The maintenance of genomic stability in normal SCs is essential to preserve the integrity of cell lineages. Efficient DNA damage repair and cell cycle control can be maintained in SCs after oncogenic transformation, as indicated by glioblastoma SC resistance to IR.13
Here, we show that NSCLC-SCs are considerably more resistant to chemotherapeutic drugs than their differentiated progeny. During exposure to chemotherapy, NSCLC-SCs undergo a growth arrest process readily reversible upon drug removal. In the clinical setting, this behavior could be associated with tumor recurrence observed in NSCLC patients treated with chemotherapy, whose survival is extremely poor. The analysis of the molecular mechanisms involved in such chemoresistance showed that upon DNA damage NSCLC-SCs undergo cell cycle arrest preferentially in S or G2
/M phases, thus allowing DNA repair and successful cell duplication. The checkpoint kinase Chk1 has a major role in the DNA damage response and acts as a key regulator of genomic integrity.30
For this reason Chk1 represents a critical therapeutic target for cancer treatment.22, 23, 31, 32, 33, 34
Our results show that Chk1 activation is essential for drug resistance in NSCLC-SCs. Treatment of NSCLC-SCs with gemcitabine, cisplatin or paclitaxel results in a strong activation of Chk1, considerably higher than in differentiated non-tumorigenic cells, indicating that the DNA damage machinery is more robust in NSCLC-SCs than in their progeny.
In human cancers, the high p53 mutation rates result in reliance on S and G2 checkpoints, controlled by Chk1 and Chk2, to repair DNA damage and promote cell survival. We observed that Chk1 activation is an early response to DNA damage even in p53-wild type NSCLC-SCs. After chemotherapy treatment in NSCLC-SCs, Chk2 phosphorylation and p53 activation in p53 proficient cells occurred days after Chk1 activation, suggesting that Chk1 acts as a major DNA damage checkpoint in NSCLC-SCs, regardless of the p53 status. Accordingly, we observed that Chk1 inhibitors sensitize NSCLC-SCs to chemotherapy by altering the DNA damage response and favoring the incidence of aberrant endomitosis with subsequent cell death. Our results indicate that the Chk1-Cdc25-Cdc2-cyclin B1 pathway is efficiently triggered after drug-induced DNA damage in NSCLC-SCs, as demonstrated by the ability of Chk1 inhibitors to reduce Cdc25 and Cdc2 phosphorylation and promote cyclin B1 translocation to the nucleus.
AZD7762 is a new inhibitor of Chk1 and Chk2, currently in phase I clinical trial in combination with chemotherapy. This drug has been shown to enhance the response to chemotherapy and radiotherapy in preclinical models of colorectal, lung and pancreatic cancer.25, 35, 36
Unlike cancer cell lines, CSCs produce tumor xenografts that recapitulate the original tumors and appear a promising tool to study human tumors and devise more effective therapies.29
Using NSCLC xenografts generated by CSCs, we found that AZD7762 increases considerably the anti-tumor effect of chemotherapy.
Compelling evidence indicates tumor regrowth in NSCLC patients following chemotherapy withdrawal.37, 38, 39
We found that the interruption of co-treatment did not correspond to a rapid rebound in tumor growth, suggesting that co-administration of the Chk1 inhibitor AZD7762 and either gemcitabine or cisplatin could be exploited to devise more effective therapeutic approaches for NSCLC. Moreover, the significant reduction in the number of clonogenic cells in tumor xenografts treated with the combined therapy suggests that such treatment affects the survival of NSCLC-SCs, which are largely spared by chemotherapy alone.
In conclusion, here we show for the first time that primary NSCLC-SCs survive during the course of chemotherapy by exploiting an efficient DNA damage response, which can be prevented by the use of drugs that target Chk1. This distinctive property, which was not found in differentiated NSCLC cells, may explain the inefficacy of chemotherapy in eradicating lung cancer and the consequent poor clinical outcome of NSCLC patients. Inhibition of Chk1 sensitized CSCs to chemotherapy-induced DNA damage and dramatically reduced their survival in vitro and in vivo. Together our results suggest the hypothesis that Chk1 inhibition might improve the progression-free survival of NSCLC patients during chemotherapy treatment. Due to the number of Chk1 inhibitors currently undergoing early clinical trials, these observations argue in favor of a future clinical evaluation of Chk1 inhibitors in combination with chemotherapy as a cancer-SC-directed therapy, whereas providing substantial preclinical support for future phase II clinical trials with the combination of chemotherapy and Chk1 inhibitors for the treatment of NSCLC.