Translation initiation is regulated in response to nutrient availability and mitogenic stimulation and is coupled with cell cycle progression and cell growth. Several alterations in translational control occur in cancer. Variant mRNA sequences can alter the translational efficiency of individual mRNA molecules, which in turn play a role in cancer biology. Changes in the expression or availability of components of the translational machinery and in the activation of translation through signal transduction pathways can lead to more global changes, such as an increase in the overall rate of protein synthesis and translational activation of the mRNA molecules involved in cell growth and proliferation. We review the basic principles of translational control, the alterations encountered in cancer, and selected therapies targeting translation initiation to help elucidate new therapeutic avenues.

The fundamental principle of molecular therapeutics in cancer is to exploit the differences in gene expression between cancer cells and normal cells. With the advent of cDNA array technology, most efforts have concentrated on identifying differences in gene expression at the level of mRNA, which can be attributable either to DNA amplification or to differences in transcription. Gene expression is quite complicated, however, and is also regulated at the level of mRNA stability, mRNA translation, and protein stability.

The power of translational regulation has been best recognized among developmental biologists, because transcription does not occur in early embryogenesis in eukaryotes. For example, in Xenopus, the period of transcriptional quiescence continues until the embryo reaches midblastula transition, the 4000-cell stage. Therefore, all necessary mRNA molecules are transcribed during oogenesis and stockpiled in a translationally inactive, masked form. The mRNA are translationally activated at appropriate times during oocyte maturation, fertilization, and early embryogenesis and thus, are under strict translational control.

Translation has an established role in cell growth. Basically, an increase in protein synthesis occurs as a consequence of mitogenesis. Until recently, however, little was known about the alterations in mRNA translation in cancer, and much is yet to be discovered about their role in the development and progression of cancer. Here we review the basic principles of translational control, the alterations en countered in cancer, and selected therapies targeting translation initiation to elucidate potential new therapeutic avenues.

Mechanism of Translation Initiation

Translation initiation is the main step in translational regulation. Translation initiation is a complex process in which the initiator tRNA and the 40S and 60S ribosomal subunits are recruited to the 5′ end of a mRNA molecule and assembled by eukaryotic translation initiation factors into an 80S ribosome at the start codon of the mRNA (Fig. 1). The 5′ end of eukaryotic mRNA is capped, i.e., contains the cap structure m7GpppN (7-methylguanosine-triphospho-5′-ribonucleoside). Most translation in eukaryotes occurs in a cap-dependent fashion, i.e., the cap is specifically recognized by the eIF4E,3 which binds the 5′ cap. The eIF4F translation initiation complex is then formed by the assembly of eIF4E, the RNA helicase eIF4A, and eIF4G, a scaffolding protein that mediates the binding of the 40S ribosomal subunit to the mRNA molecule through interaction with the eIF3 protein present on the 40S ribosome. eIF4A and eIF4B participate in melting the secondary structure of the 5′ UTR of the mRNA. The 43S initiation complex (40S/eIF2/Met-tRNA/GTP complex) scans the mRNA in a 5′→3′ direction until it encounters an AUG start codon. This start codon is then base-paired to the anticodon of initiator tRNA, forming the 48S initiation complex. The initiation factors are then displaced from the 48S complex, and the 60S ribosome joins to form the 80S ribosome.

Unlike most eukaryotic translation, translation initiation of certain mRNAs, such as the picornavirus RNA, is cap independent and occurs by internal ribosome entry. This mechanism does not require eIF4E. Either the 43S complex can bind the initiation codon directly through interaction with the IRES in the 5′ UTR such as in the encephalomyocarditis virus, or it can initially attach to the IRES and then reach the initiation codon by scanning or transfer, as is the case with the poliovirus (1).

Regulation of Translation Initiation

Translation initiation can be regulated by alterations in the expression or phosphorylation status of the various factors involved. Key components in translational regulation that may provide potential therapeutic targets follow.

eIF4E.

eIF4E plays a central role in translation regulation. It is the least abundant of the initiation factors and is considered the rate-limiting component for initiation of cap-dependent translation. eIF4E may also be involved in mRNA splicing, mRNA 3′ processing, and mRNA nucleocytoplasmic transport (2). eIF4E expression can be increased at the transcriptional level in response to serum or growth factors (3). eIF4E overexpression may cause preferential translation of mRNAs containing excessive secondary structure in their 5′ UTR that are normally discriminated against by the translational machinery and thus are inefficiently translated (47). As examples of this, overexpression of eIF4E promotes increased translation of vascular endothelial growth factor, fibroblast growth factor-2, and cyclin D1 (2, 8, 9).

Another mechanism of control is the regulation of eIF4E phosphorylation. eIF4E phosphorylation is mediated by the mitogen-activated protein kinase-interacting kinase 1, which is activated by the mitogen-activated pathway activating extracellular signal-related kinases and the stress-activated pathway acting through p38 mitogen-activated protein kinase (1013). Several mitogens, such as serum, platelet-derived growth factor, epidermal growth factor, insulin, angiotensin II, src kinase overexpression, and ras overexpression, lead to eIF4E phosphorylation (14). The phosphorylation status of eIF4E is usually correlated with the translational rate and growth status of the cell; however; eIF4E phosphorylation has also been observed in response to some cellular stresses when translational rates actually decrease (15). Thus, further study is needed to understand the effects of eIF4E phosphorylation on eIF4E activity.

Another mechanism of regulation is the alteration of eIF4E availability by the binding of eIF4E to the eIF4E-binding proteins (4E-BP, also known as PHAS-I). 4E-BPs compete with eIF4G for a binding site in eIF4E. The binding of eIF4E to the best characterized eIF4E-binding protein, 4E-BP1, is regulated by 4E-BP1 phosphorylation. Hypophosphorylated 4E-BP1 binds to eIF4E, whereas 4E-BP1 hyperphosphorylation decreases this binding. Insulin, angiotensin, epidermal growth factor, platelet-derived growth factor, hepatocyte growth factor, nerve growth factor, insulin-like growth factors I and II, interleukin 3, granulocyte-macrophage colony-stimulating factor + steel factor, gastrin, and the adenovirus have all been reported to induce phosphorylation of 4E-BP1 and to decrease the ability of 4E-BP1 to bind eIF4E (15, 16). Conversely, deprivation of nutrients or growth factors results in 4E-BP1 dephosphorylation, an increase in eIF4E binding, and a decrease in cap-dependent translation.

p70 S6 Kinase.

Phosphorylation of ribosomal 40S protein S6 by S6K is thought to play an important role in translational regulation. S6K −/− mouse embryonic cells proliferate more slowly than do parental cells, demonstrating that S6K has a positive influence on cell proliferation (17). S6K regulates the translation of a group of mRNAs possessing a 5′ terminal oligopyrimidine tract (5′ TOP) found at the 5′ UTR of ribosomal protein mRNAs and other mRNAs coding for components of the translational machinery. Phosphorylation of S6K is regulated in part based on the availability of nutrients (18, 19) and is stimulated by several growth factors, such as platelet-derived growth factor and insulin-like growth factor I (20).

eIF2α Phosphorylation.

The binding of the initiator tRNA to the small ribosomal unit is mediated by translation initiation factor eIF2. Phosphorylation of the α-subunit of eIF2 prevents formation of the eIF2/GTP/Met-tRNA complex and inhibits global protein synthesis (21, 22). eIF2α is phosphorylated under a variety of conditions, such as viral infection, nutrient deprivation, heme deprivation, and apoptosis (22). eIF2α is phosphorylated by heme-regulated inhibitor, nutrient-regulated protein kinase, and the IFN-induced, double-stranded RNA-activated protein kinase (PKR; Ref. 23).

The mTOR Signaling Pathway.

The macrolide antibiotic rapamycin (Siralimus; Wyeth-Ayerst Research, Collegeville, PA) has been the subject of intensive study because it inhibits signal transduction pathways involved in T-cell activation. The rapamycin-sensitive component of these pathways is mTOR (also called FRAP or RAFT1). mTOR is the mammalian homologue of the yeast TOR proteins that regulate G1 progression and translation in response to nutrient availability (24). mTOR is a serine-threonine kinase that modulates translation initiation by altering the phosphorylation status of 4E-BP1 and S6K (Fig. 2; Ref. 25).

4E-BP1 is phosphorylated on multiple residues. mTOR phosphorylates the Thr-37 and Thr-46 residues of 4E-BP1 in vitro (26); however, phosphorylation at these sites is not associated with a loss of eIF4E binding. Phosphorylation of Thr-37 and Thr-46 is required for subsequent phosphorylation at several COOH-terminal, serum-sensitive sites; a combination of these phosphorylation events appears to be needed to inhibit the binding of 4E-BP1 to eIF4E (25). The product of the ATM gene, p38/MSK1 pathway, and protein kinase Cσ also play a role in 4E-BP1 phosphorylation (2729).

S6K and 4E-BP1 are also regulated, in part, by PI3K and its downstream protein kinase Akt. PTEN is a phosphatase that negatively regulates PI3K signaling. PTEN null cells have constitutively active of Akt, with increased S6K activity and S6 phosphorylation (30). S6K activity is inhibited both by PI3K inhibitors wortmannin and LY294002 and by mTOR inhibitor rapamycin (24). Akt phosphorylates Ser-2448 in mTOR in vitro, and this site is phosphorylated upon Akt activation in vivo (3133). Thus, mTOR is regulated by the PI3K/Akt pathway; however, this does not appear to be the only mode of regulation of mTOR activity. Whether the PI3K pathway also regulates S6K and 4E-BP1 phosphorylation independent of mTOR is controversial.

Interestingly, mTOR autophosphorylation is blocked by wortmannin but not by rapamycin (34). This seeming inconsistency suggests that mTOR-responsive regulation of 4E-BP1 and S6K activity occurs through a mechanism other than intrinsic mTOR kinase activity. An alternate pathway for 4E-BP1 and S6K phosphorylation by mTOR activity is by the inhibition of a phosphatase. Treatment with calyculin A, an inhibitor of phosphatases 1 and 2A, reduces rapamycin-induced dephosphorylation of 4E-BP1 and S6K by rapamycin (35). PP2A interacts with full-length S6K but not with a S6K mutant that is resistant to dephosphorylation resulting from rapamycin. mTOR phosphorylates PP2A in vitro; however, how this process alters PP2A activity is not known. These results are consistent with the model that phosphorylation of a phosphatase by mTOR prevents dephosphorylation of 4E-BP1 and S6K, and conversely, that nutrient deprivation and rapamycin block inhibition of the phosphatase by mTOR.

Polyadenylation.

The poly(A) tail in eukaryotic mRNA is important in enhancing translation initiation and mRNA stability. Polyadenylation plays a key role in regulating gene expression during oogenesis and early embryogenesis. Some mRNA that are translationally inactive in the oocyte are polyadenylated concomitantly with translational activation in oocyte maturation, whereas other mRNAs that are translationally active during oogenesis are deadenylated and translationally silenced (3638). Thus, control of poly(A) tail synthesis is an important regulatory step in gene expression. The 5′ cap and poly(A) tail are thought to function synergistically to regulate mRNA translational efficiency (39, 40).

RNA Packaging.

Most RNA-binding proteins are assembled on a transcript at the time of transcription, thus determining the translational fate of the transcript (41). A highly conserved family of Y-box proteins is found in cytoplasmic messenger ribonucleoprotein particles, where the proteins are thought to play a role in restricting the recruitment of mRNA to the translational machinery (4143). The major mRNA-associated protein, YB-1, destabilizes the interaction of eIF4E and the 5′ mRNA cap in vitro, and overexpression of YB-1 results in translational repression in vivo (44). Thus, alterations in RNA packaging can also play an important role in translational regulation.

Three main alterations at the translational level occur in cancer: variations in mRNA sequences that increase or decrease translational efficiency, changes in the expression or availability of components of the translational machinery, and activation of translation through aberrantly activated signal transduction pathways. The first alteration affects the translation of an individual mRNA that may play a role in carcinogenesis. The second and third alterations can lead to more global changes, such as an increase in the overall rate of protein synthesis, and the translational activation of several mRNA species.

Variations in mRNA Sequence

Variations in mRNA sequence affect the translational efficiency of the transcript. A brief description of these variations and examples of each mechanism follow.

Mutations.

Mutations in the mRNA sequence, especially in the 5′ UTR, can alter its translational efficiency, as seen in the following examples.

c-myc.

Saito et al. proposed that translation of full-length c-myc is repressed, whereas in several Burkitt lymphomas that have deletions of the mRNA 5′ UTR, translation of c-myc is more efficient (45). More recently, it was reported that the 5′ UTR of c-myc contains an IRES, and thus c-myc translation can be initiated by a cap-independent as well as a cap-dependent mechanism (46, 47). In patients with multiple myeloma, a C→T mutation in the c-myc IRES was identified (48) and found to cause an enhanced initiation of translation via internal ribosomal entry (49).

BRCA1.

A somatic point mutation (117 G→C) in position −3 with respect to the start codon of the BRCA1 gene was identified in a highly aggressive sporadic breast cancer (50). Chimeric constructs consisting of the wild-type or mutated BRCA1 5′ UTR and a downstream luciferase reporter demonstrated a decrease in the translational efficiency with the 5′ UTR mutation.

Cyclin-dependent Kinase Inhibitor 2A.

Some inherited melanoma kindreds have a G→T transversion at base −34 of cyclin-dependent kinase inhibitor-2A, which encodes a cyclin-dependent kinase 4/cyclin-dependent kinase 6 kinase inhibitor important in G1 checkpoint regulation (51). This mutation gives rise to a novel AUG translation initiation codon, creating an upstream open reading frame that competes for scanning ribosomes and decreases translation from the wild-type AUG.

Alternate Splicing and Alternate Transcription Start Sites.

Alterations in splicing and alternate transcription sites can lead to variations in 5′ UTR sequence, length, and secondary structure, ultimately impacting translational efficiency.

ATM.

The ATM gene has four noncoding exons in its 5′ UTR that undergo extensive alternative splicing (52). The contents of 12 different 5′ UTRs that show considerable diversity in length and sequence have been identified. These divergent 5′ leader sequences play an important role in the translational regulation of the ATM gene.

mdm.

In a subset of tumors, overexpression of the oncoprotein mdm2 results in enhanced translation of the mdm2 mRNA. Use of different promoters leads to two mdm2 transcripts that differ only in their 5′ leaders (53). The longer 5′ UTR contains two upstream open reading frames, and this mRNA is loaded with ribosomes inefficiently compared with the short 5′ UTR.

BRCA1.

In a normal mammary gland, BRCA1 mRNA is expressed with a shorter leader sequence (5′UTRa), whereas in sporadic breast cancer tissue, BRCA1 mRNA is expressed with a longer leader sequence (5′ UTRb); the translational efficiency of transcripts containing 5′ UTRb is 10 times lower than that of transcripts containing 5′ UTRa (54).

TGF-β3.

TGF-β3 mRNA includes a 1.1-kb 5′ UTR, which exerts an inhibitory effect on translation. Many human breast cancer cell lines contain a novel TGF-β3 transcript with a 5′ UTR that is 870 nucleotides shorter and has a 7-fold greater translational efficiency than the normal TGF-β3 mRNA (55).

Alternate Polyadenylation Sites.

Multiple polyadenylation signals leading to the generation of several transcripts with differing 3′ UTR have been described for several mRNA species, such as the RET proto-oncogene (56), ATM gene (52), tissue inhibitor of metalloproteinases-3 (57), RHOA proto-oncogene (58), and calmodulin-I (59). Although the effect of these alternate 3′ UTRs on translation is not yet known, they may be important in RNA-protein interactions that affect translational recruitment. The role of these alterations in cancer development and progression is unknown.

Alterations in the Components of the Translation Machinery

Alterations in the components of translation machinery can take many forms.

Overexpresssion of eIF4E.

Overexpression of eIF4E causes malignant transformation in rodent cells (60) and the deregulation of HeLa cell growth (61). Polunovsky et al. (62) found that eIF4E overexpression substitutes for serum and individual growth factors in preserving viability of fibroblasts, which suggests that eIF4E can mediate both proliferative and survival signaling.

Elevated levels of eIF4E mRNA have been found in a broad spectrum of transformed cell lines (63). eIF4E levels are elevated in all ductal carcinoma in situ specimens and invasive ductal carcinomas, compared with benign breast specimens evaluated with Western blot analysis (64, 65). Preliminary studies suggest that this overexpression is attributable to gene amplification (66).

There are accumulating data suggesting that eIF4E overexpression can be valuable as a prognostic marker. eIF4E overexpression was found in a retrospective study to be a marker of poor prognosis in stages I to III breast carcinoma (67). Verification of the prognostic value of eIF4E in breast cancer is now under way in a prospective trial (67). However, in a different study, eIF4E expression was correlated with the aggressive behavior of non-Hodgkin’s lymphomas (68). In a prospective analysis of patients with head and neck cancer, elevated levels of eIF4E in histologically tumor-free surgical margins predicted a significantly increased risk of local-regional recurrence (9). These results all suggest that eIF4E overexpression can be used to select patients who might benefit from more aggressive systemic therapy. Furthermore, the head and neck cancer data suggest that eIF4E overexpression is a field defect and can be used to guide local therapy.

Alterations in Other Initiation Factors.

Alterations in a number of other initiation factors have been associated with cancer. Overproduction of eIF4G, similar to eIF4E, leads to malignant transformation in vitro (69). eIF-2α is found in increased levels in bronchioloalveolar carcinomas of the lung (3). Initiation factor eIF-4A1 is overexpressed in melanoma (70) and hepatocellular carcinoma (71). The p40 subunit of translation initiation factor 3 is amplified and overexpressed in breast and prostate cancer (72), and the eIF3-p110 subunit is overexpressed in testicular seminoma (73). The role that overexpression of these initiation factors plays on the development and progression of cancer, if any, is not known.

Overexpression of S6K.

S6K is amplified and highly overexpressed in the MCF7 breast cancer cell line, compared with normal mammary epithelium (74). In a study by Barlund et al. (74), S6K was amplified in 59 of 668 primary breast tumors, and a statistically significant association was observed between amplification and poor prognosis.

Overexpression of PAP.

PAP catalyzes 3′ poly(A) synthesis. PAP is overexpressed in human cancer cells compared with normal and virally transformed cells (75). PAP enzymatic activity in breast tumors has been correlated with PAP protein levels (76) and, in mammary tumor cytosols, was found to be an independent factor for predicting survival (76). Little is known, however, about how PAP expression or activity affects the translational profile.

Alterations in RNA-binding Proteins.

Even less is known about alterations in RNA packaging in cancer. Increased expression and nuclear localization of the RNA-binding protein YB-1 are indicators of a poor prognosis for breast cancer (77), non-small cell lung cancer (78), and ovarian cancer (79). However, this effect may be mediated at least in part at the level of transcription, because YB-1 increases chemoresistance by enhancing the transcription of a multidrug resistance gene (80).

Activation of Signal Transduction Pathways

Activation of signal transduction pathways by loss of tumor suppressor genes or overexpression of certain tyrosine kinases can contribute to the growth and aggressiveness of tumors. An important mutant in human cancers is the tumor suppressor gene PTEN, which leads to the activation of the PI3K/Akt pathway. Activation of PI3K and Akt induces the oncogenic transformation of chicken embryo fibroblasts. The transformed cells show constitutive phosphorylation of S6K and of 4E-BP1 (81). A mutant Akt that retains kinase activity but does not phosphorylate S6K or 4E-BP1 does not transform fibroblasts, which suggests a correlation between the oncogenicity of PI3K and Akt and the phosphorylation of S6K and 4E-BP1 (81).

Several tyrosine kinases such as platelet-derived growth factor, insulin-like growth factor, HER2/neu, and epidermal growth factor receptor are overexpressed in cancer. Because these kinases activate downstream signal transduction pathways known to alter translation initiation, activation of translation is likely to contribute to the growth and aggressiveness of these tumors. Furthermore, the mRNA for many of these kinases themselves are under translational control. For example, HER2/neu mRNA is translationally controlled both by a short upstream open reading frame that represses HER2/neu translation in a cell type-independent manner and by a distinct cell type-dependent mechanism that increases translational efficiency (82). HER2/neu translation is different in transformed and normal cells. Thus, it is possible that alterations at the translational level can in part account for the discrepancy between HER2/neu gene amplification detected by fluorescence in situ hybridization and protein levels detected by immunohistochemical assays.

Components of the translation machinery and signal pathways involved in the activation of translation initiation represent good targets for cancer therapy.

Targeting the mTOR Signaling Pathway: Rapamycin and Tumstatin

Rapamycin inhibits the proliferation of lymphocytes. It was initially developed as an immunosuppressive drug for organ transplantation. Rapamycin with FKBP 12 (FK506-binding protein, Mr 12,000) binds to mTOR to inhibit its function.

Rapamycin causes a small but significant reduction in the initiation rate of protein synthesis (83). It blocks cell growth in part by blocking S6 phosphorylation and selectively suppressing the translation of 5′ TOP mRNAs, such as ribosomal proteins, and elongation factors (8385). Rapamycin also blocks 4E-BP1 phosphorylation and inhibits cap-dependent but not cap-independent translation (17, 86).

The rapamycin-sensitive signal transduction pathway, activated during malignant transformation and cancer progression, is now being studied as a target for cancer therapy (87). Prostate, breast, small cell lung, glioblastoma, melanoma, and T-cell leukemia are among the cancer lines most sensitive to the rapamycin analogue CCI-779 (Wyeth-Ayerst Research; Ref. 87). In rhabdomycosarcoma cell lines, rapamycin is either cytostatic or cytocidal, depending on the p53 status of the cell; p53 wild-type cells treated with rapamycin arrest in the G1 phase and maintain their viability, whereas p53 mutant cells accumulate in G1 and undergo apoptosis (88, 89). In a recently reported study using human primitive neuroectodermal tumor and medulloblastoma models, rapamycin exhibited more cytotoxicity in combination with cisplatin and camptothecin than as a single agent. In vivo, CCI-779 delayed growth of xenografts by 160% after 1 week of therapy and 240% after 2 weeks. A single high-dose administration caused a 37% decrease in tumor volume. Growth inhibition in vivo was 1.3 times greater, with cisplatin in combination with CCI-779 than with cisplatin alone (90). Thus, preclinical studies suggest that rapamycin analogues are useful as single agents and in combination with chemotherapy.

Rapamycin analogues CCI-779 and RAD001 (Novartis, Basel, Switzerland) are now in clinical trials. Because of the known effect of rapamycin on lymphocyte proliferation, a potential problem with rapamycin analogues is immunosuppression. However, although prolonged immunosuppression can result from rapamycin and CCI-779 administered on continuous-dose schedules, the immunosuppressive effects of rapamycin analogues resolve in ∼24 h after therapy (91). The principal toxicities of CCI-779 have included dermatological toxicity, myelosuppression, infection, mucositis, diarrhea, reversible elevations in liver function tests, hyperglycemia, hypokalemia, hypocalcemia, and depression (87, 9294). Phase II trials of CCI-779 have been conducted in advanced renal cell carcinoma and in stage III/IV breast carcinoma patients who failed with prior chemotherapy. In the results reported in abstract form, although there were no complete responses, partial responses were documented in both renal cell carcinoma and in breast carcinoma (94, 95). Thus, CCI-779 has documented preliminary clinical activity in a previously treated, unselected patient population.

Active investigation is under way into patient selection for mTOR inhibitors. Several studies have found an enhanced efficacy of CCI-779 in PTEN-null tumors (30, 96). Another study found that six of eight breast cancer cell lines were responsive to CCI-779, although only two of these lines lacked PTEN (97) There was, however, a positive correlation between Akt activation and CCI-779 sensitivity (97). This correlation suggests that activation of the PI3K-Akt pathway, regardless of whether it is attributable to a PTEN mutation or to overexpression of receptor tyrosine kinases, makes cancer cell amenable to mTOR-directed therapy. In contrast, lower levels of the target of mTOR, 4E-BP1, are associated with rapamycin resistance; thus, a lower 4E-BP1/eIF4E ratio may predict rapamycin resistance (98).

Another mode of activity for rapamycin and its analogues appears to be through inhibition of angiogenesis. This activity may be both through direct inhibition of endothelial cell proliferation as a result of mTOR inhibition in these cells or by inhibition of translation of such proangiogenic factors as vascular endothelial growth factor in tumor cells (99, 100).

The angiogenesis inhibitor tumstatin, another anticancer drug currently under study, was also found recently to inhibit translation in endothelial cells (101). Through a requisite interaction with integrin, tumstatin inhibits activation of the PI3K/Akt pathway and mTOR in endothelial cells and prevents dissociation of eIF4E from 4E-BP1, thereby inhibiting cap-dependent translation. These findings suggest that endothelial cells are especially sensitive to therapies targeting the mTOR-signaling pathway.

Targeting eIF2α: EPA, Clotrimazole, mda-7, and Flavonoids

EPA is an n-3 polyunsaturated fatty acid found in the fish-based diets of populations having a low incidence of cancer (102). EPA inhibits the proliferation of cancer cells (103), as well as in animal models (104, 105). It blocks cell division by inhibiting translation initiation (105). EPA releases Ca2+ from intracellular stores while inhibiting their refilling, thereby activating PKR. PKR, in turn phosphorylates and inhibits eIF2α, resulting in the inhibition of protein synthesis at the level of translation initiation. Similarly, clotrimazole, a potent antiproliferative agent in vitro and in vivo, inhibits cell growth through depletion of Ca2+ stores, activation of PKR, and phosphorylation of eIF2α (106). Consequently, clotrimazole preferentially decreases the expression of cyclins A, E, and D1, resulting in blockage of the cell cycle in G1.

mda-7 is a novel tumor suppressor gene being developed as a gene therapy agent. Adenoviral transfer of mda-7 (Ad-mda7) induces apoptosis in many cancer cells including breast, colorectal, and lung cancer (107109). Ad-mda7 also induces and activates PKR, which leads to phosphorylation of eIF2α and induction of apoptosis (110).

Flavonoids such as genistein and quercetin suppress tumor cell growth. All three mammalian eIF2α kinases, PKR, heme-regulated inhibitor, and PERK/PEK, are activated by flavonoids, with phosphorylation of eIF2α and inhibition of protein synthesis (111).

Targeting eIF4A and eIF4E: Antisense RNA and Peptides

Antisense expression of eIF4A decreases the proliferation rate of melanoma cells (112). Sequestration of eIF4E by overexpression of 4E-BP1 is proapoptotic and decreases tumorigenicity (113, 114). Reduction of eIF4E with antisense RNA decreases soft agar growth, increases tumor latency, and increases the rates of tumor doubling times (7). Antisense eIF4E RNA treatment also reduces the expression of angiogenic factors (115) and has been proposed as a potential adjuvant therapy for head and neck cancers, particularly when elevated eIF4E is found in surgical margins. Small molecule inhibitors that bind the eIF4G/4E-BP1-binding domain of eIF4E are proapoptotic (116) and are also being actively pursued.

Exploiting Selective Translation for Gene Therapy

A different therapeutic approach that takes advantage of the enhanced cap-dependent translation in cancer cells is the use of gene therapy vectors encoding suicide genes with highly structured 5′ UTR. These mRNA would thus be at a competitive disadvantage in normal cells and not translate well, whereas in cancer cells, they would translate more efficiently. For example, the introduction of the 5′ UTR of fibroblast growth factor-2 5′ to the coding sequence of herpes simplex virus type-1 thymidine kinase gene, allows for selective translation of herpes simplex virus type-1 thymidine kinase gene in breast cancer cell lines compared with normal mammary cell lines and results in selective sensitivity to ganciclovir (117).

Translation is a crucial process in every cell. However, several alterations in translational control occur in cancer. Cancer cells appear to need an aberrantly activated translational state for survival, thus allowing the targeting of translation initiation with surprisingly low toxicity. Components of the translational machinery, such as eIF4E, and signal transduction pathways involved in translation initiation, such mTOR, represent promising targets for cancer therapy. Inhibitors of the mTOR have already shown some preliminary activity in clinical trials. It is possible that with the development of better predictive markers and better patient selection, response rates to single-agent therapy can be improved. Similar to other cytostatic agents, however, mTOR inhibitors are most likely to achieve clinical utility in combination therapy. In the interim, our increasing understanding of translation initiation and signal transduction pathways promise to lead to the identification of new therapeutic targets in the near future.

Fig. 1.

Translation initiation in eukaryotes. The 4E-BPs are hyperphosphorylated to release eIF4E so that it can interact with the 5′ cap, and the eIF4F initiation complex is assembled. The interaction of poly(A) binding protein with the initiation complex and circularization of the mRNA is not depicted in the diagram. The secondary structure of the 5′ UTR is melted, the 40S ribosomal subunit is bound to eIF3, and the ternary complex consisting of eIF2, GTP, and the Met-tRNA are recruited to the mRNA. The ribosome scans the mRNA in a 5′→3′ direction until an AUG start codon is found in the appropriate sequence context. The initiation factors are released, and the large ribosomal subunit is recruited.

Fig. 1.

Translation initiation in eukaryotes. The 4E-BPs are hyperphosphorylated to release eIF4E so that it can interact with the 5′ cap, and the eIF4F initiation complex is assembled. The interaction of poly(A) binding protein with the initiation complex and circularization of the mRNA is not depicted in the diagram. The secondary structure of the 5′ UTR is melted, the 40S ribosomal subunit is bound to eIF3, and the ternary complex consisting of eIF2, GTP, and the Met-tRNA are recruited to the mRNA. The ribosome scans the mRNA in a 5′→3′ direction until an AUG start codon is found in the appropriate sequence context. The initiation factors are released, and the large ribosomal subunit is recruited.

Close modal
Fig. 2.

Regulation of translation initiation by signal transduction pathways. Signaling via p38, extracellular signal-related kinase, PI3K, and mTOR can all activate translation initiation.

Fig. 2.

Regulation of translation initiation by signal transduction pathways. Signaling via p38, extracellular signal-related kinase, PI3K, and mTOR can all activate translation initiation.

Close modal
3

The abbreviations used are: eIF4E, eukaryotic initiation factor 4E; UTR, untranslated region; IRES, internal ribosome entry site; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; S6K, ribosomal p70 S6 kinase; mTOR, mammalian target of rapamycin; ATM, ataxia telangiectasia mutated; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog deleted from chromosome 10; PP2A, protein phosphatase 2A; TGF-β3, transforming growth factor-β3; PAP, poly(A) polymerase; EPA, eicosopentaenoic acid; mda-7, melanoma differentiation-associated gene 7.

1

F. M. is supported by The University of Texas M. D. Anderson Cancer Center Physician-Scientist Program and by NIH Grant 1KO8-CA 91895-01. K. K. H. is supported by Department of Defense Award DAMD-17-97-1-7162.

We thank Gayle Nesom from The University of Texas M. D. Anderson Cancer Center Department of Scientific Publications for editorial assistance and Dr. Elmer Bernstam for assistance with manuscript preparation.

1
Pestova, T. V., Kolupaeva, V. G., Lomakin, I. B., Pilipenko, E. V., Shatsky, I. N., Agol, V. I., and Hellen, C. U. Molecular mechanisms of translation initiation in eukaryotes.
Proc. Natl. Acad. Sci. USA
,
98
:
7029
–7036, 
2001
.
2
Rosenwald, I. B., Kaspar, R., Rousseau, D., Gehrke, L., Leboulch, P., Chen, J. J., Schmidt, E. V., Sonenberg, N., and London, I. M. Eukaryotic translation initiation factor 4E regulates expression of cyclin D1 at transcriptional and post-transcriptional levels.
J. Biol. Chem.
,
270
:
21176
–21180, 
1995
.
3
Rosenwald, I. B., Hutzler, M. J., Wang, S., Savas, L., and Fraire, A. E. Expression of eukaryotic translation initiation factors 4E and 2α is increased frequently in bronchioloalveolar but not in squamous cell carcinomas of the lung.
Cancer (Phila.)
,
92
:
2164
–2171, 
2001
.
4
Darveau, A., Pelletier, J., and Sonenberg, N. Differential efficiencies of in vitro translation of mouse c-myc transcripts differing in the 5′ untranslated region.
Proc. Natl. Acad. Sci. USA
,
82
:
2315
–2319, 
1985
.
5
Kozak, M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes.
Proc. Natl. Acad. Sci. USA
,
83
:
2850
–2854, 
1986
.
6
Koromilas, A. E., Lazaris-Karatzas, A., and Sonenberg, N. mRNAs containing extensive secondary structure in their 5′ non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E.
EMBO J.
,
11
:
4153
–4158, 
1992
.
7
Rinker-Schaeffer, C. W., Graff, J. R., De Benedetti, A., Zimmer, S. G., and Rhoads, R. E. Decreasing the level of translation initiation factor 4E with antisense RNA causes reversal of ras-mediated transformation and tumorigenesis of cloned rat embryo fibroblasts.
Int. J. Cancer
,
55
:
841
–847, 
1993
.
8
Kevil, C. G., De Benedetti, A., Payne, D. K., Coe, L. L., Laroux, F. S., and Alexander, J. S. Translational regulation of vascular permeability factor by eukaryotic initiation factor 4E: implications for tumor angiogenesis.
Int. J. Cancer
,
65
:
785
–790, 
1996
.
9
Nathan, C. A., Franklin, S., Abreo, F. W., Nassar, R., De Benedetti, A., and Glass, J. Analysis of surgical margins with the molecular marker eIF4E: a prognostic factor in patients with head and neck cancer.
J. Clin. Oncol.
,
17
:
2909
–2914, 
1999
.
10
Fukunaga, R., and Hunter, T. MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates.
EMBO J.
,
16
:
1921
–1933, 
1997
.
11
Waskiewicz, A. J., Flynn, A., Proud, C. G., and Cooper, J. A. Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.
EMBO J.
,
16
:
1909
–1920, 
1997
.
12
Wang, X., Flynn, A., Waskiewicz, A. J., Webb, B. L., Vries, R. G., Baines, I. A., Cooper, J. A., and Proud, C. G. The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways.
J. Biol. Chem.
,
273
:
9373
–9377, 
1998
.
13
Pyronnet, S., Imataka, H., Gingras, A. C., Fukunaga, R., Hunter, T., and Sonenberg, N. Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E.
EMBO J.
,
18
:
270
–279, 
1999
.
14
Kleijn, M., Scheper, G. C., Voorma, H. O., and Thomas, A. A. Regulation of translation initiation factors by signal transduction.
Eur. J. Biochem.
,
253
:
531
–544, 
1998
.
15
Raught, B., and Gingras, A. C. eIF4E activity is regulated at multiple levels.
Int. J. Biochem. Cell Biol.
,
31
:
43
–57, 
1999
.
16
Takeuchi, K., Shibamoto, S., Nagamine, K., Shigemori, I., Omura, S., Kitamura, N., and Ito, F. Signaling pathways leading to transcription and translation cooperatively regulate the transient increase in expression of c-Fos protein.
J. Biol. Chem.
,
276
:
26077
–26083, 
2001
.
17
Kawasome, H., Papst, P., Webb, S., Keller, G. M., Johnson, G. L., Gelfand, E. W., and Terada, N. Targeted disruption of p70(s6k) defines its role in protein synthesis and rapamycin sensitivity.
Proc. Natl. Acad. Sci. USA
,
95
:
5033
–5038, 
1998
.
18
Christie, G. R., Hajduch, E., Hundal, H. S., Proud, C. G., and Taylor, P. M. Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner.
J. Biol. Chem.
,
277
:
9952
–9957, 
2002
.
19
Hara, K., Yonezawa, K., Weng, Q. P., Kozlowski, M. T., Belham, C., and Avruch, J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism.
J. Biol. Chem.
,
273
:
14484
–14494, 
1998
.
20
Graves, L. M., Bornfeldt, K. E., Argast, G. M., Krebs, E. G., Kong, X., Lin, T. A., and Lawrence, J. C., Jr. cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.
Proc. Natl. Acad. Sci. USA
,
92
:
7222
–7226, 
1995
.
21
Merrick, W. C., and Hershey, J. W. B. The pathway and mechanism of eukaryotic protein synthesis. In: J. W. B. Hershey and M. B. Mathews (eds.),
Translational Control
, pp.
31
–69. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 
1996
.
22
Kimball, S. R. Eukaryotic initiation factor eIF2.
Int. J. Biochem. Cell Biol.
,
31
:
25
–29, 
1999
.
23
Jagus, R., Joshi, B., and Barber, G. N. PKR, apoptosis and cancer.
Int. J. Biochem. Cell Biol.
,
31
:
123
–138, 
1999
.
24
Thomas, G., and Hall, M. N. TOR signalling and control of cell growth.
Curr. Opin. Cell Biol.
,
9
:
782
–787, 
1997
.
25
Gingras, A. C., Raught, B., and Sonenberg, N.
Regulation of translation initiation by FRAP/mTOR. Genes Dev.
,
15
:
807
–826, 
2001
.
26
Gingras, A. C., Gygi, S. P., Raught, B., Polakiewicz, R. D., Abraham, R. T., Hoekstra, M. F., Aebersold, R., and Sonenberg, N. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.
Genes Dev.
,
13
:
1422
–1437, 
1999
.
27
Kumar, V., Pandey, P., Sabatini, D., Kumar, M., Majumder, P. K., Bharti, A., Carmichael, G., Kufe, D., and Karbhanda, S. Functional interaction between RAFT1/FRAP/mTOR and protein kinase Cδ in the regulation of cap-dependent initiation of translation.
EMBO J.
,
19
:
1087
–1097, 
2000
.
28
Yang, D. Q., and Kastan, M. B. Participation of ATM in insulin signalling through phosphorylation of eIF-4E-binding protein 1.
Nat. Cell. Biol.
,
2
:
893
–898, 
2000
.
29
Liu, G., Zhang, Y., Bode, A. M., Ma, W. Y., and Dong, Z. Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in response to UVB irradiation.
J. Biol. Chem.
,
277
:
8810
–8816, 
2002
.
30
Neshat, M. S., Mellinghoff, I. K., Tran, C., Stiles, B., Thomas, G., Petersen, R., Frost, P., Gibbons, J. J., Wu, H., and Sawyers, C. L. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR.
Proc. Natl. Acad. Sci. USA
,
98
:
10314
–10319, 
2001
.
31
Sekulic, A., Hudson, C. C., Homme, J. L., Yin, P., Otterness, D. M., Karnitz, L. M., and Abraham, R. T. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells.
Cancer Res.
,
60
:
3504
–3513, 
2000
.
32
Scott, P. H., and Lawrence, J. C., Jr. Attenuation of mammalian target of rapamycin activity by increased cAMP in 3T3–L1 adipocytes.
J. Biol. Chem.
,
273
:
34496
–34501, 
1998
.
33
Reynolds, I. T., Bodine, S. C., and Lawrence, J. C., Jr. Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load.
J. Biol. Chem.
,
277
:
17657
–17662, 
2002
.
34
Peterson, R. T., Beal, P. A., Comb, M. J., and Schreiber, S. L. FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions.
J. Biol. Chem.
,
275
:
7416
–7423, 
2000
.
35
Peterson, R. T., Desai, B. N., Hardwick, J. S., and Schreiber, S. L. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin-associated protein.
Proc. Natl. Acad. Sci. USA
,
96
:
4438
–4442, 
1999
.
36
McGrew, L. L., Dworkin-Rastl, E., Dworkin, M. B., and Richter, J. D. Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element.
Genes Dev.
,
3
:
803
–815, 
1989
.
37
Sheets, M. D., Wu, M., and Wickens, M. Polyadenylation of c-mos mRNA as a control point in Xenopus meiotic maturation.
Nature (Lond.)
,
374
:
511
–516, 
1995
.
38
Varnum, S. M., and Wormington, W. M. Deadenylation of maternal mRNAs during Xenopus oocyte maturation does not require specific cis-sequences: a default mechanism for translational control.
Genes Dev.
,
4
:
2278
–2286, 
1990
.
39
Gallie, D. R. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency.
Genes Dev.
,
5
:
2108
–2116, 
1991
.
40
Sachs, A. B., and Varani, G. Eukaryotic translation initiation: there are (at least) two sides to every story.
Nat. Struct. Biol.
,
7
:
356
–361, 
2000
.
41
Wolffe, A. P., and Meric, F. Coupling transcription to translation: a novel site for the regulation of eukaryotic gene expression.
Int. J. Biochem. Cell Biol.
,
28
:
247
–257, 
1996
.
42
Evdokimova, V. M., Wei, C. L., Sitikov, A. S., Simonenko, P. N., Lazarev, O. A., Vasilenko, K. S., Ustinov, V. A., Hershey, J. W., and Ovchinnikov, L. P. The major protein of messenger ribonucleoprotein particles in somatic cells is a member of the Y-box binding transcription factor family.
J. Biol. Chem.
,
270
:
3186
–3192, 
1995
.
43
Matsumoto, K., Meric, F., and Wolffe, A. P. Translational repression dependent on the interaction of the Xenopus Y-box protein FRGY2 with mRNA. Role of the cold shock domain, tail domain, and selective RNA sequence recognition.
J. Biol. Chem.
,
271
:
22706
–22712, 
1996
.
44
Evdokimova, V., Ruzanov, P., Imataka, H., Raught, B., Svitkin, Y., Ovchinnikov, L. P., and Sonenberg, N. The major mRNA-associated protein YB-1 is a potent 5′ cap-dependent mRNA stabilizer.
EMBO J.
,
20
:
5491
–5502, 
2001
.
45
Saito, H., Hayday, A. C., Wiman, K., Hayward, W. S., and Tonegawa, S. Activation of the c-myc gene by translocation: a model for translational control.
Proc. Natl. Acad. Sci. USA
,
80
:
7476
–7480, 
1983
.
46
Nanbru, C., Lafon, I., Audigier, S., Gensac, M. C., Vagner, S., Huez, G., and Prats, A. C. Alternative translation of the proto-oncogene c-myc by an internal ribosome entry site.
J. Biol. Chem.
,
272
:
32061
–32066, 
1997
.
47
Stoneley, M., Paulin, F. E., Le Quesne, J. P., Chappell, S. A., and Willis, A. E. c-Myc 5′ untranslated region contains an internal ribosome entry segment.
Oncogene
,
16
:
423
–428, 
1998
.
48
Paulin, F. E., West, M. J., Sullivan, N. F., Whitney, R. L., Lyne, L., and Willis, A. E. Aberrant translational control of the c-myc gene in multiple myeloma.
Oncogene
,
13
:
505
–513, 
1996
.
49
Chappell, S. A., LeQuesne, J. P., Paulin, F. E., de Schoolmeester, M. L., Stoneley, M., Soutar, R. L., Ralston, S. H., Helfrich, M. H., and Willis, A. E. A mutation in the c-myc-IRES leads to enhanced internal ribosome entry in multiple myeloma: a novel mechanism of oncogene de-regulation.
Oncogene
,
19
:
4437
–4440, 
2000
.
50
Signori, E., Bagni, C., Papa, S., Primerano, B., Rinaldi, M., Amaldi, F., and Fazio, V. M. A. A somatic mutation in the 5′UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency.
Oncogene
,
20
:
4596
–4600, 
2001
.
51
Liu, L., Dilworth, D., Gao, L., Monzon, J., Summers, A., Lassam, N., and Hogg, D. Mutation of the CDKN2A 5′ UTR creates an aberrant initiation codon and predisposes to melanoma.
Nat. Genet.
,
21
:
128
–132, 
1999
.
52
Savitsky, K., Platzer, M., Uziel, T., Gilad, S., Sartiel, A., Rosenthal, A., Elroy-Stein, O., Shiloh, Y., and Rotman, G. Ataxia-telangiectasia: structural diversity of untranslated sequences suggests complex post-transcriptional regulation of ATM gene expression.
Nucleic Acids Res.
,
25
:
1678
–1684, 
1997
.
53
Brown, C. Y., Mize, G. J., Pineda, M., George, D. L., and Morris, D. R. Role of two upstream open reading frames in the translational control of oncogene mdm2.
Oncogene
,
18
:
5631
–5637, 
1999
.
54
Sobczak, K., and Krzyzosiak, W. J. Structural determinants of BRCA1 translational regulation.
J. Biol. Chem.
,
277
:
17349
–17358, 
2002
.
55
Arrick, B. A., Grendell, R. L., and Griffin, L. A. Enhanced translational efficiency of a novel transforming growth factor β3 mRNA in human breast cancer cells.
Mol. Cell. Biol.
,
14
:
619
–628, 
1994
.
56
Myers, S. M., Eng, C., Ponder, B. A., and Mulligan, L. M. Characterization of RET proto-oncogene 3′ splicing variants and polyadenylation sites: a novel C-terminus for RET.
Oncogene
,
11
:
2039
–2045, 
1995
.
57
Byrne, J. A., Tomasetto, C., Rouyer, N., Bellocq, J. P., Rio, M. C., and Basset, P. The tissue inhibitor of metalloproteinases-3 gene in breast carcinoma: identification of multiple polyadenylation sites and a stromal pattern of expression.
Mol. Med.
,
1
:
418
–427, 
1995
.
58
Moscow, J. A., He, R., Gudas, J. M., and Cowan, K. H. Utilization of multiple polyadenylation signals in the human RHOA protooncogene.
Gene (Amst.)
,
144
:
229
–236, 
1994
.
59
Senterre-Lesenfants, S., Alag, A. S., and Sobel, M. E. Multiple mRNA species are generated by alternate polyadenylation from the human calmodulin-I gene.
J. Cell. Biochem.
,
58
:
445
–454, 
1995
.
60
Lazaris-Karatzas, A., Montine, K. S., and Sonenberg, N. Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5′ cap.
Nature (Lond.)
,
345
:
544
–547, 
1990
.
61
De Benedetti, A., and Rhoads, R. E. Overexpression of eukaryotic protein synthesis initiation factor 4E in HeLa cells results in aberrant growth and morphology.
Proc. Natl. Acad. Sci. USA
,
87
:
8212
–8216, 
1990
.
62
Polunovsky, V. A., Rosenwald, I. B., Tan, A. T., White, J., Chiang, L., Sonenberg, N., and Bitterman, P. B. Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc.
Mol. Cell. Biol.
,
16
:
6573
–6581, 
1996
.
63
Miyagi, Y., Sugiyama, A., Asai, A., Okazaki, T., Kuchino, Y., and Kerr, S. J. Elevated levels of eukaryotic translation initiation factor eIF-4E, mRNA in a broad spectrum of transformed cell lines.
Cancer Lett.
,
91
:
247
–252, 
1995
.
64
Kerekatte, V., Smiley, K., Hu, B., Smith, A., Gelder, F., and De Benedetti, A. The proto-oncogene/translation factor eIF4E: a survey of its expression in breast carcinomas.
Int. J. Cancer
,
64
:
27
–31, 
1995
.
65
Li, B. D., Liu, L., Dawson, M., and De Benedetti, A. Overexpression of eukaryotic initiation factor 4E (eIF4E) in breast carcinoma.
Cancer (Phila.)
,
79
:
2385
–2390, 
1997
.
66
Sorrells, D. L., Black, D. R., Meschonat, C., Rhoads, R., De Benedetti, A., Gao, M., Williams, B. J., and Li, B. D. Detection of eIF4E gene amplification in breast cancer by competitive PCR.
Ann. Surg. Oncol.
,
5
:
232
–237, 
1998
.
67
Li, B. D., McDonald, J. C., Nassar, R., and De Benedetti, A. Clinical outcome in stage I to III breast carcinoma and eIF4E overexpression.
Ann. Surg.
,
227
:
756
–761; discussion, 761–763, 
1998
.
68
Wang, S., Rosenwald, I. B., Hutzler, M. J., Pihan, G. A., Savas, L., Chen, J. J., and Woda, B. A. Expression of the eukaryotic translation initiation factors 4E and 2α in non-Hodgkin’s lymphomas.
Am. J. Pathol.
,
155
:
247
–255, 
1999
.
69
Fukuchi-Shimogori, T., Ishii, I., Kashiwagi, K., Mashiba, H., Ekimoto, H., and Igarashi, K. Malignant transformation by overproduction of translation initiation factor eIF4G.
Cancer Res.
,
57
:
5041
–5044, 
1997
.
70
Eberle, J., Krasagakis, K., and Orfanos, C. E. Translation initiation factor eIF-4A1 mRNA is consistently overexpressed in human melanoma cells in vitro.
Int. J. Cancer
,
71
:
396
–401, 
1997
.
71
Shuda, M., Kondoh, N., Tanaka, K., Ryo, A., Wakatsuki, T., Hada, A., Goseki, N., Igari, T., Hatsuse, K., Aihara, T., Horiuchi, S., Shichita, M., Yamamoto, N., and Yamamoto, M. Enhanced expression of translation factor mRNAs in hepatocellular carcinoma.
Anticancer Res.
,
20
:
2489
–2494, 
2000
.
72
Nupponen, N. N., Porkka, K., Kakkola, L., Tanner, M., Persson, K., Borg, A., Isola, J., and Visakorpi, T. Amplification and overexpression of p40 subunit of eukaryotic translation initiation factor 3 in breast and prostate cancer.
Am. J. Pathol.
,
154
:
1777
–1783, 
1999
.
73
Rothe, M., Ko, Y., Albers, P., and Wernert, N. Eukaryotic initiation factor 3 p110 mRNA is overexpressed in testicular seminomas.
Am. J. Pathol.
,
157
:
1597
–1604, 
2000
.
74
Barlund, M., Forozan, F., Kononen, J., Bubendorf, L., Chen, Y., Bittner, M. L., Torhorst, J., Haas, P., Bucher, C., Sauter, G., Kallioniemi, O. P., and Kallioniemi, A. Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis.
J. Natl. Cancer Inst. (Bethesda)
,
92
:
1252
–1259, 
2000
.
75
Topalian, S. L., Kaneko, S., Gonzales, M. I., Bond, G. L., Ward, Y., and Manley, J. L. Identification and functional characterization of neo-poly(A) polymerase, an RNA processing enzyme overexpressed in human tumors.
Mol. Cell. Biol.
,
21
:
5614
–5623, 
2001
.
76
Scorilas, A., Talieri, M., Ardavanis, A., Courtis, N., Dimitriadis, E., Yotis, J., Tsiapalis, C. M., and Trangas, T. Polyadenylate polymerase enzymatic activity in mammary tumor cytosols: a new independent prognostic marker in primary breast cancer.
Cancer Res.
,
60
:
5427
–5433, 
2000
.
77
Janz, M., Harbeck, N., Dettmar, P., Berger, U., Schmidt, A., Jurchott, K., Schmitt, M., and Royer, H. D. Y-box factor YB-1 predicts drug resistance and patient outcome in breast cancer independent of clinically relevant tumor biologic factors HER2, uPA and PAI-1.
Int. J. Cancer
,
97
:
278
–282, 
2002
.
78
Shibahara, K., Sugio, K., Osaki, T., Uchiumi, T., Maehara, Y., Kohno, K., Yasumoto, K., Sugimachi, K., and Kuwano, M. Nuclear expression of the Y-box binding protein, YB-1, as a novel marker of disease progression in non-small cell lung cancer.
Clin. Cancer Res.
,
7
:
3151
–3155, 
2001
.
79
Kamura, T., Yahata, H., Amada, S., Ogawa, S., Sonoda, T., Kobayashi, H., Mitsumoto, M., Kohno, K., Kuwano, M., and Nakano, H. Is nuclear expression of Y box-binding protein-1 a new prognostic factor in ovarian serous adenocarcinoma?
Cancer (Phila.)
,
85
:
2450
–2454, 
1999
.
80
Bargou, R. C., Jurchott, K., Wagener, C., Bergmann, S., Metzner, S., Bommert, K., Mapara, M. Y., Winzer, K. J., Dietel, M., Dorken, B., and Royer, H. D. Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression.
Nat. Med.
,
3
:
447
–450, 
1997
.
81
Aoki, M., Blazek, E., and Vogt, P. K. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt.
Proc. Natl. Acad. Sci. USA
,
98
:
136
–141, 
2001
.
82
Child, S. J., Miller, M. K., and Geballe, A. P. Cell type-dependent and -independent control of HER-2/neu translation.
Int. J. Biochem. Cell Biol.
,
31
:
201
–213, 
1999
.
83
Jefferies, H. B., Reinhard, C., Kozma, S. C., and Thomas, G. Rapamycin selectively represses translation of the “polypyrimidine tract” mRNA family.
Proc. Natl. Acad. Sci. USA
,
91
:
4441
–4445, 
1994
.
84
Terada, N., Patel, H. R., Takase, K., Kohno, K., Nairn, A. C., and Gelfand, E. W. Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins.
Proc. Natl. Acad. Sci. USA
,
91
:
11477
–11481, 
1994
.
85
Jefferies, H. B., Fumagalli, S., Dennis, P. B., Reinhard, C., Pearson, R. B., and Thomas, G. Rapamycin suppresses 5′TOP mRNA translation through inhibition of p70s6k.
EMBO J.
,
16
:
3693
–3704, 
1997
.
86
Beretta, L., Gingras, A. C., Svitkin, Y. V., Hall, M. N., and Sonenberg, N. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation.
EMBO J.
,
15
:
658
–664, 
1996
.
87
Hidalgo, M., and Rowinsky, E. K. The rapamycin-sensitive signal transduction pathway as a target for cancer therapy.
Oncogene
,
19
:
6680
–6686, 
2000
.
88
Hosoi, H., Dilling, M. B., Shikata, T., Liu, L. N., Shu, L., Ashmun, R. A., Germain, G. S., Abraham, R. T., and Houghton, P. J. Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells.
Cancer Res.
,
59
:
886
–894, 
1999
.
89
Huang, S., and Houghton, P. J. Resistance to rapamycin: a novel anticancer drug.
Cancer Metastasis Rev.
,
20
:
69
–78, 
2001
.
90
Geoerger, B., Kerr, K., Tang, C. B., Fung, K. M., Powell, B., Sutton, L. N., Phillips, P. C., and Janss, A. J. Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor/medulloblastoma models as single agent and in combination chemotherapy.
Cancer Res.
,
61
:
1527
–1532, 
2001
.
91
Gibbons, J. J., Discafani, C., Peterson, R., Hernandez, R., Skotnicki, J., and Frost, P. The effect of CCI-779, a novel macrolide anti-tumor agent, on the growth of human tumor cells in vitro and in nude mouse xenografts in vivo.
Proc. Am. Assoc. Cancer Res.
,
40
:
301
, 
1999
.
92
Hidalgo, M., Rowinsky, E., Erlichman, C., Marshall, B., Marks, R., Edwards, T., and Buckner, J. J. A Phase I and pharmacological study of CCI-779 cycle inhibitor.
Ann. Oncol.
,
11
(Suppl. 4):
133
, 
2001
.
93
Alexandre, J., Raymond, E., Depenbrock, H., Mekhaldi, S., Angevin, E., Paillet, C., Hanauske, A., Frisch, J., Feussner, A., and Armand, J. P. CCI-779, a new rapamycin analog, has antitumor activity at doses inducing only mild cutaneous effects and mucositis: early results of an ongoing Phase I study. Proceedings of the 1999 AACR-NCI-EORTC International Conference,
Clin. Cancer Res.
,
5
(Suppl.):
3730s
, 
1999
.
94
Chan, S., Johnston, S., Scheulen, M. E., Mross, K., Morant, A., Lahr, A., Feussner, A., Berger, M., and Kirsch, T. First report: a Phase 2 study of the safety and activity of CCI-779 for patients with locally advanced or metastatic breast cancer failing prior chemotherapy.
Proc. Am. Soc. Clin. Oncol.
,
21
:
44a
, 
2002
.
95
Atkins, M. B., Hidalgo, M., Stadler, W., Logan, T., Dutcher, J. P., Hudes, G., Park, Y., Marshall, B., Boni, J., and Dukart, G. A randomized double-blind Phase 2 study of intravenous CCI-779 administered weekly to patients with advanced renal cell carcinoma.
Proc. Am. Soc. Clin. Oncol.
,
21
:
10a
, 
2002
.
96
Smith, S. G., Trinh, C. M., Inge, L. J., Thomas, G., Cloughsey, T. F., Sawyers, C. L., and Mischel, P. S. PTEN expression status predicts glioblastoma cell sensitivity to CCI-779.
Proc. Am. Assoc. Cancer Res.
,
43
:
335
, 
2002
.
97
Yu, K., Toral-Barza, L., Discafani, C., Zhang, W. G., Skotnicki, J., Frost, P., and Gibbons, J. J. mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer.
Endocr. Relat. Cancer
,
8
:
249
–258, 
2001
.
98
Dilling, M. B., Germain, G. S., Dudkin, L., Jayaraman, A. L., Zhang, X., Harwood, F. C., and Houghton, P. J. 4E-binding proteins, the suppressors of eukaryotic initiation factor 4E, are downregulated in cells with acquired or intrinsic resistance to rapamycin.
J. Biol. Chem.
,
277
:
13907
–13917, 
2002
.
99
Guba, M., von Breitenbuch, P., Steinbauer, M., Koehl, G., Flegel, S., Hornung, M., Bruns, C. J., Zuelke, C., Farkas, S., Anthuber, M., Jauch, K. W., and Geissler, E. K. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor.
Nat. Med.
,
8
:
128
–135, 
2002
.
100
Lane, H. A., Schell, C., Theuer, A., O’Reilly, T., and Wood, J. Antiangiogenic activity of RAD001, an orally active anticancer agent.
Proc. Am. Assoc. Cancer Res.
,
43
:
184
, 
2002
.
101
Maeshima, Y., Sudhakar, A., Lively, J. C., Ueki, K., Kharbanda, S., Kahn, C. R., Sonenberg, N., Hynes, R. O., and Kalluri, R. Tumstatin, an endothelial cell-specific inhibitor of protein synthesis.
Science (Wash. DC)
,
295
:
140
–143, 
2002
.
102
Caygill, C. P., Charlett, A., and Hill, M. J. Fat, fish, fish oil and cancer.
Br. J. Cancer
,
74
:
159
–164, 
1996
.
103
Falconer, J. S., Ross, J. A., Fearon, K. C., Hawkins, R. A., O’Riordain, M. G., and Carter, D. C. Effect of eicosapentaenoic acid and other fatty acids on the growth in vitro of human pancreatic cancer cell lines.
Br. J. Cancer
,
69
:
826
–832, 
1994
.
104
Noguchi, M., Minami, M., Yagasaki, R., Kinoshita, K., Earashi, M., Kitagawa, H., Taniya, T., and Miyazaki, I. Chemoprevention of DMBA-induced mammary carcinogenesis in rats by low-dose EPA and DHA.
Br. J. Cancer
,
75
:
348
–353, 
1997
.
105
Palakurthi, S. S., Fluckiger, R., Aktas, H., Changolkar, A. K., Shahsafaei, A., Harneit, S., Kilic, E., and Halperin, J. A. Inhibition of translation initiation mediates the anticancer effect of the n-3 polyunsaturated fatty acid eicosapentaenoic acid.
Cancer Res.
,
60
:
2919
–2925, 
2000
.
106
Aktas, H., Fluckiger, R., Acosta, J. A., Savage, J. M., Palakurthi, S. S., and Halperin, J. A. Depletion of intracellular Ca2+ stores, phosphorylation of eIF2α, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole.
Proc. Natl. Acad. Sci. USA
,
95
:
8280
–8285, 
1998
.
107
Mhashilkar, A. M., Schrock, R. D., Hindi, M., Liao, J., Sieger, K., Kourouma, F., Zou-Yang, X. H., Onishi, E., Takh, O., Vedvick, T. S., Fanger, G., Stewart, L., Watson, G. J., Snary, D., Fisher, P. B., Saeki, T., Roth, J. A., Ramesh, R., and Chada, S. Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.
Mol. Med.
,
7
:
271
–282, 
2001
.
108
Su, Z. Z., Madireddi, M. T., Lin, J. J., Young, C. S., Kitada, S., Reed, J. C., Goldstein, N. I., and Fisher, P. B. The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice.
Proc. Natl. Acad. Sci. USA
,
95
:
14400
–14405, 
1998
.
109
Saeki, T., Mhashilkar, A., Chada, S., Branch, C., Roth, J. A., and Ramesh, R. Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro.
Gene Ther.
,
7
:
2051
–2057, 
2000
.
110
Pataer, A., Vorburger, S. A., Barber, G. N., Chada, S., Mhashilkar, A. M., Zou-Yang, H., Stewart, A. L., Balachandran, S., Roth, J. A., Hunt, K. K., and Swisher, S. G. Adenoviral transfer of the melanoma differenti ation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR).
Cancer Res.
,
62
:
2239
–2243, 
2002
.
111
Ito, T., Warnken, S. P., and May, W. S. Protein synthesis inhibition by flavonoids: roles of eukaryotic initiation factor 2α kinases.
Biochem. Biophys. Res. Commun.
,
265
:
589
–594, 
1999
.
112
Eberle, J., Fecker, L. F., Bittner, J. U., Orfanos, C. E., and Geilen, C. C. Decreased proliferation of human melanoma cell lines caused by antisense RNA against translation factor eIF-4A1.
Br. J. Cancer
,
86
:
1957
–1962, 
2002
.
113
Polunovsky, V. A., Gingras, A. C., Sonenberg, N., Peterson, M., Tan, A., Rubins, J. B., Manivel, J. C., and Bitterman, P. B. Translational control of the antiapoptotic function of Ras.
J. Biol. Chem.
,
275
:
24776
–24780, 
2000
.
114
D’Cunha, J., Kratzke, M. G., Alter, M. D., Polunovsky, V. A., Bitterman, P. B., and Kratzke, R. A. Over-expression of the translational repressor 4E-BP1 inhibits NSCLC tumorigenicity in vivo.
Proc. Am. Assoc. Cancer Res.
,
43
:
816
–817, 
2002
.
115
DeFatta, R. J., Nathan, C. A., and De Benedetti, A. Antisense RNA to eIF4E suppresses oncogenic properties of a head and neck squamous cell carcinoma cell line.
Laryngoscope
,
110
:
928
–933, 
2000
.
116
Herbert, T. P., Fahraeus, R., Prescott, A., Lane, D. P., and Proud, C. G. Rapid induction of apoptosis mediated by peptides that bind initiation factor eIF4E.
Curr. Biol.
,
10
:
793
–796, 
2000
.
117
DeFatta, R. J., Li, Y., and De Benedetti, A. Selective killing of cancer cells based on translational control of a suicide gene.
Cancer Gene Ther.
,
9
:
573
–578, 
2002
.