bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3992: Proteomic profiling of breast tumors using a microfluidic quantitative immunohistochemistry platform
Authors: Kim Minseok S. , Kwon Seyong , Lee Jungmin , Lee Eun-Sook , Park Je-Kyun
Published: February 2011

In breast cancers, one of the most troublesome characteristics for cure is the heterogeneity in point of genetic alterations, treatment-response variability, and histopathological features. This characteristic urgently requires personalized tailored therapy, getting out of a simple and widespread prescription. Therefore, the investigation of multiple biomarkers is inevitable for complete recovery. Immunohistochemistry (IHC) has been a major diagnostic method in various malignancies and many studies showing the relationship between immunohistochemical profiles and molecular classification support that IHC might play a significant role in subclassification of breast cancer patients for personalized medicine. However, conventional IHC method is not adequate for examination of multiple biomarkers since it requires consumption of many tissue slices and proportionally increases diagnostic cost which can give a big burden to patients. In addition, subjective decision for biomarker expression is another major problem. Therefore, an innovative multiplexed IHC platform satisfying not only investigation of multiple biomarkers with reducing consumption of tissue slice but also quantification for the biomarkers is the current demand for personalized medicine.

Here, a novel microfluidic multiplexed and quantitative IHC platform was developed for proteomic profiling of breast tumors. The large-scale microfluidic device was designed to have 20 microchannels, and multilayer soft lithography and two-step lithography technologies were used for fabrication. Fluidic resistance was considered for equivalent flow rate of individual reaction channel. Ten biomarkers known as predictive and prognostic indicators in breast cancers, including SMM-HC, CK5, CK14, E-Cadherin, p53, ER, p63, PR, HER2, and Ki-67, were examined on four breast cancer cell lines (MCF-7, SK-BR-3, AU-565, and HCC70) to confirm the massive multiplexed IHC. Results showed that the platform successfully realized the expressions of each biomarker on a cellblock at the same time and proteomic profiling for 10 biomarkers could be quantitatively analyzed from the image analysis. The platform was also applied to a breast cancer tissue sample and the result was corresponded to the results of conventional IHC method. This microfluidic platform is expected to realize precise subtyping of cancer patients by the proteomic profiling even though the size of tumor samples acquired by operation has been getting smaller due to early detection and neoadjuvant 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 3992.
Abstract 3995: Differential expression of CD10 immunohistochemistry in stroma and epithelium of prostatic adenocarcinoma
Authors: Fahim Lisa , Lockwood Gina , Fleshner Neil , Sweet Joan
Published: February 2011

Introduction:

CD10 is a transmembrane metallo-endopeptidase involved in cleavage and inactivation of certain peptide hormones important for signal transduction.

CD10 is strongly expressed by normal prostatic luminal epithelial cells. Immunohistochemical (IHC) studies have shown expression of CD10 in prostatic adenocarcinoma. (PCa). Loss of CD10 expression is a common, early event in human prostate cancer (PCa); however, CD10 positive cancer cells frequently appear in lymph node metastasis.

Only a few studies are available on the expression of CD10 in the prostatic stromal and cancer cells. We investigated the IHC expression of CD10 in epithelium and stroma of PCa using TMAs.

Material and Methods:

Prostate tissue from 72 radical prostatectomies was used to construct two TMAs; 35 case of Gleason score (GS) 6(3+3) and 37 cases of high grade (HG) prostate cancer (GS 8, 9, 10) were included. Each case was represented by three, 1mm cores for a total of 105 cores for the GS6 and 111 cores for HG prostate carcinomas. TMAs were stained with H&E and IHC was performed for CD10. Digital images of the TMA slides were scanned using the T3 ScanScope by Aperio Technologies. Epithelial and stromal expression of CD10 was scored as negative or positive.

Results:

CD10 was expressed in epithelium and stroma of PCa. CD10 had heterogeneous expression within individual patients in both epithelium and stroma. This heterogeneity was not significantly different between GS6 and HG prostate cancer.

CD10 was differentially expressed in epithelium of GS6 and HG prostate cancer. HG prostate cancer showed significantly more epithelial staining with CD10 (p=0.001) and less stromal staining with CD10 (p=0.003) compared to GS6.

Conclusion:

GS6 and HG prostate cancer demonstrate a reverse relationship between CD10 expression in the epithelium and the stroma.

Epithelial CD10 expression is an unfavorable risk factor in prostate cancer. Prostate tumors expressing high levels of epithelial CD10 correlate with a poor disease outcome. CD10 may be important in the biology of the tumor stromal microenvironment and its contribution to tumor progression requires further investigation.

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 3995.
Abstract 4: 5′ - 3′ UTR interactions regulate p53 mRNA translation and provide a target for modulating p53 induction after DNA damage
Authors: Chen Jing , Kastan Michael
Published: February 2011

Induction of p53 protein is a significant determinant of cellular outcome following exposure to DNA damage or other cellular stresses. Though the increase of p53 protein level after DNA damage is largely attributed to an increase in its protein half-life, optimal induction of p53 protein after DNA damage requires RPL26-mediated increases in p53 mRNA translation (Takagi et al., 2005). We report here the existence of a double-strand RNA region containing complementary sequences of the 5′- and 3′- untranslated regions (UTR) of p53 mRNA that is critical for its translational regulation by RPL26. Mutating as few as three bases in either of the two complementary UTR sequences to disrupt this UTR interaction abrogates the ability of RPL26 to bind to p53 mRNA and to stimulate p53 translation. Compensatory mutations that restore the above UTR interaction rescue RPL26 binding as well as translational regulation of p53 by RPL26. Short, single-strand oligonucleotides containing critical sequences from this RNA interaction region inhibit the binding of RPL26 to p53 mRNA and reduce p53 induction and p53-mediated cell death after several different types of DNA damage and cellular stress. These observations demonstrate a novel mechanism of regulating cellular protein levels and provide a reagent (oligonucleotides) that can be used to modulate stress induction of p53 in cells, a potentially clinically useful intervention. These mechanistic insights also set the stage for identifying small molecules other than oligonucleotides which could modulate p53 induction by targeting this mechanism.

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 4.
Abstract 40: Developmental profile and regulation of the glycolytic enzyme hexokinase 2 and its association with aerobic glycolysis
Authors: Wolf Amparo M. , Agnihotri Sameer , Munoz-Gajadhar Diana M. , Hawkins Cynthia , Guha Abhijit
Published: February 2011

Proliferating embryonic and tumor tissues rely on aerobic glycolysis, or the metabolism of glucose to lactate under oxygenated conditions, to assist in the synthesis of biosynthetic precursors necessary for proliferation. The reliance on aerobic glycolysis may be mediated by the expression of specific metabolic enzymes. Mammalian Hexokinases HK1 and HK2 are 100kDa proteins that phosphorylate glucose to glucose-6-phosphate as the first step of the glycolytic pathway. In normal adult tissues, HK1 is ubiquitously expressed but is particularly prominent in the brain and kidney. HK2 is generally expressed at low levels within adipose tissue and skeletal tissue and negligently in normal brain. In this study, we wished to determine the ontogeny of these isoforms within the developing brain and determine how their expression relates to the extent of aerobic glycolysis in Glioblastoma Multiforme (GBM), a highly aggressive malignant brain tumor. Mining of existing published microarray data found strong expression of HK1 in 1, 2, 4 cell stage but a switch to stronger expression of HK2 in the blastocyst stage, previously reported to rely heavily on aerobic glycolysis. We performed HK2 immunohistochemistry in mice from gestational age E12, E16, post natal 1 month and 2 months. Our results demonstrated an age and cell specific HK1 and HK2 immunoreactivity in embryonic brain tissue with decreased expression of HK2 post-natally. Subsequently, we investigated the expression of HK1 and HK2 isoforms in a panel of GBM cell lines with varying levels of dependence on aerobic glycolysis, as measured by lactate levels and O2 consumption. HK2 but not HK1 expression was higher in GBM cells that had low O2 consumption and high extracellular lactate levels, supporting an association of HK2 with aerobic glycolysis. As HK2 expression is nearly silent in adult brain but expressed in fetal tissue and GBM cells, we hypothesized that DNA methylation/demethylation events may be playing important in its regulation. Adult normal human brain and GBM cell lines that had no HK2 expression were found to be methylated at CpG islands within intron 1 by bisulfite sequencing in contrast to fetal tissue and HK2-expressing GBM cells. The degree of methylation correlated with transcript expression in GBM cell lines. Treatment of U343 and A172 cell lines with 5-azacytidine restored HK2 transcript expression, supporting that HK2 maybe epigenetically regulated. Overall, our results demonstrate that the expression of the HK2 isoform, in contrast to HK1, may be particularly important in tissues relying on aerobic glycolysis for proliferation including embryonic tissue and GBMs.

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 40.
Abstract 4002: C-terminal phosphorylation of human GSTP1 by EGFR and site-directed mutation allows JNK reactivation in glioma cells
Authors: Okamura Tatsunori , Ali-Osman Francis
Published: February 2011

In malignant gliomas, the c-jun N-terminal kinase (JNK) pathway regulates tumor growth and resistance to apoptosis, and correlates with histologic grade and epidermal growth factor receptor (EGFR) tyrosine kinase expression. We recently reported that glutathione S-transferase P1 (GSTP1), a major metabolizing and stress response signaling protein that is frequently overexpressed in brain tumors, is a downstream target of EGFR and undergoes EGFR-dependent tyrosine phosphorylation, in vitro and in vivo, resulting in an enhancement of its enzymatic function and increased drug resistance. Although the dimeric GSTP1 exists in a reversible equilibrium with its monomeric form, the monomeric GSTP1 binds to JNK and acts as an endogeneous JNK inhibitor. In our previous study, the EGFR-phosphorylation of GSTP1 shifted the GSTP1 dimer-monomer equilibrium toward the monomeric state and the c-Jun phosphorylation by JNK was enhanced by phosphorylated-GSTP1 and suppressed by unphosphorylated-GSTP1. EGFR activation resulted in dissociation of GSTP1 from the GSTP1-JNK1 complex in a cell-free system and in glioma cells. The C-terminal domain of GSTP1 is reported to be involved in the interaction with JNK. Accordingly, we synthesized the C-terminal domain peptides of human GSTP1 containing the phospho-acceptor Tyr198, unique to hominidae among species, by EGFR (Y198WT) and its phenylalanine-replaced mutant peptide (Y198F). Both the C-terminal domain peptides significantly suppressed the c-Jun phosphorylation by JNK1 in a cell-free system. The binding assay using the synthetic peptides showed that the C-terminal domain peptides of human GSTP1 bound to JNK1 with a lower affinity when Tyr 198 was phosphorylated by EGFR than when it was unphosphorylated. Mutagenesis in human glioma cells revealed that the EGF-induced c-Jun phosphorylation by JNK decreased in the GSTP1-Y198F mutant, whereas increased in the phospho-mimic GSTP1-Y198D mutant, compared with vector control. Together these results indicate that in malignant glioma cells the C-terminal tyrosine phosphorylation of human GSTP1 by EGFR leads to dissociation of GSTP1 from the GSTP1-JNK1 complex. Thus, the GSTP1-JNK1 direct interaction rather than JNK upstream signaling elicits reactivation of JNK. This regulation of JNK signaling function by the EGFR-GSTP1 crosstalk, defines a novel signaling network with a potential to regulate the biology and therapeutic outcome in malignant gliomas. Supported by NIH grants RO1 CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236.

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 4002.
Abstract 4008: p75NTR is overexpressed in vestibular schwannomas and protects cells from apoptosis due to suppressed c-Jun N-terminal kinase (JNK) activity
Authors: Yue WeiYing , Woodson Erika , Tryggvason Geir , Clark Jason J. , Hansen Marlan R.
Published: February 2011

Vestibular schwannomas (VSs) represent benign Schwann cell (SC) neoplasms arising from the eighth cranial nerve and account for 8-10% of all intracranial neoplasms. Denervation by axotomy leads to increased p75NTR expression, JNK activation, and eventual death of SCs. In contrast to SCs, VS cells survive in the absence of axons and here we investigated the interactions of p75NTR and JNK signaling on the survival response of cultured primary human VS cells. Using qRT-PCR and western blotting, we find that p75NTR is over-expressed in VS cells compared with normal nerve. However, in contrast to normal SCs, treatment of cultured VS cells with pro-NGF, a high affinity ligand for p75NTR, failed to induce apoptosis. We confirmed that VS cells express sortilin, a co-receptor with p75NTR to bind pro-NGF and induce apoptosis. We further show that VS cells display persistent JNK phosphorylation and activity compared with normal SCs. Treatment of VS cultures with the JNK inhibitors, I-JIP or SP600125, results in decreased proliferation, increased apoptosis, and increased accumulation of mitochondrial superoxides. Overexpression of mitochondrial superoxide dismutase prevents apoptosis due to suppressed JNK. These results suggest that persistent JNK contributes to the survival of VS cells, at least in part, by suppressing accumulation of mitochondrial superoxides. Treatment of VS cultures with proNGF protected the cells from apoptosis due to JNK inhibition. This protective effect is specific to proNGF and not seen with other neural derived growth factors such as neuregulin-1 (NRG-1). Indeed treatment with NRG-1 increased apoptosis in cultures with suppressed JNK activity. Further, VS cells lack Trk expression supporting the notion that the protective effect of proNGF is mediated by p75NTR. These results suggest that, in contrast to normal SCs which undergo apoptosis in response to p75NTR and JNK signaling, these signals promote VS cell survival. Such differences may account for the ability of VS cells to proliferate and survive in the absence of axons.

Support: DOD NF050193 and NIDCD R01DC009801

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 4008.
Abstract 4009: Identification of Y726 and Y793 as novel phosphorylation sites in human Flt3 and their role in signaling
Authors: Razumovskaya Elena , Sun Jianmin , Rönnstrand Lars
Published: February 2011

Fms-like tyrosine kinase-3 (FLT3), a growth factor receptor expressed in hematopoietic progenitor cells. FLT3 mutations, ITD and D835, are found in approximately 30% of AML patients and correlate with a poor prognosis, thus making receptor a potential therapeutic target. Flt3 mutations cause constitutive activation of the receptor, leading to alteration in signal transduction. The aims of this study are to analyze the kinetics and specificity of FLT3 autophosphorylation in wild-type FLT3 and FLT3 mutants, to identify new phosphorylation sites in FLT3 receptor and to determine related signaling pathways.

Using a panel of “homemade” phosphospecific antibodies directed against potential tyrosine phosphorylation sites in FLT3, we identified three novel phosphorylation sites and studied the kinetics and specificity of phosphorylation in living cells.

Our results show that activation of the wild-type Flt3 receptor is ligand- dependent and responding in a time dependent manner in contrast to FLT3-ITD and D835Y that are constitutive active and ligand-independent. Eight phosphorylated tyrosine residues (pY589, pY591, pY599, pY726, pY768, pY793, pY842 and pY955) were shown to have different phosphorylation characteristics in the wild-type receptor compared to the mutated receptors. Furthermore, we showed that Y726, Y793 and Y842 are novel phosphorylation sites of FLT3 receptor. Further study on stable transfected cells with Y-to-F mutations (726F and 793F), showed that these sites might be important for cell survival, proliferation and transformation. We could also see decreased phosphorylation of Erk1/2 and SHP2 in mutants compare to wild-type FLT3 or FLT3-ITD. Potential binding partners for Y726 and Y793 were predicted by using synthetic phosphopeptides for probing of SH2 domain arrays. The candidate binding partners will be verified as true binding partners using co-immunoprecipitation experiments.

From this study we could observe not only quantitative changes but also qualitative differences in the phosphorylation patterns of wild-type and mutated FLT3 receptors. We have also identified novel phosphorylation sites in FLT3 receptor that can be involved in different signaling pathways. Using this data we can further study the mechanisms of signaling pathways of the Flt3 receptor that are involved in many biological responses and understand the mechanism by which FLT3-ITD and -D835Y functions in pathological conditions.

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 4009.
Abstract 401: CD5+CD23+ leukemic cell populations in Tcl1 transgenic mice show significantly increased proliferation and Akt phosphorylation
Authors: Efanov Alexey A. , Zanesi Nicola , Maximov Vadim , Nazaryan Natalya , Santanam Urmila , Palamarchuk Alexey , Croce Carlo M. , Pekarsky Yuri
Published: February 2011

B-cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia. Deregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene in mouse B-cells causes a CD5 positive leukemia similar to aggressive human B-CLLs. We recently reported that TCL1 expression in B-CLL is regulated by miR29 and miR181. To determine whether treatment with microRNAs can inhibit

B-CLL in mice by targeting TCL1 we generated TCL1 transgenic mice using a construct containing 3’ and 5’ UTRs of TCL1 under B-cell specific Eμ promoter (Eμ-TCL1fl). At the age of 12-18 months these mice showed B-CLL like disease. Immunophenotyping revealed accumulation of CD5+CD23+B220+ population in spleens and lymph nodes. Our results show that CD5+CD23+ B-cell populations from Eμ-TCL1fl mice actively proliferate and display significantly increased levels of phospho-Akt. Eμ-TCL1fl mice showed immunological abnormalities similar to human B-CLL, including hypoimmunoglobulinemia, abnormal levels of cytokines, and impaired immune response. These findings revealed biochemical and immunological similarities between Tcl1 driven B-CLL in mice and human B-CLL.

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 401.
Abstract 4011: CYP3A4 over-expression promotes ER-positive breast cancer cell growth, in part, through Stat3 activation
Authors: Guo Zhijun , Mitra Ranjana , Potter David A.
Published: February 2011

Cytochrome P450 3A4 (CYP3A4) is an important monooxygenase that metabolizes a wide range of xenobitics including many drugs and environmental chemicals, as well as endogenous substances such as steroids. Expression of CYP3A4 has been observed in breast tumors, but its studies in breast cancer cells have mainly focused on carcinogen bioactivation, hormone metabolism and chemotherapy resistance. Whether CYP3A4 has a direct effect on breast cancer progression is unknown. To address these questions, we performed RNAi knock down studies, demonstrating roles for CYP3A4 in proliferation, motility, and clonogenicity of the MCF7 line. CYP3A4 knock down also inhibited phosphorylation of key regulatory proteins such as c-Src and STAT3, implicating its importance in breast cancer progression. To further study the effect and mechanisms of CYP3A4 in breast cancer progression, stable CYP3A4 over-expressing MCF7 cell lines were isolated and characterized. CYP3A4 over-expressing clones exhibited a higher proliferation rate than the vector control clone, consistent with the flow cytometry cell cycle analysis in which CYP3A4 over-expressing clones exhibited a markedly higher S phase component. Apoptosis was not increased due to CYP3A4 over-expression. The CYP3A4 over-expressing clones adhere poorly to plastic and require poly-D-lysine coated tissue culture plate to grow robustly. Adhesion to fibronectin was also reduced as the result of over-expression. In immunofluorescence microscopic study, the highest CYP3A4 over-expressing clone displayed disorganized focal adhesions, potentially explaining the poor adhesion. Consistent with the knock down study, CYP3A4 over-expressing clones exhibited elevated phosphorylation of both c-Src and Stat3 that could be responsible for the observed promotion of proliferation. These results support the model that CYP3A4 is an important signaling protein, linked to the c-Src/Stat3 pathway that influences breast cancer progression.

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 4011.
Abstract 4015: The role of IL-6/STAT3 axis in the transformation of Epstein-Barr Virus (EBV) infected-immortalized nasopharyngeal epithelial cells
Authors: ZHANG GUITAO
Published: February 2011

The role of IL-6/STAT3 axis in the transformation of Epstein-Barr Virus (EBV) infected-immortalized nasopharyngeal epithelial cells.

Zhang Guitao, Tsang Chi Man, Chen Honglin, Tsao Sai Wah.

Nasopharyngeal carcinoma (NPC) is rare throughout most parts of the world but common in certain geographic area, such as Southern China including Hong Kong. The etiology is based on intricate interactions among environmental factors, genetic susceptibility and Epstein-Barr virus (EBV) infection. Elevated antibody titers to EBV antigens are regularly observed in NPC patients. However, little information is available concerning the role of EBV in the early stage of NPC carcinogenesis. Our laboratory has established and characterized immortalized nasopharyngeal epithelial cells lines as premalignant nasopharyngeal cell models to examine for the transformation property of EBV infection in early stage of NPC development. Our results show that EBV infection activates STAT3 in immortalized nasopharyngeal epithelial cells. We had previously reported that the EBV-encoded LMP1 could upregulate IL-6 secretion to activate STAT3 signaling. Interestingly, we observed that activation of STAT3 in EBV-infected immortalized nasopharyngeal epithelial cells is highly susceptible to the induction by inflammatory cytokines, notably IL-6. Using quantitative real-time PCR, up-regulation of LMP1 was observed after IL-6 treatment on a time course dependent manner. An analysis of IL-6 receptor (IL-6R) indicated that EBV-infected nasopharyngeal epithelial cells have elevated level of IL-6R expression compared with uninfected cells. Activation of STAT3 signaling in immortalized nasopharyngeal epithelial cells by expressing a flag-tagged constitutively active STAT3-C confers transformed properties to cells. Our results support a model that EBV infection may predispose the aberrant STAT3 signaling for inflammatory cytokines involving the activation of LMP1/IL-6/STAT3 axis in premalignant nasopharyngeal epithelial cells. Enhanced STAT3 activation in EBV-infected epithelial cells by stromal inflammatory cytokines could result in irregular-gene expressions and cell signaling essential for malignant transformation, which may represent a crucial function of EBV infection in the development of nasopharyngeal carcinoma.

(This project is supported by The Research Grant Council (Hong Kong) and Area of Excellence on Nasopharyngeal Carcinoma Research Center (Hong Kong)).

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 4015.
Abstract 4017: Identifying Protein-Protein Interactions in Single Protein Complex level by Microchannel Device
Authors: Chou Chao-Kai , Jing Nan , Yamaguchi Hirohito , Tsou Pei-Hsiang , Kameoka Jun , Hung Mien-Chie
Published: February 2011

Cancer is the most threaten disease in this country and deregulations of signal pathways are often responsible for the cancer progression. Many mysteries of cancer related signal pathways have been revealed by current molecular biology research and the detection of protein-protein interactions is one of the critical techniques. The general techniques for protein-protein interactions detection include immuno-precipitation, immuno-fluorescence staining, and fluorescence resonant energy transfer. These techniques can detect specific protein in cultured cell and in tissue and have been applied in most biomedical research. However, all of them have various limitations. For instance, only two molecules could be examined in one experiment and the detection usually require long processing time with complicated procedures and large sample amount. Moreover, the detection of signals currently is still relied on X-ray film exposure or eye-direct observation that may limit the sensitivity and accuracy. We are developing a microchannel detection system in order to avoid these defects and to provide rapid and accurate detection in breast cancer signal transduction research. The principle is that nanochannel fluidics could only allow a single protein complex to pass through the detection point of fluorescence spectroscopy during examination and the result from the single protein complex could provide us most direct information of protein-protein interaction. Since the detection level is down to the single protein complex, it could highly increase the detection sensitivity. Currently, we have successful fabricated different size of microchannels and we recognized that top-down fabrication approach on transparent silicon based wafers could provide the best quality nanochannels. The channel was than integrated to the fluorescence spectroscopy for single fluorescence detection. To test the performance of the device, fluorescence antibodies were loaded into the channel and we successfully detected a single fluorescence signal during the experiment. Subsequently, we detect demonstrated that epidermal growth factor receptor (EGFR) interact with its downstream proteins Src and STAT3 in a single complex after epidermal growth factor (EGF) stimulation. However, only 1.8% of EGFR interact with both Src and STAT3 at the same time which suggests that EGFR/Src/STAT3 is a transition product in the dynamic of EGFR signal transduction. Thus, our system provides the interaction information from a single protein complex with the ratio of interactive proteins to dissect the signal transduction in dynamic aspect and will offer an effective technology.

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 4017.
Abstract 4018: Crosstalk between the pleiotrophin-anaplastic lymphoma kinase axis and the protein tyrosine phosphatase ζ signaling pathways
Authors: Kodack David P. , LaConti Joseph , Noda Masaharu , Wellstein Anton
Published: February 2011

Pleiotrophin (PTN) is a heparin-binding growth factor that can support tumor growth, invasion, angiogenesis, and metastasis. The mechanism of action (receptor signaling pathway) responsible for mediating PTN's biological effects, however, is still controversial. Here we show that an antibody targeting the ligand-binding domain of anaplastic lymphoma kinase (ALK) is capable of blocking the signaling effects of PTN in a glioblastoma cell line that expresses high levels of ALK. This corroborates recent findings by Stylianou, et al. (2009), who showed disruption of PTN activity with a single chain variable IgG fragment that binds to the ALK ligand-binding domain. Furthermore, expression of protein tyrosine phosphatase receptor ζ (Ptprz) in cells resulted in crosstalk with the PTN-ALK signaling axis at the level of p38 MAPK. While PTN increased p38 MAPK phosphorylation, the presence of Ptprz decreased its phosphorylation, both at its basal as well as PTN-enhanced levels. Enhancement of p38 MAPK phosphorylation by PTN is possibly mediated through activation of MKK4, as it was phosphorylated in the presence of PTN. On the other hand, the mechanism for Ptprz-mediated suppression of p38 MAPK is not yet clear, as there was no change in phosphorylation of MKK3, MKK4, or MKK6 after its over-expression. These findings suggest that ALK is playing a direct role in PTN activity, and that the Ptprz signaling pathway may act in a separate but inter-connecting fashion. Parallel targeting of both the ALK and Ptprz signaling pathway may be necessary for the treatment of tumors (glioblastoma) expressing both membrane 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 4018.
Abstract 402: Role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in chemotherapy induced myeloid leukemia
Authors: Potla Ratnakar , Jaiswal Anil K.
Published: February 2011

Individuals with NQO1P187S mutation have no detectable NQO1 protein levels. These individuals have been extensively predicted to be at high risk for developing chemotherapy-induced leukemia. We have studied the effect of Etoposide, a DNA Topoisomerase II inhibitor used in many chemotherapeutic regimens, on NQO1 gene deleted (NQO1-/-) mice. NQO1-/- mice are born with a myelodysplastic bone marrow. Flow cytometric analysis of blood, bone marrow and spleen showed an increase in megakaryocytic and myeloid cell lineages in NQO1-/- mice. A spectrum of responses ranging from leukopenia to myeloid leukemia were observed with increasing drug concentration by Flow cytometry and confirmed by histochemistry in NQO1-/- mice. C/EBPalpha plays a key role in the myeloid lineage commitment of hematopoietic progenitor cells. NQO1 protects C/EBPalpha from 20S proteasomal degradation in in vitro assays. Altered C/EBPalpha levels are responsible for the preferential increase in myeloid cell lineage in NQO1-/- mice.

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 402.
Abstract 4023: Novel role of neurofibromin in transport of RNA granules
Authors: Arun Vedant , Wiley Joseph , Karim Zia , Guha Abhijit
Published: February 2011

Neurofibromatosis 1 (NF1) is the most common tumor predisposing syndrome in humans, with an incidence of 1:3500 live births. Neurofibromin, encoded by Nf1, acts as a p21-Ras-GAP to directly interact with and inactivate p21-Ras, through its’ GAP Related Domain (GRD). Evidence suggests that non-Ras-GAP functions mediated through interactions with domains outside of the GRD are of importance. By making a number of NF1-GST domain constructs coupled with differential Mass Spec analysis, we identified LRPPRC and Dynein as previously unreported NF1-TBD interacting proteins. LRPPRC was of interest as it is mutated in the Leigh Syndrome French Canadian (LSFC) variant, a cytochrome-oxidase deficiency syndrome, characterized by neurodegeneration, developmental- mental- and growth-retardation, thereby having some similarities with non-tumor manifestations of NF1. We have established that LRPPRC and neurofibromin interact in vitro by immunoprecipitation and reverse bait immunoprecipitation. Use of a novel Proximity Ligation Assay (PLA) was used to further confirm the interaction in situ in cell lines and clinical tissue cryosections. Towards elucidating the functional relevance of the interaction, we have established that the two proteins complex with microtubules and motor proteins, which indicates a role in microtubule-dependant intracellular transport. Since LRPPRC binds with mRNA and is involved in its nucleocytosolic shuttling, we hypothesize that the cargo transported by this complex could be RNA granules. Mass Spec analysis of NF1 immunoprecipitates revealed a number of proteins that are a part of the RNA granule complex which include RNA binding proteins and proteins involved in mRNA translation. By performing RNA Immunoprecipitation (RIP) with NF1 and LRPPRC antibodies, we have demonstrated that the two proteins do complex with 18S and 28S rRNA which is indicative of the presence of intact mRNA. Towards identifying the specific messages transported by the NF1-LRPPRC RNA granule complex we are performing RIP - microarray gene expression profiling (CHIP). The resultant candidate mRNAs will be further investigated to understand the importance of this novel interaction and novel mechanism towards some of the overlap of neurological and developmental manifestations between LSFC and NF1.

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 4023.
Abstract 4029: Sox9 functions downstream of Hedgehog-Gli1 in epithelial transformation
Authors: Deng Wentao , Ruppert J. Michael
Published: February 2011

The Sonic Hedgehog (Shh) signaling pathway plays critical roles in cell fate specification during embryonic and fetal development and in adult tissues. The extracellular morphogen, Shh, signals through Gli family zinc finger transcription factors to regulate the expression of downstream target genes. Constitutive Shh-Gli signaling is implicated in the pathogenesis of several human cancers, including cutaneous basal cell carcinoma (BCC), prostate cancer and medulloblastoma. How Gli can signal through downstream genes to induce malignant transformation remains poorly understood.

In this work, we performed microarray analysis to reveal the gene expression profile after tetracycline-regulated induction of Gli1 for 3 or 6 hours in cultured epithelial cells, identifying multiple known Shh target genes. Among the most rapidly induced genes was Sox9, a member of SRY family of transcription factors. Sox9 is essential for male sex determination and is a master regulator of chondrogenesis. Sox9 is also well established as a Shh-Gli target gene, but how it functions in Shh-Gli signaling is not well understood. We found that Sox9 mRNA and protein levels are greatly increased after short-term or long-term Gli1 expression in a rat culture model of epithelial cells, termed RK3E, and in mouse skin. As shown by reporter assays, Sox9 transcriptional activity was increased by approximately 18-fold in RK3E cells transformed by Gli1. To enable knockdown studies in rat cells, we defined the full-length rat Sox9 coding sequence. Subsequently, suppression of rat Sox9 by siRNA indicated an essential role for this Gli-regulated gene in malignant transformation of RK3E in vitro. Specifically, shRNA plasmids that suppress Sox9 could dramatically reduced the transforming activity of Gli1. In contrast, there was little effect on other transforming oncoproteins including H-Ras and ErbB2. These results establish Sox9 induction as an early response to Gli1, and demonstrate that induction of Sox9 is essential for transformation by Gli1. Further characterization of the Sox9 knockdown phenotype is in progress and will be presented.

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 4029.
Abstract 403: Deletion of HBZ alters lymphomagenesis in HTLV-1 infected humanized SCID mice
Authors: Crawford Lindsey , Banerjee Prabal , Lairmore Michael , Green Patrick , Feuer Gerold
Published: February 2011

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of Adult T-cell Leukemia (ATL), an aggressive, mature 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 ATL development. We have previously demonstrated that HTLV-1 is capable of infecting human hematopoietic progenitor and stem cells (CD34+ HPCs). Humanized NOD/SCID (HU-SCID) mice generated by injection with HTLV-1-infected CD34+ HPCs demonstrate persistent infection and reproducibly develop CD4+ T-cell lymphomas (∼80%) with clinical characteristics associated with ATL. The HTLV-1 bZIP protein (HBZ) is consistently expressed in all ATL cells and has been implicated in the maintenance of leukemogenesis. HBZ has previously been demonstrated to modulate Tax activity and to interact with the cellular factors CREB, Jun and p53. Transduction of HBZ into CD34+ HP/HSCs using a lentivirus vector suppresses clonogenic colony formation in vitro. Moreover, infection of CD34+ HP/HSCs with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ) results in elevated clonogenic colony formation in vitro, suggesting that HBZ suppresses hematopoiesis. To establish the role of HBZ in HTLV-1 replication and leukemogenesis in vivo, HU-SCID mice were infected with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ). HTLV-1ΔHBZ-infected HU-SCID mice developed lymphoproliferations that were phenotypically characterized as a mixture of immature lymphoid progenitors (CD90+) and mature B-cells (CD19+) starting at ∼10 weeks post-reconstitution, in contrast to wild-type HTLV-1 infection. Lymphoproliferations induced by HTLV-1ΔHBZ successfully engraft and expand in naïve NOD/SCID mice when injected into the peritoneal cavity and leukemic cells and lymphoma cells express HTLV-1 gp46env and p19gag. FACS-purified CD90+CD19− HTLV-1ΔHBZ infected cells also successfully engraft in naïve NOD/SCID mice and mature into CD19+ cells suggesting that these cells may be leukemic progenitors with the capability of maturing in vivo. In addition, a majority of HTLV-1ΔHBZ-infected HU-SCID mice also develop a progressive, flaccid hind limb paralysis with features similar to the neurological disorder, HAM/TSP. These results suggest that HBZ is dispensable for HTLV-1 replication and modulates lymphomagenesis in the HU-SCID model. The humanized SCID mouse provides a novel in vivo platform for analysis of the role of HTLV-1 genes in viral replication and pathogenesis.

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 403.
Abstract 4033: MiR-21 upregulation in breast cancer cells leads to PTEN loss and Herceptin resistance
Authors: Rehman Sumaiyah K. , Huang Wen-Chien , Yu Dihua
Published: February 2011

MicroRNAs (miRNA) are a class of small 22-nucleotides endogenous non-coding RNAs which serve as regulators of gene expression by targeting specific messenger RNAs (mRNA). Aberrant expression of certain microRNAs can lead to therapeutic drug resistance, such as Cisplatin resistance by miR-214 targeting PTEN. Our lab previously found that PTEN loss confers Herceptin resistance in ErbB2-overexpressing breast cancers. Other studies in hepatocellular carcinoma have shown that miR-21 targets the 3′UTR of PTEN mRNA leading to downregulation of PTEN protein levels. Based on these findings, we hypothesized that overexpression of miR-21 may confer Herceptin resistance in breast cancer patients by downregulation of PTEN.

To test this hypothesis, mir-21 and/or control miRNA were transiently transfected and expressed in ErbB2-overexpressing AU565 and SKBr3 breast cancer cell lines, followed by Herceptin treatment. We found that cells with increased miR-21 had reduced PTEN expression and were significantly more resistant to Herceptin treatment versus control miRNA transfected cells. Conversely, when we knocked down miR-21 in high ErbB2 expressing BT474 cells, the cells showed increased PTEN expression and became more sensitive to Herceptin treatment versus control miRNA transfected cells. To determine the clinical relevance of our findings, we detected miR-21 level in patients’ breast cancers with ErbB2 expression that were treated with Herceptin and we found that high miR-21 level significantly correlated with patients’ poor response to Herceptin treatment and disease progression. We are currently identifying other targets of miR-21 to better understand the mechanisms of miR-21 mediated-Herceptin resistance, which may provide new insight on how to overcome Herceptin resistance.

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 4033.
Abstract 4036: The microRNA profile of prostate carcinoma obtained by deep sequencing reveals potential miRNA targets such as myosin VI
Authors: Szczyrba Jaroslaw , Löprich Elke , Wach Sven , Jung Volker , Unteregger Gerhard , Barth Stephanie , Grobholz Rainer , Wieland Wolf , Stöhr Robert , Hartmann Arndt , Wullich Bernd , Grässer Friedrich
Published: February 2011

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify and characterize the underlying molecular mechanisms, we performed microRNA (miRNA) profiling of primary prostate cancers and non-cancer prostate tissue. Furthermore, using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs.

Material and Methods: We performed comparative miRNA expression profiling in ten prostate cancer specimens and ten non-cancer prostate tissue samples using deep sequencing of cDNA libraries. The deregulation of selected miRNAs was validated in established prostate cancer cell lines by Northern blotting as well as in 26 paired samples of prostate cancer tissue and adjacent normal tissue by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The regulative capabilities of selected miRNAs on potential target genes were analyzed in vitro using reporter gene constructs and transfection of miRNA expression vectors

Results: Using deep sequencing we found that 16 miRNAs were up-regulated more than 1.5-fold and 17 miRNAs were down regulated more than 1.5-fold. The most pronounced deregulation was noted for miRNAs miR-375 and miR-200c which showed an up-regulation of more than 4.5-fold and miR-143 and miR-145 which were down regulated 4-fold each. The differential expression of these miRNAs could be confirmed by Northern blot analysis of established prostate cancer cell lines. By analyzing 26 primary prostate cancer samples by qRT-PCR we could detect a significant deregulation of the miRNAs miR-375, miR-143 and miR-145 (p<0.05, paired students T-test). Independent target prediction databases (TargetScan, PicTar) indicate, that the 3′ untranslated region (3′UTR) of the Myosin VI gene is a regulative target for both miR-143 and miR-145. This prediction could be confirmed using a luciferase reporter gene vector containing the Myosin VI 3′UTR. Furthermore, transient expression of miR-143 and miR-145 reduced the amount of Myosin VI protein by 20% and 48% respectively.

Conclusion: Our findings indicate that specific miRNAs are reproducibly found to be deregulated in different cohorts of prostate cancer specimens using independent experimental methods. This is the first report to show that miRNAs miR-143 and miR-145 are capable of negatively regulating the protein expression of Myosin VI.

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 4036.