Crosstalk between the mitochondrial fission protein, Drp1, and the cell cycle is identified across various cancer types and can impact survival of epithelial ovarian cancer patients

Abstract

Mitochondrial metabolic reprogramming is a hallmark of tumorigenesis. Although mitochondrial function can impact cell cycle regulation it has been an understudied area in cancer research. Our study highlights a specific involvement of mitochondria in cell cycle regulation across cancer types. The mitochondrial fission process, which is regulated at the core by Drp1, impacts various cellular functions. Drp1 has been implicated in various cancer types with no common mechanism reported. Our Drp1-directed large-scale analyses of the publically available cancer genomes reveal a robust correlation of Drp1 with cell-cycle genes in 29 of the 31 cancer types examined. Hypothesis driven investigation on epithelial ovarian cancer (EOC) revealed that Drp1 co-expresses specifically with the cell-cycle module responsible for mitotic transition. Repression of Drp1 in EOC cells can specifically attenuate mitotic transition, establishing a potential casual role of Drp1 in mitotic transition. Interestingly, Drp1-Cell-Cycle co-expression module is specifically detected in primary epithelial ovarian tumors that robustly responded to chemotherapy, suggesting that Drp1 driven mitosis may underlie chemo-sensitivity of the primary tumors. Analyses of matched primary and relapsed EOC samples revealed a Drp1-based-gene-expression-signature that could identify patients with poor survival probabilities from their primary tumors. Our results imply that around 60% of platinum-sensitive EOC patients undergoing relapse show poor survival, potentially due to further activation of a mitochondria driven cell-cycle regime in their recurrent disease. We speculate that this patient group could possibly benefit from mitochondria directed therapies that are being currently evaluated at various levels, thus enabling targeted or personalized therapy based cancer management.

Keywords: Drp1; cancer; cell cycle; genome analyses; mitochondria.

Figure 1. Identification of Drp1 co/anti-expression modules across TCGA tumor types

A. Box plot of Drp1 expression (RNA-seq) in tumor tissues across the TCGA cancer types. B. Bar plot representing patient frequency with more than one copy of DNM1L gene (based on GISTIC scores) in tumor tissues across the TCGA cancer types. C. Heat map of the functional REACTOME pathways correlating positively (left) or negatively (right) with Drp1 expression (X axes) in 31 TCGA cancer types (Y axes), as identified by GSEA analyses of the Drp1 correlating genes. Heat map represents negative log converted p values of the individual REACTOME pathways (within the cut of p and q ≤0.05). D. Heat map of the top level REACTOME pathways (X axes) depicted in C. Heat map represents median NES scores of the identified pathways (see Supplementary Figure S2). Top-level pathways present in REACTOME but not in MsigDB are boxed. * depicts re-assigned “Ribosome” pathway. Arrows indicate the pathways common in majority of the cancer types.

Figure 2. Elevated expression of Drp1 due to gene duplication in epithelial ovarian tumors

A. Density plots depicting the frequency distribution of Drp1 expression in the primary epithelial ovarian tumors (red) in comparison to all other primary tumors of TCGA (black). B. Linear regression analysis of Drp1 expression (plotted on a log scale) and raw GISTIC scores in individual EOC patients (red dots). Regression coefficient (R²), p value and correlation mentioned. C. Box-plot of Drp1 expression at various thresholded GISTIC levels in EOC patients; median Drp1 expression values mentioned on top. N denotes sample size.

Figure 3. Drp1 co-expresses with cell cycle genes, independent of gene duplication events, and supports proliferation of the EOC cells

A. Venn diagram depicting the overlap in the leading edge genes of the REACTOME top-level pathways correlating positively (Up) or negatively (Down) with Drp1 expression in the primary EOC tumors of TCGA. Numbers signify the total number of leading edge genes. B. Pie chart representing distribution of Drp1 co-expressing REACTOME Cell Cycle leading edge genes in various phases of cell cycle (G1 phase, G1/S transition, S phase, G2/M transition and M (mitosis)). The Drp1 co-expressing genes involved in proteasomal function that are assigned to all cell cycle phases were excluded in this classification. C. Venn diagram depicting the overlap in the leading edge genes of the REACTOME top-level pathways correlating positively (Up) with Drp1 expression in the TCGA EOC patients with no amplification of DNM1L gene in their primary tumors; no statistically significant negatively correlated pathways were detected. Numbers signify the total number of leading edge genes. D. Bar plot showing the distribution of cells in G1, S and G2-M phases in A2780 cell line stably expressing Drp1shRNA2 as normalized by the distribution of A2780 cell line stably expressing a non-targeting (NT) shRNA. E. Bar plot showing the cell proliferation rate of A2780 cell line stably expressing Drp1shRNA2 as normalized by that of A2780 cell line stably expressing a non-targeting (NT) shRNA. n denotes number of genes; N denotes number of patients. * denotes p value < 0.05 in Student's t-test.

Figure 4. Drp1 expression and its correlation with cell cycle module vary with chemosensitivity

A. Heat map and hierarchical clustering of the genes correlating positively or negatively with Drp1 expression in the primary EOC tumors of TCGA. Heat map represents gene to gene correlation matrix (in the form of distance) where the color scale depicts distance between the genes. The GSEA output is superimposed on the heatmap in the form of color-coded ticks representing the Drp1 correlating genes in various REACTOME pathways as labeled. B. Box plot of Drp1 expression in the primary tissues of sensitive, recurred-resistant and resistant EOC patients of TCGA. Median values of Drp1 expression mentioned on the top. p values signifies significance in permutation test. C. Heat map and hierarchical clustering (like in A) in Sensitive patients. D. Heat map and hierarchical clustering (like in A) in Recurred-Resistant patients. E. Heat map and hierarchical clustering (like in A) in Resistant patients. n denotes number of genes; N denotes number of patients. Extra description of the pathways in parenthesis is obtained from the REACTOME pathway hierarchy (see relevant supplementary table in each case).

Figure 5. Drp1 co-expresses with cell cycle genes in relapsed tumors of a particular group of EOC patients

A. Drp1 expression of various ICGC recurred-resistant EOC patients (color coded) in matched primary and relapsed disease. Lines connect the same patients. Patient with dashed line has been dropped from further analyses (please see relevant text in the Results section). The numbers on the right signify percentage increase (+) or decrease (−) in Drp1 expression in the relapsed samples in comparison to the primary. B. Barplots showing mean Drp1 expression in the primary and relapsed samples of Drp1-High and Drp1-Low patient groups of ICGC. * denotes p value < 0.05 in Student's t-test. C. Barplots showing the Normalized Enrichment Score (NES) of the pathways uniquely altered in the primary or relapsed samples between the Drp1-Hogh and Drp1-Low patient groups. The top-level REACTOME pathways are mentioned in the figure index. Extra description of the pathways in parenthesis is obtained from the pathway hierarchy (see relevant supplementary Table in each case), D. Barplots showing the Normalized Enrichment Score (NES) of the pathways uniquely altered in the Drp1-High or Drp1-Low patient groups between their relapsed and primary samples. The top-level REACTOME pathways are mentioned in the figure index. Extra description of the pathways in parenthesis is obtained from the REACTOME pathway hierarchy (see relevant supplementary Table in each case).

Figure 6. Drp1 based classification of the primary tumors of recurred-resistant EOC patients

A. NMF consensus plot of 100 runs clustering the TCGA recurred-resistant EOC patients into TCGA-RR-70 (bottom right, red) and TCGA-RR-48 (top left, red) groups based on a Drp1-based-gene-expression-signature (basis). The color coded patients are the core of the TCGA-RR-70 (brown) or TCGA-RR-48 (orange) groups based on their weights. See Supplementary Table 10. B. Unrooted dendrogram showing hierarchical clustering of the TCGA-RR-70, TCGA-RR-48, ICGC-Drp1-High and ICGC-Drp1-Low patient groups. Bootstrap values on the nodes indicate branching confidence. C. Survival plot of the TCGA-RR-70 and TCGA-RR-48 patient groups after diagnoses of the disease as EOC. Dashed lines indicate mean survival probability. D. Survival plot of the TCGA-RR-70 and TCGA-RR-48 patient groups after commencement of chemotherapy. Dashed lines indicate mean survival probability. p values denote significance with a chi-square test.