Investigating Hypoxic Tumor Physiology Through Gene Expression Patterns

Investigating hypoxic tumor physiology through gene expression patterns

Nicholas C Denko*,1, Lucrezia A Fontana1, Karen M Hudson1, Patrick D Sutphin1, Soumya Raychaudhuri2, Russ Altman2 and Amato J Giaccia1

1Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA; 2Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA

Clinical evidence shows that tumor hypoxia is an relyupon oxygen-dependent energyproduction suffer independent prognostic indicator of poor patient outcome. when oxygen falls below a certain threshold level. Hypoxic tumors have altered physiologic processes, However, too much oxygen can also result in the including increased regions of angiogenesis, increased generation of toxic, damaging radical byproducts. The local invasion, increased distant metastasis and altered net effect of these opposing pressures necessitates apoptotic programs. Since hypoxia is a potent controller the evolution of molecular mechanisms to regulate the of gene expression, identifying hypoxia-regulated genes is cellular availabilityand usage of oxygen. a means to investigate the molecular response to hypoxic The response to low-oxygen conditions has been stress. Traditional experimental approaches have identi- identified in both procaryotes and eucaryotes. Although fied physiologic changes in hypoxic cells. Recent studies the mechanisms for gene regulation in response to low- have identified hypoxia-responsive genes that may define oxygen conditions are diverse, parallel systems have the mechanism(s) underlying these physiologic changes. evolved in different organisms that are transcriptionally For example, the regulation of glycolytic genes by responsive to low-oxygen. This convergent evolution hypoxia can explain some characteristics of the Warburg suggests that the abilityto respond to low-oxygen effect. The converse of this logic is also true. By conditions offers a competitive Darwinian advantage. identifying new classes of hypoxia-regulated gene(s), we Although the magnitude of the ‘hypoxic’ response may can infer the physiologic pressures that require the differ between unicellular and multicellular organisms, induction of these genes and their protein products. theyboth share the principal goal of maintaining the Furthermore, these physiologically driven hypoxic gene critical energylevels required for homeostasis, while expression changes give us insight as to the poor outcome extracellular oxygen concentrations decrease. While this of patients with hypoxic tumors. Approximately 1–1.5% review is focused on the transcriptional changes in the of the genome is transcriptionally responsive to hypoxia. pathophysiologic environment of hypoxic tumor cells, However, there is significant heterogeneity in the tran- the reason whyhypoxicgene regulation evolved is scriptional response to hypoxia between different cell obviouslyin response to a more physiologic condition of types. Moreover, the coordinated change in the expression hypoxia such as occurs during embryonic development of families of genes supports the model of physiologic (Chen et al., 1999a) or during wound healing (Warren pressure leading to expression changes. Understanding the et al., 2001). evolutionary pressure to develop a ‘hypoxic response’ provides a framework to investigate the biology of the hypoxic tumor microenvironment. Mechanisms of gene regulation in response to hypoxia Oncogene (2003) 22, 5907–5914. doi:10.1038/sj.onc.1206703

Keywords: hypoxic gene induction; HIF-1; VHL Hypoxia/anoxia-responsive gene expression has been well characterized in procaryotic organisms such as bacteria as well as in lower and higher eucaryotes such as yeast and mammals, respectively. Unicellular organisms have less control over environmental oxygen, so their hypoxia response is designed to utilize efficiently Introduction the decreased levels of oxygen. The facultative anaerobic bacterium Escherichia coli utilizes the fumarate, nitrate Evolution of the transcriptional ‘hypoxic response’ reduction (FNR) protein, which is a bifunctional protein Molecular oxygen is beneficial to many organisms due that acts both as a hypoxic sensor and a hypoxia- to its role in efficient energyproduction. Organisms that responsive transcription factor. Under aerobic conditions, FNR exists as an inactive apoprotein, but under hypoxia, it forms an active homodimer that is dependent *Correspondence: NC Denko; E-mail: [email protected] Supplemental data is available at: http://cbrl.stanford.edu/hypoxia/ on a redox-sensitive 4Fe–4S cluster (Green et al., 2001). welcome.htm In contrast, one mechanism that yeast Saccharomyces cerevisiae uses for hypoxic gene expression is a heme- Functions of hypoxia responsive genes NC Denko et al 5908 regulated repressive system based on the ROX1 blood cells, especially in times of systemic hypoxia, such (repressed in oxygen) molecule. Under aerobic condi- as during travel to high altitudes (Caro, 2001). The tions, abundant heme activates the HAP1 complex that enhancer element responsible for EPO induction under in turn transactivates the ROX1 gene. ROX1 protein hypoxia was identified (Imagawa et al., 1991), and the blocks expression of a large set of hypoxia-responsive protein that bound to this hypoxia-responsive element genes (Becerra et al., 2002). Under hypoxic conditions, was purified, cloned, and found to be a heterodimer heme-deficient HAP1 represses ROX1 transcription, (Wang and Semenza, 1993). One subunit of the dimer and the hypoxia-responsive genes are derepressed was constitutivelyexpressed in cells (HIF-1 b or ARNT), (Zitomer et al., 1997). These are two examples of the and the other component was oxygen labile and diverse mechanisms that have evolved in single-cell stabilized in cells exposed to low oxygen (HIF-1a) procaryotes and lower eucaryotes to respond to low- (Jiang et al., 1996). The molecular mechanism of HIF oxygen concentrations. activation has been recentlyelucidated, largelybecause The mammalian transcriptional response to hypoxia of the functional interaction between HIF-1a and the is considerablymore complicated, relying on multi- tumor suppressor protein von Hippel–Lindau (VHL). protein complexes to regulate several transcription Under normoxic conditions, HIF-1a is rapidlyde- factors, the most well studied being (hypoxia-inducible graded, with a half-life of a few minutes. In contrast, factor 1 (HIF-1)). General stress-responsive transcrip- under hypoxic conditions, HIF-1a becomes stabilized tion factors such as AP-1, NF-kB and Egr1 have also (Maxwell et al., 1999), associates with the HIF-1b been reported to be regulated byhypoxiaand/or subunit, translocates to the nucleus, and binds in a reoxygenation (Faller, 1999; Ten and Pinsky, 2002). sequence-specific manner to a hypoxia-responsive ele- However, the sensitivityof these factors to mild ment in target genes to activate transcription. The hypoxia, and the duration of their transcriptional regulation of HIF-1a stabilityhas been shown to rely response is much less than that of HIF-1. The ‘general heavilyupon the VHL tumor suppressor protein stress responsive’ transcription factors are therefore not (Maxwell et al., 1999). Under normoxia, HIF-1a is well suited to regulating gene expression in the rapidly hydroxylated at proline 564 (Ivan et al., 2001; chronicallyhypoxic tumor microenvironment. Further- Jaakkola et al., 2001) bya novel familyof proline more, the number of genes that are upregulated bynon- hydroxylases (Bruick and McKnight, 2001; Epstein HIF mechanisms in response to chronic hypoxia seems et al., 2001). The hydroxylated form of HIF-1a binds to be small compared to the HIF-responsive genes. to the VHL protein that is part of the multiprotein In contrast to gene induction byhypoxia, several complex containing elongins B and C and CUL1 (Hon mechanisms of gene repression byhypoxiahave also et al., 2002; Min et al., 2002). This complex acts to been reported. Repression can contribute to the hypoxic ubiquitinate HIF-1a and target it to the proteasome for response bydownregulating such genes as the anti- degradation (Maxwell et al., 1999). Under hypoxia, angiogenic thrombospondin (TSP) genes (Tenan et al., HIF-1a is not hydroxylated (Chan et al., 2002), does not 2000). The TSP genes function to block new blood vessel bind to VHL, and becomes stabilized. formation, so theyare presumablydownregulated by Studies of the VHL tumor suppressor have identified hypoxia in order to stimulate angiogenesis (Laderoute a role for hypoxia-regulated genes in modifying et al., 2000). Recent in vitro data have suggested that malignant transformation in human tumors (Kondo hypoxic downregulation of gene expression can be et al., 2002). VHL has been identified as a classic tumor through the induction of the (negative cofactor 2 suppressor gene that requires the loss of both alleles to (NC2)) transcriptional repressor (Denko et al., 2003). generate disease (Friedrich, 1999). The von Hippel– NC2 has been shown to block gene expression by Lindau syndrome is a result of the germline loss of one regulating core promoter action through binding to the VHL allele, with the loss of the second allele resulting in TATA-associated factor TBP (Kamada et al., 2001). In high-frequencyrenal cell carcinoma, pheochromocyto- addition, the P53 tumor suppressor gene has been found mas and hemangioblastomas (Friedrich, 1999). In the to associate with the transcriptional corepressor mSin3A absence of VHL, HIF-1a exhibits increased stabilityin under hypoxia, and may also be involved in repressing a normoxic conditions and stimulates constitutive expres- subset of genes in these conditions (Koumenis et al., sion of HIF-responsive genes under normoxic condi- 2001). Further studyis needed to define all the genes tions (Wykoff et al., 2000). Thus, it is thought that one that are repressed in response to hypoxia, and that are factor contributing to VHL’s role as a tumor suppressor regulated through the activityof either P53, NC2, or is its abilityto downregulate the expression of hypoxia- other mechanisms (Yun et al., 2002). responsive genes under normoxic conditions (Kondo et al., 2002). The importance for HIF-1-regulated genes in driving tumor development is supported bystudies on murine HIF-1 regulation in mammalian cells tumor cell lines that are deficient in either HIF-1a (Ryan et al., 1998) or HIF-1b (Maxwell et al., 1997). Both HIF1 was identified through the investigation of the tumors that are derived from HIF-1a and HIF-1b- hypoxia-responsive gene erythropoetin (EPO) (Gold- deficient cells exhibit reduced aggressiveness in allo- berg et al., 1988). EPO is the growth factor responsible grafted tumor formation in immune-deficient mice for stimulating the bone marrow to produce new red (Maxwell et al., 1997; Ryan et al., 1998). The reduced

Oncogene Functions of hypoxia responsive genes NC Denko et al 5909 tumor formation of HIF-deficient tumor cells has been formation of new blood vessels is unable to keep pace attributed to several factors, including decreased levels with growing tumor cells. The result is a constant of vascular endothelial growth factor (VEGF) secretion production of hypoxia-dependent changes that are never that leads to decreased levels of angiogenesis in vivo able to establish a uniform normoxic environment. The (Grunstein et al., 1999). continuous subversion of the normal hypoxic response is what gives the hypoxic tumor its unique phenotype (Denko and Giaccia, 2001). Understanding the normal Physiologic versus pathophysiologic hypoxia hypoxic response can therefore give insight into how it can lead to the pathophysiologic effects. Mice in which HIF-1a has been deleted fail to develop beyond day 10 (Iyer et al., 1998; Ryan et al., 1998). This observation suggests that HIF-1a or hypoxia-regulated Tumor hypoxia and clinical correlates genes are necessaryfor normal embryonic development. One target hypoxia-responsive gene that has been Tumor oxygenation can be directly measured in vivo in hypothesized to be in large part responsible for these patients bymicroelectrodes or PET imaging through effects is the vascular endothelial growth factor A hypoxic marker binding (Hockel and Vaupel, 2001). (VEGFA) gene. Embryos deficient in a single copy of Numerous clinical studies have demonstrated that the VEGFA do not develop (Carmeliet et al., 1996; Ferrara pretreatment oxygenation status of solid tumors can be et al., 1996). The direct link between hypoxia and VEGF used to stratifypatients prospectively.These studies is found when the HRE of VEGF is specifically indicate that mean oxygen status below 10 mmHg in the mutated, and mice also show a partial embryonic tumor predicts for poor survival in patients with tumors lethalityat day10 (Oosthuyse et al., 2001). These of the head and neck (Nordsmark et al., 1996), or cervix animal studies reinforce an essential role for hypoxia- (Hockel et al., 1996) or in soft tissue (Brizel et al., 1996). responsive genes during embryonic development. Phy- The poor prognosis of hypoxic tumors is independent of siologic hypoxia occurs during development, wound treatment modality, with patients treated surgically healing, exposure to increased elevation, or other suffering a similarlypoor outcome as patients treated transient vascular alterations (Elson et al., 2000). These with radiotherapy(Hockel et al., 1996). Most impor- examples are conditions in which the hypoxic state tantly, the clinical data suggest that hypoxic tumors elicits a response and that response in turn ‘cures’ the represent a biologicallymore aggressive tumor, in hypoxic insult. Wounding causes vascular damage that addition to one that is more resistant to oxygen- leads to the induction of VEGF, the reestablishment of dependent therapies such as radiation (or some che- blood flow through new vessels and a return to a motherapies) (Teicher, 1994). normoxic state (Figure 1). In contrast to the physiologic hypoxia described above, tumor hypoxia is an example of a chronic, pathophysiologic condition. The difference lies in the Target genes regulated by hypoxia fact that the response is unable to resolve completelythe hypoxic insult (Dvorak, 1986) (Figure 1). Despite What are the specific genes that are responsible for the hypoxia-induced VEGF production in the tumor, the hypoxic tumor phenotype, and how do they give the hypoxic tumor its increased predisposition to invade, metastasize and apoptose? Several groups have used genomic approaches to identifygene expression profile changes in hypoxia (Denko et al., 2000a; Koong et al., 2000; Wykoff et al., 2000; Scandurro et al., 2001). Thus far, the different cell types examined in vitro, the relative level and duration of hypoxia used, and the resultant gene profiles have underscored the heterogeneityof the induced genes. An analysis of these various studies indicates that there is a core set of genes that are induced consistentlybyhypoxiaand a large number of genes that exhibit cell-type-specific induction. The differential response of various cell types emphasizes the specialized roles for the different cellular components of the solid tumor. Some of the more specific examples of cell-type- dependent hypoxia-responsive genes include the production of tyrosine hydroxylase (TH) by cells of the carotid body(Millhorn et al., 1996), or the production of EPO bycells of the juxtaglomerular apparatus (Fandreyand Bunn, 1993). However, some genes are induced by Figure 1 Model showing comparison of physiologic stress hypoxia in a large number of diverse cell types, such as response to pathophysiologic stress response VEGF or glucose transporter 3 (GLUT3). Members of

Oncogene Functions of hypoxia responsive genes NC Denko et al 5910 the family of glycolytic enzymes are also induced in almost all cell types, even if the specific members may not be induced in all (Seagroves et al., 2001). The cellular need to generate energyseems to be an obvious universal response to hypoxia, while production of TH or EPO is required in a more specialized cellular context. One example of expression profiling cells treated in vitro with hypoxia is shown in Figure 2. The hypoxic profile of six cell types, representing normal cervical and dermal keratinocytes (NCK, NDK), normal stromal fibroblasts (NCF) and transformed keratinocytes (Siha, C33a, FaDu), is shown. In this cDNA arrayof 6800 genes, 110 genes were found to be hypoxia-responsive, with 84 being induced, 24 being repressed and three showing a mixed response of induction in some cells with repression in others. Extrapolating from these data, approximately1.5% of the genome is transcriptionally responsive to hypoxia. This frequency of hypoxia- responsive genes agrees with previous findings using a much smaller array(Koong et al., 2000). The hypoxia-responsive genes identified in this experiment are ordered in Figure 2 byboth cell type and induction pattern for displaypurposes. A red signal represents induction, and green represents repression, with maximal changes of approximately10-fold. Using this set of genes, cell types were ordered for the similarityof hypoxic response byprinciple component analysis (Raychaudhuri et al., 2000). PCA orders the cell lines as depicted in Figure 2 in the order of similarity and shows that the normal keratinocytes are most closelyrelated, the normal fibroblasts more distantly related and the tumor lines even more distant from the normal keratinocytes. These two axes, the similarity of cell type in the x-dimension, and the level of induction in the y-dimension describe the representation of the ‘hypoxiatome,’ as depicted in Figure 2. Figure 3 shows a panel of selected genes used as probes for Northern blot comparison of techniques. Interestingly, the differences in hypoxia-responsive profiles cannot be simplyexplained bythe fact that the cells were geneticallyunstable tumor cells. The normal stromal and normal epithelial cells also show differences in their hypoxic profiles. These data underscore the importance of studying hypoxic gene expression in a cell-type-dependent context. From this sample, we can see that some genes are widelyinducible, such as NIP3L, IGFBP3 and Dec1, but manygenes are inducible onlyin certain cellular contexts, such as placental growth factor or ephrin A1. Identifying the additional factors regulating hypoxic gene responsiveness will continue to be a major point of investigation.

" Figure 2 Expression proﬁle changes in response to long-term hypoxia. The subset of genes with robust expression changes was selected as described in the text. Expression ratios were converted into log base 2 values; these are plotted in matrix form, with cell lines listed across the top and gene name and accession number on the side. Boxes are colored to indicate relative expression levels. The red values indicate induction, while green values indicate repression with maximal changes of approximately10-fold

Oncogene Functions of hypoxia responsive genes NC Denko et al 5911 Table 1 Categorization of selected hypoxia-regulated genes by function Metabolism References

Glucose transporter 1, 3 Ebert et al. (1995), O’Rourke et al. (1996) 6-Phosphofructo-2 kinase Ebert et al. (1996) Phosphoglycerate kinase Salceda et al. (1996) Aldolase A, C Ebert et al. (1996), Semenza et al. (1994) Triose phosphate isomerase Niitsu et al. (1999) Lactate dehydrogenase A, B Semenza et al. (1994) Glycogen branching enzyme This work Solute carrier family6, 16 This work Carbonic anhydrase IX Ivanov et al. (2001)

Angiogenesis VEGF A, B C D Enholm et al. (1997), Shweiki et al. (1992) VEGF R1 Plate et al. (1993) Placental growth factor Gleadle et al. (1995) Angiopoietin 2 Mandriota and Pepper (1998) Adrenomedullin Nakayama et al. (1998) Endothelin 1, Endothelin 2 Kourembanas et al. (1993), Koong et al. (2000) Ephrin A1 This work Nitric oxide synthase Melillo et al. (1995) COX1 COX2 This work, Schmedtje et al. (1997) Thrombospondin 1, Tenan et al. (2000), this work Thrombospondin 2

Tissue remodeling Lysyl oxidase This work PLOD2 This work Integrin 5alfa Suzuma et al. (1998) Plasminogen activator Pinsky et al. (1998) inhibitor 1 Urokinase plasminogen Graham et al. (1998) activator receptor LDLR related protein Koong et al. (2000) Tissue factor Yan et al. (1998) Mucin 1 This work

Apoptosis BNIP3 BNIP3L Bruick (2000) IGFBP1, 3 Tazuke et al. (1998), Koong et al. (2000) Pim1, Pim-2 This work Bcl-w like This work

Proliferation/differentiation BTG1 This work Cyclin G2 Wykoff et al. (2000) Figure 3 Northern analysis of hypoxic induction in several test DEC1/stra13 Wykoff et al. (2000) genes. The same series of cells lines were treated with hypoxia in Adipophilin Saarikoski et al. (2002) vitro, and Northern blot was used to determine hypoxic gene p21 CDKI Denko et al. (2000b) regulation. Representative genes are shown for several of the functional classes (GAPDH, glyceraldehye 3-phosphate dehydro- Gene expression genase; G6PI, glucose 6-phosphate isomerase; GBE, glycogen Earlygrowth response 1 Yan et al. (1999) branching enzyme; VEGF, vascular endothelial growth factor; p35srj Bhattacharya et al. (1999) PGF, placental growth factor; EphA1, ephrin A1; PLOD2, lysyl ETS-1 Oikawa et al. (2001) hydroxylase 2; PAI-1, plasminogen activator inhibitor 1; TF, tissue Mxi-1 This work factor; IGFBP3, insulin-like growth factor binding protein 3; GRP78 GRP94 ORP150 Ozawa et al. (2001), Roll et al. BNIP3, 19 K interacting protein; DEC1, differentiated in (1991) embryonic chondrocytes; BTG1, B-cell translocation breakpoint Prolyl-4-hydroxylase Epstein et al. (2001) gene)

Coordinated regulation suggests functional categories of response induced genes into functional categories. Table 1 is a compilation of data from our lab as well as data from Based on the published function(s) of the various the literature. The six largest functional groups are hypoxia-responsive genes, we grouped the most robustly shown and represent genes involved in metabolism/

Oncogene Functions of hypoxia responsive genes NC Denko et al 5912 transport, angiogenesis, tissue remodeling, apoptosis, and BNIP3L (Bruick, 2000). While the kinetics of proliferation/differentitation and gene expression. In BNIP3 induction correlate with apoptosis, there are addition to the five major categories, there are many several characteristics of its expression pattern that still interesting genes that fall into smaller functional need to be reconciled. For example, VHL-negative cells categories such as mRNA processing (RNAse L, are able to withstand constitutive expression of these CDC-like kinase 1, CCR4-associated protein), or genes supposedlyapoptogenic molecules (Sowter et al., 2001), with unknown functions (HIG2, RTP, RAIG3, and normal fibroblasts can induce high levels of BNIP3 TGFBind, VitDind68). Some genes, such as PAI-1, expression in the absence of apoptosis (Figures 2 and 3, can be placed in multiple categories of tissue remodeling and data not shown). Normoxic expression of BNIP3/ and angiogenesis. BNIP3L in the VHL-negative tumor cells could lead to the selection of cells with a second site mutation somewhere downstream in the apoptotic signaling pathway(Graeber et al., 1996). Hypoxia and metastasis Alternatively, it is possible that BNIP3/BNIP3L have different functions under hypoxia from those reported in The physiological demands of hypoxia and the response transfection studies under normoxia (Chen et al., to those demands in the various cell types result in 1999b). We could therefore gain new insight into the different gene expression patterns. For example, lysyl function(s) of BNIP3/BNIP3L bythis supposed dis- hydroxylase (PLOD2, Figures 2 and 3) is normally crepancybetween hypoxicinduction and apoptosis. required for collagen maturation and is therefore While BNIP3/BNIP3L induced byhypoxiacould still be normallyonlyexpressed in fibroblasts (Wang et al., targeted to the mitochondria just as the normoxic 2000). However, under hypoxia, both primary and experiments report (Chen et al., 1999b), their biochem- transformed keratinocytes induce high levels of PLOD2 ical roles under hypoxia may be different. It is possible expression, while fibroblasts exhibit little change in their that the mitochondrion has physiological pressures PLOD2 mRNA levels. The same environmental signal under hypoxia that require BNIP3/BNIP3L expression, elicits two different responses in the two cell types. and it is onlyunder normoxic conditions that its While the reason for PLOD2 upregulation in hypoxic overexpression is apoptogenic. epithelial cells is unclear, we hypothesize that it is related to the wounding/re-epithelialization response of the keratinocytes. The intimate interaction between stromal and epithelial cells in the hypoxic solid tumor combines Additional levels of regulation to regulate PLOD2 expression and, presumably, collagen maturation. The alterations in the extracellular Finally, there are genes that are induced by hypoxia in matrix of hypoxic tumors could in turn contribute to some cell types, while they are repressed by hypoxia in hypoxia-induced metastasis (Denko & Giaccia, 2001; De other cell types with unknown factors controlling this Jaeger et al., 2001). regulation, such as ornithine decarboxylase (ODC) and Furthermore, the hypoxic profiles of the untrans- glucose-regulated protein 78 (GRP78). These examples formed cells are more closelyrelated to each other than make it clear that the definition of a hypoxia-responsive to the tumor cells. For example, while tumor cells gene is cell-type dependent. There are also multiple exhibit a significant downregulation in the basal level of levels of gene regulation within the same family. For PAI-1 expression (Figure 2), hypoxic upregulation is example, lactate dehydrogenase isoform A (LDHA) is found both in the untransformed cells and in the tumor stronglyinduced in all cell lines, while LDH B is cells (Figures 2 and 3). Likewise, hypoxia can upregulate repressed in all the cell lines tested (Figure 2). Thus, we another member of the plasmin pathway, urokinase- have examples of added complexityto hypoxic gene type plasminogen activator receptor, which has also expression, multiple levels of control of hypoxic been implicated in metastasis (Postovit et al., 2002). The response between cells of different origins and multiple induced response, irrespective of the transformed levels of control between members of a single gene phenotype, suggests that the physiological demands family. upon the cells have a dominant effect on their expression patterns. Thus, one can see how alteration of the extracellular protease plasmin byPAI-1 byboth Conclusions hypoxia and transformation could also combine to contribute to hypoxia-induced metastasis (Denko and We can infer that physiologic changes in the hypoxic Giaccia, 2001; De Jaeger et al., 2001). tumor drive gene expression changes, and these in turn result in the different cellular effects within that tumor. For example, low oxygen stimulates angiogenesis, and Apoptotic response to hypoxia the establishment of new vessels requires tissue remodeling. The genes involved in tissue remodeling maythen be P53 independent, hypoxia-induced apoptosis is thought part of the explanation as to whyhypoxic tumors are to relyupon the hypoxicupregulation of certain more likelyto be locallyinvasive or distantlymetastatic. proapoptotic genes, such as BH3-containing BNIP3 The expression changes in the lysine hydroxylase gene

Oncogene Functions of hypoxia responsive genes NC Denko et al 5913 PLOD2 in keratinocytes and tumor cells is an example and BNIP3L are robustlyinduced in all hypoxic cells of how gene expression profiling can allow us to infer regardless of their apoptotic potential makes one new insights about tumor physiology in hypoxic tumors. question their role in hypoxia-induced apoptosis. The Keratinocytes are able to play an active role in collagen targeting of BH3-onlymolecules to the mitochondria maturation in the hypoxic solid tumor instead of the mayrepresent a survival response to impaired mito- fibroblast that is traditionallythought to serve this chondrial function under hypoxic conditions. The function. The interaction between hypoxia, fibroblasts function of the BH3 proteins maybe verydifferent in and tumor cells in tissue remodeling represents a cell- this environmental context than under control condi- context-dependent regulation of gene expression to tions. We therefore need to design our functional assays result in a coordinated tissue response. Understanding to take into account the conditions in which the gene that tumor cells are an active participant in this process products are expressed. mayinfluence the development of targeted antimeta- static chemotherapeutics in the future. Acknowledgements Furthermore, we maylearn how the biochemical This work was supported in part byVarian Biosynergyand characterization of hypoxic gene products may also be NIH Grant CA67166. LF was supported bya predoctoral influenced by the cellular physiology. For example, fellowship from Fondazione Italiana per la Ricerca sul seeing that the potentiallyapoptogenic genes BNIP3 Cancero.

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