In developed countries, colorectal cancer (CRC), or rather its progression to metastatic disease, accounts for 25% of tumour deaths . Characterising the metastases associated processes is therefore of crucial importance for identifying ways of earlier and more sensitive diagnosis, more defined prognosis, and possibly in the selection of patients for targeted therapies.
A variety of genes have been described and extensively investigated in the literature as key candidates in the tumorigenesis and progression of colorectal carcinoma, including APC, p53, K-ras, BRAF, DCC, MSH, EGFR, SFK, TGFR2, SMAD4, etc. [2–5].
Each individual step of the metastatic cascade is the result of complex molecular interactions, regulated by several already identified and/or unidentified genes . One of the candidates of key importance has been CD44. In fact, it is one of the most investigated molecules in the metastatic process of several malignancies [7–13], among them that of colorectal cancer [14–16].
CD44 was first described to be a lymphocyte homing receptor . The role of CD44 was later proven in fetal arteriogenesis , as well.
Furthermore, expression and function of various isoforms of CD44 were detected in chronic inflammatory diseases (rheumatoid arthritis, ulcerative colitis, resolution of lung inflammations, etc.) [19–21].
A number of studies demonstrated the expression of CD44s (standard version) on most tissue types of epithelial origin (stomach mucous membrane, small intestinal mucosa, prostate, ductal epithelium of breast, skin, hair follicules, transitional cell membrane of utgenital tract) [22–26]. Regarding colonic mucosa, immunohistochemical examinations demonstrated that CD44s (identified with antibody against the standard region of the molecule) is located at the level of basal crypts, similar to the “intestinal stem cells” of the small intestine sitting between Paneth-cells [27–29]. This means that proliferative cells of colonic mucosa, as well as cells in the basal segment of differentiation zone are able to provide CD44s expression, while differentiated mucosal cells don’t show membrane positivity on immunostaining any more . This fact gains special importance when considering the change of ‘CD44’ expression characteristics from the normal, the dysplastic and the malignant colorectal tissues .
Additionally, and probably not independently form the above mentioned findings, recent studies demonstrated ‘CD44’ as a potential tumour stem cell marker (in colorectal, gastric and pancreatic adenocarcinoma, as well as breast cancer and other cancers) [32–39].
With the presence of CD44 protein in so many tissues, consideration has to be given to its physiological and pathological functions.
‘CD44’ has a variety of biological functions such as cell-cell, cell-ECM interactions, tumour cell migration [40, 41] or even chemoresistance [16, 41–50]. This multitude of representations and functions is less surprising when considering that through its 9 variable exons (v2-v10), hundreds of different isoforms can be transcribed and translated from the single CD44 gene resulting in a mixture of glycoproteins with potentially different functions [51–53]. Talking about ’CD44’ and studying its ’over expression’ therefore is rather debatable and not as straight-forward as it was previously thought. A basic understanding and consideration of alternative splicing should be warranted when dealing with this versatile gene. For the same reason, when using probes (i.e. primers or antibodies) against the standard region of CD44 (CD44s), one will detect a mixture of isoforms as they all share the same standard region [54, 55].
Determining the role of ’CD44’ is further complicated by the difficulties to identify all the isoforms potentially characterizing certain cell or tissue types. The trend is to focus on the expression of a single variable exon, which, of course, will only show the summation of isoforms containing the exon in question. However, these isoforms might only share a single variable exon, therefore they can be of different functions. In addition, several other CD44 variant isoforms can be present within the same cell, adding further functions to ’CD44’ [56–59]. Furthermore, different cells of a tumour can express various, possibly different sets of CD44 isoforms. As most of our tests examine a group of cells from the tissue at the same time, the results are summated, failing to represent the cell-to-cell differences in details . In colorectal carcinoma the most studied variant exons / protein domains are v3 and v6.
CD44v3 is the heparane-sulphate proteoglycane domain of CD44, however, the v3-containing isoforms of CD44 have hyaluronate binding potential and may contain a chondroitin-sulphate proteoglycan domain as well. Consequently, CD44v3 can bind different heparin-binding growth factors (GF), such as HBEGF, VEGF, HGF, βFGF, KGF and amphiregulin [60, 61].
CD44v6 functions as receptor of HGF/SF cooperating with c-met [61, 62], regulating EMT and taking part in the positive feedback loop of K-ras activation driving both Ras signal transduction pathway and CD44-splicing machinery [3, 63, 64]. Additionally, this is the domain most frequently found to be associated with metastatic phenotype in the literature .
Unsurprisingly, the studies of detecting ‘v3’ and ‘v6’ under different experimental and clinical settings do not lead to coherent results, and can even be contradictory. This also can be observed in the literature regarding colorectal cancer [14, 66–71].
Recently, characteristic CD44 isoform patterns of different normal tissue types have been described in the literature [24–26, 72, 73] and our earlier studies described CD44 isoform patterns characteristic of different tumour types, respectively (data publication in progress, Rásó-Barnett et al.). Most of the literature agrees that ‘CD44’ plays an important role during tumour progression based on ‘overexpression’ of specific variant regions containing isoforms at mRNA and/or protein level. However, no data is available concerning the pattern of CD44 isoforms and its modulation during tumour progression. Our previous study on CD44 alternative splice patterns showed no qualitative change of the melanoma specific ‘CD44-fingerprint’, while the expression level of all isoforms uniformly increased during the metastatic process (data publication in progress, Rásó-Barnett et al.).
The aim of our research was to find answers to the following two questions: (1) Is there a colorectal carcinoma specific CD44 isoform expression combination like to one identified in human melanoma? If there is such pattern, is it qualitatively / quantitatively stable during tumour progression? (2) What is the background of the incoherent quantitative results on the expression of the most investigated v3 and v6 variable exons / protein domains?
To answer the first question, we designed primer pairs covering the entire variable region (primers A-B, see Methods) for our quantitative and qualitative PCR reactions. The expressed, i.e. transcribed CD44 isoforms were identified using direct sequencing and next-generation sequencing.
Due to the large number of potential isoforms, we used a series a PCR reactions to create the CD44 fingerprint, a simple, easy to handle representation of the expressed CD44 isoforms. We used this fingerprint to track the changes of CD44 expression during tumour progression [41, 55, 74–76].
To answer our second question, we designed a complex experimental animal model to examine the functional role CD44v3 and CD44v6 containing isoforms play in metastasis formation. The main question was whether or not there is a change in the expression of these variable exons that could be associated to the metastatic process of human colorectal carcinoma. We implanted the same colorectal cancer cell population orthotopically to form real metastases in liver, as well as heterotopically. We performed two types of heterotopic implantation: the first involved subcutaneous implantation into permissive and non-permissive hosts, the second was direct implantation into the spleen so that the implanted cells could directly colonize the liver. The latter method created a new type of secondary tumour, which albeit is in the same localization as the liver metastases of the true metastatis model, has not been through the metastatic cascade. This experimental setup allowed to functionally differenciate between phases of metastatic process and host related changes, regarding the expression activity of the examined variable CD44 exons.