It takes two to tango in the microenvironment!

Using mortal non-tumorigenic human mammary epithelial cells and fibroblasts, Fordyce and colleagues show that an epithelial stress response promotes pro-tumorigenic changes in mammary fibroblasts. Fibroblast reprogramming was dependent on activin A or prostaglandin E2 produced by epithelial cells and, in turn, promoted enhanced migration of epithelial cells. These events in epithelial cells in vitro, including telomere loss, heightened DNA damage response, and activin A expression, are observed in breast ductal carcinoma in situ lesions surrounded by stroma bearing hallmarks of activated fibroblasts and immune and endothelial cell infiltration. Thus, reciprocal epithelial-stromal interactions facilitate progression to malignancy and occur even at the earliest stages of mammary tumorigenesis.

Extensive evidence supports an integral role for crosstalk between tumor cells and neighboring stromal cells, which promotes the growth of epithelial cancers and their progression toward increasing malignancy. Th e traditional view of these interactions posits that stromal components, including fi broblasts, endothelial cells, and a subset of immune cells, condition the microenvironment to favor tumor growth and metastasis by secreting growth factors, angiogenic factors, cytokines, and proteases. However, recent studies have convincingly demonstrated reciprocal crosstalk between the epithelial and stromal compartments, whereby tumor cells engage in paracrine signaling to increase the pro-tumorigenic properties of immune cells and fi broblasts within the stromal microenvironment. Th e article by Fordyce and colleagues [1] in a recent issue of Breast Cancer Research demon strates convincingly that stromal-epithelial interactions, enhancing the acquisition of malignancy, occur even at the initial stages of carcinogenesis.
Previous work by the Tlsty laboratory has identifi ed activin A, a member of the transforming growth factorbeta (TGF-β) family, as an integrator of tumor/stromal crosstalk [2]. Th ey showed that, while human immortalized mammary epithelial cells (HMECs) undergo senescence in response to DNA damage or telomere erosion, HMECs lacking an intact p16/Rb pathway are rendered hyper-proliferative in response to such genotoxic insults as a result of activin A-induced cyclooxygenase-2 (COX2) expression [2]. In their article in Breast Cancer Research, the authors demonstrate that epithelial-derived activin A is suffi cient to increase the tumor-promoting properties of primary mammary fi broblasts in a COX2-dependent manner. Th ese properties include increased depostion of extra cellular matrix components and elevated production of growth factors and infl ammatory cytokines and are highly reminiscent of a cancer-associated fi broblast (CAF) phenotype [1]. In contrast, a DNA damage stimulus applied directly to fi broblasts is suffi cient to induce activin A-driven COX2 pro-infl ammatory responses but cannot promote extracellular matrix depo si tion from primary mammary fi broblasts. Th ere fore, cell-extrinsic stress signals emanating from immorta lized mammary epithelial cells are required to fully elicit a CAF-like phenotype in neighboring fi broblasts. Th ese data suggest that immortalized mammary epithelial cells that have bypassed the p16/Rb senescence checkpoint are ex quisitely sensitive to DNA damage-induced oncogenic transformation as a consequence of both increased genomic instability and the acquisition of a pro-tumorigenic stromal microenvironment. It is likely that such epithelial stress-induced functions will extend to components of the immune lineage. Indeed, CAFs can promote a nuclear factor-kappa B (NF-κB)-driven pro-infl ammatory response that facilitates the transition of hyperplastic lesions to overt carcinoma [3]. Moreover, activin A was recently shown to favor diff erentiation of macrophages with M 1type infl ammatory properties, whereas inhibition of activin A signaling promotes M 2 macrophage polarization [4]. Indeed, macrophage infi ltration into mammary tumors is required for the angiogenic switch and supports breast cancer metastasis [5].

Abstract
Using mortal non-tumorigenic human mammary epithelial cells and fi broblasts, Fordyce and colleagues show that an epithelial stress response promotes pro-tumorigenic changes in mammary fi broblasts. Fibroblast reprogramming was dependent on activin A or prostaglandin E 2 produced by epithelial cells and, in turn, promoted enhanced migration of epithelial cells. These events in epithelial cells in vitro, including telomere loss, heightened DNA damage response, and activin A expression, are observed in breast ductal carcinoma in situ lesions surrounded by stroma bearing hallmarks of activated fi broblasts and immune and endothelial cell infi ltration. Thus, reciprocal epithelial-stromal interactions facilitate progression to malignancy and occur even at the earliest stages of mammary tumorigenesis.
Observations generated by the Tlsty laboratory suggest that the combined loss of tumor suppressors, such as p16INK4a, and stress signals induced from a DNA damage response are suffi cient to convert primary epithelial cells into tumor promoters. In contrast, nontrans formed mammary epithelial cells undergo senescence under these conditions [2]. When mixed with ErbB2-transformed mammary epithelial cells, normal mammary epithelial cells have the inherent ability to inhibit the tumorigenic phenotype whereby they secrete soluble factors that allow ErbB2-transformed mammary cells to reconstitute a normal, diff erentiated mammary gland instead of forming overt mammary tumors [6]. ErbB2-expressing cells isolated from these chimeric ductal structures retain their tumor-forming properties when injected into epithelium-free mammary fat pads [6], highlighting that signals from a normal mammary microenvironment, composed of stromal, epithelial, and host-mediated signals, may combine to suppress the cancer phenotype.
Fordyce and colleagues [1] demon strate that activin A secreted from immortalized mammary epithelial cells following genotoxic stress is suffi cient to induce a fi brotic response in neighboring fi broblasts. In a similar study, reactive oxygen species (ROS) released from cancer cells stimulated an oxidative stress response in adjacent fi broblasts and, in turn, increased their replicative potential by inducing a metabolic shift toward aerobic glycolysis [7]. Indeed, DNA damage induced by chemotherapeutic agents or telomere dysfunction is known to increase ROS production within tumor cells. Th is suggests that intra cellular stress responses that occur within tumor cells, and perhaps immortalized epithelial cells, result in the elaboration of extracellular signals in the form of cyto kines and free radicals, which activate a pro-tumori genic stroma. In turn, oxidative stress within the CAFs can feed back onto the cancer cells to potentiate genomic instability, further increasing their malignant properties [8]. Th ese observations have important clinical implica tions as residual ductal carcinoma in situ (DCIS) lesions that remain following neoadjuvant chemotherapy may paradoxically potentiate mammary tumorigenesis. Indeed, the presence of adjacent DCIS lesions in patients with human epidermal growth factor receptor 2-positive (HER2 + ) invasive ductal carcinoma (IDC) showed an inferior response rate to chemotherapy plus trastuzumab compared with patients who present with IDC only [9]. Hence, activin A produced following chemotherapyinduced DNA damage may increase the tumorigenicity of resident DCIS lesions or render remaining IDC tumors more refractory to treatment.
Th e study by Fordyce and colleagues [1] focused primarily on the contribution of chemo therapy or telomere dysfunction to trigger double-stranded breaks within mammary epithelial cells, leading to the observed fi brotic response in neighboring fi bro blasts. Supporting this, quantitative measurements of telomere fusions, indicative of genomic instability, have established their presence at comparable levels in DCIS and IDC lesions but absence in normal breast [10]. It is likely that mammary epithelial cells will adopt other strategies to initiate stress-induced desmoplasia of mammary fi broblasts. For instance, human telomerase (hTERT) has been associated with additional roles beyond telomere maintenance in inhibiting intracellular stress signals. Indeed, a fraction of hTERT resides within the mito chondria, where it functions to directly limit ROS production in response to stimuli that promote oxidative stress by increasing the pool of available anti-oxidants [11]. Moreover, Fordyce and colleagues [1] demon strate that hTERT overexpression in p16-defi cient immortal ized mammary epithelial cells suppresses the ability of mammary fi broblasts to deposit extracellular matrix and produce pro-infl ammatory cytokines. Th us, elevated hTERT expression in HMECs may limit desmoplasia both by stabilizing telomeres and by inhibiting mito chondrial ROS production. Finally, the increased replica tive capacity of DCIS lesions may itself contribute to the evolution of a protumorigenic stroma. For example, hyper-proliferation of cells within pre-neoplastic tissue results in reduced intracellular nucleo tide pools, which in turn can lead to replication-induced DNA damage and increased genomic instability [12]. Th erefore, the increased metabolic demands of DCIS lesions that have lost G 1 or G 2 cell cycle checkpoints (or both) may render them extremely adept at stress-induced conditioning of a tumorigenic stroma. In light of these observations, defi ning the integrity of cell cycle check points within DCIS lesions may be an important diag nostic tool prior to initiating neoadjuvant chemotherapy.