Tissue transglutaminase expression is necessary for adhesion, metastatic potential and cancer stemness of renal cell carcinoma

Abstract

Tissue transglutaminase (TG2) is the ubiquitously expressed member of transglutaminase family and shown to play a critical role in the development and progression of drug resistance malignancies. We have previously showed the association of TG2 upregulation with progression and metastasis of renal cell carcinoma (RCC) and low disease-free survival. In the present study we further investigate the role of TG2 in cell adhesion, migration and invasion of RCC by silencing TG2 expression in Caki-2 and A-498 primary site and Caki-1 and ACHN metastatic site RCC cell lines. Downregulation of TG2 expression led up to a 60% decrease in actin stress fiber formation and adhesion to β 1 integrin (ITGB1) substrates fibronectin, collagen type I and laminin in both primary and metastatic site RCC cell lines. In addition, treatment with siRNAs against TG2 impaired the migration capacity and cellular invasiveness of ITGB1 substrates in all 4 RCC cell lines. Lastly, the knockdown of TG2 in metastatic Caki-1 cells diminished the expression of CD44, CD73-and CD105 cancer stem cell-like markers. We conclude, for the first time, that TG2 expression is critical for cancer cell adhesion, migration, invasiveness and cancer cell-stemness during RCC progression and dissemination. Therefore, combined targeting of TG2 with drugs widely used in the treatment of RCC may be a promising therapeutic strategy for RCC.

Keywords: cancer cell stemness; cell adhesion; invasion; renal cell carcinoma; tissue transglutaminase.

Figure 1.

Down-regulation of TG2 reduces actin stress fiber formation in the primary site and metastatic RCC cell lines. (A) Efficiency of siRNA transfection on downregulation of TG2 in Caki-2 and A-498 primary RCC line lines and Caki-1 and ACHN metastatic RCC lines was analyzed by Western blot using actin as the control loading protein. (B) Changes in actin cytoskeleton organization in RCC cell lines in response to TG2 silencing was examined under florescence microscopy using an FITC filter. Bars, 10 µm. Following the treatment of RCC cell lines with NS siRNA and siRNAs against TG2 (siR1 and siR6), cells were seeded on tissue culture plastic for 60 mins and actin stress fibers were stained using FITC-phalloidin as described in Experimental Procedures. The downregulation level of TG2 was determined with gene expression analysis run in parallel with each experiment. (C) Non-overlapping images of 10 random fields/sample were acquired and the number of cells with actin stress fibers were scored. Data values represent the mean percentage of cells with formed actin stress fibers from 3 independent experiments, which were expressed as the percentage of control values. Mean percentage value of stress fiber positive cells treated with NS was used as 100% for each cell line.

Figure 2.

TG2 is required to support cell adhesion and spreading of the primary site RCC cell lines Caki-2 and A-498 on integrin β1 substrates FN, Col 1, LM. The percentage of attached cells or spread cells ± _SD shown were from the mean of at least 3 independent experiments, performed in triplicate. As described in Experimental Procedures, NS siRNA, siRNA 1 (siR1) and siRNA 6 (siR6) treated Caki-2 cells seeded on (A) FN, (C) Col1, (E) LM and A-498 cells seeded on (B) FN, (D) Col1, (F) LM were allowed to attach for the indicated early (30 mins for FN and LM, and 60 mins for Col1) and the late (60 mins for FN and LM, and 90 mins for Col1) time points. Cell attachment or spreading of control cells (cells treated with NS siRNA) on integrin β1 substrates recorded for the late time point represents 100%. Bars, 20 µm.

Figure 3.

TG2 is also necessary for the cell adhesion and spreading of the metastatic RCC cell lines Caki-1 and ACHN on integrin β1 substrates FN, Col 1, LM. Means of at least 3 independent experiments, performed in triplicate was used to calculate the percentage of attached cells or spread cells ± _SD. NS siRNA, siRNA 1 (siR1) and siRNA 6 (siR6) treated Caki-1 cells seeded on (A) FN, (C) Col1, (E) LM and ACHN cells seeded on (B) FN, (D) Col1, (F) LM were allowed to attach for indicated early (30 mins for FN and LM, and 60 mins for Col1) and late (60 mins for FN and LM, and 90 mins for Col 1) time points as described in the Experimental Procedures. Cell attachment or spreading obtained at the late time point for cells treated with NS siRNA on integrin β1 substrates was taken as 100%. Bars, 20 µm.

Figure 4.

Silencing of TG2 delays or impairs the migration of RCC cell lines (A) Caki-2 (B) A-498, (C) Caki-1, and (D) ACHN in wound scratch assay. Percentage wound closure rate was expressed as the percentage of non-silencing RNA (NS) treated cells for each RCC cell line. Data values represent average of 3 independent experiments. Non-overlapping images of the whole wound area for each well were captured at the time of 0, 24 and 48 hours, as described in Experimental Procedures. A complete wound closure was detected for each cell line at 48 hours. Representative images were shown for each RCC cell line at the time of 0 and 48 hours after wounding. Bar, 500 µm.

Figure 5.

TG2 downregulation reduces the invasion of β1 integrin substrates by RCC cell lines. NS siRNA, siRNA 1 (siR1) and siRNA 6 (siR6) treated RCC cell lines seeded in AIM-V medium on (A) FN, (B) Col 1, and (C) LM coated transwells and allowed to migrate through β1 integrin substrates for indicated early (3 hours for FN, 6 hours for Col1 and 10 hours for LM) and late (6 hours for FN, 10 hours for Col1 and 14 hours for LM) time points as described under “Experimental Procedures.” Images of migrated cells were captured from 10 different non-overlapping areas for each point and analyzed using Scion Image analysis. Percentage of migrated cells was calculated by considering the number of control NS cells migrated at the late time point as 100%. The percentage of migrated cells ± _SD shown were from the mean of at least 3 independent experiments.

Figure 6.

Decrease in TG2 expression in Caki-1 cells results in the loss of cell surface CSC markers. (A) TG2 gene knockdown was achieved by using shRNA lentiviral particles and silencing efficiency was determined by Western bot using actin as the loading control protein. Flow cytometric analysis of CD markers on Caki-1 scrambled control (light-shaded histograms) and TG2-shRNA transduced cells (dark-shaded histograms). (B) Caki-1 scrambled cells incubated with isotype-matched FITC- and PE-IgG were used as negative control. Cells were either stained with FITC- or PE- conjugated (C) haematopoietic and (D) mesenchmal markers represented below x-axis as FL1 and FL2, respectively. Background fluorescence was detected with FITC- or PE-conjugated isotype control mouse IgG antibodies.