Tissue transglutaminase (TG2) constitutively suppresses autophagy in pancreatic cancer cells

Abstract

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Elevated expression of tissue transglutaminase (TG2) in cancer cells has been implicated in the development of drug resistance and metastatic phenotypes. However, the role and the mechanisms that regulate TG2 expression remain elusive. Here we provide evidence that Protein Kinase C-delta (PKCδ) regulates TG2 expression, which in turn inhibits autophagy, a type II programmed cell death, in pancreatic cancer cells that are frequently insensitive to standard chemotherapeutic agents. Rottlerin, a PKCδ-specific inhibitor, downregulated the expression of TG2 mRNA and protein and induced growth inhibition without inducing apoptosis in pancreatic cancer cells. No sign of apoptosis was detected by Annexin V, PARP cleavege and PI staining (lack of sub-G1 cell population) assays. Inhibition of PKCδ by rottlerin or knock-down of TG2 protein by a TG2-specific siRNA resulted in a marked increase in autophagy demonstrated by presence of autophagic vacuoles in the cytoplasm detected by phase contrast microscopy and tranmission electron microscopy, formation of the acidic vesicular organelles by acridine orange staining, and a marked induction membrane association of microtubule-associated protein light chain 3 (LC3)-II by Western blotting, important hallmarks of autophagy. Furthermore, inhibition of TG2 by Rottlerin or by the siRNA led to accumulation of green flourescent protein taged LC3-II (GFP-LC3-II) in autophagosomes in pancreatic cancer cells transfected with GFP-LC-3(GFP-ATG8) expression vector. Treatment of TG2 negative cells with rottlerin under optimal conditions failed to produce any increase in LC3-II accumulation, indicating that the induction of autophagy by Rottlerin involved TG2. Knockdown of Beclin-1, a specific autophagy promoting protein and the product of Becn1 (ATG6), inhibited rottlerin- and TG2 siRNA-induced autophagy, indicating that Beclin-1 is required for the process. A specific mTOR inhibitor, rapamycin, failed to induce autophagy in these cells, indicating that rottlerin-induced autophagy is not mediated by mTOR. These results revealed that PKCdelta plays a critical role in the expression of TG2, which in turn regulates autophagy. In conclusion, these results suggest a novel mechanism of regulation of TG2 and autophagy in pancreatic cancer cells.