金沢大学十全医学会雑誌98

第131 巻第3号

98 −103（2022）

【総説】
第20 回

高安賞最優秀論文賞受賞

論文「Treatment of RB1-intact hepatocellular carcinoma with CDK4/6
inhibitor combination therapy」
2021 年 4 月 19 日掲載
Hepatology. 2021 April 19; 74(4): 1971-199.
Jindan Sheng, Susumu Kohno, Nobuhiro Okada,Nobuyuki Okahashi, Kana Teranishi, Fumio Matsuda,
Hiroshi Shimizu, Paing Linn, Naoko Nagatani, Minako Yamamura,
Kenichi Harada, Shin-ichi Horike, Hiroshi Inoue, Seiji Yano,
Sharad Kumar, Shunsuke Kitajima, Itsuki Ajioka
and Chiaki Takahashi
盛 金丹 (Jindan Sheng せい

Introduction
Synthetic cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors keep retinoblastoma 1 (RB1) tumor
suppressor gene product in an unphosphorylated form
for long time, causing cellular senescence, apoptosis
and enhanced immunogenicity in RB1-intact tumor
cells. These medications are now approved forpatients in advanced stages of hormone receptor (HR)positive; HER2-negative breast cancers. Aromataseinhibitors or estrogen receptor (ER) antagonist
fluvestrant doubled progression-free survival in thesepatients2). These agents are tested in clinical trials
for a variety of advanced solid tumors including
liver, lung, colon and brain malignancies1). However,single administration of these agents mostly resulted
in suboptimal outcomes in these cancers.
RB family member genes are rarely mutated ordeleted in hepatocellular carcinoma (HCC). However,many lines of evidence indicate that the loss ofRB function is mechanistically involved in thedevelopment of HCC. The HCV-derived NS5B
protein accelerates the degradation of RB1, as well
as RBL1 and RBL2, by increasing the recruitmentof the E6AP ubiquitin ligase 3). The constitutiveきんたん )production of preS2 and HBx generated from HBV
activates cyclin-CDK complexes, causing RB1
inactivation that is reversible 4). Based on thesefindings, HCC has been supposed to be one of themost promising targets of CDK4/6 inhibitors.
In the study reviewed here, we first demonstrated
that loss of all Rb family members in Trp53 -/mouse liver resulted in development of liver tumor
reminiscent to human HCC. Then we discovered thatre-expression of RB1 sensitized these tumors to a CDK
4/6 inhibitor, palbociclib. In multiple human HCC
cell lines, an unphosphorylatable thus constitutively
activated version of RB1 mutant (RB7LP) elicitedeffects similar to palbociclib, i.e., partial cellularsenescence and partial apoptosis. By screening
chemical compounds that enhance the efficacy of
RB7LP in HCC, we discovered an IKKβ inhibitor,
Bay11-7082. This agent significantly enhanced
RB7LP function to increase IKKα/β phosphorylationand following NF-κB activation. Subsequently,
palbociclib in combination with Bay11-7082 wasfound to be much more successful than single
administration in the treatment of hepatoblastoma
and HCC. Furthermore, blockade of IKK-NF-κB
or AKT pathways significantly enhanced therapeutic

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effects of palbociclib in RB1-intact K-Ras mutant
lung and colon cancers. Therefore, we propose that
CDK4/6 inhibitors have a good potential to curevarieties of RB1-intact malignancies including HCC
and KRAS cancers when combined with a suitablekinase inhibitor5).

Results
RB pathway inactivation drives HCC
We employed the hydrodynamic injection method
to transduce Rb1lox/lox; Rbl1-/-; Rbl2lox/lox; Trp53-/(QKO) mice with the PiggyBac transposon vector
construct (pCMV-Puro-Cre-IRES2-GFP) and pCMVhyPBase vector expressing a transposase in order toinvestigate the role of RB1 in the development ofHCC. Multiple liver tumors were observed in QKO

mice typically within 2.5 months after injection
with no overt expression of Rb1, Rbl1, Rbl2, orp53. Rb1lox/lox; Rbl1-/-; Rbl2lox/lox; Trp53+/+ (TKO) micedeveloped tumor typically after 12 months following
injection as described previously by other researchers.
Liver tumors developed in injected QKO mice
exhibited high cellularity and frequent expressionof Ki67 and alpha feto protein (AFP). Then, weestablished primary HCC cell lines from the livertumor. We introduced hemagglutinin (HA)-tagged
wild-type RB1 or V5-tagged RB7LP mutant into theQKO mouse-derived primary HCC cells. Both thewild-type RB1 and the RB7LP mutant showed
inhibition of cell proliferation, but RB7LP wassuperior to wild-type RB1 in terms of growth
suppression (Figure 1).

Figure 1. RB pathway inactivation drives HCC.
(A) The strategy used to generate QKO mice. (B) A representative image of liver tumor developed in QKO mouse. (C)Representative images of H & E staining (upper) and immunohistochamical (IHC) analysis of the indicated proteins (middle andlower) of the liver of a control mouse or a tumor developed in a QKO mouse 3 month after Cre-GFP-puro injection. (D) WST-8-based
assessment of growth property in primary HCC cells transduced with the indicated protein.

100RB7LP mimics the action of CDK4/6 inhibitors
We found that wild-type RB1 and the RB7LP
mutant induced partial cellular senescence and partial
apoptosis in QKO mouse-derived primary HCC cell.
Furthermore, we observed that compared with RB1
negative cells, RB1 positive cell lines are moresensitive to the palbociclib treatment. Next, we treatedan RB1-intact human hepatoblastoma cell line HepG2
with palbociclib. It showed an effect similar to theRB1-reconstituted mouse-derived primary HCC cell.
Then, we employed a Tet-ON system to overexpress
the RB7LP mutant in various human HCC cell lines.
We found that the RB7LP mutant induced partial
cellular senescence and partial apoptosis in thesecells as well (Figure 2).

Figure 2. RB7LP mimics the action of CDK4/6 inhibitors(A, F, G, J, K) Representative images of senescence-associated β-galactosidase (SA-β-gal) staining in the indicated cells and SA-β-gal staining and quantification. (B, D, E, I) Immunoblotting (IB) of the indicated proteins in primary HCC and HepG2 cells
infected with the lentivirus expressing the indicated proteins.

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IKKβ inhibition sensitizes hepatoblastoma andHCC cells to CDK4/6 inhibitors
Palbociclib when singly administrated exhibiteda significant but an unsatisfactory therapeutic effecton HA-RB1(+) QKO HCC and HepG2 cells. We
screened chemical libraries seeking for compounds
that enhance the efficacy of RB7LP in HepG2 cells.
We detected Bay11-7082, known as a chemical
inhibitor for IκB kinase β (IKKβ), which appeared
to synergize with either palbociclib or RB7LP toinduce apoptosis. Furthermore, RB7LP increased
IKKα/β phosphorylation and following activationof NF-κB in DOX (+) HepG2 cells. The Luciferase
assay revealed that IKKβ inhibitors reduced NF-κBactivation. We then treated HepG2 by palbociclib
together with an NF-κB inhibitor, which significantly
promoted cell apoptosis. In addition, combined use ofpalbociclib and IKKβ inhibitor exhibited synergistic
effects in human HCC cell lines both in vitro and in
immuno-deficient mice (Figure 3).

Figure 3. IKKβ inhibition sensitizes hepatoblastoma and HCC cells to RB7LP and a CDK4/6 inhibitor.
(A) Screening of a chemical library that covers 80 kinase inhibitors was performed to seek the compounds that selectively kill
HepG2 cells in which RB7LP was overexpressed. (B, C, F, G, H) Representative results of flow cytometr y analysis andquantification of apoptotic cells treated in the indicated manners. (D) IB of the indicated proteins in HepG2 cells infected with thelentivirus expressing the indicated proteins. (E) The NF-kB activity assessed by a luciferase reporter assay in HepG2 cells treatedwith the indicated chemicals. (I, J) Combination index was assessed in the indicated cells treated with the indicated chemicals.
(K, L) A representative result of xenograft assay.

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The mechanism whereby palbociclib IKKα/β
phosphorylation
To determine the mechanism whereby palbociclib
increases IKKα/β activities in RB1-intact cells, wemeasured the concentrations of nucleotides andmetabolites (ADP, ATP, AMP, GDP, GMP, G1P, G6P,F6P, NAD and NADH), and found that inductionof RB7LP reduced the abundance of all of them,implying nucleotide deficiency. Furthermore, weobserved increased expression of p53 and its target
genes, higher expression of γ-H2AX and increased

phosphorylation of CHK2 following RB7LP induction.
These findings suggest that RB7LP induces DNA
damage in cells. Next, we detected a similar metabolic
shift was achieved by the treatment with palbociclib.
In addition, we found that supplementation of dNTPs
antagonized the activation of γ-H2AX caused either
by RB7LP induction or palbociclib treatment. We
therefore reasoned increased IKKα/β phosphorylation
and NF-kB activity by nucleotide deficiency owing toRB1 hypophosphorylation followed by DNA damage
responses (Figure 4).

Figure 4. The mechanism whereby palbociclib and RB7LP increases IKKa/b phosphorylation.
(A, D) The cellular concentration of the indicated nucleotides and metabolites was determined in the indicated cells treated with theindicated chemicals. (B) GSEA results for hallmark gene sets in DOX (+) versus DOX (-) cells. (C, E, F) IB of the indicated proteinsin HepG2 cells infected with the lentivirus expressing the indicated proteins. (G) Luciferase reporter assay in indicated cells.

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Current Perspectives. Breast Cancer (Dove Med Press) 12: 45-56,2020
3 ) Munakata T, Liang Y, Kim S, McGivern DR, Huibregtse J,Nomoto A, Lemon SM. Hepatitis C virus induces E6AP-dependentdegradation of the retinoblastoma protein. PLoS Pathog 3(9);1335-1347, 2007
4 ) Hsieh YH, Su IJ, Wang HC, Tsai JH, Huang YJ, Chang WW,
Lai MD, et al. Hepatitis B virus pre-S2 mutant surface antigen
induces degradation of cyclin-dependent kinase inhibitor p27Kip1
through c-Jun activation domain-binding protein 1. Mol Cancer
Res 5(10); 1063-1072, 2007

Figure 5. Schematic presentation of our hypothesis.

5 ) Sheng J, Kohno S, Okada N, Okahashi N, Teranishi K,Matsuda F, Shimizu H, et al. Treatment of Retinoblastoma 1-Intact
Hepatocellular Carcinoma With Cyclin-Dependent Kinase 4/6Conclusion
Synthetic CDK4/6 inhibitors limit RB1 phosphorylationinducing G1 arrest, cellular senescence and apoptosis.
On the other hand, these agents cause DNA damage
response following nucleotide deficieny and stimulate
the survival signals depending on the IKK-NF-κBpathway. Our findings suggest that the use of asynthetic CDK4/6 inhibitor in combination with aninhibitor of the IKK-NF-κB pathway may provide
a therapeutic benefit to HCC patients (Figure 5).
We also proposed that synthetic CDK4/6 inhibitors
are applicable to KRAS mutated cancers whencombined with an appropriate kinase inhibitor5).

References1 ) Schettini F, De Santo I, Rea CG, De Placido P, Formisano L,Giuliano M, Arpino G, et al. CDK 4/6 Inhibitors as Single Agent inAdvanced Solid Tumors. Front Oncol 8; 608, 20182 ) Iorfida M, Mazza M, Munzone E. Fulvestrant in Combination
with CDK4/6 Inhibitors for HER2- Metastatic Breast Cancers:

Inhibitor Combination Therapy. Hepatology 74(4); 1971-1993, 2021

ProfileEDUCATION
Bachelor’s degree, Jilin Medical University,Jilin
September 2008 ̶ July 2013
Master’s degree, Beihua University, Jilin
September 2013 ̶ July 2016
Outstanding Master Graduates ( 2016)
MD.PhD, Kanazawa University, Kanazawa
October 2017 ̶ October 2021
EMPLOYMENT HISTORY
Resident doctor at Shanghai CHANGHAIHOSPITAL, Shanghai
June 2013 ̶ July 2016
Assistant Professor at Kanazawa UniversityCancer Research Institute, KANAZAWA
October 2021 ̶ January 2022
Special Associate Research Fellow at
Shanghai Cancer Institute，Shanghai Jiao
Tong University School of Medicine andRenji Hospital.
April 2022-Current