Downregulation of HER2 by RIG1 involves the PI3K/Akt pathway in ovarian cancer cells

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Down-regulation of HER2 by RIG1 Involves the PI3K/Akt Pathway in Ovarian Cancer Cells Chien-Chih Ou1, Shih-Chung Hsu2, Yin-Hui Hsieh3, Wan-Ling Tzou3, Tzu-Chao Chuang4,5, Jah-Yao

Liu6*, and Ming-Ching Kao3, 7*

1Graduate Institute of Medical Sciences and 3Graduate Institute of Biochemistry, National Defense

Medical Center, Taipei, Taiwan 2The Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan

4Department of Chemistry and 5Graduate Institute of Life Sciences, Tamkang University, Tamsui, Taipei, Taiwan

6Department of Obstetrics & Gynecology, Tri-Service General Hospital, Taipei, Taiwan 7Graduate Institute of Clinical Medical Science, China Medical University College of Medicine,

Taichung, Taiwan

Running Title: Down-regulation of HER2 by RIG1 *Corresponding author: Dr. Ming-Ching Kao, Graduate Institute of Clinical Medical Science, China Medical University College of Medicine, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402, ROC, E-Mail: [email protected]; or Dr. Jah-Yao Liu, Department of Obstetrics & Gynecology, Tri-Service General Hospital, No. 325, Sec. 2, Cherng-Kung Road, Neihu, Taipei, Taiwan 114, ROC.

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Abstract

Interferon-γ (IFN-γ) is known to down-regulate HER2 oncoprotein (p185HER2 or briefly p185) in

prostate cancer cells. We demonstrate that the IFN-γ-induced retinoid-inducible gene 1 (RIG1) acts as

a transrepressor of p185. Furthermore, we exhibit that RIG1 down-regulates the activated

(phosphorylated) form of p185 and PI3K/Akt and the mammalian target of rapamycin (mTOR),

downstream substrates of HER2. We also elucidate that heregulin (HRG) specifically restores the

activation of p185 and Akt after their activities are reduced by RIG1. Additionally, expression of

vascular endothelial growth factor (VEGF) increases through the HER2 and Akt/mTOR signaling

pathways, indicating that VEGF is down-regulated by RIG1 within the cell. These findings suggest

that RIG1 plays a role in IFN-γ-mediated therapy by down-regulating p185 and its downstream

PI3K/Akt/mTOR/VEGF signaling pathway. These results may provide a new therapeutic mechanism

for the clinical use of IFN-γ and RIG1.

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Introduction

Interferon-γ (IFN-γ), one of three IFN cytokines, inhibited the proliferation and regulation of

oncogenes and tumor suppressor genes (1, 2). Receptor-bound IFN-γ induces JAK1 and JAK2

phosphorylation and activates STAT1, resulting in translocation to the nucleus and the turning on of

downstream genes. Although many actions of IFN-γ have been studied, the underlying mechanisms

are still not well known.

Retinoid-inducible gene 1 (RIG1), also known as tazarotene-induced gene 3 (TIG3) or retinoic acid

receptor responder 3 (RARRES3), belongs to the class II tumor suppressor gene HREV107 family and

is one of the IFN-γ downstream genes (3-5). RIG1 is expressed in high levels in well-differentiated

skin and colon mucosal tissues but at low levels in cancer tissues derived from the skin, liver,

colorectum, and biliary tract (6, 7). Overexpression of RIG1 suppresses colony formation in T47D

breast cancer cells and HaCaT keratinocytes. Ectopic RIG1 expression leads to cellular apoptosis and

suppression of growth in cancer cells and human keratinocytes (3, 8, 9, 10). RIG1 also facilitates the

terminal stages in keratinocyte differentiation by activating type I transglutaminase (11). Previous

studies have demonstrated that RIG1 decreases protein levels and activities of Ras, one of the HER2

downstream proteins, while interacting with Ras protein in the cytoplasmic compartment in HtTA

cervical cancer cells (9, 12). However, the molecular mechanisms responsible for the effects of RIG1

remain unclear.

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The HER2 gene, also known as neu or erbB2, encodes a 185 kDa transmembrane receptor tyrosine

kinase belonging to the epidermal growth factor receptor (EGFR) family (13-18). Overexpression of

p185 is found in about 30% of human breast cancers and in many other cancer types (19, 20). The

p185 phosphorylates downstream substrates and activates a variety of signaling cascades, including

the phosphatidylinositol-3 kinase (PI3K)/Akt and Ras/MAPK pathways. These regulatory signal

cascades promote cell survival and tumor growth and metastasis (20-22).

We demonstrated previously that SV40 LT425 (LT425) acts as a transforming and metastasis

suppressor of HER2 oncogene (23, 24). Microarray and proteomic analysis revealed that both of

IFN-γ and RARRES3 genes are up-regulated in LT425 stable transfectants (Kao et al., unpublished

data). Through a literature search and database comparison, we found that IFN-γ down-regulates p185

expression in prostate cancer cells and induces RIG1 expression in ovarian cancer cells (2, 5). Thus,

we hypothesize that IFN-γ-induced RIG1 may contribute to repression of HER2. We also investigate

the effects of RIG1 on the HER2 signaling cascade.

Materials and Methods

Cell Culture and Plasmids — SKOV-3, OVCAR-3, and TOV-21G human ovarian cancer cell lines

were grown in McCoy’s 5A, RPMI 1640 (Gibco BRL), and MCDB 105/Medium 199

(Sigma-Aldrich) media, respectively, supplemented with 10-20% fetal bovine serum (FBS, HyClone).

BT-474, MCF-7/HER (MCF-7 of a HER2-transfected stable line), and MCF-7 human breast cancer

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cell lines were grown in DMEM/F12 medium supplemented with 10% FBS. Cells were grown at 37

°C in a humidified incubator with 5% CO2. The plasmids pRIG1-EGFP and pRIG1-myc were kindly

provided by Dr. SY Jiang (Department of Education and Research, Buddhist Tzu Chi General

Hospital, Taipei, Taiwan, ROC).

Cell Transfection — A liposome-mediated transfection method was used to transfect cells. In

brief, siRNAs (Dharmacon and Ambion) or plasmids were diluted in Opti-MEM (Gibco BRL)

medium and then mixed with Lipofectamine 2000 (Gibco BRL) at room temperature for 20 min. The

lipoplex complex was added to the cells for 6 h, and the cells refreshed with complete medium.

Western blot analysis — Cell lysates were prepared and sedimented as described previously (23).

The supernatant was used for SDS-PAGE analysis. Sixty micrograms of each cell sample was loaded

onto the NuPAGE 4-12% Bis-Tris Gel (NP0322, Invitrogen). After electrophoresis, the gels were

blotted onto nitrocellulose (NC) membranes. The NC membranes were subjected to blocking with 5%

skim milk in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween-20) for 2 h at room

temperature. The membranes were then incubated with the primary anti-neu antibody (Ab-3; 1:1000

dilution; Oncogene Science), anti-phospho-neu (Tyr1248) antibody (Ab-18; 1:500 dilution;

NeoMarkers), anti-Akt antibody (1:1000 dilution; Cell Signaling), anti-phospho-Akt (Ser473)

antibody (1:500 dilution; Cell Signaling), anti-GFP antibody (Santa Cruz Biotechnology), anti-PI3K

antibody (Cell Signaling), anti-phospho-(Tyr) p85 PI3K antibody (Cell Signaling), anti-mTOR

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antibody (Cell Signaling), anti-phospho-mTOR (Ser2448) antibody (Cell Signaling), anti-β-actin

(Chemicon, 1:5000 dilution) antibody, or other antibodies (Cell Signaling) in fresh 5% skim

milk-TBST buffer for 1 h at room temperature or 4 °C overnight. The membranes were washed and

incubated with the secondary antibody of HRP-conjugated goat anti-mouse or goat anti-rabbit IgG

(1:1000 dilution; Santa Cruz Biotechnology). Visualization was performed in an Amersham ECL

chemiluminescence system. Bands were quantified by Adobe Photoshop software. IFN-γ, heregulin

(HRG), and all chemicals were purchased from Sigma-Aldrich.

RNA isolation and reverse transcription polymerase chain reaction (RT-PCR) — Total RNAs were

extracted using TRIzol solution (Invitrogen). cDNA was prepared by incubation at 37 °C for 1 h in 20

μl of a mixture containing 3 μg total RNA, 1 μl Moloney murine leukemia virus reverse transcriptase

(Promega), 1 μl dNTP, 1 μl oligo-dT, and 1 U RNaseout recombinant RNase inhibitor. PCR was

performed in 25 μl of a reaction mixture containing 100 ng cDNA, and 200 nM each of HER2 (sense:

5′-CCAGAGCAAGAGAGGTATCC-3′; antisense: 5′-CTGTGGTGGTGAAGCTGTAG-3′), RIG1

(sense: 5′-ACCAGACCTCCTCTTGGC-3′; antisense: 5′-GAAGGGGCAGATGGCTGT-3′), or

β-actin (sense: 5′-CCAGAGCAAGAGAGGTATCC-3′; antisense:

5′-CTGTGGTGGTGAAGCTGTAG-3′) primers. The PCR products were quantified by ethidium

bromide staining on a 0.8% agarose gel.

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Quantitative PCR — Quantitative PCR was performed on a LightCycler (Roche Diagnostics)

using SYBR Green I as a double-strand DNA-specific binding dye and continuous fluorescence

monitoring. Amplification was carried out in a total volume of 20 μl containing 0.5 μM of each

primer, 4 μM MgCl2, 2 μl LightCycler DNA Master SYBR Green I (containing 1.25 U Taq

polymerase, 10× Taq buffer [500 μM KC1 and 100 μM Tris-HC1, pH 8.3], 2 μM of each dNTP, and

10× SYBR Green; Roche Diagnostics), and 1 μl of cDNA prepared as described above. The PCR

primers were identical to those used for semiquantitative RT-PCR. Melting curve analysis of

amplification products was performed at the end of each PCR reaction by cooling the samples to

40 °C and then increasing the temperature to 95 °C at 0.2 °C/s.

Fluorescence-activated Cell Sorter (FACS) analysis — The FACS analysis of p185 was performed

as described (25). In brief, PE-conjugated anti-HER2 monoclonal antibody (Becton Dickinson) was

diluted with washing buffer (0.1% NaN3 and 1.0% FBS in PBS) and the cell pellets were resuspended

to a concentration of 2 × 107 cells/ml. The cell–antibody complex were incubated at 4 °C for 30 min

in the dark and then washed twice with washing buffer. The supernatant was removed after each

centrifugation. Five hundred microliters of washing buffer was added to transfer the cell pellets to

Falcon round bottom tubes (Becton Dickinson), and the cells were analyzed using either a FACS

Caliber or FACS Vantage SE (Becton Dickinson).

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ELISA Assay— Sorted cells (1 × 106) were plated in a 60-mm tissue culture plate and allowed to

attach overnight. The cells were then washed and incubated in serum-free medium for 24 h. The

serum-free conditioned medium was collected and centrifuged at 1000 x g for 15 min to remove

debris. ELISA assay was performed using the VEGF Quantikine ELISA kit (R&D Systems) (26).

HER2 Promoter Activity Assay — Cells were cotransfected with pNulit (kindly provided by Dr.

MC Hung, Department of Molecular and Cellular Oncology, University of Texas MD Anderson

Cancer Center, Houston, Texas) and pCMV-β-gal plasmids and various amounts of pRIG1-myc. Cell

lysates were prepared before the luciferase activity assay by following the manufacturer’s (Promega)

instructions. The measured luciferase activity levels were normalized against the β-galactosidase

(β-gal) standard. The β-gal activity was measured by mixing equal amounts of cell extract and the 2×

assay buffer (120 mM Na2HPO4, 80 mM NaH2PO4, 2 mM MgCl2, 100 mM β-mercaptoethanol, and

1.33 mg/ml orthonitrophenyl galactoside). After incubation at 37°C, the enzyme activity of β-gal was

determined at A420 nm (27).

Statistical Analysis — Data were analyzed by using the paired t-test for comparison of

independent means.

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Results

Down-regulation of p185 by IFN-γ induced RIG1

IFN-γ has been shown to have inhibitory effects on p185 in prostate cancer cells (2). Thus, it is

reasonable to ask whether IFN-γ down-regulates p185 in ovarian and breast cancer cell lines. As

expected, IFN-γ down-regulated p185 expression in SKOV-3, OVCAR-3, and TOV-21G ovarian

cancer cell lines, as well as in BT-474, MCF-7/HER, and MCF-7 breast cancer cell lines (Figure 1A).

IFN-γ also down-regulated p185 expression in a dose- and time-dependent manner in

HER2-overexpressing SKOV-3 cells, as shown in the western blot analysis (Figures 1B and 1C). We

then examined whether IFN-γ could up-regulate RIG1 gene expression in SKOV-3 cells. Since there

is no effective anti-RIG1 antibody available, we used a quantitative RT-PCR approach to measure the

RIG1 mRNA levels. As expected, increasing RIG1 mRNA levels after treatment with increasing

doses of IFN-γ were observed. The level of RIG1 mRNA increased more than two folds after

treatment with 1000 U/ml of IFN-γ (Figure 2A). The suppressive effects of IFN-γ on p185 expression

by induction of RIG1 were examined by treating several ovarian cancer cell lines with different doses

of IFN-γ (100 and 1000 U/ml). After IFN-γ-treatment for 24 h, cells were transfected with RIG1

siRNA or negative control siRNA. In cells treated with 1000 U/ml of IFN-γ, RIG1 siRNA restored the

IFN-γ-mediated repression of p185 level to 290% in TOV-21G and 51% in OVCAR-3 cell lines. In

addition, the p185 level in SKOV-3 cells could be restored by 40% by RIG1 siRNA with 100 U/ml of

IFN-γ treatment (Figure 2B). These results were also revealed by different set of RIG1 siRNA

(Supplement 1). Taken together, these data suggest that IFN-γ down-regulates p185 by induction of

RIG1 (Figures 1 and 2).

To further explore whether RIG1 is an important suppressor of p185 in SKOV-3 cells, the protein

levels of p185 detected with the PE-conjugated anti-HER2 monoclonal antibody were analyzed by

FACS. We transfected the pEGFP vector only, or pRIG1–EGFP into SKOV-3 cells, and then

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measured the red fluorescence (p185) and green fluorescence (pEGFP or pRIG1–EGFP) in the cells.

RIG1 could significantly down-regulate p185 expression by about 52% after 24 h of transfection of

RIG1 (Figure 3A). At the same time, we isolated the successfully transfected RIG1-overexpressing

cells 18 h and 24 h after plasmid transfection (Figure 4A). Western blot analysis demonstrated that

these FACS-sorted RIG1-overexpressing cells were able to effectively reduce the p185 level by 24 h

(Figure 4A). In addition, phospho-p185 (the activated form of p185) was remarkably down-regulated

by RIG1 at both 18 and 24 h (Figure 4A). These results suggest that RIG1 down-regulates p185

protein levels and suppresses p185 activation.

RIG1 represses the HER2 gene activity

To unravel the mechanisms of down-regulation of p185 by RIG1, we assayed the mRNA levels of

different sorted transcripts. As shown in Figure 3B, consistent with the p185 protein levels, the HER2

mRNA levels were down-regulated by RIG1 at 24 h but not at 18 h. RIG1 obviously reduced HER2

RNA by more than 50% at 24 h (Figure 3B, right). The transcriptional activity of the HER2 promoter

was also analyzed. In transient expression assays, using NIH3T3 (Figure 3C) and SKOV-3 (Figure

3D) as recipient cells, cotransfection of pNulit and pCMV-β-gal and different amounts of pRIG1-myc

for 24 h led to a significant decrease in luciferase activity (Figures 3C and 3D, left), which was

normalized by β-gal. The HER2 promoter inhibitory rates increased in relation to an increasing

amount of RIG1, and an inhibition rate of more than two times was observed at a ratio of

pNulit:pRIG1-myc as low as 1:1 (Figures 3C and 3D, right). Taken together, these data suggest that

down-regulation of p185 protein by RIG1 occurs because of repression of HER2 promoter activity.

Down-regulation of phospho-PI3K/Akt and phospho-mTOR by IFN-γ and RIG1

Among HER2 stimulated intracellular signaling pathways, two main pathways, the Ras/MAPK

and the PI3K/Akt cascades, were shown to be activated by HER2 (22). RIG1 was reported to inhibit

the Ras/MAPK pathway by suppressing the activity of Ras and Ras protein in HtTA cervical cancer

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cells (12). Here, we examined whether RIG1 could interfere with the Ras/MAPK pathway in ovarian

cancer cells. As demonstrated in Figure 4A, both Ras and ERK proteins were not altered in

RIG1-overexpressing cells. The activation of ERK was not significantly reduced after ectopic

expression of RIG1 (Figure 4A). We then investigated if RIG1 affected the PI3K/Akt signaling

cascade. After 18 and 24 h of transfection with RIG1, phospho-PI3k, phospho-Akt and

phospho-mTOR were all dramatically down-regulated (p < 0.01). More than 40% of inhibition rate

was observed at 24 h (Figure 4A). In addition, a new finding that RIG1 has an inhibitory effect on

mTOR protein (Figure 4A) suggests that mTOR is another target for RIG1 inhibition. These data

clearly demonstrate that RIG1 exerts inhibitory effects on

phospho-PI3K/phospho-Akt/phospho-mTOR signaling pathway. This is the first report showing that

RIG1 could affect PI3K/Akt pathway.

To further evaluate the effects of IFN-γ on the PI3K/Akt pathway, SKOV-3 cells were treated with

1000 U/ml of IFN-γ. After 24 h of IFN-γ administration, the expression of p185, phospho-p185,

phospho-PI3K, phospho-Akt, and VEGF all remarkably decreased, but the protein levels of PI3K and

Akt did not change (Figure 4B). These results were identical to those obtained for RIG1 ectopic

expression, as shown in Figure 4A. In summary, these data demonstrate that both IFN-γ and RIG1 can

abrogate the HER2 and PI3K/Akt pathways.

Down-regulation of VEGF by RIG1

HER2-overexpressing tumors tend to be more angiogenic than other tumors (28). VEGF is one of

the most potent inducers of angiogenesis (28). The HER2 and PI3K/Akt signaling pathway has been

implicated in the regulation of VEGF through activation of mTOR/p70S6K (26). Thus, we proposed

that RIG1 down-regulated VEGF by inactivating mTOR. As expected, western blot analysis of

intracellular VEGF demonstrated that RIG1 down-regulated VEGF at 18 and 24 h after transfection

(Figure 5A). To further explore the participation of VEGF in the medium, we used ELISA to quantify

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and compare the VEGF secreted into serum-free conditioned medium after 24 h of incubation with

different transfectants. As shown in Figure 5B, RIG1 significantly reduced the secreted VEGF level

by about 50%, indicating that RIG1 could decrease VEGF expression by down-regulating the HER2

and PI3K/Akt/mTOR signaling pathway.

Heregulin restores the RIG1-induced decrease in phospho-p185 and phospho-Akt

HRG is a member of the neuregulin family of ligands that interact with and activate EGFR family

members. We found that RIG1 suppresses the activation of p185 and wondered whether HRG could

reactivate p185. As shown in Figure 6A, phospho-p185 was down-regulated by RIG1 at 24 h and

HRG restored phospho-p185 at this time point. Since RIG1 down-regulated both p185 levels and

phospho-p185 expression, HRG might not effectively restore the phosphorylation levels to the vector

control level. In addition, HRG restored the down-regulated phospho-Akt to the control level,

implying that RIG1 acts as a suppressor of the HER2 and PI3K/Akt pathway. HRG was added to the

cells before they were applied to FACS so VEGF expression would not have been increased by HRG

(Figure 5A). We also found p185 protein expression slightly decreased 24 h after addition of HRG

(Figure 6A, lower panel). This finding is consistent with a previous report that ligand can induce

endocytosis and degradation of HER2 (32).

Discussion

Type II IFNs possess antiproliferative effects in various tumor cell types. The binding of IFN-γ to

its cognate receptor activates the JAK/STAT signal transduction pathway, in which STAT1 molecules

are phosphorylated and translocated to the nucleus where they bind to IFN-γ-activated sites and

activate transcription of target genes. Here, we demonstrated that IFN-γ suppresses p185 expression in

both ovarian and breast cancer cell lines. The phosphoprotein levels of p185, PI3K, and Akt, and the

protein levels of p185 and VEGF were all down-regulated by IFN-γ treatment (Figure 4B). The

repressive effect of IFN-γ-induced RIG1 on p185 expression was verified by the RIG1 siRNA.

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Treatment with 1000 U/ml of IFN-γ together with RIG1 siRNA restored the IFN-γ-mediated

repression of the p185 level to 290% in TOV-21G cells and to 51% in OVCAR-3 cells (Figure 2B) ,

whereas p185 was only partially restored by RIG1 siRNA in SKOV-3 cells after treatment with 1000

U/ml of IFN-γ (data not shown). This phenomenon was also observed in the HER2-overexpressing

breast cancer BT-474 cell line when treated with 1000 U/ml of IFN-γ (data not shown). However, the

p185 level in both SKOV-3 and BT-474 cells could be restored by RIG1 siRNA under a low dose of

IFN-γ (100 U/ml). Therefore, inhibition of RIG1 appeared insufficient to restore the repression of

p185 expression after a high dose of IFN-γ treatment. It has been reported that down-regulation of

p185 by a high dose (5000 U/ml) of IFN-γ occurs through the interaction of phosphorylated STAT1

with p300 (2). Thus, RIG1 may play an important role after IFN-γ treatment, but other mechanisms

may be involved in the regulation of p185, especially with a high dose of IFN-γ.

Using a transient transfection method and cell sorter, we isolated RIG1-overexpressing SKOV-3

cells. Inside these cells, both p185 protein and its phosphorylated form were down-regulated. We also

showed that repression of p185 protein was caused by the inhibition of HER2 promoter activities

(Figures 3C and 3D). Therefore, we propose that RIG1 may possess phosphatase activities, and the

interaction and colocalization of RIG1 with p185 in the cytoplasmic compartment may contribute to

the decreased p185 activation (data not shown). In addition to down-regulation of phospho-p185, the

amount of activated PI3K, Akt and mTOR decreased 18 h after transfection of RIG1 (Figure 4A).

Thus, IFN-γ induced RIG1 may play an important role in the HER2 and PI3K/Akt pathways (Figure

6B).

RIG1 belongs to the HREV107 family of class II tumor suppressor genes. A notable feature of

class II tumor suppressor is loss of the growth-constraining function in tumor cells because of blocked

expression, but not because of DNA mutation or deletion, such as p53 (29, 30). Previous studies have

shown the induction of RIG1 protein by retinoids (3, 4), but the mechanisms of induction of RIG1 by

IFN-γ are not well understood. Evidence suggests that the RIG1 promoter contains a retinoic acid

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response element (RARE), which is also referred to as a putative p53 response element (p53RE) (31).

The presence of p53RE suggests that the class II tumor suppressor, RIG1, might be a direct

transcriptional target of the class I tumor suppressor p53. However, SKOV-3 is a p53-null cell line,

meaning that the regulation of RIG1 by IFN-γ is p53-independent. Treatment of A27/80 and

OVCAR-3 human ovarian carcinoma cells with IFN-γ can stimulate HREV107-1 expression, which is

directly related to activation of IFN regulatory factor-1 (IRF-1), indicating that HREV107-1 is a target

of IRF-1 and is involved in IFN-γ-induced cell death (5). In this study, we demonstrated that RIG1

mRNA levels increased by more than two folds after administration of IFN-γ for 2 h (Figure 2A). This

result confirmed an earlier report that IRF-1 is detectable within 3 h after IFN-γ administration (5).

Thus, the mechanisms responsible for the IFN-γ induction of RIG1 probably involve IRF-1 and

should be investigated further.

It has been hypothesized that RIG1 is involved in a negative feedback loop that normally controls

the level of MAPK activation (33). Overexpression of RIG1 down-regulates MAPK activities in

HtTA cervical and T47D breast cancer cells, and enhances MAPK-mediated suppression of RIG1

(33). In our study, the activity of ERK is not significantly reduced by RIG1. Therefore, we focused on

PI3K/Akt pathway. As shown in Figure 4A, PI3k/Akt activities were dramatically reduced (p < 0.01)

after ectopic expression of RIG1. In SKOV-3 cells, Akt signaling is dominantly activated as a result

of increased HER2 activation and in the absence of p53 and PTEN (34, 35, 43, 44). p53 and PTEN are

known to repress PI3K/Akt activities (34, 35, 43, 44). Although MAPK is also activated in ovarian

carcinoma cells, it is not the major pathway for RIG1-mediated suppression of HER2 signaling in

SKOV-3 cells in our study.

HER2 overexpression increases the metastatic potential of human cancer cells (20, 21). Patients

with HER2-overexpressing tumors have increased incidences of metastasis and a poor survival rate

compared with HER2 normal patients (19). Angiogenesis is a key component of cancer metastasis

(36) and is tightly controlled under normal physiological conditions. However, in pathological

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diseases such as cancer, the fine balance between proangiogenic and antiangiogenic factors is

disrupted. VEGF is one of the most potent inducers of angiogenesis and induces endothelial cell

proliferation and migration (28). VEGF expression in human breast cancers correlates with increased

microvessel density and reduced survival (37). HER2 has been implicated in the regulation of VEGF,

which is activated by the mTOR/p70S6K pathway. mTOR is a 289 kDa phosphoinositide

kinase-related serine/threonine kinase (38). Through the formation of multimolecular complexes,

rictor or raptor, the evolutionarily conserved TOR pathway controls an array of fundamental cell

functions, such as translation initiation, protein stability, transcription of ribosome and stress response

genes, ribosomal biogenesis, and tRNA synthesis, thereby playing a central role in the regulation of

cell growth, proliferation, and survival (39, 40). Several pharmaceutical and biotechnology companies

are actively pursuing the clinical development of inhibitors of the mTOR pathway for cancer

therapies, such as rapamycin. Our data show that RIG1 effectively down-regulates both

phospho-mTOR and mTOR protein (Figure 4A), suggesting mTOR as a target for RIG1 inhibitory

effects. mTOR localizes in the endoplasmic reticulum and Golgi apparatus (GA) membranes (41, 42).

Coincidentally, RIG1 also resides in the GA membrane (12). Further investigations may be needed to

understand whether RIG1 associates with mTOR in the GA membrane and whether this regulates its

protein level. In addition, activation of mTOR/p70S6K increases angiogenesis and spontaneous

metastasis by increasing VEGF synthesis. We found that RIG1 significantly reduced VEGF

expression in serum-free conditioned medium and intracellular protein levels (Figure 5), implicating a

role for RIG1 in decreasing angiogenesis in tumor growth. This is the first report that RIG1 exerts its

function to down-regulate mTOR and VEGF (Figure 6B). However, the underlying mechanisms

remain to be elucidated.

Acknowledgements

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We wish to thank Drs. SY Jiang (Department of Education and Research, Buddhist Tzu Chi

General Hospital, Taipei, Taiwan, ROC) and MC Hung (Department of Molecular and Cellular

Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas) for providing plasmids

as well as Dr. JJ Yen (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC) for

assisting FACS sorting experiments. This work was supported in part by the “C.Y. Foundation” and

the National Science Council, Taiwan, ROC, granted to MC Kao (NSC 95-2320-B-039-047).

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Figure Legends

Figure 1 Down-regulation of p185 expression by IFN-γ in ovarian and breast cancer cell lines. (A)

SKOV-3, OVCAR-3, and TOV-21G human ovarian cancer cell lines, and BT-474, MCF-7/HER, and

MCF-7 human breast cancer cell lines were treated with 1000 U/ml of IFN-γ or media alone for 72 h.

Cell lysates were immunoblotted with antibodies specific for neu or β-actin. (B) SKOV-3 cells were

treated with 1000 U/ml of IFN-γ or media for the indicated time. Densitometric analysis of the

percentage decrease was determined using Adobe Photoshop software. (C) SKOV-3 cells were treated

with the indicated doses of IFN-γ for 72 h. Western blot and densitometric analysis were performed as

described above. p185 expression was down-regulated by IFN-γ in SKOV-3 cell line in a dose- and

time-dependent manner. The experiment was repeated three times. The data are presented as the mean

± SE. *, p < 0.05. **, p < 0.01.

Figure 2 (A) Up-regulation of RIG1 expression by IFN-γ treatment in SKOV-3 ovarian cancer cells.

Cells were harvested after 2 h incubation with the indicated doses of IFN-γ. The PCR products were

stained with ethidium bromide on a 0.8% agarose gel (upper panel). The q-PCR quantification was

performed by adding the CT values of RIG1 cDNA and normalizing them to GAPDH cDNA (Roche)

(lower panel). (B) RIG1 siRNA restored the reduction of p185 caused by IFN-γ. Cells were treated

with 1000 or 100 U/ml of IFN-γ for 24 h and then transfected with negative control siRNA (NCi) or

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RIG1 siRNA (RIG1i). Cell lysates were immunoblotted with antibodies specific for neu or β-actin

(left). RT-PCR for RIG1 shows the inhibitory effect of RIG1 siRNA (right).

Figure 3 Down-regulation of the HER2 gene and protein by RIG1. (A) The amount of p185 in

RIG1-containing or vector control cells was quantified by flow cytometry using PE-conjugated

anti-HER2 monoclonal antibody. The mean fluorescence intensity was 237.27 for the vector and

116.98 for RIG1. The 52% (exponential scale) reduction of fluorescence by RIG1 was quantified

using the GeoMean fluorescence parameter provided with CELLQUEST software. (B) SKOV-3 cells

were transfected and sorted at the indicated time. mRNA quantification was performed as described

previously. The data are presented as the mean ± SE. NIH3T3 (C) and SKOV-3 (D) cell lines were

cotransfected with 2 μg of pNulit with different amounts of RIG1 plasmid DNA (0 μg, 0.5 μg, 1 μg,

1.5 μg, or 2 μg on the abscissa). The experiments were repeated four times. The relative light units

(RLU) activity of luciferase was normalized relative to β-galactosidase activity (left), and the

inhibition of promoter activities was also determined (right).

Figure 4 Down-regulation of p185 and phospho-PI3K/Akt/mTOR by IFN-γ and RIG1. (A) RIG1 or

vector transfectants or SKOV-3 parental cells were collected 18 and 24 h after transfection.

Successfully transfected cells were sorted by FACS Vantage SE. Western blot analysis of cell lysates

was performed with antibodies recognizing p185, phospho-p185-Y1248 (p-p185), p85 PI3K,

phospho-Y p85 PI3K (p-PI3K), Akt, phospho-Akt-S473 (p-Akt), Ras, ERK,

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phospho-ERK-T202/Y204 (p-ERK), mTOR, phospho-mTOR-S2448 (p-mTOR), GFP (RIG1), and

β-actin proteins in the transfected cells. The densitometric analysis was done as previously. (B)

SKOV-3 cells were treated with 1000 U/ml of IFN-γ or media alone. Cells were harvested after 24 h,

and western blot analysis of cellular proteins was performed as described previously. The experiments

were repeated three times. The data are presented as the mean ± SE. Both IFN-γ and RIG1 could

down-regulate p185 and its signaling pathway.

Figure 5 Down-regulation of VEGF by RIG1. (A) Protein lysates were prepared using similar

procedures from the vector control and RIG1 transfectants. Antibodies specific to VEGF (A-20) and

β-actin were applied. (B) Cells were seeded overnight, and then incubated for 24 h in serum-free

medium. VEGF concentration was measured by Quantikine ELISA kits. RIG1 produced the lowest

VEGF levels.

Figure 6 (A) Heregulin restored the RIG1-induced reduction of phospho-p185 and phospho-Akt. Cells

were transfected for the indicated time, 5 nM of HRG was added to the cells for 30 min before

harvest, and the cells were applied to the FACS Vantage SE. HRG specifically restored the activated

levels of p185 and Akt after 24 h of RIG1 transfection (upper left). Densitometry was performed as

described above (lower panel). Treatment with 5 nM HRG for 30 min was enough to increase

phospho-p185 but not p185 (upper right). (B) A schematic model of the effects of IFN-γ and RIG1 on

the regulation of HER2.

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Downregulation of HER2 by RIG1 involves the PI3K/Akt pathway in ovarian cancer cells

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