bluuebunny/crossref_metadata_2025 huggingface dataset:
Profiles of metabolic protein expressions in proteomic and phospho‐proteomic analysis of low folate modulates energy metabolism bioenergetics in colon cancer
Authors: Lan Wen‐Yu , Ku Wei‐Chi , Huang Rwei‐Fen Syu.
Published: November 2023

Previous studies we found that low folate microenvironment promotes cancer stem cell properties. Induction of cancer stem cells is associated with energy metabolism. How folate nutritional status induces cancer stem cell‐like potentials and energy switch mechanisms remains unclear. Aims of this study were to investigate profiles of metabolic protein expressions in proteomic and phospho‐proteomic analysis of low folate modulates energy metabolism bioenergetics in colon cancer. Human colon cancer cell line SW480 were cultured in medium containing 2.2 μM folate (control (C) group), 10 nM folate (low folate (LF) group) 4 days and 8 days. SW480 cells (control and LF) were scraped in a lysis buffer containing 4% SDS and protease inhibitor cocktails. After acetone precipitation, total proteins were digested by trypsin followed by dimethyl labeling at the peptide level. The labeled peptides were combined and analyzed by nanoLC‐MS/MS. The peptides were separated by MonoCap C18 High Resolution Ultra 2000 (0.1 × 2000 mm) column and analyzed by Thermo LTQ‐Orbitrap XL mass spectrometer. Peptide identification and quantification were performed by MaxQuant packages. Bio‐informatics analyses were performed by Perseus and GproX program. These proteomic analysis data observed regulated proteins related to energy metabolism, lipid metabolism, and TOR signaling. Among these proteins, mTOR, pyruvate dehydrogenase‐phosphatase 1, ADP/ATP translocase 3 were over 2 fold‐changes in LF group. ATP synthase was under 2 fold‐changes in LF group. Using phospho‐proteomic analysis data revealed that MAPK1, IRS2 were over 2 fold‐changes in LF group. Akt, S6K1, and pyruvate dehydrogenase protein phosphorylation were significantly up‐regulated in LF group. Overall protein and phosphorylated proteins related to metabolism were overlap and associated with cancer stem cell‐like potentials. And there were onco‐spheroid formation cells in LF treated group. These findings demonstrated that proteomic analysis was applied to metabolic protein expressions of low folate modulates energy metabolism bioenergetics in colon cancer.
Changes in Blood Folate Concentrations in Response to Intake of Folic Acid – a Systematic Review and Meta‐analysis
Authors: Crider Krista S , Devine Owen , Qi Yan Ping , Yeung Lorriane , Sekkarie Ahila , Zaganjor Ibrahim , Berry Robert J
Published: November 2023

Background

The threshold for optimal red bloodcell (RBC) folate concentration for the prevention of neural tube defects wasset at 400ng/ml (906 nmol/L) by the World Health Organization (WHO) in 2015. After countries determine the distribution of RBC folate concentrations among reproductive age women, it will be critical to know how much additional folic acid intake from either a supplement or a fortification program is needed to reach the optimal concentrations specified in the WHO guideline. It is currently recommended that women consume 400 micrograms (mcg) of folic acid a day either through a supplement, fortified foods, or both.

Objective

The objective of this project was to determine how much blood folate concentrations increase in response to increasing doses of folic acid after three months of intake (as when taking a supplement) and then again after intake at steady state (similar to fortification).

Results

To accomplish this we conducted a systematic review of the literature for studies that included a known dose of folic acid taken continuously for ≥28 days. We searched for publications in Medline, Embase, CINAHL, Cochrane Library, POPLINE, and ClinicalTrials.gov. Of the 13,682 abstracts reviewed, 516 were selected for full‐text review and data from 100 articles were abstracted. Using a Bayesian meta‐analytic approach, one‐compartment physiologically based pharmacokinetic(PBPK) models describing folate uptake and elimination were used to estimate the steady‐state concentrations of both serum and RBC folate levels at varying levels of folic acid supplementation. Preliminary estimates showed that at approximately 400 mcg folic acid/day, RBC folate concentration increased 85% [Uncertainty Interval:75%, 97%] at steady state (after more than about 30 weeks of intake).

Conclusions

These results suggest that at there commended intake of folic acid to prevent neural tube defects (400 mcg/day) at steady state, RBC folate concentrations almost double. Additional analyses are underway to determine the impact of different doses of folic acid on serum and RBC folate concentrations.
The Role of Lipocalin‐2 (Lcn2) in Acetaminophen Induced Acute Liver Failure
Authors: Paul Christina , Gajjar Nisha , Bhave Vishakha
Published: November 2023

Acetaminophen (APAP) toxicity is the number one cause of acute liver failure, and treatment options are limited. The initial toxicity is caused by the bio‐activation of the drug to the reactive metabolite, N‐acetyl‐p‐benzoquinone imine (NAPQI). However, progression of injury still occurs even after all of the drug has been eliminated from the body. APAP overdose involves sterile inflammation. Lipocalin‐2 is an innate immune protein that is known to be up‐regulated in various disease models and has been observed to have a protective as well as destructive role dependent on the model analyzed. Studies have shown that Lcn2 acts as an anti‐inflammatory modulator in low doses of toxicity, when pro‐mitogenic signaling is activated. In contrast, these repair and regeneration pathways are shown to be inhibited in a high dose model of toxicity, which ultimately leads to acute liver failure. The role of Lcn2 in a high dose model of toxicity has not yet been investigated. This study aims to investigate the role of Lcn2 in drug induced acute liver failure (APAP overdose) model and the mechanism of its involvement. An in vivo model of acute liver failure will be utilized and Lcn2−/− mice will be used as our intervention. We hypothesize that Lcn2−/− mice will be protected from APAP induced acute liver failure. Time course studies will be done over a period of 0–96 hours post APAP overdose. Liver and serum will be harvested at each time point and extent of injury will be assessed via ALT/AST assays and H&E staining. Bio‐activation studies will be done to determine any differences in the metabolism of the drug between the treatment groups, which include protein adduct formation and glutathione depletion and replenishment. Pro‐ and anti‐inflammatory cytokines will be measured to elucidate the role of Lcn2 in mediating progression of injury. Proliferating cell nuclear antigen (Pcna) staining will be done as a marker of liver regeneration. Our preliminary data demonstrate 20% lethality in Lcn2−/− mice and 60% lethality in WT mice subjected to APAP overdose. In conclusion, the protection of Lcn2−/− mice from APAP overdose indicates that Lcn2 may play a role in mediating progression of injury. Further experiments will be done to elucidate the mechanism of action.

Support or Funding Information

Philadelphia College of Osteopathic Medicine‐Division of Research
Transcriptional Response in a Rat Model of Liver Ischemia‐Reperfusion Injury
Authors: Zabala Valerie , Thevenot Paul , Cohen Ari , Gruppuso Philip A. , Sanders Jennifer A.
Published: November 2023

Ischemia‐reperfusion (I/R) is central to the pathophysiology of liver injury caused by acute hepatic traumas, including liver transplantation. Elevated levels of reactive oxygen species (ROS) govern the magnitude of liver injury and tissue damage. Therapies that reduce the ROS burden are under investigation to determine their ability to improve transplant outcomes including those associated with extended criteria donors. However, the pathways and upstream regulators that drive ROS‐mediated responses remain unknown. In this study, we have utilized a rodent model of 70% partial transient warm hepatic ischemia to characterize transcriptional changes in response to ischemic insult and during early reperfusion. Blood supply to the left and medial lobes was clamped for 30 minutes while the right lobes remain perfused. Animals were sacrificed at the end of ischemia (no reperfusion), and after thirty minutes or six hours of reperfusion. The ischemic lobes and the non‐ischemic lobes were each compared to tissue from animals that underwent sham surgery at each time. Total RNA was prepared for microarray and analyzed using Affymetrix Rat Transcriptome 1.0 arrays. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) of the 24,536 quantified genes revealed a great overlap in biological pathways and upstream regulators that changed in both the ischemic and non‐ischemic lobes when compared to shams. This implies a significant proportion of changes in the ischemic‐reperfused lobes were attributable to the effects of anesthesia and/or surgery. We found 1575 differentially expressed genes (FDR<0.05) in the ischemic‐reperfused group at 30 minutes and 2672 genes at 6 hours. However, 1067 and 1646 of the altered genes at 30 minutes and 6 hours, respectively, also changed in the non‐ischemic lobes, suggesting systemic factors play in role in the response to I/R. Among those genes unique to the ischemic‐reperfused group at thirty minutes, the expression of several immediate early genes (Egr‐1, Fos‐b, Jun‐b) increased over five‐fold. This response could indicate that induction of AP‐1 (activator protein‐1) in response to hypoxia plays a role in promoting cell survival. Further investigation of AP‐1 activity may identify new pharmacological targets to decrease hepatic ischemia‐reperfusion injury. This immediate early gene response, which is characteristic of liver regeneration after partial hepatectomy, has not been previously identified in models of hepatic ischemia‐reperfusion.

Support or Funding Information

This work was supported by NIGMS grant 5T32GM077995 from the National Institutes of Health, a Pharmacia Predoctoral Fellowship, and the Graduate Research Fellowship Program of the National Science Foundation (NSF).

Valerie Zabala is a Howard Hughes Medical Institute Gilliam Fellow.
Leukocyte Specific Protein‐1 (LSP1) Functions as a Suppressor of Proliferation During Liver Regeneration After Partial Hepatectomy (PHx) and in Primary Hepatocytes
Authors: Koral Kelly , Bowen William , Haynes Meagan , Mars Wendy , Michalopoulos George
Published: November 2023

Leukocyte specific protein‐1 (LSP1), an f‐actin binding protein expressed in hematopoietic cells and hepatocytes, is the gene with the highest copy number variation in human hepatocellular carcinoma. Our previous work has demonstrated that loss of LSP1 expression leads to increased proliferation and migration of rat hepatocellular carcinoma cells. We have also demonstrated that overexpression of LSP1 in mouse liver through hydrodynamic tail vein injection of LSP1 plasmid DNA results in a significant decrease in proliferation on day 2 after partial hepatectomy (PHx). To further characterize the role of LSP1 in liver regeneration, we performed 2/3 PHx on both a global LSP1 knockout mouse as well as a hepatocyte specific LSP1 transgenic mouse model and analyzed proliferation using ki67 immunohistochemistry as well as western blot analysis of ERK signaling. Since expression of LSP1 leads to decreased proliferation, we hypothesized that the knockout mouse model would exhibit increased proliferation and activation of ERK whereas the transgenic mouse model would display decreased proliferation and pERK expression. LSP1 knockout mice displayed an increase in Ki67 labeling on day 4 after PHx and western blot analysis shows increased pERK expression after PHx in comparison to controls. In the transgenic mouse model, a significant decrease in Ki67 positive hepatocytes was observed on day 4 after PHx as well as decreased pERK expression. Transgenic mice also displayed decreased liver to body weight ratios on day 7 following PHx. Hepatocytes isolated from knockout mice exhibited increased proliferation in the absence of growth factors compared to controls whereas transgenic mouse hepatocytes displayed decreased BrdU incorporation in both the presence and absence of growth factors. LSP1 knockout hepatocytes and transgenic hepatocytes also displayed increased and decreased pERK expression, respectively. Our results demonstrate that LSP1 functions as an inhibitor of proliferation in primary hepatocytes as well as during liver regeneration following PHx. Future studies aim to address the signaling pathways downstream of LSP1 that effect proliferation during liver regeneration as well as how LSP1 functions in hepatocarcinogenesis mechanisms and sensitivity to sorafenib.

Support or Funding Information

Menten Endowment, University of Pittsburgh
Mass spectrometry analysis of the phosphoproteome in an in vitro lipotoxicity model
Authors: Khachatoorian Ronik , Cohn Whitaker , Ganapathy Ekambaram , Liu Natalie , Vu James , Lu Nu , Whitelegge Julian , French Samuel W
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is estimated to affect between 75 to 100 million individuals in the United States and is predicted to become the most common indication for liver transplantation. Here, we established a lipotoxicity model in Huh7 cells exposed to palmitic or oleic acid to identify kinase pathway alterations that may be related to NAFLD. We demonstrated increased phosphorylation of JNK and induction of GRP78 and HSP70 by Western analyses. We then assayed changes in the phosphoproteome of Huh7 cells in response to lipid treatment. Briefly, Cells were treated with lipid (75 μM of palmitic or 300 μM of oleic acid) or DMSO control (three replicates for each) for 72 hours. Subsequently, cells were harvested and lysed and the extracted proteins were subjected to trypsin treatment and differentially labeled. Phosphopeptide enrichment was performed by Immobilized Metal Affinity Chromatography (IMAC) followed by liquid chromatography/tandem mass spectrometry (LC/MS‐MS). Phosphopeptides with a fold change of at least 1.2 were considered for data analysis. Differentially phosphorylated peptides were subjected to Pathway Commons analysis by WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/) to identify the pathways affected. Treatment with palmitic acid resulted in dramatic changes in the phosphoproteome, while oleic acid had a milder effect. The results identified several pathways previously shown to be involved in steatosis including TRAIL, mTOR, PI3K, uPA, and VEGF as well as specific proteins known to be involved in steatosis such as GRB14, JUND, MAP3K7, FGFR4, YAP1, and TRIM28. We also identified novel proteins not previously implicated in lipotoxicity including COPA, NF1, RAF1, MTDH, HMGA1, and RPS6KA3. The increased phosphorylation of JNK and altered phosphorylation of MAP3K7, RAF1, and RPS6KA3 highlights the role of the MAPK/ERK pathway in steatosis. We also identified altered phosphorylation of the tumor suppressors NF1, PDCD4, and TLE4 and the proto‐oncogenes RAF1, MTDH, JUND, FGFR4, RPS6KA3, YAP1, and HMGA1 raising the possibility of their role in steatosis‐related carcinogenesis. These results may provide further insights into the pathology of fatty liver disease and potential therapeutic interventions.
Mice Lacking Liver‐Specific [beta]‐Catenin Develoop Steatohepatitis and Fibrosis After Iron Overload
Authors: Preziosi Morgan , Singh Sucha , Valore Erika , Jung Chun‐Ling , Nagarajan Shan , Ganz Tom , Monga Satdarshan
Published: November 2023

Background & aims

Iron overload disorders such as hereditary hemochromatosis and iron‐loading anemias are a common cause of morbidity from liver diseases and are risk factors for hepatic fibrosis and hepatocellular carcinoma (HCC). Once iron‐induced damage occurs, treatment options are limited, partly because of the lack of animal models recapitulating human disease, since mice do not exhibit fibrosis or HCC following chronic iron overload.

Methods

A high iron containing diet was fed to normal mice to assess any effect on Wnt signaling. Iron overload diet was also administered to liver‐specific β‐catenin knockout (KO) and control (CON) mice for three months. To ameliorate an oxidant‐mediated component of tissue injury, N‐Acetyl‐L‐(+)‐cysteine (NAC) was added to drinking water of mice on iron overload diet.

Results

KO on iron diet (KO+Fe) exhibited remarkable steatosis, inflammation, hepatic fibrosis, regenerating nodules, and sequelae associated with increased oxidative stress. We also noted increased activation of AKT and NFkB pathways in KO+Fe. Addition of NAC to drinking water protected KO+Fe from hepatic steatosis, injury and fibrosis, and suppressed phosphorylation of AKT and NFkB.

Conclusions

We provide evidence that lack of hepatic b‐catenin predisposes mice to hepatic injury and fibrosis following iron overload thus phenocopying disease in patients. This is chiefly due to lack of antioxidant response in the absence of b‐catenin demonstrating its role in hepatic redox homeostasis. Lastly, antioxidant therapy may have a chemopreventive role in the management of chronic iron overload.
Proteomic Analysis of Focal Lesions in a Rat Model of Progenitor Marker‐Positive Hepatocellular Carcinoma
Authors: Michael Adeola O Adebayo , Ahsan Nagib , Francois‐Vaughan Heather , Zabala Valerie , Post Stephanie , Brilliant Kate E , Salomon Art , Gruppuso Philip , Sanders Jennifer A
Published: November 2023

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR), markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt‐Farber) model of HCC in the rat. In the present study, we characterized the proteome of persistent, pre‐neoplastic focal lesions in rats subjected to the Solt‐Farber protocol. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and sacrificed 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to animals that underwent a protocol to activate hepatic progenitor cells. Normal rat liver was used as an additional control. Regions of formalin‐fixed, paraffin‐embedded tissue were obtained by laser capture microdissection. Peptides were generated and analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), which yielded 11,070 unique peptides representing 2,227 proteins. Peptides that showed increased abundance in placebo foci represented known HCC markers (glutathione S‐transferase‐P, epoxide hydrolase, 6 others) and potential markers (aflatoxin aldehyde reductase, carbonic anhydrase 2, 11 others). Peptides derived from cytochrome P450 enzymes were generally reduced. Neither placebo nor rapamycin samples showed similarity with the progenitor cell samples, indicating that the foci in this model, while considered to be progenitor marker‐positive, do not show evidence of persistent progenitor cells. Pathway analysis, based on non‐parametric identification of affected peptides, was consistent with an effect of rapamycin to reduce dedifferentiation in foci. Finally, we have demonstrated that the combination of LCM and LC‐MS/MS is an effective approach to characterize focal liver lesions.

Support or Funding Information

NIH R01DK100301 NIH R01HD024455Rhode Island Hospital Department of Pediatrics
Understanding the contribution of macrophage‐specific Wnts to hepatic tumorigenesis and cancer progression
Authors: Preziosi Morgan , Monga Satdarshan
Published: November 2023

Hepatocellular carcinoma (HCC) incidence and mortality continue to rise, and treatment options have not advanced passed liver transplant or resection. Considering HCC occurs in a fibrotic background in up to 90% of cases, understanding the kinetics of injury progression to tumorigenesis and tumor advancement will serve significant therapeutic potential. Aberrant Wnt signaling has been observed in HCC and may be an attractive therapeutic target considering there are Wnt inhibitors in phase I clinical trials (NCT01351103, NCT01608867) for solid tumors. However, a major concern with non‐discriminatory Wnt inhibition is associated toxicity, and the repercussions of inhibiting all canonical and noncanonical Wnt signaling. Therefore, we seek to identify the source of Wnts required for tumorigenesis, and hypothesized that macrophage‐specific Wnts are important for this process. We subjected macrophage‐specific wntless knockout animals (KO), unable to secrete Wnts from macrophages, to the carcinogenesis model DEN/CCl4. Intriguingly, KO displayed a lower tumor burden and liver weight to body weight ratio than control (CON) mice. CON had larger lung metastases suggesting more aggressive disease, overall implying that macrophages secrete Wnts necessary for complete tumorigenesis. In addition to identifying a mechanism for decreased tumor burden, we will assess whether macrophage Wnts contribute to tumor development in the absence of hepatic injury and fibrosis using hydrodynamic tail vein injections to insert oncogenes using sleeping beauty transposons. Our lab has previously shown this robust model to cause HCC in an otherwise healthy liver. These studies will enable us to identify the role of macrophage‐specific Wnts in the tumor microenvironment present in a fibrotic liver, and in tumor development in the absence of a priming environment.
Analysis of Thermography and Breast Cancer Prevention Among Minority Women and Other Ethnic Groups
Authors: New Katina M , Einstein George P , Tulp Orien L
Published: November 2023

Breast cancer is often a devastating disease that affects millions of women, regardless of race, ethnic group or socioeconomic status annually. The goal of this research is to demonstrate the high‐risk breast patterns as detected via Infrared Thermography and how the thermographic patterns can be improved with lifestyle and other changes, thereby decreasing the individual's risk for developing late stage breast cancer. Results of data obtained from fifteen women who underwent breast thermography, via a digital infrared imaging camera called the FLIR T420 were compared via t‐test. The goal of the study was to stratify patients on the scale of fibrocystic or precancerous breasts into none, mild, moderate and severe categories, and to identify high risk, moderate risk, and low risk groups based on a risk assessment questionnaire, blood tests for vitamin D 25‐OH, and testing for genetic mutation of the Methylenetetrahydrofolate reductase [MTHFR] gene. From this risk stratification we then determined the need for incorporating a prevention protocol which included: 1] education on diet, 2] exercise and prevention of exposure, 3] detoxification of environmental toxins; 4] education on stress relief; 5] education on breast self‐examination; 6] iodine and vitamin D supplementation; and 7] local application of Frankincense oil to the breast to help attenuate inflammatory processes. Patients were re‐evaluated after the initial exam at intervals of three months, six months and one year. This follow up monitored the benefits of the protocol and documented the progression of decrease in high risk factors for each patient. All 15 participants complied with all treatment regimens throughout the study. Participants exhibited marked improvements in breast health at the conclusion of the study. The results of this study indicate that beast examination via therrmography is effective in the aid of breast cancer prevention. These results indicate that thermography is an effective tool that should be utilized more by physicians and other health care providers to assist with early detection and prevention of breast cancer. Also, the results of this study support the encouragement of routine exercise, maintaining a BMI less than 30 and strategies that assist in stress management. Early MTHFR genetic testing will also help determine if the patient is at an increased risk for developing breast cancer prior to the onset of symptoms.

Support or Funding Information

Supported by Institutional Resources of USAT and Einstein Medical Institute.
Nuclear Trafficking of ABCB1‐Daunorubicin Vesicles Initiated by Sphingomyelinase Reverts Multidrug Resistance in Chinese Hamster Fibroblasts
Authors: Lee Wing‐Kee , Kolesnick Richard N
Published: November 2023

Effective cancer chemotherapy treatment is hampered by multidrug resistance (MDR), defined by upregulation of ABCB1, which effluxes drugs. Intracellular ABCB1 also sequesters anthracycline drugs in acidic vesicles preventing nuclear targeting. Reports indicate sphingolipids affect vesicular trafficking. We hypothesized sphingomyelinase (SMase) activity might mobilize ABCB1/drug vesicles to revert MDR. Using drug‐resistant ADX fibroblasts expressing ABCB1‐EGFP, time‐lapse confocal microscopy revealed daunorubicin (DNR) sequestration in ABCB1‐EGFP‐containing vesicles within 5min. In ADX/ABCB1‐EGFP loaded with DNR (20μM,2h) followed by drug washout, exogenous bacterial (b)SMase, which hydrolyzes plasma membrane sphingomyelin and initiates sphingolipid signaling, induced rapid ABCB1‐EGFP/DNR vesicle movement to the nucleus, where DNR was deposited, while ABCB1‐EGFP remained in nuclear membranes. Immunoblots of isolated nuclei and nuclear envelope membranes purified by sucrose density gradient confirmed these observations. In DNR‐exposed ADX cells, bSMase‐induced ABCB1 translocation to the nucleus peaked at 1h, and was detectable at 4h but not at 8h, suggesting transient incorporation of ABCB1 into the nuclear membrane. No nuclear ABCB1 was observed in drug sensitive DC3F cells loaded with an isotoxic DNR dose (0.2μM, 2h), indicating ABCB1 nuclear translocation by bSMase is MDR specific. Cell viability studies by MTT assay demonstrate augmented DNR toxicity in the presence of bSMase and can be enhanced by co‐incubation with the ABCB1 inhibitor, PSC833/valspodar (1μM), to prevent cellular drug efflux, in an additive manner. Intriguingly, in the postnuclear supernatant of DNR‐treated ADX cells, ABCB1 primarily sediments at 8000g, but shifts to 18000g at 4h after bSMase suggesting altered vesicular density or ABCB1 transfer to a distinct vesicular pool. In conclusion, SMase activity mobilizes ABCB1/DNR vesicles to the nucleus where DNR is deposited and reverses MDR.

Support or Funding Information

The work was supported by the Max Kade Foundation and Intramural Research Program at Witten/Herdecke University (IFF2016‐20) (W.K.L).
Optimization of Schwann Cell Differentiation from Porcine Stem Cells
Authors: Schomberg Dominic , Shanmuganayagam Dhanansayan
Published: November 2023

Swine as a translational animal model of cancer is becoming increasingly recognized for its value in closing the gap between basic research and the development of clinically effective therapies. While rodent models have helped elucidate the complexities of many mechanistic pathways of cancer, they often have physiological and genetic differences that greatly limit the translatability of results to humans. The recognition of the value of swine models has also created a need for swine‐based in vitro cell culture platforms for mechanistic studies and drug screening. Such platforms are particularly sought after for tackling Schwannomas (Schwann cell based tumors) that are associated with diseases like neurofibromatosis 1 (NF1), a genetic disease caused by a mutation of the neurofibromin 1 (nf1) gene. These tumors are often benign, however, a small portion of them become malignant, degenerating into a form of cancer called neurofibrosarcoma. Patients with NF1 often develop neurofibromas from Schwann cells that can become malignant peripheral nerve sheath tumors, a tumor type that carries poor prognosis. Schwann cells, like most cells of a neuronal lineage, are challenging to culture in vitro. This has slowed progress of understanding the mechanisms responsible for these types of cancers. We therefore aim to develop an in vitro culture platform using swine cells to overcome these obstacles. This necessitated the optimization of culturing conditions to differentiate porcine stem cells into Schwann cells and then develop tumorigenic stem cells by the manipulation of the nf1 gene.

We isolated adipose‐derived mesenchymal stem cells and testis‐derived spermatogonial stem cells for use in our experiments. Culturing parameters manipulated include temperature, matrix composition, growth factor levels, feeder cell status, and passaging conditions. In the subsequent phase, different stem cell transfection methods were studied with the goal of delivering CRISPR/Cas9 components targeting the nf1 gene. Techniques compared include lipofection using Lipofectamine 2000 and 3000 and electroporation using the Gene Pulser II or Neon Transfection System. Our findings will help researchers establish a more robust in vitro model for culturing Schwann cells from swine stem cells to study cancer development.

Support or Funding Information

The research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF Network, NF Central Plains, NF Michigan, NF Northeast, and Washington State NF Family.
African Americans with Pancreatic Ductal Adenocarcinoma Exhibit Gender Differences in Kaiso Expression
Authors: Mukherjee Angana , Jones Jacqueline , Yates Clayton
Published: November 2023

Kaiso, a bi‐modal transcription factor, regulates gene expression and is elevated in breast, prostate, and colon cancers. Depletion of Kaiso in other cancer types leads to a reduction in markers for the epithelial– mesenchymal transition (EMT), however, its clinical implications in pancreatic ductal adenocarcinoma (PDCA) have not been widely explored. PDCA is rarely detected at an early stage but is characterized by rapid progression and invasiveness. Herein, we report the significance of the subcellular localization of Kaiso in PDCAs from African Americans. In the overall patient population, we observed a higher expression of cytoplasmic Kaiso in high‐grade pancreatic tumors compared to low‐grade pancreatic tumors (P = 0.0435). Kaiso expression was higher in the cytoplasm of invasive and metastatic pancreatic cancers (P < 0.0001). In males, cytoplasmic and nuclear expression of Kaiso correlated with cancer grade and lymph node positivity (P = 0.0013; P = 0.0112 and P = 0.0177, respectively). In male and female patients, cytoplasmic Kaiso expression correlated with invasiveness (P = 0.0101). Further, analysis of the largest PDCA dataset available on ONCOMINE shows that with an increase in Kaiso expression, there is an overall increase in Zeb1, which is the inverse for E‐cadherin. Hence, these findings suggest an oncogenic role for Kaiso in the progression of PDCAs, involving the EMT markers, E‐cadherin and Zeb1.

Support or Funding Information

This work was supported by the Department of Defense Prostate Cancer Research Program (PC120913) [CY], G12 RR03059‐21A1 (NIH/RCMI) [CY], (NIH/NCI) 1 R21 CA188799‐01 [CY] and U54 CA118623 (NIH/NCI) [CY], a pilot project on Pancreatic SPORE 2 P50 CA101955‐11 [CY], Statistical analysis was performed by Tuskegee University Statistical Core.
Aspirin attenuates melanoma tumor growth via PGF2α‐SOX‐2 dependent pathways
Authors: Thyagarajan‐Sahu Anita , Sahu Ravi P
Published: November 2023

Introduction

Exposure to pro‐oxidative stressors including cancer therapies via its ability to generate reactive oxygen species (ROS) produce potent oxidized lipid mediator, platelet‐activating factor (PAF) and PAF‐like agonists. We have shown that these PAF‐agonists significantly: 1) augmented the growth of subcutaneously implanted murine B16F10 melanoma tumors in a PAF‐receptor (PAF‐R) dependent manner; 2) impeded the efficacy of cancer chemotherapeutic agents and radiation therapy in murine melanoma models and; 3) detected in the perfusates and tumors collected from chemotherapy and radiation therapy‐treated melanoma and non‐melanoma cancer patients. As melanoma tumors often acquire resistance to the known chemotherapy, radiation therapy and even novel targeted therapeutic regimens, it is necessary to investigate an alternative approach for its intervention. Aspirin (ASN) has classically been used as an anti‐inflammatory drug and also possesses anti‐cancer activity in‐part via its ability to inhibit enzyme cyclooxygenases. While anti‐carcinogenic activity of ASN has been shown in other cancers, little is known about its effects in melanoma.

Objectives

The current studies were sought to investigate the mechanism and test the hypothesis if ASN‐mediated anti‐melanoma activity is via inhibition of PAF‐R activation.

Methodology

Cell proliferation and survival assay, apoptosis by caspase 3/7 luminescence assay, immunoblotting, gene array, quantitative real time PCR and flow cytometry studies were performed. These assays were conducted in stably PAF‐R‐expressing (B16‐PAFR) and deficient melanoma (B16‐MSCV) cells and C57BL/6‐wild‐type (WT) and PAF‐R knockout (PAFR−/−) mice.

Results and Discussion

We demonstrate that ASN inhibits the survival of B16‐PAFR and B16‐MSCV cells at similar rates in a time and dose dependent manner via inducing apoptosis as measured increased caspase 3/7 activity. These ASN‐mediated effects were blocked by PGF2α addition but not by PGE2. Importantly, ASN‐mediated significant suppression of murine B16‐PAFR and B16‐MSCV melanoma tumor growth was independent to both cellular and stromal PAF‐R expression. Moreover, gene array analysis of melanoma tumors revealed an inhibition of SOX‐2 oncogene by ASN. Furthermore, PGF2α add back upregulated SOX‐2 and rescued melanoma cells from ASN‐mediated inhibition of cell survival and induction of apoptosis.

Conclusion

ASN targets PGF2α and downstream SOX‐2 to inhibit melanoma tumor growth in a PAF‐R independent pathway and provide the rationale for its implication as a novel melanoma therapy.

Support or Funding Information

NIH K22 ES023850 and WSU‐RIF 190002
MOLECULAR ANALYSIS OF THE BLOOD‐TUMOR BARRIER IN BRAIN METASTASIS FROM BREAST CANCER
Authors: Lyle L. Tiffany , Duchnowska Renata , Lockman Paul , Adkins Chris , Shareef Afroz , Sechrest Emily , Hua Emily , Liewehr David , Steinberg Seth , Kloc Wojciech , Nayyar Naema , Brastianos Priscilla , Patricia Steeg , Gril Brunilde
Published: November 2023

Brain metastases of breast cancer are increasing in incidence; chemotherapy is generally ineffective due to poor overall uptake, and heterogeneous permeability. Herein, functional components of the blood‐brain barrier (BBB)/blood‐tumor barrier (BTB) were characterized in highly permeable and poorly permeable experimental brain metastases from breast cancer. Brain seeking variants of 3 breast cancer cell lines, MDA‐MB‐231‐BR6, JIMT‐1‐BR3, and SUM190‐BR3 were injected into the left cardiac ventricle of mice to produce brain metastases. Mice were injected with 3kd Texas Red dextran and sterilely perfused; highly permeable metastases were identified by exudation of red dye. Immunofluorescence expression of BTB functional components was quantified in uninvolved brain, highly permeable metastases, and poorly permeable metastases. We report a significant increase in endothelial cell and neuroinflammatory components in brain metastases compared to uninvolved brain in all 3 models. There was no trend in these markers in highly permeable versus poorly permeable metastases. Interestingly, increased expression of the desmin+ subpopulation of pericytes was associated with highly permeable metastases compared to poorly permeable metastases in the 231‐BR6 (p=0.0002), JIMT‐1‐BR3 (p=0.004), and SUM190‐BR3 (p=0.008) models. This increase was accompanied by a decrease in CD13+ pericytes in the 231‐BR6 (p=0.014), JIMT‐1‐BR3 (0.002), and SUM190‐BR3 (p=ns) models. Findings for both pericytes populations were corroborated in human craniotomy specimens. Preliminary functionality experiments demonstrated increased permeability in an in vitro model of the BTB with desmin+ pericytes compared to CD13+ pericytes. These alterations provide testable hypotheses for the development of new targeted therapeutic strategies to increase chemotherapeutic delivery to brain metastatic lesions.
Expression of matricellular protein CCN1 in the tumor stroma is required for melanoma metastasis
Authors: Leask Andrew , Postovit Lynne , Quensel Katherine , Hutchenreuther James
Published: November 2023

Metastatic melanoma has an extremely poor prognosis. BRAF inhibitors are used to treat a subset of patients but eventually patients acquire resistance to this therapy. An increasing body of evidence suggests that an activated tumor stroma is responsible for the acquisition of resistance to chemotherapy. Herein, we find that he secreted matricellular protein cyr61(CCN1) is overexpressed in human melanoma in a fashion that correlates negatively with clinical outcome, positively with the extent of tumor stroma development and not at all with BRAF mutational status. We have used a fibroblast‐specific type I collagen promoter‐driven cre recombinase that is tamoxifen dependent to generate C57BL6 mice deleted or not for CCN1 in fibroblasts. CCN1‐deficicent mice display progressive reduction in skin thickness. When injected subcutaneously, B16(F10) cells show reduced metastasis to the lung. Tumor growth is unaffected. This phenotype is linked with reduction in expression of collagen‐modifying enzymes LOX, P4H and PLOD2 as well as a decrease in insoluble collagen protein expression as visualized by a hydroxyproline assay and by trichrome staining. Type I collagen mRNA expression is unaffected.. Thus, CCN1 is required for proper induction of a tumor stroma and hence for metastasis and thus should be further evaluated as a possible therapeutic target for BRAF inhibitor‐resistant melanoma.

Support or Funding Information

CIHR, Canadian Cancer Society
Assessing the effects of TIMP2 knockout on lung cancer cell lines cultured in 3D
Authors: Peeney David
Published: November 2023

Tissue inhibitor of matrix metalloproteinases (TIMPs) are a small family of endogenous proteins that classically function to inhibit metalloproteinase activity. Since the original description of this protein family in the 80s and 90s, various MMP‐independent biological functions of TIMPs have been described. This built the impression that MMP/TIMP ratios may play an important role in tissue homeostasis, an idea which is supported by the observation that altered MMP/TIMP expression ratios are often associated with a number of human conditions such as cancer, cardiovascular and CNS disease. TIMP2 is the most abundantly expressed protein in this family and has previously been shown to interact with several membrane proteins including MT1‐MMP, insulin‐like growth factor‐1 receptor (IGF‐I‐R) and alpha3 beta1 integrin (α3β1) to mediate downstream intracellular signaling. In addition, TIMP2 has been shown to inhibit growth factor stimulated proliferation, angiogenesis and tumor cell invasion and metastasis, highlighting the potential for TIMP2‐based cancer bio‐therapies that can be used in conjunction with conventional treatments. Recent studies in our lab highlight that syngeneic lung tumors (LL2 cells; Lewis lung carcinoma) developed in C57BL mice harboring a loss‐of‐function mutation in TIMP2 are significantly larger than tumors grown in their WT counterparts. To gain a deeper understanding of the role of TIMP2 in tumor initiation and progression we have used CRISPR‐Cas9 to develop stable TIMP2 knockout (T2KO) human lung cancer cell lines. Although indistinguishable in 2D culture, T2KO tumor cells display a morphologically distinct phenotype when grown in spheroids. Preliminary data show that, when grown in spheroids, T2KO cells exhibit enhanced EGFR activation in comparison to WT cells. By assessing the functional characteristics and gene expression of T2KO cells grown in 3D culture conditions we hope to gain further insight into the biological functions of TIMP2 and to provide a mechanistic link between the loss of TIMP2 activity and enhanced tumor formation that is observed in our mouse model.
Enlarged endosomes and altered transferrin recycling in breast cancer cells
Authors: Tubbesing Kate Elizabeth , Malhotra Aditi , Rudkouskaya Alena , Barroso Margarida
Published: November 2023

Understanding how the endocytic pathway is regulated in different types of cancer cells remains a critical biological question with far reaching implications in the development of receptor‐targeted cancer therapies. To address this problem, we have characterized the regulation and organization of the endocytic pathway in triple‐negative breast cancer cells (MDA‐MB‐231), luminal A breast cancer cells (T47D) and non‐cancerous breast epithelial cells (MCF10A). We have focused this study on the well‐known endocytic pathway of transferrin (Tf) and the Rab‐GTPases, which are known regulators of the early, late and recycling endocytic compartments. Super‐resolution microscopy and quantitative confocal morphological analysis have been used to characterize the enlarged Tf and early endosome antigen‐1 (EEA1) positive endosomes in breast cancer cell lines. These enlarged Tf and EEA1 positive endosomes are also observed in human breast cancer tumor xenografts. Our Tf recycling assay shows that T47D cells retain significantly more Tf, compared to MDAMB231 or MCF10A cells. However, the observed Tf recycling behavior in different cancer cells does not correlate with the protein expression levels of transferrin receptor or different endogenous Rab‐GTPases evaluated. Interestingly, the colocalization of EEA1 with a panel of rabs differs between cell lines. This data indicates that differences observed in Tf recycling and endosomal morphology in different cancer cell lines may be dependent on the organization of the Rab‐GTPases, which alters the regulation of the endocytic pathway. Currently, we are performing quantitative live‐cell airyscan microscopy experiments to address the link between the altered endosomal morphology, receptor trafficking and localization of over‐expressed Rab‐GTPases. This research is essential to the optimization of receptor‐targeted anti‐cancer therapies, as the regulation of the endocytic pathway determines the retention, release and ultimate efficiency of the anti‐cancer therapy.

Support or Funding Information

Albany Medical College internal funds
MiR‐105/93‐3p promotes metastasis, chemoresistance and stemness in TNBC and circulating miR‐105/93‐3p act as predictive biomarker for TNBC chemoresistance
Authors: Li Hao‐Yi , Lu Pei‐Jung
Published: November 2023

Triple negative breast cancer (TNBC) is defined as lack of ER, PR and HER2 receptor and accounts for 15–20% of all breast cancers. The treatment for TNBC is a big challenge due to lack of hormone and target therapy and neoadjuvant chemotherapy only can achieve 20%–30% pathological complete response in TNBC patients. In the current study, we identified four highly expressing oncomiRs, miR‐301b, miR‐181a‐3p, miR‐105 and miR‐93‐3p that were significantly associated with poor survival in TNBC patients. To explore the oncogenic roles of four oncomiRs described above, these miRNAs were ectopically expressed in MB‐231/HCC70 cells and silenced in HCC1937/BT‐549 cells, respectively, for examining proliferation, metastasis, chemoresistance and stemness. MiR‐105/93‐3p overexpressing cells showed Cisplatin resistance and high metastatic ability whereas silence of miR‐105/93‐3p can decrease Cisplatin resistance, metastatic ability and stemness. Bioinformatics analysis showed that Wnt/b‐catenin is one of the most potential miR‐105/93‐3p downstream pathways. Ectopic overexpression of miR‐105 or miR‐93‐3p actives Wnt/b‐catenin signaling by TOP/FOP reporter assay. Bioinformatics, RNA‐IP and immunoblotting showed that SFRP1 is directly targeted and down‐regulated by miR‐105 and miR‐93‐3p. In addition, restoration of SFRP1 can overcome miR‐105/93‐3p mediated cisplatin resistance. High expression of miR‐105/93‐3p and low expression of SFRP1 strongly associated with TNBC poor survival even after chemotherapy treatment. The expression of miR‐105 and miR‐93‐3p inversely correlate with SFPR1 protein level in TNBC N‐T pair. Notably, circulating miR‐105 and miR‐93‐3p significantly higher expression in TNBC than non‐TNBC patients' serum and normal serum. Collectively, our data conducted that miR‐105/93‐3p activate Wnt/b‐catenin signaling through down‐regulate SFRP1 to promote TNBC poor survival and circulating miR‐105/93‐3p may act as predictive biomarker for TNBC poor survival.

Support or Funding Information

MOST‐105‐2320‐B‐006‐054‐
Quercetin inhibition of TNFα‐induced CCL28 release from human triple negative breast cancer (MDA‐MB‐468) cells
Authors: Buchanan Timonia F , Bauer David , Soliman Karam F
Published: November 2023

Quercetin is a natural chemopreventative flavonoid found in fruits, vegetables, and herbs, which exerts antitumor, antiinflammatory, and antioxidant effects. A hallmark of cancer is the recruitment of regulatory T cells (Tregs), which promotes hypoxia‐driven angiogenesis. In this study, we evaluated the effects of quercetin on the attenuation of the release of tumor necrosis factor‐alpha (TNF‐α)‐induced tumor promoting chemokines in MDA‐MB‐468 triple negative breast cancer cells, with a focus on those associated with Tregs recruitment. Initial studies were performed using antibody microarrays which specifically show that TNF‐α (40ng/mL)‐induced protein expression of CNTF, IGFBP‐1, IL‐5, IL‐6, PARC, TNF‐beta, BTC, CCL28, GITR, GRO, GRO‐alpha, HCC‐4, IL‐8, oncostatin M, sTNF‐RI, VEGR‐D, were downregulated by a sublethal concentration of quercetin (40μM). Furthermore, the obtained data show that quercetin downregulated TNF‐α‐induced CCL28 release by 60% as it was further validated by ELISA. To date, there is little known about the presence of CCL28 in breast cancer. These findings suggest that quercetin can modulate CCL28 release and inhibit the recruitment of Tregs, a pivotal factor in tumor cell growth.

Support or Funding Information

This research was supported by the National Institute for Minority Health and Health Disparities of the National Institutes of Health through Grant Number G12 MD007582 and Grant Number P20 MD006738.