Neuropilin-2RegulatesEndosomeMaturationand EGFR Trafﬁcking to Support … · Therapeutics, Targets, and Chemical Biology Neuropilin-2RegulatesEndosomeMaturationand EGFR Trafﬁcking

Therapeutics, Targets, and Chemical Biology

Neuropilin-2 Regulates EndosomeMaturation andEGFR Trafficking to Support Cancer CellPathobiologySamikshan Dutta1, Sohini Roy1, Navatha S. Polavaram1, Marissa J. Stanton1, Heyu Zhang2,Tanvi Bhola1, Pia H€onscheid3, Terrence M. Donohue Jr.1,4,5, Hamid Band6,Surinder K. Batra1,6, Michael H. Muders1,3, and Kaustubh Datta1,6

Abstract

Neuropilin-2 (NRP2) is a non-tyrosine kinase receptor fre-quently overexpressed in various malignancies, where it hasbeen implicated in promoting many protumorigenic behaviors,such as imparting therapeutic resistance to metastatic cancercells. Here, we report a novel function of NRP2 as a regulator ofendocytosis, which is enhanced in cancer cells and is oftenassociated with increased metastatic potential and drug resis-tance. We found that NRP2 depletion in human prostate andpancreatic cancer cells resulted in the accumulation of EEA1/Rab5-positive early endosomes concomitant with a decrease inRab7-positive late endosomes, suggesting a delay in early-to-late endosome maturation. NRP2 depletion also impaired theendocytic transport of cell surface EGFR, arresting functionally

active EGFR in endocytic vesicles that consequently led toaberrant ERK activation and cell death. Mechanistic investiga-tions revealed that WD-repeat– and FYVE-domain–containingprotein 1 (WDFY1) functioned downstream of NRP2 to pro-mote endosome maturation, thereby influencing the endoso-mal trafficking of EGFR and the formation of autolysosomesresponsible for the degradation of internalized cargo. Overall,our results indicate that the NRP2/WDFY1 axis is required formaintaining endocytic activity in cancer cells, which supportstheir oncogenic activities and confers drug resistance. Therefore,therapeutically targeting endocytosis may represent an attrac-tive strategy to selectively target cancer cells in multiple malig-nancies. Cancer Res; 76(2); 418–28. �2015 AACR.

IntroductionNeuropilins (NRP) are transmembrane, nontyrosine kinase

receptors. Often, they function as coreceptors to modulate var-ious cellular pathways including angiogenesis, cellular commu-nication, and migration (1, 2). Neuropilin-2 (NRP2), a memberof the NRP family of receptors, has a similar molecular mass andstructural domain to its family member neuropilin-1 (NRP1;ref. 3). In addition to its role in neuronal development, NRP2is important for the development of capillaries and lymphaticvessels (4, 5). The known binding ligands for NRP2 are VEGF-C,VEGF-D, VEGF-A, and semaphorin-3F (6). Importantly, NRP2 isalso expressed in various human cancer tissues and cancer cell

lines (1, 3, 5, 7–10), and it is implicated in promoting theirproliferation, survival, and migration (11). It is also important inmaintaining the tumor initiating population of breast cancer(12). Interestingly, NRP2, but not NRP1, maintains its proteinlevel during metabolic stress, such as nutrient starvation andhypoxia, suggesting that it has a potential role in stress (13). Wehave previously observed a survival-promoting role of NRP2 incancer cells during therapeutic stress (14, 15). Our findingscorroborated an earlier report, where depletion of NRP2 in coloncancer cells increased their death during hypoxia (16).

In this article, we report a novel function of NRP2 in cancercells. Our results suggested the role of NRP2 in regulating thematuration of endocytosis. Although this function can providethe underlying mechanism of the role of NRP2 in regulatingautophagy during therapeutic stress (14, 15, 17), NRP2-regulatedendosomematuration is also important for the proper function ofcell surface receptors, which require endocytic trafficking tomain-tain optimum activity. Previously NRP1 was indicated to regulateendocytosis of tyrosine kinase receptors such as VEGFR2 (18, 19).It has been shown that upon ligand binding, VEGFR2 and NRP1undergo endocytosis as a complex. NRP1 has a C-terminal PDZ-binding site, which helps its interaction with a protein calledsynectin that links VEGFR2–NRP2 complex to myosin-VI motorproteins. NRP1 therefore helps VEGFR2-containing endosomesto move away from the plasma membrane. NRP1 also helpsendocytosis of CendR peptides or membrane lytic peptides suchas K8L9 and melittin (19–21). Recently, NRP1 has also beenimplicated in the internalization of the Epstein–Barr virusinto the nasopharyngeal epithelial cells (22). In all these

1Biochemistry and Molecular Biology, University of Nebraska MedicalCenter, Omaha, Nebraska. 2Department of Urologic Research, Bio-chemistry, Mayo Clinic College of Medicine, Rochester, Minnesota.3Institute of Pathology, University Hospital Carl Gustav Carus, TU,Dresden, Germany. 4Department of Internal Medicine, University ofNebraska Medical Center, Omaha, Nebraska. 5Omaha VA MedicalCenter, Omaha, Nebraska. 6Buffett Cancer Center, Eppley CancerInstitute, University of Nebraska Medical Center, Omaha, Nebraska.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

CorrespondingAuthor: Kaustubh Datta, University of Nebraska Medical Center,Durham Research Center II, Room 4022, 985870 Nebraska Medical Center,Omaha, NE 68198-5870. Phone: 402-559-7404; Fax: 402-559-6650; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-1488

�2015 American Association for Cancer Research.

CancerResearch

Cancer Res; 76(2) January 15, 2016418

on May 20, 2021. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 11, 2015; DOI: 10.1158/0008-5472.CAN-15-1488

http://cancerres.aacrjournals.org/

circumstances, the receptors, peptides and the virus particles thatare endocytosed, directly interact with NRP1 at the cell surface,which facilitates their internalization during endocytosis. Thisprocess is distinctly different from our current findings whereNRP2 axis is involved in thematuration of late endocytic vesicles.We have also identified WD-repeat– and FYVE-domain–contain-ing protein 1 (WDFY1) as a downstream of the NRP2 axis. Fewstudies have been reported on WDFY1. A recent study indicatedWDFY1 as a potential candidate of chronic pancreatitis (23).Interestingly, WDFY1 has been shown to recruit signalingadaptor TRIF to Toll-like receptors (TLR3 and TLR4), and therebypotentiating signaling necessary for the initiation of innateimmune system (24). WDFY1 has also been considered as apotential biomarker for Alzheimer's disease (25) and can beinvolved in placental development and for the maintenance ofhematopoietic stem cells (26). In this study, we are reporting anovel NRP2–WDFY1 axis, which are important for the mainte-nance of endocytic activity in cancer cells. Recent evidence indi-cates that altered endocytosis promotes aberrant cell surfacereceptor signaling in cancer cells, either by expediting their recy-cling to the cell surface or by enabling a different receptorsignaling program from the endosomal compartment (27, 28)We specifically focused on how the NRP2 axis regulates thetrafficking of EGFR in cancer cells. Interestingly, our resultsindicated that the inhibition of the NRP2 axis would lead to theaccumulation of ligand-stimulated EGFR in early endosomes andthen initiate an apoptosis-promoting signal. Therefore, inhibitorsof NRP2 should show a potent antitumor effect in cancer cellswith activated EGFR signaling.

Materials and MethodsCell culture, plasmid constructs, and transfection reagents

Twometastatic human prostate cancer cell lines [PC3 (CRL1435;ATCC) and Du145 (HTB81; ATCC)] and one pancreatic cancercell line CaPan1 (HTB-79, ATCC; kind gift from Dr. MichaelHollingsworth, University of Nebraska Medical Center, Omaha,NE) that express high levels of NRP2 and VEGF-C were used forthe experiments described here. Wild-type WDFY1 (RC509030;Origene), NRP2 (RC220706, Origene) plasmids were used to over-express these proteins in cancer cells. To deplete the VEGF-C, NRP2,andWDFY1, siRNAs against VEGF-C (L-012071-00-0020; Dharma-con RNA Technologies; #LQ-012071-00-0005; Qiagen), NRP2(L-017721-00-0010 and LU-017721-00-005; Dharmacon), andWDFY1 (L-017721-00-0010; Dharmacon) were transfected in can-cer cells using Dharmacon reagents (T-2005-02; Dharmacon).

Western blot, ELISA, isolation of membrane proteinWestern blot and ELISA were performed using established

techniques (see Supplementary materials). For isolation ofcell membrane fractions, cells were washed and homogenized inice-cold lysis buffer (25 mmol/L HEPES, 100 mmol/L NaCl,1 mmol/L ethylenediaminetetraacetic acid, pH 7.4, and proteaseinhibitor cocktail) in a glass Dounce homogenizer for 30 to 40vertical strokes. The homogenized lysate was then subjected tocentrifugation (800 g for 10 minutes) to remove cell debris andisolate the nucleus (pellet). The supernatant was then subjectedto ultracentrifugation (108,000 g for 1 hour at 4�C) andmembrane (pellet) and cytosolic (supernatant) fractionswere collected. The pellet was resuspended in urea buffer(70 mmol/L Tris–HCl, pH 6.8, 8 mol/L urea, 10 mmol/L

n-ethylmaleimide, 10 mmol/L iodoacetamide, 2.5% SDS and0.1 mol/L dithiothreitol) at 37�C for 15 minutes. Proteins wereanalyzed using Western blot with the following antibodies:LC3B (2775; Cell Signaling Technology), Rab5 (2143; CellSignaling Technology), Rab7 (9367; Cell Signaling Technolo-gy), Caveolin (3238; Cell Signaling Technology), Myc-Tag(2276, Cell Signaling Technology), EGFR (4267, Cell SignalingTechnology), phosphor-ERK (4370, Cell Signaling Technolo-gy), ERK (4695, Cell Signaling Technology), PARP (9532, CellSignaling Technology), EH-domain–containing protein 4(EHD4; ab153892, Abcam), NRP2 (#sc-13117; Santa CruzBiotechnology; AF2215, R&D System), Rho-GDI (sc-360; SantaCruz), Cathepsin B (sc-6493; Santa Cruz), Cathepsin D (sc-10725; Santa Cruz), Cathepsin L (sc-6498; Santa Cruz), EEA1(sc-33585; Santa Cruz), phosphor-EGFR 1173 (sc-12351, SantaCruz), PIKfyve (sc-100408; Santa Cruz), WDFY1 (123058;GeneTex Inc. and SAB2106120, Sigma-Aldrich).

Confocal and electron microscopyCells were grown on poly-DL-lysine–coated coverslips (BD

Biosciences) for 72 hours before fixation and confocal analysis.Cells were rinsed with Dulbecco's Phosphate-Buffered Saline(DPBS; Invitrogen), followed by fixation with 4% paraformalde-hyde at room temperature for 10minutes. Later, cellswerewashedwith DPBS and treated with ice-cold methanol for 20 minutes at�20�C. Finally, cells were blocked using 1% BSA and 0.2%saponin. All confocal images were captured using a Zeiss 710Confocal Laser ScanningMicroscope (equipped with four lasers),and data were analyzed and processed with the Zeiss Zen 2010software.

For electron microscopy, cells were cultured in 25-mm poly-D/L-lysine–coated coverslips (BD Biosciences) and fixed with2% glutaraldehyde (Sigma-Aldrich) containing 2% para-form-aldehyde solution in 0.1 mol/L Sorensen's Phosphate Buffer(Sigma-Aldrich) for 30 minutes at room temperature followedby vigorous washing with 0.2 mol/L HEPES, pH 7.4, at roomtemperature for 1 hour. Samples were post-fixed in 1% osmiumtetroxide, aqueous solution (Sigma-Aldrich), for 30 minutes.Samples were washed 3 times in buffer, followed by dehydra-tion with 50%, 70%, 90%, 95%, and 100% ethanol. Thedehydrated samples were then passed through a series ofgraded Araldite/ethanol mixtures, (1:2, 1:1, and 2:1; Sigma-Aldrich), before being embedded onto Araldite blanks andstored at 65�C overnight for polymerization. Next, cell culturecolonies were excised and adhered to an Araldite blank blockfor thin sectioning. Thin sections were collected onto 200 meshcopper grids and stained with 1% uranyl acetate (Sigma-Aldrich) and Reynolds lead citrate (Sigma-Aldrich). Sectionswere examined on an FEI Tecnai G2 TEM (FEI Company)operated at 80 kV.

ResultsInhibition of the VEGF-C/NRP2 axis prevents the formation ofautolysosomes from autophagosomes

We previously reported regulation of autophagy by NRP2 incancer cells (15, 17). To explain the underlying mechanism,autolysosome formation was monitored in human prostatecancer PC3 cell lines stably expressing LC3B-tagged GFP-mCherry protein following depletion of NRP2. An appro-ximately 52% reduction in the formation of autolysosomes

NRP2 Axis and Endosome Maturation

www.aacrjournals.org Cancer Res; 76(2) January 15, 2016 419




(Red Puncta) was observed in NRP2-depleted cells comparedwith scrambled siRNA-transfected cells (Fig. 1). This becameeven more apparent after continuous monitoring of autopha-gosome and autolysosome formation in the stable clones usingtime-lapse video microscopy (Supplementary video S1). More-over, transmission electron microscopy analysis (Supplemen-tary Fig. S1A) revealed that after NRP2 depletion, the number ofelectron-dense autophagosomes (indicated by yellow arrows)increased compared with controls. These results suggest thatinhibition of the NRP2 axis delays the formation of autolyso-some and thus, slows the lysosomal degradation of the seques-tered cargo.

Effect of expression of WDFY1 on the formation ofautolysosomes

We previously reported that expression of WDFY1 increasesafter depletion of either NRP2 or its ligand VEGF-C (15). Wehypothesized that WDFY1 acts downstream of NRP2 in regu-lating the formation of autolysosomes. Simultaneous deple-tion of NRP2 and WDFY1 partially rescued defective autop-hagosomal maturation, as detected using confocal imaging(Fig. 1). Western blot measured the autophagic flux andverified the findings of the confocal imaging (SupplementaryFig. S1B). Our findings therefore indicate that WDFY1, as adownstream target of the VEGF-C/NRP2 axis, prevents theformation of autolysosomes.

The role of the NRP2/WDFY1 axis in endosome maturationPC3 cells showed thepresence ofWDFY1 inEEA1-positive early

endosomes (Supplementary Fig. S1C); a similar observation wasalso previously reported for WDFY1 (29). We hypothesized thattheNRP2/WDFY1 axis regulates endosomematuration and there-by regulates the formation of autolysosome. An increase in EEA1-positive puncta in NRP2 and VEGF-C–depleted cells wereobserved, suggesting an increase in early endosome number(Fig. 2A). Although the circularity of the puncta remainedunchanged, mean diameter of the individual EEA1-positive

puncta often increased in siNRP2- and siVEGF-C–treated cells(Fig. 2B), indicating a defect in the maturation of EEA1-positiveearly endosomes. Simultaneous depletion of NRP2 and WDFY1partially restored the defect in EEA1-positive puncta, thus sup-porting the hypothesis that WDFY1 acts as downstream of NRP2in regulating endocytosis (Fig. 2A and B). Similar to EEA1,depletion of NRP2 increased the number and size of puncta thatwere positive for Rab5, another early endosome marker (Supple-mentary Fig. S1D). Once again, simultaneous depletion of NRP2and WDFY1 restored the wild-type phenotype for Rab5-positivevesicles. Similar to PC3, Capan1 (pancreatic cancer cell line) andDu145 (prostate cancer cell line), which maintain higher expres-sion of endogenous NRP2, also showed similar defect in earlyendosomes following NRP2 depletion (Supplementary Fig. S2Aand S2B). Once again, simultaneous depletion of NRP2 andWDFY1 rescued the defects caused by NRP2 depletion alone(Supplementary Fig. S2B). To rule out the off-target effect ofsiRNAs, we exogenously expressed NRP2 in siNRP2-transfectedcells and recovered the wild type phenotype of early endosomes(Supplementary Fig. S2A and S2C). Overexpression of full lengthNRP2 in siNRP2-transfected cells downregulates the WDFY1protein in the basal level (Supplementary Fig. S2D), indicatingthat upregulation of WDFY1 abrogates the endosomal functionand acts downstream of NRP2 axis.

To determine if deregulation of the NRP2 axis can affect lateendosomes, vesicles that were positive for the late endosomemarker Rab7 were examined. Interestingly, depletion of NRP2 orVEGF-C reduced the Rab7-positive puncta (Fig. 2C). The controlphenotype was once again restored either after simultaneousdepletion of NRP2 and WDFY1 or by overexpressing full lengthNRP2 in all the cell lines (Fig. 2C and Supplementary Fig. S3A–S3C). Knockdown efficiency of NRP2 in PC3 cells was shownin Fig. 2D.

To confirm the observations made during immunostaining,immunoblot analysis of the total cellular andmembrane fractionwas conducted to identify functionally active endosomalmarkers.Here, after depleting either VEGF-C or NRP2 (Fig. 3A), an

Figure 1.Simultaneous depletion of WDFY1 and NRP2 rescue autophagy inhibition. Autophagic activity was detected using confocal microscopy for PC3 cells thatstably expressed GFP-mCherry-LC3B plasmids after 48 hours of transfection either with scrambled, siNRP2 or with siNRP2 and siWDFY1 simultaneously. Thepercentage of autophagosomes (yellow puncta; autophagosomes vs. sum of autolysosmes and autophagosomes per field) was quantified and is representedgraphically. Scale bar, 20 mm. DAPI was used for nuclear staining. Images within the boxed region were magnified to show the distribution of autophagosomes(yellow) and autolysosomes (red).

Dutta et al.

Cancer Res; 76(2) January 15, 2016 Cancer Research420




increased level ofmembrane-associated EEA1 and a simultaneousdecrease in membrane-bound Rab7 were observed. No change inthe total cellular content of EEA1 and Rab7 proteins occurred(Supplementary Fig. S4A–S4C shows the efficiency of knockdownof VEGF-C and NRP2 in PC3). A similar increase in the level inmembrane-bound EEA1 was observed using two independentVEGF-C and NRP2 siRNAs (Supplementary Fig. S4D and S4G).Results from RT-PCR and ELISA show the relative knockdownefficiency of the individual siRNAs (Supplementary Fig. S4E, S4F,and S4H). Similar to PC3, Du145 showed the similar phenotypein immunoblot where membrane-bound EEA1 increases andRab7 decreases following the knockdown of NRP2 (Supplemen-tary Fig. S4I).

Finally, following NRP2 knockdown, membrane recruitmentof FYVE-domain–containing phosphoinositide kinase (PIKfyve)was also assessed. PIKfyve is recruited during the maturation ofearly to late endosomes and synthesizes PtdIns-3, 5-P2 fromPtdIns-3-P (30). Both immunoblot and immunostaining analysesshowed a decrease in active membrane-bound PIKfyve (Fig. 3Band C and Supplementary Fig. S5A). However, the total PIKfyve

remain unchanged (Supplementary Fig. S5B and S5C). Together,these results suggest that inhibition of the NRP2 axis alters thetransition of early to late endosomes. Notably, autophagosomesoften merge with late endosomes to form amphisomes prior tofusing with lysosomes (31). We therefore propose that, followinginhibition of the NRP2 axis, the decrease in late endosomesinterferes with the crosstalk between autophagosomes andmatureendosomes, which in turn, delays the formation of autolysosomes.

Depletion of NRP2 in cancer cells and delayed transfer ofcathepsin cargos from early to late endosomes

To further validate that depletion of the NRP2 axis affectsendosome maturation, the Golgi-to-lysosome trafficking of var-ious cathepsins was examined. Cathepsin precursors are synthe-sized in the endoplasmic reticulum (ER), trafficked to the Golgiapparatus, and reach the lysosomes through the early–late endo-some–lysosome maturation process (32). We postulated that,following disruptionof theNRP2 axis, a decrease in thenumber oflate endosomes will enhance the accumulation of cathepsin-containing cargo in early endosomes. Following knockdown of

Figure 2.Depletion of VEGF-C/NRP2 inhibits early to late endosome maturation. A, immunostaining of early endosome marker EEA1 (green) following the depletionof VEGF-C and NRP2 in PC3 cells. Recovery experiments with simultaneous depletion of NRP2 and WDFY1 were also conducted under each condition.Immunostaining data were quantitated using image J software and are represented as a bar graph. � , statistically significant differences (P ¼ 0.006 and P ¼ 0.02,respectively, for siNRP2 and siVEGF-C compared with control). Inset, the magnified image of each panel. B, following NRP2 or VEGF-C depletion or doubleknockdown with siNRP2 and siWDFY1, EEA1 puncta was determined. The diameter of the EEA1 puncta was calculated using image J software and is representedas a graph (P < 0.00001 for all). C, immunostaining for the late endosome marker Rab7 (green) after depletion of VEGF-C or NRP2 alone, or simultaneousdepletion of NRP2 andWDFY1, in PC3 cells. Quantitation of staining data is represented graphically. � , statistically significant differences in Rab7 puncta (P¼0.0001and 0.04 for siNRP2 and siVEGF-C, respectively). Magnified areas are represented under each panel. Scale bars, 20 mm in length for all immunostaining images.DAPI was used for nuclear staining. D, Western blot showing the NRP2 depletion in PC3 following siNRA transfection.






the NRP2 axis, a significant increases in cathepsins in EEA1-positive vesicles (yellow and greenish-yellow puncta) wereobserved (Fig. 4A and B and Supplementary Fig. S6A and S6B).In contrast, simultaneous knockdown of NRP2 and WDFY1 inPC3 andDu145 cells resulted in reduced accumulation of cathep-sin-positive puncta in early endosomes (Fig. 4A and B andSupplementary Fig. S6A–S6C).

Cathepsins become mature and catalytically active within theacidic pH of the late endosome and lysosome (32). Therefore, weexpected a reduction in themature cathepsins in the cell followingNRP2depletion.Western blot analyses revealed decreased levels ofmature cathepsin D and B following the depletion of NRP2 (Fig.4C), indicating that maturation of cathepsin was hindered. Inter-estingly, simultaneous depletion of NRP2 andWDFY1 rescued thelevel of mature cathepsins (Fig. 4C). Overall, these results indicatethat blocking of early to late endosome maturation inhibits thedelivery of certain lysosomal enzymes like cathepsins (33).

The underlying mechanism of NRP2/WDFY1 axis–regulatedendosomal trafficking

The EHD4protein is a regulator of endocytosis and is located inearly endosomes (34). Endogenous depletion of EHD4 has beenshown to result in a similar increase in EEA1- and Rab5-positiveearly endosomes, with a concomitant defect in trafficking of cargofrom early to late endosomes and lysosomes (34). Consideringthat a similar defect in endocytosis was observed following thedepletion ofNRP2, the potential crosstalk betweenNRP2 axis and

EHD4 was examined in prostate and pancreatic cancer cells. Adecrease of membrane-bound EHD4 occurred when NRP2 wasdepleted (Fig. 5A); the total level of EHD4 remained unchangedwithin the cancer cells (Fig. 5B). Further, immunostaining ofEHD4 showed a significant decrease in EHD4-positive peripheralpuncta following the depletion of NRP2 in both PC3 (Fig. 5C andD) and Capan1 (Fig. 5E) cell lines. The EHD-positive puncta wererescued either by simultaneous depletion of NRP2 and WDFY1(Fig. 5C) or overexpression of full length NRP2 (Fig. 5D and E).Our results indicate that NRP2 maintains WDFY1 expression inthe cell and thereby regulates the level of EHD4 in themembrane,thus influencing the endocytic pathway in cancer cells.

Depletionof theNRP2axis and altered trafficking of cell surfacereceptors

Cancer-promoting functions of several cell surface receptorsdepend on their ability to endocytose within the cell. EGFR is onesuch tyrosine kinase growth factor receptors, which is often over-expressed and mutated in cancer cells. EGFR controls cancer cellproliferation, survival, invasion, angiogenesis, and metastasis(7, 35). In a recent report, it has been suggested that EGF-inducedchemotactic migration of metastatic cancer cells depends on theirability to coordinate the endocytosis of EGFR (36). Functionallyactive endocytosis can carefully regulate the availability of EGFRand other cell adhesion molecules on the cell surface, which isrequired for the directionality and magnitude of the migration ofmetastatic cancer cells. Therefore inhibition of endocytosis

Figure 3.Depletion of VEGF-C/NRP2 inhibitsthe transition to late endosome. A,membrane fractions were analyzedfor EEA1 and Rab7 following VEGF-C/NRP2 depletion in PC3 cells. Caveolinwas used as a loading control for themembrane fraction. B, immunoblotwas performed to analyze membranePIKfyve following NRP2 depletion. C,immunostaining of PIKfyve (green)was carried out following thedepletion of VEGF-C/NRP2. Recoveryof PIKfyve-positive puncta wasperformed via simultaneous depletionof NRP2 and WDFY1. Insets, themagnified images of the cells. DAPIwas used for nuclear staining.Quantitation of total PIKfyvefluorescence intensity is representedgraphically. � , significant statisticaldifferences (P¼ 0.00012, P¼ 0.0005for siNRP2 and siVEGF-C samples,respectively, compared with control).Scale bar, 10 mm.

Dutta et al.





imparts a negative effect on chemotactic cell migration. Interest-ingly, NRP2 is also known in regulating themigration of neuronaland endothelial cells (37). Many metastatic cancer cells thatexpress significantly high level of NRP2 also showed higheractivity for EGFR such as metastatic prostate cancer cells, glio-blastoma, and breast cancers (38–40). NRP is also known tomodulate the downstream signaling axis of EGFR (41). Wetherefore speculate that the implication of inhibiting the NRP2axis would be to deregulate the endocytic process of EGFR incancer cells and therefore hinder its tumorogenic activity.

An EGFR internalization assay was performed in PC3 cellsfollowing a brief exposure of its ligand EGF and the role of NRP2axis in EGFR endocytosis was investigated. As shown in Fig. 6A–C,EGF exposure to PC3 cells led to internalization of EGFR incontrol cells; over time, the internalized EGFR was transportedto lysosomes and then mostly degraded within 5 hours after EGFstimulation. However, in NRP2 knockdown cells, a sufficientamount of EGFR still remained after 5 hours chase followingEGF exposure, indicating a delayeddegradationof EGFR (Fig. 6A).Immunoblot analysis indicated that these internalized EGFRremained phosphorylated (Tyr 1173) and thus active even after5 hours of EGF stimulation following NRP2 depletion (Fig. 6B).Moreover, we found that most of these EGFR-positive vesicles

remained colocalized within EEA1 after 5 hours of EGF stimula-tion following the overexpression of WDFY1 (Fig. 6C andSupplementary Fig. S7A), suggesting an extended presence ofEGFR in the early endosomes. Similar results were observed inCaPan1 (Fig. 6D) and Du145 cells (Supplementary Fig. S7B),which further supported the results obtained from PC3 cells.

In order to test whether inhibition of NRP2 can delay thetrafficking of other receptors, we monitored the transport oftransferrin (TF) receptor following the NRP2 knockdown. Similarto EGFR, TF receptor was present in the EEA1-positive (Fig. 6E)early endosomes for prolonged period. Our result, therefore,suggested that depletion of the NRP2 axis affects the recycling ofTF receptor to the plasma membrane.

In summary, our results indicate that depletion of NRP2interferes with the cargo sorting at early endosomes and therebyinhibits both lysosomal delivery and trafficking of cell surfacereceptors.

Sustained EGF-induced ERK phosphorylation and cell deathfollowing NRP2 depletion

To understand the effect of prolonged growth factor exposureon cellular signaling, PC3 and CaPan1 cells were incubated withEGF at 20 ng/mL concentration for 48 hours. Under these

Figure 4.Depletion of NRP2 inhibits endocytic trafficking of cathepsins. Colocalization of cathepsin (red) with early endosomal marker EEA1 (green) was analyzed witheither depletion of NRP2 alone or depletion of WDFY1 and NRP2 simultaneously for cathepsin D (A) and cathepsin B in PC3 cells (B). Scale bar, 50 mm. DAPIwas used for nuclear staining. Quantitation of the immunostaining data is represented graphically. � , significant statistical difference with P values < 0.001.Inset was magnified to show the colocalization of cathepsins with EEA1. C, immunoblot was performed for the analysis of maturation of cathepsins in PC3lysates following the depletion of NRP2 alone or simultaneous depletion of NRP2 and WDFY1.






conditions, we observed an increased ERK phosphorylation inboth the cell lines, following the depletion of either NRP2 oroverexpressing WDFY1 (Fig. 7A–C). Previous reports indicatedERK phosphorylation by functionally active EGFR when presentin early endosomes for an extended time period (36). We alsoobserved a significant increase in active EGFR (pEGFR 1173) inEEA1-positive early endosomes located in the perinuclear regionin NRP2 knockdown PC3 and CaPan1 cells (Fig. 7D and E),suggesting the presence of endosomal active EGFR over a pro-longed period due to the depletion of the NRP2 axis. Earlierreports indicated that prolong intracellular EGFR signaling wasoften responsible for the induction of apoptosis (36, 42); wherehyperactivation of ERK was described as an underlying mecha-nism of apoptosis induction. Therefore, we tested whether NRP2depletion in EGF-stimulated cancer cells can induce death. Fol-lowing EGF stimulation, we observed a significant increase inapoptotic death in NRP2-depleted cancer cells (Fig. 7F andG). Anincrease in PARP cleavage underNRP2-depleted condition furtherconfirmed induction of apoptosis in EGF-stimulated cancer cells(Fig. 7Hand I).Overall our results suggest that inhibition ofNRP2

in prolonged EGF-stimulated cancer cells arrests the phosphory-latedEGFR in early endocytosed vesicles, inducing ERK-driven celldeath.

DiscussionOverall, we propose a novel function of the NRP2 axis in

cancer cells. The increases in EEA1- and Rab5-positive punctaand simultaneous decreases in Rab7-positive puncta in siNRP2-or siVEGF-C–treated cells suggest a defect in the maturation ofearly endosomes. This was further supported by observationsthat depletion of the NRP2 axis caused accumulation of cathep-sin-containing cargos in early endosomes, and decreased PIK-fyve recruitment to the membrane. Autophagosomes oftenmerge with late endosomes to form amphisomes prior to fusingwith lysosomes (31). We, therefore, propose that, followinginhibition of the NRP2 axis, the decrease in late endosomesinterferes with the crosstalk between autophagosome andmatured endosomes, which, in turn, delays the formation ofautolysosomes.

Figure 5.Mechanism of endocytic regulation. A and B, membrane (A) and total (B) fraction of EHD4 was analyzed following NRP2 depletion in PC3 cells. C, immunostainingof EHD4 (green) following depletion of NRP2 alone or double depletion of NRP2 and WDFY1 simultaneously. DAPI was used for nuclear staining. Scale bar,20 mm. Using image J software, intensity of the green puncta was calculated for each assay condition. Quantitation is represented graphically. � , significantstatistical differences (P < 0.0001 in both cases). Immunostaining of EHD4 was performed following the overexpression of full length NRP2 in NRP2-depletedsample for both PC3 (D) and Capan1 (E). Inset was magnified and split into different channels to represent the staining of NRP2 (green) and EHD4 (red) inthe cells. Scale bar, 20 mm. DAPI was used for nuclear staining.

Dutta et al.





We hypothesized that WDFY1 acts as a downstream reg-ulator of the NRP2 axis as its expression increases afterdepletion of either VEGF-C or NRP2 (15). The ability ofsimultaneous depletion of NRP2 and WDFY1 to restore thedefect in endosome maturation supports our hypothesis.Although, limited information is currently available onWDFY1, a study by Arisi and colleagues suggested thatWDFY1 is a potential biomarker for Alzheimer's disease(25). Because deregulation of autophagy is linked to neuro-degenerative diseases such as Alzheimer's (25), the potentialrole of WDFY1 in inhibiting autophagy, as suggested here,may explain this connection.

The decrease of membrane-bound EHD4 suggests that thefunction of EHD4 becomes defective after the NRP2 axis isinhibited. Considering that EHD4 is an important regulator inthe transport of cellular cargo from early to late endosome(34, 43), the ability of NRP2 to regulate the function of EHD4can be considered as an underlyingmechanism for how theNRP2axis regulates endocytic maturation. The rescue of the EHD4-positive puncta by simultaneous depletion of NRP2 andWDFY1 indicates that WDFY1 is important for the cellular reg-ulation of EHD4. We speculate that an optimum level of EHD4is needed in the membrane for its proper function, and theNRP2/WDFY1 axis thereby regulates the membrane level of

Figure 6.Depletion of NRP2 arrests cell surface receptors in early endosomes following brief stimulation with EGF. Following activation with EGF (20 ng/mL for 15 minutes at37�C), EGFR (green) degradation was monitored over time. A, immunostaining was performed to monitor the EGFR trafficking at various time intervalsfollowing NRP2 depletion in PC3 cells. Scale bar, 20 mm. DAPI was used for nuclear staining. Insets, themagnified images of cells at each time point. B, PC3 cells weredoped with 20 ng/mL EGF for 15 minutes at 37�C. Following this incubation, media was replaced with normal growthmedia for chasing the status of phospho-EGFRand total EGFR at various time intervals. PC3 cells were lysed at each time point and immunoblot was performed to compare the status of phospho-EGFRand total EGFR following depletion of NRP2. C, following WDFY1 overexpression, EGFR degradation was monitored for more than 5 hours time period afterEGF activation. Early endosome marker EEA1 was counterstained with EGFR (Supplementary Fig. S3A). Colocalization of internalized EGFR with EEA1 wasquantified in control and WDFY1 overexpressed samples at various time points following the EGFR chase and is represented here as a bar graph. � , significantstatistical difference (P ¼ 0.0014). D, similar to PC3 cells, EGFR degradation was monitored in Capan1 cells following the short exposure to EGF (20 ng/mLfor 15 minutes at 37�C) at various time points. Scale bar, 10 mm. DAPI was used for nuclear staining. E, TF receptor (tagged with Alexa 633 fluorophore)internalization analyzed following 1 hour serum starvation in PC3 cells. Cells were incubated with the labeled-TF (20 ng/mL) for 5 minutes at 37�C, after whichthe media containing the TF was replaced with the normal growth media. Recycling pathway of TF was monitored using early endosome marker EEA1 (green).Colocalization of TF and EEA1 was quantified and represented graphically. Line graph indicates the pattern of EEA1 and TF colocalization after 45 minutesof chase. DAPI was used for nuclear staining. Scale bar, 50 mm. Inset was magnified to show the trafficking of TF at various time points. � , the differenceswere significant (P ¼ 0.003, 0.005, and 0.0001 for 10, 20, and 45 minutes, respectively).






EHD4, which influences the transition from early to late endo-somes. WDFY1 strongly interacts with the PtdIns-3-P, whichis an important membrane lipid of endocytic vesicles. It is pos-sible that increased presence of WDFY1 in the membraneinhibits the recruitment of EHD4, a hypothesis needs to betested in future studies. As the NRP2–WDFY1–EHD4 axis has noinfluence on cargo delivery to early endosomes, they becomeenriched with the incoming cargos with large vesicular structure.Therefore, NRP2 depletion can cause significant defect in receptortrafficking especially in cancer cells. Due to delayed turnover,endocytosed receptors can be activated in early endosomes for aprolonged period, which can promote an aberrant receptorsignaling.

We have shown that tyrosine kinase receptor, EGFR is presentin the early endosomal compartment of cells for an extendedperiod following the depletion of NRP2. Sustained activation ofEGFR in this early endosomal compartment has been shown tostimulate apoptosis and therefore can be detrimental to meta-static cells (36). Currently, several anti-EGFR drugs are approvedby the FDA; however, a mixed outcome has been reported. It hasnow been well documented that spatial localization of EGFR isimportant for maintaining metastatic behavior of cancer cells.

Plasma membrane-bound EGFR has been shown to promotethe cell survival by activating antiapoptotic AKT pathway as wellas induce the migration and proliferation by upregulating theRas–MAPK pathway. The maintenance of this spatial distribu-tion is also important for maintaining its Ras-driven tumorige-nicity. However, the internalized EGFR or endocytosed EGFRreverting the signaling cascade and is responsible for cellularapoptosis. It has been shown that, therapeutic efficacy of Citux-imab, an anti-EGFR drug, induces EGFR endocytosis to perturbits cytotoxic effect. Moreover, chemotherapeutic efficacy wasincreased in the presence of internalized EGFR and the drugsthat induce or arrest internalized EGFR actually had a betterefficiency in chemotherapy outcome (35, 36, 44). In this con-text, we here showed that depletion of NRP2 arrested theinternalized EGFR in early endosomes. It thereby caused hyper-activation of ERK signaling and induced cell death. Thus, NRP2depletion can be a potential mode of therapy for cancers withprominent EGFR signaling cascade.

The endocytic process is enhanced and altered in cancer cells,thus facilitating tumor growth, the epithelial–mesenchymal tran-sition, evasion of apoptosis, and metastasis (28, 45). Rapiddegradation of E-cadherin, aberrant trafficking, and recycling of

Figure 7.Prolonged exposure to EGF following NRP2 depletion induces cell death. Immunoblot was performed to analyze ERK phosphorylation following either NRP2depletion or overexpressing WDFY1 under continuous exposure to EGF (20 ng/mL at 37�C for 48 hours) in both PC3 (A and B) and Capan1 (C) cells. Colocalizationof phosphorylated EGFR 1173 (red) with EEA1 (green) was analyzed following 48 hours exposure to EGF in both PC3 (D) and Capan1 (E) cells under NRP2knockdown conditions. Inset was magnified and split into corresponding channels to represent the individual and merged images. DAPI was used for nuclearstaining. Scale bar, 20 mm. Cell death was assayed using confocal microscopy, following EGF exposure for indicated time points using YO-Pro, propidiumiodide, and Hoechst dyes in PC3 (F) and Capan1 (G) cells. Immunoblot was performed for cleaved PARP to analyze the induction of cellular apoptosis uponEGF exposure in the NRP2 depletion state for both PC3 (H) and Capan1 (I) cells.

Dutta et al.





integrins are a few of the underlying mechanisms through whichendocytosis promotes oncogenesis and metastasis. Recent evi-dence indicates that altered endocytosis in cancer cells alsopromotes aberrant tyrosine kinase and G-protein–coupled recep-tor signaling, either by recycling them at a faster rate through thecell surface or enabling altered receptor signaling from the endo-somal compartment (27, 28, 46). Thus, it is important to under-stand the mechanisms by which the NRP2 axis regulates endo-cytosis in cancer cells and the effect that the NRP2 axis has onregulating endocytic trafficking of other tyrosine kinase andG-protein–coupled receptors, thereby modifying their functions.Considering that regulation of the endosomal pathway in cancercells is an emerging topic in immunotherapy and nanoparticledelivery, understanding the functional significance of the NRP2–WDFY1 axis is expected to help further therapeutic strategies.

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

Authors' ContributionsConception and design: S. Dutta, H. Zhang, H. Band, M.H. Muders, K. DattaDevelopment of methodology: S. Dutta, H. Zhang, H. Band, K. DattaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Dutta, S. Roy, N.S. Polavaram, H. Zhang, T. BholaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Dutta, S. Roy, N.S. Polavaram, M.J. Stanton,H. Zhang, T.M. Donohue Jr., H. Band, M.H. Muders, K. Datta

Writing, review, and/or revision of the manuscript: S. Dutta, S. Roy,N.S. Polavaram, M.J. Stanton, H. Zhang, P. H€onscheid, T.M. Donohue Jr.,H. Band, S.K. Batra, M.H. Muders, K. DattaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Dutta, K. DattaStudy supervision: S. Dutta, K. Datta

AcknowledgmentsThe authors thank Janice A. Taylor and James R. Talaska at the Confocal Laser

ScanningMicroscope Core Facility at the University of NebraskaMedical Center(UNMC) for providing assistance and the Nebraska Research Initiative (NRI)and the Eppley Cancer Center for their support of the core facility. They alsothank Tom Barger from UNMC Electron Microscopy Core Facility for assistingwith electron microscopy. The authors kindly thank Melody A. Montgomery atthe University of Nebraska Medical Center (UNMC) Research Editorial Officefor the professional editing of this manuscript.

Grant SupportThis study was supported by the following grants: NIH grant CA140432,

CA182435A (K. Datta); CA 163120 (K. Datta and S.K. Batra); CA RO1138791 (S.K. Batra); and Else Kroener Fresenius Stiftung 2012_A169(M.H. Muders).

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 indicatethis fact.

Received June 10, 2015; revised September 22, 2015; accepted October 23,2015; published OnlineFirst November 11, 2015.

References1. Bagri A, Tessier-Lavigne M, Watts RJ. Neuropilins in tumor biology. Clin

Cancer Res 2009;15:1860–4.2. Uniewicz KA, Fernig DG. Neuropilins: a versatile partner of extracellular

molecules that regulate development and disease. Front Biosci 2008;13:4339–60.

3. DallasNA,GrayMJ, Xia L, Fan F, vanBurenG2nd,Gaur P, et al.Neuropilin-2-mediated tumor growth and angiogenesis in pancreatic adenocarcino-ma. Clin Cancer Res 2008;14:8052–60.

4. Caunt M, Mak J, Liang WC, Stawicki S, Pan Q, Tong RK, et al. Blockingneuropilin-2 function inhibits tumor cell metastasis. Cancer Cell 2008;13:331–42.

5. YasuokaH, KodamaR, TsujimotoM, YoshidomeK,AkamatsuH,NakaharaM, et al. Neuropilin-2 expression in breast cancer: correlation with lymphnode metastasis, poor prognosis, and regulation of CXCR4 expression.BMC Cancer 2009;9:220.

6. Karpanen T, Heckman CA, Keskitalo S, JeltschM, Ollila H, Neufeld G, et al.Functional interaction of VEGF-C and VEGF-D with neuropilin receptors.FASEB J 2006;20:1462–72.

7. Fritz WA, Lin TM, Cardiff RD, Peterson RE. The aryl hydrocarbon receptorinhibits prostate carcinogenesis in TRAMP mice. Carcinogenesis 2007;28:497–505.

8. Goel HL, Chang C, Pursell B, Leav I, Lyle S, Xi HS, et al. VEGF/neuropilin-2 regulation of Bmi-1 and consequent repression of IGF-IRdefine a novel mechanism of aggressive prostate cancer. Cancer Discov2012;2:906–21.

9. Beierle EA, Dai W, Langham MR Jr., Copeland EM 3rd, Chen MK. VEGFreceptors are differentially expressed by neuroblastoma cells in culture. JPediatr Surg 2003;38:514–21.

10. Handa A, Tokunaga T, Tsuchida T, Lee YH, Kijima H, Yamazaki H, et al.Neuropilin-2 expression affects the increased vascularization and is aprognostic factor in osteosarcoma. Int J Oncol 2000;17:291–5.

11. Goel HL, Mercurio AM. VEGF targets the tumour cell. Nat Rev Cancer2013;13:871–82.

12. Goel HL, Pursell B, Chang C, Shaw LM, Mao J, Simin K, et al. GLI1regulates a novel neuropilin-2/alpha6beta1 integrin based autocrinepathway that contributes to breast cancer initiation. EMBO Mol Med2013;5:488–508.

13. Bae D, Lu S, Taglienti CA, Mercurio AM. Metabolic stress induces thelysosomal degradation of neuropilin-1 but not neuropilin-2. J Biol Chem2008;283:28074–80.

14. Muders MH, Zhang H, Wang E, Tindall DJ, Datta K. Vascular endothelialgrowth factor-C protects prostate cancer cells from oxidative stress by theactivation of mammalian target of rapamycin complex-2 and AKT-1.Cancer Res 2009;69:6042–8.

15. Stanton MJ, Dutta S, Zhang H, Polavaram NS, Leontovich AA, Hon-scheid P, et al. Autophagy control by the VEGF-C/NRP-2 axis in cancerand its implication for treatment resistance. Cancer Res 2013;73:160–71.

16. Gray MJ, Van Buren G, Dallas NA, Xia L, Wang X, Yang AD, et al.Therapeutic targeting of neuropilin-2 on colorectal carcinoma cellsimplanted in the murine liver. J Natl Cancer Inst 2008;100:109–20.

17. StantonMJ, Dutta S, PolavaramNS, Roy S,MudersMH,Datta K. Angiogenicgrowth factor axis in autophagy regulation. Autophagy 2013;9:789–90.

18. Lanahan A, Zhang X, Fantin A, Zhuang Z, Rivera-Molina F, Speichinger K,et al. The neuropilin 1 cytoplasmic domain is required for VEGF-A-dependent arteriogenesis. Dev Cell 2013;25:156–68.

19. Zhang X, Lanahan AA, Simons M. VEGFR2 trafficking: speed doesn't kill.Cell Cycle 2013;12:2163–4.

20. PangHB, BraunGB, Friman T, Aza-Blanc P, RuidiazME, Sugahara KN, et al.An endocytosis pathway initiated through neuropilin-1 and regulated bynutrient availability. Nat Commun 2014;5:4904.

21. Kohno M, Horibe T, Ohara K, Ito S, Kawakami K. The membrane-lyticpeptides K8L9 and melittin enter cancer cells via receptor endocytosisfollowing subcytotoxic exposure. Chem Biol 2014;21:1522–32.

22. Wang HB, Zhang H, Zhang JP, Li Y, Zhao B, Feng GK, et al. Neuropilin 1 isan entry factor that promotes EBV infection of nasopharyngeal epithelialcells. Nat Commun 2015;6:6240.

23. Ulmasov B, Oshima K, Rodriguez MG, Cox RD, Neuschwander-Tetri BA.Differences in the degree of cerulein-induced chronic pancreatitis inC57BL/6 mouse substrains lead to new insights in identification of poten-tial risk factors in the development of chronic pancreatitis. Am J Pathol2013;183:692–708.

24. Hu YH, Zhang Y, Jiang LQ,Wang S, Lei CQ, SunMS, et al.WDFY1mediatesTLR3/4 signaling by recruiting TRIF. EMBO Rep 2015;16:447–55.






25. Arisi I, D'OnofrioM, Brandi R, Felsani A,Capsoni S,DrovandiG, et al. Geneexpression biomarkers in the brain of a mouse model for Alzheimer'sdisease: mining of microarray data by logic classification and featureselection. J Alzheimers Dis 2011;24:721–38.

26. Sferruzzi-Perri AN, Macpherson AM, Roberts CT, Robertson SA. Csf2 nullmutation alters placental gene expression and trophoblast glycogen celland giant cell abundance in mice. Biol Reprod 2009;81:207–21.

27. Mellman I, Yarden Y. Endocytosis and cancer. Cold Spring Harb PerspectBiol 2013;5:a016949.

28. Mosesson Y,Mills GB, Yarden Y. Derailed endocytosis: an emerging featureof cancer. Nat Rev Cancer 2008;8:835–50.

29. Ridley SH, Ktistakis N, Davidson K, Anderson KE, Manifava M, Ellson CD,et al. FENS-1 and DFCP1 are FYVE domain-containing proteins withdistinct functions in the endosomal and Golgi compartments. J Cell Sci2001;114(Pt 22):3991–4000.

30. ShishevaA. PtdIns5P: news and views of its appearance, disappearance anddeeds. Arch Biochem Biophys 2013;538:171–80.

31. Fader CM,ColomboMI. Autophagy andmultivesicular bodies: two closelyrelated partners. Cell Death Differ 2009;16:70–8.

32. Brix K, Dunkhorst A, Mayer K, Jordans S. Cysteine cathepsins: cellularroadmap to different functions. Biochimie 2008;90:194–207.

33. Capony F, Braulke T, Rougeot C, Roux S, Montcourrier P, Rochefort H.Specific mannose-6-phosphate receptor-independent sorting of pro-cathepsin D in breast cancer cells. Exp Cell Res 1994;215:154–63.

34. Sharma M, Naslavsky N, Caplan S. A role for EHD4 in the regulation ofearly endosomal transport. Traffic 2008;9:995–1018.

35. Tomas A, Futter CE, Eden ER. EGF receptor trafficking: consequences forsignaling and cancer. Trends Cell Biol 2014;24:26–34.

36. Rush JS, Quinalty LM, Engelman L, Sherry DM, Ceresa BP. Endosomalaccumulation of the activated epidermal growth factor receptor (EGFR)induces apoptosis. J Biol Chem 2012;287:712–22.

37. Miao HQ, Klagsbrun M. Neuropilin is a mediator of angiogenesis. CancerMetastasis Rev 2000;19:29–37.

38. Chang YS, Chen WY, Yin JJ, Sheppard-Tillman H, Huang J, Liu YN.EGF receptor promotes prostate cancer bone metastasis by down-regulating miR-1 and activating TWIST1. Cancer Res 2015;75:3077–86.

39. Padfield E, Ellis HP, Kurian KM. Current therapeutic advances targetingEGFR and EGFRvIII in glioblastoma. Front Oncol 2015;5:5.

40. Del Vecchio CA, Jensen KC, Nitta RT, Shain AH, Giacomini CP, WongAJ. Epidermal growth factor receptor variant III contributes to cancerstem cell phenotypes in invasive breast carcinoma. Cancer Res 2012;72:2657–71.

41. Rizzolio S, Rabinowicz N, Rainero E, Lanzetti L, Serini G, Norman J, et al.Neuropilin-1-dependent regulation of EGF-receptor signaling. Cancer Res2012;72:5801–11.

42. Shaughnessy R, Retamal C, Oyanadel C, Norambuena A, Lopez A, Bravo-Zehnder M, et al. Epidermal growth factor receptor endocytic trafficperturbation by phosphatidate phosphohydrolase inhibition: new strategyagainst cancer. FEBS J 2014;281:2172–89.

43. George M, Ying G, Rainey MA, Solomon A, Parikh PT, Gao Q, et al. Sharedas well as distinct roles of EHD proteins revealed by biochemical andfunctional comparisons in mammalian cells and C. elegans. BMC Cell Biol2007;8:3.

44. Ardito CM, Gruner BM, Takeuchi KK, Lubeseder-Martellato C, TeichmannN, Mazur PK, et al. EGF receptor is required for KRAS-induced pancreatictumorigenesis. Cancer Cell 2012;22:304–17.

45. Lanzetti L, Di Fiore PP. Endocytosis and cancer: an `insider' network withdangerous liaisons. Traffic 2008;9:2011–21.

46. Pelekanos RA, Ting MJ, Sardesai VS, Ryan JM, Lim YC, Chan JK, et al.Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymalstem/stromal cells. BMC Cell Biol 2014;15:15.


Dutta et al.




2016;76:418-428. Published OnlineFirst November 11, 2015.Cancer Res Samikshan Dutta, Sohini Roy, Navatha S. Polavaram, et al. to Support Cancer Cell PathobiologyNeuropilin-2 Regulates Endosome Maturation and EGFR Trafficking

Updated version

10.1158/0008-5472.CAN-15-1488doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2015/11/11/0008-5472.CAN-15-1488.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/76/2/418.full#ref-list-1

This article cites 46 articles, 13 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/76/2/418.full#related-urls

This article has been cited by 8 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/76/2/418To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-15-1488

http://cancerres.aacrjournals.org/content/suppl/2015/11/11/0008-5472.CAN-15-1488.DC1

http://cancerres.aacrjournals.org/content/76/2/418.full#ref-list-1

http://cancerres.aacrjournals.org/content/76/2/418.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/76/2/418


Documents

Neuropilin-2RegulatesEndosomeMaturationand EGFR Trafﬁcking to Support … · Therapeutics, Targets, and Chemical Biology Neuropilin-2RegulatesEndosomeMaturationand EGFR Trafﬁcking