Our results indicate that tumor cells already carrying critical genetic alterations can remain dormant or be triggered to proliferate by changes occurring in their microenvironment. Thus, tumor dormancy may be maintained in an initially non-permissive microenvironment, but transition to a proliferative state may result from extrinsic changes within the microenvironment. This study demonstrates in vivo
for the first time how changes in the ECM composition produced by stromal cells can initiate the transition of solitary dormant cells to metastatic proliferation. Several gene expression studies have identified ECM-related genes (including Col-I) in prognostic signatures for poor outcome, metastases and tumor recurrence (13
). In this study, we demonstrate that an in vivo
fibrotic environment that contains high amounts of Col-I, as well as other ECM proteins, may induce the transition from a dormant-to-proliferative state through Intβ1. Mice with fibrotic lungs displayed greatly incrased numbers and sizes of proliferative, metastatic lesions compared to mice without fibrosis.
Intβ1 is the primary receptor that interacts with Col-I. Previous studies have reported Intβ1’s important role in different stages of tumor progression (31
). We demonstrated that Intβ1 is a key regulator in the switch from dormancy to metastatic growth. Reduced expression of Intβ1 in D2.0R cells disseminated to pre-existing fibrotic lungs resulted in a 5-fold reduction in proliferative metastatic lesions compared to control cells. Similarly, in the context of a normal lung environment, loss of Intβ1 expression in metastatic D2A1 cells resulted in a 9-fold reduction of metastatic lesions and lower tumor burden compared to D2A1-sh-NT cells. Metastatic lesions arising from D2A1 cells in non-fibrotic lungs were associated with significant deposition of Col-I suggesting that the metastatic D2A1 cells are able to induce a Col-I stromal response to stimulate their growth, whereas D2.0R cells require an exogenous fibrotic stimulus to initiate their proliferative response. Previous studies in head and neck carcinoma have demonstrated critical cross-talk between urokinase receptor and Intβ1 in tumor dormancy (36
). We have determined that Col-I increases Intα1 mRNA, but not α2 or α7 integrin in D2.0R cells in vitro
, suggesting that α1β1 is the most likely candidate Col-I receptor on D2.0R cells (data no shown).
Based upon our previous study, fibronectin may also contribute to the dormant-to-proliferative switch in this in vivo
fibrosis model system since fibronectin also activates Intβ1 in D2.0R and D2A1 cells (20
). While we do not exclude the influences of other factors in the microenvironment of the fibrotic lungs, such as a possible role of TGFβ in sustaining/enhancing the proliferation of the emerging micrometastasis, our results using the 3D in vitro
model system for dormancy (20
) demonstrated that a signaling cascade induced by Col-I is sufficient to trigger the dormant–to-proliferative switch of D2.0R cells through activation of Intβ1, Src, FAK, and ERK leading to phosphorylation of MLC by MLCK, and actin stress fiber formation - all critical for the proliferative response.
Our previous study demonstrated the critical role of actin stress fiber formation in the transition from dormancy to proliferative growth. In this study, we have further demonstrated that actin stress fiber formation is regulated through Col-I activation and is dependent upon Intβ1, FAK, Src, and ERK, a key regulator of the cell cycle and MLCK (29
), which phosphorylates MLC leading to actin stress fiber formation. Furthermore, we demonstrated that activated ERK is critical for actin stress fiber formation through phosphorylation of MLC by MLCK, required for the transition from quiescence to proliferation.
Our results are consistent with several clinical correlations between enriched stromal Col-I and cancer recurrence and reduced survival. Women with high breast density, associated with enriched stromal Col-I have an increased risk for local recurrence after mastectomy or radiotherapy (38
). Fibrotic foci (FF), are found around some invasive ductal carcinomas and their recurring lesions residing at lymph nodes and bones. Notably, patients with FF are at higher risk of developing bone and lymph node metastasis, and disease recurrence (17
). Osteosclerotic breast cancer bone metastases exhibit marrow fibrosis and new bone formation (40
). Therefore, enriched stromal Col-I in a more fibrotic microenvironment or induced by the residing tumor cells may provide a fertile ‘soil’ for the transition from dormancy to metastatic growth.
In summary, we have demonstrated for the first time to our knowledge, how fibrosis with Col-I enrichment may serve as a critical element in modifying the ‘soil’ of metastatic sites and activating dormant tumor cells to proliferate. These results suggest that inhibiting the interaction between dormant tumor cells and growth-promoting changes in the ECM that signal through Intβ1 may be an important new avenue to prevent disease recurrence.