Mary H. Barcellos-Hoff, R.L. Henshall-Powell, M.J. Bissell, and B. Parvin
Lawrence Berkeley National Laboratory, Life Sciences Division
We have proposed that the ability of radiation to induce altered microenvironments affects the frequency and features of neoplastic progression. Thus, we have sought to characterize the irradiated microenvironment and determine how these events contribute to mammary carcinogenesis. By using imaging bioinformatics to analyze mouse and human models of breast cancer we have now examined cell adhesion molecules (CAMs) critical for tissue-specific organization and function. We found that 1) radiation-induced microenvironments can contribute to neoplastic potential of unirradiated non-malignant murine mammary cells; 2) radiation-induced microenvironments are different in mice that are sensitive versus resistant to mammary carcinogenesis; and 3) cell-cell and cell-extracellular matrix interactions of irradiated human mammary epithelial cells (HMEC) are persistently dysfunctional in a manner that heralds malignant progression. As previously shown for microenvironment remodeling in vivo, HMECs are sensitive to doses in the cGy range. In each of these studies, we found that transforming growth factor-$ (TGF-$) is a critical player.
Single cells of the non-malignant HMEC cell line, HMT-3522, were irradiated with a low dose (0.1-2 Gy) at the time of plating into reconstituted basement membrane. TGF-$ (400 pg/ml) was added to some cultures to mimic the presence of an irradiated stroma. The multicellular organization of colonies arising from irradiated, TGF$-treated cells displayed pronounced acinar disorganization in comparison to colonies from sham controls or following single treatments. Surprisingly, we also found that the number of cells per colony increased in colonies surviving 2 Gy that were cultured in the presence of TGF-$ (13.8 + 2.3 vs 9.7+1.8 S.E. sham).
E-cadherin immunofluorescence intensity at day 10 of HMT-3522 colonies arising from cells irradiated at day 0 with graded doses of ((-radiation. We then examined cell adhesion systems necessary for the assembly of cells into tissue-specific structures. E-cadherin is a crucial epithelial adhesion molecule that links cells via an homophilic extracellular domain and is anchored intracellularly to the cytoskeleton via dynamic interactions with the catenins. Low E-cadherin immunoreactivity in breast cancer is associated with poor prognosis, while restoration of E-cadherin reverts the invasive phenotype of cancer cells. E-cadherin was localized using immunofluorescence, confocal microscopy and image analysis. Colonies from irradiated cells cultured in the presence of TGF-$ showed a dramatic loss of E-cadherin immunoreactivity ( P=<0.0001). E-cadherin immunoreactivity was decreased in colonies exposed at day 0 with doses as low as 0.25 Gy (Figure 1). Consistent with this finding, immunobloting of total protein showed that both E-cadherin and it's partner, $-catenin, were decreased in irradiated cells, which was augmented by TGF-$ treatment. These data suggest that very low doses of radiation may further compromise multicellular organization and growth restraints in preneoplastic cells, which may already have less E-cadherin.
To evaluate cell adhesion to the extracellular matrix, integrin receptors were also localized. Integrins form heterodimers consisting of an " and $ subunit, to bind to matrix proteins. HMT-3522 colonies exhibit basolateral $1 integrin, but colonies arising from irradiated cells showed significantly increased $1-integrin immunoreactivity that was distributed throughout the cytoplasm. TGF-$ treatment did not affect $1 integrin in the absence of prior irradiation. In contrast, the immunoreactivity of "6-integrin, which partners with $4 integrin, was decreased in colonies generated from irradiated cells or cultured in the presence of TGF-$.
Gap junctions are mediated by connexins, a family of proteins that modulate the transfer of molecules between cells, are implicated in the bystander effect and whose loss correlates with breast cancer metastatic potential. Connexin 43 localized as distinct aggregates between cells in HMT-3522 colonies. The number of connexin-43 aggregates per colony was dramatically decreased in colonies arising from irradiated cells, regardless of TGF-$ exposure.
Together these data demonstrate that colonies arising from irradiated cells exhibit a consistent phenotype consisting of inappropriate intercellular adhesion, deranged extracellular adhesion molecules, loss of gap junction proteins, and disorganized tissue-specific organization. This phenotype is augmented by the presence of TGF-$, which itself is rapidly and persistently activated in irradiated tissue. These data are remarkable in that the phenotype is inherited by the daughters of individually irradiated cells, suggesting that radiation induces a heritable derangement of pathways affecting cell adhesion, extracellular matrix interactions, polarity and cell-cell communication.
From these preliminary studies, and others in the literature, we conclude that radiation exposure not only affects the composition of the microenvironment, but also persistently alters cell interactions with the microenvironment. These fundamental cellular changes occur in a characteristic fashion that produce an irradiated phenotype. Some aspects of the irradiated phenotype appear to be due to altered intracellular signaling that can result in a heritable phenotypic changes. Others may be mediated by extracellular signaling that affects how cells communicate. Our global hypothesis is that multicellular responses directed via extracellular signaling affect the probability of malignant progression following radiation exposure. In normal tissues, a major role of extracellular signaling is inhibition of carcinogenesis by elimination of abnormal cells and suppression of neoplastic behavior.