MDA-231 Cells Research Articles

Abstract B099: Characterization of monoclonal antibodies generated to the 287-302 amino acid loop of the human epidermal growth factor receptor

Abstract Dysregulation of the epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of malignancies including glioblastoma and various epithelial cancers. Oncogenesis occurs from overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287-302 loop from the extracellular domain is exposed completely on EGFRvIII (deletion of exons 2-7), partially exposed on some cancers but cryptic on normal cells expressing WT levels of EGFR. We report on the generation of antibodies to this loop. A peptide corresponding to amino acids 286-303 was synthesized and then coupled chemically to Keyhole Limpet Hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V-regions were cloned and sequenced. Receptor binding was characterized using ELISA and flow cytometry. Monoclonal antibodies (n=7) reactive for the 287-302 loop were characterized further. Each one reacted with EGFRvIII but not EGFR WT. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be potently cytotoxic for MDA-468, A431 and F98-EGFRvIII-expressing cells. We conclude that immunization with a peptide corresponding to a cryptic EGFR epitope can produce cancer-binding antibodies. By way of example, the 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be developed as a therapeutic agent for clinical use. Citation Format: Eric Chun Hei Ho, Antonella Antignani, Robert Sarnovsky, David J FitzGerald. Characterization of monoclonal antibodies generated to the 287-302 amino acid loop of the human epidermal growth factor receptor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B099. doi:10.1158/1535-7163.TARG-19-B099

A cost-effective, analytical method for measuring metabolic load of mitochondria

Analysis of cellular energetics is central to understanding metabolic diseases including diabetes and cancer. The two most commonly used methods to monitor cellular respiration are the Seahorse-XF system, and Glo™ assays, which are considered “gold standards”. These commercial methods measure energetics indirectly and require considerable financial investment. Here we describe an alternative assay that enables accurate quantification of NADH turnover and that is affordable. This method measures resazurin reduction to resorufin at rising concentrations in the presence of purified mitochondrial extracts until NADH becomes a rate-limiting factor. This indicates the maximal level of NADH turnover in each sample and therefore infers metabolic activity. Here we compare MRC5, MCF7 and MDA231 cell lines which have differing metabolic profiles.

2005P - Aptamer-mediated exosomes detection for early breast cancer identification

BackgroundIncreasing evidence indicates that the release of exosomes by tumor cells play a key role in tumor progression, drug resistance, immune surveillance escape, angiogenesis, tumor invasion, and metastasis. For this reason, cancer-derived exosomes are emerging as very interesting targets for early diagnosis and therapy in cancer, including breast cancer. Indeed, progresses in developing nucleic acids-based therapeutic compounds, including antisense DNAs, aptamers, short interfering/microRNAs, and short activator RNAs, have attracted great interest as emerging platforms for precise cancer treatment. MethodsWe have recently developed a novel differential SELEX (Systematic Evolution of Ligands by Exponential enrichment) strategy by using exosomes purified from epithelial BC primary cells in the positive selection step and exosome-derived from primary normal epithelial breast cell lines in the negative selection step. ResultsThree aptamers have been shown to be enriched in different families, and their ability to selectively bind BC cells-derived exosomes has been proven. Interestingly, two of them can selectively bind triple negative-derived exosomes, compared to more differentiated BC cells. To optimize the best aptamers, shortened version of aptamers (about 35mer) have been generated and their binding ability has been tested. Moreover, the aptamers showed no binding affinity for lung cancer and glioblastoma-derived exosomes. We also tested by binding assay the ability of one of the identified aptamers, named ex-50sh, to selective recognize exosomes isolated from serum of breast cancer patients. Proteomic analysis of the putative target is in progress. Immunofluorescent experiments showed that the short aptamers can also block the uptake of MDA-231-derived exosomes on MDA-231 cell lines. Moreover, ex-50sh is able to block the EMT transformation induced by MDA-231 exosomes in low malignant MCF7. ConclusionsAltogether, the selected aptamers will provide new insights in the molecular characterization of breast cancer exosomes and, most importantly, innovative tools for early breast cancer diagnosis. Legal entity responsible for the studyGerolama Condorelli. FundingUniversity of Naples Federico II. DisclosureAll authors have declared no conflicts of interest.

Abstract 2091: Lysosomal targeting of doxorubicin induces different membrane permeabilization and cytotoxicity in two breast cancer cell lines

Abstract Background: Lysosomal targeting has been investigated for the treatment of cancer but details of the resulting cytotoxicity remain unclear. The induced lysosomal membrane permeabilization (LMP) has been shown to induce apoptosis and cytotoxicity. The purpose of this study is to elucidate details of doxorubicin (DOX) induced LMP in breast cancer cells and identify correlations with cytotoxicity. Methods: A gelatin – doxorubicin conjugate (GDOX) to target lysosomes was synthesized by EDC chemistry in formamide and purified by precipitation and desalting. GDox and free Dox uptake studies at 10 µM or its equivalent were conducted in MCF7 and MDA-MB-231 (triple negative breast cancer-TNBC) cells in growth media. Intracellular localization was followed by fluorescent markers. LMP was shown by cytosolic release of 10 kD Dextran-Alexa Fluor 488 previously loaded into lysosomes. Intra-lysosomal release of Dox from GDOX was examined by rupture of lysosomes and nuclear accumulation of released Dox followed by fluorescent microscopy and ImageJ. Released Dox accumulation in the nucleus without lysosome rupture was analyzed by UHPLC. Viability, growth inhibition profiles, and IC50 values were determined by the MTT procedure and GraphPad Prism 7. Results: GDox localized substantially to the lysosomes in both MCF7 and TNBC cells while free DOX localized to the nucleus, as expected. GDOX induced LMP by 24 hrs in 100% of the TNBC cells but only 20% of the MCF7 cells even up to 48 hrs. Intra-lysosomal DOX release in the MCF7 cells was notable only by 24 hrs and was estimated by fluorescent intensity (300,000 integrated density units per cell) at one-half the amount of free DOX accumulation in the nucleus. Nuclear accumulation of released DOX escaping intact lysosomes in MCF7 cells by 24 hrs was one-half that at 48 hrs which reached 0.13 µg Dox per 106 cells by 48 hrs representing only one-tenth that achieved by free DOX. MCF7 cell viability in GDOX was reduced to 62±3% of controls by 24 hr but reached 19±4% by 48 hrs compared to 11±1% for free Dox. The IC50 values of GDOX after 48 hr were 1.3±0.7 µM and 4.5±2 µM in TNBC and MCF7 cells, respectively. Conclusions: Taken together these results indicate that LMP is not induced by pooling of released DOX in MCF7 lysosomes nor does LMP contribute to cytotoxicity in these cells. The lysosomal accumulation of GDOX in MDA cells, in contrast to MCF7 cells, leads to extensive LMP which may contribute to the greater cytotoxicity in these cells compared to the MCF7 cells. The small amount of released DOX accumulating in the MCF7 nuclei may induce a nuclear pathway of cytotoxicity in these cells. Acknowledgements: Financial support is appreciated from the Agnes Varis Trust for Women’s Leadership and Health, King Abdul-Aziz University, and NIH R15 CA135421. Citation Format: Mohammed Alvi, Rachel Nicoletto, Bayan A. Eshmawi, Clyde M. Ofner. Lysosomal targeting of doxorubicin induces different membrane permeabilization and cytotoxicity in two breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2091.

Abstract 4518: IL-4/IL-13 stimulated tumor-associated macrophages enhance breast cancer cell invasion through Rho-GTPase signaling

Abstract The high metastatic potential of inflammatory and other aggressive breast cancer is the major determinant of mortality. Recently, it has become clear that signals from the tumor microenvironment (TME) harbor the capacity to enhance cancer cell dissemination. Of the various cells that comprise the TME, macrophages are the most abundant in solid tumors. Therefore, we aimed to better understand the relationship between macrophage-breast cancer cell crosstalk. Intriguingly, in our previous work we showed that inflammatory breast cancer (IBC) cells are hyper-migratory in response to macrophage conditioned media. Additionally, we found this process is regulated by the Rho-GTPase, RhoC. Recent advances in tumor associated macrophage (TAM) biology have led to the understanding that TAM populations in the TME are diverse, primarily due to directional differentiation promoted by unique secreted components from the various cells in the TME. Therefore, this study aimed to understand which subpopulation of M2-like TAMs had the greatest impact on breast cancer cell aggressiveness. Our data show that stimulation of the triple negative breast cancer (TNBC) cell model, MDA-MB-231, and the IBC TNBC cell line, SUM-149, with media extracts from IL-4/IL-13 stimulated M2 macrophages (M2a) elicit the strongest migratory and invasive responses. Subsequently, we stimulated RhoA or RhoC knockout (CRISPR) MDA-231 and SUM-149 cells with M2a conditioned media to address their role in regulating migratory/invasive responses. In brief, we find that RhoA and RhoC, in part, regulate M2a-TAM induced responses. Secretome analysis of M2a-TAM conditioned media reveals high levels of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 18 (CCL-18). Functional studies suggest that VEGF and CCL-18 synergistically enhance cellular invasiveness. Moreover, pretreatment with the ROCK inhibitor Y-27632 or GSK429286A drastically inhibited VEGF, CCL-18, or M2a induced migratory responses. These data suggest that the Rho-GTPases regulate M2a-mediated breast cancer cell invasion and potentially offer a novel approach for the treatment of metastatic breast cancer through potential prevention of progression. Citation Format: Andrew C. Little, Pragathi Pathanjeli, Zhifen Wu, Liwei Bao, Laura Goo, Joel A. Yates, Sofia D. Merajver. IL-4/IL-13 stimulated tumor-associated macrophages enhance breast cancer cell invasion through Rho-GTPase signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4518.

Abstract 4433: Novel and highly selective CDK9 inhibitors suppress proliferation of triple negative breast cancer (TNBC) cells in vitro

Abstract This study evaluates the efficacy of two newly developed selective CDK9 inhibitors (CDK9i) across a panel of TNBC cell lines. MDA-MB-453, MFM-223, MDA-MB-468 and MDA-MB-231 TNBC cells were treated with increasing concentrations of two novel and highly selective CDK9 inhibitors and the effect on proliferation, apoptosis and expression of CDK9 targets determined. MDA-MB-453 and -468 cells showed significant growth inhibition with as little as 150nM of CDK9i, evident 3 days after commencement of treatment. Both MDA-MB-231 and MFM-223 cells were less sensitive to the CDK9 inhibitors, with MDA-231 cells requiring at least 300nM to suppress growth. MFM-223 cells did not demonstrate any growth inhibition after 7 days of culture with CDK9i concentrations up to 1.2uM. Protein expression of CDK9 targets, including RNA Polymerase II (RNAPII), phosphorylated-RNAPII, the proto-oncogene C-MYC, and apoptotic marker cleaved caspase-3, were examined by Western blot after optimal CDK9i exposure across each cell line. CDK9i suppressed phosphorylated-RNAPII, but not total RNAPII, indicative of targeted CDK9 inhibition. The master transcription factor C-MYC, which is highly expressed in TNBC, was downregulated, and cleaved-caspase-3 was upregulated with CDK9i treatment. These data demonstrate cell specific efficacy of novel CDK9 inhibitors in cell line models of TNBC via transcriptional suppression of proto-oncogenes and upregulation of apoptotic pathways. Future studies will identify molecular markers of response to CDK9 inhibition and evaluate these novel inhibitors in TNBC patient derived xenografts. Citation Format: Jean M. Winter, Ebtihal H. Mustafa, Shudong Wang, Luke A. Selth, Theresa E. Hickey, Wayne D. Tilley. Novel and highly selective CDK9 inhibitors suppress proliferation of triple negative breast cancer (TNBC) cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4433.

Abstract 1525: Kaiso influences immune signaling of breast cancer exosomes

Abstract Introduction: Exosomes are communication vesicles between tumor cells and immune cells. However, the mechanism underlining this cell-cell communication is not well understanding, particularly in African American (AA) breast cancer patients. Kaiso, a bi-modal transcription factor is highly expressed in AA patients and high Kaiso expression correlates with aggressiveness and the disparity in survival outcomes compared to European American (EA) patients. However, the biological consequences of Kaiso in immune signaling of breast cancer exosomes has not been studied. Herein we demonstrate the biological role of Kaiso in immune signaling in breast cancer exosomes. Methods: We utilized Nanostring immune profiling technology along with multiple in vitro and in vivo assays to study the role of Kaiso in breast cancer immune escape. Results: Nanostring pan cancer immune profiling showed that EA breast cancer exosomes exhibited higher expression of TILs markers, T cell activation markers and CD8+T Cells markers compared to AA, while we observed an increase in the expression of anti-phagocytic molecule CD47 in breast cancer patient exosomes of AA compared to EA. In addition to that CD47 and SIRP-α (Signal Regulatory Protein) are highly expressed in Kaiso-scrambled MDA-MB-231 cells (sh-SCR) and exosomes, whereas THBS1, which is a regulator of CD47 expression and is regarded as angiogenesis inhibitor is significantly increased in sh-Kaiso MDA-231 cells and exosomes. Additionally, we observed that Kaiso directly binds methylated sequences in the promoter region of CD47 and THBS1 by ChIP assay. Furthermore, in vivo sh-Kaiso cells injected into athymic mice exhibited delayed tumor formation after four weeks with smaller tumor size as compared to sh-SCR cells, and we observed higher expression of THBS1 with lower expression of CD47 and SIRP-α molecules by IHC and exosomes isolated from invivo tumors, indicating that Kaiso is associated with macrophage mediated immune escape. Conclusion: Our findings demonstrate the role of kaiso in immune signaling through exosomes which may be related with more aggressive cancer phenotype in breast cancer specially in African Americans. Citation Format: Md Shakir U. Ahmed, Brittany Jenkins, Benjamin Adu-Addai, Balasubramanyam Karanam, Melissa B. Davis, William E. Grizzle, Honghe Wang, Clayton C. Yates. Kaiso influences immune signaling of breast cancer exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1525.

Association of SOX9 expression with sensitivity to CDK7 inhibition and overall survival in triple-negative breast cancer.

1065 Background: Triple-negative breast cancer (TNBC) has been shown to display cyclin-dependent kinase 7 (CDK7)-dependent transcriptional addition and is sensitive to a CDK7 inhibitor THZ1. As tumor heterogeneity is well known, we sought to identify predictive markers of sensitivity to CDK7 inhibition in TNBC. Methods: Published mRNA microarray data was analyzed to identify genes associated with sensitivity of TNBC cells to the CDK7 inhibitor THZ1. Cell survival assay after THZ1 treatment was performed using Cell Counting Kit-8. Expression of SOX9 was examined by Western blot in cell lines and IHC in breast cancer tissue microarray. Patient survival analysis was done using the Kaplan-Meier method. Results: Analysis of published mRNA microarray data showed that SOX9 was overexpressed in the MDA-468 and BT-549 TNBC cell lines, which were sensitive to the CDK7 inhibitor THZ1 and showed decreased SOX9 expression with THZ1 treatment. We treated 10 TNBC cell lines with various concentrations of THZ1 and found that these cell lines displayed a wide spectrum of sensitivity to THZ1, with an IC50 ranging from 20-30 nM in sensitive lines (MDA-468 and MDA-231) to ~300 nM in insensitive lines (BT-20 and MDA-157). There was a strong correlation of SOX9 expression by Western blot and sensitivity to THZ1 in these cell lines (Pearson correlation, r = 0.82, P= 0.01). IHC of two tissue microarrays containing 278 breast cancer samples showed that SOX9 expression was significantly higher in TNBC (n = 59) compared to hormone positive breast cancer (n = 173; P< 0.01). Among the 59 TNBC cases, 43 had high SOX9 expression and 16 had low SOX9 expression. Patients with high or low SOX expression had similar grade, T stage and nodal involvement (All P> 0.20). Finally, with standard of care treatment, TNBC patients with high SOX9 expression had a significantly worse overall survival (OS) compared to patients with low SOX9 expression (2 year OS rate 100% vs 92.9%, 5 year OS rate 93.8% vs 83.3%, log rank P= 0.03). Conclusions: SOX9 expression level in TNBC cell lines was associated with sensitivity to the CDK7 inhibitor THZ1. TNBC patients with high expression of SOX9 had worse OS, and may benefit from CDK7 inhibition as a novel therapy.

The Laminin-α1 Chain-Derived Peptide, AG73, Binds to Syndecans on MDA-231 Breast Cancer Cells and Alters Filopodium Formation.

Breast cancer is one of the most common forms of cancer affecting women in the United States, second only to skin cancers. Although treatments have been developed to combat primary breast cancer, metastasis remains a leading cause of death. An early step of metastasis is cancer cell invasion through the basement membrane. However, this process is not yet well understood. AG73, a synthetic laminin-α1 chain peptide, plays an important role in cell adhesion and has previously been linked to migration, invasion, and metastasis. Thus, we aimed to identify the binding partner of AG73 on breast cancer cells that could mediate cancer progression. We performed adhesion assays using MCF10A, T47D, SUM1315, and MDA-231 breast cell lines and found that AG73 binds to syndecans (Sdcs) 1, 2, and 4. This interaction was inhibited when we silenced Sdcs 1 and/or 4 in MDA-231 cells, indicating the importance of these receptors in this relationship. Through actin staining, we found that silencing of Sdc 1, 2, and 4 expression in MDA-231 cells exhibits a decrease in the length and number of filopodia bound to AG73. Expression of mouse Sdcs 1, 2, and 4 in MDA-231 cells provides rescue in filopodia, and overexpression of Sdcs 1 and 2 leads to increased filopodium length and number. Our findings demonstrate an intrinsic interaction between AG73 in the tumor environment and the Sdcs on breast cancer cells in supporting tumor cell adhesion and invasion through filopodia, an important step in cancer metastasis.

Regulation of death receptor signaling by the autophagy protein TP53INP2.

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway.

Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer

The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the uptake mechanisms by which cancer cells acquire vitamin C has remained unclear. The aim of this study is to characterize how breast cancer cells acquire vitamin C. For this, we determined the expression of vitamin C transporters in normal and breast cancer tissue samples, and in ZR-75, MCF-7, MDA-231 and MDA-468 breast cancer cell lines. At the same time, reduced (AA) and oxidized (DHA) forms of vitamin C uptake experiments were performed in all cell lines.We show here that human breast cancer tissues differentially express a form of SVCT2 transporter, that is systematically absent in normal breast tissues and it is increased in breast tumors. In fact, estrogen receptor negative breast cancer tissue, exhibit the most elevated SVCT2 expression levels. Despite this, our analysis in breast cancer cell lines showed that these cells are not able to uptake ascorbic acid and depend on glucose transporter for the acquisition of vitamin C by a bystander effect. This is consistent with our observations that this form of SVCT2 is completely absent from the plasma membrane and is overexpressed in mitochondria of breast cancer cells, where it mediates ascorbic acid transport. This work shows that breast cancer cells acquire vitamin C in its oxidized form and are capable of accumulated high concentrations of the reduced form. Augmented expression of an SVCT2 mitochondrial form appears to be a common hallmark across all human cancers and might have implications in cancer cells survival capacity against pro-oxidant environments.

Long non-coding RNA ZEB2-AS1 promotes the proliferation, metastasis and epithelial mesenchymal transition in triple-negative breast cancer by epigenetically activating ZEB2.

The triple‐negative breast cancer is the most malignant type of breast cancer. Its pathogenesis and prognosis remain poor despite the significant advances in breast cancer diagnosis and therapy. Meanwhile, long noncoding RNAs (LncRNAs) play a pivotal role in the progression of malignant tumors. In this study, we found that LncRNA‐ZEB2‐AS1 was dramatically up‐regulated in our breast cancer specimens and cells (MDA231), especially in metastatic tumor specimens and highly invasive cells, and high lncRNA‐ZEB2‐AS1 expression is associated with clinicopathologic features and short survival of breast cancer patients. LncRNA‐ZEB2‐AS1 promotes the proliferation and metastasis of MDA231 cells in SCID mice. Thus, it is regarded as an oncogene in triple‐negative breast cancer. It is mainly endo‐nuclear and situated near ZEB2, positively regulating ZEB2 expression and activating the epithelial mesenchymal transition via the PI3K/Akt/GSK3β/Zeb2 signaling pathway. Meanwhile, EGF‐induced F‐actin polymerization in MDA231 cells can be suppressed by reducing lncRNA‐ZEB2‐AS1 expression. The migration and invasion of triple‐negative breast cancer can be altered through cytoskeleton rearrangement. In summary, we demonstrated that lncRNA‐ZEB2‐AS1 is an important factor affecting the development of triple‐negative breast cancer and thus a potential oncogene target.

Abstract P2-01-08: The potential role of CTNND1 (catenin (cadherin-associated protein), Delta 1) in breast cancer bone metastasis

Abstract Background: The bone is a frequently visited site by breast cancer cells. Most women who die of metastatic breast cancer would already have bone metastases, whether they are micro- or macro-metastases. Metastatic bone metastasis from breast cancer is mostly osteolytic, with reasons unclear and little in vitro and in vivo studies exploring the osteolytic nature of bone metastasis. In the present study, we investigated the potential role of CTNND1, Catenin (Cadherin-Associated Protein) Delta 1, in the context of bone metastasis of breast cancer. Materials and Methods: In order to identify potential genes involved in bone metastasis, we established a novel in vitro model named Bone Matrix Extract (BME) which was extracted from human femur and used to mimic the bone environment. Full profile of gene expression in response to BME was conducted using Ampliseq technology. Potential genes associated with bone metastasis was examined in a clinical breast cohort containing both cancer and normal tissues (n = 103), collected immediately following surgery. Gene transcript levels were quantified using QPCR and analysed against patient's pathological information and clinical outcome. We generated a series of cell models by knocking down and over-expressing one of the most relevant genes, CTNND1, using siRNA, sh-RNA, ribozyme transgenes and insertion of full coding sequence containing plasmids. Function assays including Matrigel based-adhesion, cancer cell-osteobalstic cell contact, proliferation, transwell invasion and migration were used to investigate the changes of biological features after interfering with CTNND1 expression in relation to BME / co-culture models. Results: CTNND1 was down regulated in all breast epithelial cells following BME treatment at both mRNA and protein level. From the clinical cohort, we found that compared with benign tissue, breast cancer tissues had significantly decreased CTNND1 transcript expression. Reduced CTNND1 was associated with advanced TNM stage and poor distant metastasis, local recurrence and bone metastasis. We went on to knockdown CTNND1 by siRNA, ribozyme as well as lenti-shCTNND1 transfection in MCF-10A and MDA-231 cells and overexpressed CTNND1 in MCF-7 cells. In vitro study demonstrated that knockdown of CTNND1 expression led to decreased capacity of Matrigel-adhesion, migration and invasion but increased cancer cell-osteobalstic cell adhesion. No effects were observed on cell proliferation after altering CTNND1 expression, in the presence or absence of BME. Conclusions: In this initial study on CTNND1 in breast cancer, our current data suggests that lower transcript expression of CTNND1 associates with a poorer patient prognosis. CTNND1 reduction may play a role in the progression of breast cancer bone metastasis. Citation Format: Gong C, Andrew A, Feng Y, Owen S, Liang G, Davies E, Song E, Jiang W. The potential role of CTNND1 (catenin (cadherin-associated protein), Delta 1) in breast cancer bone metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-08.

Abstract P4-06-07: Activity of nivolumab alone or in combination with targeted therapies in a humanized BLT-mouse model of human breast cancer

Abstract Background: Recent advances in the field of cancer immunotherapy have increased demand for reliable preclinical models to inform patient selection and rational drug combination strategies. The development of the bone marrow-liver-thymus (BLT) mouse may provide the opportunity to study the complex interactions of human tumor and host immune systems in vivo. Other models are limited by the rapid onset of graft versus host disease (GVHD) and a lack of orderly maturation and trafficking of human T and B cells. In BLT mice, implantation of human fetal liver and thymus fragments beneath the kidney capsule of NSG (NOD/SCID/IL-2RΥ-/-) mice followed by engraftment of matched ex vivoexpanded CD34+ cells supports the production of an almost complete human immune system. In this study, we used this model to assess the efficacy of the anti-PD-1 therapeutic antibody, nivolumab, in combination with targeted therapeutics in specific breast cancer sub-types. Materials and Methods: For triple negative breast cancer (TNBC), the activity of nivolumab was assessed in combination with the PARP1/2 inhibitor, talazoparib, in humanized BLT mice. Xenografts were established by subcutaneous injection of 5.0x106MDA-231 (TNBC) cells. Mice (n=5) were randomized into treatment groups as follows; 1) Vehicle control (PBS), 2) nivolumab (10mg/kg QW), 3) talazoparib (0.33mg/kg Q5/2D) and 4) nivolumab+talazoparib. After 21 days of treatment, tumor tissue, serum and PBMCs were collected for biomarker analysis. Results: Successful reconstitution of mature human T and B cells was confirmed in BLT mice 12-weeks post engraftment of donor tissue and CD34+ hematopoietic stem cells. MDA-231 cells injected subcutaneously into the flank of these mice formed palpable tumors (150-200mm3) within 9 days of injection. For vehicle control treated mice, tumors grew (2.5-fold) throughout the 21-day study. Single agent nivolumab induced significant tumor growth inhibition (TGI) relative to vehicle control treated mice at Day 21. Single agent talazoparib also induced comparable levels of TGI as did the combination of nivolumab plus talazoparib. Nivolumab treated mice continued to gain weight throughout the study without overt signs of toxicity. Reversible weight loss was observed in the talazoparib and combination treated arms. Overt signs of GVHD were not observed in any of these animals. Preliminary tissue analysis identified high levels of cell surface PD-L1 protein in control treated MDA-231 xenografts. Further analysis of the treated tumors will provide valuable insight into the mechanism of action of this class of molecule. We are establishing xenograft models of hormone receptor (ER+) positive breast cancer to measure the activity of nivolumab+/-CDK4/6-inhibition in humanized BLT mice and these data will also be presented. Discussion: The data presented here highlight the potential of the PD-1 antibody nivolumab to have activity in TNBC. Furthermore, these findings illustrate the potential of the humanized BLT-mouse to model responses to immune check-point in the preclinical setting. Expanded use of this model may help to identify response biomarkers and inform design of combination therapies using immune oncology molecules and approved targeted therapies. Citation Format: O'Brien NA, Luo T, Ayala R, Salgar S, Conklin D, McDermott M, Kitchen S, Rezek V, Horak C, Dugan U, Slamon DJ. Activity of nivolumab alone or in combination with targeted therapies in a humanized BLT-mouse model of human breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-07.

Inertial focusing of cancer cell lines in curvilinear microchannels

Circulating tumor cells (CTCs) are rare cancer cells, which originate from the primary tumors and migrate to the bloodstream. Separation of CTCs from blood is critical because metastatic CTCs might hold different genomic and phenotypic properties compared to primary tumor cells. In this regard, accurate prognosis and effective treatment methods are necessary. For this purpose, focusing biological particles and cells using microfluidic systems have been implemented as an efficient CTCs enumeration and enrichment method. Passive, continuous, label-free and parallelizable size-dependent focusing based on hydrodynamic forces is preferred in this study to sort cancer cells while avoiding cell death and achieving high throughput. The focusing behavior of MDA-MB-231 (11–22 μm), Jurkat (8–17 μm), K562 (8–22 μm), and HeLa (16–29 μm) was examined with respect to different Reynolds numbers and Dean numbers. The effect of curvature on cell focusing was carefully assessed. The focusing positions of the cells clearly indicated that isolations of MDA cells from MDA-Jurkat cell mixtures as well as of HeLa cells from HeLa-Jurkat cell mixtures were possible by using the curvilinear channels with a curvature angle of 280° at the Reynolds number of 121. © 2019 The Author(s)

KIF11 Functions as an Oncogene and Is Associated with Poor Outcomes from Breast Cancer

PurposeThe study aimed to search and identify genes that were differentially expressed in breast cancer, and their roles in cancer growth and progression.Materials and MethodsThe Gene Expression Omnibus (Oncomine) and The Cancer Genome Atlas databases (https://cancergenome.nih.gov/) were screened for genes that were expressed differentially in breast cancer and were closely related to a poor prognosis. Gene expressions were verified by quantitative real-time polymerase chain reaction, and genes were knocked down by a lentivirus-based system. Cell growth and motility were evaluated and in vivo nude mice were used to confirm the in vitro roles of genes. Markers of epithelial-to-mesenchymal transition and the associations of KIF11 with the classical cancer signaling pathways were detected by Western blot.ResultsA series of genes expressed differentially in patients with breast cancer. The prognosis associated with high KIF11 expression was poor, and the expression of KIF11 increased significantly in high stage and malignant tumor cells. Inhibiting KIF11 expression in lentivirus-suppressed cells revealed that KIF11 inhibition significantly reduced cell viability and colony formation, inhibited migration and invasion, but promoted apoptosis. The sizes and weights of KIF11-inhibited tumors in nude mice were significantly lower than in the negative controls. Western blot showed that E-cadherin in breast cancer was significantly upregulated in KIF-inhibited cells and tumor tissues, whereas N-cadherin and vimentin were significantly downregulated. BT549 and MDA231 cells with KIF11 knockdown exhibited decreased ERK, AMPK, AKT, and CREB phosphorylation.ConclusionKIF11 acts as a potential oncogene that regulates the development and progression of breast cancer.

Engineering synucleinopathy-resistant human dopaminergic neurons by CRISPR-mediated deletion of the SNCA gene.

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory‐generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host‐to‐graft transfer of α‐synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α‐synuclein, in a clinical‐grade hESC line to generate SNCA +/− and SNCA −/− cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α‐synuclein preformed fibrils (PFFs) to seed the formation for Lewy‐like pathology as measured by phosphorylation of serine‐129 of α‐synuclein (pS129‐αSyn). Wild‐type neurons were fully susceptible to the formation of protein aggregates positive for pS129‐αSyn, while SNCA +/− and SNCA −/− neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n‐mediated gene editing confers a measure of resistance to Lewy pathology.

Reversal of Chemoresistance in Leukemia Cells Using Synthetic Bisbenzylisoquinoline Derivatives

IntroductionChemoresistance represents a major problem for the treatment of leukemia patients, which frequently manifests in the multidrug resistance (MDR) phenotype1. The drug refractoriness to structurally related and unrelated drugs can be caused by the P-glycoprotein (P-gp), an efflux pump belonging to the superfamily of ATP-binding cassette (ABC) proteins1, 2. Multiple large clinical studies have evaluated the effectiveness of potential P-gp inhibitors, but all of them failed clinically, except for the alkaloid tetrandrine (TET)3. Given the high structural complexity and the pulmonary toxicity of the plant-derived TET in animal studies4, we are seeking to develop new bisbenzylisoquinoline derivatives (BBIQDs) with similar potency and reduced toxicity to overcome P-gp-mediated chemoresistance in leukemia.MethodsCalcein-AM, Cell-TiterBlue® cell viability and propidium iodide cell death were performed as described by the manufacturers. TUNEL assays were conducted as described previously5. For the assessment of in vivo toxicity, female C57BL/6-Tyr mice (Envigo) were treated with 57 or 90 nmol/kg of the corresponding P-gp inhibitor on three consecutive days.ResultsBBIQDs were synthesized and screened for their potential to inhibit P-gp-mediated drug efflux using calcein-AM as model substrate in vincristine-resistant leukemia cells (VCR-R CEM). The in vitro characterization revealed small molecule hits with a similar potency compared with the reference molecule TET. Consequently, the lead compounds were able to overcome chemoresistance to vincristine (VCR) when applied as a combination therapy, as assessed by cell viability and cell death assays. More specifically, the usage of P-gp inhibitors as add-on to VCR enabled a decrease of the IC50 value from 0.73 µM to up to 0.03 µM, corresponding to a maximum dose modifying factor of 24. Additionally, the ability of BBIQDs to reverse chemoresistance to imatinib in MDA cells overexpressing the breast cancer resistance protein (BCRP), another major efflux transporter responsible for MDR in leukemia6, 7, was tested. As no relevant chemosensitization was observed here, we concluded that our BBIQD hits are specific inhibitors of P-gp. Furthermore, in vivo toxicity was evaluated in a mouse model, showing that the BBIQDs were well-tolerated at the highest tested dose of 90 nmol/kg. Additionally, TUNEL assays on organ slides indicated lack of toxicity in lungs, heart and brain. Finally, the P-gp inhibitors will be tested for their efficacy to overcome chemoresistance in vivo using a zebra fish leukemia model.ConclusionTaken together, we have identified derivatives of the alkaloid TET with a similar potency to inhibit P-gp mediated drug efflux. Remarkably, the application of the BBIQDs as add-on to VCR was able to reverse chemoresistance in P-gp-mediated, VCR-resistant leukemia cells, while no toxicity issues were observed in a mouse model.ReferencesMarin, J.J., Briz, O., Rodriguez-Macias, G., Diez-Martin, J.L. & Macias, R.I. Role of drug transport and metabolism in the chemoresistance of acute myeloid leukemia. Blood Rev30, 55-64 (2016).Joshi, P., Vishwakarma, R.A. & Bharate, S.B. Natural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancer. Eur J Med Chem138, 273-292 (2017).Kelly, R.J. et al. A pharmacodynamic study of the P-glycoprotein antagonist CBT-1(R) in combination with paclitaxel in solid tumors. Oncologist17, 512 (2012).Jin, H. et al. Pulmonary toxicity and metabolic activation of tetrandrine in CD-1 mice. Chem Res Toxicol24, 2142-2152 (2011).Leanza, L. et al. Direct Pharmacological Targeting of a Mitochondrial Ion Channel Selectively Kills Tumor Cells In Vivo. Cancer Cell31, 516-531 e510 (2017).Burger, H. et al. Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump. Blood104, 2940-2942 (2004).Mao, Q. & Unadkat, J.D. Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport–an update. AAPS J17, 65-82 (2015). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Antiproliferative Activity of Combined Biochanin A and Ginsenoside Rh₂ on MDA-MB-231 and MCF-7 Human Breast Cancer Cells.

Breast cancer is the most frequently diagnosed cancer in women worldwide. The antiproliferative activities of biochanin A (BA) and ginsenoside Rh2 were determined by evaluating their inhibitory effect on MDA-MB-231 human breast cancer cell proliferation. The combination of BA with Rh2 was also assessed. In MDA cells, combination treatment led to a decrease in the EC50 values of BA and Rh2 to 25.20 μM and 22.75 μM, respectively. In MCF-7 cells, the EC50 values of combined BA and Rh2 decreased to 27.68 μM and 25.41 μM, respectively. BA combined with Rh2 also improved the inhibition of MDA-MB-231 and MCF-7 cell migration and invasion compared to the individual compounds. Western blot analysis demonstrated upregulation in p-p53, p-p38, and p-ASK1 proteins while levels of TRAF2 were downregulated. These results suggest that BA combined with Rh2 exhibits synergistic effects against MDA-MB-231 and MCF-7 cell proliferation.

BIOACTIVE MARINE-DERIVED COMPOUNDS AS POTENTIAL ANTICANCER CANDIDATES

Objective: The objective of this research was to evaluate for the 1st time the anticancer activities of sarcophytol M (1), alismol (2), alismoxide (5), 7β-acetoxy-24-methylcholesta-5-24(28)-diene-3,19-diol (7), erythro-N-dodecanoyl-docosasphinga-(4E,8E)-dienine (8), and 24 methylcholesta- 5,24(28)-diene-3β,7β,19-triol (9). Compounds were isolated from the soft coral Lithophyton arboreum and tested in liver (HepG2), lung (A549), and breast (MDA) cancer cell line.Methods: Anticancer activities of the compounds were tested using (XTT) 2,3-bis-(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, Na2) in vitro assay in order to estimate the cytotoxicity and to determine the IC50s. The free radical scavenging activity of the compounds were measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH•). All compounds were screened at 100 μg/ml while the most potent active compounds were assayed at lower concentrations.Results: Compounds (7) and (9) showed a strong cytotoxic effect with IC50 of 6.07, 8.5 μg/ml in HepG2, 6.3, 5.5 μg/ml in MDA cells, and 5.2, 9.3 μg/ml in A549 cancer cell lines, respectively. In addition, moderate cytotoxicity was shown by compound (2) (IC50 16.5, 15, and 13 μg/mL) in HepG2, MDA, and A549 cancer cell lines, respectively.Conclusion: The results obtained in this research work indicated a promising potential cytotoxicity of compounds (7) and (9) compared to its safety margins in Vero cells, and the expected cytostatic effect of compound (2) can be used in drug cocktails for the treatment of the major cancer types’ lung, breast, and liver cancer.