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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.

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c-Kit is repressed after ectopic p53 and miR-34a expression in colorectal cancer cell lines(A) Western blot analysis of c-Kit protein levels after induction of p53 in colorectal cancer cell lines SW480 and DLD-1. α-tubulin served as a loading control. (B) Scheme of the c-Kit mRNA and conservation of the putative miR-34 seed-matching sequences, which are represented as grey vertical bars. Detailed sequences of the two sites and phylogenetic homologies are shown below. Potential base pairing is shaded in grey. (C) qPCR analysis of pri-miR-34a and c-Kit mRNA levels in the colorectal cancer cell lines SW480 and DLD-1 after induction of p53 by addition or withdrawal of doxycycline (DOX) for 72 hours. Results were normalized to β-actin mRNA. (D) qPCR analysis of c-Kit mRNA in the colorectal cancer cell lines SW480 and DLD-1 carrying the inducible pRTR/pri-miR-34a vector after addition of doxycycline. E) Detection of c-Kit protein by Western blot analysis after induction of pri-miR-34a in the indicated cells. α-tubulin served as a loading control. C+D: results represent the mean +/−S.D. (n=3).
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Figure 1: c-Kit is repressed after ectopic p53 and miR-34a expression in colorectal cancer cell lines(A) Western blot analysis of c-Kit protein levels after induction of p53 in colorectal cancer cell lines SW480 and DLD-1. α-tubulin served as a loading control. (B) Scheme of the c-Kit mRNA and conservation of the putative miR-34 seed-matching sequences, which are represented as grey vertical bars. Detailed sequences of the two sites and phylogenetic homologies are shown below. Potential base pairing is shaded in grey. (C) qPCR analysis of pri-miR-34a and c-Kit mRNA levels in the colorectal cancer cell lines SW480 and DLD-1 after induction of p53 by addition or withdrawal of doxycycline (DOX) for 72 hours. Results were normalized to β-actin mRNA. (D) qPCR analysis of c-Kit mRNA in the colorectal cancer cell lines SW480 and DLD-1 carrying the inducible pRTR/pri-miR-34a vector after addition of doxycycline. E) Detection of c-Kit protein by Western blot analysis after induction of pri-miR-34a in the indicated cells. α-tubulin served as a loading control. C+D: results represent the mean +/−S.D. (n=3).

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)

c-Kit is repressed after ectopic p53 and miR-34a expression in colorectal cancer cell lines(A) Western blot analysis of c-Kit protein levels after induction of p53 in colorectal cancer cell lines SW480 and DLD-1. α-tubulin served as a loading control. (B) Scheme of the c-Kit mRNA and conservation of the putative miR-34 seed-matching sequences, which are represented as grey vertical bars. Detailed sequences of the two sites and phylogenetic homologies are shown below. Potential base pairing is shaded in grey. (C) qPCR analysis of pri-miR-34a and c-Kit mRNA levels in the colorectal cancer cell lines SW480 and DLD-1 after induction of p53 by addition or withdrawal of doxycycline (DOX) for 72 hours. Results were normalized to β-actin mRNA. (D) qPCR analysis of c-Kit mRNA in the colorectal cancer cell lines SW480 and DLD-1 carrying the inducible pRTR/pri-miR-34a vector after addition of doxycycline. E) Detection of c-Kit protein by Western blot analysis after induction of pri-miR-34a in the indicated cells. α-tubulin served as a loading control. C+D: results represent the mean +/−S.D. (n=3).
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Figure 1: c-Kit is repressed after ectopic p53 and miR-34a expression in colorectal cancer cell lines(A) Western blot analysis of c-Kit protein levels after induction of p53 in colorectal cancer cell lines SW480 and DLD-1. α-tubulin served as a loading control. (B) Scheme of the c-Kit mRNA and conservation of the putative miR-34 seed-matching sequences, which are represented as grey vertical bars. Detailed sequences of the two sites and phylogenetic homologies are shown below. Potential base pairing is shaded in grey. (C) qPCR analysis of pri-miR-34a and c-Kit mRNA levels in the colorectal cancer cell lines SW480 and DLD-1 after induction of p53 by addition or withdrawal of doxycycline (DOX) for 72 hours. Results were normalized to β-actin mRNA. (D) qPCR analysis of c-Kit mRNA in the colorectal cancer cell lines SW480 and DLD-1 carrying the inducible pRTR/pri-miR-34a vector after addition of doxycycline. E) Detection of c-Kit protein by Western blot analysis after induction of pri-miR-34a in the indicated cells. α-tubulin served as a loading control. C+D: results represent the mean +/−S.D. (n=3).
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