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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 orGOLPH3 siRNA and then fixed and stained with anti-GOLPH3 and anti-GM130 antibodies, and phalloidin. Scale bar, 20 mm. 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 fixedand stained with anti–b-catenin antibody and phalloidin. Scale bar, 20 mm. D, the indicated siRNA-transfected MDA-MB-231 cells were seeded ontoMatrigel invasion chambers. Eighteen hours after seeding, the cells were fixed and stained with phalloidin (top). The number of invasive cells thatpassed through each Matrigel chamber was counted (bottom). The data shown are the mean (SEM) values of three independent experiments. �, P < 0.001.Scale bar, 100 mm. E, the indicated siRNA-transfected MCF7 cells were analyzed in a migration assay. The data shown are the mean (SEM) values offour independent experiments. �, P < 0.01; ��, P < 0.005. Scale bar, 100 mm.

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Sac1 knockdown increased PI(4)P levels in the Golgi apparatusby approximately 2-fold, compared with that in control MCF7cells (Fig. 2G and H). Further knockdown of PI4KIIIb, whichgenerates PI(4)P in the Golgi apparatus, in SAC1-depletedMCF7 cells restored the formation of cell–cell adhesion (Sup-plementary Fig. S3B and S3C), indicating that PI(4)P promotescell migration by disrupting cell–cell adhesion.

PI(4)P levels in the Golgi apparatus during breast cancerprogressionTGFb and TNFa are known to induce EMT in several

cancer cell lines (25, 26). Treatment with TGFb and TNFaefficiently altered the normal epithelial morphology ofMCF7 cells, producing a migratory phenotype. TGFb- andTNFa-treated MCF7 cells also exhibited an increase ofapproximately 2-fold in PI(4)P levels in the Golgi apparatus(Fig. 3A and B). We then compared PI(4)P levels at the Golgiapparatus in highly invasive (MDA-MB-231 and Hs578t) andweakly invasive (MCF7 and T-47D) breast cancer cell lines.In highly invasive cell lines, PI(4)P levels in the Golgiapparatus were significantly higher than those in weaklyinvasive cell lines (Fig. 3C and D). Moreover, PI4KIIIbexpression was higher in late-stage human breast cancertissues (metastatic, stages III and IV) than in early-stagetissues (nonmetastatic, stages I and IIa; Fig. 3E). In contrast,SAC1 expression decreased in stages III and IV human breastcancer tissues (metastatic; Fig. 3E). These data collectivelysupport the idea that there is a strong correlation betweenGolgi PI(4)P and breast cancer malignancy.

PI4KIIIb knockdown decreases PI(4)P levels in the Golgiapparatus and affects cell–cell adhesion and invasionTo further confirm the correlation between PI(4)P levels

in the Golgi apparatus and cell–cell adhesion, we examinedthe effect of silencing PI4KIIIb, which produces PI(4)P at theGolgi apparatus, in MCF7 cells. PI4KIIIb knockdowndecreased PI(4)P levels in the Golgi apparatus by half inMCF7 cells (Supplementary Fig. S3D–S3F). Because tightcell–cell adhesion already exhibit in MCF7 cells, furtherformation of cell–cell adhesion was not induced by PI4KIIIbsilencing in MCF7 cells (Supplementary Fig. S3B). We thenused highly invasive MDA-MB-231 human breast adenocar-cinoma cells that do not normally exhibit cell–cell adhesionand examined the 4 isoforms of PI4-kinase that might beinvolved in these phenotype changes. Of the four PI4-kinaseisoforms examined, attenuation of only PI4KIIIb promotedan increase in cell–cell adhesion in MDA-MB-231 cells(Supplementary Fig. S4A and S4B). Because PI4KIIIb isknown to produce PI(4)P in the Golgi apparatus, it may beinvolved in this change. In MDA-MB-231 cells, PI4KIIIbknockdown decreased PI(4)P levels in the Golgi apparatusto 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-adhesionmolecule (27, 28). PI4KIIIb-depleted MDA-MB-231 cellsformed adhesions with cadherin-11 and b-catenin withoutany changes in the expression levels of these proteins (Fig.4C and D). In addition, further transfection of PI4KIIIb-deplet-

edMDA-MB-231 cellswith Sac1 siRNAdecreased their cell–celladhesion (Supplementary Fig. S4C and S4D). Another line ofhighly invasive human ductal breast carcinoma cells, Hs578tcells, does not exhibit cell–cell adhesion under normal condi-tions. Depletion of PI4KIIIb in these cells resulted in anincrease in cell–cell adhesion (Supplementary Fig. S5A–S5C).Ht578t cells express N-cadherin and cadherin-11 but not E-cadherin (27, 28). Membrane localization of these cadherinswas induced by PI4KIIIb silencing without changes in proteinlevels (Supplementary Fig. S5A–S5C). MDA-MB-231 cell inva-sion was significantly suppressed by PI4KIIIb depletion (Fig.4E), with an increase in RhoA activity in these cells (Fig. 4F).Furthermore, these cells exhibited decreased expression ofSNAI1, an EMT marker (Fig. 4G). These data suggest thatdecrease in PI(4)P levels in the Golgi apparatus is likely toincrease cell–cell adhesion, thereby inhibiting invasion andpromotion of MET in breast cancer cells.

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

GOLPH3 is a downstream target of PI(4)P that is localized intheGolgi apparatus (9). InPI4KIIIb-depletedMDA-MB-231cells,GOLPH3wasno longer localized at theGolgi apparatus (Fig. 5A),without any significant change in its expression (SupplementaryFig. S6A). GOLPH3 expression did not change between controland SAC1 siRNA-transfected MCF7 cells (Supplementary Fig.S6B). GOLPH3 already localized at the Golgi apparatus incontrol siRNA-transfected MCF7 cells (Supplementary Fig.S6C); however, fluorescence intensity of GOLPH3 increased inSAC1-depletedMCF7cells (SupplementaryFig. S6D). These dataindicated that the PI(4)P in the Golgi apparatus regulatesGOLPH3 localization but not expression. Because it has beenreported that GOLPH3 is related to tumorigenesis (10, 15), wehypothesized that it may be involved in the regulation of cell–cell adhesion and cell migration/invasion. Attenuation ofGOLPH3 resulted in increased cell–cell adhesion and suppres-sion of the invasive phenotype, which was similar to what wasobserved during PI4KIIIb knockdown (Fig. 5B–D). In addition,further GOLPH3 depletion in SAC1-depleted MCF7 cellsdecreased migration ability (Fig. 5E) and promoted cell–celladhesion (Supplementary Fig. S6E). These data indicate thatGOLPH3 is involved in cell–cell adhesion, as well as migrationand invasion, which are dependent on Golgi PI(4)P.

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

The GFP-tagged GOLPH3 R90L mutant (GFP-GOLPH3R90L), which is unable to bind to PI(4)P (8, 9), also did notlocalize to the Golgi apparatus (Fig. 6A and SupplementaryFig. S6F). In MCF7 cells overexpressing GFP-GOLPH3, cell–cell adhesion decreased and was not altered by expression ofthe GFP-GOLPH3 R90L mutant (Fig. 6A). Cell migration wasalso promoted by overexpression of wild-type GOLPH3 inMCF7 cells but not by overexpression of the GOLPH3 R90Lmutant (Fig. 6B). In MDA-MB-231 cells, expression of theGOLPH3 R90L mutant increased cell–cell adhesion but didnot affect invasion (Fig. 6C and D), whereas expression ofwild-type GOLPH3 abolished cell–cell adhesion and enhanced

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PI(4)P Regulates Cancer Progression



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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-typeGOLPH3 (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 mm. 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 experiments. (Continued on the following page.)

Tokuda et al.





invasion (Fig. 6C and D). This enhanced invasion ability inGOLPH3-expressing cells was abolished by PI4KIIIb depletion(Fig. 6E).In addition, PI(4)P levels in the Golgi apparatus increased in

GOLPH3 wt-expressing MCF-7 and MDA-MB-231 cells but notGOLPH3 R90L-expressing MCF-7 and MDA-MB-231 cells(Fig. 7A–D). However, in GOLPH3 wt-expressing MCF7 cells,upregulation of SNAI2 expressionwas not observed (Fig. 7E). Incontrast, downregulation of SNAI2 expression was observed inGOLPH3 R90L-expressing MCF7 cells (Fig. 7E). This findingsuggests that GOLPH3 is necessary but not sufficient for EMTinduction. Finally, we tested the effect of GOLPH3 on cancermetastasis in vivo. GOLPH3 wt-expressing MDA-MB-231 cellsshowed much higher levels of metastasized to the lung thanGFP-expressing cells (Fig. 7F and G). However, GOLPH3 R90L,which is a mutant with defective lipid binding, lost its metas-tasizing activity. These data indicate that GOLPH3 functions asa regulator of cancer metastasis in breast cancer throughinteraction with Golgi PI(4)P.

DiscussionIn this study, we have shown that PI(4)P in the Golgi

apparatus is a key regulator of cancer progression. In the Golgiapparatus, SAC1 and PI4KIIIb are predominantly involved inPI(4)P metabolism. However, except for its role in the regula-tion of lipid homeostasis and sphingolipid biosynthesis, little isknown about the specific role of SAC1-dependent dephosphor-ylation of PI(4)P (29–31). We showed that PI4KIIIb and SAC1expression and PI(4)P levels in the Golgi apparatus werecorrelated with cancer progression. According to the NextBiodatabase (www.nextbio.com), SAC1 and PI4KIIIb mRNAexpression are correlated with many cancers, including breastcancer (Supplementary Tables S1 and S2). These results sup-port our hypothesis that PI(4)P at the Golgi apparatus reg-ulates cancer progression.The Golgi apparatus is a central organelle, which is involved

in protein and lipid biosynthesis and vesicular trafficking.Newly synthesized proteins and lipids are glycosylated in theGolgi apparatus. Although aberrant glycosylation and over-expression of glycolipids have been shown to be commonfeatures of malignant tumors (32, 33), the biologic significanceof these changes is not well understood. PI(4)P is enriched atthe Golgi apparatus, where PI(4)P-binding proteins that havebeen 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 transportand in the glycosylation of proteins (8, 34, 35). In yeast, Vps74,the ortholog of humanGOLPH3, 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 competeswith SAC1 for PI(4)P in the Golgi membrane, or that GOLPH3senses PI(4)P levels at the Golgi apparatus, thus regulatingSAC1 or PI4KIIIb 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 Golgiapparatus by increasing PI(4)P, leading to aberrant glycosyl-ation and overexpression of glycolipids.

EMT is an important process in cancer progression. It ischaracterized by a decrease in epithelial markers (e.g., E-cadherin) and upregulation of mesenchymal markers, includ-ing vimentin, SNAI1, and SNAI2, which together result indisruption of cell–cell adhesion. However, whether EMT/METoccurs in cancer progression remains controversial (37, 38).Weobserved that the membrane localization of cadherins andb-catenin and the expression of SNAI1 and SNAI2 are changedby PI(4)P generation at the Golgi apparatus, althoughdecreases in the expression of E-cadherin, which is a charac-teristic of complete EMT, were not detected. Malignant cancercells adopt some mesenchymal features but retain character-istics of epithelial cells (39–41), a condition that has beendescribed as incomplete EMT (42). Taken together with thefinding that the amount of PI(4)P increases at the Golgiapparatus during EMT, our data suggest that Golgi PI(4)P isnecessary but not sufficient for EMT.

Several studies have shown that GOLPH3 plays importantroles in the progression of several tumor types. For example,GOLPH3 upregulated and promotes proliferation and tumor-igenicity in breast cancer (15). Recent studies have demon-strated the clinical significance of GOLPH3 in several cancers(13, 14, 43). A very recent study reported that GOLPH3 con-tributes to the migration and invasion of glioma cells throughRhoA expression (44).We found that changes in PI(4)P levels atthe Golgi apparatus resulted in alteration of RhoA activity.RhoA activity is required for initial cell–cell adhesion forma-tion (45) and is repressed during EMT (45). Taken togetherwithour results, these findings suggest that Golgi PI(4)P andGOLPH3 regulate the cytoskeletal rearrangement of actinthrough RhoA as well as Golgi-plasma membrane transportduring tumor progression.

In conclusion, our data suggest that PI(4)P levels in the Golgiapparatus play a critical role in cancer progression by regu-lating the PI(4)P effector GOLPH3 (Supplementary Fig. S6G).In this study, we showed that changes in Golgi PI(4)P levelsand/or Golgi localization of GOLPH3 were likely to result inchanges in cell–cell adhesion and cell migration/invasion inbreast cancer cell lines. Furthermore, we showed that PI(4)P-

(Continued.) �, P < 0.0005. Scale bar, 100 mm. C, mock, GOLPH3 wt, or GOLPH3 R90L-expressing MDA-MB-231 cells were stained with an anti–b-catenin antibody and phalloidin. Scale bar, 20 mm. D, mock, GOLPH3 wt, or GOLPH3 R90L mutant-expressing MDA-MB-231 cells were seededonto Matrigel invasion chambers. Eighteen hours after seeding, the cells were fixed and stained with phalloidin. The number of invasive cells thatpassed 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 mm. E, mock or GOLPH3 wt-expressing MDA-MB-231 cells were transfected with the indicated siRNAs. Forty-eight hoursafter transfection, the cells were serum starved and seeded onto Matrigel invasion chambers. Eighteen hours after seeding, the cells were fixedand 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 mm.






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Figure 7. Binding of PI(4)P toGOLPH3 is required formetastasis in vivo. A, GFP-, GOLPH3wt-, orGOLPH3R90L-expressingMCF7 cellswere transfectedwiththe indicated siRNAs and then fixed and stained with anti-GM130 and the Alexa Fluor 647–labeled Fapp1 2 � PH domain. Scale bar, 20 mm. B, GolgiPI(4)P levels were quantified in GFP-, GOLPH3 wt-, or GOLPH3 R90L-expressing MCF7 cells. Significant increases were observed following GOLPH3 wtexpression. The data shown are the mean (SEM) values from more than 100 cells. �, P < 0.001. C, GFP-, GOLPH3 wt-, or GOLPH3 R90L-expressingMDA-MB-231 cells were transfected with the indicated siRNAs and then fixed and stained with anti-GM130 antibody and the Alexa Fluor 647–labeled Fapp12 � PH domain. Scale bar, 20 mm. D, Golgi PI(4)P levels were quantified in GFP-, GOLPH3 wt-, or GOLPH3 R90L-expressing MDA-MB-231 cells. Thedata shown are themean (SEM) values frommore than 100 cells. �,P

binding ability of GOLPH3 was essential for metastasis in vivo.Therefore, molecules that modulate the PI(4)P level in theGolgi apparatus, such as SAC1 and PI4KIIIb, may representintriguing anticancer therapeutic targets.

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

Authors' ContributionsConception and design: E. Tokuda, T. Itoh, T. TakenawaDevelopment of methodology: E. Tokuda, T. TakenawaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Tokuda, Y. Takeuchi, Y. Irino, M. FukumotoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. TokudaWriting, review, and/or revision of the manuscript: E. Tokuda, T. Itoh,J. Hasegawa, T. Ijuin, T. TakenawaStudy supervision: T. Itoh, T. Takenawa

AcknowledgmentsThe authors thank T. Oikawa (Department of Molecular Biology, Hokkaido

University Graduate School of Medicine) for the discussions and Y. Murataand T. Yanagita (Division of Molecular and Cellular Signaling, Kobe UniversityGraduate School of Medicine) for the technical advice.

Grant SupportThis study was supported by a grant-in-aid for Scientific Research (S) (grant

number 23227005 to T. Takenawa) and a grant-in-aid for Challenging Explor-atory Research (grant number 24650621 to E. Tokuda) from Japan Society forthe Promotion of Science.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received August 23, 2013; revised February 14, 2014; accepted March 3, 2014;published OnlineFirst April 4, 2014.

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Tokuda et al.




2014;74:3054-3066. Published OnlineFirst April 4, 2014.Cancer Res Emi Tokuda, Toshiki Itoh, Junya Hasegawa, et al. Cancer

Cell Adhesion and Invasive Cell Migration in Human Breast−CellPhosphatidylinositol 4-Phosphate in the Golgi Apparatus Regulates

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