Transglutaminase 2 Promotes Autophagy by LC3 Induction through p53 Depletion in Cancer Cell

Abstract

Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.

Keywords: Autophagy; Cancer cell; LC3; Transglutaminase 2; p53.

Fig. 1.

TGase 2 regulates autophagy in cancer cells. (A) Immunoblottings of autophagy markers in cancer cells showed TGase 2 is associated with autophagy in cancer. CAKI-1 and A549 cells were transfected with siRNA of TGase 2 for 24 h while MCF7 and HCT116 cells were transfected with HA-TGase 2 plasmid for 24 h. Before harvest cells, cells were treated with chloroquine (CQ, 50 µM) for 6 h under amino acid deprivation for 4 h. Whole-cell lysates were subjected to the immunoblotting with indicated antibodies. (B) Autophagy activity was regulated by TGase 2 in cancer cells. Under the same conditions as above (A), cells were stained with CYTO-ID green detection reagent for autophagy activity analysis. Fluorescence was measured at wavelength 488 nM. (C) Confocal microscopy images showed that induction of LC3 (green) is regulated by TGase 2 expression in A549 and MCF7. (D) TGase 2 knock down reduced autophagy in A549. GFP-LC3 stable expressed A549 cells were transfected with TGase 2 siRNA for 24 h. (E) TORIN-2 induced autophagy was significantly reduced by TGase 2 siRNA treatment. GFP-LC3 puncta were analyzed by live imaging using confocal microcopy. Cells were counterstained with DAPI to visualize the nuclei. Cumulative data from three independent experiments is shown here as mean ± SD (n=3). ^***p<0.001, Scale bar: 20 µm.

Fig. 2.

Inhibition of TGase 2 induces p53 stability. (A) Before harvest the samples, CAKI-1 and A549 cells were treated with GK921 (0, 250, 500, 1000 nM) for 8h, overlapped with chloroquine treatment (CQ, 50 µM) for 6 h under amino acid deprivation for 4 h. Whole-cell lysates were subjected to the immunoblotting with indicated antibodies. (B) Under the same condition as above, cells were stained with CYTPO-ID green detection reagent for autophagy activity. (C) Cells with the low level of TGase 2 such as HCT116 and MCF7 or TGase 2 transfected cells such as HCT116^TG2 and MCF7^TG2 cells were treated with GK921 (0, 250, 500, 1000 nM) for 8h, under the same condition as above. Whole-cell lysates were subjected to immunoblotting of TGase 2 as well as measuring autophagy activity using CYTO-ID green detection reagent. Cumulative data from three independent experiments is shown here as mean ± SD (n=3). ^***p<0.001.

Fig. 3.

LC transcription is suppressed by p53 in cancer cells. (A) p53 transfection suppressed LC3 expression. CAKI-1 and A549 cells were transfected with 3xFLAG p53 (p53 over-expression vectors: 0, 1, 2, 4 µg) for 24 h, and treated with chloroquine (50 µM) for 6 h under amino acid deprivation for 4 h. qRT-PCRs against p53, LC3 and β-actin were performed as described in the methods. (B) p53 knock down increased LC3 expression. MCF7 and HCT116 cells were transfected with siRNA of p53 (0, 20, 40 nM) for 24 h, and treated with chloroquine (50 µM) for 6 h. Real-time PCR against p53, LC3 and β-actin were performed as described in the methods. (C) TGase 2 knock down decreased LC3 expression. CAKI-1 and A549 cells were transfected with TGase 2 siRNA for 24 h in a dose dependent manner (0, 10, 20, 40 nM) (D) TGase 2 transfection increased LC3 expression. MCF7 and HCT116 cells were transfected with HA-TGase2 in a dose dependent manner (0, 1, 2, 4 µg). (E) TGase 2 knock down or inhibition increased p53 level. CAKI-1 and A549 cells were transfected with siRNA of TGase 2 or treated with GK921 (1 µM). (F) TGase 2 transfection decreased p53, which was reversed by TGase 2 inhibition. MCF7 and HCT116 cells were transfected with HA-TGase 2 plasmid alone or combined with GK921 (1 µM).

Fig. 4.

TGase 2 inhibition or TGase 2 knock down reversed rapamycin resistance in cancer cells. (A) TGase 2 inhibition reversed rapamycin induced autophagy in cancer cells which potentiated anti-cancer effect with rapamycin. CAKI-1 and A549 cell were treated with GK921 or Rapamycin and combination for 24h with chloroquine (50 µM) for 6 h under amino acid deprivation for 4 h. (B) TGase 2 knock down reversed rapamycin induced autophagy in cancer cells which potentiated anti-cancer effect with rapamycin. Under the same condition of (A), these cells were treated with TGase 2 siRNA or rapamycin and combination of TGase 2 siRNA and rapamycin. (C) XTT assay showed that TGase 2 inhibition reversed rapamycin resistance. Rapamycin resistant cancer cells of MCF7^RR and HCT116^RR were cultured with Rapamycin (20 µM). The cells were treated with TGase 2 inhibitor GK921, rapamycin and combination of GK921 and rapamycin for 24 h. (D) XTT assay showed that TGase 2 knock down reversed rapamycin resistance. Rapamycin resistant cancer cells of MCF7^RR and HCT116^RR were treated with siRNA of TGase 2, rapamycin and combination of siRNA of TGase 2 and rapamycin for 24 h. Cumulative data from three independent experiments is shown here as mean ± SD (n=3). ^**p<0.01, ^***p<0.001. (E) TGase 2 was increased in the drug resistant cancer cells analyzed by immunoblotting of TGase 2. Rapamycin resistant cancer cells such as MCF7^RR and HCT116^RR showed higher expression level of TGase 2 to compare to rapamycin sensitive cancer cells such as MCF7 and HCT116. (F) Summary of TGase 2 mediated regulation of autophagy.