Breast Tumor Epithelial Cells Research Articles

Abstract PS18-09: The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment

Abstract Background. Despite a recent convergence of breast cancer (BC) incidence rates between African American (AA) and European American (EA) women, AA women continue to have ~40% higher mortality rates. BC is a heterogeneous collection of diseases, where African ancestry women are disproportionately burdened with the most aggressive subtype of BC, triple negative BC (TNBC). We have previously reported that the African ancestry-specific Duffy-null allele (FY-) is a risk factor for TNBC diagnoses. FY- is a promoter variant that removes expression of the Duffy Antigen Receptor for Chemokines (DARC/ACKR1), from red blood cells (RBCs). DARC serves as a portal of entry for Plasmodium vivax malaria parasite, and FY- confers immunity, leading to fixation of FY- among Sub-Saharan African populations. DARC is an atypical chemokine receptor that functions to modulate chemokine levels in circulation and tissues to aid immune cell recruitment. DARC is also expressed on breast tumor epithelial cells, however the significance of DARC in the TNBC tumor microenvironment (TME) has not fully been explored. Leveraging our African ancestry enriched International Study for the Center of Breast Cancer Subtypes cohort and in vivo murine models, we employed multi-omics approaches to explore the function of FY- and DARC expression across cell types in the TNBC TME. Genomics. Using transcriptomic data from the TCGA BC cohort, we have previously reported that DARC expression is highly correlated with deconvoluted immune cell abundance using CIBERSORT. To build upon this finding among TNBC patients, we have analyzed RNAseq data from two cohorts of African ancestry women to explore correlation of DARC expression status and immune cell abundance. Among TNBC cases, we report that higher DARC expression was significantly associated with increased immune cell abundance, with increases in B cell, T cell and monocyte populations. Using whole genome sequencing data across 86 African ancestry cases, we are currently investigating germline FY- status with mutational signatures. Circulating biomarkers. Patient plasma was collected prior to surgery, where Luminex multiplex assays were performed to quantify levels of >40 circulating chemokines and cytokines. FY- status has been determined for ~550 patients using single-plex or multiplex PCR genotyping methods from germline saliva DNA. Our univariate analyses show several circulating chemokines, including CCL2, CXCL2, CXCL6 and CXCL11, have significant differences between FY- and non-FY- BC patients. Curation of clinicopathological variables for multivariate analysis is ongoing. Murine models. We crossed C3(1)Tag BC transgenic mice with a DARC knock-out (KO) line to generate mice lacking DARC expression that spontaneously develop tumors in the mammary fat pads. The C3(1)Tag model is reported to have similar gene expression profiles to basal-like TNBC tumors. Tumors were collected at pre-determined time points of 25 and 30 weeks, where DARC KO mice had increased tumor size and tumor burden at 30 weeks. Initial fluorescent staining revealed an increase in immune cell infiltration in the TME of DARC expressing mice compared to DARC KO mice. To further explore this, we employed multiplex staining across a representative cohort of DARC expressing and KO mice at 25 and 30 weeks to capture the specific immune cell populations infiltrating the TME. Conclusions. TNBC tumors have worse prognosis due to lack of targeted therapeutic options. Increased immunogenicity of TNBC tumors, especially among African ancestry women may be driven in part by DARC expression. Characterization of FY- status, in coordination with DARC among other cell types in the TNBC TME could present new opportunities for biomarker and/or therapeutic development to improve prognosis for these underserved populations. Citation Format: Rachel Martini, Stevens Patino, Emma Guyonnet, Brian Stonaker, Isra Elhussein, Julie Sahler, Avery August, Nancy Manley, Rick Kittles, Clayton Yates, Lisa Newman, Melissa Davis. The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment [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 PS18-09.

Endophytic Fungi from Mangrove Plant Acanthus ilicifolius L.: Antimicrobial, Anticancer, and Species Determination

The current study aimed to screen, identify and evaluate the biological activity of fungi strains from the mangrove plant Acanthus ilicifolius L, which has proven to be a rich source of fungal species. Thirteen fungi were screened from the leaves, stem bark, and roots. Each fungal strain was cultivated in rice as media, and then the secondary metabolites of the fungus were extracted with ethyl acetate. The extracts were tested for antimicrobial and cytotoxic activities. Next, a phylogenetic tree was reconstructed based on the sequence similarities of each strain. The fungal extract AIA1 displayed excellent antimicrobial activity in a concentration of 5 % against some pathogenic bacterial strains with an inhibition zone in the range of 11.87 ± 0.87 to 15.42 ± 0.51 mm. The brine shrimp lethality test (BSLT) showed that strains AIA1 and AIKB3 were toxic against Artemisia salina L with an LC50 value of less than 80 ppm. The MTT assay determined the cytotoxicity against the human ductal breast epithelial tumor (T47D) cell line. Based on molecular identification, AIA1, AIKB3, and AID1 showed maximum similarity (> 95 %) with Pestalotiopsis sp. AIA1, Trichoderma yunnanense AIKB3, and Schizopyllum commune AID1, respectively. The ethyl acetate extracts of fungi strains from the mangrove plant A. ilicifolius showed strong antibacterial and moderate cytotoxic activities, which make them good candidates for secondary metabolite isolation. HIGHLIGHTS This is the first report on exploring the endophytic fungi from the mangrove plant Acanthus ilicifolius collected from Mangrove Forest Tourism Park, West Sumatera, Indonesia Endophytic fungi strains from mangrove plant Acanthus ilicifolius showed good activity on the antimicrobial and cytotoxic assay Three potential endophytic fungi were identified asPestalotiopsis AIA1, Trichoderma yunnanense AIKB3, and Schizopyllum commune AID1 GRAPHICAL ABSTRACT

Expression and Function of StAR in Cancerous and Non-Cancerous Human and Mouse Breast Tissues: New Insights into Diagnosis and Treatment of Hormone-Sensitive Breast Cancer.

Breast cancer (BC) is primarily triggered by estrogens, especially 17β-estradiol (E2), which are synthesized by the aromatase enzyme. While all steroid hormones are derived from cholesterol, the rate-limiting step in steroid biosynthesis is mediated by the steroidogenic acute regulatory (StAR) protein. Herein, we demonstrate that StAR mRNA expression was aberrantly high in human hormone-dependent BC (MCF7, MDA-MB-361, and T-47D), modest in hormone-independent triple negative BC (TNBC; MDA-MB-468, BT-549, and MDA-MB-231), and had little to none in non-cancerous mammary epithelial (HMEC, MCF10A, and MCF12F) cells. In contrast, these cell lines showed abundant expression of aromatase (CYP19A1) mRNA. Immunofluorescence displayed qualitatively similar patterns of both StAR and aromatase expression in various breast cells. Additionally, three different transgenic (Tg) mouse models of spontaneous breast tumors, i.e., MMTV-Neu, MMTV-HRAS, and MMTV-PyMT, demonstrated markedly higher expression of StAR mRNA/protein in breast tumors than in normal mammary tissue. While breast tumors in these mouse models exhibited higher expression of ERα, ERβ, and PR mRNAs, their levels were undetected in TNBC tumors. Accumulation of E2 in plasma and breast tissues, from MMTV-PyMT and non-cancerous Tg mice, correlated with StAR, but not with aromatase, signifying the importance of StAR in governing E2 biosynthesis in mammary tissue. Treatment with a variety of histone deacetylase inhibitors (HDACIs) in primary cultures of enriched breast tumor epithelial cells, from MMTV-PyMT mice, resulted in suppression of StAR and E2 levels. Importantly, inhibition of StAR, concomitant with E2 synthesis, by various HDACIs, at clinical and preclinical doses, in MCF7 cells, indicated therapeutic relevance of StAR in hormone-dependent BCs. These findings provide insights into the molecular events underlying the differential expression of StAR in human and mouse cancerous and non-cancerous breast cells/tissues, highlighting StAR could serve not only as a novel diagnostic maker but also as a therapeutic target for the most prevalent hormone-sensitive BCs.

Investigations on the cytotoxicity and antimicrobial activities of terezine E and 14-hydroxyterezine D.

Secondary metabolites produced by endophytes are an excellent source of biologically active compounds. The newly isolated natural products terezine E and 14-hydroxyterezine D are endophytic metabolites exhibiting anticancer activity recently identified by our team (https://doi.org/10.1080/14786419.2018.1489393). In our current study, we evaluated their affinity for binding to the active site of histone deacetylase (PDB ID: 4CBT) and matrix metalloproteinase 9 (PDB ID: 4H3X) by molecular docking using AutoDock Vina software after having tested their cytotoxic activities on three cell lines (human ductal breast epithelial tumor cells (T47D)-HCC1937), human hepatocarcinoma cell line (HepG2)-HB8065), and human colorectal carcinoma cells (HCT-116)-TCP1006, purchased from ATCC, USA)). Additionally, their antimicrobial activities were investigated, and their minimum inhibitory concentration (MIC) values were determined against P. notatum and S. aureus by the broth microdilution method. Higher cytotoxicity was observed for terezine E against all tested cell lines compared to 14-hydroxyterezine D. Molecular docking results supported the high cytotoxicity of terezine E and showed higher binding affinity with 4CBT with an energy score of 9 kcal/mol. Terezine E showed higher antibacterial and antifungal activities than 14-hydroxyrerezine D: MIC values were 15.45 and 21.73 µg/mL against S. aureus and 8.61 and 11.54 µg/mL against P. notatum, respectively.

Interacting with tumor cells weakens the intrinsic clockwise chirality of endothelial cells.

Endothelial cells (ECs) possess a strong intrinsic clockwise (CW, or rightward) chirality under normal conditions. Enervating this chirality of ECs significantly impairs the function of the endothelial barrier. Malignant tumor cells (TCs) undergo metastasis by playing upon the abnormal leakage of blood vessels. However, the impact of TCs on EC chirality is still poorly understood. Using a transwell model, we co-cultured the human umbilical vein endothelial cells or human lung microvascular endothelial cells and breast epithelial tumor cell lines to simulate the TC-EC interaction. Using a micropatterning method, we assessed the EC chirality changes induced by paracrine signaling of and physical contact with TCs. We found that the intrinsic clockwise chirality of ECs was significantly compromised by the TC's physical contact, while the paracrine signaling (i.e., without physical contact) of TCs causes minimal changes. In addition, ECs neighboring TCs tend to possess a left bias, while ECs spaced apart from TCs are more likely to preserve the intrinsic right bias. Finally, we found the chirality change of ECs could result from physical binding between CD44 and E-selectin, which activates protein kinase C alpha (PKCα) and induces pseudopodial movement of EC toward TC. Our findings together suggest the crucial role of EC-TC physical interaction in EC chirality and that weakening the EC chirality could potentially compromise the overall endothelial integrity which increases the probability of metastatic cancer spread.

Abstract 6165: DARC/ACKR1 expression is associated with immune landscape changes among triple negative breast tumors

Abstract Recent work defining the immune landscape across breast cancer (BC) subtypes have revealed that TNBC tumors have increased immunogenicity, and these increases in immune cell infiltration are associated with better prognosis and outcomes. Our recent work has proposed the Duffy Antigen Receptor for Chemokines (DARC), as a potential driver of immune regulation in breast tumors. DARC/ACKR1 is an atypical chemokine receptor that promiscuously binds both pro-inflammatory CC and pro-angiogenic CXC class chemokines and plays a role in chemokine gradient establishment both in circulation, and at sites of inflammation via chemokine transcytosis. In the BC context, we and others have reported DARC/ACKR1 protein expression on breast tumor epithelial cells by IHC and have also shown a range of gene expression in breast tumors from bulk RNAseq data in the TCGA cohort. Specifically, alongside CIBERSORT deconvolution of tumor associated immune cell populations, we have shown a strong positive correlation between DARC/ACKR1 gene expression and total tumor-associated leukocyte (TAL) abundance across all BC subtypes. To investigate the role of DARC/ACKR1 specifically in the TNBC tumor microenvironment (TME), we have analyzed (1) in silico bulk RNAseq data from two independent TNBC cohorts, (2) imaging mass cytometry data from TNBC patients and (3) murine model of DARC/ACKR1 BC progression in a basal-like BC transgenic mouse model. Our in silico data shows strong positive correlation of DARC/ACKR1 expression and TAL abundance, when DARC/ACKR1 expression is both dichotomized and analyzed as a continuous variable. Immune cell populations increasing with DARC/ACKR1 expression include B cell, T cell, monocyte and macrophage populations, which was consistent with our previous findings across all BC subtypes. IMC analysis of TNBC FFPE sections to determine protein expression of various immune and structural markers on the single-cell level between DARC/ACKR1 high or low expressing tumors show cells from DARC/ACKR1 high expressing tumors clustered distinctly from DARC/ACKR1 low expressing tumors. Image analysis also revealed increased infiltration among our DARC/ACKR1 high tumors. Finally, our murine model of DARC/ACKR1 in TNBC, shows that tumor development followed similar disease progression as human BC, following early through late-stage disease. DARC/ACKR1 deficient mice were found to have larger tumor volume and more palpable tumors, where DARC/ACKR1 expressing mice show co-localization of DARC and immune cell expression. This is the first work to describe the role of DARC/ACKR1 in the TNBC TME in bulk tumor transcriptomic data, spatial single-cell protein level data, and murine BC tumor model. Further investigation of factors that drive immune response in TNBC tumors, like DARC/ACKR1, may lead the way to novel therapeutic options or biomarkers for better outcomes for women with TNBC. Citation Format: Rachel Martini, Kiel Telesford, Brittany Lord, Hiranmayi Ravichandran, Olivier Elemento, Nancy Manley, Michele Monteil, Lisa Newman, Upender Manne, Clayton Yates, Melissa B. Davis. DARC/ACKR1 expression is associated with immune landscape changes among triple negative breast tumors [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 6165.

Pt-Fe3O4, Pd-Fe3O4, and Au-Fe3O4 Nanoheterodimers and Their Efficacy as Radiosensitizers in Cancer Therapy.

The X-radiation enhancing effect of caffeic acid-functionalized Au-Fe3O4, Pt-Fe3O4, and Pd-Fe3O4 nanoheterodimers (NHDs) on 2D and 3D breast tumor (MCF-7) and healthy breast epithelial (MCF-10A) cells was comprehensively examined by performing cell viability, reactive oxygen species (ROS) detection, enzyme activity, and clonogenic assays. Intracellular NHDs were observed to cause DNA fragmentation and to enhance superoxide and hydroxyl radical formation in MCF-7 cells when exposed to a single dose of 1 Gy. MCF-7-derived multicellular tumor spheroids (MCF-7 MCTS) and MCF-10A-derived multicellular spheroids (MCF-10A MCS) were incubated with the NHDs and irradiated with five daily doses of 2 Gy. The Au-Fe3O4 and Pt-Fe3O4 NHD-loaded MCF-7 MCTS significantly decreased in volume after being exposed to fractionated irradiation, whereas intracellular Au-Fe3O4 and Pt-Fe3O4 NHDs promoted the growth of the irradiated MCF-10A MCS. Moreover, Au-Fe3O4 and Pt-Fe3O4 NHDs were shown to impede ROS formation and inhibit DNA strand breakage in the noncancerous MCF-10A cells. On the other hand, the Pd-Fe3O4 NHDs boosted X-radiation-induced damage of MCF-10 A cells, which was ascribed to impairment of the ROS scavenging enzyme activities. In summary, caffeic acid-functionalized Au-Fe3O4 and Pt-Fe3O4 NHDs performed as excellent X-radiation enhancing agents in breast tumor cells, while they protect healthy breast epithelial cells against X-radiation.

Cancer-Associated Fibroblasts in the Breast Tumor Microenvironment

Years of investigation have shed light on a theory in which breast tumor epithelial cells are under the effect of the stromal microenvironment. This review aims to discuss recent findings concerning the phenotypic and functional characteristics of cancer associated fibroblasts (CAFs) and their involvement in tumor evolution, as well as their potential implications for anti-cancer therapy. In this manuscript, we reviewed that CAFs play a fundamental role in initiation, growth, invasion, and metastasis of breast cancer, and also serve as biomarkers in the clinical diagnosis, therapy, and prognosis of this disease.

VEGF-C mediates tumor growth and metastasis through promoting EMT-epithelial breast cancer cell crosstalk.

It is well established that a subset of cells within primary breast cancers can undergo an epithelial to mesenchymal transition (EMT), although the role of EMT in metastasis remains controversial. We previously demonstrated that breast cancer cells that had undergone an oncogenic EMT could increase metastasis of neighboring cancer cells via non-canonical paracrine-mediated activation of GLI activity that is dependent on SIX1 expression in the EMT cancer cells. However, the mechanism by which these SIX1-expressing EMT cells activate GLI signaling remained unclear. In this study, we demonstrate a novel mechanism for activation of GLI-mediated signaling in epithelial breast tumor cells via EMT cell-induced production and secretion of VEGF-C. We show that VEGF-C, secreted by breast cancer cells that have undergone an EMT, promotes paracrine-mediated increases in proliferation, migration and invasion of epithelial breast cancer cells, via non-canonical activation of GLI-signaling. We further show that the aggressive phenotypes, including metastasis, imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF-C in EMT cells or by knocking down NRP2, a receptor which interacts with VEGF-C, in neighboring epithelial cancer cells. Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer, demonstrating that VEGF-C strongly correlates with activation of Hedgehog signaling and EMT in the human disease. Our study suggests that the VEGF-C/NRP2/GLI axis is a novel and conserved paracrine means by which EMT cells enhance metastasis, and provides potential targets for therapeutic intervention in this heterogeneous disease.

Platinum(II) Complexes with Bulky Disubstitute Triazolopyrimidines as Promising Materials for Anticancer Agents.

Herein, we present dicarboxylate platinum(II) complexes of the general formula [Pt(mal)(DMSO)(L)] and [Pt(CBDC)(DMSO)(L)], where L is dbtp 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine) or ibmtp (7-isobutyl-5-methyl-1,2,4- triazolo[1,5-a]pyrimidine), as prospective prodrugs. The platinum(II) complexes were synthesized in a one-pot reaction between cis-[PtCl2(DMSO)2], silver malonate or silver cyclobutane-1,1-dicarboxylate and triazolopyrimidines. All platinum(II) compounds were characterized by FT-IR, and 1H, 13C, 15N and 195Pt NMR; and their square planar geometries with one monodentate N(3)-bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine, one S-bonded molecule of dimethyl sulfoxide and one O,O-chelating malonato (1, 2) or O,O-chelating cyclobutane-1,1-dicarboxylato (3, 4) was determined. Additionally, [Pt(CBDC)(dbtp)(DMSO)] (3) exhibited (i) substantial in vitro cytotoxicity against the lung adenocarcinoma epithelial cell line (A549) (IC50 = 5.00 µM) and the cisplatin-resistant human ductal breast epithelial tumor cell line (T47D) (IC50 = 6.60 µM); and (ii) definitely exhibited low toxicity against normal murine embryonic fibroblast cells (BALB/3T3).

Hemoglobin overexpression and splice signature as new features of inflammatory breast cancer?

IntroductionInflammatory Breast Cancer (IBC) is the most aggressive form of breast carcinoma characterized by rapid onset of inflammatory signs and its molecular fingerprint has not yet been elucidated. ObjectivesThe objective of this study was to detect both gene expression levels and alternate RNA splice variants specific for IBC. MethodsW e performed splice-sensitive array profiling using Affymetrix Exon Array and quantitative RT-PCR analyses in 177 IBC compared to 183 non-IBC. We also assessed the prognostic value of the identified candidate genes and splice variants. ResultsA 5-splice signature (HSPA8, RPL10, RPL4, DIDO1 and EVL) was able to distinguish IBC from non-IBC tumors (p<10-7). This splice signature was associated with poor metastasis-free survival in hormone receptor-negative non-IBC (p=0.02), but had no prognostic value in IBC. PAM analysis of dysregulated genes in IBC compared to non-IBC identified a 10-gene signature highly predictive of IBC phenotype and conferring a poor prognosis in non-IBC. The genes most commonly upregulated in IBC were 3 hemoglobin genes able to reliably discriminate IBC from non-IBC (p<10-4). Hb protein expression in epithelial breast tumor cells was confirmed by immunohistochemistry. ConclusionIBC has a specific spliced transcript profile and is characterized by hemoglobin gene overexpression that should be investigated in further functional studies.

Three-dimensional cryogel matrix for spheroid formation and anti-cancer drug screening.

Spheroid-based systems have been developed as alternatives to two-dimensional (2D) monolayer cultures for understanding 3D cell behavior and conducting in vitro drug screening tests. However, spheroids are easily disrupted while handling and do not mimic the presence of extracellular matrix (ECM) components. To address that, we have developed a cost-effective, polyethylene glycol diacrylate (PEGDA), and gelatin methacryloyl (GELMA) based semi-synthetic cryogel matrix system, which can be used to grow spheroids and conduct studies while providing architectural support and mimicking in vivo ECM components. These matrices are macroporous and support formation of tumor-like spheroids of breast tumor epithelial (MCF-7) cells in the absence of additional growth factors otherwise required for spheroid formation. Difference in morphology of cells as a function of matrix composition and increase in size and number of spheroids as a function of time was observed. Spheroids grown in cryogel matrices showed more drug resistance than their 2D counterparts, which can partially be explained by the epithelial to mesenchymal transition (EMT) observed in spheroids. We believe that spheroids formed through these PEGDA-GELMA cryogel matrices better represent in vivo pathological conditions and can help develop cost-effective in vitro assays for screening new pharmacological drug candidates and performing cell mechanistic studies.

Development of an estrogen-dependent breast cancer co-culture model as a tool for studying endocrine disruptors

We developed an innovative co-culture system composed of Hs578t human mammary stromal-like cells and T47D hormone-dependent breast epithelial tumor cells as a representative in vitro model of the human hormone-dependent mammary tumor microenvironment. Hs578t cells expressed aromatase (CYP19) mainly via the healthy stromal CYP19 promoter I.4, but also to a lesser extent via the breast cancer-relevant promoters PII, I.3 and I.7, and produced estrogens from androgen precursors. These estrogens stimulated T47D cell proliferation and estrogen receptor-dependent expression of trefoil factor-1 (TFF1), which is known to stimulate mammary tumor cell proliferation and migration. Hs578t cells can also undergo a “promoter-switch” where the normally silent CYP19 promoters PII, I.3 and I.7 become activated, which mimics the in vivo situation in human breast cancer patients. This positive feedback loop is the hallmark of the hormone-dependent breast tumor microenvironment. Using the co-culture model we designed, we evaluated the promoter-specific expression of CYP19, expression of estrogen-dependent gene TFF1, and determined the effects exhibited by basil and fennel seed essential oils on steroidogenesis in the tumor microenvironment.

Normal epithelial and triple-negative breast cancer cells show the same invasion potential in rigid spatial confinement

The extra-cellular microenvironment has a fundamental role in tumor growth and progression, strongly affecting the migration strategies adopted by single cancer cells during metastatic invasion. In this study, we use a novel microfluidic device to investigate the ability of mesenchymal and epithelial breast tumor cells to fluidize and migrate through narrowing microstructures upon chemoattractant stimulation. We compare the migration behavior of two mesenchymal breast cancer cell lines and one epithelial cell line, and find that the epithelial cells are able to migrate through the narrowest microconstrictions as the more invasive mesenchymal cells. In addition, we demonstrate that migration of epithelial cells through a highly compressive environment can occur in absence of a chemoattractive stimulus, thus evidencing that they are just as prone to react to mechanical cues as invasive cells.

Liposomal Treatment of Cancer Cells Modulates Uptake Pathway of Polymeric Nanoparticles by Altering Membrane Stiffness.

Nanomedicines can be taken up by cells via nonspecific and dynamin-dependent (energy-dependent) clathrin and caveolae-mediated endocytosis. While significant effort has focused on targeting pathway-specific transporters, the role of nanobiophysics in the cell lipid bilayer nanoparticle uptake pathway remains largely unexplored. In this study, it is demonstrated that stiffness of lipid bilayer is a key determinant of uptake of liposomes by mammalian cells. Dynamin-mediated endocytosis (DME) of liposomes is found to correlate with its phase behavior, with transition toward solid phase promoting DME, and transition toward fluidic phase resulting in dynamin-independent endocytosis. Since liposomes can transfer lipids to cell membrane, it is sought to engineer the biophysical properties of the membrane of breast epithelial tumor cells (MD-MBA-231) by treatment with phosphatidylcholine liposomes, and elucidate its effect on the uptake of polymeric nanoparticles. Analysis of the giant plasma membrane vesicles derived from treated cells using flicker spectroscopy reveals that liposome treatment alters membrane stiffness and DME of nanoparticles. Since liposomes have a history of use in drug delivery, localized priming of tumors with liposomes may present a hitherto unexploited means of targeting tumors based on biophysical interactions.

Chromatin Immunoprecipitation of HIF-α in Breast Tumor Cells Using Wild Type and Loss of Function Models.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine whether a protein of interest binds to specific regulatory elements of the genome. Herein, we outline protocols optimized to detect binding of Hypoxia-Inducible Factor (HIF)-1α or HIF-2α to putative hypoxia response elements (HREs) within HIF target genes expressed in breast tumor epithelial cells.

Mitochondria-targeted nanocarriers using hyperbranched polycations wrapped carbon nanotubes for augment photodynamic therapeutic effects

A series of 3-heteroarylisoquinolinamines were designed, synthesized and evaluated for cytotoxicity, topoisomerases (topos) inhibitory activities and cell cycle inhibition. Several of the 3-heteroarylisoquinolines exhibited selective cytotoxicity against human ductal breast epithelial tumor (T47D) cells over non-cancerous human breast epithelial (MCF-10A) and human prostate cancer (DU145) cells. Most of the derivatives showed greater cytotoxicity in human colorectal adenocarcinoma (HCT-15) cells than camptothecin (CPT), etoposide and doxorubicin (DOX). Generally, 3-heteroarylisoquinolinamines displayed greater affinity for topo I than topo II. 3-Heteroarylisoquinolinamines with greater topo I inhibitory effect exhibited potent cytotoxicity. Piperazine-substituted derivative, 5b, with potent topo I and moderate topo II activities intercalated between DNA bases and interacted with topos through H-bonds at the DNA cleavage site of a docking model. Moreover, flow cytometry indicated that cytotoxic 3-heteroarylisoquinolinamines led to accumulation of human cervical (HeLa) cancer cells in the different phases of the cell cycle before apoptosis. Taken together, 3-heteroarylisoquinolinamines possessed potent cytotoxicity with topos and cell cycle inhibitory activities.

Abstract 497: Higher nucleolar index of nucleolin as an indicator of aberrant cellular DNA damage response (DDR)

Abstract Nucleoli provide a survival advantage for various tumor types, exemplified in prostate neoplasia, leukemia, lung, and breast carcinomas. Unregulated expression and increased nucleolar localization of many stress-responsive factors often correlate with hyper-proliferative status. Therefore, nucleolar changes in cancer cells are the first cytological indicators that can serve as an index for diagnostic and prognostic purposes. In this study, we examine an abundant nucleolar stress factor, nucleolin (NCL) for its sub-nuclear localization under normal and DNA damage conditions. NCL is a multifunctional RNA-binding phosphoprotein that plays critical role/s in many cellular processes including regulation of gene expression during normal cell cycle as well as in the cellular DNA damage response (DDR). NCL-mediated DDR involves changes in its phosphorylation status, sub-nuclear translocation and a multi-level regulation of p53-checkpoint signaling. However, the role/s of p53 status and the signaling pathways triggered during NCL-mediated stress response remain elusive, and forms the focus of this study. Taking advantage of two human ductal breast epithelial tumor cell lines with different genetic backgrounds (wild type, wt-p53 and mutant, mut-p53 L194F), we demonstrate that NCL is predominantly nucleolar in unstressed conditions irrespective of the p53 status. Upon genotoxic stress, however, wt-p53 is required for NCL to mobilize from nucleoli to the nucleoplasmic region. We observed a significant increase in nucleoplasmic index for NCL that is wt-p53 dependent and specific to the type of DNA damage. In contrast, in the presence of mut-p53, NCL failed to translocate even under various DNA damage conditions, resulting in a higher nucleolar index. Interestingly, another nucleolar factor, nucleophosmin (NPM) remained unaltered upon stress conditions even in the presence of wt-p53. This study corroborates the previous research that suggests NCL translocation requires wt-p53 and provides new insights into the role of p53-mutations (e.g. the zinc finger mutation in the central DNA binding region-L194F) in NCL-mediated DDR. Further, we also assessed the role of NCL and p53 (wt vs. mut) in regulating stress signaling (ATM/ATR and p38-kinase) pathways and gene expression in apoptosis. Together, our study elaborates a novel approach that correlates NCL sub-nuclear localization, downstream signaling pathways and gene expression during the cellular DDR. Citation Format: Iqra Nadeem, Amna Aslam, Jingyuan Wang, Anna Kozlova, Danielle Gordon, Rumsha Javed, Ruchama Steinberg, Rachele Dolce Rameau, Xinyin Jiang, Anjana D. Saxena. Higher nucleolar index of nucleolin as an indicator of aberrant cellular DNA damage response (DDR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 497. doi:10.1158/1538-7445.AM2017-497

Abstract C32: MicroRNA mediated megative regulation of caveolin 1 as a biological mechanism driving breast cancer disparities

Abstract In South Carolina, mortality differences between African American (AA) and non-Hispanic white (NHW) women breast cancer patients are amongst the highest in the country. Evidence suggests that the observed racial disparity exists independent of socioeconomic and standard of care issues, suggesting a potential biological factor may be involved. The loss of Caveolin-1 (Cav1) in the tumor stromal compartment has emerged as a novel biomarker for predicting poor clinical outcome in all of the most common subtypes of breast cancer, however the mechanism of Cav1 loss is unknown. We identified miR-510 as a novel oncomir and propose that its elevated expression in breast tumors results in stromal Cav1 loss and a subsequent worse outcome. In this study we used luciferase, western blot and quantitative PCR analysis to study Caveolin-1 as a direct target of miR-510. We used a co-culture system to assess crosstalk between epithelial and stromal compartments in vitro and a mouse model to assess this in vivo. Our research shows that Cav1 is a direct target of miR-510 and that overexpression of miR-510 leads to downregulation of Cav1 protein expression, specifically in the stromal compartment. This may be racially significant as our studies also show that miR-510 levels are elevated and Cav1 levels are reduced in AA breast cancer patients compared to their NHW counterparts. Data from our in vivo studies shows that cancer-associated fibroblasts (CAFs) isolated from miR-510 expressing epithelial derived tumors lead to more aggressive tumor growth when co-injected with breast tumor epithelial cells when compared to scrambled control CAF co-injections and breast tumor epithelial cells alone. Our results suggest that elevated miR-510 expression in breast epithelial cells leads to stromal Cav1 loss and is a mechanism driving racial disparity in breast cancer. Citation Format: David P. Turner, Brooke King, Qi Guo, Bobbie Blake, Lourdes M. Nogueira, amanda c. larue, judith d. salley, marvella e. ford, ashley evans-knowell, Victoria J. Findlay. MicroRNA mediated megative regulation of caveolin 1 as a biological mechanism driving breast cancer disparities. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C32.

The Breast Cancer Tumor Suppressor TRIM29 Is Expressed via ATM-dependent Signaling in Response to Hypoxia

Reduced ATM function has been linked to breast cancer risk, and the TRIM29 protein is an emerging breast cancer tumor suppressor. Here we show that, in cultured breast tumor and non-tumorigenic mammary epithelial cells, TRIM29 is up-regulated in response to hypoxic stress but not DNA damage. Hypoxia-induced up-regulation of TRIM29 is dependent upon ATM and HIF1α and occurs through increased transcription of the TRIM29 gene. Basal expression of TRIM29 is also down-regulated in cells expressing diminished levels of ATM, and findings suggest that this occurs through basal NF-κB activity as knockdown of the NF-κB subunit RelA suppresses TRIM29 abundance. We have previously shown that the activity of the TWIST1 oncogene is antagonized by TRIM29 and now show that TRIM29 is necessary to block the up-regulation of TWIST1 that occurs in response to hypoxic stress. This study establishes TRIM29 as a hypoxia-induced tumor suppressor gene and provides a novel molecular mechanism for ATM-dependent breast cancer suppression.