Principles of stem cell biology and cancer: future applications and therapeutics. Edited by T. Regad, T. J. Sayers and R. C. Rees. John Wiley & Sons (2015)
Part II. Cancer stem cells
The use of multiple markers to define a CSC subpopulation is, in our view, strongly recommended. A panel of markers defines haematopoietic stem cells as well. The literature is sometimes confusing, since researchers often use potential biomarkers without a specific biological motivation, employing some marker alone in some papers and in a combination in others. In this section, we discuss recent findings for four tumours: breast, colon and brain cancer and melanoma. The picture that comes out is still fuzzy and confused, but we will suggest some ideas to clarify it.
CSCs and breast cancer
Al-Hajj et al. (2003) described the presence of a tumourogenic subpopulation in eight out of nine breast cancer patients, based on the cell-surface marker expression of CD44CD24-/low. The authors isolated tumour cells from all nine patients and showed that eight generated new tumours containing additional CD44CD24-/low after serial passing (Al-Hajj et al., 2003). They also demonstrated that there is a hierarchy of breast cancer cells, with some cells proliferating extensively but the majority, derived from this population, having only limited proliferative potential in vivo (Al-Hajj et al., 2003).
Sheridan et al. (2006) showed that the CD44CD24-/low subpopulation of breast cancer cells expresses high levels of proinvasive genes and highly invasive properties, but this phenotype was not sufficient to predict capacity for pulmonary metastasis. In fact, they could not find any correlation between CD44CD24-/low phenotype and the ability to home and proliferate at sites of metastasis (Sheridan et al., 2006). One could speculate that no correlation was found because these biomarkers are not exclusively present in CSC subpopulations. Fillmore and Kuperwasser (2008) analysed eight human breast cancer cell lines for CD44, CD24 and epithelial-specific antigen (ESA) expression and showed that the percentage of CD44CD24-/low cells did not correlate with tumourogenicity. In contrast, cells characterized by CD44CD24/ESA+ can self-renew, reconstitute the parental cell line, retain BrdU label and preferentially survive chemotherapy (Fillmore and Kuperwasser, 2008). In a more recent paper, Gupta et al. (2011) studied the dynamics of phenotypic proportions in human breast cancer cell lines, using CD44high/CD24/EpCAMlow as a CSC marker. The authors showed that subpopulations of cells purified from the CSC phenotype tend to express it again in time, and speculated that these results provide an indication of phenotypic switching of cancer cells into CSCs (Gupta et al., 2011). A recently published paper showed a similarity between Her2+ intrinsic human breast cancer subtypes and mammary stem cell populations, supporting the origin of CSCs in a stem cell population (Benjamin et al., 2012). Moreover, the authors described a new signature for human CSCs, claiming that new gene sets with prognostic value could also be useful for predicting which patients will respond to certain treatment strategies (Benjamin et al., 2012). In a recent paper, Dickopf1, a protein known to negatively regulate the Wnt pathway, was shown to affect the fate decision of breast CSCs sorted according to CD44+ CD24low , driving the cells to differentiate (Agur et al., 2011). Interestingly, the results are supported by a mathematical model (Agur et al., 2011).
Aldehyde dehydrogenase (ALDH) was used as a stem cell marker in 33 human breast cell lines (Charafe-Jauffret et al., 2009). ALDH is a detoxifying enzyme that oxidizes intracellular aldehydes and is believed to play a role in the differentiation of stem cells via the metabolism of retinal to retinoic acid (Marcato et al., 2014). Interestingly, ALDH activity can be used to sort a subpopulation of cells that display stem cell properties from normal breast tissue and breast cancer (Marcato et al., 2014). ALDH activity, assessed by ADELFLUOR assay, has been successfully used to isolate CSCs from multiple myeloma and acute leukaemia, as well as from brain tumour (Fang et al., 2005; Klein et al., 2007). However, in melanoma there are contrasting results: ALDH phenotype is not associated with more aggressive subpopulations, arguing against ALDH as a ‘universal’ marker (Hadnagy et al., 2006). Another interesting pathway that has been extensively studied is the Notch receptor signalling pathway (for a recent review, see Hadnagy et al., 2006). An important point is the toxicity of these potential treatments. While the Notch pathway appears promising, it is active in other tissues, so it might have a great toxicity. Therefore, as suggested by Harrison and colleagues, it seems important to study the complexity of the Notch pathway in order to more successfully target CSCs (Harrison et al., 2010). On the other hand, in a recent study, 275 patients were analysed for CD44+ CD24-putative stem cell marker, as well as for other markers (vimentin, ostenectin, connexin 43, ADLH, CK18, GATA3, MUC1), in primary breast cancers of different subtypes and histological stages (D’Amico et al., 2013). This study reveals a high degree of diversity in the expression of several of the selected markers in different tumour subtypes and histologic stages. We would point out that the latter findings could be explained by the observation that none of these markers are really specific for CSCs.
All together, these data show that the therapeutic implications of CSCs in breast cancer are still unclear. The current understanding of the role of CSCs in clinical trials is discussed in a recent review article (Charafe-Jauffret et al., 2009).
CSCs and melanoma
Several published papers, using different putative CSC markers (CD20, CD133, ABCG2, ABCB5, CD271 and CXCR6), show that a CSC subpopulation exists in melanoma (Dou et al., 2007; Monzani et al., 2007; Schatton et al., 2008; Boiko et al., 2010; Taghizadeh et al., 2010; Zhong et al., 2010). However, Quintana et al. (2010) argued against the existence of CSCs based on the following observations: a relatively large fraction of melanoma cells (up to ~25%) initiated tumours in severely immunocompromised non-obese diabetic severe combined-immunodeficient (NOD/SCID) IL2Rγ null mice; the fraction of tumour-inducing cells depends upon assay conditions; and several putative CSC markers appear to be reversibly expressed. In conclusion, this paper suggested that the best model by which to confirm the presence of CSCs is a severe immunocompromised mouse. In a follow-up study, the same authors analysed the expression of more than 50 surface markers on melanoma cells derived from several patients (A2B5, cKIT, CD44, CD49B, CD49D, CD49F, CD133, CD166), but focusing on CD133 and CD166 (Quintana et al., 2010). Using these markers, they found no enrichment and a high frequency of tumourigenic cells (Quintana et al., 2010). In a recent paper, however, it was shown that CD133 is highly expressed in melanoma cells and is not a good marker by which to sort CSCs (Monzani et al., 2007). Moreover, Boiko et al. (2010), using the same immunocompromised mice, did not confirm Quintana et al.’s data (Monzani et al., 2007). Boiko et al. (2010) used CD271, nerve growth factor receptor, as a marker by which to identify CSCs. In 2010, our group showed for the first time that a marker connected with a functional property of stem cells, CXCR6 (connected with asymmetric division), is expressed by human melanoma cell lines and biopsies (Taghizadeh et al., 2010). The CXCR6 subpopulation showed a stronger self-renewal capability in a xenograft model (Taghizadeh et al., 2010). We also showed that CXCR6-positive cells were able to grow better when treated with the ligand (CXCL16) (Taghizadeh et al., 2010). A recent paper reports that the growth of B16-F10 melanoma cells in syngeneic mice seems to be maintained by a relatively large proportion (>10%) of tumour cells, through analysis of a side population (Zhong et al., 2010).
Finally, more recently, Kumar et al. (2012) showed that Oct4, a highly critical transcription factor in stem cells, can also promote dedifferentiation of melanoma cells to CSC-like cells. These authors also showed that hypoxia can increase the level of oct4 (Kumar et al., 2012) and therefore suggested that, since a transient expression of Oct4 protein is sufficient to induce dedifferentiation of melanoma cells and Oct4 can be regulated by hypoxia, Oct4 might mediate the effect of hypoxia on tumour progression (Kumar et al., 2012). While these results are promising, more studies are needed to elucidate the regulation and function of hypoxia in tumour progression.
CSCs and colon cancer
Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of tumour-related death in the Western world (Dallas et al., 2009). Despite the well-known genetic mutations that drive the transition from healthy colonic epithelia to dysplastic adenoma and finally to colon adenocarcinoma, current anticancer treatments are often able to eradicate the disease. Indeed, the response rate to current systemic therapies is about 50%, but resistance develops in nearly all patients (Dallas et al., 2009). CSCs are also identified from the expression of one or multiple cell-surface markers associated with cancer stemness, such as CD133 (Haraguchi et al., 2008; Puglisi et al., 2009), CD44 (Haraguchi et al., 2008; Wang et al., 2008), CD166 (Mafirgaritescu et al., 2014) or Lgr5 (Dalerba et al., 2007). More functional markers, such as Wnt activity (Vermeulen et al., 2010) and ALDH1 activity (Huang et al., 2009), have been exploited for identification of colon CSCs. However, none of the markers used to isolate stem cells in various cancerous tissues are expressed exclusively by the stem cell fraction. Indeed, most such markers are chosen either because they are expressed in normal stem cells or because they have been found to identify CSCs in other malignancies.
CSCs and brain tumours
Glioblastoma multiforme (GBM) is the most common type of primary malignant brain tumour, accounting for 55% of primary brain tumours (Legler et al., 1999). The prognosis of GBM is very poor; most patients die of tumour recurrence.
Glioma stem cells (GSCs) were among the first CSCs to be described for solid tumours (Legler et al., 1999). The existence of GSCs is now widely accepted, with the most clinically relevant features of CSCs, such as resistance to existing therapies, having been confirmed in GSCs (Singh et al., 2004; Bao et al., 2006; Bleau et al., 2009).
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