Cellular Protein Levels Research Articles

AXIN2 is a non-redundant regulator of AXIN1 stability and β-catenin in colorectal cancer cells.

AXIN proteins are major components of the β-catenin destruction complex or degradasome, which limits β-catenin nuclear translocation and Wnt signalling activation at steady state. Schmidt etal. performed quantitative analysis of cellular AXIN protein levels in human colorectal cancer cells and revealed that AXIN2 plays a non-redundant role in regulating the total AXIN pool and Wnt/β-catenin signalling activity. Tankyrase (TNKS) inhibitors failed to inhibit Wnt/β-catenin signalling in AXIN2 knockout cells, suggesting that AXIN2 is essential for TNKS inhibitors to function. Mechanistically, the authors show that AXIN2 recruits TNKS to AXIN1 and promotes TNKS-mediated degradation of AXIN1. These findings may have important implications for anti-cancer therapy by TNKS small molecule inhibitors.

Update on a brain-penetrant cardiac glycoside that can lower cellular prion protein levels in human and guinea pig paradigms.

Lowering the levels of the cellular prion protein (PrPC) is widely considered a promising strategy for the treatment of prion diseases. Building on work that established immediate spatial proximity of PrPC and Na+, K+-ATPases (NKAs) in the brain, we recently showed that PrPC levels can be reduced by targeting NKAs with their natural cardiac glycoside (CG) inhibitors. We then introduced C4'-dehydro-oleandrin as a CG with improved pharmacological properties for this indication, showing that it reduced PrPC levels by 84% in immortalized human cells that had been differentiated to acquire neural or astrocytic characteristics. Here we report that our lead compound caused cell surface PrPC levels to drop also in other human cell models, even when the analyses of whole cell lysates suggested otherwise. Because mice are refractory to CGs, we explored guinea pigs as an alternative rodent model for the preclinical evaluation of C4'-dehydro-oleandrin. We found that guinea pig cell lines, primary cells, and brain slices were responsive to our lead compound, albeit it at 30-fold higher concentrations than human cells. Of potential significance for other PrPC lowering approaches, we observed that cells attempted to compensate for the loss of cell surface PrPC levels by increasing the expression of the prion gene, requiring daily administration of C4'-dehydro-oleandrin for a sustained PrPC lowering effect. Regrettably, when administered systemically in vivo, the levels of C4'-dehydro-oleandrin that reached the guinea pig brain remained insufficient for the PrPC lowering effect to manifest. A more suitable preclinical model is still needed to determine if C4'-dehydro-oleandrin can offer a cost-effective complementary strategy for pushing PrPC levels below a threshold required for long-term prion disease survival.

LARP1 haploinsufficiency is associated with an autosomal dominant neurodevelopmental disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) that affects approximately 4% of males and 1% of females in the United States. While causes of ASD are multi-factorial, single rare genetic variants contribute to around 20% of cases. Here we report a case series of seven unrelated probands (6 males, 1 female) with ASD or another variable NDD phenotype attributed to de novo heterozygous loss of function or missense variants in the gene LARP1. LARP1 encodes an RNA binding protein that post-transcriptionally regulates the stability and translation of thousands of mRNAs, including those regulating cellular metabolism and metabolic plasticity. Using lymphocytes collected and immortalized from an index proband who carries a truncating variant in one allele of LARP1, we demonstrated lower cellular levels of LARP1 protein causing reduced rates of aerobic respiration and glycolysis. As expression of LARP1 increases during neurodevelopment, with higher levels in neurons and astrocytes, we propose that LARP1 haploinsufficiency contributes to ASD or related NDDs through attenuated metabolic activity in the developing fetal brain.

Arginyltransferase 1 (ATE1)-mediated proteasomal degradation of viral haemagglutinin protein: a unique host defence mechanism.

The extensive protein production in virus-infected cells can disrupt protein homeostasis and activate various proteolytic pathways. These pathways utilize post-translational modifications (PTMs) to drive the ubiquitin-mediated proteasomal degradation of surplus proteins. Protein arginylation is the least explored PTM facilitated by arginyltransferase 1 (ATE1) enzyme. Several studies have provided evidence supporting its importance in multiple physiological processes, including ageing, stress, nerve regeneration, actin formation and embryo development. However, its function in viral pathogenesis is still unexplored. The present work utilizes Newcastle disease virus (NDV) as a model to establish the role of the ATE1 enzyme and its activity in pathogenesis. Our data indicate a rise in levels of N-arginylated cellular proteins in the infected cells. Here, we also explore the haemagglutinin-neuraminidase (HN) protein of NDV as a presumable target for arginylation. The data indicate that the administration of Arg amplifies the arginylation process, resulting in reduced stability of the HN protein. ATE1 enzyme activity inhibition and gene expression knockdown studies were also conducted to analyse modulation in HN protein levels, which further substantiated the findings. Moreover, we also observed Arg addition and probable ubiquitin modification to the HN protein, indicating engagement of the proteasomal degradation machinery. Lastly, we concluded that the enhanced levels of the ATE1 enzyme could transfer the Arg residue to the N-terminus of the HN protein, ultimately driving its proteasomal degradation.

Machine Learning-Enhanced Estimation of Cellular Protein Levels from Bright-Field Images.

In this study, we aimed to develop a novel method for non-invasively determining intracellular protein levels, which is essential for understanding cellular phenomena. This understanding hinges on insights into gene expression, cell morphology, dynamics, and intercellular interactions. Traditional cell analysis techniques, such as immunostaining, live imaging, next-generation sequencing, and single-cell analysis, despite rapid advancements, face challenges in comprehensively integrating gene and protein expression data with spatiotemporal information. Leveraging advances in machine learning for image analysis, we designed a new model to estimate cellular biomarker protein levels using a blend of phase-contrast and fluorescent immunostaining images of epidermal keratinocytes. By iterating this process across various proteins, our model can estimate multiple protein levels from a single phase-contrast image. Additionally, we developed a system for analyzing multiple protein expression levels alongside spatiotemporal data through live imaging and phase-contrast methods. Our study offers valuable tools for cell-based research and presents a new avenue for addressing molecular biological challenges.

RIPK1 is dispensable for cell death regulation in β-cells during hyperglycemia

ObjectiveReceptor-interacting protein kinase 1 (RIPK1) orchestrates the decision between cell survival and cell death in response to tumor necrosis factor (TNF) and other cytokines. Whereas the scaffolding function of RIPK1 is crucial to prevent TNF-induced apoptosis and necroptosis, its kinase activity is required for necroptosis and partially for apoptosis. Although TNF is a proinflammatory cytokine associated with β-cell loss in diabetes, the mechanism by which TNF induces β-cell demise remains unclear. MethodsHere, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1β-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1β-KO mice were further challenged with a high-fat diet to induce hyperglycemia. For mechanistic studies, pancreatic islets were subjected to various killing and sensitising agents. ResultsInhibition of RIPK1 kinase activity (Ripk1D138N mice) did not affect the onset and progression of hyperglycemia in a type 1 diabetes model. Moreover, the absence of RIPK1 expression in β-cells did not affect normoglycemia under basal conditions or hyperglycemia under diabetic challenges. Ex vivo, primary pancreatic islets are not sensitised to TNF-induced apoptosis and necroptosis in the absence of RIPK1. Intriguingly, we found that pancreatic islets display high levels of the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) and low levels of apoptosis (Caspase-8) and necroptosis (RIPK3) components. Cycloheximide treatment, which led to a reduction in cFLIP levels, rendered primary islets sensitive to TNF-induced cell death which was fully blocked by caspase inhibition. ConclusionsUnlike in many other cell types (e.g., epithelial, and immune), RIPK1 is not required for cell death regulation in β-cells under physiological conditions or diabetic challenges. Moreover, in vivo and in vitro evidence suggest that pancreatic β-cells do not undergo necroptosis but mainly caspase-dependent death in response to TNF. Last, our results show that β-cells have a distinct mode of regulation of TNF-cytotoxicity that is independent of RIPK1 and that may be highly dependent on cFLIP.

Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation.

The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

A Flexible Fibrin‐Based Platform for Driving One‐Day Self‐Assembly of Millimeter‐Sized Cell‐Rich Spheroids and Their Applications: An Old Material with New Tricks

AbstractCell‐rich spheroids have gained increasing applications due to their ability to mimic the 3D microenvironment of real tissues. Given human organ dimensions, millimeter‐sized (>1 mm) spheroids are more clinically valuable than micro‐sized ones (<500 µm). However, constructing millimeter‐sized spheroids faces the primary challenge of core necrosis due to insufficient nutrient diffusion during long‐term in vitro cultivation (>3 days). Developing rapid spheroid fabrication strategies can address the issue but remains challenging. Here, a flexible fibrin‐based platform for rapid fabrication of the spheroids is developed using a one‐step manufacturing technology. Cell‐laden fibrin hydrogels are polymerized from cell‐containing fibrinogen by thrombin‐catalyzed reactions within minutes and cultured in an agarose‐coated plate. Benefiting from the unique viscoelasticity, softness, and cell adhesion natures of fibrin, the expression levels of several cellular junction proteins are significantly upregulated compared to the scaffold‐free spheroids. As a result, cells rapidly self‐assemble into 1–15 mm‐sized spheroids within 6–24 h, accommodating high cell density (6 × 104 cells µL−1) without leakage. The spheroid formation platform is also flexible for various cell densities and cell types. Moreover, the spheroids effectively mimic the 3D tissue niches, showing promising potential in tumor drug screening with MCF‐7 spheroids and liver regeneration with HepG2 spheroids.

Construction of An Orthotopic Xenograft Model of Non-small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities.

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model. Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease's progression and facilitate the development of potential anticancer drugs.

Osteoinductive micro-nano guided bone regeneration membrane for in situ bone defect repair

BackgroundBiomaterials used in bone tissue engineering must fulfill the requirements of osteoconduction, osteoinduction, and osseointegration. However, biomaterials with good osteoconductive properties face several challenges, including inadequate vascularization, limited osteoinduction and barrier ability, as well as the potential to trigger immune and inflammatory responses. Therefore, there is an urgent need to develop guided bone regeneration membranes as a crucial component of tissue engineering strategies for repairing bone defects.MethodsThe mZIF-8/PLA membrane was prepared using electrospinning technology and simulated body fluid external mineralization method. Its ability to induce biomimetic mineralization was evaluated through TEM, EDS, XRD, FT-IR, zeta potential, and wettability techniques. The biocompatibility, osteoinduction properties, and osteo-immunomodulatory effects of the mZIF-8/PLA membrane were comprehensively evaluated by examining cell behaviors of surface-seeded BMSCs and macrophages, as well as the regulation of cellular genes and protein levels using PCR and WB. In vivo, the mZIF-8/PLA membrane’s potential to promote bone regeneration and angiogenesis was assessed through Micro-CT and immunohistochemical staining.ResultsThe mineralized deposition enhances hydrophilicity and cell compatibility of mZIF-8/PLA membrane. mZIF-8/PLA membrane promotes up-regulation of osteogenesis and angiogenesis related factors in BMSCs. Moreover, it induces the polarization of macrophages towards the M2 phenotype and modulates the local immune microenvironment. After 4-weeks of implantation, the mZIF-8/PLA membrane successfully bridges critical bone defects and almost completely repairs the defect area after 12-weeks, while significantly improving the strength and vascularization of new bone.ConclusionsThe mZIF-8/PLA membrane with dual osteoconductive and immunomodulatory abilities could pave new research paths for bone tissue engineering.

Post-transcriptional regulation and subcellular localization of G-protein γ7 subunit: implications for striatal function and behavioral responses to cocaine.

The striatal D1 dopamine receptor (D1R) and A2a adenosine receptor (A2aR) signaling pathways play important roles in drug-related behaviors. These receptors activate the Golf protein comprised of a specific combination of αolfβ2γ7 subunits. During assembly, the γ7 subunit sets the cellular level of the Golf protein. In turn, the amount of Golf protein determines the collective output from both D1R and A2aR signaling pathways. This study shows the Gng7 gene encodes multiple γ7 transcripts differing only in their non-coding regions. In striatum, Transcript 1 is the predominant isoform. Preferentially expressed in the neuropil, Transcript 1 is localized in dendrites where it undergoes post-transcriptional regulation mediated by regulatory elements in its 3' untranslated region that contribute to translational suppression of the γ7 protein. Earlier studies on gene-targeted mice demonstrated loss of γ7 protein disrupts assembly of the Golf protein. In the current study, morphological analysis reveals the loss of the Golf protein is associated with altered dendritic morphology of medium spiny neurons. Finally, behavioral analysis of conditional knockout mice with cell-specific deletion of the γ7 protein in distinct populations of medium spiny neurons reveals differential roles of the Golf protein in mediating behavioral responses to cocaine. Altogether, these findings provide a better understanding of the regulation of γ7 protein expression, its impact on Golf function, and point to a new potential target and mechanisms for treating addiction and related disorders.

TIR predictor and optimizer: Web-tools for accurate prediction of translation initiation rate and precision gene design in Saccharomyces cerevisiae.

Translation initiation is the primary determinant of the rate of protein production. The variation in the rate with which this step occurs can cause up to three orders of magnitude differences in cellular protein levels. Several mRNA features, including mRNA stability in proximity to the start codon, coding sequence length, and presence of specific motifs in the mRNA molecule, have been shown to influence the translation initiation rate. These molecular factors acting at different strengths allow precise control of in vivo translation initiation rate and thus the rate of protein synthesis. However, despite the paramount importance of translation initiation rate in protein synthesis, accurate prediction of the absolute values of initiation rate remains a challenge. In fact, as of now, there is no available model for predicting the initiation rate in Saccharomyces cerevisiae. To address this, we train a machine learning model for predicting the in vivo initiation rate in S. cerevisiae transcripts. The model is trained using a diverse set of mRNA transcripts, enabling the comparison of initiation rates across different transcripts. Our model exhibited excellent accuracy in predicting the translation initiation rate and demonstrated its effectiveness with both endogenous and exogenous transcripts. Then, by combining the machine learning model with the Monte-Carlo search algorithm, we have also devised a method to optimize the nucleotide sequence of any gene to achieve a specific target initiation rate. The machine learning model we've developed for predicting translation initiation rates, along with the gene optimization method, are deployed as a web server. Both web servers are accessible for free at the following link: ajeetsharmalab.com/TIRPredictor. Thus, this research advances our fundamental understanding of translation initiation processes, with direct applications in biotechnology.

Abundance of the Membrane Proteome in Yeast Cells Lacking Spc1, a Non-catalytic Subunit of the Signal Peptidase Complex.

The signal peptidase complex (SPC) mediates processing of signal peptides of secretory precursors. But, recent studies show that the eukaryotic SPC also cleaves internal transmembrane segments of some membrane proteins, and its non-catalytic subunit, Spc1/SPCS1 plays a critical role in this process. To assess the impact of Spc1 on membrane proteostasis, we carried out quantitative proteomics of yeast cells with and without Spc1. Our data show that the abundance of the membrane proteome in yeast cells lacking Spc1 is in general reduced compared to that in wild-type cells, implicating its role in controlling the cellular levels of membrane proteins.

Structure-based virtual screening of novel USP5 inhibitors targeting the zinc finger ubiquitin-binding domain

The equilibrium of cellular protein levels is pivotal for maintaining normal physiological functions. USP5 belongs to the deubiquitination enzyme (DUBs) family, controlling protein degradation and preserving cellular protein homeostasis. Aberrant expression of USP5 is implicated in a variety of diseases, including cancer, neurodegenerative diseases, and inflammatory diseases. In this paper, a multi-level virtual screening (VS) approach was employed to target the zinc finger ubiquitin-binding domain (ZnF-UBD) of USP5, leading to the identification of a highly promising candidate compound 0456–0049. Molecular dynamics (MD) simulations were then employed to assess the stability of complex binding and predict hotspot residues in interactions. The results indicated that the candidate stably binds to the ZnF-UBD of USP5 through crucial interactions with residues ARG221, TRP209, GLY220, ASN207, TYR261, TYR259, and MET266. Binding free energy calculations, along with umbrella sampling (US) simulations, underscored a superior binding affinity of the candidate relative to known inhibitors. Moreover, US simulations revealed conformational changes of USP5 during ligand dissociation. These insights provide a valuable foundation for the development of novel inhibitors targeting USP5.

Abstract 559: Investigating DDX3X as a target to improve outcomes in Black and Native American endometrial cancer patients

Abstract Introduction: The goal of this project was to identify and test a new molecular target that will help reduce the worse outcomes of endometrial cancer, especially in Black and Native American patients. Endometrial cancer is rapidly on the rise in the developed world. It has overtaken many other types of cancer in both incidence and mortality. Black populations are disproportionately impacted by endometrial cancer, both in Oklahoma and across the United States. In Oklahoma specifically, Native Americans are the most heavily impacted group. Methods: Machine learning techniques were used to analyze comprehensive metabolic panel (CMP) data from electronic medical record of patients in the University of Oklahoma (OU) network of hospitals and clinics. This data was broken down by endometrial cancer status, race, and age. Plasma samples of endometrial cancer patients were collected from the OU Health Sciences Center Biobank. These samples were analyzed by mass spectrometry. Endometrial cancer cell lines Ishikawa (low-grade) and ARK-1 and ARK-4 (high grade) were cultured in EMEM. For treating the cells, we used RK-33, a small molecule inhibitor of DDX3X. MTT assays were performed in 96-well plates at increasing concentrations of RK-33 for 48 hours. Western blots were used to determine effect of the drug on cellular DDX3X and yH2AX protein levels. Results: Machine learning analysis EMR data revealed a difference between races in albumin levels of women with endometrial cancer. Black and Native American patients had lower albumin, which is known to correlate to worse outcomes. IPA analysis of plasma proteomics revealed several proteins that were differentially expressed between races. Among these was DDX3X, the X-linked form of DDX3, a DEAD-box RNA helicase which is involved in DNA damage repair. Patient data indicated that patients with high DDX3X expression tended to have lower survival rates. MTT analysis determined that Ishikawa has a lower IC50 than the high-grade cell lines, but all responded. Western blotting confirmed that treatment with RK-33 reduced the amount of DDX3X in the cells and increased y-H2AX, indicating more DNA damage. Conclusions: This study demonstrates that DDX3X is a valid molecular target in endometrial cancer. The differential expression across race groups indicates that Black and Native Americans may stand to benefit the most from a novel drug targeting this difference. Because DDX3X is involved in DNA damage repair, combining the treatment with a DNA-damaging drug may increase response to the drug as well. Citation Format: James D. Lausen, Doris M. Benbrook. Investigating DDX3X as a target to improve outcomes in Black and Native American endometrial cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 559.

Abstract 2058: Compound library screening to identify modulators of alternative splicing in non-small cell lung cancer (NSCLC)

Abstract Alternative splicing is a molecular mechanism that allows a single gene to encode multiple proteins. It is a complex and highly controlled process used to regulate normal gene expression but is often dysregulated in cancer. Compounds that can modulate alternative splicing are currently being explored as a potential new class of therapeutic agent in cancer. This highlights the need for a deeper understanding of the splicing process, its regulation, and its impact. The discovery of novel and specific tool compounds that modulate splicing would therefore not only be beneficial in investigating the regulation and dysregulation of splicing in cancer but could also be exploited therapeutically. To identify novel regulators of splicing, we generated a cell-based split luciferase screening assay based on alternative splicing of MCL-1 (myeloid cell leukemia-1 protein) pre-mRNA. The MCL1 gene usually produces an mRNA encoding a ‘long’ variant (MCL1L) that is an anti-apoptotic protein often highly expressed in cancers. However, in some circumstances, for example following genetic knockdown of splicing factors, a pro-apoptotic ‘short’ variant (MCL1S) is expressed as a result of exon-skipping. Here we engineered the human NSCLC cell line NCI-H1299 to express a luminescent-tagged version of the MCL1S splice variant to monitor its induction upon spliceosome modulation. Using this engineered cell line, we screened an unannotated library of 12,000 compounds selected to have low molecular weights and favorable properties for cellular uptake. The screen had an average Z’ value of 0.89 over 45 microplates, indicating high assay robustness. Hits were identified as any compound with a luminescence of greater than the average + two standard deviations of the screening dataset. The compounds were validated by repetition in the screening assay, giving 34 candidate hits that were then triaged by qPCR assay to confirm splicing modulation of MCL1. One interesting hit was found to increase mRNA and cellular protein levels of MCL1S and induced altered splicing across the transcriptome that was distinct from the splicing profiles of a diverse panel of splicing modulators, including compounds targeting SF3B1, CLK, and RBM39. In addition, treatment with this hit compound resulted in accumulation of the splicing factor SC35 in cytoplasmic granules, an unusual phenotype not seen with known splicing modulators, that could explain the distinct splicing modulation we have observed. Deconvolution and further characterization of screening hits and potentially unique new mechanisms of action could improve current understanding of alternative splicing and its regulation in NSCLC. The discovery of novel compounds to expand our current toolset of splicing modulators will be key in building the foundation for future splicing targeted drug discovery. Citation Format: Rachel Cooley, Marissa V. Powers, Adam G. Bond, Patrizia Jensen, Gary Newton, Andrea Scarpino, Juliane Braun, Christina Esdar, Paul A. Clarke. Compound library screening to identify modulators of alternative splicing in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2058.

Expression of HIF‑α and their association with clinicopathological parameters in clinical renal cell carcinoma.

This study aimed to assess the cellular localization and expression levels of hypoxia-inducible factor (HIF) -α proteins (specifically HIF-1α, HIF-2α, and HIF-3α) that play a role in the hypoxia pathway and to determine their correlation with clinicopathological parameters and patient survival in renal cell carcinoma (RCC). Tissue microarray (TMA) with cores from 150 clear cell RCCs and 31 non-ccRCC samples. HIF-1α, HIF-2α, and HIF-3α antibodies were used for immunohistochemistry (IHC) of TMA to evaluate the cellular localization and expression levels of HIF-α proteins, specifically in relation to the hypoxia pathway. The expression levels of the HIF-α proteins were higher in the nucleus than in the cytoplasm. Furthermore, the nuclear expression levels of all HIF-α proteins were significantly higher in clear cell RCC (ccRCC) than in non-ccRCC. Cytoplasmic HIF-3α expression was also higher in ccRCC than in non-ccRCC, whereas cytoplasmic HIF-1α and HIF-2α expression levels were similar between the different RCC types. In ccRCC, nuclear HIF-1α expression levels correlated with both nuclear HIF-2α and HIF-3α levels, whereas cytoplasmic HIF-3α expression levels were associated with HIF-1α only.In non-ccRCC, there was a positive correlation observed between nuclear HIF-1α and HIF-3α expression, but no correlation was found with HIF-2α. In patients with ccRCC, the nuclear expressions of HIF-1α and HIF-3α was significantly associated with cancer-specific survival (CSS) in univariate analysis. This association was no longer evident in multivariate analysis. Notably, there was no correlation observed between nuclear HIF-2α expression and CSS in these patients. In contrast, cytoplasmic expression levels showed no association with CSS. The expression levels of the three primary HIF-α proteins were found to be higher in the nucleus than in the cytoplasm. Furthermore, the results indicated that HIF-3α and HIF-1α expression levels were significant univariate factors associated with CSS in patients with clear cell RCC. These results highlight the critical role that HIF-3α and HIF-1α play in the hypoxia pathway.

Mild hypothermia reduces lipopolysaccharide-induced microglial activation via down-regulation of Tent5c

Microglial activation is a critical factor in the pathogenesis and progression of neuroinflammatory diseases. Mild hypothermia, known for its neuroprotective properties, has been shown to alleviate microglial activation. In this study, we explore the differentially expressed (DE) mRNAs and long non-coding RNAs (lncRNAs) in BV-2 microglial cells under different conditions: normal temperature (CN), mild hypothermia (YT), normal temperature with lipopolysaccharide (LPS), and mild hypothermia with LPS (LPS + YT). Venn analysis revealed 119 DE mRNAs that were down-regulated in the LPS + YT vs LPS comparison but up-regulated in the CN vs LPS comparison, primarily enriched in Gene Ontology terms related to immune and inflammatory responses. Furthermore, through Venn analysis of YT vs CN and LPS + YT vs LPS comparisons, we identified 178 DE mRNAs and 432 DE lncRNAs. Among these transcripts, we validated the expression of Tent5c at the protein and mRNA levels. Additionally, siRNA-knockdown of Tent5c attenuated the expression of pro-inflammatory genes (TNF-α, IL-1β, Agrn, and Fpr2), cellular morphological changes, NLRP3 and p-P65 protein levels, immunofluorescence staining of p-P65 and number of cells with ASC-speck induced by LPS. Furthermore, Tent5c overexpression further potentiated the aforementioned indicators in the context of mild hypothermia with LPS treatment. Collectively, our findings highlight the significant role of Tent5c down-regulation in mediating the anti-inflammatory effects of mild hypothermia.

Parecoxib decreases cellular growth and Bcl-2 protein levels in primary cultures of keloid fibroblasts.

Keloids seem to overexpress cyclo-oxygenase-2 (COX-2), suggesting a role in its deregulated pathway in inducing an altered epithelial-mesenchymal interaction, which may be responsible for the overgrowth of dermal components resulting in scars or keloid lesions. This study aimed to evaluate the effect of Parecoxib, a COX-2 inhibitor, on cell growth in fibroblast primary cultures obtained from human keloid tissues. Tissue explants were obtained from patients who underwent intralesional excision of untreated keloids; central fractions were isolated from keloid tissues and used for establishing distinct primary cultures. Appropriate aliquots of Parecoxib, a COX-2 inhibitor were diluted to obtain the concentration used in the experimental protocols in vitro (1, 10 or 100 μM). Treatment with Parecoxib (at all concentrations) caused a significant decrease in cellular growth from 24 hours onwards, and with a maximum at 72 hours (P < .02). Moreover, at 72 hours Parecoxib significantly reduced cellular vitality. Parecoxib treatment also induced an increase in fragmented nuclei with a maximum effect at 100 μM and a significant decrease in Bcl-2 and an increase in activated caspase-3 protein levels at 72 hours compared with control untreated cultures. Our findings suggest a potential use of the COX-2 inhibitor, Parecoxib, as the therapy for keloids.

Exploring the Interplay of RUNX2 and CXCR4 in Melanoma Progression.

Overexpression of the Runt-related transcription factor 2 (RUNX2) has been reported in several cancer types, and the C-X-C motif chemokine receptor 4 (CXCR4) has an important role in tumour progression. However, the interplay between CXCR4 and RUNX2 in melanoma cells remains poorly understood. In the present study, we used melanoma cells and a RUNX2 knockout (RUNX2-KO) in vitro model to assess the influence of RUNX2 on CXCR4 protein levels along with its effects on markers associated with cell invasion and autophagy. Osteotropism was assessed using a 3D microfluidic model. Moreover, we assessed the impact of CXCR4 on the cellular levels of key cellular signalling proteins involved in autophagy. We observed that melanoma cells express both RUNX2 and CXCR4. Restored RUNX2 expression in RUNX2 KO cells increased the expression levels of CXCR4 and proteins associated with the metastatic process. The protein markers of autophagy LC3 and beclin were upregulated in response to increased CXCR4 levels. The CXCR4 inhibitor WZ811 reduced osteotropism and activated the mTOR and p70-S6 cell signalling proteins. Our data indicate that the RUNX2 transcription factor promotes the expression of the CXCR4 chemokine receptor on melanoma cells, which in turn promotes autophagy, cell invasiveness, and osteotropism, through the inhibition of the mTOR signalling pathway. Our data suggest that RUNX2 promotes melanoma progression by upregulating CXCR4, and we identify the latter as a key player in melanoma-related osteotropism.