The key finding of this study is that hormonal variations during the estrous cycle can affect ovarian cancer growth and progression. We found that inoculation of mice during proestrus results in enhanced tumor growth. This effect was also seen in tumor-bearing ovariectomized mice treated with exogenous estrogen. Additionally, we showed that progestins could abrogate estrogen induced cancer growth. These observations are further supported by the inhibitory effects of progestins on in vivo
and in vitro
growth of some cancers, including ovarian cancer (33
). Furthermore, mechanistic studies performed in our laboratory reveal that estrogen promotes angiogenesis, endothelial cell migration and tumor cell adhesion.
An association between the reproductive cycle and malignant progression has long been suspected (1
). In the present study, we sought to identify direct and indirect processes and pathways that link the estrous cycle to malignant cell behavior in ovarian carcinomas. Both estrogen and progesterone serve as dominant reproductive hormones in the menstrual cycle and are therefore likely key mediators in this process. Proestrus is associated with higher levels of estrogen, whereas estrus is characterized by decreased estrogen (3
). Consistent with estrogen induction of VEGF production by endothelial and ovarian cancer cells (10
), we find that VEGF levels are increased at the tumor level during proestrus. In other models, estrogen-induced VEGF production was attributed to ERα and ERβ activity at the transcriptional level (40
). Specifically, it was found that ERα and ERβ could bind to estrogen response elements near the transcriptional start site of the VEGF gene (40
). Also reminiscent of previous observations in other tumor systems (41
), we found that estrogen can increase the migratory potential of tumor-associated endothelial cells and this process is mediated by activation of the MAPK signaling pathway. These two mechanisms suggest that estrogen may increase the angiogenic potential of the tumor microenvironment by increasing VEGF secretion and increasing endothelial cell migration.
In our study, we found more tumor nodules in mice inoculated during the proestrus phase of the menstrual cycle, suggesting that estrogen promotes metastatic colonization by increasing the adhesive potential of cancer cells to extracellular matrix proteins. This finding is supported by other observations related to the role of estrogen in enhancing endothelial cell adhesion to extracellular matrix proteins (42
). Although the effect of estrogen on ovarian cancer cell adhesion is not well known, it has been studied in other gynecologic diseases. In an orthotopic mouse model of endometriosis, pre-treatment with estrogen resulted in a higher percentage of animals developing endometriotic-like nodules, while treatment with progesterone alone did not affect implantation rates (43
MAPK has been suggested to play a major role in cell migration, proliferation, differentiation and survival (44
). In some experimental models, estrogen has been shown to activate MAPK (28
). For example, various studies have shown that estrogen induced porcine aortic endothelial cells (PAEC) and human umbilical vein endothelial cells (HUVEC) migration and data suggest that this effect is mediated by MAPK (31
). Thus, we examined whether estrogen treatment resulted in MAPK activation in ovarian cancer and endothelial cells. Incubation with estrogen at a dose known to reflect the physiologic conditions in the ovary was not effective in activating MAPK in either of the human ovarian cancer cells we tested (45
). However, estrogen treatment rapidly increased the levels of activated MAPK in murine ovarian endothelial cells. These data suggest that estrogen impacts tumor biology by stimulating endothelial cells in the tumor microenvironment. Our finding is further supported by data that suggests that estrogen can promote tumor growth even in ER-negative breast cancer models (17
Progesterone is the dominant hormone during the estrus phase of the menstrual cycle. Fluctuations in progesterone levels could serve as another plausible reason for the differences in ovarian tumor growth observed in our study. Progesterone has been found to cause apoptosis of ovarian cancer cells in vitro
and to reduce the tumorigenic capacity of cells inoculated into the peritoneal cavity of mice (46
). Pretreatment with progesterone has been shown to reduce the number of tumor implants and metastases, thus prolonging the animal life span (46
). Additionally, long-term progesterone treatment of ovarian cancer cells was able to suppress the transformed phenotype as indicated by the acquisition of contact inhibition and loss of anchorage-independence (48
). Our observations of decreased tumor growth in the progestin treated arms validate these findings in the literature and could serve as an explanation for the protective effect of progesterone in ovarian cancer.
Some studies have shown that estrogen can promote ovarian cancer cell proliferation, motility, invasion, and reduce apoptosis (49
). However, tamoxifen treatment is effective even in ER negative ovarian cancers, suggesting that other factors might be responsible for decreased tumor growth (53
). In contrast, progesterone treatment inhibits cell growth, invasion and promotes apoptosis (46
). Our findings extend previous work in that the effects of reproductive hormones were found to be mediated via the tumor and endothelial cells.
In summary, we have shown that the timing of ovarian cancer cell inoculation within the estrous cycle modulates the growth of ovarian cancer cells. In addition, we have identified that the reproductive hormone mileu functions as a key predictor of ovarian tumor progression. These results show that cancer cell growth and angiogenesis are highly coordinated within the estrous cycle, raising the possibility that the effectiveness of therapeutic strategies might depend in part upon when in the reproductive cycle they are used.