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Repression of CD24 surface protein expression by oncogenic Ras is relieved by inhibition of Raf but not MEK or PI3K.

Pallegar NK, Ayre DC, Christian SL - Front Cell Dev Biol (2015)

Bottom Line: In contrast, activation of the PI3K pathway downregulated mRNA expression with a partial effect on protein expression whereas activation of the RalGDS pathway only partially affected protein expression.In contrast, inhibition of Raf with sorafenib did not restore CD24 mRNA expression but significantly increased the proportion of RasV12 cells expressing CD24.Although inhibition of Raf by sorafenib only partially restored CD24 expression, sorafenib should still be considered as a potential therapeutic strategy to alter CD24 expression in CD24(-) cells, such as BCSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Memorial University of Newfoundland St. John's, NL, Canada.

ABSTRACT
CD24 is a dynamically regulated cell surface protein. High expression of CD24 leads to progression of lung, prostrate, colon, and pancreatic cancers, among others. In contrast, low expression of CD24 leads to cell proliferation and metastasis of breast cancer stem cells (BCSCs). Activating mutations in Ras are found in 30% of all human cancers. Oncogenic Ras constitutively stimulates the Raf, PI3K, and Ral GDS signaling pathways, leading to cellular transformation. Previous studies have shown that expression of oncogenic Ras in breast cancer cells generates CD24(-) cells from CD24(+) cells. However, the molecular mechanisms involved in the generation of CD24(-) cells were not determined. Here, we demonstrate that oncogenic Ras (RasV12) expression suppresses CD24 mRNA, protein, and promoter levels when expressed in NIH/3T3 cells. Furthermore, activation of only the Raf pathway was sufficient to downregulate CD24 mRNA and protein expression to levels similar to those seen in with RasV12 expression. In contrast, activation of the PI3K pathway downregulated mRNA expression with a partial effect on protein expression whereas activation of the RalGDS pathway only partially affected protein expression. Surprisingly, inhibition of MEK with U0126 only partially restored CD24 mRNA expression but not surface protein expression. In contrast, inhibition of Raf with sorafenib did not restore CD24 mRNA expression but significantly increased the proportion of RasV12 cells expressing CD24. Therefore, the Raf pathway is the major repressor of CD24 mRNA and protein expression, with PI3K also able to substantially inhibit CD24 expression. Moreover, these data indicate that the levels of CD24 mRNA and surface protein are independently regulated. Although inhibition of Raf by sorafenib only partially restored CD24 expression, sorafenib should still be considered as a potential therapeutic strategy to alter CD24 expression in CD24(-) cells, such as BCSCs.

No MeSH data available.


Related in: MedlinePlus

Inhibition of Raf is sufficient to increase CD24 cell surface protein but not mRNA expression in RasV12 cells. (A–C) Rasv12 cells were treated for 16 h with DMSO (D) or 5, 10, and 20 μM sorafenib (S). (A) Western blot analysis was performed as in Figure 4. One representative experiment from three replicates is shown. CD24 mRNA expression in Control and RasV12 was determined by (B) RT-PCR and (C) RT-qPCR as in Figure 4. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference post-hoc analysis, n = 3. (D) Surface CD24 protein was determined by flow cytometry with Control and RasV12 treated for 24 h as above. One representative histogram of isotype (Iso) and CD24-stained cells is shown. (E) Quantification of CD24 surface protein expression as mean ± s.e.m percentage of CD24+ cells. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference analysis, n = 3, different lower case letters indicate different groups at P < 0.001.
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Figure 5: Inhibition of Raf is sufficient to increase CD24 cell surface protein but not mRNA expression in RasV12 cells. (A–C) Rasv12 cells were treated for 16 h with DMSO (D) or 5, 10, and 20 μM sorafenib (S). (A) Western blot analysis was performed as in Figure 4. One representative experiment from three replicates is shown. CD24 mRNA expression in Control and RasV12 was determined by (B) RT-PCR and (C) RT-qPCR as in Figure 4. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference post-hoc analysis, n = 3. (D) Surface CD24 protein was determined by flow cytometry with Control and RasV12 treated for 24 h as above. One representative histogram of isotype (Iso) and CD24-stained cells is shown. (E) Quantification of CD24 surface protein expression as mean ± s.e.m percentage of CD24+ cells. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference analysis, n = 3, different lower case letters indicate different groups at P < 0.001.

Mentions: Since we observed that inhibition of MEK partially restored CD24 mRNA with no significant effect on protein levels we next determined if inhibition of Raf directly could restore CD24 expression levels. Raf is the major downstream target of Ras and can be directly inhibited by sorafenib, which does not inhibit MEK or ERK (Wilhelm et al., 2004). We evaluated the inhibition of Raf/MEK/ERK pathway by sorafenib at different concentrations using western blot analysis of phosphorylated ERK (P-ERK) and phosphorylated Akt (P-Akt) as measures of efficacy and specificity (Figure 5A). We found that sorafenib reduced ERK phosphorylation in RasV12 cells at all concentrations examined. We also found that both phosphorylated and total Akt was reduced with 10 and 20 μM sorafenib. Sorafenib is known to inhibit additional kinases such as EGFR, PDGFR, c-Kit and FLT-3 (Wilhelm et al., 2004; Adnane et al., 2006), therefore it is not surprising to observe inhibition of additional pathways with this inhibitor. However, we found that treatment of RasV12 cells with sorafenib had no effect on CD24 mRNA expression (Figures 5B,C).


Repression of CD24 surface protein expression by oncogenic Ras is relieved by inhibition of Raf but not MEK or PI3K.

Pallegar NK, Ayre DC, Christian SL - Front Cell Dev Biol (2015)

Inhibition of Raf is sufficient to increase CD24 cell surface protein but not mRNA expression in RasV12 cells. (A–C) Rasv12 cells were treated for 16 h with DMSO (D) or 5, 10, and 20 μM sorafenib (S). (A) Western blot analysis was performed as in Figure 4. One representative experiment from three replicates is shown. CD24 mRNA expression in Control and RasV12 was determined by (B) RT-PCR and (C) RT-qPCR as in Figure 4. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference post-hoc analysis, n = 3. (D) Surface CD24 protein was determined by flow cytometry with Control and RasV12 treated for 24 h as above. One representative histogram of isotype (Iso) and CD24-stained cells is shown. (E) Quantification of CD24 surface protein expression as mean ± s.e.m percentage of CD24+ cells. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference analysis, n = 3, different lower case letters indicate different groups at P < 0.001.
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Figure 5: Inhibition of Raf is sufficient to increase CD24 cell surface protein but not mRNA expression in RasV12 cells. (A–C) Rasv12 cells were treated for 16 h with DMSO (D) or 5, 10, and 20 μM sorafenib (S). (A) Western blot analysis was performed as in Figure 4. One representative experiment from three replicates is shown. CD24 mRNA expression in Control and RasV12 was determined by (B) RT-PCR and (C) RT-qPCR as in Figure 4. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference post-hoc analysis, n = 3. (D) Surface CD24 protein was determined by flow cytometry with Control and RasV12 treated for 24 h as above. One representative histogram of isotype (Iso) and CD24-stained cells is shown. (E) Quantification of CD24 surface protein expression as mean ± s.e.m percentage of CD24+ cells. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference analysis, n = 3, different lower case letters indicate different groups at P < 0.001.
Mentions: Since we observed that inhibition of MEK partially restored CD24 mRNA with no significant effect on protein levels we next determined if inhibition of Raf directly could restore CD24 expression levels. Raf is the major downstream target of Ras and can be directly inhibited by sorafenib, which does not inhibit MEK or ERK (Wilhelm et al., 2004). We evaluated the inhibition of Raf/MEK/ERK pathway by sorafenib at different concentrations using western blot analysis of phosphorylated ERK (P-ERK) and phosphorylated Akt (P-Akt) as measures of efficacy and specificity (Figure 5A). We found that sorafenib reduced ERK phosphorylation in RasV12 cells at all concentrations examined. We also found that both phosphorylated and total Akt was reduced with 10 and 20 μM sorafenib. Sorafenib is known to inhibit additional kinases such as EGFR, PDGFR, c-Kit and FLT-3 (Wilhelm et al., 2004; Adnane et al., 2006), therefore it is not surprising to observe inhibition of additional pathways with this inhibitor. However, we found that treatment of RasV12 cells with sorafenib had no effect on CD24 mRNA expression (Figures 5B,C).

Bottom Line: In contrast, activation of the PI3K pathway downregulated mRNA expression with a partial effect on protein expression whereas activation of the RalGDS pathway only partially affected protein expression.In contrast, inhibition of Raf with sorafenib did not restore CD24 mRNA expression but significantly increased the proportion of RasV12 cells expressing CD24.Although inhibition of Raf by sorafenib only partially restored CD24 expression, sorafenib should still be considered as a potential therapeutic strategy to alter CD24 expression in CD24(-) cells, such as BCSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Memorial University of Newfoundland St. John's, NL, Canada.

ABSTRACT
CD24 is a dynamically regulated cell surface protein. High expression of CD24 leads to progression of lung, prostrate, colon, and pancreatic cancers, among others. In contrast, low expression of CD24 leads to cell proliferation and metastasis of breast cancer stem cells (BCSCs). Activating mutations in Ras are found in 30% of all human cancers. Oncogenic Ras constitutively stimulates the Raf, PI3K, and Ral GDS signaling pathways, leading to cellular transformation. Previous studies have shown that expression of oncogenic Ras in breast cancer cells generates CD24(-) cells from CD24(+) cells. However, the molecular mechanisms involved in the generation of CD24(-) cells were not determined. Here, we demonstrate that oncogenic Ras (RasV12) expression suppresses CD24 mRNA, protein, and promoter levels when expressed in NIH/3T3 cells. Furthermore, activation of only the Raf pathway was sufficient to downregulate CD24 mRNA and protein expression to levels similar to those seen in with RasV12 expression. In contrast, activation of the PI3K pathway downregulated mRNA expression with a partial effect on protein expression whereas activation of the RalGDS pathway only partially affected protein expression. Surprisingly, inhibition of MEK with U0126 only partially restored CD24 mRNA expression but not surface protein expression. In contrast, inhibition of Raf with sorafenib did not restore CD24 mRNA expression but significantly increased the proportion of RasV12 cells expressing CD24. Therefore, the Raf pathway is the major repressor of CD24 mRNA and protein expression, with PI3K also able to substantially inhibit CD24 expression. Moreover, these data indicate that the levels of CD24 mRNA and surface protein are independently regulated. Although inhibition of Raf by sorafenib only partially restored CD24 expression, sorafenib should still be considered as a potential therapeutic strategy to alter CD24 expression in CD24(-) cells, such as BCSCs.

No MeSH data available.


Related in: MedlinePlus