cd317 activates egfr by regulating its association …...human samples and ihc staining two human...

Molecular Cell Biology

CD317 Activates EGFR by Regulating ItsAssociation with Lipid RaftsGuizhong Zhang1, Xin Li1, Qian Chen1, Junxin Li1, Qingguo Ruan1, Youhai H. Chen2,Xiaolu Yang3, and Xiaochun Wan1

Abstract

EGFR regulates various fundamental cellular processes, andits constitutive activation is a common driver for cancer. Anti-EGFR therapies have shownbenefit in cancer patients, yet drugresistance almost inevitably develops, emphasizing the needfor a better understanding of the mechanisms that governEGFR activation. Here we report that CD317, a surface mol-ecule with a unique topology, activated EGFR in hepatocel-lular carcinoma (HCC) cells by regulating its localization onthe plasma membrane. CD317 was upregulated in HCC cells,promoting cell-cycle progression and enhancing tumorigenicpotential in a manner dependent on EGFR. Mechanistically,CD317 associated with lipid rafts and released EGFR from

these ordered membrane domains, facilitating the activationof EGFR and the initiation of downstream signaling pathways,including the Ras–Raf–MEK–ERK and JAK–STAT pathways.Moreover, in HCC mouse models and patient samples, upre-gulation of CD317 correlated with EGFR activation. Theseresults reveal a previously unrecognized mode of regulationfor EGFR and suggest CD317 as an alternative target fortreating EGFR-driven malignancies.

Significance: Activation of EGFR by CD317 in hepatocel-lular carcinoma cells suggests CD317 as an alternative targetfor treating EGFR-dependent tumors.

IntroductionLiver cancer is the second leading cause of cancer-related

mortality worldwide, resulting in �800,000 deaths annually (1).Unlike most other cancers for which the mortality has declined,death rates of liver cancer cases have been rising each year overthe last 10 years in theUnited States andother countries (2, 3). Thevast majority (�90%) of liver cancers are hepatocellular carcino-ma (HCC). HCC can be treated at early stages by liver transplan-tation or surgical resection; however, the overwhelming majorityof patients are in advanced stages at the time of diagnosis with fewtreatment options. The 5-year survival remains at a dismal rate of�18% (4). Although the risk factors for HCC are well known—including chronic infection of hepatitis B and C viruses (HBV andHCV) and alcohol abuse, the molecular events driving the path-ogenesis are poorly defined (2, 3).

The EGFR (also knownas ErbB1orHER1) belongs to the EGFR/ErbB subfamily of receptor tyrosine kinases (RTK), along withErbB2/HER2, ErbB3/HER3, and ErbB4/HER4 (5). The cognate

ligands for EGFR include EGF, TGFa, and amphiregulin (AREG;ref. 6), which induce EGFR homodimerization or its hetero-dimerization, with the closely related RTKs leading to phosphor-ylation of EGFR at multiple tyrosine residues in its intracellularregion (5). This enables the recruitment of various signalingmolecules and the initiation of intracellular signaling pathways(e.g., Ras–Raf–MEK–ERK, JAK–STAT, andPI3K–AKTpathways) tomodulate proliferation, survival, mobility, metabolism, differen-tiation, and other fundamental cellular processes (7). Dysregula-tion of EGFR drives the development and progression of varioustumors (8). Therapies that inhibit EGFR, including monoclonalantibodies (e.g., trastuzumab) and small-molecule tyrosinekinase inhibitors (e.g., erlotinib), are among the most successfulexamples of targeted cancer therapies, benefiting patients withmetastatic lung, colorectal, pancreatic, or head and neck can-cers (9). However, resistance to these therapies almost invariablydevelops (9). Moreover, EGFR is overexpressed in 40% to 70% ofhuman HCCs (10). Still, EGFR inhibitors (cetuximab, gefitinib,and erlotinib) did not show significant efficacy in unselectedpatients with advanced HCC in clinical trials (11, 12). Therefore,a better understanding of the regulation of EGFR signaling iscritical for the therapy of cancer in general and HCC in particular.

EGFR is associated with lipid rafts (13). These specializedmembrane microdomains—which are enriched in cholesterol,sphingolipids, and certain proteins—are involved in intracellularsignaling, trafficking, and pathogen–host interactions. In general,lipid rafts promote interactions among signaling molecules, andtheir disruption (e.g., by cholesterol depletion) impairs receptoractivation (14).However, EGFR appears to be an exception, as it iskept in an auto-inhibitory conformation upon localization inlipid rafts (13). Release of EGFR from lipid rafts can lead to ligand-independent EGFR dimerization and auto-activation (15, 16).Given the importance of lipid rafts in EGFR activation, a salient,yet poorly understood question, is how the association of EGFRwith lipid rafts is regulated.

1Shenzhen Laboratory of Fully Human Antibody Engineering, Institute of Bio-medicine and Biotechnology, Shenzhen Institutes of Advanced Technology,Chinese Academy of Sciences, Shenzhen, People's Republic of China. 2Depart-ment of Pathology and Laboratory of Medicine, University of PennsylvaniaPerelman School of Medicine, Philadelphia, Pennsylvania. 3Department of Can-cer Biology andAbramson Family Cancer Research Institute, Perelman School ofMedicine, University of Pennsylvania, Philadelphia, Pennsylvania.

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

Corresponding Authors: Xiaolu Yang, University of Pennsylvania School ofMedicine, 421 Curie Blvd, Room 654 BRB2/3, Philadelphia, PA 19104. Phone: 215-573-6739; Fax: 215-573-6725; E-mail: [email protected];Xiaochun Wan, [email protected]

doi: 10.1158/0008-5472.CAN-18-2603

�2019 American Association for Cancer Research.

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CD317 (also known as BST2, HM1.24, or tetherin) is a type IItransmembrane protein with an unusual structure, comprising ashort cytoplasmic N-terminal region, a transmembrane (TM)domain, a coiled-coil extracellular domain, and a C-terminalglycosylphosphatidylinositol (GPI) anchor (SupplementaryFig. S1A; refs. 17, 18). As such, CD317 is double anchored intothemembrane through both the TM domain and GPI, a topologythat is unique among human proteins (19, 20). CD317 is thoughtto be inserted both inside (via the GPI anchor) and outside(via the TM domain) lipid rafts (19, 21). Thus, it may influencethe high-order structures of these lipid microdomains. CD317plays a multifaceted role in regulating host–pathogen relation-ship. It is an important restriction factor for various envelopedviruses such as HIV, HBV, and HCV, preventing their release frominfected cells (22). When physically sequestering retroviral par-ticles, CD317 also potently activates NF-kB, leading to the expres-sion of proinflammatory gene (23–26). Of note, CD317 is impli-cated in tumorigenesis. It is overexpressed in multiple myelomaand several other cancers (23, 27–30), and neutralizing mono-clonal antibodies against CD317 showed tumor toxicity in animalmodels (31, 32). Nevertheless, it remains unclear whether CD317affords a proliferative advantage to tumor cells and if so, what theunderlying molecular mechanism may be.

In this study, we investigate the role of CD317 inHCC.We findthat CD317 is upregulated in HCC samples. CD317 stronglypromotes HCC proliferation and cell-cycle progression andenhances its tumorigenicity. Interestingly, the effect of CD317 ismediated by EGFR. CD317promotes the release of EGFR from thelipid rafts, promoting its auto-activation. These results establish acritical role for CD317 in tumorigenesis and reveal a newmode ofregulation for EGFR that likely influences the pathogenesis ofHCC and other tumors.

Materials and MethodsReagents

DMEMmedium and fetal bovine serum (FBS) were purchasedfromHyClone, L-glutamine fromGibco, Annexin V-FITC/PI Apo-ptosis Detection Kit from TransGen Biotech, cell-cycle analysis kitfrom KeyGEN BioTECH, methyl-b-cyclodextrin (C4555) andcholesterol (C3045) from Sigma-Aldrich, and Erlotinib-HCl(OSI-744) from Selleckchem.

Cell cultureBel7402, HepG2, and Huh7 were purchased from Shanghai

Cell Bank of Chinese Academy of Sciences (Shanghai, China) andwere authenticated by the vendor using short tandem repeat (STR)profiling. HEK293T was purchased from ATCC and confirmedby STR (GENEWIZ). Cell lines were cultured in DMEMmedium (HyClone) supplemented with 10%FBS (HyClone) and2mmol/L L-glutamine (Gibco), and periodically authenticated bymorphologic inspection andbiomarkers detection (if applicable).All cells were used within 6 months of continuous passageand checked for the absence of Mycoplasma using detection kit(LT27-710; Lonza).

Human samples and IHC stainingTwo human hepatocellular carcinoma arrays were used in this

study. The first one contained 35 tumor samples and 8 normalliver tissues (Xi'an Alena Biotech), and the second one contained75 tumor samples (Shanghai Outdo Biotech). The other five

specimens (three HCC and two normal liver tissues) wereobtained from Second People's Hospital of Shenzhen, which wasapproved by the Research Committee of Shenzhen Institutes ofAdvanced Technology (SIAT), Chinese Academy of Sciences.

IHC staining was performed as previously described (33) usingfollowing antibodies: anti-CD317 (ab134061; Abcam), anti-pY845 EGFR (BS5013; Bioworld) or (GTX133600; GeneTex), andanti-PCNA (10205-2-AP; Proteintech). All slides were indepen-dently analyzed by two pathologists in a blinded manner andscored according to staining intensity (no staining ¼ 0, weakstaining¼ 1, moderate staining ¼ 2, strong staining¼ 3) and thenumber of stained cells (0% ¼ 0, 1%–25% ¼ 1, 26%–50% ¼ 2,51%–75% ¼ 3, 76%–100% ¼ 4). Final immunoreactive scoreswere determined by multiplying the staining intensity by thenumber of stained cells, with minimum and maximum scoresof 0 and 12, respectively (34). TheMann–WhitneyU test was usedto evaluate the statistical significance of the results.

Xenograft tumor modelsMale BALB/c nude mice at 6 to 8 weeks of age were purchased

from Guangdong Medical Laboratory Animal Center (Guangz-hou, China) and housed in the SIAT facility under pathogen-freeconditions.

To investigate the effects of CD317 on established tumorgrowth, we performed both overexpression and knockdownexperiments. For overexpression, 5 � 106 CD317-stable expres-sion HepG2 cells or control cells in 100 mL PBS containing 50%Matrigel (BD Bioscience) were injected subcutaneously intoflanks of nude mice. Tumor incidence and growth were moni-tored. Twenty-eight days later, tumor-bearing and control micewere sacrificed, and tumorswere dissected for themeasurement oftumor weights and volumes using the formula [length �(width)2]/2. For knockdown, 1.5 � 107 HepG2 cells stablyexpressing CD317 or control shRNAwere injected. Tumor growthwas monitored, and tumors were harvested at day 23. All animalexperiments were approved by the Institutional Animal Care andUse Committee at SIAT.

Bioinformatics analysis of CD317 expression in human HCCsCD317 protein expression in HCC tissues and normal tissues

was determined from thehumanprotein atlas (www.proteinatlas.org). HCC CD317 gene expression was determined throughanalysis of Mas Liver and Wurmbach Liver databases, which areavailable through Oncomine (www.oncomine.org).

Plasmids and siRNAsCD317 (the long isoform) was transiently expressed using

MigR1- or pCMV-based plasmids, or stably expressed usingPLVX-based lentiviral vectors. The full-length human CD317cDNA was generated from Jurkat cells by RT-PCR, digested withBglII and XhoI, and cloned into MigR1 or PLVX. The extracellulardomain of CD317 (ECD, amino acids: 44–159; ref. 35) wasgenerated via PCR reaction and cloned into pCMV-C-His vector.The plasmids encoding CD317 mutants in which the twoN-linked glycosylation sites (Asn-65 and Asn-92) were replacedwith Asp were generated by PCR-based site-directed mutagene-sis. The delCT and delGPI variants of CD317, which lacked theN-terminal 20 amino acids and C-terminal 19 amino acids,respectively, were fused with HA tag in the N or C terminus andcloned into pCMV-C-His or PLVX vector. siRNA-resistant (SR)CD317, delCT, and delGPI constructs, each tagged with HA, were

Activation of EGFR by CD317

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generated via PCR by making three synonymous mutations inthe siRNA recognition site of human CD317, and they are calledHA-CD317-SR, HA-delCT-SR, and HA-delGPI-SR, respectively.Specific siRNA for human CD317 and nonspecific negative con-trol were described previously (36). For stable transfection, twoshRNAs targeting human CD317 (sh317) and control shRNA(shCtrl) were cloned into pLVTHM vectors. Forward oligonucle-otide sequences for shRNAs and siRNAs were provided inSupplementary Table S1.

Transfection and lentiviral infectionTransfection of tumor cells with plasmids or siRNAs was

performed using Lipofectamine 3000 according to the manufac-turer's protocol (Invitrogen). For lentivirus production, HEK293Tcells were transfected with each lentiviral vector together withhelper plasmids Gag, Rev, and VSVG. Forty-eight to 72 hours aftertransfection, virus-containing media was collected by centrifuga-tion at 100� g for 5minutes and concentratedby centrifugation at50,000 � g for 2 hours. Cells were transduced using lentiviruseswith 6 mg/mL polybrene and selected with FACS.

Cell viability and colony formation assayTwenty-four hours following transfection, Bel7402, Huh7, or

HepG2 cells were seeded in triplicates in 96-well plates at 5,000cells/well and maintained in medium containing 10% FBS. Cellswere strained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT; Promega) at the indicated time points,and relative viable cells were determined by measuring the OD atan absorbance wavelength of 490 nm.

For colony formation assays, transfected Bel7402 or HepG2cells were plated in triplicates in six-well plate at 1,500 cells/welland maintained in medium containing 10% FBS for 7 to 10 days.Colonies were fixed withmethanol and stained with crystal violetstaining solution (Beyotime).

Cell-cycle and apoptosis analysisCell cycle and apoptosis were assessed byflow cytometry (FACS

Canto II; BD Bioscience). For cell-cycle analysis, 48 hours follow-ing transfection, Bel7402, HepG2, or Huh7 cells were seeded intriplicates in 12-well plates at 1� 105 cells/well, synchronized byserum starvation, and allowed to grow for 18 to 24 hours. Cellswere collected, washed with PBS, and fixed in cool ethanol at�20�Covernight before being stainedwith propidium iodide (PI;KeyGEN BioTECH) at room temperature for 30 minutes. Forapoptosis analysis, cells were collected, washed with PBS, dou-ble-stained with Annexin V-FITC and PI using an ApoptosisDetection Kit (TransGen Biotech) at room temperature for 15minutes in the dark.

RNA isolation and qRT-PCRTotal RNA was extracted with TRIzol reagent (Invitrogen) and

used to generate cDNA. Specific primers used for quantitative real-time PCR assays were synthesized by Invitrogen Corporation.Their sequences were shown in Supplementary Table S2.

Protein extraction and immunoblottingWhole-cell lysates were prepared by suspending cells in the

RIPA buffer (Beyotime) supplemented with 1 � complete prote-ase inhibitors mixture and 1 � phosphatase inhibitor (Roche).Protein concentration was determined by BCA assay (Pierce).Equal quantities of proteins were separated by SDS-PAGE, trans-

ferred to a PVDFmembrane, and blotted with specific antibodies.Proteins in the membrane were visualized by an enhancedChemiluminescense Detection Kit (Millipore). Rabbit antibodiesagainst the followingproteins ormodificationswere usedwith thecatalogue numbers and sources indicated: CD317 (ab134061),TGFa (ab208156; Abcam); caspase3 (9662; CST); pY1068 EGFR(BS5010), pY845 EGFR (BS5013), pY705 STAT3 (BS4181),STAT3 (AP0365), ERK1/2 (BS1112), pT202/Y204 ERK1/2(BS5016), cyclin D1 (BS6352), and p16 INK4a (BS6431), caveo-lin-1 (BS9878M; Bioworld Technology), and AREG (16036-1-AP;Proteintech). Mouse antibodies against the following proteins/epitopes were used: HA.11 (MMS-101P; Covance); Ki67 (P6834;Sigma); transferrin receptor (13-6800; Invitrogen); GAPDH(MB001; Bioworld Technology); b-actin (sc-47778) and EGFR(sc-373746; SantaCruzBiotech).HRP-conjugatedmouse anti-His(M20020) was purchased from Abmart, mouse HRP-conjugatedgoat anti-mouse IgG (074-1806) from KPL, and HRP-conjugatedgoat anti-rabbit IgG (E030120-02) from EARTHOX.

Lipid raft and non-raft proteins isolationLipid raft and non-raft proteins were isolated using the Focus

Global Fractionation Kit (G Biosciences) according to the man-ufacturer's instruction.

CoimmunoprecipitationHepG2 cells were lysed in immunoprecipitation (IP) buffer

(150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.4, 50 mmol/LEDTA, 1.0% Nonidet P-40, 1 mmol/L PMSF) supplementedwith 1 � complete protease inhibitors mixture (Roche).Extracts were assayed for protein content, using the BCA ProteinAssay Kit (Pierce) after clarification by high-speed centrifuga-tion at 4 �C. IP was performed using protein-A/G Dynabeads(Pierce, 88803). In brief, 400 mg protein-A/G Dynabeads wascoated with 1 mg specific antibody (Rabbit anti-CD317, sc-99191, or mouse anti-EGFR, sc-373746; Santa Cruz) or Igcontrol for 1 hour at room temperature with rotation. Afterremoving unbound antibody, the bead–antibody complex wasincubated with 500 mL cell lysate for 6 hours at 4 �C withrotation. The captured immunoprecipitates were washed fivetimes with IP buffer and boiled in 2� loading buffer. The elutedproteins were fractionated by SDS-PAGE and detected by West-ern blot analysis.

ELISASupernatant was collected from HepG2 or Bel7402 cells at 48

hours after transfection and kept at�80 �C. The concentration ofTGFa and AREG was measured using ELISA kits purchased fromThermoFisher Scientific and BOSTER respectively, according tothe manufacturer's instruction.

Immunofluorescence and microscopyImmunofluorescent staining was performed as previous

description (36) using the following antibodies: mouse anti-EGFR (Santa Cruz; sc-120), rabbit anti-pY1068 EGFR (BioworldTechnology; BS5010) and rabbit anti-pY845EGFR (BioworldTechnology; BS5013), rhodamine-conjugated goat anti-rabbitIgG and goat anti-mouse IgG (Molecular Probes). 4, 6-Diamino-2-phenylindole (DAPI; Roche) and Alexa Fluor 488 conjugatedCholera Toxin Subunit B (C-34775; Life Technology) wereused to label nuclei and lipid raft, respectively. Ten to fifteenhigh-powered fields were evaluated on optical microscopy

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(Olympus IX71), and fixed fluorescence images were analyzedby ImagePro Plus software (Media Cybernetics).

Statistical analysisAll analyses were performed using GraphPad Prism Software.

Data were expressed as mean � SEM. Student t test or Mann–Whitney U test was used to compare continuous data for twogroups. Pearson correlation co-efficiency was used to analyze therelationship between the phosphor-Y845 EGFR and the CD317staining levels in tissue sections. Tumor incidences of CD317-overexpression and control group were analyzed using the log-rank test. P values <0.05 were considered to be significant.

ResultsCD317 is upregulated in HCCs and enhances tumorigenicity

To investigate the role of CD317 in HCC, we examined itsexpression in38HCC tumor samples and10normal liver samplesusing IHC. As shown in Fig. 1A, the level of CD317 was signif-icantly increased in the majority of the HCC samples comparedwith the normal liver samples. A survey of a public database(Human Protein Atlas, www.proteinatlas.org) also showed thatexpression of the CD317 protein was significantly increased inHCCs (Supplementary Fig. S1B). In addition, CD317 mRNAlevels were elevated in HCCs compared with normal liver tissues(www.oncomine.org; Supplementary Fig. S1C).

To test the function of CD317 in HCC cells, we used three celllines—HepG2, Bel7402, and Huh7, which express low to highlevels of endogenous CD317 mRNA and protein (Fig. 1B;Supplementary Fig. S1D). We knocked down CD317 in HepG2and Bel7402 cells using siRNA (Fig. 1C). This led to a strongreduction in cell proliferation (Fig. 1D and E; SupplementaryFig. S1E). Conversely, we overexpressed exogenous CD317 inHepG2, Bel7402, and Huh7 cells (Fig. 1C), and observed amarked increase in proliferation (Fig. 1D and E; SupplementaryFig. S1E). Neither knockdown nor overexpression of CD317affected apoptosis (Supplementary Fig. S1F and S1G), consistentwith our previous observations (36).

To investigate the role of CD317 in tumor formation in vivo, weimplanted control and CD317-overexpressing HepG2 cells (Sup-plementary Fig. S2A) into immunodeficient nude mice. Com-pared with control cells, HepG2 cells with CD317 overexpressiongenerated tumors with an earlier onset and a faster rate (Fig. 1Fand G). By day 28 following xenograft, CD317-overexpressingcells grew into tumors that were three times as large as thosegenerated by control cells (P < 0.05, n ¼ 5–6; Fig. 1H and I;Supplementary Fig. S2B). The increased tumorigenic potential ofCD317-overexpressing cells was likely due to enhanced prolifer-ation instead of reduced apoptosis, as tumors generated fromthese cells showed increased Ki-67 levels, but unchanged caspase-3 activation, compared with tumors generated from control cells(Fig. 1J). Conversely, CD317 knockdown markedly suppressedtumor growth (Fig. 1K–M; Supplementary Fig. S2C) and reducedproliferation, characterizing by low PCNA expression (Fig. 1N).These results indicate that CD317 is required for optimal prolif-eration and tumor formation of HCC cells.

CD317 promotes cell-cycle progression in HCC cellsGiven that CD317 promotes cell proliferation, we analyzed

how it affects cell-cycle progression. Compared with their corre-sponding control cells, CD317-overexpressing HepG2, Bel7402,

and Huh7 cells displayed accelerated cell-cycle progression, asevident by a noticeable increase in S-phase cells and a concom-itant decrease in G0–G1-phase cells (Fig. 2A; SupplementaryFig. S3A). Conversely, CD317-depleted HepG2 and Bel7402 cellsshowed a significant reduction in cell-cycle progression, withfewer S-phase cells and more G0–G1-phase cells (Fig. 2B; Supple-mentary Fig. S3B).

Forced expression of CD317 in HepG2, Bel7402, and Huh7cells elevated the levels of the CDK4/6 activator cyclin D1 and theDNA polymerase cofactor PCNA, while reducing the levels of theCDK inhibitor p16 (Fig. 2C). Conversely, knockdown of CD317inHepG2 and Bel7402 cellsmarkedly decreased the expression ofcyclin D1 and PCNA, while enhancing the expression of p16(Fig. 2D).

Glycosylation and membrane localization are essential forCD317 function

CD317 is a type II transmembrane protein, with its ectodomaincontaining two N-linked glycosylation sites, Asn65 and Asn92(Supplementary Fig. S1A). Previous studies suggested thatN-linked glycosylation at these residues may be required for thecorrect folding, but not antiviral activity, of CD317 (37). However,the role of this modification in tumor cells has not been address-ed. We mutated Asn65 and Asn92 to Asp individually (N65Dand N92D, respectively) or in combination (N65D/N92D).Consistent with a previous report (37), the N65D and N92Dmutations reduced the apparent molecular weight of CD317 from30 to 36 kDa to �28 kDa, whereas the N65D/N92D mutationfurther reduced the apparent molecular weight to �21 kDa(Fig. 2E). Unlike wild-type CD317, none of the glycosylation-defective mutants affected the levels of PCNA, cyclin D1, or p16(Fig. 2E). Consistently, in contrast to wild-type CD317, cells ex-pressing these mutations showed no difference in cell-cycle pro-gression compared with the parental cells (Fig. 2F; SupplementaryFig. S3C). Additionally, we generated CD317-ECD, a truncatedmutant that contained only the ectodomain and hence was notanchored in the plasma membrane. CD317-ECD did not affectthe progression of cell cycle or the levels of cell-cycle regulatorseither (Fig. 2G and H; Supplementary Fig. S3D). Thus, bothglycosylation and membrane localization are required for thefunction of CD317 in cell-cycle progression.

CD317 activates the EGFR–STAT/ERK pathwayNext, we investigated the mechanism through which CD317

promotes cell proliferation. By analyzing various mitogenic path-ways, we observed that CD317 strongly influenced both the RAS–Raf–MEK–ERK and JAK–STAT signaling pathways. Specifically,overexpression of CD317 increased, while knocking downCD317 decreased, the activation of ERK1/2 and STAT3 inHepG2,Bel7402, and Huh7 cells (Fig. 3A). While evaluating the signalingevents upstream of both pathways, we noticed that CD317 is apotent activator for EGFR. Specifically, forced expression ofCD317 led to a strong increase in the auto-phosphorylationof EGFR at Tyr1068, as well as Src-mediated phosphorylation ofEGFR at Tyr845 (Fig. 3B). Conversely, depletion of CD317 bysiRNA resulted in anoticeable reduction in these phosphorylationevents (Fig. 3B). In contrast, CD317-N65D/N92D, the glycosyl-ationmutant that failed to accelerate cell cycle, did not affect EGFRactivation (Fig. 3B).

To confirm these results, we analyzed the levels of phosphor-ylated EGFR using immunofluorescence staining and FACS






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Figure 1.

Upregulation of CD317 expression correlates with tumorigenesis. A, CD317 upregulation in hepatocellular carcinoma. CD317 protein expression in tumors andnormal tissues from patients diagnosed with hepatocellular carcinomawas analyzed by IHC. CD317-positive cells are shown in brown. Quantitation of the CD317expression in tumors (n¼ 38) and normal tissues (n¼ 10) was performed as described in Materials and Methods. Each data point represents a CD317 expressionscore of patients. Horizontal bars, medians. Scale bar, 100 mm. B, Levels of CD317 protein in the human cell lines. C, Immunoblot analysis of HCC cells with orwithout CD317 overexpression or knockdown. D, Proliferation of HepG2, Bel7402, and Huh7 cells transiently transfected with control (MigR1) or CD317 plasmid,or with control (siCtrl) or CD317 siRNA (siCD317). � , P < 0.05; ��, P < 0.001 for CD317 vs. MigR1 group. ##, P < 0.005; ###, P < 0.001 for CD317 siRNA vs. controlsiRNA group. E, Colony formation of HepG2 and Bel7402 cells transiently transfected with MigR1, CD317, siCD317, or control siRNA. �� , P < 0.005; ��, P < 0.001.F–I, HepG2 cells transfected with control (PLVX) or CD317 plasmid were injected subcutaneously into nude mice. Shown are tumor incidences (F) and tumorgrowth (G) overtime and typical whole-body fluorescence images (H, top), tumor appearance (H, bottom), and weight (I; mean� SEM) at 28 days postinjection.�� , P < 0.005. J, Total lysates from PLVX and CD317 tumors were analyzed byWestern blotting. K–M, HepG2 cells transfected with control (shCtrl), CD317 shRNA-1 (sh317–1), or CD317 shRNA-2 (sh317-2) were injected s.c. into nude mice. Shown are tumor growth (K) overtime and typical whole-body images (L, top), tumorappearance (L, bottom), and weight (M; mean� SEM) at 23 days postinjection. N, Total lysates from shCtrl, sh317-1, and sh317-2 tumors were analyzed byWestern blotting.

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analysis. We transfected HepG2 cells with a CD317 plasmid thatcoexpressed GFP, as well as the control plasmid expressing onlyGFP. By staining cells with anti-phospho-Tyr845 (anti-pY845)and anti-pY1068 antibodies, we observed that phosphorylationof endogenous EGFR was significantly enhanced in cells withCD317 overexpression (CD317 group, GFP-positive), but notin cells without CD317 overexpression (CD317 group, GFP-negative; and control group, GFP-positive and -negative; Supple-

mentary Fig. S4A). A FACS analysis further confirmed that forcedexpression of CD317 enhanced the levels of pY845 and pY1068EGFR in HepG2 cells (Fig. 3C). Additionally, CD317 knock-down sensitized tumor cells to erlotinib treatment, as shown bya decreased IC50 in CD317-depleted tumor cells (SupplementaryFig. S4B). Moreover, tumors formed by the CD317-overexpressingHepG2 cells showed noticeably higher EGFR activation comparedwith tumors formed by control HepG2 cells (Fig. 1J), whereas

Figure 2.

CD317 accelerates cell-cycle transition in vitro. A and B, HepG2, Bel7402, and Huh7 cells were transfected with the control vector (MigR1) or either CD317 (A), orHepG2 and Bel7402 cells were transfected with control or CD317 siRNA (B). Percentage of cells in G0–G1, S, and G2–M phase was quantified (means� SEM).�� , P < 0.005; �� , P < 0.001. C and D, Expression of cell cycle-related proteins in HepG2, Bel7402, and Huh7 cells transfected with MigR1 or CD317 (C), or inHepG2, Bel7402 cells transfected with control or CD317 siRNA (D). Blots in C and Dwere exposed for different times to better show the effects of CD317overexpression or knockdown. E andG, Expression of cell-cycle regulatory proteins in HepG2 cells transfected with MigR1 vector, wild-type CD317, or theindicated CD317 mutants (E), or with the control vector pCMV and CD317-ECD-His (G). In G, the endogenous CD317 and CD317-ECD-His were detected by anti-CD317 antibody, whereas CD317-ECD-His was also detected by anti-His antibody. F and H, Cell-cycle progression of HepG2 cells transfected with MigR1 vector,wild-type CD317, or the indicated CD317 mutants (F), or with the control vector pCMV and CD317-ECD-His (H). Values represent means� SEM. �� , P < 0.005;�� , P < 0.001. The experiments were repeated at least three times with similar results.






CD317-knockdwon tumors showed substantially lower EGFRactivation compared with control tumors (Fig. 1N).

To determine whether CD317 activates RAS–Raf–MEK–MAPKand JAK–STAT pathways via EGFR, we treated HepG2 cells withthe EGFR inhibitor erlotinib at a dose that almost completelyblocked EGFR activation (Fig. 3D). Under this condition, CD317was no longer able to stimulate the activation of STAT3 and ERK(Fig. 3D, top) or alter the expression of cyclin D1, PCNA, and p16(Fig. 3D, bottom). Moreover, erlotinib effectively negatedCD317-mediated acceleration in cell-cycle progression (Fig. 3Eand Supplementary Fig. S4C) and proliferation (Fig. 3F). We alsoused siRNAs to knock down EGFR inHepG2 andHuh7 cells. Thismarkedly impaired CD317-induced activation of STAT3 and ERK(Fig. 3G and Fig. 3H, left), and effectively abrogated the effect ofCD317 on cyclin D1, PCNA, and p16 (Fig. 3H, right). Knockingdown EGFR also rendered CD317 incapable of stimulating cellcycle progression (Fig. 3I; Supplementary Fig. S4D) or prolifer-ation (Fig. 3J). Collectively, these data show that CD317 pro-motes mitogenic signaling, as well as cell-cycle progression andproliferation, via the activation of EGFR.

CD317 regulates EGFR in a lipid raft-dependent mannerCD317 did not influence the expression of EGFR (Fig. 4A and

B). Moreover, CD317 did not appear to stably associate withEGFR, as shown by a coimmunoprecipitation assay (Fig. 4C).EGFR is activated bybinding to its cognate ligands, including EGF,TGFa (5), and AREG (6). However, the expression of EGF wasminimal in HepG2 and Bel7402 cells (Fig. 4A and B). Moreover,neither CD317 overexpression (in HepG2 cells) nor knockdown(in Bel7402 cells) significantly altered the protein and mRNAlevels of TGFa or AREG (Fig. 4A and B). Moreover, knockdown ofboth TGFa and AREG did not influence CD317-mediated EGFRactivation (Supplementary Fig. S4E), suggesting that CD137-induced EGFR activation is not mediated by these EGFR ligands.

The activation of EGFR is regulated by its localization to lipidrafts (13, 15, 16). Considering that CD317 is associatedwith lipidrafts (38), we investigated whether CD317 affects the raft versusnon-raft distribution of EGFR. Using a fluorescently-tagged chol-era toxin B subunit (CTB) that specifically labels lipid rafts (39),we observed that CD317 knockdown cells exhibited high levels ofcolocalization of EGFR with lipid rafts, characterized by anincreased coefficient value between EGFR and lipid raft staining(Fig. 4D). We also detected EGFR by Western blot analysisand found that knocking down CD317 led to a dramatic re-distribution of EGFR to the lipid raft fraction (Fig. 4E). Upon theexpression of an siRNA-resistant form of CD317 in the CD317-knockdown cells, the localizationof EGFRwas essentially restoredto that in control cells (Fig. 4E), confirming the specificity of theCD317 siRNA.

The extracellular C-terminal GPI anchor of CD317 enables itsassociation with the lipid rafts, and the cytoplasmic N-terminalregion of CD317 links to the actin cytoskeleton (17, 40). Wegenerated siRNA-resistant CD317 variants, delGPI and delCT, inwhich the GPI modification signal and the cytoplasmic tail weredeleted, respectively (Fig. 4E). delCT, like the siRNA-resistant full-length CD317, was able to reverse the association of EGFR withlipid rafts in CD317 knockdown HepG2 cells, whereas delGPIshowed no such an activity (Fig. 4E). Also, delCT elicited a similareffect as the full-length CD317 in promoting EGFR activation inHepG2 cells devoid of CD317, but delGPI failed to do so (Fig. 4F).Consistently, delCT, but not delGPI, promoted cell-cycle progres-

sion (Fig. 4G; Supplementary Fig. S4F) and EGFR activation(Fig. 4H) as effectively as the full-length CD317.

Furthermore, we disrupted lipid rafts by depleting cholesterolwithmethyl-b-cyclodextrin (MbCD). In accordancewith previousreports (41), this treatment resulted in EGFR activation inHepG2,Bel7402, and Huh7 cells (Fig. 4I). Importantly, under this con-dition, neither CD317 overexpression nor CD317 depletioninfluenced the activation of EGFR in these cells (Fig. 4I). However,this effect of MbCD on CD317-mediated EGFR activation can bereverted by replenishment with cholesterol (Fig. 4I), furthersupporting the notion that lipid raft is required for effect ofCD317 on EGFR. Collectively, these data indicate that CD317regulates EGFR activation by promoting the release of EGFR fromlipid rafts.

Activation of EGFR correlates with CD317 expression in humanHCCs

To investigate the role of CD317 in EGFR activation in humantumors, we analyzed CD317 expression, EGFR activation, andhepatocyte proliferation (using nuclear PCNA as amarker) in 110HCC samples. Interestingly, the activated form of EGFR (pY845EGFR) positively correlated with the CD317 expression (rs ¼0.4370, P < 0.0001; Fig. 5A and B). Patients with low CD317expression (expression score <6, N ¼ 56) also showed reducednuclear PCNA than patients with high CD317 expression (expres-sion score�6,N¼ 54; Fig. 5B, bottom). These results suggest thatCD317 promotes HCC development through EGFR-mediatedmitogenic signals.

DiscussionEGFR was among the very first RTKs that were identified and is

frequently dysregulated in tumors (5, 42). As such, EGFRhas beenextensively studied as a prototype for RTK-mediated signaling anda target for cancer therapy (5, 9, 42). Here we reveal that theactivationof EGFR is controlled viaCD317-mediated release fromthe lipid rafts (Fig. 5C). Thisfinding has important implications inthe development of more effective treatments for EGFR-driventumors.

The activation mechanism of EGFR (and the other EGFR/ErbBfamily members) is unique among RTKs in several importantaspects. The ligands for EGFR are monomers, rather than dimers,and they promote receptor dimerization by inducing a dramaticconformational change that exposes a dimerization region (43).The subsequently EGFR activation involves the formation of anasymmetric dimer of the intracellular kinase domain, instead ofauto-phosphorylation in the activation-loop as seen in the otherRTKs (43). Importantly, EGFR is associated from lipid raft, and itsactivation requires its release from these highly organized mem-brane microdomains (15). Although the molecular basis for thisrequirement remains unclear, the dramatic conformationalchanges required for the activation of EGFRmight be constrainedin lipid rafts.

The organizational principle of lipid rafts, as well as thelocalization of proteins in andout of these ordered lipid domains,is poorly defined. Nevertheless, the ability of CD317 to releaseEGFR from lipid rafts is likely related to its unique topology.CD317 is thought to be associated with lipid rafts via the GPIanchor, but its TM domain is likely anchored outside lipidrafts (19, 21). Moreover, the rigid CD317 dimers formed by thecoiled-coil extracellular domain can further dimerize through the

Zhang et al.





Figure 3.

CD317 function is dependent on EGFR–STAT3/ERK axis. A, STAT3 and ERK1/2 phosphorylation levels in HepG2, Bel7402, and Huh7 cells transiently transfectedwith MigR1 or CD317 (left), or with control siRNA or CD317 siRNA (right). The levels of CD317, pSTAT3, total STAT3, pERK1/2, and total ERK1/2 were determinedbyWestern blot analysis. B, HepG2 cells were transfected with control MigR1 vector, CD317, or CD317 N65/92D (left). Huh7 cells were transfected with controlMigR1 vector or CD317 (middle), and Bel7402 cells were transfected with control or CD317 siRNA (right). The levels of CD317, pY845 EGFR, pY1068 EGFR, andtotal EGFR were analyzed byWestern blot analysis. C, Representative FACS graphs (left) and statistical analysis (right) of EGFR phosphorylation levels in HepG2cells transiently transfected with control MigR1 vector or CD317. Mean fluorescence intensity (MFI) values of GFP-negative and -positive cells was analyzed asindicated. � , P < 0.05; �� , P < 0.005; �� , P < 0.001. D, HepG2 cells transfected with MigR1 or CD317 were treated with or without erlotinib (50 mmol/L). Cells wereanalyzed for the activation of EGFR, STAT3, and ERK1/2 (top), and expression of cell cycle-related proteins (bottom). E and F, HepG2 cells stably infected withcontrol (PLVX) or CD317-expressing lentiviruses and Huh7 cells transfected with control (MigR1) or CD317 vectors were treated with or without erlotinib(50 mmol/L). Cell-cycle distribution (E) and cell proliferation (F) were analyzed. �� , P < 0.005; �� , P < 0.001 for CD317 vs. control group. G, Huh7 cells transfectedwith control (MigR1) or CD317 vectors were treated with control or EGFR siRNA (siEGFR). Activation of STAT3 and ERK1/2 was analyzed. H–J, HepG2 cells stablyinfected with PLVX or CD317 lentiviruses were treated with control or EGFR siRNA. Activation of EGFR, STAT3, and ERK1/2 (H, left), expression of cell cycle-related proteins (H, right), cell-cycle distribution (I), and cell proliferation (J) were analyzed. �� , P < 0.001 for CD317 vs. PLVX group.






Figure 4.

CD317 regulates EGFR in a lipid raft-dependent manner. A and B, HepG2 cells were transfected with MigR1 or CD317 plasmid (A), and Bel7402 cells weretransfected with control or CD317 siRNA (B). Forty-eight hours later, mRNA levels of CD317, EGFR, EGF, TGFa, and AREGwere determined by real-time PCR(left), and the protein levels of TGFa and AREG in culture supernatants were detected by ELISA (right). Values represent means� SEM. �� , P < 0.005. Theexperiments were repeated at least three times with similar results. C, Lysates of HepG2 cells were immunoprecipitated with anti-CD317 or anti-EGFR antibodiesas indicated. Cell lysates (input) and immunoprecipitates were analyzed byWestern blotting. D, HepG2 cells transfected with control or CD317 siRNAwereimmunostained for EGFR (red), lipid rafts (green), and DNA (DAPI, blue). Shown are representative fluorescence images and EGFR-lipid rafts colocalization plot(left), and Pearson's correlation coefficient (R) (right; mean� SEM for at least 30 cells). �� , P < 0.001. Scale bar, 20 mm. E, HepG2 cells were transfected withcontrol or CD317 siRNA, along with control (pLVX) or siRNA-resistant (SR) CD317, delCT, and delGPI plasmids. EGFR in nonlipid raft (N) and lipid raft fractionswere analyzed byWestern blot analysis. Transferrin receptor (TfR) and caveolin-1 served as loading controls for non-raft proteins and lipid raft proteins,respectively. F, EGFR activation in CD317-knockdown HepG2 cells expressing the indicated siRNA-resistant CD317 plasmids or the PLVX control vector. G and H,HepG2 cells expressing MigR1, CD317, HA-delCT-CD317 (delCT), and delGPI-CD317-HA (delGPI) (H, right) were analyzed for cell-cycle progression (G) and EGFRactivation (H, left). �� , P < 0.005; �� , P < 0.001 for CD317 or delCT vs. PLVX group. I, HepG2 and Huh7 cells were transfected with MigR1 or CD317 plasmid, andBel7402 cells were transfected with Ctrl or CD317 siRNA and treated with or without 10 mmol/L methyl-b-cyclodextrin (MbCD) for 30minutes. Thereafter,cholesterol was reloaded for 90 minutes using cholesterol-saturated MbCD (2:10 chol-MbCD, 2 mmol/L cholesterol, and 10 mmol/L MbCD) in Huh7 cells. Thelevels of CD317, pY845-EGFR, pY1068-EGFR, and total EGFR were determined byWestern blot analysis.

Zhang et al.





anti-parallel association of the N-terminal region of thedimer (17). As such, CD317 can form a tetrameric complex thatmay bring micro-lipid rafts close to each other while, at the sametime, preventing their coalescing into large assemblies. This may

facilitate the release of EGFR into the space between lipid rafts. Analternative, but notmutually exclusive, scenariomay be related tothe recently-identified palmitoylation of EGFR. Proteinsmodifiedby palmitate, like GPI-anchored proteins, are often found in lipid

Figure 5.

Upregulation of CD317 correlates with the activation of EGFR in HCC.A, Immunohistological staining of CD317, pY845-EGFR, and PCNA in HCC sections. Scalebars in black and white indicate 50 and 20 mm, respectively. B, Statistical results of data shown in A. Shown are correlation co-efficiency between the phosphor-Y845 EGFR and the CD317 staining levels in tissue sections (n¼ 110; top), and nuclear PCNA expression in CD317high (score� 6; n¼ 56) and CD317low (score < 6;n¼ 54) patients (bottom). Each data point represents a patient. Horizontal bars, medians. C, Proposed role of CD317 in HCC development. CD317 expression ismarkedly upregulated in HCC, leading to overactivation of EGFR and the downstream pathways, such as STAT3 and ERK1/2 pathways, by modulating lipid raftdynamic. STAT3 collaborates with ERK1/2 signaling to activate the expression of cyclin D1 and PCNA, and to inhibit p16 expression, which consequentlyaccelerates G1–S phase transition and cell proliferation. Therefore, CD317 promotes HCC development.






rafts. Interestingly, a recent study showed that EGFR is modifiedby the palmitoyltransferase DHHC20 in the intracellular tail,which enables the insertion of the disordered region C-terminalto the kinase domain into the plasma membrane (44). Presum-ably, the C-terminal region of EGFR may insert into lipidrafts, impeding EGFR activation. CD317 might inhibit EGFRpalmitoylation or increase its de-palmitoylation, for example,by influencing the interaction of EGFR with acyl-transferases oracyl-protein thioesterases. Regardless of the mechanism, CD317-mediated release of EGFR from lipid rafts likely represents apreviously unrecognized, important mode of regulation. Furthercharacterization of this regulationwill likely help unravel intricatemechanisms that govern the activation of EGFR and the closely-related RTKs.

Constitutive signaling emanating from EGFR and other mem-bers of the EGFR/ErbB family contributes to a wide range ofmalignancies (8). Mechanisms underlying aberrant EGFR signal-ing include increased expression of EGFR, overproduction of itsligands, andmutations of EGFR especially in the extracellular andkinase domains (5, 45). Here we find that the levels of CD317proteins are increased in HCC, and a survey of public databaseshow that CD317 transcripts are enhanced in several cancers,including pancreatic and ovarian cancers, suggesting a mecha-nism for EGFR activation that is distinct from the canonical modeof dysregulation. These cancers with high CD317 expressionshowed poor prognosis (27, 29, 46, 47), but neutralizing mono-clonal antibodies and small molecule tyrosine inhibitors of EGFRoffer marginal therapeutic benefit for these tumors. Thus, ourfindings provide a rationale for targeting CD317 as alternativetherapy strategy for EGFR-driven tumors. In this context, it isnoted that monoclonal antibodies for CD317 have been gener-ated and tested in animal models for multiple myeloma (31, 32).These antibodies may be applied to treat cancers with CD317upregulation.Moreover, as shown recently, activation of EGFR viade-palmitoylation renders tumor cells addicted to EGFR, creatinga vulnerability (44). Upregulation of CD317may induce a similar

reliance on EGFR signaling, hence sensitizing tumor cells totherapies targeting EGFR. Therefore, for tumors that are initiallyresponsive to therapies targeting EGFR, combination withCD317-targeted therapies may help resolve the problem of resis-tance in the clinic.

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

Authors' ContributionsConception and design: G. Zhang, Y.H. Chen, X. Yang, X. WanDevelopment of methodology: G. Zhang, X. LiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Zhang, X. Li, Q. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Zhang, X. Li, J. Li, X. YangWriting, review, and/or revision of the manuscript: G. Zhang, Y.H. Chen,X. Yang, X. WanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q. Chen, Q. RuanStudy supervision: X. Yang, X. Wan

AcknowledgmentsWe thank EricWitze for helpful comments on themanuscript. This studywas

supported by the National Natural Science Foundation of China (NSFC;81373112 and 81701559) to X. Wan and G. Zhang, respectively; the ChinaPostdoctoral Science Foundation (2016M602541) to G. Zhang; Special FundsforMajor Science and Technology ofGuangdongProvince (2013A022100037);Shenzhen Special Funds for Industry of the Future (Shenzhen MunicipalDevelopment and Reform Commission [2015] No. 971); and Shenzhen BasicScience Research Project (JCYJ20170413153158716) to X. Wan; and the U.S.National Institutes of Health (R01CA182675 and R01CA184867) to X. Yang.

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.

ReceivedAugust 29, 2018; revised January 24, 2019; acceptedMarch 14, 2019;published first March 19, 2019.

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