bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract PR13: Systems and structural biology approaches to elucidate new effectors in KRAS mutant tumors
Authors: Broyde Joshua , Simpson David , Wah David A. , Giorgi Federico M. , Petrey Donald , Alvarez Mariano J. , Silkov Antonina , Lachmann Alexander , Hill David E. , Vidal Marc , Jackson Peter , Honig Barry , Sweet-Cordero Alejandro , Califano Andrea
Published: May 2016

Activated KRAS interacts directly and indirectly with numerous downstream effector pathways that are still elusive or poorly characterized. Furthermore, it has become clear over the past few years that the KRAS regulatory gene network (interactome) is highly cell-context dependent and thus tumor specific. We sought to computationally elucidate novel KRAS effectors, modulators, and interactors in Lung Adenocarcinomas (LUAD) and Colon Adenocarcinomas (COAD) by integrating computational and biochemical reverse engineering methods. Specifically, results from ARACNe and MINDy based analysis of KRAS networks were combined with Protein-protein interaction (PPI) predictions based on the PrePPI algorithm. These algorithms detect, respectively, transcriptional, post-translational, and protein-protein interactions respectively. The MARINa/VIPER algorithm was used to analyze the networks resulting from these analyses to identify key modulators and effectors of KRAS activity, including modulators harboring genetic alterations that phenocopy aberrant KRAS activity. In addition, we collaborated with the Jackson (Stanford) and Vidal (Harvard) labs to include high-throughput tandem affinity mass spectrometry data and yeast 2 hybrid screens in these analyses. Validation of computationally inferred effectors, using pooled shRNA silencing assays in primary lung cancer cells isolated from a KrasLSL/p53f/f mouse model of LUAD validated novel genes as likely KRASmut effectors and putative synthetic lethal genes. At least two hairpins against RNF43, E4F1, HMCN1, MYO9A, RHOT2, SETD1B and MAP3K2 scored in this assay. These results suggest that KRAS network analyses using computational methods can identify novel members of the KRAS interactome.

Citation Format: Joshua Broyde, David Simpson, David A. Wah, Federico M. Giorgi, Donald Petrey, Mariano J. Alvarez, Antonina Silkov, Alexander Lachmann, David E. Hill, Marc Vidal, Peter Jackson, Barry Honig, Alejandro Sweet-Cordero, Andrea Califano. Systems and structural biology approaches to elucidate new effectors in KRAS mutant tumors. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR13.
Abstract A01: Innate lymphoid cells (ILCs) in murine melanoma: A new paradigm in cancer immunity
Authors: Christianson Blake , Walker James , Kocherla Marsha , Qin Xu , Yu Jack C. , Baban Babak
Published: May 2016

Introduction: Innate lymphoid cells (ILCs), have emerged as novel important immune effector cells, playing a critical role also in tumor immune-surveillance. Recently, it was shown that the number of ILCs in patients with different types of cancer is significantly reduced after chemotherapy/radiotherapy as compared to healthy donors. However, so far, no data on ILC frequency, subset distribution and function in patients with melanoma are available. Therefore, we analyzed the frequency, subset distribution and function of ILCs in a murine model of melanoma with/without treatment, a parameter that is of paramount importance to understand the role of ILCs during melanoma and to assess the impact of current treatments on this type of cancer.

Methods: C57/BL6 mice were injected with B16F10 murine melanoma cell line (1 x 106 cells/mouse, subcutaneous). Tumor growth monitored. Once tumors reached 12mm in size, the mice were sacrificed and tumor cells subjected to flow-cytometry and cell culture to identify the ILC subclasses in the tumor.

Results: Our findings establish, for the first time in melanoma at diagnosis, base-line reference values for ILC frequency and function, as well as the ILC subtype distribution.

Conclusion: Our studies extend previously published work on changes in innate lymphoid cells. Our findings will help better understanding of tumor microenvironment and its interaction with immune system. These values indicate the potential of ILCs as crucial players in the treatment of cancer, specifically melanoma.

Citation Format: Blake Christianson, James Walker, Marsha Kocherla, Xu Qin, Jack C. Yu, Babak Baban. Innate lymphoid cells (ILCs) in murine melanoma: A new paradigm in cancer immunity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A01.
Abstract A04: Hyperactive mTOR induces neuroendocrine differentiation in LNCaP prostate cancer cell with concurrent upregulation of interferon regulatory factor 1
Authors: Kanayama Mayuko , Hayano Toshiya , Maeda Tatsuya , Horie Shigeo , Aiba Atsu
Published: May 2016

The PI3K-Akt-mTOR pathway is frequently activated in prostate cancer. However, the precise function of mTOR in prostate cancer progression is yet to be elucidated. Thus, in this study, we performed a gain-of-function analysis by establishing human prostate cancer LNCaP stable line that expresses hyperactive mTOR (LNCaP-mTOR). Hyperactive mTOR induced neuroendocrine differentiation (NED) that can be suppressed by an mTOR inhibitor, rapamycin. Subsequent comprehensive mass spectrometric analysis in conjunction with cascade analysis indicates that the members of interferon regulator factor (IRF) family, especially IRF1, are key transcription factors in NED of LNCaP-mTOR. Aside from its major role in immune response, IRF1 is known to be a tumor suppressor. To further investigate the function of IRF1 in NED induced by hyperactive mTOR, we disrupted the IRF1 gene in LNCaP-mTOR by CRISPR/Cas system. The disruption of the IRF1 gene resulted in an acceleration of an NED-associated morphological change, elevation of neuroendocrine cell marker, neuron specific enolase, and a partial recovery of growth arrest. These results suggest that IRF1 partially suppresses NED at downstream of hyperactive mTOR. Together, our results indicate that two seemingly contradictory events, NED and the induction of IRF1, are induced by hyperactive mTOR, giving rise to a notion that pharmacological inhibition of mTOR can be a “double-edged sword” for prostate cancer treatments.

Citation Format: Mayuko Kanayama, Toshiya Hayano, Tatsuya Maeda, Shigeo Horie, Atsu Aiba. Hyperactive mTOR induces neuroendocrine differentiation in LNCaP prostate cancer cell with concurrent upregulation of interferon regulatory factor 1. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A04.
Abstract A05: NKG2D and CD28 receptors differentially activate mTOR to alter murine effector CD8+ T cell differentiation
Authors: McQueen Bryan , Trace Kelsey , Whitman Emily , Barber Amorette
Published: May 2016

Memory CD8+ T cells are an essential component of anti-tumor and anti-viral immunity. Activation of the mammalian/mechanistic target of rapamycin (mTOR) pathway has been implicated in regulating the differentiation of effector and memory T cells. However, the mechanisms that control mTOR activity during immunity to tumors and infections are not well known. Activation of costimulatory receptors, including CD28 and NKG2D, activate PI3K and subsequently may activate the mTOR pathway in CD8+ T cells. Therefore this study compared the activation of the mTOR signaling pathway after costimulation through CD28 or NKG2D receptors in murine effector CD8+ T cells. Compared to CD28 costimulation, activation through CD3 and NKG2D receptors had weaker activation of mTORc1 as shown by decreased phosphorylation of mTORc1 targets S6K1, ribosomal protein S6, and 4EBP-1. NKG2D costimulation also showed increased gene expression of TSC2, a negative regulator of mTORc1, whereas CD28 costimulation increased gene expression of Rheb, an activator of mTORc1, and HIF-1α and VEGFα, pro-angiogenic factors downstream of mTORc1. Strong mTORc1 activation in CD28-costimulated cells also increased expression of transcription factors that support effector cell differentiation, namely T-bet, and BLIMP1, whereas low levels of mTORc1 activation allowed for the expression of Eomes and BCL6 during NKG2D stimulation, and increased expression of central memory markers CD62L and CD127. These data show that compared to CD28, costimulation through the NKG2D receptor leads to the differential activation of the mTOR signaling pathway and potentially supports memory CD8+ T cell differentiation.

Citation Format: Bryan McQueen, Kelsey Trace, Emily Whitman, Amorette Barber. NKG2D and CD28 receptors differentially activate mTOR to alter murine effector CD8+ T cell differentiation. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A05.
Abstract A12: Chronic Coartem treatment and hepatocarcinogenesis in male Wistars rat
Authors: Aloba Martins A. , Olotu Fisayo A. , Odunola Oyeronke A. , Gbadegesin Michael A. , Owumi Solomon E.
Published: May 2016

Coartem (CA) artemether/lumefantrine) is a combination therapy widely used in sub-Saharan Africa in managing uncomplicated Plasmodium falciparum-induced malaria. According to manufacturer’s literature, no long-term carcinogenicity studies have been carried out on CA. Therapy with CA places a burden on hepatic metabolism, accumulation of its metabolites (dihyroartemisinin, desbutyl-lumefantrine) induces hepatotoxicity, altering hepatic functions and formation of DNA adducts that may have implication in hepatocarcinogenesis.

Wistar rats (n=20) were treated with CA (1, 3 and 5mg/kg) twice daily, thrice weekly for 40 consecutive days exhibited symptoms such as sneezing, lethargy with no effects on final body weight, relative liver weight or mortality during the study. Histopathological examination of treated rats’ liver revealed mild portal cellular infiltration in the group administered 1mg/kg Coartem. There was mild Kupffer cells hyperplasia in the rats that received 3mg/kg Coartem and mild to moderate portal mononuclear cellular infiltration in the group that received 5mg/kg. In addition, there was severe portal congestion and hyperplasia of the bile duct, which made the portal triad very prominent. These observations were in contrast to the negative control group with no macro /microscopic visible lesion.

Serum levels of hepatotoxicity markers (γ-glutamyl-, alanine amino-, aspartate amino- transferases and alkaline phosphatase) in CA treated group showed no significant difference (p>0.05) compared with controls. Means of urea, creatinine (in whole blood), albumin and total protein (liver homogenate) of the CA administered group were not different (p>0.05) compared with controls. CA treatment dose dependently increased the formation of micronucleated polychromatic erythrocytes (mPCEs) in rat bone marrow cells. Although this was not statistically significant (p>0.05) for the various doses of CA administered when compared with control groups.

Taken together, CA treatment (1, 3 and 5mg/kg body) for a period of 40 days had no hepatotoxic and nephrotoxic effect. Although prolonged administration may likely be toxic, Kupffer cell hyperplasia mild to moderate portal cellular infiltration of hepatic sinusoid may exacerbate drug-induced hepatocarcinogenesis over a prolonged treatment period as observed in this study. Longer-term chronic studies for 80-120 are being investigated.

Citation Format: Martins A. Aloba, Fisayo A. Olotu, Oyeronke A. Odunola, Michael A. Gbadegesin, Solomon E. Owumi. Chronic Coartem treatment and hepatocarcinogenesis in male Wistars rat. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A12.
Abstract A23: The crosstalk between developmental endothelial locus 1 (DEL-1) and cancer stem cells in murine melanoma: A potential modality of local control
Authors: Walker James , kocherla marsha , christianson Blake , qin Xu , yu Jack C. , baban Babak
Published: May 2016

Introduction: Developmental endothelial locus-1 is a homeostatic factor in the central nervous system limiting neuroinflammation and demyelination. Del-1 has an endothelial cell–restricted pattern, particularly in the embryo. However, our data now for the first time provide evidence that melanoma cells express Del-1. The relatively newly described cancer stem cells (CSCs) are tumor-initiating cells (TICs) that often survive therapy and give rise to second-line tumors by self-renewal and differentiation into multiple cell types. CSCs are proposed to persist in tumors as a distinct population and to cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially those with metastatic disease. Methods: C57/BL6 mice were injected with B16F10 murine melanoma cell line (1 x 106 cells/mouse, subcutaneous). Tumor growth monitored. Controls (Group 2) received vehicle only (PBS). Once the first tumor reached 12mm in size, the mice were sacrificed and tumor cells subjected to flow-cytometry, and statistical analysis, and p-values <0.05 considered significant. our novel findings suggest a regulatory role for Del-1 in cancer. Alteration of inflammatory indices, apoptosis, and necroptosis with ultimate affects on tumor size and growth were all may be potentially regulated partially by a Del-1 dependent mechanism. Results: Our data showed for the first time that DEL-1 was expressed by CSCs as well as typic non stem cells tumor cells in a murine melanoma model. These DEL-1-expressing CSCs may be palying a role in tumor angiogenesis. Further, DEL-1 is capable of suppressing T lymphocyte infiltration into the tumor microenvironment.

our novel findings suggest a regulatory role for Del-1 in cancer. Alteration of inflammatory indices, apoptosis, and necroptosis with ultimate affects on tumor size and growth (by affecting Cancer Stem Cells) were all may be potentially regulated partially by a Del-1 dependent mechanism. Conclusion: Our data propose a novel hypothesis that development of tumor and CSCs activity may be affected by angiogenic and regulatory nature of Del-1. Furthermore, our results suggest a therapeutic role for Del-1 as a novel modality in cancer treatment.

Citation Format: James Walker, marsha kocherla, Blake christianson, Xu qin, Jack C. yu, Babak baban. The crosstalk between developmental endothelial locus 1 (DEL-1) and cancer stem cells in murine melanoma: A potential modality of local control. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A23.
Abstract A26: Cofactor of BRCA1 as a modulator of hepatocellular carcinoma growth and migration
Authors: Zeneini Eman El , Amleh Asma
Published: May 2016

COBRA1 is one of the four subunits that make up the Negative Elongation Factor Complex (NELF) which is involved in the stalling of RNA polymerase II early during transcription elongation. As such, COBRA1 is able to regulate a substantial number of genes involved in a number of pathways, including cell cycle control, metabolism, cell proliferation and DNA repair. Even though it has been extensively studied over the years, the majority of these studies have mostly reported physiological roles. In the field of cancer, COBRA1 remains largely unexplored. The aim of our study was to investigate the functional role of COBRA1 in the tumorigenesis of hepatocellular carcinoma (HCC). We investigated the gene expression pattern of COBRA1 in HCC tumors using the publicly available Oncomine Cancer Microarray Database. Results from three different microarray datasets reveal the frequent overexpression of COBRA1 in HCC tumors versus their normal counterparts. To elucidate the biological significance for this overexpression in HCC, RNA interference was used to silence the expression of COBRA1 in the well differentiated cell line, HepG2. The silencing efficiency was confirmed by both RT-PCR and Western blot analysis. Interestingly, knockdown of COBRA1 resulted in a significant decrease in cell proliferation, accompanied by a concomitant decrease in the expression of the proliferation marker, Ki-67. A scratch wound healing assay revealed a significant decrease in the migratory potential of the HepG2 cells in culture as well upon COBRA1 knockdown. The silencing of COBRA1 was associated with a significant decrease in the expression of survivin, suggesting that survivin might be one of the mechanisms by which COBRA1 mediates its role in the tumorigenicity of HCC. Collectively, data findings presented here highlight an oncogenic role for COBRA1 in hepatocellular carcinoma. To the best of our knowledge, our study provides evidence for the first time to support a positive role for COBRA1 in the growth and migration of HCC.

Note: This abstract was not presented at the conference.

Citation Format: Eman El Zeneini, Asma Amleh. Cofactor of BRCA1 as a modulator of hepatocellular carcinoma growth and migration. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A26.
Abstract A28: New data supporting the possible evolutionary role of tumors: Evolutionary novel genes expressed in fish tumors determine progressive evolutionary characters in humans
Authors: Kozlov Andrei , Matyunina Ekaterina , Emelyanov Alexander
Published: May 2016

The origin of evolutionary novel genes in DNA of germ cells is connected with the origin and evolution of the novel organismal functions and the increase in morphological complexity of multicellular organisms. Novel gene acquire new or altered functions. Here we describe that evolutionary novel genes, expressed predominantly in transgenic fish tumors after their induced regression, determine progressive evolutionary characters and are conserved in humans. We used a sample of genes, which were activated in inducible krasV12 transgenic zebrafish tumors according to the results of RNASeq data. Genes from our sample were expressed after HCC regression upon kras inactivation. The search of orthologs by different tools, such as blastx and psiblast, with e-value cut off not less than 10-3 and query coverage more than 50%, discovered that considerable proportion of tumor-specifically expressed genes are evolutionary novel, i.e. their orthologs are not found in lamprey. 226 of these novel genes have orthologs in humans. Gene ontology (GO) data were used to analyze their functions. According to the results of GO some of the genes have functions not characteristic to fish. For example, genes ccdc40, lmx1ba, lepa, tgfbr2 and irs1 acquired functions in lung, embryonic utera, mammalian placenta and mammary gland, i.e. organs originated in taxa higher than fish. Among known functions of the human orthologues of evolutionary novel genes of zebrafish (vs. lamprey) there are some important morphogenetic functions. A large proportion of genes from our sample are connected with retina type eye, heart, lung and brain development.

Note: This abstract was not presented at the conference.

Citation Format: Andrei Kozlov, Ekaterina Matyunina, Alexander Emelyanov. New data supporting the possible evolutionary role of tumors: Evolutionary novel genes expressed in fish tumors determine progressive evolutionary characters in humans. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A28.
Abstract B07: Tumor promoting activity of SLC13A3 in metastatic prostate cells
Authors: Zhunussova Aigul , Irgebayeva Zhanar , Sen Bhaswati , Friedman Leah , Ossikbayeva Saniya , Tuleukhanov Sultan , Brooks Ari , Sensenig Richard , Orynbayeva Zulfiya
Published: May 2016

Solute carriers are membrane proteins that play an important role in cancer homeostasis by supplying cancer cells with essential building blocks and signaling molecules. Different types of membrane transporters were shown to be overexpressed in tumors in support of tumor growth and survival. Due to high affinity interaction with anticancer drugs they can also significantly contribute to the rapid chemoresistance. There is a limited number of studies on the role of solute carriers in reprogramming of cancer metabolism and signaling, especially in metastatic prostate cancer.

Our study enabled us to establish the identity of the prostate cancer solute carrier that supports uptake of succinate and possibly similar energy rich metabolites from tumor microenvironment to promote an elevated energy metabolism of cancer cells. We demonstrated that prostate metastatic DU145 cells acquire Na+-dependent dycarboxylic acid carrier SLC13A3 but normal prostate PrEC cells do not express this protein. An important finding was that only at acidic pH 6.8 prostate cancer cells are capable of consuming exogenous succinate, while physiological pH 7.4 was not favorable for this process. Importantly, succinate transported by SLC13A3 not only accelerates the oxidative phosphorylation of prostate cancer mitochondria but it is also known to stabilize onco-factor HIF-1α. The HIF-involved signaling pathways have been linked to prostate tumorigenesis and chemoresistance and therefore, targeting these pathways by inhibiting cancer specific SLC13A3 has great potential for clinical benefit. In addition, the mechanism of succinate influx via SLC13A3 transporter in metastatic prostate cancer cells could also yield a novel metabolic onco-marker and has the potential to be used for imaging-based diagnostics to detect non-glycolytic tumors.

Citation Format: Aigul Zhunussova, Zhanar Irgebayeva, Bhaswati Sen, Leah Friedman, Saniya Ossikbayeva, Sultan Tuleukhanov, Ari Brooks, Richard Sensenig, Zulfiya Orynbayeva. Tumor promoting activity of SLC13A3 in metastatic prostate cells. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B07.
Abstract B09: Differential effect of microenvironment on tumor phenotype using an immature T cell lymphoma mouse model
Authors: Lucas Philip J. , Gress Ronald E.
Published: May 2016

Human T cell lymphomas of an immature phenotype with mediastinal origin are classified as T cell lymphoblastic lymphomas (T-LBL) and comprise 85-90% of all lymphoblastic lymphomas. Treatment of T-LBL can be challenging because of the metastasizing nature of the disease, which commonly localizes to the bone marrow and can be found in other organs such as liver and spleen. We have developed a mouse T-LBL model by establishing a cell line from a spontaneous mediastinal tumor found in a T cell receptor (TCR) transgenic mouse strain. The resulting T-LBL like cell line, JMR4, has a known TCR (2C), known alloreactivity (H-2Ld), and known stimulating self-peptide (SIYR). JMR4 has an immature T cell phenotype, which includes a mixture of CD4+/CD8+, CD4lo/CD8+, CD4+/CD8lo and CD4+ cells. Other T cell markers, such as CD5, CD24 and CD69 are expressed at various levels depending on the phenotypic subset. The 2C TCR, which can be detected using the 1B2 idiotypic mAb, has been shown to be functional using flow cytometric analysis following TCR stimulation using peptide loaded thymic stromal feeder cells. In addition, JMR4 can be phenotypically altered (CD4/CD8/CD5/CD69 expression levels) differentially in vitro using the bone marrow stromal cell line OP9 and OP9DL, suggesting that it has pluripotency. Finally, when JMR4 is injected into sub-lethally irradiated mice, it develops into a multi-organ lymphoma. Interestingly, the phenotype of the JMR4 cells, based on CD4 and CD8 expression levels, is differentially regulated depending on organ type and MHC of the host, making JMR4 an ideal model to study the complex interactions of a tumor with its microenvironment. Understanding the mechanisms involved in tumor microenvironment (TME) induced changes to tumor cells should be important in understanding metastatic tumor progression and in designing new treatment regimens.

Citation Format: Philip J. Lucas, Ronald E. Gress. Differential effect of microenvironment on tumor phenotype using an immature T cell lymphoma mouse model. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B09.
Abstract B10: Cyclin D1 restrains oncogene-induced autophagy via phosphorylation of LKB1
Authors: Casimiro Mathew , Sante Gabriele Di , Loro Emanuele , Pestell Timothy , Bisetto Sara , Velasco-Velázquez Marco , Jiao Xuanmao , Li Zhiping , Wang Chenguang , Ly Daniel , Zheng Bin , Che-Hung Shen , Ertel Adam , Pestell Richard G.
Published: May 2016

Cyclin D1 is an important molecular driver of many human cancers. The cyclin D1 gene is amplified or overexpressed in up to half of human breast cancers and mammary-targeted overexpression of cyclin D1 is sufficient to induce mammary tumorigenesis in mice. Cyclin D1 encodes the regulatory subunit of the cyclin-dependent kinase (CDK) holoenzyme that phosphorylates several substrates including the retinoblastoma protein (pRb) to advance the G1S cell cycle checkpoint and promote DNA synthesis. Recent studies have implicated cell cycle regulators that govern G1S transition, in regulating cellular metabolism. Cyclin D1 phosphorylates and inactivates a nuclear encoded protein essential for mitochondrial biogenesis, Nrf1. In this manner cyclin D1 coordinates both nuclear and mitochondrial functions. Consistent with cyclin D1-dependent inhibition of mitochondrial biogenesis, pRB promotes and E2F-1 inhibits mitochondrial biogenesis. In addition to inhibiting mitochondrial biogenesis, cyclin D1 inhibits mitochondrial membrane potential, which may be in part due to binding VDAC.

Autophagy is a catabolic pathway activated in response to cellular stress such as oxidative or nutritional stress. It mitigates cellular damage via removal of insoluble and damaged proteins, lipids and organelles. Autophagy is enhanced by the phosphorylated and activated form of 5’-AMP-activated protein kinase (AMPK). We initiated the following study to directly test the effect of cyclin D1 has on cellular metabolism and autophagy. Herein, endogenous cyclin D1 restrained autophagy in breast cancer cells in tissue culture and in the mammary gland of mice. Using mammary epithelial-targeted deletion of cyclin D1 we show cyclin D1 restrains autophagy by reducing AMPK activation. Cyclin D1 may serve to couple cellular proliferation to cellular energy homeostasis.

Citation Format: Mathew Casimiro, Gabriele Di Sante, Emanuele Loro, Timothy Pestell, Sara Bisetto, Marco Velasco-Velázquez, Xuanmao Jiao, Zhiping Li, Chenguang Wang, Daniel Ly, Bin Zheng, Shen Che-Hung, Adam Ertel, Richard G. Pestell. Cyclin D1 restrains oncogene-induced autophagy via phosphorylation of LKB1. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B10.
Abstract B20: Single-cell profiling of EGFR-regulated protein changes involved in cell cycle, cell proliferation and apoptosis
Authors: Holcomb Ilona , Dakshinamoorthy Gajalakshmi , Liu Benjamin , Unger Marc , Ramakrishnan Ramesh , Gong Haibiao
Published: May 2016

Single-cell molecular analysis has become increasingly important in biomedical research, especially in the field of cancer research, where cell heterogeneity is a characteristic feature. We developed a multiplexed single-cell protein detection assay for the C1™ system that can isolate and process 96 individual cells in parallel. The assay uses oligonucleotide-conjugated antibody binders and monitors the specific oligonucleotide levels instead of protein levels. The resulting DNA amplicon is detected by qPCR on the Biomark™ HD system. Data are analyzed using Singular™ Analysis Toolset. A total of 50 antibody binders were developed for this assay to target proteins related to apoptosis, cell cycle, cell proliferation, tumor suppressors, biomarkers, stem cells, growth factors and proteins associated with specific cancers, such as breast and prostate cancers. The assay can be performed in a 48-plex format which yields over 4,600 data points in two days. Assay sensitivity and specificity were assessed using recombinant proteins and various cell lysates. To demonstrate the usefulness of this assay, a panel of antibody binders was selected to profile the changes of proteins involved in cell cycle, cell proliferation and apoptosis in single cells. We treated cells that overexpress epidermal growth factor receptor (EGFR) with either the EGF ligand or the gefitinib receptor inhibitor and analyzed the changes in protein levels. Cells without EGFR expression were used as the control. EGFR is a member of ErbB family of Type I tyrosine kinases. EGFR overexpression and its mutations are involved in a variety of cancers. While much effort has been made to investigate the consequences of EGFR activation or inhibition, hardly any information is available regarding how EGFR regulation changes protein expression in single cells. Single-cell analysis—determining protein changes and analyzing the correlation between different proteins at the single-cell level—yields valuable information that is not achievable using bulk-cell analysis.

Citation Format: Ilona Holcomb, Gajalakshmi Dakshinamoorthy, Benjamin Liu, Marc Unger, Ramesh Ramakrishnan, Haibiao Gong. Single-cell profiling of EGFR-regulated protein changes involved in cell cycle, cell proliferation and apoptosis. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B20.
Abstract B22: Screening and validation of combination therapy in T cell leukemia
Authors: Mues Marsilius , Lindenbergh Marthe F. , McManus Michael T. , Roose Jeroen P.
Published: May 2016

Oncogenic Ras mutations occur in more than 30% of metastatic cancers. There are no effective inhibitors to block oncogenic K- or N-RAS, however, blockade of Ras’ downstream RAF-MEK-ERK and PI3K-Akt-mTOR-S6 signaling cascades is currently being explored as therapy in clinical trials through the use of potent kinase inhibitors. Nonetheless, many cancers have a complex make-up of cooperating oncogenic lesions and it is therefore questionable if inhibition of single molecules in pathways will provide sufficient, long-term efficacy. To explore effective combination therapies to treat Ras-driven cancers we chose synthetic lethal screens (SLS), focusing on PI3K due to its frequent involvement in oncogenic signaling. We performed a set of SLS on T cell leukemia (T-ALL) cell lines combining specific PI3K inhibitors with high coverage shRNA libraries, initially aiming at kinases. We employed the UCSF EXPANDed RNAi library resource, which dramatically improves RNAi screening compared to commercial resources. This ultra-complex shRNA library targets each gene with >25 independent shRNAs, thus drastically minimizing experimental noise and allowing us to overcome both the common problems of high false-negative rates and high false-positive rates. Using deep sequencing of shRNAs recovered from our screening samples, we identified shRNAs that synergize with inhibitors in these drop-out SLS conditions and which represent key molecules for the survival of T-ALL lines. In addition, we also identified enriched shRNAs that confer inhibitor resistance or enhanced proliferation. To verify our screening results we used combinations of chemical inhibitors, targeting PI3K and molecules identified by our screens. To date, all inhibitor combinations tested caused strong synthetic lethality in T-ALL cells, confirming the robustness of the ultra-complex shRNA libraries used for SLS. The most promising results will be further verified in vivo, and we will expand our efforts to different leukemia and also solid cancer lines. Altogether, our high-troughput platform is a promising approach to identify effective partners for already established inhibitors, leading to novel combination therapies for the treatment of cancer.

Citation Format: Marsilius Mues, Marthe F. Lindenbergh, Michael T. McManus, Jeroen P. Roose. Screening and validation of combination therapy in T cell leukemia. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B22.
Abstract B23: Chemoproteomic profiling of native kinases during the treatment of cells with kinase inhibitors
Authors: Nomanbhoy Tyzoon K. , Brown Heidi E. , Wu Jiangyue , Vogeti Subha , Aban Arwin , Grant Wendy , Senait Alemayehu , Wu Shuzhen , Dias Christa , Sharma Geeta
Published: May 2016

Kinases are an important class of targets for a number of therapeutic indications, and currently, kinase inhibitors constitute a significant fraction of recently approved drugs, particularly for cancer. Using a probe-based chemoproteomics platform, we have demonstrated the utility of the platform to monitor the interactions between inhibitors and native kinases. Herein, we profile kinase inhibitors in cell lines either sensitive or resistant to the compound for various periods of time. We observe that at early time points, inhibitor profiles for a compound are qualitatively similar in both the sensitive and resistant cells, suggesting the compound is capable of engaging its direct targets in both cell types. In contrast, at later time points, additional kinases are observed affected by compound treatment. We infer that these kinases do not directly bind the compound but rather reflect secondary effects that result from the engagement of the primary targets (pathway effects). Significantly, the pathway effects are only observed in the sensitive cells and not the resistant cells. Kinases affected through these pathway effects include kinases known to be critical for cell cycle progression including CDK1, Aurora A and B, PLK1, and MASTL. Finally, we demonstrate that during the treatment of tumor-bearing mice with an inhibitor, in addition to inhibition of the direct targets, pathway effects are observed recapitulating the results from the cell-based studies.

Citation Format: Tyzoon K. Nomanbhoy, Heidi E. Brown, Jiangyue Wu, Subha Vogeti, Arwin Aban, Wendy Grant, Alemayehu Senait, Shuzhen Wu, Christa Dias, Geeta Sharma. Chemoproteomic profiling of native kinases during the treatment of cells with kinase inhibitors. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B23.
Abstract B31: Establishment of a 5-fluorouracil-resistant esophageal squamous cell carcinoma cells with dihydropyrimidine dehydrogenase gene amplification
Authors: Kikuchi Osamu , Baba Kiichiro , Ohashi Shinya , Muto Manabu
Published: May 2016

5-Fluorouracil (5-FU) is the key drug for the treatment of esophageal squamous cell carcinoma (ESCC), however, resistance to 5-FU remains a critical limitation to the clinical use. To clarify the mechanisms of 5-FU resistance in ESCC, we have established a 5-FU-resistant ESCC cell line TE-5R from parental TE-5 cells, by treatment with the step-wise continuous increasing concentrations of 5-FU. The half maximal inhibitory concentration of 5-FU showed that TE-5R cells were 15.6-fold resistant to 5-FU in comparison with TE-5 cells. Intracellular 5-FU concentration was significantly lower in TE-5R cells than in TE-5 cells. TE-5R cells showed regional copy number amplification of chromosome 1p including the DPYD gene, as well as high mRNA and protein expressions of dihydropyrimidine dehydrogenase (DPD), an enzyme involved in 5-FU degradation, whereas copy number of the other 5-FU metabolism-related genes in TE-5R cells did not changed from TE-5 cells. Gimeracil, a DPD inhibitor, sharply restored 5-FU resistance and increased intracellular 5-FU concentration in TE-5R cells. Next, to investigate whether other 5-FU metabolism-related genes contributed to 5-FU resistance in TE-5R cells, we added microarray gene expression assay and no significant differences in the expression of 5-FU metabolism-related genes were detected. These results indicate that 5-FU resistance in TE-5R cells is based on a DPD-dependent rapid degradation of 5-FU. Taken together, we established novel 5-FU-resistant TE-5R cells, which may have obtained 5-FU resistance with DPD copy number amplification and consequent overexpression, providing a critical insight to explore a new strategy against 5-FU-resistant ESCC using DPD inhibitor.

Citation Format: Osamu Kikuchi, Kiichiro Baba, Shinya Ohashi, Manabu Muto. Establishment of a 5-fluorouracil-resistant esophageal squamous cell carcinoma cells with dihydropyrimidine dehydrogenase gene amplification. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B31.
Abstract B43: Quantitative PCR and in-situ-hybridization analysis to determine tissue concentration and localization of MRX34
Authors: Martin Desiree , Kelnar Kevin , Bader Andreas G.
Published: May 2016

MRX34, a liposomal microRNA (miRNA)-based therapy for cancer, has recently entered clinical trials as a potential first clinical candidate in its class. MRX34 is a mimic of naturally occurring microRNA-34 (miR-34) encapsulated in a liposomal nanoparticle formulation. Preclinical animal studies have shown that intravenous delivery of MRX34 can increase miR-34 levels in liver tumor cells more than 100-fold when analyzing whole-tissue RNA extracts by quantitative PCR (qPCR). MRX34-induced tumor regression has enhanced the survival of mice by inhibiting the growth of both hepatic and non-hepatic tumors. We have established a chromogenic in situ hybridization (CISH) method to track the cellular location of the miR-34 mimic in tissues after systemic MRX34 administration. In contrast to conventional biodistribution approaches that cannot distinguish between spatial differences in tissue accumulation, CISH in conjunction with microscopy allows the detection of the miRNA mimic on a cellular level and may provide new insights into cell-type specific accumulation. Here, we evaluated the localization and tissue concentrations of systemically delivered MRX34 in mice bearing orthotopic Huh7 tumors by CISH followed by image correlation to a formalin-fixed and paraffin-embedded tissue equivalent, and isolation-free qPCR analysis. Our results show that the CISH procedure is a reproducible and robust assay capable of over 2 logs of miR-34 detection when correlated to qPCR data from matching micro-dissected samples. Systemic MRX34 delivery leads to accumulation of miR-34 mimics in tumor cells with Cmax reached approximately 2 hrs post dosing. In addition, the CISH data reveal how biodistribution data generated from whole-tissue extracts can be biased due to minute impurities and suggests that these methods should be used in combination with CISH for an accurate assessment of tissue concentrations.

Citation Format: Desiree Martin, Kevin Kelnar, Andreas G. Bader. Quantitative PCR and in-situ-hybridization analysis to determine tissue concentration and localization of MRX34. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B43.
Abstract B46: Rac1 inhibition as a therapeutic strategy in breast cancer
Authors: Senderoff Dana , Azzi Georges , Goka Erik , Lippman Marc
Published: May 2016

Over the past few decades, targeted therapies have accounted for most of the therapeutic advances in breast cancer. The purpose of this approach is to discover new, actionable therapeutic strategies for breast cancer. We previously used a forward genomic screening approach to identify novel genes capable of cooperating with HER2, and found that loss of HACE1 was prevalent in breast cancer, identifying it as a tumor suppressor gene. HACE1 had significantly lower expression in breast cancer patients compared to normals. HACE1 is an E3 ubiquitin ligase that ubiquitinates Rac1, marking it for degradation. Rac1, a Rho GTPase, is a central component of key signaling pathways in breast cancer. While we identified HACE1 in a HER2 cooperating screen, the observation that HACE1 levels were consistently low across different subtypes of breast cancer led us to suggest that Rac1 is commonly hyperactivated in breast cancer and therefore has potential as a therapeutic target. We tested a Rac1 inhibitor, EHT 1864, on 8 different breast cancer cell lines, starting with an initial soft agar assay screen for sensitivity versus resistance and found that 4 cells lines were sensitive to the Rac1 inhibitor in soft agar cloning assays (MCF7, T47D, BT549, and BT474), while 4 other cell lines were resistant (MDA231, MDA436, MDA468, HS578T). These results were confirmed in 2D proliferation assays. The majority of the cell lines that were resistant to Rac1 inhibition overexpressed EGFR, suggesting that overexpression of EGFR confers resistance to Rac1 inhibition via the MAP Kinase pathway. Isogenic cell lines (MCF7) that express multiple activating proteins of the MAPK signaling pathway including EGFR, HER2, and MEK were treated with EHT1864 to determine if MAPK signaling was capable of overcoming Rac1 inhibition. Interestingly, the expression of EGFR conferred resistance to the Rac1 inhibitor. We further showed that molecular knockdown of EGFR in HS578T cells (EGFR-overexpressing, Rac1 inhibitor-resistant cells) resensitizes this cell line to Rac1 inhibition. Pharmalogical inhibition of EGFR using Gefitinib, an EGFR tyrosine kinase inhibitor, demonstrated the same resensitization to the Rac1 inhibitor. Our results suggest that Rac1 inhibition may be a therapeutic strategy in the treatment of breast cancer, as a single agent alone or in the case of EGFR overexpressing (triple-negative) breast cancers, in combination with EGFR targeted therapies.

Citation Format: Dana Senderoff, Georges Azzi, Erik Goka, Marc Lippman. Rac1 inhibition as a therapeutic strategy in breast cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B46.
Abstract IA04: Multifaceted functions of the PD-1 pathway
Authors: Sharpe Arlene H.
Published: May 2016

The PD-1 co-inhibitory pathway is a key regulator of the critical balance between stimulatory and inhibitory signals needed for effective immune responses to microbes and tumors, as well self-tolerance. This pathway consists of the inhibitory Programmed Death (PD)-1 receptor and its two ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273). PD-1 is inducibly expressed on T cells, and its ligands have distinct expression patterns. PD-L1 is broadly expressed on hematopoietic and non-hematopoietic cells, whereas PD-L2 is inducibly expressed primarily on DCs and macrophages. In this talk, mechanisms by which the PD-1:PD-L pathway regulates optimal immune homeostasis and tolerance will be discussed. In addition, the functions of PD-1 pathway in controlling humoral immune responses will be discussed, and compared to the functions of CTLA-4 in regulating humoral immunity.

Citation Format: Arlene H. Sharpe. Multifaceted functions of the PD-1 pathway. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA04.
Abstract IA06: Angiogenesis revisited: Role and (therapeutic) implications of endothelial metabolism
Authors: Carmeliet Peter
Published: May 2016

The past 40 years of research in the angiogenesis field have focused on identifying genetic signals such as VEGF and Notch, which determine vessel sprouting. However, the role and therapeutic potential of targeting endothelial cell (EC) metabolism have been largely overlooked. We have recently reported that ECs are glycolysis addicted and that glycolysis importantly co-determinine vessel sprouting downstream of VEGF and other pro-angiogenic signals. In addition, we documented that ECs are rather unique in utilizing fatty acid-derived carbons for the de novo synthesis of deoxyribonucleotides for DNA synthesis during EC proliferation when vessels sprout. Moreover, targeting (blocking) glycolysis and fatty acid oxidation inhibit pathological angiogenesis and induce tumor vessel normalization (thereby reducing metastasis and improving chemotherapy), suggesting that these metabolic pathways are new targets for anti-angiogenic drug development without evoking systemic side effects. Furthermore, lymphatic ECs differ from other EC subtypes in their metabolic requirements for lymphangigoenesis. Since many of these metabolic targets are pharmacologically druggable, these metabolic pathways represent a new promising target for therapeutic anti-angiogenesis.

Citation Format: Peter Carmeliet. Angiogenesis revisited: Role and (therapeutic) implications of endothelial metabolism. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA06.