Project description:This West Coast Metabolomics Center pilot and feasibility project granted to Peter Kaiser (UC Irvine), aims to achieve understanding of a unique metabolic dependence of cancer cells to explore development of novel unconventional therapeutic strategies that exploit dependence of cancer cells on methyl-donor abundance. The past few years have highlighted the role of altered metabolism in cancer. While mechanistic insight into changed metabolism in cancer is very limited, the importance of the metabolic pathway surrounding homocysteine and methionine for cancer cell proliferation has been known for over 30 years. These findings, generally summarized as methionine-dependence or methionine stress sensitivity, describe the phenomenon that most cancer cells cannot proliferate in growth medium where the amino acid methionine is replaced with its direct metabolic precursor homocysteine. Importantly, non-tumorigenic cells are unaffected by replacing methionine with homocysteine in the growth medium. For the past years we have been studying methionine dependence of breast and prostate cancer and demonstrated that methionine-dependence is caused by insufficient flux through this pathway to sustain synthesis of the downstream metabolite and the principal methyl-donor S-adenosylmethionine (SAM). We have isolated rare cell clones from MDA-MB468 breast cancer cells (referred to as MB468RES) that are no longer methionine dependent and proliferate in homocysteine medium. Interestingly, MB468RES have lost their ability for anchorage independent growth, a hallmark of cancer. The MB468 and MB468RES cell line pair confirms other observations showing that methionine dependence is tightly linked to tumorigenicity. Importantly, this cell line pair is an ideal model to identify metabolite signatures linked to cancer cell methionine dependence. We propose to characterize the metabolic changes triggered by the shift from normal growth medium to homocysteine medium in MB468 breast cancer cells and the methionine stress insensitive MB468RES derivatives. In addition we have developed cancer cell lines with inducible shRNAs targeting methionine adenosyltransferase (MAT), the enzyme catalyzing synthesis of SAM from methionine and ATP. Inducible knockdown of MAT allows us to specifically reduce SAM synthesis. Our previous results suggest that SAM limitation is the critical trigger for cancer cell methionine dependence. Thus metabolite profiling using the MAT knockdown system will provide an independent dataset that together with metabolite profiles from the MB468 and MB468RES cell line pair will define critical metabolic profiles related to cancer cell methionine dependence. In the current investigation, untargeted analysis of primary metabolites and complex lipids, coupled with quantitative analysis of methionine pathway intermediates (folate and respective derivatives, s-adenosylmethoinine, s-adenosylhomocysteine, choline, betaine) and metabolic flux will be conducted on MB468, MB468RES and MB468shRNA following the switch from methionine containing media to homocysteine containing media over the course of 0, 2, 4, 8, 12, 24 and 48 hours. The primary objectives were to 1) characterize the metabolic response to methionine stress and SAM limitation and 2) correlate the metabolic signatures with cancer cell proliferation arrest and death.

Project description:In the past, animal experiments with rats, by depleting various amino acids in the diet, have shown that a deficiency of methionine inhibits tumor growth at the individual level and that methionine is important in the development and progression of cancer. This methionine is one of the essential amino acids and is the initiation codon that initiates protein translation from mRNA. Methionine is known to be converted to SAM, which is required for nucleic acid synthesis in cancer. Tumors are underdeveloped in an environment deficient in methionine, which is known as the "Hoffman effect". In fact, past reports have shown that in the absence of methionine, tumor growth is suppressed, and methionine has recently been shown to be essential in cancer stem cells or "tumor initiating cells". In addition, the focus on methionine in cancer is said to influence tumor-cell metabolism, histone patterns, and T cell immunity in the cancer microenvironment. In cancer immunity, not only methionine but also its upstream pathways are said to be involved. Tryptophan/niacin, which is located upstream of the methionine cycle, is metabolized to form nicotinamide, and MNAM is formed from the SAM and nicotinamide via nicotinamide N-methyltransferase (NNMT). NNMT and MNAM have been reported to participate in the mechanism of inhibition of the apoptosis signal-regulated kinase 1-p38 MAPK pathway, resulting in increased colon cancer cell resistance to 5-FU. NNMTs promote nicotinamide adenine dinucleotide depletion and epigenetic reprogramming, which have been implicated in the development of metabolic plasticity, circumvention of the major tumor suppressive process of cellular senescence, acquisition of stem cell properties, and resistance to therapy and poor clinical outcomes. Also, MNAM synthesized in this methionine cycle is said to suppress T cells and promote cancer. Hence, we implanted the colon cancer cell line HT-29 cultured in normal medium with all nutrients into NOD SCID mice and kept them for 2 weeks on a diet without the aforementioned methionine/tryptophan/niacin. Afterwards, tumors were removed and RNA-sequencing was performed, and a marked increase in the expression of RN7SL1, a non-coding RNA, was observed.

Dataset Information

Metabolite changes associated with methionine stress sensitivity of cancer (GC TOF MS analysis)

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