Neuropilin-2 Regulates Endosome Maturation and EGFR Trafficking to Support Cancer Cell Pathobiology Samikshan Dutta1, Sohini Roy1, Navatha Shree Polavaram1, Marissa J

Author Manuscript Published OnlineFirst on November 11, 2015; DOI: 10.1158/0008-5472.CAN-15-1488 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Neuropilin-2 Regulates Endosome Maturation and EGFR trafficking to Support Cancer Cell Pathobiology Samikshan Dutta1, Sohini Roy1, Navatha Shree Polavaram1, Marissa J. Stanton1, Heyu Zhang6, Tanvi Bhola1, Pia Hönscheid5, Terrence M. Donohue, Jr.1,2,3, Hamid Band4, Surinder K. Batra1,4, 1,5 1,4 Michael H. Muders and Kaustubh Datta

1Biochemistry and Molecular Biology, 2Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska. 3Nebraska-Western Iowa Health Care System., Omaha VA Medical Center, Buffett Cancer Center, Eppley Cancer Institute4, University of Nebraska Medical center, Omaha, Nebraska 5Institute of Pathology, Faculty of Medicine, Technische Universitaet, Dresden, Germany, 6Department of Urologic Research, Biochemistry, Mayo Clinic College of Medicine.

Running Title: NRP2 axis and endosome maturation

§Corresponding Author: Kaustubh Datta, Ph.D. Department Biochemistry and Molecular Biology, University of Nebraska Medical Center, Durham Research Center II, Room 4022 985870 Nebraska Medical Center Omaha, NE 68198-5870 Tel: (402) 559-7404 Fax: (402) 559-6650 Email: [email protected]

Keywords: WDFY1, NRP2, VEGF-C, endosome, autophagy

Conflicts of Interest: The authors have no conflicts of interest to declare.

Word count (Excluding spaces, Materials, and References):

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 11, 2015; DOI: 10.1158/0008-5472.CAN-15-1488 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract:

Neuropilin-2 (NRP2) is a non-tyrosine kinase receptor frequently overexpressed in various malignancies where it has been implicated in promoting many protumorigenic behaviors, such as imparting therapeutic resistance to metastatic cancer cells. Here, we report a novel function of NRP2 as a regulator of endocytosis, which is enhanced in cancer cells and is often associated with increased metastatic potential and drug resistance. We found that NRP2 depletion in human prostate and pancreatic cancer cells resulted in the accumulation of

EEA1/Rab5-positive early endosomes concomitant with a decrease in Rab7-positive late endosomes, suggesting a delay in early-to-late endosome maturation. NRP2 depletion also impaired the endocytic transport of cell surface epidermal growth factor receptor (EGFR), arresting functionally active EGFR in endocytic vesicles that consequently led to aberrant ERK activation and cell death. Mechanistic investigations revealed that WD-repeat and

FYVE-domain-containing protein 1 (WDFY1) functioned downstream of NRP2 to promote endosome maturation, thereby influencing the endosomal trafficking of EGFR and the formation of autolysosomes responsible for the degradation of internalized cargo. Overall, our results indicate that the NRP2/WDFY1 axis is required for maintaining endocytic activity in cancer cells, which supports their oncogenic activities and confers drug resistance. Therefore, therapeutically targeting endocytosis may represent an attractive strategy to selectively target cancer cells in multiple malignancies.

Introduction

Neuropilins (NRPs) are transmembrane, non-tyrosine kinase receptors. Often, they function as co-receptors to modulate various cellular pathways including angiogenesis, cellular communication, and migration (1,2). Neuropilin-2 (NRP2), a member of the NRP family of receptors, has similar molecular mass and structural domain compared with its family member neuropilin-1 (NRP1) (3). In addition to its role in neuronal development, NRP2 is important for the development of capillaries and lymphatic vessels (4,5). The known binding ligands for NRP2 are vascular endothelial growth factor C (VEGF-C), VEGF-D, VEGF-A, and semaphorin-3F (6).

Importantly, NRP2 is also expressed in various human cancer tissues and cancer cell lines

(1,3,5,7-10), and it is implicated in promoting their proliferation, survival, and migration (11). It is also important in maintaining the tumor initiating population of breast cancer (12). Interestingly,

NRP-2, but not NRP-1, maintains its protein level during metabolic stress, such as nutrient starvation and hypoxia, suggesting that it has a potential role in stress (13). We have previously observed a survival-promoting role of NRP-2 in cancer cells during therapeutic stress (14,15).

Our findings corroborated an earlier report, where depletion of NRP-2 in colon cancer cells increased their death during hypoxia (16).

In this paper, we report a novel function of NRP2 in cancer cells. Our results suggested the role of NRP2 in regulating the maturation of endocytosis. While this function can provide the underlying mechanism of the role of NRP2 in regulating autophagy during therapeutic stress

(14,15,17), NRP2 regulated endosome maturation is also important for the proper function of cell surface receptors, which require endocytic trafficking to maintain optimum activity.

Previously NRP1 was indicated to regulate endocytosis of tyrosine kinase receptors such as

VEGFR2 (18,19). It has been shown that upon ligand binding, VEGFR2 and NRP1 undergo endocytosis as a complex. NRP1 has a C-terminal PDZ-binding site, which helps its interaction with a protein called synectin that links VEGFR2-NRP2 complex to myosin-VI motor proteins.

NRP1 therefore helps VEGFR2 containing endosomes to move away from the plasma membrane. NRP1 also helps endocytosis of CendR peptides or membrane lytic peptides such as K8L9 and melittin (19-21). Recently NRP1 has also been implicated in the internalization of

Epstein-Barr virus (EBV) into the nasopharyngeal epithelial cells (22). In all these circumstances, the receptors, peptides and the virus particles that are endocytosed, directly interact with NRP1 at the cell surface, which facilitates their internalization during endocytosis.

This process is distinctly different from our current findings where NRP2 axis is involved in the maturation of late endocytic vesicles. We have also identified WD-repeat and FYVE-domain- containing protein 1 (WDFY1) as a downstream of NRP2 axis. Few studies have been reported on WDFY1. A recent study indicated WDFY1 as a potential candidate of chronic pancreatitis

(23). Interestingly, WDFY1 has been shown to recruit signaling adaptor TRIF to Toll-like receptors (TLR3 and TLR4), and thereby potentiating signaling necessary for the initiation of innate immune system (24). WDFY1 has also been considered as a potential biomarker for

Alzheimer’s disease (25) and can be involved in placental development and for the maintenance of hematopoietic stem cells (26). In this study, we are reporting a novel NRP2-WDFY1 axis, which are important for the maintenance of endocytic activity in cancer cells. Recent evidence indicates that altered endocytosis promotes aberrant cell surface receptor signaling in cancer cells, either by expediting their recycling to the cell surface or enabling a different receptor signaling program from the endosomal compartment (27,28) We specifically focused on how the

NRP-2 axis regulates the trafficking of EGFR in cancer cells. Interestingly, our results indicated that the inhibition of the NRP-2 axis would lead to the accumulation of ligand-stimulated EGFR in early endosomes and then initiate an apoptosis-promoting signal. Therefore, inhibitors of

NRP-2 should show a potent anti-tumor effect in cancer cells with activated EGFR signaling.

Materials and Methods

Cell culture, plasmid constructs, and transfection reagents: Two metastatic human prostate cancer cell lines [PC3 (CRL1435; ATCC, Manassas, VA) and Du145 (HTB81; ATCC)] and one pancreatic cancer cell line CaPan1 (HTB-79, ATCC; kind gift from Dr. Michael Hollingsworth,

UNMC) that express high levels of NRP2 and VEGF-C were used for the experiments described here. Wild-type WDFY1 (RC509030; Origene, Rockville, MD), NRP2 (RC220706, Origene) plasmids were used to overexpresss these proteins in cancer cells. To deplete the VEGF-C,

NRP2 and WDFY1, siRNAs against VEGF-C (L-012071-00-0020; Dharmacon RNA

Technologies, Chicago, IL; #LQ-012071-00-0005; Qiagen, Valencia, CA), NRP2 (L-017721-00-

0010 and LU-017721-00-005; Dharmacon), and WDFY1 (L-017721-00-0010; Dharmacon) were transfected in cancer cells using Dharmacon reagents (T-2005-02; Dharmacon). Western blot,

ELISA, isolation of membrane protein: Western blot and ELISA were performed using established techniques (see supplemental materials). For isolation of cell membrane fractions, cells were washed and homogenized in ice-cold lysis buffer (25 mM HEPES, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, pH 7.4, and protease inhibitor cocktail) in a glass Dounce homogenizer for 30 to 40 vertical strokes. The homogenized lysate was then subjected to centrifugation (800g for 10 min) to remove cell debris and isolate the nucleus (pellet). The supernatant was then subjected to ultracentrifugation (108,000g for 1 hour at 4°C) and membrane (pellet) and cytosolic (supernatant) fractions were collected. The pellet was resuspended in urea buffer (70 mM Tris–Hcl, pH 6.8, 8 M urea, 10 mM n-ethylmaleimide, 10 mM iodoacetamide, 2.5% SDS and 0.1 M DTT) at 37°C for 15 minutes. Proteins were analyzed using Western blot with the following antibodies: LC3B (2775; Cell Signaling Technology, Inc.;

Danvers, MA), Rab5 (2143; Cell Signaling), Rab7 (9367; Cell Signaling), Caveolin (3238; Cell

Signaling), Myc-Tag (2276, Cell Signaling), EGFR (4267, Cell Signaling), phosphor-ERK (4370,

Cell Signaling), ERK (4695, Cell Signaling), PARP (9532, Cell Signaling), EHD4 (ab153892,

Abcam), NRP2 (#sc-13117; Santa Cruz Biotechnology; Santa Cruz, CA; AF2215, R&D system,

MN, USA), Rho-GDI (sc-360; Santa Cruz), 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, Santa Cruz), PIKfyve (sc-100408; Santa Cruz), WDFY1

(123058; GeneTex Inc.; Irvine, CA and SAB2106120, Sigma-Aldrich), .

Confocal and electron microscopy: Cells were grown on poly-D/L lysine-coated cover slips (BD

Biosciences, Sparks, MD) for 72 hours before fixation and confocal analysis. Cells were rinsed with Dulbecco’s phosphate-buffered saline (DPBS; Invitrogen), followed by fixation with 4% paraformaldehyde at room temperature for 10 minutes. Later, cells were washed with DPBS and treated with ice-cold methanol for 20 minutes at -20°C. Finally, cells were blocked using 1%

Bovine Serum Albumin (BSA) and 0.2% saponin. All confocal images were captured using a

Zeiss 710 Confocal Laser Scanning Microscope (equipped with four lasers), and data were analyzed and processed with Zeiss Zen 2010 software.

For electron microscopy, cells were cultured in 25 mm poly-DL-lysine coated cover-slips (BD

Biosciences) and fixed with 2% glutaraldehyde (Sigma-Aldrich) containing 2%-paraformaldehyde solution in 0.1M Sorensen’s phosphate buffer (Sigma-Aldrich) for 30 minutes at room temperature followed by vigorous washing with 0.2 M HEPES, pH 7.4, at room temperature for 1 hour. Samples were post-fixed in 1% osmium tetroxide, aqueous solution

(Sigma-Aldrich), for 30 minutes. Samples were washed three times in buffer, followed by dehydration with 50%, 70%, 90%, 95%, and 100% ethanol. The dehydrated samples were then passed through a series of graded Araldite/ethanol mixtures, (1:2, 1:1, and 2:1) (Sigma-Aldrich), before being embedded onto Araldite blanks and stored at 65°C overnight for polymerization.

Next, cell culture colonies were excised and adhered to an Araldite blank block for thin sectioning. Thin sections were collected onto 200 mesh copper grids and stained with 1%

Uranyl Acetate (Sigma-Aldrich) and Reynolds Lead Citrate (Sigma-Aldrich). Sections were examined on an FEI Tecnai™ G2 TEM (FEI Company; Oregon, USA) operated at 80kV.

Results

Inhibition of the VEGF-C/NRP2 axis prevents the formation of autolysosomes from autophagosomes

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

NRP2-depleted cells compared with scrambled siRNA-transfected cells (Fig 1). This became even more apparent after continuous monitoring of autophagosome and autolysosome formation in the stable clones using time-lapse video microscopy (Supplementary video S1).

Moreover, transmission-electron microscopy analysis (Supplementary Fig S1A) revealed that after NRP2 depletion, the number of electron-dense autophagosomes (indicated by yellow arrows) increased compared to controls. These results suggest that inhibition of the NRP2 axis delays the formation of autolysosome and thus, slows the lysosomal degradation of the sequestered cargo.

Effect of expression of WDFY1 on the formation of autolysosomes

We previously reported that expression of WDFY1 increases after depletion of either

NRP2 or its ligand VEGF-C (15). We hypothesized that WDFY1 acts downstream of NRP2 in regulating the formation of autolysosomes. Simultaneous depletion of NRP2 and WDFY1 partially rescued defective autophagosomal maturation, as detected using confocal imaging (Fig

1). Western blot measured the autophagic flux and verified the findings of the confocal imaging

(Supplementary Fig S1B). Our findings therefore indicate that WDFY1, as a downstream target of the VEGF-C/NRP2 axis, prevents the formation of autolysosomes.

The role of the NRP2/WDFY1 axis in endosome maturation

PC3 cells showed the presence of WDFY1 in EEA1-positive early endosomes

(Supplementary Fig S1C); a similar observation was also previously reported for WDFY1 (29).

We hypothesized that the NRP2/WDFY1 axis regulates endosome maturation and thereby regulates the formation of autolysosome. A rise in EEA1-positive puncta in NRP2 and VEGF-C depleted cells were observed suggesting an increase in early endosome number (Fig 2A).

Although the circularity of the puncta remained unchanged, 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-positive early endosomes. Simultaneous depletion of NRP2 and WDFY1 partially restored the defect in EEA1-positive puncta, thus supporting the hypothesis that WDFY1 acts as downstream of NRP2 in regulating endocytosis (Fig 2A and

2B). Similar to EEA1, depletion of NRP2 increased the number and size of puncta that were positive for Rab5, another early endosome marker (Supplementary Fig S1D). Once again, simultaneous depletion of NRP2 and WDFY1 restored the wild-type phenotype for Rab5- positive vesicles. Similar to PC3, Capan1 (pancreatic cancer cell line) and Du145 (prostate cancer cell line), which maintain higher expression of endogenous NRP2, also showed similar defect in early endosomes following NRP2 depletion (Supplementary Fig S2A, S2B). Once again, simultaneous depletion of NRP2 and WDFY1 rescued the defects caused by NRP2 depletion alone (Supplementary Fig S2B). To rule out the off-target effect of siRNAs, we exogenously expressed NRP2 in siNRP2 transfected cells and recovered the wild type phenotype of early endosomes (Supplementary Fig S2A, S2C). Over-expression of full length

NRP2 in siNRP2 transfected cells down-regulates the WDFY1 protein in the basal level

(Supplementary Fig S2D), indicating that upregulation of WDFY1 abrogates the endosomal function and acts downstream of NRP2 axis.

To determine if deregulation of the NRP2 axis can affect late endosomes, vesicles that were positive for the late endosome marker Rab7 were examined. Interestingly, depletion of

NRP2 or VEGF-C reduced the Rab7-positive punctae (Fig 2C). The control phenotype was once again restored either after simultaneous depletion of NRP2 and WDFY1 or by overexpressing full length NRP2 in all the cell lines (Fig 2C and Supplementary Fig S3A-S3C).

Knockdown efficiency of NRP2 in PC3 cells was shown in Fig 2D.

To confirm the observations made during immunostaining, immunoblot analysis of the total cellular and membrane fraction was conducted to identify functionally active endosomal markers. Here, after depleting either VEGF-C or NRP2 (Fig 3A), an increased level of membrane-associated EEA1 and a simultaneous decrease in membrane-bound Rab7 were observed. No change in the total cellular content of EEA1 and Rab7 proteins occurred

(Supplementary Fig S4A; supplementary Figures S4B and S4C show the efficiency of knockdown of VEGF-C and NRP2 in PC3). A similar increase in the level in membrane-bound

EEA1 was observed using two independent VEGF-C and NRP2 siRNAs (Supplementary Fig

S4D and S4G). Results from RT-PCR and ELISA show the relative knockdown efficiency of the individual siRNAs (Supplementary Fig S4E, S4F and S4H). Similar to PC3, Du145 showed the similar phenotype in immunoblot where membrane bound EEA1 increases and Rab7 decreases following the knockdown of NRP2 (Supplementary Fig S4I).

Finally, following NRP2 knockdown, membrane recruitment of FYVE domain-containing phosphoinositide kinase (PIKfyve) was also assessed. PIKfyve is recruited during the maturation of early to late endosomes and synthesizes PtdIns-3, 5-P2 from PtdIns-3-P (30).

Both immunoblot and immunostaining analyses showed a decrease in active membrane-bound

PIKfyve (Fig 3B and 3C and Supplementary Fig S5A). However, the total PIKfyve remain unchanged (Supplementary Fig S5Band S5C). Together, these results suggest that inhibition of the NRP2 axis alters the transition of early to late endosomes. Notably, autophagosomes often

merge with late endosomes to form amphisomes prior to fusing with lysosomes (31). We therefore propose that, following inhibition of the NRP2 axis, the decrease in late endosomes interferes with the crosstalk between autophagosomes and mature endosomes, which in turn, delays the formation of autolysosomes.

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

To further validate that depletion of the NRP2 axis affects endosome maturation, the golgi-to-lysosome trafficking of various cathepsins was examined. Cathepsin precursors are synthesized in the endoplasmic reticulum (ER), trafficked to the Golgi apparatus, and reach the lysosomes through the early-late endosome-lysosome maturation process (32). We postulated that, following disruption of the NRP2 axis, a decrease in the number of late endosomes will enhance the accumulation of cathepsin-containing cargo in early endosomes. Following knockdown of the NRP2 axis, a significant increases in cathepsins in EEA1-positive vesicles

(yellow and greenish-yellow punctae) were observed (Fig 4A and 4B and Supplementary Fig

S6A and S6B). In contrast, simultaneous knock down of NRP2 and WDFY1 in PC3 and Du145 cells resulted in reduced accumulation of cathepsin-positive punctae in early endosomes (Fig

4A, 4B and Supplementary Fig S6A-S6C).

Cathepsins become mature and catalytically active within the acidic pH of the late endosome and lysosome (32). Therefore, we expected a reduction in the mature cathepsins in the cell following NRP2 depletion. Western blot analyses revealed decreased levels of mature cathepsin D and B following the depletion of NRP2 (Fig 4C), indicating that maturation of cathepsin was hindered. Interestingly, simultaneous depletion of NRP2 and WDFY1 rescued the level of mature cathepsins (Fig. 4C). Overall, these results indicate that blocking of early to late endosome maturation inhibits the delivery of certain lysosomal enzymes like cathpsins. (33)

The underlying mechanism of NRP2/WDFY1 axis-regulated endosomal trafficking

The EH-Domain Containing 4 (EHD4) protein is a regulator of endocytosis and is located in early endosomes (34). Endogenous depletion of EHD4 has been shown to result in a similar increased in EEA1- and Rab5-positive early endosomes, with a concomitant defect in trafficking of cargo from early to late endosomes and lysosomes (34). Considering that a similar defect in endocytosis was observed following the depletion of NRP2, the potential crosstalk between

NRP2 axis and EHD4 was examined in prostate and pancreatic cancer cells. A decrease of membrane-bound EHD4 occurred when NRP2 was depleted (Fig 5A); the total level of EHD4 remained unchanged within the cancer cells (Fig 5B). Further, immunostaining of EHD4 showed a significant decrease in EHD4-positive peripheral punctae following the depletion of NRP2 in both PC3 (Fig 5C, 5D) and Capan1 (Fig 5E) cell lines. The EHD-positive punctae were rescued either by simultaneous depletion of NRP2 and WDFY1 (Fig 5C) or overexpression of full length

NRP2 (Fig 5D and 5E). Our results indicate that NRP2 maintains WDFY1 expression in the cell and thereby regulates the level of EHD4 in the membrane, thus influencing the endocytic pathway in cancer cells.

Depletion of the NRP2 axis and altered trafficking of Cell surface receptors

Cancer promoting functions of several cell surface receptors depend on their ability to endocytose within the cell. Epidermal growth factor receptor (EGFR) is one such tyrosine kinase growth factor receptors, which is often overexpressed and mutated in cancer cells. EGFR controls cancer cell proliferation, survival, invasion, angiogenesis and metastasis (7,35). In a recent report, it has been suggested that EGF-induced chemotactic migration of metastatic cancer cells depends on their ability to coordinate the endocytosis of EGFR (36). Functionally active endocytosis can carefully regulate the availability of EGFR and other cell adhesion molecules on the cell surface, which is required for the directionality and magnitude of the

migration of metastatic cancer cells. Therefore inhibition of endocytosis imparts a negative effect on chemotactic cell migration. Interestingly, NRP2 is also known in regulating the migration of neuronal and endothelial cells (37). Many metastatic cancer cells which express significantly high level of NRP2 also showed higher activity for EGFR such as metastatic prostate cancer cells, glioblastoma and breast cancers (38-40). Neuropilin is also known to modulate the downstream signaling axis of EGFR (41). We therefore speculate that the implication of inhibiting NRP2 axis would be to deregulate the endocytic process of EGFR in cancer cells and therefore hinder its tumorogenic activity.

An EGFR internalization assay was performed in PC3 cells following a brief exposure of its ligand EGF and the role of NRP2 axis in EGFR endocytosis was investigated. As shown in

Figure 6A-C, EGF exposure to PC3 cells led to internalization of EGFR in control cells; over time, the internalized EGFR was transported to lysosomes and then mostly degraded within 5 hours after EGF stimulation. However, in NRP2 knockdown cells, a sufficient amount of EGFR still remained after 5hrs chase following EGF exposure; indicating a delayed degradation of

EGFR receptor (Fig 6A). Immunoblot analysis indicated that these internalized EGFR remained phosphorylated (Tyr 1173) and thus active even after 5hrs of EGF stimulation following NRP2 depletion (Fig 6B). Moreover, we found that most of these EGFR-positive vesicles remained co- localized within EEA1 after 5 hours of EGF stimulation following the overexpression of WDFY1

(Fig 6C and Supplementary Fig S7A), suggesting an extended presence of EGFR in the early endosomes. Similar results were observed in CaPan1 (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 the trafficking of other receptors, we monitored the transport of transferrin (TF) receptor following the NRP2 knockdown. Similar to

EGFR, TF receptor were present in the EEA1 (Fig 6E) positive early endosomes for prolonged period. Our result therefore suggested that depletion of NRP2 axis affects the recycling of TF receptor to the plasma membrane.

In summary, our results indicate that depletion of NRP2 interfere the cargo sorting at early endosomes and thereby inhibits both lysosomal delivery and trafficking of cell surface receptors.

Sustained EGF-induced ERK phosphorylation and cell death following NRP2 depletion.

To understand the effect of prolonged growth factor exposure on cellular signaling, PC3 and

CaPan1 cells were incubated with EGF at 20ng/ml concentration for 48hrs. Under these conditions, we observed an increased ERK phosphorylation in both the cell lines following the depletion of either NRP2 or overexpressing WDFY1 (Fig 7A-7C). Previous reports indicated

ERK phosphorylation by functionally active EGFR when present in early endosomes for an extended time period (36). We also observed a significant increase in active EGFR (pEGFR

1173) in EEA1-positive early endosomes located in the peri-nuclear region in NRP2 knockdown

PC3 and CaPan1 cells (Fig 7D-E), suggesting presence of endosomal active EGFR over a prolonged period due to the depletion of NRP2 axis. Earlier reports indicated that prolong intracellular EGFR signaling was often responsible for the induction of apoptosis (36,42); where hyper-activation of ERK was described as an underlying mechanism of apoptosis induction.

Therefore, we tested whether NRP2 depletion in EGF-stimulated cancer cells can induce death.

Following EGF stimulation, we observed a significant increase in apoptotic death in NRP2 depleted cancer cells (Fig 7F-G). An increase in PARP cleavage under NRP2 depleted condition further confirmed induction of apoptosis in EGF-stimulated cancer cells (Fig 7H-I).

Overall our results suggest that inhibition of NRP2 in prolonged EGF-stimulated cancer cells arrests the phosphorylated EGFR in early endocytosed vesicles; inducing ERK driven cell death.

Discussion

Overall, we propose a novel function of the NRP2 axis in cancer cells. The increases in

EEA1- and Rab5-positive punctae and simultaneous decreases in Rab7-positive punctae in siNRP2- or siVEGF-C-treated cells suggest a defect in the maturation of early endosomes. This was further supported by observations that depletion of the NRP2 axis caused accumulation of cathepsin-containing cargos in early endosomes, and decreased PIKfyve recruitment to the membrane. Autophagosomes often merge with late endosomes to form amphisomes prior to fusing with lysosomes (31). We therefore propose that, following inhibition of the NRP2 axis, the decrease in late endosomes interferes with the crosstalk between autophagosome and matured endosomes, which in turn delays the formation of autolysosomes.

We hypothesized that WDFY1 acts as a downstream regulator of NRP2 axis as its expression increases after depletion of either VEGF-C or NRP2 (15). The ability of simultaneous depletion of NRP2 and WDFY1 to restore the defect in endosome maturation supports our hypothesis. Although, limited information is currently available on WDFY1, a study by Arisi et al. suggested that WDFY1 is a potential biomarker for Alzheimer’s disease (25). Because deregulation of autophagy is linked to neurodegenerative diseases like Alzheimer’s (25), the potential role of WDFY1 in inhibiting autophagy, as suggested here, may explain this connection.

The decrease of membrane-bound EHD4 suggests that the function of EHD4 becomes defective after the NRP2 axis is inhibited. Considering that EHD4 is an important regulator in the transport of cellular cargo from early to late endosome (34,43), the ability of NRP2 to regulate the function of EHD4 can be considered as an underlying mechanism for how the NRP2 axis regulates endocytic maturation. The rescue of the EHD4-positive punctae by simultaneous depletion of NRP2 and WDFY1 indicates that WDFY1 is important for the cellular regulation of

EHD4. We speculate that an optimum level of EHD4 is needed in the membrane for its proper function, and the NRP2/WDFY1 axis thereby regulates the membrane level of EHD4, which

influences the transition from early to late endosomes. WDFY1 strongly interacts with the

PtdIns-3-P, which is an important membrane lipid of endocytic vesicles. It is possible that increased presence of WDFY1 in the membrane inhibits the recruitment of EHD4, a hypothesis needs to be tested in future studies. Since NRP2-WDFY1-EHD4 axis has no influence in cargo delivery to early endosomes, they become enriched with the incoming cargos with large vesicular structure. Therefore NRP2 depletion can cause significant defect in receptor trafficking especially in cancer cells. Due to delayed turn-over, endocytosed receptors can be activated in early endosomes for a prolonged period, which can promote an aberrant receptor signaling.

We have shown that tyrosine-kinase receptor, EGFR is present in the early endosomal compartment of cells for an extended period following the depletion of NRP2. Sustained activation of EGFR in this early endosomal compartment has been shown to stimulate apoptosis and therefore can be detrimental to metastatic cells (36). Currently, several anti-EGFR drugs are approved by the FDA; however, a mixed outcome has been reported. It has now been well documented that spatial localization of EGFR is important for maintaining metastatic behavior of cancer cells. Plasma membrane bound EGFR has been shown to promote the cell survival by activating anti-apoptotic AKT pathway as well as induce the migration and proliferation by upregulating the Ras-MAPK pathway. The maintenance of this spatial distribution is also important for maintaining its Ras driven tumorigenicity. However, the internalized EGFR or endocytosed EGFR reverting the signaling cascade and is responsible for cellular apoptosis. It has been shown that, therapeutic efficacy of Cituximab, an anti-EGFR drug, induces EGFR endocytosis to perturb its cytotoxic effect. Moreover, chemotherapeutic efficacy was increased in the presence of internalized EGFR and the drugs that induce or arrest internalized EGFR actually had a better efficiency in chemotherapy outcome (35,36,44). In this context, we here showed that depletion of NRP2 arrested the internalized EGFR in early endosomes. It thereby

caused hyperactivation of ERK signaling and induced cell death. Thus NRP2 depletion can be a potential mode of therapy for cancers with prominent EGFR signaling cascade.

The endocytic process is enhanced and altered in cancer cells, thus facilitating tumor growth, the epithelial-mesenchymal transition (EMT), evasion of apoptosis, and metastasis

(28,45). Rapid degradation of E-cadherin, aberrant trafficking, and recycling of integrins are a few of the underlying mechanisms through which endocytosis promotes oncogenesis and metastasis. Recent evidence indicates that altered endocytosis in cancer cells also promotes aberrant tyrosine kinase and G-protein-coupled receptor signaling, either by recycling them at a faster rate through the cell surface or enabling altered receptor signaling from the endosomal compartment (27,28,46). Thus, it is important to understand the mechanisms by which the

NRP2 axis regulates endocytosis in cancer cells and the effect that the NRP2 axis has on regulating endocytic trafficking of other tyrosine kinase and G-protein-coupled receptors, thereby modifying their functions. Considering that regulation of the endosomal pathway in cancer cells is an emerging topic in immunotherapy and nanoparticle delivery, understanding the functional significance of the NRP2-WDFY1 axis is expected to help further therapeutic strategies.

Acknowledgements: We thank Janice A. Taylor and James R. Talaska at the Confocal Laser

Scanning Microscope Core Facility at the University of Nebraska Medical Center (UNMC) for providing assistance and the Nebraska Research Initiative (NRI) and the Eppley Cancer Center for their support of the core facility. We also thank Tom Barger from UNMC Electron Microscopy

Core Facility for assisting electron microscopy. This study was supported by the following grants: NIH grant CA140432, CA182435A (K Datta); CA 163120 (K.Datta and S. Batra); CA

RO1 138791 (S.Batra); Else Kroener Fresenius Stiftung 2012_A169 (MHM). The authors kindly

thank Melody A. Montgomery at the University of Nebraska Medical Center (UNMC) Research

Editorial Office for the professional editing of this manuscript.

References:

1. Bagri A, Tessier-Lavigne M, Watts RJ. Neuropilins in tumor biology. Clin Cancer Res 2009;15(6):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. Dallas NA, Gray MJ, Xia L, Fan F, van Buren G, 2nd, Gaur P, et al. Neuropilin-2-mediated tumor growth and angiogenesis in pancreatic adenocarcinoma. Clin Cancer Res 2008;14(24):8052-60. 4. Caunt M, Mak J, Liang WC, Stawicki S, Pan Q, Tong RK, et al. Blocking neuropilin-2 function inhibits tumor cell metastasis. Cancer Cell 2008;13(4):331-42. 5. Yasuoka H, Kodama R, Tsujimoto M, Yoshidome K, Akamatsu H, Nakahara M, et al. Neuropilin-2 expression in breast cancer: correlation with lymph node metastasis, poor prognosis, and regulation of CXCR4 expression. BMC Cancer 2009;9:220. 6. Karpanen T, Heckman CA, Keskitalo S, Jeltsch M, Ollila H, Neufeld G, et al. Functional interaction of VEGF-C and VEGF-D with neuropilin receptors. Faseb J 2006;20(9):1462-72. 7. Fritz WA, Lin TM, Cardiff RD, Peterson RE. The aryl hydrocarbon receptor inhibits prostate carcinogenesis in TRAMP mice. Carcinogenesis 2007;28(2):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-IR define a novel mechanism of aggressive prostate cancer. Cancer Discov 2012;2(10):906-21. 9. Beierle EA, Dai W, Langham MR, Jr., Copeland EM, 3rd, Chen MK. VEGF receptors are differentially expressed by neuroblastoma cells in culture. J Pediatr Surg 2003;38(3):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 a prognostic factor in osteosarcoma. Int J Oncol 2000;17(2):291-5. 11. Goel HL, Mercurio AM. VEGF targets the tumour cell. Nat Rev Cancer 2013;13(12):871-82. 12. Goel HL, Pursell B, Chang C, Shaw LM, Mao J, Simin K, et al. GLI1 regulates a novel neuropilin- 2/alpha6beta1 integrin based autocrine pathway that contributes to breast cancer initiation. EMBO Mol Med 2013;5(4):488-508. 13. Bae D, Lu S, Taglienti CA, Mercurio AM. Metabolic stress induces the lysosomal degradation of neuropilin-1 but not neuropilin-2. J Biol Chem 2008;283(42):28074-80. 14. Muders MH, Zhang H, Wang E, Tindall DJ, Datta K. Vascular endothelial growth factor-C protects prostate cancer cells from oxidative stress by the activation of mammalian target of rapamycin complex-2 and AKT-1. Cancer Res 2009;69(15):6042-8.

15. Stanton MJ, Dutta S, Zhang H, Polavaram NS, Leontovich AA, Honscheid P, et al. Autophagy Control by the VEGF-C/NRP-2 Axis in Cancer and Its Implication for Treatment Resistance. Cancer Res 2013;73(1):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 cells implanted in the murine liver. J Natl Cancer Inst 2008;100(2):109-20. 17. Stanton MJ, Dutta S, Polavaram NS, Roy S, Muders MH, Datta K. Angiogenic growth factor axis in autophagy regulation. Autophagy 2013;9(5):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(2):156- 68. 19. Zhang X, Lanahan AA, Simons M. VEGFR2 trafficking: speed doesn't kill. Cell Cycle 2013;12(14):2163-4. 20. Pang HB, Braun GB, Friman T, Aza-Blanc P, Ruidiaz ME, Sugahara KN, et al. An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability. Nat Commun 2014;5:4904. 21. Kohno M, Horibe T, Ohara K, Ito S, Kawakami K. The membrane-lytic peptides K8L9 and melittin enter cancer cells via receptor endocytosis following subcytotoxic exposure. Chem Biol 2014;21(11):1522-32. 22. Wang HB, Zhang H, Zhang JP, Li Y, Zhao B, Feng GK, et al. Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells. 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 in C57BL/6 mouse substrains lead to new insights in identification of potential risk factors in the development of chronic pancreatitis. Am J Pathol 2013;183(3):692-708. 24. Hu YH, Zhang Y, Jiang LQ, Wang S, Lei CQ, Sun MS, et al. WDFY1 mediates TLR3/4 signaling by recruiting TRIF. EMBO Rep 2015;16(4):447-55. 25. Arisi I, D'Onofrio M, Brandi R, Felsani A, Capsoni S, Drovandi G, et al. Gene expression biomarkers in the brain of a mouse model for Alzheimer's disease: mining of microarray data by logic classification and feature selection. J Alzheimers Dis 2011;24(4):721-38. 26. Sferruzzi-Perri AN, Macpherson AM, Roberts CT, Robertson SA. Csf2 null mutation alters placental gene expression and trophoblast glycogen cell and giant cell abundance in mice. Biol Reprod 2009;81(1):207-21. 27. Mellman I, Yarden Y. Endocytosis and cancer. Cold Spring Harb Perspect Biol 2013;5(12):a016949. 28. Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: an emerging feature of cancer. Nat Rev Cancer 2008;8(11):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 with distinct functions in the endosomal and Golgi compartments. J Cell Sci 2001;114(Pt 22):3991-4000. 30. Shisheva A. PtdIns5P: news and views of its appearance, disappearance and deeds. Arch Biochem Biophys 2013;538(2):171-80. 31. Fader CM, Colombo MI. Autophagy and multivesicular bodies: two closely related partners. Cell Death Differ 2009;16(1):70-8. 32. Brix K, Dunkhorst A, Mayer K, Jordans S. Cysteine cathepsins: cellular roadmap to different functions. Biochimie 2008;90(2):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(1):154-63. 34. Sharma M, Naslavsky N, Caplan S. A role for EHD4 in the regulation of early endosomal transport. Traffic 2008;9(6):995-1018. 35. Tomas A, Futter CE, Eden ER. EGF receptor trafficking: consequences for signaling and cancer. Trends Cell Biol 2014;24(1):26-34. 36. Rush JS, Quinalty LM, Engelman L, Sherry DM, Ceresa BP. Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis. J Biol Chem 2012;287(1):712-22. 37. Miao HQ, Klagsbrun M. Neuropilin is a mediator of angiogenesis. Cancer Metastasis Rev 2000;19(1-2):29-37. 38. Chang YS, Chen WY, Yin JJ, Sheppard-Tillman H, Huang J, Liu YN. EGF Receptor Promotes Prostate Cancer Bone Metastasis by Downregulating miR-1 and Activating TWIST1. Cancer Res 2015;75(15):3077-86. 39. Padfield E, Ellis HP, Kurian KM. Current Therapeutic Advances Targeting EGFR and EGFRvIII in Glioblastoma. Front Oncol 2015;5:5. 40. Del Vecchio CA, Jensen KC, Nitta RT, Shain AH, Giacomini CP, Wong AJ. Epidermal growth factor receptor variant III contributes to cancer stem cell phenotypes in invasive breast carcinoma. Cancer Res 2012;72(10):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 Res 2012;72(22):5801-11. 42. Shaughnessy R, Retamal C, Oyanadel C, Norambuena A, Lopez A, Bravo-Zehnder M, et al. Epidermal growth factor receptor endocytic traffic perturbation by phosphatidate phosphohydrolase inhibition: new strategy against cancer. FEBS J 2014;281(9):2172-89. 43. George M, Ying G, Rainey MA, Solomon A, Parikh PT, Gao Q, et al. Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans. BMC Cell Biol 2007;8:3. 44. Ardito CM, Gruner BM, Takeuchi KK, Lubeseder-Martellato C, Teichmann N, Mazur PK, et al. EGF receptor is required for KRAS-induced pancreatic tumorigenesis. Cancer Cell 2012;22(3):304-17. 45. Lanzetti L, Di Fiore PP. Endocytosis and cancer: an 'insider' network with dangerous liaisons. Traffic 2008;9(12):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 mesenchymal stem/stromal cells. BMC Cell Biol 2014;15:15.

Figure Legends

Figure 1: Simultaneous depletion of WDFY1 and NRP2 rescue autophagy inhibition.

Autophagic activity was detected using confocal microscopy for PC3 cells that stably expressed

GFP-mCherry-LC3B plasmids after 48 hours of transfection either with scrambled, siNRP2 or with siNRP2 and siWDFY1 simultaneously. The percentage of autophagosomes (yellow puncta)

(autophagosomes vs. sum of autolysosmes and autophagosomes per field) was quantified and

represented graphically. Scale bar 20µm. DAPI was used for nuclear staining. Images within the boxed region were magnified to show the distribution of autophagosomes (yellow) and autolysosomes (red).

Figure 2: Depletion of VEGF-C/NRP2 inhibits early to late endosome maturation. (A)

Immunostaining of early endosome marker EEA1 (Green) following the depletion of VEGF-C and NRP2 in PC3 cells. Recovery experiments with simultaneous depletion of NRP2 and

WDFY1 were also conducted under each condition. Immunostaining data was quantitated using image J software and represented as a bar graph. Asterisks indicate statistically significant differences (p = 0.006 and p = 0.02, respectively, for siNRP2 and siVEGF-C compared to control). The inset represents the magnified image of each panel. (B) Following NRP2 or VEGF-

C depletion or double knockdown with siNRP2 and siWDFY1, EEA1 puncta was determind. The diameter of the EEA1 punctae was calculated using image J software, and represented as 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 simultaneous depletion of NRP2 and WDFY1, in

PC3 cells. Quantitation of staining data is represented graphically. Asterisks represent the statistically significant differences in Rab7 punctae (p = 0.0001 and 0.04 for siNRP2 and siVEGF-C, respectively). Magnified areas are represented under each panel. Scale bars indicate 20 µm in length for all immunostaining images. DAPI was used for nuclear staining. (D)

Western blot showing the NRP2 depletion in PC3 following siNRA transfection.

Figure 3: Depletion of VEGF-C/NRP2 inhibits the transition to late endosome. (A)

Membrane fractions were analyzed for EEA1 and Rab7 following VEGF-C/NRP2 depletion in

PC3 cells. Caveolin was used as a loading control for the membrane fraction. (B) Immunoblot

was performed to analyze membrane PIKfyve following NRP2 depletion (C) Immunostaining of

PIKfyve (green) was carried out following the depletion of VEGF-C/NRP2. Recovery of PIKfyve- positive punctae was performed via simultaneous depletion of NRP2 and WDFY1. The insets represent the magnified images of the cells. DAPI was used for nuclear staining. Quantitation of total PIKfyve fluorescence intensity is represented graphically. Asterisks indicate significant statistical differences (p = 0.00012, p = 0.0005, p = 0 .00001 for siNRP2, siVEGF-C, and

WDFY1 overexpressing samples, respectively, compared to control). The scale bar indicated is

10 µm in length.

Figure 4: Depletion of NRP2 inhibits endocytic trafficking of cathepsins. Co-localization of

Cathepsin (red) with early endosomal marker EEA1 (green) was analyzed with either depletion of NRP2 alone or depletion of WDFY1 and NRP2 simultaneously, for Cathepsin D (A) and

Cathepsin B in PC3 cells. (B) The scale bar indicated is 50µm in length. DAPI was used for nuclear staining. Quantitation of the immunostaining data is represented graphically. Asterisk indicates 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 PC3 lysates following the depletion of NRP2 alone or simultaneous depletion of NRP2 and WDFY1.

Figure 5: Mechanism of endocytic regulation: Membrane (A) and total (B) fraction of EHD4 was analyzed following NRP2 depletion in PC3 cells. (C) Immunostaining of EHD4 (green) following depletion of NRP2 alone or double depletion of NRP2 and WDFY1 simultaneously.

DAPI was used for nuclear staining. Scale bar 20µm. Using image J software, intensity of the green punctae was calculated for each assay condition. Quantitation is represented graphically.

Asterisks indicate significant statistical differences (p values <0.0001 in both cases).

Immunostaining of EHD4 was performed following the overexpression of full length NRP2 in

NRP2 depleted sample 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) in the cells. Scale bar indicates 20µm in length. DAPI was used for nuclear staining.

Figure 6: Depletion of NRP2 arrests cell surface receptors in early endosomes following brief stimulation with EGF: Following activation with EGF (20ng/ml for 15min at 37˚C), EGFR

(green) degradation was monitored over time. (A) Immunostaining was performed to monitor the

EGFR trafficking at various time intervals following NRP2 depletion in PC3 cells. The bar indicated is 20 µm in length. DAPI was used for nuclear staining. Insets represent the magnified images of cells at each time point. (B) PC3 cells were doped with 20ng/ml EGF for 15min at

37˚C. Following this incubation, media was replaced with normal growth media for chasing the status of phospho-EGFR and total EGFR at various time intervals. PC3 cells were lysed at each time point and immunoblot was performed to compare the status of phospho-EGFR and total

EGFR following depletion of NRP2. (C) Following WDFY1 overexpression, EGFR degradation was monitored over 5hrs time period after EGF activation. Early endosome marker EEA1 was counterstained with EGFR (Supplementary Fig S3A). Colocalization of internalized EGFR with

EEA1 was quantified in control and WDFY1 over-expressed samples at various time points following the EGFR chase and represented here as a bar graph. Asterisk indicates significant statistical difference (p = 0.0014). (D) Similar to PC3 cells, EGFR degradation was monitored in

Capan1 cells following the short exposure to EGF (20ng/ml for 15min at 37˚C) at various time points. Scale Bar indicates 10µm in length. DAPI was used for nuclear staining. (E) Transferrin

Receptor (TF, Tagged with Alexa 633 fluorophore) internalization analyzed following 1hr serum starvation in PC3 cells. Cells were incubated with the labeled-TF (20ng/ml) for 5min at 37˚C, after which the 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 45min of chase. DAPI was used for nuclear staining. Scale bar indicates 50 µm in length. Inset was magnified to show the trafficking of TF at various time points. Asterisks indicate the differences were significant (p=0.003, 0.005 and 0.0001 for 10, 20 and 45min respectively).

Figure 7: Prolonged exposure to EGF following NRP2 depletion induces cell death:

Immunoblot was performed to analyze ERK phosphorylation following either NRP2 depletion or overexpressing WDFY1 under continuous exposure to EGF (20ng/ml at 37˚C for 48hrs) in both

PC3 (A-B) and Capan1 (C) cells. Colocalization of phosphorylated EGFR 1173 (red) with EEA1

(green) was analyzed following 48hrs exposure to EGF in both PC3 (D) and Capan1 (E) cells under NRP2 knock down conditions. Inset was magnified and split into corresponding channels to represent the individual and merged images. DAPI was used for nuclear staining. Scale bar indicates 20µm in length. Cell death was assayed using confocal microscopy, following EGF exposure for indicated time points using YO-Pro, Propidium iodide and Hoechst dyes in PC3 (F) and Capan1 (G) cells. Immunoblot was performed for cleaved PARP to analyze the induction of cellular apoptosis upon EGF exposure in NRP2 depletion state for both PC3 (H) and Capan1 (I) cells.

Neuropilin-2 regulates endosome maturation and EGFR trafficking to support cancer cell pathobiology

Samikshan Dutta, Sohini Roy, Navatha Shree Polavaram, et al.

Cancer Res Published OnlineFirst November 11, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-1488

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2015/11/11/0008-5472.CAN-15-1488.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2015/11/11/0008-5472.CAN-15-1488. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.