Abstract
Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary and immortalized mouse PaSC, we assess the relative contributions of AMPK/mTOR signaling, autophagy and the UPR to cell fate responses during metabolic stress induced by mitochondrial dysfunction. The mitochondrial uncoupler rottlerin at low doses (0.5–2.5 μM) was added to cells cultured in 10% FBS complete media. Mitochondria rapidly depolarized, followed by altered mitochondrial dynamics and decreased cellular ATP levels. This mitochondrial dysfunction elicited rapid, sustained AMPK activation, mTOR pathway inhibition, and blockade of autophagic flux. Rottlerin treatment also induced rapid, sustained PERK/CHOP UPR signaling. Subsequently, high doses (>5 μM) induced loss of cell viability and cell death. Interestingly, AMPK knock-down using siRNA did not prevent rottlerin-induced mTOR inhibition, autophagy, or CHOP upregulation, suggesting that AMPK is dispensable for these responses. Moreover, CHOP genetic deletion, but not AMPK knock-down, prevented rottlerin-induced apoptosis and supported cell survival, suggesting that UPR signaling is a major modulator of cell fate in PaSC during metabolic stress. Further, short-term rottlerin treatment reduced both PaSC fibrogenic potential and IL-6 mRNA expression. In contrast, expression levels of the angiogenic factors HGF and VEGFα were unaffected, and the immune modulator IL-4 was markedly upregulated. These data imply that metabolic stress-induced PaSC reprogramming differentially modulates neighboring cells in the tumor microenvironment.
Highlights
Activated pancreatic stellate cells (PaSC) are the main cell type in the stroma of chronic pancreatitis and pancreatic cancer and participate in the progression of these disorders [1, 2]
To modulate cellular energy status, we treated mouse PaSC grown in 10% FBS media with the mitochondrial uncoupler, rottlerin
We and others previously showed that rottlerin, at concentrations ranging from 2.5 to 10 μM leads to rapid mitochondrial membrane depolarization in pancreatic cancer cells [24] and other cell types [27]
Summary
Activated pancreatic stellate cells (PaSC) are the main cell type in the stroma of chronic pancreatitis and pancreatic cancer and participate in the progression of these disorders [1, 2]. Stellate cell activation is accompanied by rapid cell growth, proliferation, and expansion of the mitochondria and endoplasmic reticulum (ER) networks to meet the bioenergetic and biosynthetic demands of the newly acquired secretory phenotype [1] These activities are supported by a balance between PI3K/AKT/mTOR signaling and autophagy to cope with a high demand for energy [2, 7, 8]. Autophagy-deficient hepatic stellate cells failed to acquire the activated state and displayed a reduced secretory phenotype [8] These data suggested that autophagy may modulate PaSC remodeling in the progression from a quiescent to an activated phenotype, and/or favor conversion to a secretory phenotype. Recent data indicate that mTOR and autophagy are key regulators of cellular reprogramming [11] and the hypersecretory phenotype of senescent cells [11, 12], supporting a role for these cellular programs in PaSC reprogramming
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