Identification of a functional role for the protease-activated receptor-1 in hypoxic breast cancer cells

Naldini, Antonella; Filippi, Irene; Ardinghi, C; Silini, Antonietta; Giavazzi, Raffaella; Carraro, Fabio

doi:10.1016/j.ejca.2008.10.012

Cited by 18 publications

(14 citation statements)

References 30 publications

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“…In addition to TF, hypoxic stress has been shown to modulate PAR1 expression in tumor cell lines (32,33). Furthermore, our recent study observed increased PAR1 and PAR2 staining around necrotic areas in samples from patients with GBM (22).…”

Section: Hypoxic Induction Of Par Expression In P7 and St1 Cellsmentioning

confidence: 94%

Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model

et al. 2016

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Abstract. Hypoxia and necrosis are fundamental features of glioma, and their emergence is critical for the rapid biological progression of this fatal tumor. The presence of vaso-occlusive thrombus is higher in grade IV tumors [glioblastoma multiforme (GBM)] compared with lower grade tumors, suggesting that the procoagulant properties of the tumor contribute to its aggressive behavior, as well as the establishment of tumor hypoxia and necrosis. Tissue factor (TF), the primary cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Phosphatase and tensin homolog (PTEN) loss and hypoxia are two common alterations observed in glioma that may be responsible for TF upregulation. In the present study, ST1 and P7 rat glioma lines, with different levels of aggressiveness, were comparatively analyzed with the aim of identifying differences in procoagulant mechanisms. The results indicated that P7 cells display potent procoagulant activity compared with ST1 cells. Flow cytometric analysis showed less pronounced levels of TF in ST1 cells compared with P7 cells. Notably, P7 cells supported factor X (FX) activation via factor VIIa, whereas no significant FXa generation was observed in ST1 cells. Furthermore, the exposure of phosphatidylserine on the surface of P7 and ST1 cells was investigated. The results supported the assembly of prothrombinase complexes, accounting for the production of thrombin. Furthermore, reverse transcription-quantitative polymerase chain reaction showed that CoCl 2 (known to induce a hypoxic-like stress) led to an upregulation of TF levels in P7 and ST1 cells. Therefore, increased TF expression in P7 cells was accompanied by increased TF procoagulant activity. In addition, hypoxia increased the shedding of procoagulant TF-bearing microvesicles in both cell lines. Finally, hypoxic stress induced by treatment with CoCl 2 upregulated the expression of the PAR1 receptor in both P7 and ST1 cells. In addition to PAR1, P7, but not ST1 cells, expressed higher levels of PAR2 under hypoxic stress. Thus, modulating these molecular interactions may provide additional insights for the development of more efficient therapeutic strategies against aggressive glioma.

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Section: Hypoxic Induction Of Par Expression In P7 and St1 Cellsmentioning

confidence: 94%

Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model

et al. 2016

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“…This was further validated at the protein level confirming PAR1 global downregulation in LNCaP cells treated in hypoxic conditions. However, a recent report showed that, in MDAMDB321 aggressive breast cancer cells, hypoxia (2% oxygen) enhance PAR1 expression on the basis of conventional qRT-PCR experiments using ACTB as HKG, gel analysis for transcript quantification (Naldini et al, 2009), but no reported data regarding the choice of ACTB. Nevertheless, in our hands, MCF-7 breast cancer cells appear to have the most stable HKGs in hypoxia.…”

Section: Discussionmentioning

confidence: 99%

‘Desperate house genes’: the dramatic example of hypoxia

Caradec

Sirab

Keumeugni³

et al. 2010

Br J Cancer

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BACKGROUND: Microenvironmental conditions in normal or tumour tissues and cell lines may interfere on further biological analysis. To evaluate transcript variations carefully, it is common to use stable housekeeping genes (HKG) to normalise quantitative microarrays or real-time polymerase chain reaction results. However, recent studies argue that HKG fluctuate according to tissues and treatments. So, as an example of HKG variation under an array of conditions that are common in the cancer field, we evaluate whether hypoxia could have an impact on HKG expression. METHODS: Expression of 10 commonly used HKG was measured on four cell lines treated with four oxygen concentrations (from 1 to 20%). RESULTS: Large variations of HKG transcripts were observed in hypoxic conditions and differ along with the cell line and the oxygen concentration. To elect the most stable HKG, we compared the three statistical means based either on PCR cycle threshold coefficient of variation calculation or two specifically dedicated software. Nevertheless, the best HKG dramatically differs according to the statistical method used. Moreover, using, as a reference, absolute quantification of a target gene (here the proteinase activating receptor gene 1 (PAR1) gene), we show that the conclusions raised about PAR1 variation in hypoxia can totally diverge according to the selected HKG used for normalisation. CONCLUSION: The choice of a valid HKG will determine the relevance of the results that will be further interpreted, and so it should be seriously considered. The results of our study confirm unambiguously that HKG variations must be precisely and systematically determined before any experiment for each situation, to obtain reliable normalised results in the experimental setting that has been designed. Indeed, such assay design, functional for all in vitro systems, should be carefully evaluated before any extension to other experimental models including in vivo ones.

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“…In CRC, PAR 2 activation is also protective (7,24). PAR 1 activation enhances cell survival of melanoma and breast cancer cells (38,46). Both protease receptors regulate prosurvival pathways such as ERK, Akt, and NF-B, but the final cell death or survival depends on the cell type, the amplitude, and duration of agonist exposure and the biaised agonism (35,68).…”

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confidence: 99%

PAR₂-dependent activation of GSK3β regulates the survival of colon stem/progenitor cells

Nasri

Bonnet

Zwarycz

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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Protease-activated receptors PAR1 and PAR2 play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3β (GSK3β) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR1 and PAR2 effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3β. First, PAR1 and PAR2 were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR2 activation decreased numbers and size of normal or cancerous spheroids, and PAR2-deficient spheroids showed increased proliferation, indicating that PAR2 represses proliferation. PAR2-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR2. Accordingly, active caspase-3 was strongly increased in PAR2-deficient normal spheroids. PAR2 but not PAR1 triggered GSK3β activation through serine-9 dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3β activation implicates an arrestin/PP2A/GSK3β complex that is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3β nonactive form, strengthening the role of PAR2 in GSK3β activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3β as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer.

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Identification of a functional role for the protease-activated receptor-1 in hypoxic breast cancer cells

Cited by 18 publications

References 30 publications

Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model

Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model

‘Desperate house genes’: the dramatic example of hypoxia

PAR₂-dependent activation of GSK3β regulates the survival of colon stem/progenitor cells

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Identification of a functional role for the protease-activated receptor-1 in hypoxic breast cancer cells

Cited by 18 publications

References 30 publications

Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model

Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model

‘Desperate house genes’: the dramatic example of hypoxia

PAR2-dependent activation of GSK3β regulates the survival of colon stem/progenitor cells

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PAR₂-dependent activation of GSK3β regulates the survival of colon stem/progenitor cells