Progesterone receptor membrane component 1 regulates lipid homeostasis and drives oncogenic signaling resulting in breast cancer progression

Background: PGRMC1 (progesterone receptor membrane component 1) is a highly conserved heme binding protein, which is overexpressed especially in hormone receptor-positive breast cancer and plays an important role in breast carcinogenesis. Nevertheless, little is known about the mechanisms by which PGRMC1 drives tumor progression. The aim of our study was to investigate the involvement of PGRMC1 in cholesterol metabolism to detect new mechanisms by which PGRMC1 can increase lipid metabolism and alter cancer-related signaling pathways leading to breast cancer progression. Methods: The effect of PGRMC1 overexpression and silencing on cellular proliferation was examined in vitro and in a xenograft mouse model. Next, we investigated the interaction of PGRMC1 with enzymes involved in the cholesterol synthesis pathway such as CYP51, FDFT1, and SCD1. Further, the impact of PGRMC1 expression on lipid levels and expression of enzymes involved in lipid homeostasis was examined. Additionally, we assessed the role of PGRMC1 in key cancer-related signaling pathways including EGFR/HER2 and ER alpha signaling. Results: Overexpression of PGRMC1 resulted in significantly enhanced proliferation. PGRMC1 interacted with key enzymes of the cholesterol synthesis pathway, alters the expression of proteins, and results in increased lipid levels. PGRMC1 also influenced lipid raft formation leading to altered expression of growth receptors in membranes of breast cancer cells. Analysis of activation of proteins revealed facilitated ER alpha and EGFR activation and downstream signaling dependent on PGRMC1 overexpression in hormone receptor-positive breast cancer cells. Depletion of cholesterol and fatty acids induced by statins reversed this growth benefit. Conclusion: PGRMC1 may mediate proliferation and progression of breast cancer cells potentially by altering lipid metabolism and by activating key oncogenic signaling pathways, such as ER alpha expression and activation, as well as EGFR signaling. Our present study underlines the potential of PGRMC1 as a target for anti-cancer therapy

Targeting breast cancer cells by MRS1477, a positive allosteric modulator of TRPV1 channels

There is convincing epidemiological and experimental evidence that capsaicin, a potent natural transient receptor potential cation channel vanilloid member 1 (TRPV1) agonist, has anticancer activity. However, capsaicin cannot be given systemically in large doses, because of its induction of acute pain and neurological inflammation. MRS1477, a dihydropyridine derivative acts as a positive allosteric modulator of TRPV1, if added together with capsaicin, but is ineffective, if given alone. Addition of MRS1477 evoked Ca2+ signals in MCF7 breast cancer cells, but not in primary breast epithelial cells. This indicates that MCF7 cells not only express functional TRPV1 channels, but also produce endogenous TRPV1 agonists. We investigated the effects of MRS1477 and capsaicin on cell viability, caspase-3 and -9 activities and reactive oxygen species production in MCF7 cells. The fraction of apoptotic cells was increased after 3 days incubation with capsaicin (10 μM) paralleled by increased reactive oxygen species production and caspase activity. These effects were even more pronounced, when cells were incubated with MRS1477 (2 μM) either alone or together with CAPS (10 μM). Capsazepine, a TRPV1 blocker, inhibited both the effect of capsaicin and MRS1477. Whole-cell patch clamp recordings revealed that capsaicin-evoked TRPV1-mediated current density levels were increased after 3 days incubation with MRS1477 (2 μM). However, the tumor growth in MCF7 tumor-bearing immunodeficient mice was not significantly decreased after treatment with MRS1477 (10 mg/ kg body weight, i.p., injection twice a week). In conclusion, in view of a putative in vivo treatment with MRS1477 or similar compounds further optimization is required

Treatment groups.

<p>Treatment groups.</p

Effects of MRS treatment on cell viability of cancer cell lines.

<p><b>A.</b> Cells were treated with MRS at different concentrations for 72 h. At this time point (72 h), MTT assays were performed; each dot represent mean + SD and n = 6 samples (2 independent experiments in triplicates). <b>B.</b> HODE levels in the extracts from different cells (n = 3). <b>C.</b> Cell viability in the presence of different CAPS concentrations in the absence (green curve) and presence of 2 μM MRS (red curve). MTT assays were performed; each dot represent mean + SD and n = 6 samples (2 independent experiments in triplicates) <b>D.</b> Cells were incubated with CAPS (10 μM), CapZ (0.1 mM) or both with and without MRS (2 μM) for 72 h and then MTT assays were performed. The columns represent mean + SD and n = 6 (2 independent experiments in triplicates). The letters denote the following: a—significant difference from control group, b—significant difference from CAPS group, c—significant difference from CapZ group, d—significant difference from CAPS+CapZ group, e—significant difference from MRS group, f—significant difference from MRS+CAPS group and g- significant difference from MRS+CapZ group.</p

Effects of CAPS and MRS treatment on ROS production and mitochondrial membrane depolarization in MCF7 cancer cells (mean + SD, n = 6, 2 independent experiments in triplicates).

<p>Cells were incubated with CAPS (10 μM), CapZ (0.1 mM) or both, with or without MRS (2 μM) for 72 h. Then they were subjected to the DHR 123 and JC-1 assays indicating the levels of ROS production and mitochondrial membrane potential, respectively. The letters denote the following: a—significant difference from control group, b—significant difference from CAPS group, c—significant difference from CapZ group, d—significant difference from CAPS+CapZ group, e—significant difference from MRS group, f—significant difference from MRS+CAPS group and g- significant difference from MRS+CapZ group.</p

<i>In vivo</i> effect of MRS in NSG mice bearing MCF7 cell-derived tumors.

<p><b>A)</b> No significant difference in tumor size was observed between MRS-treated (10 mg/kg body weight (b.w.), i.p., twice a week) and vehicle-treated groups. Student t-tests were used for pairwise comparisons. Bars represent SD. <b>B)</b> Photographs taken from tumors derived from vehicle- and MRS-treated mice. No obvious structural differences were observed at the macroscopic level. Size bar represents 5 mm.</p

Effect of MRS on TRVPV1-mediated Ca²⁺ signaling.

<p><b>A)</b> Schematic model of TRPV1 channel modulation by MRS in cancer cells. Homo-tetrameric TRPV1 is permeable to cations, notably Na⁺ and Ca²⁺. I) In the absence of TRPV1 agonist the channel is closed. II) Binding of a positive allosteric modulator (PAM) alone, e.g. MRS, does not activate channel opening. III) Endogenous TRPV1 agonists present in the tumor microenvironment are weak stimulators of TRPV1, possibly involved in tumor progression. IV) Exogenous agonists such as CAPS are potent TRPV1 activators. Resulting from TRPV1 hyper-activation, CAPS induces oxidative stress. V-VI) MRS amplifying the effect of both endogenous and exogenous agonists may evoke a more pronounced cytotoxic effect (oxidative stress). <b>B-I)</b> Acute effects of MRS on the intracellular Ca²⁺ regulation in various cell types. The different substances were added at the time points indicated by arrows and remained in the solution until the end of the experiments <b>B)</b> Fluorescence recordings from time-lapse videos show an increase in [Ca²⁺]_i after CAPS administration in NIH-3T3 cells stably transfected with the TRPV1 channel. Traces represent mean + SD. More than 20 single cell recordings were evaluated (n > 20). MRS added at t = 5 min evoked a second long-lasting elevation in [Ca²⁺]_i. (gray curve). In cells stimulated by CAPS only, [Ca²⁺]_i continuously decreased after the initial peak at approximately 2 min (orange curve). Statistically significant differences between the two curves (Student t-test, p<0.05) are marked with asterisks. <b>C-I)</b> Single-cell (colored traces) and average fluorescence recordings of the entire cell population (grey traces) from time-lapse videos show changes in [Ca²⁺]_i. Bars represent SD. All experiments were repeated two or more times with similar results. <b>C)</b> NIH-3T3^TRPV1 cells responded to CAPS with an increase in [Ca²⁺]_i, but not to MRS alone. <b>D)</b> Primary breast epithelial cells (prB) responded to CAPS with a brief increase in Ca²⁺]_i often lasting for less than a min. <b>E)</b> Primary breast epithelial cells did not respond to MRS1477 alone, but responded to serum re-administration. <b>F)</b> MCF7 breast cancer cell line responded to CAPS alone and <b>G)</b> to MRS1477 alone with brief Ca²⁺ transients. <b>H)</b> Neither MCF7 cell extracts nor CAPS (50 μM) evoked an elevation in Ca²⁺]_i in control (un-transfected) NIH-3T3 cells; a Ca²⁺ signal was induced by serum re-administration <b>I)</b> Application of a MCF7 cell extract increased [Ca²⁺]_i in NIH-3T3^TRPV1 cells; the Ca²⁺ signal was further increased by CAPS (50 μM). <b>J-K</b>) MRS augmented the endogenous agonist-evoked rises in [Ca²⁺]_i. The initial rise in [Ca²⁺]_i was evoked either by 13-HODE (1 μM) (<b>J</b>) or by 8,9-EET (1μM) (<b>K</b>).</p