Upon activation by phosphorylation, the half-life of the protein increases dramatically and p53 protein levels accumulate in the cell and mediate its many tumor suppression activities.
The inhibition of angiogenesis by p53 has been shown to occur through three basic mechanisms: downregulation of the hypoxia-inducible system HIF , transcriptional repression of proangiogenic genes, and transcriptional upregulation of angiostatic factors. Each of these mechanisms will be discussed in turn.
A general property of all mammalian cells is the ability to detect when they are in an environment with insufficient oxygen hypoxia. In response to hypoxia, cells activate a transcriptional program that promotes the production of new blood vessels to supply the oxygen-starved region. PHDs use molecular oxygen as a substrate, and therefore their activity is dependent on oxygen tension in the cell. Although p53 is best known to increase the expression of target genes, in some cases it has also been shown to repress genes that promote tumor growth.
The genes encoding two major secreted factors that promote angiogenesis in tumors, VEGF and bFGF, were both shown to be transcriptionally repressed by p The gene encoding cyclo-oxygenase-2 COX-2 has also been shown to be repressed by p In addition to their role in inflammation, prostanoids have been shown to mediate production of proangiogenic factors and stimulate tumor growth. As a result of their effects in promoting angiogenesis, COX-2 inhibitor compounds are being evaluated as potential cancer therapeutics.
As mentioned in the previous section, p53 is best known as a transcription factor that promotes the expression of genes that limit tumor growth. Such genes encode proteins that induce cell-cycle arrest, mediate DNA damage, and repair and initiate apoptosis.
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These effects are classified as cell-autonomous in that they act directly on the cells that express p In addition, p53 regulates many genes that exert non-cell-autonomous effects, meaning that the cells expressing p53 are stimulated to produce factors that act on other cells and potentially other cell types. An example of such an effect is when tumor cells with activated p53 induce the transcription of secreted factors that have antiangiogenic properties. Some of the most powerful endogenous angiogenesis inhibitors have been shown to be under the control of the p53 tumor suppressor.
Most angiogenesis inhibitors are proteins that are secreted into the extracellular matrix ECM and directly act upon endothelial cells to inhibit cell division or induce cell death. One of the first characterized examples of an angiogenesis inhibitor, Tsp- 1, was also one of the first proteins demonstrated to be a transcriptional target of p Tsp-1 has been shown to inhibit angiogenesis through several mechanisms, including binding to a cell surface receptor on endothelial cells called CD Like p53, the expression of Tsp-1 has been shown to be able to completely reverse the angiogenic switch and render tumors dormant.
Many angiostatic factors are proteolytic fragments derived from components of the ECM. Among the many proteins that are secreted into the ECM are the collagens. Collagens are a family of about 40 proteins that have important structural properties in multicellular organisms. With respect to angiogenesis, collagen types 4 and 18 are critical components of a specialized ECM around blood vessels called the vascular basement membrane VBM.
Increasingly, such collagens have also been shown to be a significant source of signaling molecules that can affect angiogenesis. Six different types of collagen have been demonstrated to contain antiangiogenic C-terminal domains that can be released upon proteolytic processing see Table 1. For example, endostatin is the best characterized collagen-derived antiangiogenic factor CDAF and is located at the C-terminus of collagen. The collagen family of proteins is classified by the presence of the collagen repeats comprising the amino acid sequence Gly-X-Y, where X is often a proline residue and Y is a 4-hydroxyproline.
A mature collagen molecule is formed when the collagen repeats of three separate collagen chains intertwined into a triple helix. Formation of this helical structure absolutely requires hydrogen bonding by the 4-hydroxyproline residues. Without these modified amino acids, the structure of the collagens would be unstable. For this reason, collagen production is dependent on the expression of the collagen prolyl hydroxylase enzymes in order to be formed. The most abundant collagen prolyl hydroxlase is P4HA1, which is responsible for producing the vast majority of collagen in connective tissues.
Insufficient prolyl hydroxylase activity, which occurs when its essential cofactor vitamin C is lacking from the diet, causes scurvy, demonstrating the importance of this enzyme in collagen biosynthesis. Recently, we were able to demonstrate that p53 upregulates the expression of another collagen, prolyl hydroxlase P4HA2.
Unlike P4HA1, P4HA2 is not ubiquitously expressed and is not likely to be involved in the formation of collagen in connective tissues. The overexpression of P4HA2 in lung cancer cells increased the production of collagen 4 and 18 and stimulated the release of the CDAFs tumstatin and endostatin. In addition to increasing expression of the prolyl hydroxylase enzyme required for collagen biosythesis, p53 has also been shown to upregulate expression of at least two different collagen genes.
Thus, p53 is able to initiate an entire transcriptional program that stimulates cells to secrete CDAFs to inhibit tumor angiogenesis. Since p53 upregulates a collagen prolyl hydroxylase enzyme that is a rate-limiting step in collagen biosynthesis, this mechanism has the potential of increase production of any or all of the CDAF molecules listed in Table 1.
Tumor-suppressor pathways such as p53 function in part by reducing the angiogenic potential of transformed cells.
The p53 Tumor-suppressor Gene—Inhibiting Tumor Angiogenesis
Limiting angiogenesis by p53 occurs through the dual action of inhibiting proangiogenic signals and increasing production of angiostatic factors. The major lesson that can be learned from p53 is that effectively shutting down angiogenic potential requires the execution of both of the above mechanisms. Current cancer therapies targeting angiogenesis function exclusively by inhibiting proangiogenic factors. Indeed, existing angiostatic drugs such as bevacizumab, sorafenib, and sunitinib all function through the common mechanism of inhibiting signaling through the VEGF pathway—but what of the second mechanism of increasing angiostatic factors such as the CDAFs and Tsp-1?
No significant progress has been made toward utilizing antiangiogenic factors such as Tsp-1 or CDAFs in the clinic despite years of pre-clinical data showing that these proteins are powerful antiangiogenic agents. One of the difficulties that has impeded discovery of drugs that function through antiangiogenic pathways is that the molecular mechanisms of these signals seem to be more complex than their proangiogenic counterparts.
For example, Tsp-1 has been reported to interact with several different receptors, although much of the angiostatic activity is thought to be mediated through the CD36 receptor.
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The use of Tsp-1 as a therapeutic is also complicated by the fact that it is a very large protein that would be difficult to produce in sufficient quantities for treatment. This is not the case for the CDAFs, which are relatively small 16—20kDa peptides and can be adapted almost directly as drugs. There is evidence that increasing production of CDAFs can have a significant effect on tumor growth.
Studies in mice have shown that increasing levels of endostatin by only 1. Unfortunately, when phase II clinical trials for endostatin were carried out for the treatment of advanced neuroendocrine tumors, no significant tumor regression was observed.
However, several clinical trials are being carried out in China using a modified version of endostatin called Endostar. Results of trials using chemotherapy in combination with Endostar for treatment of lung cancer have been reported to show significantly greater tumor regression than patients receiving chemotherapy alone. We have observed the p53 tumor suppressor to stimulate the release of at least two different CDAFs, endostatin and tumstatin. Among them are uncontrolled proliferation, increased survival, enhanced motility, invasiveness, metastasis, and ability to induce angiogenesis Hanahan and Weinberg, Since both p53 and Ras are key regulators of cell proliferation and survival, several studies focused on searching for molecular candidates that may link p53 and Ras in this context.
Another important feature of tumor cells is enhanced motility, which facilitates invasion and metastasis. In this context, Xia and Land found that the combination of oncogenic H-Ras and mutant p53 enhances cell motility to a much greater extent than each of the alterations alone. This was evident in several assays, including time-lapse video microscopy, wound-healing capacity, migration through Boyden chambers, and lattice structures formation in soft agar.
A supportive study was recently published by Muller et al. These two studies extend our understanding of the pRas interplay by providing evidence and molecular links that connect the p53 and Ras pathways in the context of invasion and metastasis. To prevent neoplastic transformation, normal cells harbor defense mechanisms against uncontrolled proliferation, which can be triggered by oncogene activation.
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These mechanisms involve the induction of cell-cycle arrest, senescence , or cell death, and are mediated mainly by the p53 and retinoblastoma protein pRb pathways Lin et al. Therefore, when the Ras oncogene is activated in normal cells, it will lead eventually to cell-cycle arrest and induction of the senescence program, which can be circumvented by inactivation of p53 or p16 INK4A. Using in vivo imaging , Morton et al. Furthermore, while K-Ras G12D expression in conjunction with both WT-p53 loss or mutant p53 expression promoted tumor formation, only the concomitant expression of mutant p53 RH and oncogenic K-Ras G12D promoted a highly metastatic phenotype, indicating a direct oncogenic function for mutant p Despite the fact that cellular senescence obviously protects the cell from malignant transformation , senescent cells in the periphery of a tumor can support hyper-proliferation of the tumor cells and contribute to cancer pathology.
This was suggested to be mediated by the secretion of inflammation-associated molecules from the senescent cells to the surrounding tissue. These molecules promote epithelial to mesenchymal transition and invasiveness.
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Both oncogenic Ras, which induces senescence in normal cells, and WT-p53 loss markedly amplify the secretion of these molecules upon senescence induction, leading to an amplified pro-malignant phenotype Coppe et al. These data suggest a paracrine role for p53 loss and Ras activation, by which they support malignancy in adjacent cells. One of the challenges of cancer research and, in particular, of personalized medicine , is to identify the antigens expressed by tumors.
McMurray et al. Knockdown experiments demonstrated that most of these genes are important for tumor development, as evident by attenuation of tumor growth in mice. The identified genes were classified into a broad range of functional annotations such as signal transduction, metabolism, cell adhesion, etc. More important is the fact that these genes respond synergistically to the oncogenic combination of mutant p53 RH and H-Ras V12 oncogene, suggesting that malignant cells strongly rely on the genes that are cooperatively regulated by these two oncogenic events.
Recently our laboratory developed an in vitro transformation model in which normal human fibroblasts were immortalized and were introduced with defined oncogenic alterations Milyavsky et al. Microarray analysis of cells cultures that represent defined stages of malignant progression revealed an inflammation-associated gene signature , which was synergistically induced following the concomitant expression of H-Ras G12V and the inactivation of p Accordingly, cells with increased expression of this pro-inflammatory signature demonstrated an increased ability to form tumors in mice Buganim et al.
A similar set of genes was also reported to be induced by either p53 loss or expression of oncogenic Ras in the context of premature senescence in fibroblasts Coppe et al. In this case, the expression and secretion of these proteins was suggested to promote tumorigenesis by supporting proliferation, invasiveness, and epithelial to mesenchymal transition of adjacent tumor cells.
Interestingly, this gene signature is associated not only with senescence but also with the inflammation process. Inflammation is considered to play a tumor-suppressive role in its controlled form, but to facilitate tumorigenesis when it persists in the form of chronic inflammation by promoting genome instability, cell proliferation, and angiogenesis. Among the various inducers of chronic inflammation is oncogenic stress , and accordingly, oncogenic Ras was demonstrated to promote tumorigenesis by inducing pro-inflammatory genes Schetter et al.
RhoA is a small GTPase, which functions as a main regulator of cell motility. It was recently found that H-Ras V12 positively regulates RhoA activity by recruiting it to the cell membrane, where RhoA can be activated. This in turn results in increased cell motility and invasiveness Xia and Land, While the convergence of the p53 and Ras pathways in the regulation of cancer-related processes is relatively well understood, the effects of p53 and Ras on each other are less studied.
It is well documented that oncogenic stress such as Ras oncogene expression can induce WT-p53 stabilization via the induction of p14 ARF , which in turn inhibits the negative regulator p53, HDM2. Ras can promote the expression and activation of PML and PRAK, leading to an enhancement of p53 activity via post-translational modifications.
However, even less is known about the functions exerted by p53 on Ras. Ras functions in a switch mechanism, by which it can be activated or inactivated depending on the signal sensed. When bound to GTP, Ras is found in a conformation allowing it to bind to its downstream effectors and turn on the diverse signaling pathways.
Oncogenic Ras mutants are locked in the active state, resulting in un-controlled signaling Downward, Based on these facts, our laboratory used human immortalized fibroblasts that express oncogenic H-Ras G12V and measured the effect of p53 knockdown or mutant p53 RH overexpression on Ras levels and activity. While p53 inactivation did not affect H-Ras expression, it enhanced its activity, i. As mentioned above, studies on the molecular mechanisms that underlie the counteracting role of p53 on Ras-induced transformation yielded the identification of ATF3 and BTG2 as important mediators of this p53 function Boiko et al.
It was demonstrated that knockdown of either ATF3 or BTG2 is accompanied by activation of pRb, which is associated with increased cell proliferation and represents a key event in tumorigenesis. These in turn mediate the induction of a cancer-related gene signature, which is synergistically up-regulated by oncogenic H-Ras and p53 inactivation. Thus, our findings suggest multiple mechanisms by which p53 counteracts uncontrolled activation of the Ras pathway, inhibiting Ras activity and the expression of its downstream targets. Altogether, these lines of evidence strongly suggest that the regulation of p53 on the Ras pathway underlies the synergistic effects mediated by p53 loss and Ras activation.
The presented studies in this review link two key pathways that regulate cell fate. The molecular cooperation between these pathways is manifested both in normal and transformed cells.
The p53 Tumor-suppressor Gene—Inhibiting Tumor Angiogenesis
In response to oncogenic Ras signaling, WT-p53 is stabilized, leading to premature senescence and serving as a roadblock on the slippery road to cancer. However, as frequently occurs in most cancer types, WT-p53 is inactivated, either by point mutation, deletion, or other aberrations in the p53 pathway Buganim and Rotter, This inactivation cooperates with oncogenic Ras to promote tumorigenesis by affecting virtually all the hallmark processes that are associated with tumorigenesis, including inflammation, proliferation, motility, invasion, metastasis, and angiogenesis.
The molecular mechanisms that underlie the induction of these processes are triggered by both p53 inactivation and oncogenic Ras activation. We have demonstrated for the first time that WT-p53 inhibits Ras activity, suggesting a novel tumor-suppressive function of p Thus, when p53 function is inhibited, it allows high levels of activated H-Ras. This observation can explain many processes induced by the combination of p53 loss and oncogenic Ras expression.
For example, cells expressing mutant p53 together with Ras oncogene showed accelerated motility. Considering our own data, it is also possible that inactivation of p53 results in both increased levels of activated H-Ras in conjunction with inactivation of p RhoGAP, which together lead to over-activation of RhoA, and cellular motility. The regulation of p53 on Ras and its downstream pathways can also explain the synergistic upregulation of cancer-related genes Buganim et al.
This synergism is essential for the induction and maintenance of malignancy; and therefore it is clinically important to unravel the molecular mechanisms that underlie it. A main gene group that is synergistically induced by p53 inactivation and H-Ras oncogene expression is related to inflammation, a process tightly associated with cancer development. This adds another aspect to the cross-talk between p53 loss and Ras oncogene in induction of malignancy. As p53 and Ras are two of the most altered genes in human cancer, the described studies broaden our understanding of the core molecular events leading to cellular transformation, and encourage the application of the accumulated knowledge on the cooperation between p53 and Ras for the development of novel strategies for cancer diagnosis and treatment.
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