bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3956: Hedgehog acyltransferase: A key player in Sonic hedgehog signalling in cancer cells
Authors: Jovanovic Biljana , Chang Shu-Chun , Koleva Mirella , Magee Anthony I.
Published: February 2011

Protein acyltransferases (PATs), a group of enzymes that catalyze acylation of a variety of proteins, have recently been shown to play an important part in protein sorting and signalling in the cell. In the Sonic hedgehog (Shh) signalling pathway the PAT Hedgehog acyltransferase (Hhat) is responsible for addition of a palmitic acid (C16) to the N-terminal cysteine residue of Shh. In mice, this modification has been shown to be necessary for proper packaging and release of Shh from the signalling cell as well as for transmitting a potent signal to the receiving cell, rendering Hhat crucial for a functioning Shh signal. Aberrant regulation of the Shh signalling pathway leads to a variety of malignant diseases in adults. Oncogenic activation of the Shh signalling pathway is known to arise from excessive production of the Shh signal or oncogenic mutations in the signal receiving cell. To date, drug therapies targeting the pathway in cancer have been directed at Shh-independent mutations in downstream regulators, Ptch, Smo, and Su(Fu), in the signal-receiving cell. To complement this, we hypothesize that Hhat is a potential drug target in cancers dependent on excessive Shh production for their growth. Here, we validate the potential of Hhat as a therapeutic target in cancer cell lines dependent on Shh for their growth. By knocking down Hhat in established cancer cell lines using siRNA we show that proper distribution and signalling by Shh can be inhibited. We measure the effect of the knock-down on target gene expression of the pathway (Ptch and Gli1), cancer cell growth and invasive properties of the cells. The effect is validated against inhibition of the Shh pathway by the Shh blocking antibody 5E1, and KAAD-cyclopamine, a Smo antagonist.

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 3956.
Abstract 3963: Wnt-Signaling regulates angiogenesis by regulating Neuropilin-1 expression in osteosarcoma
Authors: Ghaffar Samia , Guo Yi , McQueen Peter , Christ Alex , Tu Khoa , Xie Jun , Zi Xiaolin , Hoang Bang
Published: February 2011

Purpose of Study: To investigate the role of the Neuropilin-1 (NRP-1) in angiogenesis and regulation by Wnt-signaling pathway in osteosarcoma (OS) cell lines.

Methods: In vitro tube formation assay using HUVEC cells to examine the effect of Frzb, WIF1, and DKK3 on angiogenesis. Microarray and real-time PCR to assess the mRNA level of NRP-1 in SaOS-2 cells transfected with Dominant Negative LRP5 (DNLRP5) and PcDNA vector control. Western blots were performed to evaluate the expression of NRP-1 in 8 OS cell lines and normal osteoblast (NHOst), as well as the effect of Wnt inhibitors (DNLPR5, Frzb, WIF-1, DKK3) on NRP-1 expression in OS cell lines (LM7, 143B, and 143.98.2)

Results: In order to develop a new therapeutic target for OS, the most common primary malignant bone tumor, we examined the expression of NRP-1 and vascular endothelial growth factor (VEGF) in 8 established OS cell lines, as well as NHOst. Both VEGF and NRP-1 (a co-receptor of VEGF) were up-regulated in most OS cell lines compared with normal osteoblast, suggesting that VEGF and NRP-1 may be responsible for the excessive blood vessel formation in OS. Given our previous report that Wnt inhibitors Frzb, WIF1, DKK3, and DNLRP5, inhibited tumor growth and metastasis of OS, we hypothesize that one of the potential mechanisms may be through inhibition of tumor angiogenesis. When HUVEC assay was examined, DKK3, Frzb, WIF-1 condition medium significantly inhibited tube formation compared with control, suggesting that these Wnt inhibitors suppressed angiogenesis. Further microarray assay, real time PCR and western blots indicated that these Wnt inhibitors (DKK3, Frzb, WIF-1 and DNLRP5) dramatically down-regulated the expression of NRP-1, but VEGF level remained unchanged. These results show that inhibition of angiogenesis by Wnt signaling may be mediated by down-regulation of NRP-1 instead of VEGF.

Discussion and Conclusions: Our study suggests that the Wnt-signaling reduces tumor growth and metastasis by inhibiting angiogenesis and by down-regulating NRP-1 expression in OS. We are further studying the role of NRP-1 in osteosarcoma by knockdown of NRP1 expression in OS cells to assess tumor proliferation, metastases and angiogenesis. This may represent a novel therapeutic strategy for osteosarcoma.

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 3963.
Abstract 3965: Geldanamycin-induced heat shock protein 70 negatively regulates TGF-β signaling through interaction with TGF-β receptors
Authors: Lee Ho-Jae , Yoon Sun-Young , Nguyen Thuy Trang , Cho Han-Yang , Kim Tae-Hyun , Jeon Woo-Kwang , Kim Byung-Chul
Published: February 2011

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that plays important roles in a variety of physiological processes including cell proliferation, differentiation, and apoptosis. Dysregulation of TGF-β signaling has been implicated in the pathogenesis of a variety of diseases including cancer; therefore, pharmacological inhibitors that target the TGF-β signaling pathway might be promising drugs for disease therapy. In this study, we investigated the mechanism of inhibition of TGF-β signaling by the Hsp90 inhibitor geldanamycin (GA). Treatment with GA suppressed TGF-β signaling, as evidenced by inhibition of TGF-β-induced phosphorylation and transcriptional activity of Smad3 and decreased induction of target genes. Western blot analysis revealed that GA induced degradation of TGF-β type I and type II receptors through a proteasome-dependent pathway. Hsp70 interacted directly with TGF-β receptors following GA treatment. Notably, induction of Hsp70 by GA correlated with inhibition of TGF-β signaling. Suppression of Hsp70 expression by Hsp70 siRNA or KNK437, an inhibitor of Hsp70 synthesis, blocked the inhibition of TGF-β signaling by GA. Furthermore, down-regulation of endogenous Hsp70 enhanced TGF-β induced transcriptional activity and cell cycle arrest. These results strongly suggest that GA-induced Hsp70 acts as a negative regulator of TGF-β signaling.

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 3965.
Abstract 3979: In vitro modeling of pharmacodynamics of resveratrol-induced anti-proliferation in breast cancer cells
Authors: Li Hongyu , Melaney Elliot , Lin Sharon , Rubin Noah , Bender Charles , Meng Ran , London David , Tang Heng-Yuan , Lin Hung-Yun
Published: February 2011

We and others have shown that resveratrol, a naturally-occurring stilbene, induces p53-dependent apoptosis in human and animal cancer cell lines. The molecular basis of induction of apoptosis by resveratrol includes activation and nuclear translocation of mitogen-activated protein kinase (MAPK; ERK1/2) and MAPK-mediated Ser-15 phosphorylation of p53 (pSer15-p53). In this study, we used a newly developing perfusion bellows cell culture system to investigate resveratrol-induced apoptosis in the human estrogen receptor (ER)-negative breast cancer MDA-MB-231 cells. The cell culture system is a disposable bioreactor capable of high density cell culture for studies of anti-cancer drugs. Bellows-induced alternate flow of culture medium and air through porous matrices where cells are grown provides a relatively low shear, high aeration, foam-free culture environment. Medium level is raised and lowered to submerge and expose matrices to create a dynamic interface between air and media on the cell surface. Anchorage-dependent cells are grown on plastic flakes in the system; the flakes facilitate harvesting of whole cells for analysis. After 6 days incubation, the IC50 of resveratrol-induced apoptosis was 0.1 μM. Short term incubation with 10 μM resveratrol for 6 days inhibited cell proliferation by 75%. Using an injection system to pump media with different concentrations of resveratrol (0.5 μM to 100 μM), we were able to reproduce the resveratrol-induced anti-proliferation effect in the perfusion bellows culture system. Incubating the cells in either acidic or alkaline condition did not affect anti-proliferation induced by resveratrol. The effect of resveratrol-induced apoptosis is based on increasing transcription of pro-apoptotic p53-dependent genes such as PIG3, c-fos, c-jun and BAD. The aim of the present study is to establish the pharmacodynamics of resveratrol in the suppression or elimination of human breast cancer cells. With the use of the perfusion bellows cell culture system we will provide an efficient, convenient and financially beneficial method for determining optimal regimens for the effective suppression and elimination of these cancer cells.

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 3979.
Abstract 398: Molecular mechanisms of recurrence of PDGFB-dependent glioma in mice following inhibition of platelet-derived growth factor B expression
Authors: Liu Huan , Hitoshi Yasuyuki , Paolella Brenton , Israel Mark A.
Published: February 2011

Molecularly targeted therapies hold great promise for the treatment of cancer. Conditional transgenic mouse models offer unique opportunities for investigating whether the targeted inactivation of an oncogene is sufficient for tumor regression and for developing strategies to overcome the mechanisms by which tumors become resistant to targeted therapies. We developed transgenic mice (GFAP/tTA:TRE/PDGFB) in which human platelet-derived growth factor B (hPDGFB) is expressed in a doxycycline responsive manner. Without doxycycline these mice expressed PDGFB in the CNS and developed glioma. Doxycycline administration led to symptomatic recovery in the majority of tumor-bearing mice. However, sustained regression did not occur in 8 of 22 mice following 3 months of doxycycline treatment. In other experiments we implanted spheroid cells derived from other spontaneous mouse glioma arising in this model into the flanks of immuno-deficient mice. We initially observed a significant reduction in the size of established tumors with doxycycline administration, but residual cells from 5 out of 9 tumors began to grow rapidly following a prolonged period of the treatment. In vitro, spheroid cells derived from the relapsed tumors grew in a PDGFB-independent manner. Doxycycline addition to spheroid cultures effectively inhibited hPDGFB expression but did not inhibit proliferation or induce apoptosis. Examination of signaling pathways revealed that PDGFB receptor phopshorylation was significantly reduced by doxycycline but downstream AKT and Erk phosphorylation was sustained. Ongoing experiments are being conducted to identify upstream regulators of these signal transducers. (This work was supported in part by the Theodora B. Betz Foundation and the Andy Fund)

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 398.
Abstract 3983: Real-time imaging of human thyroid cancer progression in a fluorescent orthotopic nude mouse model
Authors: Cao Hop S. Tran , Kaushal Sharmeela , Snyder Cynthia S. , Ongkeko Weg M. , Hoffman Robert M. , Bouvet Michael
Published: February 2011

We report here a novel orthotopic mouse model of human thyroid cancer that can be imaged non-invasively with the use of red fluorescent protein (RFP).

Human thyroid cancer cell lines NPA (papillary) and KAK-1 (anaplastic) were stably transfected to express RFP or the green fluorescent protein (GFP). The cells were then injected into the thyroid glands of 8-week old athymic mice. The animals were noninvasively imaged weekly, and sacrificed when premorbid. Their tumor was subsequently resected en bloc with the respiratory system for further processing and analysis. H&E staining was performed to confirm the pathological diagnosis.

Both anaplastic and papillary thyroid cancer cell lines led to the robust development of orthotopic fluorescent tumors in nude mice. While RFP-expressing tumors were easily identified transcutaneously, autofluorescence of the skin prevented visualization of GFP-expressing tumors, making GFP a suboptimal model. Injection of 5 × 10^5 cells was as efficacious in allowing tumor development as 5 × 10^6 cells. Tumors were first visualized for both cell lines via whole-body noninvasive imaging as early as four weeks post-implantation and could be monitored over time. The time to premorbid conditions varied greatly between mice and seemed to be associated with the tumor growth pattern rather than tumor size. The earliest appearance of morbidity was five weeks post-implantation in an animal with cachexia due to local compression of the esophagus by the tumor. Other mice died at later stages from metastatic disease. The bright fluorescence of the tumors allowed identification of multiple micrometastases at necropsy in the lungs, lymph nodes, and the vascular system, lesions that were not visible under white light.

In summary, we have developed a novel orthotopic fluorescent mouse model of thyroid cancer that should serve a valuable role in the understanding of this disease and in the evaluation of new therapeutics, allowing assessment of the tumor response over time.

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 3983.
Abstract 3984: Effect of zeranol on beef skeletal muscle growth by differential image gel electrophoresis (DIGE)
Authors: Wick Macdonald , Reddish John M. , Ye Weiping , Lin Young C.
Published: February 2011

Zeranol is a resorcylic acid lactone produced by fungi of the genus Fusarium that grows on corn, wheat, barley, oats and sorghum. As such Zeranol is a virtually unavoidable contaminant of crops used to feed animals that are consumed by humans.

Zeranol has been shown to have a positive impact on muscle growth during the finishing phase of beef cattle production, was first approved and licensed for use as a growth promotant in cattle and sheep in the USA by FDA in 1969. However, the mechanism of this interaction is unknown but is likely related to changes in muscle specific proteins observable during the increased growth phase during finishing.

To determine the affect of Zeranol on beef cattle muscle growth, we performed four DIGE experiments. We compared protein expression level changes between beef cattle treated with Zeranol and untreated beef cattle. On day zero, 10 cattle were implanted with 72 mg of Zeranol pellets, the other half (n = 10) were implanted with vehicle. The beef cattle were raised at the Ohio State University Department of Animal Sciences Beef Barn in accordance with industry standards for 120 days. At 60 days, half the implanted beef cattle and half of the control beef cattle were harvested and the remaining beef cattle were harvested at day 120. Muscle samples were analyzed by DIGE and bioinformatics

Seven proteins associated with energy metabolism (creatine kinase, glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucomutase I, aconitase 2, enolase I, and triosephophate isomerase), 5 proteins with regulation of muscle contraction (slow skeletal muscle troponin T, calsequestrin I, myosin light chains 2 and 3, tropomyosin 3), 3 structural proteins (alpha actin, myosin binding protein C and kelch repeat and BTB domain), a protein associated with myotube formation, dihydrolipoamide dehydrogenase, and HSP70, a protein associated with meat quality, and albumin were identified. The differential regulation of these proteins indicates that muscle growth in cattle implanted with Z is due to fast skeletal muscle. (Supported by NIH grant R01 ES 015212).

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 3984.
Abstract 3986: Effects of immunohistochemistry on biomolecules in tissue specimens: Importance for expression-based microdissection technologies
Authors: Tangrea Michael A. , Kreitman Matthew S. , Rosenberg Alex M. , Ory Eleanor C. , Mukherjee Sumana , Eberle Franziska C. , Wallis Benjamin S. , Hanson Jeffrey C. , Rodriguez-Canales Jaime , Emmert-Buck Michael R.
Published: February 2011

Immuno-Laser Capture Microdissection (LCM) and Expression Microdissection (xMD) are techniques that rely on immunohistochemistry (IHC) to specifically define target cells for microdissection. While these techniques have been successfully applied to LCM and xMD, the full effects of IHC on the biomolecules extracted from stained samples are not well characterized. The goal of our study was to evaluate the integrity of DNA, RNA and protein biomolecules obtained from tissue sections after standard IHC or immunofluorescence (IF) techniques. Utilizing snap frozen human prostate and ovarian carcinoma specimens, as well as mouse liver samples, we evaluated biomolecule recovery from immunostained samples, including histone and pan-cytokeratins AE1/AE3 targeted antibodies. Standard horseradish peroxidase and diaminobenzidine (DAB) based IHC was used as well as Alexa Fluor 488 for indirect-IF. In general, biomolecule recovery was diminished in IHC stained tissues compared to standard hematoxylin and eosin (H&E) staining. RNA was affected the most and showed significantly reduced quantity and quality after staining. DNA was recoverable, but quantities were reduced when compared to the standard H&E. However, PCR amplification was possible with all stained samples studied. Protein analysis also demonstrated a reduction of quantity of the proteins recovered from IHC stained samples, with a variable effect depending on the protein analysis methodology. The results demonstrated that IHC techniques using DAB labeling significantly affect the extracted biomolecules. Improved staining protocols are required to address these concerns and to further the capabilities of these potentially powerful microdissection techniques.

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 3986.
Abstract 3994: Expression levels of EGFR-ligands are up-regulated in EGFR tyrosine kinase inhibitor-resistant cell line
Authors: Sakai Kazuko , Arao Tokuzo , Furuta Kazuyuki , Nagai Tomoyuki , Kudo Kanae , Kaneda Hiroyasu , Tamura Daisuke , Aomatsu Keiichi , Fujita Yoshihiko , Matsumoto Kazuko , Koizumi Fumiaki , Nishio Kazuto
Published: February 2011

Epidermal growth factor receptor (EGFR) is often highly expressed in many solid tumors and EGFR tyrosine kinase inhibitor (EGFR-TKI) demonstrated the therapeutic benefit for non small cell lung cancer in clinical setting. We previously established the EGFR-TKI resistant cell lines derived from highly parental sensitive cell line with constitutively active EGFR mutation, PC-9. EGFR-TKI-resistant cell line shows about 1000 fold resistant for EGFR-TKI compared with parental PC-9 cells. We performed the microarray and exon array analysis for these cell lines and identified that 30 genes were altered the gene expressions over 5 fold between two cell lines. There were no gene expression changes in EGFR, HER4, AKT and adaptor molecules, whereas HER2 and HER3 were down-regulated and MET were up-regulated in resistant cells. Interestingly, several ligands of EGFR such as TGF alpha, epiregulin, amphiregulin and HB-EGF were up-regulated in EGFR-TKI resistant cell lines, suggesting that the ligand expression change are associated with the phenotype of drug resistant and may be useful molecular biomarkers for EGFR-TKI treatment. We are now investigating the functional analysis for these identified genes and analysing the result of exon array.

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 3994.
Abstract 4005: JAK/STAT activation following Src inhibition is mediated by the loss of SOCS2 expression
Authors: Sen Banibrata , Peng Shaohua , Johnson Faye M.
Published: February 2011

Systemic therapy that results in both decreased invasion and significant cytotoxicity would be ideal to treat head and neck squamous cell carcinoma (HNSCC). One promising therapeutic target is Src due to its well-defined roles in both invasion and survival. STAT3 is a key mediator of the oncogenic effects of Src. However we previously found that sustained Src inhibition resulted in only transient inhibition of STAT3. Likewise, Src inhibition resulted in a universal reduction in invasion, but a more variable effect on apoptosis. Restoring STAT3 inhibition enhanced apoptosis. Previously we showed that STAT3 reactivation was mediated by JAK2 kinase activation and altered JAK2-STAT3 binding. Reactivation of JAK2/STAT3 suggests a feedback loop, possibly via the loss of a negative regulator. Of the three known feedback loops that regulate JAK/STAT function, we considered the most likely candidate to be the suppressors of cytokine signaling (SOCS) proteins, which can compete for STAT binding and inhibit JAK kinase activity. To determine if SOCS proteins are involved in JAK/STAT3 reactivation, we measured RNA levels of all 8 SOCS family members in 5 HNSCC cell lines using quantitative PCR. The expression of most of the SOCS decrease after initial Src inhibition, but recover, consistent with their regulation by STAT3. However, the expression of SOCS2 decreased in all 5 lines. Likewise, we found that the addition of a molar stoichiometric amount of recombinant SOCS2 inhibits Jak2 kinase activity in vitro. To confirm that SOCS2 regulates STAT3 activity, we knocked-down SOCS2 by siRNA which resulted in the activation of STAT3. Similarly, transient overexpression of SOCS2 resulted in significant downregulation of activated STAT3. Others have reported that STAT5 can regulate SOCS2 expression. Consistent with this, we found that Src inhibition led to durable STAT5 inhibition. Moreover, the depletion of both isoforms of STAT5 (a and b) with siRNA resulted in a dramatic downregulation of the SOCS2 expression. To determine the biological significance of SOCS2 expression, we discovered that specific SOCS2 depletion reduced HNSCC cytotoxicity following Src inhibition. Our results demonstrate that sustained Src inhibition leads to the loss of SOCS2 expression via STAT5 inhibition. The loss of SOCS2 allows the recovery of JAK2 kinase activity, JAK2/STAT3 binding, STAT3 activation, and cell survival despite durable Src inhibition. To our knowledge, this feedback pathway has never been described previously. Given that pharmacologic Src and JAK inhibitors are currently being evaluated in clinical trials, these results have significant implications for cancer therapy.

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 4005.
Abstract 4010: Adaptor protein Lnk binds to JAK3 and negatively regulates a mutant activating allele associated with megakaryocytic leukemia
Authors: Koren-Michowitz Maya , Gery Sigal , Nowak Daniel , Koeffler H. Phillip
Published: February 2011

The adaptor protein Lnk is known to associate with hematopoietic cytokine receptors such as cKIT, MPL and PDGFR, as well as, non-receptor tyrosine kinases such as JAK2, and is considered to have an inhibitory effect on these signaling pathways. JAK3 is expressed mainly in the hematopoietic system and its absence is associated with autosomal recessive severe combined immunodeficiency (SCID). Recently, activating mutations of JAK3 were described in transient myeloproliferative disorder (TMD) and acute megakaryocytic leukemia (AMKL) in Down syndrome (DS) patients as well as adult non-DS AMKL. JAK3 mutations were also rarely described in solid tumors and B-ALL. The 50% homology between JAK3 and JAK2 has led us to study the association between Lnk and JAK3.

293T cells were co-transfected with cDNAs encoding either wild-type (WT) JAK3 or JAK3 harboring an activating A572V mutation (JAK3 A572V), as well as the WT V5-tagged Lnk. Whole cell lysates were used for immunoprecipitation with either V5-tag or JAK3 antibodies. Binding of Lnk and JAK3 was detected by Western blot probed with JAK3 or V5-tag antibodies. To determine which domain of Lnk is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including a mutation in the SH2 domain (R392E), deletion of the SH2 domain (del SH2) and deletion of the PH and SH2 (del SH2/PH) domains. Our results show that WT Lnk binds to WT JAK3, as well as JAK3 A572V. The R392E and del SH2 Lnk mutants retained JAK3 binding capacity while deletion of both SH2 and PH domains of Lnk abolished JAK3 binding. In order to study the biological effect of Lnk binding to JAK3, we infected CMK cells, a megakaryocytic leukemia cell line harboring JAK3 A572V, with a bicistronic retroviral MSCV-IRES-GFP (MIG) WT Lnk vector. Effect on growth was assessed in GFP positive sorted cells by cell count and colony formation in methylcellulose. CMK cells infected with MIG WT Lnk grew slower in liquid culture and had decreased clonogenic growth in soft agar culture compared to cells infected with MIG vector alone.

In summary, we show for the first time that Lnk can bind to WT and mutant JAK3 and slow the growth of leukemic cells harboring an activating JAK3 mutation. Developing a small molecule mimetic of Lnk may have a therapeutic role in the treatment of hematopoietic malignancies associated with a variety of activated tyrosine kinase receptors and non-receptor tyrosine kinases including JAK3, as well as secondary signaling proteins.

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 4010.
Abstract 4013: Functional analysis of Bcr-Abl-dependent phosphorylation of SOCSs
Authors: Chen Jilong , Guo Guijie
Published: February 2011

Previous studies have demonstrated that constitutive activation of JAK/STAT (Janus tyrosine kinase/signal transducers and activators of transcription) signaling is required for efficient transformation by the Abelson murine leukemia virus (A-MuLV), which results from the expression of the v-Abl oncogene. Although the activation of JAK/STAT signaling pathway in cells expressing BCR-ABL still remains controversial, there is increasing evidence that deregulation of the JAK or STAT activity plays an important role in BCR-ABL malignant transformation. The suppressors of cytokine signaling (SOCS) are inhibitors of Jak kinases and as such are critically involved in a negative feedback loop of Jak/STAT signal transduction pathway. The mechanism by which Abl oncogene bypasses SOCS negative regulation remains poorly defined. Here, we found that co-expression of BCR-ABL with some SOCS proteins resulted in phosphorylation of SOCSs. Moreover, we observed that BCR-ABL-dependent phosphorylation of SOCSs altered SOCS function at least at two levels. One involves alteration of direct interaction of SOCSs with JAKs, which maintains JAK kinase activity. The other level involves inhibition of interaction of SOCS box with the Elongin BC complex, which disrupted the formation of E3 ubiquitin ligase complex that mediates proteosomal degradation of JAK kinases. Together, these results suggest that the BCR-ABL may regulate the inhibitory function of SOCSs through mediating phosphorylation of SOCS proteins.

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 4013.
Abstract 4014: Differential expression and activation of IRS1 and IRS2 in breast cancer cell lines
Authors: Porter Holly A. , Keegan Achsah D.
Published: February 2011

Numerous studies have shown that IL-4 can protect various types of tumor cells from chemotherapeutic agents including breast cancer cells. There are two main signaling pathways activated by IL-4; the STAT6, signal transducers and activators of transcription pathway, that results in expression of IL-4-responsive genes and the IRS1/2 signaling pathway that influences cellular proliferation and survival. IL-4 can act directly on many tumor cell types to prevent apoptosis. The IL-4-induced activation of STAT6 has been shown to up-regulate anti-apoptotic proteins, cFlip and BcLxL, in cancer. IRS1/2 molecules link the IL-4R and IGF-1R to signaling pathways that regulate cellular proliferation and survival, and are therefore implicated in cancer progression. Recent studies showed that IRS2 regulates mammary tumor metastasis in a mouse model of breast cancer and that knocking out IRS2 rendered the tumor cells less invasive and more susceptible to apoptosis in response to growth factor withdrawal. IRS1 was not required for metastasis and was actually suggested to be a suppressor of metastasis in breast cancer and an enhancer of proliferation. IRS2 suppressed the function of IRS1 by strongly activating the Akt/mTOR signaling pathway and inducing the serine phosphorylation of IRS1. We have found that expression of IRS1, in contrast to IRS2, leads to enhanced sensitivity to chemotherapy-induced cell death in 32D cells. Furthermore, it has been shown that MCF7 cells are more sensitive to docetaxel-induced death as compared to MDA-MB-231 cells. We propose that sensitivity of breast cancer cells to chemotherapy is in part regulated by the relative abundance of IRS1 and IRS2 and their phosphorylation status. To begin to test this hypothesis, we cultured breast cancer cells lines in the presence or absence of IL-4 and evaluated the IRS pathways. We found that MCF7 cells highly expressed IRS1 with little IRS2 while MDA-MB-231 cells expressed relatively equal amounts of IRS1 and IRS2. In MCF7 cells IL-4 induced the potent tyrosine phosphorylation of IRS1 with very little tyrosine phosphorylation of IRS2. However, in MDA-MB-231 cells, IL-4 induced the tyrosine phosphorylation of both IRS1 and IRS2 equally. However, the level of IRS1 phosphorylation in MDA-MD-231 cells was less than that observed in MCF7 cells. Taken together with previous publications, these results suggest that the differential activation status of IRS1 and IRS2 in breast cancer can lead to differential metastatic and chemosensitivity phenotypes.

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 4014.
Abstract 4022: Antioxidant-induced modification of INrf2C151 and PKCdelta-mediated phosphorylation of Nrf2S40 are both required for stabilization and nuclear translation of Nrf2 and increased drug-resistance
Authors: Niture Suryakant K. , Jain Abhinav K. , Jaiswal Anil K.
Published: February 2011

Antioxidants cause dissociation of nuclear factor erythroid 2-related factor 2 (Nrf2) from inhibitor of Nrf2 (INrf2) and so Nrf2:INrf2 can serve as a sensor of oxidative stress. Nrf2 translocates to the nucleus, binds to antioxidant response element (ARE) and activates defensive gene expression, which protects cells. Controversies exist regarding the role of antioxidant-induced modification of INrf2 cysteine 151 or protein kinase C (PKC)-mediated phosphorylation of Nrf2 serine 40 in the release of Nrf2 from INrf2. In addition, the PKC isoform that phosphorylates Nrf2S40 remains unknown. Here, we demonstrate that antioxidant-induced PKC-delta -mediated phosphorylation of Nrf2S40 leads to release of Nrf2 from INrf2. This was evident from specific chemical inhibitors of PKC isoenzymes in reporter assays, in vitro kinase assays with purified Nrf2 and PKC isoenzymes, in vivo analysis with dominant-negative mutants and siRNA against PKC isoforms, use of PKC-delta+/+ and PKC-delta −/− cells, and use of Nrf2S40 phospho-specific antibody. The studies also showed that antioxidant-induced INrf2C151 modification was insufficient for the dissociation of Nrf2 from INrf2. PKC-delta-mediated Nrf2S40 phosphorylation was also required. Nrf2 and mutant Nrf2S40A both bind to INrf2. However, antioxidant treatment led to release of Nrf2 but not Nrf2S40A from INrf2. In addition, Nrf2 and mutant Nrf2S40A both failed to dissociate from mutant INrf2C151A. Furthermore, antioxidant-induced ubiquitylation of INrf2 in PKC- delta+/+ and PKC-delta −/− cells occurred, but Nrf2 failed to be released in PKC-delta −/− cells. The antioxidant activation of Nrf2 reduced etoposide-mediated DNA fragmentation and promoted cell survival in PKC- delta+/+ but not in PKC- delta−/− cells. These data together demonstrate that both modification of INrf2C151 and PKC- delta-mediated phosphorylation of Nrf2S40 play crucial roles in Nrf2 release from INrf2, antioxidant induction of defensive gene expression, promoting cell survival, and increasing drug resistance.

J. Cell Science in press 2009.

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 4022.
Abstract 4026: Suppression of carcinogenic phenotypes by DSG3 silencing occurs via a plakoglobin-mediated signaling pathway
Authors: Chen Yin-Ju , Chiu Ching-Chi , Cheng Ann-Joy
Published: February 2011

DSG3 has been reported to act as an oncogene in head and neck cancers (HNCs), but the underlying mechanism of its oncogenic activity is unclear. In this study, we aimed to validate the proposed oncogenic function of DSG3 and investigate its downstream regulatory mechanisms. When we stably transfected HNC cells with a short hairpin DSG3-RNAi (shDSG3) to suppress DSG3 expression, reductions in cell growth (70%) and colony formation (>90%) were observed, in addition to cell cycle arrest at the G0/G1 phase. Both cell migration and invasion abilities were also decreased, to 10% and 16%, respectively, after one day. Because DSG3 is associated with plakoglobin in desmosomes, we examined whether or not the phenotypic alterations that occur following DSG3 knockdown were mediated through the plakoglobin signaling pathway. Immunoprecipitation and immunofluorescent staining analyses revealed that DSG3 knockdown disrupted the interaction of DSG3 with plakoglobin and induced the translocation of plakoglobin from the cytoplasm to the nucleus. Using a luciferase reporter assay and western blot analysis, we found that DSG3 knockdown significantly reduced TCF/LEF transcriptional activity and subsequently suppressed the expression of the downstream target genes of the TCF/LEF transcription factors, including c-myc, cyclin D1, and MMP7, which are involved in the regulation of cell proliferation and invasion. An in vivo xenograft study showed that knockdown DSG3 expression significantly inhibited tumor growth for 2 months in BALB/C nude mice and also reduces c-myc, cyclin D1 and MMP7 expression. In conclusion, we found that DSG3 has an oncogenic function in HNC. The suppression of DSG3 expression can interfere with the plakoglobin-mediated signaling pathway, subsequently leading to a reduction of pathogenicity in HNC 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 4026.
Abstract 4035: MicroRNA-221 levels correlate with aggressive and clinically recurrent prostate cancers
Authors: Gordanpour Aida , Stanimirovic Aleksandra , Nam Robert , Moreno Carlos , Leyland-Jones Brian , Sugar Linda , Seth Arun
Published: February 2011

One of the most important challenges in prostate cancer research is to identify biomarkers that are predictive of cancer aggressiveness and future tumor recurrence following radical prostatectomy. Recent studies have shown that altered expression of microRNAs (miRs) is involved in the development of prostate cancer, and among the differentially expressed miRNAs, miR-221 may be critical in metastatic induction/regulation. While the precise role of this miRNA in facilitating malignant progression remains unclear, expression of miR-221 in prostate cancer may correlate with disease state/status and serve as a surrogate biomarker for tumor recurrence and/or aggressiveness. In the present study, we sought to investigate whether miR-221 is differentially regulated in patients with aggressive and non-aggressive tumors relative to control normal prostate cell line RWPE-1 and, more specifically, whether this miRNA can be used as biomarker for disease recurrence. To address this question, we initially chose to assess the relative expression of miR-221 in a series of aggressive (n=69) and non-aggressive (n=45) primary carcinoma tissues derived from clinically resected prostates by quantitative real-time PCR. Aggressive tumors were categorized based on clinicopathological parameters such as Gleason score of 8 or higher, high PSA levels, presence of metastasis, invasion to the seminal vesicles, and recurrence after radical prostatectomy. Relative expression of mature miR-221 in tumors was quantified in comparison to control prostate epithelial cell line, RWPE-1, which was set to be 1x. Levels of miR-221 expression were found to be variable. Among all aggressive and non-aggressive cancer patients, expression of miR-221 transcript levels was differentially expressed relative to control sample. However, when we divided this group of patients on the basis of miR-221 expression levels, 72% of the patients with aggressive tumors had miR-221 less than 5-fold the RWPE-1 levels and only 28% of the patients had miR-221 expression equal to or more than 5-fold RWPE-1 levels. On the other hand, 53% of the patients with non-aggressive tumors had miR-221 less than 5-fold the RWPE-1 levels compared to 47% of the patients with miR-221 equal to or more than 5-fold RWPE-1 levels. Our results show that miR-221 is lower in majority of aggressive prostate cancer and differential expression may have utility as a biomarker for disease recurrence.

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 4035.
Abstract 4040: MicroRNAs that chemosensitize the apoptotic effect of 17-AAG
Authors: Hwang Larn , Treece Tina , Desai Neil , Trieu Vuong
Published: February 2011

Background: 17-allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone function of heat shock protein 90 (Hsp90) and promotes the proteasomal degradation of its client proteins. 17AAG also induced the mitochondrial release, accumulation of mitochondrial-derived activator of caspases, resulting in apoptosis. 17-AAG is currently in development for the treatment of cancer. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate expression of many genes and play a significant role in cancer and possibly in Hsp90 signaling. Therefore, a screen of miRNA mimics and inhibitors was conducted for chemosensitization to 17-AAG.

Methods: To identify individual microRNAs that synergizes with 17AAG, microRNA libraries were screened for apoptotic activity against HT29 cells when co-treated with 17AAG. The microRNAs were transfected into HT29 cells 48 hrs before treatment with 17AAG. After 72 hrs of 17AAG exposure, apoptosis was quantitated by the release of caspase 3/7. The effects of these miRNA on cellular protein, mRNA, and miRNA profile were also examined using microarray technology.

Results: Four miRNAs were identified which had potent chemosensitization effect on cells exposed to 17-AAG. These miRNA are different in sequence and did not synergize with one another. Alone they did not induce apoptosis; however, when combined with 17-AAG, they increased the apoptotic activity of 17-AAG up to 4 folds. Possible mechanism of action for these miRNAs was examined by microarray analysis which suggested that they act on the Hsp90/70 pathway to enhance the activity of 17-AAG.

Conclusions: Four miRNAs were identified as chemosensitizers for 17-AAG. These miRNAs could provide the basis for determination of tumor types that will be sensitive to 17-AAG, as well as a potential biomarker for predicting 17-AAG effectiveness.

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 4040.
Abstract 405: HTLV-1 infection of humanized SCID mice recapitulates adult T-cell leukemia/lymphoma (ATLL) development
Authors: Banerjee Prabal , Feuer Gerold , Crawford Lindsey , Lairmore Michael , Beilke Mark
Published: February 2011

Human T-lymphotropic virus type-1 (HTLV-1) is the etiologic agent of Adult T-cell leukemia/lymphoma (ATLL), an aggressive CD4+/CD25+ T cell malignancy. The early molecular events induced by HTLV-1 infection as well as the role of various viral genes in the induction of leukemia remain unclear, predominantly due to the lack of an animal model that recapitulates ATLL development. We have previously demonstrated that HTLV-1 is capable of infecting human hematopoietic progenitor and stem cells (CD34+ HP/HSCs) and that infection of CD34+ HPCs has dramatically different biological effects in comparison to infection of mature T lymphocytes. To determine if HTLV-1 infection of CD34+ HP/HSCs recapitulates leukemogenesis in vivo, human hematopoiesis was reconstituted in NOD/SCID mice by injection of human CD34+ HPCs infected ex vivo with HTLV-1. Humanized NOD/SCID (HU-SCID) mice infected with HTLV-1 (HTLV-1-HU-SCID) consistently developed CD4+CD25+ T cell lymphomas with clinical characteristics associated with ATLL at ∼10 weeks post-reconstitution and show significantly elevated levels of HTLV-1 infected human CD4+ T cells in the thymus, mesenteric lymph node, spleen and peripheral blood. Lymphoma cells successfully engrafted in naïve NOD/SCID mice when injected into the peritoneal cavity and maintain the expression of viral proteins, gp46env and p19gag. Moreover these infected mice showed hyperproliferation of infected human stem cells (CD34+CD38−) in the bone marrow suggesting that HP/HSCs represents viral reservoir target cells which maintain HTLV-1 infection for extended periods of time in vivo. We speculate that HTLV-1 infection of hematopoietic stem cells establishes a virally-infected “cancer stem cell” which subsequently gives rise ATL in patients. Notably, CD34+ HPCs isolated from HTLV-1 infected patient PBL demonstrate proviral integrations, suggesting that these cells harbor infection in humans. CD34+ HPCs transduced with a lentivirus vector expressing the HTLV-1 Tax gene (Tax1) also results in CD4+/CD25+ T cell leukemia/lymphoma in HU-SCID mice, suggesting that Tax1 expression is sufficient for lymphomagenesis. HTLV-1 infection of humanized NOD/SCID mice represents a novel in vivo model which recapitulates viral lymphomagenesis and provides a compelling system to investigate and characterize molecular events in human stem cells in the initiation and progression to ATLL.

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 405.
Abstract 4050: Normalization of TaqMan human microRNA array data using average expression of controls
Authors: Chandramouli Gadisetti VR , Maxwell G. Larry , Risinger John I.
Published: February 2011

The growing interest on microRNA (miRNA) expression in cancer motivates the need for improvement of technical aspects. TaqMan Human MicroRNA Arrays with comprehensive coverage of 667 unique miRNAs from Sanger miRBase are now available (Applied Biosystems, Foster city, CA) to quantitatively assay miRNA expression. A recent study reported that these qRT-PCR arrays perform better than miRNA microarrays which utilizes U6 RNA as an endogenous control for normalization. However, the choice of control for normalization varies depending on the tissue under examination. The TaqMan Low Density Arrays (TLDA) arrays A and B include six different controls with at least quadruplet assays for each. A recent study indicated that the average expression normalization including all miRNAs on array performed better than other normalization methods indicating a further need to examine normalization of TaqMan miRNA array data. The choice of a suitable control other than an average expression over the entire array is a more general approach for qRT-PCR data since constant average expression may not be valid for these arrays. Hence, we combined both concepts to determine a normalization constant. We examined miRNA expressions of 5 endometrial cancer cell lines assayed by these TaqMan Low Density Arrays for miRNA. Each cell line was assayed in triplicate. The cycle threshold (Ct) values of positive controls U6 (MammU6), RNU48, RNU44, RNU24, RNU6B, RNU43 in these were found to be in the range of 15 to 39 where RNU6B and RNU43 had low expressions. In some cases, we observed failures in endogenous control U6 and differences between arrays A and B. Therefore multiple controls were included for normalization but with appropriate weighting for highly expressed controls. This was done by calculating the average of 2−Ct values for controls that had Ct-values below a threshold level (Ct < 25) and transforming this average back to logarithmic scale to the base 2. We then compared the normalized Ct-values by this method with the normalization using geometric average method. This weighted average normalization method resulted in smaller replicate variances of controls than the geometric average method.

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 4050.