bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 99: Posttranslational modification of the RBBP6 isoform 3 is required for excessive cell proliferation
Authors: Dlamini Zodwa , Mbita Zukile
Published: August 2012

RBBP6 isoform 3 belongs to a family of RBBP6 cell cycle-related proteins. There are 3 human RBBP6 isoforms; iso 1, 2 and 3, transcribed from RBBP6 mRNA variants 1, 2 and 3 respectively. RBBP6 isoform 1 has been shown to be a pro-apoptotic protein while the function of isoform 2 has not been confirmed. Recently isoform 3 has shown to be involved in G2/M cell cycle regulation and is deregulated in human cancers. This study aimed at investigating how the RBBP6 isoform 3 regulates cell proliferation. Cell culture and transfections were used to determine the effect of RBBP6 isoform 3 on cell cycle progression. RBBP6 variant 3 mRNA was cloned into a pCDNA3.1/Zeo (+) vector and expressed in human kidney embryonic cells, Hek 293T. Cell cycle and cell counts analysis were conducted by using Flow Assorted Cell Sorting (FACS). Western blotting analysis was used to analyse RBBP6 isoform 3 using a polyclonal antibody against RBBP6 isoform 3 N-terminal region. Transfection of Hek 293T cells with RBBP6 variant 3 mRNA resulted in temporal G2/M cell cycle arrest. This cell cycle arrest ceased after 48 hours and Western blotting showed that RBBP6 isoform 3 was posttranslationally modified through either glycosylation or phosphorylation or both. Mass spectrometry confirmed that the higher molecular weight protein was indeed RBBP6 isoform 3. This higher molecular weight product was accompanied by excessive cell proliferation. This result suggests that cancer cells use this posttranslational modification to bypass G2/M cell cycle arrest. RBBP6 isoform 3 could therefore be targeted for new anti-cancer therapeutic strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 99. doi:1538-7445.AM2012-99
Abstract 994: Colorectal cancer, adenoma and normal whole genome methylation sequencing in Africa Americans
Authors: Ashktorab Hassan , Rahi Hamed , Tbaishat Rana , Daremipouran Mohammad , Smoot Duane T. , Lee Edward L. , Varma Sudhir , Sun Xueguang , Jia Xi-Yu , Chung Hunter , Bacon Eliza , Leavitt Ron , Siegel Peter , Brim Hassan
Published: August 2012

Background: Colorectal adenoma (CRA) to cancer (CRC) progression involves epigenetic changes including DNA methylation. Here we performed whole genome Bisulfite Next-Generation Sequencing (WGBNGS) on a normal, a tubular adenoma and a tumor patients’ tissue DNA to elucidate epigenetic drivers in normal to cancer progression. Patients and Methods: Genomic DNA was isolated from fresh frozen tissues from a patient with normal colon, from a tubular adenoma lesion (20X depth was performed on these DNA samples., Alignment, mapping and CpG methylation analyses were done. Pyrosequencing and immunohistochemistry (IHC) were used to confirm selected methylation target genes. Results: We identified 94 unique CpG sites hypermethylated in 4 novel genes in adenoma compared to normal and 180 unique CpG sites in 7 genes in cancer. Ingenuity pathway analysis (IPA) showed that the methylated genes including a novel gene ATXN7L1 were involved in WNT pathway, ubiquitination process and cross-talk with B-catenin, in the adenoma. Three of the hypermethylated genes in cancer were, Loc100506436: a novel gene with 17 unique CpG sites (EGFR pathway, cRNA CpG site hypermethylated is part of MEK1/2), GPNMB with 7 unique CpG sites (membrane protein likely in complexes with integrins) and TNFAIP2 with 8 unique CpG sites (its role was noted in Head and neck cancer). IHC confirmed the lack of GPNMB expression in cancer tissues. Conclusion: This work provides insight into differential CpG island methylation profiles in CRA, CRC vs. normal colon tissue and provides a window into the more complex factors, including alteration of known and novel genes as multiple CpG methylation drivers in colorectal carcinogenesis. Investigations into the possible roles of the novel genes targets in the context of early and prognostic methylation biomarker are underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 994. doi:1538-7445.AM2012-994
Abstract 998: Oncogenic potential of mitochondria regulate the cellular tumor characteristics
Authors: Ma Yewei , Gupta Vineet , Trieu Robert , Lap Lee Tin , Kaipparettu Benny A. , Wong Lee-Jun
Published: August 2012

Mitochondrial-nucleus cross-talks and mitochondrial retrograde regulation of nuclear genes can play significant role in cellular properties. Several studies reported that somatic mitochondrial DNA alterations are often detected in cancer cells. Most of such alterations are polymorphisms without functional significance. Here we use a transmitochondrial cybrid (cybrids) approach to understand the significance of mitochondrial property in regulating the oncogenic properties of a cell irrespective of its mutational and nuclear status. Cybrid models in two differently defined nuclear backgrounds were studied. In the first model, mitochondria depleted rho zero (≥0) from an aggressive osteosarcoma cell lines 143B TK− was used as a common nuclear background. 143B β0 cells were fused with mitochondria from non-cancerous breast epithelial cell line MCF10A, low metastatic breast cancer cell line MDA-MB-468 and highly metastatic 143B TK− cell lines. In the second model, we used the common nuclear background of A1N4, a non-tumorigenic immortalized breast epithelial cell. A1N4 β0 cells were fused with MCF-10A and metastatic breast cancer cells MDA-MB-231. Studies in parental mitochondrial donor cells suggested that cancer cell lines have reduced ATP synthesis, oxygen consumption and respiratory chain activities compared to non-cancerous cells. Interestingly, in spite of the common nuclear backgrounds, cybrids also showed properties similar to their parental cells under both cancerous and non-cancerous nuclear backgrounds. Functional studies in cybrids under non-cancerous nuclear background observed that fusion with cancerous mitochondria resulted in loss of growth contact inhibition, induction of colony formation potential in soft agar and increased survival potential under serum starvation. Similarly, functional studies in cybrids with cancerous nuclear background suggested that non-cancerous mitochondria could reverse different oncogenic characteristics of cancer cell including cell proliferation, viability under hypoxic condition, anti-apoptotic properties, resistance to anti-cancer drug, invasion, colony formation in soft agar, and in vivo tumor growth in nude mice. Microarray analysis of cybrids under cancerous nuclear background suggested that several oncogenic pathways are inhibited due to non-cancerous mitochondria. Interestingly, under non-cancerous nuclear background, induction of important oncogenes was observed in cybrids containing cancerous mitochondria. These results suggest that mitochondrial-nuclear cross talk have significant contribution to cancer cell properties. Also it highlights mitochondria as a promising target in cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 998. doi:1538-7445.AM2012-998
Abstract LB-119: Therapeutic sensitivity and predictive markers for CDK4/6 inhibition defined by ex vivo analyses of human tumors
Authors: Witkiewicz Agnieszka , Dean Jeffry , McClendon Amy , Knudsen Erik S.
Published: August 2012

To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g. ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-119. doi:1538-7445.AM2012-LB-119
Abstract LB-12: Building a novel multifaceted nano-carrier: In vivo and in vitro applications of multifunctional nanolipoprotein particles
Authors: He Wei , Yi Sun , Luo Juntao , Lam Kit , Murphy William , Coleman Matthew
Published: August 2012

We have developed a novel process of production and assembly of nanolipoprotein particles (NLPs) as a multifaceted reagent for cancer imaging, drug delivery, immunomodulation and stabilizing membrane protein complexes for in vitro studies. Nanolipoproteins are components of the human HDL lipoprotein complexes that could be isolated and reconstituted to form NLPs. NLPs are discoidal nanoparticles of 10∼20 nm that self-assemble around a phospholipid bilayer. This bilayer mimics closely the cell membrane can support molecules such as dyes and proteins. NLPs present distinct advantages in terms of particle size, monodispersity and consistency: the circular protein belt constrains the dimensions of the bilayer and ensures NLP particle size distributions can be monodispersed and consistent between preparations. We have demonstrated when targeting agents and imaging agents are attached to the NLPs, they were able to accumulate at the tumor region and can be used for tumor imaging; We also managed to conjugate recombinant IL2 to NLPs. Our data showed that IL2-NLP conjugates were able to stimulate immunomodulation in mice both in vivo and in vitro. NLPs also have shown great promise for solubilizing and characterizing membrane proteins and may make many more membrane protein related complexes accessible for biophysical and biochemical study. Our approach represents a unique solution to the challenge of generating soluble and functional polytopic membrane proteins, facilitating the structural and functional characterization, providing a platform for high-throughput drug screening, and potentially a new type of carrier for tumor imaging and drug delivery.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-12. doi:1538-7445.AM2012-LB-12
Abstract LB-120: Site of origin of basal cell carcinoma stromal cells
Authors: Wang Grace Y. , Wang Joy , Wang Lynn , Epstein Ervin
Published: August 2012

We have adapted a transgenic tetracycline-regulatable mouse model to identify slow dividing cells in murine basal cell carcinomas (BCCs). This system consists of 2 transgenes: histone H2B-green fluorescence protein (GFP) controlled by a tetracycline-responsive element (TRE) upstream of a CMV promoter; and a tissue specific promoter-driven tetracycline-regulatable activator protein (rTA). In the absence of rTA, the H2B-GFP expression should be silent. However, we and other labs have found that mice carrying the H2B-GFP transgene with or without the K5-rTA transgene showed significant numbers of GFP-expressing bone marrow cells, which are not regulated by doxycycline. To study BCCs, we crossed these leaky mice with our Ptch1+/− mouse and treated the offspring with ionizing radiation. Although the mice with the H2B-GFP transgene developed microscopically detectable BCCs in a manner similar to Ptch+/− parental mice, the presence of the H2B-GFP transgene significantly delayed the microscopic-to-macroscopic transition of tumors (i.e. mice develop their first macroscopic tumor at age 9-13 month for Ptch+/− vs. 13-18 months for Ptch1+/− K5rTA H2B-GFP). Moreover, approximately one third of the Ptch1+/− K5rTA H2B-GFP mice died with unknown causes before 9-month of age, the time point when we collected skin biopsies for evaluating microscopic tumors, in comparison to 10-15% mouse death in Ptch1+/− mice by this age. Studying the limited number of visible BCCs arising in Ptch1+/− H2B-GFP (no K5-rTA transgene), we found that all the tumor cells were GFP-negative; however, the stromal cells nearly uniformly expressed GFP. By contrast, microscopic BCCs had no GFP+ stromal cells. As expected, both tumor cells and stromal cells were GFP-positive in unchased Ptch1+/− K5rTA H2B-GFP mice. By immunostaining, the GFP-expressing stromal cells, unlike the tumor cells, did not express keratins 5 and 14 but were vimentin-positive, Ki67-negative and frequently located in proximity to blood vessels. Using flow cytometry analysis with a panel of cell surface markers, we further characterized the GFP-expressing tumor stromal cells and the bone marrow cells. However further assessment is needed to confirm that the BCC stromal cells are derived from the GFP-expressing bone marrow cells and to determine their potential role in the microscopic to visible tumor transition. Our findings suggest that bone marrow cells may be the cells of origin of tumor stromal cells in BCCs perhaps in other cancers in general.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-120. doi:1538-7445.AM2012-LB-120
Abstract LB-126: Cancer-retina antigens as potential therapeutic targets for immunotherapy
Authors: Jesiak Maria , Osen Wolfram , Gardyan Adriane , Soltek Sabine , Heinzelmann Anita , Bazhin Alexandr , Eichmueller Stefan
Published: August 2012

Melanoma is a fast progressing tumor, which originates from melanocytes and it is known to be highly resistant to many therapies. Although it has been proven to be very immunogenic and a number of various melanoma associated antigens are well known, nevertheless, the immune response of the patients is often not able to fight back and eliminate all tumor cells. It has been recently shown that a new class of tumor antigens, called cancer-retina antigens (CRA), apart from being expressed in a healthy retina, can be expressed at the mRNA and protein level in melanoma. The aberrant expression of transducin, cGMP-phosphodiesterase 6 (PDE6), guanylyl cyclase, recoverin and arrestin have been detected in tumor cells of melanoma patients and in tumor-bearing mice. These antigens can elicit humoral and cellular immune responses. We aim to investigate if CRA could serve as potential target antigens for immunotherapy approaches against melanoma. Among these antigens, human PDE6 was selected and analyzed for its immunogenicity in normal C57BL/6 mice and in HLA-DRB1*0301tg mice. Human (xenogenic) PDE6 alpha has been cloned and used for DNA immunization of BL/6 and HLA transgenic mice. By IFN-γ ELISPOT assay employing synthetic peptide libraries, immunogenic peptides have been identified and CRA specific T cell epitopes were determined. Further on, PDE6-specific T cells lines will be generated and their therapeutic efficiency will be checked in transplantable tumor mouse models or in transgenic mice with spontaneous melanoma formation. In addition, peripheral blood mononuclear cells (PBMCs) from tumor patients will be analyzed for the presence of specific T cells against the epitopes identified in HLA-transgenic mice. These T cells will be expanded and tested for their reactivity against HLA-matched human melanoma cell lines in vitro. Finally, both established CRA-specific T-cell lines and synthetic polypeptides will be tested as vaccines in therapeutical and preventive settings in melanoma animal tumor models. Immunized animals will be monitored for any signs of autoimmune retinopathy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-126. doi:1538-7445.AM2012-LB-126
Abstract LB-127: Identifying an IDO-expressing “fibrocyte” subset of myeloid suppressor cells in pediatric cancer patients
Authors: Zhang Hua , Mackall Crystal L.
Published: August 2012

Human myeloid derived suppressor cells (MDSC) have been described as CD14-CD11b+CD33+HLADR- cells that exert T cell suppression via ROS, ARG1 and iNOS2. Here, we report a novel myeloid subset found within mononuclear fraction elutriated from apheresis samples of pediatric cancer patients vs. healthy controls. Of 60 pediatric cancer patients studied, we frequently observed expanded numbers of CD14-CD123-CD11chi cells, which were rare and found only at low levels in healthy donors. Like previously described MDSC, these cells were CD11b+, but CD33lo/-HLADR+. In addition, these abnormal myeloid cells were CD34+CD15+CD16+CD66b+CD68+PDL1+ and IL4Rα+. Interestingly they also expressed α smooth muscle actin and collagen I/V on their surface, which are hallmarks of fibrocytes. The fibrocytes expressed high levels of indoleamine oxidase (IDO), but did not express ARG1, ARG2, iNOS2. They suppressed T cell proliferation in response to OKT3, which was partially reversed by the addition of 1MT, an inhibitor of IDO. Given the IL4Rα expression, we sought to induce these cells in vitro from normal monocytes using IL-4. Indeed, IL4 alone was sufficient to drive monocytes to differentiate into fibrocytes, which demonstrate a spindle-like shape, adhere to plastic, acquire the CD11chiCD14-CD34+CD16+collagenI+ phenotype, and express IDO. Fibrocytes have previously been implicated in chronic inflammation, angiogenesis and wound healing. We conclude that they also constitute a novel subset of myeloid derived suppressor cells in cancer patients, and could contribute to tumor progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-127. doi:1538-7445.AM2012-LB-127
Abstract LB-131: Peptide vaccine therapy for childhood gliomas: interim results of a pilot study
Authors: Pollack Ian F. , Jakacki Regina I. , Butterfield Lisa H. , Okada Hideho
Published: August 2012

Introduction: Malignant astrocytomas of the brainstem and cerebral hemispheres and multiply recurrent low-grade gliomas carry a poor prognosis despite current treatments, and new therapeutic approaches are needed. Having gained significant experience with immunotherapy for adult gliomas, we extended these insights to childhood gliomas, based on our observations regarding their profiles of glioma-associated antigen (GAA) expression. Methodology: We initiated a pilot trial of subcutaneous vaccinations with peptides for GAA epitopes emulsified in Montanide-ISA-51 given every 3 weeks for 8 courses along with intramuscular injections of poly-ICLC in HLA-A2+ children with newly diagnosed brainstem gliomas (BSG), cerebral high-grade gliomas (HGG), or recurrent gliomas. GAAs were EphA2, IL13Rα2, and survivin. Primary endpoints were safety and T cell responses against vaccine-targeted GAAs, assessed by ELISPOT and tetramer analysis. Treatment response was evaluated clinically and by MR imaging. Results: To date, 24 children have been enrolled, 13 with newly diagnosed BSG, 5 with newly diagnosed HGG, and 6 with recurrent gliomas. No dose-limiting non-CNS toxicity has been encountered. One child with a BSG had transient tumor enlargement in association with acute neurological deterioration 4 months after beginning vaccination that later regressed and culminated in a sustained partial response (PR), consistent with pseudoprogression. Two other children with BSG had symptomatic pseudoprogression, with transient neurological deterioration and tumor enlargement followed by stabilization on decreasing steroid doses. Among 22 patients evaluable for response, 4 had rapidly progressive disease, 14 had stable disease for > 3 months, 2 had PRs, 1 had an MR, and 1 had prolonged disease-free status after surgery. ELISPOT analysis, completed in seven children, showed response to IL13Rα2 in 5, EphA2 in 3, and survivin in 3. Conclusion: Peptide vaccination in children with gliomas is generally well tolerated, and has preliminary evidence of both immunological and clinical activity. Pseudoprogression can initially be difficult to distinguish from true tumor progression and aggressive management may be warranted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-131. doi:1538-7445.AM2012-LB-131
Abstract LB-134: Personalized peptide vaccination as the second line therapy for patients with chemotherapy-resistant advanced pancreatic cancer.
Authors: Sasada Tetsuro , Yutani Shigeru , Yoshitomi Munehiro , Matsueda Satoko , Komatsu Nobukazu , Itoh Kyogo
Published: August 2012

Background: Since the prognosis of advanced pancreatic cancer remains poor, development of novel therapeutic approaches, including cancer vaccine, would be highly desirable. We conducted a phase II study of personalized peptide vaccine (PPV) in chemotherapy-resistant advanced pancreatic cancer patients. Methods: Forty-one advanced pancreatic cancer patients (7 Stage IVa, 24 Stage IVb, 10 recurrence) who failed to respond to the first line chemotherapy were enrolled. Median duration of chemotherapy prior to the enrollment was 8 months. A maximum of four HLA-matched vaccine peptides showing higher antigen-specific immune responses were selected based on the titers of IgG against each of 31 different peptide candidates, and employed for subcutaneous vaccination. In order to identify potentially prognostic biomarkers for overall survival (OS), pre-vaccination clinical findings and laboratory data were retrospectively assessed and evaluated by multivariate Cox proportional hazards model in 36 patients who completed at least one cycle of 6 vaccinations. Results: Median OS from the first vaccination was 7.9 M with a one-year survival rate of 24%. Notably, median OS from the first line chemotherapy was 19.2 M. The main toxicity of PPV was skin reactions at the injection sites, but no vaccine-related serious adverse events were observed. Among the pre-vaccination factors examined, high level of serum amyloid A (SAA) and high titer of the IgG against 31 vaccine peptide candidates were significantly unfavorable for OS [hazard ratio (HR) = 1.019, P = 0.006; HR = 0.999, P = 0.0001; respectively] by the Cox multivariate analysis. The clinical stage was also associated with OS, as expected (HR = 0.496, P = 0.014). Conclusions: Even in chemotherapy-resistant advanced pancreatic cancer patients, PPV might improve the prognosis. Further randomized clinical trials of PPV would be recommended in advanced pancreatic cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-134. doi:1538-7445.AM2012-LB-134
Abstract LB-142: Induced expression and activation of oncogenes in NSCLC (non-small cell lung cancer) in the absence of activating mutations of EGFR and k-Ras
Authors: Gorgisen Gokhan , Ozes Derya , Pehlivanoglu Suray , Erdogan Abdullah , Dertsiz Levent , Ozbilim Gulay , Ozbudak Irem Hicran , Ozes Osman N.
Published: August 2012

Induced expression and activation of oncogenes in NSCLC (non-small cell lung cancer) in the absence of activating mutations of EGFR and k-Ras Background: Activating mutations in exon 18,19 and 21 of EGFR and changes in codons 12,13 and 61 of k-RAS are blamed for the development of NSCLC in 88% of patients. Therefore, it is suggested that the use of tyrosine kinase (TK) inhibitors, Erlotinib or Gefitinib, can be very useful to treat NSCLC patients. NSCLC patients harboring activating mutations of EGFR are very responsive to TK inhibitors; while, patients who do not have these mutations are unresponsive to these treatment. Therefore, it is recomended that before initiating therapy with these TK inhibitors, mutation status of EGFR must be determined. Methods: The protein lysates of tumor samples and DNA have been isolated from tumor and corresponding normal tissues of 16 NSCLC patients who underwent surgery. Genomic-DNA is used to sequence exons 18, 19 and 21 of EGFR1 as well as the second exon of k-RAS. Protein lysates have also been used to determine the expression or phosphorylation levels of EGFR and down-stream elements such as, STAT3, ERK and AKT. The expression levels of metastatic factors TWIST1 and GAPDH have also been determined. Results: Even though sequence analysis of indicated regions of EGFR and k-RAS reveal no mutation, the protein analysis of 16 tumor samples show remarkable changes in the activation or expression levels of above mentioned oncogenes. Among the patients different levels of induced phosphorylation have been found; these levels are, 2 to 23 fold induced phosphorylation of ERK, 1.5 to 20 in AKT, 2 to 10 in EGFR, and 2 to 24 in TWIST1 and 2-450 in GAPDH. These results indicate that even in the absence of any activating mutations of EGFR or k-Ras, the EGFR pathway is still very active in some NSCLC patients. Conclusion: If activating mutations of EGFR is considered the main reason for TK therapy, then TK use may not be the optimal decision to make. Results clearly indicate that NSCLC patients who do not show any activating mutations of EGFR may benefit from TK therapy. Even though, activating mutations of EGFR is the “gold standard” for TK therapy, surgery materials of NSCLC patients must be examined for oncogene activation. This is especially true for the EGFR pathway since, as shown in this work, the tumor micro-environment created by the tumor or surrounding cells can be as important as the activating mutations of EGFR.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-142. doi:1538-7445.AM2012-LB-142
Abstract LB-145: Folliculin interacts with p0071 (Plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis
Authors: Nahorski Mike S. , Seabra Laurence , Straatman-Iwanowska Ania , Wingenfeld Aileen , Reiman Anne , Lu Xiaohong , Gissen Paul , Hatzfeld Mechthild , Maher Eamonn
Published: August 2012

Inherited mutations in the folliculin gene (FLCN) cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterised. We identified plakophilin-4 (PKP4, p0071) as a potential novel folliculin interacting protein by yeast-2-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially colocalised at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalisation of E-cadherin in IMCD-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and regulation of RhoA signalling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-145. doi:1538-7445.AM2012-LB-145
Abstract LB-146: A study of p16 inactivation in lung cancer cell lines and tumor samples with a meta-analysis of the literature
Authors: Tam Kit W. , Zhang Wei , Soh Junichi , Chen Min , Sun Han , Stastny Victor , Thu Kelsie , Lam Wan , Gazdar Adi
Published: August 2012

Introduction: p16 inactivation is a common genetic alteration in lung adenocarcinoma and is often inactivated by homozygous deletion (HD) or methylation of promoter, or rarely by point mutation. While p16 methylation was reported to be linked to KRAS mutation and smoking, the association between specific p16 inactivation mechanism and other common genetic changes and smoking status remains controversial. In this study, we examined all of the three p16 inactivation mechanisms and correlated them with other common genetic chanes in lung adenocarcinoma: EGFR, KRAS, and LKB1, and smoking status. We also performed a meta-analysis to study the effect of smoking on p16 inactivation. Methods: We examined 40 cell lines and 45 tumor samples from patients with primary NSCLC, mostly adenocarcinoma. SNP and qPCR were used to detect HD. Methylation was determined by MSP analysis and mutation was identified by sequencing. We performed the meta-analysis by using Woolf's test to identify heterogeneity and followed by using a random-effect model. Results: The cell lines and tumor samples demonstrated similar results. No statistical differences were found between the two groups and the data were hence combined for analysis. p16 inactivation occurred at similar frequencies (54-57%) regardless of mutational status of EGFR, KRAS and LKB1 but the major mechanism of inactivation varied. Multivariate analysis showed that LKB1 inactivation was associated with p16 HD; p16 methylation was linked to KRAS mutation but was mutually exclusive with EGFR mutation. No significant association was established between any p16 inactivation mechanism and smoking status based on our data. HD was found to be the major mechanism of p16 inactivation among both smokers and never smokers although the rate of p16 methylation was higher in smokers. The result of our meta-analysis showed that p16 methylation was associated with smoking. The results from the literature were homogenous and the overall odds ratio from the meta-analysis was 2.01, which is significant at 0.05 level. Conclusions: p16 inactivation is a common event in lung adenocarcinoma and the inactivation mechanism of p16 is associated with other genetic changes and smoking status. Meta-analysis confirmed the association between p16 methylation and smoking. Our results indicate the presence of different pathways via p16 inactivation in the development of lung adenocarcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-146. doi:1538-7445.AM2012-LB-146
Abstract LB-148: The role of EZH2 in the regulation of the activity of matrix metalloproteinases in prostate cancer cells
Authors: Shin Yong Jae , Kim Jeong-Ho
Published: August 2012

Degradation of the extracellular matrix (ECM), a critical step in cancer metastasis, is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells, this balance is shifted towards MMPs, promoting ECM degradation. Here, we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMP genes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation of the TIMP gene promoters. Overexpression of EZH2 confers an invasive phenotype on benign prostate epithelial cells; however, this phenotype is suppressed by cooverexpression of TIMP3. EZH2 knockdown markedly reduces the proteolytic activity of MMP-9, thereby decreasing the invasive activity of prostate cancer cells. These results suggest that the transcriptional repression of the TIMP genes by EZH2 may be a major mechanism to shift the MMPs/TIMPs balance in favor of MMP activity and thus to promote ECM degradation and subsequent invasion of prostate cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-148. doi:1538-7445.AM2012-LB-148
Abstract LB-149: The OPCML tumor suppressor binds and negatively regulates the Axl receptor tyrosine kinase, sensitizing cells to foretinib in epithelial ovarian cancer (EOC)
Authors: Nawathe Sushmita Roy , Zanini Elisa , Hayward Giles , McKie Arthur B. , Gabra Hani
Published: August 2012

OPCML (opioid binding protein/cell adhesion like molecule) is a tumour suppressor gene that is silenced almost by somatic methylation in more than 80% of EOC cases, with loss of heterozygosity (LOH) occurring in 49% of cases [1,2]. OPCML binds a specific repertoire of receptor tyrosine kinases (RTKs) consisting of HER2, HER4 EphA2, FGFR1 and FGFR3. OPCML switches RTKs from clathrin to non clathrin dependent endocytosis, resulting in polyubiquitination of the RTK and targeting to the proteasome for destruction with associated signalling and growth inhibition [3]. We show that Axl, a TAM family RTK that is frequently activated in ovarian cancer [4], also interacts with and is downregulated by OPCML. Axl is downregulated following transient and stable OPCML expression in ovarian cancer lines SKOV3 and PEO1, with reciprocally increased Axl expression following stable knockdown of physiological OPCML in normal ovarian surface epithelial line OSE-C2. We performed co-IP and mammalian 2-hybrid (M 2-H) analysis between Axl and OPCML and determined that the Ig and FNIII domains are crucial to this interaction. We previously reported a complete loss of function proline 95 to arginine mutation [2]. Transfection of OPCMLWT protein in OPCML deficient cells promotes intercellular adhesion and adhesion to Collagen I and Fibronectin matrix, in association with Axl downregulation, In contrast OPCMLP95R transfection did not repress Axl and did not promote intercellular or cell to matrix adhesion. M 2-H data demonstrated that OPCMLP95R expression abolishes the interaction seen between OPCMLWT and Axl. We explored the impact of OPCML expression on cell signalling response to Foretinib (GSK1363089), a potent inhibitor of Axl [5]; OPCML led to a 3-fold sensitization to the tyrosine kinase inhibitor. The finding that OPCML, a frequently inactivated ovarian cancer tumor suppressor negatively regulates Axl, a frequently activated RTK in ovarian cancer and sensitises Axl positive OPCML deficient cells to Foretinib defines a promising predictive and therapeutic role for OPCML in ovarian cancer. References 1. Gabra et al (1996) Cancer Res 56: 950-954. 2. Sellar et al (2003) Nat Genet 34: 337-343. 3. McKie et al (Feb2012) Cancer Discovery (in press). 4. Rankin et al (2010) Cancer Res 70: 7570-7578 5. Liu et al. (2009) Cancer Res 69: 6871-6878.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-149. doi:1538-7445.AM2012-LB-149
Abstract LB-15: Alpha-2 Heremans Schmid glycoprotein (AHSG) promotes growth in head and neck squamous cell carcinoma
Authors: Thompson Pamela D. , Marshall Dana , Sawke Amos , Koumangoye Rainelli
Published: August 2012

Head and neck squamous cell carcinoma (HNSCC) is the eighth most common tumor in the world. In the United States, approximately 30,000 new cases of HNSCC are reported each year and 8,000 related deaths. HNSCC of the oral cavity is highly metastatic and has a recurrence rate of 20%-50%, with a dismal 5-year survival rate of ∼50%. There continues to be no consistent means for early detection or a reliable biomarker for the disease. This has contributed to the lack of improvement in the mortality rates of the disease, despite advances in delivery of treatment and surgical reconstruction. In a proteomic study seeking possible biomarkers for HNSCC, AHSG was detected at elevated serum levels. AHSG is a cell surface glycoprotein that is known to be synthesized by hepatocytes and found in most body fluids including serum. AHSG is also a negative acute phase function protein, which makes the results from the proteomic study, elevated levels of AHSG, intriguing. Our lab has recently shown that the lack of AHSG reduces tumor incidence and prolongs tumor latency. We now show that some HNSCC cell lines synthesize and secrete AHSG at both the message and protein levels. This is important as most cells acquire AHSG through uptake from serum. To our knowledge, this is the first report of AHSG synthesis and expression in HNSCC cell lines. We are currently depleting AHSG expression in these HNSCC cell lines in order to evaluate the effects of AHSG on the growth and metastatic properties (invasiveness and motility) in vitro and in vivo. <br

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-15. doi:1538-7445.AM2012-LB-15
Abstract LB-150: Binding selectivity of RNAs by eukaryotic initiation factor 3f in pancreatic cells
Authors: Uribe Diana J. , Wen Fushi , Shen Alex , Choi Andrw , Shi Jiaqi
Published: August 2012

Deregulation of translation is necessary for an increase in protein production that benefits cancer cells to increase proliferation and tumor growth. Eukaryotic initiation factor 3f (eIF3f), a subunit in the eIF3 protein complex, has been shown to be a regulator for translation initiation. Our group was the first to describe eIF3f as a tumor suppressor, where we show its expression is lost in melanoma and pancreatic tumor tissues. In this study, we show that eIF3f binds to various RNAs for genes involved in GTP-binding, RNA binding, RNA capping and other cellular processes, through the use of RNA-Immunoprecipitation assay followed by a microarray analysis, coined RIP-Chip in normal human pancreatic ductal epithelial (HPDE) cells. We confirmed the binding of eIF3f to these RNAs through RNA-Immunoprecipitation (RIP) assay using an antibody against eIF3f to pulldown of our sample RNA and visualized by RT-PCR analysis. To determine the role of this selective binding of eIF3f to these particular RNAs, we observed their expression in the HPDE cells, were eIF3f was stably knocked-down with lentivirus containing eIF3f-siRNA. Preliminary results demonstrate some correlation between the absence of eIF3f and the protein levels of the genes encoding for the eIF3f pulled-down RNA. Our results demonstrate that eIF3f may be involved in directly targeting specific oncogenic RNA sequences for degradation. Further investigations will be needed to elucidate the role of these genes and eIF3f expression in the transformation of pancreatic tissues to the aggressive phenotype of pancreatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-150. doi:1538-7445.AM2012-LB-150
Abstract LB-20: mTOR kinase inhibition relieves feedback suppression of receptor tyrosine kinase leading to a biphasic regulation of AKT signaling in breast cancer cells
Authors: Rodrik-Outmezguine Vanessa S. , Chandarlapaty Sarat , Pagano Nen C. , Poulikakos Poulikos I. , Scaltriti Maurizio , Baselga José , Guichard Sylvie , Rosen Neal
Published: August 2012

Activation of mitogenic signaling by receptor tyrosine kinases (RTKs) is regulated by negative feedback. Feedback is in part mediated by mTORC1/S6K dependent downregulation of IRS proteins which downregulate IGF/insulin receptor signaling. We have found that mTORC1 inhibitors or rapalogs relieve feedback inhibition of receptor tyrosine kinases and cause mTORC2-dependent phosphorylation of AKT S473 leading to activation of AKT kinase and signaling. This relief of feedback inhibition of mTORC2/AKT signaling has been associated with the limited clinical efficacy of these compounds. mTOR kinase inhibitors have now been developed to circumvent these problems; they block mTORC1 and mTORC2 and thus do not cause the mTORC2-dependent activation of AKT observed with the mTORC1 inhibitors. We now show, however, that these drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT S473 dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. Inhibition of mTOR kinase also relieves feedback suppression of RTKs leading to subsequent PI3K activation and rephosphorylation of AKT T308 sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound inhibition of mTORC1 and accumulation of activated AKT phosphorylated on T308 but not S473. The addition of RTK inhibitors can prevent this reactivation of AKT signaling and cause profound cell death and tumor regressions in vivo. These results highlight the possible need for combinatorial approaches to block feedback-regulated pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-20. doi:1538-7445.AM2012-LB-20
Abstract LB-203: Mathematical modeling of the dynamic interaction between cancers stem cells and non-stem cancer cells and its potential clinical implication
Authors: Liu Xinfeng , Kanojia Deepak , Lo Pang-Kuo , Chen Hexin
Published: August 2012

Cancer stem cells (CSCs) have been identified in primary breast cancer tissues and cell lines. The size of CSC population varies a lot among cancer tissues and cell lines but is associated with aggressiveness of breast cancer. Despite of the high tumor heterogeneity among different samples, CSCs isolated from either primary tissues or cell lines can rapidly regenerate the original heterogeneity and maintain a relatively constant proportion of CSCs. It remains largely unknown how the CSC population is maintained within a tumor. In this study, we develop a mathematical model to explore the key factors which control the size of CSC population during tumor cell growth both in vitro and in vivo. Our mathematic modeling and experimental data suggest that there is a negative feedback mechanism to control the balance between CSC and non-stem cancer cells. We further calculated how feedback sensitivities and robustness can be regulated by different intrinsic and extrinsic factors. As an example, our mathematical model predicts that overexpression of oncogeneHER2 leads to increase in the proportion of CSC via regulating the division mode or proliferation rate of CSCs. The prediction is supported by both in vitro cell culture results and the clinical data. We also show that enrichment of CSCs in suspension culture is due to both depletion of differentiated cancer cells and partial relieve of negative feedback. Mathematical modeling this culture condition reveals that the majority of tumporspheres are derived from progenitor cancer cells but not CSCs. Overall, these findings contribute to our understanding of cancer heterogeneity and may have significant implications in development and selection of anti-cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-203. doi:1538-7445.AM2012-LB-203