P2Y2 receptors mediate nucleotide-induced EGFR phosphorylation and stimulate proliferation and tumorigenesis of head and neck squamous cell carcinoma cell lines.
P2Y2 受体介导核苷酸诱导的 EGFR 磷酸化，刺激头颈部鳞状细胞癌细胞系的增殖和肿瘤发生。
- 作者列表："Woods LT","Jasmer KJ","Muñoz Forti K","Shanbhag VC","Camden JM","Erb L","Petris MJ","Weisman GA
OBJECTIVES:To assess functional expression of the P2Y2 nucleotide receptor (P2Y2R) in head and neck squamous cell carcinoma (HNSCC) cell lines and define its role in nucleotide-induced epidermal growth factor receptor (EGFR) transactivation. The use of anti-EGFR therapeutics to treat HNSCC is hindered by intrinsic and acquired drug resistance. Defining novel pathways that modulate EGFR signaling could identify additional targets to treat HNSCC. MATERIALS AND METHODS:In human HNSCC cell lines CAL27 and FaDu and the mouse oral cancer cell line MOC2, P2Y2R contributions to extracellular nucleotide-induced changes in intracellular free Ca2+ concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca2+ indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2Y2Rs using CRISPR technology or pharmacological inhibition with P2Y2R-selective antagonist AR-C118925 defined P2Y2R contributions to in vivo tumor growth. RESULTS:P2Y2R agonists UTP and ATP increased intracellular Ca2+ levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2Y2R knockout. P2Y2R-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2Y2R knockout reduced UTP-induced CAL27 cell proliferation in vitro and significantly reduced CAL27 and FaDu tumor xenograft volume in vivo. In a syngeneic mouse model of oral cancer, AR-C118925 administration reduced MOC2 tumor volume. CONCLUSION:P2Y2Rs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2Y2R signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2Y2R represents a novel therapeutic target in HNSCC.
目的: 评估 P2Y2 核苷酸受体 (P2Y2R) 在头颈部鳞状细胞癌 (HNSCC) 中的功能表达细胞系并定义其在核苷酸诱导的表皮生长因子受体 (EGFR) 反式激活中的作用。使用抗 EGFR 疗法治疗 HNSCC 受到内在和获得性耐药的阻碍。定义调节 EGFR 信号的新途径可以确定治疗 HNSCC 的额外靶点。 材料和方法: 在人 HNSCC 细胞系 CAL27 和 FaDu 和小鼠口腔癌细胞系 MOC2 中,p2Y2R 对细胞外核苷酸诱导的细胞内游离 Ca2 + 浓度和 EGFR 和细胞外信号调节激酶 (ERK1/2) 变化的贡献磷酸化测定分别采用 Ca2 + 指示剂 fura-2 和免疫印迹分析。使用 CRISPR 技术或 P2Y2R-selective 拮抗剂的药理学抑制作用对 P2Y2Rs 进行基因敲除，定义 AR-C118925 P2Y2R 对体内肿瘤生长的贡献。 结果: P2Y2R 激动剂 UTP 和 ATP 增加 CAL27 和 FaDu 细胞细胞内 Ca2 + 水平和 ERK1/2 和 EGFR 磷酸化，AR-C118925 或 P2Y2R 敲除可抑制这一反应。P2Y2R-mediated 的 EGFR 磷酸化也通过抑制 adamalysin 家族的金属蛋白酶或 Src 家族激酶而减弱。P2Y2R 敲除在体外降低了 UTP 诱导的 CAL27 细胞增殖，在体内显著降低了 CAL27 和 FaDu 肿瘤异种移植体积。在口腔癌的同基因小鼠模型中，AR-C118925 给药减少了 MOC2 肿瘤体积。 结论: P2Y2Rs 介导 HNSCC 细胞对细胞外核苷酸的反应，P2Y2R 信号的遗传或药理学阻断减弱肿瘤细胞增殖和肿瘤发生，提示 P2Y2R 代表了 HNSCC 的一个新的治疗靶点。
METHODS:INTRODUCTION:Human papillomavirus (HPV) is the most common sexually transmitted infection and is associated with several types of cancer. The number of cases of HPV-associated head and neck squamous cell carcinomas (HNSCCs), especially oropharyngeal carcinomas, has increased significantly in recent years despite decreased tobacco smoking rates. Currently, no data concerning the risk factors and prevalence of HPV in HNSCC patients in all regions of Brazil are available, making it difficult to promote advances in this field of public health. Therefore, our goal is to determine the impact of infection by HPV, including HPVs with different genotypes, on head and neck cancer and the risk factors associated with the development of head and neck cancer in all regions of Brazil. METHODS AND ANALYSIS:This is a case-control study that will include 622 patients and 622 controls from all regions of Brazil. A questionnaire will be applied to gather information on sociodemographic, behavioural and health factors. Oral, cervical or penile/scrotal, and anal specimens and serum samples will be collected from all participants. Formalin-fixed paraffin-embedded tissue from tumour biopsies will be analysed only in the case group. Molecular and serological analyses will be performed to evaluate the presence and role of HPV in the development of head and neck cancer. ETHICS AND DISSEMINATION:This project was approved by the research ethical committee of the proposing institution (Hospital Moinhos de Vento, number 2.852.060). Ethical approval from the collaborators is currently under evaluation and is not yet complete. The results of this study will be presented at meetings with the Brazilian Ministry of Health through technical reports and to the scientific community at national and international events, with subsequent publication of scientific articles.
METHODS:BACKGROUND:Factors related to head and neck cancer and the treatment of the disease can affect quality of life. The aim of this study was to determine factors associated with the severity of impact on oral health-related quality of life (OHRQoL) in survivors of head and neck cancer using a multivariate analysis. METHODS:This cross-sectional study evaluated 90 volunteers who had completed radiotherapy at least 3 months earlier. OHRQoL was assessed using oral health impact profile (OHIP-14) and the data were analyzed using robust variance poisson regression models. RESULTS:The mean total OHIP-14 score was 23.98 ± 12.55. Patients with hyposalivation had 56% higher (worse) mean OHIP-14 total scores (CI:1.11-2.18) and patients with advanced stage tumors had 31% higher mean OHIP-14 total scores (CI:1.03-1.66) in multivariate analyses. CONCLUSION:OHRQoL of survivors of head and neck cancer experienced a negative impact following radiotherapy. The impact was associated with hyposalivation and advanced stage tumors.
METHODS:BACKGROUND:To immunohistochemically evaluate the association between the presence of cancer-associated fibroblasts (CAFs) and the tumour expression of podoplanin (PDPN) in head and neck squamous cell carcinoma (HNSCC) and their association with clinicopathological variables. MATERIAL AND METHODS:A tissue microarray (TMA) with biopsy sections from patients diagnosed with HNSCC was stained with antibodies against the CAFs marker, α-smooth muscle actin (α-SMA), and PDPN. We subsequently evaluated their expression to determine the association between them and with clinicopathological variables including age, primary tumour site, TNM stage, and tumour differentiation grade. RESULTS:Positive reaction to α-SMA was observed in the tumour stroma, revealing spindle-shaped cells compatible with CAFs, which showed a high expression in 62% of cases and a significant association with laryngeal carcinomas, advanced clinical stages, and lower tumour differentiation (P ≤ 0.05). PDPN staining on tumour cells showed low expression in 72% of cases, and it was not associated with any clinicopathological variable or with the presence of CAFs. CONCLUSIONS:The presence of CAFs in the tumour stroma is related to an aggressive phenotype and could increase as the disease progresses, although based on our findings, it would have no relationship, at least directly, with the expression of PDPN.