Introduction

Small cell lung cancer (SCLC) harbors very frequent mutations in p53 and Rb, key cell cycle regulators in normal cells. In absence of such tumor suppressors, SCLC cells rely on Chk1 for cell cycle arrest in the event of DNA damage. High expression of E2F1 and Chk1 have been found in SCLCs, which is associated with poor prognosis in various solid tumors. In human SCLC cell lines, Chk1 inhibition sensitizes chemotherapy agents. The interaction between Chk1 and E2F1 is understudied. Here, we report a collaborative role of Chk1 and E2F1 in the cell cycle regulation in SCLC cells.

Methods

Three Rb-mutated human SCLC cell lines (GLC4, NCI-H128, and NCI-H209) were used. GLC4 carries mutant p53; H209 and H128 are wild type. Chk1 inhibition was achieved by either siRNA knockdown or LY2940930; E2F1 was knocked down by siRNA. Ectopic overexpression of Chk1 or E2F1 was achieved using Lonza electroporation kit. Cell viability was measured by Cell-Titer Glo assay. Cell cycle analysis was assayed by PI staining and FACS. Western blotting was used to evaluate caspase activation and other signaling proteins.

Results

Chk1 inhibition by siRNA knockdown or LY2940930 treatment enhanced cisplatin cytotoxicity in SCLC cell lines, regardless of p53 status. Also, a significant decrease of E2F1 protein was observed following Chk1 inhibition in these cells. Knockdown of E2F1 by siRNA enhanced the cytotoxic activity of cisplatin in the GLC4 and H209 cells. In GLC4 cells, Chk1 siRNA combined with cisplatin treatment caused activation of caspase-2 and caspase-3 as well as the cleavage of BID. Interestingly, knocking down caspase-2 reversed caspase-3 activation resulting from the combination of Chk1 siRNA and cisplatin, and restored the level of E2F1 protein. In contrast, ectopic overexpression of Chk1 resulted in an increase of E2F1, while increase of Chk1 expression was also noticeable upon E2F1 overexpression. It has been shown that Chk1 inhibition can abrogate cell cycle arrest and increase phospho-histone H3 expression (mitotic marker). We found that E2F1-overexpressing GLC4 and H128 cells displayed higher levels of phospho-histone H3 when treated with LY2940930, compared to that of Mock-transfected controls. Furthermore, exogenous E2F1 overexpression significantly reduced the IC50 of cisplatin (1331nM vs. 3698nM; p = 0.0028 by paired t-test) in GLC4 cells, but not that of LY2940930 (14nM vs. 19nM; p = 0.25).

Conclusions

Either downregulation of Chk1 or overexpression of E2F1 leads to cell cycle progression, resulting in more cell death when combined with DNA damaging agents. Decrease of E2F1 by Chk1 inhibition might represent a mechanism of self-protection to avoid excessive mitosis after DNA damage, and this regulation is likely involving caspase-2. Further exploration of Chk1 and E2F1 regulatory mechanisms is warranted in SCLC.

Citation Format: Wei-Hsun Hsu, In-Gyu Kim, Guanhua Rao, Justine McCutcheon, Shuo-Tse Hsu, Yu-Wen Zhang, Giuseppe Giaccone. Collaborative regulation of cell cycle progression by Chk1 and E2F1 in small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2724.