Mouse Breast Cancer Cell Line Research Articles

Anticancer activityof alkaloids of Madagascar Periwinkle Catharanthusroseus (L.)G.Don against two cancer cell line

In the present study , Madagascar Periwinkle Catharanthus roseus (L.)G.Donalkaloids were evaluated against two cancer cell lines :larynx human cancer cell line Hep2,andmousebreastcancercelllineAMN3,byusingfour concentrationsfor each cell line (25,12.5,6,25,3.125)mg/ml(in triplicate).and it has been found that alkaloids showedhighest activity against larynx human cancer cell line Hep-2 ,while exhibited weak activityagainst mouse breast cancer cell line AMN3 compared with control treatment.

Organometallic binuclear Pd(II) complex: Synthesis, crystal structure and in-vitro antitumor activity study

A novel planar chiral ferrocene cyclopalladated compound (C2) was synthesized and fully characterized. The absolute configuration of C2 has been determined by single-crystal X-ray analysis. The cytotoxic activities of C2 and cisplatin were evaluated against liver (MHCC97 and HepG2), mouse breast (4 T1) and ovarian granulosa (KGN) cancer cell lines. C2 was about 23 times more potent than cisplatin in the suppression of KGN cells.

Preclinical development of natural compound emodin as an agent to halt breast cancer metastasis

Abstract Our previous studies demonstrated that a natural compound, emodin, suppresses breast cancer growth and metastasis by modulating tumor-associated macrophages (TAMs), we aim in this study to further develop emodin as an agent to diminish breast cancer post-surgery metastatic recurrence. Using various mouse and human breast cancer cell lines, we demonstrated that emodin not only directly reversed TGF-β1-induced epithelial to mesenchymal transition (EMT), migration and invasion, but also indirectly attenuated EMT by suppressing TAM polarization and TGF-β1 production. Emodin also suppressed the stemness and progenitor properties of tumor-initiating cells (TICs). The EMT process and TIC populations in primary breast tumors were diminished by emodin in vivo, and pretreating tumor cells with emodin suppressed breast cancer lung metastasis in a stem cell-like circulating tumor cell-seeding model. In orthotopic metastasis mouse models, administration of emodin before surgery suppressed the metastatic recurrence of breast cancer and improved the overall survival. Mechanistic study revealed that emodin inhibited both canonical and non-canonical pathways of TGF-β1 signaling in breast cancer cells. Taken together, our study demonstrated that emodin can be developed as a therapeutic agent to prevent post-surgery metastatic recurrence of breast cancer by suppressing TGF-β1-mediated crosstalk between breast cancer cells and TAMs.

Dually responsive nanogels as smart carriers for improving the therapeutic index of doxorubicin for breast cancer

Nanocarriers can significantly improve the therapeutic index of drug in cancer therapy. At present, parenteral administration of free doxorubicin (Dox) to treat breast tumor produces severe side effects including cardiotoxicity. This work reports the preparation of N-isopropyacrylamide/acrylic acid nanogels (NGs), NIPA/AAc NGs, crosslinked by a disulfide-bearing monomer, which can be used as smart carriers of Dox for cancer therapy. The NGs prepared showed high load percentage of Dox through ionic interaction. The size of the NGs in simulated physiological medium was ideal to be applied as a carrier in cancer therapy. In vitro release studies demonstrated that the designed drug delivery system (DDS) can active the release of a large amount of drug in simulated intracellular medium in response to redox potential and pH, but with minimal release in simulated plasma conditions. Cellular uptake studies showed that NGs loaded with Dox can be internalized in human breast cancer cell line MDA-MB-231. Cell toxicity studies demonstrate that unloaded NGs are nontoxic for human breast cancer cell line MDA-MB-231 and mouse breast cancer cell line 4T1. In addition, it was shown that Dox-loaded NGs are more toxic for the cell than free Dox for both cell lines. In vivo studies in mice showed that Dox-loaded NGs improve the drug therapeutic index with a notable decrease in tumor size, as compared with a similar dose of free Dox. Finally, the development of this nanoformulation could improve the results and patient compliance in chemotherapy treatments of breast cancer.

Synthesis of substituted biphenyl methylene indolinones as apoptosis inducers and tubulin polymerization inhibitors

A new series of biphenyl methylene indolinones has been designed, synthesized and evaluated for their in vitro antiproliferative activity against various cancer cell lines like DU-145 (prostate cancer cell line), 4T1 (mouse breast cancer cell line), MDA-MB-231 (human breast cancer cell line), BT-549 (human breast cancer cell line), T24 (human urinary bladder carcinoma cell line), and HeLa (cervical cancer cell line). Among the series, compound 10e showed potent in vitro cytotoxic activity against HeLa and DU-145 cancer cell lines with IC50 value of 1.74 ± 0.69 µM and 1.68 ± 1.06 µM respectively. To understand the underlying mechanism of most potent cytotoxic compound 10e, various mechanistic studies were carried out on DU-145 cell lines. Cell cycle analysis results revealed that these conjugates affect both G0/G1 and G2/M phase of the cycle, tubulin binding assay resulted that compound 10e interrupting microtubule network formation by inhibiting tubulin polymerization with IC50 value of 4.96 ± 0.05 μM. Moreover, molecular docking of 10e on colchicine binding site of the tubulin explains the interaction of 10e with tubulin. Clonogenic assay indicated inhibition of colony formation by compound 10e in a dose dependent manner. In addition, morphological changes were clearly observed by AO/EB and DAPI staining studies. Moreover, ROS detection using DCFDA, JC-1, and annexin V-FITC assays demonstrated the significant apoptosis induction by 10e.

The Structural Function of Nestin in Cell Body Softening is Correlated with Cancer Cell Metastasis.

Intermediate filaments play significant roles in governing cell stiffness and invasive ability. Nestin is a type VI intermediate filament protein that is highly expressed in several high-metastatic cancer cells. Although inhibition of nestin expression was shown to reduce the metastatic capacity of tumor cells, the relationship between this protein and the mechanism of cancer cell metastasis remains unclear. Here, we show that nestin softens the cell body of the highly metastatic mouse breast cancer cell line FP10SC2, thereby enhancing the metastasis capacity. Proximity ligation assay demonstrated increased binding between actin and vimentin in nestin knockout cells. Because nestin copolymerizes with vimentin and nestin has an extremely long tail domain in its C-terminal region, we hypothesized that the tail domain functions as a steric inhibitor of the vimentin-actin interaction and suppresses association of vimentin filaments with the cortical actin cytoskeleton, leading to reduced cell stiffness. To demonstrate this function, we mechanically pulled vimentin filaments in living cells using a nanoneedle modified with vimentin-specific antibodies under manipulation by atomic force microscopy (AFM). The tensile test revealed that mobility of vimentin filaments was increased by nestin expression in FP10SC2 cells.

Effect of quercetin on the anti-tumor activity of cisplatin in EMT6 breast tumor-bearing mice.

ObjectiveThe purpose of this study was to determine the effect of quercetin on the antitumor activity of cisplatin and its side-effects.MethodsEMT6 cells, a mouse breast cancer cell line, were injected subcutaneously in mice to generate a breast tumor-bearing mouse model. Experimental groups were divided into four groups: control (C), quercetin (Q), cisplatin (CP), and cisplatin+quercetin (CP+Q).ResultsThe tumor volume of the CP+Q group was significantly lower than that of the CP group. Serum blood urea nitrogen and creatinine levels in the CP+Q group were lower than those in the CP group. Renal γ-glutamyltranspeptidase and alkaline phosphatase activities were significantly higher in the CP+Q group than in the CP group, and the content of renal thiobarbituric acid reactive substance was significantly lower in the CP+Q group than that in the CP group. These results suggested that quercetin and cisplatin synergistically increased cellular toxicity in breast cancer cells and mediated cancer growth inhibition, thereby enhancing the antitumor effect of cisplatin compared to when only cisplatin was administered. Quercetin also reduced renal toxicity, which arose as a potential a side effect of cisplatin.ConclusionThe enhanced antitumor effect of cisplatin and decreased renal toxicity after quercetin treatment suggested the applicability of quercetin as an adjuvant for chemotherapeutic agents.

Effect of Hydatid Cyst Fluid Antigens on Induction of Apoptosis on Breast Cancer Cells.

Background:Hydatid cyst is a zoonotic and parasitic disease with worldwide distribution. Anticancer effects of hydatid cyst have been shown in cell culture experiments and animal model investigations. The mechanism of anti-cancer effects of hydatid cyst fluid has not been clearly elucidated, and the induction of apoptosis may have a role in this regard. Hence, in this study, the effect of hydatid cyst fluid (HCF) on the induction of apoptosis on mouse breast cancer (4T1) cell line in cell culture medium has been investigated.Materials and Methods:Echinococcus granulosus HCF antigens including Antigen B (AgB), glycolipid, glycoprotein, and 78 KDa fractions were prepared. Breast cancer cell line (4T1) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and appropriate antibiotics. Apoptosis was determined by flow cytometry using the annexin V-fluorescein isothiocyanate apoptosis kit.Results:The 78 KDa and glycoprotein fractions induced more than 40% apoptosis. HCF and glycolipid antigens induced 39% and 34% apoptosis, respectively. However, less apoptosis observed after treatment with AgB fraction.Conclusion:Hydatid cyst antigens especially the 78 KDa and glycoprotein fractions induced apoptosis on mouse breast cancer cells.

Core needle biopsy promotes lung metastasis of breast cancer: An experimental study.

Core needle biopsy (CNB) may be used to diagnose early-stage breast cancer, but it may increase the risk of distant metastasis of tumor cells. The aim of the present study was to explore the effect of CNB on the distant metastasis of breast cancer. A total of 30 BALB/c mice were divided into two groups, namely biopsy and non-biopsy groups. The biopsy-related lung metastasis model (biopsy group) was established by the inoculation in the mammary fat pad of the mouse breast cancer cell line 4T1 combined with CNB. Flow cytometry, quantitative polymerase chain reaction analysis, morphological analysis, as well as other techniques, were used to evaluate the biological behavior of the tumors in the mouse model. A stable and reliable lung metastasis model of breast cancer was successfully established. The number of metastatic lung nodules in the biopsy group was significantly higher compared with that in the non-biopsy group (P<0.05). Compared with the non-biopsy group, the mRNA expression of transforming growth factor (TGF)-β1, SOX4 and Ezh2 in the biopsy group was significantly upregulated (P<0.05) and the number of natural killer (NK) cells detected by flow cytometry was increased, but the difference was not statistically significant (P>0.05). Therefore, CNB was found to promote the lung metastasis of breast cancer, and the underlying mechanism may be associated with epithelial-to-mesenchymal transition (EMT) mediated by the TGF-β1 signaling pathway.

Nephronectin mediates p38 MAPK-induced cell viability via its integrin-binding enhancer motif.

Nephronectin (NPNT) is an extracellular matrix (ECM) protein involved in kidney development. We recently reported intracellular NPNT as a potential prognostic marker in breast cancer and that NPNT promotes metastasis in an integrin‐dependent manner. Here, we used reverse‐phase protein array (RPPA) to analyze NPNT‐triggered intracellular signaling in the 66cl4 mouse breast cancer cell line. The results showed that the integrin‐binding enhancer motif is important for the cellular effects upon NPNT interaction with its receptors, including phosphorylation of p38 mitogen‐activated protein kinase (MAPK). Furthermore, analysis using prediction tools suggests involvement of NPNT in promoting cell viability. In conclusion, our results indicate that NPNT, via its integrin‐binding motifs, promotes cell viability through phosphorylation of p38 MAPK.

Size-selected Fe3O4-Au hybrid nanoparticles for improved magnetism-based theranostics.

Size-selected Fe3O4–Au hybrid nanoparticles with diameters of 6–44 nm (Fe3O4) and 3–11 nm (Au) were prepared by high temperature, wet chemical synthesis. High-quality Fe3O4 nanocrystals with bulk-like magnetic behavior were obtained as confirmed by the presence of the Verwey transition. The 25 nm diameter Fe3O4–Au hybrid nanomaterial sample (in aqueous and agarose phantom systems) showed the best characteristics for application as contrast agents in magnetic resonance imaging and for local heating using magnetic particle hyperthermia. Due to the octahedral shape and the large saturation magnetization of the magnetite particles, we obtained an extraordinarily high r2-relaxivity of 495 mM−1·s−1 along with a specific loss power of 617 W·gFe−1 and 327 W·gFe−1 for hyperthermia in aqueous and agarose systems, respectively. The functional in vitro hyperthermia test for the 4T1 mouse breast cancer cell line demonstrated 80% and 100% cell death for immediate exposure and after precultivation of the cells for 6 h with 25 nm Fe3O4–Au hybrid nanomaterials, respectively. This confirms that the improved magnetic properties of the bifunctional particles present a next step in magnetic-particle-based theranostics.

Abstract B10: HDAC inhibition modulates immune checkpoint pathway in triple-negative breast cancer

Abstract Background: Breast cancer remains one of the most serious and common diseases, second only to lung cancer as a leading cause of cancer death in women, accounting for 30% of new diagnoses. In particular, hormone-sensitive metastatic breast cancer and triple-negative breast cancer (TNBC) remains a therapeutic challenge. Despite aggressive therapeutic options, resistance develops in almost all patients and survival remains poor, prompting the need for novel approaches. This project focuses on the characterization of the molecular mechanisms underlying epigenetic modifiers and in particular Histone deacetylase inhibitors (HDACi) as priming modulators of immunotherapy, mainly focusing on the diversity between TNBC and hormone responsive breast cancer. HDACi represent a new class of anticancer agents that can affect multiple cancer pathways, including reverse hormone therapy resistance, resulting in prolonged antitumor responses in patients. In addition to their effects on tumor signaling, HDACi have immune modulatory functions, including modulation of regulatory T-cells, Foxp3 expression, changes in tumor-infiltrating lymphocyte composition and induction of PD-L1 expression. The PD-1/PD-L1 pathway is an immune checkpoint inhibitor pathway that under normal conditions down-regulates cytotoxic T-cell activity to maintain immune homeostasis. Cancer cells take advantage of this pathway to suppress immune control and inhibit the anti-tumor immune response by deactivating cytotoxic T-cells in the tumor microenvironment. Breast cancer is considered a less immunogenic cancer compared to melanoma and lung cancer. PD-L1 expression is mainly found in TNBC, HER2+, ER- and PR- tumors. Increased PD-L1 expression correlates with higher tumor infiltrating lymphocytes and those data together correlate with better response in breast cancer patients. PD-1/PD-L1 pathway represents one of the primary immunosuppressive drivers in multiple types of cancer. Thus, inhibiting PD-1/PD-L1 interactions may prevent T-cell suppression and reactivate immune-surveillance. Methods and Results: Evaluating PD-L1 basal expression in a different set of human and mouse breast cancer cell lines by Western blotting and real-time PCR, we found TNBC and HER2+ cells express the highest PD-L1 levels. Testing different epigenetic modifiers, we found that HDACi were able to up-regulate PD-L1 mRNA and protein expression in a time-dependent manner up to 72 hours in the TNBC MDA-MB231 cells. Co-culturing tumor cells with human peripheral blood mononuclear cell (PBMCs), we performed comprehensive flow cytometric immunophenotyping to define the role of epigenetic priming in promoting immune cell activation. We found that HDACi upregulate PD-L1 and HLA-DR expression on MDA-MB231 tumor cells independently from PBMCs presence. Opposite effects were observed with hormone responsive ER+ MCF-7 cells, where PD-L1 was not significantly increased and HLA-DR was down-regulated upon HDACi treatment. These effects were associated with reduced Foxp3 expression on CD4+ Tregs in vitro after HDACi treatment. Importantly, HDACi significantly enhanced the in vivo response to PD-1 and CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model. This was associated with increased tumor infiltrating T cells, increased survival, and tumor growth inhibition in vivo. Conclusion: Overall, this study suggests that the combination of HDACi with immune checkpoint inhibitors identify a novel therapeutic strategy and warrants further clinical evaluation for the treatment of TNBC. Citation Format: Manuela Terranova Barberio, Scott Thomas, Niwa Ali, Nela Pawlowska, Jeenah Park, Michael D. Rosenblum, Alfredo Budillon, Pamela N. Munster. HDAC inhibition modulates immune checkpoint pathway in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B10.

Abstract B084: Trop-2 plasticity is driven by epithelial-to-mesenchymal transition in prostate cancer cells

Abstract Dissemination of cancer cells to distant organs has fatal consequences to most of the patients with malignant tumors. Patients with prostate and breast cancer show apparent overlap of the most common sites of cancer metastasis, suggesting that breast and prostate tumor cells share common mechanisms of dissemination and colonization. During each step of prostate and breast cancer metastasis, malignant cells display phenotypic plasticity that is associated with the manifestation of epithelial and mesenchymal properties or an epithelial-to-mesenchymal transition (EMT). One of the molecules that most likely interlink processes of pathologic plasticity of cancer cells, their dissemination capability, and response to microenvironmental factors is Tumor-Associated Calcium Signal TransDucer 2 (Trop-2, TACSTD2). Trop-2 is a type-I transmembrane glycoprotein encoded by TACSTD2 gene often associated with tumorigenesis and cancer progression, but the data remain controversial. Trop-2 deregulation has been repeatedly proposed as an event associated with cancer progression and poor patient prognosis. Instead of this simplistic view, our results showed Trop-2 level change as a context-dependent, dynamic event associated with cancer plasticity and dissemination. Using antibody-based surface profiling of selected cancer stem-like cell markers in human and mouse prostate and breast cancer cell lines, we identified subpopulation of Trop-2+ cells within culture of metastatic prostate cell line DU-145 and similarly Trop-2+ subpopulation within mouse mammary cancer cell line 4T1. Gene expression analysis of sorted subpopulations showed significant correlation of Trop-2 with epithelial phenotype, and this finding was further validated in wide panel of human and murine cell lines and independent patient datasets. We further proved that expression of Trop-2 is regulated by EMT transcription factors and DNA methylation. Moreover, immunohistochemical analysis of Trop-2 in pairs of primary prostate tumors and lymph node metastasis showed strong association with E-cadherin and epithelial-mesenchymal plasticity in patient samples. In conclusion, we showed that Trop-2 expression associates with epithelial phenotype and can be suppressed either epigenetically or through the action of EMT master regulators. Acknowledgments: This work was supported by Ministry of Health of the Czech Republic, grant no. 15-33999A and 15-28628A, and by GAČR 15-11707S, HistoPARK (CZ.1.07/2.3.00/20.0185), and by project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123) and ICRC-ERA-HumanBridge a.k.a. REGPOT (Grant agreement no. 316 345) from the European Regional Development Fund. Citation Format: Ján Remšík, Lucia Binó, Zuzana Kahounová, Gvantsa Kharaishvili, Sarka Simeckova, Radek Fedr, Tereza Nehybova, Eva Slabáková, Lucia Knopfová, Jan Bouchal, Milan Kral, Petr Benes, Karel Soucek. Trop-2 plasticity is driven by epithelial-to-mesenchymal transition in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B084.

Regulation of the oncogenic phenotype by the nuclear body protein ZC3H8

BackgroundThe Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer.MethodsWe used RNA silencing to decrease levels of expression in mouse mammary tumor cells and overexpression of ZC3H8 in cells derived from the normal mouse mammary gland. We measured characteristics of cell behavior in vitro, including proliferation, migration, invasion, growth in soft agar, and spheroid growth. We assessed the ability of these cells to form tumors in syngeneic BALB/c mice. ZC3H8 protein was visualized in cells using confocal microscopy.ResultsTumor cells with lower ZC3H8 expression exhibited decreased proliferation rates, slower migration, reduced ability to invade through a basement membrane, and decreased anchorage independent growth in vitro. Cells with lower ZC3H8 levels formed fewer and smaller tumors in animals. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells led to an opposite effect. ZC3H8 protein localized to both PML bodies and Cajal bodies within the nucleus. ZC3H8 has a casein kinase 2 (CK2) phosphorylation site near the N-terminus, and a CK2 inhibitor caused the numerous PML bodies and ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restored PML bodies to their original state. A mutant ZC3H8 lacking the predicted CK2 phosphorylation site showed localization and numbers of ZC3H8/PML bodies similar to wild type. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable threonine showed dramatically increased numbers of smaller nuclear foci.ConclusionsThese experiments demonstrate that Zc3h8 expression contributes to aggressive tumor cell behavior in vitro and in vivo. Our studies show that ZC3H8 integrity is key to maintenance of PML bodies. The work provides a link between the Little Elongation Complex, PML bodies, and the cancer cell phenotype.

TPC2 mediates autophagy progression and extracellular vesicle secretion in cancer cells

Autophagy is an evolutionarily conserved lysosomal degradation process, and is involved in various cellular processes. Here we studied the role of two pore channel 2 (TPC2), a lysosomal non-selective Na+/Ca2+ channel, in autophagy progression. We found that TPC overexpression in 4T1 mouse breast cancer cell line or in HeLa human cervical cancer cell line inhibited the fusion between autophagosome and lysosome, resulting in the accumulation of autophagosomes accompanied with increased lysosomal pH and TFEB nuclear localization. Interestingly, we also found that extracellular vesicle (EV) secretion was markedly decreased in TPC2 overexpressing cells but was induced in TPC2 knockdown cells. In addition, migration of TPC2 knockdown cells, not TPC2 overexpressing cells, was inhibited. Taken together, these results support a role of TPC2 in autophagy progression and EV trafficking in cancer cells.

Abstract 3101: Influence of targeted knockout of the BRCA1 gene on the pharmacologic profile of the mouse breast cancer cell line EMT6 in vitro and in vivo

Abstract Around 10% of breast cancer cases are attributed to genetic disorders like mutations in BRCA-1/2 genes. Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. To study the effect of this common mutation on sensitivity towards innovative therapies in more detail, we knocked out the BRCA1 gene in the murine EMT6 breast cancer cell line. Subsequently, we analyzed the sensitivity towards PARP as well as checkpoint (CP) inhibitors in the mutated as well as the parental line in vitro and in vivo. The EMT6/BRCA1-/- cell line was created by a CRISPR/CAS9 based removal of Exon2 of the BRCA1 gene. The homozygous clone of the modified cell line was used for drug testing in comparison to the wildtype (wt) cell line. PARP inhibitors Niraparib, Rucaparib, Talazoparib and Olaparib were investigated in EMT6/BRCA1wt as well as EMT6/BRCA1-/- in vitro in 2D as well as 3D cell culture using different cell concentrations. The latter showed higher sensitivity towards Talazoparib and Olaparib in 2D assays: The respective IC50 values were 2-4 times lower in the EMT6/BRCA1-/- as compared to EMT6/BRCA1wt. In 3D assays Niraparib, Rucaparib and Talazoparib showed pronounced activity in the EMT6/BRCA1-/- : IC50 values depicted a 2-3fold difference between the two investigated lines. To further characterize the two lines, tumor growth and sensitivity towards PARP as well as CP inhibitors was determined in vivo. Tumor growth behavior of subcutaneously implanted breast cancer cells was similar in both lines: mean doubling times were 1.9 (± 0.38) days for EMT6/BRCA1-/- and 1.62 (± 0.37) days for EMT6/BRCA1wt . Both lines showed a distinct sensitivity profile towards CP inhibitors: anti-CTLA4 was more active in EMT6/BRCA1-/- : optimal T/C (test/control) value of 22% (KO line) was accompanied by 34.9% in the parental line. In contrast, anti-PD1 treatment led to optimal T/C values of 30.9% in the wt line and 54.8% in the KO line. The evaluation of Niraparib as well as Olaparib in mono-and combined therapy with anti-CTLA4 and anti-PD1 in vivo is currently under investigation. Weekly cytokine analysis in the serum of tumor bearing mice will elucidate possible interactions between PARP and CP inhibitors and give guidance to optimal combination and schedules. We expect that further evaluation of PARP inhibitors in combination with different immune modulatory interventions in our mouse breast cancer model will increase the clinical utility of this strategy for the treatment of patients with BRCA1 mutated cancers. In general, the possibility to compare a mutated vs a parental line in vivo will help to identify novel promising combination as well as patient stratification strategies for immuno-oncology. Citation Format: Anya Avrutskaya, Cordula Tschuch, William Durham, Gerhard Kelter, Astrid Jensen, Armin Maier, Aidan Synnott, Anne-Marie Zuurmond, Julia Schüler. Influence of targeted knockout of the BRCA1 gene on the pharmacologic profile of the mouse breast cancer cell line EMT6 in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3101.

Abstract 2765: Combination ONC201 and radiation therapy in the treatment of breast cancer

Abstract Purpose: Metastatic breast cancer patients continue to have poor outcomes on current treatments, and thus, novel therapies in combination with standard treatments are needed. Towards this we studied the pre-clinical effects of combining ONC201, a first-in-class imipridone (which works via activation of ER stress, upregulation of TRAIL, and activation of death receptor 5 [DR5]) with radiation therapy (RT). Since the DR5 pathway is also required for RT-induced apoptosis, we hypothesized that addition of ONC201 could increase the effectiveness of RT. Pertinent Experimental Procedures: Human and mouse breast cancer cell lines were used to study in vitro cancer cell death (by AlamarBlue and clonogenic assays). Patient-derived breast cancer tissue and mouse cell lines were used to study in vivo tumor growth kinetics. Cell lines and mice were administered radiation (0-8 Gy) delivered via a Gammacell 40 exactor. ONC201 was given at does 0-10 µM. Mechanisms of tumor cell death were elucidated using western blots of cleaved PARP (cPARP) and caspase 3 (cC3). Immune response mechanisms were elucidated using in vivo cell-specific depletion. Summary of Data: Multiple breast cancer lines (MB231, MB468, and 4T1) exhibited decreased cell viability after combination therapy compared to either single therapy. In particular, ON201 (1 µM) followed by RT (8 Gy) resulted in a decreased MB468 breast cancer cell viability compared to no treatment (36.9%, P&amp;lt;0.0001), RT alone (30.5%, p&amp;lt;0.0001) and ONC201 alone (26.9%, p&amp;lt;0.0001). A clonogenic assay using MB468 revealed a decreased surviving fraction at every RT dose (2-8 Gy). Western blot analysis revealed increasing cPARP and cC3 with increasing ONC201 (2-8 Gy). In vivo studies utilizing 4T1 cells demonstrated that combination RT (6 Gy) and ONC201 (48 mg/kg given 3x weekly) decreases tumor growth and prolongs host survival compared to either treatment alone (P&amp;lt;0.01). Depletion of NK or CD8 T cells (but not CD4 or B cells) abrogated the positive effects of ONC201 + RT. Similar efficacy of ONC201 + RT was achieved in NSG mice surgically transplanted breast cancer patient tumor sections and immune reconstituted with autologous patient immune cells. Conclusions: Treatment with combination ONC201 + RT results in decreased in vitro breast cancer cell viability and increased in vivo cancer control. Future studies are aimed at translating this combination therapy for clinical use towards improving and saving cancer patient lives. Citation Format: Sachin R. Jhawar, Devora Schiff, Hao Wu, Aditya Thandoni, Suemair Hassan, Joshua Allen, Martin Stogniew, Rohinton Tarapore, Wolfgang Oster, Mark Stein, Ann W. Silk, Sharad Goyal, Bruce G. Haffty, Andrew Zloza. Combination ONC201 and radiation therapy in the treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2765.

Abstract 4753: Bone Morphogenetic Protein 7 is a candidate promoter of cancer immune evasion

Abstract Tumors evade immune recognition and destruction by various established mechanisms, but less is known about how tumors short-circuit successful immune cell trafficking. To identify candidate proteins that inhibit immune trafficking, we developed a bioinformatics screen that utilizes an intratumoral cytotoxic T lymphocyte (CTL) gene signature that quantifies the relative abundance of tumor infiltrating CTLs. This gene signature was used as a stratification tool to examine differential gene expression between tumors of “low” versus “high” CTL status. We hypothesized that a subset of recurrently overexpressed genes in low-CTL tumors (i.e., immunologically “cold” tumors) may functionally act to limit lymphocyte infiltration into the tumor mass. A pan-TCGA analysis of 23 solid tumor types (n=8,875 tumors) revealed a number of recurrent genes highly significantly associated with low CTL infiltration. We found that 88.5% of tumor pairwise comparisons (among the 23 tumor types) showed significant overlap of top genes associated with low CTL status, consistent with the existence of conserved, tumor-agnostic mechanisms of immune evasion. From these analyses, the secreted TGFβ superfamily member, BMP7, emerged as a top-ranking candidate regulator of immune trafficking. To investigate the role of BMP7 in CTL tumor trafficking, an immunogenic variant of the 4T1 mouse breast cancer cell line, 4T1-S, was modified using CRISPR-Cas9 to inducibly express BMP7 from the ROSA26 locus. 4T1-S BMP7-inducible cells were orthotopically transplanted into Balb/C female mice and tumors were allowed to form with or without BMP7 induction. Mice were sacrificed at 2, 3, and 4 wks post-tumor inoculation, and tumors, tumor-draining LNs (TDLNs), and spleens were harvested. At both 2 wks and 3 wks, a BMP7-dependent reduction in tumor CTLs was observed by anti-CD8a IF microscopy (p=0.002 and p&amp;lt;0.001, respectively). Flow cytometric analysis confirmed the significant reduction in tumor-infiltrating CD8+ CTLs in the BMP7-induced group at 3 wks (p=0.02). BMP7 expression did not alter tumor trafficking of natural killer (NK) cells. Flow analysis also showed CTL reduction in TDLNs at 2 wks (p=0.02), and NKs were reduced in the TDLNs at 3 wks (p=0.04). Spleen sizes were also significantly smaller at all time points, indicating a systemic immunomodulatory function of tumor-derived BMP7 (p=0.002, p&amp;lt;0.001, and p=0.002 at 2, 3, and 4 wks, respectively). This study provides a framework for discovering yet unappreciated mechanisms by which tumors evade the immune system, and presents first evidence that BMP7 expression by breast tumors can function to limit CTL trafficking. Citation Format: Eric D. Routh, Julia Chifman, Ashok Pullikuth, Lance D. Miller. Bone Morphogenetic Protein 7 is a candidate promoter of cancer immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4753.

Highlights of This Issue

Chen and colleagues analyzed next generation sequencing of 216 advanced thyroid carcinomas identifying prevalent targetable MAPK pathway activating mutations. Secondary mutations in PIK3CA which may confer resistant to single agent targeted therapy were 8x more frequent compared to the thyroid TCGA. In this retrospective analysis, when targeted therapy to actionable pathway mutations was used, an improved survival was noted in patients with poorly differentiated thyroid carcinomas compared to similar tumors lacking mutations or showing only tumor suppressor mutations. The combined findings lend support for mutational testing across targetable pathways to ultimately benefit patients with advanced thyroid carcinoma through targeted therapy.Resistance to T-DM1 poses a challenge in therapy for HER2-positive breast cancer. A better understanding of the molecular mechanisms of primary and acquired resistance to T-DM1 is particularly important for the development of new therapeutic strategies. Breed and colleagues established resistant cells and identified different features of T-DM1 resistant cells. Their findings show the complexities of T-DM1 resistance in that the 2 models they studied showed little overlap in identified resistance mechanisms. The data also suggest possible therapeutic strategies using combinations with inhibitors that target signal transduction resistance pathways for overcoming T-DM1 resistance.Brain tumors are the leading cause of cancer-related deaths in children. Hence, there is an unmet need to develop novel therapies to improve outcome. Telomerase-dependent incorporation of 6-thio-dG into telomeres leads to telomere damage and cell death of primary stem-like cells derived from pediatric patients with high-risk brain tumors. The effect of 6-thio-dG is persistent after drug withdrawal. In vivo, 6-thio-dG delays tumor growth, crosses the blood-brain barrier and specifically targets tumor cells in the mouse brain. These findings suggest that 6-thio-dG is a promising novel approach to treat therapy-resistant pediatric brain tumors.5-fluorouracil (5-FU) is often given to breast cancer patients, after curative surgery of primary tumor to prevent tumor recurrence. However, 5-FU accelerated lung metastasis, when it was administered upon the complete resection of the tumors arising from the orthotopic injection of a mouse breast cancer cell line. The acceleration was associated with enhanced intrapulmonary infiltration of neutrophils, which were attracted by tumor cell-derived chemokines and expressed abundantly prokineticin-2 with a capacity to support the proliferation of prokinectin-2 receptor-expressing breast cancer cells. Thus, adjuvant chemotherapy with 5-FU may have adverse effects on some breast cancer patients.

Abstract 5153: RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression

Abstract Background: Tumor metastasis suppressors are proteins that impede secondary tumor formation by inhibiting one or more steps of the metastasis cascade without stimulating primary tumor growth. Raf-1 kinase inhibitor protein (RKIP) is a metastasis suppressor that inhibits metastasis in breast, prostate, melanoma and several other types of cancers. The molecular mechanism through which RKIP executes its anti-metastasis effects is not yet completely defined. The objective of the current study is to understand how RKIP inhibits breast cancer cell invasion and metastasis in molecular level. Given its primary function in regulating actin cytoskeleton and cell movements, Rho GTPases were studied as possible downstream effectors of RKIP. These small GTPases belong to the Ras superfamily and consist of nearly 22 members with RhoA and Rac1 as the major players affecting cell motility. Rho GTPases oscillate between a GTP-bound active form and a GDP-bound inactive form. The GDP-GTP cycle is regulated by GEFs that facilitate the exchange of bound GDP with GTP, and by GAPs that stimulate the Rho GTPase activity. Aberrantly activated GEFs are reported to drive tumorigenesis, while activated GAPs are assumed to inhibit the cancer formation and metastasis. Hence, Rho GTPases are widely accepted as oncogenes. Contrary to this notion, several recent studies suggested a possible metastasis inhibitory function for wild-type RhoA in triple negative breast cancer and several other types of cancers. Experimental procedures and results: In this study, several mouse (4T1, 168 FARN) and human (BT20, MDA-MB 231) breast cancer cell lines were used to eliminate possible cell line specific observations. Using in vitro matrigel invasion assay, we showed that RhoA, not Rac1, acts downstream of RKIP and is needed for RKIP-mediated inhibition of breast cancer cell invasion in vitro. GTPase activity pull-down assay results revealed that RKIP specifically increases RhoA activation, thus inhibiting invasiveness of these cells. Similarly, orthotopic mice tumor implantation experiments showed that ectopic expression of dominant negative RhoA in 4T1 cells confers significantly greater proclivity to metastasize in mice. These observations are consistent with RhoA's emerging role as a metastasis suppressor. In BT20 breast cancer cells, RhoA enhanced E-cadherin expression and negatively affected the cell invasiveness. Interestingly, RKIP phenocopied the RhoA effect on E-cadherin and cell invasion, suggesting that RKIP-RhoA axis inhibits breast cancer cell invasiveness by increasing E-cadherin expression. Conclusions: Our results conclude that RKIP specifically increases RhoA activation in breast cancer cells, and this activated RhoA stabilizes E-cadherin and negatively affects the invasiveness of these cells. Citation Format: Gardiyawasam Kalpana, Vu Bach, Clariza Borile, Miranda Yeung, Rafael Garcia-Mata, Kam C. Yeung. RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5153.