Angiogenesis in Cancer and Neurologic Disease
Angiogenesis is a process by which endothelial cells migrate, proliferate, and organize to form new blood vessels. Blood vessels are essentially everywhere in our body. If all the blood vessels in the body were lined up, they would circle the earth twice. The total surface area covered by endothelial cells in an adult is ~1000 m2. Angiogenesis is essential for various physiological processes, including reproduction, development and wound repair. It also features in many pathological conditions. Both excessive and insufficient angiogenesis are associated with many debilitating diseases such as cancer and neurodegeneration (Figure 1). Angiogenesis has been considered as a disease common denominator. Over 600 million patients worldwide will benefit from either antiangiogenesis or proangiogenesis therapy. Our lab focuses on cancer and neurodegeneration.
Figure 1. Examples of angiogenesis-related diseases.
Angiogenesis is a multistep process controlled by the net balance between stimulators and inhibitors. For example, tumor angiogenesis has been shown to include: (i) sprouting, (ii) intussusception, (iii) formation of extended ‘mother vessels’, (iv) ‘splitting’ of mother vessels and formation of ‘daughter vessels’, (v) vascular fusion, (vi) recruitment of circulating endothelial progenitor cells, (vii) cooption and modification of pre-existing blood vessels, and (viii) inclusion of tumor cells into the walls of vascular channels. It is now well understood that normally quiescent endothelial cells become invasive and protrude into the perivascular tissues in response to angiogenic stimuli. As the endothelial cells sprout, proteases are activated causing the surrounding basement membrane to lyse, allowing the cells to migrate. The adjacent cells divide to occupy the space created by the migrating cells. By a continuous process of penetration, migration, proliferation and differentiation, the endothelial cells eventually form a new capillary network. Smooth muscle cells are subsequently recruited to migrate along the newly formed endothelium. They proliferate and deposit extracellular matrix components for the formation of vessel walls and interact with endothelium to make a complete lining. There are therefore many molecular players in the process of angiogenesis. A concept of “angiogenic switch” referring to the onset of tumor angiogenesis, which is triggered by a surplus of endogenous angiogenic stimulators over inhibitors, has been proposed to describe a seemingly distinct event in tumor progression. It is generally believed that the ‘angiogenic switch’ in cancer is the result of a change in balance between angiogenic stimulators and inhibitors present at the site of tumor growth. Numerous angiogenic stimulators have been identified and their expression and distribution have been associated with angiogenesis-based diseases. We focus on angiogenin.
Angiogenin (ANG) was isolated in 1985 from the conditioned medium of HT-29 human colon adenocarcinoma cells based on its angiogenic activity. Structure/function studies have shown that the 123-residue protein contains a ribonucleolytic active site, a cell binding site and a nuclear localization sequence (NLS). Thus, ANG is a ribonuclease whose weak but characteristic ribonucleolytic activity is essential for angiogenesis. It is pleiotropic toward endothelial cells: it binds to the cell surface, interacts with a receptor or a binding protein on the cell surface, induces cell proliferation, activates cell-associated proteases and stimulates cell migration and invasion. It also mediates cell adhesion and promotes tube formation of cultured endothelial cells. All of these individual cellular events are considered necessary components of the process of angiogenesis.
Figure 2. Properties of angiogenin.
Role of ANG in rRNA transcription
ANG has been shown to undergo nuclear translocation in endothelial cells, in cancer cells, and in motor neurons. Nuclear translocation of exogenous ANG is very fast and occurs through receptor-mediated endocytosis, is independent of microtubules and lysosomes. ANG seems to enter the nuclear pore by the classic nuclear pore input route. Upon arriving at the nucleus, ANG accumulates in the nucleolus where ribosome biogenesis takes place. Nuclear ANG has been shown to bind to the promoter region of rDNA and stimulates rRNA transcription.
Cell growth requires the production of new ribosomes, a process involving rRNA transcription, processing of the pre-rRNA precursor and assembly of the mature rRNA with ribosomal proteins. The rate-limiting step in ribosome biogenesis is rRNA transcription, which would be an important aspect of growth control. Ribosomes are also important for maintaining a normal cell function as proteins are required for essentially all cellular activities. ANG-stimulated rRNA transcription has been shown to be permissive for other angiogenic factors to induce angiogenesis. For cancer cells and motor neurons, ANG-mediated rRNA synthesis is critical for sustained proliferation of cancer cells and for maintenance of normal physiological function of motor neurons.
Figure 3. Angiogenin stimulates rRNA transcription.
Up-regulation of ANG in cancer
ANG plays an important role in tumor angiogenesis. Its expression is up-regulated in many types of cancers, particularly in prostate cancer and brain cancer. Human ANG is progressively upregulated in prostate cancer as the disease progresses from prostate intraepithelial neoplasia (PIN) to invasive carcinoma to androgen-independent cancer. Mouse Ang is significantly up-regulated in AKT-induced PIN in the murine prostate-restricted AKT kinase transgenic (MPAKT) mice. Activated AKT promotes both cell growth and cell survival and its activity is frequently elevated in prostate cancers. A critical downstream target of AKT is mTOR that mediates PI3K- and AKT-dependent oncogenesis, especially in the pathogenesis of prostate cancer. mTOR activates S6K that phosphorylate small ribosomal protein S6 and results in enhanced translation of a specific class of mRNA termed with a TOP (a terminal oligopyrimidine track in the 5’ untranslated region) structure. This class of mRNAs includes ribosomal proteins, elongation factors, and several other proteins involved in ribosome biogenesis or in translation control. Thus, AKT activation will enhance ribosomal protein production. However, a missing link from AKT overexpression to enhanced ribosome biogenesis is how transcription of rRNA, which needs to be incorporated in an equal molar ratio, is proportionally elevated. We have recently shown that ANG-mediated rRNA transcription is this missing link that fulfills the growth requirement imposed by AKT activation.
ANG mediates both tumor angiogenesis and cancer cell proliferation
As an angiogenic protein, ANG has been shown to mediate rRNA transcription in endothelial cells. ANG-stimulated rRNA transcription is necessary for angiogenesis induced by other angiogenic factors including VEGF, bFGF, aFGF, and EGF. Thus, ANG has been proposed as a permissive factor for angiogenesis and that ANG-induced rRNA transcription is a general requirement for angiogenesis. ANG inhibitors have been shown to inhibit angiogenesis induced not only by ANG but also by other angiogenic factors. Because these angiogenic factors have been shown to play a role in tumor angiogenesis, an essential role of ANG in tumor angiogenesis can be envisioned and has been demonstrated.
ANG also plays a direct role in cancer cell proliferation by constitutive nuclear translocation in cancer cells where it also stimulates rRNA transcription. Knocking-down ANG expression in PC-3 cells decreases rRNA transcription, ribosome biogenesis, cell proliferation and tumorigenecity both in vitro and in vivo. Thus, ANG has a dual role in cancer progression. It stimulates cancer cell proliferation as well as mediates tumor angiogenesis. Consistently, ANG inhibitors including nuclear translocation blockers, enzymatic inhibitors, and lentivirus-mediated siRNA all inhibit prostate cancer progression in both xenograft and transgenic animal models.
ANG mutations in amyotrophic lateral sclerosis (ALS) patients
Since 2006, a total of 17 missense mutations (at 15 positions) in the coding region of ANG have been identified in 39 of the 4,774 ALS patients Thus, ANG seems to be the fourth member of the most frequently mutated gene found in ALS (after SOD1, FUS/TLS, TARDBP). Among ALS-associated ANG mutations, 3 occurred in the signal peptide regions and 12 in the mature protein. In the 10 sequencing efforts carried out so far, a total of 3,941 healthy controls were included and 2 mutations in the ANG gene were found in non-ALS controls.
Several mutant ANG proteins had been prepared and their ribonucleolytic, nuclear translocation capacity, and angiogenic activity have been characterized. All of them have impaired angiogenic activity, indicating that ANG mutations identified in ALS patients are associated with a functional loss of the angiogenic activity of the ANG protein due to a deficient in ribonucleolysis, nuclear translocation, or both. ANG appears as the first “loss-of-function” gene so far identified in ALS patients.
ANG is also the second angiogenic factor associated with ALS pathogenesis. The first is VEGF. The link between VEGF and ALS was uncovered when transgenic mice with a deletion in the hypoxia responsive element of VEGF gene, which results in low VEGF levels, develop an adult-onset, progressive neurodegenerative disorders similar to ALS. Studies in ALS animal models have indicated that VEGF exerts neuroprotective activities on motor neurons not only as an angiogenic factor by increasing neurovascular perfusion, but also via direct effects on the neuron themselves. Since ANG-mediated rRNA transcription is essential for VEGF to stimulate angiogenesis, it is possible that a deficiency in ANG function may also impair the physiological role of VEGF toward motor neurons.
Expression of ANG in the CNS
Mouse Ang has been reported to be strongly expressed in the developing mouse nervous system both in the brain and in the spinal cord. IHC and IF staining show that Ang expression is the strongest in the forebrain, midbrain, hindbrain and spinal cord at 9.5 day pc. At 11.5 day pc, Ang1 expression remains high in the telencephalon, mesencephalon and mylencephalon as well as in the spinal cord, spinal ganglia and choroids plexus. Until mid-gestation, Ang expression is stronger in the nervous system than in any other tissues. Co-staining with Peripherin and Islet1 demonstrated that Ang1 is expressed in mouse motor neurons.
IHC with an anti-ANG mAb indicate that human ANG is expressed in normal spinal cords obtained from fetal and adult human subjects. Strong ANG staining was observed in the motor neurons of the ventral horn of both fetal and adult human spinal cord. ANG was also detected in the extracellular matrix and interstitial tissues in all cases, consistent with it being a secreted and angiogenic protein. ANG expression in the spinal cord appears to be down-regulated as development proceeds but is still strongly expressed in the adulthood. Strong cytoplasmic and nuclear accumulation of ANG in motor neurons of both prenatal and adult spinal cords suggests a physiological role of ANG, both early in development and later in adulthood. Double IF showed that ANG is localized in both vasculatures and motor neurons of spinal cord tissues, suggesting that ANG abnormalities may have a direct (through motor neurons) and indirect (through angiogenesis) role in ALS pathogenesis. The dual role of ANG in ALS involves decreased activity in endothelial cells and motor neurons, whereas the dual role of ANG in cancer involves increased activity in endothelial cells and cancer cells.
Role of ANG in stress responses
In mammalian cells, stress-induced phosphorylation of eIF2α inhibits global protein synthesis to conserve anabolic energy for the repair of stress-induced damage. However, stress-induced translational silence is also observed in cells expressing a nonphosphorylatable eIF2α mutant (S51A), indicating the existence of a phosphor-eIF2α independent pathway of translational control. Stress-induced cleavage of tRNA is a phenomenon first described as a starvation response in Tetrahymena thermophila, and later observed in bacteria, fungi, and mammalian cells. ANG has been shown to be responsible for stress-induced cleavage of tRNA. This class of small RNA has been designated as tiRNA (tRNA-derived, stress-induced small RNA) and shown to inhibit protein translation in an eIF2α independent manner. Thus, ANG-mediated production of tiRNA is such a pathway that provides an intriguing possibility that the neuroprotective and cancer-promoting activity of ANG are related to tiRNA and other class of small RNAs which ANG help to produce. In case of ALS, accumulation of misfolded protein aggregates, a hallmark of ALS and several other neurodegenerative diseases, are known to increase the stress of endoplasmic reticulum (ER) and lead to apoptosis. In case of cancer, hypoxic stress is also known to lead to cell death. It would be an intriguing hypothesis that ANG-mediated production of tiRNAs in response to stress results in reprogramming of protein translation thereby promoting damage repairs and cell survival.
Stress-induced phosphorylation of eIF2α inhibits translation and promotes the assembly of stress granules (SGs), cytoplasmic foci at which untranslated mRNPs are transiently concentrated. SGs contain stalled 48S pre-initiation complexes that remain after elongating ribosomes have run off their transcripts. The ability of tiRNAs to inhibit translation in a phosphor-eIF2α independent manner suggests that they may also inhibit 48S scanning and that ANG may promote SG assembly. Indeed, ANG has been found to promote the formation of SG when cells are subjected to stresses. Neither WT ANG nor the inactive P112L variant (identified from an ALS patient in the North American cohort) induces SG assembly in the absence of stress. In contrast, WT ANG, but not P112L, significantly promotes the assembly of arsenite- and pateamine-induced SGs. Arsenite and pateamine induce SG assembly in an eIF-2α-dependent and -independent manner, respectively. These results therefore indicate that ANG potentiates both phosphor-eIF2α-dependent and independent inducers of SGs. WT ANG, but not P12L variant, significantly inhibits protein synthesis. The combination of ANG and SA inhibits protein synthesis more than either treatment alone. The finding that ANG inhibits protein synthesis but does not induce SG assembly suggests that a second signal is required for untranslated mRNAs to aggregate at SGs. Nevertheless, these results strongly implicate ANG as an effector of stress-induced translational repression.