Our previous work with the BK5.IGF-1 model revealed that overexpression of IGF-1 in the mammary gland induces hyperplasia of the mammary epithelium and increases susceptibility to spontaneous and DMBA-induced tumorigenesis (14
). Surprisingly, our previous studies also showed that proliferative markers are elevated only in glands from prepubertal animals (14
). Since this finding may have profound implications for understanding the role of IGF-1 in mammary tumorigenesis and identifying critical windows of enhanced susceptibility, we investigated the molecular bases of these unexpected results.
The signal transduction pathways downstream of IGF-1R are complex and have been studied extensively in cultured cells (34
). The overall consensus of these studies is that IGF-1R signals primarily through two major canonical pathways: IRS-1/PI3K/Akt and Shc/Ras/MAPK. However, since the majority of previous studies were carried out in vitro, the relative importance of the Akt and MAPK pathways in mediating IGF-1 activity in tissues and the possible contributions of the endogenous endocrine milieu remain unresolved. In addition, while PI3K/Akt signaling has been widely implicated in IGF-1–induced mitogenesis (36
), our understanding of the role of MAPK in the induction of proliferative effects is still incomplete. Some studies demonstrate the importance of the MAPK pathway in inducing cyclin D1 expression (38
). However, in MCF-7 cells, IGF-1–stimulated increases in cyclin D1 expression and cell cycle progression are inhibited by PI3K but not MEK1 (MAPK-activating kinase) inhibitors (39
The importance of ERα in IGF-1–induced Akt pathway activation and proliferative responses has also been described in cell culture models, including some studies showing that IGF-1 and E2
have additive or synergistic effects on cellular proliferation (18
). These effects are usually attributed to functional crosstalk between E2
/ERα and IGF-1 systems, which includes potentiation of IGF-1 responses by E2
), stimulation of ERα activity by IGF-1 (22
), and additive activation of common signaling pathways (21
). While both IGF-1 and E2
reportedly stimulate expression of cyclin D1 and induce cell cycle progression, results in some systems indicate that the combined activities of IGF-1 and E2
are required to achieve maximal expression (18
). However, conflicting results have also been reported. In lactotrophs in primary culture, simultaneous treatment with E2
markedly inhibits IGF-1–induced proliferation (44
), suggesting a level of tissue specificity in these responses. Recently, Cascio et al. showed that in MCF-7 cells, IGF-1–stimulated cyclin D1 mRNA expression is reduced in the presence of E2
), which is reflective of our in vivo results.
Results presented here demonstrate that the activation of specific IGF-1 signaling pathways in mammary glands was dependent on the stage of development and the endogenous hormonal milieu. In prepubertal mice, IGF-1 overexpression stimulated signaling primarily via the PI3K/Akt pathway, leading to increased cyclin D1 expression in and elevated proliferation of mammary epithelium. At puberty, an apparent “switch” occurred, favoring activation of the MAPK pathway, which did not similarly stimulate increased cyclin D1 expression or proliferation.
The present findings also indicate that the increased cyclin D1 expression in tissues from prepubertal Tg animals was due to reduced degradation. The fact that mTOR, which plays an important role in the translation of cyclin D1 mRNA (46
), was activated by Tg IGF-1 in both pre- and postpubertal mice, while cyclin D1 levels were only increased in prepubertal Tg glands, indicates that the regulation of cyclin D1 expression in this system was at the level of protein stability rather than synthesis. Further studies will be required to elucidate how signaling at different ages affects cyclin D1 protein turnover.
We speculated that the switch in the downstream IGF-1 signaling might be related to changes in ERα expression caused by the hormonal milieu, specifically by changes in the level of circulating reproductive hormones. Our in vivo experiments showed that ERα levels were relatively high in the mammary glands of the WT prepubertal mice and that paracrine overexpression of IGF-1 in the mammary gland did not reduce receptor expression. In postpubertal Tg glands, the combination of Tg IGF-1 and increased circulating levels of E2 resulted in significantly reduced ERα expression. To test our hypothesis that ERα levels determined the IGF-1 signaling pathway, we carried out an in vitro study with ERα-null NMuMG cells and an in vivo study utilizing bigenic BK5.IGF-1 × ERKO mice. Both studies demonstrated a causal relationship among ERα levels, IGF-1–induced Akt activation, and epithelial proliferation.
To explore the underlying molecular mechanisms, we investigated ERα/IRS-1 complex formation. Our results showed that Tg IGF-1 stimulated formation of ERα/IRS-1 complex only in the glands from prepubertal mice and that the complex was almost undetectable in postpubertal Tg glands. Formation of the ERα/IRS-1 complex has been reported to amplify the IGF-1 response through the IRS-1/PI3K/Akt pathway (19
), and our results show that ERα/IRS-1 complex formation controls both Akt activation and proliferation in the mammary gland. We also looked in vivo for an explanation for the lack of MAPK activation in the glands from prepubertal IGF-1 Tg mice, because Shc phosphorylation, known to initiate signaling via the MAPK pathway (48
), was elevated in these glands. Results showed that activation of the Akt pathway in prepubertal Tg glands resulted in an increase in c-Raf phosphorylation at Ser259, inhibiting transduction via the Ras/Raf/MAPK pathway. Conversely, in postpubertal Tg glands, activation of MAPK was maintained. Thus, the level of ERα expression in mammary glands indirectly regulates signaling via the MAPK pathway. A model based on these findings is presented in Figure .
Schematic diagram of IGF-1 signal transduction in mammary glands in vivo.
These results identify critical, nongenomic actions of ERα, previously unrecognized in vivo, that determine not only the predominant IGF-1 signal transduction pathway, but also the magnitude of the proliferative response to IGF-1 in the mammary gland. The lack of a proliferative response in glands from adult mice also has important ramifications for our understanding of the contributions of IGF-1 to mammary tumorigenesis. Previous results in the BK5.IGF-1 mouse and other models have shown that IGF-1 acts as a classical tumor promoter in the epidermis, where it stimulates increased proliferation following DMBA treatment (17
). However, in established mammary tumorigenesis protocols, DMBA treatment begins postpubertally, at 7–9 weeks of age (14
). At this time, cyclin D1 and BrdU incorporation are no longer elevated in the mammary epithelium of Tg mice. Thus, increased susceptibility to mammary carcinogenesis in the BK5.IGF-1 model does not proceed through a classical tumor promotion mechanism.
Several alternate mechanisms can be speculated from these results. The most likely basis for increased tumor susceptibility is that the activation of the PI3K/Akt pathway in prepubertal glands induces increased ductal proliferation and accelerated mammary development, which also results in expansion of the number of carcinogen targets, possibly stem and/or progenitor cells (51
). However, it cannot be ruled out that initiated or premalignant mammary epithelial cells responded to the proliferative effects of IGF-1 postpubertally but, due to their small numbers, were below the level of detection.
An alternate possibility is that signaling through the MAPK pathway in postpubertal glands is associated with other phenotypic alterations that contribute to mammary tumor development and progression, such as inflammation, enhanced vascularization, and/or other stromal changes (52
). Interestingly, immunostaining of p-Erk1/2 increased in both the stromal and epithelial compartments of glands from bitransgenic BK5.IGF-1 Tg/ERKO mice, supporting the contention that stromal activation also contributes to mammary tumorigenesis in this model. Further investigations into these hypothetical contributors to mammary carcinogenesis are ongoing.
Our results also provide insight into the contributions of IGF-1 to breast tumorigenesis over a lifetime. Although still somewhat controversial, epidemiological evidence indicates that exposure to high levels of IGF-1 may be a risk factor for breast, prostate, and colon cancer (2
). However, while it is well accepted that IGF-1 is a major contributor to breast cancer susceptibility (2
), results from studies assessing serum IGF-1 levels in adult breast cancer patients have been conflicting (54
). These results may provide an explanation for the apparently discordant data regarding the role of IGF-1 in breast cancer and cancer risk. Most clinical and epidemiological studies measure circulating IGF-1 levels at the time of diagnosis, which, according to our results, is not the stage at which IGF-1 has the greatest pro-tumorigenic effects.
In addition, some studies have shown that African American women, who are significantly more likely to develop aggressive forms of breast cancer at an earlier age (56
), have serum IGF-1 levels comparable to or even lower than those in women of European descent (58
). Interestingly, studies consistently show that young African American girls have significantly higher circulating levels of IGF-1 in the pre- and perimenarchal periods, compared with age-matched girls of European descent (60
). Results presented here indicate that elevated IGF-1 levels during early developmental periods, when ERα levels are relatively high, may be particularly important in determining breast cancer risk. Although speculative at this point, it is possible that early exposures to elevated levels of IGF-1 may contribute to the increased likelihood that African American women will develop aggressive, early-onset breast cancer.
These in vivo results also provide insight into the mechanisms underlying the generally accepted epidemiological findings that obesity, with the associated increases in free IGF-1, increases breast cancer risk in postmenopausal women (62
). It is well established that after menopause, levels of ERα are again elevated in breast epithelium (67
). Our current results indicate that the combination of increased ERα expression and elevated IGF-1 induced a highly proliferative mammary epithelium, which substantially increased risk.
In summary, these studies demonstrate the value of combining in vitro and in vivo models in elucidating the mechanisms of IGF-1–stimulated mammary tumorigenesis. The results show that ERα expression levels are a major determinant of the activation of IGF-1–dependent signaling pathways and pro-tumorigenic proliferation in the mammary gland. Our investigations also identified an important contribution of nongenomic actions of ERα on tumor promotion and demonstrated that early exposures to elevated IGF-1 contributed to lifetime risk. These findings are critical for elucidating the factors that contribute to breast cancer susceptibility. Results also indicate that potential preventive strategies involving modulation of diet and energy balance may need to be focused not only on post-menopausal women, but also on young and adolescents girls.