Role of CD44s and CD44v6 on human breast cancer cell adhesion, migration, and invasion

https://doi.org/10.1016/j.yexmp.2008.12.003Get rights and content

Abstract

The interaction between the transmembrane receptor CD44 on epithelial tumor cells and its ligand hyaluronan in the surrounding extracellular matrix is important in tumor progression and metastasis. CD44 is encoded by a single 20-exon gene and expressed in standard form (CD44s), as well as a myriad of CD44 variants (CD44v) generated by alternative splicing of the CD44 mRNA. Previously, we demonstrated that hyaluronan (HA) production is increased at tumor–stroma interface in invasive and metastatic human breast cancers when compared with benign or premalignant lesions. We hypothesize that CD44 expression on breast cancer cells is a major contributing factor to cell adhesion, migration and invasion. To evaluate this hypothesis we examined the effects of 3 distinct anti-CD44s and 2 anti-CD44v6 monoclonal antibodies on breast cancer cell lines that expressed high and low CD44s and CD44v6. Using these antibodies we assessed the role of CD44 in cell adhesion, cell motility, and cell invasion using immobilized HA-coated wells, wound healing assays, and modified Boyden chamber respectively. Our results showed that anti-CD44s could inhibit breast cancer cell adhesion, motility and invasion, while anti-CD44v6 inhibits cell motility. In conclusion, our data suggests that CD44s is involved in breast cancer cell adhesion, motility and invasion through interaction with HA but CD44v6 is involved only in cell motility. Furthermore we concluded that antibodies against different epitopes on CD44 mediate distinct functional effects on breast cancer cells.

Introduction

Breast cancer is one of the most common cancer among American women and world worldwide. In 2007, about 178,480 new cases of invasive breast cancer were diagnosed and approximately 40,460 women died from the disease nationwide (American, 2007). Despite improvements in treatment modalities, there is still a high failure rate mainly due to tumor invasion and metastasis. Breast cancer invasion is a three-step process characterized by altered cellular adhesion, cell motility and invasion of the extracellular matrix. Signals transmitted to the tumor cells from the peritumoral stroma will influence their activity, proliferation, and motility. This is accomplished in part through transmembrane receptors that interact with stromal extracellular matrix molecules. One of these receptors is CD44, which binds to the extracellular matrix component HA (Underhill, 1992). The engagement of CD44 with HA results in intracellular signaling that has been linked to diverse cellular functions such as adhesion, migration, and invasion, which are all important in cancer progression and metastasis (Toole, 2002, Naor et al., 1997).

CD44 (also known as homing cellular adhesion molecule, Hermes antigen, and PGP-1) is an 85–90 kDa transmembrane glycoprotein receptor comprising of four functional domains. The distal extracellular domain is primarily responsible for the binding of HA, while the proximal extracellular domain is the site of alternative splicing for CD44 mRNA that produces different isoforms of CD44. The CD44 transmembrane domain is a single 23 amino acid transmembrane domain typical of single-pass membrane glycoproteins. The intracellular domain interacts with cytoskeletal proteins and other intracellular signaling proteins. The gene encoding CD44 consists of 20 exons. In the standard form (CD44s), 10 of the 20 exons are transcribed. Multiple variant isoforms (CD44v2–v10) arise from alternate mRNA splicing of the remaining 10 exons (Tolg et al., 1993). In contrast to the standard form of CD44, which is usually ubiquitously expressed on epithelial cells and lymphocytes, CD44 variants exhibit tissue specific expression. Some of these variants, in particular splice variants of CD44v6 are associated with aggressive behavior and correlate with poor prognosis in a variety of human malignancies including breast cancer (Lopez et al., 2005, Auvinen et al., 2005, Joensuu et al., 1993).

CD44 specific monoclonal antibodies have been used extensively to understand the function of CD44 in cancer. Several monoclonal antibodies have been developed to recognize distinct epitopes on CD44. Antibody binding to specific epitopes on CD44 can clarify how different epitopes mediate distinct functional effects and influences of HA recognition (Lesley et al., 1993, Peach et al., 1993. Only CD44 antibodies that can block CD44–HA interaction are referred to as blocking antibodies and these are few in number (Zhong et al., 1995). Additionally, it has been established that HA-oligomers composed of at least 6 repeating disaccharides (HAOligo-6) is capable of binding to CD44 efficiently and blocking CD44–HA interaction (Nemec et al., 1987, Knudson et al., 1993).

In this current study we examined the effects of distinct antibodies against CD44s and one of its variants CD44v6 to understand the significance of their expression in mediating breast cancer cell adhesion, invasion, and migration.

Section snippets

Cell culture

Breast cancer cell lines MDA-MB-468, BT483 and MDA-MB-157 were acquired from the American Type Culture Collection (Rockville, MD). Breast cancer cell lines MDA-MB-231, MCF-7, and T47D were kindly provided by Dr. Hsing-Jing Kung (UC Davis). MDA-MB-468, MDA-MB-231, MCF-7, and T47D cells were cultured in Dulbecco's Modified Eagle Medium (GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (Omega Scientific, Tarzana, CA), l-glutamine [2 mM] (GIBCO), Pen Strep [100 μg/mL], and MEM

Expression of CD44s and CD44v6 on breast cancer cell lines

The expression of CD44s and CD44v6 receptor was characterized on 6 breast cancer cells lines including BT483, MDA-MB-157, MDA-MB-231, MDA-MB-468, MCF-7, and T47D. Western blotting of the breast cancer cell lines revealed that they expressed varying amounts of CD44s with a molecular weight between 80 and 90 kDa; the expected size for CD44s. The amount of CD44s varied significantly between the different cell lines. MDA-MB-468 express high levels of CD44s, while MDA-MB-157 and MDA-MB-231 expressed

Discussion

In recent years, numerous investigations have focused on the expression of CD44s and its isoforms in malignancy to further clarify their role in tumor progression and metastasis. We reported previously that in breast cancer, the tumor–stroma interface is enriched in HA, but how this could alter tumor cell functions is not well understood. Therefore, we explored the role of HA receptors, CD44s and CD44v6 on breast cancer cell adhesion, invasion, and migration.

We first characterized the

Conflict of interest statement

The authors declare that there are no conflicts of interest.

References (40)

  • AfifyA. et al.

    Expression of CD44s, CD44v6 and hyaluronan across the spectrum of normal-hyperplasia-carcinoma in breast

    Appl. Immunohistochem. Mol. Morphol.

    (2008)
  • American Cancer Society, INC, 2007. Breast Cancer Facts & Figures,...
  • AuvinenP. et al.

    Expression of CD44s, CD44v3 and CD44v6 in benign and malignant breast lesions: correlation and colocalization with hyaluronan

    Histopathology

    (2005)
  • BourguignonL.

    CD44-mediated oncogenic signaling and cytoskeleton activation during mammary tumor progression

    J. Mammary Gland Biol. Neoplasia

    (2001)
  • BourguignonL. et al.

    CD44v (3, 8–10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells

    J. Cell. Physiol.

    (1998)
  • BourguignonL. et al.

    Ankyrin–Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and m igration

    J. Cell Biol.

    (2000)
  • BourguignonL. et al.

    CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration

    J. Biol. Chem.

    (2001)
  • BrennanF.R. et al.

    CD44 expression by leucocytes in rheumatoid arthritis and modulation by specific antibody: implications for lymphocyte adhesion to endothelial cells and synoviocytes in vitro

    Scand. J. Immunol.

    (1997)
  • CampR.L. et al.

    CD44 is necessary for optimal contact allergic responses but is not required for normal leukocyte extravasation

    J. Exp. Med.

    (1993)
  • CarpenterP. et al.

    The role of hyaluronan in mesothelium-induced motility of ovarian carcinoma cells

    Anticancer Res.

    (2003)
  • Cited by (91)

    • Co-localization and crosstalk between CD44 and RHAMM depend on hyaluronan presentation

      2021, Acta Biomaterialia
      Citation Excerpt :

      The standard form of CD44 (CD44s) is widely expressed in cells of different healthy tissues. Inflammation and several types of cancer are associated with upregulation and structural changes of the extracellular domain of CD44, giving rise to so-called variants (CD44v) that promote tumor progression and metastasis [23–25]. CD44 binds HA via an amino-terminal link domain leading to ligand-induced clustering of CD44 [26,27].

    • CNTs mediated CD44 targeting; a paradigm shift in drug delivery for breast cancer

      2020, Genes and Diseases
      Citation Excerpt :

      In several research findings have demonstrated altered expression of CD44 and malfunction during inflammation response and cellular damage.2 A preponderance of research data demonstrated overexpression of CD44 in many diseases, including cancer, autoimmune disorders, and inflammatory diseases.3–6 CD44 antigen present on the cell surface plays a vital role in cell–cell interaction and downstream signaling.

    View all citing articles on Scopus
    View full text