Receptor Positive Breast Cancer Cells Research Articles

ΔNp63α induces quiescence and downregulates the BRCA1 pathway in estrogen receptor-positive luminal breast cancer cell line MCF7 but not in other breast cancer cell lines

Despite apparent resection of tumors, breast cancer patients often suffer relapse due to remnant dormant tumor cells. Although quiescence of cancer stem cells is thought as one of the mechanisms regulating dormancy, the mechanism underlying quiescence is unclear. Since ΔNp63α, an isoform of p51/p63, is crucial in the maintenance of stem cells within mammary epithelium, we investigated its roles in the regulation of dormancy in normal and malignant breast cells. Inducible expression of ΔNp63α in MCF7 estrogen receptor positive (ER+) luminal breast cancer cells led to quiescence and acquisition of progenitor-like properties. Judging from mRNA-microRNA microarray analysis, activation of bone morphogenetic protein (BMP) signaling and inhibition of Wnt signaling emerged as prominent mechanisms underlying ΔNp63α-dependent induction of quiescence and acquisition of stemness in MCF7. More interestingly, through Ingenuity Pathway analysis, we found for the first time that BRCA1 pathway was the most significantly downregulated pathway by ΔNp63α expression in quiescent MCF7 cells, where miR-205 was a downstream mediator. Furthermore, ΔNp63α-expressing MCF7 cells exhibited resistance to paclitaxel and doxorubicin. Expression of ΔNp63α in normal MCF10A basal cells increased proliferation and stemness, but did not affect more aggressive luminal (T47D) and basal (MDA-MB-231) cells with p53 mutation. Gene expression datasets analyses suggested that ΔNp63 expression is associated with relapse-free survival of luminal A/B-type patients, but not of the other subtypes. Our results established a cell type-specific function of ΔNp63α in induction of quiescence and downregulation of the BRCA1 pathway which suggested a role of ΔNp63α in the dormancy of luminal breast cancers.

Abstract B2-41: Inference of differential dependency networks between tumor and stroma by integrated UNDO-KDDN software tool

Abstract Introduction: Modeling biological networks is an important task in cancer systems biology research. Significant rewiring of molecular networks can drive key phenotypic transitions that can occur in both a tumor and its microenvironment. Tumor-stroma mixtures serve as both a major confounding factor and an underexploited information source in studying tumor systems. The co-evolution of neoplastic and stromal cells in a tumor can actively influence therapeutic response and shape resistance. Methods: We integrated unsupervised deconvolution into inference of tumor- or stroma-specific networks, for characterizing hidden crosstalk between tumor and stroma via network rewiring at hub genes. We developed a completely in silico unsupervised deconvolution method, namely UNDO, to dissect tumor-stroma mixed gene expressions in heterogeneous tumor samples. We formulated the inference of knowledge-fused differential dependency networks (KDDN) that incorporate both conditional data and prior knowledge as a convex optimization problem, and developed an efficient learning algorithm to jointly infer the conserved biological network and significant rewiring across different conditions. Results: We demonstrate the performance of UNDO over a wide range of tumor-stroma mixing proportions, validate UNDO on various biologically-mixed benchmark gene expression datasets, and further estimate tumor purity in TCGA/CPTAC datasets. The highly accurate deconvolution results obtained suggest not only the existence of cell-specific marker genes but also UNDO's ability to detect them blindly and correctly. Using KDDN, we detected that in utero estrogens induce a rewired network pattern in the mammary glands of rodent offspring that predicts for resistance to endocrine therapies. Subsequent studies have shown that tumors that arise in these mammary glands are less responsive to Tamoxifen (TAM) that represents the first study to explain why many ER+ breast cancers fail to respond (or respond and later recur) with TAM treatment. Moreover, focused on understanding how estrogen receptor-positive (ER+) breast cancer cells adapt to the stress of endocrine-based therapies, a gene network that coordinately regulates the functions of a cell that determine and execute the cell's fate decision, We followed the predictions of this topology and validated fundamentally new insights into molecular signaling, e.g., direct regulation of BCL2 by XBP1 and the requirement of NFκB for XBP1 signaling to regulate the prosurvival cell fate outcome in the cellular context of antiestrogen treatment and resistance. Conclusion: Tested on many benchmark datasets, UNDO is very effective at detecting cell-specific marker genes, estimating cell proportions and cell-specific expression profiles. Supported by a well-grounded mathematical framework, KDDN integrates the abundant biological knowledge and condition-specific experimental data to depict the overall dependency networks and their dynamics. We expect UNDO-KDDN to be a very useful tool for performing differential network analysis in many biological contexts. Citation Format: Lulu Chen, Niya Wang, Robert Clarke, Zhen Zhang, Yue Wang. Inference of differential dependency networks between tumor and stroma by integrated UNDO-KDDN software tool. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-41.

Downregulation of Choline Kinase-Alpha Enhances Autophagy in Tamoxifen-Resistant Breast Cancer Cells.

Choline kinase-α (Chk-α) and autophagy have gained much attention, as they relate to the drug-resistance of breast cancer. Here, we explored the potential connection between Chk-α and autophagy in the mechanisms driving to tamoxifen (TAM) resistance, in estrogen receptor positive (ER+) breast cancer cells (BCCs). Human BCC lines (MCF-7 and TAM-resistant MCF-7 (MCF-7/TAM) cells) were used. Chk-α expression and activity was suppressed by the transduction of shRNA (shChk-α) with lentivirus and treatment with CK37, a Chk-α inhibitor. MCF-7/TAM cells had higher Chk-α expression and phosphocholine levels than MCF-7 cells. A specific downregulation of Chk-α by the transduction of shChk-α exhibited a significant decrease in phosphocholine levels in MCF-7 and MCF-7/TAM cells. The autophagy-related protein, cleaved microtubule-associated protein light chain 3 (LC3) and autophagosome-like structures were significantly increased in shChk-α-transduced or CK37-treated MCF-7 and MCF-7/TAM cells. The downregulation of Chk-α attenuated the phosphorylation of AKT, ERK1/2, and mTOR in both MCF-7 and MCF-7/TAM cells. In MCF-7 cells, the downregulation of Chk-α resulted in an induction of autophagy, a decreased proliferation ability and an activation of caspase-3. In MCF-7/TAM cells, despite a significant decrease in proliferation ability and an increase in the percentage of cells in the G0/G1 phase of the cell cycle, the downregulation of Chk-α did not induced caspase-dependent cell death and further enhanced autophagy and G0/G1 phase arrest. An autophagy inhibitor, methyladenine (3-MA) induced death and attenuated the level of elevated LC3 in MCF-7/TAM cells. Elucidating the interplay between choline metabolism and autophagy will provide unique opportunities to identify new therapeutic targets and develop novel treatment strategies that preferentially target TAM-resistance.

Induction of USP17 by combining BET and HDAC inhibitors in breast cancer cells.

Members of the bromodomain and extra-C terminal (BET) domain protein family and the histone deacetylase (HDAC) enzyme family regulate the expression of important oncogenes and tumor suppressor genes. Here we show that the BET inhibitor JQ1 inhibits proliferation and induces apoptosis of both triple negative and estrogen receptor positive breast cancer cells. Consistent with the critical role of histone acetylation in the regulation of gene expression, treatment with JQ1 or the HDAC inhibitor mocetinostat was associated with global changes in gene expression resulting in suppression of genes involved in cell-cycle regulation. Combining JQ1 with mocetinostat, further decreased cell viability. This synergistic effect was associated with increased suppression of genes essential for cell-cycle progression. Furthermore, we detected dramatic increase in the expression of several members of the ubiquitin-specific protease 17 (USP17) family of deubiquitinating enzymes in response to the combination treatment. Increased expression of USP17 enzymes were able to attenuate the Ras/MAPK pathway causing decrease in cell viability, while, siRNA mediated depletion of USP17 significantly decreased cytotoxicity after the combination treatment. In conclusion, our study demonstrates that co-treatment with BET inhibitors and HDAC inhibitors reduces breast cancer cell viability through induction of USP17.

Low doses of bisphenol A stimulate the proliferation of breast cancer cells via ERK1/2/ERRγ signals

The effects and mechanisms of bisphenol A (BPA) on the development of breast cancer are still not well illustrated. The present study revealed that nanomolar BPA significantly promoted the proliferation of both estrogen receptor (ER) positive (MCF-7) and negative (SkBr3) breast cancer cells, which was confirmed by up regulation of proliferating cell nuclear antigen (PCNA) and Bcl-2. Neither ERα nor G-protein-coupled estrogen receptor (GPER) mediated this effect of BPA because their inhibitors had no effect on the BPA induced cell proliferation. However, silencing of estrogen related receptor gamma (ERRγ) by its specific siRNA significantly abolished BPA induced proliferation of breast cancer cells, while si-ERRα had no similar effect. Moreover, nanomolar BPA up regulated the mRNA and protein levels of ERRγ and triggered its nuclear translocation via a time dependent manner. Further studies revealed that 10−8M BPA obviously increased the phosphorylation of ERK1/2, while had no similar effect on the phosphorylation of JNK and p38 MAPK. Further, PD 98059, the inhibitor of ERK1/2, significantly abolished the BPA induced up regulation of ERRγ and proliferation of breast cancer cells. Collectively, our results revealed that nanomolar BPA can trigger the proliferation of breast cancer cells via ERK1/2/ERRγ signals. Given that nanomolar BPA has been widely detected in human tissues, the clinical relevance of BPA and breast cancer progression should be further investigated.

Abstract 1866: Constitutively active NF-κB signaling switches estrogen receptor positive breast cancer cells from a proliferative to an invasive phenotype through luminal/basal plasticity

Abstract Estrogen receptor α (ER) is a good prognostic marker expressed in ≈75% of breast tumors. Women with ER+ tumors will receive endocrine therapy, yet ≈50% will experience relapse, often with more aggressive and metastatic tumors. A growing body of evidence indicates that an inflammatory microenvironment and activation of the NF-κB pathway are highly associated with progression of ER+ tumors to more aggressive stages. However, it is not known whether activation of the canonical arm of the NF-κB pathway is sufficient to drive progression to a more aggressive phenotype. In order to study this, we developed MCF-7 and T47D breast cancer cell lines expressing a DOX-inducible, constitutively active form of IκB kinase β (CA-IKKβ), a key kinase in the canonical arm of the NF-κB pathway. Unexpectedly, we found that DOX induced CA-IKKβ inhibited 17β-estradiol (E2)-dependent proliferation. However, the combination of CA-IKKβ and E2 induced a highly migratory and invasive phenotype. Although this phenotype was accompanied by a change in cell morphology, typical markers of epithelial-mesenchymal transition were not observed. Rather, a decrease in luminal markers (e.g. Gata3, cytokeratin 8 and 18) and an increase in basal markers (e.g. p63, cytokeratin 5 and 14) were observed, suggesting luminal to basal plasticity as an underlying mechanism in the phenotype switch. In addition, we find that the invasive phenotype coincides with significantly lower ER expression, which is similar to that observed in aggressive, Luminal B, ER+ breast tumors. However, ER was still transcriptionally active since the combination of CA-IKKβ and E2 recapitulated the regulation of genes from an ER-NF-κB crosstalk signature, which we have previously reported as enriched in Luminal B tumors. In fact, both transcription factors, ER and NF-κB, proved to be necessary for the observed invasive phenotype. In order to explore the clinical relevance of our finding, we examined primary invasive breast tumors for the presence of phosphorylated IκB kinases α/β (IKKα/β), as an indicator of IKK activity. IKKα/β phosphorylation was observed at the invasive edge of the tumors whereas total IKKβ was observed throughout the tumor, further supporting a role for IKKβ activity in breast tumor invasiveness. Taken together, our findings indicate that activation of the canonical arm of the NF-κB pathway in the presence of E2 switches ER+ breast cancer cells from a highly proliferative to a highly migratory and invasive phenotype, potentially through luminal to basal plasticity. Since the most deadly aspect of breast cancer is metastasis and ≈70% of metastatic tumors are ER+, understanding the effect of the canonical NF-κB pathway can help define new specific drug targets and therapeutic strategies for the treatment of ER+ breast cancers. Citation Format: Lamiaa El-Shennawy, Jonna Frasor. Constitutively active NF-κB signaling switches estrogen receptor positive breast cancer cells from a proliferative to an invasive phenotype through luminal/basal plasticity. [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 1866. doi:10.1158/1538-7445.AM2015-1866

Abstract 2670: BKM120 and BYL719 (PI3K inhibitors) sensitize estrogen receptor positive breast cancer cells to tamoxifen

Abstract Background: Tamoxifen is currently standard first line hormonal therapy for premenopausal estrogen receptor (ER) positive metastatic breast cancer women. However, the response rate is only around 30% andprogression free survival is only around 6 months, which indicate both intrinsic resistance and acquired resistance are clinical important issues. One of the important mechanisms of hormonal therapy resistance is collateral activation of PI3K/Akt pathway. This study sought to explore whether NVP-BYL719 (a novel PI3 kinase alpha selective inhibitor) or NVP-BKM120 (a novel pan-PI3K inhibitor) may enhance the efficacy of ER positive breast cancer cells toward tamoxifen. Material and methods: Five ER positive cell lines, MCF7, T47D (PIK3CA mutant), ZR75 (PIK3CA wild type, PTEN lost), MB361 (PIK3CA mutant and HER2 amplified), and HCC1500 (PIK3CA wild type, PTEN intact) were studied. Cells and Balb/c nude mice inoculated with MCF7 cells were treated with BYL719 alone, BKM120 alone, tamoxifen alone, or either one of the PI3K inhibitors in combination with tamoxifen. Their viability was measured at 6 days with MTT assay and the combination effect was evaluated by median effect analysis. Signaling targets were determined by immunoblotting analysis. The overall survival and tumor size of the nude mice were analyzed. Results: Synergism, indicated by a combination index (CI) value <1, were seen in all five cell lines treated with BYL719 or BKM120 plus tamoxifen. By immunoblotting analysis, BYL719 alone, BKM120 alone, or either drugs in combination with tamoxifen resulted in dowregulation of the PI3K signaling pathway downstream targets, including p-AKT, p-GSK3beta, and p-p70s6k expression in MCF7 and T47D cells. When MCF7 and T47D cells were treated with tamoxifen in combination with BYL719 or BKM120, an additional increase in the subG1 population by flow cytometry analysis and increase of PARP cleavage were observed by immunoblotting analysis, indicating the increase of apoptosis cell death. When MCF7 was transfected with constitutive active (myristoylated) Akt1 construct, the inhibitory effect of BYL719 and the synergistic effect between BYL719 and tamoxifen was markedly abolished, indicating that the mechanism is mainly through the inhibition of PI3K/Akt pathway. MCF7 tumor growth in Balb/c mice was successfully slowed down by the combination of BYL719 or BKM120 with tamoxifen, as compared to that in mice treated with tamoxifen alone or control. Conclusions: Our data suggest that additional PI3K blockade might be an effective strategy to enhance therapeutic effect of tamoxifen in wild type as well as in acquired tamoxifen resistance ER positive breast cancer cell. Clinical trials along this line are warranted. (The study was supported by National Science Council grant NSC 101-2325-B-002 -059) Citation Format: I-Chun Chen, Ming Gao, Lii-Ping Hsiao, Yi-Wen Huang, Huei-Chieh Yu, Ling-Chiun Yeh, Ann-Lii Cheng, Yen-Shen Lu. BKM120 and BYL719 (PI3K inhibitors) sensitize estrogen receptor positive breast cancer cells to tamoxifen. [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 2670. doi:10.1158/1538-7445.AM2015-2670

Abstract 1207: Nrf2 suppression results in growth inhibition and enhances response to chemotherapy and radiation therapy in triple-negative breast cancer cells

Abstract Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional activator that specifically binds to the Antioxidant Response Element (ARE) to initiate expression of various anti-oxidative and anti-inflammation genes to protect cells from oxidative stress. Recently, Nrf2 has been shown to redirect glucose and glutamine into anabolic pathways to promote cell proliferation. Constitutive stabilization of Nrf2 has been observed in many human cancers and confers chemo- and radio-resistance of cancer cells. We examined Nrf2 expression and function in triple negative breast cancer cells (TNBC). mRNA and protein expression of Nrf2 were higher in the TNBC cell lines MDA-MB-231 and MDA-MB-436 compared to immortalized breast epithelial cell lines (MCF10A and MCF12A) and estrogen receptor positive breast cancer cell lines (MCF-7 and T47D). shRNA knock-down of Nrf2 in MDA-MB-231 and MDA-MB-436 TNBC cells showed decreased mRNA expression of multiple Nrf2 regulated anti-oxidant (SLC7A11, NQO-1, GCLC, GCLM, SLC7A5, etc) and pentose phosphate pathway genes (G6PD, PGD, ME-1, TKT, TALDO1, PPAT, etc), enhanced basal levels of cellular reactive oxygen species, impaired mitochondrial function as determined by measuring mitochondrial potentials in living cells, and reduced S phase entry. Cells with decreased Nrf2 had reduced cell growth in monolayer and anchorage independent growth assays. In addition, Nrf2 suppression reduced cell migration. Nrf2 down-regulated MDA-MB-231 cells also showed increased response towards ionizing radiation and mitomycin C treatment in clonogenic and soft agar assays. Furthermore, reduced Nrf2 expression decreased the number of stem-like (CD44+/CD24-) cells in MDA-MB-231 cell populations. Thus, Nrf2 regulated many aspects of the malignant phenotype in TNBC and suggest that inhibition of Nrf2 might be a therapeutic target for TNBC. Citation Format: Yuzhe Yang, Douglas Yee. Nrf2 suppression results in growth inhibition and enhances response to chemotherapy and radiation therapy in triple-negative breast cancer 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 1207. doi:10.1158/1538-7445.AM2015-1207

Abstract 4197: Mutant GATA3 drives precocious lobuloalveolar mammary development and promotes the growth of estrogen receptor positive xenograft tumors

Abstract The GATA3 transcription factor is expressed in luminal breast epithelial cells and is required for mammary gland development. Recent reports indicate that somatic mutations in GATA3 occur in up to 15% of estrogen receptor positive breast tumors. Our analysis of these published data demonstrates that the human breast tumors with GATA3 mutations are diagnosed 7 years earlier than other ER+ tumors, suggesting that these are more aggressive tumors. Previously, these mutations were proposed to be null alleles that resulted in haploinsufficiency for GATA3 in breast cancer, however this interpretation currently lacks experimental support. We report that the mutation spectrum of GATA3 in breast cancer is in sharp contrast to HDR syndrome, a true GATA3 haploinsufficiency disease. In breast cancer, there is a striking enrichment of GATA3 mutations in exons 5 and 6 (amino acids 294-444) which encode the second of two DNA binding zinc fingers and the C-terminal amino acids. The absence of whole gene deletions or additional 5′ mutations in the open-reading frame, frequently found in HDR syndrome, suggests that there is a selective pressure to mutate and retain a portion of the GATA3 in breast cancer. We hypothesize that these GATA3 mutations do not result in null alleles but instead encode proteins with residual or neomorphic functions which actively contribute to breast cancer progression. We have generated transgenic mice that express mammary-specific GATA3-336fs. Non-parous pubertal transgenic mice exhibit pronounced lobuloalveolar development compared to estrous-matched wildtype littermates. This indicates that expression of mutant GATA3 protein promotes aberrant mammary epithelial cell proliferation. We are currently testing the effect of mutant GATA3 on the kinetics of mammary tumorigenesis by crossing these mice with models of mammary cancer harboring mutant P53 and mutant PI3K. We have generated ZR751 luminal breast cancer cell lines that stably express the hotspot GATA3 mutations that affect codons 309, 336 and 409. When xenografted into the mammary fat pads of nude mice, cells with GATA3-309* and GATA3-336fs have a growth advantage, while GATA3-409fs had slightly slower growth than the parental line. Genome wide ChIP-Seq analysis shows that GATA3-409fs has a similar binding profile to wildtype GATA3, with a significant enrichment at the GATA binding motif. The GATA3-309* and GATA3-336fs mutants bind fewer sites in the genome and are highly enriched at non-GATA motifs indicating an alternate mechanism of DNA binding for target gene regulation. These data support a model in which the GATA3 mutations recently observed in breast cancer encode for dominantly-acting transcription factors. These mutations elicit proliferative phenotypes in normal mammary epithelium and promote the growth of estrogen receptor positive breast cancer cell lines. Citation Format: Natasha Chandiramani, Esther A. Peterson, Eric H. Jung, E. Charles Jenkins, Paraic A. Kenny. Mutant GATA3 drives precocious lobuloalveolar mammary development and promotes the growth of estrogen receptor positive xenograft tumors. [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 4197. doi:10.1158/1538-7445.AM2015-4197

Abstract 1692: Antibody-drug conjugates to target cell surface TACE-cleaved amphiregulin in breast cancer

Abstract Approximately 70% of breast cancer cases are Estrogen Receptor (ER) positive. In many cases, these patients are well-served by endocrine therapies such as tamoxifen and aromatase inhibitors, however the emergence of endocrine-resistant disease in up to 40% of these patients is a major clinical concern. This necessitates treatment with more toxic chemotherapies which are often successful at delaying tumor progression, but are not curative. Recent work from our group and others has demonstrated that Amphiregulin, an Epidermal Growth Factor Receptor (EGFR) ligand, is a critical effector of ER signaling in both normal development and breast cancer pathogenesis, and is expressed in both endocrine-sensitive and endocrine-resistant breast cancer. Like other EGFR ligands, Amphiregulin is proteolytically processed at the cell surface to release a soluble EGFR-binding signaling domain and a residual cell-associated stalk. In this project, we are developing antibodies that selectively recognize the residual transmembrane Amphiregulin cleavage product with the goal of developing antibody-drug conjugates with which to selectively target tumor cells with high levels of Amphiregulin shedding. We have generated six humanized antibodies that recognize the cell-associated neo-epitope revealed following the TACE-mediated proteolytic release of Amphiregulin. Using both flow Cytometry and immunofluorescence, we demonstrate that these antibodies selectively recognize cleaved over full-length Amphiregulin in estrogen receptor positive breast cancer cells. Conjugation of these antibodies with a pH-sensitive dye, pHrodo, demonstrates robust and rapid antibody internalization in live cells. Conjugation of these antibodies with monomethyl auristatin E results in the disruption of the microtubule network of breast cancer cells followed by cell death. We propose that these agents may have utility in the treatment of breast and other cancers in which Amphiregulin expression and processing play a prominent role. This study is supported by the Department of Defense Breast Cancer Research Program (W81XWH-14-1-0294). Citation Format: Edmund C. Jenkins, Kelly S. Levano, Eric H. Jung, Matthew Levy, Paraic A. Kenny. Antibody-drug conjugates to target cell surface TACE-cleaved amphiregulin in breast cancer. [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 1692. doi:10.1158/1538-7445.AM2015-1692

Abstract 818: Chronic oxidative stress induces conversion of estrogen-dependent non-aggressive breast cancer cells into estrogen-independent aggressive phenotype

Abstract We have recently reported increased growth and tumorigenic potential of estrogen receptor (ER) positive breast cancer cells with chronic exposure to oxidative stress. In this study, we evaluated mechanistic basis for chronic oxidative stress-induced aggressiveness in ER positive MCF-7 breast cancer cells. Loss of estrogen dependency is known to be associated with acquisition of aggressive form in breast cancer cells. Therefore, the objective of this study was to evaluate changes in estrogen response in ER-positive breast cancer cells exposed to chronic oxidative stress. MCF-7 cells were exposed to hydrogen peroxide (H2O2)-induced oxidative stress for chronic period (6 months). Using these cells, growth response to 17-β estradiol was evaluated by MTT assay and cell cycle analysis. Surprisingly, in response to 17-β estradiol treatment, there was no significant growth stimulation in cells exposed to chronic oxidative stress. Cell cycle analysis by flow cytometry further confirmed the cell growth data. Quantitative RT-PCR analysis also revealed significant down regulation of ER-α in chronically exposed breast cancer cells. To understand the role of epigenetic mechanism, the expression of epigenetic regulatory genes were determined at both transcript and protein level. In addition, the levels of histone modifications were also evaluated by Western blot analysis. Changes in both the expression of epigenetic regulatory genes (DNMT1, DNMT3b, HDAC1, MBD4, and HAT1) and the levels of histone modifications (H3K27 tri-methylation and H3K18 acetylation) were observed in MCF-7 cells chronically exposed to H2O2. Treatment with DNA de-methylating agent (5-aza-2′-deoxycytidine) restored the ER-α expression level, estrogen-induced growth and oxidative stress induced histone modifications. To summarize, the findings of this study suggest that chronic exposure to oxidative stress can convert estrogen-dependent non-aggressive breast cancer cells into estrogen-independent aggressive form potentially by epigenetic mechanism. Citation Format: Prathap Kumar S. Mahalingaiah, Logeswari Ponnusamy, Kamaleshwar P. Singh. Chronic oxidative stress induces conversion of estrogen-dependent non-aggressive breast cancer cells into estrogen-independent aggressive phenotype. [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 818. doi:10.1158/1538-7445.AM2015-818

Abstract 3795: Cardiac glycoside digitoxin and its monosaccharide analogue MonoD inhibit NFκB to induce apoptotic cell death in ER+ MCF7 and triple-negative MDAMB-468 breast cancer cells

Abstract Digitoxin, a cardiac glycoside well appreciated in the treatment of congestive heart failure, has been poorly understood and appreciated as an anti-cancer therapeutic due to its narrow therapeutic potential. Using a patented palladium-catalyzed de novo synthesis method, we have developed a library of digitoxin analogs, which alleviate the toxic side effects of digitoxin while maintaining its chemical activity. We have evaluated MonoD, a digitoxin analog with a single sugar moiety, in a variety of NCI-60 panel of cell lines, with a significant tumoristatic effect observed in estrogen receptor positive and triple negative breast cancer cells. Therefore, in order to determine the efficacy of digitoxin and MonoD, delineate its mode of action and identify molecular targets we chose to test this novel compound in ER+ MCF7 and TN MDAMB-468 breast cancer cells. Digitoxin and MonoD showed a dose dependent decrease in cell viability in MCF7 and MDAMB-468 cell lines. Further characterization of cell death using the Hoechst assay showed that MonoD induced significantly higher apoptosis at the same dose as digitoxin. Both drugs induce oxidative stress in the form of superoxide as assayed using Dihydroethidium (DHE). In vitro scratch migration assay showed a five-fold decrease in rate of cell migration in the presence of test drugs. MCF7 and MDAMB-468 cells were analyzed for cell cycle progression in the presence of digitoxin and MonoD. We observed that the drugs induced cell cycle arrest in G0/G1 phase at the expense of S and G2/M phases. Increased expression of p27Kip1 in the presence of drugs provided further validation, as increased p27 expression is known to block cell cycle progression through G1-S transition. In addition to cell cycle arrest, apoptosis is another widely accepted mechanism by which anti-cancer drugs inhibit tumor formation. We observed that these drugs induced apoptosis as indicated by cleaved PARP and cleaved caspase-9, in addition to down-regulation of pro-survival factor Bcl-2. NFκB has been reported to be constitutively active in many types of cancer by controlling the expression of several genes involved in cellular proliferation and apoptosis. We observed that NFκB expression was down-regulated in both cell lines with digitoxin and MonoD treatment. IKKβ, a critical kinase involved in regulating NFκB pathway showed significant down-regulation, thereby suggesting that these drugs exert their anti-cancer therapeutic potential through the inhibition of NFκB pathway. While additional work is required to establish their mode of action, we have sufficient evidence to hypothesize that down-regulation of NFκB is critical to the mode of action of these drugs. Further, it is evident from densitometric analyses of western blots, MTT and Hoechst assays that MonoD shows higher efficacy as compared to digitoxin in inducing cancer cell death. Citation Format: Yogesh Kulkarni, Vivek Kaushik, Raj Venkatadri, Clayton Wright, Juan Sebastian Yakisich, George O'Doherty, Neelam Azad, Anand Iyer. Cardiac glycoside digitoxin and its monosaccharide analogue MonoD inhibit NFκB to induce apoptotic cell death in ER+ MCF7 and triple-negative MDAMB-468 breast cancer 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 3795. doi:10.1158/1538-7445.AM2015-3795

Abstract 5562: BreastDefend enhances the effects of tamoxifen in estrogen receptor-positive human breast cancer in vitro and animal models in vivo

Abstract In this study we evaluated the combined effects of the natural product/dietary supplement BreastDefend (BD) and tamoxifen (TAM) on MCF-7 estrogen receptor-positive (ER+) human breast cancer cells in vitro and an orthotopic mouse model in vivo. BD has been studied previously in preclinical models, and shown to exert significant effects on MDA-MB-231 triple-negative breast cancer cells. The proliferation of MCF-7 cells was evaluated using MTT assays, whereas the expression of genes involved in the molecular mechanisms of cancer was evaluated using DNA microarrays. MCF-7 cells were stimulated with estradiol (10 nM) and treated with TAM (1 μM), BD (10 μg/ml), and the combination of TAM and BD for 3 days and 6 days. Compared with control, BD enhanced the anti-proliferative activity of TAM significantly (P < 0.05), and the combination of BD and TAM induced apoptosis. The results of DNA microarray analyses suggested that the pro-apoptotic activity of BD and TAM in MCF-7 cells was associated with the significant upregulation of BRAF (an oncogene that can block proliferation and induce apoptosis) and downregulation of BCL2 (an apoptosis inhibitor) and FN1 (fibronectin, a glycoprotein involved in tumorigenesis and metastasis) expression. In the orthotopic model MCF-7 cells were implanted into the mammary fat pads of female ovariectomized nude mice (n = 12-13). The mice received subcutaneous pellets that released estradiol and TAM, whereas BD was administered orally via intragastric gavage. All groups, including control, received estradiol. The combination of BD and TAM suppressed tumor size in vivo markedly compared with the BD and TAM alone groups. The inhibition of tumor growth was associated with the induction of the cell cycle inhibitor protein p21 in MCF-7-generated breast tumors. In summary, this study suggests that BD could be considered for use in clinical studies as an adjuvant therapy in conjunction with TAM for the treatment of ER+ human breast cancer. Citation Format: Shujie Cheng, Mark Alvarado, Jagadish Loganathan, Victor Castillo, Georg Sandusky, Isaac Eliaz, Daniel Sliva. BreastDefend enhances the effects of tamoxifen in estrogen receptor-positive human breast cancer in vitro and animal models in vivo. [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 5562. doi:10.1158/1538-7445.AM2015-5562

The chemomodulatory effects of resveratrol and didox on herceptin cytotoxicity in breast cancer cell lines.

Herceptin is considered an essential treatment option for double negative breast cancer. Resveratrol and didox are known chemopreventive agents with potential anticancer properties. The aim of the current study is to investigate the influence of resveratrol and didox on the cytotoxicity profile of herceptin in HER-2 receptor positive and HER-2 receptor negative breast cancer cell lines (T47D and MCF-7 cell lines, respectively). The IC50’s of herceptin in T47D and MCF-7 were 0.133 ± 0.005 ng/ml and 23.3795 ± 1.99 ng/ml respectively. Equitoxic combination of herceptin with resveratrol or didox in T47D significantly reduced the IC50 to 0.052 ± 0.001 and 0.0365 ± 0.001 ng/ml, respectively and similar results were obtained in MCF-7. The gene expression of BCL-xl was markedly decreased in T47D cells following treatment with herceptin/resveratrol compared to herceptin alone. Immunocytochemical staining of HER-2 receptor in T47D cells showed a significant reduction after treatment with herceptin/resveratrol combination compared to herceptin alone. On the contrary, herceptin/didox combination had no significant effect on HER-2 receptor expression. Cell cycle analysis showed an arrest at G2/M phase for both cell lines following all treatments. In conclusion, herceptin/resveratrol and herceptin/didox combinations improved the cytotoxic profile of herceptin in both T47D and MCF-7 breast cancer cell lines.

Deletion of the amino acid transporter Slc6a14 suppresses tumour growth in spontaneous mouse models of breast cancer.

SLC6A14 mediates Na(+)/Cl(-)-coupled concentrative uptake of a broad-spectrum of amino acids. It is expressed at low levels in many tissues but up-regulated in certain cancers. Pharmacological blockade of SLC6A14 causes amino acid starvation in estrogen receptor positive (ER+) breast cancer cells and suppresses their proliferation in vitro and in vivo. In the present study, we interrogated the role of this transporter in breast cancer by deleting Slc6a14 in mice and monitoring the consequences of this deletion in models of spontaneous breast cancer (Polyoma middle T oncogene-transgenic mouse and mouse mammary tumour virus promoter-Neu-transgenic mouse). Slc6a14-knockout mice are viable, fertile and phenotypically normal. The plasma amino acids were similar in wild-type and knockout mice and there were no major compensatory changes in the expression of other amino acid transporter mRNAs. There was also no change in mammary gland development in the knockout mouse. However, when crossed with PyMT-Tg mice or MMTV/Neu (mouse mammary tumour virus promoter-Neu)-Tg mice, the development and progression of breast cancer were markedly decreased on Slc6a14(-/-) background. Analysis of transcriptomes in tumour tissues from wild-type mice and Slc6a14-null mice indicated no compensatory changes in the expression of any other amino acid transporter mRNA. However, the tumours from the null mice showed evidence of amino acid starvation, decreased mTOR signalling and decreased cell proliferation. These studies demonstrate that SLC6A14 is critical for the maintenance of amino acid nutrition and optimal mammalian target of rapamycin (mTOR) signalling in ER+ breast cancer and that the transporter is a potential target for development of a novel class of anti-cancer drugs targeting amino acid nutrition in tumour cells.

Anti-proliferative actions of a synthetic REV-ERBα/β agonist in breast cancer cells

REV-ERBα and REV-ERBβ are nuclear receptors that are ligand-dependent transcriptional repressors. Heme is the natural ligand for these receptors, but several synthetic agonists and antagonists have been designed recently. The gene that encodes REV-ERBα, NR1D1, is closely associated with ERBB2, the gene that encodes the HER2 oncogene, which is amplified in HER2+ breast cancers. We examined the effect of a synthetic REV-ERB agonist, SR9011, on a range of estrogen receptor positive (ER+), ER−, HER2+, HER2− and triple negative breast cancer cell lines. We found that SR9011 suppressed proliferation of the breast cancer cell lines regardless of their ER or HER2 status. SR9011 had no effect on MCF10A cell proliferation. SR9011 appears to pause the cell cycle of the breast cancer cells prior to M phase. Cyclin A (CCNA2) was identified as a direct target gene of REV-ERB suggesting that suppression of expression of this cyclin by SR9011 may mediate the cell cycle arrest. These data indicate that synthetic REV-ERB ligands may hold utility in treatment of diseases associated with uncontrolled cellular proliferation such as cancer.

Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy.

Synthesis and cancer cell growth inhibitory activity of icaritin derivatives

A series of icaritin derivatives bearing carboxylic acid or carboxylic ester groups are synthesized, and their in vitro cytotoxic activity against three cancer cell lines, MCF-7, MDA-MB-435s, and A549, are evaluated by MTT assay. Several derivatives including 2h, 2j, 5b and 5d show higher cytotoxic activity than the parent compound icaritin against these cancer cell lines. Compounds 5b and 5d are even more cytotoxic to MCF-7 cells than the clinic drug tamoxifen. Moreover, compound 5b is found to be non-toxic to normal cells (Vero) and both 5b and 5d exhibit good selectivity towards estrogen receptor positive MCF-7 breast cancer cells over estrogen receptor negative MDA-MB-435s breast cancer cells. The structure activity relationship analysis has revealed that mono-substitution at either C-3 or C-7 hydroxyl group of icaritin could improve the cytotoxicity of icaritin, and the C-3 hydroxyl group may be a preferable site for chemical modification. In addition, the length, the flexibility and the additional branching substituent group of the substitution chain(s) at both C-3 and C-7 hydroxyl groups can all affect the anti-cancer activity of these derivatives.

The potential in breast tissue engineering for the combined effects of linoleic acid and tamoxifen

Literature to date has strongly suggested that linoleic acid (LA) enhances mammary cancer cell proliferation when used in the range of 0·5–20 μg/ml. In contrast, the current in vitro studies show that deoxyribonucleic acid (DNA) levels of estrogen receptor-positive (ER+) MCF7 breast cancer cells were diminished following treatment with 30 μg/ml (100 μM) LA. MCF7 cells, when treated for 6 days with LA, consumed significantly less glucose than untreated controls (P-value<0·01). MFC7 cells treated with 30 μg/ml LA and 10 μM tamoxifen had even lower levels of DNA and lower glucose uptake. The same combination treatment of tamoxifen and LA was applied to a noncancerous mammary cell line, MCF10A, and did not have a significant effect on MCF10A DNA concentrations. Linoleic acid and its byproducts are important to cell proliferation and tissue reconstruction and, in combination with tamoxifen, can provide a uniquely different approach to soft tissue engineering. That is, LA and tamoxifen-loaded scaffolds may provide a viable reconstructive approach for patients undergoing lumpectomy and mastectomy by encouraging natural breast healing while limiting the ability of ER+ cancer cells to regrow. This approach could allow the suppression of estrogen-dependent mammary carcinomas while allowing natural cell proliferation after a mastectomy or lumpectomy.

Cyclopeptide RA-V inhibits cell adhesion and invasion in both estrogen receptor positive and negative breast cancer cells via PI3K/AKT and NF-κB signaling pathways

Cyclopeptide RA-V has potent anti-tumor and anti-angiogenic activities, but its potential anti-metastatic activity is unknown. Cancer cells acquire invasive ability to degrade and adhere to extracellular matrix (ECM), allowing them to migrate to adjacent tissues and ultimately metastasize. Hence, the present study aimed to investigate the effects of RA-V on cell adhesion, migration, invasion and matrix degradation, and its underlying mechanism in two human breast cancer cell lines MCF-7 (ER-positive) and MDA-MB-231 (ER-negative). Our results demonstrated that RA-V (12.5nM) can significantly inhibit breast cancer cell adhesion and migration via interfering cofilin signaling and chemokine receptors involved in cell migration. RA-V reduced the expressions of vascular intracellular adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), focal adhesion kinase (FAK) and integrins. The activities and expressions of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) and urokinase-type of plasminogen activator (uPA) were also inhibited by RA-V. Furthermore, RA-V inhibits the expressions of EGFR, PI3K/AKT and NF-κB signaling molecules, and reduces the binding of β-estradiol to ER via affecting binding ability of ER in MCF-7 cells. RA-V inhibits breast cancer cell migration, adhesion and ECM degradation in vitro, implying that RA-V is a potential anti-metastatic agent in breast cancer, and likely acts via PI3K/AKT and NF-κB signaling pathways in both ER-positive and ER-negative breast cancer cells.