Progesterone Receptor Isoforms Research Articles

Progesterone Receptor Signaling Promotes Cancer Associated Fibroblast Mediated Tumorigenicity in ER+ Breast Cancer.

Breast cancer progression involves intricate interactions between cancer cells and the tumor microenvironment (TME). This study elucidates the critical role of progesterone receptor (PR) signaling in mediating the protumorigenic effects of cancer-associated fibroblasts (CAFs) on estrogen receptor-positive (ER+) luminal breast cancer cells. We demonstrate that CAFs produce physiologically relevant levels of estrogen and progesterone, which significantly contribute to breast cancer tumorigenicity. Specifically, CAF conditioned media (CM) promoted PR-dependent anchorage-independent growth, tumorsphere formation/stem cell expansion, and CD44 upregulation. CAF cells formed co-clusters more frequently with PR+ breast cancer cells relative to PR-null models. While both PR isoforms mediated these actions, PR-A was a dominant driver of tumorsphere formation/stemness, while PR-B induced robust CD44 expression and CAF/tumor cell co-cluster formation. CD44 knockdown impaired CAF/tumor cell co-clustering. Fibroblast growth factor 2 (FGF2), also secreted by CAFs, phosphorylated PR (Ser294) in a MAPK-dependent manner and activated PR to enhance CD44 expression and breast cancer tumorigenicity. The FGF receptor (FGFR) inhibitor PD173074 diminished CAF- and FGF2-dependent PR activation, tumorsphere formation, and co-clustering. In summary, this study reveals a novel mechanism through which stromal CAFs orchestrate elevated PR signaling in ER+ luminal breast cancer via secretion of both progesterone and FGF2, a potent activator of ERK1/2. Understanding tumor cell/TME interactions provides insights into potential therapeutic strategies aimed at disrupting PR- and/or FGF2/FGFR-dependent signaling pathways to prevent early metastasis in patients with ER+ breast cancer.

How Progesterone Receptor Expression Impacts Platinum Sensitivity in Ovarian Clear Cell Carcinoma: Insights from Clinical and Experimental Perspectives.

Ovarian clear cell carcinoma (OCCC) is often considered a relatively platinum-resistant malignancy. The aim of this study was to explore the influence of progesterone receptor (PR) expression levels on platinum sensitivity and survival outcomes in people with OCCC. A retrospective analysis was conducted with 80 people with OCCC who underwent surgery followed by adjuvant chemotherapy. PR expression was assessed via immunohistochemical (IHC) staining and quantified using the H score. The platinum sensitivity and survival outcomes of patients with weak and strong PR expression were compared. Additionally, cisplatin viability and migration experiments were conducted with OCCC cell lines (ES-2 and TOV-21G) with varying PR isoform expressions. Among the 80 patients, 62 were classified as having platinum-sensitive disease, while 18 had platinum-resistant disease. The mean total PR H- score of platinum-sensitive tumors was significantly higher than that of platinum-resistant tumors (p = 0.002). Although no significant differences in progression-free and overall survival were observed between patients with high and low PR expression, those with high PR expression tended to have longer survival. While PR protein was only weakly detectable in ES-2 and TOV-21G cells, a transfection of the PR-A or PR-B gene resulted in a strong expression of PR-A or PR-B, which led to significantly reduced proliferation and migration in ES-2 and TOV-21G cells. Furthermore, overexpression of PR-A or PR-B enhanced cisplatin cytotoxicity in these cell lines. In conclusion, strong PR expression was associated with improved platinum sensitivity and survival outcomes, consistent with our experimental findings. The potential of PR as a tumor sensitizer to cisplatin in OCCC warrants further investigation.

Early pregnancy modulates the expression of interferon-stimulated genes, progesterone receptors, and progesterone-induced blocking factor in ovine duodenum

In ruminants, the signal (interferon-tau, IFNT) for the maternal recognition of pregnancy has effects on maternal extrauterine organs, and progesterone plays a key role during pregnancy. However, it is unclear that early pregnancy has effects on the expression of interferon-stimulated genes (ISGs), progesterone receptors (PGRs), and progesterone-induced blocking factor (PIBF) in ovine small intestine. In this study, small intestines were obtained at day 16 of the estrous cycle, days 13, 16, and 25 of pregnancy, and RT-qPCR assay, Western blot, and immunohistochemistry were used to analyze the effects of early pregnancy on the expression of ISGs, PGR, and PIBF in the small intestine. The results indicated that expression levels of ISG15-conjugated protein, signal transducer and activator of transcription 1 (STAT1), myxovirus resistance protein 1, and interferon-gamma-inducible protein 10 (IP-10) at day 13 of pregnancy were the lowest. However, expression levels of PGR isoforms (60 kDa and 40 kDa), PIBF, STAT1 and 2′,5′-oligoadenylate synthetase 1 upregulated, but the expression value of IP-10 downregulated at day 25 of pregnancy compared with day 16 of the estrous cycle. In conclusion, early pregnancy modulated the expression of ISGs, PGR, and PIBF in ovine duodenum, which was implicated in immunoregulation and nutrient absorption of the small intestine in ewes.

Pregnancy influences expression of interferon-stimulated genes, progesterone receptor and progesterone-induced blocking factor in ovine thyroid.

Embryonic interferon-tau (IFNT) and progesterone affect expression of interferonstimulated genes (ISGs), progesterone receptor (PGR) and progesterone-induced blocking factor (PIBF) in the ovine thyroid. Thyroids of ewes were sampled at day 16 of nonpregnancy, days 13, 16, and 25 of pregnancy, and real-time quantitative polymerase chain reaction assay, western blot and immunohistochemistry were used to detect expression of ISGs, PGR, and PIBF. Free ISG15 protein was undetected, but ISG15 conjugated proteins upregulated at day 16 of pregnancy, and expression levels of ISG15 conjugated proteins, PGR isoform (70 kDa), PIBF, interferon-gamma-inducible protein 10 and myxovirusresistance protein 1 peaked, but expression level of signal transducer and activator of transcription 1 was the lowest at day 16 of pregnancy. In addition, the expression levels of PGR isoform (70 kDa) and signal transducer and activator of transcription 1 (STAT1) decreased, but levels of PGR isoform (43 kDa), 2',5'-oligoadenylate synthetase, IP-10 and MX1 increased at day 25 of pregnancy comparing with day 16 of the estrous cycle. Early pregnancy affects expression of ISGs, PGR, and PIBF in maternal thyroid through IFNT and progesterone, which may regulate thyroid autoimmunity and thyroid hormone secretion in ewes.

Abstract 3584: Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer

Abstract Exposure to progesterone is a recognized risk factor for breast cancer, and PGR polymorphisms are associated with various malignancies. The two progesterone receptor (PR) isoforms, full length PR-B and truncated PR-A, are expressed from the PGR gene in breast tissue and play crucial roles in normal physiology. An imbalance in the expression ratio of these isoforms, favoring increased levels of PR-A, is common in breast cancer and is associated with resistance to tamoxifen in Luminal A-type tumors. Notably, PRs have recently been implicated in promoting endocrine resistance and driving the expansion of cancer stem-like cell (CSC) populations implicated in early dissemination and maintenance of metastatic populations. Because prior gene expression studies were primarily conducted in 2D culture conditions, the isoform-specific molecular and epigenetic mechanisms underlying PR actions in advanced ER+ breast cancer remain elusive. Our goal was to define progesterone-driven gene expression in CSC cells by employing transcriptomic profiling of T47D cells cultivated in spheroid-culture conditions (3D) expressing exclusively PR-A or PR-B. Utilizing CUT&RUN, we have mapped the genomic binding sites unique to each PR isoform and identified shared sites. This genomic data has been integrated with phenotypic studies in cells expressing a single PR isoform. Our findings indicate that PR-A acts as a regulator of the cell cycle and senescence, while PR-B plays a pivotal role in cell metabolism and intracellular signaling. We find little overlap between PR-regulated gene sets derived from the same hormone-treated cells harvested from 2D relative to 3D conditions. Our genomic profiling of PRs in this 3D model system has unveiled novel strategies to combat CSC outgrowth and late recurrence in luminal breast cancer patients. This work has shifted our prior understanding of the role of PRs in gene regulation within the CSC population, and offers potential insights for therapeutic interventions in ER+ breast cancer. Citation Format: Noelle E. Gillis, Thu H. Truong, Jessica Finlay-Schultz, Carol A. Sartorius, Carol A. Lange. Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3584.

Abstract 3981: Immune targeting progesterone receptor in hormone receptor positive breast cancer

Abstract Background: Hormone receptor positive (HR⁺) breast cancer (BC) is a subtype of BC that is characterized by expression of estrogen receptor (ER) and progesterone receptor (PR). A cornerstone treatment for this subtype of BC is endocrine inhibition therapy (EIT), in which estrogen and ER are suppressed using antagonists to suppress tumor growth. However, patients eventually obtain resistance to EIT. Metastatic HR⁺BC is the deadliest form of BC, with no cure. Current therapies currently only utilize the ER-axis, with no PR-targeted therapies. Little is known about the role of PR in these BCs, but we have found evidence for an immunosuppressive role for PR in HR⁺BC. Yet, it is unclear how PR mediates immunosuppression and if this axis can be therapeutically targeted. We hypothesized that PR confers local immunosuppression through the stimulation of immunosuppressive transcriptional activity and that we can immunologically target this axis using a PR vaccine. Experimental methods: To assess the immune impact of PR, we expressed the isoforms of PR (A, B and A+B) in mouse mammary cancer cells and assessed their capacity to elicit PR signaling. We assessed the impact of PR expression on T cell activity in targeting PR⁺ mammary tumors that express GFP, along with using GFP-specific JEDI CD8⁺ T cells. We explored the ability of PR to protect against GFP xenoantigen immune responses by engrafting PR⁺ mammary cells into immune competent and deficient mice. Lastly, we engineered 1st generation adenoviral vectors encoding mouse and human PR and utilized two antiprogestins (Mifepristone and Onapristone) in vivo. Using our vectors, we vaccinated BALB/c in the footpad and assessed PR-specific immunity using IFNγ ELISPOT. Results: Using PR signaling reporters, we found canonical PR signaling activity in the absence of progesterone (P4) by PR-A and in the presence of P4 by PR-B, but the most profound activation of PR signaling was seen through combined expression of PR-A and B. While expression of these genes did not confer striking cytotoxic T cell resistance in vitro, combination of PR-A+PR-B in 67NR cells permitted successful tumor engraftment in immune competent mice, despite xenoantigen expression. GFP expression did not alter tumor growth in immunodeficient mice, suggesting a key role in modulating local anti-tumor immunity. The use of either Mifepristone or Onapristone significantly suppressed tumor engraftment in immune competent mice in vivo. We found that an Ad-PR vaccine could elicit T cell responses against human PR-specific epitopes, a potential step towards immune targeting these BCs. Conclusions: We found that murine PR-A and PR-B can confer robust P4 signaling that is linked to an immunosuppressive phenotype in vivo. We developed a PR vaccine that elicited PR-specific T cell responses in vivo. We are currently examining the mechanisms that underlie PR-mediated immunosuppression and exploring the potential of PR vaccines in HR⁺BC. Citation Format: Andrea E. Wilson, Junping Wei, Gangjun Lei, Xiao-Yi Yang, Cong-Xiao Liu, Melissa Gajda, Tao Wang, Christy Hagan, Zachary C. Hartman. Immune targeting progesterone receptor in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3981.

Novel biosensor for high-throughput detection of progesterone receptor-interacting endocrine disruptors

Progesterone receptor (PR)-interacting compounds in the environment are associated with serious health hazards. However, methods for their detection in environmental samples are cumbersome. We report a sensitive activity-based biosensor for rapid and reliable screening of progesterone receptor (PR)-interacting endocrine disrupting chemicals (EDCs). The biosensor is a cell line which expresses nuclear mCherry-NF1 and a green fluorescent protein (GFP)-tagged chimera of glucocorticoid receptor (GR) N terminus fused to the ligand binding domain (LBD) of PR (GFP-GR-PR). As this LBD is shared by the PRA and PRB, the biosensor reports on the activation of both PR isoforms. This GFP-GR-PR chimera is cytoplasmic in the absence of hormone and translocates rapidly to the nucleus in response to PR agonists or antagonists in concentration- and time-dependent manner. In live cells, presence of nuclear NF1 label eliminates cell fixation and nuclear staining resulting in efficient screening. The assay can be used in screens for novel PR ligands and PR-interacting contaminants in environmental samples. A limited screen of river water samples indicated a widespread, low-level contamination with PR-interacting contaminants in all tested samples.

The inhibitory effect of trastuzumab on BT474 triple‑positive breast cancer cell viability is reversed by the combination of progesterone and estradiol.

Breast cancer expressing the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) is known as triple-positive (TPBC). TPBC represents 9-11% of breast cancer cases worldwide and is a heterogeneous subtype. Notably, TPBC presents a therapeutic challenge due to the crosstalk between the hormonal (ER and PR) and HER2 pathways. Patients with TPBC are treated with trastuzumab (TTZ); however, several patients treated with TTZ tend to relapse. The present study aimed to investigate the effect of the PR on inhibitory effect of TTZ on cell viability. BT474 cells (a model of TPBC) and BT474 PR-silenced cells were treated with either TTZ, progesterone (Pg), the PR antagonist mifepristone (RU486) or estradiol (E2) alone or in combination for 144 h (6 days). Cell viability assays and western blotting were subsequently performed. The results showed that Pg and E2 interfered with the inhibitory effect of TTZ on cell viability and this effect was potentiated when both hormones were combined. Pg was revealed to act through the PR, mainly activating the PR isoform B (PR-B) and inducing the protein expression levels of CDK4 and cyclin D1; however, it did not reactivate the HER2/Akt pathway. By contrast, E2 was able to increase PR isoform A (PR-A) expression, which was inhibited by Pg. Notably, in most of the experiments, RU486 did not antagonize the effects of Pg. In conclusion, Pg and E2 may interfere with the inhibitory effect of TTZ on cell viability through PR-B activation and PR-A inactivation.

Steroid profile in patients with breast cancer and in mice treated with mifepristone.

Progesterone receptors (PRs) are biomarkers used as prognostic and predictive factors in breast cancer, but they are still not used as therapeutic targets. We have proposed that the ratio between PR isoforms A and B (PRA and PRB) predicts antiprogestin responsiveness. The MIPRA trial confirmed the benefit of 200 mg mifepristone, administered to patients with tumors with a high PRA/PRB ratio, but dose-ranging has not been conducted. The aim of this study was to establish the plasma mifepristone levels of patients from the MIPRA trial, along with the resultant steroid profiles, and compare these with those observed in mifepristone-treated mice using therapeutic schemes able to induce the regression of experimental mammary carcinomas with high PRA/PRB ratios: 6 mg pellets implanted subcutaneously, or daily doses of 12 mg/kg body weight. The plasma levels of mifepristone and other 19 plasma steroids were measured by liquid chromatography-tandem mass spectometry. In mifepristone-treated mice, plasma levels were lower than those registered in mifepristone-treated patients (i.e. day 7 after treatment initiation, pellet-treated mice: 8.4 ± 3.9 ng/mL; mifepristone-treated patients: 300.3 ± 31.7 ng/mL (mean ± s.d.; P < 0.001)). The increase in corticoid related steroids observed in patients was not observed in mifepristone-treated mice. The increase in progesterone levels was the most significant side effect detected in mifepristone-treated mice after 14 or 21 days of treatment, probably due to an ovarian compensatory effect not observed in postmenopausal patients. We conclude that in future clinical trials using mifepristone, the possibility of lowering the standard daily dose of 200 mg should be considered.

THU570 Isoform Specific Gene Regulation By Progesterone Receptors Drives Expansion Of Breast Cancer Stem Cell Expansion In 3D Cell Culture Models

Abstract Disclosure: N.E. Gillis: None. T.H. Truong: None. C.A. Lange: None. Exposure to progesterone is a risk factor for breast cancer, and numerous PGR polymorphisms are linked to breast, endometrial, and ovarian cancer. Two progesterone receptor (PR) isoforms are expressed from PGR in breast tissues: full-length PR-B and truncated PR-A. In normal breast epithelia, PR isoforms are equally expressed and mostly function as A:B heterodimers. An imbalance of this ratio, and thus a predominance of homodimers, is frequent in ER+ breast cancer. Luminal A-type estrogen receptor positive (ER+) tumors often exhibit an elevated PR-A:PR-B ratio that predicts resistance to tamoxifen. It has recently been shown that PRs promote endocrine resistance and drive expansion of breast cancer stem-like cell (CSC) populations. However, the isoform-specific molecular mechanisms of PR action in ER+ breast cancers remain undefined. The goal of our current work is to map the progesterone-driven gene expression in CSC cells through transcriptomic profiling of T47D cells grown in spheroid-culture conditions that express exclusively PR-A or PR-B. This transcriptomic data has been integrated with phenotypic studies of T47D and MCF7 cells that express a single isoform of PR. Our data shows PR-A is a regulator of the cell cycle and of senescence, while PR-B mediates cell metabolism and intracellular signaling. We further integrated our cell line data with transcriptomes from patient tumors that have been classified based on their PR-A:PR-B ratio and their response to treatment with anti-progestins. Our results are a first step towards re-defining the role of PRs in gene regulation within the CSC population. Presentation Date: Thursday, June 15, 2023

Aberrations in the progesterone pathway and the Th1/Th2 cytokine dichotomy - An evaluation of RPL predisposition in the northeast Indian population.

Recurrent pregnancy loss (RPL) is the spontaneous loss of two or more consecutive pregnancies prior to 20weeks of gestation, occurring in 1% of the reproductive-age population. It is a major cause of infertility in India with a staggering 7.46% prevalence rate. Blood and product of conception (POCs) from RPL cases (n=65) were enrolled for this study, along with cases of medically terminated pregnancy (MTP, n=80) and term delivery cases (n=90) as control. ELISA for progesterone and progesterone induced blocking factor (PIBF) levels was carried out, followed by mRNA expression analysis of progesterone receptor isoform B (PR-B) and its downstream immunomodulatory effectors, namely, PIBF, IL-10 and IL-12. Screening of PROGINS haplotype of PR gene and PIBF polymorphism were also conducted to correlate with their respective gene expression profiles. Serum progesterone level was found to be comparable in the RPL and MTP cases. Although the mRNA expression of PR-B was found to be downregulated in the RPL cases, no significant PROGINS haplotype was observed. Presence of a single nucleotide polymorphism (SNP) in the PIBF gene (rs1372000) was more in healthy controls compared to RPL cases. Serum PIBF levels were found to be lower in the RPL cases with a resultant increase in IL-12 and a decrease in IL-10 mRNA expression in these cases. This study indicates that progesterone, acting through PIBF, modulates the immunological state of pregnancy to be Th1-biased in RPL, indicative of a pro-inflammatory, labour-like state not desired for a healthy pregnancy.

Progesterone receptor isoform ratios influence the transcriptional activity of progestins via the progesterone receptor

Progestins (synthetic progestogens) are progesterone receptor (PR) ligands used globally by women in both hormonal contraception and menopausal hormone therapy. Although four generations of unique progestins have been developed, studies seldom distinguish between the activities of progestins via the two functionally distinct PR isoforms, PR-A and PR-B. Moreover, not much is known about the action of progestins in breast cancer tumors where PR-A is mostly overexpressed relative to PR-B. Understanding progestin action in breast cancer is crucial since the clinical use of some progestins has been associated with an increased risk of developing breast cancer. This study directly compared the agonist activities of selected progestins from all four generations for transactivation and transrepression via either PR-A or PR-B, and when PR-A and PR-B were co-expressed at ratios comparable to those detected in breast cancer tumors. Comparative dose-response analysis showed that earlier generation progestins mostly displayed similar efficacies for transactivation on a minimal progesterone response element via the PR isoforms, while most of the 4th generation progestins, similar to the natural progestogen, progesterone (P4), were more efficacious via PR-B. Most of the progestogens were however more potent via PR-A. We are the first to show that the efficacies of the selected progestogens via the individual PR isoforms were generally decreased when PR-A and PR-B were co-expressed, irrespective of the ratio of PR-A:PR-B. While the potencies of most progestogens via PR-B were enhanced when the ratio of PR-A relative to PR-B was increased, those via PR-A were minimally influenced. This study is also the first to report that all progestogens evaluated, except 1st generation medroxyprogesterone acetate and 4th generation drospirenone, displayed similar agonist activity for transrepression via PR-A and PR-B on a minimal nuclear factor kappa B containing promoter. Moreover, we showed that the progestogen activity for transrepression was significantly increased when PR-A and PR-B were co-expressed. Taken together, our results highlight that PR agonists (progestogens) do not always display the same activity via PR-A and PR-B, or when PR-A and PR-B are co-expressed at ratios mimicking those found in breast cancer tumors. These results suggest that biological responses are progestogen- and PR isoform-dependent and may differ in target tissues expressing varying PR-A:PR-B ratios.

Intrauterine administration of autologous peripheral blood mononuclear cells regulates the endometrium estrogen and progesterone receptor expression: An RCT.

Repeated implantation failure (RIF) affects 15% of women of reproductive age. There is a high endometrial expression of both estrogen receptors and progesterone receptors (PRs) during the window of implantation in women with RIF. To evaluate the effects of intrauterine administration of human peripheral blood mononuclear cells (PBMC) on estrogen receptor α (ERα) and PRs expression in the endometrium of women with RIF during the implantation window. This randomized clinical trial study was conducted on 22 women with RIF history from January 2018 to August 2019 in Erfan hospital, Tehran, Iran. Participantswere divided into 2 groups (PBMC-treated group [n = 11] and control group [n = 11]). Endometrial tissue samples were collected at the implantation window time, during the mid-secretory phase (luteinizing hormone surge +7 days) of each menstrual cycle. The quantitative real-time polymerase chain reaction technique was used to measure the mRNA levels of ERα and PRs isoforms (PR-A and PR-B) in endometrial tissues. Furthermore, the protein expression of ERα and PRs was investigated using immunohistochemical staining. PBMC treatment significantly decreased the mRNA expression of endometrial ERα and PRs isoforms at the time of the implantation window (p 0.001). Moreover, the endometrial ERα and PRs protein localization were significantly lower in PBMC-treated women compared with controls (p = 0.01, and p 0.001 respectively). The intrauterine administration of PBMC decreased the endometrial ERα and PRs expression during the window of implantation in women with RIF. This local response to PBMC therapy could promote endometrial receptivity and embryo implantation.

PRB inhibited cell proliferation through let-7b-E2F1 in breast cancer.

The presence of progesterone receptor (PR) and PR isoform B (PRB) in breast cancer is generally correlated with better clinical outcomes. Additionally, the significance of hormone-independent effects of PR/PRB correlated with better prognosis have been reported in non-small lung cancer (NSCLC). However, the detailed mechanism of that still remains unclear. In this study, we examined how microRNAs (miRNAs) could contribute to tumor inhibition via PR/PRB expression, in order to find miRNAs that have tumor-agnostic effects between breast cancer and NSCLC. We obtained miRNAs data using human tissues of breast cancer and NSCLC from The Cancer Genome Atlas database and PCR array from NSCLC patients of our cohort. Subsequently, we examined the function of the miRNA through in vitro study using breast cancer cell lines. As a result, only let-7b expression was significantly correlated with PR expression in both cancers. Additionally, the expression of let-7b significantly inhibited cell proliferation by inducing PR and PRB expression in breast cancer cell lines. However, the positive correlation of let-7b and PRB required a mediated factor, E2F1, obtained from TGCA databases analysis. In vitro experiments showed that let-7b significantly inhibited E2F1, and E2F1 significantly inhibited PRB. This study revealed that PRB inhibits the proliferation of breast cancer cells by the let-7b-E2F1 interaction. In addition, the immunohistochemical analysis in NSCLC was also consistent with these in vitro data. Our results could contribute to developing novel therapeutic strategies for patients with PR/PRB-positive cancer by targeting let7b or PRB expression in breast cancer and possibly NSCLC.

Abstract 3994: Differential regulation of hormone receptors and Ki67 in luminal breast carcinomas and tumor-adjacent mammary glands by mifepristone treatment

Abstract Since antiprogestins inhibit the growth of preclinical mammary tumor models expressing higher levels of progesterone receptor (PR) isoform A (PRA) than isoform B (PRB), we designed a window-of-opportunity trial (MIPRA; NCT02651844) to study the benefits of mifepristone (MFP) in luminal breast carcinomas from postmenopausal patients selected by their PR+ expression (&amp;gt;50%) and their PR isoform ratio (PRA/PRB&amp;gt;1.5; PRA-H). A decrease in the cell proliferation marker Ki67 was registered after treatment in 14 out of the 20 tumors evaluated. This inhibition was associated with a decrease in PR, estrogen receptor (ER) and pSer118ER evaluated by immunohistochemistry while no changes in pSer167ER expression were observed (PMID: 36269797). In selected tissues from these samples we observed that the staining intensity in trapped glands within the MFP-treated tumors did not follow a similar trend as that of tumor cells. Thus, the aim of this study was to evaluate the expression of hormone receptors and Ki67 in non-neoplastic human mammary glands (nnMG) adjacent to the PRA-H tumor tissue of postmenopausal patients, treated (n=8) or not (n=9) with MFP. No differences in PR, ER and pSer118ER expression were observed between MFP-treated nnMG and those from untreated patients. Notably, contrarily to what occurred in tumors, there was a significant decrease of pSer167ER expression (p=0.008) in the MFP-treated nnMG compared to the untreated glands, suggesting a selective modulation in the AKT-mediated activation of ER. Regarding the proliferative state of the nnMGs, there was a slight but significant increase (p=0.02) in Ki67 expression in MFP-treated vs. non-treated nnMG. Data regarding the effect of antiprogestins in normal mammary glands is limited and points, in premenopausal women, to a decrease in the Ki67 index after treatment. The basal quiescent status of nnMG in postmenopausal women may explain this slight stimulatory effect. In conclusion, our results show that MFP exerts a specific regulatory effect in PRA-H tumors that is not observed in the nnMG, probably due to the fact that nnMG have presumably equimolar levels of PRA and PRB. In addition, to counteract the possible stimulatory effect that MFP may induce on nnMG, the combination of MFP and tamoxifen treatment is suggested. Citation Format: Leo Saldain, Silvia Vanzulli, Paula Martinez Vazquez, Javier Burruchaga, Eunice Spengler, Claudia Lanari, Paola Rojas. Differential regulation of hormone receptors and Ki67 in luminal breast carcinomas and tumor-adjacent mammary glands by mifepristone treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3994.

Abstract 3084: Progesterone receptor mediated crosstalk between cancer associated fibroblasts (CAFs) and ER+/PR+ breast cancer cells through CAF-secreted FGF2

Abstract Late recurring ER+ breast cancer emerges in post-menopausal women despite low/no hormone conditions. Cancer associated fibroblasts (CAFs) represent a major player in the promotion of metastasis, stemness and chemotherapy resistance in breast cancer. Progesterone receptors (PR) are emerging as drivers of stemness and metastasis in ER+ breast cancers. We propose that CAFs provide a mechanism to turn on ligand-independent PR signaling pathways through the secretion of paracrine factors. Methods: Tumorsphere assays were used to measure stem cell expansion. Cell clustering assays were used to measure CAF/tumor cell interactions. ChIP assays were used to measure PR direct binding to stemness gene regulatory elements. Results: Using the ER(+) T47D cell series (i.e., PR-null variants or cells expressing either endogenous PR (T47DCO) or PR-A or PR-B isoforms), we observed that CAF conditioned media (CM) increased tumorsphere formation in PR+ cells relative to PR-null and controls. Similar results were obtained with MCF7L cell models. CAF CM activated MAPK/ERK kinases and induced rapid phosphorylation of PR at Ser294. Physical interaction between CAFs and PR expressing T47D and MCF7L cells in clustering assays demonstrated PR driven co-cluster formation. CAF-derived FGF2 increased PR dependent tumorsphere and co-cluster formation. Blockage of FGF2 with the inhibitor, PD173074, decreased CAF CM-induction of tumorspheres and co-cluster formation. Previous data demonstrated that the cell adhesion molecule, CD44, is implicated in CAF/BC co-cluster formation. Both FGF2 and progestins increased CD44 expression in ER+ breast cancer cell models. Chromatin immunoprecipitation (ChIP) assays demonstrated that both PR isoforms are recruited to PRE-containing regions in the CD44 gene in response to progestin. Finally, we compared the gene expression profiles of breast cancer cells from T47DCO mono-clusters and CAF/T47DCO co-clusters using Single Cell RNA Seq. Among the differentially expressed genes identified were two genes implicated in tamoxifen resistance, BAMBI and NR2F2. Conclusion: We propose a model in which clustering between CAFs and ER+/PR+ breast cancer cells is promoted by the CAF secreted factor, FGF2. FGF2 promotes PR activation and upregulation of CD44 expression, which may further increase physical cell-cell interactions. Importantly, CAF-derived FGF2 can also act through PR to initiate MAPK/ERK signaling pathways that drive stemness and endocrine therapy resistance. Our studies of the interactions between PR+ breast cancer cells with CAFs either in the primary tumor or in the circulation (as CAF/CTC clusters) suggest the need to include anti-progestins as a component of endocrine therapy in late recurring ER+ breast cancer. Citation Format: Angela K. Spartz, Caroline H. Diep, Carol A. Lange, Dorraya El-Ashry. Progesterone receptor mediated crosstalk between cancer associated fibroblasts (CAFs) and ER+/PR+ breast cancer cells through CAF-secreted FGF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3084.

Abstract 5289: Proteomic profile of breast carcinomas with unbalanced levels of progesterone receptor isoforms

Abstract Preclinical studies indicate that progesterone receptors (PR) play a relevant role in breast carcinogenesis. Even more, a misbalanced expression of PR isoforms A (PRA) and B (PRB) differentially affect breast cancer progression and only tumors with higher levels of PRA than PRB (PRA-H) respond to an antiprogestin treatment. There are controversial results regarding the prognosis of PRA-H luminal tumors compared with those with the opposite ratio (PRB-H tumors) highlighting the necessity to further understand the role of PR isoforms in tumor progression and to develop novel methods to discriminate these tumors without performing western blot studies. Along this line we have previously studied the transcriptome of PRA-H or PRB-H breast cancer samples, thus our aim is to expand these results and compare the proteome profile of PRA-H and PRB-H samples obtained from postmenopausal breast cancer patients. Nuclear (Nuc) and cytosolic (Cyt) protein fractions were obtained from 18 breast cancer samples classified as PRA-H or PRB-H and they were studied by LC-MS/MS (UltiMate 3000 LC system - Q Exactive HF-X mass spectrometer - Thermo). We observed 289 differentially deregulated proteins in Cyt (164 down and 125 up) and 301 in Nuc extracts (131 down and 170 up; logFC &amp;gt; 1, pval &amp;lt; 0.05). Gene set enrichment analysis of the Nuc fractions showed biological processes related to Cell junction organization (pval = 8,87e-05), Collagen formation (pval = 0,0005), and Cell-Cell communication processes (pval = 0,0035) in PRB-H tumors; while in PRA-H tumors, pathways related to Innate Immune System (pval = 8,8e-05), Class I MHC mediated antigen processing &amp; presentation (pval = 8e-05) and Stabilization of p53 (pval = 0,0051) processes were enriched. Similar trends were observed in Cyt fractions. Individual candidates that were overrepresented in both fractions in PRB-H tumors are BAG3 and GH3, related to cell proliferation, and VWF, related to the activation of the Akt pathway. Overrepresented proteins in both fractions of PRA-H tumors are BST2, IFT27 related to invasion, and MIF associated with poor prognosis. As we were interested in looking for differential expressed proteins that could be tested in plasma samples to discriminate both tumor types, using the Vertebrate Secretome Database we focused on those that might be secreted. Noteworthy is CBP1 which was overrepresented in the PRB-H tumors and this was in agreement with the RNA-Seq data and with results obtained using preclinical models, repurposing this protein as one candidate of concern in the search for biomarkers. Other secretory proteins of interest are SFRP2 and SERPIND1, both highly expressed in PRA-H Cyt fractions. In conclusion, this study underscores the biological differences between PRA-H and PRB-H breast carcinomas and provides candidates that deserve to be tested as surrogate markers in plasma to discriminate patients that may benefit from an antiprogestin treatment. Citation Format: Andres M. Elia, Jana Sanchez, Rui Vitorino, Leo Saldain, Paula Martinez Vazquez, Javier Burruchaga, Eunice Spengler, Javier Muñoz, Paola Rojas, Luisa Helguero, Claudia Lanari. Proteomic profile of breast carcinomas with unbalanced levels of progesterone receptor isoforms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5289.

Патогенетические особенности сигнального пути прогестерона и экспрессия PR-B при глубоком инфильтративном эндометриозе

Objective. To study the expression of progesterone receptor isoform B (PR-B) in patients with deep infiltrating endometriosis (DIE). Patients and methods. This article presents a comparative analysis of PR-B expression in eutopic and ectopic endometrial tissue samples obtained from patients with DIE (study group, n = 50) and tubo-peritoneal factor infertility (comparison group, n = 9). PR-B expression was analyzed by immunohistochemistry. Results. In the group of patients with DIE, PR-B expression in ectopic endometrial glandular and stromal cells of endometriosis lesions in the peritoneum and colon was significantly higher compared to the capsule of ovarian endometriotic cyst (p &lt; 0.05). Analysis of PR-B expression in eutopic endometrium showed no significant differences between patients in the study group and comparison group (p &gt; 0.05). Stromal cell PR-B expression in patients with infertility in the study group was significantly lower compared to fertile patients with DIE (p = 0.04). Conclusion. No decrease in PR-B expression in the endometrium of patients with DIE was found, which may indicate a favorable response to postoperative hormonal therapy in this group of patients. At the same time, we observed a decrease in PR-B expression in eutopic endometrial stromal cells in patients with DIE and infertility compared to fertile patients, which confirms the role of PR in disrupting pregnancy onset in endometriosis. Search for prognostic markers is currently implemented to develop a personalized approach to the treatment of endometriosis and individual selection of hormone therapy for preventing primary and subsequent surgical interventions and reducing severe pain syndrome in endometriosis. Key words: progesterone, PR-B expression, deep endometriosis, pain syndrome, dienogest

The transcriptional activity of progestins used in contraception and menopausal hormone therapy via progesterone receptor A is dependent on the density of the receptor

Studies directly comparing the efficacies and potencies of multiple progestins used in contraception and menopausal hormone therapy (MHT) in parallel via human progesterone receptor isoform A (PR-A) in the same model system are limited, and how these parameters are influenced by the density of PR-A are unclear. This is surprising as it is known that the expression levels of PR-A vary in different tissues and diseases. We thus determined for the first time the relative efficacies and potencies for transactivation of the natural PR ligand, progesterone (P4), the PR-specific agonist promegestone (R5020), and selected progestins from all four generations in parallel via different densities of PR-A overexpressed in the MDA-MB-231 breast cancer cell line. Comparative dose-response analysis showed that P4, R5020, the 1st generation progestins medroxyprogesterone acetate and norethisterone, 2nd generation progestin levonorgestrel, 3rd generation progestin gestodene, as well as 4th generation progestins nesterone, nomegestrol acetate and drospirenone display differential agonist efficacies and potencies via PR-A. Moreover, we showed that the agonist efficacies and potencies of the progestins via PR-A were modulated in a density- and progestin-specific manner. Our finding that the potencies of the progestins via PR-A, at all densities, do not exceed reported progestin serum concentrations in women, suggest that these progestins are likely to elicit similar effects in vivo. We are the first to report that P4 and the selected progestins display similar agonist activity for transrepression via PR-A, and that the density of PR-A enhances the transrepression activity of some, but not all progestogens. Collectively, our findings provide proof of concept that the effects of the selected progestins via PR-A is progestin-specific and dependent on the density of the receptor, suggesting differential progestin responses in women using these progestins in contraception and MHT.

The role of progesterone receptor isoforms in the myometrium

Myometrial contraction is stringently controlled throughout pregnancy and parturition. Progesterone signaling, effecting through the progesterone receptor (PR), is pivotal in modulating uterine activity. Evidence has shown that two major PR isoforms, PR-A and PR-B, have distinct activities on gene regulation, and the ratio between these isoforms determines the contractility of the myometrium at different gestational stages. Herein, we focus on the regulation of PR activity in the myometrium, especially the differential actions of the two PR isoforms, which maintain uterine quiescence during pregnancy and regulate the switch to a contractile state at the onset of labor. To demonstrate the PR regulatory network and its mechanisms of actions on myometrial activity, we summarized the findings into three parts: Regulation of PR Expression and Isoform Levels, Progesterone Receptor Interacting Factors, and Biological Processes Regulated by Myometrial Progesterone Receptor Isoforms. Recent genomic and epigenomic data, from human specimens and mouse models, are recruited to support the existing knowledge and offer new insights and future directions in myometrial biology.