Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Head and neck squamous cell carcinoma (HNSCC) still remains a lethal malignancy benefiting from the identification of the new target for early detection and/or development of new therapeutic regimens based on a better understanding of the biological mechanism for treatment. The overexpression of Her2 and Her3 receptors have been identified in various solid tumors, but its prognostic relevance in HNSCC remains controversial.</p> <p>Methods</p> <p>Three hundred eighty-seven primary HNSCCs, 20 matching metasis and 17 recurrent HNSCCs were arrayed into tissue microarrays. The relationships between Her2 and Her3 protein expression and clinicopathological parameters/survival of HNSCC patients were analyzed with immunohistochemistry.</p> <p>Results</p> <p>Her3 is detected as either a cytoplasmic or a membranous dominant expression pattern whereas Her2 expression showed uniform membranous form. In primary tumor tissues, high membranous Her2 expression level was found in 104 (26.9%) cases while positive membranous and cytoplasmic Her3 expression was observed in 34 (8.8%) and 300 (77.5%) samples, respectively. Membranous Her2 expression was significantly associated with histological grade (<it>P </it>= 0.021), as grade 2 tumors showed the highest positive expression. Membranous Her3 over-expression was significantly prevalent in metastatic tissues compared to primary tumors (<it>P </it>= 0.003). Survival analysis indicates that membranous Her3 expression is significantly associated with worse overall survival (<it>P </it>= 0.027) and is an independent prognostic factor in multivariate analysis (hazard ratio, 1.51; 95% confidence interval, 1.01-2.23; <it>P </it>= 0.040).</p> <p>Conclusions</p> <p>These results suggest that membranous Her3 expression is strongly associated with poor prognosis of patients with HNSCC and is a potential candidate molecule for targeted therapy.</p

Importance of autophagyssociated molecules in lung cancer cases

科学研究費補助金研究成果報告書研究種目: 基盤研究(C)研究期間: 2011～2013課題番号: 23590395研究代表者: 瀧北 幹子（滋賀医科大学・医学部・助教）研究分担者: 茶野 徳宏（滋賀医科大学・医学部・准教授

Association of p62/SQSTM1 Excess and Oral Carcinogenesis

<div><p>p62/SQSTM1 (sequestosome1) has never been evaluated in oral epithelium. In order to clarify the role of p62/SQSTM1 in carcinogenesis in oral epithelium, both p62/SQSTM1 and Nrf2 were immunohistochemically evaluated in 54 carcinomas and 14 low grade dysplasias. p62/SQSTM1 knockdowns were also designed in oral cancer cells, and we analyzed the Nrf2 pathway, GSH contents and ROS accumulation. The association between p62/SQSTM1 excess and prognosis was addressed in a clinical cohort of oral carcinoma cases. p62/SQSTM1 excess was more obvious in carcinomas, but Nrf2 was abundant in almost all samples of the oral epithelium. In oral carcinoma cells, p62/SQSTM1 knockdown did not affect the Nrf2-Keap1 pathway but did significantly reduce GSH content with subsequent ROS accumulation, and caused cell growth inhibition in the irradiated condition. Finally, p62/SQSTM1 excess was associated with poor prognosis in a clinical cohort. In oral epithelial carcinogenesis, p62/SQSTM1 excess played a role in GSH induction rather than Nrf2 accumulation, and may cause resistance to cytotoxic stresses such as radiation or chemotherapy. Immunohistochemical evaluation of p62/SQSTM1 may be a potential significant marker to identify early carcinogenesis, chemo-radiotherapeutic resistance or poor prognosis of oral squamous cell carcinomas.</p></div

p62/SQSTM1 excess predicts clinically worse prognoses in oral cancer cases.

<p>Forty-nine cases of oral cancer were immunohistochemically evaluated for p62/SQSTM1 and statistically analyzed relative to the clinical outcomes. Kaplan-Meier curve with log-rank tests was performed for disease-specific survival in relation to p62/SQSTM1 level. (Chi-Square value = 4.640, p = 0.0312).</p

p62/SQSTM1 was abundantly stained in oral squamous cell carcinomas.

<p>(<b>A</b>) Case-frequencies (%) of p62/SQSTM1 staining grades in oral squamous cell carcinomas (blue columns; 54 cases), low grade dysplasias (red columns; 14 cases) and non-atypical epithelia (green columns; 29 cases). (<b>B</b>) Means ± S.E. of PLA signals for p62/SQSTM1 are displayed as bar graphs. The values (RCPs/cell) are 9.95±0.89, 3.90±0.48, 2.05±0.69 and 1.95±0.30 in the highest expression grade (++) carcinomas (24 cases), other carcinomas (30 cases), low grade dysplasias (14 cases) and non-atypical epithelia (29 cases), respectively. There was a significant difference between the highest expression grade (++) carcinomas and the other categories (<i>p</i><0.0001), using one-way factorial ANOVA and multiple comparison tests accompanied by Scheffe's significance test. (<b>C</b>) Representative findings of p62/SQSTM1 staining in the highest expression grade (++) carcinoma (left), in low grade dysplasia (middle), and in non-atypical epithelium (right). Corresponding PLA signals and BlobFinder images are displayed in the middle and lower rows, respectively. Scale bar; 100 µm.</p

Nrf2 was abundantly expressed in carcinomas, low grade dysplasias, and non-atypical epithelia of oral tissue.

<p>(<b>A</b>) Case-frequencies (%) of Nrf2 staining grades in oral squamous cell carcinomas (blue columns; 54 cases), low grade dysplasias (red columns; 14 cases) and non-atypical epithelia (green columns; 29 cases). (<b>B</b>) Means ± S.E. of PLA signals for Nrf2 are displayed as bar graphs. The values (RCPs/cell) are 2.32±0.20, 2.10±0.32 and 1.24±0.17 in carcinomas (54 cases), low grade dysplasias (14 cases) and non-atypical epithelia (29 cases), respectively. There was a significant difference between carcinomas and non-atypical epithelia (<i>p</i> = 0.0029), using one-way factorial ANOVA and multiple comparison tests accompanied by Scheffe's significance test. (<b>C</b>) Representative findings of Nrf2 staining in carcinoma (left), in low grade dysplasia (middle), and in non-atypical epithelium (right). Corresponding PLA signals are displayed in the lower row. Scale bar; 100 µm. (<b>D</b>) There was a weakly positive correlation between p62/SQSTM1- and Nrf2- PLA signals (Pearson’s correlation coefficient; r = 0.245, n = 97, <i>p</i> = 0.0265). (<b>E</b>) There was a strongly positive correlation between p62/SQSTM1- and GSH-PLA signals (Pearson’s correlation coefficient; r = 0.588, n = 97, <i>p</i><0.0001).</p

p62/SQSTM1 knockdown had little effect on the Nrf2-NQO1 pathway in oral cancer cells.

<p>However, p62/SQSTM1 knockdown affected the growth of the cells. (A) p62/SQSTM1 was abundantly expressed in SAS and CAL27 oral cancer cells. HeLa, endocervical carcinoma cells; TIG-108 and 121, normal human fibroblasts. (B) p62/SQSTM1 knockdown was performed by two kinds of shRNAs (sh-p62(1) and sh-p62(2)). shRNA for luciferase was used as control knockdown (sh-control). p62/SQSTM1 expression was significantly decreased by sh-p62(1) and sh-p62(2). Under no irradiation or 10 Gy X-ray irradiation, expressions of Nrf2 (pS40), Keap1, NQO1 and HO-1 were subtly affected by p62/SQSTM1 knockdown. Similar amounts of each cell protein were loaded in each lane of the SDS-PAGE. α-tubulin was used a loading control. Left and right panels indicate the data on SAS and CAL27, respectively. Three independent experiments were repeated, and the blotting photographs are representative ones. (C) p62/SQSTM1 knockdowns and X-ray irradiation were similarly performed. No radiation (upper); The effects of shRNA (sh-p62(1) and sh-p62(2)) were partial under normal culture condition. X-ray irradiations of 5 Gy (middle) and 10 Gy (lower); The cancer cell growth was significantly inhibited by two kinds of shRNA for p62/SQSTM1. Left and middle panels indicate the data on SAS and CAL27, respectively. The data on TIG-121 are displayed as a reference at the right panels. In the WST-8 assays, mean absorbance values (OD450) ± SE are shown vertically, and the number of days after exposure to radiation is indicated horizontally. The values are derived from quadruplicate experiments (*, <i>p</i><0.05, one-way factorial ANOVA and multiple comparison tests accompanied by Scheffe's significance test).</p

Clinical parameters of the patients with low grade dysplasias.

<p>Clinical parameters of the patients with low grade dysplasias.</p