bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5129: Mapping the activity of oncogenic signaling networks with phospho-specific Liquid Tissue® mass spectrometry
Authors: Hembrough Todd A. , Thyparambil Sheeno , Liao Wei-Li , Krizman David , Darfler Marlene , Bengali Kathleen , Abdo Joe , Burrows Jon
Published: June 2012

Many of the current targeted therapies in oncology target the activity of either receptor tyrosine kinases (Her2, EGFR, IGF1R, cMet, FGFR, PGDFR) or cytoplasmic tyrosine kinases (cSrc, AurA, PI3K, MEK, Raf, Akt). The current slate of clinically useful diagnostic tests measure target expression either directly by immunohistochemistry or indirectly by extrapolation from the level of gene or mRNA expression/amplification. These assays are limited by lack of quantitation, and no assay can directly assess the activation state of the signaling pathway components. The lack of information regarding target activation and downstream signal transduction can be overcome using the Liquid Tissue®-SRM technology platform. This approach enables relative and absolute quantification of multiple proteins and their phosphorylation status directly in formalin fixed tissue.

In order to fill this diagnostic ‘gap’ we have used this platform to develop a quantitative multiplexed phospho-target assay format which measures the specific phosphorylation state of many clinically relevant oncogenic kinases (EGFR, cMet, Her3, Erk, cSrc, Mek, Akt, p70S6K) directly in FFPE tumor tissue.

This phosphopeptide multiplex assay was initially preclinically validated on the A431 tumor cell line which harbors an amplification of the EGFR gene. These cells were stimulated with a dose range of EGF (50-200ng/ml) or in a time course study (EGF 50ng/ml for 5-30min). Confluent, EGF stimulated cells were then formalin fixed, subjected to Liquid Tissue® processing, and then phospho-enriched using TiO2 magnetic beads. The resulting enriched phosphopeptides were then analyzed by mass spectrometry. This method demonstrated the feasibility, and reproducibility of this method for quantitating EGFR pY1197, EGFRpT693, AKT pS473, p-p70S6K pS447, ERK pT202/pY204.

We extended this study by performing phosphoenrichment and mass spectrometric analysis of human tumor xenografts, primary human tumor NSCLC explants and clinical trial tissue from EGFR inhibitor treated NSCLC and colorectal cancer patients. In each case we were able to enrich and measure the phosphorylation of a large set of important oncogenic kinases.

Our intention is to develop this diagnostic tool to provide a multiplex assay format in formalin fixed tissue that can be applied from preclinical to clinical studies that will impact both targeted drug development and patient stratification needs in this era of personalized healthcare.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5129. doi:10.1158/1538-7445.AM2011-5129
Abstract 5143: The role of VEGFR2 in lung cancer differs between adenocarcinoma and squamous cell carcinoma cell lines
Authors: Larrayoz Marta , Agorreta Jackeline , Pajares Maria J. , Montuenga Luis M. , Pio Ruben
Published: June 2012

Vascular endothelial growth factor A (VEGFA) is a cytokine implicated in proliferation, migration and cell survival that mediates its effects through its major receptor, VEGFR2. VEGFA is highly induced by hypoxia and is a crucial regulator of angiogenesis in both healthy and tumor tissues. For this reason, many efforts have been made to target the VEGFA/VEGFR2 pathway for the treatment of cancer. In lung cancer, an anti-VEGFA therapy (bevacizumab) was approved, in combination with platinum-based chemotherapy, as first line treatment in unresectable advanced non-small cell lung cancer (NSCLC). The expression of VEGFA has been widely reported in NSCLC, supporting the idea that VEGFA is produced by tumor cells and activates its receptors on endothelial cells, leading to vascularization of the tumor. However, it is known that NSCLC cells also express VEGFR2, although its role in tumor progression or response to treatment has not been evaluated.

The aim of the present work was to study the biological role of the autocrine/paracrine VEGFA loop in NSCLC cells, paying special attention to the differences between the two main histological subtypes. To this purpose, we assessed the expression and activation of the VEGFA-VEGFR2 pathway in NSCLC cell lines, analyzing the effect of VEGFR2 inhibition/overexpression in both adenocarcinoma (ADC) and squamous cell carcinoma (SCC) cell lines.

Human NSCLC cell lines were examined for the expression of VEGFA and VEGFR2 by real time PCR and Western blotting. All cell lines expressed VEGFA. Two out of ten ADC cell lines (NCI-H441 and NCI-H358), and two out of eleven SCC cell lines (EPLC-272H and LOU-NH91) showed detectable levels of VEGFR2. Addition of exogenous VEGFA to VEGFR2-positive cells induced phosphorylation of the receptor and activation of its intracellular signaling pathway.

To better understand the role of VEGFR2 in NSCLC, we used lentiviruses to inhibit or retroviruses to overexpress this protein in VEGFR2-positive cell lines. Changes in VEGFR2 levels had no effect in proliferation or migration in the ADC cell lines. However, in the SCC cell lines, the inhibition of the receptor increased proliferation and decreased migration. When the receptor was overexpressed in the SCC cell lines, the opposite effects were observed.

In conclusion, VEGFR2 is expressed by some NSCLC cell lines and its activation may have different effects depending on the histology of the cells. Specifically, in SCC cells the activation of VEGFR2 decreases cell growth. These data illustrate the importance of the different histologies in the study of the VEGFA biology in NSCLC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5143. doi:10.1158/1538-7445.AM2011-5143
Abstract 5167: A new panel of tissue markers for the prediction of bone metastatization from primary breast cancer
Authors: Mercatali Laura , Ibrahim Toni , Sacanna Emanuele , Scarpi Emanuela , Fabbri Francesco , Ricci Rossana , Calistri Daniele , Amadori Dino
Published: June 2012

Background: It is known that numerous molecular markers associated with proliferation, invasion and adhesion processes, growth factors, chemokines and their receptors, and molecules with bone-like phenotype are involved in metastasization to bone. Only cancer cells with specific molecular profiles are capable of reaching and colonizing bone tissue. The early prediction of relapse in this site could be used to select candidates for tailored therapy with bone-specific drugs such as bisphosphonates or RANK-L inhibitors. In the present study we aimed to identify primary tumor markers capable of predicting relapse to bone.

Material and methods: The expression of different markers was retrospectively analyzed in frozen breast cancer tissue samples from 40 patients comprising 15 cases with no evidence of disease (NEDP), 15 with bone metastases (BMP), and 10 with visceral metastases (VMP). Twelve transcripts were analyzed by Quantitative Real time PCR: trefoil factor 1(TFF1), DKK1, bone sialoprotein (IBSP), heparanase (HPSE), osteopontin (SPP1), Agr2, SPARC, CTGF, COMP, follistatin (FST), osteoprotegerin and RANK. The predictive accuracy of each marker was calculated using receiver operating characteristic (ROC) curves.

Results: The analysis of marker expression in the 3 different subgroups showed twofold higher median values of COMP and HPSE in BMP with respect to either NEDP or VMP (no significant difference). Furthermore, TFF1 median value was about sevenfold higher in BMP than in the other 2 subgroups (p=0.05). The area under the curve (AUC) was 0.73 for TFF1 and 0.62 for COMP and HPSE. In the dichotomous variable analysis, the most important result was observed for TFF1; in particular, TFF1 positivity was observed in 67% of cases in the BMP subgroup compared to only 21% and 20 % in NEDP and VMP, respectively. The combination analysis of TFF1 and other markers showed that positivity to at least one of the three markers, TFF1, DKK1, or IBSP, was observed in 80% of cases for the BMP group, and in only in 21% and 20% of cases in NEDP and VMP subgroups, respectively (p=0.041).

Conclusions: The results from this preliminary study suggest that Real-Time PCR analysis of gene expression and, in particular, the coexpression of some markers in primary breast cancer could identify patients destined to bone relapse who could benefit from adjuvant treatment with bone-specific drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5167. doi:10.1158/1538-7445.AM2011-5167
Abstract 5171: Expression and function of Snail2 in osteosarcoma
Authors: Sharili Amir Shaya S. , Allen Steve , Smith Ken , Price Joanna , McGonnell Imelda M.
Published: June 2012

Purpose: Snail2 is a well established regulator of EMT in malignant epithelial tumors, however in sarcomas it is not known if this protein is present and contributes to tumor progression. Here we examine the expression of Snail2 in clinical cases of one type of sarcoma; osteosarcoma. We hypothesize that Snail2 is expressed in osteosarcomas and that this expression correlates with tumor grade. In addition, we also hypothesized that Snail2 promotes metastasis in osteosarcomas.

Experimental design: Snail2 localization was determined in clinical biopsies of different grades of canine osteosarcomas using immunohistochemistry. The function of Snail2 in OS was evaluated using stable human and canine OS cell lines in which loss of function was achieved using small interfering RNA (siRNA) and gain of function using over-expression vectors. These lines were used to assess changes in cell motility and invasion using in vitro assays.

Results: Nuclear Snail2 staining was observed in all cases of osteosarcoma examined. Ordinal logistic regression analysis revealed that elevated Snail2 expression was significantly (P=0.014) correlated with tumor grade. Cell lines of canine (D-17) and human (Saos-2) osteosarcoma stably transfected with Snail2 siRNA showed a loss of osteoblastic morphology becoming flattened whereas those overexpressing Snail2 appeared more spindle shaped. Scratch assays showed that motility of osteosarcoma cells was correlated with levels of Snail2 expression.

Conclusion: This is the first time Snail2 has been associated with any sarcoma and this study shows that Snail2 may be a useful prognostic marker for osteosarcoma. In addition, it indicates that Snail2 may play a role in osteosarcoma metastasis. Thus it may be a potential target for clinical intervention in this disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5171. doi:10.1158/1538-7445.AM2011-5171
Abstract 5209: The roles of Notch signaling in the development of non-small cell lung cancer
Authors: Osanyingbemi-Obidi Joyce O. , Rudin Charles M.
Published: June 2012

Lung cancer is the most common cause of cancer related deaths in the United States, accounting for 29% of all cancer deaths. Approximately 85% of all lung cancers are of non-small cell histology. Although current therapies have marginally improved survival and quality of life for patients with Non-Small Cell Lung Carcinoma (NSCLC), this remains a nearly universally fatal disease. New approaches to drug development and further studies of the biology of this disease are critically needed. We hypothesized that high level Notch signaling would define a subpopulation of NSCLC cells with markedly elevated clonogenic potential, and that inhibition of Notch signaling could inhibit NSCLC tumorigenesis. To test these hypotheses and to define the potential utility of Notch inhibitors in NSCLC, we have evaluated the temporal and spatial activation of Notch signaling in a murine model of NSCLC tumorigenesis, evaluated a dominant negative transgenic mouse model, and are assessing whether Notch activation correlates with clonogenic and tumorigenic potential in human NSCLC lines. Down regulation of Notch signaling using a selective γ-secretase inhibitor (Mrk 003; Merck) was evaluated in three NSCLC cell lines (NCI-H1299, and -H1435). Cells were incubated for 96 hours with Mrk 003 at concentrations ranging from 0.1uM to 10.0μM. After 96 hours, a 60% suppression of Hes 1 expression was observed. In conjunction with these studies we have created a double transgenic model, LSL-K-rasG12D × DNMAML (dominant negative mastermind) mice. Expression of the DN-MAML gene results in inhibition of the Notch pathway. We administered adenoviral CRE intranasally to these mice and generated intraparenchymal lung tumors in both KRAS and KRAS/DN-MAML mice. Surprisingly, and in contrast to our hypothesis, tumor burden was not decreased in the double transgenic model relative to the Kras control group. Mastermind-1 may have effects other than regulation of Notch signaling, including a possible interaction with p53 that is being currently explored, and which may explain these surprising findings. Characterization of highly clonogenic progenitor populations in NSCLC, and definition of the roles of Notch pathway regulators in tumorigenesis, may aid in the development of more effective therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5209. doi:10.1158/1538-7445.AM2011-5209
Abstract 5212: Loss of G-protein coupled calcium sensing receptor augments malignancy and bestows cancer stem cell-like properties in human colon carcinoma cells
Authors: Singh Navneet , Liu Guangming , Chakrabarty Subhas
Published: June 2012

The G-protein coupled calcium sensing receptor (CaSR) is a robust promoter of differentiation in colonic epithelial cells. Loss of CaSR expression allows cancer cells to escape from CaSR control. Loss of CaSR expression augments the malignant phenotype in vitro and is associated with undifferentiated and invasive colon carcinomas in vivo (Cancer Res. 63: 67, 2003; 65: 493, 2005; Int. J. Cancer 121: 1455, 2007;126: 631, 2010; Mol. Carcinogenesis 48: 202, 2009). Interestingly, human colon carcinoma cell lines contain small subpopulations (10-20%) that do not express CaSR (termed CaSR null cells). Here, we report the isolation and characterization of CaSR null cells from the CBS and HCT116 human colon carcinoma cell lines.

We isolated CaSR null cells by magnetic cell sorting using monoclonal anti-CaSR antibody. CaSR null cells were cultured and maintained in stem cell culture medium. Biologic characterization of CaSR null cells showed that these cells were non-adherent and grew as three-dimensional spherical clusters with an increase in propensity for anchorage independent growth. CaSR null cells were highly invasive in matrigel invasion assays and resistant to treatment with cytotoxic drugs. Western analysis showed that CaSR null cells expressed a high level of thymidylate synthase and survivin which have been reported to be an underlying basis of drug resistance in colon cancer. Western analysis also showed that CaSR null cells expressed a high level of the previously reported cancer stem cell markers CD133, CD44 and Nanog. Molecular profiling by quantitative RT-PCR revealed a significant increase in the expression of epithelial-mesenchymal transition associated mRNAs in CaSR null cells. These mRNAs were N-cadherin, β-catenin, vimentin, fibronectin, FSP1, Snail1, Snail2, Twist, FOXC2 and SIP1. Interestingly, CaSR null cells re-expressed CaSR, over time, when placed in culture medium used to propagate the parental CBS and HCT116 cells. Re-expression of CaSR in CaSR null cells attenuated the expression of markers associated with the malignant phenotype.

We conclude that CaSR null cells represent a highly malignant subpopulation of colon cancer cells and that these cells may possess cancer stem cell-like properties.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5212. doi:10.1158/1538-7445.AM2011-5212
Abstract 5214: Development of a microfiltration system for the improved detection and viable capture of metastatic circulating tumor cells
Authors: Harouaka Ramdane , Zhou Mingda , Zhang Jiayun , Khan Waleed J. , Zheng Siyang
Published: June 2012

The ability of metastatic cancer to release cells that travel through the circulatory system and invade different parts of the body accounts for over 90% of cancer related deaths. The fundamental challenge with detecting these circulating tumor cells (CTCs) in blood samples is the fact that they are so rare, with only a few tumors cells occurring among billions of blood cells. Since tumor cells are almost always significantly larger and more rigid than normal blood cells, size based separation has been demonstrated to be an effective method for CTC capture and enrichment. While useful for detection, size based techniques apply concentrated stresses that affect the viability of captured cells. We have incorporated novel spring structures into our microfilter design that mitigate the stresses experienced by CTCs during filtration to encourage their health and survival. The geometric design and filtration pressures have been optimized to maximize capture efficiency, enrichment against white blood cells, and tumor cell viability. This was achieved through repeated filtrations using a model system of fluorescently labeled cells from various established tumor cell lines spiked into healthy donor blood. The viability of captured cells has been confirmed through exclusion dye testing, and through the proliferation and culture of captured cells directly on the filter surface. The successful capture and primary culture of viable CTCs from clinical patient samples will allow unprecedented analysis and genetic testing of the cells directly responsible for metastasis. This could provide the basis for externally testing tumor cell response to a variety of anticancer drugs without having to expose a patient to the unnecessary cost and toxic effects of chemotherapy, thereby improving patient outcomes through the development of personalized treatment plans.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5214. doi:10.1158/1538-7445.AM2011-5214
Abstract 5230: Development of a series of genetically fluorescent human osteosarcoma cell lines with increasing spontaneous metastatic capability
Authors: Tome Yasunori , Kimura Hiroaki , Maehara Hiroki , Kanaya Fuminori , Bouvet Michael , Tsuchiya Hiroyuki , Hoffman Robert M.
Published: June 2012

In order to investigate the molecular pathogenesis of human osteosarcoma, a reliable metastatic osteosarcoma animal model is required. Here we report the development of a series of metastatic sublines (LM1-LM4) derived from a human osteosarcoma 143B parental cell line expressing GFP in the nucleus and RFP in the cytoplasm. The sublines have increasing ability to form pulmonary metastases in nude mice after intravenous injection. In vitro proliferation rates of all sublines were similar. When the 143B parental and the metastatic sublines were transplanted orthotopically in the tibia of nude mice, all cell lines were tumorigenic. Primary tumor growth and metastases were imaged with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). LM2 and LM3 had higher metastatic capability than the parental 143B. LM1 had higher metastatic capability than LM2 or LM3. LM4 cells resulted in the most pulmonary micrometastases. The number of pulmonary metastases was 8-fold higher in LM4 cells than that with 143B parental cells. This series of metastatic sublines should be a powerful tool to investigate genetic and other factors that promote or inhibit osteosarcoma metastasis and for evaluation of novel anti-metastatic drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5230. doi:10.1158/1538-7445.AM2011-5230
Abstract 5231: Tilmaocept: A novel biotargeted agent for enhanced intraoperative discrimination of sentinel lymph nodes in breast cancer, melanoma, and head & neck SCC
Authors: Cope Frederick O. , Metz Wendy Rich , Blackburn Simon , Abbruzzese Bonnie , Orahood Richard
Published: June 2012

Sentinel lymph node (SLN) mapping and identification is a primary component of surgical therapies for breast cancers, melanomas, pediatric soft tissue sarcomas, and certain other cancers. The current standard of care for identifying SLNs to inject into a tumor (or near a tumor) a blue dye (often called “vital blue dye”), of which several are available, in conjunction with a radiolabeled (99mTc) tracer, available as either a technetium sulfur colloid or as a technetium labeled albumin formulation. The exact formulations used vary, especially between those used in the United States and Europe. All such compounds are effectively agents of passive transport in the lymphatic ducts and are “inert” with regard to any biomarker recognition specificity. Allegedly, such agents move passively from the tumor/tumor basin into lymph nodes with a presumptive tumor-node anatomic nexus and the nodes are elucidated as blue and/or radioactive, the former based on excision line-of-sight, the latter based on intraoperative evaluation with a gamma-detecting probe. But because such components do not bear any specificity, that is, bind or accumulate lymph nodes bearing targets, they either remain static at the injection site and/or quickly drain away, reducing their real-time clinical effectiveness.

A novel 99mTc-labeled, prospectively designed, low Mr molecule (99mTc DTPA Mannosyl Dextran; Tilmanocept) targeted as a ligand for the mannose binding receptor (MBR) of lymphatic reticuloendothelial cells and intended for real-time intraoperative gamma mapping of tumor-lymphatic nexuses has been synthesized and clinically tested in Phase 3 clinical studies in breast cancer and melanoma. The results of the development process and clinical testing indicate: 1) The prospective molecular designs resulted in safety and clinical performance characteristics that provided times from agent injection to patient evaluation that were significantly reduced compared to currently applied 99mTc-labeled agents; and 2) The specificity, sensitivity, and performance with regard to anatomic localization, false negative rates, and receptor retention exceed those of other colorimetric and 99mTc-labeled agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5231. doi:10.1158/1538-7445.AM2011-5231
Abstract 5243: Gene expression analysis of CRC liver metastases with different growth patterns
Authors: Van den Eynden Gert G. , Fiorentino Michelangelo , Van Laere Steven S. , Peeters Dieter S. , Trinh Xuan Bich , Dirix Luc Y. , Vermeulen Peter B.
Published: June 2012

Introduction: Liver metastases of colorectal cancer (CRC) grow according to different growth patterns (GPs) with different angiogenic properties. The aim of this study was to characterize these GPs on a molecular level using genome-wide gene expression analysis.

Materials and Methods: On 18 CRC liver metastases of which genome-wide gene expression data were available from a previous study (publicly available at GSE 10961), we assessed the GP using a haematoxylin-eosin and Gordon Sweet's reticulin stain. 7 metastases had a desmoplastic GP, 6 had a pushing GP and 3 had a replacement GP. Of 2 metastases the GP could not be assessed due to lack of sufficient material and these were excluded for further analysis. We used (hierarchical) cluster analysis, principal component analysis, significance/prediction analysis of microarrays and gene set enrichment analysis to study differences in gene expression between the different GPs.

Results: Principal component analysis and hierarchical cluster analysis showed significant gene expression differences between metastases with a desmoplastic and pushing GP (p=2.2e−16) and between metastases with a desmoplastic and replacement GP (p=2.2e−16). Gene set enrichment analysis demonstrated increased expression of biological processes, molecular functions, cellular components and KEGG pathways related to immune response, antigen processing, leucocyte activation and cell adhesion molecules in the metastases with a desmoplastic GP. Similar differences were found when metastases with a pushing GP were compared to metastases with a replacement GP and when the 3 groups were compared.

Conclusion: These results confirm on a molecular level that liver metastases of patients with CRC have a heterogeneous biology. Metastases with a desmoplastic GP appear to have upregulation of immunological processes, compared to metastases with a pushing or replacement GP. Further elucidation of these differences might lead to better understanding of host-tumour interactions in metastatic biology and new therapeutic strategies for patients with metastatic CRC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5243. doi:10.1158/1538-7445.AM2011-5243
Abstract 5250: MUC1 expression is induced by Snai1 in an epithelial mesenchymal transition renal model
Authors: Gnemmi Viviane , Bouillez Audrey , Ringot Bélinda , Zini Laurent , Copin Marie-Christine , VanSeuningen Isabelle , Leroy Xavier , Perrais Michael , Sébastien Aubert
Published: June 2012

Accumulating data suggest that epithelial mesenchymal transition (EMT) is involved in cancer progression. Sarcomatoid carcinoma represents a high-grade transformation and an EMT-like pattern. Up to 8% of conventional renal cells cancers (cRCC) present with sarcomatoid carcinoma component that portends a worse prognosis. MUC1, a membrane-bound glycoprotein is known to be over-expressed in cRCC with correlation to prognosis. Indeed, we showed that MUC1 overexpression is correlated with a metastatic phenotype using tissue microarray sampling 27 cRCC of same TNM stage. Also, we demonstrated in a series of 15 RCC with sarcomatoid component, the loss of epithelial markers and the acquisition of mesenchymal markers compared to conventional carcinomatous areas. MUC1 was significantly overexpressed along with Snai1, a major actor of EMT, in sarcomatoid component. So, we hypothesized that MUC1 could be regulated by Snai1. Bioinformatic analyses revealed two Snai1 E-boxes at -84/-72 on MUC1 promoter. Cotransfection studies of MUC1 promoter and Snai1 expression vectors in renal cells lines showed an induction of MUC1 transcriptional activity by luciferase assays. Mutagenesis site-directed of both Snai1 E-boxes in renal cells lines induced loss MUC1 transcriptional activity. By chromatin immunoprecipation assay and gel shift analysis we will demonstrate a direct interaction between Snai1 and MUC1 promoter. In conclusion, all these data revealed that MUC1, usually considered as an epithelial marker, is overexpressed during cancer linked EMT process with a direct regulation by Snai1 suggesting that MUC1 plays a role in renal cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5250. doi:10.1158/1538-7445.AM2011-5250
Abstract 5253: A model of tumor dormancy in Luminal A breast cancer
Authors: Sansone Pasquale , Shapiro Lauren , Chang Qing , Berishaj Marjan , Bromberg Jaqueline
Published: June 2012

Introduction: The clinical course and biologic features of metastasis vary extensively depending on the type of cancer. Latency is one of the most important variables in metastasis. Late-onset recurrences are frequently found in Luminal A (ER+/PR+/Her2-) breast cancer patients. In both ER+ tumors and ER+ breast cancer cell lines, tumor cells preferentially disseminate to the bone marrow where they are capable of remaining dormant. The identification of mechanisms that support the survival of disseminated cancer cells (DTCs) has clear implications for understanding the biology of latent metastatic disease.

Method: ER+ breast cancer cell lines expressing a GFP luciferase vector (for in vivo bioluminescent monitoring of tumor growth and dissemination) were orthotopically injected in the mammary fat pad (MFP) of mice. DTCs were isolated and characterized from the bone marrow and lungs after several months.

Results: The isolated DTCs from the bone and lung do not proliferate, are resistant to both chemo and hormonal therapy (HT) and are endowed with a highly invasive/disseminating capacity (loss of E-Cadherin and gain of EMT features). Moreover, these DTCs exhibit decreased expression of ER compared to the cancer cells within the primary tumor. Since ER activity is required for ER+ breast cancer cell growth in vitro and in vivo, our data suggest that loss of ER activity can be a mechanism of tumor dormancy in Luminal A breast cancer.

Conclusion: An understanding of the consequences of ER activity in terms of tumor dormancy and dissemination potential of ER+ breast cancer cells has implications for improving the treatment of latent metastatic disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5253. doi:10.1158/1538-7445.AM2011-5253
Abstract 5260: Heme oxygenase-1 is involved in prostate cancer cells-bone interaction
Authors: Ferrando Mercedes , Elguero Belén , Meiss Roberto , Tzelepi Vassiliki , De Siervi Adriana , Navone Nora , Vazquez Elba
Published: June 2012

Prostate cancer metastasis is well characterized by its tropism to the bone with metastatic lesions being typically osteoblastic. Osteoblasts produce factors that promote growth and survival of prostate cancer cells, but the molecular nature of this interaction is still unknown. Previous reports from our laboratory documented that heme oxygenase-1 (HO-1) is expressed in primary prostate carcinomas and is localized in the nucleus. Recently, the involvement of HO-1 in bone metabolism has been reported. The aim of this study is to investigate the role of HO-1 in the interaction between prostate cancer cells and bone. We analyzed the expression of HO-1 by immunohistochemistry using a tissue microarray generated from 12 castrate-resistant prostate cancer specimens (MDA Anderson Cancer Center), growing as xenografts in male CB17 SCID wild or castrated mice. Each of these tumors led to clinically relevant biological models to study human prostate cancer progression. All samples showed cytoplasmic HO-1 staining of varying intensity. Intense positive nuclear staining was also detected in 3/5 tumors with neuroendocrine morphology. HO-1 expression was correlated with other markers such as PSA, AR and Ki-67 expression. Co-cultures of the osteolytic PC3 cancer cell line with PMO (primary mice osteoblasts) demonstrated that HO-1 induction in PC3 cells abolished the PC3-induced loss of osteoblasts and significantly increased Dickkopf-1 expression in tumor cells. Furthermore, using PC3-PMO co-cultures, we found that HO-1 induction in prostate cancer cells increased mRNA levels of Runx-2 and Cyclin D1 and diminished β-catenin protein expression in PMO. These results clearly indicate the involvement of HO-1 in the interaction between prostate cancer cells and bone. The study of the molecular mechanisms of this interaction will provide effective therapeutic interventions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5260. doi:10.1158/1538-7445.AM2011-5260
Abstract 5261: Role of anterior gradient protein-2 in prostate cancer bone metastasis
Authors: Chanda Diptiman , Sawant Anandi , Isayeva Tatyana , Ponnazhagan Selvarangan
Published: June 2012

Skeletal metastasis is common in late stage prostate cancer, which leads to significant decline in the quality of life and mortality. In a recent study, we identified over-expression of anterior gradient protein (AGR2) in the PC3, bone metastatic prostate cancer cells following in vitro growth in bone marrow-conditioned medium. AGR2 is a secreted protein and originally reported for its role in the formation of forebrain and mucus-secreting cement gland in the frog Xenopus laevis. Role of AGR2 in carcinogenesis and metastasis is recently being investigated and reports indicate it is over-expressed in various adenocarcinomas and linked to poor survival. But no study has elucidated the role of AGR2 in bone metastasis. When AGR2 expression was compared between human prostate cancer cell lines obtained from bone (PC3), lymph node (LnCap) and brain (Du145) by RT-PCR and Western blot, highest level of AGR2 was observed in PC3 cells. To further confirm, PC3 cells expressing firefly luciferase were injected in athymic nude mice via intra-cardiac route and cells were retrieved from bone, adrenal, lung and heart 3 weeks later following organ dissemination. RT-PCR analysis indicated highest AGR2 expression in PC3 cells isolated from bone microenvironment, suggesting that AGR2 may have a potential role in prostate cancer bone metastasis. To correlate the relevance of this finding in humans, AGR2 immunohistochemistry was performed on human primary and metastatic prostate cancer tissue sections. Preliminary results indicate higher AGR2 expression in metastasized bone sections compared to primary prostate cancers. Additionally, AGR2 was stably silenced in the PC3 cells utilizing a short hairpin RNA and growth kinetics of the cell line was tested in vivo following intra-cardiac injection in a preclinical mouse model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5261. doi:10.1158/1538-7445.AM2011-5261
Abstract 5265: Development of a microfluidic device for tagless analysis of cell surface proteins directing lung metastasis
Authors: Vellanki Sri H. , Smith Robin L. , Tivari Samir , Kumar Rajan , Wieder Robert
Published: June 2012

Diagnostic analysis of the few tumor cells obtained by needle aspirates is currently limited to the cells’ morphologic characteristics. We are investigating tagless analysis of cell surface protein composition, when limited cell numbers are available, using a flow retardation microfluidic device. Expression of biologically significant cell surface proteins by individual cells is scored positive by achievement of flow retardation due to transient binding of cell surface proteins to cognizant antibodies immobilized on tandem patches of the channel. This permits repetitive queries of the same cell for several proteins that predict specific malignant behavior. We are adapting the model to LM2 cells, derived from lung metastases of MDA-MB-231 breast cancer cells, which selectively metastasize to the lung*. The original study identified 95 unique genes either overexpressed or underexpressed in LM2 cells compared to parental cells. Of these we identified three cell surface proteins, VCAM1, IL13RA2, and EMP1, as heterogeneously upregulated in a subset of LM2 cells and CXCR4, as downregulated in a subset of LM2 cells when compared with MDA-MB-231 cells. VCAM1 and IL13RA2 promote lung metastasis*. We sterile sorted LM2 cells into fractions expressing none, medium and high levels of these four proteins. We built a prototype microfluidic device able to retard the flow of streptavidin-coated silica oxide beads by immobilized biotin and used optical imaging and a tracking program for simultaneous analysis of multiple particles. We developed efficient and stable protein coating of microchannels and are generating patches with cognizant antibodies to these 4 antigens and to endothelial, macrophage, lymphocyte, fibroblast and endothelial cell markers as controls. We will correlate antigen expression levels with flow retardation efficiency. We will optimize shear rates affecting bond formation and shear stress affecting bond dissociation by optimizing velocity, viscosity, detergent and divalent cation concentrations to maximize cell separation. We will adapt the channel to select LM2 cells from excess controls in vitro and from tumor xenograft aspirates.

*Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL and Massague J (2005) Genes that mediate breast cancer metastasis to lung. Nature 436:518-524.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5265. doi:10.1158/1538-7445.AM2011-5265
Abstract 5270: Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer
Authors: Kang Jaeku , Kim Sun Young
Published: June 2012

Histone deacetylases (HDACs) are associated with the development and progression of cancer, but which HDAC isoforms play important roles in breast cancer metastasis is not known. Here, we identify the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) than MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5270. doi:10.1158/1538-7445.AM2011-5270
Abstract 530: Contribution of fibroblasts isolated from primary lung cancers and normal lung tissue to tumor growth
Authors: Andriani Francesca , Bertolini Giulia , Caserini Roberto , Bursomanno Sara , Gasparini Patrizia , Pastorino Ugo , Sozzi Gabriella , Roz Luca
Published: June 2012

Fibroblasts isolated from cancer specimens have been shown to possess pro-tumorigenic properties, often associated with traits reminiscent of myofibroblasts found at sites of wound healing, but little is known about the mediators of the interaction between fibroblasts and cancer cells especially in lung cancer.

To evaluate the contribution of fibroblasts to lung cancer development we established 60 fibroblast cultures from cancerous and normal lung tissue from primary lung cancer patients, including 18 pairs of normal (NF) and cancer-associated fibroblasts (CAF) from the same patient. The mesenchymal nature of the cultures was confirmed by expression of surface markers CD90, CD166, CD105 and CD73 and negativity for epithelial and hematopoietic markers (ESA and CD45). Further immunophenotyping with markers specific for fibroblasts subpopulations (FSP, vimentin, alpha-SMA, FAP) identified frequent expression of activation markers, with up to 50% of alpha-SMA+ cells. Interestingly activation properties were identified also in cultures derived from normal lung tissue.

Co-mingling of fibroblasts (NF or CAF) with A549 lung cancer cells generally resulted in increased tumor size after injection in nude mice and 2/4 of the CAF cultures tested also increased tumor take when cancer cells were injected at low doses. Human cells were identified by HLA staining in the stroma of tumors grown after co-injection confirming the contribution of the mixed fibroblasts to tumor development. Furthermore medium conditioned by fibroblasts promoted growth of lung cancer cells or immortalized bronchial epithelial cells. All the cultures tested displayed a normal karyotype suggesting that the functional properties of fibroblasts are likely to be the result of epigenetic changes.These results indicate that lung fibroblasts might play a central role in the growth and progression of lung neoplasia. Consistently with their activated phenotype also cultures from normal lung tissue of heavy smokers displayed pro-tumorigenic activity, indicating that the microenvironment of lungs heavily exposed to damage from carcinogenic substances might already be predisposed to foster the growth of transformed epithelial cells. The full dissection of this complex picture might reveal mediators of the cross-talk between stroma and cancer cells and provide novel therapeutic targets.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 530. doi:10.1158/1538-7445.AM2011-530
Abstract 5329: Degradable multimodal nanoparticles with fluorescence and magnetic resonance contrast
Authors: Papineni Rao V. , McLaughlin William , Vizard Douglas , Reddy Ram
Published: June 2012

Molecular Imaging is making rapid strides as a tool in understanding and the cure of cancer. Like no panacea, here again a need of multimodality exists, where more than one modality is becoming a necessity to cogently decipher the role or action of the biomolecule in the pathology or cure. Here, we developed a multimodal nanoparticle (NP) that can be simultaneously be imaged by optical, and magnetic resonance Imaging (MRI) modalities. In combination, the two modalities compensate each other's deficiencies in the spatial resolution, sensitivity, and the depth detection. Encapsulation of MR contrast and amine functionalization was performed in a one pot synthesis by microemulsion technique with supra magnetic iron oxide (SPIO). The hydrodynamic radii of nanoparticle and zeta potential were measured using Nicomp 380 zls particle sizer. 9L gliosarcoma tumor bearing rat model was administered with the SPIO nanoparticles and validated using 7 tesla MRI scanner. PEGylated SPIO nanoparticles after conjugation with water soluble, near-infrared (NIR) tricarbocyanine, cyclic enamine-functionalized dye, were injected intratumorally and monitored noninvasively using near-IR fluorescence imager. Steady retention in the tumor as compared to the free near-infrared (NIR) dye injected into the control mice indicates the dye-colloidal particle dwell characteristics and suitability for targeted diagnostics. In summary, we have described the development of a simple and efficient dynamic Imaging multimodal nanoparticle to be used for NIRF and MR Imaging.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5329. doi:10.1158/1538-7445.AM2011-5329
Abstract 5333: Relationship of osteosarcoma surface marker expression to human mesenchymal stem cells undergoing osteoblastic differentiation
Authors: Park Amy Y. , Tarim John , Bratman Russell E. , Gorlick Richard
Published: June 2012

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults, but it is unknown at what point, in the pathway of differentiation between a human mesenchymal stem cell (hMSC) and a mature osteoblast (OB), OS originates. Identifying the cell of origin is crucial in understanding the molecular pathogenesis of OS. These studies are hampered by the fact that the various steps in the differentiation of human MSC into its more differentiated progenitors are poorly characterized.

In this study, hMSCs were derived from the freshly obtained bone marrow of patients as well as purchased through ATCC. Flow cytometry was used to detect the surface expression phenotype of MSC markers, where CD34 and CD45 were negative and CD44, CD73, CD90, and CD105 were positive. The hMSCs were characterized and all demonstrated multilineage differentiation capacity into adipocytes, chondroblasts, and osteoblasts, which was confirmed by Oil Red O, Alcian Blue, and Alizarin Red S stains, respectively. Osteoblasts were further characterized by measuring osteocalcin protein levels using ELISA. RNA was extracted from hMSCs and OB derived from the same patient and gene expression measured by microarray on the Affymetrix Gene 1.0 ST array. A list of differentially expressed genes was generated, which were narrowed to only include surface markers. Antibodies corresponding to surface markers were used for ELISA and flow cytometry on hMSCs and OBs, to confirm the change in gene expression with differentiation. Further studies will include flow cytometry to define intermediate stages in the differentiation pathway from hMSCs to OBs and their capacity to differentiate into the various mesenchymal lineages.

It has been possible to derive hMSC with pluripotent differentiation capacity from patient bone marrows. These will be utilized to define the patterns of differentiation and ultimately relate to osteosarcoma to help define its progenitor cell. It is hoped that a clearer understanding of the progenitor cell of OS will help find prognostic factors and molecular therapeutic targets.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5333. doi:10.1158/1538-7445.AM2011-5333