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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.

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies. ispartof: SCIENCE ADVANCES vol:7 issue:19 ispartof: location:United States status: published

Adaptive responses to hypoxia are mediated by the hypoxia-inducible factor (HIF) family of transcription factors. These responses include the upregulation of glycolysis to maintain ATP production. This also generates acidic metabolites, which require HIF-induced carbonic anhydrase IX (CAIX) for their neutralisation. C-terminal binding proteins (CtBPs) are coregulators of gene transcription and couple glycolysis with gene transcription due to their regulation by the glycolytic coenzyme NADH. Here, we find that experimental manipulation of glycolysis and CtBP function in breast cancer cells through multiple complementary approaches supports a hypothesis whereby the expression of known HIF-inducible genes, and CAIX in particular, adapts to available glucose in the microenvironment through a mechanism involving CtBPs. This novel pathway promotes the survival of stem cell-like cancer (SCLC) cells in hypoxia.

Mechanisms driving adaptive developmental responses to chronic high-altitude (HA) exposure are incompletely known. We developed a novel rat model mimicking the human condition of cardiopulmonary adaptation to HA starting at conception and spanning the in utero and postnatal timeframe. We assessed lung growth and cardiopulmonary structure and function and performed transcriptome analyses to identify mechanisms facilitating developmental adaptations to chronic hypoxia. To generate the model, breeding pairs of Sprague-Dawley rats were exposed to hypobaric hypoxia (equivalent to 9,000 ft elevation). Mating, pregnancy, and delivery occurred in hypoxic conditions. Six weeks postpartum, structural and functional data were collected in the offspring. RNA-Seq was performed on right ventricle (RV) and lung tissue. Age-matched breeding pairs and offspring under room air (RA) conditions served as controls. Hypoxic rats exhibited significantly lower body weights and higher hematocrit levels, alveolar volumes, pulmonary diffusion capacities, RV mass, and RV systolic pressure, as well as increased pulmonary artery remodeling. RNA-Seq analyses revealed multiple differentially expressed genes in lungs and RVs from hypoxic rats. Although there was considerable similarity between hypoxic lungs and RVs compared with RA controls, several upstream regulators unique to lung or RV were identified. We noted a pattern of immune downregulation and regulation patterns of immune and hormonal mediators similar to the genome from patients with pulmonary arterial hypertension. In summary, we developed a novel murine model of chronic hypoxia exposure that demonstrates functional and structural phenotypes similar to human adaptation. We identified transcriptomic alterations that suggest potential mechanisms for adaptation to chronic HA.

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.

The EGLN (also called PHD) prolyl hydroxylase enzymes and their canonical targets, the HIFα subunits, represent the core of an ancient oxygen-monitoring machinery used by metazoans. In this review, we highlight recent progress in understanding the overlapping versus specific roles of EGLN enzymes and HIF isoforms and discuss how feedback loops based on recently identified noncoding RNAs introduce additional layers of complexity to the hypoxic response. Based on novel interactions identified upstream and downstream of EGLNs, an integrated network connecting oxygen-sensing functions to metabolic and signaling pathways is gradually emerging with broad therapeutic implications.

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.

Osteocytes play a critical role in mediating cell-cell communication and regulating bone homeostasis, and osteocyte apoptosis is associated with increased bone resorption. miR21, an oncogenic microRNA, regulates bone metabolism by acting directly on osteoblasts and osteoclasts, but its role in osteocytes is not clear. Here, we show that osteocytic miR21 deletion has sex-divergent effects in bone. In females, miR21 deletion reduces osteocyte viability, but suppresses bone turnover. Conversely, in males, miR21 deletion increases osteocyte viability, but stimulates bone turnover and enhances bone structure. Further, miR21 deletion differentially alters osteocyte cytokine production in the two sexes. Interestingly, despite these changes, miR21 deletion increases bone mechanical properties in both sexes, albeit to a greater extent in males. Collectively, our findings suggest that miR21 exerts both sex-divergent and sex-equivalent roles in osteocytes, regulating osteocyte viability and altering bone metabolism through paracrine actions on osteoblasts and osteoclasts differentially in males vs. females, whereas, influencing bone mechanical properties independent of sex.

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

While erythropoietin (EPO) constitutes the major treatment for anemia, a range of anemic disorders remain resistant to EPO treatment. The need for alternative therapeutic strategies requires the identification of mechanisms that physiologically restrain erythropoiesis. Here we show that P38α restrains erythropoiesis in mouse and human erythroblasts independently of EPO by integrating apoptotic signals during recovery from anemia. P38α deficiency promotes JNK activation through increased expression of Map3k4 via a negative feedback mechanism. JNK prevents Cdk1-mediated phosphorylation and subsequent degradation by Smurf2 of the epigenetic silencer Ezh2. Stabilized Ezh2 silences Bim expression and protects erythroblasts from apoptosis. Thus, we identify P38α/JNK signaling as a molecular brake modulating erythropoiesis through epigenetic silencing of Bim. We propose that inhibition of P38α, by enhancing erythropoiesis in an EPO-independent fashion, may provide an alternative strategy for the treatment of anemia., Erythropoietin (EPO) stimulates erythropoiesis and is commonly used to treat anemia. Here Hu et al. find that P38α/JNK signaling restrains erythropoiesis independently of EPO by regulating epigenetic silencing of the proapoptotic protein Bim, and thus identify putative targets for the treatment of anemic disorders resistant to EPO.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA., G.A.C. is The Alan M. Gewirtz Leukemia & Lymphoma Society Scholar. Work in G.A.C.’s laboratory is supported in part by the NIH/NCI grants 1UH2TR00943-01 and 1 R01 CA182905-01, the UT MD Anderson Cancer Center SPORE in Melanoma grant from NCI (P50 CA093459), Aim at Melanoma Foundation and the Miriam and Jim Mulva research funds, the Brain SPORE (2P50CA127001), the Center for Radiation Oncology Research Project, the Center for Cancer Epigenetics Pilot project, a 2014 Knowledge GAP MDACC grant, a CLL Moonshot pilot project, the UT MD Anderson Cancer Center Duncan Family Institute for Cancer Prevention and Risk Assessment, a SINF grant in colon cancer, the Laura and John Arnold Foundation, the RGK Foundation, and the Estate of C.G. Johnson, Jr. I.B.-N. was financed by a grant entitled Non-Invasive Intelligent Systems for Colorectal Cancer Diagnosis and Prognosis Based on circulating miRNAs Integrated in the Clinical Workflow – INTELCOR. S.M.G.D., A.L.B.A., and D.A. are supported by the São Paulo Research Foundation FAPESP under grants 2014/15968-3, 2014/20673-2, and 2014/17820-3, respectively. W.L. was partly supported by grants from The University of Texas MD Anderson Cancer Center Sheikh Ahmed Bin Zayed Al Nahyan Center for Pancreatic Cancer Research. J.A.B. was supported by the Cancer Center Support Grant (P30 CA016672), and the HP imaging program of the Small Animal Facility (SAIF) was supported by the Cancer Prevention and Research Institutes of Texas grant RP-101243P5. H.L. was supported by NIH/NCI grant R01CA175486, a grant (RP140462) from the Cancer Prevention and Research Institute of Texas, and the R. Lee Clark Fellow Award from The Jeanne F. Shelby Scholarship Fund. I.B.-N. was financed by a Fulbright fellowship and by a grant entitled Non-Invasive Intelligent Systems for Colorectal Cancer Diagnosis and Prognosis Based on circulating miRNAs Integrated in the Clinical Workflow – INTELCOR.

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor κ-light chain enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma. Although a significant amount of research has centered on Ref-1, no wide-ranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the nuclear factor erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve reactive oxygen species. The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications.

Cells adjust to hypoxic stress within the tumor microenvironment by downregulating energy-consuming processes including translation. To delineate mechanisms of cellular adaptation to hypoxia, we performed RNA-Seq of normoxic and hypoxic head and neck cancer cells. These data revealed a significant down regulation of genes known to regulate RNA processing and splicing. Exon-level analyses classified > 1,000 mRNAs as alternatively spliced under hypoxia and uncovered a unique retained intron (RI) in the master regulator of translation initiation, EIF2B5. Notably, this intron was expressed in solid tumors in a stage-dependent manner. We investigated the biological consequence of this RI and demonstrate that its inclusion creates a premature termination codon (PTC), that leads to a 65kDa truncated protein isoform that opposes full-length eIF2Bε to inhibit global translation. Furthermore, expression of 65kDa eIF2Bε led to increased survival of head and neck cancer cells under hypoxia, providing evidence that this isoform enables cells to adapt to conditions of low oxygen. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splicing potential at the RI, hypoxia-induced expression and binding of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to cotranscriptional processing lead to selective retention of a PTC-containing intron in EIF2B5., Author summary Tumor hypoxia is a negative prognostic factor for many solid cancers. Cellular adaptation to hypoxia is largely mediated by widespread changes in gene expression and enables cancer cells to adjust and survive. Recently, alternative splicing has been implicated in this process. To identify biologically impactful hypoxia-responsive isoforms, we took an unbiased approach to deeply sequence RNA of normoxic and hypoxic head and neck cancer cells. This analysis identified >1,000 mRNAs as alternatively spliced under hypoxia, including a significant enrichment of alternatively spliced genes involved in adaptation to hypoxia. Most notably, we discovered a unique retained intron in the translation initiation factor EIF2B5 that creates a premature termination codon. We show that this retained intron leads to a 65kDa truncated isoform that opposes full-length eIF2Bε to inhibit global translation and enhances survival of head and neck cancer cells under hypoxia. Strikingly, this intron and several additional hypoxia-induced retained introns were overexpressed in solid tumors relative to normal tissues. Mechanistically, we propose that intron retention under hypoxia is influenced by changes to RNA polymerase II (RNAPII) activity at weak 3′ splice sites and carry out experimental validation for the retained intron in EIF2B5 to investigate intron retention under hypoxia.

Significance: The emerging connections between an increasing number of long noncoding RNAs (lncRNAs) and oncogenic hallmarks provide a new twist to tumor complexity. Recent Advances: In the present review, we highlight specific lncRNAs that have been studied in relation to tumorigenesis, either as participants in the neoplastic process or as markers of pathway activity or drug response. These transcripts are typically deregulated by oncogenic or tumor-suppressing signals or respond to microenvironmental conditions such as hypoxia. Critical Issues: Among these transcripts are lncRNAs sufficiently divergent between mouse and human genomes that may contribute to biological differences between species. Future Directions: From a translational standpoint, knowledge about primate-specific lncRNAs may help explain the reason behind the failure to reproduce the results from mouse cancer models in human cell-based systems. Antioxid. Redox Signal. 00, 000–000.

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

Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named ‘hypoxia-induced noncoding ultraconserved transcripts' (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.

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: 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.

miR-210 is a key player of cell response to hypoxia, modulating cell survival, VEGF-driven endothelial cell migration, and the ability of endothelial cells to form capillary-like structures. A crucial step in understanding microRNA (miRNA) function is the identification of their targets. However, only few miR-210 targets have been identified to date. Here, we describe an integrated strategy for large-scale identification of new miR-210 targets by combining transcriptomics and proteomics with bioinformatic approaches. To experimentally validate candidate targets, the RNA-induced silencing complex (RISC) loaded with miR-210 was purified by immunoprecipitation along with its mRNA targets. The complex was significantly enriched in mRNAs of 31 candidate targets, such as BDNF, GPD1L, ISCU, NCAM, and the non-coding RNA Xist. A subset of the newly identified targets was further confirmed by 3′-untranslated region (UTR) reporter assays, and hypoxia induced down-modulation of their expression was rescued blocking miR-210, providing support for the approach validity. In the case of 9 targets, such as PTPN1 and P4HB, miR-210 seed-pairing sequences localized in the coding sequence or in the 5′-UTR, in line with recent data extending miRNA targeting beyond the “classic” 3′-UTR recognition. Finally, Gene Ontology analysis of the targets highlights known miR-210 impact on cell cycle regulation and differentiation, and predicts a new role of this miRNA in RNA processing, DNA binding, development, membrane trafficking, and amino acid catabolism. Given the complexity of miRNA actions, we view such a multiprong approach as useful to adequately describe the multiple pathways regulated by miR-210 during physiopathological processes.

MicroRNAs (miRs) are short non-coding transcripts involved in a wide variety of cellular processes. Several recent studies have established a link between hypoxia, a well-documented component of the tumour microenvironment, and specific miRs. One member of this class, miR-210, was identified as hypoxia inducible in all the cell types tested, and is overexpressed in most cancer types. Its hypoxic induction is dependent on a functional hypoxia-inducible factor (HIF), thus extending the transcriptional repertoire of the latter beyond ‘classic’ genes. From a clinical standpoint, miR-210 overexpression has been associated with adverse prognosis in breast tumours and been detected in serum of lymphoma patients and could serve as a tool to define hypoxic malignancies. We discuss the role of miR-210 and its emerging targets, as well as possible future directions for clinical applications in oncology and ischaemic disorders.

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.

MicroRNAs (miRNAs) are 19 – 24 nucleotide noncoding RNAs that regulate the translation and degradation of target mRNAs and are extensively involved in human cancers. One unexpected conclusion of the profiling and functional studies in tumourigenesis is that some miRNAs behave in cancer cells in a dual mode, resembling the ‘Dr Jekyll and Mr Hyde’ story, which centers on a conception of humanity as dual in nature. The authors and others have found that onco-miRNAs and suppressor-miRNAs can represent two different looks of the same gene, behaving as oncogenes or tumour suppressors depending on tissue type and specific targets. In this review, the authors analyse the regulatory mechanisms of the main miRNA genes involved in human tumourigenesis.

SummaryHematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

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

HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel–Lindau tumor suppressor protein (pVHL). We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe 2+ , this protein modification may play a key role in mammalian oxygen sensing.

The von Hippel-Lindau tumor suppressor protein (pVHL) has been shown to bind directly to the alpha subunits of the heterodimeric transcription factor HIF (hypoxia inducible factor). pVHL directs the polyubiquitination and, hence, destruction of HIF in the presence of oxygen. Loss of pVHL function leads to deregulation of HIF target genes, which play critical roles in angiogenesis.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel-Lindau protein (pVHL). Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. This is the first function to be ascribed to the pVHL beta-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.

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.

Point mutations of the gsp protooncogene (encoding the alpha-subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227--Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 10(5) infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

Point mutations of the gsp protooncogene (encoding theα -subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227→Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10–20 well differentiated epithelial colonies/dish of 105 infected cells, with an average lifespan of 15–20 population doublings, only small groups of no more than 15–50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

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.

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.

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.

To understand the mechanisms of ceramide-based responses to hypoxia, we performed a mass spectrometry-based survey of ceramide species elicited by a wide range of hypoxic conditions (0.2-5% oxygen). We describe a rapid, time-dependent, marked up-regulation of dihydroceramides (DHCs) in mammalian cells and in the lungs of hypoxic rats. The increase affected all DHC species and was proportional with the depth and duration of hypoxia, ranging from 2- (1 h) to 10-fold (24 h), with complete return to normal after 1 h of reoxygenation at the expense of increased ceramides. We demonstrate that a DHC-based response to hypoxia occurs in a hypoxia-inducible factor-independent fashion and is catalyzed by the DHC desaturase (DEGS) in the de novo ceramide pathway. Both the impact of hypoxia on DHC molecular species and its inhibitory effect on cell proliferation were reproduced by knockdown of DEGS1 or DEGS2 by siRNA during normoxia. Conversely, overexpression of DEGS1 or DEGS2 attenuated the DHC accumulation and increased cell proliferation during hypoxia. Based on the amplitude and kinetics of DHC accumulation, the enzymatic desaturation of DHCs fulfills the criteria of an oxygen sensor across physiological hypoxic conditions, regulating the balance between biologically active components of ceramide metabolism.

Mammalian gene expression patterns change profoundly in response to low oxygen levels. These changes in gene expression programs are strongly influenced by post-transcriptional mechanisms mediated by mRNA-binding factors: RNA-binding proteins (RBPs) and microRNAs (miRNAs). Here, we review the RBPs and miRNAs that modulate mRNA turnover and translation in response to hypoxic challenge. RBPs such as HuR (human antigen R), PTB (polypyrimidine tract-binding protein), heterogeneous nuclear ribonucleoproteins (hnRNPs), tristetraprolin, nucleolin, iron-response element binding proteins (IRPs), and cytoplasmic polyadenylation-element-binding proteins (CPEBs), selectively bind to numerous hypoxia-regulated transcripts and play a major role in establishing hypoxic gene expression patterns. MiRNAs including miR-210, miR-373, and miR-21 associate with hypoxia-regulated transcripts and further modulate the levels of the encoded proteins to implement the hypoxic gene expression profile. We discuss the potent regulation of hypoxic gene expression by RBPs and miRNAs and their integrated actions in the cellular hypoxic response.

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

We describe the existence in normal human primary thyroid cultures of a hitherto unrecognised sub-population of epithelial cells. This variant phenotype is characterised by squamoid morphology, absence of thyroglobulin, and an altered profile of intermediate filament expression. We suggest that these cells are derived from scattered foci of squamous metaplasia present in the normal gland. Although they are initially present at a frequency of less than 10- −4 , their very high proliferative capacity enables them to outgrow the ‘classical’ follicular cells and confers a much increased capacity for gene transduction. Recognition of these cells is therefore crucial in the interpretation of long-term thyroid culture experiments and those involving in vitro gene transfer.