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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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HPV E6 stabilizes YAP proteinEffect of YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 60 min in McCoy’s 5A with 1% serum. Data were normalized with 18S mRNA. Bars with same letters are not significantly different from each other (P > 0.05). n = 4, P = 0.9407.YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 48 h in McCoy’s 5A with 1% serum. mRNA levels were measured with real-time PCR. Data were normalized with 18S mRNA. Each bar represents mean ± SEM (n = 5). Bars with same letters are not significantly different from each other (P > 0.05) (P = 0.3233).Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide (CHX) for 4 h or 8 h. β-actin was used as a protein loading control. Confluent HT3 cells were starved for 4 h before treatment with MG132 (10 μM) or CHX (20 μg/ml) for 0, 4 or 8 h.HPV16 E6 protein prevented YAP protein from degradation. Confluent HT3 cells were starved for 4 h before treatment with or without CHX (20 μg/ml), HPV16 E6, or CHX combined with HPV16 E6 for 8 h. Treatment of starved HT3 cells with HPV16 E6 (400 nM) for 8 h suppressed degradation of YAP, but not EGFR protein.Quantitative data showing relative YAP protein levels in (D). Protein levels were normalized with β-actin and presented as ratios relative to that of control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (Ctrl vs. CHX, P = 0.0003; Ctrl vs. CHX+E6, P = 0.2665; Ctrl vs. E6, P = 0.0258).Western blot analysis showing that expression of HPV16 E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε, but decreased the protein level of SOCS6. Importantly, HPV16 E6 increased YAP phosphorylation at serine 127, but suppressed its phosphorylation at serine 397. HPV16 E6 had no effect on the level of β-TrcP, LATS1/2 and MOB1 in HT3 cervical cancer cells.Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA (siE6) reduced YAP protein levels, but increased SOCS6 protein levels.Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA (siSOCS#1 and siSOCS6#2) increased total YAP protein, enhanced phosphorylation of YAP protein at serine 127, but suppressed phosphorylation of YAP at serine 397.Data information: Quantitative data in (A) and (B) were analyzed for significance using unpaired t-test in GraphPad Prism 5 with Welch’s correction. Data in (E) were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey’s post hoc tests.
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fig10: HPV E6 stabilizes YAP proteinEffect of YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 60 min in McCoy’s 5A with 1% serum. Data were normalized with 18S mRNA. Bars with same letters are not significantly different from each other (P > 0.05). n = 4, P = 0.9407.YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 48 h in McCoy’s 5A with 1% serum. mRNA levels were measured with real-time PCR. Data were normalized with 18S mRNA. Each bar represents mean ± SEM (n = 5). Bars with same letters are not significantly different from each other (P > 0.05) (P = 0.3233).Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide (CHX) for 4 h or 8 h. β-actin was used as a protein loading control. Confluent HT3 cells were starved for 4 h before treatment with MG132 (10 μM) or CHX (20 μg/ml) for 0, 4 or 8 h.HPV16 E6 protein prevented YAP protein from degradation. Confluent HT3 cells were starved for 4 h before treatment with or without CHX (20 μg/ml), HPV16 E6, or CHX combined with HPV16 E6 for 8 h. Treatment of starved HT3 cells with HPV16 E6 (400 nM) for 8 h suppressed degradation of YAP, but not EGFR protein.Quantitative data showing relative YAP protein levels in (D). Protein levels were normalized with β-actin and presented as ratios relative to that of control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (Ctrl vs. CHX, P = 0.0003; Ctrl vs. CHX+E6, P = 0.2665; Ctrl vs. E6, P = 0.0258).Western blot analysis showing that expression of HPV16 E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε, but decreased the protein level of SOCS6. Importantly, HPV16 E6 increased YAP phosphorylation at serine 127, but suppressed its phosphorylation at serine 397. HPV16 E6 had no effect on the level of β-TrcP, LATS1/2 and MOB1 in HT3 cervical cancer cells.Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA (siE6) reduced YAP protein levels, but increased SOCS6 protein levels.Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA (siSOCS#1 and siSOCS6#2) increased total YAP protein, enhanced phosphorylation of YAP protein at serine 127, but suppressed phosphorylation of YAP at serine 397.Data information: Quantitative data in (A) and (B) were analyzed for significance using unpaired t-test in GraphPad Prism 5 with Welch’s correction. Data in (E) were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey’s post hoc tests.

Mentions: The HT3 cancer cell line, which is HPV negative (Fogh et al, 1977; Yee et al, 1985), was used to determine the mechanism underlying HPV16 E6 regulation of YAP protein level. Treatment of HT3 cells with HPV16 E6 increased YAP protein (Fig9B and D), but HPV16 E6 did not affect YAP mRNA expression (Fig10A and B). This suggests that HPV16 E6 may regulate YAP protein turnover. Treatment of HT3 cells with MG132, a potent proteasome inhibitor, for 4 h or 8 h drastically increased YAP and EGFR protein levels (Fig10C). In contrast, treatment of HT3 cells with cycloheximide (CHX), an inhibitor of eukaryotic gene translation, for 4 h or 8 h significantly reduced EGFR and YAP protein levels (Fig10C and D). This evidence suggests that YAP and EGFR proteins are continuously synthesized and degraded in a proteasome-dependent mechanism in cervical cancer cells. The addition of HPV16 E6 prevented the degradation of YAP protein in CHX-treated cells, but had little or no effect on the degradation of EGFR (Fig10D and E). These observations clearly indicate that HPV16 E6 stabilizes the YAP protein in cervical cancer cells.


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)

HPV E6 stabilizes YAP proteinEffect of YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 60 min in McCoy’s 5A with 1% serum. Data were normalized with 18S mRNA. Bars with same letters are not significantly different from each other (P > 0.05). n = 4, P = 0.9407.YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 48 h in McCoy’s 5A with 1% serum. mRNA levels were measured with real-time PCR. Data were normalized with 18S mRNA. Each bar represents mean ± SEM (n = 5). Bars with same letters are not significantly different from each other (P > 0.05) (P = 0.3233).Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide (CHX) for 4 h or 8 h. β-actin was used as a protein loading control. Confluent HT3 cells were starved for 4 h before treatment with MG132 (10 μM) or CHX (20 μg/ml) for 0, 4 or 8 h.HPV16 E6 protein prevented YAP protein from degradation. Confluent HT3 cells were starved for 4 h before treatment with or without CHX (20 μg/ml), HPV16 E6, or CHX combined with HPV16 E6 for 8 h. Treatment of starved HT3 cells with HPV16 E6 (400 nM) for 8 h suppressed degradation of YAP, but not EGFR protein.Quantitative data showing relative YAP protein levels in (D). Protein levels were normalized with β-actin and presented as ratios relative to that of control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (Ctrl vs. CHX, P = 0.0003; Ctrl vs. CHX+E6, P = 0.2665; Ctrl vs. E6, P = 0.0258).Western blot analysis showing that expression of HPV16 E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε, but decreased the protein level of SOCS6. Importantly, HPV16 E6 increased YAP phosphorylation at serine 127, but suppressed its phosphorylation at serine 397. HPV16 E6 had no effect on the level of β-TrcP, LATS1/2 and MOB1 in HT3 cervical cancer cells.Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA (siE6) reduced YAP protein levels, but increased SOCS6 protein levels.Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA (siSOCS#1 and siSOCS6#2) increased total YAP protein, enhanced phosphorylation of YAP protein at serine 127, but suppressed phosphorylation of YAP at serine 397.Data information: Quantitative data in (A) and (B) were analyzed for significance using unpaired t-test in GraphPad Prism 5 with Welch’s correction. Data in (E) were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey’s post hoc tests.
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fig10: HPV E6 stabilizes YAP proteinEffect of YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 60 min in McCoy’s 5A with 1% serum. Data were normalized with 18S mRNA. Bars with same letters are not significantly different from each other (P > 0.05). n = 4, P = 0.9407.YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 48 h in McCoy’s 5A with 1% serum. mRNA levels were measured with real-time PCR. Data were normalized with 18S mRNA. Each bar represents mean ± SEM (n = 5). Bars with same letters are not significantly different from each other (P > 0.05) (P = 0.3233).Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide (CHX) for 4 h or 8 h. β-actin was used as a protein loading control. Confluent HT3 cells were starved for 4 h before treatment with MG132 (10 μM) or CHX (20 μg/ml) for 0, 4 or 8 h.HPV16 E6 protein prevented YAP protein from degradation. Confluent HT3 cells were starved for 4 h before treatment with or without CHX (20 μg/ml), HPV16 E6, or CHX combined with HPV16 E6 for 8 h. Treatment of starved HT3 cells with HPV16 E6 (400 nM) for 8 h suppressed degradation of YAP, but not EGFR protein.Quantitative data showing relative YAP protein levels in (D). Protein levels were normalized with β-actin and presented as ratios relative to that of control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (Ctrl vs. CHX, P = 0.0003; Ctrl vs. CHX+E6, P = 0.2665; Ctrl vs. E6, P = 0.0258).Western blot analysis showing that expression of HPV16 E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε, but decreased the protein level of SOCS6. Importantly, HPV16 E6 increased YAP phosphorylation at serine 127, but suppressed its phosphorylation at serine 397. HPV16 E6 had no effect on the level of β-TrcP, LATS1/2 and MOB1 in HT3 cervical cancer cells.Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA (siE6) reduced YAP protein levels, but increased SOCS6 protein levels.Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA (siSOCS#1 and siSOCS6#2) increased total YAP protein, enhanced phosphorylation of YAP protein at serine 127, but suppressed phosphorylation of YAP at serine 397.Data information: Quantitative data in (A) and (B) were analyzed for significance using unpaired t-test in GraphPad Prism 5 with Welch’s correction. Data in (E) were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey’s post hoc tests.
Mentions: The HT3 cancer cell line, which is HPV negative (Fogh et al, 1977; Yee et al, 1985), was used to determine the mechanism underlying HPV16 E6 regulation of YAP protein level. Treatment of HT3 cells with HPV16 E6 increased YAP protein (Fig9B and D), but HPV16 E6 did not affect YAP mRNA expression (Fig10A and B). This suggests that HPV16 E6 may regulate YAP protein turnover. Treatment of HT3 cells with MG132, a potent proteasome inhibitor, for 4 h or 8 h drastically increased YAP and EGFR protein levels (Fig10C). In contrast, treatment of HT3 cells with cycloheximide (CHX), an inhibitor of eukaryotic gene translation, for 4 h or 8 h significantly reduced EGFR and YAP protein levels (Fig10C and D). This evidence suggests that YAP and EGFR proteins are continuously synthesized and degraded in a proteasome-dependent mechanism in cervical cancer cells. The addition of HPV16 E6 prevented the degradation of YAP protein in CHX-treated cells, but had little or no effect on the degradation of EGFR (Fig10D and E). These observations clearly indicate that HPV16 E6 stabilizes the YAP protein in cervical cancer cells.

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