E-cadherin function leads to the density-dependent contact inhibition of cell growth. Because cadherins control the overall state of cell contact, cytoskeletal organization, and the establishment of ...many other kinds of cell interactions, it remains unknown whether E-cadherin directly transduces growth inhibitory signals. To address this question, we have selectively formed E-cadherin homophilic bonds at the cell surface of isolated epithelial cells by using functionally active recombinant E-cadherin protein attached to microspheres. We find that E-cadherin ligation alone reduces the frequency of cells entering the S phase, demonstrating that E-cadherin ligation directly transduces growth inhibitory signals. E-cadherin binding to beta-catenin is required for cell growth inhibition, but beta-catenin/T-cell factor transcriptional activity is not involved in growth inhibition resulting from homophilic binding. Neither E-cadherin binding to p120-catenin nor beta-catenin binding to alpha-catenin, and thereby the actin cytoskeleton, is required for growth inhibition. E-cadherin ligation also inhibits epidermal growth factor (EGF) receptor-mediated growth signaling by a beta-catenin-dependent mechanism. It does not affect EGF receptor autophosphorylation or activation of ERK, but it inhibits transphosphorylation of Tyr845 and activation of signal transducers and activators of transcription 5. Thus, E-cadherin homophilic binding independent of other cell contacts directly transduces growth inhibition by a beta-catenin-dependent mechanism that inhibits selective signaling functions of growth factor receptors.
MicroRNAs (miRNAs) are a class of noncoding RNA acting at a post-transcriptional level to control the expression of large sets of target mRNAs. While there is evidence that miRNAs deregulation plays ...a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored. Here, we found a strong up-regulation of miR-21 in the kidneys of mice with unilateral ureteral obstruction and also in the kidneys of patients with severe kidney fibrosis. In addition, mouse primary fibroblasts derived from fibrotic kidneys exhibited higher miR-21 expression level compared to those derived from normal kidneys. Expression of miR-21 in normal primary kidney fibroblasts was induced upon TGFβ exposure, a key growth factor involved in fibrogenesis. Finally, ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation. As circulating miRNAs have been suggested as promising non-invasive biomarkers, we further assess whether circulating miR-21 levels are associated with renal fibrosis using sera from 42 renal transplant recipients, categorized according to their renal fibrosis severity, evaluated on allograft biopsies (Interstitial Fibrosis/Tubular Atrophy (IF/TA). Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001). By contrast, circulating miR-21 levels were not correlated with other renal histological lesions. In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = -0.398, p = 0.006). Altogether, these data suggest miR-21 has a key pathogenic role in kidney fibrosis and may represent a novel, predictive and reliable blood marker of kidney fibrosis.
The hypoxia inducible factor (HIF) signaling pathway is known as the main renal carcinogenetic pathway. MUC1, an O-glycoprotein membrane-bound mucin, is overexpressed in clear renal cell carcinomas ...(cRCC) with correlation to two major prognostic factors: tumor-node-metastasis stage and nuclear Fürhman grade. We questioned whether there is a direct link between the HIF pathway and MUC1 overexpression in renal tumors. Interestingly, we observed concomitant increase of HIF-1alpha and MUC1 in metastatic cRCC group versus nonmetastatic cRCC group. Using different renal cell models and small interfering RNA assays targeting either HIF-1alpha or YC-1, a HIF-1 pharmacologic inhibitor, we showed induction of MUC1 expression under hypoxia by a HIF-dependent mechanism. Chromatin immunoprecipitation assay showed a direct binding of HIF-1alpha at the MUC1 promoter. In addition, combined site-directed mutagenesis and gel shift assay allowed the identification of two functional putative hypoxia responsive elements at -1488/-1485 and at -1510/-1507 in the promoter. Using a rat kidney model of ischemia/reperfusion, we confirmed in vivo that clamping renal pedicle for 1 hour followed by 2 hours of reperfusion induced increased MUC1 expression. Furthermore, MUC1 knockdown induced significant reduction of invasive and migration properties of renal cancer cells under hypoxia. Altogether, these results show that MUC1 is directly regulated by HIF-1alpha and affects the invasive and migration properties of renal cancer cells. Thus, MUC1 could serve as a potential therapeutic target in cRCC.
Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the ...molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.
Renal cell carcinoma, the most common neoplasm of adult kidney, accounts for about 3% of adult malignancies and is usually highly resistant to conventional therapy. MicroRNAs are a class of small ...non-coding RNAs, which have been previously shown to promote malignant initiation and progression. In this study, we focused our attention on miR-21, a well described oncomiR commonly upregulated in cancer. Using a cohort of 99 primary renal cell carcinoma samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues whereas no significant difference was observed with stages, grades, and metastatic outcome. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, using Boyden chambers and western blot techniques, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating renal cell carcinoma chemoresistance and further showed that miR-21 silencing significantly (1) increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin, and dovitinib; (2) decreased expression of multi-drug resistance genes; and (4) increased SLC22A1/OCT1, SLC22A2/OCT2, and SLC31A1/CTR1 platinum influx transporter expression. In conclusion, our results showed that miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs. In renal cell carcinoma, targeting miR-21 is a potential new therapeutic strategy to improve chemotherapy efficacy and consequently patient outcome.
While the transmembrane glycoprotein mucin 1 (MUC1) is clustered at the apical borders of normal epithelial cells, with transformation and loss of polarity, MUC1 is found at high levels in the ...cytosol and is uniformly distributed over the entire surface of carcinoma cells, where it can promote tumor progression and adversely affects the response to therapy. Clear cell renal cell carcinoma (ccRCC), the main histotype of kidney cancer, is typically highly resistant to conventional and targeted therapies for reasons that remain largely unknown. In this context, we investigated whether MUC1 also plays a pivotal role in the cellular and molecular events driving ccRCC progression and chemoresistance. We showed, using loss- and gain-of-function approaches in ccRCC-derived cell lines, that MUC1 not only influences tumor progression but also induces a multi-drug-resistant profile reminiscent of the activation of ABC drug efflux transporters. Overall, our results suggest that targeting MUC1 may represent a novel therapeutic approach to limit ccRCC progression and improve drug sensitivity.
Variability in genes involved in drug pharmacokinetics or drug response can be responsible for suboptimal treatment efficacy or predispose to adverse drug reactions. In addition to common genetic ...variations, large-scale sequencing studies have uncovered multiple rare genetic variants predicted to cause functional alterations in genes encoding proteins implicated in drug metabolism, transport and response. To understand the functional importance of rare genetic variants in DPYD, a pharmacogene whose alterations can cause severe toxicity in patients exposed to fluoropyrimidine-based regimens, massively parallel sequencing of the exonic regions and flanking splice junctions of the DPYD gene was performed in a series of nearly 3000 patients categorized according to pre-emptive DPD enzyme activity using the dihydrouracil/uracil (UH
/U) plasma ratio as a surrogate marker of DPD activity. Our results underscore the importance of integrating next-generation sequencing-based pharmacogenomic interpretation into clinical decision making to minimize fluoropyrimidine-based chemotherapy toxicity without altering treatment efficacy.
Clear cell renal cell carcinoma (ccRCC) is the main histotype of kidney cancer, which is typically highly resistant to conventional therapies and known for abnormal lipid accumulation. In this ...context, we focused our attention on miR-21, an oncogenic miRNA overexpressed in ccRCC, and peroxysome proliferator-activated receptor-α (PPAR- α), one master regulator of lipid metabolism targeted by miR-21. First, in a cohort of 52 primary ccRCC samples, using RT-qPCR and immunohistochemistry, we showed that miR-21 overexpression was correlated with PPAR-α downregulation. Then, in ACHN and 786-O cells, using RT-qPCR, the luciferase reporter gene, chromatin immunoprecipitation, and Western blotting, we showed that PPAR-α overexpression (i) decreased miR-21 expression, AP-1 and NF-κB transcriptional activity, and the binding of AP-1 and NF-κB to the miR-21 promoter and (ii) increased PTEN and PDCD4 expressions. In contrast, using pre-miR-21 transfection, miR-21 overexpression decreased PPAR-α expression and transcriptional activity mediated by PPAR-α, whereas the anti-miR-21 (LNA-21) strategy increased PPAR-α expression, but also the expression of its targets involved in fatty acid oxidation. In this study, we showed a double-negative feedback interaction between miR-21 and PPAR-α. In ccRCC, miR-21 silencing could be therapeutically exploited to restore PPAR-α expression and consequently inhibit the oncogenic events mediated by the aberrant lipid metabolism of ccRCC.
The 11p15 mucin genes (MUC2, MUC5AC, MUC5B and MUC6) possess a cell-specific pattern of expression in normal lung that is altered during carcinogenesis. Growth factors of the epidermal growth factor ...family are known to target key genes that in turn may affect the homeostasis of lung mucosae. Our aim was to study the regulation of the 11p15 mucin genes both at the promoter and protein levels to assess whether their altered expression may represent a key event during lung carcinogenesis. Studies were performed in the mucoepidermoid NCI-H292 lung cancer cell line. Cell treatment with epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or tumor necrosis factor alpha (TNF-alpha) resulted in a dramatic increase of MUC2 and MUC5AC mRNAs levels, promoter activity, and apomucin expression, whereas those of MUC5B and MUC6 were unchanged. pGL3 deletion mutants of MUC2, MUC5AC, and MUC5B promoters were constructed and used in transient transfection assays to characterize EGF- and TGF-alpha-responsive regulatory regions within the promoters. They were located in the -2627/-2097 and -202/-1 regions of MUC2 and MUC5AC promoters, respectively. Finally, we demonstrate that transcription factor Sp1 not only binds and activates MUC2 and MUC5AC promoters but also participates to their EGF- and TGF-alpha-mediated up-regulation. We also show that Sp3 is a strong inhibitor of 11p15 mucin gene transcription. In conclusion, MUC2 and MUC5AC are two target genes of EGFR ligands in lung cancer cells, and up-regulation of these two genes goes through concomitant activation of the EGFR/Ras/Raf/Extracellular Signal-regulated Kinase-signaling pathway and Sp1 binding to their promoters.
Background
Helicobacter pylori
(Hp), which is one of the causative agents in human gastric adenocarcinoma, is known to interact with mucous gel and alter mucin gene expression. The aim of this work ...was to study, using an in vitro model of cell infection, the effects of urease, flagellin, and CagA virulence factors on the regulation of the four 11p15 mucin genes (
MUC2
,
MUC5AC
,
MUC5B
, and
MUC6
).
Methods
KATO-III and AGS gastric cancer cells were infected for 1, 3 or 6 h with Hp wild-type strains (ATCC 43504, N6, and SS1) or corresponding isogenic mutants deficient for urease subunit B, flagellin subunit A, and CagA. mRNA levels of
MUC2
,
MUC5B
,
MUC5AC
and
MUC6
were assessed by RT-PCR, and functional activity of their promoters was measured by transient transfection assays.
Results
Infection of KATO-III cells with Hp wild-type strains resulted in an early (at 1 h) transient expression of
MUC2
,
MUC5AC
, and
MUC6
mRNA concomitant with those of interleukin (IL)-1β, IL-8, and TNF-α cytokines. In these cells, the UreB
−
isogenic mutant induced strong activation of
MUC5AC
expression, and UreB-responsive elements were located in the −486/−1 region of the promoter. FlaA
−
and CagA
−
mutants had no effect on mucin gene mRNA levels in KATO-III cells. In AGS cells, Hp-responsive elements were identified in all promoters, and overexpression of NF-κB induced upregulation of
MUC5AC
promoter activity when infected with the UreB
−
isogenic mutant.
Conclusion
These results indicate that Hp infection of gastric cancer cells alters 11p15 mucin gene transcription and that
MUC5AC
downregulation is mediated by urease virulence factor.
PDF
