bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract A05: Deep immunofluorescence imaging of solvent-cleared mouse mammary glands
Authors: Polec Anna , Norum Jens Henrik , Brech Andreas , Sorlie Therese 
Published: August 2018

Abstract
We seek to trace the progression of epithelial lesions originating from Lgr5+ cells in mouse mammary glands using the 3DISCO protocol for solvent-cleared tissue. Lineage tracing is increasingly being used to probe different cell types that exist within the mammary gland. Lgr5 is a stem cell marker and it is expressed in several different tissues including colon, stomach, and breast. It has been reported that Lgr5+ cells are necessary for mammary gland organogenesis. However, the cells that initiate tumor formation have remained intangible. Lgr5+ cells reside within the basal cell population of mammary ducts. Lgr5 is a downstream target of Wnt and therefore Lgr5+ cells may have a role in breast cancer progression. Since heterogeneity of tumors is shaped by several factors, including the cell of origin, specific oncogenic events, and interactions between tumor cells and cells of the microenvironment, it is important to obtain 3-dimensional information of tumor development.
We used the 3DISCO protocol for solvent-clearing mammary fat pads for deep immunofluorescence staining of transgenic mice harboring a EGFP reporter for Lgr5+ cells (mouse: Lgr5-EGFP-Ires-CreERT2). We performed deep immunofluorescence staining with DAPI and an antibody against the basal cell marker keratin 5 (K5).
Mammary fat pads were successfully solvent-cleared and prepared for deep immunofluorescence staining. Epithelial mammary ducts were easily recognized within the fat tissue using DAPI nuclear staining and an antibody against K5. Three-dimensional confocal microscope imaging protocols for whole mammary glands, including epithelial ducts, were established. We optimized the method using tile scans covering the entire fat pad, and multiple z-stacks of selected areas of Lgr5+ cells expressing EGFP were also detected and colocalized with K5+ cells.
The whole-organ tissue clearing, assessed in the mouse mammary gland, is suitable to obtain 3-dimensional information of various cell populations using specific molecular markers. This technique enables high-resolution, 3D imaging and phenotyping of tissue and might significantly enhance our understanding of mammary gland tumorigenesis. We will use the established protocol to localize the appearance of early lesions in mouse mammary glands and determine the role of Lgr5+ cells in mammary tumor development.
Citation Format: Anna Polec, Jens Henrik Norum, Andreas Brech, Therese Sorlie. Deep immunofluorescence imaging of solvent-cleared mouse mammary glands [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A05.
Abstract A36: Chemosensitivity to trastuzumab emtansine (T-DM1) differs in naturally or transduced HER2-overexpressing human breast cancer cells
Authors: Wu Jeffrey , Muldoon Leslie , Neuwelt Edward 
Published: August 2018

Abstract
Human epidermal growth factor receptor-2 (HER2) is overexpressed in 25-30% of breast cancers and is associated with distant metastasis and poor survival. Trastuzumab emtansine (T-DM1), an antibody-drug conjugate of anti-HER2 mAb trastuzumab linked to microtubule-targeting agent mertansine, has been approved for the treatment of HER2-positive metastatic breast cancer. Chemoresistance has been a major obstacle to T-DM1 treatment, and mechanisms remain incompletely understood. We tested T-DM1 chemosensitivity in vitro and in vivo using two human breast cancer cell lines, MDA-MB-231BR-HER2 (triple-negative cells stably transfected with HER2 plasmid) and HCC1954 (natural HER2 overexpression), which show equivalent HER2 and αv integrin protein levels. MDA-MB-231BR-HER2 was strongly resistant to T-DM1, with doses as high as 1 µg/ml failing to induce apoptosis indicated by the presence of cleaved PAPR protein. In contrast, HCC1954 cells are sensitive to T-DM1, with doses as low as 10 ng/mL causing cytotoxicity in a dose-dependent manner. Cellular internalization of trastuzumab-pHrodo conjugates was significantly higher in HCC1954 than MDA-MB-231BR-HER2, but no difference was found in anti-EGFR-Ab-pHrodo conjugates internalization. Endocytosis-related proteins caveolin-1 and rab5, but not early endosome antigen, were diminished in HCC1954 cells. Consistently, single-dose treatment with T-DM1 (3.6 mg/kg, IV) significantly reduced brain tumor volume in rats inoculated with HCC1954 but not MDA-MB-231BR-HER2 cells in an intracerebral xenograft breast cancer brain metastasis model. In conclusion, our findings suggest that rab5 and caveolin-1 proteins mediating antibody internalization and/or natural vs transduced HER2 overexpression may provide possible mechanisms in resistance of T-DM1 and should be investigated when HER2-positive cancer patients fail T-DM1 HER2-targeted therapy in clinical settings.
Citation Format: Jeffrey Wu, Leslie Muldoon, Edward Neuwelt. Chemosensitivity to trastuzumab emtansine (T-DM1) differs in naturally or transduced HER2-overexpressing human breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A36.
Abstract A47: Long-term treatment of bortezomib reduced resistance to doxorubicin by reducing CerS6/GCS and elevating CerS2/GBA expressions
Authors: Shin Kim , Dongeun Kim , Inkeun Park , Woo-Jae Park 
Published: August 2018

Abstract
Multiple drug resistance (MDR) is main cause of chemotherapy failure in breast cancer. Herein, we tried to find the mechanism of MDR. Since overexpression of glucosylceramide synthase (GCS) plays an important role in development of MDR, we analyzed expression levels of many molecules from TCGA data to find a connection between GCS and other molecules. Interestingly, correlations between GCS and ceramide synthase 6 (CerS6), and between glucocerebosidase (GBA) and CerS2, 4, 5 were analyzed. To further analyze whether these correlations are true, we treated C16~C24-ceramide to MD-MBA-361 cells. C24-ceramide treatment for 48 and 72 hrs increased GBA expression, which increased susceptibility of apoptosis upon doxorubicin treatment. Moreover, C16-ceramide treatment for 16 weeks increased GCS expression, which also induced MDR1 protein expression. Recently we found that bortezomib, an FDA-approved proteasome inhibitor, increased CerS2 and decreased CerS6 expressions in a dose-dependent manner. To further understand whether bortezomib affects MDR, bortezomib was treated with doxorubicin for 6-8 months and it prevented doxorubicin-induced MDR development by reducing CerS6 and elevating CerS2. In conclusion, downregulation of CerS6 and upregulation of CerS2 is a good strategy to prevent development of MDR, and bortezomib can be used for the prevention of acquired MDR.
Citation Format: Kim Shin, Kim Dongeun, Park Inkeun, Park Woo-Jae. Long-term treatment of bortezomib reduced resistance to doxorubicin by reducing CerS6/GCS and elevating CerS2/GBA expressions [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A47.
Abstract A64: Cold-inducible RNA binding protein (CIRP) links inflammation and breast cancer
Authors: Lujan Daniel Albino , Perrone-Bizzozero Nora , Beswick Ellen J. , Kusewitt Donna F. , Hartley Rebecca S. 
Published: August 2018

Abstract
RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with the regulatory sequences in the untranslated regions of mRNAs. RBPs are often disrupted in cancer, but current knowledge of their role in regulating cancer-associated genes is largely based on in vitro studies. This study seeks to determine the in vivo role of cold-inducible RBP (CIRP), which is overexpressed in some human breast cancers. CIRP promotes cell proliferation and survival and inhibits apoptosis in vitro, as well as promotes inflammation in vivo, all hallmarks of cancer. We set out to test the hypothesis that CIRP promotes breast tumorigenesis in vivo by crossing a transgenic mouse overexpressing CIRP in the mammary epithelium with the PyMT mouse model of breast cancer. Effects on both early (7 weeks of age) and late (14 weeks) tumorigenesis were assessed. Unexpectedly, mammary glands from 7-week CIRP/PyMT mice had 50% fewer mammary intraepithelial neoplasias than those from PyMT mice. Consistent with this, histopathologic analysis showed decreased mitosis and increased apoptosis in CIRP/PyMT vs. PyMT mammary glands. Late-stage tumorigenesis was also inhibited. CIRP/PyMT mice had slower tumor growth kinetics, smaller tumors, decreased tumor burden, and decreased pulmonary metastasis compared to PyMT mice. To assess mechanism, mammary glands from 7- and 14-week mice were incubated in medium overnight followed by Luminex cytokine assays on the medium. Results showed a decrease in cytokines that promote inflammation, angiogenesis, and metastasis. Altogether, results suggest that in vivo, CIRP inhibits proliferation and promotes apoptosis to suppress early tumorigenesis, potentially via decreasing the protumorigenic inflammatory response.
Citation Format: Daniel Albino Lujan, Nora Perrone-Bizzozero, Ellen J. Beswick, Donna F. Kusewitt, Rebecca S. Hartley. Cold-inducible RNA binding protein (CIRP) links inflammation and breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A64.
Abstract B14: Sprouty4 regulates the transition to invasive breast ductal carcinoma through ERK/MAPK signaling
Authors: Brock Ethan J. , Jackson Ryan , Boerner Julie L. , Li Quanwen , Sloane Bonnie F. , Mattingly Raymond R. 
Published: August 2018

Abstract
Breast cancer is one of the most common oncologic developments in U.S. women, with approximately 25% of these cases being in situ disease (DCIS). For many years DCIS lesions have been treated using surgery and radiotherapy with the goal of decreasing the amount of late-stage disease. Unfortunately, a number of reports have since shown that early intervention is not sufficient and have highlighted the likelihood of overtreatment. This is especially true for triple-negative breast cancers, which are more strongly associated with distant recurrence, metastasis, and death compared to other types of invasive breast ductal carcinoma (IDC). These factors have generated a substantial clinical need for reliable biomarkers or molecular determinants to assess disease progression. Sprouty proteins are recognized as important regulators of ERK/MAPK signaling, and have been studied in various cancers. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling, and its loss or reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry we found that Sprouty4 expression was reduced in IDC patient tissue samples compared to normal or DCIS tissues, and that ERK/MAPK activity had an inverse relationship to Sprouty4 expression. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient overexpression of Sprouty4 reduced both ERK/MAPK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments revealed data consistent with the relocation of E-cadherin back to the cell surface and the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug produced the restoration of an epithelial-like phenotype similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 may act to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions.
Citation Format: Ethan J. Brock, Ryan Jackson, Julie L. Boerner, Quanwen Li, Bonnie F. Sloane, Raymond R. Mattingly. Sprouty4 regulates the transition to invasive breast ductal carcinoma through ERK/MAPK signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B14.
Abstract B20: Glutamine metabolic vulnerabilities define triple-negative from luminal A breast cancer subsets
Authors: Geldermalsen Michelle van , Quek Lake-Ee , Turner Nigel , Balaban Seher , Hoy Andrew , Wang Qian , Holst Jeff 
Published: August 2018

Abstract
Although a nonessential amino acid in normal cells, the demand for glutamine is dramatically increased throughout malignant transformation to support increased metabolic demands; namely, provision of catabolic substrates for ATP production and anabolic substrates for the citric acid cycle and subsequent macromolecule biosynthesis, as well as potentiating the uptake of other critical amino acids by acting as an obligate exchange substrate. Elevated expression of glutamine metabolism-related genes, MYC-driven transcriptional events, and increased consumption and reliance on glutamine are all associated with aggressive breast cancers, including the high-risk triple-negative (TN) subtype. We recently showed that in breast cancer cells, glutamine uptake by the small neutral amino acid transporter, ASCT2, is required to sustain TN cell growth in vitro and in vivo. We therefore hypothesized that highly proliferative TN breast cancers that are sensitive to ASCT2 inhibition may have unique metabolic signatures that could be additionally exploited for therapeutic purposes.
Using a targeted metabolomics approach, we combined labeled substrate tracing, liquid chromatography coupled tandem-mass spectrometry (LC-MS/MS), and gas chromatography mass spectrometry (GC-MS) to analyze intracellular levels of key tricarboxylic acid (TCA) cycle intermediates, glycolytic metabolites, fatty acid precursors, and amino acids in human breast cancer cell lines. These analyses revealed distinct metabolic effects when glutamine uptake was blocked in vitro by L-γ-glutamyl-p-nitroanilide (GPNA), a pharmacologic inhibitor of ASCT2. These data confirm a broad reliance on glutamine availability in TN breast cancers, reinforced by TCGA gene expression data showing a specific upregulation of multiple glutamine metabolism enzymes that is completely absent in the luminal A subtype. These data emphasize the link between increased glutamine metabolism and clinically aggressive breast cancers, thus highlighting the therapeutic potential of targeting glutamine metabolism pathways in these patients.
Citation Format: Michelle van Geldermalsen, Lake-Ee Quek, Nigel Turner, Seher Balaban, Andrew Hoy, Qian Wang, Jeff Holst. Glutamine metabolic vulnerabilities define triple-negative from luminal A breast cancer subsets [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B20.
Abstract B21: Improved efficacy of mitochondrial disrupting agents upon inhibition of autophagy in a mouse model of BRCA1-deficient breast cancer
Authors: Yeo Syn Kok , Paul Ritama , Haas Michael , Wang Chenran , Guan Jun-Lin 
Published: August 2018

Abstract
Breast cancer is a heterogeneous disease, and stratification of patients is fundamental to the success of treatment modalities. Breast tumors deficient in BRCA1 are mostly associated with basal-like breast cancers, and targeted therapeutics for this disease subtype are still lacking. In order to address whether autophagy inhibition will be effective in BRCA1-deficient mammary tumors, we generated mice with conditional deletion of an essential autophagy gene FIP200 along with Brca1 and Trp53, through utilization of the K14-Cre transgene. We found that FIP200 deletion suppressed tumorigenesis in the BRCA1-deficient model when compared to wild-type and heterozygous FIP200 controls. However, in contrast to previous studies in the MMTV-PyMT model, tumor growth and the distribution of histologic subtypes were not affected by loss of FIP200. Interestingly, loss of FIP200 decreased mitochondrial mass and oxidative respiratory capacity of these tumor cells, along with a decrease in the phosphorylation of mTOR substrates and transcript levels of genes involved in mitochondrial biogenesis. Importantly, we observed an increased sensitivity to mitochondrial disrupting agents upon loss of FIP200. Consequently, our data showed that combination of an autophagy inhibitor, Spautin-1, along with a mitochondrial complex I inhibitor, metformin, was more effective in limiting oxidative respiratory capacity, colony-forming ability, and tumor growth. Altogether, our results indicate that inhibition of autophagy can increase the benefits of metformin treatment in BRCA1-deficient breast cancers.
Citation Format: Syn Kok Yeo, Ritama Paul, Michael Haas, Chenran Wang, Jun-Lin Guan. Improved efficacy of mitochondrial disrupting agents upon inhibition of autophagy in a mouse model of BRCA1-deficient breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B21.
Abstract B46: Targeting EZH2 reactivates a breast cancer subtype-specific antimetastatic transcriptional program
Authors: Hirukawa Alison , Smith Harvey , Zou Dongmei , Savage Paul , Johnson Radia , Bourque Guillaume , Giguere Vincent , Basik Mark , Dufour Cathy , Park Morag , Muller William 
Published: August 2018

Abstract
Emerging evidence has illustrated the importance of epigenomic reprogramming in cancer, with altered post-translational modifications of histones contributing to the pathogenesis of many tumor types, including breast cancer. However, the contributions of histone modifiers to breast cancer progression are unclear, and how these processes vary between molecular subtypes has yet to be adequately addressed. In this study, we demonstrate that genetic or pharmacologic targeting of the epigenetic modifier Ezh2 dramatically hinders metastatic behavior in a mouse model of breast cancer reflective of the luminal B subtype. We also employ patient-derived xenografts from different intrinsic subtypes of breast cancer to confirm that pharmacologic inhibition of Ezh2 activity hinders metastasis specifically in the luminal B subtype. We further define a molecular mechanism intrinsic to the luminal B subtype whereby EZH2 maintains H3K27me3-mediated repression of the FOXC1 gene, thereby inactivating a FOXC1-driven, antiinvasive transcriptional program. We demonstrate that higher FOXC1 levels are predictive of favorable outcome specifically in luminal B breast cancer patients and establish the use of EZH2 methyltransferase inhibitors as a viable strategy to block metastasis in luminal B breast cancer, where options for targeted therapy are currently limited.
Citation Format: Alison Hirukawa, Harvey Smith, Dongmei Zou, Paul Savage, Radia Johnson, Guillaume Bourque, Vincent Giguere, Mark Basik, Cathy Dufour, Morag Park, William Muller. Targeting EZH2 reactivates a breast cancer subtype-specific antimetastatic transcriptional program [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B46.
Abstract B47: Validation of novel breast cancer drivers using mammary stem cell-based somatic mouse models
Authors: Zhang Zheng , Christin John R. , Wang Chunhui , Ge Kai , Oktay Maja , Guo Wenjun 
Published: August 2018

Abstract
Cancer genomics has provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC) organoid-based approach for rapid generation of somatic genetically engineered mouse models (GEMMs). By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K- targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study has established a robust in vivo platform for functional cancer genomics and has discovered functional breast cancer mutations.
Citation Format: Zheng Zhang, John R. Christin, Chunhui Wang, Kai Ge, Maja Oktay, Wenjun Guo. Validation of novel breast cancer drivers using mammary stem cell-based somatic mouse models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B47.