Altered expression of glycan genes in cancers induced by epigenetic silencing and tumor hypoxia: clues in the ongoing search for new tumor markers

Abstract

The glycan molecules that preferentially appear in cancers are clinically utilized as serum tumor markers. The exact reason, however, why glycans are useful as tumor markers remain elusive. Here, we will summarize lessons learned from well-established cancer-associated glycans, and propose strategies to develop new cancer markers. Our recent results on cancer-associated glycans, sialyl Lewis A and sialyl Lewis X, indicated that the repressed transcription of some glycan genes by epigenetic silencing during early carcinogenesis, and the transcriptional induction of some other glycan genes by tumor hypoxia accompanying cancer progression at locally advanced stages, are two major factors determining cancer-associated glycan expression. Multiple genes are involved in glycan synthesis, and epigenetic silencing of a part of such genes leads to accumulation of glycans having truncated incomplete structures, which are readily detected by specific antibodies. Glycans are very unique and advantageous as marker molecules because they are capable of reflecting epigenetic silencing in their structures. Transcriptional induction of some glycan genes by tumor hypoxia at the later stages produces further glycan modifications, such as an unusual increase of the N-glycolyl sialic acid residues in the glycan molecules. The entire process of malignant transformation thus creates abnormal glycans, whose structures reveal the effects of both epigenetic silencing and tumor hypoxia. The second advantage of a glycan marker over a proteinous marker is that they can reflect the plurality of genetic anomalies in a singular molecule, as it is synthesized by the cooperative action of multiple genes. Glycans are sometimes covalently bound to well-known cancer-associated proteins, such as CD44v, and this eventually contributes to a high cancer specificity and functional relevancy in cancer progression.

Figure 1

Examples of malignant transformation‐associated glycan transition of epithelial cells. (a) Sialyl Lewis A glycan transition upon malignant transformation. Cancer cells preferentially express the cancer‐associated glycan, sialyl Lewis A, whereas non‐malignant epithelial cells express the normal glycan, disialyl Lewis A, suggesting an impairment of βGlcNAc: α2‐6 sialylation by epigenetic silencing occurring during malignant transformation. Adapted from Miyazaki et al., 16 with permission. (b) Sialyl Lewis X glycan transition upon malignant transformation. Cancer cells preferentially express the cancer‐associated glycan sialyl Lewis X, while non‐malignant epithelial cells express the normal glycan, sialyl 6‐sulfo Lewis X, suggesting an impairment of sulfation at C6 position of βGlcNAc by epigenetic silencing occurring during malignant transformation. Adapted from Izawa et al., 20 with permission. Typical distribution patterns shown were obtained by immunohistochemical staining using specific anti‐glycan antibodies of consecutive sections prepared from colon cancer tissues. Ca, cancer cells; N, non‐malignant epithelial cells.

Figure 2

Clinical application of sialyl Lewis A/disialyl Lewis A glycan transition. (a) Real‐time RT‐PCR analyzes of transcription of the βGlcNAc: α2‐6 sialyltransferase gene responsible for sialyl Lewis A/disialyl Lewis A glycan transition in colon cancer tissues. Note that its transcription is significantly reduced in cancer cells in patients at relatively early as well as advanced stages. RNA samples were prepared from the cancer tissues (Ca) and non‐malignant colonic mucosa (N) of the same patients. (b) Ratio of serum concentrations of sialyl Lewis A and disialyl Lewis A glycans in patients with various disorders. Note that the ratio is high in sera from patients with malignant disorders, while it remains low in sera from patients with benign disorders, providing information for differential diagnosis of malignant and benign disorders. Adapted from Itai et al., 14 and Miyazaki et al., 16 with permission.

Figure 3

Schematic illustration of hypoxia‐induced cancer progression and its effects on glycan expression. (a) Hypoxia‐resistant cancer clones, which acquired enhanced cell mobility and cell‐adhesive glycan expression, propagate in hypoxic area of cancer nests. They eventually occupy the entire cancer cell nests, and undergo vigorous vascular infiltration. This process accompanies acceleration of abnormal glycan production in cancer cells through transcriptional induction of genes involved in glycan synthesis. (b) Enhanced expression of cancer‐associated glycans, sialyl Lewis A/X, by hypoxia in cultured cancer cells. (c) Examples of genes involved in glycan synthesis showing hypoxia‐induced transcription. (d) Real‐time RT‐PCR analyzes of transcription of hypoxia‐dependent glycan genes in colon cancer tissues. The transcription of glycan genes, which showed a clear induction by hypoxia in experiments in vitro, is elevated also in actual in vivo cancer tissues prepared from surgical specimens. Note that their transcription tends to increase preferentially in patients at relatively advanced stages, because the acquisition of hypoxia resistance is a relatively late event in cancer progression. The RNA samples were prepared from the cancer tissues (Ca) and non‐malignant colonic mucosa (N) of the same patients. (b–d) Adapted from Koike et al., 40 with permission.

Figure 4

A scheme illustrating the stepwise accumulation of structural anomalies in cell surface glycans during carcinogenesis and cancer progression. Normal epithelial cells express a glycan disialyl Lewis A having a complex carbohydrate structure. Epigenetic silencing of glycan genes occurring during carcinogenesis leads to accumulation of glycans having a truncated incomplete structure, sialyl Lewis A. Acquisition of hypoxia resistance in locally advanced cancers confers a marked increase of its expression and eventually results in the appearance of N‐glycolyl sialyl Lewis A having an abnormal sialic acid residue in its structure, which is expected to be a good marker for hypoxia‐resistant cancer cells. On the other hand, ischemic changes occurring in non‐malignant epithelial cells will lead to an increase of the normal glycan, disialyl Lewis A, and will facilitate its modification with N‐glycolyl sialic acid, that is an accumulation of N‐glycolyl disialyl Lewis A glycan, which may serve as a marker for benign ischemic diseases.

Figure 5

Schematic illustration of dual functions of CD44v carrying selectin ligand glycans. The selectin ligand glycans, sialyl Lewis A and sialyl Lewis X, are carried by cell surface glycoproteins, and their interaction with vascular selectin facilitates hematogenous metastasis (a). CD44s and CD44v on cancer cells are known to bind equally to hyaluronan in the vascular bed, and this is also implicated in hematogenous metastasis (b,c). Expression of CD44v, but not CD44s, had been known to well correlate with hematogenous metastasis clinically, but the molecular basis for the correlation of CD44v with hematogenous metastasis remained elusive, because not much difference has been noted in the hyaluronan‐binding activities between CD44s and CD44v. The CD44v molecule carrying sialyl Lewis A and sialyl Lewis X glycans has dual functions in cell adhesion; it binds to hyaluronan and can also serve as a ligand for vascular selectins (d). This could explain the preferential clinical correlation of CD44v with the frequency of hematogenous metastasis, as CD44s has much fewer potential O‐glycosylation sites compared to CD44v. The fragments of CD44v carrying sialyl Lewis A and sialyl Lewis X glycans are released during cancer cell migration by the action of metalloproteinases (e), and are detectable in the sera of patients with cancers.

Figure 6

CD44v fragments carrying sialyl Lewis A and sialyl Lewis X glycans in cultured supernatants of cancer cells and in clinical serum samples. (a) Western blotting results showing that CD44v is capable of carrying cancer‐associated glycan, sialyl Lewis A, as well as normal glycan, disialyl Lewis A. CD44 was immunoprecipitated using an anti‐CD44 antibody from culture supernatants after ionomycin treatment of cultured human colon cancer cell line SW1083 (parent), or the cells transfected with a gene for a βGlcNAc: α2‐6 sialyltransferase (transfectant), and analyzed for glycan expression by Western blotting using specific anti‐glycan antibodies. (b) Western blotting results showing that expression of sialyl Lewis X on the CD44v molecule is also enhanced by hypoxia. CD44 was immunoprecipitated using an anti‐CD44 antibody from culture supernatants of human colon cancer cell line, LS174T, cultured under normoxic or hypoxic (1% O₂) conditions. (c) Preliminary results on the levels of CD44v fragments carrying sialyl Lewis A or sialyl Lewis X glycans in sera of patients with cancers. The serum levels of CD44v fragments carrying sialyl Lewis A or sialyl Lewis X were determined by enzyme‐linked double‐determinant sandwich assays using a combination of immobilized anti‐CD44v as the catcher‐ and specific anti‐glycan antibodies as the tracer antibodies. (a,c) Adapted from Lim et al., 58 with permission.