Maria R. Kamstrup, Department of Dermatology, Bispebjerg Hospital, Bispebjerg Bakhe 23, DK-2400, Copenhagen, Denmark, Tel.: +45 3531 6005, Fax: +45 3531 6101, e-mail: firstname.lastname@example.org
Abstract: Recent experimental data offer convincing evidence for the existence of cancer stem cells in leukaemia, brain tumors and breast cancer. These cells are responsible for the maintenance of tumor growth and relapses after cytoreductive treatments. This paper provides a brief overview of current data supporting the idea of cancer stem cells in the pathogenesis of cutaneous malignancies, including skin carcinoma, malignant melanoma and cutaneous T-cell lymphoma. The characterization of putative cancer stem cells is important to develop new therapies selectively targeting these cells.
One of the most consequential concepts in recent cancer research is the stem cell theory (1–4). The fact that cancer populations are heterogenous with respect to proliferation and differentiation potential in vitro and in vivo has been acknowledged for some time (1,5,6). Two fundamentally different theories have been offered as explanation. The stochastic theory proposes that all cancer cells have an equal potential for initiating tumor formation but the likelihood of this event at a single cell is very low (7). On the contrary, the stem cell theory predicts that only a small fraction of cancer cells (cancer stem cells) holds the potential for self renewal and thereby tumor formation (8).
Cancer stem cells exhibit characteristics similar to that of normal stem cells. The definition of a stem cell relies on its capability of asymmetric division into another stem cell and one progenitor cell that further differentiates into the mature progeny comprising the adult tissue (9). Apart from self-renewal and multilineage differentiation, the stem cell can proliferate extensively. The rare population of cancer initiating cells so far identified in leukaemia, brain tumors and breast cancer possess all three features (1–4). The ‘stemness’ is conceivably maintained through deregulation of pathways such as the Notch, Hedgehog and Wnt pathways that also promote normal stem cell self-renewal (8,10,11). Recent evidence suggests an additional existence of distinct self-renewal mechanisms between normal stem cells and cancer stem cells involving the tumor suppressor protein Pten (12).
Cancer stem cells may arise from tissue-specific stem cells or from more differentiated cells that have acquired stem cell characteristics (Fig. 1). Today it is clear that within a variety of normal tissues a hierarchical system exists arising from different stem cells such as the haematopoietic or neural crest stem cells (13–15). Stem cells are long lived, and therefore have a greater risk of accumulation of mutations required for carcinogenesis (8). The relationship between normal stem cells and cancer stem cells has been most extensively studied in the haematopoietic system (for a detailed review see Ref. 16). For example, the demonstration that the hallmark of chronic myeloid leukaemic cells, the Philadelphia chromosome, is not only present in leukaemic cells, but also in all other haematopoietic lineages except mature T-lymphocytes (17–19) supports the notion that the malignant transformation can occur in a common primitive cell. Examples of possible de-differentiation of mature cells into cancer stem cells comprise studies where forced expression of leukaemia-associated genes (such as the MLL/ENL and BCR/ABL fusion genes) in committed myeloid cells in transgenic mice results in the onset of myeloid leukaemia (20,21).
The discovery that bone marrow-derived stem cells home to sites of tissue damage (22,23) opens up a third possibility for the origin of cancer stem cells. For example, in the gastrointestinal tract in mice, bone marrow-derived stem cells are recruited to the epithelia in response to chronic infection with Helicobacter felis (24). Interestingly, these bone marrow-derived cells give rise to gastric adenocarcinoma developing as a result of the chronic infection. It is conceivable that immigrating normal stem cells can fuse with mutated somatic cells giving rise to immortal, malignant cancer stem cells (25).
With regard to cutaneous malignancies, emerging evidence supports the stem cell theory in the pathogenesis of squamous cell carcinoma and malignant melanoma. Preliminary data suggest that cancer stem cells may participate in the pathogenesis of cutaneous lymphomas as well.
Cancer stem cells in non-melanoma skin cancer
Epidermal stem cells reside in the basal layer of the interfollicular epidermis and in the bulge of the hair follicle (26–29). They are likely to be founding cells for non-melanoma skin cancer.
Classical studies on chemical carcinogenesis demonstrated that papillomas and squamous cell carcinomas develop even when the time span between initiation and promotion is as long as a year suggesting persistence of the target cell in the skin (30). These initiated target cells are mitotically quiescent cells as demonstrated by their insensitivity to 5-fluorouracil, known to kill cycling cells selectively (31). When the oncogenic H-ras gene is expressed in the stem cells within the hair follicle, a spontaneous conversion to squamous carcinomas and in some cases spindle cell carcinomas often takes place (32). On the contrary, if suprabasal epidermal layers are targeted, benign papillomas develop in response to mild wounding (33). Further evidence implicating the hair follicle as a source of target cells comes from studies showing that mice, in which the interfollicular epidermis is removed by dermabrasion, have the same incidence of carcinomas as the intact mice (34). Studies by Morris support that target cells in chemical carcinogenesis in mouse models exhibit stem cell patterns (reviewed in Ref. 35) and hereby provide a possibility for the subsequent formation of a tumor with hierarchical structure in which the target of transformation retains stem cell features.
The ability to efflux the fluorescent dye Hoest 33342 is one of the features of many types of normal and cancer stem cells (36–38). Squamous cell carcinoma lines contain cells capable of excluding Hoest dye (39), but their stem cell characteristics have not been studied. In malignant epithelial cell lines, including oral squamous cell carcinoma lines, a subpopulation of cells can give rise to large colonies, the so-called holoclones (40). As holoclones are normally considered to be founded by single stem cells (41), this population could represent the cancer stem cell compartment in squamous cell carcinoma.
There is some evidence, albeit inconclusive, for the involvement of cancer stem cells in basal cell carcinoma. The Hedgehog signalling pathway is important for self renewal of normal stem cells and constitutive activation of the Hedgehog signalling results in basal cell carcinomas (11,42,43). When the pathway is blocked, basal cell carcinomas show regression. However, a small subset of long-lived, quiescent cells survives and when the pathway is reactivated, these cells maintain their capacity for tumor formation providing a link between cancer stem cells and basal cell carcinoma (44).
Bone marrow-derived cells may also contribute to epithelial cancers. CD34+ bone marrow-derived cells engraft to the bulge region in response to skin wounding and differentiate into keratinocyte stem cells (23). It appears that a homing mechanism for bone marrow-derived cells to damaged tissues could be an interaction of the integrin–ligand pair VLA-4-VCAM/fibronectin (45). A model has been proposed for epithelial cancers in which epithelial damage depletes tissue-specific stem cells and bone marrow-derived cells engraft into the novel niche serving as replacement for tissue-specific stem cells. As a result of consistent stimulation in an abnormal environment, malignant transformation subsequently occurs in the bone marrow-derived cells (24,46). In solid organ transplant recipients, haematopoietic cells from the donor are frequently detected in peripheral blood and squamous cell carcinomas have been shown to arise from such donor cells that have migrated to the skin (47).
Cancer stem cells in malignant melanoma
Melanocyte stem cells reside in the bulge region of the hair follicle (48,49), but their relevance for malignant melanoma is uncertain. However, cancer stem cells seem to be present in malignant melanoma. Fang et al. (50) demonstrated that in a subset of fresh and established melanoma cell lines, a population of cells exhibited stem cell growth pattern by forming spheres in the stem cell medium. Spheroid cells had stem cell characteristics persisting after serial passages in vitro and after transplantation in mice, sustaining the ability for differentiation and showing greater tumorigenic potential. A subset of spheroid cells consistently expressed the cell surface marker CD20+ and when isolated, these cells preferentially developed new spheres. Spheroid cells could be induced to differentiate in vitro into cells expressing markers of adipocytic, osteocytic and chondrocytic lineages suggesting a multilineage differentiation potential.
Melanoma cells are capable of expressing neural markers (51,52) and it has been speculated that the association between tumors of the nervous system and malignant melanomas in certain individuals represents an underlying abnormality in neural crest stem cells (50). The ability to efflux Hoest dye was utilized in another attempt to isolate cells with stem cell-like features (53). The ABC superfamily of active transporters responsible for the expulsion of Hoest dye (37,54), functions also as drug efflux transporters and confers protection against chemotherapy (reviewed in Ref. 55). A novel member of the ABC superfamily ABCB5, present in the side-population in the Hoest dye assay, appears to be responsible for the resistance of melanomas to the chemotherapeutic drug doxorubicin (56). The ABCB5+ stem-like tumor cells were capable of regenerating the heterogenous tumor populations in vitro and gave rise to larger clones than the ABCB5− cells. The cells also expressed the stem cell-associated surface marker CD133 like the cancer stem cells in brain tumors (3). Further insight into the pathogenesis of melanoma is provided by a study of chromosomal alterations in a patient experiencing multiple recurrences of metastatic cutaneous melanoma with temporary complete remissions (57). Cell lines obtained from each recurrence had consistent genetic traits, though other chromosomal aberrations were too divergent for the cells to derive from a sequential process. This observation suggests the existence of a common progenitor cell that gives rise to genetically unstable descendants resulting in metastatic cancer. Other researchers reported similar data supporting a clonal progression model (58,59).
Cancer stem cells in cutaneous lymphomas
Primary cutaneous T-cell lymphomas (CTCL) represent a group of extranodal non-Hodgkin T-cell lymphomas clinically originating in the skin (60,61). The cellular origin of the majority of CTCL is believed to be a skin-homing mature CD4+ T-cell. The precise aetiology is unclear, but chronic antigen stimulation is believed to be important for the neoplastic transformation (62–64).
Preliminary evidence suggests involvement of cancer stem cells in CTCL (65). At least in some patients, the appearance of CTCL is preceded by the presence of clonally related cells in other compartments, such as bone marrow. Interestingly, these bone marrow-derived cells are resistant to high-dose chemotherapy suggesting their mitotic quiescence. It has been hypothesized that CTCL may arise in bone marrow and spread via peripheral blood to the skin (66).
Certain characteristics of CTCL support the involvement of cancer stem cells, possibly arising in non-cutaneous compartments, including bone marrow (reviewed in Ref. 66). Multiple skin lesions often occur simultaneously as an initial symptom of CTCL (60). If the malignant transformation occurred in a mature skin-infiltrating T-cell and subsequently spread to other areas of the skin, one would expect that clonal T cells would be present in peripheral blood before new lesions could occur. However, in most cases clonal T cells are not detected in peripheral blood until late stages of tumor progression. Another characteristics of CTCL (mycosis fungoides) is the invariable disease relapse (67,68) despite the fact that the currently used treatment modalities are capable of achieving virtually complete eradication of the clonal T cells in the skin. Moreover, CTCL can be transferred by T-cell-depleted human bone marrow transplantation years before the malignancy arises in the skin of the donor (69). The above-mentioned finding that bone marrow-derived cells home to sites of tissue damage provides a possible explanation why mycosis fungoides is often preceded by a non-specific inflammatory stage. The stem cells immigrating into the site of cutaneous inflammation may either fuse with mutated lymphocytes in the skin or themselves undergo malignant transformation.
A growing body of evidence implicates cancer stem cells in the pathogenesis of cutaneous malignancies. The concept of cancer stem cells provides a novel framework for the understanding of cutaneous carcinogenesis and would have fundamental implications for future cancer treatment. Drug transporters in cancer stem cells probably contribute to their drug resistance (55) and recent data have indeed established a reduced drug sensitivity in putative cancer stem cells when compared with the bulk of differentiated cancer cells (56,70,71). A relative mitotic quiescence provides another mechanism by which cancer stem cells can survive traditional cytoreductive therapies (72,73). If the therapy does not target the cancer stem cells, an initial remission will inevitably be followed by a relapse. New observations such as differential sensitivity to the blockers of Akt-mTor pathway between normal and cancer stem cells (12) would provide new means for the elimination of cancer with minimal effect on normal tissue.
The authors are grateful to the Aage Bang Foundation, the Minister Erna Hamilton Foundation, the Soeren and Helene Hempel Foundation and the Jens and Maren Thestrup Foundation for financial support.