bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1063: The ErbB3/4 receptor ligand Heregulin negatively regulates ErbB2 and ErbB3 receptor levels
Authors: Awasthi Smita , Ghosh Arundhati , Wilson Gerald M. , Hassel Bret A. , Hamburger Anne W.
Published: August 2012

ErbB2 is overexpressed in 30% of breast cancers and its overexpression is associated with poor prognosis. In addition, the ErbB2/3 heterodimer forms the most potent mitogenic receptor in vitro and is key to the proliferation of breast cancer cells. Despite the importance of this heterodimer in breast cancer progression, the negative regulation of these ErbB tyrosine kinases is still poorly understood. We demonstrate here for the first time that the ErbB3/4 ligand Heregulin (HRG) reduces mRNA and protein levels of both ErbB2 and ErbB3 in human breast cancer cell lines. We have found that after 24h of HRG treatment, ErbB2 expression decreased at both the RNA and protein levels in ErbB2 overexpressing AU565, BT474 and LTLT-Ca breast cancer cell lines. In contrast, EGFR levels were unaffected by HRG treatment. An initial decline in steady state mRNA levels was first noted at 1 hour after HRG treatment. ErbB3 levels also decreased in LTLT-Ca cells after HRG treatment. Lapatinib, which inhibits ErbB2 and EGFR kinase activity and HRG induced-ErbB3 phosphorylation, diminished the HRG- induced decrease in ErbB2 and ErbB3 in AU565 and LTLT-Ca cells. This finding suggests that the kinase activity of EGFR/ErbB2 may be involved in the HRG-induced down-regulation. HRG did not change the activity of ErbB2 promoter reporter constructs spanning 215 kb to 6007 kb upstream of the ErbB2 transcription start site as indicated by luciferase assays. An ongoing study suggests that HRG could be involved in decreasing ErbB2 mRNA stability. Our findings suggest that resistance to Lapatinib may be due in part to the fact that inhibition of ErbB2/ EGFR kinase activity dampens the negative effect of HRG on ErbB2 expression, resulting in elevated levels of ErbB2 in breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1063. doi:1538-7445.AM2012-1063
Abstract 107: p53 mutation at codon 273 in colorectal cancer and colitis-derived ALDHhigh cells promotes sphere formation in vitro
Authors: Shenoy Anitha K. , Fisher Robert , Butterworth Elizabeth Ashley , Scott Edward , Huang Emina
Published: August 2012

Introduction: p53 protein is a cell cycle regulator that functions as a tumor suppressor. Mutations in the p53 gene results in uncontrolled cell division, leading to diverse types of human cancer. In the case of colorectal cancer, p53 is the last gene to be mutated in the adenoma-to-carcinoma sequence. However, in colitis, early mutations in p53 occur in the course of dysplasia-to-carcinoma transition. Analysis of these mutations in colitis and colorectal cancer can provide clues regarding the pathogenesis of colitis to cancer.Recently, we have isolated and propagated ALDHhigh colon cancer stem cells (CCSCs) from colorectal cancer patients and precursor-CCSCs (pCCSCs) from colitic patients, which may be propagated as spheres. These CCSCs and pCCSCs may be used as a model system to study the transition from colitis to cancer. To determine if any of the p53 mutations correlate with sphere formation we analyzed p53 mutations in the existing sphere isolates. Methods: We performed sequence analysis of p53 mRNA and genomic DNA on 6 ALDHhigh sphere isolates derived from human colon cancer and 2 from colitic colon. Results: 5 out of 6 (83%) colorectal cancer derived ALDHhigh sphere isolates showed mutation at 273rd codon on the 8th exon (Arg→ His), which is one of the hot spots of p53 mutations in colorectal cancer. The same mutation was also observed in the ALDHhigh sphere isolates derived from colitic patients. This mutation corresponds to the amino acid residue that is in direct contact with DNA (contact mutants), but this mutation does not result in alterations in the spatial structure of p53. However, the defect in DNA binding ability of p53 allows the cell cycle to proceed unchecked and thus this mutation correlates with sphere forming ability. Conclusions: Mutations of p53 at codon 273 on exon 8 correlates with the sphere forming ability of the ALDHhigh cells derived from human colon cancer and colitic colon. Mutational knock-in and wild-type p53 overexpression studies would be vital to further confirm the role of this mutation in sphere formation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 107. doi:1538-7445.AM2012-107
Abstract 1080: Effect of IGF2 overexpression on the tumorigenicity of human ovarian carcinoma cells
Authors: Brouwer-Visser Jurriaan , Cossio María J. , Chao Suzan K. , Huang Gloria S.
Published: August 2012

Introduction High Insulin-like Growth Factor 2 (IGF2) tumor expression correlates with reduced disease-free survival in epithelial ovarian tumors. IGF2 mRNA is also upregulated after Taxol treatment of ovarian cancer cells. The purpose of this study was to determine the effect of IGF2 overexpression on the tumorigenicity of human ovarian cancer cells. Methods HEY cells were stably transfected using lentiviral particles with IGF2 or no insert (EV) constructs. qPCR and ELISA were performed to measure the IGF2 mRNA and secreted protein expression levels, respectively. Signaling pathways were probed by immunoblotting. Tumorigenicity was evaluated in athymic nude mice by subcutaneous injection of serial cell dilutions with matrigel, followed by monitoring of in vivo tumor growth by caliper measurements. qPCR for IGF2 mRNA and immunohistochemistry for Ki67 were performed on the excised tumors. Results The IGF2 mRNA level was 4679 ±176 fold-change higher in HEY-IGF2 cells relative to HEY-EV cells (P<0.0001). The amount of IGF2 protein secreted in 48 h was 5159 ng/100,000 HEY-IGF2 cells (51.6 pg/cell), and undetectable in the media of HEY-EV cells. Tyrosine phosphorylation of the IGF1-receptor was increased 13-fold in HEY-IGF2 cells. In vitro, dependency on the IGF1-receptor signaling was unaltered, as IGF2 overexpressing cells responded similarly to the IGF1-receptor inhibitor NVP-AEW541 compared to HEY-EV cells. Following an injection of one million cells with matrigel, the mean tumor volume of xenografts after 4 weeks was 1608 ±330 mm3 versus 705 ±95 mm3 for HEY-IGF2 and HEY-EV xenografts, respectively (P=0.04, N=6 per group). Immunohistochemistry for Ki67 showed a significantly higher percentage of stained nuclei in HEY-IGF2 tumors than in HEY-EV tumors (P=0.04, N=3 per group). Discussion Stable overexpression of IGF2 in HEY ovarian cancer cells resulted in an increased xenograft growth rate. Further evaluation is underway to fully characterize the mechanisms by which IGF2 overexpression promotes tumor growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1080. doi:1538-7445.AM2012-1080
Abstract 1202: A role for adhesion in epidermal growth factor receptor (EGFR)-mitochondrial translocation
Authors: Skalski Michael , Slack-Davis Jill , Parsons Sarah J.
Published: August 2012

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src often occurs in tumors, resulting in synergistic increases in oncogenic activity and correlation with poor patient prognosis in several cancers1. We have demonstrated that overexpression of EGFR and c-Src, and stimulation with EGF results in the transient translocation of both kinases to the mitochondria, which correlates with reduced adriamycin-induced apoptosis, and lowered O2 consumption and ATP production. Translocation requires EGFR endocytosis, as well as EGFR and c-Src catalytic activity2,3. It has been suggested that to translocate to the mitochondria, EGFR is retrograde trafficked to the ER, where it is removed from the membrane and then enters the mitochondria4. Here we show that EGFR remains associated with endosomal membranes upon translocation from the plasma membrane to mitochondria, as demonstrated by the colocalizaton of EGFR, mitochondria and extracellularly applied dextran. This translocation requires the activity of integrins, as cells plated on poly-L-lysine show significantly reduced mitochondrial-EGFR in response to EGF stimulation. Alternatively, adhesion to fibronectin in the absence of other extracellular signals was sufficient to activate EGFR and cause mitochondrial translocation. Adhesion resulted in phosphorylation on Y845, Y992, and Y1068, but not Y1045 of EGFR, while EGF stimulation, even on poly-L-lysine, resulted in the phosphorylation of all these sites. pY1045 is involved in Cbl recruitment and lysosomal degradation of EGFR5, suggesting adhesion stimulates EGFR signaling and mitochondrial translocation, while preventing lysosomal degradation. Future work will examine the role of Y1045 in mitochondrial EGFR translocation, mitochondrial function and resistance to chemotherapy. References: 1. Biscardi, J. S., Tice, D. A., and Parsons, S. J. (1999) Adv. Cancer Res. 76, 61-119. 2. Demory, M. L., et. al. (2009) J Biol Chem, 284: 36592-36604. 3. Boerner, J. L., Demory, M. L., Silva, C., and Parsons, S. J. (2004) Mol Cell Biol, 24: 7059-7071. 4. Cao, X., Zhu, H., Ali-Osman, F., and Lo, L.W. (2010) Mol. Cancer. 10:26. 5. Groval, L.M., Stang, E., Sorkin, A., and Madshus, I.H. (2004) Exp Cell Res, 300:388-95.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1202. doi:1538-7445.AM2012-1202
Abstract 1209: Detection of EGFR T790M mutation in non-small-cell lung cancer patients using colony hybridization assay
Authors: Fujita Yoshihiko , Suda Kenichi , Kimura Hideharu , Matsumoto Kazuko , Arao Tokuzo , Nagai Tomoyuki , Hayashi Hidetoshi , Furuta Kazuyuki , Kato Hiroaki , Saijo Nagahiro , Yatabe Yasushi , Mitsudomi Tetsuya , Nishio kazuto
Published: August 2012

Non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations (deletion in exon 19 or L858R) show a dramatic response to EGFR tyrosine kinase inhibitors (EGFR TKIs). However, most patients have a relapse within 1 year after the initiation of therapy. An EGFR secondary mutation T790M has been observed in approximately 50 % of such lung cancers that acquire resistance to EGFR TKIs. A critical question is whether the T790M mutation occurs as a result of treatment with EGFR TKIs or whether it exists prior to treatment and is selected during the course of therapy. However, its assessment can be challenging due to limited tissue availability and low sensitivity of the previously available methods. Here, we sought to detect the T790M using colony hybridization (CH) assay. This assay is able to detect mutant allele of 0.01 % in the background of wild-type allele. We assessed the T790M mutation in pretreatment specimens from 39 EGFR-TKI-treated patients with activating EGFR mutations. The T790M mutation was detected by CH in 31 of 39 patients (79 %). Median time to treatment failure (TTF) was 9 months in patients with pretreatment T790M, whereas it was 7 months in patients without the T790M mutation (P = 0.44). When we subdivided the patients with T790M into a strong and a modest positive subgroups in terms of the frequency of positive signals observed by our colony hybridization technique, seven patients with strong positivity had a TTF significantly longer than 8 patients without T790M (P = 0.0097) and 24 patients with modest positivity (P = 0.0019). Thus, the use of our highly sensitive method showed that a subgroup of patients with the EGFR mutation harbor rare T790M alleles before exposure to EGFR TKIs. However, contrary to our expectation, such patients had longer TTF.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1209. doi:1538-7445.AM2012-1209
Abstract 1213: EGFR interacts with fatty acid synthase
Authors: Bollu Lakshmi Reddy , Ren Jiangong , Wang Jiangong , Zhang Weihua
Published: August 2012

Background: Over expression of the epidermal growth factor receptor (EGFR) and fatty acid synthase (FASN) is common in cancers. It has been found that de novo fatty acid synthesis accounts for >90% of total cellular lipids in cancer cells. In this study, we found that EGFR physically interacts with FASN and this interaction is independent of EGFR kinase activity. Methods: We created fourteen flag tagged domain deleted EGFR, and transfected them with FASN into HEK293T cells, and performed Co-immuno-precipitation (Co-IP) followed by Western blot analysis. Results: We found EGFR interacted with FASN independent of EGFR kinase activity. Fine scale domain mapping revealed that the transmembrane (TM) and intracellular domains of EGFR are necessary for the EGFR-FASN interaction. The extracelluar domains of L1 and LF are also required for a sufficient EGFR-FASN interaction. Conclusions: EGFR interacts with FASN independent of its tyrosine kinase activity and that the transmembrane and intracellular domain of EGFR is critical for the interaction between EGFR and FASN.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1213. doi:1538-7445.AM2012-1213
Abstract 1219: Defective catagen entry in EGFR deficient skin precedes cutaneous inflammation
Authors: Bichsel Kyle J. , Gaulter Jessica A. , Porto Dennis , Foote Courtney , Hansen Laura A.
Published: August 2012

Treatment of cancer patients with epidermal growth factor receptor (EGFR) inhibitors causes perifollicular inflammation in the skin. Mouse models with EGFR deficiency predicted this folliculitis and revealed that abrogation of EGFR disrupts hair follicle cycling. We hypothesized that the defect in progression through catagen resulting from EGFR deficiency causes cutaneous inflammation. EGFR regulation of the hair cycle and inflammation was investigated using skin-targeted deletion of Egfr (Krt14-Cre+/Egfrfl/fl mice). Hair cycle stage was assessed using histological criteria, analysis of follicular apoptosis and the position of the dermal papillae. Dorsal hair follicles of control mice synchronously entered catagen at 17 d and progressed to telogen by 21 d. In contrast, Egfr mutant follicles asynchronously entered catagen, with some follicles beginning catagen at 18 d or later, and others remaining in anagen through 24 d. Mast cells were examined because follicular mast cell precursors can facilitate the transition into catagen after their maturation and degranulation. Failed catagen progression preceded an inflammatory response in the mutant skin, which was characterized by a 40% or more increase in mast cell numbers beginning at 21 d. Mast cells in mutant skin were more likely to be degranulated as well. Neutrophils and macrophages increased in mutant skin by 28 d, subsequent to the increase in mast cell numbers. Transcriptional profiling was performed at 17 d, the time of normal catagen onset, using follicular RNA obtained from laser capture microdissection to identify EGFR-regulated mediators of the inflammatory response. In silico analyses of these data identified increased expression of 12 immune function genes in mutant hair follicles including the mast cell proteases Chymase and Tryptase. Additionally, the class I major histocompatibility (MHC) molecules H2-Q9, H2-Q7 and H2-D1 and class II MHC molecules H2-Eb1, H2-Dmb1 and H2-Dmb1/2 were over-expressed in mutant hair follicles consistent with a role for adaptive immunity in cutaneous inflammation following asynchronous catagen. Thus, an aberrant catagen transition and upregulation of pro-inflammatory genes preceded folliculitis in Egfr mutant skin.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1219. doi:1538-7445.AM2012-1219
Abstract 1220: ErbB4: A novel therapeutic target in glioblastoma multiforme
Authors: Donoghue Jacqueline , Kerr Lauren , Longano Anthony , Mischel Paul , Johns Terrance
Published: August 2012

Introduction: Glioblastoma Multiforme or glioma is amongst the most lethal and difficult to cancers to treat and kills 1,000 Australians and 12,000 Americans every year. The average age at diagnosis is 60 years and the death rate for glioma has remained relatively unchanged for the past 20 years; highlighting the need for new therapeutic options for its treatment. ErbB4, the fourth member of the EGFR family has both oncogenic and tumor suppressive activity dependent upon isoform expression and although ErbB4 is universally expressed in glioma, its functional role is somewhat unclear. Aims: The aim of this study was to characterize the expression of ErbB4 in a large cohort of glioma tissues at the protein and mRNA level and determine if ErbB4 expression relates to tumor biology and patient survival. Methods: Immunohistochemistry was performed on paraffin sections of glioma tissue (n=68) and normal brain (n=5) for the immunolocalisation of ErbB4 and EGFR (total and activated) and the ErbB4 ligand heregulin. Using a customized RT-qPCR assay, ErbB4 isoforms were identified in snap frozen glioma (n=23) and normal brain (n=15). Results: Patients diagnosed with glioma (n=68) had a median survival time of 15 months (range 1-120 months) with the average age of 63±2 years (range 21-80 years). ErbB4 and heregulin protein was demonstrated in all our glioma and normal brain samples. At the mRNA level however, ErbB4 expression was reduced in the glioma samples when compared to normal brain tissue. All six ErbB4 isoforms were demonstrated in all tissue samples and ErbB4 was preferentially expressed as the JMa/Cyt2 isoform in glioma. Histologically, ErbB4 activation (independent of EGFR activation) occurred in 16% of patient samples and was localized to sites like the leading edge of the tumor. The mean survival time for patients with high levels of activated ErbB4 was 12 months, while patients with low levels of activated ErbB4 had a mean survival time of 32 months. ErbB4, activated ErbB4 and heregulin were also demonstrated in the vasculature of the glioma but not normal brain. Conclusions: This study demonstrated the preferential expression of the JMa/Cyt-2 isoform of ErbB4 in glioma along with increased ErbB4 activation significantly shortening patient survival times. Due to a potential angiogenic role of ErbB4 in glioma and reduced patient survival times following ErbB4 activation, ErbB4 should be considered a new therapeutic target in glioma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1220. doi:1538-7445.AM2012-1220
Abstract 1222: Met-dependent positive and negative signaling cascades in gastric cancer cells
Authors: Lai Andrea Z. , Zhao Hong , Cory Sean , Bell Emily S. , Hallett Michael , Park Morag
Published: August 2012

Signaling by the Met, hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) activates multiple downstream signaling pathways that promote cell migration and invasive growth. Cells that overexpress and are “addicted” to Met also require Met signaling to sustain cell survival. Thus, a number of specific small-molecule inhibitors have been developed to target Met in the clinic. Although several successes of RTK-targeted therapies are acknowledged, several have limited long-term success clinically, due to development of drug resistance. Initiation of RTK signaling cascades leads to the activation of a number of downstream signaling molecules, but prolonged activation also triggers negative feedback loops that function in part to abrogate this signaling. Hence, targeted inhibition of Met, in addition to suppressing Met-dependent signaling, may also release cells from negative feedback mechanisms and allow the reactivation of signaling pathways, that may promote resistance to Met inhibition. As MET amplification occurs in 10-20% of gastric cancers, to identify core Met dependent signaling pathways activated in Met dependent cancers we have used four different gastric cancer cell lines that exhibit amplification, overexpression, and constitutive activation of Met. Upon inhibition of Met with a small-molecule inhibitor, we observe abrogation of several downstream signaling pathways at both the protein phosphorylation and transcript level. Interestingly, as Met has been demonstrated to cross-talk with the EGF receptor family, we also observe a decrease in the phosphorylation of EGFR and HER3 upon treatment with Met inhibitor, and a decrease in expression of EGFR ligands. Conversely, Met inhibition results in an elevation in expression of HER3 transcript and protein in all 4 cell lines, implicating Met signaling in HER3 repression, and inhibition of Met may release HER3 from this negative regulation. As increases in HER3 phosphorylation and expression occur in other models (such as breast or lung cancer cell lines) upon treatment with EGFR, HER2, or AKT inhibitors, and high HER3 expression is strongly associated with tumor progression and poor prognosis in gastric cancer; hence, the loss of negative regulation of HER3, downstream from Met, may ultimately contribute to clinical efficacy, or lack thereof, of Met inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1222. doi:1538-7445.AM2012-1222
Abstract 1224: PDGFR as a novel target for the treatment of malignant peripheral nerve sheath tumors
Authors: Patwardhan Parag , Tap William , de Stanchina Elisa , Schwartz Gary K.
Published: August 2012

Malignant peripheral nerve sheath tumor (MPNST) is a rare and highly aggressive tumor type that accounts for approximately 5-10% of soft tissue sarcomas. MPNSTs arise either spontaneously or in patients with type 1 neurofibromatosis (NF1). These tumors are highly resistant to chemotherapy and there are no effective treatment options. It has been demonstrated that MPNSTs with NF1 mutations are highly sensitive to the mTOR inhibitor rapamycin. However, inhibition of this pathway by rapamycin in sarcomas has been shown to release negative feedback inhibition of IGF-1R and activate AKT (pAKT). We have shown that treatment of MPNST cell lines (MPNST and ST8814) with rapamycin activates AKT, but not through IGF-1R, since IGF-1R is not expressed in these cells. This would suggest an alternate route for rapamycin induced AKT activation. To understand this process, we treated MPNST and ST8814 cells, with or without rapamycin, and used cellular lysates to determine whether the release of negative feedback by mTOR inhibition was mediated by alternate receptor tyrosine kinases (RTKs). Using RTK array profiling (R&D Systems), our results indicate that both p-PDGFRα and p-PDGFRß are activated in response to rapamycin. Using imatinib (STI571) to target both PDGFRα and PDGFRß, we show that imatinib blocks rapamycin induced reactivation of pAKT. In order to rule out off target effects of imatinib, knockdown of PDGFR using siRNA followed by treatment with rapamycin recapitulates the effects observed with imatinib treatment. The combination of imatinib and rapamycin in MPNST cells resulted in a significant decrease in cell proliferation when compared to either of the two drugs alone. Mouse xenograft studies also show that tumor growth volume is decreased significantly when xenografts are treated with imatinib in combination with rapamycin compared to either drug alone. Taken together, our data strongly suggests that in MPNST cells, PDGFR pathway is involved in rapamycin mediated release of the negative feedback loop that activates pAKT. Furthermore, the targeting of PDGFR in combination with inhibitors of mTOR could provide a new therapeutic approach for the treatment of this disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1224. doi:1538-7445.AM2012-1224
Abstract 1225: Integration of HGF/Met signaling and hypoxia response in cancer cell invasion and proliferation
Authors: Lee Young H. , Bottaro Donald P.
Published: August 2012

The cellular response to hypoxia is a major influence on the aggressiveness of primary tumors, yet much of how this response mechanism integrates with other signaling pathways is unknown. The MET proto-oncogene, which encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), is often overexpressed in cancers and can drive cell proliferation, invasion, and metastasis. Prior studies demonstrate that the Met tyrosine-kinase receptor drives cell invasion and metastasis under hypoxic conditions, and this invasive phenotype is lost if MET expression is inhibited. Hypoxia also has been shown to sensitize Met-activated pathways to drive HGF-induced invasion through an as yet undefined molecular mechanism. The high mortality rate of metastatic cancer makes it crucial to understand the functional integration of these two prevalent drivers of metastasis, hypoxia and Met signaling, and to identify ways to selectively block cancer cell invasiveness. We show that a chemically generated pseudo-hypoxic condition enhanced HGF-mediated cell migration and invasion in the papillary renal cell (PRC) carcinoma cell lines ACHN and UOK112. Enhanced HGF-driven invasiveness was associated with sensitization of the mitogen-activated protein kinase (MAPK) pathway and increased matrix metalloproteinase activity. HGF-stimulated cell proliferation, in contrast, was phosphoinositide 3-kinase (PI3K) pathway dependent and not enhanced under the same pseudo-hypoxic condition. Furthermore, MAPK activation in the presence of HGF and hypoxia negatively regulated HGF-induced Akt activation and mitochondrial reductase enzymes as detected by MTT assay. These data suggest that hypoxia promotes tumor invasion by sensitizing the MAPK pathway to HGF stimulation in PRC-derived cells while limiting and/or suppressing HGF activation of PI3K-mediated cell proliferation. Identifying the specific effectors responsible for the integration these signals will improve our understanding of pro-metastatic invasiveness and potentially identify novels targets for its selective blockade.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1225. doi:1538-7445.AM2012-1225
Abstract 1232: Overcoming resistance to EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer
Authors: Sierra J Rafael , Chang Anderson , Moffat Jason , Neel Benjamin G. , Tsao Ming-Sound
Published: August 2012

Lung cancer is the leading cause of cancer death in developed countries both for men and women; and the second most common type of cancer diagnosed, with less than 15% of patients surviving beyond 5 years. Since more than 60% of lung adenocarcinomas and more than 90% of lung squamous cell carcinomas express high levels of EGFR mRNA and protein, it has been the focus of efforts to develop new agents that target the EGFR pathway. Clinical trials have demonstrated that patient selection plays a major role for patient response. Patients that present amplification or activating mutations (L878R or exon 19 deletions) of EGFR, have higher response rates. Selection improved response rates from less than 10% to over 60-80%. Despite the promising results, all patients that receive TKI therapy develop resistance to treatment in less than one year. The mechanisms of resistance to EGFR TKi's described so far are those dependant on the overactivation/amplification of other receptor tyrosine kinases (RTK) capable to sustain anti-apoptotic signaling pathways. Our study proposes the use of genome-wide screenings using an 80,000 shRNA library on EGFR resistant cells can identify new genes that mediate the resistance to EGFR targeted therapy and provide the basis for designing new therapeutic approaches to avoid the emergence of resistance. So far, we have generated a shortlist of gene candidates that confer synergistic synthetic lethal interactions with EGFR targeted therapies and that are also highly expressed in resistant cells. We are currently overexpressing candidate genes in sensitive cells searching for those that can confer resistance to EGFR targeted therapies. Our study proposes target genes that can synergize with the current treatments to create new therapeutical strategies that can avoid the acquisition of resistance to targeted therapies. Overall, the information gained from this type of study can be applicable to tumors where ERBB receptor(s) play an important role and targeted therapies are currently used.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1232. doi:1538-7445.AM2012-1232
Abstract 1233: A triad of Brk, HER2 and STAT3 in regulating epithelial and mesenchymal transition and survival of breast cancer cells
Authors: Ai Midan
Published: August 2012

Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase recently shown to be coexpressed with human epidermal growth factor receptor-2 (HER2) at high levels in tumor specimens from patients with invasive ductal breast cancer. Brk is associated with HER2 in breast cancer cells and can directly phosphorylate signal transducers and activators of transcription-3 (STAT3). However, the biological functions of these interactions remain largely unknown. In current study, we developed a subline from HER2-transfected MCF7 breast cancer cells through in vivo selection in nude mice. Overexpression of HER2 led to a higher level of Brk in MCF7 cells and the in vivo selection resulted in a subline with markedly enhanced epithelial and mesenchymal transition (EMT) phenotype and markedly increased migration potential. We named the subline cells EMT1. Both the EMT phenotype and survival of the EMT1 cells heavily depend on Brk, because knockdown of Brk through RNA interference reversed the EMT phenotype, reduced cell migration potential and induced cell death via apoptosis in the EMT1 cells. We further found that the level of activation-specific phosphorylated STAT3 was markedly increased in the EMT1 cells compared with control vector-transfected or HER2-transfected MCF7 cells. Knockdown of Brk significantly reduced the level of STAT3 phosphorylation in the EMT1 cells. Overexpression of a constitutively active STAT3 counteracted Brk knockdown-induced inhibition of cell migration potential and Brk knockdown-induced apoptosis in the EMT1 cells. Together, these preliminary findings support a new paradigm for regulating breast cancer cell EMT and survival that involves interaction and coordination between Brk, HER2, and STAT3.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1233. doi:1538-7445.AM2012-1233
Abstract 1238: Gab1 regulates epithelial cell polarity and scattering by acting as a platform for PAR proteins
Authors: Yang Ziqiang , Neel Benjamin
Published: August 2012

Control of cell polarity plays a key role in numerous important biological processes, but the molecules and mechanisms that regulate polarity remain poorly defined. We found that the scaffolding adaptor Gab1, a component of multiple growth factor and cytokine signaling pathways, interacts with two PAR proteins, PAR1 and PAR3. Gab1 binds PAR1 and activates its kinase activity. Acting as a scaffold, Gab1 brings PAR1 and PAR3 into a transient complex, facilitating PAR3 phosphorylation by PAR1. Gab1 and PAR6 bind the PAR3 PDZ1 domain, and thereby compete for PAR3 binding. Consequently, depletion of Gab1 causes PAR3 hypo-phosphorylation and increased PAR3/PAR6 complex formation, resulting in accelerated formation of tight junctions, increased trans-epithelial electrical resistance and a shortened lateral domain in epithelial cells. Conversely, Gab1/PAR1 interaction enhances epithelial cell scattering. Thus, Gab1 is a novel regulator of epithelial cell polarity that acts as a platform for assembling and modulating PAR protein complexes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1238. doi:1538-7445.AM2012-1238
Abstract 1256: Profiling protein-coding RNA and thousands of large non-coding RNAs from nanogram amounts of total RNA using a single microarray design
Authors: Lucas Anne Bergstrom , Kulkarni Vinayak , LeProust Emily , Srinivasan Maithreyan , Fulmer-Smentek Stephanie
Published: August 2012

Large intergenic non-coding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes, yet determining the function of individual lincRNAs remains challenging. Recently, more than 8,000 human lincRNAs were annotated and cataloged from more than 4 billion RNA-Seq reads across 24 tissues and cell types by scientists at the Broad Institute of MIT and Harvard. Data from this project indicates that lincRNA expression is highly tissue-specific as compared to protein coding gene expression. As researchers continue to investigate the function of lincRNAs, there is a need for tools that can rapidly and accurately measure the expression of the recently annotated lincRNAs along with mRNA expression. We have previously developed and recently updated the content of the human SurePrint G3 microarrays so that they are comprised of all known protein-coding mRNAs and lincRNAs, to enable systematic profiling and simultaneous detection of coding and non-coding gene expression from a single sample. To demonstrate the utility of the new microarray design we used low nanogram amounts RNA from matched tumor and adjacent normal tissues to produce cyanine-labeled cRNA. The labeled cRNA was applied to the microarrays to detect differences in coding and non-coding gene expression profiles. Using the GeneSpring GX software we are able to identify differentially expressed lincRNAs and protein-coding RNAs in the tumor and normal samples in less than two days. Comparisons of probe signals from technical replicate samples demonstrated high reproducibility with wide dynamic ranges and high sensitivity. Data from the microarrays correlates well with whole transcriptome sequencing of the same matched tumor/normal samples. Using this approach we show that lincRNA expression coincides with key genes known to regulate biological processes involved in cancer progression and this work demonstrates how profiling mRNA and lincRNA from matched tumor and adjacent normal samples can enable researchers to further define the role of lincRNAs in gene regulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1256. doi:1538-7445.AM2012-1256
Abstract 126: miR-221 promotes tumorigenesis in human triple negative breast cancer cells
Authors: Yin Min-Jean , Nassirpour Rounak , Mehta Pramod P. , Baxi Sangita M.
Published: August 2012

MicroRNAs (miRNAs) are non-coding, single-stranded RNAs of ∼22 nucleotides that act as agents of the RNA interference pathway and negatively regulate their targets by either cleaving mRNA molecules or by inhibiting their translation. miRNAs are frequently located at fragile sites and genomic regions susceptible to amplification, deletion, or translocation during tumor development, and growing evidence indicates that miRNAs can function as tumor suppressors and oncogenes. Additionally, miRNA expression profiling analyses have revealed characteristic miRNA signatures in human cancers. MicroRNA-221 (miR-221) is up-regulated in many cancers including HCC, glioblastoma, pancreatic cancer, prostate cancer, and tamoxifin-resistant breast cancer. Overexpression of miR-221 has previously been shown to promote cancer cell proliferation, most likely by its ability to modulate the expression of the oncogenic proteins c-kit and cyclin dependent kinase inhibitor (CDKI) CDKN1B/p27. In this study, we demonstrate that in comparison to normal mammary tissues, miR-221 is overexpressed in triple negative breast cancer cell lines (TNBC) and downregulated in non-TNBC breast cancer cell lines. Stable knockdown of miR-221 expression reduced cell proliferation and induced apoptosis in TNBC cell lines. Overexpression of miR-221 promoted cell migration and cell invasion, while knockdown of miR-221 inhibited both cell migration and cell invasion and increased E-cadherin expression in TNBC lines but not in the non-TNBC lines. Furthermore, downregulation of miR-221 in MDA-MB-231 xenografts decreased the tumor growth rate in mice. Therefore, our results suggest that miR-221 is a potential oncomiR that promotes cell proliferation, migration, and invasion in triple negative breast cancer cells, and that knockdown of miR-221 is able to induce in vitro and in vivo anti-tumor activity in these TNBC cell lines.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 126. doi:1538-7445.AM2012-126
Abstract 1263: Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90
Authors: Gomes-DaGama Erica M. , Moulick Kamalika , Ahn James H. , Zong Hongliang , Rodina Anna , Cerchietti Leandro , Lopes-Vazquez Maria E. Caldas , Beebe Kristin , Perna Fabiana , Katerina Chatzi , Vu Ly P. , Zhao Xinyang , Zatorska Danuta , Taldone Tony , Smith-Jones Peter , Alpaugh Mary , Gross Steven S. , Pillarsetty Nagavarakishore , Ku Thomas , Lewis Jason S. , Larson Steven M. , Ross Levine , Erdjument-Bromage Hediye , Guzman Monica L. , Nimer Stephen D. , Melnick Ari M. , Neckers Len , Chiosis Gabriela
Published: August 2012

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1263. doi:1538-7445.AM2012-1263
Abstract 1288: GLI2 is a significant determinant of HH-GLI pathway activaton in Ewing sarcoma
Authors: Christensen Laura , May William A.
Published: August 2012

Ewing Sarcoma is an aggressive bone and soft tissue malignancy of children, adolescents and young adults. Non-canonical activation of the Hedgehog-GLI pathway by the Ewing specifc EWS/FLI1 transcription factor has been shown to mediate important aspects of tumor phenotype. While evidence regarding the role of EWS/FLI1 in direct activation has been published, no one has yet investigated the role of other pathway components in Ewing tumors. Physiologically, GLI2 is the principal transcriptional activator of GLI1 and substantial amounts of GLI2 can be demonstrated in Ewing tumors and cell lines. So we hypothesized that GLI2 may also function in Ewing cells to help to regulate GLI1 and Hedgehog-GLI pathway activaton. Since GLI2 can be targeted by pharmacologic means, understanding its role in Ewing tumors is of potential significance. We demonstrate that GLI2 is expressed at significant levels in Ewing tumors. Furthermore, we show by overexpression studies, that GLI2 overexpression augments expression of both GLI1 and other pathway targets in Ewing cell lines. By using shRNA, we also demonstrate that knockdown of GLI2 expression has the expected effect of diminishing GLI 1 expression in Ewing lines. Finally, we show that treatment with GLI inhibitors also diminishes GLI2 activation of GLI1. Overall, these data indicate that GLI2 in Ewing cells exists in an activator state in Ewing cells and that pharmacologic means of targeting GLI2 may be an effective strategy for targeted therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1288. doi:1538-7445.AM2012-1288
Abstract 1304: Specific MUC1 isoforms function to regulate gene expression and tumor growth in pancreatic cancer
Authors: Grandgenett Paul M. , Bunt Stephanie K. , Mohr Ashley M. , Radhakrishnan Prakash , Hamada Tomofumi , Hollingsworth Michael A.
Published: August 2012

Cancer cells, especially adenocarcinomas, express aberrant forms of mucins, as compared to their normal cell counterparts. These aberrant forms arise as a consequence of transformation-related alterations in expression of mucin mRNAs including alternative splice forms, and deregulation of enzymes that post-translationally modify mucin core proteins. Previous work has established that MUC1 is overexpressed and differentially glycosylated in adenocarcinomas and that this overexpression is associated with aggressive forms of pancreatic cancer. Most studies have focused on the full-length MUC1 isoform that contains the VNTR and all seven exons, but alternative splicing of MUC1 yields a minimum of 30 isoforms, of which several have been linked to malignancy in ovarian, cervical, and breast cancer. We evaluated expression of MUC1 alternate splice forms in a series of isogenic clonal pancreatic cancer cell lines that exhibit different properties of morphological differentiation, tumor growth and metastasis. The results revealed significant variation in splice variant expression between these isogenic cell lines and has implicated an association between isoform expression and metastatic potential. The knockdown of specific splice variants and MUC1 isoform families in S2-007 pancreatic cancer cells suggested that individual isoforms show differential capacity for regulating expression of target genes and miRNAs, which in turn led to differences in tumor growth rate and metastatic potential. Specifically, the stable knockdown of all members of the MUC1 X-family significantly altered expression of miRNAs 193b and 320 and reduced tumor growth in a pancreatic cancer animal model. MUC1 X-family also affected expression levels of several genes associated with Gemcitabine resistance and both pro and anti-inflammatory pathways that are predicted to influence disease progression and tumor associated immunosuppression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1304. doi:1538-7445.AM2012-1304
Abstract 1421: Down-regulation of neuronal repressor gene REST inhibits tumor growth in Ewing's sarcoma
Authors: Zhou Zhichao , Kleinerman Eugenie
Published: August 2012

Ewing's sarcoma is a peripheral primitive neuroectodermal tumors (PNET). The abnormal differentiation of neuronal stem cells may relate to tumorigenesis of Ewing's sarcoma. The RE1-silencing transcription factor (REST) is a neuronal repressor gene which regulates neuronal stem cell differentiation. The aim of this study is to investigate the role of REST in Ewing's sarcoma. Our results demonstrated that three human Ewing's sarcoma cell lines TC71, A4573 and SK-ES expressed high levels of REST. Six of seven tumor samples from patients with Ewing's sarcoma were shown REST positive. Transfection of EWS-FLI-1 into mesenchymal stem cells up-regulated REST expression. Down-regulation of EWS-FLI-1 using si-RNA inhibited REST expression in TC71 cells, suggesting EWS-FLI-1 fusion protein regulates REST expression in Ewing's sarcoma. The siRNA-REST stable transfected cell line was established to down-regulate REST expression in TC71 Ewing's sarcoma cells. In vivo experiments indicated that Ewing's sarcoma tumor growth was significantly inhibited in the mice injected with si-REST-TC71 cells compared to the mice injected with si-control cells. Down-regulation of REST inhibited cell proliferation and induced apoptosis in TC71 tumors. The immunohistochemical staining indicated that the tumor vessels and pericyte marker expression were reduced in si-REST tumor tissues. Inhibition of REST decreased the early neural differentiation marker MSX-1 expression and up-regulated the terminal neural differentiation marker β-Tubulin III (Tuj) expression. These data suggesting that neuronal repressor gene REST plays an important role in tumor development of Ewing's sarcoma. Inhibition of REST suppressed tumor growth and altered neuronal differentiation marker expression in Ewing's sarcoma. REST is a potential target for treatment of Ewing's sarcoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1421. doi:1538-7445.AM2012-1421