Abstract
BackgroundDormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years. We hypothesized that inflammatory cytokines produced by stromal injury can re-awaken dormant breast cancer cells.MethodsWe used an established in vitro dormancy model of Michigan Cancer Foundation-7 (MCF-7) breast cancer cells incubated at clonogenic density on fibronectin-coated plates to determine the effects of inflammatory cytokines on reactivation of dormant ER+ breast cancer cells. We measured induction of a mesenchymal phenotype, motility and the capacity to re-enter dormancy. We induced secretory senescence in murine stromal monolayers by oxidation, hypoxia and estrogen deprivation with hydrogen peroxide (H2O2), carbonyl-cyanide m-chlorophenylhydrazzone (CCCP) and Fulvestrant (ICI 182780), respectively, and determined the effects on growth of co-cultivated breast cancer cells.ResultsExogenous recombinant human (rh) interleukin (IL)-6, IL-8 or transforming growth factor β1 (TGFβ1) induced regrowth of dormant MCF-7 cells on fibronectin-coated plates. Dormant cells had decreased expression of E-cadherin and estrogen receptor α (ERα) and increased expression of N-cadherin and SNAI2 (SLUG). Cytokine or TGFβ1 treatment of dormant clones induced formation of growing clones, a mesenchymal appearance, increased motility and an impaired capacity to re-enter dormancy. Stromal injury induced secretion of IL-6, IL-8, upregulated tumor necrosis factor alpha (TNFα), activated TGFβ and stimulated the growth of co-cultivated MCF-7 cells. MCF-7 cells induced secretion of IL-6 and IL-8 by stroma in co-culture.ConclusionsDormant ER+ breast cancer cells have activated epithelial mesenchymal transition (EMT) gene expression programs and downregulated ERα but maintain a dormant epithelial phenotype. Stromal inflammation reactivates these cells, induces growth and a mesenchymal phenotype. Reactivated, growing cells have an impaired ability to re-enter dormancy. In turn, breast cancer cells co-cultured with stroma induce secretion of IL-6 and IL-8 by the stroma, creating a positive feedback loop.
Highlights
Dormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years
We demonstrated a role for fibroblast growth factor-2 (FGF-2), which is abundant on the marrow stroma extracellular matrix, in the dormancy of ER+ breast cancer cells
Reactivation of dormant Michigan Cancer Foundation-7 (MCF-7) colonies in an in vitro dormancy model Our dormancy model involves incubation of ER+ cells at clonogenic density, where the primary interaction of the cells is with the substratum instead of each other, on fibronectin-coated tissue culture plates in the presence of FGF-2 for six days [34]
Summary
Dormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years. As soon as cells with diminished cohesion enter the circulation through newly formed, leaky blood vessels [1, 2]. They undergo massive attrition, but some survive and settle in distant organs such as bone marrow, where their numbers are further reduced by a hostile environment. Dormancy occurs primarily in estrogen receptor positive (ER+) breast tumors [6]. Most recur years after adjuvant antiestrogen therapy or menopause, suggesting that estrogen deprivation may adversely affect the microenvironment’s ability to support dormancy [7, 8]
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