Breast Cancer Cell Lines BT474 Research Articles

Micromolecular methods for diagnosis and therapeutic strategy: a case study.

An intraductal carcinoma, 55 mm across, was diagnosed on a total mastectomy in a 45-year-old woman. The 2 micro-invasive areas found were too small for reliable immunostainings for estrogen, progesterone, and HER2 receptors. In the sentinel lymph-node, a subcapsular tumor embole of about 50 cancer cells was identified on the extemporaneous cryo-cut section, but not on further sections after paraffin-embedding of the sample.Considering this tumor metastatic potential, we decided to assess HER2 status on the metastatic embole using pathological and molecular micro-methods. We laser-microdissected the tumor cells, extracted their DNA, and performed droplet-digital-PCR (ddPCR) for HER2 gene copy number variation. The HER2/RNaseP allele ratio was 5.2 in the laser-microdissected tumor cells, similar to the 5.3 ratio in the HER2-overexpressing breast cancer cell line BT-474.We thus optimized the adjuvant treatment of our patient and she received a trastuzumab-based adjuvant chemotherapy.

Abstract P3-07-05: Potential of breast tumour kinase (Brk) as a therapeutic target: Brk modulates drug responses in breast cancer cell lines

Abstract Background: Breast tumour kinase (Brk/PTK6) is over-expressed in up to 86% of all breast cancers and has shown to be involved in the processes regulating tumour development and progression. A few Brk inhibitors are in development and our studies indicate potential for a Brk inhibitor to be used in combination with current breast cancer therapies. Furthermore the effect of common breast cancer therapies and Brk inhibition on the expression of ptk6 as well as it's alternatively spliced isoform (ALT-PTK6) have not been previously been shown. Using qPCR we have determined gene expression of both genes before and after treatment. Methods: Breast cancer cell lines T47D, GI101, BT474, SKBR3, MDA-MB 231, and MDA-MB 436 were treated with Brk inhibitor, Compound 4f (Mahmoud et al, 2012) and common breast cancer therapies (Taxol, Doxorubicin, Lapatinib and Tamoxifen) at concentrations ranging from 0µM to 10µM. Western blotting was carried out to determine levels of Brk and activation of Brk substrate; STAT3 after treatment with Brk inhibitor. RNA was extracted using Qiagen Mini RNeasy prep kit, cDNA synthesized using Invitrogen Superscript II Reverse Transcriptase and qPCR carried out using primers specific for PTK6 or primers that recognized both transcripts (PTK6 and ALT-PTK6) for total expression. Results: In all cell lines tested there was a moderate reduction in cell proliferation following treatment with the Brk inhibitor (4f). However a greater effect was observed in combination therapy. Triple negative breast cancer cell lines MDA-MB 231 and MDA-MB 436 were treated with Compound 4f and Taxol or Doxorubicin (n=3) resulting in modest but statistically significant reduction in cell numbers. Cell responses to Taxol in both cell lines were significantly greater in the presence of 4f over a range of doses (P between 0.05 and 0.007). Responses to Doxorubicin were also significantly improved in the presence of 4f (P<0.03 for MDA-MB-231 and P=0.03 for MDA-MB-436). Co-treatment of HER2 positive breast cancer cell lines BT474 and Sk-Br-3 with Lapatinib and 4f showed significant increase in responses over a range of doses between 0.31 and 5µM (n=3, P<0.05 for BT474 and P between 0.03 and 0.0004 for Sk-Br-3). In comparison to untreated cells, there was statistically significant reduction in ptk6 and Total gene expression observed at various time points within multiple breast cancer cell lines in response to Compound 4f treatment. Significant differences between untreated and treated cells for T47D cell line were at 8 hours post treatment (p=0.02), 2 and 4 hours post treatment in GI101 cell line (p=0.04 and p=0.02 respectively), 8 and 24 hours post treatment in Sk-Br-3 (p=0.001 and p=0.017 respectively) and 8 hours post treatment in MDA-MB 231 cell lines (p=0.03). Conclusion: Inhibition of Brk led to a decrease in ptk6 gene expression. Expression ratios of ptk6 and the short isoform ALT-PTK6 were determined and a reduction of ptk6 gene correlated with elevation of ALT-PTK6 and vice versa. Inhibition of Brk also indicated an increase in breast cancer cell sensitivity to current breast cancer therapies. Our studies thus indicate potential for inhibition of Brk kinase activity in combination with current breast cancer treatments. Citation Format: Hussain H, Harvey A. Potential of breast tumour kinase (Brk) as a therapeutic target: Brk modulates drug responses in breast cancer cell lines [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-05.

P16 promotes the growth and mobility potential of breast cancer both in vitro and in vivo: the key role of the activation of IL-6/JAK2/STAT3 signaling.

P16 is the product of cyclin-dependent kinase 2 (CDKN2A) gene and plays multi-pronged roles in the cancer progression. Breast cancer (BC) is the most commonly diagnosed cancer type among females. In the current study, the potential function of P16 in the growth and metastasis of BC was investigated. Firstly, the expression statuses of P16 in different cancer types were investigated using Oncomine database and validated with corresponding cancer cell lines. Afterwards, the expression of P16 was knocked down in BC cell line BT-549 and the effect on the cell proliferation, sensitivity to paclitaxel (TAX), apoptosis, migration, and invasion abilities was assessed using CCK-8, Edu, flow cytometry, scratch, and transwell assays, respectively. The influence of P16 inhibition and P16 overexpression on the activity of IL-6/JAK/STAT3 signaling was explored. Additionally, the effect of P16 inhibition on the tumor growth was verified with a BC xenograft mice model. The abnormal expression of P16 was detected in BC cell line BT-549 as well as colorectal cancer and osteosarcoma cell lines. The inhibition of P16 suppressed the cell proliferation, invasion, and migration abilities while induced the apoptosis and sensitivity to TAX in BT-549 cells. At molecular level, P16 knockdown inhibited the expression of IL6ST and Survivin, and the phosphorylation of JAK2 and STAT3. However, the induced expression of P16 in P16-knockdown BT-549 cells restored the activity of IL-6/JAK2/STAT3 pathway. The results of in vitro assays were confirmed with BC xenograft models: the inhibition of P16 decreased the tumor growth rate. Findings outlined in the current study demonstrated that the inhibition of P16 decreased the growth and metastasis potential of BC cells by inhibiting IL-6/JAK2/STAT3 signaling.

Abstract A151: Drug sensitivity profiling of BT-474 breast cancer cell line for identification of novel therapies against HER2-positive breast cancer

Abstract Introduction: Targeted therapy for breast cancer expressing hormone receptors such as estrogen receptor (ER) and progesterone receptor (PR), as well as human epidermal growth factor receptor (HER2), is essential. In spite of current targeted therapies, breast cancer has high susceptibility to spread and metastases often turn resistant to the used treatment. Therefore, new therapies are needed, especially when cancer becomes resistant to hormonal therapy. The aim of the study was to investigate drug sensitivity and resistance of BT-474 breast cancer cell line and to recognize possible vulnerabilities, especially in hormone therapy resistant HER2+ breast cancer. Materials and Methods: BT-474 cell line (ER+, PR+, HER2+) is originally derived from human solid, invasive ductal carcinoma of the breast. The drug sensitivity of the cell line was assessed by applying a large panel of drugs covering cancer chemotherapeutics and clinically available and emerging drugs including, e.g., conventional chemotherapy and kinase inhibitors such as HER2, pan-HER2, and EGFR-HER2 inhibitors. Altogether a panel of 525 compounds was tested in five concentrations covering a 10,000-fold drug-relevant concentration range in 384-well format. Cells were seeded to predrugged plates, followed by cell viability measurements (CellTiter-Glo) after 72 hours. Maximal and minimal responses to drugs were analyzed, the EC50 values were calculated, and Drug Sensitivity Score (DSS) was calculated for each drug as a measure of reduced viability. A selective Drug Sensitivity Score (sDSS) was calculated to identify the selective drug response pattern. Results: DSS analysis of BT-474 cell line showed sensitivity to conventional chemotherapy including paclitaxel and docetaxel and to kinase inhibitors such as EGFR inhibitors neratinib and afatinib. In the absence of supplemented estradiol, the cell line was not sensitive to hormonal therapies, including antiestrogens and selective estrogen receptor modulators. However, BT-474 cells were sensitive to HER2, pan-HER2, and EGFR-HER2 inhibitors including mubritinib, poziotinib, dacomitinib, and lapatinib with EC50 range from 2 to 98 nM. The BT-474 cells also showed sensitivity against PI3K and mTOR inhibitors, such as copanlisib and omipalisib. Conclusions: Screening of large compound libraries combined with DSS and sDSS analysis enables drug sensitivity profiling of HER2+ BT-474 breast cancer cells for identification of novel anticancer compounds against breast cancer, especially for the hormonal-resistant stage of the disease. Moreover, the assay enables repurposing of existing drugs to new indications, identification of vulnerabilities in different types of cancer cells, and functional investigation of cellular pathways behind drug sensitivity or resistance. Citation Format: Jenni Mäki-Jouppila, Tiina Kähkönen, Mari I. Suominen, Katja M. Fagerlund, Jussi M. Halleen, Jani Saarela, Jenni Bernoulli. Drug sensitivity profiling of BT-474 breast cancer cell line for identification of novel therapies against HER2-positive breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A151.

Chondroitin sulfate–mediated N-cadherin/β-catenin signaling is associated with basal-like breast cancer cell invasion

Tumor metastasis involves cancer cell invasion across basement membranes and interstitial tissues. The initial invasion step consists of adherence of the tumor cell to the extracellular matrix (ECM), and this binding transduces a variety of signals from the ECM to the tumor cell. Accordingly, it is critical to establish the mechanisms by which extracellular cues influence the intracellular activities that regulate tumor cell invasion. Here, we found that invasion of the basal-like breast cancer cell line BT-549 is enhanced by the ECM component chondroitin sulfates (CSs). CSs interacted with and induced proteolytic cleavage of N-cadherin in the BT-549 cells, yielding a C-terminal intracellular N-cadherin fragment that formed a complex with β-catenin. Of note, the cleavage of N-cadherin increased cytoplasmic and nuclear β-catenin levels; induced the matrix metalloproteinase 9 (MMP9) gene, a target of β-catenin nuclear signaling; and augmented the invasion potential of the cells. We also found that CS-induced N-cadherin proteolysis requires caveolae-mediated endocytosis. An inhibitor of that process, nystatin, blocked both the endocytosis and proteolytic cleavage of N-cadherin induced by CS and also suppressed BT-549 cell invasion. Knock-out of chondroitin 4-O-sulfotransferase-1 (C4ST-1), a key CS biosynthetic enzyme, suppressed activation of the N-cadherin/β-catenin pathway through N-cadherin endocytosis and significantly decreased BT-549 cell invasion. These results suggest that CSs produced by C4ST-1 might be useful therapeutic targets in the management of basal-like breast cancers.

Preclinical and phase I clinical studies of KW-2450, a dual IGF-1R/IR tyrosine kinase inhibitor, in combination with lapatinib and letrozole.

Background:KW-2450 is an oral dual insulin-like growth factor-1 receptor/insulin receptor tyrosine kinase inhibitor. We investigated the in vitro and in vivo preclinical activity of KW-2450 plus lapatinib and letrozole and conducted a phase I trial of the triple-drug combination in one male and 10 postmenopausal female patients with advanced/metastatic hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-positive breast cancer.Methods:A series of in vitro and in vivo animal studies was undertaken of KW-2450 in combination with lapatinib and hormonal agents. The phase I trial was conducted to establish the safety, tolerability, and recommended phase II dose (RP2D) of KW-2450 administered in combination with lapatinib and letrozole.Results:Preclinical studies showed KW-2450 and lapatinib act synergistically to induce in vitro apoptosis and inhibit growth of HER2-positive MDA-MB-361 and BT-474 breast cancer cell lines. This combined effect was confirmed in vivo using the MDA-MB-361 xenograft model. KW-2450 showed synergistic in vitro growth inhibition with letrozole and 4-hydroxytamoxifen in ER-positive MCF-7 breast cancer cells and MCF-7-Ac1 aromatase-transfected MCF-7 cells. In the phase I study, dose-limiting toxicity (DLT; grade 3 rash and grade 3 hyperglycemia, respectively) occurred in two of three patients at the dose of KW-2450 25 mg/day plus lapatinib 1500 mg/day and letrozole 2.5 mg/day. The RP2D of the triple-drug combination was established as KW-2450 25 mg/day, lapatinib 1250 mg/day, and letrozole 2.5 mg/day with no DLT at this dose level.Conclusions:The proposed phase II study of the RP2D for the triple-drug combination did not progress because of anticipated difficulty in patient enrollment and further clinical development of KW-2450 was terminated.

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases.

Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8–12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings.

MiR-193b regulates breast cancer cell migration and vasculogenic mimicry by targeting dimethylarginine dimethylaminohydrolase 1

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is responsible for metabolism of an endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), which plays a key role in modulating angiogenesis. In addition to angiogenesis, tumours can establish a vascular network by forming vessel-like structures from tumour cells; a process termed vasculogenic mimicry (VM). Here, we identified over-expression of DDAH1 in aggressive MDA-MB-231, MDA-MB-453 and BT549 breast cancer cell lines when compared to normal mammary epithelial cells. DDAH1 expression was inversely correlated with the microRNA miR-193b. In DDAH1+ MDA-MB-231 cells, ectopic expression of miR-193b reduced DDAH1 expression and the conversion of ADMA to citrulline. In DDAH1− MCF7 cells, inhibition of miR-193b elevated DDAH1 expression. Luciferase reporter assays demonstrated DDAH1 as a direct target of miR-193b. MDA-MB-231 cells organised into tube structures in an in vitro assay of VM, which was significantly inhibited by DDAH1 knockdown or miR-193b expression. Mechanistically, we found miR-193b regulates cell proliferation and migration of MDA-MB-231 cells, whilst DDAH1 knockdown inhibited cell migration. These studies represent the first evidence for DDAH1 expression, regulation and function in breast cancer cells, and highlights that targeting DDAH1 expression and/or enzymatic activity may be a valid option in the treatment of aggressive breast cancers.

MiR-1296-5p decreases ERBB2 expression to inhibit the cell proliferation in ERBB2-positive breast cancer

BackgroundThe tumor suppressive role of miR-1296 is observed in triple negative breast cancer (TNBC). However, the effect of miR-1296-5p in ERBB2-positive breast cancers remains obscure.MethodsWhether ERBB2 was the target gene of the miR-1296-5p was predicted by online software, and determined by dual-luciferase activity assay. miR-1296-5p expression levels were determined in breast cancer samples (114 breast cancer tissues and 30 adjacent normal tissues) by using qRT-PCR. The effect of miR-1296-5p and inhibition of ERBB2/mTORC1 signaling on the downstream target was assessed by Western blot. SK-BR-3 and BT-474 breast cancer cell line was transfected with miR-1296-5p mimic after which cell proliferation and apoptosis were determined by the clonogenic assay and the flow cytometry system, respectively. In addition, the chemotherapeutic drug sensitivity of SK-BR-3 and BT-474 cells transfected with miR-1296-5p mimic were determined by MTT assay.ResultsThe luciferase assay carrying ERBB2 3′-untranslated region-based reporters expressed in SK-BR-3 and BT-474 cells suggested that ERBB2 was the target gene of miR-1296-5p. MiR-1296-5p was significantly decreased in breast cancer tissues compared to adjacent normal tissues. Moreover, it was declined in ERBB2-positive breast cancer samples compared with that in ERBB2-negative breast cancer tissues. Overexpressed miR-1296-5p reduced its target protein level and mTORC1/S6 activation, inhibited the proliferation of ERBB2-positive breast cancer cells and sensitized these cells to cisplatin and 5-fluorouracil-induced apoptosis.ConclusionsOur findings suggest that miR-1296-5p is involved in the regulation of proliferation in breast cancer cells via targeting ERBB2/mTORC1 signaling pathway.

Docetaxel-loaded nanostructured lipid carriers functionalized with trastuzumab (Herceptin) for HER2-positive breast cancer cells

The aim of the present study was to prepare Herceptin targeted nanostructured lipid carriers (NLCs) of docetaxel (DTX). Herceptin was conjugated by chemical and physical methods to NLCs prepared by solvent extraction technique followed by probe sonication. Different types of fatty amines were used in construction of NLCs. The NLCs were characterized for their antibody coupling efficiency, particle size, zeta potential, polydispersity index, drug entrapment efficiency and drug release profiles. The toxicity of NLCs on MDA-MB-468 (HER2 negative receptor) and BT-474 (HER2 positive) breast cancer cell lines was evaluated by MTT assay. Also their cellular uptake was studied by flow-cytometry and fluorescent microscopy. The results showed the NLCs containing stearyl amine had the lowest particle size, the highest zeta potential and antibody coupling efficiency values. Herceptin binding to NLCs led to reduction in zeta potential and drug entrapment efficiency while, particle size increased. The NLCs containing spermine(SP) released DTX slower than other fatty amines. Non-conjugated nanoparticles containing DTX had more toxicity than the free DTX on both cell lines. Herceptin targeted NLCs caused more mortality on BT-474 cells than MDA-MB-468 cells. Flow-cytometry studies revealed enhanced cellular uptake of nanoparticles chemically conjugated by Herceptin on the BT-474 cells. DTX loaded in chemically conjugated NLCs to Herceptin showed more cytotoxic effects than the physically coated nanoparticles. The Herceptin conjugated NLCs seem promising in oriented delivery of DTX to HER2 positive breast cancer cells.

Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer activity against breast cancer cell lines

Forskolin C1-isoxazole derivatives (3,5-regioisomers) (11a–e, 14, 15a–h and 15, 16a–g) were synthesized regioselectively by adopting 1,3-dipolar cycloadditions. These derivatives were tested using estrogen receptor positive breast cancer cell lines MCF-7 and BT-474. Majority of the compounds exhibited activity against the p53-positive MCF-7 breast cancer cells but not against the p53-negative BT-474 breast cancer cells. Among forskolin derivatives, compounds 11a, 11c, 14a, 14f, 14g, 14h, 15b, 16g and 17b exhibited higher anti-cancer activity against MCF-7 cell line with an IC50≤1µM. The derivative 14f exhibited highest activity in both p53-positive (MCF-7) and p53-negative (BT-474) breast cancer cell lines with an IC50 of 0.5µM.

Abstract 1089: Anti-tumor activity of selinexor is enhanced by palbociclib in preclinical models of HER2+ breast cancer

Abstract Introduction: XPO1 (exportin-1/ CRM1) inhibitor selinexor (KPT-330) is the first-in-class, orally bioavailable, clinical stage SINE (Selective Inhibitor of Nuclear Export) compound with marked anti-tumor activity towards solid and hematological malignancies. This activity can be further enhanced by other therapeutic agents. We have previously shown strong synergistic preclinical activity of selinexor plus trastuzumab against HER2+ breast cancer. In cancer models of acquired resistance to HER2-targeted therapies, G1/S phase cell cycle regulators Cyclin D1 and CDK4/6 are inappropriately activated. We therefore investigated the combinatorial effect of selinexor plus palbociclib, a CDK4/6 inhibitor, in HER2+ breast cancer models as a treatment option for recurrent and relapsed HER2+ breast cancers. Methods: The effects of selinexor or palbociclib single agents or in combination were tested in vitro with BT474 HER2+ breast cancer cell line. Total RNA and protein was extracted from cell lysates and analyzed by qPCR and immunoblots. In vivo, a subcutaneous BT474 xenograft mouse model was treated with selinexor (5 mg/kg or 15 mg/kg; qodx3) or palbociclib (50 mg/kg or 150 mg/kg; qd) single agents or in combination. Tumor growth and body weights were monitored for 60 days. Tumors were harvested and analyzed by immunohistochemistry (IHC). Results: Selinexor plus palbociclib was highly effective in vitro and in vivo in BT474 breast cancer cells. In in vitro assays, selinexor or palbociclib single agents demonstrated inhibitory effects on cell proliferation and showed strong synergy when combined. In vivo, the combination resulted in significant survival benefit and enhanced tumor growth inhibition compared to vehicle or either single agent. IHC analysis of xenograft tumors showed synergistic inhibition of cell proliferation by selinexor plus palbociclib. The Ki67 proliferation index determined by IHC was 25% for vehicle control, 20% for selinexor, 7% for palbociclib and 2% for the combination. Based on IHC analysis, the synergistic anti-tumor activity of selinexor plus palbociclib was achieved at multiple levels of the CDK4/6 pathway. Selinexor treatment increased p21, p27 and Rb nuclear staining. Both p21 and p27 are inhibitors of CDK4/6 while Rb is a negative regulator of cell cycle progression. CDK4/6 phosphorylates and inactivates Rb, which allows cell cycle progression. In selinexor as well as palbociclib treated samples, phosphorylated Rb in the nucleus decreased, indicating a down-regulation of the CDK4/6 pathway. Conclusion: Selinexor plus palbociclib shows synergistic inhibition of cell proliferation in vivo and in vitro in HER2+ breast cancer cell line BT474 by down-regulation of CDK4/6 pathway. This combination therapy warrants further investigation as an effective treatment option for recurrent and relapsed HER2+ breast cancer. Citation Format: Hua Chang, Sharon Friedlander, Trinayan Kashyap, Boris Klebanov, Christian Argueta, Oscar A. Gonzalez, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Margaret Lee, William Senapedis. Anti-tumor activity of selinexor is enhanced by palbociclib in preclinical models of HER2+ breast cancer [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 1089. doi:10.1158/1538-7445.AM2017-1089

Abstract 5857: HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in a HER2 positive orthotopic bone model

Abstract In 2015, the estimated incidence of new breast cancer (Ca) cases in the US was 234.190 and number of deaths 40.730. Human epidermal growth factor receptor 2 (HER2) is encoded by the proto-oncogene c-erbB-2 and initiates downstream signaling pathways leading to cell proliferation and tumorigenesis. HER2 is overexpressed in several cancer types and has emerged as one of the most strongly validated targets for the treatment of breast and gastric cancer serving as both a prognostic and predictive biomarker. Given that 20% of breast Ca patients are HER2 positive and 70% of patients with metastatic disease will develop bone metastases and associated morbidities, there is still an unmet medical need for improved therapies targeting HER2. Radium-223 (Ra-223) is a novel targeted alpha therapeutic for treatment of patients with castration-resistant prostate cancer and bone metastases. Localized high energy alpha particle emission induces double-stranded DNA breaks and cellular apoptosis. Thorium-227 (Th-227) is the immediate precursor for Ra-223 which, in contrast to Ra-223, can be complexed by chelating agents at high affinity, allowing targeted delivery to tumor cells via antibodies. We describe herein the generation of a novel HER2-targeted Th-227 conjugate (HER2-TTC). HER2-TTC consists of the humanized HER2 targeting IgG1 antibody trastuzumab covalently linked via an amide bond to a 3,2-hydroxypyridino-based chelator moiety, enabling efficient radiolabeling with the alpha particle emitting radionuclide Th-227. Anonymized samples of consenting breast cancer patients were analyzed by Immunohistochemistry (IHC). The IHC data demonstrated HER2 positive expression in breast tumor and matched bone metastases, supporting the preclinical evaluation of the anti-tumor efficacy of HER2-TTC in the BT-474 orthotopic bone mouse model. HER2-TTC was prepared at high radiochemical yield and purity. When tested for binding to recombinant HER2, HER2-TTC was shown to retain comparable binding affinity to trastuzumab. In vitro cytotoxicity experiment of HER2-TTC demonstrated target mediated in vitro cytotoxicity in the pM-range on breast cancer cell line BT-474 (430 000 mAbs bound/ cell as determined by FACS). Anti-tumor efficacy of HER2-TTC was evaluated at 250 and 500 kBq/kg at a protein dose of 0.14 mg/kg. X-ray imaging, serum bone formation marker PINP, micro CT 3D reconstruction imaging and histological analysis demonstrated significantly reduced bone lesions and tumor induced bone remodeling. The promising preclinical anti-tumor activity supports the development of the HER2-TTC as a novel targeted alpha therapeutic for the treatment of patients with HER2 positive bone metastatic disease. Citation Format: Jenny Karlsson, Urs B. Hagemann, Christoph Schatz, Derek Grant, Christine Ellingsen, Alexander Kristian, Dessislava Mihaylova, Steinar R. Uran, Mari Suominen, Roger M. Bjerke, Olav B. Ryan, Carl F. Nising, Dominik Mumberg, Alan Cuthbertson. HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in a HER2 positive orthotopic bone model [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 5857. doi:10.1158/1538-7445.AM2017-5857

Abstract 471: HER2 regulates PARP-1 expression by suppressing the let-7a microRNA in HER2+ breast cancer

Abstract Background: HER2+ breast cancers are sensitive to PARP inhibition and express elevated levels of the PARP-1 protein. However, the mode of regulation of PARP-1 expression by HER2 is not well understood. MicroRNAs are small non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression. In this study, we investigate whether PARP1 expression in human breast cancer cells is regulated by HER2 regulation of microRNAs. Methods: Human HER2+ breast cancer cell lines BT-474 and SKBR3 were used in this study. MDA-MB-231 (non-HER2 expressing breast cancer cells) stably transfected with a HER2 wild-type plasmid (231 HER2) or the vector control (231 NEO) were also utilized. To identify candidate microRNAs regulated by HER2 overexpression, we performed the nCounter miRNA Expression Assay. MicroRNA and mRNA expression were validated via qRT-PCR analysis in breast cancer cell lines or patient primary tumors. Cells were also transfected with a HER2 siRNA, a let-7a mimic, and an inhibitor of let-7a as well as their respective controls. Western blot analysis and firefly luciferase assays were used to determine whether the 3’UTR of PARP1 was being directly targeted by the let-7a microRNA. Results: HER2 did not regulate PARP-1 at the mRNA level but increased PARP-1 protein in HER2+ breast cancer cells. Specifically, ectopic HER2 overexpression correlated with increased PARP-1 protein levels in the 231 HER2 cell line. Conversely, silencing HER2 reduced PARP-1 protein levels in the BT-474 and SKBR3 cell lines. NanoString nCounter analysis revealed that the HER2+ breast cancer cell lines expressed low levels of the let-7a microRNA. Further, let-7a expression was upregulated after HER2 knockdown in the two native HER2+ breast cancer cell lines. The let-7a mimic also reduced both PARP1 protein expression and luciferase activity in the 231 HER2 and BT-474 cell lines, whereas the let-7a inhibitor reversed these effects in the 231 NEO cell line. Importantly, human HER2+ breast tumors expressed higher levels of PARP-1 and lower levels of let-7a; whereas, the HER2- breast tumors expressed lower levels of PARP-1 and higher levels of let-7a. Further, overexpression of the let-7a mimic reduced cell proliferation in HER2+ breast cancer cells. Conclusions: These results suggest that let-7a regulates PARP-1 expression in HER2+ breast tumors. Let-7a may also be a potential therapeutic target and predictive biomarker of PARPi sensitivity in HER2+ breast cancer patients. Citation Format: Eddy Shih-Hsin Yang, Monicka Wielgos, Rajani Rajbhandari, Shi Wei, Susan Nozell. HER2 regulates PARP-1 expression by suppressing the let-7a microRNA in HER2+ breast cancer [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 471. doi:10.1158/1538-7445.AM2017-471

Improvement the Efficacy of Cisplatin by Niosome Nanoparticles Against human breast cancer cell line BT-20 : an in vitro study

Improvement the Efficacy of Cisplatin by Niosome Nanoparticles Against human breast cancer cell line BT-20 : an in vitro study

Abstract 3439: MicroRNA-34a epigenetically silences epithelial-mesenchymal transitions (EMT)-TFs in metastatic breast cancer cells

Abstract MicroRNA-34a (miR-34a) as key regulator of tumor suppression gene potentially plays a profound role in the tumorigenesis and progression of several cancers including breast cancer. In the present study, we invstigated the expression, targets and functional effects of miR-34a and its correlation with tumour characteristics and clinical outcome in the epithelial to mesenchymal transitions (EMT) stage of metastatic breast cancer (MBC). We focused to analyze EMT-inducing transcription factors (EMT-TFs)/miR-34a feedback loop, like TWIST1, SLUG, ZEB1/2, NOTCH1, on promotes MBC migration, invasion and metastasis. We found that miR-34a potently inhibits NOTCH1, ZEB1 protein expression activity in breast cancer cells. Importantly, the miR-34a do not have any inhibitory effects upon the SLUG and ZEB2. Using quantitative reverse transcription-PCR and western blot analysis, we showed that average pre-miR-34a expression is down regulated in human metastatic breast tissues as compared to normal human breast and negatively correlates with clinicopathological features of MBC patients. Ectopic expression of miR-34a in metastatic breast cancer cell-line BT-549 significantly inhibited cell proliferation, migrations, survival and cell invasion, but no clear effect on cancer cell growth. We provide indication that these regulatory pathways may stabilize epithelial and mesenchymal states, individually. We have consequently revealed that miR-34a is downregulated in MBC tissues and that it suppresses tumor growth by affecting several malignancy endpoints via downregulation of multiple EMT-TFs. Mutational or epigenetic inactivation of the EMT-TFs/miR-34a pathway probably alterations the equilibrium of these regulations toward EMT and the mesenchymal state and thereby contributes to metastasis. The results also suggest that miR-34a could serve as a potential therapeutic agent for MBC. Funding support: This work was supported by the NSFC (81172049, 81672887), Corresponding author email:fujunjiang@hotmail.com;fujunjiang@swmu.edu.cn Citation Format: Saber Imani, Chunli Wei, Jingliang Cheng, Junjiang Fu. MicroRNA-34a epigenetically silences epithelial-mesenchymal transitions (EMT)-TFs in metastatic 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 3439. doi:10.1158/1538-7445.AM2017-3439

Breast Cancer Migration and Proliferation Inhibitory and Antibiotic Secondary Metabolites from The Egyptian Olive Tree Endophytic Fungus Penicillium citrinum

Objectives: The main aim of this study is to discover fungal endophytes associated with the Egyptian native olive tree Olea europaea as new sources for breast cancer control and investigate the chemistry and bioactivity of the fermentation metabolites of the identified active fungal species. Methods: Screening of multiple cultured Egyptian olive tree-associated fungal endophyte extracts identified Penicillium citrinum extract in breast cancer proliferation assays. Bioassay-guided isolation of the fungus P. citrinum extract identified four known metabolites 1-4. Their structure identity was established by different 1D and 2D NMR analyses. Compounds were tested for antiproliferative activity against the human breast cancer cell lines BT-474, MDA-MB-231 and MDA-MB-468using MTT assay and antimigratory activity against the human breast cancer cell lines MDA-MB-231 using wound healing assay. The antimicrobial activity of 1-3 was assessed using modified Kirby-Bauer disc diffusion method against multiple Gram positive and negative bacteria and fungi. Results:NMR analyses identified 1 as the known mycotoxin citrinin and the three different pyrrolidine alkaloids: 2-(hept-5-enyl)-3-methyl-4-oxo-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b]-1,3-oxazine (2), scalusamide A (3) and perinadine A (4). Compounds 2-4 showed significant antiproliferative activity against the human triple negative breast cancer cell line MDA-MB-231 with IC50 values of 10.6, 14.8 and 17.7 µM, respectively. Prerinadine (3) significantly suppressed the HGF-induced cell migration in a dose dependent manner with IC50 9.7. Citrinin (1) exerted remarkable antibiotic activity against Gram (+) and Gram (-) bacteria and was even up to several-fold those of tetracycline standard. Conclusion:Egyptian olive tree endophytes are valuable sources for biologically active natural products.

Overexpression of the ATPase Inhibitory Factor 1 Favors a Non-metastatic Phenotype in Breast Cancer.

Partial suppression of mitochondrial oxidative phosphorylation and the concurrent activation of aerobic glycolysis is a hallmark of proliferating cancer cells. Overexpression of the ATPase inhibitory factor 1 (IF1), an in vivo inhibitor of the mitochondrial ATP synthase, is observed in most prevalent human carcinomas favoring metabolic rewiring to an enhanced glycolysis and cancer progression. Consistently, a high expression of IF1 in hepatocarcinomas and in carcinomas of the lung, bladder, and stomach and in gliomas is a biomarker of bad patient prognosis. In contrast to these findings, we have previously reported that a high expression level of IF1 in breast carcinomas is indicative of less chance to develop metastatic disease. This finding is especially relevant in the bad prognosis group of patients bearing triple-negative breast carcinomas. To investigate the molecular mechanisms that underlie the differential behavior of IF1 in breast cancer progression, we have developed the triple-negative BT549 breast cancer cell line that overexpresses IF1 stably. When compared to controls, IF1-cells partially shut down respiration and enhance aerobic glycolysis. Transcriptomic analysis suggested that migration and invasion were specifically inhibited in IF1-overexpressing breast cancer cells. Analysis of gene expression by qPCR and western blotting indicate that IF1 overexpression supports the maintenance of components of the extracellular matrix (ECM) and E-cadherin concurrently with the downregulation of components and signaling pathways involved in epithelial to mesenchymal transition. The overexpression of IF1 in breast cancer cells has no effect in the rates of cellular proliferation and in the cell death response to staurosporine and hydrogen peroxide. However, the overexpression of IF1 significantly diminishes the ability of the cells to grow in soft agar and to migrate and invade when compared to control cells. Overall, the results indicate that IF1 overexpression despite favoring a metabolic phenotype prone to cancer progression in the specific case of breast cancer cells also promotes the maintenance of the ECM impeding metastatic disease. These findings hence provide a mechanistic explanation to the better prognosis of breast cancer patients bearing tumors with high expression level of IF1.

Mechanical compression induces VEGFA overexpression in breast cancer via DNMT3A-dependent miR-9 downregulation

Tumor growth generates mechanical compression, which may trigger mechanotransduction in cancer and stromal cells and promote tumor progression. However, very little is known about how compression stimulates signal transduction and contributes to tumor progression. In the present study, we demonstrated that compression enhances a tumor progression phenotype using an in vitro compression model, and validated the results from the in vitro model with high- and low-compressed breast cancer tissues. Mechanical compression induced miR-9 downregulation by DNMT3A-dependent promoter methylation in the MDA-MB-231 and BT-474 breast cancer cell lines and in cancer-associated fibroblasts. The overexpression of miR-9 target genes (LAMC2, ITGA6, and EIF4E) was induced by miR-9 downregulation, which eventually enhanced vascular endothelial growth factors production. Demethylation and decompression could reverse compression-induced miR-9 downregulation and following overexpression of miR-9 target genes and VEGFA.

Abstract P2-11-09: A p53-based strategy for protecting normal breast tissue from chemotherapy-induced damage in breast conserving therapy

Abstract Introduction: Currently, the global rate of breast conserving therapy (BCT) increased steadily. Most patients with BCT are treated with chemotherapy. However, most chemotherapeutical drugs that kill cancer cells also cause undesirable injuries to normal breast tissues. Normal breast tissue damage will further cause destruction of female secondary sex characteristics, and ultimately affect the quality of patients' lives. Therefore, it is urgent to protect the normal breast tissues for BCT patients during chemotherapy. More and more evidence shows that chemotherapy-induced normal tissue damage is mainly caused by the activation of p53 pathway, which is separate from the tumor suppressor pathway of p53. Previous studies found that use of low-dose arsenic (LDA) could temporarily and reversibly suppresses p53 activation. There are recent studies showing LDA selectively protect bone marrow and gastrointestinal tract during cancer treatment. Therefore, we hypothesize that use of LDA to temporary inhibit p53 activity will be a new strategy to protect the breast normal tissues for breast conserving patients. Methods: Human breast epithelial cell line, MCF-10A, and three breast cancer cell lines MCF-7 (estrogen receptor and E6 expressed), MDA-MB-231 (triple negative and p53 mutated) and BT-474 (HER2 overexpressed and p53 mutated), were tested in this study. All cells were pretreated with either PBS or 100nM sodium arsenite for 12 hours, followed by 375 μM 5-fluorouracil (5-FU) or DMSO for 24, 48 and 72 hours. Cellular viability was determined by MTT assay and cell morphology was recorded under a light microscope. Results: Morphology changes after 5-FU treatment include: the cell density decreased, the cells became rounded in shape, the cell membrane atrophied, the cell nuclei underwent pyknosis, and the cells formed a globule with nuclear and cytoplasmic fragments surrounded by the cell membrane. LDA pretreated-MCF10A cells showed significant reduced growth inhibition by 5-FU at all detected time pointes as demonstrated by MTT assay and morphology observation. Interestingly, LDA treatment had negligible effect on survival in breast cancer cells. Table 1. Cell growth evaluated by MTT assay.Time after 5-FU treatment(h)Cell lineControl (%)LDA (%)5-FU (%)LDA+5-FU (%)24MCF-10A10099.3051.3878.28 MCF-710097.3268.5165.64 MDA-MB-23110094.9669.5669.13 BT-47410098.7971.5071.5948MCF-10A10097.3729.1347.74 MCF-710091.7649.9148.95 MDA-MB-23110093.769.048.81 BT-47410092.0460.4361.0072MCF-10A10096.3418.3443.64 MCF-710091.0142.0039.40 MDA-MB-23110093.596.445.39 BT-47410091.4635.7636.95 Conclusion: Temporary LDA pretreatment selectively protected normal tissue cells, but not tumor cells from toxicity of 5-FU chemotherapy. Our findings indicated that LDA pretreatment is a potential strategy to protect normal breast tissue during chemotherapy for BCT patients. RL and HS contributed equally to this work. Citation Format: Long M, Huang Y, Liu R, Liu R, Su H. A p53-based strategy for protecting normal breast tissue from chemotherapy-induced damage in breast conserving therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-11-09.