Targeting CTLA-4 in cancer cells induces senescence via DNA-PKcs-STING-AKT axis

Targeting CTLA-4 in cancer cells induces senescence via DNA-PKcs-STING-AKT axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting CTLA-4 in cancer cells induces senescence via DNA-PKcs-STING-AKT axis Jeon-Soo Shin, Je-Jung Lee, Woo Joong Rhee, So Young Kim, Jisun Lee Lee, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3893509/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Immune checkpoints such as cytotoxic T-lymphocyte associated protein 4 (CTLA-4), PD-1, and PD-L1 have been targeted for cancer therapy. However, the efficacy of this treatment approach remains limited. Beyond its expression on the T-cell surface, CTLA-4 is also expressed in various cancer cells and plays roles in cell proliferation, metastasis, and apoptosis. Here, we reveal that targeting CTLA-4 in melanoma cells leads to genomic instability and DNA-PKcs-STING-AKT pathway activation (via p53 and p21), which in turn blocks cell proliferation and induced senescence. Notably, DNA-PKcs orchestrates CTLA-4-depletion-induced senescence via the STING pathway regulation. To the best of our knowledge, this is the first study to report CTLA-4 leads senescence via micronuclei induction, which triggers DNA-PKcs and eventually suppresses cancer growth. These findings provide a better understanding of the mechanisms underlying CTLA-4 targeting-cancer therapy and future treatment strategies. Biological sciences/Cell biology/Senescence Biological sciences/Cancer/Cancer prevention Health sciences/Diseases/Cancer/Cancer prevention Biological sciences/Molecular biology/DNA damage and repair/DNA damage response Biological sciences/Cell biology/Cell growth/TOR signalling CTLA-4 Senescence Cancer therapy STING AKT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Cellular senescence is a permanent cell cycle arrest induced by various stimuli. Senescence is defined by morphological changes, increased senescence-associated β-galactosidase (SA-β-Gal) activity at pH 6.0, and the induced expression of cell cycle checkpoints such as p21, p16, and p27( 1 , 2 , 3 ). The expression of heterochromatin and DNA damage markers such as H3K9 trimethylation and γ-H2AX( 4 , 5 , 6 ) is also linked to senescence. Senescence can suppress growth of tumors rendered resistant to apoptosis following cancer therapy; this is achieved via blocking cell cycle progression and cell proliferation( 6 , 7 , 8 , 9 , 10 , 11 ). Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a pivotal player in regulating immune responses, is a immune checkpoint molecule expressed on the T-cell surface and functions as a negative regulator of T-cell proliferation. However, CTLA-4 is also expressed in several cancers ( 12 , 13 , 14 , 15 ), and robustly expressed in melanoma ( 16 , 17 , 18 ). The evidence of role played by CTLA-4 in tumors has sparked interest in further studies in this field. Generally, CTLA-4 accumulates in intracellular compartments; however, it transitionally moves to the cell surface in response to stimuli and is rapidly internalized( 19 ). However, the function of cytoplasmic CTLA-4 remains unclear. Thus, it is necessary to evaluate the role of CTLA-4 in cancer cells. The cyclic GMP–AMP synthase (cGAS)- stimulator of the interferon genes (cGAS-STING) signaling pathway is an important mediator of inflammation, cellular stress, tissue damage, and senescence( 20 , 21 ). Presence of DNA in the cytosol due to several reasons such as infection, separation from self-DNA damage, or slippage during replication, activates cGAS and brings it to the adaptor protein STING, then recruits TANK-binding kinase 1 (TBK1). TBK1 phosphorylates STING and interferon regulatory factor 3 (IRF3). IRF3 dimerizes and localizes in the nucleus, inducing type I interferons( 22 ). STING signaling plays a pivotal role in senescence and can be targeted for suppressing tumors via senescence induction. The serine/threonine kinase AKT enhances the expression of p53 and p21, increases cell size, and induces senescence( 23 , 24 ). Persistent activation of AKT results in cellular senescence, a tumor-suppressive mechanism. Additionally, revelation of the AKT and STING pathway interaction has expanded the role of AKT and STING-related functions in senescence( 25 , 26 ). The DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a sensor for DNA double-strand breaks (DSB), is involved in non-homologous end joining (NHEJ) and DNA damage repair (DDR) pathways. DNA-PKcs also act in STING-dependent and -independent pathways for intracellular nucleic acid recognition. Although DNA-PKcs primarily functions in the nucleus, its nonnuclear function of maintaining genomic integrity and DNA fidelity ( 27 ). In addition, DNA-PKcs kinase plays critical role in many diseases including cancers as it has been associated with various cellular processes, such as cell death, division, senescence, and metabolism( 28 ). Along with its conventional role in the DNA damage response, involvement of DNA-PKcs in STING-related pathway can bring an important paradigm shift in cancer therapy( 29 ). In this study, we aimed to determine the role of CTLA-4 in cancer. We verified the effect of silencing CTLA-4 in human and mouse melanoma cells and found that CTLA-4 depletion induced senescence. This was achieved via a signaling cascade involving genomic instability induced DNA damage, which subsequently activates DNA-PKcs-STING-AKT-p21 in human and mouse melanoma cells, eventually leading to tumor regression. To the best of our knowledge, this is the first study to report a conclusive role of CTLA-4 in cancer cell senescence. These findings can help to better understand and develop strategies for cancer therapy via CTLA-4 targeting. RESULTS CTLA-4 depletion induces senescence in melanoma cells To investigate the role of CTLA-4 in cancer, we silenced CTLA-4 either alone or in combination with doxorubicin (Dox) ( 30 , 31 ) (Fig. 1 a). Human melanoma cell line A375 showed higher expression of CTLA-4 than other cells (Supplementary Fig. 1a-d)( 17 , 32 ). Unexpectedly, CTLA-4 silencing resulted in senescence phenotype, including an increase in cell size (Fig. 1 a, b), SA-β-Gal activity proven by senescent green probe (Fig. 1 b, c), and decreased cell viability (Fig. 1 d) and colony forming assays (Fig. 1 e). In addition, western blot (WB) analysis showed increased expression of senescence markers such as p21 and p16, and the heterochromatin marker, H3K9me3, compared to the control cells (Fig. 1 f). Moreover, confocal imaging showed that p21 expression inversely correlated with that of CTLA-4 (Fig. 1 g). These effects were synergistic in both the siCTLA-4 and Dox-treated group compared to the Dox(only)-treated group. Additionally, results of fractionation assay confirmed that CTLA-4 was predominantly expressed in the cytosol (Supplementary Fig. 1e, f). We confirmed this with B16-F10 mouse melanoma cells which expressing CTLA-4 well (Supplementary Fig. 1b). We silenced CTLA-4 alone or in combination with Dox in B16-F10 cells. Depletion of CTLA-4 alone resulted in increased cell size (Fig. 2 a, b), SA-β-Gal activity (Fig. 2 b), and decreased cell viability (Fig. 2 c). Furthermore, p21, p16, and H3K9me3 protein levels increased, as shown by WB (Fig. 2 d). Moreover, p21 expression was inversely correlated with that of CTLA-4 (Fig. 2 e, f). Notably, all these effects were more significant in the siCTLA-4 and Dox combination group than in the Dox(only)-treated group. We also repeated these experiments using a different CTLA-4 specific siRNA (siCTLA-4 * ) (Supplementary Fig. 2) and the results were similar to those obtained from the siRNA sequence used in A375 cells (Fig. 1 f). Taken together, our data suggest that targeting CTLA-4 in cancer cells induces senescence and halts cancer cell proliferation, and Dox treatment enhances these outcomes synergistically. AKT pathway is required to induce CTLA-4-deficiency-induced-senescence As a well-known component of CTLA-4 related signaling, the AKT pathway is activated through the surface CTLA-4 signaling pathway( 17 , 33 ). Therefore, we investigated the status of the AKT pathway and its role in CTLA-4 deficiency-induced senescence. Ironically, phospho(p)-AKT was also upregulated along with other senescence markers, including p53, p21, p27, p16, and H3K9me3, in both CTLA-4 alone silenced and Dox combination groups (Fig. 3 a). AKT pathway-mediated senescence is known as oncogene-induced senescence (OIS)( 34 , 35 ). In addition, confocal imaging showed increased expression of p-AKT and p-mTOR, molecules acting downstream of AKT, in siCTLA-4 groups (Fig. 3 b, c). These experiments were performed in mouse melanoma cells and similar results were obtained (Fig. 3 d-f). A recent study reported an 80 kDa splicing mTOR isoform, mTORβ, which is considered an active protein kinase beyond full-length mTOR (mTORα)( 36 ). It showed an expression pattern similar to that of p-AKT in both A375 and B16-F10 cells (Supplementary Fig. 3a, b). However, CTLA-4 overexpression blocked Dox treatment -induced senescence phenotype, including morphological changes and p21, p-AKT, and p-mTOR expression in B16-F10 as well as A375 cells (Supplementary Fig. 4a-d). Taken together, we conclude that the AKT pathway is required for CTLA-4-deficiency-induced senescence. CTLA-4 deficiency causes DNA damage response DNA damage is one of the dominant causes or consequences of senescence( 2 , 11 , 37 ). So, we examined the DNA damage marker γ-H2AX, and found it was upregulated and inverse correlation with CTLA-4 expression in CTLA-4-silenced B16-F10 cells (Fig. 4 a). In addition, other DNA damage markers H3K9me3 and p53 (Fig. 1 f, 2 d, 3 a, and 3 d) were elevated along with other senescence markers. γ-H2AX expression was validated by confocal imaging in B16-F10 and A375 cells (Fig. 4 b-d). These results were confirmed in CTLA-4 knockout (KO) B16-F10 cells (Supplementary Fig. 5a). CTLA-4 KO cells showed higher sensitivity to the anticancer drugs cisplatin and Dox (Supplementary Fig. 5b, c). Micronuclei are tiny nuclear DNA, and often observed in cancer and senescence. Senescence often appears with aneuploidy induced by chromosome missegregation, which triggers the formation of cytoplasmic chromatin fragments (CCFs), resulting in and they become micronuclei( 38 ). We observed micronuclei following CTLA-4 silencing, which is colocalized with γ-H2AX (Fig. 4 d: enlarged part of Fig. 4 c) and their content was much higher in the Dox-co treated group in mouse (Fig. 4 e, f) as well as human melanoma cells (Fig. 4 g, h). Interestingly, micronuclei co-localized with γ-H2AX in the CTLA-4 silenced cells, confirming that micronuclei are a byproduct of DNA damage caused by genome instability (Fig. 4 d). In addition, Aurora B, which prevents micronuclei formation and whose downregulation induces senescence( 39 , 40 ), was downregulated by siCTLA-4 treatment (Fig. 4 i-l, Supplementary Fig. 5d ) and this effect was reverted by CTLA-4 overexpression (Fig. 4 m). These results imply that CTLA-4 depletion induced genomic instability and DNA damage response. STING signaling regulates CTLA-4-depletion-induced senescence via modulating AKT signaling pathway Micronuclei present in the cytosol, trigger cGAS-STING pathway( 41 ). cGAS binds to dsDNA in micronuclei and triggers cyclic GMP-AMP (cGAMP) synthesis. Subsequently, cGAMP attaches to STING and recruits TBK1, which in turn phosphorylates STING. Phosphorylated STING recruits interferon regulatory factor-3 (IRF3), which is then phosphorylated by TBK1 and dimerizes, followed by its nuclear translocation. Eventually, IRF3 transcribes genes encoding proteins including interferons and cytokines in the nucleus( 42 ). Therefore, we examined the effects of micronuclei on the STING pathway in the CTLA-4 silenced group. p-STING and p-IRF3 increased in a time-dependent manner, along with p-AKT and p-p53 (S15) (Fig. 5 a). Furthermore, γ-H2AX expression correlated with CTLA-4 depletion time points in both WB and confocal imaging assay (Fig. 5 a, b). However, these effects were reversed by siSTING treatment. Namely, activated AKT signaling as well as the elevation of γ-H2AX by CTLA-4 silencing, were blocked by siSTING treatment (Fig. 5 c). Notably, p-AKT expression was also observed in the confocal imaging (Fig. 5 d) and these findings were confirmed in B16-F10 cells by WB and confocal imaging (Supplementary Fig. 6a-d). Overall, our results indicated that CTLA-4 deficiency potentiates DNA damage-induced STING signaling mediated via AKT signaling. DNA-PKcs intervenes with CTLA-4-depletion-induced senescence DNA-PKcs belongs to the phosphatidylinositol 3-kinase family. DNA-PKcs forms the active DNA-PK holoenzyme with the Ku80/Ku70 heterodimer to regulate DDR following DSB. DNA-PKcs rapidly moves to the damaged sites and is activated. It then sends damage signals via p53, ultimately leading to cell cycle arrest, aging, or apoptosis. Aside from its role in the cell cycle( 43 ), DNA-PKcs keeps genome integrity( 44 ). Furthermore, DNA-PKcs recognizes cytosolic DNA and activates the cGAS-STING pathway ( 29 ). Next, we examined whether DNA-PKcs plays a role in CTLA-4-depletion-induced senescence. DNA-PKcs was upregulated together with activated STING pathway (p-STING and p-IRF3), AKT signaling markers (p-AKT and p-mTOR), and p-p53 (S15) in the CTLA-4-depleted and Dox-combined group of B16-F10 cells compared to the control and Dox-(only)-treated group (Fig. 6 a, b). Additionally, DNA-PKcs was activated (p-DNA-PKcs) by siCTLA-4 in A375 cells (Supplementary Fig. 7a, b). Surprisingly, all activated signals, including those of the STING-AKT pathway (p-STING and p-mTOR), CDK inhibitor (p21), and DNA damage marker (γ-H2AX) following siCTLA-4 treatment of B16-F10 cells were abolished by siDNA-PKcs treatment (Fig. 6 c), and these results were confirmed in A375 cells (Fig. 6 d-g). DNA-PKcs expression was elevated together with those of p-IRF3, p-STING, and p-AKT in both CTLA-4 silenced and Dox-cotreated with CTLA-4 silenced groups compared to control and only Dox-treated groups, respectively (Fig. 6 d). However, the STING pathway (p-STING and p-TBK1), AKT signaling factors (p-AKT and p-mTOR), cell cycle inhibitors (p21 and p27), and the DNA damage marker γ-H2AX activated by siCTLA-4 were blocked by siDNA-PKcs treatment (Fig. 6 f) or the specific DNA-PKcs inhibitor Nu7441 (Fig. 6 g). Interestingly, targeting only DNA-PKcs via silencing or inhibition showed a different manner compared to that observed following co-treatment with the CTLA-4 silencing group (Fig. 6 c, f, and g). Namely, targeting DNA-PKcs alone elevated AKT and p21 levels as well as STING pathway (2nd lane of 6C, 3rd lane of 6F, and 2nd -3rd lane of 6G), which may be attributed to the lack of DNA repair following DNA-PKcs silencing or inhibition( 45 ). DNA-PKcs abrogation downregulates the protein expression induced by CTLA-4 silencing, which may be due to the absence of a DNA-PKcs signaling in the STING pathway ( 46 ). Indeed, siDNA-PKcs almost abrogated CTLA-4 depletion-led senescence in A375 cells, evidenced by morphological changes (Fig. 6 h) and the degree of senescence (Fig. 6 i, j), indicating that DNA-PKcs is essential for CTLA-4-depletion induced senescence. To confirm the selectivity of DNA-PKcs for CTLA-4 depletion-induced senescence, we compared DNA-PKcs with ataxia telangiectasia mutated (ATM), which has also been implicated in DSBs( 47 ). The ATM-specific inhibitor KU-60019 showed no influence CTLA-4 depletion effect; however, DNA-PKcs inhibition by NU7441 or siRNA knockdown abrogated the CTLA-4 depletion effects (Fig. 6 g, Supplementary Fig. 7a, b). These results suggest that the effects of CTLA-4 are selectively linked with DNA-PKcs. Furthermore, immunoprecipitation (IP) assay revealed an interaction between CTLA-4 and DNA-PKcs after CTLA-4-overexpression in A375 cells (Fig. 6 k). In fact, CTLA-4 was predominantly located in the cytosol (Fig. 1 g, 2 e, Supplementary Fig. 1e, f), along with DNA-PKcs (Fig. 6 b and e), which can facilitate their interaction to detect cytosolic nucleic acids (Fig. 6 k). To further investigate the correlation of CTLA-4 with DNA-PKcs, we used The Cancer Genome Atlas (TCGA) data, and observed that the mRNA expression of PRKDC , which encodes DNA-PKcs, was negatively correlated with that of CTLA-4 (Fig. S7c). Furthermore, patients were categorized into high- (top 1/3) and low- (bottom 1/3) CTLA-4 groups based on the CTLA-4 expression level and showed an inverse correlation with non-homologous end joining pathway components (NHEJ), including DNA-PKcs, in patients with skin cutaneous melanoma (Supplementary Fig. 7d). Additionally, these features were observed in lung, cervical, head, and neck squamous cell carcinomas (Supplementary Fig. 7e). Taken together, DNA-PKcs plays an indispensable role and orchestrates CTLA-4-depletion-induced senescence of cancer cells via modulating the STING-AKT pathway axis. CTLA-4-depletion impedes tumor growth via senescence induction Finally, we performed experiments in mice to verify whether CTLA-4-depletion affected tumor growth. We generated a CTLA-4 knockout (KO) B16-F10 cell line using the CRISPR-Cas9 system. Next, C57BL/6 mice were subcutaneously injected with 5x10 5 B16-F10 WT and B16-F10 CTLA − 4 KO cells followed by intraperitoneal injections of 9 mg/kg Dox. The tumors were collected on day 16 (Fig. 7 a). Size, weight, and volume of tumors derived from mice injected with CTLA-4 KO cells, were much smaller than those of tumors derived from mice injected with wild-type (WT) cells in both DMSO- and Dox-treated groups (Fig. 7 b-d). Furthermore, SA-β-Gal staining of B16-F10 CTLA − 4 KO derived tumors was much stronger than that of B16-F10 WT derived tumors both with and without Dox treatment, confirming the effect of CTLA-4 on tumor cell senescence (Fig. 7 e). B16-F10 CTLA − 4 KO derived tumors showed higher expression of γ-H2AX and p-STING, than that in B16-F10 WT derived tumors in both the absence and presence of Dox treatment groups by WB analysis (Fig. 7 f), which was later confirmed by IHC analysis (Fig. 7 g-i). In conclusion, once CTLA-4 depletion occurs in cancers, it causes cellular senescence via DNA damage-DNA-PKcs-STING-AKT-p21 pathway, and eventually leads to tumor suppression. DISCUSSION It is important to understand the role of CTLA-4 in cancer cells, beyond in T cells, to allow targeted cancer therapy. This is the first study to demonstrate a distinctive role of CTLA-4 in cancer. The governing the immune checkpoints is a promising approach for cancer immunotherapy. Immune checkpoints PD-1, PD-L1, and CTLA-4 are the main targets for current therapeutic approaches. Although some inhibitors that target immune checkpoints expressed on the cell surface have emerged as effective candidates for certain tumors, their efficacy remains unsatisfactory. Therefore, many research groups have attempted to develop better strategies( 48 ). In the present study, we silenced the expression of CTLA-4 in human and mouse melanoma cells and observed its effects. Surprisingly, CTLA-4 silencing caused senescence, evidenced by larger and flattened morphological changes, decreased cell proliferation, increased SA-β-Gal activity, induction of cell cycle checkpoints p21 and p16, and upregulation of DNA damage and heterochromatin markers γ-H2AX and H3K9me3, respectively. Next, we elucidated the mechanism underlying senescence induced by CTLA-4 depletion. CTLA-4 deficiency in melanoma cells affected genomic stability and induced micronuclei formation via downregulating Aurora B expression( 40 , 49 ). This was sensed by DNA-PKcs, which in turn activated the STING-AKT signaling pathway and mediated senescence via p53 and p21. Our study highlights the atypical role of DNA-PKcs in CTLA-4 depletion-induced senescence. Canonically, DNA-PKcs serves as a classic component of DSB-induced DDR and maintains genome integrity( 50 ). However, in cytoplasm, DNA-PKcs acts as a DNA sensor and activates innate immunity. DNA-PKcs attaches to the cytoplasmic DNA and activates the STING pathway via STING-TBK1-IRF3 activation( 51 ). Our results showed that DNA-PKcs triggers the STING pathway, confirmed by DNA-PKcs silencing. This is consistent with a recent report in which the specific DNA-PKcs inhibitor NU7441 suppressed the activation of the STING pathway by stimulation with double-stranded DNA( 46 ). We successfully blocked the STING pathway activated by CTLA-4 deficiency via silencing DNA-PK in both human and mouse melanoma cells. Although ATM is a major component of the DNA damage response pathway, it is not involved in CTLA-4 depletion-induced senescence. In contrast, DNA-PKcs functions selectively to modulate the STING-AKT pathway required for CTLA-4 depletion-induced senescence, which was confirmed by publicly available data for patients with cervical, lung, head, and neck cancers, as well as melanoma. Furthermore, a relationship between STING and the AKT pathway has been reported, in which AKT regulates the STING pathway( 26 ). In the present study, STING regulated the AKT pathway as confirmed by the observation that phosphorylation of AKT and mTORC1 were downregulated by STING silencing. Eventually, the STING-AKT pathway axis was compromised by DNA-PK depletion, indicating that DAN-PK orchestrates CTLA-4-depletion-induced senescence by regulating the STING-AKT pathway. Taken together, we found that senescence is the dominant feature of the response of CTLA-4-depleted-melanoma cells, and is mediated by the DNA-PKcs-STING-AKT-p53-p21 axis. While several studies have reported that CTLA-4 silencing inhibits cellular proliferation ( 13 , 52 , 53 ) and CTLA-4 causes apoptosis in cancer cells( 54 ), our study is the first one to report the role of CTLA-4 as a senescence regulator in cancer. We reveal that mechanistically, CTLA-4-deficiency activates the DNA-PKcs/ STING pathway via downregulating Aurora B. Notably, a recent report suggested the positive correlation between CTLA-4 and Aurora B in an HCC patient( 55 ). To extend these findings, we investigated whether lack of CTLA-4 promotes DNA damage and found that the DNA damage marker γH2AX was significantly upregulated in the B16-F10 CTLA − 4 KO cells compared to B16-F10 WT cells when treated with anti-cancer drugs such as cisplatin and Dox; these observations strongly supports our hypothesis that lack of CTLA-4 enhances DDR and steers cells toward senescence by activating STING pathway. Although the mechanism by which CTLA-4 deficiency induces genomic instability and mechanisms underlying STING-led activation of the AKT pathway need to be verified in future studies, our study revealed the role of CTLA-4 in senescence and contributes to a better understanding of CTLA-4-targeting-mediated therapy for cancers. Collectively, our results reveal that targeting CTLA-4 in cancer cells is a potential therapeutic strategy for cancer. MATERIALS AND METHODS Cell culture, transfection, and reagents A375 human- and B16-F10 mouse-melanoma cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM). CCRF-CEM, PEER, MOLT-4 human T cells were grown in RPMI-1640. All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The CLLA-4 plasmid was obtained from Sino Biological. Plasmids and siRNA transfections were performed using FuGene HD reagent and RNAiMAX, respectively, as recommended by the manufacturers. siRNA duplexes against human CTLA-4 1ST , CTLA-4 2ND , DNA-PK, human and mouse STING, AKT, and control siRNA were purchased from Bioneer, Inc. siRNA duplexes against mouse CTLA-4, DNA-PK were obtained from Santa Cruz Biotechnology. Neutralizing anti-CTLA4 mouse antibody were purchased from BioLegend. and-Dox was obtained from Calbiochem. Nu 7441 was purchased from Selleck Chemicals. WB analysis For western blot analysis, we followed the protocol described in ( 31 ). Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against p-IRF3, STING, p-TBK1, p-AKT, AKT, p-mTOR, mTOR, β-actin, tri-H3K9, p53(S15), secondary antibodies for immunofluorescence from Cell Signaling Technologies, Inc. DNA-PKcs, γ-H2AX, p16 from Abcam. p21, p27from BD Biosciences. CTLA-4 from Proteintech, p53 from Santa Cruz Biotechnology, p-STING from Affnit were used. Antibody-antigen complexes were detected using HRP-conjugated secondary antibodies and visualized using a standard chemiluminescence method according to the manufacturer’s instructions. Immunoprecipitation. 150 ug of each protein lysate was incubated with the indicated antibodies, normal rabbit IgG or normal mouse IgG for 4 h at 4°C, followed by an incubation with 20 µl of protein A magnetic beads for 16 h at 4°C. The immune complexes were analyzed by western blot analyses with the indicated antibodies. Protein lysates were also subjected to western blot analyses with the indicated antibodies. Cell morphology analysis and SA-β-Gal staining Cellular morphologies were photographed using an inverted phase-contrast microscope. SA-β-Gal staining was carried out as recommended by the manufacturers (CELLEvent™ Senescence Green Detection Kit). Those examinations were performed on day 3 following each treatment unless otherwise indicated. Immunofluorescence For immunofluorescence analysis, we followed the protocol described in ( 31 ). Mouse experiment All animal procedures were approved by the Institutional Animal Care and Use Committee (IRB no. 2019 − 0242). We generated tumors with 5 × 10 5 B16-F1O WT and CTLA-4 KO tumors, and followed our previous procedures in ( 56 ). Photographs were acquired in randomly chosen fields per tumor section according to standard procedures. Statistical analysis Statistical analysis was performed using GraphPad Prism. The significance of the statistical differences among three or more groups was calculated using one-way analysis of variance and the Newman-Keuls test. Data is shown as the mean ± standard deviation (SD). Asterisks denote the p -values as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001. Declarations AUTHOR CONTRIBUTIONS Conception and design: Je-Jung Lee, Woo Joong Rhee, Jeon-Soo Shin. Development of methodology: Je-Jung Lee, Woo Joong Rhee, So Young Kim. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Je-Jung Lee, So Young Kim, Jisun Lee. In Ho Park. Jisun Lee, Su Ful Jung. Analysis and interpretation of data (statistical analysis, biostatistics, and computational analysis): Je-Jung Lee, Woo Joong Rhee, In Ho Park, and Jeon-Soo Shin. Writing, review, and/or revision of the manuscript: Je-Jung Lee, Woo Joong Rhee, and Jeon-Soo Shin. Administrative, technical, or material support (reporting or organizing data and constructing databases): Je-Jung Lee, Woo Joong Rhee, and In Ho Park. Study supervision: Je-Jung Lee, Woo Joong Rhee, Jeon-Soo Shin FUNDING This work was supported by the grants from the National Research Foundation of Korea (NRF), funded by the Korean government (2019R1A6A1A03032869, 2022R1A2B5B03001446, and RS-2023-00248101), Research Center Program of the Institute for Basic Science (IBS) in Korea (IBS-R026-D1), and Brain Korea 21 FOUR Project of Yonsei Advanced Medical Science Research and Education. A faculty research grant of Yonsei University College of Medicine (No. 6-2022-0115). The Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI23C1413). COMPETING INTERESTS The authors have no conflicting interests to declare. ETHICS The animals used in our study were treated in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) protocol of the Yonsei Medical University. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at……………… Correspondence and requests for materials should be addressed to Jeon-Soo Shin or Je-Jung Lee. Reprints and permission information is available at ……………… Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations DATA AVAILABILITY The data that support the findings of this study are available from the corresponding author on reasonable request. References Coppe JP, Rodier F, Patil CK, Freund A, Desprez PY, Campisi J. Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype. J Biol Chem. 2011;286(42):36396–403. 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Nature. 2017;548(7668):461–5. Basit A, Cho MG, Kim EY, Kwon D, Kang SJ, Lee JH. The cGAS/STING/TBK1/IRF3 innate immunity pathway maintains chromosomal stability through regulation of p21 levels. Exp Mol Med. 2020;52(4):643–57. Blackford AN, Jackson SP. ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. Mol Cell. 2017;66(6):801–17. Yue X, Bai C, Xie D, Ma T, Zhou PK. DNA-PKcs: A Multi-Faceted Player in DNA Damage Response. Front Genet. 2020;11:607428. An J, Huang YC, Xu QZ, Zhou LJ, Shang ZF, Huang B, et al. DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression. BMC Mol Biol. 2010;11:18. Taffoni C, Marines J, Chamma H, Guha S, Saccas M, Bouzid A, et al. DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma. EMBO J. 2023;42(7):e111961. Marechal A, Zou L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harb Perspect Biol. 2013;5(9). Morad G, Helmink BA, Sharma P, Wargo JA. Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell. 2021;184(21):5309–37. Kumari G, Ulrich T, Krause M, Finkernagel F, Gaubatz S. Induction of p21CIP1 protein and cell cycle arrest after inhibition of Aurora B kinase is attributed to aneuploidy and reactive oxygen species. J Biol Chem. 2014;289(23):16072–84. Enriquez-Rios V, Dumitrache LC, Downing SM, Li Y, Brown EJ, Russell HR, et al. DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis. J Neurosci. 2017;37(4):893–905. Ferguson BJ, Mansur DS, Peters NE, Ren H, Smith GL. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. Elife. 2012;1:e00047. Ciszak L, Frydecka I, Wolowiec D, Szteblich A, Kosmaczewska A. CTLA-4 affects expression of key cell cycle regulators of G0/G1 phase in neoplastic lymphocytes from patients with chronic lymphocytic leukaemia. Clin Exp Med. 2016;16(3):317–32. Greenwald RJ, Oosterwegel MA, van der Woude D, Kubal A, Mandelbrot DA, Boussiotis VA, et al. CTLA-4 regulates cell cycle progression during a primary immune response. Eur J Immunol. 2002;32(2):366–73. Yan Q, Zhang B, Ling X, Zhu B, Mei S, Yang H, et al. CTLA-4 Facilitates DNA Damage-Induced Apoptosis by Interacting With PP2A. Front Cell Dev Biol. 2022;10:728771. Zhao H, Wang Y, Yang Z, Wei W, Cong Z, Xie Y. High expression of aurora kinase B predicts poor prognosis in hepatocellular carcinoma after curative surgery and its effects on the tumor microenvironment. Ann Transl Med. 2022;10(21):1168. Lee JJ, Kim SY, Kim SH, Choi S, Lee B, Shin JS. STING mediates nuclear PD-L1 targeting-induced senescence in cancer cells. Cell Death Dis. 2022;13(9):791. Graphical Abstract Graphical Abstract is not available with this version CTLA-4 depletion-induced senescence in cancer. CTLA-4 depletion causes micronuclei formation derived from nuclear instability, which is due to the decrease of Aurora B by CTLA-4 reduction, and in turn, switched on DNA-PKcs by cytosolic DNA. Sequentially, canonical downstream of DNA-PK, STING signaling is activated and triggers AKT pathway, then the senescence executor p53-p21 leads the cells to the senescence, which prevents the tumor growth. Additional Declarations (Not answered) Supplementary Files ReportingSummaryforCDDisLeeetal.docx SourceDataStatisticsLeeetal.xlsx SupplemenatrayInformationLeeetal.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3893509","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":275869283,"identity":"c03cd247-a734-468e-a3ec-845f4d32532b","order_by":0,"name":"Jeon-Soo Shin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBAC9v7jzyR/ttnIsfE3H2BgbCBCC+OMHDOJxGNpxvwSxxKI1cLDJpH47FDizIYcA5K0HGDccODMN2neHXUM/O0HCGjpP/78R+K2O8wGh3u3SfOeOcwgcSaBgJaGBKBftj1jMzhwFqilDWjDDUIOA2uZd5jH4EDOM6CWOgZ5QloEwVr6DktINuSwAbUwMxgQ0iItAQrkvjQDYCAbW85tO8xjSMgvfPyQqKxv429+eONtW52c3PEDBKxBAiwSQIKHePVAwPyBJOWjYBSMglEwYgAAwM1LUk3a1ooAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8294-3234","institution":"Yonsei University","correspondingAuthor":true,"prefix":"","firstName":"Jeon-Soo","middleName":"","lastName":"Shin","suffix":""},{"id":275869284,"identity":"c28e5b5e-8ffb-4d2d-b367-8ca942c34300","order_by":1,"name":"Je-Jung Lee","email":"","orcid":"https://orcid.org/0000-0001-8439-2790","institution":"Yonsei University","correspondingAuthor":false,"prefix":"","firstName":"Je-Jung","middleName":"","lastName":"Lee","suffix":""},{"id":275869285,"identity":"48a59834-4460-4200-9be6-eb388ea78694","order_by":2,"name":"Woo Joong Rhee","email":"","orcid":"https://orcid.org/0000-0001-9690-0553","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Woo","middleName":"Joong","lastName":"Rhee","suffix":""},{"id":275869286,"identity":"a71dfc44-ce67-4775-9a24-3bc91adc1e57","order_by":3,"name":"So Young Kim","email":"","orcid":"https://orcid.org/0000-0003-1166-9853","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"So","middleName":"Young","lastName":"Kim","suffix":""},{"id":275869287,"identity":"348dd5cd-7abc-424c-898c-90c87c19f000","order_by":4,"name":"Jisun Lee Lee","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jisun","middleName":"Lee","lastName":"Lee","suffix":""},{"id":275869288,"identity":"cae64fff-9c0e-4b14-903d-59519994877a","order_by":5,"name":"Su Ful Jung","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Su","middleName":"Ful","lastName":"Jung","suffix":""},{"id":275869289,"identity":"3751fe4c-d4bf-4b28-beda-5a4a93b72873","order_by":6,"name":"In Ho Park","email":"","orcid":"https://orcid.org/0000-0003-2190-5469","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"In","middleName":"Ho","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2024-01-24 08:45:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3893509/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3893509/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51985263,"identity":"6ced9149-5562-4bfa-8a22-13f51ad47ed6","added_by":"auto","created_at":"2024-03-05 01:53:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":369522,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTLA-4-depletion induces senescence in human melanoma cells \u003c/strong\u003eA375 cells were transiently transfected with 100 nM siC or siCTLA-4 one day before treatment with or without 100 ng/ml Dox. Then morphological changes (A; scale bar: 50 mm), SA-b-Gal staining (B, C; Scale bar; 20 mm), cell viability (D), and clone formation (E) were assessed. Western blot analysis was performed with indicated antibodies (F). Confocal laser scanning microscopy assay for evaluating p21 and CTLA-4 expression were performed on day 2 post Dox treatment (G; Scale bars: 20 mm); clonogenic assay was done on day 10 post Dox treatment. The statistical significance among three groups was calculated using a one-way analysis of variance and Newman-Keuls. Quantitative data are expressed as mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure1160X230.png","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/7bd0be0924e03b640a49b746.png"},{"id":51985266,"identity":"6b46ad9e-8b47-461f-8761-0b07487acdee","added_by":"auto","created_at":"2024-03-05 01:53:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":251463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTLA-4-depletion induces senescence in mouse melanoma cells. \u003c/strong\u003eB16-F10 cells were transiently transfected with 100 nM siC or siCTLA-4 one day before treatment with or without 100 ng/ml Dox. Then morphological changes (A; scale bar: 50 μm), SA-β-Gal staining (B; scale bar: 20 μm), cell viability assay (C), western blot analysis (D), and confocal imaging assay for p21 and CTLA-4 (E; scale bar: 20 μm) were performed on day 2 post Dox treatment and quantified (F). The statistical significance among the three groups was calculated using a one-way analysis of variance and Newman-Keuls. Quantitative data are expressed as mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure2135X210.png","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/1eede6ca436a9a8759a9b154.png"},{"id":51985267,"identity":"96689174-8c08-4f73-ba55-4686e0c0b8ac","added_by":"auto","created_at":"2024-03-05 01:53:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":417265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTLA-deficiency induces senescence via the AKT pathway. \u003c/strong\u003eA375 cells were treated with 100 nM siC or siCTLA-4 one day before treatment with or without 100 ng/ml Dox. Western blot (WB) analysis was performed with the indicated antibodies on day 2 (A) and confocal imaging assay for p-AKT (B; scale bars: 20 μm) and p-mTOR (C; scale bar: 20 μm) were performed on day 2 post Dox treatment. B16-F10 cells were treated with 100 nM siC or siCTLA-4 1 day before treatment with or without 100 ng/ml Dox. Then WB was performed with the indicated antibodies on day 2 (D), confocal imaging assay for p-AKT (E; scale bar: 20 μm) and p-mTOR (F; scale bar: 20 μm) were performed on day 2 post Dox treatment.\u003c/p\u003e","description":"","filename":"OnlineFigure3170X150.png","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/7b97f09739b80134aee035a9.png"},{"id":51985270,"identity":"b9af7ae9-9f5b-4254-addf-5febb01005a1","added_by":"auto","created_at":"2024-03-05 01:53:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":246607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTLA-4-deficiency induces DNA damage response. \u003c/strong\u003eA375 and B16-F10\u003cstrong\u003e \u003c/strong\u003ecells were transiently transfected with 100 nM siC or siCTLA-4 one day before treatment with or without 100 ng/ml Dox. Then western blot (WB) analysis was performed with the indicated antibodies in B16-F10 (A), confocal imaging assay for γ-H2AX in B16-F10 (B) and A375 cells (C), micronuclei assay and γ-H2AX staining in A375 (D), B16-F10 (E, F), and A375 (G, H) cells were performed on day 2 post Dox treatment. Arrow indicates micronuclei (D, E, G). A375 and B16-F10\u003cstrong\u003e \u003c/strong\u003ecells were treated with 100 nM siC or siCTLA-4 and WB was performed 3 days later (I, J). A375 and B16-F10\u003cstrong\u003e \u003c/strong\u003ecells were treated with 100 nM siC, or siCTLA-4, or overexpressed CTLA-4 one day before Dox treatment and WB was performed on day 2 post Dox treatment (K, M). B16-F10 cells were treated with 100 nM siCTLA-4 and WB was performed at indicated times (L). Scale bars, 20 μm (B-E, G). The statistical significance among three groups was calculated using a one-way analysis of variance and Newman-Keuls. Quantitative data are expressed as mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure4200X190.png","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/01b6dcb33787359599fecc01.png"},{"id":51985268,"identity":"df52af54-de56-4d48-842b-bacaf3ca4c16","added_by":"auto","created_at":"2024-03-05 01:53:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":353005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTING signaling regulates CTLA4-depletion induced via AKT signaling modulation. \u003c/strong\u003eA375 cells transiently transfected with siCTLA-4 in a time dependent manner as indicated, and WB (A) and confocal imaging (B) analysis were carried out with the indicated antibodies. Cells were treated with 100 nM siC or siSTING one day before treatment with or without siCTLA-4. Then western blot (WB) with indicated antibodies in A375 (C) and confocal imaging assay for p-AKT in A375 cells (D) were performed on day 3 post siC or siCTLA-4 transfection. Scale bars, 20 μm (B, D). (S.E.; short exposure, L.E.; long exposure).\u003c/p\u003e","description":"","filename":"OnlineFigure5160X180.png","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/aeb546ec50720141db11bc91.png"},{"id":51985269,"identity":"4593e872-f02c-401f-9a27-967921a08c47","added_by":"auto","created_at":"2024-03-05 01:53:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":485093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA-PK mediates CTLA-4-depletion-induced senescence.\u003c/strong\u003eB16-F10 cells were treated with 100 nM siC or siCTLA-4 one day before treatment with or without 100 ng/ml Dox. Then western blot (WB) with the indicated antibodies (A) and confocal imaging assay for DNA-PK (B) were performed on day 2 post Dox treatment. B16-F10 cells were transiently transfected with 100 nM siC or siDNA-PK one day before treatment with or without siCTLA-4. Then WB with the indicated antibodies (C) was performed on day 3 post siCTLA-4 treatment. A375 cells were treated with 100 nM siC or siCTLA-4 one day before treatment with or without 100 ng/ml Dox. Then WB with indicated antibodies (D) and confocal imaging assayfor DNA-PK (E) were performed on day 2 post Dox treatment. A375 cells were treated with 100 nM siC and DNA-PK one day before treatment with or without 100 nM siCTLA-4 and WB with the indicated antibodies was performed on day 3 post siCTLA-4 treatment (F). A375 cells were treated with Nu7441, a DNA-PK inhibitor, at a low and high dose for 6 h before treatment with or without 100 nM siCTLA-4, and WB with indicated antibodies was performed on day 3 post siCTLA-4 (G). A375 cells were treated with 100 nM siC and DNA-PK one day before treatment with or without 100 nM siCTLA-4 and carried out on day 3 post siCTLA-4 treatment. Next, morphological changes (H) and SA-β-Gal staining (I, J) were performed. A375 cells were transfected with EV or CTLA-4 plasmid one day before Dox treatment, then IP for CTLA-4 and WB using the indicated antibodies was performed on day 2 post Dox treatment (K). Scale bars, 20 μm (B, E), 50 μm (H, I). (S.E.; short exposure, L.E.; long exposure). The statistical significance among the three groups was calculated using a one-way analysis of variance and Newman-Keuls. Quantitative data are expressed as mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure6190X270.png","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/0143523b1a1652caad20b02c.png"},{"id":51985271,"identity":"661eca9c-1b6f-469b-b99e-a57cb2ce21d3","added_by":"auto","created_at":"2024-03-05 01:53:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":464432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnimal experiments. \u003c/strong\u003eTumors were generated by implanting B16-F10\u003csup\u003ewt \u003c/sup\u003eor B16-F10 \u003csup\u003eCTLA-4 KO\u003c/sup\u003e cells into mice. On day 9 after implantation, mice were administered Dox (9 mg/kg) for seven days, and tumors were excised (A). Photo of the autopsied tumors (\u003cem\u003en\u003c/em\u003e = 5) (B). Tumor weight (\u003cem\u003en\u003c/em\u003e = 6) (C), and volume (\u003cem\u003en\u003c/em\u003e = 6) were measured (D). Tumors were cut in half and analyzed by SA-β-Gal staining (\u003cem\u003en\u003c/em\u003e = 3) (E). WB was performed with WT vs KO tumors with or without Dox treatment using the indicated antibodies (n=2) for each group (F). Confocal imaging assay was performed with tumors for individual CTLA-4, H3K9me3, and p-IRF3 (G-I). Scale bars, 20 μm (G-I). Statistical significance was calculated using a one-way analysis of variance and Newman–Keuls. Quantitative data are expressed as mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure7180X185.png","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/6002159e0881026bbc9d2418.png"},{"id":55387039,"identity":"6be49521-aa58-4843-bd6a-044b4448665d","added_by":"auto","created_at":"2024-04-26 14:58:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4515039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/88d10913-d8f4-4d2d-8eea-eefcd9728ff4.pdf"},{"id":51985262,"identity":"d7319bfe-632f-4656-9322-6a7bdf6f323f","added_by":"auto","created_at":"2024-03-05 01:53:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25840,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"ReportingSummaryforCDDisLeeetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/375d7429b82c552e29c9fc50.docx"},{"id":51985265,"identity":"bec27e2a-ee0d-4388-8948-0de2f0e6cd0b","added_by":"auto","created_at":"2024-03-05 01:53:06","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":112898,"visible":true,"origin":"","legend":"","description":"","filename":"SourceDataStatisticsLeeetal.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/3c0b8c826743b108e64c5474.xlsx"},{"id":51985264,"identity":"06030795-4694-4713-8a0b-678861940c27","added_by":"auto","created_at":"2024-03-05 01:53:06","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":951310,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemenatrayInformationLeeetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-3893509/v1/ae8c7b4310b77823c90b06ba.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Targeting CTLA-4 in cancer cells induces senescence via DNA-PKcs-STING-AKT axis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCellular senescence is a permanent cell cycle arrest induced by various stimuli. Senescence is defined by morphological changes, increased senescence-associated β-galactosidase (SA-β-Gal) activity at pH 6.0, and the induced expression of cell cycle checkpoints such as p21, p16, and p27(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The expression of heterochromatin and DNA damage markers such as H3K9 trimethylation and γ-H2AX(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) is also linked to senescence. Senescence can suppress growth of tumors rendered resistant to apoptosis following cancer therapy; this is achieved via blocking cell cycle progression and cell proliferation(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a pivotal player in regulating immune responses, is a immune checkpoint molecule expressed on the T-cell surface and functions as a negative regulator of T-cell proliferation. However, CTLA-4 is also expressed in several cancers (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), and robustly expressed in melanoma (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The evidence of role played by CTLA-4 in tumors has sparked interest in further studies in this field. Generally, CTLA-4 accumulates in intracellular compartments; however, it transitionally moves to the cell surface in response to stimuli and is rapidly internalized(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). However, the function of cytoplasmic CTLA-4 remains unclear. Thus, it is necessary to evaluate the role of CTLA-4 in cancer cells.\u003c/p\u003e \u003cp\u003eThe cyclic GMP\u0026ndash;AMP synthase (cGAS)- stimulator of the interferon genes (cGAS-STING) signaling pathway is an important mediator of inflammation, cellular stress, tissue damage, and senescence(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Presence of DNA in the cytosol due to several reasons such as infection, separation from self-DNA damage, or slippage during replication, activates cGAS and brings it to the adaptor protein STING, then recruits TANK-binding kinase 1 (TBK1). TBK1 phosphorylates STING and interferon regulatory factor 3 (IRF3). IRF3 dimerizes and localizes in the nucleus, inducing type I interferons(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). STING signaling plays a pivotal role in senescence and can be targeted for suppressing tumors via senescence induction.\u003c/p\u003e \u003cp\u003eThe serine/threonine kinase AKT enhances the expression of p53 and p21, increases cell size, and induces senescence(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Persistent activation of AKT results in cellular senescence, a tumor-suppressive mechanism. Additionally, revelation of the AKT and STING pathway interaction has expanded the role of AKT and STING-related functions in senescence(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a sensor for DNA double-strand breaks (DSB), is involved in non-homologous end joining (NHEJ) and DNA damage repair (DDR) pathways. DNA-PKcs also act in STING-dependent and -independent pathways for intracellular nucleic acid recognition. Although DNA-PKcs primarily functions in the nucleus, its nonnuclear function of maintaining genomic integrity and DNA fidelity (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In addition, DNA-PKcs kinase plays critical role in many diseases including cancers as it has been associated with various cellular processes, such as cell death, division, senescence, and metabolism(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Along with its conventional role in the DNA damage response, involvement of DNA-PKcs in STING-related pathway can bring an important paradigm shift in cancer therapy(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we aimed to determine the role of CTLA-4 in cancer. We verified the effect of silencing CTLA-4 in human and mouse melanoma cells and found that CTLA-4 depletion induced senescence. This was achieved via a signaling cascade involving genomic instability induced DNA damage, which subsequently activates DNA-PKcs-STING-AKT-p21 in human and mouse melanoma cells, eventually leading to tumor regression. To the best of our knowledge, this is the first study to report a conclusive role of CTLA-4 in cancer cell senescence. These findings can help to better understand and develop strategies for cancer therapy via CTLA-4 targeting.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eCTLA-4 depletion induces senescence in melanoma cells\u003c/b\u003e To investigate the role of CTLA-4 in cancer, we silenced CTLA-4 either alone or in combination with doxorubicin (Dox) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Human melanoma cell line A375 showed higher expression of CTLA-4 than other cells (Supplementary Fig.\u0026nbsp;1a-d)(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Unexpectedly, CTLA-4 silencing resulted in senescence phenotype, including an increase in cell size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b), SA-β-Gal activity proven by senescent green probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c), and decreased cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) and colony forming assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). In addition, western blot (WB) analysis showed increased expression of senescence markers such as p21 and p16, and the heterochromatin marker, H3K9me3, compared to the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Moreover, confocal imaging showed that p21 expression inversely correlated with that of CTLA-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). These effects were synergistic in both the siCTLA-4 and Dox-treated group compared to the Dox(only)-treated group. Additionally, results of fractionation assay confirmed that CTLA-4 was predominantly expressed in the cytosol (Supplementary Fig.\u0026nbsp;1e, f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe confirmed this with B16-F10 mouse melanoma cells which expressing CTLA-4 well (Supplementary Fig.\u0026nbsp;1b). We silenced CTLA-4 alone or in combination with Dox in B16-F10 cells. Depletion of CTLA-4 alone resulted in increased cell size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b), SA-β-Gal activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and decreased cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Furthermore, p21, p16, and H3K9me3 protein levels increased, as shown by WB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Moreover, p21 expression was inversely correlated with that of CTLA-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). Notably, all these effects were more significant in the siCTLA-4 and Dox combination group than in the Dox(only)-treated group. We also repeated these experiments using a different CTLA-4 specific siRNA (siCTLA-4\u003csup\u003e*\u003c/sup\u003e) (Supplementary Fig.\u0026nbsp;2) and the results were similar to those obtained from the siRNA sequence used in A375 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Taken together, our data suggest that targeting CTLA-4 in cancer cells induces senescence and halts cancer cell proliferation, and Dox treatment enhances these outcomes synergistically.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAKT pathway is required to induce CTLA-4-deficiency-induced-senescence\u003c/b\u003e As a well-known component of CTLA-4 related signaling, the AKT pathway is activated through the surface CTLA-4 signaling pathway(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Therefore, we investigated the status of the AKT pathway and its role in CTLA-4 deficiency-induced senescence. Ironically, phospho(p)-AKT was also upregulated along with other senescence markers, including p53, p21, p27, p16, and H3K9me3, in both CTLA-4 alone silenced and Dox combination groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). AKT pathway-mediated senescence is known as oncogene-induced senescence (OIS)(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In addition, confocal imaging showed increased expression of p-AKT and p-mTOR, molecules acting downstream of AKT, in siCTLA-4 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). These experiments were performed in mouse melanoma cells and similar results were obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f). A recent study reported an 80 kDa splicing mTOR isoform, mTORβ, which is considered an active protein kinase beyond full-length mTOR (mTORα)(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). It showed an expression pattern similar to that of p-AKT in both A375 and B16-F10 cells (Supplementary Fig.\u0026nbsp;3a, b). However, CTLA-4 overexpression blocked Dox treatment -induced senescence phenotype, including morphological changes and p21, p-AKT, and p-mTOR expression in B16-F10 as well as A375 cells (Supplementary Fig.\u0026nbsp;4a-d). Taken together, we conclude that the AKT pathway is required for CTLA-4-deficiency-induced senescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCTLA-4 deficiency causes DNA damage response\u003c/b\u003e DNA damage is one of the dominant causes or consequences of senescence(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). So, we examined the DNA damage marker γ-H2AX, and found it was upregulated and inverse correlation with CTLA-4 expression in CTLA-4-silenced B16-F10 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In addition, other DNA damage markers H3K9me3 and p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) were elevated along with other senescence markers. γ-H2AX expression was validated by confocal imaging in B16-F10 and A375 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-d). These results were confirmed in CTLA-4 knockout (KO) B16-F10 cells (Supplementary Fig.\u0026nbsp;5a). CTLA-4 KO cells showed higher sensitivity to the anticancer drugs cisplatin and Dox (Supplementary Fig.\u0026nbsp;5b, c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMicronuclei are tiny nuclear DNA, and often observed in cancer and senescence. Senescence often appears with aneuploidy induced by chromosome missegregation, which triggers the formation of cytoplasmic chromatin fragments (CCFs), resulting in and they become micronuclei(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). We observed micronuclei following CTLA-4 silencing, which is colocalized with γ-H2AX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed: enlarged part of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and their content was much higher in the Dox-co treated group in mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f) as well as human melanoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, h). Interestingly, micronuclei co-localized with γ-H2AX in the CTLA-4 silenced cells, confirming that micronuclei are a byproduct of DNA damage caused by genome instability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In addition, Aurora B, which prevents micronuclei formation and whose downregulation induces senescence(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), was downregulated by siCTLA-4 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei-l, Supplementary Fig.\u0026nbsp;5d ) and this effect was reverted by CTLA-4 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em). These results imply that CTLA-4 depletion induced genomic instability and DNA damage response.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSTING signaling regulates CTLA-4-depletion-induced senescence via modulating AKT signaling pathway\u003c/b\u003e Micronuclei present in the cytosol, trigger cGAS-STING pathway(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). cGAS binds to dsDNA in micronuclei and triggers cyclic GMP-AMP (cGAMP) synthesis. Subsequently, cGAMP attaches to STING and recruits TBK1, which in turn phosphorylates STING. Phosphorylated STING recruits interferon regulatory factor-3 (IRF3), which is then phosphorylated by TBK1 and dimerizes, followed by its nuclear translocation. Eventually, IRF3 transcribes genes encoding proteins including interferons and cytokines in the nucleus(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, we examined the effects of micronuclei on the STING pathway in the CTLA-4 silenced group. p-STING and p-IRF3 increased in a time-dependent manner, along with p-AKT and p-p53 (S15) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Furthermore, γ-H2AX expression correlated with CTLA-4 depletion time points in both WB and confocal imaging assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). However, these effects were reversed by siSTING treatment. Namely, activated AKT signaling as well as the elevation of γ-H2AX by CTLA-4 silencing, were blocked by siSTING treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Notably, p-AKT expression was also observed in the confocal imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) and these findings were confirmed in B16-F10 cells by WB and confocal imaging (Supplementary Fig.\u0026nbsp;6a-d). Overall, our results indicated that CTLA-4 deficiency potentiates DNA damage-induced STING signaling mediated via AKT signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA-PKcs intervenes with CTLA-4-depletion-induced senescence\u003c/b\u003e DNA-PKcs belongs to the phosphatidylinositol 3-kinase family. DNA-PKcs forms the active DNA-PK holoenzyme with the Ku80/Ku70 heterodimer to regulate DDR following DSB. DNA-PKcs rapidly moves to the damaged sites and is activated. It then sends damage signals via p53, ultimately leading to cell cycle arrest, aging, or apoptosis. Aside from its role in the cell cycle(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), DNA-PKcs keeps genome integrity(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Furthermore, DNA-PKcs recognizes cytosolic DNA and activates the cGAS-STING pathway (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNext, we examined whether DNA-PKcs plays a role in CTLA-4-depletion-induced senescence. DNA-PKcs was upregulated together with activated STING pathway (p-STING and p-IRF3), AKT signaling markers (p-AKT and p-mTOR), and p-p53 (S15) in the CTLA-4-depleted and Dox-combined group of B16-F10 cells compared to the control and Dox-(only)-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Additionally, DNA-PKcs was activated (p-DNA-PKcs) by siCTLA-4 in A375 cells (Supplementary Fig.\u0026nbsp;7a, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSurprisingly, all activated signals, including those of the STING-AKT pathway (p-STING and p-mTOR), CDK inhibitor (p21), and DNA damage marker (γ-H2AX) following siCTLA-4 treatment of B16-F10 cells were abolished by siDNA-PKcs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), and these results were confirmed in A375 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-g). DNA-PKcs expression was elevated together with those of p-IRF3, p-STING, and p-AKT in both CTLA-4 silenced and Dox-cotreated with CTLA-4 silenced groups compared to control and only Dox-treated groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). However, the STING pathway (p-STING and p-TBK1), AKT signaling factors (p-AKT and p-mTOR), cell cycle inhibitors (p21 and p27), and the DNA damage marker γ-H2AX activated by siCTLA-4 were blocked by siDNA-PKcs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef) or the specific DNA-PKcs inhibitor Nu7441 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Interestingly, targeting only DNA-PKcs via silencing or inhibition showed a different manner compared to that observed following co-treatment with the CTLA-4 silencing group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, f, and g). Namely, targeting DNA-PKcs alone elevated AKT and p21 levels as well as STING pathway (2nd lane of 6C, 3rd lane of 6F, and 2nd -3rd lane of 6G), which may be attributed to the lack of DNA repair following DNA-PKcs silencing or inhibition(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). DNA-PKcs abrogation downregulates the protein expression induced by CTLA-4 silencing, which may be due to the absence of a DNA-PKcs signaling in the STING pathway (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Indeed, siDNA-PKcs almost abrogated CTLA-4 depletion-led senescence in A375 cells, evidenced by morphological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh) and the degree of senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei, j), indicating that DNA-PKcs is essential for CTLA-4-depletion induced senescence.\u003c/p\u003e \u003cp\u003eTo confirm the selectivity of DNA-PKcs for CTLA-4 depletion-induced senescence, we compared DNA-PKcs with ataxia telangiectasia mutated (ATM), which has also been implicated in DSBs(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). The ATM-specific inhibitor KU-60019 showed no influence CTLA-4 depletion effect; however, DNA-PKcs inhibition by NU7441 or siRNA knockdown abrogated the CTLA-4 depletion effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, Supplementary Fig.\u0026nbsp;7a, b). These results suggest that the effects of CTLA-4 are selectively linked with DNA-PKcs. Furthermore, immunoprecipitation (IP) assay revealed an interaction between CTLA-4 and DNA-PKcs after CTLA-4-overexpression in A375 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek). In fact, CTLA-4 was predominantly located in the cytosol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, Supplementary Fig.\u0026nbsp;1e, f), along with DNA-PKcs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and e), which can facilitate their interaction to detect cytosolic nucleic acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek). To further investigate the correlation of CTLA-4 with DNA-PKcs, we used The Cancer Genome Atlas (TCGA) data, and observed that the mRNA expression of \u003cem\u003ePRKDC\u003c/em\u003e, which encodes DNA-PKcs, was negatively correlated with that of CTLA-4 (Fig. S7c). Furthermore, patients were categorized into high- (top 1/3) and low- (bottom 1/3) CTLA-4 groups based on the CTLA-4 expression level and showed an inverse correlation with non-homologous end joining pathway components (NHEJ), including DNA-PKcs, in patients with skin cutaneous melanoma (Supplementary Fig.\u0026nbsp;7d). Additionally, these features were observed in lung, cervical, head, and neck squamous cell carcinomas (Supplementary Fig.\u0026nbsp;7e). Taken together, DNA-PKcs plays an indispensable role and orchestrates CTLA-4-depletion-induced senescence of cancer cells via modulating the STING-AKT pathway axis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCTLA-4-depletion impedes tumor growth via senescence induction\u003c/b\u003e Finally, we performed experiments in mice to verify whether CTLA-4-depletion affected tumor growth. We generated a CTLA-4 knockout (KO) B16-F10 cell line using the CRISPR-Cas9 system. Next, C57BL/6 mice were subcutaneously injected with 5x10\u003csup\u003e5\u003c/sup\u003e B16-F10\u003csup\u003eWT\u003c/sup\u003e and B16-F10\u003csup\u003eCTLA\u0026thinsp;\u0026minus;\u0026thinsp;4 KO\u003c/sup\u003e cells followed by intraperitoneal injections of 9 mg/kg Dox. The tumors were collected on day 16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSize, weight, and volume of tumors derived from mice injected with CTLA-4 KO cells, were much smaller than those of tumors derived from mice injected with wild-type (WT) cells in both DMSO- and Dox-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-d). Furthermore, SA-β-Gal staining of B16-F10\u003csup\u003eCTLA\u0026thinsp;\u0026minus;\u0026thinsp;4 KO\u003c/sup\u003e derived tumors was much stronger than that of B16-F10\u003csup\u003eWT\u003c/sup\u003e derived tumors both with and without Dox treatment, confirming the effect of CTLA-4 on tumor cell senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). B16-F10\u003csup\u003eCTLA\u0026thinsp;\u0026minus;\u0026thinsp;4 KO\u003c/sup\u003e derived tumors showed higher expression of γ-H2AX and p-STING, than that in B16-F10\u003csup\u003eWT\u003c/sup\u003e derived tumors in both the absence and presence of Dox treatment groups by WB analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef), which was later confirmed by IHC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-i). In conclusion, once CTLA-4 depletion occurs in cancers, it causes cellular senescence via DNA damage-DNA-PKcs-STING-AKT-p21 pathway, and eventually leads to tumor suppression.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIt is important to understand the role of CTLA-4 in cancer cells, beyond in T cells, to allow targeted cancer therapy. This is the first study to demonstrate a distinctive role of CTLA-4 in cancer.\u003c/p\u003e \u003cp\u003eThe governing the immune checkpoints is a promising approach for cancer immunotherapy. Immune checkpoints PD-1, PD-L1, and CTLA-4 are the main targets for current therapeutic approaches. Although some inhibitors that target immune checkpoints expressed on the cell surface have emerged as effective candidates for certain tumors, their efficacy remains unsatisfactory. Therefore, many research groups have attempted to develop better strategies(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we silenced the expression of CTLA-4 in human and mouse melanoma cells and observed its effects. Surprisingly, CTLA-4 silencing caused senescence, evidenced by larger and flattened morphological changes, decreased cell proliferation, increased SA-β-Gal activity, induction of cell cycle checkpoints p21 and p16, and upregulation of DNA damage and heterochromatin markers γ-H2AX and H3K9me3, respectively. Next, we elucidated the mechanism underlying senescence induced by CTLA-4 depletion. CTLA-4 deficiency in melanoma cells affected genomic stability and induced micronuclei formation via downregulating Aurora B expression(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). This was sensed by DNA-PKcs, which in turn activated the STING-AKT signaling pathway and mediated senescence via p53 and p21.\u003c/p\u003e \u003cp\u003eOur study highlights the atypical role of DNA-PKcs in CTLA-4 depletion-induced senescence. Canonically, DNA-PKcs serves as a classic component of DSB-induced DDR and maintains genome integrity(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). However, in cytoplasm, DNA-PKcs acts as a DNA sensor and activates innate immunity. DNA-PKcs attaches to the cytoplasmic DNA and activates the STING pathway via STING-TBK1-IRF3 activation(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Our results showed that DNA-PKcs triggers the STING pathway, confirmed by DNA-PKcs silencing. This is consistent with a recent report in which the specific DNA-PKcs inhibitor NU7441 suppressed the activation of the STING pathway by stimulation with double-stranded DNA(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). We successfully blocked the STING pathway activated by CTLA-4 deficiency via silencing DNA-PK in both human and mouse melanoma cells.\u003c/p\u003e \u003cp\u003eAlthough ATM is a major component of the DNA damage response pathway, it is not involved in CTLA-4 depletion-induced senescence. In contrast, DNA-PKcs functions selectively to modulate the STING-AKT pathway required for CTLA-4 depletion-induced senescence, which was confirmed by publicly available data for patients with cervical, lung, head, and neck cancers, as well as melanoma. Furthermore, a relationship between STING and the AKT pathway has been reported, in which AKT regulates the STING pathway(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In the present study, STING regulated the AKT pathway as confirmed by the observation that phosphorylation of AKT and mTORC1 were downregulated by STING silencing. Eventually, the STING-AKT pathway axis was compromised by DNA-PK depletion, indicating that DAN-PK orchestrates CTLA-4-depletion-induced senescence by regulating the STING-AKT pathway. Taken together, we found that senescence is the dominant feature of the response of CTLA-4-depleted-melanoma cells, and is mediated by the DNA-PKcs-STING-AKT-p53-p21 axis.\u003c/p\u003e \u003cp\u003eWhile several studies have reported that CTLA-4 silencing inhibits cellular proliferation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) and CTLA-4 causes apoptosis in cancer cells(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), our study is the first one to report the role of CTLA-4 as a senescence regulator in cancer. We reveal that mechanistically, CTLA-4-deficiency activates the DNA-PKcs/ STING pathway via downregulating Aurora B. Notably, a recent report suggested the positive correlation between CTLA-4 and Aurora B in an HCC patient(\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). To extend these findings, we investigated whether lack of CTLA-4 promotes DNA damage and found that the DNA damage marker γH2AX was significantly upregulated in the B16-F10\u003csup\u003eCTLA\u0026thinsp;\u0026minus;\u0026thinsp;4 KO\u003c/sup\u003e cells compared to B16-F10\u003csup\u003eWT\u003c/sup\u003e cells when treated with anti-cancer drugs such as cisplatin and Dox; these observations strongly supports our hypothesis that lack of CTLA-4 enhances DDR and steers cells toward senescence by activating STING pathway.\u003c/p\u003e \u003cp\u003eAlthough the mechanism by which CTLA-4 deficiency induces genomic instability and mechanisms underlying STING-led activation of the AKT pathway need to be verified in future studies, our study revealed the role of CTLA-4 in senescence and contributes to a better understanding of CTLA-4-targeting-mediated therapy for cancers. Collectively, our results reveal that targeting CTLA-4 in cancer cells is a potential therapeutic strategy for cancer.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture, transfection, and reagents\u003c/h2\u003e \u003cp\u003eA375 human- and B16-F10 mouse-melanoma cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s medium (DMEM). CCRF-CEM, PEER, MOLT-4 human T cells were grown in RPMI-1640. All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The CLLA-4 plasmid was obtained from Sino Biological. Plasmids and siRNA transfections were performed using FuGene HD reagent and RNAiMAX, respectively, as recommended by the manufacturers. siRNA duplexes against human CTLA-4\u003csup\u003e1ST\u003c/sup\u003e, CTLA-4\u003csup\u003e2ND\u003c/sup\u003e, DNA-PK, human and mouse STING, AKT, and control siRNA were purchased from Bioneer, Inc. siRNA duplexes against mouse CTLA-4, DNA-PK were obtained from Santa Cruz Biotechnology. Neutralizing anti-CTLA4 mouse antibody were purchased from BioLegend. and-Dox was obtained from Calbiochem. Nu 7441 was purchased from Selleck Chemicals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWB analysis\u003c/h2\u003e \u003cp\u003eFor western blot analysis, we followed the protocol described in (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against p-IRF3, STING, p-TBK1, p-AKT, AKT, p-mTOR, mTOR, β-actin, tri-H3K9, p53(S15), secondary antibodies for immunofluorescence from Cell Signaling Technologies, Inc. DNA-PKcs, γ-H2AX, p16 from Abcam. p21, p27from BD Biosciences. CTLA-4 from Proteintech, p53 from Santa Cruz Biotechnology, p-STING from Affnit were used. Antibody-antigen complexes were detected using HRP-conjugated secondary antibodies and visualized using a standard chemiluminescence method according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoprecipitation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e150 ug of each protein lysate was incubated with the indicated antibodies, normal rabbit IgG or normal mouse IgG for 4 h at 4\u0026deg;C, followed by an incubation with 20 \u0026micro;l of protein A magnetic beads for 16 h at 4\u0026deg;C. The immune complexes were analyzed by western blot analyses with the indicated antibodies. Protein lysates were also subjected to western blot analyses with the indicated antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell morphology analysis and SA-β-Gal staining\u003c/h2\u003e \u003cp\u003eCellular morphologies were photographed using an inverted phase-contrast microscope. SA-β-Gal staining was carried out as recommended by the manufacturers (CELLEvent\u0026trade; Senescence Green Detection Kit). Those examinations were performed on day 3 following each treatment unless otherwise indicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eFor immunofluorescence analysis, we followed the protocol described in (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMouse experiment\u003c/h2\u003e \u003cp\u003eAll animal procedures were approved by the Institutional Animal Care and Use Committee (IRB no. 2019\u0026thinsp;\u0026minus;\u0026thinsp;0242). We generated tumors with 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e B16-F1O WT and CTLA-4 KO tumors, and followed our previous procedures in (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Photographs were acquired in randomly chosen fields per tumor section according to standard procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism. The significance of the statistical differences among three or more groups was calculated using one-way analysis of variance and the Newman-Keuls test. Data is shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Asterisks denote the \u003cem\u003ep\u003c/em\u003e-values as follows: *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: Je-Jung Lee,\u003csup\u003e\u0026nbsp;\u003c/sup\u003eWoo Joong Rhee,\u0026nbsp;Jeon-Soo Shin. Development of methodology: Je-Jung Lee,\u003csup\u003e\u0026nbsp;\u003c/sup\u003eWoo Joong Rhee, So Young Kim. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Je-Jung Lee, So Young Kim, Jisun Lee. In Ho Park.\u0026nbsp;Jisun Lee, Su Ful Jung. Analysis and interpretation of data (statistical analysis, biostatistics, and computational analysis): Je-Jung Lee,\u0026nbsp;Woo Joong Rhee, In Ho Park,\u0026nbsp;and Jeon-Soo Shin. Writing, review, and/or revision of the manuscript: Je-Jung Lee,\u0026nbsp;Woo Joong Rhee, and Jeon-Soo Shin. Administrative, technical, or material support (reporting or organizing data and constructing databases): Je-Jung Lee,\u0026nbsp;Woo Joong Rhee,\u0026nbsp;and In Ho Park.\u003c/p\u003e\n\u003cp\u003eStudy supervision: Je-Jung Lee, Woo Joong Rhee, Jeon-Soo Shin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the grants from the National Research Foundation of Korea (NRF), funded by the Korean government (2019R1A6A1A03032869, 2022R1A2B5B03001446, and RS-2023-00248101), Research Center Program of the Institute for Basic Science (IBS) in Korea (IBS-R026-D1), and Brain Korea 21 FOUR Project of Yonsei Advanced Medical Science Research and Education. A faculty research grant of Yonsei University College of Medicine (No. 6-2022-0115). The Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (grant number : HI23C1413).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicting interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animals used in our study were treated in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) protocol of the Yonsei Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary material available at\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Jeon-Soo Shin or Je-Jung Lee.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permission information\u003c/strong\u003e is available at \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note\u003c/strong\u003e Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCoppe JP, Rodier F, Patil CK, Freund A, Desprez PY, Campisi J. 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Cell Death Dis. 2022;13(9):791.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Graphical Abstract","content":"\u003cp\u003eGraphical Abstract is not available with this version\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCTLA-4 depletion-induced senescence in cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCTLA-4 depletion causes micronuclei formation derived from nuclear instability, which is due to the decrease of Aurora B by CTLA-4 reduction, and in turn, switched on DNA-PKcs by cytosolic DNA. Sequentially, canonical downstream of DNA-PK, STING signaling is activated and triggers AKT pathway, then the senescence executor p53-p21 leads the cells to the senescence, which prevents the tumor growth.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CTLA-4, Senescence, Cancer therapy, STING, AKT","lastPublishedDoi":"10.21203/rs.3.rs-3893509/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3893509/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmune checkpoints such as cytotoxic T-lymphocyte associated protein 4 (CTLA-4), PD-1, and PD-L1 have been targeted for cancer therapy. However, the efficacy of this treatment approach remains limited. Beyond its expression on the T-cell surface, CTLA-4 is also expressed in various cancer cells and plays roles in cell proliferation, metastasis, and apoptosis. Here, we reveal that targeting CTLA-4 in melanoma cells leads to genomic instability and DNA-PKcs-STING-AKT pathway activation (via p53 and p21), which in turn blocks cell proliferation and induced senescence. Notably, DNA-PKcs orchestrates CTLA-4-depletion-induced senescence via the STING pathway regulation. To the best of our knowledge, this is the first study to report CTLA-4 leads senescence via micronuclei induction, which triggers DNA-PKcs and eventually suppresses cancer growth. These findings provide a better understanding of the mechanisms underlying CTLA-4 targeting-cancer therapy and future treatment strategies.\u003c/p\u003e","manuscriptTitle":"Targeting CTLA-4 in cancer cells induces senescence via DNA-PKcs-STING-AKT axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-05 01:53:00","doi":"10.21203/rs.3.rs-3893509/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f4db59a5-6aa8-4840-9119-14e3d03a3066","owner":[],"postedDate":"March 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29087089,"name":"Biological sciences/Cell biology/Senescence"},{"id":29087090,"name":"Biological sciences/Cancer/Cancer prevention"},{"id":29087091,"name":"Health sciences/Diseases/Cancer/Cancer prevention"},{"id":29087092,"name":"Biological sciences/Molecular biology/DNA damage and repair/DNA damage response"},{"id":29087093,"name":"Biological sciences/Cell biology/Cell growth/TOR signalling"}],"tags":[],"updatedAt":"2024-04-26T14:50:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-05 01:53:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3893509","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3893509","identity":"rs-3893509","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}