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. 2017 May;19(5):439-450.
doi: 10.1016/j.neo.2017.03.001. Epub 2017 Apr 25.

DDK Promotes Tumor Chemoresistance and Survival via Multiple Pathways

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Free PMC article

DDK Promotes Tumor Chemoresistance and Survival via Multiple Pathways

Nanda Kumar Sasi et al. Neoplasia. .
Free PMC article

Abstract

DBF4-dependent kinase (DDK) is a two-subunit kinase required for initiating DNA replication at individual origins and is composed of CDC7 kinase and its regulatory subunit DBF4. Both subunits are highly expressed in many diverse tumor cell lines and primary tumors, and this is correlated with poor prognosis. Inhibiting DDK causes apoptosis of tumor cells, but not normal cells, through a largely unknown mechanism. Firstly, to understand why DDK is often overexpressed in tumors, we identified gene expression signatures that correlate with DDK high- and DDK low-expressing lung adenocarcinomas. We found that increased DDK expression is highly correlated with inactivation of RB1-E2F and p53 tumor suppressor pathways. Both CDC7 and DBF4 promoters bind E2F, suggesting that increased E2F activity in RB1 mutant cancers promotes increased DDK expression. Surprisingly, increased DDK expression levels are also correlated with both increased chemoresistance and genome-wide mutation frequencies. Our data further suggest that high DDK levels directly promote elevated mutation frequencies. Secondly, we performed an RNAi screen to investigate how DDK inhibition causes apoptosis of tumor cells. We identified 23 kinases and phosphatases required for apoptosis when DDK is inhibited. These hits include checkpoint genes, G2/M cell cycle regulators, and known tumor suppressors leading to the hypothesis that inhibiting mitotic progression can protect against DDKi-induced apoptosis. Characterization of one novel hit, the LATS2 tumor suppressor, suggests that it promotes apoptosis independently of the upstream MST1/2 kinases in the Hippo signaling pathway.

Figures

Figure 1
Figure 1
Characterization of tumors that differentially express DDK. (A) GSEA was performed using a gene expression signature differentiating CDC7-high versus CDC7-low tumors. Shown here are enriched gene sets involved in cell cycle checkpoints and drug resistance. (B) Genes with overrepresentation of mutant patients within patients groups that differentially express DDK subunits. Mutational information from the top and bottom 25% of CDC7/DBF4/DBF4B-expressing tumors was used to assess significant (P < .05, hypergeometric test) overrepresentation (blue) or underrepresentation (gray) of mutant patients. The number under each data set indicates the total number of genes with significant over- or underrepresentation of mutant patients within each cohort. (C) Mutational load (derived as the number of nonsilent mutations per 30 Mb of coding sequence) in patients with high CDC7/DBF4/DBF4B expression (top 25%, n = 122) and low CDC7/DBF4/DBF4B expression (bottom 25%, n = 122). Mann-Whitney-Wilcoxon (MW) test was used to assess statistical significance.
Figure 2
Figure 2
E2F family of transcription factors strongly bind DDK promoters. The HeLa-S3 ChIP-Seq data were obtained from ENCODE database and E2F binding analyzed using EaSeq software. (A, B, C). E2F ChIP-Seq signal intensities at the promoter regions of CDC7 (A), DBF4 (B), and MCM4 (C).
Figure 3
Figure 3
RNAi screen to identify mediators of cell death induced upon DDK inhibition. (A) Outline of the RNAi screen. (B) Scatter plot of all targeted genes. Hits with robust Z scores > = 2 are highlighted in blue. (C) List of top 41 hits from the primary screen. (D) Hits validated by secondary screens in HCC1954 cells or HeLa cells. (E) G2/M and mitotic gene sets enriched in hits validated in (D).
Figure 4
Figure 4
LATS2 mediates cell death upon DDK inhibition. (A, B) HCC1954 cells were transfected with the indicated siRNAs, 48 hours later treated with DDKi for 8 h, and harvested for Caspase 3/7 analysis (A) or Western blot (B). (C, D) HCC1954 cells were transfected with the indicated siRNAs and 72 hours later harvested for Western blot. (E) HCC1954 cells were transfected with the indicated siRNAs, 48 hours later treated with DDKi for 8 hours, and harvested for Western blot. (F) Subconfluent population of HCC1954, 786-O, and ACHN was harvested and subject to Western blot. (G) HCC1954, 786-O, and ACHN cells were plated in 96-well plates and treated with indicated drugs, and Caspase3/7 activity was measured at indicated times.

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