Therapy-resistant Breast Cancer Research Articles

A prismatic view of the epigenetic-metabolic regulatory axis in breast cancer therapy resistance.

Epigenetic regulation established during development to maintain patterns of transcriptional expression and silencing for metabolism and other fundamental cell processes can be reprogrammed in cancer, providing a molecular mechanism for persistent alterations in phenotype. Metabolic deregulation and reprogramming are thus an emerging hallmark of cancer with opportunities for molecular classification as a critical preliminary step for precision therapeutic intervention. Yet, acquisition of therapy resistance against most conventional treatment regimens coupled with tumor relapse, continue to pose unsolved problems for precision healthcare, as exemplified in breast cancer where existing data informs both cancer genotype and phenotype. Furthermore, epigenetic reprograming of the metabolic milieu of cancer cells is among the most crucial determinants of therapeutic resistance and cancer relapse. Importantly, subtype-specific epigenetic-metabolic interplay profoundly affects malignant transformation, resistance to chemotherapy, and response to targeted therapies. In this review, we therefore prismatically dissect interconnected epigenetic and metabolic regulatory pathways and then integrate them into an observable cancer metabolism-therapy-resistance axis that may inform clinical intervention. Optimally coupling genome-wide analysis with an understanding of metabolic elements, epigenetic reprogramming, and their integration by metabolic profiling may decode missing molecular mechanisms at the level of individual tumors. The proposed approach of linking metabolic biochemistry back to genotype, epigenetics, and phenotype for specific tumors and their microenvironment may thus enable successful mechanistic targeting of epigenetic modifiers and oncometabolites despite tumor metabolic heterogeneity.

Abstract PO3-26-10: Small molecule inhibitor of PELP1 exhibit cytotoxic effects on breast cancer cells by blocking ribosome biogenesis

Abstract Introduction: Breast cancer (BC) is the second-leading cause of cancer-related deaths in women. Despite responsive to endocrine therapy, a sizeable portion of estrogen receptor positive (ER+) BC goes on to develop therapy-resistant breast cancer (TR-BC). Furthermore, triple-negative breast cancer (TNBC) has a more aggressive clinical course with fewer targeted therapeutic options. The development of more effective therapies for TR-BC or TNBC represent an unmet need. Excessive ribosome biogenesis has recently been linked to BC progression, and resistance to therapy. Oncogene PELP1, which is required for ribosome biogenesis, is frequently dysregulated in BC and serves as a prognostic factor for poor BC survival and therapy resistance. We recently identified SMIP34 as a PELP1 small molecule inhibitor of its kind and it is effective against both TR-BC and TNBC. The objective of this study is to determine the mechanisms by which SMIP34 exhibit cytotoxic effects on BC cells. Methods: We have used multiple ER+, therapy resistant and TNBC models in cell viability, and apoptosis assays. Modelling studies were done based on Cryo-EM structure of the WDR18/PELP1 Rix1 complex (PDB code 7UWF). Biochemical assays include immune precipitation, reporter assays, GST pulldown, and Western Blotting. SMIP34 effect on protein synthesis was measured by using Western blot of puromycin treated total lysates for puromycinylated protein content. SMIP34 effect on protein synthesis assessed using Cayman’s Protein Synthesis Assay Kit. Ribosome biogenesis in SMIP34 treated cells was monitored using confocal microscopy using rpS6 antibody. Results: In in vitro cell based assays, SMIP34 was highly effective in reducing the cell viability, reducing colony formation and inducing apoptosis of ER+, endocrine therapy resistant, and TNBC model cells. PELP1 homodimer has recently been predicated to act as the core of the WDR18/PELP1 assembly. Our biochemical assay results suggest that SMIP34 binds to the same region involved in the PELP1 homodimerization and disrupts the formation of PELP1 homodimer and subsequently blocks the formation of WDR18/PELP1 Rix1 complex. Modeling studies predicted that altering the conformation of PELP1 aa 696-720 loop by SMIP34 binding may influence the conformation of the following C-terminal region, which may disturb the interaction of other PELP1 partners such as SENP3 and TEX10. Accordingly, the results from immunoprecipitation experiments demonstrated decreased association of Rix1 complex proteins WDR18, TEX10, and SENP3 with PELP1 under conditions of SMIP34 treatment. Further, Western blotting analysis confirmed that SMIP34 treatment decreased levels of PELP1 and its complex proteins, such as WDR18, TEX10, LAS1L, and SENP3. SMIP34 significantly reduced ribosomal biogenesis in a dose dependent manner. Additionally, puromycin labelling experiments confirmed a dose dependent decrease in new protein synthesis upon SMIP34 administration. Data from the DepMap database analysis revealed that PELP1 is required for cancer cell survival. Additionally, analysis of TCGA breast cancer datasets revealed that PELP1 expression is positively correlated with WDR18, TEX10, LAS1L, and SENP3. In in vivo xenograft assays and ex vivo explant assays using tumor tissue, SMIP34 strongly inhibited the growth of ER+, endocrine treatment resistant, and TNBC model cells. Conclusion: These findings provide solid evidence that the Rix1 complex, which is required for ribosomes production, is disturbed by SMIP34 binding to PELP1. SMIP34 represents a new therapeutic for the treatment of both ER+ and TNBC breast cancer. Supported by VA grant 1 I01 BX004545-01A1. Citation Format: Xue Yang, Khaled Mohamed Nassar, Uday Pratap, Rahul Gopalam, Behnam Ebrahimi, Durga Meenakshi Panneerdoss, Xiaonan Li, Yaxia Yuan, Daohong Zhou, Gangadhara R. Sareddy, Rajeshwar R. Tekmal, Manjeet Rao, Suryavathi Viswanadhapalli, Ratna Vadlamudi. Small molecule inhibitor of PELP1 exhibit cytotoxic effects on breast cancer cells by blocking ribosome biogenesis [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 PO3-26-10.

Abstract PO5-23-08: Adenylosuccinate lyase is essential for proliferation and mitochondrial function of endocrine therapy resistant breast cancer cells

Abstract Eighty percent of breast cancers express estrogen receptor alpha (ERα, ESR1) at the time of diagnosis. Endocrine therapy (ET) is the standard treatment that either block estrogen mediated ER activation (such as tamoxifen, fulvestrant) or suppress estrogen synthesis (such as exemestane, letrozole, or anastrozole). While ET is initially effective, emergence of ET-resistance is common and possess a major clinical challenge. Hence, there is an ongoing need to develop new treatments for ET-resistant-ERα positive breast cancer. In this study we investigated the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway as a potential target of ET-resistant breast cancer. The protein level of ADSL in two independent ET-resistant cell line model LCC9 and T47D-4HT cells were significantly higher in comparison to its parental ET-sensitive cell lines, MCF7 and T47D cells, respectively. Transient knockdown of ADSL using two independent siRNA in both LCC9 and T47D-4HT cells results in a significant decrease of cell growth, colony, and spheroid formation. Notably, reduced ADSL levels in LCC9 cells prevented the cells from progressing from G1 to S phase of cell cycle with concurrent elevation of cyclin D1/D3, CDK2/4, and cyclin E. On the other hand, in T47D-4HT cells, ADSL reduction caused cells to accumulate in S-phase of cell cycle and showed lower levels of total cyclin D1 protein. Mechanistically, in LCC9 cells (but not in T47D-4HT cells), ADSL depletion induced DNA replication stress which activates (phosphorylate) ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its major downstream effector checkpoint kinase 1 (Chk1) that in turn failed to de-phosphorylate the inhibitory-phospho-groups of cyclin-dependent kinase 2 (CDK2). In addition, ADSL depletion perturbed the mitochondrial function in both LCC9 and T47D-4HT cells. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was lower in both the cells along with the mitochondrial membrane potential. Pertinently, lower concentration of AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), a product of ADSL enzyme function, partially rescued the cell growth and restored mitochondrial membrane potential in ADSL-depleted LCC9 cells. To further understand the global effects of ADSL depletion we performed RNA-sequencing of LCC9 and T47D-4HT cells after ADSL depletion and compared with respective control siRNA transfected cells followed by gene set enrichment analysis. Intriguingly, diverse effect was observed in LCC9 and T47D-4HT cells as very few genes were found to be commonly regulated between these two cell lines. Analysis of clinical data sets revealed high level of ADSL transcript was associated with adverse progression free survival and overall survival in breast cancer patients treated with endocrine therapies. Overall, our findings demonstrate that ADSL-mediated de novo purine synthesis is critical for cellular growth, proliferation, and mitochondrial function of endocrine therapy-resistant breast cancer cells. Therefore, targeting ADSL is a novel potential therapeutic approach for ERα positive ET- resistant breast cancer. Citation Format: Anil Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Adenylosuccinate lyase is essential for proliferation and mitochondrial function of endocrine therapy resistant breast cancer cells [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 PO5-23-08.

Abstract PO3-26-11: Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors

Abstract Background: Estrogen receptor alpha (ERα) mutations are common (30-40%) in metastatic endocrine therapy-resistant breast cancers (ETR-BC), enable resistance to endocrine therapies and are the molecular drivers of ETR-BC. We had previously shown that an oligobenzamide, ERX-41, could enhance endoplasmic reticulum stress in ETR-BCs driven by mutant (MT) ERα, resulting in cancer cell death in vitro and and in vivo. To enable clinical translation of ERX-41, we performed lead optimization, followed by preclinical and IND-enabling studies. Methods: Over 2000 oligo-benzamides were designed, synthesized and tested in multiple BC models including those that express WT-ERα (MCF7, and ZR75) and BC models with acquired resistance (MCF7-Tam, and MCF7-LTLT) and engineered models that express MT-ERα (MCF7-ERα-D538G, MCF7-ERα-Y537S, ZR75-ERα-D538G, ZR75-ERα-Y537S). For our lead compound, mechanistic validation studies were performed using CRISPR LIPA mutants, RT-qPCR and Western blotting. Explants, organoids, cell line-(CDX) and patient-derived (PDX) xenografts were used to test the ex vivo and in vivo effectiveness of our lead compound as a monotherapy and in combination with abemaciclib. Results: Testing of >2000 synthesized oligo-benzamides identified a lead compound, ERX-315, that had broad and potent activity (IC50 between 20-100 nM) against both WT and MT (mutant) ERα-driven BC cells in in vitro assays. CRISPR KO of LIPA (which encodes lysosomal acid lipase (LAL) in BC cells abrogated cytotoxic response to ERX-315, validating LIPA as the molecular target of ERX-315. Ultrastructural and molecular studies confirm that ERX-315 induces significant endoplasmic reticulum stress, leading to a shutdown of de novo protein synthesis and apoptotic cell death in BC. Importantly, ERX-315 does not induce endoplasmic reticulum stress or cell death in normal cells and is non-toxic in animal models. We have shown that this capacity of ERX-315 to induce endoplasmic reticulum stress is unique among drugs targeting ERα, including selective ERα modulators and degraders, such as GDC-0180, AZD-9496 and fulvestrant. ERX-315 has potent anti-proliferative activity against MT-ERα-driven BC, as seen in genetically modified cell lines both grown as monolayer or spheroids in vitro, patient derived explants (PDEs) ex vivo and cell line derived (CDX) and patient-derived (PDX) xenografts in vivo. The combination of ERX-315 and CDK4/6 inhibitor abemaciclib was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BC cells, in vitro, in xenograft models in vivo, and in primary patient-derived explants ex vivo. We are currently optimizing both the synthesis of ERX-315 using GMP process for kilogram scale production and validated methods for pharmacokinetic studies. We have developed formulations for both intravenous and oral administration with favorable pharmacokinetic parameters and proven utility of the formulated ERX-315 against CDX and PDX tumors in vivo. Toxicity studies in dogs and rodents have demonstrated >8-fold therapeutic to toxicity window. A phase I clinical trial is planned to be open to enrollment by Q1 2024. Conclusions: We have identified a lead compound ERX-315, which represents a novel class of agent that induce catastrophic levels of ER stress resulting in cancer cell death and that can effectively work against multiple forms of ETR-BC, including those driven by MT-ERα. Preclinical studies, GMP manufacturing, formulation and IND-enabling studies are being completed in time for the commencement of the phase I clinical trial by Q1 2024. Citation Format: Suryavathi Viswanadhapalli, Karla Parra, Tae-Kyung Lee, Rahul Gopalam, Kara Kassees, Tanner Reese, Gaurav Sharma, Xihui Liu, Xue Yang, ChiaYuan Chen, Carlos Roggero, Liping Chen, Sautam Bhattacharya, Uday Pratap, Russell Hayward, Sharron Gargosky, Jung-Mo Ahn, Ganesh V. Raj, Ratna Vadlamudi. Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors [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 PO3-26-11.

Abstract 1985: Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells

Abstract Approximately 75% of breast tumors express estrogen receptors (ER+); recurrent ER+ breast cancer is generally incurable. Endocrine therapy (ET) is the mainstay of treatment for ER+ breast cancer and works by targeting either ER activity (Tamoxifen or Fulvestrant) or availability of its ligand estrogen (Letrozole, Exemestane). Unfortunately, endocrine therapies are limited by the emergence of resistance; resistance to endocrine therapies (ET) remains a significant clinical challenge. We have explored the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway, in ET-resistant breast cancer cells. Protein expression of ADSL was significantly higher in two different ET-resistant cells (LCC9 and T47D-4HT) when compared with their respective parental ET-sensitive cells (MCF-7 and T47D). siRNA mediated ablation of ADSL in LCC9 and T47D-4HT cells blocked cell proliferation and both colony and spheroid formation. Depletion of ADSL in LCC9 cells obstructed cell cycle progression from G1 to S phase and elevated the protein expression of cyclin D1/D3, CDK2/4, and cyclin E. In T47D-4HT cells, ADSL depletion led to an accumulation of cells in S-phase with reduced protein expression of cyclin D1. Notably, ADSL ablation in LCC9 cells lead to DNA replication stress, resulting in activation (phosphorylation) of ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its key downstream effector checkpoint kinase 1 (Chk1), which further limited the phosphatase activity of cdc25A leading to deactivation (phosphorylation) of Cyclin-Dependent Kinase 2 (CDK2). ADSL knockdown reduced the mitochondrial membrane potential and impaired the oxygen consumption (OCR) and extracellular acidification (ECAR) rates in both LCC9 and T47D-4HT cells. Of note, addition of AICAR to ADSL-depleted LCC9 cells significantly increased cell proliferation, colony formation, and restored mitochondrial membrane potential. RNA sequencing data revealed the effect of ADSL depletion on enrichment of different pathways in LCC9 and TH47D-4HT cells. Importantly, p53 signaling was significantly enriched in LCC9 cells but not in T47D-4HT cells after ADSL knockdown (T47D cells have a mutated p53 gene). ER+ breast cancer patients treated with ET showed high ADSL expression that is associated with poor recurrence-free survival. In summary, our findings indicate that ADSL-mediated de novo purine synthesis is crucial for cell proliferation. Therefore, targeting ADSL may be a novel therapeutic approach for ER, ET-resistant breast cancer. Citation Format: ANIL KUMAR Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells [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 1985.

275 (PB-091) Poster - Genomic and transcriptomic characterisation of endocrine therapy-resistant breast cancers: a single-institution longitudinal study

275 (PB-091) Poster - Genomic and transcriptomic characterisation of endocrine therapy-resistant breast cancers: a single-institution longitudinal study

BCAR4 Expression as a Predictive Biomarker for Endocrine Therapy Resistance in Breast Cancer

BACKGROUNDBreast cancer, particularly the estrogen receptor positive (ER+) subtype, remains a leading cause of cancer-related death among women. Endocrine therapy is the most effective treatment for ER+ breast cancer; however, the development of resistance presents a significant challenge. This study explored the role of the breast cancer antiestrogen resistance 4 (BCAR4) gene as a potential driver of resistance and a pivotal biomarker in breast cancer. PATIENTS AND METHODSThe researchers undertook a comprehensive analysis of 1743 patients spanning six independent cohorts. They examined the association of BCAR4 expression with patient outcomes across all breast cancer types and the PAM50 molecular subtypes. The relationship between elevated BCAR4 expression and resistance to endocrine therapy including AIs, the prevailing standard-of-care for endocrine therapy, was also investigated. RESULTSThis meta-analysis corroborated the link between BCAR4 expression and adverse outcomes as well as resistance to endocrine therapy in breast cancer. Notably, BCAR4 expression is predominantly clinically significant in luminal A and B subtypes. Additionally, an association between BCAR4 expression and resistance to AI treatment was discerned. CONCLUSIONThis study expands on previous findings by demonstrating that BCAR4 expression is associated with resistance to newer therapies. The identification of patients with intrinsic resistance to hormone therapy is crucial to avoid ineffective treatment strategies. These findings contribute to our understanding of endocrine therapy resistance in breast cancer and could potentially guide the development of more effective treatment strategies. Micro AbstractEstrogen receptor positive (ER+) breast cancer is a leading cause of cancer-related deaths among women due to treatment resistance. This study explored the role of the breast cancer antiestrogen resistance 4 (BCAR4) gene as a potential driver of resistance and a pivotal biomarker in breast cancer. Through an extensive study of 1743 patients across six cohorts, high BCAR4 expression was found to correlate with poor outcome, particularly in luminal A and B subtypes, with resistance to endocrine therapy including aromatase inhibitor (AI) treatments. These insights enrich our understanding of endocrine therapy resistance and may for improved therapeutic strategies.

Autotaxin and Lysophosphatidate Signaling: Prime Targets for Mitigating Therapy Resistance in Breast Cancer.

Overcoming and preventing cancer therapy resistance is the most pressing challenge in modern breast cancer management. Consequently, most modern breast cancer research is aimed at understanding and blocking these therapy resistance mechanisms. One increasingly promising therapeutic target is the autotaxin (ATX)-lysophosphatidate (LPA)-lipid phosphate phosphatase (LPP) axis. Extracellular LPA, produced from albumin-bound lysophosphatidylcholine by ATX and degraded by the ecto-activity of the LPPs, is a potent cell-signaling mediator of tumor growth, invasion, angiogenesis, immune evasion, and resistance to cancer treatment modalities. LPA signaling in the post-natal organism has central roles in physiological wound healing, but these mechanisms are subverted to fuel pathogenesis in diseases that arise from chronic inflammatory processes, including cancer. Over the last 10 years, our understanding of the role of LPA signaling in the breast tumor microenvironment has begun to mature. Tumor-promoting inflammation in breast cancer leads to increased ATX production within the tumor microenvironment. This results in increased local concentrations of LPA that are maintained in part by decreased overall cancer cell LPP expression that would otherwise more rapidly break it down. LPA signaling through six G-protein-coupled LPA receptors expressed by cancer cells can then activate virtually every known tumorigenic pathway. Consequently, to target therapy resistance and tumor growth mediated by LPA signaling, multiple inhibitors against the LPA signaling axis are entering clinical trials. In this review, we summarize recent developments in LPA breast cancer biology, and illustrate how these novel therapeutics against the LPA signaling pathway may be excellent adjuncts to extend the efficacy of evolving breast cancer treatments.

Hormone Metabolites and Herbal Bioactive Agents: Potential Drug Candidates for the Luminal A Breast Cancer subtype

Background: Hormone receptor positive, human epidermal growth factor receptor negative Luminal A breast cancer subtype responds to targeted endocrine therapy, signal transduction inhibitors and cyclin dependent kinase inhibitors. Estrogen and progesterone receptor mediated signal transduction involves receptor-DNA binding and transcriptional activation of downstream target genes. In addition to hormone receptor signaling, cellular metabolism of estradiol and progesterone exhibit distinct roles in cancer growth modulation. Targeted therapy is associated with systemic toxicity, therapy resistance and emergence of chemo-resistant cancer initiating stem cells. These limitations emphasize identification of testable drug candidates for therapy resistant breast cancer. Cellular metabolism of ovarian steroid hormones generates oxidative metabolites with distinct growth modulating effects. Anti-proliferative metabolites may represent potential drug candidates. Objectives: The objectives of the present review are to provide i) Systematic discussion of published evidence relevant to the role of ovarian steroid hormone metabolism in growth modulatory effects on cancer progression, ii) Evidence for applicability of Luminal A breast cancer and drug-resistant cancer stem cell models to identify mechanistic leads for efficacy of anti-proliferative hormone metabolites, and iii) Future research directions for clinical translatability of patient derived preclinical data. Conclusions: Collectively, present evidence validates experimental approaches to identify hormone metabolites as potential drug candidates for therapy-resistant breast cancer. .

The Involvement of Peroxiporins and Antioxidant Transcription Factors in Breast Cancer Therapy Resistance.

Breast cancer is still the leading cause of death in women of all ages. The reason for this is therapy resistance, which leads to the progression of the disease and the formation of metastases. Multidrug resistance (MDR) is a multifactorial process that leads to therapy failure. MDR involves multiple processes and many signaling pathways that support each other, making it difficult to overcome once established. Here, we discuss cellular-oxidative-stress-modulating factors focusing on transcription factors NRF2, FOXO family, and peroxiporins, as well as their possible contribution to MDR. This is significant because oxidative stress is a consequence of radiotherapy, chemotherapy, and immunotherapy, and the activation of detoxification pathways could modulate the cellular response to therapy and could support MDR. These proteins are not directly responsible for MDR, but they support the survival of cancer cells under stress conditions.

Metabolomic Rewiring Promotes Endocrine Therapy Resistance in Breast Cancer.

Increased fatty acid oxidation induced by endocrine therapy activates Src signaling to promote endocrine resistance in breast cancer, which can be overcome using clinically approved therapies targeting FAO and Src.

The role of the tumor microenvironment in endocrine therapy resistance in hormone receptor-positive breast cancer.

Endocrine therapy is the prominent strategy for the treatment of hormone-positive breast cancers. The emergence of resistance to endocrine therapy is a major health concern among hormone-positive breast cancer patients. Resistance to endocrine therapy demands the design of newer therapeutic strategies. The understanding of underlying molecular mechanisms of endocrine resistance, components of the tumor microenvironment (TME), and interaction of resistant breast cancer cells with the cellular/acellular components of the intratumoral environment are essential to formulate new therapeutic strategies for the treatment of endocrine therapy-resistant breast cancers. In the first half of the article, we have discussed the general mechanisms (including mutations in estrogen receptor gene, reregulated activation of signaling pathways, epigenetic changes, and cell cycle alteration) responsible for endocrine therapy resistance in hormone-positive breast cancers. In the latter half, we have emphasized the precise role of cellular (cancer-associated fibroblasts, immune cells, and cancer stem cells) and acellular components (collagen, fibronectin, and laminin) of TME in the development of endocrine resistance in hormone-positive breast cancers. In sum, the article provides an overview of the relationship between endocrine resistance and TME in hormone-positive breast cancers.

OR11-02 Structural Determinants of Pure Antiestrogenic Activity

Abstract Disclosure: A. Vallet: None. M. Diennet: None. K. Thiombane: None. A. Vivet: None. S. Weber: None. M. El Ezzy: None. F. Shaikh: None. J. Poupart: None. R. Mendoza-Sanchez: None. D. Schuetz: None. A. Marinier: None. G.L. Greene: None. S.W. Fanning: None. S.N. Mader: None. Inhibitory activities of antiestrogens on estrogen receptor alpha (ERα) range from mixed antagonism/agonism (selective ER modulators; SERMs) to complete antiestrogenicity associated with accelerated ERα turnover (selective ER degraders; SERDs). Using a panel of SERMs, SERDs and PROTACs, we show that antiestrogens induce variable degrees of SUMOylation of ERα and that efficient induction of SUMOylation, rather than increased suppression of coactivator recruitment or accelerated ERα degradation, correlates with suppression of ERα basal and induced transcriptional activity in reporter assays. Antiestrogens did not induce SUMOylation of ERβ and did not suppress its basal transcriptional activity in the same assays. Chimeras between the two receptors identified ERα-specific residues required for induction of SUMOylation by antiestrogens and revealed differences according to the antiestrogen structure. In addition, induction of ERα SUMOylation by antiestrogens depended on the hydrophobicity of N-terminal residues of ligand binding domain (LBD) helix H12. L536 mutations, including those occurring in endocrine therapy-resistant breast cancer, abolished both induction of ERα SUMOylation and pure antiestrogenicity. Structures of the L536S human ERα LBD bound to fulvestrant analogs and molecular dynamics simulations revealed dynamic side chain interactions with ERα H12 in the coactivator-binding groove, and predict the impact of SUMOylation-suppressing L536 mutations on these interactions. Together, our results show that antiestrogen-induced ERα SUMOylation is associated with complete suppression of its transcriptional activity and provide insights into the structural determinants of pure antiestrogenicity. Presentation Date: Friday, June 16, 2023

2-Methoxyestradiol as an Antiproliferative Agent for Long-Term Estrogen-Deprived Breast Cancer Cells.

To identify effective treatment modalities for breast cancer with acquired resistance, we first compared the responsiveness of estrogen receptor-positive breast cancer MCF-7 cells and long-term estrogen-deprived (LTED) cells (a cell model of endocrine therapy-resistant breast cancer) derived from MCF-7 cells to G-1 and 2-methoxyestradiol (2-MeO-E2), which are microtubule-destabilizing agents and agonists of the G protein-coupled estrogen receptor 1 (GPER1). The expression of GPER1 in LTED cells was low (~0.44-fold), and LTED cells displayed approximately 1.5-fold faster proliferation than MCF-7 cells. Although G-1 induced comparable antiproliferative effects on both MCF-7 and LTED cells (IC50 values of >10 µM), 2-MeO-E2 exerted antiproliferative effects selective for LTED cells with an IC50 value of 0.93 μM (vs. 6.79 μM for MCF-7 cells) and induced G2/M cell cycle arrest. Moreover, we detected higher amounts of β-tubulin proteins in LTED cells than in MCF-7 cells. Among the β-tubulin (TUBB) isotype genes, the highest expression of TUBB2B (~3.2-fold) was detected in LTED cells compared to that in MCF-7 cells. Additionally, siTUBB2B restores 2-MeO-E2-mediated inhibition of LTED cell proliferation. Other microtubule-targeting agents, i.e., paclitaxel, nocodazole, and colchicine, were not selective for LTED cells. Therefore, 2-MeO-E2 can be an antiproliferative agent to suppress LTED cell proliferation.

E2F3 induces DNA damage repair, stem-like properties and therapy resistance in breast cancer

Therapy resistance is a major hurdle to the treatment of human malignant tumors. Both DNA damage repair and stem-like properties contribute to chemoresistance and radioresistance. E2F transcription factor 3 (E2F3) is overexpressed in breast cancer tissues, and promotes proliferation of breast cancer cells. Higher E2F3 level is associated with shorter survival of breast cancer patients. Functional studies further showed that E2F3 promotes S-phage entry, DNA replication, DNA damage repair and stem-like properties. Accordingly, E2F3 knockdown sensitizes breast cancer cells to DNA-damaging agents Adriamycin, Cisplatin, Olaparib and X-ray. Forkhead box M1 (FOXM1) is a downstream molecule of E2F3 signaling, mediating the effects of E2F3 on breast cancer cells. In an m6A methyltransferase METTL14-dependent manner, YTH RNA binding protein F2 (YTHDF2) increase E2F3 mRNA stability and expression, promotes DNA damage repair and induces therapy resistance. These data demonstrate that YTHDF2-E2F3 pathway is a novel target to overcome chemoresistance and radioresistance in breast cancer.

Oxidative phosphorylation is a metabolic vulnerability of endocrine therapy and palbociclib resistant metastatic breast cancers

Resistance to endocrine treatments and CDK4/6 inhibitors is considered a near-inevitability in most patients with estrogen receptor positive breast cancers (ER + BC). By genomic and metabolomics analyses of patients’ tumours, metastasis-derived patient-derived xenografts (PDX) and isogenic cell lines we demonstrate that a fraction of metastatic ER + BC is highly reliant on oxidative phosphorylation (OXPHOS). Treatment by the OXPHOS inhibitor IACS-010759 strongly inhibits tumour growth in multiple endocrine and palbociclib resistant PDX. Mutations in the PIK3CA/AKT1 genes are significantly associated with response to IACS-010759. At the metabolic level, in vivo response to IACS-010759 is associated with decreased levels of metabolites of the glutathione, glycogen and pentose phosphate pathways in treated tumours. In vitro, endocrine and palbociclib resistant cells show increased OXPHOS dependency and increased ROS levels upon IACS-010759 treatment. Finally, in ER + BC patients, high expression of OXPHOS associated genes predict poor prognosis. In conclusion, these results identify OXPHOS as a promising target for treatment resistant ER + BC patients.

Natural products as drug candidates for breast cancer (Review).

Progression of early-stage breast cancer to advanced-stage metastatic disease represents a major cause of death in women. Long-term conventional and targeted therapy for breast cancer includes multi-drug combinations of cytotoxic chemotherapeutics and pathway-selective small molecule inhibitors. These treatment options are frequently associated with systemic toxicity, intrinsic/acquired therapy resistance and emergence of a drug-resistant cancer stem cell population. This stem cell population has a chemo-resistant, cancer-initiating, premalignant phenotype that is accompanied by cellular plasticity and metastatic potential. These limitations emphasize an unmet need to identify testable alternatives against therapy-resistant metastatic breast cancer. Natural products such as dietary phytochemicals, nutritional herbs and their constitutive bioactive agents have documented human consumption, and lack detectable systemic toxicity and resultant off-target unfavorable side effects. Because of these advantages, natural products may represent testable alternatives for therapy-resistant breast cancer. The present review discusses published evidence for growth inhibitory efficacy of natural products on cellular models for molecular subtypes of clinical breast cancer and development of drug-resistant stem cell models. Collectively, this evidence validates mechanism-based experimental approaches to screen and prioritize efficacious bioactive agents from natural products as novel drug candidates that may function as therapeutic alternatives for breast cancer.

Evaluation of PIK3CA Gene Mutations in Breast Cancer Patients Treated by Trastuzumab

Background: The phosphatidylinositol 3-kinases are known as a family of lipid kinases, playing a role in various cellular processes. A member of this family is PIK3CA which is frequently mutated in breast cancer. In this study, the association between H1047R, E542K and E545K mutations, and therapy resistance was investigated in Iranian breast cancer patients treated by Trastuzumab. Methods: Resistance and sensitive groups were chosen from Iranian patients treated by Trastuzumab. A PCR-RFLP approach was designed for detecting the H1047R mutation. Mutations in positions of codons E542K and E545K were detected using PCR-based DNA Sanger sequencing assay. The overall survival and disease-free survival were assessed. Results: A significant relationship was observed between the presence and absence of H1047R mutation and the overall survival (P = 0.025). In addition, there was a significant relationship between the response to Trastuzumab and some clinicopathological features, including the age and the status of ER/PR receptors (P-value<0.05). E542K and E545K mutations were not observed in the patients. Conclusion: It can be said that probably H1047R mutation is a prognostic marker in the Trastuzumab-based therapy resistant breast cancer. Further studies can be performed to evaluate this mutation before using Trastuzumab to predict the effectiveness of this treatment.

Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer.

e13077 Background: The past decade has seen significant advancement in increasing survival estrogen receptor alpha (ERα) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) and cyclin-dependent kinase inhibitors aided to extend overall survival of breast cancer patients. However, resistance to endocrine therapy is a common occurrence and patients will eventually succumb to their disease. Fatty acid synthase (FASN), a key enzyme in lipid biosynthesis, is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease (≥16 weeks). Methods: We generated therapy-resistant cells by long term exposure to tamoxifen (MCF7/TamR cells), and fulvestrant (MCF7/FR cells). Palbociclib-resistant (MCF7/RB1Crispr and ZR75/RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor in these cells on proliferation, viability, cell cycle, apoptosis, ERα expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate gene expression. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blot after treatment with TVB with or without the addition of palmitate or BAPTA. Results: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERα positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress. Conclusions: Our preclinical data provide evidence that FASN inhibition presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.

Abstract 3439: Evaluating nuclear receptor heterodimerization using live cell proximity assays: Implications for novel therapeutic approaches for hormone driven cancers

Abstract Breast and prostate cancer are the leading cancer diagnoses for women and men, respectively, second only to cutaneous cancers. Breast and prostate cancer arise from aberrant estrogen/estrogen receptor (ER) and testosterone/androgen receptor signaling (AR), respectively. These sex hormone receptors are members of the steroid hormone receptor family, which also includes the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and progesterone receptor (PR). Although these receptors canonically function through homodimerization, recent evidence has suggested that receptor crosstalk can amplify or dampen the actions of heterologous receptors. However, these interactions have not been extensively studied in all possible combinations of steroid hormone receptor crosstalk. In this project, we hypothesize that heterodimerization may amplify or repress the transcriptional response(s) elicited by the various receptor complexes depending upon conditions such as concentration of ligands, type of ligand, physiological states, and the types/functions of promoter regions. To test this hypothesis, we investigated the significance of ligand-induced interactions between different homo- and heterodimer combinations of AR, ARV7, GR, MR, PR, and ERα in HEK293 cells using IncuCyte live-cell imaging and NanoBRET technology. Ultimately, the combination of transfection experiments, live-cell imaging, and NanoBRET assays provided insight into which combination of steroid receptors successfully interact to amplify (or dampen) the HaloTag expression, luminescence intensity at 450nM (i.e., NanoLuc expression), and NanoBRET ratio (i.e., acceptor emission luminescence at 610nM divided by donor emission luminescence at 450nM) in HEK293 cells. Our studies revealed that heterodimerization of ERα-PR, ERα-MR, AR-GR, AR-PR, ARV7-MR, ARV7-PR, and GR-MR produced significant ligand-induced changes in signaling intensity compared to no ligand conditions for each fusion protein construct. Additionally, to demonstrate the value of the NanoBRET method for modelling clinically relevant disease conditions, we assessed the physical interaction of ERα and PR in the context of the ERα Y537S mutation - a mutation associated with endocrine therapy resistance in breast cancer that results in a constitutively active ERα. The results showed that ERα-Y537S mutation increases ERα/PR physical interaction upon PR-B stimulation in a NanoBRET proximity-based assay. Future directions include testing these interactions in breast and prostate cancer cell lines and identifying the receptors’ localization within the cell, the results of which may be of clinical importance. Citation Format: Amira Ishag-Osman, David Hosfield, Rosemary Huggins, Geoffrey Greene. Evaluating nuclear receptor heterodimerization using live cell proximity assays: Implications for novel therapeutic approaches for hormone driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3439.