Normal Human Breast Cells Research Articles

Serine protease modulation of Dependence Receptors and EMT protein expression

ABSTRACT Expression of the tumour suppressor Deleted in Colorectal Cancer (DCC) and the related protein neogenin is reduced by the mammalian serine protease chymotrypsin or the bacterial serine protease subtilisin, with increased cell migration. The present work examines whether these actions are associated with changes in the expression of cadherins, β-catenin and vimentin, established markers of the Epithelial-Mesenchymal Transition (EMT) which has been linked with cell migration and tumour metastasis. The results confirm the depletion of DCC and neogenin and show that chymotrypsin and subtilisin also reduce expression of β-catenin in acutely prepared tissue sections but not in human mammary adenocarcinoma MCF-7 or MDA-MB-231 cells cultured in normal media, or primary normal human breast cells. A loss of β-catenin was also seen in low serum media but transfecting cells with a dcc-containing plasmid induced resistance. E-cadherin was not consistently affected but vimentin was induced by low serum-containing media and was increased by serine proteases in MCF-7 and MDA-MB-231 cells in parallel with increased wound closure. Vimentin might contribute to the promotion of cell migration. The results suggest that changes in EMT proteins depend on the cells or tissues concerned and do not parallel the expression of DCC and neogenin. The increased cell migration induced by serine proteases is not consistently associated with the expression of the EMT proteins implying either that the increased migration may be independent of EMT or supporting the view that EMT is not itself consistently related to migration. (241).

The effect of size and aspect ratio of Fe-MIL-88B-NH2 metal-organic frameworks on their relaxivity and contrast enhancement properties in MRI: in vitro and in vivo studies

Metal-organic frameworks (MOFs) have shown a great potential as a novel contrast agent in magnetic resonance imaging (MRI) due to their unique properties including versatile structures, composition, and surface chemistry. Fe-MIL-88B-NH2 (MILs) was synthesized using Fe+3 ions and 2-aminoterephthalic acids (NH2-BDC) in the presence of acetic acid and Pluronic F127 under a hydrothermal process. Three MILs in different sizes were synthesized by using variant CH3COOH/Fe+3 molar ratios and denoted as MIL-S, MIL-M, and MIL-L having average sizes of 60, 350, and 730 nm, respectively. The effect of aspect ratio and particle size of MILs was investigated on proton relaxivity and contrast enhancement in aqueous, in vitro, and in vivo MR images. MRI study indicated that by increasing the MILs size, the incremental transverse relaxivity was achieved from 13.53 to 50.80 for r2 and from 38.75 to 60.25 for r2*, while the difference between r2 and r2* was decreased obviously. The r2/r1 ratios were found to be 5.80, 42.27, and 127.00 for MIL-S, MIL-M, and MIL-L, respectively. All MILs demonstrated T2-weighted MR imaging performance. Furthermore, no susceptibility artifacts were observed on T2-weighted images of MILs. By decreasing the size of MILs, r2/r1 ratio was reduced sharply and T1 contrast was also observed for MIL-S. MIL-S was used for in vitro and in vivo study due to its capability in simultaneous T1 and T2 contrast enhancements. MTT and hemolysis assays showed no significant cytotoxicity and hemolysis for MIL-S against normal human breast cell line (MCF-10A) and red blood cells, respectively, at concentrations of Fe+3 up to 300 μg/mL (p < 0.05). T1-weighted images acquired from in vitro study interestingly showed that the signal intensity of MIL-S is more than Dotarem. The in vivo experiment revealed that the signal intensity of mice liver decreased sharply after injection resulting in increase of negative contrast enhancement. The negative contrast enhancement decreased gradually by the time under T2-weighted imaging mode confirming application of MIL-S as a T2 contrast agent in biomedical applications (up to 12 h post-injection). Although a decrease of the size resulted in bright contrast in aqueous solution for MIL-S, it did not show proper efficiency for in vivo positive contrast enhancement. The advantages, including simple preparation and accessibility, acceptable potency and selectivity, blood compatibility, and suitable cytocompatibility as well as enough circulation time, make MIL-S as a dual MRI contrast agent to differentiate between low-contrast tissues for in vivo experiments.

Effects of phthalates on normal human breast cells co-cultured with different fibroblasts.

Whether or not phthalates play a role in breast carcinogenesis remains to be determined. The goal of this study was to explore the effects of phthalates on the growth of normal MCF-10A breast cells modulated by breast fibroblasts. Fibroblasts were derived from normal mammary tissue adjacent to both estrogen receptor (ER) positive and negative primary breast cancers, which were grown separately from nontumorigenic MCF-10A epithelial cells. MCF-10A co-culture cells were treated with 10 nM 17β-estradiol (E2), Butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(20ethylhexyl) phthalate (DEHP) (10 and 100 nM). After incubation for 120 hours, the cells were harvested and extracted for MTT assay. Western blot analysis was used to evaluate the proliferative pathway proteins and the effects on ER α. Only fibroblasts from ER (+) breast cancer significantly stimulated proliferation of MCF-10A cells. Exposure of the co-culture to E2, BBP, DBP, DEHP, and E2 combined with one of these phthalates resulted in significantly increased cell proliferation, as well as proliferating cell nuclear antigen (PCNA) and ER α expressions. The present study demonstrates that phthalates express a significant influence in fibroblast–epithelial interactions, similarly to the effects of E2 on breast cells. The effects of phthalates on normal breast cells depend upon ER modulating actions. In breast carcinogenesis, phthalates should be considered as having endocrine disrupting potential, even at low concentrations.

Synergistic effect of shape-selective silver nanostructures decorating reduced graphene oxide nanoplatelets for enhanced cytotoxicity against breast cancer

Graphene-based nanomaterials contain unique physicochemical properties and have been widely investigated due to a variety of applications particularly in cancer therapy. Furthermore, Ag has been known for its extensive historical background for biomedical applications. Therefore, conjugation of shape-selective Ag nanostructures with graphene may provide new horizons for pharmaceutical applications such as cancer treatments. Here we report on the synthesis of Ag nanoparticles (NPs)/reduced graphene oxide (AgNPs/RGO) conjugate nanomaterials containing various shapes of AgNPs by a novel and simple synthesis route using the deformation of dimethylformamide (DMF) as the reducing and coupling agent. The cytotoxicity and anticancer properties of AgNPs, AgNPs/RGO conjugate nanomaterials, RGO and graphene oxide (GO) were probed against MDA-MB-231 cancer and MCF-10A normal human breast cells in vitro. The AgNPs/RGO nanocomposites exhibited a strong anticancer effect by penetration and apoptosis in cancer cells as well as the lowest influence on the viability of normal cells. It was found that cancer cell viability not only depends on the geometry of Ag nanostructures but also on the interaction between AgNPs and RGO nanoplatelets. It is suggested that AgNPs/RGO conjugate nanomaterials with various shapes of AgNPs is a promising therapeutic platform for cancer therapy.

SV-BR-1-GM, a Clinically Effective GM-CSF-Secreting Breast Cancer Cell Line, Expresses an Immune Signature and Directly Activates CD4+ T Lymphocytes.

Targeted cancer immunotherapy with irradiated, granulocyte–macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study (ClinicalTrials.gov NCT00095862), a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA, HLA-DRB3, HLA-DMA, HLA-DMB), in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M, HLA-A, HLA-B), ADA (encoding adenosine deaminase), ADGRE5 (CD97), CD58 (LFA3), CD74 (encoding invariant chain and CLIP), CD83, CXCL8 (IL8), CXCL16, HLA-F, IL6, IL18, and KITLG. Moreover, both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele, raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells, thereby inducing a tumor-directed immune response. In support of this, SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43–59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus, the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together, our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells.

Preparation and mechanistic aspect of natural xanthone functionalized gold nanoparticle

Herein, a facile scale up and shape variable synthesis of gold nanoparticle (AuNP) and reaction mechanism by natural xanthone derivative (mangiferin) has been reported. Mangiferin (C19H18O11; 1,3,6,7-tetrahydroxyxanthone-C2-β-d-glucoside), a xanthone derivative is isolated from Mangifera indica L. leaves which efficiently reduces Au3+ ions to Au0 and stabilizes the formed AuNP. The structural, optical and plasmonic properties of synthesized AuNP have been investigated through different instrumental techniques like UV–Vis and FTIR spectroscopy, powder XRD, FESEM and TEM analysis. It is observed that variation of the concentration of Au3+ ions and mangiferin has a great effect on controlling size and shape of nanoparticles. The role of reaction temperature is also notable. An interesting observation is that with same concentration ratio of HAuCl4/mangiferin (0.025 mM/0.002%) at the room temperature kidney shaped AuNP is produced, whereas it is spherical at boiling temperature. Moreover, mangiferin allows high scale synthesis of AuNPs (0.025 mM to 10 mM) without changing the particles size and shape. The mechanistic investigation through UV–Vis, FTIR and GCMS analyses reveal the cleavage of glucose unit and oxidation of phenolic OH groups during AuNP formation. Non-toxicity of mangiferin conjugated AuNP on normal human breast cell line (MCF-10A) suggesting its future application as a drug delivery system and other related medicinal purposes.

Panduratin A Inhibits Cell Proliferation by Inducing G0/G1 Phase Cell Cycle Arrest and Induces Apoptosis in Breast Cancer Cells.

Because of the unsatisfactory treatment options for breast cancer (BC), there is a need to develop novel therapeutic approaches for this malignancy. One such strategy is chemotherapy using non-toxic dietary substances and botanical products. Studies have shown that Panduratin A (PA) possesses many health benefits, including anti-inflammatory, anti-bacterial, anti-oxidant and anticancer activities. In the present study, we provide evidence that PA treatment of MCF-7 BC cells resulted in a time- and dose-dependent inhibition of cell growth with an IC50 of 15 μM and no to little effect on normal human MCF-10A breast cells. To define the mechanism of these anti-proliferative effects of PA, we determined its effect critical molecular events known to regulate the cell cycle and apoptotic machinery. Immunofluorescence and flow cytometric analysis of Annexin V-FITC staining provided evidence for the induction of apoptosis. PA treatment of BC cells resulted in increased activity/expression of mitochondrial cytochrome C, caspases 7, 8 and 9 with a significant increase in the Bax:Bcl-2 ratio, suggesting the involvement of a mitochondrial-dependent apoptotic pathway. Furthermore, cell cycle analysis using flow cytometry showed that PA treatment of cells resulted in G0/G1 arrest in a dose-dependent manner. Immunoblot analysis data revealed that, in MCF-7 cell lines, PA treatment resulted in the dose-dependent (i) induction of p21WAF1/Cip1 and p27Kip1, (ii) downregulation of Cyclin dependent kinase (CDK) 4 and (iii) decrease in cyclin D1. These findings suggest that PA may be an effective therapeutic agent against BC.

Protective effects of a phenolic glycoside compound curculigoside on H2O2-induced oxidative stress and cytotoxicity in normal human breast epithelial cells

Oxidative stress has been implicated in the development of many diseases including cancer. Curculigoside, a phenolic glycoside, has been reported to protect various models against damage induced by oxidative stress. However, the protective effect of curculigoside on oxidative stress in normal human breast cells remains unclear. This study evaluated whether curculigoside protected MCF 10A human breast epithelial cells from H2O2-induced cytotoxicity and explored its underlying mechanisms. The H2O2-induced oxidative stress of cell models was demonstrated by decreased percentages of cell viability, increased levels of ROS, reduced activities in antioxidant enzymes, and inhibition of the expressions of antioxidant proteins. More importantly, pre-treatment with curculigoside significantly inhibited the H2O2-induced apoptosis in this cell model. Together, curculigoside significantly prevented the cytotoxic effects of H2O2, suggesting that it protected the cells against H2O2-induced oxidative damage. Our findings provide new insight into the cytoprotective effects of curculigoside and the possible mechanisms underlying these effects.

Breast Cancers Activate Stromal Fibroblast-Induced Suppression ofProgenitors in Adjacent Normal Tissue.

SummaryHuman breast cancer cells are known to activate adjacent “normal-like” cells to enhance their own growth, but the cellular and molecular mechanisms involved are poorly understood. We now show by both phenotypic and functional measurements that normal human mammary progenitor cells are significantly under-represented in the mammary epithelium of patients' tumor-adjacent tissue (TAT). Interestingly, fibroblasts isolated from TAT samples showed a reduced ability to support normal EGF-stimulated mammary progenitor cell proliferation in vitro via their increased secretion of transforming growth factor β. In contrast, TAT fibroblasts promoted the proliferation of human breast cancer cells when these were co-transplanted in immunodeficient mice. The discovery of a common stromal cell-mediated mechanism that has opposing growth-suppressive and promoting effects on normal and malignant human breast cells and also extends well beyond currently examined surgical margins has important implications for disease recurrence and its prevention.

DNA binding and in vitro anticarcinogenic activity of a series of newfashioned Cu(II)-complexes based on tricationic metalloporphyrin salicyloylhydrazone ligands

Four novel water-soluble Cu(II) derivatives based on the meso-10, 15, 20-Tris (N-methyl-X-pyridyl)-5-(4′-salicyloylhydrazone) metalloporphyrin ligands (X=4, M=Zn(1) Co(2); X=2, M=Zn(3), Co(4)), have been prepared and isolated. Various physicochemical techniques indicate that complex 2 interacts with calf thymus DNA stronger than the others through partial intercalation, suggesting that Co(II) has no axial ligands at porphyrin core plays a crucial role, interestingly, 2 exhibits higher DNA binding affinity compared to 4, which could be ascribed to the influences of peripheral electronic effect of porphyrin ring. Cytotoxicity studies manifest all conjugates possess superior cytotoxicity towards non-small cell lung cancer (A549) and liver hepatocellular carcinoma (HepG2) but weak toxicity for human normal breast cells (Hs 578Bst) boiled down to the tumor selectivity of porphyrin. In addition, the IC50 value of 1 is lower than its analogues against HepG2 cells when cultivated 72h, thus the effects of representative complex 1 on cell morphological and cell cycle have been tailed.

Comparative study of the effects of different radiation qualities on normal human breast cells

BackgroundAs there is a growing number of long-term cancer survivors, the incidence of carcinogenesis as a late effect of radiotherapy is getting more and more into the focus. The risk for the development of secondary malignant neoplasms might be significantly increased due to exposure of healthy tissue outside of the target field to secondary neutrons, in particular in proton therapy. Thus far, the radiobiological effects of these neutrons and a comparison with photons on normal breast cells have not been sufficiently characterised.MethodsMCF10A cells were irradiated with doses of up to 2 Gy with neutrons of different energy spectra and X-rays for comparison. The biological effects of neutrons with a broad energy distribution (<En > = 5.8 MeV), monoenergetic neutrons (1.2 MeV, 0.56 MeV) and of the mixed field of gamma’s and secondary neutrons (<En > = 70.5 MeV) produced by 190 MeV protons impinging on a water phantom, were analysed. The clonogenic survival and the DNA repair capacity were determined and values of relative biological effectiveness were compared. Furthermore, the influence of radiation on the sphere formation was observed to examine the radiation response of the potential fraction of stem like cells within the MCF10A cell population.ResultsX-rays and neutrons caused dose-dependent decreases of survival fractions after irradiations with up to 2 Gy. Monoenergetic neutrons with an energy of 0.56 MeV had a higher effectiveness on the survival fraction with respect to neutrons with higher energies and to the mixed gamma - secondary neutron field induced by proton interactions in water. Similar effects were observed for the DNA repair capacity after exposure to ionising radiation (IR). Both experimental endpoints provided comparable values of the relative biological effectiveness. Significant changes in the sphere formation were notable following the various radiation qualities.ConclusionThe present study compared the radiation response of MCF10A cells after IR with neutrons and photons. For the first time it was shown that monoenergetic neutrons with energies around 1 MeV have stronger radiobiological effects on normal human breast cells with respect to X rays, to neutrons with a broad energy distribution (<En > = 5.8 MeV), and to the mixed gamma - secondary neutron field given by interactions of 190 MeV protons in water. The results of the present study are highly relevant for further investigations of radiation-induced carcinogenesis and are very important in perspective for a better risk assessment after secondary neutron exposure in the field of conventional and proton radiotherapy.

The relationship between MMP-2 and MMP-9 expression levels with breast cancer incidence and prognosis.

The relationship between the expression levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 and breast cancer prognosis was studied. Two breast cancer cell lines (MDA-MB-231 and MCF-7) and one human normal breast cell line (HS578Bst) were investigated. Fluorescence real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect cellular mRNA and protein MMP-2 and MMP-9 expression levels. Breast cancer tissue samples from 80 patients and tumor-adjacent normal tissue samples from 40 patients were collected, and MMP-2 and MMP-9 expression in these samples were examined using immunohistochemistry (IHC). The relationship of MMP-2 and MMP-9 expression levels with breast cancer patient clinicopathological parameters and prognosis was analyzed. RT-PCR and western blot results showed that MMP-2 and MMP-9 mRNA and protein expression levels were significantly higher in MDA-MB-231 and MCF-7 cells than in HS578Bst cells. A high expression of MMP-2 and MMP-9 was found in 83.75% (67/80) and 78.75% (63/80) of breast cancer tissue samples, respectively. MMP-2 and MMP-9 expression in breast cancer tissues were significantly different from that in tumor-adjacent normal tissues (p<0.01). MMP-2 and MMP-9 expression levels in breast cancer tissues were correlated with lymph node metastasis and tumor staging. Single factor survival analysis showed that MMP-2 and MMP-9 were factors influencing breast cancer prognosis. MMP-2 and MMP-9 are highly expressed in breast cancer tissues and are closely related to lymph node metastasis and tumor staging. MMP-2 and MMP-9 can be used as reference indices for guiding breast cancer prognosis and treatment.

Elaborated DNA-binding specialities, in vitro medicated apoptosis and restrained malignancy of neotype water-soluble cationic porphyrin acylhydrazone Cu-complex

Tetrad novel Cu-complex containing multifarious meso-10,15,20-Tris (N-methyl-2′-pyridyl)-5-(4′-X-acylhydrazone) porphyrin ligands, where X respectively referred to salicylaldehyde (Cu-P1), o-Vanillin (Cu-P2), pyridine-2-carboxaldehyde (Cu-P3) and acetone (Cu-P4), have been successfully isolated and well characterized. Multiple physicochemical approaches reveal the complex Cu-P4 with preferable binding affinity than its analogues due to smaller molecular dimension. Moreover, the mechanism of the anti-neoplasm action of these conjugates have been appraised via MTT assay, manifesting all of them possess superior cytotoxicity towards A549 (non-small cell lung cancer) and HepG2 (liver hepatocellular carcinoma cells) but weak cell toxicity for Hs 578Bst (human normal breast cells) which might be boiled down to porphyrin derivatives could enrich around malignant neoplasms. The flow cytometric analysis illustrates that delegate compound Cu-P4 induced cell death predominantly through late apoptosis and arrested S phase cells.

Abstract 4915: Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells

Abstract Background and Objective: The epidemiological and experimental studies suggest that alcohol consumption is associated with increased risk for breast cancer development and metastasis. However, the mechanisms of alcohol-induced breast cancer progression and metastasis remain unknown. In this study, we examined the roles of CCN5, a tumor suppressor gene in alcohol induced breast cancer cell migration/invasion, epithelial to mesenchymal transition (EMT) and stemness. Methods: Estrogen receptor-positive (ER+) human breast cancer cell line MCF-7 and immortalized normal human epithelial breast cell line MCF-12A were treated with ethanol at various concentrations in the presence and absence of human recombinant human CCN5 protein (hrCCN5) . Invasive/migratory ability of treated and untreated cells was measured by Boyden chamber assays. The mRNA and protein expression level of CCN5 was determined by real-time qRT-PCR and Western blot. EMT and stemness markers were evaluated by Western blot. Single-cell suspensions from pre-treated cells were re-suspended at a density of 500 cells/ml mammocult media in ultralow attachment dishes. Number as well as the size of the mammosphere in specified experimental set-up was monitored and recorded alternate day for 8-10 days. Results: The studies demonstrated that alcohol promotes significantly the invasive/migratory ability and EMT phenotypes of breast cancer cells through the suppression of CCN5. Interestingly, hrCCN5 protein treatment suppresses the effect of alcohol and reprograms MET (mesenchymal to epithelial transition). In addition, alcohol-induced mammosphere formation efficiency is also suppressed by hrCCN5 protein-treatment. Conclusions: Our findings suggest that the CCN5 signaling plays a preventive role in alcohol-induced reprogramming of tumor plasticity in breast cancer cells. Citation Format: Inamul Haque, Arvind Subramanian, Vijayalaxmi Gupta, Sandipto Sarkar, Snigdha Banerjee, Sushanta K. Banerjee. Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in 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 4915. doi:10.1158/1538-7445.AM2017-4915

Abstract 5486: Oxidative stress and selective protein carbonylation in human breast cancer tissue

Abstract Protein carbonylation is an irreversible modification to the side chain of amino acid residues induced by severe oxidative stress. Reactive oxygen species (ROS) are constantly produced under normal as well as stress-induced conditions, and play a role in both cancer progression and cancer suppression. Tumor tissue are known to have higher ROS levels compared to surrounding healthy tissue but ROS-induced specific protein carbonylation and its unique role in cancer progression/suppression are poorly understood. In this study we compared the relative total and specific protein carbonylation in flash frozen human breast cancer and matched adjacent normal tissue using ELISA, two-color western blot, mass spectrometry, and immunoprecipitation approaches. To understand antioxidant capacity in tumor tissue, we analyzed superoxide dismutase (SOD) for its antioxidant activity and protein level. Our results indicate that tumor tissue has greater total protein carbonylation, lower SOD activity, lower SOD protein levels, and elevated levels of autophagy compared to matched adjacent healthy tissue. We also identified three specific proteins that showed higher level of carbonylation selectively in tumor tissue compared to adjacent normal tissue. Our findings were further confirmed using the immortalized MDA-MB-231 breast cancer cell line and MCF-12A noncancerous normal human epithelial breast cell. Identification of selectively carbonylated proteins in cancer tissue, and understanding their specific role in cancer progression may promote the development of targeted therapeutic approaches to mitigate or enhance oxidative damage of such proteins selectively in tumor tissue. Citation Format: Baikuntha P. Aryal, Ashutosh Rao. Oxidative stress and selective protein carbonylation in human breast cancer tissue [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 5486. doi:10.1158/1538-7445.AM2017-5486

Abstract 2558: MYC-dependent transformation model of triple-negative breast cancer in vivo

Abstract Recent comprehensive breast cancer studies examining mutations and genomic alterations have determined that deregulation of MYC and the PI3K pathway occur frequently during breast cancer progression and may be useful targets for therapy. As a result, there have been large efforts to develop PI3K, AKT and mTOR inhibitors (PAM inhibitors) for clinical use, however clinical trial data demonstrates that many patients treated with PAM inhibitors develop resistant disease. An alternative strategy would be to target Myc, though a lack of effective and specific inhibitors makes this difficult. To identify the core vulnerabilities in these cancers we developed an in vivo xenograft model of triple-negative breast cancer driven by deregulated PI3K signaling and MYC. We hypothesize that determining how these pathways co-operate to transform normal human breast cells into breast carcinomas will reveal a tumor progression signature and highlight new therapeutic opportunities. We developed our model using the spontaneously immortalized, basal, triple-negative MCF10A cell line. By expressing the hotspot PIK3caH1047R protein alone in MCF10A cells (MCF10.H) in addition to MYC (MCF10.HM), we can model normal/early breast cancer and invasive ductal carcinoma respectively. This is the first in vivo human model of breast cancer dependent on MYC for transformation. When injected into female NOD-SCID mice, MCF10A.H cells form organized acinar ducts embedded in extracellular matrix. MCF10A.H ducts form with hollow lumen and a single layer of myoepithelial cells, recapitulating normal human breast histology. Alternatively, MCF10A.HM cells grow as high-grade carcinomas indicative of invasive disease. MCF10A.H benign growths and MCF10A.HM tumors remain basal-like and triple-negative by immunohistochemistry. Importantly, MCF10A.HM tumors are sensitive to MYC repression and therefore may be a suitable model to evaluate direct and indirect anti-MYC therapies. Having relevant human xenograft samples representing both normal and IDC tissue, we performed RNA-seq to identify a MYC-signature driving breast cancer transformation. Our current work will involve targeting the resulting MYC-driven pathways identified by RNA-seq to therapeutically target MYC in breast cancer. Citation Format: Corey Lourenco, Manpreet Kalkat, Dharmesh Dingar, Jason De Melo, Rosemary Yu, Linda Penn. MYC-dependent transformation model of triple-negative breast cancer in vivo [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 2558. doi:10.1158/1538-7445.AM2017-2558

The size-speed-force relationship governs migratory cell response to tumorigenic factors.

Tumor development progresses through a complex path of biomechanical changes leading first to cell growth and contraction and then cell deadhesion, scattering, and invasion. Tumorigenic factors may act specifically on one of these steps or have a wider spectrum of actions, leading to a variety of effects and thus sometimes to apparent contradictory outcomes. Here we used micropatterned lines of collagen type I/fibronectin on deformable surfaces to standardize cell behavior and measure simultaneously cell size, speed of motion and magnitude of the associated traction forces at the level of a single cell. We analyzed and compared the normal human breast cell line MCF10A in control conditions and in response to various tumorigenic factors. In all conditions, a wide range of biomechanical properties was identified. Despite this heterogeneity, normal and transformed motile cells followed a common trend whereby size and contractile forces were negatively correlated with cell speed. Some tumorigenic factors, such as activation of ErbB2 or loss of the βsubunit of casein kinase 2, shifted the whole population toward a faster speed and lower contractility state. Treatment with transforming growth factor β induced some cells to adopt opposing behaviors such as extremely high versus extremely low contractility. Thus tumor transformation amplified preexisting population heterogeneity and led some cells to exhibit biomechanical properties that were more extreme than those observed with normal cells.

ARTICLE WITHDRAWN] miR-1193 Suppresses Proliferation and Invasion of Human Breast Cancer Cells Through Directly Targeting IGF2BP2.

miRNAs are involved in breast cancer initiation and progression. In this study, we investigated the role of miR-1193, a newly found and poorly studied miRNA, in the proliferation and invasion of human breast cancer cells. Our results showed that compared with the adjacent tissues and MCF-10A human normal breast cells, miR-1193 was sharply reduced in breast cancer tissues and five breast cancer cell lines, including MDA-MB-231, MDA-MB-468, MDA-MB-435, SKBR3, and MCF-7. The oligo miR-1193 mimic or anta-miR-1193 was then transfected into MDA-MB-231 and MCF-7 breast cancer cell lines. Our results showed that the miR-1193 mimic robustly increased the miR-1193 level and decreased the proliferation and invasion in MDA-MB-231 and MCF-7 cells. In contrast, anta-miR-1193 had an opposite effect on miR-1193 expression, cell proliferation, and cell invasion. Moreover, bioinformatic and luciferase reporter gene assays confirmed that miR-1193 targeted the mRNA 3′-UTR region of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), an identified proto-oncogene. miR-1193 suppressed the protein level of IGF2BP2 and the activation of the ERK and PI3K/Akt signaling pathways. Moreover, suppression could be rescued by the transfection of pcDNA-IGF2BP2. In conclusion, miR-1193 suppressed proliferation and invasion of human breast cancer cells via translational suppression of IGF2BP2.

Viscoelastic properties of normal and cancerous human breast cells are affected differently by contact to adjacent cells.

We measured the viscoelastic properties of normal and cancerous human breast epithelial cells in different states of confluency using atomic force microscopy. We found that confluent normal cells are stiffer and have lower fluidity than sparse normal cells, which appears to be governed by the formation of cell-cell contacts. Contrarily, confluent cancer cells do not stiffen and not have a decreased fluidity compared to sparse cancer cells and their viscoelastic properties are independent of cell-cell contact formation. While the observed behavior of normal cells appears to be crucial for providing the mechanical stability and therefore the integrity of the epithelial monolayer, the dysregulation of this behavior in cancer cells might be a central aspect of early stage cancer progression and metastasis in the epithelium.

Characterization of viscoelastic properties of normal and cancerous human breast cells using a confining microchannel

Biomechanical properties have been revealed as potential biomarkers for distinguishing cancer cells from normal cells. In this work, we report a novel technique using a confining microchannel for biomechanical phenotyping for floating human cells, including one normal breast cell line (MCF-10A) and two breast cancer cell lines (MCF-7 and MDA-MB-231). The floating cells move under a defined pressure profile along the microchannel, in which the cells deform dynamically under compression by the channel sidewalls. We adopt the Hertz and Tatara model to convert deformed cell shapes to cell diameters and transient stress–strain ratios. By further considering cell viscoelasticity as a standard linear solid model, we compute for whole-cell viscosity, and instantaneous and relaxed moduli. Our results show that the selected cell types have significant different viscoelastic properties. We further implement cell-type classification based on the multiple parametric biomechanical cell properties with reasonable sensitivities (>65%). Applications of the confining microchannel can be further extended for high-throughput, continuous-flow deep phenotyping of rare cells by surface functionalization for both biomechanical and biochemical biomarkers for more comprehensive and promising cell characterization.