Sialylation of Integrin β1 is Involved in Radiation-Induced Adhesion and Migration in Human Colon Cancer Cells

doi:10.1016/j.ijrobp.2009.11.022

International Journal of Radiation OncologyBiologyPhysics

Volume 76, Issue 5, April 2010, Pages 1528-1536

https://doi.org/10.1016/j.ijrobp.2009.11.022 Get rights and content

Purpose

Previously, we reported that radiation-induced ST6 Gal I gene expression was responsible for an increase of integrin β1 sialylation. In this study, we have further investigated the function of radiation-mediated integrin β1 sialylation in colon cancer cells.

Methods and Materials

We performed Western blotting and lectin affinity assay to analyze the expression and level of sialylated integrin β1. After exposure to ionizing radiation (IR), adhesion and migration of cells were measured by in vitro adhesion and migration assay.

Results

IR increased sialylation of integrin β1 responsible for its increased protein stability and adhesion and migration of colon cancer cells. However, for cells with an N-glycosylation site mutant of integrin β1 located on the I-like domain (Mu3), these effects were dramatically inhibited. In addition, integrin β1–mediated radioresistance was not observed in cells containing this mutant. When sialylation of integrin β1 was targeted with a sulfonamide chalcone compound, inhibition of radiation-induced sialylation of integrin β1 and inhibition of radiation-induced adhesion and migration occurred.

Conclusion

The increase of integrin β1 sialylation by ST6 Gal I is critically involved in radiation-mediated adhesion and migration of colon cancer cells. From these findings, integrin β1 sialylation may be a novel target for overcoming radiation-induced survival, especially radiation-induced adhesion and migration.

Introduction

Radiation therapy is a common conventional treatment modality for various human solid tumors. However, the therapeutic efficacy of radiotherapy alone for the treatment of locally or regionally advanced cancer is often limited by tumor radioresistance, system tumor progression, and local or distant metastases (1). The formation of a metastasis is one risk of clinical therapy of solid tumors, which is directly linked to the migratory potential of the cells. There is clear evidence from experimental studies for a radiation-induced metastasis to occur in vitro and in vivo after treatment with radiation therapy 2, 3, 4, 5, 6.

Aberrant cell surface oligosaccharides are highly associated with tumor invasion and metastasis. Expression of the ST6 Gal I sialyltransferase, which adds α2-6-linked sialic acids to glycoproteins 7, 8 is upregulated in several tumors, including colon cancer, and expression of this protein correlates with tumor metastasis and poor patient survival 7, 9, 10. Moreover, both in vitro and animal studies have implicated a role of ST6 Gal I in the regulation of tumor cell invasiveness and metastasis 10, 11, 12, 13.

Integrins are a family of transmembrane glycoproteins that are involved in different aspects of cell adhesion and migration. These proteins have a strong effect on survival, proliferation, and differentiation. Inasmuch as integrins not only mediate adhesion to the extracellular matrix but also regulate intracellular signaling pathways that control cytoskeletal organization, the proteins have an essential role in cell migration and invasion (14). It has been suggested that inhibition of integrin β1 dramatically enhances radiotherapy efficacy in human breast cancer (15). It has been long known that the N-glycans of the integrin β1 subunit of the integrin family of cell adhesion receptors have a different carbohydrate composition after cell transformation 9, 16, although specific changes in glycan structure have not been well defined, nor have the physiologic consequences of such changes been established. Moreover, the detailed mechanisms of how radiation affects cellular resistance and metastasis and the relationship between sialylation of integrinβ1 induced by radiation exposure and radiation resistance or radiation-induced invasion and metastasis have not been well elucidated.

In this study, we investigated for the first time, as far as we are aware, that sialylation of integrin β1 is increased by radiation exposure of cells and sialylation of integrin β1 is critical for radiation-mediated tumor adhesion and migration. Moreover, inhibition of sialylation may be a novel target to block radiation-induced adhesion and migration.

Section snippets

Cell cultures

SW480 and SW48, human colorectal carcinoma cells were cultured in Dulbecco's modified Eagle's medium supplemented with heat-inactivated 10% fetal bovine serum. Lovo cells were grown in Ham's F12 medium with 10% fetal bovine serum. GD25 cells were kindly provided by Dr. Reinhard Fässler (Max Planck Institute of Biochemistry, Department of Molecular Medicine, Germany).

Plasmids and transfection

Constructs of ST6 Gal I and neuraminidase2 (Neu2) were used as previously described (17). Predesigned small interferencing RNA

Induction of ST6 Gal I by radiation increased integrin β1 sialylation

Induction of ST6 Gal I by radiation exposure of cells increased both sialylation and protein levels of integrin β1 in SW480 and Lovo cells (Fig. 1A). The amount of sialylated integrin β1 or increase of integrin β1 protein level by radiation was dependent on endogenous sialylation patterns of integrin β1 such as the ratio of mature or precursor forms of sialylated integrin β1. The ST6 Gal I mRNA level was increased by radiation exposure as has been described previously (17). However, for

Discussion

Previously, ST6 Gal I expression and integrin β1 sialylation have been shown to be upregulated by radiation (17); therefore, one important goal of this study was to determine whether changes in radiation-mediated integrin β1 sialylation could affect integrin β1 function. Moreover, inhibition of integrin β1 sialylation is suggested to be a novel target for treatment of radiation-induced adhesion and migration.

We selected the use of colon cancer cells because sialylation patterns are frequently

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Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies and is currently the third leading cause of cancer death. The aggressiveness of PDAC stems from late diagnosis, early metastasis, and poor efficacy of current chemotherapies. Thus, there is an urgent need for effective biomarkers for early detection of PDAC and development of new therapeutic strategies. It has long been known that cellular glycosylation is dysregulated in pancreatic cancer cells, however, tumor-associated glycans and their cognate glycosylating enzymes have received insufficient attention as potential clinical targets. Aberrant glycosylation affects a broad range of pathways that underpin tumor initiation, metastatic progression, and resistance to cancer treatment. One of the prevalent alterations in the cancer glycome is an enrichment in a select group of sialylated glycans including sialylated, branched N-glycans, sialyl Lewis antigens, and sialylated forms of truncated O-glycans such as the sialyl Tn antigen. These modifications affect the activity of numerous cell surface receptors, which collectively impart malignant characteristics typified by enhanced cell proliferation, migration, invasion and apoptosis-resistance. Additionally, sialic acids on tumor cells engage inhibitory Siglec receptors on immune cells to dampen anti-tumor immunity, further promoting cancer progression. The goal of this review is to summarize the predominant changes in sialylation occurring in pancreatic cancer, the biological functions of sialylated glycoproteins in cancer pathogenesis, and the emerging strategies for targeting sialoglycans and Siglec receptors in cancer therapeutics.
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As CP and AU were confirmed to be the most efficient sialidases cleaving α2,6 sialylation on MDA-MB-231 cells, these data together indicate that decreasing α2,6 sialylation by sialidase treatment does not affect cell migration and invasion, regardless of the influence on cell adhesion. Altered glycosylation of integrins plays roles in cancer cell adhesion, migration and invasion during metastasis [2,10,19,24,34]. As one of a diverse family of sialylated integrins, α2,6 sialylated β1 integrin has been identified in many types of cancer cells, such as ovarian cancer cells (SKOV3 and PA1) [10], colon cancer cells (SW480, Lovo) [19], and melanoma cells (WM9 and WM239) [16].
To determine the role of sialylation on α5β1 and α2β1 integrins in the regulation of adhesion between breast cancer cells and extracellular matrix (ECM).
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: Supported by a Nuclear Research and Development Program of the National Research Foundation of Korea (NRF) funded by the Korean government Ministry of Education, Science and Technology (grant code M2ANA001 and M2AMA006).

: Conflict of interest: none.

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Biology ContributionSialylation of Integrin β1 is Involved in Radiation-Induced Adhesion and Migration in Human Colon Cancer Cells

Purpose

Methods and Materials

Results

Conclusion

Introduction

Section snippets

Cell cultures

Plasmids and transfection

Induction of ST6 Gal I by radiation increased integrin β1 sialylation

Discussion

Biochim Biophys Acta

Exp Cell Res

Biochim Biophys Acta

J Biol Chem

Int J Radiat Oncol Biol Phys

Biochim Biophys Acta

J Biol Chem

Cell

Biochem Biophys Res Commun

beta1 integrin as a molecular therapeutic target

Int J Radiat Biol

Does preoperative chemo-radiotherapy enhance the expression of vascular endothelial growth factor in patients with rectal cancer?

Oncol Rep

Changed adhesion molecule profile of Ewing tumor cell lines and xenografts under the influence of ionizing radiation

Anticancer Res

Gamma-irradiation induces matrix metalloproteinase II expression in a p53-dependent manner

Mol Carcinog

Adhesion molecules in radiotherapy

Radiat Res

Radiation therapy and the microenvironment

Int J Radiat Biol

Beta-galactoside alpha2,6 sialyltransferase in human colon cancer: Contribution of multiple transcripts to regulation of enzyme activity and reactivity with Sambucus nigra agglutinin

Int J Cancer

Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments: Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase

Mol Biol Cell

Cell surface sialoprotein alterations in metastatic murine colon cancer cell lines selected in an animal model for colon cancer metastasis

Cancer Res

Biology Contribution
Sialylation of Integrin β1 is Involved in Radiation-Induced Adhesion and Migration in Human Colon Cancer Cells