Activation Of CD44 Research Articles

CD44 signaling in Müller cells impacts photoreceptor function and survival in healthy and diseased retinas

Retinitis pigmentosa (RP), an inherited retinal disease, affects 1,5 million people worldwide. The initial mutation-driven photoreceptor degeneration leads to chronic inflammation, characterized by Müller cell activation and upregulation of CD44. CD44 is a cell surface transmembrane glycoprotein and the primary receptor for hyaluronic acid. It is involved in many pathological processes, but little is known about CD44’s retinal functions. CD44 expression is also increased in Müller cells from our Pde6bSTOP/STOP RP mouse model. To gain a more detailed understanding of CD44’s role in healthy and diseased retinas, we analyzed Cd44−/− and Cd44−/−Pde6bSTOP/STOP mice, respectively. The loss of CD44 led to enhanced photoreceptor degeneration, reduced retinal function, and increased inflammatory response. To understand the underlying mechanism, we performed proteomic analysis on isolated Müller cells from Cd44−/− and Cd44−/−Pde6bSTOP/STOP retinas and identified a significant downregulation of glutamate transporter 1 (SLC1A2). This downregulation was accompanied by higher glutamate levels, suggesting impaired glutamate homeostasis. These novel findings indicate that CD44 stimulates glutamate uptake via SLC1A2 in Müller cells, which in turn, supports photoreceptor survival and function.

Single-cell RNA sequencing of intestinal crypts reveals vital events in damage repair and the double-edged sword effect of the Wnt3/β-catenin pathway in irradiated mice

In this study, we executed single-cell RNA sequencing of intestinal crypts. We analyzed the differentially expressed genes (DEGs) at different time points (the first, third, and fifth days) after 13 Gy and 15 Gy abdominal body radiation (ABR) exposure and then executed gene ontology (GO) enrichment analysis, RNA velocity analysis, cell communication analysis, and ligand‒receptor interaction analysis to explore the vital events in damage repair and the multiple effects of the Wnt3/β-catenin pathway on irradiated mice. Results from bioinformatics analysis were confirmed by a series of biological experiments. Results showed that the antibacterial response is a vital event during the damage response process after 13 Gy ABR exposure; ionizing radiation (IR) induced high heterogeneity in the transient amplification (TA) cluster, which may differentiate into mature cells and stem cells in irradiated small intestine (SI) crypts. Conducting an enrichment analysis of the DEGs between mice exposed to 13 Gy and 15 Gy ABR, we concluded that the Wnt3/β-catenin and MIF-CD74/CD44 signaling pathways may contribute to 15 Gy ABR-induced mouse death. Wnt3/β-catenin promotes the recovery of irradiated SI stem/progenitor cells, which may trigger macrophage migration inhibitory factor (MIF) release to further repair IR-induced SI injury; however, with the increase in radiation dose, activation of CD44 on macrophages provides the receptor for MIF signal transduction, initiating the inflammatory cascade response and ultimately causing a cytokine release syndrome. In contrast to previous research, we confirmed that inhibition of the Wnt3/β-catenin pathway or blockade of CD44 on the second day after 15 Gy ABR may significantly protect against ABR-induced death. This study indicates that the Wnt3/β-catenin pathway plays multiple roles in damage repair after IR exposure; we also propose a novel point that the interaction between intestinal crypt stem cells (ISCs) and macrophages through the MIF-CD74/CD44 axis may exacerbate SI damage in irradiated mice.

Cholesterol crystals induce mechanical trauma, inflammation, and neo-vascularization in solid cancers as in atherosclerosis

Background and aimsCancer and atherosclerosis share common risk factors and inflammatory pathways that promote their proliferation via vascular endothelial growth factor (VEGF). Because CCs cause mechanical injury and inflammation in atherosclerosis, we investigated their presence in solid cancers and their activation of IL-1β, VEGF, CD44, and Ubiquityl-Histone H2B (Ub-H2B), that promote cancer growth. MethodsTumor specimens from eleven different types of human cancers and atherosclerotic plaques were assessed for CCs, free cholesterol content and IL1-β by microscopy, immunohistochemistry, and biochemical analysis. Breast and colon cancer cell lines were cultured with and without CCs to select for expression of VEGF, CD44, and Ub-H2B. Western blot and immunofluorescence were performed on cells to assess the effect of CCs on signaling pathways. ResultsCancers displayed higher CC content (+2.29 ± 0.74 vs +1.46 ± 0.84, p < 0.0001), distribution (5.06 ± 3.13 vs 2.86 ± 2.18, p < 0.001) and free cholesterol (3.63 ± 4.02 vs 1.52 ± 0.56 μg/mg, p < 0.01) than cancer free marginal tissues and similarly for atherosclerotic plaques and margins (+2.31 ± 0.51 vs +1.44 ± 0.79, p < 0.02; 14.0 ± 5.74 vs 8.14 ± 5.52, p < 0.03; 0.19 ± 0.14 vs 0.09 ± 0.04 μg/mg, p < 0.02) respectively. Cancers displayed significantly increased expression of IL1-β compared to marginal tissues. CCs treated cancer cells had increased expression of VEGF, CD44, and Ub-H2B compared to control. By microscopy, CCs were found perforating cancer tumors similar to plaque rupture. ConclusionsThese findings suggest that CCs can induce trauma and activate cytokines that enhance cancer growth as in atherosclerosis.

HCK promotes high-grade serous ovarian cancer tumorigenesis through CD44 and NOTCH3 signaling.

High-grade serous ovarian cancer (HGSOC) is a highly aggressive and lethal subtype of ovarian cancer. While most patients initially respond to standard-of-care treatment, the majority will eventually relapse and succumb to their disease. Despite significant advances in our understanding of this disease, the mechanisms that govern the distinctions between HGSOC with good and poor prognosis remain unclear. In this study, we implemented a proteogenomic approach to analyze gene expression, proteomic and phosphoproteomic profiles of HGSOC tumor samples to identify molecular pathways that distinguish HGSOC tumors relative to clinical outcome. Our analyses identify significant upregulation of hematopoietic cell kinase (HCK) expression and signaling in poor prognostic HGSOC patient samples. Analyses of independent gene expression datasets and immunohistochemistry of patient samples confirmed increased HCK signaling in tumors relative to normal fallopian or ovarian samples and demonstrated aberrant expression in tumor epithelial cells. Consistent with the association between HCK expression and tumor aggressiveness in patient samples, in vitro phenotypic studies showed that HCK can, in part, promote cell proliferation, colony formation, and invasive capacity of cell lines. Mechanistically, HCK mediates these phenotypes, partly through CD44 and NOTCH3-dependent signaling, and inhibiting CD44 or NOTCH3 activity, either genetically or through gamma-secretase inhibitors, can revert HCK-driven phenotypes. Implications: Collectively, these studies establish that HCK acts as an oncogenic driver of HGSOC through aberrant activation of CD44 and NOTCH3 signaling and identifies this network as a potential therapeutic opportunity in a subset of aggressive and recurrent HGSOC patients.

ITGB1BP1, a Novel Transcriptional Target of CD44-Downstream Signaling Promoting Cancer Cell Invasion.

Breast cancer (BC) is the most common malignancy worldwide and has a poor prognosis, because it begins in the breast and disseminates to lymph nodes and distant organs. While invading, BC cells acquire aggressive characteristics from the tumor microenvironment through several mechanisms. Thus, understanding the mechanisms underlying the process of BC cell invasion can pave the way towards the development of targeted therapeutics focused on metastasis. We have previously reported that the activation of CD44 receptor with its major ligand hyaluronan (HA) promotes BC metastasis to the liverin vivo. Next, a gene expression profiling microarray analysis was conducted to identify and validate CD44-downstream transcriptional targets mediating its pro-metastatic function from RNA samples collected from Tet CD44-inducedversuscontrol MCF7-B5 cells. We have already validated a number of novel CD44-target genes and published their underlying signaling pathways in promoting BC cell invasion. From the same microarray analysis, Integrin subunit beta 1 binding protein 1 (ITGB1BP1) was also identified as a potential CD44-target gene that was upregulated (2-fold) upon HA activation of CD44. This report will review the lines of evidence collected from the literature to support our hypothesis, and further discuss the possible mechanisms linking HA activation of CD44 to its novel potential transcriptional target ITGB1BP1.

CD44 Expression in Renal Tissue Is Associated with an Increase in Urinary Levels of Complement Components in Chronic Glomerulopathies.

It is suggested that activated CD44+ cells play a profibrogenic role in the pathogenesis of active glomerulopathies. Complement activation is also involved in renal fibrogenesis. The aim of the study was to evaluate the role of the activation of CD44+ cells in the kidney tissue and complement components' filtration to the urine as factors of renal tissue fibrosis in patients with glomerulopathies. In total, 60 patients with active glomerulopathies were included in our study: 29 patients with focal segmental glomerulosclerosis (FSGS), 10 patients with minimal change disease (MCD), 10 patients with membranous nephropathy (MN), and 11 patients with IgA nephropathy. The immunohistochemical peroxidase method was used to study the expression of CD44+ in kidney biopsies. Components of complement were analyzed in urine by the multiple reaction monitoring (MRM) approach using liquid chromatography. Strong CD44 expression was noted predominantly in PEC and mesangial cells (MC) in patients with FSGS, and to a lesser extent, in patients with MN and IgA nephropathy, and it was absent in patients with MCD. Expression of profibrogenic CD44+ in glomeruli correlated with the levels of proteinuria and complement C2, C3, and C9 components, and CFB and CFI in urine. The CD44+ expression scores in the renal interstitium correlated with the level of C3 and C9 components of complement in the urine and the area of tubulo-interstitial fibrosis. The strongest expression of CD44+ was found in the glomeruli (MC, PEC, and podocytes) of patients with FSGS compared with other glomerulopathies. The CD44 expression score in the glomeruli and interstitium is associated with high levels of complement components in the urine and renal fibrosis.

Small molecules targeting the NADH-binding pocket of VDAC enhance T cell cytotoxicity and selectively inhibit cancer cell proliferation.

Adoptive cell therapy involves administration of cytotoxic immune cells. Upon activation of naïve T cells, CD8+ (cytotoxic) T cells increase both mitochondrial metabolism and glycolysis. Upregulated mitochondrial metabolism is essential for the activation of effector T cells. Voltage dependent anion channels (VDAC 1/2/3) regulate the flux of most metabolites entering and exiting mitochondria. VDAC opening increases mitochondrial metabolism. NADH binding to a VDAC pocket, conserved in all isoforms, closes the channel. The small molecules SC01 and SC18 bind to the VDAC-NADH binding pocket. We hypothesize that SC01 and SC18 stabilize VDAC in an open state enhancing T cell oxidative metabolism, survival and cytotoxicity. We used melanoma epitope gp100 reactive mouse splenic CD8+ T cells, HepG2 and SNU-449 human hepatocarcinoma cancer cells, and B16 mouse melanoma cells. We determined T cell activation (CD25 expression), proliferation (Ki67), cytokine secretion (IFNγ, TNFα), cytolytic activity (granzyme-B), and expression of memory markers CD44 and CD62L, by FACS. We assessed mitochondrial membrane potential (ΔΨm) and NADH by confocal microscopy. Cell proliferation and cell death were determined by cell count and propidium iodide uptake, respectively. VDAC isoform distribution in T cells was ∼54%, 24% and 22% for VDAC 1, 2 and 3, respectively. SC18 moderately decreased T cell ΔΨm and NADH. SC01 and SC18 did not affect proliferation or activation of splenic CD8+ T cells exposed to cognate antigen (gp100). By contrast, SC18 inhibited cell proliferation in SNU-449, HepG2, and B16 cells. SC18 also increased the secretion of IFNγ, TNF-α, and granzyme-B. Memory markers determined by CD44 and CD62L co-expression were unchanged. These data suggest a two-sided antitumor property of SC18, that enhances T cell cytotoxicity and selectively inhibits cancer cell proliferation.

SIRT1, a novel transcriptional downstream target of CD44, linking its deacetylase activity to tumor cell invasion/metastasis.

Our tetracycline-off-inducible CD44 expression system previously established in mouse model, revealed that activation of CD44 with its major ligand hyaluronan (HA) promoted breast cancer (BC) metastasis to the liver. To identify the mechanisms that underpin CD44-promoted BC cell invasion, microarray gene expression profiling using RNA samples from (Tet)-Off-regulated expression system of CD44s in MCF7 cells, revealed a set of upregulated genes including, nuclear sirtuin-1 (SIRT1 also known as NAD-dependent deacetylase), an enzyme that requires NAD+ as a cofactor to deacetylate several histones and transcription factors. It stimulates various oncogenic pathways promoting tumorigenesis. This data suggests that SIRT1 is a potential novel transcriptional target of CD44-downstream signaling that promote BC cell invasion/metastasis. This review will discuss the evidence supporting this hypothesis as well as the mechanisms linking SIRT1 to cell proliferation and invasion.

Targeting Cell Adhesion Molecules in IDH Mutant Myeloid Malignances

Neomorphic mutations in the genes encoding isocitrate dehydrogenase (IDH) are common in human cancers including myeloid malignancies. The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by mutant IDH promotes leukemia development by competitively inhibiting α-ketoglutarate (α-KG)-dependent dioxygenases, causing epigenetic dysregulation of histone and DNA methylation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). FDA-approved allosteric inhibitors (ivosidenib and enasidenib targeting mutant IDH1 and IDH2 respectively) are used for treating newly diagnosed and/or relapsed/refractory AML patients with IDH mutations. However, the low response rate and primary resistance to IDHmut inhibitors are major clinical challenges. Moreover, acquired resistance to IDH inhibition can occur through multiple mechanisms, including R-2-HG-restoring second-site IDH mutations and the selection of sub-clonal mutations in RUNX1/CEBPA or RAS-RTK pathway. These findings underscore the importance of developing new and more effective approaches for the treatment of IDH-mutant MDS and AML patients. To identify mutant IDH-mediated alterations and actionable dependencies, we generated isogenic TF-1 myeloid leukemia cell lines containing endogenous, hotspot IDH mutations (IDH1R132H, IDH2R140Q, IDH2R140W, and IDH2R172K) by CRISPR/Cas9-mediated base-editing. By comparative transcriptomic (RNA-seq) and epigenomic (ATAC-seq and ChIP-seq) profiling of base-edited versus parental isogenic leukemia cells, we identified cell adhesion molecules, especially CD44, as the top upregulated genes in IDH-mutant leukemia cells. CD44 expression is significantly upregulated in IDH-mutant AML patient samples relative to normal CD34+ hematopoietic stem/progenitor cells (HSPCs) or other AML samples without IDH mutations. Increased CD44 expression is also associated with overall poorer survival and therapy resistance in independent AML cohorts. Importantly, we found that ectopic CD44 expression promotes cytokine-independent cell growth of TF-1 myeloid leukemia cells, resembling the effect observed upon expression of mutant IDH. Moreover, CD44 depletion led to decreased R-2-HG levels in base-edited IDH-mutant TF-1 leukemia cells, and impaired the propagation of IDH-mutant leukemia cells in vitro and in xenotransplanted recipient mice. These studies establish direct evidence that IDH mutations cause aberrant activation of cell adhesion molecules such as CD44, and CD44 is required for the propagation of IDH-mutant myeloid leukemia cells. Mechanistically, we found that the aberrantly activated CD44 is first cleaved by matrix metalloproteinases MMP2 and MMP9 for proteolytic release of its ectodomain from membrane, followed by γ-secretase-mediated intramembranous cleavage to produce the CD44 intracellular domain (CD44ICD). CD44ICD subsequently activates the pentose phosphate pathway (PPP) for the production of NADPH, a critical cofactor for mutant IDH enzymes to convert α-KG to R-2-HG. Hence, mutant IDH-mediated activation of CD44 functions to rewire intracellular metabolism to facilitate IDHmut-catalyzed R-2-HG production, supporting a model for mutant IDH-induced feed-forward oncogenic programs. CD44 also activates AKT/ERK signaling pathway to promote the proliferation and cytokine-independent growth of IDH-mutant leukemia cells. More importantly, primary and resistant IDH-mutant leukemia cells are sensitive to combination regimens consisting of IDHmut inhibitors and humanized monoclonal antibody specific for CD44 (RG7536) or MMP2/9 inhibitors (SB-3CT) invitro and invivo. Taken together, our findings uncover the cell adhesion molecule-mediated metabolic signaling as a new targetable dependency of IDH-mutant leukemia cells, with therapeutic implications for more effective treatment of primary and resistant IDH-mutant myeloid malignancies.

PCF11, a Novel CD44-Downstream Transcriptional Target, Linking Its 3'-End Polyadenylation Function to Tumor Cell Metastasis.

Breast Cancer (BC) is the most common and the major health issue in women worldwide. Metastasis, a multistep process, is the worst aspect of cancer and tumor cell invasion is the defining step. Tumor cell invasion requires cell adhesion molecules (CAMs), and alterations in CAMs is considered as an initiating event in metastasis. Among CAMs, CD44 is a large family of more than 100 isoform, and its precise function was initially controversial in BC. Therefore, we have previously established a (Tet)-off inducible expression system of CD44 in MCF-7 primary BC cell line, and showed that CD44 promoted BC invasion/metastasis both in vitro and in vivo. A microarray gene expression profiling revealed more than 200 CD44-downstream potential transcriptional target genes, mediating its role in BC cell invasion and metastasis. Among these CD44-target genes, the Pre-mRNA cleavage complex 2 protein (PCF11) was upregulated upon the activation of CD44 by its major ligand hyaluronan (HA); This prompted us to hypothesize PCF11 as a potential novel transcriptional target of CD44-promoted BC cell invasion and metastasis. A large body of evidence from the literature supports our hypothesis that CD44 might regulate PCF11 via MAPK/ERK pathway. This review aims to discuss these findings from the literature that support our hypothesis, and further provide possible mechanisms linking CD44-promoted cell invasion through regulation of its potential target PCF11.

The state of CD44 activation in cancer progression and therapeutic targeting.

CD44, a non-kinase transmembrane glycoprotein, is ubiquitously expressed on various types of cells, especially cancer stem cells (CSCs), and has been implicated in cancer onset and aggressiveness. The major ligand for the CD44, hyaluronan (HA), binds to and interacts with CD44, which in turn triggers downstream signaling cascades, thereby promoting cellular behaviors such as proliferation, motility, invasiveness and chemoresistance. The CD44-HA interaction is cell-specific and strongly affected by the state of CD44 activation. Therefore, the binding of HA to CD44 is essential for the activation of CD44 during which the detailed regulatory mechanism needs to be clarified. Different CD44 activation states distribute in human carcinoma and normal tissue; however, whether CD44 activation is a critical requirement for tumor initiation, progression and notorious CSC properties remains to be clarified. A deeper understanding of the regulation of CD44 activation may facilitate the development of novel targeted drugs in the future. Here, we review the current findings concerning the states of CD44 activation on the cell surface, the underlying regulatory mechanisms of CD44 activation, the known role for CD44 activation in tumor progression and CSC hallmarks, as well as the potential of HA-coated nanoparticle for targeting activated CD44 for cancer therapy.

The effect of hyaluronic acid on nucleus pulposus extracellular matrix production through hypoxia-inducible factor-1α transcriptional activation of CD44 under hypoxia

Intervertebral disc degeneration (IDD) is the leading cause of low-back pain. Implantation of hyaluronic acid (HA) is potentially a therapeutic strategy for IDD, but its pharmacological effects and mechanism under hypoxic conditions remain unclear. In this study, the expression of extracellular matrix genes and proteins were enhanced in nucleus pulposus cells (NPCs) in the presence of HA under hypoxic condition, as shown by real-time reverse transcription-polymerase chain reaction, immunofluorescence staining, and dimethylmethylene blue assays. Moreover, the expression of CD44 was increased in the presence of both HA and hypoxia compared to either alone. Using a bioinformatic database, hypoxia inducible factor-1α (HIF-1α), a key transcription factor in the hypoxic condition, was found to have 4 predicted binding sites on the CD44 promoter. CD44 expression was significantly increased by treatment with cobalt chloride or dimethyloxalylglycine. Over-expression of HIF-1α in NPCs significantly up-regulated the expression of CD44. The binding site of HIF-1α in the CD44 promoter region, was identified by promoter truncation experiments and chromatin immunoprecipitation assays. Taken together, these results indicated that hypoxic conditions positively potentiated the ability of NPCs matrix synthesis in the presence of HA, which correlated with the increasing CD44 expression by HIF-1α transcriptional activation.

KYNU, a novel potential target that underpins CD44-promoted breast tumour cell invasion.

Using a validated tetracycline‐off‐inducible CD44 expression system in mouse model, we have previously demonstrated that the hyaluronan (HA) receptor CD44 promotes breast cancer (BC) metastasis to the liver. To unravel the mechanisms that underpin CD44‐promoted BC cell invasion, RNA samples were isolated from two cell models: (a) a tetracycline (Tet)‐Off‐regulated expression system of the CD44s in MCF‐7 cells and; (b) as a complementary approach, the highly metastatic BC cells, MDA‐MB‐231, were cultured in the presence and absence of 50 µg/mL of HA. Kynureninase (KYNU), identified by Microarray analysis, was up‐regulated by 3‐fold upon induction and activation of CD44 by HA; this finding suggests that KYNU is a potential novel transcriptional target of CD44‐downtstream signalling. KYNU is a pyridoxal phosphate (PLP) dependent enzyme involved in the biosynthesis of NAD cofactors from tryptophan that has been associated with the onset and development of BC. This review will attempt to identify and discuss the findings supporting this hypothesis and the mechanisms linking KYNU cell invasion via CD44.

RUNX2 interacts with BRG1 to target CD44 for promoting invasion and migration of colorectal cancer cells

BackgroundCancer stem cells (CSCs) play an important role in tumor invasion and metastasis. CD44 is the most commonly used marker of CSCs, with the potential to act as a determinant against the invasion and migration of CSCs and as the key factor in epithelial–mesenchymal transition (EMT)-like changes that occur in colorectal cancer (CRC). Runt-related transcription factor-2 (RUNX2) is a mesenchymal stem marker for cancer that is involved in stem cell biology and tumorigenesis. However, whether RUNX2 is involved in CSC and in inducing EMT-like changes in CRC remains uncertain, warranting further investigation.MethodsWe evaluated the role of RUNX2 in the invasion and migration of CRC cells as a promoter of CD44-induced stem cell- and EMT-like modifications. For this purpose, western blotting was employed to analyze the expression of differential proteins in CRC cells. We conducted sphere formation, wound healing, and transwell assays to investigate the biological functions of RUNX2 in CRC cells. Cellular immunofluorescence and coimmunoprecipitation (co-IP) assays were performed to study the relationship between RUNX2 and BRG1. Real-time quantitative PCR (RT-qPCR) and immunohistochemistry (IHC) were performed to analyze the expressions of RUNX2, BRG1, and CD44 in the CRC tissues.ResultsWe found that RUNX2 could markedly induce the CRC cell sphere-forming ability and EMT. Interestingly, the RUNX2-mediated EMT in CRC cell may be associated with the activation of CD44. Furthermore, RUNX2 was found to interact with BRG1 to promote the recruitment of RUNX2 to the CD44 promoter.ConclusionsOur cumulative findings suggest that RUNX2 and BRG1 can form a compact complex to regulate the transcription and expression of CD44, which has possible involvement in the invasion and migration of CRC cells.

Inhibition of Y Box Binding Protein 1 Suppresses Cell Growth and Motility in Colorectal Cancer.

Although chemo- or radiotherapy is usually performed in patients with colorectal cancer, the response is highly variable in locally rectal cancer. Therefore, additional studies are needed on predictable markers and the molecular mechanisms of chemo- and radiotherapy. Y box binding protein 1 (YB1) is an oncoprotein that is aberrantly expressed in many cancers, including colorectal cancer. However, to date there are no targeting agents or strategies to inhibit YB1 expression. Here, we investigate the oncogenic function of YB1 in colorectal cancer and methods to control its expression. We observed that YB1 expression level is correlated with colorectal cancer survival rate. Moreover, YB1 overexpression was associated with colorectal cancer lymph node metastasis and invasion. We also found that radiation exposure increased YB1 expression, which led to radioresistant colorectal cancer, mediated through the activation of cancer stem cell marker CD44 and PI3K/AKT/mTOR signaling. This study revealed, by both in vitro and in vivo assays, that depletion of YB1 could reduce cell proliferation and motility in colorectal cancer. We further demonstrated that the PI3K/mTOR inhibitor BEZ235 suppressed YB1 expression and enhanced the cytotoxicity of radiation. In addition, combined treatment with BEZ235 and radiation showed a significant antitumor response in an in vivo mouse xenograft model. Taken together, our results provide evidence that the activation of YB1 is a major factor in radioresistance and suggest that targeting YB1-mediated signaling is a promising therapeutic strategy for colorectal cancer.

NNMT Involvement in CD44v3 Regulation Induced by Hepatic Stellate Cells Contributes to the Metastasis of Hepatocarcinoma Cells

The crosstalk between hepatic stellate cells (HSCs) and hepatic carcinoma cells contributes to hepatocellular carcinoma (HCC) progression, although the underlying mechanism is largely unknown. We found that activated HSCs upregulated nicotinamide (NAM) N-methyltransferase (NNMT) expression in hepatoma cells. NNMT regulates multiple metabolic pathways in the liver, and aberrantly high levels of NNMT in the HCC tissues were positively correlated with vascular invasion, serum HBV levels and distant metastasis in our study. Functional assays showed that NNMT promoted cell migration, invasion and metastasis in vitro and in vivo. Mechanistically, NNMT regulated the intracellular methylation potential which in turn altered histone H3K27 methylation pattern, resulting in the transcriptional activation of CD44. NNMT-mediated m6A modification of the CD44 mRNA resulted in the formation of the CD44v3 splice variant, while its product 1-MNA stabilized CD44 protein by preventing ubiquitin-mediated degradation. Finally, NNMT was also a target of statins which inhibited the metastatic ability of hepatoma cells. Taken together, our study provides evidence for the first time on the potential role of the NNMT/CD44v3 axis in regulating HCC metastasis, and presents NNMT as a promising prognostic biomarker and therapeutic target for HCC. Funding Statement: This study was supported by the National Natural Science Foundation of China (No.81672418 and 81871961, to Zhenyu Yin. No. 81802344,to Jie Li. No. 81572335,to Wenxiu Zhao) and the Xiamen Science and Technology Project of People-benefit (No. 3502Z20174089, to J.L.). Declaration of Interests: All of the authors have declared that no competing interests exist. Ethics Approval Statement: Specimen collection was performed after obtaining informed consent from each patient, and the utilization of the tumor materials for research purposes was carried out in accordance with the approved guidelines and approved by the Ethics Committee (No: 20111008) of Zhong Shan Hospital Xiamen University

Blockade of the AT1 receptor and silencing of CD44 gene expression are associated with inhibition of angiotensin II‐activated cardiac fibroblasts

ObjectiveWe have previously demonstrated that the stimulation of fibroblasts by angiotensin II (Ang II) contributes to cardiac fibrosis via transforming growth factor beta (TGF‐β1)‐mediated collagen production. The cellular/molecular mechanism responsible for fibroblast activation, however, is not well understood. CD44, a cell‐surface glycoprotein, is associated with cell‐cell interactions, cell adhesion and migration. This study examines the roles of Ang II AT1 receptor and CD44 gene expression in the activation of cardiac fibroblasts.MethodsCardiac fibroblasts were isolated from the neonatal rat hearts, the assays of cell viability and migration were selected to demonstrate the activation of fibroblasts, and silencing of CD44 gene expression on fibroblasts was conducted with small interfering RNA (siRNA).ResultsImmunofluorescence staining with vimentin and discoid in domain receptor‐2 revealed the purity of isolated fibroblasts. Ang □ significantly reduced the cell viability and increased the cell migration in a dose‐dependent manner with a maximal stimulation as seen at 50 nM. In coincidence with fibroblast activation, mRNA and protein level of CD44 were up‐regulated and the protein levels of TGF‐β1, Smad2/4 and collagen I were significantly increased (all p&lt;0.05 vs. unstimulated cells). Addition of Ang II AT1 receptor blocker, telmisartan into the cells abolished Ang II‐elicited stimulating effects on these parameters measured (all p&lt;0.05 vs. Ang II activated cells). Consistent with data showing that there is a linear correlation between CD44 and TGF‐β1 as demonstrated by Pearson correlation analysis (r=0.955, p&lt;0.01), gene transfection of fibroblasts with Ad‐CD44 siRNA as identified by mRNA and protein levels of CD44 demonstrated a significant attenuation in the production of collagen I, suggesting a role of CD44 in the activation of fibroblasts.ConclusionsThese results indicate that cardiac fibroblasts, in response to Ang II stimulation, express high levels of the AT1 receptor and CD44, which reduce the cell viability and facilitate cell migration. Such a signaling mechanism could be crucial for leading to the deposition of collagen and the production of cardiac fibrosis.Support or Funding InformationThis study was supported in part by grants from the Mercer University School of Medicine, the Medcen Community Health Foundation, Georgia and the National Natural Science Foundation of China (81470436).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Truncated Glioma-Associated Oncogene Homolog 1 (tGLI1) Mediates Mesenchymal Glioblastoma via Transcriptional Activation of CD44.

The molecular pathways driving mesenchymal glioblastoma (GBM) are still not well understood. We report here that truncated glioma-associated oncogene homolog 1 (tGLI1) is a tumor-specific transcription factor that facilitates GBM growth, is enriched in the mesenchymal subtype of GBM and glioma stem cells (GSC), and promotes mesenchymal GSC by upregulating transcription of CD44. In an orthotopic GBM xenograft mouse model, tGLI1-overexpressing tumors grew more aggressively with increased proliferation and angiogenesis compared with control and GLI1-overexpressing xenografts. tGLI1 was highly expressed in GBM clinical specimens but undetectable in normal brains, whereas GLI1 was expressed in both tissues. A tGLI1 activation signature (tGAS) correlated with glioma grade, tumor angiogenesis, and poor overall survival, and GBMs with high tGAS were enriched with mesenchymal GBM/GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared with the monolayer culture; mesenchymal GSC expressed more tGLI1 than proneural GSC. Ectopic tGLI1 expression enhanced the ability of mesenchymal GSC to yield neurospheres in vitro and to form tumors in mouse brains. Selective tGLI1 knockdown reduced neurosphere formation of GBM cells. tGLI1 bound to and transactivated the promoter of the CD44 gene, a marker and mediator for mesenchymal GSC, leading to its expression. Collectively, these findings advance our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSC.Significance: These findings highlight the role of a tumor-specific gain-of-function transcription factor tGLI1 in mesenchymal glioma stem cell maintenance and mesenchymal GBM growth. Cancer Res; 78(10); 2589-600. ©2018 AACR.

CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia.

We studied, in male Sprague Dawley rats, the role of the cognate hyaluronan receptor, CD44 signaling in the antihyperalgesia induced by high molecular weight hyaluronan (HMWH). Low molecular weight hyaluronan (LMWH) acts at both peptidergic and nonpeptidergic nociceptors to induce mechanical hyperalgesia that is prevented by intrathecal oligodeoxynucleotide antisense to CD44 mRNA, which also prevents hyperalgesia induced by a CD44 receptor agonist, A6. Ongoing LMWH and A6 hyperalgesia are reversed by HMWH. HMWH also reverses the hyperalgesia induced by diverse pronociceptive mediators, prostaglandin E2, epinephrine, TNFα, and interleukin-6, and the neuropathic pain induced by the cancer chemotherapy paclitaxel. Although CD44 antisense has no effect on the hyperalgesia induced by inflammatory mediators or paclitaxel, it eliminates the antihyperalgesic effect of HMWH. HMWH also reverses the hyperalgesia induced by activation of intracellular second messengers, PKA and PKCε, indicating that HMWH-induced antihyperalgesia, although dependent on CD44, is mediated by an intracellular signaling pathway rather than as a competitive receptor antagonist. Sensitization of cultured small-diameter DRG neurons by prostaglandin E2 is also prevented and reversed by HMWH. These results demonstrate the central role of CD44 signaling in HMWH-induced antihyperalgesia, and establish it as a therapeutic target against inflammatory and neuropathic pain.SIGNIFICANCE STATEMENT We demonstrate that hyaluronan (HA) with different molecular weights produces opposing nociceptive effects. While low molecular weight HA increases sensitivity to mechanical stimulation, high molecular weight HA reduces sensitization, attenuating inflammatory and neuropathic hyperalgesia. Both pronociceptive and antinociceptive effects of HA are mediated by activation of signaling pathways downstream CD44, the cognate HA receptor, in nociceptors. These results contribute to our understanding of the role of the extracellular matrix in pain, and indicate CD44 as a potential therapeutic target to alleviate inflammatory and neuropathic pain.

CD146, a novel target of CD44-signaling, suppresses breast tumor cell invasion

BackgroundWe have previously validated three novel CD44-downstream positively regulated transcriptional targets, including Cortactin, Survivin and TGF-β2, and further characterized the players underlying their separate signaling pathways. In the present study, we identified CD146 as a potential novel target, negatively regulated by CD44. While the exact function of CD146 in breast cancer (BC) is not completely understood, substantial evidence from our work and others support the hypothesis that CD146 is a suppressor of breast tumor progression.MethodsTherefore, using molecular and pharmacological approaches both in vitro and in breast tissues of human samples, the present study validated CD146 as a novel target of CD44-signaling suppressed during BC progression.ResultsOur results revealed that CD44 activation could cause a substantial decrease of CD146 expression with an equally notable converse effect upon CD44-siRNA inhibition. More interestingly, activation of CD44 decreased cellular CD146 and increased soluble CD146 through CD44-dependent activation of MMP.ConclusionHere, we provide a possible mechanism by which CD146 suppresses BC progression as a target of CD44-downstream signaling, regulating neovascularization and cancer cell motility.