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Remote solid cancers rewire hepatic nitrogen metabolism via host nicotinamide-N-methyltransferase

Mizuno, Rin 京都大学 DOI:10.14989/doctor.k24516

2023.03.23

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ARTICLE
https://doi.org/10.1038/s41467-022-30926-z

OPEN

Remote solid cancers rewire hepatic nitrogen
metabolism via host nicotinamide-Nmethyltransferase
1234567890():,;

Rin Mizuno1,2, Hiroaki Hojo1,3,4, Masatomo Takahashi 5, Soshiro Kashio6, Sora Enya3,4, Motonao Nakao5,
Riyo Konishi1, Mayuko Yoda1, Ayano Harata1, Junzo Hamanishi 2, Hiroshi Kawamoto 7, Masaki Mandai2,
Yutaka Suzuki 8, Masayuki Miura 6, Takeshi Bamba 5, Yoshihiro Izumi 5 & Shinpei Kawaoka 1,3,4,9 ✉

Cancers disrupt host homeostasis in various manners but the identity of host factors
underlying such disruption remains largely unknown. Here we show that nicotinamide-Nmethyltransferase (NNMT) is a host factor that mediates metabolic dysfunction in the livers
of cancer-bearing mice. Multiple solid cancers distantly increase expression of Nnmt and its
product 1-methylnicotinamide (MNAM) in the liver. Multi-omics analyses reveal suppression
of the urea cycle accompanied by accumulation of amino acids, and enhancement of uracil
biogenesis in the livers of cancer-bearing mice. Importantly, genetic deletion of Nnmt leads to
alleviation of these metabolic abnormalities, and buffers cancer-dependent weight loss and
reduction of the voluntary wheel-running activity. Our data also demonstrate that MNAM is
capable of affecting urea cycle metabolites in the liver. These results suggest that cancers upregulate the hepatic NNMT pathway to rewire liver metabolism towards uracil biogenesis
rather than nitrogen disposal via the urea cycle, thereby disrupting host homeostasis.

1 Inter-Organ Communication Research Team, Institute for Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan. 2 Department of Gynecology
and Obstetrics, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan. 3 The Thomas N. Sato BioMEC-X Laboratories, Advanced
Telecommunications Research Institute International (ATR), Kyoto 619-0237, Japan. 4 ERATO Sato Live Bio-forecasting Project, Japan Science and
Technology Agency (JST), Kyoto 619-0237, Japan. 5 Division of Metabolomics, Research Center for Transomics Medicine, Medical Institute of Bioregulation,
Kyushu University, Fukuoka 812-8582, Japan. 6 Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 1130033, Japan. 7 Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan. 8 Graduate School of
Frontier Science, The University of Tokyo, Chiba 277-8562, Japan. 9 Department of Integrative Bioanalytics, Institute of Development, Aging and Cancer
(IDAC), Tohoku University, Sendai 980-8575, Japan. ✉email: kawaokashinpei@gmail.com

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1

ARTICLE

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C

ancers adversely affect various host organs at a body-wide
level1,2. In the presence of solid cancers, for example,
adipose tissues exhibit enhanced lipolysis and thus
degenerate (i.e., adipose atrophy)3. Metabolism in the liver is also
adversely affected in cancer-bearing organisms despite having
been less studied than the adipose tissues in this context4–8. Such
metabolic abnormalities, often accompanied by chronic inﬂammation, systemically disrupt host homeostasis, and lead to loss of
body weight, reduced quality of life, impaired tolerance to standard anti-cancer therapies, and ultimately worsened survival1,2.
This multifactorial and systemic phenomenon is clinically
recognized as cancer cachexia, a life-threatening syndrome that
more than 50% of advanced cancer patients unfortunately suffer9.
Nicotinamide-N-methyltransferase (NNMT) is an enzyme that
transfers a methyl group from S-adenosyl methionine (SAM) to
nicotinamide (NAM)10. This reaction produces S-adenosyl
homocysteine (SAH) and 1-methylnicotinamide (MNAM). In
mice, NNMT is abundantly expressed in the liver and adipose
tissues11,12. NNMT was originally recognized as merely an NAM
clearance enzyme that discards NAM as MNAM since MNAM is
excreted in the urine10. However, this view has been challenged
by recent important studies that demonstrate the signiﬁcant roles
of NNMT in obesity11, metabolism12,13, cancers14,15, immunity16,
and lifespan17.
Despite initially being characterized as metabolically inert in
the past, MNAM is now thought to harbor various biological
functions11,12,16–20. For example, it is known that MNAM binds
and stabilizes SIRT1 thereby contributing to hepatic nutrient
metabolism12. MNAM is also considered as an anti-inﬂammatory
agent20. In addition, MNAM is metabolized by aldehyde oxidase
(AOX), leading to the production of reactive oxygen species
(ROS). In Caenorhabditis elegans (C. elegans), ROS generated by
this reaction is believed to prolong lifespan17.
Whether NNMT and MNAM are involved in cancer-induced
host pathophysiology is currently unknown. Yet, there is evidence
implicating NNMT as such a factor. The ﬁrst indication was
reported in 1998 showing that transplantation of colon cancers
into mice increased the enzymatic activity of NNMT in the
liver21. Supporting this, our transcriptome experiments demonstrated strongly induced expression of Nnmt in the liver after
transplantation of breast cancers6. A similar trend was also seen
in the livers of mice bearing genetically induced lung cancers4.
Thus, a series of non-liver solid cancers induced NNMT
expression in the liver. How the NNMT induction in the liver
affects liver physiology in the cancer-bearing condition remained
to be revealed.
In the current study, utilizing mouse genetics and multi-omics
analyses, we establish NNMT as a host factor that promotes
metabolic dysfunction of the liver in cancer-bearing animals. The
multi-omics map we construct here provides a comprehensive
picture of cancer-induced abnormalities in liver metabolism.
Moreover, our data reveal which abnormalities depend on host
NNMT (and which do not). This study not only unravels the
roles of NNMT in cancer-induced host pathophysiology but also
offers a basis to study the complex systemic syndrome that
severely impacts patients with incurable cancers.
Results
Solid cancers upregulate Nnmt expression in the liver. Our
previous research demonstrated that expression of Nnmt in the
liver is increased upon transplantation of 4T1 breast cancer6. We
conﬁrmed this observation using quantitative polymerase chain
reaction (qPCR) (Fig. 1a, b and Supplementary Data 1). The
degree of induction was correlated to the duration of time following transplantation (i.e., Day 7 < Day 14). Induction of hepatic
2

Nnmt was not restricted to the 4T1 breast cancer model as colon
cancer (Colon26), ovarian cancer (ID8-F3), and lung cancer
(LLC) all increased hepatic Nnmt mRNAs in varying degrees
(Fig. 1c). The Colon26 data were consistent with the previous
ﬁnding21. In addition, the Nnmt induction was detected in the
livers of a genetically induced lung cancer model (Supplementary
Fig. 1a)4, demonstrating that the induction was not a result of
transplantation. Therefore, upregulation of hepatic NNMT by
solid cancers appeared a general phenomenon. In this study, we
largely focused on the 4T1 breast cancer model since 4T1 was the
most prominent inducer of hepatic Nnmt.
Next, we investigated the effects of this Nnmt induction on
NNMT-related metabolites. NNMT transfers a methyl group from
S-adenosyl methionine (SAM) to nicotinamide (NAM), producing
S-adenosyl homocysteine (SAH) and 1-methylnicotinamide
(MNAM)10. Liquid chromatography coupled with tandem mass
spectrometry (LC-MS/MS) demonstrated that the steady-state
amount of MNAM was increased in the livers of 4T1-bearing
mice (Fig. 1d). On the other hand, 4T1 breast cancer did not affect
NAM, SAM, and SAH in the liver (Fig. 1d). In addition, it is known
that MNAM is further metabolized into me4PY (N-methyl-4pyridone-3-carboxamide) and me2PY (N-methyl-2-pyridone-5carboxamide) by aldehyde oxidase (AOX)10,22,23. Yet, 4T1
transplantation did not affect these two metabolites (Supplementary
Fig. 1b).
We next sought to address how 4T1 breast cancer increases
Nnmt expression in hepatocytes. For this purpose, we exploited
an approach that uses supernatant of 4T1 culture (4T1conditioned media): we treated AML12 primary hepatocyte cells
with the 4T1-conditioned media and examined the expression of
Nnmt. We found that 4T1-conditioned media was capable of upregulating Nnmt expression in AML12 (Fig. 1e). This was
accompanied by a concomitant increase in MNAM and a
decrease in SAM (Fig. 1f). 4T1 expresses a variety of cytokines
and hormones in vitro including TNFα as reported by others and
also conﬁrmed by us (Supplementary Fig. 1c)24. Our data
demonstrated that TNFα was able to increase Nnmt mRNAs
and MNAM (Fig. 1g, h). These data collectively suggested that
4T1 breast cancer activates the hepatic NNMT pathway at least in
part via soluble factors such as TNFα.
Nnmt deletion abolishes MNAM and accumulates SAM in a
cancer-bearing condition. Changes in Nnmt expression in the
liver prompted us to investigate the role of Nnmt in cancerinduced abnormalities in this organ. To address this, we generated Nnmt knockout (KO) mice using the CRISPR-Cas9 technique (Fig. 2a, b)25. We obtained mice that harbor a 25-base pair
(bp) deletion. This resulted in a premature stop codon in the
domain critical for the enzymatic activity of NNMT. In this
manuscript, we simply refer to this allele as Nnmt KO.
To characterize Nnmt KO mice, we measured the amounts of
NAM, SAM, MNAM, and SAH in the liver. In the Nnmt KO
livers, only MNAM was strongly affected by Nnmt KO; MNAM
was almost nearly abolished in Nnmt KO mice (Fig. 2c). This
indicated that MNAM is generated solely by NNMT. Moreover,
me4PY and me2PY were depleted by Nnmt KO (Supplementary
Fig. 2a). These were in contrast to NAM, SAM, and SAH were all
unaffected by loss of NNMT function. ...

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Acknowledgements

This work was supported by JSPS KAKENHI (17H06299, 18K15409, 18H04810,

20H03451, and 20H04842; S.K.), JST FOREST (JPMJFR2062; S.K.), JST Moonshot

(JPMJMS2011-61; S.K.), JST ERATO Sato Live Bio-forecasting project (JPMJER1303),

Caravel, Co., Ltd (S.K.), Ono Medical Research Foundation (S.K.), Takeda Science

Foundation (S.K.), Mochida Memorial Foundation (S.K.), The Uehara Memorial

Foundation (S.K.), Chubei Ito Foundation (S.K.), and Japan Foundation for Applied

Enzymology (S.K.). This work was also supported by JSPS KAKENHI (19K05167; Y.I.),

JST MIRAI (Y.I.), JSPS KAKENHI (JP21H04774; M. Miura), and AMED-Aging

(JP21gm5010001; M. Miura). We thank Dr. Bryce Nelson, Dr. Pieter Bas Kwak, Dr.

Hiroki Shibuya, Dr. Takeshi Watanabe, and Dr. Kosuke Yusa for critically reading the

manuscript. We also thank Dr. Gen Kondoh, Dr. Hitomi Watanabe, Hitoshi Miyachi,

and Satsuki Kitano for their help in mouse experiments.

Author contributions

S.K conceived and supervised the project and wrote the paper. R.M. performed experiments, analyzed data, constructed ﬁgures, and wrote the paper. H.H. performed

experiments and analyzed data. S.E., R.K., A.H., and M.Y. performed experiments. M.T,

S. K., M.N., M. M., T.B., and Y.I. performed metabolites measurements. Y.S. performed

RNA-seq experiments. H.K., J.H., and M. M. made a substantial contribution to the

conception of this work. All authors provided intellectual input and reviewed the paper.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s41467-022-30926-z.

Correspondence and requests for materials should be addressed to Shinpei Kawaoka.

Peer review information Nature Communications thanks Charles Brenner and the other

anonymous reviewer(s) for their contribution to the peer review of this work. Peer

reviewer reports are available.

Reprints and permission information is available at http://www.nature.com/reprints

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional afﬁliations.

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Remote solid cancers rewire hepatic nitrogen metabolism via host nicotinamide-N-methyltransferase

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