Beta-galactoside Binding Research Articles

Insights into the Role of Galectin-3 as a Diagnostic and Prognostic Biomarker of Atrial Fibrillation.

Atrial fibrillation (AF) is an irregular atrial activity and the most prevalent type of arrhythmia. Although AF is easily diagnosed with an electrocardiogram, there is a keen interest in identifying an easy-to-dose biomarker that can predict the prognosis of AF and its recurrence. Galectin-3 (Gal-3) is a beta-galactoside binding protein from the lectin family with pro-fibrotic and -inflammatory effects and a pivotal role in a variety of biological processes, cell proliferation, and differentiation; therefore, it is implicated in the pathogenesis of many cardiovascular (e.g., heart failure (HF)) and noncardiovascular diseases. However, its specificity and sensitivity as a potential marker in AF patients remain debated and controversial. This article comprehensively reviewed the evidence regarding the interplay between Gal-3 and patients with AF. Clinical implications of measuring Gal-3 in AF patients for diagnosis and prognosis are mentioned. Moreover, the role of Gal-3 as a potential biomarker for the management of AF recurrence is investigated. The association of Gal-3 and AF in special populations (coronary artery disease, HF, metabolic syndrome, chronic kidney disease, and diabetes mellitus) has been explored in this review. Overall, although further studies are needed to enlighten the role of Gal-3 in the diagnosis and treatment of AF, our study demonstrated the high potential of this molecule to be used and focused on by researchers and clinicians.

Inhibition of LPS-Induced Inflammatory Response of Oral Mesenchymal Stem Cells in the Presence of Galectin-3

Galectin-3 (GAL-3) is a beta-galactoside binding lectin produced by mesenchymal stem cells (MSCs) and other cell sources under inflammatory conditions. Several studies have reported that GAL-3 exerts an anti-inflammatory action, regulated by its natural ligand GAL-3 BP. In the present study, we aimed to assess the GAL-3 mediated regulation of the MSC function in an LPS-induced inflammation setting. Human gingival mesenchymal stem cells (hGMSCs) were stimulated in vitro with LPSs; the expression of TLR4, NFκB p65, MyD88 and NALP3 were assessed in the hGMSCs via immunofluorescence imaging using confocal microscopy, Western blot assay, and RT-PCR before and after the addition of GAL-3, both alone and with the addition of its inhibitors. LPSs stimulated the expression of TLR4, NFκB p65, MyD88 and NALP3 in hGMSCs, which was inhibited by GAL-3. The addition of either GAL3-BP or the antibody to GAL-3 were able to revert the GAL-3-mediated effects, restoring the expression of TLR4, NFκB p65, MyD88 and NALP3. GAL-3 induces the downregulation of the LPS-induced inflammatory program in MSCs.

The potential roles of galectin-3 in AKI and CKD.

Acute kidney injury (AKI) is a common condition with high morbidity and mortality, and is associated with the development and progression of chronic kidney disease (CKD). The beta-galactoside binding protein galectin-3 (Gal3), with its proinflammatory and profibrotic properties, has been implicated in the development of both AKI and CKD. Serum Gal3 levels are elevated in patients with AKI and CKD, and elevated Gal3 is associated with progression of CKD. In addition, Gal3 is associated with the incidence of AKI among critically ill patients, and blocking Gal3 in murine models of sepsis and ischemia-reperfusion injury results in significantly lower AKI incidence and mortality. Here we review the role of Gal3 in the pathophysiology of AKI and CKD, as well as the therapeutic potential of targeting Gal3.

Targeting the pentraxin 3/CD44 axis: A potential treatment for pulmonary fibrosis

Targeting the pentraxin 3/CD44 axis: A potential treatment for pulmonary fibrosis

Early Doxorubicin Myocardial Injury: Inflammatory, Oxidative Stress, and Apoptotic Role of Galectin-3.

Doxorubicin (DOXO) is an effective drug that is used in the treatment of a large number of cancers. Regardless of its important chemotherapeutic characteristics, its usage is restricted because of its serious side effects; the most obvious is cardiotoxicity, which can manifest acutely or years after completion of treatment, leading to left ventricular dysfunction, dilated cardiomyopathy, and heart failure. Galectin 3 (Gal-3) is a beta galactoside binding lectin that has different roles in normal and pathophysiological conditions. Gal-3 was found to be upregulated in animal models, correlating with heart failure, atherosclerosis, and myocardial infarction. Male C57B6/J and B6.Cg-Lgals3 <tm 1 Poi>/J Gal-3 knockout (KO) mice were used for a mouse model of acute DOXO-induced cardiotoxicity. Mice were given DOXO or vehicle (normal saline), after which the mice again had free access to food and water. Heart and plasma samples were collected 5 days after DOXO administration and were used for tissue processing, staining, electron microscopy, and enzyme-linked immunosorbent assay (ELISA). There was a significant increase in the heart concentration of Gal-3 in Gal-3 wild type DOXO-treated mice when compared with the sham control. There were significantly higher concentrations of heart cleaved caspase-3, plasma troponin I, plasma lactate dehydrogenase, and plasma creatine kinase in Gal-3 KO DOXO-treated mice than in Gal-3 wild type DOXO-treated mice. Moreover, there were significantly higher heart antioxidant proteins and lower oxidative stress in Gal-3 wild type DOXO-treated mice than in Gal-3 KO DOXO-treated mice. In conclusion, Gal-3 can affect the redox pathways and regulate cell survival and death of the myocardium following acute DOXO injury.

Galectin-9 protects humanized-ACE2 immunocompetent mice from SARS-CoV-2 infection.

SARS-CoV-2 remains a global health crisis even with effective vaccines and the availability of FDA approved therapies. Efforts to understand the complex disease pathology and develop effective strategies to limit mortality and morbidity are needed. Recent studies reveal circulating Galectin-9 (gal-9), a soluble beta-galactoside binding lectin with immunoregulatory properties, are elevated in SARS-CoV-2 infected individuals with moderate to severe disease. Moreover, in silico studies demonstrate gal-9 can potentially competitively bind the ACE2 receptor on susceptible host cells. Here, we determined whether early introduction of exogenous gal-9 following SARS-CoV-2 infection in humanized ACE2 transgenic mice (K18-hACE2) may reduce disease severity. Mice were infected and treated with a single dose of a human recombinant form of gal-9 (rh-gal-9) and monitored for morbidity. Subgroups of mice were humanely euthanized at 2- and 5- days post infection (dpi) for viral levels by plaque assay, immune changes measures by flow cytometry, and soluble mediators by protein analysis from lung tissue and bronchoalveolar Lavage fluid (BALF). Mice treated with rh-gal-9 during acute infection had improved survival compared to PBS treated controls. At 5 dpi, rh-gal-9 treated mice had enhanced viral clearance in the BALF, but not in the lung parenchyma. Increased T and dendritic cells and decreased neutrophil frequencies in the lung at 5 dpi were observed, whereas BALF had elevated levels of type-I interferons and proinflammatory cytokines. These results suggest a role for rh-gal-9 in limiting acute COVID-19. Further studies are required to determine the optimal design of gal-9 treatment to effectively ameliorate COVID-19 disease.

Galectin-3 Mitigates Cardiomyocytes Injury through Modulation of Left Ventricular Cathepsins B, D, L and S at 24-Hour Post Myocardial Infarction.

Galectin 3 (GAL-3) is a beta galactoside binding lectin that has different roles in normal and pathophysiological conditions. GAL-3 was found to be up regulated in animal models of myocardial infarction (MI). Cathepsins are intracellular lysosomal proteases that degrade proteins. The objective of his study is to investigate if high GAL-3 after myocardial infarction has a protective role on the heart through its modulation of lysosomal Cathepsins in ischemic myocardium. Male C57B6/J mice and GAL-3 knockout (KO) mice were used for permanent ligation of the left anterior descending artery of the heart to create infarction in the anterior myocardium. Hearts and plasma samples were collected 24 hours after the induction of MI and were used for enzyme linked immunosorbent assay and immunofluorescent staining. Our results show that the significant increase in GAL-3 levels in the left ventricle at 24-hour following MI is associated with significant lower levels of cathepsins B, D, L and S in GAL-3 wild MI group than GAL-3 KO MI group. We also report a significant lower plasma level of Troponin I in GAL-3 wild MI group than GAL-3 KO MI group. The increased levels of GAL-3 at 24-hour following MI regulates the process of cardiomyocytes injury through modulation of lysosomal cathepsins B, D, L and S.

Galectin-3 Possesses Anti-Necroptotic and Anti-Apoptotic Effects in Cisplatin-Induced Acute Tubular Necrosis.

Acute kidney injury (AKI) is a public health burden with increasing morbidity, mortality and health care cost. It is associated with increased risk for the development of chronic kidney disease and death. Acute tubular necrosis (ATN) is the most common cause of AKI. Apoptosis and tissue necrosis play an important role in ATN. Galectin 3 (GAL-3), a beta galactoside binding lectin, is known to have a role in inflammation, apoptosis and oxidative stress but its role in cisplatin induced acute tubular necrosis is not clearly elucidated. Male C57B6-J and C57BL-6 -GAL-3 knock-out mice were used to induce ATN using cisplatin mouse model of acute tubular necrosis. GAL-3 expression, apoptotic, necrotic and necroptotic proteins in kidneys were measured using standard histologic, immunohistochemical, and enzyme-linked immunosorbent assay techniques. Data were presented as mean ± S.E. Statistically significant differences (p<0.05) was calculated between experimental groups and corresponding control groups by one-way analysis of variance. There was a significant increase in GAL-3 in kidneys of cisplatin treated GAL-3 wild mice when compared with its control mice. In addition, there were significant higher percentage of ATN, higher levels of plasma urea and creatinine, and higher levels of cathepsin B and cathepsin D, in kidneys of cisplatin-treated GAL-3 KO mice than cisplatin-treated GAL-3 wild mice. Likewise, there were significant higher levels of necroptosis proteins RIPK1, RIPK3, and MLKL in kidneys of cisplatin-treated GAL-3 KO mice than cisplatin-treated GAL-3 wild mice. Moreover, there were significant higher levels of kidney pro-apoptotic proteins; cleaved caspase-3, cleaved PARP, TRAIL and FAS in cisplatin treated GAL-3 KO mice when compared with cisplatin treated GAL-3 wild mice. GAL-3 can affect cell survival and death through its interaction with necroptotic, apoptotic and pro-survival proteins in renal tubules during cisplatin-induced acute tubular necrosis.

Histomorphological study of malignant thyroid neoplasm in a tertiary care center and evaluation of Galectin 3 expression in papillary thyroid carcinoma

Galectin-3 is a beta-galactoside binding animal lectin, which is frequently associated with tumour progression and metastasis. In recent years, overexpression of Galectin-3 has been reported in various human cancers and more frequently in thyroid neoplasms. The aim of this study was to analyze the histomorphological characteristics of malignant thyroid neoplasms, subtype them according to the established classification system and to evaluate the expression of Galectin-3 immunostaining in papillary thyroid carcinoma. A total of 30 cases were included in the study, out of which 28 cases were papillary thyroid carcinoma and its variants and one case of medullary and anaplastic carcinoma. Majority of the papillary thyroid carcinoma cases were positive for Galectin 3 immunostaining (25/28 cases – 89%) in our study. We conclude that galectin-3 is consistently expressed in papillary carcinoma thyroid; however, there are few false-negative cases in this study and also other studies have reported Galectin 3 overexpression in non-papillary tumors. Hence, we cannot depend on Galectin 3 expression alone as a single diagnostic tool to detect papillary thyroid carcinoma.

Galectin-3: A Cardiomyocyte Antiapoptotic Mediator at 24-Hour Post Myocardial Infarction.

Galectin 3 (GAL-3) is a beta galactoside binding lectin that has different roles in normal and pathophysiological conditions. GAL-3 has been associated with heart failure and was linked to increased risk of death in a number of studies. GAL-3 was found to be up regulated in animal models of heart failure as well as myocardial infarction (MI). The objective of his study is to test if high GAL-3 after myocardial infarction has a protective role on the heart through its anti-apoptotic and anti-necrotic functions. Male C57B6/J mice and GAL-3 knockout (KO) mice were used for permanent ligation of the left anterior descending artery of the heart to create infarction in the anterior myocardium. Heart and plasma samples were collected 24 hours after the induction of MI and were used for immunohistochemistry, Tunnel procedure, electron microscopy and enzyme linked immunosorbent assay (ELISA). Our results show that the significant increase in GAL-3 levels in the left ventricle at 24-hour following MI is associated with significant lower levels of pro-apoptotic proteins; cytochrome c, Bax, annexin V, cleaved caspase-3 and a higher levels of anti-apoptotic protein Bcl2 in GAL-3 wild MI group than GAL-3 KO group. We also have identified the anti-apoptotic activity of GAL-3 is mediated through a significant increase in Akt-1, NF kappa-B and beta- catenin proteins. In addition, we have identified the antiapoptotic activity is mediated through a significant lower levels of cathepsin-D protein. We conclude that the increased levels of GAL-3 at 24-hour following MI regulate antiapoptotic mechanisms in the myocardium that will shape the future course of the disease. We also identified that the anti-apoptotic mechanisms are likely mediated through interaction of GAL-3 with Akt-1, NF kappa-B, beta- catenin and cathepsin D proteins.

EVALUATION OF GALECTIN-3 LEVELS IN OBESE ADOLESCENTS

Background: Galectin-3 is a member of the family of soluble beta-galactoside binding lectins, which is involved in inflammation, cell differentiation, adhesion, proliferation, angiogenesis and cancer cell invasion. Increased galectin-3 levels in obese patients have been shown in previous studies. In our study; we aimed to investigate the galectin-3 levels in obese adolescents for the first time in the literature.&#x0D; Materials and methods: A total of 85 adolescent patients who were admitted to our pediatrics outpatient clinic between December 19th of 2018 and January 31st of 2019 were included in our study. Among those patients 52 were obese and 33 were normal-weighted. Adolescents having body mass index (BMI) 95% percentile and above according to their age and sex were defined as obese. Enzyme-Linked Immunosorbent Assay kit was used for the measurement of galectin-3 in serum. Laboratory tests (Galectin-3, HbA1c, glucose, insulin, HOMA-IR), sex, age and BMI were statistically compared between groups.&#x0D; Results: We found no statistically significant difference in terms of gender distribution rates, age, glucose or galectin-3 levels among obese adolescent and healthy control groups (p&gt; 0.05). The BMI, HbA1c, HOMA-IR and insulin levels of the obese adolescent group were found to be significantly higher than the healthy control group (p &lt;0.05). We found no statistically significant correlation between galectin-3 levels and other parameters in obese adolescent group (p&gt; 0.05).&#x0D; Conclusion: In our study, we did not find any relationship between serum galectin-3 levels and obesity in adolescents. We believe that the effects of galectin-3 levels on obesity in adolescents should be more clearly explained by further investigations, which also measures other biomarkers of the mechanism of action.&#x0D; Keywords: Galectin-3, obesity, adolescent&#x0D; List of abbreviations: BMI: Body-Mass Index, CV: Variation coefficient, ELISA: Enzyme linked immunoassay, HOMA-IR: Homeostatic model of assessment-insulin resistance, IL: Interleukin, r: Correlation coefficient, SD: Standard deviation.

Galectins as regulators of cell survival in the leukemia niche.

The microenvironment within the bone marrow (BM) contains support cells that promote leukemia cell survival and suppress host anti-tumor defenses. Galectins are a family of beta-galactoside binding proteins that are critical components in the tumor microenvironment. Galectin 1 (LGALS1) and Galectin 3 (LGALS3) as regulators of RAS signaling intracellularly and as inhibitors of immune cells extracellularly are perhaps the best studied members for their role in leukemia biology. Interest in Galectin 9 (LGALS9) is growing as this galectin has been identified as an immune checkpoint molecule. LGALS9 also supports leukemia stem cells (LSCs) though a mechanism of action is not clear. LGALS1 and LGALS3 each participate in a diverse number of survival pathways that promote drug resistance by supporting pro-tumor molecules such BCL2, MCL-1, and MYC and blocking tumor suppressors like p53. Acute myeloid leukemia (AML) BM mesenchymal stromal cells (MSC) have protein signatures that differ from healthy donor MSC. Elevated LGALS3 protein in AML MSC is associated with refractory disease/relapse demonstrating that MSC derived galectin impacts patient survival. LGALS3 is a critical determining factor whether MSC differentiate into adipocytes or osteoblasts so the galectin influences the cellular composition of the leukemia niche. Both LGALS3 and LGALS1 when secreted can suppress immune function. Both galectins can induce apoptosis of T cells. LGALS3 also modulates T cell receptor endocytosis and impairs interferon mediated chemokine production by binding glycosylated interferon. LGALS3 as a TIM3 binding partner acts to suppress T cell function. Galectins also impact leukemia cell mobilization and may participate in homing mechanisms. LGALS3 participates in transport mechanism of integrins, receptors, and other molecules that control cell adhesion and cell:cell interactions. The diversity of these various functions demonstrate the importance of these galectins in the leukemia niche. This review will cover the role of LGALS1, LGALS3, and LGALS9 in the various processes that are critical for maintaining leukemia cells in the tumor microenvironment.

Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

Epithelial cells require a precise intracellular transport and sorting machinery to establish and maintain their polarized architecture. This machinery includes β-galactoside-binding galectins for targeting of glycoprotein to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analysed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, knockout of galectin-3 results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells.

Myocardial Ischemia Reperfusion Injury: Apoptotic, Inflammatory and Oxidative Stress Role of Galectin-3

Background/Aims: Myocardial reperfusion has the potential to salvage the ischemic myocardium after a period of coronary occlusion. Reperfusion, however, can cause a wide spectrum of deleterious effects. Galectin-3 (GAL-3), a beta galactoside binding lectin, is closely associated with myocardial infarction (MI), myocardial fibrosis and heart failure. In our study, we investigated its role in ischemia-reperfusion injuries (IR) as this phenomenon is extremely relevant to the early intervention after acute MI. Methods: C57B6/J wild type (WT) mice and GAL-3 knockout (KO) mice were used for murine model of IR injury in the heart where a period of 30 minutes ischemia was followed by 24 hours of reperfusion. Heart samples were processed for immunohistochemical and immunofluorescent labeling, morphometric analysis, western blot and enzyme-linked immunosorbent assay to identify the apoptotic, inflammatory and oxidative stress role of GAL-3. Results: Our results show that there was a significant increase in GAL-3 levels in the heart which shows GAL-3 is playing a role in the ischemia reperfusion injury. Troponin I was also significantly higher in GAL-3-KO group than wild type. Our study shows that GAL-3 is associated with an increase in the antioxidant activity in the IR injured myocardium. Antioxidant enzymes superoxide dismutase, glutathione and catalase were found to be significantly raised in the GAL-3 wild type IR as compared to the GAL-3 KO IR group. A significant increase in apoptotic activity is seen in GAL-3 KO IR group as compared with GAL-3 wild IR group. Conclusion: Our study shows that GAL-3 can affect the redox pathways, controlling cell survival and death, and plays a protective role on the myocardium following IR injury.

Abstract MP007: Higher Plasma Galectin-3 is Associated With Increased Incidence of Atrial Fibrillation: The Atherosclerosis Risk in Communities (ARIC) Study

Background: Atrial fibrillation (AF) is a common supraventricular tachyarrhythmia associated with increased risk of cardiovascular sequelae, including heart failure (HF), stroke, and premature mortality. Galectin-3, a beta-galactoside binding lectin involved in important regulatory roles in adhesion, inflammation, immunity, and fibrosis, may be relevant to AF etiology. We tested the hypothesis that plasma galectin-3 concentration would be associated positively with the incidence of AF independent of traditional AF risk factors and incident HF and coronary heart disease (CHD) in a large community-based cohort in the US. Methods: Our analysis included 8,777 participants free of AF at visit 4 (1996-98) and with measures of plasma galectin-3 from the biracial Atherosclerosis Risk in Communities (ARIC) study. Incident AF was ascertained through the end of 2013 from study visit ECGs, hospitalizations and death certificates. Multivariable Cox proportional hazards models, adjusted for AF risk factors (Model 1) plus incident HF and CHD (Model 2), were used to estimate hazards ratios for the association between galectin-3 and incident AF. Results: The mean age (SD) of participants was 62.5 (5.6) years; 58.7% were women and 21.5% blacks. During a median follow-up of 15.7 years, 1,233 incident cases of AF were observed. After adjusting for AF risk factors, participants with galectin-3 levels greater than 19.5 ng/ml had a significantly higher risk of incident AF when compared to the referent group (4.4 - 11.9ng/ml), with hazard ratios of 1.44 for the 90 th - 94.9 th percentile, and 1.61 for the 95 th - 100 th percentile (p-trend = 0.0003). The association between galectin-3 concentration and incident AF was attenuated somewhat after accounting for incident CHD and HF as time dependent variables (p-trend=0.03) (Table 1). Conclusion: Elevated plasma galectin-3 is associated independently with increased risk of incident AF.

Galectin-9 Controls the Activity of DR3 (TNFRSF25) in Suppressing Immunity

Abstract TNFR superfamily members are cell surface glycoprotein receptors that are either induced and/or constitutively expressed on various immune cell types. Previously we have shown that the membrane proximal extracellular region of the TNFR superfamily member 4-1BB, interacts with Galectin-9, and in turn facilitates oligomerization and/or clustering of 4-1BB molecules on the cell surface in a manner that allows efficient signaling. Galectin-9 is a member of the beta-galactoside binding lectin family of proteins that contain homologous carbohydrate recognition domains and typically bind to specific glycans of a glycoprotein. Consequently, a search for other TNFR superfamily glycoproteins that might be regulated by Galectin-9 revealed an interaction with the extracellular region of DR3 (TNFRSF25). DR3 is expressed on conventional CD4+ T cells and CD4+ Foxp3+ Treg cells and acts as a stimulatory receptor when ligated. Functional studies in vitro showed that the stimulatory activity of DR3 was in part dependent on Galectin-9 in that Galectin-9−/− CD4 T cells were less responsive when DR3 was ligated. Furthermore, in vivo, where an agonist of DR3 can transiently induce Treg cell expansion in wild-type mice, we found that this effect of anti-DR3 was significantly lost in Galectin-9−/− mice. Additionally, in EAE as well as Asthma models, where an agonist of DR3 can suppress disease via control of CD4+ Foxp3+ Treg cells, we found that this protective effect of anti-DR3 was lost in Galectin-9−/− mice. Thus, our data demonstrate a novel function of Galectin-9 in facilitating signaling through DR3, and suggest that Galectin-9 may allow DR3 to oligomerize and/or cluster in a manner that allows efficient engagement of intracellular signaling pathways.

Galectin-1-Induced Autophagy Facilitates Cisplatin Resistance of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers in Taiwan. Although chemotherapy is the primary treatment for HCC patients, drug resistance often leads to clinical failure. Galectin-1 is a beta-galactoside binding lectin which is up-regulated in HCC patients and promotes tumor growth by mediating cancer cell adhesion, migration and proliferation, but its role in chemoresistance of HCC is poorly understood. In this study we found that galectin-1 is able to lead to chemoresistance against cisplatin treatment, and subsequent inhibition has reversed the effect of cell death in HCC cells. Moreover, galectin-1 was found to induce autophagic flux in HCC cells. Inhibition of autophagy by inhibitors or knockdown of Atg5 cancels galectin-1-induced cisplatin resistance in HCC cells. Increase of mitophagy triggered by galectin-1 was found to reduce the mitochondrial potential loss and apoptosis induced by cisplatin treatment. Finally, using an in situ hepatoma mouse model, we clearly demonstrated that inhibition of galectin-1 by thiodigalactoside could significantly augment the anti-HCC effect of cisplatin. Taken together, our findings offer a new insight into the chemoresistance galectin-1 causes against cisplatin treatment, and points to a potential approach to improve the efficacy of cisplatin in the treatment of HCC patients.

Role of Mesenchymal Stem Cell Galectin 3 in the AML Tumor Microenvironment

Background: Mesenchymal stem cells (MSC) protect acute myeloid leukemia (AML) cells from chemotherapy. Galectin 3 (LGALS3) is a beta-galactoside binding protein that regulates cell adhesion, apoptosis, cell cycle, and mRNA processing. When secreted, LGALS3 can modulate receptor kinase activity, influence cell adhesion, and can suppress immune cells. MSC are a rich source of LGALS3. In the present study we address the role of MSC derived LGALS3 in the leukemia microenvironment.Methods: The expression of LGALS3 and 150 other proteins was examined by reverse phase protein array analysis (RPPA) in AML (N = 106) and healthy donor (N = 71) MSC. To examine the putative role of MSC-derived proteins in therapy resistance, RPPA was used to determine levels of the 151 proteins in MSC obtained at diagnosis and from relapsed/refractory patients relapse. In vitro studies were performed to examine the effects of hypoxia or co-culture with AML cell lines on LGALS3 expression by immunofluorescence (IF) microscopy and immunoblot analysis. MSC containing lentiviral control shRNA or LGALS3 shRNA were used to assess effects of LGALS3 on cell viability, apoptosis, and cell adhesion by flow cytometry assays and effects on survival signaling by immunoblot analysis.Results: LGALS3 levels were found to be higher in AML MSC compared to normal MSC (p = 0.0001). The increase in protein expression was not correlated with gene expression as mRNA levels were similar in AML and normal MSC, suggesting a post-translational mechanism. To identify MSC proteins associated with relapse or refractory status, RPPA was utilized to compare protein expression in MSC from newly diagnosed with those from relapsed and refractory (salvage) patients. RPPA identified LGALS3 as one of only three proteins increased in MSC from relapse/refractory patients. Pearson Correlation of LGALS3 with the other 150 proteins in AML MSC revealed that LGALS3 was negatively correlated with expression of integrin beta 3 and LYN. These 2 proteins were also among the 6 proteins found to have lower expression in salvage as compared to MSC from newly diagnosed patients. LGALS3 positively correlated with 13 proteins including phosphorylated beta-catenin which was also higher in salvage as compared to newly diagnosed AML MSC. . The LGALS3 promoter contains HIF1 alpha response elements. MSC from healthy donors grown in 1% oxygen displayed > 4 fold increase in LGALS3 protein after 24 hours. Hypoxic MSC exhibited a re-localization of LGALS3 from the nucleus to the cytoplasm. LGALS3 activates RAS signaling so hypoxia induced expression and nuclear export of the galectin would be expected to activate diverse survival signaling pathways and would also allow the molecule to be secreted where it could then suppress immune cells. Using in vitro co-culture experiments of MSC with human OCI-AML3 cells to mimic the leukemia microenvironment, LGALS3 levels were induced in MSC suggesting that cross talk from leukemia cells could contribute to the increased LGALS3 levels in MSC cells. The consequences of suppressing LGALS3 in normal MSC were tested using lentivral shRNA. Suppression of LGALS3 resulted in a 2-fold reduction of MYC and S473 phosphorylated AKT, and a 2-fold increase in PPP2R2A (an AKT phosphatase) compared to control shRNA. LGALS3 suppression rendered MSC less adherent to OCI-AML3 cells (~ 50% reduction in MSC with LGALS3 shRNA compared to control shRNA).Conclusions: The data presented here demonstrate that LGALS3 is elevated in AML MSC especially in relapse/refractory samples. Possible mechanisms of up-regulation of LGALS3 in AML MSC may be due to the hypoxic nature of the leukemia microenvironment and/or contact with AML cell. Hypoxia induced nuclear export of the galectin to the cytoplasm could contribute to MSC survival and anti-tumor functions (e.g. suppression of immune cells by secreted galectin). Knock down of LGALS3 in MSC suppresses AKT activation and MYC expression suggesting it has a key role in MSC cell survival. Experiments are underway to determine if MSC derived LGALS3 might impact AML cell homing (as a regulator of cell adhesion) and/or immune surveillance (as a suppressor of T and NK cells). The identification of LGALS3 as an important component of AML MSC suggests that this molecule could be a target in a tumor microenvironment based therapy concept of AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1930: Low surface Gal3 expression in breast cancer stem cells is associated with chemoresistance, tumorigenesis, and decreased overall patient survival

Abstract The development of therapeutic approaches that target specific breast cancer subtypes has improved overall survival, but some patients nevertheless become resistant to treatment and develop metastatic disease. Recent insights suggest multiple hallmarks of cancer - especially drug-resistance, unlimited self-renewal, as well as invasion and metastasis - that can be attributed to a small subpopulation frequently designated as cancer stem cells (CSC). CSCs are rare, self-renewing cells that initiate and maintain tumor growth. Galectin-3 (Gal3), a multifunctional oncogenic beta-galactoside binding lectin, was implicated in the metastatic cascade by aiding the homing of breast cancer circulating tumor cells to the metastatic niche. In addition, it was proposed that Gal3 protects breast cancer cells from mitochondria-dependent apoptosis induced by chemotherapeutic agents while concurrently promoting sensitivity to cell death ligands. Recently, we have found that in a subset of metastatic breast cancer cells (GI-LM2), cell-surface Gal3 is highly expressed compared to its non-metastatic counterpart (GI-101). Breast CSCs have been characterized by CD24-/CD44+/EpCAM+ surface marker expression in flowcytometry analysis. These subpopulations of GI-LM2 exhibit an enhanced self-renewal capability in sphere-formation assays (SFA) when compared to a similar subpopulation of GI-101. Surprisingly, depletion of Gal3 from this subset of GI-LM2 resulted in epithelial-mesenchymal-transition (EMT) - a mechanism linked to cancer stemness - as evidenced by Immunoblot and -fluorescence, resistance to the adjuvant FAC regimen (5-Fluorouracil, Doxorubicin, Cyclophosphamide), and markedly increased tumorigenicity in in vivo limited dilution assays. These findings are in keeping with results obtained from a stage 2 and 3a cohort of breast cancer patients (n=87) with axillary lymph node metastases. Gal3 expression in tissue microarrays from these patients was found to be significantly associated with the absence of lymphovascular invasion (p=0.015) as well as increased locoregional-free, disease-specific and overall survival (p=0.054, p=0.18, and p=0.019, respectively). Taken together, we present evidence in vitro, in vivo, and in clinico that lack of surface Gal3 in a subset of breast CSCs is associated with chemoresistance. This contrasts with earlier reports that Gal3 renders cancer cells chemoresistant, enhances tumorigenesis, and is associated with poor overall survival. Further studies will determine whether these finding can be applied to therapeutic stratification of breast cancer patients, or as a diagnostic tool for detection of circulating tumor cells which are frequently described as early indicators of dormant metastatic breast cancer. Citation Format: Matthias Illmer, Nachman Mazurek, James Byrd, Jody Vykoukal, Robert Scott Bresalier. Low surface Gal3 expression in breast cancer stem cells is associated with chemoresistance, tumorigenesis, and decreased overall patient survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1930. doi:10.1158/1538-7445.AM2014-1930

22. Galectin-9 is increased during cuprizone intoxication and promotes remyelination

Demyelination and axonal damage in multiple sclerosis (MS) results from inflammatory processes that are perpetuated by activated glia and infiltrating leukocytes. Galectins are beta-galactoside binding lectins, capable of modulating immune responses. The function of galectin-9 (Gal-9) upregulation in MS is important since it has been reported to promote innate immune responses while at the same time inhibit autoimmunity. Cuprizone intoxication causes reproducible demyelination of the caudal corpus callosum that is associated with reactive gliosis. Removal of cuprizone from the diet results in remyelination. Using wild-type (WT) and galectin-9 null ( Lgals9 − / −) mice we investigated the effects Gal-9 on the pathogenesis of this model of MS. Gal-9 was increased in the corpus callosum during cuprizone intoxication where it localized to both GFAP+ astrocytes and Iba-1+ microglia. However, the degree of demyelination and reactive gliosis were similar between genotypes. Likewise, reactive gliosis did not differ between WT and Lgals9 − / − mice during a time-frame corresponding to remyelination. In contrast, WT mice displayed enhance remyelination when compared to Lgals9 − / − mice as determined by oil Red O staining for myelin. Interestingly, Lgals9 − / − mice had significantly fewer mature oligodendrocytes (OLs) (CC1+) than their WT counterparts while the numbers of total OLs (Olig2+) did not differ between genotype. Finally, treatment of mixed glial cultures with recombinant Gal-9 promoted OL maturation. Together these data indicate galectin-9 promotes oligodendrocyte maturation remyelination.