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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

Oncogenic Ras downregulates CD24 expression in NIH/3T3 cells. CD24 mRNA expression in vector control (Control) and RasV12 cells was determined by (A) RT-PCR and (B) RT-qPCR. RPLP0 was used as the loading and normalization control. CD24 mRNA levels shown as mean ± s.e.m. (C) Surface CD24 protein was determined by flow cytometry in Control and RasV12 cells. One representative histogram of isotype (Iso) and CD24-stained cells is shown. (D) Quantification of CD24 surface protein expression as mean ± s.e.m percentage of CD24+ cells. Significance was determined by Student's t-test, n = 3, **P < 0.01; ***P < 0.001.
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Figure 1: Oncogenic Ras downregulates CD24 expression in NIH/3T3 cells. CD24 mRNA expression in vector control (Control) and RasV12 cells was determined by (A) RT-PCR and (B) RT-qPCR. RPLP0 was used as the loading and normalization control. CD24 mRNA levels shown as mean ± s.e.m. (C) Surface CD24 protein was determined by flow cytometry in Control and RasV12 cells. One representative histogram of isotype (Iso) and CD24-stained cells is shown. (D) Quantification of CD24 surface protein expression as mean ± s.e.m percentage of CD24+ cells. Significance was determined by Student's t-test, n = 3, **P < 0.01; ***P < 0.001.

Mentions: Previous studies have shown that activation of oncogenic Ras leads to generation of CD24−/CD44+ stem-like cells from CD24+/CD44low cells (Morel et al., 2008). To analyze the regulation of CD24 expression by oncogenic Ras we used a model system in which constitutively active H-Ras, containing a G12 to V12 mutation (RasV12), was stably expressed in the mouse embryonic fibroblast NIH/3T3 cell line (Christian et al., 2009). The level of CD24 mRNA expression in NIH/3T3 cells stably transfected with empty vector (control cells) or containing the constitutively active Ras gene was analyzed by RT-PCR and RT-qPCR (Figures 1A,B). We observed a clear suppression of CD24 mRNA expression (Figure 1A) that was over 1000-fold lower in RasV12 cells compared to control cells (Figure 1B).


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)

Oncogenic Ras downregulates CD24 expression in NIH/3T3 cells. CD24 mRNA expression in vector control (Control) and RasV12 cells was determined by (A) RT-PCR and (B) RT-qPCR. RPLP0 was used as the loading and normalization control. CD24 mRNA levels shown as mean ± s.e.m. (C) Surface CD24 protein was determined by flow cytometry in Control and RasV12 cells. One representative histogram of isotype (Iso) and CD24-stained cells is shown. (D) Quantification of CD24 surface protein expression as mean ± s.e.m percentage of CD24+ cells. Significance was determined by Student's t-test, n = 3, **P < 0.01; ***P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Oncogenic Ras downregulates CD24 expression in NIH/3T3 cells. CD24 mRNA expression in vector control (Control) and RasV12 cells was determined by (A) RT-PCR and (B) RT-qPCR. RPLP0 was used as the loading and normalization control. CD24 mRNA levels shown as mean ± s.e.m. (C) Surface CD24 protein was determined by flow cytometry in Control and RasV12 cells. One representative histogram of isotype (Iso) and CD24-stained cells is shown. (D) Quantification of CD24 surface protein expression as mean ± s.e.m percentage of CD24+ cells. Significance was determined by Student's t-test, n = 3, **P < 0.01; ***P < 0.001.
Mentions: Previous studies have shown that activation of oncogenic Ras leads to generation of CD24−/CD44+ stem-like cells from CD24+/CD44low cells (Morel et al., 2008). To analyze the regulation of CD24 expression by oncogenic Ras we used a model system in which constitutively active H-Ras, containing a G12 to V12 mutation (RasV12), was stably expressed in the mouse embryonic fibroblast NIH/3T3 cell line (Christian et al., 2009). The level of CD24 mRNA expression in NIH/3T3 cells stably transfected with empty vector (control cells) or containing the constitutively active Ras gene was analyzed by RT-PCR and RT-qPCR (Figures 1A,B). We observed a clear suppression of CD24 mRNA expression (Figure 1A) that was over 1000-fold lower in RasV12 cells compared to control cells (Figure 1B).

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