bluuebunny/crossref_metadata_2025_split huggingface dataset:
Upregulation of the Multidrug Resistance P‐glycoprotein ABCB1 by Transcription Factor Pituitary Homeobox 2 (Pitx2) in Human Colon and Kidney Cancers
Authors: Lee Wing‐Kee , Thévenod Frank 
Published: November 2023

BackgroundPituitary homeobox 2 (Pitx2) is a master regulator of organ asymmetry during development but is reactivated in neoplastic cells, contributing to cell proliferation, tumor cell invasion and cancer progression. Upregulation of the multidrug resistance P‐glycoprotein ABCB1 confers chemotherapeutic drug resistance to tumor cells, hampering effective treatment. We have previously shown that Pitx2 is upregulated in a multidrug resistant (MDR) kidney cancer cell line and serves as a positive transcriptional regulator of ABCB1 thereby increasing resistance to the anthracycline chemotherapeutic drug, doxorubicin (DOX) (Lee, W.‐K. et al. Int J Cancer 113, 556–67, 2013). The objective of this study was to determine if ABCB1 regulation by Pitx2 is also in engaged by other cancer types that exhibit intrinsic MDR.MethodsExpression of Pitx2 and ABCB1 was determined by immunofluorescence microscopy, immunoblotting and immunohistochemistry. Cell viability was assessed by MTT and clonogenic survival assays. Pitx2 transcriptional activity was measured by luciferase reporter gene assay. Isolation of nuclei was performed by Tenbroeck homogenization in hypoosmotic buffer and a discontinuous sucrose gradient.ResultsIn human colorectal adenocarcinoma Colo320DM cells, which exhibit MDR and harbor augmented ABCB1 expression, nuclear Pitx2 expression and Pitx2 reporter gene activity were significantly increased compared to less drug resistant Colo205 cells. The ABCB1 inhibitor PSC833 (0.1 μM) reversed drug resistance to 1 μM DOX after 24 h in Colo320DM cells. Further, knockdown of Pitx2 by RNAi in Colo320DM cells resulted in decreased ABCB1 protein by almost 3‐fold and diminished resistance to DOX resulting in attenuated cell survival. Moreover, the Pitx2 isoforms that are involved in regulation of ABCB1 in colon and kidney cancer cells were investigated. Both Pitx2A and Pitx2B were found to be present in the nuclear fraction of Colo320DM and MDR A498 kidney carcinoma cells. Analysis of human kidney and colon cancer tissue arrays by co‐immunostaining suggests a causal relationship between nuclear Pitx2 and ABCB1 expression.ConclusionPitx2 is a transcriptional regulator of ABCB1 in cancers of the colon and kidney suggesting a contributory role to the MDR phenotype.Support or Funding InformationFunded by an Internal Research Grant (UW/H) and ZBAF
The Role of Twist1 Phosphorylation in Tumor Angiogenesis in Lung Cancer
Authors: Mammoto Tadanori , Jiang Amanda , Jiang Elisabeth , Mammoto Akiko 
Published: November 2023

Lung cancer is the leading cause of cancer death in the US and the survival rate for advanced lung cancer patients is very low. Deregulated angiogenesis ‐the formation of new blood capillaries from existing blood vessels‐ contributes to lung cancer; however, current anti‐angiogenic therapy has potential problems such as rebound tumor growth and resistance to therapy. Here we implant fibrin gel containing either lung tumor‐derived factors or a small pellet of lung cancer cells on the mouse lung and show that a transcription factor, Twist1, mediates tumor angiogenesis through the VEGF‐VEGFR2 pathway. Conditioned media from lung cancer cells upregulate Twist1 Ser42 phosphorylation in endothelial cells (ECs), and Twist1 knockdown or inhibition of Twist1 Ser42 phosphorylation using a mutant construct (Twist1S42A) decreases VEGFR2 expression and EC sprouting induced by conditioned media in vitro. Tumor angiogenesis is also inhibited in fibrin gel implanted on Tie2‐specific Twist1 conditional knockout mouse lungs or Twist1S42A‐treated mouse lungs in vivo. These results suggest that Twist1 Ser42 phosphorylation plays a key role in tumor angiogenesis associated with lung cancer, and modulation of Twist1 Ser42 phosphorylation could be a potential therapeutic strategy for lung cancer.Support or Funding InformationBoston Children's Hospital Career Development Award
A Novel Signaling Pathway that Governs Tumor Metastasis: Ceramide Regulates Direct Crosstalk Between TGF‐ß and Sonic Hedgehog Signaling
Authors: Gencer Salih , Ogretmen Besim 
Published: November 2023

In the past twenty years, specific sphingolipids have become recognized for their participation in membrane functions and signaling events that control a wide array of cellular processes. Ceramide is considered as a main molecule of bioactive sphingolipid metabolites, are involved in signaling pathways that regulate cell proliferation, apoptosis, motility, differentiation, and angiogenesis. More recent studies have introduced bioactive sphingolipids and their metabolic enzymes take a role as regulated component in migration and cell mobility processes. However, the molecular mechanism of ceramide involved is unknown. Here, we investigated the effect of ceramide synthases (CerS) and ceramide on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4/ceramide is involved to cell migration and tumor metastasis. Here, we also have generated CerS4−/− mice for in vivo studies. Excitingly, we observed that genetically loss of CerS4 resulted in severe irreversible alopecia, which was associated with hyper‐proliferation and migration of keratinocytes. Mechanistically, we show here that genetic loss or shRNA‐mediated knockdown of CerS4 enhances cell migration by which ligand‐independent signaling of TGF‐beta receptors I and II (TßRI/II) in various cell types, including keratinocytes, mouse embryonic fibroblasts and cancer cells. Mechanisms that regulate membrane localization of TßRI/II for the regulation of cell migration and metastasis are largely unknown. We identified that alterations in ceramide metabolism by molecular knockdown and/or genetic deletion of CerS4, reducing bioactive C18‐C20‐ceramides, results in enhanced TßRI‐II signaling at the plasma membrane by inhibiting Smad7 recruitment to the receptor, leading to increased cell migration/invasion via activation of the sonic hedgehog (Shh) pathway. Mechanistically, inhibition of Smad7‐TßRI/II interaction in response to CerS4 knockdown led to the activation of the Shh signaling via enhanced association/cross‐talk between TßRI‐II and smoothened (Smo), a signaling Shh receptor, at the plasma membrane. In reciprocal studies, reconstitution of CerS4/ceramide inhibited TGF‐ß‐Shh crosstalk, reducing cancer cell migration and inhibition of the cancer cell colonization/lung metastasis in SCID mice. Moreover, knockdown of CerS4 in cancer cells, but not systemic deletion of CerS4 in mice, increased metastasis of breast cancer cells from mammary pads to the liver, which was inhibited by Smad7 expression or Smo knockdown. Thus, these data reveal that CerS4/ceramide is a novel factor that regulates the recruitment of Smad7 to the TßRI, controlling the stability/surface expression of the receptor, which modulates TßRI/II‐Smo crosstalk, controlling cell migration and cancer metastasis.Support or Funding Information1R01 DE016572
Epigenetic regulation of epithelial cellular fate by CTBP
Authors: Byun Jung S , Gardner Kevin 
Published: November 2023

Epithelial phenotype is a default state of epithelial cells. The shift between epithelial and mesenchymal phenotypes is one of the hallmarks of cancer. Epithelial cells retain incredible plasticity. They can differentiate and self‐renew during embryonic and post‐natal organ development. An essential developmental process is the ability to weaken their typical epithelial features and change to a mesenchymal state by activating epithelial to mesenchymal transition (EMT), a program that is abnormally activated during tumor progression and metastasis, yet is required for effective tissue repair. This plasticity has central implication for the dysregulation of epithelial and mesenchymal states that leads to cancer initiation and progression.C‐terminal binding protein (CTBP) is an epigenetic regulatory protein that controls gene expression in response to metabolic imbalance. It is activated in the presence of NADH to recruit and target a variety of histone modifying complexes and transcriptional regulators to chromatin, thus providing a defined network of interactions that modulate cellular programming. Recently, we have found that CTBP controls major cancer hallmark pathways including genome instability, EMT, and tumor initiating cells and stemness. In order to define the mechanisms through which CTBP controls cellular plasticity, we compared non‐tumorigenic human breast epithelial MCF10A cells with CTBP‐depleted MCF10A. Using this system, we characterized several molecular biological and biochemical properties driven by CTBP in correlation with genome‐wide profiling by ChIP‐Seq and RNA‐Seq.In this study we illustrate that CTBP depletion enhances epithelial differentiation and upregulates epithelial gene expression and CTBP overexpression shifts differentiation from epithelial to mesenchymal in mammary epithelial cells. Furthermore, we find that CTBP plays an important role in influencing epithelial plasticity mediated by DNA methylation inhibition, thus providing a conceivable opportunity for enhanced therapeutic intervention.
AA Amyloidosis In Island Foxes (Urocyon Littoralis): Pathology, Risk Factors, And The Genetic Basis For Disease
Authors: Gaffney Patricia , Witte Carmel , Clifford Deanna , Ghassemian Majid , Gaasterland Terry , Sigurdson Christina 
Published: November 2023

The island fox (Urocyon littoralis) is an endangered species that lives exclusively on the California Channel islands. Island foxes inhabit six of the eight Channel islands and have evolved into six genetically distinct, island‐specific subspecies. Systemic amyloidosis is highly prevalent in island foxes and is a threat to species recovery. I have identified the type of amyloid, described the lesions of amyloidosis in foxes, and investigated the risk and underlying molecular mechanisms of disease. Based on immunoreactivity to anti‐canine AA antibody and protein sequencing by mass spectrometry, amyloid in foxes has been identified as amyloid A (AA). AA amyloidosis occurs following prolonged elevation of the acute phase protein serum amyloid A (SAA), secondary to chronic infectious or inflammatory disease. Amyloid aggregates in tissue were most common in kidney, spleen and the oral cavity, and amyloid in foxes had a propensity for deposition along basement membranes. Sequencing of the SAA protein identified it as a 111 amino acid long protein with multiple isoforms and an amyloid‐prone, N‐terminal segment. Risk factors for disease were identified by multivariable logistic regression as older age, the San Clemente island subspecies, captivity and nephritis. Increased risk for disease in one subspecies, in addition to the lack of reports of AA amyloidosis in gray fox, the closest genetic relative to island fox, suggest a possible genetic association with disease. Examination of gene transcript levels in the SAA transcription pathway revealed SAA, IL6, IL1α, IL1β, and C/EBP‐δ transcripts were significantly elevated in island foxes with AA amyloidosis, and higher SAA transcripts correlated with more severe disease. Serum SAA protein concentrations were similar in foxes that died with and without amyloidosis, suggesting serum protein levels are not an accurate indicator of disease status. Lastly, based on sequence variation in the region of the SAA gene family in island and gray foxes, 14 SNPs unique to all island fox subspecies have been identified. Understanding the influence of genetic as well as exogenous factors on AA amyloidosis in island foxes informs not only on the disease mechanism in this endangered species, but contributes to the overall understanding of the pathogenesis of AA amyloidosis.Support or Funding InformationThis work was supported by an Academic Senate Grant from the University of California, San Diego, the Karen C. Drayer Wildlife Health Center, School of Veterinary Medicine, University of California, Davis, and the ARCS Foundation of Northern California. P.M.G was funded by The Linda Munson Fellowship for Wildlife Pathology Research administered by the ACVP/STP Coalition for Veterinary Pathology Fellows and the Peter Kennedy Fellowship in Anatomic Pathology, School of Veterinary Medicine, University of California, Davis.
Regulation of gene expression by complex RNA molecules
Authors: Pyle Anna M. 
Published: November 2023

While we typically think of proteins as highly structured molecules, we often don't think about RNAs in the same way. Recent advances in studies of ribozymes, riboswitches, and large RNA‐protein complexes such as the ribosome have changed this view and made it clear that RNA molecules can adopt elaborate structures that form enzyme active‐sites, architectural scaffolds, and ligand‐gated machines. Even messenger RNAs (mRNAs) can be highly folded, suggesting a “code within the genetic code” that contributes to regulated gene expression. Much of our knowledge about RNA structure comes from chemical probing experiments and crystallographic studies on large ribozymes, such as self‐splicing group II introns. These studies have revealed a set of building blocks from which RNA molecules can assemble into compact, well‐folded structures. RNA structural motifs can control transcription, translation and even splicing of a gene. Recently, understanding of RNA functional diversity was further expanded by the discovery that our genomes are almost completely transcribed, and that we express numerous long noncoding RNA transcripts (lncRNAs). While the function of these giants is being actively investigated, it is clear that many of them facilitate epigenetic control of gene expression. Given the significance of lncRNAs, and of other large RNAs such as viral RNA genomes, the architectural complexity of these behemoths has become an important question. Secondary and tertiary structures within large RNAs may reveal new pathways for regulating gene expression and provide new targets for the design of small molecule modulators.Support or Funding InformationHHMI and the NIH
ABSENCE OF MAST CELLS IN AN EXPERIMENTAL MODEL OF PULMONARY AND CARDIAC FIBROSIS, THE CUX‐1 MICE
Authors: Reddy Navya A. , Raza Farhan , Said Ahmed , Livingston Safia , Jacobsen David , Kearns Donovan , Baybutt Richard , Van Den Heuvel Gregory , Molteni Agostino , Hamidpour Soheila 
Published: November 2023

Both pulmonary and cardiovascular (CV) fibrosis have a significant role in clinical medicine, however the pathophysiology of fibrosis in these organs is poorly understood (1). Previous rat models of pulmonary fibrosis have shown increased staining of renin and mast cells (MC) production in pulmonary tissue following injection with Triolein (T), a substance comprising 80% of human fat and which is experimentally used to induce fat embolism (FE) (2). MCs are known to produce renin in injured pulmonary tissue, leading to Angiotensin II mediated fibroblasts proliferation (3). The angiotensin II receptor blocker losartan is associated with reduced MCs in fibrotic lungs and a reduction of fibroblasts proliferation, also suggesting that MCs may have a significant correlation with the fibrosis (4).We aimed to identify one of the mechanisms of CV fibrosis by determining if MC could be found within the lung and cardiac tissue of CUX 1 (CUT‐like homeobox) transgenic mice. Although the CUX‐1 gene is present in all metazoans, its constitutive expression such as in polycystic kidneys blocks cyclin dependent kinase inhibitors p21 and p27, leading to hepatic, pulmonary and cardiac fibrosis (5). Previous studies of CUX‐1 mice have shown severe inflammation of the pulmonary and coronary arteries with media thickening, luminal patency reduction, adventitial fibrosis and MCs presence (5).In our study, 6 wild type and 6 CUX‐1 transgenic mice were sacrificed by isoflurane anesthesia and necropsied. Lungs and heart were collected, fixed in 10% formalin and stained with H & E (morphometric scoring), Trichrome (fibrosis), May‐Grünwald‐Giemsa and CP‐11‐C‐kit (MCs). In each organ slide, 10 photographs were taken at random at 400× magnification by 2 pathologists unaware of the slide identity who also reviewed the slides (5).Mild lung and CV inflammation and fibrosis were observed in wild type mice while the CUX‐1 mice presented with very severe fibrosis, particularly in the bronchial basal membrane, the peribronchial pulmonary arteries and the coronary arteries. May‐Grünwald‐Giemsa staining showed very few MCs in the heart of wild type mice and none in the CUX‐1. MCD staining did not show MCs in either organs, not withstanding the severe damage. It remains to be seen whether the lack of MCs, as seen in this experimental model, has a pathogenetic role in the development of pulmonary and cardiac fibrosis. Further mechanisms of fibrotic damage should be explored to identify potential therapeutic options in patients with significant fibrotic conditions.Support or Funding InformationWheaton College Research Fund
Dimethyl Fumarate Ameliorates Pulmonary Hypertension in vivo and Prevents Fibrosis via βTRCP Mediated Degradation of β‐catenin and TAZ
Authors: Grzegorzewska Agnieszka , Han Rong , Seta Francesca , Stawski Lukasz , Feghali‐Bostwick Carol , Browning Jeffrey , Trojanowska Maria 
Published: November 2023

IntroductionPulmonary arterial hypertension (PAH) is a serious, progressive and fatal complication of systemic sclerosis (SSc). Oxidative stress and chronic inflammation have been shown to play important roles in pathogenesis of PAH. Dimethyl Fumarate (DMF) (Tecfidera®) is an anti‐oxidative and anti‐inflammatory agent approved by the FDA for treatment of multiple sclerosis. In this study we test DMF as a potential drug to treat PAH.MethodsDMF was administered daily via intraperitoneal injections in a chronic hypoxia mouse model of PAH in preventive and therapeutic modes as well as preventively in a bleomycin lung fibrosis model. In vitro studies were performed on human primary arterial endothelial cells and primary human lung fibroblasts from SSc patients and healthy controls. Endothelial cells were exposed to hypoxic conditions or lipopolisaccharade (LPS) in combination with DMF or vehicle treatment. Pulmonary fibroblasts were treated with transforming growth factor beta (TGFβ) and DMF or vehicle.ResultsDMF prevented and reversed hemodynamic changes and right ventricular hypertrophy as well as reduced inflammation, oxidative stress and vascular muscularization in a mouse PAH model. DMF improved the function of cultured endothelial cells by normalizing hypoxia induced gene expression of pro‐inflammatory cytokines and growth factors via inhibition of NFκB, STAT3 and JNK signaling. DMF also suppressed TGFβ dependent pro‐fibrotic gene expression in cultured human lung fibroblasts. Studies of endothelial cells and fibroblasts indicated that Nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) ‐mediated anti‐oxidative pathway is dispensable for the anti‐inflammatory and anti‐fibrogenic properties of DMF. In fibroblasts, DMF had no significant effect on canonical TGFβ signaling, but instead led to ubiquitin ligase‐ βTRCP mediated degradation of the pro‐fibrogenic transcription factors Sp1, TAZ and β‐catenin. In vivo validation of anti‐fibrotic DMF properties in a bleomycin mouse model indicated that the drug prevented development of lung fibrosis in a manner correlated with a reduction in active β‐catenin levels by immunostaining.ConclusionsIn conclusion, DMF has anti‐oxidative, anti‐inflammatory and anti‐fibrotic potential both in vitro and in vivo. Given the safety profile of Tecfidera® and its pleiotropic mechanism of action, it may provide an effective treatment for SSc‐PAH.Support or Funding InformationCORT grant NIAMS P50 AR060780‐01
R‐Spondin2 is upregulated in idiopathic pulmonary fibrosis and affects fibroblasts behavior
Authors: Munguia Adrian , Becerril Carina , Mendoza Criselda , Balderas Yalbi , Ramirez Remedios , Melendez Jorge , Pardo Annie , Selman Moises 
Published: November 2023

BackgroudIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal lung disorder of unknown etiology characterized by fibroblast activation, excessive extracellular matrix accumulation and the subsequent destruction of the lung parenchyma. The R‐spondins family (RSPO) represents a recently described group of proteins with essential activities in development. RSPO2 is expressed primarily in the lungs and mutations of this protein cause severe defects in the respiratory tract. Three proteins of the G protein coupled family (LGR4, LGR5 and LGR6) have been identified as possible receptors. RSPO2 enhances the canonical Wnt signaling (a pathway involved in IPF) resulting in activation of b‐catenin.AimTo determine the role of RSPO2 in IPF.MethodsThe expression of RSPO2 and LGR6 was explored by qPCR in IPF lungs and fibroblasts. Cell source was analyzed by immunohistochemistry. The effect of RSPO2 on lung fibroblasts proliferation was evaluated with WST1 and on gene expression by RNAseq. The effect of RSPO2 on fibroblasts and epithelial cell apoptosis was examined by flow cytometry.ResultsRSPO2 and LGR6 were significantly increased (>100‐fold) in IPF compared with normal lungs. Both molecules were localized primarily in fibroblasts and epithelial cells. RSPO2 gene expression was also increased in fibroblasts derived from IPF lungs (2−DCT 2.59−5±3.6−6 vs 1.39−5±1.35−6). In vitro, RSPO2 modified the expression of 3116 genes in IPF and 795 in normal fibroblasts. Several pathways were revealed including apoptosis, cell cycle, Wnt pathway, and extracellular matrix metabolism. Treatment of fibroblasts with RSPO2 reduced cell proliferation and induced apoptosis in both normal and IPF fibroblasts. No effect was observed in epithelial cell apoptosis.ConclusionsOur findings demonstrate that RSPO2 and its receptor LGR6 are upregulated in IPF lungs, primarily in fibroblasts and epithelial cells, although the exact role of this axis in the pathogenesis of this disease remains uncertain.Support or Funding InformationPosgrado en Ciencias Biologicas, UNAM