Induced dual-target rebalance simultaneously enhances efficient therapeutical efficacy in tumors

Zhang, Xiaoyu; Ding, Tianyi; Yang, Fan; Xu, Haowen; Zhang, Jixing; Bai, Yiran; Shi, Yibing; Yang, Jiaqi; Chen, Chaoqun; Zhang, He

doi:10.1038/s41420-024-02018-y

Induced dual-target rebalance simultaneously enhances efficient therapeutical efficacy in tumors

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Published: 23 May 2024

Volume 10, article number 249, (2024)
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Cell Death Discovery

Induced dual-target rebalance simultaneously enhances efficient therapeutical efficacy in tumors

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Xiaoyu Zhang^1,2,3^na1,
Tianyi Ding^1,2,3^na1,
Fan Yang^1,2,3,
Haowen Xu^1,2,3,
Jixing Zhang^1,2,3,
Yiran Bai^1,2,3,
Yibing Shi^1,2,3,
Jiaqi Yang^1,2,3,
Chaoqun Chen^1,2,3 &
…
He Zhang ORCID: orcid.org/0000-0001-9954-4451^1,2,3

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Abstract

Multiple gene abnormalities are major drivers of tumorigenesis. NF-κB p65 overactivation and cGAS silencing are important triggers and genetic defects that accelerate tumorigenesis. However, the simultaneous correction of NF-κB p65 and cGAS abnormalities remains to be further explored. Here, we propose a novel Induced Dual-Target Rebalance (IDTR) strategy for simultaneously correcting defects in cGAS and NF-κB p65. By using our IDTR approach, we showed for the first time that oncolytic adenovirus H101 could reactivate silenced cGAS, while silencing GAU1 long noncoding RNA (lncRNA) inhibited NF-κB p65 overactivation, resulting in efficient in vitro and in vivo antitumor efficacy in colorectal tumors. Intriguingly, we further demonstrated that oncolytic adenoviruses reactivated cGAS by promoting H3K4 trimethylation of the cGAS promoter. In addition, silencing GAU1 using antisense oligonucleotides significantly reduced H3K27 acetylation at the NF-κB p65 promoter and inhibited NF-κB p65 transcription. Our study revealed an aberrant therapeutic mechanism underlying two tumor defects, cGAS and NF-κB p65, and provided an alternative IDTR approach based on oncolytic adenovirus and antisense oligonucleotides for efficient therapeutic efficacy in tumors.

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Introduction

As a multigene disease, malignant tumors are usually accompanied by the activation of multiple oncogenes and the inactivation of various tumor suppressors [1]. Conventional single-target therapies primarily target one signaling pathway, however, tumor cells frequently acquire resistance by activating alternative pathways [2]. In lung adenocarcinoma, KRAS mutations accompanied by Stk11/Lkb1 deletion were found to promote resistance to PD-1/PD-L1 inhibitors [3]. Consequently, multitargeted therapies are emerging as a novel trend in the field of cancer therapeutics aimed at simultaneously targeting multiple signaling pathways [4, 5]. For instance, in early-stage melanoma, the pathologic response rate for PD1 therapy was only 30–33%, whereas the pathologic response rate for CTLA4 + PD1 combined therapy was 71–80% [6, 7]. In addition, drugs that simultaneously target EGFR and HER2 have been used in pancreatic ductal adenocarcinoma and have shown highly efficient antitumor effects [8]. Therefore, concurrently targeting multiple tumor defects is an effective and attractive strategy for tumor therapy.

Cyclic GMP-AMP synthase (cGAS) is located in the cytoplasm and recognizes abnormal DNA in cellular compartments, catalyzing the synthesis of cyclic GMP-AMP (cGAMP) by GTP and ATP [9]. The activation of cGAS plays a major role in preventing early neoplastic progression by upregulating the expression of various inflammatory genes, including Type 1 IFNs [10]. However, cGAS silencing suppresses the innate immune response and enhances tumor susceptibility [11]. In colorectal tumors, cGAS deficiency impairs the intestinal epithelial barrier and exacerbates inflammation, leading to a more severe stage of tumor malignancy [12, 13]. On the other hand, NF-κB is another critical transcription factor involved in controlling DNA transcription, cytokine production and cell survival [14]. NF-κB p65 is the core subunit of the NF-κB complex. Overactivation of NF-κB p65 promotes tumorigenesis and is common in most tumors [15]. In non-small cell lung cancer (NSCLC), overactivation of NF-κB p65 binds to the PD-L1 promoter and activates PD-L1 transcription, leading to immune escape [16]. It is common for cGAS inactivation and NF-κB p65 overactivation to occur simultaneously in tumors. Therefore, simultaneous reactivation of silenced cGAS and inhibition of NF-κB p65 may be a potential and interesting strategy for antitumor therapy.

Recently, human DNA and RNA viruses have been shown to be involved in the activation of host genes [17]. For instance, Rta (KSHV) induces IL10 expression through interactions with the host transcription factors SP1 and SP3 [18]. The viral bZIP transcription factor Zta directly reprograms cellular gene expression through distal elements and promotes transcription of TGFB1 genes [19]. These studies suggest that reactivation of silenced cGAS via a virus-oriented method could be an interesting approach. On the other hand, studies have reported that NKILA lncRNA could be involved in inhibiting NF-κB protein activity by interfering with the NF-κB/Snail signaling pathway in NSCLC cells [20]. Thus, inhibiting the overactivation of NF-κB p65 by using a lncRNA-oriented method is likely to be an alternative avenue for treating tumors. Therefore, we further propose the use of induced dual-target rebalance (IDTR) to simultaneously correct cGAS and NF-κB p65 abnormalities by using viral and lncRNA-oriented combinations to improve overall antitumor efficacy.

In this study, we successfully established a novel, efficient antitumor approach for IDTR based on the combination of oncolytic adenovirus and GAU1 lncRNA. We further demonstrated an alternative therapeutic mechanism underlying cGAS and NF-κB p65 abnormalities and identified a potential and interesting antitumor therapeutic strategy.

Results

Identifying tumor models for simultaneously correcting cGAS and NF-κB p65 abnormalities

To validate our hypothesis, we initially established a tumor model with both cGAS and NF-κB p65 abnormalities. We selected two representative human colorectal cancer cell lines, SW620 and LOVO, to detect the expression of cGAS and NF-κB p65. As expected, we observed that the expression of cGAS was significantly lower in SW620 and LOVO tumor cells than in normal enterocytes NCM460 (Fig. 1A). Western blot analysis also revealed that cGAS was expressed at low levels in two colorectal cancer cell lines (Fig. 1B, Lanes 2–3 and Original full length western blots image 1). Furthermore, we examined NF-κB p65 expression. As expected, real-time PCR analysis indicated a remarkable increase in the RNA level of NF-κB p65 in SW620 and LOVO tumor cells compared to NCM460 control cells (Fig. 1C). These findings were further confirmed by western blot analysis, which revealed elevated levels of NF-κB p65 protein in SW620 and LOVO tumor cells (Fig. 1D, Lanes 2–3 and Original full length western blots image 1). Taken together, these data indicate that cGAS silencing and NF-κB p65 overactivation occurred simultaneously in SW620 and LOVO tumor cells. We next used these two types of tumor cells as our tumor models for testing the IDTR strategy.

The oncolytic adenovirus H101 significantly activated cGAS expression

Next, we attempted to reactivate silenced cGAS by using the oncolytic adenovirus H101 in SW620 and LOVO tumor cells. After infecting tumor cells with a multiplicity of infection (MOI) of 100 for 72 hours, we conducted real-time PCR experiments to investigate the gene expression of cGAS. As expected, we observed significant upregulation of the RNA level of cGAS compared to that in the normal group. Specifically, cGAS expression in SW620 and LOVO cells increased 3.9-fold and 3.5-fold, respectively, after H101 treatment (Fig. 2A). We also utilized western blot techniques to assess alterations in the expression of the cGAS protein. After 72 hours of exposure to H101, the tumor cells exhibited a substantial increase in the cGAS protein level compared to that in the control group (Fig. 2B and Original full length western blots image 2). These findings indicate that oncolytic adenovirus H101 can effectively reactivate silenced cGAS in SW620 and LOVO tumor cells.

**Fig. 2: H101 activates cGAS expression and *GAU1* lncRNA inhibits over-expression of NF-κB p65.**

GAU1 can inhibit the overexpression of NF-κB p65

We further explored a lncRNA-oriented method to specifically inhibit the overexpression of NF-κB p65 in tumors. GAU1 is a long noncoding RNA that we discovered and named during our early research, and we aimed to further explore its potential in regulating NF-κB p65. We found that the GAU1 lncRNA was highly expressed in SW620 and LOVO tumor cells (Fig. 2C). To elucidate the regulatory role of GAU1 lncRNA in NF-κB p65 transcriptional regulation, we utilized CRISPR/dCas9-KRAB technology to design a gene knockdown system without deleting genomic DNA. We employed two specific sgRNAs to target SW620 and LOVO cells and successfully reduced GAU1 expression to a range of 6.3–13.7% of the original GAU1 level. Specifically, GAU1 expression in LOVO cells decreased to 9.2–13.7% of its original level, while GAU1 expression decreased to 6.3–10.5% of its original level in SW620 cells (Fig. 2D). Subsequently, we conducted real-time PCR analysis to assess the expression of NF-κB p65 following GAU1 knockdown. Intriguingly, we observed substantial 66.4% and 66.6% reductions in NF-κB p65 expression after GAU1 knockdown in LOVO and SW620 cells, respectively (Fig. 2E). Furthermore, western blot analysis revealed that the protein levels of NF-κB p65 were notably reduced after GAU1 knockdown (Fig. 2F and Original full length western blots image 2). These data suggest that GAU1 is a key factor in promoting NF-κB p65 expression and that GAU1 silencing could effectively inhibit the overexpression of NF-κB p65.

The IDTR strategy inhibits tumor growth in colorectal tumor cells

Given that oncolytic adenovirus H101 reactivated cGAS and GAU1 knockdown inhibited NF-κB p65 overexpression, we next tested the antitumor efficacy of IDTR by combining oncolytic adenovirus H101 infection and GAU1 lncRNA knockdown. We assessed tumor cell proliferation capacity in vitro using the classic CCK8 assay. As expected, we observed significant inhibition of cell growth at 72–120 hours following the combined treatment of GAU1 knockdown and H101 infection. After 120 hours of treatment, the cell growth inhibition rates of the combined group reached 62.3–71% (Fig. 3A, B, Inverted triangle) compared to those of the empty vector group of tumor cells (Fig. 3A, B, Square). In comparison, the GAU1-silenced group (Fig. 3A, B, Triangle) and the H101-treated group (Fig. 3A, B, cycle) exhibited 18.6% and 31% inhibition, respectively, after 120 hours of treatment. Furthermore, neither combined treatment nor single treatment significantly inhibited NCM460 normal enterocytes growth (Fig. 3C). Next, we tested the ability of tumor cells to form colonies in vitro using a classic colony formation assay after combined treatment with GAU1 knockdown and H101 infection. Similarly, we observed fewer and smaller cell colonies in the combined group (Fig. 3D, Lane 4) than in the group with GAU1 silencing alone (Fig. 3D, Lane 2) or the group treated with H101 alone (Fig. 3D, Lane 3). Approximately 77% fewer cell colonies were observed in the combined treatment group than in the empty vector group (Fig. 3E). We also used 5-ethynyl-2’-deoxyuridine (EdU) to label the synthesized DNA with fluorophores and observed cell growth using immunofluorescence technology. We observed 27.2% inhibition after treatment with H101 alone (Fig. 3F, G, Lane 3) and 26.6% inhibition after treatment with GAU1 alone (Fig. 3F, G, Lane 2) compared to those in the group with empty vector (Fig. 3F, G, Lane 1) in LOVO cells. However, 65% inhibition of LOVO cells was observed in the combined group (Fig. 3F, G, Lane 4). Similarly, we also observed the same trend of inhibition rates in SW620 cells (Fig. 3G, I). These data indicate that the combination of oncolytic adenovirus H101 infection and GAU1 lncRNA sgRNA significantly inhibited tumor cell growth in vitro.

The IDTR strategy decreases tumor growth in xenograft models

To examine the therapeutic efficacy of the IDTR approach in animal experiments, we chose to use a GAU1 antisense oligonucleotide (ASO) instead of a GAU1 sgRNA for increased safety and more efficient silencing. Real-time PCR analysis demonstrated that all three GAU1 ASOs reduced GAU1 expression, and GAU1 ASO-3 exhibited the most effective silencing effect in LOVO and SW620 cells (Fig. 4A). To examine whether the enhanced efficacy in vitro has a similar synergistic effect in vivo, we established a subcutaneous nude mouse xenograft model. When the volume of the tumor reached 100–150 mm³, we randomly divided the mice into four groups. The first group received an intratumor injection of PBS only as a placebo, the second group received an intratumor injection of H101 only, the third group received an intratumor injection of GAU1 ASO only, and the fourth group received an intratumor injection of a combination of H101 and GAU1 ASO. All groups were subjected to a three-day dosing regimen. The three monotreated groups received one intratumor injection every three days, and the combined group received intratumor injections of the GAU1 ASO on the first day and H101 on the second day (Fig. 4B). Compared to the PBS control group, monotreatment with either oncolytic adenovirus H101 or GAU1 ASO moderately inhibited tumor growth, resulting in approximately 33–44% inhibition of tumor growth. However, mice that received the combined treatment of GAU1 ASO and H101 demonstrated a notably greater reduction in tumor growth (n = 5, *P < 0.05) than those subjected to treatment administered individually, and the overall inhibition rate of tumor growth was approximately 88% (Fig. 4C). After 30 days of continuous treatment, the mice were sacrificed by cervical dislocation, and the tumors were removed and weighed. As expected, subcutaneous xenograft tumors in the combined group were significantly milder than those in the monotreatment groups, the tumor weight was reduced by more than 86% compared to that in the PBS group (Fig. 4D, Triangle), and one mouse tumor completely disappeared following the combination treatment (Fig. 4E). These data demonstrate that the combination of GAU1 ASO and H101 injection markedly improved the antitumor efficacy of IDTR in vivo.

There is a lack of cross-talk between GAU1 knockdown and H101 infection via the IDTR strategy

To further investigate whether cross-talk occurred between H101-induced cGAS reactivation and GAU1 knockdown-induced NF-κB p65 inhibition, we first examined the changes in cGAS expression after GAU1 knockdown. Western blot analysis showed that GAU1 knockdown did not change the expression of cGAS (Fig. 5A, Lane 3 and Original full length western blots image 3) compared to H101-induced cGAS activation (Fig. 5A, Lanes 2 and 4 and Original full length western blots image 3) in SW620 cells. Similarly, we observed the same phenomenon in LOVO cells (Fig. 5B and Original full length western blots image 3). The statistical analysis of the western blot data in triplicate further confirmed the above results (Fig. 5C). Next, we detected changes in NF-κB p65 after H101 infection. Western blot assays demonstrated that H101 infection did not change the expression of NF-κB p65 (Fig. 5D, Lane 2 and Original full length western blots image 4) compared to GAU1 knockdown-induced NF-κB p65 inhibition (Fig. 5D, Lanes 3 and 4 and Original full length western blots image 4) in SW620 cells. Similarly, we observed the same phenomenon in LOVO cells (Fig. 5E and Original full length western blots image 4), and the same trend was observed in the statistical analysis of western blot data in triplicate for the two tumor cell lines (Fig. 5F). These data indicate that there is no cross-talk between GAU1 knockdown and H101 infection in our IDTR strategy.

H101 infection increases H3K4me3 levels at the cGAS promoter

We next examined the exact mechanism underlying cGAS reactivation after oncolytic adenovirus H101 infection. Detection sites were set up at the cGAS promoter (P1–P2) and its upstream negative control site (P3–P4) (Fig. 6A). Chromatin immunoprecipitation (ChIP) assays showed that H101 treatment (MOI of 100) induced significant H3K4 trimethylation (me3) enrichment at the cGAS promoter (P1–P2) in SW620 (Fig. 6B) and LOVO tumor cells compared to the nontreated control (Fig. 6C). There was no statistically significant difference in the level of H3K4me3 within the negative control region (P3–P4) (Fig. 6D, E). Taken together, these results suggest that H101 infection activates cGAS transcription by increasing the level of H3K4me3 at the cGAS promoter in SW620 and LOVO tumor cells.

GAU1 knockdown decreases H3K27 acetylation at the NF-κB p65 promoter

We next investigated the precise mechanism underlying NF-κB p65 inhibition after GAU1 knockdown. We first investigated the location of mature GAU1 transcripts. By isolating nuclear and cytoplasmic RNA, we found that GAU1 was predominantly present in the nuclei of LOVO and SW620 cells (Fig. 7A, first panel, Lanes 1 and 3). The statistical analysis of GAU1 localization in triplicate further confirmed the above results (Fig. 7B). Considering that GAU1 is primarily localized in the nucleus, it might directly regulate the transcription of NF-κB p65 by targeting the NF-κB p65 genomic locus. To investigate whether GAU1 bound to the NF-κB p65 promoter, we detected sites within the NF-κB p65 transcriptional regulatory region, including a promoter site (P5–P6) and a negative control site (P7–P8) (Fig. 7C). Chromatin immunoprecipitation with RNA purification (ChIRP) assays were subsequently performed using biotin-labeled oligomers for chromatin fractionation. The NF-κB p65 promoter was selected as the detection site, and the GAPDH promoter was used as the negative control. ChIRP-qPCR showed that GAU1 was enriched in the NF-κB p65 promoter (P5–P6) in SW620 and LOVO cells (Fig. 7D, E, Square). However, GAU1 enrichment at the NF-κB p65 promoter significantly decreased after GAU1 knockdown (Fig. 7D,E, Triangle and Diamond). No notable difference was detected in the negative control site (P7–P8) following GAU1 knockdown in SW620 (Fig. 7F) and LOVO cells (Fig. 7G). These results show that GAU1 is a nuclear lncRNA that can directly bind to the NF-κB p65 promoter in SW620 and LOVO cells.

Fig. 7: LncRNA *GAU1* binds to the promoter of *NF-κB p65.*

We further elucidated how GAU1 could modulate NF-κB p65 transcription. We detected histone modifications at the NF-κB p65 promoter site (P5–P6) and negative control site (P7–P8) by ChIP‒qPCR (Fig. 7C). ChIP‒qPCR showed that GAU1 knockdown significantly decreased the level of H3K27 acetylation (ac) at the NF-κB p65 promoter (P5–P6) in SW620 (Fig. 8A) and LOVO (Fig. 8B) cells. We did not observe significant differences in H3K27ac in the negative control (P7–P8) (Fig. 8C, D). These data suggest that GAU1 knockdown diminishes the level of H3K27ac at the NF-κB p65 promoter.

**Fig. 8: *GAU1* knockdown decreases H3K27 acetylation at the *NF-κB p65* promoter.**

Discussion

In contrast to conventional single-target therapies, simultaneous targeting of various tumor-specific abnormalities has become an important and attractive approach for accessing antitumor therapies [21]; however, discovering suitable targets and effective methods is a global challenge for targeted tumor therapy. In this study, for the first time, we established a novel IDTR strategy for the simultaneous reactivation of silenced cGAS using the oncolytic adenovirus H101 and the inhibition of NF-κB p65 overexpression with GAU1 ASO, ultimately resulting in efficient antitumor therapeutic efficacy in vitro and in vivo (Fig. 8E). We also revealed for the first time an alternative therapeutic mechanism in which oncolytic adenovirus H101 could reactivate cGAS transcription by increasing H3K4me3 at the cGAS promoter and GAU1 knockdown could inhibit NF-κB p65 transcription by decreasing H3K27ac at the NF-κB p65 promoter, thereby providing a suitable dual-target and effective IDTR approach for promoting antitumor efficacy.

Notably, NF-κB p65 and cGAS are two frequently dysregulated cellular regulatory factors in tumors. Overexpression of NF-κB p65 and silenced expression of cGAS are often critical triggers contributing to tumor initiation, drug resistance, and treatment evasion [22,23,24]. Inhibition of NF-κB p65 overexpression could restrain tumor cell proliferation and invasion [14]. On the other hand, reactivation of cGAS expression enhances immune activity, facilitating the recognition and clearance of tumor cells and thereby increasing overall treatment efficacy [25, 26]. However, to our knowledge, reactivation of silenced cGAS expression and inhibition of NF-κB p65 overexpression have not been carried out simultaneously in tumor therapy. In this study, we chose colorectal cancer patients exhibiting simultaneous NF-κB overactivation and cGAS inactivation as the research model. We employed two classic colorectal cancer cell lines, SW620 and LOVO, and conducted both in vitro and in vivo experiments. Therefore, we successfully established a novel IDTR antitumor approach that restored NF-κB p65 and cGAS abnormalities at the same time and showed efficient therapeutic efficacy in tumors. Since each type of tumor has varying overexpressed and low-expressed targets, it is interesting to explore different combinations of over- and low-target effects by utilizing our IDTR strategy to obtain better antitumor therapeutic efficacy.

Notably, both cGAS and NF-κB p65 are key factors involved in immune regulation [27]. Activation of cGAS induces the production of type I interferons (IFNs), which initiate innate immune responses against tumor cells [28]. cGAS activation promotes the activation and recruitment of immune cells such as natural killer (NK) cells, dendritic cells (DCs), and T cells, enhancing antitumor immune responses [29, 30]. On the other hand, NF-κB p65 overactivation upregulates the expression of genes involved in cell survival, proliferation, and antiapoptotic pathways, promoting tumor cell survival and proliferation [31, 32]. Abnormal activation of NF-κB p65 also induces the production of proinflammatory cytokines and chemokines, creating a protumorigenic inflammatory microenvironment that supports tumor growth and progression [33]. Moreover, NF-κB p65 overactivation can induce the expression of immune checkpoint molecules such as PD-L1 on tumor cells or immune cells, contributing to immune evasion by suppressing antitumor immune responses [34, 35]. In this study, we found that simultaneously activating aberrantly inactivated cGAS and inhibiting the overactivation of NF-κB p65 in tumors produces a potent synergistic antitumor effect. A compelling hypothesis is that simultaneously restoring the function of these two key factors may involve the coordinated regulation of tumor immune cascade pathways, providing a more comprehensive intervention in tumor cell immune evasion mechanisms and thereby enhancing the antitumor effect. An intriguing research direction going forward is to focus on elucidating the detailed molecular mechanisms underlying the synergistic antitumor effects achieved by correcting abnormalities in both cGAS and NF-κB p65.

Studies have shown that poly(rC)-binding protein 1 (PCBP1) is recruited to the cGAS protein to increase its activity and enhance the affinity of cGAS for its ligand during viral infection [36]. Recent investigations have proposed that viral control over the host might be linked to viral transcriptional regulators (vTRs) [37]. Intriguingly, the E1A protein encoded by the adenovirus genome could interact with retinoblastoma protein (pRB) and EP300 to activate the transcription of multiple genes that promote the cell cycle and proliferation [38]. Our previous study showed that an oncolytic adenovirus called H101 has been clinically approved for the treatment of malignancies because it selectively kills tumor cells through viral oncolysis and does not have significant cytopathic effects on normal cells [21]. H101 infects tumor cells by releasing its genome, which induces tumor cell lysis and apoptosis [39]. Therefore, these studies led to the hypothesis that the oncolytic adenovirus H101 is likely to reactivate cGAS. Based on this hypothesis and subsequent validation, for the first time, we discovered that oncolytic adenovirus H101 could actually induce the reactivation of cGAS. We first revealed that cGAS reactivation was caused by increased H3K4 trimethylation of the cGAS promoter. We speculate that the dynamics of histone modification may be associated with vTRs derived from H101. However, we have yet to determine which vTRs are involved in the transcriptional regulation of cGAS, which will be an interesting avenue for our future research. It should also be emphasized that we cannot theoretically eliminate other chromatin modifications involved in cGAS reactivation in a positive or negative manner. Thus, it would be of great interest to focus on the identification of other causes to better understand the reactivation of cGAS.

NF-κB p65 proteins are mainly localized in the cytoplasm and form an inactive trimeric complex with the inhibitory protein IκB [40]. Activation of the NF-κB p65 protein occurs mainly through phosphorylation of the IκB protein, which is ubiquitinated and degraded to release the active NF-κB p65 protein [41, 42]. NKILA lncRNA can bind to NF-κB/IκB and shield the phosphorylation site of I-κB, thereby inhibiting IκB kinase (IKK)-induced I-κB phosphorylation, which in turn inhibits the activation of NF-κB proteins and suppresses cancer metastasis [43]. Many studies have focused on the regulation of NF-κB p65 protein activity by lncRNAs in the cytoplasm through influencing IκB dissociation. However, in this study, for the first time, we showed that the knockdown of the lncRNA GAU1 by utilizing ASOs or single-guide RNAs (sgRNAs) could inhibit the overexpression of NF-κB p65 in human colorectal tumors. GAU1, which was initially discovered and named in our prior research, was first identified in neuroblastoma and retinoblastoma [44]. This prompted us to delve deeper into the underlying mechanisms by which GAU1 promotes the transcriptional activation of NF-κB p65. Upon further investigation of the underlying mechanisms, we first revealed that NF-κB p65 inhibition was caused by decreased H3K27 acetylation of the NF-κB p65 promoter. Notably, the ability of GAU1 to inhibit NF-κB p65 overexpression is unlikely to be completely regulated by GAU1 due to the multiple functions of lncRNAs. Thus, further exploration of the precise role of GAU1 in human colorectal tumorigenesis is of potential interest.

Notably, in this study, the combination of H101 and GAU1 silencing was chosen as the treatment strategy for tumors. Although we observed a certain degree of tumor suppression with H101 infection, its therapeutic efficacy remains suboptimal [45]. Therefore, enhancing the therapeutic efficacy of H101 is also an important issue that needs to be considered. Concurrent administration of siGNAQ and H101 significantly impedes the proliferation of UM cells and triggers apoptosis [46]. Additionally, intratumoral injection of H101 in conjunction with concurrent chemoradiotherapy substantially enhances the therapeutic efficacy against advanced cervical cancer, leading to improved patient survival rates [47]. These studies indicate that combination therapy is an effective approach for enhancing the therapeutic efficacy of H101. In this study, we found that when H101 was combined with GAU1 silencing, the therapeutic efficacy against tumors was significantly enhanced. Compared to the monotherapy group, the combination therapy group showed a 2–3.5-fold increase in the inhibition of tumor growth. This combined approach also provides innovative insights for our subsequent development of targeted antitumor treatment strategies.

Notably, CRISPR/dCas9 is internationally recognized as a powerful gene knockdown tool that enables the establishment of stable knockdown states in cells [48]. We confirmed the effective knockdown of GAU1 in vitro, ensuring reliable experimental outcomes. However, we chose to use GAU1-ASO for the treatment of mouse xenograft tumors because it represents a safer and convenient alternative method. ASO drugs exclusively involve posttranscriptional interference, disrupting RNA levels without altering the genome, and are also recognized as clinically accepted gene silencing therapy approaches [49, 50]. This provides potential for the development of clinical-grade GAU1-ASO drugs. Intratumoral injection allows for the drug to directly target the tumor site, improving drug stability and enabling maximum therapeutic efficacy with smaller doses [51]. In contrast, intravenous administration of drugs requires circulation throughout the body to reach the tumor site, which can lead to drug degradation and accumulation at other sites. However, to ensure the effectiveness of the treatment, it is necessary to increase the dosage of the drug, potentially increasing the risk of toxicity due to higher drug concentrations.

Materials and methods

Cell culture

The human cell lines SW620 (ATCC, CCL-227), LOVO (ATCC, CCL-229), NCM460 (Shanghai Cell Bank, TCH-C450), and 293 T (ATCC, CRL-3216) were cultured in DMEM (Gibco, Waltham, MA) supplemented with 10% certified heat-inactivated fetal bovine serum (Yeasen, Shanghai, China), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. The recombinant adenovirus H101 was generously provided by Shanghai Sunway Biotech (Shanghai, China) and was stored at −80 °C as recommended.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

Total RNA was extracted from LOVO and SW620 cells using TRI-REAGENT (Invitrogen, Waltham, MA). The RNA was then reverse transcribed using qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) and analyzed with a LightCycle96 quantitative PCR system (Roche, Basel, CH). The relative mRNA expression levels of genes were calculated using the 2^-ΔΔCt method after normalization to the expression of GAPDH, which served as an internal loading control. To detect the expression levels of GAU1, cGAS, and NF-κB p65, we designed qPCR primers, which were synthesized by Sangon (Shanghai, China), according to the manufacturer’s protocol and a previous study. The primer sequences can be found in the supplementary materials (Table S1).

Western blot analysis

Cells were collected at the specified times and washed twice with PBS. The cell extracts were prepared using lysis buffer and then centrifuged at 13,000 × g for 30 minutes at 4 °C. The protein concentration was quantified using a BCA kit (Yeasen, Shanghai, China). The supernatant was collected, and 5× SDS protein loading buffer was added in proportion, followed by incubation at 100 °C for 10 minutes. Proteins were separated on a 10–12% SDS‒PAGE gel and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA). The membranes were blocked with 5% nonfat milk in Tris‐buffered saline supplemented with 0.1% Tween 20 at room temperature for 2 hours. The primary antibody was diluted with 1× TBST according to the instructions and incubated overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibodies for another 2 hours at room temperature. Immunoblots were visualized using an enhanced chemiluminescence detection kit (Yeasen, Shanghai, China) with a chemiluminescence imaging analysis system (Tanon, Shanghai, China). Relative integrated density values were calculated using ImageJ software. The following antibodies were used: anti-cGAS, cGAS (E5V3W); rabbit mAb #79978 (CST, Boston, MA); anti-NF-κB, p65 (D14E12); XP® rabbit mAb #8242 (CST, Boston, MA); and GAPDH (Sigma‒Aldrich, Louis, MI).

Chromatin immunoprecipitation (ChIP) assay

ChIP was performed using an EZ-Magna ChIP A/G kit (Millipore, Boston, MA) following the manufacturer’s instructions and a previously reported protocol. The anti-H3K27ac and H3K4me3 antibodies used for RNA-ChIP were also used for ChIP. Normal rabbit IgG was used as a negative control. Anti-H3K27ac: Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb #8173(CST, Boston, MA); Anti-H3K4me Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751(CST, Boston, MA); Anti- IgG: Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(CST, Boston, MA). The primer sequences are listed in the supplementary materials (Table S1).

Nuclear fractionation

Cells were collected at the specified times and washed twice with PBS. Subsequently, 10⁷ cells were harvested and resuspended in 1 ml of ice-cold DEPC-PBS, 1 ml of buffer C1 (1.28 M sucrose, 40 mM Tris–HCl [pH 7.5], 20 mM MgCl₂, 4% Triton X-100), and 3 ml of RNase-free water. The plates were then incubated for 15 minutes on ice. Afterward, the cells were centrifuged for 15 minutes at 3000 rpm. The resulting supernatant containing the cytoplasmic component and the pellet containing the nuclear fraction were both retained for RNA extraction.

CRISPR/dCas9-KRAB-mediated gene editing and cell transfection

Single guide RNA sequences were designed based on the promoter of the human GAU1 lncRNA and predicted using the online tool created by Prof. Zhang (http://crispr.mit.edu/). The sgRNA sequences can be found in the supplementary materials, specifically in the primer table. The targeting plasmids and packaging plasmids were cotransfected into 293 T cells using Lipofectamine 3000 (Invitrogen, Waltham, MA). After 48 or 72 hours, the supernatant was purified, ultracentrifuged, and collected for cell infection. Prior to transfection, 1 × 0⁵ LOVO and SW620 cells were cultured in 6-well plates with 2 ml of complete medium for 24 hours until they reached 50% confluence. The vectors carrying sgRNA were transfected into the LOVO and SW620 cell lines using Lipofectamine 3000 (Invitrogen, Waltham, MA) and cultured in serum-free Opti-MEM according to the manufacturer’s instructions. Puromycin (5 µg/ml) was added 72 hours after infection, and the surviving cells were collected for selection.

GAU1 antisense oligonucleotides

Cells were transfected at 60–80% confluence with 20–50 nmol/L antisense oligonucleotides (ASOs) (RiboBio, Guangzhou, China) targeting GAU1 using Lipofectamine 3000 (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. As a control, cells were transfected with scrambled ASO. Forty-eight hours after transfection, the cells were harvested for further analysis. The ASO sequences are listed in the supplementary materials (Table S1).

ChIRP assay

Biotin-labeled probes against GAU1 were designed according to an online tool (www.singlemoleculefish.com). Probes against LacZ RNA were used as negative controls. Probes against U1 were used as positive controls. A total of 2 × 10⁷ SW620 and LOVO cells were resuspended in precooled PBS buffer and crosslinked with 3% formaldehyde, and the reaction was quenched with glycine. The cells were then pelleted at 1000 × g for 10 min and resuspended in swelling buffer containing 0.1 M Tris (pH 7.0), 10 mM KOAc, and 15 mM MgOAc. 1% NP-40, 1 mM DTT, 1 mM PMSF, complete protease inhibitor, and 0.1 U/µl Superase-in for 10 min on ice. The nuclei were further resolved in 50 mM Tris 7.0, 10 mM EDTA, and 1% SDS, and DTT, PMSF and Superase-in were added. The chromatin was then sonicated to 100–500 bp in size and diluted in 500 mM NaCl, 1% SDS, 100 mM Tris 7.0, 10 mM EDTA, and 15% formamide. Prebinding probe oligos were added to streptavidin beads. The beads were mixed with the cell lysate and hybridized at 37 °C overnight on an end-to-end shaker. Subsequently, the beads were washed five times with 1 ml of prewarmed wash buffer for 5 min per wash. Then, 100 µl of elution buffer containing 50 mM NaHCO₃, 1% SDS, 200 mM NaCl, 20 U of RNase A and 20 U of RNase H was added, and the mixture was eluted at 37 °C for 1 h. Subsequently, the supernatant was collected, 10 µl of protease K (Thermo Fisher, Waltham, MA) was added, and the mixture was incubated at 55 °C for 2 h. DNA was then extracted with phenol:chloroform:isoamyl and precipitated with ethanol at −80 °C. Furthermore, the purified DNA was subjected to qPCR analysis. The primer sequences are listed in the supplementary materials (Table S1).

Cotreatment of tumor cells with GAU1 sgRNAs/ASOs and oncolytic adenovirus H101

Method 1

LOVO and SW620 cell lines stably transfected with GAU1 CRISPR/dCas9-KRAB and empty vector were constructed, 30–50% of the above cells were transferred to a 6-well plate, and the cells were infected with H101 at a MOI of 100. The control group included cells transfected with empty vector or PBS.

Method 2

LOVO and SW620 cells were combined in a 6-well plate with 30–50% Lipofectamine 3000 (Invitrogen, Waltham, MA) and transfected with 50 nmol/negative control ASO or GAU1 ASO according to the manufacturer’s instructions (Invitrogen, Waltham, MA). After incubation overnight, the cells were infected with H101 at an infection factor of 100 MOI. The control group included cells transfected with negative control ASO or PBS.

Cell counting kit-8 (CCK-8) assay

For the CCK-8 assay, the transfected cells (1 × 10³ cells/well) were seeded into 96-well plates in quintuplicate and cultured until they were entirely adherent. CCK-8 solution (10 μl, Yeasen, Shanghai, China) was added to each well at 0, 24, 48, 72, and 96 hours. After 2 hours of incubation, the absorbance at 450 nm was measured on a microplate reader (SpectraMax iD3).

Colony formation assay

The cells were also counted and adjusted to 1000 cells per well in 6-well plates and then placed into a humidified incubator for 1 week. Then, the cells were washed twice in phosphate-buffered saline (PBS), fixed with 70% methanol for 30 min and stained for 30 min with crystal violet. The colonies were counted in assays. Finally, GraphPad Prism 8.0 was used to analyze the number of cell colonies in each well.

EdU assay

For EdU labeling, 1 × 10⁵ cells were seeded in each well of a 6-well plate in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Twenty-four hours later, 10 μM EdU (Beyotime, Shanghai, China) was added to the medium. After another 72 hours, the cells were fixed for EdU staining or for further tests. For EdU staining, the cells were fixed with methanol, washed twice with phosphate-buffered saline (PBS), incubated in 3% bovine serum albumin (BSA) in PBS, and then incubated in 0.5% Triton® X-100 in PBS for 20 min at room temperature. The cells were then incubated with freshly made Click-iT reaction cocktail, which contained azide-conjugated Alexa 594 (Invitrogen, Waltham, MA), for 30 min at room temperature in the dark. The cells were further stained with 4′,6-diamidino-2-phenylindole (DAPI, for nuclear staining, 1 μg/ml, Sigma‒Aldrich, Louis, MI) and then mounted in standard mounting media. The stained cells were examined with a Nikon camera. To determine the percentage of EdU-positive cells, the number of red fluorescent (Alexa 594-stained) cells was divided by the number of blue fluorescent (DAPI-stained) cells. The experiment was performed in triplicate, and the data are presented as the average of three independent experiments.

Tumor xenograft model in nude mice

A xenograft model of LOVO cell tumors was established by injecting 5 × 10⁶ cells into the right armpit of female nude mice aged 4-6 weeks. When the tumors reached the desired mean tumor volume (100–125 mm³) (length (mm) × width (mm) ²/2), the animals were randomly divided into four groups. The GAU1 ASO + H101 group was injected with 20 µg GAU1 ASO via the intertumoral route on Days 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28, and adenovirus H101 was injected via the intertumoral route with 1 × 10⁸ PFU/mouse on Days 2, 5, 8, 11, 14, 17, 20, 23, 26, and 29. The GAU1 ASO group was injected with 10 µg of GAU1 ASO 10 times. The H101 adenoviral group was subjected to 10 intertumoral injections of H101, and the control group was injected with PBS 10 times. Tumor size was measured every 3 days with a Vernier caliper. The animal experiments were carried out in accordance with the guidelines of the SPF Animal Center of Tongji University.

Statistical analysis

All experiments were performed in triplicate, and the data are expressed as the mean ± SD. The comparative CT method was applied in the quantitative real-time RT‒PCR assay according to the delta‒delta CT method. The data are presented as the mean ± SD, the differences between two groups were calculated by unpaired two-tailed t tests, and the results were considered to be statistically significant. If P < 0.05, it is considered to have statistical difference, it is represented by ‘*’; If P < 0.01, it is indicated by ‘**’; If P < 0.001, it is indicated by ‘***’; If P < 0.0001, it is indicated by ‘****’.

Data availability

The data that support the findings of this study are available on request from the corresponding author, He Zhang, upon reasonable request. The original western blot images are provided in the Supplementary File.

References

Mars N, Koskela JT, Ripatti P, Kiiskinen TTJ, Havulinna AS, Lindbohm JV, et al. Polygenic and clinical risk scores and their impact on age at onset and prediction of cardiometabolic diseases and common cancers. Nat Med. 2020;26:549–57.
Article CAS PubMed Google Scholar
Chen Q, Xu S, Liu S, Wang Y, Liu G. Emerging nanomedicines of paclitaxel for cancer treatment. J Control Release. 2022;342:280–94.
Article CAS PubMed Google Scholar
Skoulidis F, Goldberg ME, Greenawalt DM, Hellmann MD, Awad MM, Gainor JF, et al. STK11/LKB1 mutations and PD-1 inhibitor resistance in KRAS-mutant lung adenocarcinoma. Cancer Discov. 2018;8:822–35.
Article CAS PubMed PubMed Central Google Scholar
Peng F, Liao M, Qin R, Zhu S, Peng C, Fu L, et al. Regulated cell death (RCD) in cancer: key pathways and targeted therapies. Signal Transduct Target Ther. 2022;7:286.
Article CAS PubMed PubMed Central Google Scholar
Saleem H, Kulsoom Abdul U, Kucukosmanoglu A, Houweling M, Cornelissen FMG, Heiland DH, et al. The TICking clock of EGFR therapy resistance in glioblastoma: target independence or target compensation. Drug Resist Updat. 2019;43:29–37.
Article PubMed Google Scholar
Rozeman EA, Menzies AM, van Akkooi ACJ, Adhikari C, Bierman C, van de Wiel BA, et al. Identification of the optimal combination dosing schedule of neoadjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma (OpACIN-neo): a multicentre, phase 2, randomised, controlled trial. Lancet Oncol. 2019;20:948–60.
Article CAS PubMed Google Scholar
Amaria RN, Reddy SM, Tawbi HA, Davies MA, Ross MI, Glitza IC, et al. Neoadjuvant immune checkpoint blockade in high-risk resectable melanoma. Nat Med. 2018;24:1649–54.
Article CAS PubMed PubMed Central Google Scholar
Yao H, Song W, Cao R, Ye C, Zhang L, Chen H, et al. An EGFR/HER2-targeted conjugate sensitizes gemcitabine-sensitive and resistant pancreatic cancer through different SMAD4-mediated mechanisms. Nat Commun. 2022;13 https://doi.org/10.1038/s41467-022-33037-x.
Qiu S, Zhong X, Meng X, Li S, Qian X, Lu H, et al. Mitochondria-localized cGAS suppresses ferroptosis to promote cancer progression. Cell Res. 2023;33:299–311.
Article CAS PubMed PubMed Central Google Scholar
Zhang X, Bai XC, Chen ZJ. Structures and mechanisms in the cGAS-STING innate immunity pathway. Immunity. 2020;53:43–53.
Article CAS PubMed Google Scholar
Xia T, Konno H, Barber GN. Recurrent loss of STING signaling in melanoma correlates with susceptibility to viral oncolysis. Cancer Res. 2016;76:6747–59.
Article CAS PubMed Google Scholar
Xia T, Konno H, Ahn J, Barber GN. Deregulation of STING signaling in colorectal carcinoma constrains DNA damage responses and correlates with tumorigenesis. Cell Rep. 2016;14:282–97.
Article CAS PubMed Google Scholar
Hu S, Fang Y, Chen X, Cheng T, Zhao M, Du M, et al. cGAS restricts colon cancer development by protecting intestinal barrier integrity. Proc Natl Acad Sci USA. 2021;118 https://doi.org/10.1073/pnas.2105747118.
Yu H, Lin L, Zhang Z, Zhang H, Hu H. Targeting NF-kappaB pathway for the therapy of diseases: mechanism and clinical study. Signal Transduct Target Ther. 2020;5:209.
Article CAS PubMed PubMed Central Google Scholar
Altea-Manzano P, Doglioni G, Liu Y, Cuadros AM, Nolan E, Fernandez-Garcia J, et al. A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation resulting in pro-metastatic NF-kappaB signaling. Nat Cancer. 2023;4:344–64.
Article CAS PubMed PubMed Central Google Scholar
Du F, Qi X, Zhang A, Sui F, Wang X, Proud CG, et al. MRTF-A-NF-kappaB/p65 axis-mediated PDL1 transcription and expression contributes to immune evasion of non-small-cell lung cancer via TGF-beta. Exp Mol Med. 2021;53:1366–78.
Article CAS PubMed PubMed Central Google Scholar
Liu X, Hong T, Parameswaran S, Ernst K, Marazzi I, Weirauch MT, et al. Human Virus Transcriptional Regulators. Cell. 2020;182:24–37.
Article CAS PubMed PubMed Central Google Scholar
Miyazawa M, Noguchi K, Kujirai M, Katayama K, Yamagoe S, Sugimoto Y. IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3. J Biol Chem. 2018;293:662–76.
Article CAS PubMed Google Scholar
Ramasubramanyan S, Osborn K, Al-Mohammad R, Naranjo Perez-Fernandez IB, Zuo J, Balan N, et al. Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression. Nucleic Acids Res. 2015;43:3563–77.
Article CAS PubMed PubMed Central Google Scholar
Lu Z, Li Y, Wang J, Che Y, Sun S, Huang J, et al. Long non-coding RNA NKILA inhibits migration and invasion of non-small cell lung cancer via NF-kappaB/Snail pathway. J Exp Clin Cancer Res. 2017;36:54.
Article PubMed PubMed Central Google Scholar
Zhang H, Wang H, Zhang J, Qian G, Niu B, Fan X, et al. Enhanced therapeutic efficacy by simultaneously targeting two genetic defects in tumors. Mol Ther. 2009;17:57–64.
Article PubMed Google Scholar
Volmar MNM, Cheng J, Alenezi H, Richter S, Haug A, Hassan Z, et al. Cannabidiol converts NF-kappaB into a tumor suppressor in glioblastoma with defined antioxidative properties. Neuro Oncol. 2021;23:1898–910.
Article CAS PubMed PubMed Central Google Scholar
Yu L, Liu P. Cytosolic DNA sensing by cGAS: regulation, function, and human diseases. Signal Transduct Target Ther. 2021;6:170.
Article CAS PubMed PubMed Central Google Scholar
Wu MZ, Cheng WC, Chen SF, Nieh S, O’Connor C, Liu CL, et al. miR-25/93 mediates hypoxia-induced immunosuppression by repressing cGAS. Nat Cell Biol. 2017;19:1286–96.
Article CAS PubMed PubMed Central Google Scholar
Wang Y, Luo J, Alu A, Han X, Wei Y, Wei X. cGAS-STING pathway in cancer biotherapy. Mol Cancer. 2020;19:136.
Article PubMed PubMed Central Google Scholar
Lv MZ, Chen MX, Zhang R, Zhang W, Wang CG, Zhang Y, et al. Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy. Cell Res. 2020;30:966–79.
Article CAS PubMed PubMed Central Google Scholar
Jin X, Ding D, Yan Y, Li H, Wang B, Ma L, et al. Phosphorylated RB promotes cancer immunity by inhibiting NF-kappaB activation and PD-L1 expression. Mol Cell. 2019;73:22–35.e26.
Article CAS PubMed Google Scholar
Si W, Liang H, Bugno J, Xu Q, Ding X, Yang K, et al. Lactobacillus rhamnosus GG induces cGAS/STING-dependent type I interferon and improves response to immune checkpoint blockade. Gut. 2022;71:521–33.
Article CAS PubMed Google Scholar
Marcus A, Mao AJ, Lensink-Vasan M, Wang L, Vance RE, Raulet DH. Tumor-derived cGAMP triggers a STING-mediated interferon response in non-tumor cells to activate the NK cell response. Immunity. 2018;49:754–63.e754.
Article CAS PubMed PubMed Central Google Scholar
Deng L, Liang H, Xu M, Yang X, Burnette B, Arina A, et al. STING-dependent cytosolic DNA sensing promotes radiation-induced type i interferon-dependent antitumor immunity in immunogenic tumors. Immunity. 2014;41:843–52.
Article CAS PubMed PubMed Central Google Scholar
Lwin T, Hazlehurst LA, Li Z, Dessureault S, Sotomayor E, Moscinski LC, et al. Bone marrow stromal cells prevent apoptosis of lymphoma cells by upregulation of anti-apoptotic proteins associated with activation of NF-kappaB (RelB/p52) in non-Hodgkin’s lymphoma cells. Leukemia. 2007;21:1521–31.
Article CAS PubMed Google Scholar
Sanda T, Iida S, Ogura H, Asamitsu K, Murata T, Bacon KB, et al. Growth inhibition of multiple myeloma cells by a novel IkappaB kinase inhibitor. Clin Cancer Res. 2005;11:1974–82.
Article CAS PubMed Google Scholar
Jin Y, Cai Q, Wang L, Ji J, Sun Y, Jiang J, et al. Paracrine activin B-NF-kappaB signaling shapes an inflammatory tumor microenvironment in gastric cancer via fibroblast reprogramming. J Exp Clin Cancer Res. 2023;42:269.
Article CAS PubMed PubMed Central Google Scholar
Zhou Y, Jin X, Yu H, Qin G, Pan P, Zhao J, et al. HDAC5 modulates PD-L1 expression and cancer immunity via p65 deacetylation in pancreatic cancer. Theranostics. 2022;12:2080–94.
Article CAS PubMed PubMed Central Google Scholar
Grinberg-Bleyer Y, Oh H, Desrichard A, Bhatt DM, Caron R, Chan TA, et al. NF-kappaB c-rel is crucial for the regulatory T cell immune checkpoint in cancer. Cell. 2017;170:1096–1108.e1013.
Article CAS PubMed PubMed Central Google Scholar
Liao CY, Lei CQ, Shu HB. PCBP1 modulates the innate immune response by facilitating the binding of cGAS to DNA. Cell Mol Immunol. 2021;18:2334–43.
Article CAS PubMed Google Scholar
Arvey A, Tempera I, Tsai K, Chen HS, Tikhmyanova N, Klichinsky M, et al. An atlas of the Epstein-Barr virus transcriptome and epigenome reveals host-virus regulatory interactions. Cell Host Microbe. 2012;12:233–45.
Article CAS PubMed PubMed Central Google Scholar
Ferrari R, Pellegrini M, Horwitz GA, Xie W, Berk AJ, Kurdistani SK. Epigenetic reprogramming by adenovirus e1a. Science. 2008;321:1086–8.
Article CAS PubMed PubMed Central Google Scholar
Lu W, Zheng S, Li XF, Huang JJ, Zheng X, Li Z. Intra-tumor injection of H101, a recombinant adenovirus, in combination with chemotherapy in patients with advanced cancers: a pilot phase II clinical trial. World J Gastroenterol. 2004;10:3634–8.
Article CAS PubMed PubMed Central Google Scholar
Hoffmann A, Levchenko A, Scott ML, Baltimore D. The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation. Science. 2002;298:1241–5.
Article CAS PubMed Google Scholar
Liu X, Tang R, Xu J, Tan Z, Liang C, Meng Q, et al. CRIP1 fosters MDSC trafficking and resets tumour microenvironment via facilitating NF-kappaB/p65 nuclear translocation in pancreatic ductal adenocarcinoma. Gut. 2023; https://doi.org/10.1136/gutjnl-2022-32934910.1136/gutjnl-2022-329349.
Hu Y, Zhang B, Lu P, Wang J, Chen C, Yin Y, et al. The positive regulatory loop of TCF4N/p65 promotes glioblastoma tumourigenesis and chemosensitivity. Clin Transl Med. 2022;12:e1042.
Article CAS PubMed PubMed Central Google Scholar
Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. 2017;168:707–23.
Article CAS PubMed PubMed Central Google Scholar
Chai P, Jia R, Jia R, Pan H, Wang S, Ni H, et al. Dynamic chromosomal tuning of a novel GAU1 lncing driver at chr12p13.32 accelerates tumorigenesis. Nucleic Acids Res. 2018;46:6041–56.
Article CAS PubMed PubMed Central Google Scholar
Liang M. Oncorine, the world first oncolytic virus medicine and its update in China. Curr Cancer Drug Targets. 2018;18:171–6.
Article CAS PubMed Google Scholar
Li Y, He J, Qiu C, Shang Q, Qian G, Fan X, et al. The oncolytic virus H101 combined with GNAQ siRNA-mediated knockdown reduces uveal melanoma cell viability. J Cell Biochem. 2019;120:5766–76.
Article CAS PubMed Google Scholar
Zhang X, Wang Y, Lv X, Wang F, Zhou Q, Zhang F, et al. Intratumoral injection of oncolytic virus (H101) in combination with concurrent chemoradiotherapy for locally advanced cervical cancer. Int J Gynecol Cancer. 2023;33:1051–6.
Article PubMed Google Scholar
Dominguez AA, Lim WA, Qi LS. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol. 2016;17:5–15.
Article CAS PubMed Google Scholar
Crooke ST, Baker BF, Crooke RM, Liang XH. Antisense technology: an overview and prospectus. Nat Rev Drug Discov. 2021;20:427–53.
Article CAS PubMed Google Scholar
Rinaldi C, Wood MJA. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat Rev Neurol. 2018;14:9–21.
Article CAS PubMed Google Scholar
Spalato-Ceruso M, Bouteiller F, Guegan JP, Toulmonde M, Bessede A, Kind M, et al. Pembrolizumab combined with low-dose cyclophosphamide and intra-tumoral injection of the toll-like receptor 4 agonist G100 in patients with advanced pretreated soft tissue sarcoma: results from the PEMBROSARC basket study. J Hematol Oncol. 2022;15:157.
Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (82372705, 31870748), the Shanghai Oriental Elite Project (2000152009), the National Key Research and Development Plan (2017YFE0196300), the Shanghai Natural Science Foundation (22ZR1466100), the Fundamental Research Funds for the Central Universities (22120230292), the ShuGuang Project of Shanghai Municipal Education Commission and Shanghai Education Development Foundation (17SG19), the Outstanding Yong Medical Scholar of Shanghai Municipal Commission of Health and Family Planning (2017YQ067), the Outstanding Yong Scholar Grant of Tongji University (PA2019000239), and the Startup Funding of Frontier Science Research Center for Stem Cells & Shanghai East Hospital of Tongji University (DFRC2019003).

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These authors contributed equally: Xiaoyu Zhang, Tianyi Ding.

Authors and Affiliations

State Key Laboratory of Cardiology and Medical Innovation Center, Institute for Regenerative Medicine, Shanghai East Hospital, Frontier Science Research Center for Stem Cells, School of Life Science and Technology, Tongji University, Shanghai, 200092, China
Xiaoyu Zhang, Tianyi Ding, Fan Yang, Haowen Xu, Jixing Zhang, Yiran Bai, Yibing Shi, Jiaqi Yang, Chaoqun Chen & He Zhang
Jiangxi Province Key Laboratory of Organ Development and Epigenetics, Clinical Medical Research Center, Affiliated Hospital of Jinggangshan University, Medical Department of Jinggangshan University, Ji’an, 343009, China
Xiaoyu Zhang, Tianyi Ding, Fan Yang, Haowen Xu, Jixing Zhang, Yiran Bai, Yibing Shi, Jiaqi Yang, Chaoqun Chen & He Zhang
School of Life Science, Jinggangshan University, Ji’an, 343009, China
Xiaoyu Zhang, Tianyi Ding, Fan Yang, Haowen Xu, Jixing Zhang, Yiran Bai, Yibing Shi, Jiaqi Yang, Chaoqun Chen & He Zhang

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Xiaoyu Zhang
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Tianyi Ding
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Fan Yang
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Haowen Xu
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Jixing Zhang
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Jiaqi Yang
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Chaoqun Chen
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He Zhang
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Contributions

Xiaoyu Zhang: conceptualization, investigation, methodology, experiment, data curation, writing—original draft, writing—review & editing. Tianyi Ding: conceptualization, investigation, methodology, experiment, writing—original draft, writing—review & editing. Fan Yang: validation. Jixing Zhang: methodology. Haowen Xu: resources. Yibing Shi: investigation. Yiran Bai: investigation. Jiaqi Yang: methodology. Chaoqun Chen: data curation. He Zhang: project administration, funding acquisition, conceptualization, supervision, writing—original draft. All authors have read and approved the final manuscript.

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Correspondence to He Zhang.

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Supplementary information

Original full-length western blots

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Zhang, X., Ding, T., Yang, F. et al. Induced dual-target rebalance simultaneously enhances efficient therapeutical efficacy in tumors. Cell Death Discov. 10, 249 (2024). https://doi.org/10.1038/s41420-024-02018-y

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Received: 16 February 2024
Revised: 04 May 2024
Accepted: 08 May 2024
Published: 23 May 2024
DOI: https://doi.org/10.1038/s41420-024-02018-y
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Induced dual-target rebalance simultaneously enhances efficient therapeutical efficacy in tumors

Abstract

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Introduction

Results

Identifying tumor models for simultaneously correcting cGAS and NF-κB p65 abnormalities

The oncolytic adenovirus H101 significantly activated cGAS expression

GAU1 can inhibit the overexpression of NF-κB p65

The IDTR strategy inhibits tumor growth in colorectal tumor cells

The IDTR strategy decreases tumor growth in xenograft models

There is a lack of cross-talk between GAU1 knockdown and H101 infection via the IDTR strategy

H101 infection increases H3K4me3 levels at the cGAS promoter

GAU1 knockdown decreases H3K27 acetylation at the NF-κB p65 promoter

Discussion

Materials and methods

Cell culture

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

Western blot analysis

Chromatin immunoprecipitation (ChIP) assay

Nuclear fractionation

CRISPR/dCas9-KRAB-mediated gene editing and cell transfection

GAU1 antisense oligonucleotides

ChIRP assay

Cotreatment of tumor cells with GAU1 sgRNAs/ASOs and oncolytic adenovirus H101

Method 1

Method 2

Cell counting kit-8 (CCK-8) assay

Colony formation assay

EdU assay

Tumor xenograft model in nude mice

Statistical analysis

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

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Competing interests

Additional information

Supplementary information

Supplementary information

Original full-length western blots

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