Postgenomic Global Analysis of Translational Control Induced by Oncogenic Signaling

Postgenomic global analysis of translational control induced by oncogenic signaling

Vinagolu K Rajasekhar*,1,2 and Eric C Holland*,1

1Department of Surgery (Neurosurgery), Neurology, Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021, USA; 2Department of Molecular Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021, USA

It is commonly assumed that developmental and oncogenic the parallels between development and oncogenesis also signaling achieve their phenotypic effects primarily by raise the possibility that some of the abnormalities in a directly regulating the transcriptional profile of cells. cancer cells’s proteome may be achieved by differential However, there is growing evidence that the direct effect recruitment of mRNAs to polysomes (Dua et al., 2001). on transcription may be overshadowed by differential The mRNAs that would affect the process of embryonic effects on the translational efficiency of specific existing development and oncogenesis encode proteins involved mRNA species. Global analysis of this effect using in cell-to-cell signaling, cell cycle control, and apoptosis microarrays indicates that this mechanism of controlling (Herbert et al., 2000; Polunovsky et al., 2000; Calkhoven protein production provides a highly specific, robust, and et al., 2002). This possibility is supported by the poor rapid response to oncogenic and developmental stimuli. correlation observed between protein and RNA in The mRNAs so affected encode proteins involved in cell– eucaryotic systems, where up to 20-fold changes in cell interaction, signal transduction, and growth control. protein levels can be seen without corresponding Furthermore, a large number of transcription factors alterations in mRNA abundance, and up to 30-fold capable of secondarily rearranging the transcriptional changes in mRNA levels without reflection on protein profile of the cell are controlled at this level as well. To levels (Kleijn et al., 1998; Gygi et al., 1999). what degree this translational control is either necessary While the importance of translational control in cell or sufficient for tumor formation or maintenance remains cycle, and the role of cell cycle-dependent activation of to be determined. translational initiation in transformation are known Oncogene (2004) 23, 3248–3264. doi:10.1038/sj.onc.1207546 (Lazaris-Karatzas et al., 1990; Rhoads et al., 1993; Flynn and Proud, 1996; Sonenberg and Gingras, 1998; Keywords: AKT; cancer; eiF-4E; oncogenic signal; Ras; Zimmer et al., 2000), interest in the regulatory mechan- translation isms of translational control in tumorigenesis is just being appreciated (Kleijn et al., 1998; Willis, 1999; Meric and Hunt, 2002; Watkins and Norbury, 2002; Graff and Zimmer, 2003; Ruggero and Pandolfi, 2003). Introduction Initiation of translation is the primary step that determines the rate of protein synthesis. Considerable In many ways, oncogenesis may be thought of as an progress has been made in our understanding on the abberency of development (Vogelstein and Kinzler, mechanism of translational initiation and on how 1993; Loeb et al., 2003). The mechanisms that drive modulation of general translational machinery regulates oncogenesis can be traced to an altered expression gene-specific protein synthesis (Dever, 2002). Thus, pattern of genes related to growth and development identifying the signal transduction pathways involved (Hahn and Weinberg, 2002); the ultimate effect of gene in translational control of specific mRNA species during expression is protein production, and that requires tumorigenesis is critical (Brown and Schreiber, 1996; mRNAs to be recruited to ribosomes. During early Waskiewicz et al., 1997; Proud et al., 2001; Martin, embryogenesis, rapid shifts in protein production are 2003; Shamji et al., 2003; Sonenberg and Dever, 2003; required as cells divide and differentiate in response to Gingras et al., 2004). To this end, this review will present intercellular communication. Such control may require a brief overview of the translational regulatory process, more rapid responses than can be achieved simply by emerging signaling pathways that appear to modulate differential transcription and changes in steady-state translation, and finally the prospects of global analysis total mRNA levels (Sager, 1997). Differential recruit- of translational control during induced oncogenesis. ment of existing mRNAs to polysomes could contribute to the rapid responses required in this and other signal transduction related processes (Rao et al., 1983). Thus, Translation and translational regulation in cancer

*Correspondence: VK Rajasekhar and EC Holland; The expression of many eucaryotic genes, including E-mail: [email protected] (ECH), [email protected] (VKR) some that are regulated at the transcriptional level are Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3249 also regulated at the level of translation. There are the eiF-G results in formation of a stable 48S complex. several ways that the translational efficiency of a given On most mRNAs, translation is initiated at the AUG mRNA can be modulated, such as changes in the levels codon closest to the 50end and is dependent on base and activity of protein synthesis machinery, mutations pairing with the anticodon of the Met-tRNAf and other affecting the structure of the mRNA, or altered signal initiation factors such as eiF1, eiF2, eiF5, and eiFA, transduction pathways. Unlike the prominent role of along with the hydrolysis of GTP (Sachs and Varani, base pairing between rRNA and ‘Shine Delgarno 2000; Dever, 2002). The initiation factors are then sequence preceding the initiation codon of each open released and eiF2-GDP is recycled by guanine nucleo- reading frame (ORF) in procaryotes, the first step in tide exchange factor eiF2B to eIF2-GTP. This step is eucaryotic translation, the ribosome binding to mRNA then followed by an eiF5B mediated GTP hydrolysis- in a process referred to ‘initiation of translation’, is dependent large (60S) ribosomal subunit joining. The much more complex, involving a finely tuned network of resulting 80S ribosome (upon the release of eiF5B), is initiation factors (Pestova et al., 2001). In addition to now poised to accept the first elongating aminoaceyl- initiation, elongation of the peptide chain and termina- tRNA by GTP-dependent activity of eucaryotic elongation represent the essential steps of the translation tion factor (eEF) 1. Following the peptide bond process. Various specific initiation factors, elongation formation, translocation of the ribosome relative to factors, and termination factors participate to execute the mRNA is catalysed by another GTP-dependent successfully the process of protein synthesis. activity of eEF2 bringing the next codon into position. As the termination codon is reached in this journey 0 An overview of translation towards the 3 end on the mRNA, the peptide chain releasing factors eRF1 and eRF3 bring about the Virtually all cellular mRNAs are cotranscriptionally hydrolysis of the last peptidyl-tRNA bond and release capped at their 50 termini with a characteristic 7-methyl the polypeptide. At the same time, by an unknown G(50) pppX (where X is any nucleotide) structure and regulatory mechanism, the eiF3 dissociates the ribosome many of them contain poly(A) chain at the 30end. A into its subunit particles, which become available for detailed account of various effectors mediating mRNA another round of translation. recruitment to ribosomes has been described (Gingras et al., 1999a). Binding of eucaryotic initiation factor Role of eiF-4E (eiF)-4E to the cap structure in the mRNA is the earliest event in the initiation of translation and also appears to Nearly 80–85% of the eiF-4E present in 48S initiation be the rate-limiting step in the translation per se. Then, complex is phosphorylated, in contrast to 50% phos- the eiF-4E delivers cellular mRNAs to a heterotrimeric phorylation of the uncomplexed eiF-4E. Although eiF- eiF-4F complex to facilitate the 43S ribosome recruit- 4E phosphorylation increases the rate of protein ment. Thus, in addition to eiF-4E, the eiF-4F complex is synthesis up to four-fold in the majority of cases, composed of eiF-4A (the ATP-dependent RNA helicase dephosphorylation of eiF-4E also induces eiF-4F for- facilitating secondary structure melting) and eiF-G (the mation and protein synthesis in other cases, suggesting scaffolding protein that binds eiF-4E and eiF-4A). only enhancing but nonessential function of phosphor- Association of eiF-G with eiF-4E appears to increase ylation of eiF-4E (Kleijn et al., 1998; McKendrick et al., the affinity between eiF-4E and the cap by at least 10- 2001). This contention gets support from the observa- fold. Furthermore, the eiF-G interacts with poly(A) tions that only upon binding to eiF-4G does eiF-4E binding protein (PABP), which binds the 30 end poly(A) appear to get phosphorylated to enhance translation tail of the mRNA, the eiF-4E phosphorylating kinase (Flynn and Proud, 1996; Pyronnet et al., 1999; MNK1, and the eiF3 multiprotein complex. Of interest, Waskiewicz et al., 1997). An activity-dependent switch PABP also interacts with other PABP binding proteins to cap-independent translation is also reported to be (Paips), eiF-4B, which also directly interacts with eiF-4F triggered by eiF-4E dephosphorylation (Dyer et al., and aids in the processitivity of eiF-4A, and eucaryotic 2003). It is interesting to note that there is a little or no releasing factor (eRF) 3. These functional interactions at role for phosphorylation of eiF-4E during cell differ- the two termini lead to the circularization of the mRNA, rentiation (Kleijn et al., 1998), while the phosphoryla- a functional prerequisite for efficient translation initiation is critical in cell growth (Lachance et al., 2002). tion (Sachs and Varani, 2000). The eiF-4F complex then Conversely, treatment with peptides based on conserved scans from 50 cap down stream towards AUG start eiF-4E binding motifs in eiF-4G and 4E-BPs that codon, by transiently melting secondary structures near specifically prevent engagement of eiF-4E with eiF-4G the 50 end with the help of additional RNA binding rapidly induced cell death (Herbert et al., 2000). Since proteins eiF-4B and eiF-4 H. This unwinding enables substitutions in the peptides that disrupt binding to eiF- eiF3 to access the below described preinitiation com- 4E do not cause apoptosis, an additional direct role for plex. The eiF2-GTP mediates the binding of the eiF-4E in survival unlinked to its known role in initiating methionyl-tRNA (Met-tRNAf) onto the translation is possible. It remains to be seen if the smaller (40S) subunit of the ribosome by forming a phosphorylated eiF-4E by some mechanism may confer ternary complex and this process is enhanced by specificity in enhancing the translation rates of malig- additional factors, eiF3 and eiF1A, that contribute to nancy-related mRNAs relative to others. Since a the formation of 43S preinitiation complex. Binding to fraction of eiF-4E is also colocalized in the nucleus

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3250 with splicing factors in speckles, eiF-4E has been 4E levels (DeFatta et al., 2000; Nathan et al., 2000), or suggested to involve additionally processing a specific overexpressing 4E-BP1, thereby preventing the avail- subset of mRNAs in the nucleus and/or exporting them ability of free eiF-4E to form the eiF-4F complex into the cytoplasm (Dostie et al., 2000). As nearly 50% (Rousseau et al., 1996; Graff and Zimmer, 2003), of the human transcripts involve alternative splicing suppresses tumorigenicity and angiogenesis. Dysregula- (Modrek and Lee, 2003), it is possible that enhanced tion and overexpression of several translational initia- accumulation of eiF-4E may have a causal link to the tion factors, elongation factors, and other mRNA aberrant splicing of certain growth-promoting and binding proteins related to translation-enhancing func- oncogenic mRNAs, comparable to the splicing defects tion, such as YB-1 and ribosomal proteins, have also recorded in various forms of cancer (Xu and Lee, 2003). been observed in a large number of cancers and cancer Overexpression of eiF-4E increases cyclin D1 produc- types (Table 1), underscoring the significance of trans- tion by stimulating its mRNA export (Rousseau et al., lational activation in tumor formation and invasion. 1996). It is not known if eiF-4E stimulates the mRNA export directly through its association with the cap from Cis-acting elements within an mRNA structure controlling the nucleus or indirectly by increasing the translation of translation and involvement of micro RNAs specific mRNAs. In any case, the accumulation of eiF- 4E readies certain mRNA for preferential translation Apart from the components of translational machinery, instantly upon a trigger of signaling. The S6 ribosomal the efficiency of protein synthesis can also be regulated protein (S6RP) of the 40S ribosomal subunit is by various structural features of the mRNAs, particu- implicated in RNA binding and upon phosphorylation, larly at the 50 and 30 termini (Wilkie et al., 2003). These and it is also suggested to enhance the translation of a include (1) long and structured 50-untranslated regions subset of the mRNAs that contain 50 terminal oligopyr- (UTRs), (2) unusual or multiple upstream initiation imidine tract (TOP) (Avni et al., 1997; Meyuhas, 2000). codons, (3) upstream ORFs, (4) internal ribosome entry The TOP mRNAs encode translation elongation factors sites (IRES), and (5) 50-TOP sequences. For example, and many ribosomal proteins. the majority of the growth factor-encoding transcripts contain 100–150 base long sequences in 50UTR that are Critical protein–protein interactions in translation up to 90% GC rich and have the potential to form initiation extended stem loop structures, thereby requiring the need for the eiF-4E-dependent translation initiation Two sets of protein interactions play vital roles in (Clemens and Bommer, 1999). FGF2 and c-Myc translation initiation: (1) the eiF-4E binding proteins mRNAs utilize additional and unusual initiation codons (4E-BPs) compete with eiF-4G for binding to eiF-4E such as CUG to synthesize proteins depending on and inhibit the translation initiation. Hierarchical cellular conditions (Willis, 1999). The mRNAs of many phosphorylation of 4E-BP prevents its binding to eiF- oncogenes contain long and structured areas within the 4E and thereby relieves its inhibitory effect on eiF-4E 50UTR and feature a polypyrimidine tract towards the 30 (Gingras et al., 1999b), and (2) eiF2B is a heterotrimeric end, which are utilized as IRES. Such transcripts employ guanine nucleotide exchange factor (GEF) for eiF2. In cap-independent translation initiation in the absence of contrast to monomeric GEFs for Ras and related G some or all initiation factors. Short and unstructured 50 proteins, eiF-2B is a heteromic complex of five subunits. UTRs about 40 nucleotides in length, which also bear Phosphorylation of eiF2a by eiF2a protein kinase, PKR up to 14 consecutive oligopyrimidine residues help, in converts eiF2 from a substrate to a competitive inhibitor growth-dependent translation of ribosomal proteins of eiF2B. Consequently, formation of a complex with a casual relationship to phosphorylation of S6RP between eiF2-GTP–Met.tRNA does not take place (Meyuhas, 2000). Similarly, in quiescent cells, where and global protein synthesis is inhibited. Translation eiF-4E activity is repressed and global translation inhibition of specific mRNAs such as GCN4, ATF4, declines, p27Kip1 translation is highest utilizing an and C/EBP occurs in addition to gene-specific control of IRES in the 50UTR of the mRNA (Miskimins et al., translation by specialized mRNA binding proteins 2001). Distinct adenosine uridine-rich elements (ARE) (Dever, 2002; Ostareck et al., 2001). are found in the 30 UTR of many mRNAs encoding inflammatory cytokines and growth factors, which Translation and cancer influence the stability of their own mRNAs in breast cancer (Balmer et al., 2001). Overexpression of translation initiation factors like eiF- Other distinct sequences that regulate mRNA locali- 4E and eiF-4G, or dominant-negative PKR causes zation are found in the 30UTR, which appear to function neoplastic cellular transformation in fibroblast and by interacting with transacting and noncoding small epithelial model cell systems (Lazaris-Karatzas et al., RNA, referred to as microRNA (miRNA) (Baehrecke, 1990; Clemens and Bommer, 1999; De Benedetti and 2003; Lai, 2003). The miRNAs are endogenously Harris, 1999; Graff and Zimmer, 2003). The observed encoded, produced by processing of small single- eiF-4E-induced transformation may result from in- stranded hairpin precursor transcript, and shown to creased translation of mRNAs encoding oncogenic function as antisense regulators of gene expression proteins (Rosenwald et al., 1993, 1995; Rousseau et al., (Ambros, 2003) . Recognition and translational inhibi- 1996; Shantz et al., 1996). Conversely, reducing the eiF- tion of the target mRNAs via imprecise antisense base

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3251 Table 1 Implication of factors associated with translation in oncogenesisa Factor Effect Cancer type Reference eIF2 a Protein increase Non-Hodgkin’s lymphoma Wang et al. (1999) Gastro-intestinal Lobo et al. (2000) Squamous cell lung Bauer et al. (2001) Melanoma Rosenwald et al. (2003) eIF3a (p170) Protein increase Lung, Breast, Cervical and Esophageal Pincheira et al. (2001) eIF3b (p116) Protein increase Breast Lin et al. (2001) eIF3c (p110) mRNA increase Testicular Rothe et al. (2000) eIF3e (p48) mRNA decrease Breast and Non-small cell lung Marchetti et al. (2001) eIF3h (p40) mRNA increase Prostate and Breast Nupponen et al. (1999) eIF4GI Gene amplification Squamous cell lung Bauer et al. (2002) eIF4AI mRNA increase Melanoma Eberle et al. (1997) Liver Shuda et al. (2000) eIF4E Gene amplification Head and neck, Breast Sorrells et al. (1999) Sorrells et al. (1998) mRNA increase Liver, Bladder Shuda et al. (2000) Berkel et al. (2001) Protein increase Colon, Head and Neck, Breast, Wang et al. (1999) Non-Hodgkin’s lymphoma, Larynx eIF5A2 Gene amplification Ovarian Guan et al. (2001) 4E-BP1 Protein decrease Gastrointestinal Martin et al. (2000) PKR Protein increase Breast, Colon and Melanoma Kim et al. (2000, 2002) YB-1 Protein increase Brain Grade IV Del Valle et al. (2000) Breast Bargou et al. (1997) Colorectal Shibao et al. (1999) TACC1 mRNA decrease Lung, Ovarian Conte et al. (2002) Protein decrease Uterus, Breast Conte et al. (2002) EF1 mRNA overexpression Epithelial, Mammary Kolettas et al. (1998) Edmonds et al. (1996) mRNA overexpression Oesophageal Mimori et al. (1996) mRNA overexpression Colorectal Ender et al. (1993) mRNA overexpression Gastric Mimori et al. (1995) Protein overexpression Colorectal Mathur et al. (1998) RPL 31 mRNA overexpression Colorectal Chester et al. (1989) RPL 18, 37, mRNA overexpression Colorectal Barnard et al. (1993) RPS 6 RPL 19 Protein overexpression Breast Beugnet et al. (2003) aModified after Watkins and Norbury (2002). pairing is one of the mechanisms of miRNA function be associated with various oncogenic phenotypes (Wightman et al., 1993; Olsen and Ambros, 1999; (Table 2). Seggerson et al., 2002). Extensive regulation of miRNAs occurs during brain development. Several miRNAs are Trans-acting mRNA binding factors regulating translation associated with polyribosomes in primary neurons, where they are likely to modulate translation (Krichevs- It is not possible to review comprehensively all that is ky et al., 2003). The miRNAs also associate with distinct known about 50 and 30 UTR binding proteins and how ribonucleoproteins affecting preferential translational the cognate cis-acting regulatory sequence elements in initiation (O’Donnell and Warren, 2002). These data the mRNAs react to affect the final protein production. suggest a specificity factor role in translation analogous However, identifying and understanding the regulation to the function of let-7 miRNA in repressing the of functionally related and dynamic links between 50 and translation via 30UTR of hunchback in the Caenorhab- 30 UTRs is the functional theme in the context of current ditis elegans nervous system (Abrahante et al., 2003; Lin cancer biology. For example, recent identification of et al., 2003). Interestingly, even the short interfering translational repression via a cytoplasmic polyadenyla- RNA (siRNA) mediated translational repression, which tion element (CPE) within the 30UTR of some messages also occur via partially complementary binding sites in like that of cyclin B1 opened up novel regulatory the 30UTR of mRNAs (Doench et al., 2003), addition- pathways for selective translational control influencing ally requires eiF2C translation initiation factors (Doi the eiF-4E-dependent translation initiation (Groisman et al., 2003). While some of the miRNAs such as bantam et al., 2002; Wilkie et al., 2003). A protein that binds to are implicated in cell proliferation via translational CPE (CPEB), depending on its phosphorylation status, repression due to interaction with the conserved 30UTR performs a dual control in translation, either activation of mRNAs (Brennecke et al., 2003), frequent deletions or repression. The CPEB-mediated repression is accom- and down regulation of miRNA genes for miR15 and plished while in its dephosphorylated state by an miR16 at 13q14 in human chronic lymphocytic leukemia interaction with TACC family proteins and its subse- suggest an additional tumor suppressor role for some of quent interaction with eiF-4E. The TACC protein with the miRNAs (Calin et al., 2002). Several transcripts with sequence similarity to eiF-4G and 4E-BP binds to eiF- structural features affecting translation were reported to 4E and thus prevents the formation of eiF-4F complex.

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3252 Table 2 Translationally regulated mRNAs encoding proteins related to oncogenesisa mRNA Mechanism of control Aberrant regulation in References

I Associated with cancer

a- Globin 30-UTR Erythroleukemia Henics (2000) Androgen receptor 50-UTR Prostate cancer Clemens and Bommer (1999) Apobec-1 uAUGs Colon cancer Anant et al. (2002) BRCA 1 50- UTR Breast cancer Signori et al. (2001) c-myc additional AUG, IRES, and Burkitt’s lymphoma, Clemens and Bommer (1999), mRNA degradation Bloom’s syndrome, Chappell et al. (2000) Multiple myeloma EGFRVIII Alternative splicing Lung/breast cancer Wikstrand et al. (1995) FGFR1 Alternative splicing Glioblastoma Xu and Lee (2003) FGF-2 Additional AUGs, IRES Breast carcinoma Clemens and Bommer (1999) Growth-related P23 protein 50-UTR Ehrlich ascites tumor Bohm et al. (1991) HER-2/neu Alternative splicing Ovarian carcinoma Doherty et al. (1999) Insulin-like growth factor II 50-UTR Wilm’s tumour, Rhabdosarcoma, Clemens and Bommer (1999) Embryonal carcinoma IL-15 Additional AUGs T-cell leukemia Clemens and Bommer (1999) LDHC Alternative splicing Melanoma/lung cancer Xu and Lee (2003) MUC4 Alternative splicing Pancreatic tumor Choudhury et al. (2000) NABC 1 Alternative splicing Breast cancer Xu and Lee (2003) P16 INK4A 50-UTR Melanoma Liu et al. (1999) P27 Kip1, Id4, Miz-1, Atm, Vegf, Cap-dependent translation Mouse GBM cells Rajasekhar et al. (2003) Pak3, IgfBP4, IgfBP5, Notch1 Prohibitin 30-UTR Breast cancer Manjeshwar et al. (2003) Ovarian cancer Spurdle et al. (2003) TGF-b family 50-UTR uORF Phaeochromocytoma, Clemens and Bommer (1999) Adenocarcinomas PDGF2 50-UTR Teratocarcinoma Wang and Stiles (1993) PTI-1 oncogene 50-UTR Prostate cancer Gopalkrishnan et al. (1999) Ribosomal proteins 50TOP Colorectal cancer Clemens and Bommer (1999) Elongation factors 50TOP Mammary and colon Clemens and Bommer (1999) Adenocarcinomas eIF-4G IRES (?) Squamous lung carcinoma Clemens and Bommer (1999)

II. With the potential to be associated with cancer

AML1/RUNX1 Cap and IRES Pozner et al. (2000) Apaf1 IRES Coldwell et al. (2000) ATM 50-UTR/30-UTR Savitsky et al. (1997) BDGF Additional AUGs, IRES Clemens and Bommer (1999) Bax 50-UTR Saxena et al. (2002) c-fos mRNA degradation Clemens and Bommer (1999) c-mos uORF Clemens and Bommer (1999) CDK4 50-UTR Clemens and Bommer (1999) C/EBP 50-UTR Struyk et al. (2000) 30-UTR Le Cam and Legraverend (1995) Cyclin D1 50-UTR Clemens and Bommer (1999) Cyclooxygenase 30-UTR Sawaoka et al. (2003) DNA methyl transferase 30-UTR Detich et al. (2001) ERCC1 50-UTR Yu et al. (2001) Fas/Apo-1 uORF Clemens and Bommer (1999) GDNF 50-UTR Tanaka et al. (2001) HGR-2/neu uORF Child et al. (1999) Lck proto-oncogene 50-UTR Marth et al. (1988) Mdm2 uORF Brown et al. (1999) MRP1 50-UTR Kurz et al. (2001) Ornithine decarboxylase 50-30-UTR Grens and Schefﬂer (1990) P53 50-UTR Clemens and Bommer (1999) Pim-1 50-UTR Hoover et al. (1997) PTEN 50-UTR Han et al. (2003) Retinoic acid receptor-2 uORF Clemens and Bommer (1999) Src 30-UTR Brown and Schreiber (1996) Tau 30-UTR Aronov et al. (1999) VEGF 30-UTR Abe et al. (2002)

aModiﬁed after Clemens and Bommer (1999), Watkins and Norbury (2002), and Graff and Zimmer (2003)

By contrast, in its phosphorylated state CPEB interacts cleotide element AAUAAA. The CPSF in turn interacts with a cleavage and polyadenylation speciﬁcity factor with poly (A) polymerase (PAP) catalysing the poly(A) (CPSF) that recognizes the CPE downstream hexanu- addition. A protein that binds to the newly elongating

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3253 poly(A) chain (PABP) also interacts with eiF-4G Signaling pathways that affect translational control in resulting in a complex that effectively displaces TACC oncogenesis from eiF-4E allowing the eiF-4F complex formation. As the cells begin to exit the M-phase of the cell cycle, a Signaling pathways that regulate development are phosphatase inactivates CPEB, leading to deadenylation not only frequently dysregulated in neoplastic prolifera- and loss of PABP as well as a reassociation of TACC tion but also causally related to cancer formation in with eiF-4E, and thereby translational silencing as has experimental systems. However, it is not clear how the been demonstrated for translation control of cyclin B1 specificity and complexity of cellular response to a mRNA. Apart from the dynamic regulatory control of particular signaling pathway is achieved during CPEB, its interacting partner TACC1 is downregulated oncogenesis. The growing number of indentified in several human cancers (Conte et al., 2002), which may signaling components continue to complicate the represent additional mechanisms for escaping transla- attempts to dissect the cancer-specific molecular signal- tional repression and preventing mitotic exit. Alterna- ing pathways. Although there are likely to be transla- tive pathways to the above-described cap-dependent tional effects of many if not all of the signaling pathways translation initiation also exist and utilize an IRES in involved in embryogenesis and oncogenesis, the ones the mRNAs in the absence of some or all initiation best documented to date are the tyrosine and seriene factors. kinase receptors, and cytoplasmic protein tyrosine kinases. Specific mRNAs that encode proteins involved in both development and cancer are regulated in part at the Receptor serine kinase signaling translational level Members of transforming the growth factor beta (TGF- It would not be surprising if the production of critical b) superfamily represent transmembrane receptors that proteins was regulated at many levels in order to are activated as serine kinases by docking with cognate maintain tight control over the rapid changes that occur ligands such as TGF-b and bone morphogenetic during early embryogenensis. There are many examples proteins (BMPs). There receptors signal through in- of mRNAs encoding proteins involved in signal tracellular transducers called Smads (R-smads). Most transduction and cell cycle control that are regulated cancers retain TGF-b receptors, but attenuate TGF-b- at many levels, including translation. For example, the mediated antimitogenic effects through autocrine and insulin-like growth factor (IGF) ligand signals through paracrine signaling. Translational initiation on a num- the IGF-II receptor and is mediated by a family of ber of growth receptor mRNAs, including that of TGF- binding proteins known as the IGF binding proteins b, is specifically regulated during growth, differentia- (IGFBPs) (el-Badry et al., 1991; Nielsen et al., 1995). tion, and stress (van der Velden and Thomas, 1999; van Recruitment of the mRNAs for IGFBP 4 and IGFBP5 der Velden et al., 2000). to ribosomes has been shown to be strongly dependent Apart from substantial focus on transcriptional on oncogenic signaling through the Ras and Akt events, involvement of post-transcriptional effects also pathways (Rajasekhar et al, 2003). Aberrant Notch plays a role in TGF-b-mediated oncogenesis as illu- signaling is associated with the development of human strated below. The eiF2 a subunit is associated with cancers (Allenspach et al., 2002; Nickoloff et al., 2003). kinase inactive TGF-b II R (McGonigle et al., 2002). Dysregulated expression of a series of tumor-specific Insulin via insulin growth factor receptor (IGFR) 50-deleted Notch1 mRNA transcripts in a subset of regulates the 50UTR of TGF-b 1 to induce translation- lymphoid neoplasms (Aster and Pear, 2001) as well ally synthesis of TGF-b 1 without affecting its mRNA as lack of correlation between the levels of Notch stability, at least during renal interstitial fibrosis mRNAs and proteins in different layers of basal cell (Morrisey et al., 2001), indicating that controls beyond carcinoma (Lowell et al., 2000; Nickoloff et al., 2002) transcription may participate in regulating the TGF-b suggest that some of the control over Notch protein pathway. The normal mRNA for TGF-b 3 contains a production rests at the level of translation. Furthermore, long 50UTR that exerts a potent inhibitory effect on its neoplastic transformation by Notch is independent translational efficiency and is deleted in a transcript with of transcriptional activation (Dumont et al., 2000), a considerably foreshortened 50UTR found in human suggesting involvement of post-transcriptional and breast cancer cells. This latter transcript is more actively translational elements in this process. While levels of engaged in translation on polysomes (Arrick et al., the mRNA encoding the cyclin-dependent kinase 1991). TGF-b also promotes attachment and spreading inhibitor p27Kip1 remain constant during cell cycle of malignant astrocytoma cells by increasing paxillin exit and maintenance in the quiescent state, p27 protein production through an enhancement in transla- protein accumulates to large amounts by a modulation unassociated with changes in steady-state levels of tion of translational efficiency (Agrawal et al., 1996; paxillin mRNA (Han et al., 2001). Ectopic expression of Hengst and Reed, 1996). These are but a few examples eiF-4E in human colon cancer cells promotes the TGF-b of this effect; a more complete list can be found stimulation of adhesion molecules (Rajagopal and in Table 2. In addition to translational control, Chakrabarty, 2001). Collagen mRNA translation is also expression of these genes is frequently regulated at enhanced by TGF-b in an anaplastic thyroid carcinoma other levels as well. model system (Dahlman et al., 2002). TGF-b stimulates

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3254 multiple protein interactions with a unique cis element sphorylation of the receptor subunits mediating speciﬁc in the 30UTR of the mRNA encoding the receptor for binding of cytoplasmic signaling proteins containing hyaluronan-mediated motility (RHAMM), whose ex- (SH2) and protein tyrosine kinase binding domains such pression is elevated in transformed cells (Amara et al., as growth factor receptor binding protein (Grb), 1996). Additionally, TGF-b signaling acts on the 50UTR RasGAP, and lipid kinase phosphoinositide 3-OH of cyclin-dependent kinase 4 (cdk4) to block the kinase (PI3 kinase) and activate various downstream progression through G1 (Miller et al., 2000). signaling events.

Cytoplasmic protein tyrosine kinase (CPTK) signaling Ras/Raf/Erk pathway c-Abl is one of nearly 32 known nonreceptor CPTKs, The Ras gene family represents some of the most several of which have been implicated in human cancers frequently mutated oncogenes in various human tumors. (Blume-Jensen and Hunter, 2001). c-Abl directly binds While K-ras oncogenes are found in almost 90% of to the mammalian target of rapamycin (mTOR) and pancreatic cancers, and 50% of colon cancers, and 25% inhibits its autophosphorylation, resulting in the inhibi- of adenomas, nonmutated but pathologically elevated tion of p70S6 kinase (Kumar et al., 2000). The c-Abl Ras activity is found in other tumors such as glioblas- protein consists of SH(Src-homology)3, SH2, PTK, toma. Activated Ras interacts with more than 20 DNA binding, and actin binding domains, among effectors, including Raf-1 serine/threonine kinase, PI3 others. While the c-Abl associates with ataxia–telan- kinase, and Ral GDS, which are found to participate in giectasia mutated (ATM), which may contribute to the various cancers. activation of c-Abl in response to DNA damage and Raf proteins are directly activated by Ras and relay inhibition of 50 cap-dependent translation, ATM signal information to MAPK/Erk kinase (Mek) and also independently phosphorylates 4E-BP1 (Yang and then to Erk. Mutations in Raf-1 genes were found in Kastan, 2000) with the significance to be understood. 70% of human malignant melanomas (Mercer and The transforming effects of other CPTKs, Bcr/Abl and Pritchard, 2003). Activated Erk phosphorylates various Janus PTKs (JAKs), are also mediated by Ras/Raf/Erk factors involved in transcription and translation, tissue pathway and PI3 kinase pathways (Blume-Jensen and invasion, and metastasis, apoptosis evasion, and pro- Hunter, 2001; Verma et al., 2003). In particular, Bcr/abl liferation. For example, Erk activates several transcrip- increases the translation of mdm2 mRNA by facilitating tion factors, including the Ets family members and myc, the interaction of La antigen (in tyrosine kinase- and consequently increases transcription of multiple dependent manner) with the 50UTR of the mRNA genes, some of them encoding RPTKs such as EphA2. (Trotta et al., 2003). Upregulation of eiF-4E and eiF-2a These in turn down regulate wild-type (but not was found in v-Src- and v-Abl-transformed cells oncogenic) Ras via p120RasGAP (Miao et al., 1996), (Rosenwald, 1996). These data suggest that the transla- and cMyc target genes such as eiF-4E. Erk also tional controls may be integral components of the phosphorylates eiF-4E via Mnk to enhance initiation effects achieved by CPTK signaling. of translation and/or via translational control of the antiapoptotic function (Polunovsky et al., 2000) of caspase 9 to inhibit its proteolytic cleavage (Allan Signaling networks, crosstalk, and scaffolds involving Ras et al., 2003). More interestingly, Ras/Raf-1/Erk and Akt pathways during translational control in pathway is organized by a variety of scaffolding proteins oncogenesis to insulate a specific portion of the pathway. For example, Raf kinase inhibitor protein (RKIP) negatively Owing to cell–matrix and cell–cell interactions, cells regulates the pathway by blocking the interaction perceive signals through many different growth factor between Raf-1 with Mek and is inactivated upon receptors. They integrate signal information appropri- phosphorylation by activated Erk. This positive feed- ately into various transcription, translation, and post- back loop occurs in breast and prostrate cancers, where transcriptional/translational processes to regulate even- RKIP expression was found to be downregulated tually diverse vital steps in cell growth, differentiation, (Martin, 2003). This effect is accomplished by suppres- and proliferation. Depending on developmental status sion of Ras-stimulated transformation via c-jun NH2- and cellular context, different cell types generate terminal kinase (JNK) signal transduction pathway different outcomes through a complex network (Guerra (Kennedy et al., 2003), or Raf-1-independent deregula- et al., 2003; Ptashne and Gann, 2003). tion of p38 MAPK cascade (Pruitt et al., 2002). There are many prototypic growth factor tyrosine Activation of eiF2B and phosphorylation of 4E-BPs as kinase receptors that are involved in oncogenic signal- well as eiF-4G by insulin treatment (Proud and Denton, ing, including EGFR, platelet-derived growth factor 1997; von Manteuffel et al., 1997; Kleijn et al., 1998), receptor (PDGFR), vascular endothelial growth factor phosphorylation of eiF-4B and eiF-4G via EGF- receptor (VEGFR), fibroblast growth factor receptor mediated stimulation of S6 Kinase (Wolthuis et al., (FGFR), hepatocyte growth factor receptor (HGFR), 1993; Kleijn et al., 1998), and phosphorylation of and IGFR, (Blume-Jensen and Hunter, 2001). Signaling 4E-BP1 and increase in protein synthesis (Wang and by RPTKs involves cognate ligand-induced receptor Proud, 2002) are mechanisms of Ras-mediated transla- dimerization or oligomerization, tyrosine autopho- tional control in oncogenesis.

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3255 PI3 kinase/Akt pathway and tuberin (TSC2) heterodimer proteins associated with an autosomal dominant genetic disorder, tuberous This pathway represents an important node transducing sclerosis (TSC). The TSC1/2 acts to constrain mitogen- the information of the multiple cellular signaling path- induced mTOR downstream signaling pathway (Tee ways initiated by PI3 kinase (Vivanco and Sawyers, et al., 2003) and leading to inhibition of protein 2002; Luo et al., 2003). The PI3 kinase is not only translation. Phosphorylation of the TSC2 by Akt activated by growth factor-dependent RPTK (class 1A disrupts TSC2 binding to TSC1 and relieves this subgroup) and G-protein-coupled receptors (class 1B repression (Manning and Cantley, 2003). In the absence subgroup) but also by direct interaction with oncogenic of Akt-mediated phosphorylation, TSC2 in the hetero- Ras. PI3 kinase is a heterodimeric enzyme consisting of dimer complex, through its C-terminal GAP domain, a regulatory p85 subunit and a catalytic p110 subunit. directly functions as a GTPase activating protein The p85 is encoded by three genes, a, b, and g, and (GAP) of a small G protein, Rheb (Ras homologue regulated by alternative transcript splicing. The gene is enriched in brain) (Garami et al., 2003; Inoki et al., mutated in ovarian and digestive tract tumors. The p110 2003; Stocker et al., 2003). Activated Rheb (Rheb-GTP) catalytic subunit is also encoded by three genes, a, b, requires membrane localization by farnesylation to and g and this gene is amplified in human ovarian activate specifically the mTOR/S6K/4E-BP1 pathway cancers. Upon activation, PI3 kinase phosphorylates (Tee et al., 2002). Rheb-GTP was shown to transform phosphoinosides (PI), a process reversed through the NIH3T3 cells as wild-type Raf1 or H-ras (Yee and dephosphorylation of 30 PI by the tumor suppressor Worley, 1997). Impaired Rheb function is sufficient to phosphatase (PTEN). The gene encoding PTEN is suppress the phenotypic consequences of PTEN or mutated or silenced in a wide range of human tumor TSC1/2 loss (Stocker et al., 2003). types. PI3 kinase also exists in three classes with multiple Activated mTOR regulates translation through hier- subunit isoforms. So far, only the class IA subgroup has archical phosphorylation either by activation of p70S6 been shown to be involved in oncogensis. The 30 PI- Kinase (S6K1 and S6K2), implicated in the increased dependent serine/threonine kinase, PDK1, and Akt translation of 50TOP mRNAs, and/or by inactivation of (cellular homolog of retroviral v-Akt) sequentially 4E-BP1 that inhibits cap-dependent translational initia- initiate a kinase cascade that plays a central role in tion. Recently, a regulatory associated protein of growth regulation. Of the three ubiquitously expressing mTOR, raptor (Hara et al., 2002; Kim et al., 2002), Akt isoforms, Akt1 is the dominant form in all tissues, has been identified as an mTOR scaffold protein. Akt2 is expressed in insulin-responsive tissues such as Raptor also associates with p70S6 kinase and 4E-BP1 skeletal muscle, heart, liver and kidney and Akt3 is through their respective C-terminal conserved TOR higher in the testis and brain. Although dimer- or tri- or signaling (TOS) motifs (Choi et al., 2003; Nojima et al., even multimerization of Akt is required for Akt 2003; Schalm et al., 2003). More recently, a new protein activity, it is not known if the homo or hetero- named GbL has been identified as an additional merizations of Akt plays any role in oncogenesis. Akt component of the mTOR signaling complex. GbL binds activity is indirectly inhibited by PTEN, activated by the to the kinase domain of mTOR independent of raptor Src family tyrosine kinase Lyn and a carboxy terminal and stimulates the kinase activity of raptor towards modular protein (CTMP). The chaperone heat-shock itself or towards p70S6 kinase and 4E-BP1 (Kim et al., protein (HSP90) protects Akt from dephosphorylation 2003). GbL also stabilizes the interaction between raptor by PP2A. Deregulated Akt is oncogenic; the gene is and mTOR, while facilitating a nutrient sensitive raptor- amplified in ovarian, pancreatic, and breast tumors and mTOR/GbL complex formation that decreases mTOR the activity is constitutively increased in brain and kinase activity during nutrient-limiting conditions (Kim thyroid tumors (Choe et al., 2003). Akt phosphorylates et al., 2003). This provides a mechanism by which several downstream targets and achieves many biologi- cellular responses regulate mTOR signaling to modulate cal effects related to survival, proliferation, and cell the translational control. Without affecting the 4E-BP1 growth. For example, Akt-mediated phosphorylation- phosphorylation status, Akt also induces transforma- dependent inactivation of proapoptotic BAD and tion by reducing synthesis of YB-1, a protein that binds caspase-9, or inhibition of nuclear localization of mRNA as well as inhibits both cap-dependent and forkhead transcription factor (FKHP), will facilitate IRES-dependent translation (Aoki et al., 2001; Martin survival. In addition, Akt also phosphorylates GSK3b de la Vega et al., 2001). For example, growth factor and thereby prevents it from phosphorylating and induction of cyclin D expression is controlled at the targeting cyclin D1 for degradation or WAF1 (CIP1) translational level by PI3 kinase/Akt/mTOR pathway from binding to proliferating cell nuclear antigen (Muise-Helmericks et al., 1998). PI3 kinase-dependent (PCNA). but Akt-independent pathways (such as the activation of The growth control function of Akt is largely small GTP binding proteins CDC42/Rac1, protein accomplished by regulating translation via activation kinase C, and the serum and glucocorticoid-inducible of its downstream kinase mammalian target of rapamy- kinase (SGK), or PI3 kinase-independent phosphoryla- cin (mTOR)-dependent mTOR/S6K/4E-BP1 signal tion of Akt (e.g. via elevated cyclic AMP-mediated transduction pathway. The activation of mTOR by protein kinase A) also enhance translation (Blume- Akt appears indirect and may involve a tumor Jensen and Hunter, 2001). Their effect on tumorigenesis suppressor complex composed of hamartin (TSC1) remains unknown.

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3256 Ral GDS- and Rho GTPase-mediated pathways behavior (Evers et al., 2000). Downregulation of Rac activity by oncogenic Ras results in enhanced Rho Small GTPases function as critical transducers of extra- activity and epithelial mesenchymal transition (Zondag and intracellular signals, by cycling between inactive et al., 2000). GDP and active GTP bound forms. Ral (A and B) is a Both RalGDS and Rho pathways seem to achieve member of the Ras subfamily of small GTPases and some of their onogenic effects by affecting translational functions downstream of Ras. Ras-GTP recruits guanine efficiency. Rgr, a homologue of RalGDS, induces cell nucleotide exchange factor for Ral (Ral-GEF) to the proliferation, transformation, and gene expression by plasma membrane, which then promotes membrane activating Ras-, Ral-, and Rho- mediated pathways localization of Ral (Wolthuis et al., 1998). The GEF (White et al., 1996; Hernandez-Munoz et al., 2000). proteins transduce signals by inducing the dissociation of Rgr’s human orthologue, hRGR, is also frequently GDP from the respective GTPases and thereby facilitat- altered in a subset of human T-cell malignancies. The ing the binding of GTP. Proteins of the GEF family 50 UTR of wild-type Rgr contains eight upstream activate GTPases and are therefore also called guanine start codons in a manner similar to mRNAs encod- nucleotide dissociation stimulator (GDS) family meme- ing oncoproteins and growth factors involved in ber. The guanine nucleotide dissociation stimulator of abnormal signaling in tumorigenesis. The 50 UTR exerts Ral-GDP (Ral-GDS) also interacts with Ras in a GTP- a strong inhibitory control on its own translation, and dependent manner. Therefore, Ras appears to be an activation of the Rgr oncogene to confer its transform- upstream effector of the Ral. However, Ras-independent ing ability occurs by elimination of its translational activation of the Ral-GDS/Ral effector pathway is also regulatory elements (Hernandez-Munoz et al., 2003). reported and found to induce cell transformation (Urano This is consistent with the human T-cell malignancies, et al., 1996; Hofer et al., 1998; Wolthuis and Bos, 1999; where high-level expression of truncated forms of Linnemann et al., 2002). Regulation of the Ral-GDS/ hRGR gene is observed (Leonardi et al., 2002). effector pathway by b-arrestins mediates cytoskeletal Reorganization of actin cytoskeleton is considered reorganization (Bhattacharya et al., 2002) by activation important for not only the Rho-dependent regulation of Jak/Stat3 pathway and is crucial for mouse myeloid of gene expression through factors such as serum leukemia (Senga et al., 2001). response elements in 30UTRs (Witteck et al., 2003). While Raf and PI3 kinase have emerged as critical Autoregulation in the synthesis of actin itself requires regulators of cell growth, development, and apoptosis, the 30UTR of the mRNA (Lyubimova et al., 1999), less is known about Ral-GDS or Rho. The Ral-GDS has suggesting translational control in Rho GTPase effec- been shown to induce complementary transformation tors. Further, Rho activity has also been shown to activity of Ras and it is due to a distinct activity different inhibit the translation efficiency of p27Kip1 mRNA from additive effects on Raf/Erk pathway (White et al., and the Rho responsive element lies within a 300 nt 1996). Similarly, activation of ATF2 (a basic region- region at the 30end of the mRNA (Vidal et al., 2002). leucine zipper transcription factor family member) Thus, RalGDS/Rho GTPase mediated pathways involved in oncogenic transformation and adaptive also contribute to oncogenic modulation of translational responses to viral and genotoxic stress is regulated by control. an unknown mechanism involving the cooperation between the Ras/Raf/Erk and Ral-GDS/Src/p38 pathways (Ouwens et al., 2002). The Ral-GDS/Ral and PI3 Global analysis of molecular signatures in cancer through kinase/Akt pathways mediate Ras-regulated activity of translationally regulated oncogenes choline kinase, whose increased activity is associated with human cancers (Ramirez de Molina et al., 2002). Many studies have identified the role of differential Ral-GDS/Ral effector pathway also activates phospho- gene expression in complex biological processes lipase D implicated in cancer metastasis, and critically (Pollack et al. (2002)). Amidst various available cooperates with Raf/Erk pathway to promote invasion methods such as differential display, subtractive and metastasis (Ward et al., 2001). Finally, the RalGDS/ cDNA cloning, serial analysis of gene expression Ral effector pathway additionally regulates the activity (SAGE), and comparative genomic hybridization of Ral binding protein (RalBP1), which is also a GTPase (CGH), global gene expression profiling with micro- activating protein (GAP) for Rho GTPases such as Rac1 arrays has been a very powerful tool in capturing a and Cdc42 (Jullien-Flores et al. 1995; Yamaguchi et al., comprehensive and biologically meaningful molecular 1997; Ikeda et al., 1998). Rac, cdc42, and RhoA are the signature for a given pathophysiological phenotype. key members of the family of Rho GTPases that regulate Several cancer types have been molecularly classified many important processes such as organiztion of actin based on global transcriptome analysis of total cellular cytoskeleton, gene transcription, cell cycle progression, mRNA levels. membrane trafficking, and cell transformation either by cooperation with Ras/Raf/Erk or PI3 kinase/Akt Microarray profiling of cancer and oncogenic signaling pathways (Bar-Sagi and Hall, 2000; Sahai and Marshall, 2002; Schmidt and Hall, 2002; Vial et al., Gene expression profiling of total cellular mRNA has 2003). Rac/cdc42 and Rho signaling antagonize each been successfully used to identify different cancer types, other to control cellular phenotype and migratory classify tumors, measure the effects of oncogenic

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3257 signaling pathways, and identify novel therapeutic translated by the mRNAs, it is insufficient to know targets and molecular diagnosis. Recently, gene expres- the levels of total cellular transcripts to understand a sion arrays are also developed for clinical cancer given phenotype. outcome, and patient management (Pomeroy et al., et al et al 2002; Chen ., 2003; Pedersen ., 2003; Sotiriou Using array technology to describe global recruitment of et al ., 2003). Distinct gene expression signatures help to mRNAs to ribosomes identify low-grade astrocytoma, glioblastoma, and oligodendroglioma subtypes of brain tumors (Shai The eventual outcome of gene expression, protein levels, et al., 2003; van den Boom et al., 2003). Similarly, six is regulated at many steps between the gene and the clear cell carcinomas were distinguished from other protein. To gain a global view, it is essential to know ovarian carcinomas, grade I and Grade II from Grade whether the mRNAs are being translated into cognate III serous papillary carcinoma and ovarian from breast proteins, which is in principle a given mRNA’s carcinomas (Schaner et al., 2003). Expression profiling translational state. In fact, eucaryotic gene expression also accurately identified prognostic subtypes of pedia- is significantly regulated at the level of translational tric acute lymphoblastic leukemia (Ferrando et al., 2003; initiation. Actively translating mRNAs are usually Ross et al., 2003), combinatorial versus single drug associated with multiple ribosomes and form large treatment (combination chemotherapy) responsive spe- structures called polyribosomes or polysomes. Transla- cific changes in gene expression in human leukemia tionally inactive mRNAs are sequestered as messenger (Cheok et al., 2003), human lung adenocarcinoma ribonucleoprotein (mRNP) particles or associated with subclasses (Bhattacharjee et al., 2001), central nervous a single ribosome (monosome). Thus, the degree of system embryonal tumor outcome (Pomeroy et al., mRNA recruitment to ribosomes is one determinant of 2002), distinct gene clusters between malignant and relative translational efficiency. Changes in translation normal plasma cells (De Vos et al., 2002), development initiation rates are common translation-regulating me- of diffuse large B-cell lymphoma, a subclass of non- chanisms resulting in alterations in mRNA loading on Hodgkin’s lymphoma (Alizadeh et al., 2000), clinical out ribosomes. Exploitation of this fact resulted in develop- come of breast cancer (van de Vijver et al., 2002), genes ment of a large-scale expression screening for transla- associated with prostate cancer progression (Mousses tional control that utilizes the isolation of polysomal et al., 2002), and subgroups of human melanoma RNA and its interrogation by DNA arrays. The (Bittner et al., 2000). experimental methodology involves the separation of DNA microarray gene expression profiling has also polysomes from the free mRNPs using sucrose gradient assisted in correlating signaling pathways to particular centrifugation followed by oligonucleotide microarray cancer types. For example, transcriptional profiling of hybridization. This relies on the principle that actively the head and neck squamous cell carcinoma identified translating mRNAs are recruited into polysomes and the interactions between Wnt pathway and Notch the translationally inactive mRNAs exist as free pathway components (Ha et al., 2003) and that of the cytoplasmic mRNPs with significantly lower sedimenta- sonic hedgehog (SHH) response identified a critical role tion rates. Thus, the global effect of agonists or for N-myc in neuronal precursor cell proliferation and antagonists that promote these mRNAs in and out of further the dominant-negative N-myc substantially ribosomes can be studied precisely. About 11–13% of reduced this response (Oliver et al., 2003). This work the genes were shown to be translationally regulated also identified cyclin D1 and N-myc as important (activated or repressed) by screening cDNA libraries mediators of SHH pathway. Similarly, previously upon antigenic stimulation of primary human T cells unrecognized activation of SHH pathway was identified (Mikulits et al., 2000). The early increase in protein in the derivation of medulloblastomas from cerebellar synthesis occurred prior to an increase in transcription, granule cells (Pomeroy et al., 2002). Microarray analyses and correlated with the mobilization of mRNAs into were also employed to study the effect of one or limited polysomes (Varesio and Holden, 1980) and activation of signaling intermediates of a given signal transduction translation initiation components (Cohen et al., 1990; pathway such as Ras and/or Akt (Rajasekhar et al., Welsh et al., 1996). 2003), C-Myc (Ellwood-Yen et al., 2003), and Wnt The power of polyribosomal RNA expression profil- (Willert et al., 2002) in oncogenesis. Metastasis-related ing was extended to situations where translation genes have also been identified (Ramaswamy et al., initiation factor-independent recruitment of polyribo- 2003) including an essential role of RhoC (Clark et al., somes to mRNAs occur. For example, RNA viruses 2000) in this process. proteolytically cleave eiF-G destabilizing the formation Although the above-referenced work clearly shows of eif-F complex and inhibit cap-dependent translation that this gene expression profiling of total mRNA in the host, but do not affect the viral mRNA-mediated has been used very successfully in the study of protein synthesis from their 50 noncoding region cancer, the abundance of total cellular mRNA does (50NCR). The 50NCR contains an IRES that recruits not always correlate well with steady-state levels of the 40S ribosomal subunit in the absence of intact eiF-4F. encoded proteins. Thus, microarray analysis may not Expression analysis of polysomal RNA isolated from reliably identify the post-transcriptional changes in poliovirus-infected HeLa cells facilitated the identifica- protein levels or activity. As the cellular phenotype is tion of eucaryotic mRNAs that are translated at a determined not by mRNAs, but by the proteins reduced concentrations of cap binding eiF-4F complex

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3258 (Johannes et al., 1999). Approximately 200 of the 7000 mRNAs remained associated with the polyribosomes and represented immediate early transcription factors, kinases, phosphatases, and several proto-oncogenes that have been previously identified to have roles in inflammation and angiogenesis (Iyer et al., 1999). It is interesting to note that under the same eiF-4F- limiting conditions, the mRNA encoding Cyr61, a secreted factor that can promote motility, angiogenesis, and tumor growth, was selectively loaded with polyribosomes without changes in its overall RNA abundance (Johannes et al., 1999). More interestingly, translation of another mRNA encoding Pim-1, which cooperates with c-myc in cellular transformation, was identified to be cap-independent under these conditions. Global and specific translational analysis of the RNA profile response in Saccharomyces cerevisiae to a rapid transfer from glucose to glycerol resulted in a loss of Figure 1 Normalization of polyribosomal RNA. Total cellular polysome- associated mRNAs encoding ribosomal and polysome RNAs were analysed by microarray analysis. A pair- 0 wise comparison between a total RNA and its corresponding proteins in a manner analogous to the 5 oligopyrimi- polysomal RNA was made to obtain the relative ribosomal loading 0 dine (5 TOP) sequence containing mammalian riboso- of all mRNAs, referred to normalized polyribosomal RNA (NPR) mal protein coding mRNAs upon serum starvation (Rajasekhar et al., 2003) (Kuhn et al., 2001). This observation is of particular interest as yeast ribosomal proteins do not contain 50TOP sequences and 30 untranslated regions of the genic effect of combined Ras and Akt signaling appears yeast ribosomal proteins are thought to determine the to be due to differential recruitment of existing mRNA ribosomal loading function. These studies have identi- into polysomes resulting in altered production of fied two novel genes whose transcripts are preferentially growth-regulating and oncogenic proteins (Rajasekhar mobilized into polysomes, while the overall translation et al, 2003). It is not known if these alterations in the initiation of newly synthesized and pre-existing mRNAs translational efficiency of existing mRNAs are sufficient was generally repressed after the shift in carbon source. to induce tumor formation and required for its maintenance. Polysome profiling and oncogenic signaling Global gene expression at the single cell level and Comparison of total and polysome RNA profiling has ribonomic profiling been used to normalize the loading efficiency of mRNAs to ribosomes. This approach compares microarray All of the studies listed so far have been analyses of cell profiling of both total and polysome-associated mRNAs populations; the ribosomes are assumed to be a and has been used to determine the effects on translation homogenous collection of particles. However, there is of combined Ras and Akt signaling in glia. Such no particular reason to believe that within a given cell all normalized polyribosomal RNA (NPR) values are of the ribosomes equally translate any given mRNA characteristic of a given cell type at a specific signaling species. It is at least conceivable that ribosomes exist as state. Comparison between the NPRs of two cell types a heterogeneous population and that certain ribosomes or between cells with or without signaling blockade have a predilection for certain messages. In fact, some generates comparative normalized polysomal RNA studies hint that this may be the case. Recently, mRNA (CNPR) (For details see Figure 1) (Rajasekhar et al., subsets were identified in mRNP complexes using 2003). These two signaling pathways were investigated antibodies to RNA binding proteins and genomic in glia because combined Ras and Akt signaling is found arrays. The consequent analyses is referred to as in human glioblastomas and causally related to the ‘ribonomics’ (Keene, 2001; Tenenbaum et al., 2002). formation of glioblastomas in mice (Holland et al., 2000; The ribonomic profiling reduces the complexity of Begemann et al., 2002). CNPR was measured as a standard gene expression analysis as it yields only a function of the activity of the Ras and Akt signaling subset of steady-state mRNAs. For example, using pathways in cell culture (Figure 2). At 2 h of pharma- mRNA binding proteins such as Hu, eiF-4E, and PABP, cologic signal blockade of either Ras or Akt pathways, different mRNPs were identified from P19 embryonal the fold-increase in polysome recruitment for a specific carcinoma stem cells. The mRNA profiles associated subset of mRNAs far exceeded the fold changes in the with each of these were unique and represented gene total mRNA for any gene represented on the array. The clusters that differed from total cellular RNA (Tenen- mRNAs so affected encode proteins promoting signal baum et al., 2000). transduction, cell cycle progression, cell motility, and Not only could there be different subsets of ribosomes other functions known to be involved in oncogenesis within a given cell that prefer specific mRNAs, these and/or in gliomagenesis. Therefore, part of the onco- ribosomes may physically cluster giving rise to regions

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3259

Figure 2 Regulation of translation initiation by growth factor receptor-induced signal involving activated Ras/ERK and PI3K/Akt kinase pathways. Both pathways involve sequential phosphorylation of downstream components as indicated by pink circles. Growth factor receptor stimulation of PI3 kinase results in serial activation of PDK, Akt, and mTOR, leading to phosphorylation of S6RP via S6 kinase and phosphorylation of 4EBPs. Ras sequentially activates a kinase cascade Raf1, MEK, ERK and ultimately eIF4E levels. The pathways cooperate to regulate eiF4E activity and thereby modify the translational effect of existing mRNAs, encoding growth- regulated proteins within the cell that produce certain subsets of the oncogenic pathways affects the production of speciﬁc proteome. Thus, the physical organization of the proteins by regulating the translational efﬁciency of transcripts may be critical and depend on mRNA’s their mRNAs. Components of the translation machin- stability, abundance, localization, and characteristics of ery appear to have a primary and early impact on the the other associating proteins. The proteins encoded by emergence of malignant phenotype as observed by rapid physically clustered mRNAs may participate in the changes in the relative recruitment of existing mRNAs related biological processes or structural outcomes into polysomes. This effect on translation precedes the similar to operons and their polycistronic mRNAs in transcriptional response, thus oncogenic signaling ap- procaryotic systems (Keene and Tenenbaum, 2002). It pears to put translation before transcription in oncogen- remains to be explored if the mRNPs represent some of esis (Lasko, 2003; Prendergast, 2003). By coordinately the means by which multicellular organisms preserve the modulating malignancy-related proteins, eiF-4E and its dynamics of genetic complexity. antagonists, the 4E-BPs play a pivotal role in regulating oncogenesis.

Concluding remarks Acknowledgements We thank Christina Glaster for her expertise in preparing this Translational control appears to be a major regulatory manuscript. This work was supported by NIH Grants step in normal and abnormal cell growth, cell prolifera- UO1CA894314, RO1CA94842, and RO1CA099489, and the tion, cancer initiation, and metastasis. Signaling by Seroussi, Tow, Searle, and Bressler Scholars foundations.

Oncogene Postgenomic global analysis by oncogenic signaling VK Rajasekhar and EC Holland 3260 References

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