© 2007 European Society for Medical Oncology
breast cancer |
Akt-induced endocrine therapy resistance is reversed by inhibition of mTOR signaling
1 Department of Medical Oncology
2 Department of Orthodontics
3 Department of Obstetrics and Gynecology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
* Correspondence to: Prof. L. A. deGraffenried, Division of Medical Oncology, MSC 7884, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. Tel: +1-210-567-4739; Fax: +1-210-567-6687; E-mail: degraffenri{at}uthscsa.edu
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Abstract |
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Background: Resistance to endocrine therapy is a major impediment in breast cancer therapeutics. The Phosphatidylinositol-3-OH kinase (PI3K)/Protein kinase B (Akt/PKB) kinase signaling pathway has been implicated in altering breast cancer response to multiple therapies. How Akt modulates response is an area of significant clinical relevance.
Methods: We have used an in vitro model to discern the effects of robust Akt activity on breast cancer cellular response to endocrine therapies.
Results: High levels of Akt activity confer resistance to the aromatase inhibitor Letrozole (Let) and the selective estrogen receptor (ER) down-regulator Fulvestrant (ICI). Akt-induced resistance is not due to failure of these endocrine agents to inhibit estrogen receptor 
activity. Instead, resistance is characterized by altered cell cycle and apoptotic response. Cotreatment with low concentrations of the mTOR inhibitor RAD-001 and either Let or ICI restores response of the resistant cells to levels observed in the responsive cells treated with either Let or ICI as a single agent.
Conclusions: Our preliminary findings in experiments with RAD-001 indicate that cotreatment with mTOR inhibitors and either Let or ICI reverses the Akt-mediated resistance and restores responsiveness to antiestrogens. Concurrent ER and mTOR inhibition is therefore an effective strategy to overcome growth factor-induced resistance and bears significant implications for optimal clinical development of these agents in breast cancer treatment.
Key words: Akt, breast cancer, endocrine therapy, mTOR
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introduction |
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De novo and acquired resistance to endocrine therapy are major clinical problems in the treatment of breast cancer. Several studies have now indicated that one significant mechanism by which breast cancer cells are resistant to the inhibitory effects of endocrine agents is through aberrant activation of growth factor signaling pathways. In particular, the Protein kinase B (Akt/PKB) kinase, a downstream target of PI3 kinase signaling, appears to play a critical role in drug resistance (reviewed in [1]). Both preclinical [2, 3] as well as clinical studies [4–7] have demonstrated a clear association between Akt signaling and tamoxifen (Tam) resistance. Previous studies from our laboratory [3, 8, 9], as well as from other laboratories [10], have shown that expression of a constitutively active Akt in MCF-7 cells renders these normally hormone-dependent breast cancer cells more resistant to the inhibitory effects of Tam. The specific mechanisms by which Akt activity confers resistance is an area of intense investigation since targeting this mechanism may be exploited for the treatment of resistant breast cancer.
While most studies have investigated the effects of Akt signaling on tamoxifen (Tam) resistance, which is still the most commonly used hormone therapy agent in the adjuvant setting, recent adjuvant trials have established the value of other endocrine agents, such as third-generation aromatase inhibitors in early-stage breast cancer [11]. Few studies have explored the direct effects of Akt signaling on breast cancer response to endocrine agents other than Tam. In this current study, we investigated the effects of Akt signaling on MCF-7 breast cancer response to the aromatase inhibitor, Letrozole (Let) (Novartis International AG, Basel, Switzerland) and the steroid estrogen receptor degrader (SERD) Fulvestrant (ICI) (AstraZeneca Pharmaceuticals, Wilmington, DE). In addition, previous studies have demonstrated that Akt-induced resistance to chemotherapy agents and Tam can be reversed through cotreatment with inhibitors of the mTOR kinase, such as rapamycin, or rapamycin analogues CCI-779 (Wyeth, Madison, NJ) and RAD-001 (Novartis) [3, 12]. Here, we present the results of our studies in which we evaluated the ability of RAD-001 to restore endocrine therapy sensitivity to refractory breast cancer cells with high Akt activity. We found that in vitro, breast cancer cells refractory to Let and ICI as single agents, when cotreated with low levels of RAD-001, demonstrated a dose-dependent growth inhibition response to these endocrine agents. Interestingly, these results were independent of effects on estrogen receptor (ER) activity, indicating that Akt-induced resistance is mediated, at least in part, through ER-independent mechanisms. Our results demonstrate that the combination of mTOR inhibition and endocrine agents has excellent anticancer activity in preclinical models of Akt-induced resistance and therefore may have clinical utility in treating resistant breast tumors.
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cell lines
The myrAkt1 and control MCF-7 cells lines have been previously described [3]. Briefly, the myrAkt1 MCF-7 cells express a myristoylated, constitutively active Akt1 (myrAkt
4-129) that has been previously described [13], while the control MCF-7 cells have been stably transfected with an empty pcDNA3.1 (+) vector (Invitrogen, Carlsbad, CA). Stable transfectant cell lines were maintained in the same Improved MEM [(IMEM) Invitrogen] treated with 400 mg/l G418.
growth proliferation assay
Cell growth was assessed by 3, (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazoliumbromide (MTT) (Sigma, St. Louis, MO) dye conversion at 570 nM following manufacture's instructions. Briefly, cells were seeded 5 x 103 per well in a 96-well flat bottom plate. Cells were allowed to grow 24 h, then placed in serum-starved (SS) conditions for 18 h. Cells were then treated with increasing concentrations of Tam alone, ICI alone, Let alone, or in combination with 20 nM RAD-001 (Novartis), as indicated, in the presence of 10% fetal bovine serum (FBS) supplemented with 6 ng/ml insulin. After 96 h of continuous treatment, 20 µl of MTT [5 mg/ml in phosphate-buffered saline (PBS)] was added to each well. After 3 h incubation at 37°C, cells were lysed by the addition of 0.1 N HCl in isopropanol. Presented is the combination of three independent experiments.
western blot analysis
Evaluation of the status of downstream targets of mTOR signaling was carried out on lysates from myrAkt1 and control MCF-7 cells treated 24 h with serum-free media, media with 10% FBS, 10% FBS media with 20 nM RAD-001 (Novartis), 10% FBS media with 10–7 M 4-OH Tam, 10% FBS media with 10–9 M ICI, 10% FBS media with 10–7 M Let or a combination of the endocrine agent and RAD-001. Cells were harvested in 1x lysis buffer (50 mm Tris–HCL, pH 7.5, 120 mM NaCl, 1% NP-40, 1 mM EDTA, pH 8.0, 5 mM ethylene glycol tetraacetic acid (EGTA), pH 7.5, 50 mM NaF, 40 mM ß-glycerolphosphate, 100 µM Na orthovanadate, 1 mM Benzamidine, protease inhibitor cocktail). Protein lysates were subjected to immunodetection with antibodies to ER (6F11 monoclonal antibody, Novocastra Ltd., UK), and finally actin (Santa Cruz Biotechnology, Santa Cruz, CA) for a loading control. Tumor lysates were obtained using the 1x lysis buffer described above. Antibodies for total and phosphorylated 4-EBP1 were obtained from Cell Signaling Technology. Signal detection was done using the enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL).
flow cytometry
Detection of the apoptotic effect was determined by flow cytometric analysis of 104 cells using the LysisII program on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). Briefly, 24 h after plating, cells were exposed to vehicle, ICI (10–9 M), Let (10–7 M), Tam (10–7 M), RAD-001 (20 nM), or a combination of the endocrine therapy compound with RAD-001 for 24 h in 10% FBS media. Cells were collected after treatment, washed in PBS, and after cell centrifugation, cell pellets were stained with a propidium iodide (PI) (Sigma) staining solution containing 10 µg/ml PI, 1 µg/ml of ribonuclease and 0.1% (vol by vol) Triton X-100. The cell suspension was incubated in the dark at 4°C for 18 h before analysis. Determination of DNA content was based upon a minimum total of 10 000 acquired events. Each experiment was done three times.
luciferase reporter assays
Estrogen receptor (ER) transcriptional activity was assessed using a luciferase reporter gene driven by a 3x ERE-tk promoter (a kind gift from Dr Arun Roy, UT Health Science Center at San Antonio). Transient transfections were carried out three times in triplicate wells. Cells were seeded in six-well cluster plates (Falcon, Franklin Lakes, NJ) at a density of 2 x 105 cells/well 24 h before treatment. Cells were then SS for 24 h, followed by treatment with fresh serum-free media, IMEM media containing 10% FBS, 10% FBS media with 10–7 M 4-OH Tam, 10% FBS media with 10–9 M ICI, 10% FBS media with 10–7 M Let, 10% FBS media with 20 nM RAD-001 or 10% FBS media with both the endocrine agent and RAD-001. Twenty-four hours after treatment, 3.0 µl of FuGene 6 transfection reagent (Roche, Indianapolis, IN) was used to transfect 1.0 µg of the pGL3-Basic 3x ERE-tk reporter construct. The plasmid pNull-Renilla (Promega, Madison, WI), an expression plasmid for the Renilla luciferase gene void of eukaryotic promoter or enhancer sequences, was cotransfected for transfection normalization. All transfections are reported as activity after normalization for Renilla expression. Forty-eight hours after transfection, luciferase activity was measured using the Dual Luciferase kit from Promega (Promega) as per manufacturer's instructions. Shown is a combination of three independent experiments.
statistics
For luciferase and MTT analysis, a Student' t-test was used.
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cotreatment with the mTOR inhibitor RAD-001 enhances endocrine therapy response in a model of Akt-induced resistance
In previous studies, we have observed that expression of a constitutively active Akt in MCF-7 breast cancer cells (myrAkt1 MCF-7) decreases sensitivity to the growth inhibitory effects of the antiestrogen, Tam [8, 9]. We have obtained similar results in other MCF-7 subcell lines that have high levels of Akt due to either overexpression of HER2 or expression of PTEN antisense (data not shown). To determine the specific effects of Akt signaling on breast cancer cell response to other forms of endocrine therapy, we exposed our myrAkt1 MCF-7 and pcDNA3.1 control MCF-7 cells to increasing concentrations of the SERD Fulvestrant (ICI, 10–12 M–10–8 M) and increasing concentrations of the aromatase inhibitor (Let, 10–9 M–10–6 M) (Figure 1A) in the presence of complete 10% FBS media. As with Tam, we found that the myrAkt1 MCF-7 cells (black bars) were less responsive to both ICI and Let, compared with the control MCF-7 cells (white bars). Where the control cells demonstrated a dose-dependent decrease in growth in response to both ICI and Let, the myrAkt1 cells demonstrated no significant response to ICI until 10–9 M (
25% growth inhibition) and no >20% inhibition in response to Let at any concentration This data indicates that Akt1 signaling confers resistance to multiple forms of endocrine therapy.
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Disruption of Akt signaling using the mTOR inhibitor rapamycin has been shown to be effective at reversing doxorubicin chemoresistance in lymphomas expressing Akt [12], and our group has demonstrated that use of either rapamycin or the rapamycin analogue CCI-779 (Wyeth pharmaceuticals) restores Tam sensitivity in myrAkt1 MCF-7 cells [8]. We therefore tested whether cotreatment with the RAD-001 (Novartis) would reverse Akt1-induced resistance to Tam, ICI and Let. As seen in Figure 1B, we found that as with rapamycin and CCI-779, cotreatment with RAD-001 increased the growth inhibitory effects of Tam in the myrAkt1 MCF-7 (black bars) cells from 23% to 50%. Cotreatment with RAD-001 and ICI increased response from 20% to
60% in the myrAkt1 cells, while increasing the response of the control cells (white bars) from 40% to 60%. Cotreatment of RAD-001 with Let increased the response of the myrAkt1 cells from 10% to 50% and the control cells from 20% to 50%. Contrary to what was anticipated, RAD-001 as a single-agent was more effective in the control cells, inhibiting cell growth by 50% while inhibiting myrAkt1 cell growth by only 25%. These data indicate that at least in the cells expressing high levels of Akt activity, inhibition of mTOR signaling enhances the efficacy of endocrine agents in at least an additive if not synergistic manner.
cotreatment with RAD-001 inhibits the mTOR pathway
In order to determine the mechanisms by which mTOR inhibitors reverse Akt1-induced resistance, we carried out western blot analyses of protein lysates from control and myrAkt1 MCF-7 cells exposed for 24 h to tamoxifen (T), Fulvestrant (I) or Letrozole (L) either as single agents or in combination with 20 nM RAD-001 (+ R) in media containing 10% FBS (Figure 2). Cells grown for 24 h in SS media (SS, lanes 1 and 4) or complete media containing 10% FBS (lanes 2 and 5) were used for control. We found that in agreement with previous reports, Tam actually increased the expression levels of the ER (lanes 7–10), while cotreatment with RAD-001 had little effect on ER levels. In contrast, treatment with RAD-001 significantly inhibited phosphorylation of the mTOR targets, S6 and 4E-BP (lanes 3, 6, 8, 10, 12, 14, 16, 18), indicating that even at low concentrations RAD-001 was able to inhibit mTOR activity. Recent studies indicate that inhibition of mTOR signaling reverses Akt-induced chemotherapy resistance. Intriguingly, we found that cotreatment with RAD-001 increased expression levels of the cell cycle inhibitor, p21 in the myrAkt1 MCF-7 cells (lanes 10, 14, 18), while it did not appear to effect p21 levels in the control cells, indicating a greater sensitivity to mTOR inhibition in cells expressing high levels of Akt activity.
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RAD-001 enhances the apoptotic effect of endocrine therapy
In order to determine whether the results of the MTT assays were due, at least in part, to induction of apoptosis, we carried out flow cytometric analysis (Figure 3) on the myrAkt1 (black bars) and control (white bars) MCF-7 cells that were exposed to drug treatment for 24 h in 10% FBS. While RAD-001 as a single agent at 20 nM (C + R) had little effect on the myrAkt1 cells, it did increase the percentage of control cells in apoptosis. Surprisingly, contrary to our MTT results, the myrAkt1 cells demonstrated an increase in apoptosis when exposed to 10–9 M ICI as a single agent, while the control cells did not. Combination with 20 nM RAD-001 (ICI + R) did not increase the percentage of myrAkt1 cells in apoptosis, but did increase the percentage of control MCF-7 cells in apoptosis from 15% to 27%. Conversely, 100 nM Let had little effect on the myrAkt1 MCF-7 cells as a single agent, but increased the percentage of control MCF-7 cells in apoptosis to
45%. Cotreatment with 20 nM RAD-001 (Let + R) increased the percentage of myrAkt1 apoptotic cells to 22%, while surprisingly decreasing the percentage of apoptotic control cells when compared to Let as a single agent. Tam at 10–7 M almost doubled apoptosis in the control MCF-7 cells while having no effect in the myrAkt1 cells. Cotreatment with 20 nM RAD-001 (Tam + R) increased the percentage of apoptotic control cells from 35% to 40%, and doubled the percentage of apoptotic myrAkt1 cells from 12% to 23%. These data indicate that the cotreatment with RAD-001 and Let increases cellular apoptotic response, especially in a high Akt1 activity background. Presented is the average of two independent experiments. The data from experiments with ICI and RAD 001 are conflicting and remain a topic of ongoing investigation.
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growth inhibition and apoptosis induced by cotreatment with RAD-001 are independent of effects on ER activity
Several studies have indicated that one mechanism by which Akt1 modulates Tam (and perhaps other endocrine) therapy is through hormone-independent activation of the AF-1 domain of the ER itself. In order to determine what role Akt activation of ER had on the previous results, we used an ERE-luciferase assay to measure ER activity in response to drug treatment, either alone or in combination with 20 nM RAD-001 in media containing 10% complete FBS (Figure 4). As we and others have published, high levels of Akt correlate with high ER transcriptional activity. ER activity in the control MCF-7 cells (white bars) grown for 48 h in charcoal-stripped serum (CS) was very low, and was increased six-fold by exposure to complete (estrogen containing) media (10% FBS). ER activity in the myrAkt1 MCF-7 cells (black bars) grown in CS was six-fold higher than in the control cells, and was not increased with estrogen treatment. Exposure to 20 nM RAD-001 (R) in complete media for 48 h decreased ER activity in both the control and myrAkt1 MCF-7 cells by 40% and 20%, respectively. As we have published previously, 10–7 M tamoxifen (T) decreased ER activity in the control cells by 25%, but only 10% in the myrAkt cells. Cotreatment with RAD-001 (T + R) enhanced Tam response in the control cells by 28%, but had little effect in the myrAkt1 cells. Interestingly, we observed no statistical differences in ER activity between the control and myrAkt1 cells in response to treatment with Fulvestrant (I) or Letrozole (L) either as single agents or in combination with RAD-001, indicating that the ER is still responsive to the inhibitory effects of these agents, and that the results obtained in the MTT assays were independent of effects on ER transcriptional activity. These data indicate that Akt is mediating its effects, at least in part, through non-ER-mediated pathways.
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The PKB or Akt pathway is a cell survival pathway that is activated in hormone-refractory breast cancer and appears to play a pivotal role in mediating both primary and acquired endocrine resistance. Notably, several clinical studies have demonstrated a strong correlation between activation of the Akt signaling pathway and worse outcome in breast cancer patients [4, 5, 14, 15]. Previously published results of studies from our laboratory [3, 8] and others [10, 16–18] indicate that high levels of Akt signaling confer resistance to Tam through ER
-dependent as well as independent mechanisms. In the present study, we observed that a high level of Akt signaling confers resistance to other endocrine therapies, including the aromatase inhibitor, Let, and the selective estrogen receptor degrader, ICI. Potentially conflicting results were reported in studies from Angela Brodie's laboratory indicated that the Akt pathway was not critical for development of resistance in cells grown long term in the presence of Let [19] However, more recent studies from the same laboratory have also demonstrated that suppression of the Akt and/or mTOR pathway restores sensitivity to antiestrogen therapy in breast cancer cells grown long term under estrogen-deprived conditions (personal communication and [20]), indicating that the growth conditions used experimentally significantly influence the role of the specific growth factor pathways involved in resistance. Approaches to abrogate Akt-induced endocrine resistance are therefore of high clinical relevance in breast cancer therapy. One potential approach is through targeting the mTOR pathway. Inhibition of mTOR has proven effective at enhancing Tam [3] and chemotherapy response [21] in breast cancer cells. A growing body of evidence indicates that mTOR inhibition potentates tumor response to a wide range of apoptotic and growth inhibitory agents [12, 22, 23]. In congruence with these studies, our results show that inhibition of mTOR signaling with RAD-001 enhances the efficacy of both Let as well as ICI. Our results demonstrate that inhibition of mTOR pathway by RAD-001 in myrAkt1 MCF-7 cells results in increased expression of p21cip1/waf1 and cotreatment of these cells with Let results in a significantly enhanced apoptotic response as seen on flow cytometry, when compared with the apoptosis due to either agents used singly. Additionally, the growth inhibition of myrAkt MCF-7 cells by RAD-001 occurred in a dose-dependent fashion, indicating that mTOR-mediated signaling is essential to the estrogen-dependent proliferation of these cells.
One of the principal reasons for loss of sensitivity to Tam in patients with ER-positive breast cancer is the development of acquired drug resistance. Mechanistically, multiple factors including the anomalous activation of the PI3K/Akt signaling pathway and upregulation of the mitogen-activated protein (MAP) kinase pathway have been implicated in the generation of acquired Tam resistance. Review of published literature [10, 16–18] indicates that one mechanism by which Akt confers Tam resistance is through ligand-independent activation of the ER. Our results render support to this argument, as evident from the ER transcriptional activity measured by an ERE-tk-luciferase reporter assay, being higher in the myrAkt1 MCF-7 cells as compared with the control cells. We have demonstrated that treatment with Tam, Let and ICI significantly inhibits estrogen-induced activity of this reporter in the control cells, but not in the myrAkt1 cells, and cotreatment with RAD-001 inhibits ER activity in the myrAkt1 cells to levels lower than those observed in cells grown in CS without E2 supplementation (data not shown). This indicates that Akt-mediated resistance in breast cancer is probably due to ER-dependent as well as by ER-independent mechanisms. Our group [9] and others have previously demonstrated that tumors with activation of the PI3K/Akt signaling pathway, such as those with mutations in the PTEN tumor suppressor gene, are particularly susceptible to the antiproliferative effects of rapamycin, an inhibitor of the downstream target of Akt, mTOR. Additionally, studies have shown that pharmacological inhibition of mTOR restores the susceptibility of PTEN negative/Akt positive prostate cancer cell lines to chemotherapy [24]. This current study evaluated the effects of mTOR inhibition on Akt-induced resistance to endocrine therapy. Using a syngenic pair of breast cancer cell lines differing only in their Akt expression levels, the results of our study demonstrate that inhibition of mTOR restores the susceptibility of Akt overexpressing breast cancer cell lines to the endocrine therapy. These findings are in agreement with recent studies by Lowe et al. [12] that showed that Akt-induced resistance to doxorubicin in a murine lymphoma model could be reversed by cotreatment with rapamycin.
The finding that mTOR inhibition restores the susceptibility of Akt overexpressing breast cancer cells to Tam implies that mTOR might be an important mediator of survival in Akt-induced Tam resistance. In fact, there is a growing body of evidence that mTOR inhibition potentates tumor response to a wide range of apoptotic and growth inhibitory agents [12, 22, 23]. The mechanisms by which this is mediated remain unclear. Our in vitro proliferation data indicate that RAD-001 and the endocrine agents Tam, Let and ICI have an additive, if not synergistic effect on growth inhibition of the cells expressing a constitutively active Akt.
The results of our study have significant implications in the design of clinical studies with mTOR inhibitors in patients with breast cancer, especially an orally administered agent such as RAD 001 which when combined with Tam or an aromatase inhibitor affords patients the convenience of an oral therapy. Based on our results, we propose the hypothesis that the combination of RAD 001 and endocrine therapies may hold promise as an approach to circumvent or delay the emergence of acquired endocrine resistance. A recently concluded phase IB study in patients (n = 18) with advanced breast cancer, that had not experienced objective responses after 4 months of single-agent Let therapy, established the tolerability of administering 2.5 mg of Let with 5 and 10 mg daily doses of RAD 001. Dose-limiting grade 3 thrombocytopenia was observed in one patient receiving 10 mg of RAD 001. Other commonly observed toxic effects included stomatitis (50%), anorexia (39%), fatigue (39%), headaches and skin rash (33.3%). No clinically relavant pharmacokinetic interactions were observed between RAD 001 and Let in this study. One complete response was observed in the cohort of patients receiving 10 mg of RAD 001. Stable disease lasting >4 months from study entry was observed in nine patients (50%), indicating clinically relevant antitumor activity of this combination in a Let-refractory patient population and lending clinical validation to our preclinical results. A phase II study of the combination RAD 001 administered as a 10 mg daily dose with Let 2.5 mg daily in newly diagnosed ER (+) metastatic breast cancer patients has completed accrual of 115 patients and the results are awaited. A phase III randomized study comparing the efficacy of the combination of RAD-001 and Let to Let alone in patients with advanced breast carcinoma is presently ongoing. Similar clinical trials are also ongoing with two other mTOR inhibitors in clinical development: Temserolimus (CCI-779; Wyeth Pharmaceuticals Inc.) and AP 23573 (Ariad Pharmaceutical Inc, Cambridge, MA) both as single agents and in combinations with various chemotherapeutic and endocrine therapies. If successful, potential future strategies will include evaluation of the combination in the adjuvant and neo-adjuvant setting in patients with breast carcinoma. It is foreseeable that combinations of mTOR inhibitors and endocrine therapies will yield their maximal benefit in a subset of patients. As such, it is imperative that research efforts be focused on identifying robust and reproducible correlative biomarkers of response and resistance to therapies targeting mTOR. Most clinical trials of mTOR inhibitors have relied upon two critical downstream effectors of mTOR kinase, namely a eukaryotic translation initiation factor 4E-BP1 and ribosomal protein S6 kinase (p70s6k or S6K1), as biomarkers of antitumor activity. A more comprehensive endeavor to identify valid biomarkers is presently ongoing in a neo-adjuvant study of the combination of Let and RAD 001 in newly diagnosed, locally advanced breast cancer patients, wherein tumor tissue is being analyzed at study enrollment, after 2-weeks of therapy and after surgical resection.
In conclusion, it is known that high levels of Akt activity correlate with hormone-independent growth, increased mTOR signaling activity and resistance to endocrine therapies in ER-positive breast cancers. We have shown that cotargeting the mTOR pathway and ER signaling augments the effectiveness of antiestrogenic therapies. These results have since received preliminarily validation in early phase clinical trials. Our results therefore provide a rational basis for all future clinical development of mTOR inhibitors in breast cancer. The precise mechanisms of Akt-induced aromatase inhibitor resistance and SERD resistance and the restoration of endocrine responsiveness through mTOR inhibition need to be elucidated further and are the topic of ongoing investigation.
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The authors wish to thank William Friedrichs for his invaluable assistance with the experimental procedures presented in this study. This work was supported in part by a grant to LAD by the Shelby Rae Tengg Foundation. This work was also partly supported by NIH/NCICA75019, P30 CA54174 (RRT).
Received for publication August 23, 2006. Revision received February 26, 2007. Accepted for publication March 30, 2007.
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