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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2008 Feb 9;134(8):833–839. doi: 10.1007/s00432-008-0359-5

Antineoplastic effect of rapamycin is potentiated by inhibition of IRS-1 signaling in prostate cancer cells xenografts

Josenilson C Oliveira 1, Kellen K Souza 1, Marília M Dias 1, Marcel C Faria 1, Eduardo R Ropelle 1, Marcelo B S Flores 1, Mirian Ueno 1, Lício A Velloso 1, Sara T Saad 1, Mario J A Saad 1, José B C Carvalheira 1,✉
PMCID: PMC12160750  PMID: 18264722

Abstract

Proper activation of phosphoinositide 3-kinase-Akt pathway is critical for the prevention of tumorigenesis. Recent data have characterized a negative feedback loop, wherein mammalian target of rapamycin (mTOR) blocks additional activation of the Akt/mTOR pathway through inhibition insulin receptor substrate 1 (IRS-1) function. However, the potential of IRS-1 inhibition during rapamycin treatment has not been examined. Herein, we show that IRS-1 antisense oligonucleotide and rapamycin synergistically antagonize the activation of mTOR in vivo and induced tumor suppression, through inhibition of proliferation and induction of apoptosis, in prostate cancer cell xenografts. These data demonstrate that the addition of agents that blocks IRS-1 potentiate the effect of mTOR inhibition in the growth of prostate cancer cell xenografts.

Keywords: Protein kinases, Phosphatidylinositol 3-kinase, Insulin receptor substrate 1, Rapamycin, Prostate neoplasms

Introduction

Prostate cancer is a major health problem in the world, and the available treatment options have proven to be inadequate in controlling the mortality and morbidity associated with this disease (Jemal et al. 2007; Petrylak et al. 2004; Tannock et al. 2004). Research efforts in the last decade have shown that molecular targeted-therapy is a promising approach that could expand the arsenal against prostate cancer.

The mammalian target of rapamycin (mTOR) pathway is a key regulator of cell growth and proliferation, and increasing evidence suggests that its deregulation is associated with human diseases, including cancer and diabetes (Sabatini 2006; Ueno et al. 2005). The mTOR pathway integrates signals from nutrients, energy status and growth factors to regulate many processes, including autophagy, ribosome biogenesis and metabolism (Dennis et al. 2001; Sabatini 2006). Thus, rapamycin and several analogs, such as CCI-779 and RAD001, are currently undergoing clinical evaluation as anticancer agents (Huang and Houghton 2002; Majumder et al. 2004).

The insulin and IGF-1 receptors are tyrosine kinases, which phosphorylates the insulin receptor substrate (IRS) upon ligand binding. Phosphorylated IRS, in turn, acts as a protein scaffold that activates the phosphatidylinositol (PI) 3-kinase/Akt cascade (Yenush and White 1997). The production of phosphatidylinositol 3,4,5-triphosphate (PIP3) by PI 3-kinase recruits the serine/threonine kinases PDK1 and Akt to the plasma membrane, by binding to its N-terminal pleckstrin homology (PH) domain. At the membrane, Akt is phosphorylated by PDK1-mediated phosphorylation (Lawlor and Alessi 2001). Akt phosphorylates many proteins with important physiological roles, including TSC2, inhibiting its GTPase activating protein effect towards the small G-protein Rheb (Inoki et al. 2003). The accumulation of the GTP-bound Rheb leads to the activation of mTOR through an as yet unknown mechanism.

Inactivation of the tumor-suppressor gene, PTEN, occurs in glioblastoma multiforme, endometrial cancer and prostate cancer, among others (Sansal and Sellers 2004). The suppressor-tumor function of PTEN is linked to its lipid phosphatase activity; loss of this activity leads to accumulation of its substrate, PIP3, and activation of the PI 3-kinase signaling pathway (Maehama and Dixon 1998). One consequence of PTEN loss is hyper-phosphorylation downstream Akt substrates (Cross et al. 1995; del Peso et al. 1997; Kops et al. 1999; Nakamura et al. 2000). Phosphorylation of these proteins can lead to enhanced cell survival, increased cell proliferation and altered cellular metabolism.

Recent studies have shown that many kinases, including rapamycin-sensitive enzymes, promote serine/threonine phosphorylation of IRS-1 that inhibits their function and promotes their degradation (Mothe and Van Obberghen 1996; Rui et al. 2001). Insulin- or IGF-stimulated Akt phosphorylation could be rescued by rapamycin treatment, coincident with restored IRS protein levels. The rapamycin-mediated rescue is blunted by reducing IRS-1 expression with specific siRNAs, and rendered unnecessary by overexpression of IRS-1. Thus, chronic hyperactivation of mTOR by inactivation of TSC1–TSC2 stimulates components of the protein synthesis pathway, while inhibiting the IRS branch of the insulin/IGF-1 signaling cascade (Harrington et al. 2004; Shah et al. 2004; Ueno et al. 2005).

To clarify the role of mTOR feedback in prostate cancer, we studied the potential of the IRS-1 antisense oligodeoxinucleotide (ASO) as an inhibitor of proliferation during rapamycin treatment, using the PC-3 cell line, which is reported to be PTEN-negative.

Materials and methods

Antibodies, chemicals and buffers

The reagents and apparatus for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were obtained from Bio-Rad (Richmond, CA). Tris-[hydroxymethyl]amino-methane (Tris), phenylmethylsulfonylfluoride (PMSF), dithiothreitol (DTT), Triton X-100, Tween 20 and glycerol were obtained from Sigma Chemical (St Louis, MO, USA). Dimethyl sulfoxide (DMSO) was obtained from Calbiochem (La Jolla, CA, USA). Aprotinin was obtained from Bayer (São Paulo, SP, Brazil). Ketamin was obtained from Parke-Davis (São Paulo, SP, Brazil); diazepam and sodium thiopental were obtained from Cristália (Itapira, SP, Brazil). Protein A-Sepharose 6 MB, nitrocellulose membrane (Hybond ECL, 0.45 μm) and 125I-Protein A were obtained from Amersham (Buckinghamshire, UK). Anti-IRS-1 and anti-IRS-1 phosphoserine 307-specific antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-Akt (Ser473) and anti-phospho-p70 S6K (Thr421/Ser424) antibodies were obtained from New England Biolabs (Beverly, MA, USA). Rapamycin was obtained from LC Laboratories (Woburn, MA, USA).

Phosphorothioate-modified oligonucleotides

Sense and antisense phosphorothioate oligonucleotides specific for IRS-1 (sense (SO), 5′-ACC CAC TCC TAT CCC G-3′ and antisense (ASO), 5′-CGG GAT AGG AGT GGG T-3′) were produced by Invitrogen (Carlsbad, CA). The antisense oligonucleotide sequences were submitted to BLAST analyses (http://www.ncbi.nlm.nih.gov) and matched only for the human IRS-1 coding sequence.

Cell culture

The human prostate cancer cell line PC-3 was obtained from ATCC, Philadelphia, PA, USA. Cells were cultured in RPMI containing 10% fetal bovine serum and glutamine without addition of antibiotics or fungicides; they were maintained at 37°C, 5% CO2.

Human tumor xenograft models

SCID mice were provided by the State University of Campinas–Central Breeding Center (Campinas, SP, Brazil). SCID mice, 5–6 weeks old and weighing approximately 20–25 g, were implanted with 1.0 × 106 PC-3 cells into the dorsal subcutis of male mice. Mice were weighed twice weekly, and tumor measurements were taken by calipers daily starting on day 0. These tumor measurements were converted to tumor volume (V) using the formula (V = W 2 × L × 0.52), where W and L are the smaller and larger diameters, respectively, and plotted against time. When the tumors were between 50 and 100 mm3, the animals were pair-matched into treatment and control groups. Each group contained eight mice, each of which was ear tagged and followed individually throughout the experiment. Initial doses were given on the day of pair matching (day 0). Rapamycin and IRS-1 ASO were administered via i.p. injection daily at the doses indicated. On termination, the mice were weighed and killed, and their tumors were excised. Treatment-related toxicity was evaluated by means of serial weight measurements. All experiments were approved by the Ethics Committee of the State University of Campinas.

Tumor extracts

Mice were anesthetized with sodium amobarbital (15 mg/kg body weight, i.p.), and were used 10–15 min later, i.e. as soon as anesthesia was assured by the loss of pedal and corneal reflexes. Tumors were removed, minced coarsely and homogenized immediately in extraction buffer (1% Triton-X 100, 100 mM Tris, pH 7.4, containing 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM PMSF and 0.1 mg of aprotinin/ml) at 4°C with a Polytron PTA 20S generator (Brinkmann Instruments model PT 10/35) operated at maximum speed for 30 s. The extracts were centrifuged at 15,000 rpm and 4°C in a Beckman 70.1 Ti rotor (Palo Alto, CA) for 45 min to remove insoluble material, and the supernatants of these tissues were used.

Protein analysis by immunoblotting

The whole tissue extract was treated with Laemmli sample buffer (Laemmli 1970) containing 100 mM DTT and heated in a boiling water bath for 4 min after which they were subjected to SDS-PAGE (6% bis-acrylamide) in a Bio-Rad miniature slab gel apparatus (Mini-Protean, Bio-Rad Laboratories, Inc., Richmond, CA, USA). For total extracts, similar sized aliquots (200 μg protein) were subjected to SDS-PAGE.

Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant) in a Bio-Rad miniature transfer apparatus (Mini-Protean), as described by Towbin et al. (1979), except for the addition of 0.02% SDS to the transfer buffer to enhance the elution of high molecular mass proteins. Nonspecific protein binding to the nitrocellulose was reduced by preincubating the filter overnight at 4°C in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl and 0.02% Tween 20). The nitrocellulose blot was incubated with the indicated antibodies, diluted in blocking buffer (0.3% BSA instead of nonfat dry milk) overnight at 4°C and then washed for 60 min with blocking buffer without milk. The blots were subsequently incubated with 2 μCi of 125I-protein A (30 μCi/μg) in 10 ml of blocking buffer for 2 h at room temperature and then washed again for 30 min as described above. 125I-Protein A bound to the antiphosphotyrosine and antipeptide antibodies was detected by autoradiography using preflashed Kodak XAR film with Cronex Lightning Plus intensifying screens at −80°C for 12–48 h. Band intensities were quantified by optical densitometry of developed autoradiographs (Scion Image software, Scion Corporation, Frederick, MD, USA).

Immunohistochemistry

To detect proliferating cell nuclear antigen (PCNA), microwave post fixation was carried out using a domestic oven (Panasonic Junior, Sao Jose dos Campos, SP, Brazil) at 700 W, which was delivered to slides immersed in 0.01 mol/l citrate buffer, pH 6.0, in twice doses during 7 min separated by a break of 2 min, which allowed for buffer replenishment. The slides were allowed to cool to room temperature before being removed from the oven. Sections were then incubated at room temperature for 1 h with primary antibody PC10 (Dako North America, Inc., Carpinteria, CA, USA) diluted 1:150. Biotinylated horse mouse antihuman (Dako) antibodies for PC10 were applied for 1 h at room temperature. The slides were then incubated with avidin–biotin complex (ABC) reagent (Vector) for 30 min followed by the addition of diaminobenzidine tetrahydrochloride (DAB) (Sigma) as a substrate-chromogen solution. After hematoxylin counterstaining and dehydration, the slides were mounted in Entellan (Merck, Darmstadt, Germany). The experiments were done, at least, in triplicate for each mouse.

A TUNEL apoptosis detection kit (Upstate Biotechnology, Inc., Lake Place, NY, USA) was used for DNA fragmentation fluorescence staining according to the manufacturer’s protocol. Following the extraction, the tissue extracts were fixed with 4% paraformaldehyde in 0.1 M NaH2PO4, pH 7.4, and incubated with a reaction mix containing biotin-dUTP and terminal deoxynucleotidyl transferase for 60 min. Fluorescein-conjugated avidin was applied to the sample, which was then incubated in the dark for 30 min. Positively stained fluorescein-labeled cells were visualized and photographed by fluorescence microscopy.

Statistical analysis

All groups were studied in parallel. Comparisons between different groups were performed by employing ANOVA, as appropriate. The level of significance adopted was P < 0.05.

Results

Effects of treatment with rapamycin and/or IRS-1 ASO, on Akt/mTOR signaling pathway in PC-3 xenografts

To evaluate IRS-1 ASO activity, we performed experiments using samples from PC-3 xenografts, immunoblotted with anti IRS-1 (Fig. 1a). There was an evident decrease in IRS-1 protein levels within 48 h after ASO treatment, which was maximal at 72 h after IRS-1 ASO treatment, representing a reduction in the IRS-1 protein level of ∼75% (Fig. 1a, upper panel). The ASO-mediated IRS-1 inhibition in PC-3 xenografts was dose dependent (Fig. 1b). The inhibition of IRS-1 protein levels was detected after the injection of 0.4 nmol/kg, while inhibition was observed with 0.8 nmol/kg of IRS-1 ASO, representing a reduction in the IRS-1 protein level of ∼70% (Fig. 1b, upper panel). There was no change in IRS-1 protein levels after IRS-1 sense oligonucleotide (SO) treatment (Fig. 1a, b, middle panel).

Fig. 1.

Fig. 1

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Phosphorylation status of IRS-1, Akt and p70S6k proteins after treatment with rapamycin and IRS-1 antisense oligonucleotide (IRS-1 ASO) alone, and with the combination of rapamycin and IRS-1 ASO. (a, upper panel) The time course was performed with SCID mice treated i.p. with 0.4 nmol/kg of IRS-1 ASO, as indicated. At the time-points indicated, after the treatment, they were anesthetized and a fragment from xenograft was removed. Similar treatments were carried out with the IRS-1 SO (a, middle panel). Loading control with anti-α-tubulin antibodies (a, lower panel). b In a dose response experiment, SCID mice were treated i.p. with varying doses of IRS-1 ASO or with vehicle, as indicated. After 72 h, they were anesthetized, and fragments from xenograft and liver were removed. Similar treatments were carried out with the IRS-1 SO (b, middle panel). Loading control with anti-α-tubulin antibodies (b, lower panel). c–f Representative Western blots of five independent experiments showing PC-3 lysates. The lysates were immunoblotted (IB) with anti-IRS-1 phospho-serine(307) (c), anti-IRS-1 antibodies (d), anti-phospho-Akt (pAkt) (e, upper panel), anti-Akt (e, lower panel), anti-phospho-p70S6k (p-p70S6k) (f, upper panel) and anti-p70S6k antibodies (f, lower panel). Bars represent means ± SEM. # P < 0.05 versus rapamycin and *P < 0.05 versus control

Immunoblotting experiments were conducted to determine the effects of rapamycin, IRS-1 ASO, and the combination of both on IRS-1 serine phosphorylation in PC-3 xenografts (Fig. 1c). Thus, with isolated rapamycin, there was a significant decrease in IRS-1 serine phosphorylation when compared to the control group. The isolated IRS-1 ASO inhibited partially the IRS-1 serine phosphorylation, while the simultaneous treatment with rapamycin and the IRS-1 ASO showed inhibition of IRS-1 serine phosphorylation similar to the treatment with rapamycin alone. There was an increase in IRS-1 protein levels with rapamycin treatment. On the other hand, the isolated administration of IRS-1 ASO strongly decreased IRS-1 protein levels. We observed that IRS-1 ASO blocked the effect of rapamycin on IRS-1 protein expression (Fig. 1d). Isolated rapamycin induced increases in the serine phosphorylation of Akt, compared to the control group. The IRS-1 ASO induced decreases in the phosphorylation of Akt protein; in addition, we observed that IRS-1 ASO blocked the effect of rapamycin on Akt phosphorylation (Fig. 1e). There were no changes in Akt protein levels. Conversely, the isolated treatment with rapamycin induced a great decrease in p70S6k phosphorylation; ASO treatment decreased p70S6k phosphorylation to a small extent compared to rapamycin. The association of both treatments acted synergistically in the reduction of p70S6k phosphorylation, which was significantly lower than each compound alone. There were no changes in p70S6k protein levels.

Effects of treatment with rapamycin and/or IRS-1 ASO on growth of PC-3 xenografts

As shown in Fig. 2, exposure of PC-3 xenografts to isolated IRS-1 ASO resulted in a weak decrease in tumor growth during the experimental period. Rapamycin-treated group showed an important decrease in growth, with a very low velocity of growth. Meanwhile, the combination of rapamycin and IRS-1 ASO enhanced the inhibitory effect on tumor growth, resulting in no important change in tumor volume in the frame shift period of study. No substantial weight loss was observed in any of the groups throughout the 30 days of analysis.

Fig. 2.

Fig. 2

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Growth curves for xenografts derived from PC-3 cells. Rapamycin (4 mg/kg i.p.), IRS-1 ASO (0.4 nmol) or the combination of rapamycin and IRS-1 ASO (4 mg/kg and 0.4 nmol, respectively) were injected daily in mice bearing PC-3 xenografts. Control mice received a similar schedule of the vehicle solution. Points and means for at least eight tumors

Effects of treatment with rapamycin and/or IRS-1 ASO on proliferation and apoptosis of PC-3 xenografts

We performed immunohistochemistry to detect PCNA, a proliferation index (Fig. 3a). The observed percent of PCNA-positive cells was 45.9 ± 5.2% in the control group. IRS-1 ASO and rapamycin administration significantly reduced the percent of positive cells to 36.6 ± 6.8% and 24.5 ± 4.7%, respectively (P < 0.05, for both). The group treated with the combination of rapamycin and IRS-1 ASO was found to be 4.3 ± 3.2% in positive cells. Compared to the control group, the combination of rapamycin- and IRS-1 ASO-treated group showed ±90% of reduction in positive cells (P < 0.001). IRS-1 ASO plus rapamycin enhanced the effectiveness of rapamycin by 570%.

Fig. 3.

Fig. 3

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Effects of vehicle, rapamycin (4 mg/kg i.p.), IRS-1 ASO (0.4 nmol/kg) or the combination of rapamycin and IRS-1 ASO (4 mg/kg and 0.4 nmol, respectively) on cell proliferation (a) and on apoptosis (b) in PC-3 xenografts. Bars represent means ± SEM calculated from PCNA and TUNEL positive cells counted in four fields. # P < 0.05 versus rapamycin and *P < 0.05 versus control

To analyze the mechanism responsible for the growth inhibition of PC-3 cells by rapamycin and IRS-1 ASO, the effects of rapamycin and/or IRS-1 ASO on programmed cell death was examined. Thus, DNA fragmentation was measured by TUNEL assay in PC-3 xenografts (Fig. 3b). In relation to the control group, the IRS-1 ASO- and rapamycin-treated groups showed about 4- and 23-fold greater positive cells, respectively (P < 0.05, for both). The combination of rapamycin and IRS-1 ASO promoted a 37-fold increase in apoptotic cell number, when compared to the control group (P < 0.001), and an up to 2-fold increase compared with that of rapamycin (P < 0.05).

Discussion

PTEN alterations have been robustly implicated in human prostate cancer, with PTEN deletions and/or mutations of at least one allele observed in up to 60% of primary prostate cancers, while homozygous PTEN inactivation is more frequently associated with metastatic prostate tissues (Cairns et al. 1997; Gray et al. 1995; Suzuki et al. 1998). In the present study, we show that IRS-1 ASO and rapamycin cooperatively antagonize the activation of mTOR in vivo and act synergistically in tumor suppression in PC-3 prostate cancer xenografts, which do not express PTEN. Our results show that IRS-1 ASO alone reduces the activation of PI 3-kinase pathway, while rapamycin alone reduces the activation of mTOR. Combined treatment with rapamycin and IRS-1 ASO leads to a quantitative inhibition of molecular signaling through the PI 3-kinase pathway. In accordance with these data, the proliferation of PC-3 xenografts after treatment with IRS-1 ASO combined with rapamycin was more pronounced than treatment with each alone.

Tyrosine-phosphorylated IRS-1 proteins are known to bind efficiently a number of SH2 domain-containing proteins involved in activation of downstream signaling pathways, including PI 3-K p85, GRB2, SHP-2, Nck and Crk (for review, see Saltiel and Kahn 2001). There is increasing interest in the potential role of IRS-1 in oncogenesis. Overexpression of IRS-1 in NIH3T3 fibroblasts leads to increased activation of the Ras-MAPK cascade and cell transformation (Ito et al. 1996; Tanaka et al. 1996). IRS-1 overexpression also contributes to the progression of hepatocellular carcinoma, possibly by inhibiting transforming growth factor β1-mediated apoptosis (Tanaka and Wands 1996). Although the LNCaP prostate cancer cell line does not express IRS-1 or IRS-2, introduction of either protein in combination with IGF-IR converts these cells to a more aggressive phenotype (Reiss et al. 2000). A recent study examining endogenous IRS-1 shows that it is constitutively tyrosine-phosphorylated in a wide range of human tumor samples, suggesting that IRS-1 activation may be a common phenomenon in tumors (Chang et al. 2002). Moreover, a relationship between IRS-1 activation and fusion oncoproteins has already been established. TRK-T1 (Miranda et al. 2001) and BCR-ABL (Traina et al. 2003) have both been shown to bind IRS-1 and to be associated with increased IRS-1 tyrosine phosphorylation. Therefore, IRS-1 activation may be a more general mechanism for transformation. We found that inhibition of IRS-1 by IRS-1 ASO can lead to a decrease in the growth of PC-3 xenografts. Our data demonstrate that lowering IRS-1 content leads to a decrease in the number of docking sites for maximal activation of PI 3-kinase-Akt-mTOR cascade, as shown by a reduction of Akt and p70S6k phosphorylation, respectively, further suggesting that IRS-1 is important for PC-3 xenografts growth.

The primary pathway by which most growth factors and cytokines activate mTOR and its downstream targets appears to be the PI 3-kinase/Akt pathway (for review, see Fingar and Blenis 2004). Recent reports have shown that, in absence of TSC2 or in hiperinsulinemic situations, IRS-1 increases its serine phosphorylation in parallel with a reduction in its protein levels. Importantly, all the studies found that long term treatment with rapamycin completely restores IRS-1 protein levels and the insulin/IGF-1 responsiveness of PI 3-kinase Akt pathway (Harrington et al. 2004; Shah et al. 2004; Ueno et al. 2005). In agreement with these studies, our results show an increase in IRS-1 serine phosphorylation as well as a reduction in protein levels in PC-3 xenografts; moreover, the treatment with rapamycin restored IRS-1 levels and activity in the PC-3 xenografts as demonstrated. Furthermore, PC-3 xenografts treated with both IRS-1 ASO and rapamycin exhibit a strong attenuation of the PI 3-kinase pathway. Altogether, these data suggest that loss of PTEN expression in PC-3 xenografts triggers a feedback inhibition of Akt signaling, which is reversed by rapamycin and IRS-1 ASO can overcome.

In addition to the down-regulation of the PI 3-kinase/Akt pathway, continuous treatment with IRS-1 ASO for 4 weeks resulted in significant tumor growth delay, and the treatment with IRS-1 ASO plus rapamycin strongly inhibited cell proliferation and induced apoptosis. The suppression of the PI 3-kinase/Akt pathway in these tumors might potentially result in the reduction of protein synthesis, cell growth and proliferation downstream of mTOR through inactivation of p70S6k and activation of 4E-BP1 (Fingar and Blenis 2004; Fingar et al. 2002; Jefferies et al. 1997).

Based on these results, we propose a model for the synergism between IRS-1 ASO and rapamycin in suppressing growth in PC-3 cells lacking PTEN (Fig. 4a). According to this model, rapamycin alone induces mTOR inhibition, which is rapidly followed by IRS-1 activation (Fig. 4b). IRS-1 ASO, in turn, inhibits the expression of IRS-1 and has the same role as mTOR feed back. Thus, the combination of IRS-1 ASO with rapamycin provides an additive signal inhibition, which leads to enhanced apoptosis and diminished proliferation (Fig. 4c).

Fig. 4.

Fig. 4

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Model for IRS-1 ASO and rapamycin synergism in PC-3 xenografts. a mTOR feedback in PC-3 xenografts. The activated mTOR inhibits IRS-1 signaling towards PI 3-kinase. b Rapamycin treatment. Rapamycin inhibits mTOR and activates IRS-1 signaling towards PI 3-kinase. c Treatment with combination of IRS-1 ASO and rapamycin. The simultaneous treatment with IRS-1 ASO and rapamycin delivers an additional signal, which leads to enhanced apoptosis and diminished proliferation

In conclusion, our data demonstrate that the addition of agents that block IRS-1 potentiate the effect of mTOR inhibition in the growth of PC-3 xenografts.

Acknowledgments

These studies were supported by grants from Fundacão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de desenvolvimento científico e tecnológico (CNPq). We thank Dr. Nicola Conran for English language editing.

Footnotes

Josenilson C. Oliveira and Kellen K. Souza contributed equally to this paper.

References

  1. Cairns P, Okami K, Halachmi S et al (1997) Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Res 57:4997–5000 [PubMed] [Google Scholar]
  2. Chang Q, Li Y, White MF, Fletcher JA, Xiao S (2002) Constitutive activation of insulin receptor substrate 1 is a frequent event in human tumors: therapeutic implications. Cancer Res 62:6035–6038 [PubMed] [Google Scholar]
  3. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA (1995) Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789 [DOI] [PubMed] [Google Scholar]
  4. del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G (1997) Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278:687–689 [DOI] [PubMed] [Google Scholar]
  5. Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, Thomas G (2001) Mammalian TOR: a homeostatic ATP sensor. Science 294:1102–1105 [DOI] [PubMed] [Google Scholar]
  6. Fingar DC, Blenis J (2004) Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23:3151–3171 [DOI] [PubMed] [Google Scholar]
  7. Fingar DC, Salama S, Tsou C, Harlow E, Blenis J (2002) Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev 16:1472–1487 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gray IC, Phillips SM, Lee SJ, Neoptolemos JP, Weissenbach J, Spurr NK (1995) Loss of the chromosomal region 10q23–25 in prostate cancer. Cancer Res 55:4800–4803 [PubMed] [Google Scholar]
  9. Harrington LS, Findlay GM, Gray A et al (2004) The TSC1–2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J Cell Biol 166:213–223 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huang S, Houghton PJ (2002) Inhibitors of mammalian target of rapamycin as novel antitumor agents: from bench to clinic. Curr Opin Investig Drugs 3:295–304 [PubMed] [Google Scholar]
  11. Inoki K, Li Y, Xu T, Guan KL (2003) Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 17:1829–1834 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ito T, Sasaki Y, Wands JR (1996) Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases. Mol Cell Biol 16:943–951 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, Thomas G (1997) Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO J 16:3693–3704 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ (2007) Cancer statistics, 2007. CA Cancer J Clin 57:43–66 [DOI] [PubMed] [Google Scholar]
  15. Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos JL, Burgering BM (1999) Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398:630–634 [DOI] [PubMed] [Google Scholar]
  16. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [DOI] [PubMed] [Google Scholar]
  17. Lawlor MA, Alessi DR (2001) PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci 114:2903–2910 [DOI] [PubMed] [Google Scholar]
  18. Maehama T, Dixon JE (1998) The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 273:13375–13378 [DOI] [PubMed] [Google Scholar]
  19. Majumder PK, Febbo PG, Bikoff R et al (2004) mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat Med 10:594–601 [DOI] [PubMed] [Google Scholar]
  20. Miranda C, Greco A, Miele C, Pierotti MA, Van Obberghen E (2001) IRS-1 and IRS-2 are recruited by TrkA receptor and oncogenic TRK-T1. J Cell Physiol 186:35–46 [DOI] [PubMed] [Google Scholar]
  21. Mothe I, Van Obberghen E (1996) Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action. J Biol Chem 271:11222–11227 [DOI] [PubMed] [Google Scholar]
  22. Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, Sellers WR (2000) Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Biol 20:8969–8982 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Petrylak DP, Tangen CM, Hussain MH et al (2004) Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med 351:1513–1520 [DOI] [PubMed] [Google Scholar]
  24. Reiss K, Wang JY, Romano G et al (2000) IGF-I receptor signaling in a prostatic cancer cell line with a PTEN mutation. Oncogene 19:2687–2694 [DOI] [PubMed] [Google Scholar]
  25. Rui L, Fisher TL, Thomas J, White MF (2001) Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2. J Biol Chem 276:40362–40367 [DOI] [PubMed] [Google Scholar]
  26. Sabatini DM (2006) mTOR and cancer: insights into a complex relationship. Nat Rev Cancer 6:729–734 [DOI] [PubMed] [Google Scholar]
  27. Saltiel AR, Kahn CR (2001) Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414:799–806 [DOI] [PubMed] [Google Scholar]
  28. Sansal I, Sellers WR (2004) The biology and clinical relevance of the PTEN tumor suppressor pathway. J Clin Oncol 22:2954–2963 [DOI] [PubMed] [Google Scholar]
  29. Shah OJ, Wang Z, Hunter T (2004) Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies. Curr Biol 14:1650–1656 [DOI] [PubMed] [Google Scholar]
  30. Suzuki H, Freije D, Nusskern DR et al (1998) Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues. Cancer Res 58:204–209 [PubMed] [Google Scholar]
  31. Tanaka S, Ito T, Wands JR (1996) Neoplastic transformation induced by insulin receptor substrate-1 overexpression requires an interaction with both Grb2 and Syp signaling molecules. J Biol Chem 271:14610–14616 [DOI] [PubMed] [Google Scholar]
  32. Tanaka S, Wands JR (1996) Insulin receptor substrate 1 overexpression in human hepatocellular carcinoma cells prevents transforming growth factor beta1-induced apoptosis. Cancer Res 56:3391–3394 [PubMed] [Google Scholar]
  33. Tannock IF, de Wit R, Berry WR et al (2004) Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 351:1502–1512 [DOI] [PubMed] [Google Scholar]
  34. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Traina F, Carvalheira JB, Saad MJ, Costa FF, Saad ST (2003) BCR-ABL binds to IRS-1 and IRS-1 phosphorylation is inhibited by imatinib in K562 cells. FEBS Lett 535:17–22 [DOI] [PubMed] [Google Scholar]
  36. Ueno M, Carvalheira JB, Tambascia RC et al (2005) Regulation of insulin signalling by hyperinsulinaemia: role of IRS-1/2 serine phosphorylation and the mTOR/p70 S6K pathway. Diabetologia 48:506–518 [DOI] [PubMed] [Google Scholar]
  37. Yenush L, White MF (1997) The IRS-signalling system during insulin and cytokine action. Bioessays 19:491–500 [DOI] [PubMed] [Google Scholar]

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