Chondrosarcoma of bone is a malignant cartilage-forming tumor of which distinct clinical and histological subtypes are recognized. So far, for locally advanced and metastatic chondrosarcoma no treatment options are available. Previous studies have demonstrated the presence of the ESR1 and activity of aromatase in conventional chondrosarcoma [11, 12]. Furthermore, in 2005, our group showed an effect of estrogens and the aromatase inhibitor exemestane on the proliferation of chondrosarcoma cells in vitro, indicating that chondrosarcomas might be susceptible to hormonal therapy. In that study, ESR1 and CYP19A1 mRNA expression were demonstrated in a set of 23 conventional chondrosarcomas and 7 (primary) chondrosarcoma cultures. ESR1 protein expression was demonstrated in all 23 tumors tested. Addition of 17β-estradiol, 4-androstene-3,17-dione, and exemestane showed subtle effects on the proliferation of 2 cell cultures containing ESR1 and aromatase. After addition of 4-androstene-3,17-dione, an increase in proliferation was demonstrated. Proliferation was 131% of normal proliferation which decreased to 105% after inhibition with exemestane. A cell line lacking ESR1 and CYP19A1 did not show any response.
In the current study, we aimed to gain more insight into the possibility of treating chondrosarcoma patients with hormonal therapy by further investigating the expression of the hormone receptors ESR1 and AR, and of aromatase, the enzyme that mediates the last step in the biochemical formation of estrogen, in a larger set of conventional chondrosarcomas as well as three rare chondrosarcoma subtypes. In conventional chondrosarcoma, we furthermore monitored the effect of estrogen, the estrogen precursor androstenedione and the non-aromatizable androgen dihydrotestosteron, and various known estrogen signaling-inhibiting drugs on the progression of chondrosarcoma cells in vitro.
We demonstrated expression of ESR1 in a large proportion of various types of cartilaginous tumors. In conventional central and peripheral chondrosarcoma we observed immunoreactivity against ESR1 in 81% (34 out of 42) and 81% (21 out of 26) of the tumors, respectively. These results confirm and extend 2 previous studies in which the authors demonstrated nuclear expression of ESR1 in subsets of 23 [11] and 31 [12] conventional chondrosarcomas. Grifone et al. [12] suggested a decrease or loss in ESR1 expression in the higher grade or dedifferentiated chondrosarcomas. We observed such a trend in the peripheral chondrosarcomas, where only 33% of the high grade tumors show positive staining for ESR1. However, this group included only three tumor specimens. In central chondrosarcoma no correlation with grade was observed. Aromatase protein, the enzyme responsible for the conversion of androstenedione and androgens to estrogens, was expressed in 86% and 93% of the central and peripheral chondrosarcomas respectively, suggesting that tumors are capable of metabolizing estrogens from precursors. AR is another important target for hormonal therapy in for example prostate cancer. As androstenedione is a steroid precursor for estrogens as well as androgens we also investigated the possibility of AR involvement in chondrosarcoma proliferation. However, AR nuclear protein expression was observed only in a small number of cases with very few positive cells.
Besides conventional chondrosarcoma, several rare chondrosarcoma subtypes are defined. Despite aggressive therapy, approximately 90% of the patients with dedifferentiated chondrosarcoma die with distant metastasis, within 2 years after diagnosis of the disease [6, 23]. The low-grade component and the highly malignant component display ESR1 protein expression in 72% and 86% of the samples respectively. Aromatase was observed in 97% and 89%, suggesting the presence of estrogens.
Mesenchymal chondrosarcomas are usually very aggressive with a strong tendency of local recurrence and distant metastases. Patients have a 5-year overall survival of 55% [7]. Although mesenchymal chondrosarcoma of bone is generally considered to lack sex predilection [24], Fanburg-Smith et al. [25] suggested a female predominance and raised the possibility of hormonal influence in the pathogenesis of this tumor. However, all their mesenchymal chondrosarcoma cases were ESR1 negative. In our study, in 65% (15 out of 23) of the mesenchymal chondrosarcomas the small cell component was positive for ESR1, while in 33% (5 out of 15) of the tumors also the cartilaginous areas were positive. Moreover, aromatase expression was observed in the small cells of 52% (12 out of 23) of the tumors, whereas the cartilage component demonstrated aromatase expression in 77% (10 out of 13). This might indicate that these tumors do have an active estrogen signaling pathway, which might be targetable by antiestrogens or aromatase inhibitors. Discrepant results may be explained by differences in ESR1 antibody and antigen retrieval protocols.
Clear cell chondrosarcoma is a low-grade variant of chondrosarcoma, which rarely metastasizes, but has a recurrence rate of 86% after curettage. About 15% of the patients die as a result of the disease [8]. We have observed ESR1 expression and aromatase expression each in 69% of the clear cell chondrosarcomas, suggesting that also these chondrosarcoma patients potentially might benefit from antiestrogen therapy and/or aromatase inhibition.
In vitro cell models to further study the effect of estrogen signaling on chondrosarcoma are available for conventional central chondrosarcoma only. No stimulation of proliferation of central chondrosarcoma cells was observed after addition of the non-aromatizable androgen dihydrotestosterone. This suggests no significant role for AR signaling in chondrosarcoma proliferation, which is consistent with the fact that very few tumors express AR.
In addition, in spite of positive immunohistochemical staining for ESR1 protein in all in vitro cell cultures, addition of 17β-estradiol, 4-androstene-3,17-dione or drugs targeting the estrogen-signaling pathway did not have a significant effect on the proliferation of the conventional central chondrosarcoma cell cultures. These results contradict our results published in 2005, where proliferation was stimulated by 17β-estradiol and 4-androstene-3,17-dione, and inhibited by exemestane [11]. Although we included an identical experimental set up, cell culture conditions are never 100% identical. For example, each batch of FBS contains different amounts of growth factors and other components which might influence experimental outcome. Also cell characteristics might have changed over time, resulting in passages insensitive to (anti)estrogens and aromatase inhibitors, as has been described before for certain breast cancer cell lines [26–28].
Breast cancer cell line ZR-75-1 is known to be completely dependent on estrogens for its proliferation, and proliferation can be fully inhibited by abrogating the estrogen-signaling pathway [29]. Although we previously demonstrated an effect of estrogen-signaling on chondrosarcoma cell proliferation, as compared to estrogen-dependent breast cancer cell line ZR-75-1 the effects in chondrosarcoma, if present, were very subtle. As a positive control, ZR-75-1 showed a 179% increase of proliferation upon addition of 1 nM 17β-estradiol, confirming a functional experimental setup, versus a previously demonstrated 55% increase in chondrosarcoma proliferation [11] and no significant increase in the current study. Both studies clearly indicate that, in contrast to estrogen-dependent breast cancer, chondrosarcoma proliferation is not fully dependent on estrogens.
Besides investigating estrogen dependence, we tested aromatase inhibitors which block estrogen production, and the effects of tamoxifen and fulvestrant which abrogate estrogen receptor function [30, 31]. In the estrogen-dependent ZR-75-1 breast-cancer cell line proliferation was completely inhibited upon addition of tamoxifen and fulvestrant (Figure 2B). However, in the chondrosarcoma cell cultures, estrogen-signaling inhibition caused no effects on cell proliferation, suggesting that the mechanism driving proliferation in chondrosarcoma is different from the mechanism active in estrogen-dependent breast cancer. In chondrosarcoma, effects of estrogen are much more subtle and likely depend on the tissue culture conditions used, resulting in either marginal effects (in our previous study) or no effects at all.
In addition, the median time to progression in the clinical series was five months both before and after treatment. Therefore, we can conclude that aromatase inhibition was not effective in five conventional chondrosarcoma patients, nor in a patient with dedifferentiated chondrosarcoma. Although a formal prospective phase II trial would have been more suitable to prove (in)efficacy of this concept, we were not able to gain industry support without stronger preclinical data.
Since our study is limited to the effects of estrogen signaling on conventional central chondrosarcoma only, no conclusions can be drawn about the effects of estrogen signaling in the other chondrosarcoma subtypes. However, although we demonstrated the presence of aromatase and ESR1 in a majority of various chondrosarcoma subtypes, our in vitro data on conventional chondrosarcoma and our patient trial including one dedifferentiated chondrosarcoma patient suggest that effects of estrogen-signaling inhibition in other chondrosarcoma subtypes, if present at all, will be very small and that estrogen-signaling inhibition is unlikely to play a major role in chondrosarcoma management.