Abstract
Glycogen synthase kinase-3 (GSK3) are ubiquitously expressed serine-threonine kinases involved in a plethora of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK3 inhibition triggers JNK-cJUN-dependent apoptosis in human pancreatic cancer cells. However, the comprehensive picture of downstream GSK3-regulated pathways/functions remains elusive. Herein, counterbalancing the death signals, we show that GSK3 inhibition induces prosurvival signals through increased activity of the autophagy/lysosomal network. Our data also reveal a contribution of GSK3 in the regulation of the master transcriptional regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB) in pancreatic cancer cells. Similarly to mammalian target of rapamycin (mTOR) inhibition, GSK3 inhibitors promote TFEB nuclear localization and leads to TFEB dephosphorylation through endogenous serine/threonine phosphatase action. However, GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases. Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. Quantitative mass spectrometry analyses further reveal an increased association of TFEB with nuclear proteins upon GSK3 and mTOR inhibition suggesting a positive impact on TFEB transcriptional function. Finally, a predominant nuclear localization of TFEB is unveiled in fully fed pancreatic cancer cells, whereas a reduction in TFEB expression significantly impairs their capacity for growth in an anchorage-independent manner. In addition, TFEB-restricted cells are more sensitive to apoptosis upon GSK3 inhibition. Altogether, our data uncover new functions under the control of GSK3 in pancreatic cancer cells in addition to providing key insight into TFEB regulation.
Highlights
Glycogen synthase kinase-3 (GSK3) inhibition promotes apoptosis of pancreatic cancer cells
Inhibition of autophagosome degradation by chloroquine further enhanced light chain 3B II (LC3B II) expression upon treatment with GSK3 inhibitor (Fig. 1B) or in cells transduced with a short hairpin RNA (shRNA) targeting GSK3 (Fig. 1C) suggesting that GSK3 inhibition promotes an autophagic response in pancreatic cancer cells
LC3B puncta were detected in DMSO-treated pancreatic cancer cells suggestive of a high degree of autophagy in these cells. This observation is in agreement with a previous report demonstrating an elevated level of autophagy in human pancreatic cancer cell lines that is required for their growth (26)
Summary
Glycogen synthase kinase-3 (GSK3) inhibition promotes apoptosis of pancreatic cancer cells. GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. We and others have previously demonstrated that inhibition of glycogen synthase kinase-3 (GSK3) impairs pancreatic cancer cell growth (5–10) providing a rationale for assessing the potential clinical utility of GSK3 inhibitors in PDAC patients (11). While inducing JNK-dependent apoptotic markers (8), GSK3 inhibition was found to promote a distinct autophagic response independently of the JNK-cJUN pathway Preventing this autophagic response resulted in sensitization of cells to apoptosis suggesting a prosurvival role for autophagy upon GSK3 inhibition. TFEB-depleted pancreatic cancer cells displayed increased sensitivity to apoptosis upon treatment with GSK3 inhibitors providing support for a role for TFEB in the prosurvival signals induced by GSK3 inhibitors
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