Methylseleninic Acid Superactivates p53- Senescence …cancerpreventionresearch.aacrjournals.org/content/canprevres/9/1/... · Methylseleninic Acid Superactivates p53-Senescence Cancer

Research Article

Methylseleninic Acid Superactivates p53-Senescence Cancer Progression Barrier inProstate Lesions of Pten-Knockout MouseLei Wang1, Xiaolan Guo1, Ji Wang1, Cheng Jiang2, Maarten C. Bosland3, Junxuan L€u2, andYibin Deng1

Abstract

Monomethylated selenium (MM-Se) forms that are precursorsof methylselenol, such as methylseleninic acid (MSeA), differ inmetabolismandanticancer activities in preclinical cell and animalmodels from seleno-methionine that had failed to exert preven-tive efficacy against prostate cancer inNorthAmericanmen.Giventhat human prostate cancer arises from precancerous lesions suchas high-grade prostatic intraepithelial neoplasia (HG-PIN), whichfrequently have lost phosphatase and tensin homolog (PTEN)tumor suppressor permitting phosphatidylinositol-3-OH kinase(PI3K)–protein kinase B (AKT) oncogenic signaling, we tested theefficacy of MSeA to inhibit HG-PIN progression in Pten prostate-specific knockout (KO) mice and assessed the mechanisticinvolvement of p53-mediated cellular senescence and of theandrogen receptor (AR). We observed that short-term (4 weeks)oralMSeA treatment significantly increased expression of P53 andP21Cip1 proteins and senescence-associated-b-galactosidase

staining, and reduced Ki67 cell proliferation index in Pten KOprostate epithelium. Long-term (25 weeks) MSeA administrationsignificantly suppressed HG-PIN phenotype, tumor weight, andprevented emergence of invasive carcinoma in Pten KO mice.Mechanistically, the long-term MSeA treatment not only sus-tained P53-mediated senescence, but also markedly reduced AKTphosphorylation and AR abundance in the Pten KO prostate.Importantly, these cellular and molecular changes were notobserved in the prostate of wild-type littermates which weresimilarly treated with MSeA. Because p53 signaling is likely tobe intact in HG-PIN compared with advanced prostate cancer, theselective superactivation of p53-mediated senescence by MSeAsuggests a new paradigm of cancer chemoprevention by strength-ening a cancer progression barrier through induction of irrevers-ible senescence with additional suppression of AR and AKToncogenic signaling. Cancer Prev Res; 9(1); 35–42. �2015 AACR.

IntroductionSelenium (Se) compounds have been studied for their che-

mopreventive potential in various animal models of carcino-genesis, notably mammary, colon, lung, and prostate cancer.Selenized yeast (Se-yeast) and its principal Se form Se-methi-onine (SeMet) have been tested in several human trials inNorth America for the prevention of prostate cancer (1–4).The outcomes of the Selenium and vitamin E Cancer Trial(SELECT) and other trials failed to demonstrate a preventiveefficacy of these Se forms in the Se-adequate North Americancohorts (2–4). Many, including us, have opined on the possiblereasons for such failures (5–7, 8). One key factor was the

selection of ineffective Se agents. In fact, the scarce animalefficacy data that existed prior to the initiation of these trials didnot support prostate cancer preventive efficacy of SeMet andthese negative data were not published in full-length until afterSELECT was terminated (9, 10).

Preclinical and mechanistic research has demonstrated thatSeMet has little in common with the mono-methylated methyl-selenol precursor Se forms (MM-Se), such asmethylseleninic acid(MSeA), in terms of metabolism and anticancer activities (8, 11).We have posited that the failure of SeMet should not be taken toindicate that other Se forms are ineffective for prostate cancerchemoprevention (12). Indeed, we have shown that daily orallyadministered MSeA inhibited the growth of DU145 and PC-3human prostate cancer xenografts in athymic nudemice, whereasan equal Se dose of SeMetwas inactive, in spite of SeMet leading tomuch higher retention of Se in the xenograft tumors (13).We alsoreported the efficacy of MSeA to inhibit prostate carcinogenesis inthe transgenic adenocarcinoma mouse prostate (TRAMP) model,which improved survival with no observable long-term adverseeffect (12). More efficacy and biomarker assessments in clinicallyrelevant prostate carcinogenesis models with MM-Se will beessential to evaluate their prostate cancer chemopreventionpotential in the post-SELECT era to support future translation ofthese data to humans.

The PTEN (phosphatase and tensin homolog) protein antag-onizes the phosphatidylinositol-3-OH kinase (PI3K)–proteinkinase B (AKT) signaling pathway that stimulates cancer cell

1Hormel Institute,UniversityofMinnesota, Austin,Minnesota. 2Depart-ment of Biomedical Sciences, Texas Tech University Health SciencesCenter School of Pharmacy, Amarillo, Texas. 3Department of Pathol-ogy, University of Illinois at Chicago (UIC) College of Medicine, Chi-cago, Illinois.

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

Corresponding Authors: Yibin Deng, University of Minnesota Hormel Institute,801 16th AvenueNE, Austin, MN55912. E-mail: [email protected]; and JunxuanL€u, Pennsylvania State University College of Medicine, Hershey, PA 17033.E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-15-0236

�2015 American Association for Cancer Research.

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metabolism, proliferation, and survival (14). Human PTEN losshas been identified in 45% of high-grade prostatic intraepithelialneoplasia (HG-PIN) and 70%of advanced prostate cancer (ref. 14and reference therein). Mouse genetic studies have demonstratedthat loss ofPten in prostate epithelium rapidly causesHG-PIN thatultimately progresses to invasive adenocarcinoma and metastaticdisease (15). Asmen diagnosed with HG-PIN are at increased riskof developing prostate cancer, this prostate-specific conditionalPten KO mouse model recapitulates essential characteristics ofhuman prostate carcinogenesis and is considered clinically rele-vant for studies of prostate cancer chemoprevention. In the PtenKOmodel, the sustained activation of AKT not only initiates and

perpetuates oncogenic signaling and progression pathways, but atthe same time induces cellular senescence (known as Pten-defi-ciency Induced Cellular Senescence, PICS), which acts as a for-midable barrier to restrain oncogenic progression to invasive andmetastatic disease (16, 17). Mechanistic studies suggest that PICSprimarily depends on P53 protein overexpression, which isinduced through AKT/mTOR-mediated protein synthesis andp19ARF sequestration of MDM-2, resulting in inhibition of pro-teasome-mediated P53 degradation (16).

The critical role of androgen receptor (AR) signaling inprostate cancer, even at the advanced metastatic castration-resistant stage, is well established and therapeutically exploited

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Figure 1.Oral supplement ofMSeA (3mgSe/kgbody weight, 5 days/week) for4 weeks induced p53–p21-mediatedsenescence and inhibited prostateepithelial cell proliferation in vivo.MSeAorwater treatment commencedat 12 weeks of age. A, prostate weightof Pten KO (PtenD/D) mice (n¼ 5) andWT littermates (Ptenþ/þ) (n ¼ 3).B, immunoblot analyses of Pten, p-AktSer473, P53, and P21Cip1 proteins inpooled samples of each group.Tubulin served as loading control.C, representative photomicrographsof immunohistochemical staining ofandrogen receptor (AR), Ki67, andSA-galactosidase staining of Pten KOprostate from water-treated controlmice and MSeA-treated mice.Magnification 100�. D, quantitationresults from individual mice for eachgroup are presented as mean � SEM,n ¼ 5 for PtenD/D mice and n ¼ 3 forPtenþ/þ mice. For AR and SA-galstaining, the integrated opticaldensity (IOD) was normalized tocontrol mice as 100. For Ki67,percentage of positive nuclei in theprostate epithelium was quantified.

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Cancer Prev Res; 9(1) January 2016 Cancer Prevention Research36




(18). Unfortunately, recent studies have shown that inhibitionof AR signaling by castration or antagonist drugs inadvertentlypromotes the progression of stable HG-PIN to invasive carci-nomas in Pten KO model (19), raising concerns for utilizationof these androgen deprivation strategies for chemopreventionin high-risk men and prostate cancer patients with PTENdeficiency or mutations.

In cell culture studies, we and others have shown MSeAsuppression of AR abundance and signaling in prostate cancercells (20, 21), and the phosphorylative activation of AKTSer473 (pAKT; ref. 22, 23). MSeA was recently shown to inducecellular senescence in human primary lung fibroblasts and thiscellular effect was likely mediated by ATM/P53 signaling (24,25). Therefore, in this study, we tested the hypothesis thatMSeA would inhibit Pten-deficient carcinogenesis and prostatecancer progression and assessed the mechanistic involvementof p53, PICS, AKT, and AR in conferring the hypothesizedin vivo efficacy.

Materials and MethodsGeneration of Pten KO mice

The conditional Ptenflox/flox mouse was generated as previ-ously described (26). PB-Cre4 transgenic mice were obtainedfrom the NCI Mouse Repository. Female mice carryingPtenflox/þ were crossed with male mice harboring PB-Cre4þPten-flox/þ to generate mutant mice with prostate epithelium-specificdeletion of Pten. Tail DNA was used for PCR-based genotypingas described (26). All animal protocols were approved by theUniversity of Minnesota Institutional Animal Care and UseCommittee.

Intervention experiments with MSeAIn the short-term experiment, 12-week-old Pten KO mice

(Creþ;PtenFlox/Flox, hereafter indicated as PtenD/Dmice) were ran-domly assigned, 5 mice per group, to receive water or MSeA for 4weeks by daily (5 days/week) oral application at the base of thetongue as before (12, 13). Wild-type (WT) littermates (Cre-;PtenFlox/Flox; hereafter as Ptenþ/þ; n ¼ 3) were treated identicallywith water or MSeA to provide comparison control tissues.AIN93G semipurified diet and water were provided ad libitum.Mouse body weight was monitored weekly. At necropsy, totalprostate was dissected, photographed, and weighed. One portionof prostate tissues from each mouse was fixed in formalin forhematoxylin and eosin (H&E) and immunohistochemistry stain-ing. The reminder was stored frozen at �80�C for senescence-associated b-galactosidase (SA-gal) staining and Western blotanalyses.

The long-term experiment was carried out with same design,except that the mice were 10 weeks old at the start of the MSeAand water treatments (5 days/week), lasting for 25 weeks (8mice per group). At necropsy, the genitourinary (GU) tract wascollected and weighed. Then the different prostate lobes weredissected and weighed. The prostate lobes were saved andprocessed individually for histopathology and biochemicalanalyses.

Histopathology analysisTissue processing and staining were as performed previously

(12, 26). H&E-stained lesions were verified by a pathologist (M.Bosland). The pathologic changes of all lobes of prostate wereclassified according to Shappell and colleagues (27).

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Figure 2.Effect of oral supplement of MSeA (3mg Se/kg body weight, 5 days/week)for 25 weeks on Pten KO–drivengrowths of genitourinary organs andprostate tumors. MSeA or watertreatment commenced at 10 weeks ofage. A, genitourinary (GU) tractweight of Pten KO (PtenD/D) mice(n ¼ 8) and WT littermates (Ptenþ/þ)(n ¼ 8). B, whole prostate weight ofPtenD/Dmice (n¼ 8) andPtenþ/þmice(n ¼ 8). C, individual lobe weights ofPtenD/Dmice (n¼ 8) andPtenþ/þmice(n ¼ 8). AP, anterior prostate; DLP,dorsolateral prostate; VP, ventralprostate. �P¼0.05 or two-sided t-testP values were shown.

Next-Gen Selenium Strengthens a Cancer Barrier

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Senescence-associated b-galactosidase stainingProstate tissues were embedded with optimal cutting tempe-

rature (OCT) compound and cut into 4-mm sections. The sectionswere stained for SA-gal and counterstained with eosin, asdescribed previously (26). The integrated optical density (IOD)in the prostate epithelium/lesions was quantified by ImagePro-Plus 6.3 software.

Immunohistochemistry (IHC) and immunoblot analysisIHC and immunoblot (Western) were performed as previously

described (26). Briefly, antibodies against Ki67 (NeoMarker), AR(Millipore), cleaved caspase-3, and p-AKT Ser473 (Cell SignalingTechnology) were diluted at 1:100 for IHC. Images were capturedand analyzed by ImagePro-Plus 6.3 software for integrated opticaldensity (IOD) semiquantitation. For immunoblot, the prostatetissues were homogenized in nondenaturing lysis buffer andsubjected to SDS-PAGE and blotted with antibodies againstP53, P21Cip1, p-AKT Ser473, AR, and tubulin. Pooling of prostatetissues from the short-term experiment was necessary due tolimited amount of material available.

Statistical analysesFor parametric data, themean and SEMwere calculated for each

experimental group. Differences among groups were analyzed byANOVA for more than two groups. For comparison of only twogroups, the Student t test was used. Significant differences wereaccepted at P < 0.05.

ResultsShort-term MSeA treatment of Pten KO mice led tosuperactivation of p53–p21 and cellular senescence in prostateepithelium

In the first experiment, we evaluated the effect of 4-week MSeAtreatment to identify early biochemical and cellular changes thatmight correlate and predict its long-term preventive efficacyagainst HG-PIN growth and tumor progression in Pten KO mice.As shown in Fig. 1A,MSeA treatment led to a reduction (�20%)ofprostate weight in Pten KO mice compared with the prostateweight in WT mice, which was not affected by MSeA. The proteinlevels of Pten and phospho-Akt Ser473 (p-Akt) were analyzed inpooled prostate samples of WT mice and Pten KO mice. Asexpected, Pten KO mice lacked Pten protein in the prostateand had greatly increased p-Akt expression (Fig. 1B, lanes 3, 4 vs.1, 2). Consistent with previous results (16, 17, 26), PtenKO mouse prostate showed increased basal expression of P53and P21Cip1 over the WT counterpart (Fig. 1B, lane 3 vs. lane 1).MSeA treatment of the Pten KO mice dramatically increased P53and P21Cip1, but this did not occur in WT mice, whereas p-Aktexpression was not affected in MSeA-treated KO or WT mice(Fig. 1B). In addition, there was no observable change in ARexpression determined by IHC in Pten KO mice after 4 weeks ofMSeA treatment (Fig. 1C and D).

Because increased P53 protein abundance causes PICS in thePten KO mice (16, 17), we examined SA-gal expression in situ

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Figure 3.Long-term oral MSeAsupplementation inhibited prostatecancer progression in Pten KO mice.Oral supplementation of MSeA (3 mgSe/kg body weight, 5 days/week) wasinitiated at 10 weeks of age for 25weeks. A and B, representativephotomicrographs of H&E-stainedprostate lobes of (A) Pten KO(PtenD/D) mice treated with water(Con) or MSeA and (B) WT (Ptenþ/þ)mice treated with water (Con) orMSeA. C, quantitation of anterior (AP),ventral (VP), and dorsolateral (DLP)prostate pathologic changes. Mean �SEM, PtenD/D mice (n ¼ 8), andPtenþ/þ mice (n ¼ 8). � , statisticaldifference from Pten KO control mice,P < 0.05.

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Figure 4.Effect of long-term MSeAsupplementation on p-Akt andandrogen receptor (AR), senescence,and cellular proliferative index in theanterior prostate of mice in Fig. 3.A and B, representativephotomicrographs of IHC staining ofp-Akt Ser473, AR, Ki67, and SA-galactosidase activity in anteriorprostate of (A) PtenKO (PtenD/D)micetreated with water (Con) or MSeA and(B) WT (Ptenþ/þ) mice treated withwater (Con) or MSeA. C, quantitationof changes fromA and B. Mean� SEM,PtenD/Dmice (n¼ 8) and Ptenþ/þmice(n¼ 8). The integrated optical density(IOD) was estimated by ImagePro-Plus software. � , statistical differencefrom Pten KO control mice, P < 0.05.






in frozen prostate sections (Fig. 1C). Pten KO prostate showedlow but detectable SA-gal staining (Fig. 1C). However, in theprostate of MSeA-treated Pten KO mice, the staining intensitywas remarkably elevated in the epithelial cells by as much as 4-fold, estimated by ImagePro-Plus software (Fig. 1C and D).Because senescence is an irreversible terminal proliferativearrest, we examined Ki67 as a proliferation indicator anddetected significant suppression of Ki67 labeling index (%) inprostate of the MSeA-treated Pten KO mice compared withwater-treated mice (Fig. 1C and D).

Prolonged MSeA treatment of Pten KOmice prevented prostateadenocarcinoma

The promising biochemical and cellular responses to theshort-term MSeA intervention prompted us to evaluate itschemopreventive efficacy on Pten KO HG-PIN growth andprogression in the second experiment with 25-week adminis-tration. Consistent with long-term safety of MSeA supplementin our previous study with the TRAMP model (12), no signif-icant effect of MSeA was observed on the body weight gain ofthe mice of each genotype (Supplementary Fig. S1A). As shownin Fig. 2A, long-term MSeA daily treatment did not significantlyaffect the genitourinary tract (GU) weight of the WT mice, butdecreased Pten KO–driven expansion of GU over the WTbaseline by more than 70% (Fig. 2A). Similarly, the prostateweight was not affected in the WT mice by MSeA, but wasdecreased by more than 50% in the Pten KO mice over the WTbaseline (Fig. 2B). At the gross anatomical level, blood-richprostate tumors were visible in some water-treated control PtenKO mice (Supplementary Fig. S1B). Among the different lobes,the AP exhibited the most Pten KO–driven growth (Fig. 2C;Supplementary Fig. S1C), whereas in TRAMP mice the DLPundergo the most growth (12). The prostate weight suppressingeffect of MSeA in the Pten KOmice was uniform across AP, DLP,and VP lobes (Fig. 2C; Supplementary Fig. S1C). In sharpcontrast, the weight of prostate lobes in WT mice of the MSeAand control groups was not different (Fig. 2C).

Histologically, the prostate lesions from the control Pten KOmice showed HG-PIN phenotypes in all three lobes (Fig. 3A).Notably 38% (3 out of 8 mice) of Pten KOmice progressed fromHG-PIN to invasive adenocarcinomas at termination of theexperiment at 35 weeks of age (Supplementary Fig. S1D). Incontrast, MSeA-treated mice showed dramatic histopathologicmodification, many approaching near normal appearance of theprostate of theWTmice andnoneof themwithdetectable invasiveadenocarcinoma features (Fig. 3A and C). Consistent with selec-tivity of targeting oncogenic growth, MSeA treatment of WT micedid not affect their typical normal glandular structures (Fig. 3Band 3C). These findings indicate that long-term MSeA treatmentsignificantly inhibited HG-PIN growth and progression to carci-noma in vivo.

Long-termMSeA treatment decreased p-Akt and AR abundancein Pten KO prostate epithelium

Because short-term MSeA superactivated P53/P21Cip1 andincreased senescence in the Pten KO epithelium in vivo, weexamined whether long-term MSeA was able to sustain thecellular senescence phenotype. SA-gal staining of AP lobesshowed intense senescence in the MSeA-treated Pten KO mice(Fig. 4A) with little effect in the WT mice (Fig. 4B and C). Ki67staining confirmed the paucity of proliferating cells in the MSeA-

treated Pten KO prostate epithelium (Fig. 4A and C). No appreci-able apoptosis, indicated by cleaved caspase-3, was induced byMSeA treatment in Pten KO mice (data not shown).

It is noteworthy that IHC staining intensity of p-Akt and ARproteins in the AP lobe of MSeA-treated Pten KO mice wasnoticeably decreased (Fig. 4A and C) without correspondingobservable changes in the WTmice (Fig. 4B and C). Immunoblotconfirmed the IHC results for p-AKT and AR protein abundancesuppression by MSeA in the Pten KO prostate (Fig. 5A).

DiscussionTo our best knowledge, this study is the first in which any form

of Se has been tested in the Pten KO prostate cancer mouse modelfor chemopreventive efficacy. It is also the first time that in vivosenescence was measurably increased by MSeA treatment selec-tively in the HG-PIN epithelium of Pten KO mice without anydetectable impact on the prostate of the WT mice. The observedconcomitant increase of P53 and P21Cip1 in the prostate ofMSeA-treated Pten KO mice was not evident in the prostate ofWTmice (Fig. 1B), consistent with the selective superactivation ofthis crucial senescence signaling axis. In addition to boosting andsustaining P53-P21Cip1 senescence as a cell proliferation barrier,long-term treatment with MSeA led to considerably reducedtumor burden (Fig. 2) with decreased AR abundance and phos-phorylation of Akt (Figs. 4 and 5), together contributing toeffective suppression of the progression of HG-PIN to carcinoma(shown schematically in Fig. 5B). Given that current Akt/mTOR

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Figure 5.Selective suppression effect of oral supplement of MSeA (3 mg Se/kg bodyweight, 5 days/week) for 25 weeks on androgen receptor (AR) and p-AktSer473 abundance in the Pten KO prostate. A, immunoblot analyses of AR, p-Akt, and total Akt in the anterior prostate lobe of Pten KO (PtenD/D) mice andWT littermates (Ptenþ/þ) treated with water (Con) or MSeA. Two mice pergroup were randomly chosen for analyses. Tubulin served as loading control.B, schematic illustration of temporal sequence of cellular and molecularevents in Pten KO prostate carcinogenesis and the entry points of MSeAactions.


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inhibitor drugs activate AR signaling, whereas androgen ablationand AR antagonist drugs cross-induce the Akt pathway through areciprocal feedback regulatory loop (28), the inhibition of boththe Akt and the AR signaling pathways by prolonged MSeAsupplement suggests that combined use ofMSeAwith these drugsmay mitigate their undesirable side effects and result in greaterprostate cancer risk reduction.

Human prostate cancer arises from precancerous HG-PINlesions with a prolonged clinical course, affording unique win-dows of opportunity for chemoprevention/intervention. Becausep53 signaling is more likely to be intact in precancerous lesionsthan advanced prostate cancer, the superactivation of p53-senes-cence by MSeA offers a new paradigm for prostate cancer chemo-prevention through strengthening a cancer progression barrierin the precursor lesions. Our data support that MSeA superacti-vated and sustained P53-mediated cellular senescence and sub-sequently inhibited both the Akt and the AR signaling pathwaysto suppress Pten-deficient HG-PIN progression to adenocarcino-ma (Fig. 5B). The in vivo mechanisms mediating these cellularandmolecular actions ofMSeA are currently being elucidated. Theefficacy for chemoprevention of Pten-deficient HG-PIN progres-sion by MSeA documented in this work and the previouslydemonstrated efficacy and safety of MSeA in other prostate cancermouse models (12,13) provide strong justification for furtherdevelopment of MM-Se toward human translational studies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Jiang, J. Lu, Y. DengDevelopment of methodology: L. Wang, X. Guo, J. Wang, Y. DengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Wang, X. Guo, J. Wang, J. Lu, Y. DengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Wang, X. Guo, J. Wang, C. Jiang, M.C. Bosland,J. Lu, Y. DengWriting, review, and/or revision of the manuscript: M.C. Bosland, J. Lu,Y. DengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Jiang, Y. DengStudy supervision: J. Lu, Y. Deng

Grant SupportThis workwas supported by grants R21CA155522 (to Y. Deng and J. L€u) and

R01 CA172169 (to J. L€u, Y. Deng, and M. Bosland) from the NCI, NIH.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 9, 2015; revised October 5, 2015; accepted October 20, 2015;published OnlineFirst October 28, 2015.

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Wang et al.




2016;9:35-42. Published OnlineFirst October 28, 2015.Cancer Prev Res Lei Wang, Xiaolan Guo, Ji Wang, et al.

-Knockout MousePtenProgression Barrier in Prostate Lesions of Methylseleninic Acid Superactivates p53-Senescence Cancer

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