Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC– is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC– subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

ObjectivePancreatic ductal adenocarcinoma (PDA) has among the highest stromal fractions of any cancer and this has complicated attempts at expression-based molecular classification. The goal of this work is to profile purified samples of human PDA epithelium and stroma and examine their respective contributions to gene expression in bulk PDA samples.DesignWe used laser capture microdissection (LCM) and RNA sequencing to profile the expression of 60 matched pairs of human PDA malignant epithelium and stroma samples. We then used these data to train a computational model that allowed us to infer tissue composition and generate virtual compartment-specific expression profiles from bulk gene expression cohorts.ResultsOur analysis found significant variation in the tissue composition of pancreatic tumours from different public cohorts. Computational removal of stromal gene expression resulted in the reclassification of some tumours, reconciling functional differences between different cohorts. Furthermore, we established a novel classification signature from a total of 110 purified human PDA stroma samples, finding two groups that differ in the extracellular matrix-associated and immune-associated processes. Lastly, a systematic evaluation of cross-compartment subtypes spanning four patient cohorts indicated partial dependence between epithelial and stromal molecular subtypes.ConclusionOur findings add clarity to the nature and number of molecular subtypes in PDA, expand our understanding of global transcriptional programmes in the stroma and harmonise the results of molecular subtyping efforts across independent cohorts.

Background: TatA, TatB, and TatC are subunits of the Tat translocase allowing transport of folded pre-proteins across cellular membranes Results: We identified TatC sites that interact with pre-proteins, TatA, TatB, and TatC Conclusion: The cytosolic N terminus and first cytosolic TatC loop constitute part of a twin arginine recognition site Significance: We developed a working model of how twin arginine pre-protein inserts into Tat translocase.

A priority in cancer research is to innovate therapies that are not only effective against tumor progression but also address comorbidities such as cachexia that limit quality and quantity of life. We demonstrate that TLR7/8 agonist R848 induces anti-tumor responses and attenuates cachexia in murine models of pancreatic ductal adenocarcinoma (PDAC). In vivo, tumors from two of three cell lines were R848-sensitive, resulting in smaller tumor mass, increased immune complexity, increased CD8 + T-cell infiltration and activity, and decreased Treg frequency. R848-treated mice demonstrated improvements in behavioral and molecular cachexia manifestations, resulting in a near-doubling of survival duration. Knockout mouse studies revealed that stromal, not neoplastic, TLR7 is requisite for R848-mediated responses. In patient samples, we found Tlr7 is ubiquitously expressed in stroma across all stages of pancreatic neoplasia, but epithelial Tlr7 expression is relatively uncommon. These studies indicate immune-enhancing approaches including R848 may be useful in PDAC and cancerassociated cachexia.

The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Consistent with previous in vivo data, we show that the model Tat substrate TorA-PhoA is translocated by the TatABC translocase of Escherichia coli inner membrane vesicles, only if the PhoA moiety was allowed to fold by disulfide bond formation. Although even unfolded TorA-PhoA was found to physically associate with the Tat translocase of the vesicles, site-specific cross-linking revealed a perturbed interaction of the signal sequence of unfolded TorA-PhoA with the TatBC receptor site. Some of the folded TorA-PhoA precursor accumulated in a partially protease-protected membrane environment, from where it could be translocated into the lumen of the vesicles upon re-installation of an H ؉ -gradient. Translocation arrest occurred in immediate vicinity to TatA. Consistent with a neighborhood to TatA, TorA-PhoA remained protease-resistant in the presence of detergents that are known to preserve the oligomeric structures of TatA. Moreover, entry of TorA-PhoA to the protease-protected environment strictly required the presence of TatA. Collectively, our results are consistent with some degree of quality control by TatBC and a recruitment of TatA to a folded substrate that has functionally engaged the twin-arginine translocase.

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The TatABC subunits of the twin-arginine translocation machinery allow transport of folded proteins by an unknown mechanism. Here we show that the entire surfaces of folded Tat substrates contact TatB via both of its predicted helices. Our data suggest that TatB forms an oligomeric binding site that transiently accommodates folded Tat precursors.

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