bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1961: Anti-endocrine therapy in breast cancerresistance can be circumvented by targeting ribonucleotide reductase M2 usingthe pharmaceutical agent Didox
Authors: Shah Khyati N. , Elford Howard L. , Faridi Jesika S.
Published: August 2015

Breast Cancers (BC) that are sensitive to anti-endocrine therapies such as tamoxifen develop resistance despite their initial favorable response. To gain mechanistic insight on endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of prolonged drug treatment and development of resistance. This resistance outcome is the result of the cancer cells invoking survival functions that are up-regulated leading to enhanced cell proliferation and cell motility networks. These networks were validated in a larger panel of breast cancer cells. Here, we report the activation of Growth factor signaling and IKK-βsignaling, NFκB signaling, in BC cells upon acquired resistance to ER blockade by anti-endocrine therapy. Moreover, ribonucleotide reductase (RRM2) was revealed as a key contributor to acquired tamoxifen resistance. An RNA interference screen demonstrated that suppression of RRM2 by the RR inhibitor Didox results in down-regulation of Her2 and EGFR, modulates ER-α signaling, alters NFκB activity and expression, and decreases anti-apoptotic pathways. Indeed, concomitant treatment of tamoxifen and Didox causes blockade of ER and RRM2 induced prolonged ER inhibition and severely impairs the growth of resistant tumor cells. Collectively, these results identify distinct mechanisms that mediate secondary resistance to anti-endocrine therapies, all of which reactivate RR signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that Didox and an anti-hormonal therapy should be tested in BC patients who become refractory to anti-endocrine therapies.

Citation Format: Khyati N. Shah, Howard L. Elford, Jesika S. Faridi. Anti-endocrine therapy in breast cancerresistance can be circumvented by targeting ribonucleotide reductase M2 usingthe pharmaceutical agent Didox. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1961. doi:10.1158/1538-7445.AM2015-1961
Abstract 1963: Analysis of aryl substitution and intramolecular ligand H-bonding in selective inhibitors of the MEK5/ERK5 cascade
Authors: Flaherty Patrick T. , Cavanaugh Jane C. , Gupta Mohit , Vechery Colin , Chakrabarty Suravi , Monlish Darlene , Wright Thomas
Published: August 2015

Mitogen Activated Protein Kinase (MAPK) pathways initiate with external signaling, proceed through a sequence of protein kinases, and alter cell proliferation, growth, and survival. MAPK pathways proceed through a well-ordered sequence but are interconnected and modified by the cell's internal needs. The MEK-catalyzed phosphorylation of its target ERK represents the most selective interaction of MAPK signaling cascades. The type III MEK1/2 inhibitor, Mekinist, was recently approved by the FDA for the treatment of unresectable or metastatic melanoma with BRAF V600E or V600K mutations. This success suggests inhibitors of other MEK/ERK cascades may be reasonable strategies for anti-cancer therapeutic strategies. Despite the likely involvement of MEK5 phosphorylation of ERK5 in early and triple negative breast cancers, prostate cancer, and renal cancer, little work has been focused on developing selective MEK5 inhibitors. We have previously described an initial series of anthranilic acid derivatives structurally derived from the known non-selective MEK inhibitor PD0325901. To explore structural requirements for selective inhibition of MEK5 versus MEK1/2, aryl substitution and the role of proposed internal hydrogen bonding of the ligand were examined with novel compounds. These were tested at 10 μM in MDB-MB-213 cells subsequently treated with EGF to induce MEK1/2 and MEK5 phosphorylation. Western blot analysis following electrophoresis using IR-tagged antibodies raised against pERK1/2, pERK5, ERK1/2, or ERK5 permitted determination of total and relative inhibition of MEK-mediated generation of pERK. Incubation with PD0325901 resulted in full suppression of both pERK1/2 and pERK5. For the aryl substitutions examined, omission of the halogens on the pendant phenyl group (SC-1-181) abolished MEK1/2 inhibition(0% decrease in pERK1/2) yet retained good MEK5 inhibition (82% decrease in pERK5). Disruption of an internal nitrogen-acyl hydrogen bond preferentially decreased MEK5 inhibition (SC-2-32: 40% and 16% decrease in pERK5 and pERK1/2 respectively). This establishes early evidence of significant variation in specificity of MEK type III binding sites that may be exploited as pharmacological tools and possibly as therapeutic agents for cancers overexpressing MEK5.

Citation Format: Patrick T. Flaherty, Jane C. Cavanaugh, Mohit Gupta, Colin Vechery, Suravi Chakrabarty, Darlene Monlish, Thomas Wright. Analysis of aryl substitution and intramolecular ligand H-bonding in selective inhibitors of the MEK5/ERK5 cascade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1963. doi:10.1158/1538-7445.AM2015-1963
Abstract 1967: Elucidation of signaling pathways that mediate gastrin-induced JNK activation and pGSK3β/Snail induction in gastric cancer cells
Authors: Basu Aninda , Sondarva Goutam , Santha Sreevidya , Rana Ajay , Rana Basabi
Published: August 2015

The gastrointestinal peptide hormone gastrin is known to regulate various cellular processes including proliferation, migration and metastasis in gastrointestinal cells. Our previous studies indicated that amidated gastrin (G-17) induced gastric cancer cell migration through two separate signaling axes. These revealed that inhibition of Glycogen Synthase Kinase-3β (GSK3β) was necessary to activate G17-induced migratory pathways in gastric cancer cells. Incubation of AGSE gastric cancer cells overexpressing CCK2 receptor (CCK2R) with G17 resulted in a dose and time dependent increase of GSK3βSer9 phosphorylation, indicative of an inhibition of the kinase. Treatment with G-17 was also associated with increased Snail expression, a downstream target of GSK3β and p21-activated protein kinases (PAKs). In a second pathway, G-17 also induced JNK phosphorylation through activation of Mixed Lineage Kinase 3 (MLK3). This axis also promotes gastric cancer cell migration via CCK2R. In the present study our goal was to determine whether PAKs, which are effectors for the Rho GTPases Cdc42 and Rac and are central players in cell proliferation, survival and motility, are involved in the G-17 effects on gastric cancer cells. Our studies showed that in AGSE cells when pretreated with a commercially available PAK4 inhibitor PF 3758309, G-17 was unable to phosphorylate GSK3β and G-17-induced Snail expression was markedly inhibited. G17-induced p-JNK expression was also significantly attenuated with PAK4 inhibitor pretreatment. The inhibitory effect was stronger when PAK4 inhibitor was used in combination with a pan-MLK inhibitor CEP11004. The involvement of PAK4 in G17-induced events were also confirmed by using the PAK4 siRNA. Knockdown of PAK4 inhibited G17-induced p-JNK expression which was more pronounced when both PAK4 and MLK3 were knocked down in AGSE gastric cancer cells. However, knocking down PAK4 alone does-not seem to inhibit G-17-induced p-GSK3β expression completely, which suggest the potential involvement of other PAK family members. Studies are currently underway to determine which PAK(s) is involved in mediating G17-pGSK3β-Snail axis and also to elucidate any crosstalk of PAK4 and MLK3 in this pathway. Elucidation of the signaling pathways that mediate G17-induced cellular events will be very important in designing therapeutic approaches in the future to restrict gastric cancer cell migration and metastasis.

Citation Format: Aninda Basu, Goutam Sondarva, Sreevidya Santha, Ajay Rana, Basabi Rana. Elucidation of signaling pathways that mediate gastrin-induced JNK activation and pGSK3β/Snail induction in gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1967. doi:10.1158/1538-7445.AM2015-1967
Abstract 1972: LIN28 gene targets are key mediators of breast cancer disease processes
Authors: Kinyamu Harriet K. , Yang Jun , Bennett Brian , Grimm Sara , Bushel Pierre , Archer Trevor
Published: August 2015

LIN28 is an evolutionally conserved RNA-binding protein with critical functions in developmental timing, pluripotency and reprogramming. At the molecular level, most studies indicate that LIN28 modulates gene networks and biological processes by negatively regulating miRNA biogenesis and post-transcriptionally regulating mRNA translation. In addition, increasing evidence suggests that LIN28 is an oncogene, promoting cell growth in various cancers, including breast cancer; however the molecular mechanisms underlying these effects are not known. To provide mechanistic insights into how LIN28 modulates the gene networks that control breast cancer disease processes, we integrate next generation technologies, biochemical approaches and computational analysis to identify LIN28 mRNA targets.

Our results demonstrated that endogenous LIN28 binds 843 mRNAs, ∼3.5% of the total human reference genome in breast cancer cells. Gene ontology analyses reveal that the genes targeted by LIN28 are enriched in those that control cell metabolism. Furthermore, using proteomics analysis, we identify heteronuclear ribonucleoprotein A1 (hnRNP A1), a protein with multiple roles in mRNA metabolism, as a LIN28 interacting partner. Importantly, hnRNP A1 is overexpressed and linked to increased cell proliferation and transformation in a wide variety of cancers, including breast cancer. Moreover, and consistent with the observation that majority of mRNAs bound by LIN28 are involved in metabolism, hnRNP A1 is known to regulate the expression of key enzymes that control metabolism in cancer cells. In-depth analysis of the transcriptomes of LIN28 and hNRNPA1 siRNA-treated cells reveal that these two proteins cooperate to regulate alternative splicing and the steady state mRNA expression of genes implicated in various aspects of breast cancer biology. Most notably, depleting LIN28 results in differential isoform expression of the ENAH gene, which is linked to breast cancer metastasis. In summary, by integrating genomic, biochemical and computational approaches, we identify a broad network of LIN28 targets that modulate cancer cell metabolism, cancer cell invasiveness and the immune response, processes that are hallmarks of breast cancer.

Citation Format: Harriet K. Kinyamu, Jun Yang, Brian Bennett, Sara Grimm, Pierre Bushel, Trevor Archer. LIN28 gene targets are key mediators of breast cancer disease processes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1972. doi:10.1158/1538-7445.AM2015-1972
Abstract 1975: Role and activation mechanisms of myeloid zinc finger-1 (MZF1) in ErbB2-induced breast cancer invasion
Authors: Kallunki Tuula , Brix Ditte M. , Clemmensen Knut Kristoffer B. , Tvingsholm Siri A. , Rafn Bo , Moreira José , Gromova Irina , Jäättelä Marja
Published: August 2015

ErbB2 overexpression gives rise to a highly invasive, aggressive phenotype associated with an increased activity of lysosomal cysteine cathepsins B and L and with a pericellular accumulation of lysosomes at the close vicinity of the cell membrane, enabling lysosomal exocytosis and secretion of lysosomal contents to the extracellular space. We recently showed that ErbB2 expression level correlates positively with cysteine cathepsin B and L levels in primary breast cancer and that ErbB2 regulates the expression of cathepsins B and L leading to their increased activity in several ErbB2-positive breast cancer cell lines. We found that the increased cysteine cathepsin activity is essential for the invasion of ErbB2 overexpressing breast cancer cells in 3-dimensional (3D) Matrigel cultures and that it is mediated by a novel signaling network that involves TGFβR, ERK2-MAPK, PAK4, PKCα and CDC42bpβ kinases and two transcription factors, Myeloid Zinc Finger 1 (MZF1) and ETS1, which regulate the ErbB2-induced expression of cathepsins B and L.

We currently work on understanding the mechanisms that increase the transcriptional activity of MZF1 in breast cancer. Thus far we have identified two basic mechanisms that can increase MZF1 target gene expression: cancer-induced, increased levels of MZF1 and ErbB2 signaling-induced, increased transcriptional activity of MZF1. Our results thus far show that the level of MZF1 can be directly downregulated by specific, non-coding RNAs. The cancer-induced increase in the MZF1 level is most likely due to changes in the expression of these non-coding RNAs. Preliminary data employing two-dimensional SDS-PAGE as well as the factual involvement of several kinases downstream of ErbB2 for its transcriptional activity, suggests that MZF1, a prominent phosphoprotein, is activated by phosphorylation in response to ErbB2 signaling. Towards this end we have set up various experiments to verify the phosphorylation of MZF1 in response to ErbB2 signaling. We now aim at the identification of the ErbB2-regulated phosphorylation sites. With this work we expect to collect more information about the molecular mechanisms underlying ErbB2-induced invasion. Increased knowledge of the mechanisms is expected to enable the development of novel therapies and biomarkers for aggressive, ErbB2-positive breast cancer.

Citation Format: Tuula Kallunki, Ditte M. Brix, Knut Kristoffer B. Clemmensen, Siri A. Tvingsholm, Bo Rafn, José Moreira, Irina Gromova, Marja Jäättelä. Role and activation mechanisms of myeloid zinc finger-1 (MZF1) in ErbB2-induced breast cancer invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1975. doi:10.1158/1538-7445.AM2015-1975
Abstract 1984: SOCS3 regulates IDO proteasomal degradation and inflammatory signaling in triple negative breast cancer
Authors: Ouzounova Maria , Lee EunMi , Novakovic Ena , Noonepalle Satish Kumar , Piranlioglu Raziye , Shi Huidong , Wicha Max , Korkaya Hasan
Published: August 2015

An intrinsic claudin-low subtype within the TNBC was recently identified based on low/absent expression of luminal differentiation markers, enrichment of epithelial-to-mesenchymal transition (EMT) and stem cell markers. Interleukin-6 (IL6), a potent pleiotropic cytokine, is induced in response to acute and chronic inflammation. IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the Suppressor of cytokine signaling 3 (SOCS3) mediated Stat3/NF-κB pathway. SOCS3 is a critical negative regulator of IL6-mediated pathways. We have showed that constitutive activation of inflammatory loop in transformed cells but not in their untransformed counterparts depends on proteolytic degradation of SOCS3. Several studies indicate that activation of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase (IDO) represents a key pathway suppressing anti-tumor immunity. Moreover a recent study in dendritic cells found that SOCS3 binds IDO via phosphotyrosine-containing peptides and target the IDO/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. IDO is shown to be constitutively expressed by many tumors. We analyzed a panel of breast cancer cell lines and found an inverse correlation between SOCS3 and IDO1 protein levels. Interestingly IDO protein and transcript levels were significantly increased in TNBC compared to luminal cell lines, thus confirming that IL6-JAK-STAT inflammatory loop is responsible for IDO regulation. Enforced expression of stable form of SOCS3, lacking the PEST and SOCS box domain in TNBC cell lines resulted in decreased expression of IDO. Therapeutic targeting of the IDO-IL6-SOCS3 inflammatory loop would revert the immune suppression mediated by IDO in this breast cancer subtype. These studies would provide a strong rationale for development of inflammatory pathway targeted agents for the treatment of TNBC, an aggressive disease that currently lacks molecularly targeted therapeutics.

Citation Format: Maria Ouzounova, EunMi Lee, Ena Novakovic, Satish Kumar Noonepalle, Raziye Piranlioglu, Huidong Shi, Max Wicha, Hasan Korkaya. SOCS3 regulates IDO proteasomal degradation and inflammatory signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1984. doi:10.1158/1538-7445.AM2015-1984
Abstract 1988: Role of N- ZEB1 in epithelial-mesenchymal transition (EMT)
Authors: Llorens M Candelaria , Cabanillas Ana M.
Published: August 2015

Background: ZEB1 (Zn Finger E-box Binding Homeobox) transcription factor is important in both development and disease, including the TGFβ-induced epithelial-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 contains 2 Zn finger domains (ZD1-ZD2) which bind DNA independently. ZEB1 expresses as two phosphorylated forms important for its biological role. We had characterized the role of phosphorylated C-term ZEB1. Aim: Our goal is to uncover the role of signaling pathway/s in the phosphorylation on N-term ZD1 (N-ZEB1) and characterize the functional role of N-ZEB1 in the process of EMT in mammary epithelial cells NMuMG. Results: A 728 aa fragment of N-ZEB1 was overexpressed in HEK293T cells and Immunoprecipitation with N-ZEB1 antibodies followed by western blot analysis with anti phospho kinases (AKT, MEK/ERK, PKC or PKA) substrate Antibodies. The results revealed that N-ZEB1 is solely phosphorylated by PI3K (AKT), MAPK and PKC pathways. IGF1-mediated phosphorylation can regulate nuclear localization of C-term ZEB1. Two N-ZEB1-GFP clones harboring first 490aa (eGFPZ1) and a 2nd clone of 130 aa, eGFPZ2 (included in the former) were transfected to CHO-K1 cells and treated with inhibitors and activators of kinase pathways for 15-60 minutes in serum free medium. Fluorescence microscopy techniques showed that IGF-1 induced relocation of a nuclear eGFPZ1 into the cytosol of CHO-K1 cells; on the contrary, eGFPZ2 was unresponsive to IGF-1. PMA/IONO (activator of PKC) treatment relocated both clones in the cytosol. Later on, NMuMG cells were used to investigate the role of N-ZEB1 in EMT. These epithelial cells, that can turn into mesenchymal type under TGFβ treatment, were stably transfected with eGFPZ2 or empty vector (control). The epithelial markers E-cadherin and actin assessed by Western blot and confocal microscopy were diminished or absent in eGFPZ2 cells. The activity of Matrix Metallo Proteases 2 was also increased in NMuMG-GFPZ2 cells. Also, proliferation, migration and invasion were significantly increased (P<0.001, P<0.001 and P<0.05, respectively) in NMuMG-GFPZ2 cells vs. controls. Taking together, these results show that the cells were induced to EMT by N-ZEB1.

Conclusions: We have identified a small fragment in N-ZEB1 that is able to elicit the EMT program in a TGFβ independent manner. In addition, changes in ZEB1 cellular localization induced by either PKC or IGF-1 could regulate this transcription factor biological activity.

Citation Format: M Candelaria Llorens, Ana M. Cabanillas. Role of N- ZEB1 in epithelial-mesenchymal transition (EMT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2015-1988
Abstract 199: Functional mechanism of hypoxia-inducible factor 1 alpha-mediated Dicer down-regulation
Authors: Lai Hui-Huang , Lyu Yu-Jhen , Li Jie-Ning , Su Jen-Liang , Chen Pai-Sheng
Published: August 2015

MicroRNAs (miRNAs) are generated by a multiple steps process that post-transcriptionally regulate the expression of target genes. Dysregulation of miRNAs is linked to human diseases including the development of human cancer. Abrogation of cellular global miRNAs maturation has been proposed to promote tumorigenesis and cancer metastasis. Hypoxia stress is the major cause of dysregulation of miRNAs and promotes tumor angiogenesis, metastasis and tumorigenesis which is a pivotal factor to switch on the cellular oncogenic features during tumor progression. Although hypoxia stress regulates expression of a subset of miRNAs, the upstream regulators controlling miRNAs regulation under hypoxia remain unclear. This study demonstrates the molecular mechanism of post-translational regulation of Dicer under hypoxia. Our results show that knockdown of hypoxia inducible factor 1 alpha (HIF-1α) up-regulates Dicer expression in colorectal cancer (CRC) cells. Overexpression of HIF-1α enhances ubiquitination of Dicer and subsequently promotes it recognition by autophagic receptor for autophagy-lysosomal degradation. We also find that HIF-1α enhances cancer cell migration by regulating Dicer-regulated miRNAs. In conclusion, we demonstrated that HIF-1α down-regulated Dicer and abolishes miRNA biogenesis to enhance cancer cell migration. This study may provide a potential therapeutic direction that targets the dysregulated miRNAs in tumor hypoxia for patients with poor prognosis and metastatic CRC.

Citation Format: Hui-Huang Lai, Yu-Jhen Lyu, Jie-Ning Li, Jen-Liang Su, Pai-Sheng Chen. Functional mechanism of hypoxia-inducible factor 1 alpha-mediated Dicer down-regulation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 199. doi:10.1158/1538-7445.AM2015-199
Abstract 1990: The key role of MECOM complex locus and its products in high-grade ovarian carcinoma pathogenesis and clinical outcomes
Authors: Batagov Arsen O. , Yenamandra Surya Pavan , Jenjaroenpun Piroon , Ivshina Anna V. , Kuznetsov Vladimir A.
Published: August 2015

The ecotropic virus integration site 1(Evi1) transcription factor, encoded in the MECOM complex locus, is implicated in several cancers, including ovarian cancer(OC). High-grade serous OC(HG-SOC) represents its most common and aggressive form. The pathobiological and clinical roles of MECOM and its products in HG-SOC are poorly understood.

Here, across all HG-SOC stages and grades, our meta-analysis demonstrated MECOM amplification in about 80% and EVI1 overexpression in 100% HG-SOC samples, relative to normal controls. We observed that Evi1 directly promotes double-strand DNA breaks repair via binding to DNA recombination/repair protein complexes, including a novel Rad50 isoform.

To elucidate the molecular basis and clinical significance of these findings, we studied genome-wide genome-wide Evi1-DNA binding, DNA copy number alterations, gene expression and proteome-scale Evi1 interactions, combining cell line and clinical meta-data and network/pathway analyses. Our experiments revealed major, previously unknown Evi1-binding DNA motifs specifying expression of 309 target genes, whose protein products were classified into eight functional and prognosis-significant modules implicated in embryogenesis, EMT, RNA metabolism, cancer cell survival, retinoic acid, anti-viral and therapeutic responses. The Evi1 pathway modules can collectively stratify the HG-SOC patients into four high-confidence post-surgery survival subgroups(P<1.0×10-50), over-performing previously reported HG-SOC predictors and clinical markers. This tumor stratification correlates with chemotherapy sensitivity and treatment outcomes.

Starting from early stages of HG-SOC, MECOM/Evi1 amplifies genomic instability and activates the Evi1 pathway that promotes most cancer hallmarks. Thus, MECOM/Evi1 and the Evi1 pathway provide mechanistic means for HG-SOC re-classification, introduces high-confidence early diagnostic and prognostic biomarkers and perspective therapeutic targets.

Citation Format: Arsen O. Batagov, Surya Pavan Yenamandra, Piroon Jenjaroenpun, Anna V. Ivshina, Vladimir A. Kuznetsov. The key role of MECOM complex locus and its products in high-grade ovarian carcinoma pathogenesis and clinical outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1990. doi:10.1158/1538-7445.AM2015-1990
Abstract 1995: Developing integrated single-cell metabolic/proteomic assays
Authors: Xue Min , Wei Wei , Su Yapeng , Shin Young Shik , Kim Jungwoo , Heath James R.
Published: August 2015

The technology boom of single-cell metabolomics and proteomics has led to exciting biological discoveries in the past decade. It has become widely acknowledged that resolving the intratumoral heterogeneity at single cell resolution can aid the understanding of cancer therapeutic response and progression, as well as facilitate the development of therapeutic interventions. Here we present an integrated assay that simultaneously quantifies the intracellular metabolites, glucose uptake, metabolic proteins and functional signaling proteins at the single-cell level. The measurement of metabolites is achieved through a series of competitive binding assays and is incorporated into the microfluidic-based single cell barcode chip platform. We demonstrate that this assay is capable of obtaining information from both protein and metabolic signaling pathways associated with tumor survival and progression, providing new insights into cancer heterogeneity and drug responsiveness.

Citation Format: Min Xue, Wei Wei, Yapeng Su, Young Shik Shin, Jungwoo Kim, James R. Heath. Developing integrated single-cell metabolic/proteomic assays. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1995. doi:10.1158/1538-7445.AM2015-1995
Abstract 200: Effects of H. pylori infection in regulation of miRNAs-associated with DNA mismatch repair
Authors: Santos Juliana C. , Brianti Mitsue , de Almeida Victor , Matheu Ander , Ribeiro Marcelo L.
Published: August 2015

It is well known that genetic and epigenetic inactivation of DNA mismatch repair (MMR) genes might leads to mutations in cancer-related genes and to cancer development. In gastric carcinogenesis, it has been shown that the infection by H. pylori, the major causative agent of gastric cancer, induces DNA damage and mutation and/or inhibits MMR DNA repair. Recently, it has been reported that miRNAs (miRs) have an important role in regulating genomic stability and MMR DNA repair. Thus, the aim of this work was to identify miRs regulating MMR pathway in H. pylori-associated gastric carcinogenesis

To evaluate the effects of H. pylori on miRs releated with MMR, AGS gastric carcinoma cell line and the H. pylori strains 26695, and P12 were used in co-culture experiments for 4, 8 and 12 h. As an initial screening, the AGS cells were co-cultured with H. pylori strain 26695 and subjected to Cancer PathwayFinder miRNA PCR Array (Qiagen) to determine the expression profile of 84 miRs. The expression levels of EXO1, MLH1, MSH2, MSH3, MSH6, POLD3, PMS1, and PMS2 were assessed by real-time PCR. Computational prediction of putative interaction of selected miRs to 3′UTR of MMR mRNAs were obtained from TargetScan, mirDB and MetaCore. The target validation was evaluated using mimics/inhibitors and luciferase gene reporter assays.

The results from the Cancer PathwayFinder PCR miRNA Array showded that H. pylori infection (strain 26695) modulates the expression of 23% (19/84) of the miRs after 4 and 12 h of infection, of which 89% were up-regulated and 11% down-regulated. Our data show that H. pylori infection significantly down-regulates the expression of all selected genes. The data generated from computational miR target prediction enriched with the gene expression fold induction indicated that 4 miRs (miR-150-5p, miR-155-5p, miR-3163, and miR-4775) modulated by H. pylori has at least one MMR target gene. Regarding the target validation, our results indicated that MLH1 and MSH2, are regulated by miR-150-5p. The miR-155-5p is involved in down-regulation of MLH1, MSH2, MSH3 and POLD3 expression. MSH3 is regulated by both miR-3163 and miR-4775. Briefly, this study shows that H. pylori impairs MMR DNA repair pathway thought a modulation of miR expression.

Citation Format: Juliana C. Santos, Mitsue Brianti, Victor de Almeida, Ander Matheu, Marcelo L. Ribeiro. Effects of H. pylori infection in regulation of miRNAs-associated with DNA mismatch repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 200. doi:10.1158/1538-7445.AM2015-200
Abstract 2000: Proteomic features of histological compartments in colorectal carcinoma
Authors: Slebos Robbert JC , Zimmerman Lisa J. , Manning Suzanne , Sanders Melinda E. , Shi Chanjuan , Washington M Kay , Liebler Daniel C.
Published: August 2015

Proteomic analysis of cancer tissues by mass spectrometry (MS) is complicated by the fact that histological context is lost in the analysis. Knowledge on the abundance of key proteins for the different histological compartments may allow an assessment of the contribution of each of these components to the proteomic profile obtained. We studied 18 colorectal carcinoma specimens and isolated carcinoma, necrosis, normal epithelium, stroma, smooth muscle, and lymphoid infiltrate compartments using laser capture microdissection (LCM). Proteins extracted from 60 LCM caps were subjected to short-stack, gel electrophoresis, in-gel trypsin digestion and analyzed on a Thermo-Fisher QExactive Orbitrap MS instrument. Data were searched using myrimatch and MS-GF+ on a RefSeq protein database and identified proteins were assembled using IDpicker 3. The analysis identified about 2,000 non-redundant proteins at a protein FDR<5%. Unsupervised cluster analysis of the most abundant proteins clustered each of the compartments together with few exceptions. Surprisingly, the necrotic compartment was almost identical from the carcinoma compartment, indicating the stability of the small peptides used for MS sequence identifications. Comparisons of spectral count data between each of the compartments and the remainder of the dataset by protein enrichment analyses was performed using the online tool www.webgestalt.org. The carcinoma compartment was enriched for proteins involved in nucleic acid and general metabolism processes. Smooth muscle was enriched for proteins involved in muscle contraction and cell adhesion. The lymphoid infiltrates were enriched for chromatin and nucleosome proteins, possibly indicating the relative contribution of nuclear proteins from these small cells. The stromal component was enriched for extracellular matrix proteins and proteins involved in glycosylation processes. Using data from ProteinAtlas.org, we confirmed histological associations of the most prominent protein markers with their respective histological component. For instance, COL12A1 was highly expressed in the stromal compartment, while SMTN was highly specific for smooth muscle. Expression levels characterizing each of the histological components can thus be described in protein signatures that can serve as a measure of the relative contribution of each of the components in the complex composition of a colorectal carcinoma sample.

Supported by NIH/NCI grant U24CA159988.

Citation Format: Robbert JC Slebos, Lisa J. Zimmerman, Suzanne Manning, Melinda E. Sanders, Chanjuan Shi, M Kay Washington, Daniel C. Liebler. Proteomic features of histological compartments in colorectal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2000. doi:10.1158/1538-7445.AM2015-2000
Abstract 2005: Quantitative screening of serum protein biomarkers by reversed phase protein serum arrays
Authors: Kuang Zhizhou , Huang Ruochun , Luo Shuhong , Chen Yun-Ru , Lv Zhiqiang , Zhang Zhuo , Huang Ruo-Pan
Published: August 2015

Protein biomarkers can be key indicators for cancer diagnosis, prediction, and selection of successful cancer treatments. Protein biomarkers can be identified by 2 methods: antibody arrays which profile protein expression levels from a patient's serum, or reversed phase protein arrays to measure single or limited sets of proteins from many patients’ sera. The reversed phase protein arrays have been widely used for detection of signaling molecules in cell lysates, however, this approach has been difficult to adapt to serum samples. Several years ago, we developed a sensitive method called the enhanced protein array to quantitatively measure serum protein levels from large numbers of patient samples. Here, we further refine the technology on several fronts: 1. simplifying the experimental procedure; 2. optimizing multiple parameters to make the assay more robust; 3. establishing a method for more accurate quantification. Using this technology, we quantitatively measured the protein expression levels of Nidogen-1 and CEACAM-1 from 164 serum samples from hepatocellular carcinoma (HCC) and equal number of control and found that both Nidogen-1 and CEACAM-1 expression levels are significantly increased in HCC patients. Our work reveals promise for using reversed phase protein serum arrays for biomarker discovery and validation.

Citation Format: Zhizhou Kuang, Ruochun Huang, Shuhong Luo, Yun-Ru Chen, Zhiqiang Lv, Zhuo Zhang, Ruo-Pan Huang. Quantitative screening of serum protein biomarkers by reversed phase protein serum arrays. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2015-2005
Abstract 2013: SYNTHETIC LECTINS FOR THE DETERMINATION OF BREAST CANCER SUBTYPE
Authors: O'Connell Kathleen M. , Gatrone Erin E. , Veldkamp Anna A. , Lavigne John J.
Published: August 2015

Glycans represent promising biomarkers for cancer diagnostics. Aberrant glycosylation is known to occur in cancer cells and changes continue to occur as the disease progresses. This study sought to determine if glycan ‘fingerprints’ can distinguish between differing cell types associated with cancer; specifically for the early detection and subtyping of breast cancers. Synthetic Lectins (SLs) are small peptides functionalized with boronic acids that selectively bind to glycans. Different SLs, with differing peptide backbones, acting as an array can be used to study glycans and generate glycan ‘fingerprints’ of analytes.

An array of nine SLs, present on resin beads, were incubated with fluorescently labeled membrane proteins extracted from 1.) Breast cancer cell lines 2.) Matched normal and cancerous breast tissue from patients. Binding intensity information was obtained for each sample and analyzed statistically. Linear Discriminant Analysis was used to build two models, of breast cancer cell lines, based on a) metastatic potential and b) breast cancer subtype.

Leave one out analysis determined that the models could distinguish between healthy/cancerous non-metastatic/cancerous metastatic cells with 98% accuracy and between breast cancer subtypes with 99% accuracy. Classification accuracy based on metastatic potential was validated using clinical specimens. N = 3 matched normal and cancerous tissues were analyzed and classification accuracies of 97% were obtained.

We have developed an array, which distinguishes between healthy and cancerous cells as well as breast cancer subtypes with high accuracy. Work to expand validation to a larger group of patient samples as well as testing the array using patient serum is ongoing.

Citation Format: Kathleen M. O'Connell, Erin E. Gatrone, Anna A. Veldkamp, John J. Lavigne. SYNTHETIC LECTINS FOR THE DETERMINATION OF BREAST CANCER SUBTYPE. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2013. doi:10.1158/1538-7445.AM2015-2013
Abstract 2018: Elucidating the therapeutic potential of midbrain pathways that impact breast cancer
Authors: Chandra Madeline , Lee John , Sheets Patrick L. , Prosperi Jenifer R.
Published: August 2015

Most chemotherapeutic agents for breast cancer have variable efficacy and produce unwanted side effects. While much focus is placed on dissecting the molecular mechanisms contributing to tumor development, the potential role of the brain in regulating tumor progression has not been described in detail. Previous evidence has shown that midbrain activity affects tumor growth and progression. Stimulation of the midbrain periaqueductal grey (PAG) increases metastasis in rats injected with Walker-256 carcinosarcoma cells, and the growth of prolactin-dependent mammary tumors. These findings suggest midbrain pathways play a prominent role in tumorigenesis. These studies, however, did not address the therapeutic potential of blocking PAG output for slowing tumor progression. Therefore, we are studying the mechanisms regulating midbrain-induced tumorigenesis and the therapeutic potential of altering midbrain activity to reduce metastatic activity and tumor growth.

A common challenge in neuroscience has been identifying and manipulating specific neuronal populations in the brain. However, significant advances have been made in the field of optogenetics, the integration of optics and genetics, which allows precise control of specific neural pathways in vivo using light. Optogenetics is gaining considerable clinical interest as a novel strategy for treating human neurological disorders due to success observed in rodent and non-primate human models. Therefore, optogenetic alteration of PAG activity for treating breast cancer is plausible. Non-pharmacological, therapeutic strategies aimed at manipulating circuit activity to treat mood and anxiety disorders are already creating enthusiasm. Given that many of these strategies likely impact PAG activity, there is great potential for manipulating the same pathways to treat breast cancer. Before these types of novel strategies can be implemented, we must first delineate the biology of these circuits and their therapeutic potential in animal models of cancer.

Thus far, we have identified that muscimol inhibition of PAG activity decreases breast cancer progression in a transgenic mouse model. We are continuing to investigate the distinct input/output mechanisms of the PAG that could be modified to treat breast cancer. Future studies have the potential to reveal novel avenues for targeting and preventing the development of breast cancer via the manipulability of specific neural pathways.

Note: This abstract was not presented at the meeting.

Citation Format: Madeline Chandra, John Lee, Patrick L. Sheets, Jenifer R. Prosperi. Elucidating the therapeutic potential of midbrain pathways that impact breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2018. doi:10.1158/1538-7445.AM2015-2018
Abstract 2030: FRK regulates glioma cell progression by promoting N-cadherin/β-catenin complex formation
Authors: Shi Qiong , Song Xu , Yan Haoping , Wang Jun , Zhang Weijian , Hu Jinxia , Zhou Xiuping , Yu Rutong
Published: August 2015

Fyn-related kinase (FRK), a member of Src-related tyrosine kinases, is recently reported to function as a potent tumor suppressor in several cancer types. Our previous studies has also shown that FRK over-expression inhibited the migration/invasion, proliferation and promoted apoptosis of glioma cells. However, the mechanism of FRK on glioma cell migration/invasion, proliferation and apoptosis is still not clear. In this study, we found that FRK over-expression increased the protein level of N-cadherin, but not that of E-cadherin. Meanwhile, FRK over-expression promoted β-catenin translocation to the plasma membrane, where it formed complex with N-cadherin, while inhibited β-catenin translocation to the nucleus. In addition, down-regulation of N-cadherin by siRNA promoted the migration/ invasion proliferation and inhibited apoptosis of glioma U251 and U87 cells. Interestingly, N-cadherin down-regulation abolished the effect of FRK on glioma cell migration/invasion, proliferation and apoptosis. In summary, these results indicate that FRK regulates glioma cell progression by promoting N-cadherin/β-catenin complex formation.

Citation Format: Qiong Shi, Xu Song, Haoping Yan, Jun Wang, Weijian Zhang, Jinxia Hu, Xiuping Zhou, Rutong Yu. FRK regulates glioma cell progression by promoting N-cadherin/β-catenin complex formation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2030. doi:10.1158/1538-7445.AM2015-2030
Abstract 204: Single-molecule localization microscopy analysis of a cancer metastasis-specific miRNA on the nanoscale
Authors: Abba Mohammed , Oleksiuk Olga , Tezcan Kerem , Schaufler Wladimir , Bestvater Felix , Altevogt Peter , Hafner Mathias , Cremer Christoph , Allgayer Heike
Published: August 2015

We propose a novel approach for the detection of small non-coding RNAs in cells by Single Molecule Localization Microscopy (SMLM). We describe a modified and upgraded SMLM-setup and apply this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA molecules in fixed cells with a localization accuracy of 10-15 nm, is able to quantify numbers and analyse clustering and localization in particular subcellular sites, including exosomes. We compared metastatic-site derived and primary site derived human adenocarcinoma cultured colorectal cancer (CRC) cells, and (as a proof of principle) evaluated the metastatically relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in all cell lines was very heterogeneous with the largest subpopulation of optically acquired low-metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells.

Citation Format: Mohammed Abba, Olga Oleksiuk, Kerem Tezcan, Wladimir Schaufler, Felix Bestvater, Peter Altevogt, Mathias Hafner, Christoph Cremer, Heike Allgayer. Single-molecule localization microscopy analysis of a cancer metastasis-specific miRNA on the nanoscale. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 204. doi:10.1158/1538-7445.AM2015-204
Abstract 2041: Quantitative phosphoproteomic analysis of EphA2 signaling in PC-3 prostate cancer cells
Authors: Offenhäuser Carolin , Dave Keyur A. , Jayakody Buddhika A. , Gorman Jeffrey J. , Boyd Andrew W.
Published: August 2015

The receptor tyrosine kinase EphA2 is frequently upregulated in cancer, where it plays an intriguing dichotomous role that may lend itself to therapeutic intervention. However, much remains to be understood about how ephrin-mediated activation of EphA2 signaling can reverse its tumour-promoting ligand-independent effects and instead inhibit migration and invasion. We used quantitative phosphoproteomics to analyze EphA2 signaling in PC-3 prostate cancer cells with the aim to further elucidate its downstream signaling and pinpoint phosphorylation-dependent cellular processes that may explain its dichotomous role. Stable isotope labeling by amino acids in cell culture (SILAC) combined with liquid chromatography-coupled mass spectrometry (LC-MS/MS) was used to quantify changes in relative phosphosite abundance in response to EphA2 activation. We identified 113 significantly regulated phosphosites comprising 73 phosphoproteins, including many novel and several previously described downstream effector proteins of EphA2 signaling. More than two thirds of the regulated phosphosites showed reduced phosphorylation in response to EphA2 activation. This is consistent with EphA2 activation inhibiting oncogenic signaling pathways, including Akt and ERK-MAPK signaling. Protein association analysis using the STRING database showed that a large proportion of these differentially regulated proteins are downstream targets of Akt and ERK-MAPK, respectively. Gene ontology enrichment analysis found our dataset to be strongly enriched in proteins associated with the cytoskeleton and its regulation. Overall, our data suggest that diminished phosphorylation of cytoskeletal regulators downstream of Akt and ERK-MAPK signaling may be a major mechanism by which stimulation of EphA2 can alter cell morphology and inhibit cell motility.

Citation Format: Carolin Offenhäuser, Keyur A. Dave, Buddhika A. Jayakody, Jeffrey J. Gorman, Andrew W. Boyd. Quantitative phosphoproteomic analysis of EphA2 signaling in PC-3 prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2041. doi:10.1158/1538-7445.AM2015-2041