Metazoans express three unfolded protein response transducers (IRE1, PERK, and ATF6) ubiquitously to cope with endoplasmic reticulum (ER) stress. ATF6 is an ER membrane-bound transcription factor activated by ER stress-induced proteolysis and has been duplicated in mammals. Here, we generated ATF6alpha- and ATF6beta-knockout mice, which developed normally, and then found that their double knockout caused embryonic lethality. Analysis of mouse embryonic fibroblasts (MEFs) deficient in ATF6alpha or ATF6beta revealed that ATF6alpha is solely responsible for transcriptional induction of ER chaperones and that ATF6alpha heterodimerizes with XBP1 for the induction of ER-associated degradation components. ATF6alpha(-/-) MEFs are sensitive to ER stress. Unaltered responses observed in ATF6beta(-/-) MEFs indicate that ATF6beta is not a negative regulator of ATF6alpha. These results demonstrate that ATF6alpha functions as a critical regulator of ER quality control proteins in mammalian cells, in marked contrast to worm and fly cells in which IRE1 is responsible.

Author contributions K.Y. and A.V. performed the majority of experiments and wrote the manuscript. J.Y. assisted with cloning and performed the proximity biotinylation and ubiquitylation experiments. D.E.B. and A.S.W.S. assisted with animal studies. S.G. performed immunofluorescence and analysis of patient PDAC specimens. M.K. assisted with the analysis of flow cytometry data and RNA-seq data. S.M. assisted with immunoblotting and preparing shRNAs. E.Y.L. and S.J.P. cloned fluorescent constructs. K.W.W. and G.E.K. provided PDAC patient specimens and analysis. J.D. provided GFP-NBR1 and GFP-NBR1 dUBA constructs. R.S.B. assisted with transcriptome data analysis. J.D.M. and J.A.P. performed proteomics analysis. D.T.F. provided intellectual feedback and support. R.M.P. and A.C.K. conceived the project, supervised the research, and wrote and edited the paper.Competing interests A.C.K. has financial interests in Vescor Therapeutics, LLC. A.C.K. is an inventor on patents pertaining to KRAS regulated metabolic pathways, redox control pathways in pancreatic cancer, targeting GOT1 as a therapeutic approach, and the autophagic control of iron metabolism. A.

ATF6 and XBP1 are transcription factors activated specifically in response to endoplasmic reticulum (ER) stress. Three cis-acting elements capable of binding to ATF6, XBP1 or both have been identified to date, namely ER stress-response element (ERSE), unfolded protein response element (UPRE) and ERSE-II. ERSE controls the expression of ER-localized molecular chaperones such as BiP that can refold unfolded proteins in the ER; transcription from ERSE is fully activated by ATF6 even in the absence of XBP1. In contrast, transcription from UPRE depends solely on XBP1 and it has been suggested that UPRE may control the expression of components of the ER-associated degradation system that can degrade unfolded proteins in the ER. The Herp gene, one of the most highly inducible genes under ER stress, encodes an ER membrane protein containing a ubiquitin-like domain with unknown functions, and carries ERSE-II in addition to ERSE in its promoter. In this report, we show that ERSE-II allows the NF-Y-dependent binding of ATF6 as in the case of ERSE and NF-Y-independent binding of XBP1 as in the case of UPRE, and that transcription from ERSE-II is mitigated in the absence of XBP1. Accordingly, the induction of Herp mRNA was diminished in the absence of XBP1 whereas that of BiP mRNA was not affected. These results may help in understanding the role of Herp in the quality control system in the ER.

ABSTRACT. Eukaryotic cells cope with endoplasmic reticulum (ER) stress by activating the unfolded protein response (UPR), a coordinated system of transcriptional and translational controls, which ensures the integrity of synthesized proteins. Mammalian cells express three UPR transducers in the ER, namely IRE1, PERK and ATF6. The IRE1 pathway, which is conserved from yeast to humans, mediates transcriptional induction of not only ER quality control proteins (molecular chaperones, folding enzymes and components of ER-associated degradation) but also proteins working at various stages of secretion. The PERK pathway, conserved in metazoan cells, is responsible for translational control and also participates in transcriptional control in mammals. ATF6 is an ERmembrane-bound transcription factor activated by ER stress-induced proteolysis which consists of two closely related factors, ATF6α and ATF6β, in mammals. ATF6α but not ATF6β plays an important role in transcriptional control. In this study, we performed a genome-wide search for ATF6α-target genes in mice. Only 30 of the 14,729 analyzable genes were identified as specific targets, of which 40% were ER quality control proteins, 20% were ER proteins, while the rest had miscellaneous functions. The negative effects of the absence of PERK on transcriptional induction of ER quality control proteins could be explained by its inhibitory effect on ATF6α activation. Further, proteins involved in transport from the ER are not regulated by ATF6α, and transport of folded cargo molecules from the ER was not affected by the absence of ATF6α. Based on these results, we propose that ATF6 is a transcription factor specialized in the regulation of ER quality control proteins.

We burdened mice with intraperitoneal injection of the endoplasmic reticulum stress-inducing reagent tunicamycin, and found that wild-type mice were able to recover from the insult, whereas ATF6α-knockout mice exhibited liver dysfunction and steatosis. Our results establish links between endoplasmic reticulum stress, lipid metabolism and steatosis

Dysregulated DNA methylation followed by abnormal gene expression is an epigenetic hallmark in cancer. DNA methylation is catalyzed by DNA methyltransferases, and the aberrant expression or mutations of DNA methyltransferase genes are found in human neoplasm. The enzymes for demethylating 5-methylcytosine were recently identified, and the biological significance of DNA demethylation is a current focus of scientific attention in various research fields. Ten-eleven translocation (TET) proteins have an enzymatic activity for the conversion from 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC), which is an intermediate of DNA demethylation. The loss-of-function mutations of TET2 gene were reported in myeloid malignancies, suggesting that impaired TET-mediated DNA demethylation could play a crucial role in tumorigenesis. It is still unknown, however, whether DNA demethylation is involved in biological properties in solid cancers. Here, we show the loss of 5-hmC in a broad spectrum of solid tumors: for example, a significant reduction of 5-hmC was found in 72.7% of colorectal cancers (CRCs) and 75% of gastric cancers compared to background tissues. TET1 expression was decreased in half of CRCs, and a large part of them was followed by the loss of 5-hmC. These findings suggest that the amount of 5-hmC in tumors is often reduced via various mechanisms, including the downregulation of TET1. Consistently, in the in vitro experiments, the downregulation of TET1 was clearly induced by oncogene-dependent cellular transformation, and loss of 5-hmC was seen in the transformed cells. These results suggest the critical roles of aberrant DNA demethylation for oncogenic processes in solid tissues. (Cancer Sci 2012; 103: 670-676) P atterns of DNA methylation, histone modification and chromatin structure are profoundly altered in human cancers.(1-5) In particular, aberrant promoter hypermethylation leading to inappropriate transcriptional silencing of genes, especially tumor suppressor genes, is often found in various types of human neoplasm, including colorectal and gastric cancers.(6-9) DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and it is reported that the increased level of DNMT1 is correlated with the histological grade or poor prognosis of human cancers. (10)(11)(12) In addition, a recent report demonstrated somatic mutations in the DNMT3A gene from acute myeloid leukemia patients. (13) Global loss of methylated DNA in paternal genome after fertilization suggests active DNA demethylation pathway in mammalian cells, although the molecular mechanism has been unknown for a long time. The recent discovery of ten-eleven translocation (TET) proteins those are capable of converting from 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) gave a breakthrough to the epigenetic research field. (14)(15)(16)(17)(18)(19) Following studies showed that the activationinduced cytidine deaminase family convert cytosine to uracil and 5-hmC to 5-hydroxymethyluracil, (20,21) and that TET1 mediates further oxidation of 5-h...

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Background:The renin–angiotensin system (RAS) is thought to have a role in carcinogenesis, and RAS inhibition may prevent tumour growth.Methods:We retrospectively investigated the impact of angiotensin I-converting enzyme inhibitors (ACEIs) and angiotensin II type-1 receptor blockers (ARBs) in 155 patients with pancreatic cancer receiving gemcitabine monotherapy. Patients were divided into three groups: the ACEI/ARB group (27 patients receiving an ACEI or ARB for hypertension (HT)), the non-ACEI/ARB with HT group (25 patients receiving antihypertensive drugs other than ACEIs or ARBs), and the non-HT group (103 patients receiving no antihypertensive drugs).Results:Patient characteristics were not different, except for age and HT medications. Progression-free survival (PFS) was 8.7 months in the ACEI/ARB group, 4.5 months in the non-ACEI/ARB with HT group, and 3.6 months in the non-HT group. Overall survival (OS) was 15.1 months in the ACEI/ARB group, 8.9 months in the non-ACEI/ARB with HT group, and 9.5 months in the non-HT group. The use of ACEIs/ARBs was a significant prognostic factor for both PFS (P=0.032) and OS (P=0.014) in the multivariate analysis.Conclusions:The ACEIs/ARBs in combination with gemcitabine might improve clinical outcomes in patients with advanced pancreatic cancer. Prospective trials are needed to test this hypothesis.