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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

Schematic representation of the regulation of CD24 by Ras/Raf and Ras/PI3K pathways. Rectangles represent proteins and ovals represent lipids and second messengers. Protein activation is indicated by solid arrows and inhibition indicated by solid lines with blunt ends. Potential mechanisms for cross-talk are shown in gray. The effect of U0126 and LY294002 on MEK and PI3K and on CD24 transcription is indicated by dashed lines and dash dot dash lines, respectively. The effect of sorafenib on Raf and on CD24 surface protein expression is indicated by dotted lines. The CD24 promoter is indicated with a solid line flanked by genomic DNA depicted with a dotted line. The transcription start site is indicated by a bent arrow. Abbreviations not in the main text: PLC-γ, phospholipase C- γ; PIP3, phosphotidylinositol-3,4,5-trisphosphate; DAG, diacylglycerol; Cdc42, cell division cycle 42.
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Figure 7: Schematic representation of the regulation of CD24 by Ras/Raf and Ras/PI3K pathways. Rectangles represent proteins and ovals represent lipids and second messengers. Protein activation is indicated by solid arrows and inhibition indicated by solid lines with blunt ends. Potential mechanisms for cross-talk are shown in gray. The effect of U0126 and LY294002 on MEK and PI3K and on CD24 transcription is indicated by dashed lines and dash dot dash lines, respectively. The effect of sorafenib on Raf and on CD24 surface protein expression is indicated by dotted lines. The CD24 promoter is indicated with a solid line flanked by genomic DNA depicted with a dotted line. The transcription start site is indicated by a bent arrow. Abbreviations not in the main text: PLC-γ, phospholipase C- γ; PIP3, phosphotidylinositol-3,4,5-trisphosphate; DAG, diacylglycerol; Cdc42, cell division cycle 42.

Mentions: Here, we have demonstrated that expression of oncogenic Ras is sufficient to directly downregulate expression of CD24 at the mRNA and protein levels as well as repress promoter activity. Moreover, activation of either the Raf or PI3K pathway is sufficient to downregulate CD24 expression at both the mRNA and protein levels (Figure 7). Surprisingly, inhibition of the Raf pathway, at the level of MEK or Raf, or the PI3K pathway, at the level of PI3K, either separately or together, was not sufficient to fully restore CD24 expression in RasV12 cells. However, inhibition of Raf directly was able to partially restore CD24 surface protein expression without affecting mRNA levels.


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)

Schematic representation of the regulation of CD24 by Ras/Raf and Ras/PI3K pathways. Rectangles represent proteins and ovals represent lipids and second messengers. Protein activation is indicated by solid arrows and inhibition indicated by solid lines with blunt ends. Potential mechanisms for cross-talk are shown in gray. The effect of U0126 and LY294002 on MEK and PI3K and on CD24 transcription is indicated by dashed lines and dash dot dash lines, respectively. The effect of sorafenib on Raf and on CD24 surface protein expression is indicated by dotted lines. The CD24 promoter is indicated with a solid line flanked by genomic DNA depicted with a dotted line. The transcription start site is indicated by a bent arrow. Abbreviations not in the main text: PLC-γ, phospholipase C- γ; PIP3, phosphotidylinositol-3,4,5-trisphosphate; DAG, diacylglycerol; Cdc42, cell division cycle 42.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525067&req=5

Figure 7: Schematic representation of the regulation of CD24 by Ras/Raf and Ras/PI3K pathways. Rectangles represent proteins and ovals represent lipids and second messengers. Protein activation is indicated by solid arrows and inhibition indicated by solid lines with blunt ends. Potential mechanisms for cross-talk are shown in gray. The effect of U0126 and LY294002 on MEK and PI3K and on CD24 transcription is indicated by dashed lines and dash dot dash lines, respectively. The effect of sorafenib on Raf and on CD24 surface protein expression is indicated by dotted lines. The CD24 promoter is indicated with a solid line flanked by genomic DNA depicted with a dotted line. The transcription start site is indicated by a bent arrow. Abbreviations not in the main text: PLC-γ, phospholipase C- γ; PIP3, phosphotidylinositol-3,4,5-trisphosphate; DAG, diacylglycerol; Cdc42, cell division cycle 42.
Mentions: Here, we have demonstrated that expression of oncogenic Ras is sufficient to directly downregulate expression of CD24 at the mRNA and protein levels as well as repress promoter activity. Moreover, activation of either the Raf or PI3K pathway is sufficient to downregulate CD24 expression at both the mRNA and protein levels (Figure 7). Surprisingly, inhibition of the Raf pathway, at the level of MEK or Raf, or the PI3K pathway, at the level of PI3K, either separately or together, was not sufficient to fully restore CD24 expression in RasV12 cells. However, inhibition of Raf directly was able to partially restore CD24 surface protein expression without affecting mRNA levels.

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