bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3342: Specific inhibition of IKKβ induces caspase-independent cell death in the ovarian cancer stem cells
Authors: Alvero Ayesha B. , Montagna Michele K. , Holmberg Jennie , Mor Gil
Published: February 2011

Introduction: Current studies show that the cancer stem cells represent the chemo-resistant phenotype and therefore may survive chemotherapy to eventually rebuild the tumor. Our group previously identified and characterized the ovarian cancer stem cells using CD44 and MyD88 as a marker and showed that these cells have a constitutively active IKKβ/NFκB axis. We hypothesize that specific targeting of this pathway may affect survival of the ovarian cancer stem cells.

Methods: A panel of CD44+/MyD88+ ovarian cancer stem cells were treated with increasing concentrations of the specific IKKβ inhibitor, BAY 11-7082 (0.1, 0.5 1, 5, and 10 μg/ml). Effect on cell growth was monitored using the Incucyte real time imaging system and cell viability was determined using the Celltiter 96 assay. Specific inhibition of IKKβ was determined by: (a) the effect on the mTOR pathway by analyzing levels of phospho-S6 kinase and (b) by determining the effect on TNFα-induced NFκB activity by measuring levels of IκB. Activity of caspases- 3/7, −8, and −9 was measured using Caspase-Glo assay and levels of XIAP monitored using western blot analysis. Effect on mitochondrial membrane potential was measured using flow cytometry after staining with JC1 dye. In vivo activity was monitored using a s.c. ovarian cancer xenograft model in nude mice. The effect of BAY 11-7082 (10 mg/kg every other day given i.p.) was compared to carboplatin (40mg/kg q7d given i.p.) and paclitaxel (10mg/kg q3d given i.p.).

Results: Treatment with BAY 11-7082 inhibited cell growth and decreased cell viability in all ovarian cancer stem cell cultures tested in a dose and time dependent manner with GI50 at ∼ 5μg/ml. Specific inhibition of IKKβ was observed at this dose by a decrease in phospho-p70S6 kinase and inhibition of TNFα-induced IκB degradation. Cell death was caspase-independent with no significant increase in caspases 3/7, 8, nor 9 and no effect on the levels of XIAP. Cell death was associated with loss of mitochondrial membrane potential with more than 90% of cells showing a shift in JC1 fluorescence from red to green in 4h. In vivo studies showed that BAY 11-7082 is able to inhibit tumor growth with T/C of 64%, significantly better than carboplatin and paclitaxel (T/C 87% and 74%, p=0.009 and 0.0025, respectively).

Conclusion: The capacity of the cancer stem cells to survive chemotherapy and rebuild the tumor represent a major target to inhibit recurrence in ovarian cancer patients. The demonstration that specific targeting of IKKβ can induce cell death in the ovarian cancer stem cells opens new therapeutic options for ovarian cancer patients.

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 3342.
Abstract 3347: Enrichment of a CD44+/CD24- putative cancer stem cell population drives tumor progression and growth factor independence in a mouse model of postmenopausal breast cancer
Authors: Smith Sarah M. , Chiao Lucia J. , Salcido Crystal D. , Varticovski Lyuba , Hursting Stephen D.
Published: February 2011

MMTV-Wnt-1 transgenic mice develop heterogeneous mammary tumors that accurately recapitulate human basal-like breast cancer. Additionally, targeted alterations in the Wnt signaling pathway in mammary epithelial cells preferentially induce mammary cancers from progenitor cells. Therefore, we hypothesized that MMTV-Wnt-1 mammary tumors harbor cells with cancer stem cell characteristics. To test this hypothesis, we characterized (in vitro and in vivo) two newly generated, clonal mammary epithelial cell lines (WA4 and WG4) from MMTV-Wnt-1 transgenic mouse tumors. The cell lines were analyzed using flow cytometry for CD44 and CD24 cell surface markers (a CD44+/CD24- profile is indicative of putative human and murine mammary cancer stem cells), mammosphere formation (low density seeding on non-adherent plates in stem-cell media), in vitro scratch assay (to measure wound healing), and by an in vitro Matrigel invasion chamber assay (to quantitate cell invasion). Finally, unsorted WA4 and WG4 cell lines were transplanted into syngeneic mammary fat pads of female ovariectomized (to model the postmenopausal state) C57BL/6 mice (1×10^7 cells/mouse; n=5/cell line), and tumor burden was assessed after 3 weeks. Tumors were analyzed by qRT-PCR and immunohistochemistry (IHC) for pathological and genetic markers of progression. Flow cytometric analysis demonstrated that the percentage of CD44+/CD24- cells was 62.2% in the WA4 line and 2.4% in the WG4 line. Serum starvation enriched the WA4 line for CD44+/CD24- cells (from 62.2% to 82.0%), without altering the composition of the WG4 line. Approximately 30% of unsorted WA4 cells, but <1% of unsorted WG4 cells, formed mammospheres. Additionally, the WA4 line showed increases in wound healing by scratch assay and significant increases in invasion (226-fold increase versus WG4 cells after 30 hours). In vivo, the WA4 cell line was associated with larger, more aggressive tumors on average (+/− SD) than the WG4 line (2.09 +/− 0.68 g and 0.03 +/− 0.02 g, respectively, p<0.0001). WA4 tumors were characterized by increased cellular proliferation, microvascular proliferation, and a basal appearance lacking ductal structures. WA4 tumors also demonstrated a loss of ERα/β expression (qRT-PCR), lacked IGF1-R activation (by IHC), and showed an increase in Wnt-1 progenitor signaling (qRT-PCR). In conclusion, MMTV-Wnt-1 transgenic mouse mammary tumors harbor a CD44+/CD24- putative cancer stem cell population, and an enrichment of this population corresponds with a more aggressive tumor phenotype in a mouse model of postmenopausal breast 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 3347.
Abstract 3353: The CD44 hi cell population from malignant pleural effusion derived lung tumors displays increased tumorigenic potential
Authors: Basak Saroj , Veena Mysore , Oh Scott , Srivatsan Eri , Batra Raj K.
Published: February 2011

We have developed a new model system (http://dx.plos.org/10.1371/journal.pone.0005884) to test whether we can isolate candidate Cancer Stem Cell (CSC) populations from clinical specimens and validate that they possess increased tumorigenic and/or metastogenic potential. The model utilizes primary tumor cell populations derived from human malignant pleural effusions (MPE), which are cultured in an autologous tumor microenvironment (TME). This model system maintains intratumoral heterogeneity, and contains molecular signatures, surface phenotypes, and metabolic biomarkers that are associated with candidate CSC. The current study compares isogenic primary tumor subpopulations, separated on the basis of a surface biomarker expression, in terms of differences in molecular marker expression, and bioassays that measure clonogenic efficiency and tumorigenic potential. CD44, a surface CSC-biomarker, is expressed on most of the primary tumor cells (75-95%) derived from MPE. The CD44 expressing tumor cells can be divided into two groups (CD44hi) and (CD44lo) on the basis of CD44 surface labeling intensity. The CD44hi and CD44lo tumor cells were sorted (10% of each) from total tumor cell populations by FACS and evaluated for their clonogenicity on plastic (colony forming efficiency) and for colony forming units in soft agar (anchorage independent growth). Studies show that the CD44hi tumor cells are 40-60% more efficient in forming colonies, and give rise to larger colonies than CD44lo tumor cells. Expression of other candidate CSC biomarkers was also compared in the two cell subsets to determine the molecular correlates of CD44hi versus CD44lo cells. CD44hi cells coexpress higher levels of BMI-1, hTERT, EZH2, and OCT-4 by RT-PCR than CD44lo tumor cells, but there is no apparent difference in the expression of SUZ12 between these two populations. CD44hi cells also have increased ALDH expression based on the Aldefluor assay. To determine whether these subpopulations differed in terms of tumorigenic potential, limiting dilutions of cells were heterotopically (subcutaneous flank region) injected in NOD/SCID (IL-2gRnull) mice in comparison to CD44lo tumor cells. Low numbers of CD44hi cells (down to 300 cells) efficiently formed tumors, whereas isogenic CD44lo tumor cells failed to form tumors at the same cell doses. Thus, CD44hi cells have higher tumorigenic potential than CD44lo cells, and these studies suggest that these primary tumor populations are preferentially enriched for the candidate CSC phenotype.

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 3353.
Abstract 3356: A controversial study on well reported liver cancer stem cell marker
Authors: Ko Ching-Huai , Tseng Hsiang-Wen , Chen Ting-Shou , Wang Wei-Hsin , Lin Mai-Wei , Lai Chin-Pen , Wang Chun-Chung , Sun Chung-Yuan , Tseng Shih-Heng , Li Jen-Ming , Chou Ting-Yu , Wang Ling-Mei
Published: February 2011

Recent studies suggest that the presence of cancer stem cells (CSC) could be linked with patients’ survival. The ability of cancers to grow indefinitely has fueled the idea that cancer and stem cells may have common underlying mechanisms. It has been suggested that tumors are initiated from cancer stem cells (CSCs) with proliferation potential drives the growth of cancer. CSCs are resistant to chemotherapy and radiation. However, the suggested cancer stem cell markers in human hepatocellular carcinoma cells are variable and confused. In this study, we profiled some of the most reported CSC markers, including CD133, epithelial cell adhesion molecule (EpCAM), aldehyde dehydrogenase (ALDH), CD90, CD24, c-kit, global-H and stemness genes in eight human hepatocellular carcinoma cell lines. Through specific marker expressed cell sorting by FACs aria or magnetic beads, the CSC associated drug resistance and tumorigenicity were further evaluated. However, there is no obvious difference among parental group, marker-positive group and marker-negative group in these CSC characteristics evaluated. It seems no good correlation between reported markers in liver cancer stem cells. Therefore, presence of markers alone should be taken with caution as single prognostic parameters. Through harsh culture condition, spheroid cell grew and had been isolated, which perform CSC-like properties. Moreover, forced activation of an ESC-like gene expression program can reprogram HCC cells into CSC-like cells and achieve pathologic self-renewal. The ability to create induced cancer stem cells (iCSC) may provide opportunities to better define the biology of cancer stem cells in order to trace or eliminate them in human patients.

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 3356.
Abstract 3358: Immunohistochemical features of stem cell transcription factors on colorectal carcinoma and adenoma in familial adenomatous polyposis
Authors: Tanaka Koji , Morimoto Yuhki , Toiyama Yuji , Yasuda Hiromi , Saigusa Susumu , Yokoe Takeshi , Araki Toshimitsu , Inoue Yasuhiro , Miki Chikao , Kusunoki Masato
Published: February 2011

Background: The pathogenesis of colorectal cancer in familial adenomatous polyposis (FAP) has been studied genetically. Recently, intestinal stem cell (ISC) or cancer stem cell (CSC) is being focused on colorectal carcinogenesis. The aim of this study is to investigate the immunohistochemical features of stem cell transcription factors on colorectal adenocarcinomas, adenomataous polyps, and normal crypts in patients with FAP.

Methods: Lgr5 as an ISC marker, CD133 and CD44 as CSC markers, and NANOG, OCT4, and SOX2 as embryonic stem cell transcription factors were evaluated immunohistochemically. Matched specimens of colorectal adenocarcinoma, adenomatous polyps, and adjacent normal crypts were obtained from individual FAP patients (n=4), to clarify the association between stepwise progression from normal crypt to adenomatous polyp to adenocarcinoma and expression of stem cell transcription factors.

Results: Lgr5, NANOG, OCT4, and SOX2 were mainly located in cytoplasm of cancerous and adenomatous cells. CD133 showed cytoplasmic or intraluminal membranous staining in cancerous and adenomatous cells. Immunoreactive CD44 protein was present in cell membrane or cytoplasm of cancerous and adenomatous cells. Immunoreactive positivity and intensity was greater in colorectal adenocarcinomas than in adenomatous polyps, except for NANOG. Adjacent normal crypts showed negative or weak staining of all markers. CD133, CD44, Lgr5, OCT4, and SOX2 were strongly expressed on cancerous cells at the advancing front of colorectal adenocarcinoma.

Conclusions: ISC or CSC may be associated with colorectal carcinogenesis of FAP. Stem cell transcription factors may be involved in tumor invasion of colorectal adenocarcinoma in FAP patients.

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 3358.
Abstract 3364: Evaluation of CD133/CD44 expression level in resected gastric cancer tissues after neoadjuvant chemotherapy
Authors: Yoshida Hiroshi , Nakajima Takako Eguchi , Taniguchi Hirokazu , Horita Yousuke , Masutomi Kenkichi , Shimoda Tadakazu , Yamada Yasuhide
Published: February 2011

BACKGROUND: Increasing evidence suggests that there is a small subset of cells, called cancer stem cells (CSC), that are responsible for cancer initiation, development, metastasis and treatment resistance. Although CD133 and CD44 are thought to be the putative markers to characterize CSC of various solid tumors such as brain cancer, colorectal cancer, breast cancer and prostate cancer, the link between expression status of CD133/44 and the clnical future of resected gastric cancer tissues after neo-adjuvant chemotherapy has not yet studied. We evaluated CD133/44 expression status in resected gastric cancer issue after neoadjuvant chemotherapy.

PATIENTS AND METHODS: 32 cases were selected from advanced gastric cancer specimens of patients who underwent neo-adjuvant chemotherapy (S-1 and cisplatin or irinotecan and cisplatin or S-1 monotherapy) and surgical resection between 2001 and 2007 at the National Cancer Center Hospital. Immunohistochemical staining with anti-CD133 and anti-CD44 antibody were performed with these specimens. Immunopositive cells were counted from 10 fields in the representative section and expressed as a percentage. The specimen with necrosis or disappearance of the tumor cells in more than 1/3 of the whole lesion was defined as positive histological response specimen. Correlation of immunoreactivity with clinicopathological variables was evaluated and survival curves were compared between CD133/CD44 positive and negative cases.

RESULTS: Patient characteristics: median age 57 years (range 31-74); male/female 22/10; histological type diffuse-type/intestinal-type 25/7. CD133 and CD44 expression (positive cells: 0% <) were detected in 24 specimens (75%), and 29 specimens (91%), respectively. There was no significant difference of histological response rate between the CD133 positive and negative group (17% vs 50%, P=0.152). Median survival times were 30.4months in CD133+ group and 27.7months in CD133- group (P=0.65). Median relapse free survivals were 22.1months in CD133+ group and 17.6months in CD133- group (P=0.33). Histological type of gastric cancer (intestinal type vs diffuse type) was not correlated with CD133/CD44 expression.

CONCLUSION: CD133 and CD44 were frequently expressed in gastric cancer tissues after neoadjuvant chemotherapy. This study had limited numbers of patient sample and we were unable to study the CD133/44 status before treatment. Further study to identify the usefulness of CD133 and CD44 to predict the prognosis in gastric cancer patients is warranted.

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 3364.
Abstract 3365: Identification of target antigens on breast cancer stem cells using phage-developed scFv antibodies: A Proteomic Approach
Authors: Anurag Shukla , Krag (Mentor) David N.
Published: February 2011

The term ‘cancer stem cell’ has only recently emerged, and is defined as a cancer cell that has the ability to self-renew and give rise to another malignant stem cell, as well as differentiate to give rise to the phenotypically diverse non-tumorigenic cancer cells. The ‘cancer stem cell’ hypothesis emerged from the fact that tumors contain a heterogeneous mixture of cells and that not all the cells within a tumor contribute to tumor growth. Using phage display technology, our previous studies have identified two single chain variable fragment (scFv) antibodies (clones 207, 208) that were found to specifically bind human SUM159 breast cancer stem cells (BCSC). We have also shown in a murine xenograft tumor model prepared from the implanted SUM159 cells that these scFv antibodies predominantly bind to the cells located at the periphery of the tumors. Taking in light of these discoveries, the next logical question to be investigated was, “What cellular-antigens are these scFvs binding to?” In order to answer this in the present investigation, we used proteomic approaches based on a small scale immunoprecipitation, followed by purification and mass-spectrometry-based identification of the binding target antigens. A pure population of BCSC was acquired by flow cytometry, using ALDEFLUOR®-based sorting of the SUM159 cell line. Cell lysates of the sorted BCSC were prepared and the target antigens were immunoprecipitated with scFv-207 and scFv-208 antibodies using a solid-state immunoprecipitation assay. The immunoprecipitated targets were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The target antigens were purified and mass-spectrometric techniques (LQT-MS/ESI mass-fingerprinting) were employed to compare the proteomic data to protein databases. The details of the recovery of peptide-based target antigens and their identification using the proteomic approach as outlined above, will be presented and discussed. The identification of BCSC-specific targets will be a crucial step in designing and developing better and more specific antibody-based therapies against breast 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 3365.
Abstract 3369: Discordant immunohistochemical expression patterns of four different CD133 antibody clones in glioblastoma
Authors: Hermansen Simon K. , Christensen Karina G. , Jensen Stine S. , Schrøder Henrik D. , Kristensen Bjarne W.
Published: February 2011

The putative tumor stem cell marker CD133 is the marker of choice for identifying brain tumor stem cells in gliomas, but the use of different antibody clones recognizing different epitopes with different glycosylation status, confuses the field. In this study, we sat out to highlight if current discordant CD133 observations could be a result of using different CD133 antibodies for immunohistochemical identification of CD133. Paraffin embedded sections of glioblastoma, kidney, pancreas and placenta tissue as well as glioblastoma and retinoblastoma cell lines were stained with four different CD133 antibody clones and analyzed using light microscopy. Ten consecutive sections of glioblastomas were analyzed with each of the four CD133 clones using quantitative stereology. Results revealed presence of CD133+ niches in glioblastomas, often in close relation to blood vessels, using all four antibody clones. The distribution of identified niches did, however, rarely correspond among each antibody clone. Staining of glioblastoma single and niche cells was predominantly cytoplasmatic, which is opposed to the membranous staining observed in epithelial cells in kidney, pancreas and placenta tissues. Quantitative stereology revealed vast dissimilarities regarding fractions of CD133+ niches and single cells among the CD133 antibody clones. Generally, the fraction of CD133 positivity identified by clone W6B3C1 was significantly higher than CD133 fractions identified by clones AC133, ab19898 and C24B9. Furthermore, clone W6B3C1 was the only clone to stain tumor blood vessels. In conclusion, we report that using different CD133 antibodies for immunohistochemical identification of CD133+ cells on paraffin sections will most likely cause dissimilar results. Thus, direct comparison of CD133 studies using different primary CD133 antibodies should be performed with caution.

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 3369.
Abstract 3372: Analysis of putative tumor stem cell markers in the NCI-60 tumor cell line panel
Authors: Stuelten Christina H. , Mertins Susan D. , Busch Johanna I. , Gowens Meghan , Scudiero Dominic A. , Burkett Mark W. , Hite Karen , Alley Michael , Hollingshead Melinda , Shoemaker Robert H. , Niederhuber John E.
Published: February 2011

Tumor stem cells or cancer initiating cells are tumor cells that can regenerate a recurring tumor or a metastasis. A variety of cell surface and other markers have been associated with identification and characterization of tumor stem cells. We investigated the expression of a number of these putative stem cell markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP, and aldehyde dehydrogenase) in the NCI60 tumor panel. A spectrally compatible set of labeled antibodies was designed to enable simultaneous evaluation of six markers (CD44, CD24, CD133, CD166, CD326 (EpCAM), and PgP, and forward (FSC) and side scatter (SSC). Marker expression patterns were analyzed by multiple component analysis and correlated with clonogeneic potential of cell lines (2D colony formation assay, anchorage independent colony formation in soft agar assay). Additionally sphere formation in suspension culture and the capacity of cell lines to form tumors in vivo upon injection with matrigel was assessed for colon cancer cell lines. Analysis of expression of single markers and co-expression of up to 6 markers demonstrated that all stem cell markers investigated are expressed in cell lines of the NCI-60 panel. Patterns of co-expression of these markers as defined by multiple component analysis tracked primarily with tumor type, but individual patterns were also observed to occur in multiple tumor types. In the breast cancer panel the patterns of co-expression segregated with the known molecular sub-types (basal, luminal, normal-like) of the tumor cell lines. Cells from colonosphere cultures generated from the colon tumor panel using serum-free, growth factor supplemented medium showed subtly different co-expression patterns. Both sphere-derived and bulk cultured colon tumor cells were highly tumorigenic in NOD/SCID mice and tumor formation was observed following inoculation of 10-100 cells. Only two out of seven of the colonosphere derived cell populations had enhanced tumor initiating ability, but did not share differences in stem cell marker display with respect to the unselected bulk cultures. Furthermore, marker expression patterns did not significantly correlate with in vitro colony formation (2D culture, soft agar). Our results demonstrate a high degree of complexity and heterogeneity in tumor stem cell marker display that can be influenced by culture conditions and relates to the histological classification of tumors rather than to the clonogenic potential of cells in cell lines of the NCI60 panel.

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 3372.
Abstract 3379: Role of E-cadherin in radiation resistance of human breast cancer cells
Authors: Yuan Yuhui , Liu Jenny , Meyn Raymond E. , Munshi Anupama
Published: February 2011

E-cadherin is the major adhesion protein associated with epithelial cells loss of its expression is diagnostic of the epithelial to mesenchymal transition (EMT). Our preliminary data suggest that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which typically are relatively radiosensitive. To definitively test this hypothesis we compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) were more sensitive to increasing doses of radiation and had high expression of E-cadherin. Treatment of MCF-7 cells with 100nM siRNA to E-cadherin for 72 hrs made the cells more resistant to radiation with the surviving fraction changing from 42.3% in control cells to 56.4% in siRNA treated cells. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 cells with a CDH1-expression vector and isolated stable clones. These clones were selected and tested for radiosensitivity. MDA-MB-231 cells transfected with a control vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized the cells compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. Thus, there was a general correlation between EMT, based on loss of E-cadherin expression, and radioresistance. Overall, our results suggest that E-cadherin interacts with radiation and enhances the radioresponse of human breast cancer cells. A detailed investigation of the mechanism is ongoing.

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 3379.
Abstract 3388: Selective GFP labeling of disseminated cancer with a telomerase-specific adenovirus (OBP-401) to report recurrence after resection
Authors: Kishimoto Hiroyuki , Urata Yasuo , Fujiwara Toshiyoshi , Bouvet Michael , Hoffman Robert M.
Published: February 2011

In order to achieve improved resection of disseminated cancer we used a telomerase-specific replicating adenovirus expressing GFP (OBP-401, Telomelysin®-GFP) to label tumors in vivo. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase which is highly active in cancer cells but quiescent in most normal cells. OBP-401 contains the replication cassette, in which the hTERT promoter element drives expression of E1 genes and the GFP gene. Therefore, OBP-401 can selectively replicate in and label tumor cells with GFP. We previously demonstrated that in a peritoneal carcinomatosis mouse model of colon cancer, OBP-401 administration specifically labeled tumor cells in vivo with GFP fluorescence and enabled fluorescence-guided surgical navigation (Proc. Natl. Acad. Sci. USA, 1-6, 14514-14517, 2009; Molecular Cancer Therapeutics 8, 3001-3008, 2009). However, technical difficulties in removing microscopic tumors still remained to be solved since tumor could recur after fluorescence-guided resection. In the present study, we report an additional use of OBP-401 for surgical cure of cancer. Several weeks after cytoreduction surgery with OBP-401 GFP labeling, recurrent tumor tissues were also observed by GFP expression which could be resected under fluorescence guidance. These results indicate that OBP-401 treatment enabled fluorescent detection of subsequent recurrence after cancer surgery for the potential cure of early recurrence. The results suggest future human use of OBP-401 in cancer surgery.

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 3388.
Abstract 3391: Tie2 signaling regulates osteoclastogenesis and osteolytic bone invasion of breast cancer
Authors: Min Yongfen , Ren Xiubao , Lin Charles
Published: February 2011

Breast to bone metastasis is a common occurrence in the majority of patients with advanced breast cancer. The resultant metastases are incurable, are associated with bone destruction and high rates of morbidity. Understanding the underlying mechanisms of how metastatic tumor cells induce bone destruction is critically important. We previously reported that Tie2, a receptor tyrosine kinase, is significantly increased in human breast cancer tissues compared to normal and benign breast tumors, and regulates tumor angiogenesis. In this study, we identify a new function of Tie2 in osteoclastogenesis and osteolytic bone invasion of breast cancer. Tie2 is present in hematopoietic stem/precursor cells. Genetic deletion of Tie2 or neutralization of Tie2 function using soluble Tie2 receptor impaired osteoclastogenesis in an embryonic stem (ES) cells differentiation assay. In contrast, deletion of Tie2 has no effect on osteoblastogenesis. As monocyte has the potential to become osteoclast and Tie2 is present in certain population of monocyte, we isolated Tie2+ and Tie2- monocyte. We observed a dramatic reduction of osteoclastogenesis in Tie2- monocyte compared to Tie2+ and total monocyte. These data together reveal a direct role of Tie2 signaling in osteoclast differentiation. Consistently, neutralization of Tie2 activity in vivo significantly inhibited osteolytic bone invasion and tumor growth in a mammary tumor model, which correlated with a significant reduction of osteoclast and tumor angiogenesis. Collectively, these findings identify Tie2 a therapeutic target for controlling not only tumor angiogenesis, but also osteolytic bone metastasis.

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 3391.
Abstract 3393: Identification of genes mediating early dissemination of tumor cells in breast cancer
Authors: Wikman Harriet , Müller Volkmar , Kemming Dirk , Riethdorf Sabine , Eylmann Kathrin , Kropidlowski Jolanthe , Eils Roland , Jänicke Fritz , Streichert Thomas , Brors Benedikt , Pantel Klaus
Published: February 2011

The presence of disseminated tumor cells in bone marrow (BM) at primary breast cancer diagnosis is an independent prognostic factor. We have recently shown that there is a specific gene expression signature associated with BM micrometastasis in lung cancer patients (Wrage et al., CCR 2009). Our aim was therefore, to assess whether early metastatic spread of breast tumor cells into BM is also associated with a specific genetic signature. The gene expression profiles of primary early stage breast cancer patients (T1-2, N0, M0) with (n=15) and without (n=15) early tumor cell dissemination into BM were analyzed using the Affymetrix GeneChip platform. The BM status was assessed by immunostaining the BM samples with anti-cytokeratin antibody A45-B/B3. Gene expression data were normalized using gcrma and customized chip description files were used to re-map all probes to ENSEMBL transcripts. Rank sum test analysis was used to obtain transcripts that were significantly differentially expressed between BM positive and BM negative patients. Five publicly available breast cancer and three lung cancer expression data sets were used to correlate the findings with survival information.

When using false discovery rate of 0.2 and a p-value<0.001, 28 genes were found significantly down regulated and only 4 genes significantly up regulated in the BM positive breast cancer group. The median fold-change between the BM positive and negative groups was 3.56. Quantitative RT-PCR verifications in primary tumors and metastases validated our findings.

The five large breast cancer data sets consisting of 1621 patients in total were used for in silico validations. Among the down-regulated genes 17 showed a significant association with survival in at least one data set, whereas 6 genes showed an association in at least two data sets. Among the four up-regulated genes a high expression of one gene was associated with shorter survival in two data sets whereas another gene was associated with shorter survival in one data set.

In summary, we could identify a set of potential key regulators of early hematogeneous spread of tumor cells. These potential new targets might be used to prevent metastatic relapse by eliminating DTC before the occurrence of overt metastases. Some of the identified genes are supposed to play a role only for the early steps of the metastatic cascade, i.e. dissemination, whereas others were shown also to be important for metastatic out-growth in distant organs and thus having a high impact on the survival of a patient.

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 3393.
Abstract 340: Kaempferol inhibits angiogenesis and tumorigenesis by regulating Akt-HIF, cMyc-p21, and ERRα genes
Authors: Luo Haitao , Rankin Gary , Federoff Ann , Juliano Noelle , Jiang Bing-Hua , Chen Yi C.
Published: February 2011

This study was designed to investigate kaempferol's effects on VEGF expression and angiogenesis in ovarian cancer cells and the underlying mechanisms/pathways responsible for these effects.

Kaempferol's effects on VEGF expression in ovarian cancer cells, OVCAR-3 and A2780/CP70, were studied with ELISA and a VEGF promoter reporter assay. In-vitro and in-vivo angiogenesis were studied with human umbilical vein endothelial cells and chicken embryo chorioallantoic membranes, respectively. Expression or phosphorylation of pertinent genes in ovarian cancer cells treated with kaempferol (0-160 µM) was examined with RT-qPCR, western blot, or ELISA as appropriate. To investigate kaempferol's effects on VEGF expression, the expression of certain genes was either enhanced by transfection of plasmid DNA or knocked down by transfection of siRNA, and the resultant effects evaluated by using a VEGF promoter reporter assay or RT-qPCR.

Our study found that kaempferol is effective in inhibiting VEGF expression, in-vitro and in-vivo angiogenesis, and in-vivo tumorigenesis at physiologically relevant concentrations (e.g. 40 µM). We further looked into mechanisms/pathways for these effects, and confirmed a role for Akt and HIF-1α in kaempferol's inhibition on VEGF expression. We discovered that a new HIF-1α-independent pathway is also involved in VEGF regulation. Our results showed that kaempferol executes its effects on VEGF expression through regulating ERRα but not PPARGC1A in OVCAR-3 and A2780/CP70 cells. We also identified cMyc/p21 as a novel pathway for kaempferol's inhibitory effects on VEGF expression in ovarian cancer cells.

Our study found that kaempferol is effective for angio-prevention in ovarian cancer cells, and new information was obtained on the mechanisms/pathways for kaempferol's effects. As a dietary flavonoid, kaempferol is a good candidate for angio-prevention and primary prevention of ovarian cancers, and it deserves further investigation in animal studies and human trials.

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 340.
Abstract 3402: Knockdown of TWIST1 increases chemosensitivity of osteosarcoma cells
Authors: Xu Gufeng , Shen Jianhe , Yu Alex , Wang Hongmei , Man Tsz Kwong , Lau Ching C.
Published: February 2011

Knock-down of TWIST1 increases chemosensitivity of osteosarcoma cells

Texas Children's Cancer Center; Department of Pediatrics,

Baylor College of Medicine, Houston, TX 77030

TWIST1, a basic helix-loop-helix (bHLH) transcription factor, has previously been reported to be involved in cancer metastasis, invasion, drug response, and osteoblast differentiation induced by bone morphogenic protein. Based on expression profiling, we have previously identified a chemoresistance signature in osteosarcoma that could predict at the time of diagnosis the response to neoadjuvant chemotherapy. One of the 45 genes in the chemoresistance signature is TWIST1 whose expression level was significantly higher in osteosarcomas that were subsequently found to be more chemoresistant. To investigate the involvement of TWIST1 in chemoresistance of osteosarcoma at the cellular level, we first demonstrated that the IC50 of doxorubicin but not cisplatin treatment in eight osteosarcoma cell lines correlated with the endogenous TWIST1 expression level. We then tested the hypothesis that down-regulation of TWIST1 expression through RNAi in osteosarcoma cell lines with high TWIST1 expression could restore the chemosensitivity in these cells. Using two independent shRNAs to knock-down TWIST1 in HOS, SJSA-1 or 143B cells, we were able to show that the transduced cells became more sensitive to doxorubicin treatment as compared with the parental cells or control cells that were transduced with shRNAs with scrambled sequences. The results of the cytotoxicity assay were confirmed by the clonogenic assay using single colonies of stable transfectants with HOS cells. Based on these results, we concluded that TWIST1 is involved in chemoresistance of osteosarcoma cell lines and that down-regulation of its expression through RNAi would sensitize osteosarcoma cells to doxorubicin. However, we did not observe increased chemoresistance with overexpression of TWIST1 in osteosarcoma cell lines that have low levels of TWIST1 expression, suggesting that TWIST1 alone is not sufficient to cause chemoresistance in osteosarcoma cells. Identification of downstream targets of TWIST1 that could mediate chemoresistance by comparing expression profiles of TWIST1-knockdown clones with parental and scrambled controls is ongoing. In summary, these results validate our previous finding that TWIST1 can be used as a potential marker for predicting chemoresistance in osteosarcoma and suggest that modulating TWIST1 or its downstream targets could be considered as a novel strategy to overcome chemoresistance in osteosarcoma.

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 3402.
Abstract 3403: Responsiveness to IGF-1R targeted therapy in osteosarcoma may be regulated by the 3179 G>A mutation
Authors: Sowers Rebecca , Chung So-Hak , Zhang Wendong , Kolb E. Anders , Gorlick Richard
Published: February 2011

The insulin growth factor-1 receptor (IGF-1R) has been implicated in the pathogenesis of numerous pediatric malignancies, including osteosarcoma (OS), Ewing's sarcoma, and rhabdomyosarcoma. Historically, IGF-1R targeted therapies have been hindered by IGF-1R's structural similarity to the insulin receptor as well as toxicities associated with non-specific inhibition. A preliminary study of the effects of an IGF-1R monoclonal antibody in mouse osteosarcoma xenografts indicated that while some xenografts exhibited a complete response to the IGF-1R antibody, other xenograft samples exhibited no response. A possible explanation for the difference in response across the sample population is the presence of a genetic alteration in the IGF-1R gene.

Methods: Exons 1-20 of the IGF-1R gene were sequenced in four mouse OS xenograft samples (one “high response” sample, two “intermediate response” samples, and one “low response” sample) and in 87 OS primary tissue samples.

Results: The high response xenograft sample had a sequence alteration in exon 16 (3179 G>A) that was not present in any of the low response or intermediate response xenograft samples. Of 87 OS primary tissue samples, 34 samples were homozygous wild type (GG), 33 samples were heterozygous (GA), and 20 samples were heterozygous mutant (AA). Additional sequence alterations were present in the OS primary tissue samples, but the 3179 G>A mutation was the only alteration that differed between the responsive OS xenograft sample and the non-responsive OS xenograft sample.

Conclusions: The 3179 G>A genetic alteration has been previously reported and does not represent a coding substitution; however, it may still play a role in OS tumor response, possibly through message stability or in conjunction with a series of related mutations. Due to genetic variability across cultured cell lines, in vitro functional assays will be performed on GG-expressing cell lines after the introduction of the AA mutation. Similarly, the GG mutation will be introduced to AA expressing cell lines to compare functional relevance of the 3179 G>A alteration.

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 3403.
Abstract 3409: Arsenic trioxide inhibits Ewing sarcoma growth by blocking Hedgehog/GLI pathway
Authors: Beauchamp Elspeth M. , Rodriguez Olga , Albanese Christopher , Toretsky Jeffrey A. , Üren Aykut
Published: February 2011

Ewing's Sarcoma Family of Tumors (ESFT) is an undifferentiated neoplasm of the bone and soft tissue. ESFT is characterized by a specific chromosomal translocation occurring between chromosome 22 and (in most cases) chromosome 11, which generates an aberrant transcription factor, EWS-FLI1. We have previously shown that GLI1 is a direct transcriptional target of EWS-FLI1, and is important for ESFT tumorigenicity. Thus, GLI1 is a potential therapeutic target in ESFT. Arsenic Trioxide (ATO) is an FDA approved drug used for the treatment of Acute Promyelocytic Luekemia (APL) as a second line of therapy for those patients who don't response or relapse on All-trans-retinoic acid therapy. We show that Arsenic Trioxide inhibits GLI1 transcriptional activity. Arsenic Trioxide can also decrease the expression of GLI target genes without affecting GLI1 protein levels. We also show that ATO can inhibit the growth of ESFT cell lines in vitro and in vivo in a mouse xenograft model. To further prove that ATO is causing tumor cell growth inhibition by affecting GLI, we treated a mouse medulloblastoma model that is driven by an activating Smoothened mutation SMOA1. We were able to significantly improve survival of these mice with ATO treatment. Our results establish ATO as a hedgehog pathway inhibitor, and warrant the investigation of ATO in clinical trials as a new therapy for ESFT, basal cell carcinoma and medulloblastoma patients.

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 3409.
Abstract 3416: Cetuximab promotes immunotoxicity against rhabdomyosarcoma in vitro
Authors: Herrmann Delia , Seitz Guido , Warmann Steven W. , Bonin Michael , Fuchs Jörg , Armeanu-Ebinger Sorin
Published: February 2011

Novel treatment strategies have to be evaluated to improve the prognosis of advanced stage rhabdomyosarcoma (RMS) and to deal with clinical problems such as metastatic invasion, local tumor recurrence or multidrug resistance. Immunotherapy may represent an alternative specific, effective, less toxic and more promising therapeutic approach. Potential targets for antibody therapy of RMS are currently unknown. Differential gene expression analysis between the two main histopathological subtypes - embryonal and alveolar RMS - on 12 primary RMS samples was performed with Affymetrix array technology. Statistical analysis revealed 211 up-regulated and 323 down-regulated transcripts. Ingenuity Pathways analysis based on these results indicated the epidermal growth factor receptor (EGFR) as a central molecule with a 3.9-fold higher expression in embryonal compared to alveolar RMS. Subsequently, the expression of EGFR as well as the binding of the corresponding humanized antibody Cetuximab on embryonal (RD, A-204) and alveolar (Rh30) RMS cell lines was evaluated by flow cytometry. High expression of EGFR was detected in Rh30 and RD cells, whereas Cetuximab did not bind to A-204 at a concentration of 10 µg/ml. Next, the activity of Cetuximab was quantified using proliferation assays, in which viability of the above RMS cell lines ranged between 68% and 107% and was influenced neither by Cetuximab (concentrations ranging from 1 - 100 µg/ml) nor by 10 ng/ml EGF. However, antibody-dependent cellular cytotoxicity assays with PBMCs revealed potent activity of Cetuximab even at a low concentration of 0.05 µg/ml and a 3- to 4-fold enhanced specific lysis of RMS cells expressing EGFR. These promising effects of Cetuximab must now be tested in a suitable animal model.

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 3416.