Download - Hypoxic stress proteins: Survival of the fittest

H y p o x i c Stress Proteins: Survival of t h e F i t t e s t AmatoJ. Giaccia The importance of tumor microenvironment in malignant progression has been largely ignored. Tumor cells protect themselves from changes in the microenvironment due to a decrease in nutrients and oxygen by reducing macromolecular synthesis and inducing genes that will promote angiogenesis and tissue remodeling, This ability of transformed cells to survive fluctuations in oxygen tensions is clinically important, as tumors with high hypoxic fractions respond poorly to many forms of cancer therapy. While it is commonly accepted that the decrease in molecular oxygen in hypoxic cells makes them more refractory to killing by agents such as ionizing radiation which use oxygen radical formation, the cessation of division and loss of apoptotic (cell suicide) potential in hypoxic cells are also important in

their resistance to killing by radiotherapy and chemotherapy. In this chapter, we will discuss hypoxia-induced stress proteins in regards to three clinically relevant end points: inhibition of cell proliferation, induction of apoptosis, and regulation of genes modulating angiogenesis. Although these three end points may seem unrelated, in fact, they are intimately linked with each other in the cellular response to hypoxia and malignant progression. One of the goals of this review is to inform both clinician and scientist of these interrelationships and discuss how hypoxia selects for tumors that are clonal expan- sions of cells that have lost their apoptotic ability and have switched to a proangiogenic phenotype. Copyright �9 1996 by W.B, Saunders Company

Inhibition of Cell Proliferation by Hypoxia

Insights From In Vitro Studies

I t has long been known that cells exposed to low oxygen tensions (hypoxia) stop growing) -6 This

inhibition of cell proliferation by hypoxia is not the result of a general effect on the entire cell-cycle, for example due to a lack of energy. Instead, hypoxia specifically inhibits replicon initiation in early S- phase. 7,a Because the two most critical events in the cell-cycle are S-phase where chromosomes are repli- cated and M-phase where duplicated chromosomes are segregated, it is not that surprising that hypoxia prevents DNA replicon initiation. However, it is surprising that even stringent hypoxic conditions (0.02% oxygen) do not impede cells from entering mitosis by the activation of a G~ checkpoint, a common feature of cells exposed to ionizing radiation. 9,1~ The cell-cycle response of cells exposed to hypoxia is to exhibit an immediate inhibition of replicon initiation in early S-phase and an accumulation of Gi-phase cells that cannot enter S-phase. However, S-phase replicons that initiated before exposure to hypoxia will continue to elongate, com-

From the Department of Radiation Oncology, Division ~ Radiation Biology, Stanford University School of Medicine, Stanford, CA:

Supported in part by Grant No. CA64489j%m the National Cancer Institute and ACSjunior faculty research award.

Address reprint requests to AmatoJ. Giaccia, PILD, CBRL, Rm GK220, Division of Radiation Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305-5468.

Copyright �9 1996by W..B. Saunders Company 1053-4296/96/0601-0006505.00/0

plete chromosome replication, enter mitosis, and arrest somewhere in the subsequent cell-cycle between late GI and early S-phase (Fig 1).

Little is known about why cells accumulate in the Gl/S-phase of the cell cycle when exposed to hypoxia, except that this accumulation is different than growth arrest induced by medium depletion (starvation) and DNA damaging agents such as ionizing radiation, ll One distinctive feature of growth arrest induced by hypoxia is that replicon initiation is rapidly (within minutes) resumed when cells are reoxygenated, 7 indicating that all of the essential machinery for replicon initiation is present, but cannot be used. This finding is to be contrasted with cells that require many hours to reengage in cell-cycle activity after release from medium depletion, indicating that these cells do not possess all the essential components for DNA replication and must wait until they accumulate the essential precursors to begin DNA synthesis. The induction of a G j/S-phase arrest by hypoxia has been hypothesized to protect cells from proceeding into S-phase and duplicating their genetic material under adverse conditions that could lead to genomic instability or errors in chromosome duplication. 2,6,12 Interestingly, the reversibility of the inhibition of replicon initiation by hypoxia is lost as the oxygen tension falls below 0.02%. 13

What proteins are needed for replicon initiation? Most of our understanding about eukaryotic DNA replication originates from in vitro studies on simian virus 40 (SV40) replication. I4-I6 Initiation of DNA replication occurs at specifically defined sequences called origins of replication. In the SV40 system, the replicon origin possesses two binding sites for the

46 Seminars in Radiation Oncology, Vol 6, No 1 (January), 1996:pp 46-58

Hypoxia "s Role in Cell Proliferation 47

t ~Low 02 / Inhibition of Replicon initiation

Figure 1. Cell-cycle response of cells exposed to low oxygen. The cell-cycle response of cells exposed to low oxygen conditions is to exhibit an immediate inhibition of replicon initiation in early S-phase and an accumulation of Gl-phase cells that cannot enter S-phase. However, S- phase replicons that initiated betbre exposure to hypoxia will continue to elongate, complete chromosome replication, enter mitosis, and arrest somewhere in the subsequent cell-cycle between late Gffearly S-phase, but will not initiate new replicons.

SV40 encoded protein large T antigen. Once large T antigen binds to these sites, it catalyzes an adenosine triphosphate (ATP) driven DNA unwinding reaction. Following the DNA unwinding reaction, virally encoded large T antigen interacts with the host cell's single-stranded DNA binding protein, replication protein A (RPA). RPA is composed of 70, 34, and 14 kDa molecular weight subunits.17-19 The 70-kDa protein is functionally conserved from bacteria to man and has attracted accessory subunits of 34 and 14 kDa in eukaryotic cells. Although all of the functions of these accessory subunits have not yet been identified, antibodies against the 34-kDa protein will inhibit SV40 DNA replication, indicating that it plays an essential role in DNA replication. 18 The p34 subunit is phosphorylated in a cell-cycle dependent manner and is hyperphosphorylated (a shift of 2 kDa in molecular weight) after cellular exposure to ionizing radiation 2~ and ultraviolet light. 2j The significance of RPA hyperphosphorylation by stress inducing agents is still under investigation, but recent studies seem to indicate that the hyperphosphorylation of the p34 subunit by hypoxia inhibits RPA's single-strand DNA binding activity (C. Koumenis, S. Greene, M. Tsai, R. Fischel, and AJ. Giaccia, unpub- lished observations). Therefore, as RPA binding to single-stranded DNA origins is necessary for replicon initiation, hypoxia could act to inhibit RPA binding activity by inducing its hyperphosphorylation (Fig 2). This relationship raises the intriguing and still specu-

lative possibility that RPA hyperphosphorylation inhibits replicon initiation and signals for early-S-phase arrest under hypoxic conditions. At present, the identity of the kinase (s) that induce hyperphosphorylation of RPA and the pathway that modulates this hypoxia inducible kinase activity are unknown, although several candidate kinases have been identified by in vitro studies? 2

RPA is not the only protein involved in replicon initiation that could be a target for hypoxia. Once large T antigen has catalyzed the unwinding of the

I Ill ~ III I

F i g u r e 2. Hyperphosphorylation of replication proteinA (RPA) and cell-cycle arrest by hypoxia. (A) Under normal replication conditions RPA is a high-affinity DNA single- strand binding protein and is found phospho171ated on its 34-kDa subunit. (B) When cells are exposed to low oxygen conditions, the 34 kDa subunit of RPA is found to be hyperphosphoITlated. (C) The increase in RPA's hyperphosphorylation correlates with a decrease in its ability to bind single-stranded DNA in vitro.

48 AmatoJ. Giaccia

double helix, DNA polymerase a/primase synthe- sizes primers that will permit the initiation of leading strand DNA synthesis and for initiation of each Okazaki fragment during lagging strand synthesis. 23,24 Once primers have been synthesized, replication factor C (RFC, not to be confused with RPA above) displaces pol a/primase and binds with proliferating cell nuclear antigen (PCNA) to act as a "brace-clamp" complex to recruit DNA polymerase 3. 24,25 At this point, initiation of DNA synthesis is complete and elongation of nascent DNA strands proceeds. Recent studies by several groups have shown that PCNA will bind to two different inhibitors: the cyclin-cdk inhibitor p2126 and the growth arrest and DNA damage inducible gene GADD45. 27 Although both p21 and GADD45 proteins will bind with high-affinity to PCNA, only GADD45 mRNA has been found to be inducible by low oxygen conditions. 28 The binding of PCNA with an inhibitor, such as GADD45, prevents it from binding to RFC and creating the necessary brace-clamp structure needed to interact with DNA polymerase, thereby resulting in an inhibition of DNA synthesis. To implicate GADD45 in the inhibition of replicon initiation by hypoxia, future studies will be required to determine if GADD45 protein levels are induced by hypoxia to levels that will physiologically inhibit PCNA activity.

Cell-cycle arrest induced by low oxygen conditions is independent of the activity of the p53 tumor suppressor gene (Fig 3). Previous work in our labora-

G~/S Ratio

AG1522 2184B RC.neo.1RC.IOHL-60 PC-3 DU-145 NIH 3T3

I II II 1 Wild-Type p53 WT pS3 Mutant p53

+ E6

Figure 3. The influence of p53 on cell-cycle arrest by hypoxia. Comparison of the G~ to S-phase ratio of cells under aerobic conditions or after a 14-hour exposure to hypoxia. Cells are grouped according to their p53 genotype: wild-type, mutant, or wild-type expressing HPV-E6 gene. In all human cells examined to date, the GI/S-phase ratio increases under hypoxia, indicative of a cell-cycle arrest. Cell-cycle distributions were determined as previ- ously described by Graeber et al. 11 N, Untreated cells; m, hypoxia-treated cells.

tory '1 indicated that, like ionizing radiation, low oxygen conditions caused the nuclear accumulation of p53 protein, increased p53's DNA binding, and transactivation activity in cells that are wild-type for p53, but not in cells that possess mutant p53. Unlike ionizing radiation, the accumulation of cells in Gl- phase by hypoxia was not solely dependent on wild- type p53 function at low oxygen conditions (0.02% oxygen). These results suggest that there are multiple pathways le~ding to cell-cycle growth arrest, and that the signal transduction pathway for growth arrest induced by hypoxia is at least partially distinct from the signal transduction pathway for growth arrest induced by ionizing radiation.

Detection of Growth Arrest in Experimental Mouse Tumors

The kinetics of tumor cell labeling in situ after the administration of thymidine analogs, such as Budr or Iudr, which can be detected by histochemical meth- ods, have clearly shown that tumor cells closely bordering the lumen of a blood vessel are actively proliferating. 29 Cells that are greater than 100 to 150 ~m from the blood vessel lumen do not actively incorporate thymidine analogs and are not cycling. This recent data using immunohistochemical meth- ods to detect proliferating cells supported the oxygen diffusion model by Tannock, which proposed that chronically hypoxic cells in a tumor stopped cycling and were arrested in GI/S phase? ~ Instead ofimmu- nohistochemistry, cell proliferation in Tannock's studies was assessed by the incorporation of [3HI thymidine into DNA and subsequent autoradiography. As even further proof that hy-poxic cells in vivo arrest in late Gl/early S-phase, Siemann and Keng injected Hoechst 33342 into mice bearing KHT sarcomas, dissociated the tumors and used flow cytometric analysis to examine the cell-cycle profiles of hypoxic cells and oxic cells based on the amount of Hoechst dye uptake.31 The power of this technique rests in the ability to directly compare the cell-cycle profiles of the most "dim" chronically hypoxic cells with the "brightest" oxic cells. Their results demonstrated that the most dim hypoxic cells had the smallest percentage of cells in S-phase, but the largest in G1 compared with bright oxic cells. As cells incorporated more Hoechst, which presumably reflected an increased oxygen uptake, their cell-cycle profile showed more cells in S-phase and a corresponding decrease in cells in Gi phase. Therefore, by three different but related approaches, we can conclude that tumor cells in vivo also exhibit the same biochemical events that

Hypoxia's Role in Cell Proliferation 49

lead to inhibition of replicon initiation in early S-phase.

What is the clinical significance of these results? Cell-cycle checkpoints are a mechanism the cell uses to maintain its genomic integrity. Because cells in S-phase of the cell-cycle are the most sensitive to killing by hypoxia, the early S-phase checkpoint induced by hypoxia, in part, serves to protect the cell from the deleterious effects of entering S-phase under adverse environmental conditions. Therefore, "forcing" hypoxic cells to enter S-phase and initiate DNA replication should lead to celt death. In vitro studies have shown that the supplemental addition of deoxycytidine and uridine will partially reverse the inhibition of replicon initiation by low oxygen conditions. 13 However, the use of exogenously supplied deoxynucleosides to force cells into S-phase is not a practical approach in vivo. A second approach described by Koong et aP 2 was to induce proliferative activity of hypoxic cells by treating them with mitogen stimulating factors, such as phorbol esters that will activate a family of kinases known as the mitogen activated protein kinases (MAP kinases) or extracellular signal regulated protein kinases (ERKs). These kinases respond to diverse extracellular signals, espe- cially growth factors and cytokines, and increase cell proliferation by activating the appropriate transcriptional regulatory factors. 33-35 Koong et al showed that chronically hypoxic cells were extremely sensitive to treatment by agents, such as phorbol esters and thapsigargin, which induced proliferation (Fig 4). As these studies were performed with mitogenic agents that can also act as tumor promotors, the identification of small molecules or growth-factor receptor activating-antibodies, which could be used instead of these tumor promoting agents, would be required for any application of such an approach in the clinic.

Hypoxia-Induced Apoptos is Is Enhanced by t h e Act iv i ty of t h e p53 Tumor Suppressor Gene

Role of p53 in Modulating Apoptosis in Minimally Transformed Cells

The induction of p53 by low oxygen conditions has suggested a potential role for hypoxia in tumorigenesis. If one looks at the well-studied model of colorectal tumorigenesis, it is relatively clear that p53 mutations occur late in the transformation process. 36 Therefore, at some time during the neoplastic process, a colorectal tumor will possess both homozygous wild-type and mutant p53 cells. 36 Eventually, the

tumor becomes more or less a clonal expansion of the mutant p53 tumor genotype. Thus, the question has been raised as to what physiological stimulus in a tumor selects for the growth advantage of mutant p53 tumor cells over wild-type p53 tumor cells. 37,38 Implicit in this statement is that some agent in a tumor is able to cause the activation of wild-type p53 that results in the growth restriction or elimination of wild-type p53 tumor cells. Under the same conditions, mutant p53 cells do not respond to this agent and continue growing. Although ionizing radiation is a strong inducer of wild-type p53, it is not a physiological stress that would be normally found in a developing tumor. 39 The most well-documented physiological stress, which could cause restrictive growth and elimination of wild-type, but not mutant p53 cells in a tumor, is hypoxia.

The supporting evidence for this hypothesis is as follows. Areas of low oxygen do not occur in a tumor until it contains regions further than 150 Ixm from the nearest blood vessel. 4~ Therefore, the presence of hypoxia correlates temporally with the timing of mutations in p53, a late event in several tumor types. Secondly, hypoxia, as we discussed above, is a potent inducer of wild-type p53 activity. II Thirdly, a strong relationship has been found between wild-type p53 activity and stress-induced apoptosis. 41,42 In one model system, genetically matched cell lines differing only in their p53 status were transfected with the E 1A and Ha-ras oncogenes to increase cell proliferation and then exposed to serum deprivation or chemothera- peutic agents. Cell lines homozygous ( + / + ) or heterozygous ( + / - ) for wild-type p53 died an apoptotic death when exposed to these agents both in vitro and in vivo. 41,43 In contrast, homozygous-deleted ( - / - ) p53 cells survived these cellular insults and only a small fraction of the cells died an apoptotic death. Interestingly, clones that were derived from the wild-type populations that survived these above described stresses were now found to be homozygous for mutant/inactive p53.43'Hypoxia fits the necessary criteria for a selective stress, as it induces apoptosis in a p53-dependent manner and the magnitude of this selection would be large enough to enhance the probability for mutant p53 tumor cells to become the predominant genotype in a tumor. 44 Furthermore, developing tumors derived from oncogenically transformed cells that possess wild-type p53 contain sub- stantially more apoptosis than tumors lacking wild- type p53. 44 Apoptosis in tumors that contained wild-type p53 was found distal to blood vessels and

50 AmatoJ. Giaccia

Hypoxia Alone

C

Hypoxia + Thapsigargin

Hypoxia + PDA Hypoxia + TPA

F i g u r e 4. Increased cytotoxicity of human tumor A549 cells treated with hypoxia and protein kinase C activating agents as described by Koong et al. 32 (A) Colony forming ability ofA549 cells exposed to 19 hours of hypoxia (Hypoxia Alone); (B) cells exposed to the combination of 19 hours of hypoxia and 0.5 hours to 2 Ixmol/L of thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase (Hypoxia + Thapsigargin); (C) cells exposed to 19 hours of hypoxia and 0.5 hours to the phorbol ester 4c~-phorbol-12-13-didecanoate (PDA), a phorbol ester that does not bind nor activate protein kinase C (Hypoxia + PDA); (D) cells exposed to 19 hours of hypoxia and 0.5 hours to 1 Ixmol/L of 12-O-tetradecanoylphorbol-13- acetate (TPA), a phorbol ester that binds and strongly activates PKC (Hypoxia + TPA).

co-localized with hypoxic regions 44 (Fig 5), as detected by binding of a hypoxic specific marker EF5. 45 Apoptotic cells in tumors lacking wild-type p53 showed no strong relationship with their distance from a blood vessel, and apoptosis did not colocalize with the hypoxic marker EF544 (Fig 5). These results have

important therapeutic implications. Because the same genes that regulate hypoxia-induced apoptosis also regulate radiation and chemotherapy-induced apoptosis, 41,46,47 hypoxia-mediated selection of cells with diminished apoptotic potential could also explain the poor response of solid tumors to therapy. 43

51 Hypoxia's Role in Cell Proliferation

B

Figure 5. Relationship between hypoxic and apoptotic regions in tumors differing in their p53 status. Tumors were derived from mouse embryonic fibroblasts that were transformed with adenovirus E1A and Ha-ras oncogenes. In situ detection of fragmented DNA by TUN-EL staining (fluorescein-green) of a p53 +/+ tumor (A) and a p53 - / - tumor (C). Hypoxic regions were detected by EF5 staining (orange-red) of the same p53 +/+ tumor (B) and p53 - / - tumor (D) sections. (A) and (C) show both TUNEL and EF-5 staining by using a dual-band pass fluorescence set for direct comparison of apoptotic and hypoxic regions.

Roles of Human PapiUomavirus E6 and E7 in Hypoxia-Induced Apoptosis

Genetic analysis of the human papillomavirus (HPV) genomes of high-risk types have identified two genes, E6 and E7, as being solely sufficient to cause immor- talization of keratinocytes. 48,49 The oncogenic potential of the E6 and E7 genes has been attributed in large part to their interactions with p535~ and Rb 51 proteins, respectively. Although the induction of apoptosis by hypoxia is strongly enhanced by wild- type p53 activity, it is presently unknown whether hypoxia can induce apoptosis in cells expressing the combination of E6 and E7 from high-risk types of HPV. Evidence that supports the functionally important role both proteins play in apoptosis was reported by two different groups studying retina and lens development. 52,53 In one series of experiments, transgenic mice were generated in which HPV E6, E7 or both genes were targeted to the lens. Transgenic mice that only expressed HPV E7 in the lens exhib- ited inhibition of lens differentiation, unregulated

cell proliferation, and apoptosis. In mice that expressed both E6 and E7, apoptosis was suppressed and lens tumors developed. 53 In a second series of experiments, when E7 was targeted to developing photoreceptors in the retina, mice that possessed wild-type p53 would undergo apoptosis in the retina, whereas mice lacking wild-type p53 activity would develop retinoblastoma. 52 Because in many ways E7 is functionally equivalent to the adenovirus E 1A gene in priming cells for apoptosis, its expression should make minimally transformed cells prone to apoptosis when exposed to low oxygen conditions. The unknown question is whether minimally transformed cells expressing both E6 and E7 will die by apoptosis, cell-cycle arrest, exhibit genomic instability, or a combination of all three when subjected to low oxygen conditions. We favor the hypothesis that HPV cells early in the development of cervical carci- noma will die by apoptosis if hypoxia is able to uncouple wild-type p53 from E6-mediated degrada- tion as evidenced by Graeber e t al.ll If this hypothesis

52 AmatoJ. Giaccia

proves to be correct, it may explain why the presence of hypoxia correlates with a poor outcome of cervical cancers independent of treatment modality. 54,55 As the majority of cexvical carcinomas possess a high- risk type HPV and approximately half of these tumors possess oxygen poor regions, hypoxia could select for oncogenically transformed cervical epithe- lial cells that have lost their apoptotic ability and respond poorly to therapy.

Induction of Apoptosis by p53 Independent Pathways

Although wild-type p53 is strongly associated with certain types of apoptosis induced by oncogenic overexpression, there is mounting evidence for the existence of both p53-dependent and independent pathways. 56 If p53 is a proximal step in apoptosis, then it may be possible to induce apoptosis by activating a more distal step in the apoptotic pathway. For example, androgen withdrawal by castra- tion induces apoptotic cell death of prostatic glandular cells in mice that are either homozygous wild-type or homozygous null for p53. 57 Furthermore, the induction of apoptosis is not an "all or none" phenom- enon. If one examines the difference in apoptosis induced by hypoxia between cells expressing wild- type p53 and those not expressing wild-type p53, one finds that wild-type p53 cells are extremely sensitive to induction ofapoptosis, but that a small percentage of mutant p53 cells do undergo apoptosis. This latter finding seems to be particularly true for transformed human cells. 58,59 These results indicate that stress activated apoptotic pathways in oncogenically transformed cells can be activated by other modulators in addition to wild-type p53 or different thresholds of damage are needed to induce apoptosis independent of p53. A second family of genes represented by bcl-2 or bcl-x proto-oncogenes have been shown to inhibit both p53-dependent and independent apoptosis. Mechanistically, bcl-2 protection from apoptosis was thought to involve the scavenging of free radicals, but this is certainly not the case under hypoxic conditions.60, 61

R e g u l a t i o n of A n g i o g e n i c G r o w t h Factors by Low O x y g e n C o n d i t i o n s

Tumor Angiogenesis, the Net Difference Between Negative and Positive Regulators

What is the relationship between hypoxia and angiogenesis? The answer to this question lies in understanding what induces a tumor to become angio-

genic. Human tumors are not highly angiogenic when they initially develop and, furthermore, can remain without vascularization for months to years. 62 However, for the tumor to grow and become malignant or metastatic, neovascularization is required. Therefore, a very likely hypothesis is that tumor regions poor in oxygen possess cells that secrete angiogenic mitogens to overcome their lack of oxygen. According to the current model, hypoxia would only be a potent' inducer of angiogenesis when negative growth regulators such as thrombospondin 1 (TSP1) have decreased in expression. 63 Therefore, understanding how the tumor microenvironment modulates intracellular signaling pathways involved in angiogenic growth factor expression would give us new targets in cancer therapy. As in the case with the loss of tumor suppressor genes, the ability to reacti- vate or modulate angiogenic suppressors in tumor cells may present us with a specific mechanism to inhibit tumor cell growth.

Negative Regulators of Angiogenesis The balance between positive regulators and negative regulators of angiogenesis is stringently con- trolled in normal tissues. However, in neoplastic tissue, this fine balance is tipped in the direction of neovascularization. A net increase in angiogenic growth signals is achieved by attenuation or loss of angiogenic suppressors such as TSP-1. In normal untransformed fibroblasts, TSP-1 is constitutively expressed at a high level. However, tumors derived from transformed cells possess a significantly reduced amount ofTSP-1 compared with their untransformed counterparts. Cells derived from Li-Frau- meni patients serve as a good model system to investigate the relationship between TSP1 activity and wild-type p53 activity. These individuals who are born heterozygous for wild-type p53 possess an increased risk of losing their remaining wild-type allele and developing a tumor. Using fibroblasts derived from Li-Fraumeni patients, Dameron et a164 showed that late passage fibroblasts from these individuals who lost their remaining wild-type p53 allele exhib- ited a marked decrease in TSP-1 levels. If they restored wild-type p53 activity to cells that lost their endogenous p53 activity, they could restore TSP-1 expression to similar levels found in early passage cells. More recently, it has been reported that TSP-1 possesses two p53 regulatory elements, suggesting that it can be modulated by wild-type p53 activity. In untransformed cells, little TSP-1 would be needed, as these cells are under growth factor regulation. In


contrast to untransformed fibroblasts, elevated TSP- 1 levels in minimally transformed cells, stimulated by increased levels of wild-type p5365 would help control tumor growth. Therefore, early events in oncogenic transformation seem to poise the cell to resist the switch to the angiogenesis phenotype by activating p53 to enhance the expression of anti-angiogenic agents.

Let us speculate on how hypoxia may effect malignant progression through the selection of cells that have lost wild-type p53 activity. Hypoxia activates intracellular signaling pathways needed for the transcriptional induction of positive effectors of anglo- genesis, but at the same time, it also increases the activity of anti-angiogenic regulators such as TSP- 1, presumably through the transcriptional activity of p53. To allow minimally transformed cells to com- pletely make the switch to the angiogenic phenotype, hypoxia exerts a selective pressure for the clonal outgrowth of minimally transformed cells that have lost their apoptotic ability. As a consequence of this clonal outgrowth, the expression of negative angiogenic regulators such as TSP-1 will be attenuated or lost as wild-type p53 expression is lost. Therefore, the net result of all these interactions will be the selection for transformed cells that have reduced apoptotic potential and are primed for the switch to the angiogenic phenotype (Fig 6).

Positive Regulators of Angiogenesis Compared with our dirth of knowledge about negative regulators of angiogenesis, the identity and mechanism of action of angiogenesis activators is voluminous. Positive angiogenic activators can be divided into two groups--growth factors and cytokines. Two of the most potent angiogenic polypep- tides are fibroblast growth factor (FGF) and vascular endothelial cell growth factor (VEGF). 66 Both the acidic and basic forms of fibroblast growth factor stimulate proliferation of cells that embryonically originated from mesoderm and neuroectoderm, 67,6a promote endothelial cell migration, 6a and modulate

Normal

Selection for cells [ a" ptohrtetdu;edentlal] Hyperplasia ~ Pre-Mallgnant ~ Malignant Growth ~ Metastasis

Growth l t . y . o x , a ~ l~;'re;;:~o~;'J::l

Figure 6. Schematic of how hypoxia enhances malignant progression.

protease and plasminogen expression. 69,7~ In contrast to FGF, VEGF is an endothelial cell specific growth factor. 71,72 Both FGF and VEGF activate proteases that are needed to degrade the basement membrane to allow for the formation of new blood vessels. 7~

Hypoxia increases the transcriptional activity of VEGF, 74'75 FGF, 76 and platelet derived growth factor PDGF] 7,7a Recent studies have brought forth evidence that the induction ofVEGF gene expression by hypoxia is through a nuclear protein designated hypoxia inducible factor-1 (HIF-1), 7a-al This factor was originally identifed in the transcriptional regulation of the erythoprotein (EPO) gene by hypoxia, but has since been found in many cell types that do not express EPO. a2 In addition, numerous genes encoding glycolytic enzymes such as phosphoglycerate kinase 1 (PGK1)possess HIF-1 regulatory sequences and are transcriptionally regulated by HIF-1 under hypoxic conditions, a~ Taken together, these results indicate that HIF-1 is a unique hypoxia inducible transcription factor that is ubiquitous in many different cell types and regulates a wide variety of genes involved in cellular homeostasis.

The signal transduction pathway involved in HIF- 1/VEGF induction by hypoxia is modulated by membrane associated kinases such as the src and ras proto-oncogenes. If kinase defective alleles of either src or ra t are overexpressed in cells before exposure to hypoxia, VEGF induction is inhibited a3 (N.M. Mazure, E.Y. Chen, P. Yeh, N. Mivechi, K.R. Laderoute, and AJ. Giaccia, manuscript in preparation). Because the growth factor receptor poison suramin, which is presently in clinical trials, is also able to inhibit VEGF induction by hypoxia, growth factor receptors must also play a role in signaling for VEGF induction and present a realistic target for novel therapeutic strategies. Figure 7 summarizes the possible sequential activation of kinases in the signaling pathway for VEGF induction by hypoxia. Clearly, understanding the signal transduction pathway inw)lved in VEGF, FGF, and PDGF induction would present new molecular targets for cancer therapy.

Working cooperatively with growth factors, cytokines also aid in tumor angiogenesis in two important ways. In one way, they work as paracrine effectors. For example, tumor necrosis factor (TNF)~ is induced by hypoxia in some cell types that are resident in a tumor such as mononuclear cells. 84,a5 This localized TNF~ production will then induce endothelial cells to produce basic FGF. FGF will then stimulate mitogenesis and migration of more endo-

54 AmatoJ. Giaccia

HYPOXIA

Growth Factor Receptors/Src

Ras

Pl3- Kinase

Diacylglycerol

PKC

?

HIF-1/VEGF

Suramin, Genistein

N17 Ras (dorninente negative)

Wortrnannin

Calphostin C

Figure 7. Signal transduction pathway for the induction of HIF- 1/VEGF by hypoxia.

thelial cells until neovascularization takes place. The second way cytokines such as transforming growth factor (TGF)[3 function to enhance angiogenesis is by modulating the cellular production of extracellular matrix proteins, a6

Several of the cytokines mentioned above have been shown to be induced by hypoxia. In contrast to the regulation of growth factors and glycolytic enzymes by HIF-1, cytokine regulation by hypoxia has not extensively been studied. However, as most of the cytokines implicated in angiogenesis possess regulatory sequences to which the transcription factor NF-KB binds, 87 and NF-KB is directly activated under hypoxic conditions, 88,a9 it seems highly prob- able that transcriptional regulation of cytokine gene expression by hypoxia is, in part, through NF-KB regulatory elements.

How Can Our Knowledge of the Genes Involved in Angiogenesis be Therapeutically Exploited?

One approach to this problem is to inhibit the intracellular signaling pathway involved in the activation of angiogenic growth factors or cytokines. One common feature to both VEGF induction by HIF-1 and cytokine induction by NF-KB is the requirement of ras signaling. 89,9~ In normal cells this signaling is transitory, but in tumor cells that possess mutated forms of ras, this activity is constitutively "turned on. ''91 Genetically, ras activity can be inhibited by dominant negative mutant alleles ofras that prevent transmission of extra cellular signals through ra~ to downstream effectors. 92,93 Although, the use of domi-

nant mutant alleles of ras would be impractical therapeutically, recent success has been made in inhibiting rm- activity in cell culture systems by blocking its ability to associate with the cell membrane, ras requires posttranslational modification in the form of isoprenylation by the enzyme farnesyl- protein transferase (FPTase) to associate with the cell membrane. 94,95 Several studies using different assays have shown that the present group of FPTase inhibitors are selective for farnesylated proteins and inhibit transformed cell proliferation? 6 Without question, ras represents an excellent molecular target for cancer therapy. However, the ability to selectively, and with minimal toxicity, inhibit ras activity in a tumor awaits animal testing.

A second approach that would clearly have the largest impact on cancer therapy would be to restore wild-type p53 activity. Although this idea at one time would have seemed unrealistic, several lines of evidence suggest that it may be possible to achieve. In tumors, p53 mutations do not occur randomly and are clustered within the central core region of the protein. 97,98 Several different groups have shown that specific p53 monoclonal antibodies or second site suppressor mutations can restore p53's DNA binding activity to some mutant forms of the protein. 99,1~176 Although both of these approaches would not be clinically useful, they do indicate that in some cases wild-type p53 activity can be restored to mutant forms of p53.

One possible therapeutic approach that is now being actively pursued is to develop a small organic molecule that would be able to tether mutant p53 to its binding sequence in a gene. Implicit in this approach is that p53's transcriptional enhancing is required for it to act as a tumor suppressor gene. However, p53's role as a transcriptional activator may not be necessary to induce apoptosis.101,J0~ There- fore, the heroic task of finding a small molecule that can restore p53's transcriptional activity could be complemented by hunting for small molecules that can restore p53's role in apoptosis.

C o n c l u s i o n s

Our appreciation for how the tumor microenvironment influences malignant progression is only begin- ning. Recent studies have provided us with important insights into how regions of low oxygen in a tumor can select for the clonal expansion of a transformed cell that has lost its ability to kill itself when faced with inadequate growth conditions. Al-


though hypoxia can act as a selective pressure in

exper imenta l systems both in vi tro and in vivo, it still

remains to be d e t e r m i n e d whe the r hypoxia selects

for apoptot ic deficient t u m o r cells in h u m a n neopla-

sias. This ques t ion will be best addressed in neo-

plasms that progress th rough pathologically distinct

s tages dur ing thei r deve lopment , where one can

associate the loss o fapoptos i s in hypoxic regions wi th

the acquisi t ion of genet ic a l terat ions that will de-

crease apoptot ic potent ia l such as p53 muta t ion or

bcl-2 overexpression.

Al though the inhibi t ion of cell prol i ferat ion by

hypoxia is th rough a p53 independen t pathway,

wild-type p53 ac tMty is increased by cellular expo-

sure to hypoxia. In the cellular response to hypoxia,

p53 plays several impor t an t roles. One , it signals for

the induct ion o f apoptosis when oxygen tension falls

below 0.2% oxygen. Two, it may transcript ionally

increase the expression of ant i -angiogenic factors

such as TSP- 1. Wi th both of these critical regula tory

roles, the possibility o f res tor ing wild-type p53 func-

tion would be o f p a r a m o u n t impor tance in reversing

the cellular phenotype acquired by its loss.

In conclusion, the old d o g m a tha t regions of low

oxygen tension in a t u m o r would m a k e cells refrac-

tory to killing by rad io therapy and some chemothe ra -

peut ic agents requires a m e n d m e n t . Viable hypoxic

cells that are refractory to cell killing may be a

by-product of a selective process for cells that have

lost the i r endogenous sensitivity (apoptosis) to low

oxygen conditions and at the same t ime have adopted

a pro-angiogenic phenotype. Because the same genes

that modu la te hypoxia- induced apoptosis also modu-

late radio therapy and chemotherapy- induced apopto-

sis, hypoxia-media ted selection of cells wi th dimin-

ished apoptot ic potent ia l could also explain the

resistance of some tumors to these same therapies.

A c k n o w l e d g m e n t The author thanks Luis Alvarez, Eunice Chen, Thomas Graeber, Susannah Green, Albert Koong, Constantinos Koumenis, Nath- alie Mazure, and Mitchell Tsal for their contributions to the preparation of the manuscript, and Tess Gonzales for typing the manuscript.

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