Last year, one study from Kanazawa University showed new emerging role of osteopontin in HCV-related hepatocellular carcinoma. As the study mentioned, CD44 has been identified as the most common marker for cancer stem cells (CSCs) in several human cancers. For this reason, CD44 is a lasting hot point in the field of cancer therapy. In this article, we integrate numerous studies and data to present the CD44 from the following aspects:
1. What is CD44?
CD44, also called ECMR-III, is an adhesion molecule (one type of cell-surface glycoprotein) involved in cell-cell interactions, cell adhesion and migration, such as leukocyte attachment to and rolling on endothelial cells, homing to peripheral lymphoid organs and to the sites of inflammation, and leukocyte aggregation. In humans, the CD44 antigen is encoded by the CD44 gene on Chromosome 11 . CD44 is expressed in a large amount of mammalian cell types.
2. What is The Structure of CD44?
CD44 is an 80-250 kDa type I (extracellular N-terminus) transmembrane glycoprotein. The gene for CD44 has been identified in human, mouse and rat. The human CD44 gene is located on the short arm of chromosome 11 and has a full length of about 50 kb. As the figure 1 shows:
Figure 1. The location of CD44 gene
2.1 The gene structure of CD44
The gene consists of 20 highly conserved exons, each of which varies in length from 70-210 bp, separated by introns of varying lengths. The 20 exons of CD44 gene can be divided into two major categories: constitutive exon (C) and variant splicing exon (V). There are 10 constitutive exons and they are present in all transcripts. The CD44 transcript containing only the exon is called standard CD44 (CD44S). There are also 10 mutative splicing exons, located between the 5th and 6th constitutive exons with a total length of 1245 bp.
The CD44 transcript containing the mutated splicing exon is called CD44V. The transcriptional pattern of CD44V is very complex, and it can be transcribed continuously or skipped. The number of exons involved in splicing can be more or less, so that the transcript fragments are different in length. In theory, this variant splicing of CD44 produces more than 1000 variants of CD44. In the next part, we focus on the structure of CD44S.
2.2 The Structure of CD44 Protein
The mature CD44 protein has no signal peptide region, and the molecular weight is expected to be 37.2kDa. After translation and glycosylation, the molecular weight of CD44S protein can reach 80-90kDa. As shown in the figure 2, the four functional regions include N-terminal domain, stem region, transmembrane domain and cytoplasmatic domain.
The N-terminal extracellular domain contains motifs serving as docking sites for various ligands, including hyaluronan (HA), extracellular matrix (ECM) glycoproteins and proteoglycans, growth factors, cytokines and matrix metalloproteinases .
The amino terminal domain of CD44 is separated from the plasma membrane by a short stem structure with proteolytic cleavage sites for such metalloproteinases. The cleavage of CD44 triggers the release of cells bound to HA—a process important in the regulation of cell migration .
The C-terminal cytoplasmic tail region plays a crucial role in the CD44 involvement in intracellular signal transduction. It has been shown that it binds to cytoskeletal elements such as ankyrin, the ERM proteins: ezrin, radixin, and moesin, and moesin-ezrin-radixin-like protein, it also interacts with signaling molecules, namely members of Src family kinases (SFKs), Src, Lck, Fyn and Lyn, and activators of small Rho GTPases. In addition, the cytoplasmic tail of CD44 may be cleaved off by γ-secretase and translocated to the cell nucleus, where it acts as a transcriptional regulator   .
Figure 2. the structure of CD44 protein
3. What is The Function of CD44?
The main functions of CD44 protein is acting as a receptor for hyaluronic acid (HA). HA is a linear poly-adhesive molecule, which is a component of ECM. It is present in all tissues in the form of a polymer. There are at least three HA binding sites on the hair loop of CD44, which are encoded by exon 2 and exon 5, respectively. The affinity of CD44 and HA binding is regulated by the cell. Besides, this affinity is also regulated by the extracellular segmentation of the CD44 molecule and the phosphorylation of the serine in the intracellular tail.
Moreover, CD44 is also involved in the metabolism of HA. The uptake, internalization and degradation of HA by CD44 may be closely related to the invasion of tumor cells, the ability to metastasize and the regression of inflammation.
Except for HA, the ligands of CD44 also include osteopontin, fibronectin, collagen types I and IV23 and matrix metalloproteinases (MMPs) . In term of MMPs, CD44 is more like a special platform. Because MMPs can’t interact with its substrate without CD44. The group of Yu, et al have discovered that CD44 anchored MMP-9 on the cell surface in mouse breast cancer cell lines and melanoma cell lines. When soluble CD44 was used to inhibit CD44 function, it was found that the function of MMP-9 on cell surface was also weakened.
The labeling method found that CD44 and MMP-9 co-localized on the cell surface, and this co-localization contributed to the resistance of MMP-9 to TIMP-1, which contributed to the degradation of collagen IV and promoted the invasion of tumor cells. Additionally, they further found that MMP-9 anchored to the cell surface by CD44 can also activate the inactive TGF-β precursor, which is a necessary growth factor for neovascular growth  .
4. CD44 in Cancer
As mentioned before, CD44 is a multi-structural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines to the corresponding receptors, and docking of proteases at the cell membrane. Note that all these biological properties are essential to the physiological activities of normal cells, but they are also associated with the pathologic activities of cancer cells. Here, we illustrate the relationship between CD44 and Cancer.
4.1 Tumor Development
The relationship between CD44 molecules and tumors is mainly reflected in the promotion of tumor development and invasion and metastasis. After the mice transfected the expressed soluble CD44 with mouse breast cancer cells to block CD44 function, the growth of the tumor in the tumor xenograft model was inhibited, and a large amount of apoptosis was found in the tumor cells. Although it has been reported that up-regulation of CD44 expression is an early event in the development of colon tumors, Weber et al. cross-examined CD44 knockout mice and APC mutant mice and p53 gene tml mutant mice to find CD44 in promoting tumor metastasis. The role is much more critical than promoting tumorigenesis .
4.2 Tumor Invasion and Metastasis
Early recognition of CD44's promotion of tumor metastasis comes from studies of lymphocyte migration. Early investigators transfected non-metastatic rat pancreatic cancer cells with plasmid vectors expressing CD44s and CD44v, respectively, and tumor cells expressing exon v6 and v7 sequences had metastatic potential. As mentioned above, after blocking CD44 function by soluble CD44 in mouse breast cancer cells, CD44 loses the role of anchoring MMP-9, thereby reducing the invasion and metastasis ability of tumor cells. In addition, this mechanism leads to a further decrease in TGF-β precursor activation, thereby inhibiting tumor tissue neovascularization and tissue remodeling.
5. The Clinic Significance of CD44
Tow decades ago, CD44 was began to use in clinical diagnosis and prognosis evaluation of tumors. However, due to the polymorphism of CD44, no specific CD44 phenotype associated with tumors was found.
But, it is worth noting that the detection of CD44 in body fluids leads to a higher level of diagnosis and prognosis of certain tumors. Moreover, other researchers have found that the concentration of soluble CD44 (sCD44) in body fluids is associated with some swelling and treatment response.
The mechanism of sCD44 formation is generally thought to be the result of misprocessing of tumor cells in CD44 transcription, including intron retention, exon skipping and the utility of cryptic splice sites. For this reason, some researchers have used the method of detecting abnormal retention of introns to diagnose tumors.
Additionally, the explanation for the use of recessive splicing sites in tumor cells is that the correct splicing sites can’t be utilized due to incorrect folding or coordination of proteins in tumor cells, resulting in a second selection of splicing events by cells, in tumor cells. The compromise results of the CD44 selective splicing mechanism.
6. The Latest Research Progression of CD44
The team of Calvete J et al. has revealed that the co-expression of fibroblast activation protein (FAP) and basal-type markers (CK 5/6 and CD44) are good prognosis indicators in high-grade invasive urothelial carcinoma of the bladder (UC) 
Motamedi M, et al. have found that expression of CD44, SIRT1, CXCR4, miR-21, miR-34a, and miR-451 Genes have been altered in MKN-45 Cell Line after Docetaxel treatment .
The study of Kadletz LC, et al. has suggested that AF1q expression Associates with CD44 and STAT3 and impairs overall survival in adenoid cystic carcinoma of the head and neck .
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