It is long appreciated that the prevalence of cancer in human populations is far lower than one would predict based on DNA mutation rates (23
). If oncogenic mutations are not sufficient to establish tumors, how are tumors contained, suppressed, or eliminated before they become evident? A number of tumor surveillance mechanisms have been described, including the classic molecular tumor suppressors, immune surveillance, and suppression by ECM and other microenvironmental factors. This study adds a new type of suppression to the list: factors secreted by normal differentiating cells that could kill or subdue their transformed counterparts.
We had shown previously that differentiating MECs secrete factors that allow phenotypic reversion of breast cancer cells, leading to formation of acinus-like structures and growth suppression (5
). The secreted factors could be partitioned into soluble and insoluble fractions; the former had tumor cell–killing activity and the latter had most of the “reverting” activity. Here, we identified six factors in the soluble fraction that were secreted by differentiating MECs in 3D and shown to either kill or suppress the growth of tumor cells. This collection of factors included antiangiogenic proteins (ATIII and VBP), proinflammatory cytokines (IL-1F7 and IL-25), and growth and differentiation proteins (FGF11 and BMP10). IL-25 exhibited the most potent cytotoxic activity toward breast cancer cells, whereas the other factors exhibited cytostatic activity. Here, we focused on the mechanism of action of IL-25 and its potential as a therapeutic agent in breast cancer.
IL-25 is a proinflammatory cytokine that is expressed highly in certain organs, such as testis, prostate, and spleen, and is expressed in low amounts in other organs including normal breast (11
). It is the most distant member of the IL-17 family of proteins, sharing only 16 to 30% sequence homology with the other family members (25
). It plays a role in proinflammatory responses of lymphatic, kidney, and lung cells by inducing production of T helper 2 (TH
2)–type cytokines (11
). The function of IL-25 in other tissues remains to be elucidated.
We show here that IL-25 is temporally up-regulated in developing normal mammary glands and induces caspase-mediated apoptosis of breast cancer cells without affecting nonmalignant MECs either in culture or in mice. The reason behind the resistance of nonmalignant cells to IL-25 is the differential expression of the receptor, IL-25R, high in breast cancer cells but low or absent in nonmalignant MECs (). IL-25R overexpression contributes to tumorigenic potential, as shown by the result that siRNA-induced reduction in the amounts of IL-25R impaired breast cancer cells’ anchorage-independent growth in soft agar (). Examination of breast cancer specimens showed distinct up-regulation of IL-25R in 19% of the samples, correlating strongly in those with poor prognosis ().
The exact mechanism by which IL-25R expression confers a growth advantage to breast cancer cells remains to be determined. We postulate that although IL-25R–expressing cancer cells do not express the apoptotic ligand IL-25 ( and fig. S4A
), they may express another ligand that contributes to their tumorigenic potential. Such a candidate ligand appears to be IL-17B, which binds IL-25R with a markedly lower affinity than that of IL-25 (11
). We found that IL-17B was expressed in most breast cancer cell lines that expressed high amounts of IL-25R, whereas IL-17B was absent from nonmalignant MCF10A cells (fig. S4A
). Consistently, IL-17B was up-regulated in 30% of breast cancer specimens examined (12 of 40), but undetectable in normal tissues (0 of 18) (fig. S4B
). Small hairpin RNA (shRNA)–dependent reduction of IL-17B amounts in MDA-MB468 breast cancer cells impaired their growth and invasive potentials (fig. S4, C to G
), whereas ectopic addition of IL-17B protein enhanced both potentials (fig. S4, H to J
). These results in sum suggest that IL-17B may augment the tumorigenicity of breast cancer cells in an autocrine manner. This possibility is presently under investigation. Whether IL-17B competes directly with IL-25 for receptor binding is not known. However, it is known that both IL-25 and IL-17B bind the extracellular domains of IL-25R in vitro, but that the IL-25 ligand shows a markedly higher affinity for the IL-25R than does IL-17B (11
). Accordingly, it is speculated that IL-25 binding to the receptor would outcompete IL-17B if both were present simultaneously in the same cells. Some of our data, in fact, suggest that when both ligands are present, IL-25 binding to IL-25R is dominant over IL-17B binding to IL-25R. Figure S4A
shows that IL-17B is expressed in most cancer cell lines tested. Yet, ectopic addition of IL-25 still causes the cells to apoptose. Nevertheless, the question could arise that if IL-25 has such a strong affinity for IL-25R and this binding induces apoptosis, why would tumors ever have a chance to form? We hypothesize that it is the localization and/or temporal availability of each of these ligands that accounts for the fact that IL-25R–expressing tumors grow rather than apoptose. That is, once a tumor is formed, if IL-17B were to be present in the tumor, it would act as a growth promoter. Because IL-25 is not generated by the tumor cells, IL-25–dependent apoptosis would be absent.
In lymphoid and renal cells, IL-25R activation by IL-25 induces a proinflammatory response mediated by TRAF6; in these two tissue contexts, TRAF6 associates with its cognate binding domain in IL-25R and activates nuclear factor κB (NF-κB), which in turn stimulates the transcription of genes that encode inflammatory cytokines (11
). In contrast, IL-25R activation by IL-25 in breast cancer cells causes the receptor to interact with DD adaptor proteins FADD and TRADD, rapidly activating caspases 8 and 3 sequentially for apoptotic signaling (). This action appears to be mediated by the DD-like region in the C terminus of IL-25R. Constitutive binding of TRAF6 to IL-25R confers a protective effect on breast cancer cells, inhibiting apoptosis when IL-25 is not present (). Such tissue-specific responses to IL-25/IL-25R signaling may result from additional proteins that serve as switches between TRAF6/NF-κB signals and TRADD/FADD/caspase 8 signals. This type of molecular switching has been shown previously for the related receptor, TNF-R1, which shares about 30% DD homology with IL-25R (). TNF-R1 activation by TNF-α induces both NF-κB activation and apoptosis. However, NF-κB activation can be blocked by the brain- and reproductive organ–expressed (BRE) protein, which binds the juxtamembrane cytoplasmic region of TNF-R1 and promotes apoptotic signaling (27
). It is anticipated that proteins with similar pathway-switching functions will be discovered in the IL-25 pathway.
IL-25 thus holds promise as the basis for development of novel, effective breast cancer therapeutics with broad therapeutic windows. Our demonstration of the marked effect of IL-25 administration in inhibiting tumor growth with no apparent toxicity to the normal tissues in animals () and the correlation of IL-25R expression with poor prognosis in breast cancer patients support this contention. Unlike conventional immunotherapy in which a cytokine, such as IL-2, is administered by intravenous infusion (28
) to provoke global immunologic responses (29
), a therapy that targets IL-25/IL-25R signaling would induce apoptosis specifically in cancer cells that express IL-25R. Expression of IL-25R in 19% of invasive ductal carcinomas places this receptor in a class of markers of interest for therapeutic targeting because of its prevalence, correlation with aggressiveness, and cell surface location. In these aspects, IL-25R could be compared to Her2/neu (30
). Targeting Her2/neu has become an important strategy for treating HER2+ breast cancers by means of trastuzumab, a monoclonal antibody that binds and inhibits Her2/neu, resulting in tumor regression. The findings reported here suggest that targeting IL-25R in patients bearing tumors that overexpress this receptor should similarly result in strong clinical responses. We anticipate that our present study will lead to the development of IL-25R–based diagnostics, facilitating identification of the target population, and IL-25/IL-25R–based therapeutics, such as IL-25 peptidomimetics or IL-25R antagonistic antibodies, to efficiently and specifically induce target cell apoptosis in patients with advanced breast cancer.