Development Of Endocrine Resistance Research Articles

MICAL-L2, as an estrogen-responsive gene, is involved in ER-positive breast cancer cell progression and tamoxifen sensitivity via the AKT/mTOR pathway

Endocrine treatment, particularly tamoxifen, has shown significant improvement in the prognosis of patients with estrogen receptor-positive (ER-positive) breast cancer. However, the clinical utility of this treatment is often hindered by the development of endocrine resistance. Therefore, a comprehensive understanding of the underlying mechanisms driving ER-positive breast cancer carcinogenesis and endocrine resistance is crucial to overcome this clinical challenge. In this study, we investigated the expression of MICAL-L2 in ER-positive breast cancer and its impact on patient prognosis. We observed a significant upregulation of MICAL-L2 expression in ER-positive breast cancer, which correlated with a poorer prognosis in these patients. Furthermore, we found that estrogen-ERβ signaling promoted the expression of MICAL-L2. Functionally, our study demonstrated that MICAL-L2 not only played an oncogenic role in ER-positive breast cancer tumorigenesis but also influenced the sensitivity of ER-positive breast cancer cells to tamoxifen. Mechanistically, as an estrogen-responsive gene, MICAL-L2 facilitated the activation of the AKT/mTOR signaling pathway in ER-positive breast cancer cells. Collectively, our findings suggest that MICAL-L2 could serve as a potential prognostic marker for ER-positive breast cancer and represent a promising molecular target for improving endocrine treatment and developing therapeutic approaches for this subtype of breast cancer.

Abstract PO3-14-12: SIRT3 as a new potential predictive biomarker for response to CDK4/6 inhibition in ER+/HER2- metastatic breast cancer patients

Abstract Background: Several prospective, randomized clinical trials showed that the CDK4/6 inhibitor palbociclib in combination with either letrozole or fulvestrant significantly improves progression-free survival (PFS) in patients with ER+/HER2- metastatic breast cancer (MBC). However, in some cases, there is development of endocrine resistance and ultimate disease progression. Molecular analysis performed in tissue specimens collected in the PALOMA-3 study, comparing fulvestrant and Palbociclib versus fulvestrant placebo, demonstrated that one of the genes associated with an increased clinical benefit is SIRT3 (Sirtuin 3) which is thought to control mitochondrial integrity and metabolism (Cristofanilli M. et al., Lancet Oncol. 2016). This study aimed to evaluate in preclinical models the predictive role of SIRT3 in ER+/HER2- breast tumors. Methods: Using lentiviruses we generated MCF-7 and T47D cells stably expressing either control shRNA (sh ctr) or shRNA targeting SIRT3 (shSIRT3). With these models, we studied cell viability, tumor growth, apoptosis, and autophagy in MCF-7 cells treated with fulvestrant and palbociclib as a single treatment or in combination, these either alone or in combination with shSIRT3 (knockdown). Nude mice were used for our in vivo studies, and sh ctr or shSIRT3 MCF7 cells were injected. Results: We showed in vitro that the response to palbociclib was significantly associated with levels of SIRT3 expressionas high or low in ER+ cells (p< 0.0001). Furthermore, we found that irreversible cell growth inhibition mediates the beneficial effect of palbociclib in SIRT3 high expressing cellscompared to low expression (p< 0.001).Then, we evaluated the mechanistic activity of SIRT3. We showed thatSIRT3-low expression cells induced enhanced sensitivity to palbociclib by targeting the reactive oxygen species (ROS)/autophagy axis:(i) SIRT3 regulated the mitochondrial homeostasis (e.g. glucose, lactate, pyruvate, succinates), (ii) ROS levels where lower in sh crt comparing and shSIRT3 in treated and untreated cells (p< 0.001), (iii) in terms of senescence,a higher level of positive cells for β-gal activity was found (p≤0.05) and (iv) for autophagy, our western blot analysis showed cleaved LC3 proteins. Our in vivo studies showed that SIRT3 regulated treatment response to palbociclib with a significant decrease in tumor weight and tumor volume (p< 0.01) and importantly, after immunohistochemistry analysis, we validated the role of SIRT3 in regulating the proteins involved in the autophagy/senescence balance, was mainly in epithelial cells compared to stromal cells. Conclusion: Our preclinical studies demonstrated that elevated SIRT3 expression levels sensitizes ER+/HER2- breast tumors to palbociclib treatment. This study suggested that SIRT3 expression could represent a potential predictive biomarker in HR+ MBC patients treated with Palbociclib. Citation Format: Maroua Manai, Ghada Sahraoui, Raoudha Doghri, Lorenzo Gerratana, Paolo D’Amico, Youbin Zhang, Jeannine Donahue, Ami Shah, Carolina Reduzzi, Wenan Qiang, Massimo Cristofanilli. SIRT3 as a new potential predictive biomarker for response to CDK4/6 inhibition in ER+/HER2- metastatic breast cancer patients [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 PO3-14-12.

Abstract PO4-05-07: Differential dynamics of fragmentomic and epigenetic circulating tumor DNA (ctDNA) features in first-line Palbociclib and Ribociclib treatment for Hormone Receptor (HR) positive, HER2 negative metastatic breast cancer (MBC)

Abstract Background: Cyclin-dependent kinase inhibitors (CDK4/6i) in combination with endocrine therapy (ET) represent the cornerstone for the management of HR positive, HER2 negative MBC. However, the lack of prospective cohorts comparing the distinctions among palbociclib (PAL), ribociclib (RIB) and abemaciclib (ABE) poses a significant clinical knowledge gap. The aim of this ancillary analysis was to explore the differential clinical impact of CDK4/6i by leveraging new ctDNA biomarkers in a prospective clinical study. Methods: A total of 114 patients (pts) with HR positive, HER2 negative MBC were prospectively enrolled in the multicenter MAGNETIC.1 study (CRO-2018-56) from January 2018 to January 2023. At baseline (BL) and every 3 months (mos.), ctDNA samples were collected concurrently with radiological restaging. The samples were then analyzed through methylation-specific (MS) droplet digital PCR (ddPCR), ddPCR fragmentomics, and next generation sequencing. Matched pairs of variations were tested through Wilcoxon signed rank test, and survival was analyzed by log-rank test. Results: Out of the 114 pts enrolled, 59 received PAL, 48 RIB, and 16 ABE. In the de novo subgroup, 51.4 % of pts were treated with PAL, 42.9% with RIB, and 5.7% with ABE. Among the pts who received PAL, 7 harbored an ESR1 mutation at BL (D538G: 2, H377R: 1, Y537N: 1, Y537S: 3). Additionally,12 pts had PIK3CA mutations, and 2 pts had AKT1 mutations. In the RIB group, 5 and 2 pts, respectively, had a PIK3CA- and AKT-mutated MBC, while no ESR1 mutations were detected. When comparing matched pairs of variations, it was found that at BL, at first radiological restaging (T1), and at 6 mos. after initiation of CDK4/6i (T2), only PAL showed significantly higher levels of promoter A and B methylation (promA and promB) at T1 (P = 0.0005 and P = 0.0056, respectively for promA and promB), A significant decrease in promA and promB levels was detected at T2 for PAL (P = 0.0005 and P < 0.0001 respectively for promA and promB) but not for RIB. Both PAL and RIB demonstrated a significant decrease in ctDNA short fragments at T1 (P = 0.0263 for PAL and P = 0.0001 for RIB), However, PAL showed a significant rebound at T2, with an increase in 80% of samples (P = 0.0014). The same was not seen with RIB. After a median follow-up of 34.8 mos., there were no significant differences between PAL and RIB in terms of PFS (P= 0.2573) and OS (P= 0.3783). The PFS rates at 12 and 24 mos. were 69% and 48% for PAL, and 76% and 64% for RIB. The OS rates at 12 and 24 mos. were 94% and 84% for PAL, compared to 97% and 82% of RIB, respectively. Conclusions This analysis of MAGNETIC.1 highlighted significant differences in ctDNA biomarkers between palbociclib and ribociclib, suggesting their potential impact on the development of endocrine resistance. Further biomarker-driven clinical trials leveraging this concept are needed to refine the sequencing algorithms for CDK4/6i. Citation Format: Claudia Noto, Linda Cucciniello, Elisabetta Molteni, Silvia Bolzonello, Alessandra Franzoni, Lucia Da Ros, Silvia Buriolla, Arianna Dri, Lorenzo Foffano, Simon Spazzapan, Stefania Russo, Brenno Pastò, Giulia Cudia, Giada Targato, Serena Della Rossa, Marta Bonotto, Alessandro Marco Minisini, Giuseppe Damante, Barbara Belletti, Lorenzo Gerratana, Fabio Puglisi. Differential dynamics of fragmentomic and epigenetic circulating tumor DNA (ctDNA) features in first-line Palbociclib and Ribociclib treatment for Hormone Receptor (HR) positive, HER2 negative metastatic breast cancer (MBC) [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 PO4-05-07.

TRIM33 Is a Co-Regulator of Estrogen Receptor Alpha.

Estrogen receptor alpha (ER)-positive breast cancer is responsible for over 60% of breast cancer cases in the U.S. Among patients diagnosed with early-stage ER+ disease, 1/3 will experience recurrence despite treatment with adjuvant endocrine therapy. ER is a nuclear hormone receptor responsible for estrogen-driven tumor growth. ER transcriptional activity is modulated by interactions with coregulators. Dysregulation of the levels of these coregulators is involved in the development of endocrine resistance. To identify ER interactors that modulate transcriptional activity in breast cancer, we utilized biotin ligase proximity profiling of ER interactomes. Mass spectrometry analysis revealed tripartite motif containing 33 (TRIM33) as an estrogen-dependent interactor of ER. shRNA knockdown showed that TRIM33 promoted ER transcriptional activity and estrogen-induced cell growth. Despite its known role as an E3 ubiquitin ligase, TRIM33 increased the stability of endogenous ER in breast cancer cells. TRIM33 offers a novel target for inhibiting estrogen-induced cancer cell growth, particularly in cases of endocrine resistance driven by ER (ESR1) gene amplification or overexpression.

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.

Neuronal Ptpn1 and Socs3 deletion improves metabolism but not anovulation in a mouse PCOS model.

Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility in women. Approximately half of diagnosed individuals also experience metabolic syndrome. Central and peripheral resistance to the hormones insulin and leptin have been reported to contribute to both metabolic and reproductive dysregulation. In PCOS and preclinical PCOS animal models, circulating insulin and leptin levels are often increased in parallel with the development of hormone resistance, however it remains uncertain whether these changes contribute to the PCOS state. In this study we tested whether central actions of protein tyrosine phosphatase 1B (PTP1B) and suppressor of cytokine signaling 3 (SOCS3), negative regulators of insulin and leptin signaling pathways respectively, play a role in the development of PCOS-like phenotype. A peripubertal dihydrotestosterone (DHT) excess PCOS-like mouse model was used, which exhibits both metabolic and reproductive dysfunction. Mice with knockout of the genes encoding PTP1B and SOCS3 from forebrain neurons were generated, and metabolic and reproductive function were compared between knockout and control groups. DHT treatment induced mild insulin resistance but not leptin resistance, so the role of SOCS3 could not be tested. As expected, DHT excess abolished estrous cycles and corpora lutea presence and caused increased visceral adiposity and fasting glucose levels. Knockout mice did not show any rescue of reproductive dysfunction, but did have reduced adiposity compared to control DHT mice. These data suggest that negative regulation of central insulin signaling by PTP1B is not responsible for peripubertal DHT excess-induced reproductive impairments, but may mediate its increased adiposity effects.

Abstract P5-06-04: The prognostic value of c-MET monitoring by using c-MET-enriched circulating tumor cells in HR-positive HER2-negative metastatic breast cancer

Abstract [Background] As the development of endocrine resistance and late recurrences are the major clinical concerns in hormone receptor (HR)-positive/HER2-negative metastatic breast cancer (MBC) patients, biomarkers to predict the occurrence of endocrine resistance or disease progression are crucial for improving patient outcomes. Aberrant HGF/c-MET signaling pathway has been reported to play a role in various cellular processes in cancer. Estrogen Receptor 1 (ESR1) mutations, encoding estrogen receptor α, are associated with endocrine resistance in HR+ breast cancer. PIK3CA hotspot mutations that induce hyperactivation of the PI3K are found in 30-40% of HR+ advanced breast cancers. In this context, we evaluated the prognostic values of c-MET-enriched CTC, ESR1 mutations, PIK3CA mutations, and cfDNA concentrations detected in the blood of HR+HER2- MBC patients. [Methods] MBC patients were prospectively enrolled during standard treatments at Samsung Medical Center (IRB No.2019-08-119). Circulating tumor cells (CTCs) were isolated using the GenoCTC® with c-MET-enriched or EpCAM-enriched CTC isolation kits (Genobio Corp., South Korea) from 4mL of blood each. PIK3CA and ESR1 hotspot mutations were analyzed by droplet digital PCR kits (Gencurix Inc., South Korea). cfDNA concentrations were calculated using ESR1 gene copy numbers from plasma. To compare the proportion of c-MET overexpression between primary breast tumors and metastatic sites in HR+HER2- breast cancer patients, primary breast (n=358) and metastatic sites (n=27) were independently collected. c-MET expression was evaluated by an immunohistochemistry assay using an anti-total c-MET (SP44) antibody with a Ventana Discovery XY automated system according to the manufacturer’s instruction. c-MET overexpression was defined if the staining was scored as 2+ or 3+. Progression-free survival (PFS) was defined as the time from blood draw to the first of either disease progression or death during standard therapy. [Results] Out of 93 patients with HR+ MBC, analysis was performed in 63 HR+HER2- MBC patients. Seventeen patients (27%) had one or more EpCAM-enriched CTCs, and fourteen patients (22%) had one or more c-MET-enriched CTCs detected in their blood. The median follow-up time and median time to censoring were 8.4 months and 18.7 months, respectively. According to the Kaplan-Meier survival analysis by log-rank test, c-MET-enriched CTCs, cfDNA concentrations, and ESR1 mutations were significantly associated with PFS (p=0.0026, 0.0064, and 0.011, respectively). However, PIK3CA mutations and EpCAM-enriched CTCs were not statistically significant with PFS (p=0.38 and 0.86, respectively). Multivariate analysis showed that both c-MET-enriched CTCs (HR=3.5, p=0.014) and cfDNA concentrations (HR=2.2, p=0.031) were independent predictors for PFS in HR+HER2- MBC. The proportion of c-MET overexpression was significantly higher in metastatic sites (22.2%) than in primary breast tumors (4.7%) in HR+HER2- breast cancer patients (p=0.00002). As c-MET-enriched CTCs and cfDNA concentrations were independent predictors of disease progression, patients were divided into two groups depending on the result of c-MET-enriched CTCs and cfDNA concentration. When patients with low c-MET-enriched CTC and cfDNA concentrations were classified as a low-risk group and other patients into a high-risk group, the high-risk group had a shorter PFS than the low-risk group (p=0.003). [Conclusion] This study provided c-MET-enriched CTCs and cfDNA concentrations calculated by ESR1 copy numbers in patient blood were significant independent predictors of disease progression in HR+HER2- MBC. The poor prognosis in the c-MET-enriched CTC-high group and the difference in the c-MET overexpression rate between the primary breast and metastatic sites suggested the importance of monitoring c-MET-enriched CTCs in the blood of HR+HER2- MBC patients. Citation Format: Jieun Park, Eun Sol Chang, Ji-Yeon Kim, Chaithanya Chelakkot, Minjung Sung, Ji-Young Song, Kyungsoo Jung, Na Young Kim, Hyegyeong Lee, Mi-Ran Kang, Yeon Hee Park, Young Kee Shin, Yoon-La Choi. The prognostic value of c-MET monitoring by using c-MET-enriched circulating tumor cells in HR-positive HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-04.

HAS2-Ezrin-ER axis plays a role in acquired antiestrogen resistance of ER-positive breast cancer.

The development of endocrine resistance is a major clinical problem in estrogen receptor-positive (ER+) breast cancer (BrCa) treatment, in which how cancer cells acquire resistance remains obscure. Hyaluronan synthase 2 (HAS2) is the most critical synthase in producing hyaluronan and is well known for its involvement in cancer growth, metabolism and metastasis. Recent evidence has proved that HAS2 is involved in cellular acquired resistance to drug therapy in BrCa. In this work, we first observed that HAS2 expression was decreased in the endocrine-resistant ER+ BrCa cells. Further knocking-out experiments confirmed that the loss of HAS2 in parental ER+ BrCa cells resulted in a following antiestrogen resistance. Next, we found that the HAS2-loss could induce an upregulation of Ezrin, a member of the membrane cytoskeletal protein family who plays key roles in cellular signal transduction. Notably, we identified that the increase of Ezrin induced by HAS2-loss could inhibit the ERα expression and augment antiestrogen resistance, suggesting that a HAS2-Ezrin-ER axis may be associated with the acquirement of endocrine resistance in ER+ BrCa cells. Finally, knockdown or inhibition of Ezrin could restore the sensitivity of endocrine-resistant cells to antiestrogens treatment by activating ERα signaling. Taken together, our findings unraveled a novel HAS2-Ezrin-ER route in regulating the sensitivity of ER+ BrCa cells to antiestrogens, in which Ezrin may be a potential target in endocrine therapy.

Progesterone as an Anti-Inflammatory Drug and Immunomodulator: New Aspects in Hormonal Regulation of the Inflammation.

The specific regulation of inflammatory processes by steroid hormones has been actively studied in recent years, especially by progesterone (P4) and progestins. The mechanisms of the anti-inflammatory and immunomodulatory P4 action are not fully clear. The anti-inflammatory effects of P4 can be defined as nonspecific, associated with the inhibition of NF-κB and COX, as well as the inhibition of prostaglandin synthesis, or as specific, associated with the regulation of T-cell activation, the regulation of the production of pro- and anti-inflammatory cytokines, and the phenomenon of immune tolerance. The specific anti-inflammatory effects of P4 and its derivatives (progestins) can also include the inhibition of proliferative signaling pathways and the antagonistic action against estrogen receptor beta-mediated signaling as a proinflammatory and mitogenic factor. The anti-inflammatory action of P4 is accomplished through the participation of progesterone receptor (PR) chaperones HSP90, as well as immunophilins FKBP51 and FKBP52, which are the validated targets of clinically approved immunosuppressive drugs. The immunomodulatory and anti-inflammatory effects of HSP90 inhibitors, tacrolimus and cyclosporine, are manifested, among other factors, due to their participation in the formation of an active ligand–receptor complex of P4 and their interaction with its constituent immunophilins. Pharmacological agents such as HSP90 inhibitors can restore the lost anti-inflammatory effect of glucocorticoids and P4 in chronic inflammatory and autoimmune diseases. By regulating the activity of FKBP51 and FKBP52, it is possible to increase or decrease hormonal signaling, as well as restore it during the development of hormone resistance. The combined action of immunophilin suppressors with steroid hormones may be a promising strategy in the treatment of chronic inflammatory and autoimmune diseases, including endometriosis, stress-related disorders, rheumatoid arthritis, and miscarriages. Presumably, the hormone receptor- and immunophilin-targeted drugs may act synergistically, allowing for a lower dose of each.

223P Gene expression profiles in endocrine-resistant breast cancer

Around 75% of breast cancer (BC) patients' tumors express the treatment-predictive biomarker estrogen receptor alpha (ER/ESR1) and are accordingly offered endocrine therapy. One-third of patients develop endocrine resistance, a majority with tumors that retain ER expression. For most patients, the resistance mechanism is still unknown but factors such as alterations in ER downstream signaling have been investigated. This study aims to examine the gene expression programs of endocrine-resistant tumors. A cohort of verified endocrine-resistant BC patients diagnosed in 2005-2006 and 2008-2012 was retrospectively collected at the Karolinska University Hospital in Stockholm, Sweden. Cases (N=56) were defined as patients with an ER+ and human epidermal growth factor receptor 2 (HER2)-negative primary tumor who developed an ER+/HER2- relapse during endocrine therapy. Patients with an ER+/HER2- tumor without progression or relapse at 10 years of follow-up were defined as controls (N=56). RNA was extracted from formalin-fixed paraffin-embedded tissue and analyzed using Affymetrix Clariom D microarrays. Transcriptome Analysis Console (TAC) Software, Gene Set Enrichment (GSEA) application, and R were used for gene expression data analysis. The initial analysis generated differentially expressed genes, where in general more genes were upregulated in primary tumors of cases who eventually relapse versus controls and downregulated in relapse versus primary tumors of cases. Subsequent investigation of gene sets showed upregulations in epithelial-mesenchymal transition, androgen response, MYC targets, and reactive oxygen species in primary tumors of cases versus controls. We found upregulation in metabolism-associated gene sets and downregulation of estrogen response, epithelial-mesenchymal transition, androgen response, and ESR1 regulation in relapses of cases compared to their primary tumors. This study of a unique cohort of endocrine-resistant tumors suggests that specific signaling pathways are associated with the development of endocrine resistance in BC. These gene expression signatures may guide further studies on the resistance mechanisms and develop future diagnostic tools for therapy response prediction.

Molecular crosstalk between CUEDC2 and ERα influences the clinical outcome by regulating mitosis in breast cancer.

Development of endocrine resistance in hormone-receptor-positive (HR+ve) subtype and lack of definitive target in triple-negative subtype challenge breast cancer management. Contributing to such endocrine resistance is a protein called CUEDC2. It degrades hormone receptors, estrogen receptor-α (ERα) and progesterone receptor. Higher level of CUEDC2 in ERα+ve breast cancer corresponded to poorer disease prognosis. It additionally influences mitotic progression. However, the crosstalk of these two CUEDC2-driven functions in the outcome of breast cancer remained elusive. We showed that CUEDC2 degrades ERα during mitosis, utilising the mitotic-ubiquitination-machinery. We elucidated the importance of mitosis-specific phosphorylation of CUEDC2 in this process. Furthermore, upregulated CUEDC2 overrode mitotic arrest, increasing aneuploidy. Finally, recruiting a prospective cohort of breast cancer, we found significantly upregulated CUEDC2 in HR-ve cases. Moreover, individuals with higher CUEDC2 levels showed a poorer progression-free-survival. Together, our data confirmed that CUEDC2 up-regulation renders ERα+ve malignancies to behave essentially as HR-ve tumors with the prevalence of aneuploidy. This study finds CUEDC2 as a potential prognostic marker and a therapeutic target in the clinical management of breast cancer.

Human epidermal growth factor receptor-2 and endocrine resistance in hormone-dependent breast cancer.

Endocrine therapies are the main treatment strategies for the clinical management of hormone-dependent breast cancer. Despite prolonged time to recurrence in the adjuvant setting and the initial clinical responses in the metastatic setting, many patients eventually encounter tumour relapse due to acquired resistance to these agents. Other patients experience a lack of tumour regression at the beginning of treatment indicating de novo resistance that significantly limits its efficacy in the clinic. There is compelling evidence that human epidermal growth factor receptor-2 (HER2) overexpression contributes to resistance to endocrine therapies in oestrogen receptor-positive (ER+) breast cancer. ER+/HER2+ tumours comprise about 10% of all breast cancer cases and about 60% of the whole set of HER2+ tumours. Most patients with primary ER+/HER2+ disease will receive antibody-based HER2-targeted therapy, but this is generally for no more than one year while endocrine treatment is usually for at least 5 years. A number of HER2-kinase inhibitors are also now in clinical use or in clinical trials, and the interaction of these with endocrine treatment may differ from that of antibody treatment. In this review article, we aim to summarise knowledge on molecular mechanisms of breast cancer resistance to endocrine therapies attributable to the impact of HER2 signalling on endocrine sensitivity, to discuss data from clinical trials addressing the role of HER2 in the development of endocrine resistance in the metastatic, neoadjuvant and adjuvant settings and to explore rational new therapeutic strategies.

Abstract P4-02-05: Exploiting novel models of endocrine-resistant breast cancer to identify new therapeutic targets

Abstract Introduction: While endocrine therapy is an effective, well-tolerated treatment for estrogen receptor positive breast cancer (ER+BC), a large proportion of initial responders will develop hormone therapy resistance, and relapse. A major challenge in determining the mechanisms underlying endocrine therapy resistance is our limited ability to recapitulate inter- and intra-tumour heterogeneity in vitro. In this study we developed ER+BC cells resistant to estrogen deprivation, and to palbociclib treatment, and subjected these to 2D and 3D high-throughput drug and siRNA screens, in order to elucidate the pathways underpinning the development of endocrine resistance. Methods: ER+BC cell lines modelling relapse on aromatase inhibitor therapy were derived by long-term culture in the absence of exogenous estrogen, and were termed long-term estrogen deprived (LTED). Two of these lines were then used to generate additional resistance to palbociclib, thus modelling endocrine-resistant, palbociclib-resistant disease. These lines were then subjected to screens performed in 2D and 3D. Effects of the siRNAs and drug compounds were assessed by measuring cell viability, with a robust Z score of -2 taken as a threshold of significance. Table 1 outlines the key characteristics of the cell lines, the details of the drug screens performed, and the top 3 drugs with their corresponding targets. The siRNA screens were performed in 2D and 3D with a 709 kinome library. Results: The siRNA screens highlighted PI3K-AKT-mTOR signalling as being dysregulated in multiple 3D and 2D models, while the drug screens showed that compounds targeting phosphoinositide 3-kinase alpha (PI3Kα) were significant common hits. This is consistent with the recent success of the BYLieve trial in targeting PI3Kα. A second key area of dependency was in cell cycle regulation, with CDK7 and CDK9 found to be significant hits in multiple models. The 709 kinase screens in 2D and 3D demonstrated broadly similar results (43 vs 37 median number of significant hits 2D vs 3D; Z-score <-2), while drug screening in 3D (Table 1) provided increased discrimination in identifying key vulnerabilities. CDK7 and CDK9 were selected for further study. siRNA-mediated knockdown of CDK9 had a significant effect on cell viability in endocrine-sensitive, endocrine-resistant, and palbociclib-resistant settings, whereas CDK7 knockdown did not. However, the CDK7 inhibitors THZ1 and ICEC0942, and the CDK9 inhibitors NVP-2 and AZD4573, were equally effective in endocrine-sensitive, endocrine-resistant, and palbociclib-resistant settings. Importantly, presence of a PIK3CA mutation was required for entry into the BYLieve trial, while CDK7 and CDK9 inhibitors were effective in the PIK3CA wild-type and mutant setting. Furthermore, while the presence of the ESR1 mutation is associated with poorer PFS with certain therapies, such as exemestane, the CDK7 and CDK9 inhibitors were equally effective in the ESR1 mutant and wild-type setting. Finally, combination treatment of palbociclib with CDK7 and CDK9 inhibitors showed synergism in palbociclib-resistant models. Conclusions: Targeted inhibition of CDK7 and/or CDK9 is effective in the LTED palbociclib-resistant setting, thus opening future avenues for the treatment of advanced, endocrine-resistant, palbociclib-resistant breast cancer. Table 1.Cell line characteristics and drug screen findingsCell line and key characteristics2D drug screen of 396 compounds, at 3 concentrations*3D drug screen of 70 compounds at 1 concentrationNumber of significant drug screen hits (Z score <-2). The top 3 hits and their targets are shownMCF7 LTEDWT6812PIK3CA mutantDinaciclib (CDKs 2/5/1/9)CUDC-907 (PI3Kα/HDAC)ESR1 wild typeTHZ1 (CDK7)PIK-75 HCl (PI3K)PIK-75 HCl (PI3Kα)Flavopiridol (CDKs 1/2/4/6)MCF7 LTEDY537C849PIK3CA mutantCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)Activating ESR1 mutation (Y537C)THZ1 (CDK7)BGT226 (PI3K/mTOR)PIK-75 HCl (PI3Kα)NVP-2 (CDK9)MCF7 LTEDY537C-PalboR(n/a)8As MCF7 LTEDY537CCUDC-907 (PI3Kα/HDAC)Palbociclib resistantPIK-75 HCl (PI3Kα)NVP-2 (CDK9)SUM44 LTEDY537S6717PIK3CA mutantDinaciclib (CDKs 2/5/1/9)NVP-2 (CDK9)Activating ESR1 mutation (Y537S)CUDC-907 (PI3Kα/HDAC)WYE-125132 (mTOR)E-cadherin nullBGT226 (PI3K/mTOR)CUDC-907 (PI3Kα/HDAC)HCC1428 LTED5114PIK3CA wild typeCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)ESR1 wild typeBGT226 (PI3K/mTOR)BGT226 (PI3K/mTOR)Torin-2 (mTOR)Sapanisertib (mTOR)HCC1428 LTEDPalboR(n/a)11As HCC1428 LTEDCUDC-907 (PI3Kα/HDAC)Palbociclib resistantCUDC-101 (EGFR)BGT226 (PI3K/mTOR)T47D LTED5712PIK3CA mutantCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)Loss of ER expression in adapting to LTEDDinaciclib (CDKs 2/1/5/9)PIK-75 HCl (PI3Kα)THZ1 (CDK7)CUDC-101 (EGFR)ZR75.1 LTED29(n/a)PTEN nullDinaciclib (CDKs 2/5/1/9)Loss of ER expression in adapting to LTEDTHZ1 (CDK7)Omipalisib (PI3K/mTOR)LTED: long-term estrogen deprived*Number of significant hits at 100 nM dose are shown Citation Format: Arany Soosainathan, Joanna Nikitorowicz-Buniak, Sunil Pancholi, Marjan Iravani, John Alexander, Syed Haider, Stephen R Johnston, Mitchell Dowsett, Lesley A Martin, Clare M Isacke. Exploiting novel models of endocrine-resistant breast cancer to identify new therapeutic targets [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-05.

Аммония глицирризинат в комплексном лечении стероидзависимого нефротического синдрома у детей

The development of resistance to steroid therapy and the formation of dependence (“withdrawal syndrome”) due to secondary adrenal insufficiency on the background of long-term treatment with prednisolone is a topical issue in the management of patients with nephrotic syndrome (NS). Patients and methods. This observational study included 40 children aged 5 to 10 years diagnosed with steroid-dependent nephrotic syndrome (SDNS). The patients were divided into two groups: the study group (n = 20) and the control group (n = 20). All patients received prednisolone in alternating regimens followed by its withdrawal and mofetilic acid medication; patients in the study group additionally received ammonium glycyrrhizinate (AG) over a 24-week course. The follow-up period was 24 weeks with 5 on-site visits (day 0; weeks 2, 4, 12, and 24), during which the cases of disease exacerbations, their duration, the level of proteinuria, and the development of side effects were recorded. The expression levels of ABCB1, NR3C1, CYP3A4, NR1I2 genes were assessed at the 1st and 5th visits in the study group. Results. In the first 1.5 months after prednisolone withdrawal, no child in the study group experienced a recurrence of NS against the background of ammonium glycyrrhizinate therapy, while the control group had a 25% frequency of NS recurrence (n = 5 ) ( p = 0.043). In the study group, the median length of NS remission after prednisolone withdrawal was more than 2.5 months (85.2 ± 39.9 days), while in the control group, NS recurrence occurred more than 2.5 times faster, and remission was only 33 ± 12.3 days (p = 0.009). No statistically significant differences in gene expression (ABCB1, NR3C1, CYP3A4, NR1I2) were found before and after AG treatment in patients in the study group. Conclusion. The inclusion of ammonium glycyrrhizinate during the period of prednisolone therapy withdrawal in patients with SDNS helps to reduce the recurrence rate (glucocorticosteroid withdrawal syndrome) of the disease and significantly prolongs the length of remission. The long-term use of the medication does not lead to an increase in the expression of genes associated with the development of hormone resistance. Key words: children, steroid-dependent nephrotic syndrome, prevention, prednisolone, ammonium glycyrrhizinate

In vitro breast cancer models for studying mechanisms of resistance to endocrine therapy.

The development of endocrine resistance is a common reason for the failure of endocrine therapies in hormone receptor-positive breast cancer. This review provides an overview of the different types of in vitro models that have been developed as tools for studying endocrine resistance. In vitro models include cell lines that have been rendered endocrine-resistant by ex vivo treatment; cell lines with de novo resistance mechanisms, including genetic alterations; three-dimensional (3D) spheroid, co-culture, and mammosphere techniques; and patient-derived organoid models. In each case, the key discoveries, different analysis strategies that are suitable, and strengths and weaknesses are discussed. Certain recently developed methodologies that can be used to further characterize the biological changes involved in endocrine resistance are then emphasized, along with a commentary on the types of research outcomes that using these techniques can support. Finally, a discussion anticipates how these recent developments will shape future trends in the field. We hope this overview will serve as a useful resource for investigators that are interested in understanding and testing hypotheses related to mechanisms of endocrine therapy resistance.

Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance.

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.

Combination of fulvestrant and chemotherapy in ESR1 Y537S mutant breast cancer cells and potential synergy mechanism related to p53 wildtype.

1065 Background: The acquisition of ligand-independent ESR1 mutations during endocrine therapy in metastatic ER+ breast cancer is a common mechanism of resistance to endocrine treatment, particularly aromatase inhibitors, and found in more than 30% of patients with metastatic ER+ breast cancer. Our recent work showed that the ER mutations confer resistance to currently available endocrine treatments while promoting an aberrant ER transcriptional activity that drives metastases. These results underscore the importance of targeting the ESR1 mutations even after the development of endocrine resistance. We hypothesized that during chemotherapy treatment, the ESR1 mutations remain an important driver of tumor growth and metastases and therefore drugs targeting the ESR1 mutation could enhance the efficacy of chemotherapy. In this study we investigated the combination of chemotherapy with the selective estrogen receptor degrader in the presence of WT and mutant ER. Methods: We performed synergy studies testing the combination of fulvestrant with chemotherapy treatments commonly used in ER+ metastatic breast cancer including 5FU (representing capecitabine), adriamycin and paclitaxel using MCF7 and T47D breast cancer cell lines engineered to express doxycycline inducible Y537S-ESR1 mutation. Results: We found that in MCF7 cells the combination of chemotherapy and fulvestrant was synergistic and the synergy was augmented with the induction of the Y537S mutation. In contrast, there was no synergy in T47D cells that harbor a P53 mutation. We confirmed that the synergistic activity of fulvestrant with chemotherapy is dependent on P53 by generating P53 knock-out MCF7 cells using CRISPR-cas9. Additionally, cell cycle and apoptosis analyses showed that the synergistic activity was mainly due to increased effects on G1 arrest rather than apoptosis. Conclusions: Our study indicates that chemotherapy and fulvestrant are synergistic in ER+ breast cancer and the synergy is increased in the presence of the Y537S ESR1 mutation and is dependent on P53 activity. These results support a clinical trial testing the addition of fulvestrant or other novel selective estrogen receptor degraders in patients with metastatic ER+ breast cancer who are starting chemotherapy treatment.

Abstract P6-04-23: HER2 up-regulation with neoadjuvant endocrine therapy in patients with hormone receptor-positive, HER2-negative breast cancer

Abstract Background Endocrine therapy provides significant disease-free survival (DFS) and overall survival (OS) benefit to patients with early stage hormone receptor positive (HR+) HER2 negative breast cancer. However, metastatic recurrence of endocrine-resistant disease develops in 20-25% of patients and remains a major cause of breast cancer mortality. Several mechanisms play a role in the progression and development of endocrine resistance. Understanding and characterizing the underlying molecular features of resistance for more accurate response prediction and optimal clinical management is of the utmost importance. Methods We designed an interventional study to assess tumor responses, changes in tumor proliferation and molecular biomarkers in early stage HR+ HER2- clinical node-negative breast cancer patients treated with neoadjuvant endocrine therapy for 4 weeks. The primary objective is to assess changes in HER protein expression (HER1-4) with neoadjuvant endocrine therapy; secondary objectives assess other molecular changes such as therapy-resistant CK5+ progenitor cells, iron-related proteins, Bruton’s tyrosine kinase (BTK), PD-L1, PD-L2 protein expressions, association with Ki-67 and tumor responses. Interim data on changes in HER2 protein in the first 16 patients is reported here. Optimized protocols for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to assess HER2 status in the pre and post treatment tumor specimens. This is an ongoing investigator-initiated clinical trial funded by Rock River Foundation and the Medical College of Wisconsin Cancer Center (NCT03219476). Results Median age at diagnosis was 65 (42-81) and median BMI was 28.3 (19.3-50.1). Most patients were post-menopausal (81%). Median tumor size clinically was 1.3 cm (0.5-7.7). Most tumors were low (50%) or intermediate (37.5%) grade and invasive ductal histology (62.5%). Median tumor size at surgery was 1.1 cm (0-3.9). One patient had a complete pathologic response (pCR) at surgery. Four patients had pN1 (1 lymph node involved) and one patient had pN1mic disease at surgery. There was no significant change in ER-positivity between pre and post-treatment specimens. However, a significant decrease in PR-positivity was seen in post-treatment tumors consistent with functional ER pathway disruption. On HER2 protein assessment by IHC, most patients had IHC 0 (37.5%) or IHC 1+ (56%) at diagnosis. Up-regulation in HER2 protein was seen in post-treatment tumors with 36% (n=5) IHC 2+, with one case showing Her2 gene amplification by FISH. Conclusions HER2 was up regulated in 36% of tumors after short-term neoadjuvant endocrine treatment in patients with early stage HR+ HER2- breast cancer. Short-term neoadjuvant endocrine treatment holds potential for identifying emergent endocrine resistance mechanisms and pathways. Targeted therapy with HER2-directed antibodies for such HER2 up-regulated tumors after neoadjuvant endocrine treatment may benefit patients upon disease recurrence or possibly as adjuvant combination therapy in patients with high risk of recurrence. Citation Format: Lubna N Chaudhary, Julie Jorns, MaryBeth Gonyo, Amanda Kong, Amy R Peck, Yunguang Sun, Carmen Bergom, Anjishnu Banarjee, Christopher Chitambar, Hallgeir Rui. HER2 up-regulation with neoadjuvant endocrine therapy in patients with hormone receptor-positive, HER2-negative 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 P6-04-23.

Abstract P5-05-02: Preclinical development of OP-1250, an oral complete estrogen receptor antagonist (CERAN) that shrinks ER-positive breast tumors in xenograft models

Abstract Fulvestrant (FASLODEX) is the most effective endocrine therapy for estrogen receptor positive (ER+) metastatic breast cancer (MBC). Despite this superiority, fulvestrant must be delivered by intramuscular injection and its efficacy appears limited by poor drug exposure. Thus, an oral compound that shares fulvestrant’s pharmacodynamic virtues with improved drug exposure might be both more convenient and more effective than fulvestrant. Fulvestrant development was aimed at creating a Complete Estrogen Receptor ANtagonist (CERAN) using the rodent uterine weight gain assay. It is now understood that the ability of fulvestrant to shut off both the AF1 and AF2 transcriptional activation functions of the ER underlies this activity. In contrast to CERANs, selective estrogen receptor modulators (SERMs) such as tamoxifen efficiently shut off AF2 signaling (turned on by estradiol), but incompletely antagonize AF1, a function that is turned on by multiple cell signaling pathways and shown to play a role in the development of endocrine resistance. We have developed a sensitive cell culture assay that distinguish between CERANs and SERMs. Fulvestrant is also a Selective Estrogen Receptor Degrader (SERD), and it has become widely but mistakenly assumed that such ability makes a compound a CERAN. In our assay several SERDs under clinical investigation, including AZD9496 and GDC-0810, were identified as SERMs that exhibited ER-mediated agonism on genes dependent on AF1 activity. Further, these SERM/SERDs stimulate cell proliferation of CAMA-1 and HCC-1500 breast cancer cells even in the absence of estrogen. These proliferation assays may model clinical breast cancer progression in which tumors become less dependent on estrogen and more sensitive to AF1 signaling mediated by SRC family kinases, IGFs, and FGFs. We thus predict that SERM/SERDs will be inferior in preventing and treating resistance compared to CERAN/SERDs. Guided by our cell culture assay, we have developed the CERAN/SERD OP-1250. OP-1250 completely blocks both AF2 and AF1 activity as seen in cell culture and the ovex mouse uterine weight gain assay. Unlike SERM/SERDs, OP-1250 is a potent inhibitor of CAMA1 and HCC-1500 breast cancer cell proliferation. OP-1250 also is a SERD in a large variety of cell lines including those in which some SERM/SERDs have little activity. OP-1250 achieves high and stable drug exposure in multiple animal species unlike fulvestrant and many SERMs. As might be expected for a compound with this spectrum of activities, OP-1250 shrinks tumors in xenograft models in which fulvestrant (because of limited drug exposure) and SERM/SERDs (because of residual estrogen-like action) fail to shrink the tumors. These models include HCC-1500 xenografts with a wild type ER and HCI-013 patient-derived xenografts with ESR1Y537S. These results suggest that OP-1250 has potential to be a superior compound to treat ER+ MBC, especially in patients whose tumors allow high activity of AF1 and including those with activating mutations ins ESR1. We expect to bring OP-1250 into the clinic shortly. Citation Format: Leslie Hodges-Gallagher, Richard Sun, David C Myles, Cyrus L Harmon, Peter J Kushner. Preclinical development of OP-1250, an oral complete estrogen receptor antagonist (CERAN) that shrinks ER-positive breast tumors in xenograft models [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 P5-05-02.

Abstract P1-03-06: Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics

Abstract Background: Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Hormone receptor-positive (HR+) advanced BC cancer, in particular the inflammatory BC (IBC) represents a significant clinical challenge because of the development of endocrine resistance during either adjuvant or metastatic treatment translating to poor outcome. We aim to develop unique PDX tumor models and 3D spheroid/organoid cultures that recapture advanced IBC using pleural effusions or/and circulating tumor cells (CTCs) as tumor resource and that can help understand the mechanism of IBC metastasis for new drug development. Methods: Five samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from three advanced IBC patients were obtained from Northwestern Memorial Hospital to establish IBC PDX tumor model in immunodeficient NSG female mice via subcutaneous or breast fat pad xenografting and develop the derived 3D organoid/spheroid cultures for anti-cancer drug evaluation. STR profiling against with original patient tumor DNA was conducted to confirm the tumor authentication. Results: Three developed IBC PDX tumor models (one HR+-IBC and two triple negative (TN)-IBC) were demonstrated by pathology to have highly heterogeneous characteristics and metastatic features similar to the original patient tumor. NSG mice xenografted with original TN-IBC pleural effusion, or derived spheroid cultures with varying passage numbers (<3 passages or >6 passages) manifested different inflammatory clinical symptoms. Mice xenografted with one case of original TN-IBC pleural effusion cells (~40% of tumor-associated macrophages and ~60% of tumor cells) developed palpable tumors and was shown to have the most IBC-like characteristics including reddish skin and ulcerative lesions. They were sacrificed 2 months after xenograft due to declining health. Mice xenografted with >6 passages or <3 passages of spheroid cultures manifested little to moderate skin lesions and developed tumor sizes of ~1 cm within 2-3 months while having a longer life span. Liver and lung metastases were observed in the breast fat pad of xenografted PDX tumor mice with one case of original HR+-IBC pleural effusion. 3D tumor spheroid/organoid cultures were successfully established from original IBC pleural effusion or/and IBC PDX tumor cultures. Spheroid generated from HR+-IBC pleural effusion showed phenotypic plasticity: the HR+-IBC spheroid cells display both tumor and macrophage cell characteristics and spheroid cultures can convert to adhesive cell phenotypes with addition of condition media. These spheroid/organoid cultures are used for evaluation of new drug strategies such as selectively targeting ER degraders (SERDS), CDK4/6 inhibitor, or Tyrosine kinase inhibitors for treatment of HR+-IBC or TN-IBC. Conclusions: Our results suggested 3D spheroid cultures and PDX tumor models from advanced IBC recapture aggressive features of the disease and can be an ideal model for the mechanism study of metastatic advanced IBC and preclinical testing of novel agents in IBC and endocrine resistant disease. Citation Format: Wenan Qiang, Zhangfeng Zhong, Pamela Monahan, Kristy Tse, Youbin Zhang, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Demirkan Gursel, Reiner Bleher, Jian-Jun Wei, Charles D. James, Thomas V. O’Halloran, Massimo Cristofanilli. Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics [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 P1-03-06.