bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Marginal Zinc Intake and Repletion on Essential Fatty Acid Metabolism
Authors: Suh Jung , Burke Sarah , Shigenaga Mark , Killilea David , Holland Tai , Shenvi Swapna , Sutherland Barbara , King Janet
Published: November 2023

Objective

To determine the effects of marginal zinc deficiency and repletion on essential fatty acid, sphingolipid, and lipoprotein metabolism.

Methods

Sixteen apparently healthy male subjects between ages of 18–45 were subjected to three sequential phases of dietary zinc intake modulation: Phase 1: Low Zinc intake: 6 mg/day with 1.5 g of phytate (2 wks); Phase II: Zinc Repletion: 10 mg/day with no phytate (4 wks); Phase III: Zinc Supplementation: Ad lib diet supplemented with 20 mg zinc (2 wks). Subject weight, and compliance with diet were monitored every 3–4 days during the first two phases. Plasma free fatty acids and sphingolipids were measured by mass spectrometry. FADS1 and FADS2 activities were calculated by g‐linolenic (GLA)/linolenic acid (LA) or arachidonic acid (AA)/dihomo‐g‐linolenic acid (DGLA) ratios, respectively.

Results

Plasma zinc levels remained unchanged during Phases I and II in all subjects and increased only at the conclusion of phase III. FADS1 activity significantly (p=0.005) decreased from 5.2 ± 0.5 to 3.6 ± 0.5 (mean ± SEM) following Zn depletion. In contrast, FADS2 activity was insensitive to effects of low zinc intake. As a consequence of FADS1 activity loss, concentrations of AA‐containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelins and plasmalogen antioxidant lipids decreased significantly. Furthermore, plasma concentrations of C16:0 ceramide increased following Zn depletion (p = 0.06) and was inversely correlated with FADS1 activity (p = 0.04; r2 = 0.6). Zn depletion also increased plasma triglyceride (TG) by 26 ± 15% (p< 0.05) and decreased high‐density lipoprotein (HDL) by 18.5 ± 3%; P<0.05. Interestingly, changes in FADS1 activity observed during the depletion was not normalized by dietary Zn repletion (Phase II) but, it was completely normalized with Zn supplementation (20 mg/day; Phase III).

Conclusion

Dietary Zn depletion (6 mg/day) decreases FADS1 activity and lowered AA incorporation into major lipid sub‐classes. Loss in FADS1 activity was inversely correlated with C16:0 ceramide and was further associated with dyslipidemia as evidenced by significant alterations in plasma TG and HDL. These effects were not corrected with dietary Zn repletion (10 mg/day); supplementation with 20 mg Zn/d for an additional 3 weeks was required. These data suggest that FADS1 activity may be a sensitive biomarker of inadequate zinc intake and they also implicate marginal Zn intake as a novel risk factor for dyslipidemia and insulin resistance.

Support or Funding Information

Harvest Plus, NIH S10OD0018070‐01
The effect of zinc deficiency and diet restriction on GATA family gene expression in rat's kidney
Authors: Konomi Aki , Yokoi Katsuhiko
Published: November 2023

We reported that plasma erythropoietin (EPO) concentration, kidney EPO concentration and kidney EPO mRNA level were changed by moderate zinc deficiency or diet restriction. It is not established how moderate zinc deficiency or diet restriction induces these alterations. We propose a hypothesis that moderate zinc deficiency or diet restriction affects renal GATA family genes expression. Thirty 3‐week‐old male Sprague‐Dawley rats were assigned into 3 dietary groups (n=10 each) and fed on dietary regimens for 4 weeks: control group (AIN‐93G; Control), moderate zinc‐deficient group (4.5 mg Zn/kg; zinc deficiency) and pair‐fed group (AIN‐93G; diet restriction). Kidney was separated into cortex and medulla for RNA isolation. Quantitative real time‐PCR using TaqMan proves was performed for mRNA of GATA family gene (GATA1, GATA2, GATA3, GATA4, GATA5, GATA6, Gatad1, Gatad2a and Gatad2b). Statistical analysis was performed by ANOVA and the multiple comparison test of Tukey‐Kramer. P value less than 0.05 was considered significant. Kidney medulla mRNA expression per tissue weight of GATA4 and GATA5 were significantly decreased by zinc deficiency compared with Control. GATA2, GATA3, Gatad1, Gatad2a and Gatad2b were significantly decreased by zinc deficiency and diet restriction compared with Control. Kidney cortex mRNA expression per tissue weight were not significantly different among the groups for all genes. Cortex and medulla GATA1 and GATA6 mRNA expression per tissue weight were not changed. These results suggest that kidney GATA family gene mRNA expression were decreased by zinc deficiency and diet restriction, especially in medulla.

Support or Funding Information

This work was supported by KAKENHI grant‐in‐aid for Research Activity Start‐up (21800073) and Young Scientists (B) (23700928) and (15K16216).
Wnt/Beta‐catenin and mTOR Signaling in Liver Pathophysiology
Authors: Michael Adeola O Adebayo , Pradhan‐Sundd Tirthadipa , Russell Jacquelyn , Monga Satdarshan (Paul) S.
Published: November 2023

Two distinct signaling pathways have been implicated in the pathogenesis of hepatocellular carcinoma, the mTOR and Wnt/Beta catenin pathways. At present, there is ample data on the singular role of mTOR activity and Wnt signaling in early HCC development. However, several of these studies conclude that each pathway alone is not sufficient to account for HCC initiation and development, suggesting potential interactive mechanisms between pathways. The aim of this study is to investigate signaling mechanisms of cross‐talk between mTOR signaling and Wnt/Beta catenin signaling in hepatocarcinogenesis. Previous studies in the lab have shown modulation of mTOR and target proteins in point‐mutated beta‐catenin+MET induced tumors. Based on these, we hypothesized that Wnt/Beta‐catenin and mTOR pathways may cooperate and cross‐talk during hepatic pathophysiology. We utilized beta‐catenin‐floxed and LRP5‐6 double floxed mice to interbreed to the Albumin‐Cre transgenic mice to conditionally delete these genes from hepatocytes to examine mTOR. We also used human HCC Hep3B cells either overexpressing or with silenced beta‐catenin. In vivo and in vitro studies show that beta catenin regulates mTOR in the liver. Total mTOR levels remain unaltered, but mTOR phosphorylation along with its downstream targets such as ribosomal protein S6 and 4E‐BP1 are also affected. In conclusion, our studies provide evidence of the interactions between mTOR signaling and Wnt/Beta catenin signaling pathways in both liver homeostasis and in HCC cells. These models will provide a novel opportunity to understand the role and regulation of such interactions and crosstalk.

Support or Funding Information

Post‐doctoral Trainee award T32CA186783

NIDDK 4R01DK100287‐04

NIDDK 5R01DK062277‐13
Loss of MET in the Absence of EGFR Signaling Leads to Hepatic Failure in the Resting Liver
Authors: Tsagianni Anastasia , Mars Wendy M. , Bowen William , Haynes Meagan , Paranjpe Shirish , Orr Anne , Michalopoulos George
Published: November 2023

Our recent study (Paranjpe S. et al, Hepatology 2016 Nov;64(5):1711–1724) showed that in mice, combined systemic elimination of EGFR and MET signaling abolishes liver regeneration after partial hepatectomy, leading to liver decompensation and death at 15–18 days post‐surgery. In the current study, we investigated the role of EGFR and MET in resting livers that were not stimulated by hepatectomy. Exon 16 from MET was removed using Tamoxifen IP injections in MET ff/ff:TamCre+/+ mice, preserving the protein but eliminating efficient signaling through the receptor. Canertinib diet (an irreversible pan‐ERB EGFR inhibitor) was used to achieve a complete block in EGFR signaling. Vehicle corn oil injections and normal chow were used for the control animals. Surprisingly, double inhibited mice were sickened by day 10 with most of them dead by day 14. In the double inhibited group, liver to body weight ratios decreased over time in a statistically significant way at every time point tested (days 2, 7 and 10), whereas ratios with individual treatments were not statistically altered. Further, in the combined MET and EGFR elimination group, MET protein was totally absent while EGFR protein was preserved. As Canertinib diet alone did not lead to the complete loss of MET protein, and as Canertinib completely inhibits EGFR signaling, we interpret these results to mean that EGFR signaling normally stabilizes MET protein through a mechanism involving exon 16 and that the complete loss of MET in the double‐inhibited animals is what ultimately leads to liver failure. These results underscore the fundamental contribution of the combined HGF/MET and EGFR signaling pathways to hepatic proliferation, survival and functionality.

Support or Funding Information

Menten Endowment
Integrin β6: lost in translation from mouse to human as a target for hepatic fibrosis
Authors: Saravanan Chandrassegar , Song Yanli , Wu Ping , Hu Wei , Bian Feng Bian , Heidt Analeah , Mansfield Keith
Published: November 2023

Studies in the past have shown that integrin β6 (ITGβ6), a transmembrane protein expressed on cholangiocytes, plays a role in hepatic fibrosis. For example, expression of ITGβ6 was shown to be upregulated in mouse models of biliary fibrosis and genetic ablation or pharmacologic inhibition of the protein attenuated biliary fibrosis. Likewise, ITGβ6 mRNA expression was shown to be increased in fibrotic liver from patients with primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and hepatitis C. Therefore, ITGβ6 has emerged as a promising target for hepatic fibrosis. However, few studies were focused on ITGβ6 localization in human fibrotic liver and the evidences were not compelling. Therefore, the goal of this study was to validate the expression of ITGβ6 in normal and fibrotic human and mouse formalin fixed paraffin embedded (FFPE) liver sections. Internally developed mouse monoclonal antibodies to ITGβ6 with specificity and sensitivity similar to commercially available and published antibodies (Clone‐442.5C4, EMD Millipore, Billerica, MA) were used for immunohistochemistry (IHC). An RNAscope® (Advanced Cell Diagnostics, Hayward, CA) probe set recognizing all isoforms of ITGβ6 was used for in situ hybridization (ISH). IHC experiments demonstrated that ITGβ6 protein was not detected in FFPE human liver samples from normal control (n=3), PBC (n=3), PSC (n=5), nonalcoholic steatohepatitis (NASH) (n=1), cirrhosis of unknown origin (n=3) and cirrhosis due to hepatitis C (n=1), whereas, cytokeratin 19 (cholangiocyte marker) was expressed in all the samples. In contrast, ITGβ6 protein was detected in cholangiocytes of normal mouse liver and its expression was upregulated in a bile duct ligation model. ISH experiments revealed low levels of ITGβ6 mRNA in rare cholangiocytes of some human PBC and NASH liver, but not in control liver samples, suggesting the expression of untranslated transcripts of ITGβ6 in human fibrotic liver. Together, these data indicate that ITGβ6 upregulation in mouse biliary fibrosis model is a rodent‐specific mechanism and cannot be translated to human hepatic fibrosis. Indeed, this study reinforces the value of molecular localization of targets in normal and diseased human tissues early in the drug discovery process to prevent drug attrition in clinical trials due to inadequate preclinical target validation.
Role of Na+‐K+‐ATPase in development of alcoholic fatty liver disease
Authors: Matsumoto Hiroshi , Sugimoto Kana , Yang Shipin
Published: November 2023

Patients with alcoholic fatty liver disease (AFLD) were well‐known to have low blood sodium level. Alcohol intoxication causes low blood sodium level. In the previous study, we obtained that acute and chronic alcohol consumption induced the phenomenon of low blood sodium level in rats. These findings suggested renal failure, but there was no change in renal function in human and rat. Blood sodium level is regulated by Na+‐K+‐ATPase in lots of tissues. Then, we hypothesized that regulation of Na+‐K+‐ATPase may contributes to improve the development of alcoholic organ damage. In the present study, we focused to clarify whether regulation of regulation of Na+‐K+‐ATPase improves alcoholic liver injury in rats or not. Alcoholic fatty liver disease rat model was performed by feeding a Lieber‐DeCalri diet for 4 weeks. Amiloride, lidocaine, ouibain, sanguinarine, or glibenclamide was administered i.p to rats for 2 weeks after feeding an alcohol diet. After 4 weeks feeding, blood biochemical tests were performed. Expression of mRNA and proteins was performed by qPCR and western blotting analysis. EMSA was performed. Histopathological findings showed alcoholic fatty liver changes in the liver of rats fed the alcohol diet On the other hand, administration of inhibitors of Na+‐K+‐ATPase reduced a slight improvement and decreased the NF‐kappaB activity via IRF3‐dependent pathway but increased activation of IRFy‐dependent pathway in the liver, which phenomena spotlighted in the isolated endsome from the liver, leading that Na+‐K+‐ATPase in endosome can be related to activation of innate immune system. These findings suggest that regulation of Na+‐K+‐ATPase may be one of therapies for alcoholic fatty liver disease.

Support or Funding Information

This research was supported in part by JSPS KAKENHI Grant Number 24390175.
Diagnosis of Naturally‐Occurring Nonalcoholic Fatty Liver Disease (NAFLD) in Obese Rhesus Monkeys using Dual‐Energy CT (DECT) and Histopathology Criteria
Authors: Sun Fengjiao , Liang Zhigang , Yang Zunyuan , Tang Chungui , Chen Zhengli , Shen Yubo , Yao Zunwei , Wu Mingling , Chen Yuanhai , Gao Fabao , Zeng Wen , Hansen Barbara C
Published: November 2023

Obese patients are at significant risk for the development of NAFLD. An obese NAFLD animal model that fully reflects NAFLD in obese humans can assist in the study of the mechanisms of NAFLD and of potential therapeutics to reduce NAFLD. 37 male monkeys (7–20yrs, 7–18kg; 25 of which were significantly overweight >12 kg) were grouped into moderate steatosis (Mod, n=19), mild steatosis (Mild, n=12) and no steatosis (Norm, n=6) by ultrasound evaluation. DECT was utilized for quantifying liver fat content in 37 monkeys together with the relationship of NAFLD to severity of obesity and to circulating biomarkers. Within 2 weeks after DECT, pathological diagnoses of liver needle biopsy of 24/37 monkeys were established using H&E and Masson staining. Liver sections were scored by one pathologist first by the NAFLD Activity Score (NAS) then the fibrosis score. DECT quantified 19, 12 and 6 monkeys with 10–30%, 5–10% and < 5% fat content in Mod, Mild and Norm group, respectively. The Mod and Mild groups showed increasing body weights (p<0.001), higher C‐peptide levels (p<0.01), triglyceride (not significant) and HbA1c (not significant) than the Norm group. According to the NAS, H&E results showed 9/10 in Mod were NASH (4/9 NAS scores were 5–6, 5/9 were 4); 1/10 in Mod was simple steatosis (SS). 4/8 in Mild were NASH (NAS scores all 4); 4/8 were SS. 6/6 in Norm were Normal. Masson results showed 3/4 with NAS scores 5–6 were in 2–3 stages; 1/4 was in 1a stage; 5 with NAS score 4 were in 0–1c stage; 4/4 of Mild with NAS scores 3–4 were in 1c‐3 stages; SS cases were in the 1a‐1c stage. In conclusion, DECT is important for noninvasive liver fat quantification in obese patients and the NAFLD monkey model shows the same specific liver pathology and circulating biomarkers as humans. This nonhuman primate model, permitting both repeated liver biopsies and imaging studies, is highly useful in the evaluation of potential therapeutics for human NAFLD.
Wnt7b and Wnt10a, beta‐catenin independent signaling regulates cholangiocyte proliferation and function during cholestasis
Authors: Kosar Karis , Nejak‐Bowen Kari
Published: November 2023

Cells found in biliary ducts, are believed to be bipotential cells that are capable of differentiating into both cholangiocytes and hepatocytes, leading researchers to consider them as a sort of stem cells for both the liver and biliary duct. Wnt/beta‐catenin signaling has been implicated as an important factor in hepatoblast atypical ductular reaction and in the liver stem cells of rats and in a variety of liver pathologies, including atypical ductular proliferation during hepatobiliary injury. We have previously shown that liver‐specific b‐catenin knockout (KO) mice exhibited a markedly decreased ductular response after 2 weeks of 0.1% 3,5‐diethoxycarbonyl‐1,4‐dihydro‐collidine (DDC) diet. More recently, we also identified upregulation of specific Wnt proteins in the cholangiocytes during cholestatic liver injury and that mice lacking Wnt secretion from hepatocytes and cholangiocytes (Alb‐cre Wls KO) showed fewer proliferating cholangiocytes and high mortality in response to DDC diet. Therefore, we hypothesize Wnt induces proliferation of cholangiocytes, which may alleviate some of the complications caused by cholangiopathies. Two Wnts of interest for us are Wnt7B and Wnt10A, which we have shown, through in vitro studies, promote proliferation of cholangiocytes in an autocrine manner. These Wnts are specific for cholestasis, as wild‐type (WT) mice show normal expression of Wnt7B and Wnt10A after choline deficient, ethionine‐supplemented diet, which induces hepatocyte injury and ductal hyperplasia unrelated to cholestasis. Proliferation and cell viability were unimpaired in a small cholangiocyte (sm‐cc) cell line after b‐catenin suppression in vitro under normal growth conditions. To confirm these findings, sm‐cc cells were transfected with either Wnt7B or Wnt10A, in combination with either control or b‐catenin siRNA. The absence of b‐catenin did not inhibit the Wnt 7B‐ and 10A‐induced increase in cholangiocyte proliferation. Media from sm‐cc cells transfected with Wnt7B or Wnt10A was also analyzed for cytokine production. Compared to control cells, Wnt7B and Wnt10A induced a significant increase in the expression of cytokines and growth factors such as IL‐1A, IL‐17A, and VEGF, while expression of anti‐inflammatory IL‐2 decreases in the transfected cells. Thus, b‐catenin independent Wnt signaling promotes ductular reaction during cholestasis and may also alter the phenotypic response of proliferating cholangiocytes.

Support or Funding Information

National Institute of Biomedical Imaging and Bioengineering of the National Institute of Health under Award Number T32EB0010216

UPP Academic Foundation Award 1R01DK103775
FGF19 induced hepatocarcinogenesis in mice
Authors: Tao Junyan , Singh Sucha , Chen Xin , Monga Paul
Published: November 2023

Fibroblast growth factor 19 (FGF19) overexpression is reported in a notable subset of hepatocellular carcinomas (HCC) and has been associated with larger size, early recurrence and poor prognosis in patients. HCC was also observed in transgenic mice overexpressing FGF19 in skeletal muscle. These tumors originated adjacent to central veins, which is also the area of basal activation of Wnt/beta‐catenin signaling in the liver. In the current study, we investigate the role of FGF19 during HCC development using the sleeping beauty transposon/transposase to express FGF19 in a subset of hepatocytes in mice via hydrodynamic tail vein injection (HTVI). After HTVI, mice were harvested at 14.2 weeks, 22 weeks and 25 weeks. Small tumor nodules were apparent at 14.2 weeks. H&E showed HCC nodules, and immunohistochemistry (IHC) showed these nodules to be Glycogen synthase (GS) and cyclin‐D1 positive along with abundant Ki‐67. Thus, overexpression of FGF19 in a subset of hepatocytes led to HCC development via beta‐catenin activation. This model will be of value in further investigating the mechanisms by which FGF19 induced HCC in mice and men.
Alcohol abuse promotes breast cancer development and progression via StarD10 phosphorylation
Authors: Floris Andrea , Cossu Carla , Spissu Ylenia , Tomasi Maria Lauda
Published: November 2023

Background

Epidemiological studies demonstrated that ethanol administration promotes breast cancer development and metastasis. Human breast cancer cells or mammary epithelial cells with a high expression of ErbB2 exhibited an enhanced response to ethanol‐stimulated cell invasion in vitro. In addition, ethanol stimulates cell proliferation and triggers different intracellular signaling modulating the phosphorylation status of key proteins as JNK, P38 and ERK. Post‐translational modification by phosphorylation is a common mechanism to regulate the activity of protein, increasing their local negative charge to promote conformational changes or influencing interaction with protein partners. StarD10 is a member of the start protein family well known to be regulated due to Phosphorylation on Serine 284 by Casein Kinase II (CKII); it's a lipid transfer protein with selective binding site to Phosphatidylcholine and Phosphatidylethanolamine. It was found to be co‐expressed with ErbB2 in many breast carcinoma cell lines, suggesting a selective growth advantage for tumor expressing both proteins. The aim of this study was to investigate the role of Phosphorylated StarD10 in breast cancer and its interaction with ErbB2 as consequence of ethanol administration.

Methods

The experiments were performed using MCF7, MDA‐MB231 human carcinoma cell lines treated with ethanol 100mM. Protein and mRNA levels were analyzed by western blotting/Co‐immunoprecipitation (Co‐IP) and RT‐PCR, respectively. The phosphorylation fraction of StarD10 has been detected by protein purification system columns from cell lysate.

Results

We found that ethanol treatment increases StarD10 and ErbB2 at both protein and mRNA levels in MCF7 cells. Recombinant proteins were used to demonstrate the direct interaction between StarD10 and ErbB2 and we confirmed this finding by Co‐IP. In addition, ethanol treatment resulted in increased StarD10 and ErB2 interaction. We have established that StarD10 and ErbB2 show a reciprocal regulation. Interesting, we found that both overexpression and silencing of Stard10 induce cell growth and migration suggesting an important role in maintaining cellular homeostasis and potential role of StarD10 in induced‐ethanol breast cancer development and/or progression. In addition, we found that ethanol reduces the phosphorylation status of StarD10 via downregulation of CKII, making it more active.

Conclusion

The ability of Stard10 to cooperate with ErbB receptors may be independent of its lipid binding function. This novel finding could support the hypothesis of phosphorylation regulates the interaction between ErbB2 and StarD10 and their synergistic action in the neoplastic progression of the breast cancer. This is the first report demonstrating that ethanol can modulate in dynamic manner the phosphorylation status of protein StarD10 in breast cancer cell lines.

Support or Funding Information

Supported by NIH grant 5 K01 AA022372‐04(Maria Lauda Tomasi)
Ketogenic Therapies on Cancer Cachexia in a Mouse Model of Metastatic Cancer
Authors: Koutnik Andrew P , Poff Angela M , Ward Nathan P , DeBlasi Janine , D'Agostino Dominic P
Published: November 2023

While most cancer deaths are a result of metastatic disease, 20% directly result from cachexia. Cancer cachexia is characterized by the loss of skeletal muscle mass, with or without fat loss, that cannot be fully reversed by conventional nutritional support and results in progressive functional impairment. There are many models of cancer cachexia, but none are ideal as they do not address all aspects of this multifaceted syndrome. The VM‐M3 model is a syngeneic mouse model derived from a spontaneous brain tumor which, when implanted subcutaneously, results in rapid and systemic metastasis throughout the body, including to the liver, lungs, brain, kidneys, and spleen. Male and female VM‐M3 mice were assessed for cancer cachexia phenotype. Tumor progression, organ metastasis, skeletal muscle and adipose tissue recomposition, systemic inflammation, anorexia, albumin, blood urea nitrogen, and other clinical metabolites were evaluated. VM‐M3 male and female models both present rapid systemic metastasis (31 and 32‐day mean survival) with similar progressive increases in primary tumor weight in males and females, respectively. A progressive decrease in calf skeletal muscle weight was seen from week 2 (10% and 12% reduction) to end of life (19% and 23%), with a 40% and 26% reduction in quadriceps weight at end of life for males and females, respectively. Intraperitoneal white adipose tissue displayed progressive wasting with a 20% reduction for both male and females at week 2 and an 82% and 100% reduction for male and females, respectively. Prolonged systemic inflammation was identified via elevated spleen weight (9‐ and 10‐fold increase, respectively), elevated white blood cells counts, and elevated TNF‐α and IL‐6 at week 2 and end of life. Male and female VM‐M3 mice also presented anorexia, hypoalbumin, elevated blood urea nitrogen, and reduced total protein. Dietary interventions were developed to treat this multifaceted model of metastasis induced cancer cachexia. Ketogenic diet and synthetic ketogenic agents (R,S 1,3‐butanediol acetoacetate diester) were administered via diet to determine their ability to attenuate cachexic phenotype (altered tumor progression, skeletal muscle, adipose tissue, systemic inflammation, and additional cachexic biomarkers).

Support or Funding Information

Scivation Inc.
Adjuvant Statin Therapy Efficacy is Dictated by Tumor Dormancy and Statin Lipophilicity in Ex Vivo and In Vivo Models of Metastatic Breast Cancer
Authors: Beckwitt Colin H , Clark Amanda M , Warita Katsuhiko , Oltvai Zoltan N , Wells Alan
Published: November 2023

Metastasis in breast cancer patients heralds mortality, as disseminated disease is generally chemoresistant. After tumor cells reach the ectopic tissue, they undergo an epithelial reversion to enter a period of quiescence, termed dormancy, which may last for decades before outgrowing again as mesenchymal/dedifferentiated masses. Thus, long‐term, relatively non‐toxic interventions that prevent metastatic outgrowth are needed to treat this mortal stage of tumor progression.

Epidemiological analyses have suggested that statin usage, for cardiovascular indications, is correlated with a reduction in metastatic emergent (though not in incidence of primary) breast cancer. The goal of this study is to demonstrate this is due to statins suppressing breast cancer cell proliferation, a hallmark of emergent outgrowth.

We have found that atorvastatin and simvastatin limit the growth of some cancer cell lines, but not others. The sensitive lines were marked by lacking surface E‐cadherin, the hallmark of the mesenchymal phenotype. When E‐cadherin is downregulated on epithelial tumor cells, the cells become growth inhibited by the statins. Furthermore, this is a direct effect, as we now have shown that hydrophilic statins are relatively ineffective compared to the membrane permeant lipophilic statins as tumor cells generally lack the transporters that enable these drugs to gain access to the cells.

To determine whether the statins target the emergent metastatic tumor cells, we are using an all human microphysiological system (MPS) of the most common site for metastases, the liver. Briefly, a micro‐hepatic tissue is established by seeding primary human liver cells in a porous scaffold subject to a physiological flow. RFP‐labeled breast cancer cells are seeded into these microtissues and examined weeks later. Liver function and health are monitored by clinical chemistry assays performed on supernatant samples. We have previously shown that this system robustly reproduces tumor dormancy. Initial studies suggest that statins suppress the emergence of dormant tumor cells when challenged by stressors that lead to outgrowth. Statins were further found in vivo to influence breast cancer metastasis and metastatic growth using a mouse model of spontaneous liver metastases. As 26% of adults currently take a statin for other medical conditions, these studies may suggest the best statin to use in the context of maintaining breast cancer dormancy long‐term and delaying or avoiding the morbid emergence.

Support or Funding Information

NCATS/NCI (UH3TR000496) to AW and NCI F30 (1F30CA199947), CATER Training Program (T32 EB001026), and Pitt/CMU MSTP Program (5T32GM008208) to CB
Characterization of Immunodeficient Mouse Models
Authors: McClellan Jamie , Macasocal Richard , Hare Terri , Horn Mandy
Published: November 2023

Leakiness in SCID mouse models refers to the occasionally productive VDJ rearrangement leading to clonal expansion of these limited B and T cell clones. The rate and extent of leakiness in immunodeficient models is a factor of age as well as housing environment and the background strain of the model. In the current study, serum IgG was used to determine the extent of leakiness in various immunodeficient mouse models; survival and lymphoma incidence was also analyzed. Male and female (n=23–26) C.B‐17‐SCID, NOD SCID, SCID Beige, SHrN®, Beige Nude XID and Nude mice were housed in flexible film isolators. Blood was collected and analyzed using dried blood spot technology at 12, 16, 20 and 24 weeks of age for IgG concentration analysis using multiplex fluorescent immunoassay. The leakiness threshold was set at 5 μg/mL of IgG for analysis. At 24 weeks of age, necropsies were performed to determine incidence of lymphoma. At 24 wk of age, the SHrN® model had the smallest percentage of leaky animals followed closely by the NOD SCID. Beige Nude XID mice display an age associated increase in serum IgG levels as well as an increasing percentage of animals above the 5 μg/mL threshold from 12–24 weeks of age. Nude mice also show increases in IgG from 12 to 24 weeks of age. The NOD SCID and SHrN® mice had the lowest survival rate at 24 weeks; however, survivability in all models was greater than or equal to 79%. There is a positive correlation between the percentage of leaky animals and survival in males and females across all models. The C.B17‐SCID shows low incidence of lymphoma at 24 weeks of age whereas the NOD SCID model has the highest incidence of lymphoma but when the hairless mutation is crossed onto the NOD SCID mouse to create the SHrN®, incidence of lymphoma is decreased. This is the first study reporting IgG concentration in multiple immunodeficient models to examine leakiness as well as 24 week survival and lymphoma incidence. Additional studies measuring immune cell activity and function will be useful to further characterize immunodeficient models; however, these data will be valuable information in determining the best model for xenograft research.

Support or Funding Information

This research was privately funded by Envigo.
Molecular Diagnostic Techniques Used to Confirm an Unusual Case of BAPoma
Authors: Atkinson Aaron , Linos Konstantinos , Yan Shaofeng , Tsongalis Gregory , Lefferts Joel
Published: November 2023

Study Objective

BAP1 (BRCA1‐associated protein 1) is a critical tumor suppressor that promotes genome stability and DNA damage response, in addition to other cellular processes. BAP1 maps to 3p21 and is a nuclear localized deubiquitinating hydrolase that binds BRCA1, Ino80, and Histone2A‐monoubiquitin among other proteins. Herein, we employ advanced molecular diagnostic techniques to confirm the suspected diagnosis of a BAP1‐deficient Spitzoid Tumor (BAPoma) following confounding stereotypical histology and immunohistochemical tests. Proper assessment of Bap1‐deficient tumors is important as the likelihood of tumor progression if left untreated is currently unknown, however in other cancers such as uveal melanoma BAP1‐deficient tumors are treated as higher risk.

Clinical Presentation and Methods

A 45 year old female patient presented an irregular translucent papule on her shoulder that when biopsied had hallmarks of a BAPoma including: biphasic melanocytic proliferation, and immunostaining showing BRAF V600E; p16 expression was retained. However, nuclear BAP1 immunohistochemistry was maintained. To confirm the diagnosis of BAPoma, we performed Chromosomal Microarray (CMA) analysis using Oncoscan FFPE Assay (Affymetrix); Sanger sequencing of BAP1 commonly mutated regions; and SNaPShot primer‐extension based genotyping.

Results

DNA was extracted from the tissue biopsy and used in CMA testing that revealed a heterozygous loss of 3p. To reconcile the morphologic features with both the nuclear BAP1 staining and loss of 3p, Sanger sequencing of exons 4–7 and 17 PCR products was performed. Sequencing revealed a suspected c.505C>T p.H196Y mutation that was also confirmed by primer‐specific mini‐sequencing. This BAP1 variant is likely nonfunctional and pathogenic as, in addition to H169 being among 4 conserved catalytic residues, known pathogenic H169 substitutions, including H169Y, have been found previously in uveal melanoma, mesothelioma, and bladder cancer.

Conclusions

The presence of a nonfunctional allele within the nucleus combined with loss of BAP1 heterozygosity explains the clear and characteristic BAPoma morphology by histology. While such cases are rare, this case underlines the importance of molecular diagnostics when protein expression studies do not correlate with morphology. Future molecular testing could be refined to target known missense mutations in BAP1 that do not affect nuclear localization ‐ especially those in catalytic residues such as H169.
Intrinsic Reactivation of Human Endogenous Retroviruses in the Osteosarcoma
Authors: Koks Sulev , Koks Gea , Ho Xuan Dung , Maasalu Katre , Märtson Aare
Published: November 2023

The human genome contains human endogenous retroviruses (HERVs) and they occupy around 8% of our genome. These viruses are derived from ancient infections of retroviruses that were reverse transcribed and integrated into the genome of ancestral host animals. These viral genomes were fixed and inherited for tens of millions of years ago. Almost all of the HERVs have lost their infectivity and no known HERV is presently. The role of HERVs has been shown in the different inflammatory diseases and cancers. The goal of present study was to analyse the genome‐wide transcriptional activity of HERVs in osteosarcoma. We performed RNA‐seq analysis of 36 tumor‐normal paired samples, raw sequencing data were used to identify HERV sequences and counts data as expressional activity. DESeq2 package was used for statistical analysis.

We found massive and activation of HERVs in the osteosarcoma sample. More precisely, 58 different repeat element were differently described between normal and tumor bone with the FDR levele below 0.05. The most significantly upregulated elements were satellite elements ALR, ALR/ALPHA, ALR2, ALR6 and ALRA SAT elements. In addition, over‐expression of the pericentromeric human satellite II (HSATII) and the ancient mariner transposon (HSMAR1) was found. The most down‐regulated elements were HERVK22I, LTR40A, LTR40B, LTR40C and MER87B. Up‐regulation of the satellite elements during cancer has been described in some previous studies. However, genome‐wide analysis of all repetitive elements in the human genome is novel approach and could give additional information about the oncogenesis.

Support or Funding Information

This study was supported by institutional research funding (IUT20‐46) from the Estonian Research Agency and by the European Union's Seventh Framework Programme (FP7/2007‐2013) under grant agreement no. 602398 (Hyporth).
Cervical Cancer and HPV: Dilemmas and Resolution via Biomarkers
Authors: Markovic Olivera , Markovic Nenad
Published: November 2023

Recent research reports are cooling the initial enthusiasm about HPV and cervical cancer. It has been expected since the understanding the HPV is cancer growth promoting agent and only indirectly (persistent infection) cancerogenic agent. This old/new view is expanding the value of HPV immunization (prevention of invasive cancer growth) and is

Creating a new emphasis on HPV disease detection in healthy and patients with invasive cervical cancers.

However, this emphasis is not to HPV infection detection, but to HPV disease detection. We like to remind our audience to the fact that HPV is highly contagious but weekly virulent agent, meaning that it can be present in many cases but is causing chronic or persistent disease only in few. It perfectly fits with epidemiological data. But it fits with cytopathological data, too.

HPV is found in almost all invasive cervical cancers, but few HPV positive women develop cervical cancer. However, this relation is much more statically relevant in LSIL category where HPV disease is demonstrated as koilocytosis. HPV disease is diagnosed by morphological appearance of warts and cytopathological appearance of special cells – koilocytes. It is then clear that presence of koilocytes mean worse prognosis for the patients who has HPV disease positive signs together with DNA alteration positive signs – diagnosis of LSIL.

A composite biomarker, MEDYKO (FASEB 2016, v. 30, No. 1, Suppl. 696.5), contains a metabolic marker CAP which identifies abnormal cells on Pap smears that may proceed into cervical cancer. As metabolic markers appear before morphology, the presence of CAP (cervical acid phosphatase) in epithelial cells on Pap smears are signs even earlier than morphological DNA changes, and could easily be a usable alarm of pending cervical dysplasia. Thus, CAP may widen the region of morphologically suspicious Pap test slides to categories ASC‐US, BCC and RCC; hence, to reduce the inevitable false negative rates of the Pap smear morphology.

In conclusion, a combination of three biomarkers, all visible under the microscope, CAP, HPV and DNA could improve the Pap testing making it more accurate – sensitivity increased by CAP; specificity by HPV; faster – only one quality smear is needed, and for lower cost – all three parameters are visible on the same smear. Additional advantage is amenability of those images for digital image analysis and for electronic transfer enabling mobile telemedicine based mass cervical cancer screening globally.

Support or Funding Information

Global Academy for Women's Health is supporting BioSciCon, Inc. which is sponsoring development of tools for mass cervical cancer screening globally.
Heparan Sulfate Proteoglycans Mediate Renal Carcinoma Metastasis
Authors: Qazi Henry , Shi Zhong‐Dong , Song Jonathan W , Cancel Limary M , Munn Lance L , Tarbell John M
Published: November 2023

The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive tumor cells with high metastatic potential have enhanced invasion rates in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) in the glycocalyx plays an important role in this flow mediated mechanostransduction and upregulation of invasive and metastatic potential. In this study, highly metastatic renal cell carcinoma cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chamber and microfluidic assays, we show that flow‐enhanced invasion is suppressed in HS deficient cells. To assess the ability of these cells to metastasize in vivo, parental or knockdown cells expressing fluorescence reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from the NDST1 knockdown cells compared to control cells with intact HS. The ability of these cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx in tumor progression.

Heparan sulfate knockdown induced reduction in invasive tumor colonies per tissue sample for both the single clone and combined clones models*

Single Clone Model
Combined Clones Model

Control
Knockdown
Δ%
Control
Knockdown
Δ%

Kidney
1.8
0.3
−86
0.8†
0.0
−100†

Liver
1.7
0.1
−97
2.1
0.1
−94

Lungs
14.6
0.5
−97
7.2
0.1
−98

Abdomen
3.3
0.3
−92
2.6
0.0
−100

Spleen
1.8
0.0
−100
0.4†
0.0
−100†

p < 0.005 for each comparison between the number of invasive tumor colonies formed by either the control (CtGFP) or knockdown (shORG‐8) cell lines within each tissue type**

0.005 < p < 0.05 for the samples in the combined clones models where there were fewer observed cases of invasive colonies formed by the control (CtGFP) cell line

n=165 tissue samples for the single clone control case. n=66 tissue samples for the single clone knockdown case, and n=50 for the paired cases in the combined clones model
3,4,2,4 Tetrahydroxychalcone (Butein) Inhibition of TNFα‐Induced CCL2 Release in Triple Negative Breast Cancer Cells
Authors: Horton Anisley C , Bauer David F. , Soliman Karam F.
Published: November 2023

Butein is a natural 3,4,2,4 tetrahydroxychalcone compound found in the Asian tree Rhus verniciflua, which is traditionally used for cancer treatment. Monocyte chemotactic protein‐1 (MCP‐1/CCL2) is released by tumor tissues, acting as a potent chemokine enabling the mononuclear cells to differentiate into tumor‐associated macrophages (TAMs). TAMs promote tumor progression, invasion, infiltration, and dissemination. The presence of TAMs in the tumors are associated with poor prognosis and increased mortality in breast cancers. Compounds, which can attenuate or inhibit the release of CCL2 have been shown to prevent cancer‐associated pro‐oncogenic processes. In this study; we evaluated the effects of butein on the release of TNFα ‐ induced tumor promoting chemokines in triple negative breast cancer (TNBC), MDA‐MB‐231 cells. Our studies were performed using antibody microarrays which specifically showed that TNFα (40 ng/mL) induced protein expression of CCL‐2, GM‐CSF, angiogenin, GRO‐alpha, osteoprotegerin and IL‐8, were attenuated by the administration of butein (5 μM). Moreover, the effects of butein on the attenuation of TNFα mediated CCL‐2 release were validated by ELISA (68% inhibition). These findings provide evidence to suggest that butein can modulate immunological signaling of cancer cells, which can prevent the cascades that direct TAMs infiltration, a pivotal role in tumor cell initiation, progression, invasion, and dissemination.

Support or Funding Information

Supported by NIH Grants G12 MD007582 and P20 MD 006738
Hypoxia stimulates an increase of Neuropeptide Y Y1 and Y5 receptor expression in human breast cancer cells
Authors: Medeiros Philip John , Uniacke James
Published: November 2023

A hypoxic microenvironment is a common physiological attribute in solid tumors. Low O2 in tumors promotes a malignant phenotype, associated with increased proliferative, angiogenic and metastatic potential. Accumulating evidence suggests that neuroendocrine factors and their corresponding receptors may contribute to the initiation, development and progression of various cancers. Neuropeptide Y (NPY) and its receptors have been implicated as such a factor in the progression of breast cancer. Previous studies have reported the expression of the Y1R and Y5R receptor subtypes in human breast cancer cells and tumors. We have demonstrated that in breast cancer cells, NPY stimulation of Y5R increased proliferation and migration, and induced the release of vascular endothelial growth factor. In the current study, we examined the effects of O2 concentration on NPY receptor expression in MCF‐7 and MDA‐MB‐231 cells. Using quantitative PCR (qPCR) and Western blot, we compared Y1R and Y5R mRNA and protein expression in cells exposed to 1% (hypoxia) and 21% (normoxia) O2. Y1R and Y5R mRNA was quantified after 1h, 3h, 6h, 12h and 24h of hypoxia. Relative to normoxic cells, we observed a significant increase in Y1R and Y5R mRNA after 3h's of hypoxia in both cell lines. Protein expression of Y1R and Y5R was examined after a 24h hypoxic exposure. Y1R increased 2‐fold (MDA‐MB231) compared to normoixa. Y5R expression increased 79% (MCF‐7) and 55% (MDA‐MB231) relative to normoxic cells. Previous work from our group has demonstrated that hypoxic cancer cells require eukaryotic initiation factor 4E2 (eIF4E2) to direct the cap‐dependent translation of select mRNAs, therefore we tested whether the hypoxia‐induced increases in Y1R and Y5R expression were dependent on eIF4E2. Using MDA‐MB 231 cells depleted of eIF4E2, we found that hypoxic treatment had no effect on Y1R and Y5R expression compared to normoxic cells. These data provide insight into the O2‐mediated regulation of NPY receptors in breast cancer. Taken together with our previous functional NPY receptor studies, our findings provide further evidence of pathological implications of the NPY system in breast cancer. Ongoing studies will further elucidate the transcriptional and translational regulation of NPY receptors in response to hypoxia.

Support or Funding Information

Supported by Ontario Ministry of Research and Innovation and Cancer Research Society
Uncoupling Effects of ERα on LKB1/AMPK Signaling Induced by Adiponectin in Breast Cancer Cells
Authors: Mauro Loredana , Naimo Giuseppina Daniela , Gelsomino Luca , Spina Elena , Panno Maria Luisa , Andò Sebastiano
Published: November 2023

Obesity is increasingly recognized as an oncogenic factor. Adipose tissue is an active metabolic and endocrine organ that secretes many adipocytokines, which act as key mediators in several obesity‐associated diseases. Among these adipocytokines, adiponectin has been proposed as having a key role in the pathogenesis of cardiovascular disease and type 2 diabetes along with other diseases such as obesity‐associated malignancies, including breast cancer. An inverse correlation is reported between obesity and adiponectin, for which low levels of adiponectin represent a risk factor for mammary cancer. It is emerging how the role of adiponectin on breast tumorigenesis seems to be dependent on cell phenotypes. In this concern, our in vitro and in vivo studies demonstrated that adiponectin, at relatively low concentrations, repressed growth in ERα‐negative breast cancer cells whereas increased proliferation in ERα‐positive cells. Adiponectin interacts with specific receptors (AdipoR1 and AdipoR2) and exerts its effects, including regulation of cell survival, apoptosis and metastasis, via a plethora of signaling pathways. The key molecule of adiponectin action is AMP‐activated protein kinase (AMPK), which is mainly activated by liver kinase B1 (LKB1).

Here, we investigated the effect of adiponectin on LKB1/AMPK signaling in ERα‐negative (MDA‐MB‐231) and positive (MCF‐7) breast cancer cells. Our results indicate that, in MCF‐7 cells, adiponectin was unable to phosphorylate AMPK, while, in MDA‐MB‐231 cells, we observed an increased AMPK activation, as evidenced by the enhanced phosphorylative status and kinase activity. Concomitantly, we observed a significant activation of downstream effectors, such as mTOR and P70S6K only in ERα‐positive cells, resulting in the stimulatory effect of adiponectin on proliferation of MCF‐7 cells.

The crucial role of ERα in modulating the opposite response to adiponectin, as above reported, rises from the evidence that when we knocked down ERα in MCF‐7 cells, by siRNA, AMPK phosphorylation was restored, resulting in the inactivation of mTOR and its substrate P70S6K.

Then, we investigated the molecular mechanism through which ERα was able to interfere with AMPK activation. It was extremely intriguing to observe how upon adiponectin exposure LKB1 appeared localized in the nucleus with ERα, as revealed by co‐immunoprecipitation and ligation proximity assay. This addresses how, in such circumstance, LKB1 may be recruited in the nucleus impairing its capability to couple with AMPK in the cytosol. This may explain why adiponectin at low concentrations, as observed in obese women, plays a dichotomic effect on breast cancer growth depending on ERα expression.

Support or Funding Information

A.I.R.C., Associazione Italiana Ricerca sul Cancro