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The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression.

He C, Mao D, Hua G, Lv X, Chen X, Angeletti PC, Dong J, Remmenga SW, Rodabaugh KJ, Zhou J, Lambert PF, Yang P, Davis JS, Wang C - EMBO Mol Med (2015)

Bottom Line: The Hippo signaling pathway controls organ size and tumorigenesis through a kinase cascade that inactivates Yes-associated protein (YAP).Our results suggest that YAP expression is associated with a poor prognosis for cervical cancer.Our study indicates that combined targeting of the Hippo and the ERBB signaling pathways represents a novel therapeutic strategy for prevention and treatment of cervical cancer.

View Article: PubMed Central - PubMed

Affiliation: Olson Center for Women's Health, Department of Obstetrics & Gynecology, University of Nebraska Medical Center, Omaha, NE, USA College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, China.

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Effect of YAP on the proliferation of normal and cancerous cervical cellsA, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control.B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4.G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).Data information: Data in (B), (D), and (F) were analyzed for significance using a one-way ANOVA in GraphPad Prism 5 with Tukey’s post hoc test. Data in (H) were analyzed with an unpaired t-test in GraphPad Prism 5 with Welch’s correction. Source data are available online for this figure.
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fig02: Effect of YAP on the proliferation of normal and cancerous cervical cellsA, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control.B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4.G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).Data information: Data in (B), (D), and (F) were analyzed for significance using a one-way ANOVA in GraphPad Prism 5 with Tukey’s post hoc test. Data in (H) were analyzed with an unpaired t-test in GraphPad Prism 5 with Welch’s correction. Source data are available online for this figure.

Mentions: Since YAP is overexpressed in cervical cancer, we used ME180 (HPV positive) and HT3 (HPV negative) cervical cancer cell lines to clarify the role of YAP in cervical cancer cell proliferation. We established six cell lines with differential YAP protein levels and activities: ME180-YAPS127A and HT3-YAPS127A cell lines expressing constitutively activated YAP; ME180-YAP and HT3-YAP overexpressing wild-type YAP; and ME180-MXIV and HT3-MXIV cells transfected with control vectors (MXIV). As expected, YAP was overexpressed in ME180-YAP, HT3-YAP, ME180-YAPS127A, and HT3-YAPS127A cells (Fig2A and C, Appendix Fig S3A and B). An increase in phosphorylated YAP was observed in the ME180-YAP and HT3-YAP cells, but not in ME180-YAPS127A and HT3-YAPS127A cells, consistent with the mutation of serine 127 to alanine (Fig2A and C, Appendix Fig S3A and B), which results in constitutive YAP activity (Pan, 2010). We observed that in the presence of complete medium (10% serum for HT3, 2.5% serum for ME180), the growth rate of the ME180 and HT3 cell lines was similar prior to reaching confluence. After reaching confluence (> 4 days after cell plating), cells in the control groups almost stopped proliferating. However, cells overexpressing YAP or YAPS127A continued to proliferate (Fig2B and D), with the highest growth rates observed in ME180-YAPS127A and HT3-YAPS127A cells. Interestingly, when examined under serum-reduced conditions (1% FBS), the growth rate of the ME180-YAPS127A cells was significantly higher than that of the ME180-YAP cells, while growth rate of the ME180-YAP cells was significantly higher than that of ME180-MXIV cells, even before the cell cultures reach confluence (Appendix Fig S3C). We then analyzed cell cycle progression in these cell lines after they reached confluence. The results showed that overexpression or constitutive activation of YAP increased the percentage of cells in S and G2/M phases, and reduced the proportion of cells in G1 phase in both ME180 and HT3 cervical cancer cells (Appendix Fig S4).


The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression.

He C, Mao D, Hua G, Lv X, Chen X, Angeletti PC, Dong J, Remmenga SW, Rodabaugh KJ, Zhou J, Lambert PF, Yang P, Davis JS, Wang C - EMBO Mol Med (2015)

Effect of YAP on the proliferation of normal and cancerous cervical cellsA, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control.B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4.G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).Data information: Data in (B), (D), and (F) were analyzed for significance using a one-way ANOVA in GraphPad Prism 5 with Tukey’s post hoc test. Data in (H) were analyzed with an unpaired t-test in GraphPad Prism 5 with Welch’s correction. Source data are available online for this figure.
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Related In: Results  -  Collection

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Show All Figures
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fig02: Effect of YAP on the proliferation of normal and cancerous cervical cellsA, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control.B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4.G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).Data information: Data in (B), (D), and (F) were analyzed for significance using a one-way ANOVA in GraphPad Prism 5 with Tukey’s post hoc test. Data in (H) were analyzed with an unpaired t-test in GraphPad Prism 5 with Welch’s correction. Source data are available online for this figure.
Mentions: Since YAP is overexpressed in cervical cancer, we used ME180 (HPV positive) and HT3 (HPV negative) cervical cancer cell lines to clarify the role of YAP in cervical cancer cell proliferation. We established six cell lines with differential YAP protein levels and activities: ME180-YAPS127A and HT3-YAPS127A cell lines expressing constitutively activated YAP; ME180-YAP and HT3-YAP overexpressing wild-type YAP; and ME180-MXIV and HT3-MXIV cells transfected with control vectors (MXIV). As expected, YAP was overexpressed in ME180-YAP, HT3-YAP, ME180-YAPS127A, and HT3-YAPS127A cells (Fig2A and C, Appendix Fig S3A and B). An increase in phosphorylated YAP was observed in the ME180-YAP and HT3-YAP cells, but not in ME180-YAPS127A and HT3-YAPS127A cells, consistent with the mutation of serine 127 to alanine (Fig2A and C, Appendix Fig S3A and B), which results in constitutive YAP activity (Pan, 2010). We observed that in the presence of complete medium (10% serum for HT3, 2.5% serum for ME180), the growth rate of the ME180 and HT3 cell lines was similar prior to reaching confluence. After reaching confluence (> 4 days after cell plating), cells in the control groups almost stopped proliferating. However, cells overexpressing YAP or YAPS127A continued to proliferate (Fig2B and D), with the highest growth rates observed in ME180-YAPS127A and HT3-YAPS127A cells. Interestingly, when examined under serum-reduced conditions (1% FBS), the growth rate of the ME180-YAPS127A cells was significantly higher than that of the ME180-YAP cells, while growth rate of the ME180-YAP cells was significantly higher than that of ME180-MXIV cells, even before the cell cultures reach confluence (Appendix Fig S3C). We then analyzed cell cycle progression in these cell lines after they reached confluence. The results showed that overexpression or constitutive activation of YAP increased the percentage of cells in S and G2/M phases, and reduced the proportion of cells in G1 phase in both ME180 and HT3 cervical cancer cells (Appendix Fig S4).

Bottom Line: The Hippo signaling pathway controls organ size and tumorigenesis through a kinase cascade that inactivates Yes-associated protein (YAP).Our results suggest that YAP expression is associated with a poor prognosis for cervical cancer.Our study indicates that combined targeting of the Hippo and the ERBB signaling pathways represents a novel therapeutic strategy for prevention and treatment of cervical cancer.

View Article: PubMed Central - PubMed

Affiliation: Olson Center for Women's Health, Department of Obstetrics & Gynecology, University of Nebraska Medical Center, Omaha, NE, USA College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, China.

Show MeSH
Related in: MedlinePlus