Page 57 - ebook

Therapy induced senescent tumor cells promotes
cancer stemness in breast cancer

4,5
Dong Hyun Lee 1,5 Yong Won Choi , Jang-Hee Kim 2,3,5 & Tae Jun Park 1,2,5
1 Department of Biochemistry and Molecular Biology, Ajou University School of Medicine Suwon 16499, Korea;
2 Department of Biomedical Sciences, Ajou University Graduate School of Medicine Suwon 16499, Korea;
3 Department of Pathology, Ajou University School of Medicine Suwon 16499, Korea;
4 Department of Hematology-Oncology, Ajou University School of Medicine Suwon 16499, Korea;
5 Inflamm-Aging Translational Research Center, Ajou University Medical Center Suwon 16499, Korea;
INTRODUCTION.
senescence refers to a state in which normal cells arrest growing due to various internal and external stresses. It is caused by stimuli such as DNA damage,
oncogene activation, and telomere shortening, and is known to serve as a barrier in the tumorigenesis. These phenomenon is characterized by the expression of
CDK inhibitors (p16INK4A, p21, p53), senescence associated galactosidase(SA-al-gal) staining, and senescence associated secretory protein(SASP). However,
senescence can also occur in cancer cells that are immortal and grow abnormally, and is observed in cancer patients who have undergone chemical and
radioactive chemotherapy. This is referred to as therapy induced senescence(TIS), senescent cancer cells have been reported to affect cancel metastasis,
invocation, immune cell infiltration, and tumor environment, which can exacerbate the prognosis of cancer patients. Cancer stem cells (CSCs) are a small group of
tumor populations with the ability to tumor initiation and recurrence, which express marker proteins specifically represented by differentiated cells and stem cells,
such as CD24, CD44, CD133, EpCAM. Stemness refers to the molecular process that appears in stem cells that produce self-renewal ability and differentiated
daughter cells. In cancer stem cells, the ability to stem cells and resistance to chemotherapy lead to cause recurrence of cancer. For this reason, in chemical
therapy, a treatment capable of inhibiting cancer stem cells together is required. Herein, we show that therapy induced senescence (TIS) with Doxorubicin in
various cancer cell line and expression of SASP and stem cell marker in breast cancer cell line. In a recent study, the difference in expression of SASP and CDKi
according to aging level was shown. Similarly, the grade of senescence in doxorubicin treated breast cancer cell differed senescence phenotypes not stem cell
related genes. High grade senescent cancer cells were highly expressed SASP, but not stem cell related genes. Conversely, Low grade senescent cancer cells
increased the expression of stem cell related genes, which can initiate tumor formation. In this study, it is suggested that chemotherapy induces senescence in
cancer patients, but differences in the grade of senescence can be found, and the resulting characteristics can also be expressed differently.

RESULTS.
MCF-7 MDA-MB-231 C12FDG LOW
T47D 2day 2day 3day
CON DOX 150nM CON DOX 150nM CON DOX 100nM C12FDG MED
C12FDG MEDHIGH
CON:3x10 5 cells Doxorubicin M/C C12FDG staining
DOX:6x10 5 cells 100nM, 150nM FACS C12FDG HIGH
CON DOX 150nM
Population % Population %
C12FDG L 98.1 C12FDG L 26.1
C12FDG M 1.1 C12FDG M 21.8
C12FDG MH 0.1 C12FDG MH 20.4
SW480 MKN-1 U2OS C12FDG H 0.0 C12FDG H 20.2
CON DOX 100nM CON Cisplatin 1μM CON Cisplatin 5μM
20 CON 50 40 CON
Relative mRNA expression level 10 5 DOX 150nM Relative mRNA expression level 30 20 DOX 150nM
15
C12FDG L
C12FDG L
C12FDG M
C12FDG M
C12FDG MH
C12FDG MH
0 C12FDG H 10 0 C12FDG H
p21 IL-6 IL-8 OCT3/4 SOX2 NANOG
Fig 1. Therapy-induced senescence in various cancer cell lines.
CON DOX 150nM
CON DOX 150nM 9 8
Number of colonies(x100) 2
2day 2day 3day 7 6 5
CON:4x10 4 cells Doxorubicin M/C 4 3
DOX:8x10 4 cells 100nM, 150nM 1
CON DOX 100nM DOX 150nM C12FDG L C12FDG M 0
C12FDG L C12FDG M
20
15
C12FDG MH C12FDG H C12FDG MH C12FDG H MFE(%) 10
200μm 200μm 200μm 5
0
50
5
SA-b-gal positive cells(% ) 30 20 Cell numbers 4 3 DOX con Fig 3. Distinction between senescence phenotype and stem cell capability
x 10000
40
as the grade of senescence
Dox 100nM
0 10 2 DOX 150nM
1
DISCUSSION.
0
0day 2day 4day 6day
p16 CD133 ◆ Chemotherapeutic agents can induce senescence in various cancer cell.
DOX(nM) ** * ◆ Doxorubicin can occur senescence and expresses stem cell marker protein
Relative mRNA expression level 2.5 N=4 N=4 ◆ Senescence by doxorubicin treatment can distinct in cancer population.
con 100 150 3.5 ** 2.5 * in breast cancer cell line.
CD133(133kDa) 3 Relative mRNA expression level 2
p16(16kDa) 1.5 2 1.5
α-tubulin(54kDa) 1 1 ◆ The characteristics of SASP and stemness may appear differently depending on
0.5 0.5 the senescent grade
0 0
CON Dox Dox CON Dox Dox
100nM 150nM 100nM 150nM
REFERENCE.
Fig 2. Doxorubicin induced senescence phenotype and expression of stem cell ◆ Senescence-associated reprogramming promotes cancer stemness.(M Milanovic et al. nature (2018))
marker in breast cancer cell line.
◆ Senescence-associated β- galactosidase reveals the abundance of senescent CD8+ T cells in aging
humans. (Ricardo I. Martinez-Zamudio et al. Aging cell(2020))