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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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Role of the miR-34a/c-Kit axis in the regulation of stemness markers and sphere formation(A) DLD-1 cells pRTR/pri-miR-34a were treated with DOX for 48 hours. Expression of the indicated mRNAs was analyzed by qPCR. (B) DLD-1 cells were transfected with the indicated oligonucleotides and treated with SCF (or water) and subjected to a sphere formation assay. Sphere numbers were determined after seven days for the first generation (G1) and seven days after seeding for G2. Treatment with oligonucleotides and SCF was repeated when cells were passaged. (C) Representative pictures of DLD-1 derived G1 spheres, magnification: 40x. (A,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 7: Role of the miR-34a/c-Kit axis in the regulation of stemness markers and sphere formation(A) DLD-1 cells pRTR/pri-miR-34a were treated with DOX for 48 hours. Expression of the indicated mRNAs was analyzed by qPCR. (B) DLD-1 cells were transfected with the indicated oligonucleotides and treated with SCF (or water) and subjected to a sphere formation assay. Sphere numbers were determined after seven days for the first generation (G1) and seven days after seeding for G2. Treatment with oligonucleotides and SCF was repeated when cells were passaged. (C) Representative pictures of DLD-1 derived G1 spheres, magnification: 40x. (A,B) Results represent the mean +/−S.D. (n=3) and significance was calculated applying a Student's t-test.” * “: p < 0.05.

Mentions: To investigate whether miR-34a-dependent regulation of c-Kit might affect c-Kit-induced stemness markers, miR-34a was ectopically expressed in DLD-1 cells by activation of a conditional pRTR-pri-miR-34a vector (Figure 7A). The mRNA levels of c-Kit and its published downstream effectors c-Fos and Oncostatin M were repressed upon activation of miR-34a expression. However, mRNAs representing stemness markers displayed relatively weak down-regulations upon induction of miR-34a. In order to determine whether these regulations are relevant for stemness-related properties on the cellular level, DLD-1 cells were subjected to a sphere formation assay (Figure 7B). This assay in which cells grow in suspension under non-adherent conditions can be used to evaluate self-renewing capacities of cancer stem cells and has been applied previously for analyzing the effect of miR-34 on gastric cancer stem cells [55]. In line with previous publications [56] treatment with SCF significantly stimulated sphere-formation of DLD-1 cells. In contrast, treatment with miR-34a oligonucleotides reduced the number of spheres and also suppressed SCF-induced sphere formation of DLD-1 cells (Figure 7B and C). These effects were consistently observed over two consecutive generations. Taken together, these results show that miR-34a suppresses sphere formation and therefore stemness of colorectal cancer cells by targeting c-Kit.


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)

Role of the miR-34a/c-Kit axis in the regulation of stemness markers and sphere formation(A) DLD-1 cells pRTR/pri-miR-34a were treated with DOX for 48 hours. Expression of the indicated mRNAs was analyzed by qPCR. (B) DLD-1 cells were transfected with the indicated oligonucleotides and treated with SCF (or water) and subjected to a sphere formation assay. Sphere numbers were determined after seven days for the first generation (G1) and seven days after seeding for G2. Treatment with oligonucleotides and SCF was repeated when cells were passaged. (C) Representative pictures of DLD-1 derived G1 spheres, magnification: 40x. (A,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 7: Role of the miR-34a/c-Kit axis in the regulation of stemness markers and sphere formation(A) DLD-1 cells pRTR/pri-miR-34a were treated with DOX for 48 hours. Expression of the indicated mRNAs was analyzed by qPCR. (B) DLD-1 cells were transfected with the indicated oligonucleotides and treated with SCF (or water) and subjected to a sphere formation assay. Sphere numbers were determined after seven days for the first generation (G1) and seven days after seeding for G2. Treatment with oligonucleotides and SCF was repeated when cells were passaged. (C) Representative pictures of DLD-1 derived G1 spheres, magnification: 40x. (A,B) Results represent the mean +/−S.D. (n=3) and significance was calculated applying a Student's t-test.” * “: p < 0.05.
Mentions: To investigate whether miR-34a-dependent regulation of c-Kit might affect c-Kit-induced stemness markers, miR-34a was ectopically expressed in DLD-1 cells by activation of a conditional pRTR-pri-miR-34a vector (Figure 7A). The mRNA levels of c-Kit and its published downstream effectors c-Fos and Oncostatin M were repressed upon activation of miR-34a expression. However, mRNAs representing stemness markers displayed relatively weak down-regulations upon induction of miR-34a. In order to determine whether these regulations are relevant for stemness-related properties on the cellular level, DLD-1 cells were subjected to a sphere formation assay (Figure 7B). This assay in which cells grow in suspension under non-adherent conditions can be used to evaluate self-renewing capacities of cancer stem cells and has been applied previously for analyzing the effect of miR-34 on gastric cancer stem cells [55]. In line with previous publications [56] treatment with SCF significantly stimulated sphere-formation of DLD-1 cells. In contrast, treatment with miR-34a oligonucleotides reduced the number of spheres and also suppressed SCF-induced sphere formation of DLD-1 cells (Figure 7B and C). These effects were consistently observed over two consecutive generations. Taken together, these results show that miR-34a suppresses sphere formation and therefore stemness of colorectal cancer cells by targeting c-Kit.

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