Progesterone receptor membrane component 1 is phosphorylated upon progestin treatment in breast cancer cells.

Menopausal hormone therapy, using estrogen and synthetic progestins, is associated with an increased risk of developing breast cancer. The effect of progestins on breast cells is complex and not yet fully understood. In previous in vitro and in vivo studies, we found different progestins to increase the proliferation of Progesterone Receptor Membrane Component-1 (PGRMC1)-overexpressing MCF7 cells (MCF7/PGRMC1), suggesting a possible role of PGRMC1 in transducing membrane-initiated progestin signals. Understanding the activation mechanism of PGRMC1 by progestins will provide deeper insights into the mode of action of progestins on breast cells and the often-reported phenomenon of elevated breast cancer rates upon progestin-based hormone therapy. In the present study, we aimed to further investigate the effect of progestins on receptor activation in MCF7 and T47D breast cancer cell lines. We report that treatment of both breast cancer cell lines with the progestin norethisterone (NET) induces phosphorylation of PGRMC1 at the Casein Kinase 2 (CK2) phosphorylation site Ser181, which can be decreased by treatment with CK2 inhibitor quinalizarin. Point mutation of the Ser181 phosphorylation site in MCF7/PGRMC1 cells impaired proliferation upon NET treatment. This study gives further insights into the mechanism of differential phosphorylation of the receptor and confirms our earlier hypothesis that phosphorylation of the CK2-binding site is essential for activation of PGRMC1. It further suggests an important role of PGRMC1 in the tumorigenesis and progression of breast cancer in progestin-based hormone replacement therapy.

High Level of Progesteron Receptor Membrane Component 1 (PGRMC 1) in Tissue of Breast Cancer Patients is Associated with Worse Response to Anthracycline-Based Neoadjuvant Therapy.

PGRMC1 is known to be highly expressed in breast cancer tissue and is associated with chemoresistance in breast cancer cells. However, its role in breast cancer signaling is not fully understood yet. In the present study, the expression status of PGRMC1 and its phosphorylated version (pPGRMC1) in breast cancer tissue and surrounding stroma before and after neoadjuvant therapy was examined to find a possible association to therapy response. Tissue biopsies of 69 breast cancer patients were analyzed by immunohistochemistry for expression levels of PGRMC1 and pPGRMC1. Expression status of PGRMC1 and pPGRMC1 in tumor tissue was compared with expression status of progesterone receptor (PR), estrogen receptor α (ERα), total estrogen receptor ß (ERß), ERß1, ERß2, the proliferation marker Ki-67, and human epidermal growth factor receptor 2 (HER2/neu). Correlations were calculated for expression of PGRMC1 and pPGRMC1 before and after neoadjuvant-therapy. PGRMC1 and pPGRMC1 were highly abundant in every breast cancer tissue sample. Considerably lower signals were detected in surrounding tissue. Further, PGRMC1 and pPGRMC1 abundance was found to correlate with ERß expression. A lower level of pPGRMC1 could be found in post-therapy surgical specimens compared to specimens before treatment. Interestingly, patients with high PGRMC1 tumor levels showed worse response to anthracycline-based therapy as patients with lower PGRMC1 levels. These new findings demonstrate that PGRMC1 might play an important role in progression and therapy resistance of human breast tumors and could offer an interesting target for anticancer therapy.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our findings suggest that the expression of PGRMC1 might be useful for predicting prognosis in patients with breast cancer.

Aggregation Limits Surface Expression of Homomeric GluA3 Receptors.

AMPA receptors are glutamate-gated cation channels assembled from GluA1-4 subunits and have properties that are strongly dependent on the subunit composition. The subunits have different propensities to form homomeric or various heteromeric receptors expressed on cell surface, but the underlying mechanisms are still poorly understood. Here, we examined the biochemical basis for the poor ability of GluA3 subunits to form homomeric receptors, linked previously to two amino acid residues, Tyr-454 and Arg-461, in its ligand binding domain (LBD). Surface expression of GluA3 was improved by co-assembly with GluA2 but not with stargazin, a trafficking chaperone and modulator of AMPA receptors. The secretion efficiency of GluA2 and GluA3 LBDs paralleled the transport difference between the respective full-length receptors and was similarly dependent on Tyr-454/Arg-461 but not on LBD stability. In comparison to GluA2, GluA3 homomeric receptors showed a strong and Tyr-454/Arg-461-dependent tendency to aggregate both in the macroscopic scale measured as lower solubility in nonionic detergent and in the microscopic scale evident as the preponderance of hydrodynamically large structures in density gradient centrifugation and native gel electrophoresis. We conclude that the impaired surface expression of homomeric GluA3 receptors is caused by nonproductive assembly and aggregation to which LBD residues Tyr-454 and Arg-461 strongly contribute. This aggregation inhibits the entry of newly synthesized GluA3 receptors to the secretory pathway.

May progesterone receptor membrane component 1 (PGRMC1) predict the risk of breast cancer?

OBJECTIVE: Our and other studies have pointed on an important role of progesterone receptor membrane component 1 (PGRMC1) in development of breast cancer, especially in hormone therapy. To investigate if PGRMC1 could be used to predict the risk for getting breast cancer, we assessed in tissues of patients with primary invasive breast cancer, if the expression of PGRMC1 may be associated with the expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and ki67. METHODS: Samples from 109 patients with breast cancer between the years 2008 and 2014 were obtained with the patients' consent. Each sample was evaluated for the ERα, PR, Ki67, and PGRMC1 expression by immunohistochemistry using serial sections from the ame paraffin block comparing malignant tissue to benign tissue. RESULTS: Expression of PGRMC1 is increased in tumor area compared with non-cancerous tissue and positively correlates with ERα expression (OR = 1.42 95%CI 1.06-1.91, p = 0.02). No association was obtained between expression of PGRMC1 and PR or Ki67. CONCLUSION: It can be suggested that women with breast epithelium highly expressing PGRMC1 and in interaction with ERα may have an increased risk to develop breast cancer, especially when treated with hormone therapy.

Twins in spirit - episode I: comparative preclinical evaluation of [(68)Ga]DOTATATE and [(68)Ga]HA-DOTATATE.

BACKGROUND: Recently, an intra-patient comparison demonstrated that the somatostatin (sst) ligand [(68)Ga]HA-DOTATATE ([(68)Ga]DOTA-3-iodo-Tyr(3)-octreotate) provides PET images comparable to or superior to those obtained with [(68)Ga]DOTATATE. To provide a comprehensive basis for nevertheless observed slight differences in tracer biodistribution and dosimetry, the characteristics of [(68)Ga]HA-DOTATATE were investigated in a detailed preclinical study. METHODS: Affinities of (nat)Ga-HA-DOTATATE and (nat)Ga-DOTATATE to sst1-5 were determined using membrane preparations and [(125)I]SST-28 as radioligand. Internalization into AR42J cells was studied in dual-tracer studies with [(125)I]TOC as internal reference. Biodistribution was investigated using AR42J tumor-bearing CD1 mice, and specificity of tracer uptake was confirmed in competition studies by coinjection of 0.8 mg TOC/kg. RESULTS: Sst2 affinities (IC50) of [(nat)Ga]HA-DOTATATE (1.4 ± 0.8 nM, logP: -3.16) and [(nat)Ga]DOTATATE (1.2 ± 0.6 nM, logP: -3.69) were nearly identical. Both compounds displayed IC50 > 1 µM for sst1,3,4, while sst5 affinity was markedly increased for (nat)Ga-HA-DOTATATE (102 ± 65 nM vs >1 µM for (nat)Ga-DOTATATE). [(nat)Lu]HA-DOTATATE and [(nat)Lu]DOTATATE showed slightly lower, identical sst2 affinities (2.0 ± 1.6 and 2.0 ± 0.8 nM, respectively) and sst3 affinities of 93 ± 1 and 162 ± 16 nM. Internalization of [(68)Ga]HA-DOTATATE was tenfold higher than that of [(125)I]TOC but only sixfold higher for [(68)Ga]DOTATATE and [(177)Lu]HA-DOTATATE. While [(68)Ga]HA-DOTATATE and [(68)Ga]DOTATATE had shown similar target- and non-target uptake in patients, biodistribution studies in mice at 1 h post injection (n = 5) revealed slightly increased non-specific uptake of [(68)Ga]HA-DOTATATE in the blood, liver, and intestines (0.7 ± 0.3, 1.0 ± 0.2, and 4.0 ± 0.7 %iD/g vs 0.3 ± 0.1, 0.5 ± 0.1, and 2.7 ± 0.8 %iD/g for [(68)Ga]DOTATATE). However, sst-mediated accumulation of [(68)Ga]HA-DOTATATE in the pancreas, adrenals, and tumor was significantly enhanced (36.6 ± 4.3, 10.8 ± 3.2, and 33.6 ± 10.9 %iD/g vs 26.1 ± 5.0, 5.1 ± 1.4, and 24.1 ± 4.9 %iD/g, respectively). Consequently, tumor/background ratios for [(68)Ga]HA-DOTATATE in the AR42J model are comparable or slightly increased compared to [(68)Ga]DOTATATE. CONCLUSIONS: The present preclinical data fully confirm the general biodistribution pattern and excellent in vivo sst-targeting characteristics previously observed for [(68)Ga]HA-DOTATATE in patients. The effect of slightly enhanced lipophilicity on background accumulation and normal organ dose is compensated by the high uptake of [(68)Ga]HA-DOTATATE in tumor. Thus, [(68)Ga]HA-DOTATATE represents a fully adequate, freely available substitute for [(68)Ga]DOTATATE and, given the superb sst-targeting characteristics of [(177)Lu]HA-DOTATATE in vitro, potential applicability for sst-targeted PRRT.