Overexpression Of Galectin-3 Research Articles

High glucose-induced Galectin-1 in human podocytes implicates the involvement of Galectin-1 in diabetic nephropathy.

The diabetic milieu is believed to change the activity, or result in damage of podocytes-a key component of the glomerular filtration barrier and known to secrete matrix for glomerular basement membrane. This in turn contributes to diabetic nephropathy. However, how podocyte dysfunction is triggered in diabetic nephropathy remains ambiguous. Galectin-1 belongs to Galectin family that bind to β-galactoside residues of glycosylated proteins. We explored whether Galectin-1 is dysregulated in diabetic nephropathy using three different techniques, namely real-time polymerase chain reaction, western blotting, and immunofluorescent staining, to follow the expression of Galectin-1 under high glucose levels in podocytes. High glucose consistently induced Galectin-1 expression. Immunohistochemistry using a Galectin-1-specific antibody also showed elevated Galectin-1 in renal tissues of diabetic patients with manifestation of nephropathy, indicating a correlation of Galectin-1 overexpression with diabetic nephropathy. Upregulation of Galectin-1 is associated with loss of podocin, which is important for the physiological function of podocytes and decreases in the renal tissues of diabetic nephropathy. Increased Galectin-1 is a causal event for the high glucose-induced loss of podocin, since silencing Galectin-1 in podocytes increased podocin expression in the presence of 25 mM glucose. Thus expression of Galectin-1 in diabetic nephropathy may serve as a marker and contribute to disease progression by interfering with podocin expression.

Abstract 2478: Glycosylation of tid1 regulates galectin-7 ubiquitination and localization to suppress head and neck cancer metastasis

Abstract Background: Tid1, a DnaJ cochaperone protein mainly located in mitochondria, functions as a tumor suppressor in different types of solid tumors. Recently, we first demonstrate that Tid1 is negatively associated with cell migration, tumor status, and survival prognosis in head and neck squamous cell carcinoma (HNSCC) (J Pathol 2009; 219:347-55). As a cochaperone protein like Tid1, it should be instrumental to find the Tid1 client/interacting proteins, consequently, to understand the physiological function of those Tid1 client/interacting proteins as therapeutic targets for future HNSCC treatment. Methods: First, we applied affinity chromatography and systemic proteomics analysis to identify galectin-7 as one of client/interacting proteins of Tid1. Subsequently, using the metastatic and/or non-metastatic cells determined tumorigenic properties of HNSCC with overexpression and/or knockdown of galectin-7 and/or Tid1. The nuclear and cytosolic subfractions were isolated to determine the role of Tid1 of galectin-7 nuclearization. We also attempted to correlate the expression of Tid1 and galectin-7 with tumor stage of HNSCC patient tissues by immunohistochemical assay. Finally, site-directed mutagenesis was applied to identify the novel N-link glycosylated sites of Tid1, by which to regulate the interaction and stability of galectin-7. Results: We found metastatic cells expressed lower level of Tid1 and higher level of galectin-7 than non-metastatic cells. For in vitro tumorigenic analyses, we observed that overexpression of galectin-7 resulted in elevated migration, invasion and soft agar colony formation abilities in HNSCC. Inversely, co-overexpression of Tid1 reduced the expression of galectin-7, impaired the galectin-7 mediated migration, invasion and soft agar colony formation ability of HNSCC, and retarded nuclearization of galectin-7. Moreover, knockdown of galectin-7 reduced migration, invasion and soft agar colony formation ability of HNSCC metastatic cells. Further, we found that galectin-7 ubiquitination as revealed by antiubiquitin immunoblot analysis in immunoprecipitates. Finally, the negative relationship was between Tid1 and Galectin-7 expression in HNSCC clinical specimen. We found out that Tid1 interacts with galectin-7 through N-linked glycosylation. Tid1 mutants of N-link glycosylated sites restored the level and the tumorigenic function of galectin-7. Conclusion: Our studies first pointed out that galectin-7 plays pivotal roles in HNSCC tumorigenesis. Notably, we identified galectin-7, which is the client/interacting protein of Tid1. We hypothesized that Tid1/ Hsp70 may regulate galectin-7 stability by ubiquitinylation. The molecular mechanism by which Tid1 negatively modulates the galectin-7 warrants further investigation. Citation Format: Yu-Syuan Chen. Glycosylation of tid1 regulates galectin-7 ubiquitination and localization to suppress head and neck cancer metastasis. [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 2478. doi:10.1158/1538-7445.AM2014-2478

Abstract 1352: Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation in breast cancer cells

Abstract Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 was recently shown to be specifically expressed in basal-like and HER-2 positive but not in luminal subtypes of human breast cancer. Overexpression of galectin-7 in breast cancer cells also increases lung and bone metastases. However, the molecular mechanisms used by galectin-7 to increase the dissemination of breast cancer cells remain largely unknown. Although galectins are best known for their ability to bind cell surface receptors, there is increasing evidence that their intracellular forms have important functions in cancer cells. This is particularly true in the control of apoptosis. Up to now, regulation of apoptosis by intracellular galectins has been largely attributed to their ability to translocate to mitochondria, possibly following their interaction with bcl-2. Here, we report that a mutant of galectin-7 that is unable to translocate to mitochondria and nucleus induces resistance of human breast cancer cells to apoptosis induced by etoposide or by hypoxia-mimicking conditions. Surprisingly, this mutant and the wild-type form of galectin-7 bind equally well to bcl-2 in vitro and in vivo. Interestingly, both forms decreases the translocation of p53 to the nucleus and reduce the expression of p21 following treatment with doxorubicin. These results challenges the current paradigm that mitochondrial galectins are important for resistance to apoptosis and call for a greater focus on the role of cytosolic galectins in cancer cells. Our data also indicate that targeting cytosolic galectin-7 could improve the efficacy of anti-cancer drugs for the treatment of aggressive forms of breast cancer. Citation Format: Andrée-Anne Grosset, Donald Gagné, Marilyne Labrie, Maria Vladoiu, Louis Gaboury, Nicolas Doucet, Yves St-Pierre. Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation in breast cancer cells. [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 1352. doi:10.1158/1538-7445.AM2014-1352

Galectin-3 is associated with a poor prognosis in primary hepatocellular carcinoma.

BackgroundGalectin-3, a member of the beta-galactoside-binding lectin family, is a multifunctional protein with various biological functions, including the proliferation and differentiation of tumor cells, angiogenesis, cancer progression, and metastasis. We aimed to clarify if expression of galectin-3 is related to the clinicopathological characteristics and prognosis of hepatocellular carcinoma (HCC) patients, and to explore the possible mechanisms of galectin-3 in hepatocellular carcinoma.MethodsFirst, we investigated galectin-3 mRNA and protein expression by using RT-PCR and Western blotting. Second, tissues from 165 HCC patients were used to evaluate clinicopathological characteristics and prognosis through immunohistochemical analyses. Furthermore, the functions of galectin-3 were analyzed with respect to the proliferation, cell cycle,apoptosis, migration, and invasion of HCC cell lines. Finally, we analyzed galectin-3 expression and micro-vessel density (MVD) by immunohistochemistry (IHC) to find its correlation with angiogenesis in Hepatocellular Carcinoma. Flow cytometer was used to explore apoptosis and Western-blot was used to detect the pathway proteins of apoptosis.ResultsGalectin-3 showed high expression at the mRNA and protein levels in HCC cancer tissues and cell lines. Clinicopathological analyses revealed that increased expression of galectin-3 in tumors was closely associated with a poor prognosis. Galectin-3 knockdown by siRNA significantly inhibited cell growth, migration, and invasion, and induced apoptosis in HCC cells in vitro, whereas galectin-3 overexpression promoted cell growth, migration, and invasion. Correlation analysis of galectin-3 expression and micro-vessel density (MVD) showed that galectin-3 expression in tumor cells stimulates angiogenesis. The observed regulation of cell apoptosis was accompanied by the galectin-3-mediated modulation of caspase3 signaling pathways in HCC cells.ConclusionsThese data suggest that galectin-3 plays an important part in HCC progression and may serve as a prognostic factor for HCC.

Galectin fingerprinting in naso-sinusal diseases.

Galectins, a family of endogenous lectins, are multifunctional effectors that act at various sites and can be used in immunohistochemical localization studies of diseased states. Since they form a potentially cooperative and antagonistic network, we tested the hypothesis that histopathological fingerprinting of galectins could refine the molecular understanding of naso-sinusal pathologies. Using non-cross-reactive antibodies against galectin-1, -3, -4, -7, -8 and -9, we characterized the galectin profiles in chronic rhinosinusitis, nasal polyposis, inverted papillomas and squamous cell carcinomas. The expression, signal location and quantitative parameters describing the percentage of positive cells and labeling intensity were assessed for various cases. We discovered that inverted papillomas showed a distinct galectin immunohistochemical profile. Indeed, epithelial overexpression of galectin-3 (P=0.0002), galectin-4 (P<10−6), galectin-7 (P<10−6) and galectin-9 (P<10−6) was observed in inverted papillomas compared to non-malignant diseases. Regarding carcinomas, we observed increased expression of galectin-9 (P<10−6) in epithelial cells compared to non-tumor pathologies. Our results suggest that galectin-3, -4, -7 and -9 could be involved in the biology of inverted papillomas. In addition, we observed that the expression of galectin in naso-sinusal diseases seems to be affected by tumor progression and not inflammatory or allergic phenomena.

Effect of galectin3 on proliferation and migration of esophageal cancer Eca109 cells

Objective To investigate galectin3 on proliferation and migration of esophageal cancer Eca109 cells.Methods A lentiviral vector for over-expression of RNA targeting galectin3 was designed to transfect Eca109 cancer cells following plasmid-mediated transfection manual (Eca109/Gal3 cells).Inverted fluorescence microscope was used to observe the expression of EGFP.The proliferation of Eca109 cells was measured by cell counting Kit-8 assay.Eca109 cells apoptosis was determined by Annexin-V/7-AAD doublestaining.The migration capacity of Eca109 cells was determined in transwell assays.Western blot analysis was used to measure the expression of galectin3 protein.Results Galectin-3 expression was detected in Eca109 cells,with the Galectin3 expression in Eca109/Gal3 cells much more than non-transfected cells (t =14.33,P ＜ 0.05 ; t =10.28,P =0.037).Compared with non-transfected Eca109 cells,proliferation increased significantly in Eca109/Gal3 cells (t =-17.277,P ＜ 0.05 ; t =-13.4,P ＜ 0.05).Galectin3 evidently decreased in Eca109 cell apoptosis (t =3.053,P ＜ 0.05 ; t =5.446,P ＜ 0.05).Transwell migration assay showed that a greater number of Eca109/Gal3 cells crossed the artificial basement membrane compared with non-transfected Eca109 cells and negative control Eca109 cells (t =3.465,P ＜ 0.05; t =3.252,P ＜ 0.05).Conclusion Galectin3 expression is detected in transfected esophageal cancer Eca109 cells,whose overexpression can result in enhanced proliferation,migration,invasion as well as reduced apoptosis.These data indicate that in-depth research of galectin-3 may prove to be a potential molecular target for the treatment of esophageal cancer. Key words: Esophageal neoplasms ; Galectin 3 ; RNA over-expression ; Biological function

Galectin-1 overexpression in nasopharyngeal carcinoma: effect on survival

Conclusions: Galectin-1 overexpression is significantly correlated with the survival rate of patients with nasopharyngeal carcinoma (NPC). Immunohistochemical analysis of galectin-1 expression might be useful for identifying patients with a high risk of distant metastasis and for prompting timely adjuvant systemic therapy for patients with aggressive NPC. Objectives: We examined the effect of galectin-1 on the survival rate of patients with NPC. Methods: A total of 124 patients diagnosed between 1998 and 2002 with NPC without distant metastasis were enrolled in this single-center historical prospective cohort study. Immunohistochemical analysis was used to correlate the galectin-1 expression score in the cytoplasm and the survival rate of patients with NPC. Results: Patients with NPC who overexpressed galectin-1 in cytoplasm showed more aggressive tumor growth and significantly shorter disease-specific survival (DSS) (p = 0.0037) and distant metastasis-free survival (DMFS) (p = 0.0006) than patients with NPC who did not overexpress galectin-1. Multivariate analysis showed that galectin-1 overexpression remained prognostically independent for DSS (p = 0.031, hazard ratio = 1.821), and DMFS (p = 0.005, hazard ratio = 2.417), together with the advanced III–IV stages.

The expression profiles of the galectin gene family in primary and metastatic papillary thyroid carcinoma with particular emphasis on galectin-1 and galectin-3 expression

IntroductionGalectin family members have been demonstrated to be abnormally expressed in cancer at the protein and mRNA level. This study investigated the levels of galectin proteins and mRNA expression in a large cohort of patients with papillary thyroid carcinoma and matched lymph node metastases with particular emphasis on galectin-1 and galectin-3. MethodsmRNA expression of galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were analysed by real-time polymerase chain reaction in 65 papillary thyroid carcinomas, 30 matched lymph nodes with metastatic papillary thyroid carcinoma and 5 non-cancer thyroid tissues. Galectin-1 and 3 protein expression was determined by immunohistochemistry in these samples. ResultsSignificant expression differences in all tested galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were noted for mRNA in papillary thyroid carcinomas, with and without lymph node metastasis. Galectin-1 protein was more strongly expressed than galectin-3 protein in papillary thyroid carcinoma. Galectin-1 protein was found to be overexpressed in 32% of primary papillary thyroid carcinomas. A majority of lymph nodes with metastatic papillary thyroid carcinoma (53%) had significantly increased expression of galectin-1 protein, as did 47% of primaries with metastases. Galectin-1 mRNA levels were decreased in the vast majority (94%) of primary thyroid carcinomas that did not have metastases present. Galectin-3 protein levels were noted to be overexpressed in 15% of primary papillary thyroid carcinomas. In primary papillary thyroid carcinoma with lymph node metastases, 32% had over expression of galectin-3 protein. Overexpression of galectin-3 mRNA was noted in 58% of papillary thyroid carcinomas and 64% of lymph nodes bearing metastatic papillary thyroid carcinoma. Also, primary papillary thyroid carcinoma with lymph node metastases had significantly higher expression of galectin-3 mRNA compared to those without lymph node metastases. ConclusionGalectin family members show altered expression at the mRNA level in papillary thyroid cancers. Overexpression of galectin-1 and 3 proteins were noted in papillary thyroid carcinoma with lymph node metastases. The results presented here demonstrated that galectin-1 and galectin-3 expression have important roles in clinical progression of papillary thyroid carcinoma.

Galectin-1 Upregulates CXCR4 to Promote Tumor Progression and Poor Outcome in Kidney Cancer

Galectin-1, a β-galactoside-binding lectin, is involved in many physiologic and pathologic processes, including cell adhesion, differentiation, angiogenesis, and tumor progression. However, the role of galectin-1 in kidney cancer remains elusive. This study evaluated the role of galectin-1 in the progression and clinical prognosis of renal cell carcinoma. We found significant overexpression of galectin-1 in both kidney cancer cell lines and metastatic tissue specimens from patients with renal cell carcinoma. Knockdown of galectin-1 gene expression in renal cancer cell lines reduced cell invasion, clonogenic ability, and epithelial-mesenchymal transition in vitro; reduced tumor outgrowth in vivo; and inhibited the angiogenesis-inducing activity of these cells in vitro and in vivo. Galectin-1 knockdown decreased CXCR4 expression levels in kidney cancer cells, and restoration of CXCR4 expression in galectin-1-silenced cells rescued cell motility and clonogenic ability. Additional studies suggested that galectin-1 induced CXCR4 expression through activation of nuclear factor-κB (NF-κB). Analysis of patient specimens confirmed the clinical significance and positive correlation between galectin-1 and CXCR4 expression levels and revealed concomitant overexpression of galectin-1 and CXCR4 associated adversely with overall and disease-free survival. Our findings suggest that galectin-1 promotes tumor progression through upregulation of CXCR4 via NF-κB. The coordinated upregulation of galectin-1 and CXCR4 may be a novel prognostic factor for survival in patients with renal cell carcinoma and the galectin-1-CXCR4 axis may serve as a therapeutic target in this disease.

Galectin-1 overexpression promotes progression and chemoresistance to cisplatin in epithelial ovarian cancer

This study was performed to investigate the role of galectin-1 (Gal-1) in epithelial ovarian cancer (EOC) progression and chemoresistance. Tissue samples from patients with EOC were used to examine the correlation between Gal-1 expression and clinical stage of EOC. The role of Gal-1 in EOC progression and chemoresistance was evaluated in vitro by siRNA-mediated knockdown of Gal-1 or lentivirus-mediated overexpression of Gal-1 in EOC cell lines. To elucidate the molecular mechanisms underlying Gal-1-mediated tumor progression and chemoresistance, the expression and activities of some signaling molecules associated with Gal-1 were analyzed. We found overexpression of Gal-1 in advanced stages of EOC. Knockdown of endogenous Gal-1 in EOC cells resulted in the reduction in cell growth, migration, and invasion in vitro, which may be caused by Gal-1's interaction with H-Ras and activation of the Raf/extracellular signal-regulated kinase (ERK) pathway. Additionally, matrix metalloproteinase-9 (MMP-9) and c-Jun were downregulated in Gal-1-knockdown cells. Notably, Gal-1 overexpression could significantly decrease the sensitivities of EOC cells to cisplatin, which might be ascribed to Gal-1-induced activation of the H-Ras/Raf/ERK pathway and upregulation of p21 and Bcl-2. Taken together, the results suggest that Gal-1 contributes to both tumorigenesis and cisplatin resistance in EOC. Thus, Gal-1 is a potential therapeutic target for EOC.

Galectin-3 Silencing Inhibits Epirubicin-Induced ATP Binding Cassette Transporters and Activates the Mitochondrial Apoptosis Pathway via β-Catenin/GSK-3β Modulation in Colorectal Carcinoma

Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi) on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp), MDR-associated protein (MRP) 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

Galectin-7 is epigenetically-regulated tumor suppressor in gastric cancer

Gastric cancer is the second leading cause of cancer death and remains a major clinical challenge due to poor prognosis and limited treatment options. Therefore, the basic mechanisms underlying gastric tumorigenesis deserve investigation. Although regulation of the galactoside-binding lectin galectin-7 in cancer has been studied, its role in tumor formation and progression remains controversial. In this study, we investigated galectin-7 expression and its role in gastric cancer. Immunohistochemical staining using a tissue microarray of gastric cancer patients revealed significantly low expression levels of galectin-7 in malignant tissues compared with matched normal tissues, and decreased expression of galectin-7 in malignant tissues was associated with advanced TMN stage disease (p=0.034). Importantly, low expression of galectin-7 in normal tissues was associated with a poor survival rate (p=0.0561). Over-expression of galectin-7 in AGS gastric adenocarcinoma cells suppressed cell proliferation, migration, and invasion, whereas ablation of galectin-7 in KATO III gastric carcinoma cells reversed these properties. AGS cells that overexpressed galectin-7 could not form gastric tumors in xenografted mice. More than 70% hypermethylation was observed in 7 of 9 gastric cancer cell lines tested and 5-aza-cytidine treatment lowered galectin-7 expression by reducing methylation in 24 cancer cell lines from five different organ origins. We analyzed CpG islands in the galectin-7 genomic region and detected hypermethylation at +1566bp of exon 2, the predicted p53 binding region. DNA hypermethylation of this region was also detected in gastric cancer tissues from 20 patients. Taken together, our data indicate that galectin-7 has a tumor suppressive function, and that the gene is epigenetically modified by DNA methylation and significantly down-regulated in gastric cancer. Further study of galectin-7 regulation may lead to improved gastric cancer diagnosis and therapy.

Suppression of SERPINA1-albumin complex formation by galectin-3 overexpression leads to paracrine growth promotion of chronic myelogenous leukemia cells

Galectin-3 is induced in chronic myelogenous leukemia (CML) cells by co-culture with bone marrow stromal cells, making paracrine growth promotion of CML cells in conditioned medium (CM) from galectin-3 overexpressing CML cells more potent. We used gel filtration chromatography to demonstrate that the bovine SERPINA1-fetal bovine serum albumin (BSA) complex was specifically suppressed in CM from galectin-3 overexpressing cells. The SERPINA1-BSA complex as well as human plasma SERPINA1 inhibited the growth of CML cells, while exogenous galectin-3 partly offset this effect. These findings suggest that galectin-3 overexpression promotes paracrine growth of CML cells by interfering with the action of the growth inhibitory SERPINA1-albumin complex.

FTY720 induces apoptosis of chronic myelogenous leukemia cells via dual activation of BIM and BID and overcomes various types of resistance to tyrosine kinase inhibitors

PP2A activator FTY720 has been shown to possess the anti-leukemic activity for chronic myelogenous leukemia (CML), however, the cell killing mechanism underlying its anti-leukemic activity has remained to be verified. We investigated the precise mechanisms underlying the apoptosis induction by FTY720, especially focusing on the roles of BH3-only proteins, and the therapeutic potency of FTY720 for CML. Enforced expression of either BCL2 or the dominant-negative protein of FADD (FADD.DN) partly protected CML cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways. FTY720 activates pro-apoptotic BH3-only proteins: BIM, which is essential for apoptosis by BCR-ABL1 tyrosine kinase inhibitors (TKIs), and BID, which accelerates the extrinsic apoptosis pathway. Gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the extent of cell protection was not as much as that by overexpression of either BCL2 or FADD.DN. Moreover, knockdown of both BIM and BID did not provide additional protection compared with knockdown of only BIM or BID, indicating that BIM and BID complement each other in apoptosis by FTY720, especially when either is functionally impaired. FTY720 can overcome TKI resistance caused by ABL kinase domain mutations, dysfunction of BIM resulting from gene deletion polymorphism, and galectin-3 overexpression. In addition, ABT-263, a BH3-mimetic, significantly augmented cell death induction by FTY720 both in TKI-sensitive and -resistant leukemic cells. These results provide the rationale that FTY720, with its unique effects on BIM and BID, could lead to new therapeutic strategies for CML.

Galectin-3 in pre-B acute lymphoblastic leukemia

Galectin-3 in pre-B acute lymphoblastic leukemia

Expression and Functions of Galectin-7 in Human and Murine Melanomas

The identification of galectin-7 as a p53-induced gene and its ability to induce apoptosis in many cell types support the hypothesis that galectin-7 has strong antitumor activity. This has been well documented in colon cancer. However, in some cases, such as breast cancer and lymphoma, its high expression level correlates with aggressive subtypes of cancer, suggesting that galectin-7 may have a dual role in cancer progression. In fact, in breast cancer, overexpression of galectin-7 alone is sufficient to promote metastasis to the bone and lung. In the present work, we investigated the expression and function of galectin-7 in melanoma. An analysis of datasets obtained from whole-genome profiling of human melanoma tissues revealed that galectin-7 mRNA was detected in more than 90% of biopsies of patients with nevi while its expression was more rarely found in biopsies collected from patients with malignant melanoma. This frequency, however, was likely due to the presence of normal epidermis tissues in biopsies, as shown our studies at the protein level by immunohistochemical analysis. Using the experimental melanoma B16F1 cell line, we found that melanoma cells can express galectin-7 at the primary tumor site and in lung metastasis. Moreover, we found that overexpression of galectin-7 increased the resistance of melanoma cells to apoptosis while inducing de novo egr-1 expression. Overexpression of galectin-7, however, was insufficient to modulate the growth of tumors induced by the subcutaneous injection of B16F1 cells. It also failed to modulate the dissemination of B16F1 cells to the lung.

Galectin-3 Accelerates the Progression of Oral Tongue Squamous Cell Carcinoma via a Wnt/β-catenin-Dependent Pathway

The purpose of this study was to elucidate the clinicopathological significance and mechanism of action of galectin-3 in oral tongue squamous cell carcinoma (OTSCC). Here, the expression of galectin-3 was quantified in OTSCC (n = 68) and paired OTSCC and normal surrounding tissues (n = 10) using immunohistochemical staining. Tca8113 OTSCC cells were transfected with a plasmid expressing galectin-3 cDNA or siRNA against galectin-3. Cell proliferation, migration and invasion were measured using the MTT assay, Matrigel-coated Transwell migration assay and wound healing assay. The effect of galectin-3 on the Wnt/β-catenin signaling pathway and epithelial mesenchymal transition (EMT) were investigated using a plasmid expressing the Wnt antagonist dickkopf 1 (DKK1) and Western blotting. Galectin-3 was expressed at significantly higher levels in OTSCC than the normal adjacent tissues; galectin-3 expression correlated strongly with pathological stage, pathological grade and lymph node invasion in OTSCC. Overexpression of galectin-3 promoted Tca8113 cell proliferation, migration and invasion, upregulated Wnt protein expression, activated β-catenin and induced the EMT; knockdown of galectin-3 had the opposite effects. Co-transfection of Tca8113 cells overexpressing galectin-3 with the Wnt antagonist DKK1 reduced the ability of galectin-3 to increase cell proliferation, migration and invasion, reduced upregulation of Wnt, inhibited β-catenin activation and abrogated the EMT, demonstrating that the Wnt/β-catenin signaling pathway mediated the effects of galectin-3. Galectin-3 plays an important role in the progression of OTSCC via activation of the Wnt/β-catenin signaling pathway.

Abstract IA6: Immune escape mechanisms in lymphoid malignancies.

Abstract Certain lymphoid malignancies utilize potent mechanisms to evade host anti-tumor immune responses including the AP-1-dependent overexpression of Galectin-1 (Gal1) and the genetic amplification and overexpression of the PD-1 ligands. Gal1 recognizes specific cell surface glycans (Gal1 β1 GlcNAc [N-acetyllactosamine] units on the branches of N- or O-linked glycans) on receptors such as CD45, CD43 and CD7 and induces the apoptosis of select T cell subsets – Th1, Th17 and cytotoxic T cells. Th2 cells and regulatory T (Treg) cells have different patterns of sialylation of cell surface glycoproteins, lack Gal1 ligands and resist Gal1-induced death. We recently found that malignant Reed-Sternberg (RS) cells in classical Hodgkin Lymphoma (cHL) and EBV-transformed malignant B cells in post-transplant lymphoproliferative disorders (PTLD) overexpress the secreted carbohydrate-binding lectin, Galectin-1 (Gal1). Gal1 overexpression by Hodgkin RS cells and EBV-transformed B cells promotes the immunosuppressive Th2/Treg-predominant, Th1/cytotoxic T cell-deficient micro-environment in cHL and PTLD. In both of these lymphomas with known constitutive activation of AP-1, Gal1 expression is driven, in large part, by an AP-1–dependent Gal1 enhancer. The AP-1-dependent expression of Gal1 is a useful diagnostic marker and circulating Gal1 levels have prognostic value. In addition, neutralizing Gal1 antibodies have promising immunomodulatory activity in in vitro assays and tumor models. Engagement of PD-1 receptors triggers T-cell “exhaustion” and the progressive loss of effector T-cell function and proliferative capacity. In certain cancers, the tumor cell expression of PD-1 ligands inhibits T-cell activation and promotes the apoptosis of tumor-specific T cells. We recently integrated high-resolution copy number data with transcriptional profiles and identified the PD-1 ligands, PD-L1 and PD-L2, as key targets of the 9p24.1 amplification in cHL and a related lymphoid malignancy, primary mediastinal large B-cell lymphoma (MLBCL). PD-1 ligand gene amplification was associated with increased protein expression in both lymphoid malignancies. In cHLs and MLBCLs, the extended 9p24.1 amplification region also included the JAK2 locus. JAK2 amplification increased JAK2 protein expression and activity and specifically induced PD-1 ligand transcription. Chemical inhibition of JAK2 decreased PD-1 ligand expression. These findings defined 9p24.1 amplification as a disease-specific structural alteration that increased both the gene dosage of PD-1 ligands and their induction via JAK2. Like Gal1, PD-L1 includes an AP-1-dependent enhancer which augments PD-L1 expression in lymphomas with constitutive activation of AP-1. In addition, Epstein-Barr virus (EBV) induces PD-L1 expression via AP-1 and EBV+ cHLs with diploid 9p24.1 and the majority of EBV+ PTLDs express detectable PD-L1. Therefore, AP-1- and EBV-driven malignancies with normal 9p24.1 copy numbers, including cHLs and most PTLDs, may also be amenable to PD-1 blockade. Taken together, these studies identify the PD-1 axis and Gal1 as promising therapeutic targets in certain lymphoid malignancies and define genetic bases for increased expression of these immunoregulatory molecules. For these reasons, clinical evaluation of PD-1 blockade alone and in association with JAK2 inhibition is planned and assessment of antibody-mediated Gal1 blockade is underway. Citation Format: Margaret A. Shipp. Immune escape mechanisms in lymphoid malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA6.

Epigenetic Regulation of Galectin-3 Expression by β1 Integrins Promotes Cell Adhesion and Migration

Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.

Fingolimod (FTY720) Overcomes the Resistance to Tyrosine Kinase Inhibitors Via Dual Activation of BIM and BID in Chronic Myelogenous Leukemia

Abstract 3744 [Introduction]Tyrosine kinase inhibitors (TKIs) for BCR-ABL have dramatically improved the treatment outcome of chronic myelogenous leukemia (CML) during this decade, however, the resistance to TKIs due to various molecular mechanisms, such as BCR-ABL mutations, dysfunction of cell killing mechanisms like BIM deregulation, or the supports by leukemia microenvironment factors, has remained to be overcome. We have identified that BIM, a member of BH3-only proteins, is the essential pro-apoptotic mediator under the blockade of BCR-ABL signaling (Kuroda J, PNAS, 2006). One of the attractive therapeutic strategies for complete eradication of CML cells by simultaneously targeting both TKI-sensitive and TKI-resistant CML cells may be the use of agents that can activate BIM as well as other pro-apoptotic BH3-only protein which is not utilized in apoptosis by TKIs. In this study, we assessed the therapeutic potential of the protein phosphatase 2A (PP2A) activator Fingolimod (FTY720), an immunosuppressive agent currently used for multiple sclerosis, in CML. [Materials and methods]Human CML-derived cell lines, K562, MYL, KBM5, KCL22, and their various subclones, were utilized in this study. Murine Ba/F3 lymphoid cells were transfected with either wild type or mutated bcr-abl gene. FTY720 was purchased from Cayman Chemical (Ann Arbor, MI). Cell death analyses were performed using flow cytometry. Protein levels were determined by conventional Western blotting. [Result and discussion]Treatment with FTY720 impairs phosphorylation of BCR-ABL and its downstream targets, AKT, ERK and STAT5, and inhibited the growth of CML cell lines through the induction of apoptosis. The apoptosis induced by FTY720 was accompanied by the activation of both caspase-8 and caspase-9, while enforced expression of either BCL2 or dominant-negative protein of FADD (FADD.DN) by gene introduction partly protected K562 cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways in FTY720-induced apoptosis. FTY720 treatment resulted in the activation of Bim as well as the processing of Bid into tBid, a BH3-only protein which plays crucial role in accelerating death-receptor-mediated extrinsic apoptosis pathway and is not utilized in apoptosis by TKIs. Importantly, gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the degrees of cell protection by those were not as much as the degree of cell protection by overexpression of either BCL2 or FADD.DN. Moreover, double knockdown of BIM and BID did not further protect K562 cells from apoptosis by FTY720, when compared with single knockdown of BIM. These results indicate that BIM and BID compensate each other for promoting apoptosis by FTY720 in CML cells, especially when either of them is functionally disturbed. Through these mechanisms, FTY720 overcomes TKI resistance by various mechanisms, such as ABL kinase domain (KD) mutations in BCR-ABL-transfected Ba/F3 cells (T315I, G250E, E255K or H396P) and in KBM5 cells with T315I. FTY720 also overcomes TKI resistance due to dysfunction of BIM by gene deletion polymorphism in KCL22 cells. Also, although galectin-3 overexpression has been shown to mediate bone marrow environment-mediated drug resistance in CML cells (Yamamoto-Sugitani M, PNAS, 2011), the addition of FTY720 to imatinib overcomes resistance to imatinib in galectin-3 overexpressing K562 cells and MYL cells. In conclusion, FTY720 induces apoptotic cell death in CML via dual activation of both BIM and BID, and is potent in overcoming various types of cell intrinsic and tumor microenvironment-mediated resistance to TKI. Disclosures:No relevant conflicts of interest to declare.