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YAP dysregulation by phosphorylation or ΔNp63-mediated gene repression promotes proliferation, survival and migration in head and neck cancer subsets.

Ehsanian R, Brown M, Lu H, Yang XP, Pattatheyil A, Yan B, Duggal P, Chuang R, Doondeea J, Feller S, Sudol M, Chen Z, Van Waes C - Oncogene (2010)

Bottom Line: Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP.Transfection of a YAP-serine-127-alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death.AKT and/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.

View Article: PubMed Central - PubMed

Affiliation: Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892-1419, USA.

ABSTRACT
Overexpression of the Yes-associated protein (YAP), and TP53 family members ΔNp63 and p73, have been independently detected in subsets of head and neck squamous cell carcinomas (HNSCCs). YAP may serve as a nuclear cofactor with ΔNp63 and p73, but the functional role of YAP and their potential relationship in HNSCCs are unknown. In this study, we show that in a subset of HNSCC lines and tumors, YAP expression is increased but localized in the cytoplasm in association with increased AKT and YAP phosphorylation, and with decreased expression of ΔNp63 and p73. In another subset, YAP expression is decreased but detectable in the nucleus in association with lower AKT and YAP phosphorylation, and with increased ΔNp63 and p73 expression. Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP. ΔNp63 bound to the YAP promoter and suppressed its expression. Transfection of a YAP-serine-127-alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death. Conversely, YAP knockdown enhanced cell proliferation, survival, migration and cisplatin chemoresistance. Thus, YAP function as a tumor suppressor may alternatively be dysregulated by AKT phosphorylation at serine-127 and cytoplasmic sequestration, or by transcriptional repression by ΔNp63, in different subsets of HNSCC. AKT and/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.

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AKT inhibition or overexpression of a YAP serine 127 phosphoacceptor mutant enhances nuclear localization of YAP and cell death(A) Western blot of whole cell, cytoplamsic, and nuclear extracts of UMSCC 11A cells following no treatment (−) or with AKT inhibitor AKT-X (5 μM) for 1 hour (+). Whole cell extract was probed for phospho-AKT (Ser473) and total AKT, with actin as a loading control. Cytoplasmic extract was probed for phospho-YAP (Ser127) and total YAP with actin as a loading control. Nuclear extract was probed for total YAP with Oct-1 as a loading control. (B) Western blot of nuclear extract from UMSCC 11A shown three days post-transfection with wild-type YAP 2 vector (Y2) or phosphoacceptor-site mutant YAP2 vector (Y2M). Nuclear extract was probed for total YAP with Oct-1 as a loading control. Densitometry measurements adjusted for loading revealed a quantitative increase nuclear localization of 50% with the Y2M vs. Y vector. (C) DNA cell cycle analysis of the percentage of Sub-G0/G1 cells (% Cell Death) three days post-transfection with control vector (CV), Y2 or Y2M.
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Figure 2: AKT inhibition or overexpression of a YAP serine 127 phosphoacceptor mutant enhances nuclear localization of YAP and cell death(A) Western blot of whole cell, cytoplamsic, and nuclear extracts of UMSCC 11A cells following no treatment (−) or with AKT inhibitor AKT-X (5 μM) for 1 hour (+). Whole cell extract was probed for phospho-AKT (Ser473) and total AKT, with actin as a loading control. Cytoplasmic extract was probed for phospho-YAP (Ser127) and total YAP with actin as a loading control. Nuclear extract was probed for total YAP with Oct-1 as a loading control. (B) Western blot of nuclear extract from UMSCC 11A shown three days post-transfection with wild-type YAP 2 vector (Y2) or phosphoacceptor-site mutant YAP2 vector (Y2M). Nuclear extract was probed for total YAP with Oct-1 as a loading control. Densitometry measurements adjusted for loading revealed a quantitative increase nuclear localization of 50% with the Y2M vs. Y vector. (C) DNA cell cycle analysis of the percentage of Sub-G0/G1 cells (% Cell Death) three days post-transfection with control vector (CV), Y2 or Y2M.

Mentions: To investigate the potential role of AKT activation in the phosphorylation and cellular distributon of YAP, the effect of inhibitor AKT-X previously shown to specifically inhibit both AKT phosphorylation and activation was examined (Thimmaiah et al., 2005). After exposure to 5 μM AKT-X for 1 hour, AKTserine-473 as well as YAPserine-127 phosphorylation was inhibited (Fig. 2A; upper and middle panels). The inhibition of phospho-AKT and phospho-YAP correlated with an increase in detection of YAP in the nucleus, with minimal change in the overall cytoplasmic levels of YAP (Fig. 2A; middle and lower panels). To further investigate if the limited nuclear YAP detected in cell lines with high YAP expression was due to the serine-127 phosphorylation, we transfected UMSCC-11A cells with expression vectors encoding YAP2 or YAP2-S127A mutant proteins, since YAP2 with a second WW domain can be distinguished from endogenous YAP1 by its slower mobility (Fig. 2B). An ~50% increase in nuclear localization of YAP protein was seen with mutant compared to wild-type protein when normalized to OCT-1 (Fig. 2B). We assessed the functional effect of increased nuclear S127A mutant YAP2 on viability of UMSCC cells by DNA cell cycle analysis of sub-G0/G1 fragmented DNA (a measure of % Cell Death) three days post-transfection with control vector (CV), Y2 or Y2M (Figure. 2C). The increase in nuclear YAP2 mutant was accompanied by a relatively greater increase in cell death compared with empty control or wt YAP vectors, indicating that with phospho-site inactivation and increased nuclear localization, transfected YAP2 may function as a proapoptotic factor.


YAP dysregulation by phosphorylation or ΔNp63-mediated gene repression promotes proliferation, survival and migration in head and neck cancer subsets.

Ehsanian R, Brown M, Lu H, Yang XP, Pattatheyil A, Yan B, Duggal P, Chuang R, Doondeea J, Feller S, Sudol M, Chen Z, Van Waes C - Oncogene (2010)

AKT inhibition or overexpression of a YAP serine 127 phosphoacceptor mutant enhances nuclear localization of YAP and cell death(A) Western blot of whole cell, cytoplamsic, and nuclear extracts of UMSCC 11A cells following no treatment (−) or with AKT inhibitor AKT-X (5 μM) for 1 hour (+). Whole cell extract was probed for phospho-AKT (Ser473) and total AKT, with actin as a loading control. Cytoplasmic extract was probed for phospho-YAP (Ser127) and total YAP with actin as a loading control. Nuclear extract was probed for total YAP with Oct-1 as a loading control. (B) Western blot of nuclear extract from UMSCC 11A shown three days post-transfection with wild-type YAP 2 vector (Y2) or phosphoacceptor-site mutant YAP2 vector (Y2M). Nuclear extract was probed for total YAP with Oct-1 as a loading control. Densitometry measurements adjusted for loading revealed a quantitative increase nuclear localization of 50% with the Y2M vs. Y vector. (C) DNA cell cycle analysis of the percentage of Sub-G0/G1 cells (% Cell Death) three days post-transfection with control vector (CV), Y2 or Y2M.
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Figure 2: AKT inhibition or overexpression of a YAP serine 127 phosphoacceptor mutant enhances nuclear localization of YAP and cell death(A) Western blot of whole cell, cytoplamsic, and nuclear extracts of UMSCC 11A cells following no treatment (−) or with AKT inhibitor AKT-X (5 μM) for 1 hour (+). Whole cell extract was probed for phospho-AKT (Ser473) and total AKT, with actin as a loading control. Cytoplasmic extract was probed for phospho-YAP (Ser127) and total YAP with actin as a loading control. Nuclear extract was probed for total YAP with Oct-1 as a loading control. (B) Western blot of nuclear extract from UMSCC 11A shown three days post-transfection with wild-type YAP 2 vector (Y2) or phosphoacceptor-site mutant YAP2 vector (Y2M). Nuclear extract was probed for total YAP with Oct-1 as a loading control. Densitometry measurements adjusted for loading revealed a quantitative increase nuclear localization of 50% with the Y2M vs. Y vector. (C) DNA cell cycle analysis of the percentage of Sub-G0/G1 cells (% Cell Death) three days post-transfection with control vector (CV), Y2 or Y2M.
Mentions: To investigate the potential role of AKT activation in the phosphorylation and cellular distributon of YAP, the effect of inhibitor AKT-X previously shown to specifically inhibit both AKT phosphorylation and activation was examined (Thimmaiah et al., 2005). After exposure to 5 μM AKT-X for 1 hour, AKTserine-473 as well as YAPserine-127 phosphorylation was inhibited (Fig. 2A; upper and middle panels). The inhibition of phospho-AKT and phospho-YAP correlated with an increase in detection of YAP in the nucleus, with minimal change in the overall cytoplasmic levels of YAP (Fig. 2A; middle and lower panels). To further investigate if the limited nuclear YAP detected in cell lines with high YAP expression was due to the serine-127 phosphorylation, we transfected UMSCC-11A cells with expression vectors encoding YAP2 or YAP2-S127A mutant proteins, since YAP2 with a second WW domain can be distinguished from endogenous YAP1 by its slower mobility (Fig. 2B). An ~50% increase in nuclear localization of YAP protein was seen with mutant compared to wild-type protein when normalized to OCT-1 (Fig. 2B). We assessed the functional effect of increased nuclear S127A mutant YAP2 on viability of UMSCC cells by DNA cell cycle analysis of sub-G0/G1 fragmented DNA (a measure of % Cell Death) three days post-transfection with control vector (CV), Y2 or Y2M (Figure. 2C). The increase in nuclear YAP2 mutant was accompanied by a relatively greater increase in cell death compared with empty control or wt YAP vectors, indicating that with phospho-site inactivation and increased nuclear localization, transfected YAP2 may function as a proapoptotic factor.

Bottom Line: Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP.Transfection of a YAP-serine-127-alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death.AKT and/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.

View Article: PubMed Central - PubMed

Affiliation: Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892-1419, USA.

ABSTRACT
Overexpression of the Yes-associated protein (YAP), and TP53 family members ΔNp63 and p73, have been independently detected in subsets of head and neck squamous cell carcinomas (HNSCCs). YAP may serve as a nuclear cofactor with ΔNp63 and p73, but the functional role of YAP and their potential relationship in HNSCCs are unknown. In this study, we show that in a subset of HNSCC lines and tumors, YAP expression is increased but localized in the cytoplasm in association with increased AKT and YAP phosphorylation, and with decreased expression of ΔNp63 and p73. In another subset, YAP expression is decreased but detectable in the nucleus in association with lower AKT and YAP phosphorylation, and with increased ΔNp63 and p73 expression. Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP. ΔNp63 bound to the YAP promoter and suppressed its expression. Transfection of a YAP-serine-127-alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death. Conversely, YAP knockdown enhanced cell proliferation, survival, migration and cisplatin chemoresistance. Thus, YAP function as a tumor suppressor may alternatively be dysregulated by AKT phosphorylation at serine-127 and cytoplasmic sequestration, or by transcriptional repression by ΔNp63, in different subsets of HNSCC. AKT and/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.

Show MeSH
Related in: MedlinePlus