Progesterone Receptor Protein Research Articles

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment.

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine® , 10mg/kg) was injected subcutaneously 5days before ovariohysterectomy in the treatment group (n=6); bitches without treatment served as a control group (n=6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p<.05) and ERα mRNA increased (p<.01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p<.05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p<.01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

Effect of combined administration of electroacupuncture and mesenchymal stem cells on expression of endometrium estrogen receptor and progesterone receptor in thin endometrium rats

To investigate the effect of joint administration of electroacupuncture (EA) and bone marrow mesenchymal stem cells (BMMSC) on the expression of estrogen receptor (ER) and progesterone receptor (PR) in thin endometrium rats, so as to explore its mechanisms underlying improvement of conception rate. Forty female SD rats were divided into control, model, BMMSC and EA+BMMSC groups. The thin endometrium model was established by intrauterine infusion of 95% ethanol and saline. EA (1 mA, 2 Hz/15 Hz) was applied to "Guanyuan"(CV4), unilateral "Zigong" (EX-CA1) and unila-teral "Sanyinjiao" (SP6) for 15 min, once daily for 10 d. Rats of the BMMSC and EA+BMMSC groups received injection of BMMSC suspension through caudal vein on the day of modeling, the 3rd day after surgery, and the 2nd and the 3rd estrus phases, respectively. Changes of body weight and estrus phase were continuously recorded. After three estrus cycles, uterine specimens were taken and the uterine coefficient was calculated. The immunoactivity of KI67(an antigen for cell proliferation) was detected by immunohistochemistry, and the expression of ER and PR was detected by Western blot. Compared with the control group, the uterine coefficient, and expression of endometrium ER and PR proteins were significantly decreased in the model group (P<0.001, P<0.01). In Comparison with the model group, the uterine coefficient, immunoactivity of KI67, and expression of ER and PR proteins were significantly increased in both BMMSC and EA+BMMSC groups (P<0.000 1,P<0.001, P<0.01). The expression level of PR in the EA+BMMSC group was notably higher than that in the BMMSC group (P<0.05), but no significant difference was found between the BMMSC and EA+BMMSC groups in the levels of uterine coefficient and ER expression (P>0.05). EA combined with BMMSC injection has a positive effect in promoting the proliferation of endometrium cells in rats with thin endometrium, which may be related to its effect in up-regulating the expression of ER and PR proteins.

Evaluation of pharmacodynamic (PD) and biologic activity in a preoperative window-of-opportunity (WOO) study of giredestrant (GDC-9545) in postmenopausal patients (pts) with estrogen receptor-positive, HER2-negative (ER+/HER2–) operable breast cancer (BC).

577 Background: Modulation of ER activity and/or estrogen synthesis is the mainstay therapeutic strategy in ER+ BC treatment. Giredestrant is a highly potent, nonsteroidal oral selective ER degrader (SERD) that achieves robust ER occupancy and is effective regardless of ESR1 mutation status. The first short-term preoperative WOO study (NCT03916744) of giredestrant in ER+/HER2– operable BC was designed for dose selection, while providing an early readout of PD as measured by traditional immunohistochemistry (IHC) and transcriptional profiling by assessing treatment effects in paired tumor tissue pre/posttreatment. We present an interim analysis. Methods: Pts were assigned to 14 days’ preoperative treatment with 10, 30, or 100 mg PO giredestrant QD. Pts had newly diagnosed, stage I–III operable, ER+/HER2– untreated BC ≥1.5 cm in diameter (by ultrasound). Modulation of ER signaling and cell proliferation were assessed using paired formalin-fixed paraffin-embedded tumor specimens collected before and after ̃14 days of study treatment. ER, progesterone receptor (PR), and Ki67 protein levels were analyzed by IHC. Change from baseline in tumor cell proliferation by Ki67 was the primary endpoint. Gene expression analysis was performed using the Illumina TruSeq RNA Access method. Results: From Jul 26, 2019 to Oct 15, 2020, 46/75 biomarker-evaluable pts were enrolled across three dose cohorts (10 mg: n = 15; 30 mg: n = 18; 100 mg: n = 13). Pt demographics and tumor characteristics were similar across cohorts. Baseline PAM50 analysis classified tumors as Luminal A (77%) or B (23%). Giredestrant treatment resulted in robust and indistinguishable PD and biologic activity at all doses. Geometric mean posttreatment proportional reduction of Ki67 was 79% (95% CI: 69–89; 10 mg: 80%; 30 mg: 76%; 100 mg: 80%), and 51% of tumors exhibited complete cell cycle arrest, defined as Ki67 ≤2.7%. Mean posttreatment proportional reductions of ER and PR H-scores were 71% (95% CI: 67–75) and 60% (95% CI: 51–70), respectively. An analysis of a predefined, experimentally derived set of 38 ER target genes (the ‘ER activity signature’), was completed for 42 paired tumor specimens. Forty-one of 42 pts (98%) showed a posttreatment reduction in ER activity with a mean proportional decrease of 79% (95% CI: 70–88). A wide range of baseline ER activity was observed with no correlation to baseline ER or PR H-score, or Ki67. There were no discontinuations due to adverse events (AEs). A single grade 3 serious AE was reported in each cohort (all assessed as unrelated to giredestrant). No grade 4 or 5 AEs were reported. Conclusions: Giredestrant was well tolerated in the preoperative setting in ER+/HER2– operable BC, and PDs were consistent with the 30 mg dose achieving maximal ER inhibition. Clinical trial information: NCT03916744.

Low-dose aspirin can downregulate progesterone resistance and increase the expression of LIF in endometriosis during the implantation window

Aim Study the effect of low-dose aspirin on the endometrial receptivity in endometriosis rat models. Materials and methods This study is to explore the expressions of progesterone receptor and LIF among three groups of endometriosis rat models: control group (n = 12), EMs group (n = 15), and aspirin group (n = 17). The expressions of progesterone receptor (PR), PRA, PRB, and leukemia inhibitory factor receptor (LIFR) in eutopic endometrium were determined using immunohistochemistry technology, western blot, and qRT-PCR. The levels of LIF in eutopic endometrium and serum were detected by western blot, qRT-PCR, and ELISA. Results The expressions of PR, PRA, and PRB protein were significantly increased in the eutopic endometrium after low-dose aspirin treatment, and the level of PRB mRNA was also increased while the ratio of PRA/PRB mRNA was decreased in the eutopic endometrium. The levels of LIF in eutopic endometrium and serum were increased compared with the untreated endometriosis rats. However, the expression of LIFR was not statistically different among the three groups. Conclusions The results suggest that the low-dose aspirin treatment could downregulate progesterone resistance and increase the expression of LIF of endometriosis rats during the implantation window, which could improve endometrial receptivity and enhance the pregnant rate of endometriosis. It may provide a potential treatment method for endometriosis-related infertility.

Mechanism of Action of Irilone as a Potentiator of Progesterone Receptor Signaling

Progesterone signaling and its proper regulation is important for reproductive function. When progesterone signaling is dysregulated, gynecological diseases can occur, for example endometriosis, uterine fibroids, and endometrial cancer. While these diseases are treated with progestin therapy, progestins can bind to multiple steroid receptors, exerting side effects of weight gain, immunosuppression, cardiovascular disease, and stroke. Discovering an alternative progestin that is selective for the progesterone receptor (PR) is ideal. One potential source of such an alternative is botanical dietary supplements, which have become increasingly popular among consumers with sales reaching $9.6 billion in 2019. Although botanical supplements are popular, the chemical structures and biological action of botanical supplements would benefit from deeper scientific investigation. Studies of Trifolium pratense L. (red clover), primarily used for the treatment of menopausal symptoms, identified phytoestrogen compounds as the chemicals that mitigate those symptoms. Interestingly, irilone, identified from red clover, potentiated progesterone signaling via a progesterone response element luciferase (PRE/Luc) assay. Potentiation is when a compound has no activity by itself but when combined with another molecule, i.e. progesterone, that compound enhances PR activity. Prior to irilone, a natural compound with the ability to potentiate progesterone signaling had not been previously reported. The purpose of this study was to determine the mechanism of action of irilone. We hypothesized that irilone was potentiating PR by blocking PR degradation and by altering PR post-translational modifications. Irilone was found to potentiate 5 nM P4 using a PRE-luciferase assay in both T47D and Ishikawa PR expressing cells. Since PR is a downstream target gene of ER, we investigated if irilone also had ER activity. Irilone increased expression of an ERE-luciferase reporter gene. Next, we investigated if irilone could stabilize PR degradation and if irilone altered PR phosphorylation via western blot. Irilone was found to increase PR protein levels, but when ER was blocked, this was mitigated. In the presence of P4, irilone did not increase phosphorylation of serine 294 on PR. Future studies will determine if irilone is altering sumoylation of PR, and if irilone can potentiate PR signaling in vivo. Determining how irilone is potentiating progesterone will help us understand PR biology and could be an effective treatment for gynecological diseases by enhancing endogenous progesterone action.

Intracellular Progesterone Receptor and cSrc Protein Working Together to Regulate the Activity of Proteins Involved in Migration and Invasion of Human Glioblastoma Cells.

Glioblastomas are the most common and aggressive primary brain tumors in adults, and patients with glioblastoma have a median survival of 15 months. Some alternative therapies, such as Src family kinase inhibitors, have failed presumably because other signaling pathways compensate for their effects. In the last ten years, it has been proven that sex hormones such as progesterone (P4) can induce growth, migration, and invasion of glioblastoma cells through its intracellular progesterone receptor (PR), which is mostly known for its role as a transcription factor, but it can also induce non-genomic actions. These non-classic actions are, in part, a consequence of its interaction with cSrc, which plays a significant role in the progression of glioblastomas. We studied the relation between PR and cSrc, and its effects in human glioblastoma cells. Our results showed that P4 and R5020 (specific PR agonist) activated cSrc protein since both progestins increased the p-cSrc (Y416)/cSrc ratio in U251 and U87 human glioblastoma derived cell lines. When siRNA against the PR gene was used, the activation of cSrc by P4 was abolished. The co-immunoprecipitation assay showed that cSrc and PR interact in U251 cells. P4 treatment also promoted the increase in the p-Fak (Y397) (Y576/577)/Fak and the decrease in p-Paxillin (Y118)/Paxillin ratio, which are significant components of the focal adhesion complex and essential for migration and invasion processes. A siRNA against cSrc gene blocked the increase in the p-Fak (Y576/Y577)/Fak ratio and the migration induced by P4, but not the decrease in p-Paxillin (Y118)/Paxillin ratio. We analyzed the potential role of cSrc over PR phosphorylation in three databases, and one putative tyrosine residue in the amino acid 87 of PR was found. Our results showed that P4 induces the activation of cSrc protein through its PR. The latter and cSrc could interact in a bidirectional mode for regulating the activity of proteins involved in migration and invasion of glioblastomas.

ARID1A and PGR proteins interact in the endometrium and reveal a positive correlation in endometriosis

Endometriosis is a disorder in which endometrial cells normally limited to the lining of the uterus proliferate outside the uterine cavity and can cause pelvic pain and infertility. ARID1A levels are significantly reduced in the eutopic endometrium from women with endometriosis. Uterine specific Arid1a knock-out mice were infertile due to loss of epithelial progesterone receptor (PGR) signaling. However, the functional association of ARID1A and PGR in endometriosis has not been studied. We examined the expression patterns and co-localization of ARID1A and PGR in eutopic endometrium from women with and without endometriosis using immunostaining and Western blot analysis. ARID1A and PGR proteins co-localized in the epithelium during the proliferative and the early secretory phases. Our immunoprecipitation analysis and proximity ligation assay (PLA) revealed physical interaction between ARID1A and PGR-A but not PGR-B in the mouse and human endometrium. ARID1A levels positively correlated with PGR levels in the eutopic endometrium of women with endometriosis. Our results bring new perspectives on the molecular mechanisms involved in endometrial receptivity and progesterone resistance in endometriosis. The interrelationship between ARID1A and PGR may contribute to explaining the non-receptive endometrium in endometriosis-related infertility.

Abstract GS4-09: Correlative studies of the breast cancer index (HOXB13/IL17BR) and ER, PR, AR, AR/ER ratio and Ki67 for prediction of extended endocrine benefit: A trans-aTTom study

Abstract Background: Several biomarkers such as estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and Ki67 have been implicated in the pathogenesis and/or as prognostic biomarkers of breast cancer, and are utilized to determine treatment. Given the heterogeneity of response to endocrine therapy, however, predictive biomarkers are critical to better individualize patient care. Previous results from the Trans-aTTom study demonstrated that the Breast Cancer Index HOXB13/IL17BR [BCI (H/I)] biomarker significantly predicts extended endocrine benefit from 10 vs 5y of tamoxifen. In this correlative study, the predictive activity of BCI (H/I) was compared with ER, PR, AR and Ki67 protein expression in node positive patients treated in the aTTom trial. Methods: Patients with available tumor tissue and biomarker analyses were included. ER, PR, AR and Ki67 were centrally assessed by immunohistochemistry (IHC) utilizing tissue microarrays. BCI gene expression analysis by RT-PCR was performed blinded to clinical outcome. Multivariate Cox models adjusting for age, tumor size, tumor grade and HER2 status were used to assess the significance of the interaction between treatment and each biomarker as continuous variables. 17-year risk of recurrence, as a function of each continuous biomarker, was estimated from Cox models in each of the 2 treatment arms. Results: Analysis of 789 HR+, N+ patients showed a weak negative correlation between BCI (H/I) and ER, PR, and AR expression whereas Ki67 and the AR/ER ratio showed no correlation (ER, cor=−0.18; PR, cor=−0.25; AR, cor=−0.14; Ki67, cor=0.04; AR/ER ratio, cor=0.02). The interaction between BCI (H/I) and extended tamoxifen treatment was significant (p=0.014). In addition, analysis of risk of recurrence as a function of continuous BCI (H/I) demonstrated that the magnitude in the reduction in recurrence risk with extended tamoxifen correlated with increasing H/I levels. In contrast, interaction P values were nonsignificant (ER, p=0.829; PR, p=0.659; AR, p=0.783; Ki67, p=0.865; AR/ER ratio, p=0.835) and the magnitude of endocrine benefit did not correlate with expression levels of any of other biomarkers. Conclusion: Results from this post-hoc analysis of the Trans-aTTom study demonstrated that whereas BCI(H/I) is a significant predictive biomarker of endocrine response, analysis of ER, PR, AR, Ki67 and AR/ER expression showed no interaction with treatment, and lacked the ability to predict benefit of extended tamoxifen in HR+ early stage breast cancer. These results add to the growing body of evidence that BCI (H/I) is distinct in its ability to predict benefit from therapy and interrogates distinct tumor biology that is not captured by other traditional biomarkers. Citation Format: Dennis C Sgroi, Kai Treuner, Yi Zhang, Tammy Piper, Ranelle Salunga, Ikhlaaq Ahmed, Lucy Doos, Sarah Thornber, Karen J Taylor, Elena F Brachtel, Sarah Pirrie, Catherine A Schnabel, Daniel W Rea, John MS Bartlett. Correlative studies of the breast cancer index (HOXB13/IL17BR) and ER, PR, AR, AR/ER ratio and Ki67 for prediction of extended endocrine benefit: A trans-aTTom study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS4-09.

Evaluation of Expression and Phosphorylation of Progesterone Receptor in Endometrial Stromal Cells of Patients with Recurrent Implantation Failure Compared to Healthy Fertile Women

Recurrent implantation failure (RIF) is the repeated failure of good-quality embryos in implantation process following several assisted reproduction cycles. Disruption of the endometrial receptivity is one of the main causes of RIF. Progesterone plays a pivotal role in the endometrial receptivity through the regulation of gene expression pattern by binding to its receptors in the endometrial cells. The aim of this study was to evaluate the expression level of progesterone receptor (PR) and its phosphorylated form in the endometrial stromal cells (eSC) of RIF patients and compare it to the eSC of healthy fertile women as control group. After isolation of the eSC from biopsy samples of RIF patients and healthy fertile women and their characterization, expression levels of PR mRNA, PR protein, and phospho-Ser294 PR protein were evaluated by quantitative real-time PCR and immunofluorescence staining, respectively. The results demonstrated a significant reduction in PR mRNA expression (P < 0.01.) and phospho-Ser294 PR protein (P < 0.05) level in RIF patients compared to the control group. These data for the first time suggest that the expression of PR and its phosphorylated form are impaired in RIF patients. Therefore, designing therapeutic methods for improving PR expression status and its regulation in the endometrium of RIF patients may help in improving the final reproductive outcomes of these cases.

Patterns of sex hormone receptor expression in stimulated endometrium from oocyte donors

In patients with high serum E2 embryo transfer is often postponed, as high E2 levels adversely affect embryo transfer outcome. We aimed to determine if stratified serum oestradiol (E2) and progesterone (P4) levels differentially affect endometrial histology and endometrial oestrogen and progesterone receptor protein levels. Endometrial biopsies were collected from oocyte donors. Samples were divided based on peak serum E2 levels into three groups: (i) low-E2 (n = 33) E2≤2999pg/mL; (ii) mid-E2 (n = 40) E2 3000–4999 pg/mL; and (iii) high-E2 (n = 15) E2≥5000 pg/mL. Oestrogen receptor alpha (ERα) and progesterone receptors A and B (PR) protein levels in endometrial stroma (S), glandular (GE) and luminal (LE) epithelia were assessed by immunohistochemistry. Samples in high-E2 group demonstrated strongest association with accelerated endometrial maturation (2 (1–2); 2 (1–3); and 3 (2.8–3) median days of advancement of endometrial maturation respectively in low, mid, high-E2 groups, p = 0.046). There were significant differences in ERα and PR immunoexpression in S, GE and LE among the groups (p < 0.05). Higher E2 levels were associated with decreased ERα expression (p < 0.017) in GE and LE, and increased PR expression in S and GE (p < 0.011 and p < 0.0001, respectively). Higher serum E2 levels were associated with impaired endometrial steroid hormone receptor expression, higher serum P4 and more advancement of endometrial maturation.

3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways.

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A “seesaw” relationship between ER/PR and Her2 pathways was observed in ER–PR–Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER–PR–Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER–PR space. Conclusions: The unique distribution of specimens in ER–PR–Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

Immunohistological expression of oestrogen receptor, progesterone receptor, mammaglobin, human epidermal growth factor receptor 2 and GATA-binding protein 3 in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) and breast cancer are common entities. Staining for oestrogen receptor (ER), progesterone receptor (PgR), mammaglobin (MAMG) and GATA-binding protein 3 (GATA3) is frequently performed to confirm a mammary origin in the appropriate diagnostic setting. However, comprehensive data on the immunohistological expression of these markers in NSCLC are limited. Therefore, the aim of this study was to analyse a large cohort of NSCLCs and correlate the staining results with clinicopathological variables. A tissue microarray was stained for ER, PgR, MAMG, human epidermal growth factor receptor 2 (HER2), and GATA3, and included 636 adenocarcinomas (ADCs), 536 squamous cell carcinomas (SqCCs), 65 large-cell-carcinomas, 34 pleomorphic carcinomas, and 20 large-cell neuroendocrine carcinomas. HER2 status was determined for immunohistochemically positive cases with chromogenic in-situ hybridisation. Markers with a proportion of ≥5% positive cases in ADC and SqCC were considered for survival analysis. Among ADCs, 62 (10%), 17 (3%), one (<1%), seven (1%), and 49 (8%) cases were positive for ER, PgR, MAMG, HER2, and GATA3, respectively. Among SqCCs, 10 (2%), 14 (3%), two (<1%) and 109 (20%) cases were positive for ER, PgR, HER2, and GATA3, but none of the samples showed positivity for MAMG. ER positivity was associated with ADC, female sex, smaller tumour size, and lower clinical stage. None of the markers had an impact on survival. We report on ER, PgR, MAMG, HER2 and GATA3 expression in a large cohort of NSCLCs. Interpretation of these markers in the differential diagnostic setting should be based on a multimarker panel.

Abstract B29: Ovarian hormones regulate C/EBPD induced EMT/MET transition in the human fallopian tube epithelia

Abstract Introduction: The histologically normal BRCA1 mutation carrier fallopian tube epithelia (FTE), compared to controls, showed that CEBPD was upregulated in the luteal phase of the ovulatory cycle. CEBPD is involved with the maintenance of genomic stability, promoting cellular differentiation, and regulating the cell cycle in response to cytotoxic stressors. In breast epithelial cells, CEBPD protein expression correlated with estrogen receptor (ER) and progesterone receptor (PR) and was found to be associated with increased progression-free survival in breast cancer patients. In fallopian tube epithelia (FTE), CEBPD was also found to modulate the epithelial-to-mesenchymal (EMT)/mesenchymal-to-epithelial transition (MET) by modulating target genes of this pathway. Given the hormonal response of this gene and its function in modulating an EMT/MET, the objective of this study was to determine whether sex hormones influence CEBPD regulation of EMT/MET in the fallopian tube and thus ovarian cancer. Methods: Fresh fallopian tube (FTE) tissues were obtained from patients approved for collection by IRB. Immunohistochemical profiling on normal fallopian tube tissue and HGSC was performed using CEBPD, ER, and PR protein markers. FTE cell lines with a p53 mutation (R175H) were subjected to estradiol (50nM and 100nM) and 4-hydroxytamoxifen (10nM) and assayed using qRT-PCR and PCR. ANOVA and t-tests were conducted in GraphPad Prism software with significance set at p&amp;lt;0.05. Results: E-cadherin in normal fallopian tube tissue was highly expressed in both the luteal and follicular phase whereas vimentin was highly expressed in the follicular phase but showed a range of expression (low expression to high expression) in the luteal phase. CEBPD overexpression increased SNAIL expression (p&amp;lt;0.0001), consistent with previous findings; treatment with 50nM estradiol (E2) resulted in increased SNAIL and SLUG mRNA expression in FTE cell lines overexpressing CEBPD (p&amp;lt;0.0001) relative to controls and decreased ZEB1 and ZEB2 mRNA expression. Addition of tamoxifen to cells overexpressing CEBPD increased SNAIL mRNA expression compared to cells without tamoxifen (p&amp;lt;0.0001); however, a combination of both tamoxifen and estradiol added to these cells decreased SNAIL expression relative to controls. IL6 mRNA expression level was increased in CEBPD overexpressing cells compared to controls (p&amp;lt;0.0001), which was further increased by E2 (p&amp;lt;0.0001). Conclusion: Together these results demonstrate a role for CEBPD in modulating EMT/MET in normal FTE in a hormonally regulated manner, which during cancer formation and spread is critical for anoikis and metastasis. Furthermore, these data will facilitate an understanding of the early events of carcinogenesis in fallopian tube epithelia. Citation Format: Ramlogan Sowamber, Leah V. Dodds, Patricia Shaw, Sophia H.L. George. Ovarian hormones regulate C/EBPD induced EMT/MET transition in the human fallopian tube epithelia [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B29.

A retrospective single-center study investigating the clinical significance of grade in triple-negative breast cancer.

e13108 Background: Triple negative breast cancer is defined as estrogen (ER), progesterone (PR), and human epidermal growth factor receptor (HER-2) proteins negative. The grade is the degree of similarity of tumor cells to normal cells under microscope and is an important biomarker of overall patient outcomes or prognosis with higher grades having a poor prognosis. As recent chemotherapy trials noted moderately undifferentiated grade 2 tumors showing higher rates of relapse, we hypothesize that grade can also be a predictive biomarker or determinant of response to specific treatment. Methods: We reviewed 305 patient charts of triple negative breast cancer patients from 2004-2017 at Windsor Regional Cancer Center analyzing the significance of grade with respect to oncological variables, survival-time, and time to relapse. Statistical analysis was performed using Fleming-Harrington, Pairwise Testing, and COX regression, where applicable. Results: Univariate analysis showed statistically significance difference in chemotherapy type (P = 0.008) and a marginal one in ER &amp; hormone therapy status (P ~0.09) between the grades. The overall survival rates were 90.12%, 64.4%, and 77.2%, for grade 1, 2, 3 respectively. The overall difference in survival among the three groups was statistically significant, based on Fleming-Harrington test (P = 0.019). Comparing only between grade 2 and grade 3, we found that after five years, grade 2 patients had a 5.5-fold increased risk of death (HR = 5.5; 95% CI 1.2-25.6) and 2-folds higher risk of relapse (HR = 1.9; 95% CI 1.1-3.2). Grade 3 does significantly better than grade 2 in time to relapse with relapse rates of 70%, 55.6 %, and 75.6%, respectively for grades 1, 2, and 3 (P = 0.04). Conclusions: Tumor grade has a significant positive predictive value in determining relapse with grade 2 tumors demonstrating poorer disease-free survival as compared to grade 1 &amp; 3, less time to relapse, and increased risk of death. This has implications in stratifying triple negative breast cancer patients by grade in future clinical trials while ongoing research yields new targets for chemotherapy.

Fractalkine Regulates HEC-1A/JEG-3 Interaction by Influencing the Expression of Implantation-Related Genes in an In Vitro Co-Culture Model.

Embryo implantation is a complex process regulated by a network of biological molecules. Recently, it has been described that fractalkine (CX3CL1, FKN) might have an important role in the feto–maternal interaction during gestation since the trophoblast cells express fractalkine receptor (CX3CR1) and the endometrium cells secrete fractalkine. CX3CR1 controls three major signalling pathways, PLC-PKC pathway, PI3K/AKT/NFκB pathway and Ras-mitogen-activated protein kinases (MAPK) pathways regulating proliferation, growth, migration and apoptosis. In this study, we focused on the molecular mechanisms of FKN treatment influencing the expression of implantation-related genes in trophoblast cells (JEG-3) both in mono-and in co-culture models. Our results reveal that FKN acted in a concentration and time dependent manner on JEG-3 cells. FKN seemed to operate as a positive regulator of implantation via changing the action of progesterone receptor (PR), activin receptor and bone morphogenetic protein receptor (BMPR). FKN modified also the expression of matrix metalloproteinase 2 and 9 controlling invasion. The presence of HEC-1A endometrial cells in the co-culture contributed to the effect of fractalkine on JEG-3 cells regulating implantation. The results suggest that FKN may contribute to the successful attachment and implantation of embryo.

Mechanism of Reproductive Effect of Acetyl acetate‐methanol Fraction of Adansonia digitata (Linn) Root Bark Extract in Female Wistar Rats

The aim of this work was to identify the exclusive components of Acetyl acetate‐methanol fraction (AAMF) of Adansonia digitata (Linn) root bark extract and to elucidate its mechanism of action in modulating hormone signaling. Gas chromatography and Mass spectrometry was done to identify the compounds present in fractions of A. digitata (Linn) root bark extract. Thirty female Wistar rats had their estrous cycles synchronized and were randomly distributed into three groups of ten rats each. Each group received 0, 150, and 300mg/kg body weight of AAMF per os from the day observed as proestrus by vaginal cytology to day‐seven. On day‐one, day‐four, day‐five, day‐six and day‐seven, blood was collected from rats in each group. Blood collected was analyzed for estrogen, progesterone and follicle stimulating hormone levels. Uterus and ovary were processed for histopathology and immunohistochemistry to study the regulation of estrogen and progesterone receptor proteins. Where a receptor protein was regulated, further investigation was done to determine its effect on the gene expression of that receptor in endometrial stromal cells through RNA extraction, OneStep reverse transcription‐pCR amplification, Agarose gel electrophoresis and quantification of pCR product. Results showed that AAMF uniquely contained 15.25% of oleic acid and caused mass atresia of antral follicles at proestrus. AAMF treated rats had significantly (p&lt;0.05) lowered serum estradiol levels at proestrus when compared to control rats and had up‐regulated estrogen receptor beta proteins in endometrium and downregulated estrogen receptor alpha (ERα) gene expression in endometrial cells. It was concluded that the oleic acid component of AAMF may have lowered estrogen level by inhibiting aromatase and hydroxysteroid 17 beta dehydrogenase enzyme activities in estrogen steroidogenesis. Also, oleic acid may have induced overexpression of Tumour Suppressor Factor (P53) which promotes follicular apoptosis and indirectly downregulates ERα when p53 activities become exacerbated. It is recommended that AAMF be investigated as a possible therapy for some ERα sensitive clinical conditions like cancer.

Immunohistochemical Expression of BRCA1 Protein, ER, PR and Her2/neu in Breast Cancer: A Clinicopathological Study.

Breast cancer is a heterogeneous hormone-dependent disease. Potential prognosis depends on the clinicopathological evaluation and assessment of other prognostic indicators. The detection of the oestrogen Receptor (ER), Progesterone Receptor (PR), Human epidermal growth factor receptor 2 (Her2/neu) and BRCA1 oncoprotein is pivotal for prognostic evaluation and to choose the appropriate post-surgical adjuvant therapy beside selecting the proper candidate for genetic counselling. Objectives:To detect the immunoexpression of the BRCA1 oncoprotein in mammary invasive ducal carcinoma and its association with the prognostic markers (ER, PR and Her2/neu hormonal receptors) and other clinicopathological parameters to improve the patients’ treatment plans. Methods:A cross-sectional study design including 83 paraffin blocks and histological slides collected from Al-Jumhoori Medical City Teaching Hospital Laboratory in Mosul and the Central Public Health Laboratory in Baghdad between the 1st of January 2010 to the 13th of March 2012 for patients diagnosed with primary invasive ductal breast carcinomas. Immunohistochemistry (IHC) using monoclonal antibodies against ER, PR, Her2/neu receptors and BRCA1 protein was performed via the fully automated immunostaining instrument ‘Ventana Benchmark’. Results:BRCA1 protein immunoexpression was detected in 20.5% of cases. It was significantly high with increasing tumour grade and stage. Although there was a trend of BRCA1 negativity toward negative ER, PR and Her2 receptors, no significant associations were observed with any of these parameters and the patients’ age. Conclusion:Altered BRCA1 expression is significantly associated with advanced tumour grade and stage. High number of cases with negative BRCA1 expression showed negative ER, PR and Her2/neu expression.

Nuclear and membrane progesterone receptors expression in placenta from early to late pregnancy in sheep: Effects of restricted nutrition and realimentation

Nutrient restriction and/or realimentation may affect several placental functions, such as expression of selected regulatory factors, blood flow and other processes in sheep and other species. To determine the effects of the plane of nutrition, nulliparous white face ewes (6–8 months) carrying singletons on day 50 of gestation were randomly assigned to two dietary treatments receiving 100% of National Research Council recommendations (control; C) or 60% of C (restricted; R). Two groups remained on C or R diets from day 50 until day 130. From day 90–130 another group of C fed ewes was switched to the R diet, and another group of R fed ewes was switched to the C diet. This resulted in 7 groups (n = 5–6 ewes/group): C (day 50, 90 and 130), R (day 90 and 130), CR (day 130) and RC (day 130). At these time points, placental tissues were collected for the evaluation of progesterone receptor (PGR) protein expression (whole tissue), and mRNA expression in maternal (caruncular, CAR) and fetal (cotyledon, COT) (separated tissues). Data were statistically analyzed using analysis of variance (SAS 9.4). Protein for PGRAB and PGRB isoforms was detected using immunohistochemistry in all placental tissues, but the pattern of expression differed depending on pregnancy stage and placental compartment (e.g., CAR vs COT). PGRAB protein expression, quantified using image analysis, was greater (P < 0.04) on day 50 than 90 or 130, and was not affected by plane of nutrition. In CAR and COT, PGRAB mRNA expression was greater (P < 0.05) on day 50 than 90 or 130. PGRB mRNA expression was greater (P < 0.03) in CAR on day 50 than 90 and 130, and was greatest (P < 0.02) in COT on day 50, less on day 130, and least on day 90. For the membrane progesterone receptors, PAQR7 (membrane PGR alpha) mRNA expression was greater (P < 0.05) on days 50 and 90 than 130 in CAR, and greater (P < 0.01) on days 50 than 90 and 130 in COT; PAQR8 (membrane PGR beta) was similar throughout pregnancy in CAR and COT, and PAQR5 (membrane PGR gamma) was greatest (P < 0.0001) on day 130 in COT, but similar throughout pregnancy in CAR. Plane of nutrition affected (P < 0.05) mRNA expression for all genes in CAR and COT throughout pregnancy. These data indicate that expression of PGR in ovine placenta is dependent on stage of pregnancy and plane of nutrition in sheep. The mechanisms of how diet and stage of pregnancy influences placental PGR expression and function remains to be elucidated.

Difficulties in the differential diagnosis of tumors in the female reproductive system

Introduction. Most pelvic tumors originate from reproductive organs. Even using the up-to-date imaging techniques, radiologists experience difficulties in determining the source of the lesion since a wide range of tumors look similar to each other on the distorted backdrop of pelvic anatomy, large invasive formations, and an active inflammatory reaction of the pelvic peritoneum.Aim: to evaluate “pitfalls” in the preoperative noninvasive diagnosis of female pelvic tumors by applying the clinical diagnostic tools.Materials and methods. Four rare clinical cases were analyzed; all of them posed difficulties in interpreting the diagnostic examination due to their atypical characteristics. There were 2 cases of ovarian cancer, initially identified by an experienced team of radiologists as benign pelvic pathology. Also, there were fibroids with degeneration, marked proliferative activity, and a massive inflammatory reaction of the peritoneum – that was diagnosed as a malignant ovarian tumor. Tumor biopsies were examined using morphological and immunohistochemical methods (with the р16, Ki-67, p53, CD 117, S 100, CD 34 markers). Immunohistochemical (IHC) studies were performed with formalin-fixed paraffin materials using the avidin-biotin-peroxidase method. Antibodies to estrogen receptor (ER), progesterone receptor (PR), cytokeratin 7 (CK7), cytokeratin 20 (CK20) and Wilms tumor protein 1 (WT1) were also used.Results. A thorough analysis of the clinical picture and a joint multidisciplinary discussion (gynecologist, oncologist, radiologist, etc.) made it possible to avoid diagnostic errors.Conclusion. These observations demonstrate the difficulties of differential diagnosis between ovarian metastases of uterine cancer and primary multiple ovarian and uterine cancer, as well between leiomyosarcoma and uterine myoma with high mitotic activity. Obviously, the change in diagnosis calls for a change in the treatment strategy.