Serum oxidative biomarkers associated with genital HPV infection and cervical lesions in women.

Human papillomavirus (HPV) infection is a major cause of cervical cancer. Studies showed HPV carcinogenesis may be induced by oxidative stress affecting the host immune system. The objective of this study is to evaluate levels of four circulating oxidative stress biomarkers associated with the HPV infection, persistence, and cervical lesion status in women. The three serum biomarkers measuring oxidative damage to biomolecules (8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine [8-oxodG] for DNA, 4-hydroxy-2-nonenal [4-HNE] for lipid, and protein carbonyl [PC] for protein) and one antioxidant (glutathione, GSH) collected from 38 women were evaluated. The PC levels were significantly higher for women with oncogenic HPV infection (p = 0.047) and persistence (p = 0.053) based on the unadjusted linear model. In particular, women with ≥3 oncogenic HPV types had a higher PC level than those without HPV infection (p = 0.041). Women with low-grade squamous intraepithelial lesions showed an elevated PC (p = 0.058). These trends remained similar after adjusting for age. The GSH levels were lower for women infected with ≥3 oncogenic HPV types based on age-adjusted results (p = 0.061). This study supported that serum PC was associated with HPV infection, persistence, and cervical lesions, so it can potentially be used to monitor HPV carcinogenesis. Further large-scale studies will be needed to confirm these findings.

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer.

BACKGROUND: Around 15-20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. METHODS: RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. RESULTS: RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. CONCLUSIONS: Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

Adverse Effects of Nutraceuticals and Dietary Supplements.

Over 70% of Americans take some form of dietary supplement every day, and the supplement industry is currently big business, with a gross of over $28 billion. However, unlike either foods or drugs, supplements do not need to be registered or approved by the US Food and Drug Administration (FDA) prior to production or sales. Under the Dietary Supplement Health and Education Act of 1994, the FDA is restricted to adverse report monitoring postmarketing. Despite widespread consumption, there is limited evidence of health benefits related to nutraceutical or supplement use in well-nourished adults. In contrast, a small number of these products have the potential to produce significant toxicity. In addition, patients often do not disclose supplement use to their physicians. Therefore, the risk of adverse drug-supplement interactions is significant. An overview of the major supplement and nutraceutical classes is presented here, together with known toxic effects and the potential for drug interactions.

Chronic Ethanol Feeding in Mice Decreases Expression of Genes for Major Structural Bone Proteins in a Nox4-Independent Manner.

Bone loss in response to alcohol intake has previously been hypothesized to be mediated by excessive production of reactive oxygen species via NADPH oxidase (Nox) enzymes. Nox4 is one of several Nox enzymes expressed in bone. We investigated the role of Nox4 in the chondro-osteoblastic lineage of the long bones in mice during normal chow feeding and during chronic ethanol feeding for 90 days. We generated mice with a genotype (PrxCre +/- Nox4 fl/fl) allowing conditional knockout of Nox4 in the limb bud mesenchyme. Adult mice had 95% knockdown of Nox4 expression in the femoral shafts. For mice on regular chow, only whole-body Nox4 knockout mice had clearly increased cortical thickness and bone mineral density in the tibiae. When chronically fed a liquid diet with and without ethanol, conditional Nox4 knockout mice had slightly reduced dimensions of the cortical and trabecular regions of the tibiae (P < 0.1). The ethanol diet caused a significant reduction in cortical bone area and cortical thickness relative to a control diet without ethanol (P < 0.05). The ethanol diet further reduced gene expression of Frizzled related protein (Frzb), myosin heavy chain 3, and several genes encoding collagen and other major structural bone proteins (P < 0.05), whereas the Nox4 genotype had no effects on these genes. In conclusion, Nox4 expression from both mesenchymal and nonmesenchymal cell lineages appears to exert subtle effects on bone. However, chronic ethanol feeding reduces cortical bone mass and cortical gene expression of major structural bone proteins in a Nox4-independent manner. SIGNIFICANCE STATEMENT: Excessive alcohol intake contributes to osteopenia and osteoporosis, with oxidative stress caused by the activity of NADPH oxidases hypothesized to be a mediator. We tested the role of NADPH oxidase (Nox) 4 in osteoblast precursors in the long bones of mice with a conditional Nox4 knockout model. We found that Nox4 exerted effects independent of alcohol intake, and ethanol effects on bone were Nox4-independent.

Liver tumorigenesis is promoted by a high saturated fat diet specifically in male mice and is associated with hepatic expression of the proto-oncogene Agap2 and enrichment of the intestinal microbiome with Coprococcus.

Liver cancer results in a high degree of mortality, especially among men. As fatty liver disease is a risk factor for development of hepatocellular carcinoma, we investigated the role of dietary fat type in tumor promotion by high-fat diets in mice after initiation with the chemical carcinogen diethyl nitrosamine. Tumor incidence and multiplicity were significantly greater in males than those in females. In males, fat type had complex effects on tumorigenesis. Preneoplastic foci were most prevalent in mice fed a polyunsaturated fat diet enriched in docosahexaenoic acid, whereas carcinomas and large visible liver tumors were significantly greater in mice fed a saturated fat diet made with cocoa butter relative to mice fed mono- or polyunsaturated fats. Different mechanisms thus seemed involved in early and late tumor promotion. The hepatic transcriptome and gut microbiome were assessed for traits associated with tumorigenesis. Hepatic expression of more than 20% of all genes was affected by sex, whereas fat type affected fewer genes. In males, the saturated fat diet induced expression of the proto-oncogene Agap2 and affected the expression of several cytochrome P450 genes, and genes involved in lipid, bile acid and fatty acid metabolism. The gut microbiome had a higher level of genus Akkermansia and a lower level of Firmicutes in females than in males. Males fed saturated fat had an altered microbiome, including an enrichment of the genus Coprococcus. In conclusion, sex and the dietary fat type affect the gut microbiome, the hepatic transcriptome and ultimately hepatic tumor growth.

Influence of Quantum Interference on the Thermoelectric Properties of Molecular Junctions.

Molecular junctions offer unique opportunities for controlling charge transport on the atomic scale and for studying energy conversion. For example, quantum interference effects in molecular junctions have been proposed as an avenue for highly efficient thermoelectric power conversion at room temperature. Toward this goal, we investigated the effect of quantum interference on the thermoelectric properties of molecular junctions. Specifically, we employed oligo(phenylene ethynylene) (OPE) derivatives with a para-connected central phenyl ring ( para-OPE3) and meta-connected central ring ( meta-OPE3), which both covalently bind to gold via sulfur anchoring atoms located at their ends. In agreement with predictions from ab initio modeling, our experiments on both single molecules and monolayers show that meta-OPE3 junctions, which are expected to exhibit destructive interference effects, yield a higher thermopower (with ∼20 µV/K) compared with para-OPE3 (with ∼10 µV/K). Our results show that quantum interference effects can indeed be employed to enhance the thermoelectric properties of molecular junctions.

Knockout of the Gsta4 Gene in Male Mice Leads to an Altered Pattern of Hepatic Protein Carbonylation and Enhanced Inflammation Following Chronic Consumption of an Ethanol Diet.

BACKGROUND: Glutathione S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild-type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4-/- mice). METHODS: Adult male WT and GSTA4-/- mice were fed chow (N = 10 to 12) or high-fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH-fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) of purified carbonylated proteins. RESULTS: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4-/- mice increased B-cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4-/- mice exhibited panlobular rather than periportal distribution of 4-HNE-adducted proteins and increased overall 4-HNE staining after EtOH binge. Comprehensive LC-MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4-/- group. CONCLUSIONS: These data suggest long-term adaptation to EtOH in WT mice does not occur in GSTA4-/- mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B-cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.

The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs.

Pancreatic angiotensin-converting enzyme 2 (ACE2) has previously been shown to be critical for maintaining glycemia and ß-cell function. Efforts to maintain or increase ACE2 expression in pancreatic ß-cells might therefore have therapeutic potential for treating diabetes. In our study, we investigated the transcriptional role of hepatocyte nuclear factor 1α (HNF1α) and hepatocyte nuclear factor 1ß (HNF1ß) in induction of ACE2 expression in insulin-secreting cells. A deficient allele of HNF1α or HNF1ß causes maturity-onset diabetes of the young (MODY) types 3 and 5, respectively, in humans. We found that ACE2 is primarily transcribed from the proximal part of the ACE2 promoter in the pancreas. In the proximal part of the human ACE2 promoter, we further identified three functional HNF1 binding sites, as they have binding affinity for HNF1α and HNF1ß and are required for induction of promoter activity by HNF1ß in insulinoma cells. These three sites are well-conserved among mammalian species. Both HNF1α and HNF1ß induce expression of ACE2 mRNA and lead to elevated levels of ACE2 protein and ACE2 enzymatic activity in insulinoma cells. Furthermore, HNF1α dose-dependently increases ACE2 expression in primary pancreatic islet cells. We conclude that HNF1α can induce the expression of ACE2 in pancreatic islet cells via evolutionarily conserved HNF1 binding sites in the ACE2 promoter. Potential therapeutics aimed at counteracting functional HNF1α depletion in diabetes and MODY3 will thus have ACE2 induction in pancreatic islets as a likely beneficial effect.

Pancreatic angiotensin-converting enzyme 2 improves glycemia in angiotensin II-infused mice.

An overactive renin-angiotensin system (RAS) is known to contribute to type 2 diabetes mellitus (T2DM). Although ACE2 overexpression has been shown to be protective against the overactive RAS, a role for pancreatic ACE2, particularly in the islets of Langerhans, in regulating glycemia in response to elevated angiotensin II (Ang II) levels remains to be elucidated. This study examined the role of endogenous pancreatic ACE2 and the impact of elevated Ang II levels on the enzyme's ability to alleviate hyperglycemia in an Ang II infusion mouse model. Male C57bl/6J mice were infused with Ang II or saline for a period of 14 days. On the 7th day of infusion, either an adenovirus encoding human ACE2 (Ad-hACE2) or a control adenovirus (Ad-eGFP) was injected into the mouse pancreas. After an additional 7-8 days, glycemia and plasma insulin levels as well as RAS components expression and oxidative stress were assessed. Ang II-infused mice exhibited hyperglycemia, hyperinsulinemia, and impaired glucose-stimulated insulin secretion from pancreatic islets compared with control mice. This phenotype was associated with decreased ACE2 expression and activity, increased Ang II type 1 receptor (AT1R) expression, and increased oxidative stress in the mouse pancreas. Ad-hACE2 treatment restored pancreatic ACE2 expression and compensatory activity against Ang II-mediated impaired glycemia, thus improving ß-cell function. Our data suggest that decreased pancreatic ACE2 is a link between overactive RAS and impaired glycemia in T2DM. Moreover, maintenance of a normal endogenous ACE2 compensatory activity in the pancreas appears critical to avoid ß-cell dysfunction, supporting a therapeutic potential for ACE2 in controlling diabetes resulting from an overactive RAS.

Impact of Alcohol on Bone Health in People Living With HIV: Integrating Clinical Data From Serum Bone Markers With Morphometric Analysis in a Non-Human Primate Model.

People living with HIV (PLWH) represent a vulnerable population to adverse musculoskeletal outcomes due to HIV infection, antiretroviral therapy (ART), and at-risk alcohol use. Developing measures to prevent skeletal degeneration in this group requires a grasp of the relationship between alcohol use and low bone mass in both the PLWH population and its constituents as defined by sex, age, and race. We examined the association of alcohol use with serum biochemical markers of bone health in a diverse cohort of PLWH enrolled in the New Orleans Alcohol Use in HIV (NOAH) study. To explore the effects of alcohol on bone in the context of HIV and ART and the role of estrogen, we conducted a parallel, translational study using simian immunodeficiency virus (SIV)+/ART+ female rhesus macaques divided into four groups: vehicle (Veh)/Sham; chronic binge alcohol (CBA)/Sham; Veh/ovariectomy (OVX); and CBA/OVX. Clinical data showed that both osteocalcin (Ocn) and procollagen type I N-propeptide (PINP) levels were inversely associated with multiple measures of alcohol consumption. Age (>50 years) significantly increased susceptibility to alcohol-associated suppression of bone formation in both female and male PLWH, with postmenopausal status appearing as an additional risk factor in females. Serum sclerostin (Scl) levels correlated positively with measures of alcohol use and negatively with Ocn. Micro-CT analysis of the macaque tibias revealed that although both CBA and OVX independently decreased trabecular number and bone mineral density, only OVX decreased trabecular bone volume fraction and impacted cortical geometry. The clinical data implicate circulating Scl in the pathogenesis of alcohol-induced osteopenia and suggest that bone morphology can be significantly altered in the absence of net change in osteoblast function as measured by serum markers. Inclusion of sophisticated tools to evaluate skeletal strength in clinical populations will be essential to understand the impact of alcohol-induced changes in bone microarchitecture. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Nox4 expression in osteo-progenitors controls bone development in mice during early life.

Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole-body Nox4 knockout mice (Nox4-/-) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4fl/fl), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4-/- mice compared with Nox4fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4-/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4-/- mice compared with control mice, and significantly lower in 32-week old Nox4-/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play an important role in osteoblast differentiation, proliferation and maturation.

Binge Ethanol Exposure in Mice Represses Expression of Genes Involved in Osteoblast Function and Induces Expression of Genes Involved in Osteoclast Differentiation Independently of Endogenous Catalase.

Excessive ethanol consumption is a risk factor for osteopenia. Since a previous study showed that transgenic female mice with overexpression of catalase are partially protected from ethanol-mediated trabecular bone loss, we investigated the role of endogenous catalase in skeletal ethanol toxicity comparing catalase knockout to wild-type mice. We hypothesized that catalase depletion would exacerbate ethanol effects. The mice were tested in a newly designed binge ethanol model, in which 12-week-old mice were exposed to 4 consecutive days of gavage with ethanol at 3, 3, 4, and 4.5 g ethanol/kg body weight. Binge ethanol decreased the concentration of serum osteocalcin, a marker of bone formation. The catalase genotype did not affect the osteocalcin levels. RNA sequencing of femoral shaft RNA from males was conducted. Ethanol exposure led to significant downregulation of genes expressed in cells of the osteoblastic lineage with a role in osteoblastic function and collagen synthesis, including the genes encoding major structural bone proteins. Binge ethanol further induced a smaller set of genes with a role in osteoclastic differentiation. Catalase depletion affected genes with expression in erythroblasts and erythrocytes. There was no clear interaction between binge ethanol and the catalase genotype. In an independent experiment, we confirmed that the binge ethanol effects on gene expression were reproducible and occurred throughout the skeleton in males. In conclusion, the binge ethanol exposure, independently of endogenous catalase, reduces expression of genes involved in osteoblastic function and induces expression of genes involved in osteoclast differentiation throughout the skeleton in males.

T-cell immunoglobulin and mucin domain 3 is upregulated in rheumatoid arthritis, but insufficient in controlling inflammation.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease, that involves both pro- and anti-inflammatory mechanisms. The purpose of the present study is to investigate T-cell immunoglobulin and mucin domain 3 (Tim-3) in RA. METHODS: Plasma levels of soluble (s) Tim-3 in early RA (n=98), were followed, to evaluate association with treatment and disease activity, acquired from a prospective collected biobank (clinicaltrials.gov (NCT00660647)). We also investigate the influence of Tim-3 on spontaneous cytokine production in synovial fluid mononuclear cells (SFMC) from RA patients after addition of neutralizing anti-Tim-3's antibodies, either alone or in combination with neutralizing anti-Programmed Cell death protein 1 (PD-1) antibodies. RESULTS: Long-time stimulated CD4 T-cells expressed high levels of Tim-3, but tended to decrease their PD-1 expression. Tim-3 expression was exclusively seen co-expressed with PD-1 by CD3, CD4, CD45RO positive cells in the inflamed RA joint. Addition of neutralizing Tim-3 antibodies increased the secretion of IFNγ and MCP-1, in SFMC cultures from RA. Whereas neutralizing anti-PD-1 antibodies showed a broader impact on cytokine production. Finally, we observed that soluble Tim-3 is increased in plasma and is associated with disease activity in early RA. CONCLUSION: Taken together, our findings indicate disease-suppressive functions of Tim-3 in RA.

The Programmed Death-1 Pathway Counter-Regulates Inflammation-Induced Osteoclast Activity in Clinical and Experimental Settings.

Objective: The programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA. Methods: Bone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores. Results: PD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro, but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression. Conclusion: The PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression.

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays.

Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 µM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 µM DX600 (>95% inhibition) but not with 1 µM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.

Glucose induces expression of rat pyruvate carboxylase through a carbohydrate response element in the distal gene promoter.

Pyruvate carboxylase is an enzyme of the so-called pyruvate cycling pathways, which have been proposed to contribute to glucose-stimulated insulin secretion in pancreatic beta-cells. In the rat insulinoma cell line 832/13, transcripts from both the distal and proximal gene promoter for pyruvate carboxylase are up-regulated by glucose, with pyruvate carboxylase being expressed mainly from the distal gene promoter. At position -408 to -392 relative to the transcription start site, the distal gene promoter was found to contain a ChoRE (carbohydrate response element). Its deletion abolishes glucose responsiveness of the promoter, and the sequence can mediate glucose responsiveness to a heterologous gene promoter. ChREBP (carbohydrate response element-binding protein) and its dimerization partner Mlx (Max-like protein X) bind to the ChoRE in vitro. ChREBP further binds to the distal promoter region at a high glucose concentration in situ. The E-box-binding transcription factors USF1/2 (upstream stimulatory factor 1/2) and E2A variant 2 [also known as E47 and TCF3 (transcription factor 3)] can also bind to the ChoRE. Overexpression of E2A diminishes the magnitude of the glucose response from the pyruvate carboxylase ChoRE. This illustrates that competition between ChREBP-Mlx and other factors binding to the ChoRE affects glucose responsiveness. We conclude that a ChoRE in the distal gene promoter contributes to the glucose-mediated expression of pyruvate carboxylase.

The Transcription Factor SLUG Uncouples Pancreatic Cancer Progression from the RAF-MEK1/2-ERK1/2 Pathway.

Activating mutations in some isoforms of RAS or RAF are drivers of a substantial proportion of cancers. The main Raf effector, MEK1/2, can be targeted with several highly specific inhibitors. The clinical activity of these inhibitors seems to be mixed, showing efficacy against mutant BRAF-driven tumors but not KRAS-driven tumors, such as pancreatic adenocarcinomas. To improve our understanding of this context-dependent efficacy, we generated pancreatic cancer cells resistant to MEK1/2 inhibition, which were also resistant to KRAS and ERK1/2 inhibitors. Compared with parental cells, inhibitor-resistant cells showed several phenotypic changes including increased metastatic ability in vivo. The transcription factor SLUG, which is known to induce epithelial-to-mesenchymal transition, was identified as the key factor responsible for both resistance to MEK1/2 inhibition and increased metastasis. Slug, but not similar transcription factors, predicted poor prognosis of pancreatic cancer patients and induced the transition to a cellular phenotype in which cell-cycle progression becomes independent of the KRAS-RAF-MEK1/2-ERK1/2 pathway. SLUG was targeted using two independent strategies: (i) inhibition of the MEK5-ERK5 pathway, which is responsible for upregulation of SLUG upon MEK1/2 inhibition, and (ii) direct PROTAC-mediated degradation. Both strategies were efficacious in preclinical pancreatic cancer models, paving the path for the development of more effective therapies against pancreatic cancer. SIGNIFICANCE: This study demonstrates that SLUG confers resistance to MEK1/2 inhibitors in pancreatic cancer by uncoupling tumor progression from KRAS-RAF-MEK1/2-ERK1/2 signaling, providing new therapeutic opportunities. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3849/F1.large.jpg.

HER2 carboxyl-terminal fragments regulate cell migration and cortactin phosphorylation.

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.

Nox4 Expression Is Not Required for OVX-Induced Osteoblast Senescence and Bone Loss in Mice.

Estrogen deficiency and aging play critical roles in the pathophysiology of bone as a result of increased oxidative stress. It has been suggested that prevention of NADPH oxidase- (Nox-) dependent accumulation of ROS may be an approach to potentially minimize bone loss caused by these conditions. Using ovariectomized (OVX) and Nox4 gene-deletion mouse models, we investigated the role of Nox4 in OVX-induced bone loss and osteoblast senescence signaling. Six-month-old WT C57Bl6 mice were allocated to a sham control group, OVX, and OVX plus E2 treatment group for 8 weeks. Decreased bone mass including BMD and BMC were found in the OVX group compared with the sham control (p < 0.05); E2 treatment completely reversed OVX-induced bone loss. Interestingly, the prevention of OVX-induced bone loss by E2 was associated with the elimination of increased senescence signaling in bone osteoblastic cells from the OVX group. E2 blunted OVX-induced p53 and p21 overexpression, but not p16 and Nox4 in bone. In addition, 8- and 11-month-old Nox4 KO female mice were OVX for 8 weeks. Significant bone loss and increased bone osteoblastic cell senescence signaling occurred not only in Nox4 KO OVX mice compared with sham-operated animals, but also in 11-month-old Nox4 KO sham mice compared with 8-month-old Nox4 KO sham mice (p < 0.05). These data suggest that Nox4-mediated ROS in bone osteoblastic cells may be dispensable for sex steroid deficiency-induced bone loss and senescence. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Potential role of gut microbiota, the proto-oncogene PIKE (Agap2) and cytochrome P450 CYP2W1 in promotion of liver cancer by alcoholic and nonalcoholic fatty liver disease and protection by dietary soy protein.

We have previously demonstrated promotion of diethylnitrosamine (DEN) initiated liver tumorigenesis after feeding diets high in fat or ethanol (EtOH) to male mice. This was accompanied by hepatic induction of the proto-oncogene PIKE (Agap2). Switch of dietary protein from casein to soy protein isolate (SPI) significantly reduced tumor formation in these models. We have linked EtOH consumption in mice to microbial dysbiosis. Adoptive transfer studies demonstrate that microbiota from mice fed ethanol can induce hepatic steatosis in the absence of ethanol suggesting that microbiota or the microbial metabolome play key roles in development of fatty liver disease. Feeding SPI significantly changed gut bacteria in mice increasing alpha diversity (P < 0.05) and levels of Clostidiales spp. Feeding soy formula to piglets also resulted in significant changes in microbiota, the pattern of bile acid metabolites and in inhibition of the intestinal-hepatic FXR/FGF19-SHP pathway which has been linked to both steatosis and hepatocyte proliferation. Moreover, feeding SPI also resulted in induction of hepatic PPARα signaling and inhibition of PIKE mRNA expression coincident with inhibition of steatosis and cancer prevention. Feeding studies in the DEN model with differing dietary fats demonstrated tumor promotion specific to the saturated fat, cocoa butter relative to diets containing olive oil or corn oil associated with microbial dysbiosis including dramatic increases in Lachnospiraceae particularly from the genus Coprococcus. Immunohistochemical analysis demonstrated that tumors from EtOH-fed mice and patients with alcohol-associated HCC also expressed high levels of a novel cytochrome P450 enzyme CYP2W1. Additional adoptive transfer experiments and studies in knockout mice are required to determine the exact relationship between soy effects on the microbiota, expression of PIKE, CYP2W1, PPARα activation and prevention of tumorigenesis.