• Aucun résultat trouvé

Myostatin Gene Inactivation Prevents Skeletal Muscle Wasting in Cancer

N/A
N/A
Protected

Academic year: 2021

Partager "Myostatin Gene Inactivation Prevents Skeletal Muscle Wasting in Cancer"

Copied!
14
0
0

Texte intégral

(1)

Myostatin Gene Inactivation Prevents Skeletal Muscle

Wasting in Cancer

Yann S. Gallot1, Anne-Cecile Durieux1, Josiane Castells1, Marine M. Desgeorges1, Barbara Vernus2, Lea Plantureux1, Didier Remond3

, Vanessa E. Jahnke1, Etienne Lefai4, Dominique Dardevet3, Georges Nemoz4, Laurent Schaeffer5, Anne Bonnieu2, and Damien G. Freyssenet1

Abstract

Cachexia is a muscle-wasting syndrome that contributes significantly to morbidity and mortality of many patients with advanced cancers. However, little is understood about how the severe loss of skeletal muscle characterizing this condition occurs. In the current study, we tested the hypothesis that the muscle protein myostatin is involved in mediating the pathogenesis of cachexia-induced muscle wasting in tumor-bearing mice. Myostatin gene inactivation prevented the severe loss of skeletal muscle mass induced in mice engrafted with Lewis lung carcinoma (LLC) cells or in ApcMin/þmice, an established model of colorectal cancer and cachexia. Mechanistically, myostatin loss attenuated the activation of musclefiber proteolytic pathways by inhibiting the expression of atrophy-related genes, MuRF1 and MAFbx/Atrogin-1, along with autophagy-related genes. Notably, myostatin loss also impeded the growth of LLC tumors, the number and the size of intestinal polyps in ApcMin/þmice, thus strongly increasing survival in both models. Gene expression analysis in the LLC model showed this phenotype to be associated with reduced expression of genes involved in tumor metabolism, activin signaling, and apoptosis. Taken together, our results reveal an essential role for myostatin in the pathogenesis of cancer cachexia and link this condition to tumor growth, with implications for furthering understanding of cancer as a systemic disease. Cancer Res; 74(24); 7344–56. 2014 AACR.

Introduction

Cancer cachexia is a wasting syndrome characterized by the uncontrolled loss of body weight that results from depletion of adipose tissue and skeletal muscle, while the nonmuscle protein compartment is relatively preserved (1). Tumor- and host-derived factors induce cachexia in up to 80% of patients with advanced cancers, particularly in pancreatic and gastric cancers (2). Cancer cachexia accounts for 20% of cancer-related deaths (3), and dramatically contributes to reducing the quality of life, increasing toxicity from chemotherapy, and decreasing survival of patients with cancer (4).

Different strategies have been developed to limit cancer cachexia, but much interest has been devoted to the

thera-peutic potential of inhibiting type-II activin receptor (ActRII)-mediated signaling triggered by activins and myostatin (Mstn; reviewed in ref. 5). Mstn is a highly conserved member of the TGF-b superfamily, mainly secreted from skeletal muscle fibers (6). Mstn acts in an autocrine/paracrine manner by binding to ActRIIA and ActRIIB, which then recruit and activate type-I activin receptors, also known as activin receptor-like kinase (Alk) 4 and 5. This, in turn, causes phosphorylation of Smad2 and Smad3, the association with Smad4 into a Smad2/3/4 complex, which then enters into the nucleus to trigger gene transcription.

Mstn is a master regulator of skeletal muscle mass. Mstn gene inactivation induces skeletal muscle hypertrophy (6, 7), whereas forced overexpression of Mstn induces skeletal mus-cle atrophy (8–10). An increase in Mstn expression is the molecular signature of multiple conditions leading to skeletal muscle wasting (5), including cancer cachexia (11–16). In mouse models of cancer cachexia, administration of a soluble form of ActRIIB preserves skeletal muscle mass (14, 15), restores muscle strength (15, 16), and increases lifespan (15). However, a shortcoming of targeting ActRIIB ligands has been the difficulty in discriminating the respective effects of Mstn and activins (15), whose expression is also upregulated in many catabolic disease states, and likely contributes to the wasting syndrome (5).

In this study, we set out (i) to determine the molecular mechanisms by which Mstn gene inactivation may alter the course of cancer cachexia and (ii) to examine the possible

1Laboratoire de Physiologie de l'Exercice, Universite de Lyon, Saint

Eti-enne, France.2INRA UMR 866 Dynamique Musculaire et Metabolisme,

Montpellier, France.3INRA UMR 1019, Unite de Nutrition Humaine,

Cler-mont-Ferrand, France.4

INSERM U1060, INRA USC1235, CarMeN Labo-ratory, Universite de Lyon, Oullins, France.5

CNRS UMR 5239, Laboratoire de Biologie Moleculaire de la Cellule, Ecole Normale Superieure de Lyon, Lyon, France.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Corresponding Author: Damien G. Freyssenet, Laboratoire de Physiolo-gie de l'Exercice, Faculte de Medecine, 15 rue Ambroise Pare, 42023 Saint-Etienne Cedex 2, France. Phone: 14-77; Fax: 033-4-77-42-14-78; E-mail: damien.freyssenet@univ-st-etienne.fr

doi: 10.1158/0008-5472.CAN-14-0057

(2)

Animals

All experiments were conducted in accordance with the European Community guidelines for the care and use of laboratory animals for scientific purposes. Constitutive C57BL/6 Mstn knockout (Mstn/) mice were previously described as MstnD/Dmice (7). Thefloxed Mstn allele (where the third exon of the Mstn gene isflanked with a pair of loxP sites) has been deleted by transiently expressed Cre recombi-nase at the zygote stage. C57BL/6 ApcMin/þ male mice were purchased from Jackson Laboratory. To generate Apc-Min/þ/Mstn/ mice, ApcMin/þ males were first mated to Mstn/females. ApcMin/þMstnþ/male mice from the prog-eny were then mated to Mstn/females. ApcMin/þMstnþ/þ, ApcMin/þMstnþ/, and ApcMin/þMstn/mice were identified by genotype analysis.

Culture of Lewis lung carcinoma cells and inoculation LLC cells (ATCC) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37C and 5% CO2in air. Mstn/(14.54 0.80 months; n ¼ 11) and wild-type (14.60 0.95 months; n ¼ 12) male mice were subcu-taneously inoculated with 5  106 LLC cells in 150mL of sterile DPBS. Control Mstn/(14.67 1.13 months; n ¼ 8) and wild-type (14.66  0.95 months; n ¼ 9) male mice received sterile DPBS. Mice were allowed food and water ad libitum. Body weight, food intake, and tumor size were measured daily.

Survival rate

Survival rate was determined in wild-type (15.06  0.62 months; n¼ 20) and Mstn/(15.15 0.16 months; n ¼ 17) mice inoculated with LLC cells. Body weight, food intake, and tumor size were measured daily until death.

Survival rate was also determined in ApcMin/þMstnþ/þ(n¼ 30), ApcMin/þMstnþ/(n¼ 15), and ApcMin/þMstn/(n¼ 12) male mice. ApcMin/þMstnþ/þwas bred until death. Survival rates of ApcMin/þMstnþ/ and ApcMin/þMstn/ mice were determined until the age of 45 weeks.

Removal of tissues

Mice were anesthetized (i.p. injection of 90 mg/kg ketamine and 10 mg/kg xylazine) 35 days after the inoculation of LLC cells. Extensor digitorum longus, gastrocnemius, soleus, and tibialis anterior muscles, as well as tumor and visceral adipose tissues were rapidly excised and weighed. Mice were then killed by cervical dislocation.

Plasma amino acid level

Plasma (250mL) was homogenized in 50 mL of sulfosalicylic acid solution (1 mol/L in ethanol with thioglycolate 0.5 mol/L)

derivation with ninhydrin. Real-time PCR

Total RNA was collected from gastrocnemius muscle and tumor tissue by using the RNeasy Fibrous Tissue Mini Kit (Qiagen). RNA (400 ng) was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). The selected forward and reverse primer sequences are listed in Supplementary Table S1. Real-time PCR was performed in a 20-mL final volume using the SsoFast EvaGreen Super mix (Bio-Rad). Fluorescence intensity was recorded using a CFX96 Real-Time PCR Detec-tion System (Bio-Rad). Data were analyzed using the DDCt method of analysis. Reference genes (hypoxanthine guanine phosphoribosyl transferase, ribosomal protein large P0, and a-tubulin) were used to normalize the expression levels of genes of interest (8).

Protein extraction

Gastrocnemius muscles and tumor tissues were homoge-nized (1:20 dilution wt:vol) in an ice-cold 50 mmol/L Tris HCl buffer (pH 7.4) containing 100 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 50 mmol/L b-glycerophosphate, 50 mmol/L sodium fluoride, 1 mmol/L sodium orthovana-date, 120 nmol/L okadaic acid, and 1% Triton X-100, and then centrifuged at 12,000 g for 10 minutes at 4C. Protein concentration of the supernatant was determined at 750 nm (Bio-Rad).

Protein analyses

For Luminex analysis, fluorescent capturing beads cou-pled to antibodies directed against IkB-a, IkB-aSer32/Ser36, and p70S6KThr421/Ser424(Bio-Rad) were incubated for 2 hours with 50mL of protein fractions (1:10 dilution vol:vol) in 96-well plates. Samples were then washed, incubated with biotinylated antibodies for 30 minutes, followed by the incubation with a streptavidin–phycoerythrin solution for 10 minutes. The analysis consisted of a double-laser fluo-rescence detection, which allowed simultaneous identi fica-tion of the target protein through the red fluorescence emission signal of the bead and quantification of the target protein through thefluorescence intensity of phycoerythrin. Fluorescence intensities were recorded on a Bio-Plex 200 System instrument (Rad). Data were analyzed using Bio-Plex Manager software.

For Western blot analysis, 50mg of proteins were resolved on 12.5% SDS-polyacrylamide gels, then blotted onto 0.45 mm nitrocellulose membranes (GE Healthcare Life Sciences), and incubated overnight with the appropriate antibody. Antibodies against 4E-BP1 (1:500 vol/vol), NF-kB p65 (1:600), NF-kB p65Ser536

(1:200), ribosomal protein (rp) S6 (1:600), rpS6Ser235/236 (1:800), Smad2/3 (1:500), and

(3)

Smad2Ser465/467/3Ser423/425 (1:200) were from Cell Signaling Technology. Antibodies against Bax (1:1,000) and Bcl-2 (1:800) were from Santa Cruz Biotechnology. Antibodies against Atg5–Atg12 complex (1:800), FoxO3aSer253 (1:500), LC3b (1:800), anda-tubulin (1:2,000) were from Sigma-Aldrich. Incubation with horseradish peroxidase-conjugated secondary antibody (Dako) allowed the chemiluminescent detection of immunocomplexes (GE Healthcare Life Sciences). Labeled Western blot analyses were quantified using ImageJ software analysis (NIH).a-tubulin immunoblot analyses were used to check for equal protein loading.

Caspase-3 enzyme activity wasfluorometrically (lexc¼ 380 nm andlexc¼ 460 nm) determined on tumor protein extracts by using Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Bachem) as afluorescent substrate (19).

Count of intestinal polyps in ApcMin/þMstnþ/þ and ApcMin/þMstn/ mice

The small intestine was carefully dissected distally to the stomach and proximally to the cecum, and was cut into three equal sections (upper, middle, and lower intestine). The colon was also removed. All sections wereflushed with PBS, opened longitudinally, andfixed in 10% paraformalde-hyde for 24 hours. Sections were then rinsed with PBS (3 12 hours) and stained in 0.1% methylene blue for 3 hours. Polyps were counted under a dissecting microscope and were categorized as<1 mm, 1 to 2 mm, 2 to 3 mm, and >3 mm diameter.

Statistical analysis

Statistical comparisons were assessed across multiple con-ditions using a two-way ANOVA, with the Student–Newman– Keuls posthoc test used to identify specific differences between means (GraphPad Prism 5.03). Student's t test was used for comparisons between two conditions. Difference in survival rates was determined by thec2test. To account for differences in tumor weight at death between wild-type and Mstn/ tumor-bearing mice, an analysis of covariance was performed (SuperANOVA; Abacus Concepts). Thea-level of significance was set at 0.05 for all comparisons. Data are presented as mean SE.

Results

Mstn gene inactivation prevents loss of skeletal muscle mass in LLC tumor-bearing mice

Body mass, fat mass, gastrocnemius muscle mass, and pro-tein content were all decreased in wild-type tumor-bearing mice (Fig. 1A–D). In contrast, Mstn loss attenuated the decrease in body mass (Fig. 1A), fat mass (Fig. 1B), and completely prevented loss of skeletal muscle mass (Fig. 1C) and protein content (Fig. 1D). Protection against muscle wasting in Mstn/tumor-bearing mice was also illustrated by the preservation of plasma amino acid concentration (Fig. 1E and Supplementary Fig. S1). Prevention of skeletal muscle mass loss was also observed in extensor digitorum longus, tibialis anterior, and soleus muscles of Mstn/tumor-bearing mice (Supplementary Fig. S2). Importantly, caloric intake was

not different between groups (Supplementary Fig. S3). Mstn loss thus protects hind limb skeletal muscle against atrophy during cancer cachexia.

Mstn gene inactivation decreases LLC tumor growth Tumor growth was observed in 100% of mice, but tumor weight was significantly lower in Mstn/mice compared with wild-type mice (Fig. 2A). Importantly, Mstn per se did not alter the in vitro proliferative characteristics of LLC cells (Supplementary Fig. S4A–S4C). Expression of angiogenesis and tumorigenesis markers was therefore determined in tumor tissue. mRNA levels of vascular endothelial growth factor (VEGF) A, a major driver of tumor angiogenesis and growth (20), glucose transporter 1 (GLUT1), the predomi-nant glucose transporter in many types of cancer cells (21), and hypoxia-inducible factor (HIF)-1a, a transcriptional activator of VEGF and GLUT1 gene expression (22), were significantly lowered in the tumors of Mstn/mice (Fig. 2B). Targeting activin signaling has recently been shown to inhibit angiogenesis and tumorigenesis (23). Activins are homo- or heterodimeric proteins of variousb-subunit iso-forms referred to as activin A (bA-bA), activin AB (bA/bB), and activin B (bB/bB; ref. 24). In the present study, mRNA levels of bothbA and bB subunits were significantly lower in the tumors of Mstn/mice (Fig. 2C). Expression of both type II (ActRIIB) and type I (Alk4) receptors, which trans-duce activin signal (24), was also lowered in the tumors of Mstn/mice (Fig. 2C).

The possibility that Mstn loss reduces tumor growth by increasing apoptosis of tumor cells was also investigated. Consistent with this hypothesis, mRNA level of TWEAK, an inducer of apoptosis (25), Bax protein content, Bax-to-Bcl-2 protein ratio, and caspase-3 enzyme activity were higher in the tumors of Mstn/mice (Fig. 2D–G and Supplementary Fig. S4D). Altogether, these results indicate that Mstn loss represses the expression of genes involved in angiogenesis and tumorigenesis, and increases apoptosis.

Mstn gene inactivation attenuates the activation of proteolytic pathways in skeletal muscle of LLC tumor-bearing mice

Mstn loss can prevent muscle wasting by regulating the balance between protein synthesis and/or degradation. We first explored the regulation of the Akt/mTOR pathway, a crucial regulator of skeletal muscle protein synthesis whose activation prevents muscle atrophy in vivo (26, 27). However, phosphorylation levels of downstream effectors of the path-way, including p70S6K, rpS6, and the translation repressor 4E-binding protein (4E-BP) 1, were all significantly decreased in response to tumor growth in the gastrocnemius muscle of both wild-type and Mstn/mice (Fig. 3).

We next investigated whether Mstn loss in tumor-bearing mice was associated with an inhibition of the ubiquitin-proteasome pathway. Increase in the expression of muscle-specific E3 ubiquitin ligases (MAFbx/Atrogin-1 and MuRF1) induced by tumor growth in the gastrocnemius muscle of wild-type mice was completely blunted in Mstn/ tumor-bearing mice (Fig. 4A and B). Transcript level of forkhead

(4)

box O (FoxO) 3, a transcription factor involved in the expression of MAFbx/Atrogin-1 and MuRF1 (28, 29), was also markedly reduced in Mstn/tumor-bearing mice (Fig. 4C). However, FoxO3 phosphorylation level remained unchanged (Fig. 4D). Active phosphorylated form of p65, a constituent of NF-kB transcription factor involved in the

expression of MuRF1 (30), was also 3-fold lower in Mstn/ tumor-bearing mice compared with wild-type tumor-bear-ing mice (Fig. 4E). Phosphorylation of IkBa on Ser32 and Ser36 allows the release of NF-kB by IkBa. IkBa phosphor-ylation, which was markedly increased in the gastrocnemius muscle of wild-type mice in response to tumor growth, Figure 1. Mstn gene inactivation prevents loss of skeletal muscle mass in tumor-bearing mice. A, changes in body weight during the course of cancer cachexia. B, visceral fat mass. C, gastrocnemius muscle mass. D, gastrocnemius muscle protein content. E, plasma concentration of total amino acids. Wild-type and Mstn/mice were inoculated with LLC cells or received DPBS. Tissues were removed 35 days after the inoculation of LLC cells. Data are means SE (n ¼ 8–12/group)., P< 0.05;, P< 0.01;, P< 0.001.

(5)

remained unchanged in Mstn/tumor-bearing mice (Fig. 4F). Accordingly, transcript levels of TWEAK, a skeletal muscle-wasting cytokine (31), and TRAF6, an E3 ubiquitin ligase and adaptator protein required for the activation of NF-kB in response to TWEAK (32), were downregulated in Mstn/tumor-bearing mice (Fig. 4G and H).

We also monitored the expression of autophagy-related genes (33, 34). Cytoplasmic and membrane-bound forms of microtubule-associated protein light chain 3 (LC3) b, a pro-totypical marker of autophagy (35), were significantly increased in gastrocnemius muscle of wild-type tumor-bearing mice. No variation was observed in Mstn/tumor-bearing mice (Fig. 5A). Ulk1 mRNA level and Atg5–Atg12 protein complex were also lowered in Mstn/mice in response to

tumor growth compared with wild-type mice (Fig. 5B and C). Collectively, all thesefindings indicate that Mstn loss attenu-ates the activation of proteolytic pathways in skeletal muscle during cancer cachexia.

Mstn/activin signaling in skeletal muscle of LLC tumor-bearing mice

Elevated expression of activins promotes muscle wasting and cachexia by upregulating the expression of MAFbx/ Atrogin-1 (36), and activation of activin signaling are also critical in triggering cachexia (5, 37). We therefore deter-mined whether Mstn loss possibly altered activin signaling. Surprisingly, Mstn transcript level remained unchanged in wild-type tumor-bearing mice (Fig. 6A). Transcript levels of

Figure 2. Mstn gene inactivation reduces tumor growth and expression of genes involved in tumor metabolism and

angiogenesis, and activin signaling while increasing apoptosis. A, tumor weight. B, VEGF-A, GLUT1, and HIF-1a transcript levels in the tumors of wild-type and Mstn/ mice. C, activinbA, activin bB, ActRIIB, and Alk4 transcript levels in the tumors of wild-type and Mstn/

mice. D, TWEAK transcript level in the tumors of wild-type and Mstn/

mice. E and F, immunoblot analysis of Bax protein content (E) and Bax-to-Bcl-2 protein ratio (F) in the tumors of wild-type and Mstn/mice. G,fluorometric

measurement of caspase-3 enzyme activity in the tumors of wild-type and Mstn/mice. Wild-type and Mstn/mice were inoculated with LLC cells. Tissues were removed 35 days after the inoculation of LLC cells. Data are means SE (n ¼ 8–12/group).

, P< 0.05;, P< 0.01; , P< 0.001.

(6)

activinbA and ActRIIB were decreased in response to tumor growth both in wild-type and Mstn/mice (Fig. 6B and C), but transcript levels of Alk4 and Alk5 were reduced in Mstn/ mice by tumor growth (Fig. 6D and E). Finally, Smad2/3 phosphorylation and Smad2/3 protein content were similar in wild-type and Mstn/mice, and remained unchanged in response to tumor growth (Fig. 6F). There-fore, Mstn loss does not seem to markedly alter Mstn/ activin signaling in skeletal muscle 35 days after inoculation of LLC cells.

Mstn gene inactivation dramatically prolongs survival of LLC tumor-bearing mice andApcMin/þmice

LLC tumor-bearing mice manifested a lethal wasting syndrome characterized by progressive weight loss (Fig. 1A) and death (Fig. 7A). Notably, Mstn loss was associated with a significant prolongation of animal survival (Fig. 7A). We therefore examined the efficacy of Mstn gene inactiva-tion in ApcMin/þmouse, an established model of colorectal cancer (17, 38) and cachexia (18) that carries a mutation in the adenomatous polyposis coli (Apc) gene, leading to mul-tiple intestinal neoplasia (Min; ref. 17). In human, mutation in Apc gene is responsible for the initiation and development

of familial adenomatous polyposis (38), an autosomal dom-inant inherited disorder characterized by cancer of the large intestine. The advantage of ApcMin/þ mice is the regular progression of tumor growth and muscle wasting (18) that is more closely related to human cancer cachexia, compared with the inoculation of tumor cells, which triggers extremely fast tumor growth and muscle wasting. On heterozygous Mstnþ/ and homozygous Mstn/ genetic backgrounds, survival of ApcMin/þ mouse was remarkably prolonged (Fig. 7B). When 100% of ApcMin/þMstnþ/þ mice had died 29 weeks after birth, about 90% of ApcMin/þMstnþ/ and ApcMin/þMstn/mice were still alive. Of note, wild-type and Mstn/mice had similar lifespan under normal conditions (Supplementary Fig. S5A). Increase in the survival rate of ApcMin/þMstn/mice was associated with a reduction in the number and size of intestinal polyps (Fig. 7C and D and Supplementary Fig. S5B). Development of polyposis is usu-ally associated with anemia and bloody feces (17). Measure-ment of hematocrit is therefore a good criterion to assess the health status of the animals. Although hematocrit was collapsed in ApcMin/þMstnþ/þmice, it was completely pre-served in ApcMin/þMstn/mice (Fig. 7E). Finally, Mstn loss on the ApcMin/þbackground attenuated the decrease in body Figure 3. Akt/mTOR pathway is

inhibited in gastrocnemius muscle of wild-type and Mstn/mice during cancer cachexia. A, immunoblot analysis of 4E-BP1 phosphorylation on Thr37 and Thr46. B, Luminex analysis of p70S6K phosphorylation on Thr421 and Ser424. C, immunoblot analysis of rpS6 phosphorylation on Ser235 and Ser236 and rpS6 protein content. Wild-type and Mstn/mice were inoculated with

LLC cells or received DPBS. Tissues were removed 35 days after the inoculation of LLC cells. Data are means SE (n¼ 8–12/group)., P< 0.05; , P< 0.01;, P< 0.001.

(7)

Figure 4. Mstn gene inactivation attenuates the activation of ubiquitin-proteasome pathway and NF-kB pathway in gastrocnemius muscle during cancer cachexia. A–C, transcript levels of MAFbx/Atrogin-1 (A), MuRF1 (B), and FoxO3 (C). D, immunoblot analysis of FoxO3 phosphorylation on Ser253. E, immunoblot analysis of NF-kB p65 phosphorylation on Ser536 and NF-kB p65 protein content. F, Luminex analysis of IkBa phosphorylation on Ser32 and Ser36 and IkBa protein content. G and H, transcript levels of TWEAK (G) and TRAF6 (H). Wild-type and Mstn/

mice were inoculated with LLC cells or received DPBS. Tissues were removed 35 days after the inoculation of LLC cells. Data are means SE (n¼ 8–12/group)., P< 0.05;, P< 0.01;, P< 0.001.

(8)

mass (Fig. 7F), and completely prevented the loss of skeletal muscle mass (Fig. 7G).

Discussion

Cancer-associated cachexia is a multifactorial devastating syndrome that increases toxicity from chemotherapy, increases surgery risk, reduces physical performance and patient's quality of life, and ultimately decreases survival in patients with cancer (4). Although muscle wasting, the key feature of cancer-associated cachexia, is recognized as a factor responsible for the death of many patients with cancer (3), it remains a poorly understood process whose molecular mechanisms are only beginning to emerge. The present study clearly indicates that targeting Mstn is an effective strategy to prevent skeletal muscle wasting, to slow tumor growth, and to increase survival rate in LLC tumor-bearing mice and ApcMin/þ mice.

Mechanistically, Mstn can promote muscle catabolism by repressing protein synthesis (9, 39) and increasing protein degradation (39). Ourfindings showing that Mstn loss did

not attenuate the inhibition of the Akt/mTOR pathway induced by tumor growth, but drastically attenuated the activation of proteolytic pathways, clearly indicate that Mstn loss provides protection against cancer-induced mus-cle wasting by repressing the activation of proteolytic path-ways. However, we cannot exclude the possibility that protein synthesis could be regulated differently earlier during the time course of cancer cachexia. The mechanisms signaling the cachectic state have been controversial, but cancer cachexia is considered to result, at least in part, from interactions between the host and the tumor through the production of tumor- and host-derived cachectic factors (2). In the present study, transcript levels of TWEAK, a powerful skeletal muscle-wasting cytokine (31), and that of TRAF6, its downstream effector (32), were markedly reduced in skel-etal muscle of Mstn/tumor-bearing mice. Furthermore, we also observed that circulating levels of catabolic cyto-kines are lowered in Mstn/mice compared with wild-type mice under normal conditions (Supplementary Fig. S6). Therefore, Mstn loss could contribute favorably to regulat-ing the level of cachectic factors. However, additional Figure 5. Mstn gene inactivation

attenuates the activation of autophagy-lysosome pathway in gastrocnemius muscle during cancer cachexia. A, immunoblot analysis of LC3b-I and LC3b-II protein content. B, Ulk1 transcript level. C, immunoblot analysis of Atg5–Atg12 protein complex. Wild-type and Mstn/mice were inoculated with LLC cells or received DPBS. Tissues were removed 35 days after the inoculation of LLC cells. Data are means SE (n ¼ 8–12/group).

, P< 0.05;, P< 0.01; , P< 0.001.

(9)

investigations will be necessary to clearly determine the role of Mstn in the regulation of the production of cachectic factors.

Myostatin and activins signal by binding to ActRIIA or ActRIIB, which then recruits and activates type-I activin receptors, Alk 4 and 5. Neither transcript levels of activinbA, Figure 6. Mstn/activin signaling in gastrocnemius muscle during cancer cachexia. A, Mstn transcript level. B–E, transcript levels of activinbA (B), ActRIIB (C), Alk4 (D), and Alk5 (E). F, immunoblot analysis of Smad2/3 phosphorylation on Ser465/467 and Ser423/425 and Smad2/3 protein content. Wild-type and Mstn/mice were inoculated with LLC cells or received DPBS. Tissues were removed 35 days after the inoculation of LLC cells. Data are means SE (n¼ 8–12/group)., P< 0.05; , P< 0.01;, P< 0.001.

(10)

ActRIIB, Alk4 and Alk5, nor Smad2/3 phosphorylation, were consistently regulated in wild-type and Mstn/ mice in response to tumor growth, suggesting that myostatin/activin signaling was not impaired 35 days after inoculation of LLC cells. However, it is possible that myostatin/activin signaling was differently regulated between wild-type and Mstn/ mice earlier during the time course of tumor growth. In support of this assumption, myostatin expression also

remained unchanged in skeletal muscle of wild-type mice in response to tumor growth at day 35, an observation that contrasts with all the studies that measured myostatin expres-sion in response to tumor growth, but at earlier time points (11–16). Therefore, a stimulation of myostatin/activin signal-ing in wild-type mice could occur earlier dursignal-ing the time course of tumor growth. Finally, a similar level of Smad 2/3 phos-phorylation was observed between wild-type and Mstn/ Figure 7. Mstn gene inactivation

prolongs lifespan of LLC tumor-bearing mice and ApcMin/þmice. A, survival rate of wild-type and Mstn/

tumor-bearing mice. B, Kaplan–Meier plot showing that Mstn gene inactivation dramatically prolongs lifespan of ApcMin/þMstnþ/and ApcMin/þMstn/mice. C,

representative longitudinal sections of intestine stained with methylene blue showing multiple intestinal polyps in ApcMin/þMstnþ/þ. D, the number

and size of intestinal polyps are reduced in ApcMin/þMstn/mice.

E, hematocrit in ApcMin/þMstnþ/þ and ApcMin/þMstn/mice. F, Mstn

gene inactivation prevents body weight loss in ApcMin/þmice. G, prevention of skeletal muscle wasting in ApcMin/þMstn/mice. Mice were 23.3 0.9 weeks old at the time of analysis. Data are means  SE (n ¼ 4–7/group)., P< 0.01; , P< 0.001.

(11)

mice at day 35, suggesting that other signaling influences may exert a stimulatory effect on this pathway in Mstn/mice.

Preservation of skeletal muscle mass in Mstn/ tumor-bearing mice and ApcMin/þMstn/mice is in line with studies showing that sequestration of extracellular activins and Mstn by administration of a soluble form of ActRIIB exerts antic-atabolic effects in mice models of cancer-cachexia (15, 16). However, our study extends these observations by showing that Mstn per se is a critical determinant of muscle mass homeostasis during cancer cachexia. One may question wheth-er the double-muscling phenotype of Mstn/mice is the basis for protection against muscle wasting. Previous studies have clearly demonstrated that the blockade of ActRIIB signaling at the time of tumor development in mice also provides protec-tion against cancer cachexia (15, 16). Although the strategy used in these studies inhibits both Mstn and activin signaling, these data strongly suggest that Mstn inhibition confers resis-tance to skeletal muscle atrophy. This also raises the question of whether blocking activin may provide additional protection against cancer cachexia. An increase in activin level has been reported in many catabolic states, including cancer cachexia (5). Increasing circulating level of activin A in mice also promotes muscle wasting, an effect that is not dependent on Mstn signaling (36). Furthermore, inhibition of ActRIIB sig-naling leads to muscle hypertrophy both in wild-type and Mstn/mice (40). Similar results have also been obtained by using an anti-ActRII antibody (41). Together, these data sup-port an imsup-portant role of activins in the regulation of skeletal muscle mass during cachexia. However, soluble ActRII can also be a target for other ligands, such as bone morphogenetic proteins (42), whose positive action on skeletal muscle homeo-stasis has been recently demonstrated (43, 44). Therefore, the beneficial effect of inhibiting both Mstn and activins through an ActRII blockade could be counterbalanced by the inhibition of bone morphogenetic proteins. Specific inhibition of Mstn or activins in the context of cancer cachexia should allow dis-criminating between the respective effects of Mstn and activins.

One strikingfinding of the present study was the reduction in tumor growth in Mstn/ mice, an effect that can be attributed to a decrease in tumor metabolism and angiogen-esis, and an increase in apoptosis. Tumor growth was also markedly slowed in ApcMin/þMstn/mice, together with a preservation of body weight and skeletal muscle mass. Although a reduction in tumor growth has not been system-atically observed (14, 15), the currentfindings are consistent with a previous study demonstrating that administration of a soluble ActRIIB slowed LLC tumor growth in mice (16). Our data are also further supported by genetic and pharmacologic studies showing that reduction/inhibition of ActRII signaling slows tumor development in gonads (45–48). Taken together, our results identify Mstn as a potential growth-promoting factor in intestinal tumor progression. Therefore, the current findings have implications in the design of new drugs and strategies for the treatment of tumors of the intestine and colon cancer.

It is assumed that up to 20% of cancer-related mortalities may derive from cachexia rather than direct tumor burden (2).

Here, we clearly show that prevention of muscle wasting and reduction in tumor growth in Mstn/mice was associated with an increase in survival rate. The reduction in tumor growth probably contributes to increasing the survival rate of Mstn/tumor-bearing mice. However, this antitumori-genic effect alone does not explain the prolonged survival of LLC tumor-bearing mice: when a covariance analysis was used to account for differences in tumor weight, survival rate was still significantly increased in Mstn/tumor-bearing mice. An association between preservation of skeletal muscle mass, reduction in tumor growth, and an increase in survival rate does not prove causality, but mechanistic links between these factors are biologically possible. Skeletal muscle constitutes an important reservoir of amino acids. An increase in the circu-lating pool of amino acids, due to excessive skeletal muscle protein degradation, may increase the availability of glucose precursors for liver gluconeogenesis, and ultimately provide energy in glucose form for tumor metabolism (49). Alterna-tively, acute respiratory failure is a frequent fatal event in patients with cancer (2). Preservation of muscle mass and function would therefore be beneficial by maintaining respi-ratory muscle function and could thus contribute to prolong-ing lifespan. Finally, preservation of skeletal muscle mass is also critical to limit the decrease in physical activity that occurs with the progression of the disease, and ultimately to delay whole body deconditioning.

The present study indicates that preserving skeletal muscle mass should be one of the goals of interventions aimed at prolonging survival in patients with advanced cancers. How-ever, further studies will be necessary to precisely determine the antitumorigenic function of Mstn inhibition. We also need more information about the circulating level of Mstn and other ActRII ligands (activins, inhibins, bone morphogenetic pro-teins) in patients with cancer. Possible differences in the expression of Mstn and other ActRII ligands between cancer types should be also determined. Finally, cachexia is not only encountered in cancer, but also in numerous diseases, includ-ing sepsis, chronic heart failure, renal failure, pulmonary diseases, prolonged immobilization, and neurogenic atrophy. Thus, the development of knowledge and strategies for the treatment of cancer cachexia may also be applicable to other pathologic situations associated with muscle wasting and excessive Mstn signaling.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design:Y.S. Gallot, D.G. Freyssenet

Development of methodology:Y.S. Gallot, A.-C. Durieux, D.G. Freyssenet Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.):Y.S. Gallot, A.-C. Durieux, J. Castells, M.M. Desgeorges, B. Vernus, L. Plantureux, D. Remond, V.E. Jahnke, D. Dardevet, G. Nemoz, A. Bonnieu, D.G. Freyssenet

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis):Y.S. Gallot, A.-C. Durieux, D. Remond, V.E. Jahnke, D. Dardevet, G. Nemoz, D.G. Freyssenet

Writing, review, and/or revision of the manuscript:Y.S. Gallot, E. Lefai, G. Nemoz, L. Schaeffer, D.G. Freyssenet

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):Y.S. Gallot, J. Castells, D.G. Freyssenet Study supervision:Y.S. Gallot, D.G. Freyssenet

(12)

References

1. Fearon K, Strasser F, Anker SD, Bosaeus I, Bruera E, Fainsinger RL, et al. Definition and classification of cancer cachexia: an international consensus. Lancet Oncol 2011;12:489–95.

2. Tisdale MJ. Mechanisms of cancer cachexia. Physiol Rev 2009; 89:381–410.

3. Tisdale MJ. Cancer cachexia. Curr Opin Gastroenterol 2010;26: 146–51.

4. Tisdale MJ. Cachexia in cancer patients. Nat Rev Cancer 2002;2: 862–71.

5. Han HQ, Zhou X, Mitch WE, Goldberg AL. Myostatin/activin pathway antagonism: molecular basis and therapeutic potential. Int J Biochem Cell Biol 2013;45:2333–47.

6. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 1997;387:83– 90.

7. Grobet L, Pirottin D, Farnir F, Poncelet D, Royo LJ, Brouwers B, et al. Modulating skeletal muscle mass by postnatal, muscle-specific inac-tivation of the myostatin gene. Genesis 2003;35:227–38.

8. Durieux AC, Amirouche A, Banzet S, Koulmann N, Bonnefoy R, Pasdeloup M, et al. Ectopic expression of myostatin induces atrophy of adult skeletal muscle by decreasing muscle gene expression. Endocrinology 2007;148:3140–7.

9. Amirouche A, Durieux AC, Banzet S, Koulmann N, Bonnefoy R, Mouret C, et al. Down-regulation of Akt/mammalian target of rapamycin signaling pathway in response to myostatin overexpression in skeletal muscle. Endocrinology 2009;150:286–94.

10. Zimmers TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tom-kinson KN, et al. Induction of cachexia in mice by systemically admin-istered myostatin. Science 2002;296:1486–8.

11. Costelli P, Muscaritoli M, Bonetto A, Penna F, Reffo P, Bossola M, et al. Muscle myostatin signalling is enhanced in experimental cancer cachexia. Eur J Clin Invest 2008;38:531–8.

12. Liu CM, Yang Z, Liu CW, Wang R, Tien P, Dale R, et al. Myostatin antisense RNA-mediated muscle growth in normal and cancer cachex-ia mice. Gene Ther 2008;15:155–60.

13. Bonetto A, Penna F, Minero VG, Reffo P, Bonelli G, Baccino FM, et al. Deacetylase inhibitors modulate the myostatin/follistatin axis without improving cachexia in tumor-bearing mice. Curr Cancer Drug Targets 2009;9:608–16.

14. Benny Klimek ME, Aydogdu T, Link MJ, Pons M, Koniaris LG, Zimmers TA. Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia. Biochem Biophys Res Commun 2010;391:1548–54.

15. Zhou X, Wang JL, Lu J, Song Y, Kwak KS, Jiao Q, et al. Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival. Cell 2010;142:531–43.

16. Busquets S, Toledo M, Orpi M, Massa D, Porta M, Capdevila E, et al. Myostatin blockage using actRIIB antagonism in mice bearing the Lewis lung carcinoma results in the improvement of muscle wasting and physical performance. J Cachexia Sarcopenia Muscle 2012;3:37– 43.

17. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 1990;247:322–4. 18. Baltgalvis KA, Berger FG, Pena MM, Davis JM, Muga SJ, Carson JA. Interleukin-6 and cachexia in ApcMin/þ mice. Am J Physiol Regul Integr Comp Physiol 2008;294:R393–401.

19. Berthon P, Duguez S, Favier FB, Amirouche A, Feasson L, Vico L, et al. Regulation of ubiquitin-proteasome system, caspase enzyme

activi-ties, and extracellular proteinases in rat soleus muscle in response to unloading. Pflugers Arch 2007;454:625–33.

20. Rapisarda A, Melillo G. Role of the VEGF/VEGFR axis in cancer biology and therapy. Adv Cancer Res 2012;114:237–67.

21. Macheda ML, Rogers S, Best JD. Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer. J Cell Physiol 2005;202:654–62.

22. Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology 2004;19:176–82.

23. Hu-Lowe DD, Chen E, Zhang L, Watson KD, Mancuso P, Lappin P, et al. Targeting activin receptor-like kinase 1 inhibits angiogenesis and tumorigenesis through a mechanism of action complementary to anti-VEGF therapies. Cancer Res 2011;71:1362–73.

24. Antsiferova M, Werner S. The bright and the dark sides of activin in wound healing and cancer. J Cell Sci 2012;125:3929–37.

25. Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, Hession C, et al. TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis. J Biol Chem 1997;272:32401–10. 26. Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, Stitt TN, et al.

Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/ Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol 2001;3: 1009–13.

27. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, et al. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 2001;3:1014–9.

28. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, et al. Foxo transcription factors induce the atrophy-related ubiquitin ligase atro-gin-1 and cause skeletal muscle atrophy. Cell 2004;117:399–412. 29. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, et al.

The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 2004;14:395–403.

30. Cai D, Frantz JD, Tawa NE Jr, Melendez PA, Oh BC, Lidov HG, et al. IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell 2004;119:285–98.

31. Mittal A, Bhatnagar S, Kumar A, Lach-Trifilieff E, Wauters S, Li H, et al. The TWEAK-Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice. J Cell Biol 2010;188:833–49. 32. Paul PK, Gupta SK, Bhatnagar S, Panguluri SK, Darnay BG, Choi Y,

et al. Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice. J Cell Biol 2010;191:1395–411.

33. Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, et al. FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab 2007;6:458–71.

34. Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, et al. FoxO3 coordinately activates protein degradation by the autophagic/lyso-somal and proteaautophagic/lyso-somal pathways in atrophying muscle cells. Cell Metab 2007;6:472–83.

35. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012;8:445–544.

36. Chen JL, Walton KL, Winbanks CE, Murphy KT, Thomson RE, Makanji Y, et al. Elevated expression of activins promotes muscle wasting and cachexia. FASEB J 2014;28:1711–23.

37. Matzuk MM, Finegold MJ, Mather JP, Krummen L, Lu H, Bradley A. Development of cancer cachexia-like syndrome and adrenal tumors in inhibin-deficient mice. Proc Natl Acad Sci U S A 1994;91:8817–21.

(13)

38. Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, Albertsen H, et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 1991;66:589–600.

39. Lokireddy S, Mouly V, Butler-Browne G, Gluckman PD, Sharma M, Kambadur R, et al. Myostatin promotes the wasting of human myo-blast cultures through promoting ubiquitin-proteasome pathway-mediated loss of sarcomeric proteins. Am J Physiol Cell Physiol 2011;301:C1316–24.

40. Lee SJ, Reed LA, Davies MV, Girgenrath S, Goad ME, Tomkinson KN, et al. Regulation of muscle growth by multiple ligands signaling through activin type II receptors. Proc Natl Acad Sci U S A 2005;102:18117–22.

41. Lach-Trifilieff E, Minetti GC, Sheppard K, Ibebunjo C, Feige JN, Hartmann S, et al. An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atro-phy. Mol Cell Biol 2014;34:606–18.

42. Souza TA, Chen X, Guo Y, Sava P, Zhang J, Hill JJ, et al. Proteomic identification and functional validation of activins and bone morpho-genetic protein 11 as candidate novel muscle mass regulators. Mol Endocrinol 2008;22:2689–702.

43. Sartori R, Schirwis E, Blaauw B, Bortolanza S, Zhao J, Enzo E, et al. BMP signaling controls muscle mass. Nat Genet 2013;45:1309–18.

44. Winbanks CE, Chen JL, Qian H, Liu Y, Bernardo BC, Beyer C, et al. The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass. J Cell Biol 2013;203:345–57.

45. Cipriano SC, Chen L, Kumar TR, Matzuk MM. Follistatin is a modulator of gonadal tumor progression and the activin-induced wasting syndrome in inhibin-deficient mice. Endocrinology 2000; 141:2319–27.

46. Coerver KA, Woodruff TK, Finegold MJ, Mather J, Bradley A, Matzuk MM. Activin signaling through activin receptor type II causes the cachexia-like symptoms in inhibin-deficient mice. Mol Endocrinol 1996;10:534–43.

47. Li Q, Kumar R, Underwood K, O'Connor AE, Loveland KL, Seehra JS, et al. Prevention of cachexia-like syndrome development and reduc-tion of tumor progression in inhibin-deficient mice following adminis-tration of a chimeric activin receptor type II-murine Fc protein. Mol Hum Reprod 2007;13:675–83.

48. Kumar TR, Palapattu G, Wang P, Woodruff TK, Boime I, Byrne MC, et al. Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis. Mol Endocrinol 1999;13:851–65.

49. Argiles JM, Lopez-Soriano FJ. The energy state of tumor-bearing rats. J Biol Chem 1991;266:2978–82.

(14)

2014;74:7344-7356. Published OnlineFirst October 21, 2014.

Cancer Res

Yann S. Gallot, Anne-Cécile Durieux, Josiane Castells, et al.

Updated version

10.1158/0008-5472.CAN-14-0057

doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2014/10/21/0008-5472.CAN-14-0057.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/74/24/7344.full#ref-list-1

This article cites 49 articles, 12 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/74/24/7344.full#related-urls

This article has been cited by 8 HighWire-hosted articles. Access the articles at:

E-mail alerts

Sign up to receive free email-alerts

related to this article or journal.

Subscriptions

Reprints and

.

pubs@aacr.org

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site.

Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.

http://cancerres.aacrjournals.org/content/74/24/7344

Figure

Figure 1. Mstn gene inactivation prevents loss of skeletal muscle mass in tumor-bearing mice
Figure 2. Mstn gene inactivation reduces tumor growth and expression of genes involved in tumor metabolism and
Figure 3. Akt/mTOR pathway is inhibited in gastrocnemius muscle of wild-type and Mstn / mice during cancer cachexia
Figure 4. Mstn gene inactivation attenuates the activation of ubiquitin-proteasome pathway and NF-kB pathway in gastrocnemius muscle during cancer cachexia
+4

Références

Documents relatifs

Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial. Alemtuzumab for

ﺔﻴﻟﺎﺘﻟﺍ ﺕﺎﻴﻁﻌﻤ ﻥﻋ ﺕﻴﻭﺼﺘﻟﺍ ﻡﻭﻴ ﺏﺭﻐﻤﻟﺍ ﻲﻓ ﺔﻴﺴﺎﻴﺴﻟﺍ ﺔﻁﻴﺭﺨﻟﺍ ﺕﻔﺸﻜ ﺩﻗﻭ :.. 161 1 ﺱﺎﻤﺤ ﻙﺎﻨﻫ ﻥﻜﻴ ﻡﻟ ـ لﺠ ﻥﺃ لﻭﻘﻟﺍ ﺯﺎﺠ ﺎﻤﺒﺭ لﺒ ،ﺕﺎﺒﺎﺨﺘﻨﻻﺍ ﻩﺫﻫ ﺀﺍﺯﺇ ﻙﻟﺫ ﻲﻠﺠﺘ

COMBINED SANGER AND NGS SEQUENCE ANALYSIS OF THE MYOSTATIN GENE (MSTN) IN THE CAMELUS DROMEDARIUS SPECIES..

MBLR matrices can be represented using the cluster tree modelization typically used in the H literature; this provides a convenient tool to formalize the key difference between the

لﻫﺎﻧﻣﻟا بﺣﺎﺻ لﺎﻗ &#34; : فدارﻣ ردﺻﻣ ﺔﻐﻠﻟا ﻲﻓ وﻫ ةءارﻘﻠﻟ (...) اذﻫ نﻣ لﻘﻧ مﺛ مﻠﺳو ﻪﯾﻠﻋ ﷲا ﻰﻠﺻ ﻲﺑﻧﻟا ﻰﻠﻋ لزﻧﻣﻟا زﺟﻌﻣﻟا مﻼﻛﻠﻟ ﺎﻣﺳا لﻌﺟو يردﺻﻣﻟا ﻰﻧﻌﻣﻟا

ةيضايرلا جماربلا نع نيلوئسملاو نيبردملاو نييضايرلا نم ريبك عاطق نيب ةرشتنم تاداقتعلاا هذه نأ فسلأا كولسلل يطعت لا ةيضايرلا ةيدنلأا مظعم نأ

In this arti- cle, we study two level systems governed by the con- trolled Lindblad equation, we will compute all the pos- sible states to which the systems can be driven at arbi-

(A) Principal component analysis (PCA) on expression of 622 genes from 24 individuals with latent M.tb infection (LTBI) and 24 with active TB disease (TB) after whole