bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Ribonucleoside Analog, 8-Chloro-Adenosine, Inhibits the mTOR Pathway and Induces Autophagy.
Authors: Stellrecht C. , Shentu S. , Gandhi V. , Gandhi V. 
Published: December 2010

Abstract
In contrast to deoxyribose or arabinose containing nucleoside analogs that are currently established for cancer therapeutics, 8-chloro-adenosine (8-Cl-Ado) possesses a ribose sugar. This unique nucleoside analog is RNA directed and is in a phase I clinical trial for hematological malignancies. Preclinically, it has been also found to be cytotoxic to a number of epithelial and neuronal cell lines. Previously, we demonstrated that in the breast cancer cell lines, MCF-7 and BT-474, 8-Cl-Ado accumulates as a triphosphate reaching its plateau by 6 h of treatment with 10 μM 8-Cl-Ado. This accumulation was coupled with a parallel decrease in the level of the endogenous ATP pool by 60 and 40%. These changes in nucleoside levels then lead to ∼50% inhibition of RNA synthesis. The net effect of these metabolic changes results in a growth inhibition of ∼90% after 5 d treatment with 10 μM 8-Cl-Ado as measured by MTS assay. This growth inhibition was not due to a cell cycle arrest. Additionally, flow cytometry analysis of annexin V staining showed only a modest induction of apoptosis first detected within 24 h of treatment and reached ∼30% by 5 d. Because the low level of apoptosis can not account for high percent of growth inhibition suggests alternative mechanism(s) for the cytotoxic activity of 8-Cl-Ado. Depletion of the cellular ATP pool is often associated with the activation of AMP-activated protein kinase (AMPK) through its phosphorylation on Thr172. Correspondingly, we demonstrated that 8-Cl-Ado treatment of MCF-7 and BT-474 cells induced AMPK Thr172 phosphorylation within 7 h. Once activated, AMPK alters various energy regulating signaling cascades which includes the mTOR pathway. Inhibition of mTOR leads to a decrease of energy depletion by reducing protein translation and promotes nutrient restoration through the induction of autophagy. 8-Cl-Ado treatment of MCF-7 and BT-474 cells was associated with a decrease in the phosphorylation of the mTOR translation targets, 4E-BP1 and p70S6K within 8 h of treatment. Likewise, there was an increase in the conversion of microtubule-associated protein 1 light chain 3B (LC3B)-I to its lipidated form, LC3B-II, which is an autophagosome marker. Consistent with the immunoblot analysis, fluorescence microscopic analysis of 8-Cl-Ado-treated MCF-7 cells and BT-474 cells expressing a GFP-LC3 construct also demonstrated an increase in the formation of autophagosomes. Finally, acridine orange staining of 8-Cl-Ado treated MCF-7 and BT-474 cells demonstrated the formation autophagolysosomes. This was further confirmed in MCF-7 cells by monodansylcadaverine staining. In conclusion, our data indicates that 8-Cl-Ado treatment induces autophagy in breast cancer cells which in part elicits its growth inhibitory effects on these cells. Based on this biological activity, we are planning to test 8-Cl-Ado in the clinic for patients with breast cancer.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6116.
Proteomic Discrimination of Well- from Poorly-Differentiated Breast Carcinomas.
Authors: Ellsworth R. , Seeley E. , Ellsworth D. , Deyarmin B. , Hooke J. , Sanders M. , Caprioli R. , Shriver C. 
Published: December 2010

Abstract
Background: Pathological grade is a useful prognostic factor for stratifying breast cancer patients into favorable (well-differentiated tumors) and less favorable (poorly-differentiated tumors) outcome groups. The current system of tumor grading, however, is highly subjective and a large proportion of tumors are characterized as intermediate-grade, making determination of optimal treatments difficult.Methods: Primary breast tumor specimens from patients diagnosed with well- (n=27) and poorly-differentiated (n=51) invasive ductal carcinoma were obtained from patients enrolled in the Clinical Breast Care Project. Frozen tissues were sectioned and mounted on gold coated MALDI target plates for protein expression profiling. Hematoxylin and eosin (H&E) stained slides were prepared from serial sections for histological characterization. MALDI matrix was deposited as individual spots on the tissue sections in a histology directed manner to assay specific areas and tissue types of interest. Mass spectral data were then acquired from multiple sites across each tissue section.Results: 129 features were observed in well-differentiated and 132 in poorly-differentiated tumors. While the majority of features detected were similar between the two groups, 6 protein features were expressed at significantly lower and 12 at significantly higher levels in the poorly-differentiated tumors, including increased expression of Calgranulin A and Calgizzarin.Conclusions: Protein expression differences detected here suggest that well- and poorly-differentiated invasive breast tumors are molecularly distinct diseases and that these protein changes may contribute to the structural integrity of the tumor cell. In particular, calgranulin A and calgizzarin are members of the S100 protein family, and function in processes such as cell proliferation and differentiation. Further refinement of this differentiation protein signature may not only improve our understanding of the biological processes involved with tumor grade but provide pathologists with new molecular tools to classify breast tumors and reduce the subjectivity associated with current grading criteria.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6126.
The Role of Stromal Cells in Tumor Metastases.
Authors: Pieters A. , Duda D. , Steller E. , Fukumura D. , Jain R. 
Published: December 2010

Abstract
Background: In 1889, Stephen Paget hypothesized that metastatic cells - the “seeds” - only grow in the secondary sites - the “soil” - with a permissive microenvironment. Here, we show that the “seeds” can carry their own “soil” - activated fibroblasts from primary site of metastatic tumors. Moreover, we report that carryover of these non-malignant host-derived cells during metastatic cell colonization in the lungs can increase cancer's metastatic efficiency.Materials and Methods: We use multiple animal models and two metastatic tumor cell lines (LLC1 and LA-P0297) to study the composition, origin and role of fragments shed by tumors in the metastatic process.Results: we have shown that, in addition to single cells (81%), tumors shed fragments consisting of both tumor and host cells. We show that these fragments are more viable in the circulation compared to tumor cells alone (p&lt;0.05). When we selectively deplete the host cells in the tumor microenvironment the spontaneous formation of metastases is significantly reduced three weeks after primary tumor resection compared to control mice (p&lt;0.05). Next, survival was significantly increased in mice with metastases after co-implantation of carcinoma-associated fibroblasts (CAFs) isolated from breast cancer patients together with LLC1 cells, but had no effect in mice implanted with LLC1 cells alone (p&lt;0.01) Lastly, we have assessed the nature of these host stromal cells using immunohistochemistry and RT-PCR and indentified the majority of them as being of mesenchymal/fibroblast lineage.Discussion: We found that tumor-associated stromal cells can escape primary tumors along with cancer cells, survive in blood circulation and in metastatic nodules. Pre-existence of a tissue-like structure in the form of heterotypic cell fragments may increase the viability of cancer cells in blood circulation and modulate metastasis efficiency. How this mechanism contributes to the organo-tropism of certain metastatic tumors is not known. But demonstration of the involvement of some of the same tumor “soil” in both local and distant tumor invasion has important conceptual and clinical implications for colonization during metastasis progression.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6151.
Implication of a Pro-Apoptotic Protein, Prostate Apoptosis Response-4 (Par-4) in Breast Cancer.
Authors: Ye L. , Mansel R. , Jiang W. 
Published: December 2010

Abstract
Background: Par-4, Prostate Apoptosis Response-4, is pro-apoptotic in certain cell types and has been implicated in the development and progression of solid tumours. However, the role of Par-4 in breast cancer remains unclear. The current study investigated the role played by Par-4 in breast cancer.Methods: The expression of Par-4 was examined in a breast cancer cohort using quantitative real time PCR and immunohistochemistry (IHC). The biological functions of Par-4 in breast cancer cells were tested by creating breast cancer cell lines that had differential Par-4 expression. The cell functions were examined using a variety of in vitro assays, including growth, migration and invasion. The implication of Par-4 in breast cancer was further tested in an in vivo tumour model.Results: A marked decrease of both Par-4 mRNA and protein was seen in breast tumours compared with background tissues. Par-4 staining was cytoplasmic in both mammary epithelial cells and cancer cells in the tissues. The level of Par-4 transcripts in primary tumours was decreased in the patients with poor prognosis and worse clinical outcome. The overall survival was poor in the patients with lower Par-4 transcript level, p=0.0082 compared with the patients who had higher expression. Patients with higher expression level also had longer disease free survival (133.1 months (95% CI, 121.1-145.1 months)), p=0.0597 compared with patients who had lower expression (118.4 months (95% CI, 103.2-133.5 months)). Over-expression of Par-4 in MDA-MB-231 cells (MDA-MB-231Par4exp) resulted a reduction of cell growth, migration and invasion in vitro. The invaded cell number of MDA-MB-231Par4exp was 17.9±2.5, p&lt;0.01 compared with both MDA-MB-231WT(30.8±2.6) and MDA-MB-231pEF(34.1±6.5) controls. This also led to a decreased in vivo tumour growth, in an athymic mice model.Conclusions: Taken together, decreased Par-4 expression in breast cancer correlates with poor prognosis and shorter survival. Par-4 suppresses growth, migration and invasiveness of breast cancer cells in vitro, and also inhibits in vivo tumour growth. It suggests that Par-4 plays critical roles in the progression of breast cancer.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6155.
Tumor Invasion and Metastasis Initiated by mir-106b in Breast Cancer by Targeting BRMS1 and RB.
Authors: Pan S. , Yu F. , Gong C. , Song E. 
Published: December 2010

Abstract
Background: MicroRNAs (miRNAs) are noncoding RNAs that have recently been demonstrated to have an important role in tumor metastasis. Mir-106b family is reported to promote the proliferation of cancer as an oncogene. However, whether mir-106b has roles in breast cancer metastasis and EMT remains unknown. Therefore, functional characterization of mir-106b in breast cancer cells would potentially facilitate our understanding of the development of breast cancer.Methods: Mir-106b expression and location in primary breast cancer samples from patients with metastasis were compared to the samples without metastasis by in situ hybridization analysis. Differentially expressed miRNAs in MDA-MB-231 breast cancer cell line were quantified by qRT-PCR and Northern Blot followed by scratch assays, migration assay, Boyen chamber assay and invasion assay to determine the significance of mir-106b in metastasis. Lentivirus-mediated miRNA transduction was used to determine the significance of mir-106b function. Target scan and luciferase reporter assay were used to identify the targets of mir-106b. Mir-106b targets' gene expression (BRMS1 and RB) was determined by qRT-PCR and western blot. The expression of E-cadherent by immunofluorescence and Western Blot were used to determine the regulation of mir-106b on the effect of TGF- β induced EMT.Results: Mir-106b was dramatically increased in primary breast cancer samples from patients with metastasis compared to the samples without metastasis, which is negatively correlated with patients' prognosis. MDA-MB-231 cells transfected with lentivirus-mir106b had a higher ability of invasion and migration. Conversely, transfecting mir-106b ASO into MDA-MB-231 cells decreased the invasion and migration ability in vitro. Silencing BRMS1 (breast cancer metastasis suppressor 1) and RB (retinoblastoma), which were proved to be new targets of mir-106b by luciferase assay, showed the similar results with mir-106b over-expressing. Furthermore, the expression of mir-106b in MCF10A cells was increased during TGF-βinduced EMT, while TGF-β-induced EMT was inhibited in MCF10A cells transfected with mir-106b ASO. Furthermore, we found that mir106b-expressing MDA-MB-231 cells gave rise to more tumors and metastasized more frequently in vivo.Conclusion: mir-106b can promote breast cancer invasion and metastasis by targeting BRMS1 and RB. Mir-106b mediates TGF-β-induced EMT, which is an early process of tumor metastasis. Therefore, mir-106b may be an effective maker to predict prognosis of breast cancer patients, and a new targets to treat breast cancer.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6157.
The Role of Dragon (Repulsive Guidance Molecule-B, RGM-B) in Human Breast Cancer.
Authors: Li J. , Ye L. , Sanders A. , Mansel R. , Jiang W. 
Published: December 2010

Abstract
Introduction: Repulsive guidance molecules (RGMs) are involved in embryonic development and iron homeostasis. RGM-A mediates repulsive axonal guidance and neural tube closure, and RGM-C is mutated in juvenile hemochromatosis. RGM-B, also known as Dragon, is a myelin-derived inhibitor of axon growth in the central nervous system. The RGM family was also identified as co-receptors of bone morphogenetic proteins (BMPs), a group of proteins that are involved in development of bones, the differentiation and progression of cancer. However, the role of RGMs played in breast cancer remains unclear. In the present study, we examined the pattern of expression of the RGM family in human breast cancer cells and investigated the impact of Dragon (RGM-B) on BMP-induced cell function in breast cancer cells.Material and Methods: Conventional RT-PCR was performed to screen the expression of RGMs in human breast cancer samples and a range of breast cancer cell lines. Dragon/RGM-B ribozyme transgenes were generated and in order to knock down the Dragon transcript. Subsequently, MDA-231RGMB-Knock-down variants were created by way of the transgenes. A series of cell function assays were employed to investigate any biological effects upon RGM-B knockdown on the breast cancer cells as well as cell's response to recombinant BMP proteins.Results: RGM-A and RGM-C transcripts were barely detectable in breast cancer cell lines (MDA-MB-231 and MCF-7) and tissues. However RGM-B transcripts were expressed in both cell lines. Using anti-RGM-B transgenes, MDA-231RGMB-Knock-down variant cell lines, in which RGM-B transcripts were knocked down, were created. Compared with wild type and control transfection, MDA-231RGMB-Knock-down variants displayed a significant increase in both the adhesiveness and cells growth (p &lt; 0.05, vs the respective controls, for adheresion and for cell growth). Interestingly, RGM-B knockdown did not have any significant effects on the invasiveness of the cells. Finally, wild type breast cancer cells and the RGMB knock down variant cell lines showed a similar response to the treatment with rhBMP-11, indicating that RGM-B is less critical in BMP-11 mediated cell functions.Conclusion: The present study is the first to examine the role of RGM-B in breast cancer and has demonstrated that knock-down of RGMB could enhance breast cancer cells' ability to grow and attach, indicating that RGM-B may act as an inhibitor in breast cancer. This property is unique to RGMB, as this ability is not associated with any other member in the RGM family.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6158.
Role for CD44 in Enhancing Invasion, Migration, and Growth of Triple Negative (TN) Breast Cancer Cells.
Authors: Iida J. , Nesbella M. , Lehman J. , Mural R. , Shriver C. 
Published: December 2010

Abstract
Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and her-2/neu receptors. It is widely recognized that TN breast cancers have a poorer prognosis than any other subtypes of breast caner. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of growth and invasion in the tissues provides insight into developing novel approaches to lower the mortality from TN breast cancer.Neoplastic epithelial cells in breast carcinomas interact with various components in the tissue microenvironment including extracellular matrix (ECM) and mesenchymal cells. Recent studies identified CD44 as a metastasis-related molecule with multiple functions by promoting cell-cell and cell-ECM interactions. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. In the standard form (CD44s), 10 of the 20 exons are translated. Multiple variant isoforms exist (CD44v1-10) which arises from alternate mRNA splicing of the remaining 10 exons. In contrast to the ubiquitous expression of the standard form of CD44, splice variants are highly restricted in their expression in normal or malignant tissues. Indeed, CD44 variants containing v3, v5, v6, v7-8, v10 exons are expressed in malignant breast cancer tissues. However, there is limited information regarding the biological functions of these exons to promote tumor invasion and metastasis. The goal of this study is to evaluate specific exon(s) of CD44 expressed on TN breast cancer cells for promoting tumor progression and metastasis.In order to approach this goal, we utilized three TN cell lines (HCC38, HCC1937, and HCC1806) as model systems to evaluate CD44 in regulating invasion, migration, and growth in extracellular matrix (ECM) environments. Reverse transcriptase (RT)-PCR analysis using exon specific primers indicate that these cells expressed CD44v8-v10 and CD44s. We demonstrated that an inhibitory antibody against exon v10 of CD44 significantly inhibited b1 integrin-mediated migration and invasion into Matrigel and type I collagen gel. Importantly, this antibody also inhibited three dimensional (3D) growth which is a b1 integrin-independent process. The significant inhibition of these processes was also achieved when a FLAG-fusion exon v10 peptide (FLAG-v10, in which FLAG is tagged at the N-terminal of the peptide) was used as an inhibitor, implying that this exon would function to assemble molecular complexes on TN breast cancer cells that facilitate invasion, migration, and growth. Thus, these results suggest that generation of small synthetic molecules that block the functions of exon v10 of CD44 is promising approaches to inhibit invasion and metastasis of TN breast cancer cells.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6161.
Radiation-Induced S-Nitrosylation of ER-α Predisposes MCF-7 Breast Cancer Cells to Aggressive Invasion and Migration.
Authors: Tamfu R. , Natarajan A. , Yu H. , Mohan S. , Shanmugasundaram K. , Natarajan M. , Natarajan M. , Natarajan M. 
Published: December 2010

Abstract
The objective of this study is to decipher the potentials of breast cancer cells that survive radiation exposure at clinical doses to acquire invasive and metastatic determinants. In this study we exposed estrogen receptor positive (MCF-7) and negative (MDAMB-231) breast cancer cells to clinical doses of low LET radiation including doses used as single fraction (2Gy), cumulative dose (10Gy) and scattered dose (10cGy). The cells were exposed to 137Cs source at a dose rate of 1.27 Gy/min. The mock irradiated cells were used as controls. The mRNA transcript level, protein expression and protein-protein interaction were analyzed by QPCR/RT-PCR, immunoblotting, immunoprecipitation and mammalian two-hybrid system, respectively. Intracellular nitric oxide (NO) levels were determined by immunofluorescence and electron spin resonance spectroscopy (ESR) using DAF-FM and Fe-MGD as NO traps, respectively. NO-dependent ER-α s-nitrosylation was determined through immunoprecipitating s-nitrosylated proteins from the cell lysates with an s-nitrosylated-cysteine IgG antibody followed by ER-α western blot analysis. Transactivation of ER-α was measured by EMSA and luciferase reporter assay. Cell invasion and migration were examined by co-culture system using thin inserts.Radiation induces eNOS expression and activation through the phosphorylation of eNOS at Ser1177 site resulting in increased bioavailability of NO in a dose- and time-dependent manner. The three-fold bioavailability of NO s-nitrosylates ER-α leading to a two-fold binding of ER-α to ERE. Paradoxically, this binding did not translate into the transcriptional activation of ER-α dependent gene regulation. Altered structure due to s-nitrosylation of ER-α contributes to enhanced cell invasion and cell migration. In consistent with these results, there was an associated 2.3-fold increase in the expression of MMP-2 and MMP-9 and corresponding TIMP-1 decreased expression leading to a 3.5-fold increase in MMP activity. Radiation, while controlling tumor growth, could simultaneously play a significant role in breast cancer relapse and metastasis through the activation of eNOS and the generation of NO. The bioavailability of NO s-nitrosylates ER-α leading to re-defining the genomic functions of ER-α and thereby impart metastatic transformation potentials to ER-α positive breast cancer cells.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6162.