New Insight on a Possible Mechanism of Progestogens in Terms of Breast Cancer Risk

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Horm Mol Biol Clin Invest 2011;6(1):185–192 2011 by Walter de Gruyter • Berlin • New York. DOI 10.1515/HMBCI.2010.082

New insight on a possible mechanism of progestogens in terms of breast cancer risk

Hans Neubauer1,a, Rong Chen2,a, Helen Schneck1, gestins that are used for hormone therapy are different in Thomas Knorrp3, Markus F. Templin3, Tanja Fehm1, their proliferative effects on MCF-7 and MCF-7/PGRMC1- Michael A. Cahill4, Harald Seeger1,QiYu2 and 3HA cells. Progesterone appears to act neutrally, whereas Alfred O. Mueck1,* MPA, NET and DRSP trigger proliferation and thus might increase breast cancer risk. The data presented are very 1Department of Obstetrics and Gynecology, University of important in terms of the positive results of progestogens and Tuebingen, Tuebingen, Germany breast cancer risk in clinical studies so far. 2Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Beijing, China Keywords: progestogens; progesterone receptor membrane 3Natural and Medical Sciences Institute at the University component 1; proliferation; signal cascade. of Tuebingen, Reutlingen, Germany 4School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia Introduction Abstract Hormone therapy and oral contraception are still an impor- Objectives: Progestogens influence mammary gland devel- tant risk factor for breast cancer. Evidence is accumulating opment and probably breast cancer tumorigenesis by regu- that progestogens can play a crucial role. Progesterone recep- lating a broad spectrum of physiological processes. We tor membrane component 1 (PGRMC1) is expressed in investigated receptor membrane-initiated actions of proges- breast cancer w1, 2x. It could be important in tumorigenesis togens in MCF-7 breast cancer cells overexpressing proges- and thus could increase breast cancer risk. Little is known terone receptor membrane component 1 (PGRMC1). about downstream signaling pathways w3, 4x. Design: MCF-7 cells were stably transfected with PGRMC1 The role of progestogen addition to estrogen therapy in expression plasmid (MCF-7/PGRMC1-3HA) and overex- postmenopause has come under scrutiny since the results of pression of PGRMC1 was verified by immune fluorescent the Women’s Health Initiative (WHI) mono arm were pub- analysis and Western blot. To test the effects of progestogens lished compared to the WHI combined arm w5, 6x. The WHI on cell proliferation, MCF-7 and MCF-7/PGRMC1-3HA trial used the combination of conjugated equine estrogens cells were stimulated with a membrane-impermeable proges- plus medroxyprogesterone acetate (MPA). In contrast to the terone: BSA-fluorescein-isothiocyanate conjugate (P4-BSA- WHI combined arm, in the estrogen only arm no increase FITC), unconjugated progesterone (P4), medroxyproges- but rather a reduction of breast cancer risk was found, which terone acetate (MPA), norethisterone (NET) and drospire- was significant for patients with more than 80% adherence none (DRSP). Furthermore, reverse phase protein technology to study medication w7x. This result indicates a negative was applied to identify modified downstream signaling. effect of progestogens with regard to breast cancer risk. Results: Progesterone did not elicit any proliferative effect However, it remains unclear whether the combination of on MCF-7/PGRMC1-3HA cells. By contrast, P4-BSA-FITC, estrogens with synthetic progestins and/or natural progester- DRSP, MPA and NET significantly triggered proliferation one can elicit the same increased risk. Thus, many questions of MCF-7/PGRMC1-3HA cells, the effect being more pro- concerning the extrapolation of the WHI results to all syn- nounced for NET. Almost no effect of progestogens on thetic progestogens and to natural progesterone remain proliferation was observed in MCF-7 cells. In MCF-7/ unanswered. Of special note is one cohort study using PGRMC1-3HA cells, expression of Erk1/2 was significantly micronized progesterone in combination with estrogens, reduced by 40% compared to MCF-7 cells. which detected no increase in breast cancer risk when com- Conclusions: Our data indicate that PGRMC1 mediates a bining transdermal (patches) or percutaneous (gels) estradiol progestogen-dependent proliferative signal in MCF-7 cells. therapy with progesterone w8x. In 80,377 women breast can- Of significant interest is that progesterone and synthetic pro- cer risk was increased with oral synthetic progestogens, but not with progesterone and dydrogesterone. The mean dura- a These authors contributed equally. tion of hormone therapy (HT) was 7 years. This study had *Corresponding author: Professor Alfred O. Mueck, MD, a mean follow-up of 8.1 years. PharmD, PhD, University Women’s Hospital, Department of Progestogens are conventionally thought to act via the Endocrinology and Menopause, Head, Center of Women’s Health activation of the intracellularly located progesterone recep- BW, Head, Calwer Strasse 7, D-72076 Tuebingen, Germany E-mail: [email protected] tors (PRs), PR-A and PR-B. Several in vitro studies indicate Received October 15, 2010; accepted December 13, 2010; that progestogens can exert an antiproliferative effect by acti- previously published online March 8, 2011 vation of these receptors in human breast cancer cells w9–11x.

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186 Neubauer et al.: Progestogens and possible mechanism of breast cancer risk

These data are in contrast to the above mentioned clinical selection of stable integration events. Transfection rates were meas- data. Other data suggested a proliferative effect of synthetic ured by cotransfection of a GFP-expressing plasmid and immune progestogens w12, 13x. Thus, the mechanisms by which pro- fluorescence analysis. After 2 weeks single colonies had formed and gestogens act on human breast cells remain unclear. limiting dilutions were performed three times to select for colonies Recent experimental data revealed that in addition to the grown from a single cell. Stable transfection was verified by PCR using chromosomal intracellular-located receptors, PGRMC1 is associated with a w x DNA and primers spanning intron 1 to distinguish integrated membrane-associated progesterone receptor activity 3 . PGRMC1 cDNA from the chromosomal sequence. The sequences PGRMC1 was originally cloned from the endoplasmatic of the primers were 59-CTGCTGCATGAGATTTTCACG-39 hybrid- reticulum from porcine hepatocytes w14x. It contains several izing to nucleotides 71–91 of PGRMC1 open reading frame and 59- predicted motifs for protein interactions and overlapping GCATAGTCCGGGACGTCATA-39 hybridizing to the sequence sites for phosphorylation, for which phosphorylation status coding for the HA tag. PCR products were sequenced. might correlate with its localization in the cell w3, 15, 16x. PGRMC1 has been detected in several cancers and cancer Dissolution of progestins cell lines, e.g., breast cancer w2, 17x. It is overexpressed in lung cancer and colon cancer w3x. Progesterone (P4), MPA, norethisterone (NET) and progesterone-3- There is a long-standing link between PGRMC1 and pro- (O-carboxymethyl) oxime: BSA-fluorescein-isothiocyanate conjugate (P4-BSA-FITC) were purchased from Sigma (Munich, gesterone signaling. However, because bacterially expressed Germany) and drospirenone (DRSP) from Schering (Berlin, Ger- PGRMC1 does not bind to progesterone w18x, and because –4 many). P4-BSA-FITC was dissolved in H2O and stored as 10 M the majority of PGRMC1 is not localized to the plasma at 48C for less than 1 week, whereas the other compounds were membrane w1, 19, 20x it is now tentatively assumed that dissolved in ethanol, stored as 10–2 Mat–208C and further diluted PGRMC1 does not bind P4 by itself w3x, but requires an during experiments to a final ethanol concentration of less than unknown protein that is associated only in partially purified 0.01%. PGRMC1 preparations w21x. PGRMC1-associated progesterone binding is functionally important in cancer cells because Cell proliferation progesterone inhibits apoptosis in granulosa cells, and this antiapoptotic activity requires PGRMC1 w21, 22x. However, All proliferation assays were conducted using stripped medium, i.e., it is unclear how PGRMC1 transduces antiapoptotic signal- phenol red-free medium with charcoal/dextran-treated fetal bovine serum (Thermo, Karlsruhe, Germany). Approximately 10,000 cells ing by progesterone. Expression of PGRMC1 has been iden- were seeded in normal medium per well in 96-well plates. On the tified in several subcellular compartments including cell next day the medium was changed to stripped medium and the cells membrane, cytoplasm, endoplasmatic reticulum and nucleus were incubated at 378C for 2 days. Afterwards, stripped medium (reviewed in w3x). Swiatek-De Lange et al. reported that containing progestogens at concentrations ranging from 10–9 Mto PGRMC1 localizes to the plasma membrane and microsomal 10–5 M was added. Cell proliferation was measured by MTT assay fraction of retinal cells w23x. Based on these data in this after 4 days of incubation as described earlier w17x. In brief, yellow study, we investigated receptor membrane-initiated actions of MTT w3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- progesterone and various synthetic progestins in MCF-7 mide, a tetrazolex was reduced to purple formazan in the mitochon- breast cancer cells overexpressing PGRMC1 as well as the dria of living cells. The absorbance of this colored solution can be influence of the overexpression of PGRMC1 on the activa- quantified by measuring at a certain wavelength (usually between tion of important signaling proteins. 500 and 600 nm) by a spectrophotometer. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable (living) cells. In in-house experiments we have validated the MTT Methods assay against the BrdU assay. For kinetic studies the same cell lines were incubated with all Cell cultures progestogens mentioned above at 10–6 M for 6 days. Medium was changed every 2 days and proliferation was measured daily by the MCF-7, a human estrogen receptor (ER) positive primary breast MTT assay. cancer cell line, was purchased from American Type Culture Col- lection (ATCC). Cells were routinely cultured in RPMI-1640 medi- Reverse phase protein microarrays (RPPMs) um containing 10% (v/v) heat inactivated fetal calf serum, 25 mM

HEPES and 1% penicillin/streptomycin at 378C in a humid 5% CO2 Protein expression profiling was performed using RPPMs and the atmosphere. planar waveguide-based ZeptoMARK platform (Zeptosens, Witters- wil, Switzerland) w24x. For analysis, flash frozen cell pellets were Transfection of MCF-7 cells lysed. Using the NanoPlotter 2 (GeSim, Grosserkmannsdorf, Ger- many) samples (400 pl) were spotted onto ZeptoMARK Protein MCF-7 cells were stably transfected with expression vector pc- Microarray Chips (Zeptosens). On each chip, six identical arrays DNA3.1 containing heme agglutinin-tagged (3HA) PGRMC1 using each containing 80 samples in quadruplicate were generated. After lipofectamine 2000 (Invitrogen, Karlsruhe, Germany), in accor- spotting, the microarrays were blocked with 3% BSA, washed thor- 5 dance with the manufacturer’s recommendation. A total of 5=10 oughly with double distilled H2O, dried in a stream of nitrogen and cells were transfected and plated with RPMI medium for 24 h. Then, stored in the dark at q48C until use. The immobilized protein con- the medium was changed to RPMI complete medium containing tent of a given spot in a microarray was determined by protein 100 mg/mL hygromycin B. Cells were cultured for 2 weeks for staining with a reactive fluorescent dye that binds to primary amines

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Neubauer et al.: Progestogens and possible mechanism of breast cancer risk 187

of the immobilized proteins (Alexa647-NHS-ester; Invitrogen, Immune fluorescence analysis Carlsbad, CA, USA) according to manufacturer’s protocol for label- ing proteins. Detection of proteins was performed using a direct Immune fluorescence analysis was performed as described earlier two-step immunoassay using diluted primary antibody and fluores- w17x. Rabbit anti-HA (1:100) (Santa Cruz) was used as primary cence-labeled anti-species secondary antibodies in assay buffer. antibody and goat anti-rabbit AlexFluor 594 (1:100, Invitrogen, Blank incubations (i.e., assays with no primary antibody) were per- Karlsruhe, Germany) was used as secondary antibody. formed under identical conditions. After washing images of the microarrays were taken using the ZeptoREADER imager (Zepto- Statistical analysis sens) and analyzed using the array analysis software ZeptoVIEW Pro 2.0 (Zeptosens). Signal intensity for each spot was determined All proliferation experiments were done in triplicate and were as background-corrected mean intensity with the local background repeated at least three times, with each experiment yielding essen- subtracted from the spot intensity. Each of the measured values was tially identical results. Statistical analysis was done by analysis of referenced against adjacent reference spots as implemented in the variance with the logarithmized values followed by Dunnett’s pro- software package giving referenced fluorescence intensity (RFI) cedure from triplicates of at least three independent experiments. units that are independent on image exposure time. The overall a level was set at 0.05. Fluorescence signals (RFI) for four replicate spots were averaged and the blank signal determined for these spots was subtracted to correct the assay signals for the contribution of non-specific binding Results of the secondary antibody, yielding blank-corrected MFI signals. In a final step, the signal intensity was normalized against the protein Transfection of MCF-7 cells with PGRMC1 coding content in the spot by using the signal intensities from the on-chip expression plasmid protein determination. Therefore, the calculated signal intensities correspond to normalized, blank-corrected MFI values (NFIs). Stan- Transfection rates measured by cotransfection of a GFP- dard deviations were calculated according to standard error propa- expressing plasmid were around 40%–50% (data not shown). gation rules from the standard deviations of raw, blank and protein After subcloning by limiting dilution technology, PGRMC1- stain signals. NFI values were used for all subsequent statistical 3=HA transfected MCF-7 cells were prepared for cytospins analysis. and immune fluorescence analysis was performed using anti- HA antibody indicating that the transfected cell line was Western blot analysis almost devoid of non-transfected MCF-7 cells (Figure 1A). Western blot analysis for the PGRMC1-3HA fusion pro- Cells were washed twice with ice-cold phosphate buffered saline tein resulted in a single band at approximately 30 kDa, which (PBS) and lysed in M-PER mammalian protein extraction reagent is the predicted size of 28 kDa for PGRMC1 plus approxi- containing Halt Protease Inhibitor Cocktail according to the manu- mately 3 kDa for the three HA tags (Figure 1B). The untrans- facturer’s protocol (both from Pierce, Rockford, IL, USA). Protein fected MCF-7 cells produce only a very faint signal at concentrations were determined using the BCA Protein Assay Kit 28 kDa indicating a weak intrinsic PGRMC1 expression. (Pierce). In total, 25 mg of protein extract was loaded per lane onto a 10% polyacrylamide gel and separated by electrophoresis. The gel Preincubation of the PGRMC1-specific antibody with syn- was blotted onto a Hybond ECL nitrocellulose membrane (Amers- thetic PGRMC1 protein abolished both the exogenously and ham, Piscataway, NJ, USA) at 15 V for 90 min using a semi-dry the endogenously PGRMC1 signal, confirming the specific- blot system. The membrane was blocked for 2 h at room tempera- ity of the antibody (Figure 1B). Similar signal intensities for ture using 5% dried low fat milk powder dissolved in TBST buffer the housekeeping protein actin (approximately 42 kDa) indi- (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). Then, cate loading of equal amounts of total protein. PCR products the first antibody was incubated overnight at 48C. After washing for PGRMC1 from genomic DNA of the transfected cell three times with TBST, the second antibody was incubated for 2.5 h lines provided the expected signal in the agarose gel (data at room temperature. Chemiluminescence was generated using the not shown). Sequencing of PCR products confirmed the ECL Western Blotting Analysis System (Amersham). The signals PGRMC1 wild-type sequence. At the end of the experiments were measured and quantified with a Lumi-Imager and LumiAnalyst the high purity of the MFC-7/PGRMC1-3HA cells was val- 3.1 software (Boehringer, Mannheim, Germany). Western blot anal- idated by immune fluorescence analysis of cytospins and ysis was used to measure PGRMC1 and Erk1/2 expression. The respective first antibodies were: rabbit PGRMC1 antibody (G21, sc- by sequencing PCR products. These results indicated that 133906, 1:200, Santa Cruz, CA, USA) and rabbit Erk1/2 polyclonal MFC-7/PGRMC1-3HA cells are highly pure, overexpress antibody (1:1000) (Cell Signaling Technology, Danvers, MA, USA). PGRMC1 wild-type protein and can be used for functional Goat anti-rabbit horseradish peroxidase (HRP, 1:1000, Santa Cruz) assays. was used as secondary antibody. Exploration of signal proteins

Preparation of cytospins To explore signal proteins regulated by PGRMC1 expression or proteins potentially involved in downstream PGRMC1 For preparation of cytospins, 5=104 cells were resuspended in 500 mL PBS spun onto slides using Cytospin 2 centrifuge (Shan- signaling, reverse phase protein array technology was don, Waltham, MA, USA) at 1000 rpm (130=g) for 5 min. Then, applied. With that purpose, MCF-7 and MCF-7/PGRMC1- the cytospins were dried overnight at room temperature and stored 3HA cells were stimulated with P4, P4-BSA-FITC, MPA and at –208C until further use. Cytospins were prepared as described NET and the expression levels of 15 signal transduction pro- earlier w17x. teins were determined: Akt, Bad, Bax, c-Fos, c-Jun, Caspase

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188 Neubauer et al.: Progestogens and possible mechanism of breast cancer risk

w26x) and based on its potential involvement in apoptosis and proliferation. Stimulation did not change the expression levels of these proteins in four replicate experiments. Only the basal expression, i.e., without stimulation, of Erk1/2 protein was significantly downregulated by approximately 40% in MCF-7/ PGRMC1-3HA cells compared to MCF-7 cells (Figure 2A). This result was confirmed by Western blot analysis using independent cell lysates (Figure 2B).

Synthetic progestins increase proliferation of PGRMC1 overexpressing MCF-7 cells

Because Erk1/2 are members of the mitogen-activated protein kinase superfamily that can mediate cell proliferation, we first investigated potential differences in the basal proliferation of MCF-7 and MCF-7/PGRMC1-3HA cells. The latter showed a significant higher proliferation after day 7 Figure 1 Overexpression of PGRMC1 in MCF-7 cells. (Figure 3A). Dose-dependent effects on cell proliferation of Immunofluorescence analysis of cytospin preparations from MCF- P4, P4-BSA-FITC, MPA, NET or DRSP were then deter- 7 (left) and MCF-7/PGRMC1-3HA (right) cells. PGRMC1-3-HA is mined using the MTT assay (Figure 3B). Between 10–9 M detected with anti-HA antibody conjugated with Alexa 594. Cell –5 nuclei are stained with DAPI. Magnification: 100=, size bar: and 10 M P4 did not increase proliferation of either MCF- 10 mm. Western blot analysis of lysates prepared from MCF-7 and 7 or MCF-7/PGRMC1-3HA cells. However, proliferation of MCF-7/PGRMC1-3HA cells. PGRMC1-3HA is detected with a MCF-7/PGRMC1-3HA cells was significantly increased polyclonal rabbit anti-PGRMC1 antibody followed by anti-rabbit when treated with P4-BSA-FITC or the synthetic progesto- horseradish peroxidase antibody. Signals are visualized with chem- gens: for P4-BSA-FITC at concentrations from 10–7 Mto iluminescence. (Left panel) PGRMC1-3HA is detected at approxi- 10–5 M with a maximal effect at 10–6 M, for NET reaching mately 30 kDa. A faint signal for the endogenously expressed its maximal effect compared to untreated control at 10–7 M, PGRMC1 is visible in lysates from MCF-7 and MCF-7/PGRMC1- for MPA at concentrations higher than 10–6 M, and for DRSP 3HA cells. In the right panel no signals are detected. Here, the at concentrations higher than 10–7 M. The effect of NET was primary antibody was preincubated with recombinant PGRMC1. significantly different to that of DRSP at the concentrations (Upper panels) The same lysates were incubated with an actin spe- –9 –8 cific antibody for loading control. of 10 and 10 M and to the effect of MPA at the concentrations of 10–9,10–8 and 10–7 M. DRPS showed a significant stronger effect compared to MPA at the concentration 3, Cyclin D1, Cyclin E2, Erk1/2, IkB, Ki67, mTOR, PCNA, of 10–7 M. PI3K and PKC. The clinically relevant blood concentrations for the pro- Because there are only scarce data about potential down- gestogens most commonly used for HT, MPA and NET are stream signaling pathways, proteins were selected based on in the range of 4=10–9 Mto10–8 M for MPA w27x and published data (IkB, Akt w25x, PI3K, PKC, Erk1/2 w23x, Bad around 10–8 M for NET w28x. However, higher concentrations might be required in vitro in short-time tests in which the reaction threshold can only be achieved with supraphys- iological dosages. Higher concentrations can also be reached in vivo in the vessel wall or organs compared to the concentrations usually measured in the blood. No effects were observed in MCF-7 cells within the investigated concentration ranges for all the progestogens used in this experiment. For further kinetic experiments, 10–6 M was chosen for all progestogens. In comparison to all other synthetic progestogens tested, NET significantly increased proliferation almost to maximum even at 10–9 M, the lowest concentration that we tested. Taken together, the results strongly suggested that some progestogens elicit a PGRMC1- Figure 2 MCF-7/PGRMC1 cells express lower levels of Erk1/2. dependent proliferative response. Reverse phase analysis of lysates from MCF-7 and MCF-7/ To determine time-dependent proliferative effects of pro- PGRMC1-3HA cells. In lysates from 31 MCF-7 cells and 31 MCF- 7/PGRMC1-3HA cells total Erk1/2 protein expression was reduced gestogens, a kinetic analysis over 6 days was performed by approximately 40%. Western blot analysis of lysates from MCF- (Figure 3C). MCF-7 and MCF-7/PGRMC1-3HA cells were 7 and MCF-7/PGRMC1-3HA cells. Expression of Erk1/2 is reduced incubated with P4, P4-BSA-FITC, DRSP, MPA and NET at by approximately 43% in MCF-7/PGRMC1-3HA cells. Actin sig- 10–6 M and proliferation was determined by MTT assay. The nals indicate equal loading. results indicate that P4-BSA-FITC, DRSP, MPA and NET

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Neubauer et al.: Progestogens and possible mechanism of breast cancer risk 189

Discussion

Hormones, acting through their receptors, drive the proliferation of several tumor types, including breast, ovarian and prostate tumors. Progesterone-regulated responses are mediated by an array of progesterone receptors that include the classic nuclear PR-A and PR-B receptors and splice variants of each. During the past years, evidence has accumulated that at least three PR groups exist which could be potentially involved in membrane-initialized progesterone signaling: (a) intracellular PRs localizing at the plasma membrane, (b) PGRMC1, and (c) members of the newly described family of the G-protein-coupled seven transmembrane domain 7TMPRb membrane progestin receptors, with the latter recently questioned whether these are really progestin receptors w29x. As demonstrated w30x, 5% of classical PR-A in Xenopus oocytes binds to the plasma membrane where it associates with Erk2 to induce PI3K signaling. This observation was supported by reports of membrane localization of Xenopus PR-A in transfected Cos-7 cells w25x. However, to date, association of PR-A with plasma membranes has only been demonstrated in reproductive tissues. Membrane-associated PRs such as PGRMC1 induce gene expression by signaling cascades that originate at the cell membrane and ultimately activate transcription factors. PGRMC1 contains a cytochrome b5 domain fold and belongs to the so-called membrane-associated progesterone receptor protein family that is widespread in eukaryotes w3, 31x. PGRMC1 is overexpressed in breast tumors and in cancer cell lines from the colon, thyroid, lung and cervix w1x. Recently, we have demonstrated that PGRMC1 is present in human breast cancer tissues w2, 17x. PGRMC1 is also present in ovarian cancer tissues, whereby in advanced stages a higher expression of PGRMC1 is observed compared to the classic progesterone receptors A and B w20x.

Figure 3 (A) Basal proliferation (extinction at 550 nm) of MCF- Signal transduction of PGRMC1 7 and MCF-7/PGRMC1-3HA cells after day 1 (D1), day 3 (D3) and day 7 (D7). The difference after D7 was statistically significant There are limited data concerning PGRMC1-activated sig- (p-0.05) (means"SD). (B) Titration of progesterone and synthetic naling pathways. PGRMC1 plays a role in regulating protein progestogens. MCF-7 and MCF-7/PGRMC1-3HA (WT12) cells kinase-associated signaling in which PGRMC1 increases Akt were incubated with progesterone (P4), P4-BSA-FITC, DRSP, MPA activation and IkB phosphorylation leading to NFkB acti- and NET from 10–5 Mto10–9 M in 10-fold dilution steps. Cell vation w32x. Akt is phosphorylated by the PDK1 protein proliferation was measured after 4 days. Data were normalized to kinase, and there is a putative PDK1 binding region on " unstimulated controls (means SD; **p-0.01 vs. controls). (C) PGRMC1 w3x. The exact mechanism through which Kinetic analysis of proliferation. MCF-7 and MCF-7/PGRMC1- PGRMC1 activates Akt is unknown. Because PGRMC1 has 3HA (WT12) cells were incubated with progesterone (P4), P4-BSA- several potential binding sites for interacting proteins it FITC, DRSP, MPA and NET at 10–6 M. Cell proliferation was might act as a type of adaptor protein, providing docking measured daily for 6 days (D1–D6). Data were normalized to unstimulated controls (means"SD; *p-0.05; **p-0.01 vs. controls). sites for proteins that activate Akt, such as PDK1. Membrane-impermeable progesterone conjugate induces calcium influx and subsequent phosphatidylinositol-3-kinase- increased proliferation in MCF-7/PGRMC1-3HA by approx- mediated phosphorylation of PKC and Erk1/2 w23x.A imately 3.5- to 4-fold on day 6 which is highly significant putative Erk2 consensus site at T177 in PGRMC1 w3x has compared to the simultaneously cultured untreated control been demonstrated to be phosphorylated in vivo w33, 34x. cells. No effects on proliferation were observed for P4, Erk2 is induced by PGRMC1 and could therefore be DRSP, MPA and NET in MCF-7 cells. Only the membrane- involved in feedback inhibition of signaling through the impermeable P4-BSA-FITC caused a marginal increase of adjacent putative SH2 protein interaction motif centered on proliferation in the parental MCF-7 cells by approximately the amino acids Y190 and S180. S180 appears to be consti- 1.5-fold compared to the control cells. tutively phosphorylated in non-stimulated MCF-7 cells w18x

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190 Neubauer et al.: Progestogens and possible mechanism of breast cancer risk

and is presumed to require the activation of a phosphatase molar doses, which have been used in these experiments for the interaction of putative SH2 domain proteins with w35x. Given that the dissociation constant of the progestins Y179 w2x. Unlike PGRMC1 phosphorylated at Serine 180, for the PR-A/-B is 1–5 nM w36x and for PGRMC1 is in the tyrosine phosphorylated PGRMC1 is not located in the endo- 0.20–0.3 mM range w14x, which is well within the levels of plasmic reticulum w3, 16x. P4 in serum and in follicular fluid w37x, in MCF-7 cells the The mechanisms of the proliferative effect observed in classical PR-A/-B receptors are perhaps activated preferen- MCF-7/PGRMC1-3HA cells are currently unknown. Reverse tially by gestagens inducing an antiproliferative signal. This protein array technology revealed that basal Erk1/2 levels concept is supported by a previous observation that at micro- appear to be reduced by approximately 40% in MCF-7/ molar doses P4 inhibits granulosa cell and spontaneously PGRMC1-3HA cells compared to MCF-7 cells. Stimulation immortalized granulosa cell mitosis w38x. of cells with progestogens does not alter Erk1/2 expression. Interestingly, NET exerts its activity on proliferation In rat neural progenitor cells, Liu et al. found that P4 induced already at the lowest concentration tested (10–9 M, Figure a PGRMC1-dependent cell proliferation which required the 3B), whereas DRSP and MPA increase proliferation only at Erk signaling pathway w34x. higher concentrations (10–7 M and 10–6 M). This suggests that NET binds PGRMC1 with the highest affinity, followed Synthetic progestins increase proliferation of by DRSP and MPA. Compared to PR-A/-B this is different PGRMC1 overexpressing MCF-7 cells because the latter binds MPA better than NET w39x. These results indicate that HT including NET might result in an Since the results of the WHI mono arm were published, indi- increased risk for breast cancer development. Indeed, some cating a negative effect of progestins on breast cancer risk, studies in which norethisterone- or levonorgestrel-derived the molecular pathway responsible for this effect and the progestogens were continuously administered a significantly many questions on the extrapolation of the WHI results to higher risk for breast cancer was observed than for contin- all synthetic progestins and to natural progesterone remain uously administered progesterone-derived progestogens w40, unknown. Here, we present the first results suggesting that 41x. In one study, the use of norethisterone acetate was signaling of synthetic progestins via PGRMC1 could be one accompanied with a higher risk after 5 years of use (2.03, explanation. We have chosen two synthetic progestins that 1.88–2.18) than that of medroxyprogesterone acetate (1.64, are widely used in hormone therapy, i.e., MPA and NET, as 1.49–1.79) w41x. It is known that NET can be converted in well as a new synthetic progestin, i.e., DRSP, which might vivo into ethinylestradiol w42x. With regard to this conversion differ in its behavior to MPA and NET because of a different influencing the observed NET effect is currently unknown chemical structure. and is under investigation. The synthetic progestins MPA, NET and DRSP signifi- Serum concentrations of progesterone during HT are in cantly induced a relatively large proliferative effect in MCF- the range of 10–8 Mto10–7 M, i.e., concentrations which 7 cells that overexpress PGRMC1. For P4, however, no such do in fact have an effect in our experiments. However, exper- effect was found. Because progesterone and the synthetic imental investigations can only point to mechanism(s) and progestins used in HT are able to activate PR-A/-B and clearly cannot replace clinical studies. In agreement with our PGRMC1 simultaneously, our data suggest that in vivo the results, NET alone at 10–6 M has been found to stimulate balance of the expression levels of both receptors might the growth of ER positive MCF-7 and T47DA18 human influence whether epithelial cells proliferate or not in the breast cancer cells, but not ER negative MDA-MB-231, BT- presence of progestogens. Therefore, it might be instructive 20 and T47DC4 human breast cancer cells w43x. Varying to determine the expression ratio of PGRMC1 and PR-A/-B results regarding the effects of MPA on breast cancer cells before HT. have been published, but it seems to induce a modest and Interestingly, P4-BSA-FITC is able to induce a marginal statistically significant cell growth at 10–6 M in a specific proliferative signal in MCF-7 cells (Figure 3B). P4-BSA- subgroup of MCF-7 cells w8x. FITC is thought to be unable to cross the plasma membrane and can therefore only bind to membrane associated progesterone receptors. MCF-7 cells express endogenous PGRMC1 at very low amounts (Figure 1) which can transduce the Conclusion weak proliferative signal because the classical PR-A/-B response is not triggered. The synthetic progestins and P4 The effect of progestins on breast cancer tumorigenesis can bind to all progesterone receptors expressed by MCF-7 cells. depend on the specific progestin used for hormone therapy Binding to PR-A/-B might transduce an antiproliferative sig- and the expression of PGRMC1, PR-A and PR-B in the tar- nal, countermanding the proliferative signal induced by low get tissue. However, in terms of the clinical situation it levels of PGRMC1. By contrast, in MCF-7/PGRMC1-3 HA remains unknown how uniformly PGRMC1 is expressed in cells the exogenously expressed PGRMC1 might overrule the normal breast epithelial cells between patients. Thus, the antiproliferative effect of PR-A/-B. In several human screening, which might be based on determining the expres- ovarian surface epithelial cell lines, P4 inhibits their prolif- sion ratio of PGRMC1 and PR in cells from nipple aspirate eration w35x. Because these cells express PR-A/-B it has been fluid, might be of interest to identify women who show an assumed that the actions of P4 are mediated via these recep- increased expression of PGRMC1 and who might thus be tors. However, P4 exhibits antimitotic action only at micro- susceptible for breast cancer development under HT w44x.

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Neubauer et al.: Progestogens and possible mechanism of breast cancer risk 191

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