LY294002 Enhances Expression of Proteins Encoded by

Feb 1, 2013 - Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklouho-Maclay Street, Moscow, 117997, ...
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LY294002 Enhances Expression of Proteins Encoded by Recombinant Replication-Defective Adenoviruses via mTOR- and Non-mTORDependent Mechanisms Mikhail V. Shepelev,*,† Elena V. Korobko,† Tatiana V. Vinogradova,‡ Eugene P. Kopantsev,‡ and Igor V. Korobko†,‡ †

Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, Moscow, 119334, Russia Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklouho-Maclay Street, Moscow, 117997, Russia

‡

S Supporting Information *

ABSTRACT: Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replicationdefective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirusdelivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery. KEYWORDS: mTOR, LY294002, adenovirus, transgene expression

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INTRODUCTION Adenoviral vectors have remained for a long time in a focus as a gene therapy tool. Three recombinant adenoviruses has been approved for clinical use to treat cancer including conditionally replicating oncolytic adenovirus (Oncorine) and replicationdeficient adenovirus for p53 tumor suppressor production (Gendicine) approved by the Chinese SFDA (State Food and Drug Administration), and Cerepro, herpes simplex virus thymidine kinase (HSVtk)-expressing replication-deficient adenovirus, which has been granted an orphan drug status in Europe and USA for malignant glioma treatment. Yet numerous results obtained through therapeutic adenovirus development, clinical trails and their clinical use revealed that their application as a single agent is not very efficient while the efficiency is achieved by combining them with other anticancer treatments.1−3 This conclusion necessitates identification of treatments which would exert the most potent action when combined with therapeutic adenoviruses designed to treat cancer. An attractive possibility consists in using adjuvant anticancer treatments which would not only act against the tumor cells but also enhance adenovirus performance per se. To date, several examples of such synergistic treatments have been identified which, besides their antitumor action, directly affect adenovirus© XXXX American Chemical Society

delivered transgene expression or replication of oncolytic adenoviruses. These include irradiation,1 topoisimerase4,5 or histone deacetylase inhibitors,6 gemcitabine,7 microtubulestabilizing agents.8 However for novel rationally designed drugs targeting specific molecular pathways the feasibility of combination with adenoviruses remains largely unexplored. As drugs of this type rapidly pave the way into clinical practice, it challenges delineation of molecular pathways which targeting would be beneficial to combine with therapeutic adenoviruses. Akt signaling pathway is one of the attractive targets in oncology, and Akt pathway inhibitors for cancer treatment are being actively developed now9 with some (e.g., rapalogues) already used to treat cancer. Thus it is tempting to investigate if adenoviral therapy would benefit from inhibiting Akt signaling pathway components. Moreover, adenoviral infection was reported to activate pro-survival Akt signaling in tumor cells10 thus providing another rationale to combine therapeutic adenoviruses with inhibitors of Akt pathway. In this work we studied effects of several inhibitors targeting different Received: June 8, 2012 Revised: January 11, 2013 Accepted: February 1, 2013

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obtain replication-defective adenoviruses. Aliquots of viruscontaining medium were stored at −70 °C. Virus titers determined using plaque titration method were ∼1 × 108 pfu/ mL for Ad-EGFP and Ad-hTERT-HSVtk, and ∼5 × 107 pfu/ mL for Ad-CMV-HSVtk. For infection, cells were plated at a density 1 × 104 cells/cm2 for NCI-H1299, A549 and Calu-I cells, and 2.5 × 104 cells/cm2 for NCI-H358 cells. Cells were infected the next day with adenoviruses with multiplicity of infection (MOI) 10 for Ad-EGFP and Ad-CMV-HSVtk, and MOI 200 for Ad-hTERT-HSVtk. To address the cytotoxic activity of Ad-hTERT-HSVtk adenovirus in the presence of HSVtk-convertable prodrug gancyclovir, Calu-I cells were plated in 96-well plates at 2000 cells/per well (day 0). The next day (day 1) cells were infected with Ad-hTERT-HSVtk adenovirus at MOI 5. After 24 h (day 2), medium was replaced with fresh medium with or without 30 μM gancyclovir (Sigma) and with or without addition of 25 μM LY294002. After 24 h (day 3) medium was replaced with fresh medium with or without 30 μM gancyclovir, but contained no LY294002. Twenty-four hours later (day 4) the medium was changed as above. 72 h later (day 7) the cell viability was assessed using colorimetric MTS-based CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Data from three independent wells for each experimental point were averaged and presented as mean relative cell viability ± standard deviation (SD) after normalization to viability of cells infected with adenovirus and not treated by gancyclovir and LY294002 which was taken as 1.00. Western Blot Analysis. For Western blot analysis of Addelivered transgene expression, cells were plated in 24-well plates and infected with adenovirus. Twenty-four hours after infection medium was changed for a fresh medium containing inhibitors. After 20 h of incubation with inhibitors cells were lysed in 1× SDS−PAGE loading buffer. Proteins were separated by SDS−PAGE and transferred to Hybond-P membrane (GE Healthcare, Little Chalfont, Buckinghamshire, U.K.). Western blot analysis was done with primary goat polyclonal anti-thymidine kinase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antiEGFP antibodies, clone 3A9 (Proteinsynthesis, Moscow, Russia), rabbit anti-phospho-Thr389 and anti-pan S6 kinase 1, anti-phospho-Ser473 and anti-pan Akt antibodies (Cell Signaling, Danvers, MA), and mouse monoclonal anti-α-tubulin antibodies, clone DM1A (Sigma, St. Louis, MO) followed by appropriate secondary (anti-mouse and anti-rabbit, GE Healthcare, Little Chalfont, Buckinghamshire, U.K.; or anti-goat, Santa Cruz Biotechnology, Santa Cruz, CA) horseradish peroxidaseconjugated antibodies. Immobilon Western Chemiluminiscent HRP Substrate (Millipore, Billerica, MA) was used for detection, and ChemiDoc XRS gel documentation system (BioRad, Hercules, CA) was used to acquire images. Flow Cytometry Analysis of EGFP Expression. For flow cytometry analysis cells in 6-well plates were infected with AdEGFP and treated with inhibitors as described above but in the presence of 50 ng/mL of doxycycline to induce moderate EGFP expression. Cells were harvested by trypsinization, washed in phosphate-buffered saline (PBS) and fixed in 3% freshly prepared PBS-buffered paraformaldehyde prior to flow cytometry analysis. Samples were analyzed on a Cytomics FC500 MPL flow cytometer (Beckman Coulter, Brea, CA). The mean fluorescence of samples outside the area of background fluorescence defined by uninfected cells was used to estimate EGFP expression in samples.

components of Akt signaling pathway, on expression of proteins encoded by replication-deficient recombinant adenoviruses. Although inhibiting Akt activity itself failed to improve adenovirus performance, we identified kinase inhibitor 2-(4morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) as a potent enhancer of adenovirus (Ad)-encoded protein expression, partially through inhibition of mammalian target of rapamycin (mTOR) protein kinase. Moreover, we showed that combination of LY294002 with topoisomerase inhibitors results in their synergistic effect on boosting Ad-encoded transgene expression.

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EXPERIMENTAL SECTION Inhibitors. Wortmannin, etoposide and camptothecin were supplied by Sigma (St. Louis, MO). Compound 401 and DMNB (4,5-dimethoxy-2-nitrobenzaldehyde) were from Santa Cruz Biotechnology (Santa Cruz, CA). TBCA ((E)-3-(2,3,4,5tetrabromophenyl)acrylic acid), Akt inhibitor X, LY303511, rapamycin, PIM1/2 kinase inhibitors V and VI, and Ku-63794 (mTOR inhibitor IV) were obtained from Calbiochem (La Jolla, CA). LY294002 was supplied by Calbiochem (La Jolla, CA) or Promega (Madison, WI). Stock solutions of wortmannin (1 mg/mL), etoposide (50 mM), camptothecin (10 mM), Compound 401 (10 mM), DMNB (100 mM), TBCA (50 mM), LY294002 (10 mM), LY303511 (10 mM), PIM1/2 kinase inhibitor V (50 mM) and VI (50 mM), and Ku63794 (25 mM) were prepared in DMSO and stored in singleuse aliquots at −70 °C. InSolution GSK3β inhibitor VIII was supplied as 25 mM solution by Calbiochem (La Jolla, CA) and stored in single-use aliquots at −70 °C. Akt inhibitor X stock solution (25 mM in water) was stored in single-use aliquots at −70 °C. Rapamycin was dissolved in methanol at 100 μg/mL and stored at −20 °C. Cells and Cell Culture. Non-small cell lung carcinoma NCI-H1299 (ATCC #CRL-5803), epidermoid carcinoma of lung Calu-I (ECACC #93120818), bronchioalveolar carcinoma of lung NCI-H358 (ECACC #95111733) and lung adenocarcinoma A549 (ATCC #CCL-185) cells were cultured in DMEM/F12 (1:1) (HyClone, South Logan, UT) supplemented with 10% FBS (HyClone, Cramlington, U.K.), penicillin (100 U/mL) and streptomycin (100 μg/mL). Adenoviral Vectors and Infection. Recombinant adenoviruses expressing HSVtk or EGFP (enhanced green fluorescent protein) were generated using the Ad-Easy system (Stratagene, La Jolla, CA) according to the instructions of the manufacturer. Briefly, the expression cassettes containing HSVtk cDNA under control of immediate/early CMV or hTERT (human telomerase reverse transcriptase) (nt −206... +37) promoter (cloning details are available by request) were subcloned to pShuttle vector (Stratagene, La Jolla, CA). To generate shuttle vector containing EGFP cDNA under control of tetracycline-regulated promoter, two expression cassettes were subcloned to pShuttle. The first expression cassette contained EGFP cDNA under control of tetracyclineresponsive elements and minimal CMV promoter derived from pBI-EGFP plasmid (Clontech, Mountain View, CA). The second expression cassette coding for tetracycline-responsive transcription factor under control of CMV promoter was subcloned from pTet-On plasmid (Clontech, Mountain View, CA). Homologous recombination of pShuttle-based plasmids was performed with Ad5 backbone (pAd-Easy; Stratagene, La Jolla, CA), and the resulting plasmids were linearized with PacI restriction endonuclease and transfected into HEK293 cells to B

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RESULTS

LY294002 Enhances Adenovirus-Encoded EGFP Expression in NCI-H1299 Cells. As an experimental model, we used NCI-H1299 cells infected with replication-deficient adenovirus encoding EFGP under control of tetracyclineinducible promoter (Ad-EGFP). To pinpoint critical targets in Akt signaling pathway affecting Ad-encoded transgene expression, we inhibited Akt upstream regulators, phosphoinositide 3-kinases (PI3Ks), with wortmannin and LY294002, the latter also directly inhibiting downstream Akt target mTOR.11 In addition, we inhibited Akt activity with Akt inhibitor X, and two principal downstream targets of Akt, protein kinase GSK3β and mTOR signaling complex 1 (mTORC1), with GSK3β inhibitor VIII and rapamycin, respectively. Among inhibitors tested, only treatment with LY294002 has resulted in profound increase in EGFP expression in Ad-EGFP infected cells (Figures 1a and 2a). LY294002 Acts through PI3K-Independent Mechanism. Although LY294002 is generally considered as a PI3K inhibitor, it was shown to inhibit activities of many “off-target” molecules.11−14 The lack of effect of wortmannin which at the concentration used (200 nM) efficiently targets PI3Ks but not mTOR, ATM, ATR or DNA-PK11,15 indicates that the ability of LY294002 to boost EGFP expression does not rely on the PI3K inhibition. Indeed, treatment of Ad-EGFP infected cells with LY294002 structural analogue, which lacks activity toward PI3Ks, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one (LY303511), 16 also resulted in upregulation of EGFP expression although the effect of LY303511 was less pronounced than that of LY294002 (Figures 1b and 2a and Supplemental Figure 1 in the Supporting Information). Finally, the effect of LY294002 was dose-dependent with maximal enhancement of EGFP expression reached at 50 μM LY294002 (Supplemental Figure 2 in the Supporting Information), a concentration significantly higher than that required to inhibit PI3K activities (IC50 ∼ 3−5 μM).11,17 Together these data indicate that PI3K inhibition does not underlie enhanced expression of adenovirus-encoded EGFP. LY294002 Acts through mTOR-Dependent and mTOR-Independent Mechanisms. Besides being PI3K inhibitor, LY294002 has a potency to inhibit a number of protein kinases which include mTOR, DNA-dependent protein kinase (DNA-PK),11 casein kinase II (CKII)14 and Pim-1,18 with other potential targets likely to exist.12 To reveal inhibition of what substrate(s) is responsible for the effect of LY294002 we assayed effects of various chemicals inhibitors with specificities overlapping that of LY294002. Treatment of cells with TBCA, a CKII inhibitor,19 has resulted only in marginal enhancement of EGFP expression achieved at TBCA concentration of 50 μM (Figures 1b and 2a and Supplemental Figure 3 in the Supporting Information). This suggests that CKII inhibition by LY294002 might be partially responsible for the enhancement of Ad-delivered EGFP expression although the contribution of this activity to the overall effect of LY294002 seems to be negligible. However, treatment of AdEGFP infected cells with LY294002 or LY303511 together with TBCA has resulted in suppression of both LY294002- and LY303511-induced upregulation of EGFP expression (Figure 1c) (LY303511 also inhibits CKII but with the effect being significant only at a concentration of 100 μM,20 which is higher than used in our experiments; see Supplemental Figure 1 in the Supporting Information). This result argues that CKII

Figure 1. Effects of different inhibitors on Ad-encoded EGFP expression. NCI-H1299 cells infected with Ad-EGFP adenovirus were treated with indicated inhibitors or their combinations. EGFP protein was detected by Western blotting with anti-EGFP antibodies. Membranes were also probed with anti-α-tubulin antibodies to monitor total protein loading. (a) GSK3β inhibitor VIII (10 μM; GSK inh), Akt inhibitor X (10 μM; Akt inh X), LY294002 (50 μM; LY2), wortmannin (200 nM; WM), rapamycin (500 ng/mL; Rap); (−) untreated cells. (b) LY294002 (25 μM; LY2), LY303511 (25 μM; LY3), TBCA (50 μM; TBCA), Compound 401 (50 μM; 401), wortmannin (20 μM; WM); (−) untreated cells. (c) LY294002 (25 μM; LY2), LY303511 (25 μM; LY3), TBCA (50 μM; TBCA) alone or in indicated combinations. (d) Ku-63794 (0.5, 2.5, 12.5, or 50 μM as indicated); (−) untreated cells. (e) Compound 401 (50 μM; 401) alone or together with wortmannin (20 μM; WM). (f) LY303511 (25 μM; LY3) and wortmannin (20 μM; WM) alone or in combination. (g) LY303511 (25 μM; LY3) and Compound 401 (50 μM; 401) alone or in combination.

inhibiting activity of LY294002 is required for its effect on Ad-encoded transgene expression. Another protein kinase targeted by LY294002 is DNA-PK. To address the question if inhibition of DNA-PK is responsible for the observed effect of LY294002 we assayed the effect of DMNB, a specific and potent (IC50∼15 μM) DNA-PK inhibitor.21 DMNB failed to enhance EGFP expression even at 50 μM concentration (Figure 2a and Supplemental Figure 4 in the Supporting Information) thus indicating that inhibition of DNA-PK does not affect Ad-delivered transgene expression. While showing no effect at 200 nM concentration, wortmannin in a dose-dependent manner upregulated EGFP expression in Ad-EGFP-infected NCI-H1299 cells with maximal effect observed at concentration 20 μM (Figure 1b and Supplemental Figure 5 in the Supporting Information). At C

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Figure 2. Effects of inhibitors and their combinations on Ad-encoded EGFP expression quantified by flow cytometry. (a) Graph showing mean EGFP fluorescence of NCI-H1299 cells infected with Ad-EGFP and treated with rapamycin (20 ng/mL; Rap), DMNB (50 μM; DMNB), Akt inhibitor X (20 μM; Akt inh X), LY294002 (25 μM; LY2), LY303511 (25 μM; LY3), Compound 401 (50 μM; 401), TBCA (50 μM; TBCA), etoposide (10 μM; Eto) or their indicated combinations relative to mock-treated Ad-EGFP infected cells (−). (b) Example of histogram plot showing fluorescence of NCI-H1299 cells not infected with adenovirus (control), cells infected with Ad-EGFP and left untreated (not treated) or incubated with 25 μM LY294002 alone (LY294002) or in combination with 10 μM etoposide (LY294002 + etoposide).

higher resulted in increased EGFP expression in Ad-EGFP infected NCI-H1299 cells (Figure 1d). Importantly, at these lower concentrations treatment with Ku-63794 resulted in nearly complete inhibition of Akt phosphorylation at Ser473 directed by mTOR signaling complex 2 (mTORC2),27 indicating inhibitory effect of Ku-63794 on mTOR (Supplemental Figure 7 in the Supporting Information). Finally, downregulation of mTOR with shRNA resulted in increased EGFP expression after Ad-EGFP infection (Supplemental Figure 8 in the Supporting Information) thus supporting the conclusion that mTOR inhibition is required for enhancing expression of adenovirally encoded transgene. Summarizing, LY294002 affects Ad-encoded transgene expression in part through direct mTOR inhibition. In addition, our results suggest that the effect of wortmannin at high concentration can be attributed to its targeting of mTOR rather than ATM/ATR. In line with that, combined treatment of cells with Compound 401 and wortmannin did not result in further increase in Ad-delivered EGFP expression (Figure 1e) thus suggesting their common target(s) in this process and favoring the hypothesis that the effect of wortmannin is due to direct inhibition of mTOR activity. Finally, wormannin further potentiated the effect of LY303511 (Figure 1f), indicating that these two inhibitors act through different molecular pathways to enhance Ad-encoded transgene expression. This apparently contradicts the reported inhibitory activity of LY303511 toward mTOR.20 However when we assayed activation of S6 kinase 1 (S6K1), an mTORC1 target, we found that in cells treated with LY303511 no suppression of S6K1 phosphorylation on mTOR-dependent site Thr389 had occurred while treatment with mTOR inhibitor Compound 401 (as well as LY2094002 and wortmannin) resulted in complete suppression of S6K1 phosphorylation (Figure 3a).

the concentration much higher than required to inhibit PI3Ks, wortmannin was reported to suppresses ATM, ATR, DNAPK15 and mTOR protein kinases,11 and one of these activities might be responsible for the improved transgene expression. However results of our experiments with DMNB ruled out the possibility that DNA-PK suppression affects Ad-encoded transgene expression. As LY294002 was reported not to affect ATM and ATR protein kinase activities22 but does target mTOR,11 it seems plausible that mTOR inhibition by wortmannin might be responsible for increased EGFP expression in Ad-infected cells. Indeed, treatment of AdEGFP infected cells with 2-(morpholin-1-yl)pyrimido[2,1α]isoquinolin-4-one (Compound 401), an mTOR and DNAPK inhibitor,23,24 did result in increase in EGFP expression (Figures 1b and 2a). As inhibition of DNA-PK does not affect EGFP expression, the effect of Compound 401 is likely to be determined by its ability to inhibit mTOR and/or other common targets for these compounds. Analysis of potential common protein kinase targets for LY29400213 and Compound 40124 revealed that Pim1 can be inhibited by both compounds. Therefore we investigated effects of specific Pim1/ 2 kinase inhibitiors V and VI.25 Treatment with either inhibitor failed to affect expression of adenovirally delivered EGFP transgene in cells in concentrations up to 50 μM (Supplemental Figure 6 in the Supporting Information) thus ruling out the possibility that Pim1 inhibition is involved in augmented transgene expression. To ultimately confirm that mTOR inhibition is required for enhancing adenovirally encoded transgene expression, we treated cells with highly selective mTOR inhibitor Ku-63794, showing little or no activity toward more than 200 protein and lipid kinases and with much reduced potency against MKK1 and PI3Kα (∼9 μM in in vitro assay).26 Treatment with Ku-63794 at concentration 2.5 μM or D

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Figure 3. LY303511 does not inhibit mTOR. NCI-H1299 cells infected with Ad-EGFP adenovirus were treated with wortmannin (20 μM; WM), LY303511 (25 μM; LY3), LY294002 (25 μM; LY2) or left untreated (−). Results of Western blot analysis with antibodies detecting total levels of S6 kinase 1 (S6K1) (a; pan-S6K1), Akt (b; pan-Akt), S6K1 phosphorylated at Thr389 (a; P-Thr389-S6K1) and Akt phosphorylated at Ser473 (b; P-Ser473-Akt) are shown.

Figure 4. Topoisomerase inhibitors potentiate effect of LY303511 and Compound 401 on expression of Ad-encoded EGFP. NCI-H1299 cells infected with Ad-EGFP adenovirus were treated with (a) LY303511 (25 μM; LY3), Compound 401 (50 μM; 401) and etoposide (10 μM; Eto) or with (b) LY303511 (25 μM; LY3), etoposide (10 μM; Eto) and camptothecin (0.1 μM; CPT) in indicated combinations. Results of Western blot analysis with anti-EGFP antibodies are shown. Membranes were also probed with anti-α-tubulin antibodies to monitor total protein loading.

This indicates that under these conditions LY303511 fails to inhibit mTOR activity in NCI-H1299 cells. The failure of LY303511 to target mTOR is further supported by the lack of suppression of Akt phosphorylation at Ser473 directed by mTOR signaling complex 2 (mTORC2)27 while treatment with LY294002, wormannin or Compound 401 resulted in decreased phosphorylation of Akt at this site (Figure 3b). Taken together, these data indicate that LY303511 does not inhibit mTOR activity under the experimental conditions used, and its synergistic effect with wortmannin on upregulation of Ad-encoded EGFP expression could be explained by combination of non-mTOR LY303511 activities and direct inhibition of mTOR by wortmannin. In support of this conclusion, combination of LY303511 and Compound 401 has resulted in higher level of EGFP expression in Ad-EGFP infected cells than either inhibitor alone (Figure 1g) which came close to the level of expression achieved in cells treated with LY294002 (Figure 2a). This suggests that while Compound 401 stimulates transgene expression through inhibiting mTOR, LY303511 inhibits mTOR-independent targets of LY294002, together mimicking LY294002 action. Synergistic Effect of Topoisomerase Inhibitors with LY303511, Compound 401 or LY294002. We have demonstrated that LY294002 has the potency to increase Adencoded EGFP expression which is likely to be due to mTOR inhibition mimicked by Compound 401, with other mTORindependent target inhibition mimicked by LY303511, both of which alone are also capable of increasing expression of Addelivered EGFP in NCI-H1299 cells. So far several other treatments have been shown to exert a similar effect on Adencoded transgene expression. Among them, inhibitors or topoisomerases I and II, topotecan and etoposide, respectively, were reported to improve expression of proteins encoded by recombinant adenoviruses.4,5 In line with this, both etoposide and camptothecin, a water-insoluble analogue of topotecan, were able to upregulate EGFP expression in Ad-EGFP-infected cells (Figure 4). We have questioned if combination of topoisomerase inhibitors and LY294002, LY303511 or Compound 401 would further potentiate expression of EGFP in Ad-EGFP infected cells. As evident from Figures 2a and 4a, combined treatment with LY303511 or Compound 401 and etoposide has resulted in further increase in EGFP expression. A similar synergistic effect was observed for combination of LY303511 and camptothecin (Figure 4b). Thus inhibitors of both topoisomerase I and II are capable of potentiating effects of

LY303511 and Compound 401. In line with this, simultaneous treatment of Ad-infected cells with LY294002 and etoposide has led to significant upregulation of Ad-encoded EGFP expression (Figures 2a,b), making their combination the most potent to increase transgene expression in cells infected with recombinant adenovirus. Promoter, Transgene and Cell Line Specificity of LY294002 and LY303511 Action. Next we questioned if the observed effects of inhibitors are restricted to EGFP transgene which expression is driven by tetracycline-inducible promoter in NCI-H1299 cells. We first assayed if treatment with LY294002 and LY303511, alone or in combination with etoposide, will enhance transgene expression in NCI-H358, A549 and Calu-I cells infected with Ad-EGFP. Similarly to NCI-H1299 cells, treatment with LY294002 or LY303511 upregulated EGFP expression in all three cell lines (Figure 5).

Figure 5. Effects of LY294002 and LY303511 on Ad-encoded EGFP expression is not restricted to NCI-H1299 cells. Indicated cells (NCIH358, A549 and Calu-I) were infected with Ad-EGFP adenovirus and treated with LY294002 (25 μM; LY2), LY303511 (25 μM; LY3) and etoposide (10 μM; Eto) in indicated combinations. EGFP protein was detected by Western blotting with anti-EGFP antibodies. Membranes were also probed with anti-α-tubulin antibodies to monitor total protein loading.

Moreover, their combination with etoposide further increased the level of Ad-encoded EGFP expression with the most pronounced effect observed for Calu-I and NCI-H358 cells (Figure 5). To address the question about promoter and transgene specificity of the observed effect, we assayed the expression of Ad-encoded HSVtk under control of various promoters. Both E

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LY294002 and LY303511 improved expression of HSVtk in cells infected with adenovirus encoding HSVtk under control of CMV immediate/early promoter (Ad-CMV-HSVtk; Figure 6a).

Figure 7. LY294002 augments gancyclovir-dependent cell death of Ad-hTERT-HSVtk-infected Calu-I cells. Calu-I cells were infected by Ad-hTERT-HSVtk adenovirus and treated (LY294002) or not treated (None) with LY294002 in the presence (+Ganc) or absence (−Ganc) of gancyclovir as described in Experimental Section, and viable cells were quantified with MTS tetrazolium compound-based colorimetric test. Data are presented as an average relative cell viability in three independent wells for each experimental point ± SD after normalization to viability of cells infected with adenovirus and not treated by gancyclovir and LY294002 which was taken as 1.00.

Figure 6. LY294002 and LY303511 upregulate expression of HSVtk under control of different promoters. NCI-H1299 cells were infected with (a) Ad-CMV-HSVtk or (b) Ad-hTERT-HSVtk adenoviruses and treated with (a) LY294002 (25 μM; LY2), LY303511 (25 μM; LY3) or left untreated, or with (b) and LY294002 (25 μM; LY2), LY303511 (25 μM; LY3) and etoposide (10 μM; Eto) in indicated combinations. HSVtk was detected in cell lysates by Western blotting with antithymidine kinase antibodies (HSVtk). Membranes were also probed with anti-α-tubulin antibodies to monitor total protein loading.

A similar effect was observed when CMV promoter was replaced by hTERT promoter (Ad-hTERT-HSVtk). Importantly, the effect of both inhibitors was potentiated in the presence of etoposide (Figure 6b). Together, these results demonstrate that the revealed potentiation of Ad-encoded transgene expression by LY294002 and LY303511 is not limited to the NCI-H1299 cell line and the combination of EGFP transgene and tetracycline-inducible promoter but is also observed in other cell lines and for different Ad-encoded promoter/transgene combinations. LY294002 Synergistically Augments GancyclovirInduced Cell Death in HSVtk-Expressing Cancer Cells after Adenoviral HSVtk Transgene Delivery. We have shown that LY294002 is a powerful inducer of recombinant protein expression delivered by adenoviral vectors, including therapeutically relevant HSVtk used in anticancer therapy in conjunction with gancyclovir prodrug to kill tumor cells. To address the question if LY294002 enhancement of adenovirusencoded HSVtk transgene expression would potentiate cancer cell killing capacity of adenovirus encoding HSVtk in the presence of gancyclovir, we have analyzed the effect of adjuvant LY294002 treatment on Ad-hTERT-HSVtk adenovirus cytotoxic effect in the presence of gancyclovir in Calu-I lung cancer cells. In the absence of LY294002, Ad-hTERT-HSVtk infection resulted in marginal inhibition of cell growth upon addition of gancyclovir under the experimental settings used, indicating low level of HSVtk expression which is not high enough to cause significant cell death (Figure 7). Transient treatment with LY294002 for 24 h after adenoviral infection in the absence of gancyclovir resulted in modest (20%) suppression of cell growth, which should be considered as an effect attributed to LY294002 action alone owing to the lack of adenovirusmediated toxicity in the absence of gancyclovir. However, when Ad-hTERT-HSVtk-infected cells were treated by LY294002 for 24 h to boost transgene expression, and cultured in the presence of gancyclovir, cell viability dropped at the end of incubation down to 40% (Figure 7). Thus, LY294002 enabled HSVtk protein level increase in infected cells which becomes sufficient to mediate massive gancyclovir-dependent cell death.

This result directly demonstrates that combined use of adenovirus-mediated therapeutic transgene delivery and LY294002 (and, owing to similarity of the effects, other compounds and their combinations tested) to increase transgene expression provides a valuable option to improve therapeutical effect of recombinant adenoviruses.

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DISCUSSION The current concept of using adenovirus-based drugs to treat cancer is their combination with adjuvant treatments which allows gaining maximal benefits from the adenovirus-based therapeutics.1−3 However adjuvant treatment should not suppress but preferably potentiate therapeutic adenovirus performance. Akt signaling pathways is one of the attractive targets to treat cancer.9 Moreover, infection with adenoviruses triggers activation of Akt signaling pathway thus potentially counteracting anticancer effect of adenoviral drug.10 Indeed, infection of cells with Ad-EGFP adenovirus resulted in activation of Akt signaling as evidenced by the increased level of Akt protein kinase phosphorylated at Ser473 in cells infected by the adenovirus at high multiplicity of infection (Supplemental Figure 9 in the Supporting Information), thus reconfirming previously reported results of Flaherty et al.10 As currently implied methods of recombinant adenovirus administration in cancer treatment remain to be local delivery with consequent locally high multiplicity of infection, this would promote local activation of pro-oncogenic Akt signaling and potentially hamper therapeutical adenovirus efficiency. Results of our study showed that inhibiting Akt signaling pathway (either upstream of Akt with LY294002 and wortmannin or directly by Akt inhibitor X) does not affect expression of Ad-delivered transgene. This validates the use of Akt pathway inhibitors to complement adenovirus-based anticancer therapy both to avoid unwanted effect of adenoviral infection on Akt pathway activation and to combine their antitumor activities to maximize efficiency of treatment. Besides combining antitumor effects of adenovirus-based drug and conventional cancer treatments to maximize treatF

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treatment to improve therapeutic adenovirus performance. Indeed, it was shown that combination of irradiation with inhibitors of DNA repair pathway (PARP, ATM and DNA-PK) resulted in increased Ad-encoded transgene expression compared with irradiation alone.1 At the same time, treatments with inhibitors alone had no effect, which is in line with no effect of DNA-PK inhibitor DMNB on Ad-encoded EGFP transgene expression observed by us. Moreover, the lack of effect of DNA repair pathway inhibitors on Ad-encoded transgene expression further supports our suggestion that the effect of wortmannin at high concentration is not due to ATM/ ATR inhibition but relies on suppression of mTOR. Summarizing, we have revealed the property of LY294002 to boost Ad-encoded transgene expression which is seemingly in part due to mTOR inhibition, and partially due to mTOR- and PI3K-independent effects of LY294002 which can be emulated by LY303511. These findings broaden the list of candidate anticancer treatments to be used in conjunction with adenovirus-based therapy to further potentiate its efficiency. As LY294002 and LY303511 are prototypes for anticancer drug development, and various mTOR inhibitors are currently under development for cancer treatment, our data have important implications for development of future therapeutic strategies with adenoviral gene delivery. This is further stratified by the revealed synergistic effect on Ad-delivered transgene expression brought by combination of the above treatments and topoisomerase inhibitors.

ment efficiency, it is tempting to use an adjuvant therapy which would also boost Ad-delivered transgene expression to further improve the overall efficiency. The well-described examples of such adjuvant treatments are irradiation,1 etoposide4 or histone deacetylase inhibitors.6 Viability of this concept has been experimentally proven in animal models with the examples of etoposide,4 irradiation28 and DNA repair inhibitors when combined with irradiation,29 thus demonstrating benefits from using adjuvant treatments capable to enhance adenovirusencoded therapeutic transgene expression along with adenovirus-based therapy. In line with this concept, we have also demonstrated that the revealed capacity of LY294002 to augment expression of adenovirus-encoded transgene could significantly potentiate therapeutical effect of HSVtk-encoding adenovirus in the presence of gancyclovir on a model of cancer cells in culture. Clearly, broadening the list of treatments with similar properties would allow selecting the most appropriate one based on the individual tumor sensitivity. In this work, we identified LY294002 as a potent enhancer of Ad-encoded transgene expression which was demonstrated in several lung tumor cell lines and for different promoter/transgene combination including promising for therapeutic application combination of hTERT promoter and HSVtk transgene. We found that the effect of LY294002 is partially due to mTOR inhibition, with mTOR-independent effects emulated by treatment with LY303511. Identification of LY294002, LY303511 and mTOR inhibitors as enhancers of Ad-encoded transgene expression validates the use of these drugs in combination with therapeutic adenoviruses in cancer treatment as they all by themselves target tumor cells. Although LY294002 itself has poor pharmacological properties, its RGDS-peptide coupled derivative SF1126, developed by Semaphore Pharmaceuticals, has shown promising results in preclinical studies and phase I clinical trial.30,31 Akin, LY303511 shows antitumor activity in vitro and in in vivo models20,32,33 and is being developed by Emiliem, Inc., under the name EM101 as an anticancer drug.34 Finally, a number of mTOR inhibitors are under development with several being in clinical trials as agents to treat cancer.9,35 Thus, when approved, combination of these drugs and tumor-targeting adenoviruses is a ready-to-go solution with the predicted improvement in cancer treatment efficiency. Although we have revealed that the effect of LY294002 is partially due to inhibition of mTOR activity, there are mTORindependent mechanisms of LY294002 action which are yet to be explored. The involvement of mTOR inhibition in boosting Ad-encoded transgene expression is supported by the observed effect of Compound 401, an mTOR and DNA-PK inhibitor, with the concomitant lack of effect of DNA-PK inhibitor DMNB. mTOR exerts its action as a component of two signaling complexes, mTORC1 and mTORC2. While Compound 401 is an ATP-competitive mTOR inhibitor and suppresses activities of both mTOR signaling complexes,23,24 rapamycin, which has no effect on Ad-encoded transgene expression, targets only mTORC1. This suggests that inhibition of mTORC2 but not of mTORC1 is responsible for boosting Ad-encoded protein production although this remains to be confirmed experimentally. Finally, we found that combination of LY294002, LY303511 or Compound 401 with etoposide or camtothecin, agents known to have the similar effect on Ad-encoded transgene expression, results in further increase in expression. This observation provides a basis for their combined use as adjuvant

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental Figures 1−7, showing the effects of treatment with different inhibitors on EGFP expression in Ad-EGFP infected NCI-H1299 cells; Supplemental Figure 8, showing the effect of shRNA mediated mTOR knock-down on EGFP expression in Ad-EGFP infected NCI-H1299 cells; and Supplemental Figure 9, demonstrating activation of Akt signaling in NCI-H1299 cells upon infection with Ad-EGFP adenovirus. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Laboratory of Molecular Oncogenetics, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Str., Moscow, 119334, Russia. Phone: +7-499-135-9970. Fax: +7499-135-4105. E-mail: [email protected]. Notes

Elena V. Korobko and Igor V. Korobko declare the following competing financial interest(s): The method of enhancing production of proteins encoded by replication-defective recombinant adenoviruses using LY294002, LY303511 or their derivatives is a patent pending in Russian Federation. Mikhail V. Shepelev, Tatiana V. Vinogradova, and Eugene P. Kopantsev declare no competing financial interest(s).

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ACKNOWLEDGMENTS We thank Denis Logunov and Maxim Shmarov (Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia) for production of recombinant adenoviruses. This work was supported by the Molecular and Cell Biology program of the Presidium of Russian Academy of Sciences. This work was also G

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supported by the Russian Federal Agency for Science and Innovation (Contract #02.522.11.2005).

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ABBREVIATIONS USED

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REFERENCES

mTORC1/2, mTOR signaling complex 1/2; HSVtk, herpes simplex virus thymidine kinase; Ad, adenovirus; EGFP, enhanced green fluorescent protein; PI3K, phosphoinositide 3-kinase; IC50, inhibitory concentration50; CKII, casein kinase II; TBCA, (E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid; DMNB, 4,5-dimethoxy-2-nitrobenzaldehyde; hTERT, human telomerase reverse transcriptase

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