Regulation Of Cell Signaling Research Articles

Abstract 3528: Influence of obesity-associated intra-tumor microbes on exacerbating cancer severity

Abstract Background: Despite there being a well-established connection between the gut microbiome and metabolic disease in humans, intra-tumor microbes and the mechanisms by which they regulate cell signaling, inflammation, and adipocyte growth to exacerbate disease severity in cancer patients also suffering from obesity remains largely unclear. In this study, we identified microbes to be distinctly abundant in cancer patients who are obese and correlated microbe abundance to patient survival, clinical variables, and immunological genes and pathways, in order to mechanistically explain how differential microbe abundance may influence clinical outcome. Methods: Microbial reads were aligned and extracted from raw whole-transcriptome RNA-sequencing data of cancer patient samples using Pathoscope 2.0 software. The Kruskal-Wallis test was used to correlate body mass index (BMI)-associated microbes to clinical variables. Reactome FIViz and Gene Set Enrichment Analysis were used to calculate pathway enrichment. Results: We identified specific microbes, including Pseudomonas fluorescens SBW25 and Enterobacter cloacae, and chemokine and interleukin-related genes to be potential determining factors of disease severity among cancer patients in BMI-associated groups. Gene set enrichment analysis revealed that microbes abundant in cancer tissue in obese patient samples, including Pseudomonas baetica in liver cancer patients, were significantly associated with the upregulation oncogenic, cell migration-related signaling pathways. Intra-tumor microbes from obese patient samples were also found to correlate with chemokine signaling and TFR2/NFkB-related genes, both of which have well-established roles in inflammatory activity. Conclusions: Our study significantly advances the understanding of the microbiome composition of the tumor microenvironment in patients who are obese and microbes’ relationship with clinical and immunologic variables, particularly inflammatory-related genes and pathways. We uncovered unknown mechanisms of the microbiome-immune interaction and obtained definitive data on microbiome dysbiosis in patients with obesity as a key determinant of severity of cancer, including microbe regulation of inflammasome activity. While deeper sequencing, more rigorous contamination correction, and in vitro and in vivo experiments are necessary to fully elucidate how microbe species can effectively act as therapeutic agents in probiotic and prebiotic therapies to reduce insulin resistance, inflammation, and glucose levels, our results are essential for guiding this future research. Citation Format: Aditi Gnanasekar, Neil Shende, Jaideep Chakladar, Wei T. Li, Lindsay M. Wong, Michael Karin, Weg M. Ongkeko. Influence of obesity-associated intra-tumor microbes on exacerbating cancer severity [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 3528.

Abstract 5822: Leptin modulated microRNA-628-5p targets Jagged1 and inhibits prostate cancer hallmarks

Abstract Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer deaths in men. Obesity is a major risk factor for prostate cancer. Leptin is an obesity hormone that controls body weight by regulating energy intake and expenditure, and clinical studies have demonstrated a correlation between blood leptin levels and PCa development. MicroRNAs (miRs) are master regulators of cancer cell signaling, including in advanced PCa. We previously identified miR-628-5p as a potential biomarker in PCa patients that was downregulated in serum samples of men with PCa (Tumor Biol., 2014, 35: 4867). In this study, we investigated the link between the obesity hormone leptin and the functional role of miR-628-5p in PCa cell lines. We demonstrate that leptin downregulates the expression of miR-628-5p and increases cell proliferation and migration in PCa cells. Enforced expression of miR-628-5p inhibited cell proliferation in PCa cells. Further, miR-628-5p reduced PCa cell survival/migration/invasion/spheroids formation and cell stemness. Mechanistically, miR-628-5p binds with JAG1-3’UTR and inhibits the expression of Jagged-1 (JAG1). JAG1 inhibition by miR-628-5p also downregulated Notch signaling, decreased the expression of snail/slug, and modulated epithelial-mesenchymal transition and invasiveness in PC3 cells. Furthermore, miR-628-5p increased sensitivity towards enzalutamide and docetaxel by inducing cell apoptosis. Collectively our data suggested that miR-628-5p is a key regulator of PCa carcinogenesis modulated by adipokine leptin and offers a novel therapeutic opportunity to inhibit the growth of advanced PCa. Citation Format: Leslimar Rios Colon, Juliet Chijioke, Suryakant Niture, Zainab Afzal, Qi Qi, Anvesha Srivastava, Malathi Ramalinga, Habib Kedir, Patrice Cagle, Elena Arthur, Gagan Deep, Simeng Suy, Sean Collins, Deepak Kumar. Leptin modulated microRNA-628-5p targets Jagged1 and inhibits prostate cancer hallmarks [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 5822.

Changes of serum protein N-glycosylation in cancer.

Glycosylation is a posttranslational modification responsible for many bio-logical processes including protein-protein interactions, cell signaling or cell cycle regulation. Changes in glycosylation of serum proteins reflects the status of tissues and cells in the organism and therefore can be used as markers for dia-gnosis of cancer, its progression and determination of its subtypes. N-glycan profiling is often used for characterization of N-glycosylation changes. It is based on the measurements of N-glycans released from the serum proteins. Beside the N-glycan profiling, glycoproteomic approach is emerging as it preserves the information about glycan composition, original protein, and its glycosylation sites. This review covers existing works describing the changes in serum protein N-glycosylation in various cancer types. Attention was paid to both the glycomic and glycoproteomic approaches. The last part of the review shortly presents the analytical methods used for these analyses.

Abstract 3674: Differential expression of stress-survival pathway genes related to Hispanic colorectal cancer disparities

Abstract Colorectal cancer (CRC) accounts for 12% and 8% of all estimated new cases of cancer and 11% and 9% of all cancer deaths in Hispanic men and women, respectively. Stress-survival pathways have been implicated in tumorigenesis of CRC and in providing resistance to chemotherapy-induced cell-death by suppressing reactive oxygen species (ROS), repairing DNA damage, and by regulating cell signaling pathways. However, studies related to the role of these pathways in CRC from the Hispanic health disparities perspective, are limited. Essential to improving treatment, prognosis, and detection strategies is the identification and validation of new ethnicity-specific transcriptomic markers within the stress-survival pathways as effective predictors of CRC. In this study, we have explored the role of stress-survival pathway genes in Hispanic and Non-Hispanic White (NHW) CRC tissues. We have used microarray and RNA-seq datasets obtained from the gene expression omnibus (GEO) and the cancer genome atlas (TCGA) to identify 28 genes associated with CRC. These genes were screened for transcript level expression in one normal colon and two CRC cell lines, and in cDNA arrays containing tumor (n=40) from different stages, and control (n=8) samples. The protein level expressions were evaluated by immunohistochemistry (IHC) in CRC tissue microarrays (TMAs) containing different stages of tumor (n = 108) and control (n = 12) tissues. All 28 genes were also analyzed for their transcript level expressions in Hispanic and NHW tissues. Genes related to apoptosis like FOXM1, CDK1, CDK4, Chek1, and MCM10 showed consistent upregulation in CRC cell lines, cDNA arrays and TMAs when compared to normal samples. We could also detect a correlation between the differential expression of these genes and the tumor stages. CDK4, Chek1, FOXM1, and CDC25, were observed to be downregulated in the Hispanic CRC tissues. On the other hand, oxidative stress markers were not seen to be differentially expressed in the two CRC cell lines when compared to the normal cell line. However, some of the known oxidative stress markers like PRDX4, GPX1, and SOD2 were found to be upregulated in Hispanics when compared to NHW tissues. Our results suggest that some of these stress-survival pathway genes could be explored as interesting candidates for ethnicity-specific CRC biomarkers and as novel therapeutic targets in the future. Citation Format: Aditi Kulkarni, Urbashi Basnet, Abhijeet R. Patil, Rebecca DeJesus, Sourav Roy. Differential expression of stress-survival pathway genes related to Hispanic colorectal cancer disparities [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 3674.

First person – Sophie Frampton

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Sophie Frampton is first author on ‘ Modelling the structure of Short Gastrulation and generation of a toolkit for studying its function in Drosophila’, published in BiO. Sophie is a postdoc in the lab of Hilary Ashe at The University of Manchester, UK, investigating the processes governing tissue patterning and mechanisms of cell signal regulation.

73-OR: Loss of NADPH Oxidase–Derived Superoxide Impairs Autoreactive T Cell Trafficking into Islets to Delay Type 1 Diabetes Development

Type 1 diabetes (T1D) and β-cell destruction is mediated by proinflammatory cytokines, reactive oxygen species (ROS) , and immune cells. NADPH oxidase (NOX) -derived superoxide, a precursor for many ROS, regulate cell signaling, cellular differentiation, survival, and autoimmune responses in T1D. We previously discovered that NOX-derived superoxide promote chemokine synthesis and skew macrophages and T cells towards a proinflammatory response in T1D. Yet, it remains unknown if NOX is necessary for diabetogenic T cell trafficking into islets. Using non-obese diabetic (NOD) mice lacking NOX-derived superoxide (NOD.Ncf1m1J ) , we tested the hypothesis that loss of NOX-derived superoxide delays T1D by impairing autoreactive T infiltration into islets. Female NOD.Ncf1m1J mice (n=20) had a significant delay in spontaneous T1D development, as only 20% became diabetic by 40-weeks of age compared to 90% NOD mice (n=23) . Flow cytometry analysis of 12-week-old NOD.Ncf1m1J mice revealed a significant reduction in pancreatic CD4 T cells expressing CD69+ (5.5-fold) , CCR2+ (1.9-fold) and CCR5+ (2.1-fold) compared to NOD. NOD.Ncf1m1J islets also had significantly fewer total CD4+ T cells (1.8-fold) , CD25+ CD4+ T cells (2.0-fold) , naïve CD4+ T cells (2.0-fold) , and effector CD4+ T cells (2.2-fold) compared to NOD. Utilizing tetramer staining to identify autoreactive T cells, we observed a significant reduction in NOD.Ncf1m1J islet-infiltrating CD4+ T cells specific for insulin (Ins) (2.6-fold) , Ins-chromogranin A hybrid peptide (HIP) (3.0-fold) , and Ins-islet amyloid polypeptide HIP (2.6-fold) compared to NOD. Our findings provide evidence that loss of NOX-derived superoxide can impair autoreactive T cell activation and islet-infiltration as a possible mechanism to delay T1D development. Future studies will determine if NOX regulates islet chemokine synthesis to mediate autoreactive T infiltration into islets. Disclosure S.I.Blum: None. J.Barra: None. R.L.Baker: None. H.M.Tse: None. Funding Translational and Molecular Sciences T32 (GM109780) , Immunologic Diseases and Basic Immunology T32 (AI007051) , National Institutes of Health NIDDK F31 (DK130551) , National Institutes of Health NIDDK R (DK126456) , National Institutes of Health NIDDK RO1 (DK127497) .

Interplay between Caveolin-1 and body and tumor size affects clinical outcomes in breast cancer

BackgroundCaveolin-1 (CAV1) is associated with cholesterol-rich membrane raft domains and is a master regulator of cell signaling and membrane transport. Here, we investigated CAV1’s role in cellular compartments of breast cancer in relation to signaling pathways, clinicopathological features, and clinical outcomes. MethodsCAV1 levels were evaluated with immunohistochemistry in cytoplasm of invasive tumor cells and stromal cells in tumor tissue microarrays from a cohort of 1018 breast cancer patients (inclusion 2002–2012, Sweden). Cytoplasmic and stromal CAV1 were categorized as positive/negative and strong/not strong, respectively. CAV1 expression in relation to clinical outcomes was assessed with Cox regression. Investigations into CAV1 functional pathways was conducted in the STRING, GOBO, and TCGA databases. ResultsCAV1 expression was associated with non-luminal subtypes, cell cycle control, inflammation, epithelial-mesenchymal transition, and the IGF/Insulin system. Generally, CAV1 was not associated with recurrence risk. Stromal CAV1’s impact on recurrence risk was modified by BMI ≥25 kg/m2 (Pinteraction = 0.002), waist ≥80 cm (Pinteraction = 0.005), and invasive tumor size (pT2/3/4) (Pinteraction = 0.028). In low-risk patients only, strong stromal CAV1 significantly increased recurrence risk (HRsadj ≥1.61). In all patients, positive cytoplasmic CAV1 conferred >2-fold risk for contralateral disease HRadj 2.63 (95% CI 1.36–5.10). Strong stromal CAV1 conferred nearly 2-fold risk for locoregional recurrence HRadj 1.88 (95% CI 1.09–3.24). ConclusionsCAV1’s prognostic impact depended on its localization, anthropometric, and tumor factors. Stromal CAV1 predicted high recurrence risk in a group of supposedly ‘low-risk’ patients. Cytoplasmic CAV1 predicted metachronous contralateral disease. If confirmed, CAV1 could be used as treatment target and for risk-stratification.

LGR4, a G Protein-Coupled Receptor With a Systemic Role: From Development to Metabolic Regulation

Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4/GPR48), a member of the GPCR (G protein-coupled receptors) superfamily, subfamily B, is a common intestinal crypt stem cell marker. It binds R-spondins/Norrin as classical ligands and plays a crucial role in Wnt signaling potentiation. Interaction between LGR4 and R-spondins initiates many Wnt-driven developmental processes, e.g., kidney, eye, or reproductive tract formation, as well as intestinal crypt (Paneth) stem cell pool maintenance. Besides the well-described role of LGR4 in development, several novel functions of this receptor have recently been discovered. In this context, LGR4 was indicated to participate in TGFβ and NFκB signaling regulation in hematopoietic precursors and intestinal cells, respectively, and found to be a new, alternative receptor for RANKL (Receptor Activator of NF kappa B Ligand) in bone cells. LGR4 inhibits the process of osteoclast differentiation, by antagonizing the interaction between RANK (Receptor Activator of NF kappa B) and its ligand-RANKL. It is also known to trigger anti-inflammatory responses in different tissues (liver, intestine, cardiac cells, and skin), serve as a sensor of the circadian clock in the liver, regulate adipogenesis and energy expenditure in adipose tissue and skeletal muscles, respectively. The extracellular domain of LGR4 (LGR4-ECD) has emerged as a potential new therapeutic for osteoporosis and cancer. LGR4 integrates different signaling pathways and regulates various cellular processes vital for maintaining whole-body homeostasis. Yet, the role of LGR4 in many cell types (e.g. pancreatic beta cells) and diseases (e.g., diabetes) remains to be elucidated. Considering the broad spectrum of LGR4 actions, this review aims to discuss both canonical and novel roles of LGR4, with emphasis on emerging research directions focused on this receptor.

LINCing Senescence and Nuclear Envelope Changes.

The nuclear envelope (NE) has emerged as a nexus for cellular organization, signaling, and survival. Beyond its role as a barrier to separate the nucleoplasm from the cytoplasm, the NE’s role in supporting and maintaining a myriad of other functions has made it a target of study in many cellular processes, including senescence. The nucleus undergoes dramatic changes in senescence, many of which are driven by changes in the NE. Indeed, Lamin B1, a key NE protein that is consistently downregulated in senescence, has become a marker for senescence. Other NE proteins have also been shown to play a role in senescence, including LINC (linker of nucleoskeleton and cytoskeleton) complex proteins. LINC complexes span the NE, forming physical connections between the cytoplasm to the nucleoplasm. In this way, they integrate nuclear and cytoplasmic mechanical signals and are essential not only for a variety of cellular functions but are needed for cell survival. However, LINC complex proteins have been shown to have a myriad of functions in addition to forming a LINC complex, often existing as nucleoplasmic or cytoplasmic soluble proteins in a variety of isoforms. Some of these proteins have now been shown to play important roles in DNA repair, cell signaling, and nuclear shape regulation, all of which are important in senescence. This review will focus on some of these roles and highlight the importance of LINC complex proteins in senescence.

Mitochondrial Inhibition by Sodium Azide Induces Assembly of eIF2α Phosphorylation-Independent Stress Granules in Mammalian Cells.

Mitochondrial stress is involved in many pathological conditions and triggers the integrated stress response (ISR). The ISR is initiated by phosphorylation of the eukaryotic translation initiation factor (eIF) 2α and results in global inhibition of protein synthesis, while the production of specific proteins important for the stress response and recovery is favored. The stalled translation preinitiation complexes phase-separate together with local RNA binding proteins into cytoplasmic stress granules (SG), which are important for regulation of cell signaling and survival under stress conditions. Here we found that mitochondrial inhibition by sodium azide (NaN3) in mammalian cells leads to translational inhibition and formation of SGs, as previously shown in yeast. Although mammalian NaN3-induced SGs are very small, they still contain the canonical SG proteins Caprin 1, eIF4A, eIF4E, eIF4G and eIF3B. Similar to FCCP and oligomycine, other mitochodrial stressors that cause SG formation, NaN3-induced SGs are formed by an eIF2α phosphorylation-independent mechanisms. Finally, we discovered that as shown for arsenite (ASN), but unlike FCCP or heatshock stress, Thioredoxin 1 (Trx1) is required for formation of NaN3-induced SGs.

The glycosaminoglycan interactome 2.0

Glycosaminoglycans (GAGs) are complex linear polysaccharides, which are covalently attached to core proteins (except for hyaluronan) to form proteoglycans. They play key roles in the organization of the extracellular matrix, and at the cell surface where they contribute to the regulation of cell signaling and of cell adhesion. To explore the mechanisms and pathways underlying their functions, we have generated an expanded dataset of 4,290 interactions corresponding to 3,464 unique GAG-binding proteins, four times more than the first version of the GAG interactome (Vallet, Clerc, and Ricard-Blum. J Histochem Cytochem 69: 93-104, 2021). The increased size of the GAG network is mostly due to the addition of GAG-binding proteins captured from cell lysates and biological fluids by affinity chromatography and identified by mass spectrometry. We review here the interaction repertoire of natural GAGs and of synthetic sulfated hyaluronan, the specificity and molecular functions of GAG-binding proteins, and the biological processes and pathways they are involved in. This dataset is also used to investigate the differences between proteins binding to iduronic acid-containing GAGs (dermatan sulfate and heparin/heparan sulfate) and those interacting with GAGs lacking iduronic acid (chondroitin sulfate, hyaluronan, and keratan sulfate).

Preferential redox regulation of cysteine-based protein tyrosine phosphatases: structural and biochemical diversity.

Protein phosphorylation is a major post-translational modification involved in cell signalling that regulates many physiological and pathological processes. Despite their biological importance, protein phosphatases are less studied than protein kinases. Importantly, the activity of Cys-based protein tyrosine phosphatases (PTPs) can be regulated by reversible oxidation. The initial two-electron oxidation product of the active site Cys is a sulfenic acid (Cys-SOH) that can then undergo distinct outcomes, such as the disulfide bond or a sulfenyl amide formation. Here, we review the biochemical and structural features of PTPs to find patterns that might specify their oxidation products, aiming to get insights into redox regulatory mechanisms. Initially, the structure and biochemistry of PTP1B is presented. Then, we describe structural aspects that are relevant for substrate recognition and catalysis. Notably, all PTPs contain critical Cys residues for the catalysis of dephosphorylation that is prone to oxidative inactivation, which are frequently found oxidized in cells under physiological conditions, such as upon growth factor stimuli. However, direct oxidations of Cys residues in PTPs by H2 O2 are rather slow. Therefore, we discuss possible mechanisms that may account for this apparent contradiction between biological and chemical redox aspects of PTPs. Furthermore, we performed a systematic analysis of the distance between active site cysteine and its backdoor cysteine with the attempt to analyse the preference between disulfide bond formation or sulfenyl amide interaction upon oxidation. In summary, PTPs have been showing many possibilities to auto-protect from irreversible oxidation, which is important for cell signalling regulation.

The sialyltransferase ST6Gal1 is upregulated in a cytokine‐mediated manner during pancreatitis

Pancreatitis is a life‐threatening medical disorder that occurs when abnormally activated pancreatic enzymes damage surrounding pancreatic acinar cells, leading to the release of several pro‐inflammatory cytokines. Subsequently, inflammation of the pancreas promotes the dedifferentiation of acinar cells towards a ductal‐like, progenitor phenotype, to heal from pancreatic injury, in a process known as acinar to ductal metaplasia (ADM). Recent studies by our group suggest that the sialyltransferase, ST6Gal1 promotes ADM. ST6Gal1 adds sialic acid to N‐glycosylated surface receptors and thereby regulates cell signaling and phenotype. In the current study, we report the novel finding that ST6Gal1 is upregulated in the pancreatic tissues of patients with chronic pancreatitis and mice with experimental pancreatitis, whereas ST6Gal1 expression is undetectable in normal acinar cells. These results are the first to show stark modulation of ST6Gal1 within pancreatic inflammation. While the upregulation of ST6Gal1 has been noted across multiple cancers, the mechanisms by which ST6Gal1 expression is modulated within an inflammatory milieu are not well understood. To evaluate inflammation‐induced ST6Gal1 upregulation, we injected mice with cerulein to evoke pancreatitis. We first examined levels of pro‐inflammatory cytokines through immunohistochemistry in cerulein‐treated mice and observed striking increases in the release of both IL‐6 and IL‐1β. Accordingly, we proceeded to treat multiple pancreatic acinar cell line models with IL‐6 and IL‐1β to assess their respective capability to upregulate ST6Gal1. These two cytokines stimulated increases in ST6Gal1 mRNA and protein in all cell models. It's known that there are three major isoforms of ST6Gal1, each driven by a different promoter; thus, we investigated which of the three isoforms was increased following cytokine treatment. We found that the isoform driven by the P3 promoter, a ubiquitously expressed isoform known as “YZ”, was the only isoform seen to be upregulated following cytokine treatment. To further understand the mechanisms by which IL‐6 and IL‐1β mediate their upregulation of ST6Gal1, we used an online transcription factor binding predictor to assess which transcription factors bind to the P3 promoter of the YZ isoform of ST6Gal1. On this P3 promoter, we found binding sites for STAT3, which is activated by IL‐6, and NF‐κB, which is activated by IL‐1β. Furthermore, we observed STAT3 and NF‐κB pathway activation following cytokine treatment. Importantly, STAT3 and NF‐κB inhibitors blocked cytokine‐induced ST6Gal1 upregulation in multiple cell models, thus confirming that IL‐6 and IL‐1β mediated their upregulation of the YZ isoform through the STAT3 and NF‐κB pathways, respectively. Taken together, these results not only show ST6Gal1 modulation within pancreatitis but detail the mechanisms by which this occurs, thus providing significant insight into an area of research that has not been well characterized.

Development of highly potent and selective GRK5 inhibitors

G protein‐coupled receptor kinases (GRKs) regulate cell signaling by triggering receptor desensitization via phosphorylation on G protein‐coupled receptors (GPCRs). (1) The seven human GRKs (GRK1−GRK7) are classified into three subfamilies: GRK1 (GRK1 and GRK7), GRK2 (GRK2 and GRK3), and GRK4 (GRK4, GRK5, and GRK6). GRK2 and GRK5 are the most abundant in cardiovascular tissue, where they are potential targets for treatment of cardiovascular disease. GRK5 also undergoes Ca2+/calmodulin‐dependent nuclear localization, where it phosphorylates histone deacetylase 5 (HDAC5), inducing an increase in transcription of cardiac hypertrophy‐related genes. In GRK5‐knockdown mice, cardiomyocytes are protected from hypertrophy; however, the specific roles of GRK5 in heart failure and hypertrophic cardiomyopathy are still unclear. Furthermore, GRK5 is required for cancer progression in various cancer types. Depletion of GRK5 has been shown to suppress prostate cancer, breast cancer, and non‐small‐cell lung cancer. (2) Therefore, targeting GRK5 can also be a chemotherapeutic strategy. Here we are testing a series of inhibitors for GRK5/6 derived from the indolinone scaffold, utilizing Cys474 residue unique in GRK5/6 to enhance selectivity by covalent capture. An immediate goal is to facilitate the development of more selective inhibitors via rational design by determining x‐ray crystal structures of inhibitor complexes with GRK5. Recently, we have crystallized a hyper‐phosphorylated GRK5, and the active site structure suggested the possibility for compound soaking. Overall, discovery of the inhibitors can significantly facilitate understanding and treatment of cardiovascular diseases and cancer.1. Z. Yang et al., Phosphorylation of G Protein‐Coupled Receptors: From the Barcode Hypothesis to the Flute Model. Molecular Pharmacology 92, 201‐210 (2017).2. L.‐P. Jiang et al., GRK5 functions as an oncogenic factor in non‐small‐cell lung cancer. Cell Death Dis 9, 295 (2018).

Characterizing the transcriptional signature regulated by the AHR ligands TCDD and kynurenine in colon cancer cells

AHR was first identified as the receptor that binds to the pollutant TCDD, which causes tumorigenesis. Since its identification, AHR has been credited to play roles in multiple aspects of carcinogenesis such as increased cellular proliferation, immune evasion, and metastasis. A wide variety of tumors express increased AHR in comparison to the tissue of origin, thus suggesting a positive correlation with tumorigenesis. While most of the research has been focused on the role of activated AHR, a topic that has not received enough attention is whether activation of AHR by different agonists leads to different transcriptional outputs. In this study, we investigated the immediate transcriptional output of colon cancer cell lines exposed to TCDD and Kyn in an AHR‐dependent manner. We found that the expression of CYP1A1, ALDH1A3, ABCG2, ADGFR1, SCIN, and ITPR1 were commonly activated by TCDD, and Kyn, suggesting that these are canonical targets of AHR in colon cancer cells. The activation of expression of CYP1A1, ALDH1A3 and ABCG2 suggests that xenobiotic response pathways are induced upon AHR activation, independent of its ligands. TCDD, which can induce carcinogenesis in various rodent models, induced a larger group of 40 genes, including several potential oncogenes, regulators of cell proliferation, cytoskeletal organization, and signaling. Additionally, we further investigated the effects of SCINin colonic cancer cells. SCINdisrupted proliferation of colon cancer cells when silenced. Gain of function of this gene in colon has not been studied. Our study indicates that this gene can be critical for attachment and activation of the Wnt pathway. Genes activated by AHR are crucial for multiple aspects of carcinogenesis and suggests that targeting AHR by using AHR antagonists could have therapeutic value by limiting cell growth.

Identification of Signaling and Functional Adaptations of Hematopoietic Stem/Progenitor Cells via Generation of a Low Oxygen Landscape

Hematopoietic stem and progenitor cells (HSC/HPC) reside in low O2 (1‐4%) bone marrow (BM) niches which provide critical signals for stem cell maintenance and function. Since the technology to directly compare broad signaling/functional differences of live HSC/HPC maintained in their native low O2 environment did not previously exist, historic studies have been performed in ambient air (~21% O2), utilizing whole (W) BM or fixed cells leading to a lack of mechanistic insight into endogenous signaling pathways/functional outcomes dependent on relevant microenvironmental O2. Using our novel technology to study live HSC/HPC in continuous low O2, we generated the first reference low O2 landscape of differential phenotypic/signaling/functional regulation in live HSC/HPC. Murine BM was harvested from long bones, enriched for SCA1+ cells, sorted for HSC/HPC (LSKCD150/LSK) fractions at continuous 3% O2 then split to remain in low O2 or be exposed to room air (60 minutes) with paired comparisons performed on the divided samples. Statistical analyses (t‐test or ANOVA) are based on multiple experiments (n>3/experiment). All subsequent comparisons are between 3% O2 samples compared to room air exposed samples. Live cell analysis demonstrated enhanced phenotypic marker expression and frequency in low O2 of HSC (2‐fold), HPC (2‐fold), B‐cell‐ (B220) and erythroid‐lineage (Ter119) cells under low O2 (p<0.05) relative to both historic data (utilizing WBM, fixed cells) and our room air data and identified masking of phenotypic difference in low O2 upon cell fixation. Transcriptional analyses of HPC detected 23,611 total genes; 1,239 were identified as differentially expressed (DEGs). HSC analyses detected 13,719 total genes with 135 identified DEGs (FDR<0.05) in low O2. DAVID analysis of bulk mRNA sequencing identified differential pathway regulation of HPC (324 increased GO pathways, 230 decreased) and HSC (73 increased, 30 decreased) with significant enhancements of genes responsible for calcium regulation, transmembrane transport, and cell differentiation observed in low O2. Proteomic analysis of HPC detected 2,352 expressed proteins; 99 identified as differentially expressed (FDR<0.05) and DAVID analysis highlighting enhancements in metabolism, cell signaling, and membrane regulation (p<0.05) in low O2. Single cell sequencing of sorted HPC identified over 18,000 total genes in both air and low O2 with GSEA of significantly detected genes (p<0.05) demonstrating differential regulation of cell cycle, metabolism, protein stabilization, enzyme binding/kinetics and calcium pathways in low O2. Functional analyses of HPC maintained at native low O2 revealed enhanced maintenance of HSC/HPC over 3‐4 re‐platings and altered fate decisions resulting in enhanced numbers of B‐cell‐, myeloid‐ (CD11b) and erythroid‐lineage (p<0.05). These data provide novel insight into HSC/HPC endogenous signaling and function via generation of the first low O2 landscape and identified unique populations and differentially regulated pathways/gene signatures in low O2 vs. room air. Thus, highlighting the critical importance of native experimental conditions on data generation and interpretation in studies of HSC/HPC with potential broad implications for other stem cell populations.

Characterization of Protein Tyrosine Phosphatase 1B activator peptides in cells

The Protein Tyrosine Phosphatase (PTP) family of phosphatases regulates cell signaling through dephosphorylation of serine, threonine and tyrosine residues of downstream effector proteins. The role of PTPs is very well known in normal cell functions, as well as in disease states such as diabetes and cardiovascular diseases. We previously reported on the role of the phosphotyrosine recognition loop (PTBL) of PTP1B in protein‐protein interactions (Londhe et al.2020, Nat. Chem. Biol., 16(2):122‐125) and showed that interfering with protein‐protein interactions between PTP1B and 14‐3‐3zusing R18, a pan‐14‐3‐3 inhibitor, destabilizes the reversibly oxidized form of PTP1B (PTP1B‐OX) and activates the phosphatase. We rationally designed peptides using phosphotyrosine recognition loop of PTP1B to determine whether selective and effective activation of PTPs was possible. We first performed a structural analysis to compare the changes occurring in PTP1B‐OX compared to its reduced form. Using this approach, we observed profound conformational changes in the phosphotyrosine recognition loop (PTBL) of PTP1B and rationally designed peptides comprised of the sequence of amino acids of the newly exposed loop, with a TAT (transactivator of transcription) peptide sequence in amino or carboxy‐terminal to facilitate cell permeability. Targeting the PTP1B‐OX‐14‐3‐3z interaction using R18 and TAT‐PTBL peptide led to disruption in protein‐protein interaction and maintained PTP1B in its active form. Interestingly, destabilizing the PTP1B‐OX form by treating cells with rationally designed PTP1B activator peptides also decreased EGFR phosphorylation at PTP1B sites. In cells stimulated with exogenousH2O2 to bypass the NOX‐mediated PTP1B oxidation, the pretreatment with R18 and TAT‐PTBL prevented the interaction between PTP1B‐OX and 14‐3‐3z. Then we observed that our TAT‐PTBL peptide was not interfering with NOX‐mediated reactive oxygen species (ROS) generation, whereas R18, a pan‐14‐3‐3 inhibitor, decreased ROS production significantly. Finally, exposing A431 epidermoid carcinoma cells to PTP1B activator peptides prevented EGFR phosphorylation and impaired colony formation. Collectively our data suggest that the biological activity of PTP1B activator peptides is likely to be therapeutically useful, and could translate into drug leads.

Dissecting the Role of AXL in Cancer Immune Escape and Resistance to Immune Checkpoint Inhibition.

The development and implementation of Immune Checkpoint Inhibitors (ICI) in clinical oncology have significantly improved the survival of a subset of cancer patients with metastatic disease previously considered uniformly lethal. However, the low response rates and the low number of patients with durable clinical responses remain major concerns and underscore the limited understanding of mechanisms regulating anti-tumor immunity and tumor immune resistance. There is an urgent unmet need for novel approaches to enhance the efficacy of ICI in the clinic, and for predictive tools that can accurately predict ICI responders based on the composition of their tumor microenvironment. The receptor tyrosine kinase (RTK) AXL has been associated with poor prognosis in numerous malignancies and the emergence of therapy resistance. AXL is a member of the TYRO3-AXL-MERTK (TAM) kinase family. Upon binding to its ligand GAS6, AXL regulates cell signaling cascades and cellular communication between various components of the tumor microenvironment, including cancer cells, endothelial cells, and immune cells. Converging evidence points to AXL as an attractive molecular target to overcome therapy resistance and immunosuppression, supported by the potential of AXL inhibitors to improve ICI efficacy. Here, we review the current literature on the prominent role of AXL in regulating cancer progression, with particular attention to its effects on anti-tumor immune response and resistance to ICI. We discuss future directions with the aim to understand better the complex role of AXL and TAM receptors in cancer and the potential value of this knowledge and targeted inhibition for the benefit of cancer patients.

Inhibition of SHP2 and SHP1 Protein Tyrosine Phosphatase Activity by Chemically Induced Dimerization.

Protein tyrosine phosphatases (PTPs), the enzymes that catalyze the dephosphorylation of phosphotyrosine residues, are important regulators of mammalian cell signaling, whose activity is misregulated in numerous human diseases. PTPs are also notoriously difficult to selectively modulate with small molecules, and relatively few small-molecule tools for controlling their activities in the context of complex signaling pathways have been developed. Here, we show that a chemical inducer of dimerization (CID) can be used to selectively and potently inhibit constructs of Src-homology-2-containing PTP 2 (SHP2) that have been engineered to contain dimerization domains. Our strategy was inspired by the naturally occurring mechanism of SHP2 regulation, in which the PTP activity of SHP2’s catalytic domain is autoinhibited through an intramolecular interaction with the protein’s N-terminal SH2 (N-SH2) domain. We have re-engineered this inhibitory interaction to function intermolecularly by independently fusing the SHP2 catalytic and N-SH2 domains to protein domains that heterodimerize upon the introduction of the CID rapamycin. We show that rapamycin-induced protein dimerization leads to potent inhibition of SHP2’s catalytic activity, which is driven by increased proximity of the SHP2 catalytic and N-SH2 domains. We also demonstrate that CID-based inhibition of PTP activity can be applied to an oncogenic gain-of-function SHP2 mutant (E76K SHP2) and to the catalytic domain of the SHP2’s closest homologue, SHP1. In sum, CID-driven inhibition of PTP activity provides a broadly applicable tool for inhibiting dimerizable forms of the SHP PTPs and represents a novel paradigm for selective PTP inhibition through inducible protein–protein interactions.

Noggin proteins are multifunctional extracellular regulators of cell signaling.

Noggin is an extracellular cysteine knot protein that plays a crucial role in vertebrate dorsoventral patterning. Noggin binds and inhibits the activity of bone morphogenetic proteins via a conserved N-terminal clip domain. Noncanonical orthologs of Noggin that lack a clip domain (“Noggin-like” proteins) are encoded in many arthropod genomes and are thought to have evolved into receptor tyrosine kinase ligands that promote Torso/receptor tyrosine kinase signaling rather than inhibiting bone morphogenic protein signaling. Here, we examined the molecular function of noggin/noggin-like genes (ApNL1 and ApNL2) from the arthropod pea aphid using the dorso-ventral patterning of Xenopus and the terminal patterning system of Drosophila to identify whether these proteins function as bone morphogenic protein or receptor tyrosine kinase signaling regulators. Our findings reveal that ApNL1 from the pea aphid can regulate both bone morphogenic protein and receptor tyrosine kinase signaling pathways, and unexpectedly, that the clip domain is not essential for its antagonism of bone morphogenic protein signaling. Our findings indicate that ancestral noggin/noggin-like genes were multifunctional regulators of signaling that have specialized to regulate multiple cell signaling pathways during the evolution of animals.