Pain assessment in animal models: do we need further studies?

In the last two decades, animal models have become important tools in understanding and treating pain, and in predicting analgesic efficacy. Although rodent models retain a dominant role in the study of pain mechanisms, large animal models may predict human biology and pharmacology in certain pain conditions more accurately. Taking into consideration the anatomical and physiological characteristics common to man and pigs (median body size, digestive apparatus, number, size, distribution and communication of vessels in dermal skin, epidermal–dermal junctions, the immunoreactivity of peptide nerve fibers, distribution of nociceptive and non-nociceptive fiber classes, and changes in axonal excitability), swines seem to provide the most suitable animal model for pain assessment. Locomotor function, clinical signs, and measurements (respiratory rate, heart rate, blood pressure, temperature, electromyography), behavior (bright/quiet, alert, responsive, depressed, unresponsive), plasma concentration of substance P and cortisol, vocalization, lameness, and axon reflex vasodilatation by laser Doppler imaging have been used to assess pain, but none of these evaluations have proved entirely satisfactory. It is necessary to identify new methods for evaluating pain in large animals (particularly pigs), because of their similarities to humans. This could lead to improved assessment of pain and improved analgesic treatment for both humans and laboratory animals.

Introduction
To date, the majority of publications on pain research have focused on humans. The greatest limitation of this human approach is the fact that these studies are primarily aimed at characterizing states of pain, and only a small percentage directly test the anatomical, biochemical, or pathophysiological mechanisms of pain. 1 If we are to make significant advances in our understanding and treatment of pain, animals provide an important resource for predicting analgesic efficacy, which can lead to the development of clinical drugs. 2 The last two decades have witnessed an evolution of animal models which has improved our understanding of the pathophysiology of inflammation, peripheral nerve disease, and bone infiltration by cancer cells. 3 The animal models have also encouraged multidisciplinary analysis in the field of pain studies. Although the relative importance of genetic variability in human pain perception remains unclear, it is well-known that rodent populations display large and heritable differences in both nociceptive and analgesic sensitivity. 4 Improving our understanding of the genetic bases of pain-related traits may have important scientific and clinical implications, and could facilitate the development of novel analgesic strategies or improve treatment of pain using conventional therapies. The first evidence has recently been presented of epigenetic regulation in animals as a key factor in controlling states of pain. 5 Other "-omics" techniques are currently being developed, such as metabolomics, which has recently been associated with pain in the rat model. 6 Behavior resulting from pain can even be measured in decerebrate animals. 7,8 Withdrawal from algogenic stimulus (spinal reflex), 9 abdominal stretching and jumping (bulbspinal reflex), 10 vocalization, biting, guarding, scratching, and licking (innate behaviors) are the most frequently used parameters in this type of evaluation. 7,8 Unfortunately, some of these behaviors may be affected by a number of conditions (eg, reflexive withdrawal by surgical damage to motor neurons) which could affect the objectivity of the measurements, 9,11 as well as their relevance to clinical pain. 12 Despite the many studies conducted on small animals, more efforts are necessary to develop tools for identifying pain and evaluating its intensity and form in large animals. Further research is needed on pain mechanisms and their phylogenetic bases across farm animal species. 13

Materials and methods
In order to assess published data on pain in large animals, we conducted a literature search on PubMed by using the keywords: "animal model sheep and pain", "animal model horse and pain", "animal model cattle and pain", "animal model cow and pain", "animal model pony and pain", and "animal model pig and pain". We searched a full range of articles that explain how the authors assessed pain in these animal models.
For "pig" we conducted a further search for clinical trials only, with the words "pig and pain" and "pig and castration". This additional search for "pig" reflected the importance of this animal model in the scientific community, despite the fact that other species are also used. We searched "pig and castration" because castration is one of the most common operations to cause pain in this animal, so analyzing the evaluation of pain in this context could help us assess pain in pigs more effectively.
To summarize the results of the literature search, we used tables with four columns containing the following information: the first author as reported in the article, the publication year, the animal model used, and a brief description of how pain was evaluated.
We listed the articles in the table in order of publication year, from newest to oldest. For each species, we also grouped together the articles which evaluated pain in similar ways, and in the description, we ordered the methods used from most frequent to least frequent.

Large animal studies -the current situation
There are a number of reasons why rodent models have dominated the study of pain mechanisms: they cost less, are easier to manage ethically, and there is a large historical database of previous research with which authors can compare new findings. However, large animal models may predict human biology and pharmacology more accurately for a number of pain conditions. Validated pain models in these animals could facilitate the development of new and efficient analgesic drugs with few side effects, which could also be used in humans. In fact, the phylogenetic proximity of humans and large animals plays a key role for several reasons: 14 1) species-specific variations in sequence patterns which result in different affinity or potency of the target; 2) they share a greater sequence homology than small animals and humans; 3) they experience different evolutionary pressure and therefore express pain in different ways; and 4) they have a different drug metabolism (large animals better predict human metabolism). 15 Unfortunately, a universal method for identifying and recording pain objectively in large animal models has not yet been developed.
In studies involving cattle, locomotor function is used as the main indicator for pain. Bruijnis et al used a fivepoint scale, in which scores 1 (discomfort) and 2 (severe discomfort) represented a subclinical disorder visible on close inspection, where scores 3 (pain), 4 (severe pain), and 5 (very severe pain) represented a clinical disorder which causes lameness. 16,17 O'Driscoll et al used a locomotion score which considered four characteristics (spine curvature, tracking, head carriage, and abduction/adduction), each of which was evaluated on a five-point scale, from less severe to very severe. 18 Rajkondawar et al compared a gait score (GS), a fivepoint score from "sound" to "severely lame", and a lesions score (LS), in which lesion descriptions (such as "sole ulcer", "interdigital dermatitis", "puncture wound of the sole", and "hemorrhage") were associated with a score. This led to a better descriptor of lameness in cows. 19 The authors found that LS was a better descriptor of lameness than GS. Thoefner et al, in a study on heifers, considered lameness an indicator of pain, as well as other clinical signs, such as claw inflammation (warmth and increased pulsation), cardiovascular function, and gastrointestinal status. 20 Newby et al, in a study for evaluating the effects of a label dose of ketoprofen after left displaced abomasums surgery in dairy cattle, used physiological (respiratory rate, heart rate, rumen motility, and rectal temperature) and behavioral

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Pain assessment in animal models (bright/quiet, alert, responsive, depressed, unresponsive) indicators of pain, 21 whereas Saeed et al, after median sternotomy in calves, evaluated pain only by indirect signs such as heart rate, respiratory rate, and the animal's ability to change posture. 22 Finally, Coetzee et al, in a study to evaluate plasma concentration of substance P and cortisol after castration or simulated castration in calves, also considered behavioral changes such as vocalization (scored on a scale of 0 (no vocalization) to 3 (continuous vocalization), and attitude or temperament, scored on a scale of 0 (unchanged from premanipulation behavior) to 3 (violent escape behavior) ( Table 1). 23 Pippi et al analyzed three kinds of pain (superficial, deep, and visceral) to test analgesic drugs in ponies. The test for superficial pain used a heat source on a skin area, the test for deep pain used a current stimulus through a heating device on the surface of the radius, and the visceral test used a pressure stimulus through a rubber balloon into the cecum. In all the cases, the animal's reaction was movement away from the source in the superficial and deep tests, and a strong movement in the visceral test. This movement was recorded using an accelerometer. 24 Boatwright et al, in a study comparing two drugs for analgesia in a model of abdominal pain in ponies, used a cumulative pain score, a numerical ranking based only on physical criteria (kicking, pawing, head movement) with a score ranging from 0 (no pain) to 12 (maximum pain). 25 Clinical signs of colic in ponies were used by Roelvink et al to evaluate pain in a comparative study of two analgesic and spasmolytic drugs. These signs (head shaking, kicking the abdomen, flehmen, stretching, pawing the ground and looking at the abdomen, yawning, restlessness, and leaning against the stocks) were evaluated before and after the administration of the drugs. 26 Fikes et al compared lidocaine and xylazina as epidural analgesics in ponies; to evaluate pain, they observed the reaction to a pin prick adjacent to the anus. Movements, such as attempts to kick or turn the head towards the stimulus site, were considered positive signs of pain. 27 Epidural analgesia in ponies with carpal synovitis was reported by Freitas et al. Pain was assessed mainly through a lameness score, where 0 signified absence of visible lameness and 4 indicated severe lameness. Clinical signs such as heart rate, systolic arterial pressure, respiratory rate, body temperature, and intestinal motility were also evaluated during the experiment ( Table 2). 28 In sheep, pain has largely been evaluated on the basis of escape-avoidance responses to different noxious stimuli. Dolan et al assessed withdrawal responses after formalin injection (into interdigital space) and mechanical stimulation with a pneumatic device. 29 Wilkes et al used a stiff von Frey filament tip connected to an anesthesiometer on the hind limb of the animal, exerting increasing pressure until it elicited a withdrawal response. 30 Stubsjøen et al inflated a tourniquet to a pressure of 300 mmHg (or until sheep showed signs of aversion) and Ong et al used four different electrical stimuli to produce a response. 31,32 Mather et al measured the pain threshold with a pneumo-mechanical pressure device on the animal's foreleg. 33 Some authors have used clinical signs and measurements (heart rate, blood pressure, eye temperature, rectal body temperature, electromyography), behavioral measures (appetite,

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Gigliuto et al vocalization, lip-licking, teeth-gnawing, ear posture, reaction to the environment, social isolation, feeding behavior), and motor function (abnormalities in gait, righting reflex, limping) as direct and indirect signs of pain. [34][35][36][37][38][39] Welsh et al compared a visual analog scale (VAS) and a numerical rating scale (NRS), developed by two veterinarians, to assess lameness in sheep. They found that, although the NRS and VAS are both repeatable and reproducible but not interchangeable, the VAS is intrinsically more sensitive. 40 An evaluation of electroencephalography (EEG) changes in young lambs before, during, and after seven treatments (tail-docking, castration, sham-shearing, formalin injection, mulesing, ear-tagging, handling) was reported by Jongman et al, 41 and showed a good correlation between EEG and painful procedure (Table 3). In studies involving horses as animal models, lameness is considered the main indicator of pain. Buchner et al used kinematic patterns of head and trunk, as well as the body centre of mass, to evaluate adaptations in movement during experimental lameness. 42,43 This indicator was applied to assess repair and the evolution of pain after removing calcified cartilage. 44 Other authors used a lameness score associated with other signs. Collier et al, for example, analyzed whether the surgical site was hot, painful, or swollen, 45 and an increase in heart rate has also been used as a good indicator of the severity of lameness. [46][47][48][49] Cornelissen et al, using a five-point scale of lameness, considered indirect clinical and behavioral signs of pain such as heart and respiratory rate, rectal temperature, demeanor, appetite, and circumference and distension of the fetlocks; they also noted changes in local temperature by palpation. 50 Cayzer et al measured responses to pain through flexion tests and joint palpation, and noted the degree of swelling, scoring each indicator on a five-point scale. 51 Some authors have detected pain by monitoring mainly behavioral responses. Bussières et al, for example, used a composite pain scale (CPS) with multifactorial numerical rating, which was also used by Van Loon et al. 52,53 The CPS considers physiological data (heart rate, respiratory rate, digestive sounds, rectal temperature), response to treatment (response to palpation of the painful area and interactive behavior), and behavior (sweating, appearance, posture, kicking at abdomen, pawing on the floor, head movement, appetite). In the authors' opinion, these indicators are the most effective in identifying orthopedic pain. Scantlebury et al studied the incidence of recurrent colic in horses and the risk factors involved, using specific indicators of colic as signs of pain, including pawing, violent rolling, lying still, getting up and down, kicking belly, vocalization, rolling eyes, rapid breathing, and irritability. Other clinical signs, such as heart and respiratory rate, temperature, and borborygmi were considered. 54 Clinical observations such as resting, respiratory rate, rectal temperature, carpal flexion angle, carpal circumference, carpal hyperthermia, and signs of carpal pain were scored on a scale from 0 (no warmth/signs of pain) to 2 (marked warmth/signs of pain). 55 Miller et al considered indirect signs of pain, and evaluated the quality of analgesia during anesthesia in horses, using EEG, electrocardiography, heart rate, and blood pressure. The authors found that associating EEG and standard clinical parameters led to a better understanding of anesthetic management. Haussler

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Pain assessment in animal models limb, or stepping away from the pressure source. 56,57 Finally, Fureix et al hypothesized that chronic pain (for the presence of vertebral problems) in horses is associated with increased aggression towards humans. 58 The authors found that chronic pain may behave similarly to acute pain, and therefore, it is important to include chronic pain as an influential factor in interpersonal relations and aggressive behavior (Table 4).

Focusing on pigs
In studies on pain, pigs seem to be the most suitable animal model. They share a number of anatomical and physiological characteristics with man. Moreover, their median body size facilitates the collection of samples such as biopsies, body fluids, and blood samples, which can be managed conveniently in standard facilities. Moreover, pigs are omnivorous, and anatomically, have a similar digestive apparatus to humans. The skin of pigs is similar to human skin in that it has little hair, and the pigmentation changes in different breeds. 59 The similarities between the skin of pigs and humans has been demonstrated in a number of studies, particularly when the animal is on average 12±4 weeks of age and the body weight is 20-27 kg. 60 Number, size, distribution, and communications of vessels in dermal porcine skin are similar to those in human skin, 61 as are tissue turnover time and keratinous proteins in the epidermis. 62 Transmission electron microscopy shows that the epidermal-dermal junctions in pigs and humans are similar, as well as immunoreactivity in the peptide nerve fibers, for example calcitonin gene related peptide, vasoactive intestinal polypeptide (VIP), and substance P. 63,64 Changes in distribution and axonal excitability of nociceptive and non-nociceptive fiber classes are also similar in humans and pigs, which means that this animal model can be used to study the modulation of excitability in these C-fiber classes. 65 Unfortunately, pain in pigs can only be estimated by responses to nociceptive stimuli and none of these are ideal. Any reactions which are monitored are similar to other animal models in that they are almost always motor responses ranging from spinal reflexes to complex behaviors. Motor responses, however, are not the only ones analyzed. In fact, Kluivers-Poodt et al, who studied the effect of pain relief in piglets, used vocalization as an indicator. 66 Using the common classification system described by Weary et al, 67 they divided the calls produced by piglets into "high calls" ($1,000 Hz) and "low calls" (,1,000 Hz), and these variations may reflect not only gross quantitative differences, but also the intensity and nature of the pain. The calls are registered to provide a record of their characteristics, using measures such as temporal parameters (eg, call rate and duration), waveform parameters (ie, peak-to-peak), and spectrum-based parameters (including peak amplitude and frequency, main frequency, and band width).
Other authors have used vocalization in association with other behaviors, but have evaluated quantitative characteristics, rather than qualitative ones as described above. Walker et al assessed the reaction of piglets after castration by monitoring the presence and degree of movements, and the presence or absence of vocalization. 68 Reyes et al modified a method used by Firth and Morton, which considers behavioral parameters such as vocalization, lameness, aggression, restlessness, posture, isolation, appearance, sling time, agitation, and posture. [69][70][71] Possible scores ranged from 10 (no pain) to 35 (maximum pain). Other clinical parameters were  73 Sutherland et al, in their study on reactions in pigs after castration with or without anesthesia, analyzed behavior such as lying down, nursing, sitting, standing, and walking. In terms of behavior indicating pain, the authors analyzed sitting, huddling, scooting, and stress vocalization, with an automatic system for monitoring stress calls. 74 Murison et al, in their study on pain behavior after laryngeal transplant, used a combination of locomotion scores to assess pain (the willingness of the pig to lie down, to stretch its neck and to approach its food, or how it moved), wound palpation (gently increasing pressure until the animal responded by turning away or grunting, for example), and other behavior (grunting, willingness to play with carers, appetite, or nesting behavior). Numerical scores were used, from 0 (no pain) to 10 (worst pain imaginable). 75 In a study to evaluate the response to doses of ketoprofen, Mustonen et al examined clinical signs (locomotion, general temperature, respiratory rate, and general behavior) to form a total clinical score. 76 The same authors used a five-grade lameness scale to measure the efficacy of oral ketoprofen, recording lameness scores before and after the treatments as an index of pain relief. 77 Friton et al also used lameness as a primary parameter. 78 In a study to evaluate the safety and efficacy of meloxicam in non-infectious locomotor disorders in pigs, the authors used clinical examination classifications to describe and categorize "lameness at rest" and "lameness while walking", as well as "feed intake" and "behavior". Other authors have analyzed pain indicators in terms of behavior such as inactivity, huddling up, trembling, tail-wagging, scratching, stiffness, sleep spasms, recumbency, coprophagy, aggression, depression, head-pressing, changes in activity, nursing, lying, body movement, muscle-twitching, and withdrawal. [79][80][81][82][83][84] These analyses allowed the authors to evaluate pain relief in pigs, distress after surgical procedures (such as castration), and the effects of some analgesics. Finally, some authors have used different methods, such as assessing postoperative analgesia after femoral fracture in pigs using a modified VAS for five different conditions: VAS1, observation; VAS2, contact; VAS3, ambulation; VAS4, vocalization; and VAS5, overall. The VAS chart consisted of a 100 mm horizontal line, where the left side represented no pain and the right side the highest level of pain. 85 Haga et al analyzed EEG, mean arterial blood pressure, and pulse rates as indicators of pain in post-castration piglets. The results showed significant differences between animals which had received intrafunicular or intratesticular lidocaine and the control group which had not received it. 86 Rukwied et al analyzed axon reflex vasodilatation by laser Doppler imaging as an indirect sign of C-fiber activation after irradiating pig skin to create erythema and applying mechanical (with von Frey needles) and thermal stimuli (with increased skin temperature to 45°C or 47°C). 87 To measure the concentration of substance P and VIP involved in the transmission of pain, Cornefjord et al applied an ameroid constrictor at the root of the spinal nerve, and measured the gradual reduction in diameter of tissue samples from the nerve root (cranial to constrictor), and from the dorsal root (ganglion). 88 The results showed that there was an increase of substance P but no significant differences in VIP concentration (Table 5).
Reactions analyzed in the studies of all species include: 1) responses organized by centers which are "low" in the hierarchy of the central nervous system (termed "pseudoaffective reflexes") -these include neurovegetative reactions (tachycardia, hyperpnea, arterial hypertension, etc), basic motor responses (contractures, withdrawal, etc), and vocalization; 89,90 and 2) more complex responses integrated by higher nervous centers, which include conditioned motor responses after a period of learning such as behavioral reaction (escape, avoidance, aggression, etc) or modifications of behavior (social, food, sleep, etc).
However, none of these evaluations are entirely satisfactory. They display a number of weaknesses, some of which are due to the types of stimuli or how they are applied, the plasticity of the animal or the way the data has been measured. Specific protocols will therefore need to be developed to improve estimates of pain in animal models, and in future, in patients who are unable to express their pain verbally, such as infants and comatose or cognitively impaired patients (Table 6). [91][92][93]

Future pain models in pigs
When we choose the animal species for a scientific study, it is important to consider the similarity of organs and tissues between the animal and humans. It is not possible, therefore, to make a general statement about animal models. 94 For example, some experts suggest that non-human primates should be used where there is no alternative, such as in studies involving neuroscience and brain function, or in diseases common to   -Number, size, distribution, and communications of dermal vessels -Tissue turnover time -Keratinous proteins in the epidermis -epidermal-dermal junctions 6. Immunoreactivity in the peptide nerve fibers 7. Similarities in nociceptive and non-nociceptive fiber classes man and other primates (HIV/AIDS or tuberculosis). On the other hand, dogs are preferred in the development of antiulcer drugs because of the similarity between the human and canine gastric mucosal membrane. 95 In terms of pain and its evaluation, it is important to emphasize that each species manifests pain in its own way, according to the behavioral repertoire of the species, and a particular reaction does not necessarily suggest that the animal is experiencing pain. Moreover, no single behavior is a definite sign of pain, and each reaction must be assessed in context (Committee on Regulatory Issues in Animal Care and Use, 2000). 96 In fact, vocalization in piglets is not necessarily an indicator of pain. Piglets run a natural risk of being crushed by their mother, and they have developed a low threshold for screaming in order to alert her. Piglets will scream simply if they are picked up, but this does not mean they are feeling any pain. Equally, it is not true to say that piglets never feel pain when they scream. It only means that vocalization, as a single indicator, is not a sensitive parameter of pain.
Conversely, if it is difficult to find an indicator of pain, we can analyze signs of well-being that suggest the absence of pain, such as play (nudging and running, grabbing, biting chains, playful fighting), vocalization (calling the piglet or conversing in a group), strong growth, and good health. [97][98][99] Overall, pigs seem to be the most suitable animal model for studying pain. In fact, there are a number of reasons for choosing pigs. Firstly, their similarities with humans in terms of size, anatomy, and genetics make pigs suitable for studies where results can be reproduced in humans. Secondly, pigs are easily managed in the animal facility, which optimizes the conditions for study. Thirdly, the ethics committee is more likely to approve studies involving pigs as opposed to other

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Gigliuto et al animals such as dogs or primates, and finally, pigs are most frequently used by the scientific community because they are likely the most practical for follow-up studies.
This evidence therefore indicates that pigs represent the best models for studying and assessing postoperative pain in humans.

Conclusion
It is necessary to identify new methods for evaluating pain (particularly postoperative pain) in large animals, and pigs represent the most suitable model because of their similarities to humans. The aim would be to transfer the results to human patients, to develop better pain evaluation and treatment. This would be especially useful in non-verbal patients to prevent complications in short-and long-term therapies due to incorrect diagnosis on the basis of pain.
Teamwork will be essential in this endeavor: biologists, anesthetists, surgeons, pain therapists, and biomedical engineers will need to work together "from bench to bedside", each contributing their specific skills to the task.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry Medicine

text:
Sj7170, a Unique Dual-function Peptide with a Specific α-Chymotrypsin Inhibitory Activity and a Potent Tumor-activating Effect from Scorpion Venom*

Background: Sj7170 is a new peptide from scorpion venom. Results: Sj7170 specifically inhibits the activity of α-chymotrypsin. Sj7170 promotes glioblastoma tumorigenesis and metastasis by up-regulating cyclin D1 and Snail. Conclusion: Sj7170 not only specifically inhibits α-chymotrypsin activity but also promotes the tumorigenesis and metastasis of glioblastoma. Significance: Sj7170 is a unique dual-function peptide with a protease inhibitory activity and a potent tumor-activating effect. A new peptide precursor, termed Sj7170, was characterized from the venomous gland cDNA library of the scorpion Scorpiops jendeki. Sj7170 was deduced to be a 62-amino acid peptide cross-linked by five disulfide bridges. The recombinant Sj7170 peptide (rSj7170) with chromatographic purity was produced by a prokaryotic expression system. Enzyme inhibition assay in vitro and in vivo showed that rSj7170 specifically inhibited the activity of α-chymotrypsin at micromole concentrations. In addition, Sj7170 not only promoted cell proliferation and colony formation by up-regulating the expression of cyclin D1 in vitro but also enhanced tumor growth in nude mice. Finally, Sj7170 accelerated cellular migration and invasion by increasing the expression of the transcription factor Snail and then inducing the epithelial-mesenchymal transition. Moreover, Sj7170 changed cell morphology and cytoskeleton of U87 cells by the GTPase pathway. Taken together, Sj7170 is a unique dual-function peptide, i.e. a specific α-chymotrypsin inhibitor and a potent tumorigenesis/metastasis activator. Our work not only opens an avenue of developing new modulators of tumorigenesis/metastasis from serine protease inhibitors but also strengthens the functional link between protease inhibitors and tumor activators.

A new peptide precursor, termed Sj7170, was characterized from the venomous gland cDNA library of the scorpion Scorpiops jendeki. Sj7170 was deduced to be a 62-amino acid peptide cross-linked by five disulfide bridges. The recombinant Sj7170 peptide (rSj7170) with chromatographic purity was produced by a prokaryotic expression system. Enzyme inhibition assay in vitro and in vivo showed that rSj7170 specifically inhibited the activity of ␣-chymotrypsin at micromole concentrations. In addition, Sj7170 not only promoted cell proliferation and colony formation by up-regulating the expression of cyclin D1 in vitro but also enhanced tumor growth in nude mice. Finally, Sj7170 accelerated cellular migration and invasion by increasing the expression of the transcription factor Snail and then inducing the epithelial-mesenchymal transition. Moreover, Sj7170 changed cell morphology and cytoskeleton of U87 cells by the GTPase pathway. Taken together, Sj7170 is a unique dual-function peptide, i.e. a specific ␣-chymotrypsin inhibitor and a potent tumorigenesis/metastasis activator. Our work not only opens an avenue of developing new modulators of tumorigenesis/metastasis from serine protease inhibitors but also strengthens the functional link between protease inhibitors and tumor activators.
Human glioblastoma is the most frequent primary malignant glioma brain tumor in adults and is one of the most aggressive malignancies. Approximately half of the newly diagnosed glioblastoma patients are in the late stages and have a poor prognosis. Current therapy with surgery, radiation, and chemotherapy, if ever, rarely cures the disease and infrequently prolongs life for more than 1 year. The median survival rate for patients with glioblastoma is less than 1 year, and the average life expectancy remains ϳ14 -18 months (1).
Glioblastomas have the following four characteristics: rapidly dividing cells, higher increasing vascularity, invasion into normal brain tissue, and a strong resistance to death-inducing effects. The diffusely infiltrative nature of glioblastoma is one of the major obstacles to its successful surgical control. The specific molecular mechanisms underlying the invasive behavior of glioblastomas remain largely unclear (2). Therefore, the development of effective modulators (activators or inhibitors) against glioblastomas will help to reveal the specific molecular mechanism of tumorigenesis, which certainly contributes to finding methods and cures for glioblastoma diagnosis and therapy.
Serine protease inhibitors (SPIs) 4 are widely found in a variety of organisms in nature, not only in the bodies of various animals but also in the secretions of parasites, hematophagous invertebrates, amphibian skins, and the venom glands of poisonous animals. SPIs are very fundamental tools because they act as modulators by playing key roles in regulating the activities of numerous target serine proteases, blocking these in emergency cases, and signaling receptor interactions or clearance.
SPIs are classified into several families based on sequence homology, location of reactive site, structural characteristics, and mechanism of action. Generally, they are grouped into the Kunitz family, potato 1 family, and potato 2 family, Kazal family, Bowman-Birk family, and Ascaris family. They control com-plement activation and a variety of other physiological functions, such as blood coagulation, fibrinolysis, inflammation, tumor cell metastasis, apoptosis, and others. Several serine protease inhibitors from human or other organisms have evolved functions that do not involve protease inhibition (3). For example, TFPI-2, a Kunitz-type serine protease inhibitor, has been described as a tumor suppressor gene in several types of cancers, including glioma. Its expression was absent in five of the nine investigated high grade glioma cell lines (4). SPIs are often overexpressed in different tumor types, suggesting that the overexpression of these inhibitors might favor tumor progression (5).
Indeed, it has been demonstrated that the overexpression of a number of SPIs from the Serpin and Kunitz families results in the enhancement of cancer cell malignancy. However, all of these SPIs are secreted by endogenous human cells, and none of chymotrypsin-inhibitory peptides or Ascaris-type SPIs were found to promote malignancy of cancer cells, especially glioma cells.
Here, we describe a new Ascaris-type serine protease inhibitor that was first discovered from the venom of the scorpion Scorpiops jendeki. We also demonstrate that the recombinant peptide Sj7170 (rSj7170) effectively promoted the proliferation of glioma U87 cells in vitro and tumor growth in vivo. This effect could be suppressed by knockdown of the expression of cyclin D1, indicating that the proliferation triggered by Sj7170 occurs through the cyclin D1-Rb-E2F pathway. In addition, Sj7170 also enhanced the migration and invasion of U87 cells by inducing cellular EMT progress. The cell motility induced by Sj7170 also could be inhibited by knockdown of the expression of the EMT transcription factor Snail. Finally, we confirmed that Sj7170 changed morphology of U87 cells and rearranged cytoskeleton by GTPase pathway. Overall, this study describes a new tumor modulator of serine protease inhibitor from animal venom and provided a potential molecular tool for cancer research.

EXPERIMENTAL PROCEDURES
cDNA Library Construction and Screening-The venomous gland cDNA library of the scorpion S. jendeki was constructed as described in our previous work (6,7). Some new and randomly selected colonies were sequenced using the ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA). Sequences were identified for open reading frames using the ORF finder program (www.ncbi.nlm.nih.gov). Signal peptide was removed using the SignalP 4.0 Server. Sequences of Ascaris-type toxins were obtained by searching the data of this venomous gland cDNA library and the GenBank TM National Center for Biotechnology Information database (www.ncbi. nlm.nih.gov) using the Basic Local Alignment Search Tool algorithm. Sequence alignment was performed using Clustal_X 1.83 software followed by manual adjustment.
Construction of Sj7170 Expression Vector-Expression vector pET-28a was used to produce the recombinant Sj7170 (rSj7170) in Escherichia coli. The cDNA sequence of Sj7170 from the venom gland cDNA library of the scorpion S. jendeki was used as the template for the generation of fragments using PCR. The PCR product of Sj7170 cDNA was digested with NcoI and XhoI and inserted into the NcoI-XhoI cutoff pET-28a expression vector. After confirmation by sequencing, the recombinant plasmid pET-28a-Sj7170 was transformed into E. coli Rosetta (DE3) cells for expression.
Expression and Purification of Sj7170-Transformed cells containing the expression plasmid pET-28a-Sj7170 were cultured at 37°C in Luria-Bertani (LB) medium with 30 g/ml kanamycin. Protein synthesis was induced by the addition of 10 mM isopropyl ␤-D-thiogalactoside (IPTG) when the absorbance at 600 nm reached 0.8 -1.0. After 4 h of continued growth at 37°C, cells from 1 liter of culture were harvested by centrifugation. The cell pellet was resuspended in phosphate-buffered saline (PBS) buffer and lysed by sonication on ice. rSj7170 was exclusively accumulated in inclusion bodies. The insoluble inclusion bodies were washed twice with washing buffer (1-2% Triton X-100 in PBS) and denatured in 2.5 ml of denaturation solution (6 M guanidinium HCl, 0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, 30 mM reduced glutathione). The rSj7170 was then reactivated by 100-fold dilution in renaturation solutions (0.2 M ammonium acetate at pH 8.0 containing 0.2 mM oxidized glutathione and 0.5 M arginine) at 16°C for 24 h. The soluble material was then desalted and enriched using centrifugal filter devices (Sartorius Stedim Biotech, Germany, cutoff value Ͼ3 kDa). The renatured peptide was finally purified by HPLC on a C18 column (10 mm ϫ 250 mm, 5 m; Elite-HPLC, China) with a constant flow rate of 5 ml/min. Peaks were detected at a wavelength of 230 nm. The fraction containing rSj7170 was collected manually and lyophilized immediately. The molecular mass of the purified rSj7170 was further analyzed by MALDI-TOF-MS (Voyager-DESTR; Applied Biosystems). The secondary structures of Sj7170 and its mutants were analyzed by CD spectropolarimetry. All purified peptides were dissolved in water at a concentration of 0.2 mg/ml. Spectra from 250 to 190 nm were recorded at 25°C with a scan rate of 50 nm/min on a Jasco-810 spectropolarimeter. The final CD spectra were obtained by averaging three scans and subtracting the signal from a water blank.
Serine Protease Inhibition Activity Assays in Vitro-The inhibitory activity of rSj7170 was tested by measuring the hydrolysis of synthetic chromogenic substrates in the presence of serine proteases. Trypsin (bovine pancreatic trypsin; Chymotrypsin Activity Assay in Vivo-Clear-bottom, whitewalled 96-well microtiter cell culture plates (Greiner Bio-One North America Inc., Monroe, NC) were seeded with U87 cells as indicated and treated with epoxomicin or rSj7170. Proteasome inhibition was measured using the proteasome Glo reagent according to the manufacturer's instructions (Promega). In brief, tumor cells were treated with epoxomicin or rSj7170 at different concentrations and incubated for 2 h. After incubation, cells were incubated for 15 min with 100 l of proteasome Glo reagent, which permeabilizes cells and contains the bioluminescent substrates succinyl-LLVY-amino luciferin to determine the chymotrypsin-like activities, respectively. The luminescence was measured with a Dynex MLX luminometer.
Cell Line and Cell Culture-The human glioblastoma cell line U87 was purchased from American Type Culture Collection (ATCC). The cell was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (1000 units/ml), and streptomycin (1000 g/ml) in a humidified incubator at 37°C with 5% CO 2 . Cell culture dishes and plates were obtained from Wuxi NEST Biotechnology Co. Ltd. (Wuxi, China).
Cell Proliferation Assay-The effects of rSj7170 on cell proliferation were characterized by cell counting. To assess cellular proliferation, a growth curve was generated by seeding 5 ϫ 10 4 cells/well into a 24-well tissue culture plate. Six groups of cells were treated with 0, 2, 4, 6, 8, and 10 M rSj7170 for 48 h and counted using a hemocytometer. The seventh group of cells was treated with 10 M rSj7170 and counted daily over a 3-day period.
Plate Colony Formation Assay-For the plate colony formation assay, the cells were plated into a 6-well tissue culture plate (500 cells per well) and incubated at 37°C for 14 days. The resulting colonies were rinsed with 1ϫ PBS, fixed with methanol for 10 min, and stained with crystal violet. The colonies were photographed and counted for three independent experiments. The colony index of rSj7170-treated cells was derived using the following formula: colony index (%) ϭ (the number of colonies in rSj7170-treated group)/(the number of colonies in the untreated group) ϫ 100. As for the untreated U87 cells, the colony index was regarded as 100%.
Cell Cycle Analysis-The cells were plated into a 6-well tissue culture plate at concentrations determined to yield 70% confluence within 48 h. The cells were harvested using 0.25% trypsin and subsequently pelleted by centrifugation at 2000 rpm for 5 min. The pellet was fixed in 70% ethanol at 4°C overnight. Next, the cells were washed with 1ϫ PBS, resuspended in propidium iodide solution (50 g/ml) containing RNase, and incubated at room temperature in the dark for 30 min. The cell suspensions were filtered through a 50-m nylon mesh, and the cell cycle profiles were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences). The cell cycle distribution (G 1 , G 2 /M, or S phase) was determined using FlowJo software (Tree Star, Ashland, OR).
shRNA Preparation and Cell Transfection-shRNA sequences of cyclin D1 and Snail were designed by the software of siRNA Sequence-Selector and then synthesized ( Table 1). The fragments digested by endonuclease BbsI and BamHI were inserted into the vector pGPU/GFP/Neo (Shanghai GenePharma Co. Ltd. China) to generate pGPU/GFP/Neo-cyclin D1 and pGPU/ GFP/Neo-Snail. Transient transfection was performed at ϳ80% confluency in 6-well plates (NEST) using FuGENE HD (Roche Applied Science) according to the manufacturer's instructions. Briefly, a total of 5 ϫ 10 5 cells were seeded into each well in DMEM containing 10% fetal bovine serum the day before transfection. Four micrograms of the purified shRNA expression vectors (shcyclin D1-326, shcyclin D1-526, shcyclin D1-719, or shcyclin D1-901) were transfected into U87 cells with 10 l of FuGENE HD reagent. After 48 h of transfection, RT-PCR and Western blotting were done to assess the efficiency of cyclin D1 knockdown. G418 is an analog of neomycin. For stable transfection, the optimal concentration of G418 for selection was determined by titration before transfec- tion. About 2 ϫ 10 4 cells were seeded into each well of a 24-well dish (NEST). After 24 h, cells were cultured with G418 concentrations of 0, 200, 400, 600, 800, 1000, 1200, and 1400 g/ml.
The lowest concentration of G418 initiated cell death in ϳ7-9 days and killed all cells within 2 weeks. After 24 -48 h transfection as described above, cells were cultured with 10% FBS at the optimal concentration of G418. After 2 weeks, resistant cell clones were picked and transferred to 24-well plates and gradually expanded to 6-well plates and 1-cm dishes. At 90% confluence, cyclin D1 expression was measured with Western blotting.
Real Time Quantitative PCR-Total RNA was reverse-transcribed into cDNA using SuperScript II (Invitrogen). Real time quantitative PCR was performed using an ABI 7500 real time PCR system (Applied Biosystems, Foster City, CA ) and the SYBR Green real time PCR Master Mix (TOYOBO, Osaka, Japan). Primers used for real time quantitative PCR are shown in Table 2. Each PCR was performed in triplex tubes, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) being used as an endogenous control to standardize the amount of sample RNA.
In Vivo Xenograft Tumor Study-Male BALB/c nude mice (6 weeks old) were obtained from the Disease Prevention Center of Hubei Province, China. Each mouse was inoculated in the right axilla with 1 ϫ 10 6 U87 cells suspended in 0.1 ml of PBS. At 1 week later, mice with tumors reached 200 Ϯ 100 mm 3 in the rSj7170 group (n ϭ 4). Tumor volumes and body weights were measured every day. All mice were maintained for 30 days before they were sacrificed to collect tumors. Tumor volume was calculated using the following formula: tumor volume ϭ 1/2 ϫ (longer diameter) ϫ (shorter diameter) 2 . All animal experiments were approved by the Animal Research Ethics Board of Wuhan University in China and were in compliance with institutional guidelines on the care of experimental animals.
Immunohistochemistry (IHC) of Tumor Xenografts-To assess cyclin D1 expression in xenograft tumors, rabbit monoclonal anti-cyclin D1 antibody (diluted to 1:50, Cell Signaling Technology) was used in IHC. Specimens of U87 xenografts were fixed in 4% paraformaldehyde and embedded with paraffin. All immunohistochemistry staining assays were performed by Hubei BIOS Biological Technology Co., Ltd. (Wuhan, China), following a standard procedure. The expression signal in tumor tissue was observed and analyzed under microscopy.
Cell Migration and Invasion Assay-The cell migration assay was performed using a Transwell Assay Chamber (PET tracketched membrane; BD Biosciences). For the invasion assays, the cells were seeded into BioCoat Matrigel Invasion Chambers (BD Biosciences). The untreated U87 clones (5 ϫ 10 4 cells) maintained in DMEM supplemented with 0.1% FBS were seeded in the top chamber, and DMEM supplemented with 10% FBS was added to the bottom chamber. For both assays, the cells were allowed to migrate or to invade. After incubation for 24 h at 37°C, the cells on the top side of the membranes were removed by cotton swab, and those on the bottom side were fixed and stained with crystal violet. Migrated and invaded cells were fixed and stained after 24 h. Six randomly selected fields per well were photographed, and the numbers of migrated or invaded cells were counted. The extent of cell migration was expressed as a migration index. The migration index of rSj7170-treated cells was calculated using the following formula: migration index (%) ϭ (number of cells that migrated through the insert membrane in rSj7170-treated group)/(number of cells that migrated through the insert membrane in the untreated group) ϫ 100. As for the untreated cells, the migration index was regarded as 100%. The invasion indices of the rSj7170-treated cells and the untreated cells were calculated in the same way.
Immunofluorescent Staining-Cells were grown on sterile coverslips up to 50% confluence. Coverslip-grown cells were washed twice with 1ϫ PBS and fixed with 4% paraformaldehyde for 15 min. The cells were first incubated with primary antibodies at a 1:100 dilution in 0.05% Triton X-100 and 1% BSA in 1ϫ PBS at 4°C overnight. After thorough washing, the cells were incubated for 1 h with fluorescence-conjugated secondary antibodies (TRITC-conjugated sheep anti-rabbit). The nuclei were labeled blue with 4Ј,6-diamidino-2-phenylindole (DAPI). The coverslips were mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The fluorescence signals were visualized using confocal laser microscopy (Fluoview FV1000; Olympus, Tokyo, Japan).
Statistical Analysis-Mean values were calculated from at least three independent experiments and expressed as mean Ϯ S.D. Student's t test was used to evaluate statistically significant differences, which were considered at p Ͻ 0.05 (two-sided).

Sequence Analysis of Sj7170, a Member of the Ascaris-type
Toxin Family-Using random screening and bioinformatics analysis of the S. jendeki venom gland cDNA library, a number of new scorpion venom peptides were characterized. After searching for homologs in the GenBank TM NCBI database, a new venom peptide termed Sj7170 (GenBank TM accession number GH547578) was found. Sj7170 has a precursor nucleotide sequence of 459 nucleotides including three parts as follows: 5Ј-untranslated region (UTR), open reading frame (ORF), and 3Ј-UTR. The lengths of 5Ј-and 3Ј-UTR parts are 95 and 96 nucleotides long, respectively. The ORF region of 261 nucleotides encodes a precursor polypeptide of 86 amino acid residues, including a 24-residue signal peptide and a 62-residue mature peptide, with the unique feature of 10 cysteine residues (Fig. 1A).
Expression and Purification of rSj7170-The nucleotide sequence encoding mature Sj7170 peptide was inserted the NcoI-XhoI cut of the vector pET-28a (ϩ) to generate the recombinant expression vector pET-28a/Sj7170. After induction of the E. coli Rosetta (DE3) cell culture with IPTG, the rSj7170 peptide was found exclusively in inclusion bodies. Using a refolding protocol described under "Experimental Procedures," soluble rSj7170 peptide was recovered ( Fig. 2A). After concentration, the soluble material was separated by reverse phase (RP)-HPLC. The peak eluting at 19.5 min corresponding to rSj7170 peptide was collected (Fig. 2B)   7241.51 Da, consistent with the calculated value (Fig. 2C). Both MALDI-TOF-MS and SDS-PAGE analyses showed that rSj7170 was expressed and purified successfully. Subsequently, the purified rSj7170 peptide was quantified by the BCA protein assay kit (Thermo Fisher Scientific) and stored at Ϫ20°C after freeze-drying. The rSj7170 peptide yield was 6 mg/ml LB media. We prepared the polyclonal antibody of Sj7170 in Beijing Perfect Biotechnology, Ltd. (China). The titer of antibody was tested by Western blot (Fig. 2D) because the numbers of scorpion S. jendeki are very scarce. So the venom of S. jendeki is also too hard to obtain. Thus, we chose other two homologous species Mesobuthus martensii and Heterometrus petersii, which were obtained easily. We used Western blot to detect whether Sj7170 or homolog existed in the venom of these two species of scorpion. The results are shown in Fig. 2D; Sj7170 peptide actually exists in scorpion venom.
Serine Protease Inhibitory Activity of Sj7170 in Vitro and in Vivo-The purified rSj7170 peptide was assayed for inhibitory activity against chymotrypsin, trypsin, and elastase by measuring the inhibition of hydrolysis of synthetic chromogenic substrates by serine proteases. The results showed that the rSj7170 peptide inhibited ␣-chymotrypsin with a 1:1 stoichiometric ratio (Fig. 3A), but it exhibited no inhibitory effect on trypsin and elastase even at a high concentration (data not shown). Furthermore, the inhibitory constant (K i ) of the chymotrypsin-rSj7170 complex was determined by Lineweaver-Burk plots and further slope replotting, yielding a K i value of 1.0 ϫ 10 Ϫ7 M (Fig. 3, B and C).
To investigate further the chymotrypsin-like activity of rSj7170 in vivo, we tested the effectiveness of rSj7170 in a cell culture-based system. As shown in Fig. 3D, rSj7170 inhibited the chymotrypsin-like activity in a dose-dependent manner. Epoxomicin as a positive control showed a stronger inhibitory effect, compared with rSj7170. These results suggest the conclusion that rSj7170 was confirmed to be a potent ␣-chymotrypsin inhibitor in vitro and in vivo.
Sj7170 Promotes Cell Proliferation and Colony Formation-To investigate the cellular effects of Sj7170 on glioma U87 cells, we treated U87 cells using dose-and time-dependent assays. U87 cells were treated with rSj7170 at various concentrations (0, 2, 4, 6, 8, and 10 M) for 48 h and with 10 M for various timings (24, 48, and 72 h). The results showed that rSj7170 significantly increased cell proliferative capacity in a dose-and time-dependent manner, compared with control cells (Fig. 4, A  and B). Furthermore, in vitro colony formation assays demonstrated that the frequencies of colony formation of the rSj7170treated U87 cells are markedly higher than those of control cells (p Ͻ 0.01; Fig. 4C). To further probe the molecular mechanism of its proliferative effect on U87 cells, cell cycle analysis was performed. The results indicated that the treatment of rSj7170 peptide resulted in an increased S phase population and a concomitantly decreased G 1 population (Fig. 4D).
Sj7170 Promotes U87 Cell Proliferation by Up-regulating cyclin D1 through Rb/E2F Signaling-To explore the mechanism of the effect of Sj7170 on the cell cycle, we detected the expression levels of cell cycle-related proteins by Western blot   analysis, including cyclin D1, cyclin D2, cyclin E1, cyclin E2, CDK2, CDK4, CDK6, Rb2/p130, E2F1, E2F5, P21, and P27 (Fig.  5A). The results revealed that the expression levels of cyclin D1 and E2F1 were significantly increased in rSj7170-treated U87 cells. Consistently, the expression levels of Rb2/p130 and E2F5 in U87 cells were reduced by rSj7170. As is well known, the activity of cyclin D1 is required for cell cycle G 1 /S transition. This protein has been shown to interact with tumor suppressor protein Rb, and its expression is regulated positively by Rb. The growth inhibitory activity of Rb2/p130 also correlated with its E2F-binding capacity. E2F is an activator, but E2F5 is an inhibitor (14). The variations of expression levels of cyclin D1, E2F1, Rb2/p130, and E2F5 were consistent with the results of cell cycle analysis. These results indicate that Sj7170 promotes U87 cells proliferation and G 1 /S phase transition by cyclin D1-Rb-E2F signaling.
To determine whether Sj7170 targeted to cyclin D1 protein, four different GenePharma Supersilencing TM cyclin D1 shRNAs (pGPU6/GFP/Neo-cyclin D1-326, -719, -901, and -526) were employed to down-regulate the cyclin D1 expression as a validation. Real time PCR and Western blot analysis showed that all the four shRNAs led to a significant decrease in mRNA and protein levels of cyclin D1 (Fig. 5B). rSj7170 (10 M) was used to treat U87 cells whose cyclin D1 was silenced, and the cell numbers were counted. As shown in Fig. 5C, rSj7170 did not induce the proliferation of U87 cells after knockdown of cyclin D1. These results suggest that Sj7170 promotes U87 cells proliferation by up-regulating cyclin D1 through Rb/E2F signaling. Cyclin D1 is in fact a predominantly cytoplasmic protein in mammalian cancer cell lines. Cytoplasmic sequestration may additionally serve to regulate cyclin D1 activity. Immunofluorescence results revealed that Sj7170 entered into cells and located in the cytoplasm (Fig. 5D). So the subcellular localizations of both cyclin D1 and Sj7170 were already in the cytoplasm. These data suggested cyclin D1 as a crucial target is a response gene for Sj7170 that drives the G 1 /S progression of U87 cells.
Sj7170 Promotes Tumor Growth in Vivo Tumor Xenograft Model-To further investigate the effect of Sj7170 on the progression of tumors, we detected the proliferated activity of rSj7170 against U87 xenografted tumor in vivo. Control U87 cells (n ϭ 4) and rSj7170-treated U87 cells (n ϭ 4) were used to inject mice, respectively. The growth of tumors was monitored every 3 days, and tumors were excised and weighed 32 days after injection. As shown in Fig. 6A, the tumor volumes significantly increased during the 5-week follow-up period, suggesting that rSj7170 effectively promotes the growth of U87 tumor engraftments. After 32 days, tumor growth was completely enhanced in each mouse (100%) inoculated with rSj7170treated U87 cells (Fig. 6B). The average tumor weights of the untreated and rSj7170-treated mice were 0.4 Ϯ 0.21 and 2.1 Ϯ 0.4 g, respectively (Fig. 6C). After acquired specimens of U87 xenografts, IHC analysis of samples, including both the control group and the rSj7170-treated group, revealed a higher expression level of cyclin D1 in the rSj7170-treated group, compared with the untreated group (Fig. 6D). Consistently with the results of cell proliferation in vitro, cyclin D1 also plays an important role in tumorigenesis in vivo. Furthermore, we obtained tumor tissue to detect the cyclin D1 expression by Western blot analysis. The result is shown in Fig. 6E. Consistently with the IHC analysis, the rSj7170-treated group revealed a higher expression level of cyclin D1, compared with the untreated group. All these results demonstrate the tumor-activated function of Sj7170 in U87 cells.
Sj7170 Enhances Cell Migration and Invasion of U87 by EMT-To determine the role of Sj7170 in metastasis, we examined cell migration using a transwell assay chamber with PET track-etched membranes. Cells that migrated toward the bottom side of the membrane were fixed and stained with crystal violet. The results showed that rSj7170 resulted in markedly increased cellular motility, as the numbers of migrating rSj7170-treated cells were significantly higher than those of the untreated cells (Fig. 7A). In addition, we examined the cellular invasive activity of rSj7170 using invasion chamber assays with a Matrigel basement membrane matrix. Consistent with the migration results, rSj7170 significantly enhanced cell invasion capacity by ϳ20-fold compared with the control cells (p Ͻ 0.01; Fig. 7B). Taken together, these results indicate that Sj7170 promotes cell metastasis.
EMT is an important mechanism associated with cancer invasiveness and metastasis (15). To examine whether the Sj7170-induced cell migration and invasion were associated with EMT, we first assessed the status of epithelial and mesenchymal cell markers by real time PCR, Western blot, and immunofluorescence analyses. As shown in Fig. 7, C and D, mRNA and protein levels of the epithelial cell markers E-cadherin were down-regulated in the rSj7170-treated U87 cells. Correspondingly, the control U87 cells expressed low levels of the mesenchymal cell markers vimentin and Snail. Transcription factor Twist expression had no change. Cell metastasis-related proteins MMP2 and MMP9 also were up-regulated by Sj7170. Consistent with the results of real time PCR and Western blot, the down-regulation of E-cadherin and up-regulation of vimentin were also observed by immunofluorescence analysis in the rSj7170-treated U87 cells (Fig. 7, E and F). In particular, the vimentin intermediate filaments localized in a concentrated and polarized pattern in rSj7170-treated U87 cells.
Sj7170 Increases Cell Motility by Up-regulating Snail-To further confirm the relationship between cell motility and EMT, three different GenePharma Supersilencing TM Snail shRNAs (pGPU6/GFP/Neo-Snail-142, -771, and -822) were employed to down-regulate the Snail expression as a validation. Real time PCR and Western blot analysis showed that all the four shRNAs led to a significant decrease in mRNA and protein levels of Snail (Fig. 8, A and B). rSj7170 (10 M) was used to treat U87 cells whose Snail was silenced, and the cell migration and invasion were assayed. As shown in Fig. 8, C and D, rSj7170 did not induce the motility of U87 cells after knockdown of Snail. In other words, when EMT progress was inhibited, Sj7170 lost the ability to enhance cell motility. Therefore, we conclude that Sj7170 promotes cell migration and invasion by inducing EMT through increasing the expression of Snail.
Sj7170 Leads to Morphological Changes and Actin Cytoskeleton Rearrangement by Perturbing Rho GTPase Signaling-Our initial observations suggested that Sj7170 induced morphological changes in U87 cells. Untreated U87 cells were relatively small, morphologically heterogeneous, and loosely attached, with many cells eventually rounding up. In contrast, rSj7170treated U87 cells were significantly larger, more homogeneous, firmly attached, and well spread (Fig. 9A). In addition, rSj7170 decreased the tumor sphere number of U87 cells in a dose-dependent manner (Fig. 9B). These results suggested that Sj7170 induced a profound cytoskeletal rearrangement. Therefore, we evaluated the organization of the actin cytoskeleton of rSj7170treated and -untreated U87 cells. F-actin staining indicated that polymerized actin was mostly located within the cell periphery in rSj7170-treated U87 cells, which were largely devoid of actin stress fibers (Fig. 9C). Small GTPases of the Rho-GTPase family (RhoA, Rac1, and Cdc42) are known to act directly on the cytoskeleton and are responsible for the development of membrane ruffles, stress fibers, lamellipodia, and filopodia (16). In this study, the protein levels of RhoA, Rac1, and Cdc42 were significantly increased in rSj7170-treated U87 cells, compared with the control U87 cells (Fig. 9D). These results indicate that Sj7170 promotes the rearrangement of actin cytoskeleton through the modulation of Rho GTPase signaling.

DISCUSSION
Despite the progress in understanding carcinogenesis, cancer is still a major cause of morbidity and mortality worldwide. The development of novel therapies based on biologically active peptides and protein has emerged as a new strategy to defeat cancer. Venom-producing animals are usually known solely for the negative effects they cause after accidental contact with humans. They carry a variety of toxins with different physiological activities that cause mild symptoms. Even though the effects of the envenomation might lead to a negative reputation, these animals are also seen as a rich source of pharmacologically active principles, and many of their toxins have been the subject of research projects aiming at the development of new molecules for the diagnosis, treatment, and cure of some types of disease (17). Among the animals that produce pharmacologically active molecules capable of interfering in human cellular physiology, the highlights are venomous animals, such as scorpions, bees, wasps, spiders, snakes, and conus. The substances found in the venom of these animals present great potential as anti-tumor or pro-tumor agents (18 -22).
Venom from these animals may hold the promise for curing many types of malignancies, especially after analyzing the results from studies that show a complete remission of tumor cells after treatment with molecules derived from animal venom. However, studies focusing on the mechanism by which these venoms act are still very recent, and much has yet to be found out about these molecules. The first clinical trials against cancer using synthetic peptides derived from animal venom (called Capoten, copied from a pit viper venom peptide) are beginning to show results. As more positive results are obtained, researchers and patients find reasons to believe that these small substances found in nature may have extraordinary applications.
Developing venoms to kill or paralyze prey provides an important means for venomous animals to interact with their environment. Under the natural selection pressure, venomous animals strive to construct more efficient toxins so as to be evolutionarily successful. It was hypothesized that the evolutionary process of the animal venom system includes a series of important events, including recruitment of existing ancestor genes, gene duplications, and mutations, eventually forming diverse types of toxins. Peptide protease inhibitors are considered to be the earliest protein toxins recruited from fluid protein (23). Several protease inhibitors have been isolated from venomous animals, mainly sea anemones, snakes, and anurans. Nevertheless, the characterization of protease inhibitors from scorpions, spiders, and Hymenopterans has recently attracted increasing interest from the scientific community. Protease inhibitors can be primarily grouped as serine, cysteine, aspartic, and metalloprotease inhibitors because of targeting the four major protease classes.
Many known protease inhibitors from venomous animals are characterized as the group of SPIs, the largest and most widely distributed superfamily of protease inhibitors. Based on their possession of conserved functional motifs, SPIs can be subdivided in many classes, in which the Kunitz-type inhibitor class was the best characterized. Most of SPIs discovered thus far from animal venom belong to the Kunitz-type class, such as AEAPI and AXAPI from sea anemone, dendrotoxin E from snake, BSTI from anuran, HWTX-XI from spider, As-fr-19 from Hymenopteran, and Hg1 from scorpion.
In this study, a new venom peptide, termed Sj7170, was characterized from the venom of the scorpion S. jendeki. Sj7170 shares high homology with the known Ascaris-type peptides from other phyla and possesses the same cysteine framework stabilized by five disulfide bridges as them. Based on a protease inhibition assay in vitro and in vivo, Sj7170 was confirmed to be a potent ␣-chymotrypsin inhibitor. These results suggest the idea that Sj7170 from the venom of the scorpion S. jendeki is a typical Ascaris-type serine protease inhibitor. Thus, this work enriches the member and type of SPIs from animal venoms.
Ascaris-type peptides constitute an important class of SPIs. But compared with other types of serine protease inhibitors, only 20 Ascaris-type peptides have been characterized from animals and fewer Ascaris-type peptide inhibitors have been reported from venomous animals. To date, the first identified Ascaris-type scorpion toxin is SjAPI with ␣-chymotrypsin and elastase inhibitory activities from S. jendeki (8). Although both Sj7170 and SjAPI are characterized from the venom of the scorpion S. jendeki, Sj7170 has a specific inhibitory activity against ␣-chymotrypsin, different from SjAPI with ␣-chymotrypsin and elastase inhibitory activities.
The Ascaris-type peptide was almost isolated from the parasitic worm A. suum and has been implicated in the survival of the parasite within the host by inhibiting the activities of exogenous host proteases (13). In a different way, Ascaris-type SPIs from animal venoms were deduced to inhibit the proteases of their preys or predators, thereby protecting other toxic peptides to avoid degradation and inactivation. Almost all Ascaristype SPIs have a common structural characteristic with four short ␤-strands arranged in two approximately perpendicular ␤-sheets and stabilized by five disulfide bridges: Cy-I-Cys-VII, Cys-II-Cys-VI, Cys-III-Cys-V, Cys-IV-Cys-X, and Cys-VIII-Cys-IX (24). Consistent with the previous results, Sj7170 shares the similar structural characteristic with Ascaris-type SPIs. 5 Tumor progression is generally associated with extensive tissue remodeling to provide a proper environment for tumor growth, angiogenesis, invasion, and metastasis of cancer cells. An impressive amount of data reveals that, among many factors, proteases expressed by cancer cells are key players in this process. Indeed, because of their abilities to activate and release cytokines and growth factors and to degrade components of the extracellular matrix, proteases are necessary to provide optimal conditions for growth and invasion of cancer and endothelial cells.
Because protease inhibitors can inhibit these proteases, they are therefore considered to counteract tumor progression and metastasis. Moreover, there is growing evidence that serine protease inhibitors may even inhibit the malignancy of cancer cells.
Conversely, SPIs are often overexpressed in different tumor types, suggesting that overexpression of these inhibitors might favor tumor progression. Indeed, a number of SPIs from Serpin and Kunitz families have been reported to result in enhancement of cancer cell malignancy. For example, serpinE2, a serine protease inhibitor, is a novel target of ERK signaling involved in human colorectal tumorigenesis. Other oncogenic pathways have been associated with induction of serpinE2 expression. MET and PTEN deletion can up-regulate the expression of ser-pinE2 (25). SPINK1, a Kazal type 1 serine protease inhibitor, acts as a growth factor involved in cancer progression and local invasion through EGF receptor signaling and its downstream signaling pathway MAPK/ERK (26). After the discovery of the Sj7170 peptide, our pursuit of the potential underlying molecular mechanisms of Sj7170 in tumor progression revealed a significant divergence from other SPIs. In in vitro and in vivo proliferations, cyclin D1 is co-localized in the cytoplasm of U87 cells with Sj7170 and acts as a crucial target for Sj7170 that promotes the proliferation of U87 cells and tumor growth in  nude mice. Previously, none of the Ascaris-type and endogenous SPIs have yet been shown to promote malignancy of cancer cells; the discovery of Sj7170 expends the diversity of tumorigenesis/metastasis activators among SPIs, which possibly helps to point out the functional evolution of SPIs with tumorigenesis or metastasis activation. The majority of cancer-related deaths result from tumor metastasis, rather than from effects of the primary tumors. Tumor metastasis includes a series of biological processes that move tumor cells from the primary neoplasm to the distal metastatic nodules and acquires the ability to proteolytically remodel the extracellular matrix to coordinate cell adhesion and contractility. EMT was originally identified as a crucial differentiation and morphogenetic process during embryogenesis; it has now been found to be important in cancer progression and metastasis (27). Many molecular mechanisms contribute to EMT progress. These mechanisms usually involve growth factors/cytokines, their cognate receptors, and downstream signaling molecules. Protease activities have been implicated in EMT by activating growth factors and their receptors and cleaving cellular adhesion molecules (28). Recently, in addition to MMPs, the membrane-bound serine protease TMPRSS4 and the membrane-associated serine protease inhibitor HAI-1/ SPINT1 were reported to initiate EMT (29,30).
In our study, we show for the first time that exogenous serine protease inhibitor Sj7170 increases the capacity of migration and invasion of cancer cells by inducing EMT and up-regulating Snail expression. During the EMT, cell-cell junctions are disrupted, the actin cytoskeleton is extensively reorganized, and the cells acquire increased migratory characteristics. Interestingly, we also observed that Sj7170 alters the morphology and motility of glioma cell U87 by activating the Rho GTPase pathway. Altogether, cyclin D1 and Snail expression and Rho GTPase activity both have a significant regulatory role within the context of tumorigenesis and metastasis of tumor cells by inducing EMT. Mutants and other chymotrypsin inhibitor assays also demonstrate that tumorigenic activity of Sj7170 was not induced by protease inhibitory activity, and the two had no necessary associations (data not shown). All these results illustrate that Sj7170, as a serine protease inhibitor, is not just a growth factor but has more active roles in cancer development.
In summary, a new Ascaris-type serine protease inhibitor Sj7170 was identified from the venom of the scorpion S. jendeki. The recombinant Sj7170 peptide was expressed in a prokaryotic expression system and purified by HPLC and affinity chromatography. We found that rSj7170 played an important role in the tumorigenesis and metastasis of glioma U87 cells by accelerating cellular proliferation and colony formation in vitro and in vivo, significantly enhancing cell invasiveness by inducing EMT and changing cell morphology and cytoskeleton through the cyclin D1 and Rho GTPase pathway. Furthermore, Sj7170 has functions in promoting cell proliferation and enhancing cell migration and invasion independent of anti-protease activity. There are no relations between the two functions. Thus, Sj7170, as a unique dual-function peptide, may be a pro-cancer agent or a tumor activator of glioblastoma. This study provides a new understanding of the natural peptide library of scorpion venom, supports new findings of tumorigenesis/metastasis modulators from serine protease inhibitors, and strengthens the functional link between serine protease inhibitors and tumorigenesis/metastasis activators, which help to further reveal the cancer development mechanism and provide a possible breakthrough of cancer treatment.


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FOXO1 downregulation contributes to the oncogenic program of primary mediastinal B-cell lymphoma.

Recently we have shown that the transcription factor FOXO1, highly expressed in B cells, is downregulated in classical Hodgkin lymphoma (cHL). As primary mediastinal B cell lymphoma (PMBL) has similarities with the cHL transcription program we investigated FOXO1 expression in this entity. By using immunohistochemistry we found that FOXO1 was absent or expressed at low levels in 19 of 20 primary PMBL cases. PMBL cell lines reproduce the low FOXO1 expression observed in primary cases. By analyzing gene expression profiling data we found that FOXO1 expression inversely correlated with JAK2 in PMBL cases. Targeting JAK2 activity by the small molecular weight inhibitor TG101348 resulted in upregulation of FOXO1 mRNA and protein expression in MedB-1 and U2940 cell lines, and the MYC inhibitor 10058-F4 increased FOXO1 mRNA in MedB-1 cells. Moreover, in MedB-1 cells FOXO1 expression was strongly upregulated by the inhibitor of DNA methylation 5-aza-2-deoxycytidine and by the histone deacetylase inhibitor trichostatin A. Since FOXO1 promoter was unmethylated, this effect is most likely indirect. FOXO1 activation in the FOXO1-negative MedB-1 cell line led to growth arrest and apoptosis, which was accompanied by repression of MYC and BCL2L1/BCLxL. Thus, FOXO1 repression might contribute to the oncogenic program and phenotype of PMBL.

INTRODUCTION
PMBL is a distinct subtype of diffuse large B cell lymphoma (DLBCL) sharing several morphological and molecular similarities with classical Hodgkin lymphoma (cHL). It comprises about 2% of NHL and preferentially affects young females [1]. Although PMBL belongs to the most curable lymphoma subtypes [2], delayed late complications of chemo-and radiotherapy remain a main challenge. With respect to complications of chemotherapy such as cardiomyopathy after high anthracycline doses and mediastinal irradiation, further investigation of the PMBL oncogenic program to find specific molecular targets is warranted.
The oncogenic program of PMBL is similar to that of cHL. Both entities rely on constitutive activation of JAK-STAT and NF-κB signaling [3]. The genomic amplifications on 9p and 2p16 involving JAK2 and REL genes, respectively, are recurrent features of PMBL and cHL [4,5]. Furthermore, suppressor of cytokine signaling 1 (SOCS1), a negative regulator of JAK/STAT signaling, is recurrently mutated in both entities leading to increased phosphorylation of the JAK2 downstream targets STAT5 and STAT6 [6]. STAT transcription factors, in turn, induce transcription of genes responsible for proliferation and survival including MYC and BCLx L /BCL2L1 [7]. In addition, JAK2 and histone demethylase KDM4/JMJD2C can directly activate MYC transcription in PMBL and cHL cell lines [8].
Despite these similarities, PMBL principally differ from cHL, e.g. in terms of maintenance of major parts of the B cell differentiation program. The characteristic trait of cHL is almost complete loss of the B cell phenotype, whereas PMBL express most of the B cell-specific transcription factors including POU2AF1/BOB.1/ OBF1, POU2F2/OCT2, PU.1, PAX5, BCL6 and B cell surface differentiation markers CD19, CD20, and CD79a [9]. However, PMBL like cHL typically lacks surface immunoglobulins [10].
Recently, we have shown that the forkhead O family transcription factor FOXO1, which is highly expressed in B cells, is downregulated in Hodgkin and Reed-Sternberg (HRS) cells of cHL. Interestingly, all NHL subtypes tested including follicular lymphoma, marginal zone B-cell lymphoma, DLBCL, marginal zone B lymphoma of mucosa-associated lymphoid tissue, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, and Burkitt lymphoma expressed FOXO1 protein at levels comparable with those of normal B cells [11]. FOXO family transcription factors have been shown to act as tumor suppressors regulating expression of proapoptotic and antiproliferative genes [12]. FOXO1 plays a critical role in establishing and maintaining the B cell specific differentiation program, but it is also responsible for cell death due to an inappropriate BCR signaling [13,14]. The best-studied mechanism of FOXO inactivation is phosphorylation followed by nuclear export and proteolytic degradation. AKT, ERK, and IKK kinases are known to phosphorylate FOXO proteins thereby contributing to cell proliferation and survival [15][16][17][18]. Constitutive activation of PI3K/AKT and ERK pathways is typical for many lymphoma subtypes [19,20]. In addition, FOXO1 mutations were detected in 7% of all NHLs [21] and in 8.6% cases of DLBCL. These mutations did not influence FOXO1 mRNA and protein levels [22]. In cHL high expression of specific miRNAs, chromosomal deletions, and constitutive activity of AKT and ERK signaling pathways contribute to almost complete repression of FOXO1 [11].
Considering that PMBL resembles cHL in various aspects, we asked whether it also expresses low levels of FOXO1 and which role FOXO1 might play in PMBL. By using immunohistochemistry we found that most PMBL cases were either low or negative for FOXO1. We identified FOXO1 as a tumor suppressor in PMBL and revealed mechanisms responsible for its repression.

FOXO1 is repressed in PMBL
To clarify the expression status of FOXO1 in PMBL we analyzed 20 clinically and morphologically validated PMBL cases using immunohistochemistry (IHC). In 15% of cases FOXO1 was absent, in 80% of cases only weak staining was observed, and one case (5%) was scored as strongly positive ( Figure 1A). Further, we measured expression of FOXO1 mRNA in an independent PMBL cohort and in two samples of CD19 + cells isolated from hyperplastic human tonsils ( Figure 1B). The expression of FOXO1 mRNA in PMBL samples significantly varied but in all cases it was substantially lower than in normal tonsillar B cells. There is a scarcity in cell lines representing PMBL, the only three available cell lines are MedB-1, Karpas1106, and U2949. We therefore analyzed FOXO1 expression in these three PMBL cell Objective: Plan-Neofluar 40×/0.75 NA. (B) FOXO1 mRNA expression is downregulated in PMBL cases. RNA was isolated from CD19 + tonsillar cells and from frozen PMBL cases, and FOXO1 expression was measured by Q-RT-PCR and analyzed by delta Ct method. Here and in all other Q-RT-PCR experiments the house keeping gene RPL13A was used as reference and the data are shown as mean±SD of at least 2 independent Q-RT-PCR runs that included 3 technical replicates each. lines using Q-RT-PCR, immunoblot, and IHC ( Figure  2). The levels of FOXO1 mRNA in all PMBL cell lines were significantly lower than in CD19 + /CD10 + tonsillar cells representing the germinal (GC) population ( Figure  2A). The highest FOXO1 mRNA levels were detected in Karpas1106, followed by U2940 and MedB-1. Of note, FOXO1 mRNA expression levels in Karpas1106 and U2940 cells were similar to that in PMBL cases with highest FOXO1 levels, whereas expression of FOXO1 in MedB-1 cells was somewhat lower than in PMBL cases with lowest expression ( Figure 1B and Figure 2A). The FOXO1 protein levels in the PMBL cell lines correlated well with the mRNA data. Karpas1106 expressed the highest and MedB-1 expressed the lowest levels of FOXO1 ( Figure 2B). To further corroborate similarities between clinical PMBL cases and cell lines we also used IHC to analyze FOXO1 expression in paraffin embedded pellets of the cell lines ( Figure 2C). The cell lines matched well to three groups of primary PMBL tumors which were identified on the basis of FOXO1 expression: FOXO1 negative -MedB-1, weakly positive -U2940, and strongly positive Karpas1106.

Contribution of genetic and epigenetic factors to FOXO1 repression in PMBL
To clarify the genetic mechanisms of FOXO1 repression we first sequenced both FOXO1 exons in all three PMBL cell lines ( Figure S1). We did not find any missense mutations in the transcribed region and the promoter region of FOXO1. Next, we analyzed publically available human genome tiling array data from 31 PMBL cases [28]. We found five samples with copy number aberrations affecting FOXO1 but none of them was limited to FOXO1 locus. Specifically, we found one sample with a single allele loss of FOXO1 and four cases displaying a single allele gain, which was based on trisomy in two cases. Therefore, FOXO1 copy number aberrations in PMBL are biased to gains rather than to losses and therefore do not contribute to FOXO1 downregulation in this lymphoma subtype (Table S1).
Epigenetic factors are often involved in repression  of tumor suppressor genes. To test the contribution of DNA methylation to the FOXO1 repression we treated PMBL cell lines with the DNA methyltransferase inhibitor 5-aza-dC. We observed an increase of the FOXO1 mRNA levels in all cell lines with highest upregulation in MedB-1 cells (6.6 fold) whereas in Karpas1106 and U2940 FOXO1 levels were only slightly increased (1.9 and 2 fold, respectively) ( Figure 3A). Interestingly, 5-aza-dC increased expression of FOXO1 on protein levels only in MedB-1 ( Figure 3B), but not in Karpas1106 and U2940 cell lines (data not shown). Given that histone deacetylation can also be responsible for gene silencing, we treated MedB-1 cells with the histone deacetylase inhibitor TSA. In fact, TSA treatment resulted in a dose dependent increase of FOXO1 protein levels ( Figure  3C). Finally, to find out whether the positive effect of 5-aza-dC on FOXO1 expression can be explained by hypermethylation of the FOXO1 promoter, we analyzed the methylation status of 26 CpG-dinucleotides located in the vicinity of the transcription start site ( Figure S2). Surprisingly, the MedB-1 promoter was unmethylated like in normal B cell subtypes (data not shown), suggesting that 5-aza-dC and TSA affect FOXO1 expression indirectly.
The absence of promoter methylation indicated "open" chromatin structure of the FOXO1 promoter in MedB-1 cells. To clarify whether this assumption is true, we overexpressed a constitutively active mutant of FOXO1 fused in frame with the estrogen receptor ligand-binding domain (FOXO1ER) in MedB-1 cells. We measured the expression level of endogenous FOXO1 mRNA due to the fact that FOXO1 promoter harbors FOXO binding motifs and is regulated in a positive feed-back loop manner in normal cells [29]. Stimulation with 4-OHT for 24 h increased the levels of FOXO1 mRNA 8.3 fold in comparison with non-treated control. Thus, the FOXO1 promoter has functionally "open" chromatin structure and can be activated by unknown epigenetically silenced transcription factors. Taken together, our results suggest that down-regulation of FOXO1 is not due to genetic aberrations in PMBL. Additionally, epigenetic events may play an indirect role in FOXO1 repression.

FOXO1 expression in PMBL is regulated by a complex network including JAK2 and MYC
We hypothesized that FOXO1 down-regulation in PMBL may result at least in part from transcriptional repression. To find factors that might be responsible for the repression of FOXO1, we mined available GEP data of 31 PMBL cases to identify genes whose expression negatively correlates with FOXO1 expression (Table S2). Among these genes was JAK2. Given that JAK2 inhibition blocks FOXO1 nuclear export [30] and due to the critical role of JAK2 in survival and proliferation of PMBL and cHL cell lines [8] we used correlation analysis to prove the correlation between FOXO1 and JAK2 expression. We actually found a significant negative correlation between JAK2 and FOXO1 in PMBL samples ( Figure 4A). Since JAK2 maintains proliferation of PMBL and cHL cell lines by induction of MYC [8], we also investigated correlation between these genes. In fact, we found a positive correlation between MYC and JAK2 ( Figure  4A). Given reciprocal interactions between FOXO1 and MYC [31], we assessed their expression and found a significant negative correlation ( Figure 4A). Next, we asked whether the correlations between FOXO1, JAK2, and MYC expression, found in primary PMBL tumors also holds true in PMBL cell lines. Therefore, we measured the mRNA and protein expression levels of these three factors in MedB-1, Karpas1106, and U2940 cells by Q-RT-PCR and by immunoblot. The expression of MYC and JAK2 positively correlated at the mRNA level in all cell lines confirming the results from the primary cases. Moreover, a positive correlation was observed at the protein level where the highest expression of both genes was detected in MedB-1 cells ( Figure 4B,C,D). Like in PMBL primary cases, expression of FOXO1 inversely correlated with expression of MYC and JAK2 in PMBL cell lines (Figure 2A,B and Figure 4B,C,D). Thus, PMBL cell lines are similar to original tumors in terms of FOXO1, JAK2, and MYC expression. Since JAK2 and FOXO1 correlated negatively, we investigated whether inhibition of JAK2 activity is able to increase FOXO1 levels. The physiological effect of JAK2 inhibition was controlled by analyzing the phosphorylation status of STAT6, a known JAK2 target in PMBL [32].
In all cell lines treatment with JAK2 inhibitor mildly increased FOXO1 mRNA expression ( Figure  4E). Interestingly, this weak transcriptional upregulation resulted in upregulation of FOXO1 protein level in MedB-1 and U2940, but not in Karpas1106 cell line. The efficiency of JAK2 inhibition by TG101348 was proven by a concentration-dependent decrease of STAT6 phosphorylation in all cell lines ( Figure 4E). Given reciprocal interactions between MYC and FOXO1 we asked whether MYC downregulation might also increase FOXO1 expression. We found that the small molecular weight inhibitor 10058-F4 induced a significant and dosedependent upregulation of FOXO1 mRNA specifically in MedB-1 cells ( Figure 4F). Taken together our results suggest that the JAK2/MYC axis contributes to FOXO1 repression in PMBL.

FOXO1 is a tumor suppressor in PMBL
To investigate the functional role of FOXO1 repression in PMBL we overexpressed a constitutively active inducible variant of FOXO1 (FOXO1ER) in the FOXO1 negative cell line MedB-1 ( Figure 5A). Activation of FOXO1 by 4-OHT led to a decrease of cell viability in comparison to untreated control. 4-OHT did not influence proliferation of MedB-1 cells expressing empty vector ( Figure 5B). The decrease of viable cells can be explained by a block of proliferation due to accumulation of cells in G 1 -phase ( Figure 5C). FOXO1-induced cell death was associated with activation of caspase 3 ( Figure 5D and Figure S3). Therefore, FOXO1 repression in PMBL contributes to the maintenance of the oncogenic program in this lymphoma type.

FOXO1 activation represses genes responsible for proliferation and survival in PMBL
Given that proliferation of PMBL depends on expression of the protooncogene MYC [8], and that constitutive JAK-STAT activation is responsible for expression of the antiapoptotic gene BCL2L1 [33] in PMBL, we analyzed the effect of FOXO1 on expression of MYC and BCL2L1 in MedB-1 cells. As a control for physiological effects of FOXO1 we measured the expression of the known direct FOXO1 target gene BCL6 [34]. FOXO1 activation almost completely abolished MYC protein expression, although MYC mRNA levels were only moderately affected ( Figure 6A,B). Expression of BCL2L1 protein was also strongly downregulated ( Figure 6B), while BCL6 was upregulated. ( Figure  6C,B). Therefore, repression of MYC and BCL2L1 might contribute to the tumor suppressor effects of FOXO1 in PMBL.

DISCUSSION
We found that FOXO1 is downregulated at both the mRNA and protein levels in PMBL. In 95% of PMBL cases analyzed by IHC FOXO1 expression was low or absent, and in all samples analyzed by Q-RT-PCR FOXO1 mRNA was expressed at substantially lower levels than in normal B cells. Several functional mechanisms including repression by JAK2 and MYC, as well as deregulation of a positive feedback mechanism regulating FOXO1 promoter activity, contribute to FOXO1 repression in PMBL. Activation of FOXO1 in MedB-1 cells led to growth arrest and apoptosis which was associated with repression of the protooncogene MYC and the antiapoptotic protein BCL2L1 ( Figure 6D).
PMBL is similar to cHL in molecular aspects [3,4]. Therefore, downregulation of FOXO1 in PMBL provides additional evidence of the close similarities of cHL and PMBL oncogenic programs. At the same time our finding raises the question of heterogeneity of PMBLs. Although virtually all PMBL expressed FOXO1 mRNA and protein at lower levels than normal B cells, there were substantial differences among primary cases and cell lines. In PMBL complete repression of FOXO1 was observed only in 15 % of cases, whereas only 3% of the cases were FOXO1positive in cHL [11]. The observed heterogeneity in FOXO1 expression is in line with reported morphologic and molecular differences among PMBL cases [35,36]. Of note, this heterogeneity cannot be explained by genetic aberrations. We did not find FOXO1 mutations in PMBL cell lines. We have proved that chromosomal aberrations are not responsible for FOXO1 repression in PMBL, therefore our data are in line with the recent next-generation sequencing study which also did not reveal FOXO1 genetic aberrations in 10 primary PMBL cases [37]. Epigenetic silencing is certainly involved in FOXO1 repression in some cases of PMBLs but it is likely to play an indirect role because we were not able to detect methylation of the promoter CpG-island in the FOXO1-negative cell line. Interestingly, although FOXO1 is frequently repressed in different tumor types, promoter hypermethylation has never been observed by other studies [38,39].
We identified JAK2 as a factor contributing to FOXO1 repression in PMBL. The ability of JAK2 to repress FOXO was shown in different cellular models. In neuronal cells JAK2 activation led to nuclear export of FOXO1 [30]. Mechanistically, the repression of FOXO1 can be explained by activation of PI3K-AKT and ERK pathways by JAK2 [40]. This, in turn, results in phosphorylation, nuclear export, and degradation of FOXO transcription factors [17,41]. It is conceivable that the upregulation of FOXO1 protein by the JAK2 inhibitor is due to repression of FOXO1 inactivating pathways. The increase in FOXO1 transcription by repression of JAK2 and the negative correlation between JAK2 and FOXO1 mRNA expression in PMBL samples might be explained by positive autoregulatory feedback loop controlling FOXO1 transcription [29].
We further observed that a small molecular weight MYC inhibitor reactivates FOXO1 transcription in the FOXO1-negative cell line MedB-1. Considering that MYC was shown to compete with FOXO3 for PUMA, GADD45A, and p27/CDKN1B promoters [15,42], it is conceivable that MYC can also interfere with FOXO1 binding on the FOXO1 promoter thus disrupting a positive autoregulatory mechanism.
FOXO transcription factors simultaneously activate numerous proapoptotic and antiproliferative pathways. Therefore it is hard to determine single factors responsible for FOXO-induced cell death and growth arrest [15]. In the present study we analyzed only FOXO targets which are known to play a critical role in the maintenance of PMBL. We found that FOXO1 represses the protooncogene MYC and inhibitor of apoptosis BCL2L1 in MedB-1 cells. FOXO proteins were shown to repress MYC at the mRNA and protein levels by induction of miR-145, miR-34b, and miR-34c [43,44]. In addition, FOXO3 inhibits MYC protein stability by decreasing phosphorylation [45]. Given that FOXO1 activation in MedB-1 cells led to strong inhibition of MYC protein but relatively mild reduction of MYC mRNA level, the inhibition of translation and protein stability might be critical.
We have shown that FOXO1 repression contributes to the maintenance of PMBL and that constitutive expression of JAK2 and MYC is involved in FOXO1 downregulation. We identified the protooncogene MYC and the antiapoptotic gene BCL2L1 as FOXO1 repression targets in PMBL.

DNA methylation and mutational analysis
For analysis of the methylation status of the FOXO1 promoter CpG island we used pyrosequencing [11]. The mutational status of FOXO1 coding exons was analyzed by direct sequencing as we described earlier [11].

Cell death and proliferation analysis
Apoptosis was assessed by flow cytometry using annexin V-PE and 7-amino-actinomycin D co-staining [11]. For cell-cycle analysis we used PI staining [11].

Human material, immunohistochemistry
Twenty cases of PMBL were included in this study. Lymphoma diagnosis was in accordance with the current World Health Organization classification [10]. As control, we used samples of non-neoplastic tonsils. All PMBL samples were drawn from our archive of formalin-fixed, paraffin-embedded tissues and pseudonymized to comply with the German law for ethical usage of archival tissue for clinical research (Deutsches Ärzteblatt 2003; 100 A1632). Approval for these studies was obtained from the University of Ulm ethics board. The CD19 + tonsillar cells and B-cell subtypes were isolated as described earlier [25]. For immunostaining, deparaffinized tissue sections were heat-denatured in a pressure cooker and incubated with rabbit monoclonal antibody against FOXO1 used for immunoblot (1:25 dilution). Bound antibody was labeled using EnVision System (Dako, Jena, Germany). Peroxidase activity was visualized by the substrate 3-amino-9-ethylcarbazole (0.1 mg/mL in 0.17 M sodium acetate, pH 5.2 plus 0.01% H 2 O 2 ). The images were acquired as described earlier [11].

Gene expression and DNA copy number analysis
Gene expression profiling and CGH data of 31 PMBL cases were mined from GEO database (http://www. ncbi.nlm.nih.gov/geo/; GSE 11318; 20.08.2013). Gene expression data were analyzed with help of Genesifter software (Perkin Elmer, Seattle, WA). Copy number aberrations were detected using the R package on the original publication by Olshen and Venkatraman [26].

Correlation analysis of FOXO1, MYC, and JAK2 expression
The data were mined from GEO database (GSE11318; http://www.ncbi.nlm.nih.gov/geo/, 10.08.2013). To analyze FOXO1 expression we used the probe set 202724_s_at. In our pilot experiments we found that FOXO1 expression levels in B-cell lymphomas and B cell subtypes (GEO data set GDS3516, 18.02.2011) assessed with help of 202724_s_at and 202723_s_at probe sets perfectly correlated with the results of our Q-RT-PCR, immunoblot, and immunohistochemistry in the relevant tissues [11]. We have chosen 202724_s_at probe set because it had highest Jetset score (overall score 0.443). The scoring is based on assessment of specificity, coverage of all transcripts, and probability of transcription of target [27]. The MYC probe set 202431_s_at we used was the only true MYC probe set on Affymetrix Human Genome U133 Plus 2.0 Array (overall score 0.328). JAK2 is represented by two probe sets 205842_at and 205841_at. Both JAK2 probe sets revealed a statistically significant negative correlation between FOXO1 and JAK2 expression. When we assessed correlation between MYC and JAK2 expression both probe sets revealed a positive correlation, but only 205842_at yielded statistically significant results and was chosen for analysis. Statistical significance of the correlation was measured by Statistics Calculators software (http://www.danielsoper.com/ statcalc3/calc.aspx?id=44; 13.08.2013).

ACKNOWLEDGMENTS
We thank Anita Kick, Elena Kelsch, Julia Kiedaisch, and Iwona Nerbas for excellent technical assistance and Beatrix Schwarz for help in preparation of the manuscript. We are grateful to TG Unterman for donation of the FOXO1 construct. This study was supported in part by grant 110564 from the Deutsche Krebshilfe eV (to TW and AU), and by the National Natural Science Foundation of China (No. 81101788 to HG, and No. 81201867 to LX). OR was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG, RI 1915/1-1-2).

Author contribution statement
LX, OR, FL, GH, JF, CDW, FG, MJV, and SB performed the research; KH statistically analyzed the experimental data; OR, PM, TW, AU designed the study and wrote the paper.