bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4070: Mutant tristetraprolin enhances growth factor downregulation and inhibition of cell growth in malignant glioma
Authors: Suswam Esther A. , Shacka John J. , Zhang Xiaowen , Lu Liang , Li Xuelin , Zheng Lei , Si Ying , King Peter H.
Published: February 2011

Background: Tristetraprolin (TTP) negatively regulates short-lived mRNAs by binding to AU-rich elements in the 3′ untranslated region (3′UTR). A number of mRNA targets, including vascular endothelial growth factor (VEGF), and interleukin (IL)-8, play an important role in tumor progression. We have shown previously that TTP downregulates IL-8 and VEGF in malignant glioma (MG) cells via RNA destabilization. In primary glioblastoma tumors, TTP is heavily phosphorylated which may render the protein less active. Hypothesis: We postulate that the destabilizing effect of TTP on target growth factor mRNAs is held in check by hyperphosphorylation which leads to altered subcellular localization and protein stability. Methods: Wild-type TTP and two mutant forms in which select serines were exchanged with alanines were cloned with a Flag epitope into pTRE2 and pEGFP vectors. U251 Tet On MG cells were transfected with pTRE2–TTP plasmids to produce stable clones. TTP expression was induced using doxycycline. Cellular location was assessed by fluorescence microscopy and Western blotting. Protein stability was measured by assessment of TTP levels following cycloheximide treatment. VEGF and IL-8 mRNA and protein levels were measured by qRT-PCR and ELISA. For assessment of RNA half-lives, cells were treated with actinomycin D. Cell growth, proliferation and cell viability were assessed using biochemical assays, soft agar, and Trypan Blue method. Results: Both IL-8 and VEGF mRNA levels were significantly lowered in the mutant TTP clones compared to wild-type. Half-lives of these mRNAs were also shortened, and ELISA analysis showed a correspondingly greater decrease in protein expression. There was a marked impact on cellular phenotype with greater inhibition of proliferation and loss of cell viability. A Soft agar colony formation was decreased by five-fold in mutant clones versus wild-type and a greater than ten-fold decrease versus the parent cell line. With TNF-α, there was a significant shift of mutant TTP to the nuclear compartment compared to WT TTP. Treatment with cycloheximide revealed that the WT TTP was less stable compared to dephosphorylated mutant. Conclusion: Removal of key serine residues from TTP enhances its function as an RNA destabilizer of VEGF and IL-8 mRNs and as a negative regulator of tumor cell growth in MG.

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 4070.
Abstract 4072: Overexpression of FOXO1 increased sensitivity to Tamoxifen in MCF7 cells, and is associated with estrogen receptor α (ERα) expression
Authors: Shang Xiying , Wu Yanyuan , Sarkissyan Marianna , Koeffler H Phillip , Vadgama Jaydutt V.
Published: February 2011

The transcription factor FOXO1, which belongs to the Forkhead family, plays an important role in promoting cellular apoptosis in cancer cells. Clinical data from our laboratory suggested that breast cancer subtypes vary in their FOXO1 expression, and this difference may contribute to differential response to drug therapy. Our goal is to investigate the mechanisms associated with FOXO1 expression and anti-estrogen therapy in ERα positive breast cancer cells (MCF7 cells). First, we examined the FOXO1 expression in different Akt transfected clones, and the results showed that FOXO1 expression decreased in MCF7/AA cells (active Akt), but significantly increased in MCF7/DN cells (dominant negative Akt). Next, we examined FOXO1 expression in the Tamoxifen resistant cells (MCF7/TAMR cells). FOXO1 expression decreased in MCF7/TAMR cells, compared to MCF7/wildtype cells. To further explore the association between FOXO1 expression and the response to Tamoxifen in MCF7 cells, we established FOXO1 overexpression clones (MCF7/FOXO1), and checked the cell growth rate under Tamoxifen treatment. The results showed that MCF7/FOXO1 cells had better response to Tamoxifen, when compared with the vector transfected cells (MCF7/pcDNA3). Since Tamoxifen is an antagonist of the ERα receptor, we examined the relationship between ERα and FOXO1. We used the ERα knockdown cells (MCF7/antisense hERα cells) as a model, and found the high Akt, but low FOXO1 expression in these cells. In summary, our study demonstrates that FOXO1 is inversely related to Akt and ERα and improves sensitivity of Tamoxifen treatment in MCF7 breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4072.
Abstract 4082: U6 upregulation in cancer
Authors: Hwang Larn , Lin Xiaoqin , Desai Neil , Trieu Vuong
Published: February 2011

Background: U6 snRNA is a non-coding RNA that is a component of the splicesome, which is involved in RNA splicing of the pre-mRNA. In view of the critical function performed by U6 snRNA in RNA splicing and therefore cellular proliferation, it is possible that U6 would play a role in cancer development. In this study, U6 levels were examined across various tumor types.

Methods: Detection of U6 snRNA in various normal and tumor tissue was performed with Tissue Microarray by In Situ Hybridization technique. The 5′-DIG-labeled U6 LNA probe (sequence 5′-CACGAATTTGCGTGTCATCCTT-3′) from Exiqon was used for this study and the Vector Red was used as substrate. The U6 stained tissue microarray was then scanned by GenePix 6.0 (Molecular Devices) at 532 nm to detect the staining intensity from each tissue sample. Each sample on the TMA was also visually scored by the intensity on a scale of 0 to 4. A quantitative real-time PCR assay was also used to confirm the overexpression of U6 snRNA in breast and other cancers.

Results: A baseline study using tumor microarrays and visual scoring suggested that U6 snRNA levels were increased in tumors relative to normal tissues. This was confirmed using the rapid U6 snRNA ISH for colon carcinoma, melanoma, pancreatic carcinoma, renal carcinoma, and lung carcinoma. In all cases, U6 levels were significantly elevated in tumor versus normal by t-test. By RT-PCR, U6 snRNA levels were also found to be increased relative to the corresponding normal tissue in carcinoma of the breast, colon, kidney, liver, lung, and ovary. No increase over the corresponding normal tissue was found in prostate and thyroid carcinomas. In breast carcinoma, there was no correlation between U6 snRNA levels and estrogen receptor status, progesterone receptor status or erb-B2 expression levels.

Conclusions: Using a scanning system and tumor tissue array, we were able to demonstrate the use of microarray scanner to quantitate ISH signal. Application of this technology to U6 snRNA demonstrated that U6 is significantly elevated in most cancer types examined.

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 4082.
Abstract 4083: Long stress-responsive non-coding transcripts identified in a whole genome screen have altered expression in cancer
Authors: Silva Jessica M. , Perez Damon S. , Pritchett Jay , Halling Merideth , Smith David I.
Published: February 2011

The number of non-coding transcripts (NCTs) exceeds the number of transcripts that code for protein. Their diverse and important functions are now being identified and utilized in the lab as well as in the clinic. Utilizing the Affymetrix Gene Chip Human Tiling 1.0R Array Set, we analyzed the transcription across the entire genome in both normal human bronchial epithelial cells (NHBE) alone and cells exposed to the tobacco carcinogen (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), NNK. We focused our efforts on the identification and function of novel long non-coding transcripts that had increased expression after exposure to NNK. We report on the identification of 12 long NNK-induced non-coding transcripts (NiTs) that are 300 nucleotides and larger in length. We first validated that these transcripts were indeed stress responsive using quantitative real-time RT-PCR (qPCR) and by Northern blot analysis revealed that these transcripts were between two and four kilobases in length. We also show using qPCR that these transcripts are more abundantly expressed in normal rapidly growing tissues or tissues that are under cellular stress. We show that there is altered expression of NiTs in a panel of lung cancer cell lines and many of these transcripts had increased expression in many breast cancer cell lines. We further analyzed one of these transcripts, NiT5 and identified it to be a 2.5 kb antisense, polyadenylated transcript by RLM-RACE and by Northern Blot analysis. Isolating nuclear and cytoplasmic fractions in BT474 cells, we identified Nit5 to be localized in the nucleus and found it to be transcribed by RNA polymerase III. NiT5 also has increased expression in breast, ovarian and cervical cancer-derived cell lines relative to normals from these three sites, but not increased expression in lung cancer cell lines. In summary, our results suggest that these novel long non-coding transcripts play an important role in stress also may play a role in 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 4083.
Abstract 410: Expression of class 3 semaphorins in prostate cancer and modulation by hypoxia
Authors: Williamson Magali , Blanc Veronique , Nariculam Joseph , Freeman Alex , Masters John
Published: February 2011

Class 3 semaphorins are secreted proteins that act as guidance cues for migrating cells via their transmembrane receptors, plexins. Semaphorins have a role in cancer affecting tumour growth both directly and indirectly, by affecting angiogenesis. The aims of this study were to determine if semaphorins have a potential role in prostate cancer and if their expression is regulated by hypoxia.

Here we show that semaphorins and their transmembrane receptors, plexins and neuropilins, are widely expressed in prostate cancer cell lines and tissue with a significant overexpression of Sema3E in tumour tissue.

Co-expression of semaphorins and their receptors by tumour cells suggests that semaphorins may act through an autocrine or paracrine mechanism in prostate cancer cells and have a direct effect on prostate tumour progression. We found that Sema3E affected integrin-mediated adhesion to fibronectin of prostate cancer cells, either enhancing or inhibiting adhesion, depending on cellular context. Sema3E also inhibited the migration of prostate cancer cells.

As well as affecting tumour progression directly, class 3 semaphorins act as angiogenesis factors. We found that expression of several semaphorin genes is affected by growth in hypoxic conditions. Notably, expression of sema3C is upregulated and Sema3A and Sema3E are down regulated by hypoxia in prostate and colon cancer cells. The down-regulation of sema3A and 3E by hypoxia is consistent with a role for Sema3A and 3E as anti-angiogenic factors in prostate cancer. These results indicate a potential role for Sema3E in prostate 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 410.
Abstract 4101: Fibroblast growth factor receptor (FGFR) 2 IIIc, is a novel molecular target for suppressing pancreatic cancer cell growth and migration
Authors: Ishiwata Toshiyuki , Matsuda Yoko , Kawahara Kiyoko , Yamamoto Tetsushi , Teduka Kiyoshi , Uchida Eiji , Korc Murray , Naito Zenya
Published: February 2011

Background: Fibroblast growth factor receptor (FGFR) isoforms IIIb and IIIc arise as a consequence of alternative splicing of the carboxy-half of the third immunoglobulin (Ig)-like domain, with IIIb isoforms being mainly expressed in epithelial cells and IIIc isoforms expressed mainly in mesenchymal cells. In pancreatic ductal adenocarcinoma (PDAC), the FGFR1 IIIb isoform mediates inhibition of cancer cell proliferation, migration, and invasion, while FGFR1 IIIc mediates cancer cell proliferation. We have previously reported that the FGFR2 IIIb isoform, also known as keratinocyte growth factor receptor/KGFR, is expressed in PDAC, and its expression correlates with VEGF-A expression and venous invasion. Furthermore, there was a significant correlation between co-expression of FGFR2 IIIb and KGF, and a poor therapeutic response. Single nucleotide polymorphisms (SNPs) of the FGFR2 gene are associated with breast and endometrial cancers, and missense mutations or copy number gains of the FGFR2 gene occur in breast and gastric cancers. A class switch of FGFR2IIIb to FGFR2IIIc is reportedly associated with a more-malignant phenotype in prostate and bladder cancers. Accordingly, in the present study we examined the role of FGFR2 IIIc in PDAC. Methods: FGFR2 IIIc expression levels were examined in six pancreatic cancer cell lines using real-time PCR and western blot analyses. Immunohistochemical analysis using anti-FGFR2 IIIc antibodies was performed on 51 pancreatic cancer patients, and FGFR2 IIIc mRNA expression was confirmed by in situ hybridization analysis. FGFR2 IIIc stably transfected KLM-1 cells were prepared and the growth rates in vitro and in vivo were compared with mock cells. The effects of specific anti- FGFR2 IIIc antibodies on the growth and migration of PANC-1 cells were also examined. Results: FGFR2 IIIc was localized in cancer cells in 37 of 51 PDAC samples (73%), and the expression correlated with retroperitoneal invasion. FGFR2 IIIc expression was detected in all six pancreatic cancer cell lines, and the expression level was high in PANC-1 cells and low in KLM-1 cells. The growth rates of FGFR2 IIIc stably transfected KLM-1 cells were higher than those of mock cells in vitro and in the subcutaneous tissue of nude mice, whereas anti-FGFR2 IIIc antibodies inhibited the growth and migration of PANC-1 cells. Conclusion: These findings indicate that FGFR2 IIIc is commonly overexpressed in PDACs, where it promotes cancer cell growth and migration. Thus, FGFR2 IIIc may serve as a novel therapeutic target in PDAC.

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 4101.
Abstract 4104: Protease-activated receptor 2, a KLK14 target in colon cancer cells
Authors: Gratio Valérie , Loriot Céline , Oikonomopoulou Katerina , Chung Hyunjae , Diamandi Eleftherios P. , Hollenberg Morley D. , Darmoul Dalila
Published: February 2011

Serine proteinases signal through proteinase-activated receptors (PARs). Our studies have implicated PAR1 and PAR4 (thrombin receptors) and PAR2 (trypsin receptor) in human colon cancer growth. However, endogenous activators of PARs in colon tumors are still unknown. Recently, members of the kallikrein-related peptidase (KLK) family have been shown to activate PARs in many cell systems. Thus, our aim was to determine if KLK14, a KLK family member, is expressed in colonic tumors and to see if this KLK can activate PARs in human colon cancer cells. The presence of KLK14 in colon cancer cell lines was assessed by RT-PCR, ELISA and immunofluorescence, and its expression in human colonic tissue was assessed by immunohistochemistry. The effects of recombinant KLK14 in parallel with PAR1/2 agonists (SFLLRN-NH2, an activating peptide for both PAR1 and PAR2; TFLLR-NH2, a selective activating peptide for PAR1 and the selective PAR2 agonists, 2-furoyl-LIGRLO-NH2; SLIGRL-NH2) were assessed for calcium flux, receptor internalization and ERK1/2 phosphorylation in a human colon cancer-derived HT29 cell line that expresses PAR1 and PAR2. We found that: a) KLK14 mRNA (RT-PCR) was present in 16 out of 16 human colon cancer cell lines, b) KLK14 protein is present (ELISA) in HT29 conditioned media and other colon cancer cell lines; c) there was strong staining of KLK14 in the cytoplasmic compartment of HT29 cells and of human colonic tumors (immunofluorescence and immunohistochemistry; paraffin sections) d) KLK14 (0.1 µM) caused prompt increases in intracellular calcium ([Ca2+]i) in HT29 cells (Microspectrofluorimetry with Fura 2/fluo-3). The KLK14-induced Ca2+-flux was abrogated after an initial challenge of the cells with SFLLRN-NH2 (100 µM), which desensitizes PAR1 and PAR2 simultaneously. Desensitization with 2-furoyl-LIGRLO-NH2 (10 µM) a potent PAR2 agonist, attenuated most of the Ca2+ flux induced by KLK14, whereas responses to TFLLR-NH2, the PAR1 agonist, were minimally affected. Consistently, a first challenge with thrombin did not affect KLK14-induced Ca2+ flux. These results strongly suggest that KLK14 activates preferentially PAR2 in HT29 cells. e) KLK14 initiates a dramatic loss of cell surface PAR2 (microscopic analysis) due to its internalization and f) KLK14 induces a rapid (within 5-10 min) and significant ERK1/2 phosphorylation in HT29 cells. This KLK14-PAR signaling pathway may represent a novel therapeutic target for colon tumorigenesis.

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 4104.
Abstract 4106: Methods for the development of carcinoid cell lines and carcinoid associated fibroblasts
Authors: Eric Sceusi L. , Rashid Asif , Zhou Yunfei , Samuel Shaija , Fan Fan , Xia Ling , Ye Xiang-Chang , Lu Jia , Tozzi Federico , Gaur Puja , Yao James , Meyn Raymond , Ellis Lee M.
Published: February 2011

Background: Research in carcinoid and neuroendocrine tumors (NETs) is limited by the lack of existing midgut derived cell lines. The two existing NET cell lines, BON and H727, are foregut derived. Foregut NETs are phenotypically distinct from midgut NETs, thus the development of a midgut derived NET cell line is of critical importance. Human NETs contain areas of stroma that likely effect carcinoid cell growth. In other types of cancer, cancer associated fibroblasts (CAFs) have been shown to secrete factors that mediate tumor cell growth. In our preliminary studies, cultured human NET cells showed better attachment to plates when they were in close association with fibroblasts. Unfortunately, the relatively slow growth of NET cells allows fibroblast overgrowth and ultimately detachment of the cancer cells. We have developed a novel approach to processing and culturing NET cells through the isolation of autologous CAFs and their use as irradiated feeder layers.

Methods: Human NET surgical specimens are procured under sterile conditions and processed in our lab. The tissue is digested to a single cell suspension. Anti-fibroblast labeled beads (Miltenyi Biotec) are used to separate CAFs from tumor cells. CAFs are then cultured and irradiated to produce feeder layers for the tumor cells. The NET cells are initially maintained on extracellular matrix protein coated plates and then plated on top of the CAF monolayer.

Results: CAFs in culture can survive 40 Gy of irradiation and will not proliferate after this treatment. These cells were viable at 12 days, at which point they were split. These irradiated fibroblasts were able to reattach to standard culture flasks and were viable, but did not proliferate after splitting, while the non-irradiated CAF controls had reached confluence by day 12 of this second passage. We currently have 5 patient NET samples that have been processed and plated in vitro on autologous CAFs. NET cells can then be separated from the irradiated fibroblasts using standard trypsinization followed by fibroblast separation using anti-fibroblast beads as described above. The tumor cells can then be used for analysis or further culturing. Early observations shows that the cancer cells appear more elongated and flattened when adjacent to fibroblasts, possibly indicating better attachment and survival.

Conclusions: While NET cells remain difficult to culture, we have developed a system that utilizes autologous CAFs in our efforts to potentially establish new NET cell lines for in vitro and in vivo studies.

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 4106.
Abstract 4108: Syndecan-1 overexpression enhances colon cancer growth by modulating heparan sulfate binding growth factors sensitivity
Authors: Hayashi Hiroaki , Minamiguchi Kazuhisa , Kanoh Akira , Ishida Keisuke , Terada Tadashi , Matsuo Kenichi
Published: February 2011

Syndecan-1 is a multifaceted transmembrane heparan sulfate proteoglycan known for its significant role in a wide range of cellular processes by interacting with a multitude of extracellular components. Although loss of syndecan-1 correlates well with the gain of cancerous characteristics in a wide range of cancers, increased expression of syndecan-1 also corresponds to adverse outcomes in some cancers. The biological function is yet to be completely understood in most of malignancies, including colon cancer. In the present study, to evaluate biological functions of syndecan-1, its expression level was altered, and subsequent outcomes were examined using human colon cancer LoVo cells. Ectopic expression of syndecan-1 in LoVo cells (LoVo/SDC1) led to larger tumor formation than did control LoVo cells (LoVo/mock) in mice. Lower concentrations of HB-EGF and HGF could activate their receptors in LoVo/SDC1 than in LoVo/mock. And, the prolonged phosphorylation of EGFR induced by HB-EGF was observed in LoVo/SDC1. Furthermore, HB-EGF more effectively promoted cell growth of LoVo/SDC1 than that of LoVo/mock. Blockade of syndecan-1 using small-interfering RNA (siRNA) gene knockdown canceled sensitization of LoVo/SDC1 to HB-EGF and diminished LoVo/SDC1 cell growth stimulated by HB-EGF. These results suggest that syndecan-1 overexpression may contribute to heparan sulfate binding growth factors sensitization and their promoting effects to cancer growth. Finally, we examined the effects of syndecan-1 on chemotherapy in the presence of heparan sulfate binding growth factors. In comparison to LoVo/mock, LoVo/SDC1 was resistant to some cytotoxic drugs (paclitaxel, 5-FU, or CPT-11) in the presence of HB-EGF or HGF. Blockade of syndecan-1 using siRNA diminished chemotherapy resistance of LoVo/SDC1 caused by HB-EGF. These findings demonstrate that syndecan-1 confers chemotherapy resistance in colon cancer through concomitant heparan sulfate binding growth factors sensitization. In summary, our results identify syndecan-1 as a functional coreceptor for heparan sulfate binding growth factors that activate their receptors and promote their downstream pathway in colon cancer cells. Syndecan-1 overexpression may contribute to tumor growth and the chemotherapy resistance characteristic of colon cancer. Therefore, therapeutic invention aimed at inhibiting this molecule may be useful in cancer treatment.

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 4108.
Abstract 4111: Induction of CXCL8 by TNFalpha and LIF (Leukemia Inhibitory Factor) in pancreatic carcinoma cells: Impact of CXCL8 as an autocrine growth factor
Authors: Kamohara Hidenobu , Hiyoshi Yoshiharu , Kurashige Junji , Kinoshita Koichi , Iwatsuki Masaaki , Baba Hideo
Published: February 2011

Pancreatic carcinoma is one of the most lethal in the gastroenterogical malignant tumors. Chronic inflammation cause to cancer development and progression. CXCL8 is a CXC chemokine, which play an important role in neutrophil chemotaxis and activation. We previously reported that CXCL8 was produced by a variety of human carcinoma cells and tissues and that CXCL8 also promoted proliferation in pancreatic carcinoma cells. In the present study, we analyzed whether various cytokines affect cell proliferation by CXCL8 induction in pancreatic carcinoma cells.

Eight examined pancreas carcinoma cell lines expressed CXCL8, CXCR1 CXCR2 and TNFRII mRNA constitutively in RPMI1640 medium without FBS. TNFalpha, LIF, IL1beta, IL6, CXCL8, or IFNbeta enhanced the expression of CXCL8 mRNA, but IL10 did not in Hs-700T cells. Especially, TNFalpha was a robust inducer. Actinomycin D suppressed and cycloheximide augmented CXCL8 mRNA which was induced by TNFalpha, LIF or not. The half-life of CXCL8 mRNA was 36.5 min by TNFalpha and 35.2 min without stimulation. That indicated the stability of CXCL8 mRNA was not affected by the stimulation of TNFalpha. Nextly, we examined the effect of CXCL8 on cell growth in Hs700T cells. Addition of recombinant TNFalpha or CXCL8 did not promoted cell growth of Hs700T. High amount of CXCL8 induced by TNFalpha or CXCL8 did not reponse to cell growth. On the other hand, LIF promoted cell growth after activation of STAT3 phosphrylation in Hs-700T cells by IP-Western blotting. The cell growth mechanism by LIF would depend on STAT3 activation. Anti-CXCL8 IgG significantly suppressed cell growth Hs-700T cells. Optimized concentration of CXCL8 would act as an autocrine growth factor in Hs-700T cells, which expressed CXCL8 mRNA highly without stimulation. Curcumin (Diferuloylmethane), NF-kappaB inhibitor, suppressed cell proliferation in Hs-700T cells.

These results suggest that CXCL8 play a pivotal role in development and progression of pancreatic cancer, and its expression is regulated by inflammatory cytokines. High concentration of CXCL8 produced by cancer cells could affect neigboring cells in pancreatic tumor microenvironment.

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 4111.
Abstract 4127: EGFR and K-Ras mutations and resistance of lung cancer to the IGF-1R tyrosine kinase inhibitors
Authors: Kim Woo-Young , Jin Quanri , Prudkin Ludmilla , Kim Jin-Soo , Morgillo Floriana , Feng Lei , Kim Edward S. , Hennessy Bryan , Lee Ju-Seog , Mills Gordon , Lee J. J. , Glisson Bonnie , Lippman Scott M. , Wistuba Ignacio I. , Lee Ho-Young
Published: February 2011

Background: The majority of patients with non-small cell lung cancer (NSCLC) has responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). We investigated (1) the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and (2) the molecular determinants of resistance to IGF-1R TKIs. Methods: Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in NSCLC tissue microarrays. The antitumor effects of IGF-1R TKIs (PQIP, OSI906), either alone or in combination with small-molecular inhibitors or siRNA targeting K-Ras or MAPK/extracellular signal-regulated kinase kinase (MEK) were analyzed in vitro and in vivo in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-transformed human bronchial epithelial (HBE) cells and in NSCLC cells with variable histologic features and mutations in EGFR or K-Ras. Results: pIGF-1R/IR expression in NSCLC specimens was positively correlated with presence of a history of tobacco smoking, squamous cell carcinoma, mutant (mut) K-Ras, and wild-type (wt) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF-1R TKIs exhibited significant antitumor activity in NNK-transformed HBE cells and in NSCLC cells harboring wt EGFR and wt K-Ras, but not those with mutations in these genes. Introduction of mut K-Ras attenuated the effects of IGF-1R TKIs on wt K-Ras-expressing NSCLC cells. Conversely, inactivation of MEK restored sensitivity to IGFR-TKI in cells carrying mut K-Ras. Conclusions: The mutation status of both EGFR and K-Ras could be a predictive marker for response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC to IGF-1R TKIs. This work was supported by NIH grants R01 CA-109520-01 and CA-100816 (all to H-YL.) and in part by DOD grant W81XWH-04-1-0142 VITAL and W8XWH-06-1-0303 BATTLE (W-K H.)

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 4127.
Abstract 4158: The p53 gain-of-function mutation p53R172H promotes oral cancer progression in response to DNA damage
Authors: Acin Sergio , Caulin Carlos
Published: February 2011

Oral cancer is one of the most common cancers worldwide, presenting high mortality rates and a severe impact on the quality of life of patients and survivors. Oral cancers arise from the accumulation of genetic alterations. Some of the most prevalent mutations found in patients with oral cancer involve mutations in Ras and p53 genes, although the mutation rates exhibit geographical variation, most likely as a result of different exposure to carcinogens. Most p53 mutations result in the expression of mutant forms of p53, rather than inactivation of the p53 gene. To determine the role of these mutations during oral cancer development we generated mice in which the endogenous K-rasG12D allele and p53 gain-of-function mutation p53R172H could be activated exclusively in the oral epithelium. We found that the p53R172H mutation, but not deletion of p53, cooperates with K-rasG12D to initiate oral tumor formation, accelerates oral tumor growth and promotes progression to squamous cell carcinoma. Expression profiling of the tumors that developed in these mice revealed that cyclin B1 was overexpressed in the oral tumors that expressed p53R172H compared to tumors with no p53. To expand the functional studies, we generated cell lines from these mouse oral tumors. Similar to the primary tumors, cell lines derived from the tumors expressing p53R172H generated tumors that grew faster upon injection in nude mice than cell lines that lacked p53. Using these cell lines we found that the expression of cyclin B1 was dramatically induced in the p53R172H cells in response to DNA damage, suggesting that cyclin B1 may contribute to the oncogenic properties p53R172H in oral tumor cells in response to DNA damage. Notably, we observed DNA damage in the oral tumors, as determined by staining for γ-H2AX. Since these tumors were initiated by oncogenic K-ras, we speculated that oncogene activation induced DNA damage in the tumors, thereby allowing the induction of cyclin B1 expression. Consistent with this hypothesis we found that suppressing K-ras in cells that express p53R172H resulted in significant reduction in cyclin B1 expression, indicating that p53R172H can induce the expression of cyclin B1 in response to oncogenic K-ras. In addition, we found increased DNA damage in the oral tumors that expressed p53R172H compared to tumors that lack p53, suggesting that p53R172H may modulate the DNA damage response. Together, these findings indicate that p53R172H may promote oral tumor progression in response to DNA damage, and suggest that attenuation of the DNA damage response may contribute to the oncogenic properties of mutant p53.

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 4158.
Abstract 4170: Primary Orthotopic Hepatocellular Carcinoma Models for Evaluation of Anticancer Drugs
Authors: Chen Dawei , Chen Taiping , Wery Jean-Pierre , Chen Yiyou
Published: February 2011

Human primary tumor model represents better alternatives for preclinical drug evaluation than cell lined derived xenograft models, due to its preservation of tumor tissue organization and pathology. At the same time, orthotopic xenograft models have also been used to better mimic the human patho-physiologic conditions such as vascularization, tumor expansion, and local microenvironmental factors. Here we combined the orthotopic transplantation method with our primary HCC models. The initial tumor inoculate was derived from a HCC patient tumor which was maintained subcutaneously in nude mice with limited passages. Tumor fragment was then anchored to the anterior hepatic lobe of nude mice. The Plasma AFP level of the orthotopic tumor bearing mice were determined once per week in order to correlate with the tumor volume. We have applied several standard care anticancer drugs to evaluate this model. Cisplatin (5mg/kg, q3d x 4) and Sorafinib (50mg/kg, qd x14), were dosed for 2 weeks after randomized grouping based upon the serum AFP levels of tumor bearing mice. The procedure of HCC fragments inoculation for this orthotopic model was successful and efficient. The tumor take rate of transplantation is over 90%. AFP level in tumor bearding mice plasma could be used as a biomarker to monitor tumor burden. These results indicated that primary orthotopic HCC model is a powerful tool for the evaluation of the drugs targeting heptocellular carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4170.
Abstract 4171: The role of the Src kinase family in determining three-dimensional epithelial cell morphology of colorectal cancer cell lines
Authors: Hellwig Sabine , Smithgall Thomas E.
Published: February 2011

Members of the Src kinase family are involved in numerous cellular signaling pathways that regulate cellular growth, survival, differentiation, adhesion and migration. Src family kinases (SFKs) associate with cellular membranes and function as molecular switches at the interface of various extracellular input signals (e.g., growth factors, cell-matrix adhesion) and intracellular signaling cascades. Deregulation of SFK expression levels and activity is implicated in a variety of human pathologies, including cancer, neurodegeneration, epilepsy and progression of HIV/AIDS. Elevation of c-Src activity is a common event in human colon cancer, and increases in c-Src activity are often associated with disease progression. In addition, upregulation of other Src-family members has been observed in colon cancer (e.g., c-Yes, Lck), suggesting that deregulation of SFKs may contribute to colorectal cancer progression.

The three-dimensional culture of epithelial cells in a laminin-rich extracellular matrix (lrECM) has become increasingly popular in investigating the integration of cellular signaling pathways. Because the growth of cells in lrECM more closely recapitulates the microenvironment encountered by cells in vivo, three-dimensional cell culture is particularly useful in elucidating the biological roles of molecular components, such as SFKs, that function at the interface of extracellular and intracellular signals.

In this study we use a three-dimensional culture system to study the role of SFKs in determining epithelial cell morphology of colorectal cancer cells. We tested a panel of ten commonly studied colorectal cancer cell lines for their ability to form organized cysts with established apical-basal polarity when grown in Matrigel in the presence or absence of broad-spectrum SFK inhibitors. We observed that Caco-2 and T84 cells, two lines capable of polarized cyst formation, show distinct sensitivity to SFK inhibition in three-dimensional culture but not in monolayer growth. Surprisingly, in Caco-2 cells, but not in T84 cells, global inhibition of SFK activity appears to interfere with cyst polarization, suggesting that one or more SFKs contribute to epithelial cell polarity. In addition, using RT-PCR and immunoblot techniques we demonstrate that SFK expression profiles are altered in Matrigel culture compared to monolayer culture. By comparing SFK expression profiles in cell lines displaying polarized versus unpolarized cell morphology we seek to identify individual members of the SFK family that contribute to three-dimensional epithelial morphology.

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 4171.
Abstract 4177: Inhibition of tumor malignancy by anti-angiogenic therapies in orthotopic mouse models of hepatocellular carcinoma
Authors: Li Gang , Hu-Lowe Dana , Christensen James , Pocalyko David
Published: February 2011

Hepatocellular carcinoma (HCC) is the most common liver malignancy, and the third most common cause of cancer-related mortalities worldwide. Most cases occur in Asia, and in China alone an estimated 251,000 male and 95,000 female patients are diagnosed annually. Although significant advances have been made with the use of certain chemotherapeutic and targeted therapeutic agents in HCC treatment, the prognosis for most liver cancer patients remains poor. To address this significant unmet medical need, Pfizer Oncology has set out to strengthen its research and drug discovery activities targeting HCC. Here we report the establishment of two series (cell line-derived and primary human tumor-derived) of orthotopic mouse models of HCC, and their validation with anti-angiogenic compounds sutent and axitinib. In both series of models, tumor fragments obtained from in vivo expansion were surgically implanted into the left lobe of mouse liver to mimic the natural microenvironment and preserve the interaction between tumor cells and the stroma components. Both sutent and axitinib demonstrated significant anti-tumor activity, as well as metastatic potential at therapeutic doses. The anti-tumor activity of these agents are associated with reduction of CD31 in the tumor. Both sutent and axitinib were well tolerated by the animals during the course of the studies. In addition, plasma samples were collected at different time points for α-feto-protein (AFP) measurement. A significant correlation between tumor size (or weight) and serum AFP level was demonstrated, which indicated that circulating AFP can be utilized as a non-invasive biomarker for HCC burden. These results support anti-angiogenesis as a valid approach for effective treatment of HCC.

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 4177.
Abstract 42: Biological significance of the lipogenic enzyme acetyl-CoA carboxylase in the control of metastatic properties of murine and human transformed cells
Authors: Scott Kristen E. , Wheeler Frances B. , Ntambi James M. , Seals Darren F. , Kridel Steven J.
Published: February 2011

In recent years, it has been determined that the fatty acid synthesis pathway is upregulated in several types of cancers of epithelial origin at both transcriptional and translational levels. Unlike normal cells, cancer cells rely on this pathway to provide the fatty acid precursors needed for synthesizing lipids that preferentially segregate into the liquid ordered microdomains of the plasma membrane. Acetyl-CoA carboxylase (ACC) is the ATP dependent and rate limiting step of fatty acid synthesis and catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. Interestingly, overexpression of this enzyme increases with severity of breast cancer patient samples and cell lines, indicating a possible role for ACC in the metastatic process. Preliminary findings from our lab indicate that ACC is important for the proteolytic invasion and migration of transformed cells independently of cell death. Moreover, ACC is critical for formation of F-actin rich membrane structures known as podosomes, which have been implicated in the metastatic process in recent years. Restoration of podosomes in cells with ACC inhibition was achieved with the addition of the soluble fatty acid, C18:1 (oleate). In contrast, inhibition of the fatty acid synthetic enzyme, fatty acid synthase (FASN), or the Δ9 desaturase, stearoyl-CoA desaturase 1 (SCD-1), does not affect podosome formation. However, inhibition of FASN resulted in attenuated proteolytic invasion and migration of transformed cells with a phenotype similar to cells with ACC inhibition although not quite to the same degree. In addition to regulation at transcriptional and translational levels, ACC activity is also regulated by phosphorylation/dephosphorylation by AMP activated protein kinase (AMPK). Decreased ACC phosphorylation at serine 79 (S79) has been shown to correlate with increased disease severity and reduced disease free survival of lung adenocarcinoma patients. This indicates that the phosphorylation status of ACC may be an important mechanism that cancer cells manipulate to successfully achieve metastasis. Therefore, induction of phosphorylation, and inactivation of ACC, either by activation of AMPK or by direct mutation of S79 may be sufficient to reduce invasion and migration of cancer cells. Taken together, investigating ACC phosphorylation status and analyzing the resulting phenotype will advance the mechanistic understanding of how cancer cells metastasize and possibly provide new therapeutic avenues to explore in the treatment of disseminated disease.

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 42.
Abstract 4206: Utilizing a Drosophila high throughput screening technology for cancer drug discovery
Authors: Gracheva Elena M. , Martinez Eduardo J. , Thompson Jill , Thompson Dominick , Cagan Ross L.
Published: February 2011

MEDROS has developed unique high-throughput 96-well drug screening methods to model human diseases in the context of a whole organism, the fruit fly Drosophila melanogaster. Our approach is designed to address disease complexity and whole animal drug pharmacology, aspects poorly addressed by in vitro or cell screening methods.

Here we focus on cancer, a complex disease that will benefit from the powerful tools available in Drosophila. We have designed a cancer fly model where the human oncogenes Ras and Src, centrally important in a variety of major human cancers, are targeted together to the retinal epithelium. We utilize inducible systems to calibrate their activation, generating reliably metastatic tumors that progress in a manner that is readily screened. In one iteration, these ‘tumors’ lead to organismal lethality that we have developed into a robust high-throughput screen. The kinase inhibitor Sorafenib rescued this phenotype, allowing flies to survive to adulthood and providing important validation of our screening platform. In a second iteration we permit Ras and Src to more mildly transform the eye epithelium to generate a ‘rough’ or ‘tumorous’ eye. We demonstrate that these phenotypes provide a second, more sensitive assay of Ras/Src oncogenic activity; again, Sorafenib rescued these cancer-like phenotypes. This work has important implications regarding novel drug screening methods for cancer and provides useful insights about the metastatic process.

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 4206.
Abstract 4216: Activation of Akt and β-catenin pathways are required for the sustention of cancer stem cell-like characteristics generated by epithelial-mesenchymal transition
Authors: Li Junlin , Evers Mark B. , Zhou Binhua P.
Published: February 2011

Approximately 90% of cancer deaths are caused by metastasis. The increased motility and invasiveness of tumor cells is reminiscent of the processes at the epithelial-mesenchymal transition (EMT), which is essential for morphogenetic events in embryonic development, tissue remodeling, and wound healing. EMT not only confers tumor cells with a distinct advantage for metastatic dissemination but also provides cancer stem cell-like characteristics for their proliferation and survival. However, the molecular mechanism for maintenance of these cancer stem cell-like traits by EMT remains unclear. In this study, we generated two stable transfectants in breast cancer MCF7 and cervical cancer Hela cell lines with expression of Twist, a key transcriptional factor of EMT. We found that expression of Twist induced a morphological EMT changes in these cells by downregulating the expression of E-cadherin and ZO-1 and by increasing vimentin and N-cadherin expression. We also showed that the CD44highCD24low subpopulation in these stable transfectants, which is associated with cancer stem cells properties, was significantly increased. Consistent with these findings, expression of stem cell marker ALDH1 was also elevated in these cells. The increased stem cell-like traits in Twist expression clones correlated with the tumorsphere formation in these cells. Strikingly, we found that β-catenin and Akt pathways were activated in these Twist-expressing transfectants. The nuclear accumulation of β-catenin correlated with the expression of CD44. Knockdown of β-catenin expression or inhibition of the Akt pathway significantly decreased the expression of CD44. Together, our results indicate that the activation of β-catenin and Akt pathway are required for the sustention of cancer stem cell-like traits generated by EMT.

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 4216.