RNA sequencing of tumors from mice fed diets excluding methionine/tryptophan/niacin (Part 2)
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. Overall design: All experiments in the present study were approved by the Osaka University Animal Experiments Committee, the formal ethics committee for institutional animal study at Osaka University, Japan, under the approval No. 30-011-053, and the experiments were conducted in accordance with the Osaka University Regulations on Animal Experiments. Five-week-old wild-type NOD SCID mice, derived from a mouse strain with a naturally occurring mutant, exhibiting an autosomal recessive inheritance pattern, and showing severe immunodeficiency due to the absence of T and B cells, which results in minimal rejection of xenogeneic cells and tissues, were procured from Japan SLC, Inc. All mice were female. Mice were randomly divided into 5 mice per cage. Given that not only methionine but also its upstream pathways are involved in cancer immunity. Tryptophan/niacin, which is located upstream of the methionine cycle, is metabolized to form nicotinamide, which is related to the methionine pathway, and 1-methyl-nicotinamide (MNAM) is formed from S-adenosylmethionine (SAM) synthesized from methionine, via nicotinamide N-methyltransferase, and thus MNAM synthesized in this methionine cycle is said to suppress T cells and promote cancer, we therefore subcutaneously implanted HT-29 cultured in regular medium containing all nutrients into the backs of 2 x 105 NOD SCID mice, and the mice were then divided into four groups: those kept on normal food, those kept on food excluding methionine only, and those kept on food excluding niacin/tryptophan, and those kept on mice without methionine/tryptophan/niacin. And, they were reared for 2 weeks (except for 08_T_zen (GSM7873764) and 09_T_3ketsu (GSM7873765), described in each Sample section). Special diets for the mice were custom-ordered with as much appearance and taste as possible. Rearing conditions were such that the floor was changed daily to minimize the intake of excretory-derived amino acids. Room temperature was maintained around 23°C and lights were turned on in 12-hour cycles. Although our initial plan was to euthanize the mice if the tumor size exceeded 150 mm3 or if the mouse's body weight increased by more than 20% within a week, we in practice successfully completed the intended three-week experimental period without encountering any breaches of the restrictions pertaining to tumor size or body weight. At the conclusion of the animal experiment, we adhered to the following ethical guidelines: For mice weighing around 20g, the concentration of carbon dioxide used for scarification was 40%, with a flow rate of 0.5L/min for an inhalation time of 10 minutes; For smaller mice weighing 16g or less, the concentration of carbon dioxide was reduced within the range of 30% to 40%, with a lower flow rate between 0.2L/min to 0.5L/min for a shorter inhalation time between 5 to 10 minutes, to minimize the stress and pain to the mice during the procedure. Tumors were then removed and evaluated for RNA expression and type by RNA sequence. After confirming the cessation of mouse respiratory movements, the tumor was promptly excised within 30 minutes in a well-maintained environment with laboratory air conditioning and a clean experimental table in the laboratory. Subsequently, the tumor was immersed in RNA LATER reagent (Sigma-Aldrich), and RNA extraction was performed using RNAeasy kit (Qiagen), followed by RNA sequencing analysis.