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저작자표시-비영리-변경금지 2.0 대한민국

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Master’s Thesis in Biomedical Sciences

cGAS/STING/TBK1/IRF3 innate immunity pathway can regulate chromosomal instability in a p21-

dependent manner

Graduate School of Ajou University Department of Biomedical Sciences

Abdul Basit

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cGAS/STING/TBK1/IRF3 innate immunity pathway can regulate chromosomal instability in a p21-

dependent manner

Supervised by

Jae-Ho Lee, M.D., Ph.D.

I submit this thesis as the

Master’s thesis in Biomedical Sciences.

February, 2021

Graduate School of Ajou University Department of Biomedical Sciences

Abdul Basit

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The Master’s Thesis of Abdul Basit in Biomedical Sciences is hereby approved.

Thesis Defense Committee Chair (Seal) Prof. Jae-Ho Lee

Member (Seal) Prof. Hye Seong Cho

Member (Seal) Prof. Kyeong Sook Choi

Graduate School of Ajou University

January 7

th

, 2021

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i Abstract

Chromosomal instability (CIN) in cancer cells has been reported to activate the cGAS- STING innate immunity pathway via micronuclei formation, thus affecting tumor immunity and tumor progression. However, converse effects of the cGAS/STING pathway per se on CIN have not yet been investigated. I addressed this issue using knockdown and add-back of each component of the cGAS/STING/TBK1/IRF3 pathway, and monitored the extent of CIN by measuring micronuclei formation after release from nocodazole-induced mitotic arrest. Interestingly, knockdown of cGAS (cyclic GMP-AMP synthase) along with induction of mitotic arrest in HeLa and U2OS cancer cells clearly resulted in an increased micronucleus phenotype and chromosome missegregation. Knockdown of STING (stimulator of interferon genes), TBK1 (TANK binding kinase-1), or IRF3 (interferon regulatory factor-3) also increased micronuclei formation. Moreover, transfection of cGAMP, the product of cGAS enzymatic activity, as well as add-back of cGAS WT (but not catalytic-dead mutant cGAS), or WT or constitutively active STING (but not an inactive STING mutant) rescued the micronuclei phenotype, demonstrating that all components of the cGAS/STING/TBK1/IRF3 pathway play a role in preventing CIN. Moreover, p21 levels were decreased in cGAS- , STING-, TBK1- and IRF3-knockdown cells in association with precocious G2/M transition and an enhanced micronuclei phenotype. Overexpression of p21 or inhibition of CDK1 in cGAS-depleted cells reduced micronuclei formation and abrogated precocious G2/M transition, indicating that the decrease in p21 and subsequent precocious G2/M transition is the main mechanism underlying the induction of CIN by defective cGAS/STING signaling.

Innate immunity, cGAS/STING/TBK1/IRF3 pathway, Chromosomal instability, p21, Cell- cycle progression

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ii

TABLE OF CONTENTS

ABSTRACT . . . i

TABLE OF CONTENTS . . . . . . ii

LIST OF FIGURES . . . iv

I. INTRODUCTION . . . . . . . 1

II. MATERIALS AND METHODS . . . 14

A. Antibodies . . . 14

B. Cell culture . . . 14

C. Synchronization and drug treatment . . . 15

D. Plasmids and transfection experiments . . . 16

E. Knock-down experiments . . . 16

F. Immunoblotting . . . .. . . 17

G. Immunocytochemistry . . . 18

H. Time-lapse analysis . . . . . . 18

I. Mitotic index . . . .. . . 19

J. Statistical analysis . . . .. . . 19

III. RESULTS . . . . . . . . . . 20

A. Investigating cGAS/STING pathway to choose best suited cancer cell-line for my experimental setting . . . 20

B. cGAS add-back can rescue cells from cGAS-depletion induced CIN . . . 22

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iii

C. cGAS down-regulation augments chromosomal

missegregations . . . 26

D. Micronuclei formation is regulated by enzymatic activity of cGAS . . . 29

E. STING plays mediating role to maintain chromosomal stability . . . 32

F. Depletion of TBK1 or IRF3 also enhanced CIN phenotypes . . . . 35

G. p21 expression levels are dependent on cGAS/STING pathway . . . 37

H. p21 as a principal regulator in maintaining chromosomal integrity via cGAS/STING pathway . . . . . . 42

IV. DISCUSSION . . . 47

V. CONCLUSION . . . 52

REFERENCES . . . 53

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iv

LIST OF FIGURES

Fig. 1. Detailed classification of immune system . . . 2

Fig. 2. Graphical representation of cGAS/STING/TBK1/IRF3 signaling pathway . . . 4

Fig. 3. Causes of genomic instability and its outcomes . . . 6

Fig. 4. Types of chromosomal instability . . . 8

Fig. 5. Cell-cycle and cell-cycle checkpoints . . . 10

Fig. 6. Cell-cycle regulators and their roles during each cell-cycle phase . . . 12

Fig. 7. Expression levels of cGAS/STING pathway in stable cancer cell lines . . . 21

Fig. 8. cGAS rescued micronucleated cells . . . 23

Fig. 9. Accurate chromosomal segregations are dependent on cGAS . . . 27

Fig. 10. cGAS activity is essential to maintain chromosomal integrity . . . 30

Fig. 11. cGAS activates STING to control chromosomal stability . . . 33

Fig. 12. TBK1 and IRF3 depletion also induces chromosomal

instability . . . 36

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v

Fig. 13. cGAS/STING pathway regulates p21 expression levels during

cell cycle progression . . . 39

Fig. 14. Ectopic expression of p21 or inhibiting cell-cycle progression at

G2 phase enhances chromosomal stability . . . . 43

Fig. 15. cGAS down-regulation results in early mitotic progression

leading to mitotic defects . . . 49

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1 I. Introduction

A. Immunity and its types:

Immunity is defined as an organism’s ability to synthesize and recruit specific antibodies or sensitized white blood cells to counter a particular infection or toxin. It is broadly classified into two types: 1) Adaptive immunity and; 2) Innate immunity.

Adaptive immunity is an antigen-dependent immune response. It is much more complex as compared to innate immune response. The antigen is first processed and recognized. After completion of antigen recognition, an army of antigen-specific immune cells is synthesized by adaptive immune system to target the specific antigen and removing it from the body. Moreover, adaptive immunity also stores a "memory"

to efficiently counter any infection caused by the same antigen in the future. Innate immunity is defined as a non-specific protection mechanism that may activate immediately or within hours after an antigen’s appearance in the body. These mechanisms encompass various lines of defenses including physical barriers like skin, chemical barriers like interferons & chemokines present in the blood stream, and immune cells that eradicate foreign invading cells causing infection in the body.

Innate immune response activates depending on the chemical properties of any antigen (1).

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2

Figure 1. Detailed classification of immune system

Immune system basically provides ability to defend against invasions caused by foreign entities. Immediately after being attacked by an invading pathogen, immune system protects the body utilizing non-specific defense mechanisms termed as innate immune responses. If the infection is prolonged then it starts generating a swarm of white blood cells targeting that specific antigen and eradicating any harmful effects caused by it. This response usually activates after 1~2 weeks of infection and is therefore termed as adaptive immune response.

Immunity

Innate immunity Anatomical

barriers

Phagocytic barriers

Blood

proteins Cytokines

Adaptive immunity Passive

immunity Naturally acquired

Artificially acquired

Active immunity Naturally acquired

Artificially acquired

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3

cGAS/STING/TBK1/IRF3 pathway and tumorigenesis:

There are several mechanisms being part of innate immune system that are involved in defeating attacking pathogens immediately after infection. cGAS/STING/TBK1/IRF3 pathway is one of those mechanisms that acutely and non-specifically detects any dsDNA in the cytosol of the cell and initiate an immune response. Cyclic GMP-AMP synthase (cGAS) acts as a pattern-recognition receptor that detects pathogen-associated molecular patterns or damage-associated molecular patterns in the form of dsDNA in the cytosol and binds with it non-specifically. This initiates some conformational changes in the cGAS structure enabling it to synthesize a second messenger known as cyclic GMP-AMP (cGAMP) utilizing AMP and GMP as substrates. This second messenger then translocates and binds with an endoplasmic reticulum transmembrane protein known as Stimulator of Interferon Genes (STING). This activates STING causing it to localize in the Endoplasmic reticulum Golgi intermediate compartments (ERGIC). During STING translocalization, it recruits TANK-binding kinase-1(TBK1) which phosphorylates itself as well as STING. Moreover, TBK1 also phosphorylates Iκβα, an inhibitor of NF-κβ, thus marking it for degradation by proteasomes and releasing NF-κβ. On the other hand, IRF3 is also phosphorylated by TBK1 and phosphorylated IRF3 then binds with another phospho-IRF3 monomer to form a dimer.

After dimerization IRF3 together with NF-κβ translocates inside the nucleus to initiate the transcription of interferons and cytokines.

Role of cGAS-STING pathway on tumorigenesis has been a controversial matter. Some reports suggest that activation of cGAS-STING pathway keeps the immune response under sub-optimal levels within the tumor micro-environment thus enabling tumor cells to bypass anti-tumor immunity and metastasize to distant sites of the body (2). On the contrary, some reports support the fact that enhancing cGAS-STING pathway activation by using chemotherapy or radiotherapy can not only increase the survival rate of cancer patients but it can also be used as a more specific and targeted therapy against cancer cells causing less damage to normal cells (3).

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Figure 2. Graphical representation of cGAS/STING/TBK1/IRF3 signaling pathway This figure summarizes the initiation, propagation and termination of cGAS/STING/TBK1/IRF3 signaling pathway once it detects dsDNA whether originated from self-DNA or foreign DNA after infection.

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5 Chromosomal Instability and tumorigenesis:

Genomic instability is considered as one of the hallmarks of cancer. The two most prominent causes of genomic instability induction are chromosomal segregation errors and compromised DNA damage repair pathways. Genomic instability can give rise to two different phenotypes. One being structural chromosomal instability and other one is termed as numerical chromosomal instability. In structural CIN, different anomalies can be observed as cell cycle progresses especially during mitosis in the form of lagging chromosomes, chromatin bridges and multipolar division. These anomalies give rise to micronucleated-, binucleated- and multinucleated-cells. Whereas in numerical CIN, no observable changes in cell morphology but rather a change in its genomic content in the form of aneuploidy can be seen. In some cases, numerical CIN can also result in structural CIN by inducing micronuclei formation as cells continue to replicate.

There are several reports extensively linking CIN to tumorigenesis indicating CIN as one of the key factors involved in regulating cancer progression, invasion, metastasis as well as providing immunity against anti-tumor defenses. In 2005, Franziska et al. provided a mathematical framework to establish the fact that one or few neutral CIN genes are enough to initiate tumorigenesis in a pathway where two tumor suppressor genes (TSGs) are eliminated in rate limiting situations (4). Another report provided the mechanism by which chromosomal unstable cells delaminate from epithelium, form actin-based cellular protrusions giving rise to membrane blebs thus invading neighboring cells (5). Recently new advancements in the studies pertaining relationship between CIN and Cancer suggest that tumor cells adopt themselves by deleting tumor suppressor genes due to recurrent and persistent chromosomal instability. Moreover, CIN initiates anti-tumor immunity inside the tumor microenvironment but keeps it below a threshold level so that tumor cells can easily bypass anti-tumor immunity (6),(7).

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Figure 3. Causes of genomic instability and its outcomes

Both, defects during chromosome segregation and/or DNA damage accompanied with impaired DNA damage repair, give rise to genomic instability. Genomic instability is apparent in the form of chromosomal instability (CIN) and/or DNA mutations.

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Relationship between CIN and cGAS-STING pathway in cancer

cGAS-STING pathway has been first attributed to CIN since its role in detecting dsDNA inside ruptured micronuclei has been established. Thus, activating downstream signaling to elicit anti-tumor immune response against chromosomal unstable cells (8, 9). But whether this response is beneficial to eradicate cancer is of controversary. Some reports suggested that cGAS-STING pathway is activated in CIN cells which in turn recruit interleukins, cytokines and chemokines at suboptimal levels within the tumor microenvironment and augments tumor metastasis via activation of the noncanonical NF-κβ pathway (6, 10). Contrary to these reports, some researchers argue that cancer cells with abnormal number of chromosomes such that it elevates cGAS-STING-IRF3 proteins levels will show enhanced cGAS- STING pathway activation. This will lead to mitochondrial outer-membrane permeabilization and promotes apoptotic cell death. Thus, suggesting a beneficial role of cGAS-STING pathway activation via CIN in eradication of tumor cells (11).

However, all these reported mechanisms suggest an indirect relationship between cGAS-STING pathway and CIN in tumorigenesis where CIN in the form of micronuclei leads to cGAS activation. I performed series of experiments to elucidate a mechanism by which activation of cGAS-STING pathway at basal levels directly minimizes the possibility of structural CIN as the cell cycle progresses without involvement of immune response.

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8 Figure 4. Types of chromosomal instability

Numerical and Structural CIN will give rise to Aneuploidy and Micronuclei phenotype, respectively, both of which can induce cytosolic DNA formation and activate cGAS/STING innate immunity pathway.

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9 Cell cycle and cell cycle checkpoints

Cells go through a series of intricate and regulated events to replicate themselves into two normal daughter cells. These events combined are called cell cycle. Cell cycle broadly consists of two phases; 1. Interphase and, 2. Mitosis. Interphase is further subcategorized into four phases; 1. G0 (quiescent phase or resting phase), 2. G1 (growth phase during which cells prepare necessary proteins required for normal cell division), 3. S (DNA replication phase) and, 4. G2 (DNA damage repair and growth phase to prepare cells for normal and even distribution of genetic material into two daughter cells). At the end of the interphase, cell is committed to enter mitotic phase of the cell cycle. During mitosis, cells undergo through following phases; 1. Prophase (condensation of chromosomes and nuclear envelope breaks down), 2. Prometaphase (kinetochores are apparent and mitotic spindle microtubules attach to kinetochores), 3. Metaphase (chromosomes arrange at the metaphase plate and spindle assembly checkpoint is satisfied), 4. Anaphase (centromeres are split into two during early anaphase and in late anaphase two sister chromatids have now reached their respective poles and constriction ring or pinching of cell membrane starts to appear) and, 5. Telophase (nuclear envelope starts to form around separated chromosomes and constriction of cell membrane completes in early phase whereas in late telophase two daughter cells completely separate having same structures and number of chromosomes as parent cell).

This whole process of division of one parent cell to two daughter cells is regulated and checked at three timepoints during cell cycle. First checkpoint ensures whether cells have all the necessary raw materials in the form of proteins, nutrients, etc., before committing to cell division. This checkpoint is named as G1/S checkpoint. Cells encounter second checkpoint before entering mitotic phase of cell cycle named as G2/M checkpoint.

This checkpoint confirms that DNA damage during replication is properly repaired and all the proteins and nutrients required for mitosis are sufficient. Last checkpoint known as spindle assembly checkpoint (SAC) verifies proper attachment of kinetochores to mitotic spindle microtubules and alignment of chromosomes at the metaphase plate. Once this checkpoint is satisfied, cells are safe to undergo anaphase.

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10 Figure 5. Cell-cycle and cell-cycle checkpoints

Cell cycle includes interphase and mitotic phase. G1, S and G2 phases are collectively known as interphase. Cell cycle progression is further regulated by three distinct checkpoints. G1/S checkpoint ensures whether cells should progress towards S phase. G2/M checkpoint confirms proper DNA replication before entering mitotic phase. Spindle-assembly checkpoint (SAC) or metaphase checkpoint checks for proper chromosome spindle attachment before anaphase begins.

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11 Cell cycle regulators

Cell cycle checkpoints are further regulated by cell cycle control system which consists of positive cell cycle regulators and negative cell cycle regulators. Positive cell cycle regulators include protein kinases known as cyclin-dependent kinases (CDKs). CDKs bind with regulator proteins called cyclins in a rate limiting step.

Specific Cyclin-CDK complexes, formed during each cell cycle phase, are then phosphorylated by CDK-activating kinases (CAK). Phosphorylated Cyclin-CDK complexes then phosphorylate target proteins to help in the progression of cell cycle.

Negative cell cycle regulators comprise of retinoblastoma protein (Rb), p53, INK family (inhibitors of kinases) and CIP family (CDKs inhibitory proteins). They are also known as CDK inhibitors (CKI). Rb protein binds with transcription factors like E2F in unphosphorylated form. Rb protein upon phosphorylation by Cyclin-CDK complex, detaches thus releasing the bound transcription factor to initiate DNA replication. p53 protein detects DNA damage before, during and after DNA replication in S phase. Once it detects any DNA damage it recruits DNA damage response elements and also trigger upregulation of p21 protein. p21 protein then halts cell cycle progression by inhibiting Cyclin-CDK complex formation. p21 and p57 belong to CIP family. Whereas, INK family containing p19 and p15 protein phosphatases is involved in regulation of positive cell cycle regulators.

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Figure 6. Cell-cycle regulators and their roles during each cell-cycle phase

Cell cycle checkpoints are further regulated by cell cycle regulators that can act as agonists or antagonists during cell cycle progression. Namely, cyclins (Cyc-D, Cyc- E, Cyc-A, Cyc-B) and cyclin-dependent kinases (CDK-1, CDK-2, CDK-4/6) are positive cell-cycle regulators. Whereas, p21, wee1, Rb protein and p53 are some of the negative cell-cycle regulators.

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Previously reported effects of downstream components of cGAS-STING pathway on cell cycle progression

There have been numerous reports arguing over the fact whether downstream proteins involved in cGAS-STING signaling play any role in cell cycle regulation and chromosomal instability. In 2019, a report elaborated that upon activation of STING by 2’3’-cGAMP, it activates downstream pathway where NF-κβ is released and moves inside nucleus and enhances p53 transcription. p53 upregulates p21 levels thus halting the cell cycle due to DNA damage (12). Another report mentioned that TANK binding kinase-1 (TBK1) binds with centrosomal proteins, CEP170 and NuMA, and phosphorylates them during mitosis. This in turn leads to enhanced microtubule stability during mitosis thus decreases incidence of mitotic defects (13).

Then there were some other reports illustrating the role of IRF3 in cell cycle progression. They mentioned that once IRF3 phosphorylates and transports inside nucleus. Once inside nucleus, it induces p53-dependent growth inhibition of cancer cells (14, 15).

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14 Materials and methods:

A. Antibodies

The following antibodies were used: mouse monoclonal antibodies to α- tubulin (Santa Cruz, sc23948), GAPDH (Santa Cruz, sc32233), phospho-H2A.X (Ser139) (EMD Millipore, 05-636), anti-ᵧ-tubulin (Sigma-Aldrich, T5326) and IRF3 (Abcam, ad50772) at a 1:1000 dilution in PBS with 3% BSA; rabbit monoclonal antibodies to cGAS (Cell signaling, #15102S), STING (Cell signaling, 13647), phospho-IRF3 (Ser386) (Abcam, ab76433), phospho-IRF3 (Ser396) (Cell signaling, #29047S), phospho-TBK1 (Ser172) (Cell signaling, 5483S) and TBK1 (Cell signaling, 3504S) at a 1:1000 dilution in PBS with 3% BSA. Horseradish- peroxidase conjugated anti-mouse (G21040) and anti-rabbit (G21234) antibodies were obtained from Invitrogen (at a 1:3000 dilution in TBST). The following fluorochrome-conjugated secondary antibodies were used: anti-mouse Alexa-488 (Invitrogen, A11059), anti-rabbit Alexa-488 (Invitrogen, A11034), and anti-mouse Cy3 (Jackson ImmunoResearch, 715-165-151) at a 1:500 dilution in PBS with 3%

BSA.

B. Cell culture

All cells used herein were purchased from American Type Culture Collection (Manassas, VA, USA) unless said otherwise, and cultured in Dulbecco's modified Eagle's medium (DMEM)/high glucose medium (Gibco, 31600-034) supplemented

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with penicillin/streptomycin (Gibco BRL, 15240-062) and 10% (V/V) fetal bovine serum (FBS) (Gibco BRL, 16000-044) at 37 °C in a humidified incubator with 5%

CO2. The hTERT-immortalized retinal pigment epithelial cell line, hTERT RPE-1, was obtained from the ATCC, and cultured in DMEM/F12 supplemented with penicillin/streptomycin, 10% FBS and 0.01 mg/mL hygromycin B (Sigma-Aldrich, H0654). The cGAS-deficient HeLa cell line was generated through TALEN- mediated gene disruption (16).

C. Synchronization and drug treatment

To synchronize the cells at G1/S phase by double-thymidine block (DTB), cells were grown on coverslips and incubated in growth medium containing 1 mM thymidine (Sigma, T9250) for 16 h. Cells were then released from the thymidine block by washing with thymidine-free medium (first release) and cultured in growth medium for 8 h. Subsequently, cells were subjected to the second thymidine block for an additional 16 h. For G2/M phase arrested cells, cells were synchronized by double- thymidine block as described above and then cells that were previously arrested with thymidine for 16 hours were washed with thymidine-free medium and cultured in complete medium for 7 h (for HeLa cells) or 8 h (for U2OS). Then, the cells were cultured again in 9 μM RO3306 (Enzo, ALX-270_463) containing medium to arrest at the G2/M transition for 2 h (for HeLa cells) or 3 h (for U2OS). For mitotic arrest, cells were synchronized at prometaphase with 100 ng/ml nocodazole (Sigma, M1404) for 16 h. To inhibit protein degradation, cells were treated with 10 μM

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MG132 (Sigma, C2211) for 8 h after STING knock-down. For inhibition of protein translation, cells were transfected with siRNA targeting STING and then treated with 10 μg/mL cycloheximide (Sigma, C7698) for indicated time intervals.

D. Plasmids and transfection experiments

DNA encoding human cGAS and human STING were cloned by PCR and subcloned into N-Myc-pcDNA3.1(+) Myc-HisC (Thermo Fisher scientific) and N-Flag- pcDNA3.1(+) Myc-HisC plasmids, respectively. Plasmid encoding HA-p21 was a gift from Dr. Jaewhan Song (Yonsei University, Korea). RFP-cGAS (plasmid # 86676) was purchased from Addgene. The MYC-cGAS (E225A, D227A) mutant, MYC-cGAS (si-non-targeting) mutant, Flag-STING (R284M) mutant and Flag- STING (L374A) mutant was constructed using a Muta-DirectTM site directed mutagenesis kit (iNtRON Biotechnology, #15071). 2’3’-cGAMP was purchased from Invitrogen. HeLa cells were transfected using Neon electroporation (Invitrogen), Lipofectamine® 2000 transfection reagent (Invitrogen) or ViaFectTM transfection reagent (Promega) according to manufacturer’s protocol.

E. Knock-down experiments

High-performance liquid chromatography-purified (>97% pure) small interfering RNA (siRNA) oligonucleotides targeting cGAS, STING, TBK1 and IRF3 were purchased from Thermo Fisher. The sequences of the sense strands of the siRNA oligonucleotides were as follows: cGAS #2, 5’-CGUGAAGAUUUC UGCACCU-

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3’; 5’-AGGUGCAGAAAUCUUCACG-3’; cGAS #4, 5′-

GCAAAAGUUAGGAAGCAAC-3′; 5′-GUUGCUUCCUAACUUUUGC-3′;

STING, 5’-UCAUAAACUUUGGAUGCUA-3’; 5’-

UAGCAUCCAAAGUUUAUGA-3’; TBK1, 5’-GGAGACAACAACAAGACAU-

3’; 5’-AUGUCUUGUUGU UGUCUCC-3’; IRF3, 5’-

GGAAGACAUUCUGGAUGAG-3’; 5’-CUCAUCCAGAAUGUCUUCC-3’;

CDKN1A, 5’-CAAGGAGUCAGACAUUUUA-3’; 5’-

UAAAAUGUCUGACUCCUUG-3’. Cells were transfected with 10 nM of either the cGAS #2, cGAS#4, STING, TBK1, IRF3, CDKN1A (p21) or control siRNA oligonucleotides using Oligofectamine® Transfection Reagent (Invitrogen) according to the manufacturer’s protocol.

F. Immunoblotting

Conventional immunoblotting was performed as previously described using the corresponding antibodies. Briefly, cell lysates (30 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were then transferred to polyvinylidene fluoride membranes. After blocking for 1 h at room temperature (RT) with TBS containing 0.1% (V/V) Tween–20 and 5% (W/V) non-fat milk, membranes were incubated with the corresponding primary antibodies at 4 °C, followed by washing with TBS containing 0.1% Tween-20 and incubation with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Amersham

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Biosciences, Piscataway, NJ) for 1 h at RT. Detection was carried out using ECL reagents (Amersham Biosciences) and exposure of the membranes to X-ray film.

G. Immunocytochemistry

Mitotic cells were split onto poly-L-lysine (PLL, P6282, Sigma-Aldrich)-coated slides. Cells were grown on coverslips and fixed in 100% methanol for 15 min at - 20°C. Fixed cells were pre-incubated in blocking solution (3% bovine serum albumin in PBS), followed by incubation with primary antibodies at 4 °C overnight. Cells were then washed three times in PBS with shaking and probed with fluorophore (Cy3 or Alexa Fluor 488)-conjugated anti-mouse or anti-rabbit secondary antibodies.

After washing three times with PBS, DAPI (Invitrogen, D3571) was used for DNA counterstaining. Three washes with PBS were followed by mounting in the mounting solution (Biomeda, M01). The samples were examined under a fluorescence microscope (Axio Imager M1, Carl Zeiss).

H. Time lapse analysis

HeLa cells were transfected with the siControl or sicGAS and treated with nocodazole (100 ng/mL) for 16 h. Then, cells at prometaphase were collected by shake-off and seeded (1 × 104 cells/well) in a four-well glass dish (Thermo Scientific™ Nunc™Lab-Tek II Chambered Coverglass, MA, USA) and incubated overnight in standard culture conditions, for the estimation of mitosis duration along with visualization of chromosomal unstable phenotypes with time-lapse

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photomicroscopy. To visualize chromosomes, cells were incubated with 1 μg/mL Hoechst 33342 (Thermo Scientific™ Hoechst® 33342, MA, USA) for 30 min.

Fluorescence images were acquired every 5 min for 24 h while using a Nikon eclipse Ti camera (Tokyo, Japan) with a 40× dry Plan-Apochromat objective. Images were captured with an iXonEM +897 Electron Multiplying charge-coupled device camera (Teledyne Princeton Instruments, Trenton, NJ, USA) and analyzed in the Nikon Imaging Software (NIS)-elements advanced research (AR) (Nikon, Tokyo, Japan).

I. Mitotic index

Mitotic cells were stained with aceto-orcein solution in 60% acetic acid (Merck, ZC135600) to visualize the condensed chromosomes. To determine the mitotic index, the percentage of mitotic cells with condensed chromosomes was quantified under a light microscope.

J. Statistical analysis

Most data are presented as the means ± standard deviations (SDs). Each experiment was performed with triplicates. Statistical differences were analyzed by Student’s t- test, and asterisks (*) indicate significant differences: *P < 0.05; **P < 0.01; and

***P < 0.005.

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20 Results

A. Investigating cGAS/STING pathway to choose best suited cancer cell-line for my experimental setting

To establish my experimental setting, I detected cGAS/STING levels in four cell lines: HEK293T (human embryonic kidney cell line), U2OS (human osteosarcoma cell line), hTERT-RPE1 and HeLa. Protein levels of both cGAS and STING were detectable in HeLa and U2OS cells, as opposed to hTERT-RPE1 or HEK293T cells (Fig. 7a). To confirm cGAS localization to micronuclei, as previously reported in cGAS-positive cell lines, I treated the above-mentioned cell lines with nocodazole (100 ng/ml) for 16 h, thus inducing mitotic arrest. These cell lines were then released from the arrest for 10 h. After that time interval, I observed the cells in subsequent interphase (Fig. 7b, c). Whether HeLa cells have intact and functional cGAS/STING/TBK1/IRF3 pathway, I transfected empty pcDNA vector and checked phospho-TBK1 and phospho-IRF3 levels to confirm activation of cGAS-STING pathway (Fig. 7d). The obtained results supported the fact that HeLa cells are the first choice to investigate the effect of cGAS/STING pathway on CIN directly without the participation of the immune system.

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Figure 7. Expression levels of cGAS/STING pathway in stable cancer cell lines

(a) Protein expression levels were analyzed by western blot using antibodies against cGAS, STING & GAPDH in hTERT-RPE1, U2OS, HEK293T & HeLa cells. (b) Representative confocal images of cGAS localized micronuclei (yellow arrow head) and micronuclei without cGAS localization (white arrow head) 10 h after release from nocodazole (100ng/ml) treatment for 16 h. (c) Percentage of cells showing cGAS positive micronuclei and cGAS negative micronuclei after 10 h release from nocodazole treatment in HeLa, U2OS, hTERT-RPE1 & HEK293T cells. The results are given as the mean ± SD from three independent experiments (n = 300). ***P < 0.001 by Student’s t-test. (d) cGAS-STING pathway activation after pcDNA transfection was analyzed in HeLa cells by western blot using antibodies against cGAS, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3 &

GAPDH.

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B. cGAS add-back can rescue cells from cGAS-depletion induced CIN

I transfected two different small interfering RNAs, to exclude the possibility of false positive result, in Hela cells to deplete cGAS. Then I estimated the degree of CIN by observing micronuclei formation in siRNA transfected cells 10 h after release from nocodazole-induced mitotic arrest (100 ng/ml nocodazole for 16 h) under fluorescence microscope (Fig. 8a). Intriguingly, the proportion of micronucleated cells were markedly increased in cGAS-depleted cells in comparison with control siRNA-transfected cells upon immunocytochemical analysis (Fig. 8b), indicating that chromosomal stability within cycling cells is essentially maintained by cGAS. I further checked fraction of micronucleated cells in wild-type (WT) HeLa and cGAS-knockout HeLa cells 10 h after release from nocodazole-induced mitotic arrest. Same as cGAS-knock down HeLa cells, CIN phenotype was markedly increased in cGAS-knockout HeLa cells because the percentage of micronucleated cells was approximately two times higher than that of WT HeLa cells (Fig. 8c). When cGAS-depleted cells were transfected with WT cGAS cDNA, the decrease in the number of micronucleated cells proved that regulation of chromosomal stability is cGAS-dependent (Fig. 8d). Moreover, introduction of plasmid encoding WT cGAS via transfection of cGAS-knockout HeLa cells also resulted in reduction of micronuclei formation (Fig. 8e). Whether this is a cell-type specific response or not, I also depleted cGAS in U2OS cells by transfecting them with siRNA targeting cGAS. Similar to cGAS-knockdown HeLa cells, this resulted in the elevation of micronucleated cells (Fig. 8f). The results of this analysis serve as a basic pillar to support the fact that cGAS is in fact involved in the regulation of chromosomal stability.

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25 Figure 8. cGAS rescued micronucleated cells

(a) Generalized experimental scheme utilized to identify the role of cGAS-STING pathway upon induction of chromosomal instability. (b) After 30 h of cGAS siRNA transfection, nocodazole treatment (100ng/mL) was applied for 16 h. Fraction of mitotic cells obtained by shake off were reseeded on a cover slip and remaining cells were subjected to Western blotting by indicated antibodies (top panel). Quantification of the percentage of micronucleated cells was performed by immunocytochemistry (bottom panel) along with representative fluorescent images showing micronuclei. (c) Wild-type HeLa and cGAS -/- HeLa cells were treated with 100ng/mL nocodazole for 16 h. Mitotic cells were collected by shake-off and subjected to Western blotting (top panel) together with immunocytochemical analysis (bottom panel) along with representative fluorescent images showing micronuclei. (d) Co-transfection of sicGAS with control vector or MYC-cGAS (si non-targeting) in HeLa cells for 20 h and then arrested at prometaphase by nocodazole treatment for 16 h. Western blotting with indicated antibodies was performed to confirm cGAS knock-down and cGAS add-back (top panel).

Immunocytochemistry was performed to evaluate micronucleated cells after 10 h release from nocodazole-induced mitotic arrest (bottom panel) along with representative fluorescent images showing micronuclei (white arrow head denotes micronuclei). (e) Transfection of MYC-cGAS plasmid to cGAS -/- HeLa cells for 12 h before nocodazole treatment for 16 h. Mitotic cells were collected and reseeded for 10 h to quantify number of cells containing micronuclei by immunocytochemistry (bottom panel) along with representative fluorescent images showing micronuclei and their cell lysates were used for Western blotting with indicated antibodies (top panel). (f) U2OS cells were transfected with sicGAS for 30 h pre-treatment with nocodazole for 16 h. Mitotic cells were subjected to Western blotting to check cGAS and GAPDH (loading control) protein levels (top panel) as well as ICC to enumerate percentage of cells with micronuclei (bottom panel) along with representative fluorescent images showing micronuclei.

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C. cGAS down-regulation augments chromosomal missegregations

Chromatin bridges and lagging chromosomes, which may partly arise from multipolar division during mitotic phase, are primarily two basic chromosome segregation errors that give rise to micronuclei formation (17). I tried to establish the relationship between these two phenotypes and chromosomal missegregations by counting number of anaphase cells, released after 1 h from nocodazole-induced mitotic arrest, displaying lagging chromosomes, chromatin bridges, or multipolar division (Fig. 9a). The results obtained favored the fact that cGAS-knockdown cells exhibited more chromosomal segregation errors as compared to the control cells (Fig. 9b). When I transfected siRNA-resistant WT MYC-cGAS cDNA in cGAS- knock-down cells, cGAS protein levels were restored and the ability to ensure proper chromosomal segregation was regained (Fig. 9c). Additionally, upon comparing cGAS-knockout and WT HeLa cells during chromosomal segregation by observing cells 1 h after release from mitotic arrest displayed increased chromosomal missegregations in cGAS-knockout cells than WT cells (Fig. 9d). However, when cGAS protein levels were restored via transfection in cGAS-knockout HeLa cells, chromosomal segregation defects were rescued (Fig. 9e). The results of the findings support the fact that normal chromosome segregation during mitosis is cGAS- dependent and cGAS acts as a suppressor of CIN during cell-cycle progression.

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Figure 9. Accurate chromosomal segregations are dependent on cGAS

(a) Representative confocal images of mitotic cells after 1 h release from nocodazole- induced mitotic arrest. Cells showing bipolar division, multipolar division, lagging chromosome, and chromatin bridge (from left to right). (b) After 30 h of sicGAS transfection, mitotic cells were collected by shake-off after 16 h treatment with nocodazole and seeded on a PLL-coated cover slip. After 1 h, cells were fixed with 100% ice-chilled methanol and ICC was performed. Cells showing indicated chromosomal segregation errors were calculated as percentage. The results are given as the mean ± SD from three independent experiments (n=300). ***P < 0.001 by Student’s t-test. (c) Co-transfection of sicGAS with pcDNA or MYC-cGAS (si non-targeting) in HeLa cells for 20 h before nocodazole treatment for 16 h. Cells arrested in mitosis were collected and reseeded on PLL- coated cover slip. After 1 h, percentage of micronucleated cells was quantified using ICC.

The results are given as the mean ± SD from three independent experiments (n=300). ***P

< 0.001 by Student’s t-test. (d) cGAS -/- and Wild-type HeLa cells were treated with nocodazole for 16 h. Then, mitotic cells were collected by shake-off and seeded on PLL- coated cover slip for 1 h before performing ICC to analyze number of cells showing indicated chromosomal missegregations. The results are given as the mean ± SD from three independent experiments (n=300). ***P < 0.001 by Student’s t-test. (e) cGAS -/- HeLa cells were transfected with pcDNA or MYC-cGAS and then treated with nocodazole for 16 h to induce mitotic arrest. After release from nocodazole treatment, mitotic cells were seeded on PLL-coated coverslip for 1 h and then subjected to ICC to quantify percentage of cells with chromosomal segregation errors. The results are given as the mean ± SD from three independent experiments (n=300). *P < 0.05, ***P < 0.001 by Student’s t-test.

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D. Micronuclei formation is regulated by enzymatic activity of cGAS

After confirming cGAS role in regulating chromosomal stability, I checked whether cGAS enzymatic activity is essential for this function of cGAS. Therefore, I performed add-back of WT cGAS or catalytic-dead cGAS mutant plasmids via transfection in cGAS-knockout HeLa cells and treated them with nocodazole for mitotic arrest as described previously. I confirmed that cGAS catalytic-dead mutant was in fact catalytic dead as it was not able to induce IRF3 phosphorylation by performing western blot analysis, and thus unable to activate cGAS/STING pathway.

Unlike WT cGAS transfected cells, cells with cGAS CD mutant were inefficient in rescuing cells with micronuclei when released from nocodazole arrest for 10 h and then undergo immunocytochemical analysis (Fig. 10a). Cells showing micronuclei formation were significantly reduced when I transfected cGAS-knockout cells with cGAMP, supporting previously obtained results (Fig. 10b). Last but not the least, cGAMP transfection to cGAS-knockdown HeLa cells also led to decreased micronuclei formation as compared to the vehicle control transfected cGAS- knockdown cells (Fig. 10c). All in all, it is safe to say that these results clearly indicate that inhibition of micronuclei formation is dependent on synthesis of cGAMP by cGAS as cell-cycle progresses, hence proving the necessity of cGAS enzymatic function in decreasing the incidence of CIN phenotype in successive cell cycles.

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Figure 10. cGAS activity is essential to maintain chromosomal integrity

(a) cGAS -/- HeLa cells were transfected with indicated plasmids for 6 h and then treated with nocodazole for 16 h. Then, mitotic cells were collected by shake-off and subjected to Western blotting (left panel) with indicated antibodies accompanied by ICC (right panel) to count percentage of cells showing micronuclei after 10 h release from nocodazole 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) cGAS -/- HeLa cells were transfected with or without cGAMP using Lipofectamine® 2000 transfection reagent for 6 h. Then, the cells were arrested at M phase via nocodazole for 16 h. Fraction of mitotic cells were seeded on a cover slip to enumerate the cells showing micronuclei phenotype after 10 h from release by ICC (right panel) along with representative fluorescent images showing micronuclei (white arrow head denotes micronuclei) and other fraction was subjected to Western blotting with indicated antibodies (left panel). The results are given as the mean ± SD from three independent experiments (n=300). ***P < 0.001 by Student’s t-test. (c) HeLa cells transfected with sicGAS for 20 h were transfected with or without cGAMP for 6 h and then subjected to nocodazole treatment for 16 h. After release from nocodazole drug, Western blotting (left panel) with indicated antibodies and ICC (right panel) was performed to quantify cells displaying micronuclei phenotype after 10 h from release. The results are given as the mean ± SD from three independent experiments (n=300). ***P < 0.001 by Student’s t-test.

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E. STING plays mediating role to maintain chromosomal stability

cGAMP, synthesized by cGAS, binds with STING and results in the activation of downstream signaling. To check either STING is required to regulate chromosomal stability or not, HeLa cells were transfected with siRNA targeting STING (siSTING) and monitored for micronuclei formation post-treatment with nocodazole-induced mitotic arrest. As predicted, STING-knockdown HeLa cells displayed an increase in the number of micronuclei formation as compared to siControl-transfected HeLa cells, indicating STING-dependent regulation of chromosomal stability after cGAS activation (Fig. 11a). Whether STING activity is required for its proposed function, I transfected STING-knockdown HeLa cells with WT STING, STING constitutively active mutant, or an inactive STING mutant plasmid DNA. According to western blot analysis, both WT STING and constitutively active STING mutant transfected STING-depleted HeLa cells showed phosphor-IRF3 band as opposed to the inactive STING mutant transfected STING-depleted HeLa cells. Interestingly, micronuclei formation was enhanced in inactive STING mutant transfected cells whereas WT STING and constitutively active STING mutant displayed reduction in micronuclei formation (Fig. 11b). Moreover, when I transfected these same STING mutants and empty vector into cGAS-depleted Hela cells, I observed an increase in the number of micronucleated cells in both inactive STING mutant- and empty vector-transfected cells. Opposingly, micronucleated cells were significantly reduced in case of both WT STING and constitutively active STING mutant-transfected cells (Fig. 11c). These findings taken together, suggest that activation of STING is necessary to regulate chromosomal stability via cGAS.

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Figure 11. cGAS activates STING to control chromosomal stability

(a) HeLa cells transfected with siRNA against STING for 30 h and then treated with nocodazole for 16 h. Mitotic cells were subjected to Western blotting (left panel) with cGAS, STING and α-tubulin antibodies and ICC (right panel) for analyzing percentage 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 t-test. (b) HeLa cells were co-transfected with siSTING and different indicated plasmids for 20 h and then arrested via nocodazole treatment. After 16 h, cells were released and Western blotting (left panel) with indicated antibodies was performed. Percentages of cells with micronuclei were counted by performing ICC (right panel) after 10 h release from mitotic arrest 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. (c) cGAS -/- HeLa cells were transfected with indicated plasmids for 12 h before nocodazole treatment for 16 h. After 10 h release from mitotic arrest, cells were subjected to Western blotting with indicated antibodies and ICC to calculate percentage of cells showing micronuclei formation after nocodazole release 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.

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F. Depletion of TBK1 and IRF3 also enhanced CIN phenotypes

To establish whether downstream components of cGAS/STING pathway, TBK1 and IRF3, play a role in maintaining chromosomal stability, I transfected HeLa cells with siTBK1 and observed micronuclei formation. TBK1-knockdown HeLa cells displayed increased percentage of micronucleated cells in comparison with the siControl transfected cells (Fig. 12a), indicating the possibility that TBK1 is required for proper chromosome segregation. I then depleted IRF3 using RNA interference and treated IRF3-knockdown HeLa cells with nocodazole to induce mitotic arrest.

Similar to TBK1-depleted HeLa cells, decrease in the IRF3 levels predisposed the cells towards enhanced micronuclei formation as compared to the cells with normal IRF3 levels (Fig. 12b). The data collected so far, indicates that cGAS/STING/TBK1/IRF3 pathway as a whole affect chromosomal stability during cell-cycle progression.

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Figure 12. TBK1 and IRF3 depletion also induces chromosomal instability

(a) HeLa cells were transfected with siRNA targeting TBK1 for 30 h and treated with nocodazole for 16 h. After 16 h, cells were released and subjected to Western blotting with indicated antibodies. Mitotic cells collected after nocodazole treatment was reseeded and ICC was performed to evaluate the percentage of cells with 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. (b) IRF3 was down-regulated using RNA- interference in HeLa cells for 30 h before treatment with nocodazole for 16 h. After 16 h, cells were released to collect mitotic cells and subjected to ICC (right panel) for evaluating percentage of cells with micronuclei along with representative fluorescent images showing micronuclei (white arrow head denotes micronuclei). Western blotting was performed with indicated antibodies to confirm IRF3 knock-down via siRNA transfection (left panel).

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G. p21 expression levels are dependent on cGAS/STING pathway

The question as to how cGAS/STING/TBK1/IRF3 pathway regulates chromosomal segregation remained unanswered. So, by reviewing some recent reports, I came across two possible mechanisms by which cGAS-STING pathway can contribute towards chromosomal stability. Both suggested mechanisms roughly provided the idea that this pathway can either directly affect mitotic phase or interphase of the cell cycle, leading to chromosomal segregation defects. The first possible scenario as to how this pathway maintains chromosomal stability states that cGAS-STING pathway affects centrosome number, since they mention that TBK1 binds with centrosomal proteins and affect microtubule stability (13). Unfortunately, in my experimental setting, STING knock-down or add-back had no significant effect on centrosome number (Fig. 13a, b). The second possible scenario proposed that p21 levels are regulated via activation of STING/TBK1/ NF-B/ p53 axis (12). Now, p21 has been known to halt cell-cycle progression especially during G2/M transition, it means that upon p21 down-regulation as a consequence of STING depletion will induce early G2/M transition and affect successive mitotic events as well. To check whether cGAS is the upstream signaling protein involved in regulation of p21 expression levels during cell- cycle progression, I checked p21 levels in cGAS-depleted HeLa cells. Significant reduction in p21 levels was apparent in cGAS-depleted cells as compared to WT HeLa cells (Fig. 13c). Furthermore, STING depletion in HeLa cells also resulted in down-regulation of p21 levels (Fig. 13d), as previously reported (12). Since, IRF3

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has been reported to aid transcriptional activation of p53 and p53-dependent cell- cycle inhibition (14, 15), I down-regulated IRF3 in HeLa cells using RNA interference and checked p21 levels. Interestingly, p21 levels were significantly decreased in IRF3 depleted HeLa cells, proposing IRF3 as downstream signaling protein involved in regulating p21 levels in my experimental setting (Fig. 13e). To investigate whether reduction in p21 expression levels is dependent on p53 or not, I transfected WT HeLa cells with sip53. Depletion of p53 resulted in reduction of p21 levels accompanied by significant rise in micronuclei phenotype, proposing that p53 regulates p21 levels in these cells (Fig. 13f). Then, I down-regulated cGAS or p53 or both using RNA interference, and found out that 1) p53 levels are independent of cGAS levels, and 2) although p53 is not depleted, p21 levels were significantly reduced after just cGAS depletion, suggesting that p21 depletion in cGAS-knock down cells is not dependent on p53 (Fig. 13g). Due to decrease in p21 levels, cells would undergo early mitotic entry thus suffer from chromosomal segregation errors possibly either because of insufficient DNA damage repair or uncoordinated mitotic entry (18-20). Upon comparing extent of DNA damage among cGAS knock-down or cGAMP-transfected HeLa cells, there was no significant change (Fig. 13h, i), however, when I calculated mitotic index after double-thymidine block release, I found out that STING knock-down HeLa cells repeatedly displayed an hour earlier G2/M transition as compared to WT HeLa cells (Fig. 13j). This possibly meant that increase in p21 levels after cGAS/STING pathway activation mainly occur during G2 phase, thus allowing cells enough time to get ready for entering into mitotic phase with sufficient nutrients and proteins.

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Figure 13. cGAS/STING pathway regulates p21 expression levels during cell-cycle progression

(a) Representative confocal images of a mitotic cell during metaphase showing bipolar orientation of centrosomes (white arrow head) after staining with anti-gamma-tubulin antibody. (b) Quantification of cells with less than or equal to 2 centrosomes (black bar) or more than 2 centrosomes (gray bar) after co-transfection with siSTING accompanied with or without ectopic expression of STING wild-type, STING constitutively-active & STING inactive mutant forms. (c) sicGAS transfection to HeLa cells for 48 h and then subject to western blot with indicated antibodies. (d) HeLa cells transfected with siRNA against STING for 48 h and then cell lysates were subjected to Western blotting with antibodies against p21, STING and GAPDH. (e) IRF3 knock-down using RNA-interference for 48 h before collecting cell lysates. Cell lysates were subjected to Western blotting with indicated antibodies. (f) p53 knock-down using sip53 in HeLa cells and checking micronuclei after 10 h release from Nocodazole by ICC (right panel) and checking p21 levels using indicated antibodies by Western Blot (left panel). The results are given as the mean ± SD from three independent experiments (n=300). ***P < 0.001 by Student’s t-test. (g) HeLa cells were transfected with sip53 or sicGAS or both and then micronuclei formation was quantified after release from Nocodazole using ICC (right panel). cGAS, p53 and p21 levels were checked by indicated antibodies using Western Blot. The results are given as the mean ± SD from three independent experiments (n=300). ***P < 0.001 by Student’s t-test. (h) Representative images of a cell showing gamma-H2A.X foci formation upon staining with gamma-H2A.X antibody after cGAS knock-down with or without cGAMP transfection. (i) Percentage of cells showing gamma-H2A.X foci formation with or without cGAMP transfection in cGAS-depleted HeLa cells. (j) HeLa cells were transfected with siSTING and then subjected to double-thymidine block with 2mM thymidine for 40 h. 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. The results are given as the mean ± SD from three independent experiments (n = 300).

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H. p21 as a principal regulator in maintaining chromosomal integrity via cGAS/STING pathway

Whether reduction of p21 levels due to impaired cGAS/STING pathway resulted in the escalation of CIN phenotypes (i.e., micronuclei), I depleted p21 by transfecting siRNA targeting p21 and calculated percentage of micronucleated cells. In fact, p21 down-regulation strongly augmented the micronuclei formation when released from nocodazole-induced mitotic arrest as compared to the control cells (Fig. 14a), which was further supported by early mitotic entry after synchronized cells at G1/S phase were released from double-thymidine block (Fig. 14b). Then, I checked if p21 overexpression can rescue the cells from CIN (i.e., micronuclei formation) induced after cGAS down-regulation.

Interestingly, p21 overexpression in cGAS-depleted cells significantly reduced the number of micronucleated cells as compared to empty pcDNA vector-transfected cGAS-depleted cells (Fig. 14c). Moreover, overexpression of p21 was also able to rescue the cGAS-depleted cells from precocious G2/M transition (Fig. 14g). In the same manner, ectopic expression of p21 was also able to strongly reduce the percentage of micronucleated cells in cGAS-knockout HeLa as compared with the cGAS-knockout cells transfected with empty pcDNA vector (Fig. 14d).

Hypothetically, it means that by delaying mitotic entry under similar conditions will result in reduced chromosomal instability. To confirm this, I synchronized cGAS- depleted HeLa cells with double-thymidine block and then released for 6~7 hours before treatment with RO3306, an inhibitor of cyclin-dependent kinase 1 (CDK1), to prolong G2 phase and determined the extent of CIN caused by cGAS-depletion.

In fact, RO3306 treatment not only strongly rescued cGAS-depleted cells from micronuclei formation (Fig. 14e), but also abrogated the early mitotic onset (Fig.

14f). All in all, the obtained data describes the phenomenon that how attenuation of cGAS/STING pathway give rise to CIN due to early G2/M transition via reduction in p21 levels.

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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 t-test. (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

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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).

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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

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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.

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