Accumulation of Dephosphorylated 4EBP After Mtor Inhibition with Rapamycin Is Sufficient to Disrupt Paracrine Transformation By

SHORT COMMUNICATION Accumulation of dephosphorylated 4EBP after mTOR inhibition with rapamycin is sufﬁcient to disrupt paracrine transformation by the KSHV vGPCR oncogene

D Martin, Q Nguyen, A Molinolo and JS Gutkind

Dysregulation of the PI3K/Akt/mTOR pathway is one of the most frequent events in human cancer. However, the clinical benefits of PI3K/Akt/mTOR inhibitors have not yet achieved their predicted potential in many of the most prevalent human cancers. Of interest, treatment of Kaposi’s sarcoma (KS) patients with rapamycin provided the first evidence of the antineoplastic activity of mTOR inhibitors in humans, becoming the standard of care for KS arising in renal transplant patients. Thus, the study of KS may provide a unique opportunity to dissect the contribution of specific mTOR downstream targets to cancer development. The KS- associated herpesvirus (KSHV) is the etiological agent for KS, and the KSHV-encoded oncogene viral-G protein-coupled receptor (vGPCR) promotes the potent activation of the PI3K-Akt-mTOR pathway by both direct and paracrine mechanisms. We focused on a direct target of mTOR, EIF4EBP1/2/3 (4EBP), which inhibits the translation of eukaryotic initiation factor 4E (eiF4E)-bound mRNAs. 4EBP phosphorylation by mTOR relieves its inhibitory activity, hence resulting in increased eiF4E-dependent mRNA translation. We developed a paracrine transformation model, recapitulating the cellular composition of KS lesions, in which vGPCR-expressing cells promote the rapid proliferation of endothelial cells, thus expressing KSHV-latent genes by the release of growth factors. Using this model, we show here that the accumulation of dephosphorylated 4EBP in response to rapamycin or by the expression of an mTOR- insensitive mutant of 4EBP1 is sufficient to disrupt the eiF4E function downstream of mTOR to a similar extent than the mTOR catalytic inhibitor Torin2 and to halt KS development. We also provide evidence that eiF4E contributes to paracrine neoplastic, signaling through the release of pro-angiogenic factors that are acting on endothelial cells, expressing KSHV-latent genes. These findings may provide a preclinical platform and the rationale for the development of novel mTOR, inhibiting agents that may selectively disrupt the mTOR-4EBP interaction for the treatment of KS and other tumor lesions, exhibiting hyperactive mTOR pathway function.

Oncogene (2014) 33, 2405–2412; doi:10.1038/onc.2013.193; published online 27 May 2013 Keywords: Kaposi’s sarcoma; mTOR; PI3K; 4EBP; rapamycin; cancer

INTRODUCTION the treatment of advanced renal cell carcinoma,5 targeting the The mTOR signaling circuitry has a key role in a wide array of mTOR pathway has not yet achieved its expected clinical activity 6 biological processes.1 This serine–threonine kinase represents a in some cancers, displaying mTOR overactivity. A notable point of convergence of growth factor receptors and multiple exception is the activity of mTOR inhibitors in Kaposi’s sarcoma 7 oncogenes that activate the PI3K-Akt pathway and, as such, (KS) patients. This aggressive vascular tumor develops in dysregulation of mTOR represents one of the most frequent immunocompromised patients including AIDS and organ events in human cancer.1,2 mTOR exists in two molecular transplant recipients as a result of the opportunistic infection or complexes, the rapamycin sensitive mTOR complex 1 (mTORC1) reactivation of the KS-associated herpesvirus (KSHV, also termed and the insensitive mTOR complex 2, each one with specific roles HHV-8), which induces atypical vascular proliferation in the skin and targets.1 mTORC1 is positively regulated by growth signals and viscera.8 Molecular dissection of the oncogenic drivers in KS and nutrients, with two extensively studied downstream effectors, unveiled the potent activation of the PI3K-Akt-mTOR pathway p70 S6 kinase (p70S6K, RPS6K1/2) and the EIF4EBP1/2/3 family downstream of a KSHV-encoded oncogene, v-G protein-coupled (4EBP). RPS6K phosphorylates ribosomal protein S6, leading to receptor (vGPCR),9 in endothelial cells. Treatment of patients increased ribosomal biogenesis and overall increase in protein developing KS in immunosuppressed renal transplanted patients translation,3 whereas the phosphorylation of 4EBP prevents its provided the first evidence of the antineoplastic activity of interaction with the eiF4E, enabling its participation in the mTOR inhibitors in humans,10,11 and is now the standard of care formation of the initiation complex eiF4F and the translation of for this group of KS patients. Thus, the study of this rare neoplasia 7mGTP-cap containing mRNAs.4 may provide a unique opportunity to dissect the specific Rapamycin and its analogs (rapalogs) are allosteric inhibitors of contribution of mTOR downstream effectors. By the use of mTORC1 widely used in the clinic. Although recently approved for a paracrine transformation model and the expression of an

Oral and Pharyngeal Cancer Branch; National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA. Correspondence: Dr JS Gutkind, Oral and Pharyngeal Cancer Branch; National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 30 Room 320, 30 Convent Drive MSC 4340, Bethesda, MD 20892-4340, USA. E-mail: [email protected] Received 1 November 2012; revised 26 March 2013; accepted 1 April 2013; published online 27 May 2013 The mTOR/4EBP axis regulates Kaposi’s sarcoma paracrine transformation D Martin et al 2406

Figure 1. Antitumor properties of Rapamycin in experimental KS. (a) SV40-immortalized murine endothelial cells (SVEC4-10) were purchased from ATCC (Manassas, VA, USA). SVEC cells were cultured in DMEM 10% fetal bovine serum supplemented with antibiotics, 5% CO2 at 37 1C. SVEC vGPCR cells were generated by infection with an elongation factor 1 alpha (EF-1a)-driven N-terminal HA-tagged vGPCR-expressing lentiviral vector and selected by fluorimeter-activated cell sorting (FACS) against the HA epitope. SVEC vGPCR cells were starved overnight and treated with increasing concentrations of rapamycin, as indicated, for 1 h and lysed. A representative WB (procedure described in17) shows a dose-dependent reduction in pS6 (rabbit polyclonal; Cell Signaling, Danvers, MA, USA) expression that is already evident at doses as low as T46 2nM and almost complete 5–10 nM. Non-phosphorylated 4EBP (rabbit polyclonal; Cell Signaling) also accumulates in a dose-dependent fashion. Control, parental cell line SVEC. (b). SVEC cells lines were used to induce endothelial tumor xenografts in athymic mice as described previously.9 One million SVEC vGPCR or SVEC cells were injected into both flanks of female athymic (nu/nu) nude mice (Harlan Sprague- Dawley, Frederick, MD, USA), 5–6 weeks of age and weighing 18–20 g (n ¼ 16 tumors). Tumors were allowed to develop to a mean weight of B100 mg and then treated with Rapamycin (LC Laboratories, Woburn, MA, USA; 5 mg/kg, daily i.p.) or vehicle until the end of the study. Tumor mass was determined from tumor volume ((LW2/2); where L and W represent the length and the width of the tumor) by conversion to weight (mg) assuming unit density. Data points represent mean tumor mass for each group±s.e.m. Rapamycin effectively inhibited tumor growth, with significant differences seen already at day 7 after treatment initiation. (c) Animals from the SVEC vGPCR control and rapamycin treatment groups were killed after 48 h of the beginning of the rapamycin treatment. Animals were injected with BrdU (5 mg/kg i.p.) 2 hours prior to euthanasia to label proliferating cells. Paraffin sections of the tumors were reacted as described previously27 with antibodies against the mTOR activity marker pS6 (used 1:100) and the proliferation marker BrdU (Rat monoclonal, Accurate Chemical & Scientific Corporation, Westbury, NY, USA). An atypical proliferation of spindle-shaped cells with occasional vascular differentiation is shown in the haematoxylin and eosin staining (H&E) image. (d) pS6 expression is dramatically decreased following rapamycin treatment, as it is proliferation, as judged by the algorithm- based quantification (Aperio ScanScope software, Vista, CA, USA) of representative fields. (e) Tumor lysates from samples collected in parallel as in c, analyzed by western blot showed the decrease in p4EBPT37/46 (Rabbit polyclonal, Cell Signaling) following rapamycin, as well as the accumulation of non-phosphorylated form of 4EBPT46.

Oncogene (2014) 2405 – 2412 & 2014 Macmillan Publishers Limited The mTOR/4EBP axis regulates Kaposi’s sarcoma paracrine transformation D Martin et al 2407 mTOR-insensitive mutant of 4EBP1, we show here that the expressed in cells that fail to undergo a lytic cycle.12 vGPCR accumulation of dephosphorylated 4EBP in response to induces chemokine, cytokine and growth factor secretion, thereby rapamycin is sufﬁcient to disrupt the eiF4E function downstream promoting the growth of other naive and latently infected of mTOR, and to halt KS development. We also provide evidence endothelial cells in a paracrine fashion,13 thus giving raise to the that eiF4E contributes to paracrine neoplastic signaling through characteristic angioproliferative lesions of the KS. Although the release of pro-angiogenic factors, acting on endothelial cells inefﬁcient intratumoral vascular networks in cancer may limit expressing KSHV-latent genes. the appropriate delivery of therapeutic agents,14 the direct exposure of endothelial-derived KS cells to the bloodstream circumvents these limitations, thus creating an excellent scenario RESULTS AND DISCUSSION for the study of therapeutic molecular mechanisms. Rapamycin The KSHV vGPCR oncogene encodes a constitutively active GPCR induced a marked decrease in the phosphorylation of the that is expressed during the lytic viral activation or aberrantly downstream target S6, as well as an accumulation of non-

Figure 2. For caption please see next page.

& 2014 Macmillan Publishers Limited Oncogene (2014) 2405 – 2412 The mTOR/4EBP axis regulates Kaposi’s sarcoma paracrine transformation D Martin et al 2408 T46 phosphorylated 4EBP at doses as low as 2 nM, with almost induction of proliferation by increasing concentrations of vGPCR- complete inhibition between 5 and 10 nM (Figure 1a), which are conditioned media. We challenged this paracrine-induced pro- the trough blood levels aimed for in KS treatment.10 Rapamycin liferation model in a mouse xenograft system. SVEC KS-LT cells effectively halted the growth of SVEC vGPCR xenografts failed to induce tumor growth when combined with a defined (Figure 1b) concomitant with a dramatic decrease in phospho- proportion (10%) of SVEC cells (Figure 2f). Conversely, when mixed S6 (pS6) staining and cell proliferation as depicted by with 10% of SVEC vGPCR cells, mimicking the proportion of Bromodeoxyuridine (BrdU) incorporation, as we have previously vGPCR-expressing cells present in human KS lesions,13 tumors reported15 (Figures 1c and d), and an increase in apoptosis were formed, where most of the cells expressed the latent as detected by caspase 3 activation (Supplementary Figure S1). transcript LANA, closely resembling the abundance of LANA the Rapamycin also decreased the amount of phospho-4EBP most common diagnostic marker for KSHV infection in human KS (p4EBP), one of the mTORC1 targets, albeit incompletely (Figure 2g). These results support the relevance of a paracrine as previously reported in other cancer models.16 However, neoplasia mechanism in KS, defining two distinct promoting and under these conditions, we observed a substantial accumulation proliferating cellular compartments, cooperating in the formation of non-phosphorylated 4EBP (Figure 1e), thus raising the of the KS lesion. possibility that 4EBP may still represent a biologically relevant We next explored the contribution of mTOR to paracrine target in KS. neoplasia, with a focus on 4EBP, as the ineffective inhibition of KS tumoral mechanisms involve the paracrine induction of cell phosphorylation of 4EBP has been often associated with proliferation.9 Several models have documented the contribution therapeutic failure of rapamycin.19 We engineered a 4EBP of a small number of vGPCR-expressing endothelial cells to the mutant protein, where the residues T37, T46, S65 and T70 were proliferation of adjacent endothelial cells, expressing other KSHV mutated to alanine (4EBP-M). This mutant lacks the ability to be viral genes.9,17,18 Here, we developed a simple paracrine regulated by mTOR, and hence it is expected to mimic the transformation reporter system by engineering endothelial cells, disruption of the mTORC1 complex function by rapamycin on the expressing the three major KSHV-latent transcripts, vCyclin, vFlip downstream regulation of eiF4E function. Indeed, 7mGTP pull- and LANA, which we named SVEC KS-LT. The expression of these down experiments (scheme in Figure 3a) revealed that expression latent transcripts was confirmed by western blot and of 4EBP-M in SVEC vGPCR and SVEC RheB, both of which have immunofluorescence (Figures 2a and b, and Supplementary elevated mTOR activity, prevented the association of eiF4E and Figure S2) against the corresponding epitopes for the tagged eiF4G, effectively disrupting the formation of the translation- vCyclin and vFlip, and for the protein product for LANA. Despite the initiating eiF4F complex, to a degree similar to that induced by expression of these potentially transforming genes, basal cell rapamycin or the mTOR catalytic inhibitor Torin220 (Figure 3a). proliferation was not altered when compared with control Although the expression of 4EBP-M completely blocked eiF4E/ parental cells. However, vGPCR-expressing cells displayed a eiF4G association and reduced 4EBP phosphorylation, it only twofold increase in basal proliferation (Figure 2c). Interestingly, moderately affected S6 phosphorylation in SVEC vGPCR and SVEC when exposed to SVEC vGPCR-conditioned media, both parental RheB cells (Figure 3b). Moreover, expression of 4EBP-M dramati- control and SVEC KS-LT cells showed increased proliferation, the cally reduced the proliferation of both SVEC vGPCR and SVEC RheB latter to a much greater extent (Figure 2d). This hypersensitivity to cells in vitro (Figure 3c) and their tumorigenic potential and vGPCR-induced secretions was further depicted by the robust proliferation rate in vivo (Figures 3d and e), suggesting that the

Figure 2. Characterization of a cell line expressing the KSHV three major latent transcripts. (a) SVEC cells were stably transfected (ExGen 500, Fermentas, Hanover, MD, USA) with a naturally occurring bicistronic28 DNA encoding an N-terminal AU5-tagged vCyclin and C-terminal HA- tagged viral FLICE (FADD (Fas-associated death domain)-like IL1 beta-converting enzyme)-inhibitory protein (vFlip) proteins.9 These cells where further infected with a lentivirus encoding the KSHV ORF 73 (LANA) under the control of the elongation factor 1 alpha (EF-1a) promoter and denominated SVEC KS-LT. As a control, cell lysates from 293T transiently transfected with the bicistronic, as well as LANA encoding vectors was used. Expression of the different proteins in pooled cultures was confirmed by western blot. Mouse monoclonal anti-HA (clone 12CA5) and anti-AU5 (clone MMS-135R) were used and purchased from Covance Research Products, Denver, PA, USA. Rat monoclonal anti-ORF73 (KSHV LANA) was purchased from Advanced Biotechnologies (Columbia, MD, USA). (b) Immunocytochemistry depicting the expression and localization of the latent transcripts in SVEC KS-LT. Briefly, cells were plated onto poly-D lysine-coated coverslips for 24 h washed once with cold PBS and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature (RT). Cells were washed three times with PBS and permeabilized for 5 min with PBS containing 0.5% NP-40. Cells were washed again twice and incubated in blocking solution (3% BSA in PBS) for 20 min. Slides were incubated with the primary antibodies as indicated diluted 1:500 in blocking solution for 1 h at RT. After this incubation, the coverslips were washed with PBS and incubated with the appropriate Alexa546–conjugated secondary antibodies supplemented with Phalloidin-Alexa488 (Invitrogen, Carslbad, CA, USA) in blocking solution for 1 h at RT. Cells were then washed with PBS and nuclei stained with 1 mg/ml TO-PRO3, washed with PBS and distilled water, and mounted using FluorSafe mounting medium (EMD, Gibbstown, NJ, USA). Imaging was performed on a Zeiss LSM 700 confocal microscope. Bar ¼ 10 mm. (c) Cells were cultured in standard conditions at subconfluency, serum starved overnight, and cell proliferation was determined by EdU incorporation assay. Briefly, cells cultured in poly-D-lysine-treated coverslips, starved as indicated above and treated for 20 h. Then 10 mM EdU (Click-it kit, Invitrogen) was added to the cultures for 4 h and fixed in 4% paraformaldehide (Electron Microscopy Sciences, Fort Washington, PA, USA) in PBS. Nuclei where counterstained using Hoechst 33342 (Invitrogen) and visualized on an Axio Imager Z1 microscope equipped with ApoTome system controlled by the AxioVision software (Carl Zeiss, Thornwood, NY, USA). Fields were visually scored for the proportion of EdU positive nuclei (nX300) with respect to the total number of cells as determined by nuclear 4’,6-diamidino-2-phenylindole (DAPI) staining. Bars represent the average proportions±s.e.m. with respect to SVEC (Control) cells, ***Pp0.001. (d) SVEC KS-LT were cultured at subclonfluency, starved and stimulated for 24 h with conditioned supernatant collected in serum-free conditions from SVEC (SVEC-CM) or SVEC vGPCR (SVEC vGPCR CM) cultures. EdU incorporation assays were performed during the last 4 h of the 24 h period. Panels show a representative field depicting EdU incorporating nuclei (red) overlaid on DAPI nuclear staining (blue). Inset values represent the average percentage±s.e.m. of a total of nine fields obtained from three independent preparations. (e) SVEC KS-LT cells were cultured for 24 h in the presence of increasing concentrations of SVEC (SVEC- CM) or SVEC vGPCR (vGPCR-CM)-conditioned media diluted in DMEM medium. Data points represent the average percentage±s.e.m. of EdU incorporating cells, microscopically evaluated from nine fields from three independent preparations with respect to untreated cells (0% conditioned medium). (f) In vivo tumorigenesis study for paracrine neoplasia. Briefly, 9 Â 105 SVEC KS-LT cells were mixed with either 1 Â 105 SVEC (red squares) or SVEC vGPCR cells (black circles), and injected s.c. in the flanks of athymic nude mice. Data points represent the average tumor volume ±s.e.m. (n ¼ 10). (g) Tumor histology and LANA immunostaining of tissue sections obtained from SVEC KS-LT/vGPCR mixed tumors from the experiment in panel f, as well as from a human KS, showing a similar proportion of LANA-expressing cells.

Oncogene (2014) 2405 – 2412 & 2014 Macmillan Publishers Limited The mTOR/4EBP axis regulates Kaposi’s sarcoma paracrine transformation D Martin et al 2409

Figure 3. A dominant-positive 4EBP mutant prevents eiF4E-mediated vascular endothelial growth factor (VEGF) translation and tumorigenesis. (a) Left panel, scheme of the 7mGTP pull down depicting the regulation of complex formation by mTOR and 4EBP. Briefly, cells were starved and treated as described above, were quickly washed in ice-cold PBS and lysed in pull-down buffer (1 ml of 50 mM HEPES; pH 7.5), 150 mM NaCl, 10% glycerol, 0.3% (w/v) CHAPS, 1.5 mM MgCl2,1mM EGTA, 100 mM NaF, 500 mM sodium orthovanadate, 10 mg/ml aprotinin and 10 mg/ml leupeptin and 1 mM PMSF) and incubated at 4 1C for 2 h with 7mGTP-Sepharose beads (Amersham, Piscataway, NJ, USA). Beads were then washed three times with pull-down buffer and resuspended in Laemmli sample buffer and resolved by SDS–PAGE. Right panel, SVEC vGPCR and SVEC RheB cells were infected with lentiviral vectors encoding a 4EBP1 (T36A, T45A, S65A, T70A) mutant protein (4EBP-M). A representative 7mGTP pull-down experiment is shown depicting the prevention of eiF4G association to eiF4E by the expression of 4EBP-M, Rapamycin and Torin2. (b) Exponentially growing cell cultures were starved as indicated in the experimental procedures and then treated with 100 nM rapamycin for 1 h and lysed. The panel shows a representative western blot analysis of SVEC vGPCR and SVEC RheB cells, in which the expression of 4EBP-M completely blocked eiF4E/eiF4G association and reduced 4EBP phosphorylation. S6 phosphorylation was not significantly affected. (Figure 3b). (c) Cell proliferation assay. Exponentially growing cultures were starved overnight and media was replaced with EdU containing DMEM and incubated for an additional 4 h. The bars represent the mean percentage±s.e.m. of EdU incorporating cells normalized to control (SVEC) cells (n ¼ 9). (d) Tumor xenograft generated from cells as indicated in the figure were implanted into the flanks of nude mice and allowed to grow. Data points represent the mean tumor volume±s.e.m. (n ¼ 10). (e) Animals from the experiment in panel d were injected with BrdU 4 h prior to euthanasia. Representative H&E and BrdU immunohistochemistry showing the differences in cell proliferation are depicted.

4EBP signaling branch downstream of mTOR is required for their vGPCR-conditioned media to stimulate the proliferation of SVEC oncogenic properties. KS-LT cells (Figures 4a and b). In this regard, VEGF-A is a potent Expression of 4EBP-M not only reduced the growth of endothelial speciﬁc growth factor that contributes to KS develop- vGPCR-expressing cells, but also reduced the ability of SVEC ment, and the VEGF expression levels are reduced by rapamycin

& 2014 Macmillan Publishers Limited Oncogene (2014) 2405 – 2412 The mTOR/4EBP axis regulates Kaposi’s sarcoma paracrine transformation D Martin et al 2410

Figure 4. 4EBP mutant inhibits vGPCR-induced paracrine transformation. (a) SVEC KS-LT cells were stimulated overnight with conditioned media collected from SVEC vGPCR and RheB cultures, stably expressing 4EBP-M as indicated in the figure. Representative pictures of Click-it EdU incorproration assays are shown. (b) The quantification of the assay depicted in a is shown. Nine fields were visually scored for positive EdU-incorporating nuclei. The bars represent the percentage of positive nuclei±s.e.m. with respect to control (SVEC-CM-treated) cells. (c) VEGF levels were determined by enzyme-linked immunosorbent assay (ELISA) in 24 h old cell supernatants collected in triplicate from tissue cultures as indicated using a Mouse VEGF ELISA kit from RayBiotech (Norcross, GA, USA, cat. no. ELM-VEGF-001) following the manufacturer’s recommendations. Bars represent mean ±s.e.m. Total RNA was isolated from the samples in panel d. Murine vegfa mRNA levels were determined by quantitative PCR. The bars represent the average VEGF-A mRNA expression level normalized to the house keeping gene GAPDH±s.e.m.. (e and f) KC and IL6 levels were determined by ELISA assays in 24-h old cell supernatants collected in triplicate from tissue cultures as indicated using a Mouse KC and IL6 ELISA kits from RayBiotech (cat. no. CODE: ELM-KC-001 and ELM-IL6-001, respectively) following the manufacturer’s recommendations. Bars represent mean±s.e.m. (g) In vivo paracrine transformation assay. Tumor xenografts were generated from cell mixes containing 9 Â 105 SVEC KS-LT and 1 Â 105 SVEC vGPCR (red circles) or SVEC vGPCR 4EBP-M cells (pink squares). Tumor xenografts were generated in the flanks of athymic nude mice by subcutaneous injection. Representative fields include H&E and LANA, pS6 and BrdU immunostainings. BrdU quantification is showed in the panel on the right. (h) Tumor volume was calculated as described in Figure 1b. Data points represent mean±s.e.m. (n ¼ 10). (i) Scheme depicting the emerging proposed model.

treatment.21,22 Inhibition of eiF4E by 4EBP-M was concomitant potent angiogenic cytokines involved in KS, interleukin (IL) 6 and with a decrease secretion of VEGF-A in the conditioned media KC/Cxcl1, the latter the murine homolog of IL8/Gro-a, were both (Figure 4c), even in the absence of a signiﬁcant alteration in the also reduced by 4EBP-M expression (Figures 4e and f, respec- levels of VEGF-A mRNA (Figure 4d), compatible with a decrease in tively). The biological impact of 4EBP-M in paracrine signaling was translation of VEGF-A mRNA. In addition, the secretion of two reﬂected in the xenograft model in vivo, where 4EBP-M expression

Oncogene (2014) 2405 – 2412 & 2014 Macmillan Publishers Limited The mTOR/4EBP axis regulates Kaposi’s sarcoma paracrine transformation D Martin et al 2411 rendered SVEC vGPCR cells unable to support the growth of SVEC development of novel mTOR inhibiting agents that may KS-LT cells, thus resulting in decreased overall levels of pS6 and selectively disrupt the mTOR-4EBP interaction for the treatment VEGF staining and BrdU incorporation, (Figure 4g) and a dramatic of KS, as well as other tumor lesions, exhibiting hyperactive mTOR impairment of tumor growth (Figure 4f). pathway function. The molecular mechanism whereby rapamycin exerts its antitumoral effects remains unclear, given the emerging complex- 1 ity of the mTOR network. In our experimental model, rapamycin CONFLICT OF INTEREST treatment induced tumor regression concomitant with a dramatic decrease in pS6, which has served as a prototypical mTOR activity The authors declare no conflict of interest. marker, but only a moderate decrease in p4EBP was achieved. The perceived resistance of 4EBP to complete dephosphorylation upon rapamycin treatment in this and other systems has ACKNOWLEDGEMENTS prompted the denomination of rapalogs as incomplete mTOR This research was supported by the National Institutes of Health Intramural AIDS- inhibitors,19 thereby prompting the development of a novel class Targeted Antiviral Program and the National Institute of Dental and Craniofacial of mTOR catalytic inhibitors in the last few years.20 Interestingly, Research. We apologize to colleagues whose primary research papers may not have however, the availability of commercial antibodies detecting the been cited due to space constraints. non-phosphorylated 4EBP, revealed the accumulation of the dephosphorylated form of this protein in vitro and in vivo after rapamycin treatment. Moreover, rapamycin was able to disrupt REFERENCES the interaction of eiF4G and eiF4E to a similar degree than that 1 Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012; achieved by the potent second generation mTOR catalytic 149: 274–293. 20 inhibitor Torin2. This in turn suggests that despite the 2 Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as presence of considerable amounts of p4EBP remaining after regulators of growth and metabolism. 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Oncogene (2014) 2405 – 2412 & 2014 Macmillan Publishers Limited