Table 1.
Biological pathways with enrichment of differentially expressed kinases.
Table 2.
Function of the 10 most overexpressed kinases.
Fig 1.
Increased expression spindle assembly checkpoint components and its regulator TTK in PDAC.
(A) IPA software identified prominent cellular functions that were significantly affected by differentially expressed kinases between normal and primary PDAC. (B) Box and whisker plot of median, upper, and lower quartiles of mRNA expression of core components and regulators of the spindle assembly checkpoint. Asterisk represent the p-value of the Mann-Whitney test (ns: p≥0.05, **: p≤0.01, ***: p≤0.001, ****: p≤0.0001). (C) Expression of TTK in a panel of patient samples. N = normal pancreas and T = primary tumor. (D) Expression of TTK in immortalized pancreas epithelium (HPNE and HPDE) and PDAC cell lines.
Fig 2.
Ingenuity pathway analysis of differentially expressed kinases in primary PDAC compared to normal pancreas identified by unbiased methods.
All 106 differentially regulated kinases were analyzed by integrated pathway analysis using IPA. The posttranslational modification cell cycle, cellular assembly and organization, one of the most dominant networks, is depicted here. Signaling pathways are colored according to expression, green representing down-regulation and red representing up-regulation with the expression fold change represented by more intense colors.
Fig 3.
Genetic and pharmacologic inhibition of TTK decrease growth of PDAC cell lines.
(A) Immunoblot analysis of HPAC and PANC-1 cell extracts showing protein level of TTK in control mismatch siRNA (siMM) and a TTK siRNA (siTTK) pool 48 h after transfection. (B) Growth of HPAC and PANC-1 PDAC cell lines transfected with control siMM and siTTK show reduced viability with TTK depletion. Cells were measured for proliferation at 48, 72, and 120 h as indicated. (C) Growth of HPAC and PANC-1 PDAC cell lines treated with DMSO control or 2 μM AZ3146. Cells were measured for proliferation at 48, 72, and 120 h as indicated. (D) Representative images of colony formation of the PANC-1 cell line in soft agar. (E) Quantitation of colony formation in soft agar of the HPAC and PANC-1 cell lines after transfection of either control or TTK targeted siRNA. Samples normalized to control. (F) Quantitation of colony formation in soft agar of the HPAC and PANC-1 cell lines after with continuous treatment with vehicle (DMSO) or AZ3146. Normalized to DMSO control. Asterisk represent the P-value of the two-sided T-test (*:≤0.05, P **: P≤0.01). Results representative of at least 2 independent experiments.
Fig 4.
TTK inhibition overrides the SAC mediated cell cycle arrest and leads to aberrant cell cycle progression, multi-nucleation and apoptosis.
(A) Immunoblot of HPAC and PANC-1 PDAC cell lines arrested in mitosis by treatment with nocodazole. Cells were then treated with 2 μM AZ3146 for 4 h and probed for expression of cyclin B1. (B) Representative flow cytometry plots of the cell cycle of HPAC and PANC-1 cell lines of 2 experiments. Cells were transfected with control or TTK targeted siRNA. 72 h post transfection cells were fixed and stained with propidium iodide. DNA content was assessed by flow cytometry. (C) Quantitation of B showing distribution of cells in each phase of the cell cycle. (D) Confocal microscopy of the PANC-1 cell line stably expressing a GFP-Histone 2B construct to visualize DNA. Chromosomal instability is visible in cells depleted of TTK in the form of multi- and micro-nucleation. (E) Quantitation of cells with multi- or micro-nucleated phenotypes. (F) Scatterplots showing induction of apoptosis with depletion of TTK. PANC-1 cells were transfected with control or a TTK targeted siRNA pool. 72 h post transfection cells were harvested and stained with the apoptotic marker Annexin V and the counterstained with propidium iodide to visualize necrotic cells. (G) quantitation of the apoptotic induction of cells used in F.
Table 3.
Predicted TTK phosphorylation substrate with known mitotic roles.
Fig 5.
Usp16 is a TTK phosphorylation substrate.
(A) In vitro kinase assay measuring TTK dependent phosphorylation by 32P incorporation measured by liquid scintillation counts. Representative of 2 independent experiments. (B) Exogenously expressed FLAG-Usp16 was immunoprecipitated from DMSO and AZ3146 treated mitotic 293FT cells, digested with trypsin and enriched for phosphopeptides. Phosphorylated residues of Usp16 were identified by mass spectrometry. Spectral counts of representative individual experiments are shown. (C) Immunoblot analysis of 293FT cells transiently transfected with control GFP, GFP-Usp16, GFP-Usp16 3xA (phosphodeficient mutant) or GFP-Usp16 3xE (phosphomimetic mutant) and treated with control DMSO or MG-132. (D) Densitometry of (C). (E) RT-PCR of Usp16 using 2 independent Taqman probes from cells used in (C), normalized to β-actin and represented as percent of WT-Usp16.