Normal Breast Epithelial Cell Line Research Articles

Abstract P5-04-02: The KRAS-variant induces an EMT in normal breast epithelial cells

Abstract Background: The KRAS-variant is a functional, microRNA binding site variant in the 3'untranslated regions (3'UTR) of the KRAS oncogene, which predicts increased cancer risk for certain populations of patients, including an increased risk of triple negative breast cancer [1], and multiple primary breast cancers [2]. Tumors in KRAS-variant patients exhibit altered biology with a KRAS-addicted signature, an estrogen negative basal-like gene expression pattern, and a unique response to cancer therapy. Our goal was to better understand the underlying normal cellular biology associated with the KRAS-variant using an isogenic normal epithelial cell line model. Methods: Perfectly matched, isogenic normal breast epithelial cell lines with (MCF10AKRAS+/-, MT1 and MT2) versus without the KRAS-variant (MCF10AKRAS-/-, WT) were created and used in these studies [2]. The underlying molecular changes leading to EMT, stem cell phenotypes and altered microRNA expression were evaluated by targeted qRT-PCR, immunofluorescence, immunoblotting, immunoprecipitation, flow cytometry, global microRNA array and human phospho-kinase array analysis. Results: We found that KRAS-variant MT cells display a mesenchymal phenotype. We confirmed the downregulation of epithelial markers (E-cadherin and Occludin), upregulation of mesenchymal markers (Vimentin, Fibronectin and N-cadherin) and transcription factors (SNAIL and ZEB1) involved in EMT, as well as decreased level of miR-200c (1000-fold), consistent with EMT induction in the presence of the KRAS-variant. We found that KRAS-variant MT cells also exhibited elevated TGFß, consistent with our findings in KRAS-variant patients [3]. We further found that TGFβ signaling in KRAS-variant MT cells appears to lead to EMT through non-canonical signaling through MAPK (ERK) and PI3K (AKT) and AKT/GSK3β/Snail pathways. Conclusions: KRAS-variant MT normal epithelial cells exhibit an EMT phenotype and genotype, apparently through a non-canonical TGFß/AKT-mediated pathway. This is the first evidence to our knowledge of the powerful impact that a 3'UTR germ-line mutation can have on epithelial normal cellular biology, opening up numerous avenues for further elucidation of germ-line human variants in biology and their clinical applications to breast cancer risk.

MicroRNA-223 Targeting STIM1 Inhibits the Biological Behavior of Breast Cancer

Background/Aims: To investigate the cellular effects and clinical significance of microRNA-223 (miR-223) in breast cancer by targeting stromal interaction molecule1 (STIM1). Methods: Breast cancer cell lines (T47D, MCF-7, SKB-R3, MDA-MB-231 and MDA-MB-435) and a normal breast epithelial cell line (MCF-10A) were prepared for this study. MiR-223 mimics, anti-miR-223 and pcDNA 3.1-STIM1 were transiently transfected into cancer cells independently or together, and then RT-qPCR was performed to detect the expressions of miR-223 and STIM1 mRNA, dual-luciferase reporter assay was conducted to examine the effects of miR-223 on STIM1, Western blotting was used to measure the expressions of the STIM1 proteins, MTT and Trans-well assays were performed to detect cell proliferation and invasion. Finally, the correlation of miR-223 and STIM1 was investigated by detecting with ISH and IHC in breast cancer specimens or the corresponding adjacent normal tissues. Results: Compared with normal cells and tissues, breast cancer tissues and cells exhibited significantly lower expression of miR-223, but higher expression of STIM1. MiR-223 could inhibit the proliferation and invasiveness of breast cancer cells by negatively regulating the expressions of STIM1. Reimplantation with STIM1 partially rescued the miRNA-223-induced inhibition of breast cancer cells. Clinical data revealed that high expression of STIM1 and miR-223 was respectively detrimental and beneficial factor impacting patient’s disease-free survival (DFS) rather than overall survival (OS). Moreover, Pearson correlation analysis also confirmed that STIM1 was inversely correlated with miR-223. Conclusion: MiR-223 inhibits the proliferation and invasion of breast cancer by targeting STIM1. The miR-223/STIM1 axis could possibly be a potential therapeutic target for treating breast cancer patients.

TRNA-Derived Fragments as Novel Predictive Biomarkers for Trastuzumab-Resistant Breast Cancer

Background/Aims: Resistance to trastuzumab remains a common challenge to HER-2 positive breast cancer. Up until now, the underlying mechanism of trastuzumab resistance is still unclear. tRNA-derived small non-coding RNAs, a new class of small non-coding RNA (sncRNAs), have been observed to play an important role in cancer progression. However, the relationship between tRNA-derived fragments and trastuzumab resistance is still unknown. Methods: We detected the levels of tRNA-derived fragments expression in normal breast epithelial cell lines, trastuzumab-sensitive and -resistant breast cancer cell lines using high-throughput sequencing. qRT-PCR was conducted to validate the differentially expressed fragments in serums from trastuzumab-sensitive and -resistant patients. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the power of specific tRNA-derived fragments. Progression-free survival (PFS) was analyzed using Cox-regression. Results: Our sequence results showed that tRNA-derived fragments were differentially expressed in the HBL-100, SKBR3, and JIMT-1 cell lines. tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN were found significantly upregulated in trastuzumab-resistant patients compared to sensitive individuals, and the ROC analysis showed that tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN were correlated with trastuzumab resistance. In a multivariate analysis, higher levels of tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN expression were associated with significantly shorter PFS in patients with metastatic HER-2 positive breast cancer. Conclusion: Our results suggest that tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN play important roles in trastuzumab resistance. Patients with high levels of tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN expression benefitted less from trastuzumab-based therapy than those that express lower-levels of these molecules. tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN may be potential biomarkers and intervention targets in the clinical treatment of trastuzumab-resistant breast cancer.

Abstract A178: Targeting the ATM-mediated DNA damage response pathway for cancer treatment

Abstract Epigenetic modulators have emerged as potent therapeutic agents for treatment of patients with cancers. We have developed dual-function small molecules targeting activation of ATM, the DNA damage response transducer, ataxia telangiectasia mutated gene product, and inhibition of histone deacetylases (HDACi). The goal is to develop a single-molecule therapeutic that optimizes the protective effects of ATM activation in normal tissue while targeting HDAC inhibition to sensitize cancerous tissue to radiation. The candidate compound SP-1-161 is a hydroxamic acid that was rationally designed by optimizing ATM activation in the cap domain. In vitro assays against a panel of Class I and Class II HDAC enzymes identified SP-1-161 as a pan-HDAC inhibitor with nanomolar potency (IC50 = 8 nM). Western analysis confirms that SP-1-161 increases acetylated histone H3/H4 and α-tubulin and activates ATM (p-ATM/S1987) in MCF7 cells. The cytotoxicity values (IC50) were in low micro-molar ranges in normal breast epithelial (184A1) and breast cancer (MCF7) cell lines. Radiation clonogenic survival assays demonstrated that the compound protected nonmalignant 184A1 cells (from D0 = 1 Gy to D0 =1.4 Gy) while increasing sensitivity of malignant MCF7 cells to IR (from D0=1.6 Gy to D0=1.12 Gy). Furthermore, in vivo plasma pharmacokinetics following oral administration revealed the area under the curve of 415.79 ng/ml × h with the half-life 1.94 h. Together, our results demonstrate that SP-1-161 is an unprecedented radiation chemotherapeutic agent for treating cancers and protecting normal cells, potentially by targeting the DDR signaling pathways. Citation Format: Scott Grindrod, Alfredo Velena, Mira Jung. Targeting the ATM-mediated DNA damage response pathway for cancer treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A178.

A novel small molecule inhibitor of CD151 inhibits proliferation of metastatic triple negative breast cancer cell lines

The large extracellular loop (LEL) of CD151 is receiving much attention and as such described as a novel potential therapeutic target of metastatic cancers due to presence of functionally important sites. In the present study, three dimensional structure of large extracellular loop of CD151 (LEL) was modeled and validated by RAMPAGE. Virtual screening predicted small molecule inhibitor, NSC 146268 (2-thio-6-azauridine) as potential lead molecule targeting LEL of CD151. The 2-thio-6-azauridine (TAU) presented a specific effect on viability of triple negative breast cancer (TNBC) cell lines, MDA-MB 231 and MDA-MB 468 with IC50 value 6.0 and 6.2 μg/mL, respectively and least effect on normal breast epithelial cell line (IC50 value 48 μg/mL). It also exhibited significant anti-proliferative activity comparable to paclitaxel with arrest of cell cycle at G1 phase and induction of apoptosis. PathDIP database predicted cell junction pathways as a possible target of CD151. The protein levels of CD151 together with cell junction scaffold proteins, CD46, E-cadherin, β-catenin and ZO-1 were decreased with TAU at 50% inhibitory concentration. The present results showcase the CD151 as a potential target together with TAU as possible lead molecule of triple negative breast cancer cell lines.

Identification of Novel Bisbenzimidazole Derivatives as Anticancer Vacuolar (H⁺)-ATPase Inhibitors.

The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I–IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a–e). All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7) and ovarian cancer (A2780, Cis-A2780, and PA-1) cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 μM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.

Solubility of acyclovir in nontoxic and biodegradable ionic liquids: COSMO-RS prediction and experimental verification

The pharmaceutical industry faces a challenge to find potential solvents for drug molecules that are sparingly soluble in water and conventional organic solvents. Recently, ionic liquids (ILs) have attracted great attention as pharmaceutical solvents owing to their unique physicochemical and biological properties. In this study, the solubility of the sparingly soluble drug molecule acyclovir (ACV) in a wide variety of ILs was investigated by conductor-like screening model for real solvents (COSMO-RS) calculations. The predicted solubilities were validated by experimental measurements, and good agreement was found between the predicted and experimental results. The solubility of ACV was greatly affected by the structure of ILs, particularly the anionic moiety. Among the various ILs tested, ACV showed excellent solubility in ammonium-based ILs with an acetate anion. In vitro cytotoxicity of ILs to the MCF-10 normal breast epithelial cell line and cancer cell lines (MDA MB 231 and MCF 7) was investigated. The ammonium-based ILs showed higher IC50 values than the imidazolium-based ILs with the acetate anion. Biodegradability results showed that diethylammonium acetate, triethylammonium acetate, and choline acetate ILs have high levels of biodegradation under aerobic conditions and can be classified as readily biodegradable. These findings will be useful for the design of IL-based drug delivery carriers that can act as versatile and efficient drug delivery systems for sparingly soluble drug molecules.

Structure-based development of novel triazoles and related thiazolotriazoles as anticancer agents and Cdc25A/B phosphatase inhibitors. Synthesis, in vitro biological evaluation, molecular docking and in silico ADME-T studies

Synthesis of twenty nine new 1,2,4-triazoles and some derived thiazolothiadiazoles (structurally-relevant to some reported triazoles with anticancer and/or Cdc25A/B inhibitory activities) is described in this study. The obtained NCI's in vitro antitumor data revealed that five analogs (12, 15, 18, 19 and 22) displayed considerable tumor percentage growth inhibitory activity (GI%), among which the analog 18 possessed a special antitumor potential and spectrum. Additionally, the same five analogs showed a marginal GI effect on the normal breast epithelial cell line MCF-10A indicating higher selectivity towards cancer cells. The same active analogs 12, 15, 18, 19 and 22 were further assessed for their in vitro Cdc25A/B phosphatase inhibitory activity (a possible antitumor target), where 18 and 22 displayed a distinctive inhibitory affinity towards the Cdc25B isozyme (6.7 and 8.4 μM, respectively). Docking of 12, 15, 18, 19 and 22 with the active site of human Cdc25B phosphatase enzyme demonstrated superior binding profile by the top-scoring analog 18 relative to a reported Cdc25 phosphatase ligand. In silico calculations of molecular properties revealed that all of the active compounds comply with Lipinski's RO5 and Veber's criteria for good bioavailability suggesting good drug-likeness upon oral administration with a predicted high safety profile.

Functional characterization of lysine-specific demethylase 2 (LSD2/KDM1B) in breast cancer progression.

Flavin-dependent histone demethylases govern histone H3K4 methylation and act as important chromatin modulators that are extensively involved in regulation of DNA replication, gene transcription, DNA repair, and heterochromatin gene silencing. While the activities of lysine-specific demethylase 1 (LSD1/KDM1A) in facilitating breast cancer progression have been well characterized, the roles of its homolog LSD2 (KDM1B) in breast oncogenesis are relatively less understood. In this study, we showed that LSD2 protein level was significantly elevated in malignant breast cell lines compared with normal breast epithelial cell line. TCGA- Oncomine database showed that LSD2 expression is significantly higher in basal-like breast tumors compared to other breast cancer subtypes or normal breast tissue. Overexpression of LSD2 in MDA-MB-231 cells significantly altered the expression of key important epigenetic modifiers such as LSD1, HDAC1/2, and DNMT3B; promoted cellular proliferation; and augmented colony formation in soft agar; while attenuating motility and invasion. Conversely, siRNA-mediated depletion of endogenous LSD2 hindered growth of multiple breast cancer cell lines while shRNA-mediated LSD2 depletion augmented motility and invasion. Moreover, LSD2 overexpression in MDA-MB-231 cells facilitated mammosphere formation, enriched the subpopulation of CD49f+/EpCAM- and ALDHhigh, and induced the expression of pluripotent stem cell markers, NANOG and SOX2. In xenograft studies using immune-compromised mice, LSD2-overexpressing MDA-MB-231 cells displayed accelerated tumor growth but significantly fewer lung metastases than controls. Taken together, our findings provide novel insights into the critical and multifaceted roles of LSD2 in the regulation of breast cancer progression and cancer stem cell enrichment.

Abstract 3215: Therapeutic potential of dual-function small molecules for breast cancers

Abstract Epigenetic silencing of tumor suppressors is an important mechanism in tumor initiation and progression. A tumor suppressor gene, BRCA1, is part of complex molecules in the repair of DNA double-strand breaks (DSBs), and loss of BRCA1 contributes to both sporadic and inherited breast tumor progression. Furthermore, recent studies have shown that the ataxia-telangiectasia mutated (ATM) activity is abnormal in breast cancer. Therefore, we tested the hypothesis that the dual-functional drug that fused the HDAC inhibitor with an ATM activator may enhance killing of breast cancer (BC) cells by increasing ATM activation. In addition, the expression of ATM may enhance the synergistic antitumor effects of HDAC inhibitors in combination with radiotherapy. To determine the HDAC inhibitory potency, SP-1-303 was screened against a panel of Class I and Class II HDAC enzymes in vitro and identified as a pan-HDAC inhibitor (IC50=120 nM). Western blot confirmed that SP-1-303 increased acetylated histone H3/H4, α-tubulin, and phosphorylation of ATM within 30 min - 4 h in a time-dependent manner. Using two ER positive BC cell lines (MCF7 and T-47D), two triple negative breast cancer (TNBC) cell lines (MDA-MB-231 and HCC 1937), and one normal breast epithelial cell line as a control (MCF10A), the effects of SP-1-303 on cell growth and cell death were examined by performing cytotoxicity assays and flow cytometry. SAHA and Tamoxifen were used as controls. SP-1-303 conferred the values of cytotoxicity (IC50) for the ER positive BC cells in a range of 0.25-0.4 μM, while the values are in a range of 1-3 μM for TNBC and 12 μM for normal breast epithelial cells, respectively. Flow cytometry (FACS) analyses demonstrated significant increases in G1 arrest (81%) in MCF7 cells and a further enhancement in the G1 phase (91%) in combination with radiation. The data implies the mechanism of action from this drug—cancer cells are being arrested in the G1 phase of the cell cycle, a radiation sensitive phase, suggesting that SP-1-303 may have a synergistic effect with radiation on cell killing. Taken together, SP-1-303 had the largest impact on ER positive breast cancer over TNBC. These data imply that an increased cell death is occurring in ER positive breast cancers from these dual-action molecules through HDAC inhibition and increased ATM activation, suggesting that SP-1-303 alone or its combination with radiotherapy may improve clinical efficacy. Citation Format: Nicole Nicholas, Alfredo Velena, Scott Grindrod, Mira Jung. Therapeutic potential of dual-function small molecules for breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3215. doi:10.1158/1538-7445.AM2017-3215

Abstract 512: Knockdown of the spindle assembly checkpoint gene Bub1b results in increased cell death and cell cycle impairment in breast cancer

Abstract Genomic instability is an enabling hallmark of cancer that provides cancer cells a replicative advantage. Accumulation of genomic aberrations can also compromise the genomic integrity and put cells under mitotic stress. Maintaining balance between the instability that gives cancer cells a replicative advantage and the instability that could lead them to mitotic catastrophe is crucial for survival of cancer cells. Mitotic catastrophe can be caused by rapid progression through mitosis before crucial checkpoints are met, resulting in cell death. To overcome this challenge, cancer cells may acquire overexpression of spindle assembly checkpoint (SAC) genes, which can prevent mitotic catastrophe that would occur if they undergo mitosis prematurely. Bub1b is an important part of SAC and inhibits the onset of anaphase until all chromosomes are aligned at the metaphase plate. Our analysis of clinical datasets show Bub1b expression is elevated in breast cancer, compared to normal breast, and is exemplified by a pattern of increasing overexpression in more aggressive variants, such as triple negative breast cancer (TNBC). Bub1b overexpression also correlates with decreased overall survival in patients. Expression analysis of breast cancer cell lines corroborates this clinical data. We hypothesize that the requirement for Bub1b expression indicates a vulnerability of rapidly proliferating breast cancers cells, and the inhibition of Bub1b will result in cell death through mitotic catastrophe. Using RNA interference with siRNAs we knocked down Bub1b expression in a variety of breast cancer cells. This resulted in significant decrease in cellular viability and clonogenicity in soft agar. Furthermore, analyses of apoptosis using Annexin V and PI costaining showed that Bub1b knockdown results in significantly increased apoptosis and cell death, especially in the TNBC cell line MDA-MB-468. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. Bub1b knockdown resulted in silencing of SAC activity, as measured by Cyclin B levels, and also significantly impaired cell cycle progression in breast cancer cell lines. Additionally, knockdown of Bub1b in breast cancer cell lines demonstrated an additive effect when combined with DNA-damaging agents. Our data support that Bub1b is a critical player in breast cancer cell survival, by enabling them to overcome mitotic stress. We demonstrate that impairment of Bub1b has detrimental effects on cell cycle and progression of mitosis. Further investigation of the role of Bub1b in promoting successful chromosome alignment and proliferation of breast cancer cells with genomic instability could provide a novel pathway on how cancer overcomes the mitotic stress caused by genomic instability. These studies may also lead to novel therapeutic strategies that combine SAC inhibitors with standard genotoxic treatments. Citation Format: Dilara Koyuncu, Erik T. Goka, Philip C. Miller, Marc E. Lippman. Knockdown of the spindle assembly checkpoint gene Bub1b results in increased cell death and cell cycle impairment in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2017-512

Abstract 4054: Development of dual function small molecules as therapeutic agents for cancer treatment

Abstract Inhibitors of histone deactylases (HDACs) are emerging as potent anti-cancer agents for treatment of patients with hematopoietic and solid tumors. However, most of these compounds exhibit limitations, including off-targets and toxicity. To improve the efficacy and target specificity of drugs, we developed the compound, SP-1-161 conferring HDAC inhibition activity and ataxia-telangiectasia mutated (ATM) activation. The purpose of this study is to develop a therapy that optimizes the protective effects of ATM activation in normal tissue with the sensitizing effects of HDAC inhibition in cancerous tissue into a single molecule. The compound SP-1-161 was rationally designed by optimizing ATM activation by the cap domain of a hydroximic acid, screened against a panel of Class I and Class II HDAC enzymes, and identified as a pan-HDAC inhibitor with nanomolar potency (IC50 = 8 nM). Western analysis confirms that SP-1-161 increases acetylated histone H3/H4 and α-tubulin and ATM activation in MCF7 cells. Phosphorylated ATM was gradually increased within 1 - 4 hrs in a time-dependent manner. SP-1-161 was then tested in normal breast epithelial (184A1) and breast cancer (MCF7) cell lines to determine its cytotoxicity and effect on radiation clonogenic survival in combination with graded radiation exposure. The data showed that the cytotoxicity values (IC50) were low micro-molar ranges. Furthermore, SP-1-161 protected 184A1 cells (from D0 = 1 Gy to D0 = 1.4 Gy) while increasing sensitivity of MCF7 cells to IR (from D0=1.6 Gy to D0=1.12 Gy). Together, our results demonstrate that SP-1-161 is an unprecedented radio-chemo therapeutic agent for treatment of cancers while protecting normal cells. Citation Format: Scott Grindrod, Alfredo Velena, Mira Jung. Development of dual function small molecules as therapeutic agents for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2017-4054

MiR-106b and miR-93 regulate cell progression by suppression of PTEN via PI3K/Akt pathway in breast cancer

Accumulating evidences have revealed that dysregulated microRNAs (miRNAs) involve in the tumorigenesis, progression and even lead to poor prognosis of various carcinomas, including breast cancer. MiRNA-106b-5p (miR-106b) and miRNA-93-5p (miR-93) levels were confirmed to be significantly upregulated in breast cancer clinical samples (n=36) and metastatic cell line (MDA-MB-231) compared with those in the paired adjacent tissues and normal breast epithelial cell line (MCF-10A). Moreover, further research stated that the capability of migration, invasion and proliferation changed along with the altered expression of miR-106b and miR-93 in breast cancer. PTEN, the tumor-suppressor gene, was discovered to be reduced in breast cancer tissues or MDA-MB-231 cells with high levels of miR-106b and miR-93, which were inversely expressed in PTEN overexpression tissues or cells. Based on the investigation, miR-106b and miR-93 induced the migration, invasion and proliferation and simultaneously enhanced the activity of phosphatidylinositol-3 kinase (PI3K)/Akt pathway of MCF-7 cells, which could be blocked by upregulation of PTEN. Furthermore, suppression of PTEN reversed the function induced by anti-miR-106b and anti-miR-93 in MDA-MB-231 cells, indicating that PTEN was directly targeted by these miRNAs and acted as the potential therapeutic target for breast cancer therapy. In short, reductive PTEN mediated by miR-106b and miR-93 promoted cell progression through PI3K/Akt pathway in breast cancer.

Abstract P6-10-06: Inducing immune responses to tumor associated carbohydrate antigens by a carbohydrate mimetic peptide vaccine: Clinical experience in phase I and phase II trials

Abstract Active immunization of cancer patients to induce de novo functional anti-tumor immune responses is an alternative/complementary approach to chemotherapy. Tumor vaccines hold the potential to deliver durable, specific and systemic anti-tumor responses in patients. We have been developing active vaccination strategies targeting tumor associated carbohydrate antigens (TACAs) using carbohydrate mimetic peptides. TACAs play roles in initiation and metastasis of cancer and considered as common targets shared by many tumor types. TACA support cell survival that can be interrupted by anti-carbohydrate antibodies. An early-phase 3+3 clinical trial was conducted to evaluate the feasibility, safety and immune functionality of a carbohydrate mimetic-peptide (CMP) vaccine referred to as P10s, which can induce TACA reactive, proapoptotic antibodies. In this trial a dose-escalation trial of vaccine plus adjuvant was conducted in two cohorts of 3 subjects each. Patients were restricted to females of all races with histologically or cytologically confirmed stage IV breast cancer who had stable disease and a positive recall-antigen response. P10s was synthesized with the Pan-T-cell epitope PADRE and formulated at 300 and 500 µg/injection with MONTANIDE™ ISA 51 VG for the 1st and 2nd cohorts, respectively. Doses of the appropriate formulation of the vaccine were administered to research participants subcutaneously on weeks 1, 2, 3, 7 and 19. Blood samples were collected at various time points and tested for presence and functionality of antibodies. Antibody response to P10s and in particular against the ganglioside GD2 was measured by ELISA. Binding of pre-immune and post-immune sera was assessed against breast cancer cell lines. Vaccination generates IgG response with serum antibodies capable of inhibiting tumor growth in spheroid culture of breast cancer cell lines. The vaccine induced antibodies in all 6 subjects, displaying significant cytotoxic activity against several representative human breast-cancer cell lines. Caspase 3 was involved in the postimmune serum-mediated apoptosis. No cytotoxicity toward a normal breast epithelial cell line was detected. Apoptosis and caspase 3 activation seems to be involved in anti-tumor cell activity. Immunization with the P10s vaccine was found to be safe and tolerable, and induces functional antibodies that potentially have a cell-death-mediated therapeutic benefit. Incubation of spheroids with post-immune serum further sensitized cells to drugs, improving the efficacy of drug treatment at lower doses. The data suggest that the vaccine-induced anti-tumor immune response in combination with standard of care chemotherapy may further improve clinical outcome. Consequently, we are testing the vaccine in a Phase II study in the neoadjuvant setting. 5 Cohorts of 5 patients each administered with the vaccine at different schedules of chemotherapy are being assessed for immune response to the vaccine as in the Phase I study and if the combination approach contributes to a difference in pathological complete response (PCR) from chemotherapy alone. Citation Format: Kieber-Emmons T, Hutchins LF, Emanuel PD, Pennisi A, Siegel E, Jousheghany F, Karbassi BM, Makhoul I. Inducing immune responses to tumor associated carbohydrate antigens by a carbohydrate mimetic peptide vaccine: Clinical experience in phase I and phase II trials [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 P6-10-06.

Astrocyte elevated gene-1 promotes the proliferation and invasion of breast cancer cells by activating the Wnt/β-catenin signaling pathway

Astrocyte elevated gene-1 (AEG1) was identified to be overexpressed in breast cancer, and to be associated with the development of breast cancer. In the present study, AEG1 was identified as highly expressed in the MCF-7, MDA-MB-231 and SK-BR-3 breast cancer cell lines and was detected in the MCF-10A normal breast epithelial cell line. The present study established an AEG1-knockdown MCF-7 cell line to investigate the expression status of certain cancer-associated proteins. Western blotting demonstrated that AEG1 may affect cancer cell proliferation and invasion via activating the Wnt/β-catenin signaling pathway, a hypothesis that has been supported by cell function tests. The results of the present study demonstrated that when AEG1 was significantly overexpressed in breast cancer cells it promoted cell proliferation and invasion via activating the Wnt/β-catenin signaling pathway. Therefore, AEG1 may serve as a novel therapeutic target in breast cancer.

Evaluation of Cytotoxic and Genotoxic Effects of Euphorbia Triaculeata Forssk. Extract

Objective:To evaluate the cytotoxic and genotoxic activity of Euphorbia triaculeata Forssk. plant extract from Jazan region, Saudi Arabia, in an in vitro cancer model, which could be beneficial in anticancer therapy against human breast cancer cell line (MCF-7), prostate cell line (PC-3), human hepatocellular carcinoma cell line (HEPG2) and normal breast epithelial cell line (MCF-10A). The human foreskin fibroblast cell line, (Hs68), was also included in the cell panel. Doxorubicin and 5-Flurouracil, broad-spectrum anticancer drugs, were used as the positive control.Methods:Cytotoxicity of Euphorbia triaculeata plant extract was investigated by employing MTT assay and the genotoxicity was assessed by using comet assay.Results:Both toxicity tests exhibited significant toxicity results. In the comet assay, the Euphorbia triaculeata extract exhibited genotoxic effects against MCF-7 DNA and PC 3 but not on HEPG2 cell lines in a time-dependent manner by increasing the mean percentage of DNA damage. Euphorbia triaculeata extract showed significant toxicity against cancer cells. Comparison with positive control signifies that cytotoxicity exhibited by methanol extract might have moderate activity.Conclusion:The present work confirmed the cytotoxicity and genotoxicity of Euphorbia triaculeata plant. However, the observed toxicity of this plant extract needs to be confirmed by additional studies. Based on our results, further examination of the potential anticancer properties of Euphorbia triaculeata plant species and the identification of the active ingredients of these extracts is warranted.

Expression of metallothionein3 in ductal breast cancer.

Metallothionein3 (MT-3) has the ability to regulate the growth of nerve cells, but the significance of MT-3 expression outside the central nervous system and its participation in carcinogenesis have not yet been clarified. The aim of our study was to investigate the expression of MT-3 in ductal breast cancer and to determine its relationship with well-defined clinicopathological factors in this type of tumor. The study was conducted on 134 cases of invasive ductal breast carcinoma (IDC), 42 samples of non-malignant breast tissue (NMBT), and 26 cases of mastopathy. Moreover, selected breast cancer cell lines (MCF-7, SKBR-3, MDA-MB-231, BO2) and normal human breast epithelial cells (hTERT-HME1) were used. The expression of MT-3 was examined on the protein level using immunohistochemistry and on the mRNA level using real-time PCR. It was shown that the MT-3 protein in cells of IDC and mastopathy appeared in the cytoplasm as well as in the cell nuclei. Both the cytoplasmic and nuclear expression of MT-3 was significantly lower in IDC than in the mastopathies (p<0.0001 and p<0.001). However, no significant correlation was demonstrated between the level of MT-3 protein and the studied clinicopathological factors. The mRNA expression of MT-3 in IDC was also lower than in non‑malignant breast tissue (p<0.0001). Furthermore, in the cases of IDC with lymph node metastasis, the level of MT-3 mRNA was significantly lower than in the cases without metastasis (p=0.0199). The expression of MT-3 mRNA in breast cancer cell lines was significantly lower than in the normal human breast epithelial cell line (p<0.001). These results suggest that MT-3 may play a role in the malignant transformation of breast epithelial cells and in tumor progression.

The role of semaphorin 4D in tumor development and angiogenesis in human breast cancer.

BackgroundSemaphorin 4D (Sema4D) is highly expressed in certain types of tumors and functions in the regulation of tumor angiogenesis and growth. However, it is still not clear regarding the roles of Sema4D in breast cancer. This study was designed to explore the effects of Sema4D on proliferation, cell cycle progression, apoptosis, invasion, migration, tumor growth, and angiogenesis in breast cancer.Materials and methodsThe expression level of Sema4D was investigated in MCF10A, 184A1, HCC1937, MDA-MB-468, MDA-MB-231, Hs578T, BT474, MCF-7, and T47D breast cancer cell lines by Western blotting analysis. Sema4D downregulation or overexpression was established by infection with lentiviruses-encoding Sema4D short hairpin RNA (shRNA) or Sema4D. To evaluate the effects of Sema4D on cell proliferation, cell cycle progression, apoptosis, invasion, and migration of MDA-MB-231 and MDA-MB-468 cells, methods including MTT assay, flow cytometry, wound healing assay, and transwell experiments were applied. BALB/c nude mice were injected with MDA-MB-231 cells, which were respectively infected with lentiviruses-encoding Sema4D, Sema4D shRNA, and GFP, followed by tumor angiogenesis assay.ResultsSema4D was expressed at higher levels in breast cancer cell lines compared with the normal human breast epithelial cell lines, especially in MDA-MB-231 and MDA-MB-468 cells. Cell proliferation ability was remarkably inhibited in Sema4D downregulated condition, whereas the proportions of cells in the G0/G1 phase and apoptosis increased in MDA-MB-231 and MDA-MB-468 cells. In addition, the invasion and migration abilities of these cells were obviously reduced. Xenograft growth as well as angiogenesis was inhibited when infected with lentiviruses-encoding Sema4D shRNA in vivo.ConclusionDownregulation of Sema4D had notable influence on cell proliferation ability, invasion, migration, and apoptosis of both MDA-MB-231 and MDA-MB-468 cells. Furthermore, infection with lentiviruses-encoding Sema4D shRNA obviously inhibited tumor growth and angiogenesis in BALB/c nude mice. Our results showed that Sema4D may represent a novel therapeutic target for human breast cancer.

Expression status of cyclase-associated protein 2 as a prognostic marker for human breast cancer

Cyclase-associated protein2(CAP2) protein is reported to be upregulated in hepatocellular carcinoma(HCC). However, data regarding its expression pattern and clinical relevance in breast cancer are unknown. The aim of this study was to investigate CAP2 expression and its prognostic significance in breast cancer. CAP2 expression at the mRNA and protein levels was examined by real‑time quantitative-polymerase chain reactionand western blotting in 10paired breast cancer tissues and adjacent normal tissues. The expression level of CAP2 protein in normal breast epithelial cells and breast cancer cell lines was quantified by western blotting. CAP2 protein expression was analyzed in paraffin‑embedded breast cancer samples, paired adjacent non‑tumor and normal breast tissues by immunohistochemicalanalysis. Statistical analyses were also performed to evaluate the clinicopathological significance of CAP2 expression. The results showed that the expression of CAP2 mRNA and protein was higher in breast cancer than that noted in the adjacent normal tissues in 10paired samples. The expression level of CAP2 protein in breast cancer cell lines was higher than that in normal breast epithelial cells. In paraffin‑embedded tissue samples, the expression of CAP2 was higher in breast cancer than that found in the adjacent non‑cancerous tissues and normal breast tissues. Compared with the adjacent non‑cancerous tissues, overexpression of CAP2 was detected in 29.4% (37/126) of the patients. Overexpression of CAP2 was significantly associated with progesterone receptor(PR) expression (p<0.05), and decreased overall survival(OS) (p<0.05). In multivariate analysis, expression of CAP2 was an independent prognostic factor for OS [hazard ratio(HR), 4.821; 95% confidence interval(CI), 2.442‑9.518; p<0.001]. CAP2 is upregulated in breast cancer and is associated with the expression of PR and patient survival. CAP2 may serve as a prognostic indicator for patients with breast cancer.

Sef Regulates Epithelial-Mesenchymal Transition in Breast Cancer Cells.

Sef (similar expression to fgf), also know as IL17RD, is a transmembrane protein shown to inhibit fibroblast growth factor signaling in developmental and cancer contexts; however, its role as a tumor suppressor remains to be fully elucidated. Here, we show that Sef regulates epithelial-mesenchymal transition (EMT) in breast cancer cell lines. Sef expression was highest in the normal breast epithelial cell line MCF10A, intermediate expression in MCF-7 cells and lowest in MDA-MB-231 cells. Knockdown of Sef increased the expression of genes associated with EMT, and promoted cell migration, invasion, and a fibroblastic morphology of MCF-7 cells. Overexpression of Sef inhibited the expression of EMT marker genes and inhibited cell migration and invasion in MCF-7 cells. Induction of EMT in MCF10A cells by TGF-β and TNF-α resulted in downregulation of Sef expression concomitant with upregulation of EMT gene expression and loss of epithelial morphology. Overexpression of Sef in MCF10A cells partially blocked cytokine-induced EMT. Sef was shown to block β-catenin mediated luciferase reporter activity and to cause a decrease in the nuclear localization of active β-catenin. Furthermore, Sef was shown to co-immunoprecipitate with β-catenin. In a mouse orthotopic xenograft model, Sef overexpression in MDA-MB-231 cells slowed tumor growth and reduced expression of EMT marker genes. Together, these data indicate that Sef plays a role in the negative regulation of EMT in a β-catenin dependent manner and that reduced expression of Sef in breast tumor cells may be permissive for EMT and the acquisition of a more metastatic phenotype. J. Cell. Biochem. 117: 2346-2356, 2016. © 2016 Wiley Periodicals, Inc.