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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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Knockdown of YAP alters gene expression, increases cell proliferation and migration, and decreases programmed cell death and cisplatin cytotoxicity(A) QRT-PCR demonstrates knockdown efficiency of YAP mRNA days 1–4, and YAP protein (inset) day 3 post transfection of UMSCC 11A with YAP siRNA. (B) QRT-PCR probe for pro-apoptotic p53AIP and BAX, anti-apoptotic BCL-XL, and pro-angiogenic VEGF genes two days post-YAP knockdown. (C) MTT assay showing increased density of UMSCC-11A after knockdown of YAP. (D) Left panel: DNA cell cycle analysis of the percentage of sub-G0/G1 DNA (% Cell Death) by UMSCC-11A one and three days post-YAP siRNA treatment. Right panel: Flow cytometric analysis of the percentage apoptosis as indicated by the percentage of annexinV and propidium iodide double positive cells, two days post-transfection with indicated siRNA. (E) Wound healing/cell migration time course assay of the effects of YAP knockdown in UMSCC 11A. Left panel: measurements of the scratch/wound, using borders highlighted by the white lines in the timecourse images (right panel). (F) MTT assay of UMSCC-11A cell density after knockdown of YAP and treatment with 5 μM of cisplatin. * indicates significant difference (student t-test, p< 0.05).
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Figure 5: Knockdown of YAP alters gene expression, increases cell proliferation and migration, and decreases programmed cell death and cisplatin cytotoxicity(A) QRT-PCR demonstrates knockdown efficiency of YAP mRNA days 1–4, and YAP protein (inset) day 3 post transfection of UMSCC 11A with YAP siRNA. (B) QRT-PCR probe for pro-apoptotic p53AIP and BAX, anti-apoptotic BCL-XL, and pro-angiogenic VEGF genes two days post-YAP knockdown. (C) MTT assay showing increased density of UMSCC-11A after knockdown of YAP. (D) Left panel: DNA cell cycle analysis of the percentage of sub-G0/G1 DNA (% Cell Death) by UMSCC-11A one and three days post-YAP siRNA treatment. Right panel: Flow cytometric analysis of the percentage apoptosis as indicated by the percentage of annexinV and propidium iodide double positive cells, two days post-transfection with indicated siRNA. (E) Wound healing/cell migration time course assay of the effects of YAP knockdown in UMSCC 11A. Left panel: measurements of the scratch/wound, using borders highlighted by the white lines in the timecourse images (right panel). (F) MTT assay of UMSCC-11A cell density after knockdown of YAP and treatment with 5 μM of cisplatin. * indicates significant difference (student t-test, p< 0.05).

Mentions: To directly characterize the functional role of endogenous YAP, we inhibited YAP gene expression by siRNA in UMSCC-11A cells. Efficient inhibition of YAP mRNA was observed for up to four days, and knockdown of YAP protein was also confirmed three days post-transfection (Fig. 5A). YAP inhibition was accompanied by effects on important genes implicated in the malignant phenotype of HNSCC (Fig. 5B), including a significant decrease in mRNA expression of p53/p73 targets p53AIP1 and p21, which function to promote apoptosis and inhibit cell growth, as well as an increase in the expression BCL-XL and VEGF, which promote cell survival and angiogenesis, respectively (Bancroft et al., 2002; Lee et al., 2008; Oda et al., 2000).


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)

Knockdown of YAP alters gene expression, increases cell proliferation and migration, and decreases programmed cell death and cisplatin cytotoxicity(A) QRT-PCR demonstrates knockdown efficiency of YAP mRNA days 1–4, and YAP protein (inset) day 3 post transfection of UMSCC 11A with YAP siRNA. (B) QRT-PCR probe for pro-apoptotic p53AIP and BAX, anti-apoptotic BCL-XL, and pro-angiogenic VEGF genes two days post-YAP knockdown. (C) MTT assay showing increased density of UMSCC-11A after knockdown of YAP. (D) Left panel: DNA cell cycle analysis of the percentage of sub-G0/G1 DNA (% Cell Death) by UMSCC-11A one and three days post-YAP siRNA treatment. Right panel: Flow cytometric analysis of the percentage apoptosis as indicated by the percentage of annexinV and propidium iodide double positive cells, two days post-transfection with indicated siRNA. (E) Wound healing/cell migration time course assay of the effects of YAP knockdown in UMSCC 11A. Left panel: measurements of the scratch/wound, using borders highlighted by the white lines in the timecourse images (right panel). (F) MTT assay of UMSCC-11A cell density after knockdown of YAP and treatment with 5 μM of cisplatin. * indicates significant difference (student t-test, p< 0.05).
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Figure 5: Knockdown of YAP alters gene expression, increases cell proliferation and migration, and decreases programmed cell death and cisplatin cytotoxicity(A) QRT-PCR demonstrates knockdown efficiency of YAP mRNA days 1–4, and YAP protein (inset) day 3 post transfection of UMSCC 11A with YAP siRNA. (B) QRT-PCR probe for pro-apoptotic p53AIP and BAX, anti-apoptotic BCL-XL, and pro-angiogenic VEGF genes two days post-YAP knockdown. (C) MTT assay showing increased density of UMSCC-11A after knockdown of YAP. (D) Left panel: DNA cell cycle analysis of the percentage of sub-G0/G1 DNA (% Cell Death) by UMSCC-11A one and three days post-YAP siRNA treatment. Right panel: Flow cytometric analysis of the percentage apoptosis as indicated by the percentage of annexinV and propidium iodide double positive cells, two days post-transfection with indicated siRNA. (E) Wound healing/cell migration time course assay of the effects of YAP knockdown in UMSCC 11A. Left panel: measurements of the scratch/wound, using borders highlighted by the white lines in the timecourse images (right panel). (F) MTT assay of UMSCC-11A cell density after knockdown of YAP and treatment with 5 μM of cisplatin. * indicates significant difference (student t-test, p< 0.05).
Mentions: To directly characterize the functional role of endogenous YAP, we inhibited YAP gene expression by siRNA in UMSCC-11A cells. Efficient inhibition of YAP mRNA was observed for up to four days, and knockdown of YAP protein was also confirmed three days post-transfection (Fig. 5A). YAP inhibition was accompanied by effects on important genes implicated in the malignant phenotype of HNSCC (Fig. 5B), including a significant decrease in mRNA expression of p53/p73 targets p53AIP1 and p21, which function to promote apoptosis and inhibit cell growth, as well as an increase in the expression BCL-XL and VEGF, which promote cell survival and angiogenesis, respectively (Bancroft et al., 2002; Lee et al., 2008; Oda et al., 2000).

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