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 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

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

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

GSH cannot be induced by the irradiation, and ROS accumulate in p62/SQSTM1 knockdown cells.

<p>(<b>A</b>) GSSG (upper) and GSH (lower) levels of shRNAs (sh-control, sh-p62(1) and sh-p62(2); blue, red and brown columns, respectively) -treated cells are indicated by bar chart. In each graph, the left 3 columns indicate the data of no radiation. The middle 3 and right 3 columns indicate the data of 5-10 Gy X-ray irradiation followed by 24h culture. Although GSH was maintained in sh-control cells, it was significantly reduced in sh-p62(1) and sh-p62(2) cells. The values are derived from triplicate experiments (*, <i>p</i><0.05, one-way factorial ANOVA and multiple comparison tests accompanied by Scheffe's significance test). (<b>B</b>) ROS levels were detected with CellROX® Green reagent in sh-control and sh-p62 cells, under no radiation (upper) and 5 (middle) - 10 (lower) Gy X-ray irradiation. ROS levels in p62/SQSTM1 knockdown cells (red lines) were higher than in control cells (blue lines). Numbers indicate mean fluorescent values. (<b>C</b>) DNA contents were evaluated with Hoechst 33342 reagent in sh-control and sh-p62 cells, under no radiation (upper) and 5 (middle) - 10 (lower) Gy X-ray irradiation. The more cells were irradiated, the more prominently >G2/M phase cell fractions were accumulated. The accumulation was more prominent in p62/SQSTM1 knockdown cells (sh-p62; red lines) than in the control cells (sh-control; blue lines). Sub-G1 apoptotic fractions could not be detected. Left and right panels indicate the data on SAS and CAL27 cells, respectively.</p

Clinical parameters of the patients with low grade dysplasias.

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

Additional file 2: of Phase II study of bevacizumab, cisplatin, and docetaxel plus maintenance bevacizumab as first-line treatment for patients with advanced non-squamous non-small-cell lung cancer combined with exploratory analysis of circulating endothelial cells: Thoracic Oncology Research Group (TORG)1016

Table S1. CEC data and treatment efficacy of 35 patients whose CEC count was measured on days 1 and 8. Abbreviations: EGFR, epidermal growth factor receptor; CEC, circulating endothelial cell; PR, partial response; SD, stable disease; PFS, progression-free survival; OS, overall survival. (DOCX 22 kb