Autophagy is a conserved cellular process that functions as a first-line defense to restrict the growth of invading parasitic bacteria. As an intracellular pathogen, Salmonella (S) Typhimurium invades host cells through two Type III secretion systems (T3SS) and resides in the Salmonella-containing vacuole (SCV). When the SCV membrane is perforated and ruptured by T3SS-1, a small portion of the Salmonella egresses from the SCV and replicates rapidly in the nutrient-rich cytosol. Cytosolic Salmonella and those residing in the membrane-damaged SCV are tagged by ubiquitination and marked for autophagy through the ubiquitin-binding adaptor proteins such as p62, NDP52, and optineurin. Prior studies suggest that transient intracellular amino-acid starvation and subsequent inactivation of the mechanistic target of rapamycin (mTOR), a key molecule that phosphorylates Unc-51 like autophagy activating kinase (ULK1) and inhibits its activity, can trigger autophagy in S. Typhimurium-infected cells. Other studies suggest that energy stress in S. Typhimurium-infected cells leads to AMP-activated protein kinase (AMPK) activation and autophagy. In the present study, we report that autophagy was rapidly induced in S. Typhimurium-infected cells, as evidenced by increased LC3 lipidation and decreased p62 levels. However, S. Typhimurium infection drastically increased AKT phosphorylation but decreased S6K1^T389, 4E-BP^T37/46, and ULK1^S757 phosphorylation, suggesting that mTOR activation by AKT is subverted. Further studies showed that AMPK was activated in S. Typhimurium-infected cells, as evidenced by increased ULK1^S317 and ACC^S79 phosphorylation. AMPK activation was mediated by Toll-like receptor-activated TAK1. Functional studies revealed that AMPK and TAK1 inhibitors accelerated S. Typhimurium growth in HeLa cells. Our results strongly suggest that TAK1 activation leads to AMPK activation, which activates ULK1 by phosphorylating ULK1^S317 and suppressing mTOR activity and ULK1^S757 phosphorylation. Our study has unveiled a previously unrecognized pathway for S. Typhimurium-induced autophagy.

Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1^S757phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPK^T172and ULK1^S555phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1^G93Aprotein aggregates with autophagosomes and accelerated SOD1^G93Aprotein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.

The sonic hedgehog (Shh) pathway plays important roles in tumorigenesis, tumor growth, drug resistance, and metastasis. We and others have reported earlier that this pathway is highly activated in thyroid cancer. However, its role in thyroid cancer stem cell (CSC) self-renewal and tumor development remains incompletely understood. B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and SRY-Box Transcription Factor 2 (SOX2) are two CSC-related transcription factors that have been implicated in promoting CSC self-renewal. The objective of our current investigation was to determine the role of the Shh pathway in regulating BMI1 and SOX2 expression in thyroid cancer and promoting thyroid tumor growth and development. Here we report that inhibition of the Shh pathway by Gli1 siRNA or by cyclopamine and GANT61 reduced BMI1 and SOX2 expression in SW1736 and KAT-18 cells, two anaplastic thyroid cancer cell lines. The opposite results were obtained in cells overexpressing Gli1 or its downstream transcription factor Snail. The Shh pathway regulated SOX2 and BMI1 expression at a transcriptional and post-transcriptional level, respectively. GANT61 treatment suppressed the growth of SW1736 CSC-derived tumor xenografts but did not significantly inhibit the growth of tumors grown from bulk tumor cells. Clinicopathological analyses of thyroid tumor specimens by immunohistochemical (IHC) staining revealed that BMI1 and SOX2 were highly expressed in thyroid cancer and correlated with Gli1 expression. Our study provides evidence that activation of the Shh pathway leads to increased BMI1 and SOX2 expression in thyroid cancer and promotes thyroid CSC-driven tumor initiation. Targeting the Shh pathway may have therapeutic value for treating thyroid cancer and preventing recurrence.