Breast Cancer Model System Research Articles

Cytotoxic Effects of Plant Secondary Metabolites and Naturally Occurring Bioactive Peptides on Breast Cancer Model Systems: Molecular Mechanisms

Breast cancer is the second leading cause of death among women, and the number of mortal cases in diagnosed patients is constantly increasing. The search for new plant compounds with antitumor effects is very important because of the side effects of conventional therapy and the development of drug resistance in cancer cells. The use of plant substances in medicine has been well known for centuries, but the exact mechanism of their action is far from being elucidated. The molecular mechanisms of cytotoxicity exerted by secondary metabolites and bioactive peptides of plant origin on breast cancer cell lines are the subject of this review.

Isolation and Characterization of Exosomes from Cancer Cells Using Antibody-Functionalized Paddle Screw-Type Devices and Detection of Exosomal miRNA Using Piezoelectric Biosensor.

Exosomes are small extracellular vesicles produced by almost all cell types in the human body, and exosomal microRNAs (miRNAs) are small non-coding RNA molecules that are known to serve as important biomarkers for diseases such as cancer. Given that the upregulation of miR-106b is closely associated with several types of malignancies, the sensitive and accurate detection of miR-106b is important but difficult. In this study, a surface acoustic wave (SAW) biosensor was developed to detect miR-106b isolated from cancer cells based on immunoaffinity separation technique using our unique paddle screw device. Our novel SAW biosensor could detect a miR-106b concentration as low as 0.0034 pM in a linear range from 0.1 pM to 1.0 μM with a correlation coefficient of 0.997. Additionally, we were able to successfully detect miR-106b in total RNA extracted from the exosomes isolated from the MCF-7 cancer cell line, a model system for human breast cancer, with performance comparable to commercial RT-qPCR methods. Therefore, the exosome isolation by the paddle screw method and the miRNA detection using the SAW biosensor has the potential to be used in basic biological research and clinical diagnosis as an alternative to RT-qPCR.

Elucidating the Role of MicroRNA-18a in Propelling a Hybrid Epithelial-Mesenchymal Phenotype and Driving Malignant Progression in ER-Negative Breast Cancer.

Epigenetic alterations that lead to differential expression of microRNAs (miRNAs/miR) are known to regulate tumour cell states, epithelial-mesenchymal transition (EMT) and the progression to metastasis in breast cancer. This study explores the key contribution of miRNA-18a in mediating a hybrid E/M cell state that is pivotal to the malignant transformation and tumour progression in the aggressive ER-negative subtype of breast cancer. The expression status and associated effects of miR-18a were evaluated in patient-derived breast tumour samples in combination with gene expression data from public datasets, and further validated in in vitro and in vivo breast cancer model systems. The clinical relevance of the study findings was corroborated against human breast tumour specimens (n = 446 patients). The down-regulated expression of miR-18a observed in ER-negative tumours was found to drive the enrichment of hybrid epithelial/mesenchymal (E/M) cells with luminal attributes, enhanced traits of migration, stemness, drug-resistance and immunosuppression. Further analysis of the miR-18a targets highlighted possible hypoxia-inducible factor 1-alpha (HIF-1α)-mediated signalling in these tumours. This is a foremost report that validates the dual role of miR-18a in breast cancer that is subtype-specific based on hormone receptor expression. The study also features a novel association of low miR-18a levels and subsequent enrichment of hybrid E/M cells, increased migration and stemness in a subgroup of ER-negative tumours that may be attributed to HIF-1α mediated signalling. The results highlight the possibility of stratifying the ER-negative disease into clinically relevant groups by analysing miRNA signatures.

In vivo evaluation of mebendazole and Ran GTPase inhibition in breast cancer model system.

Aim: To investigate the therapeutic potential of mebendazole (MBZ)-loaded nanostructured lipid carriers (NLCs). Methodology: NLC-MBZ was prepared and characterized to evaluate the in vitro and in vivo anticancer effects and the inhibitory effect on RanGTP and its potential as an antimetastatic treatment in vivo. Results: NLC-MBZ exhibited a size and charge of 155±20nm and-27±0.5mV, respectively, with 90.7% encapsulation. Free MBZ and NLC-MBZ had a 50% inhibitory concentrationof 610 and 305nM, respectively, against MDA-MB-231 cell lines. NLC-MBZ decreasedtumor size, suppressed tumor lung metastases, and lowered the expression of CDC25A, SKP2, RbX1and Cullin1while boosting the Rb proteins. Conclusion: NLC-MBZ displayedantiangiogenic potential and resulted in a reduced rate of lung metastasis in vivo.

A core-satellite micellar system against primary tumors and their lymphatic metastasis through modulation of fatty acid metabolism blockade and tumor-associated macrophages.

Lymph nodes (LNs) are the initial sanctuary of various metastatic tumor cells, and thus a precise lymphatic drug delivery strategy is necessary for the effective inhibition of metastasis. However, the complex biological barriers have restrained the drug delivery to tumor-draining lymph nodes (TDLNs). Metastatic tumor cells would undergo metabolic adaptation towards fatty acid oxidation (FAO) upon reaching the lipid-rich LNs. Herein, to inhibit primary tumors and their lymphatic metastasis, a core-satellite matrix metalloproteinase 2 (MMP-2) responsive micellar system was developed for sequential delivery of paclitaxel (PTX) and the metabolism-regulating drug etomoxir (ET) to tumors and TDLNs, respectively. Upon arrival at the tumor microenvironment (TME), the small satellite micelle encapsulating ET was detached from the core micelle in response to MMP-2, which not only drained to TDLNs via tumor-draining lymphatic vessels and inhibited the FAO of metastatic tumor cells, but also blocked M2-like macrophage polarization in the TME. Meanwhile, the core micelle containing PTX could largely accumulate in the TME and kill tumor cells. In an orthotopic 4T1 breast cancer model, the tumor and TDLN dual-targeted core-satellite micellar system effectively inhibited the growth of the primary tumor and alleviated immune suppression by blocking macrophage polarization. More importantly, tumor lymphatic metastasis was suppressed through FAO metabolic regulation. This strategy provides a promising approach for TDLN targeted therapy against breast cancer and its lymphatic metastasis.

Metastasis suppressor NME1 in exosomes or liposomes conveys motility and migration inhibition in breast cancer model systems.

Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis.

Abstract 1005: Ecological interactions in breast cancer: Cell cooperation within heterogeneous populations promotes growth and survival under drug pressure

Abstract The interplay of positive and negative interactions between drug sensitive and resistant cells determines the efficacy of treatment in heterogeneous cancer cell populations. While competition between different cell lineages within tumors has been considered in cancer evolution and progression, cooperative interactions receive far less study. We have developed in vitro ER+ breast cancer model systems to investigate cell-cell communication in heterogeneous cancer populations under selective drug pressure and probe for differences in growth, resistance, and interactions, such as cooperation, between cell types. Using cells resistant and sensitive to ribociclib (CDK 4/6 inhibitor), we have compared cell population growth in monoculture and coculture under different treatments. Furthermore, with the application of mathematical models, we have unraveled the contributions of proliferation rates, treatment effects, competition, and cooperation. Under ribociclib treatment, resistant cells confer an increased survival benefit to sensitive cells when cocultured together. Treatment-conditioned media taken from monocultured resistant cells and used to supplement sensitive cells reproduces this advantage, suggesting that secretion of some factor from resistant cells manifest the effect. Resistant cells may promote this growth by supplying estradiol as LC-MS/MS assays detected increased estradiol levels in coculture media compared with monoculture media under ribociclib pressure. This suggests estrogen-dependent sensitive cells benefit from the increase in local estradiol from resistant cells under treatment in coculture, resulting in cooperation. scRNA sequencing data between populations also revealed broad dysregulation of cell cycle pathways in resistant cells with cocultured sensitive cells shifting toward this resistant phenotype. Western blot detection in conjunction with scRNAseq data also supports an increased expression of estradiol conversion enzyme, HSD17β1, in resistant cells and increased expression of estradiol inactivating enzyme, HSD17β8, in sensitive cells shedding mechanistic insight into the estrogen-induced survival benefit provided by resistant cells. Proliferation of drug sensitive cells by resistant cells in co-culture is also enhanced through addition of estradiol, and blocked by small molecule inhibitors targeting the estrogen signaling pathway. These studies show that cooperation imparts drug resistance to normally sensitive cells and promotes survival, which highlights how interactions within heterogenous cell populations can shape the dynamics of growth and resistance. From a therapeutic standpoint, uncovering the mechanisms and consequences of cooperative cell-cell interactions may lead to strategic advancements for promoting drug response by targeting these influential interactions. Citation Format: Rena Emond, Jason Griffiths, Vince K. Grolmusz, Rachel Sousa, Jinfeng Chen, Eric M. Castaneda, Andrea H. Bild, Frederick R. Adler. Ecological interactions in breast cancer: Cell cooperation within heterogeneous populations promotes growth and survival under drug pressure [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 1005.

Rapamycin Plus Doxycycline Combination Affects Growth Arrest and Selective Autophagy-Dependent Cell Death in Breast Cancer Cells.

Metabolic alteration is characteristic during tumour growth and therapy; however, targeting metabolic rewiring could overcome therapy resistance. mTOR hyperactivity, autophagy and other metabolic processes, including mitochondrial functions, could be targeted in breast cancer progression. We investigated the growth inhibitory mechanism of rapamycin + doxycycline treatment in human breast cancer model systems. Cell cycle and cell viability, including apoptotic and necrotic cell death, were analysed using flow cytometry, caspase activity measurements and caspase-3 immunostainings. mTOR-, autophagy-, necroptosis-related proteins and treatment-induced morphological alterations were analysed by WesTM, Western blot, immunostainings and transmission electron microscopy. The rapamycin + doxycycline combination decreased tumour proliferation in about 2/3rd of the investigated cell lines. The continuous treatment reduced tumour growth significantly both in vivo and in vitro. The effect after short-term treatment was reversible; however, autophagic vacuoles and degrading mitochondria were detected simultaneously, and the presence of mitophagy was also observed after the long-term rapamycin + doxycycline combination treatment. The rapamycin + doxycycline combination did not cause apoptosis or necrosis/necroptosis, but the alterations in autophagy- and mitochondria-related protein levels (LC3-B-II/I, p62, MitoTracker, TOM20 and certain co-stainings) were correlated to autophagy induction and mitophagy, without mitochondria repopulation. Based on these results, we suggest considering inducing metabolic stress and targeting mTOR hyperactivity and mitochondrial functions in combined anti-cancer treatments.

Supplementation with Fermented Barley Extract Prevents Mammary Epithelial Cell Invasion in an Early Breast Cancer Model.

Diet-based prevention of malignant transformation contributes to the maintenance of quality of life by avoiding a battle against cancer. Invasion is one of the features of malignant breast cancer, and the prevention of invasion may reduce breast cancer malignancy. A recently established early breast cancer model system showed mammary ductal dysplasia with invasion in mice. This study utilized the model system and investigated the effect of fermented barley extract (FBE), a food material. The elastic fiber layer is the outermost layer of the mammary duct. A reduction in the elastic fiber layer was observed in the mammary glands of the model system, whereas supplementation with 8% FBE containing water prevented this reduction. Moreover, we found that FBE supplementation prevented mammary epithelial cell invasion. Based on our findings, FBE might be a candidate material for a diet-based prevention of early breast cancer invasion.

Development and evaluation of polycaprolactone based docetaxel nanoparticle formulation for targeted breast cancer therapy

The present study describes the preparation, characterization, and applications of newly developed polymeric poly-ɛ-caprolactone (PCL)/pluronic F108 nanoparticles, loaded with anticancer drug, and near infrared (NIR) dye for assessing the localization in the breast cancer model system. Docetaxel and NIR dye, “1,1′-dioctadecyl- 3,3,3′,3′- tetramethylindotricarbocyanine iodide (DiR)” loaded, poly-ɛ-caprolactone (PCL)/pluronic F108–based nanoparticles were formulated by nanoprecipitation method. This formulation was then characterized using various physicochemical methods including size distribution, zeta potential, surface morphology, drug entrapment efficiency, loading capacity, differential scanning calorimetry, in vitro release, and kinetics are performed. In vitro cell proliferative studies of human breast cancer BT-474 cells were carried out to evaluate the anticancer efficacy of the nanoparticles. The role of pluronic F108 was also evaluated in cell uptake of the nanoparticles. Drug- and NIR dye–loaded nanoparticles localization at tumor site were studied using noninvasive infrared dye imaging. The optimized nanoparticle formulation had a particle size of 100–300 nm, poly dispersity index of 0.156 and zeta potential of − 7.37 mV ± 4.93 mV. Drug release from the nanoparticle system followed Higuchi kinetics, suggesting diffusion mediated drug release. DSC studies suggest that the drug is either in amorphous or disordered crystalline state in the nanoparticle formulation. The increased accumulation of the PCL/ F108 nanoparticles in vivo suggests its potential as a superior targeted drug delivery system.

Abstract A131: Fulvestrant resistance in an MCF-7 model for breast cancer is associated with complete loss of ER pathway activity and gain of MAPK-AP1 pathway activity

Abstract Background: 10-15 signal transduction pathways govern major cellular processes, e.g. cell division, differentiation and migration, both in physiology and pathophysiology. They are frequently abnormally active in cancer and can drive cancer growth and metastasis. Resistance to targeted drugs directed towards specific signaling pathways like the ER pathway, can be mediated by induction of activity of other signaling pathways. The past decade we developed a new method to quantitatively measure functional activity status of signal transduction pathways in individual cell and tissue samples, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the pathway-associated transcription factor1,2,3. Assays have been developed for androgen (AR) and estrogen receptor (ER), Hedgehog (HH), Wnt, TGFb, Notch, NFkB, PI3K, JAK-STAT 1/2 and 3, and MAPK-AP1 pathways. They can be performed simultaneously on a single cell or tissue sample using Affymetrix HG-U133 Plus 2.0 microarray or qPCR, and provide quantitative pathway activity scores expressed on a log2odds scale. Method. Pathway analysis using these assays was performed on public Affymetrix expression microarray data (GSE74391,4) from ER-positive MCF-7 cells that were either sensitive or resistant to fulvestrant (n=26). Results. Resistance to fulvestrant was associated with complete loss of functional ER pathway activity (difference 16 log2odds score, p=0.00029) and gain of MAPK-AP1 pathway activity (difference 5.8 log2odds, p=0.00029) in ER positive MCF-7 cells. In the resistant MCF-7 cells, PI3K and MAPK-AP1 pathway activity were negatively correlated (Pearson 0.652, p=0.002), suggesting mutually exclusive pathway activation. Conclusion. Our pathway assays enabled identification of the previously unidentified mechanism of resistance to fulvestrant in a cell culture model system for breast cancer, demonstrating that: (1) ER positivity is a prerequisite, but not sufficient, for ER pathway activity, in agreement with our earlier clinical studies1,5,6;; (2) MAPK pathway activity may confer resistance to hormonal therapy; (3) in the absence of MAPK-AP1 pathway activity, the PI3K pathway may be an alternative resistance pathway; (3) signalling pathway assays are well suited to (quantitatively) investigate activity of signal transduction pathways which may confer resistance to a (targeted) drug treatment.

Implementation of Compact Ultra wide Band Antenna Design for Breast Cancer Tumor detection

One of the applications of UWB antenna is for detecting a cancer tumor according to breast cancer model system. The absolute dimensions of antenna are 44x30mm2 having a thickness of 1.6 mm. It involves Ultra wide band of 80% frequency range of 3 GHz–12 GHz for the FCC band. The proposed antenna resulted high gain and omni-directional radiation patterns and a considerable impedance matching. The optimized functioning of the antenna is illustrated by the simulation results. This antenna has been implemented in a designed system model with dielectric properties of a human breast capable to detect peculiar bodies. The tumor is detected and examined by the received Proportions and Positional coordinates, accessed by the application of UWB antenna. The accurate position coordinates of the tumor inside the breast are obtained at the places with the least Specific absorption rate (SAR). The antenna was used to localize the tumor precisely which shows the excellent functioning of the antenna and device. The proposed device is simulated by using Ansoft HFSS software and also CST Microwave studio simulator developed the breast model.

Unjamming and collective migration in MCF10A breast cancer cell lines

Each cell comprising an intact, healthy, confluent epithelial layer ordinarily remains sedentary, firmly adherent to and caged by its neighbors, and thus defines an elemental constituent of a solid-like cellular collective [1,2]. After malignant transformation, however, the cellular collective can become fluid-like and migratory, as evidenced by collective motions that arise in characteristic swirls, strands, ducts, sheets, or clusters [3,4]. To transition from a solid-like to a fluid-like phase and thereafter to migrate collectively, it has been recently argued that cells comprising the disordered but confluent epithelial collective can undergo changes of cell shape so as to overcome geometric constraints attributable to the newly discovered phenomenon of cell jamming and the associated unjamming transition (UJT) [1,2,5–9]. Relevance of the jamming concept to carcinoma cells lines of graded degrees of invasive potential has never been investigated, however. Using classical in vitro cultures of six breast cancer model systems, here we investigate structural and dynamical signatures of cell jamming, and the relationship between them [1,2,10,11]. In order of roughly increasing invasive potential as previously reported, model systems examined included MCF10A, MCF10A.Vector; MCF10A.14-3-3ζ; MCF10.ErbB2, MCF10AT; and MCF10CA1a [12–15]. Migratory speed depended on the particular cell line. Unsurprisingly, for example, the MCF10CA1a cell line exhibited much faster migratory speed relative to the others. But unexpectedly, across different cell lines higher speeds were associated with enhanced size of cooperative cell packs in a manner reminiscent of a peloton [9]. Nevertheless, within each of the cell lines evaluated, cell shape and shape variability from cell-to-cell conformed with predicted structural signatures of cell layer unjamming [1]. Moreover, both structure and migratory dynamics were compatible with previous theoretical descriptions of the cell jamming mechanism [2,10,11,16,17]. As such, these findings demonstrate the richness of the cell jamming mechanism, which is now seen to apply across these cancer cell lines but remains poorly understood.

Folate-coated, long-circulating and pH-sensitive liposomes enhance doxorubicin antitumor effect in a breast cancer animal model

Long circulating pH-sensitive liposomes have been shown to effectively deliver doxorubicin (DOX) to tumors and reduce its toxic effects. Folic acid receptors are upregulated in a wide variety of solid, epithelial tumors, including breast cancer. In order to improve liposomal endocytosis and antitumor activity, folic acid has been added to nanoparticles surfaces to exploit overexpression of folate receptors in tumor cells. The purpose of this study was to evaluate the antitumor activity in vitro and in vivo of long circulating pH-sensitive folate-coated DOX-loaded liposomes (SpHL-DOX-Fol) in a 4T1 breast cancer model system in vitro and in vivo. Biodistribution studies were performed and in vivo electrocardiographic parameters were evaluated. A higher tumor uptake for radiolabeled SpHL-Fol (99mTc-SpHL-Fol) 4 h after intravenous administration was observed in comparision with non-folate-coated liposomes (99mTc-SpHL). Antitumor activity showed that SpHL-DOX-Fol treatment led to a 68% growth arrest and drastically reduce pulmonary metastasis foci. Additionally, eletrocardiographic parameters analysis revealed no dispersion in the QT and QTc interval was observed in liposomal treated mice. In summary, this novel multifunctional nanoplatform deomonstrated higher tumor uptake and antitumor activity. SpHL-DOX-Fol represents a drug delivery platform to improve DOX tumor delivery and reduce dose-limiting toxicity.

MiR-23b and miR-27b are oncogenic microRNAs in breast cancer: evidence from a CRISPR/Cas9 deletion study

BackgroundAltered expression of microRNAs (miRNAs) is known to contribute to cancer progression. miR-23b and miR-27b, encoded within the same miRNA cluster, are reported to have both tumor suppressive and oncogenic activity across human cancers, including breast cancer.MethodsTo clarify this dichotomous role in breast cancer, miR-23b and miR-27b were knocked out using CRISPR/Cas9 gene knockout technology, and the role of endogenous miR-23b and miR-27b was examined in a breast cancer model system in vitro and in vivo.ResultsCharacterization of the knockout cells in vitro demonstrated that miR-23b and miR-27b are indeed oncogenic miRNAs in MCF7 breast cancer cells. miR-23b and miR-27b knockout reduced tumor growth in xenograft nude mice fed a standard diet, supporting their oncogenic role in vivo. However, when xenograft mice were provided a fish-oil diet, miR-27b depletion, but not miR-23b depletion, compromised fish-oil-induced suppression of xenograft growth, indicating a context-dependent nature of miR-27b oncogenic activity.ConclusionsOur results demonstrate that miR-23b and miR-27b are primarily oncogenic in MCF7 breast cancer cells and that miR-27b may have tumor suppressive activity under certain circumstances.

MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors

BackgroundNon-coding RNAs and especially microRNAs have been discovered to act as master regulators of cancer initiation and progression. The aim of our study was to discover and characterize the function of yet functionally uncharacterized microRNAs in human breast carcinogenesis.MethodsIn an unbiased approach, we utilized an established model system for breast cancer (BC) stem cell formation (“mammosphere assay”) to identify whole miRNome alterations in breast carcinogenesis. Clinical samples of BC patients were used to evaluate the human relevance of the newly identified miRNA candidates. One promising candidate, miR-1287-5p, was further explored on its impact on several hallmarks of cancer. The molecular mode of action was characterized by whole transcriptome analysis, in silico prediction tools, miRNA-interaction assays, pheno-copy assays, and drug sensitivity assays.ResultsAmong several other microRNAs, miR-1287-5p was significantly downregulated in mammospheres and human BC tissue compared to normal breast tissue (p < 0.0001). Low expression levels were significantly associated with poor prognosis in BC patients. MiR-1287-5p significantly decreased cellular growth, cells in S phase of cell cycle, anchorage-independent growth, and tumor formation in vivo. In addition, we identified PIK3CB as a direct molecular interactor of miR-1287-5p and a novel prognostic factor in BC. Finally, PI3Kinase pathway chemical inhibitors combined with miR-1287-5p mimic increased the pharmacological growth inhibitory potential in triple negative BC cells.ConclusionOur data identified for the first time the involvement of miR-1287-5p in human BC and suggest a potential for therapeutic interventions in difficult to treat triple negative BC.

Tylophorine Abrogates G2/M Arrest Induced by Doxorubicine and Promotes Increased Apoptosis in T47D Breast Cancer Cells

Background:The effects of tylophorine, a natural alkaloid found in Tylophora indica, administered as a single compound or in combination with doxorubicin on cell cycling and apoptosis were assessed in T47D breast cancer cells, selected as a model system for breast cancer.Methods:Cell cycle distribution and apoptosis were examined by flow cytometry. Caspase 3 and 9 expression was determined by immunocytochemistry.Result:We found that tylophorine did not significantly influence the cell cycle distribution of T47D cells. However, the alkaloid did prevent accumulation of cells in the G2/M phase. In addition, tylophorine increased the number of apoptotic cells. Expression of proapoptotic proteins (caspases 3 and 9) was up-regulated upon administration of tyloporine alone or in combination with doxorubicin.Conclusions:Tylophorine alone or in combination with doxorubicin induced apoptosis in T47D breast cancer cells through modulation of the cell cycle and affecting the expression of caspases 3 and 9.

Curcumin exerts its antitumor effects in a context dependent fashion

Proteome profiling profoundly contributes to the understanding of cell response mechanisms to drug actions. Such knowledge may become a key to improve personalized medicine. In the present study, the effects of the natural remedy curcumin on breast cancer model systems were investigated. MCF-7, ZR-75-1 and TGF-β1 pretreated fibroblasts, mimicking cancer-associated fibroblasts (CAFs), were treated independently as well as in tumor cell/CAF co-cultures. Remarkably, co-culturing with CAF-like cells (CLCs) induced different proteome alterations in MCF-7 and ZR-75-1 cells, respectively. Curcumin significantly induced HMOX1 in single cell type models and co-cultures. However, other curcumin effects differed. In the MCF-7/CLC co-culture, curcumin significantly down-regulated RC3H1, a repressor of inflammatory signaling. In the ZR-75-1/CLC co-culture, curcumin significantly down-regulated PEG10, an anti-apoptotic protein, and induced RRAGA, a pro-apoptotic protein involved in TNF-alpha signaling. Furthermore, curcumin induced AKR1C2, an important enzyme for progesterone metabolism. None of these specific curcumin effects were observed in single cell type cultures. All high-resolution mass spectrometry data are available via ProteomeXchange with the identifier PXD008719. The present data demonstrate that curcumin induces proteome alterations, potentially accounting for its known antitumor effects, in a strongly context-dependent fashion. Biological significanceBetter means to understand and potentially predict individual variations of drug effects are urgently required. The present proteome profiling study of curcumin effects demonstrates the massive impact of the cell microenvironment on cell responses to drug action. Co-culture models apparently provide more biologically relevant information regarding curcumin effects than single cell type cultures.

Limited fibrosis accompanies triple-negative breast cancer metastasis in multiple model systems and is not a preventive target.

The lysophosphatidic acid receptor 1 (LPAR1) is mechanistically implicated in both tumor metastasis and tissue fibrosis. Previously, metastasis was increased when fulminant fibrosis was first induced in mice, suggesting a direct connection between these processes. The current report examined the extent of metastasis-induced fibrosis in breast cancer model systems, and tested the metastasis preventive efficacy and fibrosis attenuation of antagonists for LPAR1 and Idiopathic Pulmonary Fibrosis (IPF) in breast and ovarian cancer models. Staining analysis demonstrated only focal, low-moderate levels of fibrosis in lungs from eleven metastasis model systems. Two orally available LPAR1 antagonists, SAR100842 and EPGN9878, significantly inhibited breast cancer motility to LPA in vitro. Both compounds were negative for metastasis prevention and failed to reduce fibrosis in the experimental MDA-MB-231T and spontaneous murine 4T1 in vivo breast cancer metastasis models. SAR100842 demonstrated only occasional reductions in invasive metastases in the SKOV3 and OVCAR5 ovarian cancer experimental metastasis models. Two approved drugs for IPF, nintedanib and pirfenidone, were investigated. Both were ineffective at preventing MDA-MB-231T metastasis, with no attenuation of fibrosis. In summary, metastasis-induced fibrosis is only a minor component of metastasis in untreated progressive breast cancer. LPAR1 antagonists, despite in vitro evidence of specificity and efficacy, were ineffective in vivo as oral agents, as were approved IPF drugs. The data argue against LPAR1 and fibrosis as monotherapy targets for metastasis prevention in triple-negative breast cancer and ovarian cancer.

UFH-001 cells: A novel triple negative, CAIX-positive, human breast cancer model system

ABSTRACTHuman cell lines are an important resource for research, and are often used as in vitro models of human diseases. In response to the mandate that all cells should be authenticated, we discovered that the MDA-MB-231 cells that were in use in our lab, did not validate based on the alleles of 9 different markers (STR Profile). We had been using this line as a model of triple negative breast cancer (TNBC) that has the ability to form tumors in immuno-compromised mice. Based on marker analysis, these cells most closely resembled the MCF10A line, which are a near diploid and normal mammary epithelial line. Yet, the original cells express carbonic anhydrase IX (CAIX) both constitutively and in response to hypoxia and are features that likely drive the aggressive nature of these cells. Thus, we sought to sub-purify CAIX-expressing cells using Fluorescence Activated Cell Sorting (FACS). These studies have revealed a new line of cells that we have name UFH-001, which have the TNBC phenotype, are positive for CAIX expression, both constitutively and in response to hypoxia, and behave aggressively in vivo. These cells may be useful for exploring mechanisms that underlie progression, migration, and metastasis of this phenotype. In addition, constitutive expression of CAIX allows its evaluation as a therapeutic target, both in vivo and in vitro.