Breast Cancer Cell Lines BT474 Research Articles

Abstract LB-B11: Assessment of HER3 status during lapatinib treatment in HER3-positive breast cancer using 89Zr-anti-HER3 mAb

Abstract Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with the HER-targeting tyrosine kinase inhibitor lapatinib can lead to a rapid compensatory increase in expression, signaling activity and relocalization of HER3 to the plasma membrane, which may attenuate the response to lapatinib. This might imply a potential role for a more dynamic assessment of HER3 tumor status using molecular imaging techniques, such as positron emission tomography (PET), instead of immunohistochemical HER3 staining on tumor biopsies. Here, we explored the feasibility of a dynamic assessment of HER3 status during lapatinib treatment in human breast cancer xenografts using zirconium-89 labeled anti-human HER3 monoclonal antibody (mAb) as a potential tracer for animal PET imaging. The anti-human HER3 mouse mAb MAB3481 was used for all experiments. The effect of lapatinib treatment on HER3 expression and HER3 mAb internalization in human breast cancer cell lines SKBR3 and BT474 was determined using flow cytometry. Biodistribution was performed using 89Zr-anti-HER3 mAb in mice bearing BT474 or SKBR3 tumors. Mice received daily vehicle or a lapatinib dose of 25, 50 or 100 mg/kg orally. A tracer dose of 89Zr-anti-HER3 mAb combined with the aspecific tracer 111In-IgG was injected 3 days after treatment. Ex vivo organ distribution assessment of 89Zr-anti-HER3 mAb was performed 6 days after tracer injection. Ex vivo tumor analysis using western blotting, ELISA and immunohistochemistry were performed to measure HER3 levels. In vitro, lapatinib treatment resulted in a ∼2-fold increase in membranous HER3 expression and HER3 internalization in SKBR3 and BT474 tumor cells. 89Zr-anti-HER3 mAb tumor uptake was significantly higher compared to 111In-IgG uptake in BT474 (P < 0.01), demonstrating HER3 specific tumor uptake. SKBR3 xenografts did not show HER3 specific uptake, which was likely caused by the poor viability of the tumors. HER3 upregulation was observed in BT474 xenografts after lapatinib treatment for 9 days. The enhanced HER3 expression was related to a 76 ± 9% increase in 89Zr-anti-HER3 mAb tumor uptake at 25 mg/kg and 92 ± 27% at 50 mg/kg lapatinib. Lapatinib at the highest concentration (100 mg/kg) strongly inhibited tumor growth and did not increase tumor uptake of the tracer. In conclusion, HER3-specific uptake of 89Zr-anti-HER3 mAb was shown in breast cancer xenografts. HER3 upregulation after lapatinib treatment was related to an enhanced 89Zr-anti-HER3 mAb uptake in these xenografts. These promising data warrant future dynamic assessment of HER3 status with 89Zr-anti-HER3 mAb PET imaging. Citation Format: Arjan Kol, Martin Pool, Steven de Jong, Elisabeth GE de Vries, Marjolijn N. Lub-de Hooge, Anton GT Terwisscha van Scheltinga. Assessment of HER3 status during lapatinib treatment in HER3-positive breast cancer using 89Zr-anti-HER3 mAb. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B11.

Downregulation of caveolin-1 upregulates the expression of growth factors and regulators in co-culture of fibroblasts with cancer cells

Reduced expression levels of caveolin-1 (Cav-1) in tumor stromal fibroblasts influences the occurrence and progression of tumors, particularly in breast cancer, but the relevant molecular mechanism is unclear. The present study aimed to clarify the potential mechanism underlying the promotion of tumor growth by reduced Cav-1 expression levels, by investigating Cav-1-targeted molecules in fibroblasts and breast cancer cells. The expression of growth factors in the ESF fibroblast cell line transfected with Cav-1 small interfering RNA (siRNA) was examined. The expression of apoptotic regulators in the BT474 breast cancer cell line that was co-cultured with the fibroblasts, was also investigated. The transfection of Cav-1-targeting siRNA in ESF cells resulted in efficient and specific inhibition of Cav-1 expression. The downregulation of Cav-1 increased the expression and secretion of stromal cell-derived factor-1 (SDF-1), epidermal growth factor (EGF) and fibroblast-specific protein-1 (FSP-1) in ESF cells. This resulted in the accelerated proliferation of the breast cancer cells. Tumor protein 53-induced glycolysis and apoptosis regulator (TIGAR) was upregulated in the BT474 cells under the condition of co-culture with Cav-1 siRNA fibroblasts, while levels of reactive oxygen species (ROS) were decreased, resulting in apoptosis inhibition in the breast cancer cells. These results demonstrated that the downregulation of Cav-1 promoted the growth of breast cancer cells through increasing SDF-1, EGF and FSP-1 in tumor stromal fibroblasts, and TIGAR levels in breast cancer cells. To the best of our knowledge, the present study supports the hypothesis that Cav-1 possesses tumor-suppressor properties, with the mechanism of Cav-1-dependent signaling involving the regulation of SDF-1, EGF, FSP-1 and TIGAR.

Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition.

Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15-40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity.

Prolactin-Induced Protein regulates cell adhesion in breast cancer

Prolactin-Induced Prolactin (PIP) is widely expressed in breast cancer and has key cellular functions in this disease that include promoting invasion and cell cycle progression. Notably, we have recently identified a strong association between PIP-binding partners and a number of cell functions that are involved in cell adhesion. Therefore in this study, we investigated the effect of PIP on the regulation of cell adhesion using PIP-silencing in breast cancer cell lines T-47D, BT-474, and MFM-223. Our findings suggest that PIP expression is necessary for cell adhesion in a process that shows variation in the pattern of PIP regulation of cell-matrix and cell–cell adhesions based on the types of adhesion surface and breast cancer cell line. In this respect, we observed that PIP-silencing markedly reduced cell adhesion to uncoated plates in all three cell lines. In addition, in T-47D and MFM-223 cells fibronectin matrix induced baseline adhesion and reversed the PIP-silencing mediated reduction of cell adhesion. However, in BT-474 cells we did not observe an induction of baseline adhesion by fibronectin and PIP-silencing led to a marked reduction in cell adhesion to both uncoated and fibronectin-coated plates. Furthermore, we observed a significant reduction in cell–cell adhesion of BT-474 cell line following PIP-silencing. To explain an underlying mechanism for PIP regulation of cell adhesion, we found that PIP expression is necessary for the formation of α-actinin/actin-rich podosomes at the adhesion-sites of breast cancer cells. In summary, this study suggests that PIP expression regulates the process of cell adhesion in breast cancer.

Abstract 1729: Discovery of a synthetic small molecule inhibitor of HSP90 for cancer therapy

Abstract HSP90 is a major heat shock protein involved in the proper folding of its client proteins including many onco-proteins. HSP90 is elevated in tumor cells with a more active configuration, making it a potential target for cancer drug development. Utilizing structure-base design, we discovered a small molecule compound that inhibits HSP90 activity with an IC50 at low- nanomolar concentration. Cell proliferation was inhibited by this compound at a IC50 of single-digit nanomolar concentration across many different cancer cell lines. Mechanism of action studies demonstrated that this compound could induce HER-2 degradation in the breast cancer cell line BT-474 in vitro at concentrations between 100-300 nM. The compound showed an ideal pharmacological kinetic feature of less exposure and fast clearance in rat retina after intravenous administration, thus reducing the possibility of ocular toxicity as demonstrated by some other earlier HSP90 inhibitors. The compound also caused tumor growth inhibition in the xenograft tumor models, correlating with its target inhibition activity in these tumors. These data supports further development of this compound as a cancer therapeutic agent. Citation Format: Rudi Bao, Zhongzong Pan, Zhiming Zhao, Hongping Yu, Yaochang Xu. Discovery of a synthetic small molecule inhibitor of HSP90 for cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1729. doi:10.1158/1538-7445.AM2015-1729

Abstract 1971: ESE-1 controls transformation properties in HER2+ breast cancer cells, and predicts poor prognostic status and survival in breast cancer patients

Abstract The ETS transcription factor ESE-1 belongs to the top 5% of all overexpressed genes in ductal and lobular breast carcinomas. We have previously established that cytoplasmic expression of ESE-1 initiates transformation in benign MCF10A and MCF12A mammary epithelial cells, while nuclear ESE-1 maintains the transformed phenotype in luminal MCF7 and ZR-75-1 breast cancer cells. ESE-1 is also a transcriptional regulator of HER2 gene expression, and is highly expressed in HER2+ BT474 and SKBR3 cells. In this study we have delineated how ESE-1 controls transformation properties in HER2+ breast cancer cells, and have validated the clinical significance of ESE-1 overexpression in breast cancer patients. Here we show that ESE-1 not only maintains transformation properties in HER2-dependent, Herceptin (anti-HER2) sensitive HER2+ breast cancer cell lines BT474 and SKBR3, but also in HER2-independent, Herceptin-resistant clones of BT474 and SKBR3 cells, named BT474/HR20 and SKBR3/pool2 respectively. Using crystal violet growth assays, BrdU labeling, 2D clonogenic assays, and 3D colony formation in soft agar we show that knocking down ESE-1 inhibits proliferation, clonogenicity, and anchorage independent growth in SKBR3, BT474, BT474/HR20, and SKBR3/pool2 cell lines in-vitro. Mechanistically, ESE-1 knocked down Herceptin sensitive cells evinced a delay in cell cycle progression through G1, along with down-regulation of cyclin D1. Long-term stable inhibition of ESE-1 in these cells resulted in down-regulation of pAKT, which was not always correlated with inhibition of HER2 and pHER2. In the HER2-independent BT474/HR20 and SKBR3/pool2 cells, stable ESE-1 knockdown inhibited pAKT independent of HER2 down-regulation. Evaluation of the clinical significance of ESE-1 expression was done by IHC analysis on 319 patient tumor samples. While ≥ 20% nuclear ESE-1 expression associated with aggressive LN metastasis, ≥30% of cytoplasmic ESE-1 associated with poor prognosis in LN+ breast cancer patients. High cytoplasmic ESE-1 significantly associated with 27% of ER-negative, 35% of PR-negative, and 25% of HER2+ metastatic tumors. Log-rank analysis of disease free survival (DFS) in LN+ patients revealed that patients with ≥ 30% cytoplasmic ESE-1 had half the length of median DFS compared to LN+ patients with ≤ 30% cytoplasmic ESE-1 (p = 0.027). Analysis in all patients irrespective of their prognostic status depicted that patients expressing ≥ 20% nuclear ESE-1 or ≥ 30% cytoplasmic ESE-1 associated with poor median DFS compared to patients with < 20% nuclear ESE-1 or < 30% cytoplasmic ESE-1. Overall we established that ESE-1 is critical in both HER2-dependent, and HER2-independent tumorigenesis and provided novel mechanistic insights into ESE-1's mode of transformation. Finally, the data warrants a global role for ESE-1 as a prognostic marker in all breast cancer patients. Citation Format: Adwitiya Kar, Susan Edgerton, Ann Thor, Arthur Gutierrez-Hartmann. ESE-1 controls transformation properties in HER2+ breast cancer cells, and predicts poor prognostic status and survival in breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1971. doi:10.1158/1538-7445.AM2015-1971

Abstract 2314: d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells

Abstract The transmembrane tyrosine kinase receptor HER2, overexpressed in ∼20% of human breast cancers (BCs), identifies an aggressive tumor subtype and is reportedly an important regulator of breast cancer-initiating cell activity (BCIC). We found that ∼90% of HER2+ BC patients constitutively express a splice isoform of wild-type HER2 (WTHER2) gene characterized by the lack of exon 16 (d16HER2), a deletion that promotes the generation of a particularly aggressive HER2 isoform and that forms stable and constitutively activated d16HER2 homodimers. Our comparison of the tumorigenic potential of the human d16HER2 and WTHER2 genes in the corresponding transgenic mouse models revealed a significantly shorter tumor latency period (p< 0.001) and a higher tumor incidence in the d16HER2 mice (p<0.001), suggesting a role for this variant in HER2-driven activation of BCICs. In this context, our preliminary analyses of HER2-positive primary mammary tumor cell lines MI6 and MI7 derived from spontaneous transgenic d16HER2 mice showed a significantly higher mammosphere-forming efficiency and higher levels of stem cell marker transcripts, including CD44, Wnt, Notch and Bmi1, compared to transgenic mouse WTHER2 tumor cells (WTHER2_1 and WTHER2_2). Mammospheres generated from human HER2-overexpressing breast cancer cell lines BT474 and MDA-MB-361, which also express the d16HER2 variant, exhibited an increase in the relative abundance of d16HER2 mRNA compared with that in the same parental cells cultured in adhesion conditions, as indicated by qPCR analyses. Experiments in mice injected into the mammary fat pad with the d16HER2- and WTHER2-positive cell lines at different serial dilutions indicate a consistently higher “stemness potential” of MI6 and MI7 cells compared to WTHER2_1 and WTHER2_2 cells, strongly suggesting that the d16HER2 variant plays a greater role than WTHER2 in regulating BCICs of HER2-driven mammary tumors. Supported by Minister of Health and AIRC Citation Format: Lorenzo Castagnoli, Ada Koschorke, Gaia C. Ghedini, Lorenzo Galvani, Valentina Ciravolo, Cristina Ghirelli, Arianna Palladini, Alessia Lamolinara, Manuela Iezzi, Pier Luigi Lollini, Tiziana Triulzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2015-2314

Abstract 2703: Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106

Abstract Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that is overexpressed in about 20% of breast cancers and most often in the more aggressive inflammatory breast cancer (IBC). Lapatinib is one of the most potent small molecule inhibitors of HER2. Although Lapatinib has shown to be an effective targeted therapy, development of drug resistance stands as a major problem. Here we show that the anti-cancer activity of the novel fatty acid synthase inhibitor HS-106 can re-sensitize resistant cell lines to Lapatinib. Lapatinib resistant SKBR3 and BT474 breast cancer cell lines were treated with increasing concentrations of HS-106 either alone or in combination with Lapatinib and their effects on cell proliferation were assessed using the DNA dye Hoechst 33342. The ability of HS-106 and Lapatinib drug combination to induce apoptosis were measured in Caspase 3/7 activity assay using the fluorogenic Caspase substrate (DEVD)2 R110. Their effect on apoptosis was further confirmed in Lapatinib resistant BT474 cells using flow cytometry based Annexin V assay. In addition, the effect of HS-106 on the expression levels and phosphorylation of the kinases involved in Her2 pathway including Her2, AKT and ERK were determined. The fatty acid synthase inhibitor HS-106 significantly inhibits the proliferation of Lapatinib resistant cell lines with concentrations. Interestingly, when combined with Lapatinib, the drugs synergize to dramatically affect cell growth. This anti-proliferative activity of HS-106 was found to be the result of induction of apoptosis, Caspase 3/7 activity was found to increase in a dose dependent manner following HS-106 treatment. Low concentrations of Lapatinib and HS-106, which does not induce Caspase activity when administrated separately, were found to significantly induce Caspase activity when combined together. These results were confirmed with the Annexin V assay. By examining the expression levels of HER2 pathway proteins we found a decrease in HER2 levels which was reflected on the levels of the downstream kinases AKT and ERK when treating with high concentrations of HS-106. In a previous work, HS-106 showed a good tolerability and anti-cancer activity in the MMTV neu+ mouse model. By showing potent efficacy against Lapatinib resistant breast cancer cell lines, our data suggest that HS-106 is an excellent drug candidate to advance into translational studies for the treatment of Lapatinib resistant breast cancer. Citation Format: Yazan Alwarawrah, Philip Hughes, Rachid Safi, Donald P. McDonnell, Neil L. Spector, Timothy A. Haystead. Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2015-2703

Poly(ADP-ribose) polymerase as a novel regulator of 17β-estradiol-induced cell growth through a control of the estrogen receptor/IGF-1 receptor/PDZK1 axis.

BackgroundWe and others have extensively investigated the role of PARP-1 in cell growth and demise in response to pathophysiological cues. Most of the clinical trials on PARP inhibitors are targeting primarily estrogen receptor (ER) negative cancers with BRCA-deficiency. It is surprising that the role of the enzyme has yet to be investigated in ER-mediated cell growth. It is noteworthy that ER is expressed in the majority of breast cancers. We recently showed that the scaffolding protein PDZK1 is critical for 17β-estradiol (E2)-induced growth of breast cancer cells. We demonstrated that E2-induced PDZK1 expression is indirectly regulated by ER and requires IGF-1 receptor (IGF-1R).MethodsThe breast cancer cell lines MCF-7 and BT474 were used as ER(+) cell culture models. Thieno[2,3-c]isoquinolin-5-one (TIQ-A) and olaparib (AZD2281) were used as potent inhibitors of PARP. PARP-1 knockdown by shRNA was used to show specificity of the effects to PARP-1.ResultsIn this study, we aimed to determine the effect of PARP inhibition on estrogen-induced growth of breast cancer cells and examine whether the potential effect is linked to PDZK1 and IGF-1R expression. Our results show that PARP inhibition pharmacologically by TIQ-A or olaparib or by PARP-1 knockdown blocked E2-dependent growth of MCF-7 cells. Such inhibitory effect was also observed in olaparib-treated BT474 cells. The effect of PARP inhibition on cell growth coincided with an efficient reduction in E2-induced PDZK1 expression. This effect was accompanied by a similar decrease in the cell cycle protein cyclin D1. PARP appeared to regulate E2-induced PDZK1 at the mRNA level. Such regulation may be linked to a modulation of IGF-1R as PARP inhibition pharmacologically or by PARP-1 knockdown efficiently reduced E2-induced expression of the receptor at the protein and mRNA levels.ConclusionsOverall, our results show for the first time that PARP regulates E2-mediated cell growth by controlling the ER/IGF-1R/PDZK1 axis. These findings suggest that the relationship between ER, PDZK1, and IGF-1R may be perturbed by blocking PARP function and that PARP inhibitors may be considered in clinical trials on ER(+) cancers.

Can Plants’ Ability for DNA Repair and Stress Defense be Used against Patients’ Circulating Tumor Cells?

Introduction: It was suggested that specific plants may reduce cancer's resistance to chemotherapy. Resistance inhibits apoptosis, as well as other fundamental anti-cancer protective mechanisms. Soy bean has been found to reduce cellular stress and repair DNA damage caused by drought or parasites, and can transfer this defense mechanism to other plant species as well. The aim of this study is therefore to conduct a systematic comparison of the effect of soy bean formulation (FSWW08) on gene expression in in vitro human breast cancer cell line, and in in vivo in blood circulating tumor cells (CTC), after oral consumption of FSWW08 by patients suffering from breast-, ovarian-, and prostate cancer. Method: In vitro gene expressions studies were conducted with the human breast cancer cell line BT-474 that was exposed to doxorubicin or FSWW08, either alone or in combination. Ovarian-, prostate-, and breast cancer patients received FSWW08 for 30 days. CTC were extracted from their blood according to an established protocol. Gene expression evaluations were conducted before and after treatment. Results: In vitro, the multi-drug resistance (MDR) protein was reduced by FSWW08, but was increased by doxorubicin. The combination of FSWW08 and doxorubicin, however, showed a protective effect against the increase of MDR in physiologic concentrations, increased, however, also in high experimental concentrations of both agents. The expression of several cancer-related protective genes, such as tumor suppressor factors p21, p38 and p53, was improved by FSWW08 in vitro and in vivo, which helped cell differentiation and new tissue formation. Additionally, the BAX/Bcl2 ratio was improved, in vitro, as well as gene expression of estrogen receptor beta, NF-κB, MAP kinase, c-JUN, and matrix metalloproteinase 9, together with an increase of VEGF expression in vivo in CTC. Conclusion: It was demonstrated that FSWW08 improved the gene functions related to DNA repair and stress in human blood CTC and resistance marker, in vitro, when applied in combination with doxorubicin. As such, larger in vitro and in vivo clinical studies that investigate single botanical compounds from other plants, are warranted.

Synthesis and biological evaluation of 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine derivatives as novel PI3Kδ inhibitors

Abstract An efficient synthesis of novel 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine scaffold has been designed and deveopled. A series of 5-phenylurea derivatives was synthesized using this method. Their cytotoxic activities against breast cancer cell line BT-474 were evaluated by CCK-8 assay. Most of them showed potent anti-proliferative activities, of which compound 20 and 21 exhibited IC50s of 1.565 μmol/L and 1.311 μmol/L, respectively. Furthermore, compound 20 and 21 also showed potent inhibitory activities against PI3Kδ with IC50s of 0.286 μmol/L and 0.452 μmol/L, respectively. These results indicate that these 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine derivatives are novel antitumor agents through the inhibition of PI3Kδ.

The potential utility of acetyltanshinone IIA in the treatment of HER2-overexpressed breast cancer: Induction of cancer cell death by targeting apoptotic and metabolic signaling pathways

Increased lipogenesis and protein synthesis is a hallmark of cancer cell proliferation, survival, and metastatic progression and is under intense investigation as a potential antineoplastic target. Acetyltanshinone IIA (ATA) is a compound that was obtained from chemical modifications of tanshinone IIA (TIIA), a potent anticancer agent extracted from the dried roots of the Chinese herbal medicine Salvia miltiorrhiza Bunge. A previous investigation indicated that ATA is more effective in inhibiting the growth of breast cancer especially cells with HER2 overexpression. However, the molecular mechanism(s) mediating this cytotoxic effect on HER2-positive breast cancer remained undefined. Studies described here report that ATA induced G1/S phase arrest and apoptosis in the HER2-positive MDA-MB-453, SK-BR-3, and BT-474 breast cancer cell lines. Mechanistic investigations revealed that the ATA-induced apoptosis effect is associated with remarkably down-regulation of receptor tyrosine kinases (RTKs) EGFR/HER2 and inhibition of their downstream pro-survival signaling pathways. Interestingly, ATA was found to trigger oxidative and endoplasmic reticulum (ER) stresses and to activate AMP activated protein kinase (AMPK) leading to inactivation of key enzymes involved in lipid and protein biogenesis. Intraperitoneal administration of ATA significantly inhibited the growth of MDA-MB-453 xenografts in athymic mice without causing weight loss and any other side effects. Additionally, transwell migration, invasion, and wound healing assays revealed that ATA could suppress tumor angiogenesis in vitro. Taken together, our data suggest that ATA may have broad utility in the treatment of HER2-overexpressed breast cancers.

Time-lapse imaging assay using the BioStation CT: a sensitive drug-screening method for three-dimensional cell culture.

Three-dimensional (3D) cell culture is beneficial for physiological studies of tumor cells, due to its potential to deliver a high quantity of cell culture information that is representative of the cancer microenvironment and predictive of drug responses in vivo. Currently, gel-associated or matrix-associated 3D cell culture is comprised of intricate procedures that often result in experimental complexity. Therefore, we developed an innovative anti-cancer drug sensitivity screening technique for 3D cell culture on NanoCulture Plates (NCP) by employing the imaging device BioStation CT. Here, we showed that the human breast cancer cell lines BT474 and T47D form multicellular spheroids on NCP plates and compared their sensitivity to the anti-cancer drugs trastuzumab and paclitaxel using the BioStation CT. The anticancer drugs reduced spheroid migration velocity and suppressed spheroid fusion. In addition, primary cells derived from the human breast cancer tissues B58 and B61 grown on NCP plates also exhibited similar drug sensitivity. These results were in good agreement with the conventional assay method using ATP quantification. We confirmed the antitumor effects of the drugs on cells seeded in 96-well plates using the BioStation CT imaging technique. We expect this method to be useful in research for new antitumor agents and for drug sensitivity tests in individually-tailored cancer treatments.

English

The identification of medicinal plants and derived natural products for development as anti-cancer agents is of long standing interest. We have investigated the anti-proliferative properties of Lupinus angustifolius on breast cancer and, in particular, whether the extracts of roots and shoots of L. angustifolius can be considered as candidates for primary and secondary cancer prevention. Ethanolic extracts of roots and shoots of L. angustifolius were analysed for their substance classes by pyrolysis-field ionization mass spectrometry. Various concentrations of these extracts (0.1, 1, 10, 25, 50, 100 and 200 µg/ml, incubation period: 24 h) were studied with respect to their effects on cell proliferation and cytotoxicity against the breast cancer cell lines MCF-7 (ER-α+, ER-β+) and BT20 (ER-α-, ER-β-). L. angustifolius ethanolic root and shoot extracts inhibited cell proliferation in the MCF-7 and BT20 cells; root extract: strongest inhibition MCF-7 (200 μg/ml 71.03 ± 12.62%); BT20 (200 μg/ml 99.72 ± 6.48%), IC50-values: MCF-7 (52.3 ± 9.39 μg/ml); BT20 (66.86 ± 7.02 μg/ml); shoot extract: strongest inhibition MCF-7 (200 μg/ml 64.7 ± 10.07 %); BT20 (200 μg/ml 86.32 ± 9.19%), IC50-values: MCF-7 (18.06 ±4.49 μg/ml); BT20 (70.27 ± 0.76 μg/ml). Thus, extracts of L. angustifolius roots and shoots have anti-tumour activity against receptor-positive and receptor-negative breast cancer cell lines.   Key words: Anti-tumour activity, Lupinus angustifolius, breast cancer, MCF-7, BT20

ABL tyrosine kinase inhibition variable effects on the invasive properties of different triple negative breast cancer cell lines.

The non-receptor tyrosine kinase ABL drives myeloid progenitor expansion in human chronic myeloid leukemia. ABL inhibition by the tyrosine kinase inhibitor nilotinib is a first-line treatment for this disease. Recently, ABL has also been implicated in the transforming properties of solid tumors, including triple negative (TN) breast cancer. TN breast cancers are highly metastatic and several cell lines derived from these tumors display high invasive activity in vitro. This feature is associated with the activation of actin-rich membrane structures called invadopodia that promote extracellular matrix degradation. Here, we investigated nilotinib effect on the invasive and migratory properties of different TN breast cancer cell lines. Nilotinib decreased both matrix degradation and invasion in the TN breast cancer cell lines MDA-MB 231 and MDA-MB 468. However, and unexpectedly, nilotinib increased by two-fold the invasive properties of the TN breast cancer cell line BT-549 and of Src-transformed fibroblasts. Both display much higher levels of ABL kinase activity compared to MDA-MB 231. Similar effects were obtained by siRNA-mediated down-regulation of ABL expression, confirming ABL central role in this process. ABL anti-tumor effect in BT-549 cells and Src-transformed fibroblasts was not dependent on EGF secretion, as recently reported in neck and squamous carcinoma cells. Rather, we identified the TRIO-RAC1 axis as an important downstream element of ABL activity in these cancer cells. In conclusion, the observation that TN breast cancer cell lines respond differently to ABL inhibitors could have implications for future therapies.

Highlights from the latest articles in nano-oncology.

Highlights from the latest articles in nano-oncology.

Promestriene Affects <i>GREB</i>1 Expression in Estrogen Sensitive Breast Cancer Cells

Promestriene (3-propyl ethyl, 17B-methyl estradiol) is a synthetic estrogen analogue with reported minimal systemic absorption which has been suggested for topical treatment of vaginal atrophy. Promestriene’s ability to stimulate proliferation and estrogen responsive gene expression was analyzed in estrogen receptor (ER+) positive breast cancer cell lines MCF-7, T-47D, and BT-474 using CFSE flow cytometric analysis, and quantitative RT-PCR analysis of GREB1 RNA expression, an estrogen responsive gene involved in estrogen receptor alpha expression. In estrogen replete conditions, Promestriene did not stimulate proliferation even at high concentrations (100,000 pg/ml). However, anti-estradiol depletion allowed low dose Promestriene (2 - 10 pg/ml) to stimulate GREB1 expression in all three cell lines at levels equal to that induced by estradiol (BT-474) or significantly higher than estradiol (MCF7 and T-47D). These findings suggest that Promestriene has the potential to support estrogen like cell signaling, a possible contraindication for use in treatment of vaginal atrophy associated with breast cancer aromatase inhibitor therapy.

TXNIP interaction with the Her-1/2 pathway contributes to overall survival in breast cancer.

Previous studies have indicated that Her-2 induction causes a strong decrease in thioredoxin interaction protein (TXNIP) in breast cancer cells. However, little is known regarding the prognostic value of TXNIP in clinical breast cancer patients with anti-Her-2 treatment. Using a tissue microarray, we detected TXNIP and p27 expression in breast cancer tissue, as well as corresponding noncancerous tissues. We found that TXNIP expression was associated with better overall survival (OS) in these 150 breast cancer patients and that TXNIP and Her-2 expression status were significantly inversely correlated (r=-0.334, P<0.001). These results were validated in another 101 breast cancer tissue samples (r=-0.422, P<0.001). Moreover, TXNIP expression increased significantly following treatment of the human breast cancer cell lines BT474 and SK-BR-3 with a Her-1/2 inhibitor. Furthermore, TXNIP transfection induced p27 expression and G1 cell cycle arrest and apoptosis. Taken together, our findings suggest that TXNIP plays a critical role in anti-Her-1/Her-2 treatment and may be a potential prognostic marker in breast cancer.

REGγ regulates ERα degradation via ubiquitin–proteasome pathway in breast cancer

REGγ is a proteasome coactivator which regulates proteolytic activity in eukaryotic cells. Abundant lines of evidence have showed that REGγ is over expressed in a number of human carcinomas. However, its precise role in the pathogenesis of cancer is still unclear. In this study, by examining 200 human breast cancer specimens, we demonstrated that REGγ was highly expressed in breast cancers, and the expression of REGγ was positively correlated with breast cancer patient estrogen receptor alpha (ERα) status. Moreover, the expression of REGγ was found positively associated with poor clinical features and low survival rates in ERα positive breast cancer patients. Further cell culture studies using MCF7 and BT474 breast cancer cell lines showed that cell proliferation, motility, and invasion capacities were decreased significantly by REGγ knockdown. Lastly, we demonstrated that REGγ indirectly regulates the degradation of ERα protein via ubiquitin–proteasome pathway. In conclusion, our findings provide the evidence that REGγ expression was positively correlated with ERα status and poor clinical prognosis in ERα positive breast cancer patients. As well, we disclose a new connection between the two molecules that are both highly expressed in most breast cancer cases.

TRAIL-mediated apoptosis in breast cancer cells cultured as 3D spheroids.

TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44hiCD24loALDH1hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway. We show that inhibition of the COX-2/PGE2 pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44hiCD24lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance.