Evidence to support the CSC hypothesis has been derived primarily from studies of a variety of leukemias and solid human tumors in which FACS sorted cell populations were transplanted into immunocompromised mice. In a recent report, however, based upon the study of three syngeneic mouse lymphoma tumor models (34
), the authors concluded that the majority of the cells isolated from these relatively homogeneous mouse tumors were able to initiate tumor formation. These results contrasted with those of John Dick and colleagues (5
), who had identified a rare leukemia initiating population of “cancer stem cells” based primarily on xenografts of AML. Kelly et al. (34
) questioned whether the overall microenvironment in xenograft models using immunocompromised mice might have accounted for the differences observed in these studies, thus casting doubt on the “cancer stem cell” hypothesis. However, an alternative interpretation is that these apparently disparate results most likely reflect the importance of selecting appropriate genetically engineered heterogeneous mouse models in which to perform these studies. The present study using the heterogeneous p53 null mouse mammary tumor model provides direct evidence using limited dilution transplantation experiments for the existence of a tumor-initiating subpopulation of CSCs in a syngeneic mouse model.
The anti-CD44 antibody successfully employed for the isolation of tumor-initiating cells from breast cancer cells present in pleural effusions (7
) did not facilitate the isolation of a tumor-initiating subpopulation in the p53 null mouse model. This may reflect differences in the epitopes recognized by the specific anti-CD44 antibody expressed in human breast cancer as compared to mouse mammary tumor cells, possibly due to alternative splicing or post-translational modifications (35
). CD44 has been identified as an important regulator of AML stem cell homing (36
). However, the functional importance of CD44 in mouse mammary tumor-initiating cells has yet to be determined. While human breast cancer CSCs have been defined operationally as CD24−/LOW
, the tumor-initiating population of p53 null tumors was predominantly CD24H
, although some activity was observed in the Lin−
subpopulation. Unlike in human, where CD24 was only expressed on luminal epithelial cells (37
), in mice, the metastasis-associated CD24, a glycosyl phosphatidylinositol linked membrane protein, is expressed on major hematopoietic lineages, developing neural and epithelial cells, and has been suggested to be a marker for luminal MECs (38
). The significance of this observation remains to be determined, but may be a consequence of the possible origin of these tumors from a bipotent progenitor as discussed below.
Breast cancer is not a disease that is driven through a simple mechanism, but through a complex set of changes (both genetic and epigenetic) in many pathways. BMI1 has been reported to play an important role in maintaining stem cell self-renewal. Up-regulation of Bmi-1
within the tumorigenic subpopulation may partially explain the increased mammosphere formation efficiency of such cells, but this remains to be demonstrated directly (32
). BMI1 can be recruited to histone H3 by EZH2, which was also increased in this tumorigenetic Lin−
subpopulation. Overexpression of Ezh2
maintained long-term repopulating potential by preventing exhaustion of hematopoietic stem cells (40
). Kamminga et al. identified 46 genes, categorized in three groups, that intereacted with Ezh2
in epigenetic chromatin modification of hematopoietic stem cells. Among these, 15 genes are among the up-regulated gene list in the present study, suggesting a similar mechanism may be active.
Bao et. al. (41
) showed that following radiation, DNA damage checkpoint proteins were more activated in tumor cells bearing CD133+
(a marker for both neural and brain CSCs) than in CD133−
tumor cells, suggesting that such tumor-initiating cells could be responsible for the recurrence of tumors following radiation therapy. Specific inhibitors of the checkpoint kinases, CHK1 and CHK2, were shown to sensitize CD133+
tumor cells to radiation treatment. Cell cycle checkpoint, DNA damage response and repair proteins are highly up-regulated in Lin−
tumor-initiating cells. Interestingly, the proportion of Lin−
tumor-initiating cells appears to increase upon successive mammosphere passaging, suggesting that the loss of p53 may promote symmetric division and expansion of this subpopulation. Taken together with the observation of radiation resistance of the mammospheres, a more efficient DNA damage repair mechanism may exist in Lin−
cells as compared to the other subpopulations. Ongoing studies are directed at testing this hypothesis.
There are controversial reports as to ER status of mammary gland stem cells. Stem cells are believed to be slowly proliferating cells, and it has been reported that majority of long-lived MECs that retained the tritiated thymidine incorporation are ER+
luminal cells (42
). “Side population” (SP) cells, a small percentage of cells with mammary stem cell properties, proportionally, contained six times as many ER+
cells as non-SP cells (43
), though so far no in vivo
transplantation experiments have supported the self-renewal property of the SP population. In contrast, two independent studies have reported that ER+
cells exhibit few stem cell properties, and that instead the basal population, which is enriched in mouse mammary stem cells, did not express ERα (44
). In agreement with these results, the mouse mammary gland MRU Lin−
(MEC) cells exhibit basal features, with increased expression of K5, K14, and decreased expression of ERα. However, Lin−
cells from p53 null mammary tumors, contain some ERα positive cells and cells with a mixed basal and luminal lineage (data not shown) supporting the hypothesis that they may have been derived from bipotent progenitors present in normal epithelium. The loss of p53 followed by other genetic changes appears to result in the deregulation of stem cell self-renewal and possibly an expansion of this progenitor population, which would then result in both basal and luminal, as well as ER+
cells in the tumorigenic Lin−
subtypes. Therefore, the cell of origin for these tumors might be a bipotent progenitor, which possibly may be ER positive.
In summary, this study has identified a tumorigenic subpopulation within the p53 null mammary tumors as supported by both in vivo transplantation and in vitro mammosphere assay, and has identified genes that are preferentially expressed in the putative mouse mammary tumor-initiating cell population. The correlation of mammosphere with their tumorigenic outgrowth potential validates the use of the assay as in vitro surrogate, and suggests that it may be used for high throughput screens of small molecules and RNAi to identify pathways which are essential for the self-renewal of these cells. This may allow us, to identify new CSC markers, to test the functional importance of these markers in a syngeneic mouse model, and ultimately to improve the prognosis and treatment of breast cancer.