Desmoid and Oestrogens

Mechanism of Action of Oestrogens

The oestrogenic bio-effects are mediated by specific receptor subtypes, ERa and ERp, which exhibit a tissue-specific distribution. The unbound ERs exist as inactive intracellular receptors expressed in reproductive and not reproductive tissues. The mechanism of action is switched on after the interaction of the receptors with the ligand(s), which could be either the steroidal oestrogen, the polyphenolic phyto-oestrogens or the synthetic SERMs (selective estrogen receptor modulators). The latter exhibit either agonist or antagonist oestrogenic actions. The first generation of SERMs are represented by the triphenylethylenes tamoxifen and toremifene, which show agonist properties in the bone and uterus, but an antagonist action on the breast. The last generation of SERMs, represented by raloxifene and its ana-logues—LY-353381, EM-800 and CP-336156—are benzothiophene derivatives, showing anti-oestro-genic effects on the breast and uterus, but an oestrogen agonist effect on bone and cardiovascular systems.

The ER expression was shown by the authors both in DT tissues and in desmoid-derived cells in culture [41, 42]. Furthermore, we studied the gene expression of ERs in primary cultures obtained from desmoid tumoral tissue. The results showed that, in all the cultures, ERa and ERp were variably expressed (Picariello et al., 2006, personal communication); in fact, in some patients the ERa expression was predominant in respect to ERp. The extremely variable expression of the two ERs in these primary cell cultures underlines the individual differences among patients.

To evaluate if oestrogen could influence the DT derived cell growth, primary cell cultures obtained were cultured in presence or in absence of different concentrations of 17pE2, from 1 pM to 1 ^M concentration for 1 week (Fig. 5) [41]. The results showed that 1 nM concentrations of 17pE2 stimulated cell proliferation in five of the seven cultures analyzed and the effect induced by oestrogen on cell proliferation was different from one cell culture to another. Any influence on cell proliferation by the oestrogen was observed in two of the seven cultures. These results confirmed that oestrogen induced a prolifera-tive effect in the different cell cultures with a cell-specific potency. This could be due to the ER expression pattern and to the different 17pE2 potency in transac-tivating gene expression controlled by ERa and ERp.

In those cultures, where the increase of cell growth was great, there was a higher expression of ERap than ERp, while in those cultures where the effect of oestrogens was slight or absent, a comparable expression of the two ERs was evident. Therefore, the characteristic pattern of ER expression in each culture could be responsible for the cell culture-specific proliferative potency of 17pE2 which displays a greater ERa selectivity with a higher potency in transactivating gene expression controlled by ERa rather than ERp.

We also studied the effect of SERMs on the proliferation of desmoid cell cultures and the putative mechanism of action of SERMs. Firstly, we observed that tamoxifen inhibited the proliferative effect of 17pE2 [41]. Subsequently, we cultured desmoid cells in the presence of different concentrations of an analogue of raloxifene, LY117018 for one week (Fig. 6) [42]. LY117018 induced a dose-dependent inhibition of cell proliferation at the pharmacological dose of 5 ^M concentration independently of a greater or lesser expression of each ER. This suggests that pharmacological concentration of raloxifene, as well as other SERM molecules (e.g. tamoxifen and toremifene), could directly inhibit the proliferation and function of DT cells acting through or independently of ERs. To investigate if the inhibition of cell proliferation by LY117018 was dependent on the apoptotic cell death process, the desmoid cells were stimulated with the drug and then the internucleoso-mal fragmentation of genomic DNA was evaluated. The results showed that DT derived cell cultures stimulated with 5 ^M of raloxifene did not develop the typical nucleosomal ladder pattern of DNA degradation, clearly exhibited by the positive control (HL-60 cells stimulated for 4 h with 2.5 ^g/ml camp-tothecin; personal observations). These data suggest

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Fig. 5. Effects of 17ßE2 on cell growth of desmoid tumour cells. Cells were plated in phenol-red-free growth medium supplemented with 1% charcoal stripped foetal calf serum, containing different concentrations of 17ßE2. After 1 week cells were counted. Results were expressed as % of control values of three separate experiments. *p<0.05

Fig. 6. Effects of LY117018, an analogue of raloxifene, on cell growth of desmoid cells. Cells were plated in growth medium containing different concentrations of LY117018. After 1 week cell number was evaluated. Results were expressed as % of control values of three separate experiments. *p<0.05

that the biological effect exerted by SERM molecules on DTs is mediated only in part by their binding to both ERa and ERp, but is essentially due to a cytotoxic and cytostatic mechanism. The hypothesis to be evaluated is that raloxifene as well as triphenylethyl-enes reduce the synthesis of TGFp1, a potent growth stimulator of mesenchymal cells. Recent studies have shown increased 17pE1 production, 17pE1 mRNA expression and 17pE1 receptor number in desmoid cells in culture compared with normal fibroblasts [43]. This growth factor enhances organic macromol-ecule accumulation in the extra-cellular matrix (ECM) via a reduction of matrix metalloproteinases (MMPs), a family of zinc-dependent neutral endopeptidases which are involved in the degradation of ECM, and an increase in the natural tissue inhibitors of MMPs. The growth of DTs is favoured by a decreased collagen degradation rather than by an increase of collagen synthesis. It has been shown that the presence of 1 ^M toremifene in the cultural medium inhibited 17pE1 activity and consequently reduced collagen accumulation by increasing collagen degradation.

Treatment with SERMs

Anti-oestrogen therapy has been largely based on triphenylethylenes such as tamoxifen or its chlorinated derivate toremifene since its first use by Waddell in 1983 [44]. As for the use of NSAIDs, medical treatment with SERMs is empirical and controversial, being based largely on anecdotal reports and small, poorly controlled studies, most of which are retrospective [45]. The dose commonly used is 30 mg/day which is accompanied by a positive effect in about 50% of the cases. Other authors have employed higher doses of tamoxifen (120-200 mg/day) obtaining a

Fig. 6. Effects of LY117018, an analogue of raloxifene, on cell growth of desmoid cells. Cells were plated in growth medium containing different concentrations of LY117018. After 1 week cell number was evaluated. Results were expressed as % of control values of three separate experiments. *p<0.05

cessation of growth in 63-77% of the patients [42, 47]. However, the best outcome was observed in the group of patients who received high-dose tamoxifen in combination with sulindac 300 mg. Development of ovarian cysts is a frequent side effect of tamoxifen treatment in the female patients. The response to tamoxifen is usually gradual and slow so that the achievement of a partial or complete regression lasts several months or years. If DTs are particularly aggressive with rapid growth, the effect of SERMs could be negative in the first months because the time period required for the action can be prolonged. It is not clear how long the treatment should be once a complete regression has been obtained. The likelihood of accelerated DT growth on the cessation of tamoxifen treatment should justify a prolonged or indefinite treatment [46]. However, the risk of endometrial cancer with 20 mg per day of tamoxifen has been recognized for many years [48, 49]. Conversely, lack of worrying endometrial stimulation seen with triphenylethylenes confers to raloxifene a more favourable profile, particularly in the long-term use of the drug. We studied the effect of 120 mg daily (a dosage double than recommended for prevention of osteoporotic fractures) of raloxifene on progression of DT and of mesenteric fibromatosis by evaluation of lesion size and symptoms in 13 FAP patients. The patients had a significant response to raloxifene therapy with complete remission in five cases and partial remission in five other cases [50]. None of the patients experienced major side-effects and no significant changes in biochemical parameters or endometrial thickness were observed. It is important to bear in mind that raloxifene was efficacious even if previous treatments with tamoxifen and sulindac had failed. Tables 4 and 5 focuses our personal experience respectively on chemoprophylaxis and on drug therapy.

Table 4. Chemoprophylaxis of desmoids: personal experience in familial adenomatous polyposis

Type of lesions

Patients

Drug

Duration, mean range (months)

Length of follow-up mean range (months)

Absence of progression

Regression Recurrence

DPL

20

Tamoxifen/

65 (6-168)

79(10-168)

lSb

2a

raloxifen

Abdominal wall D

lS

Tamoxifen/

59 (2-120)

84 (2-154)

l3c

2

raloxifen

D, Desmoid; DPL, Desmoid precursor lesion

"Intraoperative evaluation for second surgery; bNo evidence at clinical examination or at CT scan; cNo evidence of D at clinical examination or at US/CT scan

D, Desmoid; DPL, Desmoid precursor lesion

"Intraoperative evaluation for second surgery; bNo evidence at clinical examination or at CT scan; cNo evidence of D at clinical examination or at US/CT scan

Table 5. Drug therapy of desmoids: personal experience in familial adenomatous polyposis

Type of desmoids

Patients

(range)

Lenght of follow-up months (range)

Progression

Regression

Stable

Mesentery D

S

Tamoxifen

65 (2-lS6)

57 (2-156)

2a

6

0

Raloxifen

Retroperitoneal

l

Tamoxifen

24

24

0

2

0

fibrosis

l

Raloxifen

4S

192

Abdominal wall D

2

Tamoxifen

l4

14

2a

l

Raloxifen

44

60

l

"Voluntary drop-out in one case

"Voluntary drop-out in one case

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