In this report, we have demonstrated that CSCs isolated from metastatic breast tumor cells indeed show significantly higher tumorigenic as well as metastatic abilities compared to that from low-metastatic cells. CD44+/CD24−/ESA+ cells have both tumor initiating ability in vivo and self-renewal capability in vitro, and these are the most critical characteristics to define cancer stem cells. Theoretically, metastatic tumor cells should have these characteristics because metastasized tumor cells must re-initiate their growth at the distant organs. However, these markers per se are not necessarily correlated with the aggressiveness of the cancer cells, and it is likely that these markers and “stemness” are pre-requisites for metastatic tumor cells but not sufficient conditions for metastasis. In fact, we have shown in this report that HAS2 is a necessary factor to endow CSC with metastatic phenotype. Our results also indicate that the HAS2 gene is significantly up-regulated in the metastatic CSCs and that hyaluronan plays a critical role in generating a favorable microenvironment by promoting the interaction of TAM and CSCs followed by secretion of PDGF-BB by TAMs. This growth factor then activates other stromal cells that in turn augment the growth of CSCs by secreting FGF7 and FGF9 in the bone. Therefore, HAS2 plays a pivotal role in orchestrating the cascade event to structure the niche of CSCs in the bone (). Importantly, our results clearly indicate that blocking HAS2 expression in CSCs by a small molecule serves as an effective strategy for the treatment of metastatic disease.
Aberrant expression of hyaluronan in tumor cells and adjacent stroma has been known to be linked to tumor progression and poor survival of cancer patients (17
). In animal experiments, ectopic expression of HAS2 was indeed shown to promote invasiveness and metastatic ability of various cancer cell lines (19
). HAS2 is responsible for the synthesis of large molecular weight hyaluronan and is involved in a variety of cellular functions including proliferation, differentiation and inflammation (23
). One of the major receptors of hyaluronan is CD44 which is abundantly expressed on monocytes and macrophage lineage, and therefore, CSCs with a high level of HAS2 are likely to interact with other CD44-positive cells in the microenvironment which may endow CSCs with growth advantage. We have indeed shown that the metastatic CSCs which express high-level of HAS2 directly interact with TAMs to promote secretion of PDGF-BB. This result is consistent with the previous finding that monocytes are often recruited preferentially to stromal regions in hyaluronan-rich tumors (24
) and that hyaluronan treatment of the monocytes can induce M2 conversion into TAMs (25
). Therefore, the direct interaction of CSCs and TAMs is the critical step for remodeling the tumor microenvironment, and how TAMs promote CSCs growth is an intriguing question. Although previous reports indicate that TAMs secrete various cytokines and growth factors that augment the tumor growth (14
), we found that neither CM of TAMs nor PDGF-BB alone showed little effect on the growth of CSCs in vitro
. However, TAMs significantly enhanced the growth of CSCs in the animal, suggesting that the effect of TAMs on CSCs is indirect and other environmental factors or cells are involved. We indeed found that PDGF-BB secreted from TAMs was able to activate mesenchymal stem cells, osteoblasts, as well as bone stromal cells that in turn promoted the growth and self renewal of CSCs by secreting other growth factors and cytokines such as FGF7 and FGF9. It should be noted that PDGF-BB was also shown to stimulate both osteoblasts and osteoclasts and hence promote bone metastasis of tumor cells (26
). It is also known that breast cancer patients who have high expression of PDGF in the tumor are associated with poor prognosis (29
). Collectively, our results indicate that the initial contact of CSCs and TAMs and the following secretion of PDGF-BB triggers a series of cascade events for remodeling the microenvironment which involves mesenchymal stem cells, osteoblasts and bone stromal cells through intrinsic autocrine factors in a positive feedback loop. Furthermore, we have shown that PDGF-BB indeed activates these stromal cells to secrete FGF7 and FGF9 that are capable of enhancing self-renewal of CSCs in the bone microenvironment. Note that fibroblast growth factors are commonly used to expand the embryonic stem cells (30
), suggesting that these factors have important roles in self-renewal of stem-like cells. More recently, FGF9 was found to promote the growth of CSC population in primary breast cancers (31
). We have also shown that tumor growth is significantly enhanced by co-inoculation of stromal cells into the tibial bones, while the knockdown of these growth factors in the stromal cells significantly suppressed tumor growth. Therefore, HAS2 and its product, hyaluronan, are considered as master regulators for generating metastatic niche for CSCs. In this context, it is of significant interest to understand how the expression of HAS2 in CSCs is controlled by the environmental factors of niche. HAS2 has previously been shown to be up-regulated by TGF-beta and osteopontin that play roles in bone regeneration (32
). We have also shown here that BMP7 can significantly enhance the expression of HAS2 through activation of SMAD1 pathway as shown in Figure S7
. Notably, BMP7 is one of the critical factors for bone regeneration and it is highly up-regulated in metastatic breast cancer (34
), although the function of BMP7 is context dependent and it can serve as both promoter and suppressor depending on the stage and type of tumor (37
). These results imply that bone regenerative conditions may favor creating propitious microenvironment for the growth of metastatic CSCs in the bone, although this idea needs to be further tested.
As we have shown that HAS2 and PDGF play major roles in generating the auto-looped microenvironment for CSCs, intervention of the HAS2-PDGF axis offers a window of therapeutic opportunity for metastatic disease. We have indeed shown that 4-MU can significantly suppress the incidence of metastasis and growth of CSCs in the bone due to specific inhibition of HA synthesis in our animal model. 4-MU is a natural compound and abundantly exists in many edible plants such as broccoli and celery (38
) and the anti-cancer activity of this compound has been observed in prostate cancers and melanomas (39
). In fact, 4-MU is already approved by FDA and is currently under phase II clinical trial for the treatment of Hepatitis B and C infections. Therefore, 4-MU and a combination with other inhibitors for the auto-loop network of the CSCs microenvironment are considered to be promising therapeutic and preventive measures for metastatic breast cancer.