Increased expression of CD133 and reduced dystroglycan expression are strong predictors of poor outcome in colon cancer patients

The cancer stem cell (CSC) model of tumorigenesis postulates that only a small number of cancer cells are able to both self renew and give rise to a differentiated progeny. CSC are believed to be responsible for the primary disease as well as its recurrence and metastasis. Thus, it is expected that their evaluation in clinical samples might provide useful information for a better prediction of disease aggressiveness and evolution. Although phenotypic characterisation of colon CSCs is still controversial, CD133 is presently considered a useful marker to identify CSC in colorectal cancers and its detection has been used to evaluate the prognostic significance of CSC in colon cancer patients[1‐3].

Dystroglycan (DG) is a non-integrin adhesion molecule expressed in a wide variety of tissues at the interface between the basement membrane and the cell membrane[4]. It is formed by two subunits, the α (extracellular) and β (transmembrane) subunits which bind to the major ECM components and proteins involved in signal transduction and cytoskeleton organization, respectively. DG has been implicated in several cell functions (i.e., growth control, differentiation, shape change and movement) which are all relevant in the process of tumour development and metastasis[4‐7]. We and others demonstrated that DG expression, and mainly α-DG, is reduced or lost in a variety of human cancer cell lines and primary tumours and overall, the available findings indicate that loss of DG expression is a frequent event in human malignancies and might play an important role in human tumour development and progression[4‐6, 8‐10].

In the present study, CD133 and DG expression levels were analyzed by immunostaining in specimens of human primary colon cancers from a large group of patients with a long term follow-up and their relation with traditional prognostic indicators and with the clinical outcome of the patients was evaluated.

In this study, the expression of the surface markers CD133 and α-DG was evaluated in a subset of colon cancers and their potential prognostic significance was investigated.

We and others previously reported that loss of the α subunit of the DG complex (α-DG) is a frequent event in human cancers[6, 8, 10, 12, 14‐16]. We also demonstrated, by western blot analysis, that α-DG is frequently reduced in colon cancer tissues compared to normal adjacent normal tissues while the β subunit did not display significant variations between normal and tumour tissues[12]. In this study, we further analyzed the DG involvement in colon tumorigenesis and confirmed, by immunohistochemistry, that detection of α-DG is frequently reduced or lost in the majority of cancer tissues compared to adjacent normal tissues (Figure2) and that loss of α-DG correlates with a worse prognosis (Figure3). These findings are in agreement with the proposed tumour-suppressor function of the protein[17] and with previous observations in several human malignancies[5, 18‐20]. The functional inactivation of the DG complex in tumour cells has been mainly attributed to post-translation mechanisms which cause the loss and/or an altered glycosylation of the extracellular α-DG[21‐25]. Since DG subunits are encoded by a single gene and are formed upon cleavage of a precursor protein[6, 26], our previous findings that β-DG subunit is detectable in most of the colon cancers in which α-DG was not detectable[12] suggest that, as reported in other types of human malignancies, this lack of detection is likely not due to loss of gene expression but to a specific posttranscriptional mechanism affecting α-DG processing in colon cancer cells.

The DG complex connects the ECM network to the cytoskeleton and is likely involved in the regulation of signaling pathways[6]. Thus, regardless of the underlying molecular mechanisms, loss of a functional α-DG subunit can play an important role in the tumorigenesis process by compromising the formation of strong contacts between ECM and the cytoskeleton of cells resulting, as for integrins, in less sticky tumour cells able to move unhindered in the extracellular matrix, thus predisposed to invade surrounding tissue and metastasize[6, 17]. It will be of interest to evaluate DG expression in the entire process of human colon tumorigenesis (i.e., from early to metastatic lesions).

CD133 has been reported to be a CSC marker in colorectal cancer[27, 28], and, although some doubts have been arisen about its ability to specifically identify tumour-initiating cells[29], it has been widely used to identify and analyze CSC in colorectal cancers. We were able to detect CD133 staining in the majority (78%) of colon cancers analyzed although with a high heterogeneity in term of percentage of positive cells (range 0-80%) whose increase was associated with an increased risk of recurrence and death for the disease (Table2 and Figure3). These findings are in agreement with previous evidence suggesting a potential prognostic role of the protein in colon cancer patients. Indeed, it has been reported that CD133 expression levels correlate with patients survival in colorectal cancers[1‐3, 30, 31] although available data on the presence of CD133+ cells in human colorectal cancers are not always consistent in term of distribution and percentage of positive cells. Several factors might explain such discrepancies: i) inadequate patient cohort; ii) mixed tumour stages; iii) different criteria used to identify positive staining; iv) different cut-off used to discriminate positive and negative tumours; v) different antibodies used for the analysis, with the latter being, in our opinion, the most important factor. Different antibodies have been indifferently used in different studies for the detection of the CD133 molecule. In our opinion this can be a highly confusing factor. Indeed, we previously demonstrated, by western blot analysis, that CD133 is expressed at various levels in colon cancers[32, 33] and that different results can be obtained by using different antibodies[34] and similar observations have been also reported by other Authors[35, 36]. The observation that high CD133 expression has been reported to be a negative prognostic factor for colorectal cancers in several studies using different antibodies strongly suggests an important prognostic significance of its detection[1, 2, 37]. In our study, CD133 also confirmed to be an independent risk factor for a shorter disease-free and overall survival in a multivariate analysis (Tables 4 and5). These findings are consistent with similar results reported in other human cancers and warrant studies on larger cohorts of patients to further evaluate its suitability as a prognostic marker in the clinical management of colon cancer patients.

We observed an unexpected behaviour of CD133 expression which tended to be higher in the lowest grade/stage tumours than in more advanced lesions. Although not expected, this distribution is consistent with previous findings in a mouse model of colon carcinogenesis[38] and in human primary colon cancers[39]. Indeed, in mouse colon carcinogenesis we observed a significantly increased expression of CD133, assessed by immunohistochemistry, in early neoplastic lesions which tended to decrease with tumour development, although remaining always higher in cancer than in normal adjacent tissues[38] and an increased CD133 expression, assessed using a quantitative reverse-transcription PCR, was reported in Dukes A compared to Dukes B and C colon cancers[39]. These findings are in agreement with the proposed ability of the protein to specifically identify tumour initiating cells, important for the growth of both primary and recurrent/metastatic cancers[40] and thus mainly involved in the most active phases of tumour development, i.e., in early lesions (low grade and low stage cancers) as well as in metastatic lesions. Consistent with this hypothesis, CD133 expression has been reported to be highly expressed in colon cancers with early liver metastases and to be a potential biomarker for the early liver metastases[41] and we also previously reported an increased percentage of CD133+ cells, assessed by flow cytometry, in metastatic vs primary colon cancers,[42]. It will be of interest to evaluate the immunohistochemical CD133 expression in the entire process of human colon tumorigenesis (i.e., from early to metastatic lesions) and evaluate how it correlates with tumour development.

An unexpected finding of the present study was the observed inverse relationship between CD133 and α-DG expression (Table2). The significance of this observation is unknown since no data are available up to date linking the two molecules. It is of interest that DG expression increases with cell differentiation while CD133 expression decreases in differentiated cells[7, 33, 43‐45] thus suggesting a potential functional link between the two molecules. Further studies will be required to fully understand the biological significance of the observed relationship between the two molecules.

To our knowledge, this is the first study analyzing the immunohistochemical expression of both CD133 and α-DG, two surface molecules previously reported to be altered in human colorectal cancers, in a large series of colon cancer patients. Our results demonstrate that an inverse relationship exists between the two molecules (Table2) and that CD133 expression is an independent risk factor associated with patient survival in multivariate analyses (Tables 4 and5). The role of CD133 as a biomarker for CSC is still debated[46]. Regardless of its significance as a CSC marker, however, our results suggest that evaluation of CD133 staining might be useful to identify colon cancer patients at high risk of recurrence and death. Thus, we believe, as previously reported, that it will be important to define standardized procedures and reagents to evaluate expression of this molecule in clinical samples[34]. Afterwards, a prospective multicenter evaluation of CD133 immunostaining on a larger population of surgically resected colon cancers is warranted to allow a conclusive and definitive assessment of its suitability in predicting tumor aggressiveness and outcome in colon cancer patients.