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Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator.

ABSTRACT: Notch, a type 1 transmembrane protein, plays a key role in the development of many tissues and organ types. Aberrant Notch signaling, found in a wide variety of human cancers, contributes to tumor development. Because Notch1 was found to be overexpressed in prostate cancer (PCa) cells and human PCa tissue, we therefore tested our hypothesis that overexpression of Notch1 in PCa promotes tumor invasion.Notch1 expression was evaluated in human PCa cells and human PCa tissues. PCa cells were transiently transfected with Notch1-specific small interfering RNAs in concentrations ranging from 30 to 120 nmol/L and subsequently evaluated for effects on invasion and expression analysis for molecules involved in invasion.Small interfering RNA-mediated knockdown of Notch1 in PC3 and 22Rnu1 PCa cells dramatically decreased their invasion. Focused cDNA array revealed that Notch1 knockdown resulted in significant reduction in the expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) gene transcripts. These data were further verified by reverse transcription-PCR, real-time reverse transcription-PCR, and immunoblot analysis. Knockdown of Notch1 was also observed to significantly reduce the mRNA expression and protein levels of uPA and its receptor uPAR. A significant reduction in MMP9 expression in Notch1 knockdown cells suggested a role for Notch1 in augmenting MMP9 transcription.Our data show the involvement of Notch1 in human PCa invasion and that silencing of Notch1 inhibits invasion of human PCa cells by inhibiting the expression of MMP9 and uPA. Thus, targeting of Notch1 could be an effective therapeutic approach against PCa.

SUBMITTER: Bin Hafeez B

PROVIDER: S-EPMC2951134 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator.

Bin Hafeez Bilal B Adhami Vaqar Mustafa VM Asim Mohammad M Siddiqui Imtiaz A IA Bhat Kumar M KM Zhong Weixiong W Saleem Mohammad M Din Maria M Setaluri Vijayasaradhi V Mukhtar Hasan H

Clinical cancer research : an official journal of the American Association for Cancer Research 20090101 2

<h4>Purpose</h4>Notch, a type 1 transmembrane protein, plays a key role in the development of many tissues and organ types. Aberrant Notch signaling, found in a wide variety of human cancers, contributes to tumor development. Because Notch1 was found to be overexpressed in prostate cancer (PCa) cells and human PCa tissue, we therefore tested our hypothesis that overexpression of Notch1 in PCa promotes tumor invasion.<h4>Experimental design</h4>Notch1 expression was evaluated in human PCa cells a ...[more]

PMID: 19147749

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Project description:Data from clinical studies, cell culture, and animal models implicate the urokinase (uPA)/Plasminogen (Plg) system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/Plg stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression to clarify mechanisms of uPA/Plg-accelerated atherosclerosis and aneurysm formation. Microarray studies were performed to identify potential mediators of uPA-accelerated atherosclerosis. These studies identified S100A8 and S100A9 mRNA as the most highly upregulated transcripts in uPA-overexpressing macrophages; upregulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also upregulated in aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is upregulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Bioinformatics analysis of the microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm—in a second animal model—that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease. Six independent biological replicate RNA samples were prepared from thioglycollate-elicited peritoneal macrophages from mice of two different genotypes: SR-uPA+/0 transgenic (overexpress uPA in macrophages) and nontransgenic, respectively), for a total of 12 independent RNA samples. Both transgenic and nontransgenic mice were Apoe-/- and were fed Western diet from 5-15 weeks before peritoneal macrophages were elicited. In addition, from the six samples of each genotype, a pooled sample was prepared by combining the six genotype-specific samples. Each of the two pooled samples was assayed on the BeadChip in two technical replicates, for a total of 16 hybridizations performed using two Illumina Mouse Ref-8 v1.1 chips.

Project description:BackgroundThe urokinase plasminogen activator receptor (uPAR) is associated with poor prognosis in oral squamous cell carcinoma (OSCC), and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells' expression level of uPAR affected the activity of gelatinolytic enzymes.MethodsThe Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM) proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography.ResultsWe found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes.ConclusionsAlthough high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational modification of uPAR.

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Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator.

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Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator.

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