Cancer stem cells; Hierarchical and stochastic

The existence and important role of cancer stem cells appears to support many of the clinical observations on malignant cells and their responses to treatment. Whilst their existence was originally suggested in the 19^th century [10], cancer stem cells were first identified in the 1990s, in acute myeloid leukemia (AML) [11, 12]. Further studies have now observed cancer stem cells in a wide range of malignancies including glioblastoma, breast, endometrial, pancreatic, prostate, lung, colon cancers and a range of other tumor types [13–17]. Overall cancer stem cells are characterised as having distinct key properties including: self renewal, the ability to differentiate, active anti-apoptotic pathways, the expression of CD44, aldehyde dehydrogenase, CD133 and other markers also expressed by normal tissue specific somatic stem cells [18].

The importance of tissue specific somatic stem cells on cancer biology and therapeutics is twofold. Firstly, the existence of tissue specific stem cells allows for cells to be sufficiently long lived to be able to accumulate the number of mutations required to develop the malignant phenotype [18]. The second characteristic of tissue specific stem cells is that they have an intrinsically biologically damage resistant phenotype that allows the tissue specific stem cells to be resistant to natural adverse challenges. This phenotype is that result of two processes, the first the presence of multiple systems including ATP-binding cassette (ABC) transporter proteins, that help protect the stem cell from any genotoxic agents [19, 20] and secondly the low level of apoptotic response to any DNA damage results in the resistance to chemotherapy treatment that may kill cancer cells and more mature normal healthy cells.

The resistance to chemotherapy of the normal tissue specific somatic stem cells appears to be routinely shared by the stem cells that have undergone malignant transformation and have become cancer stem cells [21]. It is believed that the natural resistance of the cancer stem cells to chemotherapy treatment may contribute to the underlying chemotherapy resistance of the common malignancies [14, 22, 23].

The main cancer stem cell model relates to cancer stem cells that form a hierarchy in giving rise to the tumour cells, following the linear progression from somatic stem cell to cancer stem cell and then to cancer cell. However there is also evidence that cells with some similar properties of stemness can arise from within the tumour cell pool in the stochastic cancer stem cell model. However these cancer stem cells having a different aetiology to hierarchical stem cells and the potential to have different biological characteristics. These two approaches to cancer stem cells are not mutually exclusive with stochastic cancer stem cells being reported in colon cancer that is also supported by a hierarchical cancer stem system [24].

The malignant phenotype and inhibition of developmental progression

By definition the development of the malignant phenotype dramatically affects the biology of the cell with a major impact on the inhibition of normal patterns of cellular differentiation, senescence, ageing and programmed cell death [25]. The result of these processes is to lead to the accumulation of excess cells, a lack of control of numbers, abnormal development patterns and the inhibition of apoptosis that is normal part of the natural history of healthy cells.

The most dramatic impact of the onset of the malignant phenotype on altering cellular development can be seen in B and T cell malignancies which arise at a number of differing points along the normal but complex linear lymphocyte development pathways. In these malignancies, the malignant cells appear to become developmentally frozen with no further progression in the normal inear development of these cells from the time of the onset of the malignant phenotype [26]. As a result B cell malignancies that arise at differing points in B cell development can give rise to a wide spectrum of malignancies ranging from acute lymphocyctic leukemia (ALL), through chronic lymphocytic leukemia (CLL), mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), Hodgkin’s disease, follicular lymphoma, and on to multiple myeloma. The actual diagnosis and treatment related prognosis being dependant on the point in B cell development when the malignant phenotype is established and further cellular development halted [27].

More recently we have postulated how the impact of the onset of malignancy on freezing the cells normal developmental progression may leave malignant cells developing at certain normally transient but key developmental points with fixed heightened physiological pro-apoptotic potential [3]. For each of the chemotherapy curable malignancies there appears to be a close association with a unique physiological genetic event and their naturally associated relevant pro-apoptotic pathways. These events are nuclear fusion for gestational trophoblast tumors, meiosis for germ cell tumours, VDJ recombination for ALL and anaplastic lymphoma kinase (ALK) + ve T cell NHL, somatic hypermutation (SHM) for DLBCL, Hodgkin’s disease and Burkitt’s lymphoma and gastrulation for the rare childhood malignancies. Based on the assumption that the onset of malignancy occurs closely related to these events, we have postulated the persistence of the naturally upregulated pro-apoptotic machinery associated with these events remaining active and that this may be a likely major contributor to the ease of cure of these malignancies with chemotherapy treatment [4].

Cancer cell of origin, ABC transporter proteins and chemotherapy sensitivity

Alongside the major contribution made to chemotherapy responses of the chemotherapy curable malignancies by the apoptotic sensitivity associated with their associated unique genetic developments, the ABC transport proteins also plays an important role in determining chemotherapy responses [28, 29].

A number of studies have indicated that the expression of this family of molecules is associated with a reduction in the efficacy of chemotherapy as a result of the increased efflux of drugs from cancer cells [30, 31]. However despite this important role, clinical developments to reverse this impact have not to date been of significant success [32–35].

The relationship between stem cells, the cancer cell of origin, the expression of the ABC system and the response to chemotherapy treatment in the chemotherapy curable malignancies helps to define the unique biology of these cells.

The ABC protein system is widely expressed in conventional tissue specific stem cells, where it plays a key role in protecting these cells from genotoxic stress [36]. Similarly in epithelial cancer stem cells, which are derived from tissue specific stem cells, ABC proteins are documented to be persistently expressed [37–39]. In the majority of solid tumours further increases in ABC protein expression levels have been associated with declining sensitivity to chemotherapy [40, 41].

However, as previously discussed, in the chemotherapy curable malignancies, there are no standard hierarchical cancer stem cells derived from the tissue specific stem cells and each of these malignancies arises from a transient developmental cell. It is apparent that these transient developmental cells have very differing patterns of ABC protein expression compared to tissue specific stem cells and this status appears to impact significantly on the expression of ABC proteins in malignancies derived from them.

In normal trophoblast cells, assessment of the expression and functional activity of ABC transporter genes in murine embryos indicate that activity of these efflux systems is not present immediately post nuclear fusion but only becomes active by day 6 [42]. Pathological studies demonstrate that in molar pregnancies there is only focal and much lower levels of the multi-drug resistance transporter ABCB5 expression than is seem in normal first trimester placentas [43]. This observation suggests that the failure of molar pregnancies to successfully undergo nuclear fusion and subsequent cellular differentiation is associated with an inhibition of the cellular development that would normally include the normal increase in expression of ABC proteins.

There is less data on the expression of the ABC family proteins in choriocarcinoma and placental site trophoblast tumours, however the limited data indicates that the majority of cases of choriocarcinoma or PSTT do not express ABCB5 [43].

B and T cell malignancies arise from differing transient cell types occurring along the lymphocyte development pathways [26]. The level of ABC protein expression at these points can vary significantly and this can impact on the lasting phenotype and chemotherapy sensitivity of the resultant malignancies. In normal B cells, studies have indicated high levels of functional activity for the efflux pump systems in HSCs but that these levels decline sharply in cells as they become committed as 34+/38+, 34+/33+, or 34+/10+ progenitor cells [44]. A wider review of ABC protein expression indicates that their activity varies across B cell development from a peak as HSC then falling during B cell development and then rising again in plasma cells [45].

Data suggests that this variation in ABC protein activity during normal B cell development is reflected in the various malignancies arising along the course of lymphocyte development. A number of studies have indicated that there the expression of ABC family members is significantly lower in both B cell and T cell ALL than in HSCs [46]. This reduction in the ABC activity marks that the ALL cells have, on entering VDJ rearrangement, moved away from the phenotype of the chemotherapy resistant HSC and the low level of ABC protein expression may in part be linked to the sensitivity of ALL to chemotherapy.

In Hodgkin’s lymphoma which is believed to arise from germinal centre B cells, the majority of cases do not express ABC proteins. Of note a more recent study has indicated that the approximately 30% of Hodgkin’s cases that do express the ABCC1 protein have a tendency to a worse prognosis [47]. Similarly in DLBCL expression of the ABC system is highly variable between cases and the outcome is worse in patients with higher levels of expression [48].

In contrast in CLL, levels of ABC proteins are higher than in DLBCL [49] and myeloma which develops from cells in the final stage of B cell development expresses ABC proteins at higher levels which can rise further on exposure to chemotherapy [50].

In testicular cancer characteristically the majority of ABC proteins are expressed at only very low levels, with significant levels of expression only seen in tumours that have demonstrated chemotherapy resistance [51]. The presumed cell of origin of testicular cancer is intraepithelial germ cell neoplasia which arises from a defect in gonocyte development. There is limited information on the expression of ABC proteins in these cells, but these cancer precursor cells are, unlike hierarchical cancer stem cells, very sensitive to chemotherapy [52] and it is likely that low or absent ABC expression will occur in these unique cells.

Reviewing the chemotherapy curable malignancies as a group it is apparent that they have this additional shared characteristic of generally low levels of ABC protein expression. This finding is intrinsically linked to particular cells of origin of these malignancies being transient developmental cells. Each of these developmental cells themselves have low ABC expression, rather than the high levels that are seen in hierarchical cancer stem cells. The relative role that these low levels of ABC expression plays compared to the impact of the unregulated apoptotic pathways associated with the unique genetic events occurring in these cells is an area to debate and hopefully one with interesting data to come.