T47D Cell Lines Research Articles

Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway.

The Cancer Genome Atlas (TCGA) data indicate that high MDM2 expression correlates with all subtypes of breast cancer. Overexpression of MDM2 drives breast oncogenesis in the presence of wild-type or mutant p53 (mtp53). Importantly, estrogen-receptor positive (ER+) breast cancers overexpress MDM2 and estrogen mediates this expression. We previously demonstrated that this estrogen-MDM2 axis activates the proliferation of breast cancer cell lines T47D (mtp53 L194F) and MCF7 (wild-type p53) in a manner independent of increased degradation of wild-type p53 (ie, p53-independently). Herein we present data supporting the role of the estrogen-MDM2 axis in regulating cell proliferation and mammary tissue architecture of MCF7 and T47D cells in a p53-independent manner. Inducible shRNA mediated MDM2 knockdown inhibited colony formation in soft agar, decreased mass size and induced lumen formation in matrigel and also significantly reduced mitosis as seen by decreased phospho-histone H3 positive cells. The knockdown of MDM2 in both cell lines decreased Rb phosphorylation and the level of E2F1 protein. This signaling was through the estrogen receptor because fulvestrant (a selective estrogen receptor degrader) decreased MDM2 protein levels and decreased phosphorylation of Rb. Taken together these data indicate that in some ER+ breast cancers the estrogen-MDM2-Rb-E2F1 axis is a central hub for estrogen-mediated p53-independent signal transduction. This is the first indication that estrogen signaling utilizes the estrogen-MDM2 axis to provoke phosphorylation of Rb and increase E2F1 while promoting abnormal mammary architecture.

Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models

BackgroundMutations in the estrogen receptor alpha (ERα) 1 gene (ESR1) are frequently detected in ER+ metastatic breast cancer, and there is increasing evidence that these mutations confer endocrine resistance in breast cancer patients with advanced disease. However, their functional role is not well-understood, at least in part due to a lack of ESR1 mutant models. Here, we describe the generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations, Y537S and D538G.MethodsGenome editing was performed using CRISPR and adeno-associated virus (AAV) technologies to knock-in ESR1 mutations into T47D and MCF7 cell lines, respectively. Various techniques were utilized to assess the activity of mutant ER, including transactivation, growth and chromatin-immunoprecipitation (ChIP) assays. The level of endocrine resistance was tested in mutant cells using a number of selective estrogen receptor modulators (SERMs) and degraders (SERDs). RNA sequencing (RNA-seq) was employed to study gene targets of mutant ER.ResultsCells with ESR1 mutations displayed ligand-independent ER activity, and were resistant to several SERMs and SERDs, with cell line and mutation-specific differences with respect to magnitude of effect. The SERD AZ9496 showed increased efficacy compared to other drugs tested. Wild-type and mutant cell co-cultures demonstrated a unique evolution of mutant cells under estrogen deprivation and tamoxifen treatment. Transcriptome analysis confirmed ligand-independent regulation of ERα target genes by mutant ERα, but also identified novel target genes, some of which are involved in metastasis-associated phenotypes. Despite significant overlap in the ligand-independent genes between Y537S and D538G, the number of mutant ERα-target genes shared between the two cell lines was limited, suggesting context-dependent activity of the mutant receptor. Some genes and phenotypes were unique to one mutation within a given cell line, suggesting a mutation-specific effect.ConclusionsTaken together, ESR1 mutations in genome-edited breast cancer cell lines confer ligand-independent growth and endocrine resistance. These biologically relevant models can be used for further mechanistic and translational studies, including context-specific and mutation site-specific analysis of the ESR1 mutations.

ANTICANCER ACTIVITY OF Β-SITOSTEROL FROM PLECTRANTHUS AMBOINICUS (LOUR. SPRENG.) LEAVES: IN VITRO AND IN SILICO STUDIES

Objective: β-sitosterol is the steroid compound which is an important nutrient in the diet meal, hydrophobic and soluble in organic solvents and considered as a good biomarker due to its biological activity.Methods: In vitro study was using 2,5-diphenyl tetrazolium bromide method towards T47D, MCF-7, HeLa, and WiDr cell lines. In silico docking using PLANTS program and visualized by Yasara program. The model of three dimension enzyme structures used in this research were epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K), estrogen receptor-alpha (ER-α), ER-beta (ER-β), and human EGFR 2 (HER-2). Two and three dimensions of β-sitosetrol, ZSTK474, and tamoxifen as the standard were generated using Marvin Sketch program.Results: β-sitosterol was found to have inhibitory concentration 50% of 0.55; 0.87; 0.76, and 0.99 mM. β-sitosterol and ZSTK474 were inhibited EGFR and PI3K with docking score −92.8195; −91.7920 and −91.7470; −94.7491 β-sitosterol and tamoxifen were inhibited ER-α, ER-β and HER-2 with docking score −78.5570; −89.535, −68.7717; −52.008 and −90.4908; −50.5576, respectively.Conclusion: Based on the results above that shows β-sitosterol provide effective as anticancer.Keywords: β-sitosterol, Inhibitor, Anticancer, In vitro, In silico.

CHARACTERIZATION OF NANOPARTICLES PRODUCED BY CHLOROFORM FRACTION OF KAEMPFERIA ROTUNDA RHIZOME LOADED WITH ALGINIC ACID AND CHITOSAN AND ITS BIOLOGICAL ACTIVITY TEST

Objective: The main objectives of this research are to characterize of nanoparticles produced by chloroform fraction of K. rotunda loaded with alginic acid and combination alginic acid-chitosan, and its biological activity test. Methods: Chloroform fraction of K. rotunda was loaded on alginic acid and combination of alginic acid-chitosan nanoparticles by ionic gelation method in various compositions. Characterizations of the products were investigated in particle size, zeta potential, and morphology by Scanning Electron Microscopy (SEM). The biological activity of the products as an antioxidant was tested by the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The cytotoxic effect was analysed using MTT [3-(4,5 dimethyltiazol-2-yl)-2,5-diphenyltetrazoilium bromide] assay.Result:The nanoparticles alginic acid can be synthesized at the optimal mass ratio range of alginic acid : CaCl2 of 10 :1 (% w/v),the percentage nanoparticle products was100%, the size range of the nanoparticles were 87 to 584 nm, with a zeta potential of -39.0 mV, and the morphology shows a spherical shape and smooth surface. Furthermore, nanoparticles result from the combination of alginic acid-chitosan at the optimal mass ratio range of alginic acid : chitosan of 10 :1 (% w/v) and added calsium ion at 0.015% w/v, the percentage nanoparticle products was100%, the size range of the nanoparticle were 87 to 877 nm, with a zeta potential of -27.1 mV, and the morphology shows a form of rectangular beam.Conclusion: The nanoparticle products of chloroform fraction of K. rotunda loaded alginic acid and combination alginic acid-chitosan were successfully obtained by ionic gelation method. The nanoparticle products show lower activity in antioxidant and cytotoxic effect against human breast cancer T47D cell lines than the starting material chloroform fraction of K. rotunda.Keywords: Alginic acid, Chitosan, Nanoparticles, Kaempferia rotunda, Antioxidant, Cytotoxic effect, Human breast cancer T47D cell lines.

Activation of Akt characterizes estrogen receptor positive human breast cancers which respond to anthracyclines.

Anthracyclines are key components of human breast cancer chemotherapy. Here, we explored the role of Akt signaling in anthracycline resistance.The antitumor activity of doxorubicin and Akt inhibitor A-443654 alone or combined was examined in estrogen receptor (ER) positive and negative human breast cancer cell lines. Further, we examined mRNA changes induced by anthracyclines in locally advanced breast cancers biopsied before and after treatment in two clinical trials.Doxorubicin increased Akt phosphorylation in ER positive MCF7 and T47D cell lines, with no effect in ER negative MDA-MB231 breast cancer cells. A-443654 was significantly more cytotoxic in doxorubicin-resistant compared to doxorubicin-naïve MCF7. This difference was not observed in MDA-MB231. Among 24 patients, AKT1 gene expression increased 24 hrs after the initial epirubicin exposure in ER positive tumors responding to therapy (n=6), as compared to ER positive non-responders (n=7) or ER negative tumors (n=11). In contrast, AKT1 mRNA changes after 16 weeks of doxorubicin were unrelated to clinical response and ER status (n=30).In conclusion, rapid Akt activation was observed in ER positive breast cancers which responded to anthracyclines. Increased cytotoxicity of A-443654 in doxorubicin-resistant MCF7 cells indicates a possible role for Akt inhibitors in ER positive breast cancers where chemoresistance evolves.

Differing Mechanisms of Death Induction by Fluorinated Taxane SB-T-12854 in Breast Cancer Cells

Classical taxanes are routinely used in cancer therapy. In this study, mechanisms involved in death induction by the novel fluorine-containing taxane SB-T-12854 were investigated. We employed breast cancer SK-BR-3, MCF-7 and T47D cell lines to assess activation of individual caspases, changes in the expression of proteins of the Bcl-2 family, and the release of pro-apoptotic factors from mitochondria into the cytosol after SB-T-12854 treatment. Caspase-2, -8, and -9 were activated in SK-BR-3 and MCF-7 cells. Only caspase-8 was activated in T47D cells. Caspase-7 and -6 were activated in all tested cells while caspase-3 was activated only in SK-BR-3 cells. Pro-apoptotic Bad protein seems to be important for cell death induction in all tested cells. Anti-apoptotic Bcl-2 and pro-apoptotic Bim, Bok, Bid and Bik seem to be also associated with cell death induction in some of the tested cells. The mitochondrial apoptotic pathway was significantly activated in association with the release of cytochrome c and Smac from mitochondria, but only in SK-BR-3 cells, not in MCF-7 and T47D cells. Cell death induced by SB-T-12854, in the tested breast cancer cells, differs regarding activation of caspases, changes in levels of pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family and activation of the mitochondrial apoptotic pathway.

Involvement of miR-106b in tumorigenic actions of both prolactin and estradiol.

Prolactin promotes a variety of cancers by an array of different mechanisms. Here, we have investigated prolactin's inhibitory effect on expression of the cell cycle-regulating protein, p21. Using a miRNA array, we identified a number of miRNAs upregulated by prolactin treatment, but one in particular that was strongly induced by prolactin and predicted to bind to the 3′UTR of p21 mRNA, miR-106b. By creating a p21 mRNA 3′UTR-luciferase mRNA construct, we demonstrated degradation of the construct in response to prolactin in human breast, prostate and ovarian cancer cell lines. Increased expression of miR-106b replicated, and anti-miR-106b counteracted, the effects of prolactin on degradation of the 3′UTR construct, p21 mRNA levels, and cell proliferation in breast (T47D) and prostate (PC3) cancer cells. Increased expression of miR-106b also stimulated migration of the very epithelioid T47D cell line. By contrast, anti-miR-106b dramatically decreased expression of the mesenchymal markers, SNAIL-2, TWIST-2, VIMENTIN, and FIBRONECTIN. Using signaling pathway inhibitors and the 3′UTR construct, induction of miR-106b by prolactin was determined to be mediated through the MAPK/ERK and PI3K/Akt pathways and not through Jak2/Stat5 in both T47D and PC3 cells. Prolactin activation of MAPK/ERK and PI3K/Akt also activates ERα in the absence of an ERα ligand. 17β-estradiol promoted degradation of the construct in both cell lines and pre-incubation in the estrogen antagonist, Fulvestrant, blocked the ability of both prolactin and 17β-estradiol to induce the construct-degrading activity. Together, these data support a convergence of the prolactin and 17β-estradiol miR-106b-elevating signaling pathways at ERα.

Anti-tumor effects of everolimus and metformin are complementary and glucose-dependent in breast cancer cells

BackgroundClinical efficacy of the mTOR inhibitor everolimus is limited in breast cancer and regularly leads to side-effects including hyperglycemia. The AMPK inhibitor and anti-diabetic drug metformin may counteract everolimus-induced hyperglycemia, as well as enhancing anti-cancer efficacy. We investigated the glucose-dependent growth-inhibitory properties of everolimus, metformin and the combination in breast cancer cell lines.MethodsThe breast cancer cell lines MCF-7, MDA-MB-231 and T47D were cultured in media containing 11 mM or 2.75 mM glucose with 21% or 1% oxygen. Everolimus and metformin treated cells were subjected to cytotoxicity and clonogenic assays, western blotting, FACS and metabolic measurements.ResultsEverolimus was less effective in MCF7 cells under low glucose conditions compared to high glucose conditions (IC50 of >50 nM vs 29.1 ± 1.4 nM) in a short-term survival assay, while sensitivity of MDA-MB-231 and T47D cells to everolimus was lost under low glucose conditions. In contrast, metformin was more effective in low than in high glucose conditions in MCF7 (IC50 of 1.8 ± 1.2 mM vs >5 mM) and MDA-MB231 cells (1.5 ± 1.3 mM vs 2.6 ± 1.2 mM). Metformin sensitivity of T47D cells was independent of glucose concentrations. Everolimus combined with metformin additively inhibited cell survival, clonogenicity, mTOR signaling activity and mitochondrial respiration. These effects were not the result of enhanced autophagy or apoptosis induction. Similar results were observed under hypoxic conditions.ConclusionMetformin-induced effects are additive to the anti-proliferative and colony inhibitory properties of everolimus through inhibition of mitochondrial respiration and mTOR signaling. These results warrant further in vivo investigation of everolimus combined with metformin as a putative anti-cancer therapy.

Dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines: Topoisomerase I and IIα dual inhibitors with DNA non-intercalative catalytic activity

With the aim to develop novel antiproliferative agents, a new series of eighteen dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines were systematically designed, prepared, and investigated for their topoisomerase (topo) I and IIα inhibitory properties and antiproliferative effect in three different human cancer cell lines (HCT15, T47D, and HeLa). Compounds 22–30 which possess a meta- or para-phenol on 2-, or 6-position of central pyridine ring showed significant dual topo I and topo IIα inhibitory activities with strong antiproliferative activities against all the tested human cancer cell lines. However, compounds 13–21 which possess an ortho-phenol on 2-, or 6-position of central pyridine ring did not show significant topo I and topo IIα inhibitory activities but displayed moderate antiproliferative activities against all the tested human cancer cell lines. Compound 23 exhibited the highest antiproliferative potency as much as 348.5 and 105 times compared to etoposide and camptothecin, respectively, in T47D cancer cell line. The structure-activity relationship study revealed that the para position of a hydroxyl group at 2-and 6-phenyl ring and chlorine atom at the para position of 4-phenyl ring of the central pyridine exhibited the most significant topo I and topo IIα inhibition, which might indicate introduction of the chlorine atom at the phenyl ring of 4-pyridine have an important role as dual inhibitors of topo I and topo IIα. Compound 30 which showed the most potent dual topo I and topo IIα inhibition with strong antiproliferative activity in T47D cell line was selected to perform further study on the mechanism of action, which revealed that compound 30 functions as a potent DNA non-intercalative catalytic topo I and IIα dual inhibitor.

The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer

Cell lines are often used as in vitro tools to mimic certain types of in vivo system; several cell lines, including MCF-7 and T47D, have been widely used in breast cancer studies without investigating the cell lines' characteristics. In this study, we compared the genome-wide binding profiles of ERα, PR, and P300, and the gene expression changes between MCF-7 and T47D cell lines that represent the luminal A subtype of breast cancer. Surprisingly, several thousand genes were differentially expressed under estrogenic condition. In addition, ERα, PR, and P300 binding to regulatory elements showed distinct genomic landscapes between MCF-7 and T47D cell lines in the same hormonal states. In particular, the T47D cell line was markedly susceptible to progesterone, whereas the MCF-7 cell line did not respond to progesterone in the presence of estrogen. Consistently, changes in the expression level of the PR-target gene, STAT5A, were only observed in the T47D cell line, not the MCF-7 cell line, when treated with progesterone. Overall, the results highlight the importance of selecting appropriate cell lines for breast cancer studies and suggest that T47D cell lines can be an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer.

Alpha2-adrenoceptor agonists trigger prolactin signaling in breast cancer cells

Breast cancer is the most frequent malignancy among women worldwide. We have described the expression of α2-adrenoceptors in breast cancer cell lines, associated with increased cell proliferation and tumor growth. A mitogenic autocrine/paracrine loop of prolactin (Prl) has been described in breast cancer cells. We hypothesized that the α2-adrenergic enhancement of proliferation could be mediated, at least in part, by this Prl loop.In both T47D and MCF-7 cell lines, the incubation with the α2-adrenergic agonist dexmedetomidine significantly increased Prl release into the culture medium (measured by the Nb2 bioassay), this effect being reversed by the α2-adrenergic antagonist rauwolscine. No change in Prl receptors (PrlR) was observed by RT-qPCR in these cell lines. In IBH-6 cells a decrease in Prl secretion was observed at the lower dexmedetomidine concentration.The signaling pathways involved in ovine Prl (oPrl) and dexmedetomidine action were also assessed. Both compounds significantly activated STAT5 and ERK in all three cell lines. In T47D and MCF-7 cell lines also AKT was activated by both Prl and dexmedetomidine. We therefore describe the STAT5 phosphorylation by an α2-adrenergic agonist, dexmedetomidine.In T47D cells, the α2-adrenergic stimulation of cell proliferation is probably mediated, at least in part, by the Prl autocrine/paracrine loop, because this effect is abrogated by the specific PrlR antagonist Δ1–9-G129R-hPrl. The implication of Prl loop describes a novel mechanism of action of this GPCR.

Evaluation of Anticancer and Antimicrobial Activity of Arborinine from Glycosmis pentaphylla

The natural product arborinine, isolated from the plant Glycosmis pentaphylla was evaluated for its anticancer activity against five human cancer cell lines viz. human colon cancer cell line COLO-205, human ovarian cancer cell line OVCAR-3, human cervical cancer cell line SiHa, human breast cancer cell lines T-47D and MDA-MB-435 by SRB assay. The results were compared against a standard anticancer drug Adriamycin. Arborinine shows good activity on COLO-205, OVCAR-3 and T-47D with GI50 value <10 μg/ml in all the three experiments. It also shows mild to moderate antimicrobial activity against Staphylococcus aureus, Klebsiella pneumonia, Bacillus subtilis and Candida albicans strains in Agar well diffusion method.

Abstract P3-03-12: CDK4/6 inhibitor resistant ER-positive cells remain dependent on estrogen signalling and retain sensitivity to endocrine therapy

Abstract Background: Dysregulation of the cyclin D-CDK4/6-Rb pathway is a frequent feature of ER positive breast cancers and has been linked with endocrine resistance. Several randomised trials have shown that CDK4/6 inhibitors in combination with endocrine treatment can substantially improve the outcome of patients with metastatic ER positive breast cancer. With increased clinical use of CDK4/6 inhibitors acquired resistance is emerging as a major clinical challenge. It is critical to understand the sensitivity and responsiveness of these resistant cells to estrogen to consider continued use of endocrine therapy in combination with CDK4/6 inhibition. Results: Long-term culture of the ER positive cell lines T47D and MCF7 with increasing doses of drug generated distinct clones with acquired resistance to either palbociclib or ribociclib. ESR1 expression was stable across all resistant clones. All clones continued to be reliant on estrogen for cell growth, with estrogen deprivation inducing cell death. Resistant clones still responded to estrogen stimulation. Estradiol treatment induced at least 2-fold transcriptional upregulation of pS2 and c-myc; the induction was inhibited by tamoxifen and fulvestrant treatment. Both palbociclib and ribociclib resistant clones remained sensitive to endocrine therapy, with tamoxifen IC50 values equivalent to the parental T47D and MCF7 cell lines. Mechanisms of resistance were different between the different CDK4/6 inhibitors and within the two cell lines. Palbociclib resistant MCF7 and T47D clones all increase cyclin E protein levels, but only T47D transcriptionally upregulated cyclin E. MCF7 clones transcriptionally upregulated cyclin D, with corresponding protein increase. Therefore, CDK2/cyclin E complexes seem to drive progression in MCF7 cells, whilst CDK2/cyclin D complexes function in T47D cells. Ribociclib resistant clones presented with unchanged or even decreased CDK2 expression, but demonstrated notable increase in E2F1, indicating the requirement of a non-canonical CDK-cyclin pairing independent of CDK2. Cell lines did not exhibit complete cross resistance to all CDK4/6 inhibitors. All ribociclib-resistant T47D and MCF7 clones conferred cross-resistance to palbociclib. The majority of clones were also resistant to abemaciclib, although one T47D clone retained abemaciclib sensitivity. All palbociclib-resistant clones were equally resistant to ribociclib. Conversely, whilst one T47D ribociclib clone exhibited marked cross resistance to abemaciclib, the majority of clones remained sensitive to treatment with abemaciclib. This highlights potential different resistant mechanisms for abameciclib independent characterised cell cycle changes for palbociclib and ribociclib. Conclusions: Cells resistant to CDK4/6 inhibitors remain dependent on estrogen signalling and retain sensitivity to endocrine therapy. We continue to show that resistance is mediated by non-canonical CDK-cyclin pairings. Palbociclib and ribociclib resistant clones confer cross-resistance to both Palbociclib and ribociclib but incomplete cross-resistance to abemaciclib. Citation Format: Lenihan C, Bouchekioua-Bouzaghou K, Abdulghani R, Chupin J, Shia A, Schmid P. CDK4/6 inhibitor resistant ER-positive cells remain dependent on estrogen signalling and retain sensitivity to endocrine 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 P3-03-12.

Abstract P2-04-18: JAK2 and PD-L1 amplification enhance the dynamic expression of PD-L1 in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is a heterogeneous disease. Amplification of chromosome 9p24.1 encoding JAK2 and PD-L1 has been reported in up to 25% of TNBC and is associated with poor clinical outcome. In lymphoma, JAK2 is a transcriptional activator of both PD-1 ligands, and chromosome 9p copy number gain has been associated with therapeutic activity of nivolumab. We evaluated the interaction of JAK2 and PD-L1 expression in TNBC. Methods: 9p24.1 amplification in 4 TNBC cell lines (MDA-MB-231, MDA-MB-436, HCC1937, and HCC70) was measured using array comparative genomic hybridization (aCGH). Amplification was defined as aCGH log2ratio&amp;gt;2.0. Cell surface expression of PD-L1 was detected by flow cytometry and compared with the median fluorescence intensity (MFI) of isotype control Ig. To selectively inhibit JAK2, lentiviral vectors encoding two different shRNA were generated. JAK2, pSTAT1 and pSTAT3 expression were measured by immunoblot. The effects of the anti-JAK1/2 inhibitor ruxolitinib and interferon-gamma (IFN-γ) induction of PD-L1 was measured. Results: 9p24.1 copy number loss was measured in MDA-MB-231 (log2ratio =-1), neutral in HCC1937 ((log2ratio =0), gained in MDA-MB-436 ((log2ratio =+1) and amplified in HCC70 ((log2ratio =+2). No correlation was observed between PD-L1 expression and the 9p24.1 amplification, with the MFI ratio range from 5 to 16.5, mean 8.3 by flow cytometry. TNBC cell lines had higher baseline expression of PD-L1 compared to the ER+ cell lines T47D and MCF-7 (ratio, 0 p=0.1). Low dose IFN-γ (1.0-10.0 ng/ml) rapidly induced expression of PD-L1 in MDA-MB-231 (1.5 fold increase) and HCC70 (3.3 fold increase) with significant activation of the JAK2/STAT1 pathway. The induction of pSTAT1 and PD-L1 expression by IFN-γ was blocked with low dose (1mM) JAK1/2 inhibitor ruxolitinib. Knockdown of JAK2 with shRNA (&amp;gt;80%) did not impact PD-L1 baseline expression in MDA-MB-231 and HCC70 but abrogated IFN-γ –mediated induction of PD-L1 and the phosphorylation of STAT1. Conclusion: These data suggest that TNBC cell lines have baseline PD-L1 expression, but the cells with 9p24.1 amplification are highly sensitive to PD-L1 induction with IFN-γ which can be abrogated with inhibition with a JAK1/2 inhibitor or shRNA. Synergistic inhibition of PD1/PD-L1 and JAK2 may have therapeutic efficacy in the subset of TNBC with 9p24.1 amplification, and the dynamic effects of tumor PD-L1 expression in response to local inflammation should be considered in the evaluation of PD-1/PD-L1 checkpoint blockade. Citation Format: Chen M, Pockaj B, Andreozzi M, Barrett MT, Ocal IT, McCullough AE, Krishna S, Anderson KS. JAK2 and PD-L1 amplification enhance the dynamic expression of PD-L1 in triple negative breast cancer [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 P2-04-18.

Abstract P1-08-04: SOX9 is a critical regulator of triple-negative breast cancer cell growth and invasion

Abstract Background: SRY (Sex Determining Region Y)-related HMG-box (SOX) genes belong to a super-family of genes, which is characterized by a homologous sequence called the HMG-box residing on the Y-chromosome. There are 20 SOX genes present in humans and mice. We performed a siRNA screen of SOX transcription factors, and found that SOX9 was essential for breast cancer cell growth. The SOX9 protein recognizes the sequence CCTTGAG along with other members of the HMG-box class DNA-binding proteins and has been shown to be required for development, differentiation and lineage commitment. Moreover, SOX9 is expressed in adenocarcinomas, and is highly expressed in the most aggressive cancers. Our previous data shows SOX9 is highly expressed in “triple negative breast cancer” (TNBC) than in non-TNBC. Thus, we hypothesized that the SOX9 transcription factor acts as an essential molecule regulating TNBC growth and invasion. To test the hypothesis, we used SOX9-overexpressed, or SOX9-knockdown/knockout breast cancer cell models to determine whether SOX9 is necessary and/or sufficient to regulate TNBC cell proliferation, migration and invasion. Methods: We measured the cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell migration &amp; invasion assays in ER-positive (MCF7 and ZR75-1) and ER-negative (MDA231 and MDA468) breast cancer cells. DOX-inducible SOX9-knockout cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs was used to knockdown SOXs in the different breast cancer cells. Protein and mRNA levels of SOX9 in TNBC, non-TNBC, immortalized human breast epithelial cell lines were examined by western blotting and qRT-PCR assay. Results: Knockdown of SOXs by siRNA caused decreased cell proliferation of MDA231 by ≥50% and of MDA468 by 30%-50% in siSOX4, siSOX6, siSOX9, siSOX10 and siSOX11 treatment groups (but not in siSOX8 and siSOX17 treatment groups). However, in MCF7 and T47D cell lines, treatment with siRNA to these SOX factors did not cause significant cell growth reduction. We demonstrated that SOX9 is more highly expressed in TNBC cells at both the mRNA and protein levels. Knockdown of SOX9 decreased cell migration and invasion of MDA231 to 25% and 50% respectively. The same effect also was observed in MDA468 cells, with approximately a 50% decline in migration and invasion. In SOX9-knockout MDA231, MDA468, and LM2 cells, cell proliferation, migration, and invasion were significantly reduced. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. We are now conducting in vivo studies to determine the effect of SOX9 on breast cancer cell metastasis. Conclusion: SOX9 is a critical regulator of TNBC cell proliferation, migration and invasion. These studies suggest that regulating SOX9 transcription factor and its signaling pathway will be a promising therapeutic strategy to treat TNBC and prevent metastasis. This work was supported by a Susan G. Komen Scientific Advisory Board Grant, SAB1600006 (PB), and a grant from the Breast Cancer Research Foundation 2015-2016 BCRF grant(PB), and by the Charles Cain Endowment (PB). Citation Format: Ma Y, Shepherd J, Mazumdar A, Zhao D, Bollu L, Hill J, Zhang Y, Brown P. SOX9 is a critical regulator of triple-negative breast cancer cell growth and invasion [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 P1-08-04.

Abstract P1-08-06: BC200 lncRNA is involved in the progression of triple negative breast cancer

Abstract Long non coding RNAs (lncRNAs) have been identified as regulators of the cell cycle, apoptosis, and DNA damage among other processes that if deregulated, may lead to cancer by acting as proto-oncogenes, tumor suppressor genes, and drivers of metastatic transformation. Using RNA sequencing we have identified 42 differentially expressed lncRNAs from a healthy cohort of parous vs. nulliparous women. After bioinformatics and RT-qPCR analysis, we have focused on a vaguely studied lncRNA called BC200 that is highly expressed in the nulliparous postmenopausal breast tissue. It is known that BC200 lncRNA is overexpressed in invasive and pre-invasive breast cancer; however, its functional role in the initiation and progression of breast cancer is poorly understood. In the present work we provide insight on the role of BC200 in the context of luminal and triple negative breast cancer (TNBC). We have confirmed that BC200 is highly expressed in breast cancer tissue and in widely used breast cancer cell lines such as MCF7, T47D, MDAMB231, and Hs578T. Using a lentiviral system we successfully obtained cell lines which stably express BC200. Overexpression of BC200 increases proliferation, migration, and invasion potential in vitro in the cell lines tested, specifically luminal T47D and TNBC MDAMB231. Xenograft studies performed in the mammary fat pad of female SCID mice confirm the role of BC200 as a tumor promoter. Tumors in mice injected with MDAMB231 cells overexpressing BC200 were 4.5 times bigger than tumors in the control group in only 6 weeks when injecting 1 million cells. Moreover, we have determined, using reverse transcriptase PCR targeting genes less than 200 kb from the start site of BC200, that when BC200 is overexpressed, CALM2 is downregulated in both T47D and MDAMB231 cell lines. CALM2 or Calmodulin is a calcium binding protein that plays a role in signaling pathways, cell cycle progression, proliferation, and apoptosis. Mutations in CALM2 are associated with increased risk of breast cancer. Our positive results on Cis regulation are being expanded using chromatin isolation by RNA immunoprecipitation to determine BC200's genome wide regulation. These results demonstrate the participation of BC200 lncRNA in the progression of breast cancer. Notably, BC200 regulates nearby genes that have an implication in cancer progression. BC200, identified in the normal breast tissue of nulliparous women, not only plays a key role in breast cancer progression but also provides a new insight in the preventive role of pregnancy by the downregulation of the expression of this lncRNA in the normal parous breast. [This work was supported by the NCI (National Cancer Institute) Core Grant CA06927 to Fox Chase Cancer Center and generous support from Christian - Diane Martin, the Flyers Wives, and Joseph - Barbara Breitman to Dr. J. Russo, MD]. Citation Format: Barton M, Santucci-Pereira J, Su Y, Russo J. BC200 lncRNA is involved in the progression of triple negative breast cancer [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 P1-08-06.

Abstract S4-01: A novel BRD4 inhibitor enhances endocrine therapy efficacy and circumvents endocrine-resistance in estrogen receptor-positive breast cancer models

Abstract Background: The selective estrogen receptor (ER) down-regulator (SERD) fulvestrant (Ful) antagonizes ER activity and degrades ER protein in a dose-dependent manner in the preclinical and clinical settings, though its efficacy is limited by an incomplete abolition of ER protein levels. Therefore additional strategies are needed to achieve a more complete suppression of ER level and activity. The bromodomain-containing protein 4 (BRD4), a member of the BET family that is required both for ESR1 gene expression and for ER-mediated gene transcription, represents an attractive therapeutic target for ER+ breast cancer (BC). Here, we investigated the efficacy of the novel BRD4 inhibitor GS-626510 (GS-6510) in a panel of ER+ BC parental and endocrine-resistant (EndoR) cell lines and in a patient-derived xenograft (PDX) model. Materials and Methods: The effects of GS-6510 (25nM – 290nM) alone or in combination with endocrine therapies were tested in ER+ MCF7, T47D, and ZR75-1 cell lines, as well as in their derivatives made resistant to estrogen (E2) deprivation (EDR), tamoxifen (TamR), or Ful (FulR). Cell growth (by methylene blue) after 6 days of GS-6510 and protein levels (by Western blot) after 2 days of GS-6510 were assessed. The in vivo efficacy of GS-6510, Ful, and the combination was tested in the ER+ HCBx34 PDX model. The mRNA levels of genes associated with cell cycle, ER signaling, and endocrine-resistance were assessed using the NanoString platform. Results: A dose-dependent inhibitory effect of GS-6510 was observed in all of the experimental settings. At a clinically relevant dose, GS-6510 reduced E2-stimulated cell growth and enhanced the efficacy of endocrine therapies in all parental cell lines. In the endocrine-sensitive HCBx34 PDX model, GS-6510 reduced tumor growth and, in combination with Ful, induced tumor regression and inhibited the expression of ER-dependent and cell cycle related genes including CCND1, MYC, and BCL2. Notably, the addition of GS-6510 to EndoR cell models that continue to rely on and express ER (MCF7 EDR and TamR) led to a substantial inhibition in cell growth (85%; 98%, respectively). Though the combination of GS-6510 and Ful was not associated with a significantly greater cell growth inhibition compared to GS-6510 alone in these models, a better suppression of ER levels was observed. Interestingly, GS-6510 also remained effective in EndoR models that exhibited an ER-independent growth, including all FulR lines, though its efficacy varied among the different cell lines and resistant derivatives. Conclusion: Our findings suggest that the epigenetic regulator BRD4 is a suitable target for therapeutic intervention in ER+ BC. The anti-tumor efficacy of GS-6510 in endocrine sensitive and especially in ER-dependent EndoR models is worthy of further clinical investigation. The growth inhibitory effects observed in some of the ER-independent EndoR models suggests that additional genes/pathways involved in endocrine resistance could be affected by GS-6510. Identifying these pathways and determining their predictive role are needed to guide patient selection for future clinical trials. Citation Format: De Angelis C, Nardone A, Cataldo ML, Fu X, Trivedi M, Yi S, Breckenridge D, Chamnsess GC, Vitorino P, Osborne CK, Schiff R. A novel BRD4 inhibitor enhances endocrine therapy efficacy and circumvents endocrine-resistance in estrogen receptor-positive breast cancer models [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 S4-01.

Abstract P3-03-08: A large-scale functional screen to identify resistance mechanisms to selective estrogen receptor degraders fulvestrant and GDC-810 in ER+ breast cancer

Abstract Therapies that target the estrogen receptor provide clinical benefit and improved survival for patients with estrogen receptor-positive (ER+) breast cancer, yet drug resistance remains a challenging problem, leading to disease relapse and mortality. In recent years, the selective estrogen receptor degrader (SERD) fulvestrant has become an important therapeutic option for patients with resistant ER+ metastatic breast cancer, and newer oral SERDs such as GDC-810 are currently being tested in clinical trials. The mechanisms of intrinsic and acquired resistance to SERDs remain to be fully elucidated. We conducted a large-scale lentiviral open reading frame (ORF) screen to identify genes whose overexpression confers drug resistance to either fulvestrant or GDC-810 in the ER+ breast cancer cell line T47D. The lentivral ORF expression library used in this study consists of 16,544 barcoded ORFs, including 2,767 ORFs with mutations. The initial screen yielded 72 genes resulting in resistance to fulvestrant and 85 genes resulting in resistance to GDC-0810, with 44 genes overlapping. The top ranked-genes included multiple genes belonging to the PI3K/Akt, ERbB/HER, and FGF/FGFR pathways as well as genes involved in cell cycle progression. Fibroblast growth factor receptor 1 (FGFR1) amplifications are frequently observed in patients with ER+ breast cancer, and have previously been implicated in resistance to endocrine therapies. Several FGFs (FGF3, FGF6, FGF10, and FGF22) were among the top-ranked resistance genes for both fulvestrant and GDC-0810, suggesting that activation of the FGFR signaling pathway may render cells resistant to fulvestrant and GDC-810. In the presence of FGF2, overexpression of FGFR1 in ER+ breast cancer cells resulted in resistance to both fulvestrant and GDC-0810. The ability of an FGFR inhibitor to overcome FGFR-mediated resistance to SERDs is being tested. Additional potential resistance genes identified in the ORF screen are also being validated. In summary, a whole-genome functional resistance screen has identified several candidate genes and pathways that may cause resistance to fulvestrant and GDC-810. Several of these candidates, such as FGFR1, are also found in patients who develop resistance to SERDs, suggesting rational combination therapies to overcome or preempt SERD resistance. Citation Format: Mao P, Quartey Q, Cohen O, Piccioni F, Wagle N. A large-scale functional screen to identify resistance mechanisms to selective estrogen receptor degraders fulvestrant and GDC-810 in ER+ breast cancer [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 P3-03-08.

Abstract PD1-04: CSMD1 SNPs selectively affect anastrozole response in postmenopausal breast cancer patients

Abstract BACKGROUND: Based on prospective clinical trials, there is no evidence for differences in efficacy between the 3 aromatase inhibitors (AIs) anastrozole, exemestane, and letrozole. The purpose of this study was to identify germline genetic variants associated with response to AIs and to help identify novel mechanisms associated with drug disease efficacy. METHODS: A genome-wide association study (GWAS) was performed for 624 patients (Steroids 2015;99:32-38) to identify SNPs associated with estrogen level change in women with estrogen receptor (ER) positive breast cancer treated with anastrozole. Replication of associated SNPs was performed in a GWAS from the MA.27 trial that compared adjuvant anastrozole and exemestane treatment of post-menopausal women with ER+ breast cancer. Functional studies were subsequently performed to determine SNP effects and underlying mechanisms. RESULTS: Our initial GWAS identified SNPs within CSMD1 that were associated with changes in estrogen levels during anastrozole therapy. An additional SNP in CSMD1 was also associated with breast cancer events in CCTG MA.27. Functionally, we showed that CSMD1 regulates CYP19 expression in a SNP-, and in an anastrozole- dependent fashion. These phenomena were not observed for either letrozole or exemestane. In MA.27, an anastrozole- specific effect was also seen with the minor allele having a protective effect on time to distant metastasis (HR=0.49, p=0.00259), but this was not the case for exemestane (HR=0.71, p=0.111). Our in vitro functional studies indicated that overexpression of CSMD1 sensitized anastrozole or letrozole resistant cells to anastrozole but not to the other two AIs. The SNP in CSMD1 that was associated with increased CSMD1 and CYP19 expression levels increased anastrozole sensitivity, but not letrozole or exemestane in lymphoblastoid cell lines (LCLs) homozygous for either WT or variant CSMD1 SNP genotypes. Based on these observations, we explored whether anastrozole has additional mechanisms beyond its function as a CYP19 inhibitor. Utilizing an estrogen response element (ERE) luciferase reporter assay in a CYP19 CRISPR knockout breast cancer T47D cell line and a surface plasmon resonance (SPR) assay, we found that anastrozole can also function as an ERα agonist, and can bind to, and result in, proteasome dependent ERα degradation, especially in the presence of E2. Treatment of these CYP19 CRISPR knockout cells with anastrozole in the presence of increasing concentrations of E2 results in greater sensitivity compared with anastrozole alone, while the addition of E2, as expected, does not improve letrozole or exemestane sensitivity. These same observations were also seen in letrozole and anastrazole resistant cells. CONCLUSIONS: Our findings suggest that anastrozole might be more effective than letrozole or exemestane in patients with the CSMD1 SNP. Furthermore, anastrozole can function as an ERα agonist, binding to ERα and resulting in its degradation, especially in the presence of E2. These findings should help to make it possible to develop precision endocrine therapies for women who are candidates for AIs. Citation Format: Cairns J, Ingle J, Dudenkov T, Kalari K, Buzdar A, Kubo M, Robson M, Ellis M, Goss P, Shepherd L, Goetz M, Weinshilboum R, Wang L. CSMD1 SNPs selectively affect anastrozole response in postmenopausal breast cancer patients [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 PD1-04.

Antiproliferation effect of imatinib mesylate on MCF7, T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR-β, PDGF-BB, c-Kit and SCF genes.

Recent cancer molecular therapies are targeting main functional molecules to control applicable process of cancer cells. Attractive targets are established by receptor tyrosine kinases, such as platelet-derived growth factor receptors (PDGFRs) and c-Kit as mostly irregular signaling, which is due to either over expression or mutation that is associated with tumorigenesis and cell proliferation. Imatinib mesylate is a selective inhibitor of receptor tyrosine kinase, including PDGFR-β and c-Kit. In this research, we studied how imatinib mesylate would exert effect on MCF7 and T-47D breast cancer and MCF 10A epithelial cell lines, the gene and protein expression of PDGFR-β, c-Kit and their relevant ligands platelet-derived growth factor (PDGF)-BB and stem cell factor (SCF). The MTS assay was conducted in therapeutic relevant concentration of 2–10 µM for 96, 120 and 144 h treatment. In addition, apoptosis induction and cytostatic activity of imatinib mesylate were investigated with the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and cell cycle assays, respectively, in a time-dependent manner. Comparative real-time PCR and Western blot analysis were conducted to evaluate the expression and regulation of imatinib target genes and proteins. Our finding revealed that imatinib mesylate antiproliferation effect, apoptosis induction and cytostatic activity were significantly higher in breast cancer cell lines compared to MCF 10A. This effect might be due to the expression of PDGFR-β, PDGF-BB, c-Kit and SCF, which was expressed by all examined cell lines, except the T-47D cell line which was not expressed c-Kit. However, examined gene and proteins expressed more in cancer cell lines. Therefore, imatinib mesylate was more effective on them. It is concluded that imatinib has at least two potential targets in both examined breast cancer cell lines and can be a promising drug for targeted therapy to treat breast cancer.