The Role of Membrane-Initiated Signalling in Progestin-Induced Growth Inhibition in Mammary Epithelial and Breast Cancer Cells

Abstract

Progesteronin synteettistä muotoa progestiinia käytetään laajalti rintasyövän hoidossa, osana hormonikorvaushoitoa ja hormonaalista ehkäisyä. Viimeaikaiset tutkimukset hormonikorvaushoidoista kuitenkin osoittavat, että progestiini lisää rintasyövän riskiä. Tämän johdosta tutkimukset, jotka selvittävät progestiinin tapaa vaikuttaa solujen kasvuun ovat ajankohtaisia.

Tässä työssä tutkittiin progestiinin vaikutusmekanismia käyttäen mallina rintasyöpäsoluja. Havaitsisimme progestiinin säätelevän solukalvon pintareseptorin GPR30:en (G protein-coupled receptor) ilmentymistä lähetti-RNA tasolla. GPR30:en ilmentyminen esti sekä rintasyöpäsolujen että ei-pahanlaatuisten maitorauhassolujen jakautumista. Osoitimme käyttäen ns antisense tekniikkaa, että GPR30:en ilmentyminen oli kriittinen progestiinin kyvylle estää solujen kasvua. Kun GPR30:en lähetti-RNA taso väheni, myös progestiinin vaikutus kasvuun heikkeni. Tutkimuksessamme progestiini esti MAPK (mitogen-activated protein kinase) solun signaalinvälitysjärjestelmän aktivaatiota GPR30 reseptorin kautta. GPR30:lla havaitsimme myös olevan vaikutusta geenien luennan aktiivisuuteen ja tätä säätelevän molekyylin TIF2 cofactorin ilmentymisen määrään.

Työ osoittaa yhteneväisen vaikutusmekanismin solun pinnan reseptorin GPR30:en ja solun tumareseptoriin vaikuttavan progestiinin kesken. Tulokset viittaavat siihen, että progestiinin vaikutus soluihin ainakin osittain välittyisi solun pintareseptorin GPR30:en kautta. Työ tuo esiin uuden mahdollisen vaikutusmekanismin, jolla steroidihormonit säätelevät solujen jakautumista. Löytämällä reseptoriin sitoutuva molekyyli voidaan varmistaa nyt saadut tulokset ja mahdollisesti tätä molekyyliä käyttäen estää solujen jakautumista maitorauhassoluissa välttäen progestiinin kasvua lisäävä vaikutus.

1. Progestins and progesterone enhanced the expression of GPCR mRNA at 24-48 h in different breast cancer cell lines. Other steroid hormones did not upregulate GPCR30 mRNA expression, and the expression was abrogated by anti-progestin RU486 suggesting progestin specificity in the mRNA regulation. Although a number of progestin target genes have been described, the result presented provides an interesting new insight into progestin signalling, since GPCR30 gene has been suggested to be critical for estrogen-mediated MAPK activation.

2. Transient expression of GPCR30 inhibited proliferation of MCF-7 breast cancer cells, and GPCR30 antisense enhanced proliferation. In immortalized HME cells stable expression of GPCR30 inhibited proliferation. We have characterized GPCR30 as a growth inhibitory receptor for the first time. The results are consistent with the evidence that other family members of GPCR30 inhibit growth, particularly the rat homologue of GPCR30.

3. Progestin-induced growth effects: cyclin D1 down-regulation, G1 phase arrest and inhibition of cell proliferation were diminished in MCF-7 cells when GPR30 expression was down-regulated by the antisense, suggesting that GPCR30 is critical for a progestininduced growth effect. Additionally, the growth-inhibitory response by progestin correlated with GPCR30 expression level. In view of numerous contradictory findings on the effect of progestin on cell growth, it is hypothesized that progestin drives cells to a decision point at the G1/S boundary and thereafter induces cellular changes which determine the ultimate response of the cells. Our results thus point to a gene which explains some of the effects of progestin on the cell cycle. It is also possible that GPCR30 and progestin have a cross-talk cell signalling pathways.

4. Progestin inhibited ERK activity through GPCR30 in MCF-7 breast cancer cells. The antisense, cell growth and ERK activation studies suggested that MAPK inactivation is associated with growth inhibition. A role of MAPK activation in progestin-induced growth inhibition has previously been suggested. However, MAPK inactivation by GPR30 or progestin has not previously been established. Our data further imply an important role of the MAPK pathway in progestin signalling in breast cancer cells.

5. Stable expression of GPCR30 in immortalized human mammary epithelial cells downregulated the expression of cofactor TIF2 as verified by RT-PCR analysis and immunoblotting. The regulation of cofactors has been established only in very few studies. Our result provides evidence that this can happen.

6. Stable and transient expression of GPCR30 abolished the ability of glucocorticoid to enhance transcription from the cognate hormone response element of GR, suggesting that GPCR30 interferes with the transcriptional activity of the steroid hormone. GPCR30 has not been reported to regulate the transcriptional activation of steroid hormones. Thus our result provides an interesting new insight into the regulation of steroid receptor activity.

7. Interference in GR activity by GPCR30 was seen to be associated with inhibition of cell proliferation. It has previously been shown that inhibition of ER activity and TIF2 down-regulation leads to growth inhibition. This observation provides further evidence that inhibition of TIF2 expression results in diminution of cell proliferation. It might also suggest a pathway by which GPCR30 affects the cell cycle.