bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3285: Phenotypic characterization of circulating tumor cells (CTCs) in triple negative breast cancer patients
Authors: Kallergi Galatea , Agelaki Sofia , Mathioudaki Argyri , Mavroudis Dimitris , Georgoulias Vassilis
Published: February 2011

Background: Molecular classification of breast cancer revealed that there are five different subtypes related to clinical outcome:. luminal A, luminal B, normal-like, ERBB2-positive and basal-like. Triple negative patients (ER-negative, PR-negative, HER2-negative) belong to the basal-like subtype. Their tumors commonly express EGFR and are associated with poor prognosis. Circulating Tumor Cells (CTCs) have been proposed as a “real time liquid biopsy” in breast cancer patients. CTCs are associated with disease relapse and could serve as a target for molecular cancer therapies. The aim of the present study was, for the first time, the phenotypic characterization of CTCs in triple negative breast cancer patients. Methods: We evaluated peripheral blood mononuclear cells (PBMC) cytospins from 40 triple negative patients and found CTCs in thirty one of them (15 early and 16 metastatic). The expression of Cytokeratins (CK), Estrogens Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor (HER2) in CTCs was assessed using double immunofluorescent staining and confocal laser scanning microscopy and the Ariol system. PBMCs were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with ER, PR, EGFR or HER2 antibodies, respectively. Results: Our results demonstrated that ER, PR, EGFR and HER2 were expressed in 58%, 45%, 55% and 58% of the examined CK-positive patients. The respective proportions for early versus (vs) metastatic patients were 67% vs 50% for ER, 53% vs 38% for PR, 60% vs 44% for EGFR and 60% vs 56% for HER2. In addition ER, PR, EGFR and HER2 were expressed in 5.6%, 79.7%, 80.7% and 13.4%, respectively, of the total examined CTCs. Triple staining experiments with the Ariol system revealed no co-expression of CK/EGFR/ER in CTCs; however, there was only one patient with CTCs co-expressing CK/HER2/ER. Conclusions: ER is expressed but in a minority of CTCs in triple negative breast cancer patients though the majority of CTCs revealed expression of PR and EGFR. However the clinical significance of these findings remain unknown and need further evaluation.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3285.
Abstract 3287: Investigating the role of PTX3 in cervical cancer
Authors: Wada Yumi , Akutagawa Osamu , Nishi Hirotaka , Higuma Chinatsu , Terauch Fumitoshi , Isaka Keiichi
Published: February 2011

Tumor progression requires tumor angiogenesis, as newly-formed blood vessels supply necessary oxygen and nutrition to the growing tumor. Many unknowns remain in our understanding of the cancer metastasis process.

Pentraxin3 (PTX3) is categorized as a long pentraxin in the pentraxin family and is produced by macrophages and vascular endothelial cells. PTX3 concentrations reportedly increase with exposure to IL-1 and TNF-α. Recently, PTX3 has gained attention as a potential marker that reflects inflammation in blood vessels, as well as that which may predict the onset of various diseases, including vascular diseases.

The present study examined whether or not an analysis of PTX3 expression may contribute to a mechanistic understanding of carcinogenesis (metastasis) of cervical cancer.

Subjects and Methods:

Cervical Cancer Cell Lines

A comparative study of PTX3 was performed at the protein level and the RNA level by cultivating each of the cervical cancer cell lines CasKi and SiHa under normal conditions and under hypoxic conditions.

Clinical Tissue Samples

Normal cervical tissues (n=7) and cervical cancer tissues (n=20) were collected from subjects after an explanation of the study was given and consent was obtained. PTX3 expression in both groups was compared.

All measurements were taken using ELISA, Western blot, and real time RT-PCR methods.

Results:

Increased PTX3 expression was observed both at the protein level and at the RNA level in cervical cancer cell lines grown under hypoxic conditions. Conversely, in clinical samples, we observed many samples for which PTX3 expression decreased in cervical cancer samples, contrary to our expectations.

Discussion:

These results indicate a potential role of PTX3 in the carcinogenesis mechanism of cervical cancer. Further, PTX3 may exhibit cytoprotective effects, which may have played a role in our present study. Future investigation of these results is necessary.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3287.
Abstract 3292: Discordance between receptor status in primary and metastatic breast cancer: No discordance for HER-2 based on IHC and FISH
Authors: Lee Soohyeon , Koo Ja Seung , Park Jong-Pil , Park Byeong-Woo , Kim Seung-Il , Park Seho , Moon Yongwha , Roh Jae Kyung , Kim Joohang , Jung Hyun Cheol , Sohn Joohyuk
Published: February 2011

Background: The assessment of tumor receptor profile (ER, PR, and HER-2) is necessary to select patients who are candidate for hormonal and anti-HER-2 therapy. The occurrence of changes in tumor receptor profile (ER, PR and HER-2) between primary and metachronous metastatic tissue has been recognized, which might have significant implications in terms of treatment. The primary aim of this analysis was to compare ER, PR and HER-2 status in primary tumor versus metastatic sites in breast cancer patients.

Materials and Methods: Thirty patients with available paired samples from both primary tumors and metastases were included in this study and re-analyzed by an independent and blind pathologist. HER-2 status was evaluated by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH); ER and PR status were assessed by IHC.

Results: The median age was 40.3 years (range: 27.6∼87.4 years) and 25(83%) patients were premenoposal status. For paired metastases, a change in ER status was observed in 3 cases (10%): 2 cases from positive to negative, while 1 case converted from negative to positive and a change in PR status was observed in 4 cases (13.3%). HER-2 status was not changed in all cases based on IHC and FISH technique. In addition, there was a strong correlation of amplification index (AI) between primary and metastatic tissue based on FISH test (p<0.0001).

Conclusions: This study suggests that while there was around 10% of discordance rate for hormone receptor status, HER2 receptor discordance was not found between primary and metastatic breast cancer samples.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3292.
Abstract 33: Cancer-associated IDH1 mutations produce 2-hydroxyglutarate
Authors: Fantin Valeria R. , Dang Lenny , White David W. , Gross Stefan , Bittinger Mark A. , Driggers Edward M. , Jang Hyun Gyung , Jin Shengfang , Keenan Marie C. , Marks Kevin M. , Yen Katharine E. , Ward Patrick S. , Prins Robert M. , Liau Linda M. , Bennett Bryson D. , Rabinowitz Joshua D. , Cantley Lewis C. , Thompson Craig B. , Heiden Matthew Vander , Su Shinsan M.
Published: February 2011

Mutations in the enzyme isocitrate dehydrogenase 1 (IDH1) are a common feature of most gliomas and secondary glioblastomas, as well as approx 10% acute myeloid leukemias. This event results in loss of the enzyme's ability to catalyze conversion of isocitrate to α -ketoglutarate. However, these mutations are all heterozygous and occur at a single amino acid residue of the IDH1 active site consistent with an enzymatic gain of function rather than a simple loss of function. To test this hypothesis we characterized mutant IDH1 (IDH1m) biochemically. We have shown that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2-HG). Patients with an inherited, neurometabolic disorders called 2-hydroxyglutaric aciduria exhibit an accumulation of 2-HG in their CNS, and an increased risk of developing malignant brain tumors. Similarly, in human malignant gliomas harboring IDH1 mutations, we find elevated levels of 2-HG. Altogether our data demonstrate that the IDH1 mutations result in production of 2-HG, and suggest that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 33.
Abstract 3318: Molecular targeting of brain tumor stem cells
Authors: Eyler Christine E. , Forrester Michael T. , MacSwords Jennifer M. , Wu Qiulian , LaFiura Katherine M. , Lathia Justin D. , Sloan Andrew E. , Lee Jeongwu , McLendon Roger E. , Hjelmeland Anita B. , Rich Jeremy N.
Published: February 2011

Although current therapeutic strategies for malignant gliomas are non-specifically directed against the tumor bulk, increasing evidence suggests that a single glioma contains a range of phenotypically disparate cell populations with differential capacity for tumor maintenance and initiation. Recent work in brain tumors suggests the existence of cellular sub-populations (known as cancer stem cells, or CSCs) that possess stem cell-like behaviors and a potent capacity for tumor initiation in transplantation assays relative to non-CSCs. Specifically targeting the highly tumorigenic CSCs has the potential to improve the prognosis of malignant glioma patients, but many molecularly-directed anti-CSC agents may also compromise the survival of normal neural stem cells due to molecular similarities between CSCs and normal stem cells. Here we demonstrate that malignant glioma CSCs express high levels of a particular protein relative to non-CSCs. Glioma CSC growth and tumorigenic capacity were abrogated by multiple strategies interfering with the expression and activity of this protein, including RNA interference, pharmacological inhibition, and heterologous expression of a prokaryotic enzyme that specifically consumes the enzymatic product of this critical protein. Importantly, tumor growth was inhibited by systemic treatment of xenograft-bearing mice with a well-studied small molecule inhibitor of this protein that has already demonstrated low toxicity in clinical trials for other diseases. Though the activity of this protein appears critical for the maintenance of highly tumorigenic glioma CSCs, our examination of both normal human neural progenitor cells and neural stem cells derived from genetically disrupted mice indicate no substantial role for this protein in normal neural stem cells. Our findings identify a novel glioma therapeutic target with a negligible role in normal neural stem cells, suggesting clinical utility for selective inhibitors of this CSC-specific molecular target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3318.
Abstract 3325: Cancer stem cell like properties of 5-fluorouracil resistant Panc-1 cell
Authors: Izumiya Motoko , Higuchi Hajime , Kabashima Ayano , Igarashi Toru , Nakamura Shoko , Funakoshi Shinsuke , Adachi Masayuki , Takaishi Hiromasa , Hibi Toshifumi
Published: February 2011

Cancer stem cells (CSCs), a small fraction of tumor-initiating cells, are known to be resistant to chemotherapy-induced apoptosis. CSCs are considered to be responsible for self-renewal/differentiation, invasion, and metastasis of malignant tumor. We have previously reported that pancreatic cancer derived side population (SP) cells, a cancer stem cell enriched fraction, were more resistant to chemotherapeutic agents and more likely to cause epithelial-to-mesenchymal transition (EMT) compare to major population (MP) cells. Therefore, we formulated the HYPOTHESIS that 5-fuluorouracil (5-FU) resistant population cells are enriched with CSCs. The AIM of this study is to evaluate whether apoptosis-resistant cancer cells have CSC-like properties, i.e., self-renewal, local invasion and EMT. METHODS: Seventy to eighty percent confluent pancreatic cancer cells, PANC-1, were incubated in the presence of 5-FU (30mg/ml) for 24 hours, and further incubated for one to five weeks without 5-FU. To assess the capacity of self-renewing, cells were planted using a method for anchorage-independent culturing to form spheroids. EMT was induced with TGF-beta (7.5 ng/ml, for 3 days), then evaluated mRNA expression by real time PCR for E-cadherin, Snail, and vimentin. E-cadherin protein level was also examined for immunoblot analysis. Activity of local invasion was analyzed by Matrigel invasion assay. To evaluate tumorigenicity, 5-FU resistant cells were injected into NOD/SCID mice. RESULTS: 5-FU induced significant apoptosis in PANC-1 cells, reducing the number of surviving cells to less than one percent. The survived cells showed a colony-forming activity as they started to re-proliferate at four weeks after 5-FU removal. There was a group of cells among the 5-FU resistant surviving cells, that gradually regains sensitivity to 5-FU by re-plating. The frequency of cells that were capable of initiating spheres was higher in 5-FU resistant cells and they over expressed stem cell marker genes, Oct 4 and Nanog. Incubation with TGF-beta induced EMT-associated gene alterations. These alterations were greater in 5-FU resistant surviving cells as compared to untreated cells. Matrigel invasion activity of apoptosis-resistant PANC-1 cells was greater by seven fold than controlled cells. In addition, in vivo tumor experiments showed 5-FU resistant cells to be more tumorigenic than conventional Panc-1 cells. In CONCLUSION, apoptosis-resistant cancer cells have CSC-like properties, i.e., initiating sphere formation, expressing stem cell genes, and respond to EMT stimulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3325.
Abstract 3334: Telomerase activity corresponds to the level of aggressiveness of ovarian cancer stem cells and are a potential site of targeted therapy
Authors: Meng Erhong , Taylor Brandon A. , Shevde Lalita A. , Rocconi Rodney P.
Published: February 2011

Objective:

Cancer stem cells possess the important property of self-renewal. Telomerase activity (TA) is often used as a molecular marker for cancer aggressiveness and allows propagation of cancer cells by adding telomere repeats which can result in unlimited cell replication. As such, the potential of telomerase in maintaining self-renewal properties of cancer stem cells have been proposed. Our objectives were to determine the TA of ovarian cancer stem cells (OSC) and the effectiveness of targeting telomerase for cancer therapy.

Methods

Ovarian cancer cell lines (ES2, TOV112D, and SKOV3) were grown in non-adherent growth conditions to form spheroid forming cells for numerous successive spheroid generations. Flow cytometry determined the CD44+/CD24- phenotype consistent with cancer stem-cells. Histologic subtypes were chosen to correspond to decreasing levels of aggressiveness: ES2- clear cell; SKOV3- papillary serous; and TOV112D- endometrioid. TRAPEZE RT Telomerase Detection Kit utilized flourescently labeled primers to detect and quantify telomerase activity by RT-PCR. Results were confirmed with luciferase reporter plasmid containing promoter of human telomerase reverse transcriptase (hTERT). Each cancer cell line was treated with 10, 20, 50, and 100uM of a small molecule telomerase inhibitor BIBR1532 measured by cell proliferation survival assay for a total of 72 hours.

Results

All ovarian cancer cell lines readily formed spheroids in non-adherent growth conditions with flow cytometry demonstrating the enrichment of cancer stem cell phenotype: CD44+/CD24-. TA was significantly increased in spheroid forming cells enriched for OSC in both ES2 (5-fold increase) and SKOV3 (6-fold increase) (p < 0.001), however TOV112D showed a decrease in TA. Similarly, ES2 and SKOV3 showed significant TA by luciferase assay with 400% and 1,700%, respectively (p < 0.001), while TOV112D had a decrease in TA. In all cell lines studied, treatment with BIBR1532 demonstrated significant cell death in a dose-dependent fashion with a 5-fold cell kill and only 20% of cancer cell survival at 72 hours.

Conclusions

Telomerase activity appears to increase with ovarian cancer stem cells and supports the hypothesis that it could be important in the self-renewal properties of cancer stem cells. Inhibition of telomerase with a competitive small molecule demonstrated significant response in-vitro and could represent novel targeted therapy for ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3334.
Abstract 3343: Targeting thyroid cancer stem cells with PPARγ agonists
Authors: Vu-Phan Dang L. , Koenig Ron , Wicha Max
Published: February 2011

Ligand activation of PPARγ is reported to inhibit proliferation and promote differentiation in many tissue types, including thyroid. Nonetheless, clinical trials treating thyroid cancer with the PPARγ agonist rosiglitazone yielded disappointing results when the measured outcomes were reacquisition of radioiodine uptake and overall survival. We report here evidence that PPARγ agonists preferentially target cancer stem cells and may still be useful for treatment of thyroid cancer in a different context, especially in combination with reagents that can target the remaining bulk of the tumor.

We used for our studies FTC236 and FTC238, two related cell lines derived from follicular thyroid carcinoma metastases to lymph node and lungs. Both cell lines had robust PPARγ expression and harbored no known PPARγ fusion protein.

Stem cell populations within the cell lines were measured using the Aldefluor (Aldf) assay for Aldehyde Dehydrogenase activity. Both cell lines have 5-10% Aldf+ cells. Populations which contained Aldf+ were the only ones capable of forming passageable sphere colonies in serum free suspension cultures and the only ones capable of forming tumors in NOD/SCID mice. Furthermore, FTC populations grown as spheres had six times more Aldf+ cells than those grown on plastic; therefore serum free suspension cultures was an effective way to enrich for FTC stem cells.

FTC cells grown on plastic expressed PPARγ at five times the level seen in FTC spheres. We activated PPARγ signalling in FTC cells by addition of rosiglitazone or CAY10410, a chemical analogue of the natural PPARγ agonist 15-deoxy-Δ12,14-Prostaglandin-J2. At concentrations which exhibited less than 50% growth inhibitory effects, PPARγ agonists reduced the Aldf+ percentage of the treated population by 90%. Addition of PPARγ agonists into suspension cultures at the same concentrations reduced sphere formation by 80%.

We identified the Aldefluor positive fraction as the tumorigenic and sphere-forming cancer stem cell population within our FTC cell lines. This population exhibited reduced PPARγ expression and was depleted upon treatment with PPARγ agonists. We hypothesize that downregulation of PPARγ signalling is required for maintenance of the stem cell phenotype and that induced activation of PPARγ is a possible strategy for targeting thyroid cancer stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3343.
Abstract 3344: Two different stem cell populations exist in breast cancer to control tumor initiation
Authors: Grant Denise , Ma Jun , Sell Stewart , Glinsky Gennadi
Published: February 2011

The cancer stem cell theory states that a determinate number of cells within a tumor have unique, stem cell-like features to regenerate the entire tumor mass. However, a cancer stem cell may need the right microenvironment to have its greatest tumor initiation potential. We have documented the presence of two stem cell populations within a tumor to yield consistent and most aggressive recapitulation of tumor initiation in vivo. To isolate cancer stem cells, we used the MMTV-PyMT transgenic model of breast cancer in mice to generate stable cell lines from spontaneous breast tumors. Stable in vitro cultures of FACS analyzed cancer stem cells and tumor-residing mesenchymal stem cells were established and tested for tumor initiating ability using two methods of implantation: subcutaneous (s.c.) injection and cleared mammary fat pad (CMFP) injection. To determine the limit of tumor initiation, a highly malignant cancer stem cell culture (419) was injected s.c. This cell culture produced tumors with 1×104, 1×103, and 1×102 cells with 100%, 93%, and 33% tumorigenicity, respectively. In comparison, 100% tumorigenicity was obtained with 1×102 419 cells implanted in the CMFP, a significant increase compared to s.c. injection (Chi-squared, p<0.001). S.c. injection of a less malignant cancer stem cell culture (145) produced tumors less efficiently, with 100% tumorigenicity with 1×104 cells but 13% tumorigenicity with 1×103 cells. To increase tumorigenicity at low cell numbers, cancer stem cell cultures were combined with tumor-residing mesenchymal stem cells just prior to injection. Cancer stem cell cultures were s.c. injected with 1×105 tumor-residing mesenchymal stem cells (383) at the tumor limiting dose, 1×101 for 419 and 1×103 for 145; tumorigenicity increased 160% in the highly malignant cells (419) and 133% in the less malignant cells (145). The tumor-residing mesenchymal stem cells increased tumorigenicity significantly compared to cancer stem cell injection alone (Chi-squared, p<0.002). Control experiments using non-tumorigenic mesenchymal cells did not produce any increase in tumorigenicity. Genome-wide expression profiling experiments were carried out to demonstrate the clinical relevance of the varying malignancy settings in the cancer stem cell and tumor-residing mesenchymal stem cell cultures. Our results suggest that the cellular composition of the local microenvironment within the breast tumors is the major determinant of efficiency of tumor initiation. The addition of tumor-residing mesenchymal stem cells markedly increases the tumor initiation potential of breast cancer stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3344.
Abstract 335: Therapeutic targeting of EphB4 with a novel monoclonal antibody
Authors: Spannuth Whitney A. , Krasnoperov V. , Liu R. , Nick Alpa M. , Lin Yvonne G. , Merritt William , Coleman Robert L. , Gill Parkash S. , Sood Anil K.
Published: February 2011

Objective: EphB4 is a transmembrane receptor tyrosine kinase that plays an important role in neural plasticity and angiogenesis. Its biological and clinical relevance in ovarian cancer is the focus of this study.

Methods: Benign ovarian (ML5 and ML10) and ovarian cancer (Hey, SKOV3ip1, A2780-par, A2780-cp20, and IGROV-af1) cell lines, 7 normal ovarian and 85 invasive ovarian cancer samples were evaluated for EphB4 expression by Western blot or immunohistochemistry. Univariate and multivariate analyses were performed for clinical correlation. EphB4 blockade was accomplished using a novel EphB4-targeted monoclonal antibody (EphB4-131). Systemic delivery of EphB4-131 antibody was utilized in vivo to target EphB4 in orthotopic mouse models of ovarian cancer.

Results: By Western blot analysis, all five malignant cell lines overexpressed EphB4, whereas the two benign cell lines had low EphB4 expression. All of the normal ovarian tissues had low or absent EphB4 expression. Among the invasive ovarian carcinomas (mean age of patients was 59.4 years), 73 (86%) had some level of EphB4 expression and overexpression (moderate or strong) was noted in 58% of all samples. EphB4 overexpression significantly correlated with advanced stage (III or IV; p<0.001) and presence of ascites (p<0.001). In multivariate analysis, EphB4 overexpression (p=0.04) and stage (p=0.03) were independent predictors of poor survival. EphB4-131 antibody led to decreased EphB4 expression in A2780-cp20 and IGROV-af1 cell lines, lasting for over 5 days. Compared to controls, EphB4-131 antibody alone significantly decreased tumor growth in A2780-cp20 (83%, p<0.01) and IGROV-af1 (80%, p<0.001) orthotopic ovarian cancer models. Combination therapy with EphB4-131 antibody and docetaxel resulted in the greatest tumor reduction in both A2780-cp20 and IGROV-af1 cell lines (94-98% reduction versus controls; p<0.01).

Conclusions: EphB4 overexpression is predictive of poor clinical outcome in ovarian cancer patients and is associated with aggressive features. Therapeutic targeting of EphB4 with the novel antibody EphB4-131 is an attractive strategy for further development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 335.
Abstract 3359: Glucose regulated protein78, a putative head and neck cancer initiating cells marker
Authors: Wu Meng-Ju , Lo Jeng-Fan , Yu Cheng-Chia
Published: February 2011

Background: Head and neck squamous cell carcinoma (HNSCC) is a highly lethal cancer that contains cellular and functional heterogeneity. Previously, we enriched a subpopulation of highly tumorigenic head and neck cancer initiating cells (HN-CICs) from HNSCC cells (HNSCCs) using tumor sphere formation approach. GRP78, a stress-inducible endoplasmic reticulum (ER) chaperone, has been reported to play a role in the maintenance of embryonic stem cells and was up-regulated in enriched HN-CICs. However, the molecular mechanisms mediated by GRP78 to operate the physiological characteristics in HN-CICs remain unclear. Herein, we determined the critical role of GRP78 in the maintenance of stemness characteristics and tumorigenic phenotype in HNSCCs.

Methods: First, Affymatrix microarray and two-dimensional differential gel electrophoresis (2-D DIGE) proteomic analyses were applied to identify GRP78 and membrane associated GRP78 (memGRP78) as a putative marker of HN-CICs. Flow cytometry cell sorting was used to isolate memGRP78+ cells and memGRP78− cells for evaluating CICs properties. Down-regulation of GRP78 expression in HNSCCs and HN-CICs were achieved by lentiviral-mediated small hairpin RNA interference (shRNAi), and the stemness characteristics in GRP78-knockdown HN-CICs were elucidated. In addition, in vitro tumorigenic properties between GRP78-knockdown and control HN-CICs were determined. The in vivo tumorigenicity of GRP78-knockdown HN-CICs was evaluated by xenotransplantation assay. Finally, we examined the correlation on expression of GRP78 and stemness genes in HNSCC patient tissues by immunohistochemical staining.

Results: We identified that GRP78/memGRP78 was significantly up-regulated in enriched HN-CICs. Subsequently, memGRP78+ HNSCCs displayed CICs properties. Lentiviral mediated knockdown of GRP78 significantly reduced expression of stemness genes in HN-CICs and HNSCCs, but enhanced the differentiation capability of HN-CICs. Overall, down-regulation of GRP78 lessened tumorigenicity of HN-CICs both in vitro and in vivo.

Conclusion: Our studies first elucidated the crucial role of GRP78 in the maintenance of stemness and tumorigenicity of HN-CICs. Notably, targeting GRP78 signaling might be a potential therapeutic target for HNSCC by eliminating CICs and memGRP78 could be a novel surface biomarker for isolation HN-CICs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3359.
Abstract 3360: Inhibition of ALDH activity prevents the maintenance and expansion of cancer stem cell-like population in a mouse model of breast cancer
Authors: Kim Ran-Ju , Park Jeong-Ran , Roh Kyung-Jin , Nho Kyoung-Jin , Kim Soo-Rim , Lee Yoo-Kyung , Nam Jeong-Seok
Published: February 2011

The cancer stem cell (CSC) hypothesis proposes that CSCs are the root of cancer and cause cancer metastasis and recurrence. High activity of aldehyde dehydrogenase (ALDH) has been recognized as a marker for normal and malignant stem cells. It has been also known that ALDH activity is important for multiple biological activities including drug resistance, cell differentiation, and oxidative metabolism. In this study, we focused on the molecular mechanism of ALDH activity in CSC dynamics. To address the underlying mechanism, we have used the 4T1 mouse model of breast cancer. We found that ALDHpos cells showed a high increase of the stem cell-like properties, such as sphere forming ability, expression of stem markers, and tumor initiation ability in vivo, when compared with the counterparts. Blockade of ALDH activity suppressed these properties of CSC. Especially, the high activity of ALDH was shown to enhance CSC properties through up-regulating Oct-4 expression that is dependent on oxygen-related transcription factor. Therefore, these findings suggest that the selective targeting of ALDH activity may be useful for stem specific cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3360.
Abstract 3377: CXCL13-directed therapy against bone metastasis in hormone-refractory prostate cancer
Authors: Lillard James W. , Singh Rajesh , Singh Shailesh , Sharma Praveen K.
Published: February 2011

Metastasis is responsible for most prostate cancer (PCa) related deaths; therefore, therapies designed to prevent or abrogate the spread of cancer cells and prognostic tests to better predict outcomes are greatly needed. Chemokines and their receptors are thought to play key roles in this multi-factorial process. We have recently reported, chemokine receptor CXCR5 and its ligand CXCL13 expression positively correlates with prostate tumor staging. We have also noted differential expression of CXCR5 by PCa cell lines (LNCaP, C4-2B, DU145, and PC3). By virtue of the presence of elevated CXCL13 levels in patient serum, CXCL13 secretion by bone marrow endothelium, and expression of CXCR5 on the surface of PCa cells, we hypothesized that gradient-dependent CXCR5-CXCL13 interactions are responsible in part for PCa cell bone metastases and inhibition of this axis may prove beneficial for prevention of bone metastasis and tumor progression. Luciferase-expressing PC3 cells were injected into the right carotid artery or directly into the tibia of nude mice. Before and after the development of bone metastases, groups were treated with control or anti-CXCL13 antibodies every third day and monitored for tumor progression or regression by non-invasive bioluminescent and microCT in vivo imaging. We demonstrate CXCL13 is present in bone marrow of xenograft tumor-bearing mice following intra-cardiac /intra-tibial injection of PC3 cells. This hormone-refractory PCa cell line established skeletal (osteolytic) metastases and tumor growth, but CXCL13 blockade significantly delayed prostate tumor formation, osteolysis, and spread to bones as well as growth, than compared to mice treated with control antibody. In conclusion, our study shows that the CXCL13-CXCR5 axis supports PCa bone metastasis and growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3377.
Abstract 3385: A craniectomy open window model for longitudinal subcellular imaging of cancer cell nuclear-cytoplasmic dynamics in the brain
Authors: Momiyama Masashi , Endo Itaru , Hoffman Robert M.
Published: February 2011

Brain metastasis is a lethal step in breast and other cancers. However, there has not been a method to image brain metastasis at the single-cell level in vivo. In order to visualize metastatic single cancer cell nuclear-cytoplasmic dynamics in the brain, dual-color Lewis Lung carcinoma (LLC) cells, labeled with GFP in the nucleus and RFP in the cytoplasm, were injected into the right internal carotid artery of nude mice. A craniectomy open window was made over the right parietal bone. The animals were imaged at the subcellular level through the craniectomy open window with the Olympus OV100 Small Animal Imaging System under high magnification. Metastatic dual-color LLC cells could be visualized at the subcellular level in the brain. Metastatic colony progression, tumor micro vessels, and cancer cell mitosis apoptosis were imaged. Imaging could be carried out for at least two weeks through the craniectomy window. The novel mouse model for subcellular imaging of cancer cells in the brain will enable a deeper understanding of brain metastases and cancer cell nuclear-cytoplasmic dynamics in the brain.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3385.
Abstract 3386: In vivo imaging of inflammation- and tumor-induced lymph node lymphangiogenesis by immuno-positron emission tomography
Authors: Mumprecht Viviane , Honer Michael , Vigl Benjamin , Proulx Steven , Trachsel Eveline , Kaspar Manuela , Banziger-Tobler Nadja , Schibli Roger , Neri Dario , Detmar Michael
Published: February 2011

Metastasis to regional lymph nodes is a prognostic indicator for cancer progression. There is a great demand for sensitive and non-invasive methods to detect metastasis to the lymph nodes. While conventional in vivo imaging approaches have focused on the detection of cancer cells, lymphangiogenesis within tumor draining lymph nodes might be the earliest sign of metastasis. In mouse models of lymph node lymphangiogenesis, we found that systemically injected antibodies to lymphatic epitopes accumulated in the lymphatic vasculature in tissues and lymph nodes. Using a 124I-labeled antibody against the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), we imaged, for the first time, inflammation-and tumor-draining lymph nodes with expanded lymphatic networks in vivo by positron emission tomography. This method might open up new avenues for the early detection of metastases and the images obtained might be used as biomarkers for the progression of diseases associated with lymphangiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3386.
Abstract 3389: SLPI promotes EMT in HaCaT cells
Authors: ElMasri Wafic M. , Yu Minshu , Virador Victoria M. , Kohn Elise C.
Published: February 2011

The majority of solid tumors are carcinomas. To invade, carcinomas must lose cell-cell adhesion and acquire motility: a process termed epithelial-to-mesenchymal transition (EMT). EMT is a highly conserved cellular program that enables polarized, immotile epithelial cells to convert to motile mesenchymal-like cells. Secretory leukocyte protease inhibitor (SLPI; 12-14 kD) is genomically, transcriptionally, and translationally upregulated in epithelial ovarian cancer. SLPI is anti-inflammatory, mediates wound healing, and stimulates cellular proliferation, angiogenesis, invasion, and metastasis. We previously showed that SLPI binds to and protects progranulin (pgrn), a survival factor for ovarian cancer, from elastase-mediated degradation. We also showed that overexpression of SLPI mediates paclitaxel resistance in 1A9 and HeyA8 ovarian cancer cells. We hypothesize that SLPI promotes metastasis via induction of EMT, which can be modulated by inhibition of its protease inhibitory (PI) activity or binding to pgrn. We stably transfected immortalized human skin keratinocytes (HaCaT cells) with wild type SLPI and three mutants: F-SLPI [loss of PI activity], KRK-SLPI [loss of pgrn binding], and F-KRK-SLPI [double mutant]. Overexpression of SLPI or the mutants did not transform the immortalized HaCaT cells; however, it increased colony proliferation, anchorage-independent growth, and resistance to anoikis. The SLPI mutants that do not bind pgrn were more aggressive. Transfection with SLPI and its mutants markedly decreased cell-cell adhesion, altered cellular adhesions to extracellular matrix components, and led to increased cell motility and wound healing in transfected HaCaT cells. SLPI and its mutants enhanced TGF-β induced EMT and effectively blocked TGF-β mediated growth inhibition of HaCaT cells. Thus, SLPI overexpression advances an EMT phenotype in immortalized HaCaT epithelial cells. We continue elucidating the role of SLPI in EMT and the mechanism of its interaction with proganulin for future therapeutic targeting in epithelial ovarian carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3389.
Abstract 3392: Heat shock protein 27 increases metastasis of human head and neck squamous cancer cells in vitro
Authors: Zhu Zhenkun , Xu Xin , Yu Yanke , Graham Martin , Liu Gangli , Prince Mark , Carey Thomas , Sun Duxin
Published: February 2011

The small heat shock protein 27 (Hsp27) is a molecular chaperone that is involved in a variety of cellular functions in cancer cells. The purpose of this research is to study the function of Hsp27 in metastasis of head and neck squamous cancer cells. The expression of Hsp27 in primary and metastatic head and neck cancer cells was determined using real-time PCR and western blotting. The siRNA knockdown of Hsp27 was performed in metastatic head and neck cancer cells using in vitro transfection. The proliferation of the primary and metastatic head and neck cancer cells were evaluated using MTS proliferation assay. The metastatic potential was determined using both migration assay and invasion assay in metastatic head and neck cancer cells after siRNA silencing of Hsp27. MTS proliferation showed that both primary (UM-SCC-22A) and metastatic (UM-SCC-22B) head and neck cancer cells have similar proliferation rate from 24-48 hrs. However, the metastatic head and neck cancer cells (UM-SCC-22B) showed more than 2-fold migration ability and more than 3-fold invasion ability than primary head and neck cancer cells (UM-SCC-22A). Real-time PCR demonstrated that Hsp27 mRNA is 22-fold higher in metastatic UM-SCC-22B than primary UM-SCC-22A. Similarly, Western blotting exhibited that primary UM-SCC-22A did not show any detectable levels of Hsp27, while metastatic UM-SCC-22B showed significant high levels (> 40-fold) of Hsp27. siRNA of Hsp27 knocked down expression of Hsp27 by more than 5-fold in mRNA level and 23-fold in protein levels in metastatic head and neck cancer cells UM-SCC-22B. Furthermore, siRNA knockdown of Hsp27 decreased metastasis of UM-SCC-22B by more than 50% in cell invasion assay. These data suggest that Hsp27 may regulate metastatic potential of head and neck squamous cancer cells. Silencing Hsp27 may decrease metastatic potential of head and neck squamous cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3392.
Abstract 3395: Role of ErbB3 signaling in tumor cell motility and metastasis
Authors: Smirnova Tatiana , Wyckoff Jeffrey , Segall Jeffrey E.
Published: February 2011

The major cause of death for most cancers, including that of the breast, is from metastasis. One of the trends in anticancer therapy research has recently been inhibiting the epidermal growth factor receptor (EGFR, ErbB) family that is part of the subclass I of the receptor tyrosine kinase superfamily. ErbB receptors are involved in activating such key pathways as PI3-kinase and Ras/MAP kinase. Close to 30% of highly invasive human breast cancers show elevated expression/activity of the ErbB2 receptor, a marker for poor prognosis and decreased disease-free survival. The preferred heterodimerization partner of ErbB2 is ErbB3. Previous data from our laboratory has shown that increasing expression of ErbB3 caused a significant increase in intravasation and metastasis without affecting primary tumor growth/formation. The ligand for ErbB3 is Heregulin Beta 1 (HRGβ1) and its binding leads to heterodimerization with other ErbB's, followed by activation of signaling pathways that result in cell chemotaxis and movement. Knowing that ErbB3 is important for metastasis, our goal is to determine which signaling pathway downstream of ErbB3 is responsible for metastasis as well as what cellular property (e.g. motility or chemotaxis) and which step of metastasis is affected (invasion, intravasation, lung seeding).

Stably transduced MTLn3 cells (rat mammary adenocarcinoma cell line) overexpressing WT and mutant versions of ErbB3 defective in PI3K, MAPK and Grb7 signaling were used for in vitro and in vivo experiments examining the role of ErbB3 signaling pathways. WT and mutant versions of ErbB3 were compared in their ability to chemotax in vitro. Orthotopic injection of the WT and mutant MTLn3 cell lines into the mammary fatpads of SCID mice has allowed us to examine their in vivo invasion potential by measuring the ability of cells to migrate out of the primary tumor towards a gradient of HRGβ1, intravasation - number of cells able to enter blood stream, and live imaging using multiphoton microscopy which will allow to see tumor cell polarization and movement towards blood vessels. In addition, tail vein injection tested the ability of cells to seed the lungs.

While previous data from our laboratory has shown that ErbB3 overexpression significantly enhances metastasis, it is still not known how. Preliminary results from these experiments suggest a key role for tyrosine residues on the ErbB3 receptor that couple to the PI3-kinase pathway in tumor cell motility and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3395.
Abstract 3413: Tumor-specific transcriptional control networks regulating DNA replication genes in Ewing's sarcoma
Authors: Smrzka Oskar W. , Pöhler Christina , Kauer Maximilian , Westermann Frank , Kovar Heinrich
Published: February 2011

Surveillance of DNA replication licensing is often perturbed in cancer cells allowing for genomic aberrations. Although this process contributes to cellular transformation, it is not well understood to what extent and how oncogenes might deregulate genes involved in DNA replication and in the control of replication licensing. Previous findings on the EWS-FLI1 fusion oncogene (which is the genetic hallmark Ewing's sarcoma), and our recent findings on MYC targets in this aggressive childhood tumor suggested that both these oncogenes constitute a regulatory feed-back loop. Our gene expression data from Ewing's cells in which MYC had been knocked down, we propose that MYC mediates at least part of the oncogenic EWS-FLI1 functions since it controls most essential biological functions required for cell proliferation. Gene expression correlation and gene set enrichment analysis in tumors and cell lines suggests the MYC/EWS-FLI1 loop as a central regulator in a regulatory network that controls DNA replication. To further narrow down the target gene repertoire and to identify direct transcriptional targets, we performed a genome wide chromatin-immunoprecipitation study using an anti MYC antibody in combindation with ChIP/CHIP analysis. We further extended our model whereby the core EWS-FLI1/MYC feedback loop could be linked to other candidate transcriptional regulators of DNA replication. Several replication licensing genes including MCM, ORC or licensing control genes such as CDT1 and CDC6 were found to be under direct and/or indirect control of the MYC/EWS-FLI1 loop and their associated transcription factor network. A subset of DNA replication genes is particularly deregulated in Ewing's cells when compared to mesenchymal progenitor cells that are considered the tissue of origin of Ewing's sarcoma. When comparing Ewing's sarcoma with other childhood tumors such as e.g. neuroblastoma, we found Ewing's-specific versus more general regulatory connections between the MYC/EWS-FLI1 loop and DNA replication genes. Together, our data provide first insight into a newly defined MYC/EWS-FLI1-driven regulatory network underlying transcriptional DNA replication control.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3413.