Acceleration of Sialyl Lewisx/a Expression Associated with Cancer Progression

Cell surface carbohydrate determinants undergo drastic changes during malignant transformation. It is well known that expression of sialyl Lewisx and sialyl Lewisa determinants is markedly enhanced in cancer cells. These determinants serve as ligands for selectins, the cell adhesion molecules present on endothelial cells, and they also mediate hematogenous metastasis of cancers.

Normal epithelial cells express various carbohydrate determinants, some of which have structures more complex than that of sialyl Lewisx/a. Good examples are sialyl 6-sulfo Lewisx and 23, 26 disialyl Lewisa determinants, which are the further modified forms of sialyl Lewisx/a determinants (1). These complex determinants are present on non-malignant gut epithelial cells and mediate interaction of epithelial cells with mucosal mesenchymal cells expressing Siglecs and other carbohydrate-binding molecules (2). Expression of genes responsible for the modifications of these determinants is impaired at the early stages of cancer initiation, mainly due to epigenetic silencing through promoter DNA methylation or histone deacetylation. Appearance of sialyl Lewisx/a determinants in early stage cancers is the result of "incomplete synthesis" of more complex determinants on normal epithelial cells. "Incomplete synthesis" is a classical concept proposed to characterize cancer-associated alteration of cell surface carbohydrates in the early 1980's.

Progression of cancer is a long process, sometimes spanning a number of years. Later, in the more advanced stages, cancer cells accumulate genetic abnormality and more malignant cancer cells evolve according to the principle of survival of the fittest, and such cells have higher infiltrative and metastatic activities. Expression of sialyl Lewisx/a determinants is further accelerated during the course of cancer progression.

One of the selection mechanisms for more malignant cells during cancer progression is their resistance adaptation against hypoxic conditions. Because of the uncontrolled proliferation of cancer cells, delivery of oxygen is significantly reduced in solid tumors, and some of the cancer cells are always subjected to a hypoxic environment (Fig. 1). When the normal cells are exposed to such an environment, the -chain of a transcription factor, hypoxia inducible factor (HIF), which is usually rapidly degraded in proteasome in the cytoplasm under normoxic conditions, undergoes a nuclear transition. After forming the heterodimer with the -chain constitutively present in the nucleus, it triggers the transcription of several important cellular genes which help the cells cope with the hypoxic environment. This is a reversible change in normal cells. In cancers, however, this process leads to the evolution of cancer cell clones that acquire constitutive and irreversible expression of HIF through the accumulation of genetic abnormality in oncogenes or anti-oncogenes.

One of the consequences of sustained HIF expression is a particular deviation in the intracellular carbohydrate metabolism, a metabolic shift from oxidative to elevated anaerobic glycolysis (the Warburg effect), which is correlated with the increased gene expression of some glycolytic enzymes and glucose transporters including GLUT1. Another consequence is the facilitated production of vascular endothelial growth factor (VEGF), which supports tumor angiogenesis.

Fig.1. Suggested mechanisms for increased expression of sialyl Lewisx/a during hypoxia-induced cancer progression.

Recently, we found a significant induction of sialyl Lewisx and sialyl Lewisa expression on epithelial cells cultured under hypoxic conditions, together with a concomitant increase of E-selectin binding activity (3). Participation of HIF in the process, direct or indirect, is clear since the introduction of dominant negative HIF to the cells completely abrogated these induced effects. A significant induction of transcription of genes for GLUT1, UDP-galactose transporter (UGT1), a fucosyltransferase (FUT7), a sialyltransferase (ST3O) and some other genes closely related to the carbohydrate metabolism was detected after hypoxic culture by DNA microarray technique and RT-PCR (3).

These results strongly suggest that augmentation of sialyl Lewisx/a expression on cancer cells is intimately related to the process of cancer progression, and the more malignant cancer cells tend to have enhanced expression of these carbohydrate determinants. This mechanism seems to contribute to the further enhancement of sialyl Lewisx/a expression on advanced cancer cells, which had already been predisposed to express these determinants by epigenetic gene silencing in the early stages of carcinogenesis. Expression of genes for GLUT1, UGT1, FUT7, and ST3O is significantly elevated in cancer cells prepared from surgical specimens of colon cancers, compared to non-malignant colonic epithelial cells taken from the same patients, especially those in the advanced stages such as Dukesí C and D (3, 4). This suggests that the HIF-induced transcription of these genes indeed takes place in the actual cancers of patients.

Interaction of cancer cells with endothelial cells mediated by selectins and sialyl Lewisx/a determinants may have patho-physiological relevance not only in the hematogenous metastasis of cancer as formulated earlier but also in the blood-vessel formation of tumors. We assessed the possible significance of this cell adhesion system in tumor vascularization in a recent study by employing an in vivo model using a cultured endothelial cell line, which expressed selectins and could adhere to human cancer cells expressing sialyl Lewisx/a (5). When human cancer cells and the endothelial cell line cells at a ratio of 10:1 were subcutaneously co-transplanted into the back of nude rats, the tumors formed were extensively vascularized throughout by the blood vessel-like structures formed by the endothelial cells, the lumen of which contained the host blood cells. The administration of anti-Lewisx/a antibodies resulted in a marked reduction in the size of tumors which were not vascularized and were accompanied by independent tiny remnant clumps composed of endothelial cells. These results served to corroborate the hypothesis that cell adhesion mediated by selectins and sialyl Lewisa/x determinants is significantly involved in tumor vascularization.

Cancer cells try to survive hypoxic conditions by acquiring sustained expression of HIF, thus altering their glucose metabolic pathway from aerobic to anaerobic, and also by inducing endothelial cell growth with VEGF. The HIF-induced enhancement of sialyl Lewisx/a expression on cancer also seems to be a link in the chain of these unfolding events, given that sialyl Lewisx/a determinants promote tumor vascularization (6).

Reiji Kannagi (Molecular Pathology, Aichi Cancer Center)
References (1) Kannagi R, Izawa M, Koike T, Miyazaki K, Kimura N: Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis. Cancer Sci., 95, 377-384, 2004
(2) Miyazaki K, Ohmori K, Izawa M, Koike T, Kumamoto K, Furukawa K, Ando T, Kiso M., Yamaji T, Hashimoto Y, Suzuki A, Yoshida A, Takeuchi M, Kannagi R: Loss of disialyl Lewisa, the ligand for lymphocyte inhibitory receptor sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) associated with increased sialyl Lewisa expression on human colon cancers. Cancer Res., 64, 4498-4505, 2004
(3) Koike T, Kimura N, Miyazaki K, Yabuta T, Kumamoto K, Takenoshita S, Chen J, Kobayashi, M, Hosokawa M, Taniguchi A, Kojima T, Ishida N, Kawakita M, Yamamoto H, Takematsu H, Suzuki A, Kozutsumi Y, Kannagi R: Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates. Proc. Natl. Acad. Sci. USA, 101, 8132-8137, 2004
(4) Kumamoto K, Goto Y, Sekikawa K, Takenoshita S, Ishida N, Kawakita M, Kannagi R: Increased expression of UDP-galactose transporter mRNA in human colon cancer tissues and its implication in synthesis of Thomsen-Friedenreich antigen and sialyl Lewis A/X determinants. Cancer Res., 61, 4620-4627, 2001
(5) Tei K, Kawakami-Kimura N, Taguchi O, Kumamoto K, Higashiyama S, Taniguchi N, Toda, K, Kawata R, Hisa Y, Kannagi R: Roles of cell adhesion molecules in tumor angiogenesis induced by cotransplantation of cancer and endothelial cells to nude rats. Cancer Res., 62, 6289-6296, 2002
(6) Kannagi R: Molecular mechanism for cancer-associated induction of sialyl Lewis X and sialyl Lewis A expression -The Warburg effect revisited. Glycoconjugate J., 20, 353-364, 2004
Links GP-C03 Nucleotide Sugar Transporters and Synthesis of Glycoconjugates (Nobuhiro Ishida)
GD-A07 Carbohydrate Determinants Involved in Inflammation and Homing of Lymphocytes (Reiji Kannagi)
GP-A06 Malignant Transformation and Mucins (Hiroshi Nakada)
LE-A05 Cell Adhesion Molecules: Selectins (Yasuyuki Imai)
Dec. 28, 2004

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