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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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miR-34a inhibits basal and SCF-induced migration and invasion of Colo320 cells(A) Colo320 CRC cells were transfected either with a control (ctrl) or a miR-34a oligonucleotide for 48 hours. qPCR analysis was used to determine c-Kit mRNA levels. (B) Detection of c-Kit protein levels by Western blot analysis 48 hours after oligonucleotide transfection. α-tubulin served as loading control. (C) Colo320 cells were transfected with the indicated oligonucleotides and simultaneously treated with SCF (or water) for 24 hours. Thereafter cells were seeded into Boyden chambers to determine migration. (D) Determination of invasion using a Boyden chamber assay. Cells were treated as in (C). (A,C,D) Results represent the mean +/−S.D. (n=3) and significance was calculated applying a Student's t-test.” * “: p < 0.05.
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Figure 5: miR-34a inhibits basal and SCF-induced migration and invasion of Colo320 cells(A) Colo320 CRC cells were transfected either with a control (ctrl) or a miR-34a oligonucleotide for 48 hours. qPCR analysis was used to determine c-Kit mRNA levels. (B) Detection of c-Kit protein levels by Western blot analysis 48 hours after oligonucleotide transfection. α-tubulin served as loading control. (C) Colo320 cells were transfected with the indicated oligonucleotides and simultaneously treated with SCF (or water) for 24 hours. Thereafter cells were seeded into Boyden chambers to determine migration. (D) Determination of invasion using a Boyden chamber assay. Cells were treated as in (C). (A,C,D) Results represent the mean +/−S.D. (n=3) and significance was calculated applying a Student's t-test.” * “: p < 0.05.

Mentions: Another function of the SCF/c-Kit axis is an enhancement of migration, which was previously observed after treatment of the CRC cell line Colo320 with SCF [45]. Therefore, we analyzed whether transfection of Colo320 cells with miR-34a may interfere with the effects of SCF. First, regulation of c-Kit by miR-34a was confirmed on mRNA and protein level (Figure 5A and B). As expected, transfection of Colo320 cells with miR-34a oligonucleotides for 48 hours led to a decrease of c-Kit on the mRNA and protein levels. As determined by a modified Boyden chamber assay, the ectopic expression of miR-34a resulted in a decrease in cell migration (Figure 5C). Conversely, when cells were treated with SCF, the number of migrating cells doubled, confirming results described by Yusada et al. [45]. Combined treatment of Colo320 cells with SCF and miR-34a mimetics significantly reduced migration approximately to the levels observed in the controls. miR-34a had similar inhibitory effects on basal invasion and SCF-induced invasion of Colo320 cells (Figure 5D). A likely explanation for these effects is that miR-34a interferes with SCF signaling by suppressing the expression of the c-Kit receptor which could render cells refractory to SCF.


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 inhibits basal and SCF-induced migration and invasion of Colo320 cells(A) Colo320 CRC cells were transfected either with a control (ctrl) or a miR-34a oligonucleotide for 48 hours. qPCR analysis was used to determine c-Kit mRNA levels. (B) Detection of c-Kit protein levels by Western blot analysis 48 hours after oligonucleotide transfection. α-tubulin served as loading control. (C) Colo320 cells were transfected with the indicated oligonucleotides and simultaneously treated with SCF (or water) for 24 hours. Thereafter cells were seeded into Boyden chambers to determine migration. (D) Determination of invasion using a Boyden chamber assay. Cells were treated as in (C). (A,C,D) Results represent the mean +/−S.D. (n=3) and significance was calculated applying a Student's t-test.” * “: p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: miR-34a inhibits basal and SCF-induced migration and invasion of Colo320 cells(A) Colo320 CRC cells were transfected either with a control (ctrl) or a miR-34a oligonucleotide for 48 hours. qPCR analysis was used to determine c-Kit mRNA levels. (B) Detection of c-Kit protein levels by Western blot analysis 48 hours after oligonucleotide transfection. α-tubulin served as loading control. (C) Colo320 cells were transfected with the indicated oligonucleotides and simultaneously treated with SCF (or water) for 24 hours. Thereafter cells were seeded into Boyden chambers to determine migration. (D) Determination of invasion using a Boyden chamber assay. Cells were treated as in (C). (A,C,D) Results represent the mean +/−S.D. (n=3) and significance was calculated applying a Student's t-test.” * “: p < 0.05.
Mentions: Another function of the SCF/c-Kit axis is an enhancement of migration, which was previously observed after treatment of the CRC cell line Colo320 with SCF [45]. Therefore, we analyzed whether transfection of Colo320 cells with miR-34a may interfere with the effects of SCF. First, regulation of c-Kit by miR-34a was confirmed on mRNA and protein level (Figure 5A and B). As expected, transfection of Colo320 cells with miR-34a oligonucleotides for 48 hours led to a decrease of c-Kit on the mRNA and protein levels. As determined by a modified Boyden chamber assay, the ectopic expression of miR-34a resulted in a decrease in cell migration (Figure 5C). Conversely, when cells were treated with SCF, the number of migrating cells doubled, confirming results described by Yusada et al. [45]. Combined treatment of Colo320 cells with SCF and miR-34a mimetics significantly reduced migration approximately to the levels observed in the controls. miR-34a had similar inhibitory effects on basal invasion and SCF-induced invasion of Colo320 cells (Figure 5D). A likely explanation for these effects is that miR-34a interferes with SCF signaling by suppressing the expression of the c-Kit receptor which could render cells refractory to SCF.

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