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Published OnlineFirst November 14, 2010; DOI: 10.1158/1078-0432.CCR-10-1994

Clinical
Canceresearch
cer Therapy: Preclinical

027 Prevents Pulmonary Metastasis in Experimental and

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ntaneous Mouse Metastasis Models

r Santel, Manuela Aleku, Nadine Röder, Kristin Möpert, Birgit Durieux, Oliver Janke, Oliver Keil,
Endruschat, Sibylle Dames, Christian Lange, Mona Eisermann, Kathrin Löffler, Melanie Fechtner, Fisch, Christiane Vank, Ute Schaeper, Klaus Giese, and Jörg Kaufmann
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pose: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph nodetasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate theacologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in fournt preclinical mouse models.erimental Design: Atu027 compared with vehicle or control small interfering RNA lipoplexes wasin two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneoustasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedulested low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment.ry tumor growth and lung metastasis were measured, and tissues were analyzed by immunohisto-stry and histology. In vitro studies in human umbilical vein endothelial cells were carried out toe an insight into molecular changes on depletion of PKN3, in support of efficacy results.ults: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, forma-f spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as wellmice with resected primary mammary fat pad tumors. In addition, we provide evidence that anse in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may changehelial function, resulting in reduced colonization and micrometastasis formation.clusion: Atu027 can be considered as a potent drug for preventing lung metastasis formation,
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which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy.Clin Cancer Res; 16(22); 5469–80. ©2010 AACR.

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interference (RNAi) therapeutics have been hailedotential new drug modality with the opportunity ofssing novel, nondruggable targets for combating aspectrum of diseases such as respiratory infections,olic disease, or cancer (1). Recently, a small num-f new RNAi-based drugs have been translated intol testing. Atu027 poses a novel investigational ther-ic agent, currently being tested in a Phase I clinicaloncology (2). This drug is composed of a liposo-formulated small interfering RNA (siRNA) target-e expression of PKN3 specifically in the vascular

or details, see refs. 3, 4). This targeting en-ually all vascular beds of different organs

the porgancells mestablescapevasionextracthe syvasaticirculinto ttion aforma

: Silence Therapeutics AG, Berlin, Germany

ry data for this article are available at Clinical Cancerttp://clincancerres.aacrjournals.org/).

uthor: Jörg Kaufmann, Silence Therapeutics AG,rasse 10, Otto-Warburg-Haus 80, 13125 Berlin,49-30-9489-2833; Fax: 49-30-9489-2801; E-mail:-therapeutics.com.

0432.CCR-10-1994

ssociation for Cancer Research.

ls.org

Research. on August 8, 20clincancerres.aacrjournals.org ed from

ing the tumor vasculature. Hence, an antiangio-mode of action was proposed resulting in preven-f tumor invasion and metastasis (2).pite substantial progress in oncology toward suc-l treatment of certain operable tumor entities, metas-is still the primary cause of severe morbidity andlity in cancer. The occurrence of metastasis in cancerts is often linked to poor prognosis and remains ange for therapeutic intervention. The metastatic pro-epends on multiple parameters, such as the compo-of the tumor microenvironment, the properties ofrimary tumor, and the compatibility of the distantthat influences the metastatic growth (5, 6). Cancerust pass through a sequence of different steps to

ish macroscopic metastases. These steps include theof a cancer cell from the tumor tissue through in-into adjacent host tissue, penetration through the

ellular matrix/basement membrane, and entry intostemic bloodstream (intravasation). Following intra-on, additional steps such as distribution via theation, arrest in the microvasculature, extravasationhe parenchyma of distant organs, and cell prolifera-
t these ectopic sites occur resulting ultimately in thetion of secondary colonies (“colonization”). Finally,
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20. © 2010 American Association for Cancer

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Translational Relevance

The development of RNA interference therapeuticsholds great promise in finding new treatment opportu-nities in oncology. Atu027, a novel RNA interference–based therapeutic modality based on liposomal smallinterfering RNA targeting PKN3, has recently entered aPhase I clinical trial in patients with advanced solidtumors and exemplifies a successful translational“bench-to-bedside” approach. This study underscoresthe initial findings on the antimetastatic pharmacolog-ic activity of Atu027, affecting both lymphatic and he-matogenous dissemination. Furthermore, these resultsprovide new insights in the mechanisms of action aswell as in its pharmacologic potential, which is rele-vant information for future Phase II clinical trial designand conduct.

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tases become established after outgrowth of micro-tases into angiogenic macroscopic tumors at thedary organ sites (7). Novel cancer therapeutics couldt all these different steps during metastasis as tore with cancer cell dissemination and even more im-tly with the progressive outgrowth of macroscopictases. This can be achieved by targeting the meta-behavior of the disseminating cancer cell itself buty modulating the host's environment, such as thetory system, the immune system or the quality ofstructures at the secondary metastatic site (“meta-niche”; ref. 8). In this context, changes in vasculareability, hemostasis, as well as the premetastaticformation contribute profoundly to the coloniza-nd outgrowth of metastases (5, 8, 9). For example,enance of vascular barrier function is important forlled paracellular transport between the bloodstreame tissue parenchyma. Therefore, changes in the bar-nction have a direct implication for tumor cell entryhe bloodstream as well as during extravasation intodary sites during hematogenous metastasis. Altera-in the vascular permeability through destabilizationrcellular adhesion within an endothelial monolayereen suggested to stimulate inflammatory processesetastasis (10, 11). The vascular endothelial growth-A (VEGF-A) protein (also known as vascular perme-factor; refs. 12, 13) is a prominent cytokine, whichtes endothelial cell proliferation during angiogene-

d increases also in vascular permeability (14). VEGFound to enhance junctional permeability by VE-rin (a main adherens junctions protein) internaliza-rough Src kinase (Src) activation associated with theembly of endothelial adherens junctions in cultured15–17). Interestingly, Src as a downstream effectorGF signaling was shown to affect directly vascular
ability and metastasis in vivo. In Src-KO mice, per-ility of the vasculature was reduced and the number
centra96 ho

ancer Res; 16(22) November 15, 2010

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g metastases was significantly decreased as assessedexperimental lung metastasis model (18).3, a member of the AGC kinase family [a serine/

nin kinase subfamily of which members sharerities in the catalytic kinase domain to PKA (alson as PKAC), cyclic guanosine 3′,5′-monophosphate–dent protein kinase, and protein kinase C; ref. 19],een validated as a promising novel therapeutic tar-prostate cancer cells for inhibiting tumor progres-nd lymph node metastasis formation (20). Theseloss-of-function studies have revealed that PKN3tes malignant cell growth downstream of chroni-activated phosphoinositide 3-kinase (PI3K) path-20). Recently, PKN3 has also been considered astable therapeutic target for modulating tumor-ated angiogenesis, because loss of function analysisAtu027 in cultured primary endothelial cells re-an essential role of PKN3 for endothelial tube

tion and migration (2). Additional preclinicalacologic studies with Atu027 showed its inhibito-ect on invasive tumor growth and regional lymphmetastasis in orthotopic pancreatic and prostate can-ouse models. Finally, systemic i.v. administration of7 has been shown to suppress PKN3 expression in a-mediated and dose-dependent manner in rodentsonhuman primates (2). In this manuscript, we fur-valuated the pharmacologic activity of Atu027 onmetastasis in both experimental and spontaneousnary metastasis models.

rials and Methods

ration of siRNA lipoplexes and siRNA moleculessiRNA lipoplexes were prepared as described previ-by mixing liposomes containing the novel cationicAtuFECT01, the neutral helper lipid DPhyPE, andEGylated lipid DSPE-mPEG with siRNA at a finalntration of 0.375 mg/mL siRNA and 2.89 mg/mLlipid (3, 4). Physicochemical properties of Atu027les and PKN3 siRNA sequence are as published (2).llowing siRNAs were used as negative controls: lucif-(Luc, unrelated control; ref. 4) and “Pool” (whichto an equimolar mixture of 30 different controls). All siRNA molecules were stabilized againstases by incorporation of alternating 2′-O-methyl-tides (21).

ro transfection and Western blot analysisman umbilical vein endothelial cell (HUVEC) cellspurchased from Lonza and cultured as recom-ed. Transfection and subsequent immunoblottingcarried out as described previously (22). Briefly,C cells were transfected ∼12 hours after cell seed-nd different amounts of Atu027 or control siRNAex solution diluted in 10% serum–containing me-were added to the cells to achieve transfection con-
tions in a range of 1 to 40 nmol/L siRNA. At 24 tours after transfection, cells were lysed and subjected
Clinical Cancer Research



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Atu027 Inhibits Pulmonary Metastasis

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munoblotting. The following monoclonal or poly-l antibodies were used for Western blot analysis:TEN, anti-VEGF receptor-2 (VEGFR-2), anti-AKT,RC, anti-β-catenin, anti-MEK1/2 (all Cell Signaling),KN3 (20), anti-FAK (Upstate), anti-Tie2 (Santa, anti-Redd1 (23), and anti-JAK1 (Becton Dickinson).nofluorescence analysis with HUVEC was done ased previously (22).

r syngraft and xenograft studies in miceerimental lung metastasis models. B16V or Lewis lungoma (LLC) cells (1 × 106) resuspended in 200 μLere injected into the tail vein of 8-week-old BDF1Harlan). siRNA lipoplex treatment started 1 day af-or cell challenge. After necropsy, metastasis to the

was evaluated qualitatively by macroscopic photo-s and quantitatively by determining lung weight.ntaneous lung metastasis models. MDA-MB-435 orMB-231 cells (5 × 106) resuspended in 40 or 50 μLere injected into the mammary fat pad (m.f.p.) ofk-old female scid/bg mice (Taconic). Treatment withlipoplexes started after tumors became established.r volume was determined by caliper measurementlculated according to the formula length × width2/2.ropsy, metastasis burden refers to relative increaseal lung weight. Alternatively, tumors were resectedys after orthotopic transplantation of MDA-MB-435Mice were randomized into four groups accordingsplantation tumor volume and subsequently treatediRNA lipoplexes. Atu027 and negative control siRNAexes were given i.v. by low-pressure, low-volume tailnjection at 300 μL/30 g mouse of siRNA lipoplexon containing 0.28 mg/mL siRNA and 2.17 mg/mLiluted in 270 mmol/L sucrose (equivalent to a dosemg/kg siRNA and 21.7 mg/kg lipid). All animalments were done according to approved protocolscompliance with the guidelines of Landesamt für

dheit und Soziales Berlin.

logy, immunohistochemistry, and microscopye were sacrificed, and tissue samples of tumor andwere instantly fixed in 4.5% buffered formalin forurs and processed for paraffin embedding. Paraffinns (3 μm) from different areas of each tumor samplegenerated and mounted onto glass slides for histo-ssessment. For chromogenic staining, tissue sectionsprocessed for immunohistochemistry according toard protocols with purified antihuman vimentinclonal antibody (Santa Cruz), mouse monoclonali-67 (DAKO), and rat anti-CD34 (Cedarlane Labs).ody binding was visualized using NovaRED Sub-(Vector Laboratories). Nuclei were counterstainedematoxylin (Chroma). Sections were examined withs Axioplan microscope. The number of microvesselsetermined as previously described (2). For confocalsis of tissue sections, paraffin-embedded tissue
ns were (double-)stained by immunofluorescenceing to standard protocols (3, 24) with a rat mono-
mRNAcies w

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l anti-CD34, rabbit polyclonal anti-VE-cadherinan), or rabbit polyclonal anti-smooth muscle actina). Antibody binding was visualized using Alexaabeled secondary antibodies (Invitrogen). Nucleicounterstained with SYTOX green dye (Invitrogen)-PRO3. Microscopic analysis of microvasculatureone with a Zeiss LSM510 Meta confocal microscopeouble/triple staining recorded using the MultiTrack. The Zeiss LSM 510 Ver. 3.0 software was used forimensional color-coded height projection of fluores-intensity maxima.

tification of micrometastasis in the lungue sections stained with antihuman vimentin mono-l antibody were examined with a Zeiss Axioplan lightscope. Three areas with high density of metastaticies instead of large uncountable metastatic lesionseach lung section were identified and micrometas-(defined as vimentin-positive foci) were analyzed.J software (Ver. 1.42q NIH) was used to quantifytasis in lung sections. For ImageJ software–basedification of micrometastatic density, images wereken at 20× magnification by light microscopy. Themetastatic burden was expressed as number oftasis present in the defined area of 700.43 μm ×6 μm.

noblotting and quantitative reversecription-PCR (TaqMan) analysis of-lung tissue lysates from micelysis of protein and mRNA expression was carrieddescribed previously (3, 24). The sequence of the

rs in TaqMan can be obtained on request.

ticsa are presented as means ± SEM as indicated for each. Statistical significance of differences was deter-by the Mann-Whitney U test.

lts

7, a liposomal siRNA formulation targetingexpression in the lung vascular

thelium, prevents the formationerimental lung metastasis027 suppresses PKN3 mRNA levels by RNAi in vitrovivo, exhibiting an IC50 of 5 to 10 nmol/L (in vitro)

ermined in various cell culture systems (2). In addi-substantial knockdown of PKN3 in the vascular en-lium of the lungs in rodents and nonhumantes has been previously reported for Atu027, whenistered systemically (bolus or infusion; ref. 2). Asarized previously, different minimal effective dosesobserved after 4 hours of continuous i.v. infusionsree different species (mouse, rat, and cynomolgusey), and these effective doses for obtaining a PKN3
knockdown have been correlated in all three spe-
ith a Cmax in plasma of above 5 nmol/L (mouse)

Clin Cancer Res; 16(22) November 15, 2010 5471



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0 nmol/L (rat, cynomolgus), respectively (2).stingly, a comparable minimal efficacious doseg/kg) for pharmacologic inhibition of tumor pro-

on and lymph node metastasis (orthotopic PC-3l) has been established previously in appropriate
te cancer mouse models (2). These and other phar-ogy studies formed the basis for the ongoing Phase I
mentacell li



l trial for Atu027 (http://clinicaltrials.gov/ct2/show/0938574?term=atu027&rank=1).ause pulmonary vascular endothelium exhibitedt knockdown of PKN3 after Atu027 treatment, wed its effect on pulmonary metastasis in two experi-

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20. © 2010 American Associ

Atu027 efficacy onary metastasis inental metastasis models.elanoma cells (A-C) or LLC-F) were injected i.v. andwith Atu027 as indicatedB, E, inhibitory effect ofon total pulmonary tumordetermined by overall

ung weight. P valueslculated accordingn-Whitney test. C, F,copic pictures of cancercted lungs from each

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in injections to enable lung metastasis during ak time period (Fig. 1). This route of transplantationucing the cancer cells directly into the lung micro-ature circumvents the initial steps of metastasis, inular the intravasation of disseminating tumor cells.se both models lead very rapidly to moribund-ent mice, we immediately treated the animalsAtu027 after tumor cell challenge (for schedules,g. 1A and D). In comparison to the control groupd with the vehicle (270 mmol/L sucrose), tumorh in the lung was significantly inhibited in the7-treated mice in both models as indicated by thescopic pictures (Figs. 1C and F) and by the lungt measurement relative to the body weight (Fig. 1B; a normal mouse lung represents ca. 0.6% of bodyt; data not shown). These results suggest that Atu027ent reduced the establishment of hematogenousetastases.

ition of pulmonary metastasis after adjuvantpy with Atu027uvant therapy refers to additional supportive treat-
after primary cancer care, such as tumor surgery orion, suppressing the risk of cancer recurrence. We
day 7the re

s from indicated treatment groups. Tumor cells were visualized by anti-Ki-67 (huon paraffin-embedded tissue sections. Three representative examples per treat

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the MDA-MB-435 xenograft–based, spontaneousetastasis model to evaluate the therapeutic efficacy027 on metastasis after primary tumor resection.enograft was chosen for assessing the general effects027 treatment on tumor cell dissemination from thery tumor site (here m.f.p.) via the hematogenousto the lung as a remote target organ. In contrast toove-discussed experimental model, these spontane-ng models resemble more authentically the multipleduring metastasis, so that critical parameters such asnt host-tumor interaction, organ-specific outgrowth,rug exposure inflicting metastasis can be addressed.irror the clinical situation of an adjuvant therapyach more closely, treatments with Atu027 or vehiclese) and two different control siRNA lipoplexesetails, see Materials and Methods: siRNAPool andcontrol) began on d5 after resection of the establishedry tumors (d35 post implantation) at the m.f.p. site2A). Treatments twice weekly continued over ak period as indicated in Fig. 2A. Before resection,ean tumor volume of all cohorts had reachedm3 on day 35 (data not shown). By contrast on

2, the total tumor burden in the lung, measured bylative lung weight, was significantly reduced in the

Inhibition of pulmonary metastasis by Atu027 in a spontaneous metastasis model in an adjuvant treatment setting. A, experimental design ofntaneous lung metastasis model (MDA-MB-435, m.f.p.). B, lung metastasis burden on day 72 in respective treatment groups shown as mean valuesweight from n = 9 to 10 animals. P values were calculated according to Mann-Whitney test. C, histochemical analysis of metastasis in lung

man cell proliferation marker for specific detection of tumor cells)ment group are shown. Scale bar shown for all panels.




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7 treatment group when compared with the twol siRNA lipoplexes and the vehicle-treated groupsB). In addition, inhibition of lung metastasis wasd by histology/immunohistochemistry, visualizingerating metastasized human tumor cells throughi-67 staining (Fig. 2C) compared with control. Theis of the stained sections revealed a strong reductionmetastatic load to various areas of the lungs in7-treated mice (Fig. 2C). These results underscoreitial observation in the experimental lung metastasisls (Fig. 1) and show that Atu027 treatment selective-ibits pulmonary metastasis also in a more clinicallynt model for spontaneous lung metastasis.

7 suppresses spontaneous lung metastasis butrimary tumor growth in m.f.p. xenograft models027 inhibits hematogenous metastasis to the lungut affecting primary tumor growth. Therapeutic effi-f Atu027 was evaluated in a xenograft mouse modelontaneous lung metastasis using two highly meta-
cell lines [MDA-MB-435 (Figs. 2 and 3) and-MDA- analys
mor cell implantation. Representative pictures of immunohistochemically stained lu. Tumor tissue in the lung was visualized by staining with anti human-Ki-67 antibo



ow the pharmacologic effect of Atu027 on spontane-ng metastasis, we established primary MDA-MB-435rs in the m.f.p. of immune compromised mice andd the animals (n = 18/19) with Atu027 or sucrose,45-day period by i.v. bolus injection (twice weekly),g on day 12 post cell implantation. Tumor growthonitored during the treatment period, and pulmo-etastasis was analyzed at different time points postent by determining lung weights as indirect measureerall lung tumor burden (see experimental schedule. 3A). Interestingly, the tumor growth at the implan-site was not dramatically reduced through repeated7 treatment when compared with the sucrose vehiclel group (Fig. 3B; for MDA-MB-231 model in Supple-ry Fig. S1B).ontrast, the formation of spontaneous pulmonarytasis differed significantly between sucrose (vehicle)tu027-treated animals, when comparing lung weighttwo later time points after treatment (Fig. 3C). Thisvation was confirmed by immunohistochemical
is of lung sections using the human cancer cell pro-
1 (Supplementary Fig. S1)] at the m.f.p. (25, 26). liferation marker Ki-67. In control animals, the area of

Atu027 inhibits pulmonary metastasis. A, experimental setup of the spontaneous MDA-MB-435 metastasis model. B, m.f.p. tumor growth curvecle/sucrose (circle) and Atu027 (triangle). Group mean volumes of tumors with SEMwere plotted over time. The treatment period is highlighted in gray.rams showing mean lung weight as percentage of body weight for the vehicle (white column) and Atu027 (black column) at indicated time points

ng lobe sections from tissue samples of corresponding treatmentdy. P values were calculated according to Mann-Whitney test.




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erative metastatic lesions increased massively overinvading almost the entire area of the inspected lungat the end point of the experiment (day 76: 76 daysmplantation; 19 days post treatment). In contrast, inns from Atu027-treated mice, only smaller metastatics were scattered across the entire lung parenchymaC).

7 impairs establishment of pulmonarymetastasest, we studied the quality and appearance of metasta-ore closely at three different time points after end ofent (days 58, 68, and 76) by visualizing vimentin-ve tumor cells by immunohistochemistry. The overaller of detectable individual tumor cell colonies in7-treated animals was lower than in the sucrose con-oup (Fig. 4A). This observation has led us to speculatetu027may affect early colony formation ofmetastaticcells in the lung. As shown in Fig. 3C, no significantnce in lung weight as an indirect measure for metas-as found at the time point (day 58) immediatelyhe end of the treatment. However, quantificationividual metastatic clusters defined as all smallnohistochemistry-detectable vimentin-positive can-ll foci in a size range from 50 to 2,000 μm2 (seeials and Methods; Fig. 4C) showed the sharp reduc-micrometastasis formation after Atu027 treatmentfirst time point (day 58; Fig. 4B). This result wasevealed when looking at distinct micrometastaticrs, comprising individual tumor cells and small clus-nging in size from 10 to 100 cells instead of out-metastatic lesions (Fig. 4C, left: category 1-4). Foree analyzed categories, formation of early microme-s was already decreased in analyzed sections from7-treated mice in contrast to control at earliest time(Fig. 4C). Again, from these data, we conclude that7 treatment inhibits profoundly cancer cell dissem-n, resulting in a smaller number of early-formed me-c colonies. In summary, these observations advocateharmacologic effect on both, colonization (dissem-n and extravasation) of metastasizing tumor cells atcondary sites, which is likely to affect consequenttatic outgrowth.upport of these results, we also observed a similaron micrometastasis and macrometastasis formationusing the slower metastasizing human breast cancere MDA-MB-231 in the m.f.p. xenograft model (Sup-ntary Fig. S1A). Again, tumor size at the primary siteot significantly differ between the treatment groupslementary Fig. S1B), but the overall metastatic tumorn in the target organ lung was significantly decreasedAtu027 cohort compared with vehicle control group,ting that the antimetastatic effect of Atu027 is notdent on a particular cell line but seems to act onng as a secondary site for metastasis (SupplementaryC). Furthermore, Atu027-treated mice exhibited less
ized micrometastases, suggesting again a therapeuticon metastatic colonization.
lipopany st

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en together, these data revealed a pharmacologicof Atu027 on pulmonary metastasis inhibition independent models rather than on tumor progres-t the primary site (m.f.p.).

sment of different treatment schedulestu027discussed above, adjuvant treatment with Atu027s an option in preventing metastasis to remote sec-y organ sites. In support for future clinical trials with regards to treatment regimens (number ofand duration), we set out to compare four differentent schedules with Atu027 in the above-describedaneous MDA-MB-435 lung metastasis mouse xeno-odel. We compared the therapeutic effect of eightgiven either once (regimen R1) or twice weeklyen R2) over an 8- or 4-week period, respectively.er regimen (R4) included 16 doses over a treatmentof 8 weeks (twice weekly) or for comparison over aek period with two 4-week treatments separated byeek intermission (R3: 8 + 8 doses; see schematic. 5A).shown in Fig. 5B, the comparison of these differentens in this mouse model shows an improved thera-effect on metastasis with an expanded dosing (8 ver-treatments: R2, R4). Interestingly, the group treateduously for 8 weeks (R4) showed the same inhibitorywhen compared with a group treated in two 4-weekwith one intermission (R3). These data suggest thatre regimen with separated treatment cycles, resem-rather a schedule used in chemotherapy, may beption, but a continuous adjuvant treatment mighte reasonable for increasing the therapeutic benefit.ver, it should be recognized that the mouse modelives only limited insight into pharmacologic rele-of these different schedules, so that it remains diffi-extrapolate to clinically relevant treatment regimens

man patients.

tion of PKN3 resulted in elevated levels ofdherin: Implications for Atu027-mediateditory effect on metastasisin vivo observations made in the lung metastasis

ls prompted us to elucidate the potential molecularanisms, underlying the prevention of spontaneousmetastasis on Atu027 treatment. We first analyzedle antiangiogenic effects of Atu027 with respect tovascular density (MVD) and endothelial cell remo-g of the vasculature (namely vessel maturation/te coverage) in respective m.f.p. tumors. Histologicment with respect to MVD as well as the quality ofr vasculature (pericyte coverage) was carried out onns from the primary breast tumors (MDA-MB-231DA-MBA-435; see experiments discussed above).l tumor tissue sections from Atu027-treated miceespective control sections (vehicle or control siRNA
lex treated) were analyzed, which did not revealriking differences in MVD and tumor vessel quality



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Atu02HUVEpossibtransfeffectssignalkinaseanalyPKN3with ccells;proteiknockAtu02

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ar (examples given in Supplementary Fig. S2). Thisation is consistent with a lack in antitumor efficacy.first attempt of deciphering molecular effects of7 on the cellular level, we did in vitro studies inC, studying PKN3 loss of function and subsequentle molecular downstream events. HUVEC cells wereected with Atu027 to generate PKN3 loss-of-functionand analyzed for changes in protein levels of selecteding mediators (receptor and nonreceptor tyrosines) as well as some structural proteins. Western blotsis of protein extracts from HUVEC depleted ofrevealed elevated levels of VE-cadherin comparedontrol samples (untreated, siRNALuc lipoplex-treatedSupplementary Fig. S3). An increase in VE-cadherinn levels correlated in a dose-dependent fashion with

of quantification for each category (right diagrams; white columns, vehiclehitney test.

down efficacy as shown by titration of different7 concentrations (Fig. 6A). Of note, the loss of

cadhemulat



did not affect VE-cadherin mRNA levels, as revealedqMan PCR analysis in HUVECs transfected with7, suggesting that transcription of VE-cadherin wasffected (Fig. 6B). Moreover, loss of PKN3 did note levels of all other analyzed proteins, such as Src,-catenin, MEK1/2, AKT, JAK1, VEGFR-2, and PTENlementary Fig. S3). Interestingly, some othermarkers,ing the receptor tyrosine kinases, Tie2 and VEGFR-2,ow an increase in expression levels with increasedxic condition as a result of increased cell densitylementary Fig. S3; e.g., see also protein levels of theia-responsive Redd1 protein; ref. 23). Additionally,alyzed VE-cadherin levels in transfected confluentC by confocal microscopy to confirm the observedation. Immunofluorescence staining with anti-VE-

l; black columns, Atu027). P values were calculated according to

Atu027 impairs micrometastasis formation. A, histochemical detection of micrometastasis development over time in lung sections fromd treatment groups of the MDA-MB-435 metastasis model (see Fig. 3A). B, the diagram summarizes the quantification of vimentin-positiveetastatic colonies in lung sections from two time points after treatment. Individual micrometastatic colonies were counted in three fields per sectionGroup mean value of counts. C, quantification of separate micrometastatic colony categories (left immunohistochemical panels) as indicated.

rin antibody showed VE-cadherin membrane accu-ion with increasing intensity (by means of plotting




color-red, hmanncell-cemembFur

MicepulmolevelscussePKN3on VEHowenary bAtu02the dassessevidenVE-cacadhelungs

the mdothevesseling etumoa robNo

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Fig. 5.Atu027xenogrused inB, effecon lungMean vstatisticdue toaccord


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coded fluorescence intensity data: blue, low intensity;igh intensity) in an Atu027 concentration-dependenter (Fig. 6C). Elevated anti-VE-cadherin staining atll adhesion is reflected by an increase in red-coloredrane areas.thermore, we aimed to confirm these findings in vivo.treated i.v. with Atu027 exhibited a decrease in thenary expression of PKN3 on mRNA and protein(Supplementary Figs. S4A and B; ref. 2). As dis-d above for the in vitro situation, depletion ofmRNA in the pulmonary vasculature had no effect-cadherin mRNA levels (Supplementary Fig. S4C)ver, elevated expression of VE-cadherin in pulmo-lood vessels was identified in lung sections from7-treated mice (Fig. 6D). It should be noted thatata represent merely qualitative rather quantitativement and that VE-cadherin upregulation was nott in all analyzed sections. Unfortunately, the anti-dherin antibody did not allow assaying for VE-
rin levels in protein extracts derived from mouse. Recently, enhanced expression of VE-cadherin,
that Aulates

ing to Mann-Whitney test.

acrjournals.org


ain junctional protein of adherence junctions in en-lial cells (27), was reported as a marker for tumornormalization (28). Preliminary attempts of show-nhanced VE-cadherin protein expression in ther vasculature of Atu027-treated mice did not revealust VE-cadherin staining (data not shown).netheless, it remains conceivable that loss-of-PKN3on in endothelial cells might result in increased lev-VE-cadherin, suggesting consequences for endotheli-erens junction integrity in the control of vascularability or outside-in signaling properties for diversear beds in Atu027-treated animals.

ssion

his study, we aimed to give a deeper insight into theacologic activity and the possible underlying molec-echanisms of Atu027, a novel investigational drugtly in Phase I clinical trial for oncology. We propose
tu027, as a novel liposomal RNAi therapeutic, mod-the properties of the vascular endothelium, which
Effect of different treatment regimens ontherapeutic efficacy. A, schematic ofaft model and the treatment regimensthis animal study (regimens R1-R4).t of different Atu027 treatment regimensmetastasis compared with control.alues of relative lung weight as well asally significant increase in lung weightmetastasis. P values were calculated




ultimatasis.Atu02mightof thetoo. Pcularmetastasis.vasculbasaltion (

tumo(extraendotorganvascultemicboththelestumointrav

Fig. 6.with AtVE-cadshowedRelativelipoplexon coaare shown, indicating different VE-cadherin levels by the degree of red membrane staining at cellular adhesion sites. D, two examples for pulmonary venousvessels ared w

Santel et al.

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tely leads to the prevention of hematogenous metas-The data we provided suggest a model whereby7-mediated reduction of endothelial PKN3 expressionlead to elevated VE-cadherin levels in the vasculaturelung as secondary organ site and possibly tumor,erhaps, this change in VE-cadherin levels reduces vas-leakage and the intravasation and extravasation oftatic tumor cells, resulting in the prevention of metas-Metastatic tumor cells need at first to overcome thear endothelium of blood vessels and their underlying

with increased VE-cadherin endothelial staining (arrows) are shown comp

membrane at the tumor site for entering the circula-intravasation). To establish second site metastatic

the apversus



rs, the spread cancer cells need to successfully invadevasate) from the circulation by again crossing thehelial barrier into the adjacent parenchyma of a given(e.g., lung or liver). Finally, metastases initiate neo-arization (angiogenesis; refs. 29, 30). Therefore, sys-action of Atu027 on diverse vascular beds can affectentry and exit steps of metastatic cancer cells. Never-s, we cannot definitively discriminate whether ther cell dissemination is blocked at the stage of eitherasation or extravasation. However, the results from

ith control.

Atu027 alters VE-cadherin levels. A, concentration-dependent effect of Atu027 on VE-cadherin, as revealed by transiently transfecting HUVECu027 and control siRNALuc lipoplex (Luc, luciferase). Corresponding protein extracts were probed in a Western blot experiment for PKN3 andherin; PTEN was used as loading control. Tested siRNA concentrations are indicated: ut, untreated cell extracts. B, Atu027-transfected HUVECconcentration-dependent PKN3-mRNA knockdown but no changes in VE-cadherin mRNA levels as measured by TaqMan real-time PCR.amount of mRNA levels of the respective target normalized to PTEN-mRNA are shown for indicated siRNA concentrations (gray columns, siRNALuc

; black columns, Atu027) transfected in HUVEC. C, HUVECs transfected with Atu027 or siRNALuc lipoplex (Luc, luciferase) were grown to confluencyted slides, stained by immunofluorescence with anti-VE-cadherin, and subsequently analyzed by confocal microscopy. Representative pictures

plied two different metastasis models (experimentalspontaneous) may shed some light on this aspect,




implyperimof tumafter A(Fig. 1spontzationsugge(Figs.taneomerelHowethe eamendactionsophimodecer celrespecas it winsteatumortationmenevaluagrowtrelevaThe

primalishedderiverelevamadegraft mkinaseVEGFMDA-depeninhibimacolis alsSrc-KOtumoKO aIntereangioPHD2Syngeimpai(28).VE-cadvesselupregrestorwithinbut atighteinhibi

At pmetaswherein theenousorganand borthoevidennodeeffectsuppotheliaassocfluidmetasthe heby Atactiviantiasorafeas revto thebinatmothpotenaspecemergagainsOu

in thetreatmthe frmice)tumorapidment,targetcurrendoesissuesther eTak

in moventindirectand psettinpeutictionincludtastaspotenby ounarytransp


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ing Atu027 effects on both metastasis steps. The ex-ental model mimics already successful intravasationor cells, so that the observed inhibition of metastasistu027 treatment points to impaired extravasation). On the other hand, the data obtained with theaneous model indicate a reduced metastatic coloni-(reduced number of micrometastasis) and therefore

sts an impairment of the intravasation step as well2-4; Supplementary Fig. S1). The here applied spon-us metastasis models bear the following caveat thaty the end point effect of the drug can be evaluated.ver, it would be attractive to obtain insights intorly steps of the metastasis process, which would tre-ously contribute to the understanding of the mode ofof Atu027 during hematogenous metastasis. Moresticated imaging methods and mouse metastasisls, for example, could help to track the fate of the can-ls in real time during all steps of metastasis also witht to their interaction with the vascular endothelium,as shown in recent studies (30, 31). Furthermore,d of using cultured human cancer cells for orthotopicimplantation, so-called surgical orthotopic implan-(32), tumor fragments from surgical cancer speci-from patients can be used for implantation tote the efficacy of a novel drug modality on tumorh and metastasis in a more “clinically accurate andnt” model.observed lack of antitumor effect of Atu027 on

ry tumor growth resembles results from other pub-tumor inhibition studies with, e.g., MDA-MB-435–d tumors, suggesting the absence of tumor growthnt angiogenesis, and support previous findingsin orthotopic pancreatic and prostate cancer xeno-odels (2). For example, treatment with the multi-inhibitor E7080 directed against VEGFR-2 and

R-3 or with bevacizumab showed no significantMB-435 tumor inhibition whereas the lymph vesseldent MDA-MB-231 m.f.p. mouse model revealedtion of tumor growth (33). Furthermore, the phar-ogic effect for Atu027 in the here described studyo reminiscent of Src kinase inactivation. Like in

mice, metastasis inhibition occurred althoughr growth and MVD remained unchanged betweennd control (wild-type and heterozygotes; ref. 18).stingly, a recent report describes a very similar anti-genic effect of haplodeficient expression of thegene in the vascular endothelium on metastasis.nic tumors implanted in PHD2+/− mice showedred metastasis rather than reduced tumor growthMoreover, on the molecular level, HIF-inducedherin expression was shown along with improvedmaturation, whereas MVD was not affected. Again,ulated VE-cadherin may not only account fored endothelial normalization and vessel functionthe tumor as speculated by Mazzone et al. (28)

lso contribute to enhanced cell-cell adhesion and
ning of endothelial monolayer, thereby eventuallyting metastasis.
situatwe co

acrjournals.org


resent, it remains to be clarified whether this anti-tatic effect of Atu027 is restricted to the lung tissue,in strong RNAi-mediated PKN3 suppression occurspulmonary endothelium (2) or whether hematog-metastasis can also be reduced in other pivotalsites of metastasis, such as liver, kidney, brain,ones. Previous findings with Atu027 applied intopic pancreas and prostate cancer models providedce of suppressed local invasion as well as lymphmetastasis (2). The here proposed modulatoryof Atu027 on the vascular endothelium could alsort the initial finding in a way that improved endo-l barrier function virtually results in less tumor-iated swelling/edema development or interstitialpressure and the reduced possibility of lymphatictasis. Because the density of tumor vasculature inre described models were not significantly affectedu027 treatment (data not shown), the therapeuticty of Atu027 stands in sharp contrast to classicalngiogenic therapeutics such as bevacizumab ornib, both acting on tumor vessel size and numberealed in preclinical models (34, 35). The differencese classical antiangiogenic drugs supports the com-ion of Atu027 not only with conventional che-erapeutics but also other antiangiogenic agents totiate effective cancer treatment (36). The lattert is of importance especially with respect to theing tendency of tumors to develop resistancet antiangiogenic therapy (37–39).r results regarding treatment schedule and frequencymouse model system suggest that a prolongedent period might have a clinical benefit. However,equency of treatment (twice weekly schedule inis difficult to predict for the human system becauser development in mouse models progresses veryly and is likely to require a more frequent treat-enabling an RNAi-mediated target inhibition incell structures with high proliferation rates. Thet ongoing Atu027 Phase I study (NCT00938574)include a repeated treatment period to assess safetybut the optimal treatment regimen needs to be fur-valuated in future Phase II studies.en together, the here presented results with Atu027use models suggest a therapeutic benefit in pre-g metastasis to the lung. This observation hasimplications for future therapeutic applicationsroposes that Atu027 when given in an adjuvantg might be a promising new antimetastatic thera-drug. This approach will require a specific selec-

of patients with potential high metastatic risk,ing patients with evidence of inoperable microme-es or lack of clinically detectable metastasis. Thistial adjuvant treatment is supported in particularr results showing profound inhibition of pulmo-metastasis after resection of the breast xenograftlantation tumor (Fig. 2), modeling the clinical
ion of cancer patients post surgery. Therefore,nclude from our current preclinical studies that



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7 represents a new therapeutic modality for inhi-of pulmonary metastasis and may therefore be

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sure of Potential Conflicts of Interest

uthors are employees of and have stock options in Silence Thera-
AG. O. Keil, A. Santel, and J. Kaufmann are named inventors on
bilising modifications of synthetic siRNAs in mammalian cells.cleic Acids Res 2003;31:2705–16.pert K, Hajek P, Frank S, Chen C, Kaufmann J, Santel A. Loss of

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owledgments

hank Hüseyin Aygün (BioSpring) for siRNA synthesis.

Support

desministerium für Bildung und Forschung grant 0315156 PTJ.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

pplications directed to PKN3. O.
Keil and J. Kaufmann are namedrs on patent applications directed to AtuPLEX.
Received 07/26/2010; revised 09/14/2010; accepted 09/16/2010;published OnlineFirst 11/09/2010.

rencesitehead KA, Langer R, Anderson DG. Knocking down barriers:vances in siRNA delivery. Nat Rev Drug Discov 2009;8:129–38.ku M, Schulz P, Keil O, et al. Atu027, a liposomal small interferingA formulation targeting protein kinase N3, inhibits cancer pro-ssion. Cancer Res 2008;68:9788–98.ntel A, Aleku M, Keil O, et al. RNA interference in the mouse vas-ar endothelium by systemic administration of siRNA-lipoplexes forcer therapy. Gene Ther 2006;13:1360–70.ntel A, Aleku M, Keil O, et al. A novel siRNA-lipoplex technologyRNA interference in the mouse vascular endothelium. Gene Ther6;13:1222–34.uyen DX, Bos PD, Massague J. Metastasis: from dissemination toan-specific colonization. Nat Rev Cancer 2009;9:274–84.eg PS. Tumor metastasis: mechanistic insights and clinical chal-ges. Nat Med 2006;12:895–904.ler IJ. The pathogenesis of cancer metastasis: the “seed and soil”othesis revisited. Nat Rev Cancer 2003;3:453–8.aila B, Lyden D. The metastatic niche: adapting the foreign soil.t Rev Cancer 2009;9:285–93.ce JA, Pollard JW. Microenvironmental regulation of metastasis.t Rev Cancer 2009;9:239–52.MP, Park SI, Kopetz S, Gallick GE. Src family kinases as med-

ors of endothelial permeability: effects on inflammation andtastasis. Cell Tissue Res 2009;335:249–59.onan DM, De Lerma Barbaro A, Vannini N, Mortara L, Albini A.lammation, inflammatory cells and angiogenesis: decisions andecisions. Cancer Metastasis Rev 2008;27:31–40.orak HF. Discovery of vascular permeability factor (VPF). Exp Cells 2006;312:522–6.gy JA, Dvorak AM, Dvorak HF. VEGF-A and the induction of path-gical angiogenesis. Annu Rev Pathol 2007;2:251–75.is SM, Cheresh DA. Pathophysiological consequences of VEGF-uced vascular permeability. Nature 2005;437:497–504.is S, Cui J, Barnes L, Cheresh D. Endothelial barrier disruption byGF-mediated Src activity potentiates tumor cell extravasation andtastasis. J Cell Biol 2004;167:223–9.vard J, Gutkind JS. VEGF controls endothelial-cell permeability bymoting the β-arrestin-dependent endocytosis of VE-cadherin. Natll Biol 2006;8:1223–34.vard J, Patel V, Gutkind JS. Angiopoietin-1 prevents VEGF-uced endothelial permeability by sequestering Src through mDia.v Cell 2008;14:25–36.scuoli ML, Nguyen M, Eliceiri BP. Tumor metastasis but not tumorwth is dependent on Src-mediated vascular permeability. Blood5;105:1508–14.arce LR, Komander D, Alessi DR. The nuts and bolts of AGC pro-kinases. Nat Rev Mol Cell Biol 2010;11:9–22.nders F, Mopert K, Schmiedeknecht A, et al. PKN3 is required forlignant prostate cell growth downstream of activated PI 3-kinase.BO J 2004;23:3303–13.auderna F, Fechtner M, Dames S, et al. Structural variations and

1 function alters OPA1 processing and changes mitochondrialmbrane organization. Exp Cell Res 2009;315:2165–80.hwarzer R, Tondera D, Arnold W, Giese K, Klippel A, Kaufmann J.DD1 integrates hypoxia-mediated survival signaling downstreamphosphatidylinositol 3-kinase. Oncogene 2005;24:1138–49.ku M, Fisch G, Mopert K, et al. Intracellular localization of lipo-xed siRNA in vascular endothelial cells of different mouse tissues.crovasc Res 2008;76:31–41.ce JE, Polyzos A, Zhang RD, Daniels LM. Tumorigenicity and me-tasis of human breast carcinoma cell lines in nude mice. Cancers 1990;50:717–21.ce JE, Zhang RD. Studies of human breast cancer metastasising nude mice. Cancer Metastasis Rev 1990;8:285–97.stweber D. VE-cadherin: the major endothelial adhesion moleculentrolling cellular junctions and blood vessel formation. Arteriosclerromb Vasc Biol 2008;28:223–32.zzone M, Dettori D, Leite de Oliveira R, et al. Heterozygous defi-ncy of PHD2 restores tumor oxygenation and inhibits metastasisendothelial normalization. Cell 2009;136:839–51.o D, Nolan DJ, Mellick AS, Bambino K, McDonnell K, Mittal V.dothelial progenitor cells control the angiogenic switch in mouseg metastasis. Science 2008;319:195–8.nast Y, von Baumgarten L, Fuhrmann M, et al. Real-time imagingeals the single steps of brain metastasis formation. Nat Med 2010;:116–22.ffman RM. The multiple uses of fluorescent proteins to visualizencer in vivo. Nat Rev Cancer 2005;5:796–806.ffman RM. Orthotopic metastatic mouse models for anticancerg discovery and evaluation: a bridge to the clinic. Invest Newgs 1999;17:343–59.tsui J, Funahashi Y, Uenaka T, Watanabe T, Tsuruoka A, Asada M.lti-kinase inhibitor E7080 suppresses lymph node and lung metas-es of humanmammary breast tumor MDA-MB-231 via inhibition ofcular endothelial growth factor-receptor (VEGF-R) 2 and VEGF-R3ase. Clin Cancer Res 2008;14:5459–65.KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growth

tor-induced angiogenesis suppresses tumour growth in vivo.ture 1993;362:841–4.L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK

thway, inhibits tumor angiogenesis, and induces tumor cell apo-sis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res06;66:11851–8.J, Waxman DJ. Combination of antiangiogenesis with chemo-rapy for more effective cancer treatment. Mol Cancer Ther08;7:3670–84.rgers G, Hanahan D. Modes of resistance to anti-angiogenicrapy. Nat Rev Cancer 2008;8:592–603.os JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG,rbel RS. Accelerated metastasis after short-term treatment withotent inhibitor of tumor angiogenesis. Cancer Cell 2009;15:

2–9.os JM, Lee CR, Kerbel RS. Tumor and host-mediated pathways ofistance and disease progression in response to antiangiogenic
rapy. Clin Cancer Res 2009;15:5020–5.



2010;16:5469-5480. Published OnlineFirst November 14, 2010.Clin Cancer Res Ansgar Santel, Manuela Aleku, Nadine Röder, et al. Spontaneous Mouse Metastasis ModelsAtu027 Prevents Pulmonary Metastasis in Experimental and

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