A novel functional interplay between Progesterone Receptor-B and PTEN, via AKT, modulates autophagy in breast cancer cells.

The tumour suppressor activity of the phosphatase and tensin homologue on chromosome 10 (PTEN) is subject of intense investigative efforts, although limited information on its regulation in breast cancer is available. Herein, we report that, in breast cancer cells, progesterone (OHPg), through its cognate receptor PR-B, positively modulates PTEN expression by inducing its mRNA and protein levels, and increasing PTEN-promoter activity. The OHPg-dependent up-regulation of PTEN gene activity requires binding of the PR-B to an Sp1-rich region within the PTEN gene promoter. Indeed, ChIP and EMSA analyses showed that OHPg treatment induced the occupancy of PTEN promoter by PR and Sp1 together with transcriptional coactivators such as SRC1 and CBP. PR-B isoform knockdown abolished the complex formation indicating its specific involvement. The OHPg/PR-B dependent induction of PTEN causes the down-regulation of PI3K/AKT signal, switching on the autophagy process through an enhanced expression of UVRAG and leading to a reduced cell survival. Altogether these findings highlight a novel functional connection between OHPg/PR-B and tumour suppressor pathways in breast cancer.

Progesterone increases hepatic lipid content and plasma lipid levels through PR- B-mediated lipogenesis.

Progesterone (P4) is a crucial reproductive hormone that acts as a precursor for all other endogenous steroids. P4 modulates transcriptional activity during reproduction by binding to progesterone receptors (PR). However, the physiological role of P4 in the liver is understudied. P4-mediated lipid metabolism in the liver was investigated in this study, as P4 facilitates insulin resistance and influences energy metabolism. While exogenous lipids are mainly obtained from food, the liver synthesizes endogenous triglycerides and cholesterol from a carbohydrate diet. Hepatic de novo lipogenesis (DNL) is primarily determined by acetyl-CoA and its biosynthetic pathways, which involve fatty acid and cholesterol synthesis. While P4 increased the hepatic levels of sterol regulatory element-binding protein 1 C (SREBP-1 C), peroxisome proliferator-activated receptor-gamma (PPARγ), acetyl-CoA carboxylase (ACC), and CD36, co-treatment with the P4 receptor antagonist RU486 blocked these proteins and P4-mediated lipogenesis. RNA sequencing was used to assess the role of P4 in lipogenic events, such as fatty liver and fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism. P4 induced hepatic DNL and lipid anabolism were confirmed in the liver of ovarian resection mice fed a high-fat diet or in pregnant mice. P4 increased lipogenesis directly in mice exposed to P4 and indirectly in fetuses exposed to maternal P4. The lipid balance between lipogenesis and lipolysis determines fat build-up and is linked to lipid metabolism dysfunction, which involves the breakdown and storage of fats for energy and the synthesis of structural and functional lipids. Therefore, P4 may impact the lipid metabolism and reproductive development during gestation.

Additional confirmation of successful cell suspension fractionation of metastatic axillary node tissue.

We present herein an extension to our recently developed and published method termed "Fractionation of Nodal Cell Suspension" (FNCS). The method enables efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic axillary lymph node (mALN) tissue, using the entire nodule. For the purpose of the present study, a case of invasive lobular breast cancer (BC) patient with pT2N3aMx status and defined primary tumor markers (ERα 8, PR-B 8, and HER2 score 0) was available. Initially, the mALN tissue of this patient was analyzed by immunohistochemistry (IHC), and a positive correlation of nodal ERα, PR-B and HER2 biomarkers to those of the primary tumor was obtained. Subsequently, the mALN was FNCS fractionated into N and C, and Western blot (WB) analysis demonstrated a single band for ERα, PR-B and nuclear loading control (HDAC1) in nuclear, but not in the cytosolic compartments, confirming the efficiency of our fractionation protocol. At the same time, HER2 bands were not observed in either compartment, in accordance with HER2 negativity determined by IHC in both primary tumor and mALN tissue. In conclusion, by confirming the nuclear expression of ERα and PR-B biomarkers in metastatic loci, we demonstrate the purity of the FNCS-generated compartments - the protocol that offers a reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of novel biomarkers in the whole mALN of BC patients.

Thermosensitive and biodegradable hydrogel encapsulating targeted nanoparticles for the sustained co-delivery of gemcitabine and paclitaxel to pancreatic cancer cells.

Pancreatic cancer represents a life threatening disease with rising mortality. Although the synergistic combination of gemcitabine and albumin-bound paclitaxel has proven to enhance the median survival rates as compared to gemcitabine alone, their systemic and repeated co-administration has been associated with serious toxic side effects and poor patient compliance. For this purpose, we designed a thermosensitive and biodegradable hydrogel encapsulating targeted nanoparticles for the local and sustained delivery of gemcitabine (GEM) and paclitaxel (PTX) to pancreatic cancer. GEM and PTX were loaded into PR_b-functionalized liposomes targeting integrin α5ß1, which was shown to be overexpressed in pancreatic cancer. PR_b is a fibronectin-mimetic peptide that binds to α5ß1 with high affinity and specificity. The PR_b liposomes were encapsulated into a poly(δ-valerolactone-co-D,L-lactide)-b-poly(ethylene glycol)-b-poly(δ-valerolactone-co-D,L-lactide) (PVLA-PEG-PVLA) hydrogel and demonstrated sustained release of both drugs compared to PR_b-functionalized liposomes free in solution or free drugs in the hydrogel. Moreover, the hydrogel-nanoparticle system was proven to be very efficient towards killing monolayers of human pancreatic cancer cells (PANC-1), and showed a significant reduction in the growth pattern of PANC-1 tumor spheroids as compared to hydrogels encapsulating non-targeted liposomes with GEM/PTX or free drugs, after a one week treatment period. Our hybrid hydrogel-nanoparticle system is a promising platform for the local and sustained delivery of GEM/PTX to pancreatic cancer, with the goal of maximizing the therapeutic efficacy of this synergistic drug cocktail while potentially minimizing toxic side effects and eliminating the need for repeated co-administration.

Altered expression of progesterone receptor isoforms A and B in human eutopic endometrium in endometriosis patients.

Recent data implicate an altered expression of progesterone receptor isoform A (PR-A) and B (PR-B) in the endometrium of endometriosis patients. This prospective exploratory study aimed to precisely determine the PR-A and PR-B expression using immunohistochemical techniques in eutopic endometrium of women with endometriosis compared with disease-free women throughout the menstrual cycle. All symptomatic patients underwent laparoscopy for the diagnosis of endometriosis and histological confirmation of the disease (EO) whereas controls were proven disease-free (CO). In CO samples (n=10) an increased expression of PR-A and PR-B during the proliferative to early secretory phase and a decreased expression of both receptor isoforms during the mid to late secretory phase was ascertained in accordance with previous studies. In patients with endometriosis (n=16) no cycle dependent pattern of PR-A and PR-B expression was identified in contrast to patients without endometriosis. Moreover, in EO samples a huge variety of inter- and intra-individual differences in PR-A and PR-B expression were detected. These data provide further evidence that dysregulation of the PR-A and PR-B expression might contribute to the pathophysiology of endometriosis.

Revisiting the roles of progesterone and allopregnanolone in the nervous system: resurgence of the progesterone receptors.

Progesterone is commonly considered as a female reproductive hormone and is well-known for its role in pregnancy. It is less well appreciated that progesterone and its metabolite allopregnanolone are also male hormones, as they are produced in both sexes by the adrenal glands. In addition, they are synthesized within the nervous system. Progesterone and allopregnanolone are associated with adaptation to stress, and increased production of progesterone within the brain may be part of the response of neural cells to injury. Progesterone receptors (PR) are widely distributed throughout the brain, but their study has been mainly limited to the hypothalamus and reproductive functions, and the extra-hypothalamic receptors have been neglected. This lack of information about brain functions of PR is unexpected, as the protective and trophic effects of progesterone are much investigated, and as the therapeutic potential of progesterone as a neuroprotective and promyelinating agent is currently being assessed in clinical trials. The little attention devoted to the brain functions of PR may relate to the widely accepted assumption that non-reproductive actions of progesterone may be mainly mediated by allopregnanolone, which does not bind to PR, but acts as a potent positive modulator of γ-aminobutyric acid type A (GABA(A) receptors. The aim of this review is to critically discuss effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABA(A) receptors, with main focus on the brain.

Dissection of androgen receptor-promoter interactions: steroid receptors partition their interaction energetics in parallel with their phylogenetic divergence.

Steroid receptors comprise a homologous family of ligand-activated transcription factors. The members include androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and progesterone receptor (PR). Phylogenetic studies demonstrate that AR, GR, MR, and PR are most closely related, falling into subgroup 3C. ER is more distantly related, falling into subgroup 3A. To determine the quantitative basis by which receptors generate their unique transcriptional responses, we are systematically dissecting the promoter-binding energetics of all receptors under a single "standard state" condition. Here, we examine the self-assembly and promoter-binding energetics of full-length AR and a mutant associated with prostate cancer, T877A. We first demonstrate that both proteins exist only as monomers, showing no evidence of dimerization. Although this result contradicts the traditional understanding that steroid receptors dimerize in the absence of DNA, it is fully consistent with our previous work demonstrating that GR and two PR isoforms either do not dimerize or dimerize only weakly. Moreover, both AR proteins exhibit substantial cooperativity between binding sites, again as seen for GR and PR. In sharp contrast, the more distantly related ER-α dimerizes so strongly that energetics can only be measured indirectly, yet cooperativity is negligible. Thus, homologous receptors partition their promoter-binding energetics quite differently. Moreover, since receptors most closely related by phylogeny partition their energetics similarly, such partitioning appears to be evolutionarily conserved. We speculate that such differences in energetics, coupled with different promoter architectures, serve as the basis for generating receptor-specific promoter occupancy and thus function.