The translation initiation factor EIF4E5 from Leishmania: crystal structure and interacting partners.

The mRNA cap-binding protein, eIF4E, mediates the recognition of the mRNA 5' end and, as part of the heterotrimeric eIF4F complex, facilitates the recruitment of the ribosomal subunits to initiate eukaryotic translation. Various regulatory events involving eIF4E and a second eIF4F subunit, eIF4G, are required for proper control of translation initiation. In pathogenic trypanosomatids, six eIF4Es and five eIF4Gs have been described, several forming different eIF4F-like complexes with yet unresolved roles. EIF4E5 is one of the least known of the trypanosomatid eIF4Es and has not been characterized in Leishmania species. Here, we used immunoprecipitation assays, combined with mass-spectrometry, to identify major EIF4E5 interacting proteins in L. infantum. A constitutively expressed, HA-tagged, EIF4E5 co-precipitated mainly with EIF4G1 and binding partners previously described in Trypanosoma brucei, EIF4G1-IP, RBP43 and the 14-3-3 proteins. In contrast, no clear co-precipitation with EIF4G2, also previously reported, was observed. EIF4E5 also co-precipitated with protein kinases, possibly associated with cell-cycle regulation, selected RNA binding proteins and histones. Phosphorylated residues were identified and mapped to the Leishmania-specific C-terminal end. Mutagenesis of the tryptophan residue (W53) postulated to mediate interactions with protein partners or of a neighbouring tryptophan conserved in Leishmania (W45) did not substantially impair the identified interactions. Finally, the crystal structure of Leishmania EIF4E5 evidences remarkable differences in the eIF4G interfacing region, when compared with human eIF4E-1 and with its Trypanosoma orthologue. Mapping of its C-terminal end near the cap-binding site also imply relevant differences in cap-binding function and/or regulation.

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression.

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.

Pre-clinical study of drug combinations that reduce breast cancer burden due to aberrant mTOR and metabolism promoted by LKB1 loss.

Cancer therapies that simultaneously target activated mammalian target of rapamycin (mTOR) and cell metabolism are urgently needed. The goal of our study was to identify therapies that effectively inhibited both mTOR activity and cancer cell metabolism in primary tumors in vivo. Using our mouse model of spontaneous breast cancer promoted by loss of LKB1 expression in an ErbB2 activated model; referred to as LKB1-/-NIC mice, we evaluated the effect of novel therapies in vivo on primary tumors. Treatment of LKB1-/-NIC mice with AZD8055 and 2-DG mono-therapies significantly reduced mammary gland tumorigenesis by inhibiting mTOR pathways and glycolytic metabolism; however simultaneous inhibition of these pathways with AZD8055/2-DG combination was significantly more effective at reducing tumor volume and burden. At the molecular level, combination treatment inhibited mTORC1/mTORC2 activity, selectively inhibited mitochondria function and blocked MAPK pro-survival signaling responsible for the ERK-p90RSK feedback loop. Our findings suggest that loss of LKB1 expression be considered a marker for metabolic dysfunction given its role in regulating AMPK and mTOR function. Finally, the outcome of our pre-clinical study confirms therapies that simultaneously target mTORC1/mTORC2 and glycolytic metabolism in cancer produce the best therapeutic outcome for the treatment of patients harboring metabolically active HER2 positive breast cancers.

Omega-3 polyunsaturated fatty acid promotes the inhibition of glycolytic enzymes and mTOR signaling by regulating the tumor suppressor LKB1.

The omega-3 polyunsaturated fatty acids (ω3PUFAs) are a class of lipids biologically effective for the treatment of inflammatory disorders, cardiovascular disease and cancer. Patients consuming a high dietary intake of ω3PUFAs have shown a low incidence of metabolic disorders, including cancer. Although the effects of ω3PUFAs intake was shown to be involved in the prevention and treatment of these diseases, the underlying molecular mechanisms involved are not well understood. Here, we show that ω3PUFA, docosahexaenoic acid (DHA) enhanced the tumor suppressor function of LKB1. We observed that when LKB1 expressing cells are treated with DHA, there is an increase in LKB1 activity leading to phosphorylation of AMPK and inhibition of mTOR signaling. Abrogation of LKB1 in MCF-7 cells by siRNA reversed this phenotype. Furthermore, cellular metabolism was altered and ATP levels were reduced in response to DHA treatment, which was further attenuated in cells expressing LKB1. More importantly, in mammary epithelial cells expressing LKB1, the rate of glycolysis was decreased as a result of diminished expression of glycolytic enzymes. Functionally, these events lead to a decrease in the migration potential of these cells. Overall, our discovery shows for the first time that LKB1 function is enhanced in response to ω3PUFA treatment, thereby resulting in the regulation of cell metabolism.

Loss of LKB1 expression reduces the latency of ErbB2-mediated mammary gland tumorigenesis, promoting changes in metabolic pathways.

The tumor suppressor kinase LKB1 is mutated in a broad range of cancers however, the role of LKB1 mammary gland tumorigenesis is not fully understood. Evaluation of human breast cancer tissue microarrays, indicate that 31% of HER2 positive samples lacked LKB1 expression. To expand on these observations, we crossed STK11 (fl/fl) mice with mice genetically engineered to express activated Neu/HER2-MMTV-Cre (NIC) under the endogenous Erbb2 promoter, to generate STK11 (-/-/) NIC mice. In these mice, the loss of lkb1 expression reduced the latency of ErbB2-mediated tumorigenesis compared to the latency of tumorigenesis in NIC mice alone. Analysis of STK11(-/-/)NIC mammary tumors revealed hyperactivation of mammalian target of rapamycin (mTOR) through both mTORC1 and mTORC2 pathways as determined by the phosphorylation status of ribosomal protein S6 and AKT. Furthermore, STK11(-/-/)NIC mammary tumors had elevated ATP levels along with changes in metabolic enzymes and metabolites. The treatment of primary mammary tumor cells with specific mTOR inhibitors AZD8055 and Torin1, that target both mTOR complexes, attenuated mTOR activity and decreased expression of glycolytic enzymes. Our findings underscore the existence of a molecular interplay between LKB1-AMPK-mTORC1 and ErbB2-AKT-mTORC2 pathways with mTOR at its epicenter, suggestive that loss of LKB1 expression may serve as a marker for hyperactivated mTOR in HER2 positive breast cancer and warranting further investigation into therapeutics that target LKB1-AMPK-mTOR and glycolytic pathways.