Transglutaminase 2 is associated with adverse colorectal cancer survival and represents a therapeutic target

Molecular markers for predicting prognosis of colorectal cancer (CRC) patients are urgently needed for effective disease management. We reported previously that the multifunctional enzyme Transglutaminase 2 (TGM2) is essential for CRC cell survival by inactivation of the tumor suppressor p53. Based on these data, we determined the clinical relevance of TGM2 expression and explored its potential as prognostic marker and therapeutic target in CRC. We profiled TGM2 protein expression in tumor samples of 279 clinically characterized CRC patients using immunohistochemical staining. TGM2 expression was upregulated in matched tumor samples in comparison to normal tissue. A strong TGM2 expression was associated with advanced tumor stages and predicted worse prognosis regarding progression-free and overall-survival, even at early stages. Inhibition of TGM2 in CRC cell lines by the inhibitors LDN27219 and Tyrphostin resulted in a strong reduction of cancer cell proliferation and tumorsphere formation in vitro by induction of p53-mediated apoptosis. Primary patient-derived tumorsphere formation was significantly reduced by inhibition of TGM2. Treatment of mice with TGM2 inhibitors exhibited a significant deceleration of tumor progression. Our data indicate that high TGM2 expression in CRC is associated with worse prognosis and may serve as a therapeutic target in CRC patients with strong TGM2 expression.


Clinical tissue samples
A total of 279 patients with CRC were enrolled and underwent surgery at the University Hospital Frankfurt between January 2008 and July 2018.Tumor tissue samples of all patients were obtained from the biobank of the Goethe University Frankfurt Cancer Center's Tissue Procurement Facility.The use of the samples for research purposes was approved by the institutional ethics review board and all patients gave written consent before operation.Tumor samples were dissected and identified by two pathologists to confirm the pathologic nature and histologic grading of the tumor.The surgical specimens were fixed in formalin.Most patients were followed-up every 3-6 months after surgery, including tumor marker testing (serum carcinoembryonic antigen, CEA) and imaging (abdominal ultrasonography, chest radiography or computed tomography).Postoperative adjuvant chemotherapy was recommended in patients with stage III and in stage II tumors with risk factors, according to the German Colorectal Cancer Society Guidelines.Clinicopathological features were evaluated according to the International Union for Cancer Control Tumor Nodular Metastasis Classification (TNM Classification of Malignant Tumors, 7 th Edition).The study was approved by the institutional ethics review board (Number: SGI-04-2014).Written informed consent was obtained from all participants prior to inclusion in the study in accordance with the Declaration of Helsinki and local laws and regulations.

Slide digitalization and production of a human tissue micro array (TMA)
Hematoxylin and eosin stains were digitalized using a 20x brightfield slide scanner (Pannoramic Scan II, 3D Histech, Budapest, Hungary).Annotations of defined regions of interest were set to a core diameter of 1 mm.The TMA was produced using the TMA Grand Master (3DHistech, Budapest, Hungary) and matching settings of 1 mm core diameter.Via slide overlay function, digital annotations were matched to the donor tissue and transferred to an empty recipient paraffin matrix.In summa, three TMAs were produced, each containing well characterized areas of the tumor and tumor adjacent physiological tissue.

Isolation of primary cells from patient specimens
Fresh human colon cancer and adjacent normal mucosa tissue were obtained from patients undergoing surgical resection at Goethe University Hospital Frankfurt or at Bethanien-Hospital (Frankfurt, Germany), who had given informed consent.All tissues were collected under protocols approved by the ethics committee of the University Hospital Frankfurt.All samples were characterized by a pathologist.Briefly, solid tissues were minced into small fragments, washed with PBS containing penicillin/streptomycin followed by enzymatic dissociation with 200 U/ml Collagenase type III, 100 U/ml Dispase and 100 U/ml DNase I (all Worthington Biochemical Corp., Lakewood, USA).During enzymatic incubation, cell suspension was subjected to MACS tissue-dissociator (Miltenyi Biotec, Bergisch-Gladbach, Germany) under a defined program every 15 minutes.The digested material was filtered and contaminating red blood cells were removed by osmotic lysis using 0.83% ammonium chloride solution with 0.1mM EDTA for 10 minutes at 37°C.Magnetic cell separation was performed to obtain an epithelial cell-enriched suspension using the human Tumor Cell Isolation Kit from Miltenyi Biotec (Germany) according to the manufacturer's instructions.After magnetic sorting, the viability was assessed using trypan blue exclusion and purified cells were resuspended in serum-free DMEM/F12 (Gibco Fisher Scientific, Schwerte, Germany) supplemented with 20 ng/ml epidermal growth factor (order code # E9644-.2MG) and fibroblast growth factor (order code #F0291-25UG, both purchased from Sigma Aldrich, Taufkirchen, Germany), 2 % N2 supplement (Thermo Fisher Scientific Inc, Waltham, MA, USA), 20 mM HEPES, and 50 U/ml penicillin/streptomycin.The purity of the isolated cells was verified by flow cytometry.
All experiments were performed using cell lines which had been passaged <25 times.

Sphere formation assay
Cancer cell line cells or freshly isolated CRC cells were cultured in serum-free medium described above at a density of 5 000 cells per well in ultra-low-attachment 24-well plates (Corning Inc., Corning, NY, USA).Plates were scored microscopically after 7 and 14 days using Axio Observer Z-1 microscope (Carl Zeiss, Jena, Germany).

In vitro drug treatment
LDN27219 and Tyrphostin 47 (both Sigma Aldrich, St. Louis, USA) were dissolved in dimethyl sulfoxide (DMSO) and stored at -20°C. 3 000 SW480 and CaCo2 cells per well in 96-well plates were cultured with or without LDN27219 (1 or 10µM) or Tyrphostin 47 (10 or 100µM) for 24-72 hours.Proliferation was assessed using 3-(4,5-dimethylthiazol-2yl)-2,5diphenyltetrazoliumbromide (MTT) assay.Further, the effect of TGM2 inhibition on tumorsphere formation was investigated in SW480, CaCo2 and primary CRC cells using sphere formation assay.Apoptosis was determined using AnnexinV/7AAD staining (BD Becton Dickinson, Heidelberg, Germany), according to the manufacturer's instructions in SW480 and CaCo2 cells after treatment with LDN27219 and Tyrphostin for 72 hours.In order to determine p53 activity under TGM2 inhibition, phosphorylated p53(S15) was detected by IHC and flow cytometry.For IHC, SW480 cells were cultured in 8-well chamber slide (Corning) and treated with LDN27219 or Tyrphostin for 24 to 72 hours.Subsequently, cells were washed and fixed with 10% formalin solution (Sigma-Aldrich).IHC staining with phosphorylated p53(S15) antibody was performed as described previously in the section Immunohistochemistry. Finally, flow-cytometric assessment of phosphorylate p53(S15) was performed.SW480 cells treated with LDN27219 or Tyrphostin for 24 to 72 hours, were harvested and fixed with 4% formaldehyde, followed by permeabiliziation with ice-cold methanol for 1 hour.Subsequently, cells were washed and stained with FITC-conjugated anti-p53S15 antibody for 1 hour at room temperature.The cells were analyzed on a FACSCanto II (Becton Dickinson, Heidelberg, Germany).DMSO treated cells served as control.

In vivo xenograft experiments
All animal experiments were performed according to protocols approved by the state government.NOD.CB17-Prkdc scid /J (NOD-SCID) mice (Jackson Laboratory, Maine, USA) were used for experiments at 6-8 weeks of age.LDN27219 and Tyrphostin 47 (both Sigma Aldrich, St. Louis, USA) were dissolved in dimethyl sulfoxide (DMSO).5x10 4 living SW480 cells were subcutaneously injected into the flank of NOD-SCID mice.
When tumor size reached 0.2-0.3cm in diameter, LDN27219 (25 mg/kg) was administered orally and Tyrphostin 47 (2,2mg/kg) intraperitoneally three times a week and compared with a DMSO control.Tumor growth was measured twice weekly using a caliper, and mice were sacrificed when tumor size reached a diameter of 1.0 cm.Tumors were harvested and lysates were prepared for transglutaminase activity assay and protein expression analysis by Simple Western technology.

Transglutaminase activity assay
Transamidase activity of TGM2 was assessed in SW480 cells treated with LDN27219, Tyrphostin 47 or DMSO as well as in corresponding xenograft tumors using Tissue Transglutaminase Microassay kit (order code # T055, Zedira, Darmstadt, Germany) following the manufacturer's instructions.Briefly, cells or minced tumor tissue were lysed on ice in M-PER Mammalian extraction Reagent (Thermo Fisher Scientific) and protein concentration was determined subsequently.Protein samples were incubated with reaction buffer containing calcium, DTT and biotin-pepT26 in the wells of the microtiter plate.For negative control, EDTA was added.The assay uses biotin-pep26 as the first substrate and an amine donor/acylacceptor as a second substrate.In the presence of active TGM2, the γ-carboxamide of the glutaminyl residue of biotin-pepT26 is incorporated into the amine substrate to form biotinylated isopetide bound.Enzymatic reaction is determined by its interaction with streptavidin labelled peroxidase.A substrate solution for peroxidase was added for color development.The color intensity was measured on the Infinite 200 microplate reader (Tecan, Germany).

Protein expression analysis by Simple Western technology
Protein expression was detected by Simple Western TM assays using the Wes TM System following the manufacturer's protocol (Bio-Techne, Wiesbaden, Germany).The automated capillary western blot assay incorporates protein separation based on size performed in glass capillaries and immobilization of the proteins directly onto the capillary walls, followed by immuno-probing and chemiluminescent detection.This method was used to analyze protein expression in tumor lysates.Briefly, tumor tissues were lysed by m-PER mammalian extraction buffer containing 1x Halt protease inhibitor (Thermo Fisher Scientific) and homogenized by Precellys homogenizer (Peqlab Biotechnologie, Erlangen, Germany).Protein concentration was determined using Coomassie plus (Bradford) assay (Thermo Fisher Scientific).0.05 µg tumor lysates were diluted with 0.1 × Sample Buffer, combined with Fluorescent Master Mix (containing sample buffer, fluorescent standard, and DTT) and denaturated at 95 °C for 5 minutes.The denatured samples, blocking reagent, primary antibodies, HRP-conjugated secondary antibodies and chemiluminescent substrate were then applied on a Protein Simple 12-230 kDa capillary cartridge separation module (Bio-Techne).The primary antibody dilution was 1:100 to 1:50.The following primary antibodies were used: anti-p53 (clone 1C12, #2524) and anti-phospho-p53(S15) (clone 16G8, #9286, both Cell Signaling Technology, Danvers, Massachusetts, USA).Beta Actin (#MAB8929, Cell Signaling Technology) served as loading control.A biotinylated ladder provided molecular weight standards for each assay.After plate loading, the separation electrophoresis and immunodetection steps take place in the fully automated capillary system.An optimized defined run setting was used.Protein expression was measured at exposure times between 15 and 16 seconds.Quantification of chemiluminescence was based on peak area after correction for a baseline signal.Molecular weight, the area under the peak, signal-to-noise ratio, peak height, and peak width were reported for each named peak.
The area under the peak represents the signal intensity of the immuno-detected protein.Data were generated by the Compass software for Simple Western instruments.

Statistical analysis
The level of TGM2 expression in CRC and normal colorectal mucosa, as well as the relationship between TGM2 expression level and clinicopathological factors were analyzed using Wilcoson's matched paired and Chi-squared (χ 2 )-test.
For baseline characteristics the follow-up started with the date of first diagnosis.End of followup was the date of death or the last patient visit.Categorical variables were described in frequencies and percentages.Continuous variables were represented as a mean and its standard deviation (SD).Categorical variables were compared by the Chi-squared (χ 2 )-test or Fisher's exact test, as needed.χ 2 -Test was performed over three groups of TGM2 expression.