Clinical significance of mTOR, ZEB1, ROCK1 expression in lung tissues of pulmonary fibrosis patients

We enrolled 30 pulmonary fibrosis patients from January 2005 to May 2010 at Seoul National University Hospital. The study included patients who underwent surgical lung biopsy, and were histologically diagnosed with usual interstitial pneumonia (UIP). The pathological diagnosis was undertaken using previously established criteria, and clinical diagnosis was judged according to the international guidelines [8]. Two normal lung tissues were obtained from certified tissue bank to compare with UIP lungs. The demographic characteristics, pulmonary function, and chest computed tomography (CT) scan images of the study patients were reviewed. The survival data of the patients were obtained from the Ministry of Public Administration and Security. A part of the patient data was reported in a previous study [9]. This study was approved by the Institutional Review Board of the Seoul National University Hospital (H-1105-099-363).

The expression of mTOR, ZEB1, and ROCK1 was evaluated by immunohistochemical staining of formalin-fixed and paraffin-embedded lung tissue sections, which were cut to a thickness of 4 μm. After deparaffinization in xylene and rehydration using graded alcohol to water, these slides were soaked in Target Retrieval Solution (Dako, Carpenteria, CA, USA), and then placed in a water bath at 97°C for 20 min to enable antigen retrieval. The sections were then immersed for 5 min in distilled water containing 3% hydrogen peroxidase to block endogenous peroxidase activity. The sections were incubated for 30 min with primary antibodies for mTOR, ZEB1, and ROCK1 diluted 1:200, 1:100 and 1:50, respectively. The primary rabbit monoclonal mTOR and ROCK1 antibodies were purchased from Abcam (Cambridge, MA, USA). The primary goat polyclonal ZEB1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Dako EnVision⁺ System that contains a horseradish peroxidase-labeled polymer was used along with LSAB®2 (Dako, Carpenteria, CA, USA), a streptavidin-peroxidase conjugate that serves as the secondary antibody. Routinely processed tissue sections of prostate cancer, breast cancer, and thyroid cancer were used as positive staining controls for mTOR, ZEB1, and ROCK1 staining, respectively. Staining without the primary antibody was also performed to verify staining specificity.

Two pathologists reviewed the histology slides independently and without access to the clinical data. The fibrosis score was analyzed using the hematoxylin-and-eosin-stained lung section slides. The extent of fibrotic lesions was scored as 0 (0-10%), 1 (10-25%), 2 (25-50%), 3 (50-75%), or 4 (75-100%). The severity of fibrosis was scored from 0 (normal lung) to 8 (total fibrosis) by determining the average of 10 distinct microscopic fields at 200× magnification in accordance with the Ashcroft scoring system (Figure 1A,B) [10]. Finally, fibrosis scores were calculated by multiplication of the extent of fibrotic lesions to Ashcroft scores. The expression of mTOR, ZEB1, and ROCK1 was scored using both the intensity of staining and the percentage of positively stained alveolar epithelial cells. The total expression score was calculated by multiplying intensity score (0, negative; 1, weak; 2, moderate; 3, strong) to the score of the positively stained cells (0, <10%; 1, 10–49%; 2, 50–75%; 3, >75%). We randomly selected 10 microscopic fields under 100× magnification. Each field was individually assessed and the expression score was determined by obtaining the mean score of all the fields. Finally, the expression level was graded as negative (0), intermediate (1–3), or strong (≥4) based on the total expression score (Figure 1C-H).

Honeycombing on the chest CT scan images was defined as air-filled cysts with thick-walled, between the sizes of several millimeters to several centimeters in diameter. A previously published method [11] was used to assess the honeycombing score. Briefly, the whole lung was divided into 6 lobes (right upper, right middle, right lower, left upper, lingular division, and left lower lobe), and the extent of the honeycombing lesion was assessed as 0 (absent), 1 (0–25%), 2 (25–50%), 3 (50–75%), or 4 (<75%) in each lobe. The honeycombing score was the final sum of all 6 lobes.

The differences in patients’ characteristics, on the basis of the expression of mTOR, ZEB1, and ROCK1, were analyzed by using the Mann-Whitney Test for continuous variables and Fisher’s exact test for categorical variables using SPSS 18.0 statistical software. Pearson’s test was used to verify the association between fibrosis score and mTOR, ZEB1, and ROCK1 expression. A Kaplan-Meier curve was plotted for survival analysis. A p-value of <0.05 was considered as significant.

The median age of the patients was 57 years (range, 38–74 years), including 14 male patients (46.7%; Table 1). Four patients had connective tissue disease (CTD), however histologic patterns were UIP. Sixteen patients were on steroid or immunosuppressive treatment. The mean forced vital capacity (FVC) and carbon monoxide diffusion capacity (DL_CO) of the patients was 75% and 65%, respectively. The patient survival rate during the follow-up period (median, 40 months; range, 5–74 months) was 80% (24 patients survived). Baseline clinical characteristics were not different between the patients with IPF and CTD-ILD (Table 2).

The mTOR and ZEB1 were mainly expressed in the hyperplastic alveolar epithelial cells and in some mesenchymal cells of UIP lungs (Figure 1C,D,E,F). The ROCK1 expression was detected in the hyperplastic alveolar epithelial cells, some mesenchymal cells, macrophages, and lymphocytes of the UIP patients (Figure 1G,H). The mTOR, ZEB1 expression was not detected in the control normal lung tissue, except in the bronchial epithelial cells (Figure 2A, E). ROCK1 expression was not detected in the normal lung tissue alveolar epithelial cells, but detected in bronchial epithelial cells and surrounding smooth muscle cells (Figure 2I). Cells overlying fibroblastic foci showed weak staining, however mesenchymal cells of the fibroblastic foci did not show mTOR, ZEB1 and ROCK1 expression (Figure 2C, G, K). Staining was weak or absent in normal area of UIP lungs (Figure 2B, F, J).

The correlation of the 3 markers with each other was evaluated (Tables 3, 4 and 5). The mTOR strong expression group showed higher ROCK1 expression scores (p = 0.006); however, ZEB1 expression scores were not related to the mTOR scores (Table 3).

The strong mTOR expression group had significantly higher (p = 0.034) histological fibrosis scores than those of the negative and intermediate expression groups (Table 3). In addition, the annual decline in FVC and DL_CO was higher in the strong mTOR expression group than that in the negative and intermediate expression groups (Table 2). PaO₂ was lower in strong expression group than in the negative and intermediate expression groups (p = 0.023). The strong mTOR expression group had significantly higher ROCK1 expression scores than those of the negative and intermediate expression groups. The mTOR expression score significantly correlated with the fibrosis score (r = 0.441, p = 0.015; Figure 3A). However, mTOR expression did not correlate with the honeycombing score, FVC, and DLCO. During the study period, no fatality was observed in the mTOR-negative group. In contrast, the mTOR-positive (intermediate and strong) group had 6 fatalities. (Figure 4A; p = 0.325).

Patients with positive (intermediate and strong) ZEB1 expression showed higher fibrosis scores, greater annual rate of decline in DL_CO (% predicted), and higher mTOR scores than that of patients with negative ZEB1 expression (Table 4). The ZEB1 expression score did not significantly correlate with the fibrosis score (Figure 3B), honeycombing score, FVC, and DLCO. Similar to the findings related to mTOR lung tissue expression, no fatality was observed in the ZEB1-negative group (Figure 4B; p = 0.326).

Pulmonary function, honeycombing, and histological fibrosis scores were not different among ROCK1 negative, intermediate, and strong expression groups (Table 5). However, ROCK1 expression was associated with fibrosis score (r = 0.441, p = 0.024; Figure 3C). Survival was not significantly associated with the ROCK1 expression (Figure 4C).