Figure 1.
High expression of TTK in human triple-negative breast cancers.
(A and B) RNA and protein levels of TTK in human breast cancers from omic data selected from the Institut Curie Human Tumor database. Box-plots illustrate the logarithmic (log2) transformed levels of TTK RNA (A) and protein (B) expression in triple-negative breast cancer (TNBC), Her2-overexpressing/ER-negative tumors (Her2), luminal A (LA) and luminal B (LB) tumors, and in normal breast tissues (N). (C) Correlation between TTK protein and RNA levels. Omic data obtained in panels A and B reveal the consistency of TTK measurements between RNA and protein levels as quantified by Spearman correlation within the entire tumor population, within TNBC (O, red filled), Her2 (Δ), LB (X) and LA (+) tumors. (D) Quantitative analysis of the percentage of TTK positive cells measured by immunohistochemistry (IHC) in human breast cancer tissues. We present the results as the percentage of cells positively stained for TTK within fractions of samples in each BC sub-types, with TNBC in red, Her2 in purple, LB in green and LA in blue. (E) Cellular detection of TTK within breast cancer biopsies by IHC. We give a representative example of TTK staining on tissue microarrays (TMA) for the different breast tumor subtypes (TNBC, Her2, LB and LA) and for healthy breast tissues (N). Moreover, we indicate for each the percentage of TTK-positive cells. For all images, the scale bar is 20 µm (F) Correlation between TTK and Ki67 RNA levels within the TNBC subtype. (G) Genomic alterations at TTK locus in human breast cancers. Box-plots illustrate gain or loss of TTK DNA copy number from our breast cancer cohort with TNBC, Her2, LA and LB tumors. (H) Correlation between the levels of TTK transcript and TTK DNA copy number. Omic data obtained in panels A and G reveal the consistency of TTK measurements between RNA and DNA copy number, with TNBC (O, red filled), Her2 (Δ), LB (X) and LA (+) tumors.
Figure 2.
Correlation between TTK mRNA levels and overall survival in human triple-negative breast cancers.
(A) Distribution of TTK mRNA levels in TNBC patients. We present the distribution of the logarithmic (log2) transformed levels of TTK RNA expression within the TNBC subgroup. (B) Univariate Kaplan–Meier curves of the overall survival in patients with TNBC divided into two groups, according to TTK mRNA expression dichotomized at the median value (≤9.28 and >9.28) to illustrate our data using the Wald test. We indicate the p values from Kaplan–Meier curves (hazard ratio = 3.33; 95% confidence interval = 0.9−12.39; p = 0.072, logrank test) and from the Cox regression model (p = 0.006).
Figure 3.
Expression of TTK protein in human cell lines derived from breast tissues.
In left part, we show logarithmic (log2) transformed levels of TTK protein expression in our breast cell line collection. Breast cell lines are classified as basal-A, basal-B and luminal as in [41]. In addition, we present the ER/PR expression and the Her2 overexpression status as in [41]. We indicate in bold the cell lines used in our study. In the right part, the box-plots illustrate the logarithmic (log2) transformed levels of TTK protein within the breast cell line subgroups.
Figure 4.
Viability of triple-negative breast cancer cell lines is impaired upon TTK depletion.
(A to D) Cell growth arrest after depletion of TTK. We transfected established TNBC cell lines, MDA-MB-468 (A), HCC70 (B) and MDA-MB-231 (C), and non-malignant MCF10A cells (D) with control siRNA (Ctrl; black bars) or two siRNAs against TTK (TTK_6, grey bars and TTK_7; dark grey bars). We analyzed their capacity to grow at the indicated time points by MTT assay. Each value in the histograms corresponds to the percentage of cell viabilty relative to the control and represents the mean of at least three independent experiments. (E to G) Impairment of clonogenicity after depletion of TTK. We transfected TNBC cells, MDA-MB-468 (E), HCC70 (F) and MDA-MB-231 (G) with the indicated siRNAs, and analyzed their capacity to form colony in soft-agar (same representations as in panels A-D). The error bars represent the standard deviation of the mean and asterisks, the p values from Student’s t test (*p<0.05; **p<0.01; ***p<0.001).
Figure 5.
Depletion of TTK in triple-negative breast cancer cells affects cell cycle progression.
(A to D) Flow cytometry analysis of the cell cycle distribution after TTK depletion. We transfected TNBC cells, MDA-MB-468 (A), HCC70 (B) and MDA-MB-231 (C), and normal MCF10A cells (D) with the indicated siRNAs, as in Figure 4. We monitored the cell cycle status of siRNA-treated cells by flow cytometry analysis following propidium iodide (PI) staining, 120 h for TNBC cells and 72 h for normal cells after siRNA-transfection. We also indicated for each profiles the percentage of sub-G1 cells on the left bars and the percentage of aneuploid cells on the right bars. (E to H) Quantification of the cell cycle distribution after TTK depletion corresponding to the panels A to D for MDA-MB-468 (E), HCC70 (F), MDA-MB-231 (G), and MCF10A (H) cells, with the percentages of cells in sub-G1 phase (right dark hatched), G1 phase (dark gray), S phase (light greys), G2+ M phase (grey) and aneuploid population (left black hatched).
Figure 6.
Induction of apoptosis by depletion of TTK in triple-negative breast cancer cells.
We transfected MDA-MB-468 (A), HCC70 (B) and MDA-MB-231 (C) and MCF10A (D) cells with the indicated siRNAs. We analyzed the proportion of living and dead cells by FACs following PI and annexin V staining. Histograms with the percentage of apoptotic cells are presented, and are the mean of two to three independent experiments. We show the standard deviation of the mean by error bars and the p values from Student’s t test by asterisks (*p<0.05; **p<0.01).
Figure 7.
Activation of caspase 3/7 and cleavage of PARP following depletion of TTK in triple-negative breast cancer cells.
(A to D) Increased caspase 3/7 activity in TTK-depleted cells. At the indicated time we measured caspase 3/7 activity in TNBC cells, MDA-MB-468 (A), HCC70 (B) and MDA-MB-231 (C), and normal MCF10A cells (D) following siRNA-transfection performed as in Figure 4. Each value in the histograms corresponds to the fold change in caspase 3/7 activity and represents the mean of three independent experiments. The error bars represent the standard deviation of the mean and the asterisks, the p values from Student’s t test (*p<0.05; **p<0.01; ***p<0.001). (E to H) Increased cleavage of caspase 7 and PARP in TTK-depleted cells. We transfected MDA-MB-468 (E), HCC70 (F), MDA-MB-231 (G) and MCF10A (H) cells with the indicated siRNAs. At the indicated time points, we harvested the cells and performed immunoblotting with antibodies against (i) cleaved PARP, which recognizes both uncleaved (PARP) and cleaved (c-PARP) forms, (ii) cleaved caspase 7 (c-casp7) and (iii) phosphorylated H2AX (γH2AX). We used an anti-TTK antibody to confirm TTK depletion and β-actin as a loading control.