Adaptation and survival to strenuous environmental conditions are evolutionary conserved mechanisms that allow organisms to produce progeny. In general, adaptation to stress promotes the repair of protein and DNA damage and must occur rapidly. Thus, translation based responses have great potential to maintain or quickly increase the levels of important regulatory proteins when cells are experiencing stress1-2. Regulation of the translation machinery during toxic conditions, such as endoplasmic reticulum (ER) stress, can arrest cell growth and promote the repair of damage or promote cell death. Similarly, translational regulation has recently been associated with the DNA damage response, and appears to promote efficient cell cycle arrest and DNA repair after damage1. Our overarching hypothesis proposes that stress-induced regulatory programs operating at the mRNA translation level have tumor suppressive / growth inhibitory effects. This hypothesis is based on our recent findings in colon and head and neck squamous carcinoma cell models that translational regulatory mechanisms inhibit tumor growth. SW620 (p53wt, K-Rasmut) colon carcinoma cells3-4, show loss of the gene encoding TRM9 (KIAA1456)3 which encodes a tRNA methyltransferase that modifies the uridine wobble base of tRNAARG(UCU) and tRNAGLU(UUC). TRM9 is a highly conserved activity that completes the formation of 5-methylcarbonylmethyluridne (mcm5U) modifications1,3. The yeast homolog of TRM9 enhances the translation of transcripts over-represented with specific ARG and GLU codons. Genes for key translation, DNA damage and ER stress proteins have an over-representation of these codons. Hence Trm9 deficient cells display environmental stress phenotypes that were attributed to a deficiency in translation elongation of specific transcripts1,3. Recently, we found that the TRM9 transcript was undetectable in aggressive colon (SW620) and head and neck (T-HEp3) carcinoma cells. Re-expressed TRM9 in SW620 colorectal cancer cells resulted in a severe suppression of their tumorigenic potential in xenograft studies. Similarly, a variant of T- HEp3 that reprograms into a non-tumorigenic phenotype (D-HEp3) has detectable levels of the TRM9 transcript. This further suggests a tumor suppressor / tumor growth inhibitory role for TRM9. In D-HEp3 cells, which express TRM9, loss of tumorigenicity is functionally linked to activation of an ER stress response characterized by attenuation of translation initiation dependent on PERK and eIF2? phosphorylation4. Further, ER stress inducing drugs, which activate the ER kinase PERK and induce growth arrest5, result in up-regulation of TRM9 expression in various cell lines. This suggests that growth inhibitory signals initiated by ER stress may be functionally linked to TRM9 induction. In agreement, ER stress signaling through PERK and other ER stress pathways is tumor suppressive in melanocytes6 and mammary epithelial cells7 and inhibitory of tumor growth of T-HEp3 and SW620 (similar to TRM9 re-expression) cells. We hypothesize that growth suppressive signals induced by ER stress require TRM9-catalyzed tRNA modifications for efficient translation. Hence TRM9 is required for regulating the levels of proteins that promote growth arrest, repair damage and/or induce apoptosis, thus placing TRM9 in a tumor suppressive / growth inhibitory role after environmental stress. We propose the following specific aims:
SPECIFIC AIM1. To determine the extent and mechanism by which TRM9 is down-regulated in tumors. In colon carcinoma cells TRM9 down-regulation appears to be linked to promoter sequence methylation. Our results suggest that in HEp3 carcinoma cells TRM9 is epigenetically silenced. We will explore whether transcriptional or translational repression and/or promoter methylation are common mechanisms resulting in TRM9 loss in a panel of colon, head and neck, and breast cancer cells.
SPECIFIC AIM2. To analyze the tumor suppressor / growth inhibitory pathways regulated by TRM9. Using SW620 and T-HEp3 cells as models, we will explore how TRM9 re-expression suppresses tumorigenicity. We will determine in vitro and in vivo whether specific proteins that regulate the cell cycle machinery (cyclin D1/D3, CDK4) and/or apoptosis (p53) are subject to translational regulation by TRM9. These studies will be used to determine whether TRM9 is a regulator of a sub-program of growth arrest and/or apoptosis responsible for tumor growth inhibition.
SPECIFIC AIM3. To determine whether a cross talk between ER-stress signaling and TRM9 regulates translational programs and tumor growth suppression. We will explore whether ER stress induced growth arrest and/or cell death requires TRM9 activity. We will also specifically modulate PERK signaling, a major regulator of translation initiation and a growth suppressor, with an Fv2E-PERK dimerizing system. This will allow us to determine whether TRM9 is required for efficient translation of genes expressed during an unfolded protein response and required for growth suppression.
The proposed studies will be used to analyze the tumor suppressor / growth inhibitory function of tRNA methyltransferase9 (TRM9) in colon, breast, head and neck cancers. Specifically, the influence of TRM9 on the regulation of cell cycle and apoptotic proteins, as part of the ER stress response, will be ascertained to define signal transduction pathways associated with tumor suppression. The long term potential of the proposed research is significant, as understanding the mechanisms by which TRM9 inhibits tumor growth may provide novel therapeutic opportunities to restore growth-restricting programs in cancer cells.
|Sosa, Maria Soledad; Parikh, Falguni; Maia, Alexandre Gaspar et al. (2015) NR2F1 controls tumour cell dormancy via SOX9- and RAR?-driven quiescence programmes. Nat Commun 6:6170|
|Gu, Chen; Begley, Thomas J; Dedon, Peter C (2014) tRNA modifications regulate translation during cellular stress. FEBS Lett 588:4287-96|
|Avivar-Valderas, A; Wen, H C; Aguirre-Ghiso, J A (2014) Stress signaling and the shaping of the mammary tissue in development and cancer. Oncogene 33:5483-90|
|Bragado, Paloma; Estrada, Yeriel; Parikh, Falguni et al. (2013) TGF-?2 dictates disseminated tumour cell fate in target organs through TGF-?-RIII and p38?/? signalling. Nat Cell Biol 15:1351-61|
|Avivar-Valderas, A; Bobrovnikova-Marjon, E; Alan Diehl, J et al. (2013) Regulation of autophagy during ECM detachment is linked to a selective inhibition of mTORC1 by PERK. Oncogene 32:4932-40|
|Sosa, Maria Soledad; Bragado, Paloma; Debnath, Jayanta et al. (2013) Regulation of tumor cell dormancy by tissue microenvironments and autophagy. Adv Exp Med Biol 734:73-89|
|Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro et al. (2013) A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-?. EMBO Mol Med 5:366-83|
|Bragado, Paloma; Sosa, Maria Soledad; Keely, Patricia et al. (2012) Microenvironments dictating tumor cell dormancy. Recent Results Cancer Res 195:25-39|
|Towns, William L; Begley, Thomas J (2012) Transfer RNA methytransferases and their corresponding modifications in budding yeast and humans: activities, predications, and potential roles in human health. DNA Cell Biol 31:434-54|
|Kim, Ryung S; Avivar-Valderas, Alvaro; Estrada, Yeriel et al. (2012) Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer. PLoS One 7:e35569|
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