STIL Overexpression Is Associated with Chromosomal Numerical Abnormalities in Non-Small-Cell Lung Carcinoma Through Centrosome Amplification

Abstract

STIL is a regulatory protein essential for centriole biogenesis, and its dysregulation has been implicated in various diseases, including malignancies. However, its role in non-small-cell lung carcinoma (NSCLC) remains unclear. In this study, we examined STIL expression and its potential association with chromosomal numerical abnormalities (CNAs) in NSCLC using The Cancer Genome Atlas (TCGA) dataset, immunohistochemical analysis, and in vitro experiments with NSCLC cell lines designed to overexpress STIL. TCGA data revealed upregulated STIL mRNA expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), the two major subtypes of NSCLC. Immunohistochemical analysis of cases from our hospital (LUAD, n = 268; LUSC, n = 98) revealed STIL protein overexpression. To elucidate the functional role of STIL, an inducible STIL-overexpressing H1299 NSCLC cell line was generated. Overexpression of STIL in these cells promoted centrosome amplification, leading to chromosomal instability. Finally, analysis of arm-level chromosomal copy number alterations from the TCGA dataset revealed that elevated STIL mRNA expression was associated with CNAs in both LUAD and LUSC. These findings suggest that STIL overexpression is associated with CNAs in NSCLC, likely through centrosome amplification, which is linked to chromosomal instability and might represent a potential therapeutic target for NSCLC treatment.

Keywords: STIL; centrosome amplification; chromosomal instability; chromosomal numerical abnormality; lung adenocarcinoma; lung squamous cell carcinoma; non-small-cell lung carcinoma; overexpression.

Figure 1

Overexpression of STIL mRNA and protein in primary NSCLC. (A) STIL mRNA overexpression in LUAD and LUSC based on the TCGA database analysis. Statistical comparison between non-cancerous tissues (N) and cancerous tissues (T) was performed using the Mann–Whitney U test, with p-values and median expression levels reported. (B) STIL protein overexpression in LUAD and LUSC was assessed by IHC analysis using a rabbit anti-STIL polyclonal antibody on samples obtained from our institution. Statistical comparisons of STIL expression between non-cancerous lung alveolar tissue and LUAD, as well as between non-cancerous lung alveolar tissue and LUSC, were conducted using the Mann–Whitney U test, with median expression levels and p-values reported. (C) Representative IHC images illustrating STIL protein expression in primary LUAD and LUSC. Scale bar = 100 μm.

Figure 2

Centrosome amplification induction by STIL overexpression in the H1299 NSCLC cell line. (A) Detection of STIL protein levels in two cumate-inducible stable H1299 NSCLC cell lines, termed STIL-1 and STIL-2. Both cell lines were engineered to express FLAG-STIL in the presence of cumate. Increased STIL protein expression was shown via Western blot analysis using anti-STIL antibody. Two empty vector-transposed cell lines, designated Empty-1 and Empty-2, were used as controls. GAPDH expression served as the internal loading control. The protein band intensities and the expression ratio of STIL to GAPDH are presented. (B,C) Effect of STIL overexpression on centrosome amplification in H1299 cells. Cumate-inducible stable H1299 cell lines overexpressing STIL and corresponding empty vector controls were treated with cumate for 72 h. Following fixation, the cells were immunostained with an anti-centrin antibody (red) to visualize the centrioles, and the nuclei were counterstained with DAPI (blue). The percentage of cells containing more than four centrin foci (centrioles) was quantified and is presented in the bar graph in panel (B). Data represent the mean ± standard error from three independent experiments. Statistical significance was assessed using an unpaired t-test. Representative immunofluorescence images are shown in panel (C). Insets in the middle panels are magnified in the right panels. Scale bar = 10 μm (left and middle panels); 2.5 μm (right panels).

Figure 3

Induction of CIN by STIL overexpression in the H1299 NSCLC cell line. Stable H1299 NSCLC cell lines designed to express STIL by cumate treatment and empty vector-transposed H1299 cell lines were treated with or without cumate for 6 days. Stable H1299 lung cancer cell lines, either transposed by an empty vector or engineered to express STIL upon cumate induction, were treated with or without cumate for 6 days. Metaphase spreads were then prepared, and FISH analysis was performed using a Cy3-labeled pan-centromeric DNA probe to quantify the number of chromosomes. The results are presented as a box plot, and statistical comparison between the groups was conducted using the Mann–Whitney U test. * indicates p < 0.0001, and NS indicates no significant difference. Representative images are shown in the panels below the graph.

Figure 4

Association between STIL expression levels and CNA status in primary NSCLC. Copy number alteration data for whole chromosomal genes in LUAD and LUSC were retrieved from the TCGA dataset. Using the data, chromosomal arm-level copy number status was constructed for each case, and then, based on the results, each case was categorized as either CNA-low or CNA-high. STIL mRNA expression levels were also obtained from TCGA. A Mann–Whitney U test was performed to compare STIL expression levels between the CNA-low and CNA-high groups. The p-values and median expression levels are presented.