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Morphoproteomic and Molecular Concomitants of an Overexpressed and Activated mTOR Pathway in Renal Cell Carcinomas

Address correspondence to Robert E. Brown, M.D., at his present address: Department of Pathology, University of Texas Houston Medical School, 6431 Fannin Street, Room 2.286, Houston, TX 77030, USA; tel 713 500 5442; fax 713 500 0732; e-mail robert.brown{at}uth.tmc.edu.

	Abstract

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CCI-779 (temsirolimus), an ester of rapamycin and an inhibitor of the mammalian target of rapamycin (mTOR), is currently in phase II trials for treatment of patients with solid cancers. The mTOR functions as a checkpoint for cell proliferation, with upstream Akt and downstream p70S6K serving as its most important mediators. The aim of this study was to evaluate the expression and activation of the Akt-mTOR-p70S6K pathway in renal cell carcinoma (RCC), seeking to strengthen the rationale for targeted therapy of RCC using rapamycin or a rapamycin analogue. Tissue microarray sections containing 128 primary RCCs, 22 metastatic RCCs, and 24 non-neoplastic (normal) kidneys (NK) were immunostained with monoclonal antibodies to phosphorylated (p)-Akt (Ser473), p-mTOR (Ser2448), and p-p70S6K (Thr389). Western blotting was performed on 3 cases of clear cell RCC (CRCC) and the corresponding non-neoplastic (normal) renal tissues using the same antibodies. The immunostain scoring system included: (a) location; (b) distribution; and (c) intensity. The normal kidneys provided baseline scores for comparison. Expression of p-Akt, p-mTOR, and p-p70S6K was seen in 100% (n = 24) of NKs and nearly 100% (n = 150) of both primary and metastatic RCCs. The p-p70S6K was located in the nucleus in both NKs and RCCs. The p-Akt was observed in the nucleus and cytoplasm of NKs and in the nucleus and cytoplasm/ membrane (plasmalemma) of RCCs. The p-mTOR was identified in the membrane of NKs and the membrane/nucleus of RCCs. The levels of expression of p-p70S6K, p-mTOR, and p-Akt were significantly higher in RCC than in NK in the overall pattern (intensity and distribution, p <0.05). Western blotting also showed higher expression of p-p70S6K, p-mTOR, and p-Akt in RCCs compared to the corresponding normal kidney tissues (p <0.05). These findings indicate that correlative over-expression and activation of p-Akt, p-mTOR, and p-p70S6K are commonly observed in RCCs. After considering these findings in the context of other established protein circuitries and pathways in RCC, we propose therapeutic approaches that incorporate rapamycin-like agents and other small molecule inhibitors in a combinatorial fashion in future clinical trials for RCC.

Keywords: rapamycin, p-Akt, p-mTOR, p-p70S6K, renal cell carcinoma

	Introduction

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Renal cell carcinoma is one of the most common malignant tumors in the United States, accounting for approximately 2% of all cancers. In 1998, more than 30,000 new cases of renal cell carcinoma were diagnosed, resulting in 12,000 deaths from the tumors in the United States alone [1]. The incidence of this cancer has continued to rise over the past two decades [1,2]. Renal cell carcinoma is notorious for distant metastases that occur years after the initial diagnosis and treatment. Lung, lymph node, liver, bone, and brain are the most common sites of metastasis [1]. For those RCC patients with local recurrence, advanced stage, or metastasis, in addition to the conventional chemotherapy, high-dose interleukin-2 and/or interferon-alpha [3,4], anti-vascular endothelial growth factor (VEGF) antibody [5], and nonmyeloablative allogeneic transplantation [6], showed only modest effects. Recently, CCI-779 (temsirolimus), an analogue of rapamycin and an inhibitor of mTOR, has been tested in phase II clinical trials for treatment of patients with solid cancers, including RCC [7–11]. Importantly, the pharmacodynamic effects of CCI-779 can be effectively determined by monitoring p70S6 kinase activity in peripheral blood mono-nuclear cells [10].

The mTOR functions as a checkpoint for cell growth and proliferation, an upstream Akt and a downstream p70S6K being the two most important mediators. Akt, also called protein kinase B (PKB), belongs to the serine/threonine protein kinase family, It has been implicated in the pathogenesis and progression of many human malignant tumors, such as prostate, breast, lung, ovary, and thyroid cancers, by regulating some key steps that control the balance of cell survival and apoptosis [12–17]. One of the functions of Akt is activation and phosphorylation of mTOR; subsequently, activated mTOR regulates p70S6K activation and phosphorylation [18].

Activation of Akt has been reported in some high-grade and high-stage RCC [19]. In addition, PTEN (phosphatase and tensin homologue deleted on chromosome 10) protein, a negative regulator of p-Akt, and PTEN tumor-suppressor gene that is frequently inactivated or mutated in various human carcinomas, have reduced expression in some RCC [20–23]. Alteration of PTEN gene may be associated with tumor progression and poor outcome [22]. However, the status of activation and phosphorylation of the Akt-mTOR-p70S6K pathway has not been well documented in renal cell carcinoma. Since rapamycin and its analogues are inhibitors of mTOR, it appears crucial to demonstrate and confirm the presence of the Akt-mTOR-p70S6K pathway in renal cell carcinoma; that is the subject of this report.

	Materials and Methods

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Archival materials  One hundred and fifty (150) cases of renal epithelial neoplasms were retrieved from the archives at the Department of Laboratory Medicine at Geisinger Medical Center and the Department of Pathology at Northwestern Memorial Hospital. The tissues were made anonymous and disassociated from any clinical data. The study protocol was approved by the Institutional Review Boards of Geisinger Medical Center and Northwestern Memorial Hospital. The cases included 70 cases of conventional RCC, 40 papillary renal cell carcinomas (PRCCs), 18 chromophobe renal cell carcinomas (ChRCCs), and 22 metastatic RCCs. Twenty-four cases of normal renal tissue (NK) were also included. Tissue microarray blocks were constructed with one 1.5 mm or two 1.0 mm tissue cores from each case as previously reported [24].

Immunohistochemical procedures and analyses.  Immunohistochemical stains were performed on formalin-fixed and paraffin-embedded 4 µm tissue microarray sections. To minimize the staining background using monoclonal antibodies against phosphorylated (p)-Akt (Ser 473), p-mTOR (Ser 2448), and p-p70S6K (Thr 389), a semiautomatic method for analysis of phosphorylated protein antibodies was developed. All primary antibodies were purchased from Cell Signaling Technology (Beverly, MA). In brief, paraffin-embedded tissues were sectioned, placed on glass slides, and dried at 60°C for 1–2 hr. They were then deparaffinized and subjected to antigen retrieval. Slides were placed in a 0.1 M citric acid (Sigma-Aldrich), 0.1 M sodium citrate (Fisher) solution and heated in a microwave oven. Once boiling was reached, the slides were heated for 10 min, followed by a 20 min cooling period outside the microwave oven. The slides were then placed in 0.05 M Tris-HCl, 0.05% Tween-20 (TBST buffer) for 5 min (Tris-HCl in packs from DAKO Cytomation, Tween-20 from EM Science). The tissue on the slides was treated with 3% H₂0₂ (McKesson General Medical) for 5 min, and then rinsed with TBST buffer. A few drops of diluted normal blocking serum (Vectastain kit, Vector Labs) were placed on the tissue and incubated with primary antibody overnight at 4°C. The following day, the tissues were rinsed well with TBST buffer for a minimum of 5 min. The rest of the staining procedure was performed on a DAKO Autostainer. The machine was programmed to treat each slide with diluted biotinylated secondary antibody solution (Vectastain Kit) for 30 min. The slides were rinsed with TBST buffer and incubated with Vectastain Elite Antigen Binding Complex Reagent (Vectastain Kit) for 30 min. The slides were rinsed with TBST buffer and incubated with DAB solution (3,3'-diaminobenzidine chromogen solution, DAKO EnVision+ System Kit) for 10 min. The slides were removed from the Autostainer and rinsed with distilled water. They were counterstained with Gill II hematoxylin (Thermo/Shandon) for 20 sec, treated with xylene (Fisher) for 10–15 sec, and cover-slipped. The scoring system for immunostained tissues was as follows: (a) location–nucleus, cytoplasm, membrane, or a combination of two or three locations; (b) distribution–an estimated percentage of staining on each case; and (c) intensity–weak (1+), moderate (2+), or strong (3+). The normal kidneys served as a baseline for comparison.

Statistics.  A combined score was calculated using the staining intensity score (1+, 2+ or 3+) multiplied by the estimated percentage of positive staining. The overall score of staining intensity and average percentage was recorded in each group as mean ± SD. One-way ANOVA was used to determine the overall difference between the mean intensity scores of normal kidney and those of each group for p-mTOR, p-p70S6K, and p-Akt.

Western blotting.  Three cases of clear cell RCC and the corresponding normal renal tissues were included. Western blotting was performed as previously described [25]. In brief, frozen tissue blocks containing renal tumors and the corresponding normal tissue comprised of renal cortex were retrieved from the Division of Laboratory Medicine’s tissue archive, thawed, minced, and then lysed in the lysis buffer. Tissue homogenates from each case were electrophoresed on 6 or 10% sodium dodecyl sulfate-polyacrylamide gel (SDS PAGE). The fractionated proteins were transferred onto polyvinylidene diflouride (PVDF) membranes and the membranes were incubated with the same antibodies to phosphorylated (p)-Akt (Ser473), p-mTOR (Ser2448), and p-p70S6K (Thr389) used for immunohistochemistry. The appropriate secondary antibody conjugated with horseradish peroxidase was used. Immunoreactive proteins were visualized by an enhanced chemiluminescence-Western blotting system (Amersham Pharmacia Biotech). Antibodies to ß-actin (Sigma; 1:3000 dilution) were employed to evaluate the evenness of the protein loading.

	Results

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Expression of p-Akt, p-mTOR, and p-p70S6K was seen in 100% (n = 24) of NKs and nearly 100% (n = 150) of both primary and metastatic RCCs (excepting 4 cases of chromophobe RCC that were negative for p-Akt). The p-p70S6K was located in the nucleus in both NKs and RCCs. The p-Akt was observed in the nucleus and cytoplasm of NKs and the nucleus and cytoplasm/membrane of RCCs. The p-mTOR was identified in the membrane of NKs and the membrane and/or nucleus of RCCs. The levels of expression of p-p70S6K, p-mTOR, and p-Akt were significantly higher in RCCs compared to NKs in the overall expression pattern (intensity and distribution, p <0.05).

Fig. 1 shows immunoreactivity of p-Akt (Ser 473), p-mTOR (Ser 2448), and p-p70S6K (Thr 389) in RCC (left-hand frames), and in normal kidney (right-hand frames), respectively. The details of immunostaining results on the location, intensity, and average distribution of positively stained cases of p-Akt, p-mTOR, and p-p70S6K are summarized in Tables 1, 2, and 3. Overall results of the combined scores (intensity x estimated percentage) for each group with statistical analysis are listed in Table 4.

View larger version (149K): [in this window] [in a new window]   Fig. 1. Renal cell carcinoma (left-hand frames) depicting the relatively uniform expression of activated Akt (phosphorylated at serine 473), mTOR (phosphorylated at serine 2448), and p70S6K (phosphorylated at threonine 389) with predominantly cytoplasmic, plasmalemmal, and nuclear compartmentalizations of the intense chromogenic signals, respectively. Contrast with variable and/or focal and generally less intense signal for the corresponding protein analytes in the normal kidney is depicted in the right-hand frames and indicates overexpression of the constitutively activated Akt/mTOR/p70S6K pathway in renal cell carcinoma. Molecular correlates by Western blotting are depicted in Fig. 2.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Western blot showing p-mTOR (Ser 2448), and p-p70S6K (Thr 389), and actin in 3 clear cell renal cell carcinomas (M) and the 3 corresponding non-neoplastic renal tissue (B). The lane with actin shows the equal loading of proteins in each lane. The ratio of average band densities of M to B is 5.78 for p-mTOR (p <0.05) and 7.32 for p-p70S6K (p <0.05). The ratio for p-Akt (Ser 473) is 1.66 (p <0.05). The results for p-Akt are not illustrated here. Morphoproteomic correlates are depicted in Fig. 1.

	Discussion

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In support of our observations concerning a constitutively activated mTOR pathway in human RCC are various molecular, experimental, and scintigraphic concomitants. These include: (a) the demonstration by Chuang and co-workers [41] of the overexpression of glutathione s-transferase (GST)- in the majority of primary and metastatic clear cell RCC, given that GST- can be increased through PI3-K /Akt /mTOR /p70S6K signaling [42]; (b) the efficacy of rapamycin as an inhibitor of human renal cancer pulmonary metastasis in a xenograft model [43]; and (c) the study by Thomas et al [44] showing that loss of the von Hippel-Lindau tumor suppressor gene (VHL) sensitizes kidney cancer cells to the mTOR inhibitor, CCI-779, in vitro and in mouse models, and that VHL-deficient tumors show increased uptake of a positron emission tomography (PET) tracer, fluorodeoxy-glucose (FDG), in an mTOR-dependent manner.

This latter observation raises the question of other mechanisms that may influence the sensitivity or resistance of RCC to rapamycin or its analogues. To put this in perspective, although we have demonstrated constitutive activation of the mTOR pathway in 100% of the cases in this large series of RCCs, the response rate of patients with advanced RCC to CCI-779, a rapamycin analogue, included a 7% objective response rate and minor responses in 26% in a randomized phase II study [7]. Potential indicators of the relative sensitivity of a given RCC to rapamycin might include one or more of the following: (a) the absence of PTEN, which normally suppresses tumor formation by restraining the PI3-K/Akt pathway [8,20–23]; (b) the absence of the tuberous sclerosis complex-1 and -2 gene products [45–47], which could result in enhanced mTOR signaling by removing a counter-regulatory mechanism to mTOR-induced translational synthesis of anti-apoptotic and growth-promoting proteins; and (c) the loss of the von Hippel-Lindau tumor suppressor protein that Liu et al [48] demonstrated in 22% of metastatic RCC’s but not in primary RCC. Additional consideration should be given to the expression or state of activation of other protein analytes and pathways in RCC that could either: (a) interact with and promote mTOR signaling; (b) function independently of mTOR signaling to promote tumorigenesis of RCC; (c) act in a counter-regulatory manner to oppose rapamycin; or (d) act in collaboration with rapamycin to downregulate mTOR signal transduction. Specific examples of the latter protein circuitry that we and others have identified collectively in RCC include the ras/ prenylation/Raf kinase/extracellular signal-regulated kinase (ERK) pathway, bcl-2, nuclear factor-kappaB (NF-B), and peroxisome proliferator–activated receptor (PPAR).

To expand on these, activated ERK 1/2 in RCC, as evidenced by phosphorylation on threonine 202/tyrosine 204 and nuclear translocation, has been noted previously by one of us [REB, 49] and confirmed in a small number of RCC’s in this study (data not shown). Such activation was accompanied by immunopositivity for p21ras and the alpha subunit common to farnesyl and geranylgeranyl transferases, upstream components in the ras/ prenylation/Raf kinase signaling pathway. Phosphorylated-ERK acts in a collaborative fashion with mTOR to promote p70S6K signaling but also acts independently to inhibit 4E-BP1 and to stimulate eukaryotic initiation factor-4E (eIF-4E)-induced, translational synthesis of proteins that promote tumorigenesis [49].

Bcl-2, an anti-apoptotic protein, has been shown to be overexpressed in RCC [50–52]. The expression of bcl-2 has been associated with resistance to rapamycin [53]. Because the mTOR pathway counterbalances the anti-apoptotic potential of bcl-2 by effecting phosphorylative inactivation of this protein [54–56], the use of rapamycin or a rapamycin analogue alone may be counterproductive in RCCs with high bcl-2 expression.

One of the mechanisms known to facilitate the expression of bcl-2 is through transcriptional activation at the genomic level by NF-B [57]. Oya and co-workers [58] have reported that increased NF-B activation is related to tumor development of renal cell carcinoma. Our preliminary data in this same series of cases have demonstrated the activation and overexpression of p-NF-Bp65, phosphorylated at serine 536. Notably, An et al [59] have shown that the proteasome inhibitor, bortezomib (Velcade PS-341), promoted maximal apoptosis of RCC in an NF-B dependent fashion and Fahy et al [60] have successfully targeted bcl-2 overexpression in various human malignancies through NF-B inhibition using bortezomib. Finally, activation of the NF-B dependent pathway reflects convergent but also independent signaling through Akt, immunophilins/mTOR, and ERK pathways [49]. Therefore, rapamycin-like agents may be only partly effective in down-regulating the NF-B pathway in RCC.

Inoue and co-workers [61] have demonstrated the expression of PPAR in primary renal cell carcinoma and have shown an inhibitory effect of the PPAR ligand, pioglitazone, on the growth of renal cell carcinoma cell lines. This growth inhibitory effect by PPAR ligands may be due in part to the synthesis of p21waf, which would antagonize the mTOR pathway by blocking G1 cell cycle progression [62]. Similarly, activation of PPAR could lead to downregulation of the anti-apoptotic protein, bcl-2 [62]. Recently, Han et al [63] reported that rosiglitazone, another synthetic ligand for PPAR, reduced the phosphorylation of Akt and increased PTEN protein expression in non-small cell lung carcinoma cells; this was accompanied by inhibition of proliferation. Additionally, the inhibitory effect of rosiglitazone on tumor cell growth in their study was enhanced by the mTOR inhibitor, rapamycin.

Fortunately, pharmaceutical agents are either available or in clinical trials to target mTOR and these associated pathways in RCC. These include: CCI-779, a rapamycin analogue, which showed, as previously stated, a 7% objective response rate and minor responses in 26% of patients with advanced RCC in a randomized phase II study [7]; BAY 43-9006 (sorafenib, Nexavar), a Raf kinase inhibitor, which resulted in stabilization of disease in 30% of patients and a response of >25% reduction in the tumor in 40% of patients with RCC in a phase II study [64]; and bortezomib (Velcade), a proteasome inhibitor, known to interfere in NF-B activation, which produced a partial response in 11% and stable disease in 38% of patients with advanced renal cell carcinoma in a phase II trial [65]. The limited response to individual small molecule inhibitors speaks to the complexity of the protein circuitry in RCC and suggests that combinatorial therapy with inhibitors of the Akt/mTOR/p70S6K, the ras/prenylation/Raf kinase/ERK, and the NF-B pathways may be necessary to slow the growth and effect apoptosis in RCC [49,66,67]. Fig. 3 illustrates the established protein circuitries in RCC and how they relate to the mTOR pathway in RCC, along with some potential small molecule inhibitors.

View larger version (82K): [in this window] [in a new window]   Fig. 3. A composite of the complex protein circuitries identified by the authors and others [8,49,68–72] utilizing immunohistochemistry in cases of renal cell carcinoma (RCC) and depicting the interrelationships with the mTOR pathway. Specifically, downstream signaling by the tyrosine kinases, PDGFR-alpha, EGFR, and IGF-1R and their ligands, proceeds through the PI3-K/Akt and ras/prenylation(FTa/GGTa)/Raf kinase/ERK 1/2 pathways. The former leads to phosphorylative activation of mTOR and its effector p70S6K and the latter (p-ERK 1/2) converges on p70S6K promoting G1 cell cycle progression. Bipathway signal transduction also converges on phosphorylative activation of NF-kappaBp65 with nuclear translocation contributing to tumoral proliferation, chemoresistance, and anti-apoptosis, including the synthesis of bcl-2. Notably, HIF-1alpha induced by mTOR signaling is relevant to VEGF production and autocrine stimulation of TGF-alpha/EGFR signaling in RCC [72–76]. Clinical trials utilizing a rapamycin analogue (CCI-779), or BAY43-9006 (Raf kinase inhibitor), or Velcade (proteasome inhibitor) individually have resulted in some, but limited, clinical successes [7,64,65]. Preclinical ligands such as pioglitazone (Actos) to activate PPARgamma in renal cell carcinoma leading to events that counteract the mTOR pathway by interfering in G1 cell cycle progression [61,62] or upregulate PTEN [63] may be readily applicable to clinical trials. Abbreviations: PDGF: platelet-derived growth factor; TGF: transforming growth factor; EGFR: epidermal growth factor receptor; IGF: insulin-like growth factor; PI3-K: phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homologue deleted on chromosome 10; TSC: tuberous sclerosis complex; mTOR: mammalian target of rapamycin; VHL: von Hippel-Lindau protein; FT: farnesyl transferase; GGT: geranylgeranyl transferase; ERK: extracellular signal-regulated kinase; IKK: inhibitor kappa B kinase: I-B; inhibitor-kappaB; NF-B: nuclear factor-kappaB; HIF: hypoxia-inducible factor;VEGF: vascular endothelial growth factor; PPAR: peroxisome proliferator-activated receptor; GST-: glutathione s-transferase-alpha.

	Acknowledgments

 
The authors thank Laurie Kneller and Glen Kauwell for expert technical assistance and Sharon Coup-Stroh for secretarial support and help with the graphics.

	Footnotes

 
^* Presented in part at the Annual Meeting of the United States and Canadian Association of Pathologists in San Antonio, TX, during March 2005.

	References

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1. Renshaw AA, Richie JP. Subtype of renal cell carcinoma: Different onset and sites of metastatic disease. Am J Clin Pathol 1999;111:539–543.[Medline]
2. Chow WH, Devesa SS, Warren JL, Fraumeni JF Jr. Rising incidence of renal cell cancer in the United States. JAMA 1999;281:1628–1631.[Abstract/Free Full Text]
3. Fisher RI, Rosenberg SA, Fyfe G. Long-term survival update for high-dose recombinant interleukin-2 in patients with renal cell carcinoma. Cancer J Sci Am 2000(Suppl);6:S55–S57.[Medline]
4. Steineck G, Strander H, Carbin BE, Borgstrom E, Wallin L, Achtnich U, Arvidsson A, Soderlund V, Naslund I, Esposti PL, et al. Recombinant leukocyte interferon alpha-2a and medroxyprogesterone in advanced renal cell carcinoma. A randomized trial. Acta Oncol 1990;29:155–162.[Medline]
5. Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL, Steinberg SM, Chen HX, Rosenberg SA. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. NEJM 2003;349:427–434.[Abstract/Free Full Text]
6. Childs R, Chernoff A, Contentin N, Bahceci E, Schrump D, Leitman S, Read EJ, Tisdale J, Dunbar C, Linehan WM, Young NS, Barrett AJ. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. NEJM 2000;343:750–758.[Abstract/Free Full Text]
7. Atkins MB, Hidalgo M, Stadler WM, Logan TF, Dutcher JP, Hudes GR, Park Y, Liou SH, Marshall B, Boni JP, Dukart G, Sherman ML. Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol 2004;22:909–918.[Abstract/Free Full Text]
8. Xu G, Zhang WH, Bertram P, Zheng XF, McLeod H. Pharmacogenomic profiling of the PI3K/PTEN-AKT-mTOR pathway in common human tumors. Int J Oncology 2004;24:893–900.[Medline]
9. Harding MW. Immunophilins, mTOR, and pharmacodymic strategies for a targeted cancer therapy. Clin Cancer Res 2003;9:2882–2886.[Free Full Text]
10. Peralba JM, deGraffenried L, Friedrichs W, Fulcher L, Grunwald V, Weiss G, Hidalgo M. Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients. Clin Cancer Res 2003;9:2887–2892.[Abstract/Free Full Text]
11. Galanis E, Buckner JC, Maurer MJ, Kreisberg JI, Ballman K, Boni J, Peralba JM, Jenkins RB, Dakhil SR, Morton RF, Jaeckle KA, Scheithauer BW, Dancey J, Hidalgo M, Walsh DJ; North Central Cancer Treatment Group. Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: A North Central Cancer Treatment Group Study. J Clin Oncol 2005;23: 5294–5304.[Abstract/Free Full Text]
12. McCarty M. Targeting multiple signaling pathways as a strategy for managing prostate cancer: multifocal signal modulation therapy. Integr Cancer Ther 2004;3:349–380.[Abstract/Free Full Text]
13. David O, Jett J, LeBeau H, Dy G, Hughes J, Friedman M, Brody AR. Phospho-Akt overexpression in non-small cell lung cancer confers significant stage-independent survival disadvantage. Clin Cancer Res 2004; 10:6865–6871.[Abstract/Free Full Text]
14. Mukohara T, Kudoh S, Matsuura K, Yamauchi S, Kimura T, Yoshimura N, Kanazawa H, Hirata K, Inoue K, Wanibuchi H, Fukushima S, Yoshikawa J. Activated Akt expression has significant correlation with EGFR and TGF-alpha expression in stage I NSCLC. Anticancer Res 2004;24:11–18.[Abstract/Free Full Text]
15. Miyakawa M, Tsushima T, Murakami H, Wakai K, Isozaki O, Takano K. Increased expression of phosphorylated p70S6 kinase and Akt in papillary thyroid cancer tissues. Endocr J 2003;50:77–83.[Medline]
16. Zhou X, Tan M, Stone Hawthorne V, Klos KS, Lan KH, Yang Y, Yang W, Smith TL, Shi D, Yu D. Activation of Akt/mammalian target of rapamycin/4E BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers. Clin Cancer Res 2004;10:6779–6788.[Abstract/Free Full Text]
17. deGraffenried LA, Friedrichs WE, Russell DH, Donzis EJ, Middleton AK, Silva JM, Roth RA, Hidalgo M. Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt activity. Clin Cancer Res 2004;10:8059–8067.[Abstract/Free Full Text]
18. Gingras AC, Raught B, Sonenberg N. Regulation of translation initiation by FRAP/mTOR. Genes Dev 2001;15:807–826.[Free Full Text]
19. Horiguchi A, Oya M, Uchida A, Marumo K, Murai M. Elevated Akt activation and its impact on clinico-pathological features of renal cell carcinoma. J Urol 2003;169:710–713.[Medline]
20. Cantley LC, Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphinositide 3-kinase/AKT pathway. PNAS USA 1999;96:4240–4245.[Abstract/Free Full Text]
21. Myers MP, Pass I, Batty IH, Van der Kaay J, Stolarov JP, Hemmings BA, Wigler MH, Downes CP, Tonks NK. The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. PNAS USA 1998;95:13513–13518.[Abstract/Free Full Text]
22. Shin Lee J, Seok Kim H, Bok Kim Y, Cheol Lee M, Soo Park C. Expression of PTEN in renal cell carcinoma and its relation to tumor behavior and growth. J Surg Oncol 2003;84:166–172.[Medline]
23. Brenner W, Farber G, Herget T, Lehr HA, Hengstler JG, Thuroff JW. Loss of tumor suppressor protein PTEN during renal carcinogenesis. Int J Cancer 2002; 99:53–57.[Medline]
24. Chuang ST, Chu P, Sugimura J, Tretiakova MS, Papavero V, Wang K, Tan MH, Lin F, Teh BT, Yang XJ. Overexpression of glutathione s-transferase alpha in clear cell renal cell carcinoma. Am J Clin Pathol 2005; 123:421–429.[Abstract/Free Full Text]
25. Gu Y, Lin Q, Childress C, Yang W. Identification of the region in Cdc42 that confers the binding specificity to activated Cdc42-associated kinase. J Biol Chem 2004; 279:30507–30513.[Abstract/Free Full Text]
26. Withers DJ, Ouwens DM, Nave BT, van der Zon GC, Alarcon CM, Cardenas ME, Heitman J, Maassen JA, Shepherd PR. Expression, enzyme activity, and sub-cellular localization of mammalian target of rapamycin in insulin-responsive cells. Biochem Biophys Res Commun 1997;241:704–709.[Medline]
27. Ferrari S, Thomas G. S6 phosphorylation and the p70s6k/p85s6k. Crit Rev Biochem Mol Bio 1994;29:385–413.[Medline]
28. Kozma SC, Thomas G. p70s6k/p85s6k: mechanism of activation and role in mitogenesis. Semin Cancer Biol 1994;5:255–260.[Medline]
29. Reinhard C, Fernandez A, Lamb NJ, Thomas G. Nuclear localization of p85s6k: functional requirement for entry into S phase. EMBO J 1994;13:1557–1565.[Medline]
30. Edelmann HM, Kuhne C, Petritsch C, Ballou LM. Cell cycle regulation of p70S6 kinase and p42/p44 mitogen-activated protein kinases in Swiss mouse 3T3 fibroblasts. J Biol Chem 1996;271:963–971.[Abstract/Free Full Text]
31. Kim SJ, Kahn CR. Insulin stimulates p70 S6 kinase in the nucleus of cells. Biochem Biophys Res Commun 1997;234:681–685.[Medline]
32. Bachman RA, Kim JH, Wu AL, Park IH, Chen J. A nuclear transport signal in mammalian target of rapamycin is critical for its cytoplasmic signaling to s6 kinase 1. J Biol Chem 2006;281:7357–7363.[Abstract/Free Full Text]
33. Cao X, Kambe F, Moeller LC, Refetoff S, Seo H. Thyroid hormone induces rapid activation of Akt/protein kinase B-mammalian target of rapamycin-p70S6K cascade through phosphatidylinositol 3-kinase in human fibroblasts. Mol Endocrinol 2005;19: 102–112.[Abstract/Free Full Text]
34. Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 1999;344: 427–431.[Medline]
35. Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA, Thomas G. Phosphorylation and activation of p70s6k by PDK1. Science 1998;279:707–710.[Abstract/Free Full Text]
36. Dennis PB, Pullen N, Kozma SC, Thomas G. The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases. Mol Cell Biol 1996;16:6242–6251.[Abstract/Free Full Text]
37. Valentinis B, Navarro M, Zanocco-Marani T, Edmonds P, McCormick J, Morrione A, Sacchi A, Romano G, Reiss K, Baserga R. Insulin receptor substrate-1, p70S6K, and cell size in transformation and differentiation of hemopoietic cells. J Biol Chem 2000;275:25451–25459.[Abstract/Free Full Text]
38. Ali SM, Sabatini DM. Structure of S6 kinase 1 determines whether raptor-mTOR or rictor-mTOR phosphorylates its hydrophobic motif site. J Biol Chem 2005;280: 19445–19448.[Abstract/Free Full Text]
39. Brown, RE, Zhang PL, Lun M, Pellitteri PK, Law A, Wood GC, Kennedy TL. Morphoproteomic and pharmacoproteomic rationale for mTOR effectors as therapeutic targets in head and neck squamous cell carcinoma. Ann Clin Lab Sci 2006;36:273–282.[Abstract/Free Full Text]
40. Weng QP, Kozlowski M, Belham C, Zhang A, Comb MJ, Avruch J. Regulation of p70S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies. J Biol Chem 1998;273:16621–16629.[Abstract/Free Full Text]
41. Chuang ST, Chu P, Sugimura J, Tretiakova MS, Papavero V, Wang K, Tan MH, Lin F, Teh BT, Yang XJ. Overexpression of glutathione s-transferase alpha in clear cell renal cell carcinoma. Am J Clin Pathol 2005; 123:421–429.[Abstract/Free Full Text]
42. Kim SK, Abdelmegeed MA, Novak RF. Identification of the insulin signaling cascade in the regulation of alpha-class glutathione s-transferase expression in primary cultured rat hepatocytes. J Pharmacol Exp Therap 2006;316:1255–1261.[Abstract/Free Full Text]
43. Luan FL, Ding R, Sharma VK, Chon WJ, Lagman M, Suthanthiran M. Rapamycin is an effective inhibitor of human renal cancer metastasis. Kidney Internat 2003; 63:917–926.[Medline]
44. Thomas GV, Tran C, Mellinghoff IK, Welsbie DS, Chan E, Fueger B, Czernin J, Sawyers CL. Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med 2006;12:122–127.[Medline]
45. Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, Blenis J. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. PNAS USA 2002;99:13571–13576.[Abstract/Free Full Text]
46. Kenerson HL, Aicher LD, True LD, Yeung RS. Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. J Cancer Res 2002;62:5645–5650.
47. Mak BC, Yeung RS. The tuberous sclerosis complex genes in tumor development. Cancer Invest 2004;22: 588–603.[Medline]
48. Liu H, Zhang K, Blasick T, Zhang PL, Ahmed M, Lin F. Loss expression of VHL protein in metastatic renal cell carcinoma associated with overexpression of p53. Lab Invest 2006:86(Suppl 1):147A (Abstract #680).
49. Brown RE. Morphoproteomics: exposing protein circuitries in tumors to identify potential therapeutic targets in cancer patients. Exp Rev Proteomics 2005;2:337–348.
50. Huang A, Fone PD, Gandour-Edwards R, White RW, Low RK. Immunohistochemical analysis of BCL-2 protein expression in renal cell carcinoma. J Urol 1999; 162:610–613.[Medline]
51. Tomita Y, Bilim V, Kawasaki T, Takahashi K, Okan I, Magnusson KP, Wiman KG. Frequent expression of Bcl-2 in renal cell carcinomas carrying wild-type p53. Int J Cancer 1996;66:322–325.[Medline]
52. Itoi T, Yamana K, Bilim V, Takahashi K, Tomita F. Impact of frequent Bcl-2 expression on better prognosis in renal cell carcinoma patients. Br J Cancer 2004;90: 200–205.[Medline]
53. Aguirre D, Boya P, Bellet D, Faivre S, Troalen F, Benard J, Saulnier P, Hopkins-Donaldson S, Zangemeister-Wittke U, Kroemer G, Raymond E. Bcl-2 and CCND1/CDK4 expression levels predict the cellular effects of mTOR inhibitors in human ovarian carcinoma. Apoptosis 2004;9:797–805.[Medline]
54. Calastretti F, Rancati F, Ceriani MC, Asnaghi L, Canti G, Nicolin A. Rapamycin increases the cellular concentration of the bcl-2 protein and exerts an anti-apoptic effect. Eur J Cancer 2001:37;2121–2128.[Medline]
55. Asnaghi L, Calstretti A, Bevilacqua A, D’Agnano I, Gatti G, Canti G, Delia D, Capaccioli S, Nicolin A. Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt. Oncogene 2004:23;5781–5791.[Medline]
56. Asnaghi L, Bruno P, Priulla M, Nicolin A. mTOR: a protein kinase switching between life and death. Pharm Res 2004:50;545–549.
57. Ichikawa H, Takada Y, Murakami A, Aggarwal BB. Identification of a novel blocker of I kappa B alpha kinase that enhances cellular apoptosis and inhibits cellular invasion through suppression of NF-kappa B-regulated gene products. J Immunol 2005:174;7383–7392.[Abstract/Free Full Text]
58. Oya M, Takayanagi A, Horiguchi A, Mizuno R, Ohtsubo M, Marumo K, Shimizu N, Murai M. Increased nuclear factor-kappa B activation is related to the tumor development of renal cell carcinoma. Carcinogenesis 2003;24:377–384.[Abstract/Free Full Text]
59. An J, Sun Y, Fisher M, Rettig MB. Maximal apoptosis of renal cell carcinoma by the proteasome inhibitor bortezomib is nuclear factor-kappaB dependent. Mol Cancer Ther 2004;3:727–736.[Abstract/Free Full Text]
60. Fahy BN, Schlieman MG, Mortenson MM, Virudachalam S, Bold RJ. Targeting BCL-2 overexpression in various human malignancies through NF-kappaB inhibition by the proteasome inhibitor bortezomib. Cancer Chemother Pharmacol 2005;56:46–54.[Medline]
61. Inoue K, Kawahito Y, Tsubouchi Y, Kohno M, Yoshimura R, Yoshimura T, Sano H. Expression of peroxisome proliferator-activated receptor in renal cell carcinoma and growth inhibition by its agonists. Biochem Biophys Res Commun 2001;287:727–732.[Medline]
62. Yang FG, Zhang ZW, Xin DQ, Shi CJ, Wu JP, Guo YL, Guan YF. Peroxisome proliferator-activated receptor ligands induce cell cycle arrest and apoptosis in human renal carcinoma cell lines. Acta Pharmacol Sinica 2005; 26:753–761.[Medline]
63. Han S, Roman J. Rosiglitazone suppresses human lung carcinoma cell growth through PPAR (gamma)-dependent and PPAR (gamma)-independent signal pathways. Mol Cancer Ther 2006;5:430–437.[Abstract/Free Full Text]
64. Ahmad T, Eisen T. Kinase inhibition with BAY 43-9006 in renal cell carcinoma. Clin Cancer Res 2004;10:6388S–63892S.[Abstract/Free Full Text]
65. Kondagunta GV, Drucker B, Schwartz L, Bacik J, Marion S, Russo P, Mazumdar M, Motzer RJ. Phase II trial of bortezomib for patients with advanced renal cell carcinoma. J Clin Oncol 2004;22:3720–3725.[Abstract/Free Full Text]
66. Gemmill RM, Zhou M, Costa L, Korch C, Bukowski RM, Drabkin HA. Synergistic growth inhibition by Iressa and Rapamycin is modulated by VHL mutations in renal cell carcinoma. Br J Cancer 2005;92:2266–2277.[Medline]
67. Gollob JA. Sorafenib: scientific ratuionales for single-agent and combination therapy in clear-cell renal cell carcinoma. Clin Genitourin Cancer 2005;4:167–174.[Medline]
68. Sulzbacher I, Birner P, Traxler M, Marberger M, Haitel A. Expression of platelet-derived growth factor-alpha receptor is associated with tumor progression in clear cell renal cell carcinoma. Am J Clin Path 2003;120:107–112.[Medline]
69. Uhlman Dl, Nguyen P, Manivel JC, Zhang G, Hagen K, Fraley E, Aeppli D, Niehans GA. Epidermal growth factor receptor and transforming growth factor alpha expression in papillary and nonpapillary renal cell carcinoma: correlation with metastatic behavior and prognosis. Clin Cancer Res 1995;1:913–920.[Abstract]
70. Schips L, Zigeuner R, Ratscheck M, Rehak P, Ruschoff J, Langner C. Analysis of insulin-like growth factors and insulin-like growth factor I receptor expression in renal cell carcinoma. Am J Clin Path 2004;122:931–937.[Medline]
71. Rosendahl A, Forsberg G. Influence of IGF-IR stimulation or blockade on proliferation of human renal cell carcinoma cell lines. Int J Oncol 2004;25:1327–1336.[Medline]
72. Treins C, Giorgetti-Peraldi S, Murdaca J, Monthouel-Kartmann MN, Van Obberghen E. Regulation of hypoxia-inducible factor (HIF)-1 activity and expression of HIF hydroxylases in response to insulin-like growth factor I. Mol Endocrin 2005;19:1304–1317.[Abstract/Free Full Text]
73. Nakamura H, Makino Y, Okamoto K, Poellinger L, Ohnuma K, Morimoto C, Tanaka H. TCR engagement increases hypoxemia-inducible factor-1 alpha protein synthesis via rapamycin-sensitive pathway under hypoxic conditions in human peripheral T cells. J Immun 2005; 174:7592–7599.[Abstract/Free Full Text]
74. Phillips RJ, Mestas J, Gharaee-Kermani M, Burdick MD, Sica A, Belperio JA, Keane MP, Strieter RM. Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non-small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1 alpha. J Biol Chem 2005;280:22473–22481.[Abstract/Free Full Text]
75. Abraham RT. MTOR as a positive regulator of tumor cell responses to hypoxia. Curr Topics Microb Immunol 2004;279:299–319.
76. Gunaratnam L, Morley M, Franovic A, de Paulsen N, Mekhail K, Parolin DA, Nakamura E, Lorimer IA, Lee S. Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(–/–) renal cell carcinoma cells. J Biol Chem 2003;278:44966–44974.[Abstract/Free Full Text]

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