Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic, aggressive cancer that frequently progresses and spreads by metastasis to the liver1. Cancer-associated fibroblasts, the extracellular matrix and type I collagen (Col I) support2,3 or restrain the progression of PDAC and may impede blood supply and nutrient availability4. The dichotomous role of the stroma in PDAC, and the mechanisms through which it influences patient survival and enables desmoplastic cancers to escape nutrient limitation, remain poorly understood. Here we show that matrix-metalloprotease-cleaved Col I (cCol I) and intact Col I (iCol I) exert opposing effects on PDAC bioenergetics, macropinocytosis, tumour growth and metastasis. Whereas cCol I activates discoidin domain receptor 1 (DDR1)-NF-κB-p62-NRF2 signalling to promote the growth of PDAC, iCol I triggers the degradation of DDR1 and restrains the growth of PDAC. Patients whose tumours are enriched for iCol I and express low levels of DDR1 and NRF2 have improved median survival compared to those whose tumours have high levels of cCol I, DDR1 and NRF2. Inhibition of the DDR1-stimulated expression of NF-κB or mitochondrial biogenesis blocks tumorigenesis in wild-type mice, but not in mice that express MMP-resistant Col I. The diverse effects of the tumour stroma on the growth and metastasis of PDAC and on the survival of patients are mediated through the Col I-DDR1-NF-κB-NRF2 mitochondrial biogenesis pathway, and targeting components of this pathway could provide therapeutic opportunities.

ATF4 suppresses hepatocarcinogenesis by inducing SLC7A11 (xCT) to block stress-related ferroptosis.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC), a leading cause of cancer-related death, is associated with viral hepatitis, non-alcoholic steatohepatitis (NASH), and alcohol-related steatohepatitis, all of which trigger endoplasmic reticulum (ER) stress, hepatocyte death, inflammation, and compensatory proliferation. Using ER stress-prone MUP-uPA mice, we established that ER stress and hypernutrition cooperate to cause NASH and HCC, but the contribution of individual stress effectors, such as activating transcription factor 4 (ATF4), to HCC and their underlying mechanisms of action remained unknown. METHODS: Hepatocyte-specific ATF4-deficient MUP-uPA mice (MUP-uPA/Atf4Δhep) and control MUP-uPA/Atf4F/F mice were fed a high-fat diet to induce NASH-related HCC, and Atf4F/F and Atf4Δhep mice were injected with diethylnitrosamine to model carcinogen-induced HCC. Histological, biochemical, and RNA-sequencing analyses were performed to identify and define the role of ATF4-induced solute carrier family 7a member 11 (SLC7A11) expression in hepatocarcinogenesis. Reconstitution of SLC7A11 in ATF4-deficient primary hepatocytes and mouse livers was used to study its effects on ferroptosis and HCC development. RESULTS: Hepatocyte ATF4 ablation inhibited hepatic steatosis, but increased susceptibility to ferroptosis, resulting in accelerated HCC development. Although ATF4 activates numerous genes, ferroptosis susceptibility and hepatocarcinogenesis were reversed by ectopic expression of a single ATF4 target, Slc7a11, coding for a subunit of the cystine/glutamate antiporter xCT, which is needed for glutathione synthesis. A ferroptosis inhibitor also reduced liver damage and inflammation. ATF4 and SLC7A11 amounts were positively correlated in human HCC and livers of patients with NASH. CONCLUSIONS: Despite ATF4 being upregulated in established HCC, it serves an important protective function in normal hepatocytes. By maintaining glutathione production, ATF4 inhibits ferroptosis-dependent inflammatory cell death, which is known to promote compensatory proliferation and hepatocarcinogenesis. Ferroptosis inhibitors or ATF4 activators may also blunt HCC onset. IMPACT AND IMPLICATIONS: Liver cancer or hepatocellular carcinoma (HCC) is associated with multiple aetiologies. Most HCC aetiologies cause hepatocyte stress and death, as well as subsequent inflammation, and compensatory proliferation, thereby accelerating HCCdevelopment. The contribution of individual stress effectors to HCC and their underlying mechanisms of action were heretofore unknown. This study shows that the stress-responsive transcription factor ATF4 blunts liver damage and cancer development by suppressing iron-dependent cell death (ferroptosis). Although ATF4 ablation prevents hepatic steatosis, it also increases susceptibility to ferroptosis, due to decreased expression of the cystine/glutamate antiporter SLC7A11, whose expression in human HCC and NASH correlates with ATF4. These findings reinforce the notion that benign steatosis may be protective and does not increase cancer risk unless accompanied by stress-induced liver damage. These results have important implications for prevention of liver damage and cancer.

Pericentral hepatocytes produce insulin-like growth factor-2 to promote liver regeneration during selected injuries in mice.

Liver regeneration (LR) happens after various types of injuries. Unlike the well-studied LR caused by partial hepatectomy (PHx), there is accumulating evidence suggesting that LR during other injuries may result from unknown mechanisms. In this study, we found that insulin-like growth factor 2 (IGF-2) was drastically induced following the liver injuries caused by tyrosinemia or long-term treatments of CCl4 . However, this was not observed during the early phase of acute liver injuries after PHx or single treatment of CCl4 . Remarkably, most IGF-2-expressing hepatocytes were located at the histological area around the central vein of the liver lobule after the liver injuries caused either in fumarylacetoacetate hydrolase-deficient mice or in CCl4 chronically treated mice. Hepatocyte proliferation in vivo was significantly promoted by induced IGF-2 overexpression, which could be inhibited by adeno-associated virus-delivered IGF-2 short hairpin RNAs or linsitinib, an inhibitor of IGF-2 signaling. Proliferating hepatocytes in vivo responded to IGF-2 through both insulin receptor and IGF-1 receptor. IGF-2 also significantly promoted DNA synthesis of primary hepatocytes in vitro. More interestingly, the significantly induced IGF-2 was also found to colocalize with glutamine synthetase in the region enriched with proliferating hepatocytes for the liver samples from patients with liver fibrosis. CONCLUSION: IGF-2 is produced by pericentral hepatocytes to promote hepatocyte proliferation and repair tissue damage in the setting of chronic liver injury, which is distinct from the signaling that occurs post-PHx. (Hepatology 2017;66:2002-2015).

Structures, microfabrics and textures of the Cordilleran-type Rechnitz metamorphic core complex, Eastern Alps.

Rechnitz window group represents a Cordilleran-style metamorphic core complex, which is almost entirely located within nearly contemporaneous Neogene sediments at the transition zone between the Eastern Alps and the Neogene Pannonian basin. Two tectonic units are distinguished within the Rechnitz metamorphic core complex (RMCC): (1) a lower unit mainly composed of Mesozoic metasediments, and (2) an upper unit mainly composed of ophiolite remnants. Both units are metamorphosed within greenschist facies conditions during earliest Miocene followed by exhumation and cooling. The internal structure of the RMCC is characterized by the following succession of structure-forming events: (1) blueschist relics of Paleocene/Eocene age formed as a result of subduction (D1), (2) ductile nappe stacking (D2) of an ophiolite nappe over a distant passive margin succession (ca. E-W to WNW-ESE oriented stretching lineation), (3) greenschist facies-grade metamorphism annealing dominant in the lower unit, and (4) ductile low-angle normal faulting (D3) (with mainly NE-SW oriented stretching lineation), and (5) ca. E to NE-vergent folding (D4). The microfabrics are related to mostly ductile nappe stacking to ductile low-angle normal faulting. Paleopiezometry in conjunction with P-T estimates yield high strain rates of 10- 11 to 10- 13 s- 1, depending on the temperature (400-350 °C) and choice of piezometer and flow law calibration. Progressive microstructures and texture analysis indicate an overprint of the high-temperature fabrics (D2) by the low-temperature deformation (D3). Phengitic mica from the Paleocene/Eocene high-pressure metamorphism remained stable during D2 ductile deformation as well as preserved within late stages of final sub-greenschist facies shearing. Chlorite geothermometry yields two temperature groups, 376-328 °C, and 306-132 °C. Chlorite is seemingly accessible to late-stage resetting. The RMCC underwent an earlier large-scale coaxial deformation accommodated by a late non-coaxial shear with ductile low-angle normal faulting, resulting in subvertical thinning in the extensional deformation regime. The RMCC was rapidly exhumed during ca. 23-18 Ma.

The lateral boundary of a metamorphic core complex: The Moutsounas shear zone on Naxos, Cyclades, Greece.

We describe the structure, microstructures, texture and paleopiezometry of quartz-rich phyllites and marbles along N-trending Moutsounas shear zone at the eastern margin of the Naxos metamorphic core complex (MCC). Fabrics consistently indicate a top-to-the-NNE non-coaxial shear and formed during the main stage of updoming and exhumation between ca. 14 and 11 Ma of the Naxos MCC. The main stage of exhumation postdates the deposition of overlying Miocene sedimentary successions and predates the overlying Upper Miocene/Pliocene conglomerates. Detailed microstructural and textural analysis reveals that the movement along the Moutsounas shear zone is associated with a retrograde greenschist to subgreenschist facies overprint of the early higher-temperature rocks. Paleopiezometry on recrystallized quartz and calcite yields differential stresses of 20-77 MPa and a strain rate of 10-15-10-13 s-1 at 350 °C for quartz and ca. 300 °C for calcite. Chlorite geothermometry of the shear zone yields two temperature regimes, 300-360 °C, and 200-250 °C. The lower temperature group is interpreted to result from late-stage hydrothermal overprint.

Photo-aged non-biodegradable and biodegradable mulching film microplastics alter the interfacial behaviors between agricultural soil and inorganic arsenic.

The impacts of microplastics (MPs) prevalent in soil on the transport of pollutants were urged to be addressed, which has important implications for ecological risk assessment. Therefore, we investigated the influence of virgin/photo-aged biodegradable polylactic acid (PLA) and non-biodegradable black polyethylene (BPE) mulching films MPs on arsenic (As) transport behaviors in agricultural soil. Results showed that both virgin PLA (VPLA) and aged PLA (APLA) enhanced the adsorption of As(Ⅲ) (9.5%, 13.3%) and As(Ⅴ) (22.0%, 6.8%) due to the formation of abundant H-bonds. Conversely, virgin BPE (VBPE) reduced the adsorption of As(Ⅲ) (11.0%) and As(Ⅴ) (7.4%) in soil owing to the "dilution effect", while aged BPE (ABPE) improved arsenic adsorption amount to the level of pure soil due to newly generated O-containing functional groups being feasible to form H-bonds with arsenic. Site energy distribution analysis indicated that the dominant adsorption mechanism of arsenic, chemisorption, was not impacted by MPs. The occurrence of biodegradable VPLA/APLA MPs rather than non-biodegradable VBPE/ABPE MPs resulted in an increased risk of soil accumulating As(Ⅲ) (moderate) and As(Ⅴ) (considerable). This work uncovers the role of biodegradable/non-biodegradable mulching film MPs in arsenic migration and potential risks in the soil ecosystem, depending on the types and aging of MPs.

Effects of Early Life Oral Arsenic Exposure on Intestinal Tract Development and Lipid Homeostasis in Neonatal Mice: Implications for NAFLD Development.

BACKGROUND: Newborns can be exposed to inorganic arsenic (iAs) through contaminated drinking water, formula, and other infant foods. Epidemiological studies have demonstrated a positive association between urinary iAs levels and the risk of developing nonalcoholic fatty liver disease (NAFLD) among U.S. adolescents and adults. OBJECTIVES: The present study examined how oral iAs administration to neonatal mice impacts the intestinal tract, which acts as an early mediator for NAFLD. METHODS: Neonatal mice were treated with a single dose of iAs via oral gavage. Effects on the small intestine were determined by histological examination, RNA sequencing, and biochemical analysis. Serum lipid profiling was analyzed by fast protein liquid chromatography (FPLC), and hepatosteatosis was characterized histologically and biochemically. Liver X receptor-alpha (LXRα) knockout (Lxrα-/-) mice and liver-specific activating transcription factor 4 (ATF4)-deficient (Atf4ΔHep) mice were used to define their roles in iAs-induced effects during the neonatal stage. RESULTS: Neonatal mice exposed to iAs via oral gavage exhibited accumulation of dietary fat in enterocytes, with higher levels of enterocyte triglycerides and free fatty acids. These mice also showed accelerated enterocyte maturation and a longer small intestine. This was accompanied by higher levels of liver-derived very low-density lipoprotein and low-density lipoprotein triglycerides, and a lower level of high-density lipoprotein cholesterol in the serum. Mice exposed during the neonatal period to oral iAs also developed hepatosteatosis. Compared with the control group, iAs-induced fat accumulation in enterocytes became more significant in neonatal Lxrα-/- mice, accompanied by accelerated intestinal growth, hypertriglyceridemia, and hepatosteatosis. In contrast, regardless of enterocyte fat accumulation, hepatosteatosis was largely reduced in iAs-treated neonatal Atf4ΔHep mice. CONCLUSION: Exposure to iAs in neonatal mice resulted in excessive accumulation of fat in enterocytes, disrupting lipid homeostasis in the serum and liver, revealing the importance of the gut-liver axis and endoplasmic reticulum stress in mediating iAs-induced NAFLD at an early age. https://doi.org/10.1289/EHP12381.

Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness.

Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is unclear. Fructose-1,6-bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis, and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin dependent. Independently of its catalytic activity, FBP1 prevents insulin hyperresponsiveness by forming a stable complex with AKT, PP2A-C, and aldolase B (ALDOB), which specifically accelerates AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:PP2A-C:ALDOB:AKT complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, an FBP1-derived complex disrupting peptide reverses diet-induced insulin resistance.

PIDDosome-SCAP crosstalk controls high-fructose-diet-dependent transition from simple steatosis to steatohepatitis.

Sterol deficiency triggers SCAP-mediated SREBP activation, whereas hypernutrition together with ER stress activates SREBP1/2 via caspase-2. Whether these pathways interact and how they are selectively activated by different dietary cues are unknown. Here, we reveal regulatory crosstalk between the two pathways that controls the transition from hepatosteatosis to steatohepatitis. Hepatic ER stress elicited by NASH-inducing diets activates IRE1 and induces expression of the PIDDosome subunits caspase-2, RAIDD, and PIDD1, along with INSIG2, an inhibitor of SCAP-dependent SREBP activation. PIDDosome assembly activates caspase-2 and sustains IRE1 activation. PIDDosome ablation or IRE1 inhibition blunt steatohepatitis and diminish INSIG2 expression. Conversely, while inhibiting simple steatosis, SCAP ablation amplifies IRE1 and PIDDosome activation and liver damage in NASH-diet-fed animals, effects linked to ER disruption and preventable by IRE1 inhibition. Thus, the PIDDosome and SCAP pathways antagonistically modulate nutrient-induced hepatic ER stress to control non-linear transition from simple steatosis to hepatitis, a key step in NASH pathogenesis.

Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.

Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice.

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.

CRISPR/Cas9 - Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms.

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fah-/- liver failure mice, offering an efficient approach for precisely targeted gene therapies.

Homology-mediated end joining-based targeted integration using CRISPR/Cas9.

Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.