Progesterone Receptor Membrane Component 1 Overexpression Research Articles

PGRMC1 promotes NSCLC stemness phenotypes by disrupting TRIM56-mediated ubiquitination of AHR

Cancer stem cells (CSCs) are responsible for tumor chemoresistance, and the aryl hydrocarbon receptor (AHR) is indispensable for maintaining CSC characteristics. Here, we aimed to investigate how the interaction between progesterone receptor membrane component 1 (PGRMC1) and AHR contributes to the maintenance of CSC phenotypes in non-small cell lung cancer (NSCLC). Clinical data and tissue microarray analyses indicated that patients with elevated PGRMC1 expression had poorer prognoses. Moreover, PGRMC1 overexpression enhanced CSC phenotypes and chemotherapy resistance in vitro and in vivo by modulating AHR ubiquitination. We then determined the specific interaction sites between PGRMC1 and AHR. Mass spectrometry screening identified tripartite motif containing 56 (TRIM56) as the E3 ligase targeting AHR. Notably, PGRMC1 overexpression inhibited the interaction between TRIM56 and AHR. Overall, our study revealed a regulatory mechanism that involves PGRMC1, AHR, and TRIM56, providing insights for developing CSC-targeting strategies in NSCLC treatment.

Progesterone receptor membrane component 1 facilitates Ca2+ signal amplification between endosomes and the endoplasmic reticulum

Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca2+ signaling, where local Ca2+ released via endolysosomal ion channels is amplified by ER Ca2+-sensitive Ca2+ channels into global Ca2+ signals. Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca2+ crosstalk between endosomes and the ER remain poorly defined. Here, we identify progesterone receptor membrane component 1 (PGRMC1), an ER transmembrane protein that undergoes a unique heme-dependent dimerization, as an interactor of the endosomal two pore channel, TPC1. NAADP-dependent Ca2+ signals were potentiated by PGRMC1 overexpression through enhanced functional coupling between endosomal and ER Ca2+ stores and inhibited upon PGRMC1 knockdown. Point mutants in PGMRC1 or pharmacological manipulations that reduced its interaction with TPC1 were without effect. PGRMC1 therefore serves as a TPC1 interactor that regulates ER-endosomal coupling with functional implications for cellular Ca2+ dynamics and potentially the distribution of heme.

Insight into mitochondrial dysfunction mediated by clozapine-induced inhibition of PGRMC1 in PC12 cells

Clozapine is usually considered as the last resort for treatment-resistant schizophrenia (TRS). However, it shows limited efficacy in cognition improvement. Moreover, the metabolic side effects induced by clozapine can aggravate cognitive impairment, which is closely related to its neurotoxicity. Nevertheless, the mechanisms underlying clozapine’s neurotoxicity remain largely elusive. In this study, PC12 cells were simultaneously treated with different concentrations (0 μM, 10 μM, 20 μM, 40 μM and 80 μM) of clozapine and AG205 which functions as a blocking reagent of progesterone receptor membrane component 1 (PGRMC1). In addition, we examined the effect of PGRMC1 in clozapine-induced neurotoxicity through overexpressing or downregulating PGRMC1. Molecular docking and surface plasmon resonance (SPR) analysis indicated that clozapine and AG205 inhibited the binding of endogenous progesterone to PGRMC1. The results showed that high concentration of clozapine and AG205 induced a significant increase in cytotoxicity, reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) collapse, all of which were worsened as concentration increases, while overexpression of PGRMC1 reverted the above toxic effect of clozapine on PC12 cells. Furthermore, clozapine and AG205 also downregulated the expression of PGRMC1, glucagon-like peptide-1 receptor (GLP-1R) and mitofusin2 (Mfn2). Interestingly, overexpression of PGRMC1 could revert these effects. Our data suggest that overexpression of PGRMC1 in PC12 cells prevents and restores clozapine-induced oxidative and mitochondrial damage. We propose PGRMC1 activation as a promising therapeutic strategy for clozapine-induced neurotoxicity to facilitate the relief of neuronal damage.

PGRMC1 promotes triple-negative breast cancer cell growth via suppressing ferroptosis

Objective Triple-negative breast cancer (TNBC) is the most malignant form of breast cancer with increasing incidence and mortality worldwide. The progesterone receptor membrane component-1 (PGRMC1) is a well-identified hormone receptor with unknown functions in TNBC. The current study aims to explore the involvement of PGRMC1 in regulation of glutathione metabolism and ferroptosis during development of TNBC, providing new therapy options for TNBC patients. Methods Bioinformatic analysis, cell proliferation assay, western blot assay and other biochemistry methods were performed in TNBC cells. Results Our results revealed that the expression of PGRMC1 is higher in TNBC than the other subtypes of breast cancer. Interestingly, as an iron binding protein, increased PGRMC1 expression in TNBC cells leads to resistance to ferroptosis inducer. On the contrary, silenced PGRMC1 expression enhanced sensitivity of MDA-MB231 cells to Erastin. Mechanistically, overexpression of PGRMC1 decreased the intracellular free iron concentration, which was reduced by AG205 treatment. Conclusions PGRMC1 increases the possibility of TNBC development through binding to intracellular iron and suppressing ferroptosis, providing the molecular basis of combined treatment for TNBC.

Ultrasound-triggered microbubble destruction enhances the radiosensitivity of glioblastoma by inhibiting PGRMC1-mediated autophagy in vitro and in vivo

BackgroundUltrasound-triggered microbubble destruction (UTMD) is a widely used noninvasive technology in both military and civilian medicine, which could enhance radiosensitivity of various tumors. However, little information is available regarding the effects of UTMD on radiotherapy for glioblastoma or the underlying mechanism. This study aimed to delineate the effect of UTMD on the radiosensitivity of glioblastoma and the potential involvement of autophagy.MethodsGL261, U251 cells and orthotopic glioblastoma-bearing mice were treated with ionizing radiation (IR) or IR plus UTMD. Autophagy was observed by confocal microscopy and transmission electron microscopy. Western blotting and immunofluorescence analysis were used to detect progesterone receptor membrane component 1 (PGRMC1), light chain 3 beta 2 (LC3B2) and sequestosome 1 (SQSTM1/p62) levels. Lentiviral vectors or siRNAs transfection, and fluorescent probes staining were used to explore the underlying mechanism.ResultsUTMD enhanced the radiosensitivity of glioblastoma in vitro and in vivo (P < 0.01). UTMD inhibited autophagic flux by disrupting autophagosome-lysosome fusion without impairing lysosomal function or autophagosome synthesis in IR-treated glioblastoma cells. Suppression of autophagy by 3-methyladenine, bafilomycin A1 or ATG5 siRNA had no significant effect on UTMD-induced radiosensitization in glioblastoma cells (P < 0.05). Similar results were found when autophagy was induced by rapamycin or ATG5 overexpression (P > 0.05). Furthermore, UTMD inhibited PGRMC1 expression and binding with LC3B2 in IR-exposed glioblastoma cells (P < 0.01). PGRMC1 inhibitor AG-205 or PGRMC1 siRNA pretreatment enhanced UTMD-induced LC3B2 and p62 accumulation in IR-exposed glioblastoma cells, thereby promoting UTMD-mediated radiosensitization (P < 0.05). Moreover, PGRMC1 overexpression abolished UTMD-caused blockade of autophagic degradation, subsequently inhibiting UTMD-induced radiosensitization of glioblastoma cells. Finally, compared with IR plus UTMD group, PGRMC1 overexpression significantly increased tumor size [(3.8 ± 1.1) mm2 vs. (8.0 ± 1.9) mm2, P < 0.05] and decreased survival time [(67.2 ± 2.6) d vs. (40.0 ± 1.2) d, P = 0.0026] in glioblastoma-bearing mice.ConclusionUTMD enhanced the radiosensitivity of glioblastoma partially by disrupting PGRMC1-mediated autophagy.

Progesterone Receptor Membrane Component-1 May Promote Survival of Human Brain Microvascular Endothelial Cells in Alzheimer's Disease.

Cerebrovascular changes occur in Alzheimer's disease (AD). The progesterone receptor membrane component-1 (PGRMC1) is a well identified hormone receptor with multiple functions in AD. This study aims to explore the involvement of PGRMC1 in the regulation of vascular endothelial function, providing new therapy options for AD. Single-cell sequencing revealed that the expression of PGRMC1 is lower in AD. By bioinformatics analysis, we found PGRMC1 was associated with regulation of cell proliferation, angiogenesis and etc. To understand the functional significance of PGRMC1, knockdown and overexpression were performed using human brain microvascular endothelial cells (HBMVECs), respectively. Cell proliferation assay, migration assay, tube formation assay were performed in experiments. We demonstrated that the overexpression of PGRMC1 promoted the cellular processes associated with endothelia cell proliferation, migration, and angiogenesis, significantly. In conclusion, PGRMC1 may contribute to the modulation of HBMVECs function in AD. This finding may offer novel targets for AD treatment.

PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling.

Simple SummaryCombined menopausal hormone therapy is associated with increased breast cancer risk in postmenopausal women. In our previous studies, progesterone receptor membrane component 1 (PGRMC1) was shown to play a role in progestins’ elicitation of enhanced proliferation of breast cancer cells. Here we describe a potential mechanism by which PGRMC1 contributes to breast cancer progression via interaction with prohibitins, inhibiting their function as transcriptional repressors. This facilitates estrogen receptor alpha (ERα) transcriptional activity and enhances oncogenic signaling upon treatment with certain progestins, including norethisterone and dydrogesterone. Our data underline the contribution of PGRMC1 to especially hormone receptor positive breast cancer pathogenesis and demonstrate the need for further studies to understand its role in cancer.In previous studies, we reported that progesterone receptor membrane component 1 (PGRMC1) is implicated in progestin signaling and possibly associated with increased breast cancer risk upon combined hormone replacement therapy. To gain mechanistic insight, we searched for potential PGRMC1 interaction partners upon progestin treatment by co-immunoprecipitation and mass spectrometry. The interactions with the identified partners were further characterized with respect to PGRMC1 phosphorylation status and with emphasis on the crosstalk between PGRMC1 and estrogen receptor α (ERα). We report that PGRMC1 overexpression resulted in increased proliferation of hormone receptor positive breast cancer cell lines upon treatment with a subgroup of progestins including norethisterone and dydrogesterone that promote PGRMC1-phosphorylation on S181. The ERα modulators prohibitin-1 (PHB1) and prohibitin-2 (PHB2) interact with PGRMC1 in dependency on S181-phosphorylation upon treatment with the same progestins. Moreover, increased interaction between PGRMC1 and PHBs correlated with decreased binding of PHBs to ERα and subsequent ERα activation. Inhibition of either PGRMC1 or ERα abolished this effect. In summary, we provide strong evidence that activated PGRMC1 associates with PHBs, competitively removing them from ERα, which then can develop its transcriptional activities on target genes. This study emphasizes the role of PGRMC1 in a key breast cancer signaling pathway which may provide a new avenue to target hormone-dependent breast cancer.

Progesterone receptor membrane component 1 promotes the growth of breast cancers by altering the phosphoproteome and augmenting EGFR/PI3K/AKT signalling.

BackgroundIncreased expression of the progesterone receptor membrane component 1 (PGRMC1) has been linked to multiple cancers, including breast cancer. Despite being a regulatory receptor and a potential therapeutic target, the oncogenic potential of PGRMC1 has not been studied.MethodsThe impact of PGRMC1 on breast cancer growth and progression was studied following chemical inhibition and alteration of PGRMC1 expression, and evaluated by using online-based gene expression datasets of human breast cancer tissue. MTS, flow cytometry, qPCR, Western blotting, confocal microscopy and phosphoproteome analysis were performed.ResultsWe observed higher PGRMC1 levels in both ER-positive ZR-75-1 and TNBC MDA-MB-468 cells. Both chemical inhibition and silencing decreased cell proliferation, induced cell-cycle arrest, promoted apoptosis and reduced the migratory and invasive capabilities of ZR-75-1 and MDA-MB-468 cells. Further, phosphoproteome analysis demonstrated an overall decrease in activation of proteins involved in PI3K/AKT/mTOR and EGFR signalling pathways. In contrast, overexpression of PGRMC1 in non-malignant MCF10A cells resulted in increased cell proliferation, and enhanced activity of PI3K/AKT/mTOR and EGFR signalling pathways.ConclusionsOur data demonstrate that PGRMC1 plays a prominent role in regulating the growth of cancer cells by altering the PI3K/AKT/mTOR and EGFR signalling mechanisms in both ER-positive and TNBC cells.

Abstract P2-04-02: Progesterone receptor membrane component 1 - A novel key regulator in lipid homeostasis drives oncogenic signaling resulting in breast cancer progression

Abstract 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 cancer carcinogenesis. Nevertheless, little is known about the mechanisms by which PGRMC1 drives tumor progression. Based on our previous studies on the involvement of PGRMC1 in cholesterol metabolism we describe a potential mechanism by which PGRMC1 can increase lipid metabolism and alter EGFR signaling. Methods: To obtain PGRMC1 overexpressing cell lines, MCF7, T47D and MDA cells were stably transfected with expression plasmid pcDNA3.1. containing HA-tagged PGRMC1. As a control, cells transfected with the empty plasmid were used.To investigate the role of PGRMC1 in breast cancer progression, expression of genes involved in lipid metabolism were quantified by qPCR. Subsequent lipid and cholesterol levels as well as lipid raft expression were measured by flow cytometry in various breast cancer cell lines overexpressing PGRMC1 compared to empty vector control. Since many studies show that EGFR is enriched in lipid rafts, we further investigated alteration of EGFR/HER2 signaling through PGRMC1 overexpression via western blot and immunofluorescence analyses. The impact of lipid inhibition on tumor progression was analyzed by viability assays. Results: PGRMC1 overexpression resulted in higher levels of mRNA coding for proteins responsible for lipid homeostasis (SREPF1, SREBF2), lipid uptake (LDLR) and lipid synthesis (FASN, HMGCS, SCD, ACAT) leading to higher levels of neutral lipids, estradiol and cholesterol. Furthermore, PGRMC1 overexpressing hormone receptor positive cells show increased lipid raft formation with higher expression of EGFR/HER2 and altered EGFR/HER2 signaling. PGRMC1 overexpressing cells are more sensitive to a simvastatin treatment, which indicates a higher dependence of PGRMC1 overexpressing cells on cholesterol synthesis. Conclusion: Cholesterol and lipid metabolism play an important role in breast carcinogenesis. Our recent studies underline the effects of PGRMC1 on increasing lipid synthesis and uptake thereby altering oncogenic signaling. PGRMC1 overexpressing breast cancer cells could be particularly suitable for a statin treatment. Citation Format: Hannah Asperger, Marina Ludescher, Nadia Stamm, Jan-Philipp Cieslik, Ute Hofmann, Ulrich Zanger, Zaklina Kovacevic, Des Richardson, Eugen Ruckhäberle, Tanja Fehm, Dieter Niederacher, Hans Neubauer. Progesterone receptor membrane component 1 - A novel key regulator in lipid homeostasis drives oncogenic signaling resulting in breast cancer progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-04-02.

Abstract P6-05-08: Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers

Abstract Introduction: Increased expression of progesterone receptor membrane component 1 (PGRMC1), has been linked to breast cancer cell proliferation and tumor growth via epidermal growth factor receptor (EGFR) dependent interaction. As estrogen receptor positive breast cancers receive endocrine therapy and triple negative breast cancers (TNBC) rely on chemotherapeutic agents and or small molecule inhibitors. PGRMC1 could be a potential therapeutic target for both estrogen receptor positive and TNBCs, however its mechanism of action remains elusive. We, aim to understand the signaling mechanism behind PGRMC1 and its potential as viable target for the treatment of breast cancers. Materials and Methods: A panel of non-malignant and malignant breast cell lines were cultured and screened for PGRMC1 expression. PGRMC1 overexpressing breast cancer cell lines were treated with AG-205 (PGRMC1 inhibitor) and PGRMC1 targeting siRNAs. MTS, qRT-PCR, Western blot, immunofluorescence, flow cytometry assays and phospho explorer antibody array analysis were performed. Results: Increased PGRMC1 mRNA and protein levels were observed in estrogen receptor positive ZR-75-1 and TNBC MDA-MB-468 cells, these results were validated and compared to online RNA-seq based gene expression analysis of breast cell lines and breast tumor data sets. Data from online databases also demonstrated PGRMC1 overexpression in multiple breast cancer subtypes (Luminal, Basal B and Basal A). Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal tissue. Both AG-205 treatment and PGRMC1 silencing decreased cell proliferation, induced cell cycle arrest at the G0/G1 phase, promoted apoptosis and reduced the capability of the cells to migrate and invade. Phospho-specific antibody array data demonstrated overall downregulation of the EGFR/PI3K/AKT signaling mechanisms following AG-205 and PGRMC1 silencing. Alteration in the expression of key markers of cell proliferation, apoptosis and cell cycle revealed that PGRMC1 inhibition decreases breast cancer proliferation. Conclusion: Our data demonstrate that PGRMC1 plays a prominent role in regulating breast cancer growth and progression by altering the EGFR/PI3K/AKT signaling mechanisms. Citation Format: Diego A Pedroza, Alejandra Bencomo, Adriana Galvez, Ramadevi Subramani, Venkatesh Rajamanickam, Rajkumar Lakshmanaswamy. Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-08.

Abstract 846: Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers

Abstract Introduction: The role of progesterone and its receptors in breast cancer progression continue to be studied but remain controversial. Progesterone membrane receptors with the ability to regulate kinase signals, mediating breast cancer proliferation have been demonstrated. Increased expression of the progesterone receptor membrane component 1 (PGRMC1), a heme - binding protein with the ability to interact and stabilize epidermal growth factor receptor (EGFR) is frequently found in breast cancer tissue. The basis of the signaling mechanisms of progesterone membrane receptors remain largely unknown. Both the nuclear and membrane progesterone receptors could play a significant role in the development and progression of breast cancers and both could become viable therapeutic options. We, aim to investigate the molecular circuitry of PGRMC1 in both ER-positive and Triple Negative Breast Cancers (TNBCs). Materials and Methods: Human breast tissues were utilized to identify the expression of PGRMC1 along with a panel of normal and breast cancer cell lines. PGRMC1 overexpressing ZR-75-1 and MDA-MB-468 breast cancer cell lines were selected and treated with AG-205 (PGRMC1 inhibitor) and PGRMC1 siRNAs, while PGRMC1 was overexpressed in MCF10A non-malignant breast epithelial cells. MTS, qRT-PCR, Western blot, immunofluorescence, immunohistochemistry and flow cytometry were performed to study cell proliferation, apoptosis and key markers involved in these processes. In silico analysis utilizing publicly available gene expression datasets were also performed. Results: Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal breast tissue. Increased PGRMC1 expression was observed specifically in ZR-75-1 and MDA-MB-468 cells by qRT-PCR, Western blot and immunofluorescence, these results were validated and compared to microarray-based gene expression analysis of breast cell lines and breast tumor data sets. Both AG-205 and PGRMC1 targeted siRNAs decreased cell proliferation in ZR-75-1 and MDA-MB-468. Minimal effects of AG-205 were observed in MCF10A non-malignant breast epithelial cells. AG-205 treatment and silencing of PGRMC1 induced apoptosis in both cancer cell lines. Furthermore, PGRMC1 overexpression transformed MCF10A cells into a malignant phenotype. Key markers of cell proliferation (pAKT, CCND1, pEGFR, pmTOR) and apoptosis (PTEN, Bcl2, Bax,) revealed that PGRMC1 inhibition decreases proliferation while overexpression promotes tumorigenesis. Conclusion: Our data demonstrates that PGRMC1 plays a prominent role in both ER-positive and TNBCs. These initial findings uncover the potential of PGRMC1 as an oncogene and therapeutic target for breast cancer patients who overexpress this gene. Citation Format: Diego A. Pedroza, Ramadevi Subramani Reddy, Adriana Galvez, Rajkumar Lakshmanaswamy. Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 846.

Progesterone receptor membrane component 1 as a potential prognostic biomarker for hepatocellular carcinoma.

AIMTo investigate the clinicopathological significance of progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 in hepatocellular carcinoma (HCC).METHODSWe performed immunohistochemical staining to evaluate the estrogen receptor (ER), progesterone receptor (PR), PGRMC1, and PGRMC2 in a clinical cohort consisting of 89 paired HCC and non-tumor liver samples. We also analyzed HCC data (n = 373) from The Cancer Genome Atlas (TCGA). We correlated the expression status of PGRMC1 and PGRMC2 with clinicopathological indicators and the clinical outcomes of the HCC patients. We knocked down or overexpressed PGRMC1 in HCC cell lines to evaluate its biological significance in HCC cell proliferation, differentiation, migration, and invasion.RESULTSWe found that few HCC cases expressed ER (5.6%) and PR (4.5%). In contrast, most HCC cases expressed PGRMC1 (89.9%) and PGRMC2 (100%). PGRMC1 and PGRMC2 exhibited significantly lower expression in tumor tissue than in non-tumor tissue (P < 0.001). Lower PGRMC1 expression in HCC was significantly associated with higher serum alpha-fetoprotein expression (P = 0.004), poorer tumor differentiation (P = 0.045) and liver capsule penetration (P = 0.038). Low PGRMC1 expression was an independent predictor for worse disease-free survival (P = 0.002, HR = 2.384, CI: 1.377-4.128) in our cases, as well as in the TCGA cohort (P < 0.001, HR = 2.857, CI: 1.781-4.584). The expression of PGRMC2 did not relate to patient outcome. PGRMC1 knockdown promoted a poorly differentiated phenotype and proliferation of HCC cells in vitro, while PGRMC1 overexpression caused the opposite effects.CONCLUSIONPGRMC1 is a non-classical hormonal receptor that negatively regulates hepatocarcinogenesis. PGRMC1 down-regulation is associated with progression of HCC and is a poor prognostic indicator.

The presence of a membrane-bound progesterone receptor induces growth of breast cancer with norethisterone but not with progesterone: A xenograft model

ObjectivesDuring menopausal hormone therapy (MHT) a possible increase in breast cancer risk is thought to depend mainly on the progestogen component. In vitro studies have shown that the progesterone receptor membrane component 1 (PGRMC1) is important for tumor proliferation induced by progestogens. The primary aim of this study was to compare for the first time the natural progestogen, progesterone (P), with a synthetic progestogen, norethisterone (NET), using a xenograft model. MethodsMCF7 cells, transfected with PGRMC1 plasmid or empty vector, were injected into nude mice and estradiol (E2) pellets were implanted. After 12days, NET or P or placebo pellets were implanted. Tumor volumes in all groups (6 mice/group) were monitored for 6–7 weeks. Immunohistochemical expression of PGRMC1 and KI-67 was assessed. These experiments were repeated using T47D cells. ResultsCompared with the control condition, E2 and sequential E2/NET combination increased xenograft tumor growth with MCF7 and T47D cells that transgenically expressed PGRMC1 (p<0.01); progesterone did not increase growth. Breast cancer cells transfected with empty vectors did not respond to either progestogen. Comparing KI-67 and PGRMC1 expression, the Pearson correlation was r=0.848, p=0.002. ConclusionsE2 plus NET increases tumor growth in human breast cancer cells overexpressing PGRMC1, but there is no change with progesterone. To our knowledge, this is the first comparison of both progestogens in vivo using nude mice, which are frequently used in xenograft models. Clinical trials are needed to determine whether women with overexpression of PGRMC1 are at increased risk of breast cancer if NET instead of progesterone is used in MHT.

Increased expression of progesterone receptor membrane component 1 is associated with aggressive phenotype and poor prognosis in ER-positive and negative breast cancer.

Expression of progesterone receptor membrane component 1 (PGRMC1) has been shown to be higher in breast cancer than normal tissue. We have previously shown that certain progestogens strongly stimulate proliferation of breast cancer cells overexpressing PGRMC1, and therefore hypothesize that PGRMC1 may play a critical role in breast cancer progression. Because little information is available if expression of PGRMC1 is also associated with worse prognosis for breast cancer patients, in this study we investigated the clinicopathologic significance of PGRMC1 expression in breast cancer tissue. Expression of PGRMC1 was analyzed by immunohistochemical staining of primary tumor tissues obtained from 69 breast cancer patients. A labeling score was developed, and results were correlated with tumor size, lymph node metastasis, and clinical outcome. Overexpression of PGRMC1 is correlating with larger tumor size and lymph node metastasis. Kaplan-Meier survival curves indicate that patients with PGRMC1 tumors have poorer disease-free and overall survival independent from the estrogen receptor status than breast cancer patients with PGRMC1 tumors. Our findings suggest that the expression of PGRMC1 might be useful for predicting prognosis in patients with breast cancer.

PGRMC1 contributes to doxorubicin-induced chemoresistance in MES-SA uterine sarcoma.

Chemotherapy is one of the major categories of medical oncology and a primary tumor treatment; however, the effectiveness of chemotherapy is restricted by drug resistance. Overcoming resistance to chemotherapy and investigating molecular targeted therapies are challenges currently faced during resistance management. Progesterone receptor membrane component 1 (PGRMC1) is an adapter protein mediating cholesterol synthesis, steroid signaling, and cytochrome p450 activation. Attention has recently focused on the role of PGRMC1 in cell survival, anti-apoptosis, and damage response. In the present study, we used knockdown and overexpression approaches in the following set of uterine sarcoma models to further evaluate the role of PGRMC1 in drug resistance: the doxorubicin-sensitive MES-SA cells and the doxorubicin-resistant MES-SA/DxR-2 µM and MES-SA/DxR-8 µM cells (with different levels of doxorubicin resistance). PGRMC1 repressed doxorubicin-induced cytotoxicity and exhibited an anti-apoptotic effect; it also promoted cell proliferation and cell cycle progression to the S phase. Of note, PGRMC1 overexpression led to the epithelial-mesenchymal transition (EMT) of the sensitive MES-SA cells, thus facilitating their migration and invasion. The combination of PGRMC1 knockdown and the P-glycoprotein inhibitor verapamil significantly decreased the viability of P-glycoprotein-overexpressing MES-SA/DxR-8 μM cells after doxorubicin treatment. Taken together, our results show that PGRMC1 contributed to chemoresistance through cell proliferation, anti-apoptosis, and EMT induction, leading to the suggestion that PGRMC1 may serve as a therapeutic target in combination with an inhibitor in different drug resistance pathways and indicating the usefulness of predictive resistance biomarkers in uterine sarcoma.

Abstract 356: Photoacoustic imaging of pancreatic cancer proliferation via sigma-2 receptor/PGRMC1-eYFP

Abstract Sigma-2 receptor binding sites, recently identified as progesterone receptor membrane component 1 (PGRMC1) (Nature Communications, 2011; 2:380), are highly expressed in tumor cells. We have previously reported that measuring the sigma-2 receptor expression is a novel strategy for evaluating the proliferative status of tumors in vivo using the non-invasive imaging technique, positron emission tomography (PET). Our group has developed a series of sigma-2 receptor radioligands as PET imaging probes, one sigma-2 receptor radiotracer [18F]ISO-1 is under clinical evaluation for imaging the proliferation status in the patients with different types of cancers. PGRMC1 depleted human ovarian SKOV-3 cancer cells have been reported to lack tumor formation in mice and PGRMC1 depleted tumors displayed poor microvasculature system (Endocrinology, 2009; 150:4846-4854). To further understand the regulation of PGRMC1 in cell cycle and cancer proliferation, we have stably transfected PGRMC1-eYFP, enhanced yellow fluorescent protein conjugated PGRMC1, into human pancreatic BxPC3 cancer cells and those cells were transplanted into immunodeficient nude mice to grow into solid tumors; subsequently, a traditional fluorescence imaging system and an optical-resolution photoacoustic microscopy (OR-PAM) were utilized for in vivo monitoring of BxPC3 cancer cells growth and observing the surrounding microvasculature development for those tumors under conditions of sigma-2 receptor/PGRMC1 overexpression. Tumor tissue sections were prepared and in vitro fluorescence microscopy analysis of PGRMC1-eYFP cells revealed that PGRMC1-eYFP overexpressed cells form cluster patterns which may represent the proliferation priority created by PGRMC1 overexpression. The subcellular localization of PGRMC1-eYFP appeared to be similar to that of sigma-2 receptors reported previously by our group using the sigma-2 receptor fluorescent probes (Cancer Res 2007; 67, 6708-6716). PGRMC1-eYFP BxPC3 cells were surrounded by fully developed microvasculature system. Further application of this in vivo imaging platform for evaluating the sigma-2 receptor pharmacology and imaging proliferation is ongoing in our group. (Supported by CA 102869 and CA136398). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 356. doi:1538-7445.AM2012-356

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

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). 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. 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. 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.

Regulation of Ovarian Cancer Cell Viability and Sensitivity to Cisplatin by Progesterone Receptor Membrane Component-1

Progesterone (P4) influences ovarian cancer cells by an unknown mechanism. The objective was to determine whether P4 acts through progesterone receptor membrane component-1 (PGRMC1) in ovarian cancers. Archival tissue and cDNA provided by OriGene were used for expression studies. In vitro experiments were conducted with Ovcar-3 cells. PCR, Western blot, and immunohistochemistry were used to measure expression of PGRMC1 and nuclear progesterone receptor (PGR). PGRMC1's role in regulating the viability of ovarian cancers was assessed by overexpressing PGRMC1, depleting PGRMC1 using small interfering RNA, and attenuating PGRMC1's action with a blocking antibody. Apoptosis was determined by 4',6'-diamino-2-phenylindole staining. PGRMC1 mRNA increased and PGR mRNA decreased in advanced stages of ovarian cancer. Unlike PGR, PGRMC1 was expressed in virtually every cancer cell within the tumor. A similar relationship between PGRMC1 and PGR was observed in Ovcar-3 cells. In these cells P4 suppressed apoptosis induced by either serum withdrawal or cisplatin (CDDP). Moreover, in the presence of P4, the following occurs: 1) overexpression of PGRMC1 reduces the effectiveness of CDDP, 2) depletion of PGRMC1 with small interfering RNA enhances the effects of CDDP, and 3) PGRMC1 antibody treatment increases the apoptotic response to CDDP. These findings indicate that PGRMC1 plays an important role in promoting ovarian cancer cell viability and that attenuating PGRMC1's action makes the ovarian cancer cells more sensitive to CDDP. These data suggest that targeted depletion of PGRMC1 could be useful as an adjunct to CDDP therapy.

Progesterone Membrane Receptor Component 1 Expression in the Immature Rat Ovary and Its Role in Mediating Progesterone’s Antiapoptotic Action

Progesterone receptor membrane component-1 (PGRMC1) interacts with plasminogen activator inhibitor RNA binding protein-1 (PAIRBP1), a membrane-associated protein involved in the antiapoptotic action of progesterone (P4). In this paper, the first studies were designed to assess the ovarian expression pattern of PGRMC1 and PAIRBP1. Western blot analysis revealed that spontaneously immortalized granulosa cells (SIGCs) as well as granulosa and luteal cells express both proteins. Luteal cells were shown to express more PGRMC1 than granulosa cells. Immunohistochemical studies confirmed this and demonstrated that PGRMC1 was present in thecal/stromal cells, ovarian surface epithelial cells, and oocytes. PAIRBP1 was also expressed in thecal/stromal cells and ovarian surface epithelial cells but not oocytes. Furthermore, PAIRBP1 and PGRMC1 were detected among the biotinylated surface proteins that were isolated by avidin affinity purification, indicating that they localized to the extracellular surface of the plasma membrane. Confocal microscopy revealed that both of these proteins colocalize to the plasma membrane as well as the cytoplasm. The second studies were designed to assess PGRMC1's role in P4's antiapoptotic actions. These studies showed that overexpression of PGRMC1 increased 3H-P4 binding and P4 responsiveness. Conversely, treatment with a PGRMC1 antibody blocked P4's antiapoptotic action. Taken together, the present findings indicate that both PAIRBP1 and PGRMC1 show a similar expression pattern within the ovary and colocalize to the extracellular surface of the plasma membrane. At the plasma membrane, these two proteins interact to form a complex that is required for P4 to transduce its antiapoptotic action.