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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 induces expression of stemness markers and correlates with their expression in primary CRC(A) DLD-1/pRTR-c-Kit cells were treated with DOX for 48 hours. Expression of the indicated mRNAs was determined by qPCR. Results represent the mean +/−S.D. (n=3).” * “: p < 0.05. (B) mRNA expression data derived from primary CRC samples (n = 196) were obtained from the public database TCGA [54]. Correlation coefficients and p-values were calculated applying the Spearman correlation algorithm. Scatter plots show the respective correlations. Both, the c-Kit levels on the x-axes and the y-axes of the OLFM4, CD44, Lgr5 and SCF mRNA expression values are provided as log10 scale.
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Figure 6: Ectopic c-Kit induces expression of stemness markers and correlates with their expression in primary CRC(A) DLD-1/pRTR-c-Kit cells were treated with DOX for 48 hours. Expression of the indicated mRNAs was determined by qPCR. Results represent the mean +/−S.D. (n=3).” * “: p < 0.05. (B) mRNA expression data derived from primary CRC samples (n = 196) were obtained from the public database TCGA [54]. Correlation coefficients and p-values were calculated applying the Spearman correlation algorithm. Scatter plots show the respective correlations. Both, the c-Kit levels on the x-axes and the y-axes of the OLFM4, CD44, Lgr5 and SCF mRNA expression values are provided as log10 scale.

Mentions: Since SCF is involved in the regulation of hematopoietic stem cells [46] and recent studies indicated a role for c-Kit in stemness in ovarian cancer [19], we asked whether c-Kit influences the expression of stemness markers in colorectal cancer cells. Therefore, c-Kit was ectopically expressed in DLD-1 cells and changes in mRNA expression were determined by qPCR. As positive controls for c-Kit-mediated gene regulations, expression of its downstream effectors c-Fos and Oncostatin M [47, 48], was analyzed. As markers for CRC stemness the expression of CD44, CD133, Lgr5, Nanog, Nanog P8, β-catenin, Sox2, BMI-1 and OLFM4 was determined [49-53]. As expected, c-Fos and Oncostatin M were induced after c-Kit activation (Figure 6A). Besides CD44 and BMI-1, all analyzed stemness markers were significantly up-regulated by c-Kit. CD44 was repressed and BMI-1 remained unchanged. These results indicate that c-Kit might enhance stemness of colorectal cancer cells. Next, we determined whether an association between c-Kit and stemness markers may also be present in clinical samples obtained from CRC patients as well. Therefore, expression data of 196 colorectal tumors from the public database TCGA (The Cancer Genome Atlas, [54]) was analyzed (Figure 6B). From these analyses, the expression of OLFM4, CD44, β-catenin, BMI-1 and Lgr5 mRNA emerged as significantly associated with elevated c-Kit mRNA expression (p ≤ 0.05). Additionally, elevated c-Kit ligand (SCF/stem cell factor) expression correlated with high receptor expression. This confirms previous results describing a co-expression of receptor and ligand [22-25]. Collectively, these associations suggest that c-Kit or/and presumably factors regulating c-Kit, as miR-34, might play a role in the regulation of stemness in colorectal cancer.


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 induces expression of stemness markers and correlates with their expression in primary CRC(A) DLD-1/pRTR-c-Kit cells were treated with DOX for 48 hours. Expression of the indicated mRNAs was determined by qPCR. Results represent the mean +/−S.D. (n=3).” * “: p < 0.05. (B) mRNA expression data derived from primary CRC samples (n = 196) were obtained from the public database TCGA [54]. Correlation coefficients and p-values were calculated applying the Spearman correlation algorithm. Scatter plots show the respective correlations. Both, the c-Kit levels on the x-axes and the y-axes of the OLFM4, CD44, Lgr5 and SCF mRNA expression values are provided as log10 scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Ectopic c-Kit induces expression of stemness markers and correlates with their expression in primary CRC(A) DLD-1/pRTR-c-Kit cells were treated with DOX for 48 hours. Expression of the indicated mRNAs was determined by qPCR. Results represent the mean +/−S.D. (n=3).” * “: p < 0.05. (B) mRNA expression data derived from primary CRC samples (n = 196) were obtained from the public database TCGA [54]. Correlation coefficients and p-values were calculated applying the Spearman correlation algorithm. Scatter plots show the respective correlations. Both, the c-Kit levels on the x-axes and the y-axes of the OLFM4, CD44, Lgr5 and SCF mRNA expression values are provided as log10 scale.
Mentions: Since SCF is involved in the regulation of hematopoietic stem cells [46] and recent studies indicated a role for c-Kit in stemness in ovarian cancer [19], we asked whether c-Kit influences the expression of stemness markers in colorectal cancer cells. Therefore, c-Kit was ectopically expressed in DLD-1 cells and changes in mRNA expression were determined by qPCR. As positive controls for c-Kit-mediated gene regulations, expression of its downstream effectors c-Fos and Oncostatin M [47, 48], was analyzed. As markers for CRC stemness the expression of CD44, CD133, Lgr5, Nanog, Nanog P8, β-catenin, Sox2, BMI-1 and OLFM4 was determined [49-53]. As expected, c-Fos and Oncostatin M were induced after c-Kit activation (Figure 6A). Besides CD44 and BMI-1, all analyzed stemness markers were significantly up-regulated by c-Kit. CD44 was repressed and BMI-1 remained unchanged. These results indicate that c-Kit might enhance stemness of colorectal cancer cells. Next, we determined whether an association between c-Kit and stemness markers may also be present in clinical samples obtained from CRC patients as well. Therefore, expression data of 196 colorectal tumors from the public database TCGA (The Cancer Genome Atlas, [54]) was analyzed (Figure 6B). From these analyses, the expression of OLFM4, CD44, β-catenin, BMI-1 and Lgr5 mRNA emerged as significantly associated with elevated c-Kit mRNA expression (p ≤ 0.05). Additionally, elevated c-Kit ligand (SCF/stem cell factor) expression correlated with high receptor expression. This confirms previous results describing a co-expression of receptor and ligand [22-25]. Collectively, these associations suggest that c-Kit or/and presumably factors regulating c-Kit, as miR-34, might play a role in the regulation of stemness in colorectal cancer.

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