bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A039: The role of long noncoding RNAs in epithelial to mesenchymal transition and cancer stem cells
Authors: Herschkowitz Jason I. , Coarfa Cristian , Prat Aleix , Toneff Michael J. , Hoadley Katherine A. , Dinger Marcel E. , Mattick John S. , Mani Sendurai A. , Perou Charles M. , Rosen Jeffrey M.
Published: May 2015

The claudin-low subtype is generally triple (ER, PR, HER2) negative and there are currently no targeted agents directed at them. These tumors express low levels of tight and adherens junction genes including claudin 3 and E-cadherin, and high levels of markers associated with epithelial-mesenchymal transition (EMT) including Snail, Twist, and Zeb1/2. Claudin-low tumors are also enriched in signatures derived from human tumor-initiating cells and a sorted population enriched for human mammary stem cells. miRNAs are differentially expressed in claudin-low tumors including low expression of the miR-200 family - regulators of EMT and stemness. MiR-200 overexpression in claudin-low cell lines causes them to lose this classification and to adopt an expression profile of another subtype. While there has been considerable emphasis on miRNAs, our knowledge is still lacking about the role of long noncoding RNAs (lncRNAs) that comprise the majority of the mammalian transcriptome. Here, we have examined the expression profiles of >17,000 lncRNAs in a large set of breast tumors. Like mRNAs and miRNAs, lncRNAs differ dramatically in expression across subtypes and can be used for classification. LncRNAs that are differentially regulated in cell lines induced to undergo EMT are associated with claudin-low tumors and we have identified some of these lncRNAs as potential regulators of the EMT/CSC phenotype. We have begun to study the subcellular localization and potential function of a couple of these candidate lncRNAs using RNA FISH and siRNA knockdown respectively. These results suggest major roles for noncoding RNAs in claudin-low breast tumors and in the regulation of breast cancer stem cells.

Citation Format: Jason I. Herschkowitz, Cristian Coarfa, Aleix Prat, Michael J. Toneff, Katherine A. Hoadley, Marcel E. Dinger, John S. Mattick, Sendurai A. Mani, Charles M. Perou, Jeffrey M. Rosen. The role of long noncoding RNAs in epithelial to mesenchymal transition and cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A039.
Abstract A055: A novel cell growth control pathway repressed by the oncogene TBX2 in breast cancer cells
Authors: D'Costa Zenobia Carmel , Higgins Catherine A. , Williams Richard , Mullan Paul B.
Published: May 2015

Purpose: To identify a therapeutic target downstream of TBX2 which may be used to treat this aggressive subset of breast cancers

T-box 2 (TBX2) is a transcriptional repressor known to drive breast cancer proliferation. It is known to switch off a number of key cell cycle regulatory molecules such as p21WAF1 and p14ARF. A breast cancer Disease Specific Array identified >700 potential TBX2 regulated genes. We have validated one particular target, Cystatin 6 (CST6), to be consistently repressed by TBX2 in a panel of breast cancer cell lines. CST6 is a putative breast tumor suppressor and we observe that exogenous expression of CST6 resulted in cell death of breast cancer cells but not in non-tumorigenic breast cell lines. Mechanistically, we show that TBX2 transcriptionally represses CST6 in an EGR-1 dependent manner rather than through direct CST6 promoter binding.

CST6 is a cysteine protease inhibitor known to repress several members of Clan CA (papain family) and Legumain (LGMN, an asparginyl endopeptidase from the Clan CD family). LGMN has previously been reported to be highly expressed in breast cancers compared to normal breast tissue. Through a range of genomic approaches we showed that LGMN was responsible for driving the proliferation of breast cancer cell lines. Consequently, knockdown of LGMN by siRNA resulted in cell death in breast cancer cell lines overexpressing TBX2. We are in the process of generating small molecule inhibitors against LGMN. Preliminary studies have shown promise with growth inhibitory effects on breast cancer cells but not in non-tumorigenic MCF10A breast cells. LGMN inhibitors may be useful for targeted treatment for breast and other tumors types showing high levels of LGMN expression where it is known to play roles in control of ECM remodeling/turnover, tumor growth and in the development of metastases. LGMN is also known to process and activate a number of downstream oncogenic proteins and we show that one of these proteins, matrix metalloproteinase-2 (MMP2), may contribute to the aggressive traits of LGMN-expressing breast cancer cells.

Conclusions: We have determined a proliferative/invasive pathway employed by breast cancer cells and identified a protein downstream ‘LGMN’ which may be therapeutically targeted to prevent oncogenesis in a subset of breast cancers.

Citation Format: Zenobia Carmel D'Costa, Catherine A. Higgins, Richard Williams, Paul B. Mullan. A novel cell growth control pathway repressed by the oncogene TBX2 in breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A055.
Abstract A077: Post-transcriptional regulation of CXCR4 by the tristetraprolin/HuR axis in breast cancer
Authors: Al-Souhibani Norah A. , Al-Ghamidi Maha , Al-Ahmadi Wijdan , Khabar. Khalid SA
Published: May 2015

Chemokine receptors can direct tumors to distant organs and tissues along a gradient of their respective ligands. One such chemokine receptor is CXCR4 which has been implicated in breast cancer metastasis, notably to the lung, bone, and lymph nodes, where its chemokine ligand, CXCL12, is highly expressed. Expression of the receptor or its ligand is associated with a poor prognosis. Here, we report that aberrations in the post-transcriptional regulation of the CXCR4/CXCL12 signaling pathway play a pivotal role in the metastatic phenotype of breast cancer cells.

We show, utilizing 3′UTR reporter assays, that CXCR4 3′UTR contains an AU-rich element that confers instability to its mRNA and presents it as a prospective target for RNA binding proteins. Indeed, immunoprecipitation of TTP- and HuR-bound mRNA and RNA shift revealed CXCR4 mRNA is a novel target of both proteins suggesting post-transcriptional regulation of the chemokine receptor. We demonstrate correlation between the aberrant TTP/HuR axis and increased CXCR4 expression and metastasis in Tumor Cancer Genome Atlas (TCGA) breast cancer patient data. Furthermore, real-time PCR and western blotting techniques showed that CXCR4 mRNA and protein levels were significantly higher in the invasive breast cancer cell line MDA-MB-231 compared to the non-invasive MCF7 or normal cell lines. Using MDA-MB-231 cells as a model of invasive breast cancer, we attempted to normalize the aberrant TTP/HuR axis by a miR-29a inhibitor which led to de-suppression of TTP expression. This triggered increased instability of CXCR4 mRNA, a down regulation of CXCR4 expression, and a subsequent reduction in the migratory potential of MDA-MB-231 cells. The anti-invasive and anti-migratory effect was similarly obtained by knockdown of HuR and CXCR4. Further regulation of CXCR4 by TTP and HuR was substantiated by mRNA decay assays and 3′UTR reporter experiments. Our findings indicate that CXCR4 is a novel target of post-transcriptional regulation by the RNA binding proteins TTP and HuR and that restoration of a normal balance between TTP and HuR may offer a novel therapeutic approach in the control of metastatic breast cancer.

Citation Format: Norah A. Al-Souhibani, Maha Al-Ghamidi, Wijdan Al-Ahmadi, Khalid SA Khabar. Post-transcriptional regulation of CXCR4 by the tristetraprolin/HuR axis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A077.
Abstract A082: Study of the effects of localized collagen manipulation on the behavior of breast cancer cells
Authors: Clark Ashley N. , Williams James , Padgen Michael , Keely Patricia , Castracane James
Published: May 2015

The tumor microenvironment is complex and heterogeneous, comprised of the extracellular matrix, various cell types, blood vessels, and soluble factors. Multiple interactions occur between tumor cells and their environment which affect cellular behavior and the composition of the tumor microenvironment. Changes to the composition of collagen within the tumor microenvironment have been linked to an increased risk of developing breast carcinoma and an increased invasiveness of breast cancer cells. The goal of this study is to understand how selected, well-controlled manipulations of the collagen composition alter the behavior of an invasive human breast cancer cell line, MDA-MB-231. A BioMEMS device will be loaded with molecules that alter collagen and inserted into a 3-D gel containing MDA-MB-231 cells, to establish a localized gradient of these molecules. The compositional changes include increased collagen concentration through stimulating cellular deposition of collagen, increased collagen crosslinking and inhibition of a collagen cross-linker produced by tumor cells. Confocal reflectance microscopy, atomic force microscopy, and environmental scanning electron microscopy are utilized to characterize the changes induced and the rates at which the changes occur. Changes in gene expression are investigated to determine the pathways each of the collagen manipulations induce which result in the observed changes to cellular behavior. The observed alterations in cellular phenotype resulting from these collagen changes will help to better understand what degree of changes occur within breast carcinoma resulting in cancer progression.

Citation Format: Ashley N. Clark, James Williams, Michael Padgen, Patricia Keely, James Castracane. Study of the effects of localized collagen manipulation on the behavior of breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A082.
Abstract A085: miR-222/221 in aggressive breast cancer
Authors: Leva Gianpiero Di , Piovan Claudia , Cheung Douglas , Briskin Daniel , Kumar Arpan , Nuovo Gerard , Fassan Matteo , Coppola Vincenzo , Garofalo Michela , Croce Carlo
Published: May 2015

MicroRNAs (miRNAs) are small non coding RNAs that regulate gene expression at post-transcriptional level through translational inhibition and/or degradation of mRNA target genes. Recent evidence points to a widespread role for miRNAs in the initiation and progression of tumorigenesis in a plethora of tissues including the mammary gland. Aberrant miRNA expression profiles have been described in breast cancer specimens compared to normal tissues, discriminating breast tumors with different clinico-biological phenotypes. In our previous publications, we have demonstrated that miR-222/221 cluster is highly expressed in aggressive breast cancer and high levels of miR-222/221 may confer a proliferation advantage to cancer cells and resistance to therapeutic agents by repressing several genes such as ERα;, CDKN1B, BIM, FOXO3, CAV1, PTEN. To demonstrate the in vivo role of miR-222/221 cluster in breast cancer initiation and progression, we have genetically deleted miR-222/221 gene in the MMTV-PyVT mouse model that develops multifocal mammary adenocarcinoma and lung metastatic lesions in 80% of the population. The tumor latency and multiplicity were assessed and none of these parameters were significantly affected by miR-222/221 ablation. Interestingly, lack of miR-222/221 impairs the development of lung metastasis with a reduction of the metastatic load of the lung. Moreover, metastases arising from miR-222/221 deficient cells present a greater cytoarchitectural similarity and a reduced rate of mitosis and apoptosis. We also show that loss of miR-222/221 modulates the transcriptome and miRNA expression profiles of mouse breast tumors and cancer cells, stimulating a stronger estrogen receptor alpha transcriptional network and a suppression of the cancer stem cells population.

Citation Format: Gianpiero Di Leva, Claudia Piovan, Douglas Cheung, Daniel Briskin, Arpan Kumar, Gerard Nuovo, Matteo Fassan, Vincenzo Coppola, Michela Garofalo, Carlo Croce. miR-222/221 in aggressive breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A085.
Abstract A087: Angiogenin (ANG) localized at the leading edges of breast cancer cells regulates plasmin formation by plasminogen activation complex (PAC) and cell migration
Authors: Dutta Sujoy , Bandyopadhyay Chirosree , Wilson Lydia , Warshall Case , Bottero Virginie , Johnson Karen E. , Chandran Bala
Published: May 2015

Angiogenin (ANG), a 14-kDa pro-angiogenic protein, had been shown to play a role in cell migration and tumor invasion processes, which involve proteolytic cleavage of plasminogen to generate plasmin. However, the mechanism by which ANG regulates plasmin formation and cell migration was not known. Our studies detected elevated levels of secreted ANG and cell surface-bound ANG in highly invasive metastatic breast cancer cells. By immunofluorescence and immunoprecipitation analysis, ANG was detected at leading edges of the cell surfaces where it colocalized and interacted with members of the plasminogen activation complex (PAC) such as Annexin A2 (A2), Calpactin (S100A10) and Urokinase plasminogen receptor (uPAR). Analysis of lipid raft (LR) and non-lipid raft (NLR) regions of the cell membranes showed the predominance of ANG, A2 and S100A10 in the LR regions. In contrast, uPAR was detected only in the NLR fractions, suggesting that ANG interacts with uPAR at junctions of LR and NLR regions. ANG knockdown in T47D and MDA-MB-231 breast cancer cell lines did not affect the cellular expression of A2, S100A10 and uPAR but decreased cell migration and plasmin formation. Neutralization of ANG with monoclonal antibodies similarly decreased the migration of MDA-MB-231 cells. In the presence of ANG on cell surfaces, uPAR was observed to interact with UPA, which is known to be necessary for proteolytic cleavage of plasminogen by UPA to form plasmin. However, in the absence of ANG, uPAR interacted exclusively with PAI, which is known to inhibit plasmin formation by blocking UPA mediated proteolytic cleavage of plasminogen. Assessment of phosphorylation states of cellular FAK and Src kinases under these conditions further showed that both FAK and Src are dephosphorylated in the absence of ANG. Taken together, our results identified a novel regulatory role of ANG as a bridging protein that facilitates the interaction of uPAR with UPA, which leads to plasmin formation and cell migration necessary for tumor invasion and metastasis of breast cancer cells. These studies also suggest that ANG could serve as a bio-marker for identifying the leading edges of metastatic breast cancer cells and targeting ANG could be a novel method to block breast cancer invasiveness.

Citation Format: Sujoy Dutta, Chirosree Bandyopadhyay, Lydia Wilson, Case Warshall, Virginie Bottero, Karen E. Johnson, Bala Chandran. Angiogenin (ANG) localized at the leading edges of breast cancer cells regulates plasmin formation by plasminogen activation complex (PAC) and cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A087.
Abstract A097: Inhibition of the Na+/H+ exchanger (NHE1) increases susceptibility to paclitaxel in invasive breast cancer cells
Authors: Amith Schammim Ray , Wilkinson Jodi Marie , Fliegel Larry
Published: May 2015

The pH gradient in normal cells is tightly controlled by the activity of various pH regulatory membrane proteins including the Na+/H+ exchanger, NHE1. NHE1 becomes constitutively active in a neoplastic milieu, dysregulating pH homeostasis and altering the survival, differentiation, and proliferation of cells, thus causing them to become tumorigenic. In breast cancer cells, cytoplasmic alkalinization as a result of NHE1 hyper-activity results in an acidic tumor microenvironment that facilitates aggressive cellular proliferation, migration, and invasion leading to tumor metastasis. The pathophysiological role of NHE1 in tumor progression with regards to ion flux is well known, however, the manipulation of pH in and around tumor cells has only recently been considered as a strategy in augmenting cancer therapy. We studied the effect of NHE1 inhibitors EMD87580 ((2-methyl-4,5-di-(methylsulfonyl)-benzoyl)-guanidine) and HMA (5-(N, N-hexamethylene)-amiloride), either alone or in combination with paclitaxel (Taxol), on NHE1 exchanger activity, cell viability, proliferation, migration, and invasive potential, in a highly invasive breast cancer cell line MDA MB 231. We found that cells treated with EMD or HMA in combination with paclitaxel at low doses (0.1nM to 1nM), were significantly more susceptible to cell death than cells treated with paclitaxel or inhibitors alone. While no discernible differences between treatments were observed in cell proliferation rates, a significant reduction in the rate of migration and invasion was observed in cells treated with paclitaxel and either EMD or HMA. To highlight the importance of NHE1 function, we also generated an MDA-MB-231NHE1- knockout cell line for comparison with the wild-type cells that endogenously express NHE1. The NHE1 knockout cell line showed no demonstrable Na+/H+ exchange activity, and much lower rates of proliferation, migration and invasion compared to wild-type cells. Our results demonstrate, for the first time, that inhibition of NHE1 potentially increases the susceptibility of invasive breast cancer cells to paclitaxel-mediated cell death at doses much lower than the established IC50 of paclitaxel in MDA MB 231 cells. Since pH regulation appears to play an integral role in the switch from a normal to a neoplastic microenvironment, these data lend further credence to the importance of NHE1 as a potential target in breast cancer chemotherapy.

Supported by the Canadian Breast Cancer Foundation.

Citation Format: Schammim Ray Amith, Jodi Marie Wilkinson, Larry Fliegel. Inhibition of the Na+/H+ exchanger (NHE1) increases susceptibility to paclitaxel in invasive breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A097.
Abstract A098: Contribution of macrophages to FGFR-dependent breast cancer
Authors: Brady Nicholas J. , Farooqui Mariya , Bohrer Laura , Schwertfeger Kaylee
Published: May 2015

Genomic profiling studies have demonstrated that a number of potentially targetable pathways are capable of contributing to breast cancer, including the fibroblast growth factor receptor (FGFR) pathway. Our studies focus on understanding the mechanisms through which FGFR1 activation in tumor cells leads to pro-tumorigenic changes in the microenvironment. Specifically, using an in an in vivo model of fibroblast growth factor receptor 1 (FGFR1)-induced mammary tumorigenesis, we have found that following FGFR1 activation in mammary epithelial cells, macrophages are recruited to the hyperplastic regions where they are required for angiogenesis and epithelial cell proliferation. To identify the factors that contribute to the pro-tumorigenic microenvironment, we have developed two- and three-dimensional co-culture in vitro models to mimic what is observed in vivo. Using these in vitro models, we have identified that soluble factors derived from FGFR1-activated epithelial cells lead to the upregulation of target genes in macrophages that promote tumor formation, invasion, and migration. Findings from the in vitro studies are further assessed using a mouse mammary tumor model utilizing orthotopic transplants into immunocompetent mice. These model systems will help us identify novel regulators of mammary tumorigenesis and provide new insight into the mechanisms by which macrophages promote tumor formation.

Citation Format: Nicholas J. Brady, Mariya Farooqui, Laura Bohrer, Kaylee Schwertfeger. Contribution of macrophages to FGFR-dependent breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A098.
Abstract A102: Osteopontin produced by myeloid cells determines the outcome of breast cancer metastases
Authors: Colombo Mario P. , Chiodoni Claudia
Published: May 2015

The matricellular protein osteopontin (OPN) combines regulatory and structural functions in the extracellular matrix and, when produced by tumor cells, concurs to progression, cell invasion and metastasis. Little is known about its activity when produced by host cells including that component of tumor stroma. Combining gene silencing in tumor cells and mice deficient for OPN we have dissected the role of tumor and host produced OPN in breast cancer metastasis using 4T1 and BJMC3879 spontaneously metastatic models. While tumor produced OPN favors lung metastasis promoting tumor cell survival in the blood stream, stromal OPN greatly affects the immusuppression at the metastatic site. Immunosuppression is often associated with the expansion of a heterogeneous myeloid population called myeloid derived suppressor cells (MDSC) comprising a granulocytic (Gr-1high) and a monocytic (Gr-1int/low) subset, the latter endowed with higher immune suppressive capacity and characterized by OPN expression. Analysis of MDSC showed that inhibition of lung metastases in OPN-deficient mice goes along with a skewing toward the Gr1high subset, whereas the Gr1int/low subset is more expanded in wild type mice. Moreover, the latter subset, in lungs of tumor-bearing OPN-deficient animals, is less suppressive than its wild type counterpart, and associated with a lower expression of arginase-1, IL-6 and phospho-STAT3. Our results find correlation with human tumors and underscore that different mechanisms characterize tumor- and host-produced OPN activities in breast cancer metastasis and reveal a new function for host-derived OPN as regulator of the local immune response in the metastatic lung niche.

Citation Format: Mario P. Colombo, Claudia Chiodoni. Osteopontin produced by myeloid cells determines the outcome of breast cancer metastases. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A102.
Abstract A103: The neurotransmitter norepinephrine and β2-AR activation of MB-231 breast cancer cells alters fibrillar collagen structure
Authors: Dawes Ryan P. , Burke Kathleen A. , Stastka Petr , Brown Edward B. , Madden Kelley S.
Published: May 2015

The impact of psychosocial stress on breast cancer pathogenesis has become an area of intense research in the past several years. Sympathetic nervous system (SNS) activation and release of the catecholamine norepinephrine (NE) is a major stress pathway that promotes breast cancer pathogenesis. We have evidence that SNS activation can regulate breast tumor growth and metastasis by altering the tumor extracellular matrix (ECM), specifically fibrillar collagen. Alterations in fibrillar collagen microstructure can be detected using second harmonic generation multiphoton laser scanning microscopy (SHG MPLSM). This non-linear optical technique takes advantage of endogenous light scattering by non-centrosymmetric structures, including collagen. Stress-induced changes to the tumor collagen microstructure can be quantified by measuring the ratio of forward- to backward-scattered light (F/B ratio), as well as the directional coherence of resident collagen fibers. Here, we demonstrate that NE can directly stimulate breast cancer cells and tumor stromal cells to elicit changes in collagen microstructure. In these experiments, cells were pretreated with NE, washed to remove NE, and placed in 3D-type I collagen matrices. Pretreatment of MDA-MB-231 metastatic breast cancer cells with NE significantly lowered the F/B ratio and decreased fiber directional coherence of type I collagen gels. Treatment of MB-231 cells with the β2-adrenergic receptor (β2-AR) selective agonist, terbutaline, mimicked the effects of NE. Furthermore, NE pretreatment of the β-AR-negative metastatic breast cancer line, 4T1, did not elicit alterations in SHG F/B ratio or fiber coherence in collagen gels, compared to control cells. These results suggest that NE acts through tumor cell β2-AR to alter collagen microstructure. Future experiments will elucidate the molecular mediators of this collagen reorganization and determine the impact of such changes on tumor metastatic capacity. These experiments will provide new insights into mechanisms underlying stress-induced tumor pathogenesis that may reveal new therapeutic avenues for the treatment of breast cancer.

Citation Format: Ryan P. Dawes, Kathleen A. Burke, Petr Stastka, Edward B. Brown, Kelley S. Madden. The neurotransmitter norepinephrine and β2-AR activation of MB-231 breast cancer cells alters fibrillar collagen structure. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A103.
Abstract A121: Retinoic acid signaling and initiation of mammary gland development
Authors: Cho Kyoung-Won , Moon Woo-Kyung
Published: May 2015

Mammary glands are skin appendages whose development is initiated through continuous cell-cell interactions between ectoderm and adjacent mesenchyme. Considerable progress has been made in uncovering the molecular basis of these interactions in mammary gland formation in mouse embryos, including the network of initiating signals comprising Fgfs and Wnts, Bmps involved in gland positioning and transcription factors, Tbx3 and Lef1, essential for mammary gland development. Here we provide evidence that retinoic acid signaling may also be involved. Raldh2 is expressed in E10-E10.5 mouse embryos in ventral hypaxial extensions of somites adjacent to the flank region where mammary buds 2, 3 and 4 develop. This pattern of expression overlaps with that of Fgf10, known to be required for mammary gland formation. Manipulation of retinoic acid levels in organ cultures of E10.5 mouse embryo flank by adding either retinoic acid or citral, a retinoic acid synthesis inhibitor, resulted in changes in expression of genes involved in retinoic acid homeostasis and also showed that retinoic acid signaling is required for Tbx3 expression while high levels of retinoic acid signaling inhibit Bmp4 expression and repress Wnt signaling. The results of experiments using RNAi against Tbx3 and Wnt10b suggest that manipulating RA signaling in the mammary gland forming regions. Furthermore, RA affects expression of genes involved in mammary gland initiation. We produce a molecular model for mammary gland initiation incorporating retinoic acid signaling.

Citation Format: Kyoung-Won Cho, Woo-Kyung Moon. Retinoic acid signaling and initiation of mammary gland development. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A121.
Abstract A129: Contributions of RON downstream signaling to breast cancer progression
Authors: Faham Najme , Bieniasz Magdalena , Welm Alana
Published: May 2015

Since its discovery in 1993, various investigations have explored different roles of RON receptor tyrosine kinase in cancer pathogenesis. RON (also known as MST1R), a receptor tyrosine kinase with significant homology to c-Met, exhibits increased expression in at least 47% of human breast cancers, and deregulated expression of the RON signaling axis has been correlated with reduced metastasis-free survival. RON is known to activate multiple signaling pathways in vitro, including MAPK, phosphatidylinositol-3 kinase (PI3K), Src tyrosine kinase, phospholipase C-γ (PLC-γ) and β-catenin, largely through direct binding of SH2-domain containing adaptor proteins to the C-terminal docking site. However, the relationships of each of these signal transduction pathways to specific biologic responses relevant to cancer progression and metastasis are still poorly defined. To this end, we have taken advantage of a strategy used for c-Met in order to increase the binding affinity of individual adaptors to the docking site, which promotes signaling through one pathway at the expense of the others. The C-terminal part of RON contains two tandem tyrosines in a degenerate motif that when phosphorylated bind different SH2-containing molecules in a competitive manner. To amplify the signaling through particular pathways, we mutated the multifunctional docking site of RON to optimal binding motifs for PI3K, Src, Grb2 or PLC-γ. Mutations were introduced to the docking site using pMSCV retroviruses expressing human wild type Ron cDNA. As a negative control, two essential tyrosines in the docking site were replaced by phenylalanine to eliminate the transforming capability of RON. Viral transduction of the generated mutants or wild type RON was performed in MCF-7 and T47D breast cancer cells and LoVo colon cancer cells. Analysis of mRNA and protein showed the same expression levels among the mutants and wild type infected cells, indicating similar stability of the mutants compared with WT-RON. We report an analysis of RON signaling through the aforementioned pathways in the presence and absence of its ligand, macrophage stimulating protein (MSP). Our data indicate that the MAPK, PI3K, PLC-γ and Src pathways are differentially activated in the various cellular contexts we tested. Moreover, the activation of each signaling pathway occurs with different kinetics and with differential responses to MSP concentration.

Citation Format: Najme Faham, Magdalena Bieniasz, Alana Welm. Contributions of RON downstream signaling to breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A129.
Abstract A132: The HER4 intracellular domain (4ICD) functions as a potent estrogen receptor coactivator mediating estrogen stimulated proliferation of breast tumor cells
Authors: Han Wen , Jones Frank
Published: May 2015

Among women with breast cancer more than 70% present with tumors expressing the estrogen receptor (ER) and will be treated with a therapeutic combination that includes endocrine therapy. Unfortunately, over 50% of these women fail therapy and develop incurable and often lethal metastatic disease. Disengaging ER interactions with essential transcriptional coactivators represents an effective approach to suppress ER driven breast cancer. To this end we have identified the HER4 intracellular domain (4ICD) as a potent estrogen receptor coactivator modulating estrogen induced gene expression and mediating estrogen stimulated proliferation of breast tumor cells. In multiple clinical studies, HER4 expression is significantly associated with ER positive tumors with up to 90% of ER positive breast tumors coexpressing HER4. These clinical observations imply a strong tumor cell selection for HER4 and ER coexpression. Estrogen stimulation of the ER and HER4 positive MCF-7 breast tumor cell line results in proteolytic processing of HER4 to release 4ICD. Significantly, estrogen stimulates nuclear translocation and binding of the 4ICD/ER complex to promoters containing estrogen response elements. Here we performed microarray gene expression analysis comparing MCF-7 cells with endogenous HER4 expression to MCF-7 cells where HER4 was stably suppressed. We found that estrogen significantly stimulated expression (2 fold and above) of 786 genes and 399 (51%) of these genes also required HER4 expression. Furthermore, chromatin immunoprecipitation (ChIP) analysis of two ER-inducible genes, progesterone receptor (PgR) and stromal-derived factor 1 (SDF-1) shows that 4ICD binds to PgR and SDF-1 gene promoters suggesting that it is required for efficient promoter binding of ER to certain ER-mediated genes. These results clearly indicate that 4ICD is an important ER coactivator that enhances ER promoter interactions. Finally, we have evidence that HER4/4ICD is required for estrogen stimulated tumor cell proliferation raising the possibility that disrupting the interaction between 4ICD and ER would represent a novel therapeutic approach to suppress breast tumorigenesis. In the future, we propose to identify additional transcription factors recruited with the ER/4ICD genomic complex.

Citation Format: Wen Han, Frank Jones. The HER4 intracellular domain (4ICD) functions as a potent estrogen receptor coactivator mediating estrogen stimulated proliferation of breast tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A132.
Abstract A133: The DNA cytosine deaminase APOBEC3B drives mutagenesis in breast cancer
Authors: Harris Reuben
Published: May 2015

A typical breast cancer accumulates hundreds to thousands of somatic mutations. Breast cancer mutation spectra are often dominated by C-to-T transition mutations, which occur in a mostly dispersed manner but can also occur in localized clusters called kataegis. Several lines of evidence indicate that the innate immune DNA cytosine deaminase APOBEC3B is the primary cause of these mutations. First, APOBEC3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Second, endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Third, APOBEC3B knockdown causes a corresponding decrease in genomic uracil concentrations, mutation frequencies, and C-to-T mutation levels in breast cancer cell lines. Fourth, the biochemical signature of APOBEC3B is over-represented in breast cancer mutation spectra. Fifth, breast carcinomas that express high levels of APOBEC3B have twice as many mutations as those expressing low levels. Finally, high levels of APOBEC3B associate with more aggressive disease including shorter durations of survival. All of these data are consistent with a model in which APOBEC3B-catalyzed DNA cytosine deamination provides a chronic source of DNA damage and mutagenesis in breast carcinomas that may help to explain how some tumors evolve rapidly and manifest gross heterogeneity.

Citation Format: Reuben Harris. The DNA cytosine deaminase APOBEC3B drives mutagenesis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A133.
Abstract A136: Deconstructing stemness from the fetal mammary perspective
Authors: Spike Benjamin T. , Engle Danielle D. , Lin Jennifer C. , Kelber Jonathan A. , La Justin , Cheung Samantha K. , Booker Evan , Rodewald Rose , Dravis Christopher , Gray Peter C. , Wahl Geoffrey M.
Published: May 2015

Somatic stem cells govern the development, maintenance and regeneration of tissues, and their dysregulation is associated with diverse pathologies including cancer. Given the significance of these cells both biologically and therapeutically, it is critical to elucidate the signaling mechanisms that dictate their behavior. Our recent work on the fetal mammary gland has dissected complementary roles for the stem cell compartment and stromal cues in fetal mammary stem cell function. We also identified significant similarities between the gene expression profiles of some of the most aggressive and untreatable forms of human breast cancer and those of the mouse fetal stem cell enriched mammary epithelial and stromal fractions. These similarities suggest that stem cell regulatory mechanisms operative during organogenesis, including those specified by the stromal niche, may serve as novel therapeutic targets in these cancers.

We also discovered significant gene expression heterogeneity within the fetal stem cell compartment when analyzed at single cell resolution. While such heterogeneity likely reflects the natural diversity of stem and non-stem cell states comprising the system, it confounds the interpretation of expression profiles from mixed populations and the delineation of stem cell specific and niche specific molecular components. Our current objective is to utilize genome wide single cell RNA-Sequencing to hasten the identification of functionally relevant, stem cell specific regulators and markers in the context of this multi-cellular/multi-signal system. Among other candidates, we have identified the Cripto/GRP78 signaling axis as a critical regulator of mammary stem cell activity, and have developed novel molecular genetic tools to temporally control the activity of candidate stem cell regulators in vivo in normal and neoplastic contexts. Integration of these tools promises to deliver a new molecular understanding of stem cell biology in the mammary gland and other systems and of stem cell-like cancer cells.

Citation Format: Benjamin T. Spike, Danielle D. Engle, Jennifer C. Lin, Jonathan A. Kelber, Justin La, Samantha K. Cheung, Evan Booker, Rose Rodewald, Christopher Dravis, Peter C. Gray, Geoffrey M. Wahl. Deconstructing stemness from the fetal mammary perspective. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A136.
Abstract B056: p70S6K activity drives local relapse in breast cancer
Authors: Segatto Ilenia , Berton Stefania , Sonego Maura , Massarut Samuele , Perin Tiziana , Baldassarre Gustavo , Belletti Barbara
Published: May 2015

For early breast cancer (EBC) patients local relapse represents what mostly influences disease outcome. Notably, 90% of local recurrences occur at or close to the same quadrant of the primary cancer. Surgery itself and the consequent process of wound healing have been proposed to stimulate local recurrences via pathway(s) still to be clarified. Our previous studies implicated p70S6K pathway in breast cancer cell response to post-surgical inflammation, supporting the hypothesis that it may be crucial also for breast cancer recurrence.

We designed an in vivo experimental model resembling the course of human BC, in which MDA-MB-231 breast cancer cells were bilaterally injected in nude mice mammary fat pads and, when primary tumors were grown, masses were surgically removed under anesthesia. After recovering, mice were followed up to detect appearance of local relapse. Using this model, we dissected the role of p70S6K during breast cancer growth and recurrence by modulating its activity using both genetic and pharmacologic approaches.

Our results showed that p70S6K positively contributed to proliferation and survival programs in breast cancer cell lines. In vivo, the analysis of the impact of p70S6K on primary tumor growth highlighted that a robust p70S6K signaling was required for the initiation of the tumor masses. But, more importantly, interfering with p70S6K activity strongly impaired local relapse. A three-day schedule of peri-operative treatment using specific pharmacological inhibition of p70S6K1 was sufficient to reduce by 83% the rate of local recurrence. We showed that inhibition of p70S6K activity induced apoptosis in cells challenged to survive in a hostile environment, supporting the idea that p70S6K signaling is necessary for the survival of isolated cancer cells in breast microenvironment, following surgery. The significance of our results was confirmed in human EBC specimens, proving that p70S6K activity is robustly induced by surgery, also in human patients.

Taken together, our results provide a biological rationale for peri-operative treatment targeting p70S6K pathway, directed to compensating the harmful consequences of surgery and to restraining local recurrence in early breast cancer patients.

Citation Format: Ilenia Segatto, Stefania Berton, Maura Sonego, Samuele Massarut, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. p70S6K activity drives local relapse in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B056.
Abstract B063: Tumor targeting and diagnostic applications of glycosylated nanotubes
Authors: Singh Ravi N.
Published: May 2015

The goal of this research is to develop a new agent based on multiwalled carbon nanotubes (MWCNT) for the diagnosis and monitoring of advanced breast cancer. MWCNT consist of sheets of graphene carbon rolled into multiwalled tubes and possess many properties that make them extremely useful in biomedical applications, including targeted molecular imaging. Foremost is their ability to deliver large numbers of imaging agents per each targeted molecular recognition event, which can improve the sensitivity of imaging; secondly, they can deliver several different types of imaging agents (such as magnetic resonance imaging or photoacoustic agents) to perform multimodality imaging; thirdly, they can be used for therapeutic applications including chemotherapeutic drug or gene delivery, and as mediators for photothermal cancer therapy. As early and accurate diagnosis is still the most effective approach to treating cancer, improvements in the use of imaging technology to detect and monitor cancer will have a dramatic effect on human health. Breast cancer cells have alterations in the normal metabolism of the sugar, glucose, and exhibit increased glucose uptake and decreased glucose clearance. The hypothesis to be tested is that MWCNTs targeted to glucose transporters will be taken up by cancer cells with high efficiency, and that such MWCNT can be engineered to be effective cancer diagnostic agents. To this end, we have chemically engineered MWCNTs to display deoxyglucose on their surface, which rendered them water soluble, and targets them to cancer cells. It is anticipated that this agent will serve as the basis for a multimodal platform for future combined cancer therapy and diagnostic applications. We now report on our initial findings with this agent, and describe: 1) a new, glycosylated MWCNT which can be traced by radiolabelling; 2) data on clinically-relevant issues relating to blood compatibility, and preclinical data on toxicity (clot formation), biodistribution, blood, urinary, and fecal clearance of intravenously injected glycosylated MWCNT, all of which will be essential for any future translational applications; 3) a new nanomedical platform technology, developed specifically to target and diagnose invasive breast cancer, but with broad applicability to many types of cancer.

Citation Format: Ravi N. Singh. Tumor targeting and diagnostic applications of glycosylated nanotubes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B063.
Abstract B068: Inhibition of GSK-3: A novel therapeutic approach for breast cancer treatment
Authors: Ugolkov Andrey , Gaisina Irina , Malin Dmitry , Kulesza Peter , Kozikowski Alan , Cryns Vincent , Mazar Andrew
Published: May 2015

NF-kappaB is known to regulate human breast cancer progression, metastasis and chemoresistance. Glycogen Synthase Kinase-3beta (GSK-3beta), a serine/threonine protein kinase, is an essential positive regulator of NF-kappaB transcriptional activity and thereby GSK-3beta may represent a potential therapeutic target in human breast cancer. Our objectives were to determine the expression pattern of GSK-3beta in human breast cancer, to assess the effect of GSK-3 inhibition on breast cancer cell proliferation, survival and chemoresistance, and to evaluate our lead GSK-3 inhibitors alone and in combination with chemotherapeutic drugs for the targeted therapy of breast cancer. Using nuclear/cytosolic fractionation, Western immunoblotting and immunohistochemical staining, we observed aberrant nuclear accumulation of GSK-3beta in five breast cancer cell lines and in 7 of 8 primary human breast carcinomas and corresponding lymph node metastases whereas no detectable expression of GSK-3beta was found in benign breast tissue. Pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of breast cancer cells. Our in vitro comparison studies demonstrated that our lead GSK-3 inhibitor 9-ING-41 was significantly (30 times) more effective than GSK-3 inhibitor LY2090314 (Eli Lilly), currently in mid-stage clinical trials, in terms of growth inhibition of breast cancer cells. Inhibition of GSK-3 resulted in decreased expression of the NF-kappaB target genes Bcl-2 and XIAP and induction of apoptosis. Moreover, our lead GSK-3 inhibitor 9-ING-41 and doxorubicin acted synergistically to suppress growth of breast cancer cells. Our preliminary in vivo experiments demonstrated that monotherapy with our lead GSK-3 inhibitor 9-ING-41 (20 mg/kg) for 48 hours inhibited proliferation (as shown by BrDU assay) and induced apoptosis (as shown by TUNEL assay) in MDA-MB-468 orthotopic xenograft tumors. Moreover, in vivo toxicology study demonstrated that intravenous injections of 9-ING-41 did not adversely affect mouse complete blood counts or cause overt pathological changes in mouse organs. Our results identify GSK-3beta as a new potential drug target in breast cancer and suggest that inhibition of GSK-3 is a novel therapeutic approach to overcome NF-kappaB-mediated chemoresistance in breast cancer.

Citation Format: Andrey Ugolkov, Irina Gaisina, Dmitry Malin, Peter Kulesza, Alan Kozikowski, Vincent Cryns, Andrew Mazar. Inhibition of GSK-3: A novel therapeutic approach for breast cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B068.