42
H. p21 as a principal regulator in maintaining chromosomal integrity via
43
44
45
Figure 14. Ectopic expression of p21 or inhibiting cell-cycle progression at G2 phase enhances chromosomal stability
(a) Transfection of sip21 in HeLa cells for 30 h and treatment with nocodazole for 16 h.
After nocodazole release, cells were subjected to Western blotting (top panel) with p21 and GAPDH (loading control) antibodies. Mitotic cells were utilized to perform ICC and amount of micronucleated cells was calculated (bottom panel) along with representative fluorescent images showing micronuclei (white arrow head denotes micronuclei). The results are given as the mean ± SD from three independent experiments (n = 300). ***P
< 0.001 by Student’s t-test. (b) Same as in (a). Mitotic cells as indicated above were collected and stained with aceto-orcein to visualize cells with condensed chromosome.
Percentage of mitotic cells with condensed chromosome was calculated at indicated time points. The results are given as the mean ± SD from three independent experiments (n = 300). (c) Co-transfection of sicGAS with or without p21 plasmid was performed for 30 h. Then cells were treated with nocodazole for 16 h before implying Western blotting (top panel) with indicated antibodies as well as ICC (bottom panel) for enumeration of micronucleated cells along with representative fluorescent images showing micronuclei (white arrow head denotes micronuclei). The results are given as the mean ± SD from three independent experiments (n = 300). ***P < 0.001 by Student’s ttest. (d) cGAS -/- HeLa cells were transfected with p21 plasmid for 12 h and then treated with nocodazole for 16 h. After 16 h, cells are subjected to Western blotting (top panel) with indicated antibodies and mitotic cells collected upon nocodazole release were reseeded on a cover slip to perform ICC. Immunocytochemical analysis (bottom panel) was used
46
to detect and count percentage of cells showing micronuclei along with representative fluorescent images showing micronuclei (white arrow head denotes micronuclei). The results are given as the mean ± SD from three independent experiments (n = 300). ***P
< 0.001 by Student’s t-test. (e) HeLa cells transfected with sicGAS and synchronized by double thymidine block for 40 h were released and then treated with RO3306 for 3 h after 7 h release from DTB. Mitotic cells were collected by shake off at 9 & 10 h with or without RO3306 treatment and reseeded on a cover slip to analyze cells showing chromosomal instability phenotype i.e. micronuclei by immunocytochemistry (bottom panel) along with representative fluorescent images showing micronuclei (white arrow head denotes micronuclei). Western blotting was performed with indicated antibodies to confirm cGAS down-regulation (top panel). (f) Same as indicated in (e). After 6 h release from DTB, quantification of the percentage of mitotic cells with condensed chromosome was performed by aceto-orcein staining at indicated time points with or without RO3306 treatment for 3 h after 7 h release from DTB. The results are given as the mean ± SD from three independent experiments (n = 300). (g) Cells were co-transfected with sicGAS and pcDNA (control vector) or p21 and then subjected to DTB. After release from DTB, cells were harvested at indicated time points and stained with aceto-orcein to count mitotic cells with condensed chromosomes. The results are given as the mean
± SD from three independent experiments (n = 300).
47 Discussion
The cGAS/STING pathway has been extensively studied as a part of the innate immune system. But reports on its impacts on cell-cycle progression are sparse, and little or nothing is known about its effects on chromosomal segregation. My findings provide the first evidence for a role for the cGAS/STING pathway in maintaining chromosomal homeostasis as a cell undergoes cell division. There have been reports of a STING role in maintaining chromosomal stability via the NF-B/p53/p21 axis and a TBK1 role in regulating chromosomal segregation during mitosis through binding to Cep170 and NuMA (12, 13). Another report also suggested that IRF3 overexpression causes cell-cycle arrest at the G1/S phase, resulting in inhibition of DNA synthesis (21). Some recent reports have suggested that cGAS detection of DNA in ruptured micronuclei can elicit an immune response that helps eliminate cells with CIN phenotypes, thus decreasing CIN indirectly (11, 22). But there have been no reports on possible direct contributions of the cGAS/STING/TBK1/IRF3 pathway to CIN. My findings suggest the first mechanism by which the cGAS-STING pathway directly regulates CIN without involvement of the immune system, and demonstrate that all components of the cGAS/STING/TBK1/IRF3 signaling pathway function together to maintain chromosomal stability.
Theoretically, the cGAS/STING pathway might affect chromosomal stability through actions during interphase that subsequently induce CIN during mitosis or
48
through direct effects on mitotic progression. In terms of the first of these two possibilities, there is literature support for the conclusion that, in interphase cells, cGAS is capable of detecting dsDNA inside micronuclei with fragile envelopes and subsequently eliciting a downstream pathway that induces transcription of inflammatory cytokines and chemokines via the transcription factors, IRF3 and
NF-B. The net effect of the operation of this pathway is to recruit cytotoxic T cells to the tumor micro-environment and promote apoptotic cell death, thereby indirectly down-regulating cells with CIN. It has also been reported that IRF3 can induce transcription of p53, resulting in up-regulation of p21, which arrests cell-cycle progression (14, 15). Here, I clearly showed that IRF3-dependent down-regulation of p21 is involved in cGAS-depletion–induced micronuclei formation via precocious entry into mitosis (Figs. 13 and 14). In terms of possible direct effects of the cGAS/STING pathway during mitosis, the cGAS/STING pathway might affect microtubule stability or spindle assembly, given that TBK1 is known to interact with the centrosome proteins, Cep170 and NuMA, to regulate mitotic progression (13).
The cGAS/STING pathway may also be involved in various mitotic events, including cytokinesis, mitotic checkpoint function and mitotic cell death, as well as progression at prometaphase (Fig. 15). However, dissecting the direct role of the cGAS/STING pathway in mitotic progression through regulation of the G2/M transition independent of p21 will require additional and more precisely designed studies.
49
Figure 15. cGAS down-regulation results in early mitotic progression leading to mitotic defects
(a) Depletion of cGAS using RNA interference and then comparing mitotic duration of siControl- or sicGAS-transfected cells by time-lapse analysis. (b) Determining time interval from mitosis onset to metaphase in siControl- or sicGAS-transfected cells by time-lapse analysis. (c & d) Quantification of cells undergoing mitotic defects i.e., cytokinesis failure
& mitotic slippage after cGAS-depletion. (e) Percentage of cells showing cell death in cGAS-positive and cGAS-negative cells after release from nocodazole treatment for 16 h.
50
Various outcomes have been attributed to down-regulation of p21 during the interphase of cell-cycle progression. The first is that p21 down-regulation can cause forced mitotic entry of S phase–arrested cells, since p21 is no longer available to inhibit complex formation between cyclin B1 and CDK1, resulting in premature mitotic entry before DNA synthesis is complete. This gives rise to abnormal mitotic phenotypes with dispersed chromosomes and disorganized bipolar spindle assembly.
These mitotic cells will “slip through” this forced mitosis, leading to gross micro-nucleation and apoptotic cell death (20, 23). The second outcome follows from the fact that p21 is considered the sole regulator of the G2/M DNA-damage checkpoint.
When p21 is depleted, cells with DNA double-strand breaks in the preceding S phase can transit into M phase without proper DNA damage repair because p21 can no longer inhibit the phosphorylation of CDK1 at threonine 161 necessary to enforce the G2 DNA-damage checkpoint, leading to chromosome missegregation events and micronuclei formation (24-27). This is consistent with my observations, given that p21 down-regulation was able to enhance micronuclei formation and also support precocious G2/M transition, giving rise to CIN. However, I could not detect an increase in gamma-H2AX foci to prove my hypothesis that decrease in p21 levels can result in inefficient DNA damage repair in preceding interphase leading to enhanced micronuclei formation in my system (Fig. 13h, i).
There are two theoretically possible mechanisms by which a decrease in the activity of the cGAS/STING/TBK1/IRF3 pathway might down-regulate p21. One is that the
51
decrease in p21 levels is a consequence of transcriptional changes that would otherwise be controlled by cGAS/STING pathway, which is now depleted, a possibility based on the fact that p21 levels are mainly regulated at the transcriptional level via various mechanisms. This mechanism predicts the possible involvement of IRF3 and/or NF-B transcriptional activity. The second possible mechanism is that changes in p21 levels that occur after depletion of the cGAS-STING pathway are attributable to post-translational changes. The half-life of p21 in actively dividing cells has been reported to be 20–60 min (28). Three E3 ubiquitin ligase complexes, SCF Skp2, CRL4Cdt2 and APC/CCdc20, are involved in p21 degradation at specific stages of the cell cycle. Since APC/CCdc20 is known to be involved in p21 degradation during G2 and M phases, it may be involved in p21 down-regulation here (29).
Moreover, there are two other reports suggesting that post-translational modification of p21 protein—acetylation and deubiquitylation mediated by Tip60 and USP11-deubuquitylase, respectively—regulates cell-cycle progression and DNA-damage responses by increasing p21 expression levels (30, 31). These observations suggest the need for additional studies on the possible association between the cGAS/STING/TBK1/IRF3 axis and post-translational modification of p21 protein.
My ongoing efforts are focused on deciphering the precise mechanism underlying cGAS/STING pathway-dependent regulation of p21 levels.
52 Conclusion
By performing these experiments, I provided the first reported evidence that cGAS/STING pathway can indeed regulates chromosomal stability directly without involvement of immune response. Thus, proposing for the first time another previously unknown role of cGAS/STING/TBK1/IRF3 innate immunity pathway during cell-cycle progression. I have provided reproducible and tangible results supporting the fact that cGAS/STING pathway regulates p21 levels that are otherwise necessary for proper cell-cycle progression. Whether p21 expression levels are controlled transcriptionally or by post-translational mechanisms, is still to be further investigated by intricately designed experimental techniques.
53 References
1. Dempsey PW, Vaidya SA, Cheng G. The art of war: Innate and adaptive immune responses. Cell Mol Life Sci. 2003;60(12):2604-21.
2. Zheng Z, Jia S, Shao C, Shi Y. Irradiation induces cancer lung metastasis through activation of the cGAS-STING-CCL5 pathway in mesenchymal stromal cells. Cell Death Dis. 2020;11(5):326.
3. Marill J, Mohamed Anesary N, Paris S. DNA damage enhancement by radiotherapy-activated hafnium oxide nanoparticles improves cGAS-STING pathway activation in human colorectal cancer cells. Radiother Oncol.
2019;141:262-6.
4. Michor F. Chromosomal instability and human cancer. Philos Trans R Soc Lond B Biol Sci. 2005;360(1455):631-5.
5. Benhra N, Barrio L, Muzzopappa M, Milan M. Chromosomal Instability Induces Cellular Invasion in Epithelial Tissues. Dev Cell. 2018;47(2):161-74 e4.
6. Bakhoum SF, Cantley LC. The Multifaceted Role of Chromosomal Instability in Cancer and Its Microenvironment. Cell. 2018;174(6):1347-60.
7. Tijhuis AE, Johnson SC, McClelland SE. The emerging links between chromosomal instability (CIN), metastasis, inflammation and tumour immunity. Mol Cytogenet. 2019;12:17.
8. de Oliveira Mann CC, Kranzusch PJ. cGAS Conducts Micronuclei DNA Surveillance. Trends Cell Biol. 2017;27(10):697-8.
54
9. Mackenzie KJ, Carroll P, Martin CA, Murina O, Fluteau A, Simpson DJ, et al. cGAS surveillance of micronuclei links genome instability to innate immunity.
Nature. 2017;548(7668):461-5.
10. Bakhoum SF, Ngo B, Laughney AM, Cavallo JA, Murphy CJ, Ly P, et al.
Chromosomal instability drives metastasis through a cytosolic DNA response.
Nature. 2018;553(7689):467-72.
11. Zierhut C, Yamaguchi N, Paredes M, Luo JD, Carroll T, Funabiki H. The Cytoplasmic DNA Sensor cGAS Promotes Mitotic Cell Death. Cell.
2019;178(2):302-15 e23.
12. Ranoa DRE, Widau RC, Mallon S, Parekh AD, Nicolae CM, Huang X, et al.
STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability. Cancer Res. 2019;79(7):1465-79.
13. Pillai S, Nguyen J, Johnson J, Haura E, Coppola D, Chellappan S. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis. Nat Commun. 2015;6:10072.
14. Kim TK, Lee JS, Jung JE, Oh SY, Kwak S, Jin X, et al. Interferon regulatory factor 3 activates p53-dependent cell growth inhibition. Cancer Lett.
2006;242(2):215-21.
15. Kim TK, Lee JS, Oh SY, Jin X, Choi YJ, Lee TH, et al. Direct transcriptional activation of promyelocytic leukemia protein by IFN regulatory factor 3 induces the
55
p53-dependent growth inhibition of cancer cells. Cancer Res. 2007;67(23):11133-40.
16. Kwon D, Park E, Kang SJ. Stimulator of IFN genes-mediated DNA-sensing pathway is suppressed by NLRP3 agonists and regulated by mitofusin 1 and TBC1D15, mitochondrial dynamics mediators. FASEB J. 2017;31(11):4866-78.
17. Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, et al. Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis. 2011;26(1):125-32.
18. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, et al.
Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science.
1998;282(5393):1497-501.
19. Moran DM, Gawlak G, Jayaprakash MS, Mayar S, Maki CG. Geldanamycin promotes premature mitotic entry and micronucleation in irradiated p53/p21 deficient colon carcinoma cells. Oncogene. 2008;27(42):5567-77.
20. Aarts M, Sharpe R, Garcia-Murillas I, Gevensleben H, Hurd MS, Shumway SD, et al. Forced mitotic entry of S-phase cells as a therapeutic strategy induced by inhibition of WEE1. Cancer Discov. 2012;2(6):524-39.
21. Kim TY, Lee KH, Chang S, Chung C, Lee HW, Yim J, et al. Oncogenic potential of a dominant negative mutant of interferon regulatory factor 3. J Biol Chem. 2003;278(17):15272-8.
56
22. Mitchison TJ, Pineda J, Shi J, Florian S. Is inflammatory micronucleation the key to a successful anti-mitotic cancer drug? Open Biol. 2017;7(11).
23. Lewis CW, Jin Z, Macdonald D, Wei W, Qian XJ, Choi WS, et al. Prolonged mitotic arrest induced by Wee1 inhibition sensitizes breast cancer cells to paclitaxel.
Oncotarget. 2017;8(43):73705-22.
24. Hirai H, Iwasawa Y, Okada M, Arai T, Nishibata T, Kobayashi M, et al.
Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents. Mol Cancer Ther. 2009;8(11):2992-3000.
25. O'Connell MJ, Raleigh JM, Verkade HM, Nurse P. Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J.
1997;16(3):545-54.
26. De Witt Hamer PC, Mir SE, Noske D, Van Noorden CJ, Wurdinger T. WEE1 kinase targeting combined with DNA-damaging cancer therapy catalyzes mitotic catastrophe. Clin Cancer Res. 2011;17(13):4200-7.
27. Hauge S, Macurek L, Syljuasen RG. p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775. Cell Cycle. 2019;18(8):834-47.
28. Abbas T, Dutta A. p21 in cancer: intricate networks and multiple activities.
Nat Rev Cancer. 2009;9(6):400-14.