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Supplement: Nutrition and Gene Regulation

Translational Regulation of Gene Expression by -3 Fatty Acids^1,2

Laboratory for Translational Research and Department of Medicine, Harvard Medical School and Brigham and Woman’s Hospital, Boston, MA 02115

³To whom correspondence should be addressed. E-mail: jose_halperin{at}hms.harvard.edu.

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

 
The incidence of some cancers shows dramatic variations around the world that cannot be explained by ethnic or racial differences. Observational studies point to a negative correlation between consumption of fish and incidence of breast and prostate cancer. In vitro and animal model studies indicate that (-3) PUFAs present at high concentrations in marine animals inhibit proliferation of cancer cells and growth of tumors. However, how these fatty acids inhibit cell proliferation and tumor growth is a matter of considerable debate. In this review we summarize our recent work indicating that Ca⁺⁺ depletion mediated phosphorylation of the alpha subunit of eIF2 and subsequent inhibition of translation initiation account for the anti-cancer activity of (-3) PUFAs.

KEY WORDS: • (-3) PUFA • translation initiation • ternary complex • cancer

The old dictum "nature vs. nurture" summarizes a dilemma that has historically permeated the scientific discourse even in modern times. It is evident that in addition to heredity, environmental factors in general and nutritional factors in particular critically influence the incidence and prevalence of a large variety of diseases including cancer, and therefore the overall health status of a population. However, understanding the complex interaction between nutrients and genes requires the knowledge and tools provided by modern molecular biology. We now know that the composition of diets critically influences the expression of many genes (1–3). Of particular interest are the long chain (-3) PUFAs since they represent one dietary component that appears to have a significant impact on the expression of specific genes (2,4,5). This article summarizes recent discoveries in the field of gene-specific regulation by (-3) PUFAs with particular emphasis on their potential use for cancer therapy and prevention.

The incidence of some cancers has increased dramatically in the last century. Several reasons, such as increased exposure to environmental carcinogens, increased life expectancy with the consequent higher risk of developing acquired genetic abnormalities, as well as the development of better detection systems, are usually advanced in attempts to explain this phenomenon. Several, albeit not all epidemiological, studies conducted in the second half of the past century pointed to a possible connection between fish consumption and the risk of cancer in general, and of prostate and breast cancer in particular [see (6) for a comprehensive review]. For example, Eskimo populations in Alaska and some Japanese populations in Northern Japan who consume diets based almost exclusively on fish seem to have a very low incidence of cancer, which increases with the "westernization" of their diet (7–12). The association between fish intake and cancer risk has been strengthened by studies like the one recently published by Dr. E. Giovannucci (13), from the Harvard School of Public Health. The authors followed a cohort of 50,000 men in the Health Professionals Follow-Up Study for almost 10 years, and studied the correlation between the risk of prostate cancer and food consumption, assessed by food frequency questionnaires validated several times with blood measurements. Remarkably, the study showed that men consuming fish more than 3 times a week almost halved the incidence of metastatic, aggressive prostate cancer, while the incidence of prostate cancer in general was not affected (13). Also, significantly lower levels of (-3) PUFAs were found in the serum of patients with prostate cancer compared to normal subjects (14), and a significantly reduced (-3)/(-6) PUFA ratio was found in the prostates of cancer patients compared to those with benign hyperplasia (15). One possible interpretation of these results is that consumption of fish, probably due to its high content of (-3) PUFA, influences the aggressiveness and metastatic potential of prostate cancer but not the incidence of prostate cancer itself. While informative, these observational population studies do not allow one to establish cause-effect relationships nor to isolate a single variable. Indeed, although other suspected dietary and nondietary factors were controlled for in the epidemiological studies, it cannot be concluded with certainty that an unconsidered factor associated with fish consumption did not account for the observed negative association between fish consumption and prostate cancer. Nevertheless, these epidemiological studies fueled the idea that (-3) PUFAs have anti-cancer effects and triggered substantial experimental work showing that (-3) PUFAs inhibit cancer cells’ proliferation in vitro and tumor growth in animal models of experimental cancer and perhaps in humans (16–24).

In our own research, the (-3) PUFA eicosapentaenoic acid [eicosapentaenoic acids (EPA)⁴], a major component of fish oils, inhibited the proliferation of a large panel of cancer cells in vitro, albeit with different efficiencies. In addition, EPA displays anti-cancer activity in animal models of cancer. Below we first summarize the anti-cancer activity of EPA in 2 cancer mouse models used in our laboratory and then discuss the role of gene-specific regulation in the anti-cancer activity of EPA.

EPA inhibits tumor growth in animal models

The anti-cancer action of EPA in vivo was tested in a syngenic and orthotopic model of mouse KLN-205 squamous cell carcinoma. DBA mice were injected with a total of 2.5 x 10⁵ KLN-205 cells into the right ventral area. Animals with visible tumors were divided into experimental and control groups and were administered either EPA (treatment, 2.5 g/kg/d) or an isocaloric amount of corn oil (control, 2.5 g/kg/d), a source of (-6) PUFAs, and the tumor size was recorded biweekly. EPA significantly reduced the tumor growth (Fig. 1A) (25).

View larger version (63K): [in this window] [in a new window]   FIGURE 1 EPA inhibits growth of KLN tumors and cyclin D1 expression in vivo. a. KLN-205 mouse squamous cell carcinoma cells were implanted subcutaneously in DB/2J mice. Treatment with either EPA (2.5 g/kg/d) or corn oil (2.5 g/kg/d) administered daily by gavage was started 4 d after injection. Each point represents the mean of tumor volume measured at the end of each week (n = 10). b. Tumor sections were immunostained with monoclonal anti-cyclin D1 antibody and horseradish peroxidase conjugated secondary antibodies [from (25)].

The molecular mechanism of the anti-cancer activity of EPA

Several hypotheses, including stabilization of membranes or liposomes and inhibition of the 5- reductase, have been advanced to explain the mechanism of the anti-cancer action of (-3) PUFAs. However, the molecular mechanism of the anti-cancer activity of (-3) PUFAs remained poorly understood until recently when we showed that inhibition of translation initiation mediates the anti-cancer effect of EPA. Below we briefly describe the translation initiation process, and then regulation of gene-specific expression by EPA at the level of translation initiation.

Translation initiation

In the initiation phase of mRNA translation, the translation initiation factor eIF2 forms a ternary complex with GTP and the initiating methionyl-tRNA (Met-tRNA_i). The eIF2 · GTP · met-tRNA_i ternary complex recruits the 40S ribosomal subunit forming the 43S preinitiation complex, which then binds to the mRNA cap with the help of other translation initiation factors. The preinitiation complex scans the 5' untranslated region (5'UTR) of mRNA for the initiator AUG codon, a process that requires the participation of several translation initiation factors including the RNA helicase eIF4A. At the AUG codon, the 60S ribosomal subunit is recruited to form the 80S ribosome. Concomitantly, GTP that was associated with the eIF2 is hydrolyzed to GDP. This GDP remains associated with the eIF2 and in order to initiate a new round of translation it must be exchanged for GTP. The GDP-GTP exchange is catalyzed by the multi-subunit guanine nucleotide exchange factor eIF2B and is inhibited when the alpha subunit of eIF2 (eIF2) is phosphorylated. Phosphorylated eIF2 has a much higher affinity for and inhibits the function of eIF2B because when bound to phosphorylated eIF2, eIF2B cannot catalyze the GDP-GTP exchange (26). Because the stoichiometric ratio of eIF2B to eIF2 in the cytosol is quite low, i.e., molecules of eIF2 are far more abundant than molecules of eIF2B, even partial phosphorylation of eIF2 is sufficient to decrease the availability of eIF2B necessary to re-cycle the eIF2 · GDP into the functional eIF2 · GTP. For this reason, even partial phosphorylation of eIF2 reduces the overall rate of translation initiation (27,28).

The eIF2 kinases are activated by perturbations in the synthesis, folding or transport of the proteins (29,30) and by depletion of endoplasmic reticulum (ER) Ca⁺⁺ stores. Indeed, it is well established that partial depletion of ER Ca⁺⁺ stores rapidly activates eIF2 kinases phosphorylating eIF2 and limiting the rate of translation initiation (27,31), however, the exact mechanism by which reduction of intracellular Ca⁺⁺ activates eIF2 kinases is not clearly understood. Figure 2 summarizes our current understanding of translation initiation and the proposed site of EPA action.

View larger version (42K): [in this window] [in a new window]   FIGURE 2 The translation initiation cascade. See text for details. The site of EPA action is indicated by the arrow. See Figure 6 and text for the molecular basis of depletion of the ternary complex by EPA.

EPA has a dual effect on intracellular homeostasis. On the one hand EPA induces a Ca⁺⁺ release from the intracellular Ca⁺⁺ stores (Fig. 3A), and on the other hand it inhibits Ca⁺⁺ influx through store-operated Ca⁺⁺ channels (SOC) in the plasma membrane. These SOC normally open in response to release of Ca⁺⁺ from internal stores and Ca⁺⁺ influx through SOC refills the Ca⁺⁺ stores, thus maintaining cellular Ca⁺⁺ homeostasis. By releasing Ca⁺⁺ from the ER stores while simultaneously closing SOC, EPA would partially deplete intracellular Ca⁺⁺ stores (25). These effects require peroxidation of EPA as they are blocked by vitamin E.

View larger version (17K): [in this window] [in a new window]   FIGURE 3 EPA releases Ca++ from ER stores and closes SOC. Fura-2 loaded 3T3 cells were treated with EPA in the presence or absence of vitamin E (A) in Ca++-free media, or with thapsigargin in Ca++-containing media to open SOC and then treated with or without EPA (B). ER targeted cameleon expressing cells were treated with EPA excited at 440 nmol/L and FRET was measured by determining the emission ratio at 530 nmol/L (YFP) vs. 480 nmol/L (CFP) (C). A and B from (25).

At the molecular level inhibition of translation initiation by EPA is most likely mediated by PKR-dependent phosphorylation of eIF2. This conclusion is based on the finding that EPA causes phosphorylation of eIF2 and inhibits translation initiation in wild-type cells but not in cells expressing either the dominant negative PKR mutant (PKR-K296) or the phosphorylation resistant mutant of eIF2 (eIF2-51A). The mutant cells are also resistant to the inhibitory effect of EPA on protein synthesis and cell growth (25).

Phosphorylation of eIF2 results in preferential downregulation of oncogenes and G1 cyclins

It is well established that several structural features influence the translational efficiency of individual mRNAs. For example, long and complex 5'UTRs are associated with inefficient translation probably because in the presence of stable secondary structures ribosomes cannot scan the entire 5'UTR to reach the AUG initiation codon with high efficiency (32,33). In contrast, mRNAs with simple, less structured 5'UTRs are translated more efficiently. Interestingly, the 5'UTR of 90% of vertebrate mRNAs encoding for most "housekeeping" proteins are between 10 and 200 bases long, mostly without a complex secondary structure and are efficiently translated ("strong" mRNAs). On the other hand, most mRNAs encoding for cell growth regulatory proteins or proto-oncogenes contain atypical 5'UTRs, which are >200 bases long and complex (34), that restrict their translational efficiency and render their translation highly dependent on the activity of translation initiation factors ("weak" mRNAs) (34). This translational inefficiency of proteins that regulate cell proliferation probably plays a crucial role in the maintenance of proper restraints on cell growth; unrestricted translation due to overexpression or disregulation of translation initiation factors increases the expression of oncogenes and results in malignant transformation. For the reasons summarized above, pharmacological or dietary interventions that restrict the rate of translation initiation by targeting translation initiation factors (such as eIF2) preferentially decrease the expression of growth-promoting proteins and oncogenes and can thereby inhibit the growth and metastatic potential of cancers (35–37).

We have shown that EPA-mediated phosphorylation of eIF2, which limits the rate of translation initiation, results in a preferential translational inhibition of G1 cyclins including cyclin D1, cyclin E, and cyclin A. Figure 4A shows that EPA inhibits the synthesis and expression of cyclin D1, cyclin E, and Ras, while having a minimal effect on the synthesis and expression of housekeeping proteins such as ß-actin or ubiquitin. Figure 4B shows that cyclin D1 expression is downregulated at the level of translation. In this experiment, cells were made quiescent by serum withdrawal for 18 h and then stimulated with the growth factor bFGF. The figure shows that in quiescent cells there is no cyclin D1 mRNA. Eight hours after mitogenic stimulation with bFGF, the expression of cyclin D1 mRNA is fully induced, and the cyclin D1 protein is synthesized at high level. In contrast, cells stimulated with bFGF in the presence of EPA show full expression of cyclin D1 mRNA but reduced synthesis of cyclin D1 protein (Fig. 4B). This experiment confirms that EPA inhibits cyclin D1 synthesis and expression at the level of translation. Importantly, this experiment also shows that EPA does not inhibit the bFGF induced mitogenic signal upstream from the transcriptional activation of cyclin D1. Furthermore, EPA also downregulated cyclin D1 expression in the tumors in vivo (Fig. 1B). Taken together, these data indicate that EPA preferentially inhibits the translation of cell cycle regulatory but not of housekeeping proteins that may account for the potent anti-cancer effects of EPA with low toxicity. Downregulation of G1 cyclins by EPA causes cell cycle arrest in the G1 phase, as would have been expected from an agent that inhibits expression of G1 cyclins (25).

View larger version (41K): [in this window] [in a new window]   FIGURE 4 EPA inhibits synthesis and expression of growth-regulatory proteins at the level of translation initiation. A. Exponentially growing NIH 3T3 cells were pulsed with [35S]Met-Cys with or without 30 mmol/L EPA for 1 h, and equal protein-containing cell lysates immunoprecipitated with anti-cyclin D1, cyclin E, Ras, ß-actin, or ubiquitin antibodies. Immunocomplexes were separated by SDS-PAGE and visualized by PhosphorImager (synthesis) or by Western blot (expression). B. Quiescent NIH 3T3 cells were stimulated with bFGF (5 µg/L)/0.1% calf serum for 8 h with or without 30 mmol/L EPA, total RNA was extracted and Northern blotted with a cyclin D1-specific probe using 18S mRNA as loading control. Parallel cultures were pulse-labeled with [35S]Met-Cys and cell lysates were immunoprecipitated with anti-cyclin D1 antibodies. C. Cellular extracts were separated by sucrose density gradient centrifugation, RNA was extracted from the eluted fractions (from bottom to top 1–15) and Northern blotted with an anti-cyclin D1 specific probe [adapted from (25)].

As we have discussed, treatment of cells with EPA limits the availability of the eIF2 · GTP · Met-tRNA_i ternary complex and decreases the overall rate of translation initiation. Scarcity of the ternary complex has different consequences for the translation of the various mRNAs. Under conditions of restricted ternary complex availability, the translation of mRNAs coding for housekeeping proteins such as ß-actin and ubiquitin is minimally reduced, while the translation of mRNAs coding for oncogenes such as cyclin D1 is dramatically reduced. Interestingly, the translational efficiency of another subset of mRNAs is significantly enhanced when the ternary complex is restricted. The mRNAs that are translated with higher efficiency when the ternary complex is limited include the activating transcription factor-4 (ATF-4) mRNA that regulates transcription of members of the ER-stress response gene cluster (38,39). The mRNA encoding for the ATF-4 is more efficiently translated under conditions of the limited ternary complex availability. This is because the presence of several upstream open reading frames (uORF) in the 5'UTR of its mRNA render translation of ATF-4 mRNA highly inefficient when the ternary complex is abundant but significantly more efficient when the ternary complex is scarce. When the 43S preinitiation complex binds to the 5' end of the ATF-4 mRNA, it scans the 5'UTR and initiates translation at the AUG codon of the first uORF, synthesizing a small peptide encoded by this uORF. By recognizing the initiation codon of the uORF, the ribosomal machinery is primed to also recognize the stop codon and dissociate. However, a fraction of the 40S ribosomal subunit remains associated with the mRNA and continues scanning towards the 3' end. If the ternary complex is abundant and can be recruited by this scanning 40S subunit, the AUG codon of the next uORF will be recognized. At the stop codon of the second uORF, the ribosome will dissociate and an even smaller fraction of the 40S ribosomal subunit that remains associated with the mRNA will recruit a new ternary complex and reinitiate protein synthesis at the AUG codon of the third uORF. Because at each stop codon the ribosome dissociates and only a small fraction of 40S ribosome remains associated with the mRNA, after the translation of the third uORF there is practically no scanning 40S subunit left on the mRNA. As a result, although the ternary complex is abundant, the translation of the bona fide ORF is very inefficient (as ribosomes fall off before they can reach it). In contrast, when the ternary complex is scarce, the probability that the ribosomal machinery would translate the second or third uORF is reduced and the likelihood for the 43S subunit to reach and translate the bona fide ORF of ATF4 is enhanced several-fold (38). As a consequence of the translational upregulation of ATF-4 expression under conditions of limited ternary complex availability, several ATF-4 target genes such as BiP and pro-apoptotic transcription factor CHOP are transcriptionally upregulated when stimuli like EPA induce phosphorylation of eIF2. Consistently, we have shown that treatment of cells with EPA induces the expression of both BiP and CHOP (Fig. 5). These results indicate that prolonged treatment of cancer cells with EPA could induce apoptosis thus contributing to its anti-cancer properties.

View larger version (58K): [in this window] [in a new window]   FIGURE 5 EPA induces expression of ATF-4 responsive genes. Exponentially growing NIH 3T3 cells were treated with the indicated concentration of EPA for 8 h, cell lysates were separated by SDS-PAGE and immunoblotted with anti-CHOP, anti-BiP and anti-ß-actin antibodies.

View larger version (58K): [in this window] [in a new window]   FIGURE 6 Diagram of EPA’s mechanism of action. EPA depleted internal Ca++ stores causing phosphorylation of eIF2 and inhibiting the activity of eIF2B thus abrogating regeneration of the eIF2 · GTP ·Met-tRNAi ternary complex.

	ACKNOWLEDGMENTS

 
The authors are grateful to Katie Hazard for editing the manuscript.

	FOOTNOTES

 
¹ Presented at the 6th Postgraduate Course on Nutrition entitled "Nutrition and Gene Regulation" Symposium at Harvard Medical School, Boston, MA, March 13–14, 2003. This symposium was supported by Conrad Taff Nutrition Educational Fund, ConAgra Foods, GlaxoSmithKline Consumer Healthcare, McNeil Nutritionals, Nestle Nutrition Institute, The Peanut Institute, Procter & Gamble Company Nutrition Science Institute, Ross Products Division–Abbott Laboratories, and Slim Fast Foods Company. The proceedings of this symposium are published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were: W. Allan Walker, Harvard Medical School, George Blackburn, Harvard Medical School, Edward Giovanucci, Harvard School of Public Health, Boston, MA, and Ian Sanderson, University of London, London, UK.

² Some of the data presented here were published in Cancer Research (2000) 60: 2919–2925. This research was supported by NIH grants 5 U19 CA87427 and 5 R01 CA78411 to J.A.H.

⁴ Abbreviations used: ATF-4, activating transcription factor 4; eIF, eukaryotic translation initiation factor; EPA, eicosapentaenoic acid; ER, endoplasmic reticulum; PKR, protein kinase r, double stranded RNA dependent protein kinase; SOC, store-operated channel; uORF, upstream open reading frame; UTR, untranslated region (of mRNA).

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