Although aromatase inhibitors are standard endocrine therapy for postmenopausal women with early-stage metastatic estrogen-dependent breast cancer, they are limited by the development of drug resistance. A better understanding of this process is critical towards designing novel strategies for disease management. Previously, we demonstrated a global proteomic signature of letrozole-resistance associated with hormone-independence, enhanced cell motility and implications of epithelial mesenchymal transition (EMT). Letrozole-resistant breast cancer cells (LTLT-Ca) were treated with a novel phytoalexin, glyceollin I, and exhibited morphological characteristics synonymous with an epithelial phenotype and decreased proliferation. Letrozole-resistance increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression (4.51-fold), while glyceollin I treatment caused a −3.39-fold reduction. Immunofluorescence analyses resulted of glyceollin I-induced increase and decrease in E-cadherin and ZEB1, respectively. In vivo studies performed in ovariectomized, female nude mice indicated that glyceollin treated tumors stained weakly for ZEB1 and N-cadherin and strongly for E-cadherin. Compared to letrozole-sensitive cells, LTLT-Ca cells displayed enhanced motility, however in the presence of glyceollin I, exhibited a 68% and 83% decrease in invasion and migration, respectively. These effects of glyceollin I were mediated in part by inhibition of ZEB1, thus indicating therapeutic potential of glyceollin I in targeting EMT in letrozole resistant breast cancer.
Triple negative breast cancer (TNBC) is subtype of breast disease devoid of the estrogen, progesterone, and Her2/neu receptors which are targets for pharmacological intervention. There is a need for novel anti-breast cancer agents that target TNBC. Therefore, novel isochalcone DJ52 was evaluated using the alamar blue dye exclusion assay, the luciferase colony assay, and xenograft models to determine its efficacy and potency. DJ52 significantly decreased proliferation of cells measured by using the alamar blue dye method and produced IC50 values of DJ52, DJ56, and DJ82 at 10-6M, 10-5M, and 10-5M, respectively. In vivo studies were conducted by injecting MDA-MB-231 cells into SCID mice to determine tumor regression was measured over 20 days. DJ52 at 50mg/kg caused significant decrease in tumor volume (p value <.05) by nearly 50% compared with the control with vehicle alone. These data suggest that DJ52 has merit for further evaluation as a novel anticancer agent.
Introduction: Triple negative breast cancer (TNBC) is one of the most aggressive forms of five major types of breast cancer. Because TNBC mortality disproportionately affects premenopausal African American women, a health disparity exists within this group. Our research has provided insight on how the noncancerous microenvironment plays a role in TNBC proliferation, migration, and invasion. Exosomes, extracellular vesicles (30-300 nm) produced ubiquitously by mammalian cells, are secreted by noncancerous breast epithelial cells, and have been noted to enhance proliferation, migration and invasion in TNBC. Therefore, the purpose of this study is to determine if exosome-free conditioned media (EFCM), which contains noncancerous MCF10A exosomes, enhances proliferation, migration and invasion in TNBC. Methods: MDA-MB-231 and MCF10A breast epithelial cells were used as cell line models. Proliferation was measured by the alamar blue proliferation assay. The transwell migration assay was used to assess migration, and the Matrigel invasion assay was used to measure migration. ExoELISA assays were also used to detect exosomes in exosome-free conditioned media (EFCM) from MCF10A cells. MCF10A cells were serum-starved for 24 hours, using DMEM/F-12 with 0.5% standard or exosome-depleted FBS (edFBS), to produce exosomal conditioned media (CM) and EFCM. This media was harvested and sterile filtered before use. MDA-MB-231 cells were treated with EFCM in each of the bioassays, and RPMI-1640 was used as a control. Results: MCF10A exosomes introduced via EFCM enhanced proliferation, migration and invasion of MDA-MB-231 cells. Conclusions: Taken together, our findings demonstrate that secreted factors from noncancerous MCF10A cells that have been stressed as a direct result of serum starvation are exosomes. These exosomes recruit TNBC cells to increase proliferation, migration, and invasion. The role of these exosomes in the noncancerous microenvironment is poorly understood, but these data suggest that exosomes from the noncancerous microenvironment have potential to be used in targeted therapies against triple negative breast disease. Citation Format: Letitia A. Yearby, Terri Cunningham, Jacenta Matthews, KiTani Parker Lemieux. Exosomes secreted from the noncancerous microenvironment promote proliferation, migration, and invasion in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4094.
Background. Triple negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer and disproportionally affects African American women. This subtype of breast cancer is characterized by the absence of the estrogen, progesterone, and HER2/neu receptors. Our research has provided insight on understanding that the microenvironment, specifically noncancerous cells, play in enhancing cancer progression and metastasis. However, the role of adjacent noncancerous cells in the metastatic phenomenon is poorly understood. Therefore, the aims of this project were: 1) to determine if conditioned media from MCF-10A cells would enhance proliferation of MDA-MB-231 and MDA-MB-468 cells and 2) to evaluate the ability of the effects of migration of the conditioned media on the MDA-MB-231 and MDA-MB-468 cells. Methods. Crystal violet and alamar blue dye exclusion assays were performed to measure proliferation in the different medias. The trans well migration assay was used to evaluate motility enhancement based on the media used, either conditioned media and the Cellometer was used to evaluate whether the MDA-MB-468 and MCF-10A cells shared the same microenvironment. Results. Conditioned media is made from base medium with a concentration of 1.0 g/L glucose with .5% FBS exposed to non-cancerous MCF-10A cells that have been driven into crisis. This conditioned media encouraged robust and continuous proliferation of the tumorigenic MDA-MB-231 and MDA-MB-468 cell lines. HGCM was extracted in our lab using sterile technique and reused to analyze the proliferative effects on cancerous and non-cancerous cells. MDA-MB-231 cells demonstrated significant proliferation when compared to the control. MDA-MB-468 cells demonstrated a similar response. The trans well migration assay demonstrated that the GFP labeled MDA-MB-468 cells had a higher percentage of motility than with MCF-10A cells in standard media. Conclusions: Taken together, these data indicate that factors secreted into the microenvironment from noncancerous cells may contribute to the mobility of TNBC cells and the conditioned media contains unknown factors that promote the cell mobility of the MDA-MB-231 and MDA-MB-468 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2424. doi:1538-7445.AM2012-2424
IntroductionTriple negative breast cancer (TNBC) is one of the most aggressive forms of five major types of breast cancer. Because TNBC mortality disproportionately affects premenopausal African American women, a health disparity exists within this group. Our research has provided insight on how the noncancerous microenvironment plays a role in TNBC proliferation, migration, and invasion. Exosomes, extracellular vesicles (30–300 nm) produced ubiquitously by mammalian cells, are secreted by noncancerous breast epithelial cells, and have been noted to enhance proliferation, migration and invasion in TNBC. Therefore, the purpose of this study is to determine if exosome‐free conditioned media (EFCM), which contains noncancerous MCF10A exosomes, enhances proliferation, migration and invasion in TNBC.MethodsMDA‐MB‐231 and MCF10A breast epithelial cells were used as cell line models. Proliferation was measured by the alamar blue proliferation assay. The transwell migration assay was used to assess migration, and the Matrigel invasion assay was used to measure migration. ExoELISA assays were also used to detect exosomes in exosome‐free conditioned media (EFCM) from MCF10A cells. MCF10A cells were serum‐starved for 24 hours, using DMEM/F‐12 with 0.5% standard or exosome‐depleted FBS (edFBS), to produce exosomal conditioned media (CM) and EFCM. This media was harvested and sterile filtered before use. MDA‐MB‐231 cells were treated with EFCM in each of the bioassays, and RPMI‐1640 was used as a control.ResultsMCF10A exosomes introduced via EFCM enhanced proliferation, migration and invasion of MDA‐MB‐231 cells.ConclusionsTaken together, our findings demonstrate that secreted factors from noncancerous MCF10A cells that have been stressed as a direct result of serum starvation are exosomes. These exosomes recruit TNBC cells to increase proliferation, migration, and invasion. The role of these exosomes in the noncancerous microenvironment is poorly understood, but these data suggest that exosomes from the noncancerous microenvironment have potential to be used in targeted therapies against triple negative breast disease.Support or Funding InformationThis research was supported by the National Institute on Minority Health and Health Disparities (NIHMHHD) of the National Institutes of Health under award number U54MD008149, sub‐award 13–14‐MB‐G007RN0A‐XU‐KPL. Technical support was provided by use of the Cellular and Molecular Biology Core facilities, supported by funding from the Louisiana Cancer Research Consortium (LCRC) and the NIH‐RCMI grant #8G12MD007595‐04 from the NIMHHD.
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