Significant Differences In Gene Expression Research Articles

Loss of Caveolin-1 Expression in Tumor Cells is Associated with Increased Aggressiveness and Cell Invasion in Oral Squamous Cell Carcinoma.

Changes in Caveolin-1 (CAV-1) expression are related to tumorigenesis. The aim of this study was to evaluate the role of CAV-1 in tumor progression in oral squamous cell carcinoma (SCC) tissue samples and the effect of CAV-1 silencing on two oral tongue SCC (OTSCC) cell lines (SCC-25, from a primary tumor, and HSC-3 from lymph node metastases). Mycroarray hybridization, mRNA expression, and immunohistochemistry were performed on OSCC tissue samples and corresponding non-tumoral margin tissues. The effects of CAV-1 silencing (siCAV-1) on cell viability, membrane fluidity, on the expression of epithelial to mesenchymal transition (EMT) markers and on cell migration and invasion capacity of OTSCC cell lines were evaluated. Microarray showed a greater CAV-1 expression (1.77-fold) in OSCC tumors than in non-tumoral tissues and 2.0-fold more in less aggressive OSCCs. However, significant differences in CAV-1 gene expression were not seen between tumors and non-tumoral margins nor CAV-1 with any clinicopathological parameters. CAV-1 protein was localized both in carcinoma and in spindle cells of the tumor microenvironment (TME), and CAV-1 positive TME cells were associated with smaller/more aggressive tumors, independent of the carcinoma cells' expression. Silencing of CAV-1 increased cell viability only in SCC-25 cells. It also stimulated the invasion of HSC-3 cells and increased ECAD and BCAT mRNA in these cells; however, the protein levels of the EMT markers were not affected. Decreased expression of CAV-1 by tumor cells in OSCC and an increase in the TME were associated with increased cell invasiveness and tumor aggressiveness.

Molecular profiling of a real-world breast cancer cohort with genetically inferred ancestries reveals actionable tumor biology differences between European ancestry and African ancestry patient populations

BackgroundEndocrine-resistant HR+/HER2- breast cancer (BC) and triple-negative BC (TNBC) are of interest for molecularly informed treatment due to their aggressive natures and limited treatment profiles. Patients of African Ancestry (AA) experience higher rates of TNBC and mortality than European Ancestry (EA) patients, despite lower overall BC incidence. Here, we compare the molecular landscapes of AA and EA patients with HR+/HER2- BC and TNBC in a real-world cohort to promote equity in precision oncology by illuminating the heterogeneity of potentially druggable genomic and transcriptomic pathways.MethodsDe-identified records from patients with TNBC or HR+/HER2- BC in the Tempus Database were randomly selected (N = 5000), with most having stage IV disease. Mutations, gene expression, and transcriptional signatures were evaluated from next-generation sequencing data. Genetic ancestry was estimated from DNA-seq. Differences in mutational prevalence, gene expression, and transcriptional signatures between AA and EA were compared. EA patients were used as the reference population for log fold-changes (logFC) in expression.ResultsAfter applying inclusion criteria, 3433 samples were evaluated (n = 623 AA and n = 2810 EA). Observed patterns of dysregulated pathways demonstrated significant heterogeneity among the two groups. Notably, PIK3CA mutations were significantly lower in AA HR+/HER2- tumors (AA = 34% vs. EA = 42%, P < 0.05) and the overall cohort (AA = 28% vs. EA = 37%, P = 2.08e−05). Conversely, KMT2C mutation was significantly more frequent in AA than EA TNBC (23% vs. 12%, P < 0.05) and HR+/HER2- (24% vs. 15%, P = 3e−03) tumors. Across all subtypes and stages, over 8000 genes were differentially expressed between the two ancestral groups including RPL10 (logFC = 2.26, P = 1.70e−162), HSPA1A (logFC = − 2.73, P = 2.43e−49), ATRX (logFC = − 1.93, P = 5.89e−83), and NUTM2F (logFC = 2.28, P = 3.22e−196). Ten differentially expressed gene sets were identified among stage IV HR+/HER2- tumors, of which four were considered relevant to BC treatment and were significantly enriched in EA: ERBB2_UP.V1_UP (P = 3.95e−06), LTE2_UP.V1_UP (P = 2.90e−05), HALLMARK_FATTY_ACID_METABOLISM (P = 0.0073), and HALLMARK_ANDROGEN_RESPONSE (P = 0.0074).ConclusionsWe observed significant differences in mutational spectra, gene expression, and relevant transcriptional signatures between patients with genetically determined African and European ancestries, particularly within the HR+/HER2- BC and TNBC subtypes. These findings could guide future development of treatment strategies by providing opportunities for biomarker-informed research and, ultimately, clinical decisions for precision oncology care in diverse populations.

Propensity of Patient-Derived iPSCs for Retinal Differentiation: Implications for Autologous Cell Replacement.

Prior to use, newly generated induced pluripotent stem cells (iPSC) should be thoroughly validated. While excellent validation and release testing assays designed to evaluate potency, genetic integrity, and sterility exist, they do not have the ability to predict cell type-specific differentiation capacity. Selection of iPSC lines that have limited capacity to produce high-quality transplantable cells, places significant strain on valuable clinical manufacturing resources. The purpose of this study was to determine the degree and root cause of variability in retinal differentiation capacity between cGMP-derived patient iPSC lines. In turn, our goal was to develop a release testing assay that could be used to augment the widely used ScoreCard panel. IPSCs were generated from 15 patients (14-76 years old), differentiated into retinal organoids, and scored based on their retinal differentiation capacity. Despite significant differences in retinal differentiation propensity, RNA-sequencing revealed remarkable similarity between patient-derived iPSC lines prior to differentiation. At 7 days of differentiation, significant differences in gene expression could be detected. Ingenuity pathway analysis revealed perturbations in pathways associated with pluripotency and early cell fate commitment. For example, good and poor producers had noticeably different expressions of OCT4 and SOX2 effector genes. QPCR assays targeting genes identified via RNA sequencing were developed and validated in a masked fashion using iPSCs from 8 independent patients. A subset of 14 genes, which include the retinal cell fate markers RAX, LHX2, VSX2, and SIX6 (all elevated in the good producers), were found to be predictive of retinal differentiation propensity.

Cathepsin S Upregulation Measured in the Peripheral Blood Mononuclear Cells Prior to Surgery Points to Postoperative Pain Development in Patients with Hip Osteoarthritis.

Disability caused by hip osteoarthritis has increased due to population aging, obesity, and lifestyle behaviors. Joint failure after conservative therapies results in total hip replacement, which is considered to be one of the most successful interventions. However, some patients experience long-term postoperative pain. Presently, there are no reliable clinical biomarkers for the prognosis of postoperative pain prior to surgery. Molecular biomarkers can be considered as intrinsic indicators of pathological processes and as links between clinical status and disease pathology, while recent innovative and sensitive approaches such as RT-PCR have extended the prognostic value of clinical traits. In light of this, we examined the importance of cathepsin S and proinflammatory cytokine gene expression in peripheral blood in addition to the clinical traits of patients with end-stage hip osteoarthritis (HOA) to predict postoperative pain development prior to surgery. This study included 31 patients with radiographic Kellgren and Lawrence grade III-IV HOA who underwent total hip arthroplasty (THA) and 26 healthy volunteers. Before surgery, a visual analog scale (VAS), DN4, PainDETECT, and the Western Ontario and McMaster Universities osteoarthritis index scores were used for pain and function assessment. Three and six months post-surgery, VAS pain scores of 30 mm and higher were reported. The intracellular protein levels of cathepsin S were measured using ELISA. The expression of the cathepsin S, tumor necrosis factor α, interleukin-1β, and cyclooxygenase-2 genes in peripheral blood mononuclear cells (PBMCs) was assessed using quantitative real-time RT-PCR. Pain persisted in 12 (38.7%) patients after THA. Patients who developed postoperative pain demonstrated significantly higher cathepsin S gene expression in the PBMCs and higher rates of neuropathic pain based on the DN4 testing compared to the other HOA subjects that were examined. No significant differences in proinflammatory cytokine gene expression were noted in either patient cohort prior to THA. The development of postoperative pain in patients with hip osteoarthritis might be associated with disturbances in pain perception, while increased expression of cathepsin S in the peripheral blood prior to surgery may serve as its prognostic biomarker and could be used in clinical settings to improve medical service for patients with end-stage hip OA.

Dynamics of Gene Expression Profiling and Identification of High-Risk Patients for Severe COVID-19.

The clinical manifestations of SARS-CoV-2 infection vary widely, from asymptomatic infection to the development of acute respiratory distress syndrome (ARDS) and death. The host response elicited by SARS-CoV-2 plays a key role in determining the clinical outcome. We hypothesized that determining the dynamic whole blood transcriptomic profile of hospitalized adult COVID-19 patients and characterizing the subgroup that develops severe disease and ARDS would broaden our understanding of the heterogeneity in clinical outcomes. We recruited 60 hospitalized patients with RT-PCR-confirmed SARS-CoV-2 infection, among whom 19 developed ARDS. Peripheral blood was collected using PAXGene RNA tubes within 24 h of admission and on day 7. There were 2572 differently expressed genes in patients with ARDS at baseline and 1149 at day 7. We found a dysregulated inflammatory response in COVID-19 ARDS patients, with an increased expression of genes related to pro-inflammatory molecules and neutrophil and macrophage activation at admission, in addition to an immune regulation loss. This led, in turn, to a higher expression of genes related to reactive oxygen species, protein polyubiquitination, and metalloproteinases in the latter stages. Some of the most significant differences in gene expression found between patients with and without ARDS corresponded to long non-coding RNA involved in epigenetic control.

Effects of cadmium and zinc on gene expression of novel molecular biomarkers in the mangrove oyster Crassostrea gasar

Metal contamination impacts various aquatic species, and mollusk bivalves are appropriate sentinel organisms in coastal pollution assessment. Metal exposure can disrupt homeostasis, alter gene expression, and harm cellular processes. However, organisms have evolved mechanisms to regulate metal ions and counteract their toxicity. This study examined the effect of acute cadmium (Cd) and zinc (Zn) on metal-related gene expression in gills of Crassostrea gasar following 24 and 48 h of laboratory exposure. We focused on Zn transport, metallothionein (MT), glutathione (GSH) biosynthesis, and calcium (Ca) transporter genes to understand the underlying Cd and Zn-accumulating mechanisms that prevent metal toxicity. Our findings revealed increased Cd and Zn levels in oyster gills, with significantly higher accumulation after 48 h. C. gasar accumulated high Cd concentrations even in scarce conditions and increased Zn levels, suggesting a strategy to cope with toxicity. While no significant gene expression differences were observed after 24 h, the increased metal accumulation after 48 h led to upregulation of CHAC1, GCLC, ZnT2, and MT-like genes in oysters exposed to Cd, and increased ZnT2-like expression following exposure to higher Cd/Zn mixtures. We found evidence of oysters may mobilize metal-related genes to mitigate Cd-induced toxicity by both chelating metals and/or reducing their intracellular concentrations. The observed genes upregulation also indicates their sensitivity to changes in metal bioavailability. Overall, this study offers insights into oyster mechanisms for coping with metal toxicity and suggests ZnT2, MT, CHAC1, and GCLC-like as molecular biomarkers for monitoring aquatic metal pollution using C. gasar as sentinel species.

Multi-omics Analyses Identify Galectin-1 as a Potential Novel Mediator of Heart Failure with Preserved Ejection Fraction in Mice and Humans

Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome where patients present with symptoms of volume overload despite having a normal systolic function. HFpEF is associated with elevation of inflammatory biomarkers, but the precise pathways leading to cardiac dysfunction is not known. In a deoxycorticosterone acetate (DOCA)-salt model of hypertension and HFpEF, we performed CITE-Seq analysis on total leukocytes isolated from the heart, which revealed significant differential gene expression primarily in the myeloid population. We then took the top 20 genes that were differentially expressed between myeloid cells from DOCA-salt vs sham treated mice and performed a genetic analysis called PrediXcan in Vanderbilt’s DNA databank, BioVU. Genetically predicted expression of two genes were associated with a HFpEF phenotype in humans. Of these, the gene encoding galectin 1, Lgals1, had the lowest p-value (0.02) and highest beta coefficient (0.32) corresponding to an odds ratio for HFpEF of 1.38. The role of galectin-1 in heart failure is unknown, we hypothesized that overexpression of Lgals1 exacerbates HFpEF. We found that siRNA knockdown of Lgals1 in bone marrow derived macrophages stimulated with lipoprotein saccharide and IFNγ, they expressed significantly more inflammatory genes such as IL-1α (p&lt;0.0001), IL-6 (p=0.0219), iNOS (p=0.043), and TNFα (p=0.0031) using a 2-way ANOVA followed by Sidak’s multiple comparison test. Next, we explored the role of Lgals1 in the pathophysiology of HFpEF using Lgals1-/- mice. Surprisingly, despite both in vitro and in vivo data supporting a role of galectin-1 in HFpEF, Lgals1-/- mice did not show a significant difference in HFpEF phenotype (blood pressure, exercise tolerance, echocardiography, heart weight, or pulmonary congestion) after DOCA-salt treatment compared to littermate Lgals1+/+ controls in either males or females (p &gt; 0.05 using Student’s t-test for all parameters measured, n=10-12). One hypothesis for this negative result is that Lgals1 is ubiquitously expressed, and a total body knockout may have compensatory mechanisms. We therefore created Lgals1 floxed mice and future studies will determine the effect of macrophage specific deletion of galectin-1 on HFpEF. In conclusion, while multi-omics approaches in mice and humans identify galectin-1 as a novel mediator of HFpEF, further studies in mice are needed to confirm its roles as a potential therapeutic target. 1R01HL161212-01A1, 19EIA34480023,,19IPLOI34760558, and 1T32HL144446-01 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute ischemic stroke: how to investigate the association between disease etiology and gene expression profiles

Background: Acute ischemic stroke (AIS) represents one of the principal causes of neurological morbidity and mortality worldwide. For a prompt and efficient cerebral blood restoration, intravenous treatment with rt-PA is often combined with mechanical thrombectomy (MT) which provides cerebral thrombi (CT) as study material, allowing the investigation of its cellular composition, morphological and histopathological features. Indeed, the determination of stroke etiology, typically defined by the TOAST classification, is paramount for prognostic factors, outcome, and management of the event. Aim of the study is therefore to highlight and analyze gene expression profiles in thrombotic tissue and peripheral blood (PB) in the comparison between strokes of cardioembolic (CE) and atherosclerotic (LAA) origin. Methods: We performed gene expression profiles of 92 patients. CT were stored in RNA later and RNA was extracted by PAX gene blood miRNA kit. The global gene expression profile was assessed by Affymetrix technology using GeneChip Human Transcriptome Array 2.0 combined with Affymetrix Transcriptome Analysis Console (TAC) Software. Results: Currently, we focused our attention on CT data analysis. The analysis revealed a significant difference (p-value&lt;0.05 and FoldChange=2 as threshold) in gene expression when comparing LAA and CE stroke. In particular, from CT of atherosclerotic origin emerges an overexpression of 1766 genes. Prominent among them are genes such as MMP-9, TGFB, TGFBR and CXCL1, primarily involved in neutrophil-mediated immunity, Blood-Brain Barrier (BBB) disruption processes, and associated with atherosclerotic plaque instability and related to poor neurological outcome, suggesting a deleterious role in human brain injury. As concerns CE patients, 57 genes mainly involved in transcriptional regulatory processes turn out to be significantly overexpressed. Conclusions: Transcriptome profiling is a powerful weapon for revealing expression patterns associated with complex disorders. The variation of gene expression profiles confirmed and extended several known pathophysiological mechanisms and may be one way of delineating different stroke etiology.

Physiological traits and expression profile of genes associated with nitrogen and phosphorous use efficiency in wheat

Nitrogen (N) and phosphorous (P) play a very important role in the growth and development of wheat as well as major constituents of biological membranes. To meet the plant's nutritional demand these nutrients are applied in the form of fertilizers. But the plant can utilize only half of the applied fertilizer whereas the rest is lost through surface runoff, leaching and volatilization. Thus, to overcome the N/P loss we need to elucidate the molecular mechanism behind the N/P uptake. In our study, we used DBW16 (low NUE), and WH147 (high NUE) wheat genotypes under different doses of N, whereas HD2967 (low PUE) and WH1100 (high PUE) genotypes were studied under different doses of P. To check the effect of different doses of N/P, the physiological parameters like total chlorophyll content, net photosynthetic rate, N/P content, and N/PUE of these genotypes were calculated. In addition, gene expression of various genes involved in N uptake, utilization, and acquisition such as Nitrite reductase (NiR), Nitrate transporter 1/Peptide transporter family (NPF2.4/2.5), Nitrate transporter (NRT1) and NIN Like Protein (NLP) and induced phosphate starvation (IPS), Phosphate Transporter (PHT1.7) and Phosphate 2 (PHO2) acquisition was studied by quantitative real-time PCR. Statistical analysis revealed a lower percent reduction in TCC, NPR, and N/P content in N/P efficient wheat genotypes (WH147 & WH1100). A significant increase in relative fold expression of genes under low N/P concentration was observed in N/P efficient genotypes as compared to N/P deficient genotypes. Significant differences in physiological data and gene expression among N/ P efficient and deficient wheat genotypes could be useful for future improvement of N/P use efficiency.

P158 Defining distinct fibroblast populations in systemic sclerosis skin using functional characterization and single-cell RNA sequencing

Abstract Background/Aims Fibroblasts isolated by traditional explant culture from skin biopsies in systemic sclerosis (SSc) have a profibrotic and “migratory” phenotype. Single cell RNAseq (scRNAseq) studies suggest other fibroblast subpopulations may be important in SSc pathogenesis. We have isolated a second fibroblast population termed “resident” from whole skin in SSc. We used bulk and scRNAseq to further characterise these populations. Methods Forearm skin from dcSSc (n = 3) and HC (n = 3) was biopsied. “Migratory” fibroblasts were isolated by standard explant culture. The remaining biopsy fragments underwent collagenase digestion to yield a second “resident” fibroblast population. Functional characterization included 3-D collagen gel contraction, migratory scratch-wound assays and western blot comparing expression of pro-collagen I, CTGF and αSMA. Significant difference in gene expression was defined by fold change≥1.5, and adjusted p-value&amp;lt;0.05. scRNAseq was performed on 12 SSc whole skin biopsies, and 3 HC, using the 10X Genomics platform. Results Both SSc fibroblast populations showed a fibrotic phenotype with overexpression of COL1, CTGF and αSMA compared to HC, but with reduced expression αSMA in resident compared to migratory SSc fibroblasts (p &amp;lt; 0.05). RNAseq confirmed 1483 genes being significantly differentially expressed between the SSc fibroblast populations (Table 1). All four populations clustered separately on hierarchical clustering. 10 fibroblast clusters were identified using scRNAseq of whole skin. Of these, cluster 0 and 4 showed greater expression of COL10A1, and STC2 in keeping with migratory fibroblasts. Increased expression of CCL2, and EGR1 were seen in clusters 3 and 6, representing the resident fibroblast population. Gene set enrichment analysis (GSEA) confirmed concordance between KEGG pathway upregulation of the migratory and resident fibroblast clusters and bulk RNAseq analysis. Resident fibroblasts showed upregulation of complement and coagulation genes, and JAK/STAT signalling pathway. There was upregulation of glycolysis gluconeogenesis, and PPAR signalling in migratory fibroblasts. There was agreement in GSEA findings between relevant scRNAseq clusters and bulkRNAseq for resident and migratory subpopulations. Conclusion We have isolated for the first time two distinct profibrotic fibroblast populations from SSc skin with altered expression of key genes associated with SSc pathogenesis. Future work will define their role in SSc pathogenesis, potentially facilitating a more targeted treatment approach. Disclosure K.E. Clark: None. S. Xu: None. V.H. Ong: None. C.D. Buckley: None. C. Denton: None.

Early events marking lung fibroblast transition to profibrotic state in idiopathic pulmonary fibrosis

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is an age-associated progressive lung disease with accumulation of scar tissue impairing gas exchange. Previous high-throughput studies elucidated the role of cellular heterogeneity and molecular pathways in advanced disease. However, critical pathogenic pathways occurring in the transition of fibroblasts from normal to profibrotic have been largely overlooked.MethodsWe used single cell transcriptomics (scRNA-seq) from lungs of healthy controls and IPF patients (lower and upper lobes). We identified fibroblast subclusters, genes and pathways associated with early disease. Immunofluorescence assays validated the role of MOXD1 early in fibrosis.ResultsWe identified four distinct fibroblast subgroups, including one marking the normal-to-profibrotic state transition. Our results show for the first time that global downregulation of ribosomal proteins and significant upregulation of the majority of copper-binding proteins, including MOXD1, mark the IPF transition. We find no significant differences in gene expression in IPF upper and lower lobe samples, which were selected to have low and high degree of fibrosis, respectively.ConclusionsEarly events during IPF onset in fibroblasts include dysregulation of ribosomal and copper-binding proteins. Fibroblasts in early stage IPF may have already acquired a profibrotic phenotype while hallmarks of advanced disease, including fibroblast foci and honeycomb formation, are still not evident. The new transitional fibroblasts we discover could prove very important for studying the role of fibroblast plasticity in disease progression and help develop early diagnosis tools and therapeutic interventions targeting earlier disease states.

Integrative analysis of the association between circadian rhythm and lupus nephritis.

Lupus nephritis (LN) is an autoimmune nephropathy associated with systemic lupus erythematosus. Circadian rhythms are involved in the development of several diseases, especially inflammation-related diseases, but their relationship with LN is unclear. This was an integrative bioinformatics study. The expression profile from glomeruli, tubular interstitium and renal whole tissue samples was used to assess the expression levels and relevance of circadian rhythm-related genes. To screen for circadian rhythm-related signatures, we employed the LASSO and SVM-RFE algorithms. A consensus clustering algorithm was used to classify LN patients into two circadian rhythm patterns (cluster A and cluster B). We made immune cell infiltration analysis. We used the weighted gene co-expression network analysis (WGCNA) algorithm to identify cluster-specific differentially expressed genes. Nephroseq data were used to observe the relationship between genes and renal function. We found more significant differences in circadian rhythm-related gene expression in LN glomeruli compared with tubulointerstitial and whole-kidney tissues. We established a circadian rhythm-related signature consisting of eight genes that can easily distinguish LN from healthy individuals. Patients in cluster A were associated with B-cell-dominated immunity, whereas patients in cluster B were associated with T-cell-dominated immunity. As most of the patients with proliferative LN combined with membranous LN belonged to cluster B, patients in cluster B may have more severe renal pathology compared with patients in cluster A. Fifteen circadian rhythm-related genes associated with LN and LN typing were screened using the WGCNA algorithm, with COL1A2 and DOCK2 associated with renal prognosis. This study found that circadian rhythms are associated with the occurrence of LN, providing new ideas for the development of new LN treatment options from the perspective of circadian rhythms.

Membrane Adaptations and Cellular Responses of Sulfolobus acidocaldarius to the Allylamine Terbinafine.

Cellular membranes are essential for compartmentalization, maintenance of permeability, and fluidity in all three domains of life. Archaea belong to the third domain of life and have a distinct phospholipid composition. Membrane lipids of archaea are ether-linked molecules, specifically bilayer-forming dialkyl glycerol diethers (DGDs) and monolayer-forming glycerol dialkyl glycerol tetraethers (GDGTs). The antifungal allylamine terbinafine has been proposed as an inhibitor of GDGT biosynthesis in archaea based on radiolabel incorporation studies. The exact target(s) and mechanism of action of terbinafine in archaea remain elusive. Sulfolobus acidocaldarius is a strictly aerobic crenarchaeon thriving in a thermoacidophilic environment, and its membrane is dominated by GDGTs. Here, we comprehensively analyzed the lipidome and transcriptome of S. acidocaldarius in the presence of terbinafine. Depletion of GDGTs and the accompanying accumulation of DGDs upon treatment with terbinafine were growth phase-dependent. Additionally, a major shift in the saturation of caldariellaquinones was observed, which resulted in the accumulation of unsaturated molecules. Transcriptomic data indicated that terbinafine has a multitude of effects, including significant differential expression of genes in the respiratory complex, motility, cell envelope, fatty acid metabolism, and GDGT cyclization. Combined, these findings suggest that the response of S. acidocaldarius to terbinafine inhibition involves respiratory stress and the differential expression of genes involved in isoprenoid biosynthesis and saturation.

Abstract 132: The molecular basis of an evolutionary arms race between cancer cells and their host

Abstract Genetic and nongenetic changes can drive abnormal characteristics of cancer cells, including resistance to tumor growth. Through selective breeding of animals with the slowest growing tumor at 11 days following implantation over ten generations, we evolved resistance to tumor growth in two contrasting strains of mice: severe combined immunodeficiency disease (SCID) and immuno-competent C57BL/6. We found that the two strains evolved different strategies of resistance to the cancer. In the immunocompromised mice, skin fibroblasts infiltrated into and around the tumor to impede growth, while an enhanced immune response to tumor antigens, slowed tumor growth in the immunocompetent mice. However, when the experiment was extended beyond 11 days, tumor growth rapidly increased so that, at 28 days, it was comparable in size to the control animals. We investigated the molecular-level mechanisms underlying these resistance traits in SCID mice. We identified transcriptomic changes using RNA-Seq in both skin and tumor samples to compare the evolved mice (EV) with the original strain (WT) at 11 days and 28 days. We used DESeq2 and two-way ANOVAs on the normalized data to identify significant differences in gene expression. We adjusted p-values for multiple testing with the Benjamini-Hochberg false discovery correction and used a q value of less than 0.1 (and/or a greater than two-fold change) to determine the significance for differential expression levels. Comparing transcriptomic data from skin fibroblasts from evolved (EV) and non-evolved (WT) SCID mice at day 11 following implantation (i.e., the point of maximal tumor suppression) revealed 98 differentially expressed genes (DEGs). EV SCID fibroblasts showed increased expression of MicroRNAs, including MiR-7 and Mir5125, and acid metabolic pathways, such as Carbonic anhydrase III. A comparison of the tumor cells from day 11 and day 28 demonstrated substantial epigenetic evolution with 1353 DEGs. The tumor cells at day 28 adapted to the host response by increasing expression of genes for extracellular matrix (ECM) remodeling, mainly the metalloproteases gene family. Here we used selective breeding to allow evolution to reveal potential host strategies to suppress tumor growth. However, we find that, over time, tumor cells can evolve successful counter strategies to overcome host resistance. Our results provide novel insights into key dynamics of somatic evolution during carcinogenesis and highlight the critical roles of epigenetic modification and inheritance in these somatic eco-evolutionary dynamics. Citation Format: Arig A. Ibrahim-Hashim, Robert A. Gatenby, Christina L. Richards, Joel S. Brown. The molecular basis of an evolutionary arms race between cancer cells and their host [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 132.

Abstract 1244: Examining the differential roles of OSM and IL-6 in varying subtypes of breast cancer for novel therapeutic treatment

Abstract Breast cancer remains the second leading cause of cancer-related death in women, in large part due to distant metastasis to organs such as bone, lung, liver, and brain. Our laboratory has demonstrated that the IL-6-family cytokine oncostatin M (OSM) increases circulating tumor cell (CTC) numbers and metastases to lung in vivo, and that OSM stays active when bound to proteins in the extracellular matrix, thus “bioaccumulating” in the tumor microenvironment. These published findings are supported by our results demonstrating that OSM strongly promotes expression and secretion of IL-6 from breast cancer cells. Interestingly, the increased production of IL-6 by OSM occurs only in estrogen receptor-negative (ER-) cells. It is well known that patients with ER- breast cancer, specifically triple negative breast cancer (TNBC; ER- PR- HER2-), have limited therapeutic options. However, the divergent role OSM plays in ER+ and ER- breast cancer and the mechanism by which this occurs has yet to be fully understood. Thus, we aimed to fully elucidate the difference between OSM in ER+ versus ER- breast cancer. First, we performed RNA-Seq analysis on OSM-treated human ER+ T47D cells and ER- TNBC MDA-MB-231 cells and discovered significant differences in gene expression between both cell lines upon treatment with OSM, including alteration in DNA-replication and cell cycle pathway genes. Next, to better understand the mechanism by which OSM-mediated IL-6 secretion is repressed, we utilized a computational modeling approach and evaluated the direct inhibition of STAT3 and ER to identify theoretical dimerization complex for future studies. This research aims to elucidate the unique role OSM plays in ER+ and ER- breast cancer and will provide a molecular understanding of how this occurs and will provide detailed information on which patients will benefit most from anti-OSM therapeutics. Citation Format: Cody L. Wolf, Ken Tawara, Cheryl L. Jorcyk. Examining the differential roles of OSM and IL-6 in varying subtypes of breast cancer for novel therapeutic treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1244.

Abstract 1904: Somatic copy number alterations and gene expression signatures are associated with aggressive tumor biology in African Americans and European Americans with renal cell carcinoma

Abstract Background: Renal cell carcinoma (RCC) is the deadliest of all urologic cancers in the US. RCC survival is highly dependent on histology. Survival rates range between 55-60% for clear cell RCC (ccRCC) and 80-90% for papillary RCC (pRCC) patients. African Americans (AAs) have lower survival rates when compared to their European American (EA) counterparts for both histologies (ccRCC 67.1 vs 72.6, pRCC 80.5 vs 87.5). RCC survival disparities may be accredited to population differences in tumor biology, such as somatic copy number alteration (SCNA) profiles of tumor suppressor and oncogenes, and differential gene expression signatures with associated cellular pathway dysregulation. SCNAs are the most common structural change in the human genome and ccRCC and pRCC-specific SCNAs have been associated with clinical outcomes. ClearCode34, a 34 gene expression signature, was developed to classify ccRCC tumors into two distinct ccRCC subtypes (ccA and ccB). The ccB subtype is associated with worse survival and features like increased tumor size, grade, and metastasis rates. We hypothesize there are more aggressive tumor biology phenotypes in AA RCC patients compared to their European counterparts. Methods: Comparative genome-wide, chromosomal, and gene SCNA analyses were performed in AA and EA ccRCC and pRCC patients in TCGA using Partek Genomics Suite 7 (1-way ANOVA, P&amp;lt;0.05). Prediction analysis of microarray in R was used to classify ccRCC patients into ccA and ccB subtypes by race based on the ClearCode34 gene expression signature (t-test, P&amp;lt;0.05). Differentially expressed genes (DEGs) between AA and EA patients were identified using Partek Genomics Suite 7 (1-way ANOVA, P&amp;lt;0.05). Gene Set Enrichment Analysis (GSEA) was performed using a pre-ranked gene list (Fold changes &amp;gt;2 or &amp;lt;-2, P&amp;lt;0.01) and two Molecular Signatures Database collections (C6 oncogenic signatures, C7 immunologic signatures). Results: AA-enriched SCNVs were identified on chromosomes 1, 3, 6, 7, and 12 in ccRCC patients, and chromosomes 1, 4, 6, 7, 8, 10, 16 and 20 in pRCC patients. Six cytobands and genes experienced a SCNV across both histologies (1p21.1, 1p21.3, 6p12.1, 6p12.3, 6p21.1, 6q12), BMP5 (on 6p12.1), CD2AP (on 6p12.3), ENPP4 (on 6p21.1), and SLC44A3-AS1 (on 1p21.3). AAs and EAs had significantly different frequencies of ccA (AA 33.9% vs EA 52.2%) and ccB subtypes (AA 66.1% vs EA 47.8%). Unique DEGs were found between AA and EA ccA and ccB patients (131 ccA only, 165 ccB only), which corresponded with up- and down-regulation in kidney cancer with oncogenic KRAS (C6) and various immunologic pathways (C7). Conclusions: Population-specific SCNAs, ccB subtypes, and pathways may help explain differences in RCC tumor biology amongst AA and EA patients. Future Directions: Replicate these findings in the Cooperative Human Tissue Network/Geisinger validation cohort. Citation Format: Samantha Greenberg, Alex Bart, Destiny Ortiz Fernandez, Heinric Williams, Khadijah A. Mitchell. Somatic copy number alterations and gene expression signatures are associated with aggressive tumor biology in African Americans and European Americans with renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1904.

Abstract 375: Smoking induces Wee1 expression through miRNA deregulation, promoting docetaxel resistance in esophageal adenocarcinoma

Abstract Esophageal Adenocarcinoma (EAC) is the most common subtype of Esophageal Cancer in Western countries, and its incidence rate has increased over the past few decades. The overall five-year survival rate of EAC is less than 20%. Smoking is a major risk factor for EAC development. WEE1 kinase plays a crucial role in the cell cycle by regulating the G2/M checkpoint, providing a time frame for the cells to repair DNA damage. Thus, targeting WEE1 using an inhibitor could potentiate the effect of chemotherapeutic drugs. This study investigated how smoking induces WEE1 protein expression, promoting docetaxel resistance in EAC. IHC staining for WEE1 in the normal esophagus and EAC tissue sections revealed significant over-expression of WEE1 protein in EAC. Nicotine, Nicotine derived Nitrosamine Ketone (NNK), and 2% cigarette smoke extract (CSE) treatment induced WEE1 expression with a concomitant increase in CDC2 phosphorylation (p-CDC2, Y15), a well-established read-out of WEE1 activity in EAC cells. In addition, we found that smoking upregulates WEE1 expression by decreasing miR-195-5p expression levels in EAC. Based on the differential gene expression signatures in EAC cell lines FLO1 and OE33 following WEE1 knockdown, drug and small molecule induced gene expression signatures analysis, L1000 fireworks display (L1000FWD), predicted that docetaxel is one of the best drug candidates which can synergize with the WEE1 inhibitor MK1775. Smoking-induced docetaxel resistance in EAC cells measured by cell viability assay. Down-regulating WEE1 expression with siRNA or pharmacological inhibition using MK1775 significantly sensitized EAC cells to docetaxel treatment, as evidenced by a remarkable decrease in the IC50 value of docetaxel. Inhibition of WEE1 combined with docetaxel can be considered an ideal therapeutic strategy due to its superior anti-tumor efficacy compared to the standard single-agent treatment. In conclusion, our data provide a firm rationale for the clinical combination of docetaxel with MK1775 in EAC patients. Citation Format: Krishnapriya Thangaretnam, Islam MD Obaidul, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, Zheng Chen. Smoking induces Wee1 expression through miRNA deregulation, promoting docetaxel resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 375.

Abstract 5280: O-GlcNAcylation of progesterone receptor promotes mammary tumorigenesis

Abstract The role of hormone receptors in breast cancer has been well established as a key contributor to breast cancer tumorigenesis. Estrogen receptor and progesterone receptors are present in nearly 70% of breast cancers. The role of estrogen receptor and estrogens in breast cancer has been extensively studied and targeted effectively through endocrine therapy, however the impact of progesterone and the progesterone receptor (PR) independent of estrogen is not well understood. We have previously demonstrated that PR promotes tumor growth and drives an immune suppressive environment, therefore understanding the biology of PR in breast cancer is critical. We have also demonstrated that PR attenuates type 1 interferon signaling via inhibition of STAT1 phosphorylation and increased degradation of STAT2. Post-translational modifications such as phosphorylation and SUMOylation can influence PR target gene promoter selectivity thereby altering its function. Our recent work indicates PR is modified by O-GlcNAc, a single N-acetyl-glucosamine sugar that cycles on and off and serine or threonine amino acids in nuclear, cytoplasmic and mitochondrial proteins. Levels of total O-GlcNAc staining are higher in breast cancer tissue compared to adjacent normal tissue. Active O-GlcNAcylation, as evidenced by immunohistochemistry staining of O-GlcNAc-transferase (OGT), is elevated in patients with PR+ tumors compared to PR-. Using T47D breast cancer cells (an ER/PR-positive tumor line), mass spectrometry analysis revealed an O-GlcNAc site at S499 on PR. We then used a naturally occurring PR-negative variant of T47D cells to introduce stable expression of a mutant PR with serine to alanine substitution at the O-GlcNAc site (S499A), thereby blocking O-GlcNAc flux, in addition to wt PR as a control. RNA-Seq analysis of these cells following treatment with progesterone revealed several significant differential gene expression patterns, including rescue of PR dependent interferon signaling attenuation. T47D-mutant PR cell lines implanted into immune deficient mice had significantly decreased growth as compared to WT-PR control tumors. In syngeneic mice harboring PR-positive and PR-negative tumors, loss of O-GlcNAc flux and increased O-GlcNac levels increased tumor growth only when tumors were expressing PR, corroborating the influence of O-GlcNAc in immunodeficient mice. Together these findings indicate O-GlcNAcylation of PR is potentially a mechanism of PR driven breast cancer tumorigenesis and immune evasion. Citation Format: Harmony Ivanna Saunders, Sean M. Holloran, Gloria Trinca, Chad Slawson, Christy Hagan. O-GlcNAcylation of progesterone receptor promotes mammary tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5280.

Overexpression of the PTEN Gene in Myocardial Tissues of Coronary Bypass Surgery Patients.

Coronary artery disease is a complex disorder that causes death worldwide. One of the genes involved in developing this disease may be PTEN. This study aimed to investigate the PTEN gene and protein expression in tissue and blood samples taken from coronary bypass surgery patients. Molecular studies were performed at Erciyes University Genome and Stem Cell Center (GENKOK). Right atrial appendage and blood samples were taken from the central vein of 22 coronary bypass surgery patients before starting and ending cardiopulmonary bypass. PTEN expression was determined using quantitative real-time PCR and western blot analysis. The significance level was accepted as p<0.05. There was no significant difference in the PTEN gene expression in blood samples taken before and after cardiopulmonary bypass. However, a substantial increase in both protein and gene expression levels of P-PTEN and PTEN was observed in the tissue samples. Myocardial expression of the PTEN gene was significantly increased at the end of the cardiopulmonary bypass. PTEN gene expression in the post-cardiopulmonary bypass period was increased when compared to the pre-bypass period, but it was insignificant when compared to healthy controls. This study first revealed the role of the PTEN gene by analyzing both mRNA and protein expression in coronary bypass patients, appearing in both myocardial tissue and blood samples. Increased levels of PTEN may be a marker in myocardial tissue for patients with coronary artery disease.

Identification of a pediatric acute hypoxemic respiratory failure signature in peripheral blood leukocytes at 24 hours post-ICU admission with machine learning.

There is no generalizable transcriptomics signature of pediatric acute respiratory distress syndrome. Our goal was to identify a whole blood differential gene expression signature for pediatric acute hypoxemic respiratory failure (AHRF) using transcriptomic microarrays within twenty-four hours of diagnosis. We used publicly available human whole-blood gene expression arrays of a Berlin-defined pediatric acute respiratory distress syndrome (GSE147902) cohort and a sepsis-triggered AHRF (GSE66099) cohort within twenty-four hours of diagnosis and compared those children with a PaO2/FiO2 < 200 to those with a PaO2/FiO2 ≥ 200. We used stability selection, a bootstrapping method of 100 simulations using logistic regression as a classifier, to select differentially expressed genes associated with a PaO2/FiO2 < 200 vs. PaO2/FiO2 ≥ 200. The top-ranked genes that contributed to the AHRF signature were selected in each dataset. Genes common to both of the top 1,500 ranked gene lists were selected for pathway analysis. Pathway and network analysis was performed using the Pathway Network Analysis Visualizer (PANEV) and Reactome was used to perform an over-representation gene network analysis of the top-ranked genes common to both cohorts. Changes in metabolic pathways involved in energy balance, fundamental cellular processes such as protein translation, mitochondrial function, oxidative stress, immune signaling, and inflammation are differentially regulated early in pediatric ARDS and sepsis-induced AHRF compared to both healthy controls and to milder acute hypoxemia. Specifically, fundamental pathways related to the severity of hypoxemia emerged and included (1) ribosomal and eukaryotic initiation of factor 2 (eIF2) regulation of protein translation and (2) the nutrient, oxygen, and energy sensing pathway, mTOR, activated via PI3K/AKT signaling. Cellular energetics and metabolic pathways are important mechanisms to consider to further our understanding of the heterogeneity and underlying pathobiology of moderate and severe pediatric acute respiratory distress syndrome. Our findings are hypothesis generating and support the study of metabolic pathways and cellular energetics to understand heterogeneity and underlying pathobiology of moderate and severe acute hypoxemic respiratory failure in children.