mir-205 targets pten and phlpp2 to augment akt...

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miR-205 targets PTEN and PHLPP2 to augment AKT signaling and drive malignant

phenotypes in non-small cell lung cancer

Junchao Cai1, 3, §, Lishan Fang1, 3, §, Yongbo Huang1, 3, Rong Li1, 3, Jie Yuan3, 4,

Yi Yang2, 3, Xun Zhu1, 3, Baixue Chen1, 3, Jueheng Wu1, 3, Mengfeng Li1, 3 *

Departments of 1Microbiology and 2Pharmacology, Zhongshan School of Medicine, Sun

Yat-Sen University; 3Key Laboratory of Tropical Disease Control (Sun Yat-Sen University),

Ministry of Education; 4Key Laboratory of Functional Molecules from Oceanic

Microorganisms (Sun Yat-sen University), Department of Education of Guangdong Province,

Guangzhou, Guangdong 510080, China

*Correspondence to:

Mengfeng Li, MD, Ph.D.

Departments of Microbiology, Zhongshan School of Medicine, Sun Yat-Sen University, 74

Zhongshan Road II, Guangzhou, Guangdong 510080, China. Voice: +86(20)87332748; Fax:

+86(20)87331209; E-mail: [email protected]

§ These authors contributed equally to this work.

Running Title: miR-205 activates AKT signaling by targeting PTEN and PHLPP2.

Keywords: miR-205; Lung cancer; Proliferation; Angiogenesis; NF-κB

The authors disclose no potential conflicts of interest.

Research. on October 14, 2018. © 2013 American Association for Cancercancerres.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 July 15, 2013; DOI: 10.1158/0008-5472.CAN-13-0297

http://cancerres.aacrjournals.org/

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Abstract

AKT signaling is constitutively activated in various cancers, due in large part to loss of

function in the PTEN and PHLPP phosphatases that act as tumor suppressor genes. However,

AKT signaling is activated widely in non-small cell lung cancers (NSCLC) where genetic

alterations in PTEN or PHLPP genes are rare, suggesting an undefined mechanism(s) for their

suppression. In this study, we report upregulation of the oncomir miR-205 in multiple

subtypes of NSCLC, which directly represses PTEN and PHLPP2 expression and activates both

the AKT/FOXO3a and AKT/mTOR signaling pathways. miR-205 overexpression in NSCLC

cells accelerated tumor cell proliferation and promoted blood vessel formation in vitro and in

vivo. Conversely, RNAi-mediated silencing of endogenous miR-205 abrogated these effects.

The malignant properties induced by miR-205 in NSCLC cells were reversed by AKT inhibitors,

FOXO3a overexpression, Rapamycin treatment, or restoring PHLPP2 or PTEN expression.

Mechanistic investigations revealed that miR-205 overexpression was a result of

NF-κB-mediated transactivation of the miR-205 gene. Taken together, our results define a

major epigenetic mechanism for suppression of PTEN and PHLPP2 in NSCLC, identifying a

pivotal role for miR-205 in development and progression of this widespread disease.




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Introduction

Lung cancer remains a leading cause of cancer-related deaths worldwide, and among all lung

cancer cases, over 80% are non-small cell lung cancer (NSCLC), including squamous cell

carcinoma, adenocarcinoma, adenosquamous cell carcinoma, large cell carcinoma and several

other histologic types (1, 2). The overall five-year survival rate for NSCLC, with all stages

and subtypes combined, remains as low as 15% (1). Despite advances in surgical therapy,

radiotherapy, chemotherapy and recently developed targeting therapy, such as anti-epithelial

growth factor receptor (EGFR) and anti-vascular endothelial growth factor (VEGF) targeting

strategies, the mortality of the disease has not been markedly reduced thus far (2, 3). The

suboptimal efficacies of currently available therapeutic approaches to the treatment of lung

cancer is largely attributable to difficulties in dealing with the malignant phenotype of the

disease, namely, its highly uncontrolled characteristics of overactivated proliferation,

neovascularization, invasiveness and metastasis supported and/or promoted by deregulated

molecular signaling pathways within lung cancer cells (4).

Indeed, mounting evidence has demonstrated that the poor prognosis of NSCLC patients and

therapeutic failure are associated with a number of abnormally activated signaling pathways,

among which PI3K/AKT signaling represents one of the most important regulatory pathways

for the malignancy (5, 6). Notably, aberrant Akt activation is a poor prognostic factor for

NSCLC of all stages and contributes to resistance to first-generation single-agent targeting

therapy, such as gefitinib, a tyrosine kinase inhibitor clinically used for NSCLC patients with

EGFR overactivation (7, 8). Biologically, activated AKT confers NSCLC cells resistance to

chemotherapy and radiation, and promotes cancer cell survival (9, 10), and by contrast,

chemically synthetic compounds inhibiting AKT activation induce apoptosis of NSCLC cells in

vitro as well as in vivo (11). Moreover, AKT signaling contributes to oncogenesis through

activating multiple downstream effector molecules (12). Of note, activated AKT




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phosphorylates tumor suppressor FOXO3a and impairs the transcription of its target genes

related to cell growth arrest such as p21, inactivation of which has also been implicated in the

promotion of tumor angiogenesis (13, 14). In addition, mTOR, another substrate subjected to

phosphorylation by AKT, enhances phosphorylation of S6K1 and 4E-BP1 (15) and plays crucial

roles in the regulation of ribosomal protein synthesis, for example, production of cyclin D1 and

VEGF-A at both transcriptional and translational levels (16, 17). It has been found that

mediated by the above molecular mechanisms, both AKT/FOXO3a and AKT/mTOR pathways

underlie lung cancer development and progression (18, 19). Thus, inhibitors targeting these

pathways might represent potentially applicable therapeutic agents against NSCLC.

Under physiological conditions, PI3K/AKT signaling is sophistically regulated by both

positive signals, such as signals emitted from growth factor-activated receptor tyrosine kinases

(RTK) (12, 20), and negative regulators, mainly including phosphatases PTEN and PHLPP.

PTEN, a well-known tumor suppressor, for instance, is essential for both normal lung

morphogenesis and the prevention of lung carcinogenesis (21). Lack of PTEN expression

represents a common event in a variety of tumor types. Unlike frequently observed mutations,

chromosomal deletion and/or loss of heterozygosity (LOH) in other cancer types, however, it

appears that loss of PTEN in NSCLC has not been associated with genomic alterations but

instead, more likely due to epigenetic silencing (22). PHLPP2, a recently characterized

member of the PHLPP family and suppressor of PI3K/AKT signaling, inhibits cell cycle

progression and promotes apoptosis in cells of various cancer types, including lung cancer cells

(23), albeit the expression profile of PHLPP2 in lung cancer tissues remains unclear.

Knowledge of how these negative regulators are suppressed in NSCLC so that AKT signaling is

aberrantly activated might provide a new basis for future targeting therapies against the disease.

In the framework of gene expression regulation, it is widely recognized that

miRNA-mediated post-transcriptional repression plays important roles, largely attributed to the




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capability of a miRNA to inhibit multiple target mRNAs through binding to their 3’UTRs.

Previously, upregulated miR-205, which is located at lung cancer-associated genomic

amplification region 1q32.2, was implicated, respectively, in 104 cases of primary lung cancers

and 38 stage-I and -II NSCLC specimens (24, 25). Moreover, miR-205 was reported to be

expressed at higher level in squamous cell lung carcinoma than other types of NSCLC (26).

Nevertheless, whether miR-205 is mechanistically associated with NSCLC progression remains

unknown. In the current study, we identify that miR-205 is highly expressed in multiple

subtypes of NSCLC tissues and causes simultaneous downregulation of PHLPP2 and PTEN,

leading to activation of both AKT/FOXO3a and AKT/mTOR pathways, consequently leading to

accelerated proliferation and enhanced angiogenesis in NSCLC. We have also found that the

observed overexpression of miR-205, at least partly, is ascribed to transactivation by the NF-κB

transcription factor.




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Materials and Methods

Cell culture

NSCLC cell lines NCI-H460 (H460) (large cell carcinoma), NCI-H596 (H596)

(adenosquamous carcinoma), A549 (adenocarcinoma) and Calu-1(squamous cell carcinoma), as

well as NCI-H292 and NCI-H1299 were purchased from Shanghai Institutes of Biological

Sciences (Shanghai, China) and maintained in the DMEM medium (Invitrogen, Carlsbad, CA)

supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 1%

penicillin/streptomycin (Invitrogen). Primary normal lung epithelial cells (NLEs) were

obtained according to our previous report (27). In brief, surgically resected specimens of

normal lung tissue were promptly removed and transported aseptically in Hanks' solution

(Invitrogen, Carlsbad, CA) supplemented with 100 units/ml penicillin, and 100 µg/ml

streptomycin (Invitrogen) and 5 µg/ml gentamycin (Invitrogen). The tissue specimens were

then incubated with 1.5 units/ml dispase (Roche Molecular Biochemicals, Indianapolis, IN) at

4 °C overnight, and the epithelium was dissected and incubated with trypsin (Invitrogen). The

reaction was stopped with soybean trypsin inhibitor (Sigma, Saint Louis, MI) and centrifuged,

followed by resuspension in keratinocyte-SFM medium (KSFM) supplemented with 40 µg/ml

bovine pituitary extract, 1.0 ng/ml EGF, 100 units/ml penicillin, 100 µg/ml streptomycin, 5

µg/ml gentamycin and 100 units/ml nyastatin (Invitrogen). The BEAS-2B immortalized

human bronchial epithelial cell line (Shanghai Institutes of Biological Sciences, Shanghai,

China) was cultured in LHC-9 medium as instructed by the provider. All cell lines were

authenticated by short tandem repeat fingerprinting at IDEXX RADIL (Columbia, MO, USA)

and Services at SYSU Forensic Medicine Lab (Guangzhou, China).

Tissue specimens

All clinical NSCLC specimens conducted in this study were histopathologically and

clinically diagnosed at the Sun Yat-sen University Cancer Center during the period of 2009 to




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2011. Clinicopathological classification of staging and subtypes of the tumor samples were

characterized according to the current International Union Against Cancer (UICC) standard.

Normal lung tissue specimens were taken from a standard distance (3cm) from the margin of

resected neoplastic tissues of NSCLC patients who underwent surgical lung resection. For the

use of these clinical materials for research purposes, prior patients’ consents and approval from

the Institutional Research Ethics Committee were obtained. Clinical information of 60 cases

of NSCLC patient specimens is presented in Supplemental Table 1.

Western blotting analysis

Western blotting analysis was performed using anti-phospho-AKT (ser473), anti-AKT,

anti-FOXO3a, anti-phospho-S6K1 (Thr389), anti-S6K1 and anti-4E-BP1(Epitomics,

Burlingame, CA), anti-phospho-FOXO3a (ser253), and phospho-4E-BP1 (Ser65) (Cell

signaling, Danvers, MA), anti-p21, anti-cyclinD1 and anti-PTEN (BD PharMingen, San Diego,

CA), anti-PHLPP2 (Abcam, Cambridge, MA) antibodies. Blotted membranes were stripped

and re-blotted with an anti-GAPDH, β–actin or p84 monoclonal antibody (Sigma, Saint Louis,

MO) as loading control. Surgically resected tumor tissues and corresponding adjacent normal

lung tissues were homogenized and the nuclear proteins were extracted using CelLytic Nuclear

Extraction Kit (Sigma) according to the manufacturer’s instructions. Relative protein levels

were quantified by densitometric scanning, and the relative gray values of protein were

calculated as Band Intensity of Protein of Interest / Band Intensity of Loading Control.

Xenografted tumor model in vivo and immunohistochemical analysis (IHC)

H460-vector and H460-miR-205 cells (2x106) were subcutaneously inoculated into the dorsal

thighs of individual female BALB/C nude mice (n = 5/group). Tumor lengths (L) and widths

(W) were measured every three days using a digital caliper, and tumor volumes were calculated

using the equation volume (mm3) = L*W2/2. After 19 days, mice were anesthetized and

sacrificed, and tumors were removed, photographed, weighed and sectioned (5µm in thickness),




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followed by immunostaining. Following deparaffinization, sections were IHC analyzed using

antibodies for Ki-67, CD31, VEGF-A, PHLPP2 and PTEN, respectively, and subsequently were

pathologically confirmed for the tumor phenotype and specific immunostaining. The resultant

immunostaining images were captured using the AxioVision Rel.4.6 computerized image

analysis system (Carl Zeiss, Oberkochen, Germany). Briefly, the stained sections were

evaluated at 200x magnification, and 10 representative staining fields of each section were

analyzed to calculate the mean optical density (MOD), which denotes the strength of staining

signals as measured by positive pixels. The microvessel density (MVD) was measured

according to the Weidner’s method (28). Every endothelial cell cluster of positive CD31

immunoreactivity, clearly separated from tumor cells, was counted as an individual vessel.

The immunostaining of p65, PHLPP2 and PTEN in 60 cases of formalin-fixed,

paraffin-embedded sections was evaluated and scored by two independent observers as

described in supplementary information.

Statistical analysis

Comparative analysis between groups for statistical significance was performed with

two-tailed paired Student’s t test. The correlation between miR-205 expression and nuclear

p65 level or PHLPP2 and PTEN protein levels in frozen and paraffin-embedded NSCLC tumor

tissues was respectively calculated using chi-square test and Pearson test. All error bars

represent mean ± SD derived from three independent experiments. P < 0.05 was considered

statistically significant in all cases.




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Results

miR-205 inhibited expression of PHLPP2 and PTEN and bound to their 3’UTRs.

To identify miRNAs that might contribute to activation of Akt signaling, we began our study

with searching for those miRNAs that potentially target PTEN, a major negative regulator of the

pathway, using the TargetScan database. Consequently, miR-205 was predicted to target

putative binding sites in the 3’UTR of PTEN mRNA, as well as that of PHLPP2 (Fig. 1A). Of

particular interest, miR-205 is located in a lung cancer-associated amplification region and

previously found to be upregulated in NSCLC tissues as aforementioned. Furthermore, we

found that transfection of miR-205 mimic oligonucleotides drastically reduced, but

anti-miR-205 (Anti-miR) oligonucleotides increased, the protein levels of both PHLPP2 and

PTEN, as compared to those in control oligonucleotides (NC) transfection in H460 and H596

NSCLC cells, which showed moderately enhanced expression of miR-205 compared to primary

normal lung epithelial cells (NLEs) and immortalized human bronchial epithelial cell line

BEAS-2B. These results were also validated in two other subtypes of NSCLC cells, namely,

A549 and Calu-1, which expresses slightly and markedly higher, respectively, level of miR-205

than NLEs and BEAS-2B, in response to transfection of miR-205 mimic and Anti-miR

oligonucleotides, respectively. (Fig. 1B and Fig. S1). To further assess whether miR-205 and

mRNA of PHLPP2 or PTEN interact and form miRNP complexes, RNA-immunoprecipitation

(RIP) analysis performed by pulling down Ago1 showed that far more PHLPP2 and PTEN

transcripts were assembled into miR-205 mimic oligonucleotides-containing miRNPs than into

NC oligonucleotides-containing miRNPs, due to binding of miR-205 with the mRNA 3’UTR

regions (Fig. 1C). Moreover, we separately cloned 3’UTRs of PHLPP2 and PTEN into a

luciferase reporter plasmid to determine the direct inhibitory binding of miR-205 to the 3’UTRs.

As shown in Fig. 1, D and E, miR-205 mimic oligonucleotides transfection imposed a

dose-dependent reduction in the normalized luciferase activities of both reporters, which were




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dose-dependently increased by antagonizing endogenous miR-205. The suppressive effects of

miR-205 mimic oligonucleotides on luciferase activities were completely deprived by

introduction of 4-nucleotide mutations in miR-205, suggesting an importance of appropriate

binding of miR-205 to the target 3’UTRs. The engineered levels of miR-205 expression in the

above transfection with miR-205 mimic or anti-miR oligonucleotides were confirmed (Fig. 1F).

Taken together, these data strongly suggested that miR-205 posttranscriptionally inhibited

protein expression of PHLPP2 and PTEN through directly binding to the 3’UTRs of their

mRNAs.

Ectopic miR-205 expression in NSCLC cells stimulated proliferation and angiogenesis.

To investigate whether miR-205 is biologically relevant to the development of malignant

phenotype of NSCLC cells, we stably overexpressed miR-205 in H460, H596 and A549

NSCLC cell lines (Fig. S2A) and performed gain-of-function experiments in vitro. As

analyzed by MTT assay and shown in Fig. 2A, miR-205 transduction significantly accelerated

the proliferation of H460, H596 and A549 cells by 58.3%, 48.5% and 44.6%, respectively, as

compared to control vector transduction. Additionally, clonogenic and anchorage-independent

growth assays revealed that H460-miR-205, H596-miR-205 and A549-miR-205 cells formed

more and larger-sized colonies in either 2-D or 3-D setting than their corresponding control

cells (Fig. 2, B and C). Furthermore, serum-free conditioned medium collected from cultured

miR-205-overexpressing cells of various subtypes stimulated HUVEC to form markedly longer

and more well-organized network of tubule structure than those from control vector cells (Fig.

2D), accompanied by formation of more secondary- and third-order vessels from preexisting

vessels as determined by chicken chorioallantoic membrane (CAM) assay (Fig. 2E). Taken

together, our data suggested a functionally important role of miR-205 in promoting the

proliferative and angiogenic phenotype of NSCLC cells.

Silencing endogenous miR-205 abrogated the abilities of NSCLC cells to proliferate and to




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induce vascular growth.

To further investigate whether endogenous miR-205 contributes to maintenance of the

proliferative and pro-angiogenic properties of NSCLC cells, loss-of-function studies were

performed by employing anti-miR-205 oligonucleotides to silence miR-205 expression (Fig.

S2B). As shown in Fig. 3A, transfection with anti-miR-205 oligonucleotides suppressed cell

proliferation by 43.1%, 34.5% and 38.9%, respectively, in H460, H596 and Calu-1 cells. In

parallel, silencing miR-205 remarkably compromised the tumorigenic abilities of all cell lines

as demonstrated by clonogenic and anchorage-independent growth assays (Fig. 3, B and C).

Furthermore, knockdown of miR-205 completely abrogated the ability of NSCLC cells of

various subtypes to induce tube formation of cultured HUVEC and formation of lower-order

blood vessels within CAMs (Fig. 3, D and E). Together with the above overexpression

experiments, these data strongly suggested an essential contribution of endogenous miR-205 to

the tumor proliferation and angiogenesis in NCSLC.

miR-205 promoted NSCLC tumor growth and tumor blood vessels growth in vivo.

To confirm whether the biological effects of miR-205 observed in cultured cells is relevant to

NSCLC tumor growth in vivo, H460-vector and H460-miR-205 cells, respectively, were

subcutaneously inoculated into BALB/C athymic mice. As shown in Fig. 4A, tumors formed

by miR-205-overexpressing cells grew more rapidly than those by vector-control cells within

the first week following inoculation, and the difference in average tumor volume between

experimental and control animals continued to increase for 2.5 folds at the experimental

endpoint (19 days). In parallel, increases in sizes and weights of tumors excised from animals

of the miR-205 overexpression group were also observed as compared to those of the control

group (Fig. 4, B and C). Notably, high degrees of neovascularization were seen on the surface

of miR-205-overexpressing tumors, whereas control tumors displayed nearly avascular surface,

as shown in Fig. 4D, strongly suggesting that miR-205 enhanced tumor angiogenesis.




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Consistent with the above observations, as shown in Fig. 4E, the proportion of proliferative

Ki67-positive cells increased from 44.6% in control tumors to 89.4% in

miR-205-overexpressing tumors, and there were more intratumoral microvessels formed with

higher microvascular densities (MVD) in miR-205-overexpressing tumors, as assessed by

CD31 staining, than the control tumors. Moreover, significant upregulation of VEGF-A

expression was shown in miR-205-transduced tumors, which simultaneously presented lower

protein levels of PHLPP2 and PTEN than control tumors, as confirmed by quantifying staining

intensities of these proteins (Fig. 4E). Collectively, these in vivo data strongly demonstrated a

biological role of miR-205 as a promoter of tumor growth and inducer of angiogenesis in

NSCLC.

Both AKT/FOXO3a and AKT/mTOR signaling contributed to miR-205-mediated

malignant phenotype of NSCLC cells.

We next examined the role of miR-205-mediated inhibition of PTEN and PHLPP2 in the

development and maintenance of the malignant phenotype of NSCLC cells. Of note, ectopic

miR-205 expression in H460, H596 and A549 cells remarkably increased the level of

phosphorylated AKT, resulting in enhanced phosphorylation of FOXO3a, S6K and 4E-BP1,

while knockdown of miR-205 in H460, H596 and Calu-1 cells robustly suppressed

phosphorylation of AKT, FOXO3a, S6K and 4E-BP1 (Fig. 5A), indicating that miR-205 indeed

activated the AKT/FOXO3a and AKT/mTOR pathways. Furthermore, decreased p21 and

increased cyclin D1 expression could be caused by miR-205 overexpression, whereas opposite

effects on the regulation of p21 and cyclin D1 were found at both the mRNA and protein levels

when miR-205 was knocked down (Fig. 5B). In addition, as determined by real-time PCR and

ELISA, VEGF-A expression was shown to be upregulated in miR-205 overexpressing cells and

downregulated in miR-205 knocked-down cells (Fig. 5C), providing a molecular basis for the

regulatory effects of miR-205 on NSCLC cell proliferation and angiogenesis.




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We next separately tested the effect of specific blockage of AKT/FOXO3a and AKT/mTOR

pathways on the pro-proliferative and pro-angiogenic functions of miR-205. As shown in Fig.

5D, treatment with a specific PI3K inhibitor, LY294002, or an AKT inhibitor drastically

retarded the proliferation of miR-205-overexpressing cells, such as H460-miR-205 and

H596-miR-205 cells, and considerably weakened the abilities of these miR-205-transduced

cells to induce HUVEC tube formation. Similarly, potentiation of the proliferation and

pro-angiogenic ability of H460 and H596 cells by miR-205 were markedly eliminated in

response to Rapamycin inhibition of the mTOR pathway (Fig. 5E). Furthermore,

overexpression of FOXO3a repressed the growth of, and HUVEC tube formation induced by,

H460-miR-205 or H596-miR-205 cells (Fig. 5F). The effectiveness of treatment with

LY294002, AKT inhibitor, Rapamycin or FOXO3a overexpression was confirmed, as shown in

Fig. 5G. These data suggested that miR-205 promoted the proliferative and angiogenic

phenotype of NSCLC cells by simultaneously activating both AKT/FOXO3a and AKT/mTOR

pathways.

Repression of PHLPP2 and PTEN in NSCLC cells was essential for miR-205-induced

proliferation and neovascularization.

Based on the indispensable role of AKT activation in the biological functions of miR-205, we

then asked whether sustained repression of PHLPP2 and PTEN was essential. Indeed,

re-expression of the open reading frame (ORF, without 3’-UTR) of PHLPP2 or PTEN partially,

but significantly, impaired the abilities of miR-205-transduced cells to proliferate and to induce

tube formation of HUVEC as assessed by MTT assay and HUVEC/Matrigel assay, respectively

(Fig. 6, A and B). By contrast, in miR-205 knocked-down cells, namely, Calu-1-Anti-miR,

which expressed increased level of PHLPP2 and PTEN relative to its parental cells,

silencingPHLPP2 or PTEN significantly promoted their proliferation and induced tube

formation of HUVEC (Fig. 6, C and D). Unexceptionally, restoration of PHLPP2 or PTEN




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protein expression greatly diminished phosphorylation of AKT, FOXO3a, S6K1 and 4E-BP1 in

both H460-miR-205 and H596-miR-205 cells (Fig. 6E), accompanied by elevated p21, reduced

cyclin D1 transcription, and decreased VEGF-A secretion (Fig. 6, F and G). Conversely,

silencing PHLPP2 or PTEN in Calu-1-Anti-miR cells exhibited opposite effects (Fig. 6, E, F

and G). These data supported that downregulation of PHLPP2 and PTEN was essential for

miR-205-induced increase of cell-proliferative activity and angiogenesis-inducing ability in

NSCLC.

NF-κB-transactivated miR-205 expression correlated with PHLPP2 and PTEN expression.

Previous array-based miRNA profiling has demonstrated that miR-205 expression is

upregulated in human NSCLC tissues. Indeed, we confirmed the upregulation of miR-205 in

22 frozen NSCLC tumor lesions consisting of 6 cases of adenocarcinoma (AD), 6 cases of

squamous cell carcinoma (SCC), 6 cases of large cell carcinoma (LCC) and 4 cases of

adenosquamous carcinoma (ASC) paired with non-cancerous adjacent tissues, among which

miR-205 was markedly highly expressed in SCC compared to other subtypes (Fig. 7A). To

elucidate the mechanism underlying the upregulation of miR-205 in NSCLC, we employed the

UCSC genome browser tool and identified that putative binding sequences for NF-κB p65

subunit were present at a ENCODE H3K4Me1 site, an indicator for the presence of active

chromatin region and transcription enhancer(s), located 3.5kb upstream to the miR-205 gene

locus (Fig. 7B). Moreover, overexpression of p65 or treatment with TNF-α to activate NF-κB

transcriptional activity robustly elevated pri-miR-205 expression, and in contrast, p65 siRNA or

a dominant-negative mutant IκBα, which blocked NF-κB activation, abated pri-miR-205

expression (Fig. 7B), suggesting that NF-κB signaling contributed to the modulation of

miR-205 transcription. In further support of this notion, ChIP analysis revealed enhanced

binding of p65 with the putative binding site located in the miR-205 upstream region, as

compared to that with the control sequence, when detected by primers spanning irrelevant




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sequences (Fig. 7C). Of note, when a 500bp DNA fragment covering the putative p65 binding

site was cloned upstream to luciferase reporter gene and transfected into cells overexpressing

p65, significantly increased luciferase activity was achieved, whereas luciferase activity was not

changed when linked to mutated putative p65 binding sequence or irrelevant control sequence

(Fig. 7D), suggesting that miR-205 could be directly transactivated by the NF-κB transcription

factor in NSCLC cells.

In an effort to reveal the clinical relevance of above identified correlation, we found that in 8

frozen primary NSCLC tissues, a high miR-205 level was linked to diminished expression of

PHLPP2 and PTEN and increased nuclear p65 abundance as compared with those in 2

non-cancerous lung tissue specimens (Fig. 7E). Furthermore, examination of miR-205

expression and PHLPP2 and PTEN expression, as well as p65 localization was conducted in a

cohort of 60 cases of paraffin-embedded archived human NSCLC specimens (Supplementary

Table 1). The high (> median, n=30) and low (< median, n=30) miR-205 expression group

were identified. Of note, 63.3% of 30 specimens with high miR-205 expression exhibited

strong nuclear localization of p65, in contrast to the mainly cytoplasmic p65 staining in 73.3%

of 30 specimens expressing low level of miR-205 (P = 0.004, r = 0.369). Moreover, in the

high miR-205 expression group, 63.3% and 66.7% of cases, respectively, showed low levels of

PHLPP2 and PTEN, which were highly expressed, respectively, in 63.3% and 70.0% of

specimens with low miR-205 expression (P = 0.039, r = -0.265 and P = 0.004, r = -0.367,

respectively) (Fig. 7, F and G). Taken together, these data suggest that the experimental

demonstration that NF-κB-transactivated miR-205 overexpression negatively regulates PHLPP2

and PTEN is clinically relevant in NSCLC.




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Discussion

In the current study, we have demonstrated a tumor-promoting role of miR-205 in NSCLC,

overexpression of which robustly promotes cell proliferation and vascular growth both in vitro

and in vivo. In contrast, inhibition of endogenous miR-205 remarkably abrogates the abilities

of NSCLC cells of various tissue subtypes to proliferate and to induce angiogenesis. At the

molecular level, both the AKT/FOXO3a and AKT/mTOR pathways contribute to

miR-205-mediated malignant phenotype of NSCLC cells, likely mediated by suppressing

PHLPP2 and PTEN expression. Of note, the close correlation between high miR-205

expression and low expression of PHLPP2 and PTEN, as well as with the malignant properties

of NSCLC tumors, were also confirmed in xenoplanted tumors and in clinical NSCLC samples,

suggesting a possible role of miR-205 in the development and progression of NSCLC.

It has been previously indicated that miR-205 might represent a specific biomarker in lung

squamous cell carcinoma (SCC), a subtype of NSCLC (26). Interestingly, our current study on

one hand confirms a higher level of miR-205 expression in SCC tissues than that in other

NSCLC subtypes, and on the other hand verifies that the non-SCC NSCLC subtypes also

overexpress miR-205 as compared with their adjacent non-cancerous tissues, suggesting that

miR-205 overexpression might indeed be an indicator for the presence and/or progression of

NSCLC. Notably, the enhanced expression of miR-205 in multiple subtypes of NSCLC may

be attributed, at least in part, to direct transactivation of NF-κB. It is widely recognized that

NF-κB signaling plays a key role in stimulating oncogenesis, and that its activation can be

triggered by carcinogenic or tumor-promoting factors (29, 30). Our study uncovers a novel

mechanism, mediated by miR-205, linking NF-κB overactivation to supporting the malignant

phenotype in NSCLC, highlighting the importance of miRNA in the NF-κB-promoted

oncogenic cascades, and such a finding is of particular significance in the interest of developing

interventional strategies against NF-κB signaling, as direct NF-κB inhibitors may cause severe




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adverse pharmacological effects due to the important roles of NF-κB in a broad array of

physiological processes (31). In this context, more experimental investigation regarding the

therapeutic efficacy of anti-miR-205 approaches is warranted.

It should be noted that miR-205 can be upregulated or downregulated in different cancer

types or subtypes, as previous studies have suggested. For example, miR-205 has been

reported to be highly expressed in normal mammary gland stem cells and triple-positive breast

cancer tissues but present low-level expression in metastatic samples (32-34). In addition,

miR-205 is markedly upregulated in specimens of cervical cancer but in contrast,

downregulated in those of prostate cancer (35, 36). In the scenario of lung cancer

development and progression, however, several previous studies have identified an upregulation

of miR-205, which is localized at 1q32.2, a lung cancer-associated genomic amplification

region (24-26). In support of the tumor-promoting role of miR-205 in NSCLC, our current

study provides comprehensive evidence for the functional, mechanical as well as clinical

significance of this molecule. These findings together indicate that the regulation of miR-205

expression, as well as its biological functions, might be tissue- and cancer-type dependent.

Thus, it remains important to thoroughly understand the molecular mechanisms mediating the

differential biological effects and targets of miR-205 in NSCLC and other cancer types.

Importantly, two bona fide target genes of miR-205, PTEN and PHLPP2, have been

identified by our study, both of which are phosphpatases essential for specific and effective

termination of the oncogenic AKT signaling. PTEN, of note, controls normal lung

morphogenesis and is essential for prevention of lung carcinogenesis in mice (21). Indeed,

low PTEN expression correlates with poor prognosis in NSCLC patients, and nearly 70% of

NSCLC cases have been detected to display reduced or lost PTEN expression (37, 38).

Genetic alterations, including LOH or inactivating mutation of PTEN, however, are rare in

NSCLC, identified in only approximately 10% of NSCLC tumors (39, 40), and promoter




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methylation of PTEN is found in only 24%-26% of NSCLC tumors (22, 41), suggesting that

other suppressive mechanism(s) also plays important roles in the cancer type. Thus, our

current finding adds an important dimension of regulatory mechanism underlying the inhibition

of PTEN in NSCLC. On the other hand, PHLPP2, which has been postulated to act as a tumor

suppressor that inhibits cell growth and promotes apoptosis in cancer cell lines (23), is located

at 16q22.3, a region that undergoes LOH in breast and ovarian cancers, prostate cancer and

hepatocellular carcinoma (42). Nonetheless, expression status and biological function of

PHLPP2 in lung cancer remains unclear. Our data show that experimental overexpression of

PHLPP2 in NSCLC cells significantly antagonizes the stimulatory effect of miR-205 on cell

growth and vascularization, silencing of which rescues the compromised abilities of miR-205

knocked-down NSCLC cells to proliferate and to induce angiogenesis, and that PHLPP2 is

downregulated in NSCLC in a manner inversely correlated with high miR-205 expression,

underscoring the importance of this phosphatase in the cancer type. Interestingly, we also

observe that neither PTEN nor PHLPP2 restoration could completely reverse the malignant

properties of NSCLC cells induced by miR-205, suggesting that a simultaneous repression of

PTEN and PHLPP2 may be necessary for the resultant oncogenic effects of miR-205.

In summary, this study provides a miR-205-mediated mechanism for the frequent activation

of AKT signaling in NSCLC pathogenesis, causing uncontrolled proliferation and neoplastic

angiogenesis. It is well documented that AKT activation confers survival disadvantage in

NSCLC patients (37, 43), and that kinase inhibitors targeting PI3K/AKT signaling or/and

mTOR pathway exhibit promising antitumor activities in preclinical trials but probably induce

upstream RTK activity and reversely activate AKT signaling in a wide spectrum of tumor types,

including NSCLC, resulting in compromised therapeutic efficacy, suggesting that effective

therapy may require combined inhibitors targeting both the upstream signaling and the

downstream specific pathways (44-46). Therefore, the feature that miR-205 inhibition almost




19

shuts down upstream signaling of AKT activation and simultaneously suppresses both

AKT/mTOR and AKT/FOXO3a pathways makes it a reasonable candidate target for therapeutic

intervention in NSCLC.




20

Grant support

This work was supported by the Ministry of Science and Technology of China grant (No.

973-2011CB11305); The Natural Science Foundation of China (No. 81071647, 81071762,

30900415, 81272339, 81272417); Guangdong Recruitment Program of Creative Research

Groups (No. 2009010058, 2010B030600003); National Science and Technique Major Project

(No. 201005022-2, 2012ZX10004213, 311030).The Key Science and Technique Research

Project of Guangdong Province (12B292060029).




21

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Figure Legends

Figure 1. miR-205 directly targets PHLPP2 and PTEN genes in NSCLC cells. (A)

Predicted target sequences in 3’UTRs of PHLPP2 and PTEN bound to miR-205. (B) Western

blotting analysis of PHLPP2 and PTEN expression in indicated cells. (C) Quantitation of

recruited mRNAs of PHLPP2 and PTEN to miRNP complex immunoprecipitated with Ago 1,

examined by RIP analysis. IgG immunoprecipitation was used as a negative control. (D and

E) Luciferase assays of pGL3-PHLPP2-3’UTR or pGL3-PTEN-3’UTR reporter in indicated

cells transfected with increasing amounts (20 and 50 nM) of indicated oligonucleotides.

Sequence of miR-205 mutant is shown. (F) Validation of the levels of miR-205 expression in

indicated cells examined by real-time RT-PCR. *: P<0.05; **: P<0.01.

Figure 2. Ectopic miR-205 expression in NSCLC cells accelerates proliferation and

induces angiogenesis. (A) MTT assay reveals cell growth curves of H460, H596 and A549

cells. (B) Representative micrographs (left) and relative quantification (right) of crystal

violet-stained cell colonies analyzed by colony formation assay for 12 or 15 days. (C)

Representative micrographs (left) and quantification (right) of colonies larger than 50µm or

containing more than 100 cells. (D) Representative micrographs (left) and relative

quantification (right) of tube length by HUVEC tube formation assay. (E) Representative

micrographs of induced neovessels by CAM assay. *: P<0.05; **: P<0.01.

Figure 3. Silencing endogenous miR-205 abrogates the abilities of NSCLC cells to

proliferate and to induce vascular growth. (A) MTT assay reveals cell growth curves of

H460, H596 and Calu-1 cells. (B) Representative micrographs (left) and relative




25

quantification (right) of colonies analyzed by colony formation assay for 10 or 15 days. (C)

Representative micrographs (left) and quantification (right) of colonies that larger than 25µm or

containing more than 50 cells. (D) Representative micrographs (left) and relative

quantification (right) of tube length by HUVEC tube formation assay. (E) Representative

micrographs of induced neovessels by CAM assay. *: P<0.05; **: P<0.01.

Figure 4. Overexpressing miR-205 in NSCLC cells promotes tumor growth and blood

vessel formation in vivo. (A) Tumor growth curves in mice (n = 5/group) inoculated with

indicated cells at indicated days. (B) At the experimental endpoint, tumors were dissected and

photographed as indicated. (C) Each tumor formed by indicated cells was weighed. (D)

Stereoscopic microscope revealed surface blood vessels of indicated tumors. (E) H&E

staining confirms tumor cells in slices of indicated tumor sections and IHC stained for Ki-67,

CD31, VEGF, PHLPP2, and PTEN are quantified by staining intensity. Levels of miR-205

expression in indicated tumor tissues are confirmed by real-time PCR. For A and E, data are

presented as mean ± SD (n = 5 mice/group). *: P<0.05.

Figure 5. miR-205 activates both AKT/FOXO3a and AKT/mTOR pathways to exert its

biological effects on NSCLC cells. (A) Western blotting analysis of phospho-AKT, total

AKT (T-AKT), phospho-FOXO3a, total FOXO3a (T- FOXO3a), phospho-S6K1, total S6K1,

phospho-4E-BP1 and total 4E-BP1 in indicated cells. (B) Protein expression and relative

mRNA quantitation of p21 and cyclin D1 in indicated cells. (C) Relative VEGF-A mRNA

expression examined with real-time RT-PCR and VEGF-A protein secretion measured with

ELISA for indicated cells. (D) miR-205-transduced cells were pretreated with PI3K inhibitor

LY294002 or AKT inhibitor III, and their abilities to proliferate and induce HUVEC tube




26

formation were suppressed. (E) Suppressive effect of Rapamycin treatment on the malignant

phenotype of miR-205-transduced cells. (F) Suppressive effect of Foxo3a overexpression in

miR-205-transduced cells on cell growth and HUVEC tube formation. (G) Western blotting

analysis shows the effect of LY294002, AKT inhibitor III, Rapamycin or Foxo3a

overexpression. *: P<0.05.

Figure 6. Restoration of PHLPP2 and PTEN inverses miR-205-induced proliferation

and tube formation. (A) Growth rates of indicated miR-205-overexpressing cells transduced

with PHLPP2, PTEN or control vector. (B) Representative micrographs (left) and relative

quantification (right) of lengths of tubes by HUVEC tube formation assay. (C) Growth rates

of indicated miR-205 knocked-down cells, silenced with two different siRNAs against PHLPP2

or PTEN, respectively. (D) Representative micrographs (left) and relative quantification (right)

of lengths of tubes by HUVEC tube formation assay. (E) Western blotting analysis of

indicated proteins in indicated cells. (F) Measurement of p21 and cyclin D1 mRNAs and level

of secreted VEGF-A in indicated cells. (G) Western blotting confirmation of re-expression of

PHLPP2 or PTEN, as well as depletion of PHLPP2 or PTEN in indicated cells. *: P<0.05.

Figure 7. miR-205 overexpression by NF-κB transactivation correlates with low

expression of PHLPP2 and PTEN in NSCLC tumor tissues. (A) Relative expression of

miR-205 in twenty-two NSCLC tumor tissue specimens compared to their corresponding

adjacent non-cancerous tissues. (B) Schematic diagram of predicted p65 binding site within a

3.5kb region upstream to the miR-205 gene locus and relative expression of pri-miR-205 in

response to NF-κB activation (p65 overexpression or TNFα treatment) or inactivation (p65

siRNA or IκB α-mut transfection). (C) ChIP enrichment assay confirms that NF-κB subunit




27

p65 binds to the predicted promoter site of miR-205. (D) Luciferase activity of reporter

constructs spanning putative p65 binding site or control irrelevant sequences. (E) Correlation

of miR-205 overexpression with nuclear p65 abundance and PHLPP2 and PTEN

downregulation in indicated NSCLC tumor tissues. p84 and GAPDH were used as loading

controls. AD, adenocarcinoma; LCC, large cell carcinoma; ASC, adenosquamous carcinoma;

SCC, squamous cell carcinoma. (F) miR-205 expression level correlates with p65 subcellular

localization and expression of PHLPP2 and PTEN. Two representative cases (Low and High

miR-205) are shown. (G) Percentage of specimens showing cytoplasmic/nuclear p65 staining,

low- or high expression of PHLPP2 and PTEN in patient specimens, respectively, with low and

high miR-205 expression. *: P<0.05.




Published OnlineFirst July 15, 2013.Cancer Res Mengfeng Li, Junchao Cai, Lishan Fang, et al. and drive malignant phenotypes in non-small cell lung cancermiR-205 targets PTEN and PHLPP2 to augment AKT signaling

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