Aberrant expression of sialidase and cancer progression

Abstract

Aberrant sialylation is closely associated with the malignant phenotype of cancer cells including metastatic potential and invasiveness. However, its biological significance and molecular mechanisms have yet to be fully elucidated. To determine causes and consequences, we have focused attention on mammalian sialidases, which cleave sialic acids from gangliosides and glycoproteins. The four types of human sialidases identified to date behave in different manners during carcinogenesis. One, found in the lysosomes, shows down-regulation in cancers, promoting anchorage-independent growth and contributing to metastatic ability, while another, found in the plasma membranes, exhibits marked up-regulation, resulting in suppression of apoptosis. The present review summarizes mostly our results on aberrant expression of sialidases and their possible roles in cancer progression.

Introduction

Sialidases are glycosidases catalyzing the removal of α-glycosidically linked sialic acid residues from the terminal positions of the carbohydrate groups of glycoproteins and glycolipids, which is the initial step in the degradation of these glycoconjugates. The sialic acids are considered to play important roles in various biological processes largely in two ways, one related to their hydrophilic and acidic properties exerting physicochemical effects on glycoconjugates to which they are attached, and the other as recognition sites or in an opposing fashion as masking sites.^1)–3) The removal of sialic acids catalyzed by a sialidase, therefore, greatly influences many biological processes through changing the conformation of glycoproteins and through recognition and masking of biological sites of functional molecules. In fact, sialidases of mammalian origin have been implicated not only in lysosomal catabolism but also in regulation of important cellular events including cell differentiation, cell growth and apoptosis.³⁾

Aberrant glycosylation is a characteristic feature of cancer cells, and in particular, alterations in sialylation during malignant transformation have been proposed to be closely associated with malignant phenotype in terms of metastatic potential and invasiveness. In the 1960’s and 1970’s, the subject of cell surface sialic acids in cancer cells received great attention: a large number of studies suggested that the increase in negative surface charge determining electrophoretic mobility was correlated with reduced adhesiveness of tumor cells, whereas decrease in the surface charge on incubation with bacterial sialidase caused suppression of malignancy. A general increase in sialylation is often found in cell surface glycoproteins of malignant cells,⁴⁾ and altered sialylation of glycolipids is also observed as a ubiquitous phenotype, associated with the appearance of tumor-associated antigens, aberrant adhesion, and blocking of transmembrane signaling.⁵⁾ However, drawing definite conclusions regarding physiological links between sialic acid contents and malignant properties is difficult due to controversial experimental results. To cast further light on the causes of such aberrant sialylation and the consequences, our studies have focused on mammalian sialidases, which regulate the cellular sialic acid contents and function of glycoconjugates by desialylation. Sialidase expression levels indeed change in response to various cellular phenomena and especially in relation to cancer progression.^6),7)

Mammalian sialidases

Existence of four sialidases

Four types of mammalian sialidases have been identified and characterized to date, designated as Neu1, Neu2, Neu3 and Neu4. The first three are localized predominantly in the lysosomes, cytosol and plasma membranes, respectively, and the fourth recently identified sialidase, Neu4, has been suggested to exist in lysosomes, or in mitochondria and certain intra-membranous components. The properties of the four forms are briefly described and compared in Table 1. All contain several Asp boxes (-Ser-X-Asp-X-Gly-X-Thr-Trp-) and the Arg-Ileu-Pro sequence, which are the conserved sequences found in sialidases from microorganisms,⁸⁾ despite the fact that the mammalian enzymes do not exhibit any other sequence similarities to microbial sialidases. Among human sialidases, the overall amino acid identity of NEU1 to the other sialidases is relatively low (19–24%), while NEU2, NEU3 and NEU4 show 34–40% homology to each other. Regarding comparative expression levels of human sialidases, NEU1 generally shows the highest expression, 10–20 times higher than those of NEU3 and NEU4, while NEU2 expression is extremely low, only four- to ten-thousandth of the NEU1 value at the most in a wide range of tissues, as assessed by quantitative real time RT-PCR using a standard curve for each cDNA,⁹⁾ although these profiles differ among the human, rat and mouse.

Table 1.

Four types of mammalian sialidases

	Neu1	Neu2	Neu3	Neu4
Majora subcellular localization	Lysosomes	Cytosol	Plasma membrane	Lysosomes41) Mitochondria9) Intracellular membranes9),42)
Good substrates	Oligosaccharides Glycopeptides	Oligosaccharides Glycoproteins Gangliosides	Gangliosides	Oligosaccharides Glycoproteins Gangliosides
Optimal pH	4.4–4.6	6.0–6.5	4.6–4.8	4.4–4.5
Total amino acids
(human)	415	380	428	496 (484)
(mouse)	409	379	418	478
Chromosome location
(human)	6p 21.3	2q 37	11q13.5	2q37.3
(mouse)	17	1	7	10
Possible function	Degradation in lysosomes	Myoblast differentiation	Neural differentiation	Apoptosis
	Immune function Elastic fiber assembly	Neural differentiation	Apoptosis Adhesion
Frequent changes in cancer
References for gene cloning	(10–15)	(20–25)	(27, 29, 30)	(9, 40–42)

Sialidase Neu1

Taking advantage of SM/J inbred strain of mice carrying a defective sialidase allele, the mouse Neu1 gene^10)–12) was mapped near the H-2D end of the a major histocompatibility complex (MHC) on chromosome 17 by linkage analysis, a region which is syntenic to human MHC on chromosome 6. The human lysosomal sialidase,^13)–15) NEU1, has been extensively investigated as a target in sialidosis. It was found that NEU1 is associated with a protective protein (carboxypeptidase A) and β-galactosidase as a complex in lysosomes, and dissociation of the complex leads to sialidase inactivation.¹⁶⁾ NEU1 features a lysosomal C-terminal targeting motif and evidence has demonstrated that a protective protein is responsible for NEU1 transport to lysosomes. However, recent observations have revealed an intracellular distribution of the sialidase localizing at plasma membranes as well as lysosomes under certain conditions. Neu1 possesses narrow substrate specificity, with oligosaccharides and glycopeptides serving as good substrates and is involved in cellular signaling for immune responses and elastic fiber assembly during the transportation to plasma membranes, as well as glycoconjugate catabolism in lysosomes. During PMA-induced monocyte differentiation, Neu1 is up-regulated and targeted together with carboxypeptidase to MHC II-positive vesicles that merge later with the plasma membrane.¹⁷⁾ Facilitation of elastic fiber assembly is probably through desialylation of microfibrillar glycoproteins and other adjacent matrix glycoconjugates.^18),19)

Sialidase Neu2

Cytosolic sialidase Neu2 was the first example of a mammalian sialidase for which cDNA cloning was achieved from rat skeletal muscle.²⁰⁾ The homologues were cloned from cDNA libraries of CHO,²¹⁾ mouse brain,^22),23) and rat thymus²⁴⁾ and from a genomic library of human skeletal muscle,²⁵⁾ showing high amino acid identity (98–70%) to the rat gene. Unlike Neu1 sialidase, Neu2 is able to hydrolyze glycoprotein, oligosaccharides and gangliosides at near neutral pH. The three-dimensional structure of human NEU2 has recently been determined by X-ray crystallography²⁶⁾ and compared with that of S. typhimurium sialidase, providing evidence for a canonical six-blade beta-propeller with the active site in a shallow crevice. There exist residues recognizing the N-acetyl and glycerol moieties of 2-deoxy-2,3-dehy-dro-N-acetylneuraminic acid (DANA), as observed for viral and bacterial sialidases, and Neu2 is thought to participate in muscle cell and neuronal differentiation.

Sialidase Neu3

The plasma membrane-associated sialidase Neu3 was first cloned from a bovine brain library,²⁷⁾ based on peptide sequence information obtained with the purified enzyme protein,²⁸⁾ and later from the human genome data base.^29),30) The catalyzed hydrolysis is essentially specific for gangliosides other than GM1 and GM2, and addition of Triton X-100 is needed to obtain the maximum activity in vitro. The major subcellular localization of the bovine sialidase proved to be plasma membranes on Percoll density gradient centrifugation of cell homogenates and by immuno-fluorescence staining. With administration of the radiolabeled ganglioside GD1a to murine Neu3-transfected cells, Neu3 was shown to hydrolyze ganglioside substrates in intact living cells at a neutral pH mainly through cell-to-cell interactions.³¹⁾ The murine Neu3 can also regulate cell apoptosis in human fibroblasts by producing ceramide from GM3 in plasma membranes through detachment of sugar units.³²⁾ Unlike the bovine and mouse Neu3 sialidases, the human ortholog NEU3 is not always detected on the cell surface but may exist in other cellular membrane components and can be moved to and concentrated at the leading edges in response to growth stimuli.³³⁾ Recent analysis of membrane topology has suggested that the sialidase might be localized partially on the cell surface as a peripheral membrane protein and also in endosomal structures.³⁴⁾ Neu3 participates in neurite formation in mice²³⁾ and in human neuroblastoma cells,³⁵⁾ and in regulation and regeneration of rat hippocampus neurons.^36),37) It is located in rafts of neuroblastoma cells³⁸⁾ and in caveolae of HeLa cells,³⁹⁾ closely associated with caveolin-1. In response to growth stimuli such as EGF treatment, human NEU3 mobilizes to membrane ruffles together with Rac-1, a small G protein participating in actin reorganization and cell motility, and enhances cell movement.³³⁾ Recent our observations have further provided evidence that NEU3 regulates transmembrane signaling by interacting with signaling molecules including caveolin-1, Rac-1, interim β4, Grb-2 and EGFR as well as by modulation of gangliosides as an enzyme.⁴⁰⁾

Sialidase Neu4

The fourth sialidase, Neu4, was only recently identified based on cDNA sequences in public databases.^9),41)–43) With regard to subcellular localization of the human ortholog, NEU4, two different descriptions have been reported on the basis of gene transfection studies: one featuring targeting to the lysosomal lumen,⁴²⁾ and the other to mitochondria⁹⁾ and intracellular membranes.^9),43) NEU4 appears to consist of iso-forms which differ in their possession of 12 N-terminal amino acid residues which act in mitochondrial targeting. The isoforms are also differentially expressed in a tissue-specific manner, brain, muscle and kidney containing both, and the liver and colon possessing predominantly the short form, as assessed by RT-PCR.⁴⁴⁾ The iso-forms possess broad substrate specificity, including activity towards mucin. The biological functions of Neu4 are not clear at present, but several possibilities have been suggested. Expression in cells of sialidosis patients results in clearance of storage materials from lysosomes and thus the human ortholog NEU4 may be useful for novel therapeutic purposes. Furthermore, NEU4 may be involved in cell apoptosis or neural differentiation,⁴⁴⁾ based on the observation that the long form with the mitochondrial targeting signal probably regulates the level of GD3, which is known to be an apoptosis-related ganglioside.

Aberrant expression of sialidase in cancer

Endogenous sialidases in cancer

In the 1970–1980’s, several observations on alteration of endogenous sialidase activity in cancer suggested that this family of enzymes might be related to tumorigenic transformation and tumor invasiveness. For example, Schengrund et al.⁴⁵⁾ described increased sialidase activity toward gangliosides in BHK-transformed cells, and Bosmann et al.⁴⁶⁾ observed elevated sialidase activity in human cancer tissues with fetuin as a substrate. Loss of cell density-dependent suppression of membrane-bound sialidase activity for gangliosides is observed in 3T3-transformed cells.⁴⁷⁾ In the human promyelocytic leukemia cell line HL-60, stimulation of sialidase activity toward 4MU-NeuAc occurs during cell differentiation into granulocytes by retinoic acid or DMSO.⁴⁸⁾

However, it has remained uncertain whether the activities are due to the same or different types of sialidase. We have therefore made attempts to isolate and characterize sialidases from rat tissues, and have presented evidence for the existence of the four types of sialidases differing in their subcellular localization and enzymatic properties, including the substrate specificity.^49)–51) Biochemical characterization of the four sialidases suggested that each might play a unique role depending on its particular subcellular localization and catalytic properties. In fact, recent advances in the molecular cloning of mammalian sialidases have confirmed the existence of the four forms and their biological roles, as described above. Using a differential assay procedure for each form, we observed that intra-lysosomal and membrane-bound sialidase activities were elevated, whereas cytosolic sialidase activity was reduced, in rat hepatomas as compared with normal liver.⁵²⁾ In mouse epidermal JB6 cells exposed to TPA and in anchorage-independent transformants, we also found lysosomal sialidase activity to be decreased while plasma membrane-associated sialidase activity was increased as compared with that in untreated JB6 cells.⁵³⁾ Reduction in lysosomal sialidase also occurred in rat 3Y1 fibroblasts after src-transformation, levels of activity inversely correlating with the metastatic potential. Furthermore, v-fos transfer to these transformed cells induced an even more severe decrease in the sialidase activity with acquisition of high lung metastatic ability. Various lysosomal enzymes other than sialidase were not appreciably affected by the transformation, suggesting that the alteration occurs specifically in sialidase. Since metastatic potential did not parallel the sialic acid levels, it is likely that altered sialidase expression is more important for metastasis in transformed cells (Fig. 1).⁵⁴⁾

Fig. 1.

Inverse relationship between Neu1 expression and metastatic potential.^54),55) Lysosomal sialidase activity was measured in rat 3Y1 transformants (a). The activity was decreased in rat 3Y1 fibroblasts after src-transformation, and v-fos transfer resulted in a more severe decrease in the activity with acquisition of high metastatic ability. Lysosomal sialidase activity (b) and NEU1 mRNA (c) levels were compared in mouse adenocarcinoma colon 26 cells of different metastatic potential, and found to be inversely correlated with their metastatic potential.

Sialidases Neu1 and Neu2 in cancer

After Neu1 gene was cloned, we measured its activity and mRNA level in mouse adenocarcinoma colon 26 cells of different metastatic potential⁵⁵⁾ as well as in the rat 3Y1 transformants described above.⁵³⁾ A good inverse relationship between Neu1 expression level and matastatic ability was found in both cases (Fig. 1). We then investigated how sialidase expression influences metastasis by introducing a cytosolic sialidase (Neu2) cDNA, with broad substrate specificity, encompassing both glycoproteins and gangliosides, into a B16–BL6 mouse melanoma varient subclone derived from B16 melanoma known to be highly invasive and metastatic.⁵⁶⁾ Intravenous injection of stable transfectants into syngeneic mice resulted in marked decrease in experimental pulmonary metastasis, invasiveness and cell motility but no change in cell growth or cell attachment to fibronectin, collagen type VI or laminin. Analysis of the molecular mechanisms showed that sialidase overexpression did not lead to any significant changes in cell surface or intracellular glycoproteins, while there were a decrease in ganglioside GM3 and an increase in lactosylceramide as assessed by thin layer chromatography. When the sialidase gene was transfected into highly metastatic mouse colon 26 adenocarcinoma cells, changes in the sialyl Le^x level were observed in addition to marked suppression of metastasis.⁵⁵⁾ Compared to low metastatic NL4 and NL44 cell lines, highly metastatic NL17 and NL22 cells exhibit low expression of Neu1 sialidase, accompanied by higher levels of sialyl Le^x and GM3. NL17 stable transfectants show marked inhibition of lung metastasis, invasion and cell motility with a concomitant decrease in sialyl Le^x and GM3 levels, in line with spontaneously low metastatic sublines having a relatively high level of endogenous sialidase. Treatment of the cells with antibodies against sialyl Le^x and GM3 affected cell adhesion and/or cell motility, providing evidence that desialylation of these molecules, as targets of sialidase, is involved in the suppression of metastasis. However, the highly metastatic cells exhibited rather decreased sialic acid contents, both total and cell surface, as compared to the low metastatic cells, consistent with the sialidase activity. The results together indicate that the sialidase level is a determining factor affecting metastatic ability, irrespective of the sialic acid contents. In addition, Neu2 may participate in cell apoptosis, Tringali et al. reporting that Neu2 gene introduction into leukemic K562 cells induced increased sensitivity to apoptotic stimuli by impairing Bcr-ABl/Src kinase signaling.⁵⁷⁾

To investigate whether overexpression of Neu1 sialidase can reverse metastatic ability, we next introduced a rat lysosomal sialidase gene into Bl6-BL6 melanoma cells.⁵⁸⁾ As expected, sialidase-over-expressing cells showed suppression of experimental pulmonary metastasis and tumor progression. In contrast to the case with Neu2 sialidase, the transfectants exhibited reduced anchorage-independent growth and increased sensitivity to apoptosis, induced by suspension culture or serum depletion in vitro, but no significant alterations in invasiveness, cell motility, or cell attachment. The results indicate that the sialidase affects malignant properties including the metastatic ability of cancer cells, in a manner different from that of Neu2. We next evaluated human ortholog NEU1 expression levels in human colon cancer by real time RT-PCR and by activity assays using 4MU-NeuAc as the substrate. The mRNA level showed a tendency towards decrease in cancer tissues as compared with that in the adjacent non-cancerous mucosa.⁵⁹⁾ Over-expression of the human sialidase gene NEU1 resulted in similar alterations of cancer cells to those observed in the murine cells, with suppressed cell migration and invasion in human colon adenocarcinoma HT-29 cell, whereas its knockdown resulted in the opposite effects. When NEU1-over-expressing cells were injected transsplenically into mice, the in vivo liver metastatic potential was significantly reduced.⁶⁰⁾

Sialidase Neu3 in cancer

Our investigation of plasma membrane-associated sialidase revealed that NEU3 mRNA levels were increased 3- to 100-fold in human colon cancer tissues compared to adjacent non-tumor mucosa, and associated with significant elevation of sialidase activity in the tumors (Fig. 2a).⁶¹⁾In situ hybridization showed the high sialidase expression in epithelial elements of adenocarcinomas (Fig. 2b). To understand the significance of the increased expression, cultured human colon cancer cells were treated with sodium butyrate, and changes in expression during differentiation and apoptosis were observed (Fig. 2c). NEU3 level was down-regulated by the treatment while NEU1 was up-regulated. Transfection of the NEU3 gene into cancer cells inhibited apoptosis accompanied by increased Bcl-2 and decreased caspase expression (Fig. 2d), while knock down of this gene with a short interfering RNA (siRNA) resulted in enhanced apoptosis. Colon cancer tissues exhibit marked accumulation of lactosylceramide, a possible NEU3 product, and addition of the glycolipid to cultures reduced the numbers of apoptotic cells in response to sodium butyrate treatment. These results indicate that high expression of NEU3 in cancer cells leads to protection against programmed cell death. In colon cancer cells, NEU3 differentially regulates cell proliferation through integrin-mediated signaling depending on the extracellular matrix.⁶²⁾ The sialidase further causes increased adhesion to laminins and consequent cell proliferation, but rather decrease in cell adhesion to fibronectin, collagen I and IV. Triggered by laminins, NEU3 can clearly stimulate phosphorylation of focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK), without any activation of fibronectin. NEU3 markedly enhances tyrosine phosphorylation of intergrin β4 only on laminin-5, with recruitment of Shc and Grb-2, and is co-immunoprecipitated by anti-integrin β4 antibody, suggesting that the association of NEU3 with integrin β4 might facilitate promotion of integrin-derived signaling on laminin 5.

Fig. 2.

Increased NEU3 expression and its apoptosis suppression in colon cancer.⁶¹⁾ NEU3 mRNA level was measured by quantitative RT-PCR in colon cancers and noncancerous mucosa (closed and open columns, respectively) (a) and in situ hybridization analysis was performed in colon cancer tissues (b). Apoptosis induced by sodium butyrate (NaBT) treatment was assessed by flowcytometry analysis with annexin V in mock- and NEU3-transfectants (c). Altered expression of apoptosis-related molecules was observed in NEU3 overexpressing cells (d).

NEU3 mRNA expression is also significantly increased in renal cell carcinomas (RCCs), correlating with elevation of interleukin (IL)-6, a pleio-tropic cytokine that has been implicated in immune responses and the pathogenesis of several cancers, including RCCs.⁶³⁾ In human RCC ACHN cells, IL-6 treatment has been shown to enhance NEU3 promotor luciferase activity 2.5-fold and endogenous sialidase activity significantly. NEU3 gene transfection or IL-6 treatment both result in suppression of apoptosis and promotion of cell motility, exerting synergistic effects in combination. NEU3 was found to hardly affect MAPK or IL-6-induced STAT3 activation but promoted the PI3K/Akt cascade in both IL-6 dependent and independent ways. Furthermore, IL-6 promoted Rho activation and the effect was potentiated by NEU3, leading to increased cell motility that was affected by LY294002, a PI3K inhibitor. NEU3 silencing by siRNA resulted in the opposite: decreased Akt phosphorylation and inhibition of Rho activation. As also described for colon tumors, gycolipid analysis showed decrease in ganglioside GM3 and increase in lactosylceramide after NEU3 transfection, these lipids apparently affecting cell apoptosis and motility. Thus, NEU3 activated by IL-6 stimulates IL-6-mediated signaling largely via the PI3K/Akt cascade in a positive feedback manner and contributes to expression of a malignant phenotype in RCCs. In addition, increase in the NEU3 mRNA level is often detected in prostate cancer, and shows a significant correlation with the histological differentiation grade (Kawamura S. et al., in preparation). Immunohistochemical examination of ovarian clear cell adenocarcinomas reveals NEU3 expression to be positive in 77.5% of all 71 patients of ovarian clear cell adenocarcinomas, and furthermore, a high level of NEU3 expression is significantly correlated with the T3 factor (T: tumor size) of the pTNM classification (cancer stage classification) according to the results of univariate and multivariate analyses.⁶⁴⁾

To define further the molecular mechanisms of NEU3 effects and its possible targets, the encoding gene was silenced by siRNA or overexpressed in human cancer cells.⁶⁵⁾ NEU3 silencing caused apoptosis without specific stimuli, accompanied by decreased Bcl-xl and increased mda7 and GM3 synthase mRNA levels in HeLa cells (Fig. 3a and b), whereas overexpression resulted in the opposite. Human colon and breast carcinoma cell lines, HT-29 and MCF-7 cells, appeared to be similarly affected by treatment with the NEU3 siRNA, but interestingly non-cancerous human WI-38 and NHDF fibroblasts and NHEK keratinocytes showed no significant changes (Fig. 3c). NEU3 siRNA was found to inhibit Ras activation and NEU3 over-expression to stimulate it with consequent influence on ERK and Akt. Ras activation by NEU3 was largely abrogated by PP2 (a src inhibitor) or AG1478 (an EGFR inhibitor), and in fact, siRNA introduction reduced phosphorylation of EGFR while overexpression promoted its phosphorylation in response to EGF (Fig. 4). To summarize these observations on NEU3 in cancer, the sialidase activates molecules including EGFR, FAK, ILK, Shc, integrin β4 and also Met, often up-regulated in carcinogenesis, and may thus cause accelerated development of malignant phenotypes in cancer cells.

Fig. 3.

Induction of apoptosis by siRNA-mediated NEU3 silencing in carcinoma cells but not in normal cells.⁶⁵⁾ After transfection of NEU3 siRNA, scrambled (Sc) or non-specific control siRNA, TUNEL assay (a) and MTT assay were performed in HeLa cells and (b) noncancerous keratinocytes (c).

Fig. 4.

A possible mechanism of apoptosis regulation by NEU3 in cancer cells.^40),65) NEU3 overexpression suppresses and its silencing accelerates apoptosis of cancer cells through modulation of EGF receptor phosphorylation and Ras activation.

Immunohistochemical analysis of surgical specimens using anti-NEU3 monoclonal antibody confirmed NEU3 upregulation in several human cancers. In colon cancer tissues, clear positive signals were observed in the carcinomatous parts, while non-cancerous mucosa was hardly stained. In prostate cancer, the intensity of the histochemical staining showed a positive relationship with the Gleason score, which reflects the pathological progression stage. In addition, our preliminary experiments with nude mice bearing tumor cells showed a significant tumor reduction on treatment with the NEU3 specific siRNA (Sato, I. et al., in preparation). These results indicate that the sialidase could indeed be a useful target for cancer diagnosis and therapy.

Sialidase Neu4 in cancer

When NEU4 mRNA levels were compared between human colon cancer and adjacent non-cancerous tissues, marked decrease in expression was noted in the tumors,⁵⁹⁾ in clear contrast to the NEU3 case. Levels were not significantly correlated with the histological differentiation or the pathological stage, but the T/NT (tumor to non-tumor expression ratio) value remained significant at p = 0.025 with degree of venous invasion (v) between v 0 (n = 28) and v1-3 (n = 13). In cultured human colon cancer cells, the enzyme was up-regulated in the early stage of apoptosis induced by either the death ligand TRAIL, or serum-depletion. Transfection of NEU4 gene into DLD-1 and HT-15 colon adenocarcinoma cells resulted in acceleration of apoptosis and decreased invasiveness and cellular motility. siR-NA-mediated NEU4 targeting, on the other hand, caused a significant inhibition of apoptosis and promotion of cellular invasiveness and motility. Lectin blot analyses revealed that desialylated forms of approximately 100-kDa glycoproteins were prominently increased with peanut agglutinin (PNA) in the NEU4-transfectants, whereas only slight changes in glycolipids were detected by thin layer chromatography. These results suggest that NEU4 plays important roles in the maintenance of normal mucosa, mostly through desialylation of glycoproteins and that down-regulation may contribute to invasive properties and protect against programmed cell death in colon cancers. Table 2 summarizes possible roles of the four sialidases in cancer progression as described above.

Table 2.

Roles of mammalian sialidases in cancer

Sialidase	References
Neu1	(58, 60)
in vivo
Suppression of metastasis and tumor growth (murine melanoma B16-BL6 cells and human colon HT-29 cells)
in vitro
decreased anchorage-independent growth and increased sensitivity to apoptosis (murine melanoma B16-BL6 cells)
decreased cell invasion and motility (human colon HT-29 cells)
Neu2	(55–57)
in vivo
suppression of metastasis (murine melanom B16-BL6 cells)
suppression of metastasis (colon adenocarcinom 26 cells)
in vitro
decreased cell invasion and motility (murine melanoma B16-BL6 cells) possible target: GM3
decreased cell invasion and motility (murine colon adenocarcinoma 26 cells) possible target:GM3 and sialylLex
increased sensitivity to apoptosis (human leukemic K562 cells)
Neu3	(61–65)
in vivo
no significant changes in metastasis (murine melanoma B16-BL6 cells)
in vitro
inhibition of differentiation (human colon cancer cells)
decreased sensitivity to apoptosis (human colon cancer cells)
increased adhesion to laminins and activation of integrin β4-mediated signaling (human colon cancer DLD-1cells)
increased cell motility and invasion and activation of IL-6-mediated signaling (human renal cell carcinoma ACHN cells)
activation of EGF receptor signaling (human cervical carcinoma HeLa cells)
Neu4	(59)
in vitro
increased sensitivity to apoptosis and decreased invasiveness and cellular motility (human colon adenocarcinoma DLD-1 and HT-15cells)

The alterations of malignant phenotypes described above were observed as the results of overexpression or silencing of the respective sialidase genes.

Future perspectives

In conclusion, investigation of mammalian sialidases has clarified some of the molecular bases of aberrant sialylation. We have documented that the expression level of NEU1 is a critical factor for metastasis, and NEU3 up-regulation is essential for survival of cancer cells, and that alteration in sialidase expression may be a defining factor for cancer progression, irrespective of sialic acid contents. Sialidase alterations, therefore, open up potential applications in cancer cure and diagnosis. As illustrated in Fig. 5, down-regulation of NEU3 expression by treatment with the specific siRNA, antibody or inhibitor may lead to prevention of cancer progression. In particular, taking advantage of its limited effects on normal cells, NEU3 siRNAs causing apoptosis in cancer cells could offer a useful tool for cancer therapy.

Fig. 5.

Functional relationship of three sialidases in human cancer cells and a possible role of NEU3 as a potential target for cancer diagnosis and therapy.

Profile

Taeko Miyagi graduated from Tohoku University, School of Medicine in 1968 and received her Ph.D. from Tohoku University, Graduate School of Medicine in 1973. She continued her research mainly on sialyltransferase and sialidase as a Research Associate and then as an Associate Professor with Prof. Shigeru Tsuiki at the Division of Biochemistry in the Institute of Development, Aging and Cancer, Tohoku University. In 1993 she moved to Miyagi Cancer Center Research Institute as a Head of the Division of Biochemistry and in 2004 she became Director of the Research Institute. For the past decade, she has focused her research on structure and function of mammalian sialidases and their carcinogenic alterations. Her wish is to apply the sialidase works for cancer diagnosis and therapy.