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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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Ectopic c-Kit overrides miR-34a-dependent inhibition of Erk signaling and colony formation in soft agar(A) DLD-1/pRTR-pri-miR-34a cells were treated with DOX for the indicated time-points. The indicated proteins were detected by Western blot analysis. α-tubulin served as a loading control. (B) Western blot analysis of the indicated SW480 and DLD-1 cells after oligonucleotide transfection for 48 hours and addition of DOX for 24 hours. α-tubulin served as a loading control. le = long exposure, se = short exposure. (C) Soft agar colony formation assay. The indicated cells were treated as described under (B). 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 3: Ectopic c-Kit overrides miR-34a-dependent inhibition of Erk signaling and colony formation in soft agar(A) DLD-1/pRTR-pri-miR-34a cells were treated with DOX for the indicated time-points. The indicated proteins were detected by Western blot analysis. α-tubulin served as a loading control. (B) Western blot analysis of the indicated SW480 and DLD-1 cells after oligonucleotide transfection for 48 hours and addition of DOX for 24 hours. α-tubulin served as a loading control. le = long exposure, se = short exposure. (C) Soft agar colony formation assay. The indicated cells were treated as described under (B). Results represent the mean +/−S.D. (n=3) and significance was calculated applying a Student's t-test.” * “: p < 0.05.

Mentions: In order to determine the consequences of a miR-34a-mediated decrease in c-Kit protein we analyzed Erk signaling, which is known to be activated by c-Kit. Indeed, the levels of phosphorylated Erk were decreased following the induction of ectopic miR-34a expression in DLD-1 cells, which was accompanied by a reduction in the levels of c-Kit protein, whereas the total amount of Erk protein levels was not affected (Figure 3A). Next, we determined whether the miR-34a-mediated decrease in Erk-phosphorylation was dependent on the down-regulation of c-Kit. Therefore, we generated pools of DLD-1 and SW480 cells carrying a conditional expression vector for c-Kit lacking its original 3'-UTR and therefore a miR-34 seed matching sequence. Ectopic miR-34a expression resulted in decreased levels of Erk-phosphorylation in both cell lines, while the control oligo had no effect (Figure 3B). Notably, induction of ectopic c-Kit largely reversed the effect of miR-34a on phosphorylated Erk, whereas the amount of total Erk was not affected. Collectively, these results show that the down-regulation of c-Kit is necessary for the inhibitory effects of miR-34a on mitogenic Erk signaling.


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)

Ectopic c-Kit overrides miR-34a-dependent inhibition of Erk signaling and colony formation in soft agar(A) DLD-1/pRTR-pri-miR-34a cells were treated with DOX for the indicated time-points. The indicated proteins were detected by Western blot analysis. α-tubulin served as a loading control. (B) Western blot analysis of the indicated SW480 and DLD-1 cells after oligonucleotide transfection for 48 hours and addition of DOX for 24 hours. α-tubulin served as a loading control. le = long exposure, se = short exposure. (C) Soft agar colony formation assay. The indicated cells were treated as described under (B). 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 3: Ectopic c-Kit overrides miR-34a-dependent inhibition of Erk signaling and colony formation in soft agar(A) DLD-1/pRTR-pri-miR-34a cells were treated with DOX for the indicated time-points. The indicated proteins were detected by Western blot analysis. α-tubulin served as a loading control. (B) Western blot analysis of the indicated SW480 and DLD-1 cells after oligonucleotide transfection for 48 hours and addition of DOX for 24 hours. α-tubulin served as a loading control. le = long exposure, se = short exposure. (C) Soft agar colony formation assay. The indicated cells were treated as described under (B). Results represent the mean +/−S.D. (n=3) and significance was calculated applying a Student's t-test.” * “: p < 0.05.
Mentions: In order to determine the consequences of a miR-34a-mediated decrease in c-Kit protein we analyzed Erk signaling, which is known to be activated by c-Kit. Indeed, the levels of phosphorylated Erk were decreased following the induction of ectopic miR-34a expression in DLD-1 cells, which was accompanied by a reduction in the levels of c-Kit protein, whereas the total amount of Erk protein levels was not affected (Figure 3A). Next, we determined whether the miR-34a-mediated decrease in Erk-phosphorylation was dependent on the down-regulation of c-Kit. Therefore, we generated pools of DLD-1 and SW480 cells carrying a conditional expression vector for c-Kit lacking its original 3'-UTR and therefore a miR-34 seed matching sequence. Ectopic miR-34a expression resulted in decreased levels of Erk-phosphorylation in both cell lines, while the control oligo had no effect (Figure 3B). Notably, induction of ectopic c-Kit largely reversed the effect of miR-34a on phosphorylated Erk, whereas the amount of total Erk was not affected. Collectively, these results show that the down-regulation of c-Kit is necessary for the inhibitory effects of miR-34a on mitogenic Erk signaling.

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