Receptor Positive Breast Cancer Cells Research Articles

8550 Processing Technique Affects Aromatase Expression in Fat Grafts for Breast Reconstruction

Abstract Disclosure: S. Arbuiso: None. Y. Chen: None. N.A. Vernice: None. A. Zhang: None. I.J. Rhodes: None. C.C. Alston: None. S. Hanif: None. S. Liu: None. P. Bhardwaj: None. D. Janhofer: None. G.G. Black: None. D.M. Otterburn: None. K.A. Brown: None. Introduction: Autologous fat grafting is commonly used in breast reconstruction as it is associated with good cosmetic outcomes, high patient satisfaction, and minimal risks. Adipose tissue is known to support cancer growth via the secretion of adipokines and the modulation of estrogen levels due to aromatase expressed in adipose stromal cells. A higher body mass index (BMI) and postmenopausal status are associated with higher levels of aromatase. With novel processing techniques being developed to increase fat graft retention, there is a need to better understand how these will affect the biology of lipoaspirates for women who have previously had breast cancer. This study aims to assess how different processing techniques used in fat grafting (1) impact aromatase gene expression, (2) affect breast cancer cell proliferation, and (3) affect the cellular composition of the engrafted lipoaspirate. Methods: Patients who underwent breast reconstruction for breast cancer treatment or for prophylactic measures, and were receiving autologous fat grafting, were enrolled in a prospective randomized controlled trial (NCT04891510). Patients were randomized to one of three processing techniques: standard decantation, active filtration, and low-pressure decantation. Conditioned media (CM) was created from each sample. Aromatase mRNA was measured in the lipoaspirate using qRT-PCR for aromatase (CYP19A1) and related to patient BMI or menopausal status. The effect of CM on the proliferation of MCF7 cells (estrogen receptor positive (ER+) breast cancer cell line) was measured using CyQUANT. Formalin-fixed lipoaspirate samples were embedded in HistoGel and paraffin. Adipocyte diameter was measured in hematoxylin and eosin (H&E) stained sections using ImageJ. Results: Aromatase mRNA levels were correlated with BMI and were higher in postmenopausal women (p≤0.05), with the standard decantation group demonstrating a stronger correlation with BMI. Samples processed via active filtration were associated with weaker correlations between aromatase gene expression and BMI and menopausal status. CM induced MCF7 cell proliferation to a similar extent to cells plated in fetal bovine serum-containing media. There was no difference in proliferation patterns between treatment groups. Upon histological assessment, adipocytes were intact, and adipocyte diameter was significantly correlated with patient BMI. Conclusion: Lipoaspirate may contain factors that promote breast cancer cell proliferation, unaffected by processing technique. Caution should be used to ensure local control of the cancer has been achieved prior to reconstruction. Our data suggest that active filtration selects for a lower percentage of adipose stromal cells, which may be beneficial for patients who previously had ER+ breast cancer. Presentation: 6/1/2024

Polyoxometalate inhibition of SOX2-mediated tamoxifen resistance in breast cancer

BackgroundIncreased cancer stem cell (CSC) content and SOX2 overexpression are common features in the development of resistance to therapy in hormone-dependent breast cancer, which remains an important clinical challenge. SOX2 has potential as biomarker of resistance to treatment and as therapeutic target, but targeting transcription factors is also challenging. Here, we examine the potential inhibitory effect of different polyoxometalate (POM) derivatives on SOX2 transcription factor in tamoxifen-resistant breast cancer cells.MethodsVarious POM derivatives were synthesised and characterised by infrared spectra, powder X-ray diffraction pattern and nuclear magnetic resonance spectroscopy. Estrogen receptor (ER) positive breast cancer cells, and their counterparts, which have developed resistance to the hormone therapy tamoxifen, were treated with POMs and their consequences assessed by gel retardation and chromatin immunoprecipitation to determine SOX2 binding to DNA. Effects on proliferation, migration, invasion and tumorigenicity were monitored and quantified using microscopy, clone formation, transwell, wound healing assays, flow cytometry and in vivo chick chorioallantoic membrane (CAM) models. Generation of lentiviral stable gene silencing and gene knock-out using CRISPR-Cas9 genome editing were applied to validate the inhibitory effects of the selected POM. Cancer stem cell subpopulations were quantified by mammosphere formation assays, ALDEFLUOR activity and CD44/CD24 stainings. Flow cytometry and western blotting were used to measure reactive oxygen species (ROS) and apoptosis.ResultsPOMs blocked in vitro binding activity of endogenous SOX2. [P2W18O62]6− (PW) Wells-Dawson-type anion was the most effective at inhibiting proliferation in various cell line models of tamoxifen resistance. 10 µM PW also reduced cancer cell migration and invasion, as well as SNAI2 expression levels. Treatment of tamoxifen-resistant cells with PW impaired tumour formation by reducing CSC content, in a SOX2-dependent manner, which led to stem cell depletion in vivo. Mechanistically, PW induced formation of reactive oxygen species (ROS) and inhibited Bcl-2, leading to the death of tamoxifen-resistant cells. PW-treated tamoxifen-resistant cells showed restored sensitivity to tamoxifen.ConclusionsTogether, these observations highlight the potential use of PW as a SOX2 inhibitor and the therapeutic relevance of targeting SOX2 to treat tamoxifen-resistant breast cancer.

Endocrine therapy for early breast cancer in the era of oral selective estrogen receptor degraders: challenges and future perspectives.

Growth and survival of hormone receptor positive breast cancer cells are dependent on circulating hormones (e.g., estrogen and progesterone). Endocrine therapy improved outcomes in both early and advanced hormone receptor positive breast cancer. These treatments include drugs with different mechanisms of action, namely selective estrogen receptor modulators (SERM), aromatase inhibitors, and selective estrogen receptor degraders (SERDs). SERDs represent estrogen receptor antagonists, favoring its degradation and thus interfering with proliferation genes transcription and activation. Fulvestrant is the first approved SERD, administered intramuscularly for treating advanced breast cancer. Oral SERDs have been tested to overcome the limitation of the intramuscular administration, and to increase SERD bioavailability. Recently, an oral SERD, Elacestrant, has been approved by the Food and Drug Administration (FDA) for patients carrying an ESR1 mutation. In fact, oral SERDs seem to be effective in tumors harboring ESR1 mutations, a well known mechanism of resistance to endocrine therapy (especially aromatase inhibitors). More recently, oral SERDs have been tested in patients with early hormone receptor positive breast cancer, although their impact on survival and in this curative setting compared to standard endocrine therapy still needs to be elucidated. The best timing and duration of SERD administration and specific biomarkers in (neo)adjuvant setting remain largely unknown.

Metabolic and Oxidative Stress Management Heterogeneity in a Panel of Breast Cancer Cell Lines.

Metabolic alterations are recognized as one of the hallmarks of cancer. Among these, alterations in mitochondrial function have been associated with an enhanced production of Reactive Oxygen Species (ROS), which activate ROS-regulated cancer cell signaling pathways. Breast cancer is the main cancer-related cause of death for women globally. It is a heterogeneous disease with subtypes characterized by specific molecular features and patient outcomes. With the purpose of identifying differences in energy metabolism and the oxidative stress management system in non-tumorigenic, estrogen receptor positive (ER+) and triple negative (TN) breast cancer cells, we evaluated ROS production, protein enzyme levels and activities and profiled energy metabolism. We found differences in energetic metabolism and ROS management systems between non-tumorigenic and cancer cells and between ER+ and TN breast cancer cells. Our results indicate a dependence on glycolysis despite different glycolytic ATP levels in all cancer cell lines tested. In addition, our data show that high levels of ROS in TN cells are a result of limited antioxidant capacity in the NADPH producing and GSH systems, mitochondrial dysfunction and non-mitochondrial ROS production, making them more sensitive to GSH synthesis inhibitors. Our data suggest that metabolic and antioxidant profiling of breast cancer will provide important targets for metabolic inhibitors or antioxidant treatments for breast cancer therapy.

Dual-targeting class I HDAC inhibitor and ATM activator, SP-1-303, preferentially inhibits estrogen receptor positive breast cancer cell growth.

Dual-targeting chromatin regulation and DNA damage repair signaling presents a promising avenue for cancer therapy. Applying rational drug design, we synthesized a potent dual-targeting small molecule, SP-1-303. Here, we report SP-1-303 as a class I isoform selective histone deacetylase (HDAC) inhibitor and an activator of the ataxia-telangiectasia mutated protein (ATM). In vitro enzymatic assays demonstrated selective inhibition of HDAC1 and HDAC3. Cellular growth inhibition studies show that SP-1-303 differentially inhibits growth of estrogen receptor positive breast cancer (ER+ BC) cells with effective growth inhibition concentrations (EC50) for MCF-7 and T47D cells ranging from 0.32 to 0.34 μM, compared to 1.2-2.5 μM for triple negative breast cancer cells, and ~12 μM for normal breast epithelial cells. Western analysis reveals that SP-1-303 decreases estrogen receptor alpha (ER-α) expression and increases p53 protein expression, while inducing the phosphorylation of ATM and its substrates, BRCA1 and p53, in a time-dependent manner in ER+ BC cells. Pharmacokinetic evaluation demonstrates an area under the curve (AUC) of 5227.55 ng/ml × h with an elimination half-life of 1.26 h following intravenous administration in a rat model. Collectively, SP-1-303 emerges as a novel second generation class I (HDAC1 and HDAC3) selective HDAC inhibitor, and ATM activator, capable of modulating ER expression, and inhibiting growth of ER+ BC cells. Combined targeting of class I HDACs and ATM by SP-1-303 offers a promising therapeutic approach for treating ER+ breast cancers and supports further preclinical evaluation.

Abstract PO4-06-10: Targeting necroptosis in breast cancer induced by Ophiobolin A

Abstract Abstract Necroptosis or programmed necrosis is a form of cell death distinct from apoptosis. Necroptotic cells display the character of rounding of the cell, gain in cell volume, rupture of plasma membrane and release of intra-cellular contents. Although few key necroptosis regulators including receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like pseudokinase (MLKL) has been identified, their role/mechanism in inducing cell death in triple-negative breast cancer (TNBC) which lack RIPK3 expression remains elusive. Ophiobolin A is a compound naturally made by fungus and potently kills cancer cells especially TNBC. Our current study investigated the cell death mechanism(s) induced by Ophiobolin A (natural product) in breast cancer cell lines. The mechanism of cell death elicited by OpA was analyzed by applying inhibitors for specific pathways including apoptosis, necroptosis, ferroptosis and paraptosis, by analyzing markers of cell death using flow cytometry and western blotting. The formation of necrosomes was analyzed with exogeneous expression of RIPK3 tagged with green fluorescence protein (GFP) in HeLa, MDA-MB-231, MDA-MB-468 cell lines which lacks endogenous RIPK3 expression and in MCF7 estrogen receptor (ER) positive cell line. OpA induced necrosome formation was identified by formation of puncta in TNBC and HeLa cells which lack RIP3 expression, however, necrosomes were not detectable in ER positive cells. Clinical relevance of RIPK3 and MLKL has been analyzed using bioinformatic tools. Effect of OpA In vivo has been analyzed using Immune compromised SCID mice. The data obtained in the present study indicates that OpA has an increased anticancer effect in TNBC compared to the estrogen receptor (ER) positive breast cancer cells. Cells having undergone EMT were shown to have more sensitivity towards OpA when compared to uninduced cell lines, which is sensitive to Necrostatin-1, an inhibitor of necroptosis. However, apoptosis, ferroptosis and paraptosis inhibitors failed to rescue cell death which clearly confirms that OpA induces necroptosis cell death pathway in EMT undergone cells. OpA treatment significantly increased puncta in TNBC compared non-TNBC cells, further confirming the necroptotic cell death in TNBC. Bioinformatic analysis revealed that RIPK3 is negatively corelated with breast cancer and has least over-all survival in patients who lack RIPK3 expression. OpA treatment showed a significant tumor regression in tumor bearing SCID mice. All together our findings identify that OpA induce necroptosis in EMT-enriched TNBC. The study further needs an in-depth analysis of molecular mechanism induced by OpA and involvement of EMT in necroptotic cell death. Citation Format: Tolulope Ojo, Santhalakshmi Ranganathan. Targeting necroptosis in breast cancer induced by Ophiobolin A [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 PO4-06-10.

Abstract PO2-27-04: Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer

Abstract Fibroblast Growth Factor Receptor 1 (FGFR1) has emerged as a critical player in various cancer types, including breast cancer. FGFR1 alterations, including gene amplification, overexpression, and activating mutations, have been identified in subsets of breast cancers, contributing to aberrant signaling pathways that promote tumor growth, angiogenesis, and metastasis. Our current research endeavors are directed towards unravelling the intricate mechanistic underpinnings of FGFR1's dual impact on breast tumor progression, specifically its contribution to endocrine resistance and its regulatory role in innate immune sensing and subsequent induction of Type 1 interferons (IFNs) Our investigative efforts have previously established a direct association between FGFR1 and endocrine resistance in hormone receptor positive T47D breast cancer cells. Moreover, a remarkable and distinct inverse correlation linking FGFR1 expression to the Type 1 IFN response was observed. We noticed that in vitro stimulation of T47D cells with Type 1 interferons induces the expression of FGFR1 and ESR1, thereby suppressing the interferon-stimulated genes. Interestingly, we observed that the FGFR1-mediated suppression of the Type 1 interferon response is associated with intracellular nucleic acid sensors, specifically toll-like receptors TLR7 and TLR9. Furthermore, we found that the inhibition of FGFR kinase enhances the expression of IFN-alpha, IFN-beta, TLR7, and TLR9 in T47D cells when stimulated with the TLR7 agonist loxoribine. These results strongly suggest that FGFR1 functions as an innate checkpoint and may be exploited by tumor cells to dampen Type 1 interferon responses, thereby inhibiting antitumor immunity in hormone receptor-positive breast cancer. Citation Format: Sushil Dhakal, Torbjørn Amundsen Lien, Jens Henrik Norum, Helga Bergholtz, Helene Midtun Flatekvål, Therese Sørlie. Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer [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 PO2-27-04.

A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

Progestins and breast cancer hallmarks: The role of the ERK1/2 and JNK pathways in estrogen receptor positive breast cancer cells

Progestins used in hormonal contraceptives and menopausal hormone therapy (MHT) have been linked to increased breast cancer risk. Whether the association holds for all progestins is unclear and the underlying mechanisms remain poorly understood. We directly compared the effects of four progestins (medroxyprogesterone acetate (MPA), norethisterone acetate (NET-A), levonorgestrel (LNG) and drospirenone (DRSP)) to each other and the natural progestogen progesterone (P4) on selected cancer hallmarks. To provide mechanistic insight into these effects, we assessed the role of the progesterone receptor (PR), and the extracellular signal-related kinase (ERK1/2) and c-Jun N terminal (JNK) signaling pathways. We showed that the increased proliferation of the luminal T47D breast cancer cell line by P4 and all progestins, albeit to different extents, was inhibited by PR knockdown and inhibition of both the ERK1/2 and JNK pathways. While knockdown of the PR also blocked the upregulation of MKI67 and CCND1 mRNA expression by selected progestogens, only a role for the ERK1/2 pathway could be established in these effects. Similarly, only a role for the ERK1/2 pathway could be confirmed for progestogen-induced colony formation, whereas both the ERK1/2 and JNK pathways were required for cell migration in response to the three older progestins implicated in the etiology of breast cancer, MPA, NET-A and LNG. Together our results show that all the progestins elicit their effects on cell proliferation via a mechanism requiring the PR, ERK1/2 and JNK pathways. While the ERK1/2 and JNK pathways are also required for increased cell migration by the older progestins, only a role for the ERK1/2 pathway could be established in their effects on colony formation. Notably, the cytoplasmic PR was not needed for activation of the ERK1/2 pathway by the progestogens. Given that DRSP showed significantly lower proliferation than MPA and NET-A, and that it had no effect on breast cancer cell migration and colony formation, hormonal formulations containing the newer generation progestin DRSP may provide a better benefit/risk profile towards breast cancer than those containing the older generation progestins.

Single Live Cell Imaging of Multidrug Resistance Using Silver Ultrasmall Nanoparticles as Biosensing Probes in Triple-Negative Breast Cancer Cells.

Silver ultrasmall nanoparticles (Ag UNPs) (size < 5 nm) were used as biosensing probes to analyze the efflux kinetics contributing to multidrug resistance (MDR) in single live triple-negative breast cancer (TNBC) cells by using dark-field optical microscopy to follow their size-dependent localized surface plasmon resonance. TNBC cells lack expression of estrogen (ER-), progesterone (PR-), and human epidermal growth factor 2 (HER2-) receptors and are more likely to acquire resistance to anticancer drugs due to their ability to transport harmful substances outside the cell. The TNBC cells displayed greater nuclear and cytoplasmic efflux, resulting in less toxicity of Ag UNPs in a concentration-independent manner. In contrast, more Ag UNPs and an increase in cytotoxic effects were observed in the receptor-positive breast cancer cells that have receptors for ER+, PR+, and HER2+ and are known to better respond to anticancer therapies. Ag UNPs accumulated in receptor-positive breast cancer cells in a time-and concentration-dependent mode and caused decreased cellular growth, whereas the TNBC cells due to the efflux were able to continue to grow. The TNBC cells demonstrated a marked increase in survival due to their ability to have MDR determined by efflux of Ag UNPs outside the nucleus and the cytoplasm of the cells. Further evaluation of the nuclear efflux kinetics of TNBC cells with Ag UNPs as biosensing probes is critical to gain a better understanding of MDR and potential for enhancement of cancer drug delivery.

Exosomes isolated from metabolically unhealthy normal weight and overweight phenotypes deteriorate the ER/PR positive breast cancer behavior.

Obesity has been linked to a higher risk of postmenopausal breast cancer Yet, research indicates an opposite correlation between obesity and premenopausal breast cancer risk. Various obesity phenotypes based on metabolic health could play a significant part. This study aims to assess how plasma exosomes taken from women with varying obesity phenotypes impact MCF-7 cell migration, matrix metalloproteinase-2 activity, and apoptosis. The characterization of isolated exosomes and their internalization into MCF-7 cells was evaluated. The treatment of MCF-7 cells with exosomes isolated from different groups was done. Migration, the activity of MMP-2, mRNA expression of Bax and Bcl-2, protein expression of p-53 and Thr55 p-p53, and apoptosis were assessed. Isolated exosomes from unhealthy obese individuals increase MCF-7 cell migration. Regarding MMP activities, unhealthy normal weight and overweight and healthy obese groups isolated exosomes increase the MMP-2 activity than the treated group with exosomes isolated from counterpart groups. Furthermore, unhealthy normal weight and overweight and healthy obese obtained exosomes decrease apoptosis compared to counterpart groups. Altogether, plasma exosomes derived from both unhealthy individuals with normal weight and overweight status, as well as those with unhealthy obesity, negatively impacted the behavior of estrogen/progesterone receptor-positive breast cancer cells. The online version contains supplementary material available at 10.1007/s40200-023-01295-1.

Social isolation and breast cancer.

Although the role of life-stressors in breast cancer remains unclear, social isolation is consistently associated with increased breast cancer risk and mortality. Social isolation can be defined as loneliness or an absence of perceived social connections. In female mice and rats, social isolation is mimicked by housing animals one per cage. Social isolation causes many biological changes, of which an increase in inflammatory markers and disruptions in mitochondrial and cellular metabolism are commonly reported. It is not clear how the two traditional stress-induced pathways, i.e., hypothalamic-pituitary axis (HPA), resulting a release of glucocorticoids from the adrenal cortex, and autonomic nervous system (ANS), resulting a release of catecholamines from the adrenal medulla and post-ganglionic neurons, could explain the increased breast cancer risk in socially isolated individuals. For example, glucocorticoid receptor activation in estrogen receptor positive breast cancer cells inhibits their proliferation, and activation of β-adrenergic receptor (β-AR) in immature immune cells promotes their differentiation towards anti-tumorigenic T cells. However, activation of HPA and ANS pathways may cause a disruption in the brain-gut-microbiome axis, resulting gut dysbiosis. Gut dysbiosis, in turn, leads to an alteration in the production of bacterial metabolites, such as short chain fatty acids, causing a systemic low-grade inflammation and inducing dysfunction in mitochondrial and cellular metabolism. A possible causal link between social isolation -induced increased breast cancer risk and mortality and gut dysbiosis should be investigated, as it offers new tools to prevent breast cancer.

Cyclopia extracts act as selective estrogen receptor subtype downregulators in estrogen receptor positive breast cancer cell lines: Comparison to standard of care breast cancer endocrine therapies and a selective estrogen receptor agonist and antagonist.

Breast cancer is the most diagnosed type of cancer amongst women in economically developing countries and globally. Most breast cancers express estrogen receptor alpha (ERα) and are categorized as positive (ER+) breast cancer. Endocrine therapies such as, selective estrogen receptor modulators (SERMs), aromatase inhibitors (AIs), and selective estrogen receptor downregulators (SERDs) are used to treat ER+ breast cancer. However, despite their effectiveness, severe side-effects and resistance are associated with these endocrine therapies. Thus, it would be highly beneficial to develop breast cancer drugs that are as effective as current therapies, but less toxic with fewer side effects, and less likely to induce resistance. Extracts of Cyclopia species, an indigenous South African fynbos plant, have been shown to possess phenolic compounds that exhibit phytoestrogenic and chemopreventive activities against breast cancer development and progression. In the current study, three well characterized Cyclopia extracts, SM6Met, cup of tea (CoT) and P104, were examined for their abilities to modulate the levels of the estrogen receptor subtypes, estrogen receptor alpha and estrogen receptor beta (ERβ), which have been recognized as crucial to breast cancer prognosis and treatment. We showed that the Cyclopia subternata Vogel (C. subternata Vogel) extracts, SM6Met and cup of tea, but not the C. genistoides extract, P104, reduced estrogen receptor alpha protein levels while elevating estrogen receptor beta protein levels, thereby reducing the ERα:ERβ ratio in a similar manner as standard of care breast cancer endocrine therapies such as fulvestrant (selective estrogen receptor downregulator) and 4-hydroxytamoxifen (elective estrogen receptor modulator). Estrogen receptor alpha expression enhances the proliferation of breast cancer cells while estrogen receptor beta inhibits the proliferative activities of estrogen receptor alpha. We also showed that in terms of the molecular mechanisms involved all the Cyclopia extracts regulated estrogen receptor alpha and estrogen receptor beta protein levels through both transcriptional and translational, and proteasomal degradation mechanisms. Therefore, from our findings, we proffer that the C. subternata Vogel extracts, SM6Met and cup of tea, but not the C. genistoides extract, P104, selectively modulate estrogen receptor subtypes levels in a manner that generally supports inhibition of breast cancer proliferation, thereby demonstrating attributes that could be explored as potential therapeutic agents for breast cancer.

Abstract P2-26-02: Valproic acid induces apoptosis and mitochondrial perturbations in breast cancer cells

Abstract Introduction: Breast cancer is the second leading cause of death among women after lung cancer. Despite the increase and improvement in preventive screening, breast cancer still remains a threat, both for pre- and postmenopausal women, due to the rapid development of resistance to drug treatments, which recalls the complicity of multiple factors. Breast cancer prognosis can be correlated with specific histological subtypes and their receptor status. Depending on stage and invasiveness, ER + and PR + breast cancers are treated with surgery and adjuvant chemotherapy, which includes endocrine-based agents, such as SERMS (Tamoxifen), SERDs (Fulvestrant) and aromatase inhibitors (Letrazole and Anastrazole). Resistance to endocrine therapy can be explained by various mechanisms, such as mutations of the estrogen receptor, epigenetic modifications of the same receptor, alterations in hormone synthesis and metabolism, aberrant activation of signaling transduction pathways (up-regulation of PI3K, MAPK, CDKs). To reduce pharmacological resistance, it is possible to use histone deacetylase inhibitors, such as valproic acid, as the epigenetic alterations play an important role in the oncogenesis of breast cancer. Epigenetic modifications of genes are a mechanism used by cancer cells to silence the expression of tumor suppressor genes, driven by epigenetic mutations of regulator factors. The valproic acid, used as an antiepileptic drug and for the treatment of bipolar disorders, is also a selective inhibitor of class I and class II HDAC isoforms, involved in the progression of different types of tumors. In this study, we tested the effects of valproic acid on the signaling involved in apoptosis and in the generation of Reactive Oxygen Species (ROS) in MCF-7 breast cancer cells. Methods: MTT assay was performed to evaluate cell proliferation. Cell cycle, ROS levels and apoptosis were analyzed by flow cytometry, while protein levels were detected by Western Blotting (WB). Results: Cell treatment with valproic acid reduced cell proliferation and induced G0/G1 cell cycle arrest. In addition, the drug enhanced the generation of ROS by the mitochondria and addressed breast cell death by apoptosis. Indeed, the valproic acid was found to downregulate the anti-apoptotic marker Bcl-2 and to upregulate the expression of pro-apoptotic markers, such as Bax and Bad, leading to release of cytochrome C into the cytosol and PARP cleavage. The drastic reduction in mitochondrial membrane potential and in mitochondrial mass was observed following treatment with valproic acid, indeed the ratio between the two parameters, index of functionality per mitochondrion, resulted to be lowered, thus emphasizing the metabolic perturbation in the cells. Conclusion: In summary, our results have demonstrated that in estrogen receptor positive breast cancer cells the valproic acid is a suitable drug to arrest cell growth, to address apoptosis and mitochondrial perturbations, all factors that are important in determining cell fate and health. This study encourages that valproic acid, as an epigenetic drug, is a promising class of antineoplastic agent to be further explored also in combination with other classical chemotherapeutics in breast tumors. Citation Format: Alessandro Paolì, Martina Forestiero, Giuseppina Daniela Naimo, Loredana Mauro, Maria Luisa Panno, Francesca Giordano. Valproic acid induces apoptosis and mitochondrial perturbations in breast cancer cells [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 P2-26-02.

Abstract P6-14-03: Oestrogen represses Noggin expression by interfering BMP/Smad signalling in ER positive breast cancer cells

Abstract Background Bone morphogenetic proteins (BMPs) are members of Transforming Growth Factor β (TGF-β) superfamily. BMPs are actively involved in the disease progression and bone metastasis of breast cancer. As a natural antagonist of BMP, Noggin plays important roles in the regulation of BMP signalling. It can facilitate spread of breast cancer cells to bone and subsequent colonisation and formation of osteolytic bone lesions. The present study aimed to investigate the regulatory mechanism for Noggin expression in oestrogen receptor (ER) positive breast cancer cells. Method Noggin expression was analysed in The Cancer Genome Atlas (TCGA ER (+) n=763, ER (-) n=216) and E-MTAB-6703 (ER (+) n=733, ER (-) n=421) cohorts. Correlation between Noggin and ER was evaluated using Spearman test. The expression of Noggin in an ER positive breast cancer cell line MCF-7 was determined by depriving the cells from oestrogen using phenol red-free DMEM supplemented with 10% charcoal stripped foetal calf serum or adding 10-10M 17-β-oestradiol. Activation of Smad-1/5/8 and involvement of BMP receptors in the oestrogen repressed Noggin expression were further examined using recombinant human BMP7 and a BMP receptor inhibitor (LDN-193189). Influence of Noggin on cellular functions was evaluated in MCF-7 cells with Noggin overexpression using a lentiviral Noggin expression vector. Results Noggin expression was negatively correlated with ERα in both TCGA BRCA (r=-0.162, p&amp;lt;0.01, n=1093) and E-MTAB-6703 (r=-0.078, p&amp;lt;0.01, n=2302) cohorts. The expression of Noggin was increased in the MCF-7 cells upon a deprivation from oestrogen which was further validated by adding 17-β-oestradiol. This is in line with the increased expression of Noggin observed in the MCF-7 cells with ER silencing (GSE27473). Furthermore, an increased level of phosphorylated Smad1/5/8 was seen in the MCF-7 being hungered from oestrogen which was prevented by adding 17-β-oestradiol and LDN-193189, respectively. As a result, the oestrogen hunger induced Noggin expression was also decreased by adding with 17-β-oestradiol and LDN-193189. To further investigate the influence of oestrogen on BMP-Smad signalling regulated Noggin expression, Noggin expression and phosphorylation of Smad1/5/8 and Smad3 were determined in MCF-7 cells which were treated with rhBMP7 and in combination with 17-β-oestradiol and LDN-193189, respectively. BMP7 induced Noggin expression and activation of Smad1/5/8 can be diminished by 17-β-oestradiol and LDN-193189. Noggin overexpression in MCF-7 cells resulted in an increase of proliferation. Conclusion Noggin expression can be repressed by oestrogen through an inference of the BMP- Smad signalling. Overexpression of Noggin promoted proliferation of MCF-7 cells. Further investigation is required to clarify the exact role of Noggin in ER positive breast cancer and its implication in the disease progression and current therapies. Citation Format: Ming Liu, Lin Ye, Wen G. Jiang. Oestrogen represses Noggin expression by interfering BMP/Smad signalling in ER positive breast cancer cells [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 P6-14-03.

Abstract C096: Obese adipose derived secretome stimulates triple negative breast cancer via stimulation of the mTOR pathway

Abstract Background: Breast cancer remains the most common cancer amoung women with multipe risk factors including smoking, genetics, environmental factors, and obesity. Smoking and obesity are the top two risk factors for development of breast cancer, with smoking increasing the risk of development by 21% and obesity by 20-40%. Obesity increases the risk for all breast cancer subtypes, including triple negative breast cancer (TNBC) that is null for estrogen and progesterone receptors and the tyrosine kinase receptor, HER2. To this end, recent retrospective patient studies have shown that the majority of patients with TNBC were obese at the time of diagnosis. In addition, obese patients had a higher TNBC tumor grade and a higher staging. However, the signaling mechanisms that promote the progression of TNBC in obesity are not well understood. We published that adipose tissue (AT) derived secretome (ADS) in obesity promotes the invasiveness of estrogen receptor positive breast cancer cells. Based on these prior findings, we hypothesize ADS in obesity acts on TNBC cells to induce mTOR activity and in turn stimulates TNBC cell migration and invasiveness. Methods: De-identified peritumor (PT) breast AT samples were obtained with patient consent at the time of surgery for breast cancer treatment. Breast AT samples were cultured in serum free culture medium for 24 hours. After the incubation, the AT conditioned media was clarified by centrifugation. For this study, considered BMI less than 30 as lean-ADS (LADS) and BMI of 30 or greater as obese-ADS (OADS). ADS diluted 1:10 in serum free culture medium was applied to human MDA-MB-231 and MDA-MB-436 TNBC cells. Cell migration was assayed by the scratch assay. Images were taken at time 0, and 24 hours. Analysis was performed using image J. Western blot studies were performed to assay the levels of total and/or phosphorylated proteins. Results: OADS stimulated a statistically significant increase (1.5 fold) in TNBC cell migration compared with LADS. The increase in TNBC cell migration was associated with a significant increase (1.5 fold) in phosphorylated S6 in TNBC cells treated with OADS relative to LADS. Conclusion: Increases in mTOR activity in TNBC cells in response to OADS is associated with increased TNBC cell migration. Citation Format: Cora E. Miracle, Travis Salisbury, Chelsea McCallister. Obese adipose derived secretome stimulates triple negative breast cancer via stimulation of the mTOR pathway [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C096.

ADİPONEKTİN’İN MCF-7 PROLİFERASYONU VE GİRELİN SEVİYESİ ÜZERİNE ETKİSİ

Amaç&#x0D; Lokal ve sistemik faktörlerin, östrojen reseptörü pozitif&#x0D; meme kanseri riskinde artış olan postmenopozal&#x0D; obez kadınlarda meme kanseri hücrelerinin büyümesini&#x0D; yönlendirdiği gösterilmiştir. Postmenapozal&#x0D; dönemdeki obez kadınlarda, dolaşımdaki girelin hormonunun&#x0D; azaldığı, bazı tümör hücreleri tarafından&#x0D; üretildiği ve ayrıca tümör büyümesini etkilediği gösterilmiştir.&#x0D; Bu çalışmada, adiponektin uygulamasının&#x0D; östrojen reseptörü pozitif MCF-7 meme kanseri hücre&#x0D; hattındaki rolünü ve girelin seviyeleri üzerine olan etkilerini&#x0D; araştırmayı amaçladık.&#x0D; Gereç ve Yöntem&#x0D; MCF-7 meme kanseri hücre hattı 2 boyutlu kültürde&#x0D; çoğaltıldı. MCF-7 meme kanseri hücrelerine artan&#x0D; konsantrasyonlarda adiponektin (50-5000 ng/mL) ile&#x0D; muamele edilerek inhibisyon dozu belirlendi. MCF-7&#x0D; meme kanseri hücre hattına 50, 100, 250 ve 500 ng/&#x0D; mL konsantrasyonda adiponektin uygulanarak 24, 48&#x0D; ve 72. Saatlerde örneklerde girelin seviyeleri enzim&#x0D; bağlı immünosorbent analizi (ELISA) ile ölçüldü.&#x0D; Bulgular&#x0D; 24, 48 ve 72. saate alınan MCF-7 meme kanseri hücre&#x0D; hattı kültür örneklerinde 50, 100, 250 ve 500 ng/mL&#x0D; adiponektin konsantrasyonuna bağlı olarak proliferasyonun&#x0D; azaldığı bulundu. 24, 48 ve 72. saate alınan&#x0D; MCF-7 meme kanseri hücre hattı kültür örneklerinde&#x0D; 50, 100, 250 ve 500 ng/mL adiponektin konsantrasyonuna&#x0D; bağlı olarak girelin miktarında artış oluştuğu&#x0D; bulundu.&#x0D; Sonuç&#x0D; Çalışmamızda MCF-7 meme kanseri hücre hattında&#x0D; (ER/PR pozitif) anti-kanserojenik özelliği kanıtlanmış&#x0D; olan ve aynı zamanda obezite ile negatif korelasyon&#x0D; gösteren adiponektinin, girelin ile karşılıklı etkileşimlerinin,&#x0D; ER pozitif meme kanserlerindeki rolünü ortaya&#x0D; koymuş olduk.

Advantages and Disadvantages of Two &lt;i&gt;In Vitro&lt;/i&gt; Assays in Evaluating Aromatase Activity: “A Cell-Based and a Cell-Free Assay”

Aromatase is an enzyme that catalyzes the conversion of androgens to estrogens. While inhibition of aromatase is a useful approach for treating breast cancer, it may also have toxicological consequences due to its endocrine disrupting/modulating effect. In this study, sensitivity and performance of two in vitro assays -a cell free and a cell-based- for evaluating aromatase activity were investigated by testing known aromatase inhibitors and partial validation of the methods was performed. Advantages and disadvantages of these methods are also discussed. Aromatase activity was evaluated via two in vitro models; direct measurement with a cell-free assay using a fluorescent substrate and recombinant human enzyme and indirect evaluation with a cell-based assay where cell proliferation was determined in estrogen receptor positive human breast cancer cells (MCF-7 BUS) in the absence of estrogen and the presence of testosterone. In the cell-free direct measurement assay, reference compounds ketoconazole and aminoglutethimide have been shown to inhibit the aromatase enzyme with half-maximal inhibitory concentration (IC50) values concordant with literature. In cell-based indirect measurement assay, only ketoconazole dose-dependently inhibited cell proliferation with 3.47 x 10-7 M IC50. Inter-assay and intra-assay reproducibility of both methods was found to be within acceptable deviation levels. Both methods can be successfully applied. However, to evaluate the potential aromatase activity of the novel compounds in vitro, it seems better to perform both the cell-based and the cell-free assays that allows low-moderate biotransformation and eliminate cytotoxicity potential, respectively.

Multi-Layered PLGA-PEI Nanoparticles Functionalized with TKD Peptide for Targeted Delivery of Pep5 to Breast Tumor Cells and Spheroids.

Peptide-based therapy is a promising strategy for cancer treatment because of its low drug resistance. However, the major challenge is their inability to target cancer cells specifically. So, a targeted nano-delivery system that could deliver therapeutic peptides selectively to cancer cells to stimulate their action is highly desirable.This study aims to deliver the antitumor peptide, Pep5, to breast tumor cells selectively using a targeting peptide functionalised multi-layered PLGA-PEI nanoparticles. In this study, Pep5 entrapped PLGA-PEI (Pep5-PPN) dual layered nanoparticles were developed. These nanoparticles were decorated with TKD (Pep5-TPPN) on their surface for site-specific delivery of Pep5 to breast tumor cells. The particles were then characterized using various instrumental analyses. In vitro cytotoxicity of the particles was evaluated in estrogen receptor positive (ER+ve) and triple negative breast cancer (TNBC) cells. An ex vivo tumor spheroid model was used to analyze the antitumor activity of the particles. Uniformly round Pep5-TPPN particles were synthesized with an average diameter of 420.8 ± 14.72 nm. The conjugation of PEI over Pep5-PLGA nanoparticles shifted the zeta potential from -11.6 ± 2.16 mV to +20.01 ± 2.97 mV.In vitro cytotoxicity analysis proved that TKD conjugation to nanoparticles enhanced the antitumor activity of Pep5 in tested breast cancer cells. Pep5-TPPN induced cytoskeletal damage and apoptosis in the tested cells, which showed that the mechanism of action of Pep5 is conserved but potentiated. Active targeting of Pep5 suppressed the tumor growth in ex vivo spheroid models. A multi-layered nanoparticle functionalized with dual peptide was fabricated for active tumor targeting, which stimulated Pep5 activity to reduce the tumor growth in vitro and ex vivo.

Abstract 5450: New se-nsaid hybrid molecules as potent and selective cytotoxic agents against breast cancer cells

Abstract Breast cancer is currently the most frequently diagnosed cancer and one of the leading causes of mortality among women worldwide. The introduction of a selenium atom (Se) in the structure of organic molecules has been demonstrated to be a valid approach in the design of novel chemotherapeutic agents with several biological effects, including antitumoral activity. In addition, several epidemiological, preclinical, and clinical studies support the chemopreventive and chemotherapeutic potential use of non-steroidal anti-inflammatory drugs (NSAIDs).On the basis of these findings, and taking into account the results of previous studies by our research group, we have developed a new series of twenty-five NSAID analogs bearing Se in the form of selenoesters modulated with different functional groups. The new compounds were tested in four cancer cell lines (HTB-54, DU-145, HT-29 and MCF-7) and two non-malignant cells (184B5 and BEAS-2B). The cytotoxicity of the five most potent and selective molecules was further studied in other cancer cell lines as well. Compounds 4a and 4d stood out as the most selective compounds with a selectivity index at least 5-fold higher in breast cancer cells. Compound 4d showed IC50 values below 10 µM and was found to be more potent than 4a in almost every cell line tested. Additionally, 4d effectively induced apoptosis in triple negative (MDA-MB-231) and estrogen receptor-positive (MCF-7) breast cancer cell lines. Taken together, this study supports the design of new derivatives containing Se as potential anticancer agents. Citation Format: Sandra Ramos-Inza, Ignacio Encío, Asif Raza, Arun Sharma, Carmen Sanmartín, Daniel Plano. New se-nsaid hybrid molecules as potent and selective cytotoxic agents against breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5450.