Research ArticleCancer

Cell type–specific abundance of 4EBP1 primes prostate cancer sensitivity or resistance to PI3K pathway inhibitors

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Science Signaling  17 Nov 2015:
Vol. 8, Issue 403, pp. ra116
DOI: 10.1126/scisignal.aad5111

Targeting drug-resistant prostate tumors

The activity of the PI3K-AKT-mTOR signaling pathway is often increased in various cancer types. Unfortunately, the development of resistance to PI3K pathway inhibitors is common. In a transgenic mouse model of prostate cancer, Hsieh et al. found that cell type–specific resistance was mediated by the abundance of 4EBP1, an mTOR target that inhibits protein synthesis. Compared with basal cells, luminal prostate epithelial cells had increased expression of 4EBP1, decreased protein synthesis rates, and decreased sensitivity to the mTOR inhibitor MLN0128. In both mice and patients with prostate cancer, treatment with a PI3K pathway inhibitor increased the proportion of luminal tumor cells that had high abundance of 4EBP1. Because decreasing 4EBP1 abundance suppressed resistance to MLN0128, the findings suggest that co-targeting 4EBP1 may improve therapeutic outcomes for prostate cancer patients.

Abstract

Pharmacological inhibitors against the PI3K-AKT-mTOR (phosphatidylinositol 3-kinase–AKT–mammalian target of rapamycin) pathway, a frequently deregulated signaling pathway in cancer, are clinically promising, but the development of drug resistance is a major limitation. We found that 4EBP1, the central inhibitor of cap-dependent translation, was a critical regulator of both prostate cancer initiation and maintenance downstream of mTOR signaling in a genetic mouse model. 4EBP1 abundance was distinctly different between the epithelial cell types of the normal prostate. Of tumor-prone prostate epithelial cell types, luminal epithelial cells exhibited the highest transcript and protein abundance of 4EBP1 and the lowest protein synthesis rates, which mediated resistance to both pharmacologic and genetic inhibition of the PI3K-AKT-mTOR signaling pathway. Decreasing total 4EBP1 abundance reversed resistance in drug-insensitive cells. Increased 4EBP1 abundance was a common feature in prostate cancer patients who had been treated with the PI3K pathway inhibitor BKM120; thus, 4EBP1 may be associated with drug resistance in human tumors. Our findings reveal a molecular program controlling cell type–specific 4EBP1 abundance coupled to the regulation of global protein synthesis rates that renders each epithelial cell type of the prostate uniquely sensitive or resistant to inhibitors of the PI3K-AKT-mTOR signaling pathway.

INTRODUCTION

The PI3K-AKT-mTOR (phosphatidylinositol 3-kinase–AKT–mammalian target of rapamycin) signaling pathway is altered in 100% of advanced human prostate cancer patients, which is a disease that arises from the prostatic epithelium composed of two distinct epithelial cell types, luminal and basal epithelial cells (1). Both cell types can transform and develop into tumors in the context of various oncogenic stimuli. For example, loss of PTEN (phosphatase and tensin homolog), the tumor suppressor and negative regulator of the PI3K-AKT-mTOR signaling pathway, leads to tumor development in either cell type in mouse models of prostate cancer (2). Others have shown that overexpression of the kinase AKT and the transcription factor MYC in normal basal epithelial cells leads to the formation of a luminal-like prostate cancer (3). Moreover, loss of PTEN within a prostate luminal epithelial stem cell population also leads to tumorigenesis in vivo (4). These findings demonstrate that multiple cancer-initiating cell types exist within the prostate and that tumor initiation can be driven by oncogenic PI3K-AKT-mTOR activity. However, an important unanswered question is whether all prostate tumor epithelial cell types are equally sensitive to inhibitors of the PI3K pathway or are specific cell types primed for drug resistance. This is a critical question because an emerging problem shared by all PI3K pathway inhibitors is drug resistance, which is significantly stifling the clinical success of this class of therapeutic agents.

The kinase mTOR promotes mRNA translation by converging on the eIF4F (eukaryotic translation initiation factor 4F) cap-binding complex, which is a critical nexus that controls global protein synthesis as well as the translation of specific mRNA targets (57). All eIF4F complex members including the cap-binding protein and oncogene eIF4E (8, 9), the scaffolding molecule eIF4G (10), and the RNA helicase eIF4A (11) are required for cap-dependent translation. The eIF4F complex is negatively regulated by a critical interaction between eIF4E and the tumor suppressor eIF4E-binding proteins (4EBPs), which are phosphorylated and inhibited by mTOR (6, 12). Using unique mouse models of prostate cancer, we addressed the important question of cell type specificity and translation control in tumor initiation, cancer progression, and drug resistance, and found that 4EBP1 activity is not only a marker of PI3K-AKT-mTOR signaling but is also critical for prostate cancer initiation and maintenance as well as the therapeutic response. We found that a specific population of tumor-forming luminal epithelial cells, which exhibit high transcript and protein levels of 4EBP1 and low protein synthesis rates, is remarkably resistant to inhibition of the PI3K-AKT-mTOR signaling pathway. Furthermore, we found that elevated 4EBP1 expression is necessary and sufficient for drug resistance. Using patient samples acquired from a phase 2 clinical trial with the oral pan-PI3K inhibitor BKM120, we found that a high amount of 4EBP1 protein was a characteristic of posttreatment prostate cancer cells. Together, our findings reveal a normal cellular program characterized by high 4EBP1 abundance and low protein synthesis rates in luminal epithelial cells that can be exploited by prostate cancer to direct tumor growth in the context of PI3K pathway inhibition.

RESULTS

Luminal epithelial cells with increased 4EBP1 abundance define a PI3K-AKT-mTOR pathway inhibitor–resistant cell type in vivo

PI3K-AKT-mTOR pathway inhibitors have demonstrated significant preclinical efficacy in prostate cancer preclinical trials; however, drug resistance inevitably develops (13). Multiple prostate epithelial cell types have been implicated in tumorigenesis, including luminal epithelial cells and basal epithelial cells (2); however, it is unknown if both cell types are equally sensitive to PI3K-AKT-mTOR pathway inhibition or if specific cell types are more resistant than others. We previously conducted a preclinical trial with the adenosine 5′-triphosphate (ATP) site mTOR inhibitor MLN0128 (7) in mice that develop prostate cancer through loss of the tumor suppressor PTEN in both basal and luminal epithelial cells (herein referred to as PTENL/L) (14). Although we observed a decrease in the amount of prostate tumors, we also observed that a significant number of tumors remained despite an 8-week therapeutic course with MLN0128 (7). To characterize the prostate cancer epithelial cell types prone to drug resistance, we quantified the number of basal epithelial cells and luminal epithelial cells that remained in PTENL/L mice treated with MLN0128 (7). After an 8-week treatment course with MLN0128 or vehicle, the tumors that remained in PTENL/L mice treated with MLN0128 were enriched for CK8+ luminal epithelial cells over CK5+ basal epithelial cells (Fig. 1A and fig. S1, A and B). These findings suggest that tumor-forming luminal epithelial cells are more resistant than basal epithelial cells to inhibition of the PI3K-AKT-mTOR signaling pathway.

Fig. 1 Luminal epithelial prostate cancer cells are enriched upon ATP site inhibition of mTOR and are characterized by increased 4EBP1 protein abundance and lower baseline protein synthesis rates in vivo.

(A) Schematic of MLN0128 preclinical trial. Age-matched 9- to 12-month-old PTENL/L mice were treated with vehicle or MLN0128 (1 mg/kg) daily for 8 weeks (upper panel). Quantification for ratio of CK8+ luminal epithelial cells over CK5+ basal epithelial cells in vehicle- or MLN0128-treated PTENL/L mice (lower panel; n = 6 mice per condition; *P < 0.05, t test). (B) Representative Western blots for 4EBP1, eIF4A, eIF4E, eIF4G, phospho-4EBP1 (Thr37/46), phospho–rpS6 (ribosomal protein S6) (Ser240/244), and rpS6 in basal epithelial and luminal epithelial cells from PTENL/L mice treated with and without MLN0128 (1 mg/kg) daily for 4 days by oral gavage. (C) Representative Western blot for 4EBP1 in basal epithelial and luminal epithelial cells from wild-type (WT) mice (left panel). Quantification of 4EBP1 densitometry in both WT and PTENL/L basal and luminal epithelial cells (right panel; n = 3 mice per genotype; *P = 0.04, t test). (D) 4EBP1 mRNA expression in WT and PTENL/L basal and luminal epithelial cells (n = 2 to 3 mice per genotype; *P = 0.002, t test). All samples were first normalized to actin. (E) Representative [35S]methionine incorporation run on the same gel in sorted basal and luminal epithelial cell types from WT and PTENL/L mice (left panel). Quantification of [35S]methionine incorporation by densitometry in sorted basal and luminal epithelial cell types from WT and PTENL/L mice (right panel; n = 3 mice per genotype; *P < 0.0001, **P = 0.002, t test). Data are means ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; n.s., not significant.

Next, we sought to determine the molecular features of resistant luminal epithelial cells that differentiate them from basal cells. The 4EBP1-eIF4E axis is a critical downstream target of mTOR that mediates the therapeutic efficacy of ATP site mTOR inhibitors (15). Hence, we sought to determine the abundance of 4EBP1 and each of the eIF4F components (eIF4E, eIF4A, and eIF4G) in both luminal and basal epithelial cells. To this end, we sorted luminal and basal epithelial cells from wild-type and PTENL/L mice treated with and without MLN0128 and Western-blotted for pathway components. We used the cell surface marker and integrin CD49f and the murine stem cell marker Sca-1 to distinguish luminal epithelial cells from basal epithelial cells (16). In particular, wild-type and PTENL/L basal epithelial cells are CD49f- and Sca-1–high, and luminal epithelial cells are CD49f positive and Sca-1–low, which was confirmed by quantitative polymerase chain reaction (qPCR) analysis of their respective markers on sorted cell populations (fig. S2, A and B) (16). Loss of PTEN and hyperphosphorylation of AKT were confirmed by qPCR, Western blot analysis, and immunofluorescence in PTENL/L basal and luminal epithelial cells (fig. S3, A to C). Among all the translation initiation components analyzed, only 4EBP1 protein levels were increased in PTENL/L luminal epithelial cells in both MLN0128-treated and untreated mice (Fig. 1, B and C). Notably, the phosphorylation of 4EBP1 was similar in basal and luminal epithelial cells and therefore do not contribute to the differences seen in total 4EBP1 abundance (Fig. 1B and fig. S3D). Moreover, we did not observe nuclear sequestration of 4EBP1 or cytoplasmic foci of 4EBP1 in luminal cells compared to basal cells (fig. S3E). The abundance of eIF4A and eIF4E was equivalent between treated and nontreated luminal and basal epithelial cells (Fig. 1B), whereas the abundance of eIF4G exhibited only a mild decrease in luminal epithelial cells compared to basal epithelial cells (Fig. 1B). Together, these findings reveal that luminal epithelial cells, which we found to be prone to drug resistance, are characterized by high abundance of the translational repressor 4EBP1.

Prostate epithelial cells have distinct amounts of 4EBP1 mRNA expression that underlies cell type–specific protein synthesis rates within the prostate

We next asked whether the higher amounts of 4EBP1 in prostate luminal epithelial cells were the result of PTEN loss or instead reflect a normal expression program. Unexpectedly, we found that 4EBP1 protein abundance was considerably higher in wild-type luminal epithelial cells compared to that in wild-type basal epithelial cells (Fig. 1C). To determine at what level of gene expression 4EBP1 is regulated in luminal and basal epithelial cells, we conducted qPCR analysis of 4EBP1 expression in both cell types from wild-type and PTENL/L mice. 4EBP1 mRNA expression was three- to fivefold higher in normal and PTENL/L luminal epithelial cells compared to basal epithelial cells, revealing differential transcriptional regulation of 4EBP1 in distinct tumor-prone prostate epithelial cell types (Fig. 1D).

Next, we sought to determine the functional relevance of increased 4EBP1 transcript and protein amounts in luminal epithelial cells. 4EBP1 is a negative regulator of cap-dependent translation and has been shown to decrease the rate of protein synthesis (12). We therefore conducted [35S]methionine incorporation assays on fluorescence-activated cell sorting (FACS)–sorted basal and luminal epithelial cells from wild-type and PTENL/L mice to determine the baseline mRNA translation rates in each cell type. The rates of de novo protein synthesis between the two cell types were significantly different, with basal epithelial cells exhibiting a markedly greater amount of protein synthesis than luminal epithelial cells (Fig. 1E). To determine whether other translation components besides 4EBP1 may contribute to the difference in protein synthesis rates between basal and luminal epithelial cells, we conducted a candidate protein expression analysis of major regulators of translation, including the phosphorylation of eEF2 (eukaryotic elongation factor 2), eIF2α, rpS6 and the abundance of the translation initiation inhibitors 4EBP2 and PDCD4 (programmed cell death protein 4). The phosphorylation of eEF2 and eIF2α was similar in both cell types in the PTENL/L with no changes in total abundance of eEF2 or eIF2α (fig. S4). Phosphorylation of rpS6, which has an inhibitory effect on protein synthesis (17), was decreased in luminal epithelial cells compared to basal epithelial cells (Fig. 1B). The abundance of both 4EBP2 and PDCD4 was decreased in PTENL/L luminal epithelial cells compared to that in basal epithelial cells (fig. S4). Hence, the phosphorylation status of eEF2, eIF2α, and rpS6 and the protein abundance of 4EBP2 and PDCD4 cannot account for the difference in protein synthesis rates observed between PTENL/L basal and luminal epithelial cells.

Despite having similarly increased abundance of 4EBP1 as PTENL/L luminal epithelial cells, wild-type luminal epithelial cells displayed the lowest protein synthesis rates compared to all other cell types studied. This suggests that other factors besides 4EBP1 abundance may further limit wild-type luminal epithelial cell protein synthesis rates. For example, we observed that eIF2α phosphorylation was slightly increased in wild-type luminal epithelial cells compared to basal epithelial cells (fig. S4). This increase was not observed in PTENL/L luminal cells and therefore cannot account for drug resistance or the low protein synthesis rates in the transformed setting (fig. S4). Thus, prostate epithelial cell types were distinguished by their innate protein synthesis rates, which inversely correlated with 4EBP1 transcript and protein amounts and may underlie MLN0128 resistance. Moreover, these findings suggest that a transcriptional program, which dictates the cell type–specific expression of 4EBP1, may control the differential protein synthesis rates between basal and luminal epithelial cells. The molecular program that governs prostate epithelial cell–specific 4EBP1 expression remains a question for future investigation.

The 4EBP1-eIF4E axis is a critical driver of prostate cancer initiation and maintenance downstream of mTOR

Although in vitro work has demonstrated the importance of eIF4E activity in prostate cancer cell lines (1820), little is known about the role of 4EBP1 in prostate cancer initiation and progression in vivo. Given the striking difference in 4EBP1 expression within the normal prostate, we next asked whether eIF4E hyperactivity is a critical driver of prostate cancer development as well as the therapeutic response or resistance of specific epithelial cell types. To directly address this question, we developed a genetic model in which eIF4E activity can be inhibited within all prostate epithelial cell types in an inducible fashion. In this model (Fig. 2A), the probasin (PB) promoter drives Cre recombinase expression (21) and prostate-specific recombination of a lox-stop-lox (LSL) element, which precedes a reverse tetracycline-controlled transactivator gene (rtTA) within the Rosa26 locus (22). In the presence of the tetracycline analog doxycycline, the rtTA drives the expression of a tetracycline-responsive mutant form of 4EBP1 (TetO-4EBP1M) in which all the mTOR-sensitive phosphorylation sites have been mutated to alanine. We previously used the mTOR-insensitive mutant 4EBP1M to decrease eIF4E hyperactivation in vivo in hematologic cancers (15). Using this mouse model, we studied the effects of the TetO-4EBP1M in normal prostate epithelial cells. We induced the expression of 4EBP1M in PB-CreTg/+;Rosa-LSL-rtTAKI/KI;TetO-4EBP1M/M (herein referred to as 4EBP1M) mice at weaning before the onset of male puberty for 4 to 5 weeks with doxycycline (fig. S5A) (23). We verified transgene expression and found that the prostate glands of 4EBP1M-induced mice exhibited no histological differences compared to wild-type mice (Fig. 2B and fig. S5, B and C). Thus, 4EBP1M expression does not affect normal prostate gland development or histological features of a mature murine prostate.

Fig. 2 Inhibition of eIF4E activity does not affect WT prostate epithelial cell maintenance but suppresses prostate tumor initiation and progression in the setting of PTEN loss in vivo.

(A) Schematic representation of the prostate-specific and doxycycline-inducible PTENL/L;4EBP1M mouse model, in which the addition of doxycycline (dox) to the drinking water (at 2 g/liter) induced the expression of the 4EBP1M transgene. (B) Representative hematoxylin and eosin (H&E) staining of ventral prostate glands from 4EBP1M mice after 4 weeks with or without doxycycline in their drinking water. Scale bar, 100 μm. (C) Representative H&E staining of WT, PTENL/L, and PTENL/L;4EBP1M prostates after 4 to 5 weeks of exposure to doxycycline after weaning. Percent high-grade prostatic intraepithelial neoplasia (PIN)–positive glands in PTENL/L and PTENL/L;4EBP1M mice (n = 3 mice per genotype, *P = 0.03, t test). Scale bar, 500 μm. (D) Fold change in TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling)–positive cells in WT, PTENL/L, and PTENL/L;4EBP1M prostates after 4 to 5 weeks of exposure to doxycycline after weaning (n = 3 mice per genotype; *P = 0.004, **P < 0.0001, t test). (E) Representative PTENL/L;4EBP1M prostate with or without exposure to doxycycline for 8 weeks starting at age 6 to 8 months. Mice were at 8 to 10 months of age at necropsy. (F) Quantification of mouse prostate weights between PTENL/L and PTENL/L;4EBP1M exposed to doxycycline for 8 weeks (n = 4 to 5 mice per genotype; *P = 0.02, t test). (G) Representative ultrasounds of PTENL/L and PTENL/L;4EBP1M anterior prostates before and after 8 weeks of exposure to doxycycline. (H) Quantification of tumor area in PTENL/L and PTENL/L;4EBP1M anterior prostates before and after 8 weeks of exposure to doxycycline (n = 4 to 5 mice per genotype; *P = 0.003, t test). (I) Fold change in TUNEL-positive (apoptotic) cells in PTENL/L and PTENL/L;4EBP1M (n = 3 mice per genotype; *P = 0.0006, t test). Data are means ± SEM.

To genetically determine the role of eIF4E hyperactivity toward prostate tumorigenesis and cancer progression, we developed the PB-CreTg/+;PTENL/L;Rosa-LSL-rtTAKI/KI;TetO-4EBP1M/M mouse model (herein referred to as PTENL/L;4EBP1M). In this system, loss of the PTEN tumor suppressor drives PI3K-AKT-mTOR signaling, hyperactivation of eIF4E, and subsequent development of preinvasive PIN lesions by 10 weeks of age (14). Administration of doxycycline induces the expression of the 4EBP1M transgene (Fig. 2A), thereby decreasing eIF4E hyperactivation (15). Expression of 4EBP1M within the prostate did not affect the ability of mTOR to phosphorylate other downstream targets such as the p70S6K1 and p70S6K2 target rpS6 (fig. S5D). PTENL/L and PTENL/L;4EBP1M mice were placed on doxycycline for 4 to 5 weeks immediately after weaning and analyzed for the effects on tumor initiation (fig. S5A). Loss of PTEN caused a twofold increase in prostate size that was significantly blunted in PTENL/L;4EBP1M mice (fig. S5E). At a histological level, the 4EBP1M transgene markedly inhibited the development of PIN in PTENL/L;4EBP1M mice (Fig. 2C), which complements a previous finding that eIF4E phosphorylation is necessary for prostate cancer development (24). Next, we sought to delineate the cellular mechanism underlying the tumor suppressive effects of 4EBP1M expression. Confirming previously published work, PTENL/L mice exhibited increased baseline amounts of both proliferation and apoptosis compared to wild-type mice (Fig. 2D and fig. S5F) (14). However, inhibition of eIF4E hyperactivity substantially increased apoptosis by fivefold (Fig. 2D), while having no effect on cell proliferation (fig. S5F), demonstrating that increased activity of eIF4E promotes a prosurvival program that is critical for tumor initiation.

Given the significant therapeutic potential of targeting eIF4E hyperactivity in established cancers, we used the inducible nature of the PTENL/L;4EBP1M mouse model to test the effect of eIF4E inhibition on tumor maintenance. PTENL/L and PTENL/L;4EBP1M mice were aged to 7 to 9 months, corresponding to the development of large prostate tumors that can be visualized by ultrasound. At that point, mice were imaged before and after 8 weeks of doxycycline administration (fig. S5G). By the end of the trial, PTENL/L;4EBP1M mouse prostates were 50% smaller than those of PTENL/L mice (Fig. 2, E and F). Moreover, whereas PTENL/L mice exhibited a twofold increase in tumor area, PTENL/L;4EBP1M tumors exhibited no growth but instead a slight decline in tumor area (Fig. 2, G and H), which was associated with a threefold increase in apoptosis (Fig. 2I). Together, these findings provide in vivo evidence that eIF4E hyperactivity is critical for prostate cancer initiation and maintenance, and provide clinical precedence for therapeutically targeting upstream regulators of the translation initiation machinery, such as mTOR, in epithelial cancers.

PTENL/L luminal epithelial cells are resistant to inhibition of eIF4E activity

Our genetic studies reveal that despite the marked effect of restraining eIF4E hyperactivity on prostate cancer development, this effect is incomplete, and certain prostate cancer cell populations continue to grow (Fig. 2G). This genetic effect was mirrored by what we observed after MLN0128 treatment, where a population of luminal epithelial cells was enriched after treatment (Fig. 1A). We therefore asked whether a specific population of prostate epithelial cells in PTENL/L;4EBP1M mice may be less sensitive to the expression of the 4EBP1M transgene. To address this question, we determined the change in the number of PTENL/L luminal epithelial cells compared to PTENL/L basal epithelial cells after the induction of the 4EBP1M by both immunofluorescence and FACS. In the setting of PTEN loss (fig. S3, A and B), both basal and luminal epithelial cell populations increased in total number compared to wild-type cells and were characterized by increased entry into the cell cycle (fig. S6A), demonstrating the mitogenic effects of increased PI3K-AKT-mTOR pathway activation (Fig. 3A). Strikingly, however, we found that luminal epithelial cells were specifically enriched upon inhibition of eIF4E by the 4EBP1M, which phenocopied our pharmacological findings with MLN0128 (Figs. 1A and 3B, and fig. S6, B to D). These findings suggest that eIF4E may be critical to transform basal epithelial cells, whereas luminal cells do not rely on increased eIF4E activation for their growth.

Fig. 3 PTENL/L luminal epithelial cells are enriched upon expression of 4EBP1M.

(A) Fold change in basal and luminal epithelial cell numbers upon PTEN loss in WT and PTENL/L mice (n = 7 to 9 mice per genotype; *P = 0.005, **P = 0.01, t test). (B) Ratio of CK8+ luminal epithelial cells over CK5+ basal epithelial cells in PTENL/L and PTENL/L;4EBP1M mice (n = 6 mice per condition; *P = 0.001, t test). (C) Percent 5-bromo-2′-deoxyuridine (BrdU) incorporation by FACS of the basal and luminal epithelial cell types from mice exposed to doxycycline (administered in the drinking water at 2 g/liter) at weaning for a total of 4 to 5 weeks. Upper: Representative BrdU dot plot. Lower: Quantification of percent BrdU-positive basal and luminal cells in each mouse strain (n = 5 to 6 mice per genotype, t test). FSC, forward scatter. (D) 7AAD/annexin FACS for apoptosis in basal and luminal epithelial cell types from mice exposed to the doxycycline regimen upon weaning for a total of 4 to 5 weeks. Upper: Representative 7AAD/annexin zebra plot. Lower: Quantification of 7AAD/annexin double-positive cells in basal or luminal cells in each mouse strain (n = 6 to 7 mice per genotype; *P = 0.03, t test). All mice were at 8 to 10 weeks of age at necropsy. Data are means ± SEM.

To elucidate the cellular mechanism that enables this divergent response in basal and luminal epithelial cells to inhibition of eIF4E activity, we conducted in vivo analysis of cell proliferation and apoptosis. Induction of the TetO-4EBP1M transgene did not affect cell proliferation as determined by BrdU incorporation in any of the cell types in PTENL/L mice (Fig. 3C). However, 4EBP1M expression increased apoptosis in basal cells but did not affect luminal epithelial cells (Fig. 3D and fig. S6E). Hence, despite having tumorigenic potential in the context of PTEN loss, luminal epithelial cells are not dependent on eIF4E activity for cell survival, which underscores a surprising specificity in cell identity and sensitivity to inhibition of eIF4E activity in prostate cancer. These findings are consistent with the overall marked increase in 4EBP1 transcript expression and decreased protein synthesis rates in luminal compared to basal epithelial cells. Together, these findings suggest that the high protein synthesis rates normally exhibited by prostate basal cells prime them to be more vulnerable to eIF4E down-regulation in the oncogenic setting. These findings have further therapeutic implications because cells that normally exhibit higher 4EBP1 transcript expression may be more resistant to pharmacological inhibition of the PI3K signaling pathway.

Increased 4EBP1 abundance controls the rate of protein synthesis and drives resistance to PI3K-AKT-mTOR pathway inhibition in human prostate cancer cells

To determine whether increased abundance of 4EBP1 primes cells for resistance to PI3K-AKT-mTOR pathway inhibition, we developed a human prostate cancer cell line characterized by knockdown of PTEN and overexpression of large/small T antigen and human telomerase reverse transcriptase (hTERT) in primary prostate epithelial cells (PTEN KD LHS PrECs) (fig. S7A) (25). These cells exhibit important similarities with human prostate cancer, including the expression of luminal epithelial markers and the ability to form colonies in clonogenic growth assays (fig. S7, A and B). Similar to our pharmacologic and genetic studies, these cells also exhibited a heterogeneous response to treatment with MLN0128 characterized by populations of sensitive and primary resistant cells. To determine whether 4EBP1 abundance defines the resistant cell type, single-cell clones were isolated, characterized for their baseline 4EBP1 protein abundance, and treated with MLN0128 to determine the effects on cell survival by propidium iodide staining and annexin V analysis by FACS. We found that despite having similar loss of PTEN, individual clones had varying amounts of 4EBP1 mRNA and protein amounts (Fig. 4A and fig. S7C). Moreover, we observed that clones with lower 4EBP1 protein abundance were more sensitive to MLN0128 treatment, whereas those with high 4EBP1 abundance were resistant (Fig. 4, A and B). Next, we sought to determine whether high 4EBP1 protein abundance primed cells for resistances to MLN0128. To this end, we knocked down 4EBP1 using pooled small interfering RNA (siRNAs) to amounts comparable to those observed in the “low 4EBP1” clones (Fig. 4C). Remarkably, knockdown of 4EBP1 in resistant cells increased their sensitivity to MLN0128 (Fig. 4D). Moreover, enforced expression of wild-type 4EBP1 conferred resistance to a previously sensitive clone exhibiting low 4EBP1 abundance (fig. S7, D and E). At a molecular level, decreasing 4EBP1 mRNA and protein to amounts equivalent to those in sensitive cells appeared to increase de novo protein synthesis (Fig. 4E) and increased the formation of the eIF4E complex (Fig. 4F). Hence, high 4EBP1 abundance directly inhibited the activity of eIF4E by skewing the amount of eIF4G and eIF4A bound to eIF4E, thereby lowering the amount of protein synthesis and conferring resistance to PI3K-AKT-mTOR pathway inhibitors. These findings implicate 4EBP1 as a marker and mediator of drug resistance.

Fig. 4 Increased abundance of 4EBP1 is required to maintain resistance to PI3K pathway inhibitors and is a marker of resistant cells in human prostate cancer.

(A) Representative Western blot analysis from two experiments for PTEN and 4EBP1 in PTEN KD LHS PrEC clones and control LHS cells. (B) Analysis of apoptosis by propidium iodide/annexin V staining in PTEN KD LHS PrECs after 12-hour exposure to MLN0128 or vehicle (–MLN0128) (n = 5 replicates in two independent experiments; *P < 0.0001, **P = 0.04, t test). (C) Representative Western blot of 4EBP1 in PTEN KD LHS PrECs with and without si4EBP1. (D) Analysis of apoptosis by propidium iodide/annexin V staining of PTEN KD LHS PrECs after transfection with a 4EBP1-targeted siRNA pool (n = 8 replicates in two independent experiments; *P < 0.0001, **P = 0.005, t test). (E) Representative autoradiograph (left) and quantification of [35S]methionine incorporation assays in PTEN KD LHS PrEC clones upon silencing 4EBP1 [n = 3 independent experiments; *P < 0.05, ANOVA (analysis of variance)]. (F) Representative cap-binding assay in PTEN KD LHS clones upon silencing 4EBP1. Data are means ± SEM.

High 4EBP1 abundance is associated with resistance to PI3K inhibitors in prostate cancer patients

Next, we sought to determine whether increased 4EBP1 abundance is a common feature of resistance to inhibitors of the PI3K-AKT-mTOR signaling pathway in prostate cancer patients. To this end, tissues obtained from prostate cancer patients enrolled in an ongoing, phase 2 clinical trial with the oral pan-PI3K inhibitor buparlisib (BKM120) were used to determine total abundance of 4EBP1 before and after PI3K-AKT-mTOR pathway inhibition (www.clinicaltrials.gov; NCT01695473). In this neoadjuvant trial, newly diagnosed prostate cancer patients are treated with 100 mg of buparlisib daily for 14 days before radial prostatectomy (Fig. 5A). Paired diagnostic prostate core biopsy and radical prostatectomy tissue specimens were collected from each patient for pharmacodynamic evaluation. PI3K pathway inhibition was confirmed by phosphorylated AKT immunohistochemistry in nearly all (seven of nine) patients (fig. S8A). The two patients who did not exhibit significant differences had low baseline phosphorylated AKT abundance in the pretreatment setting (fig. S8A). Prostate-specific antigen (PSA) is a biomarker commonly used to monitor treatment response or disease progression. We found that no patients experienced an objective PSA response, defined as a >30% decrease in PSA serum concentration, to BKM120 during the course of the trial (fig. S8B), and this was associated with a significant enrichment of high 4EBP1–expressing luminal epithelial cells (CK8+) posttreatment compared to pretreatment specimens (Fig.5, B and C). Thus, increased 4EBP1 abundance may characterize a specific epithelial cell type with intrinsic resistance to PI3K-AKT-mTOR pathway inhibition in prostate cancer patients.

Fig. 5 Increased 4EBP1 abundance is associated with drug resistance in prostate cancer patients.

(A) Schematic of the phase 2 neoadjuvant BKM120 clinical trial conducted at the University of California, San Francisco (UCSF). (B) Representative H&E, CK8, and 4EBP1 immunofluorescence images of a patient tumor before and after treatment with the PI3K inhibitor BKM120. Far right: Magnified insets from the 4EBP1 images. Scale bars, 50 and 10 μm, respectively. (C) Quantification of the percentage of luminal epithelial cells with low or high abundance of 4EBP1 for each patient as well as the average for all of the patients before (pre) and after (post) treatment with BKM120 (*P < 0.0001, t test).

DISCUSSION

The PI3K pathway drives tumor growth in prostate cancer, but resistance is often observed to PI3K pathway inhibitors. We found a cell type–specific intrinsic resistance mechanism to PI3K pathway inhibitors in luminal epithelial prostate cells that was mediated by increased abundance of the translation repressor 4EBP1. Compared to luminal cells, basal epithelial cells in the prostate had considerably lower mRNA and protein abundance of 4EBP1, greater protein synthesis activity, and increased MLN0128 sensitivity. Thus, contrary to the expected outcome of increased PI3K signaling resulting in equivalent eIF4E activation across all prostate epithelial cell types, our data uncovered cell type specificity in 4EBP1 transcript abundance that primes cells for drug sensitivity or resistance. In the future, it will be interesting to determine the molecular basis for the increased 4EBP1 expression in luminal epithelial cells compared to basal epithelial cells and whether these differences arise from transcription factor promoter binding (26, 27), chromatin remodeling, or mRNA degradation rates.

The clinical implication of these findings is the in vivo realization of a critical mode of resistance to PI3K pathway inhibitors other than those that have been previously proposed. For example, direct mutations of PI3K pathway components have been described to confer resistance to PI3K-mTOR inhibitors in prostate cancer (28), as have parallel mitogenic signaling pathways driven by the androgen receptor (29, 30). In addition to pathway-mediated mechanisms of resistance, the Brugge laboratory has demonstrated that the physical location of a cancer cell within a three-dimensional culture can determine its sensitivity to PI3K pathway inhibitors (31). However, unlike these observations, which demonstrate adaptive resistance mechanisms, our findings illustrate the central importance of cell identity dictated by the status of the 4EBP1-eIF4E axis and protein synthesis rates to determine intrinsic resistance to PI3K-AKT-mTOR inhibition. This may be a cellular mechanism akin to oncogene addiction where the genetic makeup of the cell dictates its drug sensitivities (32).

Loss of 4EBP1 or increased eIF4E, which is associated with increased protein synthesis, has been previously shown to confer drug-resistant phenotypes in cell culture and xenografts (3337). In the context of our in vivo studies, it is therefore intriguing to speculate as to why cancer-prone cells with high 4EBP1 abundance associated with lower protein synthesis rates escape target inhibition. Specific cell types, such as luminal epithelial cells with high 4EBP1 abundance, may harbor more quiescent features associated with lower protein synthesis and therefore may be less sensitive to therapeutic agents that impinge on protein synthesis control coupled to growth and survival. Together, our findings along with previously published reports (3337) suggest that there is both a lower and upper threshold of protein synthesis rates that can prime cells for drug resistance. Thus, 4EBP1 abundance, which our study here indicated is cell type–specific (in the prostate) and correlated with mRNA translation, is a distinguishing factor that might be used to predict sensitivity or resistance to PI3K pathway inhibitors in patients.

MATERIALS AND METHODS

Mice

PTENL/L and Rosa-LSL-rtTAKI/KI mice where obtained from Jackson Laboratories. PB-CreTg/+ mice were obtained from the Mouse Models of Human Cancers Consortium. All mice were maintained in the C57BL/6 background. The TetO-4EBP1M/M was generated as previously described (15). Mice were maintained under specific pathogen–free conditions, and experiments were performed in compliance with institutional guidelines as approved by the Institutional Animal Care and Use Committee of UCSF. Doxycycline (Sigma) was administered in the drinking water at 2 g/liter.

Cell lines and reagents

The LHS PrECs were provided by P. Febbo (UCSF) and were cultured as previously described (25). A PTEN-targeted short hairpin RNA (shRNA) sequence 5′-GACTTAGACTTGACCTATATT-3′ (TRCN0000355842, Broad Institute) was cloned into the pLKO.1 vector and overexpressed using standard lentiviral packaging constructs. The PTEN shRNA virus was infected into LHS PrECs. Single-cell clones were selected using trypsin-soaked sterile cloning discs (Sigma). MLN0128 was provided by K. Shokat (UCSF) and used at 1 mg/ml in the preclinical trial and at 100 nM in the cell lines. 4EBP1 siRNAs were obtained from Thermo Scientific (ON-TARGETplus SMARTpool human eIF4EBP1 siRNA, catalog no. L-003005-00). Lipofectamine 2000 (Life Technologies) was used to transfect LHS PrECs with siRNA. The wild-type V5-tagged 4EBP1 construct (pLX304-4EBP1-V5) was provided by P. Paddison (Fred Hutchinson Cancer Research Center).

Clonogenic assay

In brief, 5000 PTEN-deficient LHS cells were plated on to a 35-mm six-well plate. The covering medium was changed every 2 or 3 days during culture. After 14 days, the cells were fixed and stained with crystal violet solution (10% acetic acid, 10% ethanol, and 0.06% crystal) and then visualized. Colonies were enumerated and averaged over three independent experiments.

Prostate tissue processing

Whole mouse prostates were removed from wild-type, 4EBP1M, PTENL/L, and PTENL/L;4EBP1M mice, microdissected, and frozen in liquid nitrogen. Frozen tissues were subsequently manually disassociated using a biopulverizer (BioSpec) and additionally processed for protein and mRNA analysis as described below.

Prostate epithelium single-cell dissociation

Wild-type, 4EBP1M, PTENL/L, and PTENL/L;4EBP1M mouse prostates were retrieved, microdissected, and minced using a scalpel. Tissue chunks were incubated in collagenase I (1 mg/ml; Life Technologies) in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), l-glutamine, and penicillin/streptomycin for 1 to 2 hours at 37°C. This was followed by further dissociation with 0.05% trypsin, which was quenched using fully supplemented DMEM and 500 U of deoxyribonuclease (DNase) (Roche). Cells were manually dissociated with an 18-gauge followed by a 20-gauge needle and syringe. Cells were counted using a hemocytometer.

Western blot analysis

Western blot analysis was performed as previously described (15) with antibodies specific to eIF4A (Cell Signaling), eIF4G (Cell Signaling), eIF4E (BD Biosciences), PTEN (Cell Signaling), 4EBP1 (Cell Signaling), phosphorylated 4EBP1 Thr37/46 (Cell Signaling), AKT (Cell Signaling), phosphorylated AKT Ser473 (Cell Signaling), rpS6 (Cell Signaling), phosphorylated rpS6 Ser240/244 (Cell Signaling), PTEN (Cell Signaling), GAPDH (Cell Signaling) and β-actin (Sigma), phosphorylated eEF2 Thr56 (Cell Signaling), eEF2 (Cell Signaling), PDCD4 (Cell Signaling), phosphorylated eIF2α Ser51 (Cell Signaling), eIF2α (Cell Signaling), and 4EBP2 (Cell Signaling). Densitometry analysis was completed using ImageJ (http://imagej.nih.gov/ij/).

qPCR analysis

RNA was isolated using the manufacturer’s protocol for RNA extraction with TRIzol reagent (Invitrogen) using the Pure Link RNA Mini Kit (Invitrogen). RNA was DNase-treated with PureLink DNase (Invitrogen). DNase-treated RNA was transcribed to complementary DNA (cDNA) with SuperScript III First-Strand Synthesis System for Reverse Transcription PCR (Invitrogen), and 1 microliter of cDNA was used to run each SYBR Green detection qPCR assay (SYBR Green Supermix and MyiQ2, Bio-Rad). Primers were used at 200 nM. Oligomer sequences are in table S1.

FACS of distinct prostate epithelial populations

Live prostate epithelial cells were counted and labeled with CD49f–phycoerythrin (PE) (eBioscience), Sca-1–PE-Cy7 (BioLegend), CD31–eFluor 450 (eBioscience), CD45–eFluor 450 (eBioscience), and Ter119–eFluor 450 (eBioscience). Data were acquired using a BD FACSCanto (BD Biosciences) and analyzed with FlowJo (v.9.4.10). To calculate the absolute number of cells per populations, we multiplied the percentage of each cell populations with the total number of cells. To sort the basal and luminal populations, dissociated epithelial cells were stained as above and sorted on a BD FACSAria III (BD Biosciences).

Prostate immunofluorescence and analysis

Prostates were dissected from mice and fixed in 10% formalin overnight at 4°C. Tissues were subsequently dehydrated in ethanol (Sigma) at room temperature, mounted into paraffin blocks, and sectioned at 5 microns per tissue slice. Specimens were deparaffinized and rehydrated using CitriSolv (Fisher) followed by serial ethanol washes. Antigen unmasking was performed on each section using citrate (pH 6; Vector Laboratories) in a pressure cooker at 125°C for 10 to 30 min. Sections were washed in distilled water followed by two washes in tris-buffered saline (TBS). The sections were then incubated in 5% goat serum, 1% bovine serum albumin in TBS for 1 hour at room temperature. Various primary antibodies were used including those specific for cytokeratin 5 (Covance), cytokeratin 8 (Abcam), total 4EBP1 (Cell Signaling), phosphorylated AKT Ser473 (Cell Signaling), and phosphorylated 4EBP1 Thr37/46 (Cell Signaling), which were diluted in blocking solution and incubated on sections overnight at 4°C. Specimens were then washed in TBS and incubated with the appropriate Alexa 488– and Alexa 594–labeled secondary antibodies (Invitrogen) at 1:500 for 2 hours at room temperature. A final set of washes in TBS was completed at room temperature followed by mounting with HardSet Mounting Medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories). A Zeiss Axio Imager M1 was used to image the sections. Individual prostate epithelial cells and cancer cells were quantified by mean fluorescence intensity using the Zeiss AxioVision (Release 4.8) densitometric tool. To determine the ratio of luminal epithelial cells to basal epithelial cells, 10 to 15 images were taken for each pharmacologic or genetic manipulation and analyzed. Specifically, a mask for CK5+ basal epithelial cells and CK8+ luminal epithelial cells was drawn for each image and an area was calculated, thereby providing an amount of each cell type (ImageJ). To calculate the ratio, luminal epithelial areas were divided by basal epithelial areas and graphed (GraphPad Inc.).

Hematoxylin and eosin staining

Paraffin-embedded prostate specimens were deparaffinized and rehydrated as described above (see immunofluorescence section), stained with hematoxylin (Thermo Scientific), and washed with water. This was followed by a brief incubation in differentiation ready-to-use solution (VWR) and two washes with water, followed by two 70% ethanol washes. The samples were then stained with eosin (Thermo Scientific) and dehydrated with ethanol, followed by CitriSolv (Fisher). Slides were mounted with Cytoseal XYL (Richard-Allan Scientific).

Apoptosis analysis

TUNEL staining of optimal cutting temperature–embedded prostate was conducted per the manufacturer’s protocol (Roche). Apoptosis analysis of live prostate epithelial cells was conducted by first labeling single-cell prostate isolates with CD49f-PE (eBioscience), Sca-1–PE-Cy7 (BioLegend), CD31–eFluor 450 (eBioscience), CD45–eFluor 450 (eBioscience), and Ter119–eFluor 450 (eBioscience). This is followed by labeling with annexin V–allophycocyanin (APC) (BD Pharmingen) and 7-AAD (BD Pharmingen) following the manufacturer’s instructions (BD Biosciences). Data were acquired using a BD FACSCanto (BD Biosciences) and analyzed with FlowJo (v.9.4.10).

Cell proliferation analysis by BrdU incorporation in vivo

Two hundred microliters of BrdU (10 mg/ml) (BD Pharmingen) was administered by intraperitoneal injection in mice 48 and 24 hours before they were euthanized. Prostates were retrieved and dissociated to single cells as above. Staining for BrdU was conducted using the manufacturer’s protocol (BD Pharmingen). In short, cells were labeled with CD49f-PE (eBioscience), Sca-1–PE-Cy7 (BioLegend), CD31–eFluor 450 (eBioscience), CD45–eFluor 450 (eBioscience), and Ter119–eFluor 450 (eBioscience), subsequently fixed, and permeabilized with BD Cytofix/Cytoperm buffer. Cells were treated with DNAse to expose the BrdU epitopes and were stained with APC–anti-BrdU antibody. Data were acquired using a BD FACSCanto (BD Biosciences) and analyzed with FlowJo (v.9.4.10).

Ultrasound imaging of the mouse prostate

A Vevo 770 ultrasound imaging system (VisualSonics) was used to image PTENL/L and PTENL/L;4EBP1M anterior prostates before and after doxycycline treatment. Areas of interest were measured by assessing the largest area (height and width) of each mass within the anterior prostate.

Ex vivo [35S]methionine labeling in primary prostate epithelial cells

Primary basal and luminal epithelial cells from wild-type and PTENL/L mice were dissociated and sorted as described above. For each cell type, 40,000 cells were incubated with 33 mCi of [35S]methionine for 1.5 hours in methionine-free DMEM (Invitrogen) plus 10% dialyzed FBS and l-glutamine. Cells were prepared using a standard protein lysate protocol, resolved on a 10% SDS–polyacrylamide gel, and transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membrane was exposed to autoradiography film (Denville) for 24 hours and developed. Densitometry analysis was completed using ImageJ (http://imagej.nih.gov/ij/). For the LHS PTEN KD PrECs, 250,000 cells were plated and incubated as described above.

Neoadjuvant BKM120 prostate cancer clinical trial

A phase 2 prospective pharmacodynamic study of buparlisib (BKM120) (38), an oral pan-class PI3K inhibitor, in patients with high-risk, localized prostate cancer was conducted at UCSF (NCT01695473). The primary study objective was to determine the proportion of men with downstream target inhibition of PI3K in prostate tumor tissue, as measured by immunohistochemistry. Eligible subjects had localized adenocarcinoma of the prostate that were candidates for and had selected radical prostatectomy as the primary treatment. Subjects had high-risk disease, defined as Gleason ≥8 and ≥2 discrete core biopsies containing ≥20% cancer or Gleason 4 + 3 and ≥50% of core biopsies containing cancer. Patients were required to have adequate diagnostic core biopsy specimens for pharmacodynamic evaluation; patients who did not have adequate specimens for evaluation were required to undergo a pretreatment biopsy for tissue acquisition. Patients received buparlisib 100 mg daily for the 14 days preceding radical prostatectomy, with the final dose taken on the night before surgery. The study was conducted in accordance with International Conference on Harmonization good clinical practice standards, with approval by the institutional review board at UCSF. All patients provided written informed consent to participate.

Cap-binding assay

Cells were lysed in buffer A [10 mM tris-HCl (pH 7.6), 140 mM KCl, 4 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, and protease inhibitors supplemented with 1% NP-40], and cell lysates (250 μg of protein in 500 μl) were incubated overnight at 4°C with 50 μl of the mRNA cap analog m7GTP-Sepharose (Jena Bioscience) in buffer A, under constant and gentle agitation. The protein complex Sepharose beads were washed with buffer A supplemented with 0.5% NP-40, and the eIF4E-associated complex was resolved by SDS–polyacrylamide gel electrophoresis and Western blotting.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/8/403/ra116/DC1

Fig. S1. Absolute quantification of basal epithelial cells and luminal epithelial cells after treatment with MLN0128.

Fig. S2. qPCR phenotyping of distinct sorted epithelial cell populations in wild-type and PTENL/L mice.

Fig. S3. Efficiency of PTEN deletion and the phosphorylation and localization of 4EBP1 in basal and luminal epithelial cells in vivo.

Fig. S4. Western blot analysis of translation initiation factors and regulators in wild-type and PTENL/L basal and luminal epithelial cells.

Fig. S5. 4EBP1M does not affect normal prostate homeostasis but impedes prostate cancer initiation and progression.

Fig. S6. Effect of PTEN loss and 4EBP1M expression on absolute number of basal and luminal epithelial cells.

Fig. S7. Expression of 4EBP1 endows resistance to MLN0128 in LHS PTEN KD cell lines.

Fig. S8. Phosphorylated AKT (Ser473) immunohistochemistry of prostate tumors and serum PSA concentrations from patients before and after treatment with BKM120.

Table S1. qPCR oligonucleotide sequences.

REFERENCES AND NOTES

Acknowledgments: We thank M. Barna for critical reading of the manuscript and J. Shen for editing; B. Hann of the UCSF Preclinical Therapeutics Core for technical advice regarding ultrasound imaging; J. Gordon of the UCSF Laboratory for Cell Analysis for technical advice with FACS; M. Meng and M. Cooperberg for enrolling patients onto the BKM120 trial; A. Foye, A. Toschi, and J. Youngren for BKM120 clinical trial tissue acquisition; Goldberg-Benioff Program in Cancer Translational Biology for their support and commitment to prostate cancer research; and L. Wang and Y. Liu for technical support. We also thank P. Paddison (Fred Hutchinson Cancer Research Center) for gifting the pLX304-4EBP1-V5 construct, P. Febbo (UCSF) for the LHS PrECs, and K. Shokat (UCSF) for MLN0128. Funding: A.C.H. is supported by a Burroughs Wellcome Fund Career Award for Medical Scientists, by the Prostate Cancer Foundation, and by the NIH (1K08CA175154). A.C.H. is a V Foundation for Cancer Research scholar. D.R. is supported by the NIH (1R01CA154916 and 1R01CA184624) and a Stand Up to Cancer/Prostate Cancer Foundation Prostate Dream Team Translational Cancer Research Grant. This research grant is made possible by the support of the Movember Foundation. Stand Up to Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. D.R. is a Leukemia and Lymphoma Society Research Scholar. Author contributions: A.C.H. and D.R. conceived of and designed all experiments. A.C.H., L.W., H.G.N., and M.P.E. conducted all experiments. W.K. was the lead investigator for the BKM120 phase 2 clinical trial. P.R.C. provided prostate cancer specimens through the BKM120 clinical trial. A.C.H. and D.R. analyzed the data and wrote the manuscript. All authors discussed results and edited the manuscript. Competing interests: The authors declare that they have no competing interests.
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