Regulation Of Cell Signaling Research Articles

Rediscovering Primary Cilia in Pancreatic Islets.

Primary cilia are microtubule-based sensory and signaling organelles on the surfaces of most eukaryotic cells. Despite their early description by microscopy studies, islet cilia had not been examined in the functional context until recent decades. In pancreatic islets as in other tissues, primary cilia facilitate crucial developmental and signaling pathways in response to extracellular stimuli. Many human developmental and genetic disorders are associated with ciliary dysfunction, some manifesting as obesity and diabetes. Understanding the basis for metabolic diseases in human ciliopathies has been aided by close examination of cilia action in pancreatic islets at cellular and molecular levels. In this article, we review the evidence for ciliary expression on islet cells, known roles of cilia in pancreas development and islet hormone secretion, and summarize metabolic manifestations of human ciliopathy syndromes. We discuss emerging data on primary cilia regulation of islet cell signaling and the structural basis of cilia-mediated cell crosstalk, and offer our interpretation on the role of cilia in glucose homeostasis and human diseases.

Chaperone-Dependent Degradation of Cdc42 Promotes Cell Polarity and Shields the Protein from Aggregation.

Rho GTPases are global regulators of cell polarity and signaling. By exploring the turnover regulation of the yeast Rho GTPase Cdc42p, we identified new regulatory features surrounding the stability of the protein. We specifically show that Cdc42p is degraded at 37 °C by chaperones through lysine residues located in the C-terminus of the protein. Cdc42p turnover at 37 °C occurred by the 26S proteasome in an ESCRT-dependent manner in the lysosome/vacuole. By analyzing versions of Cdc42p that were defective for turnover, we show that turnover at 37 °C promoted cell polarity but was defective for sensitivity to mating pheromone, presumably mediated through a Cdc42p-dependent MAP kinase pathway. We also identified one residue (K16) in the P-loop of the protein that was critical for Cdc42p stability. Accumulation of Cdc42pK16R in some contexts led to the formation of protein aggregates, which were enriched in aging mother cells and cells undergoing proteostatic stress. Our study uncovers new aspects of protein turnover regulation of a Rho-type GTPase that may extend to other systems. Moreover, residues identified here that mediate Cdc42p turnover correlate with several human diseases, which may suggest that turnover regulation of Cdc42p is important to aspects of human health.

The role of O-GlcNAcylation mediated by OGT during tooth development.

To understand the mechanisms underlying tooth morphogenesis, we examined the developmental roles of important posttranslational modification, O-GlcNAcylation, which regulates protein stability and activity by the addition and removal of a single sugar (O-GlcNAc) to the serine or threonine residue of the intracellular proteins. Tissue and developmental stage-specific immunostaining results against O-GlcNAc and O-GlcNAc transferase (OGT) in developing tooth germs would suggest that O-GlcNAcylation is involved in tooth morphogenesis, particularly in the cap and secretory stage. To evaluate the developmental function of OGT-mediated O-GlcNAcylation, we employed an in vitro tooth germ culture method at E14.5, cap stage before secretory stage, for 1 and 2 days, with or without OSMI-1, a small molecule OGT inhibitor. To examine the mineralization levels and morphological changes, we performed renal capsule transplantation for one and three weeks after 2 days of in vitro culture at E14.5 with OSMI-1 treatment. After OGT inhibition, morphological and molecular alterations were examined using histology, immunohistochemistry, real-time quantitative polymerase chain reaction, in situ hybridization, scanning electron microscopy, and ground sectioning. Overall, inhibition of OGT resulted in altered cellular physiology, including proliferation, apoptosis, and epithelial rearrangements, with significant changes in the expression patterns of β-catenin, fibroblast growth factor 4 (fgf4), and sonic hedgehog (Shh). Moreover, renal capsule transplantation and immunolocalizations of Amelogenin and Nestin results revealed that OGT-inhibited tooth germs at cap stage exhibited with structural changes in cuspal morphogenesis, amelogenesis, and dentinogenesis of the mineralized tooth. Overall, we suggest that OGT-mediated O-GlcNAcylation regulates cell signaling and physiology in primary enamel knot during tooth development, thus playing an important role in mouse molar morphogenesis.

Deciphering Cell-Cell Interactions with Integrative Single-Cell Secretion Profiling.

Cell-cell interactions are the fundamental behaviors to regulate cellular activities. A comprehensive evaluation of intercellular interactions requires direct profiling of various signaling behaviors simultaneously at the single-cell level, which remains lacking. Herein, an integrative single-cell secretion analysis platform is presented to profile different secreted factors (four proteins, three extracellular vesicles (EV) phenotypes), spatial distances, and migration information (distances and direction) simultaneously from high-throughput paired single cells using an antibody-barcode microchip. Applying the platform to analyze the tumor-stromal and tumor-immune interactions with the human oral squamous cell carcinoma (OSCC) cell lines and primary OSCC cells reveals that the initial distances between cells would determine their migratory distances and direction to approach stable organization. The cell-cell in close proximity enhances protein secretions while attenuating EV secretions. Migration has a more profound correlation with protein secretions than EV secretions, in which absolute migration distance affects protein secretions significantly but not the direction. These findings highlight the significance of spatial organization in regulating cell signaling behaviors and demonstrate that the integrative single-cell secretion profiling platform is well-suited for a comprehensive dissection of intercellular communication and interactions, providing new avenues for understanding cell-cell interaction biology and how different signaling behaviors coordinate within the tumor microenvironment.

Meta-analysis of commonly mutated genes in leptomeningeal carcinomatosis.

Leptomeningeal carcinomatosis (LMC) is a rare type of cancer that settles at the meninges through metastasis of non-small cell lung cancer (NSCLC), breast cancer and melanoma. The molecular mechanism underlying LMC is not known, therefore molecular studies investigating the development of LMC are needed. Here, we aimed to identify commonly mutated genes in LMC caused by NSCLC, breast cancer, and melanoma using an in-slico approach and their interactions using integrated bioinformatic approaches/tools in this meta-analysis. We conducted a meta-analysis using information from 16 studies that included different sequencing techniques of patients with LMC caused by three different primary cancers: breast cancer, NSCLC, and melanoma. All studies that assessed mutation information from patients with LMC were searched in PubMed, from their inception to February, 16 2022. Studies that performed NGS on LMC patients with NSCLC, breast cancer, or melanoma were included, while studies that did not apply NGS to CSF samples, did not provide information on altered genes, were reviews, editorials, or conference abstracts, or whose main goal was the detection of malignancies were all excluded. We identified commonly mutated genes in all three types of cancer. Next, we constructed a protein-protein interaction network, then performed pathway enrichment analysis. We searched National Institutes of Health (NIH) and Drug-Gene Interaction Database (DGIdb) to find candidate drugs. We found that TP53, PTEN, PIK3CA, IL7R, and KMT2D genes were commonly mutated genes in all three types of cancer via our meta-analysis that consisted out of 16 studies. Our pathway enrichment analysis showed that all five genes were primarily associated with regulation of cell communication and signaling, and cell proliferation. Other enriched pathways included regulation of apoptotic processes of leukocytes and fibroblasts, macroautophagy and growth. According to our drug search we found candidate drugs; Everolimus, Bevacizumab and Temozolomide, which interact with these five genes. In conclusion, a total of 96 mutated genes in LMC were investigated via meta-analysis. Our findings suggested vital roles of TP53, PTEN, PIK3CA, KMT2D, and IL7R, which can provide insight into the molecular basis of LMC development and paving the door to the development of new targeted medicine and will encourage molecular biologists to seek biological evidence.

Epigenetic regulation of stem cells in lung cancer oncogenesis and therapy resistance.

Epigenetics plays an important role in regulating stem cell signaling, as well as in the oncogenesis of lung cancer and therapeutic resistance. Determining how to employ these regulatory mechanisms to treat cancer is an intriguing medical challenge. Lung cancer is caused by signals that cause aberrant differentiation of stem cells or progenitor cells. The different pathological subtypes of lung cancer are determined by the cells of origin. Additionally, emerging studies have demonstrated that the occurrence of cancer treatment resistance is connected to the hijacking of normal stem cell capability by lung cancer stem cells, especially in the processes of drug transport, DNA damage repair, and niche protection. In this review, we summarize the principles of the epigenetic regulation of stem cell signaling in relation to the emergence of lung cancer and resistance to therapy. Furthermore, several investigations have shown that the tumor immune microenvironment in lung cancer affects these regulatory pathways. And ongoing experiments on epigenetics-related therapeutic strategies provide new insight for the treatment of lung cancer in the future.

A transcriptome analysis of basal and stimulated VWF release from endothelial cells derived from patients with type 1 VWD.

Type 1 von Willebrand disease (VWD) is associated with a reduction in qualitatively normal von Willebrand factor (VWF). Current diagnostic guidelines only take into consideration the contribution of basal VWF levels, despite a lack of correlation with bleeding severity. Defects in stimulated VWF release, which occurs after hemostatic challenge, may contribute to bleeding in type 1 VWD, but the pathogenic mechanisms are poorly defined. In this study, a layered multiomic approach including messenger RNA (mRNA) and microRNA (miRNA) sequencing was used to evaluate transcriptome-wide differences between type 1 VWD- and control-derived endothelial colony forming cells (ECFCs) during basal and stimulated VWF release. ECFCs from 8 patients with type 1 VWD and 4 other patients were included in this study as controls. VWF protein analysis revealed heterogenous responses to stimulation among type 1 VWD and control ECFCs. During basal VWF release, 64 mRNAs and 7 miRNAs were differentially regulated between type 1 VWD and control ECFCs, and 65 putatively pathogenic miRNA-mRNA interactions were identified. During stimulated VWF release, 190 mRNAs and 5 mRNAs were differentially regulated between type 1 VWD and control ECFCs, and 110 putatively pathogenic miRNA-mRNA interactions were identified. Five gene ontology terms including coagulation, regulation of cell shape, and regulation of cell signaling were also differentially regulated in type 1 VWD ECFCs during stimulated release. To our knowledge, we have shown for the first time that transcriptome-wide differences exist between type 1 VWD and control ECFCs. These differences may contribute to bleeding in type 1 VWD, and further investigation may reveal novel biomarkers and therapeutic targets.

Abstract CT048: A Phase II trial of JDQ443 in KRAS G12C-mutated NSCLC with PD-L1 expression <1% or PD-L1 expression≥1% and an STK11 co-mutation

Abstract Background Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC). KRAS G12C, the most frequent KRAS variant, is found in ~13% of patients (pts) with NSCLC. KRAS is a GTPase that regulates cell signaling pathways necessary for proliferation, differentiation, and survival. KRAS mutation reduces the intrinsic GTPase activity of the enzyme, allowing for the accumulation of active, GTP-bound KRAS and hyperactivation of downstream signaling, driving tumorigenesis. JDQ443 is a potent, selective KRASG12C inhibitor that irreversibly traps KRASG12C in its inactive, GDP bound state and blocks downstream signaling. In preliminary data from the Phase Ib part of the KontRASt-01 study (NCT04699188), JDQ443 showed promising antitumor activity and an acceptable safety profile in previously treated pts with KRAS G12C-mutated advanced NSCLC. Pts with KRAS G12C-mutated NSCLC currently receive the same first-line (1L) treatment as those without driver mutations, consisting of immunotherapy alone or combined with chemotherapy; however, ~30% of pts with NSCLC present with programmed death-ligand 1 (PD-L1) expression <1%, and ~10-20% of pts harbor an STK11 mutation, both indicators of poor response to immunotherapy. Therefore, alternative 1L treatment options are needed for these pts. Of note, PD-L1 expression and STK11 mutation do not affect responsiveness to KRASG12C inhibitors, raising interest in the evaluation of these targeted therapies as 1L alternatives to immunotherapy for pts with KRAS G12C-mutated NSCLC. Methods KontRASt-06 (NCT05445843) is an open-label, Phase II, single-arm, multicenter study evaluating JDQ443 monotherapy (200 mg JDQ443 twice daily in 21-day cycles) as a 1L treatment for two cohorts of adult pts with locally advanced or metastatic, KRAS G12C-mutated NSCLC. Cohort A (n=90) includes pts whose tumors have PD-L1 expression <1%, regardless of STK11 mutation status, while Cohort B (n=30) includes pts whose tumors have PD-L1 expression ≥1% and an STK11 co-mutation. Local testing for PD-L1 status and KRAS and STK11 mutations is accepted; KRAS and STK11 mutations may be assessed in blood samples. A tissue sample is required for retrospective biomarker status confirmation and exploratory study. The study is currently enrolling pts into both cohorts. The primary endpoint is the overall response rate (ORR) per RECIST version 1.1, assessed by a blinded independent review committee, in Cohort A. Key secondary endpoints are ORR in Cohort B and duration of response in both cohorts. Other secondary endpoints include progression-free survival, overall survival, safety, pharmacokinetics, and pt-reported outcomes. A comprehensive biomarker strategy aims to investigate predictors of treatment response and resistance in the study population. Citation Format: Colin R. Lindsay, Daniel Tan, Shweta Malhotra, Rafael Caparica, Aislyn D. Boran, Gilberto Castro, Sabine Glaser, Rajwanth R. Veluswamy, Enriqueta Felip. A Phase II trial of JDQ443 in KRAS G12C-mutated NSCLC with PD-L1 expression <1% or PD-L1 expression≥1% and an STK11 co-mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT048.

Caveolin-1 genotypes as predictor for locoregional recurrence and contralateral disease in breast cancer

PurposeCaveolin-1 (CAV1) has been implicated in breast cancer oncogenesis and metastasis and may be a potential prognosticator, especially for non-distant events. CAV1 functions as a master regulator of membrane transport and cell signaling. Several CAV1 SNPs have been linked to multiple cancers, but the prognostic impact of CAV1 SNPs in breast cancer remains unclear. Here, we investigated CAV1 polymorphisms in relation to clinical outcomes in breast cancer.MethodsA cohort of 1017 breast cancer patients (inclusion 2002–2012, Sweden) were genotyped using Oncoarray by Ilumina. Patients were followed for up to 15 years. Five out of six CAV1 SNPs (rs10256914, rs959173, rs3807989, rs3815412, and rs8713) passed quality control and were used for haplotype construction. CAV1 genotypes and haplotypes in relation to clinical outcomes were assessed with Cox regression and adjusted for potential confounders (age, tumor characteristics, and adjuvant treatments).ResultsOnly one SNP was associated with lymph node status, no other SNPs or haplotypes were associated with tumor characteristics. The CAV1 rs3815412 CC genotype (5.8% of patients) was associated with increased risk of contralateral breast cancer, adjusted hazard ratio (HRadj) 4.26 (95% CI 1.86–9.73). Moreover, the TTACA haplotype (13% of patients) conferred an increased risk for locoregional recurrence HRadj 2.24 (95% CI 1.24–4.04). No other genotypes or haplotypes were associated with clinical outcome.ConclusionCAV1 polymorphisms were associated with increased risk for locoregional recurrence and contralateral breast cancer. These findings may identify patients that could derive benefit from more tailored treatment to prevent non-distant events, if confirmed.

From TNF-induced signaling to NADPH oxidase enzyme activity: Methods to investigate protein complexes involved in regulated cell death modalities

The formation of molecular complexes is a key feature of intracellular signaling pathways which governs to the initiation and execution of dedicated cellular processes. Tumor Necrosis Factor (TNF) and Reactive Oxygen Species (ROS) function as signaling molecules and are both involved in balancing cell fate decision between cell survival or cell demise. As master regulators of cell signaling, they are also instrumental in controlling various cellular processes towards tissue homeostasis, innate immunity and inflammation. Interestingly, TNF and ROS are interlinked and involved in regulating each other’s production via the engagement of molecular signaling complexes. This relationship calls for detailed reviewing of both TNF-induced and ROS-producing molecular complexes in the context of regulated cell death (RCD) modalities. Here, we outline biotechnological approaches that were used to investigate the TNF- and, concerning ROS, the NADPH oxidase-related molecular complexes with an emphasis on different regulated cell death modalities. This systematic review highlights how the cell death field has benefited from both biochemical and live-cell fluorescence imaging approaches. This knowledge and established workflows are highly generalizable, can be of a broader use for any protein-complex studies, and well suited for addressing new challenges in signaling dynamics. These will help understand molecular signaling complexes as ensembles organized into signaling platforms, most likely the key sites of signaling dynamics integration toward cell fate regulation.

Abstract 3132: CLUMPS-PTM: Spatial clustering of post-translational modifications across cancer types

Abstract Post-translational modifications (PTMs) play key roles in regulating cell signaling in health and disease processes. Recent advances in mass spectrometry enable comprehensive investigation of PTMs. However, identifying functionally relevant PTMs and protein domains implicated in disease remains a key challenge. We present CLUMPS-PTM, an algorithm for spatial clustering of PTMs to propose functional domains. We apply CLUMPS-PTM to the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset (one of the largest proteogenomic pan-cancer PTM datasets) comprising 1110 samples spanning 11 tumor types. First, we map over 14K phospho-sites and 13K acetyl-sites detected in the CPTAC dataset to protein data bank (PDB) files. We expand this to the predicted AlphaFold proteome and recover additional 12K phospho-sites and 10K acetyl-sites exceeding model confidence (pLDDT) of 70%. Consistent with previous research, phospho-sites are found on residues with lower AlphaFold prediction confidence than acetyl-sites due to their abundance in unstructured domains (p-value < 1e-4). To identify functionally relevant protein domains in the pan-cancer context, we characterize the 1110 samples by applying SignatureAnalyzer, a Bayesian non-negative matrix factorization method, to discover multi-omic patterns. This analysis identifies strong signatures associated with DNA damage response (DDR) and stratifies the CPTAC cohort into DDR-high (n=442) and DDR-low (n=668) groups. Next, we applied CLUMPS-PTM to differentially modified phospho/acetyl-sites between these groups. We identified 22 proteins (including GMPS, NCBP1, and ANPEP (CD13)) with significant clusters of differentially phosphorylated sites (q-value < 0.1) in DDR-high samples. ANPEP is a zinc-dependent aminopeptidase with a significant cluster (q-value = 0.0129) of differentially phosphorylated sites in the ERAP1-C domain that faces peptidase active sites known to have proliferative activity via the MAPK pathway. CLUMPS-PTM also identified a significant cluster of acetyl-sites on ARID1A (a SWI/SNF chromatin remodeler known to be mutated in cancer) in DDR-high samples (q-value = 0.085). The ARID1A cluster is on the C-terminal tail of the protein within the glucocorticoid receptor (GR) domain, an upstream regulator of proteins that increase catabolism, reduce inflammation, and increase cell survival. Increased acetylation may block a ubiquitination site at the C-terminus of the protein, resulting in increased protein levels, likely due to reduced ARID1A degradation, and increased GR signaling. Overall, CLUMPS-PTM is an open-source tool that allows near proteome-wide spatial analysis with the growing availability of PTM data. We anticipate that it will be useful to the broader proteomic community for the discovery of novel targets and generation of insights into functional mechanisms. Citation Format: Shankara K. Anand, Yifat Geffen, Yo Akiyama, Francois Aguet, Gad Getz. CLUMPS-PTM: Spatial clustering of post-translational modifications across cancer types [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 3132.

Abstract 2566: Blocking the functional domain of TIP1 upregulates midkine via the β-catenin/Wnt signaling pathway

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortalities world-wide. Targeted therapy has been a growing topic of investigation to improve therapeutic efficacy for NSCLC. A few targeted therapies that exploit aberrant protein expression profiles have been approved for NSCLC. However, marginal improvement in efficacy observed with these therapeutic approaches highlights the need for discovery of additional therapeutic targets. Tax interacting protein 1 (TIP1) is a molecular target that is overexpressed on the surface of various cancers and correlates with poor overall patient survival. The functional domain of TIP1 (PDZ domain) caps the C-terminus of many cellular proteins that regulate cell signaling, cancer development, and progression. We found that antibodies against the PDZ domain of TIP1 induced cytotoxicity of lung cancer cells but not normal cells. A quantitative mass spectrometry-based approach identified a ten-fold increase in Midkine protein expression on blockade of TIP1. Midkine is a heparin-binding growth factor that is involved in cancer progression, therapy resistance, and immune suppression. This study tested the hypothesis that Midkine is upregulated by the anti-PDZ/TIP1 antibody via the β-catenin/Wnt signaling pathway, which subsequently modulates downstream signaling mechanisms. In co-immunoprecipitation studies we found a direct interaction of TIP1 with β-catenin on the cell surface. Anti-TIP1 antibody treatment led to nuclear translocation of β-catenin in NSCLC cells. We used a luciferase based TOPFlash/FOPFlash reporter assay to study the transcriptional activity of the canonical β-catenin/Wnt pathway following TIP1 blockade. Reporter assays showed a three to four-fold increase (p<0.0001) in the transcriptional activity of β-catenin following TIP1 blockade in NSCLC cells. Genetic silencing of β-catenin inhibited Midkine mRNA transcription (p<0.005), protein induction and its extracellular release (p<0.0001) following anti-TIP1 antibody treatment. In conclusion, the mechanism of Midkine release following TIP1 blockade involves the β-catenin/Wnt signaling pathway. Ongoing studies are evaluating the dual-targeting of TIP1 and Midkine for enhanced efficacy of NSCLC treatment. Citation Format: Minakshi Saikia, Abhay K. Singh, Nathan Cheung, Dennis E. Hallahan, Vaishali Kapoor. Blocking the functional domain of TIP1 upregulates midkine via the β-catenin/Wnt signaling pathway [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 2566.

Oryzalin impairs maternal-fetal interaction during early pregnancy via ROS-mediated P38 MAPK/AKT and OXPHOS downregulation.

Oryzalin is a dinitroaniline pesticide for the control of weed growth via suppression of microtubule synthesis. There are studies about the deleterious effects of dinitroaniline pesticides on the reproductive system. Therefore, we attempted to demonstrate the toxic mechanisms of oryzalin on early pregnancy using porcine uterine epithelial cells (pLE) and trophectoderm (pTr) cells. According to our results, the viability and proliferation of pLE and pTr cells were suppressed in response to oryzalin exposure, and cell cycle progression was affected. Additionally, oryzalin induced apoptotic cell death and impaired mitochondrial membrane polarity in pLE and pTr cells. Moreover, we confirmed that oryzalin significantly downregulated adenosine triphosphate (ATP) production via the oxidative phosphorylation system and upregulated reactive oxygen species (ROS) generation in both pLE and pTr cells. The oryzalin-induced ROS generation was mitigated by N-acetylcysteine, a ROS scavenger, and further upregulation of phosphor-P38 MAPK/AKT/P70S6K protein expression was ameliorated in both pLE and pTr cells. We also confirmed that the suppression of migration and proliferation in oryzalin-treated pLE and pTr cells was restored upon oxidative stress mitigation. In summary, we revealed that the cytotoxic mechanisms of oryzalin-induced implantation failure were mediated by ROS-induced intracellular signaling regulation and migratory potential in pLE and pTr cells.

Extracellular Vesicles Act as Carriers for Cargo Delivery and Regulate Wnt Signaling in the Hepatocellular Carcinoma Tumor Microenvironment.

As the primary type of liver cancer, hepatocellular carcinoma (HCC) causes a large number of deaths every year. Despite extensive research conducted on this disease, the prognosis of HCC remains unclear. Recently, research has largely focused on extracellular vesicles (EVs), and they have been found to participate in various ways in the development of various diseases, including HCC, such as by regulating cell signaling pathways. However, recent studies have reported the mechanisms underlying the regulation of Wnt signaling by EVs in HCC, primarily focusing on the regulation of the canonical pathways. This review summarizes the current literature on the regulation of Wnt signaling by EVs in HCC and their underlying mechanisms. In addition, we also present future research directions in this field. This will deepen the understanding of HCC and provide new ideas for its treatment.

Gene expression changes in therapeutic ultrasound-treated venous leg ulcers.

Low-frequency, low-intensity ultrasound has been previously shown to promote healing of chronic wounds in humans, but mechanisms behind these effects are poorly understood. The purpose of this study was to evaluate gene expression differences in debrided human venous ulcer tissue from patients treated with low-frequency (20 kHz), low-intensity (100 mW/cm2) ultrasound compared to a sham treatment in an effort to better understand the potential biological mechanisms. Debrided venous ulcer tissue was collected from 32 subjects one week after sham treatment or low-frequency, low-intensity ultrasound treatment. Of these samples, 7 samples (3 ultrasound treated and 4 sham treated) yielded sufficient quality total RNA for analysis by ultra-high multiplexed PCR (Ampliseq) and expression of more than 24,000 genes was analyzed. 477 genes were found to be significantly differentially expressed between the ultrasound and sham groups using cut-off values of p < 0.05 and fold change of 2. The top differentially expressed genes included those involved in regulation of cell metabolism, proliferation, and immune cell signaling. Gene set enrichment analysis identified 20 significantly enriched gene sets from upregulated genes and 4 significantly enriched gene sets from downregulated genes. Most of the enriched gene sets from upregulated genes were related to cell-cell signaling pathways. The most significantly enriched gene set from downregulated genes was the inflammatory response gene set. These findings show that therapeutic ultrasound influences cellular behavior in chronic wounds as early as 1 week after application. Considering the well-known role of chronic inflammation in impairing wound healing in chronic wounds, these results suggest that a downregulation of inflammatory genes is a possible biological mechanism of ultrasound-mediated venous chronic wound healing. Such increased understanding may ultimately lead to the enhancement of ultrasound devices to accelerate chronic wound healing and increase patient quality of life.

The inflammatory macrophages repress the growth of bone metastatic human prostate cancer cells via TNF-α and IL-6 signaling: Involvement of cell signaling regulator regucalcin

Macrophages in the cancer microenvironments may play a regulatory role in the progression and metastasis of prostate cancer cells. However, the crosstalk between macrophages and prostate cancer cells is poorly understood. This study elucidates whether inflammatory macrophages regulate the proliferation and death of human prostate cancer cells in vitro. The RAW264.7 mouse macrophages were cocultured with PC-3 or DU-145 wild-type cells by using a Transwell chamber in vitro. RAW264.7 cells were cocultured with PC-3 or DU-145 cells in the presence of lipopolysaccharide (LPS). This coculturing blocked the proliferation and accelerated the death of cancer cells. Interestingly, cancer cell proliferation was repressed and death was promoted by the addition of the conditioned medium obtained from RAW264.7 cells treated with LPS. Culturing with LPS mostly augmented the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture medium of RAW264.7 cells. The effects of the conditioned medium on the proliferation and death of PC-3 or DU-145 cells were blocked by NF-κB or STAT3 signaling inhibitors. Moreover, the effects of the conditioned medium on the proliferation and death of prostate cancer cells were not expressed in regucalcin-overexpressing cancer cells that diminish the levels of NF-κB p65 and STAT3. Culturing with extracellular TNF-α, IL-6, or regucalcin triggered inhibition of the proliferation of PC-3 wild-type cells. The levels of regucalcin in PC-3 cells were elevated by TNF-α or IL-6 stimulation. This study demonstrates that inflammatory macrophages triggered the loss of prostate cancer cells via the signaling process of NF-κB, STAT3, or regucalcin.

Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages.

Irisin is a hormone‑like myokine that regulates cell signaling pathways and exerts anti‑inflammatory effects. However, the specific molecular mechanisms involved in this process are currently unknown. The present study explored the role and mechanisms underlying the functions of irisin in alleviating acute lung injury (ALI). The present study used MH‑S, an established murine alveolar macrophage‑derived cell line, and a mouse model of lipopolysaccharide (LPS)‑induced‑ALI to examine the efficacy of irisin against ALI invitro and invivo, respectively. Fibronectin typeIII repeat‑containing protein/irisin was expressed in the inflamed lung tissue, but not in normal lung tissue. Exogenous irisin reduced alveolar inflammatory cell infiltration and pro‑inflammatory factor secretion in mice following LPS stimulation. It also inhibited the polarization of M1‑type macrophages and promoted the repolarization of M2‑type macrophages, thus reducing the LPS‑induced production and secretion of interleukin (IL)‑1β, IL‑18 and tumor necrosis factor‑α. In addition, irisin reduced the release of the molecular chaperone heat shock protein90 (HSP90), inhibited the formation of nucleotide‑binding and oligomerization domain‑like receptor protein3 (NLRP3) inflammasome complexes, and decreased the expression of caspase‑1 and the cleavage of gasdermin D (GSDMD), leading to reduced pyroptosis and the accompanying inflammation. On the whole, the findings of the present study demonstrate that irisin attenuates ALI by inhibiting the HSP90/NLRP3/caspase‑1/GSDMD signaling pathway, reversing macrophage polarization and reducing the pyroptosis of macrophages. These findings provide a theoretical basis for understanding the role of irisin in the treatment of ALI and acute respiratory distress syndrome.

Survey of genomic and physiological characteristics for survival in lymphoma: The NCI genomic data portal

Lymphoma represents a myriad collection of neoplasms that impact lymphocytes. This cancer often involves disrupted cytokine, immune surveillance, and gene regulatory signaling, sometimes with expression of Epstein Barr Virus (EBV). We explored mutation patterns for People experiencing Lymphoma (PeL) in the National Cancer Institute (NCI) Genomic Data Commons (GDC), which contains detailed, deidentified genomic data on 86,046 people who have/had cancer with 2,730,388 distinctive mutations in 21,773 genes. The database included information on 536 (PeL), with the primary focal sample being the n = 30 who had complete mutational genomic data. We used correlations, independent samples t-tests, and linear regression to compare PeL demographics and vital status on mutation numbers, BMI, and mutation deleterious score across functional categories of 23 genes. PeL demonstrated varied patterns of mutated genes, consistent with most other cancer types. The primary PeL gene mutations clustered around five functional protein groups: transcriptional regulatory proteins, TNF/NFKB and cell signaling regulators, cytokine signaling proteins, cell cycle regulators, and immunoglobulins. Diagnosis Age, Birth Year, and BMI negatively (P < 0.05) correlated with Days to Death, and cell cycle mutations negatively correlated (P = 0.004) with survival days (R2 = 0.389). There were commonalities in some PeL for mutations across other cancer types based upon large sequence length, but also for 6 small cell lung cancer genes. Immunoglobulin mutations were prevalent but not for all cases. Research indicates a need for greater personalized genomics and multi-level systems analysis to evaluate facilitators and barriers for lymphoma survival.

Analysis of the Interactome of the Toxoplasma gondii Tgj1 HSP40 Chaperone.

Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein-protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.

Abstract P6-11-02: Mitochondria-Nuclear Crosstalk Regulates the WNT Pathway in Triple-Negative Breast Cancer

Abstract Compared to hormone receptor (HR) positive breast cancer (BC), basal or triple-negative BC (TNBC) suffers a poor prognosis caused by a limited understanding of its driver signaling pathways. We used the transmitochondrial cybrid approach to understand the metabolic reprogramming and mitochondria-nuclear crosstalk in metastatic TNBC. In cybrids, mitochondria from cancer and benign cells are compared under a commonly defined nuclear background. Using cybrid-based discovery and its validation in cell lines, patient-derived xenografts, and clinical data, we have previously reported the activation of fatty acid oxidation (FAO) in metastatic TNBC. Recently, we developed a unique gene (DEG) signature by integrating gene expression data from cybrids with different omnibus databases such as TCGA and METABRIC. Our DEG could effectively distinguish the PAM50 subtypes of BC. Further analyses nominated negative regulation of the canonical WNT signaling pathway as one of the major pathways altered by mitochondrial character. The WNT pathway is known to regulate cell signaling, metabolism, epithelial-mesenchymal transition (EMT), and cancer stemness. Cybrids with benign mitochondria showed transcriptional activation of negative WNT regulators, including DKK1, SOST, DAB2, and CAV1. Citrate is a key metabolite from the tricarboxylic acid cycle (TCA cycle) and is an intermediate of many other metabolic pathways. Analysis of TNBC cybrids, cell lines, and BC tissues suggest that in TNBC, citrate levels are increased with the activation of CPT1A, the rate-limiting enzyme of FAO. We confirmed the reduction of the beta-catenin protein in TNBC cells after pharmacological and genetic inhibition of CPT1A and mitochondrial citrate transporter (SLC25A1). We further analyzed the gene set enrichment analysis (GSEA) using gene expression data developed from short and long-term CPT1A inhibited TNBC cells and observed that the FAO alters polycomb repressive complex 2 (PRC2) activity. PRC2 is a critical modulator of the H3K27 methylation pattern as well as pathways related to cancer stemness, including WNT/beta-catenin and Hedgehog (SHH) pathways. These results suggest that the mitochondrial FAO transcriptionally inhibits WNT antagonist genes through repressive histone modification and consequently activates the canonical WNT pathway and cancer stemness. Overall, our current observations provide new insights into the regulation WNT pathway by mitochondria-nuclear crosstalk in TNBC. Citation Format: Sukjin Yang, Jun Hyoung Park, Suna Kim, Meron Ghidey, Benny Kaipparettu. Mitochondria-Nuclear Crosstalk Regulates the WNT Pathway in Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-02.