Scott W. Blume

Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo.

The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Sp1 to both decanucleotide Sp1 binding sequences which are located 49 and 14 base pairs upstream of the transcription start site. The more distal of the two binding sites exhibits a somewhat higher affinity for Sp1. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Sp1 binding. Promoter-dependent in vitro transcription of a DHFR template is selectively inhibited by mithramycin when compared to the human H2b histone gene. A similar effect is also noted in vivo. Mithramycin treatment of MCF-7 human breast carcinoma cells containing an amplified DHFR gene induces selective inhibition of DHFR transcription initiation, resulting in a decline in DHFR mRNA level and enzyme activity. This selective inhibition of DHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells.

The ELAV RNA-stability factor HuR binds the 5′-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5′-untranslated region (5′-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5′-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3′-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5′-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5′-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5′-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5′-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5′-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.

The 5′‐untranslated RNA of the human dhfr minor transcript alters transcription pre‐initiation complex assembly at the major (core) promoter

The human dhfr minor transcript is distinguished from the predominant dhfr mRNA by an ∼400 nucleotide extension of the 5′‐untranslated region, which corresponds to the major (core) promoter DNA (its template). Based on its unusual sequence composition, we hypothesized that the minor transcript 5′‐UTR might be capable of altering transcription pre‐initiation complex assembly at the core promoter, through direct interactions of the RNA with specific regulatory polypeptides or the promoter DNA itself. We found that the minor transcript 5′‐UTR selectively sequesters transcription factor Sp3, and to a lesser extent Sp1, preventing their binding to the dhfr core promoter. This allows a third putative transcriptional regulatory protein, which is relatively resistant to sequestration by the minor transcript RNA, the opportunity to bind the dhfr core promoter. The selective sequestration of Sp3 > Sp1 by the minor transcript 5′‐UTR involves an altered conformation of the RNA, and a structural domain of the protein distinct from that required for binding to DNA. As a consequence, the minor transcript 5′‐UTR inhibits transcription from the core promoter in vitro (in trans) in a concentration‐dependent manner. These results suggest that the dhfr minor transcript may function in vivo (in cis) to regulate the transcriptional activity of the major (core) promoter.

Alterations in RNA-binding activities of IRES-regulatory proteins as a mechanism for physiological variability and pathological dysregulation of IGF-IR translational control in human breast tumor cells

The type I insulin‐like growth factor receptor (IGF‐IR) is integrally involved in the control of cellular proliferation and survival. An internal ribosomal entry site (IRES) within the 1,038 nucleotide 5′‐untranslated region of the human IGF‐IR mRNA helps to provide the tight control of IGF‐IR expression necessary for maintenance of normal cellular and tissue homeostasis. The IRES maps to a discrete sequence of 85 nucleotides positioned just upstream of the IGF‐IR initiation codon, allowing the ribosome to bypass the highly structured regions of the 5′‐UTR as well as the upstream open reading frame. The authenticity of the IGF‐IR IRES has been confirmed by its sensitivity to deletion of the promoter from a bicistronic reporter construct, and its resistance in a monocistronic reporter construct to co‐expression of a viral 2A protease. We previously characterized HuR as a potent repressor of IGF‐IR translation. Here we demonstrate that hnRNP C competes with HuR for binding the IGF‐IR 5′‐UTR and enhances IRES‐mediated translation initiation in a concentration‐dependent manner. We observed changes in binding of hnRNP C versus HuR to the IGF‐IR 5′‐UTR in response to physiological alterations in cellular environment or proliferative status. Furthermore, we have found distinct alterations in the pattern of protein binding to the IGF‐IR 5′‐UTR in human breast tumor cells in which IGF‐IR IRES activity and relative translational efficiency are aberrantly increased. These results suggest that dysregulation of the IGF‐IR IRES through changes in the activities of RNA‐binding translation‐regulatory proteins could be responsible for IGF‐IR overexpression in a proportion of human breast tumors. J. Cell. Physiol. 217: 172–183, 2008.

The human IGF1R IRES likely operates through a Shine-Dalgarno-like interaction with the G961 loop (E-site) of the 18S rRNA and is kinetically modulated by a naturally-polymorphic polyU loop

IGF1R is a proto‐oncogene with potent mitogenic and antiapoptotic activities, and its expression must be tightly regulated to maintain normal cellular and tissue homeostasis. We previously demonstrated that translation of the human IGF1R mRNA is controlled by an internal ribosome entry site (IRES), and delimited the core functional IRES to a 90‐nucleotide segment of the 5′‐untranslated region positioned immediately upstream of the initiation codon. Here we have analyzed the sequence elements that contribute to the function of the core IRES. The Stem2/Loop2 sequence of the IRES exhibits near‐perfect Watson–Crick complementarity to the G961 loop (helix 23b) of the 18S rRNA, which is positioned within the E‐site on the platform of the 40S ribosomal subunit. Mutations that disrupt this complementarity have a negative impact on regulatory protein binding and dramatically decrease IRES activity, suggesting that the IGF1R IRES may recruit the 40S ribosome by a eukaryotic equivalent of the Shine–Dalgarno (mRNA–rRNA base‐pairing) interaction. The homopolymeric Loop3 sequence of the IRES modulates accessibility and limits the rate of translation initiation mediated through the IRES. Two functionally distinct allelic forms of the Loop3 poly(U)‐tract are prevalent in the human population, and it is conceivable that germ‐line or somatic variations in this sequence could predispose individuals to development of malignancy, or provide a selectable growth advantage for tumor cells. J. Cell. Biochem. 110: 531–544, 2010.

Oncogenes, Malignant Transformation, and Modern Medicine

During the past decade there have been remarkable strides in the understanding of the basic mechanism of cancer. It is now clear that there is a set of genes, known as oncogenes, that can cause cells to become malignant if their expression is altered, either by mutation or overexpression. The products of these genes include growth factors, growth factor receptors, signal tranduction proteins, and DNA binding proteins. The normal cellular counterparts of these genes play very important roles in the regulation of growth and proliferation by normal cells. Another set of genes, anti-oncogenes, also play an important role in preventing abnormal cell proliferation. The remarkable explosion of understanding of the pathophysiology of malignancy has led to a common unifying concept of malignant transformation that applies to all tumors. It is likely that these new insights will lead to improved and more specific treatments for malignant disease in the next decade.

mrtl—A translation/localization regulatory protein encoded within the human c-myc locus and distributed throughout the endoplasmic and nucleoplasmic reticular network

mrtl (myc‐related translation/localization regulatory factor) is a previously uncharacterized protein synthesized from the first open reading frame contained within the human c‐myc P0 transcript, ∼800 nucleotides upstream of the Myc coding sequence. The mrtl protein, 114 amino acids in length, is projected to contain an N‐terminal transmembrane domain and a highly charged C‐terminal interaction domain with homology to numerous RNA‐binding proteins. Using monoclonal antibodies raised against the hydrophilic C‐terminal domain, endogenous mrtl was visualized in human breast tumor cell lines and primary mammary epithelial cells at the nuclear envelope and contiguous endoplasmic/nucleoplasmic reticulum. mrtl colocalizes and coimmunoprecipitates with translation initiation factor eIF2α and the 40S ribosomal protein RACK1, and appears capable of binding specifically to the c‐myc RNA. Inducible ectopic overexpression of wild‐type mrtl interferes with the function of endogenous mrtl, which results in loss of Myc from the nucleus. Furthermore, treatment of cells with a peptide derived from the C‐terminal domain displaces endogenous mrtl and causes a dramatic reduction in total cellular Myc protein levels. Together with our previous work demonstrating complete loss of tumorigenicity in association with ectopic expression of the c‐myc P0 5′‐UTR (containing the mrtl coding sequence), these results suggest that mrtl may serve an important function in regulating Myc translation and localization to the nucleus, perhaps ultimately contributing to the role of the c‐myc locus in oncogenesis. J. Cell. Biochem. 105: 1092–1108, 2008.

Small molecule inhibitors of IRES-mediated translation

Many genes controlling cell proliferation and survival (those most important to cancer biology) are now known to be regulated specifically at the translational (RNA to protein) level. The internal ribosome entry site (IRES) provides a mechanism by which the translational efficiency of an individual or group of mRNAs can be regulated independently of the global controls on general protein synthesis. IRES-mediated translation has been implicated as a significant contributor to the malignant phenotype and chemoresistance, however there has been no effective means by which to interfere with this specialized mode of protein synthesis. A cell-based empirical high-throughput screen was performed in attempt to identify compounds capable of selectively inhibiting translation mediated through the IGF1R IRES. Results obtained using the bicistronic reporter system demonstrate selective inhibition of second cistron translation (IRES-dependent). The lead compound and its structural analogs completely block de novo IGF1R protein synthesis in genetically-unmodified cells, confirming activity against the endogenous IRES. Spectrum of activity extends beyond IGF1R to include the c-myc IRES. The small molecule IRES inhibitor differentially modulates synthesis of the oncogenic (p64) and growth-inhibitory (p67) isoforms of Myc, suggesting that the IRES controls not only translational efficiency, but also choice of initiation codon. Sustained IRES inhibition has profound, detrimental effects on human tumor cells, inducing massive (>99%) cell death and complete loss of clonogenic survival in models of triple-negative breast cancer. The results begin to reveal new insights into the inherent complexity of gene-specific translational regulation, and the importance of IRES-mediated translation to tumor cell biology.

IRES inhibition induces terminal differentiation and synchronized death in triple-negative breast cancer and glioblastoma cells

Internal ribosome entry site (IRES)-mediated translation is a specialized mode of protein synthesis which malignant cells depend on to survive adverse microenvironmental conditions. Our lab recently reported the identification of a group of compounds which selectively interfere with IRES-mediated translation, completely blocking de novo IGF1R synthesis, and differentially modulating synthesis of the two c-Myc isoforms. Here, we examine the phenotypic consequences of sustained IRES inhibition in human triple-negative breast carcinoma and glioblastoma cells. A sudden loss of viability affects the entire tumor cell population after ∼72-h continuous exposure to the lead compound. The extraordinarily steep dose-response relationship (Hill-Slope coefficients −15 to −35) and extensive physical connections established between the cells indicate that the cells respond to IRES inhibition collectively as a population rather than as individual cells. Prior to death, the treated cells exhibit prominent features of terminal differentiation, with marked gains in cytoskeletal organization, planar polarity, and formation of tight junctions or neuronal processes. In addition to IGF1R and Myc, specific changes in connexin 43, BiP, CHOP, p21, and p27 also correlate with phenotypic outcome. This unusual mode of tumor cell death is absolutely dependent on exceeding a critical threshold in cell density, suggesting that a quorum-sensing mechanism may be operative. Death of putative tumor stem cells visualized in situ helps to explain the inability of tumor cells to recover and repopulate once the compound is removed. Together, these findings support the concept that IRES-mediated translation is of fundamental importance to maintenance of the undifferentiated phenotype and survival of undifferentiated malignant cells.

Translational Dysregulation in Cancer: Molecular Insights and Potential Clinical Applications in Biomarker Development

Although transcript levels have been traditionally used as a surrogate measure of gene expression, it is increasingly recognized that the latter is extensively and dynamically modulated at the level of translation (messenger RNA to protein). Over the recent years, significant progress has been made in dissecting the complex posttranscriptional mechanisms that regulate gene expression. This advancement in knowledge came hand in hand with the progress made in the methodologies to study translation both at gene-specific as well as global genomic level. The majority of translational control is exerted at the level of initiation; nonetheless, protein synthesis can be modulated at the level of translation elongation, termination, and recycling. Sequence and structural elements and epitranscriptomic modifications of individual transcripts allow for dynamic gene-specific modulation of translation. Cancer cells usurp the regulatory mechanisms that govern translation to carry out translational programs that lead to the phenotypic hallmarks of cancer. Translation is a critical nexus in neoplastic transformation. Multiple oncogenes and signaling pathways that are activated, upregulated, or mutated in cancer converge on translation and their transformative impact “bottlenecks” at the level of translation. Moreover, this translational dysregulation allows cancer cells to adapt to a diverse array of stresses associated with a hostile microenviroment and antitumor therapies. All elements involved in the process of translation, from the transcriptional template, the components of the translational machinery, to the proteins that interact with the transcriptome, have been found to be qualitatively and/or quantitatively perturbed in cancer. This review discusses the regulatory mechanisms that govern translation in normal cells and how translation becomes dysregulated in cancer leading to the phenotypic hallmarks of malignancy. We also discuss how dysregulated mediators or components of translation can be utilized as biomarkers with potential diagnostic, prognostic, or predictive significance. Such biomarkers have the potential advantage of uniform applicability in the face of inherent tumor heterogeneity and deoxyribonucleic acid instability. As translation becomes increasingly recognized as a process gone awry in cancer and agents are developed to target it, the utility and significance of these potential biomarkers is expected to increase.