP family

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    Chimaerins are a family of diacylglycerol- and phorbol ester-regulated GTPase activating proteins (GAPs) for the small G-protein Rac. Extensive evidence indicates that these proteins play important roles in development, axon guidance, metabolism, cell motility, and T cell activation. Four isoforms have been reported to-date, which are products of CHN1 (Ī±1- and Ī±2-chimaerins) and CHN2 (Ī²1- and Ī²2-chimaerins) genes. Although these gene products are assumed to be generated by alternative splicing, bioinformatics analysis of the CHN2 gene revealed that Ī²1- and Ī²2-chimaerins are the products of alternative transcription start sites (TSSs) in different promoter regions. Furthermore, we found an additional TSS in CHN2 gene that leads to a novel product, which we named Ī²3-chimaerin. Expression profile analysis revealed predominantly low levels for the Ī²3-chimaerin transcript, with higher expression levels in epididymis, plasma blood leucocytes, spleen, thymus, as well as various areas of the brain. In addition to the prototypical SH2, C1, and Rac-GAP domains, Ī²3-chimaerin has a unique N-terminal domain. Studies in cells established that Ī²3-chimaerin has Rac-GAP activity and is responsive to phorbol esters. The enhanced responsiveness of Ī²3-chimaerin for phorbol ester-induced translocation relative to Ī²2-chimaerin suggests differential ligand accessibility to the C1 domain.Fil: Zubeldia Brenner, Lautaro. Consejo Nacional de Investigaciones CientĆ­ficas y TĆ©cnicas. Oficina de CoordinaciĆ³n Administrativa Ciudad Universitaria. Instituto de QuĆ­mica BiolĆ³gica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de QuĆ­mica BiolĆ³gica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Gutierrez Uzquiza, Alvaro. University of Pennsylvania; Estados UnidosFil: Barrio Real, Laura. University of Pennsylvania; Estados UnidosFil: Wang, Hongbin. University of Pennsylvania; Estados UnidosFil: Kazanietz, Marcelo Gabriel. University of Pennsylvania; Estados UnidosFil: Coluccio Leskow, Federico. Consejo Nacional de Investigaciones CientĆ­ficas y TĆ©cnicas. Oficina de CoordinaciĆ³n Administrativa Ciudad Universitaria. Instituto de QuĆ­mica BiolĆ³gica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de QuĆ­mica BiolĆ³gica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad Nacional de LujĆ”n. Departamento de Ciencias BĆ”sicas; Argentin

    C3G down-regulation enhances pro-migratory and stemness properties of oval cells by promoting an epithelial-mesenchymal-like process

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    International audiencePrevious data indicate that C3G (RapGEF1) main isoform is highly expressed in liver progenitor cells (or oval cells) compared to adult mature hepatocytes, suggesting it may play an important role in oval cell biology. Hence, we have explored C3G function in the regulation of oval cell properties by permanent gene silencing using shRNAs. We found that C3G knock-down enhanced migratory and invasive ability of oval cells by promoting a partial epithelial to mesenchymal transition (EMT). This is likely mediated by upregulation of mRNA expression of the EMT-inducing transcription factors, Snail1, Zeb1 and Zeb2, induced in C3G-silenced oval cells. This EMT is associated to a higher expression of the stemness markers, CD133 and CD44. Moreover, C3G down-regulation increased oval cells clonogenic capacity by enhancing cell scattering. However, C3G knock-down did not impair oval cell differentiation into hepatocyte lineage. Mechanistic studies revealed that HGF/MET signaling and its pro-invasive activity was impaired in oval cells with low levels of C3G, while TGF-Ī² signaling was increased. Altogether, these data suggest that C3G might be tightly regulated to ensure liver repair in chronic liver diseases such as non-alcoholic steatohepatitis. Hence, reduced C3G levels could facilitate oval cell expansion, after the proliferation peak, by enhancing migration

    HGK promotes metastatic dissemination in prostate cancer

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    Abstract Metastasis is the process of cancer cell dissemination from primary tumors to different organs being the bone the preferred site for metastatic homing of prostate cancer (PCa) cells. Prostate tumorigenesis is a multi-stage process that ultimately tends to advance to become metastatic PCa. Once PCa patients develop skeletal metastases, they eventually succumb to the disease. Therefore, it is imperative to identify essential molecular drivers of this process to develop new therapeutic alternatives for the treatment of this devastating disease. Here, we have identified MAP4K4 as a relevant gene for metastasis in PCa. Our work shows that genetic deletion of MAP4K4 or pharmacological inhibition of its encoded kinase, HGK, inhibits metastatic PCa cells migration and clonogenic properties. Hence, MAP4K4 might promote metastasis and tumor growth. Mechanistically, our results indicate that HGK depleted cells exhibit profound differences in F-actin organization, increasing cell spreading and focal adhesion stability. Additionally, HGK depleted cells fails to respond to TNF-Ī± stimulation and chemoattractant action. Moreover, here we show that HGK upregulation in PCa samples from TCGA and other databases correlates with a poor prognosis of the disease. Hence, we suggest that it could be used as prognostic biomarker to predict the appearance of an aggressive phenotype of PCa tumors and ultimately, the appearance of metastasis. In summary, our results highlight an essential role for HGK in the dissemination of PCa cells and its potential use as prognostic biomarker

    Transcriptional Regulation of Oncogenic Protein Kinase CĻµ (PKCĻµ) by STAT1 and Sp1 Proteins

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    Background: PKCĻµ, a kinase widely implicated in tumorigenesis and metastasis, is overexpressed in many cancers. Results: Transcription factors Sp1 and STAT1 control the expression of PKCĻµ in cancer cells. Conclusion: Up-regulation of PKCĻµ is mediated by dysregulated transcriptional mechanisms. Significance: Our results may have significant implications for the development of approaches to target PKCĻµ and its effectors in cancer therapeutics.Fil: Wang, HongBin. University of Pennsylvania; Estados UnidosFil: Gutierrez Uzquiza, Alvaro. University of Pennsylvania; Estados UnidosFil: Garg, Rachana. University of Pennsylvania; Estados UnidosFil: Barrio Real, Laura. University of Pennsylvania; Estados UnidosFil: Abera, Mahlet B. University of Pennsylvania; Estados UnidosFil: Lopez Haber, Cynthia. University of Pennsylvania; Estados UnidosFil: Rosemblit, Cinthia. University of Pennsylvania; Estados UnidosFil: Lu, Huaisheng. University of Pennsylvania; Estados UnidosFil: Abba, MartĆ­n Carlos. Consejo Nacional de Investigaciones CientĆ­ficas y TĆ©cnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias MĆ©dicas. Centro de Investigaciones InmunolĆ³gicas BĆ”sicas y Aplicadas; ArgentinaFil: Kazanietz, Marcelo G.. University of Pennsylvania; Estados Unido
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