Fibroid explants reveal a higher sensitivity against MDM2-inhibitor nutlin-3 than matching myometrium

Uterine fibroids are by far the most common gynaecological tumors at all. In premenopausal women incidences of 60-70% have been reported [1, 2] Although only a minority of the leiomyomas become symptomatic the presence of symptomatic leiomyomas is still the leading cause for hysterectomy worldwide. Despite their high prevalence the treatment options besides surgical removal by hysterectomy or tumor enucleation are still limited. Treatment by GnRH agonists as well as antagonists can induce shrinkage of fibroids but re-growth of the tumors usually occurs after termination of the therapy [3, 4]. Thus, intervention at the hormonal level is as a rule only recommended to reduce tumor size pre-operatively [5]. Another alternative represents embolization of the fibroids but the recurrence of myoma-related symptoms is not a rare finding after that treatment as well [6]. Thus, therapies aimed at permanent shrinkage of the fibroids still remain a challenge. Recently, we have presented evidence that in leiomyoma development the overexpression of p14 ^Arf drives a negative feedback-loop between p53 and MDM2 that governs the fate of the individual fibroid. Compared to matching myometrial tissue the myomas display a significantly higher expression of one of the genes of the senescence associated Ink4a/Arf locus i.e. p14 ^Arf [7]. It is not clear yet if this elevated expression solely results from an enhanced proliferative activity of the fibroid compared to its tissue of origin or if the same oncogenic stimuli triggering the leiomyoma growth do simultaneously stimulate p14 ^Arf as an oncogene-induced senescence-like mechanism. However, whatever is the cause of the p14 ^Arf overexpression it activates a p53-MDM2 negative feedback-loop [8] that may govern a delicate balance of the fibroids between proliferative activity and senescence [7]. This makes antagonizing MDM2 an interesting approach towards the growth control of fibroids. Accordingly, we have used nutlin-3, a known MDM2 inhibitor, to antagonize its activity in cell cultures from fibroids. Interestingly, we were able to show that antagonizing MDM2 induces the activity of genes associated with senescence (p21) as well as those with apoptosis (BAX) in leiomyoma cells in vitro [9]. Also, the MDM2 inhibitor drastically reduced cell proliferation as indicated by a decreasing level of Ki-67 expression. Nevertheless, the question arises if fibroids display a higher sensitivity than matching myometrium as can be suggested from their higher expression of p14 ^Arf [7] or the spontaneous senescence observed in fibroids [10]. Herein, we have used tissue explants taken from leiomyomas and matching myometrium to analyze the effects of an MDM2 antagonist and possible different sensitivities of the fibroids and their matching tissue of origin.

Recently, we have shown that compared to myometrium leiomyomas express higher levels of p14^Arf, an important upstream regulator of p53 [7]. Herein, this observation has been confirmed on a much larger series of cases. In addition, a marked heterogeneity of the p14^Arf expression was noted when comparing UL from one patient. Most likely due to the well-documented positive feedback-loop that exists between p53 and MDM2 [14, 15], the fibroids do also express higher levels of MDM2 leading to a positive correlation between p14 ^Arf and MDM2 expression [7] that may keep fibroids in a delicate balance between growth and senescence. This finding has been confirmed here based on a larger series and makes MDM2 an interesting target molecule for the growth control of leiomyomas. Accordingly, we were able to demonstrate that in leiomyoma cells in vitro treatment with nutlin-3, a small-molecule inhibitor of MDM2, activates the expression of canonical groups of genes associated with senescence and apoptosis downstream of p53 [9]. Herein, we were able to show that as a rule tissue explants taken from leiomyomas display a higher sensitivity after treatment with nutlin-3 than do those from matching myometrium. Of note, nutlin-3 increased the expression of p53-dependent marker genes associated with senescence as well as with apoptosis. As a rule for both genes i.e. p21 and BAX leiomyoma tissue turned out to be more sensitive than myometrial tissue. We assume that the higher expression of p14 ^Arf in the fibroids [7] is the most likely explanation for this different sensitivity. When interpreting leiomyomas as the result of proliferation of a stem-cell like population it seems reasonable to assume that this proliferation is accompanied by activation of the p53-pathway via p14^Arf to protect the cellular genome [12]. Vice versa, functional p53 activates the positive feedback-loop with MDM2 that may be essential for the development of fibroids. Likewise, disturbing this balance by MDM2 inhibitors can be assumed to cause senescence as well as apoptosis of the leiomyoma cell population because p53 still remains intact with a strongly decreased MDM2 activity.

The present study has limitations which are mainly related to the use of an in vitro model. Cell cultures from fibroids can easily be set up without major problems of overgrowing of normal cells but the cells have only a limited growth potential and, as an even more serious reason for concern, rapidly lose characteristic features of leiomyomas in vivo. E.g. the estrogen receptor level of fibroid cells rapidly declines in vitro [16]. Immortalization of the cells is possible e.g. by using SV40-large T-antigen or the SV40 early region, but with these cells experiments are still facing the problem of drastic changes compared to the normal cells [17]. Though the explants cultures as used here may reflect the in vivo situation better than isolated cells it is well known that explant cultures as well loose characteristics of the tissue in vivo e.g. a decrease the expression of estrogen receptors after a short time in vitro has been noted [18] that was within the time-range used here for the nutlin-3 experiments. Nevertheless, the higher sensitivity of the leiomyoma tissue against the inhibition of MDM2 compared to surrounding myometrium corresponds to a higher in vivo expression of p14^Arf and thus likely exists in vivo as well. Then, it may have considerable therapeutical implications. In UL antagonizing MDM2 seems to be a way to induce growth arrest as well as apoptosis. Both can be expected to irreversibly impair tumor growth and to decrease the tumor size, respectively. Interestingly, estrogens are known as negative regulators of p53 [19]. Thus, it seems reasonable to speculate that changes of the behaviour of fibroids following changes of the hormonal milieu as in particular their shrinkage are at least in part also due to skewing the balance towards p53. Accordingly, a combination e.g. of a GnRH antagonist and a MDM2 antagonist may be a favourable approach for the treatment of fibroids. In summary, the results of the present study strengthen the idea that senescence and apoptosis play an important role in the growth control of fibroids and that their induction may offer interesting approaches for the therapy of these frequent tumors.

Based on their expression of p14^Arf we have concluded that as a rule leiomyomas represent a cell population of advanced senescence compared to matching myometrial tissue. Accordingly, fostering the p14^Arf - p53 network by nutlin-3, a known MDM2 antagonist, increases the expression of pro-senescence and pro-apoptotic genes in tissue explants from myometrium as well as from leiomyomas with the latter displaying a significantly higher sensitivity than the matching normal tissue. In summary, though the data need confirmation in vivo they point to members of the p53-network as to potential novel therapeutic targets for the treatment of uterine fibroids.