Comparative effects of free doxorubicin, liposome encapsulated doxorubicin and liposome co-encapsulated alendronate and doxorubicin (PLAD) on the tumor immunologic milieu in a mouse fibrosarcoma model.

Background: We have previously shown that alendronate, an amino-bisphosphonate, when reformulated in liposomes, can significantly enhance the efficacy of cytotoxic chemotherapies and help remodel the immunosuppressive tumor microenvironment towards an immune-permissive milieu resulting in increased anticancer efficacy. In addition, we have previously shown that the strong metal-chelating properties of alendronate can be exploited for nuclear imaging of liposomal biodistribution. To further improve anticancer efficacy, a pegylated liposome formulation co-encapsulating alendronate and doxorubicin (PLAD) has been developed. In this study, we examined the effects of PLAD on the tumor immunologic milieu in a mouse fibrosarcoma model in which the tumor microenvironment is heavily infiltrated with tumor-associated macrophages (TAM) that are associated with poor prognosis and treatment resistance. Methods: Doxorubicin biodistribution, characterization of the tumor immunologic milieu, cellular doxorubicin uptake, and tumor growth studies were performed in Balb/c mice bearing subcutaneously implanted WEHI-164 fibrosarcoma cells treated intravenously with PLAD, pegylated liposomal doxorubicin (PLD), free doxorubicin, or vehicle. Results: PLAD delivery resulted in a high level of tumor doxorubicin that was 20 to 30-fold greater than in free doxorubicin treated mice, and non-significantly higher than in PLD treated mice. PLAD also resulted in increased uptake in spleen and slightly lower plasma levels as compared to PLD. Importantly, our results showed that PLAD, and to a lesser extent PLD, shifted cellular drug uptake to TAM and to monocytic myeloid-derived suppressor cells (MDSC), while there was no drug uptake in neutrophilic MDSC or lymphoid cells. Free doxorubicin cellular drug uptake was below detectable levels. PLAD, and to a lesser extent PLD, also induced significant changes in number and functionality of tumor-infiltrating TAM, MDSC, Treg, NKT, and NK cells that are consistent with enhanced antitumor immune responses in the tumor microenvironment. In contrast, free doxorubicin induced moderate changes in the tumor microenvironment that could promote (decreased Treg) or be detrimental to antitumor immune responses (decreased M1 TAM and NK cells). These immune modulatory effects are reflected in the therapeutic study which showed that PLAD and PLD inhibited tumor growth and significantly prolonged survival, while free doxorubicin showed little or no anticancer activity. Conclusion: We show that liposomal delivery of doxorubicin not only alters pharmacokinetics, but also dramatically changes the immune modulatory activity of the drug cargo. In addition, our data support that the PLAD nanotheranostic platform further enhances some immune changes that may act in synergy with its cytotoxic chemotherapy effects.

A First-in-Human Phase 1 Randomized Single and Multiple Ascending Dose Study of RPh201 in Healthy Volunteers.

RPh201 is a drug extracted from gum mastic that has been studied for its anti-inflammatory and antibacterial properties. Preclinical studies of RPh201 demonstrated neuroprotective and neuroenhancing effects. Toxicology studies in animals did not reveal safety concerns or genotoxic effects. This single-center, phase 1, randomized, placebo-controlled, double-masked study in healthy volunteers assessed the safety and tolerability of RPh201, and determined the highest tolerated dose. There were 2 parts: a single ascending dose (SAD) stage, followed by a multiple ascending dose (MAD) stage. Three dosing arms were included in each stage (5 mg, 10 mg, and 20 mg). Safety data in the lower dosing arms were evaluated before higher doses were initiated. Eighteen participants were randomized in the SAD stage: 12 to RPh201 (4 at each dose) and 4 to placebo. Twenty-one participants were randomized in the MAD stage, of which 13 received RPh201. All 18 participants in the SAD stage completed treatment. Sixteen of the 21 participants in the MAD stage completed treatment. The most frequently reported adverse events were local injection site pain and erythema. No deaths or adverse events related to changes in vital signs or electrocardiograms were reported. No occurrences of suicidal behavior or ideation were reported.

Randomized Controlled Phase 2a Study of RPh201 in Previous Nonarteritic Anterior Ischemic Optic Neuropathy.

BACKGROUND: No proven treatment exists for nonarteritic anterior ischemic optic neuropathy (NAION), either in the acute or late phase. OBJECTIVE: To assess safety and changes in visual function and structure after RPh201/placebo treatment in participants with previous NAION. DESIGN AND SETTING: Phase 2a, single-site, prospective, randomized, placebo-controlled, double-masked trial (registration NCT02045212). MAIN OUTCOMES MEASURES: Early Treatment Diabetic Retinopathy Study best-corrected visual acuity (BCVA), visual fields, retinal nerve fiber layer, and visual evoked potential at weeks 13, 26, and after a 13-week wash-out ("off-drug") period; and safety. STUDY POPULATION: Twenty-two participants aged 18 years or older with previous NAION. INTERVENTION(S): RPh201 (20 mg) or placebo (cottonseed oil vehicle) administered subcutaneously twice weekly at the study site. RESULTS: Thirteen men and 9 women were randomized, of which 20 completed all visits. The mean (±SD) age was 61.0 ± 7.6 years. In a post hoc analysis, after 26 weeks of treatment, BCVA improved by ≥15 letters in 4/11 (36.4%) eyes with RPh201, compared to 1/8 (12.5%) eyes with placebo (P = 0.24). Overall, 7/11 (63.6%) of participants on RPh201 showed some improvement in BCVA, compared with 3/8 (37.5%) on placebo (P = 0.26). Improvement in BCVA from a calculated baseline was 14.8 ± 15.8 letters for RPh201 and 6.6 ± 15.3 for placebo (P = 0.27). Of the 154 adverse effects (AEs), 52 were considered related to the study procedures/treatment. Across the study and 1,017 injections, the most frequently reported AE was injection site pain (23 events in 5 participants). There were no clinically significant changes in vital signs or laboratory values. CONCLUSIONS: This Phase 2a was designed to assess safety, feasibility, and explore potential efficacy signals in treating previous NAION with RPh201. No safety concerns were raised. The results support a larger trial in patients with previous NAION.

Toxicity and Toxicokinetic Study of Subcutaneously Administered RPh201 in Minipigs.

Mastic gum extracts are widely used as herbal remedies and are being tested for several clinical indications. Nevertheless, information on their safety is limited. RPh201 is an extract of the mastic gum, formulated and stabilized in a proprietary method, which is being developed as a novel drug candidate for neurological indications. The aim of this study was to assess the systemic toxic potential of RPh201, administered twice weekly by subcutaneous injections to minipigs, after 39 weeks of administration followed by a recovery period of 6 weeks. No clinical or dose-related signs were observed, but treatment-related findings were seen at the injection sites of the high-dose animals, composed of abscesses, chronic inflammation, and subcutaneous fibrosis. Abscesses >30 mm in size, graded as marked severity, were confined to the high-dose group and were considered as adverse. Minimal-slight subcutaneous and lymph nodes abscesses seen in control, low, and intermediate doses, related to the vehicle (cottonseed oil), were not considered as adverse. Additionally, minimal-to-slight cystic spaces or vacuolation related to the vehicle were observed in the skin, lymph nodes, kidney, and lungs. These findings were considered not to be adverse. The no-observed-adverse-effect level was considered to be 12.5 mg/kg/occasion.

Toxicity and toxicokinetic study of RPh201 in Sprague-Dawley rats.

Mastic gum is used for health products and in the food industry, and is being tested for several clinical indications. Nevertheless, information on its safety is scarce. Our aim was to test the local and systemic toxicity of RPh201, a botanical extract of gum mastic, and to assess the toxicokinetic profile of the mastic gum constituents masticadienonic acid (MDA) and isomasticadienonic acid (IMDA). 340 Sprague-Dawley rats were administered twice weekly subcutaneously with placebo or different doses of RPh201 for 6 months with an interim group at 3 months and a 4-week recovery group. No systemic toxicity was observed with RPh201. Local injection site reactions were observed in all animals, with comparable severity and frequency in the placebo and high dose groups. However, given the relative increase in tissue reaction in the high dose group, these changes were attributed to RPh201 administration. Nevertheless, considering the minor local irritation effects and clear trend for reversibility, the effects were not judged to be adverse. The toxicokinetic study revealed that the MDA and IMDA exposure increased with dose and the increase was supra-proportional on all days. This study supports a "no observed adverse effect level" (NOAEL) of 300 mg/kg body weight in Sprague-Dawley rats.

Caspr and caspr2 are required for both radial and longitudinal organization of myelinated axons.

In myelinated peripheral axons, Kv1 potassium channels are clustered at the juxtaparanodal region and at an internodal line located along the mesaxon and below the Schmidt-Lanterman incisures. This polarized distribution is controlled by Schwann cells and requires specific cell adhesion molecules (CAMs). The accumulation of Kv1 channels at the juxtaparanodal region depends on the presence of Caspr2 at this site, as well as on the presence of Caspr at the adjacent paranodal junction. However, the localization of these channels along the mesaxonal internodal line still persists in the absence of each one of these CAMs. By generating mice lacking both Caspr and Caspr2 (caspr(-/-)/caspr2(-/-)), we now reveal compensatory functions of the two proteins in the organization of the axolemma. Although Kv1 channels are clustered along the inner mesaxon and in a circumferential ring below the incisures in the single mutants, in sciatic nerves of caspr(-/-)/caspr2(-/-) mice, these channels formed large aggregates that were dispersed along the axolemma, demonstrating that internodal localization of Kv1 channels requires either Caspr or Caspr2. Furthermore, deletion of both Caspr and Caspr2 also resulted in widening of the nodes of Ranvier, suggesting that Caspr2 (which is present at paranodes in the absence of Caspr) can partially compensate for the barrier function of Caspr at this site even without the formation of a distinct paranodal junction. Our results indicate that Caspr and Caspr2 are required for the organization of the axolemma both radially, manifested as the mesaxonal line, and longitudinally, demarcated by the nodal domains.

Identification of Tmem10/Opalin as an oligodendrocyte enriched gene using expression profiling combined with genetic cell ablation.

Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, which allows efficient and rapid propagation of action potentials. However, little is known about the molecular mechanisms operating at the onset of myelination and during maintenance of the myelin sheath in the adult. Here we use a genetic cell ablation approach combined with Affymetrix GeneChip microarrays to identify a number of oligodendrocyte-enriched genes that may play a key role in myelination. One of the "oligogenes" we cloned using this approach is Tmem10/Opalin, which encodes for a novel transmembrane glycoprotein. In situ hybridization and RT-PCR analysis revealed that Tmem10 is selectively expressed by oligodendrocytes and that its expression is induced during their differentiation. Developmental immunofluorescence analysis demonstrated that Tmem10 starts to be expressed in the white matter tracks of the cerebellum and the corpus callosum at the onset of myelination after the appearance of other myelin genes such as MBP. In contrast to the spinal cord and brain, Tmem10 was not detected in myelinating Schwann cells, indicating that it is a CNS-specific myelin protein. In mature oligodendrocytes, Tmem10 was present at the cell soma and processes, as well as along myelinated internodes, where it was occasionally concentrated at the paranodes. In myelinating spinal cord cultures, Tmem10 was detected in MBP-positive cellular processes that were aligned with underlying axons before myelination commenced. These results suggest a possible role of Tmem10 in oligodendrocyte differentiation and CNS myelination.

Altered adenosine-to-inosine RNA editing in human cancer.

Adenosine-to-inosine (A-to-I) RNA editing was recently shown to be abundant in the human transcriptome, affecting thousands of genes. Employing a bioinformatic approach, we identified significant global hypoediting of Alu repetitive elements in brain, prostate, lung, kidney, and testis tumors. Experimental validation confirmed this finding, showing significantly reduced editing in Alu sequences within MED13 transcripts in brain tissues. Looking at editing of specific recoding and noncoding sites, including in cancer-related genes, a more complex picture emerged, with a gene-specific editing pattern in tumors vs. normal tissues. Additionally, we found reduced RNA levels of all three editing mediating enzymes, ADAR, ADARB1, and ADARB2, in brain tumors. The reduction of ADARB2 correlated with the grade of malignancy of glioblastoma multiforme, the most aggressive of brain tumors, displaying a 99% decrease in ADARB2 RNA levels. Consistently, overexpression of ADAR and ADARB1 in the U87 glioblastoma multiforme cell line resulted in decreased proliferation rate, suggesting that reduced A-to-I editing in brain tumors is involved in the pathogenesis of cancer. Altered epigenetic control was recently shown to play a central role in oncogenesis. We suggest that A-to-I RNA editing may serve as an additional epigenetic mechanism relevant to cancer development and progression.

A central role for Necl4 (SynCAM4) in Schwann cell-axon interaction and myelination.

Myelination in the peripheral nervous system requires close contact between Schwann cells and the axon, but the underlying molecular basis remains largely unknown. Here we show that cell adhesion molecules (CAMs) of the nectin-like (Necl, also known as SynCAM or Cadm) family mediate Schwann cell-axon interaction during myelination. Necl4 is the main Necl expressed by myelinating Schwann cells and is located along the internodes in direct apposition to Necl1, which is localized on axons. Necl4 serves as the glial binding partner for axonal Necl1, and the interaction between these two CAMs mediates Schwann cell adhesion. The disruption of the interaction between Necl1 and Necl4 by their soluble extracellular domains, or the expression of a dominant-negative Necl4 in Schwann cells, inhibits myelination. These results suggest that Necl proteins are important for mediating axon-glia contact during myelination in peripheral nerves.

Hepcidin, a key regulator of iron metabolism, is transcriptionally activated by p53.

Hepcidin is an iron-regulatory protein that is upregulated in response to increased iron or inflammatory stimuli. Hepcidin reduces serum iron and induces iron sequestration in the reticuloendothelial macrophages - the hallmark of anaemia of inflammation. Iron deprivation is used as a defense mechanism against infection, and it also has a beneficial effect on the control of cancer. The tumour-suppressor p53 transcriptionally regulates genes involved in growth arrest, apoptosis and DNA repair, and perturbation of p53 pathways is a hallmark of the majority of human cancers. This study inspected a role of p53 in the transcriptional regulation of hepcidin. Based on preliminary bioinformatics analysis, we identified a putative p53 response-element (p53RE) contained in the hepcidin gene (HAMP) promoter. Chromatin immunoprecipitation (ChIP), reporter assays and a temperature sensitive p53 cell-line system were used to demonstrate p53 binding and activation of the hepcidin promoter. p53 bound to hepcidin p53RE in vivo, andthis p53RE could confer p53-dependent transcriptional activation. Activation of p53 increased hepcidin expression, while silencing of p53 resulted in decreased hepcidin expression in human hepatoma cells. Taken together, these results define HAMP as a novel transcriptional target of p53. We hypothesise that hepcidin upregulation by p53 is part of a defence mechanism against cancer, through iron deprivation. Hepcidin induction by p53 might be involved in the pathogenesis of anaemia accompanying cancer.

Apaf1 in chronic myelogenous leukemia (CML) progression: reduced Apaf1 expression is correlated with a H179R p53 mutation during clinical blast crisis.

Chronic myelogenous leukemia (CML) is a stem cell disorder that eventually progresses to a blast crisis phase (BC) characterized by distorted apoptotic pathways. The exact mechanism leading to failure in apoptotic pathways during CML progression is unclear. In view of the central role of p53 and apaf1 in the apoptotic machinery we examined six human paired chronic and BC phases samples for their expression. Real-time PCR (RQ-PCR) experiments showed an elevation of p53 mRNA in all patients during transition to BC. However, elevation of apaf1 during BC was observed in five patients only. In contrast, one patient displayed a significant 11.5-fold reduction of apaf1 expression during the transition to BC. No apaf1 promoter methylation was observed. The reduced apaf1 expression was accompanied by a trans-dominant point mutation (H179R) in one p53 allele and the loss of the other. This mutant p53, when tested using functionality assays, was unable to activate apaf1, consequently explaining the reduced expression observed in this patient. Furthermore, the same mutant failed to activate either genes involved in apoptotic or cell cycle arrest pathways, and can be considered as a complete loss of function mutation. This specific mutation was reported in several types of cancer, but was not implicated in CML. To conclude, in this study we have demonstrated mRNA elevation of p53 and apaf1 during CML blast crisis, indicating that genes and proteins involved in cellular apoptosis might be involved in disease progression/response to therapy. Moreover, the mutated p53 discovered in the patient exhibiting lowered apaf1 expression provides, in a clinical case, the first correlation between p53 and apaf1 transcription regulation in humans.

Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera.

Beta-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of beta-thalassaemia, we observed that the liver expressed relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in beta-thalassaemia might suppress liver hepcidin expression. Sera from beta-thalassaemia and haemochromatosis (C282Y mutation) patients were compared with those of healthy individuals regarding their capacity to induce changes the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from beta-thalassaemia major patients induced a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared with sera from healthy individuals. A significant correlation was found between the degree of downregulation of HAMP and HFE2 induced by beta-thalassaemia major sera (r = 0.852, P < 0.0009). Decreased HAMP expression was also found in HepG2 cells treated with sera from beta-thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients induced an increase in HAMP expression, which correlated with transferrin (Tf) saturation (r = 0.765, P < 0.0099). Our results suggest that, in beta-thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption in these patients.

mRNA expression of iron regulatory genes in beta-thalassemia intermedia and beta-thalassemia major mouse models.

beta-Thalassemia is an inherited anemia in which synthesis of the hemoglobin beta-chain is decreased. The excess unmatched alpha-globin chains accumulate in the growing erythroid precursors, causing their premature death (ineffective erythropoiesis). Clinical features of beta-thalassemia include variably severe anemia and iron accumulation due to increased intestinal iron absorption. The most anemic patients require regular blood transfusions, which exacerbate their iron overload and result in damage to vital organs. The hepatic peptide hepcidin, a key regulator of iron metabolism in mammals, was recently found to be low in the urine of beta-thalassemia patients, compared with healthy controls, despite their iron overload. In our work, we measured by RQ-PCR the liver mRNA expression of hepcidin and other iron regulatory genes in beta-thalassemia major mouse model (C57Bl/6 Hbb(th3/th3)), and compared it with beta-thalassemia intermedia mouse model (C57Bl/6 Hbb(th3/+)) and control mice. We found decreased expression of hepcidin and TfR2 and increased expression of TfR1 and NGAL in the beta-thalassemia mouse models, compared with the control mice. Significant down-regulation of hepcidin expression in beta-thalassemia major, despite iron overload, might explain the increased iron absorption typically observed in thalassemia.

Identification of novel cell-adhesion molecules in peripheral nerves using a signal-sequence trap.

The development and maintenance of myelinated nerves in the PNS requires constant and reciprocal communication between Schwann cells and their associated axons. However, little is known about the nature of the cell-surface molecules that mediate axon-glial interactions at the onset of myelination and during maintenance of the myelin sheath in the adult. Based on the rationale that such molecules contain a signal sequence in order to be presented on the cell surface, we have employed a eukaryotic-based, signal-sequence-trap approach to identify novel secreted and membrane-bound molecules that are expressed in myelinating and non-myelinating Schwann cells. Using cDNA libraries derived from dbcAMP-stimulated primary Schwann cells and 3-day-old rat sciatic nerve mRNAs, we generated an extensive list of novel molecules expressed in myelinating nerves in the PNS. Many of the identified proteins are cell-adhesion molecules (CAMs) and extracellular matrix (ECM) components, most of which have not been described previously in Schwann cells. In addition, we have identified several signaling receptors, growth and differentiation factors, ecto-enzymes and proteins that are associated with the endoplasmic reticulum and the Golgi network. We further examined the expression of several of the novel molecules in Schwann cells in culture and in rat sciatic nerve by primer-specific, real-time PCR and in situ hybridization. Our results indicate that myelinating Schwann cells express a battery of novel CAMs that might mediate their interactions with the underlying axons.

Role of iron in inducing oxidative stress in thalassemia: Can it be prevented by inhibition of absorption and by antioxidants?

The pathophysiology of thalassemia is, to a certain extent, associated with the generation of labile iron in the pathological red blood cell (RBC). The appearance of such forms of iron at the inner and outer cell surfaces exposes the cell to conditions whereby the labile metal promotes the formation of reactive oxygen species (ROS) leading to cumulative cell damage. Another source of iron accumulation results from increased absorption due to decreased expression of hepcidin. The presence of labile plasma iron (LPI) was carried out using fluorescent probes in the FACS. RNA expression of hepcidin was measured in two models of thalassemic mice. Hepcidin expression was also measured in human hepatoma HepG2 cells following incubation with thalassemic sera. LPI was identified and could be quantitatively measured and correlated with other parameters of iron overload. Hepcidin expression was downregulated in the livers of thalassemic mice, in major more than in intermedia. Thalassemic sera down regulated hepcidin expression in HepG2 liver cells. A possible way to decrease iron absorption could be by modulating hepcidin expression pharmacologically, by gene therapy or by its administration. Treatment with combination of antioxidants such as N-acetylcysteine for proteins and vitamin E for lipids in addition to iron chelators could neutralize the deleterious effects of ROS and monitored by quantitation of LPI.

Exploring the role of hepcidin, an antimicrobial and iron regulatory peptide, in increased iron absorption in beta-thalassemia.

To develop new treatments for beta-thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp-knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of beta-thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.

Identification of RNA editing sites in the SNP database.

The relationship between human inherited genomic variations and phenotypic differences has been the focus of much research effort in recent years. These studies benefit from millions of single-nucleotide polymorphism (SNP) records available in public databases, such as dbSNP. The importance of identifying false dbSNP records increases with the growing role played by SNPs in linkage analysis for disease traits. In particular, the emerging understanding of the abundance of DNA and RNA editing calls for a careful distinction between inherited SNPs and somatic DNA and RNA modifications. In order to demonstrate that some of the SNP database records are actually somatic modification, we focus on one type of these modifications, namely A-to-I RNA editing, and present evidence for hundreds of dbSNP records that are actually editing sites. We provide a list of 102 RNA editing sites previously annotated in dbSNP database as SNPs, and experimentally validate seven of these. Interestingly, we show how dbSNP can serve as a starting point to look for new editing sites. Our results, for this particular type of RNA editing, demonstrate the need for a careful analysis of SNP databases in light of the increasing recognition of the significance of somatic sequence modifications.

Suppressors of cytokine signaling 4 and 5 regulate epidermal growth factor receptor signaling.

Suppressors of cytokine signaling (SOCS) are Src homology-2-containing proteins originally identified as negative regulators of cytokine signaling. Accumulating evidence indicates a role for SOCS proteins in the regulation of additional signaling pathways including receptor tyrosine kinases. Notably, SOCS36E, the Drosophila ortholog of mammalian SOCS5, was recently implicated as a negative regulator of the Drosophila ortholog of EGFR. In this study, we aimed at characterizing the role of SOCS5 in the negative regulation of EGFR. Here we show that the expression of SOCS5 and its closest homolog SOCS4 is elevated in cells following treatment with EGF, similar to several negative feedback regulators of EGFR whose expression is up-regulated upon receptor activation. The expression of SOCS5 led to a marked reduction in EGFR expression levels by promoting EGFR degradation. The reduction in EGFR levels and EGF-induced signaling in SOCS5-expressing cells requires both the Src homology-2 and SOCS box domains of SOCS5. Interestingly, EGFR is degraded by SOCS5 prior to EGF treatment in a ligand- and c-Cbl-independent manner. SOCS5 can associate with EGFR and can also bind the ElonginBC protein complex via its SOCS box, which may recruit an E3 ubiquitin ligase to promote EGFR degradation. Thus, we have characterized a novel function for SOCS5 in regulating EGFR and discuss its potential role in controlling EGFR homeostasis.

Evolution of p53 in hypoxia-stressed Spalax mimics human tumor mutation.

The tumor suppressor gene p53 controls cellular response to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Inactivation of p53, found in 40-50% of human cancers, confers selective advantage under hypoxic microenvironment during tumor progression. The mole rat, Spalax, spends its entire life cycle underground at decidedly lower oxygen tensions than any other mammal studied. Because a wide range of respiratory adaptations to hypoxic stress evolved in Spalax, we speculated that it might also have developed hypoxia adaptation mechanisms analogous to the genetic/epigenetic alterations acquired during tumor progression. Comparing Spalax with human and mouse p53 revealed an arginine (R) to lysine (K) substitution in Spalax (Arg-174 in human) in the DNA-binding domain, identical to known tumor associated mutations. Multiple p53 sequence alignments with 41 additional species confirmed that Arg-174 is highly conserved. Reporter assays uncovered that Spalax p53 protein is unable to induce apoptosis-regulating target genes, resulting in no expression of apaf1 and partial expression of puma, pten, and noxa. However, cell cycle arrest and p53 stabilization/homeostasis genes were overactivated by Spalax p53. Lys-174 was found critical for apaf1 expression inactivation. A DNA-free p53 structure model predicts that Arg-174 is important for dimerization, whereas Spalax Lys-174 prevents such interactions. Similar neighboring mutations found in human tumors favor growth arrest rather than apoptosis. We hypothesize that, in an analogy with human tumor progression, Spalax underwent remarkable adaptive p53 evolution during 40 million years of underground hypoxic life.