Cultured Breast Cancer Cells Research Articles

Fluorescence Lifetime Imaging of a Caspase-3 Apoptosis Reporter.

Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used. Changes in FRET by fluorescence lifetime imaging microscopy (FLIM) were measured. Unlike FRET measurements based on fluorescence intensity, lifetime measurements are independent of reporter concentration and scattering of light in tissue, making FLIM a robust method for imaging in 3D environments. Apoptosis of breast cancer cells in 2D culture, spheroids, and in vivo murine breast tumor xenografts in response to a variety of genetic and pharmacologic methods implicated in apoptosis of cancer cells was studied. This approach for quantifying apoptosis of cancer cells is based on caspase-3 activity at single-cell resolution using FLIM. © 2017 by John Wiley & Sons, Inc.

Glutaminolysis drives membrane trafficking to promote invasiveness of breast cancer cells

The role of glutaminolysis in providing metabolites to support tumour growth is well-established, but the involvement of glutamine metabolism in invasive processes is yet to be elucidated. Here we show that normal mammary epithelial cells consume glutamine, but do not secrete glutamate. Indeed, low levels of extracellular glutamate are necessary to maintain epithelial homoeostasis, and provision of glutamate drives disruption of epithelial morphology and promotes key characteristics of the invasive phenotype such as lumen-filling and basement membrane disruption. By contrast, primary cultures of invasive breast cancer cells convert glutamine to glutamate which is released from the cell through the system Xc- antiporter to activate a metabotropic glutamate receptor. This contributes to the intrinsic aggressiveness of these cells by upregulating Rab27-dependent recycling of the transmembrane matrix metalloprotease, MT1-MMP to promote invasive behaviour leading to basement membrane disruption. These data indicate that acquisition of the ability to release glutamate is a key watershed in disease aggressiveness.

Therapy response testing of breast cancer in a 3D high-throughput perfused microfluidic platform

BackgroundBreast cancer is the most common invasive cancer among women. Currently, there are only a few models used for therapy selection, and they are often poor predictors of therapeutic response or take months to set up and assay. In this report, we introduce a microfluidic OrganoPlate® platform for extracellular matrix (ECM) embedded tumor culture under perfusion as an initial study designed to investigate the feasibility of adapting this technology for therapy selection.MethodsThe triple negative breast cancer cell lines MDA-MB-453, MDA-MB-231 and HCC1937 were selected based on their different BRCA1 and P53 status, and were seeded in the platform. We evaluate seeding densities, ECM composition (Matrigel®, BME2rgf, collagen I) and biomechanical (perfusion vs static) conditions. We then exposed the cells to a series of anti-cancer drugs (paclitaxel, olaparib, cisplatin) and compared their responses to those in 2D cultures. Finally, we generated cisplatin dose responses in 3D cultures of breast cancer cells derived from 2 PDX models.ResultsThe microfluidic platform allows the simultaneous culture of 96 perfused micro tissues, using limited amounts of material, enabling drug screening of patient-derived material. 3D cell culture viability is improved by constant perfusion of the medium. Furthermore, the drug response of these triple negative breast cancer cells was attenuated by culture in 3D and differed from that observed in 2D substrates.ConclusionsWe have investigated the use of a high-throughput organ-on-a-chip platform to select therapies. Our results have raised the possibility to use this technology in personalized medicine to support selection of appropriate drugs and to predict response to therapy in a real time fashion.

Independently Tuning the Biochemical and Mechanical Properties of 3D Hyaluronan-Based Hydrogels with Oxime and Diels-Alder Chemistry to Culture Breast Cancer Spheroids.

For native breast cancer cell growth to be mimicked in vitro as spheroids, a well-defined matrix that mimics the tumor microenvironment is required. Finding a biomimetic material for 3D cell culture other than Matrigel has challenged the field. Because hyaluronan is naturally abundant in the tumor microenvironment and can be chemically modified, we synthesized a hyaluronan (HA) hydrogel with independently tunable mechanical and chemical properties for 3D culture of breast cancer cells. By modifying HA with distinct bioorthogonal functional groups, its mechanical properties are controlled by chemical cross-linking via oxime ligation, and its biochemical properties are controlled by grafting bioactive peptides via Diels-Alder chemistry. A series of hydrogels were screened in terms of stiffness and peptide composition for cancer spheroid formation. In the optimal hydrogel formulation, the 3D breast cancer spheroids showed decreased drug diffusion into their core and upregulation of cellular multidrug-resistant efflux pumps similar to what is observed in drug-resistant tumors. Our results highlight the potential of these tunable and well-defined gels in drug screening assays.

Induction of a novel isoform of the lncRNA HOTAIR in Claudin-low breast cancer cells attached to extracellular matrix.

Elevated overexpression of the lncRNA HOTAIR mediates invasion and metastasis in breast cancer. In an apparent paradox, we observed low expression of HOTAIR in the invasive Claudin‐low MDA‐MB‐231 and Hs578T cells in two‐dimensional culture (2D). However, HOTAIR expression exhibited robust induction in laminin‐rich extracellular matrix‐based three‐dimensional organotypic culture (lrECM 3D) over that in 2D culture. Induction of HOTAIR required intact ECM signaling, namely integrin α2 and SRC kinase activity. Moreover, invasive growth was suppressed by HOTAIR‐specific siRNA. Induction of HOTAIR in lrECM 3D culture resulted from the activation of a novel isoform of HOTAIR (HOTAIR‐N) whose transcription is started from the first intron of the HOXC11 gene. The HOTAIR‐N promoter exhibited increased trimethylation of histone H3 lysine 4, a histone marker of active transcription, and binding of BRD4, a reader of transcriptionally active histone markers. Inhibition of BRD4 substantially reduced the expression of HOTAIR in lrECM 3D culture. In summary, our results indicate that HOTAIR expression is activated by BRD4 binding to a novel HOTAIR‐N promoter in Claudin‐low breast cancer cells that are attached to ECM. Induction of HOTAIR is required for invasive growth of Claudin‐low breast cancer cells in lrECM 3D culture.

Hydrophilic interaction liquid chromatography tandem mass spectrometry analysis of malonyl-coenzyme A in breast cancer cell cultures applying online solid-phase extraction.

Cofactors such as coenzyme A and its derivatives acetyl-coenzyme A and malonyl-coenzyme A are involved in many metabolic pathways. Due to trace level concentrations in biological samples and the high reactivity of cofactors, a fast, sensitive, and selective method for quantification is mandatory. In this study, online solid-phase extraction was coupled successfully to hydrophilic interaction liquid chromatography with tandem mass spectrometry for isolation of analytes in complex matrix and quantification by external calibration. Online solid-phase extraction was carried out by application of a weak anion-exchange column, whereas hydrophilic interaction liquid chromatography separation was performed on an amide modified stationary phase. Sample preparation of the extracts before the analysis was reduced to a centrifugation and dilution step. Moreover, the applied online solid-phase extraction significantly reduced matrix effects and increased the signal-to-noise ratio. The limit of detection and the limit of quantification were in the lower nanomolar range. Finally, the applicability of this method was demonstrated on MCF-7 breast cancer cell cultures, a commonly used model system, where acetyl-coenzyme A and malonyl-coenzyme A were determined using standard addition procedure in concentrations of 1.98μM and 41nM, respectively.

Effects of the Gold Nanoparticles (AuNPs) on the Proliferation and Morphological Characteristics of Human Breast Cancer Cells (MCF-7) in Culture

Ultrastructural characteristic and morphological changes of untreated and treated breast cancer MCF-7 cells were observed by energy-filtered transmission electron microscope (EFTEM). Morphological observation of MCF-7 after being treated with 13 nm, 50 nm, and 70 nm AuNPs, were looking unhealthy and dying out of the populace, the observed cells were more reduced and dying as treatment with 50 nm and 70 nm AuNPs. Cells detachment, clumping, shrunken, and dispersed cells in the culture medium and floating cells were also observed. The observed morphological changes increase in 50 nm and 70 nm AuNPs than in 13 nm AuNPs, which is less toxic to MCF-7 cells. The presented morphological analysis has established that 13 nm AuNPs showed less toxic to MCF-7 breast cancer cells. Whereas, control cells of MCF-7 were treated with only complete culture media, despite the duration of treatment, whereby the cells maintained most of their morphological features and observed to have a typical morphology of healthy cells that are well attached to the surface. These results indicate that AuNPs were clustered in the cells and there was no significant difference between images of different sizes of AuNPs observed in the cells, because the AuNPs always clustered together inside the cells.

HIF-1α Promotes Breast Cancer Cell MCF-7 Proliferation and Invasion Through Regulating miR-210

Many malignant tumors grow in hypoxic condition, which is associated with tumor growth, invasion, and metastasis. MicroRNAs are of great significance in the development of multiple malignant tumors. This study cultured breast cancer cell MCF-7 under the condition of different concentrations of oxygen, to test cell proliferation and invasion, and detect miR-210 expression, aiming to analyze the influence of hypoxia on breast cancer cell behaviors as well as miR-210 expressions. Breast cancer cell MCF-7 was cultured under normoxia, hypoxia, or anaerobic conditions for 12, 24, or 48 hours. Cell proliferation was detected by MTT assay. Cell invasion and migration were tested by transwell assay. HIF-1α mRNA and miR-210 expressions were determined by real-time polymerase chain reaction. MCF-7 cell proliferation was gradually increased following time extension (p < 0.05). MCF-7 cell exhibited higher proliferation, invasion, and migration activities in hypoxic and anaerobic groups compared with those in normoxic group during the same time period. HIF-1α mRNA and miR-210 were significantly upregulated in anaerobic group compared with those in other groups (p < 0.05). HIF-1α mRNA and miR-210 were obviously elevated at 12, 24, and 48 hours (p < 0.05). MCF-7 cell proliferation was increased, invasion and migration were enhanced, with upregulated expression of HIF-1α mRNA and miR-210 in the hypoxic and anaerobic group following time extension.

Printing of small molecular medicines from the vapor phase

There is growing need to develop efficient methods for early-stage drug discovery, continuous manufacturing of drug delivery vehicles, and ultra-precise dosing of high potency drugs. Here we demonstrate the use of solvent-free organic vapor jet printing to deposit nanostructured films of small molecular pharmaceutical ingredients, including caffeine, paracetamol, ibuprofen, tamoxifen, BAY 11-7082 and fluorescein, with accuracy on the scale of micrograms per square centimeter, onto glass, Tegaderm, Listerine tabs, and stainless steel microneedles. The printed films exhibit similar crystallographic order and chemistry as the original powders; controlled, order-of-magnitude enhancements of dissolution rate are observed relative to powder-form particles. In vitro treatment of breast and ovarian cancer cell cultures in aqueous media by tamoxifen and BAY 11-7082 films shows similar behavior to drugs pre-dissolved in dimethyl sulfoxide. The demonstrated precise printing of medicines as films, without the use of solvents, can accelerate drug screening and enable continuous manufacturing, while enhancing dosage accuracy.

Stromal Stiffness Increases YAP-Dependent Mediated Cell Survival of Breast Cancer Cells

The Extracellular Matrix (ECM) provides biochemical and biophysical cues that activate key molecular pathways that direct cell fate in cancer cell development. These cues are primarily transduced through the integrin family of receptor ECM stiffness, which are known to enhance integrin activity and enhance the malignant behavior of tumors by activating the YAP-TAZ transcription factors. The stroma of cancerous tissues is a potent and active mitigator of malignant transformation. The mechanism by which stromal tension acts to promote resistance has yet to be identified. In the present study, we investigate whether ECM stiffness mediates therapy resistance in premalignant invasive breast cancer tumors by regulating integrin-dependent YAP activity. We used 3D organotypic cultures of invasive breast cancer cells (T4-2 WT, and T4-2 YAP-KO cells) cultured on polyacrylamide gels of varying stiffness (Low [400 Pa], and High [4K Pa]). To determine whether stromal stiffness increases YAP-mediated cell survival, we monitored proliferation (Ki-67) and cell survival (TUNEL) post-irradiation treatment (IR, 8 Gy). Additionally, we looked at the direct effect of YAP activation on tumor grow by injecting T4-2 WT or T4-2 YAP-KO breast cancer cells in Neu mice treated either with a YAP-inhibitor verteporfin (100mg/kg) or a control (IPTG, 10mM/M) for two weeks (5 treatments). Our study shows that irradiated breast cancer cells grown on stiff ECM in vitro lead to a significant increase in YAP nuclear localization. Furthermore, using ANOVA analysis, we observed a significant elevated cell proliferation (P < 0.05) and enhanced cell survival (P < 0.05) leading to the activation of the YAP-TAZ signaling pathways. Our results also showed that inhibition of YAP signaling in stiff ECM cultures treated with IR resulted in a significant reduction of proliferation of breast cancer cells cultured in vitro as well as a reduction of tumor growth in mice (P < 0.05). Overall, these results provide evidence that stiff micro-environmental conditions of breast cancer cells are a potent mitigator of therapy resistance whose effects are mediated through YAP activity.

Effects of Chronic and Intermittent Calorie Restriction on Adropin Levels in Breast Cancer

ABSTRACTAdropin is a peptide hormone that has been implicated in insulin resistance and as a potential regulator of growth. The aim of this study is to determine the effect of calorie restriction on circulating levels of adropin in the MMTV-TGFα breast cancer mouse model and investigate the effects of adropin peptide on the viability of MCF-7 and MDA-231 breast cancer cells in culture. Ten-week-old mice were assigned to either ad libitum-fed (AL), chronic calorie-restricted, or intermittent calorie-restricted groups. Concentrations of serum adropin were measured using an enzyme-linked immunosorbent assay. Results showed an inverse correlation between serum adropin levels and mouse age that was attenuated by calorie restriction. In the AL group the level of adropin was significantly lower at week 50 compared to levels at week 10. However, among the calorie-restricted groups, serum levels of adropin remained high at week 50. The cell-line-specific effects were observed after treatment of cancer cell lines with a series of adropin concentrations (5, 10, 25, 50 ng/mL). Flow cytometry analysis showed that MCF-7 cells entered the early phase of apoptosis after treatment with 50 ng/mL for 24 h. Adropin may be involved in the protective effects that calorie restriction has on breast cancer risk.

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

Lattice light sheet imaging of membrane nanotubes between human breast cancer cells in culture and in brain metastases

Membrane nanotubes are cytosolic protrusions with diameters <1 µm that extend between cells separated by tens of µm. They mediate several forms of intercellular communication and are upregulated in diverse diseases. Difficulties in visualizing and studying nanotubes within intact tissues have, however, prompted skepticism regarding their in vivo relevance, and most studies have been confined to cell culture systems. Here, we introduce lattice-light sheet imaging of MDA-MB-231 human breast cancer cells genetically engineered to brightly express membrane–targeted GFP as a promising approach to visualize membrane nanotubes in vitro and in situ. We demonstrate that cultured cells form multiple nanotubes that mediate intercellular communication of Ca2+ signals and actively traffic GFP-tagged membrane vesicles along their length. Furthermore, we directly visualize nanotubes in situ, interconnecting breast cancer cells in live acute brain slices from an experimental mouse model of breast cancer brain metastasis. This amenable experimental system should facilitate the transition of the study of intercellular communication by membrane nanotubes from cell culture to the whole animal.

Antibody and Antibody Mimetic Immunotherapeutics

Antibody and Antibody Mimetic Immunotherapeutics

Electrospinning PCL Scaffolds Manufacture for Three-Dimensional Breast Cancer Cell Culture.

In vitro cell culture is traditionally performed within two-dimensional (2D) environments, providing a quick and cheap way to study cell properties in a laboratory. However, 2D systems differ from the in vivo environment and may not mimic the physiological cell behavior realistically. For instance, 2D culture models are thought to induce cancer stem cells (CSCs) differentiation, a rare cancer cell subpopulation responsible for tumor initiation and relapse. This fact hinders the development of therapeutic strategies for tumors with a high relapse percentage, such as triple negative breast cancer (TNBC). Thus, three-dimensional (3D) scaffolds have emerged as an attractive alternative to monolayer culture, simulating the extracellular matrix structure and maintaining the differentiation state of cells. In this work, scaffolds were fabricated through electrospinning different poly(ε-caprolactone)-acetone solutions. Poly(ε-caprolactone) (PCL) meshes were seeded with triple negative breast cancer (TNBC) cells and 15% PCL scaffolds displayed significantly (p < 0.05) higher cell proliferation and elongation than the other culture systems. Moreover, cells cultured on PCL scaffolds exhibited higher mammosphere forming capacity and aldehyde dehydrogenase activity than 2D-cultured cells, indicating a breast CSCs enrichment. These results prove the powerful capability of electrospinning technology in terms of poly(ε-caprolactone) nanofibers fabrication. In addition, this study has demonstrated that electrospun 15% PCL scaffolds are suitable tools to culture breast cancer cells in a more physiological way and to expand the niche of breast CSCs. In conclusion, three-dimensional cell culture using PCL scaffolds could be useful to study cancer stem cell behavior and may also trigger the development of new specific targets against such malignant subpopulation.

Enhanced efficacy of anti-miR-191 delivery through stearylamine liposome formulation for the treatment of breast cancer cells

MicroRNAs are gaining rapid attention as promising targets for cancer treatment; however, efficient delivery of therapeutic miRNA or anti-miRNA into cancer cells remains a major challenge. Our previous work identified miR-191 as an oncogenic miRNA overexpressed in breast cancer that assists in progression of malignant transformation. Thus, inhibition of miR-191 using antisense miR-191 (anti-miR-191) has immense therapeutic potential. Here, we have developed a stearylamine (SA) based cationic liposome for delivery of miR-191 inhibitor (anti-miR-191), and studied its efficacy in breast cancer cells (MCF-7 and ZR-75-1) in culture. SA liposomes alone inhibited cancer cell growth with lesser IC50s (50% inhibitory concentration) values as compared to normal mouse fibroblast cells (L929). The efficient delivery of anti-miR-191 in SA liposome complex was found to be highly effective in killing the cancer cells than a comparable dose of SA free anti-miR-191 liposome complex. The formulation also showed negligible cytotoxicity in human erythrocytes. Combined treatment of SA liposome with anti-miR-191 markedly enhanced apoptotic cell death and suppressed the migration of cancer cells in vitro. Notably, anti-miR-191 loaded SA liposome complex increased chemosensitivity of breast cancer cells to currently used anti-cancer drugs (doxorubicin or cisplatin) in free form. Our work demonstrates that anti-miR-191 loaded in SA liposome complex has promising clinical application for breast cancer therapy.

Abstract 5265: Breast cancer stem cell inhibition by benzyl isothiocyanate is hampered by induction of Kruppel-like factor 4

Abstract Breast cancer is a leading cause of cancer-related deaths among women in the United States. Over the last decade, breast cancer stem cells (bCSC) have been identified and characterized as a key player in breast cancer progression and recurrence. Benzyl isothiocyanate (BITC) is a highly promising cancer chemopreventive constituent of edible cruciferous vegetables such as garden cress. We have shown previously that BITC inhibits bCSC in cultured breast cancer cells (MCF-7 and SUM159 cells) and in spontaneous tumors of mouse mammary tumor virus-neu (MMTV-neu) mice in vivo. Krüppel-like factor 4 (KLF4), a zinc finger transcription factor, is known as both oncogene and tumor suppressor in various types of human cancers and plays an important role in reprogramming differentiated somatic cells into induced pluripotent stem cells. The present study demonstrates that KLF4 impedes bCSC inhibition by BITC. Exposure of breast cancer cells (MCF-7, MDA-MB-231, and SUM159) to pharmacological concentrations of BITC (2.5 and 5 µM) resulted in induction of KLF4 protein, which was accompanied by increase of its transcription. In addition, BITC treatment increased nuclear level of KLF4 in breast cancer cells. KLF4 protein expression was also higher in tumors form BITC-treated MMTV-neu mice compared with the control group. Ectopic expression of KLF4 in MCF-7 cells conferred marked protection against BITC-mediated inhibition of bCSC as evidenced by aldehyde dehydrogenase 1 (ALDH1) activity. Consistent with these observations, BITC-mediated inhibition of bCSC in MCF-7, MDA-MB-231 and SUM159 cells was significantly augmented by RNA interference of KLF4 as evidenced by ALDH1 activity and frequency of mammospheres. KLF4 overexpression also increased cell migration capacity of MCF-7 cells. Furthermore knockdown of KLF4 augmented BITC-mediated inhibition of cell migration in SUM159 cells. These results suggest that anticancer activity of BITC may be enhanced by KLF4 inhibition or knockdown in breast cancer cells. This study was supported by the grant RO1 CA129347-09 awarded by the National Cancer Institute. Citation Format: Su-Hyeong Kim, Yong Wan, Shivendra V. Singh. Breast cancer stem cell inhibition by benzyl isothiocyanate is hampered by induction of Kruppel-like factor 4 [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 5265. doi:10.1158/1538-7445.AM2017-5265

Abstract 4851: NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells

Abstract Background: NOTCH oncogenic signaling induces epithelial-to-mesenchymal transition (EMT) and tumor stemness that play a critical role in driving tumor progression. Following interaction of NOTCH receptors (NOTCH 1, 2, 3, and 4) with their ligands (DELTA-LIKE 1, 3, 4 and JAGGED 1, 2), γ- secretase complex performs an intra-membrane cleavage releasing the NOTCH intracellular domain (NNICD). NNICD translocates to the nucleus and induces the expression of HEY1 and HES1 transcription factors that in turn orchestrate NOTCH-mediated EMT and stemness reprogramming. Materials and Methods: Parental MCF-7 and unique metastatic MCF-7RAF-1 xenografts with constitutively active MAPK signaling (D’Assoro et al., Oncogene 2014: 33:599-610) were used to define the extent to which increased expression of NOTCH signaling was linked to distant metastases. SKY analysis was performed to assess the grade of chromosomal instability (CIN) in tumor xenografts. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of CD24 luminal marker was characterized by FACS analysis and immunofluorescence. Global transcriptome analysis was performed using Affymetrix® microarray. Highly invasive MDA-MB 231 breast cancer cells were used to perform a tail vein cancer cell-seeding assay. The LY411575 γ-secretase inhibitor (Pan-NOTCH inhibitor) was used to block NOTCH signaling in MDA-MB 231 cells. Results: Metastasis Initiating Cells (MICs) isolated from the lungs of nude mice carrying MCF-7RAF-1 xenografts exhibited a higher degree of genomic stability than primary tumor xenografts that was characterized by the absence of centrosome amplification and non-clonal chromosomal abnormalities. MICs also showed higher mammosphere forming capacity that was linked to lower expression of luminal marker CD24. Significantly, global transcriptome analysis showed that expression of NOTCH3 and HES1 stemness genes was increased in MICs. To corroborate the role of NOTCH signaling in inducing metastatic seeding, MDA-MB 231 breast cancer cells were pre-treated with 1 µM of LY411575 After 72 hours viable cells were injected into the tail vein of immune-compromised mice to develop lung metastases. While animals injected with untreated MDA-MB 231 cells developed lung metastases, those injected with the LY411575-treated MDA-MB 231 cells failed to develop lung metastatic lesions. Conclusions: These preliminary findings demonstrate the role of NOTCH stemness signaling in promoting metastatic seeding of breast cancer cells. Since NOTCH signaling is a “druggable target”, these results also provide the rationale to develop novel NOTCH-targeted therapies to inhibit tumor stemness and early onset of distant metastases. Citation Format: Alexey Leontovich, Jeffrey Salisbury, Candace L. Haddox, Lisa Mills, Tufia Haddad, James McCubrey, Matthew Goetz, James Ingle, Evanthia Galanis, Antonino B. D'Assoro. NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells [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 4851. doi:10.1158/1538-7445.AM2017-4851

Abstract 5942: Collagen proly-hydroxylase regulates breast cancer progression and chemoresistance

Abstract Collagen prolyl hydroxylation (P4H), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. Upregulation of collagen prolyl-4-hydroxylase in cancer cells is required for breast cancer promotion. P4HA1 mRNA levels were significantly upregulated in triple-negative breast cancer (TNBC) compared to other cancer subtypes. Increased mRNA levels of P4HA1 correlated with poor clinical outcome in breast cancer patients. Silencing P4HA1 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen. We found that activation of the P4HA1 in TNBC cells enhanced cancer cell stemness. We also found that knockdown of P4HA1 inhibited mammary tumor growth and metastasis to lungs in xenograft models. Inhibition of P4HA1 activity with the P4H inhibitor sensitized TNBC to the docetaxel treatment in the patient-derived organoid (PDO) and xenograft models. Therefore, these results suggest the crucial role of P4HA1 in breast cancer progression and that targeting collagen P4H is a promising strategy to inhibit tumor progression and to sensitize TNBC to chemotherapy. Citation Format: Gaofeng Xiong, Ren Xu. Collagen proly-hydroxylase regulates breast cancer progression and chemoresistance [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 5942. doi:10.1158/1538-7445.AM2017-5942

Abstract LB-171: Throwing the 'book' at the cancer cell speciifically: A new paradigm and a new strategy for cancer prevention and cure

Abstract There are numerous reports of natural compounds that act on cancer pathways. These compounds work at high micromolar concentrations that cannot be achieved under in vivo conditions. Toxic side effects are a concern when such high concentrations are used. Also synergism between different natural compounds have been explored using 2 or 3 compounds together, but these have still not been able to kill 100% of the cancer cells. To develop the new paradigm, we considered general features common to several cancers viz. proliferation (due to cell cycle changes), survival of cancer cell &amp; metastasis controlling pathways. In addition pathways involved in programmed cell death (apoptosis) were given consideration. We chose 6 natural compounds &amp; created a ‘phytochemical super cocktail’ capable of killing 100% of breast cancer cells (MCF7, MDA-MB-231) in tissue culture, by simultaneously affecting all of the above mentioned pathways( J.Cancer 4, 703-715, 2013 ). The super cocktail had no effect on normal cells ( mesenchymal stem cells, NIH 3T3 cells, normal mammary epithelial cells). However, many of these compounds are lipophilic and not hydrophyllic, thus having poor absorption resulting in low bioavailability. By inclusion in the super cocktail, we demonstrated that the synergism between compounds can be extended to a degree wherein bioavailable/bioachievable levels also become effective levels that can kill cancer cells, without affecting normal cells. This represents a new strategy to achieve the effects predicted by the paradigm which can be used now to prevent/cure cancers. We now have demonstrated that it can be applied to several cancers; as examples, we have successfully killed BRCA1 mutation carrying breast cancer cells (SUM-149, separate abstract submitted).using the super cocktail. Here we show that it can kill prostate cancer cells(PC3), &amp; non-small cell lung cancer cells in a dose dependent manner, using proliferation assay. These data will be presented to demonstrate the usefulness of this new approach immediately to prevent/cure cancers. We have already shown the efficacy of C-phycocyanin, one the compounds in the super cocktail, to prevent mammary tumor formation in rats ( induced by dimethyl benzanthracene). Inclusion of spirulina extract in the diet reduced the mortality from 85% to about 13% (Am.Jour.Pathol 184, 296-303, 2014 ) &amp; induced lysis of formed tumors. However C-phycocyanin, the active compound of spirulina, by itself was not fully effective in killing human breast cancer cells, unlike the super cocktail. This indicates a greater in vivo efficacy of the super cocktail &amp; we are planning to evaluate the same in the mouse xenograft model. At this writing, 750 women (normal and breast cancer bearing) have used the super cocktail as a nutritional supplement over the past 2 years without a single report of adverse reactions or toxicity. Support: Louisiana Cancer Research Consortium. Citation Format: Madhwa H. Raj, Shubha K. Ireland, Hector Biliran, Shailaja G. Raj. Throwing the 'book' at the cancer cell speciifically: A new paradigm and a new strategy for cancer prevention and cure [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 LB-171. doi:10.1158/1538-7445.AM2017-LB-171