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1 Phosphatidylinositol 4-phosphate in the Golgi apparatus regulates cell-cell adhesion, and invasive cell migration in human breast cancer Emi Tokuda 1 , Toshiki Itoh 2, 3 , Junya Hasegawa 2, 4 , Takeshi Ijuin 1 , Yukiko Takeuchi 1 , Yasuhiro Irino 5 , Miki Fukumoto 1 , and Tadaomi Takenawa 1 1 Integrated Center for Mass Spectrometry, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan, 2 Divisions of Membrane Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan, 3 present address: Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan, 4 present address: Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan, 5 Department of Evidence-based laboratory Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan on July 23, 2020. © 2014 American Association for Cancer Research. cancerres.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 April 4, 2014; DOI: 10.1158/0008-5472.CAN-13-2441

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Phosphatidylinositol 4-phosphate in the Golgi apparatus regulates

cell-cell adhesion, and invasive cell migration in human breast cancer

Emi Tokuda1, Toshiki Itoh2, 3, Junya Hasegawa2, 4, Takeshi Ijuin1, Yukiko Takeuchi1,

Yasuhiro Irino5, Miki Fukumoto1, and Tadaomi Takenawa1

1 Integrated Center for Mass Spectrometry, Kobe University Graduate School of

Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan, 2 Divisions of

Membrane Biology, Kobe University Graduate School of Medicine, 7-5-1

Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan, 3 present address: Biosignal

Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo,

657-8501, Japan, 4 present address: Laboratory of Intracellular Membrane Dynamics,

Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamadaoka, Suita,

Osaka, 565-0871, Japan, 5 Department of Evidence-based laboratory Medicine, Kobe

University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo,

650-0017, Japan

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Correspondence author: Tadaomi Takenawa: Kobe University, 7-5-1 Kusunoki-cho,

Chuo-ku, Kobe, Hyogo, 650-0017, Japan. Phone: 81-78-382-5410; E-mail:

[email protected]

Running title: PI(4)P regulates cancer progression

Precis: Findings suggest that invasive progression of breast cancer cells may be

directed by formation of a procancerous phospholipid in the Golgi apparatus.

Keywords: phosphatidylinositol 4-phosphate, Golgi apparatus, GOLPH3, cell-cell

adhesion, cell invasion

Authors’ Contributions

Conception and design: E. Tokuda, T. Itoh and T. Takenawa

Development of methodology: E. Tokuda and T. Takenawa

Acquisition of data (provided animals, acquired and managed patients, provided

facilities, etc.): E. Tokuda, J. Hasegawa, T. Ijuin, Y. Takeuchi, Y. Irino and M. Fukumoto

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational

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analysis): E. Tokuda

Writing, review, and/or revision of the manuscript: E. Tokuda, T. Itoh, J. Hasegawa, T.

Ijuin, and T. Takenawa

Study supervision: T. Takenawa

Disclosure of Potential conflicts of interest

The authors disclose no potential conflicts of interest.

Grant Support

This study was supported by a Grant-in-Aid for Scientific Research (S) (Grant Number

23227005 to T.T.), and a Grant-in-Aid for Challenging Exploratory Research (Grant

Number 24650621 to E.T.) from Japan Society for the Promotion of Science.

Main text: 4993 words, Abstract: 164 words, 7 figures

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Abstract

Downregulation of cell-cell adhesion and upregulation of cell migration play critical

roles in the conversion of benign tumors to aggressive invasive cancers. In this study,

we show that changes in cell-cell adhesion and cancer cell migration/invasion capacity

depend on the level of phosphatidylinositol 4-phosphate (PI4P) in the Golgi apparatus

in breast cancer cells. Attenuating SAC1, a PI4P phosphatase localized in the Golgi

apparatus, resulted in decreased cell-cell adhesion and increased cell migration in

weakly invasive cells. In contrast, silencing phosphatidylinositol 4-kinase III�

(PI4KIII�), which generates PI4P in the Golgi apparatus, increased cell-cell adhesion

and decreased invasion in highly invasive cells. Furthermore, a PI4P effector, Golgi

phosphoprotein 3 (GOLPH3), was found to be involved in the generation of these

phenotypes in a manner that depends on its PI(4)P-binding ability. Our results provide a

new model for breast cancer cell progression in which progression is controlled by PI4P

levels in the Golgi apparatus.

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Introduction

Phosphoinositides are lipid constituents of plasma and organelle membranes

and comprise seven different phosphorylated versions. The levels of various

phosphorylated phosphoinositides within the cell are regulated by multiple

phosphoinositide kinases and phosphoinositide phosphatases (1, 2). PI(4)P is a

relatively abundant phosphoinositide required for the maintenance and function of the

Golgi apparatus, including intracellular trafficking (3). The PI(4)P level at the Golgi

apparatus is controlled by phosphatidylinositol 4-kinases (PI4Ks) and SAC1

phosphoinositide phosphatase. The PI4K isoforms PI4KII� and PI4KIII� are known to

localize to the Golgi apparatus and to be involved in membrane transport from the

trans-Golgi network to the plasma membrane (4, 5). In contrast, SAC1, which

dephosphorylates PI(4)P at the Golgi apparatus, is involved in the same membrane

transport pathway and in protein glycosylation (6).

Several PI(4)P-binding proteins, including FAPP, OSBP, CERT, and GGA,

have been identified (7). One PI(4)P effector, GOLPH3, maintains a tensile force on the

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Golgi apparatus and plays a role in the Golgi secretory pathway (8, 9). Recently,

GOLPH3 was shown to be involved in cancer progression. Human chromosome 5p13,

on which the GOLPH3 gene is located, is amplified in several cancers (10), and

expression of GOLPH3 is increased in several cancer cell lines and in some cancer

patients (11-14). GOLPH3 enhances cell proliferation and tumorigenicity (10, 15).

However, its role in cell migration and invasion remains unknown.

To metastasize from their location within a primary tumor to the surrounding tissue,

tumor cells undergo several processes, including loss of cell-cell adhesion and gains in

motile and invasive potential (16). This temporary and reversible phenotype is referred

to as epithelial-mesenchymal transition (EMT). Following invasion or distal metastasis,

metastatic tumor cells revert to an epithelial phenotype in the so-called

mesenchymal-epithelial transition (MET). EMT/MET conversions are recognized as

critical events in cancer progression (17).

To identify the physiological role of PI(4)P in the Golgi apparatus during

tumor progression, we investigated the effects of PI(4)P metabolism on cell-cell

adhesion and cell migration in breast cancer cells. PI(4)P in the Golgi apparatus was

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found to regulate cell-cell adhesion, cell migration, invasion, and metastasis, which are

essential for breast cancer progression through regulation of GOLPH3 localization to

the Golgi apparatus. Based on these results, we propose a novel mechanism for the

development of cancer progression.

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Materials and Methods

Reagents and antibodies

Mouse anti-E-cadherin, �-catenin, PI4KIII�, and GM130 monoclonal antibodies were

purchased from BD Biosciences. Rat anti-E-cadherin monoclonal, mouse anti-GAPDH

monoclonal, rabbit anti-SAC1, rabbit anti-GOLPH3, and anti-cadherin 11 antibodies

were purchased from Takara Bio, Sigma-Aldrich, Proteintech, Epitomics, and Cell

Signaling Technology, respectively. All fluorescent reagents and antibodies were

purchased from Life Technologies. Purified phosphatidylethanolamine (PE) and

phosphatidylcholine (PC) were purchased from Avanti Polar Lipids, Inc. Purified

phosphoinositides were obtained from CellSignals Inc. and human breast cancer tissue

protein lysates from OriGene Technologies.

Plasmids

Human Fapp1 2 × PH, Grp1 PH, PLC�1 PH, and Hrs1 FYVE cDNA fragments were

subcloned into the pGEX-6P1 vector (GE Healthcare) as described previously (18).

GOLPH3 was generated by polymerase chain reaction (PCR) amplification with

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MDA-MB-231 cDNA as a template and subcloned into the pRetroQ-AcGFP1-C1 vector

(Clontech Laboratories, Inc.). The GOLPH3 R90L mutant was generated from

GOLPH3-wt DNA using site-directed mutagenesis.

Cell culture

The human breast cancer cell lines MCF7, MDA-MB-23, and Hs578t and GP2-293

were maintained in DMEM medium supplemented with 10% FBS. T-47D cells were

maintained in RPMI medium supplemented with 10 μg/ml insulin and 10% FBS. The

cell lines were obtained from American Type Culture Collection. The authenticity of all

cell lines was confirmed by short tandem repeat profiling by National Institute of

Biomedical Innovation in Japan.

RNA interference (RNAi)

Stealth siRNAs targeting phosphoinositide phosphatases, SAC1 (HSS117880 and

HSS117881), PI4KIII� (HSS108028 and HSS108029), and GOLPH3 (HSS127330 and

HSS127331) were purchased from Life Technologies. The stealth siRNAs were

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transfected into cells with Lipofectamine RNAiMAX (Life Technologies); experiments

were performed 72 h after transfection.

Production of retrovirus-infected cell lines

Retroviruses with the VSV-G envelope were packaged and produced according to

manufacture’s instruction (Clontech Laboratories, Inc.). Cells were infected with

retroviruses in the presence of 8 μg/m1 polybrene. The infected cells were selected by

adding 1 μg/ml puromycin to the medium for 2 weeks.

Immunofluorescence analysis

Cells grown on coverslips were fixed with 3.7% formaldehyde in PBS and

permeabilized using 0.2% Triton X-100 or 20 μM digitonin in PBS and blocked with

1.5% BSA in PBS before incubation with primary antibodies diluted in blocking buffer.

Immunofluorescence analysis was performed as previously described (19).

Quantification of Golgi PI(4)P levels

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Recombinant GST-conjugated proteins were expressed in the Rosseta-gami

B(DE3)pLyS bacterial strain (Merck) and purified on glutathione-Sepharose 4B (GE

Healthcare) according to the manufacturer’s instructions. GST was removed by

on-column cleavage with PreScission protease (GE Healthcare). Recombinant Fapp1 2

× PH domain protein was labeled using the Alexa Fluor 647 Protein Labeling Kit (Life

Technologies) and used as a fluorescent probe for detecting Golgi PI(4)P. Cells were

fixed with 3.7% formaldehyde in PBS, permeabilized using 20 μM digitonin in PBS,

and stained as described under “immunofluorescence analysis.” PI(4)P levels were

quantified using FluoView software. For quantifying the amount of PI(4)P at the Golgi

apparatus, the fluorescence intensity of AF647-labeled Fapp1 2 × PH was normalized to

that of GM130, a Golgi apparatus marker.

Matrigel invasion assay

The indicated siRNA-transfected or the indicated retrovirus-infected MDA-MB-231

cells were plated in a BioCoat Matrigel invasion chamber (BD Biosciences) and

cultured for 18 h. The cells were fixed with 3.7% formaldehyde in PBS, and the

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invading cells were stained with phalloidin.

Migration assay

The indicated siRNA-transfected MCF7 or T-47D cells, or the indicated

retrovirus-infected MCF7 cells were seeded. Forty-eight hours after transfection or

seeding, the cells were serum-starved for 16 h. The migration assay was performed for

24 h.

Subcellular fractionation

Cells were resuspended in Triton X-100 buffer (20 mM Tris-HCl [pH 7.5], 150 mM

NaCl, 2% glycerol, 0.1% Triton X-100, 1 mM CaCl2, and Complete Mini [Roche

Diagnostics]). The cell lysate was centrifuged at 20,630 g for 15 min at 4°C. The

supernatant was collected as a Triton X-100 soluble fraction. The pellet was sonicated

in SDS buffer (20 mM Tris-HCl [pH 7.5], 1% SDS, and 10% glycerol) and collected as

a Triton X-100 insoluble fraction.

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Real-time PCR

Total RNA was isolated from cells using TRIzol (Life Technologies) and used as a

template for cDNA synthesis with the PrimeScript II 1st-strand cDNA Synthesis Kit

(Takara). Real-time PCR was performed using a TaqMan probe in an ABI 7500

real-time PCR system (Life Technologies). The data were normalized to those obtained

for GAPDH.

RhoA activation assay

Cells were transfected with the indicated siRNAs. Seventy-two hours after transfection,

a RhoA activation assay was performed using a G-LISA RhoA Activation Assay

Biochem Kit (Cytoskeleton, Inc.). The data were normalized to total RhoA.

Tumor tail vein injection

Experiments were performed with 7-week-old female BALB/c-nu/nu mice (Charles

River Laboratories). All experiments were approved by the Institutional Animal Care

and Use Committee in Kobe University and were carried out in accordance with the

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institute’s guidelines. Cells suspended in Hanks’ Balanced Salt Solution (Gibco) were

injected intravenously (1 × 106 cells/mouse). Three weeks after injection, the mice were

euthanized and their lungs were fixed with Bouin’s Fluid (Wako Pure Chemical

Industries). The number of lung surface metastatic foci was counted.

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Results

Effect of knockdown of various phosphatases on cell-cell adhesion

Non-invasive MCF7 human breast cancer cells normally exhibit tight cell-cell

adhesion. To obtain insights into the role of phosphoinositide metabolism in the

regulation of cell-cell adhesion, we investigated the effect of siRNA-mediated depletion

of all phosphoinositide phosphatase isoforms in MCF7 cells (Fig. 1). Among siRNAs

targeting 22 phosphoinositide phosphatases, transfection of those targeting PTEN,

INPP4B, SAC1, INPP5D (SHIP1), and PIB5PA (PIPP) resulted in decreases in cell-cell

adhesion (Fig. 1, asterisk). PTEN, INPP4B, INPP5D, and PIB5PA have been previously

reported to exhibit tumor-suppressive activity through dephosphorylation of PI(3,4,5)P3

or PI(3,4)P2 and subsequent inhibition of PI3K signaling (20-22).

SAC1 knockdown increases PI(4)P levels in the Golgi apparatus and affects

cell-cell adhesion and cell migration

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SAC1, which dephosphorylates PI(4)P to PI in the Golgi apparatus, has not

been reported to be involved in cancer progression. Therefore, we decided to further

study whether this phosphoinositide phosphatase is involved in cancer progression.

Transfection of MCF7 cells with SAC1-targeting siRNA did not alter E-cadherin

or��-catenin protein levels in these cells (Fig. 2A), although it did result in reduced

cell-cell adhesion (Fig. 2B). Similar results were observed in T-47D human ductal

breast epithelial tumor cells, which normally exhibit cell-cell adhesion (Supplementary

Fig. 1A and B). We therefore used subcellular fractionation to monitor the association of

the actin cytoskeleton with E-cadherin and �-catenin in these cells. The amount of

E-cadherin and �-catenin in the Triton X-100-insoluble fraction decreased in

SAC1-depleted cells (Figure 2C). Consistent with the previous finding that cell

migration increases as a consequence of the disappearance of cell-cell adhesion (23),

SAC1 depletion promoted MCF7 cell (Fig. 2D) and T-47D cell (Supplementary Fig.

1C) migration and caused reduction in cell-cell adhesion in both cell types. SAC1

depletion seemed to induce stress fiber formation at the cell periphery in MCF7 cells

(Supplementary Fig. 3A), with an increase in RhoA activity in these cells (Fig. 2E).

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Furthermore, SAC1 depletion induced expression of SNAI2, an EMT marker in MCF7

cells (Fig. 2F). These results indicate that SAC1 partly regulates EMT, which is

characterized by changes in the localization of E-cadherin and �-catenin at cell-cell

adhesion sites, actin cytoskeletal rearrangement, and expression of EMT markers.

Because Sac1 dephosphorylates PI(4)P in the Golgi apparatus, we quantified

the levels of cellular phosphoinositides using a liposome overlay assay. No change in

the total amount of phosphoinositides was detected in Sac1-depleted cells

(Supplementary Fig. 2A and B). Therefore, we measured relative PI(4)P levels in the

Golgi apparatus using Alexa Fluor 647 (AF647)-labeled Fapp1 PH domains (Fapp1 2 ×

PH) that specifically recognize PI(4)P with high affinity (18). PI(4)P localized at the

Golgi apparatus of HeLa cells can be efficiently detected by this fluorescent probe after

permeabilization of the cells with digitonin (Supplementary Fig. 2C) (24). The

specificity of the fluorescence signals was confirmed by pre-incubation of the probe

with PC/PE/PI(4)P liposomes, which completely blocked PI(4)P detection in the Golgi

apparatus (Supplementary Fig. 2C). Using this method, we found that Sac1 knockdown

increased PI(4)P levels in the Golgi apparatus by approximately two-fold, compared to

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that in control MCF7 cells (Fig. 2G and H). Further knockdown of PI4KIII�, which

generates PI(4)P in the Golgi apparatus, in SAC1-depleted MCF7 cells restored the

formation of cell-cell adhesion (Supplementary Fig. 3B and C), indicating that PI(4)P

promotes cell migration by disrupting cell-cell adhesion.

PI(4)P levels in the Golgi apparatus during breast cancer progression

TGF� and TNF� are known to induce EMT in several cancer cell lines (25,

26). Treatment with TGF� and TNF� efficiently altered the normal epithelial

morphology of MCF7 cells, producing a �migratory phenotype. TGF�- and

TNF�-treated MCF7 cells also exhibited an increase of approximately two-fold in

PI(4)P levels in the Golgi apparatus (Fig. 3A and B). We then compared PI(4)P levels at

the Golgi apparatus in highly invasive (MDA-MB-231 and Hs578t) and weakly

invasive (MCF7 and T-47D) breast cancer cell lines. In highly invasive cell lines, PI(4)P

levels in the Golgi apparatus were significantly higher than those in weakly invasive

cell lines (Fig. 3C and D). Moreover, PI4KIII� expression was higher in late-stage

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human breast cancer tissues (metastatic, stages III and IV) than in early-stage tissues

(non-metastatic, stages I and IIa) (Fig. 3E). In contrast, SAC1 expression decreased in

stages III and IV human breast cancer tissues (metastatic) (Fig. 3E). These data

collectively support the idea that there is a strong correlation between Golgi PI(4)P and

breast cancer malignancy.

PI4KIII� knockdown decreases PI(4)P levels in the Golgi apparatus and affects

cell-cell adhesion and invasion

To further confirm the correlation between PI(4)P levels in the Golgi

apparatus and cell-cell adhesion, we examined the effect of silencing PI4KIII�, which

produces PI(4)P at the Golgi apparatus, in MCF7 cells. PI4KIII� knockdown decreased

PI(4)P levels in the Golgi apparatus by half in MCF7 cells (Supplementary Fig. 3D-F).

Because tight cell-cell adhesion already exhibit in MCF7 cells, further formation of

cell-cell adhesion was not induced by PI4KIII� silencing in MCF7 cells (Supplementary

Fig. 3B). We then used highly invasive MDA-MB-231 human breast adenocarcinoma

cells that do not normally exhibit cell-cell adhesion and examined the four isoforms of

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PI4-kinase that might be involved in these phenotype changes. Of the four PI 4-kinase

isoforms examined, attenuation of only PI4KIII� promoted an increase in cell-cell

adhesion in MDA-MB-231 cells (Supplementary Fig. 4A and B). Because PI4KIII� is

known to produce PI(4)P in the Golgi apparatus, it may be involved in this change. In

MDA-MB-231 cells, PI4KIII� knockdown decreased PI(4)P levels in the Golgi

apparatus to one-third of the control level (Fig. 4A and B).

MDA-MB-231 cells have been reported to utilize cadherin-11 rather than

E-cadherin and N-cadherin as a cell-adhesion molecule (27, 28). PI4KIII�-depleted

MDA-MB-231 cells formed adhesions with cadherin-11 and �-catenin without any

changes in the expression levels of these proteins (Fig. 4C and D). In addition, further

transfection of PI4KIII�-depleted MDA-MB-231 cells with Sac1 siRNA decreased their

cell-cell adhesion (Supplementary Fig. 4C and D). Another line of highly invasive

human ductal breast carcinoma cells, Hs578t cells, does not exhibit cell-cell adhesion

under normal conditions. Depletion of PI4KIII� in these cells resulted in an increase in

cell-cell adhesion (Supplementary Fig. 5A - C). Ht578t cells express N-cadherin and

cadherin-11 but not E-cadherin (27, 28). Membrane localization of these cadherins was

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induced by PI4KIII� silencing without changes in protein levels (Supplementary Fig.

5A - C). MDA-MB-231 cell invasion was significantly suppressed by PI4KIII�

depletion (Fig. 4E), with an increase in RhoA activity in these cells (Fig. 4F).

Furthermore, these cells exhibited decreased expression of SNAI1, an EMT marker (Fig.

4G). These data suggest that decrease in PI(4)P levels in the Golgi apparatus is likely to

increase cell-cell adhesion, thereby inhibiting invasion and promotion of EMT in breast

cancer cells.

GOLPH3 is involved in PI(4)P-dependent changes in the phenotype

GOLPH3 is a downstream target of PI(4)P that is localized in the Golgi

apparatus (9). In PI4KIII�-depleted MDA-MB-231 cells, GOLPH3 was no longer

localized at the Golgi apparatus (Fig. 5A), without any significant change in its

expression (Supplementary Fig. 6A). GOLPH3 expression did not change between

control and SAC1 siRNA-transfected MCF7 cells (Supplementary Fig. 6B). GOLPH3

already localized at the Golgi apparatus in control siRNA-transfected MCF7 cells

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(Supplementary Fig. 6C); however, fluorescence intensity of GOLPH3 increased in

SAC1-depleted MCF7 cells (Supplementary Fig. 6D). These data indicated that the

PI(4)P in the Golgi apparatus regulates GOLPH3 localization but not expression.

Because it has been reported that GOLPH3 is related to tumorigenesis (10, 15), we

hypothesized that it may be involved in the regulation of cell-cell adhesion and cell

migration/invasion. Attenuation of GOLPH3 resulted in increased cell-cell adhesion and

suppression of the invasive phenotype, which was similar to what was observed during

PI4KIII� knockdown (Fig. 5B-D). In addition, further GOLPH3 depletion in

SAC1-depleted MCF7 cells decreased migration ability (Fig. 5E) and promoted cell-cell

adhesion (Supplementary Fig. 6E). These data indicate that GOLPH3 is involved in

cell-cell adhesion, as well as migration and invasion, which are dependent on Golgi

PI(4)P.

PI(4)P-binding ability of GOLPH3 plays an important role in cancer progression

The green fluorescent protein (GFP)-tagged GOLPH3 R90L mutant

(GFP-GOLPH3 R90L), which is unable to bind to PI(4)P (8, 9), also did not localize to

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the Golgi apparatus (Fig. 6A and Supplementary Fig. 6F). In MCF7 cells

overexpressing GFP-GOLPH3, cell-cell adhesion decreased and was not altered by

expression of the GFP-GOLPH3 R90L mutant (Fig. 6A). Cell migration was also

promoted by overexpression of wild-type GOLPH3 in MCF7 cells but not by

overexpression of the GOLPH3 R90L mutant (Fig. 6B). In MDA-MB-231 cells,

expression of the GOLPH3 R90L mutant did not affect cell-cell adhesion or invasion

(Fig. 6C and D), whereas expression of wild-type GOLPH3 abolished cell-cell adhesion

and enhanced invasion (Fig. 6C and D). This enhanced invasion ability in

GOLPH3-expressing cells was abolished by PI4KIII� depletion (Fig. 6E).

Additionally, PI(4)P levels in the Golgi apparatus increased in GOLPH3

wt-expressing MCF-7 and MDA-MB-231 cells but not GOLPH3 R90L-expressing

MCF-7 and MDA-MB-231 cells (Fig. 7A-D). However, in GOLPH3 wt-expressing

MCF7 cells, upregulation of SNAI2 expression was not observed (Fig. 7E). In contrast,

downregulation of SNAI2 expression was observed in GOLPH3 R90L-expressing

MCF7 cells (Fig. 7E). This finding suggests that GOLPH3 is necessary but not

sufficient for EMT induction. Finally, we tested the effect of GOLPH3 on cancer

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metastasis in vivo. GOLPH3 wt-expressing MDA-MB-231 cells showed much higher

levels of metastasized to the lung than GFP-expressing cells (Fig. 7F and G). However,

GOLPH3 R90L, which is a mutant with defective lipid binding, lost its metastasizing

activity. These data indicate that GOLPH3 functions as a regulator of cancer metastasis

in breast cancer through interaction with Golgi PI(4)P.

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Discussion

In this study, we have shown that PI(4)P in the Golgi apparatus is a key

regulator of cancer progression. In the Golgi apparatus, SAC1 and PI4KIII� are

predominantly involved in PI(4)P metabolism. However, except for its role in the

regulation of lipid homeostasis and sphingolipid biosynthesis, little is known about the

specific role of SAC1-dependent dephosphorylation of PI(4)P (29-31). We showed

that PI4KIII� and SAC1 expression and PI(4)P levels in the Golgi apparatus were

correlated with cancer progression. According to the NextBio database

(www.nextbio.com), SAC1 and PI4KIII� mRNA expression are correlated with many

cancers, including breast cancer (Supplementary Tables 1 and 2). These results support

our hypothesis that PI(4)P at the Golgi apparatus regulates cancer progression.

The Golgi apparatus is a central organelle that is involved in protein and lipid

biosynthesis and vesicular trafficking. Newly synthesized proteins and lipids are

glycosylated in the Golgi apparatus. Although aberrant glycosylation and

overexpression of glycolipids have been shown to be common features of malignant

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tumors (32, 33), the biological significance of these changes is not well understood.

PI(4)P is enriched at the Golgi apparatus, where PI(4)P-binding proteins that have been

shown to be involved in vesicular transport, lipid transfer, and glycosylation are located.

(1). A Golgi PI(4)P effector, GOLPH3, is involved in Golgi-plasma membrane transport

and in the glycosylation of proteins (8, 34, 35). In yeast, Vps74, the ortholog of human

GOLPH3, binds directly to Sac1 (36). We found that expression of GOLPH3 wt, but not

GOLPH3 R90L, resulted in an increase in PI(4)P levels in the Golgi apparatus. These

data suggest that GOLPH3 overexpression competes with SAC1 for PI(4)P in the Golgi

membrane, or that GOLPH3 senses PI(4)P levels at the Golgi apparatus, thus regulating

SAC1 or PI4KIII� localization or activity. We observed that PI(4)P regulated GOLPH3

localization to the Golgi apparatus. These result suggest that GOLPH3 is localized to

the Golgi apparatus by increasing PI(4)P, leading to aberrant glycosylation and

overexpression of glycolipids.

EMT is an important process in cancer progression. It is characterized by a

decrease in epithelial markers (e.g., E-cadherin) and upregulation of mesenchymal

markers, including vimentin, SNAI1, and SNAI2, which together result in disruption of

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cell-cell adhesion. However, whether EMT/MET occurs in cancer progression remains

controversial (37, 38). We observed that the membrane localization of cadherins and

�-catenin and the expression of SNAI1 and SNAI2 are changed by PI(4)P generation at

the Golgi apparatus, although decreases in the expression of E-cadherin, which is a

characteristic of complete EMT, were not detected. Malignant cancer cells adopt some

mesenchymal features but retain characteristics of epithelial cells (39-41), a condition

that has been described as incomplete EMT (42). Taken together with the finding that

the amount of PI(4)P increases at the Golgi apparatus during EMT, our data suggest that

Golgi PI(4)P is necessary but not sufficient for EMT.

Several studies have shown that GOLPH3 plays important roles in the

progression of several tumor types. For example, GOLPH3 upregulated and promotes

proliferation and tumorigenicity in breast cancer (15). Recent studies have demonstrated

the clinical significance of GOLPH3 in several cancers (13, 14, 43). A very recent study

reported that GOLPH3 contributes to the migration and invasion of glioma cells through

RhoA expression (44). We found that changes in PI(4)P levels at the Golgi apparatus

resulted in alteration of RhoA activity. RhoA activity is required for initial cell-cell

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adhesion formation (45) and is repressed during EMT (45). Taken together with our

results, these findings suggest that Golgi PI(4)P and GOLPH3 regulate the cytoskeletal

rearrangement of actin through RhoA as well as Golgi-plasma membrane transport

during tumor progression.

In conclusion, our data suggest that PI(4)P levels in the Golgi apparatus play a

critical role in cancer progression by regulating the PI(4)P effector GOLPH3

(Supplementary Fig. 6G). In this study, we showed that changes in Golgi PI(4)P levels

and/or Golgi localization of GOLPH3 were likely to result in changes in cell-cell

adhesion and cell migration/invasion in breast cancer cell lines. Furthermore, we

showed that PI(4)P-binding ability of GOLPH3 was essential for metastasis in vivo.

Therefore, molecules that modulate the PI(4)P level in the Golgi apparatus, such as

SAC1 and PI4KIII�, may represent intriguing anticancer therapeutic targets.

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Acknowledgements

We are grateful to T. Oikawa (Department of Molecular Biology, Hokkaido University

Graduate School of Medicine) for the discussions and to Y. Murata and T. Yanagita

(Division of Molecular and Cellular Signaling, Kobe University Graduate School of

Medicine) for the technical advice.

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Figure Legends

Figure 1. Phosphoinositide phosphatase knockdown.

MCF7 cells were transfected with siRNA targeting the indicated phosphoinositide

phosphatases. Seventy-two hours after transfection, the cells were fixed and stained

with phalloidin. Scale bar = 20 �m.

Figure 2. SAC1 knockdown decreases cell-cell adhesion and promotes cell

migration.

(A) The indicated siRNA-transfected MCF7 cells were lysed and immunoblotted with

the indicated antibodies.

(B) The indicated siRNA-transfected MCF7 cells were fixed and stained with

anti-E-cadherin antibody and phalloidin. Scale bar = 20 �m.

(C) The indicated siRNA-transfected MCF7 cells were fractionated into Triton

X-100-soluble and Triton X-100-insoluble fractions. Each fraction was

immunoblotted with the indicated antibodies.

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(D) The indicated siRNA-transfected MCF7 cells were analyzed using a migration

assay. The data shown are the mean (SEM) values of three independent experiments.

*, P < 0.001; **, P < 0.005. Scale bar = 100 �m.

(E) MCF7 cells were transfected with the indicated-siRNAs. RhoA activity was

measured using a G-LISA RhoA activation assay. The data shown are the mean

(SEM) values of four independent experiments. *, P < 0.01.

(F) MCF7 cells were transfected with indicated-siRNAs. The SNAI2 mRNA level was

quantified by real-time PCR. The data shown are the mean (SEM) values of three

independent experiments. *, P < 0.003.

(G) Golgi PI(4)P levels were quantified in the indicated siRNA-transfected MCF7 cells.

The data shown are the mean (SEM) values from more than 100 cells measured. *,

P < 0.001.

(H) The indicated siRNA-transfected MCF7 cells were fixed and stained with the

anti-GM130 antibody and Alexa Fluor 647-labeled Fapp1 2 × PH domain. Scale bar

= 20 �m.

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Figure 3. EMT-like phenotypes are affected by PI(4)P levels in the Golgi

apparatus.

(A) MCF7 cells, not treated or treated with 2 ng/ml TGF� + 5 ng/ml TNF�, were fixed

and stained with anti-GM130 antibody together with Alexa Fluor 647-labeled

Fapp1 2× PH domain. Scale bar = 20 �m.

(B) Quantification of Golgi PI(4)P levels in MCF7 cells before and after EMT induction.

The data shown are the mean (SEM) values from more than 100 cells. *, P < 0.001.

(C) Each breast cancer cell line was fixed and stained with anti-GM130 antibody and

the Alexa Fluor 647-labeled Fapp1 2 × PH domain. Scale bar = 20 �m.

(D) Golgi PI(4)P levels were quantified in breast cancer cell lines. The data shown are

the mean (SEM) values from more than 100 cells measured. *, P < 0.001.

(E) Human breast cancer tissue lysates were immunoblotted with the antibodies against

PI4KIII� and SAC1. Lysates isolated from five non-metastatic and metastatic breast

cancer subjects were tested.

Figure 4. PI4KIII� knockdown induces cell-cell adhesion and suppression of

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42

invasion.

(A) The indicated siRNA-transfected MDA-MB-231 cells were fixed and stained with

anti-GM130 antibody and Alexa Fluor 647-labeled Fapp1 2 × PH domain. Scale bar

= 20 �m.

(B) Golgi PI(4)P levels were quantified in control- or PI4KIII�-silenced MDA-MB-231

cells. The data represent the mean (SEM) values from more than 100 cells. *, P <

0.001.

(C) The indicated siRNA-transfected MDA-MB-231 cells were fixed and stained with

anti-�-catenin, anti-cadherin-11, and phalloidin. Scale bar = 20 �m.

(D) Control- or PI4KIII�-siRNA-transfected MDA-MB-231 cells were lysed and

immunoblotted with the indicated antibodies.

(E) The indicated siRNA-transfected MDA-MB-231 cells were seeded onto Matrigel

invasion chambers. Eighteen hours after seeding, the cells were fixed and stained

with phalloidin (lower panels). The number of invasive cells that passed through

each Matrigel chamber was counted (upper panel). The data represent the mean

(SEM) values of three independent experiments. *, P < 0.001. Scale bar = 100 �m.

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(F) MDA-MB-231 cells were transfected with the indicated-siRNAs. RhoA activity was

measured using a G-LISA RhoA activation assay. The data shown are the mean

(SEM) values of four independent experiments. *, P < 0.02; **, P < 0.002.

(G) MDA-MB-231 cells were transfected with indicated-siRNAs. The SNAI1 mRNA

level was quantified by real-time PCR. The data shown are the mean (SEM) of three

independent experiments. *, P < 0.001; **, P < 0.03.

Figure 5. GOLPH3 is required for regulation of cell-cell adhesion and for

migration/invasion ability.

(A) MDA-MB-231 cells were transfected with control or GOLPH3 siRNA and then

fixed and stained with anti-GOLPH3 and anti-GM130 antibodies, and phalloidin.

Scale bar = 20 �m.

(B) The indicated siRNA-transfected MDA-MB-231 cells were lysed and

immunoblotted with GOLPH3 antibodies.

(C) The indicated siRNA-transfected MDA-MB-231 cells were fixed and stained with

anti-�-catenin antibody and phalloidin. Scale bar = 20 �m.

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44

(D) The indicated siRNA-transfected MDA-MB-231 cells were seeded onto Matrigel

invasion chambers. Eighteen hours after seeding, the cells were fixed and stained

with phalloidin (upper panels). The number of invasive cells that passed through

each Matrigel chamber was counted (lower panel). The data shown are the mean

(SEM) values of three independent experiments. *, P < 0.001. Scale bar = 100 �m.

(E) The indicated siRNA-transfected MCF7 cells were analyzed in a migration assay.

The data shown are the mean (SEM) values of four independent experiments. *, P

< 0.01; **, P < 0.005. Scale bar = 100 �m.

Figure 6. Binding of PI(4)P to GOLPH3 is required for regulation of cell-cell

adhesion and cell migration/invasion.

(A) GFP (mock), GFP-tagged wild-type GOLPH3 (GOLPH3 wt), or the GFP-tagged

GOLPH3 R90L mutant (GOLPH3 R90L) was overexpressed in MCF7 cells. Cells

were stained with an anti-E-cadherin antibody and phalloidin. Scale bar = 20 �m.

(B) Mock, GOLPH3 wt, or GOLPH3 R90L-expressing MCF7 cells were used for a

migration assay. The data shown are the mean (SEM) values of six independent

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45

experiments. *, P < 0.0005. Scale bar = 100 �m.

(C) Mock, GOLPH3 wt or GOLPH3 R90L-expressing MDA-MB-231 cells were

stained with an anti-�-catenin antibody and phalloidin. Scale bar = 20 �m.

(D) Mock, GOLPH3 wt, or GOLPH3 R90L mutant-expressing MDA-MB-231 cells

were seeded onto Matrigel invasion chambers. Eighteen hours after seeding, the

cells were fixed and stained with phalloidin. The number of invasive cells that

passed through the Matrigel chambers was counted. The data shown are the mean

(SEM) values of four independent experiments. *, P < 0.005; **, P < 0.0005. Scale

bar = 100 �m.

(E) Mock or GOLPH3 wt-expressing MDA-MB-231 cells were transfected with the

indicated siRNAs. Forty-eight hours after transfection, the cells were

serum-starved and seeded onto Matrigel invasion chambers. Eighteen hours after

seeding, the cells were fixed and stained with phalloidin. The number of invasive

cells that passed through the Matrigel chambers was counted. The data shown are

the mean (SEM) of four independent experiments. *, P < 0.0005. Scale bar = 100

�m.

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Figure 7. Binding of PI(4)P to GOLPH3 is required for metastasis in vivo.

(A)GFP, GOLPH3 wt, or GOLPH3 R90L-expressing MCF7 cells were transfected with

the indicated siRNAs and then fixed and stained with anti-GM130 and the Alexa

Fluor 647-labeled Fapp1 2 × PH domain. Scale bar = 20 �m.

(B) Golgi PI(4)P levels were quantified in GFP, GOLPH3 wt, or GOLPH3

R90L-expressing MCF7 cells. Significant increases were observed following

GOLPH3 wt expression. The data shown are the mean (SEM) values from more

than 100 cells. *, P < 0.001.

(C) GFP, GOLPH3 wt, or GOLPH3 R90L-expressing MDA-MB-231 cells were

transfected with the indicated siRNAs and then fixed and stained with anti-GM130

antibody and the Alexa Fluor 647-labeled Fapp1 2 × PH domain. Scale bar = 20 �m.

(D)Golgi PI(4)P levels were quantified in GFP, GOLPH3 wt, or GOLPH3

R90L-expressing MDA-MB-231 cells. The data shown are the mean (SEM) values

from more than 100 cells. *, P < 0.001.

(E) Total RNA was isolated from GFP or GFP-tagged GOLPH3 stable MCF7 cells. The

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SNAI2 mRNA level was quantified by real-time PCR. The data shown are the mean

(SEM) values of three independent experiments. *, P < 0.0001.

(F) Lung surface metastatic foci were counted. The data shown are the mean (SEM)

values from seven (mock and GOLPH3 wt) or eight (GOLPH3 R90L) mice. *, P <

0.04; **, P < 0.02.

(G)Representative images of the lungs with metastatic foci.

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cont

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Figure 2

A

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0.7

0.8

0.9

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10

15

20

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30

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Figure 3

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Figure 4

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P le

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Figure 5

A

CD

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β-catenin actin

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10

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20

35

30

40

5

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ratio

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siRNA

cont

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cont

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Figure 6

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1800

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10

70

20

80

30

40

50

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Figure 7

A B

C

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wt

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mock wt R90L

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Published OnlineFirst April 4, 2014.Cancer Res   Emi Tokuda, Toshiki Itoh, Junya Hasegawa, et al.   human breast cancerregulates cell-cell adhesion and invasive cell migration in Phosphatidylinositol 4-phosphate in the Golgi apparatus

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