Remarkable progress has been made in defining a new understanding of the role of mRNA translation and protein synthesis in human cancer. Translational control is a crucial component of cancer development and progression, directing both global control of protein synthesis and selective translation of specific mRNAs that promote tumour cell survival, angiogenesis, transformation, invasion and metastasis. Translational control of cancer is multifaceted, involving alterations in translation factor levels and activities unique to different types of cancers, disease stages and the tumour microenvironment. Several clinical efforts are underway to target specific components of the translation apparatus or unique mRNA translation elements for cancer therapeutics.

Inflammatory breast cancer (IBC) is the most lethal form of primary breast cancer. IBC lethality derives from generation of tumour emboli, which are non-adherent cell clusters that rapidly spread by a form of continuous invasion known as passive metastasis. In most cancers, expression of E-cadherin, an epithelial marker, is indicative of low metastatic potential. In IBC, E-cadherin is overexpressed and supports formation of tumour emboli by promoting tumour cell interactions rather than adherence to stroma. E-cadherin, a surface component of adherens junctions, is anchored by interaction with p120 catenin (p120). We show that the unique pathogenic properties of IBC result in part from overexpression of the translation initiation factor eIF4GI in most IBCs. eIF4GI reprograms the protein synthetic machinery for increased translation of mRNAs with internal ribosome entry sites (IRESs) that promote IBC tumour cell survival and formation of tumour emboli. Overexpression of eIF4GI promotes formation of IBC tumour emboli by enhancing translation of IRES-containing p120 mRNAs. These findings provide a new understanding of translational control in the development of advanced breast cancer.

As a step toward developing poliovirus as a vaccine vector, poliovirus recombinants were constructed by fusing exogenous peptides (up to 400 amino acids) and an artificial cleavage site for viral protease 3Cpro to the amino terminus of the viral polyprotein. Viral replication proceeded normally. An extended polyprotein was produced in infected cells and proteolytically processed into the complete array of viral proteins plus the foreign peptide, which was excluded from mature virions. The recombinants retained exogenous sequences through successive rounds of replication in culture and in vivo. Infection of animals with recombinants elicited a humoral immune response to the foreign peptides.

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The poly(rC)-binding proteins (PCBP1 and PCBP2) are RNA-binding proteins whose RNA recognition motifs are composed of three K homology (KH) domains. These proteins are involved in both the stabilization and translational regulation of several cellular and viral RNAs. PCBP1 and PCBP2 specifically interact with both the 5-element known as the cloverleaf structure and the large stem-loop IV RNA of the poliovirus 5-untranslated region. We have found that the first KH domain of PCBP2 (KH1) specifically interacts with the viral RNAs, and together with viral protein 3CD, KH1 forms a high affinity ternary ribonucleoprotein complex with the cloverleaf RNA, resembling the full-length PCBP protein. Furthermore, KH1 acts as a dominant-negative mutant to inhibit translation from a poliovirus reporter gene in both Xenopus laevis oocytes and HeLa cell in vitro translation extracts.Translation in eukaryotic cells is a highly regulated process involving a complex protein machinery. There is increasing evidence that translation of several mRNAs is determined by the specific and regulated interaction of certain proteins with RNA elements in the 3Ј-and 5Ј-untranslated regions (for reviews, see Refs. 1 and 2). Although many of these cis-acting RNA elements have been defined, only a few trans-acting regulatory proteins are known, and the mechanisms by which they regulate translation are poorly understood.As with cellular messages, translation of viral RNA is also subjected to complex regulation. For example, for positive strand RNA viruses, the genomic RNA is utilized as a template for translation, RNA replication, and formation of new virions; hence, the usage of the RNA must be regulated. For poliovirus, this regulation seems to be dependent on signals within the 5Ј-UTR.1 Whereas the majority of cellular mRNAs depend on the 5Ј-cap structure to initiate translation, poliovirus initiates translation internally via a cap-independent mechanism from an RNA element termed the internal ribosomal entry site (IRES) located within the 5Ј-UTR. Computer modeling and biochemical analysis suggest that the secondary structure of the poliovirus 5Ј-UTR is composed of six distinct domains, stem-loops I-VI (see Fig. 1A). In addition to the canonical initiation factors, poliovirus translation initiation requires additional host cell factors, some of which appear to interact directly with the viral RNA (reviewed in Refs. 3 and 4). Among these essential factors are PCBP1 and PCBP2, two closely related poly(rC)-binding proteins, (also referred to as hnRNPs E1 and 2 or ␣CP1 and ␣CP2). These proteins facilitate viral translation through the interaction with both the first stemloop domain (which folds into a cloverleaf-like structure) and stem-loop IV of the poliovirus 5Ј-UTR (5-8). Both PCBP1 and PCBP2 form a low affinity complex with the cloverleaf RNA, but together with viral protein 3CD (the precursor of the viral polymerase 3D and the viral protease 3C), they are incorporated into a high affinity ternary ribonucleoprotein complex (9, 10). Ternary compl...

Introduction Inflammatory breast cancer (IBC) is an aggressive and rare cancer with a poor prognosis and a need for novel targeted therapeutic strategies. Preclinical IBC data demonstrates strong activation of the PI3K/mTOR and JAK/STAT pathways, expression of inflammatory cytokines and tumor associated macrophages (TAMs). Methods Archival tumor tissue from three disease types (IBC treated with neoadjuvant chemotherapy (NAC) (n=45); invasive ductal carcinoma (IDC) treated with NAC (n=24; ‘treated IDC’); and untreated IDC (n=27; ‘untreated IDC’)) was analyzed for the expression of biomarkers pS6 (mTOR), pJAK2, pSTAT3, IL6, CD68 (monocytes, macrophages) and CD163 (TAMs). Surrounding non-tumor tissue was also analyzed. Results Biomarker levels and surrogate activity by site-specific phosphorylation were demonstrated in the tumor tissue of all three disease types but were highest in IBC and treated IDC and lowest in untreated IDC for pS6, pJAK2, pSTAT3 and IL6. Of 37 IBC patients with complete biomarker data available, 100% were pS6 positive and 95% were pJAK2 positive. In non-tumor tissue, biomarker levels were observed in all groups but were generally highest in untreated IDC and lowest in IBC, except for JAK2. Conclusions IBC and treated IDC display similar levels of mTOR and JAK2 biomarker activation, suggesting a potential mechanism of resistance after NAC. Biomarker levels in surrounding non-tumor tissue suggest that the stroma may be activated by chemotherapy and resembles the oncogenic tumor-promoting environment. Activation of both pS6 and pJAK2 in IBC may support dual targeting of mTOR and JAK/STAT pathways, and the need for prospective studies to investigate combinatorial targeted therapies in IBC.

Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector to engender mucosal immunity. Replication-competent chimeric viruses can be constructed by fusing foreign antigenic sequences to several positions within the poliovirus polyprotein. Artificial cleavage sites ensure appropriate proteolytic processing of the recombinant polyprotein, yielding mature and functional viral proteins. To study the effect of the position of insertion, two different recombinant polioviruses were examined. A small aminoterminus insertion delayed virus maturation and produced a thermosensitive particle. In contrast, insertion at the junction between the P1 and P2 regions yielded a chimeric poliovirus that replicated like the wild type. Eight different chimeras were constructed by inserting simian immunodeficiency virus (SIV) sequences at the P1/P2 junction. All recombinant viruses replicated with near-wild-type efficiency in tissue culture cells and expressed high levels of the SIV antigens. One of the inserted fragments corresponding to gp41 envelope protein was N-glycosylated but was not secreted. Inserted sequences were only partially retained after few rounds of replication in HeLa cells. This problem could be remedied to some extent by altering the sequences flanking the insertion point. Reducing the homology of the direct repeats by 37% decrease the propensity of the recombinant viruses to delete the insert. To determine the immunogenic potential of the recombinants, mice susceptible to poliovirus infection were inoculated intraperitoneally. The antibody titers elicited against Gag p17 depended on the viral doses and the number of inoculations. In addition, recombinants which display higher genetic stability were more effective in inducing an immune response against the SIV antigens, and inoculation with a mix of recombinants carrying different SIV antigens (a cocktail of recombinants) elicited humoral responses against each of the individual SIV sequences.

mTOR coordinates growth signals with metabolic pathways and protein synthesis and is hyperactivated in many human cancers. mTOR exists in two complexes: mTORC1, which stimulates protein, lipid, and ribosome biosynthesis, and mTORC2, which regulates cytoskeleton functions. While mTOR is known to be involved in the DNA damage response, little is actually known regarding the functions of mTORC1 compared to mTORC2 in this regard or the respective impacts on transcriptional versus translational regulation. We show that mTORC1 and mTORC2 are both required to enact DNA damage repair and cell survival, resulting in increased cancer cell survival during DNA damage. Together mTORC1 and -2 enact coordinated transcription and translation of protective cell cycle and DNA replication, recombination, and repair genes. This coordinated transcriptional-translational response to DNA damage was not impaired by rapalog inhibition of mTORC1 or independent inhibition of mTORC1 or mTORC2 but was blocked by inhibition of mTORC1/2. Only mTORC1/2 inhibition reversed cancer cell resistance to DNA damage and replicative stress and increased tumor cell killing and tumor control by DNA damage therapies in animal models. When combined with DNA damage, inhibition of mTORC1/2 blocked transcriptional induction more strongly than translation of DNA replication, survival, and DNA damage response mRNAs.KEYWORDS breast cancer, DNA damage, mTOR, protein synthesis, translational control, DNA damage response, protein synthesis, transcriptional control T he mammalian target of rapamycin (mTOR) is a downstream kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway that integrates signals from growth factors and nutrients to regulate key metabolic and macromolecular processes and is dysregulated in many human cancers (1-3). mTOR exists in two complexes, mTOR complex 1 (mTORC1) and mTORC2, which mediate different functions defined by their molecular composition. In response to nutrient levels, growth factors, and other mitogenic signals, mTORC1 regulates protein synthesis, lipid synthesis, and ribosome biogenesis (1, 3). mTORC1 includes the proteins mTOR, Raptor, and G␤L, among others (4), and is responsible for the phosphorylation (inactivation) of the negative regulator of cap binding protein eukaryotic translation initiation factor 4E (eIF4E) known as 4E-BP1. 4E-BP1 binds and blocks the activity of the translation initiation factor eIF4E by competing for interaction with translation initiation factor eIF4G, a molecular scaffold upon which the 40S ribosome and translation factors assemble (5). mTORC2 includes the proteins mTOR, Rictor, and G␤L, among others. mTORC2 regulates cytoskeleton