Cancer Research Audrey Hepburn Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reagan-Shaw, S.
Right arrow Articles by Ahmad, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reagan-Shaw, S.
Right arrow Articles by Ahmad, N.
[Cancer Research 66, 1062-1069, January 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

RNA Interference–Mediated Depletion of Phosphoinositide 3-Kinase Activates Forkhead Box Class O Transcription Factors and Induces Cell Cycle Arrest and Apoptosis in Breast Carcinoma Cells

Shannon Reagan-Shaw1 and Nihal Ahmad1,2,3

1 Department of Dermatology; 2 University of Wisconsin Comprehensive Cancer Center; 3 Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin

Requests for reprints: Nihal Ahmad, Department of Dermatology, University of Wisconsin, Medical Science Center 25B, 1300 University Avenue, Madison, WI 53706. Phone: 608-263-5359; Fax: 608-263-5223; E-mail: nahmad{at}wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Breast cancer is one of the most common malignancies affecting women in the Western world and one in seven women is predicted to develop invasive breast cancer in their lifetime. Breast cancer arises following the accumulation of a series of somatic changes often including deregulation of key signal transduction pathways. The phosphoinositide 3-kinase (PI3K) pathway has been shown to be activated in breast cancer and overexpression of PI3K is sufficient to confer a malignant phenotype. Activation of the PI3K pathway serves to repress forkhead box class O (FoxO) transcription factor–mediated growth arrest and apoptosis. In this study, we used small interfering RNA (siRNA) to knockdown PI3K in three breast cancer cell lines representing different stages of cancer development. Transfection of PI3K siRNA in breast cancer cells resulted in a significant decrease in cell viability and induction of apoptosis irrespective of their estrogen receptor {alpha} (ER{alpha}) or ErbB2 status. PI3K depletion also resulted in a significant G1 phase cell cycle arrest in ER{alpha}-positive breast cancer cells. Further, our data showed that PI3K knockdown resulted in a significant activation of FoxO; interestingly, a simultaneous knockdown of FoxO1a rescued the cells from apoptosis. Furthermore, the downstream effects of FoxO activation were found to be inhibition of cyclin-dependent kinase 4, cyclin-dependent kinase 6, and cyclin D1, and accumulation of p27/Kip1. Thus, we suggest that (a) PI3K plays a critical role in breast cancer development and (b) gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K could be developed for the management of breast cancer. (Cancer Res 2006; 66(2): 1062-9)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
In the year 2005, an estimated 211,240 new cases of invasive breast cancer were expected to occur in women in the United States (1). In addition to invasive cancer, 58,940 new cases of in situ breast cancer were also expected to occur in women in the United States (1). Conventional chemotherapy regimens for the treatment of breast cancer have limited efficacy and are associated with significant toxicity (2, 3). It is, therefore, necessary to intensify our efforts to better understand the mechanism of breast cancer development and to develop novel targeted approaches for the management of this disease. An example of a target-specific molecular therapy is trastuzumab, which alters the function of the ErbB2 (encoding Her-2/neu) proto-oncogene overexpressed in a portion of breast cancers and can be safely combined with several chemotherapeutic agents to increase the time to progression, quality of life, and overall survival for some patients (2, 4). Truly, an increasing knowledge about the genetic control of cellular proliferation and the modulation of the signaling pathways that are aberrant in breast cancer has the potential to provide an effective and better approach for its management.

Phosphoinositide 3-kinases (PI3K) represent a family of intracellular signaling proteins that control a variety of important cellular functions, including proliferation, apoptosis, and migration (5). Overexpression of PI3K in cultured nonmalignant human mammary epithelial cells (HMEC) is sufficient to confer a malignant phenotype (6). In addition, PI3K has been found to be constitutively up-regulated in a substantial fraction of human breast cancers (7). Evidence has shown that deregulation of PI3K signaling is a feature of many common cancers, either by loss of the suppressor protein PTEN or by constitutive activation of PI3K isoforms or downstream elements, such as Akt and mTOR (8). The deregulation of PI3K signaling promotes not only cell survival and proliferation but also cytoskeletal deformability and motility, key elements in tumor invasion (8). In addition, the PI3K pathway is implicated in many aspects of angiogenesis, including up-regulation of angiogenic cytokines due to tumor hypoxia or oncogene activation and endothelial cell responses to them (8). A more complete understanding of the role of the PI3K pathway in cancer may lead the way to the development of more potent and selective inhibitors, which should be a useful adjunct to conventional therapies, potentially interfering with tumor progression at several pivotal points, in particular cell survival, invasion, and angiogenesis.

Components of the PI3K pathway present promising targets for therapeutic intervention for several reasons. First, this pathway serves to inhibit many tumor suppressor–like proteins that negatively regulate cell survival, proliferation, and growth (5, 9). Thus, blocking this pathway could, therefore, inhibit the proliferation of tumor cells and sensitize them toward apoptosis. Second, many components in the PI3K pathway are kinases, which are ideal for the development of small molecule inhibitors (10–12). Third, as hyperactivation of the PI3K-Akt pathway is found in a wide range of neoplasms, including breast cancer, drugs inhibiting this pathway are likely to have broad applications for treating many cancers (5, 8).

The PI3K pathway regulates Forkhead box class O (FoxO) family of transcription factors. This family of transcription factors has been shown to be functional in breast cells, and activated PI3K and Akt have been shown to induce FoxO1a-FKHR phosphorylation and nuclear exclusion (13). Studies in mammalian cells have shown that the overproduction of FoxO1a (FKHR), FoxO3a (FKHRL1), and FoxO4 (AFX) induces either cell cycle arrest or apoptosis (14–16). Activation of PI3K controls cell cycle entry by inactivating the FoxO forkhead transcription factors, which have been shown to regulate expression of p27/Kip1 (17), cyclin D1, and cyclin E (18).

Based on these facts, in this study, we investigated the hypothesis that a targeted depletion of PI3K will eliminate breast cancer cells via modulation of FoxO transcription factors and the associated antiproliferative signaling. To evaluate the proposed hypothesis, we used the RNA interference technique using small interfering RNA (siRNA) targeted at the p85 regulatory subunit of PI3K to selectively knockdown PI3K in human breast carcinoma cell lines representing different stages of disease progression. Our data clearly showed that a siRNA-mediated depletion of PI3K resulted in a significant (a) decrease in cell viability; (b) induction of apoptosis; (c) G1 phase arrest; (d) activation of FoxO1a, Foxo3a, and FoxO4; (e) inhibition of cyclin-dependent kinase (cdk) 4, cdk6, and cyclin D1; and (f) up-regulation of p27/Kip1 in human breast carcinoma cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell Culture
Normal HMECs, purchased from Cambrex Bio Science (Walkersville, MD), were grown in mammary epithelial cell growth medium obtained from the vendor. Three human breast carcinoma cell lines differing in estrogen receptor (ER) status as well as ErbB2 expression were used in this study. The cell lines Mcf-7:SW8 (ER{alpha}-positive, normal ErbB2) and Mcf-7:Her18 (ER{alpha}-positive, overexpressed ErbB2) were grown in RPMI 1640 [American Type Culture Collection (ATCC), Manassas, VA] and the cell line Sk-Br-3 (ER{alpha}-negative, overexpressed ErbB2) was grown in McCoy's 5a (ATCC, Manassas, VA) medium. The cells were maintained in 10% fetal bovine serum (FBS) and 1% antibiotics at the standard cell culture conditions (37°C, 5% CO2 in a humidified incubator).

SiRNA-Mediated RNA Interference
The PI3K siRNA SMARTpool (Dharmacon, Lafayette, CO) used in this study contained four pooled siRNA duplexes with "UU" overhangs and a 5' phosphate on the antisense strand. Thus, a mixture of several siRNAs ensures an effective depletion of PI3K gene in the cells. Custom synthesized siRNA for human FoxO1a, corresponding to nucleotides 961 to 979 of the coding region (GAGCGTGCCCTACTTCAAG), was prepared by Dharmacon and was used previously (19). This sequence targets FoxO1a with 100% identity, FoxO3a with 17 of 19 nucleotides, and FoxO4 with 14 of 19 nucleotides of the siRNA sequence. A nonrelated control siRNA pool (Dharmacon) that lacks identity with known gene targets was used as a control for nonsequence-specific effects.

The cells were plated in six-well plates with 3.5 x 105 per well. Twenty-four hours later, the cells were transfected with siRNA using OligofectAMINE reagent (Invitrogen, Carlsbad, CA) as described by the vendor. Briefly, for PI3K knockdown, the cells were treated with 100 nmol/L siRNA (PI3K SMARTpool or nonspecific siRNA pool). For the simultaneous knockdown of PI3K and FoxO, the cells were treated with a combination of PI3K SMARTpool and FoxO siRNA (at 50 or 100 nmol/L concentrations) with appropriate controls (nonspecific siRNA pool or PI3K SMARTpool alone or FoxO siRNA alone). For this purpose, OligofectAMINE reagent was diluted with serum-free medium in two thirds the transfection volume for 10 minutes. Then, the diluted OligofectAMINE was added to the diluted siRNA and incubated at room temperature for 20 minutes. The culture medium was aspirated from the cells and the cells were washed with serum-free medium and the siRNA-OligofectAMINE complex was added dropwise to the cells and incubated at 37°C for 4 hours, at which time one-third volume medium with 30% FBS was added and the cells were incubated further for another 20 hours (for a total incubation of 24 hours). At this time, the cells were ready for collection for further experiments.

Preparation of Protein Lysates
Total protein lysate. Following the transfection of cells with PI3K siRNA, as described above, the medium was aspirated the cells were washed with ice-cold PBS [10 mmol/L (pH 7.4)]. Ice cold radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, and 1% NP40] with freshly added phenylmethylsulfonyl fluoride (PMSF, 1 mmol/L) and 10 µg/mL protease inhibitors (Protease Inhibitor Cocktail Set III, Pierce, Rockford, IL) was added to the plates. The cells were then scraped and the cell suspension was transferred into a microfuge tube on ice for 15 minutes with occasional vortexing, ensuring a complete cell lysis. The cell suspension was cleared by centrifugation at 14,000 x g for 15 minutes at 4°C and the supernatant (total cell lysate) was either used immediately or stored at –70°C.

Nuclear and cytoplasmic protein lysates. Following the transfection of cells with PI3K siRNA, as described above, the medium was aspirated the cells were washed with ice-cold PBS. Ice-cold lysis buffer [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, and 1 mmol/L DTT] with freshly added PMSF (1 mmol/L) and 10 µg/mL protease inhibitors (Protease Inhibitor Cocktail Set III, Pierce) was added to the cells on ice for 15 minutes with occasional vortexing, ensuring a complete cell lysis. The cells were cleared by centrifugation at 14,000 x g for 2 minutes at 4°C and the supernatant (cytoplasmic protein lysate) was stored at –70°C. The cell pellet was resuspended in ice-cold Nuclear Extraction buffer [20 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, and 1 mmol/L DTT] with freshly added PMSF (2 mmol/L) and 10 µg/mL protease inhibitors (Protease Inhibitor Cocktail Set III, Pierce) and incubated on ice for 30 minutes with occasional vortexing. The cells were cleared by centrifugation at 14,000 x g for 10 minutes at 4°C and the supernatant (nuclear protein lysate) was either used immediately or stored at –70°C.

Western Blot Analysis
The protein concentration was determined using the BCA Protein Assay (Bio-Rad Laboratories, Hercules, CA) as per the protocol of the manufacturer. For immunoblot analysis, 30 µg protein were subjected to SDS-PAGE (using 10-15% Tris-HCl gel). The protein was transferred onto a nitrocellulose membrane and blocked with TBS-Tween (0.1%) plus 5% dry milk. The membrane was probed with an appropriate primary antibody followed by a secondary horseradish peroxidase–conjugated antibody. The membrane was detected by freshly prepared chemiluminescent solution [100 mmol/L Tris-HCl (pH 8.5), 0.018% H2O2 v/v, 1.25 mmol/L luminol, and 225 nmol/L coumaric acid]. The following antibodies were used: PI3K p85, Akt1/PKB{alpha}, phospho-Akt1/PKB{alpha} (Ser473), FoxO3a (FKHRL1), and phospho-FoxO3a (FKHRL1, Ser253), purchased from Upstate Biotechnology (Waltham, MA); cdk4, cdk6, and p27/Kip1, purchased from Biosource International (Camarillo, CA); FoxO1a (FKHR), phospho-FoxO1a (FKHR, Ser256), FoxO4 (AFX), phospho-FoxO4 (AFX, Ser193), and cyclin D1, purchased from Cell Signaling Technology (Beverly, MA); 14-3-3, purchased from BD PharMingen (San Diego, CA); TATA binding protein, purchased from Novus Biologicals (Littleton, CO) and ß-actin, purchased from Sigma Chemical Company (St. Louis, MO). The quantification of protein was done by a digital analyses of protein bands (TIFF images) using UN-SCAN-IT software (Silk Scientific, Orem, UT).

Apoptosis and Cell Cycle Analysis by Flow Cytometry
The extent of apoptosis and cell cycle distribution was assessed with the APO-BrdUrd terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) Apoptosis Assay kit (Molecular Probes, Eugene, OR) as per the protocol of the manufacturer. Briefly, at 24 hours posttransfection, the culture medium was collected. The cells were gently trypsinized and added to the culture medium and pelleted by centrifugation. The pellet was washed with PBS, counted, and the cells (1 x 106) were fixed overnight in ethanol (90%). The cells were washed and labeled with UTP-BrdUrd overnight, washed again with PBS, and incubated with Alexa 488 Anti-BrdUrd antibody followed by counterstaining with propidium iodide. Cells were analyzed using a FACScan benchtop cytometer (BD Biosciences, San Jose, CA) in the University of Wisconsin Comprehensive Cancer Center Flow Cytometry Facility in the University of Wisconsin. The analyses were done using Cell Quest software (BD Biosciences) for apoptosis and ModFit LT software (Verity Software House; Topsham, ME) for cell cycle analysis. When analyzing the cell population with ModFit LT software, the sub-G0 population (apoptotic and cell debris) was excluded as much as possible to accurately quantitate the cell population in the other phases.

Statistical Analysis
The results are expressed as the mean ± SE. Statistical analyses of the data between PI3K-depleted cells and untreated cells were done by Student's t test. P <0.01 was considered statistically significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
This study was an effort to investigate the hypothesis that targeted depletion of PI3K will eliminate breast cancer cells via modulation of FoxO transcription factors and the associated antiproliferative signaling. For this purpose, we used a unique set of three human breast carcinoma cell lines (see details in Materials and Methods) differing in ER{alpha} and ErbB2 (Her-2/neu) status, which are classifiers for tumors, and are accepted as accurate representations of cancer cells in vivo (20). The ER remains arguably the most important growth factor receptor identified for breast cancer and adjuvant hormonal therapies, whether by ovarian ablation or with tamoxifen, and ER status has a bigger effect on recurrence and survival than any other treatment (21–23). On the other hand, ErbB2 is overexpressed in 20% to 25% of breast cancers and is associated with a poor prognosis (24). Further, studies have suggested considerable cross-talk between the ErbB2 and ER{alpha} signaling, which provides the rationale for combining therapies that target ER{alpha} and ErbB2 or their downstream mediators (25–27). Studies have shown that ErbB2 overexpression acts as a trigger for the activation of the PI3K/Akt pathway in many cell types (28–30). Breast cancer cells with high ErbB2 levels show constitutively high Akt activity that has been suggested to increase stress-induced apoptosis resistance (31–33). Based on these studies, it is believed that PI3K could be a useful therapeutic target for the management of breast cancer; however, selective inhibition of PI3K in carcinoma cells has been problematic.

To study the effect of a targeted knockdown/depletion of PI3K in human breast cancer cells, we used RNA interference technique using siRNA. In the recent past, the PI3K inhibitors LY294002 and wortmannin, which target the catalytic site of p110 in PI3K, have been crucial in deciphering the roles of PI3Ks in cellular processes (34, 35). However, because the PI3K catalytic domain is highly conserved among PI3K family members, neither compound discriminates among the various forms of PI3K (such as the class-1b PI3K downstream of G protein–coupled receptors) and thus affects many cellular processes (36). An alternative and potentially more selective approach is to block the phosphotyrosine binding of the p85 SH2 domains, hence preventing the recruitment and activation of class-1a PI3K by growth factor receptor tyrosine kinases. In our experiments, for an efficient depletion of PI3K, we used a siRNA SMARTpool that is a mixture of four siRNAs targeted at different areas of the coding region in the p85 regulatory subunit of PI3K.

As shown by Western blot analysis, PI3K was abundantly present in all the three breast carcinoma cells used in our study and was barely detectable in HMEC (Fig. 1A), and siRNA-mediated targeted depletion of PI3K resulted in a significant inhibition (64-75%) of PI3K protein levels in all breast cancer cell lines (Fig. 1B) with no apparent effect in HMEC (data not shown). As the first step of our study, we assessed the effect of siRNA-mediated depletion of PI3K on the viability of the cells. As shown by the trypan blue exclusion analysis, our data clearly showed that depletion of PI3K resulted in a significant decrease in cell viability (40-54%) in all the three breast carcinoma cell lines irrespective of their association with either ER{alpha} or ErbB2 (Fig. 1C). Interestingly, treatment with PI3K siRNA did not decrease the viability of normal HMEC and transfection with nonsense siRNA did not affect the viability of any cells (data not shown).


Figure 1
View larger version (48K):
[in this window]
[in a new window]
  		Figure 1. Targeted depletion of PI3K by siRNA results in significant reduction in the viability and increase in apoptosis of breast carcinoma cells. Following transfection of cells (HMEC, Mcf-7:SW8, Mcf-7:Her18, and Sk-Br-3) with PI3K siRNA, the PI3K protein levels were detected by Western blot analysis (A) and quantitated by densitometric analysis of protein bands (B). Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. The effect of PI3K depletion on cell viability (C) was measured using trypan blue exclusion analysis. Cell viability data is expressed as the percentage viable cells of the total number of cells. The extent of apoptosis was assessed with the APO-BrdUrd TUNEL assay kit. The fragmentation of DNA in apoptotic cells is measured by BrdUrd incorporation, which is visualized by conjugation to an Alexa Fluor 488 dye–labeled anti-BrdUrd antibody. BrdUrd incorporation was analyzed with a flow cytometer (D) followed by a computational analysis (E) of cells staining positive for BrdUrd. Columns, mean of three experiments; bars, SE. *, P < 0.01. From a representative experiment repeated thrice with similar results. Details of the experiments are given in Materials and Methods.

 
Because studies have suggested that (a) cancer cells frequently recruit the PI3K/Akt pathway to evade cell death and (b) PI3K inhibition may be associated with an induction of apoptosis in cancer cells (35, 37, 38), we determined whether the observed PI3K depletion–caused loss of cell viability was mediated via the apoptotic death of breast cancer cells. For this purpose, we used a TUNEL assay kit that used BrdUrd incorporation to measure apoptosis by flow cytometry. This assay is based on the principle that when DNA strands are cleaved (apoptosis), a large number of 3'-hydroxyl ends are exposed, which are detected using an Alexa Fluor 488 dye–labeled anti-BrdUrd monoclonal antibody. This kit also uses propidium iodide staining for determining total cellular DNA content. Our data showed that the targeted depletion of PI3K resulted in a significant induction of apoptosis (50-84% in transfected cells versus 1-5% in controls) irrespective of the ER{alpha} or ErbB2 association of the cells (Fig. 1D and E). On the other hand, the transfection of HMEC with PI3K siRNA did not induce apoptosis. Also, the transfection of breast cancer cells or HMEC with nonsense siRNA had no effect on apoptosis (data not shown).

Because PI3K pathway is intimately associated with the regulation of cell cycle, we next determined the effect of PI3K depletion on cell cycle distribution using the same kit. As shown in Fig. 2, the depletion of PI3K resulted in a significant increase in the accumulation of ER{alpha}-positive breast carcinoma cells (Mcf-7:SW8 and Mcf-7:Her18) in G1 phase of the cell cycle; however, the increase in G1 cell population was not significant in ER{alpha}-negative Sk-Br-3 cells. The observed increase in G1 cell population in the breast carcinoma cells was accompanied by a reduction of cells in the S phase (Fig. 2A and C). Our results are consistent with prior studies where the treatment of cells with PI3K inhibitor LY294002 has been shown to induce a G1 arrest and reduction in the S-phase fraction of cells in breast carcinoma BT-474 (39) and osteosarcoma Saos-2 (38) cells.


Figure 2
View larger version (46K):
[in this window]
[in a new window]
  		Figure 2. PI3K depletion results in a significant G1 arrest in breast carcinoma cells. Following transfection of cells (Mcf-7:SW8, Mcf-7:Her18, and Sk-Br-3) with PI3K siRNA, the cell cycle distribution was assessed using the APO-BrdUrd TUNEL assay kit. The FAC profiles (A), indicating the positions of G0-G1, S, and G2-M, are shown along with computational analysis of the cell population in G0-G1 phase (B) and the cells in each phase (C). Columns, mean of three experiments; bars, SE. *, P < 0.01. A, representative experiment repeated thrice with similar results. Details of the experiments are given in Materials and Methods.

 
It is known that following growth factor receptor stimulation, PI3K mediates an increase in 3-poly-phosphoinositides, which in turn activate downstream effectors, such as Akt-PKB (40, 41). We, therefore, evaluated the effect of PI3K depletion on the activation of Akt. As shown by Western blot analysis, PI3K depletion resulted in significant decrease in the protein levels of phosphorylated Akt1/PKB{alpha} in all the three breast carcinoma cell lines studied (Fig. 3). The PI3K depletion also resulted in a significant reduction in total Akt1/PKB{alpha} protein levels in Mcf-7:SW8 cells (Fig. 3). However, the decreases in total Akt1/PKB{alpha} protein levels in ErbB2-overexpressing cell lines was not statistically significant, although a decreasing trend was consistently observed in repeat experiments. This may be associated with the fact that breast cancer cells with high ErbB2 levels (Mcf-7:Her18 and Sk-Br-3) possess high levels of constitutive Akt (31–33).


Figure 3
View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. PI3K depletion results in an inhibition of Akt activation in breast carcinoma cells. Following transfection of cells (Mcf-7:SW8, Mcf-7:Her18, and Sk-Br-3) with PI3K siRNA, the protein levels of Akt1/PKB{alpha} and phospho-Akt1/PKB{alpha} were assessed by Western blot analysis (A) and quantitated by densitometric analysis (B) of protein bands. Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. Representative experiment repeated thrice with similar results. Columns, mean relative density normalized to ß-actin from three experiments; bars, SE. *, P < 0.01. Details of the experiments are given in Materials and Methods.

 
Studies have suggested that PI3K-mediated Akt activation in mammals negatively regulates the FoxO subfamily of forkhead transcription factors (15, 18, 42, 43) and the direct phosphorylation by Akt inhibits transcriptional activation of FoxO factors, causing their displacement from the nucleus into the cytoplasm (44, 45). Studies in mammalian cells have shown that the overproduction of FoxO1a (FKHR), FoxO3a (FKHRL1), and FoxO4 (AFX) induces either cell cycle arrest or apoptosis (14–16) via transcriptional modulations in a variety of genes, including p27/Kip1 (17) and cyclin D1 (18). Jackson et al. (13) have shown the phosphorylation and nuclear exclusion of the FoxO1a-FKHR following epidermal growth factor treatment in human breast cancer cells by a PI3K-dependent mechanism. In the present study, we found that targeted siRNA depletion of PI3K in breast carcinoma cells resulted in a significant increase in nuclear FoxO1a, FoxO3a, and FoxO4 proteins in ER{alpha}-positive human breast carcinoma Mcf-7:SW8 and Mcf-7:Her18 cells, but not in ER{alpha}-negative Sk-Br-3 cells (Fig. 4A and B). Our data also showed that PI3K depletion resulted in a significant decrease in the phosphorylated forms of FoxO1a, FoxO3a, and FoxO4 in the cytosol of ER{alpha}-positive Mcf-7:SW8 and Mcf-7:Her18 cells. However, the levels of the phosphorylated FoxO proteins were not affected by PI3K depletion in the ER{alpha}-negative SK-Br-3 cells (Fig. 4A and C).


Figure 4
View larger version (57K):
[in this window]
[in a new window]
  		Figure 4. PI3K depletion results in an accumulation of nuclear FoxO proteins with a decrease in cytoplasmic phosphorylated FoxO proteins in ER{alpha}-positive breast carcinoma cells. Following transfection of cells (Mcf-7:SW8, Mcf-7:Her18, and Sk-Br-3) with PI3K siRNA, the protein levels in nuclear protein lysates of FoxO1a, FoxO3a, and FoxO4 were assessed by Western blot analysis (A) and quantitated by densitometric analysis of protein bands (B). Equal loading was confirmed by stripping the blot and reprobing it for TATA binding protein (TBP). The protein levels in cytoplasmic protein lysates of phospho-FoxO1a, phospho-FoxO3a, phospho-FoxO4, and 14-3-3 were assessed by Western blot analysis (A) and quantitated by densitometric analysis of protein bands (C). Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. Representative experiment repeated thrice with similar results. Columns, mean relative density normalized to the loading control from three experiments; bars, SE. *, P < 0.01. Details of the experiments are given in Materials and Methods.

 
The 14-3-3 protein family represents an important group among proteins recognizing phosphorylated targets and 14-3-3 protein has been shown to bind to FoxO factors in a phosphorylation-dependent manner (46). Thus, 14-3-3 protein masks the nuclear localization signal on the FoxO proteins, thereby sequestering the phosphorylated FoxO to the cytoplasm of the cell (46). Therefore, we also determined the effect of PI3K depletion on the levels of 14-3-3 protein. The cytoplasmic protein levels of 14-3-3 was found to be significantly decreased in ER{alpha}-positive Mcf-7:SW8 and Mcf-7:Her18 cells, but remained unaffected in ER{alpha}-negative SK-Br-3 cells (Fig. 4A and C). Taken together, the observed increase in nuclear FoxO proteins with a concurrent decrease in phosphorylated FoxO and 14-3-3 proteins indicates that depletion of PI3K causes an activation of FoxO transcription factors. However, it is an interesting observation that the ER{alpha} as well as ErbB2 associations of breast cells may result in a differential modulation of FoxO transcription factors.

We next designed an experiment to establish a cause-and-effect association between FoxO activation and induction of apoptosis/cell cycle arrest in breast cancer cells. For this purpose, we did experiments to study the effect of a simultaneous knockdown of FoxO1a (with PI3K) on apoptosis and cell cycle distribution in breast cancer cells. We found that siRNA-mediated depletion of PI3K and FoxO resulted in an inhibition of PI3K and phospho-FoxO1a protein levels, respectively, in all breast cancer cell lines (data not shown). Interestingly, a simultaneous knockdown with FoxO1a was found to reverse PI3K depletion–mediated accumulation of ER{alpha}-positive breast carcinoma cells (Mcf-7:SW8 and Mcf-7:Her18) in G1 phase of the cell cycle, but did not have a significant effect in ER{alpha}-negative Sk-Br-3 cells (Fig. 5A). Further, the simultaneous depletion of PI3K and FoxO significantly (partially) rescued all three cancer cell lines from apoptosis, irrespective of the ER{alpha} or ErbB2 association of the cells (Fig. 5B). It is important to mention here that the siRNA sequence used in our study targets FoxO1a with 100% identity, FoxO3a with 17 of 19 nucleotides, and FoxO4 with 14 of 19 nucleotides of the siRNA sequence. Further, we observed some toxicity in cells treated with the high concentrations of siRNA—200 nmol/L nonsense siRNA or 100 nmol/L PI3K plus 100 nmol/L FoxO siRNA (data not shown). Thus, our data clearly establish a causal connection between FoxO activation and induction of apoptosis and cell cycle arrest in human breast cancer cells.


Figure 5
View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. Simultaneous depletion of PI3K and FoxO by siRNA results in a rescue from apoptosis and G1 cell cycle arrest in breast carcinoma cells. Following transfection of cells (Mcf-7:SW8, Mcf-7:Her18, and Sk-Br-3) with PI3K and/or FoxO siRNA, the extent of apoptosis and the cell cycle distribution was assessed with the APO-BrdUrd TUNEL assay kit. A, the FAC profiles indicating the positions of G0-G1, S, and G2-M were used for a computational analysis of the cell population in G0/G1 phase. B, BrdUrd incorporation was analyzed with a flow cytometer followed by a computational analysis of cells staining positive for BrdUrd. Columns, mean of three experiments; bars, SE. *, P < 0.01. Representative experiment repeated thrice with similar results. Details of the experiments are given in Materials and Methods.

 
Studies have shown that breast cancer cells with high ErbB2 levels show constitutive Akt activation, which has been suggested to increase stress-induced apoptosis resistance (31–33). In addition, a correlation was found between expression of FoxO3a in the cytoplasm and phosphorylated Akt in 30% of human primary breast tumors, suggesting an Akt-independent mechanism of regulating FoxO3a localization (47). I{kappa}B kinase has been shown to physically interact with, phosphorylate, and inhibit FoxO3a independent of Akt and causes proteolysis of FoxO3a via the ubiquitin-proteasome–dependent pathway (47). Thus, it is possible that in the ER{alpha}-negative SK-Br-3 cells, which overexpress ErbB2, the forkhead transcription factors are regulated by I{kappa}B kinase. Further investigation is required to examine this hypothesis.

Progression through the G1-S transition is mainly regulated through the sequential activation of cdk4, cdk6, and later cdk2 by D-type cyclins in mid-to-late G1 (48). The Cip/Kip family of cyclin kinase inhibitors bind all cdks and may prevent their activation or directly inhibit their kinase activity (48). Treatment with PI3K inhibitor LY294002 in breast carcinoma BT-474 cells has been shown to markedly reduce cyclin D1 levels and down-regulate cdk4 and increase p27 levels (39). Therefore, we evaluated the effect of targeted depletion of PI3K on these important cell cycle regulatory molecules in human breast carcinoma cells representing different stages of disease progression. Targeted depletion of PI3K resulted in a significant depletion of cdk4, cdk6, and cyclin D1 protein levels in all three breast carcinoma cell lines (Fig. 6A and B). PI3K depletion also resulted in a significant accumulation of p27/Kip1 proteins in ER{alpha}-positive Mcf-7:SW8 and Mcf-7:Her18 cell as shown by Western blot analysis, but not in ER{alpha}-negative Sk-Br-3 cells (Fig. 6A and C). These events are consistent with observed accumulation of cells in G1 phase of the cell cycle (Fig. 2). The down-modulation of cyclin D1 and accumulation of p27/Kip1 proteins in ER{alpha}-positive cell lines is also consistent with the observed accumulation of FoxO proteins (Fig. 4).


Figure 6
View larger version (54K):
[in this window]
[in a new window]
  		Figure 6. PI3K depletion disrupts mitotic cell cycle progression in breast carcinoma cells. Following transfection of cells (Mcf-7:SW8, Mcf-7:Her18, and Sk-Br-3) with PI3K siRNA, the protein levels of cdk4, cdk6, and cyclin D1 were assessed by Western blot analysis (A) and quantitated by densitometric analysis of protein bands (B). The protein levels of cyclin kinase inhibitors p27-Kip1 were assessed by Western blot analysis (A) and quantitated by densitometric analysis of protein bands (C). Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. Representative experiment repeated thrice with similar results. Columns, mean relative density normalized to ß-actin from three experiments; bars, SE. *, P < 0.01. Details of the experiments are given in Materials and Methods.

 
Taken together, our data suggested that a targeted silencing of p85 subunit of PI3K in human breast cancer cells causes an activation of FoxO transcription factors, which, in turn, activates transcription of target genes such as those involved in cell cycle regulation and apoptosis (e.g., cyclin kinase inhibitors-cyclin-cdk machinery). This series of events ultimately lead to a G1 phase cell cycle arrest and apoptosis of human breast carcinoma cells. Our study also suggested that PI3K inhibition–mediated cell cycle arrest and apoptosis does not depend on the endogenous levels of ER{alpha} or ErbB2 in cancer cells. However, because PI3K depletion caused an activation of all three members of the forkhead family of transcription factors in ER{alpha}-positive breast carcinoma cells irrespective of their ErbB2 expression levels, it is conceivable that gene therapeutic approaches or pharmacologic inhibitors aimed at the p85 regulatory subunit of PI3K may be developed for the management of breast cancer.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank undergraduate students Jorien Breur and Iulia Dorneanu for their technical assistance and Dr. Janet Mertz (Department of Oncology, University of Wisconsin, Madison, WI) for the Mcf-7:SW8 and Mcf-7:Her18 cell lines.

Received 3/25/05. Revised 9/ 1/05. Accepted 10/28/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Jemal A, Murray T, Ward E, et al. Cancer statistics. CA Cancer J Clin 2005;55:10–30.[Abstract/Free Full Text]
  2. Biganzoli L, Minisini A, Aapro M, Di Leo A. Chemotherapy for metastatic breast cancer. Curr Opin Obstet Gynecol 2004;16:37–41.[CrossRef][Medline]
  3. Foggi P, Amodio A. Molecular therapy of breast carcinoma in the advanced phase. Tumori 2003;89:189–91.[Medline]
  4. Spigel DR, Burstein HJ. Trastuzumab regimens for HER2-overexpressing metastatic breast cancer. Clin Breast Cancer 2003;4:329–37.[Medline]
  5. Fresno Vara JA, Casado E, de Castro J, et al. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 2004;30:193–204.[CrossRef][Medline]
  6. Zhao JJ, Gjoerup OV, Subramanian RR, et al. Human mammary epithelial cell transformation through the activation of phosphatidylinositol 3-kinase. Cancer Cell 2003;3:483–95.[CrossRef][Medline]
  7. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489–501.[CrossRef][Medline]
  8. Brader S, Eccles SA. Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion and angiogenesis. Tumori 2004;90:2–8.[Medline]
  9. Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell 2003;4:257–62.[CrossRef][Medline]
  10. Chang F, Lee JT, Navolanic PM, et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia 2003;17:590–603.[CrossRef][Medline]
  11. Harris TK. PDK1 and PKB/Akt: ideal targets for development of new strategies to structure-based drug design. IUBMB Life 2003;55:117–26.[Medline]
  12. Mills GB, Kohn E, Lu Y, et al. Linking molecular diagnostics to molecular therapeutics: targeting the PI3K pathway in breast cancer. Semin Oncol 2003;30:93–104.[Medline]
  13. Jackson JG, Kreisberg JI, Koterba AP, Yee D, Brattain MG. Phosphorylation and nuclear exclusion of the forkhead transcription factor FKHR after epidermal growth factor treatment in human breast cancer cells. Oncogene 2000;19:4574–81.[CrossRef][Medline]
  14. Alvarez B, Martinez A, Burgering BM, Carrera AC. Forkhead transcription factors contribute to execution of the mitotic programme in mammals. Nature 2001;413:744–7.[CrossRef][Medline]
  15. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999;19:857–68.
  16. Nakamura N, Ramaswamy S, Vazquez F, et al. Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Biol 2000;20:8969–82.[Abstract/Free Full Text]
  17. Medema RH, Kops GJ, Bos JL, Burgering BM. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 2000;404:782–7.[CrossRef][Medline]
  18. Rena G, Guo S, Cichy SC, Unterman TG, Cohen P. Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B. J Biol Chem 1999;274:17179–83.[Abstract/Free Full Text]
  19. Potente M, Fisslthaler B, Busse R, Fleming I. 11,12-Epoxyeicosatrienoic acid-induced inhibition of FOXO factors promotes endothelial proliferation by down-regulating p27Kip1. J Biol Chem 2003;278:29619–25.[Abstract/Free Full Text]
  20. Lacroix M, Leclercq G. Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res Treat 2004;83:249–89.[CrossRef][Medline]
  21. Mamounas EP. Present state and future prospects: a review of cooperative groups' adjuvant and neoadjuvant trials in breast cancer. Clin Breast Cancer 2001;2 Suppl:S20–30.
  22. Pritchard KI. The best use of adjuvant endocrine treatments. Breast 2003;12:497–508.[CrossRef][Medline]
  23. Tamaki Y, Miyoshi Y, Noguchi S. Adjuvant hormonal therapy. Breast Cancer 2002;9:185–9.[Medline]
  24. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–12.[Abstract/Free Full Text]
  25. Kurokawa H, Arteaga CL. ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanisms. Clin Cancer Res 2003;9:511–5S.
  26. Schiff R, Massarweh S, Shou J, Osborne CK. Breast cancer endocrine resistance: how growth factor signaling and estrogen receptor coregulators modulate response. Clin Cancer Res 2003;9:447S–54S.[Abstract/Free Full Text]
  27. Sledge GW, Jr., Miller KD. Exploiting the hallmarks of cancer: the future conquest of breast cancer. Eur J Cancer 2003;39:1668–75.[CrossRef][Medline]
  28. Contessa JN, Hampton J, Lammering G, et al. Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells. Oncogene 2002;21:4032–41.[CrossRef][Medline]
  29. Hii CS, Moghadammi N, Dunbar A, Ferrante A. Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signaling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family. J Biol Chem 2001;20:27246–55.
  30. Motoyama AB, Hynes NE, Lane HA. The efficacy of ErbB receptor-targeted anticancer therapeutics is influenced by the availability of epidermal growth factor-related peptides. Cancer Res 2002;62:3151–8.[Abstract/Free Full Text]
  31. Bacus SS, Altomare DA, Lyass L, et al. AKT2 is frequently upregulated in HER-2/neu-positive breast cancers and may contribute to tumor aggressiveness by enhancing cell survival. Oncogene 2002;21:3532–40.[CrossRef][Medline]
  32. Muthuswamy SK, Li D, Lelievre S, Bissell MJ, Brugge JS. ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Nat Cell Biol 2001;3:785–92.[CrossRef][Medline]
  33. Zhou BP, Hu MC, Miller SA, et al. HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-{kappa}B pathway. J Biol Chem 2000;275:8027–31.[Abstract/Free Full Text]
  34. Berrie CP. Phosphoinositide 3-kinase inhibition in cancer treatment. Expert Opin Investig Drugs 2001;10:1085–98.[CrossRef][Medline]
  35. Bondar VM, Sweeney-Gotsch B, Andreeff M, Mills GB, McConkey DJ. Inhibition of the phosphatidylinositol 3'-kinase-AKT pathway induces apoptosis in pancreatic carcinoma cells in vitro and in vivo. Mol Cancer Ther 2002;1:989–97.
  36. Meier TI, Cook JA, Thomas JE, et al. Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms. Protein Expr Purif 2004;35:218–24.[CrossRef][Medline]
  37. Kim S, Kang J, Qiao J, et al. Phosphatidylinositol 3-kinase inhibition down-regulates survivin and facilitates TRAIL-mediated apoptosis in neuroblastomas. J Pediatr Surg 2004;39:516–21.[CrossRef][Medline]
  38. Ren S, Gao C, Zhang L, Koike K, Tsuchida N. PI3K inhibitors changed the p53-induced response of Saos-2 cells from growth arrest to apoptosis. Biochem Biophys Res Commun 2003;308:120–5.[CrossRef][Medline]
  39. Lenferink AE, Busse D, Flanagan WM, Yakes FM, Arteaga CL. ErbB2/neu kinase modulates cellular p27(Kip1) and cyclin D1 through multiple signaling pathways. Cancer Res 2001;61:6583–91.[Abstract/Free Full Text]
  40. Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem 1998;67:481–507.[CrossRef][Medline]
  41. Vanhaesebroeck B, Waterfield MD. Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 1999;253:239–54.[CrossRef][Medline]
  42. Biggs WH, III, Meisenhelder J, Hunter T, Cavenee WK, Arden KC. Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci U S A 1999;96:7421–6.[Abstract/Free Full Text]
  43. Kops GJ, de Ruiter ND, Vries-Smits AM, et al. Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 1999;398:630–4.[CrossRef][Medline]
  44. Birkenkamp KU, Coffer PJ. Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors. Biochem Soc Trans 2003;31:292–7.[Medline]
  45. Burgering BM, Medema RH. Decisions on life and death: FOXO forkhead transcription factors are in command when PKB/Akt is off duty. J Leukoc Biol 2003;73:689–701.[Abstract/Free Full Text]
  46. Brunet A, Kanai F, Stehn J, et al. 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. J Cell Biol 2002;156:817–28.[Abstract/Free Full Text]
  47. Hu MC, Lee DF, Xia W, et al. I{kappa}B kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 2004;117:225–37.[CrossRef][Medline]
  48. Reed SI. Control of the G1-S transition. Cancer Surv 1997;29:7–23.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
R. J. Crowder, C. Phommaly, Y. Tao, J. Hoog, J. Luo, C. M. Perou, J. S. Parker, M. A. Miller, D. G. Huntsman, L. Lin, et al.
PIK3CA and PIK3CB Inhibition Produce Synthetic Lethality when Combined with Estrogen Deprivation in Estrogen Receptor-Positive Breast Cancer
Cancer Res., May 1, 2009; 69(9): 3955 - 3962.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reagan-Shaw, S.
Right arrow Articles by Ahmad, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reagan-Shaw, S.
Right arrow Articles by Ahmad, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online