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

Clinical parameters of the patients with low grade dysplasias.

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

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

Hepatocellular carcinoma occurring in a Crohn’s disease patient

We report a case of hepatocellular carcinoma (HCC) occurring in a patient with Crohn’s disease (CD) without chronic hepatitis or liver cirrhosis, and review the clinicopathological features of HCC in CD patients. A 37-year-old Japanese man with an 8-year history of CD and a medication history of azathioprine underwent resection of a liver tumor. The histopathology of the liver tumor was pseudoglandular type HCC. In the non-neoplastic liver, focal hepatocyte glycogenosis (FHG) was observed, however, there was no evidence of liver cirrhosis or primary sclerosing cholangitis. Only nine cases of HCC in CD patients have been reported previously in the English-language literature. Eight of 10 cases (including the present case) had received azathioprine treatment, and four of these cases also showed FHG, which is considered a preneoplastic liver lesion, within the non-neoplastic liver. Although the precise mechanism of the development of HCC in CD patients is controversial, these results suggest that azathioprine therapy and FHG in the non-neoplastic liver contribute to the development of HCC. These findings also indicate that it is important to survey CD patients treated with prolonged azathioprine therapy for potential liver tumors