Adaptive Cell Responses Research Articles

LAMP-1 Chimeric to HIV-1 p55Gag in the Immunization of Neonate Mice Induces an Early Germinal Center Formation and AID Expression.

Neonates have a limited adaptive response of plasma cells, germinal center (GC) B cells, and T follicular helper cells (TFH). As neonatal vaccination can be an important tool for AIDS prevention, these limitations need to be overcome. Chimeric DNA vaccine encoding p55Gag HIV-1 protein conjugated with lysosomal-associated membrane protein 1 (LAMP-1) has been described as immunogenic in the neonate period. Herein, we investigated the immunologic mechanisms involved in neonatal immunization with a LAMP-1/p55Gag (LAMP/Gag) DNA vaccine in a C57BL/6 mouse background. Neonatal LAMP/Gag vaccination induced strong Gag-specific T-cell response until adulthood and elevated levels of anti-Gag IgG antibodies. We also demonstrated for the first time that the immunogenicity of the neonatal period with LAMP/Gag is due to the induction of high-affinity anti-p24 IgG antibodies and long-term plasma cells. Together with that, there is the generation of early TFH cells and the formation of GC sites with the upregulation of activation-induced cytidine deaminase (AID) enzyme mRNA and protein expression in draining lymph nodes after neonatal LAMP/Gag vaccination. These findings underscore that the LAMP-1 strategy in the chimeric vaccine could be useful to enhance antibody production even in the face of neonatal immaturity, and they contribute to the development of new vaccine approaches for other emerging pathogens at an early stage of life.

Inhibition of Autophagy Negates Radiofrequency-Induced Adaptive Response in SH-SY5Y Neuroblastoma Cells.

In the last years, radiofrequency (RF) has demonstrated that it can reduce DNA damage induced by a subsequent treatment with chemical or physical agents in different cell types, resembling the adaptive response, a phenomenon well documented in radiobiology. Such an effect has also been reported by other authors both in vitro and in vivo, and plausible hypotheses have been formulated, spanning from the perturbation of the cell redox status, to DNA repair mechanisms, and stress response machinery, as possible cellular mechanisms activated by RF pre-exposure. These mechanisms may underpin the observed phenomenon, and require deeper investigations. The present study aimed to determine whether autophagy contributes to RF-induced adaptive response. To this purpose, SH-SY5Y human neuroblastoma cells were exposed for 20 h to 1950 MHz, UMTS signal, and then treated with menadione. The results obtained indicated a reduction in menadione-induced DNA damage, assessed by applying the comet assay. Such a reduction was negated when autophagy was inhibited by bafilomycin A1 and E64d. Moreover, CRISPR SH-SY5Y cell lines defective for ATG7 or ATG5 genes did not show an adaptive response. These findings suggest the involvement of autophagy in the RF-induced adaptive response in human neuroblastoma cells; although, further investigation is required to extend such observation at the molecular level.

Abstract 5684: Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress

Abstract Cancer cells survive under extreme environments that alter protein homeostasis, thereby provoking cellular stress responses, such as the unfolded protein response (UPR) and the integrated stress response (ISR). These pathways increase the expression of molecular chaperones to prevent the accumulation of toxic proteins in the cell. One molecular chaperone in particular, Hsp70, plays a central role in both cellular protection and the response to proteotoxic insults. This is perhaps most evident in cancer cells, some of which overexpress Hsp70 and thrive even when harboring high levels of misfolded, aggregated, and/or unassembled protein complexes. In an effort to define how cells compensate for compromised proteostasis, we examined the adaptive response in breast cancer cells after challenge with a specific Hsp70 inhibitor. We first showed that established breast cancer cell lines bin into classes based on their sensitivity to the drug. We next discovered that resistant cells have higher autophagy levels compared to the more sensitive lines. Moreover, gene and protein expression studies and imaging methods showed that autophagy was further induced by Hsp70 inhibition in resistant breast cancer cells. Consistent with the fact that Hsp70 normally targets aberrant proteins for degradation, our data indicated that resistance to compromised Hsp70 activity arose from elevated levels of autophagy. We next showed that Hsp70 inhibition specifically triggers the ISR pathway. Unexpectedly, protection against the Hsp70 inhibitor was dependent on the ISR sensor, GCN2, which is most commonly associated with the amino acid starvation response. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating the PERK pathway. These data reveal an unexpected route through which cancer cells can adapt to chaperone targeted-proteotoxicity and position GCN2 and autophagy as critical compensatory mechanisms for cancer cells when proteostasis is compromised. Citation Format: Sara Sannino, Jeffrey L. Brodsky. Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5684.

Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences

Aggressive breast cancer variants, like triple negative and inflammatory breast cancer, contribute to disparities in survival and clinical outcomes among African American (AA) patients compared to White (W) patients. We previously identified the dominant role of anti-apoptotic protein XIAP in regulating tumor cell adaptive stress response (ASR) that promotes a hyperproliferative, drug resistant phenotype. Using The Cancer Genome Atlas (TCGA), we identified 46–88 ASR genes that are differentially expressed (2-fold-change and adjusted p-value < 0.05) depending on PAM50 breast cancer subtype. On average, 20% of all 226 ASR genes exhibited race-related differential expression. These genes were functionally relevant in cell cycle, DNA damage response, signal transduction, and regulation of cell death-related processes. Moreover, 23% of the differentially expressed ASR genes were associated with AA and/or W breast cancer patient survival. These identified genes represent potential therapeutic targets to improve breast cancer outcomes and mitigate associated health disparities.

Hydrogen Peroxide Stimulates Dihydrotestosterone Release in C2C12 Myotubes: A New Perspective for Exercise-Related Muscle Steroidogenesis?

Skeletal muscle is a tissue that has recently been recognized for its ability to produce androgens under physiological conditions. The steroidogenesis process is known to be negatively influenced by reactive oxygen species (ROS) in reproductive Leydig and ovary cells, while their effect on muscle steroidogenesis is still an unexplored field. Muscle cells are continuously exposed to ROS, resulting from both their metabolic activity and the surrounding environment. Interestingly, the regulation of signaling pathways, induced by mild ROS levels, plays an important role in muscle fiber adaptation to exercise, in a process that also elicits a significant modulation in the hormonal response. The aim of the present study was to investigate whether ROS could influence steroidogenesis in skeletal muscle cells by evaluating the release of testosterone (T) and dihydrotestosterone (DHT), as well as the evaluation of the relative expression of the key steroidogenic enzymes 5α-reductase, 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, and aromatase. C2C12 mouse myotubes were exposed to a non-cytotoxic concentration of hydrogen peroxide (H2O2), a condition intended to reproduce, in vitro, one of the main stimuli linked to the process of homeostasis and adaptation induced by exercise in skeletal muscle. Moreover, the influence of tadalafil (TAD), a phosphodiesterase 5 inhibitor (PDE5i) originally used to treat erectile dysfunction but often misused among athletes as a “performance-enhancing” drug, was evaluated in a single treatment or in combination with H2O2. Our data showed that a mild hydrogen peroxide exposure induced the release of DHT, but not T, and modulated the expression of the enzymes involved in steroidogenesis, while TAD treatment significantly reduced the H2O2-induced DHT release. This study adds a new piece of information about the adaptive skeletal muscle cell response to an oxidative environment, revealing that hydrogen peroxide plays an important role in activating muscle steroidogenesis.

BNIP3 mediates the different adaptive responses of fibroblast-like synovial cells to hypoxia in patients with osteoarthritis and rheumatoid arthritis

BackgroundHypoxia is one of the important characteristics of synovial microenvironment in rheumatoid arthritis (RA), and plays an important role in synovial hyperplasia. In terms of cell survival, fibroblast-like synovial cells (FLSs) are relatively affected by hypoxia. In contrast, fibroblast-like synovial cells from patients with RA (RA-FLSs) are particularly resistant to hypoxia-induced cell death. The purpose of this study was to evaluate whether fibroblast-like synovial cells in patients with osteoarthritis (OA-FLSs) and RA-FLSs have the same adaptation to hypoxia.MethodsCCK-8, flow cytometry and BrdU were used to detect the proliferation of OA-FLSs and RA-FLSs under different oxygen concentrations. Apoptosis was detected by AV/PI, TUNEL and Western blot, mitophagy was observed by electron microscope, laser confocal microscope and Western blot, the state of mitochondria was detected by ROS and mitochondrial membrane potential by flow cytometry, BNIP3 and HIF-1α were detected by Western blot and RT-qPCR. The silencing of BNIP3 was achieved by stealth RNA system technology.ResultsAfter hypoxia, the survival rate of OA-FLSs decreased, while the proliferation activity of RA-FLSs further increased. Hypoxia induced an increase in apoptosis and inhibition of mitophagy in OA-FLSs, but not in RA-FLSs. Hypoxia led to a more lasting adaptive response. RA-FLSs displayed a more significant increase in the expression of genes transcriptionally regulated by HIF-1α. Interestingly, they showed higher BNIP3 expression than OA-FLSs, and showed stronger mitophagy and proliferation activities. BNIP3 siRNA experiment confirmed the potential role of BNIP3 in the survival of RA-FLSs. Inhibition of BNIP3 resulted in the decrease of cell proliferation, mitophagy and the increase of apoptosis.ConclusionIn summary, RA-FLSs maintained intracellular redox balance through mitophagy to promote cell survival under hypoxia. The mitophagy of OA-FLSs was too little to maintain the redox balance of mitochondria, resulting in apoptosis. The difference of mitophagy between OA-FLSs and RA-FLSs under hypoxia is mediated by the level of BNIP3 expression.

The RelB-BLNK Axis Determines Cellular Response to a Novel Redox-Active Agent Betamethasone during Radiation Therapy in Prostate Cancer.

Aberrant levels of reactive oxygen species (ROS) are potential mechanisms that contribute to both cancer therapy efficacy and the side effects of cancer treatment. Upregulation of the non-canonical redox-sensitive NF-kB family member, RelB, confers radioresistance in prostate cancer (PCa). We screened FDA-approved compounds and identified betamethasone (BET) as a drug that increases hydrogen peroxide levels in vitro and protects non-PCa tissues/cells while also enhancing radiation killing of PCa tissues/cells, both in vitro and in vivo. Significantly, BET increases ROS levels and exerts different effects on RelB expression in normal cells and PCa cells. BET induces protein expression of RelB and RelB target genes, including the primary antioxidant enzyme, manganese superoxide dismutase (MnSOD), in normal cells, while it suppresses protein expression of RelB and MnSOD in LNCaP cells and PC3 cells. RNA sequencing analysis identifies B-cell linker protein (BLNK) as a novel RelB complementary partner that BET differentially regulates in normal cells and PCa cells. RelB and BLNK are upregulated and correlate with the aggressiveness of PCa in human samples. The RelB-BLNK axis translocates to the nuclear compartment to activate MnSOD protein expression. BET promotes the RelB-BLNK axis in normal cells but suppresses the RelB-BLNK axis in PCa cells. Targeted disruptions of RelB-BLNK expressions mitigate the radioprotective effect of BET on normal cells and the radiosensitizing effect of BET on PCa cells. Our study identified a novel RelB complementary partner and reveals a complex redox-mediated mechanism showing that the RelB-BLNK axis, at least in part, triggers differential responses to the redox-active agent BET by stimulating adaptive responses in normal cells but pushing PCa cells into oxidative stress overload.

Adaptive response analysis of colorectal cancer cells to low-dose oxaliplatin as a tool to deciphering mechanisms of synergistic drug interaction.

3091 Background: Using an in vitro dose matrix approach, we previously showed in multiple colorectal cancer cell lines a striking cytotoxic synergism between oxaliplatin at very low concentrations and the ATR inhibitor VE-822. We confirmed this finding in vivo, and, surprisingly, in this setting the oxaliplatin-induced cell addiction to VE-822 persists over several days after oxaliplatin elimination. We tried to elucidate the molecular mechanism of the latter phenomenon. Methods: We evaluated by RNAseq the gene expression changes induced in vitro by low-dose oxaliplatin in the colorectal cancer cell line HCT-116 after 24 and 48 hours of treatment. In order to untangle the functional significance of the adaptive response to oxaliplatin, we performed on the RNAseq data an extensive gene set enrichment analysis (GSEA) using gene set from all Molecular Signature Database v7.4 collections with the exception of C7. For ontology-based gene set collections, we clusterized the enriched gene sets using the semantic similarity methodology in order to increase the readability of global functional response. Results: Extensive GSEA showed that after 24 hours of oxaliplatin treatment cancer cells upregulate several gene sets involved in aspecific responses to cellular stress or to various type of extracellular stimulations, including other organisms, oxygen-containing compounds, abiotic stimuli and hypoxia. In addition, several gene sets involved in proteolysis and autophagy are upregulated, suggesting a rewiring of cell machinery. After 48 hours of oxaliplatin treatment, we observed the activation of ribosome function, mitochondrial assembly and synthesis of aminoacids and ribonucleosides. Finally, a widespread negative enrichment of gene sets involved in DNA repair-related was detected both at 24 and at 48 hours, with a far greater negative enrichment at 48 hours, which suggest a commitment of cancer cell to a major limitation of DNA repairing capability lasting several days following a DNA damaging insult. Analysis of leading edge genes from the DNA repair gene sets showed a profound repression both at 24 and 48 hours of the transcripts of BRCA1, BRCA2, ATM, CHK1, WEE1, BARD1, BRIP1, NEHJ1, RAD51, XRCC2, CLSPN, GEN1, DNA2, EXO1, TOPBP1, POLE, RMI1. Interestingly, ATR mRNA was minimally repressed both at 24 and at 48 hours, which could explain the long-standing in vivo dependence of cancer cell to ATR after a brief oxaliplatin exposure. Conclusions: Extensive GSEA was able to elucidate the molecular mechanism underlying synergistic interaction between oxaliplatin and VE-822. The impact of profiling cancer cell adaptive responses by extensive GSEA should be further evaluated in the setting of rational development of drug combinations.

Oxidative potential and in vitro toxicity of particles generated by pyrotechnic smokes in human small airway epithelial cells

Pyrotechnic smokes are widely used in civilian and military applications. The major issue arise from the release of particles after smoke combustion but the health risks related to their exposure are poorly documented whereas toxicity of airborne particles on the respiratory target are very well known. Therefore, this study aimed to explore the in vitro toxicity of the particle fraction of different pyrotechnic smokes.Particles from a red signalling smoke (RSS), an hexachloroethane-based obscuring smoke (HC-OS) and an anti-intrusion smoke (AIS) were collected from the cloud. RSS particles displayed the highest organic fraction (quinones and polycyclic aromatic hydrocarbons) of the three samples characterized. AIS particles contained K and cholesterol derivatives. HC-OS particles were mainly metallic with very high concentrations of Al, Fe and Ca. Intrinsic oxidative potential of smoke particles was measured with two assays. Depletions of DTT by RSS particles was greater than depletion obtained with AIS and HC-OS particles but depletion of acid ascorbic (AA) was only observed with HC-OS particles. In vitro toxicity was assessed by exposing human small airway epithelial cells (SAEC) to various concentrations of particles. After 24 h of exposure, cell viability was not affected but significant modifications of mRNA expression of antioxidant (SOD-1 and -2, catalase, HO-1, NQO-1) and inflammatory markers (IL-6, IL-8, TNF-α) were observed and were dependent on smoke type. Particles rich in metal, such as HC-OS, induced a greatest depletion of AA and a greatest inflammatory response, whereas particles rich in organic compounds, such as RSS, induced a greatest DTT depletion and a greatest antioxidant response.In conclusion, the three smoke particles have an intrinsic oxidative potential and triggered a cell adaptive response. Our study improved the knowledge of particle toxicity of pyrotechnic smokes and scientific approach developed here could be used to study other type of particles.

Mito-Q promotes porcine oocytes maturation by maintaining mitochondrial thermogenesis via UCP2 downregulation

Mitochondrial thermogenesis is an adaptive response of cells to their surrounding stress. Oxidative stress is one of the common stresses during in vitro maturation (IVM) of oocytes, which leads to mitochondrial dysfunction. This study aimed to probe the effects of the mitochondria-targeted antioxidant Mito-Q on oocyte development and unravel the role of Mito-Q in mitochondrial ATP production and thermogenesis regulation. Our results showed that Mito-Q had a positive effect on porcine oocytes maturation and subsequent embryo development. During oocytes IVM, Mito-Q could reduce ATP levels and ROS, increase lipid droplets accumulation, induce autophagy, and maintain mitochondrial temperature stability. Moreover, in metaphase II (MII) oocytes, Mito-Q would induce mitochondrial uncoupling manifested by decreased ATP, attenuated mitochondrial membrane potential (MMP), and increased mitochondrial thermogenesis. Notably, the expression of mitochondrial uncoupling protein (UCP2) was significantly reduced in oocytes treated with Mito-Q. Further study indicated that specific depletion of UCP2 in oocytes also resulted in increased thermogenesis, decreased ATP and declined MMP, suggesting that UCP2 downregulation may participate in Mito-Q-induced mitochondrial uncoupling. In summary, our data demonstrate that Mito-Q promotes oocyte maturation in vitro and maintains the stability of mitochondrial thermogenesis by inhibiting UCP2 expression.

Abstract B011: P300 histone acetyltransferase mediates glioma stem cell adaptive response to therapeutic stress

Abstract Glioma stem-like and tumor cells exhibit phenotypic plasticity in response to environmental and therapeutic stress leading to tumor recurrence, but the underlying mechanisms remain largely unknown. In this study, we employed single-cell and whole transcriptomic analyses to uncover that radiation-stress induces a dynamic shift in functional states of glioma cells allowing for acquisition of vascular endothelial-like and pericyte-like cell phenotypes. These vascular-like cells provide trophic support to promote proliferation of tumor cells, and their selective depletion results in reduced tumor growth post-treatment in vivo. Mechanistically, the acquisition of vascular-like phenotype is driven by increased chromatin accessibility and H3K27 acetylation in specific vascular gene regions allowing for their increased expression post-treatment. Blocking P300 histone acetyltransferase activity using a small molecule inhibitor C646 or gene knockdown reverses the epigenetic changes induced by radiation, inhibits the adaptive conversion of GSC into vascular-like cells, reduces tumor growth and enhances animal survival. Our findings highlight an important role for P300 in mediating adaptive response of glioma stem cells to therapeutic-stress, and opens a new therapeutic avenue for preventing glioma recurrence. Citation Format: Sree Deepthi Muthukrishnan, Riki Kawaguchi, Pooja Nair, Alvaro Alvarado, Harley Kornblum. P300 histone acetyltransferase mediates glioma stem cell adaptive response to therapeutic stress [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B011.

Physics of Brain Cancer: Multiscale Alterations of Glioblastoma Cells under Extracellular Matrix Stiffening.

The biology and physics underlying glioblastoma is not yet completely understood, resulting in the limited efficacy of current clinical therapy. Recent studies have indicated the importance of mechanical stress on the development and malignancy of cancer. Various types of mechanical stress activate adaptive tumor cell responses that include alterations in the extracellular matrix (ECM) which have an impact on tumor malignancy. In this review, we describe and discuss the current knowledge of the effects of ECM alterations and mechanical stress on GBM aggressiveness. Gradual changes in the brain ECM have been connected to the biological and physical alterations of GBM cells. For example, increased expression of several ECM components such as glycosaminoglycans (GAGs), hyaluronic acid (HA), proteoglycans and fibrous proteins result in stiffening of the brain ECM, which alters inter- and intracellular signaling activity. Several mechanosensing signaling pathways have been identified that orchestrate adaptive responses, such as Hippo/YAP, CD44, and actin skeleton signaling, which remodel the cytoskeleton and affect cellular properties such as cell–cell/ECM interactions, growth, and migration/invasion of GBM cells. In vitro, hydrogels are used as a model to mimic the stiffening of the brain ECM and reconstruct its mechanics, which we also discuss. Overall, we provide an overview of the tumor microenvironmental landscape of GBM with a focus on ECM stiffening and its associated adaptive cellular signaling pathways and their possible therapeutic exploitation.

Role of PTP1B in the Regulation of Cholesterol Homeostasis

Protein tyrosine phosphatase 1B (PTP1B), encoded by the PTPN1 gene, is a well‐known negative regulator of insulin signaling. Since insulin, via the activation of the insulin receptor, is involved in cholesterol synthesis by stimulating genes like HMG‐CoA reductase (HMGCR), we enquired whether PTP1B also played a role in the regulation of cholesterol homeostasis. Interestingly, knocking out PTP1B in hepatocytes, where most of the circulating cholesterol is synthesized, generates mice that have reduced circulating cholesterol levels. Moreover, SNPs in the human PTPN1 gene are associated with higher levels of low‐density lipoprotein cholesterol, total plasma cholesterol, and with the insulin sensitivity index and fasting glucose levels. As a first step, to better understand the function of PTP1B in cholesterol homeostasis, we used Mouse Embryonic Fibroblast (MEFs) cells derived from WT or PTP1B KO mice and measured cholesterol levels utilizing an Amplex red assay. As previously observed in liver PTP1B KO mice, we observed a ~ 50% decrease in cholesterol levels in PTP1B KO cells when compared to WT cells. Supporting this, Western blot analysis also indicated that proteins regulating cholesterol homeostasis are differently expressed in WT and KO MEFs at basal. We observed decreased expression of LDLR, PCSK9, HMGCR, SREBP1 and Cav‐1 in PTP1B KO cells. In addition, cholesterol treatment also showed a decreased adaptive response in PTP1B KO cells. Interestingly, Src, a substrate of PTP1B, was hyperphosphorylated in PTP1B KO MEFs treated with cholesterol and these cells were more sensitive to cholesterol‐induced toxicity as indicated by caspase‐3 cleavage at lower concentrations of cholesterol. Quantitative mass spectrometry analysis using isobaric tag for relative and absolute quantitation (iTRAQ) further revealed that several proteins involved in cholesterol homeostasis (e.g. NPC2, NPC1L1, DHCR7, NCEH1, ApoA, ApoB, ApoC, ApoE) were overexpressed in PTP1B KO MEFs. We also observed that proteins involved in retinoic acid homeostasis, which have also been involved in the regulation of insulin resistance and lipid homeostasis, were overexpressed in PTP1B KO MEFs. We are currently validating these findings and exploring underlying mechanisms by which PTP1B gene inactivation regulates cholesterol homeostasis in mammalian cells.

Acidosis regulation of epithelial barrier function and immune signaling

A common but often underappreciated aspect of the inflammatory microenvironment is extracellular acidification. As with other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease (IBD) patients. Colonic pH as low as 4 have been reported in IBD patients. In contrast, the pH of a healthy colon is between a pH of 7.0‐7.4. Recently, we identified an adaptive tissue response in intestinal epithelial cells (IEC) which buffered extracellular acidification during neutrophil transepithelial migration, suggesting pH homeostasis is an important aspect of acute inflammation. Extensions of these studies identified a previously undescribed CREB‐dependent pathway for sensing extracellular pH and a unique acidosis gene signature in IEC. The full impact of acidosis on IEC function remains unclear. To better understand the impact of chronic extracellular acidification on IEC function, we investigated changes in IEC immune signaling and barrier function in the setting of acute and chronic exposure to acidosis. We initially examined barrier function in T84 IEC cultured under acidic conditions and observed a significant decrease in barrier function, as determined by transepithelial electrical resistance (TER) and paracellular flux, in cells exposed to acidosis in a pH range of 6.0 – 7.4. Interestingly, following acute acidification, treatment with low pH for less than 8hr, barrier function recovered within 16hr. However, following chronic acidification, treatment with low pH beyond 16hr resulted in an appreciable barrier dysfunction for several days. We extended these studies to examine expression and localization of tight‐junction proteins in IEC exposed to acidosis and observed a time and dose‐dependent decrease in ZO‐1 and E‐cadherin expression, with almost complete loss of expression after 72hr at a pH of 6.0. Likewise, expression and localization of claudins 1, 2, and 4 decreased over time. In parallel to a loss of barrier function, IEC exposed to chronic acidosis revealed a prominent increase in the expression of inflammatory markers including several chemokines (IL‐8, CCL20, TNF) as well as anti‐microbial peptides (mucins and beta‐defensins).Thus, chronic extracellular acidosis induces the loss of IEC tight junction function, while inducing the expression of several pro‐inflammatory chemokines and the production of various anti‐microbial peptides. These results identify extracellular acidification as significant signaling mechanism which induces a loss of epithelial features and the acquisition of a more immune‐like phenotype. Furthering our understandings of the mechanisms involved in acidosis induced phenotype switching in IEC has the potential to uncover novel therapeutic drug targets.

Ultrasensitive Hypoxia Sensing at the Single-Molecule Level via Super-Resolution Quantum Dot-Linked Immunosandwich Assay

Activated hypoxia-inducible factor-1alpha (HIF-1α) plays an important role in the adaptive response of tumor cells to oxygen changes through the transcriptional activation of genes that regulate important biological processes required for tumor survival and progression. In this study, we developed an ultrasensitive hypoxia sensor based on read-out with quantum dots on a gold nanodisc (quantum dot-linked immunosandwich assay, QLISA) with excellent selectivity for HIF-1α. The immunoassay platform was established by comparing the immune response results using Qdot525 as a detection nanoprobe instead of a fluorescent dye (Alexa488) (fluorescent-linked immunosandwich assay, FLISA). In addition, using three-dimensional total internal reflection fluorescence microscopy, the platform was optically sectioned along the z-axis at 10 nm intervals to compare the height difference between the nanodisc and the nanoprobe following the QLISA and FLISA procedures and to localize the target location. Here, the super-resolution QLISA (srQLISA)-based hypoxia sensor exhibited high accuracy and precision for the detection of HIF-1α-extracted samples in cancer spheroids compared with the super-resolution FLISA (srFLISA) method. The developed nanobiosensor method demonstrated a wide dynamic linear detection range of 32.2 zM-8.0 pM with a limit of detection of 16 zM under optimal experimental conditions for HIF-1α, an approximate 106-fold enhanced detection sensitivity compared with the conventional enzyme-linked immunosorbent assay method based on absorbance. The detection of HIF-1α using the newly developed srQLISA sensor allows for independently predicting tumor progression and early cancer onset increases in the microvasculature density of tumor lesions.

CD38 DEFICIENCY PROTECTS MICE FROM AGE-RELATED SPONTANEOUS OA DEVELOPMENT

Purpose: CD38 is recently shown to be the main NADase in mammalian tissue, which contributes substantially to degradation of cellular nicotinamide adenine dinucleotide (NAD+), a key metabolite involved in cellular energy metabolism and adaptive responses of cells to bioenergetics and oxidative stress. CD38 expression and activity are increased during aging, which contributes to age-related decline of NAD+. In our previous studies, we observed an age-related increase in expression of CD38 in human knee cartilage, and that increased CD38 expression in chondrocytes is associated with reduced NAD/NADH levels and increased catabolic responses to pro-inflammatory cytokine IL-1β.

C-MYC-USP49-BAG2 axis promotes proliferation and chemoresistance of colorectal cancer cells in vitro

Deubiquitinases (DUBs) play critical roles in tumorigenesis and are emerging as potential therapeutic targets. However, it remains less clear which DUBs may play important roles and represent a realistic vulnerability for a particular type of tumor. Here we revealed that Ubiquitin Specific Peptidase 49 (USP49) is transcriptionally activated by c-MYC in colorectal cancer (CRC), and CRC patients with elevated USP49 levels exhibited significantly shorter survival. Knockdown of USP49 markedly inhibited CRC cell proliferation, colony formation, and chemotherapy resistance in vitro. Investigation of mechanisms unravels that USP49 deubiquitinates and stabilizes Bcl-2-Associated Athanogene 2 (BAG2), a well-known protein that antagonizes apoptosis and enables adaptive response of CRC cells. This study identified a novel mechanism by which USP49 promotes CRC cell survival by stabilizing BAG2 through the c-MYC-USP49-BAG2 axis, indicating that USP49 may become a potential therapeutic target for CRC.

The Role of Nuclear Factor Kappa B (NF-κB) in the Immune Response against Parasites.

The immune system consists of various cells, organs, and processes that interact in a sophisticated manner to defend against pathogens. Upon initial exposure to an invader, nonspecific mechanisms are raised through the activation of macrophages, monocytes, basophils, mast cells, eosinophils, innate lymphoid cells, or natural killer cells. During the course of an infection, more specific responses develop (adaptive immune responses) whose hallmarks include the expansion of B and T cells that specifically recognize foreign antigens. Cell to cell communication takes place through physical interactions as well as through the release of mediators (cytokines, chemokines) that modify cell activity and control and regulate the immune response. One regulator of cell states is the transcription factor Nuclear Factor kappa B (NF-κB) which mediates responses to various stimuli and is involved in a variety of processes (cell cycle, development, apoptosis, carcinogenesis, innate and adaptive immune responses). It consists of two protein classes with NF-κB1 (p105/50) and NF-κB2 (p100/52) belonging to class I, and RelA (p65), RelB and c-Rel belonging to class II. The active transcription factor consists of a dimer, usually comprised of both class I and class II proteins conjugated to Inhibitor of κB (IκB). Through various stimuli, IκB is phosphorylated and detached, allowing dimer migration to the nucleus and binding of DNA. NF-κB is crucial in regulating the immune response and maintaining a balance between suppression, effective response, and immunopathologies. Parasites are a diverse group of organisms comprised of three major groups: protozoa, helminths, and ectoparasites. Each group induces distinct effector immune mechanisms and is susceptible to different types of immune responses (Th1, Th2, Th17). This review describes the role of NF-κB and its activity during parasite infections and its contribution to inducing protective responses or immunopathologies.

Abstract P3-14-18: Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences

Abstract Background &amp; Hypothesis: The death rate is higher in Black (B) patients (40%), and, in particular, is two-fold higher among young B women (&amp;lt;50 years) compared to White (W) women with breast cancer. Aggressive breast cancer variants, like hormone receptor-negative and inflammatory breast cancer (IBC), contribute to a race-related survival gap and poor clinical outcomes in African American (AA) patients compared to Whites (W) in the United States. We previously identified the dominant role of XIAP in a tumor cell adaptive stress response (ASR) that promotes nuclear transcription factor (NFκB)-mediated proliferative, invasive, and immunosuppressive signaling in breast cancer progression. Our current work sought to address the urgent need to interrogate molecular mechanisms driving the disparity in both AA and IBC tumor biology by investigating gene expression differences between AA and White tumors and Triple-negative breast cancer (TNBC) and non-TNBC tumors, thereby identifying common genes which may be modifiable risk factors and targets that can be modulated for therapeutic benefits. Methods: We used The Cancer Genome Atlas (TCGA) breast cancer data to conduct a comparative analysis of the expression of 226 ASR genes in breast cancer molecular subtypes, normal-adjacent tissues, and in AA and W patients. The ASR gene list includes 101 XIAP-related, 11 NFκB targets, 10 MNK targets, 33 Oxidative Stress Response (OSR)-related, 13 TGFβ-related, 6 JAG1-Notch targets, and 52 immune-related, in addition to 14 genes that belong to more than one ASR set. In the present study, we focused on primary tumor samples [1090 Primary Solid Tumor] and normal tissue samples adjacent to the tumors [113 Solid Tissue Normal]. Within these, our sample set included 559 lumA, 207 lumB, 82 Her2, 190 Basal, and 40 Normal-like. Racial designations in TCGA are based on patient self-identification. In the present study, the race-related analysis focused on AA and W breast cancer patient datasets from TCGA (179 AA and 744 W). It is important to note that among the 113 Normal-adjacent samples, there are 105 W and only 6 AA samples. Results: Analysis of the ASR gene score in different Breast cancer subtypes revealed that ASR genes associated with oxidative stress and immune response pathways score highly in the basal breast cancer subtype. We also observed differential scores between samples of AA or W. The differential gene expression analysis identified 46-88 genes in the ASR set to be differentially expressed (≥2 fold-change and adjusted p-value &amp;lt;0.05) in breast cancer subtypes when comparing each subtype to normal-adjacent tissue or comparing the subtypes to each other. Pathway analysis of the enriched genes revealed the involvement of the cell cycle, the DNA damage response, the signal transduction, and the regulation of cell death-related processes. Moreover, on average, 20% of the ASR genes showed race-related differential expression. Furthermore, a number of ASR that were differentially expressed in breast cancer subtypes and significantly associated (hazard ratio &amp;gt;1.5 and p-value &amp;lt;0.05) with AA and/or W patient survival. For example, CDC45, MCM3, and CDKN2D were uniquely associated with poor survival in AA, but not in W. Conclusion: Within invasive breast cancer, factors that regulate tumor cell ASR reveal subtype and race-specific differences in expression. Understanding their function and downstream signaling will elucidate the intrinsic mechanisms that drive racial disparities in breast cancer outcomes and have the potential to reveal race-related biomarkers and therapeutic targets. Support: NCI-P20 NCCU-Duke Cancer Disparities Translational Research Partnership (SP; GRD, KPW, JF), DoD-W81XWH-17-1-0297 (GRD), Duke Bridge Funds (GRD). Citation Format: Muthana Al Abo, Larisa Gearhart-Serna, Steven Van Laere, Jennifer Freedman, Steven Patierno, Eun-Sil Hwang, Savitri Krishnamurthy, Kevin Williams, Gayathri Devi. Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-18.

Antigen-Presenting Cells in Psoriasis.

Psoriasis is classically considered a chronic inflammatory skin disorder, however the identification of autoantigens in its pathogenesis established it as a T cell mediated autoimmune disease. As such professional antigen-presenting cells (APCs) are key players in the development of lesions. APCs in the skin include dendritic cells, Langerhans cells and monocytes/macrophages. In addition, epidermal keratinocytes and dermal mast cells are also endowed with antigen-presenting capacity. Skin APCs have central role in the maintenance of cutaneous immune homeostasis, as well as in initiating and sustaining inflammation under pathologic conditions. In this review we discuss the functional specialization of human skin APCs that promote T cell activation and adaptive immune response during psoriasis initiation and onset.