Murine Mammary Tumor Cell Line Research Articles

Abstract P5-01-10: A panel of murine mammary tumor cell lines to study immune responses to epitopes created by different mutagens including human APOBEC3 enzymes

Abstract Background: Tumor mutation loads are a strong predictor of immunotherapy benefit. This is evidenced by studies on skin, lung, and colon cancer, where well-defined processes elicit high mutation burdens and checkpoint therapies often provoke strong anti-tumor immune responses. In comparison, only a small number of breast cancer patients have responded to checkpoint therapies such as anti-PD-L1 and anti-PD-1 monoclonal antibodies. Approximately one-fourth of primary breast cancer and one-third of metastatic tumors have a significant APOBEC mutation burden defined as C-to-T and C-to-G mutations in TCA or TCT trinucleotide motifs, and many tumors have >10,000 of these APOBEC signature single base substitution mutations. However, to our knowledge, systematic immunotherapy studies focusing on this unique, hypermutated subset of breast cancers have yet to be done. Methods: To address the potential relationship between different checkpoint therapies and APOBEC mutation burden, we created a panel of >30 mammary tumor cell lines derived from fully immune-proficient C57BL/6 animals. At least 6 of these cell lines are immortalized, readily engineered using standard molecular biology procedures, and capable of re-engraftment and tumor formation in immune proficient C57BL/6 animals. Two lines were engineered to inducibly express human APOBEC3A, subjected to 1- to 20-rounds of sublethal mutagenesis, and tested systematically for checkpoint therapy responsiveness. Results: Two cell lines were engineered to inducibly express APOBEC3A or a catalytic mutant (E72A). Clones were characterized for inducible APOBEC3A expression and associated DNA damage responses. After varying rounds of mutagenesis, clones were engrafted into the subcutaneous flank or mammary fat pad of C57BL/6 animals. Immunohistochemical and flow cytometry analysis revealed a robust intratumoral CD8 T cell population including a subset with PD-1 expression. Additionally, engrafted tumor cells expressed high levels PD-L1. Experiments are ongoing and immunotherapy responses will be presented. Conclusions: We have isolated a panel of C57BL/6 mammary tumor-derived cell lines and demonstrated re-engraftment into immunocompetent animals. These cell lines constitute a platform for studying the relationship between different mutagens including APOBEC and immunotherapy responses. Citation Format: Jordan Naumann, Michael Carpenter, Prokopios Argyris, William Brown, Reuben Harris. A panel of murine mammary tumor cell lines to study immune responses to epitopes created by different mutagens including human APOBEC3 enzymes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-10.

Evaluation of the oncolytic property of recombinant Newcastle disease virus strain R2B in 4T1 and B16-F10 cells in-vitro

Recombinant Newcastle disease virus vectors have gained a lot of interest for its oncolytic virus therapy and cancer immune therapeutic properties due to its selective replication to high titers in cancer cells. The aim of this study was to find out the oncolytic effects of mesogenic recombinant NDV strain R2B-GFP on murine mammary tumor cell line 4T1 and murine melanoma cell line B16-F10. The anti-tumor effects of R2B-GFP virus were studied via expression of virus transgene GFP in cancer cells, evaluating its cytotoxicity and cell migration efficacies by MTT and wound healing assays respectively. In addition, the underlying apoptotic mechanism of R2B-GFP virus was estimated by TUNEL assay, colorimetric estimation of Caspase-3, 8 and 9 and the estimation of Bax to Bcl-2 ratio. The results showed a significant decrease in viability of both 4T1 and B16-F10 cells infected with R2B-GFP virus at 0.1 and 1 MOI. R2B-GFP virus could significantly induce apoptosis in the 4T1 and B16-F10 cells as compared to the uninfected control. Further, a flow cytometry analysis on apoptotic cells percentage and mitochondria membrane permeability test was also studied in R2B-GFP virus treated 4T1 and B16-F10 cell lines. The R2B-GFP virus caused an increase in loss of mitochondrial membrane permeability in both 4T1 and B16-F10 cells indicating the involvement of mitochondrial regulated cell death. Thus, the recombinant virus R2B-GFP virus proved to be a valid candidate for oncolytic viral therapy in 4T1 and B16-F10 cells.

Abstract B27: Modeling the impact of miR-200s on mammary tumor initiation and progression in vitro and in vivo

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that regulate mRNA translation through inhibiting the translation of mRNAs into proteins or through degrading mRNAs. Each miRNA potentially targets hundreds of mRNAs, and thus an individual miRNA can regulate entire gene networks. The miR-200 family consists of 5 miRNAs (miR-141, miR-200a, miR-200b, miR-200c, miR-429) organized into two clusters: the miR-200c/141 cluster and the miR-200b/200a/429 cluster. The miR-200s regulate a variety of cellular functions, with the best characterized being their ability to target mesenchymal transcription factors and thus maintain an epithelial phenotype. Given that miR-200s maintain an epithelial phenotype, we evaluated their role in mesenchymal mammary tumors. All five miR-200s were expressed at significantly lower levels in the murine mesenchymal mammary tumor cell line RJ423 and the human mesenchymal breast cancer cell line MDA-MB-231 compared to epithelial tumor cell lines. To evaluate the function of miR-200s, the miR-200c/141 and miR-200b/200a/429 clusters were re-expressed in the mesenchymal mammary tumors. Re-expression of the miR-200c/141 cluster partially restored an epithelial phenotype in MDA-MB-231 cells while the miR-200b/200a/429 cluster partially restored an epithelial phenotype in RJ423 cells. Re-expression of the miR-200c/141 cluster in MDA-MB-231 cells and re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed tumor growth following intramammary injection and altered the tumor microenvironment. To identify genes regulated by miR-200 expression, RNA sequencing was performed on control and miR-200 re-expressing cells grown in 2D culture and following intramammary injection. Interestingly, the in vivo environment dramatically altered the gene repertoire regulated by the miR-200s. Hierarchical clustering revealed that alterations in gene expression induced by miR-200 re-expression in vitro poorly correlated with the alterations in gene expression induced by miR-200 in vivo. Since miR-200 re-expression significantly decreased tumor growth in vivo, a transgenic model was used to determine whether miR-200s could also impact tumor initiation. Using doxycycline-inducible miR-200 expression in combination with the mammary tumor oncogene, IGF-IR, we found that overexpression of the miR-200b/200a/429 cluster dramatically reduced mammary tumor incidence (100% vs. 19%). These data show the potent impact miR-200s have on mammary tumorigenesis and that gene expression profiling of 2D cultures is a poor predictor of genes regulated by miR-200s in vivo. Citation Format: Katrina L. Watson, Kaitlyn Simpson, Majesta Roth, Courtney Martin, Garret Conquer-Van Heumen, Roger A. Moorehead. Modeling the impact of miR-200s on mammary tumor initiation and progression in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B27.

Abstract P6-06-06: Platelet derived growth factor-b (PDGFB) promotes breast cancer progression

Abstract Objectives of the Study: Platelet-derived growth factor (PDGF) mediated signaling is pro-tumorigenic in many types of cancer, including breast cancer. However, the requirement of individual PDGF ligands in mediating breast cancer progression remains unclear. Our evaluation of publicly available breast cancer patient datasets shows that high primary tumor expression of PDGFB correlates with reduced overall survival (OS) and reduced metastasis free survival (MFS). This is in contrast to the lack of prognostic power of PDGFA and PDGFC and the association with improved OS and MFS for PDGFD. Based on our analysis of patient datasets, we set out to test the requirement of PDGFB in mammary tumor growth. Methods Used: Gain-of-function and loss-of-function experiments were performed wherein PDGFB was either overexpressed or knocked-down in mammary tumor cells. Manipulated tumor cells were injected directly into the mammary fat pads of adult female mice, and tumor growth was monitored over time. PDGFB is produced by tumor cells whereas the corresponding receptor, PDGFRβ, is expressed by mesenchymal cells in the stroma. As another way to mimic oncogenic PDGFB-to-PDGFRβ signaling in the breast, we developed a mouse model of stroma-specific PDGFRβ activation using the Fsp-cre transgene. We evaluated changes mammary gland development upon mesenchymal specific PDGFRβ activation, and performed orthotopic injections with mammary tumor cells in these animals to test the functional role of receptor activation in mammary tumor growth. Results and Conclusions: We evaluated expression of PDGF ligands in FVB/N murine mammary tumor cell lines and found that the PDGFB is dramatically higher in DB7 tumor cells compared to other syngeneic cell lines. In the high-PDGFB expressing DB7 cells, we used shRNA technology to knockdown the ligand. At the same time, we overexpressed the ligand in an isogenic cell line that expresses low levels of PDGFB. These cells were injected orthotopically into the mammary fat pads of adult female mice, and in both cases, expression of PDGFB dictated tumor growth. There was a significant reduction in tumor growth with shRNA-mediated knockdown of PDGFB whereas overexpression of the ligand accelerated tumor growth. In our mouse model of mesenchymal-specific PDGFRβ activation, we reveal that activation of the receptor exerts pro-proliferative signals on adjacent mammary epithelial cells and accelerates orthotopic tumor growth of PDGFB-expressing cells. These findings indicate that PDGFB-to-PDGFRβ signaling is a viable therapeutic target for breast cancer. In fact, treatment with the PDGFR inhibitor, imatinib, impedes tumor cell proliferation when mouse mammary fibroblasts are co-injected orthotopically with DB7 cells. Significance: The requirement of other PDGF ligands in breast cancer remains to be evaluated, but our data support a pro-tumorigenic role for PDGFB in the breast. Importantly, PDGFR inhibitors are being used in clinical trials for several cancer types. Our data advocate for (1) the potential utility of PDGFB as a prognostic biomarker and (2) the pre-clinical evaluation of PDGFR inhibitors in breast cancer models. Citation Format: Katie A Thies, Anisha M Hammer, Sarah A Steck, Manjusri Das, Raleigh D Kladney, Steven T Sizemore, Michael C Ostrowski, Gina M Sizemore. Platelet derived growth factor-b (PDGFB) promotes breast cancer progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-06.

Abstract 2554: Branched chain amino acid transaminase 1 in claudin-low breast cancer

Abstract Breast cancer, the most commonly diagnosed cancer in women, can be classified into five distinct subtypes. One subtype, claudin-low breast cancer, accounts for approximately 7% of the breast cancer cases and these tumors are aggressive and highly metastatic. RNA sequencing of human claudin-low breast cancers by other groups and RNA sequencing of a murine claudin-low mammary tumor cell line by our group has revealed that Bcat1 is significantly up-regulated in this breast cancer subtype. Bcat1 regulates the metabolism of branched chain amino acids and has been linked to numerous pathologies including heart disease, diabetes, and cancer. Based on this data we hypothesized that the expression of Bcat1 in claudin-low mammary tumors is driving the aggressive and metastatic nature of this cancer subtype and disrupting Bcat1 will deter these features. Elevated expression of Bcat1 in the murine claudin-low cell line RJ423, compared to the murine luminal mammary tumor cell line RJ345, has been confirmed at the mRNA and protein level. Bcat1 has been transiently down-regulated ~70% in RJ423 cells using siRNA and this suppression of Bcat1, contrary to the anticipated result, showed no effect on proliferation based on phospho-histone H3 immunofluorescence. However, when the cell population doubling time was investigated, a significant difference was seen between the RJ423 cell line and the cell lines with suppressed Bcat1 expression. To elucidate these contradicting results, cell cycle analysis using Bromodeoxyuridine and 7-AAD through flow cytometry is currently underway. RJ423 stably expressing Bcat1 shRNA and RJ345 stably expressing a Bcat1 expression vector are currently being selected. Further in vitro and in vivo studies will utilize these stably transfected cell lines. This study will determine whether further investigation into the effects of Bcat1 on claudin-low human breast cancer is prudent and if Bcat1 may be used as a therapeutic target. Citation Format: Lisa C. Reynen, Robert Jones, Roger Moorehead. Branched chain amino acid transaminase 1 in claudin-low breast cancer [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 2554.

Identification of S100A14 as a metastasis-promoting molecule in a murine organotropic metastasis model.

Cancer metastasis shows great diversity in target organs, routes and molecular mechanisms depending on the type of cancer and even on the individual patients. To identify key molecules involved in metastasis, we constructed a murine model system including multiple sublines with different organotropism and pathways of metastasis. We selected metastatic sublines from a murine mammary tumor cell line MCH66. Using this model, we extracted metastasis-related molecules by gene expression screening methods and verified their metastasis-promoting effects by gene knockdown or overexpression experiments. For the candidates promoting metastasis, we analyzed molecular functions involved in metastasis: cell growth, motility and invasive activity. We established a metastasis model including low metastatic sublines (66C8, 66LM, 66-4) and highly metastatic counterparts with various organotropism, such as to the lung (66Lu10), liver (HM-KAN5) and general organs (66HM and its clones: HM1-6 and HM1-7). The sublines basically exhibited the invasion-independent metastasis pathway characterized by endothelial cell-covered tumor emboli, whereas 66HM and HM-KAN5 showed an alternative metastasis pathway based on invasion in part and in whole, respectively. Comprehensive gene analysis extracted several molecular candidates responsible for metastasis. S100A14 was identified as one of the promissing candidates promoting lung-metastasis, which was verified by gene knockdown experiments in vivo. In addition, in vivo and in vitro functional analyses demonstrated that S100A14 enhanced scattering, motility and invasiveness of mouse tumor cells. Our model system may be adaptable to the diversity of metastasis in human cancers and useful for exploring the molecular mechanism responsible for metastasis.

Abstract 2059: Alcohol enhances estrogen-responsive breast cancer neovascularization

Abstract The pleiotropic effects of estrogen in breast cancer development have been observed at both the initiation and progression phases. Interestingly, alcohol, as a dietary component, has been observed to possess estrogenic activity, but the synergistic interaction between alcohol and estrogen and its cellular targets remains undefined. Our previous study identified neovascularization as a cellular target of estrogen that was mediated by the mobilization of bone marrow-derived endothelial progenitor cells (BM-EPCs) and their subsequent incorporation within the tumor mass. The present study was undertaken to define the synergistic interaction between estrogen and alcohol using a cell culture model. To examine the effects of both estrogen and alcohol on breast cancer progression, we developed an in vitro model using the murine mammary tumor cell line, TG1-1, and the murine endothelial cell line, SVEC4-10. Neovascularization was assayed by the tubulogenesis and scratch wound assays as well as quantitating the expression of various provasculogenic markers. Conditioned media, generated by culturing the murine mammary cancer cell line, TG1-1, in estrogen and ethanol, was observed to enhance tubule formation and migration of the murine endothelial cell line, SVEC4-10. In addition, the TG1-1 conditioned media enhanced the prosurvival MAP kinase-signaling pathway in SVEC4-10 cells. Presumably, the observed enhancement of both endothelial cell tubulogenesis and migration is in part attributed to provasculogenic proteins secreted by TG1-1 cells. In order to address this, Western blot analysis using TG1-1 cell lysates indicated an increase in VEGF, MMP-9, and eNOS expression in presence of alcohol and estrogen. As a whole, our data suggest that both estrogen and alcohol function to enhance expression of key provasculogenic molecules in breast cancer cells, which in turn modulates the morphologic, migratory, and survival properties of endothelial cells. Citation Format: Natalie C. Cruz, Rachana Maniyar, Robert Suriano. Alcohol enhances estrogen-responsive breast cancer neovascularization [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 2059.

The miR-200b/200a/429 cluster prevents metastasis and induces dormancy in a murine claudin-low mammary tumor cell line

The miR-200 family of microRNAs consisting of miR-141, miR-200a, miR-200b, miR-200c and miR-429 are emerging as important regulators of breast cancer progression. This family of microRNAs maintain mammary epithelial identity and downregulation of miR-200 expression has been associated with epithelial-to-mesenchymal transition in mammary tumors. Therefore, re-expression of one or more miR-200 family members in mammary tumor cells with mesenchymal characteristics may restore an epithelial phenotype including growth and metastasis suppression. To test this hypothesis, the miR-200b/200a/429 cluster was re-expressed in a murine claudin-low cell line, RJ423. Re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed the expression of Vim, Snai1, Twist1, Twist2 and Zeb1, reverted RJ423 cells to a more epithelial morphology and significantly inhibited proliferation in vitro. Moreover, the miR-200b/200a/429 cluster prevented lung metastasis in an experimental metastasis model and although tumor initiation was not prevented, re-expression of the miR-200b/200a/429 cluster induced a dormancy-like state where mammary tumors failed to grow beyond ~150 mm3 or grew extremely slowly following intra-mammary injection. These dormant tumors contained elevated levels of collagen and were highly vascularized. Therefore, re-expression of the miR-200b/200a/429 cluster in the claudin-low mammary tumor cell line, RJ423, is sufficient to alter cell morphology, impair metastasis and induce tumor dormancy.

Cytotoxic effect of crab shell extracts on different tumor cell lines

Crab, a kind of marine animal that has a unique status in evolution, has many bioactive substances with special functions. In the present study, we investigated the antitumor effects of four different extracts derived from crab shell (aqueous and methanolic extracts of burned and normal crab shell powder). Aqueous and methanolic extracts of burned crab shell powder were shown to be cytotoxic and inhibit the in vitro proliferation of human laryngeal carcinoma tumor cell lines (Hep-2) and human Rhabdomyosarcoma cell line (RD) and one murine mammary adenocarcinoma tumor cell line (AMN3), in dose and time dependant manner. These results suggest that the burned crab shell powder have an antitumor effect that needs more investigations.

Abstract 3911: Stromal platelet derived growth factor receptor-beta (PDGFRbeta) promotes breast brain metastasis

Abstract The platelet derived growth factor (PDGF) pathway is a prime example of tumor-stroma signaling in a number of cancer types. Others have shown that PDGF receptors are expressed in breast fibroblasts and pericytes while PDGF ligands are often expressed in breast cancer cells and tumor-associated endothelium; however, how PDGF signaling mediates breast cancer initiation, progression and metastasis remains unclear. Importantly, our evaluation of publicly available datasets revealed that PDGFB expression correlates with breast cancer patient metastatic recurrence leading to the hypothesis that PDGF-B to PDGFR signaling promotes metastatic progression of breast cancer. Given that PDGF-B preferentially activates PDGFRβ, we established an in vivo system to investigate this pathway during breast cancer progression. We utilized a mesenchymal-specific promoter to drive Cre recombinase and conditionally activate PDGFRβ by way of the endogenous Pdgfrb promoter (hereafter “PDGFRβ mutant”). A murine mammary tumor cell line which expresses high levels of PDGF-B was injected either by tail vein or intracranially to evaluate metastatic seeding and distant tumor growth. Following tail vein injection of tumor cells, we observed 50% incidence of brain metastases in the PDGFRβ mutant mice while no brain lesions were seen in the controls. There was no difference in incidence of lung, liver or bone metastases (other common sites of breast cancer metastasis). Not surprisingly, larger tumors formed in the brains of PDGFRβ mutant mice when cells were injected intracranially. Brains were stained for phospho-PLCγ as a way to confirm activation of PDGFRβ. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of breast brain metastases. Furthermore, this study highlights a role for stromal activation of PDGFRβ in the brain microenvironment and during metastatic progression. For the 20-30% of patients that develop breast cancer brain metastases, the one-year survival rate is sadly less than 20%, and how the brain microenvironment contributes to metastatic seeding and subsequent growth of tumor cells remains poorly understood. To confirm translational relevance, we analyzed a small cohort of matched primary breast tumors and brain metastases for PDGFRβ expression observing strong stromal staining in fibroblasts and pericytes within and around all of the primary tumors similar to previous studies. Importantly, high PDGFRβ expression was found in the perivasculature of all associated brain metastases suggesting a functional role in the establishment or growth at this site. Combined, our findings strongly suggest that high primary tumor expression of PDGF-B/PDGFRβ might define a subset of breast cancer patients predisposed to brain metastases. These patients may benefit from therapeutic targeting of PDGFR signaling as a means to thwart metastatic seeding in the brain. Citation Format: Katie A. Thies, Anisha M. Hammer, Anthony J. Trimboli, B. Eason Hildreth, Luke O. Russell, Chelsea M. Bolyard, Raleigh D. Kladney, Steven T. Sizemore, Robert Pilarski, Lynn Schoenfield, Jose Otero, Arnab Chakravarti, Balveen Kaur, Gustavo Leone, Michael C. Ostrowski, Gina M. Sizemore. Stromal platelet derived growth factor receptor-beta (PDGFRbeta) promotes breast brain metastasis [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 3911. doi:10.1158/1538-7445.AM2017-3911

Abstract IA09: Translational control of the tumor microenvironment

Abstract Translational control has emerged as a critical determinant in tumorigenesis. The mRNA cap-binding protein eIF4E is an oncoprotein that plays an important role in cancer initiation and progression. We recently demonstrated that eIF4E phosphorylation on serine 209 by the Map Kinase Integrating Kinases (MNK1/2) promotes epithelial to mesenchymal transition, invasion and metastasis. We recently discovered a critical importance of eIF4E phosphorylation in the tumor microenvironment (TME) for the metastatic process. To investigate the role of eIF4E phosphorylation in the TME, we utilized a syngeneic model in which a murine mammary tumor cell line (66cl4) was orthotopically injected into mice bearing the non-phosphorylatable eIF4E ^S209A mutation and their WT counterparts. Tumor growth was monitored by caliper measurements, and tissues were collected for histological assessment and immunohistochemistry. The onset of tumor formation was significantly delayed in eIF4E ^S209A mice. However, established tumors showed no differences in growth, proliferation, apoptosis, vascularization or immune infiltration. Strikingly, eIF4E ^S209A mice were nearly completely resistant to the formation of lung metastases from primary mammary tumors. Metastatic potential in this model was correlated with neutrophil accumulation in the spleen, blood and lungs of WT mice, which was significantly reduced in eIF4E ^S209A mice. Furthermore, treatment of tumor-bearing WT mice with an inhibitor of MNK1/2 significantly reduced lung metastasis without affecting primary tumor growth. Considering that eIF4E phosphorylation in both the TME and cancer cells promotes metastasis, our findings support the development of MNK inhibitors for metastasis prevention. Citation Format: Nathaniel Robichaud, Qianyu Guo, Sonia del Rincon, Wilson H. Miller, Peter Siegel, Brian Hsu, Nahum Sonenberg. Translational control of the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA09.

Abstract 760: Identifying candidate genes that regulate tumor cell dormancy

Abstract Tumor cells disseminate from the primary tumor site early in the disease process. These disseminated tumor cells may remain dormant for years and later become proliferative to cause clinical recurrence and morbidity. The goal of this study is to better understand the biological and genetic mechanisms driving this dormant to proliferative switch. We have developed a 3D culture system using the dormant murine mammary tumor cell line, D2.0R. D2.0R cells remain dormant when cultured on basement membrane extract (BME), and exit dormancy when supplemented with collagen-1 (col-1). We have characterized the gene expression profile of these cells exiting dormancy in 3D through microarray analysis, and developed a fourteen-gene dormancy signature by profile cross-referencing this expression profile to a tumor cell dormancy signature developed by Kim, R.S. et al (PLOS ONE 2012). We hypothesize that knocking down expression of genes upregulated in proliferative cells will hinder the ability of D2.0R cells to break dormancy in 3D culture supplemented with collagen-1. We have validated three genes by qPCR and are screening these upregulated genes for biological effects on proliferation by short-hairpin RNA or molecular inhibition of D2.0R cells in our 3D model. We conclude that this signature will provide useful insight into genetic drivers of the dormant to proliferative switch and potential candidates as therapeutic targets in recurrent disease. Citation Format: Ryan J. Nini, Lara H. El Touny, Meera Murgai, Rosandra N. Kaplan, Jeffrey E. Green. Identifying candidate genes that regulate tumor cell dormancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 760.

Targeting cell-bound MUC1 on myelomonocytic leukemias and leukemic stem cells: Therapeutic implications.

e14003Background: Monocytic neoplasms,include chronic myelomonocytic leukemia (CMML), acute myelomonocytic and monocytic leukemia, and monocytic sarcoma. Cell surface molecules overexpressed by leukemic cells represent potential disease-specific therapeutic targets..MUC1 has an extracellular domain containing tandem repeats of a 20 amino acid-long sequence, a transmembrane domain, and a short cytoplasmic tail. Cleavage of MUC1 yields an extracellular alpha subunit containing the tandem repeat and a smaller beta subunit. Anti-MUC1 Abss reported to date target the highly immunogenic tandem repeat of the MUC1 alpha chain. However the alpha chain is cell-bound only intermittently to MUC1+ cells. In contrast, the MUC1 SEA domain remains fixed to the cell surface. Methods: A series of 22 AML samples (AML0 = 2, AML1 = 2, AML2 = 10, AML4 = 1, AML5 = 5, AML6 = 2) collected either at the time of diagnosis or at relapse were analysed for MUC1 expression by flow cytometry. A murine mammary tumor cell line transfected...

Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemias and Leukemic Stem Cells: Therapeutic Implications

Monocytic neoplasms comprise a heterogeneous group of hematologic malignancies including chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5), and monocytic sarcoma. Monocytic or granulomonocytic hyperplasia is a finding frequently-if not invariably-shared by these different entities, as is a poor therapeutic outcome in the absence of hematopoietic stem cell transplantation. Cell surface molecules aberrantly expressed or overexpressed by leukemic cells represent potential disease-specific therapeutic targets. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a molecule. MUC1 consists of an extracellular domain containing 20 to 125 tandem repeats of a 20 amino acid-long sequence, followed by a transmembrane domain and a short cytoplasmic tail leading to intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit containing the tandem repeat array bound in a strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic tandem repeat of the MUC1 alpha chain. Because the alpha chain binds the cell-bound domains of MUC1 only intermittently in an 'on-and-off' manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on AML cells by flow cytometry. A series of twenty-two AML samples (blood-derived n=12; bone marrow-derived n=10; AML0=2, AML1=2, AML2=10, AML4=1, AML5=5, AML6=2) collected either at the time of diagnosis or at relapse were analysed for MUC1 expression by flow cytometry. A murine mammary tumor cell line stably transfected with human MUC1 DNA served as control. Blasts cells from 5 AML samples highly expressed MUC1, and significantly, all were of monocytic or myelomonocytic lineage (AML4=1, AML5=4). Leukemic stem cells (CD34pos or CD34neg linneg) from the MUC1+ AMLs were examined and likewise found to express MUC1. In addition, AML cell lines MV411, MOLM14, and SHI-1 derived from monocytic leukemic lineage clearly expressed cell surface MUC1, while non- monocytic leukemic cell lines U937, K562, and HL60 had little or no expression. Normal monocytes and monocytes derived from patients with activated monocytosis were also found to express MUC1. Based on these findings we examined MUC1 expression in a series of myelomonocytic leukemia (CMML and JMML). In fifteen CMML samples examined (type 1 n=11, type 2 n=4) (blood n=7, BM n=7) 92%-100% (median 99.7%) of CD14+CD56+ CMML cells bound mAb DMB-5F3 to cell-surface MUC1. CD14+CD16+CD56+ blast cells from 2 pts with JMML were also found to express MUC1 (between 64% and 71 % positive). Based on these findings we conclude that expression of MUC1 is restricted to monocytic and myelomonocytic leukemias and that MUC1 represents an effective target for leukemic immunotherapy. Significantly, anti-MUC1 mAb also targets monocytic leukemic stem cells, reinforcing its therapeutic potential. The fact that the anti-MUC1 antibody DMB-5F3 can enter cells and thereby ferry Ab-bound toxin opens the way for us to demonstrate leukemic cell killing with anti-MUC1 mAb-immunotoxin conjugates. DisclosuresMoreau:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 3250: Prostaglandin E2 receptor EP1 suppresses breast cancer metastasis

Abstract Cyclooxygenases (COX-1 and COX-2) catalyze the formation of prostaglandins and play a role in the pathogenesis of breast cancer. Prostaglandin E2 (PGE2), the chief COX product in tumors, is the predominant protumorigenic prostanoid and mediates biological effects by binding to each of four EP receptors (EP1-4). Each receptor is coupled to different intracellular signaling pathways; and EP1 is coupled to calcium mobilization and PKC activation. Our published studies indicate that EP1 was detected in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas, and overall survival for women with tumors negative for nuclear EP1 was significantly worse than for women with any nuclear EP1 expression. Pharmacologic antagonism or reduction of EP1 expression increased metastatic capacity in a murine model of metastatic breast cancer. These data support our hypothesis that EP1 functions as a metastasis suppressor. We now report that murine metastatic mammary tumor cell lines 410.4 and 66.1 have decreased EP1 mRNA expression compared to the non-metastatic cell line 410. Western blot analysis of total and subcellular fractions of EpH4 (normal, immortalized mammary epithelial) and malignant 410, 410.4 and 66.1 cell lines demonstrates that EP1 protein is present in the total and cytoplasmic fractions for all cell lines examined. Nuclear EP1 is detected in EpH4 and non-metastatic 410 cells; however, very little to no nuclear protein is detected in metastatic cell lines 410.4 and 66.1. The absence of nuclear EP1 in metastatic cell lines is consistent with the prognostic data that overall survival for women with tumors negative for nuclear EP1 was significantly worse. Previously, we determined that reduction of EP1 expression leads to increased metastatic capacity; therefore, we investigated the effect of EP1 overexpression on lung colonization in 410.4 and 66.1 cells via tail vein injection in a syngeneic murine model of breast cancer. In 410.4 cells, EP1 overexpression leads to a 57%-97% decrease in metastasis, and in 66.1 cells EP1 overexpression resulted in a 10% - 38% decrease in lung tumor burden compared to vector control mice. The inverse correlation between EP1 expression and metastatic capacity supports our hypothesis that EP1 functions as a metastasis suppressor. We explored potential mechanisms leading to the alteration in the metastatic behavior in response to manipulation of the expression of EP1. We previously published that an EP1 antagonist altered adhesion to laminin in 410.4 cells. Overexpression of EP1 altered the expression of several integrin receptors in 410.4 and 66.1 cell lines including integrins alpha-V, alpha-6 and alpha-3. Also, overexpression of EP1 led to altered adhesion of these cells to several extracellular matrices including fibronectin, collagen, laminin and fibrinogen. Alteration of the adhesive properties of these cells by EP1 could contribute to the metastasis suppressor function of the EP1 receptor. Citation Format: Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Olga Goloubeva, Amy Fulton. Prostaglandin E2 receptor EP1 suppresses breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3250. doi:10.1158/1538-7445.AM2015-3250

The calcineurin/NFAT pathway is activated in diagnostic breast cancer cases and is essential to survival and metastasis of mammary cancer cells.

Nuclear factor of activated T cells 1 (NFAT1) expression has been associated with increased migratory/invasive properties of mammary tumor-derived cell lines in vitro. It is unknown, however, if NFAT activation actually occurs in breast cancer cases and whether the calcineurin/NFAT pathway is important to mammary tumorigenesis. Using a cohort of 321 diagnostic cases of the major subgroup of breast cancer, we found Cn/NFAT pathway activated in ER−PR−HER2− triple-negative breast cancer subtype, whereas its prevalence is less in other subgroups. Using a small hairpin RNA-based gene expression silencing approach in murine mammary tumor cell line (4T1), we show that not only NFAT1 but also NFAT2 and their upstream activator Cn are essential to the migratory and invasive properties of mammary tumor cells. We also demonstrate that Cn, NFAT1 and NFAT2 are essential to the tumorigenic and metastatic properties of these cells in mice, a phenotype which coincides with increased apoptosis in vivo. Finally, global gene expression analyses identified several NFAT-deregulated genes, many of them being previously associated with mammary tumorigenesis. In particular, we identified the gene encoding a disintegrin and metalloproteinase with thrombonspondin motifs 1, as being a potential direct target of NFAT1. Thus, our results show that the Cn/NFAT pathway is activated in diagnostic cases of breast cancers and is essential to the tumorigenic and metastatic potential of mammary tumor cell line. These results suggest that pharmacological inhibition of the Cn/NFAT pathway at different levels could be of therapeutical interest for breast cancer patients.

Mass spectrometry based quantitative proteomics and integrative network analysis accentuates modulating roles of annexin-1 in mammary tumorigenesis.

Annexin-1 (ANXA1) is known to be involved in important cellular processes and implicated in cancer. Our previous study showed its roles in cell migration and DNA-damage response processes in breast cancer initiation. In order to understand its roles in tumorigenesis, we extended our studies to analyze tumors derived from polyomavirus middle T-antigen ANXA1 heterozygous (ANXA1(+/-) ) and ANXA1 null (ANXA1(-/-) ) mice. We performed quantitative comparison of ANXA1(+/-) and ANXA1(-/-) tumors employing reductive dimethyl labeling quantitative proteomics. We observed 253 differentially expressed proteins (DEPs) with high statistical significance among over 5000 quantified proteins. Combinatorial use of pathway and network-based computational analyses of the DEPs revealed that ANXA1 primarily modulates processes related to cytoskeletal remodeling and immune responses in these mammary tumors. Of particular note, ANXA1(-/-) tumor showed reduced expression of a known epithelial-to-mesenchymal transition (EMT) marker vimentin, as well as myosin light-chain kinase, which has been reported to induce Rho-kinase mediated assembly of stress fibers known to be implicated in EMT. Integrative network analysis of established interactome of ANXA1 alongside with DEPs further highlights the involvement of ANXA1 in EMT. Functional role of ANXA1 in tumorigenesis was established in invasion assay where knocking down ANXA1 in murine mammary tumor cell line 168FARN showed lower invasive capability. Altogether, this study emphasizes that ANXA1 plays modulating roles contributing to invasion-metastasis in mammary tumorigenesis, distinctive to its roles in cancer initiation.

Attenuation of TGF-β signaling supports tumor progression of a mesenchymal-like mammary tumor cell line in a syngeneic murine model

Previous studies have suggested that TGF-β functions as a tumor promoter in metastatic, mesenchymal-like breast cancer cells and that TGF-β inhibitors can effectively abrogate tumor progression in several of these models. Here we report a novel observation with the use of genetic and pharmacological approaches, and murine mammary cell injection models in both syngeneic and immune compromised mice. We found that TGF-β receptor II (TβRII) knockdown in the MMTV-PyMT derived Py8119, a mesenchymal-like murine mammary tumor cell line, resulted in increased orthotopic tumor growth potential in a syngeneic background and a similar trend in an immune compromised background. Systemic treatment with a small-molecule TGF-β receptor I kinase inhibitor induced a trend towards increased metastatic colonization of distant organs following intracardiac inoculation of Py8119 cells, with little effect on the colonization of luminal-like Py230 cells, also derived from MMTV-PyMT tumors. Taken together, our data suggest that the attenuation of TGF-β signaling in mesenchymal-like mammary tumors does not necessarily inhibit their malignant potential, and anti-TGF-β therapeutic intervention requires greater precision in identifying molecular markers in tumors with an indication of functional TGF-β signaling.

Mechanisms of antitumor and immune-enhancing activities of MUC1/sec, a secreted form of mucin-1

Mucin 1 (MUC1) is a polymorphic type 1 transmembrane protein found on the apical surface of normal cells lining the lumen of ducts and glands. Mucins are thought to provide mucosal protection from environmental exposures and carcinogens. An altered form of the MUC1 glycoprotein, which is hypoglycosylated, is expressed in several types of human cancers. In our laboratory, we have found that transfection of a murine mammary tumor cell line with a human secreted isoform of MUC1 rendered these DA-3 cells (DA-3/sec) incapable of growing in intact BALB/c mice. In contrast, implantation of DA-3 cells transfected with the human transmembrane isoform of MUC1 (DA-3/TM), resulted in tumor formation and ultimately death of the animals, similar to the DA-3 parental line. Importantly, inoculation of the DA-3/sec cells in immunodeficient nude mice resulted in tumor formation, indicating that the MUC1/sec molecule's antitumor activity is immunologically controlled. In this review, we summarize the studies we have performed to elucidate possible mechanisms for the immune-mediated antitumor effect of MUC1/sec and/or a unique peptide present in this mucin. Understanding these mechanisms may provide new immunotherapeutic approaches that could be used to target different types of cancer.

Abstract PR13: Interleukin-4 receptor α chain regulates the growth of primary and metastatic mammary tumors

Abstract Interleukin-4 (IL-4), a cytokine produced mainly by immune cells, may promote the growth of epithelial tumors by mediating increased proliferation and survival. The IL-4 Receptor (IL-4R) is overexpressed in human and murine epithelial mammary cancers, and may be a promising target for anti-tumor therapy. Here, we show that loss of IL-4Rα, a component of the IL-4 receptor, in two murine mammary tumor cell lines (R221A and 4T1), reduces tumor cell survival and outgrowth ability, and results in attenuated growth at primary and metastatic sites. RNA interference was used to knock down the expression of IL-4Rα in both cell lines. In support of our hypothesis, reduced IL-4Rα expression in R221A cells nearly abolished tumor take after orthotopic mammary injection in vivo, decreased the median latency of 4T1 primary tumors, and resulted in a significant reduction in tumor size at endpoint. In addition, reduced IL-4Rα expression in both cell lines resulted in a decrease [>2.4 fold] in lung and [>2 fold] liver metastatic tumor burden following experimental metastasis assays in vivo. The decrease in metastatic ability of IL-4Rα knockdown cells was attributed to 1) reduced tumor cell survival as determined by seeding ability (number of tumors) and an increase in apoptosis by caspase-3 positivity, and 2) a reduction in proliferation as determined by outgrowth ability (tumor size) and a decrease in Ki67 positivity. In addition, an overall increase in immune infiltrates (macrophages, neutrophils, and T lymphocytes) within IL-4Rα knockdown tumors was observed, indicating that enhanced clearance of knock down tumor cells could also contribute to the reduction in knock down tumor size visualized at endpoint. Finally, it was determined by western blot analysis that IL-4 ligand stimulates the phosphorylation of AKT (s373) and ERK in R221a and 4T1 cells in vitro. Immunohistochemical analysis of R221a and 4T1 lung metastases showed significantly reduced levels of pAKT (s472) and pERK in IL-4Rα knock down tumors compared to control tumors, implicating a possible role for AKT and ERK in mediating pro-tumor effects IL-4Rα signaling. Collectively, our results demonstrate that the IL-4 receptor promotes the growth of mammary cancer cells at primary and metastatic sites, possibly through activated AKT and ERK signaling, and suggests that available drugs targeting IL4-Rα signaling may have potential for treating primary breast cancers and limiting metastatic disease. This abstract is also presented as poster C51. Citation Format: Katherine T. Venmar, Daniel Hwang, Ashley Dozier, Kathy Carter, Sareena Gillani, Barbara Fingleton. Interleukin-4 receptor α chain regulates the growth of primary and metastatic mammary tumors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR13.