Altered Cell-Cycle Control, Inflammation, and Adhesion in High-Risk Persistent Bronchial Dysplasia

Abstract

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell-cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G₂-M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention.Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell-cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971-83. ©2018 AACR.

Figure 1.

Summary of specimens analyzed and differentially expressed genes identified in persistent versus regressive bronchial dysplasia (BD). A) Representative H&E images (400X) from each of the four classifications of biopsy sites according to baseline and follow-up histology scores at a specific site (ND = non-dysplasia). B) Graphical representation of all sites included in gene expression analysis comparing persistent (left) and regressive (right) sites. Lines connect baseline (gray dots) and follow-up (black dots) histologic scores (1 = normal, 2 = reserve cell hyperplasia, 3 = squamous metaplasia without atypia, 4 = mild bronchial dysplasia, 5 = moderate dysplasia, 6 = severe dysplasia and 7 = carcinoma in-situ) for consecutive biopsies from a single site within the airway of an individual. C) Venn diagram showing overlap of genes that distinguish persistent from regressive sites (light gray circle) and those associated with histology score regardless of outcome (dark gray circle).

Figure 2.

Pathway and network analyses showing major cellular processes associated with persistence of BD. A) Pathway analysis showing top 20 pathways with significant alteration of activity in persistent versus regressive BD. Red indicates that the majority of the persistence related genes associated with a given pathway are up-regulated, green indicates predominant down-regulation. Lines connecting pathways indicate that genes are present in both pathways. Intensity of color indicates degree of significance as described in color bar. B) Inflammation associated, differentially expressed genes are predominantly down-regulated and are strongly associated with T-helper lymphocyte and dendritic cell networks. C) The majority of cell cycle progression network associated genes are up-regulated and PLK1 shows the most numerous regulatory interactions. D) The majority of morphology of epidermis network associated genes are up-regulated and include a number of keratins and desmosomal (desmoglein, desmocollins, plakins) genes.

Figure 3.

PLK1 overexpression is associated with persistence of BD. A) Biopsy sites with PLK1 overexpression (PLK1 OE) show higher histology scores in follow-up biopsies than those with underexpression (PLK1 UE). Baseline histologic scores were not significantly different between OE and UE groups (PLK1 OE=5.14, PLK1 UE=4.75; p=0.11). B) PLK1/Pan-keratin (Pan-Ker) dual immunofluorescence showing representative frequent moderate (i.e. white arrows) and strong (i.e. red arrow) nuclear and cytoplasmic positivity for PLK1 in persistent BD (PBD) that is not seen in regressive BD (RBD). Magnification 600X. C) Cultures of persistent bronchial dysplasia (BD) show higher PLK1 expression than regressive BD. Diamonds represent the mean of triplicate measurements of a single cell line. Horizontal bars indicate mean PLK1 level for the entire group. Vertical bars indicate standard error of mean (based on all included replicates). D) Western blot of PLK1 on protein lysates from normal, RBD and PBD derived cultured cells. By densitometry of duplicates from each case, PBD shows increased PLK1 levels vs. RBD (normalized mean 1.93 vs. 0.99, p=0.02). Student’s T-test, *p<0.01

Figure 4.

PLK1 inhibition inhibits cell cycle progression and induces apoptosis of persistent BD (PBD). A) Flow cytometric analysis of cell cycle fraction in PLK1 inhibitor, volasertib (PLK1i), treated cultured normal bronchial epithelial (Nl BE) and PBD derived cells showing an increase in the fraction of cells in the S (diagonal lines) and G2 (gray peak to the right) phase of the cell cycle in PBD but not normal BE or regressive BD (RBD) derived cells. B) The proportion of PBD cells arrested at the G2/M checkpoint is > 4-fold higher in PLK1 treated versus vehicle (DMSO) alone treated cells and significantly greater than that seen in normal and RBD cells (three replicates each of one normal, two RBD and one PBD cell lines). C) Apoptotic activity and inhibition of proliferation are increased in PBD treated with 100 nM but not in normal BE or RBD (RBD), although a lesser degree of induction of apoptosis is noted in RBD. Caspase activity is normalized to that measured in vehicle only treated parallel cultures (sixteen replicates each of one normal, two RBD and two PBD cell lines). Cell numbers in analyses of proliferation are expressed as ratios compared to initial seeding of 100,000 cells for each cell line/condition (three replicates each). Student’s T-test, *p < 0.05.

Figure 5.

A) H&E based mild to severe inflammation scores are more frequent in regressive than persistent BD. B) Macrophage and T-lymphocyte markers are more strongly expressed in regressive than persistent BD, though only macrophage marker CD68 shows a trend toward statistical significance (C - representative CD68 immunostains). D) Dual immunofluorescence stains of representative persistent and regressive BD. While the majority of macrophages lack reactivity for M1 marker HLADRA (red arrows, upper middle), in regressive BD nearly all show dual positivity for CD68 and HLADRA (see inset, bottom middle) and additionally, membranous HLADRA staining is seen in epithelial cells (green arrows). Regulatory T-lymphocytes co-expressing CD4 and FoxP3 (Tregs, green arrows, upper and lower right panels) are more abundant in persistent as compared to regressive BD (see inset, upper right). Overall, the numbers of M1 macrophages and Tregs in these biopsies were 63.3% (19 dual positive/30 CD68 positive) and 33.7% (4 dual positive/57 CD4 positive) for persistent BD versus 73.1% (19/26) and 7.0% (28/83) for regressive BD, respectively. Student’s T-test: *p<0.05; **p=0.05–0.15.

Figure 6.

Expression of desmosomal components is increased in persistent BD. A) Immunofluorescence shows low desmoglein 3 (DSG3) expression in stable reserve cell hyperplasia (left) with increased expression in high grade regressive BD and strongest expression in persistent high grade BD (right). B) A similar pattern of junctional plakoglobin (PG) expression is noted in stable reserve cell hyperplasia (left) with more pronounced increased expression in high grade regressive BD and strongest expression in persistent high grade BD (right). Positive controls = invasive skin SCC (DSG3) and lung SCC (PG) and negative controls = persistent HGDs with secondary Ab only. C) Increased DSG3 expression is seen as histology progresses from normal bronchial epithelium (Nl BE, n=6) to low grade bronchial dysplasia (LGD, n=4) and high grade dysplasia (HGD, n=8). There is a trend toward increased expression of PG in LGD vs normal BE. Bars represent standard error of mean. D) More frequent overexpression of DSG3 in persistent (n=8) versus regressive BD (n=12 [10 for PG]) using an immunofluorescence score (I-score) cut-off of 50 and a trend toward overexpression of PG in persistent BD using a cut-off score of 100. Magnification = 200X. Student’s T-test (C) and Chi-square (D): *p<0.05, **p=0.5–0.15.