The Genomic Landscape and Pharmacogenomic Interactions of Clock Genes in Cancer Chronotherapy

Abstract

Cancer chronotherapy, treatment at specific times during circadian rhythms, endeavors to optimize anti-tumor effects and to lower toxicity. However, comprehensive characterization of clock genes and their clinical relevance in cancer is lacking. We systematically characterized the alterations of clock genes across 32 cancer types by analyzing data from The Cancer Genome Atlas, Cancer Therapeutics Response Portal, and The Genomics of Drug Sensitivity in Cancer databases. Expression alterations of clock genes are associated with key oncogenic pathways, patient survival, tumor stage, and subtype in multiple cancer types. Correlations between expression of clock genes and of other genes in the genome were altered in cancerous versus normal tissues. We identified interactions between clock genes and clinically actionable genes by analyzing co-expression, protein-protein interaction, and chromatin immunoprecipitation sequencing data and also found that clock gene expression is correlated to anti-cancer drug sensitivity in cancer cell lines. Our study provides a comprehensive analysis of the circadian clock across different cancer types and highlights potential clinical utility of cancer chronotherapy.

Keywords: cancer; chronotherapy; circadian rhythms; clinical actionable genes; clock genes; drug; pharmacogenomics.

Figure 1. The transcriptional dysregulation of clock genes in cancer

(A) Upregulation (red) and downregulation (blue) patterns of clock genes across different cancer types (Y-axis) compared to paired normal samples (FC > 1.5; t-test corrected p < 0.05). X-axis indicates 14 core clock genes (red) and 37 clock-associated genes (black). The color intensity indicates the fold-change, the point size indicates the significance of p. Upper bars show the frequency of cancer types, with upregulation (red) and downregulation (blue) for each clock gene. (B) Dysregulation of core clock genes in clock transcriptional loops (red, upregulation; blue, downregulation).

Figure 2. Functional effects of clock genes in oncogenic pathways

(A) The proportion of cancer types with core clock genes significantly associated to activation (red) or inhibition (blue) of the ten key signaling pathways in 31 cancer types. (B) The total number of activation (red) or inhibition (blue) pathways in ten key signaling pathways across 31 cancer types, which are associated with individual or paralog clock genes. See also Figure S2.

Figure 3. Mutational landscape of clock genes in cancer

(A) Heatmap shows the number of mutations (number in cell) and frequency of mutations (color-scale) for each clock gene in each cancer type. X-axis and Y-axis labels ordered by the sum of clock gene mutations in each cancer type and the sum of mutations in all cancer types across clock genes, respectively. Core clock genes are marked in red. (B) Box plot shows the number of mutations of core clock genes across cancer types, with outliers shown as dots. (C) Kaplan-Meier curves show overall survival between samples with (red) or without (blue) mutations in core clock genes in BRCA. (D) Kaplan-Meier curves of BRCA stratified by PER1/2/3 mutation. See also Figure S3.

Figure 4. Disruption of circadian rhythms in cancer

(A) The number of genes which negatively correlated to clock genes (R ≤ −0.5 and FDR < 0.05, number in cell) decreased (blue) or increased (red) in tumor samples compared to paired normal samples for each clock gene in each cancer type. Pearson correlation is corrected by tumor purity in cancer samples. Core clock genes are marked in red. (B) Circadian oscillating genes as determined by JTK_CYCLE and MetaCycle::meta2d (P < 0.05) in MCF10A (left), and disrupted oscillation of these genes in MCF7 (right). Red, high expression; blue, low expression. X-axis displayed time points after serum shock. (C) Time-dependent relative expression of clock genes ARNTL, CSE1L, GSK3B, NR1D2, PARP1 and PER1 in MCF10A (blue) and MCF7 (red). See also Figure S4.

Figure 5. Clinical relevance of clock genes across cancer types

(A) Clinically relevant clock genes across different cancer types. The red and blue boxes indicate high and low expression in tumors associated with worse overall survival times (log-rank test FDR < 0.05), respectively. The green box shows clock genes with significantly differential expression among tumor stages (FC > 1.5; ANOVA FDR < 0.05). The arrows represent the upregulation or downregulation of clock genes in later stages (fold change of transcriptional expression between stage III/IV and stage I/II larger than 1.5). The gold box indicates significant differential expression of clock genes among tumor subtypes (FC > 1.5; ANOVA FDR < 0.05). (B) Kaplan-Meier curves of multiple cancer types stratified by median expression levels of clock genes. See also Figure S5.

Figure 6. The expression of many clock genes and clinically actionable genes is associated with each other in cancer cells

(A) Correlation of transcriptional expression between clock genes and clinically actionable genes. Pearson correlation coefficients |R| > 0.3; FDR < 0.05; blue: negative correlation; red: positive correlation; color scale: number of cancer types with negative or positive correlation between clock genes and clinically actionable genes. X-axis (clinically actionable genes) is ordered by the number of positively correlated clock genes minus the number of negatively correlated clock genes. Y-axis (clock genes) is ordered by the total number of correlated clinically actionable genes. If the number of cancer types is less than five, the fill color of cell is white. Triangles highlight genes discussed in the main text. Bold boxes highlight the protein-protein interactions of actionable genes and clock genes based on the experimental evidence from BioGRID and HPRD. X marks ChIP-seq evidence for interaction between clinically actionable genes and clock genes. Upper blue and red bars indicate the number of drugs that directly target (blue bars) or correlate with (red bars) actionable genes. (B) Transcript levels of clinically actionable genes (CAGs) in primary wild-type (WT) and CRY2 knock-down (CRY2 −/−) MEFs at different time points. Upper top 10 genes are positively correlated with CRY2 in cancer, while the bottom 10 genes are negatively correlated. Error bar indicates standard deviation (SD) for three biological replicates. See also Figure S6.

Figure 7. Drug effects of clock genes in chronotherapy

(A) Correlation between drug sensitivity (AUCs) and gene expression of clock genes in at least 10 clock genes (green: negative correlation; magenta: positive correlation; size: p-value). Black triangles indicate compound verified in chronotherapy. Color bars in Y-axis indicate drugs (right) and their targeted pathways (left). (B) Correlation between clock genes and known drugs that target the genes (green: negative correlation; magenta: positive correlation), which corresponds to significant correlation between drug sensitivity and clock genes. See also Figure S7.