Limits...
Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness.

Siemens H, Jackstadt R, Kaller M, Hermeking H - Oncotarget (2013)

Bottom Line: The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit.Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells.Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University München, D-80337 Munich, Germany.

ABSTRACT
The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches.

Show MeSH

Related in: MedlinePlus

miR-34a directly targets c-Kit and mediates c-Kit repression by p53(A) Scheme of constructs used for dual luciferase assays. The positions of the putative miR-34 seed-matching sequences in the c-Kit 3'-UTR are depicted as a grey vertical bars, their mutations as crosses. Sequences of the respective targeted mutations are given below. (B) Dual reporter assay in SW480 cells transfected with miR-34a/b/c mimics or control oligonucleotide and the indicated 3'-UTR-reporter constructs for the human c-Kit 3'-UTR. Data are represented as mean ± SD (n = 3). (C) DLD-1/tTA-p53 cells were either transfected with a control oligonucleotide, miR-34a or an antagomiR directed against miR-34a for 24 hours either in the presence or absence of DOX (without or with ectopic p53). Expression of the indicated proteins was detected by Western blot analysis. α-tubulin served as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3824539&req=5

Figure 2: miR-34a directly targets c-Kit and mediates c-Kit repression by p53(A) Scheme of constructs used for dual luciferase assays. The positions of the putative miR-34 seed-matching sequences in the c-Kit 3'-UTR are depicted as a grey vertical bars, their mutations as crosses. Sequences of the respective targeted mutations are given below. (B) Dual reporter assay in SW480 cells transfected with miR-34a/b/c mimics or control oligonucleotide and the indicated 3'-UTR-reporter constructs for the human c-Kit 3'-UTR. Data are represented as mean ± SD (n = 3). (C) DLD-1/tTA-p53 cells were either transfected with a control oligonucleotide, miR-34a or an antagomiR directed against miR-34a for 24 hours either in the presence or absence of DOX (without or with ectopic p53). Expression of the indicated proteins was detected by Western blot analysis. α-tubulin served as a loading control.

Mentions: Since a recent study reported a p53-dependent regulation of c-Kit, which occurred in the absence of p53 binding to the c-Kit promoter in mice [32], we hypothesized that miR-34 could be the mediator of this effect. In order to investigate this putative connection we used two different systems to conditionally express p53: SW480 cell pools transfected with the doxycycline (DOX) -inducible vector pRTR expressing the p53 open reading frame (ORF) and a DLD-1 single cell clone harboring a p53 allele under control of the tet-off system [36, 37]. Although the endogenous levels of c-Kit were lower in SW480 cells than in DLD-1 cells, activation of p53 in both cellular systems resulted in the down-regulation of c-Kit protein expression (Figure 1A). Since miRNAs were shown to mediate gene repression by p53 we examined the c-Kit 3'-UTR using the Target-Scan algorithm [38]. Thereby we identified two potential miR-34 seed-matching sequences in the 3'-UTR of c-Kit (Figure 1B). While the first site (which is a perfect match to the miR-34a 8-mer seed-matching sequence) is relatively conserved among different species, the second site seems to be less conserved. In line with previous reports, expression of the primary miR-34a transcript was induced and the c-Kit mRNA was repressed after p53 activation in both SW480 and DLD-1 cells (Figure 1C). Since the expression of miR-34b and miR-34c is at least 100 fold lower than that of miR-34a [39-41] in CRC cells and cell lines we focused our further studies on miR-34a. Notably, the ectopic expression of miR-34a driven by a conditional, episomal vector was sufficient to reduce c-Kit expression at the mRNA and protein levels in SW480 and DLD-1 cells (Figure 1D and 1E). Similar results were obtained with the CRC cell line HCT15 harboring the same miR-34 expression vector, though miR-34a mediated regulation was not as pronounced as in the other two cell lines (Supplemental Figure 1A and B). In order to determine whether miR-34 directly binds to the seed-matching sequences mentioned above we placed the c-Kit 3'-UTR (including the two potential binding sites) downstream of a luciferase open reading frame (Figure 2A). In a dual-reporter luciferase assay miR-34a as well as miR-34b and c significantly decreased the activity of this reporter (Figure 2B). When the seed-matching sequence in site 1 was mutated, the reporter was resistant to down-regulation by miR-34a, whereas mutation of site 2 did not affect the miR-34-mediated down-regulation of the reporter. Mutation of both sites led to resistance against miR-34a regulation. These results indicate that the miR-34 family directly targets the c-Kit 3'-UTR via site 1. This result is in accordance with the higher degree of conservation of site 1 when compared to site 2. Furthermore, p53-mediated down-regulation of c-Kit in DLD-1 cells could be prevented by simultaneous transfection of an antagomiR directed against miR-34a (anti-miR-34a), while additionally transfected miR-34a further enhanced the repression of c-Kit when p53 was activated concomitantly (Figure 2C). Taken together, miR-34a therefore mediates the repressive effects of p53 on c-Kit expression by directly targeting the c-Kit 3'-UTR via a single conserved seed-matching sequence.


Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness.

Siemens H, Jackstadt R, Kaller M, Hermeking H - Oncotarget (2013)

miR-34a directly targets c-Kit and mediates c-Kit repression by p53(A) Scheme of constructs used for dual luciferase assays. The positions of the putative miR-34 seed-matching sequences in the c-Kit 3'-UTR are depicted as a grey vertical bars, their mutations as crosses. Sequences of the respective targeted mutations are given below. (B) Dual reporter assay in SW480 cells transfected with miR-34a/b/c mimics or control oligonucleotide and the indicated 3'-UTR-reporter constructs for the human c-Kit 3'-UTR. Data are represented as mean ± SD (n = 3). (C) DLD-1/tTA-p53 cells were either transfected with a control oligonucleotide, miR-34a or an antagomiR directed against miR-34a for 24 hours either in the presence or absence of DOX (without or with ectopic p53). Expression of the indicated proteins was detected by Western blot analysis. α-tubulin served as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824539&req=5

Figure 2: miR-34a directly targets c-Kit and mediates c-Kit repression by p53(A) Scheme of constructs used for dual luciferase assays. The positions of the putative miR-34 seed-matching sequences in the c-Kit 3'-UTR are depicted as a grey vertical bars, their mutations as crosses. Sequences of the respective targeted mutations are given below. (B) Dual reporter assay in SW480 cells transfected with miR-34a/b/c mimics or control oligonucleotide and the indicated 3'-UTR-reporter constructs for the human c-Kit 3'-UTR. Data are represented as mean ± SD (n = 3). (C) DLD-1/tTA-p53 cells were either transfected with a control oligonucleotide, miR-34a or an antagomiR directed against miR-34a for 24 hours either in the presence or absence of DOX (without or with ectopic p53). Expression of the indicated proteins was detected by Western blot analysis. α-tubulin served as a loading control.
Mentions: Since a recent study reported a p53-dependent regulation of c-Kit, which occurred in the absence of p53 binding to the c-Kit promoter in mice [32], we hypothesized that miR-34 could be the mediator of this effect. In order to investigate this putative connection we used two different systems to conditionally express p53: SW480 cell pools transfected with the doxycycline (DOX) -inducible vector pRTR expressing the p53 open reading frame (ORF) and a DLD-1 single cell clone harboring a p53 allele under control of the tet-off system [36, 37]. Although the endogenous levels of c-Kit were lower in SW480 cells than in DLD-1 cells, activation of p53 in both cellular systems resulted in the down-regulation of c-Kit protein expression (Figure 1A). Since miRNAs were shown to mediate gene repression by p53 we examined the c-Kit 3'-UTR using the Target-Scan algorithm [38]. Thereby we identified two potential miR-34 seed-matching sequences in the 3'-UTR of c-Kit (Figure 1B). While the first site (which is a perfect match to the miR-34a 8-mer seed-matching sequence) is relatively conserved among different species, the second site seems to be less conserved. In line with previous reports, expression of the primary miR-34a transcript was induced and the c-Kit mRNA was repressed after p53 activation in both SW480 and DLD-1 cells (Figure 1C). Since the expression of miR-34b and miR-34c is at least 100 fold lower than that of miR-34a [39-41] in CRC cells and cell lines we focused our further studies on miR-34a. Notably, the ectopic expression of miR-34a driven by a conditional, episomal vector was sufficient to reduce c-Kit expression at the mRNA and protein levels in SW480 and DLD-1 cells (Figure 1D and 1E). Similar results were obtained with the CRC cell line HCT15 harboring the same miR-34 expression vector, though miR-34a mediated regulation was not as pronounced as in the other two cell lines (Supplemental Figure 1A and B). In order to determine whether miR-34 directly binds to the seed-matching sequences mentioned above we placed the c-Kit 3'-UTR (including the two potential binding sites) downstream of a luciferase open reading frame (Figure 2A). In a dual-reporter luciferase assay miR-34a as well as miR-34b and c significantly decreased the activity of this reporter (Figure 2B). When the seed-matching sequence in site 1 was mutated, the reporter was resistant to down-regulation by miR-34a, whereas mutation of site 2 did not affect the miR-34-mediated down-regulation of the reporter. Mutation of both sites led to resistance against miR-34a regulation. These results indicate that the miR-34 family directly targets the c-Kit 3'-UTR via site 1. This result is in accordance with the higher degree of conservation of site 1 when compared to site 2. Furthermore, p53-mediated down-regulation of c-Kit in DLD-1 cells could be prevented by simultaneous transfection of an antagomiR directed against miR-34a (anti-miR-34a), while additionally transfected miR-34a further enhanced the repression of c-Kit when p53 was activated concomitantly (Figure 2C). Taken together, miR-34a therefore mediates the repressive effects of p53 on c-Kit expression by directly targeting the c-Kit 3'-UTR via a single conserved seed-matching sequence.

Bottom Line: The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit.Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells.Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University München, D-80337 Munich, Germany.

ABSTRACT
The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches.

Show MeSH
Related in: MedlinePlus