1 University of Texas Health Science Center at San Antonio, San Antonio, TX; 2 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD; 3 Saint Louis University, St Louis, MO, USA
* Correspondence to: Dr Manuel Hidalgo, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, Room 1M88, Baltimore, MD 21231, USA. Tel: +1-410-502-7149; Fax: +1-410-614-9006; Email: mhidalg1{at}jhmi.edu
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Abstract |
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Key words: breast cancer, PI3K/Akt, PTEN
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Introduction |
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Although the intracellular functions of PTEN are complex and only partially deciphered, it appears that one of the most relevant consequences of the PTEN-defective phenotype is the activation of the PI3K/Akt signaling pathway. Akt mediates multiple intracellular functions pertaining to cell proliferation and apoptosis and has been implicated in chemoresistance in colon, bladder and ovarian cancers [79
]. In addition, recent studies suggest that pharmacological or genetic modulation of Akt activity alters the response of non-small cell lung cancer (NSCLC) cell lines to conventional chemotherapy [10
]. Although the signaling pathways downstream from Akt are not totally elucidated, it is well established that mTOR (also known as FRAP and RAFT) is one of the most relevant mediators of Akt functions [11
]. mTOR is activated in response to Akt phosphorylation, resulting in the phosphorylation of p70S6 kinase and 4E-BP1, and, consequently, increases the translation of mRNAs of proteins involved in the regulation of the cell cycle [12
]. Recently, PTEN-negative cell lines have been shown to have elevated p70S6 kinase activation that is abrogated in the presence of the mTOR inhibitor CCI-779, an ester of rapamycin, supporting the notion that mTOR is an important downstream signaling mediator in PTEN-negative tumors [13
, 14
]. Inhibition of mTOR by rapamycin results in cell cycle arrest, p53-dependent and -independent apoptosis and tumor growth inhibition [15
17
]. CCI-779, an ester of rapamycin, is currently in clinical development for the treatment of cancer. Preliminary data from ongoing clinical trials indicate that CCI-779 is well tolerated and has significant antitumor activity in a variety of tumor types, including breast cancer [18
].
We have been interested in developing specific treatments for patients with PTEN negative tumors. In this study, we assessed the response of a series of breast cancer cell lines with varied PTEN levels to the mTOR inhibitor rapamycin and other inhibitors of the PI3K/Akt pathway. We found that PTEN status negatively correlated with sensitivity to the growth inhibitory effects of rapamycin and the PI3 kinase inhibitor LY294002. These findings support previous observations and suggest that inhibitors of mTOR could prove especially effective in the treatment of tumors with a PTEN-negative phenotype.
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Materials and methods |
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Western blot analysis
Cells were seeded in 6-well plates at a density of 2 x 105 cells per well. Cells were serum-starved for 18 h, then stimulated with 10% FBS for the indicated times. Cells were then harvested in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors and 0.1 M sodium orthovanadate in PBS) and 40 µg samples of protein extract were resolved on a polyacrylamide gel. These were subsequently transferred from the gel to nitrocellulose membranes and subjected to immunodetection with antibodies against PTEN (Sigma), phosphorylated and total Akt, phosphorylated and total p70S6 kinase (Cell Signaling Technology, Beverly, MA) and finally actin (Santa Cruz Biotechnology, Santa Cruz, CA) for a loading control. Signal was detected using the enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL).
Growth proliferation assay
Cell growth was assessed by MTT [3,(4,5-dimethylthiazol-2-l)2,5-diphenyltetrazoliumbromide] (Sigma) dye conversion at 570 nm following the manufacturer's instructions. Briefly, cells were seeded 5 x 103 per well in a 96-well flat bottom plate. Cells were allowed to grow for 24 h, then placed in serum-starved conditions for 18 h. Cells were then treated for 96 h with indicated concentrations of LY294002, rapamycin and PD98059 (all from Sigma) in the presence of 10% FBS. 20 µl of MTT (5 mg/ml in PBS) was then added to each well. After 3 h incubation at 37°C, cells were lysed by the addition of 0.1 N HCl in isopropanol.
Expression vectors and constructs
The expression plasmid for PTEN, CMV-PTEN, has already been described [19]. For the antisense (AS) PTEN plasmid, forward (5'-GGTTCGAACGGCGGCTGCAGCTCCA-3') and reverse (5'-CCAGATCTTTTTCAGTTTATTCAAGTTTATTTTC-3') primers were designed with HindIII and XbaI restriction digest sites, respectively, for ease of cloning, and used to amplify a 1.7 kb fragment of the human PTEN cDNA from the CMV-PTEN plasmid. This cDNA fragment was cloned into the pCDNA3.1() expression vector (Invitrogen, Carlsbad, CA) in the antisense orientation. All constructs were confirmed by dideoxy sequencing.
Generation, selection and analysis of stable transfectants
MCF-7 cell lines were transfected with the antisense PTEN constructs or with an empty plasmid as a control, using FuGene 6 (Boehringer Mannheim, Indianapolis, IN). One day after transfection, cells were placed into the selection medium containing 1.0 mg/ml G418 (Gibco/BRL). Twenty-one days after selection, individual G418-resistant colonies were subcloned. PTEN protein expression was analyzed by western blot using the above described PTEN antibody. Several clones were selected based upon PTEN expression levels. Figures are shown with data from two representative clones of several selected.
Flow cytometry
Flow cytometry analysis was performed as previously described [20], with the following modifications: the propidium iodide was in the presence of PBS with 25 mg/ml RNase A and 0.5% Triton X-100.
Data analysis
Each growth inhibition or flow cytometry experiment was repeated 56 times and the average data is presented. For growth inhibition studies, values were expressed as a percentage of untreated controls from which IC50 concentrations were calculated and mean values compared using non-parametric tests for two or multiple unrelated samples. The same methodology was used to compare the flow cytometry analysis data.
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Results |
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PTEN levels modify the response to treatment with PI3K/Akt pathway inhibitors
Because there are many biological differences between the breast cancer cell lines used in these studies in addition to the differences observed in expression levels of PTEN that could affect their response to signal transduction inhibitors, we genetically manipulated the existing breast cancer cell lines to generate syngenic pairs of cell lines differing only in their PTEN status (Figure 2). In these experiments, the PTEN-positive MCF-7 cell line was transfected with either an empty expression vector (MCF-7 control) or an antisense PTEN to develop MCF-7 cell lines with greatly reduced PTEN expression (MCF-7 AS PTEN clone 3 and MCF-7 AS PTEN clone 9). We used the MCF-7 cell line because these more closely reflect the ER-positive, wild-type p53 status reflected in clinical samples. Several clones were selected based upon inhibition of PTEN expression levels. The data presented in Figures 2, 3 and 4 using AS PTEN clones 3 and 9 are representative of experiments done with several of the clones. In these antisense PTEN cells, Akt became constitutively phosphorylated and displayed heightened response to treatment with 10% FBS. In both cases, total Akt protein was not affected. Downstream of Akt, phosphorylated levels of p70s6k also increased, though not as dramatically as the Akt. These results demonstrated that inhibition of PTEN expression directly affected both the constitutive as well as the inducible phosphorylation status of key downstream targets.
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Genetic manipulation of PTEN expression levels results in altered cell cycle distributions in response to PI3 kinase pathway inhibitors
Since the PTEN/Akt pathway has previously been described as important for cell proliferation [22], we next investigated what proliferative changes would be induced by alteration of PTEN expression levels (Figure 4). Flow cytometry analysis of these cells indicated a higher S-fraction in the AS PTEN 3 MCF-7 cells compared to both the control MCF-7 cells and the AS PTEN 9 cells (54% compared to 48%, P
0.04). While the differences are not dramatic, even small differences over a short period of time in vitro could result in much more pronounced long-term, in vivo differences. In response to exposure to 20 nM rapamycin, 55% of the control MCF-7 cells compared to 63% of the AS PTEN 3 and 67% of the AS PTEN 9 cells were found to be in the G1 + G2 phase. Exposure of the cells to 5 µM LY294002 inhibited the cell cycle inhibition in the antisense PTEN cells (87% and 91% for clones 3 and 9, respectively), with only 67% of the MCF-7 control cells found in G1 and G2 phases at the same concentration.
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Discussion |
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Importantly, from a clinical perspective, our proliferation data, in which our PTEN antisense MCF-7 cell lines were growth inhibited by both LY294002 and rapamycin to a much greater degree than our PTEN-positive control cells, indicate that PTEN-negative breast cancer tumors may be more sensitive to the antiproliferative effects of PI3K inhibition. Similar results have been observed by other groups in different cancer models [13, 14
], as well as in the breast in response to mTOR inhibition [21
], indicating that this is probably a general, tumor type-independent, observation. Furthermore, introduction of a constitutive active Akt into PTEN-negative prostate cancer cells further increased the susceptibility of these cells to CCI-779 [13
]. In addition, treatment of PTEN heterozygous mice with CCI-779 resulted in decreased tumor size and neoplastic proliferation [14
]. Overall, these data indicate that inhibition of the PI3 kinase pathway has the potential to be active in this aggressive tumor type and that this may be a biologically-related, tissue-type independent finding. Rapamycin analog compounds are already in clinical development for the treatment of patients with cancer [18
]. CCI-779 has already completed phase I evaluation in patients with advanced cancer and is currently in a broad phase II developmental phase. The agent has demonstrated significant antitumor activity in patients with various tumor types such as breast cancer, non small cell lung cancer, soft tissue sarcomas and renal cell cancer among others. We have used rapamycin for in vitro studies and CCI-779 for in vivo studies and have obtained similar results with the two compounds [26
], indicating that our findings with rapamycin would translate to results that could be anticipated with CCI-779 or the Novartis compound, RAD001.
The most relevant question based on the data presented in this paper, together with recent published information, is how to translate these observations to the clinic. At least two distinct strategies are possible for the development of clinical trials. First, the use of PTEN mutations or Akt activation as markers for upfront selection of patients. The second strategy will consist of not selecting patients for trial participation but evaluating the presence of PTEN mutations/Akt activation in a retrospective way to determine if a correlation exists between these molecular markers and indices of activity that will provide the rationale to explore in prospective clinical investigations the activity of CCI-779 in patients with Akt-hyperactive/PTEN-defective tumors.
In summary, constitutive signaling through the PI3K/Akt pathway in PTEN-defective tumors results in a strong dependence upon this pathway for growth and survival that can be exploited therapeutically. The data from this study indicate that tumors with PTEN mutations that result in higher Akt phosphorylation are more susceptible to the antiproliferative effects of inhibition of the PI3K/Akt pathway. Collectively, these data indicate that inhibition of PI3K/Akt signaling could be a potential strategy to treat patients with PTEN-negative tumors.
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Acknowledgements |
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Presented at the 92nd Annual Meeting of the American Association for Cancer Research, New Orleans, LA, USA, April 2000.
Received for publication August 21, 2004. Revision received March 11, 2004. Accepted for publication May 17, 2004.
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References |
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