Kwansei Gakuin University
Report of Research Outcome
2023/02/06

To President
Department： Science and Technology
Position： Postdoctoral fellow
Name： Ferbian Milas Siswanto
I report the outcome of the research as follows.
□Sabbatical leave with grantName of theFund/Prog ra m

□KGU Joint Research□Sabbatical leave with no grant□Individual Special Research⊠Postdoctoral f ellow※ Please report by desi gnated f orm as f or “Inte rnational ResearchCollaboration”.

ResearchThe me

New biological f unctions and regulation of Parkin under hypoxia and oxidativestressResearchImaoka La b., Graduat e School of Science and Technology,Site/Venue

Kwansei Gakuin Unive rsity

Researchperiod2022/04/01

2023 /03/31 （～12months）◆ Summary of the research outcome （ appr ox . 2 ,5 00 wo r ds）
Please write down the outcomes in detail regarding the research theme above.
All m etazoans require a sufficient suppl y of ox ygen (O 2 ), an essential substrate invar ious m etabolic proc esses, bioenergetics, and cellular signaling. O 2 depr ivation or
hypoxia (0.1–1% O 2 ) is a fundam ental feature of phys iological and pathoph ys iologicalconditions, such as respirator y and circulator y disorders, as well as cancer. In solid
tum ors, rapidl y prolifer ating cancer cells caus e tum or m ass outgr owth and a shortage ofblood s uppl y, r esulting in the form ation of a hypoxic region. H ypoxia pla ys a s ignificant
role in tum or developm ent, contr ibutes to therapeutic resistance, and is a criticalm icroenvir onm ental factor pr om oting m etastatic progression, ther eb y ser ving as aprognostic factor as well as a com pelling therapeutic target. Central to the m olecularand cellular r esponses to hypox ia, h ypoxia-inducible factors (HIFs) sense andorchestrate var ious m etabolic adaptation pathways and the transcriptional cascades ofstress factors regulating c ell death or s urvival. The tr anscriptional activit y of HIF isregulated b y various factors, inc luding r eduction/oxidation (redox) factor 1 (Ref-1). Dueto its centr al r ole in hypox ia r esponses, HIF and its target genes have em erged asprom ising therapeutic targets for solid tum or therapy. However, HIF inhibitors have not1

yet been approved for the tr eatm ent of cancer patients due to lim ited s electivit y,specificit y, and ther apeutic efficac y or safet y.
Oxidative stress refer s to excessive levels of reactive ox ygen species ( ROS)
com pared to antiox idant defense. ROS repr esents highly reactive ox ygen-containing
chem ical interm ediates, such as superox ide anion r adical (O 2 · − ), hydr ox yl radical ( · O H),
singlet ox ygen ( 1 O 2 ), and hydrogen per oxide ( H 2 O 2 ). RO S ar e generated endogenous ly
as b yproducts of nor m al m etabolism and, at low concentrations, are r equired forcellular signaling. Upon exceeding a threshold level, ROS leads to c ell death in norm alcells. However, in pathologic al conditions, ox idative str ess leads to the developm ent of
a diseased state, including carcinogenesis and neur odegenerative diseases ( i.e.,
Park inson’s). The elevated m etabolism driven by aberrant cell growth in cancer caus es
excess production of ROS. To adapt to constant high ROS levels, tum or cells optim ize
the activity of antioxidant transcription factors. This particular feature has been widel y
exploited in efforts to s earch for new anticanc er treatm ents. In Park inson’s disease, theclearance ability of oxidative stress-induc ed dam aged and unwanted proteins isim paired. Park in im pairm ent is strongly associated with oxidative stress-inducedPark inson’s, but its role in the ox idative stres s pathwa y in cancer is currently unk nown.
Park in is an E3 ubiquitin ligas e belonging to the RING-between-RING fam ily thatcatal yzes the covalent attac hm ent of ubiquitin onto substrate proteins andsubsequentl y leads to its degradation via the ubiquitin-pr oteasom e s ystem , a m ajor
intr acellular pr otein degradation m achiner y. Park in is involved in num erous cellular
processes, partic ularl y pathwa ys which are tightly c onnected to m itochondrial qualit ycontrol. In the present stud y, we found that Park in protein levels were elevated anddepleted under hypox ia and ox idative stress, respectivel y. These changes were foundto be critical for canc er cell sur vival under h yp oxic and oxidative stress conditions.
Under h ypoxia, we found that Park in m RNA and protein levels wer e elevated in a
HIF-1α-dependent m anner. Stabilized HIF- 1α under h ypoxia tr anslocates to thenucleus, where it binds to the h ypoxia res pons e elem ent (HRE) located in the -107/-103
bp of the Parkin proxim al prom oter, increas ing transactivation of Parkin and its protein
levels. The elevation of Park in under h ypoxia is cr itic al for cancer survival underhypoxia, as Park in k nock down reduces the viabilit y of h ypoxia-tr eated Hep3B c ells. W edeterm ined that upon activation, Park in is res ponsible for ubiquitination andproteasom al degradation of sequestos om e 1 (SQSTM1/p62). p62 sequesters andnegativel y regulates extracellular signal- regulated k inase ( ERK), reduces O 2
consum ption, decreases CO2 releas e, and lowers ener gy ex penditure; ther efore, thesuppression of p62 b y Park in is a critical step in res pons e to hypox ia. These findings
show the potential of targeting Park in/p62 as a novel strategy to eradicate hypox ic solidtum ors.
Under ox idative str ess, we found that Park in m RNA and protein levels wer e
depleted. Mechanistic ally, we found that H 2 O 2 activates NF-κ B. The RelA/p65
com ponent of the NF-κB pathwa y inhibits the expression of Parkin, lik ely by interferingwith ATF4 activity. Next, we found that Park in is responsible for the ubiquitination andproteasom al degr adation of Ref-1; thus, H 2 O 2 -suppr essed Parkin express ion is
respons ible for the elevated Ref-1. Liver cancer patients with high Ref-1, low Park in,and high RelA ex pression had a lower probability of survival. Additionall y, theexpression levels of Park in and RelA were negativel y and positively correlated with
Ref-1 levels, res pectivel y, in the TCGA liver cancer cohort. W e concluded that anincreas e in Ref-1 via inhibition of Park in is critical for c ancer cell sur vival underoxidative str ess. Thes e res ults show the potential of the Park in/Ref-1 ax is as aprognostic factor and therapeutic str ategy to eradicate liver cancer.
2CONFERENCE
1. Sisw anto FM , Im aok a S. Yeast β- glucan poss esses a resistance-m odif ying propert yby m odif ying the levels of drug-m etabolizing enzym es thr ough the NF-k B and Nrf2
pathwa ys. The 37th Annual Meeting of the Japanese Society for the Study ofXenobiotics. Yok oham a, Japan. 7- 10 Nov 2022.
PUBLIC ATIONS
2. Sisw anto FM , Mitsuoka Y, Nak am ura M, Ogur o A, Im aok a S. Nrf2 and Park in-Hsc70
regulate the expr ession and protein stabilit y of p62/SQSTM1 under hypoxia. Sc iRep. 2022 Dec 8;12(1):21265. doi: 10.1038/s41598-022-25784-0. PMID: 36481701;
PMCID: PMC9731985.
3. Sisw anto FM , Oguro A, Im aok a S. Nrf2 regulates the expr ession of CYP2D6 byinhibiting the activit y of Krüppel- Lik e Factor 9 (KLF9) . Genes to Cells. Underrevision.
4. Sisw anto FM , Ok ugawa K, Tam ura A, Im aok a S. H ydr ogen per oxide activates
Ref-1/APE1 via NF-κB and Park in: role in liver cancer resistance to oxidative stress.
Manuscript In preparation.

Deadline： W ithin two months af ter f inishing the research pe riod.
Sabbatical leave with grant: Sub mit this report t o President with conf irmation by the dean ofschool you belong to.
※ Postdoctoral f ellow is required to sub mit this report with conf irmation by the de an of graduate

school bef ore the end of emplo yment pe riod.
W here to submit： Orga nization f or Research a nd Develop ment and Ou treach (N UC)◆ W e put this report on t he web of KGU. If there is any proble m abo ut it because of diff iculties on
your research, please le t us know.

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