Here we show for the first time that dsDNA damage and telomere malfunction in human breast epithelial cells results in a p53- and activin A-dependent COX-2 induction. By identifying signaling events leading to COX-2 induction, this study complements our previous work establishing a direct link between COX-2 and malignant phenotypes (3
). Strikingly, COX-2 expression, and its associated phenotypes, are not confined to the initial cell with telomere malfunction, but are also induced in cells in absence of DNA damage through the cell non-autonomous action of activin A (). While induction of this pathway is self-limiting, i.e. leading to cell cycle arrest, in HMEC (intact p16/Rb pathway), it is not in vHMEC (silenced p16), where cells continue to proliferate. Finally, we demonstrate in vivo
that high COX-2 expression is associated with high levels of γH2AX, TRF2 and activin A in a pilot cohort of DCIS lesions.
Our study highlights the coordinated action of p53, activin A and p38 in inducing COX-2 following dsDNA damage. To our knowledge this is the first report of an activin A-dependent COX-2 induction in human cells. A similar observation has been reported in rat macrophages (36
). Multiple stimuli can induce COX-2 including mitogens, cytokines, hormones, irradiation, oncogene activation and inflammation (33
). Many of these factors induce p53, which can in turn increase COX-2 expression. Interestingly, COX-2 induction decreases p53-dependent apoptosis in vHMEC, suggesting that COX-2 represses p53 function (38
). This feed-back loop may explain the biphasic activin A and COX-2 induction in response to DNA damage described here.
Activin A is a TGF-β superfamily member whose signaling requires receptor dimer formation and downstream effectors including Smads, MAPKs, ERK, and p38 among others. The effects of activin A signaling are cell context-dependent. For example, through interaction with p53, activin A can induce either dorsal or ventral mesoderm formation during Xenopus embryogenesis (39
), erythroid differentiation (40
) or cell cycle arrest via p21 induction and CDK4 down-regulation (41
In breast tumor cell lines, activin A induces growth arrest and inhibits HGF-induced tubule formation in primary mammary epithelial cells grown in a collagen matrix (32
). However, the role of activin A in breast tumorigenesis remains ambiguous. Activin A mRNA and protein are frequently up-regulated in DCIS and in IDC compared to normal breast. Moreover, breast tumors with local recurrence or metastasis to lymph nodes have the highest levels of activin A expression (43
) highlighting the potential predictive value of activin A as biomarker. Gains in chromosomes 3p, 7p, 12q and 15q, which contain the genes encoding the type II receptor, activin A, two other activin family members and Smad 3, are often observed in breast cancer (45
). Last, activin A overexpression increases tumor volume by inhibiting apoptosis in mouse xenographs (49
). This paradox is reminiscent of that described for TGF-β. The induction of growth arrest described here in HMEC (intact p16) and elsewhere (32
) supports that activin A acts as a barrier to tumor initiation. In contrast, the observation that activin A is frequently up-regulated in IDC and metastatic breast lesions suggests that activin A, like TGF-β, may facilitate tumorigenesis in the context of impaired growth inhibitory response (for example due to loss of p16 in vHMEC) by decreasing immune response and altering the tumor microenvironment. Despite the similarity between activin A and TGF-β, these proteins are not synonymous; since vHMEC arrest in response to TGF-β (4
). Inhibiting activin A, either through induction of physiological regulators (follistatin, inhibin A or follistatin-related protein FLRG) or use of inhibitors (SB432542 or type I and II receptor antibodies), is an attractive therapeutic approach to ablate the COX-2 overexpression triggered by DNA damage, although side effects of such therapies remain to be investigated.
DNA damage is an early and nearly universal event in epithelial cancers. It is well appreciated that in the absence of cell cycle checkpoints (e.g. p53 or p16/Rb), DNA damage generates genomic instability and, consequently, may result in random loss of tumor suppressors, gain of oncogenes and clonal expansion. Thus, DNA damage indirectly contributes to tumorigenesis through generation of genomic instability. Here we show that DNA damage may also directly contribute to tumorigenesis through a separate mechanism, the specific induction of activin A and COX-2. Since activin A and the prostaglandins are secreted, DNA damage in one cell could drive tumorigenic phenotypes in an adjacent p16-compromised precursor cell or lead to proliferative arrest in an adjacent p16-intact cell. We postulate that these cell non-autonomous effects might provide a proliferative advantage to precursor lesions and facilitate the expression of pre-malignant phenotypes. Understanding how DNA damage contributes to cell autonomous and cell non-autonomous events will further elucidate the tumorigenic process. For example, it may provide novel insights into the ecology of breast tissues and cell-cell interactions that modulate early events in malignancy, identify people with an increased propensity to develop aggressive tumors, or finally, provide an opportunity to prevent the progression of a precursor lesion to a fully tumorigenic state.