Cancer Letters 339 (2013) 144–151

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Cancer Letters

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Recombinant leukemia inhibitory factor suppresses human medullarythyroid carcinoma cell line xenografts in mice

0304-3835/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.07.006

Abbreviations: MTC, medullary thyroid carcinoma; LIF, leukemia inhibitoryfactor; MEN, multiple endocrine neoplasia; RET, rearranged during transfection;MALDI-TOF, matrix-assisted laser desorption/ionization-time-of-flight; PBS, phos-phate-buffered saline.⇑ Corresponding author. Address: Department of Biochemistry, Medical College

of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. Tel.: +1(414) 955 4098; fax: +1 (414) 955 6510.

E-mail address: jipark@mcw.edu (J.-I. Park).

Dmytro Starenki, Nishant K. Singh, Davin R. Jensen, Francis C. Peterson, Jong-In Park ⇑Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA

a r t i c l e i n f o

Article history:Received 13 May 2013Received in revised form 2 July 2013Accepted 8 July 2013

Keywords:Medullary thyroid carcinomaRETLeukemia inhibitory factorSTAT3

a b s t r a c t

Medullary thyroid carcinoma (MTC) is a neoplasm of the endocrine system, which originates from para-follicular C-cells of the thyroid gland. For MTC therapy, the Food and Drug Administration recentlyapproved vandetanib and cabozantinib, multi-kinase inhibitors targeting RET and other tyrosine kinasereceptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor.Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the develop-ment of additional therapeutic modalities that have distinct activity. Previously, we reported that expres-sion of activated Ras or Raf in the human MTC cell lines, TT and MZ-CRC-1, can induce growth arrest andRET downregulation via a leukemia inhibitory factor (LIF)-mediated autocrine/paracrine loop. In thisstudy, we aimed to evaluate bacterially-produced recombinant human LIF for its efficacy to suppresshuman MTC xenografts in mice. Here, we report that, consistent with its effects in vitro, locally or system-ically administered recombinant LIF effectively suppressed growth of TT and MZ-CRC-1 xenografts inmice. Further, as predicted from its effects in TT and MZ-CRC-1 cell cultures in vitro, recombinant LIF acti-vated the JAK/STAT pathway and downregulated RET and E2F1 expression in tumors in mice. Theseresults suggest that LIF is a potent cytostatic agent for MTC cells, which regulates unique mechanismsthat are not targeted by currently available therapeutic agents.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Medullary thyroid carcinoma (MTC) is a neoplasm of the endo-crine system, which originates from parafollicular C-cells of thethyroid gland [1]. MTC occurs either sporadically or in hereditaryforms, i.e., familial MTC and multiple endocrine neoplasia (MEN)type 2 syndrome. MTC progresses slowly and is relatively rare,comprising about 5% of all thyroid cancers. Nevertheless, MTCcan be fatal. Currently, the only curative therapy for MTC is surgicalresection, which is not effective for metastatic or recurring MTC. Itis, therefore, necessary to develop additional therapeutic means,which requires identification of a mechanism exploitable to controlMTC cell growth/survival. Although recent studies report thedetection of additional oncogenic alterations (e.g., RasG12V) inMTC at lower frequencies [2–4], MTC is mainly caused by alteredactivity of the receptor tyrosine kinase, rearranged during transfec-

tion (RET). For example, various activation mutations occur in thecell surface receptor domain or the cytoplasmic kinase domain ofRET in about 95% of hereditary MTC and about 50% of sporadicMTC cases (reviewed in [5]). Accordingly, RET is a key primary tar-get for the design of a therapeutic strategy for MTC. The Food andDrug Administration recently approved vandetanib (trade nameCaprelsa, AstraZeneca) and cabozantinib (Cometriq, Exelixis), themulti-kinase inhibitors targeting RET and other tyrosine kinasereceptors activated by vascular endothelial growth factor, epider-mal growth factor, or hepatocyte growth factor for the treatmentof inoperable progressive MTC [6,7]. Nevertheless, these drugsare not always effective, demanding additional therapeutic strate-gies [6–8]. In this regard, a cytokine that inhibits oncogenic RETexpression in MTC cells may be useful.

It is now well understood that oncogenic stress can elicitgrowth inhibitory responses in different cell types, as an innate tu-mor defense mechanism [9–11]. Therefore, an emerging questionis whether these phenomena can be exploited for therapy. Previ-ously, we reported that expression of activated Ras or Raf in thehuman MTC cell lines, TT and MZ-CRC-1, can induce growth arrestand RET downregulation via a leukemia inhibitory factor (LIF)-mediated autocrine/paracrine loop [12,13]. LIF is a multifunctionalcytokine of the interleukin-6 family (reviewed in Ref. [14]). In MTCcells, LIF mediated growth inhibition via activation of the JAK/STAT

D. Starenki et al. / Cancer Letters 339 (2013) 144–151 145

pathway through the LIFR-gp130 receptor and subsequent down-regulation of RET and E2F1 [12,13] and induction of IFI16 [15].E2F1 is a critical transcription factor involved in S-phase cell cycleprogression [16] and IFI16 is a member of the interferon-inducibleHIN200 nuclear protein family, which is well known for its growthinhibitory effects in cells [15,17]. Of note, LIF sufficiently inducedgrowth arrest of TT and MZ-CRC-1 cells in vitro, presenting animportant possibility that LIF has a potential as a therapeutic agentfor MTC. It is therefore necessary to test this possibility in a phys-iologically more relevant MTC model.

In this study, we evaluated bacterially-produced recombinant hu-man LIF for its efficacy to suppress human MTC xenografts in mice.Consistent with its effects in vitro, recombinant LIF induced growthinhibitory responses in MTC xenografts in mice. Further, as predictedfrom it effects in MTC cell culture in vitro, recombinant LIF activatedJAK/STAT pathway and downregulated RET and E2F1 expression inMTC xenografts in mice. These results suggest that LIF is a potent cyto-static agent for MTC cells, which regulates unique mechanisms that arenot targeted by currently available therapeutic agents.

2. Materials and methods

2.1. Cell culture and reagents

Two well characterized human MTC lines, TT and MZ-CRC-1, were used in thisstudy. TT was obtained from ATCC and MZ-CRC-1 was provided by Dr. Robert Gagel(MD Anderson). Generation of TT-GAS3 cell line that stably expresses the STAT3 lucif-erase reporter construct, (GAS)3-Luc, was previously described [12]. Maintenance ofthese cell lines was previously described [18,19]. Briefly, TT and TT-GAS3 were main-

Fig. 1. Production of recombinant LIF. (A) Schematic of SUMO-LIF. (B) Silver staining of pof purified LIF. (D) The STAT3 activity reporter cell line TTGAS3 was treated with indicateddetermining the luciferase activity. Equal doses of commercially available human LIF (C

tained in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 16% fetal bovineserum (FBS), 100 units of penicillin and 100 lg of streptomycin per ml. MZ-CRC-1was maintained in high-glucose DMEM (Invitrogen) supplemented with 10% FBS inculture dishes coated with rat collagen (Sigma, St. Louis, MO). The control humanLIF was obtained from EMD Millipore (Billerica, MA). Silver stain kit was purchasedfrom Thermo Scientific (Rockford, IL).

2.2. Recombinant LIF

The recombinant human LIF used in this study was produced using the tan-dem hexa-histidine (His)-sumoylation (SUMO) tagging system, as previously de-scribed [20]. Briefly, LIF cDNA was cloned into a modified pQE30 vector(Qiagen, Germantown, MD) to generate a tandem hexa-His tagged SUMO-LIF fu-sion protein. BL21 E. Coli containing the pREP4 plasmid (for lac I expression) weretransformed with this pQE30-SUMO-LIF plasmid. One liter cultures in Luria–Ber-tani media were grown at 37 �C to A600 of 0.7. Expression of SUMO-LIF was in-duced with 1 mM isopropyl-b-D-thiogalactopyranoside for 20 h at 15 �C. Cellpellets collected by centrifugation were resuspended in 20 ml of lysis buffer(50 mM Tris-Cl, 300 mM NaCl, pH 7.8) and lysed using a French Press(1000 psi). The supernatant containing soluble SUMO-LIF was loaded onto Ni–NTA agarose resin (Clontech), washed with 50 mM Tris-Cl/300 mM NaCl/10 mMimidazole (pH 7.8) and eluted with 50 mM Tris-Cl/300 mM NaCl/400 mM imidaz-ole (pH 7.8). Elutions containing hexa-His tagged SUMO-LIF were digested withUlp1 (Ubiquitin-like specific protease 1/SUMO-Protease 1) leaving the native N-terminus of mature LIF. After dialysis, the cleaved hexa-His tagged SUMO andLIF were loaded onto Ni–NTA agarose resin and the flow-through containing LIFwas collected. LIF was further purified by reverse phase HPLC purification to>98% homogeneity. The purity of the final product was estimated by SDS–PAGEand silver staining. The identity of LIF was confirmed by matrix-assisted laserdesorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. Thein vitro biologic activity of LIF was analyzed in a reporter assay using TT-GAS3 cellline, containing the (GAS)3-Luc reporter. Luciferase activity was measured usingthe Luciferase Assay System (Promega, Madison, WI) and data were normalizedaccording to the manufacturer’s instructions.

urified fractions resolved by SDS–PAGE. (C) MALDI-TOF mass spectrometry analysisdoses of recombinant human LIF produced in this study (SUMO-LIF) for 48 h before

ont. LIF) were used for comparison. Data are mean ± SD (n = 3).

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2.3. Immunoblot analysis

Cells were lysed in 62.5 mM Tris (pH 6.8)-2% SDS mixed with the proteaseinhibitor cocktail (Sigma) and briefly sonicated before determining the proteinconcentration using the BCA reagent (Pierce, Rockford, IL). 50 lg of protein wasresolved by SDS–PAGE and transferred to a polyvinylidene difluoride membranefilter (Bio-Rad, Hercules, CA). Membrane filters were blocked in 0.1 M Tris (pH7.5)–0.9% NaCl–0.05% Tween 20 with 5% nonfat dry milk, and incubated withappropriate antibodies. Antibodies were diluted as follows: ERK1/2, 1:2500; phos-pho-ERK1/2 (Thr202/Tyr204), 1:1000; p27, 1:2000; glyceraldehyde-3-phosphatedehydrogenase (GAPDH), 1:5000; RET, 1:1000; IFI16, 1:1000 (Santa Cruz Biotech-nology); phospho-RET (Tyr1062), 1:1000 (R&D Systems); phospho-STAT3(Tyr705), 1:2000; phospho-STAT3 (Ser727), 1:2000; STAT3, 1:2000 (Cell Signal-ing); E2F-1 1:1000 (NeoMarkers). The Supersignal West Femto and Pico chemilu-minescence kits (Pierce) were used for visualization of the signal. Images ofimmunoblots were taken and processed using ChemiDoc XRS + and Image Lab3.0 (Bio-Rad).

2.4. Tumor xenograft studies

1 � 107 TT or 1.5 � 107 MZ-CRC-1 cells in 200 ll Hank’s Balanced Salt Solutionwere inoculated subcutaneously into the rear flanks of 6-week-old female athymicnude (nu/nu) mice (Charles River Laboratories). Once palpable, tumors were mea-

Fig. 2. Effects of local administration of LIF on MTC tumor xenografts. Athymic mice bearLIF was administered s.c. at the area adjacent to tumors every 2 days beginning at day(details in Section 2). (A) Changes in tumor sizes were determined at the indicated timetumors collected at the end of the treatment. (D) Body weight changes of animals at th

sured using Vernier calipers at intervals indicated in the text. Tumor volumes werecalculated using the formula: length �width � height � 0.5236. Mice were sortedinto groups of 4 to achieve equal distribution of tumor size in all treatmentgroups. Lyophilized recombinant LIF was dissolved in sterile nonpyrogenic salineat 0.2 mg/ml and administered every 2 days (total 10 doses). For subcutaneous(s.c.) administration, LIF-treated groups for each cell line received recombinantLIF (0.5 mg/kg per dose), and control groups received only the vehicle (sterile sal-ine), injected s.c. in the area adjacent to tumors. For systemic administration, LIF-treated groups for each cell line received recombinant LIF (1 mg/kg per dose), andcontrol groups received only the vehicle, sterile phosphate-buffered saline (PBS),injected into tail vein (i.v.) of each mouse. At the end of the experiments, animalswere euthanized by CO2 asphyxiation. All animal studies were performed accordingto protocols approved by the Institutional Animal Care and Use Committee atMedical College of Wisconsin.

2.5. Analysis of tumors

Tumors were homogenized in buffer containing 20 mM Tris (pH 7.5), 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 mixed with the protease inhibitorcocktail (Sigma), briefly sonicated and cleared by centrifugation. Measurement ofprotein concentration and immunoblot analyses were performed as describedabove.

ing TT and MZ-CRC-1 xenografts were treated with ten doses of LIF (0.5 mg/kg/dose).15 after tumor implantation. The control group was treated with the vehicle only-points. (B) Images of tumors collected at the end of the treatment. (C) Weights of

e end of treatment. Data are mean ± SEM (n = 4), *p < 0.05, **p < 0.01, t-test.

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2.6. Statistical analysis

Two-tailed unpaired Student’s t-test was used to assess the statistical signifi-cance of data sets. P value <0.05 was considered statistically significant.

3. Results

3.1. Production and validation of recombinant LIF

We produced a recombinant human LIF from bacteria using thetandem hexa-histidine-sumoylation tagging system (Fig. 1A). Anal-ysis of the final purification fraction by SDS–PAGE and silver stain-ing showed only one protein band on the gel, indicating the purityof our recombinant LIF (Fig. 1B). Analysis of this recombinant LIFby MALDI-TOF mass spectrometry identified the purified proteinas monomeric LIF (Fig. 1C). We then determined activity of thepurified LIF in a reporter assay using TT-GAS3 cell line, whichwas stably transfected with the (GAS)3-Luc reporter. Our recombi-nant LIF increased the activity of the luciferase reporter activity aseffectively as a commercially available LIF when used at an equiv-alent dose (Fig. 1D), confirming the in vitro biological activity of thepurified LIF.

3.2. Local administration of LIF effectively suppresses growth of MTCxenografts in mice

We first evaluated the potential of recombinant human LIF tosuppress TT and MZ-CRC-1 xenografts in mice. These cell linesare the most thoroughly characterized human MTC lines and, thus,

Fig. 3. Effects of systemic administration of LIF on MTC tumor xenografts. (A) Athymic midose) via tail vein injection every 2 days beginning at day 20 after tumor implantation. Thafter the termination of LIF treatment. Data are mean ± SEM (n = 4). (B) Individual monitoanimals at the end of the treatment. Data are mean ± SEM (n = 4).

have been extensively used for the evaluation of different thera-peutic agents [21–23]. TT contains an extracellular domain muta-tion (RETC634W) whereas MZ-CRC-1 contains a mutation in thekinase domain of Ret (RETM918T), representing two different mech-anisms of oncogenic RET activation in MEN2A and MEN2B tumors,respectively. TT and MZ-CRC-1 cells were subcutaneously injectedinto the flanks of immune-compromised nude mice. After 15 days,when tumors became palpable, we began treating mice by inject-ing 10 lg LIF (0.5 mg/kg) or same volume of the vehicle, PBS, intothe tumor sites. These treatments were performed every 2 days for20 days. We found that recombinant LIF treatment substantiallysuppressed growth of TT and MZ-CRC-1 xenografts (Fig. 2A). Whentumor weights were measured at the end of the treatment, recom-binant LIF-treated TT and MZ-CRC-1 tumors showed 81% and 93%reduction in size relative to the PBS-treated control tumors, respec-tively (Fig. 2B and C). Importantly, during these treatments, we didnot observe any apparent adverse effects of LIF on mice except thatLIF induced mild decreases in body weight (Fig. 2D).

3.3. Systemic administration of LIF effectively suppresses growth ofMTC xenografts in mice

Observing positive effects from the local administrationexperiment above, we next investigated whether TT andMZ-CRC-1 tumor growth could also be suppressed by a systemicadministration of recombinant LIF. For this, when TT andMZ-CRC-1 cells developed palpable tumors in the flanks of nudemice, LIF was administered by tail vein injection at a dose of1 mg/kg every 2 days for a total of 10 doses. This amount of LIF

ce bearing TT and MZ-CRC-1 xenografts were treated with ten doses of LIF (1 mg/kg/e control group was treated with the vehicle only. Mice were observed until 40 daysring of tumor relapse of LIF-treated TT xenografts in (A). (C) Body weight changes of

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(20 lg per dose) is twice as much as that used for the local admin-istration. Indeed, this systemic administration of LIF suppressedgrowth of MTC tumors very effectively (Fig. 3A). We observedthat growth inhibitory effects of LIF were more significant in TTtumors than in MZ-CRC-1 tumors; the tumor relapse of TT wasnot observed for about 3 weeks after cessation of the treatment(Fig. 3A, left panel). Intriguingly, two of four TT tumors did notrelapse after cessation of the treatment, as monitored for up to40 days post-treatment (Fig. 3B). In contrast, MZ-CRC-1 tumorsrelapsed shortly after the treatment (Fig. 3A, right panel). When

Fig. 4. Effects of LIF on cell signaling in MTC tumor xenografts. TT (A and B) and MZ-CRC-1described in Section 2. The lysates were examined by Western blotting for expression of t(100 ng/ml, 2 days) were loaded on the two right hand side lanes for comparison. (B and D1. Mean values of the control vs LIF-treated tumors were compared to calculate p-value

tumor suppressive effects were compared during the treatmentperiods (20 days), the systemic administration was as effective asthe local administration (compare Fig. 3 with Fig. 2).

Consistent with the local administration, the systemicadministration of LIF did not induce any apparent adverse effectson mice, except that LIF induced mild decreases in body weight(Fig. 3C). Loss of body weight was previously noted as a harmlessside effect of LIF [24]. These data suggest that LIF may beuseful for the suppression of MTC without significant systemictoxicity.

(C and D) xenografts from seven and eight mice, respectively, were homogenized ashe indicated proteins. Total cell lysates of TT and MZ-CRC-1 cultures treated with LIF) Total optical density was measured from the Western blot images of RET and E2F-by t-test.

Fig. 5. Effects of MEK/ERK inhibition on STAT3 activation in MZ-CRC-1 cells. MZ-CRC-1 cells were treated with increasing doses of the MEK1/2 inhibitor, AZD 6244,for 48 h in the absence/presence of LIF (100 ng/ml). Total cell lysates wereexamined by Western blotting for expression of the indicated proteins.

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3.4. Recombinant LIF consistently activates the JAK/STAT pathway anddownregulates RET and E2F1 in MTC xenografts in mice

In TT and MZ-CRC-1 cultures in vitro, LIF mediates growth inhi-bition via activation of the JAK/STAT pathway, which is accompa-nied by downregulation of RET and E2F1, and induction of IFI16[12,13,15]. To determine whether recombinant LIF consistently in-duces these effects in the tumor xenografts in mice, we analyzedTT and MZ-CRC-1 tumors collected after the local administrationof LIF (N = 4 per group). Western blot analyses of tumor extracts re-vealed that LIF induced significant elevation of STAT3 phosphory-lation at Tyr705 in all LIF-treated TT and MZ-CRC-1 tumors(Fig. 4A and C). We previously showed that STAT3 phosphorylationat Tyr705, a key mechanism for activation of STAT3 as a transcrip-tion factor [25], is necessary for LIF-mediated growth inhibition[12,13]. Moreover, recombinant LIF significantly downregulatedRET and E2F1 in all TT and MZ-CRC-1 xenografts (Fig. 4A and C;densitometry shown in Fig. 4B and D). These effects of LIF onMTC cells in an in vivo microenvironment are consistent with ourpreviously observed in vitro effects of LIF.

Nevertheless, not all LIF effects on MTC cells were consistentbetween in vitro and in vivo conditions. Although LIF consistentlyinduced STAT3 phosphorylation at Ser727 in TT tumors, it was

Fig. 6. Effects of long term LIF treatment on MTC cells in vitro. TT and MZ-CRC-1 cells werexamined by Western blot analysis for expression of the indicated proteins.

not observed in MZ-CRC-1 tumors (Fig. 4A and C). Importantly,we previously demonstrated that Ser727 phosphorylation of STAT3is not necessary for LIF to mediate growth inhibition in TT cells[12]. Although LIF induced ERK1/2 phosphorylation in MZ-CRC-1tumors, LIF did not induce ERK1/2 phosphorylation in TT tumors(Fig. 4A and C). Of note, inhibition of ERK1/2 phosphorylation usingthe MEK1/2 inhibitor, AZD6244, did not affect LIF-induced RETdownregulation or STAT3 phosphorylation in MZ-CRC-1 cellsin vitro (Fig. 5), indicating that MEK/ERK activation is not necessaryfor LIF to mediate growth inhibition. This is consistent with ourprevious observation in TT cells [12]. In addition, unexpectedly,LIF-mediated IFI16 regulation was not detected in both MTC tu-mors (Fig. 4A and C). These differences between in vitro andin vivo settings were not due to the different LIF treatment sched-ules. We observed that in vitro responses of TT and MZ-CRC-1 cellsto LIF could be sustained for a prolonged time period, as deter-mined by monitoring cells in cultures treated with 10 or 100 ng/ml of LIF every 2 days for 20 days (Fig. 6). These data demonstratethat, while the key mechanisms required for LIF to induce tumorsuppression are conserved between in vitro and in vivo, not allMTC cell responses to LIF are conserved between in vitro andin vivo microenvironments.

4. Discussion

This study demonstrates that administration of recombinanthuman LIF can suppress growth of the human MTC xenografts inmice. MTC is generally not responsive to classic chemo- or radia-tion-therapies. Current targets for the development of novel ther-apeutics for surgically incurable progressive MTC have focusedon inhibition of the receptor tyrosine kinases, including RET andreceptors of vascular endothelial growth factor, epidermal growthfactor, or hepatocyte growth factor. Although the multi-kinaseinhibitors, vandetanib and cabozantinib, have been shown to beeffective in about 50% or 47% of progressive MTC cases, respec-tively [7,6,26,27], these kinase inhibitors also cause multiple sideeffects. For example, significant dose reduction or discontinuationof the treatment was reported in 28% of the drug-treated patients[26]. Therefore, additional therapeutic options for MTC are re-quired. Importantly, when compared to the effects of vandetanibthat we recently reported using the same preclinical model [19],LIF treatment appears to be similarly effective as this tyrosine ki-nase inhibitor in suppressing TT xenografts (compare Figs. 2 and3 with Fig. 5 of the Ref. [19]).

e cultured for 20 days in the presence of LIF (10 or 100 ng/ml). Total cell lysates were

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Consistent with its previously shown in vitro effects [12,13,28],LIF induced STAT3 activation in MTC xenografts in mice. However,LIF-mediated regulation of the MEK/ERK pathway was not consis-tent between in vitro and in vivo experimental settings of TT andMZ-CRC-1 growth. Importantly, our previous data [12,13,28] andcurrent study indicates that MEK/ERK activation is not necessaryfor LIF to mediate RET downregulation in these MTC cells. Our pre-vious and current studies characterize STAT3 as an upstream regu-lator of RET in MTC cells, which is contrary to a previouscharacterization of STAT3 as a downstream mediator of RET-MEN2A in NIH3T3 fibroblasts [29]. Along with STAT3 activation,LIF consistently induced downregulation of RET-MEN2A and RET-MEN2B in MTC xenografts in mice. These observations supportthe capability of LIF to induce downregulation of the RET onco-genes in MTC cells. LIF also induced downregulation of the S-phasetranscription factor, E2F1, which may underlie an important mech-anism for LIF-induced cytostatic effects. Currently, we do not knowwhether these two LIF-induced mechanisms are connected to eachother. This will have to be addressed in our future study for betterunderstating of how LIF mediates growth inhibition in MTC cells.Although STAT3 activation and RET downregulation were consis-tent between the in vitro and in vivo experimental settings, notall cellular responses to LIF were conserved between the two set-tings. For example, LIF did not consistently regulate IFI16 expres-sion in the MTC xenografts, suggesting that there is a significantdifference between MTC cell responses under in vitro conditionsand in in vivo microenvironments.

There are a few interesting questions as to the effects of LIF onMTC cells. First, why does LIF induce cytostatic effects in MTCcells? Our previous studies characterized STAT3 activation inMTC cells as a key mechanism for LIF to downregulate RET andto induce cytostatic effects [12,13,28], which is contrasted to thecharacterization of STAT3 as an oncogene in different tumor types[30–32]. Of note, a recent study has demonstrated that STAT3 has atumor suppressive role in papillary thyroid cancer [33]. In conjunc-tion with this, our study may suggest that STAT3 has a tissue typespecific role and that STAT3 activity is growth inhibitory in thyroidepithelium. Second, is LIF-induced growth inhibitory signaling spe-cific to MTC cells? It has been reported that different cell typesundergoing Ras/Raf-induced growth inhibition secrete soluble fac-tors that can help reinforce cellular growth inhibitory responses[34–36]. These factors were cell type-specific and include insu-lin-like growth factor binding protein-7, plasminogen activatorinhibitor-1, interleukin-8, and growth-regulated oncogene-a. Wepreviously showed that LIF is induced upon Raf activation in differ-ent cell types, including MTC cells, a pheochromocytoma line(MPC8622), human small cell lung carcinoma cell lines (NCI-H209 and DMS53), and a human prostate carcinoma line (LNCaP)[12,28]. Intriguingly, LIF induced growth inhibitory responses onlyin MTC and pheochromocytoma cell lines [12,28]. Considering thatpheochromocytoma often arises in association with MEN2B [37],one may speculate that the growth inhibitory effects of LIF are spe-cific to certain cell types, possibly associated with MEN type 2syndrome.

Various cytokines have been tested for their efficacy in cancertherapy [38]. Some of those factors stimulate the immune systemor modulate the microenvironment of tumor cells while others(e.g., tumor necrosis factor-related apoptosis inducing ligand) tar-get tumor cells directly and induce cell death. Although LIF has notbeen tested in cancer, it has been tested for other pathophysiolog-ical conditions, including a few neurological disorders (e.g., amyo-trophic lateral syndrome and radiation- or chemotherapy-inducedneuropathy) [39–41]. Moreover, LIF has been shown to be rela-tively nontoxic in a few clinical settings [42]. For example, recom-binant LIF proteins (e.g., rhuLIF/emfilemin/AM424) were tested inphase 2 clinical trials at a relatively high dose (4 lg/kg) without

inducing adverse effects or adverse changes in the patients’ qualityof life [41]. These previous studies support the potential advantageof LIF to be easily introduced into a clinical application. In conclu-sion, our study suggests that LIF may have potential for MTCtherapy.

Conflict of interest

None declared.

Acknowledgements

We thank Dr. Robert Gagel for MZ-CRC-1 and Dr. Brain Volkmanfor generous support for protein purification. This work wassupported by American Cancer Society (RSGM-10-189-01-TBE),National Cancer Institute (R01CA138441), and FAMRI YoungInvestigator Award (062438) to J.P.

References

[1] R.M. Tuttle, D.W. Ball, D. Byrd, G.H. Daniels, R.A. Dilawari, G.M. Doherty, et al.,Medullary carcinoma, J. Natl. Compr. Canc. Netw. 8 (2010) 512–530.

[2] N. Agrawal, Y. Jiao, M. Sausen, R. Leary, C. Bettegowda, N.J. Roberts, et al.,Exomic sequencing of medullary thyroid cancer reveals dominant andmutually exclusive oncogenic mutations in RET and RAS, J. Clin. Endocrinol.Metab. 98 (2013) E364–E369.

[3] A. Boichard, L. Croux, A. Al Ghuzlan, S. Broutin, C. Dupuy, S. Leboulleux, et al., J.Clin. Endocrinol. Metab. 97 (2012) E2031–E2035.

[4] R. Ciampi, C. Mian, L. Fugazzola, B. Cosci, C. Romei, S. Barollo, et al., Evidence ofa low prevalence of RAS mutations in a large medullary thyroid cancer series,Thyroid 23 (2013) 50–57.

[5] M. Ichihara, Y. Murakumo, M. Takahashi, RET and neuroendocrine tumors,Cancer Lett. 204 (2004) 197–211.

[6] N. Degrauwe, J.A. Sosa, S. Roman, H.A. Deshpande, Vandetanib for thetreatment of metastatic medullary thyroid cancer, Clin. Med. Insights Oncol.6 (2012) 243–252.

[7] M. Nagilla, R.L. Brown, E.E. Cohen, Cabozantinib for the treatment of advancedmedullary thyroid cancer, Adv. Ther. 29 (2012) 925–934.

[8] S.A. Wells Jr., B.G. Robinson, R.F. Gagel, H. Dralle, J.A. Fagin, M. Santoro, et al.,Vandetanib in patients with locally advanced or metastatic medullary thyroidcancer: a randomized, double-blind phase III trial, J. Clin. Oncol. 30 (2012)134–141.

[9] M. Serrano, A.W. Lin, M.E. McCurrach, D. Beach, S.W. Lowe, Oncogenic rasprovokes premature cell senescence associated with accumulation of p53 andp16INK4a, Cell 88 (1997) 593–602.

[10] J. Zhu, D. Woods, M. McMahon, J.M. Bishop, Senescence of human fibroblastsinduced by oncogenic Raf, Genes Dev. 12 (1998) 2997–3007.

[11] A.W. Lin, M. Barradas, J.C. Stone, L. van Aelst, M. Serrano, S.W. Lowe, Prematuresenescence involving p53 and p16 is activated in response to constitutiveMEK/MAPK mitogenic signaling, Genes Dev. 12 (1998) 3008–3019.

[12] J.I. Park, C.J. Strock, D.W. Ball, B.D. Nelkin, The Ras/Raf/MEK/extracellularsignal-regulated kinase pathway induces autocrine-paracrine growthinhibition via the leukemia inhibitory factor/JAK/STAT pathway, Mol. CellBiol. 23 (2003) 543–554.

[13] D. Arthan, S.K. Hong, J.I. Park, Leukemia inhibitory factor can mediate Ras/Raf/MEK/ERK-induced growth inhibitory signaling in medullary thyroid cancercells, Cancer Lett. 297 (2010) 31–41.

[14] C.J. Auernhammer, S. Melmed, Leukemia-inhibitory factor-neuroimmunemodulator of endocrine function, Endocrine Rev. 21 (2000) 313–345.

[15] E.J. Kim, J.I. Park, B.D. Nelkin, IFI16 is an essential mediator of growthinhibition, but not differentiation, induced by the leukemia inhibitory factor/JAK/STAT pathway in medullary thyroid carcinoma cells, J. Biol. Chem. 280(2005) 4913–4920.

[16] N.B. La Thangue, The yin and yang of E2F-1: balancing life and death, Nat. CellBiol. 5 (2003) 587–589.

[17] D. Choubey, R. Deka, S.M. Ho, Interferon-inducible IFI16 protein in humancancers and autoimmune diseases, Front. Biosci. 13 (2008) 598–608.

[18] J.I. Park, C.J. Strock, D.W. Ball, B.D. Nelkin, Interleukin-1beta can mediategrowth arrest and differentiation via the leukemia inhibitory factor/JAK/STATpathway in medullary thyroid carcinoma cells, Cytokine 29 (2005) 125–134.

[19] D. Starenki, J.I. Park, Mitochondria-targeted nitroxide, mito-CP, suppressesmedullary thyroid carcinoma cell survival in vitro and in vivo, J. Clin.Endocrinol. Metab. 98 (2013) 1529–1540.

[20] M. Love, J.L. Sandberg, J.J. Ziarek, K.P. Gerarden, R.R. Rode, D.R. Jensen, et al.,Solution structure of CCL21 and identification of a putative CCR7 binding site,Biochemistry 51 (2012) 733–735.

[21] J.W. de Groot, I. Plaza Menacho, H. Schepers, L.J. Drenth-Diephuis, J. Osinga, J.T.Plukker, et al., Surgery 139 (2006) 806–814.

D. Starenki et al. / Cancer Letters 339 (2013) 144–151 151

[22] R. Morisi, M. Celano, E. Tosi, S. Schenone, M. Navarra, E. Ferretti, et al., Growthinhibition of medullary thyroid carcinoma cells by pyrazolo-pyrimidinederivates, J. Endocrinol. Invest. 30 (2007) RC31-4.

[23] I. Plaza-Menacho, L. Mologni, E. Sala, C. Gambacorti-Passerini, A.I. Magee, T.P.Links, et al., Sorafenib functions to potently suppress RET tyrosine kinaseactivity by direct enzymatic inhibition and promoting RET lysosomaldegradation independent of proteasomal targeting, J. Biol. Chem. 282 (2007)29230–29240.

[24] J.O. Jansson, S. Moverare-Skrtic, A. Berndtsson, I. Wernstedt, H. Carlsten, C.Ohlsson, Leukemia inhibitory factor reduces body fat mass in ovariectomizedmice, Eur. J. Endocrinol. 154 (2006) 349–354.

[25] J.F. Bromberg, M.H. Wrzeszczynska, G. Devgan, Y. Zhao, R.G. Pestell, C.Albanese, et al., Stat3 as an oncogene, Cell 98 (1999) 295–303.

[26] S.A. Wells Jr., B.G. Robinson, R.F. Gagel, H. Dralle, J.A. Fagin, M. Santoro, et al.,Vandetanib in patients with locally advanced or metastatic medullary thyroidcancer: a randomized, double-blind phase III trial, J. Clin. Oncol.: OfficialJournal of the Am. Soc. Clin. Oncol. 30 (2012) 134–141.

[27] P. Schoffski, R. Elisei, S. Müller, M.S. Brose, M.H. Shah, L.F. Licitra, B. Jarzab, V.Medvedev, M. Kreissl, B. Niederle, E.E.W. Cohen, L.J. Wirth, H.Y. Ali, C. Hessel, Y.Yaron, D.W. Ball, B. Nelkin, S.I. Sherman, M. Schlumberger, EXAM study group.An international, double-blind, randomized, placebo-controlled phase III trial(EXAM) of cabozantinib (XL184) in medullary thyroid carcinoma (MTC)patients (pts) with documented RECIST progression at baseline, J. Clin.Oncol. 30 (2012). Abstract 5508.

[28] J.I. Park, J.F. Powers, A.S. Tischler, C.J. Strock, D.W. Ball, B.D. Nelkin, GDNF-induced leukemia inhibitory factor can mediate differentiation via the MEK/ERK pathway in pheochromocytoma cells derived from nf1-heterozygousknockout mice, Exp. Cell Res. 303 (2005) 79–88.

[29] J.J. Schuringa, K. Wojtachnio, W. Hagens, E. Vellenga, C.H. Buys, R. Hofstra,et al., MEN2A-RET-induced cellular transformation by activation of STAT3,Oncogene 20 (2001) 5350–5358.

[30] R. Garcia, T.L. Bowman, G. Niu, H. Yu, S. Minton, C.A. Muro-Cacho, et al.,Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participatesin growth regulation of human breast carcinoma cells, Oncogene 20 (2001)2499–2513.

[31] J.I. Song, J.R. Grandis, STAT signaling in head and neck cancer, Oncogene 19(2000) 2489–2495.

[32] A. Subramaniam, M.K. Shanmugam, E. Perumal, F. Li, A. Nachiyappan, X. Dai,et al., Potential role of signal transducer and activator of transcription (STAT)3signaling pathway in inflammation, survival, proliferation and invasion ofhepatocellular carcinoma, Biochim. Biophys. Acta 1835 (2013) 46–60.

[33] J.P. Couto, L. Daly, A. Almeida, J.A. Knauf, J.A. Fagin, M. Sobrinho-Simoes, et al.,STAT3 negatively regulates thyroid tumorigenesis, Proc. Natl. Acad. Sci. USA109 (2012) E2361–E2370.

[34] J.C. Acosta, A. O’Loghlen, A. Banito, M.V. Guijarro, A. Augert, S. Raguz, et al.,Chemokine signaling via the CXCR2 receptor reinforces senescence, Cell 133(2008) 1006–1018.

[35] T. Kuilman, C. Michaloglou, L.C. Vredeveld, S. Douma, R. van Doorn, C.J.Desmet, et al., Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network, Cell 133 (2008) 1019–1031.

[36] N. Wajapeyee, R.W. Serra, X. Zhu, M. Mahalingam, M.R. Green, Oncogenic BRAFinduces senescence and apoptosis through pathways mediated by the secretedprotein IGFBP7, Cell 132 (2008) 363–374.

[37] N.C. Lee, J.A. Norton, Multiple endocrine neoplasia type 2B–genetic basis andclinical expression, Surg. Oncol. 9 (2000) 111–118.

[38] G. Dranoff, Cytokines in cancer pathogenesis and cancer therapy, Nat. Rev.Cancer 4 (2004) 11–22.

[39] A.M. Farese, L.A. Myers, T.J. MacVittie, Therapeutic efficacy of recombinanthuman leukemia inhibitory factor in a primate model of radiation-inducedmarrow aplasia, Blood 84 (1994) 3675–3678.

[40] J.B. Kurek, A.J. Radford, D.E. Crump, J.J. Bower, S.J. Feeney, L. Austin, et al., LIF(AM424), a promising growth factor for the treatment of ALS, J. Neurol. Sci. 160(Suppl. 1) (1998) S106–S113.

[41] I.D. Davis, L. Kiers, L. MacGregor, M. Quinn, J. Arezzo, M. Green, et al., Arandomized, double-blinded, placebo-controlled phase II trial of recombinanthuman leukemia inhibitory factor (rhuLIF, emfilermin, AM424) to preventchemotherapy-induced peripheral neuropathy, Clin. Cancer Res. 11 (2005)1890–1898.

[42] D. Metcalf, The unsolved enigmas of leukemia inhibitory factor, Stem Cells 21(2003) 5–14.