Estrogen-dependent Breast Cancer Research Articles

ROS-mediated activation and mitochondrial translocation of CaMKII contributes to Drp1-dependent mitochondrial fission and apoptosis in triple-negative breast cancer cells by isorhamnetin and chloroquine

BackgroundTriple-negative breast cancer (TNBC) is often aggressive and associated with a poor prognosis. Due to the lack of available targeted therapies and to problems of resistance with conventional chemotherapeutic agents, finding new treatments for TNBC remains a challenge and a better therapeutic strategy is urgently required.MethodsTNBC cells and xenograft mice were treated with a combination of chloroquine (CQ) and isorhamnetin (IH). Mitochondrial fission, apoptosis, and related signaling pathways were determined by flow cytometry, immunofluorescence, and related molecular biological techniques.ResultsThe inhibition of autophagy/mitophagy by CQ selectively enhances IH-induced mitochondrial fission and apoptosis in TNBC cells but not in estrogen-dependent breast cancer cells. These events were accompanied by mitochondrial translocation of Bax and the release of cytochrome c. Mechanistically, these effects were associated with oxidative stress-mediated phosphorylation of CaMKII (Thr286) and Drp1 (S616), and subsequent mitochondrial translocation of CaMKII and Drp1. The interruption of the CaMKII pathway by genetic approaches (e.g. CaMKII mutant or siRNA) attenuated combination-mediated mitochondrial fission and apoptosis. The combination of CQ/IH was a marked inhibitor tumor growth, inducing apoptosis in the TNBC xenograft mouse model in association with the activation of CaMKII and Drp1 (S616).ConclusionsOur study highlights the critical role of ROS-mediating CaMKII/Drp1 signaling in the regulation of mitochondrial fission and apoptosis induced by combination of CQ/IH. These findings also suggest that IH could potentially be further developed as a novel chemotherapeutic agent. Furthermore, a combination of IH with classic autophagy/mitophagy inhibitor could represent a novel therapeutic strategy for the treatment of TNBC.

CC chemokine ligand 2 (CCL2) stimulates local aromatase expression in mammary adipose tissue via MAPK/ERK signaling pathway

Obesity is an important risk factor for the most common type of breast cancer, i.e., estrogen‐dependent postmenopausal breast cancer. Obesity‐related inflammation, defined by enhanced tissue macrophage infiltration and increased levels of proinflammatory factors, stimulates estrogen production and, in turn, promotes tumor formation and progression. The augmented inflammatory milieu activating aromatase expression, the rate‐limiting enzyme for estrogen synthesis, is suggested to be the major mechanism behind the increased risk for breast cancer. The regulation of aromatase (CYP19A1) gene expression in breast tissue is complex and not fully understood. This study focuses on the role of CCL2, a macrophage recruiter often overexpressed in the obese and cancerous breast tissue, on the aromatase expression in mammary adipose tissue. We utilized mammary adipose tissue explants obtained from female hARO‐Luc aromatase reporter mice and cultured primary human breast adipose stromal cells (hASCs). In tissue explants, aromatase expression was measured by quantification of luciferase reporter activity, while in cultured hASCs, qPCR was used to examine expression of CYP19A1 and its specific promoter I.4 and II transcripts. Both, in mammary adipose tissue and in hASCs, CCL2 induced aromatase expression via the distal promoter I.4. Moreover, inhibitors of CCL2 receptor and MAPKs pathway, ERK1/2, blocked CCL2–induced CYP19A1 and PI.4 mRNA expression. In conclusion, these findings indicate that CCL2, an intrinsic pro‐inflammatory chemokine in adipose tissue, may stimulate aromatase expression and play an important role in controlling estrogen biosynthesis in the breast adipose stroma.Support or Funding InformationThis research is supported by the University of Turku Graduate School, the Finnish Cultural Foundation, the Turun yliopistosäätiö and the Institute of Biomedicine, University of TurkuThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

County poverty levels influence genome-wide DNA methylation profiles in African American and European American women.

Our pilot study examined global DNA methylation and telomere length (TL) using DNA from saliva samples provided by 39 participants in the Arkansas Rural Community Health (ARCH) Study. TL was quantified by qPCR, and DNA methylation and DNA methylation age was assessed using the Illumina 850K Epic BeadChip. Ingenuity Pathway Analysis (IPA) was performed to identify biological pathways that were DM between residents of counties with high or low poverty rates and by race [African American descent (AA) versus European American (EA) descent]. Among AA women, hypermethylation was more common in AA residents of counties with low compared to high poverty rates (70% vs. 30%). The top canonical pathways impacted by differential methylation were related to glucocorticoid receptor, p53, and estrogen-dependent breast cancer signaling in AA women. EA women living in low-poverty counties exhibited less hypermethylation of CpGs than those living in high-poverty counties (27% vs. 73%). The top canonical pathways were related to hereditary breast cancer, glucocorticoid receptor, androgen and PI3K/AKT signaling. Several genes involved in telomere maintenance were shown to be DM by county poverty levels. Therefore, the finding of this pilot study suggests county poverty levels may impact DNA methylation patterns in breast cancer-related pathways as well as genes involved in telomere maintenance. Larger studies should confirm our findings.

Molecular Mechanisms of Polybrominated Diphenyl Ethers (BDE-47, BDE-100, and BDE-153) in Human Breast Cancer Cells and Patient-Derived Xenografts.

Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants in household materials. Their environmental persistence has led to continuous human exposure and significant tissue levels. Three PBDE congeners (BDE-47, BDE-100, and BDE-153) have been frequently detected in human serum. Although these compounds appear to possess endocrine disrupting activity, studies are largely missing to determine the biological mechanisms of PBDEs in breast cancer cells. Here, we assessed PBDE bioactivities with three complementary strategies: receptor binding/activity assays; nonbiased RNA-sequencing analysis using an estrogen-dependent breast cancer cell line MCF-7aroERE; and in vivo assessments using patient-derived xenograft (PDX) models of human breast cancer. According to the results from in vitro experiments, the PBDE congeners regulate distinct nuclear receptor signaling pathways. BDE-47 acts as a weak agonist of both estrogen receptor α (ERα) and estrogen-related receptor α (ERRα); it could stimulate proliferation of MCF-7aroERE and induced expression of ER-regulated genes (including cell cycle genes). BDE-153 was found to act as a weak antagonist of ERα. BDE-100 could act as (1) an agonist of aryl hydrocarbon receptor (AhR), inducing expression of CYP1A1 and CYP1B1 and (2) as a very weak agonist/antagonist of ERα. In vivo, a mixture of the three congeners with ratios detected in human serum was tested in an ER+ PDX model. The mixture exhibited estrogenic activity through apoptosis/cell cycle regulation and increased the expression of a proliferation marker, Ki-67. These results advance our understanding of the mechanisms of PBDE exposure in breast cancer cells.

Tumor-Associated Macrophages Induce Endocrine Therapy Resistance in ER+ Breast Cancer Cells.

Antiestrogenic adjuvant treatments are first-line therapies in patients with breast cancer positive for estrogen receptor (ER+). Improvement of their treatment strategies is needed because most patients eventually acquire endocrine resistance and many others are initially refractory to anti-estrogen treatments. The tumor microenvironment plays essential roles in cancer development and progress; however, the molecular mechanisms underlying such effects remain poorly understood. Breast cancer cell lines co-cultured with TNF-α-conditioned macrophages were used as pro-inflammatory tumor microenvironment models. Proliferation, migration, and colony formation assays were performed to evaluate tamoxifen and ICI 182,780 resistance and confirmed in a mouse-xenograft model. Molecular mechanisms were investigated using cytokine antibody arrays, WB, ELISA, ChIP, siRNA, and qPCR-assays. In our simulated pro-inflammatory tumor microenvironment, tumor-associated macrophages promoted proliferation, migration, invasiveness, and breast tumor growth of ER+ cells, rendering these estrogen-dependent breast cancer cells resistant to estrogen withdrawal and tamoxifen or ICI 182,780 treatment. Crosstalk between breast cancer cells and conditioned macrophages induced sustained release of pro-inflammatory cytokines from both cell types, activation of NF-κB/STAT3/ERK in the cancer cells and hyperphosphorylation of ERα, which resulted constitutively active. Our simulated tumor microenvironment strongly altered endocrine and inflammatory signaling pathways in breast cancer cells, leading to endocrine resistance in these cells.

TRIGONELLA FOENUM-GRAECUM L. EXHIBITS ESTROGENIC EFFECT THROUGH PRESENILIN 2 GENE EXPRESSION IN THE BREAST CANCER CELL LINE MCF-7

Objective: The objective of this study is to investigate the estrogenic and antiestrogenic activity of Fenugreek seeds, Trigonella foenum-graecum L. in the estrogen-dependent breast cancer cell line, MCF-7, including its effect on the expression of estrogen-dependent presenilin 2 (pS2) gene.Methods: An activity guided fractionation was carried out with extracts from fenugreek seeds in MCF-7 cells. Cytotoxic activity assays were conducted with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Most fractions were also tested also tested in media with estradiol 10 nM We also analysed the expression of pS2 gene. For the analysis of pS2 gene expression we employed PCR primers for pS2 and for β-actin as a housekeeping gene using real-time polymerase chain reaction (RT-PCR).Results: Based on cytotoxic activity assay in MCF-7, the active fractions are ethyl acetic fraction and its phases ethyl acetic (EA) 2 and EA 2.2. The most active fraction was EA 2.2 (IC50=27.129 ppm), which exhibited a biphasic effect; at low concentrations, it stimulated the growth, and at high concentrations it showed strong cytotoxic effects. EA2.2 fraction in concentration 20 ppm, also could induce pS2 gene expression in media with and without estrogen.Conclusion: The most active fraction was the ethyl acetate phase and further subfractions. The most active fraction also induced the expression of pS2 gene which was studied by RT-PCR.

TRIGONELLA FOENUM-GRAECUM L. EXHIBITS ESTROGENIC EFFECT THROUGH PRESENILIN 2 GENE EXPRESSION IN THE BREAST CANCER CELL LINE MCF-7

Objective: The objective of this study is to investigate the estrogenic and antiestrogenic activity of Fenugreek seeds, Trigonella foenum-graecum L. in the estrogen-dependent breast cancer cell line, MCF-7, including its effect on the expression of estrogen-dependent presenilin 2 (pS2) gene.Methods: An activity guided fractionation was carried out with extracts from fenugreek seeds in MCF-7 cells. Cytotoxic activity assays were conducted with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Most fractions were also tested also tested in media with estradiol 10 nM We also analysed the expression of pS2 gene. For the analysis of pS2 gene expression we employed PCR primers for pS2 and for β-actin as a housekeeping gene using real-time polymerase chain reaction (RT-PCR).Results: Based on cytotoxic activity assay in MCF-7, the active fractions are ethyl acetic fraction and its phases ethyl acetic (EA) 2 and EA 2.2. The most active fraction was EA 2.2 (IC50=27.129 ppm), which exhibited a biphasic effect; at low concentrations, it stimulated the growth, and at high concentrations it showed strong cytotoxic effects. EA2.2 fraction in concentration 20 ppm, also could induce pS2 gene expression in media with and without estrogen.Conclusion: The most active fraction was the ethyl acetate phase and further subfractions. The most active fraction also induced the expression of pS2 gene which was studied by RT-PCR.

Estrogen Sulfotransferase Induction Inhibits Breast Cancer Cell Line MCF-7 Proliferation

Estrogens are known for their proliferative effects, resulting in tumorigenesis and causing cancers. The majority of breast cancers are estrogen-dependent. Estrogenb-locking drugs developed for treating estrogen-dependent breast cancers include antagonists of estrogen receptors (ERs) and inhibitors of estrogen synthesis enzymes such as aromatase and steroid sulfatase (STS). However, drugs targeting estrogen inactivation enzyme, estrogen sulfotransferase (SULT1E1), have not been developed for estrogen-dependent cancer treatment or prevention. Estrogens must be inactivated after their actions in vivo by SULT1E1, uncontrolled estrogen activity in certain tissues will cause cancers. The majority of human breast cancer cell lines are known to express very low levels of SULT1E1 compared to normal mammary cells. Therefore, gene up-regulation of SULT1E1 could provide novel possibilities for the treatment of estrogen-dependent breast and endometrial cancers. Our data suggest that certain nutritional flavonoids induce SULT1E1 and inhibit cell proliferation in estrogen-dependent breast cancer MCF-7 cells. Our results also suggest that SULT1E1 inducer has an additive or synergistic effect on the inhibition of MCF-7 cell proliferation when combined with other known breast cancer drugs. Naturally occurring flavonoids are safe and inexpensive; they could be used for the prevention of certain breast cancers and/or used as co-drug for the treatment of estrogen-dependent cancers. These results may lead to creating innovative approaches for the treatment or prevention of estrogen-dependent cancers and may lead to the discovery of novel drugs or co-drugs.

Relation of EGFR/PI3K/AKT signaling components with tamoxifen efficacy in patients with estrogen-dependent breast cancer

Background. It is generally accepted that crosstalk between the growth factor receptor and ER pathways implicated in tamoxifen resistance. The aim of the study was to examine the relationship between mRNA level, protein expression and gene polymorphism of the EGFR/PI3K/AKT signaling components with tamoxifen efficacy in patients with estrogen-dependent breast cancer. Materials and methods. The study included 95 breast cancer patients who had received adjuvant tamoxifen, of which 31 patients developed recurrence/metastasis after tamoxifen treatment (tamoxifen resistance group), 64 patients did not develop any diseases progression (tamoxifen sensitive group) during the 5 years of follow-up. Genotypes for ESR1 (rs2077647, rs2228480, rs1801132), EGFR (rs1468727, rs2227983), AKT1 (rs1130233) and PTEN (rs11202592) were analyzed using a TaqMan assay. Using reverse transcription-PCR, the relative expression of mRNA for ESR1, EGFR, AKT1 and PTEN was determined. ERα, EGFR, Akt (pS473) and PTEN expression level was evaluated using immunohistochemistry. Progression-free survival (PFS) was estimated by Kaplan – Meier analysis. Results. The minor allele of ESR1 rs2077647 was more prevalent in tamoxifen sensitive tumors compared to tamoxifen resistant tumors (p = 0.044). We found high AKT1 mRNA expression level in tamoxifen sensitive group compared with tamoxifen resistance patients (7.27 ± 5.29 and 0.02 ± 0.01, respectively, p = 0.014). ESR1 rs2228480 was significantly associated with tamoxifen resistance (p = 0.028). EGFR and Akt (pS473) protein expression level was significantly higher in the tamoxifen resistance group compared to tamoxifen sensitive breast cancer patients (p = 0.006 and 0.037, respectively). Patients carrying mutant genotypes of ESR1 rs2228480 had a poorer progression-free survival than those carrying wild and heterozygous variants (log rank p = 0.043). Positive EGFR tumor expression as well as positive Akt (pS473) expression were significantly associated with shorter PFS (log rank p = 0.014 and 0.048, respectively). Conclusion. Polymorphic sites of the ESR1 gene, AKT1 mRNA expression, EGFR expression level and Akt (pS473) protein expression can be potential molecular markers associated with tumor sensitivity/resistance to tamoxifen treatment.

Osthole prevents tamoxifen-induced liver injury in mice

Tamoxifen (TMX) is an antiestrogen drug that is used in the treatment and prevention of all stages of estrogen-dependent breast cancer. Adverse effects of TMX include hepatotoxicity. In this study, we investigated the therapeutic effects of osthole, isolated from medicinal plants especially Fructus Cnidii, on TMX-induced acute liver injury in mice. Mice were injected with osthole (100 mg/kg, ip) or vehicle, followed by TMX (90 mg/kg, ip) 24 h later. We showed that a single injection of TMX-induced liver injury and oxidative stress. Pretreatment with osthole attenuated TMX-induced liver injury evidenced by dose-dependent reduction of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Pretreatment with osthole also blunted TMX-induced oxidative stress, evidenced by significant increase of reduced glutathione (GSH) as well as reduction of malondialdehyde (MDA) and hydrogen peroxide (H2O2). Consistently, osthole significantly enhanced the expressions of antioxidant genes (GPX1, SOD2, GCL-c, and G6pdh), but suppressed those of pro-oxidant genes (NOX2 and ACOX). Furthermore, osthole inhibited the production of inflammatory cytokines, reduced the metabolic activation of TMX, and promoted its clearance. We further revealed that osthole elevated hepatic cAMP and cGMP levels, but inhibition of PKA or PKG failed to abolish the hepatoprotective effect of osthole. Meanwhile, prominent phosphorylation of p38 was observed in liver in response to TMX, which was significantly inhibited by osthole. Pretreatment with SB203580, a p38 inhibitor, significantly attenuated TMX-induced increase of ALT and AST activities, reduced oxidative stress, and reversed the alterations of gene expression caused by TMX. Moreover, pretreatment with L-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, partly reversed the effect of osthole on TMX-induced liver injury. Consistently, pretreatment with N-acetyl-L-cysteine (NAC) significantly attenuated TMX-induced increase in ALT and AST activities. Notably, both BSO and NAC had no detectable effect on the phosphorylation levels of p38. Collectively, our results suggest that osthole prevents TMX hepatotoxicity by suppressing p38 activation and subsequently reducing TMX-induced oxidative damage.

COMPARATIVE ANALYSIS OF THE EXOSOMAL CARGO OF THE ESTROGEN-RESISTANT BREAST CANCER CELLS

The exosomes involvement in the pathogenesis of tumors is based on their property to incorporate into the recipient cells resulting in the both genomic and epigenomic changes. Earlier we have shown that exosomes from different types of estrogen-independent breast cancer cells (MCF-7/T developed by long-term tamoxifen treatment, and MCF-7/M) developed by metformin treatment were able to transfer resistance to the parent MCF-7 cells. To elucidate the common features of the both types of resistant exosomes, the proteome and microRNA cargo of the control and both types of the resistant exosomes were analyzed. Totally, more than 400 proteins were identified in the exosome samples. Of these proteins, only two proteins, DMBT1 (Deleted in Malignant Brain Tumors 1) and THBS1 (Thrombospondin-1), were commonly expressed in the both resistant exosomes (less than 5% from total DEPs) demonstrating the unique protein composition of each type of the resistant exosomes. The comparative analysis of the miRNA differentially expressed in the both MCF-7/T and MCF-7/M resistant exosomes revealed 180 up-regulated and 202 down-regulated miRNAs. Among them, 4 up-regulated and 8 down-regulated miRNAs were associated with progression of hormonal resistance of breast tumors. The bioinformatical analysis of 4 up-regulated exosomal miRNAs revealed 2 miRNAs, mir- 101and mir-181b, which up-regulated PI3K signaling supporting the key role of PI3K/Akt in the development of the resistant phenotype of breast cancer cells.

WISDOM survey: attitudes and behaviors of physicians toward vulvar and vaginal atrophy (VVA) treatment in women including those with breast cancer history.

Objective:To evaluate and compare physicians’ behaviors and attitudes regarding vulvar and vaginal atrophy (VVA) treatment in menopausal women, including women with breast cancer, using an internet-based survey.Methods:The WISDOM survey queried obstetricians and gynecologists (OB/GYNs) and primary care physicians (PCPs) with 23 multipart questions assessing behaviors and attitudes towards VVA treatment.Results:Of 2,424 surveys sent, 945 (39%) responded and 644 (27%) were completed. Of the menopausal women seen by OB/GYNs and PCPs, 44% to 55% reported having VVA symptoms. Physicians prescribed VVA treatments primarily because of effectiveness. Only 34% of OB/GYNs and 17% of PCPs felt comfortable prescribing VVA therapies to women with a personal history of breast cancer. In general, the most common VVA treatment recommended by all was prescription therapy (49%; with or without other therapies) in the form of US Food and Drug Administration-approved vaginal estrogen creams. More OB/GYNs (72%) than PCPs (47%) disagreed that VVA was best treated with over the counter than prescription products. Out-of-pocket cost and fear of risks associated with estrogens were believed to be the main barriers for why women choose not to get treated and why they discontinue treatment.Conclusions:More OB/GYNs than PCPs prescribed VVA treatment, especially vaginal estrogens, for menopausal women, but both groups generally had similar attitudes and behaviors regarding VVA treatment. Physician comfort was low when prescribing to women with a history of breast cancer, despite women's health medical societies supporting vaginal estrogen use in women with a history of estrogen-dependent breast cancer who were unresponsive to nonhormonal therapies when offered in consultation with their oncologist.

Synthesis and Antiproliferative Activity of Imidazole and Triazole Derivatives of Flavonoids

Flavones can play a potential role in estrogen dependent breast cancer due to greater reactivity of imidazole and triazole heterocycles which have been investigated in this work. We emphasized on synthesis of flavones derivatives with imidazole (2a, 2b, 2c) and triazole (3a, 3b, 3c) nucleus as a fundamental hetero-aromatic system with modifications, which have been confirmed by TLC, IR, NMR and mass spectrometry data. The synthesized compound were studied as non-steroidal aromatase inhibitors and evaluated for in vitro anti-breast cancer activity against MCF-7 cell line through SRB assay. The triazole derivative 3a with nitro substitution (H-bond accepting group) was found to be more active in comparison to standard drug letrozole.

PO-471 Exosomes and transferring of hormonal resistance of breast cancer cells

IntroductionThe main goal of the present work was the study of the molecular mechanism of acquired hormonal resistance of tumour cells, in particular - the study of the intercellular interactions and exosomes and their role in the progression of the resistance.Material and methodsHormone responsive MCF-7 and hormone resistant MCF-7/T breast cancer cells were cultured in standard DMEM medium supplemented with 10% fetal calf serum. The transcriptional activities of NF-kB and AP-1 were determined by reporter luciferase assays. Then exosomes from cell supernatant were isolated by differential ultracentrifugation using standard protocol.Results and discussionsHere, using the estrogen-dependent MCF-7 breast cancer cells and estrogen-independent MCF-7/T cells we have demonstrated the ability of the resistant cells-derived exosomes to initiate the estrogen-independent growth of the parent MCF-7 cells. The subsequent analysis of the key signalling proteins revealed the ability of the exosomes of the resistant cells to inhibit the oestrogen signalling as well as to stimulate the Akt protein kinase and transcription factors AP-1 and NF-kB in the recipient cells. Importantly, the cell treatment with PI3K/Akt inhibitor wortmannin prevented the exosome-induced resistance giving the additional evidence for central role of this pathway in the mediating of exosomal resistance.The data validation was performed on the resistant subline MCF-7/M which was selected under long-term cultivation of the parent MCF-7 cells with biguanide metformin and demonstrated the cross-resistance to metformin and tamoxifen. We have shown that the exosomes of the resistant MCF-7/M cells lead to the partial resistance of the parent cells to both drugs. Moreover, the acquired resistance was characterised with the analogues signalling rearrangement: inhibition of the oestrogen signalling and activation of the Akt, AP-1 and NF-kB proteins - similar to the described above hormonal resistance. Advanced analysis of the exosomal microRNA revealed 27 microRNA differentially expressed in the exosomes of the resistant cells and associated with the progression of hormonal resistance.ConclusionTotally, we demonstrated the new mechanism of horizontal transferring of hormonal resistance by exosomes, identified the possible intercellular targets of exosomes and revealed the main features of the exosomal transcriptome. This study was supported by Russian Science Foundation, grant 14-15-00362 M.K.

Abstract 4121: Differences in optoacoustic signal reflect different characteristics of the vasculature and response to antiangiogenic therapy in breast cancer models

Abstract Optoacoustic imaging has the potential to monitor tumour neo-vascularization, essential for disease progression and also a target for therapy. While promising in some settings, anti-angiogenic therapy has yet to fulfil its promise and mechanisms of resistance are poorly understood. In breast cancer, for example, resistance has been linked to hormonal status, oxygen consumption and alternative mechanisms of neo-vasculogenesis, including vascular mimicry (VM). Here, we use two mouse xenograft models: estrogen-dependent MCF-7 and estrogen independent MDA-MB-231. We analyse their characteristics in vivo using Multispectral optoacoustic tomography (MSOT) to detect oxy- and deoxy- haemoglobin (Hb) during tumour growth. We also analysed their histological and biochemical characteristics for blood vessel density (CD31) and maturity (aSMA, PAS staining), tumour hypoxia (CAIX), angiogenesis, inflammation and VM. Anti-angiogenic therapy response was also tested using Bevacizumab (BV, 10 mg/Kg) in two cohorts. While MSOT reveals similar blood content in both tumours, estrogen-dependent MCF-7 tumours show higher blood oxygenation than estrogen-independent MDA-MB-231 (55.8±7.5 % vs. 45.6±5.1%, p=0.0008) with values similar to healthy tissue (55.8±7.5 % vs. 57.8±5.3%, p=0.917). Ex vivo histological analysis shows that while MDA-MB-231 tumours are more hypoxic compared to MCF-7, they have a higher blood vessel density, however, the vasculature of MCF-7 tumours is more mature (vessel wall thickness and aSMA coverage). Based on molecular biomarkers, MCF-7 presents a more pro-inflammatory endothelial-like response, marked by an increase of vascular endothelial growth factor (p<0.0001) of host origin (mVEGF), macrophage Nitric Oxide Sinthase (iNOS) and circulating NO, a mediator in inflammation and endothelial homeostasis (p=0.0031). VE-Cadherin of human origin is detectable in MDA-MB-231 tumours, which present CD31-absent blood vessels; together, these findings indicate presence of VM in MDA-MB-231 tumours. Interestingly, these 2 biological profiles respond differently to the anti-angiogenic drug BV: we observed a partial response in MDA-MB-231 tumours (CR=2/22, PR=3/22), but no effect in tumour size or survival was observed in MCF-7 tumours. While no change was observed in mVEGF upon BV treatment in any cohort, changes in optoacoustic signal of total Hb were observed in the MDA-MB-231 tumours after treatment. In conclusion, optoacoustic imaging delineates the vascular function of the blood vessel networks generated by angiogenesis and vascular mimicry respectively in our estrogen-dependent and estrogen-independent breast cancer xenografts. This study highlights the potential application of this technique in monitoring tumour vasculature development and in the future could be used to assess response to therapy. Citation Format: Isabel Quiros-Gonzalez, Michal Tomaszewski, Laura Ansel-Bollepalli, Sarah J. Aikten, Michael Gill, Sarah E. Bohndiek. Differences in optoacoustic signal reflect different characteristics of the vasculature and response to antiangiogenic therapy in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4121.

Abstract 3458: Neurotensin increases breast cancer proliferation through activation of AMPK

Abstract Introduction. Neurotensin (NT), a thirteen amino acid peptide predominantly localized to enteroendocrine cells of the small intestine, is released after fat ingestion. While its major role involves regulating lipid metabolism and storage, NT, acting through its high-affinity receptor (NTR1), stimulates the growth and progression of a variety of NTR1-positive cancers. Proneurotensin—a stable precursor produced in equimolar amounts as NT—has been associated with breast cancer development in women, and prior work has implicated the NT/NTR1 axis in breast cancer progression; however, very little is known about the underlying NT signaling pathways that stimulate breast cancer growth. The purpose of this study is to elucidate mechanisms by which NT leads to increased proliferation and aggression of breast cancer—with particular emphasis placed on the role of AMP-activated protein kinase (AMPK). Methods. MCF-7 (estrogen-dependent) and MDA-MB-231 (triple negative) are breast cancer cell lines that express NTR1. (i) Proliferation assays were performed by treating both cell lines with NT (0 or 100 nM) in serum-free media for 24, 48, and 72 h and then counting the viable cells. (ii) Next, to assess signaling pathways mediating the effects of NT, both cell lines were treated with NT (0 or 100 nM) in serum-free media for 15 min, 30 min, 1 h, 6 h, 12 h, and 24 h; immunoblotting was performed for phosphorylated and total forms of AMPK and its downstream effector acetyl CoA carboxylase (ACC). Results. (i) Increased proliferation of MCF-7 cells treated with NT (100 nM) was observed after serum-starvation for 48 h. While MDA-MB-231 cells treated with NT demonstrated improved survival after serum-starvation for 48 h, the effects were not significantly different. (ii) NT induced activation of AMPK and ACC in MCF-7 cells after treatment for 6 and 12 h, but these effects were not observed in MDA-MB-231 cells. Conclusions. Our findings suggest a possible linkage between NT and increased proliferation of estrogen-dependent breast cancer cells through activation of AMPK and its downstream effector proteins. These effects were minimal in NTR1-expressing triple negative breast cancer cells, suggesting that the molecular classification of the tumor plays an important role in NT signaling and downstream effects. Further delineation of the differential effects of NT in specific breast cancer phenotypes has the potential to identify novel therapeutic targets in the treatment of this disease. Citation Format: Jeremy Johnson, Jing Li, B. Mark Evers. Neurotensin increases breast cancer proliferation through activation of AMPK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3458.

Abstract 532: PELP1 and AIB1 (SRC-3) complexes promote cancer stem cell-associated phenotypes in ER+ breast cancer models

Abstract Proline, glutamic acid, and leucine rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, we sought to identify interacting partners unique to cytoplasmic PELP1 and determine the mechanisms by which these interactions promote oncogenic PELP1 signaling. We identified AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor-positive (ER+) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e., activation) of AIB1, enhanced ALDH+ tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and downregulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, inhibited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line (J110). Furthermore, in vivo syngeneic tumor studies showed that PELP1 knockdown in J110 cells resulted in increased survival of tumor-bearing mice as compared to mice injected with control cells. These studies suggest that manipulating PELP1 location or levels has the potential to mitigate tumor progression, particularly in the context of AIB1-mediated tumorigenesis. Taken together, our data demonstrate that cytoplasmic PELP1/AIB1-containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression. Citation Format: Thu H. Truong, Hsiangyu Hu, Nuri A. Temiz, Kyla M. Hagen, Brian J. Girard, Nicholas J. Brady, Kathryn L. Schwertfeger, Carol A. Lange, Julie H. Ostrander. PELP1 and AIB1 (SRC-3) complexes promote cancer stem cell-associated phenotypes in ER+ breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 532.

Raloxifene/SBE-β-CD Inclusion Complexes Formulated into Nanoparticles with Chitosan to Overcome the Absorption Barrier for Bioavailability Enhancement

Raloxifene (RXF) is a hormone-like medication used for treating postmenopausal osteoporosis and estrogen-dependent breast cancer, yet associated with bad low bioavailability due to poor solubility. This study was intended to develop cyclodextrin/chitosan nanoparticles (ccNPs) for oral delivery of RXF in order to enhance the oral bioavailability. RXF-loaded ccNPs (RXF-ccNPs) were prepared by cyclodextrin inclusion followed by complexation with chitosan. RXF-ccNPs were fully characterized by particle size, morphology and in vitro drug release. The oral delivery efficacy and transepithelial transport potential were evaluated by pharmacokinetics, in situ single-pass intestinal perfusion, cellular uptake and ex vivo imaging. The resulting RXF-ccNPs were around 165 nm in particle size with a narrow distribution. The oral bioavailability of RXF was enhanced by 2.6 folds through ccNPs compared to RXF suspensions in rats. It was shown that RXF-ccNPs could improve the intestinal permeability of RXF, increase the cellular uptake of RXF and facilitate its transport across the absorptive epithelia. The results indicate that our developed ccNPs based on sulfobutylether-β-cyclodextrin and oligochitosan are a promising vehicle to orally deliver poorly water-soluble drugs over and above RXF.

A ferutinin analogue with enhanced potency and selectivity against ER-positive breast cancer cells in vitro

Estrogen is considered a risk factor for breast cancer since it promotes breast-cell proliferation. The jaesckeanadiol-3-p-hydroxyphenylpropanoate, a hemi-synthetic analogue of the natural phytoestrogen ferutinin (jaesckeanadiol-p-hydroxybenzoate), is designed to be devoid of estrogenic activity. This analogue induces a cytotoxic effect 30 times higher than that of ferutinin towards MCF-7 breast cancer cell line. We compared these two compounds with respect to their effect on proliferation, cell cycle distribution and cancer stem-like cells in the MCF-7 cell line. Treatment with ferutinin (30 μM) and its analogue (1 μM) produced significant accumulation of cells at the pre G0/G1 cell cycle phase and triggered apoptosis. Importantly, this compound retains its anti-proliferative activity against breast cancer stem/progenitor cells that are naturally insensitive to ferutinin at the same dose. These results position ferutinin analogue as an effective compound inhibiting the proliferation of estrogen-dependent breast cancer cells and consistently targeting their stem-like cells.

Effects on estrogen-dependent and triple negative breast cancer cells growth of Ni(II), Zn(II) and Cd(II) complexes with the Schiff base derived from pyridine-2-carboxaldehyde and 5,6-diamino-1,3-dimethyluracil explored through the renin-angiotensin system (RAS)-regulating aminopeptidases

A series of Ni(II), Zn(II) and Cd(II) complexes with the Schiff base derived from the condensation 1:1 from pyridine-2-carboxaldehyde and 5,6-diamino-1,3-dimethyluracil (6-amino-1,3-dimethyl-5-[(pyridin-2-ylmethylidene)-amino]pyrimidine-2,4(1H,3H)-dione, DAAUPic) were synthesized and subsequently characterized by means of elemental analysis, FT-IR, NMR and nine of them by X-ray diffraction. Except the [Zn(μ-O,O′-AcO)(N5,N6,N1F-DAAUPicH–1)]2 and [Cd(O,O′-NO3)(μ-O4,(N5,N6,N1F)-DAAUPicH−1)(H2O)]2·2H2O dimers and the [Cd(μ-S,N-SCN)(N5,N6,N1F-DAAUPicH−1)]n chain-like polymer, all of them display monomeric molecular structures. The anticancer activity of compounds was also explored studying their effects on renin-angiotensin system (RAS)-regulating aminopeptidases on estrogen-dependent and triple negative breast cancer cell lines. At the concentrations used, some of the complexes showed different effects on (RAS) peptidases, which support the idea that their effects on cell growth/proliferation could be related to autocrine/paracrine regulatory functions of their corresponding peptide substrates.