methylseleninic acid super-activates p53-senescence...

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Methylseleninic acid super-activates p53-senescence cancer progression

barrier in prostate lesions of Pten-knockout mouse

Lei Wang1, Xiaolan Guo1, Ji Wang1, Cheng Jiang2, Maarten C. Bosland3, Junxuan Lü2* and

Yibin Deng1*

1Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN 55912

2Department of Biomedical Sciences, Texas Tech University Health Sciences Center School of

Pharmacy, 1300 S. Coulter, Amarillo, TX 79106

3Department of Pathology, University of Illinois at Chicago (UIC), College of Medicine,

Chicago, IL 60612

*Corresponding authors: Dr. Yibin Deng, University of Minnesota Hormel Institute, 801 16th

Ave NE, Austin, MN 55912. E-mail: [email protected]; Dr. Junxuan Lü, Department of

Biomedical Sciences, Texas Tech University Health Sciences Center School of Pharmacy, 1300

S. Coulter St, Amarillo, TX 79106. E-mail: [email protected];

Running title: Next-gen selenium strengthens a cancer barrier

Key words: senescence, monomethylated selenium, cancer chemoprevention

Word count (2394). Figures (5) and Supplementary Figure (1). Abstract word count (248)

for Cancer Research. on September 7, 2018. © 2015 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 28, 2015; DOI: 10.1158/1940-6207.CAPR-15-0236

http://cancerpreventionresearch.aacrjournals.org/

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Funding supports: Grants R21 CA155522 (Y. Deng, J. Lü) and R01 CA172169 (J. Lü, Y.

Deng, M. Bosland) from the National Cancer Institute, National Institutes of Health.

Disclosure of Potential Conflicts of Interest: All authors have no personal or financial

conflict of interest and have not entered into any agreement that could interfere with our access

to the data on the research or on our ability to analyze the data independently, to prepare articles,

and to publish them.




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ABSTRACT

Monomethylated selenium (MM-Se) forms that are precursors of methylselenol such as

methylseleninic acid (MSeA) differ in metabolism and anti-cancer activities in preclinical cell

and animal models from seleno-methionine that had failed to exert preventive efficacy against

prostate cancer (PCa) in North American men. Given that human PCa arises from precancerous

lesions such as high-grade prostatic intraepithelial neoplasia (HG-PIN) which frequently have

lost PTEN tumor suppressor permitting AKT oncogenic signaling, we tested the efficacy of

MSeA to inhibit HG-PIN progression in Pten prostate specific knockout (KO) mice and assessed

the mechanistic involvement of p53-mediated cellular senescence and of the androgen receptor

(AR). We observed that short-term (4 weeks) oral MSeA treatment significantly increased

expression of P53 and P21Cip1 proteins and senescence-associated-β-galactosidase staining, and

reduced Ki-67 cell proliferation index in Pten KO prostate epithelium. Long-term (25 weeks)

MSeA administration significantly suppressed HG-PIN phenotype, tumor weight, and prevented

emergence of invasive carcinoma in Pten KO mice. Mechanistically, the long-term MSeA

treatment not only sustained P53-mediated senescence, but also markedly reduced AKT

phosphorylation and AR abundance in the Pten KO prostate. Importantly, these cellular and

molecular changes were not observed in the prostate of wild type littermates which were

similarly treated with MSeA. Since p53 signaling is likely to be intact in HG-PIN compared to

advanced PCa, the selective super-activation of p53-mediated senescence by MSeA suggests a

new paradigm of cancer chemoprevention by strengthening a cancer progression barrier through

induction of irreversible senescence with additional suppression of AR and AKT oncogenic

signaling.




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INTRODUCTION

Selenium (Se) compounds have been studied for their chemopreventive potential in

various animal models of carcinogenesis, notably mammary, colon, lung, and prostate cancer.

Selenized yeast (Se-yeast) and its principal Se form Se-methionine (SeMet) have been tested in

several human trials in North America for the prevention of prostate cancer (PCa) (1-4). The

outcomes of the Selenium and vitamin E Cancer Trial (SELECT) and other trials failed to

demonstrate a preventive efficacy of these Se forms in the Se-adequate North American cohorts

(2-4). Many, including us, have opined on the possible reasons for such failures (5-7) (8). One

key factor was the selection of ineffective Se agents. In fact, the scarce animal efficacy data that

existed prior to the initiation of these trials did not support PCa preventive efficacy of SeMet and

these negative data were not published in full-length until after SELECT was terminated (9, 10).

Preclinical and mechanistic research has demonstrated that SeMet has little in common

with the mono-methylated methylselenol precursor Se forms (MM-Se), such as methylseleninic

acid (MSeA), in terms of metabolism and anti-cancer activities (8, 11). We have posited that the

failure of SeMet should not be taken to indicate that other Se forms are ineffective for PCa

chemoprevention (12). Indeed, we have shown that daily orally-administered MSeA inhibited

the growth of DU145 and PC-3 human PCa xenografts in athymic nude mice whereas an equal

Se dose of SeMet was inactive, in spite of SeMet leading to much higher retention of Se in the

xenograft tumors (13). We also reported the efficacy of MSeA to inhibit prostate carcinogenesis

in the transgenic adenocarcinoma mouse prostate (TRAMP) model which improved survival

with no observable long-term adverse effect (12). More efficacy and biomarker assessments in

clinically-relevant prostate carcinogenesis models with MM-Se will be essential to evaluate their




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PCa chemoprevention potential in the post-SELECT era to support future translation of these

data to humans.

The PTEN (phosphatase and tensin homolog) protein, antagonizes the

phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (AKT) signaling pathway that

stimulates cancer cell metabolism, proliferation and survival (14). Human PTEN loss has been

identified in 45% of high-grade prostatic intraepithelial neoplasia (HG-PIN) and 70% of

advanced PCa [(14) and reference therein]. Mouse genetic studies have demonstrated that loss

of Pten in prostate epithelium rapidly causes HG-PIN that ultimately progresses to invasive

adenocarcinoma and metastatic disease (15). As men diagnosed with HG-PIN are at increased

risk of developing PCa, this prostate specific conditional Pten KO mouse model recapitulates

essential characteristics of human prostate carcinogenesis and is considered clinically relevant

for studies of PCa chemoprevention. In the Pten KO model, the sustained activation of AKT not

only initiates and perpetuates oncogenic signaling and progression pathways, but at the same

time induces cellular senescence (known as Pten-deficiency Induced Cellular Senescence PICS),

which acts as a formidable barrier to restrain oncogenic progression to invasive and metastatic

disease (16, 17). Mechanistic studies suggest that PICS primarily depends on P53 protein

overexpression, which is induced through AKT/mTOR-mediated protein synthesis and p19ARF

sequestration of MDM-2 resulting in inhibition of proteasome-mediated P53 degradation (16).

The critical role of androgen receptor (AR) signaling in PCa, even at the advanced

metastatic castration-resistant stage, is well established and therapeutically exploited (18).

Unfortunately, recent studies have shown that inhibition of AR signaling by castration or

antagonist drugs inadvertently promotes the progression of stable HG-PIN to invasive

carcinomas in Pten KO model (19), raising concerns for utilization of these androgen




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deprivation strategies for chemoprevention in high risk men and PCa patients with PTEN

deficiency or mutations.

In cell culture studies, we and others have shown MSeA suppression of AR abundance

and signaling in PCa cells (20, 21), and the phosphorylative activation of AKT Ser473 (pAKT)

(22, 23). MSeA was recently shown to induce cellular senescence in human primary lung

fibroblasts and this cellular effect was likely mediated by ATM/P53 signaling (24)(25).

Therefore in the current study we tested the hypothesis that MSeA would inhibit Pten-deficient

carcinogenesis and PCa progression and assessed the mechanistic involvement of p53, PICS,

AKT, and AR in conferring the hypothesized in vivo efficacy.

MATERIALS AND METHODS

Generation of Pten KO mice

The conditional Ptenflox/flox mouse was generated as previously described (26). PB-Cre4

transgenic mice were obtained from the NCI Mouse Repository. Female mice carrying Ptenflox/+

were crossed with male mice harboring PB-Cre4+Ptenflox/+ to generate mutant mice with prostate

epithelium-specific deletion of Pten. Tail DNA was used for PCR-based genotyping as described

(26). All animal protocols were approved by the University of Minnesota Institutional Animal

Care and Use Committee.

Intervention experiments with MSeA

In the short-term experiment, 12-weeks old Pten KO mice (Cre+;PtenFlox/Flox, hereafter

indicated as Pten∆/∆mice) were randomly assigned, 5 mice per group, to receive water or MSeA

for 4 weeks by daily (5 days per week) oral application at the base of the tongue as before (12,

13). Wild type (WT) littermates (Cre-;PtenFlox/Flox; hereafter as Pten+/+; n=3) were treated

identically with water or MSeA to provide comparison control tissues. AIN93G semi-purified




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diet and water were provided ad libitum. Mouse body weight was monitored weekly. At

necropsy, total prostate was dissected, photographed and weighed. One portion of prostate

tissues from each mouse was fixed in formalin for H&E and immunohistochemistry staining.

The reminder was stored frozen at -80oC for senescence-associated β-galactosidase (SA-gal)

staining and Western blot analyses.

The long-term experiment was carried out with same design, except that the mice were

10-weeks old at the start of the MSeA and water treatments (5 days per week), lasting for 25

weeks (8 mice per group). At necropsy, the genitourinary (GU) tract was collected and weighed.

Then the different prostate lobes were dissected and weighed. The prostate lobes were saved and

processed individually for histopathology and biochemical analyses.

Histopathology analysis

Tissue processing and staining were as performed previously (12, 26). H&E stained lesions

were verified by a pathologist (Maarten Bosland). The pathological changes of all lobes of

prostate were classified according to Shapell et al. (27).

Senescence-associated β-galactosidase staining

Fresh prostate tissues were embedded with OCT and cut into 4 μm sections. The sections were

stained for SA-gal and counterstained with eosin, as described previously (26). The integrated

optical density (IOD) in the prostate epithelium/lesions was quantified by ImagePro-Plus 6.3

software.

Immunohistochemistry (IHC) and immunoblot analysis

IHC and immunoblot (Western) were performed as previously described (26). Briefly, Ki67

(NeoMarker), AR (Millipore), cleaved caspase 3, and p-AKT Ser473 (Cell Signaling, Beverly,

MA) were diluted at 1:100 for IHC. Images were captured and analyzed by ImagePro-Plus 6.3




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software for integrated optical density (IOD) semi-quantitation. For immunoblot, the prostate

tissues were homogenized in non-denaturing lysis buffer and subjected to SDS-PAGE and

blotted with antibodies against P53, P21Cip1, p-AKT Ser473, AR, and tubulin. Pooling of

prostate tissues from the short-term experiment was necessary due to limited amount of material

available.

Statistical analyses

For parametric data, the mean and SEM were calculated for each experimental group.

Differences among groups were analyzed by ANOVA for more than 2 groups. For comparison

of only 2 groups, student t-test was used. Significant differences were accepted at P<0.05.

RESULTS

Short-term MSeA treatment of Pten KO mice led to super-activation of p53-p21 and

cellular senescence in prostate epithelium

In the first experiment, we evaluated the effect of 4-week MSeA treatment to identify

early biochemical and cellular changes that might correlate and predict its long-term preventive

efficacy against HG-PIN growth and tumor progression in Pten KO mice. As shown in Fig. 1A,

MSeA treatment led to a reduction (~20%) of prostate weight in Pten KO mice compared to the

prostate weight in wild-type mice, which was not affected by MSeA. The protein levels of Pten

and phospho-Akt Ser473 (p-Akt) were analyzed in pooled prostate samples of WT mice and Pten

KO mice. As expected, Pten KO mice lacked Pten protein in the prostate and had greatly

increased p-Akt expression (Fig. 1B, lanes 3, 4 vs. 1, 2). Consistent with previous results (16, 17,

26), Pten KO mouse prostate showed increased basal expression of P53 and P21Cip1 over the




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WT counterpart (Fig. 1B, lane 3 vs. 1). MSeA treatment of the Pten KO mice dramatically

increased P53 and P21Cip1, but this did not occur in WT mice, while p-Akt expression was not

affected in MSeA-treated KO or WT mice (Fig. 1B). In addition, there was no observable change

in AR expression determined by IHC in Pten KO mice after 4 weeks of MSeA treatment (Fig.

1C, 1D).

Since increased P53 protein abundance causes PICS in the Pten KO mice (16, 17), we

examined SA-gal expression in situ in frozen prostate sections (Fig. 1C). Pten KO prostate

showed low but detectable SA-gal staining (Fig. 1C). However, in the prostate of MSeA-treated

Pten KO mice, the staining intensity was remarkably elevated in the epithelial cells by as much

as 4 fold, estimated by ImagePro-Plus software (Fig. 1C, 1D). Because senescence is an

irreversible terminal proliferative arrest, we examined Ki67 as a proliferation indicator and

detected significant suppression of Ki67 labeling index (%) in prostate of the MSeA-treated Pten

KO mice compared to water-treated mice (Fig. 1C, 1D).

Prolonged MSeA treatment of Pten KO mice prevented prostate adenocarcinoma

The promising biochemical and cellular responses to the short-term MSeA intervention

prompted us to evaluate its chemopreventive efficacy on Pten KO HG-PIN growth and

progression in the second experiment with 25-week administration. Consistent with long-term

safety of MSeA supplement in our previous study with the TRAMP model (12), no significant

effect of MSeA was observed on the body weight gain of the mice of each genotype (Supplement

Fig. S1A). As shown in Fig. 2A, long-term MSeA daily treatment did not significantly affect the

genito-urinary tract (GU) weight of the WT mice, but decreased Pten KO-driven expansion of

GU over the WT baseline by more than 70% (Fig. 2A). Likewise, the prostate weight was not




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affected in the WT mice by MSeA, but was decreased by more than 50% in the Pten KO mice

over the WT baseline (Fig. 2B). At the gross anatomical level, blood-rich prostate tumors were

visible in some water-treated control Pten KO mice (Supplement Fig. S1B). Among the different

lobes, the AP exhibited the most Pten KO-driven growth (Fig. 2C; Supplement Fig. S1C),

whereas in TRAMP mice the DLP undergo the most growth (12). The prostate weight

suppressing effect of MSeA in the Pten KO mice was uniform across AP, DLP and VP lobes

(Fig. 2C; Supplement Fig. S1C). In sharp contrast, the weight of prostate lobes in WT mice of

the MSeA and control groups was not different (Fig. 2C).

Histologically, the prostate lesions from the control Pten KO mice showed HG-PIN

phenotypes in all three lobes (Fig. 3A). Notably 38% (3 out of 8 mice) of Pten KO mice

progressed from HG-PIN to invasive adenocarcinomas at termination of the experiment at 35

weeks of age (Supplement Fig. S1D). In contrast, MSeA-treated mice showed dramatic

histopathological modification, many approaching near normal appearance of the prostate of the

WT mice and none of them with detectable invasive adenocarcinoma features (Fig. 3A, 3C).

Consistent with selectivity of targeting oncogenic growth, MSeA treatment of WT mice did not

affect their typical normal glandular structures (Fig. 3B, 3C). These findings indicate that long-

term MSeA treatment significantly inhibited HG-PIN growth and progression to carcinoma in

vivo.

Long-term MSeA treatment decreased p-Akt and AR abundance in Pten KO prostate

epithelium

Since short-term MSeA super-activated P53/P21Cip1 and increased senescence in the Pten-

KO epithelium in vivo, we examined whether long-term MSeA was able to sustain the cellular




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senescence phenotype. SA-gal staining of AP lobes showed intense senescence in the MSeA-

treated Pten KO mice (Fig. 4A) with little effect in the WT mice (Fig. 4B, 4C). Ki67 staining

confirmed the paucity of proliferating cells in the MSeA-treated Pten KO prostate epithelium

(Fig. 4A, 4C). No appreciable apoptosis, indicated by cleaved caspase-3, was induced by MSeA

treatment in Pten KO mice (Data not shown).

It is noteworthy that IHC staining intensity of p-Akt and AR proteins in the AP lobe of

MSeA-treated Pten KO mice was noticeably decreased (Fig. 4A, 4C) without corresponding

observable changes in the WT mice (Fig. 4B, 4C). Immunoblot confirmed the IHC results for p-

AKT and AR protein abundance suppression by MSeA in the Pten-KO prostate (Fig. 5A).

DISCUSSION

To our best knowledge, this study is the first in which any form of Se has been tested in the

Pten KO PCa mouse model for chemopreventive efficacy. It is also the first time that in vivo

senescence was measurably increased by MSeA treatment selectively in the HG-PIN epithelium

of Pten KO mice without any detectable impact on the prostate of the WT mice. The observed

concomitant increase of P53 and P21Cip1 in the prostate of MSeA-treated Pten KO mice was

not evident in the prostate of WT mice (Fig. 1 B), consistent with the selective super-activation

of this crucial senescence signaling axis. In addition to boosting and sustaining P53-P21Cip1

senescence as a cell proliferation barrier, long-term treatment with MSeA led to considerably

reduced tumor burden (Fig. 2) with decreased AR abundance and phosphorylation of Akt (Fig.

4,5), together contributing to effective suppression of the progression of HG-PIN to carcinoma

(shown schematically in Fig. 5B). Given that current Akt/mTOR inhibitor drugs activate AR




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signaling while androgen ablation and AR antagonist drugs cross-induce Akt pathway through a

reciprocal feedback regulatory loop (28), the inhibition of both Akt and AR signaling pathways

by prolonged MSeA supplement suggests that combined use of MSeA with these drugs may

mitigate their undesirable side effects and result in greater PCa risk reduction.

Human PCa arises from precancerous HG-PIN lesions with a prolonged clinical course,

affording unique windows of opportunity for chemoprevention/intervention. Since p53 signaling

is more likely to be intact in precancerous lesions than advanced PCa, the super-activation of

p53-senescence by MSeA offers a new paradigm for PCa chemoprevention through

strengthening a cancer progression barrier in the precursor lesions. Our data support that MSeA

super-activated and sustained P53-mediated cellular senescence and subsequently inhibited both

Akt and AR signaling pathways to suppress Pten-deficient HG-PIN progression to

adenocarcinoma (Fig. 5B). The in vivo mechanisms mediating these cellular and molecular

actions of MSeA are currently be elucidated. The efficacy for chemoprevention of Pten-

deficient HG-PIN progression by MSeA documented in the current work and the previously

demonstrated efficacy and safety of MSeA in other prostate cancer mouse models (12) (13)

provide strong justification for further development of MM-Se toward human translational

studies.




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Figure Legends

Figure 1. Oral supplement of MSeA (3 mg Se/kg body weight, 5 days per week) for 4 weeks

induced p53-p21-mediated senescence and inhibited prostate epithelial cell proliferation in

vivo. MSeA or water treatment commenced at 12 weeks of age. (A). Prostate weight of

Pten KO (Pten∆/∆) mice (n=5) and wild type littermates (Pten+/+) (n=3). (B) Immunoblot

analyses of Pten, p-Akt Ser473, P53 and P21Cip1 proteins in pooled samples of each

group. Tubulin served as loading control. (C) Representative photomicrographs of

immunohistochemical staining of androgen receptor (AR), Ki67 and SA-galactosidase

staining of Pten KO prostate from water-treated control mice and MSeA-treated

mice. Magnification 100x. (D) Quantitation results from individual mice for each group

are presented as mean + SEM, n=5 for Pten∆/∆ mice and n=3 for Pten+/+ mice. For AR and

SA-gal staining, the integrated optical density (IOD) was normalized to control mice as

100. For Ki-67, percentage of positive nuclei in the prostate epithelium was quantified.

Figure 2. Effect of oral supplement of MSeA (3 mg Se/kg body weight, 5 days per week) for

25 weeks on Pten-KO-driven growths of genitourinary organs and prostate tumors. MSeA

or water treatment commenced at 10 weeks of age. (A) Genitourinary (GU) tract weight of

Pten KO (Pten∆/∆) mice (n=8) and wild type littermates (Pten+/+) (n=8). (B) Whole prostate

weight of Pten∆/∆ mice (n=8) and Pten+/+ mice (n=8). (C) Individual lobe weights of

Pten∆/∆ mice (n=8) and Pten+/+ mice (n=8). AP, anterior prostate; DLP, dorsolateral

prostate; VP, ventral prostate. *P = 0.05 or 2-sided t-test p values were shown.




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Figure 3. Long-term oral MSeA supplementation inhibited prostate cancer progression in Pten

KO mice. Oral supplementation of MSeA (3 mg Se/kg body weight, 5 days per week) was

initiated at 10 weeks of age for 25 weeks. (A and B) Representative photomicrographs of

H&E stained prostate lobes of (A) Pten KO (Pten∆/∆) mice treated with water (Con) or

MSeA and (B) wild type (Pten+/+) mice treated with water (Con) or

MSeA. (C) Quantitation of anterior (AP), ventral (VP) and dorsolateral (DLP) prostate

pathological changes. Mean + SEM, Pten∆/∆ mice (n=8) and Pten+/+ mice (n=8). *Denotes

statistical difference from Pten KO control mice, p<0.05.

Figure 4. Effect of long-term MSeA supplementation on p-Akt and androgen receptor (AR),

senescence and cellular proliferative index in the anterior prostate of mice in Figure 3.

(A,B) Representative photomicrographs of IHC staining of pAkt Ser473, AR, Ki67 and

SA-galactosidase activity in anterior prostate of (A) Pten KO (Pten∆/∆) mice treated with

water (Con) or MSeA and (B) wild type (Pten+/+) mice treated with water (Con) or

MSeA. (C) Quantitation of changes from A and B. Mean + SEM, Pten∆/∆ mice (n=8) and

Pten+/+ mice (n=8). The integrated optical density (IOD) was estimated by ImagePro-Plus

software. *Denotes statistical difference from Pten KO control mice, p<0.05.




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Figure 5. Selective suppression effect of oral supplement of MSeA (3 mg Se/kg body weight, 5

days per week) for 25 weeks on androgen receptor (AR) and p-Akt Ser473 abundance in

the Pten KO prostate. (A) Immunoblot analyses of AR, p-Akt and total Akt in the anterior

prostate lobe of Pten KO (Pten∆/∆) mice and wild type littermates (Pten+/+) treated with

water (Con) or MSeA. Two mice per group were randomly chosen for analyses. Tubulin

served as loading control. (B) Schematic illustration of temporal sequence of cellular and

molecular events in Pten KO prostate carcinogenesis and the entry points of MSeA

actions.




Published OnlineFirst October 28, 2015.Cancer Prev Res Lei Wang, Xiaolan Guo, Ji Wang, et al. progression barrier in prostate lesions of Pten-knockout mouseMethylseleninic acid super-activates P53-senescence cancer

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