Loss of expression rather than cytoplasmic mislocalization of RUNX3 predicts worse outcome in non-small cell lung cancer

Abstract

Functional inactivation of human runt-related transcription factor 3 (RUNX3) through mutation or epigenetic silencing has been well-documented in many cancerous entities. In addition to gene mutation and promoter hypermethylation, cytoplasmic mislocalization has emerged as another major manifestation of RUNX3 dysfunction in malignancies including breast, colorectal and gastric cancers. The aim of the present study was to investigate whether patients with non-small cell lung cancer (NSCLC) and different RUNX3 expression patterns would have different overall survival (OS), and the associations between different patterns of clinicopathological parameters and clinical outcome. Expressions of RUNX3 and Ki-67 were immunohistochemically detected in normal lung tissue (n=5) and surgically resected tissues from NSCLC patients (n=188). The optimal cutoff of RUNX3 was determined by X-tile software associated with their survival. Apoptotic index in cancerous tissue was evaluated using the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling method. The prognostic significance of different expression patterns of RUNX3 was determined by means of Kaplan-Meier survival estimates and log-rank tests. It was revealed that loss of RUNX3 expression in NSCLC was correlated with a low cancerous apoptotic index (P<0.001), shorter OS and worse prognosis (P=0.0142), while no statistical difference of apoptotic index (P=0.73) or survival (P=0.3781) was determined between patient subgroups with different localization of RUNX3 expression, which was quite different from the situation demonstrated in other malignancies. In conclusion, loss of expression rather than cytoplasmic mislocalization of RUNX3 predicted worse outcome in NSCLC, which was quite different from what manifested in other cancer types, and thus, the underlying mechanism may deserve further investigation.

Keywords: cytoplasmic mislocalization; non-small cell lung cancer; overall survival; prognosis; runt-related transcription factor 3.

Figure 1.

Immunostaining for RUNX3 in normal lung tissue and NSCLCs. (A, C and E) H&E staining and (B, D and F) immunostaining for RUNX3 in (A, B) normal lung tissue, (C, D) ADE tissues and (E, F) SCC tissues (magnification, ×400; scale bar=20 µm). RUNX3, runt-related transcription factor 3; NSCLC, non-small cell lung cancer; H&E, hematoxylin and eosin; ADE, adenocarcinoma; SCC, squamous cell carcinoma.

Figure 2.

(A) X-tile plots of RUNX3 expression for optimal cutoff point (3; P<0.05), which is demarcated by a black circle. (B) The cutoff point was used to separate low RUNX3 expression (blue) from high expression (gray) in the expression frequency histogram of the whole sample set (n=188). (C) Kaplan-Meier curve for evaluating the survival of sample subsets defined by RUNX3 expression <3 (blue line) and >3 (gray line). (D) RUNX3 expression differed in different lung tissues. Data are presented as the mean ± standard deviation. **P<0.01 vs. NOR. RUNX3, runt-related transcription factor 3; ADE, adenocarcinoma; SCC, squamous cell carcinoma; NOR, normal tissues.

Figure 3.

Different localizations of RUNX3 expression; (A) negative, (B) nucleus, (C) cytoplasm, and (D) nucleus and cytoplasm (magnification, ×400; scale bars=20 µm). (E) Kaplan-Meier curves of overall survival in different levels of RUNX3 expression in patients with NSCLC. High expression of RUNX3 (green line) was associated with a better prognostic outcome and longer overall survival while low expression of RUNX3 (red line) was associated with poor prognosis and shorter overall survival (P=0.0142). (F) Kaplan-Meier curves of overall survival in different localization of RUNX3 expression in patients with NSCLC. No statistical significance of overall survival between nuclear expression of RUNX3 (upper, continued line) and non-nuclear expression of RUNX3 (lower, dotted line) was determined (P=0.73). Overall survival, OS; RUNX3, runt-related transcription factor 3; NSCLC, non-small cell lung cancer.

Figure 4.

(A) Kaplan-Meier curves of overall survival in different localizations of RUNX3 expression in patients NSCLC: Negative, nucleus, cytoplasm, and nucleus and cytoplasm (P=0.0495). Patients with negative expression exhibited the worst survival, while no statistical significance of overall survival was observed among nuclear, cytoplasmic and whole-cell expression of RUNX3 (P>0.05). In patients with (B) nuclear and non-nuclear expression of RUNX3 or (C) different expression levels of RUNX3, Ki-67 expression demonstrated no statistical difference (P>0.05). (D) In patients with nuclear and non-nuclear expression of RUNX3, TUNEL positive index demonstrated no statistical difference (P=0.73). (E) Apoptotic index in patients with higher expression level of RUNX3 prevailed significantly over those with low expression level of RUNX3 (P<0.001). **P<0.01. Overall survival, OS; NSCLC, non-small cell lung cancer; RUNX3, runt-related transcription factor 3; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling.

Figure 5.

Cellular dynamic parameters between patients with NSCLC and different expression levels of RUNX3. H&E staining for NSCLC tissue with (A) low and (B) high level of RUNX3. Immunostaining for Ki-67 in NSCLC tissue with (C) low and (D) high level of RUNX3. TUNEL staining for apoptotic cells in NSCLC tissue with (E) low and (F) high level of RUNX3, characterized by chromatin condensation and nuclear fragmentation, which were accompanied by cell swelling, reduction in cellular volume (pyknosis) and retraction of pseudopodes. Formation of crescentic caps of condensed chromatin at the nuclear periphery, and formation of apoptotic bodies could also be observed (indicated by arrows). (G) Negative staining for RUNX3; (H) positive staining for RUNX3 in NSCLC. Magnification, ×400; scale bars=20 µm. NSCLC, non-small cell lung cancer; RUNX3, runt-related transcription factor 3; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling.