Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers

Abstract

Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Prior work was substantially extended by showing CFI-400945 causes polyploidy, growth inhibition, and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with >4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in lung cancer cells. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was significantly inhibited at well-tolerated dosages. Phosphohistone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05). Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism cooperated with PLK4 inhibition. Taken together, PLK4 inhibition alone or as part of a combination regimen is a promising way to combat lung cancer.

Keywords: CFI-400945; PLK4 inhibitor; centriole duplication; lung cancer; polyploidy.

Fig. 1.

Antiproliferative effects and apoptosis induction by independent CFI-400945 treatments of murine and human lung cancer cells. (A) Dose–response consequences of CFI-400945 treatment in murine (ED1, LKR13, and 393P) and human (H1299, Hop62, and A549) lung cancer cells. Effects in murine immortalized pulmonary epithelial cells (C10) and human immortalized bronchial epithelial cells (BEAS-2B) are also shown. (B) Percentages of apoptotic cells after CFI-400945 treatment of murine (ED1, LKR13, and 393P) and human (H1299, Hop62, and A549) lung cancer cells. Effects on C10 and BEAS-2B cells are displayed. (C) Comparisons are presented for CFI-400945 treatments in lung cancer cells between vehicle controls, washout (CFI-400945 washout after 24 h treatment), and CFI-400945 continuously treated groups. Error bars are SD.

Fig. 2.

Polyploidy in murine and human lung cancer cells after independent CFI-400945 treatments. (A) Representative histograms are shown of flow cytometry analysis with PI staining after CFI-400945 treatment of lung cancer cells. (B) Percentages of polyploid cells are displayed after CFI-400945 treatment of murine (ED1, LKR13, and 393P) and human (H1299, Hop62, and A549) lung cancer cells. Effects on C10 and BEAS-2B cells are also displayed. Error bars are SD.

Fig. 3.

CFI-400945 treatment affects centrosome number in murine and human lung cancer cells. (A) Representative cell images are shown for cells with two centrosomes or with supernumerary centrosomes and multipolar spindles before and after CFI-400945 treatment. The blue signal is DAPI staining, the red signal displays α-tubulin staining, and the green signal shows γ-tubulin staining. (Magnification: 60×.) (B) Percentages of cell populations are displayed according to centrosome number before or after CFI-400945 treatment of murine (ED1, LKR13, and 393P) and human (H1299, Hop62, and A549) lung cancer cells. Effects on C10 murine immortalized pulmonary epithelial cells and BEAS-2B human immortalized bronchial epithelial cells are shown. (C) Average centrosome number per cell after CFI-400945 treatment is shown for murine (ED1, LKR13, and 393P) and human (H1299, Hop62, and A549) lung cancer cells. Effects on C10 and BEAS-2B cells are displayed. Error bars are SD.

Fig. 4.

In vivo antitumorigenic activity of CFI-400945 treatments. (A) Murine syngeneic 393P lung cancer cell line growth in mice treated with vehicle or CFI-400945 [3 mg/kg once per day (QD) and 7.5 mg/kg QD] is displayed. Day 0 is the treatment start date. (B) Mouse body weight changes are shown during vehicle or CFI-400945 (3 mg/kg QD and 7.5 mg/kg QD) treatments. Error bars are SD. (C) Comparison of tumor weights after treatments with vehicle or CFI-400945 (3 mg/kg QD and 7.5 mg/kg QD). Each symbol represents a single mouse. Bars represent mean value and SDs. (D) Comparison of bioluminescent signals of syngeneic lung cancers in mice treated with vehicle or CFI-400945 (3 mg/kg QD and 7.5 mg/kg QD). Representative bioluminescent images of these mice are shown over time in Upper. (E) Analysis of mitotic status by phosphohistone H3 Ser10 staining in in vivo murine lung cancers after CFI-400945 treatment. Representative phosphohistone H3 Ser10 immunostaining images are presented in Left. (Magnification: 40×.) Percentages of phosphohistone H3-positive cells and cells with aberrant mitosis in murine lung cancers after vehicle or CFI-400945 (3 mg/kg QD and 7.5 mg/kg QD) treatments appear in Right. Error bars are SD with *P < 0.05, **P < 0.01.

Fig. 5.

PLK4 expression profiles in human lung cancer cases. (A) Comparisons of PLK4 mRNA expression of lung cancers (adenocarcinoma and squamous cell carcinoma) versus adjacent normal lung tissues were explored in the TCGA database. Each symbol represents a single case. (B) Positive and negative controls are displayed for RNA ISH assays. Probes targeting PPIB and DapB were used for positive and negative controls, respectively. (C) Representative images of PLK4 RNA ISH for human lung adenocarcinomas. (D) Kaplan–Meier analysis of overall survival and progression free survival in 235 human lung cancer cases stratified by the presence or absence of PLK4 mRNA expression. (Magnification: B and C, 40×.)

Fig. 6.

Cooperative effects of CFI-400945 and seliciclib treatments. (A) Schematic of the functional relationship between supernumerary centrosomes and multipolar mitosis. (B) Effects of combination therapy of PLK4 antagonism (with CFI-400945) and CDK2 inhibition (with seliciclib) on growth of ED1 murine and Hop62 human lung cancer cells.