Transcription factor PAX6 as a novel prognostic factor and putative tumour suppressor in non-small cell lung cancer

Abstract

Lung cancer is the leading cause of cancer deaths. Novel predictive biomarkers are needed to improve treatment selection and more accurate prognostication. PAX6 is a transcription factor with a proposed tumour suppressor function. Immunohistochemical staining was performed on tissue microarrays from 335 non-small cell lung cancer (NSCLC) patients for PAX6. Multivariate analyses of clinico-pathological variables and disease-specific survival (DSS) was carried out, and phenotypic changes of two NSCLC cell lines with knockdown of PAX6 were characterized. While PAX6 expression was only associated with a trend of better disease-specific survival (DSS) (p = 0.10), the pN+ subgroup (N = 103) showed significant correlation between high PAX6 expression and longer DSS (p = 0.022). Median survival for pN + patients with high PAX6 expression was 127.4 months, versus 22.9 months for patients with low PAX6 expression. In NCI-H661 cells, knockdown of PAX6 strongly activated serum-stimulated migration. In NCI-H460 cells, PAX6 knockdown activated anchorage-independent growth. We did not observe any significant effect of PAX6 on proliferation in either of cell lines. Our findings strongly support the proposition of PAX6 as a valid and positive prognostic marker in NSCLC in node-positive patients. There is a need for further studies, which should provide mechanistical explanation for the role of PAX6 in NSCLC.

Figure 1

Validation of PAX6 antibody. (A) Two NSCLC cell lines were transfected with siRNA against human PAX6 or control scrambled siRNA. Cell lysates were tested for PAX6 using Western blotting. (B) FFPE sections of pancreas with Langerhans islands stained for PAX6.

Figure 2

Immunohistochemical staining for PAX6 in NSCLC. NSCLC tissue with negative (A), weak (B), average (C) and strong (D) staining for PAX6.

Figure 3

Disease-specific survival according to PAX6 expression. Disease-specific Kaplan-Meier survival curves according to PAX6 expression in 103 resected NSCLC patients with regional nodal metastasis (pN+). The P-value is according to the log-rank test.

Figure 4

Phenotypic effects of PAX6 knockdown on NSCLC cell lines. (A) Migration of NCI-661 cells across porous membrane. (B) Proliferative activity of NCI-H460 and (C) NCI-661 cells. (D) soft agar colony formation assay in NCI-H460 cells. PAX6 knockdown efficiency is demonstrated in Fig. 5.

Figure 5

Putative downstream target genes of PAX6 in NSCLC. PAX6 was transiently knocked down in two NSCLC cell lines. Expression of the target genes was measured by real-time qPCR. The amount of target gene was normalized to the average expression of the two human reference genes GUSB and TFRC, and is shown relative to scrambled siRNA-treated control.