Cyclin D1 and c-Myc Internal Ribosome Entry Site (IRES)-Dependent Translation is Regulated by AKT Activity and Enhanced by Rapamycin Through a p38 MAPK and

ERK-Dependent Pathway

YiJiang Shi, Anushree Sharma, Hong Wu*, Alan Lichtenstein and Joseph Gera‡

Departments of Research & Development, Molecular and Medical Pharmacology* and

Medicine, VA Greater Los Angeles Healthcare System, David Geffen School of Medicine at UCLA, Jonsson Comprehensive Cancer Center, Howard Hughes Medical Institute*,

University of California, Los Angeles, California, 91343

‡To whom correspondence should be addressed: Tel.: 818-895-9416; Fax: 818-895-

9554; E-Mail: gera@ucla.edu Keywords: translation; IRES; AKT/PKB; rapamycin; cyclin D1; c-myc Running Title: AKT-dependent cyclin D1 and c-myc IRES activity 1 The abbreviations used are: IRES, internal ribosome entry site; AKT, AKT/protein kinase B; mTOR, mammalian target of rapamycin; TSC, tuberous sclerosis complex; rRNA, ribosomal RNA; MEF, mouse embryonic fibroblast; UTR, untranslated region; ANOVA, analysis of variance.

JBC Papers in Press. Published on January 4, 2005 as Manuscript M407874200 by guest on A

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Abstract The macrolide antibiotic rapamycin inhibits the mammalian target of rapamycin

protein (mTOR) kinase resulting in the global inhibition of cap-dependent protein

synthesis, a blockade in ribosome component biosynthesis and G1 cell cycle arrest. G1

arrest may occur by inhibiting the protein synthesis of critical factors required for cell

cycle progression. Hypersensitivity to mTOR inhibitors has been demonstrated in cells

having elevated levels of AKT kinase activity, whereas cells containing quiescent AKT

activity are relatively resistant. Our previous data suggest that low AKT activity induces

resistance by allowing continued cap-independent protein synthesis of cyclin D1 and c-

myc proteins. In support of this notion, the current study demonstrates that the human

cyclin D1 mRNA 5’ untranslated region contains an internal ribosome entry site (IRES)

and that both this IRES and the c-myc IRES are negatively regulated by AKT activity.

Furthermore, we show that cyclin D1 and c-myc IRES function is enhanced following

exposure to rapamycin and requires both p38 MAPK and RAF/MEK/ERK signaling, as

specific inhibitors of these pathways reduce IRES-mediated translation and protein levels

under conditions of quiescent AKT activity. Thus, continued IRES-mediated translation

initiation may permit cell cycle progression upon mTOR inactivation in cells whose AKT

kinase activity is relatively low.

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Introduction

The global regulation of cap-dependent translation is mediated via the mTOR

signaling cascade (1-2). Activation of mTOR results in phosphorylation of the p70 S6

kinase and the translation repressor 4E-BP1, allowing the formation of functional eIF-4F

complexes resulting in cap-dependent mRNA translation initiation and ribosomal

component biogenesis (3-4). The efficiency with which an mRNA can initiate cap-

dependent translation is a function of the length and degree of secondary structure present

within the 5’ UTR, as well as, the sequence context of the initiation codon (11). Most

eukaryotic mRNAs contain 5’ UTRs with relatively short and unstructured 5’ UTRs

(< 100 nucleotides) which allows efficient cap-dependent ribosomal scanning (11).

However, some key regulators of cell proliferation and apoptosis have leaders which are

quite long, highly structured and contain many upstream AUG or CUG codons, and as a

result, are inhibitory to scanning ribosomes (12). Translation initiation in a number of

these mRNAs is achieved via IRES-mediated mechanisms (13). Protein synthesis via

this alternative form of initiation is typically favored under conditions when the default

cap-dependent pathway is inhibited (14).

The ability of AKT to regulate cap-dependent initiation is mediated via its

inhibitory effects on the mTOR inhibitor complex TSC1/TSC2 (7-9). A direct linkage

between AKT and the mTOR kinase has also been described. AKT can phosphorylate

mTOR and studies in Drosophila have demonstrated that dTOR is downstream and

epistatic to the PI3-kinase/AKT pathway (4-6). However, AKT has recently been shown

to negatively regulate the translation of the Elk-1 and Sap1a mRNAs independent of

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mTOR (52) and our prior work (10) also suggests a role for AKT in the regulation of cap-

independent translation.

Previously, we identified many mRNAs whose translation was unaffected or

induced under conditions of mTOR inhibition following rapamycin treatment (10). Many

of these transcripts remained on actively translated polysomes or shifted from

monosomal to polysomal translational states following the global inhibition of cap-

dependent translation. Interestingly, two of these mRNAs demonstrated remarkable

differential translational states depending on the AKT activity status of the cell. In cells

with relatively active AKT, the cyclin D1 and c-myc mRNAs were translationally

repressed by mTOR inhibition, while in cells containing quiescent AKT, these transcripts

were well-translated and found in polysome structures following treatment with the drug.

In this report we demonstrate that the human cyclin D1 mRNA can mediate internal

translation initiation. Furthermore, we demonstrate that both cyclin D1 and c-myc IRES

function is stimulated following rapamycin exposure in cells with quiescent AKT activity

but not in cells with activated AKT. Lastly, we show that differential AKT-dependent

cyclin D1 and c-myc IRES activity is dependent on p38 MAPK and RAF/MEK/ERK

signaling.

Experimental Procedures

Cell lines and plasmids- The U87, U87PTEN, LAPC-4puro and LAPC-4AKT cell lines have

been described previously (kind gifts of I. Mellinghoff & C. Sawyers, UCLA) (15). The

murine embryonic fibroblasts (MEFs) in which PTEN was deficient, as well as the

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parental control have also been previously described (16). The cell lines were maintained

in media supplemented with 10% fetal calf serum. We validated the expression of wild-

type cyclin D1 and c-myc mRNAs in all of these cell lines by Northern analysis and

sequencing of the 5’ UTRs of these transcripts which were identical to previously

published sequences (17). The parental construct utilized in these studies was pRF (kind

gift of A. Willis, University of Leicester, UK) (18). The 5’ UTR of the human cyclin D1

mRNA (accession no. NM053056) was amplified from total RNA. Subsequently it was

inserted into the intercistronic region of pRF to generate pRCND1F. The 396 nucleotide

c-myc (18) and 365 nucleotide p27Kip1 IRESes (19) were amplified from I.M.A.G.E.

clones 4667496 and 4298338 respectively, and also cloned into the intercistronic region

of pRF to generate pRmycF and pRp27F. The inserts were also cloned into a

promoterless version of pRF, pRF(-P) (kindly provided by J-T. Zhang, Indiana

University). All constructs were confirmed by sequencing.

Sequence Analysis and Secondary Structure Predictions- BESTFIT (GCG® Wisconsin

Package™, Accelrys) was used to compare the cyclin D1 leader to the 18S rRNA

sequences (accession no. X03205). We used the MFOLD web server to predict

secondary structures for the human cyclin D1 leader using the default settings and the

temperature fixed at 37oC (20).

Transient transfection analysis of dicistronic mRNA reporters- The reporter constructs

were transfected into cells using Lipofectamine Plus (Invitrogen) and normalized for

transfection efficiency by co-transfection with pSVβGal (Promega). Cells were

harvested 24 hours following transfection and Renilla, Firefly luciferase and β-

galactosidase activities determined (Dual-Glo Luciferase & β-Galactosidase assay

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systems, Promega). Luminescence of extracts was determined using a microplate

luminometer (Turner BioSystems, Sunnyvale, CA). Northern analysis was performed as

previously described (10), utilizing a PCR probe specific for sequences within the Firefly

luciferase open reading frame. In experiments where rapamycin (Calbiochem) was used,

cells were transfected and subsequently exposed to 10nM rapamycin for 18 hours after

which, extracts were prepared and luciferase and β-galactosidase activities were

determined.

In vitro translation of dicistronic mRNA reporters- The dicistronic plasmids were

linearized using BamHI and capped RNA transcribed in vitro (mMessage T7

Transcription kit, Ambion). Capped RNA transcripts were used to program extracts of

the indicated cell lines as previously described (21). The translation reactions were

performed with either the cap analog m7GpppG (Ambion) or GTP as indicated.

Western blotting and in vitro kinase assays- Western blotting was performed as

previously described (10). For in vitro kinase assays, MEFs treated with SB203580

(25µM) or PD98059 (25µM) for predetermined time points, harvested and lysed in

buffer contain 20mM HEPES [pH7.5]; 130mM NaCl; 25mM β-glycerolphosphate; 2mM

NaPPi; 2mM EDTA; 10% glycerol; 1mM PMSF; 2.5 µg pepstatin; 2.5µg leupeptin;

2.5µg antipain; 0.5mM dithiothreitol and 1% Triton X-100. Cleared supernatants were

subsequently incubated with either anti-p38 or anti-ERK antibody and protein A-

Sepharose overnight. Pellets were washed three times in lysis buffer and once in kinase

buffer containing 25mM HEPES [pH 7.4]; 25mM β-glycerolphosphate; 25mM MgCl2;

0.5mM EDTA and 0.5mM dithiotheitol. P38 kinase reactions were performed in the

presence of 100 µM ATP and 2 µg ATF-2 as a substrate. Phosphorylation of ATF-2 at

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Thr71 was measured by Western blot using a phospho-specific ATF-2 antibody. ERK

kinase reactions were performed using ELK-1 as a substrate and phosphorylation of

ELK-1 was detected by immunoblotting with a phospho-ELK-1 (Ser383) antibody. P38

MAPK, ERK, phosphor-ATF-2 and phosphor-ELK-1 antibodies were from Cell

Signaling. Statistical analysis was performed by using Student’s t test and ANOVA

models using Sigma Stat 3.0 (Jandel Scientific).

Results

The cyclin D1 and c-myc Leaders Function as IRESes in Transfected Cells in an AKT-

Dependent Manner.

The glioblastoma U87-MG cell line has a PTEN-null mutation with resulting

heightened AKT activity. It was stably transfected with a wild-type PTEN construct

which markedly downregulated AKT activity (15). Similarly, the LAPC-4puro prostate

cancer cell line, with relatively quiescent AKT activity was stably transfected with a

myristylated AKT allele (or empty vector control). The differential AKT/mTOR cascade

activation of these paired isogenic lines has already been described (10, 15). In addition,

for this study, we also utilized murine embryonic fibroblasts (MEFs) in which the PTEN

gene had been disrupted (16). The AKT activity in these MEFs is markedly higher as

compared to PTEN +/+ MEFs (16, 22).

To determine whether the human cyclin D1 leader could internally initiate translation,

it was cloned into the intercistronic region of a dicistronic reporter mRNA and tested in

transiently transfected cells. The c-myc leader, containing a known IRES (23), was also

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cloned into this region. The dicistronic mRNAs used in these studies contain the Renilla

and Firefly luciferases as the first and second cistrons, respectively (pRF). Since we had

previously observed a differential pattern of cyclin D1 and c-myc polysome association

in quiescent as compared to activated AKT-containing cells (10), we investigated the

ability of cyclin D1 and c-myc leaders to initiate translation internally in transiently

transfected cell lines containing differential AKT activities. We transiently transfected

these cell lines with the indicated dicistronic constructs shown in figure 1A. Expression

of the downstream Firefly luciferease in the constructs containing the cyclin D1 5’ UTR

or the c-myc IRES as compared to pRF (empty vector) was enhanced dramatically in

those cell lines containing quiescent AKT activity (U87PTEN, LAPC-4puro, PTEN +/+ MEF

in figures 1B, C and D). For example, in the U87MGPTEN, LAPC-4puro and PTEN +/+

MEF cell lines the presence of the 5’ UTR of cyclin D1 or the c-myc IRES resulted in an

~ 3 to 4 fold increase in Firefly luciferase activity. In contrast, Firefly luciferase activity

was minimally affected in the relatively “active AKT” member cell lines of these

isogenic pairs, indicating that AKT activity prevented cyclin D1 and c-myc IRES

function. Only a modest ~ 1.5 to 2 fold increase was seen in myc-IRES activity and no

increase in CCND1 IRES activity. The presence of the human p27Kip1 IRES-containing

sequences (19, 25), in the dicistronic reporter construct resulted in an ~ 10 fold increase

in Firefly luciferase activity in all of the cell lines tested irrespective of AKT activity.

This was consistent with our earlier observations that the p27Kip1 mRNA was well

translated in the face of mTOR inhibition regardless of the AKT status of the cell (10)

and indicates that p27Kip1 IRES function is independent of AKT activity. It also provides

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a control confirming that reporter expression will occur in the “high-AKT” cell lines if an

IRES is functional.

To evaluate whether the AKT-dependent enhanced translation of the downstream

cistron in the cell lines tested was the result of initiation from shorter monocistronic

transcripts or possibly from cryptic promoter activity, we analyzed the dicistronic

mRNAs via Northern blot and luciferase activities in constructs where the SV40

promoter was absent (pRF(-p), pRCD1F(-p) and pRmycF(-p)) (52). Figure 2A shows a

schematic diagram of the promoterless dicistronic constructs transfected into PTEN -/- and

PTEN +/+ MEFs. As shown in figure 2B, introduction of the promoterless constructs

resulted in minimal luciferase activities in both the PTEN -/- and PTEN +/+ MEFs,

indicating that the AKT-dependent Firefly luciferase expression from these constructs

was not the result of internal promoter activities. Furthermore, Northern analysis of

mRNAs from transfected PTEN -/- or PTEN +/+ MEFs detected only the presence of the

full-length dicistronic transcripts when probed for sequences within the downstream

Firefly luciferase open reading frame (Fig. 2C). The 5’ UTR of cyclin D1 is 208

nucleotides in length, while the c-myc IRES sequences were 396 nucleotides in length.

These data further supported the notion that the cyclin D1 5’ UTR was capable of internal

initiation and that both the IRES activities of the 5’ UTR of cyclin and the c-myc IRES

were regulated by AKT activity.

The Cyclin D1 5’ UTR and c-myc IRES Mediate Internal Initiation in an AKT-

dependent Fashion in Cell-free Extracts

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To rule out that Firefly luciferase reporter expression could be due to unusual

cryptic splicing events, we analyzed the AKT-dependent IRES activities of the cyclin D1

and c-myc mRNAs in cell-free extracts. These extracts were prepared according to

Carroll et al., and demonstrated to have high efficiencies in initiating protein synthesis

(21). Capped dicistronic mRNAs that either lacked (pRF) or contained the cyclin D1

(pRCND1F) 5’ UTR, the c-myc IRES (pRmycF), or the p27Kip1 IRES (pRp27F) were in

vitro transcribed and subsequently used to program translation in lysates from U87MG,

U87MGPTEN, LAPC-4puro, LAPC-4myrAKT, PTEN +/+ MEFs or PTEN -/- MEFs.

Translation of the parent pRF mRNA yielded Firefly luciferase activities which were

indistinguishable from the background obtained from control reaction mixtures that

lacked Firefly luciferase reporter mRNAs. In contrast, as shown in figure 3, an

equivalent amount of either pRCND1F or pRmycF mRNAs generated Firefly luciferase

activities which were ~ 2 to 4 fold higher in extracts prepared from cell lines with

relatively quiescent AKT levels. The Firefly luciferase activities generated from the

pRp27F mRNA were consistently ~ 6 fold higher in all cell extracts tested as compared

to pRF mRNA. This again was consistent with the results from the dicistronic in vivo

experiments demonstrating that the IRES-dependent translation of p27Kip1 was

independent of AKT activity.

To evaluate whether the translation of the cyclin 5’ UTR and the c-myc IRES in

these reporter mRNAs was indeed cap-independent within these cell extracts, in vitro

transcribed and capped mRNAs from pRCND1F and pRmycF were translated in the

presence of increasing concentrations of the cap analog m7GTP. This analog blocks cap-

dependent translation by binding to the initiation factor eIF-4E (24). Using mRNA in

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vitro transcribed from pRF and capped, translation of the Renilla luciferase cistron was

blocked by ~ 90-95% at 150µM of m7GpppG, but was not effected by comparable

concentrations of the non-methylated form of the analog GTP and the translation of the

Firefly cistron was unaffected by m7GpppG (data not shown). As shown in figure 4, with

both the in vitro transcribed and capped mRNAs from pRCND1F and pRmycF, the

translation of the Renilla cistron was inhibited by ~ 80-90% at concentrations of 100µM

m7GpppG or higher in all cell extracts tested. However, the translation of the Firefly

cistron remained consistent in the extracts with relatively high levels of active AKT, and

increased ~ 1.5 to 2.5 fold in extracts from the cell lines with relatively quiescent AKT

levels. This again supported the notion that sequences present within the 5’ UTR of

cyclin D1 mRNA could confer cap-independent initiation and that both this sequence and

the c-myc IRES activities were enhanced under conditions of low AKT activity.

AKT-dependent Cyclin D1 5’ UTR and c-myc IRES activity is enhanced by rapamycin

Since our previous studies (10) demonstrated a stimulation of cyclin D1 and c-myc

polysome association and protein levels by rapamycin under conditions of quiescent

AKT activity, we assessed whether cyclin D1 or c-myc IRES activity would also be

enhanced following rapamycin exposure in cells with quiescent AKT activity. To

address the AKT-dependent affects of rapamycin on cyclin D1 or c-myc IRES function,

we transfected our dicistronic constructs into the cell lines shown in figure 5 and

determined Renilla and Firefly luciferase activities prior to and following rapamycin

exposure. Renilla luciferase activity was reduced by rapamycin ~ 70 to 90 % as

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compared to values obtained in the absence of the drug for each construct tested (data not

shown). However, the results show that in the cell lines with relatively quiescent AKT

(U87PTEN, LAPC-4puro, PTEN +/+ MEFs), rapamycin treatment resulted in an ~ 3 to 6 fold

stimulation of Firefly luciferase activity as compared to values obtained for lines treated

identically containing active AKT. Interestingly, rapamycin exposure also stimulated

p27Kip1 IRES activity ~ 4 to 6 fold in all cell lines irrespective of AKT activity again,

consistent with our previous data and results from others demonstrating resistance of p27

IRESKip1 activity following exposure to the PI3-kinase inhibitor LY294002 (19).

Northern blot analysis further demonstrated that rapamycin had no affect on pRF steady-

state mRNA levels prior to and following rapamycin treatment (data not shown).

Differential AKT-dependent cyclin D1 and c-myc IRES activity is dependent on both p38

MAP Kinase and RAF/MEK/ERK signaling

Since it has been previously demonstrated that c-myc IRES function is dependent

on p38 MAPK activity during apoptosis (23) and both p38 MAPK and ERK signaling

following genotoxic stress (45) we investigated whether these cascades also contributed

to the differential AKT-dependent cyclin D1 and c-myc IRES activity we had observed.

An additional rationale was the known ability of AKT to downregulate p38 (46) and ERK

(53) activity. To determine if differential p38 or ERK signaling could be correlated with

AKT-dependent cyclin D1 or c-myc IRES activity we initially examined the activities of

these kinases in the PTEN -/- and PTEN +/+ MEFs prior to and following rapamycin

exposure. As shown in figure 6A, the basal p38 and ERK kinase activities, as

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determined by in vitro kinase assays, was approximately 3-4 fold higher in the PTEN +/+

MEFs as compared to PTEN -/- MEFs. Additionally, treatment with rapamycin resulted

in a 8 fold induction of p38 and 10 fold induction of ERK activity in PTEN +/+ MEFs

while only modestly increasing basal p38 and ERK activities (~ 1-2 fold) in the PTEN -/-

MEFs. Total p38 and ERK content in the samples was similar demonstrating that

equivalent amounts of material was immunoprecipitated (Fig. 6A). These data suggest

that p38 and ERK signaling is activated by rapamycin exposure in an AKT-dependent

manner and is consistent with the known negative regulatory effects of AKT on p38 and

on ERK (46, 53).

To investigate whether AKT-dependent cyclin D1 and c-myc IRES activity was

regulated by p38 or ERK signaling we planned to transfected PTEN +/+ and PTEN -/-

MEFs with the indicated dicistronic constructs in figure 6B and subsequently treat these

cells with the p38 inhibitor, SB203580, or the ERK inhibitor PD98058. Our preliminary

experiments demonstrated almost complete inhibition of kinase activity using these

inhibitors (data not shown). Treatment of either PTEN +/+ or PTEN -/- MEFs with

SB203580 inhibited basal p38 kinase activity by greater then 95% within 2 hours of

treatment at a concentration of 25 µM. Similarly, treatment of the MEFs with PD98058

inhibited ERK activity by greater then 92% within two hours of treatment at 25µM.

These inhibiting concentrations of SB203580 or PD98059 were found to

significantly affect AKT-dependent IRES function. The experiments in figure 6B and C

are assays performed in the absence or presence of rapamycin, respectively. As shown in

figure 6B, PTEN -/- MEFs expressed relatively little Firefly luciferase activity when

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transfected with the dicistronic constructs containing the cyclin D1 5’ UTR or the c-myc

IRES within the intercistronic regions. However, as previously observed Firefly

luciferase activity was markedly increased (~ 3-4 fold) in the PTEN +/+ MEFs transfected

with pRCND1F or pRmycF. Treatment of these cells with either SB203580 or PD98059

resulted in greater then 75% inhibition of Firefly luciferase activity. The p38 inhibitor

resulted in a modest decrease in Renilla luciferase activity (~5-10% inhibition), while the

ERK inhibitor reduced Renilla luciferase expression by 60-65% in all the constructs

tested at the concentrations used in these experiments (data not shown). Interestingly, the

p27Kip1 IRES was unaffected by treatment with either of the inhibitors and did not

demonstrate differential AKT-dependent activity as shown before.

To address whether these signaling cascades contributed to the rapamycin induced

differential cyclin D1 and c-myc IRES activity we had previously observed, we

performed the same assays in MEFs pre-treated with SB203580 or PD98058 which had

been transiently transfected with the indicated dicistronic constructs in figure 6C. As

shown in the relatively quiescent AKT containing PTEN +/+ MEFs, rapamycin induced

Firefly luciferase expression by approximately four fold relative to control experiments

without the drug. This enhancement of IRES activity was markedly inhibited by pre-

treatment with either SB203580 or PD98059. One hour pre-incubation with either of

these inhibitors resulted in greater than 80% inhibition of the rapamycin induced firefly

luciferase expression in PTEN +/+ MEFs. Renilla luciferase expression was reduced by

rapamycin (~ 65 %inhibition) in all the constructs tested and pre-treatment with

SB203580 or PD98059 in combination with rapamycin did not significantly further

reduce Renilla luciferase expression (data not shown). As before, p27Kip1 IRES activity

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was enhanced by rapamycin, irrespective of AKT activity, but was not affected by

inhibition of p38 or ERK signaling.

Differential cyclin D1 and c-myc protein levels were also assessed in the PTEN

+/+ and PTEN -/- MEFs treated with rapamycin alone and in combination with either the

p38 or ERK inhibitors. As shown in figure 7, treatment of PTEN -/- MEFs with

rapamycin resulted in downregulation of cyclin D1 and c-myc expression, however

PTEN +/+ MEFs maintain or modestly increase expression in response to the drug. This

differential response in cyclin D1 and c-myc expression is ablated by pre-treatment of the

cells with either the p38 or ERK inhibitors prior to exposure to rapamycin. As

determined by densitometry, pre-treatment with SB203580 inhibited cyclin D1 protein

levels in rapamycin treated PTEN +/+ MEFs by 7.5 fold (figure 7, compare lanes 7 and

8), while pre-treatment with the ERK inhibitor PD98059 reduced cyclin D1 expression

by 9 fold in these cells (lanes 11 and 12). Pre-treatment with either SB203580 or

PD98059 reduced c-myc protein expression to below detectable levels in quiescent AKT

containing PTEN +/+ MEFs upon rapamycin exposure (lanes 7 and 8, lanes 11 and 12).

Sequence analysis and secondary structure prediction of the Cyclin D1 5’ UTR

The 5’ UTRs of the major human cyclin D1 and c-myc mRNAs are 209 and 400

nucleotides, respectively. Both leader sequences contain the hallmarks of 5’ UTRs from

other mRNA demonstrated to exhibit IRES activity. Both leaders are relatively long,

highly structured and contain upstream AUG or CUG initiation codons (17, 26). While a

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model of the c-myc IRES structure has been described (27), the leader of the human

cyclin D1 mRNA has not been characterized in this regard.

Some mRNAs capable of internal translation initiation have been shown to contain

sequence complementarity to 18S ribosomal RNAs (28, 29). It has been proposed that

these regions of complementarity may serve as cis-acting elements involved in the direct

recruitment of ribosomal 40S subunits to mRNAs and possibly regulate cap-independent

translation (30). To address whether the cyclin D1 leader contained regions of sequence

complementarity to 18S ribosomal RNAs, we performed sequence comparisons.

Comparisons of 18S ribosomal RNAs and the cyclin D1 5’ UTR identified several

complementary sequence matches (see supplemental data). Seven of these regions

ranged from 80% to 94% similarity to 18S rRNA over 11-22 nucleotides for the cyclin

D1 leader.

A secondary structural model of the cyclin D1 5’ UTR was derived by free energy

calculations using the MFOLD algorithm (31). Thirteen structures were calculated which

ranged in initial free energies (dG) from –71.5 to –88.9 kcal/mole. The most stable

predicted structure is shown as supplemental data. The structure is highly complex with

several long stems, junctions and higher order bifurcations. Taken together, these data

support the notion that the cyclin D1 5’ UTR contains a bona fide IRES element.

Discussion

Our previous studies (10) suggested that the cyclin D1 and c-myc mRNAs are

transcripts which could be effectively translated under conditions of reduced cap-

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dependent initiation. In this report we have demonstrated that under specific signaling

conditions, the leader of the human cyclin D1 mRNA can initiate protein synthesis via an

IRES. While the cyclin encoded by the Kaposi’s sarcoma-associated herpes virus has

also been reported to contain an IRES (37), to our knowledge, this is the first report of

this mRNAs ability to initiate translation internally. We have also shown that AKT

activity regulates cyclin D1 and c-myc IRES function and demonstrated that rapamycin

increases cyclin D1 and c-myc IRES activity in an AKT-dependent manner. These

results are consistent with our previously observed AKT-dependent effects on cyclin D1

and c-myc protein synthesis following rapamycin exposure. Furthermore, we have

extended our studies by implicating the p38 MAPK and RAF/MEK/ERK signaling

cascades in the regulation of AKT-dependent cyclin D1 and c-myc IRES activity. Our

results support a working model in which the AKT-dependent control of cyclin D1 and c-

myc IRES function in response to rapamycin may regulate the expression of these critical

determinants resulting in either G1 arrest or tumor cell survival. When AKT activity is

relatively low, rapamycin treatment results in the inhibition of cap-dependent translation,

but stimulates the selective translation of cyclin D1 and c-myc via their IRESes mediated

via p38 MAPK and RAF/MEK/ERK signaling, thus maintaining expression. However,

when AKT is elevated, the rapamycin-induced inhibition of cap-dependent translation is

not associated with enhanced IRES function, most likely due to the negative regulatory

affects of AKT activity on the p38MAPK and RAF/MEK/ERK pathways, thus, cap-

independent translation is prevented and protein levels fall. This differential regulation

of cap-independent translation and overall cyclin D1/c-myc expression accounts for the

differential sensitivity of “high versus low” AKT-activity cell targets.

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An interesting question arises; under what circumstances might there be a

requirement for IRES-mediated translation initiation of cyclin D1 mRNA, particularly,

when cyclin D1 expression has been shown to be dependent on eIF-4E (38). Recent data

suggest that cyclin D1 also normally accumulates during the G2 phase of the cell cycle

and synthesis during this phase may contribute to rapidly achieving levels of cyclin D1

required for the ensuing G1 transit in actively proliferating cells (39). It has also been

recently appreciated that there is a reduction in cap-dependent protein synthesis during

the G2/M cell cycle transition (40, 41) and interestingly, it is known that both AKT

activity and protein levels transiently drop during the G2/M transition (42). While it has

been demonstrated that post-translational mechanisms contribute to the accumulation of

cyclin D1 during G2 (43, 44), it is also possible that the IRES-mediated synthesis of

cyclin D1 normally occurs during this phase of the cell cycle and supplements

expression.

Our data imply that the factor(s) responsible for AKT-dependent cyclin D1 and c-

myc IRES function are downstream of p38 and ERK. This is consistent with the results

of others who have demonstrated roles for these effectors in regulating the IRES-

mediated synthesis of c-myc during apoptosis or in response to genotoxic agents (23, 45).

It is possible that these effectors regulate cyclin D1 and c-myc IRES activity, direct or

indirectly, via phosphomodulation of an IRES trans-acting factor(s) (ITAF). Recently,

three members of the poly(rC)-RNA binding family, PCBP1, PCBP2 and hnRNPK have

been shown to be required for c-myc IRES activity and stimulate IRES-mediated

translation when overexpressed and bound to the mRNA (47). Moreover, it is known that

the activity of these proteins is regulated by phosphorylation (48-50). Alternatively, p38

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or ERK activity may lead to changes in ITAF expression thereby affecting IRES

function. Along these lines it has been demonstrated that the expression of PCBP1 under

hypoxic conditions is dependent on p38 activity in cortical neurons (54). Experiments

designed to address these questions are currently in progress.

The observation that p27Kip1 IRES activity was not AKT-dependent following

rapamycin exposure is interesting and suggests that the regulation of this IRES is similar

to but distinct from the cyclin D1 and c-myc IRESes in this setting. P27Kip1 IRES

function may be regulated by a specific ITAF(s) which enhances its function following

rapamycin exposure, but is nonresponsive to changes in p38, ERK or AKT activities.

The p27kip1 IRES has been demonstrated to be active under conditions of elevated cyclic

AMP (25) and repressed by the neuronal ELAV HuD (19), while enhancement of c-myc

IRES activity has been shown to be dependent on PCBP1, PCBP2 and HnRNPK (47). It

is certainly possible that the factors mediating cap-independent translational control of

p27kip1 are distinct from those regulating other cellular IRESes.

Our data also suggest that the ability of tumor cells to respond to mTOR inhibitors

by stimulating cap-independent mechanisms of initiation of critical cell cycle proteins

may constitute a mechanism of cellular resistance to these drugs. In particular, tumors

which have relatively little dependence on the PI3-K/AKT/mTOR signaling cascade (i.e.

low AKT activity) appear to markedly increase the cap-independent synthesis of cyclin

D1/c-myc following mTOR inhibitor exposure. Further understanding the mechanisms

regulating the expression of these determinants may assist in the development of

compounds which function in a synthetically lethal manner with mTOR inhibitors.

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Acknowledgements- We thank I. Mellinghoff, C. Sawyers, A. Willis and J-T. Zhang for

providing cell lines and plasmids. We also thank M. Rettig for helpful discussions and

critical reading of the manuscript. This work was supported by research funds of the

Veteran’s Administration including the Research Enhancement Awards Program (REAP)

entitled “Cancer Gene Medicine” and NIH CA096920.

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Figure Legends

FIG. 1. AKT-dependent cyclin D1 and c-myc IRES activity in vivo. A. Schematic diagrams of the dicistronic constructs used in this study. Constructs used are pRF, pRCD1F, which contains the 5’ UTR of human cyclin D1, pRmycF, containing the human c-myc IRES, and pRp27F, containing the human p27Kip1 IRES. B. Normalized (to values obtained for pRF alone) Renilla (white bars) and Firefly (black bars) luciferase activities of U87 and U87PTEN cells transfected with the indicated constructs. C. & D. are identical to B except for the cell line used as indicated. Transfection with each construct was performed in quadruplicate with each construct in each indicated cell line. FIG. 2. Transfection experiments with promoterless dicistronic mRNA reporter constructs in vivo. A. Diagram of promoterless constructs. pRF(-p), pRCD1F(-p) and pRmycF(-p) are identical to the constructs in FIG. 1A, except the SV40 promoter sequences have been removed (51). B. Relative luciferase activities of the indicated promoterless construct transfected into PTEN -/- MEF or PTEN +/+ MEF. C. Northern analysis of dicistronic mRNAs in PTEN -/- MEF or PTEN +/+ MEF. The positions of the 28S and 18S ribosomal RNAs are shown. FIG. 3. AKT-dependent cyclin D1 and c-myc IRES activity in cell-free extracts. In vitro transcribed dicistronic RNAs from the indicated constructs were used to program translation in cell-free extracts in the three cell line pairs shown. Renilla and Firefly luciferase activities of U87 vs. U87PTEN A, LAPC-4myrAKT vs. LAPC-4puro B and PTEN -/- MEF vs. PTEN +/+ MEF C translation extracts. Values were normalized to those obtained for pRF alone and the data are representative of three independent experiments for each cell line. FIG. 4. Effects of cap-analog on AKT-dependent cyclin D1 and c-myc IRES activity in vitro. Cell-free extracts of the three paired cell lines were programmed to translate in vitro transcribed RNA from either pRCND1F (boxes) or pRmycF (circles) in the presence of the cap analog m7GpppG. Changes in Renilla (unshaded boxes, unshaded circles) and Firefly (shaded boxes, shaded circles) luciferase activities are shown relative to activities obtained in the absence of m7GpppG. A. U87 vs. U87PTEN, B. LAPC-4myrAKT vs. LAPC-4puro, C. PTEN -/- MEF vs. PTEN +/+ MEF. Experiments were repeated three times with similar results. FIG. 5. Rapamycin stimulation of cyclin D1 and c-myc IRES activities are dependent on AKT status. The indicated cell lines were transfected with pRF, pRCD1F, pRmycF or pRp27F and treated with 10nM rapamycin. Relative fold change in Firefly luciferase activity is shown as compared to activities obtained in the absence of rapamycin and

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normalized to values obtained for pRF in each cell line. Data are representative of three similar individual experiments. FIG. 6. AKT-dependent cyclin D1 and c-myc IRES activity requires p38MAPK and ERK signaling. A. p38MAPK and ERK activities in PTEN -/- and PTEN +/+ MEFs prior to and following rapamycin exposure. In vitro kinase reactions were immunoblotted for the indicated phosphorylated substrate and total p38 or ERK protein levels. B. PTEN -/- and PTEN +/+ MEFs were transfected with the indicated dicistronic constructs and treated with either SB203580 or PD98059. Changes in Firefly luciferase (cap-independent) expression are shown. Values were normalized to pRF without treatment and were performed in triplicate. C. PTEN -/- and PTEN +/+ MEFs were transfected with the indicated dicistronic constructs and pre-treated with either SB203580 or PD98059 for 1 hour and subsequently exposed to rapamycin (rapa) as shown. Relative fold changes in Firefly (cap-independent) luciferase activities are shown as compared to activities obtained in the absence of rapamycin. Data are representative of three independent experiments. FIG. 7. Effects of p38MAPK and ERK inhibitors on AKT-dependent cyclin D1 and c-myc protein expression following exposure to rapamycin. Cyclin D1, c-myc and actin protein levels were determined in PTEN -/- or PTEN +/+ MEFs subsequent to rapamycin treatment alone or in combination with SB203580 or PD98059 as indicated. by guest on A

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Figure 2

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YiJiang Shi, Anushree Sharma, Hong Wu, Alan Lichtenstein and Joseph GeraERK-dependent pathway

regulated by AKT activity and enhanced by rapamycin through a p38 MAPK and Cyclin D1 and c-Myc internal ribosome entry site (IRES)-dependent translation is

published online January 4, 2005J. Biol. Chem. 

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