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BackgroundPancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.MethodsscRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo.ResultsDistinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat.ConclusionRef-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.

Background Approximately two thirds of patients with localized triple-negative breast cancer (TNBC) harbor residual disease (RD) after neoadjuvant chemotherapy (NAC) and have a high risk-of-recurrence. Targeted therapeutic development for TNBC is of primary significance as no targeted therapies are clinically indicated for this aggressive subset. In view of this, we conducted a comprehensive molecular analysis and correlated molecular features of chemorefractory RD tumors with recurrence for the purpose of guiding downstream therapeutic development. Methods We assembled DNA and RNA sequencing data from RD tumors as well as pre-operative biopsies, lymphocytic infiltrate, and survival data as part of a molecular correlative to a phase II post-neoadjuvant clinical trial. Matched somatic mutation, gene expression, and lymphocytic infiltrate were assessed before and after chemotherapy to understand how tumors evolve during chemotherapy. Kaplan-Meier survival analyses were conducted categorizing cancers with TP53 mutations by the degree of loss as well as by the copy number of a locus of 18q corresponding to the SMAD2, SMAD4, and SMAD7 genes. Results Analysis of matched somatic genomes pre-/post-NAC revealed chaotic acquisition of copy gains and losses including amplification of prominent oncogenes. In contrast, significant gains in deleterious point mutations and insertion/deletions were not observed. No trends between clonal evolution and recurrence were identified. Gene expression data from paired biopsies revealed enrichment of actionable regulators of stem cell-like behavior and depletion of immune signaling, which was corroborated by total lymphocytic infiltrate, but was not associated with recurrence. Novel characterization of TP53 mutation revealed prognostically relevant subgroups, which were linked to MYC-driven transcriptional amplification. Finally, somatic gains in 18q were associated with poor prognosis, likely driven by putative upregulation of TGFß signaling through the signal transducer SMAD2. Conclusions We conclude TNBCs are dynamic during chemotherapy, demonstrating complex plasticity in subclonal diversity, stem-like qualities, and immune depletion, but somatic alterations of TP53/MYC and TGFß signaling in RD samples are prominent drivers of recurrence, representing high-yield targets for additional interrogation. Electronic supplementary material The online version of this article (10.1186/s13058-019-1171-7) contains supplementary material, which is available to authorized users.

Hypoxia is arguably the first recognized cancer microenvironment hallmark and affects virtually all cellular populations present in tumors. During the past decades the complex adaptive cellular responses to oxygen deprivation have been largely elucidated, raising hope for new anti cancer agents. Despite undeniable preclinical progress, therapeutic targeting of tumor hypoxia is yet to transition from bench to bedside. This review focuses on new pharmacological agents that exploit tumor hypoxia or interfere with hypoxia signaling and discusses strategies to maximize their therapeutic impact.

Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. Hypoxic regions within tumors represent sources of aggressiveness and resistance to therapy. Although long non-coding RNAs (lncRNAs) are increasingly recognized as major gene expression regulators, their regulation and function following hypoxic stress are still largely unexplored. Combining profiling studies on early-stage lung adenocarcinoma (LUAD) biopsies and on A549 LUAD cell lines cultured in normoxic or hypoxic conditions, we identified a subset of lncRNAs that are both correlated with the hypoxic status of tumors and regulated by hypoxia in vitro. We focused on a new transcript, Nuclear LUCAT1 (NLUCAT1), which is strongly upregulated by hypoxia in vitro and correlated with hypoxic markers and poor prognosis in LUADs. Full molecular characterization showed that NLUCAT1 is a large nuclear transcript composed of six exons and mainly regulated by NF-κB and NRF2 transcription factors. CRISPR-Cas9-mediated invalidation of NLUCAT1 revealed a decrease in proliferative and invasive properties, an increase in oxidative stress and a higher sensitivity to cisplatin-induced apoptosis. Transcriptome analysis of NLUCAT1-deficient cells showed repressed genes within the antioxidant and/or cisplatin-response networks. We demonstrated that the concomitant knockdown of four of these genes products, GPX2, GLRX, ALDH3A1, and PDK4, significantly increased ROS-dependent caspase activation, thus partially mimicking the consequences of NLUCAT1 inactivation in LUAD cells. Overall, we demonstrate that NLUCAT1 contributes to an aggressive phenotype in early-stage hypoxic tumors, suggesting it may represent a new potential therapeutic target in LUADs.

Triple-Negative Breast Cancer (TNBC) accounts for approximately one-fifth of breast cancer incidence but disproportionately high mortality. Two-thirds of early-stage TNBCs are resistant to pre-surgical chemotherapy and highly prone to relapse within 3 years. Morever, no advanced therapies are indicated for patients with these cancers. We have embarked on a comprehensive genomic analysis of chemoresistant TNBC to gain an in-depth understanding of molecular entities driving chemoresistance and relapse. By collecting somatic mutation and copy number, RNA-sequencing, and outcome data in the context of a phase II post-neoadjuvant clinical trial, we have uncovered several molecular mechanisms behind these aggressive cancers. Through the analysis of matched pairs sampled before and after chemotherapy, we have discovered multiple means by which tumors are able to overcome the effects of chemotherapy including clonal evolution of high-level oncogene amplification, repression of the in situ immune system, and upregulation of the stem cell-related MEK-ERK and JAK-STAT pathways. Investigation into factors related to prognosis revealed important correlations between relapse and immune and JAK-STAT signaling. Finally, using a novel method of demarcating loss-of-function of p53, which we have termed graduated inactivation, we discovered additional associations between p53 loss and relapse, mortality, and MYC signalling. Citation Format: Hancock BA, Chen Y-H, Solzak JP, Ahmad MN, Wedge DC, Brinza D, Scafe C, Veitch J, Gottimukkala R, Short W, Atale RV, Ivan M, Badve SS, Schneider BP, Miller KD, Radovich M. Molecular regulators of resistance and relapse in chemorefractory triple-negative breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-04.

Elevated O-GlcNAcylation is associated with disease states such as diabetes and cancer. O-GlcNAc transferase (OGT) is elevated in multiple cancers and inhibition of this enzyme genetically or pharmacologically inhibits oncogenesis. Here we show that O-GlcNAcylation modulates lipid metabolism in cancer cells. OGT regulates expression of the master lipid regulator the transcription factor sterol regulatory element binding protein 1 (SREBP-1) and its transcriptional targets both in cancer and lipogenic tissue. OGT regulates SREBP-1 protein expression via AMP-activated protein kinase (AMPK). SREBP-1 is critical for OGT-mediated regulation of cell survival and of lipid synthesis, as overexpression of SREBP-1 rescues lipogenic defects associated with OGT suppression, and tumor growth in vitro and in vivo. These results unravel a previously unidentified link between O-GlcNAcylation, lipid metabolism and the regulation of SREBP-1 in cancer and suggests a crucial role for O-GlcNAc signaling in transducing nutritional state to regulate lipid metabolism.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

International audience; Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. Hypoxic regions within tumors represent sources of aggressiveness and resistance to therapy. Although long non-coding RNAs (lncRNAs) are increasingly recognized as major gene expression regulators, their regulation and function following hypoxic stress are still largely unexplored. Combining profiling studies on early-stage lung adenocarcinoma (LUAD) biopsies and on A549 LUAD cell lines cultured in normoxic or hypoxic conditions, we identified a subset of lncRNAs that are both correlated with the hypoxic status of tumors and regulated by hypoxia in vitro. We focused on a new transcript, NLUCAT1, which is strongly upregulated by hypoxia in vitro and correlated with hypoxic markers and poor prognosis in LUADs. Full molecular characterization showed that NLUCAT1 is a large nuclear transcript composed of six exons and mainly regulated by NF-κB and NRF2 transcription factors. CRISPR-Cas9-mediated invalidation of NLUCAT1 revealed a decrease in proliferative and invasive properties, an increase in oxidative stress and a higher sensitivity to cisplatin-induced apoptosis. Transcriptome analysis of NLUCAT1-deficient cells showed repressed genes within the antioxidant and/or cisplatin-response networks. We demonstrated that the concomitant knockdown of four of these genes products, GPX2, GLRX, ALDH3A1, and PDK4, significantly increased ROS-dependent caspase activation, thus partially mimicking the consequences of NLUCAT1 inactivation in LUAD cells. Overall, we demonstrate that NLUCAT1 contributes to an aggressive phenotype in early-stage hypoxic tumors, suggesting it may represent a new potential therapeutic target in LUADs.

MicroRNA molecules are small, single-stranded RNA molecules that function to regulate networks of genes. They play important roles in normal female reproductive tract biology, as well as in the pathogenesis and progression of epithelial ovarian cancer. DROSHA, DICER, and Argonaute proteins are components of the microRNA-regulatory machinery and mediate microRNA production and function. This review discusses aberrant expression of microRNA molecules and microRNA-regulating machinery associated with clinical features of epithelial ovarian cancer. Understanding the regulation of microRNA molecule production and function may facilitate the development of novel diagnostic and therapeutic strategies to improve the prognosis of women with epithelial ovarian cancer. Additionally, understanding microRNA molecules and microRNA-regulatory machinery associations with clinical features may influence prevention and early detection efforts.

Reliance on glycolysis is a characteristic of malignancy, yet the development of resistance to BRAF inhibitors in melanoma is associated with gain of mitochondrial function. Concurrent attenuation of oxidative phosphorylation and HIF-1α/PKM2-dependent glycolysis promotes a non-apoptotic, iron- and oxygen-dependent cell death that we term ferroxitosis. The redox cycling agent menadione causes a robust increase in oxygen consumption, accompanied by significant loss of intracellular ATP and rapid cell death. Conversely, either hypoxic adaptation or iron chelation prevents menadione-induced ferroxitosis. Ectopic expression of K213Q HIF-1α mutant blunts the effects of menadione. However, knockdown of HIF-1α or PKM2 restores menadione-induced cytotoxicity in hypoxia. Similarly, exposure of melanoma cells to shikonin, a menadione analog and a potential PKM2 inhibitor, is sufficient to induce ferroxitosis under hypoxic conditions. Collectively, our findings reveal that ferroxitosis curtails metabolic plasticity in melanoma.

Significance: Hypoxia is a hallmark of the tumor microenvironment and represents a major source of failure in cancer therapy. Recent Advances: Recent work has generated extensive evidence that microRNAs (miRNAs) are significant components of the adaptive response to low oxygen in tumors. Induction of specific miRNAs, collectively termed hypoxamiRs, has become an accepted feature of the hypoxic response in normal and transformed cells. Critical Issues: Overexpression of miR-210, the prototypical hypoxamiR, is detected in most solid tumors, and it has been linked to adverse prognosis in many tumor types. Several miR-210 target genes, including iron-sulfur (Fe-S) cluster scaffold protein (ISCU) and glycerol-3-phosphate dehydrogenase 1-like (GPD1L), have been correlated with prognosis in an inverse fashion to miR-210, suggesting that their down- regulation by miR-210 occurs in vivo and contributes to tumor growth. Additional miRNAs are modulated by decreased oxygen tension in a more tissue-specific fashion, adding another level of complexity over the classic hypoxia-regulated gene network. Future Directions: From a biological standpoint, hypoxamiRs are emerging modifiers of cancer cell response to the adaptive challenges of the microenvironment. From a clinical perspective, assessing the status of these miRNAs may contribute to a detailed understanding of hypoxia-induced mechanisms of resistance and/or to the fine-tuning of future hypoxia-modifying therapies. Antioxid. Redox Signal. 21, 1220–1238.

Bevacizumab, the recombinant antibody targeting vascular endothelial growth factor (VEGF), improves progression-free but not overall survival in metastatic breast cancer. To seek further insights in resistance mechanisms to bevacizumab at the molecular level, we developed VEGF and non-VEGF-driven ER-positive MCF7-derived xenograft models allowing comparison of tumor response at different timepoints. VEGF gene (MV165) overexpressing xenografts were initially sensitive to bevacizumab, but eventually acquired resistance. In contrast, parental MCF7 cells derived tumors were de novo insensitive to bevacizumab. Microarray analysis with qRT-PCR validation revealed that Follistatin (FST) and NOTCH were the top signaling pathways associated with resistance in VEGF-driven tumors (P

Pancreatic cancer is especially a deadly form of cancer with a survival rate less than 2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression, and resistance to traditional therapy is therefore critical. Reduction–oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells and pancreatic cancer-associated endothelial cells in vitro. Modulation of APE1/Ref-1 using a specific inhibitor of APE1/Ref-1′s redox function, E3330, leads to a decrease in transcription factor activity for NFκB, AP-1, and HIF1α in vitro. This study aims to further establish the redox signaling protein APE1/Ref-1 as a molecular target in pancreatic cancer. Here, we show that inhibition of APE1/Ref-1 via E3330 results in tumor growth inhibition in cell lines and pancreatic cancer xenograft models in mice. Pharmacokinetic studies also show that E3330 attains more than10 μmol/L blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NFκB, AP-1, and HIF1α that are key transcriptional regulators involved in survival, invasion, and metastasis is blocked. These data indicate that E3330, inhibitor of APE1/Ref-1, has potential in pancreatic cancer and clinical investigation of APE1/Ref-1 molecular target is warranted. Mol Cancer Ther; 10(9); 1698–708. ©2011 AACR.

Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells.

Background: Despite spectacular advances in deciphering the molecular landscape of acute myeloid leukemia (AML), the clinical course of this disease has not improved dramatically for decades. Resistance to therapy and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. Our general strategy takes into account decreased oxygen (O2) tension, a micro-environmental hallmark which remains understudied in the context of AML. While low O2 compartments such as bone marrow niches are well-recognized hosts of drug resistant AML cells, in vitro studies are still routinely performed in supra-physiologic ambient O2 concentration (21%), with potentially important consequences for clinical translatability. We hereby present an integrated molecular dissection of AML cells response to Cytarabine under O2-controlled conditions and exploit the information to develop novel synergistic/additive treatment combinations. Methods: M14, THP1, OCIAML3 and primary cells (PC) derived from AML patients of different risk groups were incubated in culture medium under 21% O2 or 1% O2 in the presence or absence of Cytarabine (Cy, 1-7.5 µM), Rosuvastatin (Ro, 10-200µM) and/or Pravastatin (Pra, 10-200µM). After 48h, proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis. Characterization of gene expression and expression levels of total and phosphorylated proteins were assessed using RNAseq and Reverse Phase Protein Arrays (RPPA), respectively. Gene Set Enrichment Analysis was performed following GSEA User Guide. Quantitative PCR reactions were performed using PowerUp SYBR Green PCR Master Mix (Applied Biosystems, USA) on 7900HT Real-Time PCR System. Intracellular cholesterol (ICC) levels were measured using WAKO Cholesterol assay kit. Results: Treatment with Cy under 1% O2 induced a specific gene signature of metabolic stress responses, including the perturbation of cholesterol homeostasis. Detailed analysis of the cholesterol pathway via RNAseq showed pathway induction by hypoxia itself and severe depletion by Cy. Focused RT-PCR assays on the cholesterol pathway components HMGCS1, MSMO1, LSS and SQLE demonstrated significant induction under 1% compared to 21% O2, and significant downregulation in response to Cy. In line with our RNAseq and PCR data, RPPA showed that critical cholesterol synthesis pathways, such as ACC1, are downregulated in response to Cy. Surprisingly, we observed that ICC levels significantly increased after 48h in response to treatment with Cy (p Summary: Treatment with Cytarabine induces a genetic signature of metabolic stress response, including the perturbation of the cholesterol biosynthesis pathway, in AML cells. Depletion of intracellular cholesterol levels is counterbalanced by upregulation of CD36 expression in Cytarabine-treated AML cells under 21% and 1% O2 conditions. Rosuvastatin exerts antileukemic activity via downregulation of intracellular cholesterol levels, induction of apoptosis and inhibition of AML cell growth. Rosuvastatin displays greater antileukemic effects than pravastatin. Rosuvastatin combined with Cytarabine acts synergistically against AML cells. Conclusions: Further investigation of Rosuvastatin as a component of anti-AML therapy is warranted. Disclosures No relevant conflicts of interest to declare.

Background: Triple-negative breast cancer (TNBC) has been plagued by the absence of targeted therapies. Discovery of therapeutic targets in TNBC has in part, been hampered by an inadequate understanding of the transcriptional biology of the normal breast as an optimal comparator. Using next-generation sequencing, we embarked on a study to compare the transcriptomes of TNBC and normal breast to comprehensively identify novel targets by analyzing all full length transcripts expressed in these tissues. Methods: Normal breast tissues from healthy pre-menopausal volunteers with no history of disease were procured from the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. To eliminate bias from stromal tissue, normal tissues were laser capture microdissected for ductal epithelium. cDNA libraries from 10 TNBC tumors and 10 normal breast tissues were sequenced on an Applied Biosystems (AB) SOLiD3 sequencer using 50bp fragment runs. For gene expression, mapping of reads to the genome was performed using the AB BioScope 1.2 Pipeline and outputs imported into Partek Genomics Suite for analysis. In Partek, mapped reads were cross-referenced against known genes from the UCSC database followed by statistical comparison of RPKM values for each gene between TNBC and normal. Dimensionality reduction analyses (PCA & Hierarchical clustering) and identification of Novel Transcribed Regions were also performed in Partek, whereas construction of gene networks was performed using Ingenuity Pathway Analysis. To identify gene fusions, partially mapped reads were interrogated utilizing a novel algorithm that searched for reads spanning exons from two different genes. Fusions that were supported by at least 3 reads (of which 2 had to be unique) were considered candidates and were subsequently validated. Results/Discussion: Sequencing produced 1.1 billion reads equaling 57.3GB of data of which 36.0GB (63%) mapped to the human genome. In comparing RPKM values between TNBC and Normal, we report 7140 RefSeq Genes, 22 pre-miRNAs, 109 lincRNA exons, and 15 ultraconserved regions that were differentially expressed between these tissues (FDR Conclusion: We report an extensive comparison of the transcriptomes of TNBC and normal ductal epithelium. We identified numerous genes previously unknown to be dysregulated in TNBC that can be utilized for therapeutic discovery. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD01-08.

Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the over-expression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of β-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-β-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two ‘oncomirs’ may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.

Background: FLT3/ITD+ AML is a highly aggressive form of leukemia that carries a dismal prognosis. Evidence suggests that this poor outcome is due to the survival of a subset of leukemic cells (LCs) that elude therapy and survive in hypoxic niches of the bone marrow (BM). Effective targeting of LCs under hypoxic conditions is thus critical to cure AML. Our study examines the anti-leukemic potential of targeting Carbonic anhydrase-IX (CA9), a transmembrane protein which functions to maintain a neutral intracellular pH (pHi) under hypoxic stress conditions, against AML cells. Targeting CA9 is of particular interest as its expression is confined to only a few normal tissues and may therefore show relatively few side effects compared to standard therapies. Methods: Molm14 (M14) and primary cells (PC) from FLT3/ITD+ AML patients were incubated under normoxic (21% O2) and hypoxic (1% O2) conditions in the presence or absence of Quizartinib (Q), Cytarabine (Cy) or the CA9 inhibitor FC531 (FC). After 48h proliferation and apoptosis were determined per MTT assays, annexin V/PI staining and FACS analysis. Cells were assessed for CA9 mRNA expression as well as pHi levels via RT-PCR and fluorescent imaging, respectively. FLT3/ITD+ AML xenografts were generated by injecting M14 cells in the tail vein of NSG mice. Tissue samples were fixed in 10% NBF, embedded in paraffin and stained with H&E and CA9 for pathologic evaluation. Results: Cy and Q were significantly less effective against M14 cells under 1% compared to 21% O2. CA9 mRNA was upregulated in M14 cells (4.5±0.9-fold; n=4, p Citation Format: Fangli Chen, Adriana L. Rogozea, Xue Wu, George Sandusky, Mircea Ivan, Heiko Konig. Targeting intracellular pH regulation as a novel approach to overcome hypoxia-mediated drug resistance in FLT3/ITD mutated AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2427.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States. Aggressive treatment regimens have not changed the disease course, and the median survival has just recently reached a year. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including hypoxia, which creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. AP Endonuclease-1/Redox Effector Factor 1 (APE1/Ref-1) is a multifunctional protein possessing a DNA repair function in base excision repair and the ability to reduce oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. Here, we explore the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling activity, which regulates the transcriptional activation of hypoxia-inducible factor 1 alpha (HIF1α). Carbonic anhydrase IX (CA9) is regulated by HIF1α and functions as a part of the cellular response to hypoxia to regulate intracellular pH, thereby promoting cell survival. We hypothesized that modulating APE1/Ref-1 function will block activation of downstream transcription factors, STAT3 and HIF1α, interfering with the hypoxia-induced gene expression. We demonstrate APE1/Ref-1 inhibition in patient-derived and established PDAC cells results in decreased HIF1α–mediated induction of CA9. Furthermore, an ex vivo three-dimensional tumor coculture model demonstrates dramatic enhancement of APE1/Ref-1–induced cell killing upon dual targeting of APE1/Ref-1 and CA9. Both APE1/Ref-1 and CA9 are under clinical development; therefore, these studies have the potential to direct novel PDAC therapeutic treatment. Mol Cancer Ther; 15(11); 2722–32. ©2016 AACR.

Hypoxia is a key feature of solid tumors that contributes to resistance to therapy and reduced overall survival. Cellular adaptation to the hypoxic tumor microenvironment involves attenuation of energy-consuming processes such as macromolecular synthesis. We have identified RNA processing as another major process which is globally downregulated under hypoxic stress. By sequencing RNA of normoxic and hypoxic head and neck cancer cells, we observed widespread repression of genes that regulate RNA splicing and processing. As a result, we observed over 1,000 changes in relative mRNA isoform expression, including a significant increase in hypoxia-induced intron retention in nearly 100 genes. Genes observed to undergo intron retention in hypoxia included major regulators of RNA processing and protein synthesis, such as the translation initiation factor EIF2B5. Surprisingly, hypoxia-induced intron retention in EIF2B5 creates a premature termination codon, which results in a truncated protein isoform of eIF2Bϵ. We provide evidence that this isoform acts in opposition to the full-length protein to inhibit translation under stringent hypoxic conditions. Moreover, this intron is expressed in solid cancers known to contain hypoxic fractions and is overexpressed in head and neck cancer in a stage-dependent manner compared to normal tissue. To investigate how this intron is retained, we are examining the role of hypoxia-mediated changes in phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Our data suggest that increased phosphorylation of the CTD may lead to transcriptional pausing and subsequent retention of intron 12 in EIF2B5. Furthermore, distinct sequence features at this locus, such as a weak splice site at the intron12:exon13 junction, may explain how this intron is selectively retained in hypoxic cells. These results implicate mRNA splicing as a key mechanism to induce alternate transcripts and subsequent protein isoforms under hypoxia that influence cellular adaptation to stress. Citation Format: Lauren K. Brady, Hejia Wang, Caleb Radens, Milan Radovich, Amit Maity, Cristina Ivan, Mircea Ivan, Yoseph Barash, Constantinos Koumenis. Transcriptome analysis of hypoxic head and neck cancer cells uncovers intron retention in EIF2B5 as a mechanism to reduce protein synthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1998. doi:10.1158/1538-7445.AM2017-1998

MicroRNAs are small regulatory RNAs that post transcriptionally control gene expression. Reduced expression of DICER, the enzyme involved in microRNA processing, is frequently observed in cancer and is associated with poor clinical outcome in various malignancies. Yet, the underlying mechanisms are not well understood. Here we identify tumour hypoxia as a regulator of DICER expression in large cohorts of breast cancer patients. We show that DICER expression is suppressed by hypoxia through an epigenetic mechanism that involves inhibition of oxygen-dependent H3K27me3 demethylases KDM6A/B and results in silencing of the DICER promoter. Subsequently, reduced miRNA processing leads to derepression of the miR-200 target ZEB1, stimulates the epithelial to mesenchymal transition and ultimately results in the acquisition of stem cell phenotypes in human mammary epithelial cells. Our study uncovers a previously unknown relationship between oxygen-sensitive epigenetic regulators, miRNA biogenesis and tumour stem cell phenotypes that may underlie poor outcome in breast cancer.

Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo, and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment.

Hypoxia is a central component of the tumor microenvironment and represents a major source of therapeutic failure in cancer therapy. Recent work has provided a wealth of evidence that noncoding RNAs and, in particular, microRNAs, are significant members of the adaptive response to low oxygen in tumors. All published studies agree that miR-210 specifically is a robust target of hypoxia-inducible factors, and the induction of miR-210 is a consistent characteristic of the hypoxic response in normal and transformed cells. Overexpression of miR-210 is detected in most solid tumors and has been linked to adverse prognosis in patients with soft-tissue sarcoma, breast, head and neck, and pancreatic cancer. A wide variety of miR-210 targets have been identified, pointing to roles in the cell cycle, mitochondrial oxidative metabolism, angiogenesis, DNA damage response, and cell survival. Additional microRNAs seem to be modulated by low oxygen in a more tissue-specific fashion, adding another layer of complexity to the vast array of protein-coding genes regulated by hypoxia.

Hypoxia is a key component of the tumor microenvironment and represents a well-documented source of therapeutic failure in clinical oncology. Recent work has provided support to the idea that noncoding RNAs (ncRNAs), and in particular microRNAs (miRNAs), may play important roles in the adaptive response to low oxygen in tumors. Specifically, all published studies agree that the induction of microRNA-210 (miR-210) is a consistent feature of the hypoxic response in both normal and transformed cells. miR-210 is a robust target of hypoxia-inducible factor (HIF), and its overexpression has been detected in a variety of diseases with a hypoxic component, including most solid tumors. High levels of miR-210 have been linked to an in vivo hypoxic signature and to adverse prognosis in breast and pancreatic cancer patients. A wide variety of miR-210 targets have been identified, pointing to roles in mitochondrial metabolism, angiogenesis, differentiation, DNA damage response, and cell survival. Such targets are suspected to affect the development of tumors in multiple ways; therefore, an increased knowledge about miR-210’s actions may lead to novel diagnostic and therapeutic approaches in the cancer field.

The tumour suppressor p53 is a crucial regulator of cell cycle arrest and apoptosis by acting as a transcription factor to regulate a variety of genes. At least in part, this control is exerted by p53 via regulating expression of numerous microRNAs. We identified two abundantly expressed microRNAs, miR-16 and miR-26a, whose expression is regulated by p53 during the checkpoint arrest induced by the genotoxic drug, doxorubicin. Importantly, among the targets of these miRs are two critical checkpoint kinases, Chk1 and Wee1. The p53-dependent augmentation of miR-16 and miR-26a expression levels led to the cell cycle arrest of tumour cells in G1/S and increased apoptosis. Strikingly, the bioinformatics analysis of survival times for patients with breast and prostate cancers has revealed that co-expression of mir-16 and miR-26a correlated with a better survival outcome. Collectively, our data provide a novel mechanism whereby p53 represses Chk1 and Wee1 expression, at least partially, via upregulation of miR-16 and miR-26a and thus sensitizes tumour cells to genotoxic therapies.

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90% of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos.

Normal thyroid follicular cells, like many highly differentiated epithelia, have limited proliferative capacity. We previously showed that this could be extended by expression of the SV40 large T oncogene, but that immortal lines always lost thyroid-specific differentiation. Detailed analysis now show that clones expressing T undergo 2 mutually exclusive fates. They either (i) remain well-differentiated, in which case they undergo irreversible growth arrest after 5 to 15 p.d., or (ii) spontaneously develop poorly differentiated sub-clones that exhibit greatly extended proliferative life spans (up to 75 p.d.). The frequency of this event (> 3 per 10(4) cell divisions) greatly exceeds that expected from somatic mutation, suggesting an epigenetic basis. This is supported by our finding of rare de-differentiated epithelial cells in normal thyroid that all generate clones with extended life spans, indistinguishable from the above, following introduction of SV40 T. Escape from early mortality in differentiated thyroid epithelium therefore requires not only loss of tumour suppressor gene function (induced here by SV40 T), but also a switch in differentiation programme, with the latter effectively converting the follicular cell into a cell type with increased intrinsic proliferative potential. The analogy between this in vitro model and the progression of thyroid cancer from the well-differentiated to the highly aggressive, anaplastic form suggests that de-differentiation may play a causal rather than a passive role in this critical switch in tumour behaviour.

Hepatocyte growth factor (HGF) has been shown to be mitogenic for a wide variety of epithelial cells, including recently, dog thyroid follicular cells. Here we have extended this work to human thyrocytes, and find that recombinant HGF stimulates DNA synthesis (proportion of cells in cell cycle S phase) in normal primary cells in monolayer, with an ED50 of ∼ 8 ng/ml and a maximum between 50 and 250 ng/ml. Stimulation was observed even in the presence of 10% fetal calf serum (previously the most potent mitogen for these cells in our hands), the maximum nuclear 3H-thymidine labelling index achieved with HGF being up to 6-fold higher than that with serum alone. A similar additive effect was observed on thyrocytes already stimulated to proliferate by expression of an activated ret oncogene. These results make HGF the most potent defined mitogen for human thyrocytes to date, and suggest that upregulated HGF met signalling may confer a significant growth advantage even in neoplastic thyroid cells, consistent with the finding of increased met expression in many thyroid carcinomas.

Half of all patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) die within a year despite extensive surgery and/or a highly aggressive chemotherapy regimen. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including reactive stroma and hypoxia. Cancer-associated fibroblasts (CAFs) contribute to tumor signaling, fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and impairs therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia to regulate intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein that possesses two major activities: an endonuclease function in DNA base excision repair and a redox signaling function that reduces oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. We explored the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1α), a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition will result in enhanced PDAC cell killing under hypoxic conditions. Methods: We performed qRT-PCR and Western Blots to confirm changes in CA9 expression in PDAC cells and CAFs following APE1/Ref-1 inhibition and hypoxia exposure. We evaluated the effects of dual-targeting APE1/Ref-1 and CA9 on acidification of tumor cells with an intracellular pH indicator. Proliferation assays were used to assess cell killing following inhibition of APE1/Ref-1 and CA9 under hypoxia. Ex vivo 3-Dimensional co-culture models including both tumor and CAFs were used to examine whether we could enhance the efficacy of APE1/Ref-1 and/or CA9 inhibition with a dual-targeting approach to kill tumor spheroids. To delineate which function of APE1/Ref-1 is critical for observed effects, we used the APE1/Ref-1 redox signaling inhibitor, APX3330, an APE1/Ref-1 repair inhibitor, ARI-3, and an APX3330 analog that does not block APE1/Ref-1 redox activity, RN7-58. To inhibit CA9, we used the sulfonamide derivative, SLC-0111 (Clinical Trial NCT02215850). Results: HIF1α-mediated induction of CA9 is significantly diminished in PDAC cells following APE1/Ref-1 redox inhibition or knock-down in both patient-derived and established cell lines, as well as pancreatic CAFs. Additionally, dual-targeting of APE1/Ref-1 redox signaling and CA9 results in acidification of PDAC cells and reduces PDAC tumor cell growth under hypoxic conditions as well as in a 3D tumor co-culture model. The results presented here demonstrate potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment. Ongoing experiments will determine the role of APE1/Ref-1 and CA9 in invasion of tumor cells exposed to hypoxic conditions, as well as the efficacy of dual-targeting APE1/Ref-1 and CA9 in in vivo models of PDAC. Consequently, our findings further demonstrate a combination approach for blocking important signaling pathways in tumor cells. Our results support our previously published studies using APX3330 and STAT3 inhibition as a dual hit strategy in PDAC cells (Cardoso, Jiang et al. 2012) and APX3330 and Avastin (bevacizumab) for HIF1α-VEGF-signaling inhibition as an anti-angiogenesis combination strategy (Luo, Delaplane et al. 2008, Jiang, Gao et al. 2011, Li, Liu et al. 2014). Continued studies centering on the APE1/Ref-1 redox signaling axis and potential clinical partners for pathway inhibition are ongoing in our laboratories. This approach is supported by the planned phase 1 clinical trial for APX3330 scheduled to begin in mid-2016. Citation Format: Derek P. Logsdon, Michelle Grimard, Safi Shahda, Nicholas Zyromski, Ernestina Schipani, Fabrizio Carta, Claudiu T. Supuran, Murray Korc, Mircea Ivan, Mark R. Kelley, Melissa L. Fishel.{Authors}. Regulation of HIF1α under hypoxia by APE1/Ref-1 impacts CA9 expression: Dual-targeting in patient-derived 3D pancreatic cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B51.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease and ∼95% die within five years, with most surviving less than six months. Targeted therapies including Gemcitabine (GemzarTM), FOLFIRINOX (5-FU/leucovorin/irinotecan/oxaliplatin), and sustained release, nab-paclitaxel (AbraxaneTM) offer modest improvement in survival, albeit at an increase in side effects and unwanted toxicities. Data is presented on redox factor-1 (Ref-1) and specific Ref-1 inhibitor APX3330. Ref-1 regulates multiple transcription factors involved in pancreatic cancer survival signaling due to its redox-coactivator activity on HIF-1α, NFkB, NRF2 and STAT3. High expression levels of Ref-1 indicate decreased survival in PDAC as well as other cancers. APX3330 has been shown in multiple in vitro and in vivo pancreatic cancer models to be effective in reducing tumor growth and metastases as a single agent. The mechanism of action has been extensively investigated and characterized for its specific activity on Ref-1, as well as its preclinical PK/PD and ADME. The safety and dose administration of APX3330 have been established by Eisai pharmaceutical company through a previous development program including toxicology, phase I, and phase II clinical evaluation in non-cancer patients in Japan. We have partnered with ApeX Therapeutics to develop APX3330 for cancer treatment (phase I trial anticipated start date early 2016). While developing APX3330 for single agent use, we studied interactions of Ref-1, APX3330, convergent pathways; i.e. HIF-1 α and STAT3, and downstream targets like CAIX. Initially, we performed in vivo studies demonstrating single and combination effects of APX3330 with Gemcitabine (Gem) showing significantly decreased tumor volume in the APX3330 and Gem combination treatments compared to the single-agents alone. We also tested single and combination studies of APX3330 in an ex vivo 3-D tumor-stroma model system using patient derived tumor cells along with patient derived cancer-associated fibroblasts (CAFs). We used the CAIX inhibitor SLC-0111 and JAK2 inhibitor, Ruxolitinib; both agents in clinical trials. In our ex vivo 3D co-culture system, APX3330 decreases the tumor area and intensity in a dose-dependent manner. The combination of APX3330 with Gem demonstrated an additive enhancement effect in the tumor. Blocking both Ref-1 redox-signaling activity with APX3330 and CAIX activity via SLC-0111 demonstrated enhanced tumor killing in our models. APX3330 along with Ruxolitinib also demonstrated enhanced tumor killing. These data demonstrate APX3330 single agent efficacy in our 3D patient PDAC model and enhanced tumor killing when pathways regulated by Ref-1, HIF-1 α and STAT3 are blocked. Additional drug combinations focused on pathways that are dependent on Ref-1 signaling will also be presented. Citation Format: Melissa L. Fishel, Derek P. Logsdon, Michelle L. Grimard, Claudiu T. Supuran, Nicholas Zyromski, Mircea Ivan, Mark R. Kelley, Fenil Shah. Targeting Ref-1/APE1 pathway inhibition in pancreatic cancer using APX3330 for clinical trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4740.

Solid tumors exhibit a range of oxygen concentrations, in part due to abnormal vasculature and limited diffusion of oxygen which lead to hypoxia. Tumor hypoxia is associated with resistance to treatment, metastasis and poor patient outcome for many types of cancer. Assessing the impact of hypoxia in the tumor microenvironment and understanding how it contributes to tumorigenesis and survival is a challenging research goal for several reasons; a chief obstacle has been developing methods to analyze the transcriptome within precise regions of the tumor microenvironment in vivo. To address this task, we used Laser-Capture-Microdissection (LCM) of tumors labeled with the hypoxia probe, EF5, to specifically isolate normoxic and hypoxic regions of tumors from a murine model of head and neck cancer. We then carried out deep RNA-sequencing of the in vivo samples and corresponding in vitro samples grown in 0.5% oxygen conditions. Here we present the first described study of the hypoxic transcriptome in tumors at base-pair resolution. Our goal is to use these data to evaluate the potential of previously unstudied hypoxia-mediated pathways, such as the regulation of mRNA splicing, to be targeted therapeutically. We identified global changes in several classes of splicing in hypoxic compared to normoxic head and neck cancer cells, including changes in expression of last exons, retained introns and 3’ untranslated regions. Many of the alternatively spliced isoforms are in pathways central to hypoxic adaptation, such as glycolysis, cell cycle and translation. Among these genes, we uncovered a previously undescribed splicing event in the master regulator of translation, EIF2B5. The full-length protein encoded by EIF2B5 (eIF2Bå), is a necessary catalytic component of the eIF2B complex, which binds eIF2á and exchanges GDP for GTP to initiate translation. Strikingly, hypoxia induces retention of intron 12 in EIF2B5, which leads to insertion of an early stop codon in frame with the coding sequence; the resulting 65kDa protein isoform lacks a functional guanine nucleotide exchange (GEF) domain compared to the full-length isoform, but retains the region known to bind eIF2B complex subunits. Thus, we predict that the 65kDa isoform of eIF2Bå, may act in opposition to full-length eIF2Bå and inhibit translation initiation in conditions of oxygen deprivation. We are investigating EIF2B5, and other select hypoxia-regulated isoforms, to evaluate how alternative splicing impacts cell growth and survival in hypoxic tumors. Citation Format: Lauren K. Brady, Rohil Shekher, Vladimir Popov, Mircea Ivan, Milan Radovich, Constantinos Koumenis. Analysis of the hypoxic transcriptome in cells and solid tumors reveals a novel spliced isoform of the key regulator of mRNA translation, eIF2B5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 733.

Background: Despite significant advances in diagnosis and treatment, breast cancer remains the leading cause of cancer-related death in women worldwide. Therefore, there is a critical need to identify molecular mechanisms responsible for cancer initiation and progression. To date, there have been attempts to identify biomarkers that can predict progression of pre-malignant lesions (i.e. DCIS) to invasive carcinoma. Our study aims to identify earliest markers of breast cancer initiation. Methods: We evaluated local (breast tissue) and systemic (serum/plasma) molecular alterations associated with breast cancer susceptibility using the unique resources available at the Komen Normal Tissue Bank at IUSCC. Human specimens (serum/plasma and breast tissue) donated by women before their cancer was clinically detectable were used to identify circulating biomarkers that are associated with breast cancer risk. Specimens from age-matched controls were used for comparison. Sera/plasma were analyzed for circulating miRNAs and telomeric level in the cell-free circulating DNA (cfDNA). We employed next generation sequencing to obtain transcriptome in the “normal” breast of women who eventually developed breast cancer and age-matched control normal breast. Results/Discussion: Serum/plasma of women who developed breast cancer showed variation in circulating miRNAs as well as telomeric cfDNA levels as compared to the healthy control group. Among 385 microRNAs detected in circulation, 10 miRNAs were present at different levels in the susceptible group. In addition, susceptible group displayed reduced levels of plasma telomeric cfDNA and telomere shortening in breast epithelium. Transcriptome analysis of microdissected breast epithelium and stroma revealed molecular alterations in the “normal” breast tissue associated with cancer development. Pathway analyses of these differentially expressed genes are underway to determine the mechanisms associated with breast cancer initiation. Conclusion/Impact: Functional analysis of biomarkers identified in this study may help in cancer risk assessment and improvement of preventive therapy. Citation Format: Natascia Marino, Mariah L. Johnson, Hiromi Tanaka, Xi Wu, Theresa Mathieson, Jill E. Henry, Mircea Ivan, Yunlong Liu, Connie Rufenbarger, Anna Maria V. Storniolo. Novel biomarkers and molecular alterations for breast cancer initiation and susceptibility. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2619.

Antiangiogenic therapy is emerging as a highly promising strategy for the treatment of ovarian cancer, but the clinical benefits are usually transitory. The purpose of this study was to identify and target alternative angiogenic pathways that are upregulated in ovarian xenografts during treatment with bevacizumab. For this, angiogenesis-focused gene expression arrays were used to measure gene expression levels in SKOV3 and A2780 serous ovarian xenografts treated with bevacizumab or control. Reverse transcription-PCR was used for results validation. The insulin growth factor 1 (IGF-1) was found upregulated in tumor and stromal cells in the two ovarian xenograft models treated with bevacizumab. Cixutumumab was used to block IGF-1 signaling in vivo. Dual anti-VEGF and IGF blockade with bevacizumab and cixutumumab resulted in increased inhibition of tumor growth. Immunohistochemistry measured multivessel density, Akt activation, and cell proliferation, whereas terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay measured apoptosis in ovarian cancer xenografts. Bevacizumab and cixutumumab combination increased tumor cell apoptosis in vivo compared with therapy targeting either individual pathway. The combination blocked angiogenesis and cell proliferation but not more significantly than each antibody alone. In summary, IGF-1 activation represents an important mechanism of adaptive escape during anti-VEGF therapy in ovarian cancer. This study provides the rationale for designing bevacizumab-based combination regimens to enhance antitumor activity. Mol Cancer Ther; 11(7); 1576–86. ©2012 AACR.

Multiple studies have consistently established that miR (microRNA)-210 induction is a feature of the hypoxic response in both normal and transformed cells. Here, we discuss the emerging biochemical functions of this miRNA and anticipate potential clinical applications. miR-210 is a robust target of hypoxia-inducible factor, and its overexpression has been detected in a variety of cardiovascular diseases and solid tumors. High levels of miR-210 have been linked to an in vivo hypoxic signature and associated with adverse prognosis in cancer patients. A wide spectrum of miR-210 targets have been identified, with roles in mitochondrial metabolism, angiogenesis, DNA repair, and cell survival. Such targets may broadly affect the evolution of tumors and other pathological settings, such as ischemic disorders. Harnessing the knowledge of miR-210's actions may lead to novel diagnostic and therapeutic approaches. © 2011 IUBMB IUBMB Life, 63(2): 94–100, 2011

Fibroblast Growth Factors (FGFs) have been implicated in malignant transformation, tumor mitogenesis, angiogenesis and chemoresistance. The aim of this study was to determine which FGFs and FGFRs play functional roles in epithelial ovarian cancer. Restriction enzyme analysis of mRNA revealed that transformation was associated with a switch in FGFR2 and FGFR3, from the IIIc to the IIIb isoform. There was widespread expression of FGFs, including FGF7, in all tissues but, FGF3 and FGF19 were expressed by malignant cell lines and cancer tissue but were not present in normal tissue. Using FGFR-specific shRNAi we demonstrated that reductions in FGFR2 inhibited proliferation of ovarian cancer cell lines in vitro (>50%, p < 0.006) and reduced cisplatin IC50 (>60%, p < 0.0001). Cell cycle analysis revealed increased cisplatin sensitivity was associated with increased G2/M arrest and increased apoptosis. FGFR2 shRNAi reduced growth rates of ovarian tumor xenografts by 20% (p < 0.006) and when combined with cisplatin caused a 40% reduction in proliferation rates (p < 0.007). In contrast, RNAi-induced reductions in FGFR1 increased SKOV3 cell numbers, with associated changes in cell cycle but had no effect on ES 2 cells. However, the cisplatin IC50 was reduced (>50%, p < 0.0001) by FGFR1 shRNAi in both cell lines and there was increased apoptosis (46–50%) compared with control cells (35%) (p < 0.004). Together our data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity. However, data on the inhibition of FGFR1 suggest that broad spectrum FGFR inhibitors may have unexpected effects on proliferation.

microRNAs (miRNAs) are master regulators of gene expression. By degrading or blocking translation of messenger RNA targets, these noncoding RNAs can regulate the expression of more than half of all protein-coding genes in mammalian genomes. Aberrant miRNA expression is well characterized in cancer progression and has prognostic implications for cancer in general. Over the past several years, accumulating evidence has demonstrated that genomic alterations in miRNA genes are correlated with all aspects of cancer biology. In this review, we describe the effects of miRNA deregulation in the cellular pathways that lead to the progressive conversion of normal cells into cancer cells as well as in cancer diagnosis and therapy in humans.

Genetic instability is a hallmark of cancer; the hypoxic tumor microenvironment has been implicated as a cause of this phenomenon. MicroRNAs (miR) are small nonprotein coding RNAs that can regulate various cellular pathways. We report here that two miRs, miR-210 and miR-373, are up-regulated in a hypoxia-inducible factor-1α–dependent manner in hypoxic cells. Bioinformatics analyses suggested that these miRs could regulate factors implicated in DNA repair pathways. Forced expression of miR-210 was found to suppress the levels of RAD52, which is a key factor in homology-dependent repair (HDR); the forced expression of miR-373 led to a reduction in the nucleotide excision repair (NER) protein, RAD23B, as well as in RAD52. Consistent with these results, both RAD52 and RAD23B were found to be down-regulated in hypoxia, but in both cases, the hypoxia-induced down-regulation could be partially reversed by antisense inhibition of miR-210 and miR-373. Importantly, luciferase reporter assays indicated that miR-210 is capable of interacting with the 3′ untranslated region (UTR) of RAD52 and that miR-373 can act on the 3′ UTR of RAD23B. These results indicate that hypoxia-inducible miR-210 and miR-373 play roles in modulating the expression levels of key proteins involved in the HDR and NER pathways, providing new mechanistic insight into the effect of hypoxia on DNA repair and genetic instability in cancer. [Cancer Res 2009;69(3):1221–9]

p53 gene mutations are the most frequent alterations in human cancers. In the published literature, a highly significant statistical difference between the prevalence of p53 mutations in haematological (H) and non-haematological (NH) malignancies can be found. However, no consistent reasons have been suggested to explain it. We propose two non-exclusive possibilities: (i) in H tumours p53 is altered with the same frequency as in NH tumours, but mechanisms other than mutations are involved and (ii) in H malignancies the prevalence of p53 mutations is much lower than in NH cancers because other genetic disturbances are involved. We hypothesized that retention of wild-type p53 in H cancers may be a consequence of: (a) the presence of telomerase activity in haematological cells and/or (b) the absence of hypoxia in the majority of H tumours.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease, and ∼95% die within five years. Treatment with chemotherapy has not changed the natural course of this disease, and just recently, with combination of chemotherapeutic agents, the median survival reached a year. Several mechanisms are proposed to play a role in the aggressive, treatment-resistant phenotype of PDAC, including adaptation to hypoxia, which leads to increased potential for metastasis and impairs the efficacy of chemotherapy and radiotherapy. Hypoxia-Inducible Factor-1α (HIF1α), a major oxygen sensor in cells, is a transcription factor that is rapidly degraded under normoxic conditions but upregulates a number of genes under hypoxic conditions that contribute to survival, metastasis, and angiogenic signaling in the tumor microenvironment. One of the most notable HIF targets is Carbonic Anhydrase IX (CA9), which promotes tumor cell survival and metastasis by maintaining a steady intracellular pH while acidifying the microenvironment, thereby encouraging epithelial-mesenchymal transition and contributing to extracellular matrix degradation. AP Endonuclease1/ Redox Effector Factor 1 (APE1/Ref-1) is a dual function protein that possesses a DNA repair function as well as the ability to reduce transcription factors and enable them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in preventing apoptosis, survival mechanisms, and hypoxia signaling, including HIF-1α. Therefore, we hypothesized that APE1/Ref-1 inhibition impairs HIF-1α-mediated signaling, and this leads to decreased survival and invasion of tumor cells exposed to hypoxic conditions. Methods: We performed co-immunoprecipitation (co-IP) studies to look at the interaction of APE1/Ref-1 with transcriptional targets, HIF-1α, STAT3, and NFκB along with RT-PCR and Western blotting to confirm expression of hypoxia signaling genes. Luciferase reporter assays were used to quantitate transcriptional activation under hypoxia. Boyden chamber was used to look at migration and invasion as well as proliferation based assays following manipulation of APE1/Ref-1 and hypoxia. Results: HIF-1α and STAT3, but not NFκB associate with APE1/Ref-1 under hypoxia. Moreover, we found that knockdown of APE1/Ref-1 protein diminishes HIF-mediated transcription in hypoxia, as shown by analysis of luciferase reporter assays. Next, we showed that, in hypoxia, APE1/Ref-1 inhibition diminishes HIF-1α-induced downstream targets including CA9 and ANGPTL4 further indicating that APE1/Ref-1 redox activity regulates HIF signaling. Importantly, we found that hypoxia, in the presence or absence of APE1/Ref-1, no longer induced CA9 mRNA levels in HIF-deficient MEFs, proving that hypoxia-dependent regulation of CA9 expression is fully mediated by HIF-1α. A blockade of both CA9 activity via small molecule and CA9 transcription via APE1/Ref-1 leads to decreased PDAC cell proliferation under hypoxia. These data indicate that APE1/Ref-1 inhibition interferes with ηψπoξια-mediated signaling and can further sensitize PDAC cells to CA9/12 inhibition even under the conditions of extreme oxygen deprivation. Ongoing experiments will determine the role APE1/Ref-1 plays in the survival and invasion of tumor cells exposed to hypoxia. Citation Format: Derek P. Logsdon, Huiwen Cheng, Meihua Luo, Safi Shahda, Yangyang Hao, Yan Tong, Zhangsheng Yu, Nicholas Zyromski, Ernestina Schipani, Yunlong Liu, Claudiu T. Supuran, Mircea Ivan, Mark R. Kelley, Melissa L. Fishel. Targeting APE1/Ref-1 results in inhibition of hypoxia signaling genes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B158.

Hypoxia is a cellular stress that influences both normal and tumor development. For many types of cancer, including head and neck carcinomas, tumor hypoxia shows a strong positive correlation with poor patient outcome. In order to gain a deeper understanding of the biology of the tumor microenvironment, we are investigating how transcription is differentially regulated in hypoxic compared to normoxic regions of tumors. Previously, we reported that the expression of over 500 genes differs between hypoxic and normoxic tumors. However, there has not yet been a global sequencing analysis of tumors to characterize hypoxia-induced differences in transcriptional regulation at base-pair resolution. To achieve this, we developed a method to isolate RNA from hypoxic regions of tumors in vivo and used this method to carry out deep RNA-sequencing of normoxic and hypoxic regions of human head and neck tumors grown in mice. Corresponding in vitro samples from cells cultured in 0.5% oxygen conditions (in which the only stress was hypoxia) were also sequenced. We used these data to examine which genes showed changes in isoform-level expression and to define hypoxia-mediated changes in splicing. The data revealed more than 1,000 hypoxia-responsive mRNA isoforms. Using a combined informatics approach to analyze both differential expression of alternatively spliced exons and entire isoforms, we observed that many hypoxia-induced isoforms showed changes in alternate last exons, retained introns and 3′ untranslated regions compared to the isoforms expressed in normoxic cells. Moreover, we observed global repression of genes involved in RNA processing and splicing, which may represent one mechanism to shift patterns in splicing and lead to expression of stress-induced isoforms. Differentially expressed isoforms include genes that play critical roles in fundamental processes involved in hypoxic adaptation, such as the translation, glycolysis and cell cycle. Therefore, we propose that alternative splicing plays a role in mediating tumor growth and survival in hypoxic conditions by regulating stress-responsive pathways. Citation Format: Lauren Brady, Vladimir Popov, Mircea Ivan, MIlan Radovich, Constantinos Koumenis. Investigating the impact of hypoxia-induced changes in splicing on tumor microenvironment. [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 3005. doi:10.1158/1538-7445.AM2015-3005

Publisher Summary This chapter focuses on oxygen sensing during the analysis of von Hippel–Lindau (VHL) hereditary cancer syndrome and the implications of oxygen sensing. VHL disease is a hereditary cancer syndrome caused by germline mutations that inactivate the VHL tumor suppressor gene that resides on chromosome 3p25. The cardinal features of this disorder are blood vessel tumors (hemangioblastomas) of the central nervous system (CNS) and retina, clear cell renal carcinomas, and pheochromocytomas. Tumor development in VHL disease is linked to somatic inactivation of the remaining wild-type VHL allele, thus depriving a susceptible cell of the wild-type VHL gene product (pVHL).The chapter discusses the expression and purification of GST–VBC complex and VBC complex, production of pure VBC complex and GST–VBC for pull down, far western blot analysis with VBC complex, i n vitro ubiquitination assay, and prolyl hydroxylation protocols

Background: Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF), had promising therapeutic efficacy in breast cancer. However, intrinsic or acquired resistance is common in the clinic. To improve our understanding of the underlying mechanisms of resistance to bevacizumab (BEV), we report the gene expression analysis of resistance to bevacizumab in a VEGF-overexpressing xenograft model of ER-positive breast cancer. Methods: We developed a nude mouse xenograft model of resistance to anti-VEGF therapy with BEV in which MCF-7 control (ML20) or MCF-7 VEGF (MV165) transfectants were implanted in mammary fat pads, allowed to grow, then treated with BEV, with collection of tumor at early or late time points (while responding (R) to or progressing (NR) on anti-VEGF therapy). To elucidate differentially expressed gene profiling associated with tumor resistance to BEV, we performed whole-genome gene expression analysis (Human WG-6v2 Expression Beadchips, Illumina) and miRNA profiling (TaqMan ***ArrayHuman MicroRNAA+B Cards Set v3.0, Applied Biosystems). Validation of the chosen genes was performed using quantitative real-time RT-PCR (qRT-PCR). Results: Gene expression analysis revealed differentially regulated genes in the MV165-NR group compared with the MV165-R group. Among the significant genes, Follistatin (FST) and HEY2 were the top genes upregulated in NR compared to R by ANOVA. Expression of HEY2 is induced by the Notch signaling pathway. Using qRT-PCR, we validated the expression of FST and Notch in our system. FST was significantly decreased (Fold change= −3.2; P=0.03) in the R group compared with vehicle in MV165 xenografts. In contrast to R group, FST was upregulated significantly (Fold change= 9.3; P=0.05) in the NR group. Notch4 displayed increased levels of expression in NR group, but it did not reach significance (P=0.23). In addition, correlation of mRNA and miRNA profiles showed that miRNAs targeting FST and Notch4 were differentially regulated in NR group compared to R group in MV165 xenograft tumors. Among the miRNAs, TGF-β-induced oncomiR miR-181a is up-regulated in NR and targets both FST and Notch4. Other miRNAs that target both Notch4 and FST include miR-1, miR-133a, miR-133b, and mir-449b. Conclusion: Our data serve as a potential mechanistic explanation for acquired resistance to bevacizumab. These data may shed light on the transitory effect of BEV observed in the E2100 firstline metastatic breast cancer trial, where VEGF-targeted therapy prolongs progression-free survival in metastatic breast cancer without improving overall survival. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-01.

Background: Anti-vascular endothelial growth factor (anti-VEGF) therapy improves disease-free but not overall survival in metastatic breast cancer. To seek further insight on resistance to anti-VEGF antibody bevacizumab (BEV) at the molecular level, we developed breast cancer xenograft models allowing comparison of tumor response at different time-points. Here we report the gene expression and miRNA analyses of response and non-response to BEV in these models. Methods: MCF-7 cells transfected with control vector (ML20) or VEGF (MV165) were implanted into the mammary fat pads of athymic mice. Tumors from short-term treatment with BEV (3 weeks; Responders to BEV, R) or long-term treatment (8 weeks; Non-Responders, NR) or with vehicle control group (V) were subjected to whole-genome gene expression analysis (Human WG-6v2 Expression Beadchips, Illumina) and miRNA profiling (TaqMan ArrayHuman MicroRNA A+B Cards Set v3.0, Applied Biosystems).Validation of the chosen genes was performed using quantitative real-time RT-PCR (qRT-PCR) and Immunohistochemistry (IHC). Results: Short-term treatment to BEV (3 weeks; 5 mg/kg, i.p./twice weekly) inhibited primary tumor growth significantly in MV165 xenografts compared with vehicle control, whereas BEV treatment did not affect the tumor growth in the ML20 model. MV165 xenografts progressed after 8 weeks of BEV treatment. Gene set enrichment analysis (GSEA) revealed that luminal A-related gene sets were enriched in MV165-R compared to MV165-NR group including DESMEDT (ESR1), SMID_Breast_Cancer_Luminal_A_up, and MASSARWEH_ Tamoxifen_Resistance_ Down. Myoepithelial-specific gene sets were upregulated in both the R and NR groups compared with the vehicle group. qRT-PCR analysis showed that estrogen receptor alpha (ESR1) representative for luminal A decreased significantly in the MV-165-NR group (P=0.001) compared to vehicle. In contrast, Cytokeratin 5 (KRT5) levels increased significantly in both R (P=0.02) and NR (P=0.03) groups. In addition, KRT14 was upregulated in R (P= 0.04) and in NR (P=0.14) group in comparison with the vehicle group, suggesting the upregulation of myoepithelial phenotype specific to BEV treated MV165 model, but not ML20 model. Similar results were obtained by IHC. Consistent with mRNA changes, ESR1 regulated miRNA such as miR-107 (P=0.007) and miRNA important in tamoxifen resistance such as mir-451 (P= 0.0003) were also altered in MV165-NR group compared to vehicle. Conclusion: These results suggest that treatment with BEV may alter the intrinsic subtypes in the presence of VEGF expression. These data may help to explain the variable results to anti-VEGF therapy based on the duration of BEV treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-04-02.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germ line mutation of the von Hippel-Lindau tumor suppressor gene (VHL). Tumors observed in this disorder include retinal and central nervous system hemangioblastomas, clear cell renal carcinomas and pheochromocytomas. The VHL gene product, pVHL, is a component of a ubiquitin ligase which targets the transcription factor known as hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. pVHL also plays roles in the control of extracellular matrix formation and cell-cycle exit. Different VHL mutations confer different site-specific risks of cancer. Type 2C VHL mutations confer an increased risk of pheochromocytoma without the other stigmata of VHL disease. Here we report that the products of such type 2C VHL alleles retain the ability to down regulate HIF but are defective for promotion of fibronectin matrix assembly. Furthermore, pVHL L188V, a well studied type 2C mutant, retained the ability to suppress renal carcinoma growth in vivo. These studies strengthen the notion that HIF deregulation plays a causal role in hemangioblastoma and renal carcinoma, and raises the possibility that abnormal fibronectin matrix assembly contributes to pheochromocytoma pathogenesis in the setting of VHL disease.

The cyclic AMP pathway is a major regulator of thyrocyte function and proliferation and, predictably, its inappropriate activation is associated with a sub-set of human thyroid tumours. Activating mutations are, however, more common in the thyrotropin receptor (TSHR) than in its downstream transducer, Galphas. To investigate whether this reflects an inherent difference in their oncogenic potency, we compared the effects of retrovirally-transduced mutant (A623I) TSHR or (Q227L) Galphas (GSP), using the rat thyroid cell line FRTL5 and primary human thyrocytes. In FRTL5, expression of GSP or mutant (m) TSHR induced a 2 - 3-fold increase in basal levels of cAMP. This was associated with TSH-independent proliferation (assessed by both cell number and DNA synthesis) and function (as shown by increased expression of thyroglobulin (Tg) and the sodium/iodide symporter). In primary cultures, expression of mTSHR, but not GSP, consistently induced formation of colonies with epithelial morphology and thyroglobulin expression, capable of 10 - 15 population doublings (PD) compared to less than three in controls. Thus, while mTSHR and GSP exert similar effects in FRTL5, use of primary cultures reveals a major difference in their ability to induce sustained proliferation in normal human thyrocytes, and provides the first direct evidence that mTSHR is sufficient to initiate thyroid tumorigenesis.

Hepatocyte Growth Factor (HGF) receptor, encoded by the protooncogene c-met, is overexpressed in many human tumours, including those of thyroid epithelium. The absence in most cases of any primary structural abnormality of the met gene suggests that overexpression is secondary to mutation of other gene(s). To test this hypothesis we investigated the effect on met expression of two activated oncogenes known to play a major role in thyroid oncogenesis, ras and ret. To minimize the possibility of unknown co-operating events, we introduced these genes directly into normal human thyrocytes in primary culture using amphotropic retroviral vectors and assessed met expression as early as possible in the resulting epithelial colonies. Double immunofluorescence revealed expression of met protein, strictly localized to cells expressing the mutant ras and ret vectors, expression in background normal cells being barely detectable. In contrast, colonies induced to proliferate at a comparable rate by a vector expressing SV40 T showed no increase in met expression. To permit quantitation by Western blotting we also extended these findings to a thyroid cell line (R18) containing a zinc-inducible mutant ras gene. Induction of the oncogene led to a fourfold increase in met protein expression. We conclude that overexpression of met is induced by activation of the ras or ret signalling pathway and not simply by deregulation of the cell cycle per se. The data suggest that the proliferative advantage conferred by these oncogenes may be in part due to the resulting sensitization of tumour epithelium to paracrine HGF secreted by stromal cells.

Human apurinic endonuclease/redox factor 1 (APE1/Ref-1) mediates the repair of baseless sites in DNA caused by alkylation and oxidative DNA damage as well as regulates the function of various transcription factors (TF) under redox signaling control (p53, NFkB, HIF-1, AP-1, and others). High levels of APE1 expression have been reported in numerous cancers (glioblastoma, ovarian, pancreatic, prostate, and others) such that APE1 is an emerging target for a variety of novel anticancer drugs. E3330 targets the redox signaling function of APE1 and its activity was studied in vivo using a glioblastoma xenograft mouse model. This preclinical xenograft model evaluated the use of dual anti-angiogenic agents, Bevacizumab (Bev) and E3330, in glioblastoma implanted Nude mice. Four mice groups were used to determine drug effects: E3330, Bev, a combination of E3330 and Bev, and an untreated vehicle control group. Immunohistochemistry (IHC) was used to predict the effectiveness of treatment for glioblastoma based on caspase-3, Ki67, and CD31 biomarker expression. IHC slides were quantified using both a traditional pathology hand count and an image system analysis. The positive pixel data closely mirrored the hand count for all three biomarkers. At the combined dosage, hand count data demonstrated that CD31 levels decreased significantly from the vehicle control group. Ki67 decreased slightly, and caspase-3 was not increased in any of the drug treatment groups. While each drug independently had anti-angiogenic effects, the combined drug group, E3330 and Bev, had anti-angiogenic effects that were greatly enhanced. These results support previous studies demonstrating the anti-angiogenic activity of E3330 and its enhancement of Bevacizumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1769. doi:1538-7445.AM2012-1769