Helen Schneck

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients

The frequently altered phosphatidylinositol‐3‐kinase (PI3K)/Akt signaling pathway is involved in the regulation of cellular processes required for breast carcinogenesis. The aim of the project was to develop a method to identify hotspot mutations in the PIK3CA gene in circulating tumor cells (CTCs) of metastatic breast cancer (metBC) patients.

Progestogens and membrane-initiated effects on the proliferation of human breast cancer cells

ABSTRACT Objectives Evidence is accumulating that progestogens may play a crucial role in the development of breast cancer under contraception and hormone therapy in reproductive and menopausal women. Progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer may be important in tumorigenesis and thus may increase breast cancer risk. The aim of this project was to investigate the influence of progesterone and nine synthetic progestins on MCF-7 breast cancer cells overexpressing PGRMC1. Methods MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12). To test the effects of progestogerone (P) and the synthetic progestins chlormadinone acetate (CMA), desogestrel (DSG), drospirenone (DRSP), dydrogesterone (DYD), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), nomegestrol (NOM) and norethisterone (NET) on cell proliferation, MCF-7 and WT-12 cells were stimulated with different concentrations (0.01–1 µmol/l). Results In MCF-7 cells, DRSP, DSG, DYD, LNG and NET increased the proliferation at 1 µmol/l, the effect being highest for NET with about 20%. In WT-12 cells, the same progestins, but additionally MPA, showed a significant increase, which was much higher (30–245%) than in MCF-7 cells. Here again, NET showed the highest proliferative effect. No effect was found for CMA, NOM and P. Conclusion Some synthetic progestins trigger a proliferative response of PGRMC1-overexpressed MCF-7 cancer cells. The effect of progestogens on breast cancer tumorigenesis may clearly depend on the specific pharmacology of the various synthetic progestins.

Nomegestrol acetate sequentially or continuously combined to estradiol did not negatively affect membrane-receptor associated progestogenic effects in human breast cancer cells

Objectives: Recently the first monophasic contraceptive pill containing estradiol has been developed which is thought to be a milestone in contraception. Nomegestrol acetate (NOM) is the progestogenic component. Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in the tissue of breast cancer patients, and can predict a progestogen dependent risk of breast cancer. Methods: MCF-7 cells were transfected with PGRMC1 expression plasmid, and were stimulated with estradiol (E2, 10−12 and 10−10 M). NOM, progesterone (P), medroxyprogesterone acetate (MPA) and norethisterone (NET) (each 10−7 M) were added sequentially or continuously. Results: E2 at 10−10 M elicited a significant increase of cell proliferation from 150 to 200%. No effect was seen at 10−12 M. Addition of the progestogens to E2 at 10−10 M had no significant effect. However, at an E2 10−12 M, NET significantly stimulated cell proliferation more pronounced in the continuous combined model. No effect was seen for NOM, P and MPA. The E2/NET combined effect could be abrogated by the addition of an estrogen receptor (ER) antagonist. Conclusion: Since NOM did not increase proliferation it may be concluded that it will be neutral in terms of breast cancer risk when combined with E2 at least in women overexpressing PGRMC1.

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

Abstract Objectives: Progestogens influence mammary gland development and probably breast cancer tumorigenesis by regulating a broad spectrum of physiological processes. We investigated receptor membrane-initiated actions of progestogens in MCF-7 breast cancer cells overexpressing progesterone receptor membrane component 1 (PGRMC1). Design: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (MCF-7/PGRMC1-3HA) and overexpression of PGRMC1 was verified by immune fluorescent analysis and Western blot. To test the effects of progestogens on cell proliferation, MCF-7 and MCF-7/PGRMC1-3HA cells were stimulated with a membrane-impermeable progesterone: BSA-fluorescein-isothiocyanate conjugate (P4-BSA-FITC), unconjugated progesterone (P4), medroxyprogesterone acetate (MPA), norethisterone (NET) and drospirenone (DRSP). Furthermore, reverse phase protein technology was applied to identify modified downstream signaling. Results: Progesterone did not elicit any proliferative effect on MCF-7/PGRMC1-3HA cells. By contrast, P4-BSA-FITC, DRSP, MPA and NET significantly triggered proliferation of MCF-7/PGRMC1-3HA cells, the effect being more pronounced for NET. Almost no effect of progestogens on proliferation was observed in MCF-7 cells. In MCF-7/PGRMC1-3HA cells, expression of Erk1/2 was significantly reduced by 40% compared to MCF-7 cells. Conclusions: Our data indicate that PGRMC1 mediates a progestogen-dependent proliferative signal in MCF-7 cells. Of significant interest is that progesterone and synthetic progestins that are used for hormone therapy are different in their proliferative effects on MCF-7 and MCF-7/PGRMC1-3HA cells. Progesterone appears to act neutrally, whereas MPA, NET and DRSP trigger proliferation and thus might increase breast cancer risk. The data presented are very important in terms of the positive results of progestogens and breast cancer risk in clinical studies so far.

Membrane-receptor initiated proliferative effects of dienogest in human breast cancer cells

Objectives: Dienogest (DNG) is already used in hormone therapy, since recently being also the progestogenic component of the first estradiol based contraceptive pill. Data on breast cancer risk are currently not available. Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in tissues of breast cancer patients and has already been proposed as a predictor for breast cancer risk. Methods: MCF-7 cells overexpressing PGRMC1 were stimulated with DNG, medroxyprogesterone acetate (MPA), norethisterone (NET) and progesterone (P) as well as sequentially and continuously combined with estradiol (E2). Results: DNG and MPA alone elicited a significant proliferation at 10–6 and 10–5 M. NET increased cell proliferation at all concentrations tested whereas P showed no effect. E2 alone elicited a significant increase at 10−10 M, no effect was seen at 10–12 M. Addition of the progestins (10–6 M) to E2 at 10−10 M had, compared to E2 only, no additional proliferating effect. However, at the low E2 concentration, DNG, MPA and NET significantly increased the E2-stimulated cell proliferation. Conclusion: DNG increased proliferation alone and in combination with low E2 concentrations. Thus a progestogen-derived breast cancer risk in the presence of low E2 concentrations cannot be excluded at least in women overexpressing PGRMC1.

Circulating Tumor Cells in Early-Stage Breast Cancer

Disseminated tumor cells (DTC) are routinely detected in bone marrow (BM) in 30-40 % of primary breast cancer patients. Positive BM status at the time of diagnosis as well as DTC persistence after therapy are strong independent prognostic factors. Since repeated BM aspirations are not well tolerated, detection of single tumor cells in peripheral blood (circulating tumor cells; CTC) have become of interest in recent years. CTC are found in 10-80 % breast cancer patients. Variability can be explained by stage of the disease and detection method. Emerging data have shown CTC to be of prognostic relevance for both, patients with primary and metastatic disease. The assessment of CTC in blood may become an important biomarker for prognostication and therapy monitoring. Determination of their molecular characteristics will enable specific targeting of minimal residual as well as metastatic disease. This review summarizes recent research and future perspectives.

Circulating Tumor Cells

Breast cancer (BC) therapy has fundamentally progressed in the last 30 years with the change from radical mastectomy to recent individualized local and systemic therapy regimens. By combining the modern treatment modalities, approximately 77 % of BC patients can be cured, still leaving potential for optimization in 23 % of cases, which will develop metastatic disease due to tumor cell dissemination despite optimal treatment. It has been known since the 19th century that most of the solid cancers shed circulating tumor cells (CTCs) into the blood circulation already at a very early stage. Based on this observation, CTCs are a surrogate marker for minimal residual disease (MRD) and precursors of metastatic disease (“seed”). Current research indicates that the phenotype and genotype differ between CTCs and primary tumor, which may result in different therapeutic responses. Therefore, characterization of CTCs may be an important step for the optimization of adjuvant and metastatic systemic treatment.