RESUMEN
Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and poses a significant health burden due to its rising incidence. Although the proto-oncogene pituitary tumor-transforming gene 1 (PTTG) predicts poor patient outcome, its mechanisms of action are incompletely understood. We show here that the protein PBF modulates PTTG function, is overexpressed in HNSCC tumors, and correlates with significantly reduced survival. Lentiviral shRNA attenuation of PTTG or PBF expression in HNSCC cells with either wild-type or mutant p53, and with and without HPV infection, led to dysregulated expression of p53 target genes involved in DNA repair and apoptosis. Mechanistically, PTTG and PBF affected each other's interaction with p53 and cooperated to reduce p53 protein stability in HNSCC cells independently of HPV. Depletion of either PTTG or PBF significantly repressed cellular migration and invasion and impaired colony formation in HNSCC cells, implicating both proto-oncogenes in basic mechanisms of tumorigenesis. Patients with HNSCC with high tumoral PBF and PTTG had the poorest overall survival, which reflects a marked impairment of p53-dependent signaling.Significance: These findings reveal a complex and novel interrelationship between the expression and function of PTTG, PBF, and p53 in human HNSCC that significantly influences patient outcome. Cancer Res; 78(20); 5863-76. ©2018 AACR.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas de la Membrana/metabolismo , Securina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Reparación del ADN , Femenino , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Estimación de Kaplan-Meier , Lentivirus/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Infecciones por Papillomavirus/complicaciones , Proto-Oncogenes Mas , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello , Análisis de Matrices Tisulares , Resultado del TratamientoRESUMEN
The PTTG1-binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P = 0.009) and protein (P < 0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione-S-transferase (GST) pull-down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (Δ126-132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53-responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (> 90%; P < 0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild-type TP53 and high PBF expression also had low p53 protein levels (P < 0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Proteínas de la Membrana/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Invasividad Neoplásica , Pronóstico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proto-Oncogenes Mas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayo de Tumor de Célula Madre , UbiquitinaciónRESUMEN
The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.