The Hippo component YAP localizes in the nucleus of human papilloma virus positive oropharyngeal squamous cell carcinoma - PubMed (original) (raw)

doi: 10.1186/s40463-017-0187-1.

Leanne Clattenburg 2, Shanmugam Muruganandan 2, Martin Bullock 3, Kaitlyn MacIsaac 2, Michael Wigerius 2, Blair A Williams 1, M Elise R Graham 1, Matthew H Rigby 1, Jonathan R B Trites 1, S Mark Taylor 1, Christopher J Sinal 2, James P Fawcett 4 5, Robert D Hart 6

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The Hippo component YAP localizes in the nucleus of human papilloma virus positive oropharyngeal squamous cell carcinoma

Faisal Alzahrani et al. J Otolaryngol Head Neck Surg. 2017.

Abstract

Background: HPV infection causes cervical cancer, mediated in part by the degradation of Scribble via the HPV E6 oncoprotein. Recently, Scribble has been shown to be an important regulator of the Hippo signaling cascade. Deregulation of the Hippo pathway induces an abnormal cellular transformation, epithelial to mesenchymal transition, which promotes oncogenic progression. Given the recent rise in oropharyngeal HPV squamous cell carcinoma we sought to determine if Hippo signaling components are implicated in oropharyngeal squamous cell carcinoma.

Methods: Molecular and cellular techniques including immunoprecipiations, Western blotting and immunocytochemistry were used to identify the key Hippo pathway effector Yes-Associated Protein (YAP) 1. Oropharyngeal tissue was collected from CO2 laser resections, and probed with YAP1 antibody in tumor and pre-malignant regions of HPV positive OPSCC tissue.

Results: This study reveals that the Scribble binding protein Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP) forms a complex with YAP. Further, the NOS1APa and NOS1APc isoforms show differential association with activated and non-activated YAP, and impact cellular proliferation. Consistent with deregulated Hippo signaling in OPSCC HPV tumors, we see a delocalization of Scribble and increased nuclear accumulation of YAP1 in an HPV-positive OPSCC.

Conclusion: Our preliminary data indicates that NOS1AP isoforms differentially associate with YAP1, which, together with our previous findings, predicts that loss of YAP1 enhances cellular transformation. Moreover, YAP1 is highly accumulated in the nucleus of HPV-positive OPSCC, implying that Hippo signaling and possibly NOS1AP expression are de-regulated in OPSCC. Further studies will help determine if NOS1AP isoforms, Scribble and Hippo components will be useful biomarkers in OPSCC tumor biology.

Keywords: HPV; Hippo; NOS1AP; Oropharyngeal squamous cell carcinoma; Scribble; YAP; p16.

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Figures

Fig. 1

Fig. 1

NOS1AP isoforms associate with YAP. a HEK293T cell lysate was Immunoprecipitated with the antibodies indicated. The resulting blot was probed with anti-YAP (arrow, left panel) and re-probed with a NOS1APc specific antibody (pep-NOS1APc) (arrow, right panel). Note, more YAP associates with the pan-NOS1AP antibody than with the NOS1APc antibody. b Rat brain cell lysate was immunoprecipitated with the indicated antibodies. The resulting blot was probed with anti-YAP antibody (arrow, left panel) and then re-probed with a NOS1APc specific antibody (pep-NOS1APc) (arrow, right panel). Asterisk denote cross reacting bands

Fig. 2

Fig. 2

NOS1APc associates with pYAP. Rat brain cell lysate was immunoprecipitated with the antibodies indicated the resulting blot was probed with the antibodies indicated (upper panel pYAP (ser 127). Lower panel, blot was re-probed with a pan-NOS1AP antibody to identify NOS1APa. Asterisks denote cross reacting band

Fig. 3

Fig. 3

NOS1AP isoforms affects cellular proliferation. Starved MCF7 cells stably expressing YFP-NOS1APa and YFP-NOS1APc incorporate less radiolabeled thymidine over a 48 and 72h period with 10% serum relative to MCF7 cells stably expressing YFP. Differences were considered significant in Students _t_-test at **P < 0.01, ***p < 0.001

Fig. 4

Fig. 4

YAP is activated in HPV+ve-OPSCC. a, b HPV+ve -OPSCC stained with anti-YAP (a) and Hoechst (b). Note: Solid line is tumor margin (a, b). Boxed regions in (a) are expanded in (c) left box and (e) right box. (c, d) Enlarged region ( left box in a) showing Yap (c) and nuclei (d). Note, Yap is mainly localized to small puncta in the cytosol in tumor margins, with some cells showing YAP accumulation in the nucleus (c, d arrow). e, f Enlarged region (right box in a) showing Yap (e) and nuclei (f). Note, Yap is localized in the nucleus in tumor (e, f, arrows). Scale bar = 50um

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