Colon cancer stem cells

Colorectal cancer (CRC) is the third most common form of cancer and the second cause of cancer-related death in many industrialized countries and is characterized by a heterogenic pool of cells with distinct differentiation patterns, leading to 655,000 deaths worldwide per year (1). Despite the emergence of new targeted agents and the use of various therapeutic combinations, none of the treatment options available is curative in patients with advanced cancer. A growing body of evidence is increasingly supporting the idea that human cancers can be considered as a stem cell disease. According to the cancer stem cell model, malignancies originate from a small fraction of cancer cells that show self-renewal and pluripotency and are capable of initiating and sustaining tumor growth (2).

        This review will focus on the biology of normal and malignant colonic stem cells, which might contribute to our understanding of the mechanisms responsible for tumor development and resistance to therapy. First, we will briefly revise the knowledge available on normal intestinal stem cells and recent advances in understanding crypt biology, which have led to new theory on the origins of colon adenomas and cancers. Then, we will summarize the evidence and current status on colon cancer stem cells, focusing on their relevance and promises for the treatment of colorectal carcinoma.

Colonic stem cell identification

The adult colonic epithelium has a well-defined architecture organized into crypts, dynamic structures which are constantly self-renewing (3). Each crypt unit is maintained by adult multipotent stem cells (SCs), located at the bottom of the structure itself, that are able to simultaneously self-renew and generate a population of transiently amplifying cells which in turn generate more the specialized intestinal epithelial mature cells. Three differentiated cell types mediate the function of colonic epithelium: the colonocytes, also termed absorptive enterocytes, the mucus-secreting goblet cells and the hormone-secreting enteroendocrine cells (Figure 1).

         Adult SCs are defined by several key functional properties including: self-renewal, potential for multilineage differentiation and tissue regeneration. Two different models have been proposed to localize the intestinal SCs; the “+4 position” model and the “stem cell zone” model (4). According to the former, the intestinal SCs are located at the +4 position relative to the bottom of the small intestine crypt, just above the non-cycling Paneth cells. These cells are quiescent, slowly cycling, label retaining cells (LRCs) and, through asymmetric division, give rise to their differentiated progeny. The more recent “stem cell zone” model states that active, rapidly cycling cells and self-renewal, termed crypt base columnar (CBC) cells, are the true intestinal SCs (Figure 2). These cells are interspersed between the Paneth cells in the small intestine or located at the very bottom of the crypt in the colon. The intestinal stem cells, both +4 cells and CBC stem cells give rise to the transitamplifying population that undergoes vigorous division and differentiation into enterocytes, goblet cells, and enteroendocrine cells as they migrate out of the crypt onto the villi (3).

Fig. 1. Colonic crypt organization. a. In the epithelial lining of normal colonic mucosa, stem cells (red) are located at the bottom of the crypts. Upon asymmetrical divisions, the daughter cells undergoing differentiation migrate upward to give rise in turns to transitamplifying (TA) precursors (light blue) and terminally differentiated cells (pink). b. Cell types in the colon epithelium. Intestinal stem cells generate three epithelial cell types: the absorptive columnar cells, the hormone-producing enteroendocrine cells, and the mucous-producing goblet cells.

Fig. 2. Models for stem cells location of stem cells in intestinal crypts. a. The “+4 position” model suggests that intestinal stem cells are quiescent, slowly cycling cells and  located just above the Paneth cells at position +4 relative to the crypt bottom (green). The most important marker that identifies these cells is Bmi-1. b. The “stem cell zone” model assumes active, rapidly cycling cells, so-called crypt base columnar (CBC) cells (red), interspersed with Paneth cells, are the true intestinal stem cells. The most important marker to identify these cells is Lgr5.

The Lgr5 gene encodes a leucine-rich repeat containing G-protein coupled receptor, also known as Gpr49. Lgr5 expression is restricted to cycling CBC cells and it has been demonstrated that Lgr5-expressing cells differentiate into the expected functional lineages of the colonic epithelium (5). Transcriptome analysis of Lgr5+ epithelial cells isolated from the bottom of the small intestinal crypts led to the identification of a gene signature for these Lgr5+ SCs. The OLFM-4 gene encodes a secreted molecule with unknown function, originally cloned from human myeloblasts (6), which is enriched in human colon crypts (7). Due to the very low expression levels of Lgr5, OLFM-4 has been recently proposed as a more faithful SC marker highly expressed in CBC cells in human small intestine and colon (8).

Colorectal cancer stem cell identification

Tumors are composed of a heterogeneous mixture of cancer cells at various levels of differentiation, very similar to the structure of an organ. Recently, the “cancer stem cell” model of tumorigenesis has proposed that within the tumor mass there is a predetermined cell population with a ‘‘stem cell’’ phenotype, able to perpetuate the cancer, while the rest of the tumor cells are incapable of self-renewal (9). Even though it has long been assumed that mutations within adult colonic stem cells may induce neoplastic transformation, the proof of existence of colorectal cancer stem cells (CRC-SCs) has been hindered in the past years by difficulties in identifying a specific biomarker for this rare cell population. Only recently, new evidence has been provided that supports the existence of CRC-SCs, confirming that the tumorigenic cell population of CRC can be isolated on the basis of the expression of specific cell surface biomarkers (9). The standard analysis to ascertain the existence of a subpopulation of cancer stem cells (CSCs) is the demonstration that these cells can transfer the tumor in immunocompromised mice and replicate the phenotypic heterogeneity of the parental tumor. Several recent studies have evaluated the functionality of specific CRC-SC biomarkers by using a combination of flow cytometry to identify a “putative” SC population and xenograft models involving immunodeficient mice to determine their tumor initiating potential (9).

In the first two studies, CD133, also known as Prominin-1, was employed to identify the tumorigenic cell population within CRC and metastatic (10, 11). When transplanted into the renal capsule of NOD/SCID mice, CD133+ cells readily developed tumors displaying morphologic features equivalent to those of the parental cancer. Tumor phenotype was further maintained upon serial transplantation (10). Similarly, in the second study, a population of CD133+ cells, accounting for approximately 2.5% of tumor cells, was isolated from colon cancer specimens and perpetuated in vitro as floating colonies or “tumor spheres” (11). CD133⁺ cells readily gave rise to tumors in mice, whereas the CD133⁻ cell population was unable to generate tumors even after serial transplantation in mice (9). Both studies demonstrated the expression of CD133 also in normal colon tissue, although at a lower frequency, reinforcing the hypothesis that CD133+ CRC-initiating cells in cancer samples might result from oncogenic transformation of normal colonic SCs.

Subsequently, one study proposed CD44 and the epithelial surface antigen (EpCAM) as CRC-SC-specific markers, with further enrichment by CD166. Purified CD44+/Ep- CAM^HIGH cells injected into NOD/SCID mice resulted in high frequency generation of tumor xenograft. In contrast, CD44-/EpCAM^LOW cells lack tumor initiating activity (12). Further subfractionation of the CD44+/EpCAM^HIGH cell population by using the mesenchymal stem cell marker CD166 increased the success of tumor xenograft.

Finally, in a more recent study, aldehyde dehydrogenase 1 (ALDH) has been proposed as a promising new marker for normal and malignant human colonic SCs (13). Flow cytometric isolation of ALDH1⁺ cancer cells and implantation of as few as 25 cells in NOD/SCID mice generate tumor xenografts. Further isolation of cancer cells using a second marker (CD44 or CD133 serially) only modestly increased enrichment based on tumor-initiating ability.

In an another study, reported that in primary colon cancer samples from humans and mice, CD133 was expressed in all epithelial, EpCAM+ cells in the malignant tissue and that CD133 expression was excluded from the non-epithelial cell components of the tumor (14). Thus, they proposed that the inability of CD133- to generate tumors could be simply due to their non-epithelial nature. Furthermore, the same authors demonstrated that both, CD133+/EpCAM+ and CD133-/EpCAM+ cell populations, isolated from liver metastasis of colon cancer, were able to generate tumors upon serial transplantation into NOD/SCID mice (14).

A comparison of expression of the three markers CD133, CD44 and CD166 that have been associated with CRC-SCs revealed that the expression of CD133 correlates with that of CD166, whereas both do not correlate with CD44, confirming that CD133 is, alone, the best marker to predict poor patient survival (15).

Identification of biomarkers for CRC-SCs will improve the understanding of the mechanism underlying tumor growth and progression. Once again, studies performed on mouse model could offer helpful suggestion. Recently, one study provided a very convincing demonstration of the origin of intestinal cancer from Lgr5⁺ CBC cells. The authors have shown that deletion of APC in Lgr5+ expressing cells leads to their transformation within days, suggesting that Lgr5 may mark not only normal intestinal stem cells, but also a limited population of CSCs (16). Simultaneously, using knock-in LacZ reporter mice within the Prominin-1 (Prom1) locus, in a study have shown that Prom1+ cells, located at the base of the crypts in the small intestine, co-express Lgr5, generate the entire intestinal epithelium and are susceptible to neoplastic transformation (17).

From a clinical point of view, a recent study showed that Lgr5 was markedly over-expressed in the majority of advanced CRCs compared with normal mucosal tissue (18). As expected, in situ hybridization analysis confirmed the expression of Lgr5 in CBC cells in both small intestine and colon. This Lgr5 expression, which was variable among CRC cases, correlated significantly with lymphatic and vascular invasion, lymph node metastasis and tumor stage, suggesting the involvement of this marker in tumor progression.

It might be summarized the markers that have been used to isolate CRC-ICs, shown in table 1 (9).

Table 1 Markers that have been proposed to characterize normal intestinal SCs and used to isolate CRC-ICs

	Marker	Function
Normal intestinal stem cells	Musashi-1	RNA-binding protein
	Hes-1	Transcriptional repressor
	EphB receptors	Cell surface receptors
	Bmi-1	Policomb-repressor protein
	Lgr5	Unknown, WNT target gene
	Aldh-1	Enzyme
Colon cancer stem cells	CD133	Unknown
	CD44	Hyaluronic acid receptor
	CD166	Adhesion molecule
	Aldh-1	Enzyme

Clinical implications of colorectal cancer stem cells

The CSC model has important implications for cancer therapy. At present, anticancer therapies for CRC include surgery, radiation, chemotherapy, and anti-VEGF or EGFR monoclonal antibodies. In most cases, many current cancer therapies target the most rapidly dividing cells, which represent the majority of the tumor cell population. Similarly, CRC-SCs have been found to be enriched in colon tumors following classical chemotherapeutic regimens and remain capable of rapidly regenerating tumor from which they were derived (19). The authors have further demonstrated that resistance is mediated, at least in part, by ALDH1 enzyme activity.

Together with resistance to chemotherapy, CSCs are frequently resistant to standard radiotherapy regimens. In this respect, it has been recently demonstrated that resistance to radiation of CD133+ glioblastoma SCs can result from elevated expression of DNA damage response genes (20). Radiotherapy for glioblastoma is associated with an increase in the proportion of the CD133+ fraction.

In another study, has recently demonstrated that the up-regulation of interleukin-4 (IL-4) in CD133+ CRC-SCs is an important mechanism that protects these tumorigenic cells from apoptosis. CD133+ CRC-SCs produce IL-4 as an autocrine growth factor promoting tumor resistance to chemotherapeutic agents such as 5-fluorouracil and oxaliplatin (21). This phenomenon was confirmed in xenografts in which the administration of anti- IL-4 antibodies significantly reduced tumor growth after chemotherapy.

In a recent study, generated CD133+ tumor sphere cultures from several colon cancer specimens and performed mass-spectrometry-based quantitative proteomics in order to identify cell surface proteins enriched on culture tumor cells (22). These cells retain the expression of cell surface markers such as CD133, CD166, CD44 and EpCAM as well as other stem cell-associated proteins including nestin, Bmi1 and Msi-1, thus confirming the value of this in vitro model for biological analysis of CSC populations as well as for drug screening experiments.

Conclusion

Increasing evidence shows that CRC-SCs may play a critical role in tumor development and progression. CRC-SC resistance to conventional therapies may explain why it is difficult to completely eradicate cancer and why recurrence is often inevitable. Thus, the identification and molecular characterization of CSCs is critical to develop therapeutic strategies that specifically target this rare population of cells and that are likely to be effective in eradicating tumors and in reducing the risk of relapse and metastasis.

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