Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

Abstract

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

Fig. 1. TGM2 protein expression and enzymatic activity are elevated in CRC.

A Representative microphotographs of immunohistochemical (IHC) stainings of TGM2 (brown) and Ki67 (red) in paired tumor and corresponding normal tissues from five CRC patients (P1-5). Scale bar, 50 µm. B Comparison of TGM2 expression in 10 colon cancer and paired adjacent noncancerous colon tissues. Data are presented as mean immunoreactive score ± SD of IHC staining. C Representative protein expression data of TGM2-isoform 1 in primary patient material showing normal tissue (N) versus the corresponding tumor tissue (T) of three different CRC patients (P1-3) via Simple Western technology. α-Tubulin served as loading control. D Quantification of TGM2 protein expression in epithelial cells of paired tumor/normal tissue samples from CRC patients (n = 8) via Simple Western technology. E TGM2 transamidation activity in epithelial cells of paired tumor/normal tissue samples from CRC patients (n = 8). Results are presented as mean ± SD. Pairwise comparisons were performed using Wilcoxon matched pairs test.

Fig. 2. Targeting TGM2 inhibits CRC cell expansion and tumorsphere formation.

A Cell expansion of lentivirally transduced SW480 or (B) HCT-116 cells with two independent shRNAs against TGM2 (shTGM2-1, shTGM2-2) or control (shSCRMBL). C Cell expansion of SW480 and (D) HCT-116 cells after transduction with CRISPR/Cas9 constructs (TGM2 gRNA) against TGM2 or non-target (NT) control. E Mean number of tumorspheres after TGM2 knockdown in SW480 and HCT-116 cells. Data are presented as mean ± SD of at least three independent experiments. F Representative microphotographs of tumorspheres of SW480 cells 14 days after transduction with shTGM2-1, shTGM2-2, or control (shSCRMBL). Shown are fluorescent (tdTOMATO) and brightfield microphotographs. Scale bar, 200 µm. *P < 0.05; **P < 0.01, Mann–Whitney U test.

Fig. 3. Targeting TGM2 blocks CRC cell tumorigenicity.

A SW480 cells were transduced with TGM2 shRNAs or control and expanded in vitro for three days. A total of, 50,000 living cells were injected subcutaneously into the flanks of NOD/SCID mice. Tumor volumes were monitored over time and determined using caliper measurements. B Comparison of growth curves of xenografts of SW480 transduced with shTGM2-1, shTGM2-2, or shSCRMBL. C After five weeks, mice were sacrificed, tumor xenografts were harvested, and tumor weight was measured. Representative tumors are shown. Data are represented as mean ± SD of xenografts in eight mice per group. ***P < 0.001, Mann–Whitney U test.

Fig. 4. The ablation of TGM2 leads to CRC cell death.

A Time-lapse imaging of SW480 cells transduced with shTGM2-1, shTGM2-2 or shSCRMBL. Shown are cumulative cell death events over time determined by single cell tracking. P value was calculated by log-rank test. B Percentage of apoptotic SW480 cells determined by Annexin V/7-AAD staining 72 hours after TGM2 knockdown. C Percentage of Caspase-3 positive SW480 cells 72 hours after TGM2 knockdown. D–F All experiments were repeated in HCT-116 cells transduced with shTGM2-1, shTGM2-2 or shSCRMBL. D Time lapse imaging showing the cumulative cell death events. E Percentage of Annexin V positive and (F) Caspase-3 positive HCT-116 cells 72 hours after TGM2 knockdown. Results are presented as mean ± SD of three independent experiments. ***P < 0.001, Mann–Whitney U test.

Fig. 5. Transamidation activity of TGM2 is predominant in CRC and essential for cancer cell survival.

A Schematic diagram of full-length TGM2 isoform 1 (I) and short or truncated isoform 2 (II) with respective exons. Positions of point mutants are indicated. B Transamidase activity assay with lysates of SW480 cells after lentiviral overexpression of TGM2-isoform 1 or 2, three days after transduction. Data are presented as mean ± SD of three independent experiments. C Transamidase activity assay with cell lysates after lentiviral overexpression of TGM2^C277S, TGM2^R580A, TGM^C277S+R580A, or vector control three days after transduction. Data are presented as mean ± SD of three independent experiments. D Cell expansion of SW480 cells lentivirally expressing TGM2 isoform 1, 2 (TGM2-Iso 1, TGM2-Iso 2) or vector control (Ctrl) or (E) TGM2 mutants TGM2^C277S, TGM2^R580A, TGM^C277S+R580A, or vector control (Ctrl). Cells were counted at day three and seven after transduction. F SW480 cells were transduced with TGM2 isoforms 1 or 2, or (G) with mutated TGM2 constructs C277S, R580A, or C277S/R580A and expanded for three days. Subsequently, cells were double transduced with CRISPR/Cas TGM2 knockout construct (TGM2 KO) or control, resulting in genetically manipulated cells with only ectopic TGM2. Transduction efficiency was evaluated by FACS and expansion kinetic monitored over 14 days. Results are presented as mean ± SD of three independent experiments. Mann–Whitney U test; **P < 0.01; ***P < 0.001.

Fig. 6. Molecular changes after TGM2 knockdown reveal p53 as central tumor suppressor.

A–C Gene expression profiling by RNA-seq of SW480 cells after transduction with either shTGM2-1 or shSCRMBL. A Unsupervised hierarchical clustering of the top 1000 differentially expressed genes (DEGs) upon TGM2 knockdown across the four biological replicates. B MA plot relating p values for all differentially expressed genes between shTGM2-1 and shSCRMBL from four biological replicates. Red dots indicate significantly regulated genes (adjusted P < 0.05). List of regulated genes is presented in Supplementary Table S2. C Scatter plot of gene set enrichment analysis of DEGs relating the Q-value for Hallmark gene-set signatures. The top 16 enriched pathways are shown (P < 0.05, Fold change ≥2). The color and size of each dot represent the Rich factor and the number of DEGs mapped to the indicated pathway, respectively. D Proteome analysis of regulated proteins involved in apoptosis upon shRNA-mediated TGM2 knockdown. Representative blot of Proteome Profiler Array™-Human Apoptosis Array analysis of SW480 cells. The regulation of protein expression of phosphorylated p53 variants is shown. E–H Quantification of p53 and phosphorylated p53 (S15, S46, and S392) upon TGM2 knockdown in SW480 (E, G) and HCT-116 (F, H) cells via Simple Western technology (n = 3; Mann–Whitney U test).

Fig. 7. Direct interaction of TGM2 and p53 in CRC.

A Representative images of proximity ligation assay (PLA) of TGM2 and p53 in SW480 cells. Cells incubated only with TGM2 antibody served as negative control (I). Protein–protein interaction of TGM2 and p53(S15) was visualized using hybridization probes labeled with Texas Red (II). Nuclei were stained with DAPI (blue). B Quantification of TGM2-p53 interaction and associated technical controls (Ctrl). Technical controls demonstrate the specificity of PLA signals. Each dot represents one cell. Mean value of PLA dots per cell is shown by the black line. C Representative images of proximity ligation assay of TGM2 and p53 in patient-derived normal epithelial cells (I) and corresponding colon cancer cells (II). D Quantification of TGM2-p53 interaction in primary patient material. (Significance was calculated using Kruskal–Wallis test). E Co-immunoprecipitation (Co-IP) of endogenous TGM2 and p53 or phosphorylated p53(S15) in SW480, HCT-116 p53 wildtype cells (wt) and HCT-116 p53 knockout cells (−/−). F Super-resolved image of a HCT-116 cell immunostained for TGM2 (red) and p53(S15) (cyan). A zoom-in of the highlighted region is shown on the right. White regions indicate overlapping signal of TGM2 and p53(S15) (yellow arrowheads). Scale bars represent 5 µm and 1 µm, respectively.

Fig. 8. TGM2 functions as a pro-survival molecule via direct interaction and inhibition of p53 signaling.

A–C HCT-116 p53 wildtype cells (wt) and HCT-116 p53 knockout cells (−/−) were transduced with either shTGM2-1, shTGM2-2, or shSCRMBL. Time-lapse imaging and proliferation assay were performed to determine a rescue from cell death upon TGM2 knockdown. A Fold change of cell number of HCT-116 p53^wt and HCT-116 p53^−/− cells upon TGM2 knockdown in comparison to shSCRMBL control determined at day three after transduction. Data are presented as mean ± SD of three independent experiments (**P < 0.01, Mann–Whitney U test). B Single cell tracking of HCT-116 p53^wt and HCT-116 p53^−/− cells after TGM2 knockdown with shTGM2-1 and (C) shTGM2-2. Cumulative cell death events are shown over time (***P < 0.001, Log-rank test). D Direct visualization of p53 activation upon TGM2 knockdown by time-lapse video-microscopy. Sequence of phase contrast images, tdTOMATO fluorescence of shTGM2-1 [1] and p53-driven destabilized GFP reporter [2], depicting the same field of view over the time course of 30 hours as indicated in the corresponding panels in I–VIII. The yellow circles designate tracked cells over time. (I–VIII) show corresponding sequence of fluorescence images taken at the same time points as the phase contrast images. (I) Shown are two representative HCT-116 cells. (II and III) 6-8 hours after lentiviral transduction of shTGM2-1 both HCT-116 cells express the red fluorescent tdTOMATO reporter, indicating a knockdown of TGM2. (IV-VI) Another 4–10 hours later both cells express the green fluorescent (GFP) p53 reporter, indicating the induction of p53 activity. (VII and VIII) About 24 hours after transduction both HCT-116 cells subsequently undergo apoptosis (white arrows). Movie S1 shows all assembled images (3 min temporal resolution) of the same sequence.