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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 and miR-34a modulate the apoptotic response to chemotherapeutic agents(A) Detection of endogenous c-Kit levels by Western blot analysis of DLD-1 and SW480 colorectal cancer cells. Detection of α-tubulin served as loading control. (B) Cells were treated with either 5-FU or Doxorubicin for 48 hours and subjected to DNA content analysis by flow cytometry. Results represent the mean +/−S.D. (n=3). (C) DLD-1 cells were transfected with the indicated oligonucleotides and simultaneously treated with 5-FU or water. The cell index, which corresponds to cell proliferation, was determined by real-time impedance measurements. (D) Cells treated as described in (C) were subjected to Western blot analysis. α-tubulin served as loading control.
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Figure 4: c-Kit and miR-34a modulate the apoptotic response to chemotherapeutic agents(A) Detection of endogenous c-Kit levels by Western blot analysis of DLD-1 and SW480 colorectal cancer cells. Detection of α-tubulin served as loading control. (B) Cells were treated with either 5-FU or Doxorubicin for 48 hours and subjected to DNA content analysis by flow cytometry. Results represent the mean +/−S.D. (n=3). (C) DLD-1 cells were transfected with the indicated oligonucleotides and simultaneously treated with 5-FU or water. The cell index, which corresponds to cell proliferation, was determined by real-time impedance measurements. (D) Cells treated as described in (C) were subjected to Western blot analysis. α-tubulin served as loading control.

Mentions: Recently, c-Kit was shown to mediate chemo-resistance in ovarian tumor initiating cells [19]. Therefore, we asked whether c-Kit might play a similar role in colorectal cancer. To address this issue DLD-1 cells, which express high levels of endogenous c-Kit, and SW480 cells expressing low levels of c-Kit were compared (Figure 4A). After treatment of both cell lines with either the anti-metabolite 5-FU or the anthracycline antibiotic Doxorubicin for 48 hours, SW480 cells showed a more pronounced apoptotic response to the treatment with both chemo-therapeutics, as determined by DNA content analysis using flow-cytometry, than DLD-1 cells (Figure 4B): upon treatment with the 5-FU the apoptotic sub-G1 fraction of cells was elevated about five times (from five to 25 %) in SW480 cells, while it tripled in DLD-1 cells (from three to nine %). Similar results were observed with Doxorubicin treatment: while SW480 cells showed a 10 fold increase (from three to 31 %) of apoptosis, apoptosis in DLD-1 cells increased about 3.5 fold (2 to 7 %). Consistently, the rate of spontaneous apoptosis in untreated cells was higher in SW480 cells than in DLD-1 cells, which therefore also inversely correlated with the level of c-Kit expression.


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 and miR-34a modulate the apoptotic response to chemotherapeutic agents(A) Detection of endogenous c-Kit levels by Western blot analysis of DLD-1 and SW480 colorectal cancer cells. Detection of α-tubulin served as loading control. (B) Cells were treated with either 5-FU or Doxorubicin for 48 hours and subjected to DNA content analysis by flow cytometry. Results represent the mean +/−S.D. (n=3). (C) DLD-1 cells were transfected with the indicated oligonucleotides and simultaneously treated with 5-FU or water. The cell index, which corresponds to cell proliferation, was determined by real-time impedance measurements. (D) Cells treated as described in (C) were subjected to Western blot analysis. α-tubulin served as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: c-Kit and miR-34a modulate the apoptotic response to chemotherapeutic agents(A) Detection of endogenous c-Kit levels by Western blot analysis of DLD-1 and SW480 colorectal cancer cells. Detection of α-tubulin served as loading control. (B) Cells were treated with either 5-FU or Doxorubicin for 48 hours and subjected to DNA content analysis by flow cytometry. Results represent the mean +/−S.D. (n=3). (C) DLD-1 cells were transfected with the indicated oligonucleotides and simultaneously treated with 5-FU or water. The cell index, which corresponds to cell proliferation, was determined by real-time impedance measurements. (D) Cells treated as described in (C) were subjected to Western blot analysis. α-tubulin served as loading control.
Mentions: Recently, c-Kit was shown to mediate chemo-resistance in ovarian tumor initiating cells [19]. Therefore, we asked whether c-Kit might play a similar role in colorectal cancer. To address this issue DLD-1 cells, which express high levels of endogenous c-Kit, and SW480 cells expressing low levels of c-Kit were compared (Figure 4A). After treatment of both cell lines with either the anti-metabolite 5-FU or the anthracycline antibiotic Doxorubicin for 48 hours, SW480 cells showed a more pronounced apoptotic response to the treatment with both chemo-therapeutics, as determined by DNA content analysis using flow-cytometry, than DLD-1 cells (Figure 4B): upon treatment with the 5-FU the apoptotic sub-G1 fraction of cells was elevated about five times (from five to 25 %) in SW480 cells, while it tripled in DLD-1 cells (from three to nine %). Similar results were observed with Doxorubicin treatment: while SW480 cells showed a 10 fold increase (from three to 31 %) of apoptosis, apoptosis in DLD-1 cells increased about 3.5 fold (2 to 7 %). Consistently, the rate of spontaneous apoptosis in untreated cells was higher in SW480 cells than in DLD-1 cells, which therefore also inversely correlated with the level of c-Kit expression.

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