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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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Genome-wide Expression Profiling Identifies Rab2A as a Pin1 Target that Promotes BCSC Expansion and EMT(A) Hierarchical cluster of the microarray data of the Lin− population of mammary epithelial cells in two pairs of WT and Pin1 KO littermates.(B) Genomic profiling identified 14 potential target genes that were downregulated in Pin1 KO MECs and neuron cells (NCs) but upregulated in mouse MaSCs or BCSCs.(C) Heatmap depicting the fold changes of 14 candidate genes, which were downregulated in Pin1 KO cells (presented by KO/WT ratio) but upregulated in either mouse MaSCs or BCSCs (presented by SC/non-SC ratio).(D) Pin1 KD reduced Rab2A mRNA in human breast cancer lines, as assayed by real-time PCR.(E and F) Pin1 activated the Rab2A promoter in a dose-dependent manner.(G–I) Both Pin1 and c-Jun bound to the Rab2A promoter, as shown by ChIP and Re-ChIP analyses. Real-time PCR data were calibrated to immunoglobulin G (IgG) control and normalized with sample inputs of chromatin harvested prior to immunoprecipitation.(J) Rab2A was knocked down in vector control and Pin1-overexpressing HMLE cells, as confirmed by immunoblot.(K and L) Rab2A KD in HMLE cells reduced the CD24−CD44+ population and suppressed the ability of Pin1 overexpression to increase the CD24−CD44+ population.(M) Rab2A KD in HMLE cells reduced mammosphere-forming activity and impaired the ability of Pin1 overexpression to increase mammosphere-forming activity.(N and O) Rab2A KD impaired the ability of Pin1 overexpression to induce the EMT in HMLE cells, as shown by cell morphology (N) or upregulation of E-cadherin and downregulation of N-cadherin, fibronectin, and vimentin by qRT-PCR (O). Scale bar represents 100 μm.In all panels, error bars represent SD of three independent experiments. See also Figures S1 and S2.
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Figure 1: Genome-wide Expression Profiling Identifies Rab2A as a Pin1 Target that Promotes BCSC Expansion and EMT(A) Hierarchical cluster of the microarray data of the Lin− population of mammary epithelial cells in two pairs of WT and Pin1 KO littermates.(B) Genomic profiling identified 14 potential target genes that were downregulated in Pin1 KO MECs and neuron cells (NCs) but upregulated in mouse MaSCs or BCSCs.(C) Heatmap depicting the fold changes of 14 candidate genes, which were downregulated in Pin1 KO cells (presented by KO/WT ratio) but upregulated in either mouse MaSCs or BCSCs (presented by SC/non-SC ratio).(D) Pin1 KD reduced Rab2A mRNA in human breast cancer lines, as assayed by real-time PCR.(E and F) Pin1 activated the Rab2A promoter in a dose-dependent manner.(G–I) Both Pin1 and c-Jun bound to the Rab2A promoter, as shown by ChIP and Re-ChIP analyses. Real-time PCR data were calibrated to immunoglobulin G (IgG) control and normalized with sample inputs of chromatin harvested prior to immunoprecipitation.(J) Rab2A was knocked down in vector control and Pin1-overexpressing HMLE cells, as confirmed by immunoblot.(K and L) Rab2A KD in HMLE cells reduced the CD24−CD44+ population and suppressed the ability of Pin1 overexpression to increase the CD24−CD44+ population.(M) Rab2A KD in HMLE cells reduced mammosphere-forming activity and impaired the ability of Pin1 overexpression to increase mammosphere-forming activity.(N and O) Rab2A KD impaired the ability of Pin1 overexpression to induce the EMT in HMLE cells, as shown by cell morphology (N) or upregulation of E-cadherin and downregulation of N-cadherin, fibronectin, and vimentin by qRT-PCR (O). Scale bar represents 100 μm.In all panels, error bars represent SD of three independent experiments. See also Figures S1 and S2.

Mentions: We have previously demonstrated a fundamental role of Pin1 in regulating human BCSCs and mouse mammary stem cells (MaSCs) (Luo et al., 2014). To elucidate the underlying molecular mechanisms, we analyzed the effects of Pin1 knockout (KO) on gene expression in mouse mammary epithelial cells (MECs). Global expression profiling of Lin− MECs from Pin1 KO and wild-type (WT) littermates identified 1,723 genes that were downregulated in both Pin1 KO mice (Figures 1A and 1B; Tables S1 and S2). To narrow down the list of Pin1-regulated genes, we compared MEC gene expression with that of neurospheres prepared from the same mice (Figure 1B; Table S3). Although comparing expression profiles of stem cells from WT and Pin1 KO mice may be a better approach, the MaSC-enriched Lin−CD24+CD29+ or Lin−CD24medCD49fhi populations are very small in Pin1 KO mice (Luo et al., 2014), which made it difficult to get enough RNA from each mouse. As an alternative approach, we re-analyzed two published expression profiling datasets of mouse MaSCs and BCSCs (Stingl et al., 2006; Zhang et al., 2008) and compared them with our expression profiling of Lin− MECs and neurons from Pin1 KO and WT mice. We identified 14 genes downregulated in both Pin1 KO MECs and neurons and upregulated in either MaSCs or BCSCs (Figure 1C; Table S4).


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)

Genome-wide Expression Profiling Identifies Rab2A as a Pin1 Target that Promotes BCSC Expansion and EMT(A) Hierarchical cluster of the microarray data of the Lin− population of mammary epithelial cells in two pairs of WT and Pin1 KO littermates.(B) Genomic profiling identified 14 potential target genes that were downregulated in Pin1 KO MECs and neuron cells (NCs) but upregulated in mouse MaSCs or BCSCs.(C) Heatmap depicting the fold changes of 14 candidate genes, which were downregulated in Pin1 KO cells (presented by KO/WT ratio) but upregulated in either mouse MaSCs or BCSCs (presented by SC/non-SC ratio).(D) Pin1 KD reduced Rab2A mRNA in human breast cancer lines, as assayed by real-time PCR.(E and F) Pin1 activated the Rab2A promoter in a dose-dependent manner.(G–I) Both Pin1 and c-Jun bound to the Rab2A promoter, as shown by ChIP and Re-ChIP analyses. Real-time PCR data were calibrated to immunoglobulin G (IgG) control and normalized with sample inputs of chromatin harvested prior to immunoprecipitation.(J) Rab2A was knocked down in vector control and Pin1-overexpressing HMLE cells, as confirmed by immunoblot.(K and L) Rab2A KD in HMLE cells reduced the CD24−CD44+ population and suppressed the ability of Pin1 overexpression to increase the CD24−CD44+ population.(M) Rab2A KD in HMLE cells reduced mammosphere-forming activity and impaired the ability of Pin1 overexpression to increase mammosphere-forming activity.(N and O) Rab2A KD impaired the ability of Pin1 overexpression to induce the EMT in HMLE cells, as shown by cell morphology (N) or upregulation of E-cadherin and downregulation of N-cadherin, fibronectin, and vimentin by qRT-PCR (O). Scale bar represents 100 μm.In all panels, error bars represent SD of three independent experiments. See also Figures S1 and S2.
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Related In: Results  -  Collection

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Figure 1: Genome-wide Expression Profiling Identifies Rab2A as a Pin1 Target that Promotes BCSC Expansion and EMT(A) Hierarchical cluster of the microarray data of the Lin− population of mammary epithelial cells in two pairs of WT and Pin1 KO littermates.(B) Genomic profiling identified 14 potential target genes that were downregulated in Pin1 KO MECs and neuron cells (NCs) but upregulated in mouse MaSCs or BCSCs.(C) Heatmap depicting the fold changes of 14 candidate genes, which were downregulated in Pin1 KO cells (presented by KO/WT ratio) but upregulated in either mouse MaSCs or BCSCs (presented by SC/non-SC ratio).(D) Pin1 KD reduced Rab2A mRNA in human breast cancer lines, as assayed by real-time PCR.(E and F) Pin1 activated the Rab2A promoter in a dose-dependent manner.(G–I) Both Pin1 and c-Jun bound to the Rab2A promoter, as shown by ChIP and Re-ChIP analyses. Real-time PCR data were calibrated to immunoglobulin G (IgG) control and normalized with sample inputs of chromatin harvested prior to immunoprecipitation.(J) Rab2A was knocked down in vector control and Pin1-overexpressing HMLE cells, as confirmed by immunoblot.(K and L) Rab2A KD in HMLE cells reduced the CD24−CD44+ population and suppressed the ability of Pin1 overexpression to increase the CD24−CD44+ population.(M) Rab2A KD in HMLE cells reduced mammosphere-forming activity and impaired the ability of Pin1 overexpression to increase mammosphere-forming activity.(N and O) Rab2A KD impaired the ability of Pin1 overexpression to induce the EMT in HMLE cells, as shown by cell morphology (N) or upregulation of E-cadherin and downregulation of N-cadherin, fibronectin, and vimentin by qRT-PCR (O). Scale bar represents 100 μm.In all panels, error bars represent SD of three independent experiments. See also Figures S1 and S2.
Mentions: We have previously demonstrated a fundamental role of Pin1 in regulating human BCSCs and mouse mammary stem cells (MaSCs) (Luo et al., 2014). To elucidate the underlying molecular mechanisms, we analyzed the effects of Pin1 knockout (KO) on gene expression in mouse mammary epithelial cells (MECs). Global expression profiling of Lin− MECs from Pin1 KO and wild-type (WT) littermates identified 1,723 genes that were downregulated in both Pin1 KO mice (Figures 1A and 1B; Tables S1 and S2). To narrow down the list of Pin1-regulated genes, we compared MEC gene expression with that of neurospheres prepared from the same mice (Figure 1B; Table S3). Although comparing expression profiles of stem cells from WT and Pin1 KO mice may be a better approach, the MaSC-enriched Lin−CD24+CD29+ or Lin−CD24medCD49fhi populations are very small in Pin1 KO mice (Luo et al., 2014), which made it difficult to get enough RNA from each mouse. As an alternative approach, we re-analyzed two published expression profiling datasets of mouse MaSCs and BCSCs (Stingl et al., 2006; Zhang et al., 2008) and compared them with our expression profiling of Lin− MECs and neurons from Pin1 KO and WT mice. We identified 14 genes downregulated in both Pin1 KO MECs and neurons and upregulated in either MaSCs or BCSCs (Figure 1C; Table S4).

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