bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5128: Physical EGFR network mapping and dynamic regulation in lung cancer cells
Authors: Li Jiannong , Bennett Keiryn , Song Lanxi , Bai Yun , Yoshida Takeshi , Edwards Arthur , Kinose Fumi , Fang Bin , Zhang Guolin , Kaupe Ines , Greben Florian , Superti-Furga Giulio , Haura Eric
Published: June 2012

Aberrant activation of Epidermal Growth Factor Receptor (EGFR) through somatic mutations plays a critical role in the development and progression in lung cancer. A better understanding of the downstream signaling network driven by EGFR mutations could uncover novel therapeutics targets and/or resistance mechanisms. We characterized a physical EGFR network by integrating (i) protein-protein interaction data derived from tandem affinity purification (TAP) and liquid chromatography mass spectrometry (LC-MS/MS) and (ii) global phospho-tyrosine protein profile identified by immunoaffinity purification of tyrosine phosphorylated peptides coupled to LC-MS/MS. All experiments were performed in lung cancer cell lines harboring activating EGFR mutations and highly sensitive to EGFR inhibitors. We identified 266 proteins in this network including other ERBB family member (ERBB2 and ERBB3), adaptors (GRB2, SHC1, MIG6 and STS1), heat shock proteins and chaperones (HSP90 isoforms, CDC37 and BIP), proteins involved in RTK endocytosis (AP family proteins), and phosphatases (PTPN12 and PTPRA). We found ERBB3 bound EGFR but not ERBB2 while PI3K subunits are exclusively in complex with ERBB3 suggesting ERBB3 is a key linkage between EGFR and downstream PI3K/AKT signaling. CDC37, as a co-chaperone of HSP90, bound multiple kinases suggesting possible functional importance in these cells. ARHGEF5, an activator of RHO pathway signaling, was found to interact with GRB2 and SHC1. MIG6 (also known as ERRFI1 and RALT), a known negative regulator of ERB signaling, interacts with numerous components of this network, including EGFR, GRB2, STS1, SHC1, ERBB2. We found that forced expression of MIG6 in PC9 and HCC827 cells inhibits EGFR-ERK signaling. Interestingly, cells maintain AKT signaling independently of EGFR through unknown mechanism. These cells become resistance to EGFR inhibitors but are sensitized to PI3K/mTOR inhibition. STS1, a tyrosine phosphatase, can also regulate EGFR signal pathway and lead to resistance to EGFR inhibitor. Updated work will be presented. Supported by the SPORE in Lung Cancer (P50-CA119997), National Functional Genomics Center, and Austrian Proteomics Platform GZ 200.145/1-VI/1/2006, part of the GEN-AU initiative of the BM:WF.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5128. doi:10.1158/1538-7445.AM2011-5128
Abstract 5131: Escape from breast cancer therapy, are neuropilins the key
Authors: Lunj Sapna , Staton Carolyn A. , Holen Ingunn , Ballmer-Hofer Kurt , Reed Malcolm W. , Brown Nicola J.
Published: June 2012

The anti-VEGF antibody Bevacizumab (Bz) inhibits VEGF binding to VEGF-R1/2 and increases progression free survival in breast cancer patients. However, eventually tumors escape treatment control and we hypothesize that this may be because Bz does not prevent VEGF from binding to its alternative receptors, the neuropilins (Np1 & Np2).

Gene expression of VEGF165, VEGF165b and the VEGF receptors, VEGF-R1/2, Np1 and Np2 in human dermal microvascular endothelial cells (ECs) and three breast cancer cell lines (MCF7, MDA-MB-231 & MDA-MB-436) was assessed using standard PCR techniques and protein expression by Western blot. The effects of 4 different Np1 binding peptides and Bz were assessed on EC differentiation (Matrigel assay of tubule formation), migration (scratch assay) and proliferation (MTS assay).

PCR revealed that VEGF and Np1/Np2 are expressed in ECs and all three breast cancer cell lines whereas VEGF-R1/2 are expressed in ECs and MDA-MB-436/231 cells only. These data were also confirmed by Western blot analysis. EC differentiation when measured as tubule number or branch points was significantly inhibited (p<0.005) by three Np1 binding peptides (p2, p7b & p10) whereas a fourth peptide (p1) had no effect. In contrast there was no significant effect on EC or breast cancer cell proliferation, although p10 caused a slight inhibition of MDA-MB-436 proliferation. Bz demonstrated a profound inhibition of EC tubule formation (Bz: 52.8± 7.2 vs. control: 76.3±1.4) but did not significantly inhibit EC or breast cancer proliferation. The effects of the peptides on migration was variable, with all four peptides having a slight inhibitory effect on EC migration, whereas only p1 and p7b slightly reduced MDA-MB-231 migration and only p10 reduced MDA-MB-436 migration. These data are currently being confirmed using the Boyden chamber, an alternative migration assay and combination studies of Bz with the most effective Np1 binding peptides are being performed

These data show that endothelial and breast cancer cells express Np1/Np2 receptors, and one Np1 binding peptide (p10: GSGSTRPPRRRR) inhibits both EC differentiation and breast cancer cell proliferation, and potentially EC and breast cancer cell migration in vitro, suggesting that this peptide may have therapeutic potential for breast cancer treatment. In vivo studies are evaluating the response to Np1 inhibition (p10) alone and in combination with Bz on angiogenesis in a breast tumor model using subcutaneous implantation of either MDA-MB-231 or MDA-MB-436 cells.

Supported by Breast Cancer Campaign

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5131. doi:10.1158/1538-7445.AM2011-5131
Abstract 5137: Pancreatic adenocarcinoma upregulated factor promotes angiogenesis and vascular permeability through modulation of endothelial cell activation
Authors: Kim Su Jin , Min Jeong-Ki , Lee Yangsoon , Koh Sang Seok
Published: June 2012

The soluble protein pancreatic adenocarcinoma upregulated factor (PAUF) is overexpressed in pancreatic cancer. We have recently reported that PAUF plays a crucial role in the progression and metastasis of pancreatic cancer cells. However its role in angiogenesis, which is crucial feature of tumor development, has not yet been understood. We here show that PAUF promotes angiogenesis and induces vascular permeability via endothelial cell activation. PAUF increased angiogenic traits of HUVEC cells such as proliferation, migration, and vascular network formation in vitro, which were abrogated by treatment with antibodies against PAUF. Consistently, PAUF stimulated the sprouting of microvessels from aortic rings ex vivo and the development of blood vessels into matrigel plug loaded with PAUF in vivo. We also found that PAUF increased permeability of endothelial cells as a result of the phosphorylation of VE-cadherin and promoted vascular leakiness of mouse skin. Intracellular signaling molecules ERK, AKT, and eNOS were activated in endothelial cell by PAUF, and chemical inhibitors for these signaling molecules inhibited PAUF-induced angiogenesis and vascular permeability. Expression analysis of several angiogenic factors indicated that these observed angiogenic effects of PAUF were independent of VEGF. We will discuss potential mechanism of angiogenesis mediated by PAUF. Collectively, our studies demonstrate PAUF as a novel angiogenic factor promoting angiogenesis and vascular permeability and suggest that targeting for PAUF can be an effective therapeutic treatment strategy to reduce tumor angiogenesis correlated with tumor progression and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5137. doi:10.1158/1538-7445.AM2011-5137
Abstract 5150: FJX1 is an important mediator of tumor biology due, in part, to its effect ontumor vascularization
Authors: Al-Greene Nicole , Lu Pengcheng , Zi Jinghuan , Jiang Aixiang , Shyr Yu , Deane Natasha , Beauchamp R. Daniel
Published: June 2012

The underlying mechanisms responsible for tumor vascularization, often correlating with poor prognosis, are an important area of cancer research. We identified Four jointed box 1 (fjx1) as a candidate gene for regulating tumor vascularization in colorectal cancer (CRC) as it was inhibited in rectal cancers (n=16) after one week treatment with Celebrex. We hypothesized that FJX1 contributes to malignant cellular behavior by enhancing tumor vascularization.

Using microarray data from 255 CRC patients we determined relative fjx1 mRNA expression levels across normal, adenoma, and stages I-IV CRC. Data were validated using quantitative RT-PCR on selected patient samples. Patients were segregated as having lower or higher than median fjx1 expression and Kaplan-Meier survival estimates were derived. Biological function was tested by overexpressing Myc-tagged human FJX1 protein in various cell lines (HEK293T, YAMC, SW480) and subjecting them to invasion and proliferation assays in vitro. SW480 derivatives were also used for flank injections to assess tumorigenesis in vivo. Tumors were analyzed via immunohistochemistry for markers of proliferation, apoptosis, and endothelial cells. Finally, conditioned media from SW480 derivatives was used in vitro to test the effects on endothelial cell (HMEC-1) invasion and tube formation.

Microarray expression data from our 255 CRC patient data set showed that fjx1 mRNA is elevated in all stages of CRC as compared to normal/adenoma tissue. Higher expression of fjx1 relative to the median was associated with significantly worse overall patient survival (p=0.0125). Elevated mRNA expression from tumor samples as compared to normal was validated by qRT-PCR within a subset of patients. Over-expression of fjx1 in HEK293T, YAMC, and SW480 cells increased invasion in vitro without altering cell proliferation. Conditioned media from SW480FJX1 cells promoted endothelial tube formation and invasion in vitro as compared to conditioned media from SW480VEC. Using an angiogenesis array and ELISA we identified and confirmed that VEGF was upregulated in SW480FJX1 conditioned media as compared to SW480VEC. Finally SW480FJX1 cells were more tumorigenic than SW480VEC in vivo, in association with increased proliferation, vascularization, and decreased apoptosis.

Taken together our results support our hypothesis that FJX1 is an important mediator of tumor biology due, in part, to its effect on tumor vascularization.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5150. doi:10.1158/1538-7445.AM2011-5150
Abstract 517: Deficiency of FAK in the bone marrow compartment enhances tumour metastasis
Authors: Batista Silvia A. , Reynolds Louise , Tavora Bernardo , Hodivala-Dilke Kairbaan
Published: June 2012

The growth of tumours depends on the tumour microenvironment. This involves both resident stromal cells such as endothelial cells and fibroblasts and also bone marrow-derived stromal cells. Focal adhesion kinase (FAK) is a downstream effector of integrins and growth factor receptors, both known regulators of the tumour stroma. Thus FAK likely plays an important role in the regulation of the tumour microenvironment.

Here we have generated adult mice RERTnERT/ERT; FAKfl/fl (UbFAKfl/fl) where FAK is deleted ubiquitously after treatment with tamoxifen (UbFAKKO). Our data demonstrate that loss of stromal FAK inhibits tumour growth even when the tumour cells themselves still express FAK. In a parallel study we have identified that endothelial FAK is required for tumour growth and angiogenesis (Tavora et al, under review).

Moreover, we show that loss of FAK in the bone marrow compartment is sufficient to enhance tumour metastasis whilst tending to inhibit primary tumour growth. Taken together our data demonstrate that bone marrow-derived FAK plays a significant but differential role in primary tumour growth and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 517. doi:10.1158/1538-7445.AM2011-517
Abstract 5170: Identification of factors secreted by melanoma exhibiting propensity for lung metastases
Authors: Huang Ruea-Yea , Odunsi Kunle , Wong Michael KK
Published: June 2012

Disseminated malignant melanoma is a result of widespread lymphatic and systemic metastases. We hypothesized that melanoma cells secret a cohort of factors that orchestrate tumor microenvironment to enable them to expand and metastasize. A proteomic approach was used to identify factors secreted by a lung metastatic and its parental primary melanoma cell lines as well as four primary cultured melanoma cell strains established from surgical material of melanoma patients. The lead factors identified were matricellular proteins, extracellular matrix proteins, tissue metalloproteinases, proteinase inhibitors, and insulin-like growth factor binding proteins. Several of these factors exhibit differential levels between the primary and the lung metastatic cell lines. To establish a melanoma lung metastasis signature, lung metastatic melanoma tumor cell lines derived from xenograph melanoma were selected. Protein secretion in culture medium by melanoma cells that exhibit a propensity to metastasize to the lung was analyzed using Western blot analysis. Our data show that decreased extracellular level of matrix metalloproteinases (MMP1, MMP2) and increased level of proteinase inhibitors (TIMP1, Glia-derived nexin) are associated with the development of melanoma lung metastases. These results suggest that these proteins can be useful markers in clinical testing for melanoma progression and as potential targets for intervening metastatic melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2011-5170
Abstract 5182: Nicotine enhanced colon cancer migration through the change of epithelial-to-mesenchymal transition biomarkers
Authors: Chang Yu-Jia , Wei Po-Li , Huang Ming-Te , Liu Jun-Jen
Published: June 2012

Long-term cigarette smoking increases the risk of colorectal cancer mortality. Tobacco's addictive toxin, nicotine, was reported to increase DNA synthesis of colon cancer cells. Since metastasis is the major cause of cancer death, the influence of nicotine on the migration of colon cancer cells remains to be determined. The influence of nicotine on the migration of colon cancer cells was evaluated using trans-well assay. Nicotine receptor-mediated migration was studied by using both inhibitors and small interfering RNA (siRNA). The role of COX-2 signal was studied using pharmacological inhibitors. The expression of epithelial mesenchymal transition (EMT) marker and COX-2 signal was evaluated using real time PCR. Nicotine enhanced DLD-1 and SW480 cell migration in dose dependent manner. We used inhibitors and siRNA to demonstrate that α7-nAChR mediates nicotine-enhanced colon cancer cell migration and upregulates fibronectin expression, which is involved in nicotine-enhanced migration. Further, COX-2 signal was induced by nicotine treatment and is involved in nicotine-enhanced fibronectin expression. We concluded that nicotine, tobacco's additive toxin, enhances colon cancer metastasis through α7-nAChR and fibronectin, a mesenchymal marker for epithelial mesenchymal transition. Furthermore, COX-2 signal was also involved in the induction of fibronectin. Therefore, smoking may play role in the progression of colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5182. doi:10.1158/1538-7445.AM2011-5182
Abstract 519: Blockade of PDGFR signaling impairs the tumor promoting effect of bone marrow-derived mesenchymal stem cells in colon cancer
Authors: Shinagawa Kei , Kitadai Yasuhiko , Ohnishi Mayu , Ohara Eiji , Tanaka Miwako , Sumida Tomonori , Tanaka Shinji , Yasui Wataru , Chayama Kazuaki
Published: June 2012

Bone marrow-derived mesenchymal stem cells (MSCs) are reported to migrate to tumor stroma as well as injured tissue. We reported recently that, in an orthotopic nude mice model of colon cancer, MSCs traveled to tumor stroma, where they differentiated into carcinoma-associated fibroblasts (CAF)-like cells. We have also found that CAFs express platelet-derived growth factor receptor (PDGFR) at a high level and that imatinib therapy targeting PDGFR in CAFs combined with administration of a cytotoxic drug significantly inhibits growth and metastasis of human colon cancer. These findings led us to examine whether the tumor promoting effect of MSCs is impaired by blockade of PDGFR signaling achieved with imatinib. KM12SM colon cancer cells alone or KM12SM cells mixed with MSCs in a 1:2 ratio were transplanted into the cecal wall of nude mice. Orthotopic transplantation of KM12SM cells mixed with MSCs, in comparison to transplantation of KM12SM cells alone, resulted in tumors of greater weight. The survival rate was significantly lower in the mixed-cell group. Co-injection of MSCs with tumor cells promotes tumor growth and metastasis of colon cancer by enhancing angiogenesis and tumor migration and invasion and by inhibiting tumor cell apoptosis. When tumor bearing animals were treated with imatinib (by gavage once daily at 50 mg/kg for 35 days), there was no significant increase in primary tumor volume or number of metastases with the KM12SM+MSC xenograft and the lower survival rate in the mixed-cell group was prolonged by the treatment. Moreover, migratory ability of MSCs to tumor stroma was impaired, and MSCs surviving in the tumor microenvironment were significantly decreased. In in vitro experiments, treatment with imatinib inhibited migration and proliferation of MSCs. Our data suggest that blockade of PDGFR signaling pathways influences the interaction between bone marrow-derived MSCs and tumor cells in the tumor microenvironment and, hence, inhibits the progressive growth of colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 519. doi:10.1158/1538-7445.AM2011-519
Abstract 5190: Biomarkers of sensitivity to PF-03084014, a γ-secretase Inhibitor, in a preclinical model of colorectal cancer
Authors: Powell Rebecca W. , Tan Aik Choon , Varella-Garcia Marileila , Touban Basel , Dasari Arvind , Puls Lauren , Pitts Todd , Kulikowski Gillian , Quackenbush Kevin , Messersmith Wells , Arcaroli John J.
Published: June 2012

Purpose: To evaluate the efficacy of PF-03084014, a γ-secretase inhibitor, in a preclinical model of colorectal cancer (CRC) and to identify biomarkers that predicts sensitivity to the compound.

Experimental Design: Fourteen patient-derived CRC explants were treated with PF-03084014 (150mg/kg/day- BID) for 28 days. Gene expression of the Notch pathway was evaluated at baseline and day 28 using RT-PCR. Additionally, baseline gene expression profiles were determined using gene array. An Aldefluor assay (stem cell technologies) was used to determine the effect of treatment on tumor initiating cells. Fluorescence in situ hybridization (FISH) was used to determine Notch 1 gene copy number.

Results: Three out of the fourteen explants showed sensitivity (cytostatic effects) to PF-03084014. Treatment reduced Notch pathway gene expression (Hes-1, Notch 1, Notch 3, and Jag1) in the sensitive tumors when evaluated by RT-PCR. Baseline gene array data showed an increase in IL-8 gene expression in resistant tumors and pathway analysis demonstrated an increase in focal adhesion, Jack-STAT, and MAPK signaling pathways in resistant explants. Evaluation of the ALDH+ cell population after 28 days of treatment showed that PF-03084014 reduced the ALDH+ cell population in the most sensitive explant (CRC001) when compared to the vehicle group. Evaluation of Notch 1 gene copy number in four CRC explants by FISH showed increased gene copy number in two sensitive explants (CRC027 and CRC001) and one intermediate explant (CRC036) and no gain in gene copy number in one resistant explant (CRC042).

Conclusion: Treatment with PF-03084014 significantly reduced tumor growth in three CRC explants. In addition, treatment inhibited expression of the Notch pathway and reduced the tumorigenic ALDH+ population. These results indicate that a subset of patients may derive benefit from treatment with PF-03084014 and biomarkers could be identified that predict sensitivity to the compound.

Acknowledgements: This work was supported by a Pfizer IIR grant.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5190. doi:10.1158/1538-7445.AM2011-5190
Abstract 5191: Dual roles for CDK8 in cancer and stem cell maintenance
Authors: McCleland Mark , Adler Adam , Firestein Ron
Published: June 2012

Cyclin Dependent Kinase 8 (CDK8) is a highly conserved member of the Mediator complex that is required for animal development and growth. Several groups have recently identified CDK8 as a colorectal oncogene (Firestein R., et al Nature 2008; Starr T. Science 2009). While, loss of CDK8 expression inhibits colon cancer cell line proliferation in vitro, the mechanism through which this occurs is unkown. In order to examine the role of CDK8 on tumor growth in vivo, we generated inducible knockdown cell lines in two colon cancer cell line models. We find that CDK8 is necessary for tumor progression in vivo and loss of CDK8 imparts a differentiated phenotype in xenografted colon cancer tumors. FACS analysis of these tumors shows that loss of CDK8 leads to a specific depletion of the tumor initating stem cell compartment. These observations were extended to mouse and human intestine, where CDK8 protein levels were found to be increased both in tumors and in the basal portion of the crypt where the intestinal stem cells reside To further explore the mechanism by which CDK8 leads to this depletion, we performed both microarray gene expression analysis and whole genome ChIP to identify CDK8 transcriptional targets. We find that many of CDK8 regulated targets overlap with gene signatures known to be upregulated in stem cells (p<1×10−45). To determine the relevance of these transcriptional targets to colon cancer, we assessed their level of expression in colon tumors and performed a loss of function screen in a panel of 12 cell lines to determine which genes are required for colon cancer proliferation. Using this approach we identify a subset of CDK8 downstream genes with dual roles in cancer and embryonic maintenance. These data show that modulation of CDK8 induces differentiation in cancer cells by potentially targeting the stem cell properties of cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5191. doi:10.1158/1538-7445.AM2011-5191
Abstract 5195: Progressive overpopulation of colonic stem cells during tumorigenesis is linked to delayed licensing of cells for DNA synthesis in the process of crypt cell maturation
Authors: Boman Bruce M. , Zhang Tao , Cofer Zenobia , Palazzo Juan , Fields Jeremy Z.
Published: June 2012

Since cancer stem cell (SC) overpopulation drives tumor growth, it is critical to identify mechanisms underlying SC overpopulation. Our hypothesis that SC overpopulation underlies development of colorectal cancer (CRC), which was based on our kinetic studies (Cancer Res 61: 3304-13, 2008), has since been supported by our biologic studies showing that ALDH1 is a specific marker for human colonic SC and can track SC overpopulation during tumor development (Cancer Res 69:3382-9, 2009). We then asked: What cellular mechanisms that normally control SC population size in colonic crypts become dysregulated during colon tumorigenesis? Accordingly, we measured the orderly, sequential phenotypic transitions in human crypts that colonocyte subpopulations undergo during their maturatio– a process involving both proliferation and differentiation. Quantitative immunohistochemical mapping of crypt cell subpopulations was done using markers for SC (ALDH1), proliferating cells (DNA-synthesis-licensing proteins: CDT1/geminin/MCM2), cell-cycle arrest (P21), and apoptosis (M30/TUNEL). Staining indices were used to evaluate changes in maturation during the step-wise progression to colon cancer. The rate of crypt cell maturation was measured using clearance of BrdU from pre-labeled wildtype (C57BL/6) and premalignant (ApcMin/+) mouse intestine. In normal human colon, the proportion of cells expressing licensing proteins was low at crypt bottom (where SC reside), high in the lower crypt where proliferating cells reside, and nil in the upper crypt (where differentiated/apoptotic cells reside). In FAP and adenomatous human crypts, SC and proliferating cell populations expanded towards the crypt top while differentiated/apoptotic subpopulations in the upper crypt contracted. ApcMin/+ mouse crypts had slower BrdU clearance than controls. Colonocytes become licensed for DNA synthesis in the transition from SC to proliferating cells; licensing is delayed in premalignant crypts. Moreover, all transitions between cell phenotypes occur more slowly in premalignant human colonic crypts, indicating that crypt cell maturation becomes progressively delayed during colon tumorigenesis. Since crypt cell maturation regulates colonic SC population size, as maturation becomes progressively delayed, crypt SC overpopulation will develop, leading to dysplasia and colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5195. doi:10.1158/1538-7445.AM2011-5195
Abstract 5201: Distinctive distribution of expression of Lgr5, an intestinal stem cell marker, during each stage of colorectal tumorigenesis
Authors: Takeda Kayoko , Kinoshita Ichiro , Shimizu Yasushi , Matsuno Yoshihiro , Shichinohe Toshiaki , Dosaka-Akita Hirotoshi
Published: June 2012

Lgr5, an orphan G-protein coupled receptor and Wnt target gene, has been identified as a murine intestinal stem cell marker. To elucidate the role of Lgr5 during each stage of human colorectal tumorigenesis, we immunohistochemically analyzed 17 low-grade, 13 high-grade intraepithelial neoplasias and 30 adenocarcinomas. While there was only sporadic expression of Lgr5 at the crypt base in normal epithelia, the increased numbers of Lgr5-positive cells were found in most tumors, with clustering of the cells to form patches. An apparent difference in their distribution was observed among the different tumorigenesis stages. Lgr5 expression in luminal surface showed a negative association with the progressive grade of the tumors (p = 0.0004), while that in the lower crypt exhibited a positive association with grade (p = 0.0001). In adenocarcinomas, Lgr5 expression in the luminal surface was negatively associated with the pStage (p = 0.02), while it was almost invariably high in the lower crypt during advanced pStage disease. These results suggest that increases in putative stem cells with the Lgr5 expression occur early during colorectal tumorigenesis and that the shifts in their distribution towards the crypt base and/or invasive tumor front play a role in the development and progression of colorectal cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5201. doi:10.1158/1538-7445.AM2011-5201
Abstract 5211: Development of a panel of colon cancer cell lines with cancer stem cell properties: a tool for cancer biology and target discovery
Authors: Roberts Penny , Young Peter , Smith Doug , Hooley Jeff , Li Jonathan , Chen Francine , Loo Deryk , Mather Jennie , Moore Paul A.
Published: June 2012

The cancer stem cell (CSC) hypothesis provides a foundation for understanding cancer development and carries significant implications relevant to drug discovery. The generation of cell lines with cancer stem cell properties would be an important advancement in the field. If CSC were capable of indefinite self-renewal without immortalization, it should be possible to develop conditions that select this minority population from a tumor for continuous culture as a line. By employing defined serum-free media and conditions that are strongly selective for fetal colon epithelial progenitor cells, we have been able to select a morphologically homogeneous minority population, hereafter we termed cancer stem-like cells (CSLC) from a repertoire of primary and metatstatic colon cancer specimens.

Six colon CSLC lines have been generated, some of which have been stably maintained in vitro for over 200 generations. When implanted subcutaneously, under the subrenal capsule, or orthotopically in immune deficient mice, all CLSC form tumors that are morphologically and phenotypically recapitulate the patient's original tumor. Furthermore, some of the lines form distant metastasis with consistent colorectal cancer morphology.

All lines express CEA; however, only a fraction expresses elevated levels of “canonical” CSC markers, including CD44 or CD133. Affymetrix U133 micro-array analysis identified a set of transcripts that were elevated in the CSLC lines compared to a series of control cell populations. These included ALDH1A1, a well known colon CSC marker, and OLFM4, a recently identified marker of normal colon crypt stem cells.

Whole-cell immunizations of mice with several lines has yielded a panel of CSLC-reactive monoclonal antibodies that recognize broadly expressed colon cancer antigens as well as surface proteins more discreetly represented within colon tumors and possibly representing CSC-specific markers.

These lines and their reactive antibodies provide the basis for developing and characterizing novel approaches aimed at targeting and the elimination of the CSC population.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5211. doi:10.1158/1538-7445.AM2011-5211
Abstract 5213: Overexpression of SIP1 and down-regulation of E-cadherin predict delayed neck metastasis in stage I/II oral tongue squamous cell carcinoma after partial glossectomy
Authors: Skakamoto Koji , Imanishi Yorihisa , Shimoda Masayuki , Kameyama Kaori , Tomita Toshiki , Ozawa Hiroyuki , Shinden Seiichi , Fujii Masato , Shibata Katsushi , Sakai Nobuya , Ogawa Kaoru
Published: June 2012

Neck lymph node involvement is considered as the most important adverse prognostic factor in head and neck squamous cell carcinoma (HNSCC). Patients with clinical stage I/II (T1-2N0M0) oral tongue carcinoma usually undergo partial glossectomy alone. However, 14 to 48% of these patients develop delayed neck metastasis (DNM) within 2 years and tend to result in unfavorable course. Clinicopathologic features and molecular biomarkers have been investigated to find the reliable predictor of DNM, however, it still remains to be elucidated. Recently E-cadherin and its transcriptional repressors are thought to play a crucial role in tumor progression and metastasis, the mechanism of which is called epithelial to mesenchymal transition (EMT). The present study was aimed to examine whether the molecular factors involved in the induction of EMT, as well as standard clinicopathologic factors, are predictive of DNM in stage I/II oral tongue squamous cell carcinoma. mRNA expressions of E-cadherin and its repressors (snail, SIP1 and twist) in 7 HNSCC cell lines were evaluated by quantitative real-time RT-PCR, which revealed significant inverse correlation between E-cadherin and SIP1 (p = 0.023). Immunohistochemical staining was performed to evaluate protein expressions of E-cadherin and its repressors in the specimens of the primary lesions of 37 stage I/II tongue carcinoma patients who had initially undergone only partial glossectomy in the Keio University Hospital without elective neck dissection. Univariate analysis showed that loss of E-cadherin (p = 0.004) and overexpression of SIP1 (p < 0.001) were significantly correlated with DNM, although no inverse correlation was found between E-cadherin and each of its repressors. Regarding other clinicopathologic factors, mode of invasion 3/4 (p = 0.048), vascular invasion (p = 0.021) and muscular invasion (p = 0,012) were also significantly correlated with DNM. Multiple logistic regression analysis including clinicopathologic and the molecular factors elucidated that overexpression of SIP1 (p = 0.005), loss of E-cadherin (p = 0.046) and vascular invasion (p = 0.024) were independently correlated with DNM. These results suggest that DNM in the stage I/II oral tongue carcinoma is closely relating to EMT, and especially SIP1 and E-cadherin are considered as the useful markers to predict development of DNM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5213. doi:10.1158/1538-7445.AM2011-5213
Abstract 5215: Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma
Authors: Goebel Steffen , Bluemke Karen , Bilkenroth Udo , Meye Axel , Fuessel Susanne , Lautenschlaeger Christine , Melchior Andres , Heynemann Hans , Fornara Paolo , Taubert Helge
Published: June 2012

Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma

The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood, so called circulating tumor cells (CTCs), for renal cell carcinoma (RCC) patients. 233 peripheral blood samples from 154 RCC patients were investigated for the presence of disseminated tumor cells (DTCs) by autoMACS technique and immunocytochemical (ICC) staining of cytokeratin. The frequency of CTCs was analyzed statistically for correlation with relevant clinical data.

Two kinds of tumor cells were detected: those with expression of cytokeratin 8/18 (CK+), but also cells without a detectable CK+ expression, which we called large blue stained (Bl+) cells with a tumor-like morphology. After following the CD45 autoMACS depletion protocol, we identified CTCs in 96 out of 233 peripheral blood samples (41%), which originated from 81 out of 154 (53%) RCC patients. A significant correlation between the detection of CTCs and positive lymph node status (p < 0.001; Chi-square test) and the presence of synchronous metastases at the time of primary tumor resection (p < 0.02; Chi-square test) was found. In a multivariate Cox's regression hazard model, presence of CK+ CTCs was significantly correlated with poor overall survival for RCC patients (RR=2.3; p < 0.05).

The presence of CTCs correlated to lymph node status and presence of synchronous metastases in RCC. It is important to evaluate CK+ and Bl+ tumor cells together to determine the role of CTCs in tumor behavior and disease progression. Detection of CK+ CTCs in peripheral blood is a significant and independent prognostic factor for RCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2011-5215
Abstract 5220: Increased expression of formyl peptide receptor (FPR) and formyl peptide receptor like-1 (FPRL1) correlates with high-risk clinical/histologic features in melanoma
Authors: Chakravarti Nitin , Peddareddigari Vijay G. R. , Warneke Carla L. , Johsnon Marcella M. , Hwu Patrick , Prieto Victor G.
Published: June 2012

Melanoma is an aggressive malignancy with a relatively high metastatic rate, responsible for almost 60% of lethal skin tumors. Despite numerous efforts, there is still a need to better understand the molecular pathways involved in malignant melanoma metastasis. FPR and its variant FPRL1, which belong to the GPCR family, have been shown to be involved in metastasis of glioma and ovarian tumor cells via activation of various signaling cascades. It is unknown whether expression of these receptors contributes to the pathogenesis of melanoma. In order to test the hypothesis that aberrant expression of these GPCRs plays a significant role in melanoma progression, we have performed semiquantitative immunohistochemical analysis of FPR and FPRL1 in benign nevi (n=49), primary melanomas with and without metastasis (n=130), and melanoma metastases (n=47 cases). A significant positive correlation was observed between the percentage of positive cells and staining intensity for FPR (p=0.001) and FPRL1 (p=0.009) with progression from benign nevi to primary melanoma. Significantly higher levels of FPR were observed in those lesions having higher Breslow thickness (p=0.044) and ulceration (p=0.019), which are well-established markers of poor clinical outcomes in patients with cutaneous melanoma. On the other hand, lesions showing a radial growth pattern expressed low levels of FPR (p=0.027). A gain in FPRL1 staining was seen in lesions displaying microscopic satellitosis (p=0.051). Disease-specific survival was significantly shorter in patients with increased cytoplasmic expression of FPR (log rank test, p=0.022). Thus, overexpression of FPR and FPRL1 correlates with disease progression and high-risk clinical and histologic features in melanoma. In addition, FPR expression is associated with shorter disease-free survival. Therefore, these markers may be an important therapeutic target in patients with melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5220. doi:10.1158/1538-7445.AM2011-5220
Abstract 5232: Lymphovascular breast carcinoma tumoral emboli beget emboli through stem-cell initiated self-budding histogenesis
Authors: Barsky Sanford H. , Ye Yin , Tellez Joseph D. , Belcher Meagan
Published: June 2012

Previous studies in our laboratory have indicated that the lymphovascular embolus is not simply a cellular fragment that detaches from the main tumor passively but represents an active clonal selection for a stem cell phenotype exhibiting enhanced stem cell signaling and survival pathways. Lymphovascular emboli exhibit increased resistance to chemotherapy and patients with large numbers of lymphovascular emboli exhibit decreased disease-free survival and poorer prognosis. Florid lymphovascular emboli is the diagnostic signature of inflammatory breast cancer (IBC) and other aggressive metastasizing cancers. The emboli have been presumed to form as a result of lymphovascular invasion but this event is thought to be rare and therefore would not alone explain the large number of emboli observed in IBC and other highly metastatic carcinomas. The reasons why some patients exhibit large numbers of lymphovascular emboli whereas other patients exhibit only rare numbers remain unknown. We wanted to specifically address this question and thus carried out both animal and in vitro imaging studies with our human xenograft model of inflammatory breast cancer, MARY-X, which exhibits florid emboli in mice and gives rise to high density spheroids in vitro. We also carried out morphometric studies on the density of human lymphovascular emboli in 25 IBC and 250 non-IBC cases and subsequent laser capture microdissection. Animal and in vitro multicolor imaging studies using anti-E-cadherin and anti-podoplanin showed evidence of self-budding histogenesis within the lymphovascular spaces with one parent embolus giving rise to daughter emboli. Correspondingly, budding spheroidgenesis was observed in vitro. Density studies of tumoral emboli in the human cases showed their numbers distributed over an exponential rather than linear range. By both RT-PCR and IHC, emboli compared to their non-embolic invasive carcinoma areas exhibited five-ten fold higher stem cell markers including Stellar, H19, Rex-1, Nestin, CD133 and Aldehyde Dehydrogenase 1 (ALDH1) as well as stem cell transcriptional determinants including OCT4, SOX2 and Nanog, and stem cell signaling pathways, eg., Bmi-1, Hedgehog and Notch3. These studies, while not explaining the genesis of the initial embolus, show conclusively that emboli beget emboli through stem-cell initiated self-budding histogenesis. It is this later process which is the more important one because it accounts for the exponential numbers of emboli seen in the human cases which exhibit aggressive behavior.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5232. doi:10.1158/1538-7445.AM2011-5232
Abstract 5246: Genomic determinants of aggressive phenotype in oral SCC
Authors: Sibson David R. , Lloyd Bryony , Woolgar Julia , Risk Janet , Shaw Richard , Dhanda Jag
Published: June 2012

Background: Oral squamous cell carcinoma (OSCC), the common form of HNSCC, affects over 600,000 cases worldwide per annum. We reviewed 561 consecutive cases in Liverpool1 finding that the most predictive marker for death was extra-capsular spread (ECS) from cervical lymph nodes (p<0.001). Overall survival at 5 years was 56% for all 561 cases, but only 24% in the ECS group independently of its site, extent or the stage of the primary tumour2. The presence of ECS predicted for recurrence, usually at the primary site, even when surgical margins were generous indicating that this aggressive phenotype is likely to be expressed in the primary tumour as much as in the metastatic cells.

Hypothesis: We hypothesised that molecular pathways represented by global gene expression signatures would be detected in the samples associated with ECS.

Methods: High density exon array expression analysis was performed on fresh frozen samples comprising T2 and T4 stage OSCC cases having no nodal involvement; positive nodes without ECS or positive nodes plus ECS picked longitudinally from a single centre series (n>200) with bias towards increasing the numbers with ECS. Contingency tests were performed to assess the significance of ECS associated pathways.

Results and Discussion: Power analysis showed that 90% of all ≥ twofold differences in gene expression between node negative & ECS cases were significant. Affected pathways included: keratinocyte differentiation, ECM remodelling, chemokines & adhesion, TGF & WNT: cytoskeletal remodelling, regulation of actin cytoskeleton by rho GTPases & cell adhesion/ histamine H1 receptor signalling/loss of cell barrier (p values:7.7×10−15 – 1.9×10−6).

A 29 gene signature able to discriminate the ECS from the non-ECS cases with 78% sensitivity and 86% specificity was found. Blocked differentiation of keratinocytes at an early (“stem cell”/ EMT) stage was suggested by the genes which included keratin 15, keratinocyte differentiation-associated protein, retinol binding protein 1, cellular serine peptidase inhibitor (Kazal type 6), and stratifin implicated in mesenchymal stem cells, basal, suprabasal expression and keratinocyte activation and differentiation. The signature may be connected with these tumours having poor prognosis.

Conclusion: This is a significant and novel finding suggesting that the pattern of metastasis in regional lymph nodes can be predicted by molecular pathways prevalent in a small oral biopsy.

1. Rogers et al. Oral Oncol 2009;45(3):201-211

2. Shaw et.al. Head Neck 2010;32(6):714-722

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5246. doi:10.1158/1538-7445.AM2011-5246
Abstract 5257: Expression and functional role of CXCR5 and CXCL13 in non-small cell lung carcinoma (NSCLC)
Authors: Singh Rajesh , Singh Shailesh , Grizzle William E. , Lillard James W.
Published: June 2012

Lung cancer is the leading cause of cancer related deaths worldwide. Chemokines and their corresponding receptor have been shown to play important role in cancer progression including lung cancer. In this regard, chemokine receptor CXCR5 has been implicated in metastatic dissemination in different models of melanoma, breast and prostate cancer. In this study we have shown that CXCR5 is highly expressed by the lung cancer cell lines and non-small cell lung carcinomas (NSCLC) tissue. CXCL13, which is the only natural ligand for CXCR5 is also expressed within the tumor. Retrospective analysis of CXCR5 expression in 45 patients specimens showed significantly higher expression of CXCR5 in Squamous cell carcinoma and Adenocarcinoma. Interestingly, nuclei of tumor cells were also positive for CXCR5 in more than 50% cases. Furthermore, serum analysis showed significantly higher serum levels of soluble CXCL13 in patients with Adenocarcinoma and Squamous cell carcinoma (n=31) compared to normal controls (n=9). The mechanism underlying these clinically and biologically important finding need to be further explored. Increased serum CXCL13 in lung cancer patients with metastatic disease required further validation as a potential therapeutic target and/or diagnostic marker for lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5257. doi:10.1158/1538-7445.AM2011-5257