In addition to maintaining the primary tumors, CSCs also are hypothesized to give rise to metastases. However, reports that identified different CSC subpopulations within primary and metastatic tumors, coupled with apparently stage-specific molecular requirements for progression, suggests that metastatic CSCs may be the children of de novo
niches, which evolve during progression of the primary tumor. For instance, only the CD133hi
fraction from human primary colon tumors, implanted subcutaneously (23
) or under the kidney capsule (22
), in NOD/SCID mice grew as xenografts. Whereas, both CD133hi
fractions of human metastatic colon cancers isolated from livers and implanted subcutaneously in NOD/SCID mice formed tumors with similar efficiency (33
). The tumors derived from the CD133lo
fraction grew relatively faster, and those from the CD133hi
fraction were the only ones to harbor both CD133+
cells. Tumor cells from both subpopulations expressed human EpCam, which verified that the tumors were of epithelial origin (33
). The discrepancy between the findings in primary and metastatic colon cancers may reflect experimental differences, but may also suggest that different cells are giving rise to the primary tumors and to the metastatic ones (reviewed in ref. 34
Examination of the stage of tumor progression at which metastasis occurs, and the molecules that were involved, provided evidence for stage-specific CSCs. In a murine model of breast cancer, it was shown that the transcription factor GATA-3, a protein essential for differentiation into the luminal lineage (35
), was required by the tumor during the earliest hyperplastic stages of growth, but as the tumors progressed through early and late adenoma stages GATA-3 was silenced by promoter methylation (37
). Metastases were observed only during the early and late adenoma stages when GATA-3 was lost, and GATA-3 expression was not detected at metastatic sites. Exogenous expression of GATA-3 in the early and late stage adenomas led to a reverted state reminiscent of early hyperplasia with no metastases, whereas knocking down GATA-3 in the early hyperplasia cells led to their death. Because there were distinctive stage-specific functional requirements for GATA-3, it seems unlikely that the same GATA-3+ CSC that established the primary tumor also established the GATA-3 null metastases. A second study also showed stage-specific requirements for growth of human and mouse breast cancer cell line-derived tumors in NOD/SCID and in syngenic immuno-competent mice, respectively. Growth of the primary tumors did not require the receptor tyrosine kinase Axl, but the protein was required for spontaneous metastasis (38
). Moreover, Axl was highly expressed in patient metastases, but not by their primaries, and its high expression was found to be an independent predictor of recurrence. These reports serve as examples whereby the molecules GATA-3 and Axl are not merely markers that delineate potentially stage-specific CSCs in breast cancers, but they also play clear functional roles in the genesis of primary or metastatic tumors.
That different CSCs may give rise to primary and to metastatic tumors does not contradict the existence of CSCs per se, but does predict that the composition of metastatic and primary CSC niches will differ. Moreover, it suggests that either the original CSC evolves throughout tumor progression, or that there should be a mechanism to generate new metastatic CSCs de novo.
Microenvironments can impose specific cell fate decisions (12
), thus a tumorigenic cell's location inside a tissue is a crucial determinant of its activity. Reacquisition of the stem cell phenotype has been observed in normal progenitors that populated vacant niches (6
); therefore, the possible consequence of CSCs usurping preexisting niches should be considered. In model systems of solid and heme malignancies, differentiation-defective stem cells were shown to be the etiological root of the diseases (16
). In Drosophila germaria
, differentiation-defective hyperplastic stem cells outcompeted normal stem cells for occupancy of their niche (; ref. 42
). Those mutant stem cells exhibited upregulation of E-cadherin, an adherens junction receptor that mediates interaction between germinal stem cells and their niche. Malcom Steinberg's differential adhesion hypothesis (43
) may explain why modulation of adherens proteins resulted in the reshuffling of CSC and normal stem cells for niche occupancy. He posited that different cell types self-organize into groups according to common levels of adherens junction proteins with an ultimate goal of reducing free-energy (43
). In the case of the germarium the mutant stem cells supplanted the normal by expressing relatively more E-cadherin, causing an energetically more favorable association with the niche. Germline mutations in E-cadherin are associated with diffuse gastric cancers, and cadherin expression is frequently misregulated in breast cancers (reviewed by Knudsen; ref. 45
). Perhaps pathological modulation of cadherin expression can play a role in early disease stages by repositioning differentiation-defective CSCs into normal stem cell niches, where they receive self-renewal and survival signals (). Cadherin modulation may occur again in later tumor stages, at least in carcinomas, when tumor cells seem to lose epithelial characteristics (e.g., loss of E-cadherin) and acquire more mesenchymal ones.
Owing to genetic instability, the tumor microenvironment is a shifting landscape that may deleteriously impose stem-like phenotypes onto malignant cells. Normally, transforming growth factor β (TGF-β) is present in the mammary stroma in an inactive form, and is cleaved into its active form following exposure to radiation, wounding, and during tumorigenesis (46
). Experiments with Rous Sarcoma virus-infected cells showed that active TGF-β at wound sites was necessary to realize the full malignant potential of a predisposed cell (47
). Similarly, progression to frank neurfibromatosis required mutations both in the epithelial cells and in the nearby stromal cells, which disrupted TGF-β and receptor tyrosine kinase c-Kit regulation (48
). A CSC-like phenotype was reportedly induced in mammary epithelial cells exposed to active TGF-β, forcing them to undergo epithelial-to-mesenchymal transition (EMT; ref. 50
), which included downregulation of E-cadherin. Induction of the stem cell-like program in nonmetastatic malignant mammary epithelial cells elicited invasive metastatic behavior (50
). Other mechanisms that may drive evolution of cellular subtypes may include increased activity by proteases during tumorigenesis. Increased expression of matrix metalloproteinase-3, which is frequently observed in breast cancers, led to genomic instability and EMT in mammary epithelial cells (51
) and tumor formation in mice (53
). Thus, a number of known tumor microenvironment components are sufficient to induce a CSC phenotype, and should be considered as potential CSC niche constituents.