Our results demonstrate that CD44 isoforms play a role in mediating HNSCC progression behaviors including tumor cell migration, proliferation, and cisplatin response. Supporting our in vitro data is the finding that the CD44 v3, -v6, and -v10 variant-containing isoforms are significantly correlated with various clinicopathologic variables including advanced T stage, regional and distant metastasis, radiation failure, perineural invasion, and shorter disease-free survival.
Proliferation, migration, and drug resistance characterize tumor progression in HNSCC cells. Oncogenic signaling and cytoskeleton functions are directly involved in these in vitro
tumor progression behaviors, which result in the observed clinical features of advanced HNSCC. Clinical features of advanced HNSCC include adjacent tissue invasion, metastasis, and treatment resistance, which lead to increased disease recurrence and decreased survival. A number of studies have aimed to identify those molecules which are expressed in epithelial tumor cells and mediate these tumor progression behaviors. Among such molecules is CD44, a family of glycoprotein receptors expressed in many tumor cells.2-10,27
As transmembrane proteins, CD44 isoforms are capable of interactions with both the cell cytoplasm and the extracellular milieu. Among the major components of the extracellular matrix are hyaluronan, and all CD44 isoforms share a common extracellular binding domain for HA. All CD44 isoforms also share common cytoplasmic domains, most notably ankyrin-binding regions, which are capable of interacting with cytoskeleton proteins to activate cell signaling pathways.27,28
CD44 isoforms also interact with other receptor and non-receptor kinases, including EGFR, ErbB2, src
, and transforming growth factor (TGF)-β.3
The ability of CD44 receptors to provide a direct link between the extracellular matrix and the cytoskeleton, coupled with their ability to interact with a multitude of signaling kinases, explains how one family of molecules is able to mediate a diversity of tumor progression functions.
The mechanism of CD44 receptors to promote tumor migration, proliferation, and survival through HA-mediated signaling has been described.4,5,8,9,13-17
HA binding to the extracellular domain of CD44 promotes interaction of CD44 with the cytoskeleton, particularly ankyrin through the conserved ankyrin-binding domain of the CD44 cytoplasmic region. This CD44-ankyrin interaction causes activation of various intracellular signaling pathways leading to the onset of cytoskeleton function.29-32
One of the important CD44-ankyrin mediated intracellular signaling pathways is the release of intracellular Ca2+
stores, which in turn, results in calmodulin-dependent activation, promoting tumor cell migration.13-16
Other CD44-ankryin mediated functions include cell adhesion and degradation of the extracellular matrix to allow tumor cell invasion.27
CD44 has also been shown to be physically linked to other receptor kinases in tumor cells including EGFR, ErbB2, and TGF-β, implicating a role for HA and CD44 in these well-described oncogenic signaling pathways.14,17,31
CD44 and ErbB2 are physically linked via disulfide bonds in ovarian tumor cells.31
HA binding to the CD44-ErbB2 complex activates ErbB2 tyrosine kinase activity and promotes ovarian tumor cell growth and migration. HA and CD44 interact with EGFR in HNSCC to promote various EGFR-mediated downstream signaling pathways.14,17
CD44-EGFR mediates extracellular signal-regulated kinase (ERK) phosphorylation, which has been shown to lead to increased tumor cell growth, migration, and chemotherapy resistance in HNSCC.
CD44 isoforms are expressed to varying degrees in both normal and tumor cells, suggesting a normal cellular function for many of the different CD44 receptor isoforms. How similar CD44-mediated signaling pathways can promote both normal and malignant cellular behaviors can be explained by several mechanisms. The mechanisms to regulate CD44 signaling include variable N-/O-linked glycosylation in the CD44 extracellular domain and modulation of the CD44 cytoplasmic domain by cytoskeletal proteins such as ankyrin. It is thought, however, that the most important mechanism that results in differing CD44-mediated signaling is by alteration of the CD44 receptor through additional exon-coded structures (via an alternative splicing process), leading to production of CD44 variant isoforms. In cancer cells, this latter mechanism can result in overexpression or novel expression of certain CD44 variant isoforms.27
The insertion of various variant exons to the extracellular component of the CD44 receptor has been shown to alter the signaling properties of the CD44 receptor by providing additional binding domains for molecules other than HA.3,27
The insertion of variant exons to the standard CD44 receptor may also result in changes to the HA-binding affinity of the CD44 isoform, further altering the receptor’s signaling behavior. We describe below some of the unique signaling properties of some of the more well-described CD44 variant isoforms.
Several CD44 variant exons are known to contain binding domains for various growth factor ligands. For example, the v3 exon (exon 7) is known to contain important glycosaminoglycan (GAG) attachment sites, and it has been postulated that tumor cells with CD44 receptors exhibiting these GAG sequences are involved with heparin binding growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and heparin binding-epidermal growth factor.8,23,24
The CD44 v3 isoform is expressed in the metastatic breast tumor cell line Met-1, where it was shown to bind VEGF, suggesting that this isoform may promote breast tumor-associated angiogenesis.8,23
CD44 v3 also appears to be linked to several other tumorigenic molecules to promote tumor cell migration and the invasive tumor cell phenotype. In particular, CD44 v3 co-localizes with the active form of matrix metalloproteinase-9 (MMP-9) in Met-1 cells, promoting degradation of the extracellular matrix to facilitate tumor cell invasion.8
This isoform also upregulates cytoskeleton function, through ankyrin, to activate the membrane-associated actomyosin contractile system and mediate tumor cell migration. The role of CD44 v3 isoforms in HNSCC progression is highlighted by studies showing an association of v3-containing isoforms with HNSCC growth, migration, and matrix metalloproteinase expression.15,33
Transfection of a CD44 v3 isoform into a non-expressing HNSCC cell line also resulted in significantly increased tumor cell migration, but not proliferation.34
In the current investigation, CD44 v3 isoforms were expressed in HSC-3 cells. While treatment of anti-CD44 v3 antibody had less influence on in vitro
proliferation or cisplatin sensitivity, tissue analysis revealed CD44 v3 isoforms were preferentially expressed in metastatic lymph nodes. Additionally, strong CD44 v3 isoform expression in primary tumors was significantly associated with advanced T stage and positive lymph nodes, but did not correlate with disease-free interval.
CD44 v6-containing isoforms also appear to promote tumor progression. Transfection of CD44 v6 converted non-metastatic rat carcinoma cells into metastatic cells, while co-injection of anti-CD44 v6 antibody into these same cells suppressed their metastatic behavior.35-36
The CD44 v6 splice variant was also found to stimulate sustained increased mitogen activated protein (MAP) kinase levels and subsequent downstream Ras signaling, resulting in increased tumor cell proliferation.35
In the current investigation, CD44 v6 isoforms were expressed in HSC-3 cells, and treatment of anti-CD44 v6 antibody resulted in decreased proliferation and increased cisplatin sensitivity. CD44 v6 isoforms were also preferentially expressed in metastatic lymph nodes, and strong expression in primary tumors was significantly associated with advanced T stage, perineural invasion, and decreased disease-free survival.
CD44 v10-containing isoforms have been studied regarding tumor progression in breast and renal cell carcinoma.23,37
Although all CD44 isoforms share HA binding domains, certain CD44 variant isoforms, such as CD44 v10, exhibit significantly reduced affinity for HA binding.27
It is thought that the reduction in HA-mediated cell adhesion in tumor cells expressing CD44 v10 may be the earliest event in the onset of tumor migration and invasion. The unique structure of CD44 v10 may also cause constitutive activation of CD44-cytoskeleton interactions which induce tumor cell migration and invasion. Studies have revealed that CD44 v10-transfected breast tumor cells display higher migration/invasion potential, produce higher levels of bFGF and interleukin-8, and exhibit more potent tumor growth potential than parental control cells.27
In the current investigation, CD44 v10 isoforms were strongly expressed in HSC-3 cells, and treatment of anti-CD44 v10 antibody resulted in moderately decreased proliferation and increased cisplatin sensitivity. CD44 v10 isoforms were also preferentially expressed in metastatic lymph nodes, and strong expression in primary tumors was significantly associated with distant metastasis, radiation failure, and decreased disease-free survival.
Considering the numerous studies regarding the unique signaling properties of CD44 variant isoforms, it is not surprising that the presence of high levels of CD44 variant isoforms is emerging as an important tumor marker in a number of solid malignancies. Overexpression of CD44 v3-containing isoforms was found to correlate with increased histologic grade and metastasis in breast cancer, increased metastasis in melanoma, and increased invasion and metastasis in colon cancer.6,9,20,23-35,38
CD44 v6 overexpression has been correlated with advanced tumor stage and poor survival in non-Hodgkin’s lymphoma and colon cancer.20
CD44 v10 correlation with renal cell carcinoma histologic grade, staging, and poor prognosis has been reported.37
The expression of CD44 variant isoforms in HNSCC has also been studied, but their role remains controversial. Whereas some studies have found a correlation between increased CD44 variant expression and HNSCC progression, other studies have reported no correlations or negative correlations. Reategui et al described a novel CD44 v3 isoform in both tissue and soluble form that correlated with HNSCC status.34
Wang et al reported that CD44 v3-containing isoforms were associated with HNSCC lymph node metastasis.15
Kawano and colleagues found correlations between CD44 v6 expression in HNSCC and tumor volume, lymph node metastasis, and shorter survival.39
However, others have reported that down-regulation of various CD44 variant isoforms correlated with a worse prognosis. Kanke et al reported that down-regulation of CD44 v2 correlated with poorer differentiation and shorter overall survival while down-regulation of CD44 v6 correlated with a higher rate of cervical metastasis.40,41
Fonseca et al found down-regulation of CD44 v3 and v6 correlated with tumor grade and pattern of invasion.7
Other groups, including Van Hal et al and Herold-Mende et al, found no correlation with CD44 splice variants and any clinicopathologic variables, and concluded that CD44 variant isoforms do not play a role in HNSCC progression.42,43
There are some possible explanations for these discrepant results. First, different studies have used different antibodies, making comparisons between research groups difficult. Certain CD44 variant domain epitopes may become hidden and not recognized by some antibodies due to post-translational changes (eg. glycosylation) which alter the 3-dimensional conformation of the protein. In addition, assessment of immunostaining positivity is dependent on what region of the tumor that is examined, and it has been reported that there are often large areas within the tumor that do not stain for many CD44 isoforms. Unlike most previous studies of HNSCC variant expression in the literature, our study included in vitro
assays and clinical tissue analysis using the same CD44 variant antibodies, and both the in vitro
and clinical specimen results in our investigation were consistent with each other. Thus, we believe our data is supportive of the notion that CD44 variant isoforms play an important role in HNSCC progression.
Recent work has shown that CD44 is preferentially expressed in tumor stem cells which have the unique ability to initiate stem cell-specific properties. Prince et al reported that CD44 is one of the important surface markers unique to cancer stem cells in HNSCC.44
The finding that CD44 is a marker for cancer stem cells provides further support to the notion that CD44 receptors are important in HNSCC progression, and suggests CD44 as a possible therapeutic target. What role the various CD44 variant isoforms might play in cancer stem cells remains largely unexplored. Studies of CD44 and cancer stem cells suggest that only a few CD44-positive cells are the critical determinants of tumor survival, which may explain the results of some studies showing minimal expression of CD44 in advanced (large volume) tumors, since these tumors may have proportionately fewer CD44-positive stem cells, but nonetheless, contain the essential tumor cells which mediate a more aggressive clinical phenotype.
Our investigation suggests an important role for CD44 variant isoforms in HNSCC progression. However, further study is warranted to better elucidate the mechanism by which CD44 v3, v6, and v10-containing isoforms mediate HNSCC progression. In addition, a search to identify other possible CD44 variant isoforms that may be important in HNSCC progression is also necessary. Our study identified isoforms in HNSCC cell cultures and tissue specimens that contained the CD44 v3, v6, or v10 exons, but we have not yet determined the exact composition of these CD44 variant isoforms. For example, it is not known whether HSC-3 cells express CD44 v3 (standard + v3 exon only) or CD44v3,8-10 (standard + v3 and v8-10 exons)--both variants have been previously reported in HNSCC--or other yet to be described v3-containing isoforms. Additional work with HSC-3 cells, including sequencing the v3-, v6-, and v10-containing isoforms that are expressed by this cell line, is planned. Similarly, for future HNSCC tissue analysis, obtaining samples that will allow use of RT-PCR techniques to clone and sequence the CD44 variant isoforms that are expressed will help elucidate the role of the unique CD44 variant isoforms that correlate with tumor progression. CD44 variant isoforms identified in clinical specimens may be transfected into HNSCC cell cultures, followed by proliferation, migration, and other assays to provide further understanding of the cellular mechanisms by which these isoforms promote tumor progression.
Our work and others suggest that CD44 variant isoforms are important tumor markers. As we learn more about the oncogenic signaling mechanisms unique to these variant isoforms, the implication that CD44 receptors may be a therapeutic target becomes apparent. Emerging techniques utilizing therapeutic siRNA and anti-sense RNAs can be applied to CD44 receptors. Future work planned includes utilizing an orthotopic nude mouse model of human HNSCC to grow CD44 variant isoform positive cancers that could then undergo anti-CD44 treatments to reduce the size and metastatic potential of the xenograft tumors.