Estrogen Receptor Positive Cell Lines Research Articles

ERα status of invasive ductal breast carcinoma as a result of regulatory interactions between lysine deacetylases KAT6A and KAT6B

Breast cancer (BC) is the leading cause of death among cancer patients worldwide. In 2020, almost 12% of all cancers were diagnosed with BC. Therefore, it is important to search for new potential markers of cancer progression that could be helpful in cancer diagnostics and successful anti-cancer therapies. In this study, we investigated the potential role of the lysine acetyltransferases KAT6A and KAT6B in the outcome of patients with invasive breast carcinoma. The expression profiles of KAT6A/B in 495 cases of IDC and 38 cases of mastopathy (FBD) were examined by immunohistochemistry. KAT6A/B expression was also determined in the breast cancer cell lines MCF-7, BT-474, SK-BR-3, T47D, MDA-MB-231, and MDA-MB-231/BO2, as well as in the human epithelial mammary gland cell line hTERT-HME1 – ME16C, both at the mRNA and protein level. Statistical analysis of the results showed that the nuclear expression of KAT6A/B correlates with the estrogen receptor status: KAT6ANUC vs. ER r = 0.2373 and KAT6BNUC vs. ER r = 0.1496. Statistical analysis clearly showed that KAT6A cytoplasmic and nuclear expression levels were significantly higher in IDC samples than in FBD samples (IRS 5.297 ± 2.884 vs. 2.004 ± 1.072, p < 0.0001; IRS 5.133 ± 4.221 vs. 0.1665 ± 0.4024, p < 0.0001, respectively). Moreover, we noticed strong correlations between ER and PR status and the nuclear expression of KAT6A and KAT6B (nucKAT6A vs. ER, p = 0.0048; nucKAT6A vs. PR p = 0.0416; nucKAT6B vs. ER p = 0.0306; nucKAT6B vs. PR p = 0.0213). Significantly higher KAT6A and KAT6B expression was found in the ER-positive cell lines T-47D and BT-474, whereas significantly lower expression was observed in the triple-negative cell lines MDA-MB-231 and MDA-MB-231/BO2. The outcomes of small interfering RNA (siRNA)-mediated suppression of KAT6A/B genes revealed that within estrogen receptor (ER) positive and negative cell lines, MCF-7 and MDA-MB-231, attenuation of KAT6A led to concurrent attenuation of KAT6A, whereas suppression of KAT6B resulted in simultaneous attenuation of KAT6A. Furthermore, inhibition of KAT6A/B genes resulted in a reduction in estrogen receptor (ER) mRNA and protein expression levels in MCF-7 and MDA-MMB-231 cell lines. Based on our findings, the lysine acetyltransferases KAT6A and KAT6B may be involved in the progression of invasive ductal breast cancer. Further research on other types of cancer may show that KAT6A and KAT6B could serve as diagnostic and prognostic markers for these types of malignancies.

7961 EMT Plasticity Induced By GRHL2 Overexpression Stimulates Dormancy And Enriches Stem Cell Characteristics In ER+ Breast Cancer Cells

Abstract Disclosure: C. Zheng: None. K.O. Allen: None. T. Liu: None. N.M. Solodin: None. K. Salem: None. A.M. Fowler: None. J. Vera: None. P.K. Tsourkas: None. S. McIlwain: None. M.S. Ozers, PhD: Owner/Co-Owner; Self; Co-Founder and CSO of Proteovista LLC. E.T. Alarid: None. Metastasis is governed by an epithelial to mesenchymal transition (EMT) - the ability for an epithelial cell to adopt a mesenchymal phenotype to promote migration and tumor progression. Though EMT can be articulated as a distinctive transition between its binary states, a cell’s capacity to become plastic, and thus, express a hybrid EMT state with both epithelial and mesenchymal markers has re-defined the literature’s understanding of metastasis. To improve our comprehension of EMT, the nuclear transcription factor responsible for driving epithelial cell fate Grainyhead-like protein 2 (GRHL2) became the protein of interest as clinical evidence suggests high GRHL2 expression is indicative of poorer prognosis in breast cancer (BC). A tetracycline-inducible GRHL2 overexpression model in the estrogen receptor (ER) positive BC MCF7 cell line was developed to understand GRHL2’s role in EMT. Surprisingly, overexpression of the EMT suppressor GRHL2 induces EMT plasticity in ER-positive BC with increased E-cadherin and vimentin expression assessed by immunocytochemistry and mRNA expression. This GRHL2 overexpression model was used to further explore the dynamic nature of EMT plasticity. Gene expression analyses was performed to assess representative genes associated with a partial EMT in purified populations of GRHL2 overexpressing cells. Gene set enrichment analysis (GSEA) determined statistically significant hallmark pathways associated with GRHL2 overexpression. Isolated RNA of GRHL2 overexpressing cells revealed increased mRNA expression of p27 and NR2F1 via RT-qPCR, consistent with a dormancy phenotype. Interestingly, mammosphere and flow cytometry further expanded on the dynamic states of EMT plasticity by emphasizing on the enrichment of stem cell characteristics with GRHL2 overexpression. Extensive GRHL2 overexpression provides cells self-renewal capacity, as examined by mammosphere formation efficiency. Flow cytometry for stem cell markers CD44 and ALDH1 expression demonstrates the increased representation of stem cell markers in EMT plastic populations. To evaluate how GRHL2 overexpression influences tumor progression in vivo, immunocompromised mice were injected with GRHL2 overexpressing cells to examine tumor growth and the potential of cells to create micro-metastases using IVIS. Tumor growth responses differed between parental and GRHL2 overexpressing xenografts, with increased GRHL2 expression decreasing tumor growth. mRNA expression of harvested tumors mirrored in vitro expression of p27 and NR2F1. Together, these data indicates that GRHL2 overexpression induces EMT plasticity, in turn stimulating dormancy and stem-cell acquisition in vitro and in vivo, allowing hybrid cells the ability to adjust to its environment. Presentation: 6/3/2024

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&amp;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

USP36 promotes tumorigenesis and tamoxifen resistance in breast cancer by deubiquitinating and stabilizing ERα

BackgroundBreast cancer is the most prevalent cancer in women globally. Over-activated estrogen receptor (ER) α signaling is considered the main factor in luminal breast cancers, which can be effectively managed with selective estrogen receptor modulators (SERMs) like tamoxifen. However, approximately 30–40% of ER + breast cancer cases are recurrent after tamoxifen therapy. This implies that the treatment of breast cancer is still hindered by resistance to tamoxifen. Recent studies have suggested that post-translational modifications of ERα play a significant role in endocrine resistance. The stability of both ERα protein and its transcriptome is regulated by a balance between E3 ubiquitin ligases and deubiquitinases. According to the current knowledge, approximately 100 deubiquitinases are encoded in the human genome, but it remains unclear which deubiquitinases play a critical role in estrogen signaling and endocrine resistance. Thus, decoding the key deubiquitinases that significantly impact estrogen signaling, including the control of ERα expression and stability, is critical for the improvement of breast cancer therapeutics.MethodsWe used several ER positive breast cancer cell lines, DUB siRNA library screening, xenograft models, endocrine-resistant (ERα-Y537S) model and performed immunoblotting, real time PCR, RNA sequencing, immunofluorescence, and luciferase activity assay to investigate the function of USP36 in breast cancer progression and tamoxifen resistance.ResultsIn this study, we identify Ubiquitin-specific peptidase 36 (USP36) as a key deubiquitinase involved in ERα signaling and the advancement of breast cancer by deubiquitinases siRNA library screening. In vitro and in vivo studies showed that USP36, but not its catalytically inactive mutant (C131A), could promote breast cancer progression through ERα signaling. Conversely, silencing USP36 inhibited tumorigenesis. In models resistant to endocrine therapy, silencing USP36 destabilized the resistant form of ERα (Y537S) and restored sensitivity to tamoxifen. Molecular studies indicated that USP36 inhibited K48-linked polyubiquitination of ERα and enhanced the ERα transcriptome. It is interesting to note that our results suggest USP36 as a novel biomarker for treatment of breast cancer.ConclusionOur study revealed the possibility that inhibiting USP36 combined with tamoxifen could provide a potential therapy for breast cancer.

Impact of steroid hormone levels on estradiol-mediated regulation of cytochrome P450 2B6 compared to 1B1 in breast cancer cells.

Pharmacogenetic variants of the steroid hormone-metabolizing enzyme cytochrome P450 2B6 (CYP2B6) were reported to be associated with breast cancer (BC) risk and prognosis. CYP2B6 expression is inducible by estradiol (E2) but induction was demonstrated only under steroid hormone-deprived medium conditions. Physiological conditions, however, even under endocrinological BC treatment, do not correspond to complete steroid hormone depletion. The aim of this study was to investigate the E2-mediated CYP2B6 and CYP1B1 regulation under various steroid hormone conditions, including physiological concentrations, in human oestrogen receptor positive (T47D, MCF-7) and negative (MDA-MB-231) BC cell lines. We confirm that steroid-deprived pre-cultivation led to CYP2B6 upregulation in T47D, but not in MCF-7. However, when pre-cultivated with steroid-containing medium CYP2B6 was downregulated in T47D and MCF-7, while the addition of physiological E2 concentrations to steroid-deprived medium resulted in a downregulation in T47D. In contrast, CYP1B1 was never downregulated in any culture condition. Thus, we show that E2-mediated CYP2B6 regulation in BC cells depends on steroid hormone exposure in a cell line-specific manner. Our data indicates the importance of being careful with conclusions drawn from CYP2B6 induction findings in vitro, as we demonstrate potential influences of hormonal changes on CYP2B6 expression, which could impact steroid hormone homeostasis and, consequently, BC risk.

Combining In Vitro, In Vivo, and Network Pharmacology Assays to Identify Targets and Molecular Mechanisms of Spirulina-Derived Biomolecules against Breast Cancer.

The current research employed an animal model of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary gland carcinogenesis. The estrogen receptor-positive human breast adenocarcinoma cell line (MCF-7) was used for in vitro analysis. This was combined with a network pharmacology-based approach to assess the anticancer properties of Spirulina (SP) extract and understand its molecular mechanisms. The results showed that the administration of 1 g/kg of SP increased the antioxidant activity by raising levels of catalase (CAT) and superoxide dismutase (SOD), while decreasing the levels of malonaldehyde (MDA) and protein carbonyl. A histological examination revealed reduced tumor occurrence, decreased estrogen receptor expression, suppressed cell proliferation, and promoted apoptosis in SP protected animals. In addition, SP disrupted the G2/M phase of the MCF-7 cell cycle, inducing apoptosis and reactive oxygen species (ROS) accumulation. It also enhanced intrinsic apoptosis in MCF-7 cells by upregulating cytochrome c, Bax, caspase-8, caspase-9, and caspase-7 proteins, while downregulating Bcl-2 production. The main compounds identified in the LC-MS/MS study of SP were 7-hydroxycoumarin derivatives of cinnamic acid, hinokinin, valeric acid, and α-linolenic acid. These substances specifically targeted three important proteins: ERK1/2 MAPK, PI3K-protein kinase B (AKT), and the epidermal growth factor receptor (EGFR). Network analysis and molecular docking indicated a significant binding affinity between SP and these proteins. This was verified by Western blot analysis that revealed decreased protein levels of p-EGFR, p-ERK1/2, and p-AKT following SP administration. SP was finally reported to suppress MCF-7 cell growth and induce apoptosis by modulating the PI3K/AKT/EGFR and MAPK signaling pathways suggesting EGFR as a potential target of SP in breast cancer (BC) treatment.

TRPV1-Dependent Antiproliferative Activity of Dioecious Maclura pomifera Extracts in Estrogen Receptor-Positive Breast Cancer Cell Lines Involves Multiple Apoptotic Pathways.

Globally, breast cancer is a significant cause of mortality. Recent research focused on identifying compounds regulating the transient receptor potential vanilloid 1 (TRPV1) ion channel activity for the possibility of developing cancer therapeutics. In this study, the antiproliferative properties and mechanisms of action through TRPV1 of Maclura pomifera, a dioecious tree native to the south-central USA, have been investigated. Male and female extracts of spring branch tissues and leaves (500 µg/mL) significantly reduced the viability of MCF-7 and T47D cells by 75-80%. M. pomifera extracts induced apoptosis by triggering intracellular calcium overload via TRPV1. Blocking TRPV1 with the capsazepine antagonist and pretreating cells with the BAPTA-AM chelator boosted cell viability, revealing that M. pomifera phytochemicals activate TRPV1. Both male and female M. pomifera extracts initiated apoptosis through multiple pathways, the mitochondrial, ERK-induced, and endoplasmic reticulum-stress-mediated apoptotic pathways, demonstrated by the expression of activated caspase 3, caspase 9, caspase 8, FADD, FAS, ATF4, and CHOP, the overexpression of phosphorylated PERK and ERK proteins, and the reduction of BCL-2 levels. In addition, AKT and pAKT protein expressions were reduced in female M. pomifera-treated cells, revealing that female plant extract also inhibits PI3K/Akt signaling pathways. These results suggest that phytochemicals in M. pomifera extracts could be promising for developing breast cancer therapeutics.

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 PO1-24-04: Gedatolisib, a pan-PI3K/mTOR inhibitor, shows superior potency and efficacy relative to other PI3K/AKT/mTOR pathway inhibitors in breast cancer models

Abstract Background: The PI3K, AKT, and mTOR (PAM) pathway is frequently activated in breast cancer (BC). Current standard-of-care therapy options for patients with advanced BC (ABC) include PAM inhibitors (PAMi), such as everolimus and alpelisib, and hormonal therapy, such as letrozole and fulvestrant. Most PAMi selectively inhibit one or only a few PAM pathway components, which can lead to drug resistance. To minimize drug resistance, a more comprehensive inhibition of the multiple PI3K isoforms and downstream mTOR complexes may be required. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, as well as mTORC1 and mTORC2, can be more effective in BC cells than a PAMi targeting single PAM pathway components. Methods: A panel of BC cell lines with mutated or non-mutated PAM pathway genes (PIK3CA, PTEN, AKT) were evaluated for their sensitivity to gedatolisib and other PAMi (PI3K-α: alpelisib; AKT: capivasertib; mTORC1: everolimus; pan-PI3K/mTOR: gedatolisib). Cell viability, growth rate inhibition, and cell death were evaluated by luciferase- and fluorescence-based assays, both in monolayer cultures and three-dimensional (3D) cultures on reconstituted basement membrane. Flow cytometry analytical assays were conducted to assess DNA synthesis (EdU incorporation), protein synthesis (OPP incorporation), and PAM pathway activity (RPS6 and 4EBP1 phosphorylation). BC xenograft studies evaluating gedatolisib in vivo were also performed. Results: Gedatolisib strongly inhibited PAM pathway activity and reduced cell viability and growth rate in the cell lines tested. Compared to the other PAMi, gedatolisib exhibited more potent and efficacious anti-proliferative and cytotoxic effects, regardless of the cell lines’ PI3K pathway mutational status. Gedatolisib effects were confirmed in 3D culture on reconstituted basement membrane, where gedatolisib inhibited tumor cell spheroid growth and induced regression. Mechanistically, gedatolisib decreased both DNA and protein synthesis more effectively than the other PAMi tested. Gedatolisib also exerted superior anti-proliferative and cytotoxic effects relative to the other PAMi tested in estrogen receptor-positive BC cell lines concomitantly treated with fulvestrant. BC xenografts confirmed growth inhibitory effects of gedatolisib in vivo. Conclusions: The pan-PI3K/mTOR inhibitor, gedatolisib, may more effectively address potential drug resistance mechanisms associated with narrowly targeted PAM inhibitors. Gedatolisib has previously demonstrated promising preliminary clinical efficacy and safety data in ABC in combination with hormonal therapy. A Phase 3 study (VIKTORIA-1) evaluating gedatolisib plus fulvestrant with and without palbociclib is underway in patients with ABC. Citation Format: Stefano Rossetti, Aaron Broege, Adrish Sen, Salmaan Khan, Ian MacNeil, Jhomary Molden, Igor Gorbatchevsky, Brian F. Sullivan, Lance Laing. Gedatolisib, a pan-PI3K/mTOR inhibitor, shows superior potency and efficacy relative to other PI3K/AKT/mTOR pathway inhibitors in breast cancer models [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 PO1-24-04.

Synthesis, biological evaluation and in-silico ADMET studies of novel chalcones and dihydro pyrazoles of 2H-Chromene as anticancer agents

2H-chromenes and chalcones are biologically important scaffolds with applications in drug discovery. Their ease of synthesis and biological importance have attracted the attention of medicinal chemists worldwide. Inspired by this, we synthesized various substituted chalcones and dihydropyrazole derivatives bearing various aryl and hetero-aryl 2H-chromene moieties. The resulting compounds were evaluated for cytotoxicity in vitro against doxorubicin in triple-negative breast cancer cell lines (MDA-MB-231), estrogen receptor-positive breast cancer cell lines (MCF-7), and human brain glioblastoma (U-87 MG) cell lines. Compounds 6b and 6j exhibited anticancer activity against triple-negative breast cancer cell line (MDA-MB-231), with IC50 values of 4.02 µM and 3.35 µM, respectively. Further investigation involving in silico pharmacokinetic (ADME) and toxicity studies also revealed pivotal features for the favourable bioavailability of these derivatives.

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.

Effects of targeting highly expressed in cancer protein 1 (Hec1) inhibitor INH 1 in breast cancer cell lines In Vitro.

In the present study, the in vitro antiproliferative effect of targeting highly expressed cancer protein 1 (Hec1) inhibitor INH1 was investigated in estrogen receptor-positive MCF-7 cell line originating from an in situ carcinoma and triple negative MDA-MB-231 cell line originating from metastatic carcinoma. Cell viability, xCELLigence RTCA DP instrument CI values, MI, BrdU proliferation assay, and AI analyses were employed for this purpose. According to the findings of the current study, INH1 altered cell proliferation by lowering cell viability, CI, MI values, and BrdU proliferation while raising AI values in both cell lines. Between the experimental and control groups, there were noticeable changes (p<0.05). These findings imply that INH1's mode of action is not dependent on the presence of estrogen receptors, making it a potentially effective therapy for breast cancer.

Nuclear isoform of RAPH1 interacts with FOXQ1 to promote aggressiveness and radioresistance in breast cancer

Radioresistance limits the efficacy of radiotherapy against breast cancer, especially the most lethal subtype of breast cancer, triple-negative breast cancer (TNBC). Epithelial-to-mesenchymal transition (EMT) is closely related to tumor radioresistance. In this work, we attempted to identify the key EMT-related transcription factor(s) that can induce radioresistance in breast cancer cells. A set of 44 EMT transcription factors were analyzed in parental and radioresistant TNBC cell lines. The function of FOXQ1, a differentially expressed transcription factor, was determined in TNBC radioresistance. FOXQ1-interacting proteins were identified by co-immunoprecipitation and mass spectrometry. Compared with parental cells, FOXQ1 was significantly upregulated in radioresistant TNBC cells. Silencing of FOXQ1 increased the radiosensitiviy of radioresistant TNBC cells both in vitro and in vivo. FOXQ1 associated with a nuclear isoform of RAPH1 (named RAPH1-i3) in radioresistant TNBC cells. Overexpression of RAPH1-i3 enhanced TNBC cell proliferation and migration, and most interestingly, induced radioresistance in parental TNBC cells when co-expressed with FOXQ1. Similar findings were observed in estrogen receptor-positive breast cancer cell lines that had co-expression of RAPH1-i3 and FOXQ1. Mechanistically, co-expression of RAPH1-i3 and FOXQ1 activated STAT3 signaling and increased the expression of CCND1, MCL1, Bcl-XL, and MMP2. Depletion of RAPH1-i3 impaired the radioresistance of radioresistant TNBC cells. Additionally, RAPH1-i3 upregulation was associated with advanced tumor stage and reduced disease-free survival in TNBC patients. These results collectively show that RAPH1-i3 interacts with FOXQ1 to promote breast cancer progression and radioresistance. RAPH1-i3 and FOXQ1 represent therapeutic targets for the treatment of breast cancer including TNBC.

Downregulation of MALAT1 in triple-negative breast cancer cells

BackgroundMALAT1 is one of the most abundant nuclear long non-coding RNAs, which has been found to be elevated in various types of cancers. However, conflicting reports on MALAT1 in breast cancer cell lines challenge understanding of MALAT1's involvement in breast cancer progression. AimMeasurement of normalized relative quantity (NRQ) of MALAT1 transcripts in cell lines representing triple-negative breast cancer (TNBC) and luminal breast cancer. Materials and methodsThe studies were performed using cell lines representing luminal breast cancer (T47D, MCF-7), TNBC (MDA-MB-468, CAL-51, MDA-MB-231), and MCF-10A cell line of normal breast epithelial cells. Total RNA was isolated from six independent cell cultures of each line, treated with DNase I, and used to synthesize complementary DNA, which was used in quantitative real-time PCR (qPCR) assays. Four MALAT1 fragments and reference genes CCSER2, ANKRD17, PUM1, GAPDH were amplified. ResultsGeometric means of the NRQ of MALAT1 in breast cancer cell lines had the shortest 95% confidence intervals when CCSER2 was used for normalization. MALAT1 major transcript levels thus estimated in TNBC cell lines were found to be statistically significantly reduced compared to levels in both MCF-10A cells and luminal breast cancer cell lines, while MALAT1 minority splice variants were found to be increased in almost all breast cancer cell lines. ConclusionCCSER2-normalized qPCR results indicate MALAT1 downregulation in cell lines representing the more aggressive breast cancer subtype compared to both the normal breast epithelial cell line and the estrogen receptor-positive breast cancer cell lines.

Orai1α and Orai1β support calcium entry and mammosphere formation in breast cancer stem cells

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1β, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1β support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1β in spheroid forming efficiency and self-renewal in breast cancer stem cells.

The TGFBI gene and protein expression in topotecan resistant ovarian cancer cell lines

PurposeThe primary limiting factor in achieving cures for patients with cancer, particularly ovarian cancer, is drug resistance. The mechanisms of drug resistance of cancer cells during chemotherapy may include compounds of the extracellular matrix, such as the transforming growth factor-beta-induced protein (TGFBI). In this study, we aimed to analyze the TGFBI gene and protein expression in different sensitive and drug-resistant ovarian cancer cell lines, as well as test if TGFBI can be involved in the response to topotecan (TOP) at the very early stages of treatment. Materials and methodsIn this study, we conducted a detailed analysis of TGFBI expression in different ovarian cancer cell lines (A2780, A2780TR1, A2780TR2, W1, W1TR, SKOV-3, PEA1, PEA2 and PEO23). The level of TGFBI mRNA (QPCR), intracellular and extracellular protein (Western blot analysis) were assessed in this study. ResultsWe observed upregulation of TGFBI mRNA in drug-resistant cell lines and estrogen-receptor positive cell lines, which was supported by overexpression of both intracellular and extracellular TGFBI protein. We also showed the TGFBI expression after a short period of treatment of sensitive ovarian cancer cell lines with TOP. ConclusionThe expression of TGFBI in ovarian cancer cell lines suggests its role in the development of drug resistance.

High-Affinity Extended Bisphosphonate-Based Coordination Polymers as Promising Candidates for Bone-Targeted Drug Delivery.

Extended bisphosphonate-based coordination polymers (BPCPs) were produced when 1,1'-biphenyl-4,4'-bisphosphonic acid (BPBPA), the analogue of 1,1'-biphenyl-4,4'-dicarboxylic acid (BPDC), reacted with bioactive metals (Ca2+, Zn2+, and Mg2+). BPBPA-Ca (11 Å × 12 Å), BPBPA-Zn (10 Å × 13 Å), and BPBPA-Mg (8 Å × 11 Å) possess channels that allow the encapsulation of letrozole (LET), an antineoplastic drug that combined with BPs treats breast-cancer-induced osteolytic metastases (OM). Dissolution curves obtained in phosphate-buffered saline (PBS) and fasted-state simulated gastric fluid (FaSSGF) demonstrate the pH-dependent degradation of BPCPs. Specifically, the results show that the structure of BPBPA-Ca is preserved in PBS (∼10% release of BPBPA) and collapses in FaSSGF. Moreover, the phase inversion temperature nanoemulsion method yielded nano-Ca@BPBPA (∼160 d. nm), a material with measurably higher (>1.5x) binding to hydroxyapatite than commercial BPs. Furthermore, it was found that the amounts of LET encapsulated and released (∼20 wt %) from BPBPA-Ca and nano-Ca@BPBPA are comparable to those of BPDC-based CPs [i.e., UiO-67-(NH2)2, BPDC-Zr, and bio-MOF-1], where other antineoplastic drugs have been loaded and released under similar conditions. Cell viability assays show that, at 12.5 μM, the drug-loaded nano-Ca@BPBPA exhibits higher cytotoxicity against breast cancer cells MCF-7 and MDA-MB-231 [relative cell viability (%RCV) = 20 ± 1 and 45 ± 4%] compared with LET (%RCV = 70 ± 1 and 99 ± 1%). At this concentration, no significant cytotoxicity was found for the hFOB 1.19 cells treated with drug-loaded nano-Ca@BPBPA and LET (%RCV = 100 ± 1%). Collectively, these results demonstrate the potential of nano-Ca@BPCPs as promising drug-delivery systems to treat OM or other bone-related diseases because these present measurably higher affinity, allowing bone-targeted drug delivery under acidic environments and effecting cytotoxicity on estrogen receptor-positive and triple-negative breast cancer cell lines known to induce bone metastases, without significantly affecting normal osteoblasts at the metastatic site.

Chayote leaves display seasonal variation in phytoestrogenic activities and phenolic content

Chayote (Sicyos edulis Jacq.) is a popular vegetable in the Philippines and has been recognized for its nutritional value and bioactive compounds, including phenolic acids found in its edible parts. Hence, this study sought to determine whether extracts from S. edulis shoots can elicit a bioactive estrogenic response. Specifically, the study determined whether the location of cultivation or monsoon season of collection would influence the estrogenic activities of S. edulis extracts. Here, we found that a specific fraction (F5.6) of S. edulis shoots isolated through solid-phase extraction (C-18 and HLB) inhibited the transactivation of an estrogen-induced luciferase reporter gene in an estrogen receptor-positive cell line T47D without affecting cell viability, independently of the farm source and the season of sample collection. This inhibition was also seen in the E2- induced expression of superoxide dismutase 2 (SOD2), an antioxidant enzyme upregulated in advanced breast cancer cells. In contrast, the phenolic content in the F5.6 fraction varied across seasons, with a higher content found during the northeast monsoon season (0.175 ± 0.027 mg GAE/mg sample) than the southwest monsoon seasons (0.043 ± 0.015 and 0.051 ± 0.012 mg GAE/mg sample). The high phenolic content increased the ratio of estrogen receptor alpha to beta transcripts and the mRNA expression level of the estrogen-responsive gene, growth regulation by estrogen in breast cancer 1 (GREB1A), in T47D cells. Overall, this study showed how seasonal variation may affect the phenolic content of S. edulis and influence the plant’s bioactivity, providing a potential cultivation strategy in obtaining extracts with the intended bioactivity.

Abstract 3696: L-arginine depletion causes different proliferation responses in breast cancer subtypes - Relation to gene expression profiles of L-arginine metabolic pathways

Abstract Metabolic fingerprinting provides insight into cancer biology, improving personalized therapeutic strategies. We sought to unravel responses of the molecular subtypes of breast cancer to L-arginine depletion in vitro. Availability of the semi-essential amino acid L-arginine limits protein synthesis and cell proliferation. L-Arginine is the substrate for nitric oxide synthases (NOS) and arginases (ARG), enzymes also involved in the regulation of cell proliferation. Clinical studies have provided contrasting results regarding the role of L-arginine in breast cancer.We studied the response of different breast cancer cell lines to L-arginine depletion and the molecular mechanisms underlying this effect. MCF-7, SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 cells were cultured under standard conditions and compared to MCF-12A normal breast epithelial cells. Cells were maintained in culture media with 200 mg/L (control) or 0 mg/L of L-arginine (depletion) for up to 240 h. Cell proliferation was analyzed and intracellular concentrations of L-arginine, L-ornithine, and L-citrulline were measured by UPLC-MS/MS every 24 h. mRNA expression of genes involved in L-arginine metabolism was quantified by qRT-PCR. MCF-12A control cells did not proliferate in the absence of extracellular L-arginine. Their intracellular L-arginine content dropped sharply, whilst L-ornithine and L-citrulline were only slightly reduced. By contrast, proliferation of BT-474, SK-BR-3, and MDA-MB-468 cells was unaffected by L-arginine depletion (p&amp;lt;0.05), whilst MCF-7 and MDA-MB-231 cells showed significantly higher proliferation rates in the absence of L-arginine (p&amp;lt;0.05). L-arginine concentration dropped sharply within 24 h in MCF-7 cells, but only by 25-50% in the other cell lines (p&amp;lt;0.01). L-ornithine dropped to almost zero within 24 h in both estrogen receptor positive cell lines (MCF-7 and BT-474), whilst being only slightly reduced in SK-BR-3 cells (p&amp;lt;0.01). L-ornithine was not significantly reduced in MDA-MB-231, but strongly reduced in MDA-MB-468 cells (p&amp;lt;0.05). L-citrulline showed no significant change in any of the cell lines. mRNA expression of NOS, ARG, and the L-arginine salvage pathway (ASS1 and ASL) was differentially expressed amongst the cell lines. SK-BR-3 showed high expression of the L-arginine salvage pathway; BT-474 had the highest expression levels of NOS and ARG2, and MDA-MB had high ARG2 but very low NOS expression. Gene expression patterns of MCF-12A and MCF-7 were very similar. In conclusion, metabolic fingerprinting reveals complex and differential regulation of L-arginine pathways in breast cancer cell lines. These analyses may help to understand differences in proliferation during L-arginine depletion, thus offering a perspective to further personalize treatment options within classical molecular breast cancer subtypes. Citation Format: Juliane Hannemann, Fiona Franke, Mariola Kastner, Rainer Böger. L-arginine depletion causes different proliferation responses in breast cancer subtypes - Relation to gene expression profiles of L-arginine metabolic pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3696.

Abstract 3789: Circular RNA expression is enriched in breast cancer extracellular vesicles and is associated with chemotherapy resistance

Abstract Up to 90% of failures in chemotherapy treatment are associated with metastatic-related drug resistance. Extracellular vesicles (EVs) are believed to play a role in the spread of chemotherapy resistance through the horizontal transfer of oncoproteins and noncoding RNA species, but the underlying mechanisms for this are not fully understood. Here, we study EV biogenesis, release, and uptake in the context of breast cancer chemoresistance. Specifically, we characterized the bioactive cargoes in EVs secreted by chemoresistant triple negative breast cancer cells and chemoresistant estrogen receptor-positive breast cancer cell lines. Preliminary data show that these EVs contain unusually high levels of circular RNAs, small RNA species formed covalently through backsplicing and are typically noncoding. When chemosensitive cells absorb these circRNA-enriched EVs, they experience a significant increase in resistance phenotypes. Moreover, knockdown of the circRNAs via antisense oligonucleotides results in decreased chemoresistance, suggesting a role of circRNAs in the development of chemotherapy resistance. Previous studies have shown that tumor-derived exosomal circRNAs may confer resistance by sponging microRNAs. We are exploring what the circRNAs do mechanistically to promote tumor survival, and clarifying these mechanisms as a whole will serve to potentially provide therapeutic targets against chemotherapy resistance and cancer recurrence. Citation Format: Harrison Ngue, Jitendra Shrestha, Hyejin Kim, Shobha Vasudevan. Circular RNA expression is enriched in breast cancer extracellular vesicles and is associated with chemotherapy resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3789.