Here we have used a xenograft model of early stage breast cancer to show that TGF-β has the potential to suppress tumorigenesis through effects at two distinct levels in the developmental hierarchy of cell types that make up the tumor parenchyma (see model in Suppl. Fig. 5
). Firstly endogenous TGF-β appears to restrict the size of the putative cancer stem cell compartment. This effect decreases the efficiency of tumor establishment, and could underlie the earlier clinical observation that reduction in TβRII expression in early hyperplastic breast lesions is associated with increased probability of subsequent development of invasive breast cancer (35
). Secondly, TGF-β acts on the proliferative progenitor cell compartment to promote differentiation to a more organized, intrinsically less proliferative state, characterized by enhanced expression of luminal markers. This effect impacts on the growth rate, bulk and histological appearance of the tumor, and could underlie the previous clinical observation that reduction in TβRII staining in invasive breast cancer, is associated with a higher mitotic index and higher tumor grade (36
). Here, we demonstrated a significant association between TβRII protein expression and luminal differentiation in 50 primary breast cancers, and in silico
datamining of a large-scale breast cancer microarray study showed TGF-β1 mRNA to be significantly upregulated in the good prognosis “luminal A” subclass and downregulated in the poor prognosis “basal-like” subclass of tumors. While direct anti-proliferative effects of TGF-β are still seen in the breast cancer model that we used here, they appear to be less important than the effects on differentiation, as overexpression of Id1 blocks the differentiating effect of TGF-β and enhances tumorigenesis, while not affecting the ability of TGF-β to inhibit cell proliferation.
TGF-βs are plausible candidates for critical regulators of cancer stem cell dynamics. TGF-β family members are enriched in all populations of normal stem cells that have been isolated so far, and likely contribute to the complex molecular network that specifies “stemness” (25
). Depending on the specific TGF-β family member and the target cell involved, TGF-β family members may either maintain pluripotency or induce commitment in germinal, embryonal and somatic stem cells (37
). In the mammary gland, putative stem cells have been identified by ultrastructural and functional criteria (reviewed in (31
)), and recently, mammary glands have been reconstituted from single mammary epithelial cells, providing definitive evidence for the existence of a multipotent mammary stem cell (38
). Although a role for endogenous TGF-β in normal mammary stem cell dynamics has not yet been demonstrated, transgenic overexpression of active TGF-β in the mouse mammary epithelium resulted in diminished regenerative capacity of the mammary gland in serial transplantation experiments, suggesting that excess TGF-β can induce premature senescence of the mammary stem cell compartment (40
). Mice overexpressing TGF-β1 in the mammary gland were relatively resistant to induction of mammary tumors (41
), as would be expected if TGF-β reduced either the normal or the cancer stem cell compartments. Conversely, mice in which the TGF-β response was compromised showed increased rates of spontaneous or chemically-induced tumorigenesis (43
). Since experimental inactivation of Bmpr1a
in the mouse intestine was recently shown to cause an expansion of the intestinal stem and progenitor cell populations, leading to intestinal polyposis (45
), other TGF-β superfamily members may suppress the early stages of tumorigenesis by similar mechanisms in other epithelial tissues.
Stem cells can undergo three different types of division. Self-renewal by symmetric division gives rise to two daughter stem cells, and occurs during allometric growth. Self-renewal by asymmetric division gives one stem cell and one committed daughter cell and is the mechanism used for tissue maintenance. Finally, the third option is symmetric division to give two committed daughter cells, which depletes the stem cell population. Since loss of TGF-β response increases the apparent size of the putative cancer stem/early progenitor cell fraction, our data suggest that local TGF-β signaling might normally reduce the probability of symmetric self-renewal of the stem cell in favor of either asymmetric self-renewal or symmetric division to two committed daughters. Alternatively, although we do not see a pro-apoptotic effect of TGF-β on the bulk cell population, conceivably TGF-β might induce apoptosis specifically of the stem cell population. These various possibilities are under active investigation. The mechanisms underlying the apparently paradoxical downregulation of Myc and upregulation of p27 in the more rapidly proliferating, poorly differentiated Ca1h tumors following TGF-β pathway blockade are also still obscure, though Myc has been shown to deplete epidermal stem cells and promote their terminal differentiation (46
The ability of TGF-β to induce differentiation of the committed progenitors, but not its ability to deplete the cancer stem/early progenitor cell population, was dependent on the down-regulation of Id1, a known TGF-β target gene (33
). Id proteins are highly expressed during embyrogenesis and have been implicated in the regulation of self-renewal and differentiation in many tissues (47
). Forced overexpression of Id1 in the SCp2 mammary epithelial cell line blocks functional differentiation in response to lactogenic hormones (48
). In breast cancer, Id1 is expressed more frequently in infiltrating ductal carcinomas than ductal carcinoma in situ
), and Id1 is a negative predictor of survival (49
). A number of oncogenes relevant to breast cancer can upregulate Id1, including ErbB2, Myc, and Ras (47
). Since oncogene overexpression frequently also upregulates TGF-β expression (50
), down-regulation of Id1 by TGF-β may be an important homeostatic mechanism to oppose these oncogenic insults early in the carcinogenic process. Once the TGF-β pathway is compromised however, the oncogene-induced increase in Id1 could block differentiation and cause expansion of the progenitor compartment, leading to a rapidly growing tumor with an aggressive histology. Overexpression of Id1 results in tumors that are 30–50% smaller than those seen with TGF-β pathway blockade (), suggesting that the differentiating effect of TGF-β could contribute at least half of its effective tumor suppressor activity in the Ca1h model.
In summary, we have used a breast cancer model system to show that loss of TGF-β response can increase the size of the putative cancer stem cell/early progenitor compartment and block further differentiation of the lineage-restricted progeny, thus promoting tumorigenesis by a mechanism that is independent of direct effects on proliferation. The data suggest that strategies to restore or enhance TGF-β response in early carcinogenesis might constitute a novel form of differentiation therapy for prevention or treatment of epithelial tumors.