Breast cancer is among the leading killers of women world-wide52
and there is a clear need for developing novel therapeutic options and preventative measures. One strategy that has been proposed for treating breast cancer is the use of selective AhR modulators (SAhRMs). The use of such compounds is based on the fact that AhR is expressed in mammary cells and that AhR activation antagonizes estrogen receptor (ER) signaling and inhibits tumor growth.8,18–21
Additionally, new evidence suggests that SAhRMs may also be effective in treating estrogen receptor-negative tumors.9
The goal of the current studies was to test whether AhR activation during pregnancy affects susceptibility to a genotoxic mammary carcinogen administered weeks later. Interestingly, we found that prior exposure to TCDD delayed tumor onset and reduced tumor incidence in adult mice. Furthermore, this beneficial effect of AhR activation occurred regardless of whether TCDD was given during pregnancy or to nulliparous animals. These interesting results provide additional support and insight for a protective role of AhR activation in mammary tumorigenesis.
In our studies, TCDD was administered 4 weeks before the animals were given DMBA and ≥ 14 weeks had elapsed by the time the first tumors formed. Therefore it was important to consider whether the beneficial effects resulted from persistent levels of TCDD in the body or from persistent changes in the fate of the mammary cells caused by previous AhR activation. To address this, we examined the level of Cyp1a1 and Cyp1b1, which are sensitive and commonly-used biomarkers of AhR activation. Our results showed that Cyp expression in the mammary gland itself had returned to background levels within the 4 weeks following TCDD treatment. Given that only minute amounts of TCDD are needed to induce Cyp1a1 and Cyp1b1,53
our results do not support the idea that residual TCDD is directly affecting tumor growth in our studies. Further support for this interpretation is provided by knowledge that the half-life of TCDD in AhRb
mice is 7–10 days,54,55
so the majority of the TCDD was cleared prior to the growth phase of the tumors. Therefore we speculate that the protection afforded by prior AhR activation results from a change in the fate of the mammary epithelial cells that causes resistance to neoplastic transformation.
While numerous studies have shown that AhR activation is protective against mammary carcinogenesis, the precise mechanism of the protection remains unclear. We began our investigation into the mechanism of delayed tumor formation by examining the impact of prior AhR activation on the initiation stage of carcinogenesis. Specifically we examined whether mice treated with TCDD four weeks prior had (i) persistent induction of metabolic enzymes that biotransform DMBA, (ii) decreased numbers of DMBA-DNA adducts, or (iii) diminished proliferation status of the mammary epithelial cells at the time of carcinogen administration. We also compared (iv) the levels of preneoplastic lesions in the mammary glands of the TCDD- and vehicle-exposed animals. Each of these four markers relevant to tumor initiation was equivalent in mice from both treatment groups, suggesting that DNA damage and tumor initiation were not diminished by prior exposure to the AhR agonist. Taken together, these data strongly suggest that prior AhR activation is not altering tumor initiation in this model.
Instead, we speculate that prior AhR activation is interfering with the second stage of chemical carcinogenesis, tumor promotion. This is supported by the findings that the level of DMBA-DNA adducts and the incidence of preneoplastic lesions was the same in the TCDD- and vehicle-treated mice, yet the ultimate development of palpable tumors was suppressed in TCDD-exposed animals. TCDD is known to influence tumor promotion in other models, and is in fact established as a tumor promoter in skin and liver.4,56,57
While these opposite effects in different tissues may at first appear to be irreconcilable, it is plausible that they reflect cell- and context-dependent differences in AhR-mediated effects on cell cycle regulation and apoptosis. For example, AhR activation is known to influence cell proliferation and apoptosis in many cell lines and tissues; however, the precise nature of the effects (increased versus
decreased) vary depending on the cell as well as by the treatment conditions.12,13,58–61
Thus in summary, because the TCDD-exposed mice in our studies had a longer
latency period and decreased
mammary tumor incidence at any given time point, it is more logical that TCDD decreases promotion in this model. Furthermore AhR-mediated suppression, or even reversal, of tumor promotion is a reasonable explanation for the regression of pre-existing mammary tumors reported previously. 18–20
While many studies in rodents show that AhR activation reduces mammary tumor growth, the complex relationship between mammary tumorigenesis, constitutive expression and function of AhR in tumors, and activation of the receptor by exogenous ligands is not fully elucidated. Studies conducted by Dave Sherr and colleagues demonstrate that AhR is overexpressed in mammary glands and tumors from DMBA-treated animals,62,63
and the receptor is also detected in many breast cancer cell lines and human tumors.64–67
Endogenous expression and activation of AhR in mammary tumors likely influences cell cycle regulation, and may promote tumor growth by stimulating cell proliferation and inhibiting apoptosis.68,69
On the other hand, AhR activation with TCDD and SAhRMs typically inhibits breast cancer cell growth in rodent models. This may be explained, at least for ER+ cancer cells, by cross-talk between AhR and ER. For example, exogenous AhR activation perturbs the expression or function of numerous genes that influence proliferation of breast cancer cells, including c-fos, TGF-β, TGF-α, and receptors for progesterone, prolactin and estrogen (reviewed in 70
). Mechanisms for this antiestrogenic activity may include competition between activated AhR and ER for shared cofactors, binding of AhR to inhibitory response elements in promoter regions of ER-inducible genes, or AhR-enhanced proteosomal degradation of the ER.70–72
In future studies we will examine the status of AhR, hormone receptors, oncogenes, and other modulators that influence growth of cancer cells. This will require collecting lesions and tumors from vehicle and TCDD pretreated mice systematically throughout the course of tumor growth, and is part of our ongoing investigation into the underlying molecular pathways that are disrupted by prior TCDD treatment to delay tumor onset.
A separate issue raised by the current studies is unrelated to the effect of AhR activation; specifically the observation that pregnancy alone did not protect against tumor formation. In other words, the tumor incidence curve for the vehicle-treated parous mice was not different from that of the vehicle-treated nulliparous animals. Nor was there a difference in tumor formation between the TCDD-parous and TCDD-nulliparous mice. This finding was somewhat surprising, since pregnancy is hypothesized to protect against breast cancer in humans and has been demonstrated in rat models.30–32
However, we are aware of only one report that has directly addressed the protective effect of pregnancy in an analogous chemical carcinogen-treated mouse model. Specifically, Medina and Smith46
found that pregnancy plus lactation reduced the incidence of DMBA-induced tumors by approximately 3-fold and lengthened the latency period by 10 weeks. Although our current study did not show evidence of protection provided by pregnancy, differences in the experimental designs may explain the different outcomes. Specifically, in the Medina and Smith study the dams nursed their pups for one week, whereas in the current study we removed the pups at birth. Given that lactation is thought to further reduce the incidence of mammary tumors above pregnancy alone,73
it is highly probable that this explains the differing outcomes in the two studies.
Our results also provide insight into understanding the mechanism of protection against breast cancer afforded by pregnancy. Specifically, we found that mammary cell differentiation alone, at least differentiation that contributes to branching and lobuloalveolar development, is not likely a causal factor in parity-induced protection against tumor formation. In this model, AhR activation during pregnancy dramatically suppressed branching, lobuloalveolar development, and milk production ( and 28,29
). However, suppressed glandular development did not correlate with increased risk of developing mammary tumors, which suggests that additional factors other than differentiation must play a role.
The chemokine CXCL12 and its receptor CXCR4 are known to function in leukocyte migration to inflammatory sites.74
With regard to tumorigenesis, CXCR4 is often over-expressed in human cancers, and CXCL12 may also play a role in survival, proliferation or angiogenesis in the primary tumor.75,76
Additionally they are believed to play a role in directing the migration of cancer cells to secondary metastatic sites.77
Hsu et al51
recently reported that TCDD treatment reduced the expression of these factors in MCF-7 cells, and postulated that this may contribute to the protective effect of AhR activation in breast cancer. A subsequent report by Hall et al
described similar suppression of CXCR4 by TCDD in additional ER+ and ER- breast cancer cell lines.67
In our studies, TCDD treatment caused a transient increase in the expression of both molecules in the mammary gland. The cause of this increase in the absence of antigen challenge is unclear, although it could reflect an enhanced inflammatory response that is commonly observed in TCDD-treated animals.78–80
Regardless, this increase was transient and did not persist at the time of carcinogen administration. There was suggestive evidence that CXCR4 expression was diminished in tumors from the TCDD-treated mice, which would be consistent with observations by Hsu et al
and Hall et al
in the cultured tumor cells. However the decrease was not technically statistically significant. Taken together our results do not provide strong support for a link between delayed tumor formation and changes in CXCL12 or CXCR4, although the potential for these molecules to influence continued tumor growth or metastasis warrants further exploration.
As discussed previously, Lamartiniere and colleagues have demonstrated that developmental
exposure to TCDD alters gland development and also increases susceptibility to mammary tumorigenesis.22,23
Thus it is reasonable that the differentiation state of cells in the mammary gland at the time of AhR activation will influence susceptibility to neoplastic transformation. Our original intention was to test whether the AhR-mediated suppression of normal pregnancy-induced mammary differentiation would likewise increase susceptibility to tumor development. However, our results did not support this hypothesis for glandular differentiation caused by pregnancy. We did not find that altered glandular development caused by TCDD influenced the development of DMBA-induced tumors; tumor development was delayed by TCDD treatment regardless of whether the animal was pregnant or not at the time of exposure.
In summary, our results show that AhR activation causes a persistent change in mammary cells that delays tumor formation, and add to the growing number of studies that have demonstrated that AhR activation reduces mammary tumor incidence or causes regression of existing tumors. In combination with other findings such as overexpression of AhR in mammary tumors,62,63,68
effects of AhR activation on cell cycle regulation and invasiveness of breast cancer cells,67,81
and the discovery that a metabolite of tamoxifen activates AhR,82
this report underscores the importance of understanding the role of the receptor in breast cancer. We hypothesize that the beneficial effect observed in our studies occurs via inhibiting tumor promotion, specifically by slowing the growth of preneoplastic lesions and initial tumors. This is promising because it provides an opportunity for biologic intervention in situations where initiation has already occurred, and provides tantalizing evidence to support continued exploration into the use of SAhRMs in breast cancer treatment and prevention.