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The Rab2A GTPase promotes breast cancer stem cells and tumorigenesis via Erk signaling activation.

Luo ML, Gong C, Chen CH, Hu H, Huang P, Zheng M, Yao Y, Wei S, Wulf G, Lieberman J, Zhou XZ, Song E, Lu KP - Cell Rep (2015)

Bottom Line: Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and β-catenin nuclear translocation.Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients.Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.

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Rab2A and Its Q58H Mutant Drive BCSC Expansion via Activating Erk1/2(A and B) Rab2A regulated Erk1/2 phosphorylation and downstream Zeb1 expression. HMLE cells stably expressing Rab2A or shRNA or control vectors were treated with EGF after serum starvation for the indicated time points to activate Erk1/2 and analyzed by immunoblot.(C and D) Rab2A Q58H mutant activated Erk1/2 faster than WT Rab2A when overexpressed at the endogenous levels after EGF treatment. Arrowhead, exogenous Flag-Rab2A; arrow, endogenous Rab2A. immunoprecipitated by Rab2A in a dose-dependent manner (Figure 4G), suggesting that Rab2A competed with MKP3 to bind Erk1/2 in vivo. However, unlike the MKP3 competition results, similar amounts of Erk1/2 were immunoprecipitated by Flag-Rab2A even though decreasing amounts of MEK1 were expressed (Figure S5B). These results may be expected, because although the docking motif of MEK is important for the ERK-MEK interaction, there are other mechanisms to ensure the activation of Erk by MEK1, such as scaffold protein facilitation and the MEK catalytic site interacting with the Erk activation loop (Roskoski, 2012).(E) Erk1 or Erk2 was knocked down by two independent lentivirus-mediated shRNAs in Rab2A-overexpressing cells.(F) KD of Erk1/2, especially Erk2, prevented Rab2A from increasing the mammosphere-forming capability.(G and H) KD of Erk1/2, especially Erk2, prevented Rab2A from increasing the CD24−CD44+ population. In all panels, error bars represent SD of three independent experiments.
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Figure 3: Rab2A and Its Q58H Mutant Drive BCSC Expansion via Activating Erk1/2(A and B) Rab2A regulated Erk1/2 phosphorylation and downstream Zeb1 expression. HMLE cells stably expressing Rab2A or shRNA or control vectors were treated with EGF after serum starvation for the indicated time points to activate Erk1/2 and analyzed by immunoblot.(C and D) Rab2A Q58H mutant activated Erk1/2 faster than WT Rab2A when overexpressed at the endogenous levels after EGF treatment. Arrowhead, exogenous Flag-Rab2A; arrow, endogenous Rab2A. immunoprecipitated by Rab2A in a dose-dependent manner (Figure 4G), suggesting that Rab2A competed with MKP3 to bind Erk1/2 in vivo. However, unlike the MKP3 competition results, similar amounts of Erk1/2 were immunoprecipitated by Flag-Rab2A even though decreasing amounts of MEK1 were expressed (Figure S5B). These results may be expected, because although the docking motif of MEK is important for the ERK-MEK interaction, there are other mechanisms to ensure the activation of Erk by MEK1, such as scaffold protein facilitation and the MEK catalytic site interacting with the Erk activation loop (Roskoski, 2012).(E) Erk1 or Erk2 was knocked down by two independent lentivirus-mediated shRNAs in Rab2A-overexpressing cells.(F) KD of Erk1/2, especially Erk2, prevented Rab2A from increasing the mammosphere-forming capability.(G and H) KD of Erk1/2, especially Erk2, prevented Rab2A from increasing the CD24−CD44+ population. In all panels, error bars represent SD of three independent experiments.

Mentions: To understand how Rab2A expands the BCSC-enriched population, we examined whether Rab2A activates Erk1/2, which is crucial for Ras to induce EMT and increase the CD24−CD44+ population (Shin et al., 2010). After serum starvation and EGF stimulation, Rab2A overexpression significantly increased Erk1/2 activation monitored by p-Erk1/2 in a time-dependent manner and also increased expression of Zeb1 (Figures 3A and 3B), a transcription factor critical for inducing the EMT and CD24−CD44+ population (Shin et al., 2010; Wellner et al., 2009). In contrast, Rab2A KD substantially impaired Erk1/2 activation (Figures 3A and 3B). We then asked whether the Q58H mutation might increase Erk1/2 phosphorylation. When expressed at the endogenous level, the Q58H mutant induced Erk1/2 activation even faster than WT Rab2A after EGF stimulation (Figures 3C and 3D). Thus, Rab2A and its Q58H mutant promote Erk1/2 activation. Next, we silenced Erk1 or 2 in Rab2A-overexpressing HMLE cells (Figure 3E). Erk1 KD only partially inhibited, but Erk2 KD substantially decreased, the ability of Rab2A overexpression to induce mammosphere formation (Figure 3F) and the CD24−CD44+ population (Figures 3G and 3H), indicating that Rab2A acts through Erk1/2 to maintain BCSC-associated properties.


The Rab2A GTPase promotes breast cancer stem cells and tumorigenesis via Erk signaling activation.

Luo ML, Gong C, Chen CH, Hu H, Huang P, Zheng M, Yao Y, Wei S, Wulf G, Lieberman J, Zhou XZ, Song E, Lu KP - Cell Rep (2015)

Rab2A and Its Q58H Mutant Drive BCSC Expansion via Activating Erk1/2(A and B) Rab2A regulated Erk1/2 phosphorylation and downstream Zeb1 expression. HMLE cells stably expressing Rab2A or shRNA or control vectors were treated with EGF after serum starvation for the indicated time points to activate Erk1/2 and analyzed by immunoblot.(C and D) Rab2A Q58H mutant activated Erk1/2 faster than WT Rab2A when overexpressed at the endogenous levels after EGF treatment. Arrowhead, exogenous Flag-Rab2A; arrow, endogenous Rab2A. immunoprecipitated by Rab2A in a dose-dependent manner (Figure 4G), suggesting that Rab2A competed with MKP3 to bind Erk1/2 in vivo. However, unlike the MKP3 competition results, similar amounts of Erk1/2 were immunoprecipitated by Flag-Rab2A even though decreasing amounts of MEK1 were expressed (Figure S5B). These results may be expected, because although the docking motif of MEK is important for the ERK-MEK interaction, there are other mechanisms to ensure the activation of Erk by MEK1, such as scaffold protein facilitation and the MEK catalytic site interacting with the Erk activation loop (Roskoski, 2012).(E) Erk1 or Erk2 was knocked down by two independent lentivirus-mediated shRNAs in Rab2A-overexpressing cells.(F) KD of Erk1/2, especially Erk2, prevented Rab2A from increasing the mammosphere-forming capability.(G and H) KD of Erk1/2, especially Erk2, prevented Rab2A from increasing the CD24−CD44+ population. In all panels, error bars represent SD of three independent experiments.
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Figure 3: Rab2A and Its Q58H Mutant Drive BCSC Expansion via Activating Erk1/2(A and B) Rab2A regulated Erk1/2 phosphorylation and downstream Zeb1 expression. HMLE cells stably expressing Rab2A or shRNA or control vectors were treated with EGF after serum starvation for the indicated time points to activate Erk1/2 and analyzed by immunoblot.(C and D) Rab2A Q58H mutant activated Erk1/2 faster than WT Rab2A when overexpressed at the endogenous levels after EGF treatment. Arrowhead, exogenous Flag-Rab2A; arrow, endogenous Rab2A. immunoprecipitated by Rab2A in a dose-dependent manner (Figure 4G), suggesting that Rab2A competed with MKP3 to bind Erk1/2 in vivo. However, unlike the MKP3 competition results, similar amounts of Erk1/2 were immunoprecipitated by Flag-Rab2A even though decreasing amounts of MEK1 were expressed (Figure S5B). These results may be expected, because although the docking motif of MEK is important for the ERK-MEK interaction, there are other mechanisms to ensure the activation of Erk by MEK1, such as scaffold protein facilitation and the MEK catalytic site interacting with the Erk activation loop (Roskoski, 2012).(E) Erk1 or Erk2 was knocked down by two independent lentivirus-mediated shRNAs in Rab2A-overexpressing cells.(F) KD of Erk1/2, especially Erk2, prevented Rab2A from increasing the mammosphere-forming capability.(G and H) KD of Erk1/2, especially Erk2, prevented Rab2A from increasing the CD24−CD44+ population. In all panels, error bars represent SD of three independent experiments.
Mentions: To understand how Rab2A expands the BCSC-enriched population, we examined whether Rab2A activates Erk1/2, which is crucial for Ras to induce EMT and increase the CD24−CD44+ population (Shin et al., 2010). After serum starvation and EGF stimulation, Rab2A overexpression significantly increased Erk1/2 activation monitored by p-Erk1/2 in a time-dependent manner and also increased expression of Zeb1 (Figures 3A and 3B), a transcription factor critical for inducing the EMT and CD24−CD44+ population (Shin et al., 2010; Wellner et al., 2009). In contrast, Rab2A KD substantially impaired Erk1/2 activation (Figures 3A and 3B). We then asked whether the Q58H mutation might increase Erk1/2 phosphorylation. When expressed at the endogenous level, the Q58H mutant induced Erk1/2 activation even faster than WT Rab2A after EGF stimulation (Figures 3C and 3D). Thus, Rab2A and its Q58H mutant promote Erk1/2 activation. Next, we silenced Erk1 or 2 in Rab2A-overexpressing HMLE cells (Figure 3E). Erk1 KD only partially inhibited, but Erk2 KD substantially decreased, the ability of Rab2A overexpression to induce mammosphere formation (Figure 3F) and the CD24−CD44+ population (Figures 3G and 3H), indicating that Rab2A acts through Erk1/2 to maintain BCSC-associated properties.

Bottom Line: Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and β-catenin nuclear translocation.Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients.Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.

Show MeSH
Related in: MedlinePlus