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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.


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Inhibition of the Raf/MEK/ERK pathway is sufficient to partially restore CD24 mRNA but not protein expression in RasV12 cells. (A–C) RasV12 cells were treated for 16 h with DMSO (D), or U0126 (U) and/or LY294002 (LY). (A) Western blot analysis was performed to detect phosphorylated ERK (P-ERK), and phosphorylated Akt (P-Akt). Total ERK and total Akt were used as loading controls. Molecular mass standards are shown in the right of each image. One representative experiment from three replicates is shown. CD24 mRNA expression in Control and RasV12 cells was determined by (B) RT-PCR and (C) RT-qPCR. RPLP0 was used as the loading and normalization control. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference post-hoc analysis, *P < 0.05. (D) Surface CD24 protein was determined by flow cytometry with Control or RasV12 cells 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 student's t-test, n = 4, −P < 0.1; **P < 0.01.
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Figure 4: Inhibition of the Raf/MEK/ERK pathway is sufficient to partially restore CD24 mRNA but not protein expression in RasV12 cells. (A–C) RasV12 cells were treated for 16 h with DMSO (D), or U0126 (U) and/or LY294002 (LY). (A) Western blot analysis was performed to detect phosphorylated ERK (P-ERK), and phosphorylated Akt (P-Akt). Total ERK and total Akt were used as loading controls. Molecular mass standards are shown in the right of each image. One representative experiment from three replicates is shown. CD24 mRNA expression in Control and RasV12 cells was determined by (B) RT-PCR and (C) RT-qPCR. RPLP0 was used as the loading and normalization control. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference post-hoc analysis, *P < 0.05. (D) Surface CD24 protein was determined by flow cytometry with Control or RasV12 cells 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 student's t-test, n = 4, −P < 0.1; **P < 0.01.

Mentions: Since we observed that both CD24 mRNA and protein is significantly and substantially downregulated by the Raf and PI3K pathways we next determined if inhibition of either or both of these pathways could restore CD24 expression in RasV12 cells at the mRNA level. The major downstream effectors of Raf are the MEK1/2 kinases (Gollob et al., 2006), which can be inhibited specifically with the chemical inhibitor U0126 (Davies et al., 2000). PI3K can be directly inhibited using LY294002 (Davies et al., 2000; Xue et al., 2007). We evaluated the inhibition of Raf/MEK/ERK and PI3K/Akt pathways using western blot analysis of phosphorylated ERK (P-ERK) and phosphorylated Akt (P-Akt), respectively (Figure 4A). We found that RasV12 cells treated with U0126 had reduced phosphorylation of ERK with no effect on Akt phosphorylation. Similarly, RasV12 cells treated with LY294002 had reduced phosphorylation of Akt with no effect on ERK phosphorylation. Treatment with both U0126 and LY294002 inhibited phosphorylation of both ERK and Akt. We found that treatment of RasV12 cells with U0126 had a significant 13-fold increase in CD24 mRNA expression (Figures 4B,C). Surprisingly, treatment with LY294002 alone or in combination with U0126 suppressed CD24 mRNA expression to below the levels seen in RasV12 cells (Figures 4B,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 the Raf/MEK/ERK pathway is sufficient to partially restore CD24 mRNA but not protein expression in RasV12 cells. (A–C) RasV12 cells were treated for 16 h with DMSO (D), or U0126 (U) and/or LY294002 (LY). (A) Western blot analysis was performed to detect phosphorylated ERK (P-ERK), and phosphorylated Akt (P-Akt). Total ERK and total Akt were used as loading controls. Molecular mass standards are shown in the right of each image. One representative experiment from three replicates is shown. CD24 mRNA expression in Control and RasV12 cells was determined by (B) RT-PCR and (C) RT-qPCR. RPLP0 was used as the loading and normalization control. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference post-hoc analysis, *P < 0.05. (D) Surface CD24 protein was determined by flow cytometry with Control or RasV12 cells 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 student's t-test, n = 4, −P < 0.1; **P < 0.01.
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Figure 4: Inhibition of the Raf/MEK/ERK pathway is sufficient to partially restore CD24 mRNA but not protein expression in RasV12 cells. (A–C) RasV12 cells were treated for 16 h with DMSO (D), or U0126 (U) and/or LY294002 (LY). (A) Western blot analysis was performed to detect phosphorylated ERK (P-ERK), and phosphorylated Akt (P-Akt). Total ERK and total Akt were used as loading controls. Molecular mass standards are shown in the right of each image. One representative experiment from three replicates is shown. CD24 mRNA expression in Control and RasV12 cells was determined by (B) RT-PCR and (C) RT-qPCR. RPLP0 was used as the loading and normalization control. Significance was determined by One-Way ANOVA with Tukey Honest Significant Difference post-hoc analysis, *P < 0.05. (D) Surface CD24 protein was determined by flow cytometry with Control or RasV12 cells 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 student's t-test, n = 4, −P < 0.1; **P < 0.01.
Mentions: Since we observed that both CD24 mRNA and protein is significantly and substantially downregulated by the Raf and PI3K pathways we next determined if inhibition of either or both of these pathways could restore CD24 expression in RasV12 cells at the mRNA level. The major downstream effectors of Raf are the MEK1/2 kinases (Gollob et al., 2006), which can be inhibited specifically with the chemical inhibitor U0126 (Davies et al., 2000). PI3K can be directly inhibited using LY294002 (Davies et al., 2000; Xue et al., 2007). We evaluated the inhibition of Raf/MEK/ERK and PI3K/Akt pathways using western blot analysis of phosphorylated ERK (P-ERK) and phosphorylated Akt (P-Akt), respectively (Figure 4A). We found that RasV12 cells treated with U0126 had reduced phosphorylation of ERK with no effect on Akt phosphorylation. Similarly, RasV12 cells treated with LY294002 had reduced phosphorylation of Akt with no effect on ERK phosphorylation. Treatment with both U0126 and LY294002 inhibited phosphorylation of both ERK and Akt. We found that treatment of RasV12 cells with U0126 had a significant 13-fold increase in CD24 mRNA expression (Figures 4B,C). Surprisingly, treatment with LY294002 alone or in combination with U0126 suppressed CD24 mRNA expression to below the levels seen in RasV12 cells (Figures 4B,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