The Lipid Kinase PI4KIIIb Is Highly Expressed in Breast ......2014/09/18 · Signal Transduction...

Signal Transduction

The Lipid Kinase PI4KIIIb Is Highly Expressed in BreastTumors and Activates Akt in Cooperation with Rab11a

Anne A. Morrow1, Mohsen Amir Alipour1, Dave Bridges2, Zemin Yao1, Alan R. Saltiel2, andJonathan M. Lee1

AbstractEmerging evidence now implicates phosphatidylinositol 4-kinases (PI4K), enzymes that generate PI(4)P from

phosphatidylinositol (PtdIns), in cancer. In this study, we investigate the role of PI4KIIIb, one of four mammalianPI4Ks, in breast cancer. Although PI4KIIIb protein levels are low in normal breast tissue, we find thatapproximately 20% of primary human breast tumors overexpress it. Expression of PI4KIIIb in breast carcinomacells leads to increased Akt activation, dependent on increased PI(3,4,5)P3 production. However, a kinase-inactiveversion of PI4KIIIb also led to increased Akt activation, and no changes in PI(4)P or PI(4,5)P2 lipid abundanceweredetected in the PI4KIIIb-overexpressing cells. This implies that PI4KIIIb regulates PI(3,4,5)P3 and Aktindependent of PI(4)P production. We find that the PI4KIIIb-binding protein, Rab11a, a small GTPase thatregulates endosomal recycling, is involved in PI4KIIIb-mediated activation of Akt, as RNAi depletion of Rab11aimpairs Akt activation. Furthermore, ectopic PI4KIIIb expression alters cellular Rab11a distribution and enhancesrecruitment of PI4KIIIb and Rab11a to recycling endosomes. This work suggests that PI4KIIIb affects PI3K/Aktsignaling through Rab11a and endosomal trafficking, independent of its lipid kinase activity. Thus, PI4KIIIb likelyplays a role in breast oncogenesis and that cooperation between Rab11a and PI4KIIIb represents a novel Aktactivation pathway. Mol Cancer Res; 1–17. �2014 AACR.

IntroductionMembrane phosphoinositide lipids, the phosphorylated

derivatives of phosphatidylinositol (PtdIns), and the kinases/phosphatases that regulate their generation have criticalroles in cancer. They do so by controlling apoptotic andproliferative pathways and stimulating cell migrationand cytoskeletal remodeling (1–5). Although the kinase andphosphatase regulators of PI(3,4,5)P3 have been well studiedin cancer, phosphatidylinositol 4-kinases (PI4K), proteinsthat generate PI(4)P from PtdIns, are now also emerging asimportant regulators of cancer (6). In particular, recent workpoints to a role for PI4KIIIb, one of four mammalian PI4Ks,in breast cancer. First, Curtis and colleagues (7) showed thatPI4KB, the 1q21 gene encoding PI4KIIIb, was frequentlyamplified in breast tumors. On the basis of a large-scaletranscriptional analysis of 2,000 tumors, the authors con-cluded that PIK4B is a novel breast cancer driver (7). Others

have also shown that 1q21 is highly amplified in breastcancers (8–10). At a functional level, we have previouslyshown that PI4KIIIb is likely to regulate breast epithelialmorphogenesis because ectopic PI4KIIIb expression dis-rupts in vitro three-dimensional acinar development inMCF10A cells (11). We have also identified PI4KIIIb asa downstream effector of eukaryotic elongation factor 1alpha 2 (eEF1A2), a transforming gene that is amplifiedand highly expressed in breast and ovarian cancer (12–15).Finally, PI4KIIIb expression has also been shown to inhibitapoptosis inMDA-MB 231 cells (16). Taken together, theseobservations suggest a likely role for PI4KIIIb in breastoncogenesis. However, the mechanism by which PI4KIIIbmight promote neoplasia is unclear.In this report, we implicate PI4KIIIb in the activation of

Akt in breast cancer cells. The Akt serine/threonine kinaseis a central regulator of multiple biologic processes, amongthem cell survival, proliferation, and growth (17, 18). Aktactivity is increased in multiple human malignancies, withaberrant activation reported in as many as 70% of breastcancers (19–23). Akt activity is regulated, in large part, byphosphatidylinositol 3-kinase (PI3K)–generated PI(3,4,5)P3 and PI(3,4)P2 phosphoinositide lipids (18, 24). PI(3,4,5)P3/PI(3,4)P2 recruit Akt to the plasma membrane throughits pleckstrin-homology (PH) domain, allowing phosphor-ylation at Thr308, within the activation loop of the protein,by 3-phosphoinositide-dependent protein kinase 1 (PDK1)and at Ser473, within the carboxyl-terminal tail, by themammalian target of rapamycin complex 2 (mTORC2)

1Department of Biochemistry, Microbiology and Immunology, University ofOttawa, Ottawa, Ontario, Canada. 2Life Sciences Institute, University ofMichigan, Ann Arbor, Michigan.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Jonathan M. Lee. Department of Biochemistry,Microbiology and Immunology, University of Ottawa, RGN 4256, 451Smyth Road, Ottawa, ON K1H 8M5, Canada. Phone: 613-562-5800, ext.8640; Fax: 613-562-5452; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-13-0604

�2014 American Association for Cancer Research.

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phosphorylates Akt (25–27). The tumor suppressors, phos-phatase and tensin homolog deleted on chromosome10 (PTEN), and inositol polyphosphate 4-phosphatase(INPP4) antagonize PI3K/Akt signaling by dephosphory-lating PI(3,4,5)P3 at the D3 position, and PI(3,4)P2 at theD4 position, respectively (28–30).In this report, we also show that approximately 20% of

primary human breast tumors exhibit increased PI4KIIIbprotein expression relative to normal breast tissue. We findthat ectopic PI4KIIIb expression leads to Akt activation in aPI(3,4,5)P3-dependent manner. Surprisingly, PI4KIIIb-mediated Akt activation is not dependent on PI4KIIIb lipidkinase activity, its interaction with the small GTPase,Rab11a seems to be critical. Rab11aa regulates slow endo-somal recycling, and has previously been shown to interactwith PI4KIIIb (31, 32). Our work suggests that PI4KIIIbhas an important role in breast cancer and that cooperationbetween Rab11a and PI4KIIIb represents a novel Aktactivation pathway.

Materials and MethodsCell cultureThe BT549 and MCF10A cell lines were purchased

from the American Type Culture Collection (ATCC).BT549 were grown in RPMI-1640 medium (Life Technol-ogies) supplemented with 10% FBS (Life Technologies),1 mmol/L sodium pyruvate (Life Technologies), 10 nmol/LHEPES buffer, and 0.023 IU/mL insulin (from bovinepancreas, # I-5500; Sigma). MCF10A cells were grown in5% horse serum (Life Technologies) supplemented with 20ng/mL epidermal growth factor, 10 mg/mL insulin, 0.5 mg/mL hydrocortisone, and 100 ng/mL cholera toxin. BT549cell lines stably expressing ectopic PI4KIIIb were generatedusing a pLXSN retroviral system as previously described(33). Full-length human wild-type (WT; MGC-1921;ATCC) and kinase-dead (KD; D656A) PI4KIIIb (gift fromA. Hausser, University of Stuttgart, Stuttgart, Germany)were cloned into the pLXSN vector in the EcoRI/XhoI andHpaI/XhoI sites, respectively, for retroviral generation.Infected cells were selected in 0.4 mg/mL G418 (BioShop).Cell lines were cultured at 37�C in humidified atmospherewith 5% CO2.

ImmunohistochemistryTumor tissue microarrays (TMA) were obtained from

US Biomax. For TMA staining, the slide was sequentiallydeparaffinized (2� xylenes, 2 � 100% EtOH, 1 � 95%EtOH, 1� 70% EtOH, and 1�H2O), boiled in 0.1 mol/Lsodium citrate (pH 6.0) for 10 minutes, and then allowed tocool in the citrate buffer for an additional 10 minutes. Slideswere then rinsed twice in PBS/0.2% Tween 20 and blockedin 10% FCS/1% BSA/PBS/0.2% Tween for 1 hour at roomtemperature in a humidified chamber. The slide was thenincubated overnight at 4�C with PI4KIIIb antibody (BDBiosciences) diluted at 1:200 in 1% BSA/PBS/0.2% Tween20. The primary antibody was then removed by three rinsesof 5 minutes each in PBS/0.2% Tween 20. Antibody

staining was detected using biotinylated VECTASTAINanti-mouse IgG antibody and either horseradish peroxidaseor alkaline phosphatase according to the manufacturer'sinstructions. Slides were counterstained using hematoxylin.Staining was visualized using a Zeiss Axio Imager.A1 micro-scope with images acquired using a Zeiss AxioCam MrRc5camera and AxioVision 4.6 software. Scoring was done usinga three-point visual system (low, moderate, and high) withrepresentative images of each shown. Counting was per-formed in triplicate with discrepancies resolved by a fourthcount.

siRNA and transfectionsPI4KIIIb-targeted siRNA sequence 1 is 50-GGAGGU-

GUUGGAGAAAGUCtt-30 (catalog number, AM51331;siRNA ID no., 283) and siRNA sequence 2 is 50-GCACU-GUGCCCAACUAUGAtt-30 (catalog number, AM51331;siRNA ID no., 184). The Rab11a-targeted siRNA sequenceused was 50-CAACAAUGUGGUUCCUAUUtt-30 (catalognumber, 442708; siRNA ID no., s16702). The Rab11b-targeted sequence is 50-CGGACGGACAGAAGCCCAA-CAAGCT-30. siRNAs and the negative control siRNA werepurchased from Life Technologies (Rab11a) or IntegratedDNA Technologies (Rab11b). siRNA transfections wereperformed in 1% serum containing growth media with 10nmol/L siRNA using Lipofectamine RNAiMAX (Invitro-gen, Life Technologies) as per the manufacturer's instruc-tions. Lysates were collected either 48 or 72 hours aftertransfection for PI4KIIIb- and Rab11-targeted siRNA trans-fections, respectively. Cells were transfected with Akt-PH-GFP, FAPP1-PH-GFP (gifts from T. Balla, NIH, Bethesda,MD), Rab11a-GFP (gift from J. Presley, McGill University,Montr�eal, QC, Canada), GALT-CFP, and PI4KIIIb-GFP(gift from A. Hausser, University of Stuttgart) using Lipo-fectamine LTX and Plus Reagents (Invitrogen, Life Tech-nologies) according to the manufacturer's instructions.

Adenoviral infectionCells were plated (5� 105 cells per 10-cm plate) 24 hours

before infection.On the day of infection, cells were placed in5 mL serum-free growth media with adenoviral particles at amultiplicity of infection (MOI) of 200. Cells were collectedfor Western blotting 24 hours after infection. The adeno-virus expressingWTPTENwas kindly provided byM.J. Lee(Wayne State University, Detroit, MI).

Western blottingCells were plated (2 � 105 cells per 6-cm plate) 48 hours

before lysate taking and serum depleted (placed in 1% serumcontaining growth media) 24 hours prior. Cells were lysedusing radioimmunoprecipitation assay (RIPA) buffer [50nmol/L Tris–Cl; pH 7.4, 1% Triton X-100, 1% sodiumdodecyl sulfate (SDS), 1 mmol/L ethylenediaminetetraaceticacid (EDTA); pH7.0, and 150mmol/LNaCl] supplementedwith protease and phosphatase inhibitor cocktail tablets(RocheDiagnostics). Protein concentrationswere determinedby Bradford protein assay (Bio-Rad). SDS sample buffer wasadded to 10mg of protein lysate, resolved by SDS-PAGE, and

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transferredontopolyvinylidenedifluoride (PVDF)membrane(Millipore). PI4KIIIb (BD Biosciences), pAkt, Akt, p-cRaf,cRaf (Cell Signaling Technology), Rab11a, Rab11b (Milli-pore), and b-actin (Sigma) antibodies were used according tothe manufacturer's instructions.

ImmunoprecipitationCells were lysed in 50 mmol/L Tris, pH 7.5, 150 mmol/L

NaCl, and 0.5% Triton X-100 supplemented with proteaseand phosphatase inhibitor cocktail tablets (Roche Diagnos-tics). Immunoprecipitation with PI4KIIIb (Millipore) wasperformedwith 5mg of antibody per 1mg/mL of lysate usingProtein A agarose beads (Millipore).

ImmunofluorescenceProtein immunostaining was performed on 1� 105 cells

plated in 6-well plates (Corning) containing glass cover-slips 24 hours later, cells were fixed with 3.7% formalde-hyde for 15 minutes and either permeabilized with 0.1%Triton X-100 for 10 minutes and blocked with 3% FBSfor 1 hour (PI4KIIIb/Giantin) or permeabilized andblocked in 0.5% saponin, 3% FBS for 1 hour [transferrinreceptor (TfR)]. Cells were then incubated with primaryantibodies overnight in 3% FBS. The following primaryantibodies were used as per the manufacturer's instruc-tions: PI4KIIIb (BD Biosciences), Giantin (Abcam), andTfR (Abcam). This was followed by 1-hour incubationwith Alexa Fluor 488– and Alexa Fluor 647–conjugatedsecondary antibodies in 3% FBS (1:200; MolecularProbes, Life Technologies) and a 20-minutes incubationwith Hoechst 33258 dye (Sigma) for DNA labeling(0.5 mg/mL). All slides were mounted using fluorescentmounting media (Dako Cytomation). Fixed cell imageswere acquired by sequential excitation using an OlympusFluoView FV1000 laser scanning confocal microscopewith an Olympus UPLSAPO 100X/1.40 oil objective.Images were collected and Pearson correlation coefficientquantification was performed using Olympus software(FV100, version 01.04a). Akt-PH-GFP reporter constructmembrane recruitment and Rab11a-GFP distributionquantification were performed using ImageJ (NIH). Brief-ly, for the Akt-PH-GFP reporter, the integrated density ofpixels for the GFP construct was measured at the cellperiphery (defined as the outer 20% area of the cell) anddivided by the total integrated density of pixels for thewhole cell. Rab11a-GFP integrated density was measuredat the Golgi, as defined by the area marked by GALT-CFPfluorescence, and converted to a proportion of the totalintegrated density of whole-cell Rab11a-GFP fluorescence.The proportion of extra-Golgi Rab11a-GFP was defined asthe difference between whole-cell and Golgi-area–integrat-ed density measurements, divided by whole-cell fluores-cence. Background was subtracted from all integrateddensity measurements. PI(4)P (Echelon Biosciences) lipidantibody staining was performed on 3 � 104 cells plated24 hours before in 4-well plates (BD Biosciences) accord-ing to the Golgi and plasma membrane–specific immu-nostaining protocols as previously described (34).

Live cell imagingFor FAPP1-PH-GFP imaging in Pik93-treated cells, 1 �

105 BT549 cells were plated on uncoated 35-mm glassbottom plates (MatTek P35G-0-20-C) 24 hours beforetransfection. Cells were transfected with FAPP1-PH-GFPusing Lipofectamine LTX as per the manufacturer's instruc-tions (Life Technologies). Twenty-four hours after transfec-tion, cells were treated with either DMSO or 250 nmol/LPIK93 in DMSO. Cell images were acquired by an ORCA-R2 Hammamatsu CCD camera in an Olympus VivaViewFLmicroscope using the 20� objective and an X-cite eXactemercury arc illuminationwith aGFPfilter cube. Imageswereprocessed in VivaView FL software. For Rab11a/GALTimaging, Vector control, WT-PI4KIIIb–expressing, andKD-PI4KIIIb–expressing BT549 cells were plated on Mat-Tek coverslips (MatTek Corporation) 24 hours beforetransfection. Cells were cotransfected with GFP-Rab11aand CFP-Galt, using FuGENE HP extreme as per themanufacturer's instructions (Roche Diagnostics). Time-lapse images were acquired 24 hours after transfection usingan inverted confocal laser scanning microscope, LSM 510(Zeiss), equipped with a stage heated to 37�C in a chambercontaining 5%CO2 (v/v)/95% (v/v) air. CFP andGFPwereexcited using 417 to 442 nm and 486 to 498 nm band-passfilters, respectively. Fluorescence emission spectra were col-lected using 455 to 475 nm (CFP) and 510 to 545 nm (GFP)band-pass filters. Images were acquired at a rate of 1/s.Captured images were converted to animation and exportedto QuickTime movie using the ImageJ software (NIHsoftware).

Competitive ELISA and HPLC analysisWedetermined the relative amount of PI(3,4,5)P3 present

in the stable cell lines using a competitive PIP3 Mass ELISAkit (Echelon Biosciences). Lipids were extracted and purifiedPIP3-containing suspensions were treated according to themanufacturer's instructions. The colorimetric signal wasmeasured at 450 nm using the Tecan Spectra plate reader.For high-performance liquid chromatography (HPLC), cellswere labeled in inositol-free media for 48 hours. Cells wereextracted and separated as previously described (35). Lipidlevels were normalized to total phosphatidylinositol levels.There were no differences in the total phosphatidylinositolbetween the groups.

ResultsHigh expression of PI4KIIIb protein in primary humanbreast tumorsTo determine whether PI4KIIIb expression might be

altered in breast cancer, we analyzed PI4KIIIb protein ex-pression in 460 primary human breast tumors [371 invasiveductal carcinomas (IDC) and 89medullary carcinomas]. Forthis purpose, we used a commercially available PI4KIIIbantibody and commercially available human breast tumortissue arrays. The antibody used for immunohistochemistrywas verified for PI4KIIIb-binding specificity, as siRNAknockdown of PI4KIIIb in human breast cancer cells

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abolished antibody staining of the protein (SupplementaryFig. S1). Staining in each tissue section was categorized aslow, moderate, or high. Representative photographs of thethree categories are shown in Fig. 1. As shown in Table 1,PI4KIIIb protein expression is detected at a low level in allnormal breast tissue samples (73 of 73). The majority ofprimary human breast tumors assayed also express PI4KIIIbprotein at this low, but detectable, level (338 of 460).

However, 27% of primary tumors assayed (81 of 371 IDCand 41 of 89 medullary carcinomas) expressed higher thannormal levels of the protein, that is, either moderate or high.These results are statistically significant, yielding a P value of0.0001 (Fisher exact test). There were no differences in theage of patients at diagnosis: 49 � 11 in both the PI4KIIIbnormal and high groups. However, in IDC, there was asignificantly increased probability of increased PI4KIIIbprotein expression in lower-grade stage I tumors (P ¼0.008, Student t test). In the PI4KIIIb-high group, 28%(21 of 75) of IDCs were stage I compared with only 10% (21of 220) in the normal population. In the high group, 61%(46 of 75) were stage II compared with 78% (172 of 220).Eleven percent (8 of 75) of the PI4KIIIb-high group werestage III, similar to the 12% (27 of 220) in the normal group.The increased expression of PI4KIIIb protein in approxi-mately one quarter of the primary tumors is consistent withthe idea that high PI4KIIIb expression has a functional rolein breast oncogenesis.

PI4KIIIb activates AktTo investigate a potential role of PI4KIIIb expression in

breast cancer cell signaling, we generated BT549 humanbreast ductal carcinoma andMCF10A immortalized humanbreast epithelial cells that ectopically express PI4KIIIb(Fig. 2A). In two independently generated BT549 andMCF10A cell lines with enhanced PI4KIIIb expression, wedetected an approximate 2-fold increase in Akt phosphor-ylation at Thr308 and at Ser473 as compared with the vectorcontrol (Fig. 2A and B). This increase in activating Aktphosphorylation was detected in the PI4KIIIb-overexpres-sing BT549 cell lines in serum-free, low serum, and fullserum media growth conditions (Supplementary Fig. S2).To determine whether this increase in Akt phosphorylationincreases intracellular activity of the protein, we probed thecell lines for the level of phosphorylation of c-Raf, a knownAkt kinase target that has been shown to shift human breastcancer cells from cell-cycle arrest to proliferation when

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Figure 1. PI4KIIIb expression in primary breast tumors in a TMA.Representative images of PI4KIIIb immunostaining in normal breasttissue and breast tumors classified as showing low, moderate, or highexpression of PI4KIIIb. Scale bars, 100 mm.

Table 1. Evaluation of PI4KIIIb expression in breast cancer

Breast tissue PI4KIIIb expressionNumber ofsamples

Percentage ofsamples

Normal (n ¼ 73) Low 73 100Moderate, high 0 0

All tumor (n ¼ 460) Low 338 73Moderate, high 122 27

IDC (n ¼ 371) Low 290 78Moderate 66 18High 15 4

Medullary carcinoma (n ¼ 89) Low 48 54Moderate 36 40High 5 6

NOTE: PI4KIIIb protein expression was categorized as either low, moderate, or high for the microarray tissue samples stained. The nvalue indicates the number of each tissue type assayed.

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Figure 2. PI4KIIIb expression regulates Akt activity. A,Western blot analysis showing levels of phosphorylation of Akt at Ser473 and at Thr308 in two independentBT549andMCF10Acell lineswith increasedPI4KIIIbproteinexpressionascomparedwithparental andvector controls.B,quantificationof the relative increase inAkt phosphorylation over total Akt levels in the PI4KIIIb-expressing cell lines as compared with the vector control. (Continued on the following page.)






phosphorylated at Ser259 (Fig. 2C; ref. 36). A 1.8-foldincrease in c-Raf phosphorylation at Ser259 was detected inthe PI4KIIIb-overexpressing BT549 cell lines relative to thevector control, indicating a higher level of Akt kinase activityfollowing PI4KIIIb overexpression (Fig. 2D). Concordantwith an increase in activation of the proproliferative kinaseAkt, as well as increased c-Raf phosphorylation, we foundthat BT549 cells overexpressing PI4KIIIb proliferate at afaster rate than their parental or vector control counterparts.Parental and empty vector control cells had doubling timesof 38.8 and 37.3 hours, respectively, whereas PI4KIIIb-overexpressing cells had a doubling time of 17.2 hours (Fig.2E). This indicates that elevated expression of PI4KIIIbincreases the in vitro proliferative capacity of breast cancercells. We then determined whether a loss of PI4KIIIbprotein expression might lead to a decrease in Akt activation.To this end, BT549 parental cells were treated with twodistinct siRNA sequences targeted to PI4KIIIb (Fig. 2F).Concomitant with a decrease in PI4KIIIb protein levels, weobserved a consistent and significant decrease (�40%–50%)in Akt phosphorylation at Ser473 and Thr308 (Fig. 2G).These results indicate that PI4KIIIb expression can regulateAkt activity.

PI4KIIIb activates Akt via PI(3,4,5)P3 signalingBecause Akt activation is dependent, in large measure, on

PI(3,4,5)P3 levels, we determined the effect of PI4KIIIbexpression on the overall cellular abundance of PI(3,4,5)P3using a competitive ELISA. As shown in Fig. 3A, weobserved a significant increase (�60%) in PI(3,4,5)P3abundance in the PI4KIIIb-overexpressing cell lines com-pared with the vector control. We then visualized theintracellular localization of PI(3,4,5)P3 using a fluorescentreporter composed of the PH domain of Akt fused to GFP(Akt-PH-GFP), which binds to PI(3,4,5)P3 and PI(3,4)P2(37). Cell lines with increased PI4IIIb expression showedvisibly increased plasma membrane recruitment of thereporter construct, indicating higher levels of PI(3,4,5)P3/PI(3,4)P2 at the cell membrane, where Akt activation isdirected by these lipid species (Fig. 3B, top).When cells weretransfected with GFP alone, no enhanced fluorescentrecruitment could be observed at the cell membrane for thePI4KIIIb-overexpressing cells as compared the vector con-trol (Fig. 3B, bottom). To quantitate this reporter recruit-ment to the plasma membrane, we measured the percentageof the total fluorescent signal that was localized to the cellperiphery (Fig. 3C). A significant increase (P< 0.01, Student

t test) in fluorescent intensity at the cell periphery wasdetected for the GFP-Akt-PH reporter in the vector controland PI4KIIIb-overexpressing cells as compared with eachcell line transfected with GFP alone (Fig. 3C). This indicatesthat the reporter is enriched in the cell periphery. Impor-tantly, in PI4KIIIb lines, 31% of total GFP-Akt-PH fluo-rescence was detected at the cell membrane compared with19% of the same reporter in the vector control line. In GFP-expressing controls in both cell lines, approximately 14% oftotal GFP fluorescence was detected at the cell membrane inboth cell lines. This significant increase (�10%; P < 0.01,Student t test) indicates that PI4KIIIb expression increasesabundance of PI(3,4,5)P3/PI(3,4)P2 at the cell membrane.BT549 cells are PTEN-null, as they contain a truncation

mutation in PTEN, which leads to the rapid degradation ofthe truncated protein (38, 39). Therefore, we next wanted todetermine the effect of re-introducing WT PTEN on Aktactivation, in the cell lines with enhanced PI4KIIIb expres-sion. Expression ofWTPTENby adenoviral infection led toa decrease in Akt phosphorylation in both the BT549 cellswith enhanced PI4KIIIb expression and the vector controlcells (Fig. 3D). PTEN expression led to a decrease in Aktphosphorylation by approximately 50% in both the vectorcontrol and PI4KIIIb-overexpressing cells (Fig. 3E). AsPTEN is responsible for dephosphorylating PI(3,4,5)P3 intoPI(4,5)P2, this result suggests that the increase in Aktactivation in the PI4KIIIb-expressing cells is dependent oncellular PI(3,4,5)P3 lipid levels.

PI4KIIIb-mediated Akt activation is independent of itskinase functionPI(3,4,5)P3 is principally generated by the sequential

phosphorylation of PtdIns, at positions D4, D5, and D3respectively, by PI4K, PI(4)P 5-kinase, and PI3K. Wetherefore hypothesized that enhanced PI4KIIIb expressionmight increase the cellular abundance of PI(3,4,5)P3 byincreasing intracellular concentrations of its precursors, PI(4)P and PI(4,5)P2. To test this idea, we measured the PIlevels in the cell lines stably ectopically expressing PI4KIIIbusing HPLC separation of 3H-labeled inositol lipids. Sur-prisingly, no significant differences in the relative abundanceof PI(4)P or PI(4,5)P2 lipids were detected in the PI4KIIIb-overexpressing cell lines as compared with the vector orparental control cell lines (Fig. 4A and B). Thus, enhancedPI4KIIIb expression seems to increase PI(3,4,5)P3 levelswithout having an impact on total cellular PI(4)P or PI(4,5)P2 abundance. To determine whether ectopic expression of

(Continued.) Data, mean � SE of the mean of three independent trials; statistical significance: �, P < 0.05 (Student t test). C, Western blot analysis showingphosphorylation of c-Raf at Ser259 in two independently derived BT549 cell lines with increased PI4KIIIb protein expression as compared withparental and vector controls. D, quantification of the relative increase in c-Raf phosphorylation at S259 over total c-Raf levels in the PI4KIIIb-expressing celllines as compared with the vector control. Data, mean � SE of the mean of two independent trials for two independent cell lines (n ¼ 4); statisticalsignificance: �, P < 0.05 (Student t test). E, PI4KIIIb-overexpressing BT549 cells (closed symbols) proliferate at a faster rate than the parental and vectorcontrol cells (open symbols). Each point represents the average fold growth of cells at a given time point over the number of cells measured at the initial timepoint (0 hours) for three independent experiments for each cell line � SD. F, Western blot analysis showing Akt phosphorylation at Ser473 and Thr308 inBT549 parental cells treated with two distinct siRNA sequences targeted to PI4KIIIb as compared with cells treated with the negative control siRNA. G,quantification of relative decrease in Akt phosphorylation in cells treated with siRNA targeted to PI4KIIIb as compared with cells treated with negativecontrol siRNA. Data, mean � SE of the mean of three independent experiments; statistical significance: �, P < 0.05 (Student t test).

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Figure 3. PI4KIIIb expression activatesAkt via PI(3,4,5)P3 signaling. A, PI(3,4,5)P3 abundance is shown as a concentration of pmol per 106 cells and is themean� SEof themeanof duplicate samples from three independent experiments in thecase of the vector control and from two independent experiments for eachof theBT549PI4KIIIb-overexpressing cell lines. Increased PI(3,4,5)P3 production in PI4KIIIb-overexpressing cell lines as compared with the empty vector control is statisticallysignificant (�, P < 0.05, Student t test). B, confocal images of BT549 vector control and PI4KIIIb-overexpressing cells transfected with the PI(3,4,5)P3/PI(3,4)P2reporter, Akt-PH-GFP (top) or GFP (bottom). Scale bars, 10 mm. C, quantification of the plasma membrane recruitment of the Akt-PH-GFP reporter in the vectorand PI4KIIIb-overexpressing cells presented as the mean � SE of the mean for 20 cells from four independent experiments. GFP alone was used as a control.Statistical significance (��, P < 0.01, Student t test) is indicated. D, Western blot analysis showing p-Akt levels in vector control and PI4KIIIb-expressing cellsinfectedwith adenovirus expressingWTPTEN as comparedwith untreated cells. E, quantification of the impact of PTEN expression on pAkt levels in vector controland PI4KIIIb-expressing cells. Data, mean � SE of the mean of three independent Western blot analyses; statistical significance: ��, P < 0.01 (Student t test).






PI4KIIIb might lead to an altered distribution of PI(4)P,cells were transfected with the PI(4)P-specific lipid reporterconstruct, GFP-FAPP1-PH (Fig. 4C).No changes in report-er construct localization were observed between thePI4KIIIb-overexpressing cells as compared with vector con-trols, indicating that ectopic PI4KIIIb expression does notlead to altered PI(4)P lipid distribution. These results weresupported by lipid antibody immunostaining performedusing organelle-specific staining protocols (34). Again theredo not appear to be any gross changes in levels or distributionpatterns of PI(4)P at the Golgi or plasma membrane inBT549 cells transfected with GFP-PI4KIIIb as comparedwith untransfected cells in the same field (SupplementaryFig. S3). Together, these results suggest that ectopicPI4KIIIb expression does not lead to a substantive changein PI(4)P lipid abundance or localization in BT549 cells.In light of these findings, we then asked whether or not the

kinase activity of PI4KIIIb was required for Akt activation.To this end, we treated the BT549 cells stably expressingectopic PI4KIIIb with a drug that inhibits PI4KIIIb, Pik93.

This drug inhibits only the PI4KIIIb isoform (and notPI4KIIIa) at a molar concentration of 250 nmol/L, thoughat this molar range Pik93 also inhibits PI3Kg (40–42). Treat-ment of cells with Pik93 had no effect on PI4KIIIb-mediatedAkt activation, as the same increase in Akt phosphorylationis observed in PI4KIIIb-expressing cells as compared withvector control cells, when treated with DMSO (vehiclecontrol) or Pik93 (Fig. 4D). To ensure that Pik93 did infact inhibit PI4KIIIb in our cell system, we followed thedistribution of the PI(4)P lipid reporter, FAPP1-PH-GFP, inBT549 parental cells treated with either DMSO or Pik93(Supplementary Fig. S4). Pik93 inhibition of PI4KIIIbhas previously been shown to lead to a loss of FAPP1-PH-GFPGolgi localization (43).We found that treatment of cellswith 250 nmol/L of Pik93 led to loss of Golgi reporterfluorescence by 30 minutes (Supplementary Fig. S4). Incontrast, FAPP1-PH-GFP remained primarily Golgi local-ized in DMSO-treated cells. This reflects the loss of PI(4)Plipid at the Golgi due to Pik93 inhibition of PI4KIIIb. Astreatment with Pik93 had no effect on PI4KIIIb-mediated

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Akt activation, this indicates that PI4KIIIb catalytic activity isnot required for its role in Akt activation.To further investigate the catalytic role of PI4KIIIb in Akt

activation, we next generated BT549 cells ectopically expres-sing a KD PI4KIIIb. KD-PI4KIIIb contains a substitutionof an aspartic acid residue for alanine at position 656,rendering it catalytically inactive (44). Previous work hasdemonstrated that the PI4K activity of KD-PI4KIIIb is

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PI4KIIIb and TfR was also detected in the WT-PI4KIIIb–overexpressing and KD-PI4KIIIb–expressing cells, as com-pared with the vector control cells (Supplementary Fig.S7C). Taken together, these data suggest that PI4KIIIb isrecruited to Rab11a containing recycling endosomes in cellswith increased PI4KIIIb expression, either WT or KD.We then examined the localization of Rab11a in the

expressing cell lines. Cells were transfected with Rab11a-GFP and Galt-CFP, to mark the Golgi. Rab11a is reportedlocalized to the trans Golgi network (TGN), early andrecycling endosomes (31). Figure 7A presents still imagestaken during live cell imaging, and show that Rab11adistribution is altered in cells ectopically expressing eitherWT- or KD-PI4KIIIb. In vector control cells, Rab11a isdetected primarily in the Golgi, in WT-PI4KIIIb–over-expressing and KD-PI4KIIIb–expressing cells, Rab11ashows a broader punctate distribution in addition to Golgilocalization. Rab11a Golgi versus extra-Golgi distributionwas quantified and showed a significant shift from Golgi toextra-Golgi localization in WT-PI4KIIIb–overexpressingand KD-PI4KIIIb–expressing cells, as compared with vectorcontrol cells (P< 0.05, Student t test; Fig. 7B). This indicatesthat with eitherWTor aKDmutant, enhanced expression ofPI4KIIIb relocalizes Rab11a from the Golgi to a broaderpunctate cellular distribution, suggesting that enhancedPI4KIIIb expression shifts Rab11a localization from theGolgi onto endosomes.To determine whether the PI4KIIIb–Rab11a interaction

might be involved in PI4KIIIb-mediated Akt activation, thevector control, WT-expressing, and KD-expressing cell lineswere treated with siRNA against Rab11a. Depletion ofRab11a protein led to a consistent and significant decrease(�50%) in Akt phosphorylation in both cell lines (Fig. 8Aand B). In these experiments, Rab11a knockdown wasapproximately 60%. Similar results were found in theMCF10A cells, where approximately 70% knockdown ofRab11a leads to a significant, approximately 60%, decreasein Akt activity (Fig. 8B). This effect is specific for Rab11abecause knockdown of the related Rab11b protein (�60%knockdown) had no discernible effect on Akt phosphoryla-tion (Fig. 8C). Furthermore, Rab11a knockdown (�60%)had no effect on the overall abundance of PI(3,4,5)P3 lipidin BT549 cells (Fig. 8D). Taken together, this work isconsistent with the idea that Rab11a is important in medi-ating the activation of Akt signaling by PI4KIIIb.To further investigate the importance of Rab11a in

breast cancer, we next determined whether there is

evidence of Rab11a overexpression during breast tumori-genesis. To this end, we measured Rab11a protein expres-sion in human breast tumor samples. As shown in Fig. 8E,Rab11a is expressed at a moderate level in 100% (18 of18) of normal breast epithelia and 97% (156 of 161)of infiltrating ductal carcinomas. The absence of breasttumors highly expressing Rab11a indicates that anyinvolvement of Rab11a in oncogenesis is likely throughexpression-independent mechanisms.

DiscussionA role for PI4Ks in cancer is now emerging (6). For

example, PI4KIIa has been reported highly expressed inmalignant melanoma, fibrosarcoma, bladder, thyroid, andbreast cancer and its expression has been shown to promotetumor angiogenesis (46). In addition, high levels ofPI4KIIIa expression are also associated with invasive andmetastatic pancreatic cancer development (47). The recentfinding that PI4KIIIb is genetically amplified in a subset ofprimary human breast tumors points to a specific role forPI4KIIIb in breast cancer (7). We have also previouslyimplicated PI4KIIIb in breast oncogenesis as the protein isbound and activated by the breast cancer oncogeneeEF1A2, and its high expression disrupts in vitro three-dimensional mammary epithelial morphogenesis (11–15).Supporting a role for PI4KIIIb in breast cancer develop-ment, we show here that PI4KIIIb protein levels areincreased in approximately 20% of primary human breasttumors assayed, consistent with the idea that highPI4KIIIb expression plays a role in breast cancer neoplasticdevelopment. Given the high, but not absolute, concor-dance between protein expression of a gene and its ampli-fication (48, 49), it is likely that the breast tumors highlyexpressing PI4KIIIb also have PI4KB gene amplifications.However, because we have not measured gene copy num-ber in our samples, it is possible that enhanced PI4KIIIbexpression is being driven through other mechanisms otherthan gene amplification.At the functional level, we found that enhanced PI4KIIIb

expression increases Akt activation in BT549 cells and theloss of PI4KIIIb by siRNA silencing decreases Akt activity.This suggests that endogenous PI4KIIIb is involved inregulating Akt activity and that increased PI4KIIIb expres-sion, as would occur in tumor cells, further augments Aktsignaling. We also show that this PI4KIIIb-dependentincrease in Akt activity itself is dependent on increased PI

Figure 5. Ectopic expression of a catalytically inactive (D656A) PI4KIIIb leads to Akt activation. A, Western blot analysis showing increased levels ofphosphorylation of Akt at Thr308 and at Ser473 in WT and two independently derived KD-PI4KIIIb–expressing BT549 cell lines as compared with the vectorcontrol. B, relative increase inAkt phosphorylation over total Akt levels is shown in theWT-PI4KIIIbandKD-PI4KIIIb–expressing cell lines as comparedwith thevector control. Data, mean�SE of themean of three independent experiments; statistical significance: �,P < 0.05 (Student t test). C, KD-PI4KIIIb–expressingBT549 cells (closed symbol-triangle-purple line) proliferate at a faster rate than vector control cells (open symbol) and at a comparable rate as WT-PI4KIIIb–expressing cells (closed symbol-diamond-blue line). D, confocal images of PI4KIIIb localization (green) in vector control, WT- PI4KIIIb–overexpressing, and KD-PI4KIIIb–expressing cells. Golgi, as identified by the cis-medial Golgi marker Giantin, is seen in red and DNA is blue. Scale bars,10 mm. E, confocal images of BT549 vector control and KD-PI4KIIIb–expressing cells transfected with the PI(3,4,5)P3/PI(3,4)P2 reporter, Akt-PH-GFP. Scalebars, 10 mm. F, quantification of the plasma membrane recruitment of the Akt-PH-GFP reporter in the vector control and KD-PI4KIIIb–expressing cellspresented as the mean � SE of the mean for 20 cells from four independent experiments. Statistical significance (��, P < 0.01, Student t test) is indicated.






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Figure 6. PI4KIIIb interacts with Rab11 in vector control, WT-PI4KIIIb–overexpressing, and KD-PI4KIIIb–expressing cells. A, coimmunoprecipitation(Co-IP) was performed on whole-cell lysates of vector control, WT-PI4KIIIb–expressing, and KD-PI4KIIIb–expressing BT549 cells. Each lanecontains 10 mg of total protein. coimmunoprecipitation was performed using a PI4KIIIb-specific antibody. Agarose beads alone were used as anegative control. Interacting proteins were identified by Western blot analysis using PI4KIIIb and Rab11a antibodies. B, top, confocal imagesof Rab11a (green) and PI4KIIIb (magenta) in the vector control, WT-PI4KIIIb–expressing, and KD-PI4KIIIb–expressing BT549 cell lines. Scale bars,5 mm. Bottom, higher magnification of yellow inset in the merge panel marking Rab11a vesicles (white arrow). Scale bars, 1 mm. C, top, confocalimages of PI4KIIIb (green) and the recycling endosome marker, TfR (magenta), in the vector control, WT-PI4KIIIb–overexpressing, and KD-PI4KIIIb–expressing BT549 cell lines. Scale bars, 5 mm. Bottom, higher magnification of yellow inset in the merge panel with TfR vesicles marked (whitearrow). Scale bars, 1 mm.

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(3,4,5)P3 production. Although PI3K-independent modesof Akt activation have been reported (50), our data supportthe hypothesis that PI4KIIIb expression affects Akt activa-tion through the canonical PI3Kpathway.We also show thatPI4KIIIb-mediated Akt activation is lost upon expression ofPTEN, the phosphatase that antagonizes PI3K signaling.This suggests that PI4KIIIb expression may affect Aktactivation only in a PTEN-null background. A loss of PTENexpression has been reported in 30% to 50% of breastcancers, with PTEN loss correlating with lymph nodemetastasis and disease-related death (51, 52). It is possiblethat a combination of enhanced PI4KIIIb expression and

PTEN deficiency may contribute to the aggressive nature ofPTEN-null breast tumors.We found that the enhanced PI(3,4,5)P3 production and

Akt activation due to increased PI4KIIIb expression isunlikely to be driven by changes in abundance or intracel-lular localization of PI(4)P and PI(4,5)P2. No changes in PI(4)P and PI(4,5)P2 lipid abundance were detected in cellsoverexpressing PI4KIIIb, nor were we able to detect sub-stantial changes in cellular lipid distribution using immu-nofluorescence. This was a surprise to us, though it remainsformally possible that a higher rate of PI4P generationoccurs, but only in a local and transitory manner. Small

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Morrow et al.





and/or compartmentalized changes in PI(4)P lipid compo-sition may not have been detectable by HPLC analysis ordramatic enough to be observable by reporter constructimaging or lipid immunostaining. However, the fact thatthe PI4KIIIb inhibitor, Pik93, had no effect on PI4KIIIb-mediated Akt activation and the ability of a catalyticallyinactive PI4KIIIb protein to activate Akt rule out a necessaryrole for PI(4)P in PI4KIIIb-dependent Akt activation.Our data suggest that PI4KIIIb activates Akt in con-

junction with Rab11a. We demonstrate that in BT549cells, Rab11a interacts with PI4KIIIb in the vector con-trol, WT-PI4KIIIb–overexpressing, and KD-PI4KIIIb–expressing cell lines. Moreover, downregulation of Rab11a(using RNAi) inhibits Akt activation. Although it ispossible that PI4KIIIb and Rab11a function in parallelpathways of Akt activity, we favor the hypothesis thatPI4KIIIb activates Akt in concert with Rab11a at theendosomal membranes. Consistent with this idea,PI4KIIIb was found to colocalize more strongly with therecycling endosomal marker, TfR, in both the WT-over-expressing and KD-PI4KIIIb–expressing cell lines. Theseresults suggest that in the overexpressing cell lines,PI4KIIIb is recruited to recycling endosomes, as theytraffic through the TGN. Coupled to our observationthat the distribution of Rab11a is altered (showing abroader punctate cellular distribution) in the WT-PI4KIIIb–overexpressing and KD-PI4KIIIb–expressingcells, suggests that enhanced expression of PI4KIIIb leadsto greater endosomal recruitment of Rab11a in BT549cells.In Drosophila, the PI4KIIIb homolog, four-wheel drive

(Fwd), has been shown to regulate Rab11a recruitmentand trafficking events. More specifically, Fwd is requiredfor Rab11a localization to secretory granules at the mid-zone of dividing spermatocyte cells, allowing successfulcompletion of cytokinesis (45). Of interest to our study, anoncatalytic role for Fwd in Rab11a regulation was deter-mined as expression of a KD mutant of Fwd, which alsobinds to Rab11, partially rescues male Drosophila sper-matocyte cytokinesis defects in fwd-null flies. Therefore,there is a conserved role for Rab11a as a downstreameffector of PI4KIIIb in the regulation of cell trafficking.We propose that PI4KIIIb-dependent recruitment of

Rab11a could enhance PI3K and Akt signaling on endo-somal vesicles exiting the TGN. Endosomal membranesare now recognized as sites of signal transduction. Anumber of receptor tyrosine kinases (RTK) have beendemonstrated to signal from endosomes (53). For example,in adipocytes, insulin-stimulated PI3K activation via phos-

phorylation of insulin receptor substrate (IRS-1) occurspreferentially in internal membranes rather than at theplasma membrane (54). Endosome-specific signaling ofEGFR has also been shown to activate PI3K/Akt signaling(55, 56). In addition, the endosomal proteins EEA1 andAppl1 (adaptor protein, phosphotyrosine interaction, PHdomain, and leucine zipper containing 1) serve as scaf-folding proteins that promote endosomal Akt recruitment,phosphorylation, and activation (57, 58). Garcia-Regaladoand colleagues (59) showed that the Lipophosphatidicacid-stimulated interaction between Rab11a and the het-erotrimeric G protein subunit Gbg on endosomes con-tributes to the recruitment and activation of PI3K and Akton these endosomal compartments. In addition, Sato andcolleagues (60) demonstrated that PI(3,4,5)P3 was rapidlyproduced at the plasma membrane in response to platelet-derived growth factor (PDGR), followed by accumulationin endomembranes of Chinese hamster ovary (CHO) cells.They further showed that PI(3,4,5)P3 was produced in situin endomembranes, a process stimulated by RTK endo-cytosis. Together, this research demonstrates an importantrole for endosome-localized PI3K/Akt activation. Webelieve that the interaction between PI4KIIIb and Rab11aat the TGN and on endosomal membranes is likelyregulating Akt activation on endosomes themselves.Because we have not observed any alterations in PI(3,4,5)P3 abundance following Rab11a knockdown, wespeculate that Rab11a may serve as an endosomal scaf-folding nexus for proteins that activate and phosphorylateAkt directly and that increased expression of PI4KIIIbincreases the number of these scaffolding complexes. RabGTPases have been previously linked to breast canceroncogenesis in their role as central regulators of celltrafficking (61–63).In conclusion, we find that PI4KIIIb expression is

increased in a subset of breast tumors, and that enhanc-ed PI4KIIIb expression in BT549 breast cancer cellsactivates Akt signaling. Enhanced PI4KIIIb expressionleads to PI3K/Akt activation independent of its kinasefunction, but likely via its interaction with the endo-somal trafficking protein Rab11a. This study reveals anovel role for PI4KIIIb expression in breast cancerpathogenesis.

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

DisclaimerThe funders had no role in study design, data collection or analysis, decision to

publish, or preparation of the article.

(Continued.) B, left, siRNA depletion of Rab11a decreases AKT activation in WT-PI4KIIIb–overexpressing MCF10A cells as compared with cells treatedwith the negative control siRNA. Right, relative decrease in Akt phosphorylation in cells treated with siRNA targeted to Rab11a as comparedwith cells treated with negative control siRNA. Data, mean � SE of the mean of three independent trials; statistical significance: �, P < 0.05 (Studentt test). C, left, siRNA depletion of Rab11b has no effect on AKT activation in WT-PI4KIIIb–overexpressing BT549 cells as compared with cellstreated with the negative control siRNA. Right, relative decrease in Akt phosphorylation in presented as mean � SE of the mean of three independenttrials. D, (A) PI(3,4,5)P3 abundance is shown as a concentration of pmol per 10

6 cells and is the mean � SE of the mean of two independent experimentsfor BT549 PI4KIIIb-overexpressing BT549 cell lines with reduced Rab11a expression. E, expression of Rab11a protein in normal human breast samplesand infiltrating ductal carcinomas.






Authors' ContributionsConception and design: A.A. Morrow, A.R. Saltiel, J.M. LeeDevelopment of methodology: A.A. Morrow, J.M. LeeAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): A.A. Morrow, M.A. Alipour, D. Bridges, J.M. LeeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): A.A. Morrow, M.A. Alipour, D. Bridges, J.M. LeeWriting, review, and/or revision of the manuscript: A.A. Morrow, Z. Yao, J.M. LeeAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): A.A. MorrowStudy supervision: Z. Yao, J.M. Lee

AcknowledgmentsThe authors thank Sandy Szeto and Dixie Pinke for helpful discussion and critical

reading of this article. The authors also thankTamas Balla, AngelikaHausser, and JohnPresley for the generous donation of plasmid constructs; Menq-jer Lee for kindly

providing the PTEN adenoviral particles used in this study; and Dr. Heidi McBrideand her research group for use of and help with confocal microscopy.

Grant SupportThis work was supported by operating funds from the Cancer Research Society

and the Canadian Breast Cancer Foundation (to J.M. Lee), a doctoral fellowshipfrom the Canadian Institutes of Health Research (to A.A. Morrow), and NIHDK61618 (to A.R. Saltiel).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received November 19, 2013; revised May 21, 2014; accepted June 5, 2014;published OnlineFirst June 24, 2014.

References1. Kolsch V, Charest PG, Firtel RA. The regulation of cell motility and

chemotaxis by phospholipid signaling. J Cell Sci 2008;121:551–9.2. Ling K, Schill NJ, Wagoner MP, Sun Y, Anderson RA. Movin' on up:

the role of PtdIns(4,5)P(2) in cell migration. Trends Cell Biol 2006;16:276–84.

3. Funamoto S, Meili R, Lee S, Parry L, Firtel RA. Spatial and temporalregulation of 3-phosphoinositides by PI 3-kinase and PTEN mediateschemotaxis. Cell 2002;109:611–23.

4. Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations ona theme. Oncogene 2008;27:5497–510.

5. Bunney TD, Katan M. Phosphoinositide signalling in cancer: beyondPI3K and PTEN. Nat Rev Cancer 2010;10:342–52.

6. Waugh MG. Phosphatidylinositol 4-kinases, phosphatidylinositol 4-phosphate and cancer. Cancer Lett 2012;325:125–31.

7. Curtis C, ShahSP,Chin SF, Turashvili G, RuedaOM,DunningMJ, et al.The genomic and transcriptomic architecture of 2,000 breast tumoursreveals novel subgroups. Nature 2012;486:346–52.

8. TirkkonenM, TannerM, KarhuR, Kallioniemi A, Isola J, Kallioniemi OP.Molecular cytogenetics of primary breast cancer by CGH. GenesChromosomes Cancer 1998;21:177–84.

9. Mesquita B, Lopes P, Rodrigues A, Pereira D, Afonso M, Leal C, et al.Frequent copy number gains at 1q21 and 1q32 are associated withoverexpression of the ETS transcription factors ETV3 and ELF3 inbreast cancer irrespective of molecular subtypes. Breast Cancer ResTreat 2013;138:37–45.

10. Larramendy ML, Lushnikova T, Bjorkqvist AM, Wistuba II, Virmani AK,Shivapurkar N, et al. Comparative genomic hybridization revealscomplex genetic changes in primary breast cancer tumors and theircell lines. Cancer Genet Cytogenet 2000;119:132–8.

11. Pinke DE, Lee JM. The lipid kinase PI4KIIIbeta and the eEF1A2oncogene co-operate to disrupt three-dimensional in vitro acinarmorphogenesis. Exp Cell Res 2011;317:2503–11.

12. Anand N, Murthy S, Amann G, Wernick M, Porter LA, Cukier IH, et al.Protein elongation factor EEF1A2 is a putative oncogene in ovariancancer. Nat Genet 2002;31:301–5.

13. Jeganathan S, Lee JM. Binding of elongation factor eEF1A2 to phos-phatidylinositol 4-kinase beta stimulates lipid kinase activity andphosphatidylinositol 4-phosphate generation. J Biol Chem 2007;282:372–80.

14. Kulkarni G, Turbin DA, Amiri A, Jeganathan S, Andrade-Navarro MA,Wu TD, et al. Expression of protein elongation factor eEF1A2 predictsfavorable outcome in breast cancer. Breast Cancer Res Treat2007;102:31–41.

15. Tomlinson VA, Newbery HJ, Wray NR, Jackson J, Larionov A, MillerWR, et al. Translation elongation factor eEF1A2 is a potential onco-protein that is overexpressed in two-thirds of breast tumours. BMCCancer 2005;5:113.

16. Chu KM, Minogue S, Hsuan JJ, Waugh MG. Differential effects of thephosphatidylinositol 4-kinases, PI4KIIalpha and PI4KIIIbeta, on Aktactivation and apoptosis. Cell Death Dis 2010;1:e106.

17. QianY,CorumL,MengQ,Blenis J, Zheng JZ, Shi X, et al. PI3K inducedactin filament remodeling through Akt and p70S6K1: implication ofessential role in cell migration. Am J Physiol Cell Physiol 2004;286:C153–63.

18. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT path-way in human cancer. Nat Rev Cancer 2002;2:489–501.

19. Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA,Watson DK,et al. Amplification of AKT2 in human pancreatic cells and inhibition ofAKT2expressionand tumorigenicity byantisenseRNA.ProcNatl AcadSci U S A 1996;93:3636–41.

20. Cheng JQ, Godwin AK, Bellacosa A, Taguchi T, Franke TF, Hamil-ton TC, et al. AKT2, a putative oncogene encoding a member ofa subfamily of protein-serine/threonine kinases, is amplified inhuman ovarian carcinomas. Proc Natl Acad Sci U S A 1992;89:9267–71.

21. Bachman KE, Argani P, Samuels Y, Silliman N, Ptak J, Szabo S, et al.The PIK3CA gene is mutated with high frequency in human breastcancers. Cancer Biol Ther 2004;3:772–5.

22. Yamamoto H, Shigematsu H, Nomura M, Lockwood WW, Sato M,Okumura N, et al. PIK3CAmutations and copy number gains in humanlung cancers. Cancer Res 2008;68:6913–21.

23. Lopez-Knowles E, O'Toole SA, McNeil CM, Millar EK, Qiu MR, Crea P,et al. PI3K pathway activation in breast cancer is associated with thebasal-like phenotype and cancer-specific mortality. Int J Cancer2010;126:1121–31.

24. Franke TF, KaplanDR, Cantley LC, Toker A. Direct regulation of the Aktproto-oncogene product by phosphatidylinositol-3,4-bisphosphate.Science 1997;275:665–8.

25. Chalhoub N, Baker SJ. PTEN and the PI3-kinase pathway in cancer.Annu Rev Pathol 2009;4:127–50.

26. Andjelkovic M, Alessi DR, Meier R, Fernandez A, Lamb NJC, Frech M,et al. Role of translocation in the activation and function of proteinkinase B. J Biol Chem 1997;272:31515–24.

27. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation andregulation of Akt/PKB by the rictor–mTOR complex. Science2005;307:1098–101.

28. Gewinner C,Wang ZC, Richardson A, Teruya-Feldstein J, Etemadmo-ghadam D, Bowtell D, et al. Evidence that inositol polyphosphate 4-phosphatase type II is a tumor suppressor that inhibits PI3K signaling.Cancer Cell 2009;16:115–25.

29. Song MS, Salmena L, Pandolfi PP. The functions and regulationof the PTEN tumour suppressor. Nat Rev Mol Cell Biol 2012;13:283–96.

30. Ivetac I, Gurung R, Hakim S, Horan KA, Sheffield DA, Binge LC, et al.Regulation of PI(3)K/Akt signalling and cellular transformationby inositol polyphosphate 4-phosphatase-1. EMBO Rep 2009;10:487–93.

31. Ullrich O, Reinsch S, Urbe S, Zerial M, Parton RG. Rab11 regulatesrecycling through the pericentriolar recycling endosome. J Cell Biol1996;135:913–24.


Morrow et al.




32. de Graaf P, Zwart WT, van Dijken RA, Deneka M, Schulz TK, GeijsenN, et al. Phosphatidylinositol 4-kinasebeta is critical for functionalassociation of rab11 with the Golgi complex. Mol Biol Cell 2004;15:2038–47.

33. Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis andoncogenesis of MCF-10A mammary epithelial acini grown inthree-dimensional basement membrane cultures. Methods 2003;30:256–68.

34. HammondGR,SchiavoG, IrvineRF. Immunocytochemical techniquesreveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2).Biochem J 2009;422:23–35.

35. Bridges D, Ma JT, Park S, Inoki K, Weisman LS, Saltiel AR. Phospha-tidylinositol 3,5-bisphosphate plays a role in the activation and sub-cellular localization of mechanistic target of rapamycin 1. Mol Biol Cell2012;23:2955–62.

36. Zimmermann S, Moelling K. Phosphorylation and regulation of Raf byAkt (protein kinase B). Science 1999;286:1741–4.

37. V�arnai P, Balla T. Live cell imaging of phosphoinositide dynamicswith fluorescent protein domains. Biochim Biophys Acta 2006;1761:957–67.

38. Li J. PTEN, a putative protein tyrosine phosphatase gene mutatedin human brain, breast, and prostate cancer. Science 1997;275:1943–7.

39. He X, Saji M, Radhakrishnan D, Romigh T, Ngeow J, Yu Q, et al. PTENlipid phosphatase activity and proper subcellular localization arenecessary and sufficient for down-regulating AKT phosphorylation inthe nucleus in Cowden syndrome. J Clin Endocrinol Metab 2012;97:E2179–87.

40. Toth B, Balla A, Ma H, Knight ZA, Shokat KM, Balla T. Phospha-tidylinositol 4-kinase IIIbeta regulates the transport of ceramidebetween the endoplasmic reticulum and Golgi. J Biol Chem 2006;281:36369–77.

41. Balla A, Kim YJ, Varnai P, Szentpetery Z, Knight Z, Shokat KM, et al.Maintenance of hormone-sensitive phosphoinositide pools in theplasma membrane requires phosphatidylinositol 4-kinase III{alpha}.Mol Biol Cell 2008;19:711–21.

42. Knight ZA, Gonzalez B, Feldman ME, Zunder ER, Goldenberg DD,Williams O, et al. A pharmacological map of the PI3-K family defines arole for p110alpha in insulin signaling. Cell 2006;125:733–47.

43. HsuNY, IlnytskaO,BelovG,SantianaM,ChenYH, TakvorianPM, et al.Viral reorganization of the secretory pathway generates distinct orga-nelles for RNA replication. Cell 2010;141:799–811.

44. Godi A, Pertile P, Meyers R, Marra P, Di Tullio G, Iurisci C, et al. ARFmediates recruitment of PtdIns-4-OH kinase-[bgr] and stimulatessynthesis of PtdIns(4,5)P2 on the Golgi complex. Nat Cell Biol 1999;1:280–7.

45. Polevoy G,Wei HC,Wong R, Szentpetery Z, Kim YJ, Goldbach P, et al.Dual roles for the Drosophila PI 4-kinase four wheel drive in localizingRab11 during cytokinesis. J Cell Biol 2009;187:847–58.

46. Li J, Lu Y, Zhang J, Kang H, Qin Z, Chen C. PI4KIIalpha is a novelregulator of tumor growth by its action on angiogenesis and HIF-1alpha regulation. Oncogene 2010;29:2550–9.

47. Ishikawa S, Egami H, Kurizaki T, Akagi J, Tamori Y, Yoshida N, et al.Identification of genes related to invasion andmetastasis in pancreatic

cancer by cDNA representational difference analysis. J Exp ClinCancer Res 2003;22:299–306.

48. Barlund M, Forozan F, Kononen J, Bubendorf L, Chen Y, Bittner ML,et al. Detecting activation of ribosomal protein S6 kinase by comple-mentary DNA and tissue microarray analysis. J Natl Cancer Inst2000;92:1252–9.

49. Farabegoli F, Ceccarelli C, Santini D, Baldini N, Taffurelli M,Marrano D,et al. c-erbB-2 over-expression in amplified and non-amplified breastcarcinoma samples. Int J Cancer 1999;84:273–7.

50. Mahajan K, Mahajan NP. PI3K-independent AKT activation in can-cers: a treasure trove for novel therapeutics. J Cell Physiol 2012;227:3178–84.

51. Depowski PL, Rosenthal SI, Ross JS. Loss of expression of the PTENgene protein product is associated with poor outcome in breastcancer. Mod Pathol 2001;14:672–6.

52. Perren A, Weng LP, Boag AH, Ziebold U, Thakore K, Dahia PL, et al.Immunohistochemical evidence of loss of PTEN expression inprimary ductal adenocarcinomas of the breast. Am J Pathol 1999;155:1253–60.

53. Murphy JE, Padilla BE, Hasdemir B, Cottrell GS, Bunnett NW. Endo-somes: a legitimate platform for the signaling train. Proc Natl Acad SciU S A 2009;106:17615–22.

54. Kelly KL, Ruderman NB. Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein(IRS-1) and localization in a lowdensitymembrane vesicle. JBiol Chem1993;268:4391–8.

55. Wang Y, Pennock S, Chen X, Wang Z. Endosomal signaling of epi-dermal growth factor receptor stimulates signal transduction path-ways leading to cell survival. Mol Cell Biol 2002;22:7279–90.

56. Vieira AV, Lamaze C, Schmid SL. Control of EGF receptor signaling byclathrin-mediated endocytosis. Science 1996;274:2086–9.

57. Nazarewicz RR, Salazar G, Patrushev N, San Martin A, Hilenski L,Xiong S, et al. Early endosomal antigen 1 (EEA1) is an obligate scaffoldfor angiotensin II-induced, PKC-alpha-dependent Akt activation inendosomes. J Biol Chem 2011;286:2886–95.

58. Schenck A, Goto-Silva L, Collinet C, Rhinn M, Giner A, Habermann B,et al. The endosomal protein Appl1 mediates Akt substrate specificityand cell survival in vertebrate development. Cell 2008;133:486–97.

59. Garcia-Regalado A, Guzman-Hernandez ML, Ramirez-Rangel I,Robles-Molina E, Balla T, Vazquez-Prado J, et al. G protein–coupledreceptor-promoted trafficking of Gbeta1gamma2 leads to AKT acti-vation at endosomes via a mechanism mediated by Gbeta1gamma2-Rab11a interaction. Mol Biol Cell 2008;19:4188–200.

60. Sato M, Ueda Y, Takagi T, Umezawa Y. Production of PtdInsP3 atendomembranes is triggered by receptor endocytosis. Nat Cell Biol2003;5:1016–22.

61. Agola J, Jim P, Ward H, Basuray S, Wandinger-Ness A. Rab GTPasesas regulators of endocytosis, targets of disease and therapeuticopportunities. Clin Genet 2011;80:305–18.

62. Cheng KW, Lahad JP, Gray JW, Mills GB. Emerging role of RABGTPases in cancer and human disease. Cancer Res 2005;65:2516–9.

63. Yoon SO, Shin S, Mercurio AM. Hypoxia stimulates carcinoma inva-sion by stabilizingmicrotubules and promoting theRab11 trafficking ofthe alpha6beta4 integrin. Cancer Res 2005;65:2761–9.






Published OnlineFirst June 24, 2014.Mol Cancer Res Anne A. Morrow, Mohsen Amir Alipour, Dave Bridges, et al. Tumors and Activates Akt in Cooperation with Rab11a

Is Highly Expressed in BreastβThe Lipid Kinase PI4KIII

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