ER Activity Research Articles

Protective Properties of FOXO1 Inhibition in a Murine Model of Non-alcoholic Fatty Liver Disease Are Associated With Attenuation of ER Stress and Necroptosis.

AimThe pathogenesis of non-alcoholic fatty liver disease is currently unclear, however, lipid accumulation leading to endoplasmic reticulum stress appears to be pivotal in the process. At present, FOXO1 is known to be involved in NAFLD progression. The relationship between necroptosis and non-alcoholic steatohepatitis has been of great research interest more recently. However, whether FOXO1 regulates ER stress and necroptosis in mice fed with a high fat diet is not clear. Therefore, in this study we analyzed the relationship between non-alcoholic steatohepatitis, ER stress, and necroptosis.Main MethodsMale C57BL/6J mice were fed with an HFD for 14 weeks to induce non-alcoholic steatohepatitis. ER stress and activation of necroptosis in AML12 cells were evaluated after inhibition of FOXO1 in AML12 cells. In addition, mice were fed with AS1842856 for 14 weeks. Liver function and lipid accumulation were measured, and further, ER stress and necroptosis were evaluated by Western Blot and Transmission Electron Microscopy.Key FindingsMice fed with a high fat diet showed high levels of FOXO1, accompanying activation of endoplasmic reticulum stress and necroptosis. Further, sustained PA stimulation caused ER stress and necroptosis in AML12 cells. At the same time, protein levels of FOXO1 increased significantly. Inhibition of FOXO1 with AS1842856 alleviated ER stress and necroptosis. Additionally, treatment of mice with a FOXO1 inhibitor ameliorated liver function after they were fed with a high fat diet, displaying better liver condition and lighter necroptosis.SignificanceInhibition of FOXO1 attenuates ER stress and necroptosis in a mouse model of non-alcoholic steatohepatitis.

Abstract P6-04-06: Protein kinase C beta 1 (PKCβ1), a novel drug target of Z-endoxifen, inhibits growth of estrogen receptor positive (ER+) breast cancer via activation of the NF-kB transcription factor RelA

Abstract Background: The first in-human phase-I clinical trial of the tamoxifen metabolite, Z-endoxifen, demonstrated promising antitumor activity in both aromatase inhibitor (AI) and tamoxifen resistant ER+ breast cancer patients (Goetz M.P., et al; JCO, 2017). By utilizing a letrozole-sensitive MCF7 aromatase expressing (MCF7AC1) cell line, and its corresponding letrozole-resistant (MCF7LR) cell line, we have shown superior antitumor and antiestrogenic activity of Z-endoxifen over tamoxifen in the AI-sensitive and resistant settings in vivo. This study also revealed that Z-endoxifen may target protein kinases in addition to estrogen receptor alpha. Follow-up studies demonstrated that Z-endoxifen, but not tamoxifen, binds to protein kinase C beta 1 (PKCβ1), a member of the serine/threonine-specific protein kinase family that regulates signaling pathways involved in cell proliferation and tumorigenic transformation (Guo C., et al; SABCS, 2017). We have further shown that PKCβ1 silencing, similar to Z-endoxifen, potently inhibits ER+ cell proliferation and additionally causes ERα turnover. Here, we report a potential mechanism by which PKCβ1 may mediate its anti-proliferative effects in ER+ breast cancer cells. Methods: The effects of siRNA-mediated PKCβ1-silencing on gene transcription were evaluated by RNAseq in MCF7AC1 cells. RNAseq results were further validated by quantitative real-time polymerase chain reaction (qRT-PCR) in PKCβ1-silenced MCF7AC1 cells as well as in ER+ T47D cells. The effects of Z-endoxifen on the mRNA expression of genes altered by PKCβ1-silencing in MCF7AC1 cells were evaluated by qRT-PCR analysis. PKCβ1 silencing effects on protein expression in MCF7AC1 cells were evaluated by immunoblot analysis. Results: RNAseq analysis of PKCβ1-silenced MCF7AC1 cells compared to non-silenced cells revealed NF-κB signaling pathway as the top biological pathway significantly altered by PKCβ1 silencing (p=5.93e-08). Enhanced mRNA expression of multiple NF-κB downstream target genes including CXCL10, RELB, MMP1 and IL6 were observed following PKCβ1 silencing. qRT-PCR analyses validated increased mRNA expression of these genes in PKCβ1-silenced MCF7AC1 and T47D cells. Notably, Z-endoxifen treatment in MCF7AC1 cells mirrored PKCβ1 silencing effects, resulting in enhanced mRNA expression of the aforementioned genes. Evaluation of the protein expression of RelA and RelB, the canonical and noncanonical NF-κB transcription factors respectively, in PKCβ1-silenced MCF7AC1 cells revealed significant upregulation of RelA (p=0.0203) but not RelB (p=0.1360) expression. ERα silencing in MCF7AC1 cells did not affect RelA protein expression. Consistent with prior reports, treatment of MCF7 cells with TNFα inhibited cell proliferation. Finally, targeted siRNA-mediated silencing of RelA in PKCβ1-silenced MCF7AC1 cells significantly attenuated PKCβ1-mediated growth inhibition (p=0.0002). Conclusion: Our findings demonstrate that both Z-endoxifen treatment and PKCβ1-silencing commonly activates a NF-κB-mediated gene signature in ER+ breast cancer cells. Notably, PKCβ1-silencing induces the protein expression of the NF-κB transcription factor RelA in an ERα-independent manner, which appears to contribute to PKCβ1-mediated growth inhibition. Current studies are ongoing to evaluate the impact of RelA silencing on Z-endoxifen mediated growth inhibition. This information will foster our understanding of the estrogenic and non-estrogenic mechanisms by which Z-endoxifen mediates its antitumor activity in ER+ breast cancer. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Elizabeth S Bruinsma, John R Hawse, Matthew P Goetz. Protein kinase C beta 1 (PKCβ1), a novel drug target of Z-endoxifen, inhibits growth of estrogen receptor positive (ER+) breast cancer via activation of the NF-kB transcription factor RelA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-06.

Abstract P6-04-26: PI3K inhibitors restored the sensitivity to anti-estrogen drugs in endocrine therapy resistant cell lines

Abstract Background The Combination of endocrine therapy with molecular targeted drugs has become one of the standard treatment option for ER positive advanced or metastatic breast cancer. The therapeutic effects of PI3K-mTOR inhibitors, which inhibit intracellular phosphorylation of signaling pathways, have been clarified in clinical trials. However, the effects of these drugs on ER signal pathways in endocrine-resistant breast cancer have not been fully elucidated. We investigated the changes in the expression and functions of ER when endocrine-resistant cell lines, established in our laboratory, were treated with PI3K-mTOR inhibitors Methods Long-term estrogen deprivation-resistant (EDR) cell lines and fulvestrant-resistant cell lines (MFR) were established from MCF-7 cells in our previous studies. These cell lines exhibited different ER expression levels, including high (EDR-1), low (EDR-2), or no expression (MFR). All these cell lines had the same PIK3CA mutations as the parental cell lines. We used buparlisib (BUP, pan-class1 PI3K inhibitor) and alpelisib (ALP, α-specific PI3K inhibitor) as PI3K inhibitors and everolimus (EVE) as an mTOR inhibitor. Tamoxifen and fulvestrant were used in anti-estrogen therapy. In this study, the procedure to examine histone modifications was CHIP-qtPCR analysis. Results EDR-1, -2 and MFR were treated with the PI3K-TOR inhibitors for 24 h. There were no changes in ER expression and ER activity on EDR-1 and MFR before and after the addition of PI3K-mTOR inhibitors. Interestingly, ER expression and ER activity in EDR-2 increased after the addition of PI3K-mTOR inhibitors. In particular, a high re-expression of ER was observed when the PI3K inhibitors were added. Next, in order to evaluate the effects of endocrine therapy on these cell lines Using BUP, ALP and EVE, each drug resistant cell lines were established from EDR-2. When the cell proliferation assay was performed in the established resistant cell line, EDR-2 which showed re-expression of ER on adding the PI3K inhibitors, showed cell growth-inhibitory effects with the addition of anti-estrogen agents. Previously, we reported that epigenetic regulation contributes to the reduction of ER expression in EDR-2. (Tsuboi et al, J Steroid Biochem Mol Biol 2017). Therefore, we investigated the possibility that epigenetic regulation could contribute to the mechanism of ER re-expression when PI3K inhibitors were used in EDR-2. It was revealed that the change of H3K27me3, one of the histonemodifications, correlated with the expression of ER. Conclusion PI3K inhibitor could restore the expression of ER and sensitivity to anti-estrogen drugs in endocrine therapy resistant cell line in which ER expression was lost during acquired resistant period. This restoration might be due to a histone modification (H3K27me3). Citation Format: Emi Tokuda, Shunta Sasaki, Kouki Tsuboi, Masafumi Iida, Toshifumi Niwa, Shigehira Saji, Shin-Ichi Hayashi. PI3K inhibitors restored the sensitivity to anti-estrogen drugs in endocrine therapy resistant cell lines [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-26.

Abstract P5-01-12: Plasma thymidine kinase 1 activity and outcome of ER+ HER2- metastatic breast cancer patients treated with palbociclib and endocrine therapy

Abstract Background: Thymidine kinase 1 (TK1) has a crucial role in DNA synthesis and cell proliferation. Prior cohort studies reported plasma TK1 activity (pTKa) as a potential prognostic biomarker in estrogen receptor-positive (ER+) HER2-negative (HER2-) metastatic breast cancer (MBC) treated by endocrine therapy. We report here the prognostic impact of pTKa, assessed in a prospective study on ER+ HER2- MBC patients treated by endocrine therapy and CDK4/6 inhibitor. Patients and methods: Patients were included after written informed consent into the prospective, ethically approved ALCINA study (NCT02866149), investigating the feasibility of the analysis of circulating tumor markers in blood in various early- or advanced-stage malignancies. Eligibility criteria were patients aged >18 years with ER+ HER2- MBC, treated at the Institut Curie with endocrine therapy (aromatase inhibitor or fulvestrant) and palbociclib. Plasma samples were obtained at baseline (prior to treatment start) and after 4 weeks of treatment. pTKa was quantified by the DiviTum® assay (Biovica, Sweden), following the manufacturer recommendations and with blinded patient characteristics and outcomes. We investigated the association between baseline and 4 weeks pTKa, patient characteristics and outcomes (progression-free survival-PFS and overall survival-OS). “Low” and “high” pTKa were defined using the median level of pTKa at each time point. Quantitative pTKa were compared using the Mann-Whitney test. Survival curves were compared by an unstratified log-rank test. Results: From 05.2016 to 08.2018, 103 patients treated with endocrine therapy and palbociclib were included. Median age was 64 years (range 26-89). Patients had received a median of 2 prior systemic therapies for MBC (range 0-10). After a median follow-up of 13.8 months (range 6-31), 73 relapses (71%) and 25 deaths (24%) were observed, with median PFS and OS of 9.6 months (95%CI[7-11.3]) and 27.9 months (95%CI[22.5-33.2]), respectively. Median baseline pTKa was 292 Du/L (range 0-27312 Du/L, IQR[89-853]). In univariate analysis, a low baseline pTKa was significantly associated with longer PFS and OS compared with high baseline pTKa (11.7 months (95%CI[7.4-16]) vs 9.1 months [4.4-14]; p=0.02, and NR vs 24.8 months [16-33]; p=0.05, respectively). After 4 weeks (N=85 patients), median pTKa was 134 Du/L (range 0-3877 Du/L, IQR[43-595]). In univariate analysis, a low pTKa at 4 weeks was also associated with longer PFS and OS compared with high pTKa (10.4 months [7.9-13] vs 4.7 months [1.2-8.3]; p=0.02, and NR vs 20 months [16-23]; p<0.001, respectively). Patients with a PFS below 6months had a significantly higher pTKa level at 4w (median 256 Du/L versus 100Du/L, p=0.046). After adjusting for prognostic factors (i.e. number of previous lines of systemic treatments, endocrine resistance, presence of visceral metastases, performance status, age, de novo MBC): baseline pTKa and 4w pTKa remained significant prognostic factors for both PFS and OS (p<0.001). Sensitivity analyses suggested that pTKa changes between baseline and 4w had no significant prognostic impact and that 4w pTKa had a superior prognostic impact than baseline pTKa. Conclusion: pTKa demonstrated its clinical validity as a new dynamic circulating prognostic marker for ER+ HER2- MBC patients treated with endocrine therapy and palbociclib. The clinical utility of this promising biomarker has to be determined in a adequately designed prospective trial of combined targeted and endocrine therapies. Citation Format: Luc Cabel, Marie Laure Tanguy, Florence Lerebours, Etienne Brain, Delphine Loirat, Mattias Bergqvist, Paul Cottu, Anne Donnadieu, Anne Bethune, Manuel Rodrigues, Jean-Yves Pierga, François-Clément Bidard. Plasma thymidine kinase 1 activity and outcome of ER+ HER2- metastatic breast cancer patients treated with palbociclib and endocrine therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-12.

Abstract ES4-1: Adaptive Resistance to Hormonal Therapy

Abstract While recurrences are delayed by the use of endocrine therapy (ET), resistance can evolve with the development of metastatic breast cancer (MBC) in about one-fourth of ER-positive patients. MBC is incurable with a median 5-year survival rate of less than 25%. We know that ET resistance can result from an adaptive process in which escape survival mechanisms are used to evade treatment. One of the best-characterized ET resistance mechanisms is transcriptional activation of growth factor receptor (GFR) signaling, and subsequent estrogen-independent activation of ER via “cross-talk” with downstream signaling from GFRs. We will discuss distinct mechanisms of ligand-independent activation of ER discovered using transcriptional profiling of metastatic, ET-resistant tumors. It is now estimated that ~20% of therapy-resistant MBCs contain ESR1 mutations. Recent data has also shown that mutations in the MAPK/RAS pathway and transcription factor regulatory programs (for example, MYC) only account for an additional 22% of recurrent breast tumors. Thus, acquired mutations alone do not account for the majority of ET resistance mechanisms in MBC. ER-positive breast cancer cell lines display profound transcriptional variability at the single cell level associated with the acquisition of therapy resistance. Recently, bulk and single cell RNA sequencing was used on sequential samples from patients with ER-positive, ET-resistant patients and identified RNA expression patterns that represented different cellular phenotypes, including an increased epithelial mesenchymal transition state that could provide a selective advantage for selective phenotypic subclones to dominate. We will focus our discussion of transcriptional reprograming as a major mechanism of ET resistance. Citation Format: S Fuqua. Adaptive Resistance to Hormonal Therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES4-1.

Abstract P5-11-06: Phase 1 study of TTC-352 in patients with metastatic breast cancer progressing on endocrine therapy

Abstract Background: TTC-352 is a selective human ER partial agonist (ShERPA) developed for treatment of ER+ breast cancer (BC). ShERPAs mimic the effects of estradiol (E2) in hormone-independent, endocrine-resistant BC cells, but since it has only partial agonist activity in ER+ cells, it could have an improved side effect profile. Thus, TTC-352 could be a promising option for endocrine-resistant BC. Methods: This is an open-label, accelerated dose escalation study with primary endpoint of maximum tolerated dose (MTD) that is evaluating up to five dose levels of TTC-352 in patients with ER+ endocrine-resistant metastatic BC (NCT03201913). The MTD of TTC-352 is determined using initial single-patient cohort escalations until grade 2 toxicity, then expansion to a modified-Fibonacci dose-escalation 3+3 design. The secondary objectives are: best response (BR), progression-free survival, overall survival, treatment tolerability, and pharmacokinetics (PK) of TTC-352. Patient population: patients with metastatic ER+ BC who received and progressed on at least two lines of endocrine therapy, with one that included a CDK4/CDK6 inhibitor, and with adequate hepatic, renal, and bone marrow function at screening. Results: This study completed enrollment of a total of 15 patients on June 5, 2019. Dose LevelTTC-352 Dose Level (mg)Number of SubjectsTwice-Daily (BID) DoseTotal Daily Dose115302230601360120141202401518036010 No dose limiting toxicity has been seen to date with any of the tested dose levels. There was one treatment-related grade 3 event of asymptomatic pulmonary embolism (30mg BID). The number and percentage (n, %) of patients with treatment-related grade 1 and grade 2 adverse events reported in two or more patients across all dose levels were diarrhea (9, 60), hot flush (4, 26), headache (3, 20), nausea (3, 20), abdominal pain (3, 20), rash (3, 20), blurred vision (2, 13) vomiting (2, 13), dizziness (2, 13), peripheral neuropathy (2, 13), breast pain (2, 13), uterine/vaginal hemorrhage (2, 13) and vaginal discharge (2, 13). BR in the 7 evaluable patients (patients with at least two 28-day cycles of treatment) showed that 3 patients had stable disease (SD) for 4, and 10 months, respectively; 1 patient continues study treatment with SD after 9 months; and 4 patients had progressive disease (PD) after completing two cycles of treatment. All 5 non-evaluable patients had PD prior to completing two cycles of treatment. PK: The intra- and inter-individual variability for AUC0-∞ hampered assessment of the relationship between dose and AUC0-∞., Half-life was 7.6-14.3 hrs. Trough TTC-352 plasma concentrations at steady-state (SS) remained well above the plasma concentrations associated with efficacy in animal models. PKC alpha expression in tumor tissue did not correlate with length of disease control with TTC-352 therapy, though the numbers are small and not all of the available specimens were metastatic. Conclusions: TTC 352 demonstrates manageable safety and early clinical evidence of antitumor activity in patients with BC progressing on endocrine therapy. Based upon SS TTC-352 plasma concentrations and tolerability, the 180mg BID dose is recommended for further testing. Citation Format: Ruth O'Regan, Randolph Hurley, Jasgit Sachdev, Jonathan Bleeker, Debra Tonetti, Gregory Thatcher, Robert Venuti, Arkadiusz Dudek. Phase 1 study of TTC-352 in patients with metastatic breast cancer progressing on endocrine therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-11-06.

Abstract P6-04-18: The regulatory role of activating transcription factor-2 (ATF2) in modulating tamoxifen resistance in estrogen-receptor positive breast cancer

Abstract Introduction: Activating transcription factor-2 (ATF2), a member of the ATF and CREB group of bZIP transcription factors regulates gene transcription by forming homodimers or heterodimers with other AP-1 family members. We have previously shown that ATF2 is important for modulating the effect of tamoxifen, whereas Thr71-ATF2 phosphorylation predicts for improved outcome of ER-positive breast cancers receiving tamoxifen. In this study we further explore the role of ATF2 in modulating breast cancer endocrine therapy resistance. Methods: ATF2 silencing: The MCF7, TamR, LCC2, LCC9 breast cancer cell lines were maintained in their appropriate growth media. Cells were transfected using RNAiMAX (ThermoFisher Scientific) with two different siRNAs and a Silencer® negative control. Gene expression was assessed by quantitative real-time PCR (RT-qPCR) (TaqMan assays, ThermoFisher Scientific) and the protein levels were analysed by western blotting. Cell growth, invasion, migration: Sulforhodamine B assay was used for cell growth determination following manufactures’ protocol. Soft-agar colony formation was performed by seeding and incubating the cells in low-melting agarose plates. The resulting colonies were 5% MTT-stained and counted in a Gel Count™ (Oxford Optronix). Invasion/migration was performed using the transwell matrigel-coated (BD Biosciences) plates and the scratch wound healing assay. All the assays were performed in triplicates in three independent experiments. Microarray profiling: Human HT12_V4 microarray (Illumina) was performed to identify genes that were differentially regulated by ATF-2 silencing in TamR cells compared to MCF-7. Data analyses including log2 transformation and quantile normalisation were performed on R Bioconductor. Differentially-expressed genes were selected using FDR correction for multiple testing with p-value cut-off <0.05. Results: ATF-2 silencing significantly inhibited the proliferation (p<0.005), colony formation (p<0.005) and migratory ability (p<0.01) of TamR, LCC2 and LCC9 cells but had a no effect on the growth and migration of MCF-7 cells. Moreover, differential gene expression analysis in MCF7 versus TamR cells revealed a shift from the ER-enriched transcription factor network towards an ATF2-enriched and TGF2β signalling pathway (p=0.025) in TamR cells suggesting alternative hormone-independent growth mechanisms. We then considered differentially-expressed genes specific to the TamR cells (not differentially-expressed in MCF7) and found a reintroduction of ER and its signalling pathway upon ATF2 knockdown along with a reduction in MYC transcriptional activity (EnrichR). RT-qPCR and western blot confirmed the increased expression of ER and its targets TFF1, GREB1, NCOA3, in TamR cells after ATF2 silencing in both the gene and protein levels. Conclusions: In conclusion, for the first time, ATF-2 was identified as a novel transcription factor that can modulate endocrine resistance by regulating ER expression and activity. This data suggests that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity. Citation Format: Athina Giannoudis, Mohammed I Malki, Hisham Mohammed, Bharath Rudraraju, Suraj Menon, Simak Ali, Jason S Carroll, Carlo Palmieri. The regulatory role of activating transcription factor-2 (ATF2) in modulating tamoxifen resistance in estrogen-receptor positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-18.

Abstract P6-05-06: Significance of prostate specific antigen as a predictor of androgen receptor activity in breast cancer

Abstract BACKGROUND: Preclinical data suggest that androgen receptor (AR) activity plays a role in breast cancer (BC). Clinical trials testing AR-targeted therapies in BC have shown clinical benefit and additional trials are currently underway. However, a method is needed to predict which patients may benefit the most. Prostate specific antigen (PSA) is the secreted product of the androgen-responsive gene kallikrein related peptidase 3 (KLK3). Serum PSA (sPSA) is an established indicator of tumor burden in prostate cancer. Recently, we found that sPSA can be detected in BC patients and is positively correlated with serum testosterone levels, AR expression in the primary tumor, and aromatase inhibitor resistance (Hanamura, et al. SABCS 2018). HYPOTHESIS: Serum PSA may reflect AR activity in BC and have utility as a predictive marker for identifying patients that would benefit from AR-targeted therapy. OBJECTIVE: To establish that PSA is regulated by AR in BC, we examined the relationship between AR and PSA in BC cell lines, BC patient derived xenograft (PDX), and publically-available datasets of patient gene expression profile. METHODS: Nine BC cell lines representing three clinical subtypes (ERα+, HER2+ and TNBC) were analyzed for response to dihydrotestosterone (DHT) and the AR antagonist enzalutamide (Enza) by crystal violet assay and further analyzed by western blot for PSA. Next, PSA was measured in conditioned media from BT474 BC cells by chemiluminescence immunoassay. Finally, HCI-009, an AR+ TNBC PDX model, grown in NOD-scid gamma mice were given vehicle control versus DHT and were used to identify AR regulated genes by RNA-sequencing, followed by verification at the protein level by IHC. Genes increased in HCI-009 from mice given DHT treatment as compared to vehicle were defined as “PDX androgen-responsive gene set” and imported into gene set enrichment analysis (GSEA). To test whether PSA reflects AR activity, two publically available datasets, The Cancer Genome Atlas (TCGA) (n=1100) and the population-based Multi center Sweden Cancerome Analysis Network (n=405), were mined for correlation between KLK3 andAR or ESR1mRNA (for comparison), and with GSEA with regard to known androgen- or estrogen-responsive gene sets. RESULTS: Although the proliferative response to DHT varied among cell lines, Enza decreased proliferation of the majority of AR+ BC lines regardless of the proliferative response to DHT. Western blot analysis showed PSA to be regulated in an AR-dependent manner (induced by DHT and suppressed by Enza). In BT474 cells, PSA secretion into the media was DHT-responsive and suppressed by Enza. Gene expression analysis of the HCI-009 PDX model showed KLK3 as one of most DHT responsive genes. IHC analysis showed PSA and other AR regulated proteins are significantly higher in tumors from mice given DHT as compared to control and that tumor growth was increased in these mice compared to control mice. In the TCGA and Sweden cohort, a stronger positive correlation was found between KLK3 and ARmRNA expression compared to KLK3 and ESR1 mRNA. An androgen-responsive gene set was significantly enriched in patients with a high KLK3, but the estrogen-responsive gene set was enriched in patients with a low KLK3. The PDX androgen-responsive gene set was also significantly enriched in patients with a high KLK3. CONCLUSIONS: Results of in vitro studies suggest that PSA reflects AR activity and the responsiveness of BC cells to AR antagonism. In comprehensive analysis of gene expression profiling, KLK3 was positively correlated with AR activity, but negatively correlated with ER activity. Together with our previous findings, these data suggest the need for further studies to determine whether sPSA or other AR-regulated secreted proteins are useful as a clinical predictive marker of response to AR targeted therapies. Citation Format: Toru Hanamura, Jessica L Christenson, Kathleen I O’Neill, Emmanuel Rosas, Nicole S Spoelstra, Michelle M Williams, Jennifer K Richer. Significance of prostate specific antigen as a predictor of androgen receptor activity in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-06.

Abstract P6-04-13: Preclinical and clinical analyses of estrogen receptor alpha mutant metastatic breast cancer

Abstract Background: Estrogen receptor alpha (ER) positive breast cancer (BC) that recurs as metastatic disease in patients on aromatase inhibitor therapy often harbors mutations in the last exon of the ESR1gene. Such mutations result in amino acid alterations in the ligand binding domain that confer a selective advantage because they render ER constitutively active under estrogen depleted conditions. Hypothesis: We hypothesized that the selective survival advantages gained by metastatic ER mutant (ERmut) tumor cells would become evident in preclinical models simulating the clinical context in which they arise (estrogen deprivation, anchorage independence or metastatic outgrowth) that could then be confirmed in ER+ patient derived xenografts (PDX) and biopsies of metastatic disease. Methods: MCF7 and T47D cells expressing flag-tagged ERmut Y537S or D538G or flag-tagged ER wild type (ERw/t) at the endogenous locus were generated using CRISPR/Cas9. These cells were studied in standard culture conditions as compared to anchorage independent soft agar or poly-HEMA coated plate assays and under long-term estrogen deprivation (LTED) conditions in vitro as well as in vivo in ovariectomized (ovx) mice in late stage metastasis models. ER+ patient derived xenografts (PDX) with naturally occurring ERmutY537S (HCI-013EI) and ERmutL536P (HCI-007) (Dr. Alana Welm) were also studied in vivo. Colony formation assays and tumor growth were evaluated in the context of treatments targeting ER (fulvestrant), androgen receptors (AR) (enzalutamide), steroid hormone synthesis (CYP17 lyase inhibitor seviteronel), or BRD4 Bromo- and Extra-terminal Domain (BET) inhibitor (BETi: JQ1). Drug synergy was determined in vitro using CalcuSyn software or in vivo by tumor caliper measurement and in vivo imaging of tumor luciferase activity. Biopsies of metastatic ER+ BC from clinical trial NCT02953860 were assayed for ESR1 exon 8 mutations using a modified Archer VariantPlex Solid Tumor Assay and immunohistochemistry performed for the four main steroid hormone receptors. Results: ERmutY537S or D538G containing MCF7 and T47D cells exhibited significantly increased anchorage independent survival, a condition in which AR is uniquely upregulated, while ER, progesterone receptors (PR), and glucocorticoid receptors (GR) are not. Furthermore, anti-androgens abrogated the ERmut survival advantage in soft agar. LTED ERmut lines expressed significantly more AR than their non-LTED predecessors or ERw/t counterparts as did ERmut PDX as compared to ERw/t PDX. MCF7 and T47D ERmutY537S and D538G demonstrated a significant increase in AR protein upon anchorage independent survival as compared to ERw/t, with ERmutD538G expressing particularly elevated AR. Biopsies of metastases from consenting patients with ER+ BC confirmed significantly higher AR protein in ERmut metastases as compared to ERw/t by IHC. Treatment of mice with ERmut PDX with combination fulvestrant and anti-androgen decreased tumor growth as compared mice given vehicle control or fulvestrant only. The BET inhibitor JQ1 synergized with anti-androgen to significantly decrease proliferation of ERmut cells. In ovx mice, only ERmut metastases were detectable, while ERw/t did not thrive at metastatic sites. ERmut cells differentially expressed AR-regulated, metastasis-promoting genes and proteins, including chitinase 3-like 1 (CHI3L1), when compared to those with ERw/t. Conclusions: AR is induced by stress encountered during disease progression and treatment, such as anchorage independence and estrogen deprivation. Targeting AR may improve the efficacy of agents such as fulvestrant that inhibit ERmut metastatic disease or delay recurrence, as may agents such as BETi that affect the activity of both ER and AR. Citation Format: Jennifer K Richer, Michelle M Williams, Nicole S Spoelstra, Spencer Arnesen, Jessica L Christenson, Kathleen O’Neill, Jordan M Reese, Zannel D Blanchard, Toru Hanamura, Britta M Jacobsen, Jason Gertz, Anthony Elias. Preclinical and clinical analyses of estrogen receptor alpha mutant metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-13.

Abstract P5-02-14: The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance

Abstract Background: Based on preclinical research, the MAPK growth factor pathway is thought to be an oncogenic pathway driving breast cancer, potentially in association with the ER pathway. In patients it is unclear how functional activity of the MAPK pathway activity relates to activity of the ER pathway to drive cancer growth. We have reported before on the development of assays, e.g. for the ER pathway, that enable quantitative measurement of activity of signal transduction pathways based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the ER transcription factor (Cancer Res., 2014, vol. 74, no. 11, pp. 2936-2945). We now present (1) development of the MAPK-AP1 pathway model, (2) MAPK-AP1 pathway activity and its relation to ER pathway activity in breast cancer; (3) emergence of MAPK pathway activity associated with hormonal therapy resistance. Methods: The MAPK-AP1 pathway model was developed and biologically validated on Affymetrix mRNA data from samples (different cell types) with a known MAPK pathway activity. MAPK-AP1 and ER pathway activity scores were measured on Affymetrix expression data of primary untreated breast cancer samples (GSE45827, n=141) and of fulvestrant sensitive and resistant MCF7 cell line clones (GSE74391). Results: In clinical breast cancer samples, average MAPK pathway activity was increased in all subtypes compared to normal breast tissue, with a large dynamic range indicating that in each subtype there are samples with high and with low pathway activity; ER pathway activity in Luminal A and B subtypes ranged from high to low, and was low in all other subtypes. MAPK-AP1 pathway activity did not correlate with ER pathway activity. Fulvestrant-sensitive MCF7 cells had high ER and low MAPK pathway activity scores, while resistant MCF7 clones exhibited increased MAPK pathway activity and complete loss of ER pathway activity, explaining therapy resistance. Discussion: The MAPK-AP1 pathway is frequently activated in all breast cancer subtypes, independent of ER pathway activity. We hypothesize that in case of resistance to hormonal therapy measurement of both pathway activities may be informative as to the underlying resistance mechanism, i.e. loss of ER pathway activity and/or gain of MAPK pathway activity. Future clinical studies are needed to confirm the role of these signaling pathways in hormonal resistance. Citation Format: Anja van de Stolpe, Laurent Holtzer, Yvonne Wesseling-Rozendaal, Wim Verhaegh. The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-14.

Abstract P1-18-07: Can some ER+/HER2+ patients be safely spared from treatment with chemotherapy plus herceptin?

Abstract Background: The ER+/HER2+ subtype accounts for up to 10% of all breast cancers (BCs) and most are treated with surgery followed by adjuvant chemotherapy with Herceptin +/- radiotherapy then adjuvant endocrine therapy (ET) to reduce the recurrence risk. Despite this it is clear that not all ER+/HER2+ patients gain benefit from the addition of chemotherapy and Herceptin. In particular, given the significant side effects associated with the chemotherapy, the risks may out way the benefit in some older patients and in those with co-morbidities. Currently there are no clinically validated tools to identify women with ER+/HER2+ BC whose risk of recurrence remains unchanged with the addition of chemotherapy plus Herceptin and who can be effectively managed with adjuvant ET alone. Using levels of IL6ST, a biomarker for ET response, ER+/HER2+ patients who gain benefit from ET can be differentiated from those for whom ET alone is not sufficient to reduce the risk of recurrence. The latter group were characterised by inactive ER signalling and active MAPK and PI3K signalling. The aim is to show that ER+/HER2+ patients predicted to respond less well to ET alone using IL6ST levels, gain additional benefit from chemotherapy plus Herceptin. Patients: • Cohort A - 32 post-menopausal women (PMW) with large ER+/HER2+ BC treated with neoadjuvant (3-6 months) then adjuvant letrozole. Neoadjuvant clinical response was assessed by changes in tumour volume. Tumour core biopsies were taken at 0, 14 days and 3 months. Gene expression analysis using Illumina HT12 whole-genome beadarrays was performed on a subset (n=17) where fresh tissue was available. Median follow-up was 7.5 years. • Cohort B - 362 women with ER+/HER2+ BC treated with surgery +/- radiotherapy followed by adjuvant endocrine therapy between 2005 and 20010. 219 also received chemotherapy plus Herceptin. Median follow-up is 9.5 years. Results: In cohort A, half (16/32) of the patients responded to ET with tumour volume reductions of >70% with neoadjuvant treatment. Innate resistance was apparent in 3 patients with continued tumour growth on ET, whereas 13 patients acquired resistance after a period of response. Neoadjuvant clinical response was predicted with 92% accuracy using levels of IL6ST. Gene expression analysis in 17 patients showed increased MAPK and PI3K pathway activity in the 9 NR compared with the 8 R tumours. In the 16/32 patients who responded well to neoadjuvant ET the actuarial recurrence rate was 0% at 5 and 10 years. The rate of recurrence in the NR was 30% at both 5 and 10 years. Samples from cohort B are currently being profiled using IL6ST in tandem with custom gene expression assays for ER, MAPK and PI3K pathway activity. Conclusions: • IL6ST levels can differentiate ER+/HER2+ BCs who respond well to ET alone and those with a poor clinical response who have a higher risk of recurrence. • NR to ET have increased expression of PI3K and MAPK pathways, consistent with active HER2 signalling. • Analysis of cohort B is underway and will elucidate the benefit in terms of recurrence-free and overall breast cancer specific survival from the addition of chemotherapy plus Herceptin to the group predicted to respond less well to ET using IL6ST. • There is potential role for IL6ST in selecting ER+/HER2+ patients that require and benefit from HER2-targeted therapies. Citation Format: Arran K Turnbull, Victoria Webber, Daniel McStay, Laura M Arthur, Carlos Martinez-Perez, James Meehan, Mark Gray, Charlene Kay, Lorna Renshaw, Jane Keys, Robert Clarke, Andrew H Sims, Olga Oikonomidou, J Michael Dixon. Can some ER+/HER2+ patients be safely spared from treatment with chemotherapy plus herceptin? [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-07.

Abstract PD7-05: A first-in-human phase I study to evaluate the oral selective estrogen receptor degrader (SERD), GDC-9545, in postmenopausal women with estrogen receptor-positive (ER+) HER2-negative (HER2-) metastatic breast cancer

Abstract Background: Modulation of estrogen activity and/or synthesis is a mainstay therapeutic strategy for ER+ breast cancer (BC). However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand-binding domain, that drive ER-dependent transcription and proliferation in the absence of estrogen. ER antagonists that robustly suppress ER signaling in both ESR1 wild-type and mutant tumors may be of substantial therapeutic benefit. GDC-9545 is a novel, potent, non-steroidal, orally bioavailable, selective ER antagonist/ER degrader (SERD) that induces tumor regression in ER+ BC patient-derived xenograft models. Methods: A phase I dose escalation study with 3+3 design was conducted in postmenopausal women with ER+ (HER2−) advanced or metastatic BC (mBC). Patients were eligible if they had advanced or mBC that had progressed while being treated with adjuvant endocrine therapy for ≥ 24 months and/or endocrine therapy in the advanced or mBC setting for ≥ 6 months. No more than 2 prior treatments for advanced or mBC. Primary objective was to determine the safety, pharmacokinetics (PK) and the recommended Phase 2 dose (RP2D) of GDC-9545. Pharmacodynamic (PD) activity was assessed with [18F]-fluoroestradiol (FES)-PET scans and, when feasible, by performing biomarker analysis of pre- and on-study tumor biopsies. Plasma samples were collected for PK analyses and imaging for response assessment (CT and bone scans) were repeated every 8 weeks. Results: From November 27 2017 to February 1 2019 patients (n = 29) with a median age of 58 years (range 42-75) and a median number of prior therapies for mBC of 2 (range 1-4) were enrolled at 4 total daily dose levels (10, 30, 90 and 250 mg) given once daily (QD) while fasting. Increases in GDC-9545 exposure were approximately dose proportional and t1/2 was ~30 hours. Treatment related adverse events (AEs) were all Grade 1 or 2. The most common treatment-related AEs in ≥10% of patients were fatigue (21%, n=6), nausea (21%, n=6), hot flushes (17%, n=5), diarrhea (17%, n=5), arthralgia (17%, n=5), constipation (10%, n=3) and dyspepsia (10%, n=3). No dose-limiting toxicities, treatment-related serious adverse events (SAEs), Grade ≥ 3 events, or fatal events have been reported. Treatment interruption was required for 3 patients: one patient for a short episode of dyspepsia, vomiting, diarrhea, and dizziness; and two patients due to unrelated SAEs. In 14 patients with FES-PET avid disease at baseline and post-baseline scans, 11 (78%) showed complete or near complete (> 90%) suppression of FES uptake to background levels, including patients with ESR1 mutations. Evidence of reduced ER, PR, and Ki67 IHC values and an ER activity signature was observed in paired pre- and on-treatment biopsies (n = 7). Twelve of 27 patients (10 mg [n=2], 30 mg [4], 90 mg [4], 250 mg [2]) had either confirmed partial responses (n = 3) or were on study ≥ 24 weeks (CBR = 44%), including patients pretreated with CDK4/6i, fulvestrant or harboring baseline ESR1 mutations. The R2PD was 100 mg QD and was selected for dose-expansion in post- and pre/peri-menopausal women with and without palbociclib, which is ongoing. Updated results will be presented. Conclusions: GDC-9545 is well-tolerated to date with PK exposure supporting QD dosing, evidence of robust PD target engagement, and encouraging anti-tumor activity in patients with advanced or ER+ mBC, including patients with ESR1 mutations. Dose expansion cohorts of GDC-9545 with or without palbociclib in post-menopausal and pre/peri-menopausal women with mBC are ongoing. Citation Format: Komal Jhaveri, Eric Paul Winer, Elgene Lim, Jose AlejandroPerez Fidalgo, Meritxell Bellet, Ingrid Alina Mayer, Valentina Boni, Jaymin M Patel, Aditya Bardia, Jose Manuel Garcia, Peter Kabos, Mary Gates, Ya-Chi Chen, Jill Fredrickson, Xiaojing Wang, Lori Sickels Friedman, Jill Spoerke, Steven Gendreau, Ciara Metcalfe, Lichuan Liu, Ching-Wei Chang, Sharareh Monemi, Monica Gonzalez, Ursula McCurry, Sandra Milan, Eric William Humke, Sherene Loi. A first-in-human phase I study to evaluate the oral selective estrogen receptor degrader (SERD), GDC-9545, in postmenopausal women with estrogen receptor-positive (ER+) HER2-negative (HER2-) metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-05.

Abstract P5-02-08: Androgen receptor pathway activity and the ratio between androgen and estrogen receptor pathway activity in breast cancer subtypes

Abstract Background: Androgen receptor (AR) immunohistochemistry staining in breast cancer has revealed frequent AR expression in all breast cancer subtypes. Activity of the AR signaling pathway can be either tumor suppressive or tumor promoting, depending on tumor context (Pharmacol Ther., In press, https://doi.org/10.1016/j.pharmthera.2019.05.005). For this reason, in an individual patient, AR phenotypic activity needs to be defined in order to choose the proper AR targeted therapy. The AR/ER protein ratio has been suggested as a biomarker to define this AR function and enable the appropriate therapy choice, i.e. androgen therapy in case of a low AR/ER protein expression ratio, and anti-androgen therapy in case of a high ratio. However, AR and ER expression are not necessarily associated with an active signaling pathway. Therefore we investigated the ratio between functional AR and ER pathway activities as this may have an advantage in guiding therapy choice. Methods: AR and ER pathway activity and AR/ER pathway activity ratio were determined on public Affymetrix HG-U133 Plus 2.0 gene expression microarray data of clinical breast cancer studies (available in the GEO database: GSE12276, GSE21653, GSE20685, GSE42568, GSE6532, GSE9195, GSE45827, GSE7307, GSE10780, GSE17907, GSE21422, GSE5764, GSE31192, GSE54002 and Array Express: E-MTAB-365; total n=2090). For this, we used previously described tests that enable quantification of functional pathway activity, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the ER and AR transcription factor (Cancer Res., 2014, vol. 74, no. 11, pp. 2936-2945; Sci Rep., 2019, vol. 9, no. 1, p. 1603). Intrinsic subtyping of all samples has been done using the same methodology as described elsewhere (Am J Pathol, 2018, vol. 188, no. 9, pp 1956-1972).Results: AR pathway activity was increased in all breast cancer subtypes compared to normal breast tissue (p<0.0001); highest AR activity scores were observed in the HER2-enriched subtype. As shown before, ER activity scores were highest in luminal A and B subtypes. The AR/ER pathway activity ratio was low in luminal A and B, and comparable with normal tissue, but high in HER2 and basal breast cancer subtypes (p<0.0001). Discussion: Results are compatible with currently available evidence and show that the AR pathway can be activated in all breast cancer subtypes. We hypothesize that its function depends on the level of co-existent ER pathway activity, i.e. a tumor suppressive function in ER pathway active breast cancer, and a tumor promoting function in ER pathway inactive breast cancer. Upon future clinical conformation, the AR/ER pathway activity ratio as determined on an individual patient tissue sample may be an improved biomarker to guide the choice of (complementary) AR pathway targeted therapy in breast cancer, i.e. androgen receptor pathway inhibitors in case of a high ratio and potential use of androgen receptor modulators in case of a low ratio. Citation Format: Anja van de Stolpe, Yvonne Wesseling-Rozendaal, Marcia Alves de Inda, Henk van Ooijen, Wim Verhaegh. Androgen receptor pathway activity and the ratio between androgen and estrogen receptor pathway activity in breast cancer subtypes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-08.

The CHORD protein CHP-1 regulates EGF receptor trafficking and signaling in C. elegans and in human cells.

The intracellular trafficking of growth factor receptors determines the activity of their downstream signaling pathways. Here, we show that the putative HSP-90 co-chaperone CHP-1 acts as a regulator of EGFR trafficking in C. elegans. Loss of chp-1 causes the retention of the EGFR in the ER and decreases MAPK signaling. CHP-1 is specifically required for EGFR trafficking, as the localization of other transmembrane receptors is unaltered in chp-1(lf) mutants, and the inhibition of hsp-90 or other co-chaperones does not affect EGFR localization. The role of the CHP-1 homolog CHORDC1 during EGFR trafficking is conserved in human cells. Analogous to C. elegans, the response of CHORDC1-deficient A431 cells to EGF stimulation is attenuated, the EGFR accumulates in the ER and ERK2 activity decreases. Although CHP-1 has been proposed to act as a co-chaperone for HSP90, our data indicate that CHP-1 plays an HSP90-independent function in controlling EGFR trafficking through the ER.

RETRACTED ARTICLE: Cyanidin-3-glucoside activates Nrf2-antioxidant response element and protects against glutamate-induced oxidative and endoplasmic reticulum stress in HT22 hippocampal neuronal cells

BackgroundCyanidin-3-glucoside (C3G), a major anthocyanin present in berries, exhibits a strong antioxidant and has been shown to possess a neuroprotection. Prolonged exposure to glutamate will lead to oxidative damage and endoplasmic reticulum stress which could play a key detrimental role in the development of neurodegenerative disorders (NDs). In the present study, we investigated the neuroprotective effect and underlying mechanisms of C3G on the reduction of oxidative/ER stress-induced apoptosis by glutamate in HT22 mouse hippocampal neuronal cells.MethodCells were pre-treated with C3G in various concentrations, followed by glutamate. Cell viability and toxicity were examined using MTT and LDH assays. The apoptotic and necrotic cell death were carried out by Annexin V-FITC/propidium iodide co-staining assays. Generation of intracellular reactive oxygen species (ROS) in cells was measured by flow cytometry using DCFH-DA probe. Expression of antioxidant genes was evaluated by Real-time polymerase chain reaction analysis. The possible signaling pathways and proteins involved were subsequently demonstrated by Western blot analysis.ResultThe pretreatment of the HT22 cells with C3G protected cell death from oxidative toxicity induced by glutamate. We demonstrated that treatment cells with glutamate caused several radical forms of ROS formation, and they were abolished by specific ROS inhibitors. Interestingly, C3G directly scavenged radical activity and inhibited intracellular ROS generation in our cell-based system. In addition, C3G pretreatment suppressed the up-regulation of specific ER proteins namely calpain, caspase-12 and C/EBP homologous proteins (CHOP) induced by glutamate-mediated oxidative and ER stress signal by up-regulating the expressions of survival proteins, including extracellular regulated protein kinase (ERK) and nuclear factor E2-related factor 2 (Nrf2). Furthermore, dramatically activated gene expression of endogenous antioxidant enzymes (i.e. superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPx)), and phase II enzymes (glutathione-S-transferases (GSTs)) was found in C3G-treated with cells.ConclusionsOur finding suggest that C3G could be a promising neuroprotectant via inhibition of glutamate-induced oxidative and ER stress signal and activation of ERK/Nrf2 antioxidant mechanism pathways.

Astragalus polysaccharide attenuates metabolic memory-triggered ER stress and apoptosis via regulation of miR-204/SIRT1 axis in retinal pigment epithelial cells.

Background: ‘Metabolic memory’ of early hyperglycaemic environment has been frequently suggested in the progression of diabetic retinopathy (DR). Retinal pigment epithelial (RPE) cells are crucial targets for DR initiation following hyperglycaemia. Astragalus polysaccharides (APS) has been long used as a traditional Chinese medicine in treating diabetes. In the present study, the preventive effects and mechanisms of APS on metabolic memory-induced RPE cell death were investigated.Methods: The expressions of miR-204 and SIRT1 were determined by reverse transcription quantitative PCR (RT-qPCR). Dual luciferase assay was applied to detect the potential targeting effects of miR-204 on SIRT1. SIRT1, ER stress and apoptosis related proteins were monitored using Western blotting. Apoptosis was assessed by TUNEL assay and Annexin V/PI staining followed by flow cytometry analysis. MiR-204 mimics and shSIRT1 were applied for miR-204 overexpression and SIRT1 knockdown, respectively.Results: High glucose exposure induced metabolic memory, which was accompanied with sustained dysregulation of miR-204/SIRT1 axis, high level of ER stress and activation of apoptotic pathway even after replacement with normal glucose. Pre-treatment with APS concentration-dependently reversed miR-204 expression, leading to disinhibition of SIRT1 and alleviation of ER stress-induced apoptosis indicated by decreased levels of p-PERK, p-IRE-1, cleaved-ATF6, Bax, cleaved caspase-12, -9, -3, and increased levels of Bcl-2 and unleaved PARP. The effects of APS on RPE cells were reversed by either miR-204 overexpression or SIRT1 knockdown.Conclusions: We concluded that APS inhibited ER stress and subsequent apoptosis via regulating miR-204/SIRT1 axis in metabolic memory model of RPE cells.

Towards Age-Related Anti-Inflammatory Therapy: Klotho Suppresses Activation of ER and Golgi Stress Response in Senescent Monocytes.

Immunosenescence in monocytes has been shown to be associated with several biochemical and functional changes, including development of senescence-associated secretory phenotype (SASP), which may be inhibited by klotho protein. To date, it was believed that SASP activation is associated with accumulating DNA damage. However, some literature data suggest that endoplasmic reticulum and Golgi stress pathways may be involved in SASP development. Thus, the aim of this study was to investigate the role of klotho protein in the regulation of immunosenescence-associated Golgi apparatus and ER stress response induced by bacterial antigens in monocytes. We provide evidence that initiation of immunosenescent-like phenotype in monocytes is accompanied by activation of CREB34L and TFE3 Golgi stress response and ATF6 and IRE1 endoplasmic reticulum stress response, while klotho overexpression prevents these changes. Further, these changes are followed by upregulated secretion of proinflammatory cytokines, which final modification takes place exclusively in the Golgi apparatus. In conclusion, we provide for the first time evidence of klotho involvement in the crosstalk on the line ER-Golgi, which may, in turn, affect activation of SASP. This data may be useful for a novel potential target for therapy in age-related and chronic inflammatory conditions.

The Role of CHK1 Varies with the Status of Oestrogen-receptor and Progesterone-receptor in the Targeted Therapy for Breast Cancer.

Objective: The therapeutic effects of the checkpoint kinase 1 (CHK1)-targeted inhibition in tumor therapy have been confirmed, but how to choose an effective application method in breast cancer with heterogeneous molecular characteristics has remained unclear.Methods: We evaluated the status of CHK1 in breast cancer using the cancer genome atlas database. Chemosensitivity and single-agent antitumor activity of CHK1 inhibition were measured by drug sensitivity assay, cell proliferation assay, cell cycle and apoptosis analysis in breast cancer with different ER/PR status. And based on the conjoint transcriptome atlas analyses, the corresponding mechanism were explored.Results: In ER-/PR-/HER2- breast cancer, CHK1 inhibition enhanced adriamycin (ADR) chemosensitivity which was mediated by the mitotic checkpoint complex (MCC)-anaphase-promoting complex/cyclosome (APC/C)-cyclin B1 axis, Msh homeobox 2 (MSX2) and Bcl-2-like protein 11 (BIM). However, in ER+/PR+/HER2- breast cancer, because of the significant suppression for centromere protein F (CENPF)-mediated transcriptional activation of CHK1 induced by ADR itself, CHK1 inhibition fails to sensitize ADR toxicity. Interestingly, CHK1 inhibition showed the single-agent antitumor activity in ER+/PR+/HER2- breast cancer which was mediated by the cyclin dependent kinase inhibitor 1A (p21), kinesin family member 11 (Eg5) and cell surface death receptor (Fas).Conclusions: CHK1's variable role determines the application of CHK1 inhibition in breast cancer with ER/PR heterogeneity.

Recent progress in selective estrogen receptor downregulators (SERDs) for the treatment of breast cancer.

Selective estrogen receptor downregulators (SERDs) are a novel class of compounds capable of reducing the ERα protein level and blocking ER activity. Therefore, SERDs are considered as a significant therapeutic approach to treat ER+ breast cancer in both early stage and more advanced drug-resistant cases. After the FDA approval of a steroidal drug, fulvestrant, as a SERD for the treatment of breast cancer in patients who have progressed on antihormonal agents, several molecules with diverse chemical structures have been rapidly developed, studied and evaluated for selective estrogen receptor downregulation activity. Here we compile the promising SERDs reported in recent years and discuss the chemical structure and pharmacological profile of the most potent compound of the considered series. Because of the availability of only a limited number of effective drugs for the treatment of breast cancer, the quest for a potent SERD with respectable activity and bioavailability is still ongoing. The goal of this article is to make available to the reader an overview of the current progress in SERDs and provide clues for the future discovery and development of novel pharmacological potent SERDs for the treatment of breast cancer.

Elacestrant (RAD1901) exhibits anti-tumor activity in multiple ER+ breast cancer models resistant to CDK4/6 inhibitors

BackgroundAddition of CDK4/6 inhibitors (CDK4/6i) to endocrine therapy significantly increased progression-free survival, leading to their approval and incorporation into the metastatic breast cancer treatment paradigm. With these inhibitors being routinely used for patients with advanced estrogen receptor-positive (ER+) breast cancer, resistance to these agents and its impact on subsequent therapy needs to be understood. Considering the central role of ER in driving the growth of ER+ breast cancers, and thus endocrine agents being a mainstay in the treatment paradigm, the effects of prior CDK4/6i exposure on ER signaling and the relevance of ER-targeted therapy are important to investigate. The objective of this study was to evaluate the anti-tumor activity of elacestrant, a novel oral selective estrogen receptor degrader (SERD), in preclinical models of CDK4/6i resistance.MethodsElacestrant was evaluated as a single agent, and in combination with alpelisib or everolimus, in multiple in vitro models and patient-derived xenografts that represent acquired and “de novo” CDK4/6i resistance.ResultsElacestrant demonstrated growth inhibition in cells resistant to all three approved CDK4/6i (palbociclib, abemaciclib, ribociclib) in both ESR1 wild-type and mutant backgrounds. Furthermore, we demonstrated that elacestrant, as a single agent and in combination, inhibited growth of patient-derived xenografts that have been derived from a patient previously treated with a CDK4/6i or exhibit de novo resistance to CDK4/6i. While the resistant lines demonstrate distinct alterations in cell cycle modulators, this did not affect elacestrant’s anti-tumor activity. In fact, we observe that elacestrant downregulates several key cell cycle players and halts cell cycle progression in vitro and in vivo.ConclusionsWe demonstrate that breast cancer tumor cells continue to rely on ER signaling to drive tumor growth despite exposure to CDK4/6i inhibitors. Importantly, elacestrant can inhibit this ER-dependent growth despite previously reported mechanisms of CDK4/6i resistance observed such as Rb loss, CDK6 overexpression, upregulated cyclinE1 and E2F1, among others. These data provide a scientific rationale for the evaluation of elacestrant in a post-CDK4/6i patient population. Additionally, elacestrant may also serve as an endocrine backbone for rational combinations to combat resistance.