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Nectin-1 Is a Marker of Thyroid Cancer Sensitivity to Herpes Oncolytic Therapy

Head and Neck Service (Y.-Y.H., Z.Y., S.-F.L., S.L., R.J.W.) and Gastric and Mixed Tumor Service (Y.F.), Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10021; and Division of Endocrinology and Metabolism (Y.-Y.H., S.-F.L.), Chang Gung Memorial Hospital, Chang Gung University, Taipei, Taiwan

Address all correspondence and requests for reprints to: Richard J. Wong, M.D., Head and Neck Service, C-1069, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021. E-mail: wongr{at}mskcc.org.

	Abstract

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Context: The prognosis for patients diagnosed with anaplastic thyroid cancer is dismal, with a median survival time of only 6 months. Novel therapies are needed for these and other thyroid cancers that are refractory to conventional therapy.

Objectives: Our goals were to assess the ability of an attenuated, replication-competent, oncolytic herpes virus (NV1023) to enter and lyse human thyroid cancers and determine whether herpes simplex virus receptor expression is a determinant of NV1023 efficacy.

Design: A panel of 12 human thyroid cancer cell lines including anaplastic, medullary, follicular, and papillary cancers were exposed to NV1023 and assessed for susceptibility to viral entry and oncolysis. The expression of herpes simplex virus glycoprotein D receptors nectin-1 and herpes virus entry mediator was assessed by quantitative fluorescence-activated cell sorter and correlated with NV1023 entry and oncolysis.

Results: There was significant variation in the ability of NV1023 to enter thyroid cancer cells as measured by lacZ expression. Thyroid cancer nectin-1 expression correlated strongly with NV1023 entry. Nectin-1 transfections and antibody receptor blocking studies validated the importance of nectin-1 for NV1023 entry. Follicular cancers were least sensitive to NV1023 oncolysis. All anaplastic, medullary, and papillary cancers tested exhibited greater than 85% cytotoxicity 7 d after exposure to NV1023 at multiplicity of infection 1, although oncolysis was variable at multiplicity of infection 0.01. Significant correlations between nectin-1 expression and NV1023 oncolysis were identified using Pearson’s coefficients.

Conclusions: NV1023 causes significant cytotoxicity of anaplastic, medullary, and papillary thyroid cancers. Nectin-1 is a novel marker of thyroid cancer sensitivity to herpes oncolytic therapy that might guide patient selection for therapy.

	Introduction

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ANAPLASTIC THYROID CANCER is considered a fatal disease, with only a 6-month median survival period (1). Other aggressive thyroid cancers such as poorly differentiated and medullary carcinomas present difficult clinical challenges. Morbidity from uncontrolled locoregional disease is severe and includes airway compromise, dysphagia, and bleeding. Novel therapies are needed to improve outcomes for these patients.

Genetically engineered viruses based on herpes simplex virus (HSV)-1 represent a new class of cancer therapeutic agents. These viruses possess a striking ability to infect and lyse a variety of malignant tumors (2, 3, 4, 5, 6, 7). Clinical trials have suggested that these viruses are safe for clinical application (8, 9, 10). Our group described the construction of the NV1023 series of viruses and showed its potent antitumoral effects in various animal models (11, 12, 13, 14). In a pilot study, we showed that NV1023 causes significant thyroid cancer regression in a murine model in vivo and demonstrates cytotoxicity in vitro (14). As a next step, we sought to examine the efficacy of NV1023 in a larger panel of human thyroid cancer cell lines and identify a potential marker of susceptibility.

Successful herpes infection of cancer cells depends on interactions between a viral particle and cell surface receptors. HSV-1 envelope glycoprotein D (gD) must interact with one of three receptors [nectin-1, herpes virus entry mediator (HVEM), 3-O-sulfated heparin sulfate] necessary for viral entry (15). We hypothesized that the gD receptor expression by thyroid cancers may be a determinant of herpes viral efficacy. Our goals were to determine the efficacy of an oncolytic HSV against a panel of human thyroid cancers and determine whether gD receptor expression by thyroid cancers is correlated with HSV therapeutic efficacy.

	Materials and Methods

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Cell lines

Twelve human thyroid carcinoma cell lines were studied: five anaplastic (DRO90–1, KAT-4, KAT-4B, KAT–4C, KAT-18), two medullary (DRO81–1, TT), a follicular (WRO82–1), a follicular undifferentiated (FRO81–2), and three papillary (NPA-187, KAT5, KAT10) lines. CHOK1 is devoid of surface nectin-1 and HVEM and was used as a control.

TT was grown in Ham’s F-12 media with L-glutamine, sodium bicarbonate, 10% fetal calf serum (FCS), and 1% penicillin and streptomycin (P&S). CHOK1 was grown in F12 with 10% FCS and 1% P&S. Other cell lines were grown in RPMI 1640 with 14 ml/liter nonessential amino acids, 10% FCS, L-glutamine, sodium pyruvate, sodium bicarbonate, and 1% P&S.

Virus

NV1023 was derived from R7020, an HSV-1 vector originally designed as an HSV-1/2 vaccine candidate. NV1023 carries a 5.2-kb fragment of HSV-2 DNA, containing HSV-2 genes US2–2 to US2–5 (gG, gD, gI, and a portion of gE) inserted in the UL/S junction (11). NV1023 is attenuated by a 15-kb deletion in the inverted repeat region, deleting UL56 and one copy of the diploid genes ICP0, ICP4, and 134.5. NV1023 contains the Escherichia coli ß-galactosidase gene (lacZ) inserted at the US10–12 locus under control of the 47 promoter and wild-type HSV-1 glycoproteins. NV1023 was provided by MediGene, Inc. (San Diego, CA).

Viral entry assays

Cells were seeded in 96-well plates at 4 x 10⁴ cells in 100 µl media. After 8 h, NV1023 in 50 µl PBS was added at a multiplicity of infection (MOI) of 0, 1, or 5 for 1 h at 4 C and then incubated for 8 h at 37 C. Viral entry was measured by assessment of lacZ expression using an enhanced ß-galactosidase assay kit (Genlantis, San Diego, CA).

Cytotoxicity assays

Cells were seeded in 12-well plates at 3 x 10⁴ cells in 1 ml media. After 6 h, NV1023 in 100 µl PBS was added at an MOI of 0, 0.01, 0.1, and 1. On d 3, 1 ml of fresh media was added. Cells were lysed with Triton X-100 (1.35%) to release lactate dehydrogenase, which was quantified daily using a Cytotox96 kit (Promega, Madison, WI).

Quantitative fluorescence-activated cell sorting (qFACS)

qFACS was used to quantify cell surface receptor number or antibody binding sites (ABS) (16). Microbeads (Quantum Simply Cellular; Sigma, St. Louis, MO) coated with varying known amounts of immunoglobulin were used as a standard reference. Samples were suspended in PBS with 3% FCS and 0.01% sodium azide. Mouse monoclonal antibodies CW10 (anti-HVEM; Santa Cruz Biotechnology, Santa Cruz, CA) or R1.302.12 (anti-nectin-1; Beckman Coulter, Fullerton, CA) conjugated with phycoerythrin were used at concentrations determined sufficient to saturate receptors and added to 3 x 10⁵ cells on ice for 1 h. Samples were fixed and run on a FACScaliber flow cytometer (Becton Dickinson, Franklin Lakes, NJ). A standard curve from the microbead populations was used to convert the mean fluorescence intensity of the cell samples to ABS per cell.

Nectin-1 transfections

The anaplastic cell line with lowest nectin-1 expression (KAT4C) and CHOK1 cells, devoid of nectin-1, were used for transfection studies. Plasmid pBG38 (gift of Claude Krummenacher, University of Pennsylvania, Philadelphia, PA) contains human nectin-1 (16). CHOK1 and KAT4C cells were transfected in 10-cm petri dishes with 0 (control), 1.5, or 10 µg of pBG38 using GenePORTER (Gene Therapy Systems). pCDNA3.1 was used to bring the total DNA amount to 10 µg for all conditions. Transfected cells underwent viral entry assays (MOI 10) or qFACS. Experiments were performed twice for verification.

Nectin-1 receptor blocking assays

Six cell lines were seeded in 96-well plates at 4 x 10⁴ cells in 100 µl media. After 8 h, cells were chilled on ice for 20 min, and monoclonal antibody R1.302.12 (anti-nectin-1) was added to final concentrations of 0, 10, and 100 µg/ml. An equal volume of NV1023 virus in culture medium was added at MOI 1, and cells were incubated at 37 C for 6 h. Viral entry assays were performed.

	Results

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Viral entry assays

Viral entry is expressed as the increase in OD between noninfected vs. infected cells at MOI 1 or MOI 5 (Fig. 1). All cell lines supported some viral entry by NV1023. At MOI 1, increases in OD ranged from 0.004 (KAT 18) to 0.036 (KAT 5). At MOI 5, increases in OD ranged from 0.045 (FRO 81–2) to 0.301 (KAT 4B; Fig. 1A). These ranges show significant variation by cell lines in permissiveness to viral entry (P < 0.05 for both MOI 1 and 5) by ANOVA.

View larger version (31K): [in this window] [in a new window]   FIG. 1. NV1023 viral entry into thyroid cancer correlates with nectin-1 expression. A, NV1023 viral entry measuring increases in OD by lacZ assay after viral exposure demonstrates a greater than 9-fold (MOI 1) and 6-fold (MOI 5) variation across the different cell lines (P < 0.05 by ANOVA for both). B, qFACS assessment of cell surface nectin-1 and HVEM expression measures the number of ABS per cell. Nectin-1 expression varies widely between the different cell lines and peak values are 5-fold higher than HVEM expression, suggesting greater functional importance. C, Correlations between nectin-1 expression or HVEM expression and NV1023 entry at MOI 1 and MOI 5. Pearson’s coefficients showed significant correlations for comparisons with nectin-1 at MOI 1 (r = 0.77, P < 0.01) and MOI 5 (r = 0.69, P < 0.05). Correlations were weaker for HVEM at MOI 1 (r = 0.66, P < 0.05) and not significant at MOI 5 (r = 0.41, data not shown). D, Transfections using 0, 1.5, or 10 µg plasmid pBG38 increase cell surface nectin-1 ABS measured by qFACS for both CHOK1 (63 ABS, 2472 ABS, 23477 ABS, respectively) and KAT4C (67808 ABS, 176808 ABS, 242808 ABS, respectively). E, NV1023 viral entry into nectin-1 transfected cells was measured by lacZ assay and reported as change in OD ± SE values. CHOK1 cells transfected with 1.5 or 10 µg of pBG38 showed significantly increased viral entry with mean OD of 0.096 ± 0.0015 and 0.356 ± 0.0015, respectively (P < 0.05 for both, t test), compared with controls (0.019 ± 0.0047). KAT4C cells showed a mean OD of 0.66 ± 0.005 and 0.8 ± 0.008, respectively (P < 0.05 for both, t test), compared with controls (0.47 ± 0.012). F, Nectin-1 antibody was used to block receptors on thyroid cancer cells with varying amounts of nectin-1 expression. Cells were exposed to NV1023 at MOI 1 and viral entry assessed by lacZ assays. At 100 µg/ml, the relative inhibition of NV1023 viral entry was minimal or absent for the low nectin-1 expressing WRO (20%) and KAT4C (2%), moderate for the moderate nectin-1 expressing KAT10 (45%) and DRO90–1 (35%), and greatest for the high nectin-1 expressing TT (85%) and KAT4 (86%).

Nectin-1 expression varied widely from undetectable (WRO) to a high of 1,233,000 ABS (KAT 5). In contrast, HVEM levels of expression were lower, ranging from 28,000 (KAT 4C) to 258,000 ABS (NPA; Fig. 1B). Nectin-1 expression was strongly correlated with NV1023 viral entry at both MOI 1 (Pearson’s coefficient r = 0.77, P < 0.01) and MOI 5 (r = 0.69, P < 0.05; Fig. 1C). HVEM showed a weaker correlation with NV1023 viral entry at MOI 1 (r = 0.66, P < 0.05) but no significant correlation at MOI 5 (r = 0.41, P = not significant).

Nectin-1 receptor transfections

The control CHOK1 cells expressed only 63 nectin-1 ABS, whereas the 1.5 µg and 10 µg transfected CHOK1 cells exhibited 2472 and 23477 ABS, respectively. The control KAT4C cells expressed a baseline nectin-1 of 67808 ABS, whereas the 1.5 µg and 10 µg transfected KAT4C cells exhibited 176808 and 242808 ABS, respectively (Fig. 1D).

Control CHOK1 cells were relatively nonpermissive to NV1023 entry with an OD of 0.019. The 1.5 µg and 10 µg nectin-1 transfected CHOK1 cells demonstrated OD increases to 0.096 and 0.356, respectively (P < 0.05 for both, t test). The control KAT4C cell lines demonstrated an OD of 0.47, with increases to 0.66 and 0.8 after 1.5 µg and 10 µg nectin-1 transfections (P < 0.05 for both, t test; Fig. 1E).

Nectin-1 receptor blocking assays

Cell lines were selected with varying nectin-1 expression. The blocking activity is expressed as the percentage of viral entry under nectin-1 antibody blockade, compared with control. At 10 µg/ml, there was no inhibition in viral entry. At 100 µg/ml, viral entry was inhibited minimally in the low nectin-1 expressing WRO (20%) and KAT4C (2%), moderately in moderate nectin-1 expressing KAT10 (45%) and DRO90–1 (35%), and significantly in the high nectin-1 expressing TT (85%) and KAT4 (86%; Fig. 1F). HVEM and 3-OS-HS may account for entry under nectin-1 blockade.

Cytotoxicity assays and nectin-1 and HVEM correlations

Results are expressed as the percentage of viable cells after NV1023 exposure, compared with untreated controls. All anaplastic, medullary, and papillary lines demonstrated greater than 85% cell lysis at MOI 1, d 6 (Fig. 2). The two follicular lines showed 40–60% viability at MOI 1, d 6. MOI 0.1 yielded similar results over a longer period. At MOI 0.01 cytotoxicity was more variable; one anaplastic (DRO90–1), one papillary (KAT10), and one follicular (WRO) cell line showed no response by d 7, with the other lines showing variable cytotoxicity.

View larger version (29K): [in this window] [in a new window]   FIG. 2. NV1023 cytotoxicity of thyroid cancer correlates with nectin-1 expression. Cytotoxicity assays were performed on thyroid cancer cell lines after exposure to NV1023. Mean cell viabilities with SE values are depicted. All anaplastic (A) and medullary (B) lines demonstrated greater than 80% cytotoxicity by d 7 after exposure to MOI 1 or 0.1. Follicular (C) lines showed a partial response to NV1023 at MOI 1. Two of three papillary (D) lines showed greater than 80% cytotoxicity at all MOIs by d 7. At MOI 0.01, there was more variation in oncolytic response by all of the cell lines. E, Correlations between nectin-1 or HVEM expression and NV1023 cytotoxicity were performed at viral concentrations and time points with the greatest variation in response (MOI 0.01, d 6; MOI 0.1, d 5; MOI 1, d 3). For nectin-1 correlations, Pearson’s coefficients at all viral concentrations were significant (r = 0.81, 0.79, 0.67, respectively; P < 0.05 for all). For HVEM, only MOI 0.01 demonstrated a significant correlation (r = 0.61, P < 0.05), whereas the other concentrations (MOI 0.1 and 1) did not show significance (r = 0.49 and 0.21, respectively; P = not significant for both; data not shown).

	Discussion

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NV1023 is a replication-competent, genetically modified, and attenuated HSV-1 that has therapeutic effects for a variety of human malignancies and a favorable safety profile in a recent clinical trial (10). The single ₁34.5-deleted NV1023 series of viruses may have more effective oncolysis than double ₁34.5-deleted vectors and can effectively treat cancers in animal models through intratumoral, lymphatic, iv, and cavitary delivery (11, 12, 13, 14, 17, 18). These viruses can also be designed to carry cytokine genes to elicit antitumoral immune effects (11, 13). Tumor specificity by oncolytic HSV-1 may be related to differential receptor expression and Ras pathway activation facilitating viral replication (19, 20).

NV1023 exhibited significant variation in its ability to enter the 12 thyroid cancer cell lines tested. A critical step for HSV-1 entry is the interaction between viral envelope gD and specific cell membrane receptors (nectin-1, HVEM, 3-OS-HS) (15). There are currently no commercially available antibodies against 3-OS-HS. Measured levels of thyroid cancer nectin-1 showed a wide natural variation. HVEM receptor levels were 5-fold lower, suggesting that HVEM does not play as important a role as nectin-1 in this setting. Correlations between nectin-1 expression and NV1023 entry were statistically significant. We have recently shown nectin-1 also correlates strongly with NV1023 cytotoxicity in human squamous carcinomas, in which peak nectin-1 expression is less than half that of the thyroid cancers examined in this study (19).

At an MOI 1, NV1023 caused near-complete cytotoxicity of all the anaplastic, medullary, and papillary thyroid cancers by d 6. The finding that all of the anaplastic cancers are susceptible to oncolysis at this modest viral dose is clinically relevant because patients with these cancers are in greatest need of novel therapies. At MOI 0.01, the oncolytic effects of NV1023 were most variable and correlations with nectin-1 expression were strongest. The ability to correlate nectin-1 with the response of a tumor to low doses of virus is likely to be clinically important. Such information may assist with viral dose selection and predict successful response to therapy.

We demonstrate that an engineered HSV-1 has significant oncolytic activity against thyroid cancer cells and identify nectin-1 as a marker of sensitivity to herpes oncolytic therapy. Nectin-1 assessment on anaplastic and other aggressive thyroid cancers might allow clinicians to identify and select patients most likely to benefit from herpes oncolytic therapy.

	Footnotes

 
R.J.W. was supported by a Clinical Innovator Award from the Flight Attendant Medical Research Institute and a Faculty Career Development Award from the American College of Surgeons and the American Head and Neck Society.

This work was presented in part as a poster at the 88th Annual Meeting of The Endocrine Society, Boston, MA, 2006.

Y.-Y.H., Z.Y., S.-F.L., S.L., Y.F., and R.J.W. have nothing to declare.

First Published Online February 27, 2007

Abbreviations: ABS, Antibody binding site; FCS, fetal calf serum; gD, glycoprotein D; HSV, herpes simplex virus; HVEM, herpes virus entry mediator; MOI, multiplicity of infection; P&S, penicillin and streptomycin; qFACS, quantitative fluorescence-activated cell sorting.

Received January 8, 2007.

Accepted February 21, 2007.

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This Article Abstract Full Text (PDF) All Versions of this Article: 92/5/1965    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huang, Y.-Y. Articles by Wong, R. J. Search for Related Content PubMed PubMed Citation Articles by Huang, Y.-Y. Articles by Wong, R. J. Related Collections Thyroid Endocrine Oncology