Shrishiv Timbalia

Introduction: Breast cancer is a leading cause of disease and mortality in women, with over 1,500,000 women diagnosed with and 500,000 dying of breast cancer worldwide annually¹. Because of breast cancer’s genetic factor and mortality rate, it is critical to discover methods for detecting and treating this disease. The mitochondrial 1-carbon synthetic pathway is important for producing anabolic biomolecules and pentose phosphate pathway intermediates, and perturbation of the 1-carbon pathway results in global cellular changes in the levels of metabolic and structural molecules^2,3. Methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) is a key enzyme in the mitochondrial 1-carbon metabolic pathway, converting methylene-THF into formyl-THF⁴. In breast cancer cells, MTHFD2 is overexpressed compared to normal tissue, raising the possibility of developing pharmacological inhibitors with therapeutic potential^5,6. Methods: MTHFD2 was knocked down in MCF-7 estrogen receptor positive breast cancer cells, and cells were subjected to growth experiments in folate or glycine depleted medium². mir-9, an miRNA implicated in tumorigenesis, was overexpressed in MCF-7 cells and genetic changes were observed using transcriptome analysis⁷. MDA-MB-231 breast cancer cells were screened using an RNAi library to observe the effect silencing on vimentin expression, and the hits were verified by assessing the ability of knockdown cells to invade a gel matrix³. Western blotting was employed to quantify the level of MTHFD2 in 698 breast cancer patient samples⁵. Purification, crystallization, and X-ray diffraction of MTHFD2 was performed and the efficacy of the inhibitor LY345899 was assessed using an enzyme inhibition assay⁶. Results: Suppression of MTHFD2 resulted in glycine auxotrophy and increased sensitivity to folate depletion². In breast cancer patient samples, MTHFD2 was one of six novel targets of mIR-9 discovered, and mIR-9 levels were found to be decreased in MCF-7 cells while MTHFD2 levels were found to be elevated⁷. Knockdown of MTHFD2 via RNAi resulted in decreased vimentin expression, and these knockdown cells failed to invade a gel matrix as quickly as control cells³.  Biochemical analysis of 698 breast cancer patient tissue samples resulted in 287 of the samples exhibiting significantly increased levels of MTHFD2⁵. The crystal structure of MTHFD2 was successfully resolved and important residues were identified. Additionally, LY345899 was capable of inhibiting MTHFD2⁶. Conclusions: MTHFD2 is involved in several metabolic processes which play a role in breast cancer proliferation^1-5,7. The crystal structure of MTHFD2 was successfully resolved and LY345899 was found to be a preliminary inhibitor of the protein, laying the foundation for more effective drug design⁶.

1. Torre, Lindsey A., Freddie Bray, Rebecca L. Siegel, Jacques Ferlay, Joannie Lortet-Tieulent, and Ahmedin Jemal. “Global Cancer Statistics, 2012.” CA: A Cancer Journal for Clinicians2 (2015): 87-108. Web.
2. Koufaris, Costas, Suchira Gallage, Tianlai Yang, Chung-Ho Lau, Gabriel N. Valbuena, and Hector C. Keun. “Suppression of MTHFD2 in MCF-7 Breast Cancer Cells Increases Glycolysis, Dependency on Exogenous Glycine, and Sensitivity to Folate Depletion.” Journal of Proteome Research8 (2016): 2618-625. Web.
3. Lehtinen, Laura, Kirsi Ketola, Rami Makela, John-Patrick Mpindi, Miro Viitala, Olli Kallioniemi, and Kristiina Iljin. “High-throughput RNAi Screening for Novel Modulators of Vimentin Expression Identifies MTHFD2 as a Regulator of Breast Cancer Cell Migration and Invasion.” Oncotarget1 (2010): 48-63. Web.
4. Nilsson, Roland, Mohit Jain, Nikhil Madhusudhan, Nina Gustafsson Sheppard, Laura Strittmatter, Caroline Kampf, Jenny Huang, Anna Asplund, and Vamsi K. Mootha. “Metabolic Enzyme Expression Highlights a Key Role for MTHFD2 and the Mitochondrial Folate Pathway in Cancer.” Nature Communications 5 (2014): n. pag. Web.
5. Liu, Feng, Yang Liu, Chuan He, Li Tao, Xiaoguang He, Hongtao Song, and Guoqiang Zhang. “Increased MTHFD2 Expression Is Associated with Poor Prognosis in Breast Cancer.” Tumor Biology9 (2014): 8685-690. Web.
6. Gustafsson, Robert, Ann-Sofie Jemth, Nina M.s. Gustafsson, Katarina Farnegardh, Olga Loseva, Elisee Wiita, Nadilly Bonagas, Leif Dahllund, Sabin Llona-Minguez, Maria Haggblad, Martin Henriksson, Yasmin Andersson, Evert Homan, Thomas Helleday, and Pal Stenmark. “Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor.” Cancer Research4 (2016): 937-48. Web.
7. Selcuklu, S. D., M. T. A. Donoghue, K. Rehmet, M. De Souza Gomes, A. Fort, P. Kovvuru, M. K. Muniyappa, M. J. Kerin, A. J. Enright, and C. Spillane. “MicroRNA-9 Inhibition of Cell Proliferation and Identification of Novel MiR-9 Targets by Transcriptome Profiling in Breast Cancer Cells.” Journal of Biological Chemistry35 (2012): 29516-9528. Web.