Epithelial Cell Line MCF-10A Research Articles

Torques within and outside the human spindle balance twist at anaphase.

At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are together required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.

Dependency of Tamoxifen Sensitive and Resistant ER+ Breast Cancer Cells on Semaphorin 3C (SEMA3C) for Growth.

Estrogen receptor positive (ER+) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy.

Proteomic analysis reveals stage-specific reprogramed metabolism for the primary breast cancer cell lines MGSO-3 and MACL-1.

Breast cancer is the most prevalent cancer in women worldwide. Its molecular subtypes are based on the presence/absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). MACL-1 and MGSO-3 are cell lines derived from primary tumor sites of patients diagnosed with luminal A subtype carcinoma (ER+/PR+/HER2-) and ductal carcinoma in situ (ER-/PR-/HER2+), respectively. However, these cell lines lost the expression of these markers over cell culturing, and both have triple-negative phenotypes (ER-/PR-/HER2-), which has the poorest prognosis. Here, we sought to study the proteome signature of MGSO-3 and MACL-1, comparing them with the epithelial cell line MCF-10A and the well-established metastatic-derived breast cancer cell line MDA-MB-231. Our results showed that proteins associated with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) were upregulated in MGSO-3 and MACL-1 cells. These cell lines also showed upregulation of pro-apoptotic proteins when compared with MDA-MB-231. The molecular differences highlighted in this study may clarify the molecular basis behind cancer cells functioning and may reveal novel signatures across the breast cancer cell models.

MiR-217-5p suppresses epithelial-mesenchymal transition and the NF-κB signaling pathway in breast cancer via targeting of metadherin.

MicroRNAs (miRNAs) have been associated with a number of human malignancies, including breast cancer (BC). However, the expression, biological function and fundamental underlying mechanism of miR-217-5p in BC remain unclear. Therefore, in the present study, the expression levels of miR-217-5p and metadherin (MTDH) were examined in BC tissues and BC cell lines using reverse transcription-quantitative PCR. Cell Counting Kit-8 assays, cell proliferation, wound healing assays, Transwell assays and western blotting were used to examine the effects of miR-217-5p on cell proliferation, migration, the epithelial-mesenchymal transition (EMT) and NF-κB signaling pathway expression. The direct relationship between miR-217-5p and MTDH was assessed using a dual-luciferase reporter assay. The results demonstrated that significantly reduced expression levels of miR-217-5p but significantly increased mRNA expression levels of MTDH were observed in BC tissues from 35 patients with BC compared with non-tumor breast tissues. Furthermore, BC cell lines SK-BR3 and BT549 expressed miR-217-5p at markedly lower levels and MTDH at markedly higher levels compared with the breast epithelial MCF10A cell line. miR-217-5p overexpression significantly inhibited cell proliferation, invasion and migration and suppressed the EMT in BC cells. miR-217-5p overexpression also inhibited the NF-κB signaling pathway by markedly decreasing p65 mRNA and protein expression levels but significantly increasing IκBα expression levels. Furthermore, miR-217-5p knockdown markedly increased MTDH mRNA and protein expression levels. The expression levels of miR-217-5p were negatively correlated with those of MTDH in BC tissues. These results suggested that restoration of MTDH expression levels could potentially attenuate the inhibitory effects of miR-217-5p overexpression on BC cell proliferation. Therefore, in conclusion miR-217-5p overexpression may inhibit cell migration, invasion, the EMT and NF-κB signaling pathway in BC via targeting of MTDH. miR-217-5p may serve as an important potential target in BC therapy.

Human Serum Albumin Decorated Indocyanine Green Improves Fluorescence-Guided Resection of Residual Lesions of Breast Cancer in Mice.

ObjectiveAchieving negative resection margin is critical but challenging in breast-conserving surgery. Fluorescence-guided surgery allows the surgeon to visualize the tumor bed in real-time and to facilitate complete resection. We envisioned that intraoperative real-time fluorescence imaging with a human serum albumin decorated indocyanine green probe could enable complete surgical removal of breast cancer in a mouse model.MethodsWe prepared the probe by conjugating indocyanine green (ICG) with human serum albumin (HSA). In vitro uptake of the HSA-ICG probe was compared between human breast cancer cell line MDA-MB-231 and normal breast epithelial cell line MCF 10A. In vivo probe selectivity for tumors was examined in nude mice bearing MDA-MB-231-luc xenografts and the FVB/N-Tg (MMTV-PyMT) 634Mul/J mice model with spontaneous breast cancer. A positive-margin resection mice model bearing MDA-MB-231-luc xenograft was established and the performance of the probe in assisting surgical resection of residual lesions was examined.ResultsA significantly stronger fluorescence intensity was detected in MDA-MB-231 cells than MCF 10A cells incubated with HSA-ICG. In vivo fluorescence imaging showed that HSA-ICG had an obvious accumulation at tumor site at 24 h with tumor-to-normal tissue ratio of 8.19 ± 1.30. The same was true in the transgenic mice model. The fluorescence intensity of cancer tissues was higher than that of non-cancer tissues (58.53 ± 18.15 vs 32.88 ± 11.34). During the surgical scenarios, the residual tumors on the surgical bed were invisible with the naked eye, but were detected and resected with negative margin under HSA-ICG guidance in all the mice (8/8). Recurrence rate among mice that underwent resection with HSA-ICG (0/8) was significantly lower than the rates among mice with ICG (4/8), as well as the control group under white light (7/7).ConclusionsThis study suggests that real-time in vivo visualization of breast cancer with an HSA-ICG fluorescent probe facilitates complete surgical resection of breast cancer in a mouse xenograft model.

Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells.

Orai1 plays a major role in store-operated Ca2+ entry (SOCE) in triple-negative breast cancer (TNBC) cells. This channel is inactivated via different mechanisms, including protein kinase C (PKC) and protein kinase A (PKA)-dependent phosphorylation at Ser-27 and Ser-30 or Ser-34, respectively, which shapes the Ca2+ responses to agonists. The Ca2+ calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca2+- and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Here, we show that the breast cancer cell lines MCF7 and MDA-MB-231 exhibit enhanced expression of Orai1 and AC8 as compared to the non-tumoral breast epithelial MCF10A cell line. In these cells, AC8 interacts with the Orai1α variant in a manner that is not regulated by Orai1 phosphorylation. AC8 knockdown in MDA-MB-231 cells, using two different small interfering RNAs (siRNAs), attenuates thapsigargin (TG)-induced Ca2+ entry and also Ca2+ influx mediated by co-expression of Orai1 and the Orai1-activating small fragment (OASF) of STIM1 (stromal interaction molecule-1). Conversely, AC8 overexpression enhances SOCE, as well as Ca2+ entry, in cells co-expressing Orai1 and OASF. In MDA-MB-231 cells, we found that AC8 overexpression reduces the Orai1 phosphoserine content, thus suggesting that AC8 interferes with Orai1 serine phosphorylation, which takes place at residues located in the AC8-binding site. Consistent with this, the subset of Orai1 associated with AC8 in naïve MDA-MB-231 cells is not phosphorylated in serine residues in contrast to the AC8-independent Orai1 subset. AC8 expression knockdown attenuates migration of MCF7 and MDA-MB-231 cells, while this maneuver has no effect in the MCF10A cell line, which is likely attributed to the low expression of AC8 in these cells. We found that AC8 is required for FAK (focal adhesion kinase) phosphorylation in MDA-MB-231 cells, which might explain its role in cell migration. Finally, we found that AC8 is required for TNBC cell proliferation. These findings indicate that overexpression of AC8 in breast cancer MDA-MB-231 cells impairs the phosphorylation-dependent Orai1 inactivation, a mechanism that might support the enhanced ability of these cells to migrate.

Unravelling the potential of seaweeds from the Black Sea coast of Romania as bioactive compounds sources. Part I: Cystoseira barbata (Stackhouse) C. Agardh

The Romanian coastlines of the Black Sea have abundant seaweed resources, but little effort has been done to investigate their biological potential. The aim of the present study was to assess the in vitro antioxidant and anti-proliferative effects of Cystoseira barbata (Stackhouse) C. Agardh (Sargassaceae), a brown alga inhabiting the Black Sea coast of Romania. The 70% acetone, methanol and water extracts of C. barbata were evaluated for their total phenolic content, antioxidant activity and anti-proliferative potential against human tumor cell lines (pulmonary A549, colon HT-29, mammary MCF-7) and the non-tumor mammary epithelial MCF-10A cell line. C. barbata 70% acetone extract (CBAE) displayed the highest antioxidant and cytotoxic activities. The mechanism of CBAE anti-proliferative activity involved initially increased intracellular ROS accumulation, followed by increased DNA content in the subG1 phase and DNA fragmentation leading to excessive apoptosis. Thus, our study provides a theoretical basis for the use of CBAE as a tumor preventive agent. Furthermore, UHPLC-DAD-QTOF-MS analysis of CBAE tentatively identified 18 phlorotannins as fucophlorethol and eckol derivatives, containing three up to seven phloroglucinol units. In conclusion, C. barbata represents a valuable source for the development of macroalgal-based products with putative use as nutraceuticals and pharmaceuticals.

Expression of miR-146a-5p in breast cancer and its role in proliferation of breast cancer cells.

In the present study, the expression level of microRNA-146a-5p (miR-146a-5p) in breast cancer tissue and cell lines was investigated and its effects on proliferation of breast cancer cells. miR-146a-5p expression was detected by reverse transcription-quantitative polymerase chain reaction in breast cancer tissues, paraneoplastic tissue (collected by The Department of Oncology of Changhai Hospital from January 2014 to June 2015), breast cancer cell line MCF-7 and normal breast epithelial cell line MCF 10A. Bioinformatics analysis was conducted to forecast target genes of miR-146a-5p, which was further verified by fluorescent reporter gene detection. The results demonstrated the expression level of miR-146a-5p in breast cancer tissue was significantly higher, compared with paraneoplastic tissue (P<0.01), and the expression level of miR-146a-5p in MCF-7 cells was significantly higher, compared with MCF 10A cells (P<0.01). Overexpression of miR-146a-5p in MCF-7 cells can promote the proliferation, and low expression miR-146a-5p in MCF-7 can inhibit the proliferation. BRCA1 was further identified as a target gene of miR-146a-5p by bioinformatics analysis and fluorescent reporter gene detection. It was concluded that miR-146a-5p is expressed in breast cancer tissue and breast cancer cell line and may regulate the proliferation of MCF-7 via BRCA1.

The IL-6/STAT3 Signaling Pathway Is an Early Target of Manuka Honey-Induced Suppression of Human Breast Cancer Cells.

There is renewed interest in the potential use of natural compounds in cancer therapy. Previously, we demonstrated the anti-tumor properties of manuka honey (MH) against several cancers. However, the underlying mechanism and molecular targets of this activity remain unknown. For this study, the early targets of MH and its modulatory effects on proliferation, invasiveness, and angiogenic potential were investigated using two human breast cancer cell lines, the triple-negative MDA-MB-231 cells and estrogen receptor-positive MCF-7 cells, and the non-neoplastic breast epithelial MCF-10A cell line. Exposure to MH at concentrations of 0.3–1.25% (w/v) induced a dose-dependent inhibition of the proliferation of MDA-MB-231 and MCF-7, but not MCF-10A, cells. This inhibition was independent of the sugar content of MH as a solution containing equivalent concentrations of its three major sugars failed to inhibit cell proliferation. At higher concentrations (>2.5%), MH was found to be generally deleterious to the growth of all three cell lines. MH induced apoptosis of MDA-MB-231 cells through activation of caspases 8, 9, 6, and 3/7 and this correlated with a loss of Bcl-2 and increased Bax protein expression in MH-treated cells. Incubation with MH induced a time-dependent translocation of cytochrome c from mitochondria to the cytosol and Bax translocation from the cytosol into the mitochondria. MH also induced apoptosis of MCF-7 cells via the activation of caspases 9 and 6. Low concentrations of MH (0.03–1.25% w/v) induced a rapid reduction in tyrosine-phosphorylated STAT3 (pY-STAT3) in MDA-MB-231 and MCF-7 cells. Maximum inhibition of pY-STAT3 was observed at 1 h with a loss of >80% and coincided with decreased interleukin-6 (IL-6) production. Moreover, MH inhibited the migration and invasion of MDA-MB-231 cells as well as the angiogenic capacity of human umbilical vein endothelial cells. Our findings identify multiple functional pathways affected by MH in human breast cancer and highlight the IL-6/STAT3 signaling pathway as one of the earliest potential targets in this process.

CRB3 downregulation confers breast cancer stem cell traits through TAZ/\u03b2-catenin

The cancer stem cell (CSC) theory depicts a special population within the cancer mass that self-renew and sustain the cancer, even if the other cells were eliminated by therapies. How CSCs acquire these unique traits is still unclear. Crumbs homolog 3 (CRB3), a member of the CRB polarity complex, has been reported to act as a tumor suppressor. Here, we detected significantly lower or negative CRB3 expression in human breast cancer tissues. Knockdown of CRB3 generated non-tumorigenic, immortalized breast epithelial cell line MCF 10A with CSC properties. Simultaneously, we found that CRB3 downregulation induced the epithelial–mesenchymal transition and activated TAZ (transcriptional co-activator with PDZ-binding motif) and β-catenin. Significantly, the activation of TAZ and β-catenin sufficed in conferring MCF 10A cells with CSC properties. This study demonstrates that cell polarity proteins may serve as a switch of the differentiated vs multipotent states in breast cancers.

Elucidating the Role of Tumor Microenvironmental Heterogeneity with a Computational Model of 3D Breast Spheroids

Tumor development and its response to therapies are highly affected by heterogeneous microenvironments, including fluctuations in chemical and physical cues, as well as in the composition of the surrounding extracellular matrix. For example, the abnormal topology of tumor vasculature and tumor tissue leads to disruption in oxygen transport and in the emergence of regions of low oxygen (hypoxic conditions), and in accumulation of acidic products and decreased pH (acidic conditions). In addition, the elevated mechanical tension is commonly observed in the tumor connective tissue (high tension conditions). All these factors affect tumor growth and efficacy of anticancer drugs. Moreover, the microenvironmental conditions can dynamically evolve after treatment that is difficult to investigate in vivo in a systematic way. To represent the complexity and dynamics of tumor microenvironment we developed a computational model for the growth of 3D organotypic cultures integrated with experimental organ-on-the-chip 3D cultures. The computational model was validated based on the data collected from cultured breast spheroids, including the non-tumorigenic epithelial MCF-10A cell line, mildly tumorigenic c-Ha-ras oncogene transfected MCF-10AT1 cell line, and a metastatic line MCF-10CA-1a. Based on the differences in these cell lines genetic profiles, cell mechanical properties, and image-guided variations in organotypic morphologies, the computational model was able to reproduce in a quantitative way the dynamics of 3D tumor spheroids’ growth under various microenvironmental conditions. This calibrated model was subsequently applied to predict tumor growth and its response to chemotherapy in heterogeneous microenvironments consisting of dynamically changing gradients of oxygen and pH, as well as tension of the surrounding microenvironment. The overarching aim of our project is to use this integrated (in silico-organ and organ-on-the-chip) approach to target the treatment of tumor 3D organotypic cultures with drugs that display different mechanism of action, such as cell cycle arrest, initiation of apoptosis, or treatment combinations. Furthermore, this approach will allow us to propose new treatment schedules and dosages that will be tested in the organ-on-the-chip experiments. The progress on this integrated model development will be presented.

Reactive oxygen species-mediated synergistic and preferential induction of cell death and reduction of clonogenic resistance in breast cancer cells by combined cisplatin and FK228

Safe and effective combination chemotherapy regimens against breast cancer are lacking. We used our cellular system, consisting of the non-cancerous human breast epithelial MCF10A cell line and its derived tumorigenic, oncogenic H-Ras-expressing, MCF10A-Ras cell line, to investigate the effectiveness of a combination chemotherapy regimen in treating breast cancer cells using two FDA-approved agents, cisplatin and FK228. Cisplatin and FK228 significantly, synergistically, and preferentially induced death and reduced drug resistance of MCF10A-Ras versus MCF10A cells. The ERK-Nox-ROS pathway played a major role in both synergistic cell death induction and GSH-level reduction, which contributed to the synergistic suppression of drug resistance in cells. Enhancement of the Ras-ERK-Nox pathway by combined cisplatin and FK228 significantly increased ROS levels, leading to induction of death, reduction of drug resistance, and induction of DNA damage and oxidation in cancerous MCF10A-Ras cells. Furthermore, synergistic induction of cell death and reduction of drug resistance by combined cisplatin and FK228 in breast cells is independent of their estrogen receptor status. Our study suggests that combined cisplatin and FK228 should be considered in clinical trials as a new regimen for therapeutic control of breast cancers.

Novel triterpenoid AECHL-1 induces apoptosis in breast cancer cells by perturbing the mitochondria–endoplasmic reticulum interactions and targeting diverse apoptotic pathways

BackgroundThe modus operandi for an anti-cancer drug must allow for an efficient discrimination system between tumorigenic and non-tumorigenic cells. Targeting ER stress and mitochondrial function in cancer cells appears to be a suitable option, as these processes are dysregulated in tumor cells. AECHL-1, a novel triterpenoid, exhibits potent anticancer activity against an array of cancer cell lines however, its mechanism of action remains elusive. MethodsMolecular targets of AECHL-1 were investigated using breast adenocarcinoma cells MCF-7, MDA-MB-231 and mammary epithelial cell line MCF 10A in vitro and xenograft tumors in SCID mice in vivo. Western blotting, flow cytometry, and immunohistochemical studies were employed to delineate the molecular pathways. ResultsAECHL-1 caused a transient elevation of ER stress proteins along with a prolonged phosphorylation of eIF2α in breast cancer cells. This was accompanied by a simultaneous release of calcium from ER stores and subsequent mitochondrial accumulation. These effects could be reversed by using ER stress inhibitors. AECHL-1 brings about mitochondria mediated, caspase independent cell death via AIF in MCF-7 cells; MDA-MB-231 succumbed to caspase dependent extrinsic pathway. Xenograft studies closely echoed our in vitro results. AECHL-1 did not alter cellular and molecular parameters in MCF 10A. ConclusionThese findings reveal that, AECHL-1 targets the Achilles Heel of cancer cell, namely dysfunctional ER and mitochondria while being non toxic to normal parenchyma and can thus be further explored as a potential chemotherapeutic intervention. General significanceAggravation of ER stress by AECHL-1 uncovers a novel pathway for selective elimination of cancer cells.

Abstract A52: The microRNA control of 17β-hydroxysteroid dehydrogenase type 1 and 2 in breast cancer

Abstract Introduction: Breast cancer is primarily a steroid hormone dependent disease, and up to 80% of all breast cancers express the estrogen receptors. Regulation of steroid hormone activity in the breast tissue is mediated by the aromatase enzyme and by the 17β-hydroxysteroid dehydrogenase (17β-HSD) enzyme family. 17β-HSD1s primary role is in catalyzing the NADH dependent reduction of esterone to estradiol. 17β-HSD2 acts in opposition to 17β-HSD1 and mediates a NAD(H) dependent conversion of estradiol to esterone. The combination of these two enzymes has been shown to be a good indicator of tamoxifen treatment benefit, as tumors with a high 17βHSD1/17βHSD2 ratio have less benefit from tamoxifen treatment. Furthermore, patients with high 17βHSD1 mRNA expression were shown to have shorter overall disease free survival and increased risk of relapse. Despite this, next to nothing is known about the control of the expression 17βHSD1 and 17βHSD2 by microRNA in breast cancer. Aim: To identify key microRNA responsible for the regulation of 17βHSD1 and 17βHSD2 in breast cancer. Materials and methods: We screened for the effect of microRNA mimics by in vitro transient transfection of breast cancer epithelial cell-lines T47D, MCF-7 and ZR75-1 as well as the epithelial cell-line MCF10A. Results: The endogenous expression of 17β-HSD type 1 in MCF-7 is low while ZR75-1 and MCF10A express moderate levels and T47D expresses high levels. Here we show that microRNA-210, 4508, 1304-3p, 6832-5p, 892a and 3674 down regulate 17β-HSD type 1 mRNA levels to 20-60% of control levels in MCF-7 and T47D. The same miRs had either a reduced effect, or no effect in ZR75-1 and MCF10A. Furthermore, we show that miR-17, 873-5p, 744-5p and 4794 result in a 2-5 fold increase in the expression of 17β-HSD type 1 ZR75-1, T47D and MCF7. This effect was reduced to 0.5 fold in MCF10A. The endogenous expression of 17β-HSD type 2 is low in MCF7, moderate in ZR75-1 and T47D and very high in MCF10A. We show that miRs 2045p, 211-5p, 5006-3p, 4251, 4745-5p, 302d-5p, 498, 1305 and 205-3p down regulates 17β-HSD type 2 expression to 10-60% of endogenous levels in ZR75-1 and MCF7, and to 30-70% of endogenous levels in MCF10A. Conclusion: These findings are the first which start to map out the microRNA regulation of 17β-HSD type 1 and 2 in breast cancer, and this may be one important mechanism by which their expression levels are regulated in the pathogenesis of breast cancer. Citation Format: Erik Hilborn, Olle Stal, Agneta Jansson. The microRNA control of 17β-hydroxysteroid dehydrogenase type 1 and 2 in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A52.

Cytotoxic effect and apoptotic activity of Parmentiera edulis DC. hexane extract on the breast cancer cell line MDA-MB-231

Parmentiera edulis is a traditional medicine plant used for different purposes in Mexico; however its biological effects and their potential mechanisms have not been sufficiently investigated. In the present work, the in vitro cytotoxic and pro-apoptotic potential effects of hexanic extract of Parmentiera edulis bark on the MDA-MB-231 breast cancer cell line were studied. The hexanic extract elicited the most pronounced cytotoxic effect in breast cancer cells, as evaluated by MTT assays performed 72 h after treatment, showing an IC50 value of 94.63 + 1.63 μg/ml. No effects were observed in the immortalized mammary epithelial MCF10A cell line and lymphocytes. The anti-proliferative activity observed following Parmentiera edulis treatment was associated with morphological changes, DNA fragmentation, phosphatidyl serine externalization and activation of caspases 3 and 9, all of them typical characteristics of apoptosis. Our findings demonstrate specific cytotoxic activity of Parmentiera edulis and the apoptosis induction through the intrinsic pathway in the breast cancer cells. Further research is necessary to find out the effective secondary metabolites responsible of cell death induction.

Targeting mitochondrial citrate transport in breast cancer cell lines.

Lipogenesis is considered to be a very important aspect of cancer metabolism and targeting de novo lipid synthesis or related pathways are among novel approaches to treat cancer. Many targets of the pathway including ATP-citrate lyase (ACLY), acetyl-CoA carboxylase and fatty acid synthase have been evaluated for their potential in cancer treatment. However the role of citrate transport protein (CTP), another important component of lipogenesis pathway, is not well known for cancer metabolism and cell survival. Here we report that while chemical inhibition of CTP reduces cytoplasmic citrate levels and limits breast cancer cell viability effectively, siRNA based inhibition had little effect on both. We also compared the effects of CTP inhibition with ACLY and found that the inhibition of ACLY reduced cytoplasmic citrate levels and limited cell viability more effectively than CTP inhibition. Finally we have demonstrated that neither cell cycle arrest nor autophagy was induced in cells treated with CTP or ACLY siRNA. Inhibitions triggered apoptosis but only slightly. Growth inhibitory effects do not occur in normal mammary epithelial MCF-10A cell line.

Abstract 5218: TGFβ regulates miR-182 control of BRCA1

Abstract Transforming growth factor beta (TGFβ) is a pluripotent cytokine whose activity is significantly increased in epithelial cancers and induced by radiation and chemotherapy. Downregulation of BRCA1 or alterations in BRCA1 related pathways may contribute to response to therapy. However, the mechanism(s) underlying BRCA1 downregulation is not well understood. Our prior work indicated that inhibition of TGFβ canonical signaling reduces BRCA1 mRNA levels (Maxwell et al., Cancer Res. 68(20):8304-11, 2008). Here, we used the non-malignant human epithelial MCF10A cell line and murine Tgfb1 heterozygote (HT) and wildtype fibroblast cell lines to investigate TGFβ regulation of BRCA1. Pharmaceutical inhibition of TGFβ type I receptor kinase or incubation with pan-isoform neutralizing antibody, 1D11, resulted in significant down regulation of both BRCA1 mRNA and protein levels in MCF10A. Likewise, Brca1 protein and mRNA was decreased by &amp;gt;50% in cell cycle synchronized Tgfb1 HT fibroblasts compared to wildtype fibroblasts. Consistent with decreased Brca1, Tgfb1 HT fibroblasts exhibited defective DNA damage response as measured by 53BP1 foci following UV radiation treatment. We next investigated mechanisms regulating protein levels in MCF10A cells, which together indicated that TGFβ signaling regulates BRCA1 protein stability, prevents its degradation, and increases BRCA1 promoter activity. Surprisingly, TGFβ treatment rapidly (&amp;lt;30 minutes) increased BRCA1 protein and mRNA, which led us to also consider control at the level of mRNA stability since BRCA1 is known to be controlled by miR-182 (Moskwa et al., Mol Cell. 41(2):210-20, 2011). TGFβ treatment downregulates miR-182, conversely, miR-182 increases when TGFβ is inhibited by either small molecule inhibition of the type I receptor kinase or by ligand capture with neutralizing antibody in human cells. miR-182 increased 18-fold in Tgfb1 HT murine fibroblasts. Notably, MCF10A do not show changes in other known miRNAs that target BRCA1 for degradation, miRNA 146a and 146b (Garcia et al., EMBO Mol Med. 3(5):279-90, 2011), are not affected by TGFβ treatment or inhibition. Transfection of Tgfb1 HT fibroblasts with a miR-182 antagomir restored BRCA1 mRNA, protein and functional deficits. Further studies show that addition of TGFβ for short periods of time affects miRNA processing by decreasing pre-miR182 up to 50%, while TGFβ inhibition affects miR-182 translation. These data indicate that TGFβ exerts control over BRCA1 at several levels: protein stability, transcriptional control of the promoter, and translational control via miR-182. As many cancer cells maintain the ability to respond to TGFβ and TGFβ inhibition is clinically viable, we propose that TGFβ inhibition provides a therapeutic opportunity by impeding BRCA1 dependent DNA damage responses. Note: This abstract was not presented at the meeting. Citation Format: Haydeliz Martinez-Ruiz, Sangeetha Vijayakumar, Mary H. Barcellos-Hoff. TGFβ regulates miR-182 control of BRCA1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5218. doi:10.1158/1538-7445.AM2014-5218

Ectopic NGAL expression can alter sensitivity of breast cancer cells to EGFR, Bcl-2, CaM-K inhibitors and the plant natural product berberine

Neutrophil gelatinase-associated lipocalin (NGAL, a.k.a Lnc2) is a member of the lipocalin family and has diverse roles. NGAL can stabilize matrix metalloproteinase-9 from autodegradation. NGAL is considered as a siderocalin that is important in the transport of iron. NGAL expression has also been associated with certain neoplasias and is implicated in the metastasis of breast cancer. In a previous study, we examined whether ectopic NGAL expression would alter the sensitivity of breast epithelial, breast and colorectal cancer cells to the effects of the chemotherapeutic drug doxorubicin. While abundant NGAL expression was detected in all the cells infected with a retrovirus encoding NGAL, this expression did not alter the sensitivity of these cells to doxorubicin as compared with empty vector-transduced cells. We were also interested in determining the effects of ectopic NGAL expression on the sensitivity to small-molecule inhibitors targeting key signaling molecules. Ectopic NGAL expression increased the sensitivity of MCF-7 breast cancer cells to EGFR, Bcl-2 and calmodulin kinase inhibitors as well as the natural plant product berberine. Furthermore, when suboptimal concentrations of certain inhibitors were combined with doxorubicin, a reduction in the doxorubicin IC50 was frequently observed. An exception was observed when doxorubicin was combined with rapamycin, as doxorubicin suppressed the sensitivity of the NGAL-transduced MCF-7 cells to rapamycin when compared with the empty vector controls. In contrast, changes in the sensitivities of the NGAL-transduced HT-29 colorectal cancer cell line and the breast epithelial MCF-10A cell line were not detected compared with empty vector-transduced cells. Doxorubicin-resistant MCF-7/DoxR cells were examined in these experiments as a control drug-resistant line; it displayed increased sensitivity to EGFR and Bcl-2 inhibitors compared with empty vector transduced MCF-7 cells. These results indicate that NGAL expression can alter the sensitivity of certain cancer cells to small-molecule inhibitors, suggesting that patients whose tumors exhibit elevated NGAL expression or have become drug-resistant may display altered responses to certain small-molecule inhibitors.

Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells reveals common expression profiles

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study, transcriptomic and proteomic changes in the estrogen receptor negative, non-tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R = 0.679, P < 0.05) and KEAP1 knockdown (R = 0.853, P < 0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism, and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and the proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and the proteomic levels. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast.

Abstract 1091: ShcA-mediated ERK signaling contributes to the tumor-promoting activity of TGF-beta in the presence of mutant p53

Abstract Transforming growth factor-beta (TGF-β) signaling has been shown to inhibit tumorigenesis at the early stage and promote metastasis at the late stage. However, biomarkers for identifying the complicated molecular alterations mediating the switch of TGF-β from a tumor suppressor to a tumor promoter still remain obscure. Published studies have shown that the presence of mutated tumor p53 can switch TGF-β to becoming tumor-promoting. However, we found that in the p53-null PC-3 prostate cancer cell line, the ectopic expression of the mutant p53 R175H (mp53) enhanced the Smad-dependent signaling, but inhibited TGF-β-induced cell migration by downregulating the active ERK. Since TGF-β is known to activate MAPK/ERK pathway to induce cell migration, EMT and survival through direct phosphorylation of adaptor protein ShcA isoform p52, we investigated how the expression of the mp53 inhibited ERK activation. We found that the expression of mp53 led to downregulation of phospho-p52ShcA and upregulation of ERK antagonist p66ShcA isoform. Interestingly, we found partial depletion of p52ShcA by using siRNA enhanced the TGF-β/Smad signaling and inhibited the TGF-β/ERK signaling in PC-3 cells and spontaneously immortalized and non-tumorigenic human mammary epithelial MCF-10A cell line, suggesting ShcA may act as an additional switcher of the TGF-β. Furthermore, reactivation of ERK by the expression of its activator, MEK1, restored active ERK level and TGF-β-induced cell migration in PC-3/mp53 cell. Those findings show that mp53 expression alone is not sufficient to promote the malignant phenotype of TGF-β in certain cells and the cooperation of mp53 and ShcA-mediated ERK signaling may switch TGF-β to be a tumor promoter. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1091. doi:10.1158/1538-7445.AM2011-1091