ER Target Research Articles

A preoperative window-of-opportunity study of oral SERD, imlunestrant, in newly diagnosed ER-positive, HER2-negative early breast cancer: Results from EMBER-2 Study.

Imlunestrant is an oral SERD with favorable safety and preliminary efficacy in patients with ABC. PD biomarker data can optimize drug dosing; herein is the PD data from the EMBER-2 study. Postmenopausal women with untreated, operable ER-positive, HER2-negative EBC were randomized to 400mg vs 800mg of imlunestrant daily for ~2 weeks before surgery. A single arm testing 200mg daily was later accrued. PD biomarker changes (ER, PR, Ki-67 by IHC, and mRNA expression of ER-related genes) were evaluated in paired tumor samples (pre-/ post-treatment). Safety and PK were also assessed. Among evaluable paired samples (n=75), PD profiles demonstrated consistent ER targeting between 400 and 800mg doses with less toxicity at the 400mg dose. Although inducing the lowest rate of CCCA, PD and PK results were similar for the 200mg dose. EMBER-2 combined with existing phase 1 data has identified 400mg as the optimal imlunestrant dose.

7993 Thienopyrimidine-Based Estrogen Receptor Modulators Adopt Unique Ligand Binding Poses to Elicit Anti-Proliferative Activities in ER+ Breast Cancer Cells

Abstract Disclosure: E.C. Fink: None. S.L. Mateus: None. N. Meganathan: None. V.K. Sammeta: None. J.D. Norris: None. D.P. McDonnell: None. T. Willson: None. S.W. Fanning: None. One in eight women will be diagnosed with breast cancer in their lifetimes. Estrogen receptor alpha (ER) drives breast cancer pathology and is expressed in approximately seventy percent of tumors. Hormone therapies are used to treat and prevent the metastasis of ER+ breast cancers and these patients have favorable 5-year survivals. However, most breast cancer patient deaths are ER+. While the next generation of antiestrogens have shown promise in the advanced setting, more work needs to be done to fully address these mortalities. ER is a ligand-dependent master transcriptional regulator, whereby ligand-specific ER conformational ensembles redirect multiple programs to produce oncogenic or therapeutic endpoints. To better understand ER structure-activity relationships, we have developed new antiestrogenic small molecules based on a thienopyrimidine scaffold. Comprehensive structural, ER activity, and cancer endpoint profiling shows that these new molecules adopt a unique ligand binding pose, which perturbs new structural elements within the orthosteric hormone binding pocket. These molecules downregulate ER target genes and halt the proliferation of ER+ breast cancer cells. Presentation: 6/3/2024

Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies.

Breast cancer therapy has been facing remarkable changes. Classic treatments are now combined with other therapies to improve efficacy and surpass resistance. Indeed, the emergence of resistance demands the development of novel therapeutic approaches. Due to key estrogen signaling, estrogen receptor-positive (ER+) breast cancer treatment has always been focused on aromatase inhibition and ER modulation. Lately, the effects of phytocannabinoids, mainly Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), have been evaluated in different cancers, including breast. However, Cannabis sativa contains more than 120 phytocannabinoids less researched and understood. Here, we evaluated, both in silico and in vitro, the ability of 129 phytocannabinoids to modulate important molecular targets in ER+ breast cancer: aromatase, ER, and androgen receptor (AR). In silico results suggested that some cannabinoids may inhibit aromatase and act as ERα antagonists. Nine selected cannabinoids showed, in vitro, potential to act either as ER antagonists with inverse agonist properties, or as ER agonists. Moreover, these cannabinoids were considered as weak aromatase inhibitors and AR antagonists with inverse agonist action. Overall, we present, for the first time, a comprehensive analysis of the actions of the phytocannabinoids in targets of ER+ breast tumors, pointing out their therapeutic potential in cancer and in other diseases.

Neoadjuvant treatment with trastuzumab and pertuzumab plus fulvestrant in older patients with HER2-positive, ER-positive early breast cancer.

13 Background: The prevalence of breast cancer in older adults (≥70 years) is increasing and worse outcomes compared to younger patients could be explained by advanced presentation, late diagnosis, deterioration of organ function, and the presence of multimorbidities. Older patients are under-represented in clinical trials. Therefore, the risks and benefits of anticancer therapy should be carefully evaluated. The CREATE-X36 and KATHERINE37 trials enrolled few older individuals but did not show any new safety concerns, older patients should be considered for trastuzumab in case of residual HER2-positive disease following neoadjuvant systemic therapy; neoadjuvant endocrine therapy for at least 4-6 months is useful for older patients who are not immediately suitable for surgery and aromatase inhibitors are favored over tamoxifen in view of better response rate. Although adjuvant trastuzumab is beneficial regardless of age, anti-HER2 (neo)adjuvant strategies remain poorly investigated in patients age 65 years or older. Pertuzumab can be considered for high-risk individuals, but diarrhoea can be debilitating in older adults, similarly with adjuvant neratinib. Age is associated with increased cardiac toxicity rates with trastuzumab,80 with 15–40% of patients requiring early discontinuation, particularly patients who are age 80 years or older and have multimorbidities. Methods: Open-label, exploratory, phase 2 study done at one center in Venezuela. Patients were eligible if they had previously untreated, histologically confirmed, unilateral, invasive, cN0, HER2-positive, ER-positive breast cancer and older >70y. Patients were treated every 3 weeks with intravenous trastuzumab (8 mg/kg loading dose followed by 6 mg/kg) and intravenous pertuzumab (840 mg loading dose in the first cycle and then at 420 mg) and intramuscular fulvestrant (500 mg) every 4 weeks for four cycles. The coprimary endpoints were change from baseline in Ki67 expression at surgery and pathological complete response. Results: Between January, 2020, and June, 2023, we enrolled 12 patients. At baseline, geometric mean Ki67 expression was 42·9 and 12·1 at time of surgery (n=8; p=0·013). A clinical objective response immediately before surgery was achieved by 10 of 12 patients. At surgery, eight patients had a pathological complete response in breast and axillary nodes. The most frequent grade 3 adverse events was diarrhea (n=4), and stomatitis. No grade 4 or serious adverse events were recorded in the study and there were no deaths. Conclusions: The combination of fulvestrant, trastuzumab, and pertuzumab had a effect on the expression of Ki67 and pCR at surgery. Triple targeting of ER, HER2, could be an effective chemotherapy-free treatment strategy for older patiens. Further clinical testing and additional molecular characterisation is necessary.

Citrus polymethoxyflavones degrade estrogen receptor-alpha (ERα) and combine with tamoxifen for the treatment of estrogen receptor-positive breast cancer

Estrogen receptor-positive (ER+) breast cancer seriously endangers the women's physical and mental health worldwide and ER targeting therapy is vital. Here, we found that a citrus polymethoxyflavones (PMFs)-rich hydrolysate (C–H) and its major components (nobiletin and 3-methoxynobiletin) potently degrade ERα protein via the ubiquitin-proteasome pathway, thereby impairing the proliferation of ER+ breast cancer cells. Moreover, our study exhibited that C–H combined with tamoxifen (TAM) inhibited the cell proliferation of ER+ breast cancer in vitro. It was further confirmed that C–H decreased tumor growth of ER+ breast cancer in tumor-bearing 129 mice in vivo and improved the efficacy of tamoxifen. Our study revealed that the citrus PMFs have potential applications as pharmaceutical and healthcare products in breast cancer treatment by targeting ERα protein degradation.

Abstract PO4-23-10: Restoring the efficacy of standard of care in treatment-refractory ER+ breast cancer by re-activation of cAMP-ROS-DNA damage axis

Abstract Estrogen receptor (ER)-positive (ER+) breast cancer has the highest incidence rate and accounts for around 75% of all cases. Endocrine therapy has been the mainstay therapy for the treatment of both early and late-stage ER+ breast cancer for decades. Although most ER+ breast cancer patients initially respond well to endocrine therapy, resistance is common. The addition of CDK4/6 inhibitors to endocrine therapy led to significant improvements in clinical outcome, and they are now considered one of the standard of care (SOC) therapies. However, a significant proportion of patients still suffer from disease relapse upon prolonged use of endocrine therapy in combination with CDK4/6 inhibitors, representing a major clinical challenge that reduces the long-term benefit and patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying novel actionable targets are urgently needed. Here, we show that endocrine therapies and CDK4/6 inhibitors cause toxic PARP1 trapping and generation of a functional BRCAness phenotype by downregulating key DNA repair proteins that ultimately result in increased histone parylation and reduced H3K9 acetylation, leading to transcriptional blockage and cell death. Mechanistically, we found that SOC therapy downregulates phosphodiesterase 4D (PDE4D), resulting in increased cAMP levels, PKA-dependent phosphorylation of mitochondrial COXIV-I, generation of mitochondrial reactive oxygen species (ROS), and DNA damage. Importantly, we identified PDE4D as a novel ER target gene that in turn stimulates ER activity in a feedforward loop in endocrine-responsive models and regulates BRCA1 expression. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun. Inhibition of PDE4D using BPN14770, the first-in-class PDE4D allosteric inhibitor that has successfully completed Phase II trials in Fragile X syndrome, or its upstream EGFR in combination with SOC therapies in drug-resistant settings, including multiple acquired SOC resistant cell line models, primary cultures and organoids of endocrine resistant ER+ PDXs reinstates PARP1 trapping and BRCAness, leading to drug sensitization in vitro and in vivo. Notably, we demonstrated that high PDE4D mRNA and protein expression is associated with dramatically worse disease-free survival and overall survival in endocrine therapy-treated ER+ breast cancer. Considering the availability of potent and non-toxic PDE4D inhibitors, clinically approved EGFR and PARP1 inhibitors, our findings have great translational potential. Citation Format: Ozge Saatci, Metin Cetin, Meral Uner, Unal Metin Tokat, Ioulia Chatzistamou, Pelin Gulizar Ersan, Elodie Montaudon, Aytekin Akyol, Sercan Aksoy, Aysegul Uner, Elisabetta Marangoni, Sajish Mathew, Ozgur Sahin. Restoring the efficacy of standard of care in treatment-refractory ER+ breast cancer by re-activation of cAMP-ROS-DNA damage axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-10.

Abstract PS15-09: Imlunestrant monotherapy and in combination with abemaciclib, with or without an aromatase inhibitor, in estrogen receptor-positive (ER+), HER2-negative (HER2-) advanced breast cancer (aBC): updated results from the EMBER study

Abstract Background: Imlunestrant is an oral, brain-penetrant selective estrogen receptor degrader, designed to deliver continuous ER target inhibition, including in ESR1-mutant breast cancer. In the first-in-human dose escalation/expansion (Phase 1a/1b) study of the EMBER trial (NCT04188548), imlunestrant showed favorable safety, pharmacokinetics, and clinical benefit in pre-treated patients with ER+, HER2- aBC when administered as monotherapy (Jhaveri, ASCO 2022) or with abemaciclib ± aromatase inhibitor (AI) (Jhaveri, SABCS 2022). Here, we report updated data from Phase 1a/1b of imlunestrant monotherapy as well as from Phase 1b of imlunestrant with abemaciclib ± AI in EMBER. Methods: Phase 1a/1b enrolled patients with ER+ aBC (prior endocrine therapy (ET) sensitivity or untreated de novo aBC). Prior aBC therapy allowance was ≤3 (no restrictions on the type of prior therapies) in Phase 1a and ≤2 (only 1 prior ET) in Phase 1b for patients receiving imlunestrant monotherapy; and ≤1 (no prior CDK4/6 inhibitors) in Phase 1b for patients randomized to receive imlunestrant + abemaciclib ± AI. Key endpoints included safety and tolerability, objective response rate (ORR: complete response (CR) or partial response (PR) in patients with measurable disease), clinical benefit rate (CBR: CR or PR, or stable disease ≥24 weeks) per RECIST v1.1, and progression-free survival (PFS). Results: As of March 2023, 114 patients received imlunestrant monotherapy (200mg n=21; 400mg (RP2D) n= 51; ≥600mg n=42), and 85 patients received imlunestrant (400mg n=80; 800 mg n=5) in combination with abemaciclib ± AI (both at labeled doses). At the RP2D, the most common all-grade/Grade 3 treatment-emergent adverse events (TEAEs) with imlunestrant monotherapy were fatigue (39%/4%), nausea (39%/2%), and diarrhea (29%/2%). With the combination of imlunestrant + abemaciclib ± AI, the most common all-grade/Grade 3 TEAEs were diarrhea (88%/9%), nausea (61%/0%), fatigue (51%/5%), and neutropenia (44%/16%). No safety signals of ocular or cardiac toxicity (bradycardia/QTc prolongation) were observed. Baseline characteristics, safety, and preliminary efficacy are presented in Table 1. Combination therapy of imlunestrant and abemaciclib ± AI improved ORR and CBR. Conclusion: With longer follow-up, imlunestrant alone or in combination with abemaciclib ± AI continues to demonstrate a tolerable safety profile along with favorable preliminary efficacy in patients with ER+, HER2- aBC. Further data will be presented at the meeting. The Phase 3, EMBER-3 study is ongoing; evaluating imlunestrant, investigator’s choice ET, and imlunestrant plus abemaciclib in ET pre-treated ER+, HER2- aBC patients (NCT04975308). Table. Baseline characteristics, safety and preliminary efficacy a, in patients with available ctDNA data. Citation Format: Komal Jhaveri, Elgene Lim, Rinath Jeselsohn, Cynthia Ma, Erika Hamilton, Cynthia Osborne, Manali Bhave, Peter Kaufman, J. Thaddeus Beck, Luis Manso, Ritesh Parajuli, Hwei-Chung Wang, Jessica Tao, Ajay Dhakal, Jean-Yves Pierga, Yen-Shen Lu, Tim Larson, Yolanda Jerez Gilarranz, Roohi Ismail-Khan, Francesca Bacchion, Claudia Karacsonyi, Yujia Li, Shawn T. Estrem, Bastien Nguyen, Muralidhar Beeram. Imlunestrant monotherapy and in combination with abemaciclib, with or without an aromatase inhibitor, in estrogen receptor-positive (ER+), HER2-negative (HER2-) advanced breast cancer (aBC): updated results from the EMBER study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS15-09.

Abstract 6236: Systems biology modeling identifies SMARCD3 as a master regulator facilitating everolimus resistance in ER+ breast cancer

Abstract Breast cancer is the most common malignancy in women and estrogen receptor positive (ER+) breast cancers represent nearly 75% of all breast tumors. Everolimus, an mTORC1 inhibitor, in combination with exemestane has been approved for patients with metastatic ER+ breast cancer. However, many patients eventually develop resistance to everolimus and leads to poor survival outcomes. Characterizing everolimus-resistant cells can reveal targetable molecular phenotypes that can eventually lead to new therapeutic targets. In this study, we elucidate systems-level phenotypic distinctions between everolimus sensitive and refractory cells. We generated several isogenic ER+ breast cancer cell lines resistant or sensitive to everolimus. We then leveraged transcriptomic profiles from sensitive and resistant cells before or after everolimus treatment to decipher the underlying mechanisms essential to the resistant state. Using linear mixed effect models, we identified several meta-phenotypes that were distinct between sensitive and resistant cell states. These included growth-factor signaling, cell cycle, metabolism, stemness, and apoptosis meta-phenotypes. Our analyses also identified strong activation of growth-factor receptors including IGF1R/INSR and ESR1 in the resistant cells, which were maintained despite everolimus treatment. Next, we adopted a transcriptional regulatory network reconstruction framework to identify the master regulators responsible for the activation of resistance meta-phenotypes. We identified SMARCD3 as the key master regulator controlling expression of genes across diverse resistance meta-phenotypes, including growth-factor signaling. Further, using transcriptomic profiles from a neoadjuvant trial of 23 post-menopausal ER+ breast cancers treated with everolimus, we found that SMARCD3 expression was elevated in resistant tumors. To confirm the role of SMARCD3 in everolimus resistance, we repressed transcription of the SMARCD3 gene using CRISPR interference to re-sensitize resistant cells to everolimus treatment. In conclusion, we used context-specific cell transcriptomic profiles and integrative systems-biology approaches to elucidate mechanisms of resistance in response to everolimus treatment in ER+ breast cancer. Our findings suggest SMARCD3 upregulation promotes resistance to everolimus treatment and may serve as a novel therapeutic target in ER+ breast cancer. Citation Format: Eric F. Medina, Patrick A. Cosgrove, Eleni Farmaki, Vince K. Grolmusz, Feng Chi, Jason I. Griffiths, Andrea H. Bild, Aritro Nath. Systems biology modeling identifies SMARCD3 as a master regulator facilitating everolimus resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6236.

Abstract 563: Targeting PDE4D reinstates toxic PARP trapping via cAMP-ROS-DNA damage axis and restores the efficacy of standard of care in treatment-refractory ER+ breast cancer

Abstract Endocrine therapies, modulating ER level and/or activity, and the CDK4/6 inhibitors are among the standard-of-care (SOC) therapies used in estrogen receptor-positive (ER+) breast cancer. However, most ER+ breast cancer patients eventually develop resistance to SOC, representing a major clinical challenge that reduces clinical outcome. Therefore, elucidating the common mechanisms of sensitivity and resistance to SOC therapies, and identifying novel actionable targets are urgently needed. Here, we demonstrated that cell death induced by endocrine therapies and CDK4/6 inhibitors involves toxic PARP1 trapping and generation of a functional BRCAness (i.e., BRCA1/2 deficiency) phenotype, leading to transcriptional blockage. We showed that this is achieved by activation of the cAMP/reactive oxygen species (ROS)/DNA damage axis upon downregulation of phosphodiesterase 4D (PDE4D). Importantly, we identified PDE4D as a novel ER target gene that in turn stimulates ER activity in a feedforward loop in endocrine-responsive models. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun. Inhibition of PDE4D using BPN14770, the first-in-class PDE4D allosteric inhibitor that has successfully completed Phase II trials in Fragile X syndrome, reinstates PARP1 trapping and BRCAness, leading to drug sensitization in vitro and in vivo. Furthermore, inhibition of EGFR or PARP1 recapitulates the effects of PDE4D targeting and overcomes SOC resistance irrespective of the BRCA1/2 status in cell line models, primary cultures and organoids of endocrine resistant ER+ PDXs. Notably, we demonstrated that high PDE4D mRNA and protein expression is associated with dramatically worse disease-free survival and overall survival in endocrine therapy-treated ER+ breast cancer. Given the availability of potent and non-toxic PDE4D inhibitors, and clinically approved EGFR and PARP1 inhibitors, our findings have great translational potential to improve the clinical outcome of refractory ER+ breast cancers. Citation Format: Ozge Saatci, Metin Cetin, Meral Uner, Unal M. Tokat, Ioulia Chatzistamou, Elodie Montaudon, Aytekin Akyol, Sercan Aksoy, Aysegul Uner, Elisabetta Marangoni, Mathew Sajish, Ozgur Sahin. Targeting PDE4D reinstates toxic PARP trapping via cAMP-ROS-DNA damage axis and restores the efficacy of standard of care in treatment-refractory ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 563.

TRPS1 modulates chromatin accessibility to regulate estrogen receptor alpha (ER) binding and ER target gene expression in luminal breast cancer cells.

Common genetic variants in the repressive GATA-family transcription factor (TF) TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over 80 percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes estrogen receptor alpha (ER) to redistribute in the genome. ER redistribution leads to both repression and activation of dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the genomic distribution of ER, both activating and repressing transcription of genes related to cancer cell fitness.

Molecular Regulation and Oncogenic Functions of TSPAN8.

Tetraspanins, a superfamily of small integral membrane proteins, are characterized by four transmembrane domains and conserved protein motifs that are configured into a unique molecular topology and structure in the plasma membrane. They act as key organizers of the plasma membrane, orchestrating the formation of specialized microdomains called "tetraspanin-enriched microdomains (TEMs)" or "tetraspanin nanodomains" that are essential for mediating diverse biological processes. TSPAN8 is one of the earliest identified tetraspanin members. It is known to interact with a wide range of molecular partners in different cellular contexts and regulate diverse molecular and cellular events at the plasma membrane, including cell adhesion, migration, invasion, signal transduction, and exosome biogenesis. The functions of cell-surface TSPAN8 are governed by ER targeting, modifications at the Golgi apparatus and dynamic trafficking. Intriguingly, limited evidence shows that TSPAN8 can translocate to the nucleus to act as a transcriptional regulator. The transcription of TSPAN8 is tightly regulated and restricted to defined cell lineages, where it can serve as a molecular marker of stem/progenitor cells in certain normal tissues as well as tumors. Importantly, the oncogenic roles of TSPAN8 in tumor development and cancer metastasis have gained prominence in recent decades. Here, we comprehensively review the current knowledge on the molecular characteristics and regulatory mechanisms defining TSPAN8 functions, and discuss the potential and significance of TSPAN8 as a biomarker and therapeutic target across various epithelial cancers.

FOXK1 promotes hormonally responsive breast carcinogenesis by suppressing apoptosis.

Globally, breast cancer constitutes the predominant malignancy in women. Abnormal regulation of epigenetic factors plays a key role in the development of tumors. Anti-apoptosis is a characteristic of tumor cells. Therefore, exploring and identifying relevant epigenetic factors that regulate the apoptosis of tumor cells is the foundation for clarifying the pathogenesis of tumors and achieving precision antitumor therapy. This study focused on exploring the epigenetic mechanism of FOXK1 in the development of estrogen receptor-positive (ER+ ) breast cancer. We used overexpressing FLAG-FOXK1 MCF-7 cells to perform silver staining mass spectrometry analysis and conducted Co-IP experiments to verify the interactions. ChIP-seq was conducted on MCF-7 cells to examine FOXK1's binding across the genome and its transcriptional target sites. To validate the ChIP-seq results, qChIP, western blotting, and quantitative polymerase chain reaction (qPCR) were performed. Through TUNEL assay, cell counting assay, colony formation assay, and the mouse xenograft models, the effect of FOXK1 on breast cancer progression was detected. Finally, by analyzing online databases, the correlation between FOXK1 and the survival of breast cancer patients was examined. FOXK1 interacts with the REST/CoREST transcriptional corepression complex to transcriptionally inhibit target genes representing the apoptotic pathway. Abnormally high expression of FOXK1 prevents the apoptosis of ER+ breast cancer cells invitro and promotes ER+ breast tumor progression invivo. Furthermore, the expression of FOXK1 is negatively correlated with the survival of ER+ breast cancer patients. FOXK1 promotes ER+ breast carcinogenesis through anti-apoptosis and acts as a potential target for ER+ breast cancer treatment.

Abstract A008: Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer

Abstract Estrogen receptor (ER)-positive (ER+) breast cancer has the highest incidence rate and accounts for around 75% of all cases. Endocrine therapy and CDK4/6 inhibitors are the standard of care (SOC) in ER+ breast cancer; however, resistance is common, and it greatly reduces patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying actionable targets are urgently needed. Here, we show that SOC therapy causes DNA damage and toxic PARP1 trapping upon generation of a functional BRCAness (i.e., BRCA1/2 deficiency) phenotype, leading to increased histone parylation and reduced H3K9 acetylation, resulting in transcriptional blockage and cell death. Mechanistically, SOC therapy downregulates phosphodiesterase 4D (PDE4D), a novel ER target gene in a feedforward loop with ER, resulting in increased cAMP, PKA-dependent phosphorylation of mitochondrial COXIV-I, ROS generation and DNA damage. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun-mediated transcription. Importantly, we demonstrated that high PDE4D mRNA and protein expression is associated with dramatically worse disease-free survival and overall survival in endocrine therapy-treated ER+ breast cancer. Notably, combining SOC with inhibitors of PDE4D, EGFR or PARP1 overcomes SOC resistance irrespective of the BRCA1/2 status in multiple acquired SOC resistant cell line models, primary cultures and organoids of endocrine resistant ER+ PDXs in vitro and in vivo, providing actionable targets for restoring SOC efficacy. Considering the availability of potent and non-toxic PDE4D inhibitors, clinically approved EGFR and PARP1 inhibitors, our findings have great translational potential. Citation Format: Ozge Saatci, Mein Cetin, Meral Uner, Unal Metin Tokat, Ioulia Chatzistamou, Pelin Gulizar Ersan, Elodie Montaudon, Aytekin Akyol, Sercan Aksoy, Aysegul Uner, Elisabetta Marangoni, Mathew Sajish, Ozgur Sahin. Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A008.

MicroRNA-190b Targets RFWD3 in Estrogen Receptor-Positive Breast Cancer.

In the year 2020, breast cancer was the most common form of cancer worldwide. Roughly 70% of breast cancers are estrogen receptor-positive (ER+). MicroRNA-190b (miR-190b) has previously been reported to be upregulated in ER+ breast cancers. Previously, we have demonstrated that miR-190b is hypomethylated in ER+ breast cancers, potentially leading to its upregulation. To further study the role of miR-190b in ER+ breast cancer and to identify its clinically relevant targets in breast cancer. Patient cohort and cell line-based RNA-sequencing analysis. The Cancer Genome Atlas was used to obtain gene expression data and clinical information on patients with breast cancer. To identify messenger RNA (mRNA) targets for miR-190b, the ER+ breast cancer cell line T-47D was used to immunoprecipitate biotin-labeled miR-190b followed by RNA sequencing. Western blot was used to confirm miR-190b target. Patient survival based on miR-190b and selected target was studied using the Cancer Genome Atlas. In this study, we confirm that miR-190b is overexpressed in breast cancer via differential expression analysis and show that high expression of miR-190b results in more favorable outcomes in Luminal A patients, hazard ratio (HR) = 0.29, 95% confidence interval [CI] = 0.12-0.71, P = .0063. MicroRNA-190b target analysis identified RING finger and WD repeat domain 3 (RFWD3) as one of miR-190b regulatory targets in ER+ breast cancer. Survival analysis of RFWD3 showed that elevated levels result in poorer overall survival in patients with Luminal A breast cancer (HR = 2.22, 95% CI = 1.33-3.71, P = .002). Gene ontology analysis of our sequencing results indicates that miR-190b may have a role in breast cancer development and/or tumorigenesis and that it may be a suitable tool in characterization between the ER+ subtypes, Luminal A, and Luminal B. We show that miR-190b targets RFWD3 in ER+ breast cancers leading to lower RFWD3 protein expression. Low levels of RFWD3 are associated with better outcomes in patients with Luminal A breast cancer but not in patients with Luminal B breast cancer. These findings provide novel insights into miR-190b role in breast cancer and that its clinical relevance is subtype specific.

Photodynamic Modulation of Endoplasmic Reticulum and Mitochondria Network Boosted Cancer Immunotherapy.

Immunogenic cell death (ICD) represents a promising approach for enhancing tumor therapy efficacy by inducing antitumor immune response. However, current ICD inducers often have insufficient endoplasmic reticulum (ER) enrichment and ineffectiveness in tumor immune escape caused by ER-mitochondria interaction. In this study, a kind of photoactivatable probe, THTTPy-PTSA, which enables sequential targeting of the ER and mitochondria is developed. THTTPy-PTSA incorporates p-Toluenesulfonamide (PTSA) for ER targeting, and upon light irradiation, the tetrahydropyridine group undergoes a photo oxidative dehydrogenation reaction, transforming into a pyridinium group that acts as a mitochondria-targeting moiety. The results demonstrate that THTTPy-PTSA exhibits exceptional subcellular translocation from the ER to mitochondria upon light irradiation treatment, subsequently triggers a stronger ER stress response through a cascade-amplification effect. Importantly, the augmented ER stress leads to substantial therapeutic efficacy in a 4T1 tumor model by eliciting the release of numerous damage-associated molecular patterns, thereby inducing evident and widespread ICD, consequently enhancing the antitumor immune efficacy. Collectively, the findings emphasize the pivotal role of photodynamic modulation of the ER-mitochondria network, facilitated by THTTPy-PTSA with precise spatial and temporal regulation, in effectively bolstering the antitumor immune response. This innovative approach presents a promising alternative for addressing the challenges associated with cancer immunotherapy.

Characterization and expression analysis of seven lipid metabolism-related genes in yellow catfish Pelteobagrus fulvidraco fed high fat and bile acid diet

SREBPs, such as SREBP1 and SREBP2, were the key transcriptional factors regulating lipid metabolism. The processing of SREBPs involved many genes, such as scap, s1p, s2p, cideb. Here, we deciphered the full-length cDNA sequences of scap, srebp1, srebp2, s1p, s2p, cideb and cidec from yellow catfish Pelteobagrus fulvidraco. Their full-length cDNA sequences ranged from 1587 to 3884 bp, and their ORF length from 1191 to 2979 bp, encoding 396–992 amino acids. Some conservative domains were predicted, including the multiple transmembrane domains in SCAP, the bHLH-ZIP domain in SREBP1 and SREBP2, the ApoB binding region, ER targeting region and LD targeting region in CIDEb, the LD targeting region in the CIDEc, the conserved catalytic site and processing site in S1P, and the transmembrane helix domain in S2P. Their mRNA expression could be observed in the heart, spleen, liver, kidney, brain, muscle, intestine and adipose, but varied with tissues. The changes of their mRNA expression in responses to high-fat (HFD) and bile acid (BA) diets were also investigated in the brain, heart, intestine, kidney and spleen tissues. In the brain, HFD significantly increased the mRNA expression of seven genes (scap, srebp1, srebp2, s1p, s2p, cideb and cidec), and the BA attenuated the increase of scap, srebp1, srebp2, s1p, s2p, cideb and cidec mRNA expression induced by HFD. In the heart, HFD significantly increased the mRNA abundances of six genes (srebp1, srebp2, scap, s2p, cideb and cidec), and BA attenuated the increase of their mRNA abundances induced by HFD. In the intestine, HFD increased the cideb, s1p and s2p mRNA abundances, and BA attenuated the HFD-induced increment of their mRNA abundances. In the kidney, HFD significantly increased the scap, cidec and s1p mRNA expression, and BA diet attenuated the increment of their mRNA expression. In the spleen, HFD treatment increased the scap, srebp2, s1p and s2p mRNA expression, and BA diet attenuated HFD-induced increment of their mRNA expression. Taken together, our study elucidated the characterization, expression profiles and transcriptional response of seven lipid metabolic genes, which would serve as the good basis for the further exploration into their function and regulatory mechanism in fish.

Cathepsin V regulates cell cycle progression and histone stability in the nucleus of breast cancer cells.

Introduction: We previously identified that Cathepsin V (CTSV) expression is associated with poor prognosis in ER+ breast cancer, particularly within the Luminal A subtype. Examination of the molecular role of the protease within Luminal A tumours, revealed that CTSV promotes tumour cell invasion and proliferation, in addition to degradation of the luminal transcription factor, GATA3, via the proteasome. Methods: Cell line models expressing CTSV shRNA or transfected to overexpress CTSV were used to examine the impact of CTSV on cell proliferation by MTT assay and flow cytometry. Western blotting analysis was used to identify the impact of CTSV on histone and chaperone protein expression. Cell fractionation and confocal microscopy was used to illustrate the presence of CTSV in the nuclear compartment. Results: In this work we have identified that CTSV has an impact on breast cancer cell proliferation, with CTSV depleted cells exhibiting delayed progression through the G2/M phase of the cell cycle. Further investigation has revealed that CTSV can control nuclear expression levels of histones H3 and H4 via regulating protein expression of their chaperone sNASP. We have discovered that CTSV is localised to the nuclear compartment in breast tumour cells, mediated by a bipartite nuclear localisation signal (NLS) within the CTSV sequence and that nuclear CTSV is required for cell cycle progression and histone stability in breast tumour cells. Discussion: Collectively these findings support the hypothesis that targeting CTSV may have utility as a novel therapeutic target in ER+ breast cancer by impairing cell cycle progression via manipulating histone stabilisation.

Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer

Resistance to endocrine therapy and CDK4/6 inhibitors, the standard of care (SOC) in estrogen receptor-positive (ER+) breast cancer, greatly reduces patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying actionable targets are urgently needed. Here, we show that SOC therapy causes DNA damage and toxic PARP1 trapping upon generation of a functional BRCAness (i.e., BRCA1/2 deficiency) phenotype, leading to increased histone parylation and reduced H3K9 acetylation, resulting in transcriptional blockage and cell death. Mechanistically, SOC therapy downregulates phosphodiesterase 4D (PDE4D), a novel ER target gene in a feedforward loop with ER, resulting in increased cAMP, PKA-dependent phosphorylation of mitochondrial COXIV-I, ROS generation and DNA damage. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun. Notably, combining SOC with inhibitors of PDE4D, EGFR or PARP1 overcomes SOC resistance irrespective of the BRCA1/2 status, providing actionable targets for restoring SOC efficacy.

Resistance to abemaciclib is associated with increased metastatic potential and lysosomal protein deregulation in breast cancer cells.

Cyclin dependent kinase 4 and 6 inhibitors such as abemaciclib are routinely used to treat metastatic estrogen receptor positive (ER+) breast cancer. However, adaptive mechanisms inhibit their effectiveness and allow for disease progression. Using ER+ breast cancer cell models, we show that acquired resistance to abemaciclib is accompanied by increase in metastatic potential. Mass spectrometry-based proteomics from abemaciclib sensitive and resistant cells showed that lysosomal proteins including CTSD (cathepsin D), cathepsin A and CD68 were significantly increased in resistant cells. Combination of abemaciclib and a lysosomal destabilizer, such as hydroxychloroquine (HCQ) or bafilomycin A1, resensitized resistant cells to abemaciclib. Also, combination of abemaciclib and HCQ decreased migration and invasive potential and increased lysosomal membrane permeability in resistant cells. Prosurvival B cell lymphoma 2 (BCL2) protein levels were elevated in resistant cells, and a triple treatment with abemaciclib, HCQ, and BCL2 inhibitor, venetoclax, significantly inhibited cell growth compared to treatment with abemaciclib and HCQ. Furthermore, resistant cells showed increased levels of Transcription Factor EB (TFEB), a master regulator of lysosomal-autophagy genes, and siRNA mediated knockdown of TFEB decreased invasion in resistant cells. TFEB was found to be mutated in a subset of invasive human breast cancer samples, and overall survival analysis in ER+, lymph node-positive breast cancer showed that increased TFEB expression correlated with decreased survival. Collectively, we show that acquired resistance to abemaciclib leads to increased metastatic potential and increased levels of protumorigenic lysosomal proteins. Therefore, the lysosomal pathway could be a therapeutic target in advanced ER+ breast cancer.

OR22-06 Functional Characterization Of X-linked Genes In Regulating Epigenetic Landscape In Endocrine-resistant Breast Cancer

Abstract Disclosure: E.I. Ramos: None. B. Yang: None. M. Sedano: None. V. Reid: None. K. Diwa: None. A. Hidalgo: None. A. Beard: None. J. Patel: None. S.S. Gadad: None. Studies show that genes transcribed from chromosome X play critical roles in several biological processes. X-linked genes are tightly regulated and expressed in tissues protected from immune surveillance. However, several of them get dysregulated in diseases such as cancer. Intriguingly, genome-wide analyses showed their presence in the previously considered gene deserts in the human genome. Their direct role in estrogen transcription in tamoxifen-resistant breast cancer and biological outcomes are still unknown. To this end, we leveraged the power of functional genomics, including RNA with 4-thiouridine (4sU) coupled with RNA-sequencing and CRISPR-based genetic screening, to identify X-linked genes with a potential role in tamoxifen-directed estrogen-regulated transcription. Through a series of phenotypic assays, we selected a poorly studied transcript, “H19X,” with limited protein-coding potential; RNA-In-Situ-Hybridization revealed its localization to the nucleus. In the current study, we have identified the H19X-controlled estrogen-dependent gene regulatory mechanisms in tamoxifen-resistant estrogen receptor-positive (ER+) breast cancer cells. We manipulated the levels of H19X in tamoxifen-resistant ER+ breast cancer cells to generate a comprehensive list of direct transcripts using 4sU-RNA-seq. Interestingly, H19X regulates the expression of estrogen-driven intergenic, divergent, and antisense transcripts whose promoters are bound by ER-alpha (ERα) binding sites. Mechanistically, ‘Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq)’ revealed that H19X modulates chromatin structure at target regions to control gene expression. In addition, ‘chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing ChIP-seq’ (H3K27ac, H3K27me3) identified a critical role for H19X in controlling the epigenetic landscape in tamoxifen-resistant ER+ breast cancer cells. Collectively, our study sheds light on the molecular functions of X-linked genes and allows us to identify new diagnostic and therapeutic targets in tamoxifen-resistant ER+ breast cancer.S.S.G. is supported by grants from a) The Cancer Prevention and Research Institute of Texas (CPRIT; RR170020); b) Lizanell and Colbert Coldwell foundation; c) The Edward N. and Margaret G. Marsh Foundation; d) The American Cancer Society Presentation: Saturday, June 17, 2023