Galectin-7 is found mainly in stratified squamous epithelia as well as in various other types of cancer cells. As with other members of the galectin family, the expression of galectin-7 has been shown to negatively regulate the development of some tumors while correlating with the progression of other tumor types. For example, up-regulation of galectin-7 is associated with rat mammary carcinomas and with progression to T-cell malignancy. Here, we provide evidence indicating that galectin-7 functions as an important molecule in the dissemination of lymphoma cells in vivo. We found that stable transfection of lymphoma cells with a plasmid encoding antisense galectin-7 cDNA significantly inhibited the dissemination and invasion of lymphoma cells to peripheral organs, thereby increasing the survival of mice. We also found that inhibition of galectin-7 in aggressive lymphoma cells correlated with a decreased invasion of tumor cells in target organs and a reduced expression of matrix metalloproteinase-9, a gene associated with a poor prognosis in non–Hodgkin's lymphoma. We finally examined the expression of galectin-7 in 50 specimens of different mature B-cell neoplasms and found high galectin-7 expression levels in a significant proportion of mature B-cell neoplasms but not in normal B cells. Taken together, these findings suggest that galectin-7 is a potential therapeutic target in the treatment of lymphoid malignancies. [Cancer Res 2007;67(6):2824–9]
Galectins constitute a family of animal lectins defined by shared consensus amino acid sequences and affinity for β-galactose–containing oligosaccharides (1). Members of the galectin family contain carbohydrate recognition domains of ∼130 amino acids, which are involved in β-galactoside binding (2–4). Some galectins are secreted by the cell, probably through a nonclassic secretory pathway, because all members do not have a signal sequence, which would be required for protein secretion through the classic secretory pathway. Thus, galectins, including galectin-1 and galectin-7, can be found in the extracellular space. Their ability to bind and cross-link to cell surface glycoconjugates, some of which are transmembrane proteins, can thus trigger a cascade of transmembrane signaling events (5–8).
In mammals, the distribution of galectins is tissue specific and their expression is developmentally regulated (1, 9). They play an important role in several physiologic processes, including embryonic development, wound healing, apoptosis, intercellular adhesion, cell migration, and immune response. They are also involved in several pathologic conditions, including infectious diseases and cancer. For example, galectin-1 and galectin-9 can induce tumor cell apoptosis when added exogenously to the cell, whereas galectin-7 and galectin-12 promote apoptosis through intracellular mechanisms (10).
Galectin-7 was initially described as a marker of the differentiation levels of keratinocytes by Madsen et al. (11). At first glance, galectin-7 should aid in the elimination of tumor cells because its expression is induced by p53 and functions as a regulator of apoptosis. A series of recent studies have indicated, however, that expression of galectin-7 was associated with tumor progression. Lu et al. (12) have first reported that galectin-7 was overexpressed in rat mammary carcinomas induced by 7,12-dimethylbenz(a)anthracene. Rorive et al. (13) have subsequently observed that galectin-7 expression was markedly down-regulated in benign thyroid tumors compared with malignant ones. Using an approach based on genomic analysis of an aggressive lymphoma variant and its nonaggressive parental cells, we found that the most prominent change among the genes tested was the strong up-regulation of galectin-7 (14). Here, we used an antisense approach to provide evidence that decreased expression of galectin-7 in aggressive murine lymphoma cells inhibits their aggressive behavior in vivo. Furthermore, examination of samples from patients with different mature B-cell lymphoproliferative disorders shows for the first time that galectin-7 is expressed at high levels in lymphoid malignancies but not in normal lymphocytes. Our data suggest that galectin-7 may represent a potential target for lymphoma therapy.
Materials and Methods
Cell lines and reagents. The aggressive 164T2S11 (S11) lymphoma cell line was established from in vivo passages of the parental 164T2 cell line and has been described elsewhere (15). This aggressive lymphoma cell line expresses αβTcR, CD3ε, TL, gp70, LFA-1, ICAM-1, and ICAM-2 and is maintained in culture without loss of aggressiveness (14, 15). Cells were maintained in RPMI 1640 complete medium [supplemented with 8% (v/v) FCS, 2 mmol/L l-glutamine, 10 mmol/L HEPES buffer, 0.1 unit/mL penicillin, 50 μg/mL streptomycin, and 55 μmol/L β-mercaptoethanol]. All products were from Life Technologies (Burlington, Ontario, Canada). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit and anti-mouse secondary antibodies were obtained from Amersham Biosciences (Piscataway, NJ). The rabbit polyclonal antibody specific for galectin-7 has been described (16). The rabbit polyclonal antibody for galectin-1 was kindly provided by Dr. Brian Key (University of Queensland, Brisbane, Queensland, Australia; refs. 17, 18). All other reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated.
Human specimens. Peripheral blood or bone marrow cells were obtained from patients with different mature B-cell lymphoproliferative diseases (LPD). These samples were collected by the Quebec Leukemia Cell Bank after informed consent. Morphology, immunophenotyping, and molecular cytogenetics of tumors were used as criteria to classify them according to the WHO classification of tumors (19). Fifty samples were analyzed: 31 chronic lymphocytic leukemias (CLL; including 1 case of transformed CLL), 2 B-cell prolymphocytic leukemias, 2 follicular lymphomas, 2 splenic marginal zone lymphomas, 5 mantle cell lymphomas, 1 lymphoplasmacytic lymphoma/Waldenström macroglobulinemia, 2 Burkitt lymphomas, and 5 B-cell LPD not classified. As controls, purified CD19+ B cells were obtained from peripheral blood mononuclear cells from 10 healthy donors using magnetic cell sorting with CD19 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and the autoMACS separator (Miltenyi Biotec).
Western blot analysis. Cells and frozen tissues were homogenized and resuspended in lysis buffer [10 mmol/L Tris (pH 7.5), 2.5% Triton X-100] containing protease inhibitors (Roche, Laval, Quebec, Canada). Equal amounts of whole-cell (50 μg) or tissue (100 μg) extracts were separated on SDS-PAGE gel and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Mississauga, Ontario, Canada). The membranes were blocked with 10% milk in PBS/0.5% Tween 20 overnight and subsequently blotted for 2 h with primary antibodies. Membranes were probed with polyclonal rabbit anti-galectin-7 (1:1,000), rabbit anti-galectin-1 (1:1,000; refs. 17, 18), or mouse anti-β-actin antibodies. Secondary antibodies consisted of HRP-conjugated donkey anti-rabbit or anti-mouse IgG. Detection was done by the enhanced chemiluminescence method (Amersham Biosciences).
RNA isolation and semiquantitative PCR. Total RNA was isolated from lymphoma cells or tissues using Trizol reagent according to the manufacturer's instructions (Invitrogen Canada, Inc., Burlington, Ontario, Canada). Total RNA (2 μg) was reverse transcribed using the OmniScript reverse transcriptase (Qiagen, Mississauga, Ontario, Canada) and PCR amplified using the following conditions: 94°C for 0.5 min, 58°C for 1 min, and 72°C for 1 min followed by a final extension step at 72°C for 10 min. The primers used for PCR amplification were the following: murine galectin-7, 5′-CCATGTCTGCTACCCATCAC-3′ (sense) and 5′-GCTTAGAAGATATTCAATGAATGC-3′ (antisense); murine matrix metalloproteinase-9 (MMP-9), 5′-CGAGTGGACGCGACCGTAGTTGG-3′ (sense) and 5′-CAGGCTGAGCACGCCATACAG-3′ (antisense); murine galectin-1, 5′-ggtggcctcggacgccaagag-3′ (sense) and 5′-ctggcttcactcaaaggccacgcac-3′ (antisense); β-actin, 5′-CATGGATGACGATATCGCTGCGC-3′ (sense) and 5′-GCTGTCGCCACGCTCGGTCAGGAT-3′ (antisense); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (sense) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ (antisense). Thirty or 35 cycles of amplification were done in a thermal cycler (model PTC-100 TM, MJ Research, Watertown, MA). PCR assays using equal amounts of RNAs that were reverse transcribed and amplified by PCR for 25 to 40 cycles with gene-specific primers confirmed that the amplification was in the linear range for each gene. Each assay was repeated two to four times to verify the accuracy of the results. As an internal control, amplification of GAPDH or β-actin mRNA was carried out by reverse transcription-PCR (RT-PCR) using specific primers. Amplified products were analyzed by electrophoresis on 1.2% agarose gels using ethidium bromide staining and UV illumination. Loading was equalized to the internal control mRNA.
Generation of stable transfectants expressing galectin-7 antisense. To obtain stable S11 transfectants expressing galectin-7 antisense, the cDNA encoding the murine galectin-7 (20) was cloned in the antisense orientation in Srα eukaryotic expression vector (kindly provided by Dr. François Denis, INRS-Institut Armand-Frappier, Laval, Quebec, Canada) using SpeI and NotI restriction enzymes to excised the cDNA, the SpeI fragment was blunted, and the galectin-7 cDNA was inserted into Srα vector using NotI and EcoRV restriction enzyme. Controls were generated using S11 lymphoma cells transfected with the empty Srα vector alone. Transfection was carried out by electroporation (Gene Pulser, Bio-Rad Laboratories) and done using the following variables: 40 μg DNA per 107 cells in PBS on ice, 960 μF, and 260 mV. After 48 h of culture in complete medium, transfected cells were allowed to grow in complete medium containing 2 μg/mL puromycin. Individual colonies were expanded, and PCR analysis was carried out on genomic DNA for assessing vector insertion. Galectin-7 expression was monitored by Western blot analysis.
Peripheral lymphoid tumor growth. Lymphoma cells (107) transfected with the empty vector (Srα) or with vectors encoding galectin-7 antisense were inoculated i.v. via the tail vein of 6- to 8-week-old C57BL/6 mice (5–10 mice per group). Animals were monitored periodically for clinical signs of lymphoid tumor growth: dyspnea, runting, and splenomegaly. When moribund, mice were sacrificed, and spleen, liver, and kidneys were examined and collected at necropsy. Biopsy fragments were fixed in formaldehyde solution and stained for histology or processed for gene expression by RT-PCR or Western blot analysis as described above.
Quantitative RT-PCR analysis. Quantitative RT-PCR analyses were carried out using the DyNAmo SYBR Green 2-Step qRT-PCR kit (New England Biolabs, Ipswich, MA). Briefly, total RNA (1 μg) was reverse transcribed using the Moloney murine leukemia virus reverse transcriptase (New England Biolabs). Real-time PCR was then done with a Rotor-Gene 3000 PCR cycler (Corbett Research, Sydney, New South Wales, Australia) using the following conditions: 40 cycles of 94°C/10 s, 64°C/30 s, and 72°C/30 s. The primers used for quantitative PCR amplification were 5′-TTGCTCCTTGCTGTTGAAGACCAC-3′ for sense human galectin-7 and 5′-AGGTTCCATGTAAACCTGCTGTGC-3′ for antisense. Galectin-7 dsDNA serial dilutions were used for establishing a standard curve ranging from 227 to 227,000 copy numbers. The results were analyzed using the Rotor-Gene 6 software. Positive/negative cutoff criteria were determined according to the maximum SDs obtained from values with the CD19+ B-cell samples.
Statistical analysis. Comparisons between different groups for tumor uptake and survival distribution (mean survival time) were made using a log-rank test. The level of significance was determined at P < 0.05. Differences between various groups for galectin-7 expression were made using a Student's t test.
Stable transfection of lymphoma cells with antisense galectin-7 RNA. To examine the role of galectin-7 in tumor progression, a cDNA fragment spanning the translational start site of the mouse galectin-7 gene was inserted into the pSRα vector in the antisense direction. The positive plasmid was verified by digestion with restriction enzymes and DNA sequence. The 164T2S11 lymphoma cells were stably transfected with either this antisense construct (S11G7AS) or a construct containing a control sequence (S11SRα). S11 is a mouse lymphoma cell line, which constitutively expresses galectin-7 (Fig. 1). The efficacy of galectin-7 antisense RNA to suppress galectin-7 at both the mRNA and protein levels was then examined by RT-PCR and Western blot in several independently derived clones. We found that galectin-7 expression was almost completely inhibited at both the mRNA and the protein levels in clones C, D, and F compared with control clones transfected with the Srα vectors without the inserted antisense sequence (Fig. 1A and B). These clones were selected for all subsequent in vivo experiments. Inhibition of galectin-7 protein expression was specific, as the antisense construct did not affect expression of galectin-1 (Fig. 1C).
Reduced MMP-9 secretion in lymphoma cells expressing lower levels of galectin-7. Previous studies have shown that members of the galectin family can bind to cell surface receptors expressed on normal or transformed lymphocytes, triggering behavioral changes and/or inducing expression of new genes (7, 21–23). For galectin-7, we have shown, specifically, that it can induce the expression of mmp-9 gene in lymphoma cells (20). Several studies have reported that release of MMP-9 correlated with the tumorigenic phenotype of various tumor cell types, the most compelling evidence of its involvement in tumor invasion being a study showing that MMP-9–specific ribozyme suppresses the metastatic phenotype of tumor cells (24). To determine whether we had achieved a functional inhibition of galectin-7 in our S11 lymphoma cells, we compared the level of mmp-9 gene expression in S11G7AS clones and control clones. Our results showed that mmp-9 gene expression was lower in lymphoma cells containing the antisense galectin-7 cDNA compared with control cells (Fig. 2), consistent with the idea that galectin-7 controls the expression of mmp-9 in lymphoma cells.
Down-regulation of galectin-7 expression results in reduced tumorigenicity of lymphoma cells. To determine whether reduced galectin-7 expression by lymphoma cells leads to impaired lymphoma dissemination and invasion in vivo, antisense-transfected or control-transfected S11 lymphoma cell clones were injected i.v. into syngeneic immunocompetent C57BL/6 mice. Our results showed that mice that expressed the galectin-7–specific antisense in S11 lymphoma cells showed a significantly delayed dissemination and growth of lymphoid tumors compared with mice whose lymphoma cells had been transfected with the control vector (Fig. 3A). Thus, the mean survival time of mice injected with the galectin-7 antisense was significantly longer (32 ± 19.96 versus 14 ± 0.53 days) than that of mice injected with control transfectants (P < 0.001). To investigate in more details the inhibitory effects of galectin-7 on metastasis of S11 lymphoma cells, analysis of galectin-7 expression in tumors collected at necropsy was carried out. We found that galectin-7 expression was substantially reduced in tumors bearing antisense cDNA relative to the controls as assessed by RT-PCR and Western blot, indicating that the levels of galectin-7 remained suppressed in S11G7AS cells in vivo (Fig. 4).
Lymphoma invasion reduced in peripheral organs by down-regulation of galectin-7. Because galectin-7 has been shown to induce mmp-9 gene expression and has previously been shown to promote lymphoma cell infiltration in peripheral organs, we compared the level of tumor cell infiltration in peripheral organs of mice injected with the S11G7AS and S11 cells transfected with the control vector by histochemical staining. As shown in Fig. 3B, we found a significant decrease in the overall infiltration of tumor cells in the kidneys of mice injected with cells containing the antisense cDNA compared with the controls. Similar results were obtained with the liver (data not shown). These results indicate that the tumor-expressed antisense cDNA for galectin-7 has an inhibitory effect on infiltration of tumor cells in target tissues and are therefore consistent with our previous data showing that galectin-7 promotes MMP-9 expression and tissue invasion (20).
Quantitative expression of galectin-7 in human lymphoma cells. Galectin-7 is expressed in human malignant thyroid tumors and type IV hypopharyngeal squamous cell carcinoma (13, 25). We have also shown that galectin-7 is expressed in murine aggressive lymphoma cells but not in normal lymphocytes (14). To study the expression of galectin-7 in human lymphoid cells, we measured the level of galectin-7 mRNA expression by quantitative RT-PCR in 50 specimens of different mature B-cell neoplasms. This group of hematologic malignancies represents over 85% of all known lymphoid neoplasms (19). Our results showed that 18 of 50 lymphoid specimens expressed high levels of galectin-7 transcripts. Eleven of 31 CLLs, 2 of 2 follicular lymphomas, 1 of 5 mantle cell lymphomas and B-cell LPD not classified, and 1 of 2 B-cell prolymphocytic leukemia, Burkitt lymphoma, and splenic marginal zone lymphoma specimens were positive. In contrast, no significant levels of galectin-7 were found in purified human CD19+ B cells isolated from peripheral blood (Fig. 5).
In the present work, experimental evidence is provided to show that galectin-7 functions as an important molecule in the dissemination of lymphoma cells in vivo. More specifically, our results showed that stable transfection of lymphoma cells with a plasmid encoding antisense galectin-7 cDNA markedly inhibited the dissemination and invasion of lymphoma cells to peripheral organs, thereby increasing the survival of mice compared with controls. Additionally, we showed that inhibition of galectin-7 in aggressive lymphoma cells correlated with a decreased invasion of tumor cells in target organs and a reduced expression of mmp-9, a gene associated with a poor prognosis in non–Hodgkin's lymphoma (26, 27). Finally, we report for the first time high galectin-7 expression levels in a significant proportion of mature B-cell lymphoid neoplasms but not in normal B lymphocytes.
Many galectins have been reported to play an important role in cancer progression (10). At first glance, because galectin-7 functions as a regulator of differentiation and apoptosis in several cell types (16, 28–30), one would expect that galectin-7 should favor the elimination of tumor cells during cancer progression. However, in addition to aggressive lymphoma cells, high levels of galectin-7 expression have been reported in chemically induced rat mammary tumors (12), in human malignant thyroid tissues (13), and in type IV hypopharyngeal squamous cell carcinoma (25). This apparent discrepancy can be explained by the fact that, depending on the cell types, galectin-7 could be preferentially found in the cytoplasm, in the nucleus, or in the extracellular space, thereby having distinct functions according to its cellular localization (29). Alternatively, galectin-7 could modulate distinct biological processes according to specific interactions with a variety of intracellular and extracellular proteins that differ between cell types. Clearly, future investigations should address this issue. In the present case, its inhibition via an antisense strategy was sufficient to severely inhibit the dissemination of metastatic lymphoma cells to peripheral tissues.
Although we were able to achieve a significant inhibition of reduction of lymphoma development in mice, a significant number of mice still developed lymphomas when injected with lymphoma cells expressing the antisense vector specific for galectin-7. Several possibilities can explain this observation. First, it is conceivable that incomplete inhibition of galectin-7 was at least in part responsible for the development of some of these lymphomas. Although we have not detected expression of galectin-7 in lymphoid tumors collected from mice at necropsy, we detected some expression of galectin-7 by RT-PCR in some lymphoid tumors collected at later times. Alternatively, it is probable that other genes can also positively contribute to the aggressive behavior of lymphoma cells. A case in point is MMP-9, which has been associated with a poor prognosis of non–Hodgkin's lymphoma (26, 27). Whether MMP-9 is essential for the protumorigenic activity of galectin-7 remains, however, to be established. Given the fact that galectin-7 is secreted by lymphoma cells and can bind several receptors expressed on the surface of stromal cells and given the fact that the tumor microenvironment plays a significant role in tumor progression (31), it is also possible that secretion of galectin-7 by aggressive lymphoma cells stimulates peritumoral cells to secrete either mmp gene or other prometastatic genes. Alternatively, galectin-7 could induce apoptosis of infiltrating T cells, a scenario similar to that observed with extracellular galectin-3, which may act as a proapoptotic factor (32). These possibilities are currently under investigation.
This study reports galectin-7 expression for the first time across a broad spectrum of B-cell lymphoid malignancies. Analysis of a larger number of samples will be necessary to establish whether expression levels of galectin-7 correlate with the most aggressive forms of the diseases or with tumor recurrence rates as recently shown for hypopharyngeal squamous cell carcinoma (25). Nevertheless, we were able to confirm that galectin-7, a gene previously considered as a keratinocyte marker and whose expression is restricted to stratified epithelia, is commonly expressed in several forms of human and mouse lymphoid disorders but not in normal lymphocytes. Questions can be asked about the mechanisms responsible for increased galectin-7 levels in lymphoid malignancies. Interestingly, four of five patients have the highest levels of galectin-7 mRNA (the two cases of CLL and the two patients with follicular lymphoma), which is at first somewhat at odds with a previous study showing that galectin-7 is under the control of p53 (33). Whether the other p53 allele is functional or is mutated remains to be tested. In fact, the association of galectin-7 with p53 should normally aid the host against tumor development. This is so neither in our case nor for head and neck squamous cell carcinomas (25) and possibly also in mammary carcinomas (12). Previous results showing that hypomethylating agents induce the expression of galectin-7 in lymphoma cells suggest that epigenetic mechanisms may play an important role in this process and/or that cell-specific transcriptional mechanisms may be involved. This issue is currently under investigation.
In summary, we now document for the first time that galectin-7 is expressed in human lymphoid malignancies and propose that it is a critical tumor-modulating gene that controls the dissemination of lymphoma cells via MMP-9. Based on the importance of these molecules and the biochemical pathways they regulate, galectin-7 may significantly affect lymphoma progression in the clinical setting.
Grant support: Fonds de la Recherche en Santé du Québec scholarship (Y. St-Pierre), Fonds de la Recherche en Santé du Québec studentship (M. Demers), Fondation Armand-Frappier studentship (K. Biron-Pain), and Réseau de Recherche sur le Cancer (J. Hébert). A. Lamarre holds the Jeanne and Jean-Louis Lévesque Chair in Immunology of the Jean-Louis Lévesque Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. François Denis for providing vectors, Dr. Brian Key for providing anti-galectin-1 antibody, Dr. Édouard F. Potworowski for reviewing the manuscript, Doris Legault and Diane Tremblay for their excellent technical support, Marie Désy for statistical analysis, and M. Giovanni D'Angelo for expert morphologic analyses of the human lymphoid specimens.