Cell-extrinsic consequences of epithelial stress: activation of protumorigenic tissue phenotypes

Abstract

IntroductionTumors are characterized by alterations in the epithelial and stromal compartments, which both contribute to tumor promotion. However, where, when, and how the tumor stroma develops is still poorly understood. We previously demonstrated that DNA damage or telomere malfunction induces an activin A-dependent epithelial stress response that activates cell-intrinsic and cell-extrinsic consequences in mortal, nontumorigenic human mammary epithelial cells (HMECs and vHMECs). Here we show that this epithelial stress response also induces protumorigenic phenotypes in neighboring primary fibroblasts, recapitulating many of the characteristics associated with formation of the tumor stroma (for example, desmoplasia).MethodsThe contribution of extrinsic and intrinsic DNA damage to acquisition of desmoplastic phenotypes was investigated in primary human mammary fibroblasts (HMFs) co-cultured with vHMECs with telomere malfunction (TRF2-vHMEC) or in HMFs directly treated with DNA-damaging agents, respectively. Fibroblast reprogramming was assessed by monitoring increases in levels of selected protumorigenic molecules with quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and immunocytochemistry. Dependence of the induced phenotypes on activin A was evaluated by addition of exogenous activin A or activin A silencing. In vitro findings were validated in vivo, in preinvasive ductal carcinoma in situ (DCIS) lesions by using immunohistochemistry and telomere-specific fluorescent in situ hybridization.ResultsHMFs either cocultured with TRF2-vHMEC or directly exposed to exogenous activin A or PGE2 show increased expression of cytokines and growth factors, deposition of extracellular matrix (ECM) proteins, and a shift toward aerobic glycolysis. In turn, these "activated" fibroblasts secrete factors that promote epithelial cell motility. Interestingly, cell-intrinsic DNA damage in HMFs induces some, but not all, of the molecules induced as a consequence of cell-extrinsic DNA damage. The response to cell-extrinsic DNA damage characterized in vitro is recapitulated in vivo in DCIS lesions, which exhibit telomere loss, heightened DNA damage response, and increased activin A and cyclooxygenase-2 expression. These lesions are surrounded by a stroma characterized by increased expression of α smooth muscle actin and endothelial and immune cell infiltration.ConclusionsThus, synergy between stromal and epithelial interactions, even at the initiating stages of carcinogenesis, appears necessary for the acquisition of malignancy and provides novel insights into where, when, and how the tumor stroma develops, allowing new therapeutic strategies.

Carcinoma-associated fibroblasts: orchestrating the composition of malignancy

“…Notably, selective knockdown of IL-6 in CAFs, but not in DCIS cells, abrogates these phenotypes. This report brings further mechanistic insights to a prior study reporting that DCIS lesions with a heightened DNA damage/activin A/ cyclooxygenase-2 (COX-2) signature were associated with a more reactive stroma (Fordyce et al 2010(Fordyce et al , 2012 and more frequent progression to invasive cancer (Kerlikowske et al 2010).…”

“…Accompanying CD36 repression is the secretion of soluble factors, such as activin A, which has been implicated as a key effector in a DNA damage-inducible secretory program that acts in a cell-extrinsic fashion to generate many of the above phenotypes ( Fig. 2; Fordyce et al 2010Fordyce et al , 2012. Extending these findings, recent evidence has been provided for CD36 repression as part of a dramatic switch in fibroblast identity.…”

Stress Signaling from Human Mammary Epithelial Cells Contributes to Phenotypes of Mammographic Density

“…Paraffin-embedded serial tissue sections, from 13 LD (25%<MD<50%) and 14 HD (MD>70%) disease-free women (Supplementary Table 1) , were assessed for γ H2AX levels by immunohistochemistry (3) and for telomere content by telomere-specific FISH (20). For immunohistochemistry, antigen retrieval was performed in citrate buffer (pH=6.0) for 30 minutes at 93°C prior to incubation with a γH2AX antibody (1:800, Millipore, #05-636) for 60 minutes at room temperature.…”

“…Immunofluorescence detection of γH2AX was performed using the γ H2AX antibody above (1:500) (20). Cell viability (fraction of live/dead cells) and apoptosis (caspase 3/7 activity) were measured using an ApoTox-Glo Triplex Assay (Promega) and proliferation (BrDU incorporation) using the Cell Proliferation Assay Kit (Cell Signaling).…”

“…Fibroblasts from reduction mammoplasties (RMF) co-cultured with DNA-damaged vHMEC induce many genes consistent with a desmoplastic phenotype (e.g. ECM and inflammatory cytokines) and activin A secretion by damaged vHMEC is necessary and sufficient for this induction (20). In vivo , DCIS lesions whose epithelial cells exhibit shorter telomeres, increased activin A and COX-2, are surrounded by activated fibroblasts and increased immune infiltrate (20), suggesting that microenvironmental alterations, mimicking aspects of desmoplasia, occur even in the absence of an invasive tumor.…”