Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy

Abstract

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Figure 1

NSCLC-SCs are resistant to conventional chemotherapeutic drugs and efficiently repair DNA damage. (a) NSCLC-SCs from five different patients and corresponding differentiated progeny treated with chemotherapeutic drugs for 96 h. Cell viability was measured by CellTiter-Glo assay. The results shown are the mean±S.D. of three independent experiments.(b) Proliferation of untreated NSCLC-SCs (control) or NSCLC-SCs exposed to chemotherapy for 6 days. Data plotted are the results ±S.D. of three independent experiments performed on four different NSCLC-SC lines.(c) Representative cell cycle profiles of control or treated NSCLC-SCs. (d) γ-H2A.X expression after 6 h and 96 h drugs treatment. β-actin was used as loading control. Lower panels indicate actin-normalized and γ-H2A.X levels for each condition. A representative of three independent experiments is shown. All experiments were performed using 5 μg/ml cisplatin, 50 μM gemcitabine and 30 ng/ml paclitaxel. ^***P-value <0.001

Figure 2

Increased cytotoxicity of chemotherapy by Chk1 inhibitors on NSCLC-SCs. (a) Activation of DNA damage proteins in untreated (control) or 6 h treated NSCLC-SC # 1 and NSCLC-SC # 3. (b) Western blot analysis for p-Chk1 in NSCLC-SC # 1, NSCLC-SC # 5, NSCLC-SC # 3 and differentiated progenies untreated or treated with gemcitabine for 12 h. (c) Modulation of phosphorylated Cdc25C (p-Cdc25) in 24-h-treated NSCLC-SC # 3. (d) γ-H2A.X expression in NSCLC-SC # 1, NSCLC-SC # 5 and NSCLC-SC # 3 treated for 96 h as indicated. In each Western blot analysis, the relative densitometric values are normalized over β-tubulin or β-actin. A representative of three independent experiments is shown. (e) Cell viability of NSCLC-SCs after treatment with chemotherapy alone or in combination with Chk1 inhibitors. Mean±S.D. from four independent experiments performed on five different NSCLC-SC lines is shown. Cells were exposed to 5 μg/ml cisplatin, 50 μM gemcitabine, 30 ng/ml paclitaxel, 20 nM SB218078 and 5 nM AZD7762, respectively. (f) Colony-forming ability assay on freshly isolated tumor cells. Mean±S.D. of 24 wells/condition obtained by treating and plating cells from four different NSCLC patients. ^***P-value <0.001

Figure 3

Chk1 inhibition induces Cdc2 activation and mitotic catastrophe in NSCLC-SCs. (a) NSCLC-SC # 1, NSCLC-SC # 2, NSCLC-SC # 3 and NSCLC-SC # 4 analyzed for p-Cdc2 and cyclin B1 expression after 96 h of indicated treatments. β-actin was used to assess equal loading. Lower panels indicate actin-normalized protein level for each condition. A representative of three independent experiments is shown. (b) Representative immunofluorescence staining for cyclin B1 localization in NSCLC-SCs after 48 h of treatments. Arrowheads indicate cytoplasmic cyclin B1 localization. Acquisition was made with a 40 × objective. (c) Representative immunofluorescence of NSCLC-SCs stained with Phalloidin and DAPI to visualize multinucleated cells. Acquisition was made with a 20 × objective. (d) Percentage of multinucleated cells estimated by counting nuclei in 100 cells on each Phalloidin-DAPI-stained slide. Arrowheads point to multinucleated cells. Mean±S.D. of three independent experiments performed on four different NSCLC-SC lines is reported. All experiments were performed with 5 μg/ml cisplatin, 50 μM gemcitabine, 30 ng/ml paclitaxel, 20 nM SB218078 and 5 nM AZD7762. ^***P-value <0.001

Figure 4

Chk1 inhibition reduces colony-forming ability of NSCLC-SCs. (a) Representative pictures of NSCLC-SC # 1 (left panel) and NSCLC-SC#3 (right panel) colonies obtained in soft agar assay under standard growth condition (control) or after treatment with cisplatin (5 μg/ml) and paclitaxel (30 ng/ml) alone or in combination with SB218078 (20 nM) and AZD7762 (5 nM). (b) Average number of colonies/plate for each indicated condition in NSCLC-SC # 1, NSCLC-SC # 2, NSCLC-SC # 3 and NSCLC-SC # 4. Mean±S.D. of three independent experiments is shown. ^**P-value <0.01, ^***P-value <0.001

Figure 5

Combination of chemotherapy and Chk1 inhibitors strongly affects tumor growth in vivo. (a) Hematoxylin and Eosin (H&E) staining of parental tumor (patient) and mouse xenograft from NSCLC-SC # 3 and NSCLC-SC # 4. Acquisition was made with a 10 × objective. (b) Growth rate of mouse xenografts generated after subcutaneous injection of NSCLC-SC # 3. Results are mean±S.D. of four independent experiments. Statistical significance at day 30 was tested by means of two-way repeated measures ANOVA with Bonferroni post-tests (cisplatin versus cisplatin+AZD7762 and gemcitabine versus gemcitabine+AZD7762). (c) Tumor mass for NSCLC-SC # 3 and NSCLC-SC # 4 xenografts measured on day 30. Mean±S.D. of three experiments is reported. Five mice for each group were treated with gemcitabine (60 mg/kg), cisplatin (3 mg/kg) and AZD7762 (10 mg/kg), as indicated, every 3 days from day 0 to day 27. ^*P-value <0.05, ^**P<0.01, ^***P<0.001

Figure 6

DNA damage, cell death detection and evaluation of clonally expanding NSCLC-SCs in tumor xenografts. (a) Representative immunohistochemistry performed on formalin-fixed paraffin-embedded tissue for γ-H2A.X, indicating an extensive necrotic area at day 30. Acquisitions were made with a 10 × and a 40 × objective on NSCLC-SC # 3 xenografts. Lower panel shows the percentage of γ-H2A.X-positive cells in NSCLC-SC # 3 and NSCLC-SC # 4 xenografts. (b) Representative Ki67 (green), Phalloidin (yellow) and TUNEL (red) triple staining acquired with a 40 × objective on NSCLC-SC # 3 xenografts sections at day 30. Lower panel shows the percentage of TUNEL positive cells assessed in three independent experiments performed on NSCLC-SC # 3 and NSCLC-SC # 4 xenografts. (c) Representative H&E performed on frozen tissue 3 weeks after treatment withdrawal (day 51). Data are representative of three independent experiments performed on mouse xenografts generated after subcutaneous injection of NSCLC-SC # 3. Right panel shows the percentage of necrotic areas in tumor xenografts of both NSCLC-SC # 3 and NSCLC-SC # 4. Acquisitions were made, respectively, with a 10 × and a 20 × objective. (d) Flow cytometry analysis for HLA I and colony-forming ability assay performed on tumor cells obtained from dissociation of NSCLC-SC # 3 and NSCLC-SC # 4 xenografts (left panel). Average number of colonies/plate for each combination of treatments (right panel). Mean±S.D. of two independent experiments with 12 wells/condition is reported. ^**P<0.01, ^***P<0.001