Adults have in situ malignancy in multiple organs by midlife [1
], but remarkably few people manifest clinical disease. We sought to understand factors involved in allowing subclinical cancers to progress to overt clinical disease. We chose our ApcMin+/−
mouse model because of the low incidence of mammary tumors in a controlled environment, and the association of clinically apparent tumor formation with increasing systemic inflammation. This pattern follows the known association of chronic inflammation with many types of human tumors. Apc mutations can also be found in human breast cancer [33
] and breast cancer cell lines [34
]. Thus the ApcMin+/−
mouse represents a model whereby the mammary tissue is at-risk for malignancy, but depending upon the local environment, cancer potential may or may not be realized.
Our experiments show that genetic changes including p53 mutations in MSC are well-tolerated in genetically normal tissues, and are not associated with an increase in cancer incidence and that p53MSC have a homing advantage over wtMSC to “at-risk” peripheral tissue by an as of yet unclear mechanism. Alone, p53MSC are unable to induce disease; however, in combination with systemic immune dysregulation, they allow neoplastic growth in at-risk mammary tissue. Restoration of immune homeostasis prevents disease despite unchanged genetic mutations within both the stromal and epithelial compartments strongly supporting the notion that immune modulation may be an effective means to prevent cancer in high-risk individuals.
Epigenetic and genetic changes within tumors, which drive proliferation and aggressive clinical behavior, have historically been assessed at the level of the cancer cell itself, with less attention given to gene alterations within the supporting stroma. Animal models of cancer typically focus on gene mutations within the epithelial cell compartment, and in many cases multiple gene alterations are required to initiate tumors. The behavior of resulting tumors differs from those found in human and rarely is the spectrum of disease caused by epithelial mutations alone as diverse as the diseases seen in humans. One way to view these discrepancies is to recognize that genes targeted to specific cell populations alter the behavior of “tumor” cells, but do not take into account the contribution of stromal cells. Several recent reports demonstrate the importance of mutations within tumor stroma and the dramatic influence these cells have on the behavior of the tumor as a whole [8
]. It is not clear, however, if the neoplastic epithelial cells influence and dictate behavior of the stroma, or vice versa, that is, if the stroma forces a neoplastic phenotype on the epithelial cell, thus creating the classical chicken and egg dilemma. Our studies using p53MSC and wtMSC in the ApcMin+/−
mouse demonstrate that stromal cell mutations accelerate tumor progression in at-risk tissue.
For the most part, it has been assumed that transformed cells within an organ proliferate and orchestrate stroma recruitment and organization via influence on local fibroblasts [35
], or through epithelial-mesenchymal transition of normal or transformed epithelial cells [38
] or by recruiting bone marrow-derived mesenchymal [39
] and other progenitor cells [42
]. Factors elaborated by stromal cells create a unique milieu that has profound effects on cancer cell proliferation, invasion, and migration [43
] similar to the contribution of activated stromal cells to wound healing. This viewpoint of stromal cell cancer cell interaction presupposes that the tumor cells orchestrate the complex interaction with the stromal cells, and these stromal cells likely undergo gene mutations as a result of the hyperproliferative environment of the tumor. This view maintains the stromal cells are under the influence and direction of the cancer cell. Our data support an alternate view that stromal cells may initiate epithelial transformation via cytokine-mediated signaling of at-risk tissue and cause the transition of preneoplastic tumor foci to overt malignant tumors by promoting tumor cell proliferation.
Though controversial, there are data suggesting that p53 mutations can be found selectively within the stromal component of breast cancer and may be associated with a more aggressive phenotype [14
]. The vast majority (95%–99%) of stromal cells within the B6Min+p53MSC and B6MinRag+p53MSC mammary tumors were host-derived, and thus wt for p53. Although rare, p53MSC were found both within tumors, and at the very edge of the epithelial cancer cells interface. Thus, sampling at any distance from the tumor itself would lead to a negative result for mutant p53, and sampling within the tumor could yield negative results due to sampling error based on the p53MSC being a small minority of cells within the stroma. Immunohistochemistry and IF analysis of our archived human breast cancer samples verifies that in 67% of cases examined, overexpression of p53 was found suggesting a mutant protein, but p53+
stromal CAF were only 1%–5% of the total stromal cells. Thus, p53CAF are <5% of the total stroma, but may have a major impact on the biology of the tumor.
Despite this controversy, our human data and compelling data from other systems warrants investigation of the association between p53MSC and breast cancer. MCF-7 mammary carcinoma cells grow faster and demonstrate a more aggressive phenotype when mixed with p53-deficient fibroblasts compared with wt fibroblasts suggesting a nonautonomous mechanism of p53-mutated stroma and transformed mammary epithelial cells [11
]. In these studies, p53-deficient fibroblasts are derived from p53 null mice. Signaling differs between p53-deleted animal models and clinically relevant p53 point mutations that occur in human disease. Our model addresses the function of clinically relevant p53 mutations [20
] in mesenchymal cells and their ability to initiate cancer in a tissue-specific fashion in an at-risk mammary carcinoma model. Our results in both the ApcMin+/−
and the MCF-7 models implicate p53 mutation in the stroma as a significant contributor to tumor cell proliferation.
MSC may acquire significant gene mutations after extended replication [13
]. Significantly, MSC acquire p53 point mutations within the DNA-binding region, consistent with point mutations commonly found in human tumors [13
], making this a highly relevant model for human cancer. MSC originating in the bone marrow circulate and engraft in peripheral tissues in low numbers as fibroblasts. During wound repair or with chronic inflammation, MSC enter solid organs in higher numbers [12
] and maintain tissue integrity through direct trans-differentiation to epithelial cells [49
] or as stromal cells [39
]. MSC may not be permanent residents within organs, but rather may reenter the circulation and travel back to the bone marrow cavity to remain quiescent, or travel to other organs. It is conceivable then that mutations acquired by MSC at one site can affect distant tissues by MSC migration.
This sets up a compelling scenario where the interplay between at-risk epithelial cells, the stromal cells that may have acquired mutations at other sites, and the local immune environment each dictates tumor outcome. In our model, mice that harbor mesenchymal p53 mutations alone remained healthy with a normal life span. Similarly, mice with an epithelial cell Apc mutation rarely developed mammary carcinoma in the absence of systemic inflammatory triggers. Combining both epithelial cell mutations and stromal mutations resulted in accelerated tumor formation. Combining stromal and epithelial mutations with dysregulated immune response as with Rag-deficient mice receiving p53MSC had profound consequences, and resulted in dramatic increase in mammary tumor incidence. This tumor phenotype could be reversed by restoring immune regulation with TREG or anti-TNF-α treatment.
p53MSC are recruited to mammary tissue by a yet unknown mechanism. p53MSC secrete many factors including MCP-1 and IL-6, which are potent inducers of inflammation. MMP-2 and −9 may further act to process epithelial-, stromal-, and inflammatory-derived growth factors and activate receptors [45
] that contribute to tumor growth and neovascularization. Under the influence of TNF-α, MSC increased ductal proliferation. Tumor formation was directly linked to TNF-α activity in vivo. Our data strongly link the synergistic requirement of epithelial mutations, stromal mutations, and dysregulated persistent inflammation with the conversion of dormant disease to active malignancy. While we do not yet have direct evidence for a similar mechanism in human disease, the indirect evidence is striking. It was recently shown that the production of induced pluripotent stem cells (iPS) cells could be dramatically simplified by disabling the p53 pathway, suggesting that the pathways used for pluripotency and cancer formation overlap [51
]. These findings suggest that cancer formation may involve a reprogramming-like mechanism of the stromal cells or the cancer cells themselves.
Cancer-associated stroma, especially stromal cells with dysregulated p53, may orchestrate local behavior of tumor cells and coordinate metastatic and invasive behavior of malignant cells [14
]. A role for TNF-α in promotion and progression of cancer is well-documented and an area of intense interest [54
]. Our findings that stromal cells assist in the conversion of dormant disease to active disease suggest that new targets for preventative therapy for at-risk human populations can be developed. One can also envision the possibility that quiescent metastatic foci, which may become active many years after seemingly curative cancer therapy, could be kept in check by therapy targeted at disrupting inflammation-stroma-cancer cell interactions.